Cell proliferation, cell cycle abnormalities, and cancer outcome in patients with Barrett's esophagus: a long-term prospective study.

PURPOSE: Elevated cellular proliferation and cell cycle abnormalities, which have been associated with premalignant lesions, may be caused by inactivation of tumor suppressor genes. We measured proliferative and cell cycle fractions of biopsies from a cohort of patients with Barrett's esophagus to better understand the role of proliferation in early neoplastic progression and the association between cell cycle dysregulation and tumor suppressor gene inactivation. EXPERIMENTAL DESIGN: Cell proliferative fractions (determined by Ki67/DNA multiparameter flow cytometry) and cell cycle fractions (DNA content flow cytometry) were measured in 853 diploid biopsies from 362 patients with Barrett's esophagus. The inactivation status of CDKN2A and TP53 was assessed in a subset of these biopsies in a cross-sectional study. A prospective study followed 276 of the patients without detectable aneuploidy for an average of 6.3 years with esophageal adenocarcinoma as an end point. RESULTS: Diploid S and 4N (G(2)/tetraploid) fractions were significantly higher in biopsies with TP53 mutation and loss of heterozygosity. CDKN2A inactivation was not associated with higher Ki67-positive, diploid S, G(1), or 4N fractions. High Ki67-positive and G(1)-phase fractions were not associated with the future development of esophageal adenocarcinoma (P=0.13 and P=0.15, respectively), whereas high diploid S-phase and 4N fractions were (P=0.03 and P<0.0001, respectively). CONCLUSIONS: High Ki67-positive proliferative fractions were not associated with inactivation of CDKN2A and TP53 or future development of cancer in our cohort of patients with Barrett's esophagus. Biallelic inactivation of TP53 was associated with elevated 4N fractions, which have been associated with the future development of esophageal adenocarcinoma.

Crypt dysplasia with surface maturation: a clinical, pathologic, and molecular study of a Barrett's esophagus cohort.

Little is known regarding the significance of esophageal biopsies that show dysplasia-like atypia limited to the bases of the crypts, without involvement of the surface epithelium in Barrett's esophagus (BE). The aim of this study was to evaluate the clinical, pathologic, immunohistochemical, and molecular characteristics of basal crypt dysplasia-like atypia (BCDA) with surface maturation in surveillance endoscopic mucosal biopsies to gain insight into its biologic significance. The Seattle Barrett's Esophagus Project is a prospective cohort study in which patients and their biopsies have been evaluated prospectively for clinical, pathologic, and molecular markers. As part of continued surveillance of the cohort, 206 consecutive BE patients were evaluated prospectively for BCDA between July 1, 2001 and August 13, 2003; 15 patients had BCDA (prevalence rate = 7.3%). These 15 patients were evaluated for clinical, pathologic, and immunohistochemical (p53 and MIB-1) features during the study period (2001-2003) as well as associations with clinical, pathologic, and molecular markers [17p(TP53) loss of heterozygosity (LOH), 9p(p16) LOH, tetraploidy, and aneuploidy] that were detected previously in the same patients in the cohort study (1983-2001). All BE patients with BCDA (male-to female ratio, 12:3; mean age, 72 years; mean length of BE, 7.0 cm; mean duration of BE, 95.1 months), except 2 (87%), had dysplasia or adenocarcinoma detected in biopsies either prior to or concurrent to the one that contained BCDA. In contrast, only 112 of 191 (59%) controls had neoplasia during the same time period (59%, P = 0.05). The difference between BCDA and controls was particularly significant with regard to the association with high-grade dysplasia (P = 0.004). Compared with adjacent nonatypical and nondysplastic (metaplastic) BE, areas of BCDA showed a significantly elevated prevalence rate of p53 positivity (60% vs. 13%, P<0.02) and a significantly elevated total crypt and basal crypt MIB-1 proliferation rate (P<0.001). Indeed, the MIB-1 proliferation rate in the basal portion of the crypts in BCDA was similar to that detected in conventional low- or high-grade dysplasia. Patients with BCDA showed a significantly increased rate of 17p(TP53) LOH (P = 0.016), aneuploidy (P = 0.004), and a trend in increased 9p(p16) LOH (P = 0.08), compared with control patients without BCDA. The clinical, pathologic, immunohistochemical, and molecular abnormalities were similar in BCDA cases that were considered low-grade versus those considered high-grade by histologic evaluation, except that high-grade cases tended to be older (79 years vs. 68 years, P = 0.06). BCDA with surface maturation, in mucosal biopsies from patients with BE, is an uncommon but significant pathologic change that shows a variety of proliferative and molecular abnormalities and has a high association with conventional dysplasia and/or adenocarcinoma. Based on these findings, BCDA warrants further investigation as a possible subtype of true dysplasia despite the morphologic appearance of surface maturation.

Mucin core polypeptide expression in the progression of neoplasia in Barrett's esophagus.

We have previously demonstrated a specific pattern of mucin (MUC) core polypeptide expression in Barrett's esophagus (BE) characterized by MUC1 and MUC6 positivity in goblet cells in a proportion of cases. The aim of this study was to determine the pattern of MUC expression associated with the development and progression of dysplasia in BE. Endoscopic mucosal biopsies from 35 patients with BE (10 with no dysplasia, 6 with indefinite for dysplasia, 12 with low-grade dysplasia [LGD], and 7 biopsies with high-grade dysplasia [HGD]) were immunostained (ABC method) with antibodies against MUC1, MUC2, MUC3, MUC5AC, and MUC6. The extent and pattern of staining for each marker was evaluated in intestinalized Barrett's epithelium and in the various grades of dysplasia. For cases with dysplasia, staining was evaluated separately in nondysplastic epithelium adjacent to (<1 cm) and distant from (>2 cm) areas of dysplasia. In nondysplastic BE, MUC1, MUC2, MUC3, MUC5AC, and MUC6 stained 40%, 100%, 100%, 100%, and 90% of cases, respectively, in goblet or nongoblet columnar epithelium. With the progression of dysplasia (from metaplasia to indefinite, LGD and HGD), there was a significant decrease in expression of MUC1, MUC2, and MUC3, and alterations in the staining patterns of MUC5 and MUC6. In fact, MUC1 and MUC3 were entirely absent from all cases of HGD. Interestingly, BE-associated adenocarcinomas showed an MUC phenotype distinct from that of HGD, with expression of MUC1 and MUC3 in 47% and 67% of cases, and expression of MUC1 in a membranous pattern. There was no significant difference in MUC staining in nondysplastic BE between patients with and without dysplasia. Alterations in MUC expression occur in the progression of dysplasia in BE. However, none of these markers helps identify a subgroup of patients at increased risk for neoplasia.

Biologic properties of columnar epithelium underneath reepithelialized squamous mucosa in Barrett's esophagus.

Chronic proton pump inhibitor (PPI) therapy may lead to partial regression of Barrett's esophagus (BE), resulting in the development of reepithelialized islands of squamous mucosa that may cover the underlying BE. The purpose of this study was to evaluate the clinical, histologic, and biologic characteristics of BE that is situated underneath squamous islands (BUSI). A total of 97 mucosal biopsies from 44 BE patients with BUSI were evaluated for a variety of histologic features (eg, type of epithelium, anatomic relationship of the underlying glands to the luminal surface, presence of adjacent mucosal glands or ducts, and the presence and degree of dysplasia), and immunostained for Ki-67, cyclin D1, and p53. BUSI was compared with adjacent areas of BE for all parameters. A clinical control group consisting of 50 BE patients without microscopic evidence of BUSI was selected for comparison of clinical and endoscopic features. The study group (34 males, 10 females; mean age, 67 years; mean length of BE, 5.5 cm) consisted of 27 (61%) and 12 (27%) patients on low- and high-dose PPI, respectively. On endoscopy, visible islands of squamous mucosa were noted in only 43% of study group patients (despite the presence of BUSI microscopically in all cases); one island was noted in 2%, multiple islands in 27%, and extensive islands in 14% of patients. The extent of squamous islands was unrelated to PPI dose. The study group was significantly more likely to have endoscopic evidence of extensive squamous islands compared with the control group (P = 0.009). Histologically, 89% of biopsies with BUSI showed intestinal-type, and 11% showed cardia-type, epithelium. Low- and high-grade dysplasia was noted in 4 (4%) and 5 (5%) biopsies, respectively. All patients with dysplasia in BUSI also showed dysplasia in other areas of the esophagus as well. Interestingly, BUSI reached the mucosal surface either by penetrating directly through, or by wrapping around, islands of squamous epithelium, in 68% of biopsies. Twenty-one percent of biopsies showed BUSI adjacent to submucosal glands or ducts. BUSI showed a significantly lower Ki-67 proliferation rate (29% vs. 49%, P < 0.001), and a lower, albeit nonsignificant, degree of cyclin D1 (16% vs. 29%) and p53 (4% vs. 17%) positivity in comparison to adjacent areas of BE. Furthermore, significantly lower proliferation rates were observed in BUSI that did not reveal an opening to the mucosal surface in comparison to foci that did. BUSI is phenotypically similar to typical surface BE but shows less severe proliferative abnormalities, particularly in buried glands that have no detectable connection to the esophageal lumen. Reduced proliferation may be due either to decreased exposure to luminal contents or to disruption of sloughing of surface epithelial cells into the crypt lumen. Prospective studies of large numbers of patients with BUSI will be required to determine the magnitude of its risk of progression to cancer.

Limited penetration of anticancer drugs through tumor tissue: a potential cause of resistance of solid tumors to chemotherapy.

PURPOSE: Potential causes of drug resistance in solid tumors include genetically determined factors expressed in individual cells and those related to the solid tumor environment. Important among the latter is the requirement for drugs to penetrate into tumor tissue and to achieve a lethal concentration in all of the tumor cells. The present study was designed to characterize further the multicellular layer (MCL) method for studying drug penetration through tumor tissue and to provide information about tissue penetration for drugs used commonly in the treatment of human cancer. EXPERIMENTAL DESIGN: EMT-6 mouse mammary and MGH-U1 human bladder cancer cells were grown on collagen-coated semiporous Teflon membranes to form MCLs approximately 200 microm thick. The properties of MCLs were compared with those of tumors grown in mice from the same cells. The penetration of drugs through the MCL was evaluated by using radiolabeled drugs or analytical methods. RESULTS: The MCL developed an extracellular matrix containing both laminin and collagen, although there were some differences in expression of extracellular matrix proteins. Electron microscopy showed rare desmosomes in both MCL and tumors. The penetration of cisplatin, etoposide, gemcitabine, paclitaxel, and vinblastine through tissue in the MCL was slow compared with penetration through the Teflon support membrane alone. CONCLUSIONS: Our results suggest limited ability of anticancer drugs to reach tumor cells that are distant from blood vessels. The limited penetration of anticancer drugs through tumor tissue may be an important cause of clinical resistance of solid tumors to chemotherapy.

High Goblet Cell Count Is Inversely Associated with Ploidy Abnormalities and Risk of Adenocarcinoma in Barrett's Esophagus.

PURPOSE: Goblet cells may represent a potentially successful adaptive response to acid and bile by producing a thick mucous barrier that protects against cancer development in Barrett's esophagus (BE). The aim of this study was to determine the relationship between goblet cells (GC) and risk of progression to adenocarcinoma, and DNA content flow cytometric abnormalities, in BE patients. EXPERIMENTAL DESIGN: Baseline mucosal biopsies (N=2988) from 213 patients, 32 of whom developed cancer during the follow up period, enrolled in a prospective dynamic cohort of BE patients were scored in a blinded fashion, for the total number (#) of GC, mean # of GC/crypt (GC density), # of crypts with ≥ 1 GC, and the proportion of crypts with ≥1 GC, in both dysplastic and non-dysplastic epithelium separately. The relationship between these four GC parameters and DNA content flow cytometric abnormalities and adenocarcinoma outcome was compared, after adjustment for age, gender, and BE segment length. RESULTS: High GC parameters were inversely associated with DNA content flow cytometric abnormalities, such as aneuploidy, ploidy >2.7N, and an elevated 4N fraction > 6%, and with risk of adenocarcinoma. However, a Kaplan-Meier analysis showed that the total # of GC and the total # crypts with ≥1 GC were the only significant GC parameters (p<0.001 and 0.003, respectively). CONCLUSIONS: The results of this study show, for the first time, an inverse relationship between high GC counts and flow cytometric abnormalities and risk of adenocarcinoma in BE. Further studies are needed to determine if GC depleted foci within esophageal columnar mucosa are more prone to neoplastic progression or whether loss of GC occurs secondary to underlying genetic abnormalities.

Extent of low-grade dysplasia is a risk factor for the development of esophageal adenocarcinoma in Barrett's esophagus.

OBJECTIVES: Previous studies that evaluated extent of high-grade dysplasia (HGD) as a risk factor for esophageal adenocarcinoma (EA) in Barrett's esophagus (BE) were conflicting, and no prior study has evaluated extent of low-grade dysplasia (LGD) as a risk factor. The aim of this discovery study was to evaluate the hypothesis that extent of LGD and HGD are risk factors for progression to EA. METHODS: We evaluated baseline biopsies from 77 BE patients with dysplasia including 44 who progressed to EA and 33 who did not progress during follow-up. The total numbers of LGD and HGD crypts were determined separately by counting all crypts and the extent of LGD, HGD, and total dysplasia were correlated with EA outcome. RESULTS: Thirty-one and 46 patients had a maximum diagnosis of LGD and HGD, respectively. When the crypts were stratified by dysplasia grade, the mean number of LGD crypts per patient was borderline higher in progressors (93.9) compared with nonprogressors (41.2, P= 0.07), and the mean proportion of LGD crypts per patient was significantly higher in progressors (46.4%vs 26.0%, P= 0.037). Neither the mean number of HGD crypts per patient (P= 0.14) nor the mean proportion of HGD crypts per patient (P= 0.20) was significantly associated with EA outcome. CONCLUSIONS: The extent of LGD is a significant risk factor for the development of EA in BE in this study. Although the presence of HGD is significantly associated with a greater relative risk for development of EA, the extent of HGD was not an independent risk factor for progression.