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 Christopher Smyrk, Jason T. Lewis, Maria Giakoumopoulos, Hatim T. Allawi, Graham P. Lidgard, Lewis Rowland Roberts & 1 others David A. Ahlquist

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

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)
JournalHepatology
DOIs
StatePublished - Jan 1 2019

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Genetic Markers
Hepatocellular Carcinoma
DNA
Area Under Curve
Homeobox Genes
Fibrosis
Confidence Intervals
C-Type Lectins
Galactosyltransferases
alpha-Fetoproteins
ROC Curve
Methylation
Alleles
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Hepatology

Cite this

Hepatocellular Carcinoma Detection by Plasma Methylated DNA : Discovery, Phase I Pilot, and Phase II Clinical Validation. / Kisiel, John B; Dukek, Brian A.; V.S.R. Kanipakam, Reddappa; Ghoz, Hassan M.; Yab, Tracy C.; Berger, Calise K.; Taylor, William R.; Foote, Patrick H.; Giama, Nasra H.; Onyirioha, Kristeen; Abdallah, Mohamed A.; Burger, Kelli N.; Slettedahl, Seth W.; Mahoney, Douglas W.; Smyrk, Thomas Christopher; Lewis, Jason T.; Giakoumopoulos, Maria; Allawi, Hatim T.; Lidgard, Graham P.; Roberts, Lewis Rowland; Ahlquist, David A.

In: Hepatology, 01.01.2019.

Research output: Contribution to journalArticle

Kisiel, JB, Dukek, BA, V.S.R. Kanipakam, R, Ghoz, HM, Yab, TC, Berger, CK, Taylor, WR, Foote, PH, Giama, NH, Onyirioha, K, Abdallah, MA, Burger, KN, Slettedahl, SW, Mahoney, DW, Smyrk, TC, Lewis, JT, Giakoumopoulos, M, Allawi, HT, Lidgard, GP, Roberts, LR & Ahlquist, DA 2019, 'Hepatocellular Carcinoma Detection by Plasma Methylated DNA: Discovery, Phase I Pilot, and Phase II Clinical Validation', Hepatology. https://doi.org/10.1002/hep.30244
Kisiel, John B ; Dukek, Brian A. ; V.S.R. Kanipakam, Reddappa ; Ghoz, Hassan M. ; Yab, Tracy C. ; Berger, Calise K. ; Taylor, William R. ; Foote, Patrick H. ; Giama, Nasra H. ; Onyirioha, Kristeen ; Abdallah, Mohamed A. ; Burger, Kelli N. ; Slettedahl, Seth W. ; Mahoney, Douglas W. ; Smyrk, Thomas Christopher ; Lewis, Jason T. ; Giakoumopoulos, Maria ; Allawi, Hatim T. ; Lidgard, Graham P. ; Roberts, Lewis Rowland ; Ahlquist, David A. / Hepatocellular Carcinoma Detection by Plasma Methylated DNA : Discovery, Phase I Pilot, and Phase II Clinical Validation. In: Hepatology. 2019.
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abstract = "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.",
author = "Kisiel, {John B} and Dukek, {Brian A.} and {V.S.R. Kanipakam}, Reddappa and Ghoz, {Hassan M.} and Yab, {Tracy C.} and Berger, {Calise K.} and Taylor, {William R.} and Foote, {Patrick H.} and Giama, {Nasra H.} and Kristeen Onyirioha and Abdallah, {Mohamed A.} and Burger, {Kelli N.} and Slettedahl, {Seth W.} and Mahoney, {Douglas W.} and Smyrk, {Thomas Christopher} and Lewis, {Jason T.} and Maria Giakoumopoulos and Allawi, {Hatim T.} and Lidgard, {Graham P.} and Roberts, {Lewis Rowland} and Ahlquist, {David A.}",
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T1 - Hepatocellular Carcinoma Detection by Plasma Methylated DNA

T2 - Discovery, Phase I Pilot, and Phase II Clinical Validation

AU - Kisiel, John B

AU - Dukek, Brian A.

AU - V.S.R. Kanipakam, Reddappa

AU - Ghoz, Hassan M.

AU - Yab, Tracy C.

AU - Berger, Calise K.

AU - Taylor, William R.

AU - Foote, Patrick H.

AU - Giama, Nasra H.

AU - Onyirioha, Kristeen

AU - Abdallah, Mohamed A.

AU - Burger, Kelli N.

AU - Slettedahl, Seth W.

AU - Mahoney, Douglas W.

AU - Smyrk, Thomas Christopher

AU - Lewis, Jason T.

AU - Giakoumopoulos, Maria

AU - Allawi, Hatim T.

AU - Lidgard, Graham P.

AU - Roberts, Lewis Rowland

AU - Ahlquist, David A.

PY - 2019/1/1

Y1 - 2019/1/1

N2 - 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.

AB - 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.

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