Yukiyasu Okamura

Adjuvant chemotherapy of S-1 versus gemcitabine for resected pancreatic cancer: a phase 3, open-label, randomised, non-inferiority trial (JASPAC 01)

BACKGROUND Although adjuvant chemotherapy with gemcitabine is standard care for resected pancreatic cancer, S-1 has shown non-inferiority to gemcitabine for advanced disease. We aimed to investigate the non-inferiority of S-1 to gemcitabine as adjuvant chemotherapy for pancreatic cancer in terms of overall survival. METHODS We did a randomised, open-label, multicentre, non-inferiority phase 3 trial undertaken at 33 hospitals in Japan. Patients who had histologically proven invasive ductal carcinoma of the pancreas, pathologically documented stage I-III, and no local residual or microscopic residual tumour, and were aged 20 years or older were eligible. Patients with resected pancreatic cancer were randomly assigned (in a 1:1 ratio) to receive gemcitabine (1000 mg/m(2), intravenously administered on days 1, 8, and 15, every 4 weeks [one cycle], for up to six cycles) or S-1 (40 mg, 50 mg, or 60 mg according to body-surface area, orally administered twice a day for 28 days followed by a 14 day rest, every 6 weeks [one cycle], for up to four cycles) at the data centre by a modified minimisation method, balancing residual tumour status, nodal status, and institutions. The primary outcome was overall survival in the two treatment groups, assessed in the per-protocol population, excluding ineligible patients and those not receiving the allocated treatment. The protocol prespecified that the superiority of S-1 with respect to overall survival was also to be assessed in the per-protocol population by a log-rank test, if the non-inferiority of S-1 was verified. We estimated overall and relapse-free survival using the Kaplan-Meier methods, and assessed non-inferiority of S-1 to gemcitabine using the Cox proportional hazard model. The expected hazard ratio (HR) for mortality was 0.87 with a non-inferiority margin of 1.25 (power 80%; one-sided type I error 2.5%). This trial is registered at UMIN CTR (UMIN000000655). FINDINGS 385 patients were randomly assigned to treatment between April 11, 2007, and June 29, 2010 (193 to the gemcitabine group and 192 to the S-1 group). Of these, three were exlcuded because of ineligibility and five did not receive chemotherapy. The per-protocol population therefore consisted of 190 patients in the gemcitabine group and 187 patients in the S-1 group. On Sept 15, 2012, following the recommendation from the independent data and safety monitoring committee, this study was discontinued because the prespecified criteria for early discontinuation were met at the interim analysis for efficacy, when all the protocol treatments had been finished. Analysis with the follow-up data on Jan 15, 2016, showed HR of mortality was 0.57 (95% CI 0.44-0.72, pnon-inferiority<0.0001, p<0.0001 for superiority), associated with 5-year overall survival of 24.4% (18.6-30.8) in the gemcitabine group and 44.1% (36.9-51.1) in the S-1 group. Grade 3 or 4 leucopenia, neutropenia, aspartate aminotransferase, and alanine aminotransferase were observed more frequently in the gemcitabine group, whereas stomatitis and diarrhoea were more frequently experienced in the S-1 group. INTERPRETATION Adjuvant chemotherapy with S-1 can be a new standard care for resected pancreatic cancer in Japanese patients. These results should be assessed in non-Asian patients. FUNDING Pharma Valley Center, Shizuoka Industrial Foundation, Taiho Pharmaceutical.

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.

Prognostic significance of postoperative complications after hepatectomy for hepatocellular carcinoma.

Hepatectomy is one of the best curative therapies for hepatocellular carcinoma (HCC). The negative impact of postoperative complications on long‐term survival has been investigated in patients with several kinds of cancers. To clarify the impact of postoperative complications on long‐term survival in patients who undergo hepatectomy for HCC, we conducted a comparative analysis of 376 patients using the modified Clavien classification and identified the predictors of postoperative complications.

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 a kinase anchor protein 12 (AKAP12) gene as a candidate tumor suppressor of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a major health problem, and identification of new tumor‐related genes is an urgent task.

Dihydropyrimidinase-like 3 is a putative hepatocellular carcinoma tumor suppressor

BackgroundPatients with hepatocellular carcinoma (HCC) may relapse after curative resection. Sensitive biomarkers for HCC are required to enhance disease management. Dihydropyrimidinase-like 3 (DPYSL3) suppresses cell proliferation and tumorigenicity of certain malignancies; however, its role in HCC is unknown.MethodsThe expression levels of DPYSL3 and genes encoding potential interacting proteins vascular endothelial growth factor (VEGF), focal adhesion kinase (FAK), ezrin, and cellular src were determined using RT-PCR. Further, we determined the methylation status of the DPYSL3 promoter in HCC cells lines and the effect of inhibiting DPYSL3 expression on their phenotype. DPYSL3 expression was determined in 151 pairs of resected liver tissues.ResultsDPYSL3 mRNA levels were down-regulated in most HCC cell lines with DPYSL3 promoter hypermethylation, and expression was restored after demethylation. DPYSL3 expression levels inversely correlated with those of VEGF and FAK. Knockdown of DPYSL3 significantly increased migration and the invasive properties of HCC cells. The mean level of DPYSL3 mRNA was significantly lower in HCC tissues compared with corresponding noncancerous tissues. The expression patterns of DPYSL3 mRNA and protein were consistent. DPYSL3 mRNA expression in HCC tissues inversely correlated with preoperative serum tumor markers and was significantly lower in patients with extrahepatic recurrences. Disease-specific and recurrence-free survival was significantly shorter in patients with down-regulated DPYSL3 expression.ConclusionsOur results indicate that DPYSL3 is a putative HCC tumor suppressor, and promoter hypermethylation potently regulates DPYSL3 transcription. Down-regulation of DPYSL3 expression in HCC tissues may serve as a predictive biomarker for HCC after curative resection.

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.

B‑cell translocation gene 1 serves as a novel prognostic indicator of hepatocellular carcinoma

Although the B‑cell translocation gene 1 (BTG1) plays an important role in apoptosis and negatively regulates cell proliferation, BTG1 expression in hepatocellular carcinoma (HCC) has not been evaluated. In this study expression analysis of BTG1 was conducted to clarify the role of BTG1 in the initiation of HCC carcinogenesis and progression. BTG1 mRNA expression levels were determined for HCC cell lines and 151 surgical specimen pairs using quantitative real‑time reverse transcription polymerase chain reaction (RT‑qPCR) assay. The mutational and methylation status of HCC cell lines were analyzed via high resolution melting (HRM) analysis and direct sequencing analysis to elucidate the regulatory mechanisms of BTG1 expression. The expression and distribution of the BTG1 protein in liver tissues were evaluated using immunohistochemistry (IHC). Decreased expression of BTG1 mRNA was confirmed in the majority of HCC cell lines (89%) and clinical HCC tissues (85%) compared with non‑cancerous liver tissues. Mutations or promoter hypermethylation were not identified in HCC cell lines. BTG1 mRNA expression levels were not influenced by background liver status. The pattern of BTG1 protein expression was consistent with that of BTG1 mRNA. Downregulation of BTG1 mRNA in HCC was significantly associated with shorter disease‑specific and recurrence‑free survival rates. Multivariate analysis of disease‑specific survival rates identified BTG1 mRNA downregulation as an independent prognostic factor for HCC (hazard ratio 2.12, 95% confidence interval 1.12‑4.04, P=0.022). Our results indicate that altered BTG1 expression might affect hepatocarcinogenesis and may represent a novel biomarker for HCC carcinogenesis and progression.

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.

Small-cell carcinoma in the common bile duct treated with multidisciplinary management

Small-cell carcinoma occasionally occurs in the gastrointestinal tract, with rare reports of it in the biliary system. When it does occur in the biliary system, it is mainly within the gallbladder, and those arising in the extrahepatic bile duct are extremely rare with only seven reported cases to date. A 62-year-old man with this unusual condition is documented. The patient was preoperatively diagnosed as having small-cell carcinoma on the basis of a biopsy specimen and was treated with multidisciplinary management, which consisted of preoperative chemotherapy, a curative resection, adjuvant chemotherapy, and radiation therapy. The patient died 23 months after the initial diagnosis (20 months after surgery). To the best of our knowledge, the survival time of this case was the longest among reported cases, and multidisciplinary management might contribute to a prolonged survival.