Patrick H. Foote

DNA Methylation and Mutation of Small Colonic Neoplasms in Ulcerative Colitis and Crohn's Colitis: Implications for Surveillance.

Background:Stool DNA testing in patients with inflammatory bowel disease (IBD) may detect colorectal cancer and advanced precancers with high sensitivity; less is known about the presence of DNA markers in small IBD lesions, their association with metachronous neoplasia, or contribution to stool test positivity. Methods:At a single center in 2 blinded phases, we assayed methylated bone morphogenic protein 3, methylated N-Myc downstream-regulated gene 4, and mutant KRAS in DNA extracted from paraffin-embedded benign lesions, and matched control tissues of patients with IBD, who were followed for subsequent colorectal dysplasia. Stool samples from independent cases and controls with lesions <1 cm or advanced neoplasms were assayed for the same markers. Results:Among IBD lesions (29 low-grade dysplasia, 19 serrated epithelial change, and 10 sessile serrated adenoma/polyps), the prevalence of methylation was significantly higher than in mucosae from 44 matched IBD controls (P < 0.0001 for methylated bone morphogenic protein 3 or methylated N-Myc downstream-regulated gene 4). KRAS mutations were more abundant in serrated epithelial change than all other groups (P < 0.001). Subsequent dysplasia was not associated with DNA marker levels. In stools, the sensitivity of methylated bone morphogenic protein 3 as a single marker was 60% for all lesions <1 cm, 63% for low-grade dysplasia ≥1 cm and 81% for high-grade dysplasia/colorectal cancer, all at 91% specificity (P < 0.0001). Conclusions:Selected DNA markers known to be present in advanced IBD neoplasia can also be detected in both tissues and stools from IBD patients with small adenomas and serrated lesions. Mutant KRAS exfoliated from serrated epithelial change lesions might raise false-positive rates. These findings have relevance to potential future applications of stool DNA testing for IBD surveillance.

Highly Discriminant Methylated DNA Markers for the Non-endoscopic Detection of Barrett’s Esophagus

BACKGROUND: Minimally invasive methods have been described to detect Barretts esophagus (BE), but are limited by subjectivity and suboptimal accuracy. We identified methylated DNA markers (MDMs) for BE in tissue and assessed their accuracy on whole esophagus brushings and capsule sponge samples. METHODS: Step 1: Unbiased whole methylome sequencing was performed on DNA from BE and normal squamous esophagus (SE) tissue. Discriminant MDM candidates were validated on an independent patient cohort (62 BE cases, 30 controls) by quantitative methylation specific PCR (qMSP). Step 2: Selected MDMs were further evaluated on whole esophageal brushings (49 BE cases, 36 controls). 35 previously sequenced esophageal adenocarcinoma (EAC) MDMs were also evaluated. Step 3: 20 BE cases and 20 controls were randomized to swallow capsules sponges (25 mm, 10 pores or 20 pores per inch (ppi)) followed endoscopy. DNA yield, tolerability, and mucosal injury were compared. Best MDM assays were performed on this cohort. RESULTS: Step 1: 19 MDMs with areas under the ROC curve (AUCs) >0.85 were carried forward. Step 2: On whole esophageal brushings, 80% of individual MDM candidates showed high accuracy for BE (AUCs 0.84‐0.94). Step 3: The capsule sponge was swallowed and withdrawn in 98% of subjects. Tolerability was superior with the 10 ppi sponge with minimal mucosal injury and abundant DNA yield. A 2‐marker panel (VAV3 + ZNF682) yielded excellent BE discrimination (AUC = 1). CONCLUSIONS: Identified MDMs discriminate BE with high accuracy. BE detection appears safe and feasible with a capsule sponge. Corroboration in larger studies is warranted. ClinicalTrials.gov number NCT02560623.

Su1914 Novel Epigenetic Markers for Detection of Esophageal Cancer: Selection by Whole Methylome Sequencing and Tissue Validation

G A A b st ra ct s pathway involved in tumor microenvironment interactions, although the role of the Act A ligand in this context has yet to be fully elucidated. Recent research has shown that myofibroblasts in the tumor microenvironment secrete high levels of Act A acting to create a reactive stroma and promote cancer cell invasion. Methods: To investigate the role of Act A in the esophageal microenvironment, Act A was stably and constitutively overexpressed in human esophageal fibroblasts, FEF-ActA. Act A expression was validated by ELISA. Premalignant esophageal keratinocytes, which express dominant-negative E-cadherin and TGFβRII (ECdnT), were compared to the esophageal squamous cell carcinoma cell line TE-11. The functional role of Act A in themicroenvironment was primarily investigated using organotypic reconstruct cultures, in which epithelial cells are grown on top of a fibroblasts embedded matrix to examine the physiological context. RNA and protein profiles were examined by qRTPCR, Western blot, and immunofluorescence staining. Results: We have shown differential invasive capabilities of epithelial cells in the presence or absence of Act A. Using FEF-ActA in organotypic cultures with premalignant ECdnT, we observed complete ablation of epithelial cell invasion. However this effect is not observed when FEF-ActA are grown with the cancer cell line TE-11. Immunofluorescence staining of organotypic cultures demonstrates that FEF-ActA exhibit downregulation of mesenchymal markers a-SMA and vimentin, as well as the invasion protein podoplanin, compared to control fibroblasts. ECdnT, when co-cultured with FEF-ActA, also showed differential localization of αv integrin along the basement membrane. Of particular interest, premalignant cultures with FEF-ActA showed reduced fibronectin staining and downregulation of MMP2 activation. Due to the differences observed in the ECM, we preformed a qRT-PCR Taqman array specifically examining genes associated with the ECM. Of 90 ECM-associated genes, 54 were upor downregulated by at least 50%. TE-11 cells co-cultured with FEF-ActA did not show these changes. Conclusions: We conclude that Act A acts to inhibit premalignant epithelial cell invasion into the stroma by preventing ECM reorganization. Therefore, in a premalignant setting, Act A works to protect the microenvironment from epithelial cell invasion. During malignancy, however, the effect of Act A is overcome, as demonstrated in cultures utilizing esophageal squamous cell carcinoma cells.

Detection of Gastric Cancer with Novel Methylated DNA Markers: Discovery, Tissue Validation, and Pilot Testing in Plasma

Purpose: Gastric adenocarcinoma is the third most common cause of cancer mortality worldwide. Accurate and affordable noninvasive detection methods have potential value for screening and surveillance. Herein, we identify novel methylated DNA markers (MDM) for gastric adenocarcinoma, validate their discrimination for gastric adenocarcinoma in tissues from geographically separate cohorts, explore marker acquisition through the oncogenic cascade, and describe distributions of candidate MDMs in plasma from gastric adenocarcinoma cases and normal controls. Experimental Design: Following discovery by unbiased whole-methylome sequencing, candidate MDMs were validated by blinded methylation-specific PCR in archival case–control tissues from U.S. and South Korean patients. Top MDMs were then assayed by an analytically sensitive method (quantitative real-time allele-specific target and signal amplification) in a blinded pilot study on archival plasma from gastric adenocarcinoma cases and normal controls. Results: Whole-methylome discovery yielded novel and highly discriminant candidate MDMs. In tissue, a panel of candidate MDMs detected gastric adenocarcinoma in 92% to 100% of U.S. and South Korean cohorts at 100% specificity. Levels of most MDMs increased progressively from normal mucosa through metaplasia, adenoma, and gastric adenocarcinoma with variation in points of greatest marker acquisition. In plasma, a 3-marker panel (ELMO1, ZNF569, C13orf18) detected 86% (95% CI, 71–95) of gastric adenocarcinomas at 95% specificity. Conclusions: Novel MDMs appear to accurately discriminate gastric adenocarcinoma from normal controls in both tissue and plasma. The point of aberrant methylation during oncogenesis varies by MDM, which may have relevance to marker selection in clinical applications. Further exploration of these MDMs for gastric adenocarcinoma screening and surveillance is warranted. Clin Cancer Res; 24(22); 5724–34. ©2018 AACR.

Abstract 4252: Accurate site prediction of gastrointestinal cancer by novel methylated DNA markers: Discovery & validation:

Purpose: Methylated DNA markers can discriminate upper from lower GI neoplasms (Gastroenterology 2013;144(5):S84). It is unknown if organ-specific molecular prediction of GI tumor site can be achieved. Experimental design: An unbiased search for neoplasm markers at each major GI organ site was conducted by reduced representation bisulfite sequencing. Top marker candidates were then blindly validated by methylation specific PCR on independent tissue DNA samples from normal mucosa [17 esophagus (E), 13 stomach (S), 33 pancreas (P), 35 bile duct/liver (BD), 21 colorectal (CR)] and neoplasia [42 E, 43 S, 36 P, 48 BD, 97 CR]. Recursive partitioning (rPart) trees modeled all stepwise combinations of single markers to classify controls vs neoplasms and then neoplasms by site (CR, gastroesophageal or pancreaticobiliary). A 2nd model was designed from tissue data for blinded clinical piloting on 42 independent plasma DNA samples (14 normal and 28 cancers (14 P, 14 CR). Results: From the top 100 markers, 95 were validated and used for rPart modeling. A 3- marker panel (chr12.133484978-5047, BMP3, chr11.123301058-255) was selected for the universal detection of GI neoplasms, as it classified neoplasms and control tissues with 95% accuracy. Two markers (chr7.25896389-501, QKI) then assigned lower vs upper GI neoplasms with 94% accuracy. Finally, 3 markers (PDGFD, ELOVL2, PCBP3) called pancreaticobiliary vs gastroesophageal neoplasms with 94% accuracy. All 8 markers applied in a single model (Table) accurately predicted control vs tumor by site [88% (p Conclusion: Panels of methylated DNA markers were identified for both universal detection of GI neoplasia and accurate prediction of tumor site. Pilot testing of these markers in plasma supports potential clinical feasibility. Citation Format: John B. Kisiel, William R. Taylor, Tracy C. Yab, Hassan M. Ghoz, Patrick H. Foote, Mary E. Devens, Douglas W. Mahoney, Thomas C. Smyrk, Lisa A. Boardman, Gloria M. Petersen, Navtej S. Buttar, Lewis R. Roberts, Graham P. Lidgard, David A. Ahlquist. Accurate site prediction of gastrointestinal cancer by novel methylated DNA markers: Discovery & validation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4252. doi:10.1158/1538-7445.AM2015-4252

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.

Sa1921 Molecular Detection of Colorectal Neoplasia: Do Markers That Target Acquired DNA Alterations in Sporadic Cases Also Discriminate Lynch Syndrome Cases?

IRS-4 was constructed. RKO cells were transfected i) with the recombinant plasmid pcDNA3IRS-4 expressing the protein, or ii) with the empty plasmid pcDNA3 as a control. The impact of IRS-4 overexpression was analyzed on cell cycle and IGF-1 signaling. Sixteen colon tumor samples and paired tumor-adjacent normal tissue were collected during surgery and processed after obtaining informed consent. We evaluated the relationship between IRS-4 level and the proteins expression involved in G1/S checkpoint in the tumor and paired normal of all the reported cases by western blot. RESULTS: As we expected, RKO cells overexpressing IRS-4 showed higher levels of this protein than the control cells. The high levels of IRS-4 caused an increase of pRb, Rb, E2F, Cyclin E and Cyclin D1 expression in RKO cells. IRS-4, pRb, Rb and E2F were highly expressed in the cancerous tissue, in comparison with the normal epithelium. We evaluated the correlations between IRS-4 expression and the level of proteins involved in the control of cell cycle. The levels of IRS4 correlated positively with pRb (rpearson=0.745, P value <0.0001), Rb (rpearson=0.810, P value <0.0001) and E2F (rpearson=0.856, P value <0.0001). Regarding to the IGF-1 pathway, the levels of IRS-4 showed a positive correlation with pIGF-1R (rpearson=0.657, P value <0.0001), pAKT (rpearson=0.537, P value <0.01), AKT (rpearson=0.520, P value <0.01) and GSK-3 (rpearson=0.911, P value <0.0001) expression. Our general finding showed that IRS4 stimulates the expression of the proteins that unlock the G1/S restriction point in the cell cycle in RKO cells. Interestingly, the proteins involved in the cell cycle increased especially in the cancerous tissue of the patients overexpressing IRS-4. CONCLUSION: Our results suggest that IRS-4 is an important protein involved in the cell cycle regulation in colon cancer cells and it could be a key protein in the CRC development.