Detection of CTNNB1 Hotspot Mutations in Cell-Free DNA from the Urine of Hepatocellular Carcinoma Patients

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. The beta-catenin gene, CTNNB1, is among the most frequently mutated in HCC tissues. However, mutational analysis of HCC tumors is hampered by the difficulty of obtaining tissue samples using traditional biopsy. Here, we explored the feasibility of detecting tumor-derived CTNNB1 mutations in cell-free DNA (cfDNA) extracted from the urine of HCC patients. Using a short amplicon qPCR assay targeting HCC mutational hotspot CTNNB1 codons 32-37 (exon 3), we detected CTNNB1 mutations in 25% (18/73) of HCC tissues and 24% (15/62) of pre-operative HCC urine samples in two independent cohorts. Among the CTNNB1-mutation-positive patients with available matched pre- and post-operative urine (n = 13), nine showed apparent elimination (n = 7) or severalfold reduction (n = 2) of the mutation in urine following tumor resection. Four of the seven patients with no detectable mutations in postoperative urine remained recurrence-free within five years after surgery. In contrast, all six patients with mutation-positive in post-operative urine recurred, including the two with reduced mutation levels. This is the first report of association between the presence of CTNNB1 mutations in pre- and post-operative urine cfDNA and HCC recurrence with implications for minimum residual disease detection.

Keywords: beta-catenin; cell-free DNA; hepatocellular carcinoma; mutation; recurrence; urine.

Figure 1

CTNNB1 exon 3 mutational frequency in HCC. Data from several studies that sequenced CTNNB1 exon 3 in patients with HCC were compiled (see Supplemental Table S1). In nearly 90% of all HCC tumors with a mutation in CTNNB1, the mutation resides within one of two hotspot regions: region 1 (codons 32–37; 54.6%) and region 2 (codons 41–45; 34.3%). The X-axis denotes the codon number and amino acid encoded by it. The box linked to an amino acid lists the reported missense mutations within the codon and the corresponding amino acid changes.

Figure 2

Analysis of CTNNB1 mutations in tissue samples from patients with different liver diseases. (A) CTNNB1 hotspot mutant allele frequencies obtained by the CTNNB1 32–37 mutation qPCR assay in liver tissues of patients with HCC, hepatitis, or cirrhosis. In the HCC subset (n = 73), 18 patients tested positive, while all of the hepatitis (n = 35) and cirrhosis (n = 35) samples tested negative. ***, p < 0.001. (B) Validation of the CTNNB1 32–37 mutation qPCR assay by Sanger sequencing. Five samples identified as positive by the qPCR assay with mutation frequencies of >15% were evaluated by Sanger sequencing. Of these five samples, three had detectable CTNNB1 mutations. When Sanger sequencing was repeated after enrichment for the mutation using the BNA^NC[NMe] in the PCR amplification reaction, CTNNB1 mutations in the other two samples were also confirmed. The right panel shows Sanger sequencing chromatograms with and without BNA^NC[NMe] enrichment. The mutation position is shaded in gray.