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The study was approved by the Research Ethics Committee of Affiliated Huai’an 1st People’s Hospital, Nanjing Medical University Huai’an, Jiangsu, P.R. China. Written Informed Consent was obtained from each patient. All specimens were handled and made anonymous according to the ethical and legal standards. This study conformed to the tenets of the Declaration of Helsinki, 1975, and that tissues were not obtained from organ transplants from imprisoned or incarcerated persons.
Thirty-two donor-matched HCC and paracarcinomatous liver tissue (PCLT) specimens were obtained from 32 patients who underwent hepatectomy at the Department of Hepatobiliary & Pancreatic Surgery at this hospital between 2008 and 2010. These patients included 26 males and 6 females with a median age of 50.28 years (range, 35-72 years). Patients who had undergone previous therapy or non-curative surgery were excluded. Clinicopathologic variables for each patient were recorded and summarized in Table 4. All specimens were fixed in 10% formalin, embedded in paraffin, and cut into 4-µm-thick serial sections for immunohistochemical staining, in addition to hematoxylin-eosin staining.
Paraffin-embedded sections were immunohistochemically stained using commercially available rabbit anti-human polyclonal antibody against CD55 (Catalog No. sc-9156; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-human polyclonal antibody against SP100 (Catalog No. sc-25569; Santa Cruz Biotechnology), rabbit anti-human polyclonal antibody against BMP-7 (Catalog No. BS3674; Bioworld Technology, Inc. MN), mouse anti-human monoclonal antibody against BMP-4 (Catalog No. AB81194; Abcam (Hong Kong) Ltd., Hong Kong, China), rabbit anti-human polyclonal antibody against MYO6 (Catalog No. ab106288; Abcam (Hong Kong) Ltd.), rabbit anti-human polyclonal antibody against INHBE (Catalog No. HPA016843; Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden), and goat anti-human polyclonal antibody against LTBR (Catalog No.: ab10493; Abcam (Hong Kong) Ltd.) were used. Immunohistochemical staining was carried out on sections using the avidin-biotin method and a commercially available kit (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA). Briefly, sections were incubated overnight at 4°C with the primary antibodies against CD55 (1:100), SP100 (1:100), BMP-7 (1:100), BMP-4 (1:40), MYO6 (1:150), INHBE (1:20), or LTBR (1:150). After sections were washed with PBS, species-appropriate biotin-labeled secondary antibodies were applied for 10 min, followed by a peroxidase-marked streptavidin (3%) for an additional 10 min. The reaction was visualized using 3, 3'-diaminobenzidine tetrahydrochloride. Nuclei were counterstained with hematoxylin. Negative controls were generated by omitting the primary antibodies. CD55, SP100, BMP-7, BMP-4, MYO6, INHBE, and LTBR expression was further confirmed by western blotting using the same antibodies. Immunoreactivity was assessed by two investigators who were blinded to the clinicopathologic data. Discrepancies were resolved by simultaneous reexamination of the slides by both investigators using a double-headed microscope. A semi-quantitative scoring system was used as previously reported[16]. Candidate marker expression was compared between HCC and PCLT tissues. The number of positive-staining cells showing immunoreactivity in ten representative microscopic fields was counted and the percentage of positive cells was calculated. The percentage of stained tumor cells was converted into a subjective score as follows: 0 score=0 % of tumor cells stained, 1=1-10% of cells stained, 2=11-50% of cells stained), 3=51-75%, and 4≥75% of cells stained. Average staining intensity was visually scored and stratified as follows: 0 (-), 1 (+), 2 (++), and 3 (+++). Final immunoreactivity scores (IRS) were obtained for each sample by multiplying the percentage and the intensity scores. Protein expression levels were further analyzed by classifying IRS values as negative (based on an IRS value equal to 0), low (IRS value ranging from 1-4), medium (IRS value ranging from 5-8), or high (IRS value ranging between 9-12).
All computations were carried out using SPSS version 13.0 software for Windows (SPSS Inc., Chicago, IL, USA). Statistical differences between the expression levels of candidate biomarkers in different clinicopathological features were evaluated by Fisher's exact test for 2×2 tables and Pearson 2 test for non- 2×2 tables[17]. Differences were considered to be statistically significant when the P value was less than 0.05.
Immunohistochemistry analysis was performed to detect the expression of LTBR, CD55, INHBE, and SP100 protein in 32 pairs of HCC and PCLT tissues. In both tumor cells and normal liver cells, positive staining for LTBR was mainly observed in the cytoplasm and/or membrane; for CD55 staining was in the membrane and/or nucleus; for INHBE, staining was in the cytoplasm; and for SP100, staining was in the nuclei. The subcellular distributions of these proteins were all consistent with the comments of HPA and UniprotKB/Swiss-Prot databases. The expression rates of LTBR, CD55, INHBE, and SP100 proteins in HCC and PCLT tissues are summarized in Table 1. There were no significant differences of LTBR, CD55, INHBE, or SP100 protein expression between HCC and PCLT tissues (P>0.05 for all comparisons).
