Lack of hotspot mutations other than TP53 R249S in aflatoxin B1 associated hepatocellular carcinoma

Introduction

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the fourth leading cause of cancer deaths worldwide [1]. Aflatoxin B1 (AFB1) is one of the major etiologic factors leading to HCC, especially in certain geographical regions. Aflatoxins are mycotoxins produced by fungal species in human foods, cereals, feeds and any dried foods at increased humidity and temperature of storage conditions. AFB1 is the best known aflatoxin, which leads to mutations that cause cancer development due to its genotoxic characteristic [2]. TP53 p.Arg249Ser (c.747G>T) somatic mutation has been shown to be a fingerprint of AFB1 related hepatocellular carcinoma [3]. However, involvement of other pathways or genes accompanying the p53 pathway in the development of AFB1-induced HCC [4] other than this mutation has not been identified yet. Here, we aimed to define the possible genetic changes taking place before or after the TP53 p.Arg249Ser (c.747G>T) mutation in hepatocarcinogenesis.

Materials and methods

The genomic DNA of three samples one archive tumor sample – T29 and two cell lines – MAHLAVU and Primary Liver Carcinoma/Poliomyelitis Research Foundation/5 (PLC/PRF/5) known to carry the AFB1 fingerprint mutation TP53 p.Arg249Ser (c.747G>T) were analyzed. The genomic DNAs were kindly provided by Prof. Dr. Mehmet Ozturk [3]. 525 cancer genes (see Supplementary Material S1) previously reported to be involved in carcinogenesis with mutations in different cancer types [5] were genotyped using custom probes (Roche/Nimblegen SeqCap EZ Choice Library, USA). Targeted sequencing protocol was performed as previously reported [6] and samples were paired-end sequenced with 100X coverage using Illumina HiSeq2500 (Illumina, USA). Data was analyzed by FASTQC (Babraham Institute, UK) and DNAnexus platform (DNAnexus, USA).

Coding regions of ARID2 and BCORL1 genes, that were shown to have mutations in HCC caused by factors other than AFB1 [7], [8], were amplified with the previously reported primers [9], [10], respectively in 50 μL reaction using GoTaq Flexi DNA Polymerase (Promega, USA, cat. no M8298) and GenomeLab DTCS – Quick Start Kit (Beckman Coulter, USA, PN 608120) was used for Sanger sequencing, according to manufacturer's instructions. The samples were run on GeXP Genetic Analysis System (Beckman Coulter, USA) and analyzed by DNASTAR software (Lasergene, USA).

In order to check if other possible hotspot mutations were reported, whole exome data of TP53 p.Arg249Ser (c.747G>T) mutation carrying cell line PLC/PRF/5 (obtained from Cancer Cell Line Encylopedia [11]) and 68 HCC samples (see Supplementary Material S2) were analyzed from COSMIC database.

Results

In this study, two cell lines and an archive tumor sample carrying the p.Arg249Ser (c.747G>T) mutation were investigated for the presence of other hotspot mutations induced by AFB1. First of all, the targeted sequencing for previously reported 525 cancer genes of tumor sample T29 and cell line MAHLAVU were performed and the data were compared. As a result, 200 common non-synonymous variations (including TP53 p.Arg249Ser (c.747G>T)) were identified (see Supplementary Material S3). 13 of these mutations were found to be G>T transversions (Table 1) which are predominant in AFB1 exposure.

In addition, Sanger sequencing for ARID2 and BCORL1 genes, shown to have mutations in virus-associated HCC, were performed for the two cell lines MAHLAVU and PLC/PRF/5. Only 4 common intronic variations [chr12: 45817941del T, chr12: 45839259T>C in ARID2 and chrX: 130022874C>T, chr X: 130050901A>G in BCORL1 (GRCh38/hg38)] were identified.

Finally, all the detected variations from the targeted and Sanger sequencing analysis were checked in the whole exome data of 68 HCC COSMIC samples and another cell line PLC/PRF/5, from Cancer Cell Line Encylopedia. According to our detailed examination of reported entries, no common variations were found in PLC/PRF/5 cell line and 68 HCC COSMIC samples using the criteria of “presence in more than three samples”.

Overall, in this study no novel fingerprint mutations related with AFB1 were detected in the analyzed 527 genes.

Discussion

Here, we aimed to define the possible genetic changes taking place in carcinogenesis of aflatoxin B1 induced HCC other than the TP53 p.Arg249Ser (c.747G>T) mutation. However, no genetic variation possibly associated with AFB1 was detected in totally 527 genes. Intronic variations detected in ARID2 and BCORL1 genes were listed as polymorphisms (rs373592222, rs112994087 in ARID2 and rs34410420, rs7060657 in BCORL1) with no reported clinical importance [12].

In our analysis, G>T transversions were especially taken into consideration because AFB1 is a carcinogen leading to major promutagenic AFB1-N7-guanine DNA adduct which results in G>T transversions when metabolized [2]. These criteria are consistent with Mutational Signature 24 for liver cancer samples with known aflatoxin exposure [5].

All the detected G>T variations common to MAHLAVU and T29 were previously reported in literature, rs28934571 being the TP53 p.Arg249Ser (c.747G>T) mutation. rs111493987 and rs28522267 variations were reported as somatic mutations in different cancer types in COSMIC database. rs1664022 (p.Arg25Leu (c.74.G>T)) and rs11528010 (p.Pro2Thr (c.4C>A)) variations were not reported previously. However, p.Arg25Cys (c.73C>T) and p.Arg25Gly (c.73C>G) variations in PDE4DIP gene were listed as somatic mutations in COSMIC as entries of endometrium/breast and prostate cancers, respectively; whereas p.Pro2Ser (c.4C>T) variation in BMPR1A gene was reported in squamous cell carcinoma. In addition, it was reported that the rs2228001 (p.Gln939Lys (c.2815C>A)) polymorphism in DNA repair XPC gene, might have an increased risk of developing HCC in case of AFB1 exposure [13]. Other detected variations rs938608, rs4842838, rs2412541, rs3745335, rs1698605, rs1613780 and rs145583085 were reported to be polymorphisms [12].

In addition, no common variations were found in the seven COSMIC HCC (see Supplementary Material S4) samples carrying another TP53 G>T hotspot mutation p.Val157Phe (c.469G>T), which was shown to be related with poor prognosis in HCC and environmental mutagen exposure in lung cancer [14].

In conclusion, TP53 p.Arg249Ser (c.747G>T) mutation still appears to be the unique hotspot mutation caused by AFB1 exposure.