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Mutation analysis of key genes in RAS/RAF and PI3K/PTEN pathways in Chinese patients with hepatocellular carcinoma

The RAS/RAF and PI3K/PTEN signaling pathways play central roles in hepatocarcinogenesis. KRAS, NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN are key cancer-related genes in the RAS/RAF and PI3K/PTEN signaling pathways. Genetic alterations in these genes often lead to the dysregulation of the two cascades. Little is known regarding the frequency of hotspot mutations in these critical components among Chinese patients with hepatocellular carcinoma (HCC). In the current study, 57 somatic hotspot mutations in 36 HCCs samples collected from Chinese patients using direct DNA sequencing method were examined. Two cases of KRAS somatic mutations (KRAS codon 61; Gln to His) were identified among 36 HCCs (5.6%). However, no mutations were found in the NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN genes. These findings indicated that point mutations in the KRAS gene, but not mutations in NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN genes, at a somatic level contribute to the abnormal activation of the RAS/RAF and PI3K/PTEN pathways in HCC.


Introduction
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, accounting for >740,000 new cases and 690,000 mortalities per year (1). Half of these new cases and mortalities were estimated to occur in China. The high rates of HCC in China are largely due to the prevalence of chronic hepatitis B virus (HBV) infection (2). The RAS/RAF and PI3K/PTEN signaling pathways play central roles in hepatocarcinogenesis (3). The aberrant activation of the RAS/RAF and PI3K/PTEN signaling pathways is associated with poor prognosis in cancer patients (4,5). HBV also utilizes the pathways for the control of hepatocyte survival and viral replication (6,7). Mutations of key components (such as RAS, RAF, PIK3CA, PIK3R1 and PTEN) in the RAS/RAF and PI3K/PTEN pathways lead to the dysregulation of the two cascades (8). The RAS family comprises three members: KRAS, NRAS and HRAS. Somatic mutations in the RAS family are common in numerous human cancer types, including pancreatic, thyroid, colorectal, liver, kidney and lung (9). BRAF is the most frequently mutated gene in the RAF family, and the BRAF mutation has been reported in 61% of melanoma, 53% of papillary thyroid cancer and 11.5% of colorectal cancer patients (10)(11)(12). The PI3K gene comprises PIK3CA, which encodes the catalytically active p110α subunit, and PIK3R1, encoding the p85α regulatory subunit (13). PIK3CA is mutated in numerous tumor types, with the frequency ranging from 4 to 32% in breast, colorectal, endometrial, brain, gastric and lung cancer (14)(15)(16)(17). PIK3R1 mutations were identified in 43% of endometrial cancer, 4% of ovarian cancer and 2% of colon cancer (18)(19). PTEN acts as a negative regulator of the PI3K pathway and PTEN mutations lead to a reduction of its phosphatase activity (20). Mutations of the PTEN gene are associated with a wide variety of human tumors (21).
Inhibitors targeting the RAS/RAF and PI3K/PTEN pathways have been developed and the clinical responses of patients were observed to differ according to the genetic alterations of the critical components of the two cascades (22). However, few data are available regarding the prevalence of KRAS, NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN mutations in Chinese patients with HCC. In the present study, we conducted mutational analysis of 57 somatic hotspot mutations in KRAS, NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN in 36 Chinese patients with HCC.

Materials and methods
Patients and tissue samples.  (Table II).
Mutation analysis of key genes in the RAS/RAF and PI3K/PTEN pathways. We analyzed hotspot-containing gene fragments of key genes in RAS/RAF and PI3K/PTEN pathways using PCR amplification followed by direct sequencing. The hotspots were listed in Table I. In all, two samples (Sample #13 and #35) had point mutations in codon 61 (Q61H) of the KRAS gene and the mutation rate was 5.6% (Fig. 1). In the two cases, codon 61 was altered from CAA, coding for Gln, to CAC, coding for His. To confirm the two mutations occurred at the somatic level, we tested codon 61 mutation status in non-tumorous tissues from the two patients. The results showed that codon 61 was wild-type in normal tissues from Sample #13 and #35 (Fig. 1)

Discussion
Targeting the RAS/RAF and PI3K/PTEN pathways are novel therapeutic strategies that may be exploited for the treatment of HCC (8). As the RAF-kinase inhibitor sorafenib has been demonstrated to be effective in the treatment of HCC, BRAF mutations have become a favored target in HCC treatment recently (25). However, the somatic mutation prevalence and distribution of the key genes in the two pathways remain largely unknown in Chinese patients with HCC. Therefore, the present   30%. One third of the twelve studies did not evaluate the KRAS codon 61 mutation status, which may cause bias in the distribution of the KRAS mutation. Three whole exome sequencing studies conducted mutational screening in all KRAS exons and found that the mutations were clustered in the hotspots (29)(30)(31).
In the current study, mutations were detected in codons 12, 13 and 61 of the KRAS gene, and two out of 36 (5.6%) HCCs harbored KRAS mutations in codon 61. Therefore, KRAS gene mutations may participate in hepatocellular carcinogenesis. The present study also investigated the hotspot mutations in NRAS and HRAS, but found no mutation in the two genes. Few studies have focused on the mutation incidence of these two RAS family members in Chinese patients with HCC. A whole exome sequencing study identified no mutation in these two genes in a Chinese population (31). Challen et al found that the frequency of NRAS mutations was 15.8%, but did not identify HRAS mutations, in British patients with HCC (32). Taketomi et al reported neither NRAS nor HRAS mutations were detected in Japanese HCC cases (33). Thus, the mutational activation of NRAS and HRAS genes is an uncommon event in the pathogenesis of HCCs.
BRAF mutations can abnormally activate downstream signaling pathways in HCC and act as indicator of cetuximab resistance in patients with colon cancer (34,35). BRAF mutations are believed to be rare in HCCs. Previously, no BRAF mutations were identified in German and Chinese populations (27,36). However, Colombino et al detected that the BRAF gene was highly mutated in ~23% of Italian HCC cases (37). In the current series, no BRAF mutations were observed, indicating that BRAF mutation does not play a major role in abnormal activation of RAS/RAF signaling pathway.
PIK3CA, PIK3R1 and PTEN are key genes in the PI3K/PTEN pathway (8). In the current study, it was found that mutations were absent in the three genes. Previously, PIK3CA was observed to be frequently mutated in Korean and Italian patients with HCC, with mutation rates of 35.6 and 28%, respectively (15,37). However, Tanaka et al did not identify PIK3CA mutations in Japanese patients with HCC, and Riener et al reported that the PIK3CA mutation incidence was 2% in Swiss patients with HCC (38,39). In two studies in Chinese patients with HCC, the mutation rates were 1.6 and 1.1% (36,40), which were similar to those of the present study. The conflicting data may be due to the different genetic backgrounds of the populations, HBV infection status and smaller sample size in the current study. PIK3R1 mutation has been found to occur infrequently in numerous cancer types, including ovarian and colon cancer (19), and the present study showed a low frequency of alteration of PIK3R1 in HCC. Inactivation of PTEN in HCC may be largely due to frequent loss of heterozygosity of the PTEN allele; the frequency was identified to be ≤44.4% (41). Wang et al investigated PTEN mutations in exons 5 and 8, but failed to detect any (42), which was in agreement with the results of the present study. Mutations in the PIK3CA, PIK3R1 and PTEN genes rarely occur in HCC, suggesting that somatic point mutations of these three genes may not play an important role in HCC in the Chinese population. However, further research is necessary to confirm these results in larger sample size.
In summary, the present study investigated the prevalence of KRAS, NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN mutations in 57 hotspot mutations. Two cases of KRAS mutation were identified among 36 HCC cases. The findings indicated that point mutations in the KRAS gene, but not mutations in the NRAS, HRAS, BRAF, PIK3CA, PIK3R1 and PTEN genes, at the somatic level contribute to the abnormal activation of the RAS/RAF and PI3K/PTEN pathways in HCC. Considering the low frequency of key genes in the RAS/RAF and PI3K/PTEN signaling pathways, other mechanisms to activate the RAS/RAF and PI3K/PTEN pathways, such as gene amplification, deletion, and aberrant methylation, may be involved in the development and progression of HCC.