Akira Ooki

Potential utility of HOP homeobox gene promoter methylation as a marker of tumor aggressiveness in gastric cancer.

HOP homeobox (HOPX) is an unusual homeobox gene encoding three spliced transcript variants, among which the only HOPX-β promoter harbors CpG islands. The characteristics of its promoter methylation was analyzed using bisulfite sequencing and quantitative-methylation-specific polymerase chain reaction (Q-MSP), and the effects of HOPX expression were also examined. HOPX-β expression was silenced in all gastric cancer cell lines tested; its expression could be restored by treatment with demethylating agent. On Q-MSP, HOPX-β hypermethylation (cut-off value of 3.55) was found in 84% (67 out of 80) of primary tumor tissues and 10% (8 out of 80) of the corresponding normal tissues and could discriminate normal from tumor tissues (P<0.0001). The prognosis of the advanced cases with HOPX-β hypermethylation was as poor as those with stage IV disease when cut-off value was set at 11.28. This finding was validated in an independent cohort of 90 advanced gastric cancers. The HOPX-β hypermethylation was also an independent prognostic factor (P=0.029) on multivariate analysis. Exogenous HOPX expression significantly inhibited cell proliferation, colony formation and invasion as well as enhanced apoptosis. Taken together, HOPX-β promoter methylation is a frequent and cancer-specific event in gastric cancer. Quantitative assessment of HOPX-β methylation has great clinical potential as a marker of tumor aggressiveness.

Phosphatase of regenerating liver‐3 as a convergent therapeutic target for lymph node metastasis in esophageal squamous cell carcinoma

Phosphatase of regenerating liver‐3 (PRL‐3) is a molecule associated with metastasis in a diverse of cancers, which, however, remains largely unknown in esophageal squamous cell carcinoma (ESCC). We examined both the clinical significance of PRL‐3 expression and its biological roles, and assessed possibilities as a therapeutic target in ESCC. PRL‐3 expression was found in 78% (69 of 88) of the primary ESCC on immunohistochemistry; it was the strong independent predictor for lymph node metastasis (LNM) on a multivariate logistic regression model (p = 0.0014, relative risk =15.20). Additionally, gene amplification was found in 3 (7.9%) of the 38 primary tumors with PRL‐3 overexpression by fluorescence in situ hybridization, but in none of the 19 tumors without it. PRL‐3 small interfering RNA robustly repressed cell proliferation, anchorage‐independent colony formation and invasion and augmented 5‐FU‐induced apoptosis in all the tested ESCC cell lines with PRL‐3 overexpression, irrespective of its gene amplification status. PRL‐3 inhibitor (1‐4‐bromo‐2‐benzylidene rhodanine) also suppressed such metastatic properties in the cell lines with PRL‐3 overexpression, but not with its low expression. Inverse effects were observed by PRL‐3 forced expression. Collectively, PRL‐3 overexpression is a frequent event associated with LNM and plays a causative role in promoting cancer progression. Moreover, the expression status may be a landmark to select patients with benefit from PRL‐3‐targeted therapy. Thus, PRL‐3 could be a convergent therapeutic target against ESCC with LNM.

Prognostic value of KRAS and BRAF mutations in curatively resected colorectal cancer.

AIM To investigate the prognostic role of KRAS and BRAF mutations after adjustment for microsatellite instability (MSI) status in Japanese colorectal cancer (CRC) population. METHODS We assessed KRAS and BRAF mutations and MSI status in 813 Japanese patients with curatively resected, stage I-III CRC and examined associations of these mutations with disease-free survival (DFS) and overall survival (OS) using uni- and multivariate Cox proportional hazards models. RESULTS KRAS and BRAF mutations were detected in 312 (38%) of 812 and 40 (5%) of 811 tumors, respectively. KRAS mutations occurred more frequently in females than in males (P=0.02), while the presence of BRAF mutations was significantly associated with the female gender (P=0.006), proximal tumor location (P<0.001), mucinous or poorly differentiated histology (P<0.001), and MSI-high tumors (P<0.001). After adjusting for relevant variables, including MSI status, KRAS mutations were associated with poorer DFS (HR=1.35; 95%CI: 1.03-1.75) and OS (HR=1.46; 95%CI: 1.09-1.97). BRAF mutations were poor prognostic factors for DFS (HR=2.20; 95%CI: 1.19-4.06) and OS (HR=2.30; 95%CI: 1.15-4.71). Neither the BRAF by MSI interaction test nor the KRAS by MSI interaction test yielded statistically significant results for DFS and OS. CONCLUSION KRAS and BRAF mutations are associated with inferior survival, independent of MSI status, in Japanese patients with curatively resected CRC.

Cancer specific promoter CpG Islands hypermethylation of HOP homeobox (HOPX) gene and its potential tumor suppressive role in pancreatic carcinogenesis.

BackgroundWe have recently identified HOP hoemobox (HOPX) as a tumor suppressor gene candidate, characterized by tumor-specific promoter DNA hypermethylation in human cancers, and it can remarkably inhibit tumors’ aggressive phenotypes. In this current study, we for the first time examined methylation level of HOPX and tested the functional relevance in pancreatic cancer (PC).MethodsClinical features of HOPX promoter hypermethylation was investigated in 89 PC tissues, and immunohistochemistry was added. We also examined its functional relevance in phenotype assays such as soft agar, proliferation, invasion, and cell cycle analysis.ResultsPC tissues had HOPX gene hypermethylation as compared to the corresponding normal pancreas tissues, and its uniqueness was robust to discriminate tumor from normal tissues (AUC = 0.85, P < 0.0001). Unexpectedly, HOPX was increased in expression in tumor tissues, and immunohistochemistry revealed its predominant expression in the Langerhans islet cells, where HOPX was reduced in expression for PC cells with promoter hypermethylation. HOPX transfectants exhibited G1 arrest with subG1 accumulation, and inhibited tumor forming and invasive ability.ConclusionDefective expression of HOPX which is consistent with promoter DNA hypermethylation may explain aggressive phenotype of pancreatic cancer, and intense expression of HOPX in the Langerhans cells may in turn uniquely contribute to pancreatic carcinogenesis.

DNA Damage-Inducible Gene, Reprimo Functions as a Tumor Suppressor and Is Suppressed by Promoter Methylation in Gastric Cancer

In several types of human cancer, the gene expression of Reprimo, a highly glycosylated protein, is frequently silenced via methylation of its promoter. The aim of this study was to characterize the epigenetic inactivation of Reprimo and its biologic function and clinical relevance in gastric cancer. The correlation between Reprimo methylation and clinical relevance was assessed in 83 primary human gastric cancer tissues. The effects of Reprimo expression were also examined using in vitro and in vivo assays. Reprimo methylation was cancer specific and frequently observed. In two gastric cancer cell lines without Reprimo methylation, we observed faint or weak Reprimo expression under normal conditions and high expression under DNA-damaging conditions. In four gastric cancer cell lines with Reprimo methylation, however, Reprimo expression remained faint even under DNA-damaging conditions, with expression being restored in combination with agents that induce demethylation. Enforced Reprimo expression robustly inhibited cell proliferation and anchorage-independent colony formation and enhanced DNA damage-induced apoptosis. Inverse effects were observed via siRNA-mediated knockdown of endogenous Reprimo. Reprimo expression inhibited tumorigenesis in vivo. Reprimo methylation was also associated with a poor response in patients with gastric cancer treated with chemotherapy (P¼ 0.028), and a poor prognosis in patients with advanced gastric cancer (P¼ 0.03). In conclusion, Reprimo expression is normally induced in response to DNA damage, acting as a novel tumor suppressor in gastric cancer. However, Reprimo methylation abrogates its expression and effects. The clinical assessment of Reprimo promoter methylation may serve not only as a predictive marker for chemotherapy, but also as a marker for tumor aggressiveness. Mol Cancer Res; 11(11); 1362–74. ©2013 AACR.

Therapeutic potential of PRL-3 targeting and clinical significance of PRL-3 genomic amplification in gastric cancer

BackgroundPhosphatase of regenerating liver-3 (PRL-3) has deserved attention as a crucial molecule in the multiple steps of metastasis. In the present study, we examined the mechanisms regulating PRL-3 expression, and assessed the clinical potential of PRL-3-targeted therapy in gastric cancer.MethodsPRL-3 genomic amplification was analyzed using quantitative-polymerase chain reaction and/or fluorescence in situ hybridization in 77 primary gastric tumors. The anticancer activity of PRL-3 inhibitor (1-4-bromo-2-benzylidene rhodanine) treatment was evaluated against cancer cells with different genetic and expression status.ResultsPRL-3 genomic amplification was closely concordant with high level of its protein expression in cell lines, and was found in 20% (8/40) among human primary tumors with its expression, which were all stage III/IV disease (40%, 8/20), but in none (0/37) among those without expression. Additionally, PRL-3 genomic amplification was associated with metastatic lymph node status, leading to advanced stage and thereby poor outcomes in patients with lymph node metastasis (P = 0.021). PRL-3 small interfering RNA robustly repressed metastatic properties, including cell proliferation, invasion, and anchorage-independent colony formation. Although neither PRL-3 genomic amplification nor expression level was responsible for the sensitivity to PRL-3 inhibitor treatment, the inhibitor showed dose-dependent anticancer efficacy, and remarkably induced apoptosis on all the tested cell lines with PRL-3 expression.ConclusionsWe have for the first time, demonstrated that PRL-3 genomic amplification is one of the predominant mechanisms inducing its expression, especially in more advanced stage, and that PRL-3-targeted therapy may have a great potential against gastric cancer with its expression.

Combined Microsatellite Instability and BRAF Gene Status As Biomarkers for Adjuvant Chemotherapy in Stage III Colorectal Cancer

The clinical relevance of combined microsatellite instability (MSI) and BRAF status for adjuvant treatment in stage III colorectal cancer (CRC) remains elusive.

Oncological outcomes of laparoscopic surgery in elderly patients with colon cancer: a comparison of patients 64 years or younger with those 75 years or older.

BACKGROUND/AIMS We compared the results of laparoscopic resection of colon cancer between patients 75 years or older and those 64 years or younger, to confirm whether this procedure is warranted in elderly patients. METHODOLOGY The study group was comprised of patients with stage I to III colon cancer treated by laparoscopic surgery from 1995 through 2006. Oncologic outcomes were compared between 74 patients 75 years or older (elderly group) and 74 patients 64 years or younger (younger group) who were matched for gender, tumor location and pathological tumor-node-metastasis (TNM) stage. RESULTS In patients with stage I or II disease, the disease-free survival rate and overall survival rate were similar in the elderly group (100% and 100%, respectively) and the younger group (95.6% and 95.8%, respectively). In patients with stage III disease, the disease-free survival rate and overall survival rate were also similar in the elderly group (76.7% and 88.5%, respectively) and the younger group (88.5% and 88.5%, respectively). CONCLUSIONS Postoperative complications and long-term oncologic outcomes were similar in elderly patients and younger patients with colon cancer who underwent laparoscopic colectomy in our hospital. These results demonstrate that laparoscopic resection of colon cancer is warranted in patients 75 years or older.

Everolimus Dramatically Improves Glycemic Control in Unresectable Metastatic Insulinoma: A Case Report

Hypoglycemia poses a significant management challenge in patients with unresectable metastatic insulinoma. A 57-year-old woman with pancreatic neuroendocrine tumor with multiple liver metastases was referred to our institution. During the clinical course of pancreatic neuroendocrine tumor, she had experienced palpitations, cold sweats and faintness between meals that indicated her tumors had attained the characteristics of an insulinoma, and her quality of life was impacted by frequent hypoglycemic episodes which could not be prevented by conventional therapies. Shortly after the approval of everolimus for pancreatic neuroendocrine tumor in Japan, we began oral administration at 10 mg per day, which produced a rapid and substantial improvement in glycemic control. The serum insulin level decreased dramatically despite the tumor size remaining stable on computed tomography evaluation. Despite a dose reduction of everolimus to 5 mg per day in response to the adverse reaction of interstitial pneumonitis and a subsequent moderate increase in the serum insulin level, the patient has maintained normoglycemia for a year. Everolimus might represent the treatment of choice for unresectable insulinoma in terms of not only tumor stabilization but also glycemic control.

Quality of Life Analysis in Patients With RAS Wild-Type Metastatic Colorectal Cancer Treated With First-Line Cetuximab Plus Chemotherapy

Micro‐Abstract The present exploratory and descriptive retrospective analysis assessed the quality of life (QoL) of CRYSTAL study patients with RAS wild‐type (wt) metastatic colorectal cancer (mCRC). Our data suggest that adding cetuximab to first‐line FOLFIRI (5‐fluorouracil, leucovorin, irinotecan) improved progression‐free survival, overall survival, and objective response rate without negatively affecting QoL in CRYSTAL study patients with RAS wt mCRC. Background: Adding cetuximab to FOLFIRI (5‐fluorouracil, leucovorin, irinotecan) significantly improved progression‐free survival (PFS), overall survival (OS), and objective response rate (ORR) in patients with KRAS or RAS (KRAS/NRAS, exons 2‐4) wild‐type (wt) metastatic colorectal cancer (mCRC) in the first‐line CRYSTAL study. The present exploratory and descriptive retrospective analysis assessed the quality of life (QoL) of CRYSTAL study patients with RAS wt mCRC—the labeled indication for cetuximab in many countries. Patients and Methods: Patient QoL was investigated using the European Organisation for Research and Treatment of Cancer QoL questionnaire core‐30 (EORTC QLQ‐C30). QoL assessments were performed at baseline, after every 8 weeks of treatment, and at the final tumor assessment. RAS wt patients were considered evaluable for QoL if they had provided ≥ 1 evaluable EORTC QLQ‐C30. Results: Of the 367 patients with RAS wt tumors, 351 were evaluable for QoL. Global health status (GHS)/QoL and the time to worsening of Eastern Cooperative Oncology Group performance status were similar between the treatment groups. However, the analysis was complicated by a large decrease in the number of evaluable patients in the FOLFIRI arm between weeks 32 and 40. The individual dimensions of interest in mCRC (eg, social functioning, fatigue, nausea/vomiting, pain, appetite loss, constipation, diarrhea, and functional difficulties) were also similar between the treatment arms. Changes in GHS/QoL and social functioning from baseline to week 8 were similar, irrespective of whether patients experienced early skin reactions. Conclusion: The findings of the present descriptive retrospective analysis suggest that adding cetuximab to first‐line FOLFIRI improves PFS, OS, and ORR without negatively affecting the QoL of CRYSTAL study patients with RAS wt mCRC.