—  SHORT COURSE #22  —

Diagnostic Problems in GI Pathology

Case 4 - Barrett's Esophagus

Lisa Yerian, John Hart and Amy Noffsinger


Introduction:
According to the Practice Parameters of the American College of Gastroenterology, the current accepted definition of Barrett's esophagus is "a change in the esophageal epithelium of any length that can be recognized at endoscopy and is confirmed to have intestinal metaplasia by biopsy" [34] . Hence, the diagnosis requires the identification of intestinal metaplasia on histologic examination. Histologic evaluation has thus become an essential tool in the diagnosis of Barrett's esophagus and assessment of dysplasia.

The major diagnostic issues related to Barrett's esophagus are:

1.) confirmation that the specimen derives from an endoscopically identified abnormality in the esophagus

2.) recognition of intestinal metaplasia

3.) assessment for and grading of epithelial dysplasia.

Diagnosis of Barrett's Esophagus
The junction between esophageal squamous mucosa and the gastric columnar mucosa is termed the squamocolumnar junction or Z-line, but this junction does not necessarily coincide with the anatomic gastroesophageal (GE) junction (where the tubular esophagus joins the stomach). The squamocolumnar junction is proximal to the GE junction in many adult patients [1, 2, 3, 24, 26, 35] . Determination of biopsy site location and the presence of a lesion is important because intestinal metaplasia is seen near the GE junction in 9-36% of adult patients without an endoscopically identifiable Barrett's segment [15, 23, 40] , and this finding does not fulfill the criteria for a diagnosis of Barrett's esophagus [34] .

Barrett's esophagus is the replacement of the normal esophageal squamous epithelium with metaplastic columnar epithelium. This metaplastic "intestinalized" or "specialized" epithelium is defined by the presence of acid mucin-containing goblet cells. In the absence of intestinal metaplasia, a biopsy containing either cardiac or fundic-type mucosa is not diagnostic of Barrett's esophagus. However, the distribution and relative proportion of goblet cells varies considerably among patients and specimens [29] , and in some cases goblet cells are scarce [14] .

Goblet cells can generally be recognized on routine H&E stained slides based on their shape and tinctorial qualities, but histochemical stains are useful in equivocal cases. Goblet cells contain acidic mucins that stain intensely with Alcian blue at pH 2.5, unlike the adjacent columnar cells, which contain neutral mucins and are Alcian blue negative [11] . Adjacent gastric foveolar-type columnar cells contain PAS-positive neutral mucins and are seen in combination with goblet cells in incomplete intestinal metaplasia - the commoner form of intestinal metaplasia in Barrett's esophagus. "Complete" intestinal metaplasia is less common and contains variable numbers of goblet cells, intestinal absorptive-type cells, and sometimes even neuroendocrine cells and Paneth cells. Histochemical stains should be carefully interpreted in conjunction with the morphology, as false positive and false negative stains occur. Gastric foveolar-type columnar cells occasionally also contain Alcian blue-positive acid mucins, although the staining intensity is less than that seen in goblet cells; these cells should not be interpreted as evidence of Barrett's esophagus [4, 27] . Further, goblet cells are singly dispersed among columnar cells, and thus a continuous row of Alcian blue positive columnar cells is also not diagnostic of Barrett's esophagus.

Dysplasia in Barrett's Esophagus
The significance of Barrett's esophagus lies in its predisposal of affected patients to esophageal adenocarcinoma. Although all patients with Barrett's esophagus are at increased risk for adenocarcinoma, patients with dysplasia arising in Barrett's esophagus, particularly high-grade dysplasia, are at higher risk for development of synchronous or metachronous adenocarcinoma, based on mapping studies and prospective analyses [10, 12, 13, 25, 31, 36, 39] . Because epithelial dysplasia may present with or without an endoscopically-identifiable abnormality, thorough biopsy sampling is required. Barrett's esophagus surveillance programs involve four-quadrant biopsies taken every 2 cm throughout the length of the Barrett's segment with additional biopsies of any endoscopic lesions [21, 34] .

All biopsy specimens exhibiting Barrett's mucosa should be assessed for dysplasia and characterized as negative for dysplasia, positive for dysplasia (low-grade or high-grade), or epithelial alterations indefinite for dysplasia. Dysplasia is recognized as neoplastic epithelium confined within the basement membrane and classified as low-grade or high-grade based on the degree of cytologic and architectural abnormalities present.

Negative for dysplasia
In nondysplastic Barrett's mucosa, the glands are round or tubular and separated by lamina propria. Epithelial cell polarity is maintained. Although the metaplastic glands often show "baseline atypia" (nuclear enlargement, slight hyperchromasia and stratification, and increased mitotic activity seen at the base of the crypts), epithelial cell maturation is seen as these features dissipate as cells progress from the base to the luminal surface. Mitotic activity may be seen at the basal aspect of the glands but should not be seen at the mucosal surface. Hence, evaluation of the surface epithelium is critical to the diagnosis of dysplasia. That said, definitive identification of surface involvement is not always possible, and in certain cases a diagnosis of dysplasia is appropriate in the absence of unequivocal surface involvement [20] . One should be very cautious when diagnosing dysplasia in this circumstance , though, and in most instances when alterations are identified at the crypt bases and the cells normal mature normally towards the mucosal surface, a diagnosis of negative for dysplasia should be applied.

Low-grade dysplasia
Low-grade dysplasia shows preservation or minimal distortion of crypt architecture with only mild gland crowding, but the glands remain separated by some fibrous stroma. There is no glandular complexity (irregularly shaped or cribriform glands). The epithelial cells are closely packed with some nuclear enlargement, overlapping nuclei, variable hyperchromasia and slightly irregular nuclear contours, but these findings are not as marked as those seen in high-grade dysplaia [8, 9, 22] . The epithelial cells overall retain their polarity, with basally located nuclei oriented perpendicular to the basement membrane. Little or no maturation is seen as the cells progress from the crypt bases to the luminal surface. Goblet cell numbers are often reduced, and dystrophic goblet cells may be present. I often characterize low-grade dysplasia as epithelium that is unequivocally neoplastic but that is insufficiently abnormal to satisfy a diagnosis of high-grade dysplasia. Low-grade dysplasia is associated with an increased risk of progression to high-grade dysplasia or invasive adenocarcinoma, and this risk is greater in cases in which there is uniform agreement for a diagnosis of low-grade dysplasia as compared to cases in which there is disagreement about the diagnosis [6, 19, 38] . That said, the natural history of low-grade dysplasia is not well-characterized, in part due to the high degree of interobserver variability related to the diagnosis [22, 30] .

High-grade dysplasia
High-grade dysplasia clearly exhibits more severe cytologic and architectural abnormalities than low-grade dysplasia, but the threshold for distinguishing low-grade and high-grade dysplasia is difficult to define. However, as this threshold corresponds to management decisions, it is a critical diagnostic task for the surgical pathologist. There is no single histologic pattern of high-grade dysplasia, and because of the varied histologic appearances, the diagnosis may be best learned not by seeing one picture of high-grade but rather by seeing a large number of cases with a wide spectrum of histologic lesions. In general, high-grade dysplasia exhibits more nuclear crowding, increased nuclear:cytoplasmic ratios, and nuclear stratification. Mitotic figures are prominent, often present at the mucosal surface, and may be atypical. Since normal epithelial cell maturation is lost, the surface epithelium is similar to that seen in deeper portions of the crypts. In addition to greater cytologic abnormalities, high-grade dysplasia exhibits also more crypt architectural complexity than low-grade dysplasia. The glands are usually more complex, irregular, branched, and/or very closely packed, cribriform or "back to back". The glands are also usually more crowded than in nondysplastic mucosa or low-grade dysplasia, with only scant or absent intervening stroma separating glands.

High-grade dysplasia is not only precursor lesion but also a marker for esophageal adenocarcinoma, although it is not entirely clear how often or how rapidly high-grade dysplasia progresses to cancer. In one large study of patients with high-grade dysplasia without concurrent cancer, 16% of patients developed carcinoma during a mean surveillance period of 7.3 years [37] , and in a prospective study of patients with a single focus of high-grade dysplasia, 53% of patients developed multifocal high-grade dysplasia or invasive carcinoma between 17 and 35 months of follow-up [42] . In some cases, the distinction between high-grade dysplasia and intramucosal adenocarcinoma (defined by the penetration of neoplastic cells through the basement membrane to infiltrate into the lamina propria or muscularis mucosae) may be difficult, particularly in a biopsy specimen, although in many centers this distinction represents a clinical management decision point [28, 32]

Indefinite for dysplasia
The diagnosis of "indefinite for dysplasia" is applied when the epithelial alterations are neither unequivocally dysplastic nor nondysplastic. Although this category should be used prudently, it represents the most accurate interpretation in many situations. For example, inflammation, mucosal injury and regenerative epithelial alterations that result from ongoing gastroesophageal reflux can result in epithelial cell changes that may be difficult or impossible to reliably distinguish from dysplasia. An "indefinite" diagnosis may also be applied when the architectural or cytologic features cannot be adequately interpreted due to fixation, sectioning or staining artifacts, poor orientation, or when the findings fall quantitatively or qualitatively short of a definitive diagnosis of dysplasia.

Complicating factors
There are many factors that make the histologic interpretation of Barrett's dysplasia subject to great difficulty. This is an innately difficult area in pathology due to the variation in histologic appearances and possible obscuring factors. Sampling error is a major issue in both clinical and research work, as dysplasia may be present diffusely or in a small focus. Adequate and standardized sampling practices are required to effectively follow a patient or to confidently assign patients into outcome groups based on dysplasia diagnoses. The difficulties in observer variation, particularly in the separation of negative, indefinite and low-grade dysplasia, have been illustrated [16, 22, 30] . For these reasons, it is recommended that biopsies are reviewed by an "experienced" pathologist for confirmation before aggressive therapy is contemplated.

References
  1. Chandrasoma P: Controversies of the cardiac mucosa and Barrett's oesophagus. Histopathology 46:361, 2005

  2. Chandrasoma PT, Der R, Ma Y, et al: Histology of the gastroesophageal junction: an autopsy study. Am J Surg Pathol 24:402, 2000

  3. Chandrasoma PT, Lokuhetty DM, Demeester TR, et al: Definition of histopathologic changes in gastroesophageal reflux disease. Am J Surg Pathol 24:344, 2000

  4. Chen YY, Wang HH, Antonioli DA, et al: Significance of acid-mucin-positive nongoblet columnar cells in the distal esophagus and gastroesophageal junction. Hum Pathol 30:1488, 1999

  5. Dar MS, Goldblum JR, Rice TW, et al: Can extent of high grade dysplasia in Barrett's oesophagus predict the presence of adenocarcinoma at oesophagectomy? Gut 52:486, 2003

  6. Dulai GS, Shekelle PG, Jensen DM, et al: Dysplasia and risk of further neoplastic progression in a regional Veterans Administration Barrett's cohort. Am J Gastroenterol 100:775, 2005

  7. Falk GW, Rice TW, Goldblum JR, et al: Jumbo biopsy forceps protocol still misses unsuspected cancer in Barrett's esophagus with high-grade dysplasia. Gastrointest Endosc 49:170, 1999

  8. Goldblum JR: Barrett's esophagus and Barrett's-related dysplasia. Mod Pathol 16:316, 2003

  9. Goldblum JR, Lauwers GY: Dysplasia arising in barrett's esophagus: diagnostic pitfalls and natural history. Semin Diagn Pathol 19:12, 2002

  10. Haggitt RC: Barrett's esophagus, dysplasia, and adenocarcinoma. Hum Pathol 25:982, 1994

  11. Haggitt RC, Reid BJ, Rabinovitch PS, et al: Barrett's esophagus. Correlation between mucin histochemistry, flow cytometry, and histologic diagnosis for predicting increased cancer risk. Am J Pathol 131:53, 1988

  12. Hameeteman W, Tytgat GN, Houthoff HJ, et al: Barrett's esophagus: development of dysplasia and adenocarcinoma. Gastroenterology 96:1249, 1989

  13. Hamilton SR, Smith RR: The relationship between columnar epithelial dysplasia and invasive adenocarcinoma arising in Barrett's esophagus. Am J Clin Pathol 87:301, 1987

  14. Harrison R, Perry I, Haddadin W, et al: Detection of intestinal metaplasia in Barrett's esophagus: an observational comparator study suggests the need for a minimum of eight biopsies. Am J Gastroenterol 102:1154, 2007

  15. Johnston MH, Hammond AS, Laskin W, et al: The prevalence and clinical characteristics of short segments of specialized intestinal metaplasia in the distal esophagus on routine endoscopy. Am J Gastroenterol 91:1507, 1996

  16. Kerkhof M, van Dekken H, Steyerberg EW, et al: Grading of dysplasia in Barrett's oesophagus: substantial interobserver variation between general and gastrointestinal pathologists. Histopathology 50:920, 2007

  17. Krishnamurthy S, Dayal Y: Pancreatic metaplasia in Barrett's esophagus. An immunohistochemical study. Am J Surg Pathol 19:1172, 1995

  18. Levine DS, Rubin CE, Reid BJ, et al: Specialized metaplastic columnar epithelium in Barrett's esophagus. A comparative transmission electron microscopic study. Lab Invest 60:418, 1989

  19. Lim CH, Treanor D, Dixon MF, et al: Low-grade dysplasia in Barrett's esophagus has a high risk of progression. Endoscopy 39:581, 2007

  20. Lomo LC, Blount PL, Sanchez CA, et al: Crypt dysplasia with surface maturation: a clinical, pathologic, and molecular study of a Barrett's esophagus cohort. Am J Surg Pathol 30:423, 2006

  21. Lunedei V, Bazzoli F, Pozzato P, et al: Endoscopic surveillance in Barrett's esophagus. Minerva Gastroenterol Dietol 48:63, 2002

  22. Montgomery E, Bronner MP, Goldblum JR, et al: Reproducibility of the diagnosis of dysplasia in Barrett esophagus: a reaffirmation. Hum Pathol 32:368, 2001

  23. Nandurkar S, Talley NJ, Martin CJ, et al: Short segment Barrett's oesophagus: prevalence, diagnosis and associations. Gut 40:710, 1997

  24. Oberg S, Peters JH, DeMeester TR, et al: Inflammation and specialized intestinal metaplasia of cardiac mucosa is a manifestation of gastroesophageal reflux disease. Ann Surg 226:522, 1997

  25. Oberg S, Wenner J, Johansson J, et al: Barrett esophagus: risk factors for progression to dysplasia and adenocarcinoma. Ann Surg 242:49, 2005

  26. Odze RD: Pathology of the gastroesophageal junction. Semin Diagn Pathol 22:256, 2005

  27. Offner FA, Lewin KJ, Weinstein WM: Metaplastic columnar cells in Barrett's esophagus: a common and neglected cell type. Hum Pathol 27:885, 1996

  28. Ormsby AH, Petras RE, Henricks WH, et al: Observer variation in the diagnosis of superficial oesophageal adenocarcinoma. Gut 51:671, 2002

  29. Qualman SJ, Murray RD, McClung HJ, et al: Intestinal metaplasia is age related in Barrett's esophagus. Arch Pathol Lab Med 114:1236, 1990

  30. Reid BJ, Haggitt RC, Rubin CE, et al: Observer variation in the diagnosis of dysplasia in Barrett's esophagus. Hum Pathol 19:166, 1988

  31. Reid BJ, Weinstein WM, Lewin KJ, et al: Endoscopic biopsy can detect high-grade dysplasia or early adenocarcinoma in Barrett's esophagus without grossly recognizable neoplastic lesions. Gastroenterology 94:81, 1988

  32. Rice TW, Mendelin JE, Goldblum JR: Barrett's esophagus: pathologic considerations and implications for treatment. Semin Thorac Cardiovasc Surg 17:292, 2005

  33. Riddell RH, Goldman H, Ransohoff DF, et al: Dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications. Hum Pathol 14:931, 1983

  34. Sampliner RE: Updated guidelines for the diagnosis, surveillance, and therapy of Barrett's esophagus. Am J Gastroenterol 97:1888, 2002

  35. Sarbia M, Donner A, Gabbert HE: Histopathology of the gastroesophageal junction: a study on 36 operation specimens. Am J Surg Pathol 26:1207, 2002

  36. Schmidt HG, Riddell RH, Walther B, et al: Dysplasia in Barrett's esophagus. J Cancer Res Clin Oncol 110:145, 1985

  37. Schnell TG, Sontag SJ, Chejfec G, et al: Long-term nonsurgical management of Barrett's esophagus with high-grade dysplasia. Gastroenterology 120:1607, 2001

  38. Skacel M, Petras RE, Gramlich TL, et al: The diagnosis of low-grade dysplasia in Barrett's esophagus and its implications for disease progression. Am J Gastroenterol 95:3383, 2000

  39. Smith RR, Hamilton SR, Boitnott JK, et al: The spectrum of carcinoma arising in Barrett's esophagus. A clinicopathologic study of 26 patients. Am J Surg Pathol 8:563, 1984

  40. Trudgill NJ, Suvarna SK, Kapur KC, et al: Intestinal metaplasia at the squamocolumnar junction in patients attending for diagnostic gastroscopy. Gut 41:585, 1997

  41. Wang HH, Zeroogian JM, Spechler SJ, et al: Prevalence and significance of pancreatic acinar metaplasia at the gastroesophageal junction. Am J Surg Pathol 20:1507, 1996

  42. Weston AP, Sharma P, Topalovski M, et al: Long-term follow-up of Barrett's high-grade dysplasia. Am J Gastroenterol 95:1888, 2000