Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high incidence and death rate in the worldwide. Because HCC often displays its’ high early-stage blood metastasis and recurrence risk, patients with HCC usually feature a poor prognosis. With advancing in the scientific technology, some new therapeutic methods, including the transarterial chemoembolization (TACE), radiofrequency ablation, microwave ablation, percutaneous ethanol ablation, targeting therapy and radioembolization, have been developed and utilized in some HCC cases. Among these therapeutic methods, TACE treatment has been reported widely to be able to effectively improve the survival status of patients with HCC, especially these with advanced-stage tumor. In China, post-operative adjuvant TACE (po-TACE) treatment has been recommended as an important post-operative adjuvant treatment for HCC patients with high risk of tumor intrahepatic metastasis. However, increasing evidence has shown that po-TACE treatment displays different therapeutic effects on patients with different genetic profiles or biomarkers. Thus, it is vital to discern specific biomarkers or genetic profiles for improving the therapeutic effects of po-TACE treatment on HCC prognosis.

Aflatoxin B1 (AFB1) is an important I-type chemical carcinogen, which can induce HCC [1]. Growing evidence has shown that AFB1-caused DNA damage, including AFB1-DNA adducts (ADAs), single-strand breaks (SSBs), and double-strand breaks (DSBs), play a vital role in the carcinogenesis of AFB1-induced HCC. DNA damage induced by AFB1, especially ADAs, will change the structures of DNA sequence and lead to mutations of some genes such as TP53 and Ras [1–3]. On the one hand, this kind of accumulating gene alterations may disrupt corresponding encoding-protein structures and functions and affect the capacity of DNA damage response (DDR), which can result in harmful effects on human health, such as aging, senescence, abnormal apoptosis status, genomic instability, and ultimately severe cancer progression in normal cells [1, 4]. On the other hand, DNA damage-linked abnormal DDR capacity occurring in cancer cells may be regarded as potential targets for cancer therapy [5–11].

However, it is unclear whether ADAs in HCC modify the effects of po-TACE treatment improving HCC survival. A retrospective study was conducted to investigate the association between the amount of ADAs in the cancerous tissues with HCC and the therapeutic effects of po-TACE treatment on HCC.

Materials and methods

Results

Po-TACE therapy improving the outcomes of HCC

Results from Kaplan-Meier survival models displayed that these HCC patients receiving po-TACE treatment featured a better OS compared to those not doing po-TACE treatment (Figure 1A) and RFS (Figure 1B). Median survival time (MST) was 23.00 months and 10.00 months for po-TACE and control group, respectively, whereas median tumor recurrence-free time (MRT) was 20.00 months and 8.00 months, respectively. Univariate Cox regression survival model analyses showed that compared with the control group, po-TACE treatment significantly decreased DR [0.63 (0.50–0.80)] and TRR [0.61 (0.48–0.78)] of patients with HCC (Table 2). Results from multivariate Cox regression survival model analyses further proved that po-TACE therapy significantly improved the survival of HCCs (Table 3).

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Figure 1. 

Po-TACE treatment significantly correlating with HCC survival. Po-TACE treatment is associated with (A) OS and (B) tumor RFS of HCC. Cumulative hazard function was plotted by Kaplan-Meier’s methodology, and P value was calculated with two-sided Log-rank tests. (+): patients receiving po-TACE treatment; (−): patients without receiving po-TACE treatment; CI: confidence interval

Table 2.

Univariate analyses of potential viables for survival of patients with HCC

Variables	OS		RFS
	DR (95% CI)	Ptrend	TRR (95% CI)	Ptrend
Age (> 48 years old vs. 48 years old)a	0.89 (0.70–1.12)	0.31	0.88 (0.69–1.12)	0.29
Gender (female vs. male)	0.91 (0.71–1.17)	0.47	0.90 (0.70–1.15)	0.39
Ethnicity (Zhuang vs. Han)	0.81 (0.64–1.02)	0.07	0.77 (0.61–1.08)	0.08
Smoking (yes vs. no)	0.76 (0.58–0.99)	0.05	0.76 (0.58–1.01)	0.06
Drinking (yes vs. no)	0.86 (0.66–1.12)	0.26	0.82 (0.63–1.08)	0.16
HBV status (positive vs. negative)	1.04 (0.81–1.33)	0.79	1.20 (0.93–1.55)	0.16
HCV status (positive vs. negative)	1.03 (0.72–1.48)	0.86	1.03 (0.71–1.50)	0.87
AFP (> 20 ng/mL vs. 20 ng/mL)	1.11 (0.88–1.41)	0.38	1.21 (0.95–1.55)	0.13
Liver cirrhosis (yes vs. no)	1.20 (0.92–1.58)	0.19	1.13 (0.85–1.49)	0.40
ES grade (high vs. low)	1.48 (1.17–1.87)	8.99 10–4	1.57 (1.24–1.99)	2.11 × 10–4
MVD (positive vs. negative)	1.39 (1.09–1.78)	7.30 10–5	1.22 (0.96–1.57)	0.11
Po-TACE treatment (yes vs. no)	0.63 (0.50–0.80)	9.80 10–5	0.61 (0.48–0.78)	5.10 × 10–5
ADAs (high vs. low)b	3.89 (2.95–5.12)	4.66 10–22	3.08 (2.36–4.02)	1.10 × 10–16

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^a Age is grouped according to the average age of patients with HCC (47.94 years old 9.98 years old); ^b ADAs are grouped according to the average ADA of patients with HCC (3.00 µmol/mol 1.51 µmol/mol DNA). P_trend: P value is calculated by trend test in the models

Table 3.

Multivariate analyses of potential viables for survival of patients with HCC

Variables	OS		RFS
	DR (95% CI)	Ptrend	TRR (95% CI)	Ptrend
Age (> 48 years old vs. 48 years old)a	0.94 (0.72–1.18)	034	0.95 (0.70–1.28)	0.25
Gender (female vs. male)	1.00 (0.77–1.29)	0.99	0.96 (0.74–1.25)	0.76
Ethnicity (Zhuang vs. Han)	0.89 (0.70–1.13)	0.34	0.79 (0.61–1.01)	0.06
Smoking (yes vs. no)	0.73 (0.52–1.02)	0.07	0.83 (0.59–1.17)	0.28
Drinking (yes vs. no)	0.95 (0.69–1.32)	0.78	0.82 (0.58–1.15)	0.25
HBV status (positive vs. negative)	0.98 (0.75–1.29)	0.90	1.16 (0.88–1.53)	0.29
HCV status (positive vs. negative)	1.01 (0.69–1.49)	0.963	1.00 (0.68–1.49)	0.99
AFP (> 20 ng/mL vs. 20 ng/mL)	1.22 (0.95–1.56)	0.13	1.31 (1.01–1.69)	0.04
Liver cirrhosis (yes vs. no)	1.02 (0.76–1.36)	0.91	0.97 (0.72–1.29)	0.81
ES grade (high vs. low)	1.31 (1.03–1.68)	0.03	1.39 (1.09–1.79)	9.14 × 10–3
MVD (positive vs. negative)	1.24 (0.95–1.62)	0.12	1.09 (0.83–1.42)	0.53
Po-TACE treatment (yes vs. no)	0.59 (0.46–0.76)	5.10 10–5	0.63 (0.49–0.82)	4.36 × 10–4
ADAs (high vs. low)b	3.69 (2.78–4.91)	2.02 10–19	2.95 (2.24–3.88)	1.52 × 10–14

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^a Age is grouped according to the average age of patients with HCC (47.94 years old 9.98 years old); ^b ADAs are grouped according to the average ADA of patients with HCC (3.00 µmol/mol 1.51 µmol/mol DNA). P_trend: P value is calculated by trend test in the models

ADAs significantly affecting HCC prognosis

The amount of ADAs was elucidated by enzyme-linked immunosorbent assay (ELISA) assay and a similar amount of ADAs was tested in cancerous tissue samples from control and po-TACE group (3.09 µmol/mol ± 1.34 µmol/mol vs. 2.92 µmol/mol ± 1.66 µmol/mol DNA) (Figure 2). To analyze the effects of ADAs on HCC survival, ADAs were divided into two groups: high level of ADAs (HADA) and low level of ADAs (LADA) according to the average amount of ADAs in tissue samples from all patients (3.00 µmol/mol 1.51 µmol/mol DNA). Results from Kaplan-Meier survival models displayed that these HCC patients with HADA in their cancerous tissues featured a poorer OS compared to those with LADA (Figure 3A) and RFS (Figure 3B). MST and MRT were 8.00 months vs. 26.00 months and 7.00 months vs. 22.00 months, respectively. Univariate Cox regression survival model analyses showed that compared with LADA, HADA significantly increased DR [3.89 (2.95–5.12)] and TRR [3.08 (2.36–4.02)] of patients with HCC (Table 2). Results from multivariate Cox regression survival model analyses further proved that ADA was a potential prognostic marker for HCC (Table 3).

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Figure 2. 

The distribution of ADAs between po-TACE group and control group. The amount of ADAs in the cancerous tissues with HCC is calculated by c-ELISA and the difference for the distribution of ADAs between two groups is tested by t test. Black lines represent the mean of ADAs in each group

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Figure 3. 

ADAs significantly correlating with HCC survival. The amount of ADAs in the cancerous tissues with HCC is calculated by c-ELISA. To analyze, the levels of ADAs are divided into two groups: LADA and HADA. ADAs are associated with (A) OS and (B) tumor RFS of HCC. Cumulative hazard function was plotted by Kaplan-Meier’s methodology, and P value was calculated with two-sided Log-rank tests

ADAs differentially modified po-TACE therapy in improving HCC prognosis

In view of some studies showing that DNA damage may act as a potential target for therapeutic intervention [5, 17, 18], the present study explored whether ADAs modified the therapeutic effects of po-TACE treatment on HCC through a stratified analysis based on the different levels of ADAs in the tissues with HCC (Table 4). The results showed that po-TACE therapy can significantly improve the outcome of these HCC patients with HADA in their cancerous tissues (MST, from 7 months to 13 months; MRT, from 6 months to 12 months) (Figure 4A and 4B). Results from multivariable Cox regression survival model analyses further proved that these patients feature a decreasing DR [0.36 (0.24–0.53)] and TRR [0.40 (0.28–0.59)] if they received po-TACE treatment (Table 4). However, this kind of improvement in the HCC prognosis was not observed among those with LADA (P > 0.05) (Figure 4C and 4D, Table 4). Taken together, these results implied that ADAs can differently modify the efficiency of po-TACE therapy in improving HCC prognosis.

Table 4.

The effects of po-TACE treatment on HCC prognosis stratified by ADAs

ADA	Po-TACE treatment	OS		RFS
		DR (95% CI)a	Ptrend	TRR (95% CI)a	Ptrend
Low	Yes vs. no	0.84 (0.58–1.20)	0.33	0.84 (0.58–1.23)	0.37
High	Yes vs. no	0.36 (0.24–0.53)	2.67 × 10–7	0.40 (0.28–0.59)	2.00 × 10–6

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^a Adjusted by age, sex, race, HBV and HCV status, smoking and drinking status, AFP, liver cirrhosis, and tumor grade. P_trend: P value is calculated by trend test in the models

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Figure 4. 

Survival analysis of po-TACE treatment in strata of ADAs. Among HCC cases with HADA, po-TACE treatment improved their (A) OS and (B) TRR. However, this treatment did not improve the (C) OS and (D) TRFS of those with LADA. Cumulative hazard function was plotted by Kaplan-Meier’s methodology, and P value was calculated with two-sided Log-rank tests. (+): patients receiving po-TACE treatment; (−): patients without receiving po-TACE treatment

The amounts of ADAs positively correlated with MVD in the tumor tissues

Because of MVD in the tumor tissues positively correlating with therapeutic effects of po-TACE on HCC [19], this study investigated the association between ADAs and MVD (Figure 5). Results exhibited that the number of ADAs in the tissues with HCC was positively related to the amount of MVD in the tumor tissues (r = 0.182, P < 0.01).

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Figure 5. 

The association between ADAs in the cancerous tissues with HCC and tumor MVD. The data were analyzed using Spearman’s correlation analysis

Discussion

To the best of authors’ knowledge, there is not a shred of evidence whether the DNA damage by AFB1 can act as a therapeutic target for HCC. On the basis of a retrospective study, this study investigated the potential association between ADA, an important DNA damage type induced by AFB, and the therapeutic efficiency of po-TACE treatment in improving HCC survival. It was found that increasing the amount of ADAs in the cancerous tissues with HCC had a significant correlation with the therapeutic efficiency of po-TACE on HCC. This study represents the first report indicating the possibility of ADA as a therapeutic target for po-TACE.

TACE treatment is an important therapeutic procedure for HCC [20]. Among known TACE techniques, conventional TACE (cTACE) and TACE with drug-eluting beads (DEB-TACE) are widely used worldwide. The cTACE, which commonly uses a mixture of lipoid and anticancer drugs (e.g., epirubicin, doxorubicin, or cisplatin), was developed and established in the late last century. In the procedure of cTACE treatment, emulsion mixtures are first injected into the tumor-feeding arteries; next, gelatin sponge or particles are administered. DEB-TACE technique has been developed, mainly because of enhancing the delivery of anti-cancer drugs, providing a better embolizing effect, and minimizing drug-related adverse events [20–22]. To date, drug-eluting beads (DEB) are designed as a kind of multifunction embolization microspheres. Furthermore, they are visible by imaging and have the capacity for loading different anticancer [20]. Evidence from in vivo and in vitro experiments and clinical trials has shown that TACE treatment can effectively improve the outcomes of patients with HCC [23, 24]. Today, TACE treatment has been recommended as the first treatment option for patients with intermediate-stage HCC (BCLC-B stage HCC) [22]. Additionally, this treatment is also widely used as a post-operative adjuvant treatment (namely po-TACE) for patients with advanced-stage HCC (BCLC-C stage HCC) [25, 26]. The present study also found that po-TACE treatment can significantly improve the survival of HCC patients after tumor resected. Compared with controls, these patients receiving po-TACE treatment featured a longer MST (10.00 months vs. 23.00 months) and MRT (8.00 months vs. 20.00 months) and a smaller DR (0.59, 95% CI = 0.46–0.76) and TRR (0.63, 95% CI = 0.49–0.82).

In the past decade, increasing evidence from clinical practice has shown that the therapeutic efficiency of po-TACE treatment can be modified by individuals’ genetic profiles and some potential biomarkers are identified for predicting the therapeutic efficiency of this treatment. This study investigated the predictive values of a DNA damage biomarker ADA for the effects of po-TACE treatment improving HCC survival through a hospital-based retrospective study and found ADAs in tumor samples with HCC can significantly modify the sensibility of HCC cases on po-TACE treatment. These patients with HADA will obtain a better survival under the conditions of po-TACE therapy than those with LADA. This may be because of the several reasons. First, increasing amount of ADAs in tumor tissues usually indicates the low capacity of detoxicating enzymes [12, 27, 28]. Evidence from molecular epidemiological studies has proved low detoxification capacity resulting from abnormal expression of detoxicating enzyme genes were positively correlated with the levels of ADAs in the peripheral blood or cancerous tissues with HCC [27, 28]. Furthermore, the downregulation of detoxicating enzyme cytochrome P450 (CYP450) 2W1 by microRNA-4651 significantly changed the efficiency of po-TACE treatment in improving HCC surviving [12]. Second, abnormal DNA repair capacity and the following DNA damage accumulation in cancer cells alters the response of cancer cells on anti-cancer drugs [5, 17, 29]. Molecular epidemiological studies conducted in high AFB1 exposure areas show that the genetic alterations in DNA repair genes (including XPC, XRCC4, XRCC1, and XPD) are not only associated with low DNA damage repair capacity and result in increasing amounts of ADAs, but also increase HCC risk and short survival of patients with HCC [15, 30–33]. For example, genetic alteration at the coding region of DNA repair gene XRCC4 increased the amount of ADAs in the HCC cells and change the sensibility of cancer cells on anticancer drug doxorubicin [34, 35]. Third, ADAs maybe affect the process of tumor angiogenesis. Evidence from clinicopathological studies has displayed that there is an increasing heterogenic expression profile of chromobox 4 (CBX4) in tumor tissular samples with HADA and this alteration statistically modifies the therapeutic efficiency of po-TACE treatment on HCC [19]. This may be result from the upregulation of CBX4 expression accelerating tumor angiogenesis and next inducing tumor proliferation and decreasing cancerous cells’ sensibility on anticancer drugs [14, 19, 36]. The present study furthermore proved that the levels of ADAs in tumor tissues were linked with tumor MVD. Similar findings are shown in genetic variations [12, 37]. Finally, ADAs can change the cancer phenotypes, such as proliferation, apoptosis, cell cycle, autophagy, and oxidative endoplasmic reticulum stress, via activating oncogenes and inducing mutations of tumor suppressor genes [2–4]. These phenotypes maybe promote tumor progression and modify the therapeutic efficiency of po-TACE treatment in improving HCC survival [38].

In conclusion, this study describes ADAs in tissues with HCC and its link with the therapeutic value of po-TACE treatment and displays that ADAs can act as a valuable biomarker for forecasting the therapeutic significance of po-TACE treatment. These findings indicate that analyzing the amount of ADAs in the cancerous tissues with HCC may help us form a management strategy to improve the survival of patients with HCC. However, the present study was based on a relatively small sample, which may limit the potential of ADAs predicting the therapeutic efficiency of po-TACE on HCC. Although the present study explored the relationship between ADAs and tumor MVD, the corresponding mechanism analyses were not finished. Thus, forthcoming studies in combination with a larger size sample and mechanism analyses will be required to fully evaluate the utility of ADAs in selecting patients for po-TACE therapy.