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Abstract

BACKGROUND:

Data about the molecular pathogenesis of hepatitis C-related hepatocellular carcinoma (HCC) are still challenging.

OBJECTIVES:

Therefore, we tried to investigate the epigenetic study of three nominated genes (APC, P14, and E-cadherin) in the pathogenesis of HCV-related HCC in Egyptian.

METHODS:

Between March 2016 and March 2017, the DNA methylation, and quantification using (epigenetic ELISA kit) for E-cadherin, APC, and P14 genes were studied in three groups of patients: HCV related liver cirrhosis without HCC group (LC-group; $n=$ 20), HCC on top of HCV-related cirrhosis (HCC-group; $n=$ 20), and a third apparently healthy control group (control-group; $n=$ 10).

RESULTS:

E-cad methylation showed non-significant differences between groups. P14 methylation was occurred only in HCC-group (45%). APC methylation was the highest in HCC group (70%). Methylation level was high in HCC group in comparison to both LC and control groups ( $P<$ 0.001). DNA methylation at a cutoff point $>$ 2.9 ng/ml predicts HCC in LC-group with 90% sensitivity and 80% specificity and at level $>$ 2.3 ng/ml had 95% sensitivity and 90% specificity in control-group. The pooled sensitivity, specificity, positive and negative predictive values and accuracy were 90%, 60%, 69.2, 85.7 and 75% respectively.

CONCLUSION:

Aberrant DNA methylation of multiple genes is associated with disease progression in HCV related cirrhosis. Moreover, early detection of promotor methylation of these may sever as good biomarker for early detection and therapeutic targets in high risk patients.

Keywords

Hepatocellular carcinoma liver cirrhosis epigenetics

1. Introduction

In the last decades, the incidence of hepatocellular carcinoma (HCC) is increasing world-wide; especially in high prevalence of hepatitis C virus (HCV) settings, like in Egypt [1]. Several mechanisms, by which HCV affects the hepatocytes and induces a hepatocarcinogenesis and the subsequent HCC, were proposed [2, 3]. Of these, the epigenetic mechanism by which an alteration(s) occur in nucleotide bases of normal DNA through methylation of cytosine-phosphate-guanine (CpG) islands in the cases with chronic inflammatory insult (e.g. chronic HCV) leading to tumor suppressor genes (TSG) silencing and/or over expression of oncogenes [3, 4]. Aberrant methylation in DNA has been found to be tumor specific with several studies tried to investigate HCC-candidate tumor suppressor genes involved in this event; such as RASSF1A, hMLH1, SOCS1, APC, P14, E-cadherin genes, etc. [5, 6]. However, the pooled figures and meta-analysis results are perplexing with varying frequencies and implications [2, 7, 8]. Also, most of methylation studies focused on the evaluation of the difference of gene expression of an existed DNA-methylation in different stages of tumor progression [9, 10]. Therefore, we tried to evaluate the frequency of DNA-methylation of three nominated genes (APC, P14, and E-cadherin) in the pathogenesis of HCV-related HCC in Egyptian patients through artificial DNA-methylation and the diagnostic yield of the level of DNA methylation.

2. Patients and methods

2.1 Study design

Between March 2016 and March 2017, a cross sectional descriptive study was conducted to evaluate the role and frequency of DNA methylation of APC, P14, and E-cadherin genes in the pathogenesis of HCV-related HCC in Egyptian patients. A total of 50 subjects were enrolled: 20 patients who were assigned to have HCV related liver cirrhosis without HCC group (LC-group; $n=$ 20), another 20 patients who assigned to have HCC on top of HCV-related cirrhosis (HCC-group; $n=$ 20), and a third apparently healthy subjects that were seronegative for both HBV and HCV that were enrolled as a control group (control-group; $n=$ 10). All cases enrolled from the outpatient clinic of Tropical Medicine and Gastroenterology Department, Assiut University, Assiut, Egypt. From each patient, a 5 ml venous blood was sampled and divided into 2 tubes; one on EDTA for molecular study and the other for serologic and other laboratory investigations.

2.2 Study definitions

The inclusion criteria were any adult patients of both sexes (18 years or older) who were seropositive for HCV and have HCV-related liver cirrhosis and are naïve to HCV treatment. The diagnosis of cirrhosis was based on combination of clinical, biochemical, and ultrasonographic findings [11]. Like-wise, the diagnosis of HCC was based on a combination of both imaging revealed the typical criteria of HCC in multi-phasic contrast computed tomography scan [12] and raised alpha-fetoprotein (AFP) [13] and in compliance to the recent international guidelines [14, 15]. HCC-patients were only early stages (stage I, II) according to the Barcelona Clinic Liver Cancer (BCLC) staging system [16]. Both LC-group and HCC group did not receive prior antiviral therapy or therapy for HCC.

2.2.1 Epigenetic study

To study the aberrant methylation of the formally nominated genes, 4 step-wise protocols were designated:

Figure 1.

Methylated and unmethylated genes sequencing.

Step 1:

DNA purification and extraction: to study the baseline unmethylated genes, DNA purification and extraction was done according to the well-standardized technique [17] using Spin Protocol technique by QIAamp ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ Spin Columns (QIAGEN Inc. CA 91355, USA) manufacturer instructions.

Step 2:

DNA Methylation: by Bisulfite Conversion Chemistry technique (Zymo-Spin ${}^{\text{TM}}$ IC Co- lumns) according to the Zymo Research Corp. manufacturer instruction protocols (EZ 96-DNA Methylation-Direct ${}^{\text{TM}}$ Kit, Cat. No D5023, USA).

Step 3:

Methylation specific PCR (MSP): Conventional PCR amplification (Verity ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ 96 – well Thermal Cycle, CA 94404, USA) in each sample by methylation specific primers was performed to analyze the promotor CpG island methylation status under 2 conditions; methylated and unmethylated genes (E-cadherin, APC, P14) using the following primers (Fig. 1).

Figure 2.

(a) Gel electrophoresis of the MSP products of E-cadherin gene, (b) Gel electrophoresis of the MSP products of P14 gene.

The PCR was carried out in a total volume of 25 $\mu$ l. The tube containing 12.5 $\mu$ l Taq DNA Polymerase Master Mix (Ampliqon A/S, Denmark), 100 ng DNA that is 1 $\mu$ l in case of unmethylated and 4 $\mu$ l in methylated DNA, lmM of each primer (forward and reverse) and 3 $\mu$ l magnesium chloride in case of methylated E-cad and APC. Then add distilled water to complete 25 $\mu$ l. PCR reaction underwent initial denaturation at 95 ${}^{\circ}$ C for 5 min, and 35 cycles of the following profile: 15 s at 94 ${}^{\circ}$ C, 30 s at 59 ${}^{\circ}$ C, and 30 s at 72 ${}^{\circ}$ C, and a final extension step at 72 ${}^{\circ}$ C for 10 min. The criterion of methylation and un-methylation status of the MSP products was 98 bp, 116 bp, 150 bp for APC, E-cad and P14 genes, respectively.

Step 4:

Quantification of DNA methylation level: The level of global DNA methylation (5me- thylcytosine) was determined by methyl flash methylated DNA quantification kit (MethylFlash ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ , colorimetric method, EPIGENTEK Group Inc. Cat No P-1034-43, USA). In this assay, DNA is bound to strip wells that are specifically treated to have a high DNA affinity. According to manufacturer instructions, 100 ng of DNA and 80 $\mu$ l of DNA binding solution were added to microplate and incubated ata 37 ${}^{\circ}$ C for 90 minutes. The 5 mC capture antibody was added for 60 minutes, followed by 50 $\mu$ l of the biotin-conjugated detection antibody for 30 minutes, and the absorbance was read at 450 nm.

2.2.2 Data management and ethical considerations

Frequencies, percentages, and means were used, as appropriate, for descriptive analysis. ANOVA test was used to compare parametric quantitative data between groups and Chi Square test was used to compare parametric qualitative data, while Mann-Whitney test was used to compare non-parametric qualitative data and fisher exact test used to compare non-parametric qualitative data. Statistical analysis was conducted by SPSS (V.19, SPSS Inc.; Chicago, IL, USA). A p-value $<$ 0.05 was considered significant. The study was conducted after approval of the Clinical Research Ethical Committee of Assiut Faculty of Medicine and was carried out according to the code of ethics of the World Medical Association (Declaration of Helsinki). All samples were obtained with a written informed consent from all patients.

3. Results

During the period of the study, a total of 50 subjects were enrolled. Table 1 revealed their demographic data.

3.1 Frequency of unmethylated genes among the studied groups

All three genes were unmethylated in control group. In LC-group, bothP14 and E-cad were unmethylated in 100% of cases and APC gene in 30% only. In HCC group, only P14 was 100% unmethylated and the E-cad and APC genes were unmethylated in 90% of cases. All details are described in Table 2 and Fig. 2a–c.

Table 1
Demographic data of study groups

Variables	Control ( $n=$ 10)	LC ( $n=$ 20)	HCC ( $n=$ 20)
Sex M: F	5 (50%):	13 (65%):	15 (75%):
	5 (50%)	7 (35%)	5 (25%)
Age (ys)	33.2 $\pm$ 8.01	56.95 $\pm$ 6.07	57.65 $\pm$ 7.74

Data was expressed in from of mean SD or frequency (percentage) as appropriate.

Table 2

The frequency of the unmethylated genes among studied groups

Genes	Control ( $n=$ 10)	LC ( $n=$ 20)	HCC ( $n=$ 20)
E-cad	10 (100%)	20 (100%)	18 (90%)
P14	10 (100%)	20 (100%)	20 (100%)
APC	10 (100%)	6 (30%)	18 (90%)

Table 3

The frequency of methylated genes among participants of the study

Genes	Control	LC	HCC	$P1$	$P2$	$P3$
	( $n=$ 10)	( $n=$ 20)	( $n=$ 20)
E-cad	3 (30%)	7 (35%)	11 (55%)	0.9	0.4	0.3
P14	0 (0%)	0 (0%)	9 (45%)	–	0.03	0.01
APC	2 (20%)	5 (25%)	14 (70%)	0.9	0.03	0.01

Data was expressed in from of frequency (percentage). P value was significant if $<$ 0.05. P1: comparison between control and LC. P2: comparison between control and HCC. P3: comparison between LC and HCC.

3.2 Results of artificial methylation

E-cad methylation showed non-significant differences between the studied groups. P14 methylation was occurred only in HCC-group (45%). APC methylation was the highest in HCC group (70%) with a significant differences between LC ( $P=$ 0.03) and control group (0.01). details are shown in Table 3.

Table 4
Comparison among the 3 study groups as regard level of methylated DNA quantification

	Control ( $n=$ 10)	LC ( $n=$ 20)	HCC ( $n=$ 20)	$P1$	$P2$	$P3$
Epigenetics (ng/ml)	1.71 $\pm$ 0.77	2.63 $\pm$ 0.36	5.48 $\pm$ 1.91	0.069	$<$ 0.001	$<$ 0.001

Data was expressed in from mean $\pm$ SDP value was significant if $<$ 0.05. P1: comparison between control and LC. P2: comparison between control and HCC. P3: comparison between LC and HCC.

Table 5

Sensitivity, specificity, PPV, NPV and accuracy of global DNA methylation for prediction of HCC

	AUC	Cutoff	Sensitivity	Specificity	PPV	NPV	Accuracy
Control group	0.93	$>$ 2.361	80%	90%	94.7%	66.7%	85%
LC group	0.93	$>$ 2.985	95%	90%	90.5%	94.7%	92.5%

AUC; Area under the curve, PPV; positive predictive value, NPV; negative predictive value, LC; liver cirrhosis, HCC; hepatocellular carcinoma.

Figure 2.

continued. (c) Gel electrophoresis of the MSP products of APC gene.

Figure 3.

ROC for prediction of HCC in patients with LC and in control.

3.3 Diagnostic yield of the quantification of DNA methylation level

A high level of methylation was detected in HCC group in comparison to both LC and control groups ( $P<$ 0.001) and also a significant high difference between LC and control ( $P=$ 0.069) (Table 4). For prediction of HCC, The level of DNA methylation at a cutoff point $>$ 2.9 ng/ml had 90% sensitivity and 80% specificity for prediction of HCC in patients LC-group while at a level $>$ 2.3 ng/ml had 95% sensitivity and 90% specificity for prediction of HCC in control-group (Table 5, Fig. 3).

Using the three methylated genes together as a biomarker for detection of HCC, the sensitivity, specificity, positive and negative predictive values and accuracy were 90%, 60%, 69.2, 85.7 and 75% respectively (Table 6).

Table 6
Sensitivity, specificity, positive and negative predictive values and accuracy of the methylation of the 3 studied genes

	Sensitivity	Specificity	PPV	NPV	Accuracy
E-cad	55%	65%	61.1	59.1	60%
P14	45%	100%	100.0	64.5	72.5%
APC	70%	75%	73.7	71.4	72.5%
Combined	90%	60%	69.2	85.7	75%

PPV; positive predictive value, NPV; negative predictive value.

4. Discussion

All epigenetic, proteomic and genomic studies evolve in a complimentary fashion with each other in the field of molecular pathology. While genomics and proteomic studies are related to the genetic structural changes, epigenetics are related to the changes in gene function without structural mis-sequencing and refer to the changes in DNA e.g. methylation, dysregulation of histone patterns, remodeling, and aberrant micro-RNAs [18, 19]. The interplay between the chronic HCV infection and its subsequent cirrhosis and HCC is still a worthy area for further research and exploration. Many reports investigated this molecular interplay and found a strong relation between HCV and the occurrence of HCC [20, 21].

All previous researches investigated the methylation arrays between normal liver and the different HCC stages and stated the potential role of DNA methylation as a crucial step in hepatocarcinogenesis [22, 23]. A wide list of genes found to be incorporated in this DNA aberration. So, we aimed to assess the frequency of DNA methylation in three nominated genes (APC, P14, and E-cad genes) in well-defined patient groups.

In our study, APC gene was found with unmethylated expression in 30% of cirrhotic liver without HCC and in with HCC, both E-cad and APC genes were unmethylated in 90% of cases. But after artificial methylation, as found in other studies, APC and P14 gene methylation were the highest among HCC patients [24]. This may convey the importance of studying the potential role of APC and P14 in cirrhotic patients to list them as a precancerous insult. Zekri et al., studied the hyper-methylation of 11 genes and found that P14, P73, APC, E-cad, and O6MGMT can independently affect progression of cases to HCC with 89% accuracy, 83.9% sensitivity and 94.7% specificity [25]. Also, in the study of Yang et al., they reported a high frequency of promotor methylation in HCC patients with also a slightly similar array of genes such as SOCS-1, GSTP, APC, E-cadherin, and p15 [26].

From our study, the quantification assessment of DNA methylation with ELISA-epigenetic kits revealed a high level of methylation in HCC group in comparison to both cirrhotic and control groups ( $P<$ 0.001). Also, its quantification a cutoff point $>$ 2.9 ng/ml had 90% sensitivity and 80% specificity for prediction of HCC in patients LC-group while at a level $>$ 2.3 ng/ml had 95% sensitivity and 90% specificity in control-group. To the best of our knowledge, there is no published study used quantified levels of DNA methylation in predication of HCC among high risk groups. This novel method may serve a benchmark in the future implications of epigenetic studies. Also, when using the pooled methylation levels of the three genes together as a biomarker for detection of HCC, it has shown a considerable sensitivity of 90% and specificity of 60% with a relatively high accuracy rate of 75%. Screening of epigenetic changes in high risk patients may help to reverse the event towards cancer prevention [27]. As well-established, epigenetic aberrations are now known to be by many of demethylating agents that was tried in therapeutic trials e.g. using 5’Azacitidine and Decitabine with promising results [28, 29].

From our point of view, our study may carry some limitations. First, the sample size is relatively small. Second, the studying of the epigenetic array was carried out in serum rather than in tissue; which is known as a gold-standard standard for methylation studies. This point was solved by many recent studies that consider the efficacy of conducting DNA methylation studies using plasma DNA is similar to that of tissue [30, 31]. Moreover, and in concordance to the recent guidelines, imaging studies alone are quite enough for establishing HCC diagnosis and hence the tissue acquisition for academic studies being so difficult.

In conclusions, aberrant DNA promotor methylation of multiple cancer related genes is associated with different stages of disease progression from HCV related cirrhosis to HCC. Moreover, detection of promotor methylation of certain genes in peripheral blood may sever as good biomarker for early detection of HCC in high risk patients.

Footnotes

Abbreviations

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