Triple Combination Array Analysis Research Articles

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.

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

BackgroundHepatocellular 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 methodsTriple-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. ResultsUsing 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. ConclusionsOur 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.

Abstract 4717: Detection of the Cyclin J (CCNJ) as a new cancer-related gene in human hepatocellular carcinoma by using a method of triple combination array analysis

Abstract Background: Hepatocellular carcinoma (HCC) has a high rate of recurrence and a poor prognosis. To detect genes correlated with HCC, we have developed a new technique, triple combination array analysis, 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 using triple combination array analysis, which identified a candidate cancer-related gene of HCC. Subsequently, samples from nine cell lines and 48 HCC patients were evaluated for their methylation and expression status of the identified gene using methylation-specific polymerase chain reaction (MSP) and quantitative real-time reverse transcriptase PCR, respectively. Results: Using the triple combination array analysis, cyclin J (CCNJ) was detected as a candidate cancer-related gene. CCNJ was located on chromosome 10q24.1 and the copy number, using SNP chip array, revealed no loss of heterozygosity. According to expression array results, The expression of CCNJ in tumor tissue decreased at two points of the expression array chip, and the decreased values of the chip were −1.3 and −2.3. CCNJ was found to be hypermethylated (methylation value 0.906, range 0-1.0) in cancer tissue compared with adjacent normal tissue (0.112) using the methylation array. Using MSP, hypermethylation of the promoter region of CCNJ was shown to occur in 37 (77.1%) tumor samples. CCNJ expression was significantly decreased in cases with methylation (p&amp;lt;0.0001). Furthermore, HCC patients with methylated CCNJ had a significantly worse prognosis for recurrence-free survival (p=0.0353) and tended to have a worse prognosis for overall survival than those with unmethylated CCNJ (p=0.0838). Conclusion: The present study indicates that triple combination array analysis is an effective method to detect novel genes related to HCC. We suggest that CCNJ acts as a cancer-related gene in HCC. Citation Format: Nao Takano, Shuji Nomoto, Mitsuhiro Hishida, Yoshikuni Inokawa, Masamichi Hayashi, Mitsuro Kanda, Yukiyasu Okamura, Yoko Nishikawa, Chie Tanaka, Daisuke Kobayashi, Suguru Yamada, Goro Nakayama, Tsutomu Fujii, Hiroyuki Sugimoto, Masahiko Koike, Michitaka Fujii, Shin Takeda, Yasuhiro Kodera. Detection of the Cyclin J (CCNJ) as a new cancer-related gene in human hepatocellular carcinoma by using a method of triple combination array analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4717. doi:10.1158/1538-7445.AM2014-4717

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

BackgroundHepatocellular 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.MethodsTriple-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.ResultsExpression 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).ConclusionsTriple-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.

STEAP4 Inactivation Correlates Poor Prognosis and might be a Possible Cause of steatotic Change in Hepatocellular Carcinoma, Detected by Triple-Combination Array Analysis

In the present study, we originally designed triple-combination array analysis to identify tumor related genes in Hepatocellular Carcinoma (HCC). Using this method, the six transmembrane epithelial antigen of prostate family 4 (STEAP4), it was reported association with obesity and insulin-resistance, was detected as a candidate tumor suppressor gene. We found 32 of 48 (66.7%) tumor tissues showed STEAP4 promoter hypermethylation, and its expression level was significantly reduced in tumor tissues (p<0.0001). Methylated cases demonstrated significantly poorer recurrence-free survival (p=0.0462) and overall survival (p=0.0411). Triple-combination array analysis represents a useful technique for identifying novel tumor-related genes in HCC.

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

BackgroundTo 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.MethodsSurgical 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.ResultsDNM3 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).ConclusionThe 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.

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

BackgroundTo 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.MethodsA 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.ResultsDCDC2 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).ConclusionsThe 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.

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.

Abstract 1152: Identification of protein tyrosine kinase 7 gene in hepatocellular carcinoma using a novel triple-combination array analysis.

Abstract Background. Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. Recent developments in the treatment of HCC still have failed to prevent HCC. Genome-wide gene-expression profile analyses can now detect many candidate genes that are modified by HCC. We have developed a new technique to identify suppressor genes of HCC: triple-combination array analysis, which combines gene expression profile, single nucleotide polymorphism array and methylation array. Methods. Triple-combination array analysis was performed on one HCC sample from a 68-year-old female patient, and one candidate of tumor suppressor gene was selected. Subsequently, we analyzed the identified gene by quantitative real-time reverse-transcriptase polymerase chain reaction (PCR) and methylation-specific PCR in samples from 48 HCC patients and in nine cell lines. Results. Using this method, protein tyrosine kinase 7 (PTK7) was detected as a candidate of tumor suppressor gene. PTK7 gene is located on 6q21.2, and expression was reduced in HCC tissues at the high level of -2.5 compared with normal tissues. PTK7 locus did not show any deletion or amplification in the SNP array. The methylation value (0-1.0) in the tumor sample was 0.825 compared to 0.047 in normal tissues, indicating high methylation in tumor tissues in the methylation array. Of the 48 clinical samples, 30 (62.5%) showed PTK7 promoter hypermethylation. The PTK7 promoter hypermethylated cases were significantly low expressions status (P=0.035), and bad prognosis in both overall survival (P&amp;lt;0.01) and recurrence-free survival (P=0.018) compared to hypomethylated cases. On the other hand, the cases, of which the PTK7 expressions in tumor tissues decreased by more than 20% degrees compared to those in normal tissues, were significantly worse prognosis in overall survival (P=0.0136) than the other cases, but not in recurrence-free survival (P=0.3102). Conclusions. We suggest that the PTK7 gene acts as a tumor suppressor gene in HCC. This present study indicates that triple-combination array is an available and convenient technique for detecting the genes with altered expression in disease. Citation Format: Mitsuhiro Hishida, Shuji Nomoto, Yoshikuni Inokawa, Masamichi Hayashi, Suguru Yamada, Tsutomu Fujii, Hiroyuki Sugimoto, Shin Takeda, Yasuhiro Kodera. Identification of protein tyrosine kinase 7 gene in hepatocellular carcinoma using a novel triple-combination array analysis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1152. doi:10.1158/1538-7445.AM2013-1152

Abstract 5535: Availability of a newly designed technique, triple-combination array analysis: Identification of estrogen receptor 1 gene in hepatocellular carcinoma using this method

Abstract Background: Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. Recent developments in the treatment of HCC have failed to treat completely or prevent HCC. Genome-wide gene-expression profile analyses can now detect many candidate genes that are modified by HCC. We have developed a new technique to identify suppressor genes of HCC: triple-combination array analysis. Methods: Triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism arrays and methylation arrays, was performed on each HCC tissue sample. Subsequently, samples from 48 HCC patients were subjected to quantitative real-time reverse-transcriptase polymerase chain reaction (PCR) and methylation-specific PCR. Results: Using this method, estrogen receptor 1 (ESR1) was detected as a candidate suppressor gene. Of the 48 clinical samples, 40 (83.3%) showed ESR1 promoter hypermethylation. In 24 (50%) HCC samples, the expression level of ESR1 gene was decreased by &amp;gt;90%, which was significantly related to hepatitis B virus, high liver damage score, and pathological invasion of the intrahepatic portal vein. Conclusions: The present study indicates that triple-combination array is an available and convenient technique for detecting the genes with altered expression in disease, and we suggest that the ESR1 gene acts as a suppressor gene in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5535. doi:1538-7445.AM2012-5535

Abstract 4586: Use of triple combination array analysis to identify STEAP family member 4 (STEAP4), a novel tumor related gene in hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide, but the molecular mechanisms underlying hepatocarcinogenesis are not clear. Array technology has made it possible to identify novel genes with alterations in liver cancer. In this study, we performed triple combination array analysis, expression profiling, karyotyping analysis using SNP array, and array-based DNA methylation profiling in the same HCC sample and attempted to find a novel tumor related gene as a prognostic marker. Materials &amp; Methods: A 68-year-old woman with chronic hepatitis C was diagnosed with HCC. The excised tumor and corresponding normal tissues were used for extraction of DNA and RNA. SNP array experiments were performed using the DNA. mRNA expression profiles were determined using the total RNA by Affimetrix HGU133A and B Gene-Chips. Bisulphite-converted genomic DNA was analyzed using Illumina's Infinium Human Methylation27 Beadchip Kit. We consequently analyzed the identified gene using quantitative RT-PCR, methylation and un-methylation-specific PCR in nine HCC cell lines and in 48 primary HCC tissues and corresponding non-cancerous tissues collected at Nagoya University Hospital, and then compared these results with clinico-pathological data. Results: STEAP4 gene, is located on 7q21.12, and expression was decreased at a Log2 ratio of –4.2 to –2.0 in cancerous tissue. There were two copies of 7q21.12, indicating no loss of heterozygosity (LOH) in the SNP array. The methylation value (0-1.0) in the tumor sample was 0.870 compared to 0.160 in non-cancerous tissue, indicating high methylation in tumor tissue in the Methylation array. Re-activation of STEAP4 expression was seen after 5-aza-dC treatment in HCC cell lines. Moreover, we found that 32 of 48 (67%) HCC samples showed promoter hypermethylation. In 32 methylated cases, the expression values of STEAP4 gene in tumor tissues by real-time RT-PCR were significantly decreased compared to corresponding normal samples (p&amp;lt;0.0001). In addition, methylated cases were significantly correlated with worse recurrence free survival (p&amp;lt;0.0001) and overall survival (p=0.0093). Conclusion: The Promoter hypermethylation of STEAP4 gene was a marker of worse prognosis in HCC. The triple combination array was a useful technique to identify the novel tumor related gene in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4586. doi:1538-7445.AM2012-4586

Abstract 322: Triple combination array analysis detects the Collagen type1 alpha1 (COL1A1) gene as a new tumor related gene in hepatocellular carcinoma

Abstract Background: Epigenetics is one of the most important mechanisms for inactivating tumor suppressor genes or tumor-related genes in various human cancers. Hepatocellular carcinoma (HCC) is one type of human cancer in which epigenetic gene suppression is frequently found. To effectively detect a tumor-related, we performed triple combination array analysis, which consists of an expression array, a single nucleotide polymorphism (SNP) array, and a methylation array of the same surgical HCC specimen. Methods: Triple array analysis of one HCC sample from a 68-year-old female patient was performed. Gene expression profiles were determined using Affymetrix HGU133A and B Gene-Chips using RNA. The SNP chip array was conducted using the standard protocol for Affymetrix Gene Chip Mapping 500K arrays. Methylation array analysis used an Illumina Infinium HumanMethylation27 Beadchip Kit. The gene encoding Collagen type1 alpha1 (COL1A1) was identified and verified using HCC cell lines and 48 primary HCC tissues from patients who had undergone hepatectomy at our hospital. Results: Expression array analysis revealed that COL1A1 gene expression was decreased in tumor tissues (Log2 Ratio -1.1). In addition, SNP array analysis showed that the COL1A1 locus, 17q21, did not show a chromosomal deletion. Importantly, the methylation value (0-1.0) in the tumor tissue was higher (0.557) than that of the adjacent normal tissue (0.008). We hypothesized that the expression of this gene was suppressed by promoter hypermethylation. The suppressed expression of the COL1A1 gene in HCC cell lines was reactivated after 5-aza-2′-deoxycytidine (5-aza-dC) treatment. Sequence analysis identified hypermethylated CpG sites in the COL1A1 promoter region. In 48 HCC samples, 21 cases (44%) also showed COL1A1 gene promoter hypermethylation. The expression values of methylated cases, examined by real-time RT-PCR, were significantly downregulated (p=0.004) compared with unmethylated cases. Downregulation of the gene's expression was significantly correlated with pathological fc positive (p=0.026) and liver damage scores (p=0.011). Conclusion: Triple combination array analysis successfully revealed that the COL1A1 gene acts as a tumor suppressor gene in HCC via promoter hypermethylation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 322. doi:10.1158/1538-7445.AM2011-322

Abstract 323: Use of triple combination array analysis to identify STEAP family member 4 (STEAP4), a novel tumor suppressor gene in hepatocellular carcinoma

Abstract Background: Hepatocellular carcinoma (HCC) is one of the most common cancers world-wide, but the molecular mechanisms underlying hepatocarcinogenesis are not clear. Array technology has made it possible to identify novel genes with alterations in liver cancer. In this study, we performed triple combination array analysis, expression profiling, karyotyping analysis using SNP array, and array-based DNA methylation profiling in the same HCC sample and attempted to find a novel tumor suppressor gene as a prognostic marker. Materials &amp; Methods: A 68-year-old woman with chronic hepatitis C was diagnosed with HCC. The tumor and corresponding normal tissues were used for extraction of DNA and RNA. SNP chip array experiments were performed according to the standard protocol of Affymetrix GeneChip Mapping 500K arrays for DNA. mRNA expression profiles were determined using Affimetrix HGU133A and B Gene-Chips. Bisulphite-converted genomic DNA was analyzed using Illumina's Infinium Human Methylation27 Beadchip Kit. We consequently analyzed the identified gene using quantitative RT-PCR, methylation and un-methylation-specific PCR in nine HCC cell lines and in 48 primary HCC tissues and corresponding non-cancerous tissues collected at Nagoya University Hospital, and then compared these results with clinico-pathological data. Results: The STEAP4 gene, is located on 7q21.12, and expression was decreased at a Log2 ratio of -4.2 to -2.0 in cancerous tissue. There were two copies of 7q21.12, indicating no loss of heterozygosity (LOH) in the SNP array. The methylation value (0-1.0) in the tumor sample was 0.870 compared to 0.234 in non-cancerous tissue, indicating high methylation in tumor tissue in the Methylation array. Re-activation of STEAP4 expression was seen after 5-aza-dC treatment in HCC cell lines. Moreover, we found that 32 of 48 (67%) HCC samples showed promoter hypermethylation. In 32 methylated cases, the expression values of STEAP4 gene in tumor tissues by real-time RT-PCR were tended to decrease compared to corresponding normal samples. In addition, methylated cases were significantly correlated with positive results for Hepatitis B and α-feto protein and had a tendency for poor prognosis (p=0.098). Conclusion: The STEAP4 gene is a tumor suppressor gene in HCC and might be a marker of poor prognosis. The triple combination array was a useful technique to identify the novel methylated tumor suppressor gene in HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 323. doi:10.1158/1538-7445.AM2011-323