Integration of tumour and viral genomic characterizations in HBV-related hepatocellular carcinomas

Abstract

Background and aim: Hepatocellular carcinoma (HCC) is the most common liver cancer. We characterised HCC associated with infection compared with non-HBV-related HCC to understand interactions between viral and hepatocyte genomic alterations and their relationships with clinical features.

Methods: Frozen HBV (n=86) or non-HBV-related (n=90) HCC were collected in two French surgical departments. Viral characterisation was performed by sequencing HBS and HBX genes and quantifying HBV DNA and cccDNA. Nine genes were screened for somatic mutations and expression profiling of 37 genes involved in hepatocarcinogenesis was studied.

Results: HBX revealed frequent non-sense, frameshift and deletions in tumours, suggesting an HBX inactivation selected in HCC. The number of viral copies was frequently lower in tumour than in non-tumour tissues (p=0.0005) and patients with low HBV copies in the non-tumour liver tissues presented additional risk factor (HCV, alcohol or non-alcoholic steato-hepatitis, p=0.006). P53 was the most frequently altered pathway in HBV-related HCC (47%, p=0.001). Furthermore, TP53 mutations were associated with shorter survival only in HBV-related HCC (p=0.02) whereas R249S mutations were identified exclusively in migrants. Compared with other aetiologies, HBV-HCC were more frequently classified in tumours subgroups with upregulation of genes involved in cell-cycle regulation and a progenitor phenotype. Finally, in HBV-related HCC, transcriptomic profiles were associated with specific gene mutations (HBX, TP53, IRF2, AXIN1 and CTNNB1).

Conclusions: Integrated genomic characterisation of HBV and non-HBV-related HCC emphasised the immense molecular diversity of HCC closely related to aetiologies that could impact clinical care of HCC patients.

Keywords: HEPATITIS B; HEPATOCELLULAR CARCINOMA.

Figure 1

HBV virus profile in tumour and non-tumour adjacent samples. (A and B) Spectra of mutation in HBS and HBX gene in tumour and paired non-tumour samples. (C) Distribution of tumour and non-tumour samples according to quantification of HBV copy/cell and determination of two groups characterised by low (<0.5 HBV copy/cell) and high (>0.5 HBV copy/cell) copy number. (D) Correlation of HBV quantification (log10 copy/cell) in tumour and paired non-tumour samples. p Values obtained from χ² (*) and Willcoxon signed rank (**) tests are shown. MHL, major hydrophilic loop; RD, regulatory domain; PSR, proline serine rich hypervariable region; KI, Kunitz domain-like; TD, transactivation domain.

Figure 2

Somatic mutation spectra of TP53 and CTNNB1 in 86 HBV-related and 90 non-HBV-related HCC. Functional domains are coloured boxes. ARM, armadillo repeat; DBD, DNA binding domain; Dim, dimerisation domain; NES, nuclear export signal; NLS, nuclear localisation signal; NTD, negative transactivation domain; TD, transactivation domain; ser, serine; HCC, hepatocellular carcinoma.

Figure 3

Significant prognostic values of TP53 mutations. (A) Disease-specific survival in patients infected by HBV with HCC mutated or wild-type to TP53 and (B) in patients with HCC, mutated or wild-type to TP53, related to other aetiologies. HCC, hepatocellular carcinoma.

Figure 4

Integration of transcriptomic profiling. (A) Transcriptomic groups (G1–G6) in HBV (white) and non-HBV-related (grey) HCC (mean with 95% CI). p Values obtained from χ² test are shown. (B) Gene's expressions in HBV- and non-HBV-related HCC (mean with 95% CI). p Values obtained from Mann–Whitney test are shown. EPCAM, epithelial cell adhesion molecule; KRT19, keratin 19; AFP, α-fetoprotein; CCNB1, cyclin B1; HCC, hepatocellular carcinoma.

Figure 5

Schematisation of the different HBV-HCC subgroups (G1–G6) defined by transcriptome analysis with their related clinical and genetic alteration. Red and green indicate overexpression and under-expression of gene, respectively, in the corresponding transcriptomic subgroup(s). p Values obtained from χ² tests are shown. IRF2, interferon regulatory factor 2; EPCAM, epithelial cell adhesion molecule; KRT19, keratin 19; AFP, α-fetoprotein; SPP1, osteopontin; TBX3, T-box protein 3; GLUL, glutamate-ammonia ligase; RHBG, Rh family, B glycoprotein; HAL, histidine ammonia-lyase; IGF2BP3, insulin-like growth factor 2 MRNA binding protein 3; NRCAM, neuronal cell adhesion molecule; NEU1, sialidase 1; BIRC5, baculoviral IAP repeat containing 5; CCNB1, cyclin B1; AURKA, aurora kinase A; UGT2B7: UDP glucuronosyltransferase 2 family, polypeptide B7; HCC, hepatocellular carcinoma.