Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma

Gengming Huang, Joseph D. Krocker, Jason L. Kirk, Shehzad Nawaz Merwat, Hyunsu Ju, Roger D. Soloway, Lucas R. Wieck, Albert Li, Anthony Okorodudu, John R. Petersen, Nihal E. Abdulla, Andrea Duchini, Luca Cicalese, Cristiana Rastellini, Peter C. Hu, Jianli Dong

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Background: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%-80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy. Methods: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. Results: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. Conclusions: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.

Original languageEnglish (US)
Pages (from-to)899-909
Number of pages11
JournalClinical Chemistry and Laboratory Medicine
Volume52
Issue number6
DOIs
StatePublished - 2014

Fingerprint

Methylation
Cyclin-Dependent Kinases
Hepatocellular Carcinoma
DNA
Biomarkers
Sensitivity and Specificity
Genetic Promoter Regions
ROC Curve
Liver
Liver Diseases
Assays
Blood
Chronic Disease
Alleles
Polymerase Chain Reaction
Testing
Serum

Keywords

  • Circulating cell-free DNA
  • DNA methylation
  • Hepatocellular carcinoma
  • INK4A
  • Pyrosequencing

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical
  • Medicine(all)

Cite this

Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma. / Huang, Gengming; Krocker, Joseph D.; Kirk, Jason L.; Merwat, Shehzad Nawaz; Ju, Hyunsu; Soloway, Roger D.; Wieck, Lucas R.; Li, Albert; Okorodudu, Anthony; Petersen, John R.; Abdulla, Nihal E.; Duchini, Andrea; Cicalese, Luca; Rastellini, Cristiana; Hu, Peter C.; Dong, Jianli.

In: Clinical Chemistry and Laboratory Medicine, Vol. 52, No. 6, 2014, p. 899-909.

Research output: Contribution to journalArticle

Huang, Gengming ; Krocker, Joseph D. ; Kirk, Jason L. ; Merwat, Shehzad Nawaz ; Ju, Hyunsu ; Soloway, Roger D. ; Wieck, Lucas R. ; Li, Albert ; Okorodudu, Anthony ; Petersen, John R. ; Abdulla, Nihal E. ; Duchini, Andrea ; Cicalese, Luca ; Rastellini, Cristiana ; Hu, Peter C. ; Dong, Jianli. / Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma. In: Clinical Chemistry and Laboratory Medicine. 2014 ; Vol. 52, No. 6. pp. 899-909.
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abstract = "Background: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60{\%}-80{\%} of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy. Methods: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. Results: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3{\%} sensitivity and 87.2{\%} specificity at 5{\%} (LOD), 39.0{\%} sensitivity and 96.5{\%} specificity at 7{\%} LOD, and 20.3{\%} sensitivity and 98.8{\%} specificity at 10{\%} LOD, respectively. Conclusions: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.",
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T1 - Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma

AU - Huang, Gengming

AU - Krocker, Joseph D.

AU - Kirk, Jason L.

AU - Merwat, Shehzad Nawaz

AU - Ju, Hyunsu

AU - Soloway, Roger D.

AU - Wieck, Lucas R.

AU - Li, Albert

AU - Okorodudu, Anthony

AU - Petersen, John R.

AU - Abdulla, Nihal E.

AU - Duchini, Andrea

AU - Cicalese, Luca

AU - Rastellini, Cristiana

AU - Hu, Peter C.

AU - Dong, Jianli

PY - 2014

Y1 - 2014

N2 - Background: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%-80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy. Methods: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. Results: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. Conclusions: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.

AB - Background: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%-80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for noninvasive diagnosis and prediction of response to therapy. Methods: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. Results: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. Conclusions: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.

KW - Circulating cell-free DNA

KW - DNA methylation

KW - Hepatocellular carcinoma

KW - INK4A

KW - Pyrosequencing

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M3 - Article

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