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

John B. Kisiel, Brian A. Dukek, Reddappa V.S.R. Kanipakam, Hassan M. Ghoz, Tracy C. Yab, Calise K. Berger, William R. Taylor, Patrick H. Foote, Nasra H. Giama, Kristeen Onyirioha, Mohamed A. Abdallah, Kelli N. Burger, Seth W. Slettedahl, Douglas W. Mahoney, Thomas C. Smyrk, Jason T. Lewis, Maria Giakoumopoulos, Hatim T. Allawi, Graham P. Lidgard, Lewis R. RobertsDavid A. Ahlquist

Research output: Contribution to journalArticlepeer-review

38 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1180-1192
Number of pages13
Issue number3
StatePublished - Mar 2019
Externally publishedYes

Bibliographical note

Funding Information:
Received April 30, 2018; accepted August 21, 2018. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.30244/suppinfo. Supported by the Maxine and Jack Zarrow Family Foundation of Tulsa Oklahoma (to J.B.K.), the Paul Calabresi Program in Clinical-Translational Research (NCI CA90628; to J.B.K.), R37 CA214679 (to J.B.K.), the Dana and Edmund Gong Foundation (to D.A.A.), and the Carol M. Gatton endowment for Digestive Diseases Research (to D.A.A.). RRBS sequencing costs, QuARTS assays, and TELQAS assays were provided by Exact Sciences (Madison, WI). © 2018 by the American Association for the Study of Liver Diseases. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.30244 Potential conflict of interest: Dr. Roberts consults for and received grants from Wako. He consults for Medscape, Axis, One Live, NACCME, and Refine. He advises for Bayer, Grail, and Tavec. He received grants from Ariad, BTG, Gilead, and Redhill. Dr. Ahlquist received grants from and holds Intellectual Property Rights for Exact Sciences. Dr. Kisiel received grants from and holds Intellectual Property Rights for Exact Sciences. Mr. Mahoney holds Intellectual Property Rights for Exact Sciences. Dr. Lidgard is employed by and owns stock in Exact Sciences. Dr. Allawi is employed by Exact Sciences. Mr. Taylor holds Intellectual Property Rights for Exact Sciences. Mr. Yab owns stock in and holds Intellectual Property Rights for Exact Sciences.

Funding Information:
The study was conducted in four sequential case-control experiments (Fig.). Briefly, we first performed discovery using reduced representation bisulfite sequencing (RRBS) and technically validated sequencing-derived candidate MDMs using methylation-specific PCR (MSP) on DNA extracted from frozen primary tumor and control samples, in addition to normal buffy coat control samples. Candidate regions were selected where there was differential hypermethylation between cases referent to liver controls and between cases and buffy coat controls; to select candidates for a circulating tumor DNA (ctDNA) assay, we assumed that background plasma DNA would primarily originate from leukocytes. Technically validated MDM candidates were then biologically validated on independent formalin-fixed, paraffin-embedded (FFPE) tissue samples. Additional MDMs from other GI cancers were also tested on the basis of (1) hypermethylation in at least two other GI cancer types, (2) area under the receiver operating characteristics curve (AUC) values in excess of 0.9, and (3) minimal cross-reactivity (<2% methylation) in leukocyte DNA.

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