Promoter methylation of CDKN2A and lack of p16 expression characterize patients with hepatocellular carcinoma

Tumor and corresponding non-neoplastic liver tissue specimens were obtained from 85 patients who underwent surgical resection. Tissue samples were stored at - 80°C until analysis.

Patients with HCC consisted of 28 men and 5 women ranging in age from 42 to 82 (median 67 ± 11.5) years. Mean tumor size was 75.1 ± 43.4 mm (range 15 to 230 mm). HCC samples were categorized according to differentiation into well (G1; seven cases), moderately (G2; nineteen cases), or poorly (G3; seven cases) differentiated types, which correspond to Edmondson's Grades I/II, III, and IV [21, 22]. One patient (HCC patient No. 10) had a hepato-cholangiocellular carcinoma (Table 1). Regular intake of more than 60 g alcohol per day over a period of more than five years was documented in ten patients. Hereditary hemochromatosis was diagnosed in one, and chronic hepatitis C was found in ten patients. One patient (HCC patient No. 22) had a coinfection of HBV and HCV. Antibodies against HBV core protein were found in two patients (HCC patients No. 4 and No. 7) without evidence of viral replication. 13 tumors were classified as cryptogenic HCC (Table 1). Locally advanced disease was found in five cases at the time of liver resection. Alfa fetoprotein (AFP) serum level was available from 21 patients, ranging from 1.3 ng/ml to 95468 ng/ml (normal range < 7.0 ng/ml). In ten patients AFP level remained normal during follow-up. Advanced liver fibrosis or liver cirrhosis (fibrosis stages F3 to F6) according to the Ishak's scoring system [23] was confirmed in twenty-three patients (Table 1).

Tissue samples were obtained from 11 patients (4 men, 7 women) with adenocarcinoma of biliary tree and gall bladder with a mean age of 60.3 ± 8.1 years (range from 42 to 73 years) and graded as well (G1; one case), moderately (G2; eight cases), or poorly (G3; two cases) differentiated tumors. Two patients (CCC patients No. 9 and No. 10) developed advanced liver fibrosis (Table 2).

Samples from seven patients (1 man, 6 women) with a benign liver tumor, i.e. focal nodular hyperplasia (n = 6) or liver adenoma (n = 1) were also studied (Table 2). Age of patients ranged from 22 to 47 (mean 40 ± 6) years. Advanced liver fibrosis was not detected in this patients' group.

Tissue samples were also obtained from 34 patients (21 men, 13 women) with liver metastasis of colorectal (n = 29), gastric (n = 2), renal (n = 1), and breast (n = 2) cancers. Age of patients ranged from 32 to 82 (median 63.9 ± 8.2) years. Liver metastases were also categorized according to differentiation into well (G1; two case), moderately (G2; twenty-five cases), or poorly (G3; seven cases) differentiated carcinomas (Table 3). Advanced liver fibrosis (Ishak's stages F3 to F6) [23] was found only in one non-cancer liver tissue (M7) (Table 3).

Only tissue slides were available for immunohistochemical analysis from further 23 patients [HCC (n = 15) (Table 1) and chronic hepatitis C (n = 8)]. All the 15 HCC patients had a HCV-related chronic liver disease, and four consumed also regularly alcohol. Core antibodies against HBV were present in two patients. Liver cirrhosis was confirmed in six samples.

The study was approved by the Ethics Committee of the Otto-von-Guericke University of Magdeburg.

Genomic DNA was extracted using proteinase K digestion and was modified by sodium bisulfit (CpGenome™ DNA Modification Kit, Q-Biogene Heidelberg, Germany) as reported previously [24, 25].

Genomic DNA was analyzed by MethyLight technique after bisulfite conversion as reported previously [19, 20]. Briefly, three oligonucleotids were used in every reaction. Two locus-specific PCR primers flanked an oligonucleotide probe with a 5' fluorescent reported dye (6FAM) and a 3'quencher dye (BHQ-1). Primer and probe sequences for CDKN2A promoter (GeneBank accession number GI 21886808) were: forward primer (bp 20012-20033) 5'-tgg-agt-ttt-cgg-ttg-att-ggt-t-3'; reverse primer (bp 20081-20060): 5'-aac-aac-gcc-cgc-acc-tcc-c-3'; oligonucleotide probe sequence (5'-3') (bp 20060-20043): 6FAM-acc-cga-ccc-cga-acc-gcg-BHQ-1. PCR reaction resulted in a 70-bp product including 8 CpG islands localized on the first exon of the gene. The gene of interest was then amplified and normalized to a reference set (β-actin = ACTB) to normalize for input DNA. The specificity of reactions for methylated DNA was confirmed using CpGenome Universal Methylated DNA [(Chemicon International Inc., CA, USA (subsidiary or Serologicals) catalog #S7821)]. TaqMan PCR reactions were performed in parallel with primers specific for the bisulfite-converted methylated sequence for a particular locus and with ACTB reference primers. The ratio between values was calculated in these two TaqMan analyses, using this approach the degree of methylation at that locus was determined. The extent of methylation at a specific locus was determined by the following formula: [(gene/actb)^sample: (gene/actb)^{SssI-treated genomic DNA}]* 100. Samples with ≥ 4% of PMR (percentage of methylated reference) were regarded as hypermethylated.

The methylation status of CDKN2A was also evaluated by MSP as described previously[18, 26]. In brief, DNA was subjected to bisulfit modification and was amplified using two primers specific for either methylated or unmethylated sequences [18]. Primer sequences for unmethylated reactions were sense: 5'-tta-tta-gag-ggt-ggg-gtg-gat-tgt-3' and anti-sense: 5'-caa-ccc-caa-acc-aca-acc-ata-a-3', which amplified a 151 bp product. Primer sequences for methylated reactions were sense: 5'-tta-tta-gag-ggt-gcg-gat-cgc-3 and anti-sense: 5'-gac-ccc-gaa-ccg-cga-ccg-taa-3', which amplified a 150 bp product. CpGenome™ Universal Methylated DNA (Q-Biogene, Heidelberg, Germany) was used as positive control. DNA from normal lymphocytes served as negative control for methylated alleles. PCR was performed as described previously [26]. PCR products were run on polyacrylamide gels, followed by silver-staining.

p16 expression was studied on formalin-fixed and paraffin embedded tissue samples. Briefly, 3-5 μm thick tissue sections were deparaffinized in xylol and rehydrated in a graded alcohol series. Endogenous biotin was blocked using the Avidin/Biotin Blocking kit (2 × 15 minutes; Vector Laboratories, Inc., Burlingame, CA). Immunostaining was performed with polyclonal anti-p16 antibody directed against the entire region of the human p16 protein (1:100; Quartett, Berlin, Germany). Incubation with the primary antibody was performed in a moist chamber at 37°C for 1 hour. Rabbit-anti-mouse IgG (30 minutes, room temperature; Vector Laboratories Inc.) served as a secondary antibody. The immunoreaction was visualized with an streptavidin-biotin complex, using the Vectastain ABC alkaline phosphatase kit (distributed by CAMON, Wiesbaden, Germany). FastRed (Zytomed, Berlin, Germany) served as chromogen. The specimens were counter-stained with hematoxylin. Omission of the primary antibody served as a negative control.

For quantification of immunohistochemical results, a numerical scoring system was applied. Only nuclear staining was considered as positive. The observed expression of p16 in hepatocytes and tumor cells was assessed using two categories. Category A documented the number of immunoreactive cells as 0 (no reactive cells), 1 (< 10%), 2 (11 to 50%), and 3 (> 50%). Category B documented the intensity of immunostaining as 0 (no immunostaining), 1 (weak), 2 (moderate), and 3 (strong). Finally, values for category A and B were added to construct an "immunoreactivity score" (IRS) ranging from 0 to 6.

PMR (percentage of methylated reference) values of Methylight assays were dichotomized for statistical purposes as reported previously [19, 20]. PMR values of tumor and non-tumor samples were compared by Kolmogorov-Smirnov test. Differences in promoter methylation rate were analysed between cancer and non-tumor tissues using Fisher's exact test. All statistical tests were two-sided, with p < 0.05 considered statistically significant difference.

Using Methylight assay, methylation status of 8 CpG islands of the first exon of CDKN2A was evaluated in 33 HCCs and the corresponding non-tumor liver samples. The mean PMR of HCC samples was 31.41 ± 27.63% (range 0.01 to 100%) (Table 1). Twenty-three patients with HCC (69.7%) showed a PMR > 4% and were regarded as hypermethylated. The CDKN2A hypermethylation positive HCCs showed a mean PMR of 42.34 ± 27.8% (range 7.91 to 100%) (Table 1). A methylation of CpG islands was detected in 4 (57%) of 7 well-differentiated, 15 (79%) of 19 moderately differentiated, and 4 (57%) of 7 poorly differentiated HCCs. The frequency of promoter hypermethylation between patients with non-HCV induced HCC and those with HCV-induced HCC was not different (69% vs. 70%; ns). The mean PMR of the corresponding non-cancer liver tissues was 1.15 ± 1.64% (range 0.01 to 22.41). All but one (HCC patient No. 21) corresponding non-tumor samples obtained from patients with HCC were found to have an unmethylated CDKN2A promoter (Figure 1).

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Next, we assessed the frequency of CDKN2A methylation in tumor and non-tumor tissue samples of 34 patients with liver metastases (Table 3) and 11 patients with adenocarcinomas of the biliary tract and gall bladder (CCC-group) as well as from 7 patients with benign liver tumors (Table 2).

None of tissue samples with benign liver pathology obtained from patients with non-HCC (altogether 59 samples including lesional samples of patients with benign liver tumors) showed CpG methylation. Liver metastases of 6 colorectal cancers (20.7%) had a mean PMR of 39.23% (range 25.18 to 52.18%). Only one tumor sample (9%) of the CCC-group had a PMR of 25.86% (Tables 2 and 3) (Figure 1). PMR in liver metastases was comparable to those results obtained from patients with HCC.

Tumor und non-tumor tissue samples from 30 patients with HCC were studied using MSP. 30% of the non-tumor and 70% of the tumor samples were found to be hypermethylated. There was no significant difference between the HCV-induced tissue probes (80%) and those from patients with HCC not related to HCV (65%) in terms of methylation of promoter region. 17 HCC samples were hypermethylated with both methods (MethyLight and MSP). None of the MSP-positive non-tumor samples showed hypermethylation analysed by MethyLight. Altogether, about half of cancer probes (21 HCCs, 1 CCC, 6 liver metastases) were found hypermethylated in MSP analysis. Among patients with MSP-positive metastatic colorectal cancer, only 2 (patients 21 and 24) showed a methylated promoter also in MethyLight analysis (Table 3). It was of interest that 32% of the non-neoplastic liver tissue samples proved methylated in MSP analysis (Figure 2).

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148 tissue sections were obtained from 81 patients [HCC (n = 36), CCC-group (n = 8), benign liver tumors (n = 4), liver metastasis (n = 25) and chronic hepatitis C (n = 8)] and were studied by immunohistochemistry for p16 protein expression. Non-cancer samples (n = 75) showed only a weak immunoreactivity for p16 (mean IRS: 1.24) in few hepatocytes (Figures 3 and 4). While samples (n = 45) without significant liver fibrosis (F0 to F2) expressed no p16 (mean IRS: 0.36), immunostaining was significantly enhanced in samples (n = 30) with advanced liver fibrosis or cirrhosis (F3 to F6) (mean IRS: 2.6) (p < 0.01) (Figure 5). Here, p16 was mainly expressed by hepatocytes lining the portal tracts and fibrous septa as well as by cells of ductular proliferations. Nuclear p16 expression was absent in the vast majority of HCC samples (Figure 3). Only 5 samples (11%, mean IRS: 0.47) obtained from moderately differentiated HCCs expressed p16 in the nucleus. In contrast, metastatic cancer probes (23/25) showed a strong p16 expression (mean IRS: 4.9) (p < 0.01) (Figure 6). A complete loss of nuclear p16 expression was seen only in two remaining samples from patients with colorectal cancer (patients 21 and 24) (Table 3). Tissue samples obtained from patients of the CCC-group (IRS: 2.6) or with benign liver tumors (IRS: 1.75) showed only a moderate p16 immunostaining.

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The difference of CDKN2A-methylation and p16 protein expression between HCCs and liver metastases was statistically significant (p < 0.01, respectively). Using the Spearman non-parametric test, no correlation between nuclear p16 expression detected by immunohistochemical staining and CDKN2A promoter methylation using Methylight assay could be shown.