A new type of protein chip to detect hepatocellular carcinoma-related autoimmune antibodies in the sera of hepatitis C virus-positive patients.

BACKGROUND: We report here a new type of protein chip to detect antibodies in sera. This chip method was used to a prototype created to detect hepatocellular carcinoma (HCC) -related autoantibodies in the sera of hepatitis C virus (HCV) infected individuals. RESULTS: Five cysteine-tagged (Cys-tag) and green fluorescent protein (GFP)-fused recombinant heat shock protein 70 (HSP70), superoxide dismutase 2 (SOD2), and peroxiredoxin 6 (PRDX6), were spotted and immobilized on maleimide-incorporated diamond-like carbon (DLC) substrates. The antibodies in diluted sera were trapped by these proteins at each spot on the chip, and visualized by a fluorescence-conjugated anti-human IgG. The total immobilized protein level of each spot was detected with anti-GFP mouse IgG and a fluorescence-conjugated secondary anti-mouse IgG. The ratio between the two fluorescence intensities was used to quantify autoantibody levels in each serum sample. Heat treatment of the chip in a solution of denaturing and reducing agents, before serum-incubation, improved autoantibody detection. We tested serum samples from healthy individuals and HCC patients using the chips. The HSP70 autoantibodies were found at high levels in sera from HCV-positive HCC patients, but not in HCV-negative sera. CONCLUSION: This protein chip system may have useful properties to capture a specific set of antibodies for predicting the onset of particular cancers such as HCC in HCV-infected individuals.

Identification of four isoforms of aldolase B down-regulated in hepatocellular carcinoma tissues by means of two-dimensional Western blotting.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal diseases, and one of the major causes of death in Japan. Our previous research of proteomics for cancerous and paired non-cancerous tissues from patients with HCC with hepatitis C virus infection (HCV-HCC) by means of the combination of two-dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) reported that four of numerous spots of weaker intensity in cancerous tissues than in paired non-cancerous tissues were identified as four isoforms of liver type aldolase (aldolase B). In the present study, two-dimensional (2-D) Western blot analysis demonstrated a significantly lower expression of four isoforms of aldolase B in cancerous than in non-cancerous tissues. CONCLUSION: Our finding of differences of expression aldolase B isoforms between cancerous and paired non-cancerous tissues for HCV-HCC may be useful for shedding light on some behaviors of aldolase B during hepatocellular carcinogenesis.

Up-regulation of 42 kDa tubulin alpha-6 chain fragment in well-differentiated hepatocellular carcinoma tissues from patients infected with hepatitis C virus.

We performed proteomic differential display analysis of hepatitis C virus-associated 21 human hepatocellular carcinoma (HCV-HCC) tissues by using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). One of the numerous spots which was located next to three spots of glutamine synthetase showed stronger intensity in well-differentiated HCC tissues compared to non-cancerous tissues. Samples from 6 out of 21 patients showed up-regulation of this spot compared to non-cancerous tissues. After in-gel digestion, MALDITOF/MS identified the spot as tubulin alpha-6 chain. Two-dimensional immunoblot analysis confirmed that this spot was indeed tubulin alpha, and this spot was stronger in cancerous tissues than in noncancerous tissues. These results suggest that tubulin alpha-6 chain is one of the candidates for biomarkers for well-differentiated HCV-associated HCC.

Screening for serological biomarkers of pancreatic cancer by two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry.

Pancreatic cancer (PC) is one of the most lethal malignant tumors because of late diagnosis and the lack of response to various therapies. To identify potential biomarkers in cancerous serum from early stage PC patients, we carried out two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare the serum proteomic profiles from 45 patients with PC and 20 healthy volunteers. Seven spots showed differential expression on 2-DE gels and two up-regulated protein spots were identified by LC-MS/MS as α-1-antitrypsin (AAT). These protein spots were also confirmed by Western blotting. This is the first time that AAT isoforms have been identified as potential serum biomarkers for PC. The serum isoforms of AAT may be clinically useful for PC diagnosis and monitoring.

Concomitant overexpression of heat-shock protein 70 and HLA class-I in hepatitis C virus-related hepatocellular carcinoma.

BACKGROUND: Human leukocyte antigen (HLA) class I expression is reportedly frequently reduced in cancer. We examined heat-shock protein (HSP) expression in hepatocellular carcinoma (HCC). PATIENTS AND METHODS: HCV-related HCC was examined in 73 patients who had undergone hepatectomy, and the relationship between HSP70 and HLA class I expressions, clinicopathological factors and survival was evaluated. RESULTS: Immunohistochemically positive results for HSP70 and HLA class I were seen in 67 (92%) and 43 cases (59%), respectively, while 38 patients (52%) were positive for both. Increased HSP70 immunoreactivity was significantly associated with high histological grade of tumor differentiation (p = 0.0179), whereas reduced HLA class I immunoreactivity was significantly associated with large tumor size (p = 0.0082). No differences were apparent between disease-free and overall survival in regard to expression levels. CONCLUSION: These results suggest that HSP70 expression may be related to tumor differentiation and HLA class I loss may occur with tumor growth in HCV-related HCC.

Improvement of early delayed gastric emptying in patients with Billroth I type of reconstruction after pylorus preserving pancreatoduodenectomy.

BACKGROUND: Early delayed gastric emptying (DGE) is the most common complication after pylorus-preserving pancreatoduodenectomy (PpPD). Recently, a vertical antecolic reconstruction for duodenojejunostomy was recommended to decrease the incidence of early DGE in patients with Billroth II-type reconstruction after PpPD. However, Billroth I-type reconstruction (B-I) after PpPD is still favored in Japan. METHODS: Twelve consecutive patients with B-I were prospectively enrolled. Our technique includes an end-to-side duodenojejunostomy and alignment of the stomach contours with fixation of the greater omentum to the abdominal wall in order to promote passage from the stomach through the jejunal loop. DGE was evaluated according to the consensus definition of the International Study Group of Pancreatic Surgery (ISGPS). RESULTS: DGE was absent, with the nasogastric tube removed within 3 days in all patients. Mean duration of nasogastric tube placement was 1.5 +/- 0.4 days. Mean maximum suction volume was 85 +/- 32 ml/day. CONCLUSION: Preliminary results were encouraging simply with relief of the outflow disturbance around the duodenojejunostomy in patients with B-I after PpPD. These findings warrant further prospective randomized trials at either multiple or high-volume centers.

Glycolysis module activated by hypoxia-inducible factor 1alpha is related to the aggressive phenotype of hepatocellular carcinoma.

An increased level of glycolysis, an intracellular hallmark of neoplasms, enables cancer cells to survive under various conditions. To elucidate the role of increased glycolysis in the progression of hepatocellular carcinoma (HCC), we investigated the associations between the expression patterns of 14 glycolysis-related genes and clinicopathologic factors in 60 HCCs by using pooled transcriptome data. We then evaluated the therapeutic efficacy of the knockdown of ENO1, which is encoded by a glycolysis-related gene, in HCC cells. Among the 14 genes, levels of 8 genes (GPI, ALDOA, TPI1, GAPD, PGK, PGAM, ENO1 and PKM), all of which can be transcriptionally activated by hypoxia-inducible factor 1alpha (HIF-1alpha), were significantly higher in HCC with venous invasion (VI) than in HCC without VI. Our cluster analysis showed that HCC patients with activation of the 8 HIF-1alpha-regulated genes had significantly shorter overall survival (P=0.023) than did HCC patients without increased expression levels of these genes. The association between the levels of ENO1 and VI was confirmed in an independent sample set of 49 HCCs by real-time reverse-transcription PCR. The knockdown of ENO1 by small-interfering RNA significantly inhibited the proliferation of an HCC cell line (HLE cells) in both the glucose-rich and glucose-free conditions, accompanied by a decreased S phase and increased G2/M phase of the cell cycle. Collectively, these data suggest that activation of an HIF-1alpha-regulated glycolysis module is closely related to the aggressive phenotype of HCC, and that ENO1, a glycolysis module gene, might serve as a new target to circumvent HCC metastasis.

Adoptive immunotherapy for pancreatic cancer: cytotoxic T lymphocytes stimulated by the MUC1-expressing human pancreatic cancer cell line YPK-1.

MUC1 is a tumor-associated antigen that is overexpressed in invasive ductal carcinomas of the pancreas (PC). MUC1-specific cytotoxic T lymphocytes (CTLs) recognize MUC1 molecules in a HLA-unrestricted manner. In this study, we performed adoptive immunotherapy (AIT) in patients with PC with CTLs stimulated by the MUC1-expressing human PC cell line YPK-1. To induce CTLs, peripheral blood mononuclear cells (PBMCs) were cultured for 3 days with inactivated YPK-1 cells and then stimulated with interleukin (IL)-2 for 7 days. The cytotoxicity of these cells against human cancer cell lines was analyzed, and a variety of antibodies were evaluated for their ability to inhibit cytotoxicity. We treated 8 patients with unresectable PC and 20 patients with resectable PC postsurgically. CTLs were induced as described above, suspended in 100 ml saline and injected intravenously. Induced CTLs were cytotoxic against 5 MUC1-expressing PC cell lines and a breast cancer cell line, regardless of the HLA phenotype. Low cytotoxicity was observed in 7 MUC1-negative cancer cell lines. Anti-CD3 monoclonal antibody (mAb) or anti-CD8 mAb strongly inhibited cytotoxicity against YPK-1, whereas anti-class I mAb showed no inhibition. YPK-1 cells incubated with anti-MUC1 mAb also showed low cytotoxicity. Clinically, the median survival time was 5.0 months for patients with unresectable PC treated with AIT. None of the 5 patients without liver metastasis showed hepatic recurrence. The median survival time was 17.8 months for 18 out of 20 patients with resectable PC who underwent curative surgery, and the 1-, 2- and 3-year survival rates after surgery were 83.3, 32.4, and 19.4%, respectively. Liver metastasis was found in only one patient and no side effects of AIT were observed. CTLs stimulated by a MUC1-expressing human pancreatic cancer cell line showed a strong tumor cytotoxic activity in a MUC1-specific and MHC-unrestricted manner. AIT with stimulated CTLs significantly suppressed the postsurgical hepatic recurrence of PC. Adjuvant immunotherapy with CTLs may be useful in the postsurgical treatment of PC.

Adoptive immunotherapy for pancreatic cancer using MUC1 peptide-pulsed dendritic cells and activated T lymphocytes.

BACKGROUND: Pancreatic cancer has a poor prognosis. The clinical efficacy of immunotherapy using both dendritic cells pulsed with MUC1 peptide (MUC1-DC) and, cytotoxic T lymphocyte (CTL) sensitized with a pancreatic cancer, YPK-1, expressing MUC1 (MUC1-CTL) was evaluated. PATIENTS AND METHODS: Twenty patients with unresectable or recurrent pancreatic cancer were enrolled. Peripheral blood mononuclear cells (PBMCs) were separated into adherent cells for induction of MUC1-DCs and floating cells for MUC1-CTLs. MUC1-DCs were generated by culture with granulocyte monocyte colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) and then exposed to MUC1 peptide and TNF-alpha. MUC1-CTLs were induced by co-culture with YPK-1 and then with interleukin-2 (IL-2). MUC1-DCs were injected intradermally and MUC1-CTLs were given intravenously. RESULTS: Patients were treated from 2 to 15 times. One patient with multiple lung metastases experienced a complete response. Five patients had stable disease. The mean survival time was 9.8 months. No grade II-IV toxicity was observed. CONCLUSION: Adoptive immunotherapy with MUC1-DC and MUC1-CTL may be feasible and effective for pancreatic cancer.

Increased expression of heat shock protein-binding protein 1 and heat shock protein 70 in human hepatocellular carcinoma tissues.

Heat shock protein-binding protein 1 (HspBP1) is a co-chaperone that inhibits heat shock 70-kDa protein (Hsp70) activity. In mouse neuroblastomas and lung tumors, the protein levels of HspBP1 and Hsp70 are elevated by a similar amount compared to non-tumor tissues. However, no studies have been reported regarding the levels of HspBP1 in human cancer tissues. Our previous proteomic study demonstrated that the expression of Hsp70 was increased in human hepatitis C virus-related hepatocellular carcinoma (HCV-HCC) tissues. Here, we investigated the expression of HspBP1 in human HCV-HCC. Immunoblotting analysis of HspBP1 and Hsp70 was performed in human HCV-HCC tissues from 20 patients. In 80% of the patients, Hsp70 increased an average of 3.55-fold, and in 50% of the patients, HspBP1 increased an average of 2.02-fold. Comparison and analysis of expression and clinical data revealed a significant difference between moderately-differentiated HCC and non-tumor tissues. In addition, there was a significant difference between the ratio of HspBP1 to Hsp70 levels and tumor size (<3 cm vs. ≥ 3cm) with larger tumors having a lower ratio. This ratio was significantly lower in moderately-differentiated HCC tissues than in non-tumor HCC tissues. In conclusion, HspBP1 was up-regulated in human HCV-HCC, an increase which correlated with the increase of Hsp70 levels. The ratio of HspBP1 to Hsp70 in HCC may provide novel information concerning the characterization of tumors, tumor progression and resistance to treatment.

Expression of tropomyosin alpha 4 chain is increased in esophageal squamous cell carcinoma as evidenced by proteomic profiling by two-dimensional electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry.

To identify proteins associated with esophageal carcinogenesis, we performed protein profiling of 16 esophageal squamous cell carcinomas (ESCCs) and paired noncancerous tissues by 2-DE and MS/MS. In cancerous tissues, three spots showed significant up-regulation in the amount of protein, while eight spots were significantly down-regulated. The identities of the spots were determined by PMF with LC-MS/MS and were confirmed by immunoblotting. The up-regulated proteins were tropomyosin alpha 4 chain, transgelin, and pyruvate kinase. The down-regulated proteins were serum albumin precursor, isoforms of annexin A1, tropomyosin beta chain, 14-3-3 protein sigma, and isoforms of serotransferrin precursor. In all 16 cases, up-regulation of the tropomyosin alpha 4 chain was confirmed by immunoblotting. Localization of the tropomyosin alpha 4 chain in ESCC cells and adjacent fibroblasts was confirmed by immunohistochemistry.

Identification of ID2 associated with invasion of hepatitis C virus-related hepatocellular carcinoma by gene expression profile.

Portal vein invasion (PVI) is a hallmark of metastatic potential of hepatocellular carcinoma (HCC) and is frequently found at a stage of moderately differentiated HCC. To identify genes involved in PVI of HCC associated with hepatitis C virus (HCV), we performed a comprehensive analysis of 12,600 genes in 35 moderately differentiated HCV-related HCCs by DNA microarray. Our supervised learning method identified 35 genes involved in PVI. Among the 35 identified genes, we focused on the inhibitor of DNA binding 2 (ID2), because it encodes a liver-rich dominant-negative helix-loop-helix protein. The microarray results for ID2 were reproduced by quantitative real-time reverse transcription (QRT)-PCR and Western blot analyses. In an independent set of HCV-related HCCs (n=28) and HCV-unrelated HCCs (n=14), our QRT-PCR showed that decrease in ID2 mRNA levels were associated with PVI in HCV-related HCC but not HCV-unrelated HCC. In conclusion, our results strongly suggest that ID2 plays an important role in PVI process of HCV-related HCC.

Proteomic analysis of autoantibodies in patients with hepatocellular carcinoma.

To detect autoantibodies that could be diagnostic markers for hepatocellular carcinoma (HCC), we analyzed serum autoantibodies comprehensively that showed immunoreactivity to proteins in tumor tissue obtained from patients with HCC. Fifteen paired samples of HCC tissue and corresponding nontumorous liver tissue as well as five normal liver tissue samples were used in the study. A combination of proteomics and SEREX (serologic analysis of recombinant cDNA expression libraries) technique was used. Tissue proteins were separated by 2-DE, transferred onto PVDF membranes, and immunoblotted with autologous sera. By comparing each immunoblot pattern, we identified four immunoreactive spots with stronger staining intensity in tumorous tissues than in corresponding nontumorous tissues and in normal liver tissues. Matched proteins on 2-DE gels were identified by LC-MS/MS. These immunoreactive proteins were heat shock 70 kDa protein 1 (HSP70), glyceraldehyde 3-phosphate dehydrogenase, peroxiredoxin, and manganese superoxide dismutase (Mn-SOD). In HCC sera, occurrences of autoantibodies against these proteins were 7/15 (46.7%), 5/15 (33.3%), 5/15 (33.3%), and 6/15 (40.0%), respectively, whereas 2/20 (10.0%), 7/20 (35.0%), 0/20 (0.0%), and 2/20 (10.0%) were in control sera. Immunoblot analysis using commercially available purified proteins was performed to confirm the specificity of autoantibodies. By statistical analysis, autoantibodies against HSP70, peroxiredoxin, and Mn-SOD showed significantly high-frequency immunoreaction in HCC sera. The three antibodies were considered patient-specific antibodies in HCC and may be candidate diagnostic biomarkers for HCC.

Increased expression and phosphorylation of liver glutamine synthetase in well-differentiated hepatocellular carcinoma tissues from patients infected with hepatitis C virus.

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers, and chronic infection with hepatitis C virus (HCV) is thought to be one of the main causes in Japan. To identify diagnostic or therapeutic biomarkers for HCC associated with HCV (HCV-HCC), we tried to elucidate the factors related to the products from cancerous tissues of HCV-infected patients. From proteomic differential display analysis of liver tissue samples from HCV-HCC cancerous tissues and corresponding non-cancerous tissues from patients, three protein spots of the same molecular mass (42 kDa), whose expression increased in well-differentiated cancerous tissues, were detected. Although their pI were different, they were identified as glutamine synthetase (GS) by PMF with MALDI-TOF MS and by Western blotting using anti-GS specific mAb. Immunohistochemical analysis showed that tumor tissue consists of two parts, GS-positive cell and GS-negative cell regions, suggesting that GS-producing cells grew in the tumor tissue as a nodule in nodules. The tryptic peptides of the most acidic GS isoform lost the signal of 899.5 Da, corresponding a peptide of SASIRIPR, and gained a signal of 1059.5 Da, which was submitted to PSD analysis. PSD analysis showed the neutral loss by elimination of two phosphate groups, supposed to be on serine residues of the 899.5-Da peptide, from serine 320 to arginine 327 in GS. PMF followed by PSD analysis is thought to be useful for the determination of phosphorylation sites of proteins showing molecular heterogeneity.

Protein level of apolipoprotein E increased in human hepatocellular carcinoma.

Many reports suggest that hepatic steatosis leads to hepatocellular carcinoma (HCC), including hepatitis C virus or non-alcoholic steatohepatitis. Proteomic study of tumor tissues from HCC patients, focusing on apolipoprotein (apo) of apoA1, apoB100 and apoE, was performed by immunoblotting. Although the significant changes of apoA1 or apoB100 could not be shown statistically, the immunoblotting showed the increase in protein level of apoE in the tumor tissues of 88% of patients without increase of apoE gene expression and serum level. These results suggest the accumulation of apoE by impaired secretion. Moreover, immunoblot analysis on two-dimensional electrophoresis showed a strong possibility that sialylated forms of apoE also were increased in tumorous tissues of HCC. ApoE level in tumorous tissues is frequently elevated and may be a good histological marker for HCC.

Current antibiotic prophylaxis in pancreatoduodenectomy in Japan.

BACKGROUND/PURPOSE: The aim of this study was to investigate the current use of antibiotic prophylaxis (AP) in association with pancreatoduodenectomy (PD) in Japan, and to determine its surgical implications. METHODS: We surveyed 2331 patients who underwent PD for treatment of disease in the periampullary region. Data, obtained during the period January 2002 through December 2003, from 111 major surgical services associated with the Japanese Society for Pancreatic Surgery, were analyzed with regard to patient characteristics, preoperative complications, AP, and postoperative morbidities. RESULTS: Eighty-five (78.7%) of the 108 eligible institutions chose a first- or second-generation cephalosporin for AP, given for a mean duration of 4.3 days. At all but 1 institution, the first dose was administered prior to surgical incision of the skin. At 42% of the institutions, an additional antibiotic was administered during surgery. The overall rate of wound infection was 6.8% of the 2266 patients for whom data were available. Preoperative jaundice was found in 55.3% of these 2266 patients, and 92.6% of these jaundiced patients were suffering from preoperative infections. In addition, those with preoperative infections were also diagnosed as having biliary infections. The number of patients with preoperative jaundice in combination with preoperative infections was significantly related to the rate of postoperative morbidity (P < 0.0001). CONCLUSIONS: Administration of AP in association with PD in Japan seems appropriate. Icteric patients with biliary infections are at high risk for postoperative morbidities and need careful monitoring after surgery.

Patterns of expression of cytochrome P450 genes in progression of hepatitis C virus-associated hepatocellular carcinoma.

Cytochrome P450 (CYP) genes are involved in the pathogenesis of hepatocellular carcinoma (HCC). To examine changes in expression of CYPs in HCC arising from hepatitis C virus (HCV)-infected liver, we used oligonucleotide array data of 27 CYPs from samples of 50 HCV-associated HCCs, five HCV-infected non-tumorous livers, and six HCV-negative normal livers. Progression of primary HCC can be characterized by decrease in the grade of tumor differentiation, increased frequency of venous invasion and increased tumor size. On the basis of tumor differentiation, the self-organizing map (SOM) classified the 27 CYPs into four groups. The first group contained 11 CYPs, including the CYP2C and CYP4F families, that showed decreased expression in parallel with progression of HCV-infected liver to HCC with less differentiation. The second group contained CYP-IID, CYP3A7 and CYP27A1, genes that showed high levels of expression specific to well differentiated HCC. The third group contained 5 sterol-metabolizing CYPs with levels lower in HCV-infected livers than in HCV-uninfected livers. The last group included the CYP2E1 and CYP3A families. Among the 27 CYPs, levels of 7 (CYP2B6, CYP-IIC, CYP2C9, CYP2C19, CYP3A5, CYP4F3 and CYP27A1) were significantly lower and levels of 2 (CYP2E1 and CYP4F2) were slightly lower in HCC with venous invasion than in HCC without venous invasion. Levels of CYP-IIC and CYP2C9 were inversely associated with tumor size. In contrast, levels of CYP51A1 were positively associated with tumor size. Our present study revealed that expression of specific CYPs was altered in conjunction with progression of HCV-associated HCC. These CYPs may serve as markers of progression and molecular targets for treatment of HCV-associated HCC.

Overexpression of alpha enolase in hepatitis C virus-related hepatocellular carcinoma: association with tumor progression as determined by proteomic analysis.

To identify proteins that could be molecular targets for diagnosis and treatment of hepatitis C virus-related hepatocellular carcinoma (HCV-related HCC), we used a proteomic approach to analyze protein expression in samples of human liver. Twenty-six pairs of tumorous and corresponding nontumorous liver samples from patients with HCV-related HCC and six normal liver samples were analyzed by two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry. One of the numerous spots that showed stronger intensity in tumorous than in nontumorous samples was identified as alpha enolase, a key enzyme in the glycolytic pathway. Expression of this protein increased with tumor dedifferentiation and was significantly higher in poorly differentiated HCC than in well-differentiated HCC. This pattern was reproduced by immunoblot analysis and immunohistochemistry. Expression of alpha enolase also correlated positively with tumor size and venous invasion. These results suggest that alpha enolase is one of the candidates for biomarkers for tumor progression that deserves further investigation in HCV-related HCC.

Sex-based molecular profiling of hepatitis C virus-related hepatocellular carcinoma.

It has been suggested that sex affects not only the incidence of hepatocellular carcinoma (HCC) but also the outcome after treatment. However, no sex-specific therapeutic targets for HCC have been identified. Identification of sex-specific genes will allow for the development of more personalized therapies. To this end, we investigated the expression of approximately 6000 genes in 50 samples of hepatitis C virus (HCV)-related HCC by oligonucleotide microarray. Our supervised learning method and subsequent random permutation test identified 27 genes that were differentially expressed in samples from male (n=34) and female (n=16) patients. Our gene selection was validated by a false discovery rate of only 0.5%. For the 27 genes, expression levels of 12 were higher and expression levels of 15 were lower in HCC samples from men than in HCC samples from women. For the cell proliferation-related genes identified, expression levels of PRDX1 were relatively high in HCC samples from men, and expression levels of PRDX3 were relatively high in HCC samples from women. The DNA microarray data for PRDX1 and PRDX3 were reproduced by reverse transcription-PCR analysis. Our results suggest that these 27 genes may serve as molecular targets or markers for sex-specific treatment of HCV-related HCC. Further studies are needed to elucidate their possible roles in male and female patients with HCV-related HCC.