Gastrointestinal Pathology, An Issue of Gastroenterology Clinics (The Clinics: Internal Medicine)

Gastroenterol Clin N Am 36 (2007) xi–xiii

GASTROENTEROLOGY CLINICS OF NORTH AMERICA

Preface

Robert D. Odze, MD, FRCPc Guest Editor

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ue to recent advancements in endoscopic and radiologic techniques, pathologists and clinicians are increasingly exposed to early neoplastic precursor lesions of the gastrointestinal (GI) tract, liver, biliary tract and pancreas. This has led to a better understanding of the molecular pathogenesis of cancer development in these organs, and in disorders such as Barrett’s esophagus and inflammatory bowel disease, has contributed to the development of models of tumor progression that may be applicable to other organ systems in humans. Scientific advancements with regard to the biological characteristics of neoplastic precursor lesions have also translated into early detection and improved patient survival as a result of use of screening and surveillance programs. In this issue of Gastroenterology Clinics of North America, internationally recognized pathologists have contributed timely reviews focused on the pathologic features, molecular pathogenesis, natural history, clinical relevance, and treatment of neoplastic precursor lesions of the GI tract, liver, biliary tract and pancreas. A cancer precursor lesion is defined as neoplastic epithelium that is confined to the basement membrane of the mucosal compartment in which it is normally located or has not invaded the surrounding tissues. In some diseases, such as Barrett’s esophagus and chronic gastritis, the dysplasia–carcinoma sequence is preceded by epithelial metaplasia. As discussed in some of the review articles in this issue, however, it is now increasingly apparent that even some types of metaplastic epithelium possess genetic, proliferation, and differentiation abnormalities that occur prior to the onset of morphologic dysplasia. In general, GI pathologists have converted to a two-tiered grading system of neoplastic precursor lesions, termed low or high grade, that define progressive stages of neoplastic development prior to tissue invasion. This grading system 0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.002

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has proved to be clinically useful and is used by the authors in this issue. In addition, in most, if not all, precursor grading systems, the term ‘‘carcinoma in situ’’ has been abandoned by pathologists and is now incorporated into the high-grade category because it has an equivalent degree of clinical relevance. As you read through this comprehensive review about neoplastic precursor lesions, it is important to realize that, regardless of the anatomic site, progression of tumor development represents a continuous/linear spectrum of advancing degrees of epithelia neoplasia, which in some instances, is difficult to compartmentalize into discrete grades. Thus, there is interobserver variability regarding interpretation of neoplastic precursor lesions by pathologists, which is important to keep in mind when evaluating pathology reports and translating the information in those reports toward patient care. It is my hope that after reading this issue of Gastroenterology Clinics of North America you will recognize several important take-home points. First is the critical role of pathologists in the diagnosis and management of patients who have neoplastic precursor lesions of the GI tract, liver, biliary tract or pancreas. However, it is also important to recognize the limitations of pathology in this regard. For a number of reasons, such as the low prevalence rate of dysplastic lesions in the general population, the relative inexperience with dysplastic lesions by most non-tertiary care–affiliated general pathologists, the difficulties in morphologic interpretation, and sampling limitations, it is highly recommended (if not mandatory) that at least two experienced, preferably GI, pathologists evaluate and agree on a specific grade of neoplastic precursor lesion before institution of patient management. The value of close cooperation, and/or face-to-face interaction, between clinicians and pathologists cannot be overemphasized with regard to the care of patients who have neoplastic precursor lesions. Second, because of inherent limitations of pathologic assessment of neoplastic precursor lesions, there is a need for better, more reliable, and reproducible biomarkers to assess risk of malignancy in affected patients. In some organs, understanding the biological characteristics and natural history of these lesions is limited by various factors, such as poor accessibility of tissue, low prevalence rate in the general population, and the relatively long period of time necessary for progression of pre-neoplasia to invasive neoplasia, and this makes the study of these lesions difficult. In some instances, such as in Barrett’s esophagus and inflammatory bowel disease, there has been much headway with regard to screening and surveillance guidelines, but in other disorders, such as chronic gastritis, precursor lesions of the liver and endocrine system, and hyperplastic/serrated precursors in the colon, surveillance guidelines are still evolving and there is need for more research in this area. Finally, although screening and surveillance for precursor lesions is both logical and scientifically valid, there is an ongoing need for prospective randomized controlled trials to determine the actual costs and benefits of surveillance programs.

PREFACE

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I would like to thank all of the authors who contributed to this issue of Gastroenterology Clinics of North America for their patience with the editorial process, for supplying their manuscripts in a timely fashion, and most importantly, for providing remarkably concise and comprehensive reviews of the key morphologic, biological, and clinically relevant aspects of pre-neoplastic lesions of the GI tract, liver, biliary tract and pancreas. As a result, much of the information provided in this issue can be used to better serve our patients directly and identify areas in need of further research. Robert D. Odze, MD, FRCPc Department of Pathology Brigham and Women’s Hospital 75 Francis Street Boston, MA 02115, USA E-mail address: [email protected]

Gastroenterol Clin N Am 36 (2007) 775–796

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Neoplastic Precursor Lesions in Barrett’s Esophagus Jason L. Hornick, MD, PhD, Robert D. Odze, MD, FRCPc* Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA

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he incidence of adenocarcinoma of the esophagus and gastroesophageal junction has increased by approximately 600% in the United States over the past 30 years [1,2]. In fact, adenocarcinoma has now surpassed squamous cell carcinoma as the most common histologic type of esophageal malignancy in the United States [1]. The precursor to esophageal adenocarcinoma is Barrett’s esophagus [3]. Barrett’s esophagus, currently defined as endoscopically apparent columnar metaplasia in the esophagus, with histologic documentation of goblet cells [4], is caused by chronic gastroesophageal reflux disease (GERD) [5]. The prevalence of Barrett’s esophagus in patients undergoing upper endoscopy for GERD is about 10%, as compared to 5% in the general population [6–10]. With symptomatic GERD affecting approximately 20% of the adult population in the United States on a weekly basis [11], Barrett’s esophagus is a relatively common disorder. Patients with Barrett’s esophagus have a 30- to 100-fold increased risk of developing adenocarcinoma, with an absolute risk of approximately 0.5% per year [2]. Traditionally, Barrett’s esophagus is separated into long-segment (>3 cm), short-segment (1–3 cm), and ultrashort-segment (<1 cm) types, depending on the length of involved mucosa [4,12]. Although length of Barrett’s esophagus is probably related to cancer risk [13–15], the biological significance of this classification is unclear. Recently, Sharma and colleagues [12] proposed an alternative consensus endoscopic classification system for Barrett’s esophagus, termed the Prague C&M criteria. This method of Barrett’s esophagus classification involves assessment of both the circumferential (C) and maximum (M) proximal extent of endoscopically visible Barrett’s esophagus. Although the reliability (j) coefficients for assessment of Barrett’s esophagus by the C&M method are high, the utility of this system awaits validation by other investigators. It is widely believed that Barrett’s esophagus–associated adenocarcinoma develops via a progressive sequence of histologic and molecular events beginning with columnar metaplasia and progressing through various grades of dysplasia *Corresponding author. E-mail address: [email protected] (R.D. Odze).

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to, ultimately, adenocarcinoma [3,4,11]. Consequently, affected patients routinely undergo endoscopic surveillance for early detection of neoplasia. Because no prospective randomized trials have addressed the issue, surveillance has not yet been proven to improve survival in patients with Barrett’s esophagus. However, several retrospective and cohort studies have shown that patients who underwent surgical resection following endoscopic detection of cancer had their tumors detected at an earlier stage and showed better longterm survival, compared with those who did not [16–19]. For instance, in one study by Streitz and colleagues [17] of 19 patients who underwent esophagectomy for adenocarcinoma detected during endoscopic surveillance, 58% had stage 0 or I tumors, compared with only 17% of patients who did not undergo surveillance. In this study, the 5-year survival rate for patients undergoing surveillance was 62%, compared with only 20% for nonsurveilled patients. Histologic evaluation of dysplasia in esophageal mucosal biopsies is the mainstay of risk assessment in the surveillance and treatment of patients with Barrett’s esophagus. The current definition of dysplasia is ‘‘unequivocal’’ neoplastic epithelium confined to the basement membrane [20,21]. In the United States, dysplasia in Barrett’s esophagus is classified as either negative, indefinite, or positive (low or high grade), based on a system initially developed in 1983 for patients with inflammatory bowel disease [20,22]. This system has been applied to Barrett’s esophagus and to most, if not all, neoplastic precursor lesions of the tubal gut. However, many pathologists in Europe and Asia prefer the recently proposed Vienna classification system [23], which uses the term ‘‘noninvasive neoplasia’’ instead of ‘‘low-’’ or ‘‘high-’’ grade dysplasia, includes a category for ‘‘noninvasive’’ carcinoma (carcinoma in-situ), and also uses the term ‘‘suspicious for invasive carcinoma’’ for biopsies that show equivocal features of tissue invasion. This article focuses on dysplasia in Barrett’s esophagus in terms of its classification, pathologic diagnostic criteria, limitations, natural history, and treatment. DEFINITION OF BARRETT’S ESOPHAGUS To establish a diagnosis of Barrett’s esophagus, the American College of Gastroenterology (ACG) guidelines require that goblet cells be identified within columnar mucosa in biopsies obtained from the esophagus above the level of the gastroesophageal junction [4]. The vast majority of esophageal adenocarcinomas arise in a background of goblet cell metaplasia [24–27], which is widely regarded as the type of epithelium at highest risk for neoplastic progression. However, cancers may also develop in goblet cell–poor or even nongoblet epithelium. A recent study by the authors’ research group [28] of 3778 biopsies from 151 Barrett’s esophagus patients, 63 of whom eventually developed cancer, showed that the goblet cell density in nondysplastic epithelium from Barrett’s esophagus patients without dysplasia was significantly higher than in nondysplastic epithelium from Barrett’s esophagus patients with dysplasia, suggesting that loss of goblet cell differentiation may be associated with neoplastic progression in Barrett’s esophagus. In fact, goblet cell density in nondysplastic

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crypts was not found to be a useful predictor of cancer risk in this retrospective case-control study. Furthermore, although goblet cells are an easily recognizable histologic marker of ‘‘intestinal’’ metaplasia, the nongoblet columnar epithelium in Barrett’s esophagus also shows physiologic properties of intestinal epithelium, such as expression of the enteric markers CDX-2, DAS-1, and MUC-2 [29]. Because Barrett’s esophagus contains an admixture of goblet cells and gastric-type mucous cells, most, if not all, cases of Barrett’s esophagus show incomplete intestinal metaplasia [30–34]. By contrast, in normal intestinal mucosa, goblet cells are present in association with absorptive enterocytes, Paneth cells, and neuroendocrine cells. Although histologic identification of intestinal (‘‘goblet’’) epithelium is required to establish a diagnosis of Barrett’s esophagus, the chance of detecting goblet cells is, essentially, proportional to the length of Barrett’s esophagus [35–37]. For instance, in a study by Oberg and colleagues [37] of 177 patients with columnar-lined esophagus, the prevalence of intestinal metaplasia (defined by the presence of goblet cells) detected on endoscopy was 30.5% in patients with 1 to 2 cm of columnar-lined esophagus, compared with 88.9% in patients with Barrett’s esophagus segments longer than 6 cm. Not surprisingly, an increase in the number of biopsies also increases the likelihood of detecting goblet cells. For example, in a recent study by Harrison and colleagues [36] of 296 endoscopies performed on 125 consecutive patients with columnar-lined esophagus (both long- and short-segment types), goblet cells were detected in 68% of endoscopies in which eight biopsies were taken, compared with only 35% if only four biopsies were analyzed. BARRETT’S ESOPHAGUS, NEGATIVE FOR DYSPLASIA Because of persistent GERD, biopsies from patients with Barrett’s esophagus are often inflamed. Consequently, the epithelium normally shows a variable degree of regenerative change [21]. Biopsies with this type of morphology are categorized as ‘‘negative for dysplasia’’ [20]. However, the spectrum of epithelial regeneration in Barrett’s esophagus is quite broad, so that at the severe end of the spectrum of regeneration, the degree of cytologic atypia may overlap with true dysplasia (see below) [21,38], particularly in areas adjacent to ulcers. In fact, both the architectural and cytologic features of nondysplastic Barrett’s esophagus also differ from ‘‘normal’’ intestinal mucosa, which complicates interpretation of regeneration versus dysplasia in mucosal biopsies [21]. For example, Barrett’s esophagus typically lacks evenly spaced test tube–like crypts characteristic of the small and large bowel, and also shows crypt budding, atrophy, branching, increased mitotic activity, hyperchromaticity, dystrophic goblet cells, and mucin depletion (Fig. 1). However, in contrast to well-developed dysplasia, the cells in nondysplastic Barrett’s esophagus maintain a low nuclear/cytoplasmic ratio, retain their polarity, and show progressive maturation (acquisition of cytoplasmic mucin) in epithelium at the mucosal surface. In fact ‘‘surface maturation’’ is, in most instances, a helpful histologic sign of

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Fig. 1. Intestinal epithelium with goblet cells in Barrett’s esophagus with regenerative changes. Note the presence of crypt distortion, crypt branching, and mild nuclear stratification, which are typical features of nondysplastic Barrett’s epithelium.

epithelial regeneration [38], except in certain circumstances discussed further below. Based on the presence of inherent histologic abnormalities in Barrett’s esophagus, it is perhaps not surprising that recent data suggests that nondysplastic Barrett’s epithelium also demonstrates many properties of ‘‘neoplasia’’ [39– 42]. For instance, molecular aberrations in the p16 gene (also known as CDKN2A and INK4A), in the form of chromosome 9p loss of heterozygosity (LOH), promoter hypermethylation, or mutations, are detected in approximately 90% of nondysplastic Barrett’s esophagus, and these changes frequently show evidence of clonal expansion throughout a Barrett’s segment [42–44]. Furthermore, mutations in the p53 tumor suppressor gene may be detected in a subset of patients with Barrett’s esophagus without dysplasia [45]. Nondysplastic Barrett’s esophagus commonly shows clonal cytogenetic abnormalities, including both numerical and structural alterations [46]. Using high-fidelity image cytometry and flow cytometry, a mild degree of aneuploidy has been detected in nondysplastic Barrett’s epithelium [47–50]. For example, in a study by Yu and colleagues [50] using high-fidelity image cytometry performed on 66 nondysplastic Barrett’s esophagus specimens, mild aneuploidy was detected in 69% of cases. Also, nondysplastic Barrett’s esophagus shows proliferative abnormalities and various degrees of loss of cell cycle regulation [51,52], both of which are cardinal features of a ‘‘neoplastic’’ process. These findings hold promise that biomarkers of malignant potential may eventually be defined in predysplastic mucosa. However, clearly, more studies are needed in this regard. BARRETT’S ESOPHAGUS, INDEFINITE FOR DYSPLASIA Pathologically, regenerating epithelium can, at times, be difficult to distinguish from true dysplasia [21]. This is particularly apparent in instances where

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significant inflammation, or ulceration, is present in the biopsy specimen. In this context, the category ‘‘indefinite for dysplasia’’ is applied [20]. Other reasons pathologists use the term ‘‘indefinite for dysplasia’’ include those related to technical issues, such as tangential or thick tissue sectioning, poor orientation, denuded surface epithelium, or marked cautery artifact. Finally, as discussed below, some cases of dysplasia are limited to the crypt bases and, thus, mimic regeneration [53]. From a clinical perspective, ‘‘indefinite for dysplasia’’ should always be considered an interim diagnosis until mucosal biopsies can be processed better or until further biopsies can be evaluated after more aggressive treatment of the patient’s reflux symptoms to decrease inflammation. BARRETT’S ESOPHAGUS, POSITIVE FOR DYSPLASIA Endoscopically, dysplasia in Barrett’s esophagus is often flat and inconspicuous. Less often, dysplasia is endoscopically detectable and may appear as a nodule, polyp, plaque, or ulcer [54]. In fact, the presence of an endoscopically recognizable mucosal abnormality in association with dysplasia has a high association with adenocarcinoma (see below) [55,56]. In Barrett’s esophagus, two general histologic types of dysplasia are recognized. These are termed ‘‘adenoma-like’’ and ‘‘nonadenoma-like’’ [21]. The former is much more common. In adenoma-like low-grade dysplasia, the crypt architecture shows few, or no, distinct changes from nondysplastic epithelium. However, cytologically, dysplastic nuclei are enlarged, elongated, and stratified, although this latter feature is normally limited to the lower half of the cell cytoplasm (Fig. 2). The presence of (1) an abrupt transition between nondysplastic and dysplastic epithelium and (2) uniform nuclear changes extending evenly from the crypt base to the mucosal surface are helpful diagnostic

Fig. 2. Low-grade dysplasia in Barrett’s esophagus. The nuclei are stratified and pencilshaped, but are limited to the lower half of the cytoplasm. The architecture is similar to that of nondysplastic epithelium.

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features of low-grade dysplasia [21,38]. Other features, including loss of polarity, mitotic figures, and pleomorphism, are typically only mild or absent. Goblet cells are usually decreased in number or entirely absent in dysplastic epithelium, and surrounding nondysplastic mucosa often shows goblet cell depletion [28], which raises questions regarding the role of goblet cells as the ‘‘field’’ upon which neoplasia develops. As mentioned above, lack of surface maturation is normally considered a cardinal feature of dysplasia [38]. However, in a recent study by Lomo and colleagues [53], 15 cases of dysplasia-like atypia all showed basal crypt involvement only, with surface maturation, suggesting that in the early stages of progression, dysplasia involves only the crypt bases (Fig. 3). In their study, 47% of patients with basal crypt dysplasia also had conventional full-crypt dysplasia, or adenocarcinoma, and a significantly higher incidence of 17p LOH and flow cytometric abnormalities (see below), compared with control patients without dysplasia. In contrast to low-grade dysplasia, high-grade dysplasia is characterized by more severe cytologic changes (Fig. 4), often with architectural abnormalities as well. High-grade dysplasia shows increased crypt complexity, crowding, irregularity, and branching, and cytologically it shows more pronounced nuclear stratification, loss of nuclear polarity, pleomorphism, nucleoli, and mitotic activity. However, neoplastic changes in Barrett’s esophagus develop and progress on a morphologic continuum and, thus, do not follow clearly definable steps, which is a limitation when pathologists try to compartmentalize dysplasia into only one of two grades. In fact, primarily because of a lack of prospective studies, no guidelines have been agreed on for the minimum number of highgrade dysplastic crypts necessary to upgrade a diagnosis of low-grade dysplasia to high-grade dysplasia in a biopsy with dysplasia [21,22].

Fig. 3. Basal crypt dysplasia in Barrett’s esophagus. Low-grade dysplasia involves only the bases of crypts, whereas the upper crypts and surface epithelium exhibit maturation.

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Fig. 4. (A) High-grade dysplasia (adenomatous-type) in Barrett’s esophagus. In contrast to low-grade dysplasia (see Fig. 2), there is a greater degree of nuclear stratification. In this example, there is minimal architectural distortion. On high-power view (B), the nuclei have more open chromatin, prominent nucleoli, and loss of polarity.

In contrast, nonadenomatous dysplasia is an uncommon and poorly studied form of dysplasia characterized by crowded glands containing cuboidal cells with a high nuclear/cytoplasmic ratio, round nuclei with irregular contours, vesicular chromatin, and prominent nucleoli (Fig. 5). This form of dysplasia is often mistaken for intramucosal adenocarcinoma. Most gastrointestinal pathologists classify this form of dysplasia as high grade [21]. However, the biological properties and natural history of nonadenomatous dysplasia are unknown. Intramucosal adenocarcinoma is defined as neoplastic epithelium that has invaded beyond the basement membrane into the surrounding lamina propria or muscularis mucosae (Fig. 6). Because the esophageal lamina propria contains lymphatic vessels, adenocarcinomas limited to the mucosa may result in lymph node metastases (approximately 5% risk). Morphologically, individual cells or small clusters of cells in the lamina propria are diagnostic of intramucosal

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Fig. 5. High-grade dysplasia (nonadenomatous-type) in Barrett’s esophagus. In contrast to adenomatous dysplasia, the cells are more cuboidal in shape, contain more irregular nuclei, and have a markedly increased nuclear/cytoplasmic ratio. The architecture is distorted with prominent back-to-back glands.

adenocarcinoma. In institutions where patients with intramucosal adenocarcinoma are managed differently from those with high-grade dysplasia, this distinction is of clinical importance. Unfortunately, distinguishing between these two diagnoses in mucosal biopsy specimens is often difficult and also suffers from a significant degree of interobserver variability [38,57]. A study by Ormsby and colleagues [57] based on a review of 75 esophagectomy specimens by two gastrointestinal pathologists and one general surgical pathologist, reported only fair interobserver agreement in distinguishing high-grade dysplasia

Fig. 6. Intramucosal adenocarcinoma. Similar to high-grade dysplasia, the nuclei show severe nuclear stratification, hyperchromasia, and loss of polarity. However, the architecture is markedly abnormal with cribriforming (arrow), which cannot be explained by preexisting Barrett’s architecture.

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from intramucosal adenocarcinoma, which also did not improve substantially upon reevaluation of the slides after uniform histologic criteria were established. INTEROBSERVER VARIABILITY IN DYSPLASIA DIAGNOSIS There is considerable interobserver variability among pathologists in the diagnosis of dysplasia in Barrett’s esophagus, particularly with regard to the indefinite and low-grade categories [22,38,58]. For example, in a study by Reid and colleagues in 1988 [22], experienced gastrointestinal pathologists showed only 60% agreement comparing biopsies that were negative for dysplasia to those that were considered indefinite or low-grade dysplasia. A more recent reproducibility study by Montgomery and colleagues [38] showed substantial agreement among pathologists for the diagnosis of high-grade dysplasia (j ¼ 0.65), but only fair agreement for low-grade dysplasia (j ¼ 0.32), and only slight agreement for indefinite cases (j ¼ 0.15). In that study, interobserver variation improved following a consensus conference; however, distinction of negative and indefinite from low-grade dysplasia categories did not. ADJUNCTIVE MARKERS OF DYSPLASIA To improve the diagnostic accuracy of dysplasia by pathologists, many types of histochemical ‘‘biomarkers’’ have been evaluated, such as markers of cell proliferation (proliferating cell nuclear antigen and Ki67), cyclin D1, and p53 [59–64]. For example, studies have shown that the extent and distribution of Ki67 staining correlates reasonably well with the grade of dysplasia [59,62,63]. In one study by Hong and colleagues [62], high-grade dysplasia was characterized by a high level of Ki67 expression in the upper portion of the crypt and in the surface epithelium, whereas nondysplastic epithelium showed Ki67 limited to the basal aspects of the crypts. Unfortunately, regenerating epithelium can also demonstrate increased cell proliferation [62], which, in some instances, approaches that seen in high-grade dysplasia. Thus, Ki67 expression is not a reliable means of confirming dysplasia. Furthermore, up to now, neither proliferating cell nuclear antigen nor Ki67 expression has been evaluated prospectively as a marker of risk of progression to adenocarcinoma. Recently, immunostaining for alpha-methylacyl-CoA racemase (AMACR) has shown promise as a marker for dysplasia [65,66]. In a study by Dorer and Odze [65], AMACR staining was negative in all cases of Barrett’s esophagus considered negative for dysplasia, whereas 38% of low-grade dysplasia, 81% of high-grade dysplasia, and 72% of adenocarcinoma cases were positive. In that study, only 3 of 14 (21%) cases of ‘‘indefinite for dysplasia’’ showed expression of AMACR, but 1 of those patients subsequently developed adenocarcinoma upon follow-up. Another recent study by Lisovsky and colleagues [66] reported similar findings. In that study, AMACR was negative in all cases considered negative or indefinite for dysplasia, whereas 11% of low-grade dysplasia, 64% of high-grade dysplasia, and 75% of adenocarcinoma cases were positive. Thus, AMACR is helpful for pathologists in distinguishing reactive

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from dysplastic epithelium in Barrett’s esophagus, but the predictive value of AMACR remains to be evaluated. RISK ASSESSMENT IN BARRETT’S ESOPHAGUS Several clinical and endoscopic parameters, such as the presence of a hiatal hernia and the length of the Barrett’s segment [13–15], have been shown to be associated with an increased risk of progression to cancer in Barrett’s esophagus. For example, a prospective endoscopic surveillance study by Iftikhar and colleagues [13] found that patients with dysplasia had a significantly longer Barrett’s segment length compared with patients without dysplasia. In another study, this one by Menke-Pluymers and colleagues [14], a doubling of the length of Barrett’s esophagus was associated with a 1.7-fold increased risk of adenocarcinoma. In a prospective study of 550 patients with Barrett’s esophagus by Weston and colleagues [15], patients without dysplasia in their index biopsy, and with less than 6 cm of Barrett’s esophagus, had a 2.4% rate of progression, compared with 6.8% for patients with greater than 6 cm of Barrett’s esophagus. Although many potential nonmorphology-based biomarkers have been studied, few, if any, have been validated as markers of risk of cancer in phase III or phase IV prospective trials. Immunostaining for p53 has been the most widely examined potential biomarker in Barrett’s esophagus [60,64,67,68]. For instance, previous studies have shown that mutations in p53, or LOH of 17p, are present in up to 75% of patients with high-grade dysplasia or adenocarcinoma [11,68–70]. P53 mutations usually result in a prolongation of the protein’s half-life, which renders it potentially detectable by immunohistochemistry. In general, the frequency of positive immunostaining for p53 has been shown to be proportional to the grade of dysplasia [61,64,68,69]. Unfortunately, p53 may be detected in up to 10% of biopsies that are histologically negative for dysplasia. In addition, its immunostaining has a high rate of both false positives and false negatives [21,69]. Thus, p53 immunostaining is not advocated as a routine marker for diagnostic use. Although p53 is not helpful for diagnosing dysplasia, several studies have suggested that p53 immunostaining may have predictive value [64,68]. For instance, in an immunohistochemistry study by Younes and colleagues [68] of 21 Barrett’s esophagus patients whose biopsy specimens were negative for p53, only 1 developed high-grade dysplasia upon follow-up. In contrast, 2 patients with p53-positive biopsies showed progression. In another study by Weston and colleagues [64], p53 immunostaining was performed on biopsies from 48 Barrett’s patients with low-grade dysplasia. In that study, 3 of 5 (60%) patients who progressed to multifocal high-grade dysplasia, a high-grade dysplasia– associated lesion, or adenocarcinoma, were positive for p53, compared with only 4 of 31 (13%) patients whose low-grade dysplasia ‘‘regressed,’’ and 3 of 12 (25%) patients who had persistent low-grade dysplasia. DNA content abnormalities, detected by flow cytometry (aneuploidy and tetraploidy) [47,48,71,72], and genetic alterations in p53 and p16 [70,73,74] are

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the most promising biomarkers studied to date. For example, a flow cytometry study by Reid and colleagues [72] in 2000 showed that patients without detectable DNA content abnormalities in their baseline biopsies had a 0% 5-year incidence of adenocarcinoma, compared with a 28% incidence rate in Barrett’s esophagus patients with either aneuploidy or increased 4N (tetraploidy). Baseline 17p (p53) LOH has also been shown to correlate with cancer progression. In a prospective study by Reid and colleagues [70] of 325 patients with Barrett’s esophagus, 20 of 54 (37%) patients with baseline 17p LOH progressed to cancer, compared with only 6 of 202 (3%) patients without 17p LOH, resulting in a relative risk of 16. Interestingly, a recent study by Galipeau and colleagues [75] showed that, in fact, a combination of biomarkers may predict cancer risk better than individual markers. In that long-term follow-up study of 243 Barrett’s patients, those without baseline genetic abnormalities had a 12% 10-year incidence of esophageal adenocarcinoma, compared with 79% of patients with 17p LOH, DNA content abnormalities, and 9p LOH. However, these methodologies are now only available at selected specialized centers. For a detailed discussion of potential nonmorphology biomarkers in Barrett’s esophagus, the reader is referred to a review article by McManus and colleagues [76]. RELEVANCE OF EXTENT OF DYSPLASIA Several recent studies suggest that not only the presence of dysplasia, but also the extent of dysplasia affects risk of adenocarcinoma in Barrett’s esophagus [55,77]. For example, in a study by Buttar and colleagues [55] of 100 Barrett’s esophagus patients with high-grade dysplasia, the risk of developing adenocarcinoma was significantly higher in patients with ‘‘diffuse’’ high-grade dysplasia (defined as high-grade dysplasia involving either more than five crypts in a single biopsy specimen or more than one biopsy fragment) compared with those with ‘‘focal’’ high-grade dysplasia (defined as dysplasia involving a single focus of five or fewer crypts). In that study, 3-year adenocarcinoma-free survival rates were significantly lower, and the relative risk of cancer was 3.7 fold higher, in patients with diffuse high-grade dysplasia compared with those with focal high-grade dysplasia. However, in a subsequent study by Dar and colleagues [78] of 42 Barrett’s esophagus resection specimens, no significant difference in the prevalence of adenocarcinoma was detected in patients with ‘‘focal’’ versus ‘‘diffuse’’ high-grade dysplasia (48% versus 67%, respectively). In that study, ‘‘focal’’ was defined as high-grade dysplasia present in any number of biopsies from only one anatomic level of the esophagus, and ‘‘diffuse’’ was defined as involvement of biopsies from more than one level of the esophagus. Unfortunately, both of these studies included patients with dysplasia-associated mucosal nodules, which has been shown to be a risk factor for adenocarcinoma. In contrast, a recent study by the authors’ research group [77] did not reveal an association between risk of cancer and extent of high-grade dysplasia, but instead detected an association between the extent of low-grade dysplasia and the development of adenocarcinoma in a case-control study of 77 Barrett’s

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esophagus patients, 44 of whom eventually developed carcinoma upon endoscopic surveillance. In that study, a detailed analysis of both the total number and percentage of dysplastic crypts (both low and high grade) were counted in baseline biopsies from all patients. When the crypts were stratified by dysplasia grade, the mean proportion of low-grade dysplastic crypts per patient (46%) was significantly higher in patients who progressed to adenocarcinoma versus those who did not (26%). When the data was analyzed by the methods used to define ‘‘focal’’ and ‘‘diffuse’’ dysplasia in the studies by either Buttar and colleagues or Dar and colleagues, diffuse high-grade dysplasia, diffuse low-grade dysplasia, and diffuse dysplasia of any grade were not associated with cancer progression. Thus, the recommendation by the ACG (see below) [4] to use extent of high-grade dysplasia, as defined by Buttar and colleagues, in determining management options is probably premature and requires further study. NATURAL HISTORY OF DYSPLASIA Combining the results of several prospective endoscopic surveillance studies of Barrett’s esophagus patients, the overall risk of cancer in patients without dysplasia, at baseline, is approximately 2% [15,72,79–82]. In contrast, the risk of cancer in patients with high-grade dysplasia ranges from 16% to 59% [15,72,79–81]. The wide range in outcome is partially explained by the separation of patients into those with ‘‘prevalent’’ versus ‘‘incident’’ dysplasia. Prevalent dysplasia is defined as dysplasia detected either at initial screening endoscopy or during the first 12 months of surveillance, which makes the assumption that some cases of baseline dysplasia are missed because of sampling error. In contrast, incident dysplasia is defined as dysplasia that develops during endoscopic surveillance. For example, in a prospective study of 327 Barrett’s esophagus patients by Reid and colleagues [72], the 5-year risk of cancer for patients with prevalent high-grade dysplasia was 59%, compared with only 31% for patients with incident high-grade dysplasia. In contrast, in a surveillance study of 1099 Barrett’s esophagus patients by Schnell and colleagues [81], 16% of patients with high-grade dysplasia (either incident or prevalent) developed cancer during surveillance. However, in that study, patients who were found to have cancer during an initial year of intensive surveillance were excluded from the analysis. In another surveillance study by Weston and colleagues [83] of 15 Barrett’s esophagus patients with high-grade dysplasia, 26.7% developed cancer between 17 and 35 months of follow-up. By reviewing prior published prospective and registry studies of patients with Barrett’s esophagus, Sampliner [4] reported an overall rate of progression from highgrade dysplasia to cancer of 22%, based on 783 patients followed for a mean of 2.9 to 7.3 years. The natural history of Barrett’s esophagus patients with low-grade dysplasia is less well understood, and this is partly related to the greater degree of interobserver variability among pathologists in establishing this diagnosis [22,38]. Furthermore, many studies have used either high-grade dysplasia or cancer as an endpoint [64,84], which also complicates interpretation of the results.

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In earlier studies, the risk of progression of low-grade dysplasia was estimated at only 2% to 12% [64,72,84]. In fact, some studies showed an even higher rate of regression [85]. However, sampling error remained a distinct limitation in these studies. For instance, in a prospective study by Reid and colleagues [72], the cumulative incidence of cancer in Barrett’s esophagus patients with baseline low-grade dysplasia was only 12%. In a study by Skacel and colleagues [84], only 2 of 25 (8%) Barrett’s esophagus patients with low-grade dysplasia developed adenocarcinoma upon follow-up. However, several more recent studies suggest a higher risk of cancer progression in patients with ‘‘definite’’ low-grade dysplasia, particularly where there has been uniform consensus among at least three gastrointestinal pathologists [77,84,86]. For example, Skacel and colleagues [84] showed that when two gastrointestinal pathologists agreed on a diagnosis of low-grade dysplasia, 41% of patients progressed to high-grade dysplasia or cancer after a mean of 11 months. By comparison, when three gastrointestinal pathologists agreed on a diagnosis of low-grade dysplasia, 80% of patients progressed to high-grade dysplasia or cancer. In another study, this one by Montgomery and colleagues [86], 47% of patients ultimately progressed to high-grade dysplasia or cancer when more than 6 of 12 gastrointestinal pathologists agreed on a diagnosis of low-grade dysplasia. Finally, in a recent study by Srivastava and colleagues [77], in which three gastrointestinal pathologists agreed on all diagnoses of lowor high-grade dysplasia in baseline biopsies from 77 Barrett’s esophagus patients, 14 (45%) of 31 patients with a maximum diagnosis of low-grade dysplasia progressed to adenocarcinoma. These findings suggest that consensus diagnosis by multiple gastrointestinal pathologists improves risk assessment and emphasize the importance of confirmation of dysplasia diagnoses by at least two experienced gastrointestinal pathologists. These aforementioned rates of progression relate primarily to detection of dysplasia in random (‘‘blind’’) biopsies in Barrett’s esophagus. In contrast, dysplasia detected in association with visible mucosal abnormalities, such as nodules, ulcers, or strictures, is associated with an increased risk of synchronous or metachronous adenocarcinoma [55,56]. For example, in a study by Buttar and colleagues [55], patients with high-grade dysplasia associated with a mucosal nodule had a significantly increased risk of cancer, compared with patients without mucosal abnormalities. In that study, 15 of 25 (60%) patients with nodularity developed adenocarcinoma, compared with only 17 of 75 (23%) patients without nodularity at endoscopy (relative risk, 3.98). In another study by Montgomery and colleagues [56], the presence of an ulcer with high-grade dysplasia increased the likelihood of detecting adenocarcinoma at the time of resection. Finally, patients with Barrett’s esophagus do not always follow a stepwise linear progression from metaplasia to low-grade dysplasia, high-grade dysplasia, and cancer. For example, in an endoscopic surveillance study by O’Connor and colleagues [82] of 136 patients with Barrett’s esophagus, 1 patient who initially did not have dysplasia developed adenocarcinoma after 7 years without

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showing dysplasia in the intervening endoscopic biopsies. In another long-term surveillance study of 1099 patients with Barrett’s esophagus by Schnell and colleagues [81] in 2001, 10 patients progressed from low-grade dysplasia directly to adenocarcinoma without showing histologic evidence of high-grade dysplasia. SURVEILLANCE FOR DYSPLASIA Most experts employ the ‘‘Seattle’’ protocol for surveillance of Barrett’s esophagus [87,88]. This includes four-quadrant biopsies using jumbo biopsy forceps obtained from every 1 to 2 cm of Barrett’s mucosa, depending upon the presence and degree of dysplasia, in addition to targeted biopsies of grossly apparent mucosal abnormalities [87–89]. Based upon available scientific evidence, the ACG and the American Gastroenterological Association have issued practice guidelines for the diagnosis, surveillance, and treatment of patients with Barrett’s esophagus. In terms of surveillance, the ACG 2002 guidelines (Box 1) [4] recommend that, in the absence of dysplasia on two consecutive endoscopies with biopsies, the follow-up endoscopy interval may be extended to 3 years. For patients with low-grade dysplasia, annual endoscopy is recommended until no dysplasia is detected. For patients with high-grade dysplasia, it is recommended that endoscopy be repeated, with special attention to mucosal irregularities to exclude carcinoma. For focal high-grade dysplasia (defined as involvement of fewer than five crypts), follow-up endoscopy may be performed Box 1: American College of Gastroenterology proposed guidelines (2002) for surveillance in Barrett’s esophagus No dysplasia in two consecutive endoscopies with systematic biopsies Follow-up endoscopy in 3 years Low-grade dysplasia Repeat endoscopy with biopsies concentrated in area of dysplasia If low-grade dysplasia is the highest grade after repeat endoscopy, follow-up with annual endoscopies until no dysplasia is detected High-grade dysplasia Repeat endoscopy with special attention to mucosal irregularities and use an intensive biopsy protocol to exclude carcinoma Confirm diagnosis of high-grade dysplasia by expert gastrointestinal pathologist Focal high-grade dysplasia (<5 crypts)a Follow-up endoscopy every 3 months Multifocal high-grade dysplasiaa Intervention a

Recommendation based on the results of one study, which has not been validated by others.

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every 3 months. For multifocal high-grade dysplasia, intervention should be considered (see below). The 2005 American Gastroenterological Association recommendations [90] for surveillance of patients with Barrett’s esophagus differ somewhat from the ACG guidelines, particularly in terms of surveillance intervals, and these are summarized in Box 2. TREATMENT OF DYSPLASIA Although esophagectomy has traditionally been considered standard therapy for Barrett’s esophagus patients with high-grade dysplasia [16–19], clinicians have recently employed endoscopic mucosal resection (EMR) [91,92] and newer mucosal ablation techniques, such as photodynamic therapy (PDT) [93,94], laser ablation [95], and argon plasma coagulation [96,97]. EMR includes submucosal tissue, which facilitates pathologic distinction between intramucosal and submucosally invasive adenocarcinoma. For instance, in a recent reproducibility study by Mino-Kenudson and colleagues [98], nine gastrointestinal pathologists reviewed mucosal biopsies and corresponding esophageal EMR specimens from 25 patients with Barrett’s esophagus. Mino-Kenudson and colleagues showed that interobserver agreement for diagnosing Barrett’s esophagus–related neoplasia was significantly higher using EMR compared with that using mucosal biopsies. Excluding submucosal invasion is critical for staging because adenocarcinoma limited to the mucosa has a low risk of lymph

Box 2: American Gastroenterological Association recommendations (2005) for surveillance in Barrett’s esophagus No dysplasia Repeat endoscopy in 1 year If no dysplasia, follow-up endoscopy in 5 years Low-grade dysplasia Repeat endoscopy in 1 year If low-grade dysplasia is confirmed by two experienced gastrointestinal pathologists, repeat endoscopy yearly If there is disagreement between pathologists on whether dysplasia is present, repeat endoscopy in 2 years High-grade dysplasia If high-grade dysplasia is confirmed by two experienced gastrointestinal pathologists, surgical or endoscopic treatment For selected patients (in particular, those who do not wish to undergo definitive treatment until cancer is diagnosed), surveillance at 3-month intervals may be offered For multifocal high-grade dysplasia, more aggressive treatment If mucosal abnormalities exist, investigate with endoscopic ultrasound and mucosal resection to exclude cancer

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node metastases (approximately 5%), compared with 20% to 30% for carcinomas that have invaded into the submucosa. Local (mucosal) therapy may be considered in selected patients for the former, but surgical resection is advisable for the latter. Among mucosal ablation techniques, PDT in particular shows considerable promise [99,100]. For example, a randomized trial by Overholt and colleagues [100] comparing PDT with the proton pump inhibitor omeprazole to omeprazole alone in Barrett’s esophagus patients with high-grade dysplasia showed a higher rate of complete ablation of high-grade dysplasia in the PDT group (77% versus 39%). For a review of these techniques, the reader is referred to an article by Johnston [101]. Following PDT, up to two thirds of patients show complete squamous reepithelialization [102]. However, up to 50% of such patients show residual Barrett’s epithelium buried underneath islands of squamous epithelium [103–105]. Buried Barrett’s epithelium also develops following standard acid suppression therapy [106–108]. In addition, some post-PDT, or post–acid suppression treated, patients harbor residual dysplastic epithelium hidden underneath squamous epithelium [94,105,108]. This is clinically important because buried Barrett’s epithelium is endoscopically inapparent and thus may be missed during endoscopic surveillance and allowed to progress [89,109,110]. However, the neoplastic potential of buried nondysplastic Barrett’s epithelium is unknown. In fact, recent studies by the authors’ group have shown that buried Barrett’s epithelium, following either chronic proton pump inhibitor therapy [108] or PDT [111], has a significantly lower crypt proliferation rate, compared with nonburied epithelium, and a significantly lower incidence of aneuploidy. These findings suggest that post-PDT buried (nondysplastic) Barrett’s epithelium may, in fact, have a lower neoplastic potential than nonburied Barrett’s epithelium. Prospective follow-up studies will be required to determine the natural history and risk of malignancy of buried Barrett’s epithelium. SUMMARY Although Barrett’s esophagus is a precursor to esophageal adenocarcinoma, not all patients with this disorder require intensive surveillance. Pathologic diagnosis and grading of dysplasia in mucosal biopsies remain the best and most widely used methods of determining which patients are at highest risk for neoplastic progression, and for selecting patients who need a more intensive surveillance program or surgical intervention. Diagnosing dysplasia suffers from considerable interobserver variability. Therefore, consultation with expert gastrointestinal pathologists to confirm the diagnosis of dysplasia before definitive management is highly advisable. Adjunctive methods to improve reproducibility, such as immunostaining for AMACR, show promise, but require confirmation in larger studies. Detection of p16, p53, and DNA content abnormalities may help identify patients at particularly high risk for progression to cancer, but these techniques are not yet widely available for routine clinical application.

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[106] Sampliner RE, Steinbronn K, Garewal HS, et al. Squamous mucosa overlying columnar epithelium in Barrett’s esophagus in the absence of anti-reflux surgery. Am J Gastroenterol 1988;83(5):510–2. [107] Sharma P, Morales TG, Bhattacharyya A, et al. Squamous islands in Barrett’s esophagus: What lies underneath? Am J Gastroenterol 1998;93(3):332–5. [108] Hornick JL, Blount PL, Sanchez CA, et al. Biologic properties of columnar epithelium underneath reepithelialized squamous mucosa in Barrett’s esophagus. Am J Surg Pathol 2005;29(3):372–80. [109] Overholt BF, Panjehpour M, Halberg DL. Photodynamic therapy for Barrett’s esophagus with dysplasia and/or early stage carcinoma: long-term results. Gastrointest Endosc 2003;58(2):183–8. [110] Sampliner RE, Fass R. Partial regression of Barrett’s esophagus—an inadequate endpoint. Am J Gastroenterol 1993;88(12):2092–4. [111] Hornick JL, Mino-Kenudson M, Lauwers GY, et al. Buried Barrett’s epithelium following photodynamic therapy shows reduced crypt proliferation and absence of DNA content abnormalities. Am J Gastroenterol 2007, in press.

Gastroenterol Clin N Am 36 (2007) 797–811

GASTROENTEROLOGY CLINICS OF NORTH AMERICA

Squamous Dysplasia and Other Precursor Lesions Related to Esophageal Squamous Cell Carcinoma Michio Shimizu, MD, PhDa,*, Shinichi Ban, MD, PhDa, Robert D. Odze, MD, FRCPcb a

Department of Pathology, Saitama Medical University International Medical Center, 1397-1 Yamane, Hidaka City, Saitama 350-1298, Japan b Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115, USA

S

quamous carcinoma is the most common malignant tumor of the esophagus worldwide. In 1997, 12,300 new cases and 11,500 deaths from this type of cancer were reported in the United States [1]. It affects men more often than women with a peak incidence in the seventh decade of life. There is a marked geographic and ethnic variation in incidence. The highest rates occur in China, Iran, South America, and South Africa [2]. In the United States, this type of cancer is more common in black versus white men [3]. This article discusses squamous dysplasia in detail, which is the most important and well described risk factor for squamous cell carcinoma of the esophagus. CLASSIFICATION OF SQUAMOUS DYSPLASIA (INTRAEPITHELIAL NEOPLASIA) In the former World Health Organization classification of esophageal and gastric tumors, dysplasia of squamous epithelium was defined as a precancerous lesion that contains both architectural and cytologic abnormalities [4]. Historically, squamous dysplasia has been classified as mild, moderate, or severe [5– 7]. Recently, however, most pathologists group mild and moderate together into a low-grade category, and refer to severe dysplasia as high-grade because of better interobserver agreement with the use of a two-tiered classification [8,9]. The term ‘‘squamous cell carcinoma in situ’’ is encompassed within the spectrum of high-grade dysplasia. With increasing grades of dysplasia, neoplastic cells involve and replace more of the squamous epithelium, but the lesion remains confined to the basement membrane. Lymph node metastases are not observed in association with dysplastic lesions. Once the neoplastic cells

*Corresponding author. E-mail address: [email protected] (M. Shimizu). 0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.005

ª 2007 Elsevier Inc. All rights reserved. gastro.theclinics.com

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invade the underlying lamina propria, the lesion is considered an invasive carcinoma. In the recent World Health Organization classification, the term ‘‘intraepithelial neoplasia’’ is preferred over ‘‘dysplasia’’ [2]. For instance, high-grade intraepithelial neoplasia is synonymous with high-grade dysplasia (or carcinoma in situ) because these conditions have the same clinical implication. It is also noteworthy that some Japanese pathologists prefer to classify ‘‘atypical’’ squamous epithelium into ‘‘nonneoplastic’’ and ‘‘noninvasive’’ carcinoma categories [10,11]. The former includes reactive epithelium, such as in esophagitis, erosions, ulcers, or hyperplasia, and the latter is divided into low-grade and high-grade noninvasive carcinoma. CLINICAL FEATURES Endoscopically, squamous dysplasia is usually flat, but may appear as areas of erythema, nodules, plaques, or erosions [12]. In a study by Dawsey and colleagues [12], the appearance of 398 biopsy sites containing squamous dysplasia or carcinoma of the esophagus were described. Eighty-one percent of moderate dysplasia, severe dysplasia, or carcinoma mucosal areas were abnormal, showing increased friability or erythema (38%), erosions (41%), or plaques or nodules (24%). A small percentage showed only irregular mucosa (6%) or small white patches (5%). Only 2% of dysplasia, or cancer, cases were endoscopically normal. Up to 96% of cases of moderate dysplasia, or worse, would have been identified if only visible lesions were targeted, which suggests that random biopsies are less valuable than targeted biopsies in this condition. On application of iodine, dysplastic areas are enhanced as unstained areas (Fig. 1A) [13–16]. Because glycogen is abundant in the superficial layers of normal esophageal squamous epithelium, lesions with loss of glycogen, such as atrophy, columnar metaplasia, esophagitis, dysplasia, or carcinoma, are

Fig. 1. (A) Endoscopic photograph of squamous dysplasia (intraepithelial neoplasia). Squamous dysplasia, is evident as an unstained area of mucosa after application of iodine. (B) Endoscopic finding by narrow-band imaging with magnifying endoscopy.

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recognized as unstained areas at endoscopy. In one study of 225 adults from Linxian, China, with cytologic evidence of dysplasia or carcinoma, unstained mucosal areas after iodine application had sensitivities of 63%, 93%, 96%, and 100% for identifying mild, moderate, and severe dysplasia (or carcinoma in situ), and early invasive carcinoma, respectively [14]. A total of 55% of moderate dysplasia cases and 23% of severe dysplasia cases were diagnosed only after application of iodine. Ultimately, however, mucosal biopsies are necessary to confirm the presence or absence of dysplasia. Recently, narrow-band imaging endoscopy has gained considerable attention for diagnosing esophageal neoplasms, such as dysplasia, squamous cell carcinoma, and Barrett’s esophagus (Fig. 1B) [17,18]. This method serves to enhance contrast between the epithelial surface and the subjacent vascular network. It is often combined with magnifying endoscopy. Although it has not been widely used for detection of early squamous cell carcinoma of the esophagus, this technique may become more useful in the near future. RISK FACTORS AND PRECURSOR LESIONS RELATED TO ESOPHAGEAL DYSPLASIA AND SQUAMOUS CELL CARCINOMA The incidence of squamous cell carcinoma of the esophagus varies geographically worldwide, and varies depending on the socioeconomic status of the population [19–21]. Alcohol consumption, smoking, diet, human papillomavirus (HPV) infection, radiation exposure, consumption of food and water rich in nitrates and nitrosamines, vitamin deficiencies, and genetic factors have all been reported as risk factors for cancer development. In addition, achalasia, Plummer-Vinson syndrome, and chronic strictures resulting from acid or lye ingestion are also predisposing conditions [21]. Although it is presumed that risk factors for dysplasia are similar to those for carcinoma, one study by Wei and colleagues [15] tested this theory. In a case sectional study of 724 adult volunteers who underwent endoscopy with Lugol’s iodine, risk factors for dysplasia were examined by logistic regression. They found that a higher number of household members, a family history of cancer, and a higher systolic blood pressure, among others, were associated with higher odds of having dysplasia. These factors were similar to those previously identified as risk factors for squamous carcinoma in this population. The role of esophagitis or basal cell hyperplasia, without dysplasia, as a risk factor for carcinoma is controversial [22–26]. Some studies suggest that prior esophagitis is a risk factor, particularly in high-risk regions. For instance, it is well known that histologic esophagitis is more prevalent in populations that have a high rate of squamous carcinoma, such as Iran and parts of China. Others showed no increase in prevalence of esophagitis in high- versus lowrisk populations, however, did find an increased rate of dysplasia and basal cell hyperplasia (which can mimic dysplasia) in high-risk groups [27,28]. In one study by Qiu and Yang [28], only 4% of patients with esophagitis, without dysplasia, developed carcinoma on follow-up. In a follow-up study by Wang

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and colleagues [23], however, these investigators found no association between esophagitis and an increased risk of carcinoma after 13.5 years of follow-up (relative risk, 0.8). In another study, esophagitis was an uncommon finding in Linxian, China, which is considered a very high-risk area for esophageal cancer [22]. In general, there is no known familial predisposition to squamous cell carcinoma of the esophagus. Tylosis, an uncommon genetic disorder characterized by hyperkeratosis of the palms and soles, is associated with a 90% incidence rate of squamous cell carcinoma of the esophagus by the age of 65 years [21,29]. Furthermore, individuals related to affected family members are also at increased risk for squamous carcinoma. HPV plays an important role in the evolution of squamous cell carcinoma of the uterine cervix. In the esophagus, however, the role of HPV infection in cancer development is controversial. HPV DNA has been isolated from esophageal squamous carcinomas at prevalence rates that vary from 0% to 66% [30–33]. Some have reported that HPV infection is not a major risk factor for esophageal squamous cell carcinoma in high-risk populations, such as China, whereas others suggest that the presence of either HPV-16 or -18 may be causally related to cancer development [34,35]. Varying rates of HPV positivity between different studies may be related to differences in techniques used to detect HPV, and to differences in patient populations. In one study by Turner and colleagues [30], HPV was detected by PCR in only 1 (2%) of 51 of squamous cell carcinomas in patients from the United States, which is considered a lower-risk region for this type of malignancy. HPV may be a causative factor in a subset of carcinomas from high-risk areas. At present, HPV has not been evaluated in dysplasia precursor lesions, although it has been detected in squamous papillomas, but these are not believed to be premalignant lesions [36]. The reported incidence of esophageal carcinoma in achalasia ranges from 0.53% to 8.6% [20]. It has been postulated that chronic irritation of the esophageal mucosa by retained food and saliva, such as in patients with achalasia, may play a role in malignant transformation. A recent study suggested that nuclear expression of Ki-67 in esophagitis and dysplasia, in achalasia, was increased in patients who developed carcinoma [37]. PATHOGENESIS Esophageal squamous cell carcinoma is believed to develop through a progression of premalignant (dysplastic) precursor lesions [9]. Squamous dysplasia of the esophagus is frequently identified in biopsy specimens in patients from China and Japan, whereas in the United States it is more commonly observed in resection specimens of esophageal squamous cell carcinoma and in patient populations at high risk for squamous cell carcinoma [12,38]. Dysplasia is found in 60% to 90% of resected cases of esophageal squamous cell carcinoma [15]. In one study, high-grade dysplasia was found in 14% and low-grade dysplasia in 20% of resection specimens with invasive squamous cell carcinoma [7]. In autopsy series, about 30% of patients with esophageal squamous cell

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carcinoma reveal squamous dysplasia. In addition, dysplasia is more frequently found in patients who are older in age [11,39,40]. Squamous dysplasia is also frequently multifocal, and in this setting is more likely to be associated with multiple carcinomas [7,16,41,42]. Unfortunately, little is known regarding the molecular pathogenesis of squamous dysplasia and squamous cell carcinoma of the esophagus. The most common alterations in squamous cell carcinoma are overexpression of cell cycle regularity proteins (cyclin D1, cyclin E) and inactivation, or loss, of tumor suppressor proteins, such as p53, Rb, and p16 in up to 80% of cases [43,44]. Most tumors also express high levels of epidermal growth factor receptor, up to 92% in some studies. Of these molecular defects, inactivating mutations of p53 occur early in neoplastic progression within squamous dysplastic precursor lesions [44]. In one study, p53 expression in severe dysplasia was similar to carcinoma in situ [16]. In one study by Kurabayashi and colleagues [45], BAX expression in dysplasia was shown to play an important role as an apoptotic factor, but it may be functionally inactive in some squamous cell carcinomas, and its absence may lead to lack of suppression of tumor progression. PATHOLOGIC FEATURES Squamous dysplasia of the esophagus is characterized by both architectural and cytologic abnormalities that vary in extent and severity according to the grade [8,9,40]. Architectural abnormalities are characterized by disorganization of the epithelium, loss of cell polarity, overlapping nuclei, and lack of surface maturation. Dysplasia often appears as either regular or irregular buds of neoplastic cells that protrude downward from the surface epithelium into the underlying lamina propria [9]. In one study by Rubio and colleagues [9], a close relationship was described between the formation of epithelial buds, particularly those that were irregular in shape, and progression to invasive carcinoma. Often ones sees a clear demarcation between dysplastic cells and nondysplastic cells in biopsy or resection specimens. Cytologic abnormalities include nuclear enlargement, nuclear hyperchromasia, nuclear pleomorphism, increased nuclear/cytoplasmic ratio, and increased mitotic activity. Low-grade dysplasia is defined as involvement of less than 50% of the thickness of the epithelium with dysplastic cells, whereas high-grade dysplasia is characterized by more than 50% involvement of the epithelium, including full-thickness involvement, which corresponds to carcinoma in situ (Figs. 2 and 3). Furthermore, dysplasia may also extend into the submucosal glands and ducts (Fig. 4), which can simulate invasive carcinoma morphologically [46]. Historically, squamous dysplasia has been subjectively divided into three grades: (1) mild dysplasia, involving the basal 25% of the epithelium; (2) moderate dysplasia, involving the lower 50% of the epithelium, with maturation in the upper half of the epithelium; and (3) severe dysplasia, involving more than 50% of the epithelium. The two-tiered system described previously, however, is preferred by most pathologists [16,39].

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Fig. 2. Low-grade dysplasia (low-grade intraepithelial neoplasia, mild dysplasia) (hematoxylin-eosin, original magnification  150). There is a clear demarcation between dysplastic epithelium and overlying nondysplastic epithelium. This lesion is considered noninvasive squamous cell carcinoma by Japanese pathologists, and low-grade dysplasia by Western pathologists.

It is well known that there are significant differences between Western and Japanese criteria for interpretation of squamous dysplasia and squamous cell carcinoma of the esophagus [8]. These differences may account for the relatively high incidence rate, and relatively good prognosis, of superficial esophageal carcinoma in Japan compared with the United States [2,8,39]. For instance, Japanese pathologists place great emphasis on nuclear findings, such as nuclear enlargement, hyperchromasia, and pleomorphism, and other findings, such as single cell dyskeratosis and the presence of sharp borders between nonneoplastic and neoplastic areas, to establish a diagnosis of carcinoma versus dysplasia [8]. In contrast, Western pathologists generally reserve the diagnosis of carcinoma for cases in which there is unequivocal morphologic evidence of invasion of cells into the lamina propria. As an example, low-grade dysplasia diagnosed by Western pathologists in a biopsy may be interpreted as ‘‘noninvasive

Fig. 3. High-grade dysplasia (hematoxylin-eosin, original magnification  200). The full thickness of the epithelium is involved by dysplastic cells. This is equivalent to carcinoma in situ.

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Fig. 4. High-grade dysplasia (hematoxylin-eosin, original magnification  60) extending into the submucosal ducts. There is no evidence of invasion into the lamina propria.

squamous cell carcinoma’’ by some Japanese pathologists. In some instances, invasion into the lamina propria may develop directly from low-grade dysplasia (Fig. 5). Early invasive carcinoma of the esophagus is not always associated with high-grade dysplasia. Occasionally, dysplasia may be present as isolated cells that spread in a horizontal pagetoid fashion within the squamous epithelium, or show extension into the underlying submucosal ducts [46–48]. This pattern is often seen in

Fig. 5. Low-grade dysplasia (hematoxylin-eosin, original magnification  100) associated with early invasive carcinoma.

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resected cases of squamous cell carcinoma. In addition, high-grade dysplasia is more frequently seen in continuity with the invasive component of squamous cell carcinoma, which supports the concept that high-grade dysplasia is a precursor to carcinoma in the esophagus. Furthermore, dysplasia is often multifocal and this helps explain the occasional occurrence of multiple carcinomas in the same patient. In a review of 205 patients with squamous cell carcinoma, 14.6% of cases showed multiple tumors [42]. More than half of the second lesions were intraepithelial carcinomas. In that study, the incidence of coexistent dysplasia contiguous to the main lesion was 42% and 100% in patients, respectively, who either did, or did not, receive preoperative irradiation. These data support the concept of a ‘‘field effect’’ in the pathogenesis of squamous cell carcinoma of the esophagus. DIFFERENTIAL DIAGNOSIS OF SQUAMOUS DYSPLASIA Benign and malignant lesions can mimic squamous dysplasia morphologically, and pathologists need to be aware of these potential diagnostic mishaps. These include squamous papilloma; pseudoepitheliomatous (regenerative) hyperplasia, induced by erosions, ulcers, or esophagitis; multinucleated change in esophagitis; and radiation or chemotherapy effect. Malignancies that mimic dysplasia include lateral spread of invasive squamous cell carcinoma and verrucous carcinomas. Squamous papillomas lack cytologic atypia and show an orderly cellular maturation from the basal layer toward the surface (Fig. 6) [36].

Fig. 6. Squamous cell papilloma (A, hematoxylin-eosin, original magnification  40; B, hematoxylin-eosin, original magnification  150). No cytologic atypia is noted, and cellular maturation from the basal layer toward the surface is seen.

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Pseudoepitheliomatous hyperplasia occurs most commonly adjacent to a healing ulcer or overlying granular cell tumors. It often shows abundant inflammatory cells and surface erosion, and may simulate invasive squamous cell carcinoma. Neither significant nuclear pleomorphism nor loss of polarity are present in pseudoepitheliomatous hyperplasia. Distinction of squamous dysplasia from regenerative or reactive squamous epithelium may sometimes be difficult, especially in biopsy specimens. Regenerative squamous epithelium lacks significant nuclear pleomorphism, overlapping, or crowding, and does not reveal abnormal mitoses (Fig. 7). In addition, surface maturation is usually present. Inflammation frequently accompanies reactive squamous epithelium, and in the presence of inflammation, a diagnosis of dysplasia must always be made with caution. In practice, when low-grade dysplasia is suspected in biopsy specimens with inflammation, some pathologists may use the term ‘‘indefinite’’ for squamous dysplasia, similar to the nomenclature in Barrett’s esophagus or inflammatory bowel disease, and recommend a follow-up biopsy after treatment of the underlying esophagitis. Recently the term ‘‘atypical regenerative hyperplasia’’ of the esophagus in endoscopic biopsies has been used to describe lesions that approach squamous cell carcinoma [49]. If the histologic changes are ambiguous, medical treatment and subsequent biopsies are recommended, particularly if there is no endoscopic or radiologic findings suggestive of a neoplasm. Not uncommonly, the basal layers of regenerating squamous epithelium may undergo multinucleation and simulate dysplasia. These changes, however, have been shown to be reactive in nature and not premalignant. After radiotherapy or chemotherapy, squamous epithelium may become atypical and mimic dysplasia or carcinoma. Atypical cells caused by radiation or chemotherapy do not show increased nucleus/cytoplasm ratio, however, but instead reveal prominent cytoplasmic vacuolization. In addition, similar

Fig. 7. Reactive squamous epithelium. Neither significant nuclear pleomorphism nor nuclear overlapping is observed. Note the neutrophilic infiltrate (A, hematoxylin-eosin, original magnification  150) and eosinophilic infiltrate (B, hematoxylin-eosin, original magnification  120). The right-side image is a case of reflux esophagitis.

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changes may be observed in stromal mesenchymal cells, which are also affected by radiation and chemotherapy [11,40]. Lateral spread of squamous cell carcinoma may mimic dysplasia particularly if the invasive component is not present in the tissue sample [48]. In these cases, nuclear hyperchromasia and pleomorphism are usually more prominent compared with high-grade dysplasia, and a clear demarcation from carcinomatous to noncarcinomatous epithelium is typically present. There are two general types of lateral spread. One shows full-thickness replacement by carcinoma (full-thickness–type) and the other reveals only basal epithelial involvement (basal layer–type). In the basal layer–type, involvement may be seen in only one or two layers of the epithelium. Verrucous carcinoma is an extremely rare form of malignancy and shows an exophytic papillary growth pattern [50]. Verrucous carcinomas are characterized by the presence of only minor cytologic atypia and prominent acanthosis. A blunt pushing invasive margin is typical, but inflammation may be noted. NATURAL HISTORY AND TREATMENT OF SQUAMOUS DYSPLASIA By definition, squamous dysplasia is confined to the epithelium, so metastases do not occur. Increasing grades of squamous dysplasia are associated, however, with a progressive increased risk of synchronous, or metachronous, squamous cell carcinoma. Unfortunately, there have been few studies that have evaluated the natural history of squamous dysplasia. Nevertheless, some studies suggest that there is a 25% and 75% incidence of carcinoma among patients with lowand high-grade dysplasia, respectively, after more than 10 years of follow-up [23,51]. For instance, in one follow-up study by Wang and colleagues [23] in 2005, of 682 endoscoped patients from a high-risk population in China, the relative risk of cancer development was 2.9 for patients with mild dysplasia (24%); 9.8 for those with moderate dysplasia (50%) (both considered low grade); 28.3 for those with severe dysplasia (74%); and 34.4 for patients with carcinoma in situ (75%). Furthermore, the outcome of severe dysplasia and carcinoma in situ were equivalent, supporting the inclusion of these two lesions into one highgrade dysplasia category. In another 15-year follow-up study of 12,693 persons in Linxian, China, screened by balloon cytology, the risk of cancer increased in parallel with the severity of cytologic diagnoses [51]. For instance, the relative risks of cancer were 1.5 for low-grade dysplasia (dysplasia 1); 1.9 for high grade (dysplasia 2); and 5.8 for cases considered suspicious for cancer. Finally, in another follow-up study from China by Qiu and Yang [28] in 1988, the rate of dysplasia was 38% in high-risk regions and 5% in low-risk regions. On follow-up of the 62 patients with dysplasia (not further subclassified), about 30% developed carcinoma between 30 and 78 months later. In this study, less than 5% of patients with esophagitis, but without dysplasia, developed cancer confirming the low risk of cancer in patients with only inflammation.

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Dysplasia may also regress or disappear, but in these situations, sampling error or interobserver variability in interpretation probably account for cases of this kind because it is widely believed that true neoplastic lesions cannot convert back to a normal biologic state. Accurate histologic grading of dysplasia is clinically relevant. Unfortunately, guidelines regarding the frequency of endoscopic surveillance have not been established for either low- or high-grade squamous dysplasia of the esophagus. Nevertheless, in general, patients with flat low-grade dysplasia unassociated with a mass lesion are usually managed with endoscopic surveillance and biopsies [16,23,28]. Some may choose mucosal resection, however, for this type of lesion. For patients with high-grade dysplasia, endoscopic mucosal resection, endoscopic submucosal dissection, or esophagectomy are all considered appropriate methods of treatment (Fig. 8). In contrast, any grade of dysplasia associated with a mass lesion should be considered a high-risk lesion, because it probably represents an invasive carcinoma. Surgical resection is considered mandatory for patients with dysplasia associated with a mass or stricture. A recent publication by Wang and colleagues [23] suggested that patients with mild dysplasia be re-evaluated periodically, or treated by chemoprevention; patients with moderate dysplasia should be followed by endoscopy more closely, or treated by endoscopic therapy; and patients with severe

Fig. 8. Specimens obtained by endoscopic submucosal dissection. The basic cut line was determined at the nearest distance to observe the area between the tumor and the surgical margin. Then the specimen was sliced serially in 2- to 3-mm widths parallel to the basic cut line.

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dysplasia or carcinoma in situ should be treated by endoscopic or surgical therapy. These recommendations, however, were entirely anecdotal. In another recent study, endoscopic mucosal resection was determined to be an effective and safe method of curative treatment in patients with high-grade dysplasia (carcinoma in situ) or intramucosal carcinoma [52]. In that study, 90% of patients with high-grade dysplasia, 100% of patients with intramucosal cancer, and 80% with superficial submucosal cancer achieved a complete response after a mean of 29.7 months of follow-up. None of the patients had a major complication, such as perforation or bleeding requiring transfusion, and only six (15%) had minor complications, such as minor bleeding or stenoses. Survival was 90% and 89% for patients with high-grade dysplasia and intramucosal cancer, respectively. In another study by Shimizu and colleagues [53] of 35 patients with highgrade dysplasia and 16 with intramucosal cancer, only two (4%) developed recurrences after endoscopic mucosal resection over a 23-month follow-up period, and in these recurrent cases, a second endoscopic resection was curative. Finally, two studies published in the Japanese literature emphasized the important role of iodine staining in management [54,55]. For instance, in one study by Shimada and colleagues [54], the risk of carcinoma in unstained areas less than 5 mm in size was 0.9%. Surveillance endoscopy was recommended at 12-month intervals for patients in this group, and 6-month intervals (or endoscopic mucosal resection) for patients with unstained areas between 5 and 10 mm in size. In that study, patients with unstained areas measuring greater than 10 mm were recommended to be treated with endoscopic mucosal resection. SUMMARY Identification of dysplasia in mucosal biopsies is the most reliable pathologic indicator of an increased risk of development of squamous cell carcinoma. The diagnosis of dysplasia may, on occasion, be difficult and close pathologistclinician interaction is highly recommended to individualize patient management appropriately. More studies are needed to define other early nonmorphologic biomarkers for risk of squamous cell carcinoma. The natural history of dysplasia is poorly understood, particularly in low-risk regions, and prospective follow-up studies are needed. References [1] Parker SL, Tong T, Bolden S, et al. Cancer statistics, 1997. CA Cancer J Clin 1997;47(1): 5–27. [2] Gabbert HE, Shimoda T, Hainaut P, et al. Squamous cell carcinoma of the oesophagus. In: Hamilton SR, Aaltonen LA, editors. World Health Organization of Tumours. Pathology and genetics. Tumours of the digestive system. Lyon (France): IARC Press; 2000. p. 9–30. [3] Brown LM, Hoover RN, Greenberg RS, et al. Are racial differences in squamous cell esophageal cancer explained by alcohol and tobacco use? J Natl Cancer Inst 1994;86(17): 1340–5.

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[4] Watanabe H, Jass JR, Sobin LH. World Health Organization. International histological classification of tumours: histological typing of oesophageal and gastric tumours. 2nd edition. New York: Springer-Verlag; 1990. p. 1–18. [5] Kuwano H, Watanabe M, Sadanaga N, et al. Squamous epithelial dysplasia associated with squamous cell carcinoma of the esophagus. Cancer Lett 1993;72(3):141–7. [6] Kuwano H, Baba K, Ikebe M, et al. Histopathology of early esophageal carcinoma and squamous epithelial dysplasia. Hepatogastroenterology 1993;40(3):222–5. [7] Kuwano H, Matsuda H, Matsuoka H, et al. Intra-epithelial carcinoma concomitant with esophageal squamous cell carcinoma. Cancer 1987;59(4):783–7. [8] Schlemper RJ, Dawsey SM, Itabashi M, et al. Differences in diagnostic criteria for esophageal squamous cell carcinoma between Japanese and Western pathologists. Cancer 2000;88(5):996–1006. [9] Rubio CA, Liu FS, Zhao HZ. Histological classification of intraepithelial neoplasias and microinvasive squamous carcinoma of the esophagus. Am J Surg Pathol 1989;13(8):685–90. [10] Watanabe G, Ajioka Y, Kobayashi M, et al. Pathological diagnosis of dysplasia in the esophageal squamous epithelium. Stomach and Intestine 2007;42(2):129–35 (in Japanese with English abstract). [11] Takubo K. Pathology of the esophagus. Tokyo: EDUCA Inc; 2000. p. 131–68. [12] Dawsey SM, Wang GQ, Weinstein WM, et al. Squamous dysplasia and early esophageal cancer in the Linxian region of China: distinctive endoscopic lesions. Gastroenterology 1993;105(5):1333–40. [13] Hashimoto CL, Iriya K, Baba ER, et al. Lugol’s dye spray chromoendoscopy establishes early diagnosis of esophageal cancer in patients with primary head and neck cancer. Am J Gastroenterol 2005;100(2):275–82. [14] Dawsey SM, Fleischer DE, Wang GQ, et al. Mucosal iodine staining improves endoscopic visualization of squamous dysplasia and squamous cell carcinoma of the esophagus in Linxian, China. Cancer 1998;83(2):220–31. [15] Wei WQ, Abnet CC, Lu N, et al. Risk factors for oesophageal squamous dysplasia in adult inhabitants of a high risk region of China. Gut 2005;54(6):759–63. [16] Saeki H, Kimura Y, Ito S, et al. Biological and clinical significance of squamous epithelial dysplasia of the esophagus. Surgery 2002;131(1):S22–7. [17] Hamamoto Y, Endo T, Nosho K, et al. Usefulness of narrow-band imaging endoscopy for diagnosis of Barrett’s esophagus. J Gastroenterol 2004;39(1):14–20. [18] Kuznetsov K, Lambert R, Rey JF. Narrow-band imaging: potential and limitations. Endoscopy 2006;38(1):76–81. [19] Ribiero U Jr, Posner MC, Safatle-Ribeiro AV, et al. Risk factors for squamous cell carcinoma of the oesophagus. Br J Surg 1996;83(9):1174–85. [20] Leeuwenburgh I, Haringsma J, Van Dekken H, et al. Long-term risk of oesophagitis, Barrett’s oesophagus and oesophageal cancer in achalasia patients. Scand J Gastroenterol Suppl 2006;243:7–10. [21] Iwaya T, Maesawa C, Ogasawara S, et al. Tylosis esophageal cancer locus on chromosome 17q25.1 is commonly deleted in sporadic human esophageal cancer. Gastroenterology 1998;114(6):1206–10. [22] Dawsey SM, Lewin KJ, Liu FS, et al. Esophageal morphology from Linxian, China: squamous histologic findings in 754 patients. Cancer 1994;73(18):2027–37. [23] Wang GQ, Abnet CC, Shen Q, et al. Histological precursors of oesophageal squamous cell carcinoma: results from a 13 year prospective follow up study in a high risk population. Gut 2005;54(2):187–92. [24] Crespi M, Munoz N, Grassi A, et al. Oesophageal lesions in northern Iran: a premalignant condition? Lancet 1979;2(8136):217–21. [25] Crespi M, Munoz N, Grassi A, et al. Precursor lesions of oesophageal cancer in a low-risk population in China: comparison with high-risk populations. Int J Cancer 1984;34(5): 599–602.

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[26] Munoz N, Crespi M, Grassi A, et al. Precursor lesions of oesophageal cancer in high-risk populations in Iran and China. Lancet 1982;1(8277):876–9. [27] Guanrei Y, Songliang Q. Endoscopic surveys in high-risk and low-risk populations for esophageal cancer in China with special reference to precursors of esophageal cancer. Endoscopy 1987;19(3):91–5. [28] Qiu SL, Yang GR. Precursor lesions of esophageal cancer in high-risk populations in Henan Province, China. Cancer 1988;62(3):551–7. [29] Marger RS, Marger D. Carcinoma of the esophagus and tylosis: a lethal genetic combination. Cancer 1993;72(1):17–9. [30] Turner JR, Shen LH, Crum CP, et al. Low prevalence of human papillomavirus infection in esophageal squamous cell carcinomas from North America: analysis by a highly sensitive and specific polymerase chain reaction-based approached. Hum Pathol 1997;28(2): 174–8. [31] Poljak M, Cerar A, Seme K. Human papillomavirus infection in esophageal carcinomas: a study of 121 lesions using multiple broad-spectrum polymerase chain reactions and literature review. Hum Pathol 1998;29(3):266–71. [32] Chang F, Syrjanen S, Shen Q, et al. Human papillomavirus involvement in esophageal carcinogenesis in the high-incidence area of China: a study of 700 cases by screening and type-specific in situ hybridization. Scand J Gastroenterol 2000;35(2):123–30. [33] Farhadi M, Tahmasebi Z, Merat S, et al. Human papillomavirus in squamous cell carcinoma of esophagus in a high-risk population. World J Gastroenterol 2005;11(8):1200–3. [34] Gao GF, Roth MJ, Wei WQ, et al. No association between HPV infection and the neoplastic progression of esophageal squamous cell carcinoma: result from a cross-sectional study in a high-risk region of China. Int J Cancer 2006;119(6):1354–9. [35] Souto Damin AP, Guedes Frazzon AP, de Carvalho Damin D, et al. Detection of human papillomavirus DNA in squamous cell carcinoma of the esophagus by auto-nested PCR. Dis Esophagus 2006;19(2):64–8. [36] Odze R, Antonioli D, Shocket D, et al. Esophageal squamous papillomas: a clinicopathologic study of 38 lesions and analysis for human papillomavirus by polymerase chain reaction. Am J Surg Pathol 1993;17(8):803–12. [37] Fujii T, Yamana Y, Sueyoshi S, et al. Histological analysis of non-malignant and malignant epithelium of the esophagus. Dis Esophagus 2000;13(2):110–6. [38] Lu XJ, Chen ZF, Guo CL, et al. Endoscopic survey of esophageal cancer in a high-risk area of China. World J Gastroenterol 2004;10(20):2931–5. [39] Dry SM, Lewin KJ. Esophageal squamous dysplasia. Semin Diagn Pathol 2002;19(1): 2–11. [40] Odze RD, Antonioli DA. Pathology of esophageal cancer. In: Rustgi AK, editor. Gastrointestinal cancers. Edinburgh (UK): Saunders; 2003. p. 253–70. [41] Morita M, Kuwano H, Yasuda M, et al. The multicentric occurrence of squamous epithelial dysplasia and squamous cell carcinoma in the esophagus. Cancer 1994;74(11): 2889–95. [42] Kuwano H, Ohno S, Matsuda H, et al. Serial histologic evaluation of multiple primary squamous cell carcinomas of the esophagus. Cancer 1988;61(8):1635–8. [43] Lin J, Beer DG. Molecular biology of upper gastrointestinal malignancies. Semin Oncol 2004;31(4):476–86. [44] Parenti AR, Rugge M, Frizzera E, et al. P53 overexpression in the multistep process of esophageal carcinogenesis. Am J Surg Pathol 1995;19(12):1418–22. [45] Kurabayashi A, Furihata M, Matsumoto M, et al. Expression of Bax and apoptosis-related proteins in human esophageal squamous cell carcinoma including dysplasia. Mod Pathol 2001;14(8):741–7. [46] Tajima Y, Nakanishi Y, Tachimori Y, et al. Significance of involvement by squamous cell carcinoma of the ducts of esophageal submucosal glands: analysis of 201 surgically resected superficial squamous cell carcinoma. Cancer 2000;89(2):248–54.

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[47] Chu P, Stagias J, West AB, et al. Diffuse pagetoid squamous cell carcinoma in situ of the esophagus: a case report. Cancer 1997;79(10):1865–70. [48] Soga J, Tanaka O, Sasaki K, et al. Superficial spreading carcinoma of the esophagus. Cancer 1982;50(8):1641–5. [49] Arista-Nasr J, Rivera I, Martinez-Benitez B, et al. Atypical regenerative hyperplasia of the esophagus in endoscopic biopsy: a mimicker of squamous esophagi carcinoma. Arch Pathol Lab Med 2005;129(7):899–904. [50] Biemond P, ten Kate FJ, van Blankenstein M. Esophageal verrucous carcinoma: histologically a low-grade malignancy but clinically a fatal disease. J Clin Gastroenterol 1991;13(1):102–7. [51] Dawsey SM, Yu Y, Taylor PR, et al. Esophageal cytology and subsequent risk of esophageal cancer: a prospective follow-up study from Linxian, China. Acta Cytol 1994;38(2): 183–92. [52] Pech O, Gossner L, May A, et al. Endoscopic resection of superficial esophageal squamouscell carcinomas: Western experience. Am J Gastroenterol 2004;99(7):1226–32. [53] Shimizu Y, Kato M, Yamamoto J, et al. Histologic results of EMR for esophageal lesions diagnosed as high-grade intraepithelial squamous neoplasia by endoscopic biopsy. Gastrointest Endosc 2006;63(1):16–21. [54] Shimada H, Makuuchi H, Machimura T, et al. Histological study of tiny iodine unstained lesions smaller than 5 mm in length. Stomach and Intestine 1994;29(9):921–30 (in Japanese with English abstract). [55] Kawachi H, Kobayashi M, Takizawa T, et al. Histopathological findings and p53 genetic abnormality in esophageal squamous intraepithelial neoplasias. Stomach and Intestine 2000;42(2):173–86 (in Japanese with English abstract).

Gastroenterol Clin N Am 36 (2007) 813–829

GASTROENTEROLOGY CLINICS OF NORTH AMERICA

Gastric Preneoplastic Lesions and Epithelial Dysplasia Gregory Y. Lauwers, MDa,*, Amitabh Srivastava, MDb a

Department of Pathology, Massachusetts General Hospital, Gastrointestinal Pathology Service, 55 Fruit Street, Warren 2, Boston, MA 02114-2696, USA b Department of Pathology, Dartmouth-Hitchcock Medical Center, 1 Medical Center Drive, Lebanon, NH 03755, USA

A

well-defined carcinogenic sequence evolving through chronic gastritis and gastric epithelial dysplasia (GED) typically precedes the development of most gastric adenocarcinomas in people. In many ways, this inflammation-metaplasia-dysplasia-cancer sequence is similar to other chronic gastrointestinal inflammatory conditions associated with an increased risk of cancer, such as gastroesophageal reflux disease and inflammatory bowel disease. Collectively, morphological alterations that precede cancer often are described under the rubric of preneoplastic states or precursors of gastric cancer. Alterations such as gastric mucosal atrophy and intestinal metaplasia, however, are merely markers of increased risk, while others, such as GED, represent a direct precursor of cancer. Morphologically, the Lauren classification, which classifies gastric cancer into intestinal and diffuse types, often is used in epidemiologic studies [1]. The more differentiated variant, the intestinal type, is linked closely with Helicobacter pylori infection and is associated with well-characterized dysplastic precursor lesions. In contrast, the precursor lesions of the diffuse type, composed of poorly differentiated discohesive cells, are understood less well. Furthermore, although H pylori infection is recognized as a causal factor, the association may not be as strong. MUCOSAL CHANGES THAT PRECEDE GASTRIC DYSPLASIA Epidemiological and morphological studies have demonstrated that intestinal gastric cancers usually are preceded by a sequence of histological events beginning with diffuse chronic gastritis and eventually leading to mucosal atrophy, intestinal metaplasia, and dysplasia [2–12]. H pylori infection is associated with the induction of chronic inflammation in gastric mucosa and with the progressive development of metaplastic changes [7]. In fact, there is evidence that DNA damage and increased mucosal proliferation secondary to H pylori

*Corresponding author. E-mail address: [email protected] (G.Y. Lauwers). 0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.008

ª 2007 Elsevier Inc. All rights reserved. gastro.theclinics.com

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infection, combined with a suitable host susceptibility phenotype (eg, genetic polymorphisms in interleukin [IL]-1B, IL-1RN, and tumor necrosis factor a [TNF-a] genes), are important factors in this progression pathway [4,6,13–15]. Gastric atrophy is defined as loss of native mucosal glandular elements and replacement by metaplastic glands, or fibrosis. Thus, it manifests as loss of either oxyntic glands in the gastric body/fundus or pyloric mucous glands in the gastric antrum (Fig. 1). Evaluation of intestinal-type gastric cancers shows evidence of mucosal atrophy even in the absence of intestinal metaplasia, suggesting that atrophy may be a better indicator of risk of cancer than intestinal metaplasia [16]. Regression of atrophy following treatment of H pylori [17–19], however, does not eliminate the risk of cancer completely. Furthermore, histologic grading of gastric atrophy suffers from a lack of interobserver reproducibility [20], and despite a recent proposal for new quantitative methods, remains to be validated in routine clinical practice [21]. Mucosal atrophy often is associated with pseudopyloric gland metaplasia in the gastric corpus mucosa, where oxyntic glands are replaced by mucous glands (Fig. 2). This form of metaplasia, which expresses a type of trefoil peptide, the spasmolytic polypeptide (termed spasmolytic polypeptide-expressing metaplasia or SPEM), has been shown to be linked more closely to gastric cancer than intestinal metaplasia [22,23]. Atrophic gastritis also leads to a reduction in serum pepsinogen levels, which are more marked for Pepsinogen I than Pepsinogen II, and result in a reduced Pepsinogen I/II ratio [24]. This surrogate marker of gastric atrophy, however, is not widely used clinically at the present time. Thus, the precise role of gastric atrophy in the pathogenesis of gastric cancer is uncertain, but may be linked to a higher gastric pH, with consequent bacterial overgrowth and nitrate reduction, which in turn may result in metaplasia. Classically, three major types of intestinal metaplasia (IM) have been described [8,25,26]. Type I (complete type) closely resembles small intestinal

Fig. 1. Intestinal metaplasia, complete type. Note the presence of goblet cells and Paneth cells.

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Fig. 2. Atrophic gastric body mucosa with loss of specialized glandular elements (oxyntic glands) and pseudopyloric metaplasia. Focal intestinal metaplasia is present in lower right corner.

mucosa and is composed of goblet cells, absorptive cells with a brush border, and Paneth cells (see Fig. 1) [25]. Biochemically, in type I IM, goblet cells secrete small intestinal sialomucin, which stains with Alcian blue (pH 2.5), but not with high-iron diamine, which highlights sulfomucins [8,25,26]. Types II and III (incomplete IM) are considered less differentiated forms of IM [9] and show numerous goblet cells, few or no Paneth cells, absence of absorptive cells, and presence of intermediate cells resembling gastric surface foveolar epithelium. Intermediate cells show hybrid cellular features at the ultrastructural level, similar to both absorptive and mucinous cells [25]. In types II and III IM, the acidic mucin content of the intermediate cells distinguishes the two subtypes: in type II IM, the mucin granules contain predominantly sialomucins, whereas in type III, the mucin granules are composed largely of sulfomucins [8]. Immunohistochemically, type I IM expresses intestinal mucins MUC2 and MUC4 in goblet cells, with little or no expression of either MUC1, MUC5AC, or MUC6 [27,28]. Types II and III also show MUC2 and MUC4 expression in goblet cells, similar to type I IM, but in addition, MUC1, MUC5AC, and to a lesser degree, MUC6 positivity is also present in both the goblet cells and the columnar cells [27,28]. The occurrence and extent of IM, and the prevalence of type III IM, increase with age [9,29]. Eventually, IM may involve most of the antrum and lesser curvature [26,30]. The exception to this rule is autoimmune gastritis, where IM normally is restricted to the corpus/fundic region. Incomplete types of IM are present in the background gastric mucosa of 75% to 95% of gastric resections for intestinal-type cancer, but in only 0% to 33% of diffuse-type gastric carcinomas [8,31]. Despite the association of IM with cancer, the presence of IM is neither sufficiently sensitive nor specific enough to guide surveillance strategies for patients with H pylori gastritis. Subtyping of intestinal metaplasia also is not

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considered clinically useful for gastroenterologists to plan surveillance for gastric cancer. The extent and severity of atrophy, and the extent of intestinal metaplasia, are far more important indicators of increased cancer risk in these patients [30]. To stratify patients appropriately using this latter approach, however, exhaustive mapping of the gastric mucosa is typically necessary [32]. Whiting and colleagues [33] reported an 11% risk of gastric cancer in patients who had gastric atrophy or intestinal metaplasia over a 10-year follow-up period. The high incidence, however, can be explained partly by the fact that only macroscopic abnormalities were biopsied. Other factors, such as ethnic background and family history, also should be considered in selecting patients for surveillance. Current recommendations include careful topographic mapping of the entire stomach in patients who ultimately are selected for surveillance endoscopy [34], along with additional biopsies from any endoscopically visible abnormalities. Although H pylori eradication therapy is recommended in these patients, cancer risk is not eliminated completely if either atrophy or intestinal metaplasia is present at the time of the index endoscopy. GASTRIC EPITHELIAL DYSPLASIA Evidence for GED as a direct precursor of gastric adenocarcinoma stems primarily from observations in surgically resected gastric cancers. In this setting, high-grade dysplasia has been identified in close proximity to 40% to 100% of early gastric cancers, and 5% to 80% of advanced adenocarcinomas [35–37]. Moreover, GED is also a marker of risk for cancer elsewhere in the gastric mucosa. Thus, GED is often present in the background mucosa distant from foci of adenocarcinoma, and removal of dysplastic lesions decreases subsequent risk of cancer. In the early 1980s, guidelines for the diagnosis and grading of GED were developed. Dysplasia was defined as ‘‘unequivocally neoplastic epithelium that may be associated with or give rise to invasive adenocarcinoma’’ [38,39]. The current two-tier scheme of low-grade and high-grade dysplasia has proven to be more reproducible than the now obsolete three-tier system of mild, moderate, and severe [40]. It also provides a clinically meaningful risk stratification method that can be used to guide patient management (Table 1) [41]. Clinical Features of Gastric Epithelial Dysplasia The prevalence of GED shows marked variation worldwide, from 9% to 20% in high-risk areas such as Colombia and China, to 0.5% to 3.75% in Western countries, where gastric cancer is less common [37,42–45]. This difference is largely a result of variations in the genetic makeup of the respective patient populations, and environmental factors, including the prevalence of H pylori infection. The risk of cancer is also variable among different strains of H pylori. For instance, cagA, vacA, or babA genotypes have been associated with increased cancer risk. The frequency of GED also differs with the underlying etiology. For instance, prevalence rates of up to 40% have been reported in patients who

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Table 1 Therapeutic guidelines recommended in the literature Conditions

Suggested recommendation

Comments

Helicobacter pylori gastritis with intestinal metaplasia

Antibiotherapy and topographic mapping of the entire stomach with biopsies of any visible abnormalities Annual endoscopic surveillance with rebiopsy

Cancer risk is not completely eliminated.

Dysplasia, low-grade

Dysplasia, high-grade

EMR and surveillance

Given the low rate of malignant transformation, surgical resection is not normally necessary Chromoendoscopy and endoscopic ultrasound help evaluate the extent and depth of invasion.

This lesion needs definitive therapy. Surgery is recommended if not amenable to endoscopic mucosal resection (EMR).

have pernicious anemia [46–48]. The increased risk of developing adenocarcinoma, however, compared with the general population, is relatively moderate in these patients [49]. Patients with familial adenomatous polyposis (FAP) are also at higher risk of developing either flat or, more commonly, polypoid (ie, adenomas) dysplasia. These typically are located in the antrum, frequently multiple, and observed in 2% to 50% of patients [50–61]. Although not universally accepted, other patient subgroups that also may be at an increased risk of gastric cancer include those with a gastric remnant status after gastrectomy, Menetrier’s disease, and Peutz-Jegher’s syndrome [62–65]. Additional studies are needed to precisely define the magnitude of risk in these patient groups. Most patients who have GED are in the sixth to seventh decade of life [66,67], with men affected more often than women (male:female ratios range from 2.4 to 3.9:1) [67,68]. Although GED may be diagnosed in any segment of the stomach, it most commonly affects the lesser curvature, particularly the antrum or the incisura angulus, in association with intestinal metaplasia (Fig. 3) [67,69]. Although the diagnosis of GED often is made on random mucosal biopsies without corresponding endoscopic abnormalities [67,68], various abnormal endoscopic patterns may be seen. These include mucosal irregularity in a background of atrophic mucosa, erosions, ulcers [67], mucosal scars [66], diffuse inflammatory changes [70], plaques, and polyps [66]. Polypoid dysplastic lesions usually are reported as gastric adenomas. Microscopic Features of Gastric Epithelial Dysplasia GED is a neoplastic epithelial proliferation characterized by variable cellular and architectural atypia. Most cases of GED have an intestinal phenotype resembling colonic adenomas. This is referred to as adenomatous dysplasia

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Fig. 3. Scanning view showing dysplastic changes arising in a background of atrophy and [63–65] intestinal metaplasia.

(or type I), which is architecturally characterized by crowded tubular glands lined by atypical columnar cells with overlapping, pencillate, hyperchromatic nuclei, pseudostratification, and inconspicuous nucleoli (Figs. 4 and 5) [71]. Other less common histologic variants of GED also have been recognized. Hyperplastic dysplasia (or type II) is usually present in nonmetaplastic foveolar or pyloric gland type epithelium [71]. It is composed of glands of variable size and shape, occasionally with cystic dilatation and papillary infoldings and serration. Cytologically, the cells in type II dysplasia are cuboidal or low-columnar in shape, contain clear or eosinophilic cytoplasm, and show round to oval, vesicular nuclei with nucleoli (Fig. 6). This variant of GED dysplasia is associated more commonly with poorly differentiated adenocarcinoma of the intestinal type [71–73]. Dysplasia also may develop in gastric hyperplastic polyps, particularly those greater than 2 cm in size. Several studies have reported that dysplastic changes occur in 1.8% to 16.4% of hyperplastic polyps [74–77]. In one study, the rate of

Fig. 4. Low-grade dysplasia, intestinal-type, characterized by limited architectural complexity.

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Fig. 5. Low-grade dysplasia, intestinal type, with characteristic pencillate, hyperchromatic nuclei, and pseudostratification.

dysplasia in hyperplastic polyps was 19.4%, but in that study, polyps less than 5 mm in size were excluded [78]. Dysplasia is exceedingly rare in sporadic fundic gland polyps, but it is prevalent in patients who have FAP. Fundic gland polyps are common in FAP, occurring in 7% to 51% of patients [50–55,57,58,60,79], and they show dysplasia in 25% to 42% of cases [58,80]. Despite rare reports of carcinoma arising in fundic gland polyps [81–83], most gastric cancers that develop in FAP are associated with adenomas, not fundic gland polyps [50,59]. Tubule neck dysplasia is an exceedingly rare type of GED thought to be a precursor of diffuse-type gastric carcinoma [84]. It occurs more commonly

Fig. 6. Low-grade dysplasia, foveolar type, with round, basal nuclei, inconspicuous nucleoli, and minimal stratification. In contrast to Fig. 5, note the abundance of clear cytoplasm in this type of dysplasia.

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in nonmetaplastic gastric epithelium. Typical features include enlarged, pale, and polygonal neoplastic cells confined to the basement membrane, occupying only the gland neck region, with sparing of both the mucosal surface and deeper glands [84,85]. As expected, it can be difficult to differentiate tubule neck dysplasia from atypical reactive changes. In familial diffuse gastric cancer syndrome, caused by germline E-cadherin/ CDH1 gene mutations, close to 50% of affected patients develop diffuse type gastric cancer. In fact, examples of signet ring cell carcinoma in situ have been reported in prophylactic gastrectomies performed in these patients [86]. These lesions spread in a pagetoid manner, showing signet ring cells between the gastric foveolar and glandular epithelium within the basement membrane, before invasion into the lamina propria [87]. Grading of Gastric Epithelial Dysplasia The identification and grading of GED are subject to interobserver variability. Distinguishing between inflammatory atypia in the setting of marked inflammation or ulceration and true dysplasia is not always straightforward for pathologists with limited experience in evaluation of gastric biopsies. Determination of the correct grade is critical, however, because it predicts both the risk of malignant transformation and the risk of developing gastric cancer elsewhere in the stomach. Low-grade dysplasia shows minimal architectural disarray, and [41,88,89] dysplastic cells show only mild to moderate cytologic atypia (see Fig. 5) [40,88]. At times, actively inflamed or regenerating mucosa also may reveal cytological atypia and increased mitotic activity that mimics dysplasia. Clues to the reactive nature of the epithelial changes include the presence of vascular congestion, a gradual rather than abrupt transition between the atypical and adjacent normal cells, and maturation of the atypical deeper glands as they reach the luminal surface [40,41]. In some cases, it may not be possible to distinguish inflammatory reactive atypia from dysplasia with complete certainty, and in this instance, a diagnosis of indefinite for dysplasia is justified. Follow-up endoscopy and targeted biopsies using newer imaging techniques, such as chromoendoscopy, may be helpful in these patients. High-grade dysplasia is characterized by epithelium with marked architectural abnormalities, such as glandular crowding, branching, and budding. Intraluminal necrotic debris is commonly present [90]. In contrast to low-grade dysplasia, dysplastic cells in high-grade dysplasia are usually cuboidal in shape rather than columnar, with a high nuclear:cytoplasmic ratio. The nuclei are usually round to oval, vesicular, with prominent nucleoli, and show distinct loss of cell polarity (Fig. 7). Mitoses are often numerous, and atypical mitoses also may be present [40,41,89]. It is worth noting that the recently proposed Vienna classification for gastric dysplasia was developed as a consensus between Western and Asian investigators [91]. The consensus view takes into account discrepancies in the reporting of dysplasia between Japanese and Western pathologists. For example,

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Fig. 7. High-grade dysplasia, foveolar (A) and intestinal type (B). Note the presence of characteristic large nuclei, with an open chromatin pattern and prominent nucleoli.

noninvasive intramucosal neoplastic lesions with high-grade cellular and architectural atypia are termed intramucosal carcinoma in Japan, whereas the same lesions are diagnosed as high-grade dysplasia by most pathologists in the West. In the Vienna classification, based on management implications, high-grade premalignant lesions without invasion of the lamina propria, and invasive adenocarcinomas confined to the lamina propria, now are placed into a single diagnostic category, because both are amenable to endoscopic mucosal resection. The World Health Organization (WHO) now recommends the terminology of low-grade and high-grade intraepithelial neoplasia and defines carcinoma as invasion of the lamina propria, or beyond. However, invasion of the lamina propria is not defined clearly in this proposed scheme [92]. Natural History and Treatment of Gastric Epithelial Dysplasia The diagnosis of gastric dysplasia alerts the gastroenterologist that the patient has an increased, albeit variable, risk of progression to gastric cancer. A review of older series shows that low-grade dysplasia regresses in 38% to 75% of cases and persists in 19% to 50% of the cases [45,66,93]. In comparison, high-grade dysplasia regresses in only 0% to 16% of cases and persists in 14% to 58% of cases. Progression to adenocarcinoma has been reported from 0% to 23% for low-grade GED, within a mean interval of 10 months to 4 years. In contrast, the rate of malignant transformation for high-grade GED ranges from 60% to 85% over a median interval of 4 to 48 months [66–68,70,94–96]. A diagnosis of carcinoma within 3 months of GED, however, is more likely to represent failure to recognize a pre-existing cancer rather than true neoplastic progression [67,68,70]. More recent studies have confirmed the low risk of progression to

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cancer in patients with low-grade dysplasia (0% to 9%) and reiterate the significant risk of malignant transformation associated with high-grade dysplasia (10% to 100%) [96,97]. The final step in the carcinogenic sequence is the development of intramucosal adenocarcinoma that may be grossly similar to highgrade dysplastic lesions. Intramucosal adenocarcinomas are distinguished from high-grade dysplasia on the basis of invasion through the epithelial basement membrane. Because intramucosal adenocarcinomas have limited metastatic potential, and less than 10% risk of nodal metastases [98], these patients can be managed similar to those with high-grade dysplasia if submucosal invasion is excluded with certainty by endoscopic ultrasound and adequate biopsy sampling. Given the low rate of malignant transformation of low-grade dysplasia, annual endoscopic surveillance with rebiopsy typically is performed, and surgical resection is usually not necessary [99,100]. It also must be emphasized that lowgrade dysplasia occurring in a background of extensive intestinal metaplasia may be associated with a higher risk of malignancy [101]. Patients who have high-grade dysplasia, large adenomatous polyps, or well-differentiated adenocarcinomas no more than 2 cm should undergo definitive therapy. Currently, chromoendoscopy and endoscopic ultrasound are used widely to evaluate the extent and depth of these lesions. Complete excision of mucosal-based lesions may be performed by endoscopic mucosal resection, obviating the need for surgical resection in many cases [102]. Mucosal lesions that are not amenable to endoscopic resection, and those with a submucosal component, are managed best with surgical resection. Molecular Biology of Gastric Neoplasia Most gastric adenocarcinomas develop as a result of a combination of predisposing environmental conditions and genetic and epigenetic abnormalities. Preneoplastic and dysplastic lesions share, albeit with a lesser frequency, many of the molecular alterations observed in gastric carcinoma. Tumor suppressor genes The APC gene is mutated in 20% to 76% of gastric adenomas and flat dysplasia [103–108]. APC mutations also have been reported in foci of incomplete intestinal metaplasia [104]. No association, however, has been found between the presence of APC mutations and either the size of the adenoma or the grade of dysplasia [103–106], suggesting that it may be an early event in gastric carcinogenesis. Mutations of the b-catenin gene are uncommon in gastric adenomas [103,105], with only one immunohistochemical study reporting abnormal nuclear accumulation in three out of eight cases [109]. p53 mutations are detected in 30% to 50% of gastric carcinomas [110,111]. Immunohistochemical studies have shown p53 nuclear staining in about one third of cases of gastric dysplasias or adenomas [110–118], usually, but not invariably, limited to areas of high-grade dysplasia [112,115,116,118,119]. Interestingly, p53 mutations also have been detected (by immunohistochemistry or molecular techniques) in H pylori gastritis and intestinal metaplasia

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(approximately 30%), indicating that p53 alterations may be an early event in gastric carcinogenesis [120–122]. Oncogenes The role of K-ras mutations in gastric adenomas is debatable. Many studies have failed to show a significant prevalence rate of K-ras mutations in either gastric adenomas or adenocarcinomas [105,106,110,123–125]. There are scant data on the involvement of other oncogenes, such as c-erb2 and c-met in dysplastic precursor lesions. Microsatellite instability Gastric carcinomas also may arise by means of the DNA mismatch repair pathway and display microsatellite instability (MSI). MSI may be present in up to 21% of gastric adenomas [105,112,117,124,126,127], whereas it is uncommon in chronic gastritis and intestinal metaplasia [128,129]. Some authors have reported an association between MSI-H and high-grade dysplasia [126]. In a recent study, MSI was reported in 20% of dysplastic precursor lesions with no associated carcinoma, again suggesting that these are early events in gastric carcinogenesis [130]. Hypermethylation Aberrant DNA methylation is present in chronic gastritis and intestinal metaplasia, and an increasing frequency of methylated promoters occurs along the multistep gastric carcinogenesis pathway leading to gastric adenocarcinoma [131–133]. Genes that are hypermethylated in gastric adenomas include, among others, those involved in cell cycle regulation (p14, p16, COX-2) [132,133], signal transduction (APC) [133], DNA repair (hMLH1 and MGMT) [132,133], and those involved in invasion and metastasis (E-cadherin and TIMP3) [132,133]. The methylation frequency for some of these genes, specifically APC, E-cadherin, MGMT, and hMLH1, is not different between precursor lesions and gastric carcinomas, suggesting that methylation may be important in the early stages of gastric carcinogenesis [131,133]. Silencing of tumor suppressor gene TFF1/pS2 by promoter hypermethylation also has been implicated in gastric cancer [134], and progressively reduced expression of pS2 has been shown in intestinal metaplasia and gastric dysplasia [135]. SUMMARY Despite the declining incidence of gastric cancer, it remains the second most common cause of cancer-related deaths worldwide. More than a decade after H pylori was designated as a definite carcinogen by the WHO, several key questions remain to be answered. Only a small minority of patients infected with H pylori eventually develops gastric cancer, and eradication of H pylori in these patients does not seem to eliminate the risk of cancer completely. The optimal surveillance strategy for patients who have H pylori gastritis remains elusive. A combination of histopathological features, serum markers such as pepsinogen I, and molecular tests that analyze host susceptibility polymorphisms and bacterial virulence factors, may allow development of strategies for early

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Gastroenterol Clin N Am 36 (2007) 831–849

GASTROENTEROLOGY CLINICS OF NORTH AMERICA

Precursors to Pancreatic Cancer Ralph H. Hruban, MDa,b,*, Anirban Maitra, MBBSa,b,c, Scott E. Kern, MDa,b, Michael Goggins, MDa,b,d a

Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA b Department of Oncology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA c The Institute for Genetic Medicine, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA d Department of Gastroenterology, The Sol Goldman Pancreatic Cancer Research Center, The Johns Hopkins Medical Institutions, Baltimore, MD, USA

I

nfiltrating ductal adenocarcinoma of the pancreas, also known as pancreatic cancer, is one of the most lethal of all cancers. In the United States it is estimated that 37,170 Americans will be diagnosed with pancreatic cancer in 2007 and that 33,370 will die from the disease [1]. One of the reasons that pancreatic cancer is so overwhelmingly lethal is that it usually is not diagnosed until after the cancer has spread to other organs [2]. The early detection and treatment of noninvasive precursor lesions arguably offers the greatest hope in the fight against this disease. Precursor lesions have been recognized in the pancreas for over a century [3], but it took many decades to define the various histologic types of precursor lesions in the pancreas. Additionally, only over the last 10 years have careful molecular analyses firmly linked histologically well-defined precursor lesions in the pancreas to invasive pancreatic cancer [3–6]. The challenge now is to translate these advances in the understanding of precursor lesions in the pancreas to better patient care—to cure pancreatic neoplasms before they progress to incurable invasive cancer. DEFINITION OF PRECURSOR Intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), and pancreatic intraepithelial neoplasia (PanIN) all meet rigorous criteria for precursor lesions. A working definition of a cancer precursor (although employing the less desirable and more deterministic term

*Corresponding author. Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Weinberg Building 2242, The Johns Hopkins Hospital, 401 North Broadway, Baltimore, MD 21231. E-mail address: [email protected] (R.H. Hruban). 0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.012

ª 2007 Elsevier Inc. All rights reserved. gastro.theclinics.com

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precancer) recently was developed by a National Cancer Institute-sponsored consensus conference [7]. Five criteria were established to define a precursor to invasive cancer, and all three lesions in the pancreas (IPMN, MCN, and PanIN) fulfill these five criteria [7]: 1. The precursor to invasive cancer must be associated with an increased risk of the cancer. 2. When a precursor to invasive cancer progresses to cancer, the resulting cancer arises from cells within the precancer. 3. A precursor to invasive cancer should differ from the normal tissue from which it arises. 4. A precursor to invasive cancer should differ from the cancer into which it develops. 5. There should be a method by which the precursor to invasive cancer can be diagnosed.

The first criterion is that a precursor to invasive cancer must be associated with an increased risk of the cancer. Although this can be hard to prove in an inaccessible organ such as the pancreas, it has been established in mouse models of PanIN and in people for both IPMNs and MCNs [4,8–14]. For example, 7 of the 197 patients who had pancreatic cystic lesions prospectively followed by Tada and colleagues developed a pancreatic malignancy with an observed incidence of pancreatic cancer (0.95% per year) 22.5 times greater than expected [14]. The second criterion established for a cancer precursor is that when it progresses to cancer, the resulting cancer arises from cells within the precursor [7]. This has been demonstrated at the molecular level for IPMN, MCN, and PanIN [15–17]. For example, Moskaluk and colleagues [17] microdissected paired PanIN lesions and infiltrating cancers from patients who had pancreatic cancer, and in one case, they were able to demonstrate identical p16/CDKN2A gene mutations in a PanIN lesion and in the patient’s paired infiltrating cancer. Third, a precursor should differ from the normal tissue from which it arises [7]. This is certainly true for IPMN, MCN, and PanIN. The epithelium in all of these lesions differs architecturally and cytologically from normal ductal epithelium in the pancreas. For example, the normal ductal epithelium is flat and composed of cuboidal cells, while IPMNs often are composed of columnar mucin-filled cells arranged in tall papillary projections. The fourth criterion for a cancer precursor is that it should differ from the cancer into which it develops [7]. By definition, this is true for IPMNs, MCNs, and PanINs. In all three lesions, the neoplastic epithelium has not penetrated through the basement membrane and, thus, has not invaded the surrounding pancreatic parenchyma. In contrast, by definition, the neoplastic cells of invasive pancreatic cancer have breached the basement membrane. The fifth and final criterion for a precursor to invasive cancer is that there be a method in which the precursor can be diagnosed. IPMNs, MCNs, and

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PanINs are all diagnosable by light microscopy. Thus, IPMNs, MCNs, and PanINs meet all five recently developed rigorous criteria for a cancer precursor (precancer) [7]. The salient features of these three neoplasms are summarized in Table 1. MUCINOUS CYSTIC NEOPLASMS Clinical Features Mucinous cystic neoplasms are defined as mucin-producing, cyst-forming epithelial neoplasms of the pancreas with a distinctive ovarian-type stroma (Figs. 1 and 2) [2]. MCNs are far more common in women than men (female to male ratio of 20 to 1), and the mean age at diagnosis is between 40 and 50 years, with a range of 14 to 95 years [2,18–21]. Patients typically present with epigastric discomfort, a sense of abdominal fullness, or an abdominal mass [20,21]. Some MCNs are discovered in asymptomatic patients imaged for other indications [22]. As expected from a precursor lesion, patients who have noninvasive MCNs are on average 5 to 10 years younger than patients who have MCNs with an associated invasive carcinoma [2,19,20,23]. Serum carcinoma antigen 19-9 (CA 19-9) levels are usually elevated only if there is an associated invasive carcinoma [24]. CT often reveals a well-demarcated thick-walled multilocular mass composed of large (1 to 3 cm) cysts [2,19,25]. The radiologic attenuation of the cysts vary. Mural nodules are more common in mucinous cystic neoplasms with an associated invasive carcinoma than in noninvasive mucinous cystic neoplasms. Endoscopic retrograde cholangiopancreatography (ERCP) usually reveals a displaced or compressed pancreatic duct, and in most cases, the cysts do not communicate with the pancreatic ducts, a feature that may be used to distinguish mucinous cystic neoplasms from intraductal papillary mucinous neoplasms.

Table 1 Common precursor lesions in the pancreas

Feature Predominant age gender Head versus body/ tail Relation of the cysts to large ducts Cyst contents Mucin oozing from ampulla Stroma Multifocal disease

Mucinous cystic neoplasm

Intraductal papillary mucinous neoplasm

Pancreatic intraepithelial neoplasia

40–50 years Female>>male Body/tail

In the 60s Male>female Head

Increases with age Male ¼ female Head>body/tail

Usually not connected Mucoid No

Always connected

N/A

Mucoid Yes

N/A No

Ovarian-type Very rare

Collagen-rich In 20% to 30%

Collagen-rich Often

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Fig. 1. Mucinous cystic neoplasm. Note the large size of the cysts and the presence of glistening mucin.

Pathologic and Molecular Features Most (90%) MCNs arise in the body or tail of the pancreas [2,26,27]. The cysts usually measure from 1 to 3 cm in size, and, as demonstrated radiographically, they do not communicate with the larger pancreatic ducts [2,19]. The cysts contain mucin (see Fig. 1) or hemorrhagic fluid, which helps explain the different attenuations observed in individual cysts on CT scanning [2]. By light microscopy, the cysts of MCNs are lined by columnar mucin-producing epithelium, which can have a broad spectrum of dysplasia (see Fig. 2) [2,26,28]. Low-grade dysplasia (MCN adenoma) is composed of uniform columnar cells with abundant supranuclear mucin. The nuclei are basally located, small and uniform in size. Neoplastic epithelium with moderate dysplasia (MCN-borderline) has, as the name suggests, a moderate degree of

Fig. 2. Photomicrograph of a mucinous cystic neoplasm. The neoplastic mucin-producing columnar epithelial cells rest on a layer of cellular ovarian-type stroma.

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architectural and cytologic atypia. The nuclei vary slightly in size and shape, and there is slight loss of nuclear polarity. MCN with high-grade (severe) dysplasia, a designation that is preferred over the term MCN carcinoma in situ, shows significant architectural and cytologic atypia. The degree of atypia in these lesions parallels that seen in invasive cancer. These lesions, however, by definition, are non-invasive. In addition to neoplastic epithelium, MCNs have a distinctive ovarian-type of stroma [2]. The stroma typically forms a band beneath the epithelium. Immunohistochemical labeling can be used to demonstrate expression of progesterone receptors, estrogen receptors, and inhibin by the stromal cells [2,29]. One third of MCNs have an associated invasive adenocarcinoma [2,19]. Invasive carcinoma is usually of the tubular or ductal type [2]. It may arise focally in an MCN, and several studies have shown that the depth of invasion is an important prognosticator [2,21]. At the molecular level, activating point mutations in the KRAS2 gene are early events in the development of MCNs, while TP53 and SMAD4 gene mutations represent late changes [2,30,31]. Aberrant methylation of the p16/CDKN2A gene occurs in a minority of MCNs [2,32]. Natural History and Treatment The disease-specific 5-year survival rate is close to 100% for patients who have a surgically resected MCN without invasive carcinoma [21,33]. The mean 5year survival rate is 50% to 60% for patients who have a surgically resected MCN with an associated invasive cancer [2,19,21,34]. MCNs highlight the importance of early detection. MCNs almost always are unifocal [2]. Therefore, noninvasive MCNs, even those with marked dysplasia, are curable neoplasms if they can be resected completely [21,33]. In contrast, almost half of patients with an invasive carcinoma associated with an MCN die of disease within 5 years [2,19,21,34]. The challenge, therefore, is to detect and remove these neoplasms before they progress to invasive carcinoma. It also follows that the term mucinous cystic carcinoma should be avoided, because it encompasses an entirely curable lesion (the mucinous cystic neoplasm with high-grade dysplasia) with a fully malignant neoplasm (an invasive carcinoma arising in association with a mucinous cystic neoplasm) [2]. Deserving special note is the misconception that some noninvasive MCNs metastasize [30]. Reports of metastasizing noninvasive MCNs almost certainly represent cases in which a resected neoplasm was sampled incompletely for histologic examination [2,33]. Dysplasia, and even invasive carcinoma, can arise focally in an MCN, and if these foci are not sampled, a malignant neoplasm may be misdiagnosed as benign [2,21,33]. Indeed, the authors recently were shown a case in which invasive carcinoma associated with an MCN was not identified until the 108th histologic section. The observation that invasive carcinomas usually arise focally within an MCN has a second important implication: that, whenever possible, partial resection of MCNs should be avoided, even if biopsies reveals only mild

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dysplasia [20,33]. Biopsy or partial resection may miss a focal invasive component. The only way to be certain that an MCN harbors an invasive carcinoma is to resect it completely and examine it thoroughly by histologic evaluation [33]. INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS Clinical Features IPMNs are grossly visible, noninvasive, mucin-producing epithelial neoplasms that usually form long finger-like papillae (Figs. 3 and 4) [2,31]. Most are at least 1 cm in size [31]. In contrast to MCNs, IPMNs, by definition, involve the main pancreatic duct or one of its branches (see Fig. 3) [2,32]. IPMNs arise in the head of the pancreas more frequently than the tail, and IPMNs lack an ovarian-type stroma [2,31,35]. IPMNs affect men slightly more often than women (male to female ratio of 3 to 2). The mean age at diagnosis is near 65 years, with a range of 25 to 95 years [2,35–37]. Common presenting signs and symptoms include abdominal pain, pancreatitis, nausea and vomiting, diabetes mellitus, weight loss, jaundice, and back pain [2,35,37,38]. With the increased use of imaging, a greater proportion of IPMNs are discovered incidentally in asymptomatic patients [22]. Serum oncoproteins, such as carcinoembryonic antigen and CA 19-9 levels, are usually normal unless the IPMN is associated with an invasive cancer [2]. CT usually reveals a dilated main pancreatic duct or a collection of cysts that represent dilated branch ducts [2]. The finding of mucin extruding from a patulous ampulla of Vater is a classic, almost diagnostic, feature at endoscopy [2]. In contrast to MCNs, ERCP demonstrates a dilated pancreatic duct and filling defects, caused by intraluminal mucous plugs or papillary projections of the neoplasm itself. Magnetic resonance cholangiopancreatography (MRCP) may demonstrate ductal dilatation and mural nodules. Similar to MCNs, the mean age of patients who have mild dysplasia is significantly younger than patients who have an associated invasive carcinoma

Fig. 3. Intraductal papillary mucinous neoplasm involving the main pancreatic duct. The duct is distended dramatically by neoplastic papillae.

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Fig. 4. Photomicrograph of an intraductal papillary mucinous neoplasm. The neoplastic mucin-producing columnar epithelial cells form long finger-like papillae. Abundant intraluminal mucin is present.

(63 years versus 68 years in one series) [36,37]. In addition, many patients who had an IPMN reported a long history of symptoms [39,40]. The patients reported in one series developed pancreatitis a mean of 43 months before they were diagnosed [39]. These observations help define the time frame for progression from a noninvasive to an invasive IPMN, and they highlight the window of opportunity that exists to cure these neoplasms if they can be detected while they are still noninvasive. Pathologic and Molecular Features Noninvasive IPMNs are subdivided grossly into two groups: main duct type and branch duct type [2,41–44]. Main duct type IPMNs, as the name suggests, predominantly involve the main pancreatic duct (see Fig. 3). Branch duct type IPMNs involve a side branch of the main duct, and, of course, mixed examples involving both the main and branch ducts occur as well. Noninvasive IPMNs are graded histologically according to the degree of architectural and cytologic atypia, into IPMN with low-grade dysplasia (IPMN adenoma), IPMN with moderate dysplasia (see Fig. 4), and IPMN with high-grade (severe) dysplasia (carcinoma in situ). Main duct IPMNs tend to have higher degrees of dysplasia and more often are associated with an invasive carcinoma compared with the branch duct type [38, 44–46]. Histologically, the papillary epithelium may show gastric mucinous cells, intestinal-type cells with goblet cells, or pancreaticobiliary-type epithelium. Often, a mixture of two or even three types of epithelium is present within a single tumor. A distinctive histologic variant of IPMN, termed intraductal oncocytic papillary neoplasm (IOPN), deserves mention. IOPNs are composed of neoplastic epithelial cells with abundant eosinophilic cytoplasm [47]. The distinctive appearance of IOPNs is a manifestation of numerous mitochondria within neoplastic cells [47]. The few IOPNs reported are insufficient to determine whether their clinical behavior differs from that of typical IPMNs.

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One third of IPMNs have an associated invasive adenocarcinoma [2,35,48,49]. Approximately, 50% of these invasive adenocarcinomas are of the colloid type (Fig. 5), and the other half are tubular/ductal adenocarcinomas [2,50]. It is important to distinguish the colloid from the tubular/ductal types of invasive carcinoma, because patients with colloid carcinoma have a better prognosis than patients with the latter [51]. IPMNs may be associated with extrapancreatic malignancies [52–54]. Twelve of 79 patients with an IPMN reported by Kamisawa and colleagues had a synchronous or a metachronous gastric cancer, and seven had colon cancer [53]. Similarly, in a series of 69 patients who had an IPMN reported by Eguchi and colleagues, 12% had a preoperative personal history of colorectal cancer, and 4% a preoperative history of gastric cancer [52]. The rate of colorectal cancer was 5.4 times greater than expected in the general population [52]. Various molecular alterations have been reported in IPMNs. Furthermore, the mutational spectrum of IPMNs differs slightly from that observed in infiltrating ductal adenocarcinoma [2]. The frequency of KRAS2 gene mutations increases with increasing degrees of dysplasia [30]. The reported frequency of TP53 and p16/CDKN2A gene inactivation varies from series to series. SMAD4 gene mutations, however, are relatively uncommon [2,51]. In contrast, LKB1, which is a gene associated with the Peutz-Jeghers syndrome, is biallelically inactivated in 25% of IPMNs, and the PIK3CA gene is mutated in 10% of IPMNs [55,56]. Natural History and Treatment Similar to MCNs, the critical prognosticators for patients with IPMNs are the presence and size of an associated invasive carcinoma [37,55]. The term intraductal papillary mucinous carcinoma should be avoided because it includes both a potentially curable lesion (the intraductal papillary mucinous neoplasm with high-grade dysplasia) and a malignant neoplasm (invasive carcinoma arising in association with an intraductal papillary mucinous neoplasm) [2]. The

Fig. 5. Photomicrograph of a colloid carcinoma of the pancreas. The neoplastic cells are embedded in copious quantities of extracellular mucin.

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overall 5-year survival rate for surgically resected patients with an invasive carcinoma arising in association with an IPMN is 45% [2,37]. Unlike MCNs, however, noninvasive IPMNs may be multifocal. Thus, patients who have surgically resected noninvasive IPMNs usually cannot be considered cured of their disease [36,48,50,56–58]. A small but significant fraction of patients develop a second pancreatic neoplasm, and some will die of their disease [36]. For example, in a series reported by Chari and colleagues [36], 5 of 60 patients who had noninvasive IPMNs treated with partial pancreatectomy recurred a median of 40 months after diagnosis. Most recurrences were caused by multifocal disease, because none of the 13 patients with noninvasive IPMNs who underwent a total pancreatomy developed a recurrence [36]. Therefore, patients who have had an IPMN resected, even a noninvasive IPMN with negative margins, should be followed carefully for evidence of metachronous disease [59,60]. Although it generally is agreed that IPMNs greater than 3 cm in size, those with a mural nodule, or those associated with dilatation of the main pancreatic duct should be resected, the most appropriate clinical management of patients with a small IPMN is controversial. This is because:   

Small pancreatic cysts are remarkably common in otherwise healthy patients. Most cysts less than 3 cm in size do not show significant dysplasia Surgical resection of pancreatic neoplasms is associated with a significant risk of mortality and morbidity [61].

For example, Schani and colleagues [61] reported that only 3 of 86 pancreatic cysts that measured less than 3 cm in greatest dimension harbored high-grade dysplasia (in situ carcinoma). Therefore, Allen and colleagues [62] recently suggested that the risk of surgical mortality is about equal to the risk of malignancy for patients with small (less than 3 cm) cysts. These data suggest that great care should be exercised before resecting smaller (less than 3 cm) pancreatic cysts. Indeed, recently proposed international guidelines advocate clinical observation with regular CT or MRI for patients with branch duct IPMNs less than 3 cm in size, unless the cysts have a mural nodule and/or are associated with significant dilatation of the main pancreatic duct [63]. The recent report described earlier from Tada and colleagues, however, suggests that there is also risk in observing patients with cystic lesions of the pancreas [14]. In that study, 7 of 197 patients who had pancreatic cysts developed pancreatic cancer upon follow-up (0.95% per year), a rate 22.5-fold greater than expected [14]. Remarkably, three of the seven cysts that progressed to cancer were no more than 1 cm in size [14]. Evidence-based criteria to determine when to follow, and when to operate, on cystic lesions in the pancreas clearly are needed. PANCREATIC INTRAEPITHELIAL NEOPLASIA Clinicopathologic Features Pancreatic intraepithelial neoplasia (PanIN) originally was recognized by Holst [3] over a century ago. It was not until this decade, however, that the PanIN

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nomenclature was developed, and careful molecular studies were performed to establish that PanINs are a precursor to invasive adenocarcinoma of the pancreas [4,64]. PanINs are noninvasive microscopic epithelial neoplasms, located in the smaller pancreatic ducts, characterized by cytologic and architectural atypia (Fig. 6) [2,31,65]. PanINs are divided into three grades based on the degree of epithelial atypia. Lesions with only minimal atypia are designated PanIN1, those with moderate atypia PanIN-2, and those with marked atypia PanIN-3 [31,65]. In addition, PanIN-1 lesions are subdivided further into flat (PanIN-1A) and papillary types (PanIN-1B) [31,65]. PanINs are remarkably common lesions [66–68]. Similar to invasive cancer, PanINs increase with age and are more common in the head than the tail of the pancreas [66,67]. PanINs are more common in the pancreas with invasive carcinoma and in those with chronic pancreatitis [66,67,69,70]. Cubilla and Fitzgerald carefully studied 227 pancreata from patients who had pancreatic cancer and 100 pancreata from patients who did not have pancreatic cancer.

Fig. 6. (A) Normal pancreatic duct. (B) PanIN-1B. (C ) PanIN-2. (D) PanIN-3. PanIN lesions are significantly smaller in size compared with the IPMN illustrated in Fig. 4, and the papillae are shorter in length.

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Fig. 6 (continued)

They found that PanIN-2 lesions were three times more common in pancreata with cancer compared with pancreata without cancer. In fact, PanIN-3 lesions were only observed in pancreata with cancer [69]. More recently, Andea and colleagues reviewed a series of 234 pancreata and identified PanIN lesions in 82% of pancreata with invasive cancer, in 60% of pancreata with chronic pancreatitis, and in 16% of otherwise normal patients [66]. PanINs also occur adjacent to other periampullary neoplasms. Agoff and colleagues identified PanINs in 40% of pancreata surgically resected for ampullary cancer, and Stelow and colleagues observed PanINs in pancreata resected for acinar cell carcinoma, mucinous cystic neoplasms, serous cystic neoplasms, well-differentiated pancreatic endocrine neoplasms, and solid–pseudopapillary neoplasms [71,72]. Molecular Features Molecular analyses have demonstrated that PanIN harbors many of the same genetic alterations found in infiltrating ductal adenocarcinoma of the pancreas [2]. These include activating point mutations in the KRAS2 gene, and inactivation of the p16/CDKN2A, TP53, and SMAD4 genes [70,73,74]. Of interest, recently developed genetically engineered mouse models in which mutant

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KRAS2 is expressed in the pancreas, develop PanIN lesions histologically identical to those in people. In fact, these mice also eventually develop invasive pancreatic cancer [9,75]. Diagnosis and Treatment Although studies of genetically engineered mice, and careful molecular and morphologic analyses clearly establish that PanINs are a precursor to invasive cancer, significant hurdles must be overcome before these lesions can be detected and treated clinically. First, the frequency and rate that PanINs progress to invasive cancer have not yet been determined. If all PanINs were to inextricably and rapidly progress to invasive cancer, then it would follow that all PanINs should be treated, whenever possible. If, on the other hand, PanINs only rarely and slowly progress to invasive cancer, then PanINs may not represent a valid target for therapy. Terhune and colleagues attempted to calculate, mathematically, the probability of a single PanIN lesion progressing to cancer [76]. Estimating that 0.8% of pancreata develop cancer, that 37.5% of the general population develops PanIN, and that the average pancreas has five foci of PanIN, they calculated that approximately 1% of PanIN lesions progress to invasive cancer [76]. These calculations are, however, based on numerous assumptions and estimates. The risk of disease progression cannot be assessed accurately until there is more evidence on the frequency and rate that PanINs progress to invasive cancer. Second, technologies need to be developed that can help detect PanIN lesions. Various molecular tests are being developed to screen for these lesions. For example, Shi and colleagues developed a novel technology, called LigAmp, that can be used to detect rare mutant KRAS2 genes shed from neoplasms, such as PanIN lesions [77]. These tests are doubtlessly years away from clinical practice, raising the question of what can be done today? A recent study by Brune and colleagues suggests that morphologic changes in the pancreatic parenchyma adjacent to PanIN lesions may be detectable using currently available imaging technologies, such as endoscopic ultrasound (EUS) [78]. Brune and colleagues demonstrated that multifocal PanINs frequently are associated with a lobulocentric form of pancreatic parenchymal atrophy, which is detectable by EUS [78]. Clearly, more work has to be done, but the authors are confident that in the near future some PanIN lesions, particularly those that are multifocal, may be detectable using a combination of molecular and imaging technologies. WHO SHOULD BE SCREENED? Even if technological challenges of screening for small microscopic lesions in an inaccessible organ, such as the pancreas, can be overcome, one still would be faced with the very real challenge of identifying the appropriate population to screen. Pancreatic cancer, although extremely deadly, is simply too uncommon to make a nonselective screening effort practical. The incidence of pancreatic cancer in the United States is 9 per 100,000 per year [79]. Enormous

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sensitivity and specificity would be needed in order for a screening test to detect a disease with the rarity of pancreatic cancer without unnecessarily alarming large numbers of nondisease carriers. A critical first step in applying a screening test for precursor lesions in the pancreas therefore would be to identify populations that harbor an increased risk of developing pancreatic cancer. Individuals with a strong family history of pancreatic cancer, and individuals with a germline genetic defect that increases their risk of pancreatic cancer may constitute such a group. To date, at least five genetic syndromes are known to increase the risk of pancreatic cancer (Table 2)[73–75,80–84]. For example, germline (inherited) mutations in the second breast cancer gene (BRCA2), are associated with a 3- to 10-fold increased risk of pancreatic cancer [81,85–88]. In addition, individuals who have a strong family history of pancreatic cancer also have an increased risk of developing pancreatic cancer [87,89–91]. The risk of pancreatic cancer is doubled in individuals who have one first-degree relative with pancreatic cancer, increasing to 6.4-fold in individuals having two first-degree relatives with pancreatic cancer. The risk reaches a 32-fold increase in individuals having three or more first-degree relatives with pancreatic cancer [90,91]. Risk-prediction models, such as the PancPRO model developed by Klein and colleagues can be used to quantify individual risk. Therefore, it could be used to determine whether an individual’s risk of developing pancreatic cancer is high enough to justify screening [92]. SCREENING FOR ASYMPTOMATIC PRECURSORS The discussion on screening up to this point has been largely theoretical. Nonetheless, a recent prospective controlled study by Canto and colleagues Table 2 Inherited genetic alterations associated with an increased risk of pancreatic cancer Individual

Gene

Relative risk (fold increase)

Risk by age 70

No history One FDR with pc Breast cancer

None Unknown BRCA1 BRCA2 p16 (CDKN2A) Unknown PRSS1 STK11/LKB1 MLH1, MSH2, others FANCC and FANC Ga

1 2.3 2.0 3.5-10 20-34 32 50-80 132 Unknown Unknown

0.5% 1.15% 1% 5% 10% to 17% 16% 25% to 40% 30% to 60% <5% Unknown

FAMMM Three FDRs with pc Familial pancreatitis Peutz-Jeghers HNPCC Young age-onset pc

Abbreviations: FAMMM, familial atypical multiple mole melanoma syndrome; FDR, first-degree relatives; HNPCC, hereditary non-polyposis colorectal cancer syndrome; pc, pancreatic cancer. a The proposed association with young onset has not been examined in follow-up literature [91]. Data from Hruban RH, Pitman MB, Klimstra DS. Tumors of the pancreas. Atlas of tumor pathology. Fourth Series, Fascicle 6th edition. Washington (DC): American Registry of Pathology and Armed Forces Institute of Pathology; 2007.

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established that high-risk populations may be screened using existing technologies [92,93]. Canto and colleagues screened 78 asymptomatic individuals with either a strong family history of pancreatic cancer or with the Peutz-Jeghers syndrome, and 149 controls, using CT and EUS [93,94]. Eight patients who had neoplastic lesions of the pancreas were identified among the cases (10% yield of screening). These included six patients with IPMNs and one patient with PanIN, who were treated surgically, and one patient with an IPMN who chose not to be treated. This latter patient later developed an invasive cancer. This study, and others like it, establish that:   

Individuals at high risk can be identified. Individuals at high risk can be screened using existing technologies. If found to have a precursor, patients can be treated before their curable pancreatic neoplasm progresses to incurable invasive cancer [93–103].

SUMMARY Three well-defined precursor lesions of adenocarcinoma of the pancreas have been identified. There is general agreement that, when possible, mucinous cystic neoplasms should be resected completely and, when resected, they should be examined completely by histology to identify or rule out small foci of infiltrating carcinoma. There is also general agreement that large (greater than 3 cm) intraductal papillary mucinous neoplasms should be resected, particularly when they contain a mural nodule or are associated with dilatation of the main pancreatic duct. The management of smaller intraductal papillary mucinous neoplasms is less clear. The risks of surgery must be balanced with the risks of leaving a cancer precursor in the patient. Even when noninvasive intraductal papillary mucinous neoplasms are surgically resected, the patient should be followed for possible multifocal pancreatic disease and perhaps even for extrapancreatic malignancies. Pancreatic intraepithelial neoplasia lesions are too small to be detected using currently available imaging methods, but improvements in imaging and a better understanding of both the molecular alterations present in PanINs and the parenchymal changes associated with PanINs may allow for their early detection in asymptomatic individuals. Screening the general population for noninvasive precursor lesions in the pancreas may not be realistic now, but practitioners are already able to identify populations at increased risk and to quantify this risk, important steps in the clinical application of any potentially clinically useful screening test. References [1] American Cancer Society. Cancer Facts & Figures 2007. Cancer, 1–52. New York: American Cancer Society; 2007. [2] Hruban RH, Pitman MB, Klimstra DS. Tumors of the pancreas. Atlas of tumor pathology. Fourth Series, Fascicle 6th edition. Washington, DC: American Registry of Pathology and Armed Forces Institute of Pathology; 2007.

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[3] Hulst SPL. Zur kenntnis der Genese des Adenokarzinoms und Karzinoms des Pankreas. Virchows Arch (B) 1905;180:288–316. [4] Hruban RH, Goggins M, Parsons JL, et al. Progression model for pancreatic cancer. Clin Cancer Res 2000;6:2969–72. [5] Brat DJ, Lillemoe KD, Yeo CJ, et al. Progression of pancreatic intraductal neoplasias to infiltrating adenocarcinoma of the pancreas. Am J Surg Pathol 1998;22(2):163–9. [6] Brockie E, Anand A, Albores-Saavedra J. Progression of atypical ductal hyperplasia/ carcinoma in situ of the pancreas to invasive adenocarcinoma. Ann Diagn Pathol 1998;2(5):286–92. [7] Berman JJ, Albores-Saavedra J, Bostwick D, et al. Precancer: a conceptual working definition. Results of a consensus conference. Cancer Detect Prev 2006;30(5):387–94. [8] Hruban RH, Iacobuzio-Donahue CA, Wilentz RE, et al. Molecular pathology of pancreatic cancer. Cancer J 2001;7(4):251–8. [9] Hingorani SR, Petricoin EF, Maitra A, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 2003;4(6):437–50. [10] Hyde GL, Davis JB, McMillin RD, et al. Mucinous cystic neoplasm of the pancreas with latent malignancy. Am Surg 1984;50:225–9. [11] Probstein JG, Blumenthal HT. Progressive malignant degeneration of a cystadenoma of the pancreas. Arch Surg 1960;81:683–9. [12] Campbell JA, Cruickshank AH. Cystadenoma and cystadenocarcinoma of the pancreas. J Clin Pathol 1962;15:432–7. [13] Williams TM, Weiner DB, Greene MI, et al. Expression of c-erbB-2 in human pancreatic adenocarcinomas. Pathobiology 1991;59:46–52. [14] Tada M, Kawabe T, Arizumi M, et al. Pancreatic cancer in patients with pancreatic cystic lesions: a prospective study in 197 patients. Clin Gastroenterol Hepatol 2006;4:1265–70. [15] McCarthy DM, Brat DJ, Wilentz RE, et al. Pancreatic intraepithelial neoplasia and infiltrating adenocarcinoma: analysis of progression and recurrence by DPC4 immunohistochemical labeling. Hum Pathol 2001;32:638–42. [16] Hustinx SR, Leoni LM, Yeo CJ, et al. Concordant loss of MTAP and p16/CDKN2A expression in pancreatic intraepithelial neoplasia: evidence of homozygous deletion in a noninvasive precursor lesion. Mod Pathol 2005;18(7):959–63. [17] Moskaluk CA, Hruban RH, Kern SE. p16 and K-ras gene mutations in the intraductal precursors of human pancreatic adenocarcinoma. Cancer Res 1997;57:2140–3. [18] Klo ¨ ppel G, Kosmahl M. Cystic lesions and neoplasms of the pancreas. The features are becoming clearer. Pancreatology 2001;1(6):648–55. [19] Le Borgne J, de Calan L, Partensky C. Cystadenomas and cystadenocarcinomas of the pancreas: a multi-institutional retrospective study of 398 cases. French Surgical Association. Ann Surg 1999;230(2):152–61. [20] Sarr MG, Carpenter HA, Prabhakar LP, et al. Clinical and pathologic correlation of 84 mucinous cystic neoplasms of the pancreas: can one reliably differentiate benign from malignant (or premalignant) neoplasms? Ann Surg 2000;231(2):205–12. [21] Zamboni G, Scarpa A, Bogina G, et al. Mucinous cystic tumors of the pancreas: clinicopathological features, prognosis, and relationship to other mucinous cystic tumors. Am J Surg Pathol 1999;23(4):410–22. [22] Fernandez-del Castillo C, Targarona J, Thayer SP, et al. Incidental pancreatic cysts: clinicopathologic characteristics and comparison with symptomatic patients. Arch Surg 2003;138(4):427–33. [23] Warshaw AL, Compton CC, Lewandrowski KB, et al. Cystic tumors of the pancreas. New clinical, radiologic, and pathologic observations in 67 patients. Ann Surg 1990;212: 432–45. [24] Bassi C, Salvia R, Gumbs AA, et al. The value of standard serum tumor markers in differentiating mucinous from serous cystic tumors of the pancreas: CEA, Ca 19-9, Ca 125, Ca 15-3. Langenbecks Arch Surg 2002;387(7–8):281–5.

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[44] Seki M, Yanagisawa A, Ohta H, et al. Surgical treatment of intraductal papillary–mucinous tumor (IPMT) of the pancreas: operative indications based on surgico–pathologic study focusing on invasive carcinoma derived from IPMT. J Hepatobiliary Pancreat Surg 2003;10(2):147–55. [45] Silas AM, Morrin MM, Raptopoulos V, et al. Intraductal papillary mucinous tumors of the pancreas. AJR Am J Roentgenol 2001;176(1):179–85. [46] Tanaka M. Intraductal papillary mucinous neoplasm of the pancreas: diagnosis and treatment. Pancreas 2004;28(3):282–8. [47] Terris B, Ponsot P, Paye F, et al. Intraductal papillary mucinous tumors of the pancreas confined to secondary ducts show less aggressive pathologic features as compared with those involving the main pancreatic duct. Am J Surg Pathol 2000;24(10):1372–7. [48] Bernard P, Scoazec JY, Joubert M, et al. Intraductal papillary–mucinous tumors of the pancreas: predictive criteria of malignancy according to pathological examination of 53 cases. Arch Surg 2002;137(11):1274–8. [49] Ban S, Naitoh Y, Mino-Kenudson M, et al. Intraductal papillary mucinous neoplasm (IPMN) of the pancreas: its histopathologic difference between 2 major types. Am J Surg Pathol 2006;30(12):1561–9. [50] Adsay NV, Adair CF, Heffess CS, et al. Intraductal oncocytic papillary neoplasms of the pancreas. Am J Surg Pathol 1996;20(8):980–94. [51] Iacobuzio-Donahue CA, Klimstra DS, Adsay NV, et al. Dpc-4 protein is expressed in virtually all human intraductal papillary mucinous neoplasms of the pancreas: comparison with conventional ductal carcinomas. Am J Pathol 2000;157(3):755–61. [52] Sohn TA, Yeo CJ, Cameron JL, et al. Intraductal papillary mucinous neoplasms of the pancreas: an increasingly recognized clinicopathologic entity. Ann Surg 2001;234(3): 313–21. [53] Cuillerier E, Cellier C, Palazzo L, et al. Outcome after surgical resection of intraductal papillary and mucinous tumors of the pancreas. Am J Gastroenterol 2000;95(2):441–5. [54] Inagaki M, Maguchi M, Kino S, et al. Mucin-producing tumors of the pancreas: clinicopathological features, surgical treatment, and outcome. J Hepatobiliary Pancreat Surg 1999;6(3):281–5. [55] Su GH, Hruban RH, Bova GS, et al. Germline and somatic mutations of the STK11/LKB1 Peutz-Jeghers gene in pancreatic and biliary cancers. Am J Pathol 1999;154(6): 1835–40. [56] Schonleben F, Qiu W, Ciau NT, et al. PIK3CA mutations in intraductal papillary mucinous neoplasm/carcinoma of the pancreas. Clin Cancer Res 2006;12(12):3851–5. [57] Adsay NV, Conlon K, Brennan MF, et al. Intraductal papillary–mucinous neoplasms of the pancreas (IPMN): an analysis of in situ and invasive carcinomas associated with 23 cases. Modern Pathology 1997;10:143A. [58] Atia GN, Brown RD, Alrashid A, et al. The role of pancreatoscopy in the preoperative evaluation of intraductal papillary mucinous tumor of the pancreas. J Clin Gastroenterol 2002;35(2):175–9. [59] D’Angelica M, Brennan MF, Suriawinata AA, et al. Intraductal papillary mucinous neoplasms of the pancreas: an analysis of clinicopathologic features and outcome. Ann Surg 2004;239(3):400–8. [60] Adsay NV, Pierson C, Sarkar F, et al. Colloid (mucinous noncystic) carcinoma of the pancreas. Am J Surg Pathol 2001;25(1):26–42. [61] Hara T, Yamaguchi T, Ishihara T, et al. Diagnosis and patient management of intraductal papillary–mucinous tumor of the pancreas by using peroral pancreatoscopy and intraductal ultrasonography. Gastroenterology 2002;122(1):34–43. [62] Milchgrub S, Campuzano M, Casillas J, et al. Intraductal carcinoma of the pancreas. Cancer 1992;69(3):651–6. [63] Navarro F, Michel J, Bauret P, et al. Management of intraductal papillary mucinous tumours of the pancreas. Eur J Surg 1999;165(1):43–8.

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[85] Surveillance, Epidemiology, and End Results (SEER) Program (www.seer.cancer.gov) SEER*Stat Databases: Incidence–SEER 11 Regs þ AK Public-Use, Nov 2003 Sub for Expanded Races (1992–2001) and Incidence–SEER 11 Regs Public-Use, Nov 2003 Sub for Hispanics (1992–2001), National Cancer Institute, DCCPS, Surveillance Research Program, Cancer Statistics Branch, released April 2004, based on the November 2003 submission, 2004. [86] Borg A, Sandberg T, Nilsson K, et al. High frequency of multiple melanomas and breast and pancreas carcinomas in CDKN2A mutation-positive melanoma families. J Natl Cancer Inst 2000;92(15):1260–6. [87] Cancer risks in BRCA2 mutation carriers. The Breast Cancer Linkage Consortium. J Natl Cancer Inst 1999;91(15):1310–6. [88] Giardiello FM, Brensinger JD, Tersmette AC, et al. Very high risk of cancer in familial PeutzJeghers syndrome. Gastroenterology 2000;119:1447–53. [89] Goldstein AM, Fraser MC, Struewing JP, et al. Increased risk of pancreatic cancer in melanoma-prone kindreds with p16INK4 mutations. N Engl J Med 1995;333(15):970–4. [90] Vasen HF, Gruis NA, Frants RR, et al. Risk of developing pancreatic cancer in families with familial atypical multiple mole melanoma associated with a specific 19 deletion of p16 (p16-Leiden). Int J Cancer 2000;87(6):809–11. [91] van der Heijden MS, Yeo CJ, Hruban RH, et al. Fanconi anemia gene mutations in youngonset pancreatic cancer. Cancer Res 2003;63(10):2585–8. [92] Pogue-Geile KL, Chen R, Bronner MP, et al. Palladin mutation causes familial pancreatic cancer and suggests a new cancer mechanism. PLoS Med 2006;3(12):e516. [93] Liede A, Karlan BY, Narod SA. Cancer risks for male carriers of germline mutations in BRCA1 or BRCA2: a review of the literature. J Clin Oncol 2004;22(4):735–42. [94] Hahn SA, Greenhalf B, Ellis I, et al. BRCA2 germline mutations in familial pancreatic carcinoma. J Natl Cancer Inst 2003;95(3):214–21. [95] Murphy KM, Brune KA, Griffin CA, et al. Evaluation of candidate genes MAP2K4, MADH4, ACVR1B, and BRCA2 in familial pancreatic cancer: deleterious BRCA2 mutations in 17%. Cancer Res 2002;62(13):3789–93. [96] Ozcelik H, Schmocker B, DiNicola N, et al. Germline BRCA2 6174delT mutations in Ashkenazi Jewish pancreatic cancer patients. Nat Genet 1997;16:17–8. [97] Hemminki K, Li X. Familial and second primary pancreatic cancers: a nationwide epidemiologic study from Sweden. Int J Cancer 2003;103(4):525–30. [98] Klein AP, Brune KA, Petersen GM, et al. Prospective risk of pancreatic cancer in familial pancreatic cancer kindreds. Cancer Res 2004;64(7):2634–8. [99] Amundadottir LT, Thorvaldsson S, Gudbjartsson DF, et al. Cancer as a complex phenotype: pattern of cancer distribution within and beyond the nuclear family. PLoS Med 2004;1(3):e65. [100] Wang W, Chen S, Brune KA, et al. PancPRO: risk assessment for individuals with a family history of pancreatic cancer. J Clin Oncol 2007;25(11):1417–22. [101] Canto MI, Goggins M, Hruban RH, et al. Screening for early pancreatic neoplasia in high-risk individuals: a prospective controlled study. Clin Gastroenterol Hepatol 2006;4(6):766–81. [102] Canto MI, Goggins M, Yeo CJ, et al. Screening for pancreatic neoplasia in high-risk individuals: an EUS-based approach. Clin Gastroenterol Hepatol 2004;2(7):606–21. [103] Brentnall TA, Bronner MP, Byrd DR, et al. Early diagnosis and treatment of pancreatic dysplasia in patients with a family history of pancreatic cancer. Ann Intern Med 1999;131(4):247–55.

Gastroenterol Clin N Am 36 (2007) 851–865

GASTROENTEROLOGY CLINICS OF NORTH AMERICA

Endocrine Hyperplasia and Dysplasia in the Pathogenesis of Gastrointestinal and Pancreatic Endocrine Tumors Guido Rindi, MD, PhDa,*, Enrico Solcia, MDb a

Department of Pathology and Laboratory Medicine, University of Parma, I-43100 Parma, Italy Department of Pathology and Genetics, University of Pavia and I.R.C.C.S, Policlinico San Matteo, I-27100 Pavia, Italy

b

N

onneoplastic endocrine growths of the gastrointestinal (GI) tract and pancreas are relatively rare lesions. This article provides a comprehensive description of reported entities according to an anatomic approach. Each section details diagnostic criteria for specific lesions, briefly outlines their relationship with tumor counterparts, and discusses, when available, supporting pathogenetic data.

STOMACH Five different endocrine cell types are present in the gastric mucosa of people (Table 1) [1–5]. The histamine-producing enterochromaffin-like (ECL) cells are the largest endocrine population in the oxyntic mucosa, whereas gastrinproducing G cells are predominant in the antrum. Serotonin-producing enterochromaffin (EC) cells and somatostatin D cells are scattered in both oxyntic and antral mucosa as minor populations. Recently, ghrelin-producing cells [6] have been described in people and correspond to the previously described P/D1 cell. It accounts for about 15% of all gastric endocrine cell types [4,6]. Cells with features of ECL and G cells are the dominant component in hyperplastic lesions of the corpus and antrum, respectively, although other endocrine cells may be observed as a minor population in nodular hyperplasia of the corpus. Of epidemiological note, non-neoplastic endocrine cell growths increasingly are observed in routine pathology practice. This finding parallels the reported incremental growth trend in incidence of gastric endocrine tumors (carcinoids) in surgical and/or endoscopic series [7–11]. Increased endoscopic surveillance for dyspepsia and Helicobacter pylori infection assessment, together with Supported in part by grant 2005069205_002 from MIUR (GR), internal university grants (GR and ES) and the Italian Ministry of Health (GR and ES).

*Corresponding author. E-mail address: [email protected] (G. Rindi). 0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.006

ª 2007 Elsevier Inc. All rights reserved. gastro.theclinics.com

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Table 1 Characteristics of gastric endocrine cells in people [3–5] Secretory granule ultrastructure Cell type

Main product

Mucosal site

%

Stains

Size (nm)

Shape

Inner structure

ECL D

Histamine Somatostatin

160–300 200–400

Round Round

Vesicular, coarsely granular core Poorly osmiophilic, homogenous

5HT

Pleomorphic

Heavily osmiophilic

Ghrelin Gastrin

Grim, CgA M-F Grim, CgA Grim, CgA

150–350

P G

40–50 10–20 10–20 10–20 10–20 10–15 40–60

Grim, CgA H-H

EC

Oxyntic Oxyntic Pyloric Oxyntic Pyloric Oxyntic Antral

100–200 150–350

Round Round

Thin-haloed Vesicular, flocculent core

Abbreviations: Grim, Grimelius silver impregnation; H-H, Hellman-Hellerstrom silver impregnation; M-F, Masson-Fontana silver impregnation; CgA, Chromogranin A immunoreactivity.

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increased clinical awareness of such lesions, may help explain this trend. A facilitating role of acid-inhibitory agents by inducing mild hypergastrinemia has also been suggested, however [12,13]. Nonantral Region Proliferative lesions of endocrine cells of the stomach are categorized, in terms of severity and capacity to progress to well-differentiated endocrine tumors, for ECL cells only (Fig. 1, Table 2) [14]. The contribution of other endocrine cells, such as EC cells, to non-neoplastic lesions has been documented [15–18], and recently, for ghrelin cells also [19–22]. ECL cell hyperplasia and dysplasia may be considered a morphological signature of hypergastrinemia. An abnormal number, and distribution, of oxyntic endocrine cells first was reported in patients with long-standing hypergastrinemia caused by chronic atrophic gastritis, either with or without pernicious anemia [23,24], or to the Zollinger-Ellison syndrome (ZES) [17,25], with or without MEN1 [26]. Hypergastrinemia is the driving force for progression of ECL cell hyperplasia to dysplasia and neoplasia, which occurs by means of a multistep process [13,14,27–29].

Fig. 1. Complex enterochromaffin-like cell hyperplasia in a patient with chronic atrophic gastritis; note the chains (arrow) and the micronodules (arrowhead). Endocrine cells stain intensely at immunohistochemistry for the vesicular monoamine transporter 2 (VMAT2), a bona fide surrogate marker for ECL cells. Immunoperoxidase, hematoxylin counterstain; original magnification  100.

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Table 2 Endocrine cell growths of the stomach (ECL cells only) Definition

Size

Normal pattern Hyperplasia Simple Linear (chain-forming) Micronodular Adenomatoid Dysplasia Dysplastic (precarcinoid) lesions Enlarging micronodule Fusing micronodule Microinvasive lesion Nodule with newly formed stroma Neoplasia Intramucosal tumor (carcinoid) Microcarcinoid, microcarcinoidosis Invasive tumor/carcinoma (carcinoid/ malignant carcinoid)

na Two standard deviations vs normal 5 cells; two chains/mm 5 cells; size 100–150 lm <500 lm

500 lm

(Data from Solcia E, Bordi C, Creutzfeldt W, et al. Histopathological classification of nonantral gastric endocrine growths in man. Digestion 1988;41;185–200.)

Enterochromaffin-like cell hyperplasia By definition, lesions that lack inherent evolutive potential are termed hyperplastic and are classified [14,30,31] as: 







Simple (diffuse), defined as an increased number (more than two times greater than normal values) of endocrine cells, otherwise retaining their normal distribution Linear or chain-forming, defined as linear sequences of at least five cells along the basement membrane and at least two chains per millimeter length of mucosa Micronodular, defined as clusters of five or more cells (size 30 to 150 lm), either within glands or the lamina propria, and at least one micronodule per mm length of mucosa Adenomatoid, defined as at least five adjacent micronodules with intervening basal membrane in the lamina propria

Enterochromaffin-like cell dysplasia By definition, ECL cell dysplasias are precarcinod lesions (size between 150 and 500 lm) that display progressive similarity with tumors and are classified as:    

Enlarging micronodules—clusters of cells greater than 150 lm in size Fusing micronodules—resulting from the disappearance of the basal membranes between adjacent micronodules Microinvasive lesions—infiltrating the lamina propria and filling the space between glands Nodule with newly formed stroma—with a microlobular or trabecular structure

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As mentioned above, ECL cell proliferative lesions are observed in conditions of long-standing hypergastrinemia. The most common underlying conditions are:  



Achlorydria associated with chronic atrophic gastritis (CAG) Functioning gastrin-producing tumors (gastrinoma) in Zollinger-Ellison syndrome (ZES), either with or without the multiple endocrine neoplasia syndrome type 1 (MEN1) Long-term proton-pump inhibitor (PPI) treatment [13,18,32,33]

ECL cell tumors (carcinoids) develop in 13% to 43% of patients with MEN1, compared with less than 1% in ZES patients without MEN1 [13]. Gastric mucosa sampling at endoscopy helps assign patients to the appropriate pathogenetic disorder. For instance, in CAG, the gastric mucosa is severely atrophic, whereas in patients taking PPIs, or with MEN1/ZES, the mucosa is usually either normotrophic or hypertrophic (MEN1-ZES) because of the longstanding trophic action of gastrin. The gastric receptor (CCKR2) exerts a pivotal role in the pathogenesis of ECL cell proliferation. For instance, CCK2R knockout mice develop hypochlorydria and hypergastrinemia (caused by G cell hyperplasia), although with paradoxical absence of ECL cell hyperplasia [34]. The acid–gastrin axis is maintained in the antrum of CCK2R knockout mice, with a reduced gastrin/somatostatin (G/D) cell ratio, whereas normal numbers of somatostatin and ghrelin cells are preserved in the corpus [35,36]. These data indicate that CCK2R is required for ECL cells to proliferate in response to gastrin. In people, hypergastrinemia induces discrete, measurable changes, including increased volume density and changes in granule ultrastructure in ECL cells of patients who have CAG [29]. In fact, reduction of hypergastrinemia, following antrectomy, causes a decrease in the number of proliferating ECL cells, which can lead to disappearance of hyperplastic changes [37,38]. Even with persistent gastrin stimulus, however, nodular endocrine hyperplasia normally does not bear significant proliferative potential. For instance, the virtual absence of mitotic activity in endocrine micronodules of CAG patients who have gastric carcinoid tumors [30,32,33,39] indicates that micronodular and adenomatoid hyperplastic endocrine lesions are mostly end-stage conditions, marking the remnants of oxyntic glands that were destroyed by inflammation. This is a phenomenon similar to that which happens to endocrine cells in patients with graft versus host disease of the colon [40]. In contrast, the malignant potential of dysplastic lesions is inferred by the observation that, with rare exceptions, they usually are detected in association with gastric carcinoid tumors in CAG or MEN-ZES patients [10,26,41]. Thus, in people, there is strong evidence that gastrin promotes ECL cell proliferation, but that further somatic hits are needed to induce ECL neoplastic transformation. The observation that the severity of ECL cell lesions parallels the degree of endoscopic sampling [42], however, suggests that the actual incidence rate of dysplastic lesions and carcinoid tumors in hypergastrinemic patients is underestimated. As a result, careful

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follow-up is advocated in patients who are taking life-long PPI agents and in whom hypergastrinemia develops [13]. Antral Region Hyperplasia of antral gastrin-producing cells (Fig. 2) occurs in conditions that promote achlorhydria, although it does not appear to increase the risk of neoplasia [43]. G cell hyperplasia is the morphologic counterpart of hypergastrinemia, and this usually observed is in patients who have hypochlorydria and gastric atrophy [44,45]. Increased numbers of G cells are characterized by palisades of cells that expand toward the upper and lower portions of the antral glands in the gland neck region. High numbers of G cells (140 to 250/mm of mucosa versus 40 to 90/mm in normal controls) usually are associated with a reduction of D cells, which results in an abnormal G/D cell ratio [46–48]. G cell hyperplasia/hyperfunction, increased G/D cell ratio, and increased gastric acid output, was described in children, either with or without H pylori infection, possibly due to abnormal G (or D) cell sensitivity to acid [49,50]. Similar findings were observed in adult patients who had H pylori gastritis [51]. Antral D-cell hyperplasia also has been described in patients who had ulcer disease in the duodenum, which also occasionally is associated with D cell hyperplasia of

Fig. 2. Hyperplastic gastrin-producing (G) cells of the antrum in a patient with chronic atrophic gastritis of the corpus; G cells are organized in palisades and colonize the upper part of glands (arrow). Immunoperoxidase, hematoxylin counterstain; original magnification  200.

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the gastric fundus [52]. Focal, deep, linear, or micronodular hyperplasia of argentaffin EC cells has been described in atrophic antral mucosa [25]. At this time, there have not been any reports of dysplastic change of endocrine cells in antral mucosa. DUODENUM Following previous sporadic reports, a recent systematic study demonstrated that duodenal gastrin cells undergo changes similar to those described for nonantral endocrine cells of the stomach in MEN1/ZES patients with gastrinoma (Table 3) [53]. Proliferating gastrin cells may be organized into patterns of diffuse, linear, and micronodular hyperplasia. Additionally, enlarged and microinvasive lesions may develop. In that study, diffuse hyperplasia was defined as two standard deviations (SDs) of gastrin cells, linear hyperplasia as five or more gastrin cells in at least two chains per mm, and nodular hyperplasia as a collection of cells that measure between 30 and 90 lm in size. Additionally, hyperplastic gastrin cell lesions showed a high Ki67 (proliferative) labeling index. In that report, lesions were not detected in patients who had gastrinoma in the absence of MEN1 [53]. In contrast, one other study reported the presence of gastrin cell hyperplasia in a series of 18 patients who had sporadic gastrinomas and H pylori gastritis, all of whom were being treated with PPIs [54]. This study confirmed the reproducibility of the proposed classification of non-neoplastic lesions of duodenal gastrin cells. In the study by Anlauf and colleagues [53], the MEN1 genetic defect was proposed to represent the driving force of these lesions. Merchant and colleagues [54] hypothesized that H pylori-gastritis-related factors, and possibly the potent antacid effect of PPIs, may have induced gastrin cell proliferation in the duodenal bulb. This phenomenon could be considered as

Table 3 Endocrine cell growths of the duodenum (G and D only) [53,56] Definition

Size

Normal pattern Hyperplasia Diffuse Linear (chain-forming) Micronodular Dysplasia Dysplastic (precarcinoid) lesions Enlarging micronodule Microinvasive lesion Neoplasia Intramucosal tumor (G or D cell tumor) Microtumor Invasive tumor/carcinoma

na

Minimum size reported for tumor 300 lm [53,56].

2SD vs normal 5 cells; two chains/mm 5 cells; size 30–90 lm 90–210 lm

210 lm–2.0 mm

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a sort of continuum with G cell lesions, which has been described in the antrum of H pylori gastritis patients. Unfortunately, G/D cell ratios were not determined in the antrum in that study [54]. It should be noted, however, that clinically silent duodenal bulb G cell tumors (in the absence of G cell hyperplasia) are a relatively frequent finding in specimens of patients either with or without peptic ulcer disease, even before the introduction of PPI therapy [55]. Nevertheless, the previously cited reports suggest that duodenal G cells may be recruited and proliferate in response to either genetic (MEN1 gene defects) or acquired (hypochlorydria) stimuli. A recent analysis of duodenal lesions in patients who had MEN1also demonstrated somatostatin-producing D cell hyperplasia that showed similar features previously demonstrated for G cells [56]. Loss of heterozygosity (LOH) for 11q13 chromosomal markers at the MEN1 gene locus occurred exclusively in G and D cell tumors but not in hyperplastic lesions. This finding suggests a different mechanism for the development of hyperplastic versus dysplastic/neoplastic lesions. Finally, the strict association of G and D cells to both the stomach and duodenum is highlighted by a single case report of G and D cell hyperplasia of these two anatomic sites in a syndrome of dwarfism, obesity, and goiter [52]. Up to now, no hyperplastic/dysplastic changes have been described as with other types of endocrine cells of the duodenum, in spite of their abundance in the normal state [57] and their occasional involvement in tumor growth [55]. PANCREAS Proliferative changes of pancreatic endocrine cells generally are classified as islet cell hyperplasia, nesidioblastosis, and islet cell dysplasia. In contrast to the stomach and the duodenum, a multistep pathogenetic sequence, from hyperplasia to neoplasia (microadenoma), has not been defined. Microadenomas, however, are defined as discrete monohormonal endocrine lesions that measure at least 500 lm in size. Islet Cell Hyperplasia This condition is defined by an expansion of the endocrine cell compartment of the pancreas to 2% to 10% of the total pancreatic mass, in adults and newborns/ infants, respectively (versus approximately 1% to approximately 3% in normal conditions) [58–60]. Large-sized islets (greater than 250 lm) characterize this condition. Normal islets typically measure less than 225 lm in size in adults. In children, 200 lm is the average size of islets [59]. By definition, in islet cell hyperplasia, the normal distribution and relative proportion of the four endocrine cell types are retained. The net mass of all endocrine cells is increased substantially, however. Islet cell hyperplasia may be associated with various disorders, such as alpha1-antitrypsin deficiency and hyperinsulinism [61,62]. Other conditions associate with hyperinsulinism and hypoglycemia in different

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clinical settings, some of which are relatively common, such as newborns from diabetic mothers, and others quite rare, such as the Beckwith-Wiedenmann syndrome (gigantism, macroglossia, visceromegaly, omphalocele, kidney dysplasia, and increased tumor incidence) [63] and leprechaunism [64]. Nesidioblastosis is a clinicopathologic condition characterized by the presence of persistent hyperinsulinemic hypoglycemia (PHH) caused by a peculiar, non-neoplastic, endocrine cell growth of the pancreas. The condition is characterized, histologically, by the presence of islet cells that bud from the pancreatic ducts, referred to as ductulo–insular complexes [65], and by clusters of normal or enlarged islets and diffuse hypertrophy of B cells [59]. Nesidioblastosis is more common in infants and newborns (congenital hyperinsulinism, CHI), and is rare in adults. In infants, this disease has an estimated incidence of 1/30000 to 1/50000 live births [66]. In adults, it is reported in 4% of PHH patients [67]. The disorder may be either focal or diffuse [68,69]. Focal nesidioblastosis corresponds to a single isolated lesion, usually located in the tail or body of an otherwise normal pancreas, that measures between 2 and 10 mm in size and is characterized by islet cell-like clusters separated by exocrine or connective tissue. B cells represent the highest proportion of islet cells, (70% to 90%). Diffuse nesidioblastosis is characterized by the presence of lesions throughout the entire pancreas. Aside from architectural changes, the most important diagnostic criterion of nesidioblastosis is hypertrophic B cells, with enlarged hyperchromatic nuclei [59,66]. Recently, this criterion also was identified in 15 adults with PHH, all of whom were investigated by morphometry [67]. Suggested major criteria for the histopathological diagnosis of PHH in adults include:    

Exclusion of concurrent insulinoma B cells with enlarged hyperchromatic nuclei Islets with normal spatial cell distribution Absence of proliferative activity of endocrine cells [67]

Islet cell dysplasia initially was described in genetically engineered mice with inheritable endocrine tumors of the pancreas [70–72]. In experimental rodents, the morphology of islet cell dysplasia depended on the specific endocrine cell. All lesions, however, were characterized by neovascular space development. This feature is present in human islet cell dysplasia also, especially in patients who had MEN1 (Fig. 3). In people, islet cell dysplasia is defined as:   

An abnormal structure of the islets with effacement of its normal microlobular pattern, often with trabecular growth A sharp increase in the presence of one specific endocrine cell type, suggestive of clonal expansion The presence of cellular atypia [59]

These lesions often were described in patients who had MEN1 and, rarely, in sporadic cases. Recent work on a series of nine patients who had MEN1 led to the identification of four putative types of dysplastic lesions, defined as

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Fig. 3. Pancreatic islet in MEN1 patient with multiple nonfunctioning microadenomas; the arrow indicates a neovascular space. Hematoxylin and eosin; original magnification  200.

atypical structures, including structurally abnormal islets (A3, reasonably consistent with the previous definition of dysplasia) and novel lesions involving acini (A1 and A4) or ducts (A2) [73]. With the exception of one type (type A4, acinar nodule), all showed LOH for MEN1 11q13 markers, suggestive of their premalignant potential. These data suggest a nonislet cell origin of pancreatic endocrine tumors, at least in MEN1. In contrast, more recent data generated by LOH and FISH analysis on four patients who had MEN1, traced the loss of the MEN1 allele to clusters of monoclonal/monohormonal cells independent of their location, within islets or ducts [74]. The latter finding suggests that tumor development in patients who have MEN1 may originate at any site within the endocrine compartment of the pancreas and more often within islets. Overall, the present state of knowledge indicates that preneoplastic lesions of the endocrine pancreas still lack a solid and widely accepted definition of their multistep growth process. REMAINDER OF THE GUT Despite the relatively high frequency of endocrine tumors in the remainder of the GI tract (ileum, appendix and rectum), hyperplastic changes of endocrine cells are rare, being reported mainly in association with carcinoid tumors [75,76]. Multiple carcinoid tumors and endocrine cell hyperplasias have been

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Fig. 4. Hyperplastic serotonin-producing EC cells (arrow) in abnormal crypts of a patient with microcarcinoid in chronic ulcerative colitis [78]. Immunoperoxidase, hematoxylin counterstain; original magnification  100. (Data from Nascimbeni R, Villanacci V, Di Fabio F, et al. Solitary microcarcinoid of the rectal stump in ulcerative colitis. Neuroendocrinology. 2005;81(6): 400–4.)

reported in ulcerative colitis and Crohn’s disease, suggesting that chronic, longstanding inflammation may represent a stimulus for endocrine cell growth (Fig. 4) [77–80]. In these instances, the proliferating cells are argentafin-positive, serotonin-producing, enterochromaffin cells. It is unknown whether other endocrine cell types participate in this process. Endocrine cell dysplasia has not been reported or defined in these other GI locations. Overall, non-neoplastic proliferative changes of the distal small intestine, appendix, and colon–rectum have not been defined systematically. References [1] Solcia E, Capella C, Vassallo G, et al. Endocrine cells of the gastric mucosa. Int Rev Cytol 1975;42:223–86. [2] Simonsson M, Eriksson S, Hakanson R, et al. Endocrine cells in the human oxyntic mucosa. A histochemical study. Scand J Gastroenterol Nov 1988;23(9):1089–99. [3] Solcia E, Rindi G, Buffa R, et al. Gastric endocrine cells: types, function, and growth. Regul Pept 2000;93(1–3):31–5. [4] Rindi G, Necchi V, Savio A, et al. Characterisation of gastric ghrelin cells in man and other mammals: studies in adult and fetal tissues. Histochem Cell Biol 2002;117(6):511–9.

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[5] Solcia E, Rindi G, Larosa S, et al. Morphological, molecular, and prognostic aspects of gastric endocrine tumors. Microsc Res Tech 2000;48(6):339–48. [6] Kojima M, Hosoda H, Date Y, et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402(6762):656–60. [7] Modlin IM, Lye KD, Kidd M. A 50-year analysis of 562 gastric carcinoids: small tumor or larger problem? Am J Gastroenterol 2004;99(1):23–32. [8] Mizuma K, Shibuya H, Totsuka M, et al. Carcinoid of the stomach: a case report and review of 100 cases reported in Japan. Ann Chir Gynaecol 1983;72(1):23–7. [9] Sjoblom SM, Sipponen P, Miettinen M, et al. Gastroscopic screening for gastric carcinoids and carcinoma in pernicious anemia. Endoscopy 1988;20(2):52–6. [10] Rindi G, Luinetti O, Cornaggia M, et al. Three subtypes of gastric argyrophil carcinoid and the gastric neuroendocrine carcinoma: a clinicopathologic study. Gastroenterology 1993;104(4):994–1006. [11] Solcia E, Fiocca R, Sessa F, et al. Morphology and natural history of gastric endocrine tumors. In: Ha˚kanson R, Sundler F, editors. The stomach as an endocrine organ. Amsterdam: Elsevier; 1991. p. 473–98. [12] Hodgson N, Koniaris LG, Livingstone AS, et al. Gastric carcinoids: a temporal increase with proton pump introduction. Surg Endosc 2005;19(12):1610–2. [13] Jensen RT. Consequences of long-term proton pump blockade: insights from studies of patients with gastrinomas. Basic Clin Pharmacol Toxicol 2006;98(1):4–19. [14] Solcia E, Bordi C, Creutzfeldt W, et al. Histopathological classification of nonantral gastric endocrine growths in man. Digestion 1988;41:185–200. [15] Solcia E, Capella C, Sessa F, et al. Gastric carcinoids and related endocrine growths. Digestion 1986;35(Suppl 1):3–22. [16] Solcia E, Capella C, Buffa R, et al. Endocrine cells of the gastrointestinal tract and related tumors. Pathobiol Annu 1979;9:163–204. [17] Bordi C, Cocconi G, Togni R, et al. Gastric endocrine cell proliferation. Association with Zollinger-Ellison syndrome. Arch Pathol 1974;98(4):274–8. [18] Bordi C, Gabrielli M, Missale G. Pathologic changes of endocrine cells in chronic atrophic gastritis. An ultrastructural study on peroral gastric biopsy specimens. Arch Pathol Lab Med 1978;102(3):129–35. [19] Solcia E, Capella C, Buffa R, et al. Identification, ultrastructure, and classification of gut endocrine cells and related growths. Invest Cell Pathol 1980;3(1):37–49. [20] Bordi C, Ferrari C, D’Adda T, et al. Ultrastructural characterization of fundic endocrine cell hyperplasia associated with atrophic gastritis and hypergastrinaemia. Virchows Arch A Pathol Anat Histopathol 1986;409(3):335–47. [21] Bordi C, Yu JY, Baggi MT, et al. Gastric carcinoids and their precursor lesions. A histologic and immunohistochemical study of 23 cases. Cancer 1991;67(3):663–72. [22] Srivastava A, Kamath A, Barry SA, et al. Ghrelin expression in hyperplastic and neoplastic proliferations of the enterochromaffin-like (ECL) cells. Endocr Pathol 2004;15(1): 47–54. [23] Rubin W. A fine structural characterization of the proliferated endocrine cells in atrophic gastric mucosa. Am J Pathol 1973;70(1):109–18. [24] Feyrter F, Klima R. [Histopathology of gastric changes in pernicious anemia]. Munch Med Wochenschr 1952;94(4):145–53. [25] Solcia E, Capella C, Vassallo G. Endocrine cells of the stomach and pancreas in states of gastric hypersecretion. Rendic R Gastroenterol 1970;2:147–58. [26] Solcia E, Capella C, Fiocca R, et al. Gastric argyrophil carcinoidosis in patients with Zollinger-Ellison syndrome due to type 1 multiple endocrine neoplasia. A newly recognized association. Am J Surg Pathol 1990;14(6):503–13. [27] Creutzfeldt W. The achlorhydria–carcinoid sequence: role of gastrin. Digestion 1988;39(2):61–79.

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[28] D’Adda T, Corleto V, Pilato FP, et al. Quantitative ultrastructure of endocrine cells of oxyntic mucosa in Zollinger-Ellison syndrome. Correspondence with light microscopic findings. Gastroenterology 1990;99(1):17–26. [29] Bordi C, D’Adda T, Azzoni C, et al. Hypergastrinemia and gastric enterochromaffin-like cells. Am J Surg Pathol 1995;19(Suppl 1):S8–19. [30] Solcia E, Fiocca R, Villani L, et al. Morphology and pathogenesis of endocrine hyperplasias, precarcinoid lesions, and carcinoids arising in chronic atrophic gastritis. Scand J Gastroenterol Suppl 1991;180:146–59. [31] Solcia E, Fiocca R, Villani L, et al. Hyperplastic, dysplastic, and neoplastic enterochromaffinlike cell proliferations of the gastric mucosa. Classification and histogenesis. Am J Surg Pathol 1995;19(Suppl 1):S1–7. [32] Solcia E, Rindi G, Fiocca R, et al. Distinct patterns of chronic gastritis associated with carcinoid and cancer and their role in tumorigenesis. Yale J Biol Med 1992;65(6):793–804 [discussion: 827–9]. [33] Lamberts R, Creutzfeldt W, Struber HG, et al. Long-term omeprazole therapy in peptic ulcer disease: gastrin, endocrine cell growth, and gastritis. Gastroenterology 1993;104(5): 1356–70. [34] Langhans N, Rindi G, Chiu M, et al. Abnormal gastric histology and decreased acid production in cholecystokinin-B/gastrin receptor-deficient mice. Gastroenterology 1997;112(1): 280–6. [35] Rindi G, Langhans N, Rehfeld JF, et al. Abnormal gastric morphology and function in CCK-B/gastrin receptor-deficient mice. Yale J Biol Med 1998;71(3–4):347–54. [36] Chen D, Zhao CM, Al-Haider W, et al. Differentiation of gastric ECL cells is altered in CCK(2) receptor-deficient mice. Gastroenterology 2002;123(2):577–85. [37] Richards AT, Hinder RA, Harrison AC. Gastric carcinoid tumours associated with hypergastrinaemia and pernicious anaemia—regression of tumors by antrectomy. A case report. S Afr Med J 1987;72(1):51–3. [38] Hirschowitz BI, Griffith J, Pellegrin D, et al. Rapid regression of enterochromaffinlike cell gastric carcinoids in pernicious anemia after antrectomy. Gastroenterology 1992;102(4 Pt 1): 1409–18. [39] Roucayrol AM, Cattan D. Evolution of fundic argyrophil cell hyperplasia in nonantral atrophic gastritis. Gastroenterology 1990;99(5):1307–14. [40] Lampert IA, Thorpe P, Van Noorden S, et al. Selective sparing of enterochromaffin cells in graft versus host disease affecting the colonic mucosa. Histopathology 1985;9:875–86. [41] Rindi G, Azzoni C, La Rosa S, et al. ECL cell tumor and poorly differentiated endocrine carcinoma of the stomach: prognostic evaluation by pathological analysis. Gastroenterology 1999;116(3):532–42. [42] Bordi C, Azzoni C, Ferraro G, et al. Sampling strategies for analysis of enterochromaffinlike cell changes in Zollinger-Ellison syndrome. Am J Clin Pathol 2000;114(3):419–25. [43] Solcia E, Capella C, Fiocca R, et al. Disorders of the endocrine system. In: Ming SC, Goldman H, editors. Pathology of the gastrointestinal tract. Philadelphia: Williams and Wilkins; 1998. p. 295–322. [44] Arnold R, Hulst MV, Neuhof CH, et al. Antral gastrin-producing G cells and somatostatin-producing D cells in different states of gastric acid secretion. Gut 1982;23(4): 285–91. [45] Arnold R, Frank M, Simon B, et al. Adaptation and renewal of the endocrine stomach. Scand J Gastroenterol Suppl 1992;193:20–7. [46] Polak JM, Stagg B, Pearse AG. Two types of Zollinger-Ellison syndrome: immunofluorescent, cytochemical, and ultrastructural studies of the antral and pancreatic gastrin cells in different clinical states. Gut 1972;13(7):501–12. [47] Friesen SR, Tomita T. Pseudo Zollinger-Ellison syndrome: hypergastrinemia, hyperchlorhydria without tumor. Ann Surg 1981;194(4):481–93.

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[48] Keuppens F, Willems G, De Graef J, et al. Antral gastrin cell hyperplasia in patients with peptic ulcer. Ann Surg 1980;191(3):276–81. [49] Oderda G, Fiocca R, Villani L, et al. Gastrin cell hyperplasia in childhood Helicobacter pylori gastritis. Eur J Gastroenterol Hepatol 1993;5:13–6. [50] Rindi G, Annibale B, Bonamico M, et al. Helicobacter pylori infection in children with antral gastrin cell hyperfunction. J Pediatr Gastroenterol Nutr 1994;18(2):152–8. [51] Liu Y, Vosmaer GD, Tytgat GN, et al. Gastrin (G) cells and somatostatin (D) cells in patients with dyspeptic symptoms: Helicobacter pylori-associated and nonassociated gastritis. J Clin Pathol 2005;58(9):927–31. [52] Holle GE, Spann W, Eisenmenger W, et al. Diffuse somatostatin-immunoreactive D cell hyperplasia in the stomach and duodenum. Gastroenterology 1986;91(3):733–9. [53] Anlauf M, Perren A, Meyer CL, et al. Precursor lesions in patients with multiple endocrine neoplasia type 1-associated duodenal gastrinomas. Gastroenterology 2005;128(5): 1187–98. [54] Merchant SH, VanderJagt T, Lathrop S, et al. Sporadic duodenal bulb gastrin cell tumors: association with Helicobacter pylori gastritis and long-term use of proton pump inhibitors. Am J Surg Pathol 2006;30(12):1581–7. [55] Capella C, Riva C, Rindi G, et al. Endocrine tumors of the duodenum and upper jejunum. A study of 33 cases with clinico–pathological characteristics and hormone content. Hepatogastroenterology 1990;37(2):247–52. [56] Anlauf M, Perren A, Henopp T, et al. Allelic deletion of the MEN1 gene in duodenal gastrin and somatostatin cell neoplasms and their precursor lesions. Gut 2007;56(5): 637–44. [57] Rindi G, Leiter AB, Kopin AS, et al. The normal endocrine cell of the gut. Changing concepts and new evidences. Ann N Y Acad Sci 2004;1014:1–12. [58] Rahier J, Wallon J, Henquin JC. Cell populations in the endocrine pancreas of human neonates and infants. Diabetologia 1981;20(5):540–6. [59] Solcia E. Capella C. Klo ¨ ppel G. 3rd series edition. Tumours of the pancreas, volume Fascicle 7. Washington, DC: Armed Force Institute of Pathology; 1997. [60] Hazelwood R. The endocrine pancreas. Englewood Cliffs (NJ): Prentice Hall; 1989. [61] Ray MB, Zumwalt R. Islet-cell hyperplasia in genetic deficiency of alpha-1-proteinase inhibitor. Am J Clin Pathol 1986;85(6):681–7. [62] Weidenheim KM, Hinchey WW, Campbell WG Jr. Hyperinsulinemic hypoglycemia in adults with islet cell hyperplasia and degranulation of exocrine cells of the pancreas. Am J Clin Pathol 1983;79(1):14–24. [63] Stefan Y, Bordi C, Grasso S, et al. Beckwith-Wiedemann syndrome: a quantitative, immunohistochemical study of pancreatic islet cell populations. Diabetologia 1985;28(12): 914–9. [64] Rosenberg AM, Haworth JC, Degroot GW, et al. A case of leprechaunism with severe hyperinsulinemia. Am J Dis Child 1980;134(2):170–5. [65] Laidlaw G. Nesidioblastoma, the islet tumor of the pancreas. Am J Pathol 1934;14: 125–34. [66] Sempoux C, Guiot Y, Jaubert F, et al. Focal and diffuse forms of congenital hyperinsulinism: the keys for differential diagnosis. Endocr Pathol 2004;15(3):241–6. [67] Anlauf M, Wieben D, Perren A, et al. Persistent hyperinsulinemic hypoglycemia in 15 adults with diffuse nesidioblastosis: diagnostic criteria, incidence, and characterization of beta cell changes. Am J Surg Pathol 2005;29(4):524–33. [68] Goossens A, Gepts W, Saudubray JM, et al. Diffuse and focal nesidioblastosis. A clinicopathological study of 24 patients with persistent neonatal hyperinsulinemic hypoglycemia. Am J Surg Pathol 1989;13(9):766–75. [69] Rahier J, Falt K, Muntefering H, et al. The basic structural lesion of persistent neonatal hypoglycaemia with hyperinsulinism: deficiency of pancreatic D cells or hyperactivity of B cells? Diabetologia 1984;26(4):282–9.

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[70] Rindi G, Bishop AE, Murphy D, et al. A morphological analysis endocrine tumour genesis in pancreas and anterior pituitary of AVP/SV40 transgenic mice. Virchows Arch A Pathol Anat Histopathol 1988;412(3):255–66. [71] Rindi G, Grant SG, Yiangou Y, et al. Development of neuroendocrine tumors in the gastrointestinal tract of transgenic mice. Heterogeneity of hormone expression. Am J Pathol 1990;136(6):1349–63. [72] Rindi G, Efrat S, Ghatei MA, et al. Glucagonomas of transgenic mice express a wide range of general neuroendocrine markers and bioactive peptides. Virchows Arch A Pathol Anat Histopathol 1991;419(2):115–29. [73] Vortmeyer AO, Huang S, Lubensky I, et al. Nonislet origin of pancreatic islet cell tumors. J Clin Endocrinol Metab 2004;89(4):1934–8. [74] Perren A, Anlauf M, Henopp T, et al. Multiple endocrine neoplasia type 1 (MEN1): loss of one MEN1 allele in tumors and monohormonal endocrine cell clusters but not in islet hyperplasia of the pancreas. J Clin Endocrinol Metab 2007;92(3):1118–28. [75] Cross SS, Hughes AD, Williams GT, et al. Endocrine cell hyperplasia and appendiceal carcinoids. J Pathol 1988;156(4):325–9. [76] Moyana TN, Satkunam N. A comparative immunohistochemical study of jejunoileal and appendiceal carcinoids. Implications for histogenesis and pathogenesis. Cancer 1992;70(5):1081–8. [77] Hock YL, Scott KW, Grace RH. Mixed adenocarcinoma/carcinoid tumour of large bowel in a patient with Crohn’s disease. J Clin Pathol 1993;46(2):183–5. [78] Nascimbeni R, Villanacci V, Di Fabio F, et al. Solitary microcarcinoid of the rectal stump in ulcerative colitis. Neuroendocrinology 2005;81(6):400–4. [79] Gledhill A, Hall PA, Cruse JP, et al. Enteroendocrine cell hyperplasia, carcinoid tumours, and adenocarcinoma in long-standing ulcerative colitis. Histopathology 1986;10(5): 501–8. [80] Matsumoto T, Jo Y, Mibu R, et al. Multiple microcarcinoids in a patient with long-standing ulcerative colitis. J Clin Pathol 2003;56(12):963–5.

Gastroenterol Clin N Am 36 (2007) 867–887

GASTROENTEROLOGY CLINICS OF NORTH AMERICA

Hepatic Precancerous Lesions and Small Hepatocellular Carcinoma Prodromos Hytiroglou, MDa,*, Young Nyun Park, MD, PhDb, Glenn Krinsky, MDc, Neil D. Theise, MDd a

Department of Pathology, Aristotle University Medical School, 54006 Thessaloniki, Greece Department of Pathology and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, CPO Box 8044, Seoul, Korea c Department of Radiology, Valley Hospital, 223 North Van Dien Avenue, Ridgewood, NJ, USA d Departments of Pathology and Medicine (Division of Digestive Diseases), Beth Israel Medical Center of Albert Einstein College of Medicine, First Avenue at 16th Street, New York, NY, 10003, USA b

M

ost hepatocellular carcinomas (HCCs) arise in livers with chronic disease, such as viral hepatitis, alcoholic disease, or hemochromatosis. In the Western world, most HCCs are detected in patients who have cirrhosis. Recent pathologic studies, based on careful dissection of cirrhotic liver specimens, have defined the morphologic features of precancerous (ie, dysplastic) lesions and small HCCs. Similarly, clinicopathologic studies have provided proof for the evolution of dysplastic lesions to HCC, while radiologic–pathologic studies have used modern imaging technology for detecting small lesions with malignant potential. At a molecular level, precancerous and early cancerous lesions arising in cirrhotic livers display various acquired genetic and epigenetic changes. Nevertheless, recent evidence suggests that the earliest molecular changes related to hepatocarcinogenesis occur by epigenetic mechanisms before the onset of cirrhosis. Rapid advances in liver surgery, pathology, and radiology in the last 20 years have created a need for clinically relevant nomenclature for dysplastic hepatic lesions and small HCCs. A standard for nomenclature was proposed by an international working party (IWP) in 1995 [1], as a remedy for the various terms that had been introduced in earlier literature. In more recent years, significant international consensus has been reached in the terminology of liver lesions as a result of several international meetings, organized by authorities from both Western countries and Japan, combined with contributions by liver

This study was supported in part by a grant from the National R&D Program for Cancer Control of the Ministry of Health and Welfare of the Republic of Korea (no. 0,620,210) to Dr. Young Nyun Park.

*Corresponding author. E-mail address: [email protected] (P. Hytiroglou). 0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.010

ª 2007 Elsevier Inc. All rights reserved. gastro.theclinics.com

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pathologists from many countries. The entities currently recognized to represent precancerous and small cancerous lesions are listed in Box 1. Dysplastic lesions consist of cells with histologic features suggestive of a precancerous nature. In the liver, dysplastic cells may form: clusters (dysplastic foci), which are detectable only upon microscopic examination, or nodules (dysplastic nodules), which are detectable macroscopically and radiologically. These two types of lesions are discussed with regard to their tissue, cell, and molecular biology and how they relate to current clinical practice. Finally, a discussion of the radiologic identification of precancerous nodules and small HCCs—both the successes and limitations, thereof—also is provided. CYTOLOGIC CHANGES IN HEPATOCARCINOGENESIS According to the IWP nomenclature [1], dysplastic foci are defined as clusters of hepatocytes with atypia, measuring less than 1 mm in diameter. Although the size criterion is arbitrary, it is related to the fact that dysplastic foci nearly always are contained within a single hepatic lobule (or cirrhotic nodule, because these lesions usually are detected in the setting of cirrhosis). Of course, these small lesions cannot be recognized grossly or radiologically, but they represent incidental findings on microscopic examination of liver biopsy or resection specimens. According to the IWP article, dysplastic foci often are composed of small or large atypical hepatocytes (ie, demonstrate small or large cell change) [1]. Small cell change (SCC), originally described as small cell dysplasia [2], is defined as hepatocytes that show decreased cytoplasmic volume, cytoplasmic basophilia, mild nuclear pleomorphism and hyperchromasia, and increased nucleocytoplasmic ratio, which gives the impression of nuclear crowding

Box 1: Hepatic precancerous lesions and small hepatocellular carcinoma Dysplastic focus 

Expansile focus of small cell change



Iron-free focus in liver with hemochromatosis

Dysplastic nodule 

Dysplastic nodule, low-grade (devoid of cytologic or architectural atypia)



Dysplastic nodule, high-grade (with cytologic or architectural atypia insufficient for a diagnosis of hepatocellular carcinoma)

Small hepatocellular carcinoma 

Early hepatocellular carcinoma (small hepatocellular carcinoma with indistinct margins)



Distinctly nodular small hepatocellular carcinoma

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(Fig. 1A). The incidence of SCC in cirrhotic livers varies considerably among different reports, from less than 1% in biopsy specimens [3], up to 50% in liver explants [4]. Le Bail and colleagues [4] found that SCC occurring in small foci, with expansile rather than widespread growth, was associated with HCC. Thus, expansile foci of SCC apparently correspond to dysplastic foci in the IWP classification. In contrast, a diffuse pattern of SCC may represent regenerating, rather than dysplastic, hepatocytes [5,6]. The difficulty in distinguishing precancerous SCC from regenerative changes may be a reason for the limited predictive value of SCC for HCC development. Some reports have shown a significant correlation between the presence of SCC and HCC [7,8], whereas others have not [3,9,10]. Large cell change (LCC), originally termed liver cell dysplasia [11], is defined as hepatocytes with both nuclear and cellular enlargement (and therefore a preserved nucleocytoplasmic ratio), nuclear pleomorphism, frequent nuclear hyperchromasia, and multinucleation (Fig. 1B). LCC is a common finding in biopsies and resection specimens of cirrhotic livers [3,4,8–10,12], and has been detected in up to 81% of cirrhotic liver explants [4]. This change usually is present diffusely within liver parenchyma, with LCC cells scattered among regenerating hepatocytes, not forming expansile subnodules (ie, not forming dysplastic foci). LCC is more prevalent in cirrhotic livers with HCC, compared with those without cancer [4,11]. Its presence has been reported to be an important independent risk factor for the subsequent development of HCC [3,8–10]. An ongoing debate relates to whether SCC and LCC represent direct HCC precursors, or innocent bystanders, pathogenetically linked to and associated with HCC, but not direct precursors. There are abundant data supporting the precancerous nature of SCC. Hepatocytes with SCC show high proliferative activity and morphologic resemblance to early HCC [2,13,14]. A histologic continuum between SCC and HCC has been described. Furthermore, hepatocytes with SCC bear chromosomal gains and losses that are also present in adjacent HCCs, but not in surrounding cirrhotic parenchyma [15].

Fig. 1. Representative examples of small cell change (A) and large cell change (B) in cirrhosis (hematoxylin–eosin, 200).

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In contrast, definitive consensus has not been reached regarding the neoplastic potential of LCC. Data, however, suggest it is an HCC-associated change. The data supporting LCC as a reactive process related to chronic injury, or senescence, include low proliferative activity and increased apoptosis [16], absence of a histologic continuum with HCC [16], association with cholestasis [17], and absence of genetic alterations present in adjacent HCCs [15]. In favor of LCC as a precancerous lesion are data showing that it has higher argyrophilic nucleolar organizer region (AgNOR) values than other hepatocytes (with the highest AgNOR values being strongly associated with HCC development during follow-up) [18], abnormal DNA content [19–21], chromosomal numerical aberrations, and chromosomal gains and losses [22,23]. In addition, Paradis and colleagues [24] reported that cirrhotic macronodules with LCC showed molecular evidence of monoclonality, a prerequisite step of hepatocarcinogenesis. Another recent study showed a progressive decrease in telomere length from nondysplastic cirrhotic liver to LCC, SCC, and HCC [25]. The expression of p21 and p16 was relatively preserved in LCC, decreased in SCC, and absent in HCC, whereas H2AX-DNA damage foci were not present in LCC, but were present in SCC and in HCC [25]. These data suggest that SCC is a precancerous lesion, characterized by inactivation of cell cycle checkpoints, short telomeres, and accumulated DNA damage. The conflicting data for LCC suggest that it may be a heterogeneous entity with two types: a type that is noninnocent, tumor-related, and another one that is innocent, nontumor-related [26]. Few articles have dealt with cytologic precancerous lesions other than SCC and LCC. Deugnier and colleagues [27] reported that iron-free foci in genetic hemochromatosis were proliferative lesions associated with a high incidence of HCC during follow-up. Foci of altered hepatocytes, similar to those reported in experimental hepatocarcinogenesis, also have been reported in human chronic liver disease and may warrant further investigation as putative preneoplastic lesions [28,29]. From a practical point of view, identification of SCC, LCC, or iron-free foci in liver biopsies may signify an increased risk of HCC over time. The presence of such lesions should be noted in pathology reports. In the case of small expansile lesions within cirrhotic, or noncirrhotic, parenchyma displaying SCC or iron resistance (in otherwise siderotic parenchyma), the term dysplastic focus is appropriate. DYSPLASTIC NODULES Dysplastic nodules (DNs) are nodular lesions that differ from the surrounding parenchyma with regard to size, color, texture, or the degree to which they bulge from the cut surface of resected specimens (Fig. 2). DNs are usually, but not always, detected in cirrhotic livers [1]. Several comprehensive reviews of the histologic features of these lesions have been published in the previous 5 years [14,29–33]. Low-grade DNs have features suggestive of a clonal cell

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Fig. 2. Two dysplastic nodules (arrows), each measuring approximately 1.0 cm in size, in a cirrhotic liver of a 59-year-old man with chronic hepatitis B.

population, such as diffuse iron or copper accumulation, or uniform steatosis in livers without fatty change, or features of chronic hepatocyte injury such as LCC. They do not, however, have cytologic or architectural atypia to suggest emerging HCC. Often, histologic distinction between large regenerative nodules and low-grade DNs is difficult, or impossible. Identification of nontriadal (unpaired) arteries in this setting suggests that the nodule in question is precancerous rather than regenerative in nature. In contrast, high-grade DNs show cytologic or architectural atypia that approach, but do not quite reach, those of HCC. Both types of lesions, in aggregate, are found in approximately 15% to 25% of cirrhotic livers [34]. The histologic changes of high-grade DNs are similar to those described in dysplastic foci, including formation of expansile subnodules with atypia. Subnodules may demonstrate SCC, iron resistance (in otherwise siderotic nodules), steatosis and/or Mallory bodies [12,34–38]. Subnodules have been referred to as nodule-in-nodule lesions, and are sometimes recognizable radiologically, particularly iron-resistant subnodules in otherwise siderotic DNs. High-grade DNs also show architectural atypia, such as thick cell plates (up to three cells thick) and occasional pseudoglandular structures. The overall cellularity (cell density) of these lesions usually is 1.3 to two times greater than that of adjacent cirrhotic parenchyma [1]. The histologic differential diagnosis between high-grade DN and well-differentiated HCC sometimes may be difficult or impossible, especially in biopsy material. Although the vascular supply of DNs is derived from portal tracts, nontriadal arteries also are detected often in these lesions [39–43]. The formation of abnormal vessels (neoangiogenesis) is caused by growth factor production by lesional cells [44], and is considered a useful feature in the histologic distinction between DNs and large regenerative nodules. Detection and characterization of nodular lesions in cirrhotic livers by dynamic CT and MRI rely heavily on the type of vascular supply they possess. DNs may be detected on gross or radiologic examination, but their definite identification is based on histologic examination. Their association with HCC

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takes two forms. First, both low- and high-grade DNs are markers of an increased risk of cancer development in the liver as a whole. In fact, HCCs do not always develop within DNs, but may be found elsewhere in the liver, independent of the latter. Second, subnodules of HCC are often seen within DNs themselves (Fig. 3). Such lesions, consisting of HCC arising within a DN, first were recognized in Japan and brought attention to DNs as a precancerous lesion [45,46]. Confirmation that DNs were precancerous nodules came from studies of autopsy livers, surgical specimens, liver explants [12,38,47–50], and most importantly, from follow-up studies of unresected lesions [51–55]. Two recent studies evaluated the natural history of large hepatic nodules in a systematic manner. Borzio and colleagues [54] followed 90 cirrhotic patients with histologically proven large regenerative nodules (n ¼ 56), low-grade DNs (n ¼ 16) or high-grade DNs (n ¼ 19), and found that the presence of high-grade DNs, or extranodular LCC, was associated with an increased hazard risk for malignant transformation (2.4 and 3.1 respectively over a mean follow-up period of 33 months). Large regenerative nodules and low-grade DNs were associated with a low propensity for HCC development. In another study, Kobayashi and colleagues [55] followed 154 patients who had histologically proven large regenerative nodules (n ¼ 99), low-grade DNs (n ¼ 42), or highgrade DNs (n ¼ 13) for a median period of 2.8 years. The hazard ratios of high-grade DN, low-grade DN, and large regenerative nodule for transformation to HCC were 16.8, 2.96, and 1.0, respectively. In this study, however, SCC was considered to be a feature of both low-grade DNs and high-grade DNs. Finally, Terasaki and colleagues [53] found that the histologic features predictive of malignant transformation of DNs included increased cellularity, clear cell change, SCC, and fatty change. SMALL HEPATOCELLULAR CARCINOMA According to the consensus nomenclature [1], small HCC is defined arbitrarily as carcinomas that measure less than 2.0 cm in diameter. Studies from Japan [48,56,57] have shown that there are two types of small HCC:

Fig. 3. Hepatocellular carcinoma (left) arising in a high-grade dysplastic nodule. (hematoxylin–eosin, 100).

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Fig. 4. (A) An early hepatocellular carcinoma (arrow) is evident as a 1.2 cm vaguely nodular, yellowish area in the liver of a 53-year-old man with chronic hepatitis B and cirrhosis. (B) Histologic examination shows thin trabecular arrangement of the neoplastic cells and fatty change. On the left, the neoplastic cells are seen to invade a portal tract (star) (hematoxylin–eosin, 200).





Vaguely nodular HCC (also known as early HCC, or HCC with indistinct margins), which is well-differentiated, lacks a fibrous capsule, and often contains portal tracts (ie, grows around preexisting portal tracts) (Fig. 4) Distinctly nodular HCC, which is well- or moderately differentiated, lacks portal tracts, and often is surrounded by a capsule (Fig. 5)

As the name implies, early HCC is considered a precursor of distinctly nodular HCC. In a series of 116 small HCCs, the mean size of early HCCs was 11.9 mm, but that of distinctly nodular HCCs was 16.0 mm [58]. Early HCCs consist of small neoplastic cells (cytologically reminiscent of SCC), arranged in irregular, thin trabeculae and pseudoglandular structures [48,56–58]. The cell density of these lesions is at least twice that of the

Fig. 5. (A) Small hepatocellular carcinoma of the distinctly nodular type (arrow), measuring 1.0 cm in greatest dimension, in the liver of a 47-year-old man with chronic hepatitis B and cirrhosis. (B) Histologic examination shows a trabecular pattern of growth. The tumor is separated from the adjacent cirrhotic parenchyma by a fibrous capsule (stars) (hematoxylin–eosin, 100).

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surrounding hepatic parenchyma. Fatty change is common. Invasion of portal tracts is often present, a helpful feature in the histologic differential diagnosis from DNs (see Fig. 4B) [58–60]. On the other hand, small HCCs of the distinctly nodular type may contain various neoplastic cell populations, often forming subnodules. Invasion of portal vein branches by tumor cells has been described in 27% of cases, and minute intrahepatic metastases have been described in 10% [56]. Nontriadal arteries are well developed in distinctly nodular HCCs, but poorly developed (ie, smaller and sparser) in early HCCs and DNs [61]. From a practical point of view, detection of a high-grade DN or small HCC, whether by radiologic or histologic methods, is considered an indication for ablation (such as percutaneous radiofrequency ablation) or surgical resection. Although the presence of HCC is taken into account when assigning priority to cirrhotic patients for liver transplant, currently there are no guidelines available for patients who have DNs. The biologic properties of DNs, however, suggest that these patients are at increased risk for HCC and, thus, at a minimum, warrant increased surveillance. MOLECULAR PATHOGENESIS OF HEPATOCELLULAR CARCINOMA The molecular changes that lead to dysplasia are initiated in chronically diseased livers, before cirrhosis is established. At that stage, alterations in gene expression are mostly quantitative, occurring by epigenetic mechanisms [62]. Alterations include increased expression of growth factors, such as transforming growth factor-a (TGF-a) and insulin-like growth factor-2 (IGF-2), which leads to accelerated hepatocyte proliferation. Promoter hypermethylation, resulting in gene inactivation, is also common in chronic hepatitis and cirrhosis [63,64]. In a study of methylation status of nine tumor-related genes, a stepwise increase in the incidence of hypermethylation was found in chronic hepatitis, cirrhosis, DNs, and HCC [64]. Telomerase, an enzyme that provides cells with the ability to sustain proliferation, is activated commonly in cirrhotic livers, DNs, and HCCs [65–67]. Cells with activated telomerase are capable of dividing continuously without attrition of their telomeres, thus escaping apoptosis. As hepatocytes proliferate, monoclonal cell populations emerge [24,68,69]. Such populations may bear molecular alterations, which provide them with a survival advantage over neighboring hepatocytes. With prolonged survival, these cells are receptive to additional alterations, ultimately leading to HCC. Various structural changes involving genes and chromosomes also have been described in precancerous lesions and HCC. In a study of loss of heterozygosity (LOH) of six microsatellite foci, the incidence of LOH was increased progressively in cirrhotic nodules, DNs, and HCCs [70]. In another study, the incidence of allelic loss of the 1p36-p34 region also was increased in the progression to carcinoma [71]. In a study of 39 microsatellite foci, LOH was detected in 45% of chronic hepatitis cases, 40% of cirrhosis cases, 98% of HCC

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cases, but not in any samples of normal livers, suggesting that genetic instability may be initiated in livers with chronic hepatitis, before the development of cirrhosis [72]. In another study of large nodules using comparative genomic hybridization, frequent chromosome 8p losses and chromosome 1q gains were detected in both DNs and HCCs [73]. Point mutations, or other types of genomic alterations, also have been identified in hepatocarcinogenesis, including those in tumor suppressor genes, such as p53, Rb1, beta-catenin, AXIN1, p16INK4A, and IGFR-II/M6PR, and oncogenes, such as PIK3CA and c-myc [62,74]. The extensive heterogeneity of genomic changes in HCCs, in association with the relatively low incidence of each one of them, suggests that HCCs are produced by genomic and epigenetic alterations compromising more than one regulatory pathway [62,75]. Indeed, there is evidence implicating different pathways in different tumors, including the p53 pathway, the Rb1 pathway, the TGF-beta pathway, the Wnt/beta-catenin pathway [75], and more recently, the RAS/MAPK pathway, the PI3 K/Akt pathway, and the Hedgehog pathway [74]. These pathways contain large numbers of molecules whose aberrant structure or expression may subvert precise control of cellular phenotype [62]. Recent large-scale studies of gene expression, by DNA microarrays and quantitative real-time polymerase chain reaction, have revealed extensive changes in hepatocarcinogenesis. Several studies have shown that there are sets of genes constituting molecular signatures that can be used to distinguish among the various types of histologically identifiable precancerous and cancerous lesions [76–79]. These studies also have led to the discovery of novel diagnostic markers that can be assessed by immunohistochemistry. For instance, heat shock protein-70 and glypican-3 are useful markers in the differential diagnosis of HCC from noncancerous lesions [76,79]. The development of HCC in cirrhosis generally is considered to be a multistep process. Early views of hepatocarcinogenesis suggested that HCCs developed from cirrhotic (regenerative) nodules composed of proliferating hepatocytes, which would enhance the possibility of mutational events, ultimately leading to HCC. This theory, however, does not explain the development of HCC in livers without cirrhosis, and some histologic features of DNs and small HCCs [30]. Following identification of a DN that contained foci of HCC in a noncirrhotic liver [80], it was realized that DNs did not necessarily derive from pre-existent cirrhotic nodules [80–82]. As a result, it was hypothesized that precancerous changes arose from molecular events, in part made possible by increased hepatocyte turnover caused by chronic injury. Chronic liver injury may produce monoclonal, preneoplastic cell populations that surround, rather than push or displace hepatic structures, such as central veins and portal tracts. Predictions that were made on the basis of this hypothesis, including a low proliferation rate, diminished apoptosis, and diminished activation of hepatic stellate cells, and, therefore, diminished scarring, were confirmed in a series of follow-up studies [83–85].

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Although this hypothesis of HCC development is specific for malignancy arising in DNs (Fig. 6A), variation of timing of progression in relationship to cirrhosis, and of stellate cell activation/scarring within the precancerous lesions, can result in a unitary hypothesis concerning the morphologic features of precancerous lesions and HCC. For example, if a preneoplastic clonal population of cells does not inhibit stellate cell activation, or if this is promoted, then the preneoplastic cells may not appear as a nodule distinct from the surrounding parenchyma. Instead, they may appear as a region of cirrhosis within which either dysplastic foci, or overt HCCs, are found (Fig. 6B). Alternatively, if the molecular events that lead to the development of dysplastic foci occur in advance of cirrhosis, then one would expect to find dysplastic foci in the early stages of chronic liver disease. In this situation, the clonally expanding, normal-appearing, preneoplastic hepatocytes may be able to acquire additional mutations leading to precirrhotic HCC. If the emergence of HCC coincides with the establishment of cirrhosis, however, then the HCC will be seen within a cirrhotic background (Fig. 6C). STEM CELLS AND HEPATOCARCINOGENESIS There are two distinct roles that stem cells play in HCC development. The first is that of an HCC initiator, such as a facultative, non-neoplastic, liver stem cell that undergoes malignant transformation. The second is that of an HCC-repopulating stem cell, such as a malignant cell that has all the functional features of a facultative stem cell (eg, self-renewal, slow cycling, production of a rapidly proliferative transit-amplifying progeny that generates the bulk of tumor cells, and resistance to chemotherapy, radiation, and hypoxia), but may be derived from malignant transformation of a facultative stem cell or from transformation of a differentiated cell population. There are few data in humans to indicate whether the cell of origin of HCC is a mature, but malignantly transformed, hepatocyte, or a transformed stem cell [86]. The existence of mixed hepatocellular-cholangiocarcinomas, however, including some that arise in DNs, is evidence in favor of a facultative stem cell origin of some hepatic malignancies [34,81,87]. Identification and characterization of facultative stem cells in the liver is difficult. As yet, there are no antigenic markers available to distinguish these cells in either humans or rodents. Most authorities believe that they are located in the most proximal branches of the biliary tree, the canals of Hering and ductules, lined by cholangiocytes [88]. These cells are positive for biliarytype cytokeratins (CKs), in particular CK19, and for epithelial cell adhesion molecule (EpCAM) [89]. Although liver stem cells are mostly, if not entirely, CK19- and/or EpCAM-positive, not all positive cells function as stem cells. These markers merely enrich for the stem cell population [90,91]. Thus, the finding that SCC is often CK19-positive [92] raises the possibility that it consists of small preneoplastic hepatocytes directly derived from

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CK19-positive cholangiocyte-like stem cells, but this is not necessarily always (or even sometimes) true. Regardless of whether HCC arises from transformed mature hepatocytes or from transformed liver stem cells, the resulting tumor contains a minute subpopulation of tumor stem cells (ie, malignant cells that have all the functional characteristics of facultative stem cells described previously) [93]. These cells probably give rise to rapidly proliferative tumor cells and also are the locus of resistance to therapeutic interventions, such as chemotherapy, radiation, and vascular ablation. As such, they may be the source of tumor recurrence. It has been shown that CD133-positive cells in some human cancer cell lines contain the subpopulation capable of tumorigenicity and clonogenicity [94,95]. CD133 (prominin-1) has been identified in cell fractions of some human HCCs [96]. Another promising group of antigens for possible stem cells are the ABC transporter proteins, including major multidrug resistance genes present in normal and injured livers [97–99]. RADIOLOGIC IMAGING OF NODULAR LESIONS IN CIRRHOTIC LIVERS Radiologic detection, and characterization, of nodular lesions in cirrhosis remains challenging. Although ultrasound is used widely for detection of HCC in noncirrhotic livers, it is less useful in patients with cirrhosis unless intravenous contrast agents are used. Furthermore, distinction between benign and malignant nodules is also challenging. The overall detection of DNs in explant studies is poor [100]. CT requires iodinated contrast agents and uses ionizing radiation, but it is widely available, and the examination can be performed in a short period of time. Similar to contrast-enhanced US, differentiation of benign regenerative nodules from small HCC is based on the presence of a blood supply from the hepatic artery (HCC enhances during the arterial phase of injection). MRI is the most sensitive modality for detection of nodular lesions in cirrhosis, but it is expensive, time-consuming, and has a high false-positive rate [101]. Newer magnetic resonance contrast agents (ferumoxides) that are taken up by Kupffer cells, however, can increase the sensitivity and specificity of MRI for detection and characterization of nodules in cirrhosis [102]. Regenerative Nodules Large regenerative nodules in cirrhotic livers show characteristic features on imaging that usually allow distinction from HCC, but not always from DNs. They invariably have a portal venous blood supply, with either minimal or no contribution from the hepatic artery. One exception are ‘‘focal nodular hyperplasia-type’’ nodules, often seen in patients with cirrhosis caused by BuddChiari syndrome, that recently have been described as a result of other, more common, etiologies also [103]. On CT and sonography, large regenerative nodules are indistinguishable from background nodules. On MRI, large regenerative nodules are usually isointense with other background nodules, on both T1 weighted and T2 weighted images. Less

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A CHRONIC DISEASE

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SPREADING MONOCLONAL POPULATION

DIMINISHED HSC ACTIVATION DEVELOPING CIRRHOSIS

DYSPLASTIC NODULE

NL or INCREASED HSC ACTIVATION DEVELOPING CIRRHOSIS

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DYSPLASTIC FOCI IN ADJACENT CIRRHOTIC NODULES

NORMAL HSC ACTIVATION

HEPATOCELLULAR CARCINOMA ARISING IN DYSPLASTIC NODULE

SINGLE OR MULTIPLE SMALL HEPATOCELLULAR CARCINOMAS

SMALL HEPATOCELLULAR CARCINOMA

CLASSIC HEPATOCELLULAR CARCINOMA

CLASSIC HEPATOCELLULAR CARCINOMA

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commonly, they may be hyperintense on T1 weighted and hypointense on T2 weighted images. Unlike some HCCs, however, large regenerative nodules are almost never hyperintense on T2 weighted images, with the noted exception of those that occur in the setting of cirrhosis caused by chronic Budd-Chiari syndrome, or those that have undergone infarction. Regenerative nodules that contain iron (siderotic nodules) have characteristic imaging features, including decreased signal intensity on both T1 and T2 weighted pulse sequences, and demonstrate a blooming effect on gradient echo pulse sequences, with longer echo times and lower flip angles [104].

m

Dysplastic Nodules As described previously, DNs receive most of their blood supply from the portal venous system. In a minority of DNs, there are enough nontriadal arteries to make arterial supply visible on radiologic examination. Arterial phase enhancement of high- and low-grade DNs has been reported with helical CT and hepatic arterial phase MRI, but this is an uncommon feature [105]. DNs typically are not identified as such by either CT or ultrasound. The signal intensity characteristics of DNs on MRI overlap significantly with those of small HCCs. A common pattern is homogeneously hyperintense on T1 weighted images and hypointense on T2 weighted images (Fig. 7). This pattern, however, may be seen with some HCCs and large regenerative nodules. One very helpful distinction between HCC and DN is that the latter is almost never hyperintense on T2 weighted images. Similar to some regenerative nodules, DNs can be siderotic, and, therefore, hypointense on both T1 and T2 weighted images [104]. Although most HCCs arise from cirrhosis on MRI, on occasion, they may be demonstrated to occur within a DN on serial imaging studies. The classic magnetic resonance appearance of these lesions is a nodule-in-nodule pattern, consisting of a high-signal intensity focus within a low-signal intensity nodule on T2 weighted images (Fig. 8) [105]. The central nodule of high-signal

Fig. 6. Emergence of hepatic nodular lesions in relation to timing and degree of hepatic stellate cell activation. Lightning bolt: first molecular events in the setting of chronic liver disease; such events continue to accumulate throughout the disease course. (A) The initial clonal expansion precedes development of cirrhosis. Because of inhibition of hepatic stellate cell (HSC) activation, the expansion takes on the appearance of a large nodule, as the rest of the liver around it becomes cirrhotic. Atypia within this expansion parallels or follows development of cirrhosis. (B) The clonal expansion is associated with normal or increased HSC activation. As a result, the expansion is obscured by scar as the entire liver progresses to cirrhosis; therefore, multiple dysplastic foci and small hepatocellular carcinomas may arise in a clustered fashion. These also may be seen in more widespread regions of the liver, depending on the size of the clonal expansion, or if multiple such expansions are happening simultaneously throughout the liver. (C) The clonal expansion and subsequent emergence of a dysplastic focus may occur either in advance, or during scarring of the liver. This process may be particularly apparent in genetic hemochromatosis, where the rapidly proliferating hepatocytes form iron-free foci.

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Fig. 7. High-risk patient with hepatitis C cirrhosis and multiple large nodules (arrows), hyperintense on unenhanced T1-weighted gradient-echo (A) and hypointense on fat-suppressed T2 weighted MRI (B). The lesions did not enhance during the hepatic arterial phase (not shown) and may represent large regenerative nodules, dysplastic nodules, or less likely, early hepatocellular carcinoma. The pattern of large nodules with T1 hyperintensity and T2 hypointensity (but without hepatic arterial phase enhancement) has been reported to represent dysplastic nodules, but in fact, is more likely to be large regenerative nodules on histologic examination.

intensity also may demonstrate enhancement during the hepatic arterial phase, consistent with enhanced neoangiogenesis (Fig. 9). Small Hepatocellular Carcinoma Small HCCs (measuring less than 2 cm) demonstrate variable patterns of signal intensity on T1 and T2 weighted magnetic resonance images [101]. The hepatic arterial blood supply of most HCCs (with the exception of some early, well-differentiated lesions which may retain some portal venous supply), however, can facilitate diagnosis. Hence, not only are dynamic contrast-enhanced images essential for the detection of small HCCs; they also may help to distinguish them from large regenerative nodules and DNs. The most common enhancement pattern of small HCCs on both CT and MRI is a diffuse, homogenous enhancement during the hepatic arterial phase, with rapid

Fig. 8. High-resolution ex vivo T2 weighted MRI shows multiple low signal nodules, some of which demonstrate central hyperintensity (arrow). This represents the nodule-in-nodule sign of a small hepatocellular carcinoma arising within a dysplastic nodule. The occurrence of multiple such lesions, as seen is this case, is exceptional.

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Fig. 9. Magnetic resonance demonstration of in vivo hepatocarcinogenesis in a 57-year-old man with cirrhosis caused by chronic hepatitis C virus infection. There are two dominant nodules at the dome of the liver that are hypointense on fat-suppressed T2 weighted (A) and hyperintense on T1 weighted (B) gradient-echo MRI. These nodules did not enhance during the hepatic arterial phase after the administration of gadolinium (not shown) and were considered suspicious for dysplastic nodules. (C) Surveillance MRI performed 1 year later shows that the medial nodule now has a focus of T2 hyperintensity (arrow) consistent with hepatocellular carcinoma arising within a dysplastic nodule (nodule-in-nodule sign). (D) Hepatic arterial phase T1 weighted magnetic resonance image now shows enhancement of this nodule (arrow), demonstrating the neoangiogenesis of a small hepatocellular carcinoma.

washout during the portal venous phase. Similarly, hepatic hemangiomas enhance strongly during the arterial phase, but remain isointense to the hepatic vasculature for multiple phases. Larger HCCs tend to display a heterogeneous or mosaic pattern of enhancement in the hepatic arterial phase and the presence

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of a capsule, or pseudocapsule, on later phases. Successfully ablated HCCs do not demonstrate hepatic arterial phase enhancement. SUMMARY Large nodules detected in cirrhotic livers by imaging include large regenerative nodules, dysplastic nodules (low- or high grade), and small HCCs. The differential diagnosis of these lesions by ultrasound, CT, or MRI may be difficult or impossible. Even biopsies may be difficult to interpret. Nevertheless, histologic evidence of a dysplastic nodule, or cytologic change suggestive of dysplasia (dysplastic focus) indicates an increased risk for carcinoma development and warrants increased surveillance. Small HCCs (measuring less than 2 cm in diameter) are of two types: Well-differentiated lesions with indistinct margins (early HCCs) Well- to moderately differentiated nodular lesions with distinct margins and features similar to those of larger HCCs

Distinctly nodular small HCCs usually contain well-developed nontriadal arteries, which facilitate their detection by contrast-enhanced imaging methods, whereas early HCCs may contain both portal tracts and nontriadal arteries, similar to dysplastic nodules. Identification of high-grade dysplastic nodules, or small HCCs, should lead to treatment by local ablation, surgical resection, or liver transplantation. References [1] International Working Party. Terminology of nodular hepatocellular lesions. Hepatology 1995;22(3):983–93. [2] Watanabe S, Okita K, Harada T, et al. Morphologic studies of the liver cell dysplasia. Cancer 1983;51(12):2197–205. [3] Borzio M, Bruno S, Roncalli M, et al. Liver cell dysplasia is a major risk factor for hepatocellular carcinoma in cirrhosis: a prospective study. Gastroenterology 1995;108(3): 812–7. [4] Le Bail B, Bernard PH, Carles J, et al. Prevalence of liver cell dysplasia and association with HCC in a series of 100 cirrhotic liver explants. J Hepatol 1997;27(5):835–42. [5] Nakanuma Y, Hirata K. Unusual hepatocellular lesions in primary biliary cirrhosis resembling but unrelated to hepatocellular carcinoma. Virchows Arch A Path Anat 1993;422(1):17–23. [6] Zhao M, Zhang NX, Du ZY, et al. Three types of liver cell dysplasia (LCD) in small cirrhotic nodules are distinguishable by karyometry and PCNA labeling, and their features resemble distinct grades of hepatocellular carcinoma. Histol Histopathol 1994;9(1): 73–83. [7] Makino Y, Shiraki K, Sugimoto K, et al. Histological features of cirrhosis with hepatitis C virus for prediction of hepatocellular carcinoma development: a prospective study. Anticancer Res 2000;20(5C):3709–15. [8] Libbrecht L, Craninx M, Nevens F, et al. Predictive value of liver cell dysplasia for development of hepatocellular carcinoma in patients with noncirrhotic and cirrhotic chronic viral hepatitis. Histopathology 2001;39(1):66–73. [9] Ganne-Carrie N, Chastang C, Chapel F, et al. Predictive score for the development of hepatocellular carcinoma and additional value of liver large cell dysplasia in Western patients with cirrhosis. Hepatology 1996;23(5):1112–8.

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Neoplastic Precursors of the Gallbladder and Extrahepatic Biliary System N. Volkan Adsay, MD Department of Pathology, Emory University School of Medicine, 1364 Clifton Road, H-180-B, Atlanta, GA 30322, USA

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ompared with other organs of the gastrointestinal tract, the clinical features, morphology, and diagnostic criteria of precursor lesions of the gallbladder and biliary tract are less well characterized [1–3]. This is partly because cancerous lesions of the biliary tract are far less common. The relative inaccessibility of the biliary tract is also a contributing factor to scientific advancement in this field. In addition, there is no effective surveillance mechanism for the biliary tract, in contrast to Barrett’s esophagus and inflammatory bowel disease, where there is ample evidence to support the dysplasiacarcinoma sequence. Such data do not exist for the biliary tract, at least not to that degree. The pathologic definition and criteria for precursor lesions of the biliary tract are based on surrogate evidence, such as (1) common association with malignancy, both spatially and epidemiologically; (2) anecdotal evidences of progression; (3) histopathologic similarities to malignancy; (4) extrapolation of experience gained in other organ systems regarding morphologic signs of dysplasia; and (5) molecular and genetic evidence of malignant transformation. Although these observations provide an acceptable substrate on which to build criteria, they nevertheless leave room for subjectivity. The clinical features, morphology, and diagnostic criteria of biliary tract precursor lesions are poorly characterized. It has been well established by epidemiologic and clinical studies, however, that the biliary tract is probably the consummate example of inflammation-associated carcinoma. The link between the development of biliary adenocarcinoma with preceding chronic inflammation has been well established, mostly by epidemiologic data showing a high incidence of gallbladder cancer in areas with a high prevalence of gallstones, and by the pathologic observation that many individual carcinomas have associated gallstones or chronic cholecystitis. Also, the risk of adenocarcinoma is relatively high in patients with primary sclerosing cholangitis (and indirectly

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with ulcerative colitis) [4–6]. The association of biliary cancers with parasitic infection and choledochal cysts is also, presumably, a reflection of an inflammation-associated carcinoma. Accordingly, one would assume that these diseases might serve as an opportunity, or a model, to study and characterize carcinogenesis and its precursor lesions. Proper identification of premalignant change in this site is difficult, however, because the border between inflammatory reactive atypia and true neoplastic transformation is somewhat subjective histologically. The data regarding diagnostic criteria, incidence, relative risk, and management of precursor lesions in the biliary tract have many uncertainties. In this article, an overview of the current literature and the author’s experience with these lesions are provided. DYSPLASTIC PRECURSORS OF THE BILIARY TRACT AND GALLBLADDER There are essentially two distinct types of premalignant lesions in the biliary tract. One is the conventional flat dysplasia, which is similar to premalignant lesions of other organs, such as Barrett esophagus and inflammatory bowel disease. The other category is benign (elevated) neoplasms of the biliary tract that may have malignant potential. This latter category may be regarded as tumoral (mass-forming) dysplasia, and as such shows many parallels with the adenocarcinoma sequence in the colon. CONVENTIONAL (FLAT) DYSPLASIA Definition and Terminology Terms for microscopic, incidental forms of preinvasive epithelial proliferations, such as epithelial atypia, atypical hyperplasia, and atypical adenomatous hyperplasia, are not encouraged. The World Health Organization has defined these lesions as ‘‘dysplasia’’ or ‘‘intraepithelial neoplasia’’ (Figs. 1–3) [3,7]. Based on the degree of cytologic and architectural atypia, dysplasia is generally graded as either low or high grade. Unfortunately, this can be highly subjective. Also, some use the terms ‘‘high-grade dysplasia’’ and ‘‘carcinoma in situ’’ (CIS) interchangeably, and do not recognize CIS as a separate diagnostic category. This author regards the spectrum of neoplastic transformation in the biliary epithelium as a continuum, with low-grade dysplasia at the lower end, high-grade dysplasia for more advanced lesions, and CIS as the uppermost end where malignant transformation is complete and the cells have almost all the cytologic characteristics of invasive carcinoma cells. The term CIS is reserved for cases in which there is definite evidence of carcinomatous change. One major problem with this approach is that cases diagnosed as CIS are classified (in the TNM system) as ‘‘Tis,’’ and are included in the ‘‘cancer’’ category, whereas slightly lesser grade lesions are dismissed as clinically insignificant [1]. Considering the level of subjectivity involved in diagnosing dysplasia, this is a major concern for the study of epidemiologic and clinicopathologic

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Fig. 1. Dysplasia (biliary intraepithelial neoplasia) of low grade (hematoxylin-eosin, original magnification  40). Pseudostratification of the cells with nuclear enlargement and clumped chromatin are evidence of neoplastic transformation.

characteristics of precursor lesions, and in determining management of individual patients. Recently, the term ‘‘biliary intraepithelial neoplasia’’ [8] was proposed for biliary dysplasia, which uses the approach used for other organs, such as the pancreas [9]. Consensus studies on biliary intraepithelial neoplasia, although illustrating the subjectivity of criteria, are nevertheless a good start toward achieving more uniform terminology and criteria for these lesions. Incidence Typically, premalignant lesions are detected incidentally during evaluation of other associated pathologic conditions [10–12]. Most occur in patients with

Fig. 2. Dysplasia of high grade (hematoxylin-eosin, original magnification  40). The nuclei are significantly enlarged, hyperchromatic, and disorganized.

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Fig. 3. Dysplasia of high grade (hematoxylin-eosin, original magnification  10). In some examples, dysplastic transformation is also manifested as architectural complexity of the epithelium, such as formation of cribriform units as seen in this example.

invasive carcinoma; it is highly uncommon to detect high-grade dysplasia and CIS in the absence of invasive carcinoma. It has been reported in 1% to 3.5% of cholecystectomies performed for gallstones [11,13]. In a recent analysis of a North American database, however, among cholecystectomies for gallstones and those performed incidentally for morbid obesity, the incidence of lowgrade dysplasia was 4% and high-grade dysplasia (or CIS) was less than 0.01%. The marked variation in frequency reported in the literature probably reflects pathologic variation in interpretation, in addition to population-related differences. Dysplasia incidence is higher in regions where biliary cancers are more common, which supports the dysplasia-carcinoma sequence. Also, there is a progressive increase in the mean age of patients with increasing degrees of neoplastic transformation, from age 50 years for low-grade dysplasia, to 58 years for high-grade dysplasia, and 64 years for invasive carcinoma. The incidence of dysplasia in surgical specimens of extrahepatic bile ducts is much lower; however, these studies are more controversial. Pathologic Features By definition, flat precursor neoplasms do not form clinically or even macroscopically identifiable lesions. Those that form grossly visible lesions are regarded as mass-forming preinvasive neoplasia, discussed later. Some types of conventional dysplasia, however, may exhibit granularity or congestion. Intraepithelial neoplasia is characterized by a disorderly intraepithelial proliferation of atypical columnar, cuboidal, or elongated biliary-type cells (see Figs. 1–3). Examples with columnar-shaped cells also tend to be associated with pseudostratification and hyperchromasia, similar to dysplasia in the colon. Nuclear enlargement and loss of polarity may help distinguish neoplasia from reactive atypia (Fig. 4). In some examples of intraepithelial neoplasia, the nuclei are enlarged, ovoid, and uniform in appearance. Others may have more pleomorphic and irregular cuboidal cells. Mitotic figures and apoptotic cells are often present. Giant

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Fig. 4. Reactive changes in biliary epithelium may be associated with pseudostratification of nuclei and mimic dysplasia (hematoxylin-eosin, original magnification  20). Reactive changes lack nuclear enlargement and chromatin changes typical of dysplasia (see Fig. 1).

cells may also be seen. Intestinal metaplasia may be present in the background. It is nearly impossible to diagnose flat precursor lesions on limited cytologic preparations like biliary brushings or washings. Lesser lesions are typically indistinguishable from reactive atypia (discussed later), and more advanced dysplasia (high-grade or CIS) is difficult to discern from invasive carcinoma cells. Having said this, there are cases in which the possibility of dysplasia can be suggested in some cases, to be corroborated with the clinical findings. Pathologic Differential Diagnosis The main differential diagnosis for dysplasia in the biliary tract is reactive epithelial atypia (Fig. 5). Biliary epithelium is notorious for undergoing marked atypia following injury, either by extrinsic causes, such as instrumentation, or by intrinsic factors, such as sclerosing cholangitis, gallstones, and parasites. Further complicating this differential is that the latter group is also a known risk factor for carcinoma, and dysplastic change may coexist with reactive atypia. In many instances, the criteria used to distinguish reactive atypia from dysplasia are highly subjective and difficult to recapitulate. This is partly because experience with these lesions is fairly limited, but also because of the nature of these lesions where the transition from regeneration to neoplasia is blurry. Other epithelial alterations that mimic dysplasia are hyperplasia and metaplasia. These generally do not create much difficulty because the cells are, by definition, normal in cytologic appearance, with the exception of intestinal metaplasia in which the cytology is inherently abnormal (Fig. 6). It is this author’s opinion that intestinal metaplasia with atypical cells represents true dysplasia. Supporting this information is the fact that intestinal metaplasia is often accompanied by frank CIS or invasive carcinoma. The same may also apply to primary hyperplasia, because this rare condition of the gallbladder is seen in patients with an anomalous union of pancreatobiliary duct who are known also to have an increased cancer risk.

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Fig. 5. Severe reactive atypia in cases of hemorrhagic or necrotizing cholecystitis may closely mimic high-grade dysplastic changes (hematoxylin-eosin, original magnification  40) (see Fig. 2).

In patients with invasive carcinoma, dysplasia may also be difficult to distinguish from retrograde mucosal involvement by invasive carcinoma, which is referred to as ‘‘cancerization’’ or ‘‘colonization’’ of surface epithelium. Although there are numerous molecular alterations that occur in biliary cells during neoplastic transformation, it is difficult to determine which are specific for carcinogenesis, or which may have use as a diagnostic marker, and whether they are an epiphenomenon. For instance, p53 overexpression or increased proliferative activity as determined by Ki-67, both of which can be analyzed by immunohistochemistry, have not been proved useful in diagnostic pathology. Clinical Significance and Natural History Intraepithelial neoplasia is present in most patients with invasive carcinoma. When intraepithelial neoplasia is encountered as an isolated finding, without invasive carcinoma, in a cholecystectomy specimen, extensive and complete sampling is warranted, particularly if the dysplasia is high grade. If no invasive carcinoma is detected, no further therapy is necessary. Surveillance, Epidemiology, and End Results Program of the National Cancer Institute data indicate, however, that a third of patients with CIS of the gallbladder die of tumor after 10 years (although all were alive at 5 years), suggesting that either a small invasive component was initially missed or patients developed a second malignancy [7]. Anecdotally, patients with a history of gallbladder CIS that developed invasive carcinoma elsewhere in the biliary tree have been documented. TUMORAL (MASS-FORMING) PRECURSOR LESIONS Mass-forming intramucosal neoplasms of the biliary epithelium are either polyps (Fig. 7) or cystadenomas. Although they are considered benign by

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Fig. 6. Pyloric metaplasia (A) is a relatively common finding in the biliary tract (hematoxylineosin, original magnification  4); however, intestinal metaplasia (B), showing goblet cells; (hematoxylin-eosin, original magnification  4) often accompanies dysplasia or invasive carcinoma.

generic classification, the occurrence of an adenoma-carcinoma sequence in these lesions has been well established and is regarded as premalignant. This category of precursor lesions is also important because they are potentially diagnosable by radiologic or endoscopic modalities, because they form clinically detectable tumoral lesions unlike the conventional precursors. Definition, Terminology, and Incidence Polypoid preinvasive lesions of the biliary tract have been termed ‘‘adenomas,’’ ‘‘papillomas,’’ ‘‘papillomatosis (adenomatosis),’’ and ‘‘papillary adenocarcinomas’’ based on their growth pattern, multiplicity, extent, or degree of malignant change [1–3]. Conceptually, these lesions represent the biliary counterpart of colonic adenomas, and a similar adenoma-carcinoma sequence. Some of the clinical and pathologic characteristics of these lesions are similar to pancreatic intraductal papillary mucinous neoplasms, which has led some authors to refer to these tumors as ‘‘biliary intraductal papillary mucinous neoplasms’’ [14–18].

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Fig. 7. (A) Pyloric gland adenoma of the gallbladder (hematoxylin-eosin, original magnification  2). (B) In some cases, carcinomatous transformation is seen in this type of lesion (hematoxylin-eosin, original magnification  20).

Adenomas and papillomas are quite uncommon. They have been reported in less than 1% of cholecystectomies. In this author’s experience, they occur in less than 0.2%. In contrast to the conventional (flat) dysplasia, mass-forming preinvasive neoplasia of the biliary tree often occur in the absence of invasive carcinoma. These also are typically detected incidentally at the time of surgery, however, particularly during surgery for gallstone disease. These lesions are much more common in the gallbladder compared with the extrahepatic bile ducts. Clinical Features Adenomas occur predominantly in women. Approximately 10% are multiple. Some may cover the entire surface of the mucosa, in which case the term ‘‘papillomatosis’’ or ‘‘adenomatosis’’ is used [19–21]. Adenomas have been reported in association with Peutz-Jeghers syndrome [22] and Gardner’s syndrome [23– 25], or anomalous union of the pancreatobiliary duct, and half are associated with cholelithiasis.

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Pathologic Features The macroscopic appearance of adenomas varies from lobular to cauliflowerlike, depending on the degree of villous architecture. Few are sessile in appearance. They occur more commonly in the body or fundus of the gallbladder, and are highly uncommon in the bile ducts. Typically, they are smaller than 2 cm in size. Not surprisingly, the risk of malignancy seems to increase with size. Based on the growth pattern, adenomas have been classified as tubular, papillary, or tubulopapillary, and based on their resemblance to different compartments of the gastrointestinal tract, as pyloric gland–, intestinal-, or biliary-type. Most are composed of tightly-packed, cytologically bland pyloric-type tubules and classified as the pyloric gland–type. These occur almost exclusively in the gallbladder [26]. Some are morphologically similar to intestinal adenomas [26]. They may have a variable degree of tubule or villous formation. Rare examples of adenomas have a more complex papillary architecture and cytologic features similar to biliary epithelium (biliary) type. Multicentric papillary adenomas within the biliary tree is designated ‘‘papillomatosis’’ [19–21], but this rare condition often displays carcinoma when current diagnostic criteria are applied. Natural History and Management Adenomas do not show the same degree of epidemiologic (geographic and population-related) association with adenocarcinomas as conventional intraepithelial neoplasia. Only a small percentage of cases with invasive adenocarcinoma have an identifiable adenoma, and the incidence of invasive carcinoma in adenomas is low. The weak association between mass-forming preinvasive neoplasia with adenocarcinoma may be attributable to three factors: (1) growth of the invasive carcinoma may replace the noninvasive component; (2) the pathogenesis is different, perhaps indicating a more indolent pathway of carcinogenesis; and (3) many cysts are detected and treated before progression to invasive carcinoma. Regardless, it has been well established that adenomas may either harbor carcinoma or be associated with carcinoma elsewhere in the biliary tract and, as such, they are regarded as dysplastic. Similar to other organ systems, there is a spectrum in the extent of carcinomatous transformation that occurs in exophytic biliary neoplasms, ranging from microscopically high-grade dysplasia (see Fig. 7B) to macroscopically visible invasive carcinoma. The terminology applied to this spectrum is problematic. When a portion of the exophytic tumor retains the features of a benign adenoma but also shows carcinomatous areas, it is reasonable to regard the process as malignant transformation of an adenoma, an uncommon but well-documented event. When the entire exophytic tumor shows significant cytologic or architectural atypia, however, it is regarded as a papillary carcinoma. These are often referred to as ‘‘noninvasive papillary carcinoma’’ if in the gallbladder or ‘‘intraductal papillary carcinoma’’ if located in the bile ducts. Conversely, in some patients with invasive adenocarcinoma,

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a residual papillary carcinoma component is identifiable. Many adenocarcinomas with a polypoid gross appearance represent this latter group. Noninvasive papillary carcinomas have an excellent prognosis if they are resected. Cases with minimal invasive carcinoma also have a favorable prognosis [7]. Once there is a significant amount of invasive tumor, the prognosis approaches that of biliary adenocarcinomas that do not arise from papillary precursor lesions. The relative frequency of carcinomatous transformation among different subsets of mass-forming intramucosal neoplasia is unknown. It seems, however, that carcinomatous transformation is quite uncommon in pyloric gland adenomas. In contrast, the association of papillomatosis with carcinoma seems quite strong. Other Types of Mass-Forming Precursor Lesions Hepatobiliary cystadenomas and cystadenocarcinomas [27,28], which are analogous to mucinous cystic neoplasms of the pancreas, are also regarded as a mass-forming preinvasive neoplasm. These are multilocular cystic lesions that occur predominantly in adult women, and exhibit pathognomonic hormone receptor expressing ovarian-type stroma [29]. The lining epithelium is composed of cuboidal to columnar cells, sometimes with abundant apical mucin. Polypoid projections may be identified in the cyst lumen. Although most hepatobiliary cystic neoplasms show benign cytoarchitectural features (ie, hepatobiliary cystadenoma), some may harbor invasive carcinoma (hepatobiliary cystadenocarcinoma). Carcinoma may be focal, and for this reason, thorough histologic examination is advised. The author regards papillary adenocarcinomas, which are tumors with prominent intraluminal growth, within the conceptual category of preinvasive neoplasia. It should be remembered, however, that these are commonly associated with invasive carcinoma and have an aggressive clinical course. As such, papillary adenocarcinoma may be the uppermost end of the spectrum in the adenoma-carcinoma sequence. Recognition of invasive carcinoma developing in papillary adenocarcinoma can be challenging. The complex architecture of the lesion, its extension to Aschoff-Rokitansky sinuses, or granulation-tissue– type changes in the stroma simulating desmoplasia may mimic invasion. SUMMARY Precursor lesions of the gallbladder and extrahepatic biliary ducts are relatively uncommon and less well studied than precursors of other components of the gastrointestinal tract. They constitute an important biologic and clinicopathologic entity, however, that needs to be fully appreciated and further characterized. References [1] Albores-Saavedra J, Scoazec JC, Wittekind C, et al. Tumours of the gallbladder and extrahepatic bile ducts. In: Hamilton SR, Aaltonen LA, editors. World Health Organization

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[2]

[3]

[4] [5]

[6]

[7] [8]

[9]

[10]

[11] [12]

[13] [14]

[15] [16]

[17] [18]

[19] [20] [21]

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classification of tumours: pathology and genetics of tumours of the digestive system. Lyon, France: IARC Press; 2000. p. 204–17. Albores-Saavedra J, Hensen DE, Klimsta DS. Tumors of the gallbladder, extrahepatic bile ducts, and ampulla of Vater: atlas of tumor pathology. Fascicle 27, series 3. Washington, DC: Armed Forces Institute of Pathology; 2000. Adsay NV. Gallbladder, extrahepatic biliary tree, and ampulla. In: Mills SE, Carter D, Greensin JK, editors. Sternberg’s diagnostic surgical pathology, 2 vol. 4th edition, Philadelphia: Lippicott Williams & Wilkins; 2004. p. 1774–828. Morowitz DA, Glagov S, Dordal E, et al. Carcinoma of the biliary tract complicating chronic ulcerative colitis. Cancer 1971;27:356–61. Mir-Madjlessi SH, Farmer RG, Sivak MV Jr. Bile duct carcinoma in patients with ulcerative colitis: relationship to sclerosing cholangitis. Report of six cases and review of the literature. Dig Dis Sci 1987;32:145–54. Morohoshi T, Kunimura T, Kanda M, et al. Multiple carcinomata associated with anomalous arrangement of the biliary and pancreatic duct system. A report of two cases with a literature survey. Acta Pathol Jpn 1990;40:755–63. Albores-Saavedra J, Murakata L, Krueger JE, et al. Noninvasive and minimally invasive papillary carcinomas of the extrahepatic bile ducts. Cancer 2000;89:508–15. Zen Y, Adsay NV, Bardadin K, et al. Biliary intraepithelial neoplasia: an international interobserver agreement study and proposal for diagnostic criteria. Mod Pathol 2007;20: 701–9. Hruban RH, Adsay NV, Albores-Saavedra J, et al. Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions. Am J Surg Pathol 2001;25:579–86. Albores-Saavedra J, Alcantra-Vazquez A, Cruz-Ortiz H, et al. The precursor lesions of invasive gallbladder carcinoma: hyperplasia, atypical hyperplasia and carcinoma in situ. Cancer 1980;45:919–27. Ojeda VJ, Shilkin KB, Walters MN. Premalignant epithelial lesions of the gall bladder: a prospective study of 120 cholecystectomy specimens. Pathology 1985;17:451–4. Yamamoto M, Nakajo S, Tahara E. Carcinoma of the gallbladder: the correlation between histogenesis and prognosis. Virchows Arch A Pathol Anat Histopathol 1989;414:83–90. Chan KW. Review of 253 cases of significant pathology in 7,910 cholecystectomies in Hong Kong. Pathology 1988;20:20–3. Kim HJ, Kim MH, Lee SK, et al. Mucin-hypersecreting bile duct tumor characterized by a striking homology with an intraductal papillary mucinous tumor (IPMT) of the pancreas. Endoscopy 2000;32:389–93. Chen TC, Nakanuma Y, Zen Y, et al. Intraductal papillary neoplasia of the liver associated with hepatolithiasis. Hepatology 2001;34:651–8. Tamada S, Goto M, Nomoto M, et al. Expression of MUC1 and MUC2 mucins in extrahepatic bile duct carcinomas: its relationship with tumor progression and prognosis. Pathol Int 2002;52:713–23. Abraham SC, Lee JH, Hruban RH, et al. Molecular and immunohistochemical analysis of intraductal papillary neoplasms of the biliary tract. Hum Pathol 2003;34:902–10. Shibahara H, Tamada S, Goto M, et al. Pathologic features of mucin-producing bile duct tumors: two histopathologic categories as counterparts of pancreatic intraductal papillary-mucinous neoplasms. Am J Surg Pathol 2004;28:327–38. Gouma DJ, Mutum SS, Benjamin IS, et al. Intrahepatic biliary papillomatosis. Br J Surg 1984;71:72–4. Sagar PM, Omar M, Macrie J. Extrahepatic biliary papillomatosis occurring after removal of a dysplastic gall bladder. HPB Surg 1993;6:219–21. Taguchi J, Yasunaga M, Kojiro M, et al. Intrahepatic and extrahepatic biliary papillomatosis. Arch Pathol Lab Med 1993;117:944–7.

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[22] Stringer MD, Ceylan H, Ward K, et al. Gallbladder polyps in children: classification and management. J Pediatr Surg 2003;38:1680–4. [23] Harned RK, Buck JL, Olmsted WW, et al. Extracolonic manifestations of the familial adenomatous polyposis syndromes. AJR Am J Roentgenol 1991;156:481–5. [24] Walsh N, Qizilbash A, Banerjee R, et al. Biliary neoplasia in Gardner’s syndrome. Arch Pathol Lab Med 1987;111:76–7. [25] Shemesh E, Bat L. A prospective evaluation of the upper gastrointestinal tract and periampullary region in patients with Gardner syndrome. Am J Gastroenterol 1985;80:825–7. [26] O’Shea M, Fletcher HS, Lara JF. Villous adenoma of the extrahepatic biliary tract: a rare entity. Am Surg 2002;68:889–91. [27] Wheeler DA, Edmondson HA. Cystadenoma with mesenchymal stroma (CMS) in the liver and bile ducts: a clinicopathologic study of 17 cases, 4 with malignant change. Cancer 1985;56:1434–45. [28] Devaney K, Goodman ZD, Ishak KG. Hepatobiliary cystadenoma and cystadenocarcinoma: a light microscopic and immunohistochemical study of 70 patients. Am J Surg Pathol 1994;18:1078–91. [29] Grayson W, Teare J, Myburgh JA, et al. Immunohistochemical demonstration of progesterone receptor in hepatobiliary cystadenoma with mesenchymal stroma. Histopathology 1996;29:461–3.

Gastroenterol Clin N Am 36 (2007) 901–926

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Neoplastic Precursor Lesions Related to the Development of Cancer in Inflammatory Bowel Disease Noam Harpaz, MD, PhD Division of Gastrointestinal Pathology, Department of Pathology, The Mount Sinai Medical Center, One Gustave L. Levy Place, New York, NY 10029, USA

C

olorectal cancer is the most serious long-term complication faced by patients with chronic inflammatory bowel disease (IBD) (ie, ulcerative colitis [UC] and colonic Crohn’s disease [CD]) [1–5]. Although IBD accounts for less than 1% of colorectal cancers in the general population, its 10- to 20-fold increased incidence in patients with extensive colitis and the relatively young mean age of its victims, more than one third of whom are below age 50 [4,6,7], combine to make cancer prevention in IBD a worthwhile and much sought-after goal. Of the various factors known to predispose to colorectal cancer in IBD, none has been more directly implicated than dysplasia of the large intestinal epithelium. The concept of a colitis–dysplasia–cancer sequence is the key to understanding the molecular pathogenesis of cancer in IBD [8], provides the rationale for research into cancer chemoprevention [9,10], and is the cornerstone of the contemporary approach to cancer-risk reduction based on endoscopic surveillance [5,11]. COLORECTAL CANCER IN INFLAMMATORY BOWEL DISEASE Incidence and Risk Factors The chief risk factors for colorectal cancer in IBD are prolonged disease duration and extensive colonic involvement [1,12]. Although published cancer rates in UC vary greatly because of methodological and possibly other factors, a recent meta-analysis has estimated their prevalence to be 5.4% for patients with pan-UC and 3.7% overall, and their mean cumulative incidence to be 2% after 10 years, 8% after 20 years, and 18% after 30 years of disease [1]. Similar rates are estimated for CD after adjustments are made for duration and extent of colonic involvement [13–17]. Other suspected or established risk factors include early disease onset (according to some studies), sustained histological [18,19] and possibly clinical [20] disease activity, concurrent primary sclerosing

E-mail address: [email protected] 0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.003

ª 2007 Elsevier Inc. All rights reserved. gastro.theclinics.com

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cholangitis [20–26], and a history of colorectal cancer in first-degree relatives [27,28]. Pathological Characteristics One of the distinctive features of cancers in IBD and a clue to an underlying precancerous field abnormality is their frequent multiplicity. Two or more synchronous cancers occur in 10% to 30% of cancer-bearing colectomy specimens [14,29–32] and three or more cancers, a rarity in the general population, occur in 9% [31]. Another distinctive feature is their gross heterogeneity, which includes plaques, nodules, strictures, villiform carpetlike growths, excavating ulcers, and, in some cases, virtually undetectable flat cancers. Additionally, cancers in IBD, if they are visible at all, usually have indistinct borders and blend macroscopically into their surroundings. Histologically, most are conventional adenocarcinomas, but more than 20% are mucinous [4]. In IBD, cancers and their dysplastic precursors invariably arise within regions of chronically diseased mucosa, whether actively inflamed or healed. However, disease involvement may not always be evident by direct endoscopic visualization because gross normalization may take place either spontaneously or as a result of therapy. Mathy and colleagues [33] described a series of 30 patients who underwent colectomy for UC-associated dysplasia or cancer, 11 of whom were diagnosed with distal colitis at preoperative endoscopy. Of this group, 4 had neoplastic lesions proximal to the area of gross involvement, yet all were found to have pancolitis based on microscopic assessment of their resection specimens [33]. DYSPLASIA AS A BASIS FOR CANCER RISK MANAGEMENT IN INFLAMMATORY BOWEL DISEASE The concept that cancers arising in patients with UC are derived from dysplastic precursor lesions was initially advanced during the 1940s and 1950s [34,35] and has been validated both in UC and CD on the basis of direct as well as circumstantial evidence. Dysplasia is observed in proximity to cancer in approximately 90% of resected colons, including virtually all cases with multiple cancers [14,29–32], and occurs in remote parts of the colon in approximately 75% of cases of UC [32,36–38] and 27% to 100% of cases of CD [30,39–42]. Of patients who undergo colectomy with a prior biopsy diagnosis of dysplasia, cancers are discovered at surgery in 20% to 50% of cases [6,43– 45]. Observations of serial endoscopic biopsies have documented neoplastic progression to cancer by way of intermediate stages of dysplasia. Finally, dysplasia and cancer in IBD harbor common genetic mutations [8]. The notion that dysplasia might be used to reduce cancer risk in patients with UC is credited to Morson and Pang, who, during the precolonoscopic era, made the initial observation that precancerous histological changes in biopsies of rectal mucosa of patients with UC are predictive of both rectal and colon cancer [35]. Since the introduction of flexible endoscopy and the inception of an endoscopic surveillance program for patients with UC at St. Mark’s Hospital in

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1971 [46], colonoscopic surveillance has gained wide acceptance and is currently the recommended standard of care for UC and CD [47–50]. However, many questions remain unanswered regarding such issues as cost, details of implementation, endoscopic techniques, patient compliance, physician compliance, and diagnostic variation among pathologists [11]. Indeed, there is currently no proof that surveillance reduces mortality or morbidity among patients at risk and, because of ethical constraints, little prospect of any randomized prospective studies to address this issue. However there is direct evidence that surveillance in IBD leads to the detection of cancers at relatively early stages and circumstantial evidence that it is an effective strategy to reduce cancer-related mortality [51]. Current United States and European guidelines for implementation of endoscopic surveillance are relatively uniform [47–49]. In summary, they suggest that patients with extensive UC or colonic CD undergo an initial screening colonoscopy with random biopsies taken every 10 cm throughout the colon, beginning 8 to 10 years after disease onset. Colonoscopic surveillance examinations should be performed at regular intervals of 1 to 2 [49] or 3 years [47,48] during the second decade and more frequently in later decades because of the possibility that the cancer hazard rate might increase over time [1]. Appropriate adjustments are recommended for patients with clinical characteristics associated with high risk, such as primary sclerosing cholangitis, or decreased risk, such as left-sided colitis. Although measures to diminish disease activity are recommended before examination to minimize reactive changes that may impede pathological interpretations of dysplasia, these ought not stand in the way of regular examinations. Biopsies should be interpreted by pathologists familiar with the criteria and classification of the IBD Morphology Study Group [52] (see below), and any findings that may significantly impact the course of management should be reviewed independently by a second pathologist. Guidelines for the management of patients with dysplasia are summarized in Table 1 and discussed below. PATHOLOGY OF DYSPLASIA IN INFLAMMATORY BOWEL DISEASE Definition of Terms Dysplastic colorectal epithelium is defined as unequivocally neoplastic epithelium that has not yet invaded beyond the basement membrane in which it originated [52]. The term intraepithelial neoplasia is used in the World Health Organization [53] and Vienna [54] nomenclature systems for gastrointestinal neoplasia, but the original terminology remains widely accepted in the United States and will be used herein. As defined by the IBD Morphology Study Group in 1983 [52], dysplasia excludes reactive epithelial changes associated with inflammation and regeneration, which are classified as negative for dysplasia, and changes of uncertain significance, which are classified as indefinite for dysplasia. Dysplasia is subdivided microscopically into low- and high-grade categories based on the severity of cytoarchitectural abnormalities, whereas

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Table 1 Classification and management of dysplasia in IBD Endoscopic appearances

Precautions

Associated cancer risk

Recommended management

Not applicable

None

Low

Continue surveillance

Indefinite for dysplasia

Normal or colitis Colitis

Visible lesion or none noted

Elevated, but undefined risk of progression to neoplasia

Repeat examination within 3–6 months or sooner (see text); consider accelerated surveillance

Sporadic adenoma

Normal

Conventional adenomatous polyp (sessile or pedunculated)

Low

Polypectomy; continue surveillance

Colitis

Conventional pedunculated adenomatous polyp

Caution commensurate with degree of clinical, endoscopic or pathologic concern; consider confirmation by second pathologist Absence of surrounding colitis should be confirmed microscopically; caution if pedicle is dysplastic; caution if patient age <40 years or if lesion recurs after polypectomy No dysplasia along pedicle

Low

Polypectomy; continue surveillance

Negative for dysplasia

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Mucosal surroundings

Diagnosis

Colitis

Dysplasia-associated lesion or mass (nonadenomalike)

Colitis

Flat low-grade dysplasia

Colitis

Conventional adenomatous polyp (ie, endoscopically resectable, domeshaped, sharp boundaries, no gross stigmata of cancer) Visible lesion, nonresectable by polypectomy, stigmata of cancer, or poorly defined boundaries No lesion noted

Flat high-grade dysplasia

Colitis

No lesion noted

No flat dysplasia in adjacent mucosa or elsewhere in colon; caution if patient age <40 years or if lesion recurs after polypectomy Confirm diagnosis with second pathologist

Low

Short-term follow-up examination; continue surveillance

>40% risk of cancer at colectomy

Colectomy

Confirm diagnosis with second pathologist

20% risk of cancer at colectomy; 50% progression to cancer or highgrade dysplasia in 5 years according to some studies >40% risk of cancer at colectomy; 25%–33% progression to cancer in 5 years if colectomy is deferred

Colectomy (alternatively, accelerated surveillance; see text)

Confirm diagnosis with second pathologist

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Adenomalike dysplastic polyp (adenomalike dysplasiaassociated lesion or mass)

Colectomy

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invasive cancer is classified separately. In summary, the standard classification system for neoplastic change in IBD comprises five major microscopic categories: negative for dysplasia, indefinite for dysplasia, low-grade dysplasia (LGD), high-grade dysplasia (HGD), and invasive adenocarcinoma. The morphological diversity of dysplasia in IBD and the need to distinguish it from reactive changes in a background of inflammation combine to make the diagnosis of dysplasia in IBD challenging and subject to interobserver variation. Microscopic Classification of Dysplasia Dysplastic colorectal mucosa is characterized by a constellation of abnormalities that include (1) atypical nuclear morphology, (2) cytoplasmic abnormalities suggesting impaired differentiation and clonality, and (3) altered growth patterns reflecting faulty control of cellular proliferation (Figs. 1 and 2). In general, dysplastic cell nuclei are characterized by large size (high nuclear/cytoplasmic ratio), crowding, stratification, clumped chromatin, excessive basophilic staining, pleomorphism, irregular nuclear membranes, enlarged nucleoli, and atypical mitotic figures. The cytoplasm commonly shows diminished, unevenly distributed, or absent mucin, which implies faulty differentiation into distinct absorptive and goblet cell lineages. Importantly, the combination of diminished mucin and altered nuclear morphology overlaps with characteristics of reactive cells, posing problems in interpretation. There may also be features suggesting clonal differentiation toward mucinous, eosinophilic, neuroendocrine, or Paneth cells. Although combined nuclear and cytoplasmic abnormalities are the general rule, there are exceptions in which one or the other feature is relatively inconspicuous. Another manifestation of clonality and poorly regulated growth is cytological uniformity within the crypts and luminal surface with absence of the base-to-surface maturation characteristic of normal or reactive epithelium. Architectural features of dysplasia include

Fig. 1. LGD in IBD. The epithelial nuclei are crowded and elongated, but confined to the basal half of each cell. Cytoplasmic mucin vacuoles are present but are small and disorganized.

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Fig. 2. HGD in IBD. The epithelial nuclei show marked stratification and the cells are piled up and focally cribriform.

villous, tubular, serrated, and mixed forms of dysplasia, which overlap histologically and macroscopically with sporadic adenomatous polyps (Fig. 3). Villous dysplasia, characterized by carpetlike growth; tall, church-spire fronds; and mucinous lining epithelium, resembles sporadic villous adenoma, but is poorly circumscribed [55–58]. Dysplasia associated with tubular and mixed tubulovillous growth patterns is associated with sessile polypoid lesions. Endoscopists and pathologists commonly face difficulty distinguishing between polypoid dysplastic lesions complicating IBD and conventional adenomas. This is discussed further below. By convention, the separation of dysplasia in IBD into low- and high-grade categories is based mainly on cytomorphological parameters related to cell polarity. An important criterion is the degree of nuclear stratification. LGD is diagnosed when the nuclei are confined to the basal half of the columnar cells. HGD is diagnosed when the nuclei are distributed haphazardly in the apical

Fig. 3. Dysplastic lesions with (A) tubulovillous and (B) villous architecture corresponding to dysplasia-associated lesions or masses (nonadenomalike).

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and basal halves. In most cases, the cytomorphology of the nuclei parallels the degree of nuclear stratification. Typically, the nuclei in LGD are uniformly enlarged, elongated, slender, and crowded, and have thin cell membranes, lightly clumped chromatin, and small nucleoli. Mitotic figures are mildly increased but morphologically typical. In HGD, one sees ovoid or round nuclei, high nuclear/cytoplasmic ratios, irregularly thickened nuclear membranes, hyperchromatic staining, coarse nuclear clumping, enlarged nucleoli, and atypical mitotic figures. A cribriform or solid growth pattern and dirty intraluminal necrosis imply a heightened likelihood of invasive cancer. Cellular polarity provides a reasonable, though less than perfect, gauge of biological derangement, since polarity is maintained even in some cancers. Biopsies with mixed features of LGD and HGD are generally assigned the higher grade. A recommendation by the IBD Morphology Study Group that a diagnosis of HGD be avoided if it occupies only one or two crypts is rather arbitrary. Therefore, any degree of HGD and its extent should be reported. Reactive and Indefinite Changes Non-neoplastic epithelium, whether normal or reactive, is categorized as negative for dysplasia. Reactive changes are common in the setting of erosions and active inflammation. Reparative colonocytes at the edges of healing erosions have a distinctive syncytial appearance and cuboidal or low columnar shapes, abundant amphiphilic or eosinophilic cytoplasm, vesicular nuclei, and prominent nucleoli. However, as they mature and differentiate, regenerative cells assume a columnar configuration and gradually produce cytoplasmic mucin. The nuclei may remain moderately enlarged and vesicular, with prominent nucleoli and frequent mitotic figures. As a result, resolving colitis may produce changes that overlap with dysplastic epithelium. To distinguish reactive from truly dysplastic epithelium, one must evaluate its cytologic features, the intensity of the surrounding inflammation, and whether or not there is surface maturation. Reactive epithelium is characterized by cytologic changes that are appropriate to the intensity of local inflammation and that dissipate near the surface (Fig. 4), whereas dysplastic epithelium is less sensitive to local variations in the inflammatory surroundings and undergoes little or no surface maturation. Certain features, such as, intense nuclear staining, macronucleoli, atypical mitotic figures, loss of cellular polarity, and dirty intraluminal necrosis are associated with dysplasia and not normally seen in reactive mucosa. It is important for pathologists to be informed when patients are treated with intravenous cyclosporine because there may be enhanced regenerative changes that mimic dysplasia [59]. Biopsies may be diagnosed as indefinite for dysplasia if they are technically inadequate or have features that cannot be classified with certainty as reactive or dysplastic. The latter problem is especially common in the setting of active colitis, but cytological deviations of uncertain significance can occur in quiescent colitis as well. The difficulty in distinguishing between reactive and dysplastic changes may be compounded by poor orientation of the biopsy,

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Fig. 4. Regenerative crypts characterized by an expanded basal zone with crowded, dark epithelial nuclei and maturation of epithelial cells toward the surface.

which precludes evaluation of surface maturation. Management guidelines for patients with a diagnosis of indefinite for dysplasia are discussed below. Macroscopic Classification of Dysplasia: Elevated Lesions Like cancer, dysplasia in IBD is grossly heterogeneous. It is classified broadly as either elevated or flat, depending on the presence or absence of an endoscopically visible lesion. Elevated lesions include pedunculated or sessile polyps, nodules, masses, clusters of polyps, smooth plaques, velvety patches, and verrucous excrescences. The sheer variety of appearances and their overlap with inflammatory lesions pose major challenges for endoscopists and account for much of the sampling variation associated with endoscopic surveillance. Although some early studies noted the existence of polypoid dysplasia in colectomy specimens of patients with UC and cancer, the first study to focus on elevated dysplasia as a distinct endoscopic entity was published by Blackstone and colleagues [60], who coined the term dysplasia-associated lesion or mass (DALM) as a collective designation for visible dysplastic lesions [60]. In their retrospective study, 7 of 12 DALMs were found to harbor invasive cancer at colectomy despite preoperative diagnoses of ‘‘severe’’ dysplasia in only 2, which implied that DALMs are an indication for colectomy regardless of the grade of dysplasia in biopsies. However, most of the DALMs in that study were irregular, nonresectable lesions, including some with endoscopic stigmata of cancer, making colectomy the only feasible form of intervention. The conclusions of this paper have been confirmed by later studies in which results compiled from multiple surveillance programs indicated a positive predictive value for cancer of greater than 40% upon colectomy [43,44]. Nevertheless, several long-term outcome studies have shown that not all elevated dysplastic lesions warrant equal concern [45,61–64]. In particular,

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patients with UC who have endoscopically resectable dysplastic polyps that resemble conventional adenomas (ie, adenomalike dysplastic polyps [adenomalike DALMs]) can be managed safely by endoscopic polypectomy alone if flat dysplasia is absent from the surrounding mucosa and remainder of the colon. For instance, in a study from The Mount Sinai Medical Center in New York, Rubin and colleagues [64] described a cohort of 48 subjects with UC or CD who underwent endoscopic polypectomy of 60 adenomalike dysplastic polyps located in diseased mucosa and 10 presumptively sporadic adenomatous polyps located in normal mucosa, all in the range of 0.5 to 3.0 cm in size. Two of the lesions showed HGD and 1 showed superficially invasive carcinoma. After a mean follow-up of 4.1 years (range, 0.8–9.6 years) and a total 103 subsequent colonoscopies, 48% of the subjects had polyp recurrences, but none were diagnosed with either cancer or flat dysplasia. Six patients referred for colectomy after multiple local recurrences had no significant findings other than a residual polyp [64]. Similar conclusions were reached in a case-control study by Engelsgjerd and colleagues from Brigham and Women’s Hospital in Boston. They followed a cohort of 24 subjects with UC harboring 26 adenomalike dysplastic polyps in diseased mucosa, 10 with sporadic adenomas and 49 non-IBD controls for a mean period of 3.1 to 3.5 years after polypectomy. In all, 58% of the patients with adenomalike polyps developed recurrent polyps, compared with 50% in the adenoma group and 39% in the controls. One patient with an adenomalike polyp was discovered to have flat LGD at colectomy, but none of the patients developed cancer [64]. In a later long-term follow-up study by the same group, based on an extended follow-up period of 6.8 years, 1 patient developed cancer. However, the diagnosis was made 7.5 years after the index polypectomy and the tumor occurred elsewhere in the colon unaccompanied by residual dysplasia in the colectomy specimen [63]. Most importantly, the polyps in these three studies were considered endoscopically indistinguishable from routine adenomas (ie, dome-shaped lesions with visually distinct borders; smooth, intact surfaces; and no stricturing or fixation to the colonic wall). A retrospective review of endoscopic surveillance at St. Mark’s Hospital by Connell and colleagues reported that of 8 subjects with UC who underwent endoscopic polypectomy of small (0.5–1.5 cm) dysplastic lesions, 3 whose polyps were surrounded by microscopic flat dysplasia all developed cancer, whereas 5 with no flat dysplasia fared well. However, a recent St. Mark’s report on surveillance in UC by Rutter and colleagues found that of the 32 subjects with 52 ‘‘adenomas’’ removed and followed for a median 4.7 years, 1 developed carcinoma in the same segment of colon where a polypectomy was performed 5 years earlier and another developed cancer in a different region from where a previous polypectomy had been performed. The significance of these observations is uncertain because the study was retrospective and the endoscopic criteria for adenomas were not defined. Indeed, one of the weaknesses of retrospective endoscopic studies was highlighted by a recent study that reported that the accuracy of endoscopic diagnosis of DALMs, adenomalike polyps, and inflammatory polyps was

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significantly higher among IBD experts than among nonexpert endoscopists in academic or private practice settings [65]. In summary, polypoid dysplastic lesions that occur in diseased mucosa are currently divided into two broad groups: (1) DALMs (nonadenomalike), which are either endoscopically nonresectable, have stigmata suggestive of malignancy, or are surrounded microscopically by flat dysplasia, and are considered an indication for colectomy; and (2) adenomalike dysplastic polyps (referred to as adenomalike DALMs by some investigators), which are endoscopically resectable and microscopically well circumscribed and may be managed conservatively [49,66–68]. Although removal of adenomalike dysplastic polyps by polypectomy should be followed by at least one precautionary short-term endoscopic reexamination before regular surveillance is resumed, the appropriate frequency of follow-up examinations in this circumstance has never been evaluated. Likewise, the appropriate management for locally recurrent dysplastic polyps is uncertain [68]. It is reasonable to infer that an adenomalike dysplastic polyp in a patient under 40 years old with extensive, longstanding colitis is related pathogenetically to the underlying colitis. Nevertheless, no clear evidence suggests that strict adherence to these recommendations carries any heightened risk of an adverse outcome. Dysplastic polyps encountered in unaffected regions of the colon, such as the proximal colon in patients with left-sided UC, are regarded as sporadic adenomas and managed accordingly, although the absence of surrounding occult colitis should be confirmed histologically [33]. Additionally, limited experience with pedunculated dysplastic polyps that have no dysplasia along their stalks suggests that they can also be managed as sporadic adenomas, even when encountered in diseased mucosa [49,66,67,69]. The distinction between DALMs (nonadenomalike) and adenomalike dysplastic polyps is mainly endoscopic because they share pathological features of dysplasia, they both exhibit mucosal expansion, and they both have a tubular, villous, or mixed architecture. (Fig. 5). Histological studies comparing various types of dysplastic polyps in IBD on the basis of individual morphological parameters have suggested that such features as a higher degree of asymmetry and architectural variability [70], a higher proportion of villous architecture, a greater density of stromal mononuclear inflammatory cells, an admixture of superficial dysplastic and nondysplastic crypts [69], and a ‘‘bottom-up,’’ as opposed to ‘‘top-down,’’ pattern of dysplasia [71], favor a UC-related dysplastic polyp, rather than a sporadic adenoma. However, in the final analysis, these criteria have not been rigorously validated and are not sufficiently objective to permit clear-cut distinctions among elevated dysplastic lesions in individual cases. Some molecular data support the hypothesis that DALMs (nonadenomalike) are distinct from sporadic adenomas [72–75]. For example, Selaru and colleagues [74], after training an artificial neural network with cDNA microarray expression profiles of 39 DALMs and sporadic neoplasms, were able to correctly classify 12 blinded lesions from the two groups on the basis of a limited set of 97 cDNA clones [74]. Odze and colleagues [73] reported that the relative rates of loss of heterozygosity of three tumor suppressor genes, 3p, the

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Fig. 5. Endoscopic appearance of elevated lesions in IBD. (A) Adenomalike dysplastic polyp characterized by its dome shape and well-defined borders. (B) DALM (nonadenomalike) characterized by its irregular shape and indistinct margins. (C) Pedunculated adenoma. (Courtesy of J. D. Waye, MD, New York, NY.)

adenomatous polyposis coli gene (APC) and p16, were lower among adenomalike dysplastic polyps and non-IBD adenomas compared with those among nonadenomalike DALMs, implying that most, if not all, adenomalike dysplastic polyps in IBD are in fact sporadic in origin [73]. Endoscopy and Flat (Occult) Dysplasia The term ‘‘flat’’ dysplasia refers to dysplasia detected in random biopsies from mucosa that endoscopically appears ordinary. Unfortunately, the term lacks uniform macroscopic criteria. Given the diversity of inflammatory changes encountered in the intestinal mucosa of patients with IBD, inconspicuous or

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subtle abnormalities are more prone to be overlooked or disregarded in IBD than they would be in a normal colon. Hence, a more appropriate term is ‘‘occult’’ dysplasia [76]. Indeed, there are retrospective endoscopic studies that suggest that truly flat dysplasia is less common than elevated dysplasia. For instance, Rutter and colleagues [77], based on a review of random and targeted surveillance biopsies performed among 525 subjects with UC over a period of 15 years, reported that 85 of 110 (77.3%) biopsies of dysplasia or cancer corresponded to macroscopically visible lesions, whereas 25 (22.7%) were invisible [77]. Similarly, Rubin and colleagues [78], reviewing surveillance biopsies performed among 46 subjects with UC over a period of 10 years, reported that 38 of 65 dysplastic lesions (58.5%) and 8 of 10 cancers (80.0%) were visible as 23 polyps and masses, one stricture, and 22 mucosal irregularities [78]. Corresponding prospective studies have not yet been reported. Endoscopic sampling error poses one of the most serious obstacles to successful outcome in endoscopic surveillance, as suggested by a high degree of inconsistency in the diagnoses resulting from serial surveillance examinations. Rubin and colleagues [79] calculated that 64 endoscopic biopsies are required to detect the highest grade of flat dysplasia in a typical colon with 95% confidence. However, only a minority of gastroenterologists adhere to a standard protocol of 4 biopsies per 10 cm [80–82]. Various means have been sought to improve both endoscopic sampling and visualization of dysplastic lesions in the hope they might improve diagnostic accuracy. Nearly 2 decades ago, Melville and colleagues [83] reported a prospective study of 82 patients with extensive UC who underwent cytologic brushing during the course of regular endoscopy. Of 5 subjects whose routine biopsies showed cancer, all were diagnosed with cancer or dysplasia by cytology, whereas 75 subjects with negative biopsies were given 72 negative and 3 indefinite cytologic diagnoses. The investigators further reported that dysplasia diagnosed in brushings appeared to be derived from larger regions of colon compared with dysplasia diagnosed in biopsies [83]. Despite these promising preliminary findings, there are no follow-up studies to evaluate this inexpensive, ‘‘lowtech,’’ technique. More recently, several reports have described the use of chromoendoscopy using indigo carmine or methylene blue dye, often combined with high-magnification endoscopy, to detect occult dysplastic lesions based on changes in the expected mucosal patterns of pits and innominate grooves (Fig. 6) [84–87] . One group has supplemented this technique with confocal endomicroscopy, a technique that permits en face microscopic visualization of the superficial region of colonic pits as a means of targeting biopsies to suspect areas [88]. All of these studies have reported an approximately three- to fivefold increased yield of dysplastic lesions compared with yields using conventional endoscopy. However, to assess whether detection of lesions will lead to commensurate clinical benefits, incorporation of these techniques into clinical practice will require additional studies, first to evaluate the additional burden of cost, time, and training, and second to determine the natural history and clinical significance of the type of lesions detected by these methods [76].

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Fig. 6. Chromoendoscopy in a patient with UC and LGD. The mucosa is shown (A) before and (B) after spraying with methylene blue dye. (Courtesy of J. F. Marion, MD, New York, NY.)

NATURAL HISTORY OF DYSPLASIA AND MANAGEMENT IMPLICATIONS The success of endoscopic surveillance depends on selection of appropriate endpoints for surgical intervention once dysplasia is diagnosed, which involves balancing the risks of potentially unnecessary surgery against those of contracting incurable cancer if intervention is delayed. Two key considerations are (1) the likelihood that a patient may be harboring undetected cancer at the time of examination and (2) the likelihood that dysplasia, whether recognized or not, will evolve into cancer during a defined follow-up interval. High-Grade Dysplasia and Dysplasia-Associated Lesion or Mass (Nonadenomalike) There is little disagreement that detection of HGD in biopsies carries sufficient risk of either concurrent cancer or progression to cancer to warrant colectomy as treatment barring any unusual mitigating circumstances. A synthesis of results from 10 surveillance programs, by Bernstein and colleagues in 1994, reported that 10 of 24 (42%) patients with HGD were found to have cancer upon colectomy, and 15 of 47 (33%) after follow-up. Similar results were reported in a recent summary of St. Mark’s Hospital’s 30-year-long surveillance program, where cancer was found in colectomy specimens of 5 of 11 (45.5%) patients diagnosed with HGD who opted for immediate surgery, and shortterm progression to cancer in 2 of 8 (25%) patients who opted for continued surveillance [6]. As discussed above, a diagnosis of nonadenomalike DALM is, likewise, regarded as an appropriate indication for colectomy regardless of whether preoperative biopsies reveal LGD or HGD. Flat Low-Grade Dysplasia The appropriate management of patients with flat (ie, non-DALM) LGD has been controversial. Some authorities advocate continued or accelerated

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surveillance [89,90], whereas others recommend immediate colectomy. Although our appreciation of the natural history of LGD is incomplete, a preponderance of recent data favors the latter view. The predictive value of flat LGD for cancer in patients who undergo colectomy has been reported as 19% in the compilation of results by Bernstein and colleagues [44] from 10 surveillance studies, as 22% in the more recent meta-analysis by Thomas and colleagues [43] of 20 surveillance studies (including 11 that were not part of the 1994 study), and 20% in a recent review of surveillance at St. Mark’s Hospital [6]. However, a much wider range of predictive values of flat LGD for progression to cancer, or HGD, among patients who opt for continued surveillance has been reported, from as low as 3% to 10% after 10 years of follow-up [89,90] to as high as 33% after 4 years [6] and 53% to 54% after 5 years [45,82]. Some of the lower progression rates among these studies might be accounted for by diagnoses originally rendered before the 1983 publication of diagnostic criteria by the IBD Morphology Study Group, especially those diagnoses made without a separate category for equivocal biopsies. Indeed, a 1994 St. Mark’s study found that reclassification of pre-1983 biopsies by two pathologists based on the newer scheme, led to a change in 5-year progression rates among patients with flat LGD from 16% to 54% [45]. Three additional points should be kept in mind. First, LGD detected at an initial screening endoscopy (prevalent dysplasia) may carry a higher risk than LGD detected during periodic surveillance (incident dysplasia) because screening is associated with a longer opportunity for neoplastic changes to accumulate, but is subject to a similar degree of endoscopic sampling error [91,92]. Second, although published surveillance guidelines recommend that patients with pathologically confirmed dysplasia who opt against colectomy should undergo surveillance on an accelerated schedule until at least two examinations are negative [47,48], experience has shown that once dysplasia, including LGD, has been diagnosed, dysplasia or cancer will usually be found at some later time despite multiple negative examinations in the interim [82,91]. Third, according to the results of a study from The Mount Sinai Hospital, there may be no significant difference in the rates of progression from flat LGD to HGD or cancer whether LGD is detected in one or in multiple biopsies [82]. Histogenesis of Cancer from Low-Grade Dysplasia The rationale for an aggressive approach to flat LGD has recently been reinforced by pathological evidence of direct progression from LGD to cancer without an obligate intermediate stage of DALM or HGD. Levi and Harpaz [93] described a series of 21 well-differentiated tubuloglandular adenocarcinomas diagnosed in 17 of 149 (11%) consecutively evaluated IBD colectomy specimens at The Mount Sinai Hospital. Nineteen cancers (92%) were accompanied exclusively by either low-grade, or indefinite, dysplasia. All 21 (100%) presented as a direct histological extension of either low-grade dysplastic crypts or, in one case, of crypts classified as indefinite for dysplasia, and there were virtually no cytological differences between the invasive glands and the

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overlying dysplasia (Fig. 7). Five (24%) cancers were flat, with no corresponding gross lesion evident even on retrospective review of the colectomy specimen. Although low grade at inception, over half of the cancers underwent histologic progression to conventional cancer. Low-grade tubuloglandular adenocarcinomas, therefore, represent a histogenetic link between LGD and invasive cancer (Fig. 8). Indefinite for Dysplasia The natural history of mucosal changes interpreted as indefinite for dysplasia is poorly defined. In a cohort study from The Mount Sinai Hospital published in abstract form, a significantly higher proportion of subjects whose biopsies were interpreted as indefinite for dysplasia, 13 of 48 (27%), progressed to neoplasia (9 LGD, 3 HGD, and 1 cancer) during a mean 54-month follow-up interval, compared with 6 of 48 (13%) matched control subjects without dysplasia (5 LGD, 1 HGD). The corresponding actuarial 5-year progression rates were 30% and 12%, respectively [94]. The results imply that patients with a biopsy diagnosis of indefinite for dysplasia require closer follow-up than patients with negative biopsies. However, because there may be diverse causes for uncertainty in biopsy interpretation, the appropriate frequency of follow-up examinations should be determined empirically based both on the pathologist’s concerns and on individual clinical factors, such as the endoscopic findings and the patient’s cancer-risk profile. Thus, whereas the usual recommendation is to repeat the examination within 3 to 6 months [5,49], there may be situations where the degree of histologic, endoscopic, or clinical concern is high enough to warrant an immediate reexamination. Diagnostic Variability and Application of Biomarkers One of the limitations of surveillance is that pathologists, including gastrointestinal pathology specialists, show only fair performance in grading dysplasia in

Fig. 7. Low-grade tubuloglandular adenocarcinoma arising from LGD. Low-grade dysplastic colonic crypts (top) give rise to invasive glands with nearly identical cytological features (bottom).

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DALM Negative for dysplasia

Indefinite for dysplasia

Low-grade dysplasia

Conventional adenocarcinoma High-grade dysplasia

Low-grade tubuloglandular adenocarcinoma Fig. 8. Cancer histogenesis in IBD usually involves intermediate stages of HGD or DALM (solid arrows). Alternatively, low-grade tubuloglandular carcinomas in IBD may arise directly from LGD or indefinite dysplasia and progress to conventional cancer (dashed arrows).

IBD in interobserver variability studies. As a rule, the highest kappa values are attained for diagnoses at the two extremes of the spectrum (ie, negative for dysplasia and HGD) and the lowest for the middle categories (ie, LGD and indefinite for dysplasia) [95–99]. Studies evaluating expert performance using static [99] and dynamic telepathology [100], vehicles that might provide access to expert consultative services, have yielded lower kappa values than those using corresponding glass slides. Riddell and colleagues, [52] anticipating in 1983 that interobserver variation might be an obstacle to the application of their newly proposed classification system for dysplasia, recommended that any diagnosis that might significantly impact patient management should be reviewed by a second pathologist. Obviously, the availability of objective immunohistochemical markers of dysplasia would go a long way toward overcoming this problem. Of markers that have been studied, most are related in some way to cancer development, either as tumor-associated antigens (eg, sialyl-Tn) [101,102] or as markers of altered cell proliferation/apoptosis (eg, Ki-67, bcl-2, survivin, YB-1) [103–105], intercellular adhesion (eg, beta catenin, E-cadherin) [75,106], or tumor suppression (eg, p53, p21) [72,107,108]. However, none have met the standards of validation needed for application in clinical practice. Even the marker studied most extensively, p53 overexpression, correlates only partly with the presence of mutations, and, furthermore, mutations can occur in inflamed, but nondysplastic, mucosa [109,110]. A recently reported marker worthy of note is alpha-methylacyl-CoA racemase (AMACR), an enzyme expressed immunohistochemically in various normal nongastrointestinal tissues, in prostatic neoplasia, and in most colorectal adenomas and adenocarcinomas. In a study by Dorer and Odze [111], AMACR was not expressed in any IBD biopsies considered negative for dysplasia, but was expressed in 25 of 26 (96%) foci of LGD, 8 of 10 (80%) foci of HGD, 10 of 14 (71%) carcinomas, and 1 of 7 (14%) foci considered to be indefinite for dysplasia.

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MOLECULAR PATHOGENESIS OF DYSPLASIA AND CANCER IN INFLAMMATORY BOWEL DISEASE There is little doubt that chronic inflammation plays a decisive role in the development of colorectal dysplasia and cancer in IBD. On a clinical level, the link between inflammation and neoplasia in IBD is implied by the classical cancer risk factors of disease extent and duration, by emerging evidence of an association between cancer risk and histological intensity of inflammation [18,20], and by evidence of chemopreventive benefit conferred by 5-aminosalicylates and possibly other anti-inflammatory drugs [10]. It is further supported by rodent and primate models of colitis demonstrating susceptibility to colorectal dysplasia and cancer. However, it is also clear that the development of dysplasia is preceded by, and is an outgrowth of, a host of histologically silent genetic derangements, and that some of the key mechanisms responsible for genomic instability predisposing to tumorigenesis are already well established by the time dysplasia is clinically detectable. Carcinogenesis in IBD is driven by a succession of mutational events that preferentially involve certain tumor-associated genes. Analogous to sporadic colorectal cancer, the patterns of gene alterations observed in approximately 85% of cancers in IBD correspond to a chromosomal instability pathway characterized by widespread gains and losses of genomic material and by inactivation of key tumor suppressor genes. The patterns associated with the other 15% of cancers in IBD correspond to a microsatellite instability pathway characterized by defective DNA mismatch repair mechanisms, instability of tandem repeat DNA sequences, and mutations of genes containing such sequences in their coding sequences. Despite the parallels between sporadic and IBD-associated neoplasms, as well as various shared genetic alterations, IBD-associated neoplasms differ in the timing and prevalence of molecular events pertaining to specific tumor-related genes [8]. For instance, inactivation of APC, among the most common and early events in sporadic carcinogenesis, and mutations of k-ras, which are common in conventional adenomas, are infrequent events in IBD. Mutations, or allelic loss, of p53, which typically herald the transition from sporadic adenoma to cancer, occur not only in IBD-associated dysplasia, but also in inflamed, non-dysplastic mucosa, albeit at a lower frequency [109,110]. Microsatellite instability in IBD-associated cancer is associated with patterns of mutational and promoter methylation profiles that are different from those for sporadic cancers [112]. Another important distinction between sporadic and IBD-associated neoplasia is that the latter typically occupies relatively large fields of dysplastic mucosa surrounded by even larger fields of genetically unstable mucosa, unlike sporadic adenomatous polyps in which neoplasia affects a confined region. As shown over a decade ago in studies that mapped mucosal aneuploidy in UC as a marker of molecular derangements, the appearance of aneuploid clones of colonocytes may precede and herald the appearance of dysplasia in IBD. Once established, aneuploidy may expand over time to involve increasingly large regions of mucosa and give rise to dysplasia [79,113].

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The normal cellular responses to inflammatory stresses, such as environmental exposure to reactive oxygen and nitrogen species, form a complex and dynamic system. The specific means by which these mechanisms might become inactivated, subverted, or overwhelmed in IBD and lead to genomic instability are equally complex and incompletely understood. Recent reviews have discussed many of these phenomena in detail, including altered telomere dynamics, increased expression of genes encoding cyclooxygenase-2 and nitric oxide synthase, mutagen formation caused by oxidative modification of DNA bases, and constitutive induction of p53-related DNA repair mechanisms bypassing the p53-G1 cell cycle checkpoint (Fig. 9) [114,115]. Once conditions have produced genomic instability, when, and by what means, do potentially neoplastic clones emerge and propagate within the colon to produce dysplasia and cancer? DNA fingerprinting, a technique that enables

Fig. 9. Proposed model by which inflammation in IBD results in carcinogenesis. MMR, mismatch repair; NSAID, nonsteroidal anti-inflammatory drug. (From Itzkowitz SH, Yio X. Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am J Physiol Gastrointest Liver Physiol 2004;287(1):G10, with permission.)

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detection of genome-wide gains and losses of genomic material, has provided insights when applied to dysplastic tissues in IBD [116,117]. In a study based on single colonic crypts from colons of patients with HGD, Chen and colleagues [117] showed that 10% to 20% of the crypt DNA was mutated at any single point in time. Surprisingly, neither the magnitude of the mutational load nor the pattern of genomic alterations differed between dysplastic and non-dysplastic crypts, suggesting that instability is widespread and precedes the development of dysplasia. Comparisons of the fingerprints of single mutated crypts with those of larger groups of surrounding crypts further suggested that approximately half of all altered crypts are in a state of clonal expansion. The investigators surmised that to maintain the overall mutational load in a steady state, at least in this precancerous stage, countervailing mechanisms must be in place, such as increased rates of luminal sloughing or cell death. The fact that such high mutational loads do not necessarily have any significant impact on cellular morphology indicates that their effects are mixed, with certain types of mutations being insignificant or silent, others conferring a growth advantage but no morphological changes, and still others giving rise to the altered morphology of dysplasia. In summary, this and other supporting evidence [8,115] suggest that dysplastic epithelium emerges from a large pool of colonocytes that have sustained significant genetic damage and exist in a quasi-steady state of mutation and clonal expansion partially balanced by selection pressures and cell loss. The mechanisms by which these changes are propagated are incompletely understood, but there is evidence that crypt fission may play a role in clonal expansion [117]. SUMMARY Dysplasia is an intermediate stage in the progression from inflammation to cancer in patients with IBD. Clinically, dysplasia is used to define appropriate endpoints for colectomy in high-risk patients undergoing endoscopic surveillance. Although the evidence that surveillance prolongs survival in this group is circumstantial, it is currently the only credible alternative to prophylactic colectomy for high-risk patients. The success of surveillance can be maximized by adherence of gastroenterologists to recommended procedural guidelines, adherence of pathologists to standardized histological criteria and nomenclature, and a joint commitment to close clinical–pathological communication. Technical enhancements to conventional endoscopy hold promise of improved efficiency and accuracy, but require further evaluation. Recognition that dysplasia is preceded by substantial genetic damage to the mucosa suggests a future role for molecular-based testing as a means of risk stratification and early detection of neoplasia in IBD. Improved understanding of the relationship between neoplasia and chronic inflammation at the cellular level is needed to unravel the pathogenesis of cancer in IBD, and is likely to have far-reaching benefits in the basic science and clinical management of other chronic inflammatory disorders with neoplastic potential.

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[47] Barthet M, Gay G, Sautereau D, et al. Endoscopic surveillance of chronic inflammatory bowel disease. Endoscopy 2005;37(6):597–9. [48] Eaden JA, Mayberry JF. Guidelines for screening and surveillance of asymptomatic colorectal cancer in patients with inflammatory bowel disease. Gut 2002;51(Suppl 5):V10–2. [49] Itzkowitz SH, Present DH. Consensus conference: Colorectal cancer screening and surveillance in inflammatory bowel disease. Inflamm Bowel Dis 2005;11(3):314–21. [50] Friedman S, Rubin PH, Bodian C, et al. Screening and surveillance colonoscopy in chronic Crohn’s colitis. Gastroenterology 2001;120(4):820–6. [51] Collins PD, Mpofu C, Watson AJ, et al. Strategies for detecting colon cancer and/or dysplasia in patients with inflammatory bowel disease. Cochrane Database Syst Rev 2006;(2) CD000279. [52] Riddell RH, Goldman H, Ransohoff DF, et al. Dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications. Hum Pathol 1983;14(11):931–68. [53] Hamilton SR, Aaltonen LA. In: Pathology and Genetics. Tumours of the Digestive System, Vol 2. Lyon: IARC; 2000. [54] Schlemper RJ, Riddell RH, Kato Y, et al. The Vienna classification of gastrointestinal epithelial neoplasia. Gut 2000;47(2):251–5. [55] Andersen SN, Lovig T, Clausen OP, et al. Villous, hypermucinous mucosa in long standing ulcerative colitis shows high frequency of K-ras mutations. Gut 1999;45(5):686–92. [56] Antonioli DA, Covell LM, Goldman H. Villous epithelial regeneration and dysplasia in ulcerative colitis. Arch Pathol Lab Med 1977;101(4):222–3. [57] Rubio CA, Johansson C, Slezak P, et al. Villous dysplasia. An ominous histologic sign in colitic patients. Dis Colon Rectum 1984;27(5):283–7. [58] Rubio CA, Befrits R, Jaramillo E, et al. Villous and serrated adenomatous growth bordering carcinomas in inflammatory bowel disease. Anticancer Res 2000;20(6C):4761–4. [59] Hyde GM, Jewell DP, Warren BF. Histological changes associated with the use of intravenous cyclosporin in the treatment of severe ulcerative colitis may mimic dysplasia. Colorectal Dis 2002;4(6):455–8. [60] Blackstone MO, Riddell RH, Rogers BH, et al. Dysplasia-associated lesion or mass (DALM) detected by colonoscopy in long-standing ulcerative colitis: an indication for colectomy. Gastroenterology 1981;80(2):366–74. [61] Medlicott SA, Jewell LD, Price L, et al. Conservative management of small adenomata in ulcerative colitis. Am J Gastroenterol 1997;92(11):2094–8. [62] Engelsgjerd M, Farraye FA, Odze RD. Polypectomy may be adequate treatment for adenoma-like dysplastic lesions in chronic ulcerative colitis. Gastroenterology 1999;117(6):1288–94 [discussion 1488–1291]. [63] Odze RD, Farraye FA, Hecht JL, et al. Long-term follow-up after polypectomy treatment for adenoma-like dysplastic lesions in ulcerative colitis. Clin Gastroenterol Hepatol 2004;2(7):534–41. [64] Rubin PH, Friedman S, Harpaz N, et al. Colonoscopic polypectomy in chronic colitis: conservative management after endoscopic resection of dysplastic polyps. Gastroenterology 1999;117(6):1295–300. [65] Farraye FA, Waye JD, Heeren TC, et al. Variability in the diagnosis and management of adenoma-like and non-adenoma like DALMs in patients with ulcerative colitis. Gastrointest Endosc, in press. [66] Harpaz N. Adenoma-like dysplastic polyps in inflammatory bowel disease. Pathol Case Rev 2004;9(4):135–41. [67] Friedman S, Odze RD, Farraye FA. Management of neoplastic polyps in inflammatory bowel disease. Inflamm Bowel Dis 2003;9(4):260–6. [68] Itzkowitz S. Polypoid dysplasia in inflammatory bowel disease: removing the bumps in the road. Inflamm Bowel Dis 2006;12(9):919–20.

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[90] Lim CH, Dixon MF, Vail A, et al. Ten year follow up of ulcerative colitis patients with and without low grade dysplasia. Gut 2003;52(8):1127–32. [91] Woolrich AJ, DaSilva MD, Korelitz BI. Surveillance in the routine management of ulcerative colitis: the predictive value of low-grade dysplasia. Gastroenterology 1992;103(2):431–8. [92] Nugent FW, Haggitt RC, Gilpin PA. Cancer surveillance in ulcerative colitis. Gastroenterology 1991;100(5 Pt 1):1241–8. [93] Levi GS, Harpaz N. Intestinal low-grade tubuloglandular adenocarcinoma in inflammatory bowel disease. Am J Surg Pathol 2006;30(8):1022–9. [94] Jani N, Kornbluth A, Croog V, et al. The fate of indefinite dysplasia in ulcerative colitis. Gastroenterology 2003;124(4 Suppl 1):A649–50. [95] Dixon MF, Brown LJ, Gilmour HM, et al. Observer variation in the assessment of dysplasia in ulcerative colitis. Histopathology 1988;13(4):385–97. [96] Dundas SA, Kay R, Beck S, et al. Can histopathologists reliably assess dysplasia in chronic inflammatory bowel disease? J Clin Pathol 1987;40(11):1282–6. [97] Eaden J, Abrams K, McKay H, et al. Inter-observer variation between general and specialist gastrointestinal pathologists when grading dysplasia in ulcerative colitis. J Pathol 2001;194(2):152–7. [98] Melville DM, Jass JR, Morson BC, et al. Observer study of the grading of dysplasia in ulcerative colitis: comparison with clinical outcome. Hum Pathol 1989;20(10): 1008–14. [99] Odze RD, Goldblum J, Noffsinger A, et al. Interobserver variability in the diagnosis of ulcerative colitis-associated dysplasia by telepathology. Mod Pathol 2002;15(4): 379–86. [100] Odze RD, Tomaszewski JE, Furth EE, et al. Variability in the diagnosis of dysplasia in ulcerative colitis by dynamic telepathology. Oncol Rep 2006;16(5):1123–9. [101] Karlen P, Young E, Brostrom O, et al. Sialyl-Tn antigen as a marker of colon cancer risk in ulcerative colitis: relation to dysplasia and DNA aneuploidy. Gastroenterology 1998;115(6):1395–404. [102] Itzkowitz SH, Young E, Dubois D, et al. Sialosyl-Tn antigen is prevalent and precedes dysplasia in ulcerative colitis: a retrospective case-control study. Gastroenterology 1996;110(3):694–704. [103] Fogt F, Poremba C, Shibao K, et al. Expression of survivin, YB-1, and KI-67 in sporadic adenomas and dysplasia-associated lesions or masses in ulcerative colitis. Appl Immunohistochem Mol Morphol 2001;9(2):143–9. [104] Wong NA, Mayer NJ, MacKell S, et al. Immunohistochemical assessment of Ki67 and p53 expression assists the diagnosis and grading of ulcerative colitis–related dysplasia. Histopathology 2000;37(2):108–14. [105] Noffsinger AE, Miller MA, Cusi MV, et al. The pattern of cell proliferation in neoplastic and nonneoplastic lesions of ulcerative colitis. Cancer 1996;78(11):2307–12. [106] Azarschab P, Porschen R, Gregor M, et al. Epigenetic control of the E-cadherin gene (CDH1) by CpG methylation in colectomy samples of patients with ulcerative colitis. Genes Chromosomes Cancer 2002;35(2):121–6. [107] Arai N, Mitomi H, Ohtani Y, et al. Enhanced epithelial cell turnover associated with p53 accumulation and high p21WAF1/CIP1 expression in ulcerative colitis. Mod Pathol 1999;12(6):604–11. [108] Harpaz N, Peck AL, Yin J, et al. P53 protein expression in ulcerative colitis–associated colorectal dysplasia and carcinoma. Hum Pathol 1994;25(10):1069–74. [109] Hussain SP, Amstad P, Raja K, et al. Increased p53 mutation load in noncancerous colon tissue from ulcerative colitis: a cancer-prone chronic inflammatory disease. Cancer Res 2000;60(13):3333–7. [110] Holzmann K, Klump B, Borchard F, et al. Comparative analysis of histology, DNA content, p53 and Ki-ras mutations in colectomy specimens with long-standing ulcerative colitis. Int J Cancer 1998;76(1):1–6.

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Gastrointestinal Polyposes: Clinical, Pathological and Molecular Features Jeremy R. Jass, MD, DSc, FRCPath, FRCPA Department of Cellular Pathology, St Mark’s Hospital & Imperial College, Watford Road, Harrow, Middlesex HA1 3UJ, UK

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condition that is rare, associated with large numbers of colorectal polyps (too many to be managed by nonsurgical approaches), precancerous, associated with life-threatening systemic manifestations (eg, fibromatosis), affects young subjects, and is inherited offers major and multilayered challenges: therapeutic, social, intellectual and emotional. It was in recognition of these daunting facts relating to the condition familial adenomatous polyposis (FAP) that a small group of surgeons, geneticists, pathologists and basic scientists established the Leeds Castle Polyposis Group (LCPG) in 1985 [1]. The focus of this group’s interest was FAP. Many seminal insights followed in quick succession: the cloning of the APC gene bringing the possibility of genetic testing, the appreciation of the central importance of the APC gene in the evolution of sporadic colorectal cancer, and the introduction of targeted chemoprevention. Other important spin-offs have been the dissemination of understanding of the condition FAP, the expansion of cancer genetics as a clinical discipline, and the fostering of productive links between clinicians and basic scientists. Parallel developments occurred through the work of the International Collaborative Group on Hereditary Non-Polyposis Colorectal Cancer (ICG-HNPCC) [2]. These groups eventually fused as the International Society for the Investigation of Gastrointestinal Hereditary Tumors (InSIGHT). Although the growth of understanding of both FAP and HNPCC (Lynch syndrome) has been remarkable, these two conditions have overshadowed other forms of intestinal polyposis and/or hereditary gastrointestinal (GI) cancer. Traditionally, pathologists have contributed to the advance of medical knowledge by demarcating clinico–pathological entities. This approach has proceeded through meticulously documenting morphological features, using this information to split off potential new pathological entities, and bringing meaning to those entities through clinico–pathological correlation. Although the diagnosis of classical FAP generally has been straightforward (based on the discovery of a prodigious number of adenomas), the phenotypes of other E-mail address: [email protected]

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intestinal polyposes are more complex: more variable polyp numbers, presence of flat adenomas, presence of two or more types of polyp in the same condition, and presence of polyps with mixed or ambiguous morphologies. It is a fact that human tissues are capable of only a limited repertoire of morphological responses to a wide range of stimuli, whether genetic or environmental. For example, the histologic features that typify juvenile and hyperplastic polyps may co-occur in inflammatory polyps. This emphasizes the importance of establishing an etiological diagnosis for intestinal polyposes. Even in the case of inflammatory polyposis complicating Crohn’s disease or ulcerative colitis, the ultimate cause is likely to be a genetic predisposition to a dysregulated inflammatory response. The link between tissue overgrowth and inflammation should come as no surprise now that the role of inflammatory signaling pathways and epithelial:stromal interactions increasingly is recognized as fundamental in tumorigenesis. This article focuses mainly on the noninflammatory epithelial polyposes, particularly the diagnostically important morphological and molecular features of the more recently recognized and/or more poorly understood conditions. One of the most important, but often neglected, of these is hyperplastic polyposis. FAMILIAL ADENOMATOUS POLYPOSIS Clinical Manifestations FAP accounts for less than 1% of colorectal cancers. This low figure is in part because of the rarity of FAP (it occurs in approximately 1 in 8000 subjects) and in part because of cancer prevention in known affected cases. An association with extracolorectal features (sebaceous cysts, bone tumors, and fibromatosis) first was noted by Gardner [3]. The list of extracolorectal features subsequently increased to include peri-ampullary adenoma and carcinoma, medulloblastoma, papillary carcinoma of thyroid, hepatoblastoma, fundic gland polyps and carcinoma of the stomach, and congenital hypertrophy of retinal pigmented epithelium (CHRPE) [4]. The presence of more than 100 colorectal adenomas traditionally has been used to distinguish the autosomal-dominant condition FAP from patients who have multiple adenomas [5]. It is wise to obtain a tissue diagnosis even when innumerable colorectal polyps are discovered in a child of an affected parent, because children may develop an unrelated and self-self lymphoid polyposis. Although most colorectal polyps are typical adenomas, hyperplastic polyps and serrated adenomas occasionally may present [6]. Genotype–Phenotype Correlations The identification of the causative gene occurred through the classical sequence of finding a large interstitial deletion in chromosome 5q in a subject with Gardner syndrome [7], confirming the 5q21 locus through linkage analysis [8], identifying the gene APC by positional cloning, and finally demonstrating a truncating mutation of APC in affected subjects [9]. The multifunctional APC protein is large and comprises several motifs and domains, allowing it

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to oligomerize and/or interact with multiple molecules, including b-catenin, a-catenin, GSK3b, axin, conductin, and tubulin [4]. Although the diagnosis of FAP may be confirmed by demonstration of a germline APC mutation, truncating APC mutations are found in only 70% to 90% of individuals or families with an FAP phenotype. Truncating mutations have been found throughout the APC gene. Most mutations are fully penetrant, but may be associated with differing severity of colorectal polyposis and differing risks of extracolorectal manifestations [10]. Mutations in the central region of APC (codons 1290 to 1400) are associated with a severe polyposis phenotype. Two codons (1061 and 1309) are mutational hotspots and account for 11% and 17% of all germline mutations, respectively. CHRPE is associated with mutations between codons 457 and 1444, while jaw osteomas and fibromatoses are more prevalent in patients with mutations occurring after codon 1400 [4]. The FAP phenotype may present in subjects without a family history of this condition. This may be because of a new mutation (which accounts for one in four new diagnoses), nonpaternity, adoption, or denial of family history. The FAP phenotype also may occur in subjects without a germline APC mutation. One explanation is the recently recognized autosomal recessive condition known as MUTYH (MYH)-associated polyposis (MAP). Just as some subjects with more than 100 colorectal adenomas may not carry an APC germline mutation, the finding of less than 100 colorectal adenomas may sometimes be explained by an APC germline mutation. One explanation is investigation of an at-risk subject at an early age, before full expression of the phenotype. The fully developed FAP phenotype usually is observed after puberty. A second explanation is an APC germline mutation that gives rise to an attenuated phenotype (attenuated FAP, AFAP). ATTENUATED FAMILIAL ADENOMATOUS POLYPOSIS, LYNCH SYNDROME, AND FLAT ADENOMAS A large multiple adenoma family was characterized by the finding of less than 100 adenomas per subject [11]. The adenomas mainly were located proximally and tended to be flat. Colorectal cancers were relatively late in onset. There was a lack of extracolorectal features. This family originally was included with a set of Lynch syndrome/HNPCC kindreds (despite the adenoma multiplicity) and probably led to the concept of flat adenomas being characteristic of Lynch syndrome/HNPCC [12]. Because adenomas in Lynch syndrome are more likely to be proximally located [13], and proximal adenomas are more likely than distal adenomas to be flat [14], there is probably a weak connection between flat adenomas and both AFAP and Lynch syndrome [15]. The more definitive connection, however, is between flatness and anatomic (proximal) location [14]. Linkage to 5q21 and the presence of pathogenic mutations in APC identified the flat adenoma syndrome as a form of AFAP and excluded the diagnosis of Lynch syndrome [11,16]. In fact, it is unusual to find multiple adenomas in subjects with Lynch syndrome [17]. In a series of 22 adenoma-positive patients with a proven

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germline mutation in a DNA mismatch repair, most subjects had only one adenoma, and only three had three adenomas [13]. The AFAP phenotype is associated with germline mutations in specific regions of the APC gene: 59 in codons 78 to 167, 39 in codons 1581 to 2843, and in exon 9 [4]. In fact, the concept that AFAP is characterized by less than 100 adenomas has been questioned following the use of dye spray during the colonoscopic examination [18]. Four subjects had a family history appropriate for FAP, but had less than 20 adenomas on standard colonoscopy. In each of the subjects, the use of dye spray highlighted over 1000 adenomas. These were sufficiently large to be recognized and counted within the subsequent colectomy specimen. Nevertheless, even smaller adenomas (microadenomas) are likely to have been overlooked. In a more recent series of genetically proven cases of AFAP, 12 of 24 subjects had more than 100 adenomas, and seven of these had more than 500 adenomas [19]. Although true AFAP does exist, these findings raise the possibility that at least some cases of AFAP may be classical FAP, in which most adenomas remain small or microscopic. It is possible that the tumorigenic effect of APC germline mutations associated with this phenotype is sufficient for adenoma initiation, but not adenoma growth. It has been suggested that the paucity of adenomas in AFAP is based on the requirement for a third hit that increases the pathogenicity of the germline mutation [19]. It is also possible, however, that the effect of the third hit is to enhance the negative regulation of Wnt signaling and, thereby, allow existing microadenomas to increase in size. MUTYH (MYH)-ASSOCIATED POLYPOSIS This type of polyposis was initially documented in a Welsh kindred in which three siblings had multiple colorectal adenomas and carcinomas, but lacked a germline APC mutation [20]. Somatic mutations in the colorectal neoplasms showed a higher than expected rate of G:C to T:A transversions, and this in turn indicated a failure to repair a promutagenic product of oxidative DNA damage: 8-oxo-7,8-dihydro29deoxyguanosine. The siblings were found to have biallelic germline mutations in one of three candidate DNA repair genes: MUTYH (MYH), which maps to chromosome 1p32-34. The other candidate genes were MTH1 and OGG1. All three siblings were compound heterozygotes for the missense mutations Y165C and G382D in MUTYH [20]. This was not an isolated finding but was confirmed in other subjects of European origin and with multiple colorectal adenomas ranging from five to several hundred [21]. Although the mutations Y165C and G382D are the most common in Europeans, a Y90X mutation occurs in subjects of Pakistani origin, whereas an E466X mutation has been found in subjects of Indian origin [22]. An in-frame deletion nt1395-7delGGA has been described in Italian populations [23]. MAP may be difficult to differentiate clinically from classical FAP, and it has been diagnosed in 7.5% of classical, but APC mutation-negative, FAP (more than 100 adenomas) [21]. MAP may present with several hundred adenomas, but not with the many thousands of adenomas that characterize the more

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severe form of FAP. In view of the lower numbers of colorectal adenomas, and presentation in the mid-50 s, MAP is particularly likely to mimic AFAP. MAP accounted for 6.4% of subjects with multiple colorectal adenomas (5 to 100) [21]. MAP is an autosomal recessive condition, and consequently it presents as a sporadic disorder, although it may affect two or more siblings. Somatic G:C to T:A transversion mutations may affect KRAS and APC [24]. One therefore might expect to observe hyperplastic polyps initiated by KRAS mutation in MAP (and adenomas), and this might assist in the distinction between MAP versus classical and attenuated FAP. Hyperplastic polyps have been noted in MAP [25]. These are usually small and distally located. In one instance, the number of hyperplastic polyps was sufficiently great to suggest a diagnosis of hyperplastic polyposis [26]. In surgical specimens containing colorectal cancers and multiple adenomas, pathologists are likely to sample only the most advanced-appearing lesions, and the presence of hyperplastic polyps may be underappreciated. A policy of deliberately targeting small polyps should be considered under such circumstances, because the finding of multiple small hyperplastic polyps would be unusual in both classical and attenuated FAP. MULTIPLE ADENOMAS The finding of multiple colorectal adenomas (5 to 100) may be explained by either AFAP or MAP, but most affected subjects will have neither APC nor MUTYH germline mutations. Multiple colorectal adenomas have been described in patients with acromegaly [27], hereditary mixed polyposis syndrome, and Bloom’s syndrome [28]. Three independent studies have identified an adenoma susceptibility locus on chromosome 9q22.32 [29–31]. At endoscopy, or when examining a surgical specimen with multiple polyps, it is advisable to sample a subset of the smaller polyps to exclude a diagnosis of hyperplastic polyposis. Some cases simply may represent the high end of the range of sporadic adenomas (although this does not preclude a genetic explanation). PEUTZ-JEGHERS SYNDROME This is an autosomal-dominant condition characterized by the development of hamartomatous polyps throughout the GI tract and muco–cutaneous pigmentation. The distinction from other polyposes is not usually problematic, but the clinical diagnosis may be less straightforward in formes frustes. Polyps are largest and most numerous in the small bowel, and the condition typically presents with obstruction or intussusception in the second or third decade of life. The polyps are typically multilobated with a papillary surface and resemble tubulovillous adenomas grossly. The epithelium covers a core of arborizing smooth muscle. There may be misplacement of mucin-secreting epithelium into the submucosa, muscularis propria, and beyond the bowel wall, causing mimicry with well-differentiated or mucinous adenocarcinoma [32]. Dysplasia is uncommon, but there is an increased risk of colorectal cancer and extracolonic malignancy. The principal sites of extracolonic tumors are the pancreas, stomach, breast, ovary (sex cord tumors), testis (Sertoli cell tumors), and cervix

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(adenoma malignum). Mutations in the LKB1 (STK11) gene on chromosome 19p13 are found in approximately 50% of affected families. The phenotype is more severe in families with a truncating mutation than a missense mutation, or when no mutation is found in LKB1 [33]. COWDEN AND BANNAYAN-RILEY-RUVALCABA SYNDROMES Cowden syndrome is a rare autosomal-dominant condition named after the family in which it first was described. It is characterized by the presence of GI, oral and cutaneous hamartomas, tumors of breast and thyroid, autoimmune thyroiditis, microcephaly, and mental impairment. The colorectal polyps have been described as hamartomatous, inflammatory, and occasionally adenomatous [34]. There is little, or no, risk of colorectal malignancy, however. There may be confusion with juvenile polyposis [35], but Cowden polyps of the colorectum are small and nodular, and the expanded lamina propria is more myofibroblastic than edematous (Fig. 1). Scattered stromal ganglion cells are not uncommon, and rare reports of juvenile polyposis with ganglioneuromatous features [36,37] may represent Cowden syndrome. The underlying mechanism is germline mutation of the PTEN gene on chromosome 10q23 [38]. PTEN mutations also account for the Bannayan-Riley-Ruvalcaba syndrome, which is phenotypically similar to Cowden syndrome, but is associated with penile speckled pigmentation and presents in infancy [39]. JUVENILE POLYPOSIS Juvenile polyps are classified as hamartomas, in which there is overgrowth of an edematous lamina propria containing glands showing cystic dilatation (mucous retention cysts) and occasionally a serrated architecture. The polyps are typically spherical with an eroded surface epithelium. Single, or small numbers of juvenile polyps are a relatively common occurrence within the colorectum of

Fig. 1. Cowden syndrome polyp of colorectum. Crypts are arranged in a loose lobular pattern and are embedded in a concentrically arranged fibrotic stroma containing scattered ganglion cells. Haematoxylin and eosin.

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young children. A rare form of juvenile polyposis occurs in infancy and is associated with diarrhea, hemorrhage, malnutrition, intussusception, and death at an early age. There is no family history. The remaining cases present in childhood or adulthood. The polyps may be limited to the colorectum or generalized throughout the GI tract [40]. Rarely, the stomach is the principal site of involvement. There may be extraintestinal defects, including abnormalities of the cranium or heart, cleft palate, polydactyly, and intestinal malrotations. Familial clustering, indicative of an autosomal dominant mode of inheritance, has been described [41]. There is an increased risk of colorectal cancer [41,42], the cumulative risk being estimated as 68% by 60 years of age [43]. The cancers are often mucinous and/or poorly differentiated [42]. The polyps in juvenile polyposis number from 5 to 200 and may differ from the classical juvenile polyp in being multilobated (Fig. 2) and showing a relative lack of expanded lamina propria [42]. The surface of these atypical juvenile polyps rarely is eroded, and there may be focal dysplasia, accounting for the increased risk of malignancy. Coexisting adenomas may occur. Most juvenile polyposis families have a germline mutation in either SMAD4/DPC4 on chromosome 18q21 [44], or the BMPR1A/ALK3 gene on chromosome 10q23 [45]. The BMP receptor 1A signals by means of SMAD4 to down-regulate cell growth and division, thereby providing an explanation for the similar phenotypes. The PTEN gene initially was linked with juvenile polyposis [35], but subsequently with Cowden syndrome. Interestingly, large deletions in 10q, implicating both BMPR1A and PTEN, have been demonstrated in infants with the rare and severe form of juvenile polyposis, suggesting a biological synergy for these genes [46]. HEREDITARY MIXED POLYPOSIS SYNDROME The term mixed relates to the variety of polyps and the presence of polyps with features reminiscent of more than one type of classic polyp. Polyps frequently have an edematous and expanded lamina propria, similar to juvenile polyps.

Fig. 2. Multilobated colorectal polyps in a patient with juvenile polyposis.

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The polyps, however, may be multilobated or villiform, resembling atypical juvenile polyps of juvenile polyposis. Glands may show a strikingly serrated architecture. The serrated epithelium may be nondysplastic, resembling that of a hyperplastic polyp, or adenomatous, resulting in an appearance reminiscent of serrated adenoma (Fig. 3). Conventional adenomas also may occur. There is an increased risk of colorectal cancer, presumably developing in the atypical juvenile polyps, serrated adenomas, or classical adenomas. The number of polyps usually ranges from 1 to 15, but rarely exceeds 50. No extracolonic features have been described [47]. The histological composition of polyps varies considerably from subject to subject, even within the same family. To date, examples of hereditary mixed polyposis syndrome HMPS have been restricted to one large and extended Ashkenazi Jewish family and smaller Ashkenazi Jewish families [48]. The HMPS locus (CRAC1) originally was mapped to chromosome 6q [49], but one of the subjects, considered originally to be unaffected, subsequently developed multiple colorectal adenomas. With the benefit of more informative markers and stringent definitions of affected status, the disease locus was mapped to 15q13-q21 [48], and in a second family to 15q14q22 [50]. The locus was then narrowed down to 15q13-q14 when information from the two families was combined [48]. HMPS may be confused with both juvenile polyposis and hyperplastic polyposis. The phenotypes of juvenile and HMPS may overlap, and at times be indistinguishable. An HMPS family from Singapore was found to have a germline mutation in the BMPR1A gene (linked with juvenile polyposis) [51]. It is possible that the underlying genetic defect in HMPS will be shown to implicate a gene that is functionally related to BMPR1A. HMPS is distinguished from hyperplastic polyposis by the relative lack of typical hyperplastic polyps and

Fig. 3. Colorectal polyp composed of serrated tubules showing mild epithelial dysplasia in patient with hereditary mixed polyposis syndrome. The lesion resembles a traditional serrated adenoma. The relative abundance of lamina propria between the serrated glands, the overall spherical contour of the polyp, and the lack of villous architectural component, however, provide a subtle distinction from the traditional serrated adenoma. Haematoxylin and eosin.

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variant hyperplastic polyps known as sessile serrated adenomas. The polyps in HMPS present as protuberant lesions that resemble adenomas grossly, regardless of the underlying histology. Most polyps in hyperplastic polyposis resemble hyperplastic polyps grossly, being sessile in shape and pale in color. HMPS appears to be inherited as an autosomal-dominant trait. Kindreds that show linkage to 15q13-14 are all of Ashkenazi Jewish origin. Most cases of hyperplastic polyposis are sporadic, and no Jewish link has been reported. HMPS and hyperplastic polyposis are unrelated conditions, and clinico–pathological distinction is normally straightforward once all clinical evidence and pathologic evidence have been gathered. HYPERPLASTIC POLYPOSIS Although hyperplastic polyps of the colorectum have been regarded for decades as clinically unimportant lesions, there are numerous reports linking hyperplastic polyposis with colorectal cancer. Hyperplastic polyposis is a rare disorder, but probably underdiagnosed. To understand the condition fully, one must accept that it is not a single disease entity, but instead it shows considerable clinical, pathological, and molecular heterogeneity. The following sections review evidence that hyperrplastic polyposis is precancerous, and explores the genetic associations underlying its clinical and pathological manifestations. Hyperplastic Polyposis: A Precancerous Condition The first report of dysplasia and colorectal cancer occurring in a background of hyperplastic polyposis was in 1979 [52]. At first this seemed to be an exception to the rule, since none of seven cases of metaplastic polyposis (synonymous with hyperplastic polyposis) from St Mark’s Hospital in London was associated with colorectal cancer [53]. In fact, the differing cancer risk with respect to case reports versus case series has continued to the present time. All of the 207 examples of hyperplastic polyposis reported up to 2006 have been either single case reports or small series of up to four cases (totalling 36 cases) [52,54–77], or larger series comprising five or more cases (totaling 171 cases) [26,53, 78–87]. Within these two groups, the frequency of patients with one or more colorectal cancers is 25/36 (69%) and 63/171 (37%), respectively. Differences in the frequency of colorectal cancers are not difficult to explain. Single case reports generally were published, because the clinical or pathological features were considered to be unusual by virtue of numerous and/or large hyperplastic polyps, proximally located polyps, transition to dysplasia, the presence of one or more colorectal cancers, and early age of onset of colorectal cancer. In contrast, larger case series have generally adopted definitions of hyperplastic polyposis in which polyps may be not be especially numerous, large, or located in proximal colon. The high frequency of colorectal cancer in case reports will be an effect of ascertainment bias. At the same time, the use of relatively nonstringent diagnostic criteria for hyperplastic polyposis in larger series may lead to an underestimation of the risk of malignancy [88].

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Serrated Adenomatous Polyposis An important breakthrough in the understanding of the heterogeneity of hyperplastic polyposis came from a description of six patients with multiple serrated adenomatous polyps, of whom four developed colorectal cancer [79]. These cases initially had been diagnosed as hyperplastic polyposis, but it was argued that the polyps appeared more like serrated adenomas than hyperplastic polyps. The polyps in question were large, sessile, showed exaggerated serration and crypt dilatation, and were hypermucinous (Figs. 4 and 5). Despite lacking the traditional cytologic features of dysplasia, these polyps were regarded as neoplasms with malignant potential. These lesions subsequently were shown to occur sporadically and since have been named sessile serrated adenomas [89]. In an editorial accompanying the paper on serrated adenomatous polyposis, it was suggested that there might be two forms of hyperplastic polyposis [90]. One form comprises small, typical hyperplastic polyps, albeit multiple, and have little associated risk of malignancy. Many of the cases in the series described by Williams and colleagues [53] and Ferrandez and colleagues [85] belong to this latter category. The other form (serrated adenomatous polyposis) features multiple and large sessile serrated adenomas, traditional serrated adenomas, mixed polyps (hyperplastic or related polyps that include a component that is unequivocally dysplastic and indicative of neoplastic progression), or conventional adenomas. This form is associated with a significant risk of colorectal cancer. Hyperplastic Polyposis: Genetic Heterogeneity The evidence for hyperplastic polyposis representing a heterogeneous condition rests primarily upon the demonstration of clinical or phenotypic heterogeneity with accompanying genetic correlations. For instance, Rashid and colleagues [82] grouped patients with hyperplastic polyps into three subsets: 1. 13 subjects (including three from one family) with hyperplastic polyposis more than 20 hyperplastic polyps)

Fig. 4. Pale sessile polyps in proximal colon from a patient with hyperplastic polyposis. The phenotype is consistent with type 1 hyperplastic polyposis as outlined in Table 1.

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Fig. 5. Hyperplastic polyp variant recently renamed sessile serrated adenoma. Crypts are hypermucinous, dilated, branched, and show exaggerated serration. This morphology frequently is seen in type 1 hyperplastic polyposis (see Table 1). Haematoxylin and eosin.

2. Five subjects with less than 20 hyperplastic polyps (1 to 14) but with at least one that measured greater than1 cm 3. Five subjects with multiple, but less than 20, hyperplastic polyps, of which none measured greater than1 cm

This study did not distinguish sessile serrated adenomas from hyperplastic polyps. KRAS mutation was most common in hyperplastic polyps from subjects with multiple hyperplastic polyps (16%), but was not found in any of the hyperplastic polyps from patients with large hyperplastic polyps. Loss of heterozygosity (LOH) in chromosome 1p32-36 occurred in 13% of hyperplastic polyps, but only in polyps from patients with hyperplastic polyposis. Five of 13 patients with hyperplastic polyposis had hyperplastic polyps with 1p LOH, which included 16 of 77 (21%) polyps from these patients. DNA microsatellite instability (MSI), at either low- (MSI-L) or high level (MSI-H), was uncommon in hyperplastic polyps (4.6%), but occurred in 22.0% of serrated polyps with dysplasia (traditional serrated adenomas or mixed polyps). One patient with both hyperplastic polyposis and colorectal cancer had MSI-H in multiple lesions (three of seven hyperplastic polyps and three of four adenomas). Other studies have documented the existence of either MSI-L or MSI-H in both sporadic and hyperplastic polyposis associated serrated polyps [67,71,91,92]. In fact, this molecular finding is more common in serrated polyps with dysplasia (mixed polyps or traditional serrated adenomas) and is, therefore, more likely to be associated with progression than with initiation of the serrated pathway [82,91]. A notable feature in the study by Rashid and colleagues [82] was the complete absence of both KRAS mutation and 1p LOH in hyperplastic polyps from subjects who had large polyps. Since the report by Rashid and colleagues [82], it has become clear that the most frequent genetic alterations in large hyperplastic polyps (sessile serrated adenomas) include both mutation of BRAF and methylation of tumor suppressor genes, such as p16INK4a, p14ARF, RASSF1, RASSF2, RASSF5, and MST-1, and the DNA

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repair genes MGMT and MLH1 [74,93–95]. Extensive DNA methylation also has been described in normal mucosa of subjects with hyperplastic polyposis [74]. Based upon the preceding descriptions of morphologic and genetic heterogeneity, two principal types of hyperplastic polyposis have been proposed (Table 1). The existence of two principal types of hyperplastic polyposis first was suggested over 10 years ago [90], but there have been few attempts to resolve hyperplastic polyposis into fully characterized entities, with differing implications for clinical management. A recent study highlights two forms of hyperplastic polyposis with concordant mutation of either BRAF or KRAS [96]. Molecular classifications, based on mutational and methylator patterns, not only serve to objectify this exercise, but may provide clues to the all-important underlying etiology. There is increasing evidence that mutation of oncogenes such as BRAF and KRAS, in isolation, is not protumorigenic at the outset, but results either in replication arrest and cell senescence or apoptosis [97]. In both serrated polyps and colorectal cancers, BRAF mutation is associated with a high-level CpG island methylator phenotype (CIMP-high), while KRAS mutation is associated with CIMP-low [93,98]. CIMP-high and CIMP-low may differ qualitatively and quantitatively [99]. It is likely that DNA methylation is of crucial importance in silencing tumor suppressor genes, such as p16INK4a (cell cycle regulator) and RASSF1 (proapoptotic), thereby directing the effects of oncogene mutation away from senescence and apoptosis toward the tumorigenic characteristics of proliferation and immortality [97]. In order for the early tumorigenic potential of the mutated forms to be realized, BRAF may be more dependent upon CIMP-high synergies, while KRAS may require only CIMPlow. Therefore, the fundamental molecular distinction between type 1 and Table 1 Hyperplastic polyposis type 1 (serrated adenomatous polyposis) and type 2 Features of serrated polyps Number Size Location Architecture Dysplasia Coexisting adenoma BRAF mutation KRAS mutation 1p LOH DNA methylation Risk of colorectal cancer Colorectal cancer with microsatellite instability high-level

Type 1

Type 2

References

Five or more Large (two >10 mm) Proximal to sigmoid Abnormal (sessile serrated adenoma) Frequent Frequent þþþ   þþþ þþþ 33%

30 or more Small (1–10 mm) Pan-colorectal Normal (hyperplastic polyp) Infrequent Infrequent þ þþ þ þ þ N/A

[82,88] [88] [88] [79,90] [74,79,83] [74,79,83] [74] [82] [82] [74] [79,90] [26,67,71,72, 74–77,82,83]

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type 2 hyperplastic polyposis may depend on a background predisposition to either CIMP-high or CIMP-low. Type 2 hyperplastic polyposis itself may be heterogeneous. Some cases may be either early or forme fruste examples of type 1 hyperplastic polyposis, while others simply may represent multiple small sporadic hyperplastic polyps in the recto–sigmoid region. Molecular studies should help in stratifying type 2 hyperplastic polyposis. For example, demonstration of polyps with BRAF mutation would point to an early or forme fruste example of type 1 hyperplastic polyposis [74]. The presence of KRAS mutation would suggest a link with small, mainly distally located, hyperplastic polyps of goblet cell type [93], whereas the demonstration of 1p LOH may suggest a third etiology [82]. Hereditary Basis of Hyperplastic Polyposis At present, hyperplastic polyposis has no known hereditary basis. There are multiple factors that point to a genetic etiology, however. These include: 1. Early age of onset. The mean age at diagnosis is 55 years, but it has occurred in an 11-year-old girl, and in a 24-year-old man (both with colorectal cancer) [59,70]. 2. Polyp multiplicity. Polyp numbers are variable, but may exceed 100. This is unlikely to be explained by environmental factors alone. 3. An increased risk of colorectal cancer, including instances of multiple colorectal cancers (up to six in a single patient) [71]. 4. Familial hyperplastic polyposis. In one family in which six members had one or more colorectal cancers, three subjects within a sibship of 10 had a phenotype consistent with hyperplastic polyposis, whereas other family members had small numbers of hyperplastic polyps [67]. Three additional families that included two or more first-degree relatives with hyperplastic polyposis, also have been described [26,82]. The rarity of familial clusters may be explained by various reasons, such as autosomal recessive inheritance, by the fact that multiple hyperplastic polyps are generally symptomless, and by the only recent acceptance that hyperplastic polyposis is a precancerous condition that may serve as an indication for screening the relatives of affected subjects. 5. Occurrence of polyps and cancers in relatives. Colorectal cancer, and/or small numbers of serrated polyps, may occur in first-degree relatives of subjects with hyperplastic polyposis [87]. Conversely, examples of hyperplastic polyposis have been described in families that meet the Amsterdam criteria, but lack germline defects in DNA mismatch repair genes [100]. Colorectal cancers in members of these families show similar molecular features to those of colorectal cancer in patients with hyperplastic polyposis: variable MSI status, BRAF mutation, and DNA methylation [100]. If hyperplastic polyposis is an autosomal recessive condition, then subjects who carry a single altered allele may develop only a small number of polyps [101]. 6. Striking differences in the incidence of hyperplastic polyposis across different patient populations. Most reports come from Western countries. It is notable that 84 of 207 (41%) reported cases are from Australia, and most of

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these are of Celtic descent [26]. In contrast, only five cases have been described in Japan. This population difference is not seen in other types of familial polyposis or colorectal cancer, although it does occur in other inherited diseases. 7. DNA methylation in normal colonic mucosa. This has been described in a small number of patients with hyperplastic polyposis who have a severe phenotype [74]. This finding raises the possibility of a genetic predisposition to DNA methylation, and, in turn, to type 1 hyperplastic polyposis [97]. 8. Germline mutation of a tumor suppressor on chromosome 1p may underlie a subset of type 2 hyperplastic polyposis cases [82]. 9. Hyperplastic polyposis in a patient with biallelic germline mutations of MUTYH (MYH) [26].

Heterogeneity of Colorectal Cancer in Hyperplastic Polyposis Colorectal cancer is a genetically heterogeneous disorder. Although genetic heterogeneity extends to cancers complicating hyperplastic polyposis, certain molecular subtypes are over-represented in this condition. For example, approximately one third of colorectal cancers associated with hyperplastic polyposis are MSI-H [26,67,71,72,74–77,82,83]. MSI-H colorectal cancers are particularly likely to be associated with type 1 hyperplastic polyposis (see Table 1), in which both polyps and cancers share the features of BRAF mutation and extensive DNA methylation. Even within this form of hyperplastic polyposis, however, the frequency of loss of expression of the DNA mismatch repair protein MLH1 within serrated polyps shows marked variation from patient to patient [74]. When hyperplastic polyps show infrequent loss of expression of MLH1, despite the presence of extensive methylation in other genes, colorectal cancers with BRAF mutation may be more likely to have evolved through pathways that do not implicate DNA mismatch repair deficiency [74]. In types of hyperplastic polyposis in which polyps are characterized by KRAS mutation and/or chromosome 1p LOH, MSI-H colorectal cancers again will be under-represented (see Table 1). EphB2, located on chromosome 1p36, recently has been highlighted as a tumor suppressor gene. EphB2 protein is implicated in the regulation of intestinal epithelial differentiation, synergizes with activated KRAS, and has been associated with the serrated pathway of colorectal tumorigenesis [102–104]. A germline variant of EphB2 was found in only one of 40 subjects with hyperplastic polyposis, however [105]. Further examples of genetic heterogeneity may be explained by alternative pathways of tumorigenesis in which traditional serrated adenomas, or even conventional adenomas, serve as cancer precursors. The latter may account for colorectal cancers with chromosomal instability and frequent loss of heterozygosity at tumor suppressor loci [72]. In summary, the genetic heterogeneity of colorectal cancer complicating hyperplastic polyposis is, to a large extent, explained by the genetic heterogeneity of hyperplastic polyposis itself.

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CAP POLYPOSIS, ERODED POLYPOID HYPERPLASIA, INFLAMMATORY MYOGLANDULAR POLYPS, AND POLYPOID PROLAPSING FOLDS A form of colorectal polyposis, with various names indicative of a background of inflammation and/or mucosal prolapse, shows a predilection for the sigmoid region [106–108]. This condition frequently is associated with diverticular disease that may be accompanied by diverticular (segmental) colitis. The polyps comprise elongated, tortuous, dilated, and sometimes serrated crypts. The surface epithelium often is eroded and capped by granulation tissue. When mucosal prolapse features as an etiological factor, there is proliferation of smooth muscle within the lamina propria. Otherwise, the lamina propria is expanded and inflamed. The condition may be confused with juvenile polyposis, hyperplastic polyposis, Peutz-Jeghers syndrome, and inflammatory bowel disease. Knowledge of the condition, the diverse histological features associated with mucosal prolapse, and the predilection for the sigmoid colon should lead to the correct diagnosis. SUMMARY The diagnosis of some forms of polyposis, most notably classical FAP and Peutz-Jeghers syndrome, is relatively straightforward. In contrast, attenuated FAP may be confused with MUTYH polyposis and Lynch syndrome. Cowden syndrome and juvenile polyposis frequently are confused with each other, and HMPS may resemble both juvenile polyposis and hyperplastic polyposis. In view of overlapping phenotypes, the ultimate diagnostic arbiter is demonstration of a causative germline mutation. A germline change, however, may not be identified in all cases, and the genetic basis for HMPS and hyperplastic polyposis is unknown at this time. Unfortunately, much of the confusion may be because of a lack of knowledge regarding the pathological characteristics of the polyps themselves. The number and type of polyps are of utmost importance in achieving an accurate diagnosis, which ultimately depends upon adequate polyp sampling and an awareness of the morphological spectrum of the various polyp types. Because each type of polyposis is rare, pathologists normally lack diagnostic expertise. Greater diagnostic precision may be achieved by assessing polyps from multiple members of the same family. Unfortunately, this is often only possible if members of the same family are followed at the same institution. Despite these difficulties, there has been considerable progress in the understanding of GI polyposis disorders over the last 20 years. The final major breakthrough will be uncovering the mechanisms that underlie hyperplastic polyposis, a condition that has been neglected until recently. References [1] Bulow S, Berk T, Neale KF. The history of familial adenomatous polyposis. Fam Cancer 2006;5:213–20. [2] Vasen HFA, Mecklin J-P, Khan PM, et al. The international collaborative group on hereditary nonpolyposis colorectal cancer (ICG-HNPCC). Dis Colon Rectum 1991;34:424–5.

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[3] Gardner EJ. Follow-up study of a family group exhibiting dominant inheritance for a syndrome including intestinal polyps, osteomas, fibromas, and epidermal cysts. Am J Hum Genet 1962;14:376–90. [4] Lipton L, Tomlinson I. The genetics of FAP and FAP-like syndromes. Fam Cancer 2006;5: 221–6. [5] Bussey HJR. Familial polyposis coli. Baltimore(MD): Johns Hopkins Press; 1975. [6] Matsumoto T, Iida M, Kobori Y, et al. Serrated adenoma in familial adenomatous polyposis: relation to germline APC gene mutation. Gut 2002;50:402–4. [7] Herrera L, Kakati S, Gibas L, et al. Brief clinical report: Gardner syndrome in a man with an interstitial deletion of 5q. Am J Med Genet 1986;25:473–6. [8] Bodmer WF, Bailey CJ, Bodmer J, et al. Localization of the gene for familial adenomatous polyposis on chromosome 5. Nature 1987;328:614–6. [9] Groden J, Thliveris A, Samowitz W, et al. Identification and characterization of the familial adenomatous polyposis gene. Cell 1991;66:589–600. [10] Olschwang S, Tiret A, Laurent-Puig P, et al. Restriction of ocular fundus lesions to a specific subgroup of APC mutations in adenomatous polyposis coli patients. Cell 1993;75: 959–68. [11] Leppert M, Burt R, Hughes JP, et al. Genetic analysis of an inherited predisposition to colon cancer in a family with a variable number of adenomatous polyps. N Engl J Med 1990;322:904–8. [12] Lanspa SJ, Lynch HT, Smyrk TC, et al. Colorectal adenomas in the Lynch syndromes. Results of a colonoscopy screening programme. Gastroenterology 1990;98: 1117–22. [13] De Jong AE, Morreau H, van Puijenbroek M, et al. The role of mismatch repair gene defects in the development of adenomas in patients with HNPCC. Gastroenterology 2004;126: 42–8. [14] Sakashita M, Aoyama N, Maekawa S, et al. Flat-elevated and depressed subtypes of flat early colorectal cancers should be distinguished by their pathological features. Int J Colorectal Dis 2000;15:275–81. [15] Lecomte T, Cellier C, Meatchi T, et al. Chromoendoscopic colonoscopy for detecting preneoplastic lesions in hereditary nonpolyposis colorectal cancer syndrome. Clin Gastroenterol Hepatol 2005;3:897–902. [16] Spirio L, Olschwang S, Groden J, et al. Alleles of the APC gene: an attenuated form of familial polyposis. Cell 1993;75:951–7. [17] Jass JR, Stewart SM. Evolution of hereditary nonpolyposis colorectal cancer. Gut 1992;33: 783–6. [18] Wallace MH, Frayling IM, Clark SK, et al. Attenuated adenomatous polyposis coli: the role of ascertainment bias through dye spray at colonoscopy. Dis Colon Rectum 1999;42: 1078–80. [19] Sieber OM, Segditsas S, Knudsen AL, et al. Disease severity and genetic pathways in attenuated familial adenomatous polyposis vary greatly, but depend on the site of the germline mutation. Gut 2006;55:1440–8. [20] Al-Tassan N, Chmiel NH, Maynard J, et al. Inherited variants of MYH associated with somatic G:C - T:a mutations in colorectal tumors. Nat Genet 2002;30:227–32. [21] Sieber OM, Lipton L, Crabtree M, et al. Multiple colorectal adenomas, classic adenomatous polyposis, and germ-line mutations in MYH. N Engl J Med 2003;348:791–9. [22] Sampson JR, Dolwani S, Jones S, et al. Autosomal recessive colorectal adenomatous polyposis due to inherited mutations of MYH. Lancet 2003;362:39–41. [23] Gismondi V, Meta M, Bonelli L, et al. Prevalence of the Y165C, G382D and 1395delGGA germline mutations of the MYH gene in Italian patients with adenomatous polyposis coli and colorectal adenomas. Int J Cancer 2004;109:680–4. [24] Lipton L, Halford SE, Johnson V, et al. Carcinogenesis in MYH-associated polyposis follows a distinct genetic pathway. Cancer Res 2003;63:7595–9.

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[25] Lipton L, Tomlinson I. The multiple colorectal adenoma phenotype and MYH, a base excision repair gene. Clin Gastroenterol Hepatol 2004;2:633–8. [26] Chow E, Lipton L, Lynch E, et al. Hyperplastic polyposis: phenotypic presentations and the role of MBD4 and MYH. Gastroenterology 2006;131:30–9. [27] Vasen HF, van Erpecum KJ, Roelfsema F, et al. Increased prevalence of colonic adenomas in patients with acromegaly. Eur J Endocrinol 1994;131:235–7. [28] Lowy AM, Kordich JJ, Gismondi V, et al. Numerous colonic adenomas in an individual with Bloom’s syndrome. Gastroenterology 2001;121:435–9. [29] Wiesner GL, Daley D, Lewis S, et al. A subset of familial colorectal neoplasia kindreds linked to chromosome 9q22.2-31.2. Proc Natl Acad Sci U S A 2003;100:12961–5. [30] Skoglund J, Djureinovic T, Zhou X-L, et al. Linkage analysis in a large Swedish family supports the presence of a susceptibility locus for adenoma in colorectal cancer on chromosome 9q22.32-31.1. J Med Genet 2006;43:e7. [31] Kemp ZE, Carvajal-Carmona LG, Barclay E, et al. Evidence of linkage to chromosome 9q22.32 in colorectal cancer kindreds in UK. Cancer Res 2006;66:5003–6. [32] Shepherd NA, Bussey HJ, Jass JR. Epithelial misplacement in Peutz-Jeghers polyps. A diagnostic pitfall. Am J Surg Pathol 1987;11:743–9. [33] Amos CI, Keitheri-Cheteri MB, Sabripour M, et al. Genotype-phenotype correlations in Peutz-Jeghers syndrome. J Med Genet 2004;41:327–33. [34] Carlson GJ, Nivatvongs S, Snover DC. Colorectal polyps in Cowden’s disease (multiple hamartoma syndrome). Am J Surg Pathol 1984;8:763–70. [35] Olschwang S, Serova-Sinilnikova OM, Lenoir GM, et al. PTEN germ-line mutations in juvenile polyposis coli. Nat Genet 1998;18:12–4. [36] Mendelsohn G, Diamond MP. Familial ganglioneuromatous polyposis of the large bowel. Report of a family with associated juvenile polyposis. Am J Surg Pathol 1984;8:515–20. [37] Weidner N, Flanders DJ, Mitros FA. Mucosal ganglioneuromatosis associated with multiple colonic polyps. Am J Surg Pathol 1984;8(10):779–86. [38] Nelen MR, Padberg GW, Peeters EAJ, et al. Localization of the gene for Cowden disease to chromosome 10q22-23. Nat Genet 1996;13:114–6. [39] Gorlin RJ, Cohen MM, Condon LM, et al. Bannayan-Riley-Ruvalcaba syndrome. Am J Med Genet 1992;44:307–14. [40] Sachatello CR, Griffen WO. Hereditary polypoid diseases of the gastrointestinal tract: a working classification. Am J Surg 1975;129:198–203. [41] Stemper TJ, Kent TH, Summers RW. Juvenile polyposis and gastrointestinal carcinoma: a study of a kindred. Ann Intern Med 1975;83:639–46. [42] Jass JR, Williams CB, Bussey HJR, et al. Juvenile polyposis—a precancerous condition. Histopathology 1988;13:619–30. [43] Murday V, Slack J. Inherited disorders associated with colorectal cancer. Cancer Surv 1989;8:139–57. [44] Howe JR, Roth S, Ringold JC, et al. Mutations in the SMAD4/DPC4 gene in juvenile polyposis. Science 1998;280:1086–8. [45] Zhou XP, Woodford-Richens K, Lehtonen R, et al. Germline mutations in BMPR1A/ALK3 cause a subset of cases of juvenile polypsis synderome and of Cowden and BannayanRiley-Ruvalcaba syndromes. Am J Hum Genet 2001;69:704–11. [46] Delnatte C, Sanlaville D, Mougenot JF, et al. Contiguous gene deletion within chromosome arm 10q is associated with juvenile polyposis of infancy, reflecting cooperation between the BMPR1A and PTEN tumor suppressor genes. Am J Hum Genet 2006;78: 1066–74. [47] Whitelaw SC, Murday VA, Tomlinson IPM, et al. Clinical and molecular features of the hereditary mixed polyposis syndrome. Gastroenterology 1997;112:327–34. [48] Jaeger EEM, Woodford-Richens KL, Lockett M, et al. An ancestral Ashkenazi haplotype at the HMPS/CRAC1 locus on 15q13-q14 is associated with hereditary mixed polyposis syndrome. Am J Hum Genet 2003;72:1261–7.

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[96] Carvajal-Carmona LG, Howarth CM, Lockett M, et al. Molecular classification and genetic pathways in hyperplastic polyposis syndrome. J Pathol 2006;212:378–85. [97] Minoo P, Jass JR. Senescence and serration: a new twist to an old tale. J Pathol 2006;210: 137–40. [98] Ogino S, Cantor M, Kawasaki T, et al. CpG island methylator phenotype (CIMP) of colorectal cancer is best characterized by quantitative DNA methylation analysis and prospective cohort studies. Gut 2006;55:1000–6. [99] Issa J-P. CIMP, at last. Gastroenterology 2005;129:1121–4. [100] Young J, Barker MA, Simms LA, et al. BRAF mutation and variable levels of microsatellite instability characterize a syndrome of familial colorectal cancer. Clin Gastroenterol Hepatol 2005;3:254–63. [101] Young J, Jass JR. The case for a genetic predisposition to serrated neoplasia of the colorectum: hypothesis and review of the literature. Cancer Epidemiol Biomarkers Prev 2006;15:1778–84. [102] Batlle E, Bacani J, Begthel H, et al. EphB receptor activity suppresses colorectal cancer progression. Nature 2005;435:1126–30. [103] Elowe S, Holland SJ, Kulkarni S, et al. Downregulation of the Ras-mitogen-activated protein kinase pathway by the EphB2 receptor kinase is required for ephrin-induced neurite retraction. Mol Cell Biol 2001;21:7429–41. [104] Laiho P, Kokko A, Vanharanta S, et al. Serrated carcinomas form a subclass of colorectal cancer with distinct molecular basis. Oncogene 2007;26:312–20. [105] Kokko A, Laiho P, Lehtonen R, et al. EPHB2 germline variants in patients with colorectal cancer or hyperplastic polyposis. BMC Cancer 2006;6:145. [106] Burke AP, Sobin LH. Eroded polypoid hyperplasia of the rectosigmoid. Am J Gastroenterol 1990;85:975–80. [107] Day DW, Jass JR, Price AB, et al. Morson and Dawson’s gastrointestinal pathology. 4th edition. Oxford: Blackwell; 2003. p. 630. [108] Nakamura S, Kino I, Akagi T. Inflammatory myoglandular polyps of the colon and rectum. A clinicopathological study of 32 pedunculated polyps, distinct from other types of polyps. Am J Surg Pathol 1992;16:772–9.

Gastroenterol Clin N Am 36 (2007) 947–968

GASTROENTEROLOGY CLINICS OF NORTH AMERICA

Hyperplastic and Serrated Polyps of the Colorectum Michael J. O’Brien, MD, MPH Boston University School of Medicine, Robinson Building, Room 904, 80 East Concord Street, Boston, MA 02118, USA

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he serrated polyp pathway is the histological expression of a molecular genetic paradigm of colorectal carcinogenesis that differs from that of the traditional adenoma–carcinoma sequence [1]. The predominant carcinomas of this pathway are those that show microsatellite instability (MSI-high) because of hMLH1 inactivation and consequent DNA mismatch repair (MMR). They additionally include some proportion of carcinomas that are microsatellite stable (MSS) or MSI-low (MSI-L) [1]. The pathway originates in a hyperplastic polyp, or precursor aberrant crypt focus (ACF), and progresses through an intermediate disordered type of hyperplastic polyp that eventually becomes dysplastic (dysplastic serrated polyp), and ultimately to carcinoma [2]. Carcinomas that develop from both the serrated polyp pathway and the conventional adenomatous polyposis coli (APC) adenoma–carcinoma sequence show histological features reflective of their respective molecular profiles and histogenesis [3]. The delineation of the phenomenon of epigenetic mutagenesis by CpGisland methylation and its key role in sporadic MSI colorectal carcinomas are at the molecular genetic core of this newly discovered serrated pathway [4–7]. CpG island methylation phenotype (CIMP) refers to nonrandom methylation of gene promoter regions that concordantly affects multiple susceptible suppressor, mutator, and other genes that may have roles in carcinogenesis [8,9]. This proclivity to epigenetic gene silencing of cancer-related genes has been demonstrated in precursor polyps and in endpoint carcinomas of the serrated polyp pathway [10–14], and it can be viewed as a molecular engine that drives serrated polyp progression. As such, it contrasts greatly with the mutagenic process of the conventional APC pathway, originally elucidated by Vogelstein and colleagues [15,16] as the molecular underpinnings of the adenoma–carcinoma sequence, in which adenoma progression to carcinoma is driven by deletions and homozygous loss of suppressor genes as a result of APC mutation-induced chromosomal instability [16–18]. E-mail address: [email protected]

0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.007

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Another recent molecular genetic advance that has illuminated that understanding of this pathway has been the discovery of the oncogene BRAF [19]. An activating mutation (BRAFV600E) of this phosphokinase of the intracellular RAS-RAF-MAP kinase signaling pathway is present in most sporadic colorectal carcinomas that exhibit CIMP-high and MSI [20–23]. Furthermore, this type of BRAF mutation also has been shown to be highly prevalent in many of the lesions that comprise the morphological spectrum of the serrated polyp pathway, including microscopic hyperplastic (serrated) ACFs and advanced dysplastic serrated polyps (serrated adenomas) [2,12,23,24]. This article reviews our current understanding of the serrated polyp pathway in molecular and clinicopathological terms. It focuses mainly on the predominant BRAF serrated pathway. A second arm of the serrated pathway is associated with KRAS mutations, and contrasts with the BRAF pathway in its association with distal rather than proximal tumors, lower levels of CpG-island methylation and MSS or MSI-L rather than MSI endpoint carcinomas [1], also is discussed. NONDYSPLASTIC SERRATED POLYPS Hyperplastic Polyps Hyperplastic polyps of the colon and rectum are diminutive (typically less than 5 mm in diameter) mucosal growths highly prevalent in Western populations [25–29]. Until recently, they have been considered entirely innocuous and unlikely precursors of colon cancer, although there have been some notable contrarian views [30,31]. Hyperplastic polyps long have been known to have an ecological association with colorectal carcinoma, tending to be more prevalent in populations with a higher incidence of cancer [32]. Specific lifestyle and dietary factors that appear to be associated with higher prevalence levels include cigarette smoking, alcohol consumption, obesity, and low folate intake [33–35], while nonsteroidal anti-inflammatory drug use, hormone replacement therapy, and high calcium intake have been linked to a reduced frequency [34]. Most factors associated with hyperplastic polyps have been more strongly linked to adenomas of the APC pathway, with the notable exception of cigarette smoking [34]. This exception has added significance in the context of recent evidence that cigarette smoking also is associated significantly with colorectal carcinomas that exhibit MSI, CIMP, and BRAF mutations [36]. Serration: A Histological Signature The primary histologic characteristic of hyperplastic polyps, which confers the alternative name of serrated polyp, is infolding of the crypt epithelium, which leads to a serrated or saw-toothed appearance in longitudinal section and a stellate appearance on cross section or with magnification chromo–endoscopy. The molecular basis for this histological signature has not been elucidated, although it frequently has been attributed to cell crowding because of failure of apoptosis or anoikis [37]. Regardless of the exact molecular mechanism, crypt serration is associated strongly with the presence of BRAF mutations

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in hyperplastic polyps and more advanced (dysplastic) serrated polyps. Hyperplastic polyps that have KRAS rather than BRAF mutations show either less, or complete absence of, crypt serration. This histological signature, however, is evident in KRAS-mutated and BRAF-mutated dysplastic polyps in this pathway, and also is recognized frequently in carcinomas that develop from serrated polyps [38–40]. In more advanced serrated neoplasms, ancillary histological criteria are recommended to reliably distinguish serration as a manifestation of the serrated polyp pathway, from spuriously serrated conventional neoplastic or reparative glands [3,38]. Serration in Aberrant Crypt Foci Aberrant crypt foci (ACF) are microscopic mucosal abnormalities, a subset of which may be precursors of colorectal carcinoma. Dysplastic ACFs, or microadenomas, have been linked to conventional adenomas and the APC pathway [41]. Nondysplastic ACFs are described as hyperplastic or heteroplastic and may be serrated or non-serrated. While dysplastic ACFs are relatively uncommon, hyperplastic ACFs are very prevalent in individuals over 50 [41]. Recently, Rosenberg and colleagues [42] reported testing the a priori hypothesis that ACFs that show a serrated or stellate morphology have BRAF mutation while nonserrated hyperplastic ACFs do not. The ACFs under study were a random sample of 55 ACFs harvested from patients undergoing screening colonoscopy. Almost two thirds of 16 serrated ACFs showed BRAF mutation, compared with only 1 of 33 nonserrated ACFs. The association of BRAFV600E mutation with serrated ACF suggests that it is an early or instigating mutation in the serrated polyp pathway. The findings also suggest that since BRAF mutated ACFs occur with such a high frequency, that only a fraction are ever likely to progress to hyperplastic polyps, or more advanced lesions of the serrated polyp pathway. Classification of Hyperplastic Polyps In retrospect, the morphological proof of concept of the serrated polyp pathway was provided in studies of hyperplastic polyposis [43–45] and notably by a small case series of this rare syndrome that described a hyperplastic polyp variant with a disordered architecture, which the authors labeled as sessile serrated adenoma [44]. Informed by their studies of hyperplastic polyposis and the recognition that a hyperplastic polyp variant could represent a morphological link between hyperplastic polyp and progression to carcinoma, Torlakovic and colleagues [46] systematically evaluated the histologic spectrum of sporadically occurring hyperplastic polyps that once were thought to represent a morphologically homogenous group. They applied cluster analysis statistical methodology to a panel of 24 different morphologic parameters in 289 hyperplastic polyps. Their analysis identified three major histological subtypes of hyperplastic or nondysplastic serrated polyps: serrated polyp with microvesicular mucin (MVSP), sessile goblet cell serrated polyp (GCSP), and serrated adenoma (SSA) (Fig. 1). (A fourth uncommon and less well-defined variant, mucin-depleted, also was described but is not discussed here.) These

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Fig. 1. Histological subtypes of hyperplastic polyps (nondysplastic serrated polyps). (A) Goblet cell serrated polyp (GCSP), showing enlarged crypts with an abundance of mature goblet cells in the upper crypts, inconspicuous serration but prominent tufting of the surface epithelium. (B) Microvesicular serrated polyp (MVSP), the prototypical hyperplastic polyp, showing elongated funnel shaped crypts with orderly maturation; serration is present in the upper and midcrypt where the predominant cell type is large and columnar with a microvesicular cytoplasm. (C) Sessile serrated adenoma (SSA), syn: serrated polyp with abnormal proliferation. Histological features typical of this subtype that are represented here are architectural disorder of the crypt bases with the formation of T- and other irregular shapes. Mature goblet cells are seen in the bases of some crypts indicating inverted maturation. The crypt at the extreme right shows an appearance resembling a disordered MVSP crypt with increased serration.

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hyperplastic polyp subcategories have been validated by later studies that have shown a robust association between these subtypes and specific molecular genetic profiles [10,12,47]. Microvesicular serrated polyp MVSP (see Fig. 1B) is the most widely distributed hyperplastic polyp variant in the colon and rectum, and, morphologically, is the prototypical hyperplastic polyp. It consists of elongated funnel shaped crypts with proliferative activity largely confined to the lower third of the crypts similar to normal colonic crypts. The upper crypts show abundant enlarged microvacuolated columnar cells mixed with fewer goblet cells. The crypts show prominent serration, most marked in the upper and middle third of the lesion, which sometimes extends, particularly in proximal lesions, to the crypt bases. BRAFV600E mutations are demonstrated in up to 80% of MVSPs [2,47]. The BRAF oncogene is a kinase normally activated by RAS as part of the RASRAF-MEK-ERK signaling pathway [48], a primeval intracellular signaling network that plays a key role in fundamental cellular processes, such as cell proliferation, differentiation, survival, and apoptosis. Oncogene activation can result in adaptive responses within the cell that include induction of a form of cell senescence distinct from replicative senescence caused by telomere shortening [49,50]. This phenomenon has been demonstrated to occur in nevi, common generally self-limited skin lesions, which share a high frequency of BRAF(V600E) mutations [51]. Although not empirically demonstrated in MVSPs, it is probable that the large microvesicular cells of the upper crypt, which long have been characterized as representing a hypermature or senescent phenotype based on ultrastructural studies [52,53], may arise as a result of such a signaling pathway interplay [54]. Senescence prevents cell transformation by up-regulating cell cycle inhibitors such p16 and p14 and proapoptotic proteins such as RASSF1 and RASSF2 [54]. Inactivation or failure of these adaptive molecular mechanisms may be the basis for transformation and progression of hyperplastic polyps to dysplasia and carcinoma. MVSPs have been shown to exhibit some increased level of susceptibility to aberrant CpG island methylation [10,55], and furthermore the CIMP status is more evident in MVSPs located in the proximal colon than in those located distally [10,12]. Genes that have been targeted in assays of CIMP status include hMLH1, MGMT (0-6 methyl guanine DNA methyl transferase, a DNA repair gene), p16 (a cell cycle inhibitor) and a number of genes of unknown function that have been shown empirically to be sensitive indicators of aberrant gene promoter methylation (MINs, methylated in neoplasia) [55]. Recent studies by Weisenberger and colleagues [8] suggested a modification of this marker set designed to optimize the sensitivity and specificity of the assessment of CIMP-high status in CRC, but these studies have not challenged the validity of the earlier markers and the insights they provided. Thus epigenetic inactivation of growth control genes provides a plausible mechanism for perturbation of the oncogene-induced senescence in BRAF mutated MVSPs, and the

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possibility of their progression to the putative next stage of serrated polyp progression, namely sessile serrated adenoma. Goblet cell serrated polyp This hyperplastic polyp variant (see Fig. 1A) is distinguished histologically by the presence of enlarged crypts with an abundance of mature goblet cells in the upper crypt and surface epithelium. Occasionally, symmetrical bifurcation of crypts in larger polyps may be seen. Serration is either inconspicuous or absent, but tufting of the surface epithelium is characteristic [46]. Up to 50% of these polyps exhibit a KRAS mutation, but BRAF mutations are seldom found [2,56]. In fact, some hyperplastic polyps may show features of both GCSP and MVSP and thus are difficult to classify [46]. An additional feature that distinguishes this type of hyperplastic polyp category is the absence of CIMP [10]. GCSPs are usually diminutive in size and found mainly in the distal sigmoid and rectum. The absence, or rarity, of an identifiable successor lesion suggests that they may be self-limited and unlikely to be the precursors of KRAS-mutated serrated adenomas [2]. Sessile serrated adenoma (syn: sessile serrated polyp, serrated polyp with abnormal proliferation) Sessile serrated adenoma (see Fig. 1C) represents the next histological step in the serrated polyp neoplasia pathway. This hyperplastic polyp variant has many characteristics of MVSPs but is distinguished from them by the presence of crypt architectural alterations that reflect disordered growth [46]. Deviations from the normal symmetrical array of crypts of MVSPs include crypt branching, dilatation, and serration that extends into the bases of the crypts [46]. The most characteristic, and recognizable, feature of these serrated polyps is the presence of inverted T- and L-shaped crypt bases, the latter often described as boot-shaped (see Fig. 1C). Additionally, the proliferation centers of the crypts, which are basally located in both MVSPs and normal crypts, may become centered higher up in the crypt epithelium. Further proliferation asymmetry is exemplified by the presence of mitoses (or MIB1 immunostaining) on one side of the crypt wall, but not the other [10,46]. Another frequently observed objective measure of disordered growth is a phenomenon referred to as inverted maturation, whereby mature goblet cells are located in the crypt bases. Focally, nuclear stratification, a mild degree of nuclear atypia or dystrophic goblet cells may be seen in the crypt bases of SSAs, but cytological dysplasia is not a feature of this type of serrated polyp [46]. SSAs share two defining molecular genetic characteristics with MVSPs, namely, BRAFV600E mutation and CIMP-H [2,23]. According to several investigators, BRAF mutations are found in approximately 75% of SSAs and conversely, as with MVSPs, KRAS mutations seldom if ever are associated with this polyp type [2,47,56]. In a study that compared hyperplastic polyp histological variants and location in the colorectum with CpG island methylation level, SSAs were found to show significantly increased methylation marker frequency compared with MVSPs [2,10]. Furthermore, although proximal MVSPs were

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characterized by higher CIMP levels than distal MVSPs, SSAs tended to be CIMP-H independent of colonic location [10]. This may indicate that high CpG island methylation levels, perhaps causing inactivation of a critical growth control gene, are a requirement for histological expression of the SSA phenotype. There is some consensus that the progression of SSA in the serrated polyp pathway is marked by the appearance of cytological dysplasia [57]. The molecular basis for this transition is poorly understood, however. Numerous studies have suggested, based on diminished immunostaining staining for hMLH1 in the crypts of these lesions, that there is an incremental acquisition of MSI status [46,58,59]. Although partial inactivation of hMLH1 may contribute to progression [3], molecular assays for MSI status in laser-microdissected serrated polyps show that MSI-H is a late development [2,60] and thus is unlikely to account for progression of SSA to histologic dysplasia. Clinicopathologic Features of Nondysplastic Serrated Polyps Distribution in the colorectum Nondysplastic serrated (hyperplastic) polyps, as a group, occur most frequently in the distal colon and rectum [61–63], although their role as potential colorectal cancer precursors is most frequently observed in the proximal colon. In one study of asymptomatic, average-risk patients undergoing colonoscopy, 57% of distal polyps compared with 25% of proximal polyps were hyperplastic [35,64]. This distribution pattern contrasts with that of adenomas, which tend to occur more evenly throughout the colon and rectum [61,65–67]. The distribution of hyperplastic polyps is more meaningful when considered in relation to histological category. For instance, GCSPs occur mainly in the rectum and are infrequent in the proximal colons, and MVSPS are also more common distally. SSAs are relatively more frequent in the proximal colon, where they comprise approximately 40% of hyperplastic polyps, whereas distally, they comprise 25% or less of the polyp total [46]. A recent survey of 190 consecutive patients who underwent magnification chromoendoscopy reported that SSAs accounted for 9% of all polyps retrieved and 23% of all hyperplastic polyps [56]. Endoscopic appearance of HPs At endoscopy, small hyperplastic polyps typically appear pale and sessile or flat, or may flatten with air insufflation [68,69]. Although it is often not possible to distinguish hyperplastic polyps from small adenomas, the former tend to be smaller in size and lighter in color [69,70]. Magnification chromoendoscopy can reveal the slightly enlarged and rounded crypt openings of GCSPs or the stellate luminal crypt contours of MVSPs or SSAs, either contrasting with the enlarged sinuous (cerebriform) or the thickened outline of the mouths of the crypts of conventional adenomas. Several reports have drawn attention to the endoscopic features of SSAs, which are sessile in appearance, smooth in contour, malleable, and yellow in color [71–74]. The latter feature is attributed to a covering of mucus that frequently obscures the underlying mucosal lesion unless it is dispersed by irrigation.

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DYSPLASTIC SERRATED POLYPS Serrated polyps that exhibit cytological dysplasia (Figs. 2 and 3) first were described definitively by Longacre and Fenoglio-Preiser [75] and were referred to as ‘‘serrated adenomas.’’ These authors also described a ‘‘mixed’’ variant that had separate areas of hyperplastic polyp and adenoma. Some authors use the term mixed or admixed serrated polyp to refer to serrated adenomas that also contain components that resemble conventional adenomatous polyps [47]. The 2000 World Health Organization (WHO) definition of serrated adenoma is a polyp characterized by the saw-tooth configuration of a hyperplastic polyp but the upper portion of the crypts and luminal surface is dysplastic [76]. The WHO classification also recognizes a mixed type. At the time these definitions were promulgated, insight into the role of serrated polyps in sporadic MSI, and other carcinomas, was evolving, and SSAs had not been characterized. The significance of serrated adenomas stems from their role as an immediate precursor of colon cancer, and, as such, they are distinguished by the presence of histologic dysplasia. That serrated adenomas are the likely progeny of hyperplastic polyps is suggested by their morphologic similarity, and, perhaps more objectively, by the concordance of key molecular genetic abnormalities in subsets of hyperplastic polyps and serrated adenomas, most notably BRAF mutations and CpG island methylation [2]. Prevalence and Clinicopathologic Characteristics of Serrated Adenomas Serrated adenomas are uncommon relative to conventional adenomas and hyperplastic polyps. In a recent colonoscopic survey, the proportion of polyps classified as serrated adenomas was 2.4%, compared with 59.7% for conventional adenomas and 38% for hyperplastic polyps, including the SSA variant (9%) [56]. Longacre and Fenoglio-Preiser’s [75] original report identified only 101 serrated adenomas (less than 0.6%) among over 18,000 polyps, and

Fig. 2. Serrated adenoma (dysplastic serrated polyp). This is an example of a dysplastic serrated polyp that has a BRAF mutation. (A) Low power magnification shows a serrated adenoma with contiguous sessile serrated adenoma (SSA). The serrated adenoma is characterized by dysplasia involving the surface and upper crypts that also exhibit a hyperplastic polyp-like serrated architecture. (B) Higher-power detail illustrates the contrast between the nondysplastic SSA and the stratified nuclei of the adjacent dysplastic serrated polyp.

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Fig. 3. Serrated adenoma (dysplastic serrated polyp)-KRAS mutation. (A) Low-power magnification reveals both a serrated dysplastic component (long arrow, lower) and a dysplastic component that resembles a conventional tubular adenoma (short arrow, higher). (B) Higher magnification contrasts serrated and nonserrated tubular glands (center). (C) Detail showing serrated dysplastic gland at left and contiguous conventional tubular adenomatous glands. At upper right, a gland cross section shows a transitional appearance.

a Japanese study reported 193 serrated adenomas (1.8%) among 10,532 polyps reviewed [77]. Serrated adenomas are estimated variously to occur in 1% to 7% of patients, and to represent 1.3% to 11% of all adenomas [78–80]. In a recent review of the literature, Huang and colleagues [80] reported that serrated adenomas are more common in men than women (approximately 2:1), and are diagnosed at a mean age at of 60 to 65 years.

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In contrast to SSAs, which occur predominantly in the proximal colon, serrated adenomas are more common in the distal colon [79,81,82]. They may be flat, sessile or polypoid, and may be tubular, tubulovillous, or villous. On one end of the morphologic spectrum, these lesions may be difficult to distinguish from SSAs, while on the opposite end, serrated adenomas may resemble conventional tubulovillous or villous adenomas closely. Oka and colleagues [83] surveyed the clinicopathologic and endoscopic features of a large series of 357 serrated adenomas; 240 (67%) were polypoid, and 127 (33%) were superficial (sessile or flat). The superficial serrated adenomas were significantly larger than polypoid serrated adenomas (mean 10.1 mm versus 6.3 mm) and were more common proximally. A tubulovillous growth pattern was common in polypoid tumors (31.5%). In terms of reproducibility of histological classification Bariol and colleagues [78] reported that two serrated polyp features, serrated component greater than 20%, and surface epithelial dysplasia were reproducible and accurate in identifying serrated adenoma using a group consensus classification as the gold standard. Recently, Snover and colleagues [57] proposed that serrated adenomas originating in SSAs in which areas of low-grade dysplasia or high-grade dysplasia (HGD) had developed should be distinguished from serrated adenomas that meet the WHO definition. The term traditional serrated adenoma was suggested as a name for the latter group of polyps. In this article, the term serrated adenoma is used to describe any dysplastic serrated polyp, and, thus, encompasses both the WHO definition of serrated adenoma and SSAs that have acquired areas of histologic dysplasia, unless specifically designated otherwise (see Figs. 2 and 3). Molecular Pathology of Serrated Adenomas The significance of serrated adenomas lies in their potential role as the immediate precursors of approximately 20% of all colorectal cancers. Further elucidation of the molecular genetics of this advanced serrated polyp, and correlation of histological features with molecular profiles and clinical outcomes, will be helpful in developing a clinically relevant pathologic classification scheme. BRAF-mutated serrated adenomas BRAF mutation is found in approximately two thirds of serrated adenomas and links this type of polyp to its presumptive precursors, SPAP, MVSP, and serrated ACFs, and to its potential successor lesion, MSI adenocarcinoma of the colon [2,47]. Contiguous SSA is encountered in approximately 50% of BRAF-mutated serrated adenomas, and these polyps exhibit similar BRAF mutations (or wild-type status) in both histological components [2]. CIMP-H status is also a molecular genetic characteristic of serrated adenomas that have a BRAF mutation [11,12,23]. Park and colleagues [11] noted concordant methylation of two or more of five assayed gene promoter sites (CIMP-high) in 68% of serrated adenomas, compared with 18% in a control series of conventional tubular adenomas. A more recent study showed

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CIMP-high was present in 80% of serrated adenomas, compared with 44% of large conventional adenomas and 10% of small conventional adenomas [2]. When a sensitivity analysis was performed, a marker level of four or more positive markers was demonstrated in 30% of serrated adenomas, compared with 0% of conventional (APC pathway) adenomas. Finally Yang and colleagues [12], using a multivariate analysis, identified the following as independent predictors of CIMP-H status among all serrated polyps: advanced histology (serrated adenoma versus hyperplastic polyps), proximal location in the colorectum, larger size (greater than 5 mm), and BRAF versus KRAS mutation. Dong and colleagues [84] used a panel of 16 genes to evaluate CpG island methylation in 42 serrated adenomas and showed that higher frequencies of marker genes showing CpG island methylation were more likely to be found in proximal, compared with distal, serrated adenomas and were also significantly associated with the development of high grade dysplasia (HGD). Goldstein studied HGD and early adenocarcinoma in eight serrated polyp resections with microsatellite unstable adenocarcinomas [60]. Among the notable findings in these, presumptively BRAF-mutated serrated polyps, was the presence of MSI-high in six of six informative areas of HGD within malignant serrated polyps. Areas of the serrated polyps that lacked HGD or carcinoma were not MSI-high. These findings are in agreement with those of a recent study [2] of MSI-high status in a large series of serrated polyps that included MVSPs, SSAs, serrated adenomas (dysplastic serrated polyps) and 11 invasive carcinomas with contiguous residual serrated adenoma. MSI was identified in 9 of 11 of the carcinomas, seven of which had BRAF mutations. It also was demonstrated in the contiguous serrated adenoma in three cases, and in areas of high-grade dysplasia. MSI was not present in MVSPs, SSAs, or in the serrated adenomas that did not have a contiguous carcinoma. There is a growing consensus that acquisition of MSI-high is usually a late event in the serrated polyp pathway, and it is likely to signal the development of HGD or carcinoma. KRAS-mutated serrated adenomas The occurrence of KRAS mutation in 20% to 30% of dysplastic serrated polyps provides the main evidence for the existence of a KRAS serrated polyp pathway. The KRAS-mutated version of this pathway, however, has proven to be a more elusive target for investigators than its BRAF-mutated counterpart. In published series, serrated polyps with dysplasia (serrated adenomas, including mixed polyps) show a frequency of KRAS mutations ranging from 15% to 36% [2,24,47,56,85]. One might expect KRAS-mutated MSS, or MSI-low carcinomas, which represent approximately 30% of all colorectal carcinomas [86], to include some proportion that have arisen from KRAS-mutated serrated adenomas. Colorectal carcinomas with KRAS mutations that develop in the conventional APC pathway can be excluded (theoretically) by identifying truncating mutations of the APC gene or mutations of beta-catenin gene. This would leave a residual group of KRAS-mutated cancers of presumed serrated histogenesis.

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KRAS-mutated colorectal carcinomas that develop in serrated adenomas are estimated to represent approximately 5% of all colorectal carcinomas, or 25% of all colorectal carcinomas of serrated polyp histogenesis (Fig. 4) [3]. As KRAS-mutated dysplastic serrated polyps progress, they appear to acquire fusion characteristics [47], a term originally coined by Jass, meaning a molecular profile that overlaps with the conventional APC pathway. Overlapping features include MSS or MSI-low, rather than MSI-high, and frequent loss of heterozygosity, reflecting some level of chromosomal instability. Low levels of CpG island methylation (CIMP-low) occur in KRAS-mutated serrated adenomas and carcinomas, but this feature also is shared with KRAS mutation bearing APC pathway carcinoma [47]. The lower level of CIMP encountered in KRAS-mutated carcinomas appears to be qualitatively and quantitatively different from CIMP-high associated with BRAF-mutated carcinomas and sporadic MSI [3]. In the setting of KRAS mutation, CIMP preferentially targets a different group of genes for promoter methylation and inactivation. One such gene associated with MSI-low status is the DNA repair gene, 0-6-methylguanine DNA methyltransferase (MGMT), whose protein product frequently

Fig. 4. Stratification of colorectal carcinomas according to histological precursor pathway (serrated polyp pathway/APC adenoma–carcinoma sequence), key mutation(s) (BRAF/ KRAS/APC), degree of CpG-island methylation (CIMP-H, CIMP-low and CIMP-negative), microsatellite instability status (MSI-H, MSI-L, and MSS), and the presence or absence of chromosomal instability (CSI/LOH/aneuploidy, CSI-negative). The relative proportions assigned to the categories within and between strata are approximations. The schema are based on data from Jass JR. Classification of colorectal cancer based on correlation of clinical, morphological, and molecular features. (Data from Jass JR. Classification of colorectal cancer based on correlation of clinical, morphological, and molecular features. Histopathology 2007;50:113–30.)

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is lost in the nuclei of KRAS-mutated advanced serrated adenomas and carcinomas [47]. It has been postulated that MGMT inactivation may contribute to point mutations because of failure of excision and repair of G;C to A;T transition, and, ultimately, to chromosomal instability promoting deletions and LOH [3]. Overlap in molecular profiles of serrated and APC pathway KRAS-mutated colorectal adenomas also may be seen at the histological level. For instance, Jass divided a series of dysplastic serrated polyps into two groups. Group A included a nondysplastic, but serrated, component, and/or dysplastic epithelium in which the architectural and cytological changes were more reminiscent of hyperplastic polyps than of an adenoma (see Fig. 2). Group B polyps comprised serrated polyps in which the epithelial dysplasia appeared adenomatous (see Fig. 3). KRAS was associated significantly with group B polyps, whereas BRAF was prevalent in group A polyps [47]. These findings are in accord with the author’s own published observations [2] and experience. Therefore KRAS serrated polyps exhibit morphological and molecular overlap with conventional colorectal adenomas. As KRAS-mutated dysplastic serrated polyps progress toward carcinoma, they may acquire a tubulovillous or villous phenotype that makes their distinction from conventional adenomas difficult or impossible. Both BRAF- and KRAS-serrated adenomas may show adenomatous transformation with the development of HGD. In BRAF-serrated adenomas, this late transformation may be associated with acquisition of MSI status [2,60]. In KRAS-mutated serrated adenomas, HGD frequently exhibits p53 mutation in association with loss of nuclear expression of MGMT [47]. Although there is cogent evidence that the immediate precursors of BRAFmutated dysplastic serrated polyps are SSAs, the immediate precursor of KRAS-mutated serrated adenomas remains an enigma [2]. Among the candidates are GCSP and null (BRAF and KRAS wild-type) MVSPs. Larger KRAS-mutated hyperplastic polyps with disordered proliferation similar to that seen in BRAF-mutated SSAs and plausible successors to GCSPs are infrequent if they occur. An alternative scenario, which also remains unproven, is that KRAS mutation is not an instigating mutation in the serrated pathway, but acquired at an intermediate stage similar to large adenomas in the APC pathway [2]. Because KRAS and BRAF are mutually exclusive, this last proposal requires that KRAS mutation develop in a null MVSP, and induce transformation, or dysplasia, in this overtly serrated lesion. Finally, there are also advocates [47] for the hypothesis that KRAS-mutated serrated polyps evolve from conventional adenomas, and that the KRAS mutation is responsible for the serration in these polyps (rather than villous change) [87]. For this third alternative to be tenable, a proportion of KRAS-mutated dysplastic serrated polyps should exhibit the specific molecular marker of conventional adenomas, namely truncating mutations of the APC gene. INHERITED PREDISPOSITION TO SERRATED CARCINOGENESIS Hyperplastic polyposis syndromes, as described elsewhere in this issue by Jass [88], may represent a model for the pathogenesis of the serrated polyp

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pathway, in the way that familial adenomatous polyposis syndrome has provided fundamental insights into the adenoma–carcinoma sequence. Hyperplastic polyposis frequently, but not invariably, (50%) exhibits familial clustering [45]; it is linked to CpG island methylation and characterized by multiple serrated polyps distributed throughout the colon, and coexisting smaller numbers of dysplastic serrated polyps and conventional adenomas [55,89]. Recent observations of affected sibships suggest a recessive or codominant mode of inheritance [90]. Sporadic MSI and or CIMP-H carcinomas frequently are associated with synchronous multiple and proximally located serrated polyps [91,92], suggesting a field phenomenon related to serrated polyp progression that favors a genetic predisposition [90]. In brief, Young and colleagues [89,90] have proposed that different levels of genetic susceptibility to CIMP may reflect the distribution of a codominant gene that impinges on CpG-island methylation susceptibility in the population. Carriers of one codominant allele may number up to 1 in 25, and such individuals are susceptible to the development of advanced BRAF-mutated serrated polyps, particularly in the proximal colon [89]. SERRATED POLYP PATHWAY-ASSOCIATED COLORECTAL ADENOCARCINOMAS Serrated polyp pathway endpoints represent an estimated 20% or more of all colorectal carcinomas (see Fig. 4) [3]. These tumors encompass several different molecular profiles. An overarching molecular signature of serrated pathway cancers is CpG island methylation, however, although this phenomenon is not unique to the serrated pathway, because it also encountered in a proportion of colorectal carcinomas of the conventional or APC pathway [14]. Within the serrated pathway of colorectal cancers, BRAF mutation represents a specific molecular marker, because it is otherwise restricted to serrated polyp precursors [2]. Furthermore, it is an early, or instigating, mutation equivalent in that role to APC mutations in the adenoma–carcinoma sequence, and it is rarely, if ever, encountered in conventional adenomas or carcinomas [2,47]. Colorectal carcinoma with BRAF mutation has been shown to be strongly associated (odds ratio greater than 200) with CIMP-high status [8,93]. Most of the 10% to 15% of sporadic colorectal carcinomas that are MSI-high prove to have BRAF mutations. Approximately 25% of sporadic MSI-high carcinomas are null but also CIMP-high. A small fraction of BRAF colorectal cancers are CIMP-high and MSS [13]. According to Samowitz and colleagues [13], patients who have tumors belonging to this category are apt to have a family history of colon cancer. KRAS is a marker for MSS serrated pathway cancers, but it is not specific, because it is found even more frequently in APC pathway cancers, and it is present in upwards of 30% of all colorectal cancers [86]. It is likely, however, that most serrated pathway cancers that are wild-type BRAF exhibit KRAS mutations if one extrapolates from the distribution of these oncogene mutations in the precursor dysplastic serrated polyps (serrated adenomas). Furthermore, these cancers are likely to be CIMP-low and either diploid or near-diploid, and also show some characteristics of the APC pathway such as LOH of suppressor genes [47].

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The APC pathway carcinomas represent the prototypical, and most prevalent colorectal carcinomas. They are distinguished by APC gene mutations and disruption of Wnt signaling. Frequently, they exhibit KRAS mutations. Invariably they demonstrate LOH and chromosomal deletions involving myriad genes including key suppressor genes, chromosomal instability, and aneuploidy [1,94]. The APC pathway also includes colorectal carcinomas with low levels of CpG island methylation (CIMP-low) and MSI carcinomas in patients who have hereditary nonpolyposis colorectal cancer [3]. These latter, representing 1% to 3% [3,95] of colorectal cancers, develop in conventional APC adenomas that acquire a mutation of the second MMR gene and consequent MSI in contrast to sporadic MSI cancers, which develop in dysplastic serrated polyps. Stratification of colorectal carcinomas according to a schema recently suggested by Jass [3] is presented in Fig. 4. CLINICAL GUIDELINES FOR MANAGEMENT AND SURVEILLANCE OF SERRATED POLYPS The most recent iteration of surveillance guidelines promulgated by the US National Taskforce on Colon Cancer [96] notes the emergence of the understanding of the serrated polyp pathway, and the likely association of advanced lesions of this pathway, such as SSA, with MSI carcinoma. In the absence of authoritative clinical studies, however, they stop short of offering specific recommendations for serrated polyp management and surveillance [96]. In a recent review, Huang and colleagues [80] noted that the current practice in the United States of removing all except the most diminutive polyps identified at colonoscopy for histologic identification makes the issue of removal of hyperplastic polyps encountered at endoscopy somewhat moot. Although it is impractical to advocate removal of every minute HP, especially those that frequently carpet the distal sigmoid and rectum, there are clinical and endoscopic features that may indicate which type of polyps warrant special attention [80]. Proximally located polyps, even those with endoscopic features reported to be highly predictive of hyperplastic histology [72,97], should be resected completely if possible, because dysplastic epithelium may be present [98]. In a discerning study performed by Rex and Ulbright in 2002 [98], step sections of a series of 51 large sessile proximally located hyperplastic polyps most (likely representing SSAs), revealed an adenomatous or dysplastic component in 4% of lesions. Goldstein, in a recent preliminary report, identified HGD in 5% of SSAs using a rigorous histological definition of SSA that required the presence of more than 50% disordered or dysmaturational crypts [99]. These preliminary studies help to establish level of risk that exists among large sessile, or flat, hyperplastic-appearing lesions of the colon, or among hyperplastic polyps with the histological features of SSAs. They also highlight the need for a uniform and reproducible pathological classification of serrated polyps. Although surveillance following removal of isolated hyperplastic polyps is not recommended [96], a strong case can be made that some level of

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surveillance of patients following removal of SSAs may be prudent, particularly if there are attendant risk factors such as large size, multiple additional hyperplastic polyps, proximal location, or if there is a family history of colorectal carcinoma [80]. Unfortunately, type and frequency of surveillance are unclear, but until further data are available, endoscopists should probably consider treating high-risk hyperplastic polyps and SSAs similar to conventional small adenomas. The time frame for progression of SSA to dysplasia and ultimately to carcinoma is not known. A notable statistic from the Rex study [98], however, was that the mean age of SSA patients was 56 years, whereas the mean age of patients with incident MSI carcinomas was approximately 75 years [100]. A more intensive surveillance and automatic or default status of advanced adenoma, may be appropriate for dysplastic serrated polyps, given the potential biological proximity of these neoplasms to microsatellite instability and the consequent possibility of more rapid progression to carcinoma.

SUMMARY The serrated polyp pathway is outlined in Fig. 5. A BRAFV600E mutation is a specific marker for a serrated polyp pathway that originates in an MVSP or a serrated ACF and has a potential endpoint in a colonic adenocarcinoma that is CIMP-high and, in most cases, also MSI. There is evidence that CIMP is the molecular engine that drives the progression through the sequential steps from MVSP and SSA, to serrated adenoma and, ultimately, to serrated carcinoma. Studies of hyperplastic polyposis syndromes suggest that the propensity to epigenetic mutations of growth control genes in BRAF-mutated hyperplastic polyps, and successor polyps, particularly those proximally located in the colon, may be governed by regulatory mechanisms that have an important inherited genetic basis, although environmental factors, such as cigarette smoking, are likely to be operative also. Progression of serrated polyps to carcinoma has been proven, but the actual level of risk for progression at the multiple steps in the pathway is unknown. The evolution of hyperplastic polyps to carcinoma is neither common, nor inevitable, and is likely to require an extended time period, perhaps at least 10 to 20 years, for it to be fully realized in susceptible individuals. A second serrated pathway, identified by mutation of KRAS in a serrated adenoma, is delineated less completely. Its endpoint is a colorectal carcinoma that is CIMP-low and MSS, and both the advanced serrated adenoma and carcinoma stages of this pathway show molecular genetic and morphologic features that overlap with those of the conventional APC pathway, such as LOH and p53 mutations. These two molecular models of colorectal carcinoma evolution provide a framework for future studies of the phenomenon of CpG-island methylation, and the specific molecular genetic events that govern transition and progression in the serrated polyp pathway. Clinical studies are needed to elucidate the timeframe for serrated neoplasia progression, and provide evidence-based guidance for risk assessment and surveillance of affected individuals.

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Fig. 5. (A) BRAF mutated serrated polyp pathway. The diagram depicts BRAFV600E as an early or instigating mutation originating in a serrated ACF; CpG-island methylation is shown to be increasing with advancing histological stage, and MSI-H is occurring late, at the interface of serrated adenoma, high-grade dysplasia (HGD), and invasive carcinoma (SA/CA). (B) KRAS mutated Serrated Polyp Pathway. KRAS mutated serrated adenoma progresses to a mixed tubulovillous adenomatous phenotype (TVA) and acquires high-grade dysplasia (HGD). The progression is contributed to by CIMP-low, followed later by the development of chromosomal instability and LOH of key suppressor genes. The interface of HGD and infiltrating carcinoma (SA/CA) is associated with p53 mutation.

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[50] Minoo P, Jass JR. Senescence and serration: a new twist to an old tale. J Pathol 2006;210: 137–40. [51] Michaloglou C, Vredeveld LC, Soengas MS, et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 2005;436:720–4. [52] Kaye GI, Fenoglio CM, Pascal RR, et al. Comparative electron microscopic features of normal, hyperplastic, and adenomatous human colonic epithelium. Variations in cellular structure relative to the process of epithelial differentiation. Gastroenterology 1973;64: 926–45. [53] Hayashi T, Yatani R, Apostol J, et al. Pathogenesis of hyperplastic polyps of the colon: a hypothesis based on ultrastructure and in vitro cell kinetics. Gastroenterology 1974;66: 347–56. [54] Minoo P, Baker K, Goswami R, et al. Extensive DNA methylation in normal colorectal mucosa in hyperplastic polyposis. Gut 2006;55:1467–74. [55] Chan AO, Issa JP, Morris JS, et al. Concordant CpG island methylation in hyperplastic polyposis. Am J Pathol 2002;160:529–36. [56] Spring KJ, Zhao ZZ, Karamatic R, et al. High prevalence of sessile serrated adenomas with BRAF mutations: a prospective study of patients undergoing colonoscopy. Gastroenterology 2006;131:1400–7. [57] Snover DC, Jass JR, Fenoglio-Preiser C, et al. Serrated polyps of the large intestine: a morphologic and molecular review of an evolving concept. Am J Clin Pathol 2005;124: 380–91. [58] Oh K, Redston M, Odze RD. Support for hMLH1 and MGMT silencing as a mechanism of tumorigenesis in the hyperplastic-adenoma-carcinoma (serrated) carcinogenic pathway in the colon. Hum Pathol 2005;36:101–11. [59] Iino H, Jass JR, Simms LA, et al. DNA microsatellite instability in hyperplastic polyps, serrated adenomas, and mixed polyps: a mild mutator pathway for colorectal cancer? J Clin Pathol 1999;52:5–9. [60] Goldstein NS. Small colonic microsatellite unstable adenocarcinomas and high-grade epithelial dysplasias in sessile serrated adenoma polypectomy specimens: a study of eight cases. Am J Clin Pathol 2006;125:132–45. [61] DiSario JA, Foutch PG, Mai HD, et al. Prevalence and malignant potential of colorectal polyps in asymptomatic, average-risk men. Am J Gastroenterol 1991;86:941–5. [62] Farraye FA, Wallace M. Clinical significance of small polyps found during screening with flexible sigmoidoscopy. Gastrointest Endosc Clin N Am 2002;12:41–51. [63] Lieberman DA, Smith FW. Screening for colon malignancy with colonoscopy. Am J Gastroenterol 1991;86:946–51. [64] Brady PG, Straker RJ, McClave SA, et al. Are hyperplastic rectosigmoid polyps associated with an increased risk of proximal colonic neoplasms? Gastrointest Endosc 1993;39: 481–5. [65] Weston AP, Campbell DR. Diminutive colonic polyps: histopathology, spatial distribution, concomitant significant lesions, and treatment complications. Am J Gastroenterol 1995;90:24–8. [66] Tedesco FJ, Hendrix JC, Pickens CA, et al. Diminutive polyps: histopathology, spatial distribution, and clinical significance. Gastrointest Endosc 1982;28:1–5. [67] Waye JD, Lewis BS, Frankel A, et al. Small colon polyps. Am J Gastroenterol 1988;83: 120–2. [68] Waye JD, Bilotta JJ. Rectal hyperplastic polyps: now you see them, now you don’t—a differential point. Am J Gastroenterol 1990;85:1557–9. [69] Chapuis PH, Dent OF, Goulston KJ. Clinical accuracy in the diagnosis of small polyps using the flexible fiberoptic sigmoidoscope. Dis Colon Rectum 1982;25:669–72. [70] Norfleet RG, Ryan ME, Wyman JB. Adenomatous and hyperplastic polyps cannot be reliably distinguished by their appearance through the fiberoptic sigmoidoscope. Dig Dis Sci 1988;33:1175–7.

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[71] Anderson JC, Pollack BJ. Predicting of hyperplastic histology by endoscopic features. Gastrointest Endosc 2000;52:149–50. [72] Rex DK, Rahmani EY. New endoscopic finding associated with hyperplastic polyps. Gastrointest Endosc 1999;50:704–6. [73] Rothstein RD, Kochman M. Large hyperplastic polyps of the right colon. Gastrointest Endosc 1998;47:211–2. [74] Langdon DE. Large hyperplastic polyps of the right colon. Gastrointest Endosc 1998;48: 659. [75] Longacre TA, Fenoglio-Preiser CM. Mixed hyperplastic adenomatous polyps/serrated adenomas. A distinct form of colorectal neoplasia. Am J Surg Pathol 1990;14:524–37. [76] Aaltonen LA, Hamilton SR. World Health Organization, International Agency for Research on Cancer. Pathology and genetics of tumours of the digestive system. Lyon, Oxford: IARC Press; Oxford University Press (distributor); 2000. [77] Iwabuchi M, Sasano H, Hiwatashi N, et al. Serrated adenoma: a clinicopathological, DNA ploidy, and immunohistochemical study. Anticancer Res 2000;20:1141–7. [78] Bariol C, Hawkins NJ, Turner JJ, et al. Histopathological and clinical evaluation of serrated adenomas of the colon and rectum. Mod Pathol 2003;16:417–23. [79] Matsumoto T, Mizuno M, Shimizu M, et al. Clinicopathological features of serrated adenoma of the colorectum: comparison with traditional adenoma. J Clin Pathol 1999;52: 513–6. [80] Huang CS, O’Brien MJ, Yang S, et al. Hyperplastic polyps, serrated adenomas, and the serrated polyp neoplasia pathway. Am J Gastroenterol 2004;99:2242–55. [81] Higuchi T, Sugihara K, Jass JR. Demographic and pathological characteristics of serrated polyps of colorectum. Histopathology 2005;47:32–40. [82] Jaramillo E, Watanabe M, Slezak P, et al. Flat neoplastic lesions of the colon and rectum detected by high-resolution video endoscopy and chromoscopy. Gastrointest Endosc 1995;42:114–22. [83] Oka S, Tanaka S, Hiyama T, et al. Clinicopathologic and endoscopic features of colorectal serrated adenoma: differences between polypoid and superficial types. Gastrointest Endosc 2004;59:213–9. [84] Dong SM, Lee EJ, Jeon ES, et al. Progressive methylation during the serrated neoplasia pathway of the colorectum. Mod Pathol 2005;18:170–8. [85] Sawyer EJ, Cerar A, Hanby AM, et al. Molecular characteristics of serrated adenomas of the colorectum. Gut 2002;51:200–6. [86] Samowitz WS, Curtin K, Schaffer D, et al. Relationship of Ki-ras mutations in colon cancers to tumor location, stage, and survival: a population-based study. Cancer Epidemiol Biomarkers Prev 2000;9:1193–7. [87] Maltzman T, Knoll K, Martinez ME, et al. Ki-ras proto-oncogene mutations in sporadic colorectal adenomas: relationship to histologic and clinical characteristics. Gastroenterology 2001;121:302–9. [88] Jass J. Gastrointestinal polyposes: a guide to diagnosis and classification based on clinical, pathological, and molecular features. Gastroenterol Clin North Am 2007, in press. [89] Young JP, Jenkins MA, Parry S, et al. Serrated pathway colorectal cancer in the population: an alternative to the adenoma–carcinoma sequence. Gut 2007;56(10):1453–9. [90] Young J, Jass JR. The case for a genetic predisposition to serrated neoplasia in the colorectum: hypothesis and review of the literature. Cancer Epidemiol Biomarkers Prev 2006;15: 1778–84. [91] Hawkins NJ, Ward RL. Sporadic colorectal cancers with microsatellite instability and their possible origin in hyperplastic polyps and serrated adenomas. J Natl Cancer Inst 2001;93:1307–13. [92] Goldstein NS, Bhanot P, Odish E, et al. Hyperplastic-like colon polyps that preceded microsatellite-unstable adenocarcinomas. Am J Clin Pathol 2003;119:778–96.

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[93] Samowitz WS. The CpG island methylator phenotype in colorectal cancer. J Mol Diagn 2007;9:281–3. [94] Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med 2004;10:789–99. [95] Samowitz WS, Curtin K, Lin HH, et al. The colon cancer burden of genetically defined hereditary nonpolyposis colon cancer. Gastroenterology 2001;121:830–8. [96] Winawer SJ, Zauber AG, Fletcher RH, et al. Guidelines for colonoscopy surveillance after polypectomy: a consensus update by the US Multi-Society Task Force on Colorectal Cancer and the American Cancer Society. CA Cancer J Clin 2006;56:143–59. [97] Sonwalker S, Rotimi O, Rembacken BJ. Characterization of colonic polyps at conventional (nonmagnifying) colonoscopy after spraying with 0.2% indigo carmine dye. Endoscopy 2006;38(12):1218–23. [98] Rex DK, Ulbright TM. Step section histology of proximal colon polyps that appear hyperplastic by endoscopy. Am J Gastroenterol 2002;97:1530–4. [99] Goldstein NS. Morphologic features of high-grade serrated dysplasia in sessile serrated adenomas without coexistent invasive adenocarcinoma. Lab Invest 2007;87(Suppl 1): A115. [100] Young J, Simms LA, Biden KG, et al. Features of colorectal cancers with high-level microsatellite instability occurring in familial and sporadic settings: parallel pathways of tumorigenesis. Am J Pathol 2001;159:2107–16.

Gastroenterol Clin N Am 36 (2007) 969–987

GASTROENTEROLOGY CLINICS OF NORTH AMERICA

Anal Intraepithelial Neoplasia and Other Neoplastic Precursor Lesions of the Anal Canal and Perianal Region Neil A. Shepherd, DM, FRCPath Department of Histopathology and Cranfield Postgraduate Medical School in Gloucestershire, Gloucestershire Royal Hospital, Great Western Road, Gloucester, GL1 3NN, UK

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pithelial tumors of the anal canal and perianal skin are relatively uncommon, accounting for only about 2% of all large intestinal malignancies [1], with an incidence of about 0.7 per 100,000 men and 0.9 per 100,000 women in the United States [2] and 0.5 per 100,000 in the United Kingdom [3]. This equates to only about 4000 cases per year in the United States [4]. There is evidence that anal squamous neoplasia is increasing in incidence, particularly in certain high-risk groups, most notably homosexuals, either with or without HIV/AIDS [5], and women with multifocal anogenital neoplasia [6]. Tumors that occur in the anal region represent a variety of histologic types [7]. The interested reader is referred to other texts for a detailed account of the microanatomy and histology of the anorectal region, which is a prerequisite for the proper understanding of the classification of epithelial lesions of this region [8,9]. Tumors that arise in the anal canal are significantly different from those in the perianal skin [10]. The most common tumor of the anal canal is nonkeratinizing variant of squamous cell carcinoma. On the other hand, neoplastic entities of the perianal region emulate their cutaneous counterparts: the most common malignancies are basal cell carcinoma and keratinizing squamous cell carcinoma. Perianal tumors usually require less aggressive treatment. However, squamous dysplasia often occurs at both sites in individual patients [7,11]. There is now a better understanding of pre-neoplastic entities that predispose to malignant tumors that occur in the anal region. Probably best understood, in terms of its causation, pathology, and natural history, is squamous dysplasia, now almost universally classified as anal intraepithelial neoplasia (AIN). On the other hand, little is known regarding pre-neoplastic conditions that lead to glandular malignancy in the anal region, and little or nothing is known about melanocytic pre-neoplastic pathology in the anal canal, despite malignant melanoma being a well-recognized, if unusual, primary tumor arising at this site.

E-mail address: [email protected] 0889-8553/07/$ – see front matter doi:10.1016/j.gtc.2007.08.001

Crown Copyright ª 2007 Published by Elsevier Inc. All rights reserved. gastro.theclinics.com

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Our lack of knowledge stems from the rarity of these tumors. The most common glandular (pre-)neoplastic condition encountered in this region is Paget’s disease, which shows characteristic clinical features, although its pathology has important differential diagnoses that usually require investigation by histochemistry and immunohistochemistry. This review focuses mainly on AIN, its cause, diagnosis, and natural history, but also touches on other rare pre-neoplastic entities that occur in the anal region. ANAL INTRAEPITHELIAL NEOPLASIA Clinical Features and Pathogenesis The incidence of AIN is unknown. However, it is generally believed that its incidence is increasing, especially in high-risk patients. High-risk patients include male homosexuals. For instance, in one study, the incidence rate of AIN was 52% in HIV-positive male homosexuals, and 17% in those who were HIV negative [12]. The same study demonstrated relatively rapid advancement of the disease to high-grade AIN within 2 years of diagnosis [12]. AIN shows epidemiological and pathological similarities with cervical intraepithelial neoplasia and vulval intraepithelial neoplasia [13,14]. In fact, women with multifocal genital intraepithelial neoplasia also demonstrate a high incidence rate of AIN (up to 20%) [15]. The association between AIN, cervical intraepithelial neoplasia, and vulval intraepithelial neoplasia is believed to be related to human papillomavirus (HPV) infection [16–18]. HPV infection was originally suspected as a major cause of anal carcinoma 40 years ago when electron microscopy demonstrated viruslike particles in anal cancer tissue [19]. Subsequently, the increased incidence rate of anal cancer in homosexuals supported a sexually transmitted agent [20]. In fact, the association of anal carcinoma with condylomata acuminata was already known to be linked to HPV infection [21]. In the late 1980s and 1990s, several studies demonstrated the presence of high-risk subtypes of HPV, by polymerase chain reaction, in anal carcinoma. Interestingly, high-risk subtypes (especially types 16 and 18) are more common in anal canal neoplasia than in perianal lesions [16]. Polymerase chain reaction techniques have shown that both AIN 3 and invasive anal cancer show very high rates of these high-risk HPV types [22–25]. Initially, HPV types 16 and 18 were most strongly implicated in the pathogenesis of anal neoplasia [22,26,27]. However, it is now known that many other high-risk HPV subtypes are involved in the genesis of anal squamous neoplasia. There is also evidence that different strains of HPV viruses may be associated with different risks of cancer [28]. HIV/AIDS is well established as a major risk factor for anal neoplasia [29–31]. In fact, both AIDS and anal warts are independent risk factors of HPV infection [5]. In one study, 92% of HIV-positive homosexuals demonstrated evidence of anal HPV infection compared with 66% of those who were HIV negative [32]. Similarly, HIV-positive women also showed higher rates of HPV infection than HIV-negative women [33,34]. Women with

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multifocal genital intraepithelial neoplasia demonstrate a 16-fold increase in the rate of AIN [33]. Immunosuppression has also been proposed as a major etiologic factor in the development of anal neoplasia. Unfortunately, there is little evidence that highly active anti-retroviral therapy (HAART) has an appreciable effect on the incidence of AIN, despite its efficacy in restoring immunological function and reducing opportunistic infections in HIV/AIDS patients [34–37]. For instance, the incidence of anal cancer continues to increase in HIV-positive homosexual males, despite widespread use of HAART in this group of individuals [38]. Anal neoplasia has also been shown to be associated with other non-HIV/ AIDS causes of immunosuppression. For instance, there is a 10- to 100-fold increased risk of anogenital neoplasia in solid organ transplant patients, with females being twice as susceptible as males [39,40]. Clinical Diagnosis AIN is more likely to develop in the upper anal canal and anal transition zone than in the lower anal canal and perianal region [10,41,42]. However, many patients show involvement of both areas, as well as the perianal skin, and this underscores the importance of widespread sampling to establish a pathologic diagnosis [10,43]. Clinically, AIN may not show any specific features. Histology and/or cytology remain the primary methods of diagnosis [28]. Various macroscopic appearances have been described for AIN, such as raised erythematous mucosa, white scaling mucosa, pigmented mucosa or ulceration. A verrucous macroscopic appearance of AIN (Fig. 1) has been associated with the highest risk of malignant transformation [44]. However, most patients with AIN do not show any gross changes, which makes early diagnosis difficult. Anal colposcopy has been advocated as a method to assess the extent of AIN [45–47]. Using techniques similar to those applied in the uterine cervix, an

Fig. 1. Macroscopic appearance of verrucous AIN 3. This is a papillomatous lesion with surface hyperkeratosis. (Courtesy of J. A. Tidy, MD, Sheffield, UK.)

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operating low-power microscope provides a visual assessment of the anus. AIN may be identified as acetowhite epithelium contrasting with iodine-positive (brown) non-neoplastic mucosa. Several studies have demonstrated good correlation between anal colposcopy findings and pathology, especially at the low and high end of the AIN spectrum (ie, normal and AIN 3) [45,48]. However, colposcopy correlates poorly with HPV infection and low-grade AIN. In contrast, many centers rely on close surveillance with anoscopy and biopsies to assess the presence and extent of AIN. Anal cytology demonstrates good sensitivity rates for the detection of AIN. As a result, some investigators recommend this technique as a method for screening [49–52]. However, a limitation of this technique is that it does not identify the extent and distribution of disease [53]. Further, there is a relatively high rate of ‘‘atypical but nonspecific’’ findings (so-called ‘‘atypical squamous cells of undetermined significance’’), which typically results in the need for further biopsies. Pathologic (Microscopic) Features Fenger and Nielsen [10,41] are credited with the first histological description of AIN. AIN demonstrates variably thickened squamous epithelium composed of neoplastic (‘‘dysplastic’’) squamous cells (Fig. 2). These cells show a high nucleo-cytoplasmic ratio and are typically orientated perpendicular to the basement membrane. They fail to demonstrate the transverse orientation of maturing cells. This feature indicates cellular ‘‘dysmaturation’’ and is the most characteristic feature of AIN, although nuclear enlargement, irregularity, and hyperchromatism become especially prominent in higher grades of AIN. AIN also shows increased mitotic activity (see Fig. 2). Abnormal cell activity is characterized by individual cell keratinization and dyskeratosis. The latter is more likely to be seen in perianal AIN and has been especially associated with Bowen’s disease or Bowenoid papulosis of the anal region, in particular.

Fig. 2. Pathology of verrucous AIN 3. The mucosa is thickened and endophytic. Cellular dysmaturation is readily apparent and there is substantial mitotic activity (bottom right).

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However, the use of the terms Bowen’s disease or Bowenoid papulosis is not recommended because AIN is not causally related to classical Bowen’s disease of the skin and the terms thus may lead to confusion. Similar to the grading system used for cervical and vulval intraepithelial neoplasia, AIN grading is based on the level of involvement by dysplastic cells in the squamous mucosa [14,41,54]. Thus, AIN 1 is defined as involvement of less than one third of the height of the mucosa, AIN 2 as more than a third, but less than two thirds, and AIN 3 as involvement of more than two thirds of the thickness of the mucosa (Fig. 3). The term carcinoma-in-situ has been used synonymously with AIN 3 for full thickness dysplasia. However, most authorities now regard this term as obsolete and incorporate carcinoma-in-situ into the AIN 3 category. High-grade AIN shows a distinct affinity for involvement of skin adnexal structures in the perianal region and of anal gland epithelium in the anal canal. Deep involvement has particular significance for AIN management because involvement of adnexal structures by AIN may extend to a depth of at least 2 mm and, thus, may not be detected, or excised, during treatment [55]. The grading of AIN remains controversial. The original ‘‘three-tiered’’ grading system of AIN 1, AIN 2, and AIN 3 is used widely, although it is associated with considerable interobserver variation [4,51,55–57]. Levels of agreement are highest for AIN 3 [4,56] but the levels of agreement for AIN 1 and AIN 2 have led some to recommend the use of a Bethesda-type ‘‘two-tiered’’ system. In this system, AIN 1 and AIN 2 are combined into low-grade AIN. Meanwhile, AIN 3 is termed high-grade AIN [47]. The histological features of AIN are often combined with those associated with HPV infection. For instance, dyskeratosis and koilocytosis are often markers of HPV infection. Adjunctive Diagnostic Techniques Most surgical pathologists use standard morphology for diagnosis of AIN and accept a certain level of observer variation. In the United Kingdom,

Fig. 3. Pathology of AIN 2 (right) contrasting with adjacent nondysplastic epithelium (left). There is nuclear enlargement and suprabasal mitotic activity.

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‘‘high-grade’’ AIN cases are discussed at a multidisciplinary team meeting. In fact, in many centers, it is highly recommended that any AIN diagnosis be approved by a second expert gastrointestinal pathologist, which is similar to the process for diagnosing dysplasia in Barrett’s esophagus or inflammatory bowel disease. Until recently, there have not been any reliable ‘‘biomarkers’’ in the diagnosis of AIN. However, some investigators have shown that Ki-67 expression is useful for the confirmation of significant AIN disease (Fig. 4). A recent study by Bean and colleagues [58] has shown that combined Ki-67 and p16 (a protein with a logical influence on AIN presence and grade) expression is both a sensitive and a reliable marker for the diagnosis of high-grade AIN. Such markers, in conjunction with standard morphology, may help improve observer reproducibility in the diagnosis of AIN. Differentiation of AIN 3 from invasive squamous cell carcinoma can also be difficult and is clinically important. Microinvasive squamous cell carcinoma is a well-accepted concept in cervical and vulval pathology. However, in the anus, it is not well recognized, and no concrete criteria have been introduced for its diagnosis and management. Unfortunately, early invasive carcinoma closely mimics tangentially sectioned dysplastic squamous mucosa, making accurate distinction of these disorders difficult at times. Molecular Characteristics The importance of HPV infection in the genesis of AIN and anal cancer has already been emphasized. HPV carcinogenicity is largely due to the production of two oncoproteins, E6 and E7 [59,60]. These proteins inhibit p53 and Rb1, two important tumor suppression proteins, and thus have a direct influence on cell cycle function [61–63]. The activity of these two proteins is increased in high-risk HPV subtypes. p53 protein is overexpressed in high-grade AIN and in invasive squamous cell carcinoma [64]. The oncoprotein c-myc also shows overexpression in AIN 3 and anal cancer [65], whereas Ki-ras mutations are restricted to anal cancers not associated with HPV infection [66].

Fig. 4. MIB1 (Ki-67) immunohistochemistry in AIN 3 showing a very high positivity rate of nuclei throughout the full thickness of the mucosa.

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Cytogenetic abnormalities, such as deletion of chromosome 11q and 3p, have also been associated with anal cancer [67]. 3q chromosomal changes have also been described [68]. Such chromosomal abnormalities are also seen in cervical carcinoma and echo the close association between anal and female genital tract neoplasia. Natural History Institution of specific guidelines for the management of AIN is difficult because the implications and natural history of AIN are poorly understood. The concept of AIN as a precursor of invasive squamous cell carcinoma of the anus is supported by the coexistence of AIN adjacent to invasive cancer and by the fact that AIN 3, in particular, shows biological and molecular properties that are similar to those of anal cancer, notably enhanced angiogenesis, increased proliferation, decreased apoptosis, p53 mutations, and, of course, a strong association with high-risk HPV subtypes. In fact, low-grade AIN (AIN 1 and AIN 2) has been associated with a considerable regression rate, up to 30% [4,15,45]. However, it has been argued that this may simply reflect inconsistent interpretation of pathology because of inexperience or poor sample quality [4]. The natural history of AIN 3, particularly in those who are HIV positive, is also poorly understood. In Scholefield’s study, 35 patients with AIN 3 underwent long-term surveillance after excision [1]. Only 6 patients (all with multifocal disease) were immunosuppressed. Three of the latter developed invasive carcinoma in the follow-up period (maximum 10 years), but none of the immunocompetent patients developed cancer. The investigators concluded that immunocompromised patients were more likely to have multifocal disease and to progress to anal cancer [1]. Thus, while the rate of progression to invasive carcinoma remains largely unknown, certain high-risk groups, such as immunosuppressed patients, seem to have a higher rate of malignancy and, thus, probably demand closer surveillance. There are few data on the appropriate management of immunocompetent patients with AIN. Treatment Given the lack of information on the natural history of AIN, the management of AIN remains controversial. Enhanced surveillance is generally considered appropriate and should be augmented in high-risk groups. Many advocate that surveillance is best achieved by ‘‘anal colposcopy’’ with targeted biopsies. However, the uptake of anal colposcopy has generally been poor, certainly in the United Kingdom, where it has been advocated for only high-risk groups. Mapping biopsies provide the most useful assessment of the overall severity, extent, and distribution of AIN disease [43,53]. Most authorities recommend complete surgical excision of AIN [1,28]. However, many of these techniques have been associated with high rates of recurrence, although it could be that some of the ‘‘recurrences’’ merely represent residual disease [28]. Techniques include local excision, wide excision with skin grafting, wide excision with flap advancement, and biopsy with ablation

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[1,69–73]. The difficulties with surgical excision are enhanced in the HIV-positive high-risk patient group, perhaps reflecting the multifocal nature of the disease. In one study, all HIV-positive patients who underwent excision of AIN showed evidence of recurrence, although some recurrent cases may represent persistent disease [73]. Thus, it is generally advised that HIV-positive patients who remain at considerable risk of progression to malignancy, despite the advent of HAART, should undergo multiple staged ablation or excision procedures combined with enhanced surveillance [28]. A management algorithm for patients with AIN is summarized in Fig. 5. Patient management should be discussed individually with the patient and in a multidisciplinary fashion. There are several alternative therapeutic methods, other than surgery, including radiotherapy (both brachytherapy and external beam), immunomodulation, and HPV therapy/vaccines [28]. However, none of these have been accepted or tested widely. The management of patients with AIN is best achieved in centers with a special interest in this disease [28]. OTHER PRE-NEOPLASTIC SQUAMOUS PROLIFERATIONS OF THE ANAL REGION Tumorlike wart virus–associated squamous proliferations, such as condyloma acuminatum and giant condyloma of Buschke and Lowenstein, can be associated with AIN. The interested reader is referred to other sources for a comprehensive description of these disorders and their malignant potential [74–78]. Use of the terms Bowen’s disease and Bowenoid papulosis should be discouraged because the terms have engendered confusion, partly because they

Fig. 5. Flow chart for the management of high-risk and low-risk AIN.

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have been regarded as specific entities, akin to their cutaneous counterparts. However, they merely reflect different types of AIN (Fig. 6) [1,28]. Perianal Bowen’s disease resembles its cutaneous counterpart, appearing as red, crusted plaquelike areas with irregular edges [72,79]. Microscopically, Bowen’s disease shows similar features to AIN, although dyskeratosis is a particular feature commonly associated with this diagnosis (see Fig. 6) [72]. In the skin, Bowen’s disease is classically related to sun exposure, whereas in the anal region, highrisk HPV-types have been detected [80,81]. This evidence suggests that anal Bowen’s disease is best regarded as a subtype of AIN [71]. Bowenoid papulosis describes a papulomacular eruptive lesion in which the features are similar to those seen in cutaneous Bowen’s disease, although the lesions are more sharply circumscribed and slightly raised in the former [82]. As with Bowen’s disease, HPV, including high-risk subtypes, has been demonstrated in Bowenoid papulosis [83], and, thus, it is likely that this ‘‘condition’’ merely represents sharply circumscribed HPV-associated AIN, more commonly seen in younger patients (compared with anal Bowen’s disease). Perhaps due to its sharp circumscription and, thus, relative ease in which it is contained or treated by minor surgery, Bowenoid papulosis shows much less propensity to progress to other forms of AIN [82]. Until there is more evidence to suggest that anal and perianal Bowen’s disease and Bowenoid papulosis represent specific entities, it is recommended that these terms be avoided. Leukoplakia is a clinical term describing white plaques in the squamous mucosa of the skin, mouth, and anal region. It may have many causes, such as infection, lichenoid pathology (including lichen planus and lichen sclerosus), and chronic irritation, especially due to hemorrhoids. There is also a specific type of leukoplakia that demonstrates significant neoplastic potential. Thus, anal leukoplakia is a poor and nonspecific term for this condition. It is better termed anal

Fig. 6. Pathology of Bowenoid AIN 3. There is surface hyperkeratosis and parakeratosis, full thickness cellular dysmaturation, prominent keratinocyte vacuolation, and dyskeratosis. The red ‘‘inclusions’’ represent dyskeratotic cells. The changes merely represent a subset of classical AIN 3.

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squamous hyperplasia, either with or without dysplasia. In keeping with the macroscopic features, there is hyperkeratosis with focal parakeratosis and acanthosis (Fig. 7). The latter is associated with an endophytic growth pattern, in which expanded and elongated rete pegs extend into the underlying submucosa/dermis (see Fig. 7). Occasionally, there are classical cytological changes of dysplasia as well [84]. Although rare, anal squamous hyperplasia is not normally associated with the usual cytologic features of neoplasia, but it is, in fact, associated with neoplastic potential. In one series from St. Mark’s Hospital in London, there was considerable malignant potential, the tumors often being well differentiated and broadly infiltrative [84]. Whether this lesion is associated with HPV remains unknown. This condition causes pathological and clinical consternation because its bland clinical appearance and pathology belie a significant neoplastic potential [85]. GLANDULAR PRE-NEOPLASTIC LESIONS OF THE ANAL REGION Rarely, benign sweat gland tumors, especially hidradenoma papilliferum, may arise in the anal region. However, on the whole, benign glandular tumors are exceptionally rare and have no recognized malignant potential. Malignant glandular tumors occur in the anal region, arising from the anal duct and gland epithelium [86–88]. These are well recognized as a complication of chronic anal fistulae [89] and a late complication of anal Crohn’s disease, either with or without fistulae [90,91]. However, little is known of the pre-neoplastic conditions that predispose to such anal adenocarcinomas. PAGET’S DISEASE Paget’s disease is the clinical manifestation of intraepidermal proliferation of neoplastic glandular cells in the skin. It is most common in the nipple of the female breast, where it represents intraepidermal extension of ductal

Fig. 7. Pathology of anal leukoplakia, often termed anal squamous hyperplasia. There is thickening of the mucosa with surface hyperkeratosis. Expanded and elongated rete pegs extend into the underlying submucosa.

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carcinoma-in-situ. However, extramammary Paget’s disease is well recognized, albeit quite rare. It is most often seen in the vulva. There remains considerable controversy concerning its optimal management, although, in the anus at least, management is essentially dependent on the underlying cause of the disease [85,92]. Clinical Features Paget’s disease of the anus occurs in either gender in almost equal proportions and is mainly a disease of the elderly. It manifests clinically as raised, red, scaling areas in the anal canal or perianal skin [93,94]. In that location, it resembles other forms of Paget’s disease, especially that of the nipple. Paget’s disease may be associated with an underlying malignancy, particularly of the rectum, sigmoid colon, or anus [92,93,95]. Thus, once a diagnosis of Paget’s is suspected or established, sigmoidoscopy should be performed to rule out an associated malignancy in the sigmoid colon or rectum. Pathology and Histogenesis Paget’s disease is characterized by an intraepidermal proliferation of large vacuolated neoplastic cells, often in clusters, but also present discretely. They are typically concentrated at the basal aspect of the epidermis where clustering is more apparent [85]. However, spread of clusters and discrete cells to the superficial parts of the epidermis also occurs, which is termed Pagetoid spread. The cells have large neoplastic-appearing nuclei and finely vacuolated cytoplasm (Fig. 8). There is intra-cytoplasmic mucin, which is diastase–periodic acid-Schiff and alcian blue positive [96]. Thus histochemical stains are valuable in helping to differentiate Paget’s disease from its mimics [85,96]. Paget’s disease should be considered when large vacuolated cells are present in the epidermis of the anal region. However, these changes should not be confused with similar reactive changes, termed Pagetoid dyskeratosis [97]. Further, HPV involvement, either with or without AIN, can mimic Paget’s disease as well.

Fig. 8. Pathology (A) and immunohistochemistry (B) of classical Paget’s disease. The cells have large nuclei and finely vacuolated cytoplasm and are predominantly basally orientated (A). They are also positive with CK7 immunohistochemistry (B).

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Paget’s disease is also characteristically associated with hyperplasia of the squamous epithelium. This is termed pseudoepitheliomatous hyperplasia [85]. Melanocytic proliferations are also potential sources of mimicry. Due to the rarity of Paget’s disease, its histogenesis is still not fully understood. However, immunohistochemistry, in particular, and careful clinical correlation have helped to clarify some aspects of the histogenesis of this disease (see Fig. 8) [98–102]. Classical Paget’s disease represents an intraepithelial proliferation of neoplastic cells arising from the ducts of adnexal structures, most notably the apocrine sweat glands of the anal region (see Fig. 8) [85]. Cells spread to the overlying epidermis and manifest a characteristic clinical appearance. Evidence for this proposed pathogenesis is supported by apocrine markers, particularly gross cystic fluid protein [100]. Paget’s disease of the anus also expresses markers, such as epithelial membrane antigen, human milk fat glycoprotein antigen, carcinoembryonic antigen, low molecular weight cytokeratins, and androgen receptors [99–101,103,104]. Some of these markers are also helpful for the diagnosis of Paget’s disease, but may not necessarily differentiate between the two main types of the disease. The apocrine source of the disease is not entirely proven. It has also been recently proposed that stem cells of the hair follicle region may be the origin of disease [105]. Although not regarded as classical Paget’s disease, a similar histologic appearance can be produced by a primary adenocarcinoma of the rectum (or occasionally the sigmoid colon or anus itself) that infiltrates the epidermis of the anus (Fig. 9) [95,106,107]. The management of this disease should be directed at the primary tumor, with treatment of the Paget’s disease largely a secondary consideration. These adenocarcinoma cells do not show expression of apocrine markers. Furthermore, primary rectal tumors show a characteristic cytokeratin immunophenotype characterized by expression of CK20, but only rarely CK7 (see Fig. 9) [108]. In contrast, classical Paget’s disease cells express CK7 and only occasionally CK20 (see Fig. 8) [95,103,109]. However,

Fig. 9. Pathology of Paget’s disease caused by spread from a primary rectal adenocarcinoma. There is more diffuse involvement of the mucosa by vacuolated cells (A), which are strongly positive for CK20 (B).

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primary anal adenocarcinoma shows an immunophenotype that closely mimics that of classical apocrine-type Paget’s disease [110]. In this situation, a panel of antibodies may be used to help in the differential diagnosis (Table 1) [95,96,103]. There are also subtle histological differences between the two major types of Pagetoid intraepithelial adenocarcinoma of the anus. For instance, primary tumors of the colorectum usually show signet ring cell morphology with more abundant intracytoplasmic mucin. Furthermore, the cells tend to invade the epidermis more discretely with less predilection for the basal aspects of the epidermis (see Fig. 8) [88,95]. Treatment The treatment of anal and perianal Paget’s disease depends on the underlying cause and, thus, accurate clinical and pathological assessment is essential. The treatment of Paget’s disease associated with primary colonic or anal adenocarcinoma is secondary to the treatment of the primary tumor. In classical Paget’s disease, local excision often results in recurrence because the disease is usually multifocal and early infiltration of the epidermis may be difficult to detect clinically. Thus wide local excision is the treatment of choice in most centers [92,111,112]. Various other treatment modalities, such as radiotherapy, photodynamic therapy and laser ablation have been attempted [113]. Many of these techniques result in recurrent disease. Most authorities recommend that wide local excision, perhaps with frozen section analysis to ensure completeness of excision, is the treatment of choice [111]. Only rarely is classical Paget’s disease associated with subsequent invasive carcinoma in the anal region [92,111]. MELANOCYTIC PRE-NEOPLASTIC LESIONS OF THE ANAL REGION Malignant melanoma is a rare tumor of the anal region with a poor prognosis [114–118]. It usually presents at a relatively advanced stage. Though it arises

Table 1 An immuno-panel for Paget’s disease of the anal region

Classical apocrine-type Paget’s disease Paget’s disease due to primary rectal/sigmoid colonic adenocarcinoma Paget’s disease due to primary anal adenocarcinoma

CK7

CK20

GCFP

EMA

CEA

þ



þ

þ

þ



þ



þ

þ

þ





þ

þ

Abbreviations: þ, usually positive; , rarely positive; , usually negative; CEA, carcinoembryonic antigen; EMA, epithelial membrane antigen; GCFP, gross cystic fluid protein.

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from melanocytes in the anal canal or perianal skin, it often presents as a lower rectal tumor. Indeed, diffuse infiltration of the rectal mucosa is common. Melanocytes are present in the upper canal, even above the dentate line, thus accounting for the common presentation of advanced malignant melanoma as an upper anal (or even lower rectal) tumor [119]. Despite this, little is known regarding pre-neoplastic melanocytic proliferations that presumably occur in the genesis of anal malignant melanoma. There are only isolated reports of an association of premalignant melanocytic proliferations with malignant melanoma [10]. SUMMARY Anal cancer is rare and this partly explains why pre-neoplastic conditions are poorly understood, especially with regard to their natural history and management. AIN is closely linked to HPV infection and is particularly common in homosexuals and immunosuppressed patients, especially those with HIV/ AIDS. Low-grade AIN is associated with a considerable rate of regression, but this probably reflects inconsistent pathological reporting. Higher grades of AIN may remain static for considerable periods of time in immunocompetent hosts, but those with HIV/AIDS show early and rapid malignant transformation. In general, most anal pre-neoplastic conditions are best diagnosed by biopsy and treated by surgical excision. There is little evidence that newer methods of ablation or other therapeutic modalities, such as radiotherapy, are more efficacious than surgical excision. After local or wide surgical excision, these conditions are associated with a high rate of local recurrence. For anal Paget’s disease, it is important to determine, at the time of diagnosis, whether it is due to a primary in-situ adnexal neoplasm of the anus, or if it is secondary to a primary carcinoma of the rectum. A better understanding of the molecular pathology of these conditions may provide more effective options for managing these diseases. References [1] Scholefield JH, Castle MT, Watson NFS. Malignant transformation of high-grade anal intraepithelial neoplasia. Br J Surg 2005;92:1133–6. [2] Maggard MA, Beanes SR, Ko KY. Anal canal cancer: a population-based reappraisal. Dis Colon Rectum 2003;46:1517–24. [3] Martin F, Bower M. Anal intraepithelial neoplasia in HIV positive people. Sex Transm Infect 2001;77:327–31. [4] Colquhoun P, Nogueras JJ, Dipasquale B, et al. Interobserver and intraobserver bias exists in the interpretation of anal dysplasia. Dis Colon Rectum 2003;46:1332–8. [5] Carter PS, de Ruiter A, Whatrup C, et al. Human immunodeficiency virus infection and genital warts as risk factors for anal intraepithelial neoplasia in homosexual men. Br J Surg 1995;82:473–4. [6] Scholefield JH, Talbot IC, Whatrup C, et al. Anal and cervical intraepithelial neoplasia: possible parallel. Lancet 1989;334:765–9. [7] Fenger C, Frisch M, Marti MC, et al. Tumours of the anal canal. In: Hamilton SR, Aaltonen LA, editors. World Health Organization classification of tumours. Pathology

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and genetics of tumours of the digestive system. Lyon (France): IARC Press; 2000. p. 146–55. [8] Fenger C. The anal transitional zone. Acta Pathol Microbiol Immunol Scand (A) 1987;95(Suppl 289):1–42. [9] Day DW, Jass JR, Price AB, et al. Normal anal region. In: Morson & Dawson’s gastrointestinal pathology. Oxford (UK): Blackwell Scientific Publications; 2003. p. 643–6. [10] Fenger C, Nielsen VT. Precancerous changes in the anal canal epithelium in resection specimens. Acta Pathol Microbiol Immunol Scand (A) 1986;94:63–9. [11] Fenger C, Nielsen VT. Intraepithelial neoplasia in the anal canal. Acta Pathol Microbiol Immunol Scand 1986;94:343–9. [12] Palefsky J, Holly E, Ralston M, et al. Anal squamous intraepithelial lesions in HIV-positive and HIV-negative homosexual and bisexual men: prevalence and risk factors. J Acquir Immune Defic Syndr Hum Retroviral 1998;17:320–6. [13] Dixon AR, Pringle JH, Holmes JT, et al. Cervical intraepithelial neoplasia and squamous cell carcinoma of the anus in sexually active women. Postgrad Med J 1991;67: 556–9. [14] Zbar AP, Fenger C, Efron J, et al. The pathology and molecular biology of anal intraepithelial neoplasia: comparisons with cervical and vulvar intraepithelial carcinoma. Int J Colorectal Dis 2002;17:203–15. [15] Scholefield JH, Hickson WGE, Smith JHF, et al. Anal intraepithelial neoplasia: part of a multifocal disease process. Lancet 1992;340:1271–3. [16] Frisch M, Glimelius B, van den Brule AJ, et al. Sexually transmitted infection as a cause of anal cancer. NEJM 1997;337:1350–8. [17] Frisch M, Fenger C, van den Brule AJC, et al. Variants of squamous cell carcinoma of the anal canal and perianal skin and their relation to human papillomaviruses. Cancer Res 1999;59:753–7. [18] Fox PA. Human papillomavirus and anal intraepithelial neoplasia [review]. Curr Opin Infect Dis 2006;19:62–6. [19] Oriel JD, Whimster IW. Carcinoma in situ associated with virus-containing anal warts. Br J Dermatol 1971;84:71–3. [20] Cooper HS, Patchefsky AS, Marks G. Cloacogenic carcinoma of the anorectum in homosexual men: an observation of four cases. Dis Colon Rectum 1979;22:470–80. [21] Metcalf AM, Dean T. Risk of dysplasia in anal condyloma. Surgery 1995;118:724–6. [22] Palmer JG, Shepherd NA, Jass JR, et al. Human papillomavirus type 16 DNA in anal squamous cell carcinoma. Lancet 1987;ii:42. [23] Youk EG, Ku JL, Park JG. Detection and typing of human papillomavirus in anal epidermoid carcinomas: sequence variation in the E7 gene of human papillomavirus type 16. Dis Colon Rectum 2001;44:236–42. [24] Evatt BL. Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridisation and the polymerase chain reaction. Am J Pathol 1992;140: 1345–55. [25] Zaki SR, Judd R, Coffield LM, et al. Human papillomavirus infection and anal carcinoma. Retrospective analysis by in situ hybridization and the polymerase chain reaction. Am J Pathol 1992;140:1345–55. [26] Palmer JG, Scholefield JH, Coates PJ, et al. Anal cancer and human papillomaviruses. Dis Colon Rectum 1989;32:1016–22. [27] Shroyer KR, Brookes CG, Markham NE, et al. Detection of human papillomavirus in anorectal squamous cell carcinoma. Correlation with basaloid pattern of differentiation. Am J Clin Pathol 1995;104:299–305. [28] Abbasakoor F, Boulos PB. Anal intraepithelial neoplasia. Br J Surg 2005;92:277–90. [29] Palefsky JM, Holly EA, Ralston ML, et al. High incidence of anal high-grade squamous intraepithelial lesions among HIV-positive and HIV-negative homosexual and bisexual men. AIDS 1998;12:495–503.

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[100] Mazoujian G, Pinkus GS, Haagensen DE. Extramammary Paget’s disease—evidence for an apocrine origin. An immunoperoxidase study of gross cystic disease fluid protein-15, carcinoembryonic antigen and keratin proteins. Am J Surg Pathol 1984;8:43–50. [101] Ordonez NG, Awalt H, Mackay B. Mammary and extramammary Paget’s disease. An immunocytochemical and ultrastructural study. Cancer 1987;59:1173–83. [102] Nakamura G, Shikata N, Shoji T, et al. Immunohistochemical study of mammary and extramammary Paget’s disease. Anticancer Res 1995;15:467–70. [103] Nowak MA, Guerriere-Kovach P, Pathan A, et al. Perianal Paget’s disease: distinguishing primary and secondary lesions using immunohistochemical studies including gross cystic disease fluid protein-15 and cytokeratin 20 expression. Arch Pathol Lab Med 1998;122: 1077–81. [104] Liegl B, Horn LC, Moinfar F. Androgen receptors are frequently expressed in mammary and extramammary Paget’s disease. Mod Pathol 2005;18:1283–8. [105] Regauer S. Extramammary Paget’s disease—a proliferation of adnexal origin? Histopathology 2006;48:723–9. [106] Wong AY, Rahilly MA, Adams W, et al. Mucinous anal gland carcinoma with perianal Pagetoid spread. Pathology 1998;30:1–3. [107] Guo L, Kuroda N, Miyazaki E, et al. Anal canal neuroendocrine carcinoma with Pagetoid extension. Pathol Int 2004;54:630–5. [108] Ramalingam P, Hart WR, Goldblum JR. Cytokeratin subset immunostaining in rectal adenocarcinoma and normal anal glands. Arch Pathol Lab Med 2001;125:1074–7. [109] Ohnishi T, Watanabe S. The use of cytokeratins 7 and 20 in the diagnosis of primary and secondary extramammary Paget’s disease. Br J Dermatol 2000;142:243–7. [110] Sasaki M, Terada T, Nakanuma Y, et al. Anorectal mucinous adenocarcinoma associated with latent perianal Paget’s disease. Am J Gastroenterol 1990;85:199–202. [111] McCarter MD, Quan SHQ, Busam K, et al. Long-term outcome of perianal Paget’s disease. Dis Colon Rectum 2003;46:612–6. [112] St Peter SD, Pera M, Smith AA, et al. Wide local excision and split-thickness skin graft for circumferential Paget’s disease of the anus. Am J Surg 2004;187:413–6. [113] Brown RSD, Lankester KJ, McCormack M, et al. Radiotherapy for perianal Paget’s disease. Clin Oncol 2002;14:272–84. [114] Morson BC, Volkstadt H. Malignant melanoma of the anal canal. J Clin Pathol 1963;16: 194–209. [115] Bolivar JC, Harris JW, Branch W, et al. Melanoma of the anorectal region. Surg Gynecol Obstet 1982;154:337–41. [116] Goldman S, Glimelius B, Pahlman L. Anorectal malignant melanoma in Sweden. Report of 49 patients. Dis Colon Rectum 1990;33:874–7. [117] Brady MS, Kavolius JP, Quan SH. Anorectal melanoma. A 64-year experience at Memorial Sloan-Kettering Cancer Center. Dis Colon Rectum 1995;38:146–51. [118] Konstadoulakis MM, Ricaniadis N, Walsh D, et al. Malignant melanoma of the anorectal region. J Surg Oncol 1995;58:118–20. [119] Clemmensen OJ, Fenger C. Melanocytes in the anal canal epithelium. Histopathology 1991;18:237–41.