Primary Motility  Disorders of the  Esophagus
 The Esophageal
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Volume: Barrett's Esophagus
Chapter: Markers

Is there any relationship between histologic findings, proliferating cell nuclear antigen expression and ploidy analysis by flow cytometry or by image analysis?

R.D. Odze (Boston)

This review will focus on the relationship between histologic findings, proliferation, and ploidy abnormalities in the progression of neoplasia in Barrett's esophagus.

In Barrett's esophagus, the esophageal mucosa is replaced by columnar epithelium which is composed of crypts and underlying glands. Many studies have documented that metaplastic epithelium in Barrett's esophagus reveals an expansion of the normal proliferative compartment that is characterized by an increase in the number of cells that are engaged in the cell cycle [1-11]. Furthermore, cell proliferation rates have been found to be higher in the intestinal type of Barrett's esophagus in comparison to the junctional, or fundic, type [1, 9]. For example, utilizing the proliferating cell nuclear antigen (PCNA) antibody, Gray et al. evaluated proliferation rates in 93 biopsies from 45 Barrett's esophagus patients without dysplasia [1]. The mean crypt PCNA labeling score was significantly higher in the specialized type of Barrett's esophagus (4.1) in comparison to the junctional (3.1) and fundic type (1.6). Surface cells were positive for PCNA only in specialized-type epithelium. Similarly, Gulizia et al. showed increased proliferative activity of intestinalized epithelium in the distal esophagus and gastroesophageal junction, including short-segment Barrett's esophagus (SSBE), characterized by an expansion of the crypt proliferative index, increased size of the proliferative zone, and the presence of surface epithelial staining, in comparison to non-intestinalized epithelium [9]. This later study was performed utilizing the Ki-67 proliferation-antigen associated marker [MIB-1] for evaluation of proliferative activity. These data indicate that cell-cycle proliferation abnormalities are early events in the progression of malignancy in Barrett's esophagus, and suggest that even patients with SSBE may be at risk for further progression.

With regard to Barrett's esophagus-associated neoplasia, many studies have shown a shift, and expansion, of the normal proliferative compartment towards the luminal surface in advancing stages of dysplasia and adenocarcinoma [2-8, 10, 11]. In low-grade dysplasia (LGD), cells with proliferative capacity have been shown to extend to higher levels of the crypt epithelium, and even on to the surface, in comparison to non-dysplastic epithelium. High-grade dysplasia typically shows even greater numbers of proliferating cells in the upper parts of the crypt and the surface epithelium in comparison to LGD and nondysplastic epithelium in Barrett's esophagus [2]. For instance, Hong et al., utilizing the Ki67 antibody in 43 Barrett's esophagus patients, measured the proliferative fraction in the surface, upper crypt, lower crypt, and glandular portion of the epithelium in normal gastric controls, non-dysplastic Barrett's esophagus, LGD and high-grade dysplasia (HGD) and adenocarcinoma. Significantly higher Ki-67 proliferative indices were noted in all anatomic zones in each higher stage of neoplasia [2]. Ki-67 positive nuclei were found predominantly in the surface epithelium and upper crypt zones in HGD, whereas LGD showed most of the Ki-67 positivity in the lower crypt zone. Similar results were documented by Yacoub et al. in a study of 25 Barrett's esophagus-associated adenocarcinomas and adjacent areas of metaplasia and dysplasia [5]. In that study, the overall crypt MIB-1 proliferation index increased progressively from Barrett's metaplasia to dysplasia and adenocarcinoma. Dysplasia could be reliably differentiated from markedly regenerative metaplastic epithelium by the presence of increased luminal crypt staining in the former in comparison to the latter.

Flow cytometry has been commonly used by investigators to evaluate cell cycle abnormalities, and DNA content, in Barrett's esophagus-associated neoplasia. The most commonly measured flow cytometric abnormalities have been S phase fraction, G2/tetraploidy cell fraction and aneuploidy, the latter defined as cells with abnormal DNA content. Several studies have documented increased G2/tetraploidy, and/or aneuploidy, in advancing stages of neoplastic progression, in parallel to increased proliferative activity [12-16].

Krishnadath et al. evaluated ploidy and proliferative activity in 73 Barrett's esophagus patients by Ki-67 immunohistochemistry, in situ hybridization, and flow cytometry [4]. Increased proliferative activity, defined as a proliferative index > 20, was noted in Barrett's metaplasia and in all stages of dysplasia and adenocarcinoma. Hyperdiploidy correlated well with advancing stages of neoplasia (Barrett's esophagus: 17%, indefinite/LGD: 40%, HGD: 71%, adenocarcinoma: 87%) and showed a high prevalence in HGD and adenocarcinoma. These authors concluded that increased proliferation is an early change, and that hyperdiploidy correlates well with advancing degrees of dysplasia in Barrett's esophagus. Gimenez et al. found aneuploidy only in cases with HGD or carcinoma, and not in cases that were either negative, indefinite, or LGD [16]. Hagget et al., in 1988, demonstrated aneuploidy in 100% of cases with either carcinoma or dysplasia (low or highgrade), 33% in those categorized as indefinite for dysplasia, and in 5% of non-dysplastic Barrett's esophagus samples [13]. However, other authors have not found such a close correlation between flow abnormalities and histologic dysplasia [17, 18]. For instance, both Fennerty et al. [17] and Sciallero et al. [18] noted aneuploidy in 32% and 17% of nondysplastic Barrett's esophagus cases, respectively, and a lack of correlation with higher stages of neoplasia. Thus, in summary, although aneuploidy is detected in most cases of HGD and carcinoma, not all tissue samples with flow abnormalities are morphologically abnormal.

Finally, one elegant study by Reid et al. used multiparameter flow cytometry in conjunction with Ki-67, to investigate the proportion of diploid and aneuploid cells in G0, G1, S and G2 phases of the cell cycle in advancing stages of Barrett's esophagus-associated neoplasia [14]. Normal control gastric mucosa showed most cells in the G0 phase, with low G1, S, G2, and total Ki-67 values. In contrast, metaplastic epithelium in Barrett's esophagus showed increased G1 cell fractions. Furthermore, dysplastic epithelium showed increased S phase fractions, and an increasing proportion of cells with aneuploidy, or both. No differences were noted between diploid and aneuploid cells in these parameters. Thus, they concluded that the neoplastic progression of Barrett's esophagus was characterized by a loss of proliferative control, characterized by early mobilization of cells from G0 to G1, ultimate loss of control of the G1/S phase transition, and accumulation of cells in the G2 phase, particularly in HGD and carcinoma. Increased S phase fractions occurred in a subset of patients with more advanced stages of neoplastic progression, i.e. HGD and adenocarcinoma, and often in association with aneuploidy.

In summary, increased cell proliferation is an early change in Barrett's esophagus. A strong correlation is noted between advancing stages of neoplasia and increased proliferative activity in this condition. In general, proliferative abnormalities correlate with DNA content abnormalities, as measured by flow cytometry, particularly in the more advanced stages of neoplasia such as HGD and adenocarcinoma. However, at present, morphologic dysplasia still seems to be the best indicator of a neoplastic risk in Barrett's esophagus. Although further studies evaluating aneuploidy in non-dysplastic cases may provide valuable information regarding high or low risk groups.


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13. Haggitt RC, Reid BJ, Rabinovitch PS, Rubin CE. Barrett's esophagus: correlation between mucin histochemistry, flow cytometry, and histologic diagnosis for predicting increased cancer risk. Am J Pathol 1988;131:53-61.

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17. Fennerty MB, Sampliner RE, Way D, Riddell R, Steinbronn K, Garewal H. Discordance between flow cytometric abnormalities and dysplasia in Barrett's esophagus. Gastroenterology 1989;87:815-820.

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Publication date: August 2003 OESO©2015