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 language | English (US) |
|---|---|
| Pages (from-to) | 899-909 |
| Number of pages | 11 |
| Journal | Clinical Chemistry and Laboratory Medicine |
| Volume | 52 |
| Issue number | 6 |
| DOIs | |
| State | Published - 2014 |
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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 journal › Article
}
TY - JOUR
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
UR - http://www.scopus.com/inward/record.url?scp=84903585680&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84903585680&partnerID=8YFLogxK
U2 - 10.1515/cclm-2013-0885
DO - 10.1515/cclm-2013-0885
M3 - Article
VL - 52
SP - 899
EP - 909
JO - Clinical Chemistry and Laboratory Medicine
JF - Clinical Chemistry and Laboratory Medicine
SN - 1434-6621
IS - 6
ER -