Droplet digital PCR detects high rate of TP53 R249S mutants in cell-free DNA of middle African patients with hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is still a major killing malignancy in sub-Saharan Africa. Lifelong intoxication with aflatoxin B1 is considered as one of the primary causes of this situation. The role of aflatoxin in HCC from a given population is commonly estimated through the prevalence of R249S mutation of TP53, a hallmark for previous exposure to the mycotoxin. However, the role of AFB1 is barely known in large part of Africa. We conducted a survey on circulating cell-free DNA from 149 patients with HCC and 213 control subjects with and without liver diseases from Cameroon and Central African Republic using droplet digital PCR technique. We observed a mutation prevalence of 24.8% (n = 37/149) in patients with tumor and 5.6% (n = 12/213) in controls (P = 2.2E-07). Patients with mutations usually displayed significantly increased circulating alpha-fetoprotein (AFP) values, high hepatitis B virus (HBV) DNA loads as well as worsened values of blood cells count. Interestingly, the fraction of droplets positive for R249S was significantly larger in patients with liver cancer (15.3 ± 3.7%) than in controls (0.5 ± 0.3%, P = 7.1E-04). Our survey indicates that AFB1 is instrumental for HCC development in Middle Africa and that droplet digital PCR might be used in the region both to diagnose HCC and to conduct public health surveys on populations at risk of chronic aflatoxin intoxication.

Circulating Tumor DNA and Hepatocellular Carcinoma

“…A recent study evaluated the frequency ctDNA mutation of TP53 R249S, well known mutation associated with aflatoxin exposure on 149 African HCC patients and 213 control subjects using ddPCR. 54 About a quarter of the HCC patients had TP53 R249S mutation, while it was seen in only 5 to 6% in control groups of individuals with chronic liver disease or healthy subjects. A similar study was conducted in the United States on 218 HCC patients and 96 Hispanic subjects with advanced fibrosis or cirrhosis.…”

Hepatitis B virus preS2Δ38–55 variants: A newly identified risk factor for hepatocellular carcinoma

“…The p53R249S allelic fraction is an integrated marker of both past AFB1 exposure and the presence of a tumour carrying this mutation, and indicative of HCC. The sensitivity and specificity of the technique used in this study to detect mutant p53 alleles are comparable with those obtained using another highly sensitive technology, the droplet digital PCR, 35 and highlight their potential in assessing this biomarker in liquid biopsy studies for identifying and confirming HCC status as reviewed in 1 study. 36 Moreover, AFB1 exposure could contribute to the low viral load observed in chronic HBV carriers in The Gambia as in vitro AFB1 exposure has been reported to reduce HBV replication.…”

Circulating tumor DNAs and non-coding RNAs as potential biomarkers for hepatocellular carcinoma diagnosis, prognosis and response to therapy

“…In the diagnostic setting, R249S mutation of the TP53 gene is hallmark of aflatoxin B1 exposure, one of the major causes of HCC in certain geographic areas. Using droplet digital PCR the authors identified a higher prevalence of this mutation in plasma cfDNAs of HCC in Cameroonian and Central African patients in comparison with control subjects with and without liver disease (almost 25% of patients with HCC and 3%-9% of non-HCC subjects were R249S carriers), suggesting a potential use of this biomarker as an early risk factor for HCC in individuals exposed to aflatoxin B1 [26] . Targeted deep sequencing was used to investigate several cancer genes involved in HCC.…”