TISSUE SPECIMENS

Twenty two HCC (mean age 59.2 (SD 9.3) years; 19 males and three females; nine HBV positive and 13 HCV positive), 17 cirrhosis (58.2 (9.7) years; 15 males and two females; seven HBV positive and 10 HCV positive), 17 chronic hepatitis (44.0 (13.7) years; nine males and eight females; seven HBV positive and 10 HCV positive), nine primary biliary cirrhosis (PBC) (55.9 (11.4) years; one male and eight females), eight autoimmune hepatitis (61.9 (4.5) years; one male and seven females), seven drug induced liver disorder (45.1 (16.2) years; two males and five females), six fatty liver (46.7 (15.3) years; two males and four females), and three normal liver (29.0 (14.0) years; two males and one female) tissue specimens were obtained surgically by needle biopsy or at autopsy. HBV and HCV infections were diagnosed by HBs antigen (LPIA-200; Diatron laboratories, Tokyo, Japan) and anti-HCV antibody (Immunocheck-HCV Ab; International Reagent, Kobe, Japan), respectively. All specimens were diagnosed pathologically and frozen at −80°C.

BISULPHITE MODIFICATION

The principles of the method have been described previously.25 In the chemical modification of cytosine to uracil by bisulphite treatment, all cytosines are converted to uracil, but those that are methylated are resistant to this modification and remain as cytosine. One can design polymerase chain reaction (PCR) primers to distinguish methylated from unmethylated DNA in bisulphite modified DNA, taking advantage of the sequence differences resulting from bisulphite modification.25 Genomic DNA (2 μg) extracted from frozen tissues by the standard phenol/chloroform procedure was denatured in 0.2 M NaOH for 15 minutes. Sodium bisulphite (Sigma Chemical, St Louis, Missouri, USA) was added to a final concentration of 3.1 M, and hydroquinone (Sigma Chemical) was added to a final concentration of 0.5 mM. The reaction was performed at 50°C for 16 hours. Modified DNA was then purified using Wizard DNA purification resin (Promega, Madison, Wisconsin, USA) according to the manufacturer's protocol and eluted with 50 μl of water. DNA was treated with NaOH (final concentration 0.3 M) for five minutes at room temperature, followed by ethanol precipitation. Modified DNA was resuspended in water and used immediately or stored at −20°C.

METHYLATION SPECIFIC PCR (MSP)

Bisulphite modified DNA was subjected to MSP using primers specific for unmethylated p16(5′-TTATTAGAGGGTGGGGTGGAT TGT-3′ and 5′-CAACCCCAAACCACAAC CATAA-3′) or methylated p16(5′-TTAT TAGAGGGTGGGGCGGATCGC-3′ and 5′-GACCCCGAACCGCGACCGTAA-3′).25PCR reactions were performed in a volume of 50 μl containing 1×PCR buffer, 0.25 mM dNTP, 1 mM of each primer, and 2.5 U of Taq polymerase. PCR conditions were as follows: one cycle of 95°C for five minutes; 35 cycles of 95°C for 30 seconds, 60°C (unmethylated) or 65°C (methylated) for 30 seconds, and 72°C for 30 seconds; and one cycle of 72°C for four minutes.25 PCR samples were then electrophoresed on a 3% agarose gel.

IMMUNOHISTOCHEMISTRY

Tissue sections (4–5 μm) were deparaffinised and incubated in target retrieval solution (Dako, Carpinteria, California, USA) at the recommended dilution and heated by steam for 15 minutes. Slides were incubated overnight at 4°C with a mouse monoclonal anti-p16 antibody (Neomarkers, Union City, California, USA) diluted to a final concentration of 4 μg/ml. Negative control slides were treated with normal rabbit immunoglobulin under similar conditions. Slides were then rinsed with phosphate buffered saline and incubated with the secondary antibody using the Dako LSAB 2 System (Dako), developed with diaminobenzidine (Kanto Chemical, Tokyo) for five minutes, and counterstained with haematoxylin for one minute.

STATISTICAL ANALYSIS

Methylation of the p16 promoter was assessed for associations with clinicopathological characteristics using the χ² or Fisher's exact test. A p value <0.05 was considered statistically significant.

METHYLATION OF THE p16 PROMOTER IN VARIOUS LIVER DISEASES

Methylation of the p16 promoter was detected in 72.7% (16/22) of HCC (fig 1A), 29.4% (5/17) of cirrhosis (fig 1B), and 23.5% (4/17) of chronic hepatitis tissue samples (fig1B, fig 2, table 1). In contrast, none of the nine PBC, eight autoimmune hepatitis, seven drug induced liver disease, six fatty liver, or three normal liver tissues showedp16 promoter methylation (fig 1C, fig 2). All methylation positive tissue samples were positive for either HBV or HCV (fig 2, table 1). Positivity for p16promoter methylation was not different between HBV and HCV positive patients in HCC (7/9 v 9/13), cirrhosis (2/7v 3/10), or chronic hepatitis (1/7v 3/10). Unmethylated bands were also detected in all methylation positive cases (fig 1A, 1B). There was no significant correlation between methylation of thep16 promoter and age, type of viral infection, tumour cell differentiation, tumour size, or histological findings in HCC (table 1).

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Figure 1

Methylation specific polymerase chain reaction (PCR) analysis and expression of p16 gene. (A) Hepatocellular carcinoma (HCC) samples. (B) Liver cirrhosis (LC) and chronic hepatitis (CH) samples. (C) Primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH), drug induced liver disease (DLD), fatty liver (FL), and normal liver (NL) samples. Unmethylated and methylated indicate methylation specific PCR analysis specific for unmethylated and methylated promoter of p16, respectively. (+), normal expression of p16; (±), partial loss of p16 expression; (−), complete loss of p16 expression.

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Figure 2

Summary of the frequencies of p16 promoter methylation. The frequency of the p16 promoter methylation is expressed as a percentage. The number of positive cases per total number of cases is also shown.

p16 EXPRESSION

All normal liver tissues (fig 3A) and those with non-viral liver diseases (fig 3B–E) showed positive nuclear staining for p16. In contrast, complete or partial loss of p16 expression was observed in 90.9% (20/22) of HCC, 47.1% (8/17) of cirrhosis, and 29.4% (5/17) of chronic hepatitis cases, all of which were associated with hepatitis virus infections (fig 3F–I). Partial loss of p16 expression (fig 3F,3G) was observed in four of the five methylation positive chronic hepatitis tissues but in only one of the 13 methylation negative chronic hepatitis tissues (p=0.002) (fig 1B). One complete (fig3H) and four partial losses of p16 expression were observed in the five methylation positive cirrhosis tissues while only three of 12 methylation negative cirrhosis tissues showed partial loss of p16 expression (p=0.009) (fig 1B). Complete (fig 3I) or partial loss of p16 nuclear staining was observed in 81.8% (18/22; 8/9 HBV positive and 10/13 HCV positive) and 9.1% (2/22; both HCV positive) of HCCs, respectively. All of the 16 methylation positive cases showed complete loss of p16 expression (fig 1A, table 1), and a significant correlation was found between methylation and complete loss of expression (p=0.002).

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Figure 3

Immunohistochemistry of p16 using the mouse anti-p16 monoclonal antibody. Strong nuclear immunostaining was detected in normal liver (A), fatty liver (B), drug induced liver disease (C), autoimmune hepatitis (D), and primary biliary cirrhosis (E) samples. (F) and (G) Partial loss of p16 expression in chronic hepatitis samples. Complete loss of p16 expression in cirrhosis (H) and hepatocellular carcinoma (I) samples.

Analysis of serial samples from individual patients with methylation positive HCC revealed that loss of p16 expression with promoter methylation occurred in 18 of 20 patients at the stage of chronic hepatitis without clinically detectable carcinoma (data not shown).