Algorithm Training and Testing for a Nonendoscopic Barrett's Esophagus Detection Test in Prospective Multicenter Cohorts.

BACKGROUND & AIMS: Endoscopic Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) detection is invasive and expensive. Nonendoscopic BE/EAC detection tools are guideline-endorsed alternatives. We previously described a 5-methylated DNA marker (MDM) panel assayed on encapsulated sponge cell collection device (CCD) specimens. We aimed to train a new algorithm using a 3-MDM panel and test its performance in an independent cohort. METHODS: Algorithm training and test samples were from 2 prospective multicenter cohorts. All BE cases had esophageal intestinal metaplasia (with or without dysplasia/EAC); control subjects had no endoscopic evidence of BE. The CCD procedure was followed by endoscopy. From CCD cell lysates, DNA was extracted, bisulfite treated, and MDMs were blindly assayed. The algorithm was set and locked using cross-validated logistic regression (training set) and its performance was assessed in an independent test set. RESULTS: Training (N = 352) and test (N = 125) set clinical characteristics were comparable. The final panel included 3 MDMs (NDRG4, VAV3, ZNF682). Overall sensitivity was 82% (95% CI, 68%-94%) at 90% (79%-98%) specificity and 88% (78%-94%) sensitivity at 84% (70%-93%) specificity in training and test sets, respectively. Sensitivity was 90% and 68% for all long- and short-segment BE, respectively. Sensitivity for BE with high-grade dysplasia and EAC was 100% in training and test sets. Overall sensitivity for nondysplastic BE was 82%. Areas under the receiver operating characteristic curves for BE detection were 0.92 and 0.94 in the training and test sets, respectively. CONCLUSIONS: A locked 3-MDM panel algorithm for BE/EAC detection using a nonendoscopic CCD demonstrated excellent sensitivity for high-risk BE cases in independent validation samples. (Clinical trials.gov: NCT02560623, NCT03060642.).

Methylated DNA markers for plasma detection of ovarian cancer: Discovery, validation, and clinical feasibility.

OBJECTIVE: Aberrant DNA methylation is an early event in carcinogenesis which could be leveraged to detect ovarian cancer (OC) in plasma. METHODS: DNA from frozen OC tissues, benign fallopian tube epithelium (FTE), and buffy coats from cancer-free women underwent reduced representation bisulfite sequencing (RRBS) to identify OC MDMs. Candidate MDM selection was based on receiver operating characteristic (ROC) discrimination, methylation fold change, and low background methylation among controls. Blinded biological validation was performed using methylated specific PCR on DNA extracted from independent OC and FTE FFPE tissues. MDMs were tested using Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) assays in pre-treatment plasma from women newly diagnosed with OC and population-sampled healthy women. A random forest modeling analysis was performed to generate predictive probability of disease; results were 500-fold in silico cross-validated. RESULTS: Thirty-three MDMs showed marked methylation fold changes (10 to >1000) across all OC subtypes vs FTE. Eleven MDMs (GPRIN1, CDO1, SRC, SIM2, AGRN, FAIM2, CELF2, RIPPLY3, GYPC, CAPN2, BCAT1) were tested on plasma from 91 women with OC (73 (80%) high-grade serous (HGS)) and 91 without OC; the cross-validated 11-MDM panel highly discriminated OC from controls (96% (95% CI, 89-99%) specificity; 79% (69-87%) sensitivity, and AUC 0.91 (0.86-0.96)). Among the 5 stage I/II HGS OCs included, all were correctly identified. CONCLUSIONS: Whole methylome sequencing, stringent filtering criteria, and biological validation yielded candidate MDMs for OC that performed with high sensitivity and specificity in plasma. Larger plasma-based OC MDM studies, including testing of pre-diagnostic specimens, are warranted.

Validation of a methylated DNA marker panel for the nonendoscopic detection of Barrett's esophagus in a multisite case-control study.

BACKGROUND AND AIMS: We previously identified a 5 methylated DNA marker (MDM) panel for the detection of nonendoscopic Barrett's esophagus (BE). In this study, we aimed to recalibrate the performance of the 5 MDM panel using a simplified assay in a training cohort, validate the panel in an independent test cohort, and explore the accuracy of an MDM panel with only 3 markers. METHODS: Participants were recruited from 3 medical centers. The sponge on a string device (EsophaCap; CapNostics, Concord, NC, USA) was swallowed and withdrawn, followed by endoscopy, in BE cases and control subjects. A 5 MDM panel was blindly assayed using a simplified assay. Random forest modeling analysis was performed, in silico cross-validated in the training set, and then locked down, before test set analysis. RESULTS: The training set had 199 patients: 110 BE cases and 89 control subjects, and the test set had 89 patients: 60 BE cases and 29 control subjects. Sensitivity of the 5 MDM panel for BE diagnosis was 93% at 90% specificity in the training set and 93% at 93% specificity in the test set. Areas under the receiver operating characteristic curves were .96 and .97 in the training and test sets, respectively. Model accuracy was not influenced by age, sex, or smoking history. Multiple 3 MDM panels achieved similar accuracy. CONCLUSIONS: A 5 MDM panel for BE is highly accurate in training and test sets in a blinded multisite case-control analysis using a simplified assay. This panel may be reduced to only 3 MDMs in the future. (Clinical trial registration number: NCT02560623.).

Analysis of DNA Methylation at Specific Loci in Stool Samples Detects Colorectal Cancer and High-Grade Dysplasia in Patients With Inflammatory Bowel Disease.

BACKGROUND & AIMS: Patients with inflammatory bowel diseases (IBDs), including ulcerative colitis and Crohn's disease, are at increased risk for colorectal cancer (CRC). Analyses of DNA methylation patterns in stool samples have been reported to detect CRC in patients with IBD. We sought to validate these findings in larger cohorts and assess the accuracy of analysis of DNA methylation patterns in stool for detection of CRC and high-grade dysplasia (HGD) normalized to methylation level at ZDHHC1. METHODS: We obtained buffered, frozen stool samples from a US case-control study and from 2 European surveillance cohorts (referral or population based) of patients with chronic ulcerative colitis (n = 248), Crohn's disease (n = 82), indeterminate colitis (n = 2), or IBD with primary sclerosing cholangitis (n = 38). Stool samples were collected before bowel preparation for colonoscopy or at least 1 week after colonoscopy. Among the study samples, stools from individuals with IBD but without neoplasia were used as controls (n = 291). DNA was isolated from stool, exposed to bisulfite, and then assayed by multiplex quantitative allele-specific real-time target and signal amplification. We analyzed methylation levels of BMP3, NDRG4, VAV3, and SFMBT2 relative to the methylation level of ZDHHC1, and compared these between patients with CRC or HGD and controls. RESULTS: Levels of methylation at BMP3 and VAV3, relative to ZDHHC1 methylation, identified patients with CRC and HGD with an area under the curve value of 0.91 (95% CI, 0.77-1.00). Methylation levels at specific promotor regions of these genes identified 11 of the 12 patients with CRC and HGD, with 92% sensitivity (95% CI, 60%-100%) and 90% specificity (95% CI, 86%-93%). The proportion of false-positive results did not differ significantly among the case-control, referral cohort, and population cohort studies (P = .60) when the 90% specificity cut-off from the whole sample set was applied. CONCLUSIONS: In an analysis of stool samples from 3 independent studies of 332 patients with IBD, we associated levels of methylation at 2 genes (BMP3 and VAV3), relative to level of methylation at ZDHHC1, with detection of CRC and HGD. These methylation patterns identified patients with CRC and HGD with more than 90% specificity, and might be used in CRC surveillance.

Hepatocellular Carcinoma Detection by Plasma Methylated DNA: Discovery, Phase I Pilot, and Phase II Clinical Validation.

Early detection improves hepatocellular carcinoma (HCC) outcomes, but better noninvasive surveillance tools are needed. We aimed to identify and validate methylated DNA markers (MDMs) for HCC detection. Reduced representation bisulfite sequencing was performed on DNA extracted from 18 HCC and 35 control tissues. Candidate MDMs were confirmed by quantitative methylation-specific PCR in DNA from independent tissues (74 HCC, 29 controls). A phase I plasma pilot incorporated quantitative allele-specific real-time target and signal amplification assays on independent plasma-extracted DNA from 21 HCC cases and 30 controls with cirrhosis. A phase II plasma study was then performed in 95 HCC cases, 51 controls with cirrhosis, and 98 healthy controls using target enrichment long-probe quantitative amplified signal (TELQAS) assays. Recursive partitioning identified best MDM combinations. The entire MDM panel was statistically cross-validated by randomly splitting the data 2:1 for training and testing. Random forest (rForest) regression models performed on the training set predicted disease status in the testing set; median areas under the receiver operating characteristics curve (AUCs; and 95% confidence interval [CI]) were reported after 500 iterations. In phase II, a six-marker MDM panel (homeobox A1 [HOXA1], empty spiracles homeobox 1 [EMX1], AK055957, endothelin-converting enzyme 1 [ECE1], phosphofructokinase [PFKP], and C-type lectin domain containing 11A [CLEC11A]) normalized by beta-1,3-galactosyltransferase 6 (B3GALT6) level yielded a best-fit AUC of 0.96 (95% CI, 0.93-0.99) with HCC sensitivity of 95% (88%-98%) at specificity of 92% (86%-96%). The panel detected 3 of 4 (75%) stage 0, 39 of 42 (93%) stage A, 13 of 14 (93%) stage B, 28 of 28 (100%) stage C, and 7 of 7 (100%) stage D HCCs. The AUC value for alpha-fetoprotein (AFP) was 0.80 (0.74-0.87) compared to 0.94 (0.9-0.97) for the cross-validated MDM panel (P < 0.0001). Conclusion: MDMs identified in this study proved to accurately detect HCC by plasma testing. Further optimization and clinical testing of this promising approach are indicated.

Placental-derived mesenchyme influences chorionic gonadotropin and progesterone secretion of human embryonic stem cell-derived trophoblasts.

Studies of early placental development in humans are difficult because of limitations on experimental material availability from the perimplantation period. We used a coculture system to determine the effects of various effector cell types on trophoblast differentiation. Enhanced green fluorescent protein (EGFP)-expressing H1 human embryonic stem cells were used in co-suspension with human term placental fibroblasts (TPFs) and dermal fibroblasts (CI2F) to form combination embryoid bodies (EBs), with the goal of recapitulating placental morphogenesis through incorporation of placental mesenchymal cells. Overall, the results demonstrated that when using mesenchymal cells for EB preparation from term placentas (TPF), combination EB-derived trophoblasts secrete higher levels of human chorionic gonadotropin (hCG) and progesterone compared to EBs made without effector cells, whereas there was no effect of dermal fibroblasts. This is due to the secretory activity of the EB-derived trophoblasts and not due to the number of differentiated trophoblasts per EB, demonstrating that nontrophoblast cells of the placenta can influence trophoblast endocrine activity.