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Clinical Cancer Research 13, 944-952, February 1, 2007. doi: 10.1158/1078-0432.CCR-06-2268
© 2007 American Association for Cancer Research
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Imaging, Diagnosis, Prognosis

CpG Island Methylator Phenotype Association with Elevated Serum {alpha}-Fetoprotein Level in Hepatocellular Carcinoma

Changsong Zhang1,2, Zhengyou Li1,3, Yue Cheng1, Fengqi Jia1, Rong Li1, Mengchao Wu1, Ke Li2 and Lixin Wei1

Authors' Affiliations: 1 Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China; 2 Department of Cancer Molecular Epidemiology, Shantou University Medical College, Shantou, Guangdong, China; and 3 Biology College, Shandong Normal University, Jinan, Shandong, China

Requests for reprints: Lixin Wei, Tumor Immunology and Gene Therapy Center, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, 225 Changhai Road, Shanghai 200438, China. Phone: 86-21-25070855; Fax: 86-21-35030398; E-mail: lixinwei{at}smmu.edu.cn.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: CpG island methylator phenotype (CIMP) involves hypermethylation targeted toward the promoters of multiple genes. To gain insight into the role of epigenetic aberration of tumor-related genes in hepatocarcinogenesis, we determined a hypermethylation profile in hepatocellular carcinoma (HCC).

Experimental Design: We examined the promoter methylation status of nine genes in 50 HCCs, 50 paired nontumor tissues, and 6 normal liver tissues by methylation-specific PCR. CIMP+ was defined as having five genes that are concordantly methylated.

Results: The frequency of promoter methylation of nine genes in 50 HCCs varied from 10% in P53 to 94% in c-Myc. The methylation status of P14, P15, P16, ER, RASSF1A, WT1, and c-Myc was significantly correlated with HCC and nontumor tissues (P < 0.05). Hypermethylation of one or more genes was found in 96% of HCC. CIMP was more frequent in HCC than in nontumor tissues (70% and 12%, P < 0.001). There is a significant association between CIMP and methylation of P14, P15, P16, ER, RSAAF1A, and WT1 (P < 0.05) and serum {alpha}-fetoprotein (AFP) level (P = 0.017). CIMP+ was more frequent in HCC with AFP ≥ 30 µg/L than those with AFP < 30 µg/L (P = 0.005). In addition, the promoter hypermethylation of P15 and P16 was associated with elevated serum AFP levels in 35 HCC samples with CIMP+ (P < 0.05).

Conclusions: Positive correlation of CIMP and AFP levels in HCC suggests that CIMP can serve as a molecular marker of late-stage HCC development.

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, and the prognosis of patients with HCC is very poor (1). {alpha}-Fetoprotein (AFP), a tumor marker for HCC, is highly expressed during fetal liver development, but is rapidly repressed within a few weeks after birth. Approximately 70% of HCC are positive for AFP, and levels of AFP increase with tumor progression (2). Expression of AFP in the adult liver is reactivated only in cases of renewed cellular proliferation, as occurs during liver regeneration and HCC (3, 4).

Promoter hypermethylation has been found to frequently occur in tumor suppressor and cancer genes involved in many different signaling pathways and to be present in a different pattern in different tumor types (5–8). Many individual genes have been reported to be abnormally methylated in HCC (9–11). Although it is important to analyze multiple genes, to have an overall picture of epigenetic alterations, there are only limited reports on such analysis in HCC (12–14). The methylation pattern of multiple genes can provide different types of useful information about the cancer cell (15). The hypermethylated subtype in tumors, also called the CpG island methylator phenotype (CIMP), in which multiple genes are concurrently methylated, is a novel marker for tumor progression and has been shown in previous research papers (16, 17). The methylator phenotype concept is important because it implies an underlying defect in the cellular machinery responsible for the generation of hypermethylation events (16–18). A new study taking an unbiased approach strongly concludes that a CIMP exists and offers new markers of cancer (19).

In this study, promoter methylation of P14 (p14ARF; ref. 20), P15 (p15INK4b; ref. 20), P16 (p16INK4a; ref. 20), P53 (21), RB1 (retinoblastoma 1; ref. 22), ER (estrogen receptor; ref. 23), WTI (Wilms tumor 1; ref. 14), RASSF1A (Ras-association domain family 1; ref. 24), and c-Myc (25) were studied in HCC because they have been found to be prone to high-frequency methylation in many malignancies. The aim of the present study was to evaluate the prognostic significance of the CIMP status and the relation between the CIMP and the serum AFP level in HCC. Promoter methylation was analyzed for the nine tumor-related genes above; we examined whether CIMP correlated with clinical features to clarify the difference between the HCC with CIMP+ and those with CIMP–.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and specimens. Tumor and corresponding noncancerous liver tissue specimens were obtained from 50 patients with HCC whose frozen tumor samples had been stored in the surgical pathology files of our hospital between August 2004 and October 2005. The frozen tissue samples were flash frozen in liquid nitrogen immediately after surgical resection until analysis.

The patients consisted of 44 men and 6 women, ranging in age from 33 to 74 years (mean ± SD, 51.66 ± 10.13 years). The diagnosis was confirmed histologically in all cases, based mainly on the examination of sections stained with H&E. All tumors were histologically diagnosed as HCC according to the Edmondson-Steiner classification system (ref. 26; 48 cases were grade III, 2 cases were grade IV). Of the 50 patients, 41 had cirrhosis. Serologic examinations indicated that 34 were both hepatitis B surface antigen positive and hepatitis C virus antibody (anti-HCV) positive. Written informed consent was obtained from each patient, and the protocol of the study was approved by the local ethics committee.

Sodium bisulfite treatment. Nontumor and tumor samples were digested overnight at 50°C with proteinase K buffered in 1% SDS (pH, 8). Genomic DNA of tumor or normal tissues was isolated by phenol-chloroform extraction and ethanol precipitation. DNA was treated with sodium bisulfite as described previously (27). Briefly, 2 µg of genomic DNA was resuspended in 50 µL of water and then denatured in 2 mol/L NaOH for 10 min at 37°C. The denatured DNA was diluted in 550 µL of freshly prepared solution containing 10 mmol/L hydroquinone (Sigma-Aldrich, St. Louis, MO) and 3 mol/L sodium bisulfite (Sigma-Aldrich). The resultant solution was covered with mineral oil and incubated for 16 h at 50°C. After incubation, the samples were desalted using a Wizard DNA Clean-Up System (Promega, Madison, WI) and treated with 3 mol/L NaOH for 5 min at room temperature. Then, 66 µL of NH4Ac and two volumes of 100% ethanol were added, and the DNA was precipitated for at least 1 to 2 h at –80°C. After precipitation, the pellets were washed with 70% ethanol, dried, resuspended in 20 µL of water, and stored at –20°C.

Methylation-specific PCR. DNA methylation was determined by methylation-specific PCR (MSP) (27). MSP distinguishes unmethylated alleles of a given gene based on DNA sequence alterations after bisulfite treatment of DNA, which converts unmethylated but not methylated cytosines to uracils. Subsequent PCR using primers specific to sequences corresponding to either methylated or unmethylated DNA sequences is then done. Primers for MSP were reported previously (14, 20–25). Primer sequences are summarized in Table 1 . The modified genomic DNA samples were PCR amplified in a total volume of 50 µL. The PCR was done in a thermal cycler. The PCR products were separated on 2% agarose gels and visualized using ethidium bromide staining.


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  		Table 1. Primer sequences for MSP analysis

 
Statistical analysis. All data were generated without the knowledge of the clinical status of the samples analyzed. Values for the clinical and biological characteristics of patients were expressed as means ± SD. Comparison was done with Student's t test (unpaired). Univariate analyses of the interaction between methylation and clinical parameters were done with Pearson's {chi}2 test, and the Fisher's exact test was used to examine the tissue samples with the low expected values. All P values presented were two sided, and a P value of <0.05 was considered statistically significant. All statistical analyses were carried out using SPSS 13.0 software (SPSS, Inc., Chicago, IL).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Frequency of CpG island hypermethylation in HCC, paired nontumor, and normal liver tissue. We examined the hypermethylation status of a panel of nine tumor-related genes: P14, P15, P16, P53, RB1, ER, WTI, RASSF1A, and c-Myc. The frequency of promoter methylation of the nine genes was determined using MSP in 50 cases of HCC, 50 cases of paired nontumor tissues, and 6 normal liver tissues. For the methylation status assay (MSP) results, the hypermethylated contains only the methylated PCR product, the partially methylated contains both methylated and unmethylated PCR products, and the unmethylated contains only the unmethylated product. Representative examples of MSP assay results are presented in Fig. 1 , and overall results are summarized in Fig. 2 and Table 2 . The frequency of promoter methylation of the gene included in the panel was P14, 88%; P15, 26%; P16, 50%; P53, 10%; RB1, 20%; ER, 60%; RASSF1A, 88%; WTI, 46%; and c-Myc, 94% of 50 HCCs. The frequency of promoter methylation of the gene was much lower in both nontumor and normal liver tissues (not including P14). There was no methylation of P15, P53, RB1, RASSF1A, WT1, and ER in normal liver tissues. The methylation status of P14, P15, P16, ER, RASSF1A, WT1, and c-Myc was significantly associated with HCC and nontumor tissues (P = 0.029; P = 0.047; P = 0.001; P = 0.010; P < 0.001; P < 0.001; and P = 0.035, respectively). Methylation was also more frequent in HCC than in nontumor tissues for P53 (10% versus 4%) and RB1 (20% versus 8%), but these changes were not statistically significant (P = 0.512, and P = 0.245, respectively).


Figure 1
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  		Fig. 1. Representative MSP experiments for methylation analysis of nine genes. PCR products amplified with unmethylated (U) and methylated (M) sequence-specific primers. Distilled water was used as negative control, DNA methylated by SssI methylase (Sss DNA) was used as positive control (pos). HCC samples (T); paired nontumor tissues (N); normal liver tissue (106). Left, gene names; Ras, RASSF1A.

 

Figure 2
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  		Fig. 2. Summary of methylation of P14, P15, P16, P53, RB1, ER, RASSF1A, WT1, and c-Myc in 50 HCC samples (A), 50 paired nontumor tissues (B), and 6 normal liver tissues (B). Filled boxes, presence of methylation; open boxes, presence of unmethylation; shaded boxes, presence of methylation and unmethylation. T, HCC samples; N, paired nontumor tissues. The numbers 101-106 were normal liver tissues. Ras, RASSF1A; Myc, c-Myc; +, CIMP-positive; –, CIMP-negative.

 

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  		Table 2. Frequency of tumor-related gene promoter methylation in HCC and nontumor tissues and its correlation with CIMP status in HCC

 
Then, we examined methylation with regard to associations between methylation of one particular gene and methylation of another gene in HCC (Table 3 ). When comparing hypermethylation of the nine genes, we found concordant hypermethylation of the P14 and RASSF1A (P = 0.001), c-Myc (P = 0.035); the methylation of P16 was associated with the methylation of P15 (P = 0.004), ER (P = 0.004), and WT1 (P = 0.047); methylation of P15 was associated with the methylation of ER (P = 0.035). In addition, we found concordant hypermethylation of the RASSF1A and c-Myc (P = 0.035). Our present data show that promoter methylation of some tumor-related genes was hypermethylation in accordance with other genes in HCC.


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  		Table 3. Association of methylation among tumor-related genes in HCC

 
Frequency of CIMP in HCC, nontumor, and normal liver tissue. We examined the promoter methylation status using MSP in nine genes and found that 96% (48:50) of HCC showed methylation of at least one gene, and only two tumors showed no methylation of any of the nine genes. A total of 2% (1:50) of HCC had one gene, 2% (1:50) had three genes, 22% (11:50) had four genes, 40% (20:50) had five genes, 24% (12:50) had six genes, and 6% (3:50) had seven genes hypermethylated (Fig. 3A ). The mean number of genes hypermethylated in each tumor was 4.82 (SD, 1.45), and the median number was 5.0. Meanwhile, a total of 4% (2:50) of paired nontumor samples had one gene, 28% (14:50) had two genes, 32% (16:50) had three genes, 20% (10:50) had four genes, 6% (3:50) had five genes, 4% (2:50) had six genes, and 2% (1:50) had seven genes hypermethylated (Fig. 3B). The mean number of genes hypermethylated in each nontumor was 3.4 (SD, 1.99), and the median number was 3.0. However, only one normal liver tissue had two genes hypermethylated in six normal liver tissues (Fig. 3C).


Figure 3
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  		Fig. 3. Number of promoter methylated genes per sample in 50 HCCs (A), 50 paired nontumor tissues (B), and 6 normal liver tissues (C), and distributions frequency of the CIMP in HCC, nontumor tissue, and normal liver tissue (D). CIMP is defined by the number of CpG islands methylated in each sample as follows: CIMP– (less than five methylated genes); CIMP+ (more than five methylated genes). CIMP+ was observed in 70% (35:50) HCC, 12% (6:50) paired nontumor tissues, and none of normal liver tissues (0:6). CIMP status is strongly associated with HCC, nontumor tissues, and normal liver tissues (P < 0.001).

 
Originally, CIMP-positive gastric cancer was defined as a tumor with methylation at more than three genes (17). Recently, CIMP was defined by the average number of methylated genes per tumor (28, 29). A new study offered new markers to define a CIMP concept, in which the threshold distinguishing CIMP+ from CIMP– samples was chosen by minimizing the within-group sum of squared errors (19). In this study, CIMP status was classified as CIMP+ samples (with five or more methylated genes) and CIMP– samples (with four or fewer methylated genes).

We investigated CIMP status in 50 HCCs, 50 corresponding nontumor tissues, and normal liver tissues. Of the HCCs, 70% (35:50) were CIMP+, 30% (15:50) were CIMP–. In contrast, CIMP+ was present in 12% (6:50) of corresponding nontumor tissues; CIMP– was 88% (44:50). Meanwhile, no CIMP+ was present in normal liver tissues (Fig. 3D). There was a statistically significant difference between CIMP status with different samples, including HCC, nontumors, and normal liver tissues (P < 0.001).

Association between CIMP and promoter methylation of tumor-related genes. To determine whether CIMP indeed affects the methylation status of known tumor-related genes important to tumor progression, promoter methylation status was analyzed for each gene, including this study. Of 35 CIMP+ HCC cases, the frequency of promoter hypermethylation of each gene was P14, 97%; P15, 37%; P16, 63%; P53, 6%; RB1, 23%; ER, 77%; RASSF1A, 97%; WTI, 54%; and c-Myc, 97% (Table 2). It was shown that the methylation of promoter CpG islands of P14, P15, P16, ER, RSAAF1A, and WT1 was far more frequently observed in HCC samples with CIMP+ than those with CIMP–, and there was a significant association between the methylation status in these genes and the CIMP status in HCC (P = 0.013; P = 0.023; P = 0.017; P = 0.001; P = 0.002; and P = 0.029, respectively). At the same time, promoter methylation of the P53 gene was less frequently observed in CIMP+ than in CIMP– (6% versus 20%) in HCC.

CIMP correlates with serum AFP level. Using statistical analysis, we examined CIMP with regard to HCC patient clinicopathologic parameters of age, gender, tumor size, smoking history, drinking alcohol, hepatitis B virus (HBV), HCV, cirrhosis, serum AFP, node number, and metastasis (Table 4 ). A significant difference between CIMP status and serum AFP level was being found in HCC (P = 0.017). The average level of serum AFP in CIMP+ was 442.87 ± 464.94 µg/L, and the average level of serum AFP in CIMP– was 142.62 ± 348.22 µg/L (Fig. 4A ). Meanwhile, we found that CIMP+ was more frequent in HCC, with AFP ≥ 30 µg/L (62.9%, 22:35) than those with AFP < 30 µg/L (37.1%, 13:35; P = 0.005, Fig. 4B). Finally, there was no statistical difference between CIMP+ and CIMP– cases in age, gender, tumor size, smoking history, drinking alcohol, HBV, HCV, cirrhosis, node number, and metastasis.


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  		Table 4. Association among CIMP status and clinicopathologic variables in HCC

 

Figure 4
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  		Fig. 4. Distribution of serum AFP levels of 50 HCC patients according to CIMP status (A). The average level of serum AFP in CIMP+ and CIMP– were 442.87 ± 464.94 and 142.62 ± 348.22 µg/L (P = 0.017), respectively. There is an association between CIMP status and serum AFP cutoff value (30 µg/L; P = 0.005; B).

 
Elevated serum AFP level correlates with the methylation status of six genes related to CIMP. We analyzed serum AFP level associated with the methylation status of six genes involved in CIMP, including P14, P15, P16, ER, RSAAF1A, and WT1. Interestingly, we found that the promoter of P15 and P16 shows more frequency hypermethylation in HCC patients with elevated serum AFP level (Table 5 ). In 35 HCC samples with CIMP+, 92.3% HCC patients with P15 hypermethylation showed elevated serum AFP levels (AFP ≥ 30 µg/L), as compared with only 45.5% of HCC patients with P15 unmethylation, showing elevated serum AFP levels (P = 0.01). Of the HCC patients with P16 hypermethylation, 86.4% showed elevated serum AFP levels, as compared with only 23.1% of HCC patients with P16 unmethylation showing elevated serum AFP levels (P < 0.001). There is a statistically significant difference between the methylation status of P15 and P16 and serum AFP level in HCC (P < 0.05). Therefore, positive correlation of CIMP and AFP levels in HCC suggests that CIMP can serve as a molecular marker of late-stage HCC development.


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  		Table 5. The methylation status of six genes related CIMP+ associated with serum AFP level in HCC samples with CIMP+

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
HCC ranks as the fifth most common cancer in the world, and most HCCs are associated with chronic HBV or HCV infections (1, 30). The development and progression of HCC is a multiple-step process, the understanding of the molecular pathways of hepatocarcinogenesis is limited, although recent molecular biological studies have led to rapid progress in the understanding of the molecular events involved. Epigenetic silencing of tumor-related genes due to the methylation of gene promoter regions plays an important role in carcinogenesis in HCC (12, 14, 31).

We have investigated the methylation profile of nine tumor-related genes and found that this CIMP is associated with the expression of AFP in 50 cases of HCC. The frequency of promoter methylation of the genes examined varied from 10% for P53 to 94% for c-Myc. We show that 96% HCC have at least one gene methylation and the concordant promoter methylation of P14, P15, P16, ER, RASSF1A, WT1, and c-Myc in HCC. These results suggested that the simultaneous hypermethylation of multiple genes may be important for tumorigenesis.

The recently described hypermethylator phenotype in colorectal cancer termed CIMP affects about half of all cases (16). The term CIMP has been used for a subset of tumors with promoter methylation in multiple genes, including colorectal, pancreatic, esophageal, and stomach tumors (16, 17, 19, 28, 32, 33). The prognostic roles of the CIMP status have been evaluated in several types of cancers. In colorectal cancer, CIMP-positive phenotype was associated with distinct clinicopathologic features, like tumor location, female sex, age, and others (34). In esophageal adenocarcinoma, CIMP was associated with poor prognosis (35). Meanwhile, the evaluation of a large, population-based sample strongly supports the biological relevance of CIMP in colon cancer (36).

However, the prognostic role of CIMP status in HCC is unclear, and there is little information about the prognostic significance of concordant gene methylation. In this study, we found that HCC patients with CIMP+ had a relatively higher serum AFP level when compared with CIMP– tumors (P = 0.017). HCC with high levels of AFP have poor prognosis and exhibit multicentric tumor lesion growths more frequently than AFP-negative HCC (37–39). In the present study, we found that CIMP+ was more frequent in HCC, with AFP ≥ 30 µg/L than those with AFP < 30 µg/L (P = 0.005). There was no statistically significant association between CIMP status and age, gender, tumor size, and other features.

Meanwhile, of the 35 HCC samples with CIMP+, we found that the promoter hypermethylation of P15 and P16 was associated with an elevated serum AFP level. Renewed AFP expression occurs only when the differentiated liver exits G0 and enters a program of resumed cellular proliferation, as a consequence of hepatic tumorigenesis, liver regeneration, or tissue damage due to chronic disease (40). The loss of component in the p16/p15/pRB pathway is frequently found in tumor progression (41, 42). The functional significance of P15 and P16 methylation could be an initiating event leading to the progressive inactivation of the cell cycle regulatory genes. A significant association between P16 methylation status in circulating DNA and the preoperative serum AFP concentration has been reported (43). Impaired P15 and P16 expression might be involved in regulating AFP expression through the inactivation of the cell cycle pathways. Functional inactivation of P15 and P16 mediated by promoter methylation may be required for the aberrant expression of AFP during hepatocarcinogenesis.

Recent studies have shown that AFP gene expression is repressed by the P53 family member in chromatin modification (44), and P53 repression AFP transcription through site-specific DNA binding within the AFP repressor domain (45). Alternatively, HNF3 binding in the AFP control region could influence nucleosome positioning and, therefore, modulate the accessibility of factors to sites within the "A" fragment, leading to a reduction in AFP promoter activity (46). The basis for this repression is not well understood, and specific trans-acting factors that govern this repression have not been identified. Our studies raise the possibility that P15 and P16 may also be involved in AFP repression. However, little is currently known about the role of P15 and P16 involved in AFP expression. Further study is necessary to clarify the link between AFP expression and methylation of P15 and P16 in hepatocarcinogenesis, and global analysis will be of increasing importance in the identification of novel hypermethylated genes in HCC.


   Acknowledgments
 
We thank Dr. Jingde Zhu for providing invaluable technical assistance, Dr. Jia He for the expert help in statistical analysis, and Hongwei Sun and Yijun Zhao for providing the patient samples.


   Footnotes
 
Grant support: Special funds for the Major State Basic Research Program of China (grant G20000057001), National Natural Science Foundation of China (grant 30471994), and the Commission of Science and Technology of Shanghai Municipality (grant 04DZ14006).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: C. Zhang and Z. Li contributed equally to this work.

Received 9/12/06; revised 10/24/06; accepted 11/16/06.


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 Materials and Methods
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