Introduction

In Mexico, hepatocellular carcinoma (HCC), follows a unique epidemiologic pattern. Male-to-female mortality ratios are close to one.¹ And Hepatitis B virus (HBV; 0.2%)² and hepatitis C virus (HCV; 0.4%)³ seroprevalence are low. However, contamination of maize tortilla, a staple food in Mexico, with aflatoxin B₁ (AFB₁), a potent liver carcinogen, has been shown to be as high as 95% in store samples in Veracruz.⁴ And Chiapas and Guatemala, together represent the region with the highest HCC burden in the Americas. We aimed to estimate AFB₁ exposure in a representative sample of adults from Chiapas.¹ To further characterize the relevance of aflatoxin in Mexico, we determined the presence of the aflatoxin signature mutation in codon 249 (R249S) of the TP53 gene⁵ in HCC tissue from patients from a cancer referral center.

Materials and methods

Results

In a representative sample of adults aged 40-59 from Chiapas (mean age, 48.3 years; SD ± 6.0;) nearly 50% were from rural areas and approximately one third indigenous. The overall prevalence of detectable AFB₁ -lys was 85.5% (95%CI 72.1, 93.1) representing 970 702 urban and rural individuals in Chiapas (table I). Prevalence appeared to be higher in younger, indigenous adults who live in rural areas. Median AFB₁ was 0.117 pg/µL (IQR, 0.300) and the geometric mean 2.03 pg AFB₁ -lys /µL (95%CI 1.11, 3.72; table II). Adduct levels were three-fold higher in men relative to women (3.68 vs.1.20 pg AFB₁ -lys /µL) and in participants living in rural as compared to those in urban areas (3.67 vs.1.22 pg AFB₁ -lys /µL).

Characteristics of the 50 HCC patients are shown in table III. The median age was 63 years (interquartile range, 20). The prevalence of chronic HBV infection (HBsAg+) was 4.0% and the prevalence of HCV infection (anti-HCV+) was 14.0%. Overweight and obesity were common in both women and men. Cirrhosis was present in 25% of the women and 42.3% of the men. We detected the TP53 R249S mutation in three patients (all from mostly rural regions) of the 50 HCCs (6.0%). Two of them were from Veracruz and one was born in Oaxaca (but lived in Mexico City). Four patients with other TP53 G>T transversions (two samples with TP53 V157F, one with TP53 V203L, and one with TP53 G245V). In total, seven (14%) of patients had mutations that could be related to AFB₁ exposure. Within this group, three tumors were from women and evidence of chronic HCV infection was present in only one patient. A preliminary analysis of mutational signatures revealed that up to 10% of somatic mutations in these tumors may be due to aflatoxin (figure 1).

* Mutations observed 1 or 2 times and likely enriched in somatic mutations were analyzed using Mutagene Identify (https://www.ncbi.nlm.nih.gov/research/mutagene/identify).

Figure 1 Frequency of mutations* observed 1 or 2 times on 50 women and men with HCC diagnosed between 2005-2015 in Mexico City 

Discussion

We found a high prevalence of exposure to aflatoxin in urban and rural Chiapas. However, circulating AFB₁ levels were moderate. Also, aflatoxin-associated TP53 R249S mutation prevalence was moderate relative to high aflatoxin exposure regions in the world.

In Chiapas, AFB₁ exposure was >85%. While the magnitude of the exposure was important (0.117 pg/µL; 2.8 pg/mg), it was not as high as in Guatemala (in the Southern border of Chiapas) where AFB₁ was detectable in all participants and the median AFB₁ -lys was 8.4 pg/mg (23.8 conversion factor from pg/uL to pg/mg).⁹ An initial AFB₁ estimate in adult indigenous women in Mexico found widespread exposure but at a more moderate level relative to very high HCC burden areas.¹⁰ While the study included women mostly in their early thirties, our results may be partly consistent with that observation. However, the exposure levels reported in our study in adults currently living in Chiapas confer increased risk of HCC.¹¹

AFB₁ undergoes enzymatic conversion in the liver by CYP3A4 to the AFB₁ -8,9-epoxide, the active metabolite that reacts with DNA to form an AFB-N7-guanine adduct. These adducts principally cause G:T/C:A mutations on the transcribed strand through the transcription-coupled pathway of nucleotide excision repair.¹² Almost two decades ago a small study in Northern Mexico documented the presence of the AFB₁ codon 249 mutations in HCC tissue (19% prevalence).¹³ In another study, this mutation was not present in any of the 69 HCC tissue samples from highly selected patient population in Mexico City most of whom with cirrhosis.¹⁴ The prevalence of this mutation in our study was lower that what was observed then and in Guatemala (24% prevalence) but higher than the 1-3% seen in low aflatoxin regions (the US, Europe, Korea).^5,15 While R249S is the dominant TP53 aflatoxin-induced mutation, this mutation has been studied almost exclusively in high HBV prevalence regions. We also saw several examples of mutation G T/C:A in V157F. This mutation is another hotspot in TP53, but has not been causally linked to aflatoxin exposure in addition, R249S and V157F have been reported to be associated with a higher stem-cell-like gene expression and poorer survival.¹⁶

Major strengths of our analyses are generalizability of AFB₁ exposure estimates to adults in Chiapas and detailed sequencing of TP53 mutations. However, AFB₁ -lys analyses were done in a limited number of participants affecting confidence on subgroup analyses. The examination of the TP53 mutations was performed in HCC cases that may not be generalizable to all patients seen in Mexico. Currently most HCC cases are diagnosed using imaging without pathology confirmation and, similar to other countries, only a fraction of HCCs were biopsied. Also, the patient population included in our study may not adequately represent the source population of HCC patients in Mexico. In conclusion as the HCC burden remains understudied, our analysis provides evidence that in Mexico AFB₁ may have a potentially important role in the burden of HCC. The five areas in Mexico with the highest HCC burden in order are Chiapas, Veracruz, Yucatán, Tabasco, and Campeche. Future research should focus in these geographic areas and accurately estimate aflatoxin exposure, identify subpopulations at risk and characterize sources of exposures.