INTRODUCTION: An automated risk prediction assay has previously been shown to objectively identify patients with nondysplastic Barrett's esophagus (NDBE) who are at increased risk of malignant progression. To evaluate the predictive performance of the assay in 76 patients with NDBE of which 38 progressed to high-grade dysplasia/esophageal adenocarcinoma (progressors) and 38 did not (nonprogressors) and to determine whether assessment of additional (spatial) levels per endoscopy and/or multiple (temporal) time points improves assay performance. METHODS: In a blinded, nested case-control cohort, progressors and nonprogressors were matched (age, sex, and Barrett's esophagus length). All random biopsy levels from the baseline endoscopy (spatial samples) and all available previous endoscopies back to 10 years before progression (temporal samples) were assayed. Because the 1:1 ratio of progressors to nonprogressors does not reflect the real-world Barrett's population, negative and positive predictive values were adjusted for prevalence. RESULTS: Seventy-six patients (58 men), mean age of 63 6 9 years, were studied. A high-risk score was associated with a prevalence-adjusted annual progression rate of 6.9%. The assay identified 31% of progressors when assessing a single biopsy level from the baseline endoscopy. Sensitivity increased to 50% and 69% in spatial and temporal analyses, respectively, while specificity remained at 95%. DISCUSSION: The assay identified a significant subset of NDBE patients who progress at a rate comparable with published estimates for expert-confirmed low-grade dysplasia. Assessing additional spatial and temporal biopsies increased the predictive accuracy, allowing for identification of most future progressors. Additional studies will evaluate the predictive performance of the assay in low-prevalence settings.