MY APPROACH Diagnosis and grading of dysplasia …Diagnosis and grading of dysplasia in Barrett’s oesophagus R D Odze ..... effort to reach a consensus on the nomenclature of gastrointestinal

MY APPROACH

Diagnosis and grading of dysplasia in Barrett’s oesophagusR D Odze. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

J Clin Pathol 2006;59:1029–1038. doi: 10.1136/jcp.2005.035337

This review focuses on the pathological features ofdysplasia in Barrett’s oesophagus. Two categorisationschemes are used for grading dysplasia in thegastrointestinal tract, including Barrett’s oesophagus. Theinflammatory bowel disease dysplasia morphology studygroup system is the one most commonly used in the USA.However, some European and most far Eastern countriesuse the Vienna classification system, which uses the term‘‘non-invasive neoplasia’’ instead of low-grade dysplasia(LGD) or high-grade dysplasia (HGD) and also uses theterm ‘‘suspicious for invasive carcinoma’’ for lesions thatshow equivocal cytological or architectural features oftissue invasion. The degree of dysplasia is based on acombination of cytological and architectural atypia.However, the precise number of HGD crypts that isnecessary to upgrade a biopsy from LGD to HGD hasnever been investigated and varies widely among expertgastrointestinal pathologists. The extent of dysplasia,particularly LGD, has also been recognised recently as animportant prognostic parameter in Barrett’s oesophagus.Other problematic areas of dysplasia interpretation includedifferentiation of regenerating epithelium versus LGD andseparating HGD from carcinoma. Dysplasia associatedwith macroscopically visible lesions, such as ulcers, nodulesor polyps, carry a high risk of synchronous ormetachronous adenocarcinoma. Recently, immunostainingfor a-methylacyl-CoA-racemase has been shown to have ahigh degree of specificity for detection of dysplasia inBarrett’s oesophagus and may be used to help distinguishnegative from positive biopsies in this condition. In thisreview, the problematic areas in dysplasia interpretationare outlined and a specific approach to these issues isdiscussed.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Correspondence to:R D Odze, Brigham andWomen’s Hospital,Harvard Medical School,75 Francis Street, Boston,MA 02115, USA;[email protected]

Accepted for publication20 November 2005. . . . . . . . . . . . . . . . . . . . . . .

Worldwide, there are two classificationsystems used for dysplasia in thegastrointestinal tract (table 1) including

Barrett’s oesophagus.1 2 In 1983, the inflamma-tory bowel disease (IBD) dysplasia morphologystudy group classified dysplasia as negative,indefinite or positive (low or high grade), whichis the system used most commonly in the USA.2

Recently, the World Health Organization pro-posed that the term ‘‘dysplasia’’ be replaced by‘‘intraepithelial neoplasia’’, but this new termhas yet to gain popularity when used in reference

to preneoplastic conditions of the gastrointest-inal tract.3 Some European and most far Easterncountries use the Vienna classification, but it hasnot gained widespread acceptance in the USA.1

The Vienna system (table 1) is similar to the oneproposed by the IBD dysplasia morphology studygroup except that it uses the term ‘‘non-invasiveneoplasia’’ instead of low-grade or high-gradedysplasia and also uses the term ‘‘suspicious forinvasive carcinoma’’ for lesions that showequivocal cytological or architectural features oftissue invasion. The Vienna system was devel-oped to reduce the widely recognised discrepan-cies in interpretation of dysplasia betweenWestern and Japanese pathologists and in aneffort to reach a consensus on the nomenclatureof gastrointestinal neoplasia. Briefly, the basis ofthe differences in interpretation relates to thefact that Western pathologists define carcinomaas lesions that show histological evidence ofpenetration beyond the basement membraneinto the lamina propria or submucosa, whereasmany Japanese pathologists diagnose carcinomaon the basis of cytological changes, in conjunc-tion with architectural changes, without theneed to accurately show invasion beyond thebasement membrane. Unfortunately, as outlinedlater, there is a high level of intraobserver andinterobserver variability with regard to thepathological categorisation of dysplasia usingboth these systems, and particularly for epitheliallesions that show borderline features betweenregeneration and low-grade dysplasia (LGD).1 4 6

Fortunately, in both systems, the detection ofclinically relevant high-grade lesions and carci-noma shows a much higher level of consistencyin interpretation among pathologists.

This review focuses on my personal approachto the diagnosis and grading of dysplasia inBarrett’s oesophagus, which is based on pub-lished data and on my experience as a highlyreferral-based and dedicated gastrointestinalpathologist with a longstanding research interestin Barrett’s oesophagus. However, as a prelude tothis review, it is worth noting that althoughmorphological detection of dysplasia in mucosalbiopsy specimens currently serves as the bestmethod (biomarker) of detecting patients atincreased risk of cancer in Barrett’s oesophagus,it has limitations.7 11 One limitation relates to theinadequacy of the currently accepted definitionof dysplasia as ‘‘unequivocal neoplastic epithe-lium confined to the basement membrane’’. In

Abbreviations: AMACR, a-methylacyl-CoA-racemase;BCDA, basal crypt dysplasia-like atypia; HGD, high-grade dysplasia; IBD, inflammatory bowel disease; LGD,low-grade dysplasia; N/C ratio, nuclear/cytoplasmicratio

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fact, it is often difficult, morphologically, to recognise‘‘atypical’’ epithelium as unequivocally neoplastic becauseof overlapping histological features with epithelial regenera-tive changes. Furthermore, it is also often difficult todetermine, with complete certainty, whether a focus ofneoplastic epithelium has, in fact, breached the basementmembrane and invaded the lamina propria. Finally, there isabundant evidence to suggest that non-dysplastic (metaplas-tic) epithelium in Barrett’s oesophagus possesses many of thebiological characteristics of ‘‘neoplastic’’ epithelium, the non-dysplastic epithelium being defined as a clonal proliferationof cells with loss of the normal capacity for regulation of cellproliferation and differentiation.7 12 Thus, dysplasia is adisease in need of redefinition, particularly as it relates tothe development of cancer in Barrett’s oesophagus.

Pathological features of dysplasia in Barrett’soesophagusNegative for dysplasiaA diagnosis of ‘‘negative for dysplasia’’ is applied to cases thatshow metaplastic columnar epithelium with regenerativechanges. Unfortunately, on occasion, epithelial regenerativechanges may be extreme, particularly in mucosa adjacent tothe neo-squamocolumnar junction or in which activeinflammation or ulceration is present. In my experience,the tendency among surgical pathologists to overinterpretregenerative changes as indicative of dysplasia is often due toa lack of awareness of the wide spectrum of ‘‘atypia’’ thatmay occur in patients with Barrett’s oesophagus, particularlythose with persistent reflux. From a biological perspective,the progression of Barrett’s oesophagus to adenocarcinoma isdriven by the evolution and proliferation of clones of cellswith accumulated genetic errors, a process referred to asgenomic instability.13 14 In fact, the process of clonal evolutionbegins early in Barrett’s oesophagus, before the phenotypicexpression of dysplasia, when metaplastic epithelial cellsacquire mutations that give them a selective proliferativeadvantage relative to genetically normal cells. Mutant cellscontaining a proliferative advantage then undergo celldivision, which ultimately results in a clone of cells thatcontain genetic lesions identical to the original progenitorcell. In this manner, morphologically non-dysplastic Barrett’soesophagus contains progeny clones with accumulatedgenetic abnormalities. For instance, a recent study15 16

reported that 85–90% of patients with Barrett’s oesophaguscontain one or more p16 mutations in non-dysplasticmucosa. In fact, mutations in the p16 gene also correlatedirectly with length of columnar mucosa in Barrett’soesophagus.15 p53 mutations may also occur in patients withBarrett’s oesophagus without dysplasia.17 18 These findings

support the hypothesis that Barrett’s oesophagus represents aneoplastic clonal proliferation rather than a polyclonalresponse to injury, as previously believed.12 19 Thus, it is notsurprising that Barrett’s oesophagus cases that are consideredto be negative for dysplasia may show a level of atypiabeyond the limits that pathologists would normally accept aspart of a tissue repair reaction. In general, non-dysplasticepithelium in Barrett’s oesophagus exhibits cytologicalfeatures characteristic of regeneration coupled with preserva-tion of crypt architecture (fig 1). However, a considerabledegree of atypia may be present in markedly reactiveepithelium. For instance, some degree of crypt budding,branching, atrophy, crowding, distortion or even cysticchange may be present, especially adjacent to or underneathareas of ulceration. In addition, nuclear stratification is oftenpresent, particularly at the bases of crypts, but occasionally atthe level of the surface epithelium as well, although usuallyto a lesser degree. Cytologically, regenerating cells containnuclei with smooth membranes, normal nuclear/cytoplasmic(N/C) ratio, and a variable number of normal mitoses, butmay also show prominent nucleoli, although without notableenlargement. In general, nuclear pleomorphism, loss of cellpolarity and markedly raised N/C ratio are features ofdysplasia and not of regenerating epithelium. However,newly formed epithelium covering a freshly ulcerated surfacemay show cells with increased N/C ratio, hyperchromaticityand slight loss of polarity and pleomorphism. In thisinstance, tufting of surface cells is often present, which helpsdeclare the benign nature of the epithelium. Nevertheless, incontrast with dysplasia, goblet cells, both normal anddystrophic, are quite common. Cytoplasmic mucin may bedepleted, particularly in areas of inflammation, but even inthese circumstances the crypt cells usually show a progressiveincrease in content of mucin in epithelium close to, or at, theluminal surface. This feature, the preservation of the N/Cratio and a decrease in the degree of nuclear stratification inthe upper levels of the crypts and in the surface epitheliumrepresent the hallmark features of surface ‘‘maturation’’,which is the most characteristic feature of non-dysplasticepithelium in Barrett’s oesophagus.

In general, nuclear and cytoplasmic changes, and stratifi-cation, are more prominent in ulcerated or actively inflamedmucosa. However, in these instances, and in contrast withdysplasia, the degree of atypia related to regenerative changesusually dissipates gradually in areas of mucosa more distantfrom the inflammatory infiltrate and represents a helpfulfeature in distinguishing it from dysplasia.

Positive for dysplasia, low and high gradeOverall, there are two general histological types of dysplasiain Barrett’s oesophagus, termed ‘‘adenoma’’ and ‘‘non-adenoma-like’’ because of the resemblance, or lack thereof,of the cytological features of the dysplastic cells to sporadiccolonic adenomas. However, non-conventional or unusualforms of dysplasia have been recently recognised in bothBarrett’s oesophagus and inflammatory bowel disease.20 22

Some of these are discussed later. ‘‘Adenomatous’’ LGD,which is the most common type of dysplasia in Barrett’soesophagus, shows crypts with relatively preserved architec-ture or perhaps only minimal distortion, and stratifiedatypical pencil-shaped nuclei limited, for the most part, tothe basal portion of the cell cytoplasm (fig 2). The nuclei inLGD are typically elongated, crowded and hyperchromatic;they show an irregular contour and a dense chromatinpattern either with or without multiple, small inconspicuousnucleoli. Dysplastic cells are mucin depleted and show adecrease in the number of goblet cells. Other features includeincreased mitoses, both typical and atypical, preservation (oronly slight loss) of cell polarity, increased N/C ratio,

Table 1 Outline and comparison of the Viennaclassification and inflammatory bowel disease (IBD)dysplasia morphology study group classification ofdysplasia in IBD

Vienna IBD study group

Negative for neoplasia/dysplasia Negative for dysplasiaIndefinite for neoplasia/dysplasia Indefinite for dysplasiaNon-invasive low-grade neoplasia (low-gradeadenoma/dysplasia)

Low-grade dysplasia

Non-invasive high-grade neoplasia High-grade dysplasiaHigh-grade adenoma/dysplasiaNon-invasive carcinoma (carcinoma in situ)Suspicious of invasive carcinoma

Invasive neoplasia Adenocarcinoma*Intramucosal adenocarcinoma IntramucosalSubmucosal carcinoma or beyond Invasive

*Not described by the IBD study group.

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particularly in the bases of the crypts, and lack of surfacematuration, which is a characteristic feature of dysplasia ingeneral. Most notably, dysplastic epithelium often shows anabrupt transition to non-dysplastic epithelium, which is a

helpful feature in distinguishing dysplasia from regeneratingepithelium.

With progression to high-grade dysplasia (HGD), thedegree of cytological and architectural complexity becomesmore advanced (table 2, fig 3). In fact, a diagnosis of HGDmay be established if either of these types of abnormalities issufficiently prominent, in the absence of the other.4 18 Typicalarchitectural abnormalities include crypt budding, branching,marked crowding (evident by a noticeable decrease in theamount of lamina propria between dysplastic crypts) or avilliform configuration of the surface epithelium. Cryptsoften have an irregular configuration and may showintraluminal papillae or bridges. However, prominent intra-luminal bridges, imparting a cribriform pattern to the glands,should raise the possibility of intramucosal adenocarcinoma.Cytologically, in contrast with LGD, HGD shows cells withmarked nuclear pleomorphism and irregularity of contour,increased N/C ratio, loss of cell polarity and a higher numberof atypical mitoses, particularly in the upper levels of thecrypts and even in the surface epithelium. Full-thicknessnuclear stratification, both in the crypt and surface epithe-lium, is a characteristic feature of HGD. Nucleoli, whenpresent, may be large in size and irregular in contour. Similarto LGD, mucin depletion is prominent and goblet cells, bothtypical and dystrophic, are markedly decreased in number orcompletely absent. In summary, the important featureshelpful in distinguishing LGD from HGD include full-thickness nuclear stratification, loss of cell polarity, particu-larly at higher levels of the crypts and surface epithelium,

A

B

C

Figure 1 (A) Intestinalised epithelium in Barrett’s oesophagus (BO) withmild regenerative changes. Note that a mild degree of crypt distortion,budding and crowding is a normal component of metaplastic epitheliumin BO. In one focus, there is mild stratification of the nuclei in the surfaceepithelium, but the nuclei are regular in shape, and the cells show arelatively low nuclear/cytoplasmic (N/C) ratio. (B) High-powerphotograph of marked regenerative changes in an area of mucosa closeto the neosquamocolumnar junction. The epithelium shows prominentstratification of the nuclei, which includes the surface epithelium as well.A few intraepithelial neutrophils are present. Key features ofregeneration in this case include tufting of the epithelial cells, low N/Cratio, prominent nucleoli and preservation of cell polarity. (C) An area ofextreme regeneration in a freshly ulcerated mucosal surface. The cryptand surface epithelium show cells with increased N/C ratio,hyperchromaticity and mucin depletion. Note that the atypia in themonolayer of epithelium covering the freshly ulcerated surface is similarto that seen in the crypt. Biopsy specimens obtained from this area of themucosa 6 months later showed no evidence of dysplasia.

A

B

Figure 2 (A) Medium power view of low-grade dysplasia in Barrett’soesophagus. The nuclei are pencil-shaped, hyperchromatic andstratified, but limited to the basal half of the cell cytoplasm, except focallyin the surface epithelium. Overall, there are minimal architecturalabnormalities. At high power (B), the nuclei show clumped chromatinand inconspicuous multiple small nucleoli.

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atypical mitoses or considerable architectural distortion inthe HGD, as outlined earlier.

With further neoplastic progression, cells may breach thebasement membrane and invade the lamina propria ormuscularis mucosa, features indicative of intramucosal

adenocarcinoma (fig 4). In some institutes, patients withintramucosal adenocarcinoma are treated more aggressively(oesophageal resection) than those with HGD (aggressivesurveillance and endoscopic mucosal ablation),7 23 25 andthus, distinction between these types of neoplastic lesionsmay be clinically important. However, in other institutes,patients with HGD or intramucosal adenocarcinoma aremanaged similarly.26 27 Therefore, it is important to have aclear understanding of the particular treatment regimensused at your institute before engaging in a laborious mentalexercise sometimes associated with this differential diagno-sis.

Non-adenomatous dysplasia is rare and is a rather poorlycharacterised entity with regard to its biological andpathological characteristics and natural history. However,most authors consider non-adenomatous dysplasia to be aform of HGD for the purpose of patient management. Non-adenomatous dysplasia is characterised by crypts that show aprominent back-to-back gland pattern and contain cells thatare more epithelioid or cuboidal-shaped, with a high N/Cratio, round or oval highly irregular-shaped nuclei, an openchromatin pattern and prominent nucleoli (fig 5). In fact, thecrypts may show little or no intervening lamina propria,which often raises the possibility of intramucosal adenocar-cinoma.

Problematic areas of Barrett’s oesophagus-associateddysplasia interpretationRegeneration versus low-grade dysplasia(‘‘indefinite for dysplasia’’)Although early studies suggested that patients with LGD didnot show a substantially higher rate of progression toadenocarcinoma than patients with biopsies considered tobe negative or indefinite for dysplasia, recent data suggestotherwise.7 Unfortunately, given the subtle gradation ofchanges that occur in the progression of dysplasia inBarrett’s oesophagus and the wide range of morphologicalpatterns of atypia related to regeneration and repair, there isa significant degree of intraobserver and interobservervariability in the diagnosis of dysplasia, particularly regardingcases at the lower end of the spectrum (ie, separatingregeneration from dysplasia), even among experiencedgastrointestinal pathologists.4 6 The highest level of variabilityoccurs in the differential of marked regenerative changesversus LGD, often necessitating an interim diagnosis ofindefinite for dysplasia. For instance, in the original study byReid et al,4 regarding interobserver variation in the diagnosis

A

B

Figure 3 (A) In contrast with low-grade dysplasia (LGD), high-gradedysplasia (HGD) shows crowded crypts with more severe nuclearstratification. The nuclei reach the luminal surface in the deeper portionsof the crypts. In addition, there is mild architectural distortion. On high-power view (B), the nuclei have a slightly more open chromatin patternwith prominent, sometimes multiple, nucleoli and a more marked loss ofcell polarity. The nuclear/cytoplasmic ratio of HGD is markedlyincreased compared with LGD.

Table 2 Cytological and architectural features of low-grade and high-grade dysplasia in Barrett’s oesophagus

Feature Low Grade High GradeCytology

q N/C ratio + ++Loss of cell polarity 2 +Mitosis + ++Atypical mitosis +/2 +Full-thickness nuclear stratification 2 +Decreased goblet cells (+/2 dystrophic) + ++Hyperchromasia + ++Multiple nucleoli +/2 +/2

Large irregular (prominent) nucleoli 2 +/2

Irregular nuclear contour + ++Nuclear pleomorphism 2 +

ArchitectureVilliform change 2 +/2

Crypt budding/branching +/2 ++Crowded (back-to-back) crypts +/2 ++Irregular crypt shapes +/2 +Intraluminal papilla/ridges 2 +/2

Lamina propria between glands + +/2

N/C, nuclear/cytoplasmic ratio; 2, absent; +/2, may be present;+, usually present.

Figure 4 High-power view of high-grade dysplasia with intramucosaladenocarcinoma. The intramucosal adenocarcinoma is characterised bya proliferation of small irregular glands with markedly atypical nucleiinfiltrating the lamina propria in a haphazard fashion that cannot beexplained on the basis of involvement of pre-existing crypts.

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of dysplasia in Barrett’s oesophagus among eight expertgastrointestinal pathologists, there was only 60% agreementin distinguishing negative from indefinite and LGD cases. Infact, in a reproducibility study by Montgomery et al,5 onlyslightly better agreement was noted among expert gastro-intestinal pathologists in separating these categories ofdysplasia. These authors also noted a high level of diagnosticdifficulty in diagnosing lesions at the lower end of thedysplasia spectrum. For instance, k values of 0.32 and 0.15,indicating only ‘‘fair’’ and ‘‘slight’’ agreement, were obtainedfor detection of LGD and indefinite for dysplasia, respectively.

In my experience, the indefinite for dysplasia category isused most often in one or more of the following threesituations:

N technical issues;

N atypia related to inflammation and ulceration; and

N dysplasia-like changes present only in the bases of thecrypts, with evidence of surface maturation.

Mucosal biopsy specimens that are sectioned in atangential manner, possess marked cautery artefact or lacksurface epithelium are often difficult to evaluate definitivelyfor dysplasia (fig 6). In a study by Baak et al28 that evaluatedBarrett’s oesophagus-associated biopsy specimens by mor-phometric analysis, 64 of 71 cases of LGD and 11 of 23 casesof HGD initially diagnosed by general pathologists weredowngraded by experts on re-evaluation of the biopsyspecimens. In fact, 46% of the experts’ ‘‘downgrades’’ were

thought to be due to technically inadequate tissue sections.Tangentially sectioned biopsy specimens that lack well-oriented crypts and surface epithelium may be difficult toevaluate for the presence or absence of surface maturation,which is an important feature to consider in the regenerationversus dysplasia differential diagnosis.

Regenerating epithelium, particularly in the setting ofactive inflammation or ulceration, may, on occasion, show aconsiderable degree of cytological atypia similar to that inLGD or even HGD. In architecturally normal biopsy speci-mens with inflammation, the lack of an abrupt transitionfrom atypical to non-atypical epithelium and the presence ofsurface maturation, combined with a lack of nuclearpleomorphism, atypical mitoses and loss of cell polarity, arehelpful features in distinguishing regenerative changes fromdysplasia. This constellation of features was also consideredhelpful in the interobserver study by Montgomery et al.5 Inthat study, the investigators thought that lesions consideredto be ‘‘indefinite’’ showed inflammation, retention of theircrypt architecture, a normal ratio of glands to lamina propria,obvious signs of surface maturation, and had nuclearchanges that ‘‘approached but did not quite reach’’ those ofLGD. In that scenario, it is reasonable to establish a diagnosisof indefinite for dysplasia, and to recommend that furtherbiopsy specimens be obtained after aggressive reflux treat-ment has been instituted and the inflammation has subsided.As noted above, inflammation-induced ‘‘atypical’’ regenera-tive changes are most pronounced at the neosquamocolum-nar junction, a region that shows a high level of cell turnover,injury and repair. Therefore, the diagnostic ‘‘threshold’’ fordysplasia should be raised when evaluating mucosal biopsyspecimens from this anatomical region.

It is commonly believed among many gastrointestinalpathologists that dysplasia, regardless of the grade, is char-acterised by total crypt and surface epithelium involvementwithout surface maturation.2 4 5 This supposition is based on thecommonly held belief that preneoplastic lesions, by definition,show a loss of differentiation capability. However, the author ofthis review has recently reported that mucosal biopsy specimensfrom patients with longstanding Barrett’s oesophagus may,rarely, show dysplastic features that are limited to the cryptbases, without involvement of the upper portions of the crypt orsurface epithelium20 (fig 7). In our study, 15 patients withBarrett’s oesophagus who had ‘‘basal crypt dysplasia-like atypia(BCDA) with surface maturation’’ were evaluated for a varietyof pathological, immunohistochemical and molecular features.

On the basis of the finding of a considerably increasedassociation with conventional dysplasia or adenocarcinoma,

A

B

Figure 5 (A) Non-adenomatous (high-grade) dysplasia in Barrett’soesophagus. In contrast with adenomatous dysplasia, the neoplastic cellsshow low columnar or cuboidal phenotype, highly irregular nuclei withmarkedly increased nuclear/cytoplasmic (N/C) ratio and marked loss ofcell polarity. High-power view of another area from the same case (B)shows a back-to-back gland pattern consisting of cuboidal cells with ahigh N/C ratio, prominent nucleoli and marked loss of cell polarity.

Figure 6 High-power view of an area considered indefinite fordysplasia. Although the crypts show some features of low-gradedysplasia, the absence of surface epithelium makes evaluation of surfacematuration impossible.


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an increased prevalence rate of p53 positivity and a markedlyincreased rate of 9pLOH, 17pLOH and aneuploidy in patientswith BCDA compared with controls, we concluded that thissubtype of ‘‘atypical’’ changes probably represents truedysplasia despite the presence of surface maturation in theselesions. In fact, in the interobserver study by Montgomeryet al5 quoted earlier, atypical lesions of this kind posed thegreatest degree of diagnostic difficulty, and the widest rangeof diagnoses, among the participating pathologists. Of course,the presence of active inflammation in association withBCDA should prompt the pathologist to use restraint, and inthis situation, it is probably wise to establish a diagnosis ofindefinite for dysplasia until further biopsy specimens can beobtained after the inflammation has subsided. Figure 8 outlinesa diagnostic algorithm that may be helpful when evaluatingdiagnostically difficult lesions in Barrett’s oesophagus.

Low-grade dysplasia versus high-grade dysplasiaAs discussed earlier, HGD is distinguished from LGD basedprimarily on the basis of the degree of architectural andcytological aberrations. Unfortunately, as dysplasia pro-gresses to cancer on a continuous scale, there are no well-defined ‘‘cut off points’’ that help separate these two types oflesions. In general, the overall grade of dysplasia isdetermined by the features of the most atypical portion ofepithelium. However, the precise number of ‘‘high-grade’’dysplastic crypts that are necessary to upgrade a biopsyspecimen from LGD to HGD has never been investigated andvaries among expert gastrointestinal pathologists. In theoriginal classification of dysplasia in the gastrointestinal tractproposed by the IBD dysplasia morphology study group in1983, the authors recommended, anecdotally, that ‘‘designa-tion of a biopsy as high grade based solely on the presence ofHGD in one or two crypts is probably not justified’’.2 In theinterobserver variability study by Reid et al,4 which includedmany of the same authors, there was considerable disagree-ment regarding the minimum area required to establish adiagnosis of HGD. This issue was not discussed in the recentstudy on Barrett’s oesophagus by Montgomery et al.5

Nevertheless, we have recent evidence to suggest thatmeasurement of the extent of dysplasia is a highly importantmethod of evaluating cancer risk in patients with Barrett’soesophagus.29 30 For instance, in a recent study by Srivastavaet al29 published in abstract form in 2005, the extent ofdysplasia, regardless of the grade, was a strong risk factor forthe development of invasive cancer in a 52-month follow-upstudy of 69 high-risk patients with Barrett’s oesophagus, 28of whom eventually developed adenocarcinoma. In thatstudy, the mean number of crypts with LGD in patientswho ultimately developed adenocarcinoma was 5.8 comparedwith only 2.6 in patients without cancer. These results aresupported by a study by Buttar et al30 in which the extent ofHGD in Barrett’s oesophagus was shown to be correlatedwith the risk of developing adenocarcinoma. In their study,cancer-free survival rates were considerably higher inpatients with ‘‘focal HGD’’ than in those with ‘‘diffuseHGD’’. However, a study by Dar et al,31 in which the extent ofHGD was evaluated on the basis of the number of levels ofthe oesophagus that contained mucosa with HGD, did notcorroborate the results of Buttar et al.30 In my practice, Ialways try to provide an objective measurement of the extentof LGD and HGD in all biopsy specimens with dysplasia.Although anecdotal, clinicians find this information usefulwhen planning treatment strategies for patients withBarrett’s oesophagus. For instance, a biopsy sign outindicating that there is ‘‘LGD and focal HGD’’ (implying thatHGD includes only a small proportion of the number ofdysplastic crypts) provides additional objective informationand may prompt more aggressive surveillance or alternativeprotocol treatments rather than oesophagectomy, which isstill the most commonly applied method of treatment forpatients having Barrett’s oesophagus with HGD.

High-grade dysplasia versus intramucosaladenocarcinomaAs mentioned earlier, the distinction between HGD andintramucosal adenocarcinoma may be difficult and is oftenclinically relevant (fig 9). Lymphatic vessels are present in the

A

B

Figure 7 Medium and (A) high-power view of an area of Barrett’s oesophagus (BO) showing basal crypt dysplasia-like atypia with surfacematuration. In this focus, the basal portion of the crypt shows dysplastic-appearing cells characterised by nuclear enlargement, stratification, loss ofpolarity and a focal gland in gland pattern. Mitoses are readily identified. The superficial portion of the crypts, however, shows evidence of surfacematuration characterised by a decrease in the size of nuclei, less stratification and progressive acquisition of mucin in the cytoplasm of cells on thesurface BO.

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lamina propria of the oesophageal mucosa. As a result, thereis a 5–8% risk of lymph node metastases in patients withtumours limited to the mucosal compartment, which is amajor reason why oesophagectomy is widely regarded as thebest method of treatment for patients having Barrett’soesophagus with intramucosal adenocarcinoma.23 26 32 Incontrast, some studies suggest that a vigorous surveillancebiopsy protocol is a practical and safe alternative tooesophagectomy for patients with HGD.24 25 In a study byOrmsby et al,23 75 oesophagectomy specimens were reviewedby two gastrointestinal pathologists and one general surgicalpathologist and classified as either, intramucosal or sub-mucosal adenocarcinoma.23 The level of interobserver agree-ment for HGD versus intramucosal adenocarcinoma was onlyfair (k= 0.42) and, most importantly, did not improve greatlyafter establishment of uniform histological criteria (k= 0.5).In my experience, there is a tendency for general pathologiststo overdiagnose intramucosal adenocarcinoma. Often, this isbecause prominent architectural aberrations, such as cryptbudding and branching, are interpreted as evidence ofinvasion, rather than simply complex in situ disease. Mypersonal practice is to diagnose intramucosal carcinoma onlywhen one or more of the following architectural features arepresent:

N single cells or small clusters of tightly compact back-to-back glands are present in the lamina propria

N there is a complex gland-in-gland or ‘‘cribriforming’’pattern present, with evidence of an expansion of thelamina propria and distortion of the surrounding crypts

N neoplastic cells or glands are present that show a back-to-back or highly irregular architectural glandular arrangement,which cannot be explained by the presence of pre-existingBarrett’s glands, as previously defined by Ormsby et al.23

The presence of necrosis or desmoplasia is evidence infavour of adenocarcinoma as well, although these featuresare rarely present in carcinomas limited to the mucosa, and,

may not be present in submucosal invasive carcinomaseither.23 When in doubt, I advocate using the term ‘‘HGDwith foci suspicious, but not diagnostic, of intramucosaladenocarcinoma’’ (which corresponds to category 4.3 in theVienna Classification System) and recommend that repeatbiopsies should be performed if this distinction is clinicallyrelevant.

Intramucosal versus submucosal adenocarcinomaOn occasion, dysplastic glands may be located in themuscularis mucosa (fig 10). In this situation, the differentia-tion of ‘‘misplaced’’ non-invasive glands from truly invasiveglands may be difficult. We must be cautious not tooverinterpret these findings, particularly if the glands containcytologically bland or only LGD changes, and the crypts are

Figure 8 Diagnostic algorithm for the differential diagnosis of epithelial atypia in Barrett’s oesophagus.

Figure 9 High-grade dysplasia in Barrett’s oesophagus with a focalarea highly suggestive of intramucosal adenocarcinoma. In the centre ofthe field is a proliferation of small glands in a back-to-backconfiguration, with little intervening lamina propria, which is difficult toexplain on the basis of involvement of pre-existing crypts. There is asuggestion of isolated single cells in the lamina propria as well.


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not particularly distorted in shape. One reason for exercisingcaution is that patients with Barrett’s oesophagus oftendevelop a new (superficial), more luminally situated, layer ofmuscularis mucosa that directly underlies the region ofmetaplastic columnar epithelium.33 Unfortunately, the natureof the stroma that lies between the original (deep) and thenew muscularis mucosa has never been investigated. As aresult, it also remains unclear whether glands that penetratethrough the new muscularis mucosa should be considered tobe intramucosal or submucosal carcinomas. However, in arecent study of 120 patients with early adenocarcinoma (13with HGD and 107 with stage T1 adenocarcinoma) of theoesophagus or gastroesophageal junction treated by oeso-phagectomy in which outcome was stratified according to thedepth of tumour invasion, patients with tumours invadingthe ‘‘new’’ muscularis mucosa had a similar outcome as thosewith invasion into the original (deep) muscularis.34 Only 1%of these patients had lymph node metastasis compared with44% of the patients with tumours that penetrated into themid or deep portion of the original (‘‘true’’) submucosa.These data suggest that the stromal space located betweenthe original and new muscularis mucosa represents ‘‘laminapropria’’ and tumours that infiltrate this region shouldprobably be considered intramucosal, from a biological pointof view. Nevertheless, further studies are needed in thisregard.

Dysplasia and macroscopically visible lesionsSeveral studies have shown that the natural history and riskof malignancy in Barrett’s oesophagus are highly dependenton the macroscopic features of the dysplastic lesions.35 39 As aresult, the diagnosis and grading of dysplasia in Barrett’soesophagus should be carried out in conjunction withknowledge of the endoscopic features of the patient.Common macroscopic lesions include ulcers, nodules(defined as an area of subtle mucosal elevation measuring,l cm in diameter) and strictures. In a study by Hillmanet al,36 patients with one or more macroscopic lesions weremore likely to develop HGD and cancer than those withoutendoscopically identifiable lesions. In a recent study byButtar et al,30 15 of 25 (60%) patients with dysplastic noduleshad cancer compared with only 17 of 25 (23%) patientswithout nodules. In fact, the risk of cancer was increased by afactor of 4 in patients who had visible nodules at endoscopy.In a study by Montgomery et al,35 a higher proportion ofpatients with Barrett’s oesophagus with ulcers had HGD oradenocarcinoma than patients without ulcers, and the

presence of an ulcer with HGD increased the likelihood ofdetecting carcinoma in a subsequent resection specimen.Mucosal nodularity offers the opportunity for endoscopicmucosal resection and the capability of assessing more tissuethan endoscopic biopsy samples.

Rarely, dysplasia in Barrett’s oesophagus may grow as awell-defined polyp with an adenoma-like appearance.37 38

However, owing to the previously reported strong associationof these lesions with HGD and adenocarcinoma, and withdysplasia in the surrounding flat dysplasia, these lesionsshould be considered ‘‘polypoid dysplasia’’ rather than‘‘adenoma’’.37 The term adenoma imparts a ‘‘benign’’connotation to the lesion and may lead to undertreatmentby polypectomy rather than oesophagectomy.

Adjunctive markers in the diagnosis of dysplasiaMany studies have evaluated the potential utility ofimmunohistochemical or molecular markers as adjunctivemethods in detecting dysplasia and in distinguishing reactivefrom dysplastic epithelium in Barrett’s oesophagus.7 11 16 28 40–49

Most of the markers investigated have been linked, in somecapacity, to the pathogenesis of cancer and include thoseassociated with control of cell proliferation, intercellularadhesion and tumour suppression, among others. Of the vastarray of markers evaluated, detection of DNA contentabnormalities by flow cytometry (aneuploidy or elevated 4Nfraction) and evaluation of mutations or loss of homozygosityof the pl6 and p53 genes are the most promising potentialmethods to help identify high-risk patients.7 50 Unfortunately,none of these methods has been shown to be particularlyuseful in differentiating non-dysplastic from dysplasticepithelium in routine pathology practice. Other methods,such as computerised quantitative pathology or measurementof proliferation indices, have also been shown to decreasediagnostic variability in this regard, but these methods havenot been standardised.28 41 Evaluation of p53 by immuno-staining remains the most controversial.7 43 51

Proponents of p53 immunostaining as an adjunctivediagnostic method state that the p53 gene is only rarelymutated in non-dysplastic epithelium and that the frequencyof mutations has been shown to increase dramatically inHGD and adenocarcinoma. Unfortunately, p53 over-expression by immunohistochemistry can be detected in upto 10% of cases considered to be morphologically negative fordysplasia.43 51 53 In addition, several studies have shown ahigh rate of false positive staining (up to 56%) in the absenceof p53 mutations, and a high frequency of false negativestaining as well (up to 30%).43 51 Non-specific binding of p53to non-p53 mutation-related antigens may also lead to falsepositive results. Furthermore, p53 results may vary substan-tially depending on the specific type of antibody used. In fact,certain types of mutations result in the production of a p53protein that does not bind to some antibodies directedagainst the wild-type protein.43 Finally, no known antibody,or combination of antibodies, can detect all p53 mutations.For these reasons, I do not advocate the use of p53immunostaining in routine practice to help differentiateregenerating from dysplastic epithelium in diagnosticallydifficult cases.

However, recently, immunostaining for a-methylacyl-CoA-racemase (AMACR), an antibody often used in the assess-ment of diagnostically difficult atypical and potentiallyneoplastic lesions of the prostate, has been shown to have ahigh degree of specificity for detection of dysplasiain Barrett’s oesophagus and inflammatory bowel disease.45

In a recent study by Dorer et al,45 AMACR was not detected inany of 36 cases with Barrett’s oesophagus without dysplasia(0%) but was positive in 38%, 81% and 72% of cases of LGD,HGD and adenocarcinoma, respectively. In that study, three

Figure 10 Mucosal biopsy from a patient with Barrett’s oesophagus(BO) showing low-grade dysplasia and the presence of atypical glandsin the superficial muscularis mucosa. The glands in the deeper portionsof the mucosa show an attenuated epithelium. However, this is notuncommon in BO and should not be interpreted as evidence ofadenocarcinoma.

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‘‘indefinite’’ cases were also positive, but in the one case inwhich follow-up information was available, adenocarcinomawas detected on follow-up. Thus, AMACR immunostainingmay represent a potentially useful adjunctive method indifferentiating reactive from dysplastic epithelium inBarrett’s oesophagus, but, in my opinion, requires validationby other investigators before use in routine clinical practice.

SUMMARYDespite the problems associated with detection and reprodu-cibility, morphological evaluation of dysplasia in mucosalbiopsy specimens still remains the mainstay of surveillanceand treatment of Barrett’s oesophagus worldwide. Carefulattention to the cytological and architectural features ofdysplasia, as outlined earlier, and recognition of the extremedegrees of regeneration that may occur in this condition, canhelp minimise observer error. Use of consultants withparticular expertise in gastrointestinal pathology is highlyrecommended before institution of definitive management inpatients with Barrett’s oesophagus with ‘‘atypical’’ biopsyspecimens. Fortunately, pathologists have a reasonably goodtrack record at detecting clinically relevant dysplasia, such asHGD and intramucosal adenocarcinoma, lesions that usuallynecessitate aggressive methods of intervention such asendoscopic mucosal resection, mucosal ablation or surgicalresection. Adjunctive diagnostic methods, such as AMACRimmunostaining, are promising, but further studies areneeded to find either non-morphology-based or morphol-ogy-based reproducible methods that can reliably helpstratify cancer risk in patients with Barrett’s oesophagus.

Competing interests: None declared.

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25 Reid BJ, Weinstein WM, Lewin KJ, et al. Endoscopic biopsy can detect high-grade dysplasia or early adenocarcinoma in Barrett’s esophagus withoutgrossly recognizable neoplastic lesions. Gastroenterology 1988;94:81–90.

26 Rice TW, Falk GW, Achkar E, et al. Surgical management of high-gradedysplasia in Barrett’s esophagus. Am J Gastroenterol 1993;88:1832–6.

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Clinical Evidence—Call for contributors

Clinical Evidence is a regularly updated evidence-based journal available worldwide both asa paper version and on the internet. Clinical Evidence needs to recruit a number of newcontributors. Contributors are healthcare professionals or epidemiologists with experience inevidence-based medicine and the ability to write in a concise and structured way.Areas for which we are currently seeking contributors:

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MY APPROACH Diagnosis and grading of dysplasia …Diagnosis and grading of dysplasia in Barrett’s oesophagus R D Odze ..... effort to reach a consensus on the nomenclature of gastrointestinal