Hiroshi Nishimiya

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.

Identification of EGFR expression status association with metastatic lymph node density (ND) by expression microarray analysis of advanced gastric cancer

Metastatic lymph node density (ND) has been reproducibly proven to be a prognostic factor in gastric cancer. The molecular mechanisms that underlie this aggressiveness are underexplored. Here, we aimed to identify molecules associated with this unique phenotype. Tumor specimens from patients with stage III gastric cancer with high or low ND (n = 4 for both) were compared at the mRNA level using Affymetrix microarray (harboring 54,675 genes). The expression data were prioritized, and genes that correlated with ND were selected. Ultimately, the EGFR was validated as such a candidate molecule in patients with primary advanced gastric cancer who underwent standard treatment (n = 167). Expression data of the microarray were prioritized based on gene expression ratio and frequency of gene expression. The first priority genes to be selected were genes that are known to be amplified in cancer, which included NKX2.1, CHST9, CTNND2, SLC25A27, FGFR2, EGFR, and PTGER1. Of these genes, the EGFR gene was of particular interest. EGFR expression in primary gastric cancer was examined using immunohistochemistry (IHC). The Students t‐test elucidated a significant difference in EGFR expression between IHC 2+/3+ and IHC 1+ according to ND (P = 0.0035). The Chi‐square test also indicated a significant difference between high and low levels of EGFR immunohistochemical staining (IHC2+/3+ and IHC1+, respectively) and ND status (P = 0.0023). According to the least squares method, as ND increased, the risk that EGFR staining levels changed from IHC 1+ to IHC 2+ also increased. In this study, we determined that high EGFR expression may underlie the aggressive mechanism of advanced gastric cancer with high ND.

Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1) Gene in Primary Breast Cancer.

Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1) gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC) with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP) in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004). Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007). Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.

The Safety of Concentrated Trastuzumab in 100 ml of Saline Solution for Administration to Patients with HER2-Positive Breast Cancer: A Phase 1 Study

Background: It is recommended that administration of trastuzumab should be carried out in a volume of 250 ml of saline solution over 90 min. Since 2011, recommendations have allowed a shortening of the administration time to 30 min at the second administration. However, the volume to be administered is still 250 ml. The purpose of this study was to evaluate the safety of trastuzumab administered in 100 ml of saline solution over 30 min. Methods: This study enrolled patients with HER2-positive breast cancer. Three dose levels of trastuzumab, each in 100 ml of saline solution, were used (2, 6 and 8 mg/kg). The primary end point was the determination of safety. Results: Nine patients were enrolled. Since no adverse events were observed, the 8 mg/kg/100 ml saline solution dose level was the recommended dose. Conclusions: A 30-min administration of trastuzumab in 100 ml of saline solution is safe in patients with HER2-positive breast cancer.

Abstract P3-09-03: Final result of randomised controlled phase II study of the efficiency of palonosetron, aprepitant, and dexamethasone for day1 with or without dexamethasone on days2 and 3

Background: Emesis is one of the major non-hematologic toxicity caused by chemotherapy. The control of Chemotherapy Induced Nausea and Vomiting (CINV) is conducted to a life lengthening. Recently, CINV is controlled by the second generation 5-HT3 receptor blocker (Palonosetron; PALO) and a NK-1 receptor antagonist (Aprepitant; APR). It has been shown that dexamethasone (DEX) with 5-HT3 receptor blocker improves acute / delayed CINV. However, it has not been determined the medication schedule for DEX with PALO and APR. The purpose of this study is evaluated the efficiency of palonosetron, aprepitant and dexamethasone for day1 with or without dexamethasone on days2 and 3. This is final result of current study. Methods: Breast cancer patients who administered anthracyclin drug regimen have been eligible from April, 2011 to June, 2013. The patients were randomised to group A (PALO/APR/DEX one day) and group B (PALO/APR/DEX three days). Eighty patients were estimate as study samples. The primary endpoint was a complete response (CR) rate of vomiting, the secondary endpoint was a complete control (CC) rate of vomiting. Results: Eighty two patients were enrolled in this study. There was not inferiority about CR rate of five days after chemotherapy (81.1% of group A, 82.1% of group B). CC rate of group A (62.2%) was better than that of group B (46.2%). However, CC rate was no significant difference between group A and B. There was no significant difference about any level of nausea between group A and B. Conclusions: One day of dexamethasone with Palonosetron and Aprepitant treatment is enough to control the emesis of high emetogenic chemotherapeutic agents. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-09-03.

Lymph node metastasis and high serum CEA are important prognostic factors in hormone receptor positive and HER2 negative breast cancer

In recent years, treatment options for breast cancer have increased, and prognosis has improved since the 1990s. The present study examined the prognosis for recurrence of breast cancer between 2006 and 2009, in comparison with the results of past treatments, and sought to guide future treatment strategies by elucidating present prognostic factors. A total of 662 patients with breast cancer stage 0-III who underwent surgery at Kitasato University Hospital between January 2006 and March 2009 were included. Cases were classified into four subtypes, based on the presence or absence of hormone receptors and human epidermal growth factor receptor 2 (HER2). Factors associated with recurrence and prognosis were then examined. The 5-year recurrence-free survival (RFS) was 94.9% and the 5-year disease-specific survival (DSS) was 98.4%. Factors related to RFS were pathological lymph node (pN) positive [hazard ratio (HR)=2.85, P=0.001], clinical lymph node (cN) positive (HR=2.28, P<0.01), and hormone receptor negative (HR=1.83, P<0.05). Factors associated with DSS were cN positive (HR=4.55, P<0.01), pN positive (HR=3.40, P<0.05), higher preoperative serum carcinoembryonic antigen (CEA) (HR=3.04, P<0.05), and hormone receptor negative (HR=2.32, P<0.05). In the hormone receptor positive HER2 negative, cN-positive/pN-positive breast cancer group, RFS and DSS were poorer compared with the other groups. In this group, preoperative high CEA level was a poor prognostic factor. The prognosis for hormone receptor positive HER2-negative breast cancer has improved significantly since the 1990s. On the other hand, the prognosis for cN-positive/pN-positive breast cancer was poor. Pre-treatment serum CEA positive cases exhibited a particularly poor prognosis.

BRCAness and Prognosis in Triple-Negative Breast Cancer Patients Treated with Neoadjuvant Chemotherapy.

BRCAness is defined as the set of traits in which BRCA1 dysfunction, arising from gene mutation, methylation or deletion, results in DNA repair deficiency. In the present study, we addressed BRCAness, therapeutic efficacy, recurrence, and survival in patients with triple negative breast cancer (TNBC) who were treated with neoadjuvant chemotherapy at Kitasato University Hospital, Japan, between April 2006 and October 2012. BRCAness was determined by preoperative core needle biopsy (CNB) specimens and surgical specimens. Assay was performed using Multiplex Ligation-dependent Probe Amplification (MLPA) with P376-B2 BRCA1ness probemix (MRC-Holland, Amsterdam, The Netherlands). The relative copy number ratio of each sample was compared to Human Genomic DNA (Promega, Madison, WI, USA) as reference samples was calculated with Coffalyser.NET default settings. The BRCAness score was calculated with the relative copy number ratio of various DNA sequences. Values of 0.5 or more were determined as the BRCA1-like Type (BRCAness) and those of less than 0.5 as the Sporadic Type to analyze pathological complete response (pCR) rate, recurrence, and survival. pCR (ypT0/Tis/N0) was observed in 15 patients (pCR rate: 37.5%). These patients had no recurrence. Twelve patients recurred, 8 died from breast cancer. The BRCA1-like Type were 22 and Sporadic Type were 18 in CNB specimens. No major differences were observed between the BRCA1-like Type and Sporadic Type with pCR rate, recurrence rate and survival. Twenty four surgical specimens of non-pCR patients were available and 9 were BRCA1-like Type, who had more recurrences (7/9 vs. 5/15), and their relapse-free survival was also lower (p<0.05) than that of Sporadic Type. Seven BRCA1-like Type patients remained BRCA1-like Type in surgical specimens, were worse in recurrence (p<0.01) and survival (p<0.05) compared with 6 patients whose BRCA status in surgical specimens turned to Sporadic Type. New clinical trials assessing the true recurrence (TR) rate of BRCA-type patients are expected since neither platinum-containing drugs nor poly (ADP-ribose) polymerase (PARP) inhibitors are effective against tumors with nonfunctional BRCA genes.

Abstract 4662: Epigenetic conversion on p53 pathway in human cancer: The clinical potential of p53 mutation status as a predictive biomarker for drug sensitivity by epigenetic treatment.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: DNA methylation plays a central role in epigenetic contribution to human cancer progression. We have mentioned actual epigenetic conversion on critical molecular pathways in human cancer (Mini-symposium on AACR 2012). Such genes included PGP9.5, NMDAR2B, DAPK, and Cyclin A1, which have been all demonstrated to be involved in apoptotic process on upstream or downstream of p53 pathway. Materials and Methods: Epigenetic profiles of PGP9.5, NMDAR2B, and Cyclin A1 were examined in 163 primary gastric cancer by Q-MSP in combination with p53 mutation status (exon 4 to 8) by SSCP. Effects of epigenetic reversion by 5-aza-2’-deoxycytidine and trichostatin A, in combination with chemotherapeutic drug, CDDP, were assessed for apoptosis by flow cytometry and p53 trans-activation ability by luciferase analysis in gastric cancer cell lines with or without p53 mutation. Results: (1) SSCP identified 44 mutations (27%) of p53 gene in primary gastric cancer. Mutation status of p53 gene was not of prognostic relevance. (2) We performed epigenetic profiles for PGP9.5, NMDAR2B, DAPK, and Cyclin A1. Methylation level is higher in primary tumors with p53 wild type than in those with p53 mutant in gastric cancer (PGP9.5>NMDAR2B>Cyclin A1>DAPK).(3) Importantly, super-high methylation level of PGP9.5, NMDAR2B, and cyclin A1 was exclusively found in primary tumors with no p53 mutation, and such cut-off TaqMeth values were 50, 163, and 133, respectively. (4) Super-high methylation was found in 14 for PGP9.5, 19 for NMDAR2B, and 8 in Cyclin A1, which are not always redundant. Super-high methylation may represent functional shut-down of expression in the individual genes. (5) In NUGC4 gastric cancer cell line, harboring wild type p53, epigenetic treatment remarkably augmented apoptosis by CDDP chemotherapy, concordant with p53 transcription activity. This finding suggested that epigenetic treatment could be additional options to classical chemotherapy more efficiently to kill cancer cells with wild type p53. (6) On the other hand, in KATO III cancer cell line, harboring null p53 (mutant), epigenetic treatment alone induced robust apoptosis, with no trans-activation of p53. Conclusion: In GI cancers, several genes involved on the p53 pathway are functionally suppressed in expression by super-dense methylation of the promoter regions, and epigenetic reversion of p53 activity may be beneficial for killing cancer cells more efficiently, if p53 is not mutated. On the other hand, cancer cell with no p53 mutation is very sensitive by epigenetic treatments, and p53 status may predict sensitivity of cancer cells by chemotherapy in combination with epigenetic treatments. Citation Format: Keishi Yamashita, Mina Waraya, Hiroshi Katoh, Akira Ooki, Hiroshi Kawamata, Kazunori Nakamura, Hiroshi Nishimiya, Akira Ema, David Sidransky, Masahiko Watanabe. Epigenetic conversion on p53 pathway in human cancer: The clinical potential of p53 mutation status as a predictive biomarker for drug sensitivity by epigenetic treatment. [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 4662. doi:10.1158/1538-7445.AM2013-4662 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.