Assessments of TP53 and CTNNB1 gene hotspot mutations in circulating
tumour DNA of hepatitis B virus-induced hepatocellular carcinoma
Background: Hepatitis B virus (HBV) infection is one of the
major causes of chronic liver disease which progresses from hepatitis to
liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). Early
detection and laboratory based screening test of HCC is still a major
challenge. HBV induces hepatocarcinogenesis through viral genome
integration, chromosomal aberrations and modulation of host signaling
pathways. Molecular alterations of cancer hallmark genes occur during
the process of hepatocarcinogenesis. These signatures may release into
the circulation through . circulating tumor DNA (ctDNA). Detection of
these mutations in ctDNA may serve as liquid biopsy marker for
screening, early detection and prognosis of HCC for which this study was
undertaken. Methods: Consecutive patients of CHB-HCC (n=80),
chronic hepatitis B (n=35) and healthy (n=15) controls were included for
blood sample collection. The ctDNA was isolated from serum.
Amplification and sequencing TP53 exon 7 and β-catenin exon 3 was
carried out for predominant mutations in the ctDNA of HCC patients.
Highly sensitive dual-probe based droplet digital PCR (ddPCR) assays
were performed for TP53 (p.R249M & p.R249S) and β-catenin (p.S45P)
driver mutations in healthy, CHB-noncirrhotic, cirrhotic and HCC
patients. Results: Both TP53 gene exon 7 and CTNNB1 gene exon 3
region was amplified and sequenced in 32 HCC patient whereas sensitive
ddPCR assay TP53 (p.R249M & p.R249S) and β-catenin (p.S45P) mutation
for all 130 subjects. In sanger sequencing TP53 c.746 G˃T, p.R249M
mutation was predominant. In ddPCR assay, 58.75% of HCC patients (n=47)
ctDNA had at least one driver mutation in the ctDNA. Combined TP53 and
CTNNB1 mutation was observed in 12.5% of HCC patients. Increased
mutation frequency was observed in CHB-cirrhotic. CHB-HCC than
CHB-noncirrhotic and healthy subjects. Percentage mutant fraction was
highest in CHB-HCC than only CHB patients. Significant association TP53.
R249M with smoking was observed in CHB-HCC patients. Poor survival was
observed in HCC patients with combined TP53 and CTNNB1 gene mutation.
Conclusion: Driver mutation screening for TP53 and CTNNB1 gene
can be done in ctDNA for early diagnosis and prognosis.