Plasma mSEPT9: A Novel Circulating Cell-free DNA-Based Epigenetic Biomarker to Diagnose Hepatocellular Carcinoma

Abstract

SummaryBackgroundPatients with cirrhosis are at high risk of hepatocellular carcinoma (HCC). The SEPT9 gene is a key regulator of cell division and tumor suppressor whose hypermethylation is associated with liver carcinogenesis. The primary aim of this study was to evaluate the diagnostic accuracy of a PCR-based assay for the analysis of SEPT9 promoter methylation in circulating cell-free DNA (mSEPT9) for diagnosing HCC among cirrhotic patients.MethodsWe report two phase II biomarker studies that included cirrhotic patients with or without HCC from France (initial study) and Germany (replication study). All patients received clinical and biological evaluations, and liver imaging according to current recommendations. The primary outcome was defined as the presence of HCC according to guidelines from the American Association for the Study of Liver Diseases. The diagnosis of HCC was confirmed by abdominal contrast-enhanced computed tomography scan and systematically discussed in a multidisciplinary consultation meeting. HCC-free cirrhotic patients were recruited if the screening abdominal ultrasound showed no evidence of HCC at the time of blood sampling for the mSEPT9 test and on the next visit six months later. The adjudicating physicians were blinded to patient results associated with the mSEPT9 test.FindingsWe included 289 patients with cirrhosis (initial: 186; replication: 103), among whom 98 had HCC (initial: 51; replication: 47). The mSEPT9 test exhibited high diagnostic accuracy for HCC diagnosis, with an area under the receiver operating characteristic curve (AUROC) of 0.944 (0.900–0.970, p < 0.0001) in the initial study (replication: 0.930 [0.862–0.971, p < 0.0001]; meta-analysis: AUROC = 0.940 [0.910–0.970, p < 0.0001], no heterogeneity: I2 = 0%, p = 0.67; and no publication bias). In multivariate logistic regression analysis, the number of positive mSEPT9 triplicates was the only independent variable significantly associated with HCC diagnosis (initial: OR = 6.30, for each mSEPT9 positive triplicate [2.92–13.61, p < 0.0001]; replication: OR = 6.07 [3.25–11.35, p < 0.0001]; meta-analysis: OR = 6.15 [2.93–9.38, p < 0.0001], no heterogeneity: I2 = 0%, p = 0.95; no publication bias). AUROC associated with the discrimination of the logistic regression models in initial and validation studies were 0.969 (0.930–0.989) and 0.942 (0.878–0.978), respectively, with a pooled AUROC of 0.962 ([0.937–0.987, p < 0.0001], no heterogeneity: I2 = 0%, p = 0.36; and no publication bias).InterpretationAmong patients with cirrhosis, the mSEPT9 test constitutes a promising circulating epigenetic biomarker for HCC diagnosis at the individual patient level. Future prospective studies should assess the mSEPT9 test in the screening algorithm for cirrhotic patients to improve risk prediction and personalized therapeutic management of HCC.

Novel Liquid Biopsy Test Based on a Sensitive Methylated SEPT9 Assay for Diagnosing Hepatocellular Carcinoma

“…The serum methylated SEPT9 CORD assay revealed significantly higher copy numbers in the HCC group than in the healthy control and CLD groups and displayed a sensitivity and specificity of 63.2% and 90.0%, respectively, for diagnosing HCC. Epi proColon has a sensitivity and specificity for HCC of 88.7% and 89.8%, respectively, based on a cutoff value of at least two positive triplicates . The proportions of tumor stages differed between our study and this previous Epi proColon report: BCLC stages 0 and A accounted for 45.6% (62 of 136) in our study and for 32.3% (31 of 96) in the previous report.…”

“…(26) Epi proColon, which targets methylated SEPT9, was the first liquid biopsy test to be approved by the US FDA for colorectal cancer screening (12) and has been reported to be useful for diagnosing HCC. (13) As an advantage over the Epi proColon assay, the CORD assay requires only a small amount of serum (0.4 mL), because it circumvents bisulfite treatment. Furthermore, the CORD assay is easier to perform, and the methylation level can be evaluated quantitatively ( Supporting Table S3).…”

“…Epi proColon (Epigenomics AG, Berlin, Germany), which was approved by the US Food and Drug Administration (FDA) in 2016, was the first commercial blood‐based test for colorectal cancer screening based on methylated DNA testing of the septin 9 gene ( SEPT9 ) . There is only one report on the efficacy of Epi proColon in HCC patients . However, Epi proColon has some disadvantages.…”

“…(12) There is only one report on the efficacy of Epi proColon in HCC patients. (13) However, Epi proColon has some disadvantages. First, a large amount of blood plasma (> 3.5 mL) is required, because the amount of cancerspecific DNA in blood is very small, and conventional DNA methylation assays require bisulfite treatment of DNA, which causes DNA degradation and loss.…”

Predicting overall survival of patients with hepatocellular carcinoma using a three‐category method based on DNA methylation and machine learning

“…8 Bisulfite sequencing analysis in the Huh2 HCC cell line showed an association between abnormal DNA methylation and abnormal DLL3 expression. 10 Several studies suggest that DNA methylation may help predict OS of HCC patients. 10 Several studies suggest that DNA methylation may help predict OS of HCC patients.…”