TP53 and RET may serve as biomarkers of prognostic evaluation and targeted therapy in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common malignancy of the liver. Genomic analysis is conducted to identify genetic alterations in driver genes which are all druggable targets for cancer therapy. In the present study, we performed an exome sequencing of 45 driver genes in 100 paired samples from HCC patients including tumors and matched adjacent normal tissues using Illumina HiSeq 2000 platform. Non-synonymous mutations were ascertained using the iPLEX MassARRAY system and Sanger sequencing. Clinicopathological relevance with genetic variations was assessed using SPSS software. The prognostic analyses of patients with gene mutation status were summarized using Kaplan-Meier curves. Sixty-one non-synonymous somatic mutations were identified in 43% of the HCC patients. The most frequent mutations were: TP53 (20%), RET (6%), PLCE1 (5%), PTEN (4%) and VEGFR2 (3%). Patients with mutations in TP53 had a lower overall survival (OS) (P=0.002) than those without mutations. Recurrent mutations in the Ret proto‑oncogene (RET) were associated with poor outcomes for both disease‑free survival (DFS) (P=0.028) and OS (P=0.001) in HCC patients. The mutational status of sorafenib-targeted genes were associated with decreased DFS (P=0.039), and decreased OS (P=0.15) without statistical significance. Mutual exclusion of TP53 and RET mutations were observed in the present study. In conclusion, patients with TP53 mutations, RET mutations and sorafenib-targeted gene mutations were demonstrated to be associated with poor HCC prognosis, which suggests that both TP53 and RET may serve as biomarkers of prognostic evaluation and targeted therapy in HCC.

Figure 1.

Overall cumulative coverage and flow-chart of the data analysis and mutation detection of genes in HCC patients. (A) A read coverage of 90% of the targeted exons was achieved as the sequencing depth was 37x. (B) In the cohort of 100 HCC patients, 321 SNVs were detected by quality filters. After excluding single nucleotide polymorphisms in dbSNP and 1K Genome, 190 mutations remained of which 119 corresponded to non-synonymous mutations. After validation, 60 somatic mutations and 58 germline mutations were finally obtained. HCC, hepatocellular carcinoma; SNVs, single nucleotide variations.

Figure 2.

Mutation frequencies in 4 categories of molecular-targeted therapy-related genes. A total of 45 genes were classified into 4 categories, including RTKs, angiogenesis, the RAS/RAF/MEK/ERK and the PI3K/AKT/mTOR pathways. Genes are indicated in pink to red. The darkness of the color is positively correlated with the percentage of tumors with genetic alterations. The mutation frequency of each gene in 100 tumors is indicated. RTKs, receptor tyrosine kinases;MEK, mitogen-extracellular activated protein kinase; PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; ERK, extracellular signal-regulated kinase.

Figure 3.

Expression of RET in HCC and peritumoral tissues with mutations in RET. (A) Immunohistochemical staining of HCC with RET mutation revealed significant (left 4 panels) or slight increase (right 2 panels) in the expression of RET in tumor tissues (upper panels) compared with peritumoral tissues (lower panels). (B) Representative expression of RET in HCC and paired peritumoral tissues using tissue microarray (magnification, ×200). High expression is defined as ≥40% staining of the tumor section, and low expression as <40%. (C) Statistical results of RET expression in HCC and paired peritumoral tissues using Student's t-test (P=0.0129). HCC, hepatocellular carcinoma.

Figure 4.

Kaplan-Meier survival estimates according to any mutations in TP53, RET and sorafenib-target genes, and mutation status of TP53 and RET in HCC patients. (A) Data are shown for the DFS of patients with and without mutations in RET (median DFS, 3.700 vs. 24.833 months, respectively, P=0.028). (B) Data are shown for the OS of patients with and without mutations in RET (median OS, 12.000 vs. 47.433 months, respectively, P=0.001). (C) Data are shown for the DFS of patients with and without mutations in TP53 (median DFS, 24.172 vs. 24.833 months, respectively, P=0.133). (D) Data are shown for the OS of patients with and without mutations in TP53 (median OS, 14.967 vs. 47.433 months, respectively, P=0.002). (E) Data are shown for the DFS (median DFS, 11.667 vs. 27.833 months, respectively, P=0.039) of patients with and without mutations in sorafenib-target genes. (F) Data are shown for the OS (median OS, 39.967 vs. 47.433 months, respectively, P=0.15) of patients with and without mutations in sorafenib-target genes. (G) Mutations of TP53 and RET are mutually exclusive. Black indicates patients with mutations and white indicates patients without mutations. SRF, sorafenib; HCC, hepatocellular carcinoma; DFS, disease-free survival; OS, overall survival.