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Barrett's 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 Barrett's 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.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.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.Flow cytometry is a useful adjunct to histology in assessing cancer risk in patients with Barrett's esophagus. Careful examination of cytometric variables revealed a better definition of those parameters that are most closely associated with increased cancer risk.