Protein tyrosine kinase 7: a hepatocellular carcinoma-related gene detected by triple-combination array.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. We developed a novel technique to identify cancer-related genes of HCC as follows: triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism arrays, and methylation arrays. MATERIALS AND METHODS: Triple-combination array analysis was performed on one HCC sample from a 68-y-old female patient, and one candidate cancer-related gene was selected. Subsequently, we analyzed the identified gene by quantitative real-time reverse-transcriptase polymerase chain reaction (PCR) and methylation-specific PCR in nine HCC cell lines and in samples from 48 HCC patients. Additionally, we evaluated gene expression by immunohistochemistry and Western blotting. RESULTS: Using this method, protein tyrosine kinase 7 (PTK7) was detected as a candidate cancer-related gene. PTK7 was revealed to be hypermethylated (methylation value 0.826, range 0-1.0) in cancer tissue, compared with that of adjacent noncancerous tissues (0.047) by methylation array. Of the 48 clinical samples, 30 HCC samples (62.5%) showed PTK7 promoter hypermethylation. Downregulation of PTK7 (expressions in tumor tissues decreased by ≥ 50% compared with the noncancerous tissues) was significantly associated with age >60 y (P = 0.030) and elevation in serum protein induced by vitamin K absence or antagonists-II (P = 0.033). Moreover, patients with downregulation were significantly inferior in overall survival (P < 0.001) than the others. CONCLUSIONS: Our data imply that PTK7 acts as a cancer-related gene and may be a potent prognostic marker for HCC. Triple-combination array analysis was once again found to be useful in identifying cancer-related genes.

Expression analysis of THOP1 in background liver, a prognostic predictive factor in hepatocellular carcinoma, extracted by multiarray analysis.

BACKGROUND: Hepatocellular carcinoma (HCC) often recurs and multicentric occurrence is more common than intrahepatic metastases after surgery. Prognostic prediction is insufficient when considering only factors in resected primary tumor. METHODS: Control samples, termed supernormal (SN) liver, were taken from 11 cases of metastatic secondary malignancies of the liver. We selected adjacent nonneoplastic liver tissue from a patient with HCC and liver cirrhosis by hepatitis C (CN) for comparison. Expression profiling and methylation arrays were performed. We identified genes showing differences in both arrays. Prognosis was predicted for 179 cases of HCC based on gene expression. RESULTS: Expression profiling showed that expression of thimet oligopeptidase (THOP1) gene was decreased 4.119-fold in CN. Methylation array showed a higher value for CN (0.869) than SN (0.488). We studied THOP1 gene expression by real-time reverse transcriptase polymerase chain reaction. The average expression level of THOP1 (THOP1 value × 10(3)/GAPDH) decreased in matching normal tissue (14.53 ± 10.14) relative to SN (78.14 ± 44.50). The group with higher than average THOP1 expression (n = 74) showed significant correlations with prolonged survival (P = 0.0383). Strongly reduced THOP1 expression (<3.0, n = 50) was shown to be an independent prognostic factor by multivariate analysis (P = 0.0024). CONCLUSIONS: Expression of the THOP1 gene in the background liver of HCC is likely to be a good biomarker for risk of HCC development. When assessing HCC, it is important to extract prognostic factors from background liver tissue as well as considering malignant factors of the primary cancer lesion.

Identification of the collagen type 1 α 1 gene (COL1A1) as a candidate survival-related factor associated with hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death especially among Asian and African populations. It is urgent that we identify carcinogenesis-related genes to establish an innovative treatment strategy for this disease. METHODS: Triple-combination array analysis was performed using one pair each of HCC and noncancerous liver samples from a 68-year-old woman. This analysis consists of expression array, single nucleotide polymorphism array and methylation array. The gene encoding collagen type 1 alpha 1 (COL1A1) was identified and verified using HCC cell lines and 48 tissues from patients with primary HCC. RESULTS: Expression array revealed that COL1A1 gene expression was markedly decreased in tumor tissues (log2 ratio -1.1). The single nucleotide polymorphism array showed no chromosomal deletion in the locus of COL1A1. Importantly, the methylation value in the tumor tissue was higher (0.557) than that of the adjacent liver tissue (0.008). We verified that expression of this gene was suppressed by promoter methylation. Reactivation of COL1A1 expression by 5-aza-2'-deoxycytidine treatment was seen in HCC cell lines, and sequence analysis identified methylated CpG sites in the COL1A1 promoter region. Among 48 pairs of surgical specimens, 13 (27.1%) showed decreased COL1A1 mRNA expression in tumor sites. Among these 13 cases, 10 had promoter methylation at the tumor site. The log-rank test indicated that mRNA down-regulated tumors were significantly correlated with a poor overall survival rate (P = 0.013). CONCLUSIONS: Triple-combination array analysis successfully identified COL1A1 as a candidate survival-related gene in HCCs. Epigenetic down-regulation of COL1A1 mRNA expression might have a role as a prognostic biomarker of HCC.

High-risk Features for Recurrence in Patients With Stage III Colorectal Cancer: A Retrospective Cohort Study.

BACKGROUND/AIM: Risk classification for recurrence in stage III colorectal cancer (CRC) is not as well established as it is for stage II. This study aimed to identify high-risk factors for stage III colorectal cancer and to investigate their clinical significance. PATIENTS AND METHODS: We retrospectively analyzed data from 120 patients with stage III CRC who had undergone curative colectomy at our institution between 2014 and 2020. We used logistic regression analysis to identify risk factors for recurrence and subsequently explored their clinical significance. RESULTS: We identified three high-risk factors in stage III CRC: preoperative bowel obstruction [odds ratio (OR)=5.39; 95% confidence interval (CI)=1.61-18.03; p=0.007], N2 disease (OR=3.12; 95%CI=1.05-9.29; p=0.041), and having fewer than 17 examined lymph nodes (OR=3.17; 95%CI=1.11-8.99; p=0.031). The prognosis of patients was clearly stratified by the number of these risk factors, and furthermore, the effectiveness of adjuvant therapy depended on their number. CONCLUSION: Tumor obstruction, N-stage, and the number of lymph nodes examined are important high-risk features for recurrence. This study provides clinicians with valuable insights to predict and stratify patient outcomes in stage III CRC.

Promoter hypermethylation of fibulin 1 gene is associated with tumor progression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide but the molecular mechanisms that underlie hepatocarcinogenesis are not fully determined. On the same surgical sample with HCC, we performed microarray-based gene expression profiling and karyotype analysis using a single nucleotide polymorphism (SNP) array. In addition, quantitative real-time reverse transcription polymerase chain reaction (PCR), methylation specific PCR (MSP) and immunohistochemical staining were conducted using specimens from 48 patients with HCC. Gene expression profiling showed the expression of fibulin 1 (FBLN1), located on 22q13, to be decreased in tumor tissue. Karyotype analysis revealed no loss of heterozygosity (LOH) since deletions were not detected in 22q, and one of the SNPs on 22q13 showed AB genotype in both cancerous tissue and in corresponding noncancerous tissue, indicating retention of heterozygosity. Quantitative real-time PCR showed FBLN1 mRNA levels in cancerous tissues to be significantly decreased compared with that in corresponding noncancerous tissues. The immunohistochemical staining results were consistent with both gene expression profiling and quantitative PCR data. Twenty-four out of 48 HCCs gave a positive result in MSP. Moreover, promoter hypermethylation of FBLN1 was significantly associated with advanced stage HCC, multiple tumors and increased tumor size. Our results indicated that FBLN1 is a novel candidate of tumor suppressor gene and that promoter hypermethylation of FBLN1 is associated with tumor progression in HCC. © 2011 Wiley-Liss, Inc.

Total pancreatectomy with segmental duodenectomy preserving right gastroepiploic vein.

We performed total pancreatectomy with segmental duodenectomy preserving the gastrocolic trunk and right gastroepiploic vein, to prevent gastric venous congestion, for treatment of pancreatic tumor. This is believed to be the first report of such a procedure. The patient was a 58-year-old man with high serum levels of carbohydrate antigen 19-9 and carcinoembryonic antigen. He was examined by abdominal ultrasonography and computed tomography, and diagnosed with cancer of the pancreatic body. No distant metastasis was found. We decided to perform distal pancreatectomy. After surgery, the cut edge of the distal pancreas was subjected to frozen section examination, and there was carcinoma in situ in the stump of the main pancreatic duct. Therefore, we cut the pancreatic head partially, twice, but carcinoma in situ remained. We additionally performed pancreatic head resection with segmental duodenectomy, with preservation of the gastroduodenal artery, right gastroepiploic artery, gastrocolic trunk and right gastroepiploic vein to prevent gastric venous congestion. The postoperative course was uneventful and the patient remains in good condition. This surgical technique is considered feasible and safe for prevention of gastric venous congestion.

Reduced-port endo-laparoscopic surgery using umbilical zigzag incision for concomitant operations: A case series.

INTRODUCTION: Recently, use of reduced-port surgery has become widespread; however, it is a difficult operation. Hachisuka et al. developed a method called the umbilical zigzag incision, which enlarges the fascial incision using only an umbilical skin incision. We believe this method will be feasible for concomitant laparoscopic surgery. We report our surgical techniques for concomitant laparoscopic surgery using an umbilical zigzag incision. METHODS: The patient who should receive more than 2 places of abdominal operation were indicated this procedure. In all cases, we made the zigzag incision in the umbilical region. After the linea alba and peritoneum were incised, the wound retractor was inserted through the incision, which enlarges the fascial opening. GelPoint was attached to the wound retractor and the operation was started. RESULTS: We could create a fascial opening of up to 6 cm with the umbilical zigzag incision, which improves the triangulation of forceps and reduces interference among the trocars. The trocars in the center of the abdomen could be utilized for almost all intraperitoneal operations. This procedure was especially useful in cases that included lymph node dissection because dissection of a malignant tumor is a delicate procedure. Furthermore, extraction of specimens and anastomosis went very smoothly because the fascial incision was large enough such that no extension of the incision was needed. No early or late postoperative complications occurred in any case. Postoperative wounds were clear and therefore patient satisfaction levels were high. CONCLUSION: Umbilical zigzag incision may be feasible especially in concomitant laparoscopic surgery.

Prognostic significance of AKR1B10 gene expression in hepatocellular carcinoma and surrounding non-tumorous liver tissue.

When assessing outcome in hepatocellular carcinoma (HCC), it is important to consider prognostic factors in background non-tumorous liver tissue as well as in the tumor, since multiple occurrence is associated with background liver status such as hepatitis. The current study aimed to elucidate molecular prognostic predictors that have an association with HCC background non-tumorous tissue. Microarray expression profiling identified aldo-keto reductase family 1, member B10 (AKR1B10) as a putative non-tumorous prognostic factor, and AKR1B10 gene expression was investigated in 158 curatively resected HCC cases by reverse transcription-quantitative polymerase chain reaction. AKR1B10 expression (AKR1B10 value/GAPDH value × 1,000) was significantly higher in tumor tissue (median, 9.2200; range, 0.0003-611.0200; n=158) than in the corresponding non-tumorous tissue (median, 0.5461; range, 0.0018-69.0300; n=158) (P<0.001). When the samples were grouped according to AKR1B10 expression in tumor tissue relative to non-tumorous tissue, tumor

High expression of Janus kinase 2 in background normal liver tissue of resected hepatocellular carcinoma is associated with worse prognosis.

When assessing hepatocellular carcinoma (HCC), it is important to examine prognostic factors in the background normal liver tissue and consider malignant aspects of the primary lesion. Candidate genes were extracted from the background normal liver samples via multiarray analysis. Control samples, termed supernormal (SN) liver, were obtained from 11 cases of metastatic liver cancer. Corresponding normal (CN) liver tissue was surgically obtained from a typical HCC patient with chronic hepatitis C background for comparison. Expression profile and methylation array demonstrated that Janus kinase 2 (JAK2) gene expression was increased by 2.378­fold in the CN tissue. Methylation array reported a lower value for CN (0.125) than SN tissues (0.748). We then investigated JAK2 expression by real-time quantitative reverse transcription-polymerase chain reaction in 100 consecutive resected HCC cases. The average expression level of JAK2 (normalized to GAPDH) was significantly lower in CN (9.24±6.43, n=100) than in SN (35.21 ± 21.38, n=11) tissues (P<0.001). As such a result was contrary to our expectation, the case used for array analysis seemed to be a rare incidence. One hundred HCC cases were subsequently divided into two groups based on JAK2 expression in the adjacent normal tissue: one consisting of the upper 70% of cases (n=70) and the other of the remaining 30% (n=30). Higher JAK2 expression in the adjacent tissue demonstrated significant correlation with worse survival (P=0.022). Furthermore, multivariate analysis identified higher JAK2 expression in the background normal liver tissue of HCC as an independent prognostic factor (P=0.032). Our findings suggest that higher JAK2 expression in the background normal liver tissue of HCC may be a good prognostic biomarker for resected HCC.

CCNJ detected by triple combination array analysis as a tumor-related gene of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has a high likelihood of recurrence and a poor prognosis. To detect cancer-related genes of HCC, we developed a new technique: triple combination array analysis, consisting of a methylation array, a gene expression array and a single nucleotide polymorphism array. A surgical specimen obtained from a 68-year-old female HCC patient was analyzed using triple combination array, which identified cyclin J (CCNJ) as a candidate cancer-related gene of HCC. Subsequently, samples from 85 HCC patients were evaluated for CCNJ promoter hypermethylation and expression status using methylation-specific PCR (MSP) and quantitative reverse transcriptase RT-PCR, respectively. CCNJ was found to be hypermethylated (methylation value, 0.906; range, 0-1.0) in cancer tissue, compared with adjacent non-cancerous tissue (0.112) using a methylation array. MSP revealed that CCNJ was hypermethylated in 67 (78.8%) of the tumor samples. CCNJ expression was significantly decreased in cases with hypermethylation (P<0.0001). Furthermore, cases with both promoter hypermethylation and decreased expression of CCNJ in the tumor tissue had a worse overall survival than the other cases (P=0.0383). In conclusion, our results indicated that CCNJ could be a novel prognostic marker of HCC, and this study indicated that triple combination array analysis was effective in detecting new tumor-related genes and their mechanisms.

Management of hepatocellular carcinoma should consider both tumor factors and background liver factors.

As progression and outcome of relapsed hepatocellular carcinoma (HCC) are distinct from those of a second primary tumor, clonal analyses of initial and recurrent HCCs are thus clinically useful. Although several studies in Japan and Taiwan had shown that the multicentric origin (MO) recurrences were more common than intrahepatic metastases (IM), a recent report from China indicated that IM cases outnumber MO recurrences. In managing HCC cases, both tumor malignancy and background liver function are important considerations (and which we characterize as tumor factors and background liver factors, respectively); they indicate both appropriate treatment, and likely post-surgical outcome. In this editorial, we explain why the report had shown such a different conclusion. We also discuss current management of HCC.

Downregulation of DENND2D by promoter hypermethylation is associated with early recurrence of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and its prognosis is poor. Novel targets for treating recurrence and progression along with associated biomarkers are urgently required. In this study, the expression and regulatory mechanism of DENN/MADD domain containing 2D (DENND2D) were investigated in an attempt to identify a tumor suppressor gene for HCC regulated by silencing through promoter hypermethylation. The levels of DENND2D expression in HCC cell lines and surgical specimens were determined using a quantitative polymerase chain reaction assay and the relationship between the expression levels of DENND2D mRNA and clinicopathological factors was evaluated. The expression and distribution of DENND2D were determined using immunohistochemistry. DNA methylation analysis was performed to determine the regulatory mechanisms of DENND2D expression in HCC. Most HCC cell lines (89%) and surgical specimens (78%) expressed lower levels of DENND2D mRNA compared with normal liver tissue. In contrast, there was no significant difference in the expression levels of DENND2D mRNA between normal tissues of HCC patients with and without cirrhosis. The expression patterns of DENND2D protein and mRNA were consistent. Patients with significantly lower levels of DENND2D mRNA in HCC tissues had remarkably earlier recurrences after hepatectomy and their prognosis worsened. The DENND2D promoter was methylated in eight out of nine HCC cell lines and DNA demethylation reactivated DENND2D mRNA expression. Hypermethylation of DENND2D was frequently detected in HCC tissues (75%) and was significantly associated with downregulation of DENND2D mRNA expression. DENND2D is a candidate tumor suppressor gene that is inactivated by promoter hypermethylation in patients with HCC and may serve as a novel biomarker of early recurrence of HCC.

Reduced expression of DENND2D through promoter hypermethylation is an adverse prognostic factor in squamous cell carcinoma of the esophagus.

Esophageal cancer ranks sixth in cancer mortality worldwide and patients with esophageal squamous cell carcinoma (ESCC) have a poor prognosis with a 5-year survival rate of less than 10%. Elucidation of the mechanisms of carcinogenesis and tumor progression in esophageal cancer is urgently required to develop targets for therapy and prognostic biomarkers. In the present study, the expression and regulatory mechanism of the differentially expressed in normal and neoplastic cells domain containing 2D (DENND2D), which is a regulator of Rab GTPases, were investigated to explore its potential as a tumor suppressor gene for ESCC. The level of DENND2D mRNA expression in ESCC cell lines and surgical specimens was determined using a quantitative real-time reverse transcription-polymerase chain reaction assay, and the relationship between the expression levels of DENND2D mRNA and clinicopathological factors was evaluated. The expression and distribution of DENND2D were determined using immunohistochemistry. DNA methylation analysis was performed to determine the regulatory mechanism of DENND2D expression in ESCC. The level of DENND2D mRNA expression was reduced in 8/9 ESCC cell lines and in 59/65 surgical specimens, and the mean expression levels were significantly lower in cancerous tissues compared to corresponding normal tissues (p<0.001). The expression pattern of DENND2D protein and mRNA was consistent. Downregulation of DENND2D mRNA in ESCC tissues was identified as an independent prognostic factor in multivariate analysis (hazard ratio, 2.194; p=0.039). The DENND2D promoter was methylated in 5/9 ESCC cell lines, and DNA demethylation reactivated DENND2D mRNA expression. Hypermethylation of DENND2D was frequently detected in ESCC tissues (64.6%) and was significantly associated with downregulation of DENND2D mRNA expression (P=0.008). Taken together, our data suggest that DENND2D is a candidate tumor suppressor gene that was inactivated by promoter hypermethylation in patients with ESCC and may serve as a novel biomarker of ESCC.

Identification of intragenic methylation in the TUSC1 gene as a novel prognostic marker of hepatocellular carcinoma.

Patients with hepatocellular carcinoma (HCC) have a poor prognosis, and novel molecular targets for treating recurrence and progression of the disease along with associated biomarkers are urgently required. In the present study, expression and the regulatory mechanism of TUSC1 (tumor suppressor candidate 1) were investigated to determine if it is a candidate tumor suppressor gene for HCC, which shows repressed transcription that involves aberrant DNA methylation. TUSC1 mRNA expression levels in HCC cell lines and 94 pairs of surgical specimens were determined using quantitative real-time reverse transcription polymerase chain reaction assay. Methylation status of HCC cell lines and clinical samples were analyzed to investigate the regulatory mechanism of TUSC1 transcription and the relationship between the methylation status of the TUSC1 gene and clinicopathological factors. The expression and distribution of the TUSC1 protein in liver tissues were determined using immunohistochemistry. A majority of HCC cell lines (89%) and surgical specimens (84%) demonstrated reduced expression levels of TUSC1 mRNA compared with paired non-cancerous liver tissues. The mean mRNA expression level in HCC was significantly lower than in corresponding non-cancerous liver. In contrast, no significant difference was found in TUSC1 mRNA expression level between adjacent normal and cirrhotic liver tissue from HCC patients. The TUSC1 protein expression pattern in HCC and liver tissues was consistent with TUSC1 mRNA expression. Twenty-nine (31%) of 94 patients showed intragenic hypermethylation of the TUSC1 gene in HCC, and hypermethylation was significantly associated with advanced pathological stage. Subsequently, patients with hypermethylation of the TUSC1 gene had a significantly poorer prognosis than patients without hypermethylation. Our results suggest that TUSC1 is a candidate tumor suppressor gene and intragenic hypermethylation is one of the suppressive mechanisms that regulate TUSC1 transcription in HCC. Intragenic methylation of the TUSC1 gene may serve as a novel prognostic marker of HCC.

Detection of doublecortin domain-containing 2 (DCDC2), a new candidate tumor suppressor gene of hepatocellular carcinoma, by triple combination array analysis.

BACKGROUND: To detect genes correlated with hepatocellular carcinoma (HCC), we developed a triple combination array consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis. METHODS: A surgical specimen obtained from a 68-year-old female HCC patient was analyzed by triple combination array, which identified doublecortin domain-containing 2 (DCDC2) as a candidate tumor suppressor gene of HCC. Subsequently, samples from 48 HCC patients were evaluated for their DCDC2 methylation and expression status using methylation specific PCR (MSP) and semi-quantitative reverse transcriptase (RT) PCR, respectively. Then, we investigated the relationship between clinicopathological factors and methylation status of DCDC2. RESULTS: DCDC2 was revealed to be hypermethylated (methylation value 0.846, range 0-1.0) in cancer tissue, compared with adjacent normal tissue (0.212) by methylation array in the 68-year-old female patient. Expression array showed decreased expression of DCDC2 in cancerous tissue. SNP array showed that the copy number of chromosome 6p22.1, in which DCDC2 resides, was normal. MSP revealed hypermethylation of the promoter region of DCDC2 in 41 of the tumor samples. DCDC2 expression was significantly decreased in the cases with methylation (P = 0.048). Furthermore, the methylated cases revealed worse prognosis for overall survival than unmethylated cases (P = 0.048). CONCLUSIONS: The present study indicates that triple combination array is an effective method to detect novel genes related to HCC. We propose that DCDC2 is a tumor suppressor gene of HCC.

Dynamin 3: a new candidate tumor suppressor gene in hepatocellular carcinoma detected by triple combination array analysis.

BACKGROUND: To identify genes associated with hepatocellular carcinoma (HCC) pathogenesis, we developed a triple combination array strategy comprising methylation, gene expression, and single nucleotide polymorphism (SNP) array analysis. METHODS: Surgical specimens obtained from a 68-year-old female HCC patient were analyzed by triple combination array, and identified Dynamin 3 (DNM3) as a candidate tumor suppressor gene in HCC. Subsequently, samples from 48 HCC patients were evaluated for DNM3 methylation and expression status using methylation specific polymerase chain reaction (PCR; MSP) and semi-quantitative reverse transcriptase (RT)-PCR, respectively. The relationship between clinicopathological factors and DNM3 methylation status was also investigated. RESULTS: DNM3 was shown to be hypermethylated (methylation value 0.879, range 0-1.0) in cancer tissue compared with adjacent normal tissue (0.213) by methylation array in the 68-year-old female patient. Expression arrays revealed decreased expression of DNM3 in cancerous tissue. SNP arrays revealed that the copy number of chromosome 1q24.3, in which DNM3 resides, was normal. MSP revealed hypermethylation of the DNM3 promoter region in 33 of 48 tumor samples. A trend toward decreased DNM3 expression was observed in patients with DNM3 promoter methylation (P = 0.189). Furthermore, patients with reduced expression of DNM3 in tumor tissues exhibited worse prognosis with decreased disease specific survival compared to patients without decreased expression (P = 0.014). CONCLUSION: The present study indicates that a triple combination array strategy is an effective method to detect novel genes related to HCC. We propose that DNM3 is a tumor suppressor gene in HCC.

Estrogen receptor 1 gene as a tumor suppressor gene in hepatocellular carcinoma detected by triple-combination array analysis.

Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. Recent developments in the treatment of HCC remain insufficient to cure unresectable disease or to prevent HCC. Consistent efforts are, therefore, needed to deepen understanding of pathogenesis of the disease. Genome-wide gene expression profile analyses can now detect various candidate genes that are modified by HCC. We have developed a new technique to identify tumor suppressor genes, triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism and methylation arrays to identify genes with altered expression. Using HCC tissue samples, triple-combination array analysis was performed to identify a candidate tumor suppressor gene. Subsequently, samples from 48 HCC patients were subjected to quantitative polymerase chain reaction (qPCR) and methylation-specific PCR to further elucidate clinical relevance of the gene. Estrogen receptor 1 (ESR1) was detected as a candidate tumor suppressor gene. Of the 48 clinical samples, 40 (83.3%) showed ESR1 promoter hypermethylation. In 24 (50%) HCC samples, the expression levels of the ESR1 gene was decreased by >90%. The decreased expression was significantly related to high liver damage score, pathological invasion of the intrahepatic portal vein, the size of tumor (>3 cm in diameter) and hepatitis B virus infection. The present study represents another example that triple-combination array is a convenient technique for detecting genes with altered expression in disease. The ESR1 gene was identified as a candidate tumor suppressor gene in HCC and further validation is warranted.

Identification of the bleomycin hydrolase gene as a methylated tumor suppressor gene in hepatocellular carcinoma using a novel triple-combination array method.

In the present study, we sought to identify novel suppressor genes of hepatocellular carcinoma (HCC) using our newly designed triple-combination array. Using this method, the bleomycin hydrolase gene (BLMH) was detected as a candidate suppressor gene. We found that 28 of 48 (58.3%) tumor tissues showed BLMH promoter hypermethylation, and its expression level was significantly reduced in tumor tissues (P=0.001). The present study suggests that our new method can detect novel genes of interest and that BLMH is a suppressor gene in HCC.