The present study was performed using Tru-cut core liver biopsies from 35 patients with chronic HCV hepatitis type 4 and 35 segmentectomy specimens of hepatocellular carcinoma complicating chronic hepatitis C. The Tru-cut core biopsies were clinically indicated and were performed as a part of the pretreatment evaluation of the grade and stage of chronic HCV in patients who were candidates for combination therapy (IFN alpha and ribavirin).
HCC specimens were also obtained from excision biopsy specimens obtained from patients with hepatitis C-related cirrhosis. At least 5 sections were collected from each tumor (to account for the heterogeneity of hepatocellular carcinomas). The cirrhosis specimens were collected from the segmentectomy specimens from tissue at the farthest point from the tumor.
The protocol of the study was reviewed and approved by the Ethics Committee of the Alexandria Faculty of Medicine.
Paraffin blocks were retrieved from the archives of the Pathology Department, Alexandria Faculty of Medicine. Confirmation of the HCV etiology of the liver disease was performed using a third-generation ELISA technique, and genotype (type 4) confirmation was performed by INNO-LiPA (17
Patients who were obese, diabetic, alcoholic or HBsAb-positive were excluded from the analysis.
Sections (5 micrometers thick) were cut from paraffin blocks and stained with H&E for histopathological examination. Masson’s trichrome stain was used to evaluate the stage. The sections were also immunohistochemically stained for p21 ras using a ready-to-use polyclonal antibody (Lab Vision) (code no. RB-1627), and the sections were then stained with the universal polyclonal kit (Lab Vision, UK) (TP-015).
The sections were stained per the manufacturer’s instructions. Antigen retrieval was performed by boiling the slides in 2% citrate buffer for 10 minutes, and the sections were then incubated with the primary antibody overnight at 4 °C. Diaminobenzidine (DAB) was used as the chromogenic substrate, and the sections were counterstained with hematoxylin. Endometrioid carcinoma was used as a positive control for p21 ras; the positive controls were included in each run. Negative controls obtained by omitting the primary antibody were also included.
Positive cytoplasmic staining was graded on a scale from 0 to 3 according to the extent of staining.
- 0: no staining;
- 1: less than 30% of the biopsy showed positive staining;
- 2: between 30-59% of the biopsy showed positive staining;
- 3: greater than 60% of the biopsy showed positive staining.
The grading and staging of the liver biopsies were performed according to the hepatitis activity index (HAI)(18
); HCC grading was performed according to the WHO classification of liver tumors (19
). All the biopsies were scored, and the scores are reported as the mean ± standard deviation of two scores given by two pathologists for each case.
Determination of the apoptotic index
The APO-BrdU Kit was used to evaluate the apoptotic index (APO-BRDU-IHC, AbD Serotec, product code APO002, Batch no. 230112, Oxford, OX5 1GE, UK).
TUNEL (terminal deoxynucleotidyl transferase-mediated d-UTP biotin nick end labeling) assay: Sections (5 µm thick) mounted on coated slides were stained according to the manufacturer’s protocol. Briefly, each section was brought to water and then left in PBS to equilibrate. The specimen was permeabilized by incubation with proteinase K at a 1:100 dilution in 10 mM Tris buffer at room temperature for 20 min. After rinsing in PBS, the endogenous peroxidase activity was quenched by incubation in H2O2 in methanol for 10 min. After rinsing, the sections were incubated with 1X reaction buffer for 30 min at room temperature. Without intermediate washes, the sections were incubated with the complete reaction mixture overnight at room temperature in a humidified chamber.
On day 2, the reaction buffer was removed, and blocking buffer was applied at room temperature for 10 minutes. The antibody solution was prepared, added and incubated for 3 hours at room temperature. After removing the excess fluid, the blocking buffer was applied. Visualization of the reaction was performed using DAB as the chromogenic substrate and hematoxylin as a counter stain.
Counting the apoptotic index
Five fields were counted per slide. Cells were considered positive for apoptosis as assessed by 2 criteria: a positive nuclear stain and, morphologic evidence of apoptotic cells.
The number of positive cells was counted according to the above-mentioned criteria, and the number of negative cells (nuclei stained blue with hemotoxylin) was also counted. The index was calculated as follows (20
No. of apoptotic cells/(no. of apoptotic cell + no. of negative cells).
Statistical analysis of the data
The data were input into the computer using IBM SPSS software package version 20.0. The qualitative data were described as the number and percent. The distributions of quantitative variables were tested for normality using the Shapiro-Wilk test and the D’Agostino test; histograms and QQ plots were used to visually assess the distribution. Parametric tests were used when the data were normally distributed, whereas non-parametric tests were used when the data were non-normally distributed. The quantitative data were described using the mean and standard deviation for normally distributed data, whereas non-normally distributed data were expressed using the median, minimum and maximum. For the non-normally distributed data, the Kruskal-Wallis test was used for comparisons between the groups, and pair-wise comparisons were assessed using the Mann-Whitney test with the Bonferroni correction. The correlations between two quantitative variables were assessed using the Spearman coefficient. For non-normally distributed data, the Mann-Whitney test (for data with a distribution that was significantly deviated from normal) was used to analyze the two independent populations.
Significant test results are quoted as two-tailed probabilities. The significance of the obtained results was judged at the 5% level.