Trunk events present minimal intra- and inter-tumoral heterogeneity in hepatocellular carcinoma [gene expression]

Introduction: According to the clonal model of tumor evolution, trunk alterations arise at early stages and are ubiquitous. Through the characterization of early stages of hepatocarcinogenesis, we aimed to identify trunk alterations in HCC and study their intra- and inter-tumor distribution in more advanced lesions. Methods: 151 samples representing the multi-step process of hepatocarcinogenesis were analyzed by targeted-sequencing and SNP array. Genes altered in early lesions [31 dysplastic nodules (DNs) and 38 small HCCs (sHCC)] were defined as trunk. The distribution of candidate trunk genes was explored in: a) different regions of large tumors [43 regions of 21 tumors, (2-3 regions/tumor)]; and b) different nodules of the same patient [39 multinodular tumors from 17 patients]. Multinodular lesions were classified as intrahepatic metastases (IMs) or synchronous tumors based on chromosomal aberrations. Results: TERT promoter mutations (10.5%) and broad copy-number aberrations in chromosomes 1 and 8 (3-7%) were identified as trunk gatekeepers in DNs and were maintained in sHCCs. Trunk drivers identified in sHCCs included TP53 (23%) and CTNNB1 (11%) mutations, and focal amplifications or deletions in known drivers (6%). Overall, TERT, TP53 and CTNNB1 mutations were the most frequent trunk events in early stages. 89% of mutations in these genes were shared between different regions of large tumors. In multinodular HCCs, 35% of patients harbored IMs. 85% of mutations in TERT, TP53 and/or CTNNB1 were retained in primary and metastatic tumors. Conclusions: Trunk events in early stages (TERT, TP53, CTNNB1 mutations) were ubiquitous across different regions of the same tumor and between primary and metastatic nodules in >85% of cases. This concept supports the knowledge that single biopsies would suffice to capture trunk mutations in HCC. 36 paraffin-embedded tumors from 16 patients presenting multinodular cases (2-3 nodules/patient) were collected and RNA was extracted. Matched non-tumor cirrrhotic tissue was also collected for all cases. Whole-genome trasncriptome analysis was done using HumanHT-12 WG-DASL V4.0 R2 expression beadchip (Illumina) according to manufacturer’s protocols. Raw data were normalized using cubic spline algorithm implemented in the Illumina Normalizer module of the GenePattern analysis toolkit (www.broadinstitute.org/genepattern) as previously described (Sia D et al, Gastroenterology, 2013).