Circulating Aflatoxin B1-Related TP53 Mutation Detected by Digital PCR in Tunisian Patients with and Without Hepatocellular Carcinoma

Abstract

Background: In the recent years, hepatocellular carcinoma (HCC) has become one of the first causes of cancer mortality in humans. Among the issues explaining this appalling situation the lack of early biomarkers occupies a prominent position. In patients with tumors, free circulating DNA originating from tumor cells represents a promising material for the early detection of the disease. In HCC, two mutations, R249S on TP53 and-124C>T in TERT promoter, are potentially present in large subsets of patients and could be considered as good candidate biomarkers. Tunisia is a subtropical country where persistent infections with hepatitis virus represent a significant burden of diseases and food contamination with mycotoxins has been consistently detected. Methods: In a pilot study, using the droplet digital PCR (ddPCR) technique, blood samples from 47 patients with HCC and 51 control individuals were evaluated for the presence of the TP53 or TERT mutants in the bloodstream. Results: We observed that 19.1% and 14.9% of the patients with HCC were carrying TP53 or TERT mutants, while 47.1% and 5.8% of controls were positive for the same biomarkers (P = 0.005 and P = 0.18, ns). Most (66.6%) HCC patients with TP53 R49S were coming from the region of Nabeul (North East). Conclusions: This preliminary analysis indicates that droplet digital might be useful for the diagnosis of HCC or screening highrisk subjects in the North African context. In addition, our results confirm previous data obtained by foodstuffs analyses suggesting that a sizeable proportion of the middle-aged Tunisian population is or has been significantly exposed to aflatoxin B1. Larger studies are now mandatory to confirm and refine these preliminary data.

Diagnostic performance of circulating tumor DNA as a minimally invasive biomarker for hepatocellular carcinoma: a systematic review and meta-analysis

“…During our study, we noticed genetic deviations such as mutations in TP53 ( Marchio et al, 2019 ; Lleonart et al, 2005 ; Marchio et al, 2018 ), CTNNB1 ( MacDonald, Tamai & He, 2009 ), and TERT ( Yang et al, 2011 ; Akuta et al, 2021 ), which have been widely studied in circulating tumor DNA in HCC patients. However, a recent study identified TTN, TMEM141, UBB, and ADGRV1 also as the most frequently mutated genes in HCC patients, making them worthy of further investigation ( Gao et al, 2021 ).…”

“…1 ). Eventually, the full text of 59 eligible articles (60 studies) was incorporated into this meta-analysis ( Huang et al, 2003 ; Wang et al, 2021 ; Wong et al, 2000 ; Wong et al, 2003 ; Chu et al, 2004 ; Lin et al, 2005 ; Yeo et al, 2005 ; Iizuka et al, 2006 ; Ren et al, 2006 ; Wang et al, 2006 ; Zhang et al, 2007 ; Chan et al, 2008 ; Chang et al, 2008 ; Igetei et al, 2008 ; Ahmed et al, 2010 ; El-Shazly et al, 2010 ; Hu et al, 2010 ; Huang et al, 2011 ; Iizuka et al, 2011 ; Yang et al, 2011 ; Chen et al, 2012 ; Huang et al, 2012 ; Mohamed et al, 2012 ; Chen et al, 2013 ; Sun et al, 2013 ; Zhang et al, 2013 ; Han et al, 2014 ; Huang et al, 2014 ; Ji et al, 2014 ; Kuo et al, 2014 ; Li et al, 2014 ; Yang et al, 2014 ; Dong et al, 2015 ; Huang et al, 2015 ; Ramadan et al, 2015 ; Teng et al, 2016 ; Dong et al, 2017 ; Hu et al, 2017 ; Mansour et al, 2017 ; Tian et al, 2017 ; Wu et al, 2017 ; Gai et al, 2018 ; Li et al, 2018 ; Oussalah et al, 2018 ; Wei et al, 2018 ; Linlin et al, 2018 ; Bai et al, 2019 ; Kisiel et al, 2019 ; Marchio et al, 2019 ; Pasha, Mohamed & Radwan, 2019 ; El-Bendary et al, 2020 ; He et al, 2020 ; Kotoh et al, 2020 ; Li et al, 2020a …”

“…In the qualitative analysis subgroup, our study enrolled a total population of 3,072 HCC patients and 3,413 control individuals (2,064 patients with benign liver disorders or liver cirrhosis, 1,001 healthy volunteers, and 308 non-cancer controls in a combined group of patients with benign liver disorders and healthy controls). Patients with chronic hepatitis, benign hepatic lesions, and cirrhosis were selected as the control group in 13 articles ( Chu et al, 2004 ; Wang et al, 2006 ; Chang et al, 2008 ; Ahmed et al, 2010 ; Huang et al, 2011 ; Huang et al, 2014 ; Ji et al, 2014 ; Huang et al, 2015 ; Oussalah et al, 2018 ; Kisiel et al, 2019 ; Qian et al, 2020 ; Akuta et al, 2021 ; Lewin et al, 2021 ) , while eight articles with only healthy control groups were chosen in this subgroup ( Yeo et al, 2005 ; Zhang et al, 2007 ; Igetei et al, 2008 ; Hu et al, 2010 ; Zhang et al, 2013 ; Kuo et al, 2014 ; Bai et al, 2019 ; Marchio et al, 2019 ) . Other articles combined healthy volunteers and chronic hepatitis or liver cirrhosis patients as the control group.…”

“…Other articles combined healthy volunteers and chronic hepatitis or liver cirrhosis patients as the control group. The majority of articles were conducted in Asia ( n = 31) ( Huang et al, 2003 ; Wong et al, 2000 ; Wong et al, 2003 ; Chu et al, 2004 ; Lin et al, 2005 ; Yeo et al, 2005 ; Wang et al, 2006 ; Zhang et al, 2007 ; Chang et al, 2008 ; Hu et al, 2010 ; Huang et al, 2011 ; Sun et al, 2013 ; Zhang et al, 2013 ; Han et al, 2014 ; Ji et al, 2014 ; Kuo et al, 2014 ; Li et al, 2014 ; Yang et al, 2014 ; Dong et al, 2015 ; Huang et al, 2015 ; Teng et al, 2016 ; Dong et al, 2017 ; Hu et al, 2017 ; Tian et al, 2017 ; Wu et al, 2017 ; Li et al, 2018 ; Wei et al, 2018 ; Bai et al, 2019 ; Liu et al, 2020 ; Qian et al, 2020 ; Akuta et al, 2021 ), five in Africa ( Igetei et al, 2008 ; Ahmed et al, 2010 ; Ramadan et al, 2015 ; Marchio et al, 2019 ; Pasha, Mohamed & Radwan, 2019 ), three in America ( Huang et al, 2014 ; Kisiel et al, 2019 ; Lewin et al, 2021 ) , and one in Europe ( Oussalah et al, 2018 ). A total of 37 studies looked at ctDNA methylation, while three evaluated single-gene mutation.…”

“…However, two studies were excluded due to the high risk of bias in patient selection and reference concerns ( Lleonart et al, 2005 ; Marchio et al, 2018 ) , and four articles ( Chan et al, 2008 ; Ahmed et al, 2010 ; Huang et al, 2012 ; Lewin et al, 2021 ) had a high risk of bias regarding patient selection and others had a risk of bias in index text ( He et al, 2020 ) or reference standards ( Huang et al, 2003 ). Additionally, due to missing information, the risk of bias could not be assessed for another 16 studies ( Yeo et al, 2005 ; Iizuka et al, 2006 ; Igetei et al, 2008 ; Chen et al, 2013 ; Li et al, 2014 ; Yang et al, 2014 ; Ramadan et al, 2015 ; Dong et al, 2017 ; Mansour et al, 2017 ; Li et al, 2018 ; Wei et al, 2018 ; Linlin et al, 2018 ; Bai et al, 2019 ; Marchio et al, 2019 ; El-Bendary et al, 2020 ; Xie et al, 2021 ). Because many case-control studies were enrolled in this meta-analysis, the selection of patients was the main bias risk across the included publications.…”