In this study, we performed comprehensive WES for 223 HCCs. The results revealed that a high frequency of mutations in genes did not always correspond to a high frequency of MMs. Instead, MMs were revealed to accumulate selectively in specific genes. The signature of mutations identified as MMs, including mutation type and functional impact, also varied compared with that of mutations identified as SM. Furthermore, the GEP results implied that MMs had a greater impact on gene expression than SMs in some oncogenes, a trend was identified that MMs led to additional up-regulation (gain-of-function) of gene expression. Consequently, MMs are not just a reflection of mutation burden but occur in specific genes and pathways and thus contribute to carcinogenesis and/or acquisition of malignant potential in HCC.
The overall landscape of MMs was recently reported through a pan-cancer analysis of 60,954 cancer samples 5. The study identified that oncogenic MMs were a relatively common driver event and thus MMs provide a novel underlying mechanism for cancer development. These observations reinforce the idea that MMs in the same oncogene cooperate to potentiate tumor-promoting activity. These findings also indicate the potential usefulness of MMs as a biomarker and a target for molecular-targeted therapy. However, the signature and clinical relevance of MMs in HCCs had remained unclear. In the present study, detailed clinical information led to the validation of the clinical significance of MMs in HCCs, and the present results identified MMs as an independent predictor for prognosis in HCC. To date, there has been no report on the clinical relevance of MMs, and therefore the present study provides further evidence of the prognostic impact of MMs.
The clinical impact of MMs in some genes in a sample led us to assess the clinical impact of MMs in individual oncogenes. Aberrant activation of WNT/β-catenin signaling is a driving molecular event in a wide range of tumors, including HCCs 11. Somatic missense mutations in exon 3 of CTNNB1 are frequently reported in HCCs (10.0–32.8% in genome-wide sequencing studies) 3 12. Consistent with previous reports, we found that mutations in CTNNB1 were frequently identified in HCC and MMs in CTNNB1 were also frequently found in 14% (11/79) of samples with at least one mutation. However, a significant prognostic difference between SM and MMs in CTNNB1 was not identified (data not shown). The conflicting prognostic impact of mutated CTNNB1 due to the bilateral nature was reported. HCC cases with the existence of an interaction between WNT activation and TGF-beta activation show poor survival, whereas HCCs harboring mutant CTNNB1 show generally favorable prognosis 13. Therefore, for further investigation, we focused on MUC16, in which MMs were identified in 15.2% (7/46) of mutated samples, as a candidate oncogene to validate the impact of MMs.
MUC16, which encodes a protein also known as ovarian carcinoma antigen CA125, has been recognized as a crucial factor in hepatocarcinogenesis 14 and its usefulness as a diagnostic and prognostic marker has been demonstrated 15. The present results demonstrated that the presence of MMs in MUC16 was associated with viral hepatitis, higher tumor markers and vascular invasion. Patient RFS was significantly worse in the group with MMs than in the group with SM, although there was no significant difference between the group with SM and the group with wild-type MUC16. The findings support the idea that MMs in the same oncogene cooperate to potentiate tumor-promoting activity.
The findings that the accumulation of MMs in specific oncogenes led us to assess the impact of MMs on phenotypes in cancer cell lines. Analysis of drug sensitivity screens in CCLE cell lines 10 revealed that cells harboring in MMs in MUC16 exhibited a higher sensitivity to regorafenib than those with no or single MUC16 mutations, indicating the potential value of MMs as a predictive marker for targeted therapies. Similarly, a previous study 5 reported that cells harboring MMs in PIK3CA exhibited a higher sensitivity to PI3K inhibitors compared with those with no or single PIK3CA mutations, suggesting that MMs may be useful as predictive markers for targeted therapies. The correlation between mutational status in an oncogene and the sensitivity to the targeted drug for the molecule should be further explored in future studies.
During recent years, new immune-modulatory agents have been introduced for HCC treatment, eventually leading to the clinical breakthrough of immune checkpoint inhibitors (ICIs) targeting programmed death-1 (PD-1), programmed death-ligand 1 (PD-L1), or cytotoxic T lymphocyte antigen-4 (CTLA-4) 16. The TMB, which correlates with MMs, has received increasing attention owing to its potential to estimate the efficacy of the response to ICI. The present results indicated that MMs were associated with the TMB to some extent; however, MMs were not just a reflection of mutation burden but occurred in specific genes and pathways through a selection mechanism to contribute to carcinogenesis and/or acquisition of malignant potential. These findings suggest the potential utility of MMs as a biomarker for ICIs. Future studies should examine the usefulness of MMs as a biomarker for ICIs.
This study had several limitations. First, the number of MMs in each specific gene was relatively small, and therefore investigation of the clinical significance of MMs in each gene could not be conducted because of under power statistics. Ideally, the clinical significance of MMs in specific candidate genes will be investigated to establish a biomarker for targeted therapy. Second, the previous study 5 reported that the proportion of MMs in cis was particularly high (86%) in oncogenes with MMs. Consistent with the previous reports, most MMs (83%) in CTNNB1 were present in cis in the present study. However, all of the MMs in MUC16 was not located in a same amplicon, therefore the allelic configuration of MMs in MUC16 could not be investigated.
In conclusions, MMs are a relatively common event that selectively occurs in specific oncogenes and is involved in aggressive malignant behavior. This is the study investigating the clinical significance of MMs in patients with HCC and provide important insights into the development of personalized treament strategies for HCCs.