**2.1 Study population**

A total of 380 patients were recruited from the affiliated hospitals of Youjiang Medical University for Nationalities and Guangxi Medical University (two main medical universities in the AFB1-exposure areas in China) between 2011 and 2013. All cases were newly diagnosed as patients using histopathological method and they had no history of radiation or chemotherapy treatment before enrollment. A total of 588 controls, who were randomly recruited from a pool of healthy individuals in the same hospitals during the same time, were all volunteers without any evidence with liver diseases. To control the effects of confounder factors such as age, gender, and race, controls were individually matched with the cases on these factors. In this study, all cases and controls had no history of hepatitis B virus (HBV) and/ hepatitis C virus (HCV) infection, whereas these subjects with positive status of serum anti-HCV and/or hepatitis B surface antigen (HBsAg) were excluded. They all agreed to participate in this investigation and did not drop out. With informed consent, all clinicopathological data, including age, gender, race, hepatitis virus B and C infection information, survival follow-up information, were collected using healthy examination or medical records. Additionally, 10 ml of peripheral blood samples for all subjects were also collected for AFB1-DNA adduct analysis. In this study, the last following-up date was set on January 31, 2019. Overall survival (OS) and tumor recurrence-free survival (RFS) status were defined according to the previously described methods [11, 14, 18]. The study protocol was approved by the ethics committees of Youjiang Medical University for Nationalities and Guangxi Medical University.

#### **2.2 AFB1-DNA adducts data**

The amount of AFB1-DNA adducts in the peripheral blood were tested using the previously published methods [8, 17]. Briefly, DNA samples were first extracted from the peripheral blood samples and adducts were next quantitated by the comparative enzyme-linked immunosorbent assay (ELISA). To investigate the association between different levels of AFB1-DNA adducts and the risk and prognosis of PHCC patients, the levels of AFB1-DNA adducts were divided into three subgroups according to the mean adduct amounts of cases and controls: low AFB1-DNA adduct level (LAL, <1.00 μmol/DNA), medium AFB1-DNA adduct level (MAL, 1.00–2.00 μmol/DNA), and high AFB1-DNA adduct level (HAL, >2.00 μmol/DNA).

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**Table 1.**

*The characteristics of subjects.*

*The Blood AFB1-DNA Adduct Acting as a Biomarker for Predicting the Risk and Prognosis…*

All statistical analyses were accomplished with SPSS statistical package (Version 18, SPSS Institute, Chicago, IL, USA). Test for the distribution of age, gender, and race between patients with PHCC and controls was accomplished using chi-square test. The effects of blood AFB1-DNA adducts on PHCC risk were evaluated using odds ratio (OR) and 95% confidence interval (CI) in the conditional logistic regression model. For survival analyses, Kaplan-Meier survival model with *Log-Rank* test and Cox regression model (the selection of significant variates based on forwardstep method with likelihood ratio test) was used to analyze the association between blood AFB1-DNA adducts and PHCC outcomes. Cumulative hazard value for the effects of adducts on the prognosis of patients with PHCC and corresponding 95% CI was calculated using hazard ratio (HR) from significant multivariate Cox regression model (including all significant variates). In this study, the *P* value <0.05 was

A total of 380 cases with PHCC and 588 controls were included in our final analyses. Baseline characteristics of all cases with PHCC and controls were summarized in **Table 1**, and results showed there were no significant distributions of age,

Total 588 100.0 380 100.0 — Gender 0.70

Age (years) 0.78

Race 0.88

Male 426 72.4 271 71.3 Female 162 27.6 109 28.7

≤35 81 13.8 53 13.9 36–40 62 10.5 38 10.0 41–45 90 15.3 55 14.5 46–50 83 14.1 43 11.3 51–55 94 16.0 56 14.7 56–60 56 9.5 42 11.1 61–65 69 11.7 49 12.9 ≥66 53 9.0 44 11.6

Han 339 57.7 221 58.2 Zhuang 249 42.3 159 41.8

**Controls PHCCs** *P*

**n % n %**

*DOI: http://dx.doi.org/10.5772/intechopen.88666*

defined as statistical significance.

**3.1 The features of study population**

gender, and race between cases and controls.

*PHCCs, patients with primary hepatocellular carcinoma.*

**3. Results**

**2.3 Statistical analysis**

*The Blood AFB1-DNA Adduct Acting as a Biomarker for Predicting the Risk and Prognosis… DOI: http://dx.doi.org/10.5772/intechopen.88666*

## **2.3 Statistical analysis**

*Aflatoxin B1 Occurrence, Detection and Toxicological Effects*

AFB1's 8,9-dihydro-8-(N<sup>7</sup>

to the risk and outcome of PHCC.

**2. Materials and methods**

**2.1 Study population**

Medical University.

>2.00 μmol/DNA).

**2.2 AFB1-DNA adducts data**

conjugating reaction) [2, 3]. During the process of AFB1's metabolism, AFB1- DNA adducts, including AFB1-formamidopyrimidine adduct (AFB1-FAPa) and

formed [2, 3]. Growing evidence has shown that AFB1-DNA adducts are usually tested in the tissue samples (such as liver and placenta tissues) of these individuals from high AFB1 exposure areas [6–10]. Recent studies have displayed that they are also found in the peripheral blood white cells of peoples who are from high AFB1 exposure areas and are associated with the time of AFB1 exposure [11–17]. However, the potential of blood AFB1-DNA adducts predicting PHCC risk and prognosis is not clear. Here, we specifically conduced a hospital-based case-control study to investigate whether blood AFB1-DNA adducts were related

A total of 380 patients were recruited from the affiliated hospitals of Youjiang Medical University for Nationalities and Guangxi Medical University (two main medical universities in the AFB1-exposure areas in China) between 2011 and 2013. All cases were newly diagnosed as patients using histopathological method and they had no history of radiation or chemotherapy treatment before enrollment. A total of 588 controls, who were randomly recruited from a pool of healthy individuals in the same hospitals during the same time, were all volunteers without any evidence with liver diseases. To control the effects of confounder factors such as age, gender, and race, controls were individually matched with the cases on these factors. In this study, all cases and controls had no history of hepatitis B virus (HBV) and/ hepatitis C virus (HCV) infection, whereas these subjects with positive status of serum anti-HCV and/or hepatitis B surface antigen (HBsAg) were excluded. They all agreed to participate in this investigation and did not drop out. With informed consent, all clinicopathological data, including age, gender, race, hepatitis virus B and C infection information, survival follow-up information, were collected using healthy examination or medical records. Additionally, 10 ml of peripheral blood samples for all subjects were also collected for AFB1-DNA adduct analysis. In this study, the last following-up date was set on January 31, 2019. Overall survival (OS) and tumor recurrence-free survival (RFS) status were defined according to the previously described methods [11, 14, 18]. The study protocol was approved by the ethics committees of Youjiang Medical University for Nationalities and Guangxi

The amount of AFB1-DNA adducts in the peripheral blood were tested using the previously published methods [8, 17]. Briefly, DNA samples were first extracted from the peripheral blood samples and adducts were next quantitated by the comparative enzyme-linked immunosorbent assay (ELISA). To investigate the association between different levels of AFB1-DNA adducts and the risk and prognosis of PHCC patients, the levels of AFB1-DNA adducts were divided into three subgroups according to the mean adduct amounts of cases and controls: low AFB1-DNA adduct level (LAL, <1.00 μmol/DNA), medium AFB1-DNA adduct level (MAL, 1.00–2.00 μmol/DNA), and high AFB1-DNA adduct level (HAL,


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All statistical analyses were accomplished with SPSS statistical package (Version 18, SPSS Institute, Chicago, IL, USA). Test for the distribution of age, gender, and race between patients with PHCC and controls was accomplished using chi-square test. The effects of blood AFB1-DNA adducts on PHCC risk were evaluated using odds ratio (OR) and 95% confidence interval (CI) in the conditional logistic regression model. For survival analyses, Kaplan-Meier survival model with *Log-Rank* test and Cox regression model (the selection of significant variates based on forwardstep method with likelihood ratio test) was used to analyze the association between blood AFB1-DNA adducts and PHCC outcomes. Cumulative hazard value for the effects of adducts on the prognosis of patients with PHCC and corresponding 95% CI was calculated using hazard ratio (HR) from significant multivariate Cox regression model (including all significant variates). In this study, the *P* value <0.05 was defined as statistical significance.
