Analysis of TP53 gene mutations demonstrates that aflatoxin is a risk factor for hepatocellular carcinoma in Guatemala

Background Guatemala has the highest incidence of hepatocellular carcinoma (HCC), the dominant type of liver cancer, in the Western Hemisphere. The major risk factors in Guatemala are not well-characterized, but the prevalence of hepatitis B (HBV) and hepatitis C virus (HCV) appear to be low, while the prevalence of aflatoxin (AFB 1 ) exposure appears to be high. To examine whether AFB 1 could be a risk factor for HCC in Guatemala, this study examined the frequency of the AFB 1 -signature mutation in the TP53 gene (R249S) as well as other somatic mutations. Methods Ninety-one formalin-fixed, paraffin-embedded (FFPE) HCC tissues were obtained from three hospitals in Guatemala City. An additional, eighteen tumor tissues preserved in RNAlater were also obtained. Targeted sequencing of TP53 was successfully performed in 89 of the FFPE samples, and a panel of 245 genes were sequenced in the RNAlater samples. Results Overall, 47% of HCCs had a TP53 mutation. The AFB 1 -signature R249S mutation was present in 24%. Among the RNAlater samples, 44% had any TP53 mutation and 33% had the R249S mutation. Other somatic mutations were identified in known HCC driver genes such as ARID1A , ARID2 , and CTNNB1 . Conclusions The presence of the TP53 R249S mutation indicates that AFB 1 is a risk factor for HCC in Guatemala. The proportion of HBV positive tumors was low, suggesting that AFB 1 is associated with HCC in the absence of concomitant HBV infection. To decrease the risk of HCC in Guatemala, AFB 1 abatements efforts are warranted. Table shows the results of the gene sequence analysis for the eighteen tumor tissues in RNAlater that were analyzed for targeted capture of the exons of 245 known cancer-related genes, compared to the genome sequence analysis results of HCCs in other populations. Among the eighteen cases, 6 (33%) had the TP53 R249S mutation (data not The proportion with any while and ARID1 and CTNNB1 FGRF3.


Introduction
Liver cancer is the seventh most commonly occurring cancer and the second leading cause of cancer mortality globally. (1) The most common histological subtype is hepatocellular carcinoma (HCC) which accounts for 80% of all liver cancers.(2) Sex-and geographicvariation in HCC incidence have been reported across regions worldwide, as has the variability in the prevalence of known risk factors. (2)(3)(4) Major risk factors for HCC, such as hepatitis B virus (HBV), hepatitis C virus (HCV), excessive alcohol consumption and aflatoxin B 1 (AFB 1 ) exposure vary between high-rate and low-rate areas. In many high-rate areas (e.g. regions in Asia and Africa), HBV and AFB 1 exposure are dominant factors, while in low-rate areas, HCV and alcohol consumption are more common. (3)(4)(5) In addition, nonalcoholic fatty liver disease (NAFLD) is beginning to play an important role as a risk factor in both high and low incidence regions. (6) In Guatemala, the estimated incidence and mortality rates of liver cancer are the highest in the Western Hemisphere (age-standardized rates (ASRs): 14.9 cases per 100,000 person-years and 14.5 deaths per 100,000 person-years), with 1,787 new cases of liver cancer estimated to have occurred in 2018. (7) A recent cross-sectional study that assessed risk factors for liver cancer in Guatemala found a very low prevalence of both HBV and HCV infection (<1%). (8) In contrast, the study found high serum levels of AFB 1albumin adducts among the participants, with significantly higher geometric mean levels among men (10.93 pg/mg albumin) than women (7.92 pg/mg albumin). (8) The results were consistent with previous evidence of high AFB 1 levels in maize samples across the country.(9) Also elevated among the study participants were the prevalence of NAFLD (60.1%), obesity (30.9%) and metabolic syndrome (64.2%).(10) AFB 1 forms DNA adducts at the N 7 position of guanine, inducing primarily G ->T transversions. One particular G ->T mutation in codon 249 (AGG to ATT, arginine to serine, R249S) of TP53 is a molecular signature of AFB 1 exposure in HCC. (11) Studies in AFB 1 endemic regions in Asia and Africa have reported a wide range in the prevalence of the R249S mutation in HCC ranging from 4.8% to 67%. (12)(13)(14)(15)(16)(17)(18)(19)

DNA extraction
One half of the tissue from each pathology slide was scraped from the slide and extracted by a phenol-chloroform procedure. FFPE blocks were sectioned in a microtome and curls (10uM) were collected for DNA extraction. Tissue stored in RNALater (ThermoFisher Scientific) was stored at -20°C until DNA was extracted using the AllPrep DNA/RNA Micro kit (Qiagen). DNA was quantitated using the PicoGreen DNA assay method (ThermoFisher Scientific).

Statistical analysis
To examine whether the prevalence of TP53 mutations varied by sex, the frequencies were evaluated for statistical significance by Fisher's exact test. SAS software v 9.4 (SAS Institute, Cary, NC) was used for the analysis.

Results
Of the 91 HCCs examined, 52 were from men and 39 were from women. The median age at diagnosis among men was 62 years (Interquartile range (IQR): 48, 73) and among women was 61 years (IQR: 52, 68). Among the persons for whom information on residence was recorded, more than 70% lived in the department of Guatemala or in contiguous departments. Among the persons for whom HBV and HCV status was recorded, only 2 were HBV positive (defined as being positive for HBsAg) and only 3 were HCV positive (defined as being positive for anti-HCV) ( Table 1)..

Prevalence of TP53 mutations in the FFPE samples:
Overall, 47% of the FFPE HCCs (42/89) had any TP53 mutation ( Table 2).. The mutation prevalence was somewhat higher in the tumors from women (58%) than in the tumors from men (39%) but, the difference was not statistically significant (p = 0.09). The prevalence of the R249S mutation was 24% with no major difference in the prevalence of the mutation in the tumors among men (22%) and women (26%) (p = 0.62). The prevalence of any G->T transversion, including the R249S mutation, was 29%, and the prevalence of any G->T transversion at a CpG site was 32%, again with no difference in the prevalence by sex.
Cancer gene sequence analysis of the tumors preserved in RNAlater: Table 3 shows the results of the gene sequence analysis for the eighteen tumor tissues in RNAlater that were analyzed for targeted capture of the exons of 245 known cancerrelated genes, compared to the genome sequence analysis results of HCCs in other populations. Among the eighteen cases, 6 tumors (33%) had the TP53 R249S mutation (data not shown). The proportion with any TP53 mutation was 44%, while the prevalence in other studies ranged between 21% and 82%. Mutations were also observed in known HCC driver genes such as ARID2 (28%), ARID1 (17%), APC (17%) and beta catenin CTNNB1 genes (17%). In addition, mutations were observed in AXIN1, SMARCA4, GNAS, RB1, FLT3M, DNMT3A, FLT3, CDKN2A, ALB, RPS6KA3, ATM and FGRF3.

Discussion
This is the first report of TP53 mutations in HCCs from northern Central America. Although the region is one of the three high-rate HCC regions in the world, it is characterized by a low prevalence of chronic HBV infection. The study found an overall TP53 mutation rate of nearly 50% in both FFPE and RNAlater samples. In addition, among all the samples, the proportion with the AFB 1 -signature mutation (R249S) was 25%.
Prior studies conducted in high-rate HCC areas have reported TP53 R249S mutation prevalences as high as 67%. (14,15,20,26) In Western Africa, a study in the Gambia The high levels of AFB 1 reported in Guatemala (8,9) are comparable to the levels found in some high-rate parts of China before the transition there from a maize-based to a ricebased diet. (28) In the current study, there were in total 24 mutations with a G->T transversion. The majority of the G->T transversions occurred in codon 249 and the rest were in codons: 157 (n = 2) and 248 (n = 1) (data not shown). These somatic mutations have been reported in HCCs previously,(29) Furthermore, one third of the HCCs have a G->T transversion at CpG sites, suggesting that DNA methylation changes could play a role in these tumors. For example, a study that characterized genome-wide DNA methylation patterns in HCC identified a large subset of CpG sites associated with HCV infection, liver cirrhosis or HCC. Furthermore, in animal studies, p53 has been implicated in the progression of steatosis to Non-alcoholic steatohepatitis (NASH), involving mechanisms such as upregulation of TP53 activity with increased mRNA levels of the p21 and p66ShC proteins, which are associated with fibrosis severity. (33) The current study also identified somatic mutations in other known HCC driver genes,  (35) with prevalences ranging from 20% to 38%. (36,37) The results of the current study are in line with prior findings as CTNNB1 mutations were identified in 17% of the HCCs. In addition, mutations in ARID1A and ARID2 which are involved in WNT (cell-differentiation) pathway activation, have also been reported. (35) De-regulation of ARID1/2 signaling appears to affect 6-18% of HCC tumors (35) similar to the mutation prevalence found in the current study (17% ARID1A and 28% in ARID2).. Furthermore, inactivating mutations in ARID2 have been found HCCs of various etiologies. An European study that conducted exome sequencing analysis of 243 HCCs, reported associations between some known risk factors and mutational patterns. (37) The study reported that alcohol-related HCCs were more likely to have mutations in CTNNB1, TERT, CDKN2A, SMARCA2 and HGF, while HBVrelated HCCs were more likely to have TP53 mutations. In contrast, no mutations were identified in either HCV-or NAFLD-related HCCs. (37) Overall, nearly 100 somatic mutations have been determined as HCC driver mutations, and approximately five to six driver mutations are considered necessary to cause cancer within a particular patient. (38) To our knowledge, the study is the first to examine mutations in HCC from Guatemala.
Other strengths include the sizable number of HCCs included, and the confirmation of all diagnoses by a liver cancer pathologist. Limitations of the study include that the tumors were not collected as part of a systematic protocol so they may not be representative of all HCCs seen at the study hospitals, or in the country. In Guatemala, it is estimated that approximately 40% of HCCs undergo are biopsied. Another limitation is that there was incomplete information available on risk factors, so it was not possible to determine the extent to which the R249S mutation corresponded to AFB 1 exposure. It was also not possible to determine HBV or HCV status of all cases. In addition, there was incomplete clinical information available on the tumors so the number of somatic mutations could not be correlated with extent of disease. Furthermore, mutations in the TERT gene, the most commonly mutated gene in HCC, (39,40) could not be examined.
In conclusion, the presence of the TP53 AFB 1 -signature mutation suggests that AFB 1 is a risk factor for HCC in Guatemala. As the prevalence of HBV was low, the current results 12 suggest that AFB 1 is associated with HCC in the absence of concomitant HBV infection. Since there is no personal information included, only de-identified data, the research was considered in the exempt category by the institutional review boards at the NIH, Human Research Protection Program.

Consent for Publication:
Not applicable

Availability of Data and Material
The dataset used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing Interests: The authors declare that they have no competing interests.