Douglas S. Levine

Predictors of Progression to Cancer in Barrett's Esophagus: Baseline Histology and Flow Cytometry Identify Low- and High-Risk Patient Subsets

OBJECTIVE:Barretts esophagus develops in 5–20% of patients with gastroesophageal reflux disease and predisposes to esophageal adenocarcinoma. The value of endoscopic biopsy surveillance is questioned because most patients do not develop cancer. Furthermore, observer variation in histological diagnosis makes validation of surveillance guidelines difficult because varying histological interpretations may lead to different estimated rates of progression. Thus, objective biomarkers need to be validated for use with histology to stratify patients according to their risk for progression to cancer.METHODS:We prospectively evaluated patients using a systematic endoscopic biopsy protocol with baseline histological and flow cytometric abnormalities as predictors and cancer as the outcome.RESULTS:Among patients with negative, indefinite, or low-grade dysplasia, those with neither aneuploidy nor increased 4N fractions had a 0% 5-yr cumulative cancer incidence compared with 28% for those with either aneuploidy or increased 4N. Patients with baseline increased 4N, aneuploidy, and high-grade dysplasia had 5-yr cancer incidences of 56%, 43%, and 59%, respectively. Aneuploidy, increased 4N, or HGD were detected at baseline in all 35 patients who developed cancer within 5 yr.CONCLUSIONS:A systematic baseline endoscopic biopsy protocol using histology and flow cytometry identifies subsets of patients with Barretts esophagus at low and high risk for progression to cancer. Patients whose baseline biopsies are negative, indefinite, or low-grade displasia without increased 4N or aneuploidy may have surveillance deferred for up to 5 yr. Patients with cytometric abnormalities merit more frequent surveillance, and management of high-grade displasia can be individualized.

An endoscopic biopsy protocol can differentiate high-grade dysplasia from early adenocarcinoma in Barrett's esophagus

BACKGROUND Surveying vs. performing resection in patients with high-grade dysplasia in Barretts esophagus is debated because of concern about the accuracy of endoscopic biopsy diagnosis. The aim of this study was to investigate the accuracy of an endoscopic biopsy protocol in patients with neoplastic abnormalities in Barretts epithelium without obvious esophageal cancer. METHODS Preoperative and postoperative diagnoses in 28 patients who underwent surgery for high-grade dysplasia or early adenocarcinoma in Barretts esophagus and compared them with 22 other patients with high-grade dysplasia who were maintained under prospective endoscopic surveillance. All 50 patients lacked gross lesions to suggest esophageal cancer. The endoscopic protocol involved rigorous, systematic acquisition of multiple, large biopsy samples. RESULTS Overall, 64% of patients had minimal but distinct endoscopic abnormalities that were targeted for biopsies. High-grade dysplasia alone (7 patients) was differentiated from early adenocarcinoma (19 patients). Two patients with preoperative diagnoses of intramucosal adenocarcinoma had high-grade dysplasia in their resection specimens. CONCLUSIONS This endoscopic protocol accurately detects and differentiates high-grade dysplasia from early adenocarcinoma in Barretts esophagus. Patients with high-grade dysplasia alone in Barretts esophagus detected by such a protocol do not necessarily require surgical resection to rule out an undiagnosed adenocarcinoma; electing for surgery should be based on other clinical considerations.

DNA aneuploidy in colonic biopsies predicts future development of dysplasia in ulcerative colitis

The objective of the present study was to determine whether abnormal epithelial DNA content (aneuploidy) in colonic biopsy specimens from ulcerative colitis (UC) patients correlated with and predicted histological progression to dysplasia. Aneuploidy was absent in 20 low-cancer risk patients. In 81 high-cancer risk patients aneuploidy correlated significantly with the severity of histological abnormality (negative, indefinite, dysplasia, or cancer). Statistically our data suggest that many more biopsy specimens than are usually taken are needed to detect focal dysplastic lesions. Prospective study of 25 high risk patients without dysplasia revealed 5 with aneuploidy, all of whom progressed to dysplasia in 1-2.5 years, whereas 19 patients without aneuploidy did not progress to either aneuploidy or dysplasia within 2-9 years. Our data indicate that aneuploidy in mucosal biopsy specimens correlates with histological grade and identifies a subset of patients without dysplasia who are more likely to develop it. It was concluded that more frequent and extensive colonoscopic surveillance of this minority subset of high risk patients and less frequent surveillance in the remaining majority may reduce cost and detect more curable lesions.

Flow-cytometric and histological progression to malignancy in Barrett's esophagus: Prospective endoscopic surveillance of a cohort

To determine whether or not flow-cytometric evidence of aneuploidy and increased G2/tetraploid fractions predispose to neoplastic progression in Barretts esophagus, 62 patients with Barretts esophagus were evaluated prospectively for a mean interval of 34 months. Nine of 13 patients who showed aneuploid or increased G2/tetraploid populations in their initial flow-cytometric analysis developed high-grade dysplasia or adenocarcinoma during follow-up; none of the 49 patients without these abnormalities progressed to high-grade dysplasia or cancer (P less than 0.0001). Neoplastic progression was characterized by progressive flow-cytometric and histological abnormalities. Patients who progressed to high-grade dysplasia and carcinoma frequently developed multiple aneuploid populations of cells that were detectable flow-cytometrically. Similarly, patients appeared to progress through a phenotypic sequence that could be recognized histologically by the successive appearance of Barretts metaplasia negative for dysplasia, abnormalities in the indefinite/low-grade dysplasia range, high-grade dysplasia, and eventually adenocarcinoma. These and prior results suggest that neoplastic progression in Barretts esophagus occurs in a subset of patients who have an acquired genomic instability that generates abnormal clones of cells, some of which have aneuploid or increased G2/tetraploid DNA contents. With continued genomic instability, multiple aneuploid subclones may evolve, one of which may ultimately acquire the ability to invade and become an early carcinoma. The combination of histology and flow cytometry can be used to identify a subset of patients with Barretts esophagus who merit more frequent endoscopic surveillance for the early detection of high-grade dysplasia or carcinoma.

Optimizing endoscopic biopsy detection of early cancers in Barrett's high-grade dysplasia

OBJECTIVE: The management of high-grade dysplasia (HGD) in Barrett’s esophagus remains controversial, in part, because of uncertainty about the ability of endoscopic biopsies to consistently detect early, curable cancers. METHODS: Here we report cancers we have diagnosed in 45 patients with Barrett’s HGD using a protocol involving serial endoscopies with four-quadrant biopsies taken at 1-cm intervals. We compare these results to a modeled endoscopic biopsy protocol in which four-quadrant biopsies are taken every 2 cm in the Barrett’s segment. RESULTS: Thirteen cancers were detected at the baseline endoscopy and 32 in surveillance. In 82% of patients, cancer was detected at a single 1-cm level of the esophagus, and in 69% the cancer was detected in a single endoscopic biopsy specimen. A 2-cm protocol missed 50% of cancers that were detected by a 1-cm protocol in Barrett’s segments 2 cm or more without visible lesions. The maximum depth of cancer invasion was intramucosal in 96% of patients. Only 39% of patients who had endoscopic biopsy cancer diagnoses had cancer detected in the esophagectomy specimen. Adverse outcomes included the development of regional metastatic disease during surveillance (1 of 32), operative mortality (3 of 36), including two patients who had their primary surgeries at other institutions, and death from metastatic disease after endoscopic ablation performed at another institution (1 of 3). CONCLUSIONS: A four-quadrant, 1-cm endoscopic biopsy protocol performed at closely timed intervals consistently detects early cancers arising in HGD in Barrett’s esophagus and should be used in patients with HGD who do not undergo surgical resection.

Predictors of progression in Barrett's esophagus III: baseline flow cytometric variables

OBJECTIVES:Barretts esophagus develops in 5–10% of patients with gastroesophageal reflux disease and predisposes to esophageal adenocarcinoma. We have previously shown that a systematic baseline endoscopic biopsy protocol using flow cytometry with histology identifies subsets of patients with Barretts esophagus at low and high risk for progression to cancer. In this report, we further examined cytometric variables to better define the characteristics that best enable DNA cytometry to help predict cancer outcome.METHODS:Patients were prospectively evaluated using a systematic endoscopic biopsy protocol, with baseline histological and flow cytometric measurements as predictors and with cancer as the outcome.RESULTS:A receiver operating curve analysis demonstrated that a 4N fraction cut point of 6% was optimal to discriminate cancer risk (relative risk [RR] = 11.7, 95% CI = 6.2–22). The 4N fractions of 6–15% were just as predictive of cancer as were fractions of >15%. We found that only aneuploid DNA contents of >2.7N were predictive of cancer (RR = 9.5, CI = 4.9–18), whereas those patients whose sole abnormality was an aneuploid population with DNA content of ≤2.7 had a low risk for progression. The presence of both 4N fraction of >6% and aneuploid DNA content of >2.7N was highly predictive of cancer (RR = 23, CI = 10–50). S phase was a predictor of cancer risk (RR = 2.3, CI = 1.2–4.4) but was not significant when high-grade dysplasia was accounted for.CONCLUSIONS:Flow cytometry is a useful adjunct to histology in assessing cancer risk in patients with Barretts esophagus. Careful examination of cytometric variables revealed a better definition of those parameters that are most closely associated with increased cancer risk.

Barrett's esophagus: Cell cycle abnormalities in advancing stages of neoplastic progression

BACKGROUND Abnormal proliferation in Barretts esophagus may predispose to the development of esophageal adenocarcinoma, but previous studies have not determined the specific cell cycle abnormalities that were associated with neoplastic progression. METHODS Ki67/DNA content multiparameter flow cytometry and DNA content flow cytometry were used to investigate G0, G1, and S phase fractions in advancing stages of neoplastic progression in Barretts esophagus. RESULTS In control biopsy specimens from gastric mucosa, G1, S phase, and total Ki67-positive proliferative fractions were low, suggesting that cells were predominantly in G0. Ki67-positive G1 fractions were increased in Barretts metaplasia. More advanced stages of neoplastic progression were characterized by a subset of biopsy specimens that had aneuploid cell populations, increased S phase fractions, or both. CONCLUSIONS The development of increased G1 fractions is an early event in Barretts metaplasia. Increased S phase fractions occur in a subset of specimens typically at more advanced stages of neoplastic progression and often in association with the development of aneuploidy. Neoplastic progression in Barretts esophagus is associated with at least three types of cell cycle abnormalities: (1) mobilization from G0 into G1; (2) loss of control of the G1/S phase transition; and (3) accumulation in G2.

Evaluation of p53 protein expression in Barrett's esophagus by two-parameter flow cytometry

Barretts esophagus is a condition in which the normal stratified squamous epithelium is replaced by metaplastic columnar epithelium that predisposes to the development of esophageal adenocarcinoma. Neoplastic progression in Barretts esophagus occurs by a multistep process associated with genomic instability and the development of aneuploid cell populations. p53 protein overexpression and allelic deletions on chromosome 17p have been shown to be present in some Barretts adenocarcinomas, but the stage in neoplastic progression at which p53 protein overexpression develops has not been investigated. To determine the stages in neoplastic progression at which p53 protein overexpression could be detected, biopsy specimens from patients with Barretts esophagus at all stages of histological progression from Barretts metaplasia negative for dysplasia to esophageal adenocarcinoma were investigated using a multiparameter flow-cytometric assay. p53 protein overexpression was found in 1 of 21 patients (5%) with Barretts metaplasia negative for dysplasia, 2 of 13 patients (15%) with Barretts metaplasia with abnormalities in the indefinite/low-grade dysplasia range, 5 of 11 patients (45%) with high-grade dysplasia, and 8 of 15 patients (53%) with Barretts adenocarcinoma (P less than 0.01). p53 protein overexpression was found in 9% of patients with Barretts esophagus who had neither high-grade dysplasia nor adenocarcinoma. Whether or not patients whose biopsy specimens show p53 protein overexpression are at increased risk for progression to adenocarcinoma can be determined by prospective endoscopic surveillance.

p53 Mutations in Barrett's adenocarcinoma and high-grade dysplasia

BACKGROUND/AIMS Allelic losses of chromosome 17p and overexpression of p53 protein have been reported in Barretts adenocarcinomas. This study aimed to determine the stage in which p53 mutations arise in neoplastic progression in Barretts esophagus and their relationship to the clonal evolution of cancer. METHODS Fourteen patients with high-grade dysplasia, adenocarcinoma, or both arising in Barretts esophagus were evaluated. Flow cytometric cell sorting was used to obtain purified populations of neoplastic cells for analysis of p53 mutations. DNA was extracted, and exons 5 through 9 of the p53 gene were amplified by polymerase chain reaction. Amplified DNA was sequenced and analyzed by automated sequencing. RESULTS Nine of the 14 patients had p53 mutations. Six of the 9 patients had regions of high-grade dysplasia that could be evaluated; all 6 had p53 mutations in high-grade dysplasia. In 3 patients, the same p53 mutations were found in both high-grade dysplasia and adenocarcinoma. All 14 patients had aneuploidy. In 4 patients, diploid cell populations could also be evaluated for p53 mutations; 3 of the 4 patients had p53 mutations in diploid cell populations. In 2 patients, the same p53 mutation was found in multiple aneuploid cell populations within a cancer. CONCLUSIONS p53 mutations occur frequently in Barretts adenocarcinomas. They develop in diploid cell populations. The same p53 mutations are then found in aneuploid cell populations in high-grade dysplasia, in cancer, and in multiple aneuploid cell populations in cancer.

C-Ki-ras mutations in chronic ulcerative colitis and sporadic colon carcinoma

Mutations in the first exon of the c-Ki-ras protooncogene were analyzed in carcinomas and dysplasias from patients with sporadic colon cancer and chronic ulcerative colitis by a combination of histological enrichment, cell sorting, polymerase catalyzed chain reaction, and direct sequencing. In contrast to sporadic colon carcinomas, where 52% (11 of 21) contained mutations in codon 12, only 1 of 28 samples of ulcerative colitis associated carcinoma or dysplasia contained a c-Ki-ras mutation, despite the presence of aneuploid cell populations. These results suggest that a different genetic pathway for tumor progression may exist between sporadic colon carcinoma and carcinomas arising in chronic ulcerative colitis.