Takashi Kawai

Comparison of Incubation Solutions Prior to the Purification of Porcine Islet Cells.

For pancreatic islet transplantation, one of the most important steps of islet isolation is islet purification. The most common method of islet purification is density gradient centrifugation because there are differences in density between islets and acinar tissue. However, the density of islets/acinar tissue depends on several conditions, such as the incubation time before purification and the osmolality of the preincubation solution. In this study, we evaluated the impact of using two different preincubation solutions before purification. We used the University of Wisconsin (UW) solution and a new preservation solution (HN-1), which we recently developed. There were no significant differences between the two solutions in terms of the islet yield, rate of viability, and purity or stimulation index after purification. There were also no differences in the attainability and suitability of posttransplantation normoglycemia. Our study shows that the HN-1 solution is equivalent to the UW solution for preincubation before islet purification.

Comparison of New Preservation Solutions, HN-1 and University of Wisconsin Solution, in Pancreas Preservation for Porcine Islet Isolation.

For pancreatic islet transplantation, maintaining organ viability after pancreas procurement is critical and a major determinant for better graft function and survival. University of Wisconsin (UW) solution is currently the gold standard for abdominal organ preservation and the pancreas in particular. However, in the use of UW preservation solution for islet transplantation, there are disadvantages to be overcome, such as the inhibition of collagenase activity during pancreatic digestion. In this study, we compared UW solution with HN-1 solution in pancreas preservation for islet isolation. Islet yield was significantly greater in the HN-1 group than the UW group both before and after purification. In the in vitro assay, the adenosine triphosphate content in cultured islets was significantly higher in the HN-1 group than in the UW group. Furthermore, in streptozotocin-induced diabetic nude mice, the islet graft function of the HN-1 group was superior to that of the UW group. We concluded that the use of HN-1 solution is a promising approach for optimal pancreas preservation in islet transplantation.

Abstract 2317: PD-1 and PD-L1 expression patterns and DNA mismatch repair status for precision management of patients with gastric cancer

BACKGROUND: Programmed cell death protein 1 (PD-1) and its ligand (PD-L1) downregulate T cell activation and are related to immune tolerance. Recently, microsatellite unstable (MSI) colorectal cancers have been shown to have good therapeutic response to PD-1 antibody immunotherapy, possibly due to release exhaustion of their ability to recognize high number of tumor neo-antigens. The aim of this study was to clarify the significance of PD-1 and PD-L1 expression, and to analyze the relationship between MSI status and MLH1 hypermethylation status in gastric cancer. METHODS: A total of 105 patients who underwent curative gastrectomy for gastric cancer were included in this study. The PD-1, PD-L1, HER2, and mismatch repair proteins (MLH1/PMS2/MSH2/MSH6) expression were examined by immunohistochemistry. MSI status were examined by analyzing three mononucleotide repeat markers. MLH1 methylation was evaluated using a highly sensitive fluorescence-based PCR assay, as we reported previously (JNCI 2009). KRAS/BRAF/PIK3CA mutations were determined by conventional Sanger sequencing. Infection of Helicobacter Pylori (H. Pylori) and EB virus (EBV) were determined by amplifying H. Pylori and EBV specific sequence by PCR, followed by conventional Sanger sequencing. RESULTS: PD-1 and PD-L1 were expressed in 40% (38/95) and 33% (31/95) gastric cancers, respectively. HER2 was expressed in 15% (14/96) gastric cancers. Infection of H.Pylori and EBV were observed in 71% (70/98) and 8% (8/98) gastric cancers, respectively. MSI was observed in 13% (13/98) gastric cancers. Among 13 MSI cancers, 11 cases (85%) showed extensive methylation in the MLH1 promoter region, suggesting their sporadic manifestation. KRAS (exon 2), BRAF (exon 15) and PIK3CA (exon 9 & 20) mutations were observed in 5% (5/98), 0% (0/98), and 4% (4/98), respectively. Among these, one case harbored simultaneous KRAS and PIK3CA mutation. Gastric cancers with KRAS/PIK3CA were more frequently MSI-positive (5/8, p = 0.003). Among eight EBV infected cancers, only one case showed MSI and MLH1 methylation. PD-1 expression was significantly correlated with PD-L1 expression (p = 0.04). Although both PD-1 and PD-L1 expression were associated with MSI (p = 0.007 and p = 0.008, respectively), only PD-L1 expression was significantly correlated with gastric cancers with MLH1 methylation (p = 0.01). While expression of immune checkpoint molecules were specific for tumors with MMR deficiency, HER2 expression was exclusively observed in MMR-proficient tumors. CONCLUSIONS: PD-1/PD-L1 expression is associated with MMR-deficient gastric cancers. In contrast, HER2 expression is observed exclusively in gastric cancers with MMR-proficiency. Our result highlight that expression analysis of genetic markers could lead the precision management of patients with gastric cancer. Citation Format: Keisuke Kimura, Takeshi Nagasaka, Yoshiko Mori, Takashi Kawai, Tomokazu Fuji, Fumitaka Taniguchi, Kazuya Yasui, Toshiaki Toshima, Yuzo Umeda, Hiroshi Tazawa, Ajay Goel, Toshiyoshi Fujiwara. PD-1 and PD-L1 expression patterns and DNA mismatch repair status for precision management of patients with gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2317.

Clinical outcomes of women with ovarian metastases of colorectal cancer treated with oophorectomy with respect to their somatic mutation profiles

We clarified the clinical prevalence of ovarian metastases from colorectal cancers (CRCs) in 296 female patients with CRC and evaluated clinical outcomes with relation to their mutational profiles, such as BRAF/KRAS mutation and microsatellite instability (MSI) status. The female CRCs were categorised into three subsets: CRCs with ovarian metastases [6.4% (n = 19), 5-year overall survival (OS) = 24.7%], CRCs with extra-ovarian metastases only [32.4% (n = 96), 5-year OS = 34.5%] and CRCs without any recurrence or metastasis [61.2% (n = 181), 5-year OS = 91.3%]. All patients with ovarian metastases underwent oophorectomy; of these, 9 who received preoperative chemotherapy had measurable metastases to extra-ovarian sites and the ovaries. Although 5 of 9 (56%) achieved partial response or complete response at extra-ovarian sites, no patient archived objective response at ovarian sites. Regarding the mutation profiles, in CRCs with extra-ovarian metastases only, the median survival time (MST) after initial treatments to progression to stage IV or recurrence was 13 [95% confidence interval (CI): 7–16 months] in BRAF-mutant and 34 months (95% CI: 22–58 months) in BRAF wild-type (P = 0.0033). Although ovarian metastases demonstrated poor response to systemic chemotherapy in CRCs with ovarian metastases, the MST after initial treatments to progression to stage IV or recurrence was 22 (95% CI: 21–25 months) in BRAF-mutant and 38 months (95% CI: 24–42 months) in BRAF wild-type (P = 0.0398). The outcomes of patients with ovarian metastases could be improved by oophorectomy regardless of their mutation profiles.

BRAF V600E mutation is a predictive indicator of upfront chemotherapy for stage IV colorectal cancer

In stage IV colorectal cancer (CRC), initial resection of the primary tumor is considered to be an important strategy for improving disease outcome. However, there is no consensus on the timing as to when the surgical intervention of the primary tumor should occur. The present study hypothesizes that genetic profiles in CRC may indicate the appropriate treatment strategies for patients with stage IV CRC, and a cohort of 113 patients with stage IV CRC resected primary lesions at various periods were analyzed for the presence of mutations in the KRAS, exon 2, and BRAF genes, exon 15, and for the microsatellite instability status of the tumor. These data were additionally correlated with various clinicopathological features. Although BRAF-mutant was revealed to be an independent negative prognostic factor in stage IV CRC (HR, 8.42; 95% confidence interval, 2.72–26.02), BRAF-mutant samples exhibited better prognoses if they were treated with chemotherapy prior to tumor resection. Thus, the presence of BRAF mutations provides a compelling rationale for the establishment of intensive upfront chemotherapy to improve survival in stage IV CRC.

Abstract 510: A novel circulating cell free DNA-based assay in colorectal cancer patients during treatment with systematic chemotherapy

Introduction: Although circulating cell-free DNA (cfDNA) in blood plasma is being touted as a frontier noninvasive approaches, its clinical utility still remains questionable. The purpose of this study was to compare the efficacy of cfDNA by comparing blood CEA levels and radiological evaluation in patients with unresectable metastatic colorectal cancer (mCRC) who received systemic chemotherapy. Experimental Procedures: In this study, we measured aberrant cancer-specific methylation in cfDNA and the concentration of cfDNA in plasma obtained following each treatment cycle of systemic chemotherapy in three patients with mCRC. To analyze aberrant cancer-specific methylation, we used a modified highly sensitive assay for bisulfite DNA followed by fluorescence-based PCR, as reported previously (JNCI 2009). This methodology can detect methylation status in eight regions, therefore both recovery score (RS) and methylation score (MS), ranged from 0-8 at a given time. We measured RS and MS two-times in each plasma specimen obtained before administration of systemic chemotherapies. Results: In this pilot study, we examined a series of blood plasma obtained from three patients who received oxaliplatin-based chemotherapy together with molecularly-targeted agents. Despite initial tumor shrinkage in the metastases, all patients ultimately developed progressive disease (PD). Patient1 had wild-type KRAS, but had developed a sigmoid colon cancer with synchronous multiple liver and lung metastases. In contrast, Patient2 had mutant KRAS with sigmoid colon cancer and synchronous multiple liver metastasis. Both patients 1 and 2, demonstrated decreasing levels of CEA after the first-line chemotherapy, along with low methylation scores and concentration of cfDNA. Interestingly, in both patients, MS and concentration level of cfDNA increased prior to radiographic documentation of PD. Patient3 harbored BRAF V600E mutation, and a cancer in the ascending colon with systemic lymph node metastasis. Although, in this case, the tumor development progressed rapidly, similar to patients 1 and 2, MS and the concentration levels of cfDNA also increased prior to radiographic documentation of rapid PD. Conclusions: Our novel DNA methylation and concentration-based monitoring assay is a novel methodology for capturing DNA methylation in circulating cell-free DNA in plasma, and is useful for the early identification of colorectal cancer patients who are at risk of developing PD prior to radiographic documentation. Citation Format: Toshiaki Toshima, Takeshi Nagasaka, Yoshiko Mori, Takashi Kawai, Tomokazu Fuji, Fumitaka Taniguchi, Keisuke Kimura, Kazuya Yasui, Hiroyuki Kishimoto, Yuzo Umeda, Hiroshi Tazawa, Ajay Goel, Toshiyoshi Fujiwara. A novel circulating cell free DNA-based assay in colorectal cancer patients during treatment with systematic chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 510.

Abstract 1078: Micro RNAs as promising therapeutic targets for anti-metastatic therapy in colorectal cancer

Background: Colorectal cancer (CRC) is the third most common cancer and is a leading cause of cancer related death worldwide and arises by accumulation of genetic and epigenetic alterations. Recently, it has been demonstrated that microRNAs (miRNAs) play critical roles in tumor progression in various cancers. In this study, we tried to find miRNAs associated with distant metastasis and poor outcome and evaluate those miRNAs as therapeutic target for advanced CRC. Methods: MiRNA array was done on CRC specimens derived from tumors with various gene status (KRAS/BRAF/microsatellite instability [MSI] status) with reference of their normal mucosal specimens. The array analysis revealed that a set of miRNAs was specifically down-regulated in CRC with BRAF V600E mutation without MSI, considered to be the poorer outcome. To examine whether the set of miRNAs causes distant metastasis or not, we investigated malignant potential of cell lines transfected and knock-downed the set of miRNAs by siRNA. Expression status of ZEB2 and Epithelial-Mesenchymal Transition (EMT) markers, E-cadherin, were evaluated. In addition, we analyzed expression level of the set of miRNAs in a cohort of 67 stage IV CRCs (TNM staging system by UICC 7th) by quantitative reverse transcription PCR using a comparative Ct method and examined association of target-miRNA fold change (tumor/normal tissue) and their clinocopathilogical findings. Patient survival analysis were performed by Cox proportional hazard model and Kaplan-Meier analysis. Results: In vitro, cell lines transfected the set of miRNAs significantly reduced their malignant potentials. In contrast cell lines knock-downed the set of miRNAs by siRNA obviously increased their malignant potentials. Finally, to confirm our results obtained from in vitro assays, we analyzed expression level of the set of miRNAs in a cohort of stage IV CRCs. Of 67 patients with stage IV CRCs, 15 patients showed KRAS mutation, 4 patients showed BRAF mutation. CRCs with the lower expression level of the set of miRNAs showed poor outcome compared with those with the higher expression level by Cox proportional hazard model and Kaplan-Meier analysis. Conclusions: Our data indicate that miRNAs associated with BRAF mutant CRCs is promising prognostic biomarkers and therapeutic targets for anti-metastatic therapy in CRC. Citation Format: Takashi Kawai, Takeshi Nagasaka, Yoshiko Mori, Tomokazu Fuji, Fumitaka Taniguchi, Keisuke Kimura, Toshiaki Toshima, Kazuya Yasui, Yuzo Umeda, Hiroshi Tazawa, Ajay Goel, Toshiyoshi Fujiwara. Micro RNAs as promising therapeutic targets for anti-metastatic therapy in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1078.

Establishment of a pancreatic stem cell line from fibroblast-derived induced pluripotent stem cells

BackgroundFor cell therapies to treat diabetes, it is important to produce a sufficient number of pancreatic endocrine cells that function similarly to primary islets. Induced pluripotent stem (iPS) cells represent a potentially unlimited source of functional pancreatic endocrine cells. However, the use of iPS cells for laboratory studies and cell-based therapies is hampered by their high tumorigenic potential and limited ability to generate pure populations of differentiated cell types in vitro. The purpose of this study was to establish a pancreatic stem cell line from iPS cells derived from mouse fibroblasts.MethodsMouse iPS cells were induced to differentiate into insulin-producing cells by a multi-step differentiation protocol, which was conducted as described previously with minor modifications. Selection of the pancreatic stem cell was based on morphology and Pdx1 expression. The pancreatic potential of the pancreatic stem cells was evaluated using a reverse transcription PCR, real-time PCR, immunofluorescence, and a glucose challenge test. To assess potential tumorigenicity of the pancreatic stem cells, the cells were injected into the quadriceps femoris muscle of the left hindlimb of nude mice.ResultsThe iPS-derived pancreatic stem cells expressed the transcription factor –Pdx1– a marker of pancreatic development, and continued to divide actively beyond passage 80. Endocrine cells derived from these pancreatic stem cells expressed insulin and pancreatic genes, and they released insulin in response to glucose stimulation. Mice injected with the pancreatic stem cells did not develop tumors, in contrast to mice injected with an equal number of iPS cells.ConclusionThis strategy provides a new approach for generation of insulin-producing cells that is more efficient and safer than using iPS cells. We believe that this approach will help to develop a patient-specific cell transplantation therapy for diabetes in the near future.

225PEXTENSIVE METHYLATION OF EPIDERMAL GROWTH FACTOR-CONTAINING FIBULIN-LIKE EXTRACELLULAR MATRIX PROTEIN 1 (EFEMP1) PROMOTER COULD PREDICT MALIGNANT FORMATION IN INTRADUCTAL PAPILLARY MUCINOUS NEOPLASMS (IPMN)

ABSTRACT Introduction: Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are pancreatic cystic tumors. Genetic and epigenetic alterations are believed to be accumulated during tumor progression. Whereas the specific genetic mutations and DNA methylation for each type of lesion have been somewhat known, the fundamental understanding for the dynamics of methylation expansion across specific CpG dinucleotides in a gene promoter during carcinogenesis remains unclear. We sought to evaluate whether the expansion of methylation in the EFEMP1 promoter could serve as a predictive biomarker for malignant IPMNs. Methods: KRAS mutations and the methylation status of two discrete regions within the EFEMP1 promoter in 65 IPMN tissues, including 30 IPMNs with low- and intermediate-grade dysplasia, 12 IPMNs with high-grade dysplasia and 23 invasive IPMN, were determined using genomic sequencing and High Sensitive Assay for bisulfate modification DNA (Hi-SA) - a modified COBRA by which fluorescence labeled DNA fragments are detected using a genetic analyzer (Nagasaka T et al., JNCI 2009), respectively. Thereafter, correlation between mutant KRAS, methylation status and various clinic-pathological features were examined. Results: KRAS mutations were detected in 42.9% of low- and intermediate-grade IPMNs, 33.3% of high-grade IPMNs and 71.4% of invasive IPMNs. All mutations were confined to codon 12 of the KRAS gene, and these mutations did not correlate with a specific histological grade or type of lesions, or any of the other clinic-pathological features. On the other hand, while the methylation of the region 1 or region 2 within the EFEMP1 promoter was observed in more than 80% of non-invasive IPMNs and invasive IPMNs, simultaneous methylation of both regions occurred in 0% of noninvasive vs. 34.7% of invasive IPMNs (p = 0.00148). These results suggest that KRAS mutations and extensive methylation within the EFEMP1 promoter were one of the early and late events in cancer progression, respectively. Conclusions: The extensive methylation in EFEMP1 promoter could be a potential predictive marker for identification of invasive IPMNs, and provides an attractive rationale for developing this molecular signature as a substrate for the development of non-invasive screening for invasive IPMNs. Disclosure: All authors have declared no conflicts of interest.

220PCYTOKERATIN 19, A NOVEL PROGNOSTIC BIOMARKER FOR HEPATOCELLULAR CARCINOMA, IS REGULATED BY DNA METHYLATION

ABSTRACT Aim: The expression of CK19 protein has recently been proposed as a novel predictor for poor prognosis following curative resection in patients with hepatocellular carcinoma (HCC). While the mechanisms underlying regulation of CK19 expression in HCC remain unclear, the presence of a CpG island within its promoter region implicates DNA methylation as a potential epigenetic process in this malignancy. Methods: A panel of 564 surgically resected HCCs at Okayama University between 2000 and 2010 were analyzed for CK19 expression and its promoter methylation status. Among all HCCs, we excluded all tumors with recurrence, cancers with TNM stage ≥IIIB, lesions with preoperative therapy, transplantation, and combined hepatocellular-cholangiocarcinomas. CK19 expression was evaluated by immunohistochemistry in FFPE tissues. CK19 promoter methylation was evaluated for region 1 and region 2 by Hi-SA - a modified COBRA by which fluorescence labeled DNA fragments are detected using a genetic analyzer (Nagasaka T et al., JNCI 2009). Molecular results were correlated with clinicopathological features, followed by Kaplan-Meier survival analysis. Results: A total of 125 cases matched for the selection criteria. Among these, 29 HCCs demonstrated positive CK19 staining, and these patients corresponded with significantly poor survival following surgery (p = 0.025), and extra-hepatic metastasis-free survival (p = 0.017). Multivariate analysis revealed that CK19-positive was an independent prognostic factor for survival, following surgery (HR2.85, 95%CI 1.21-6.41, p = 0.018). Methylation analysis revealed that methylation rates at both regions of CK19 gene were statistically higher in HCCs with loss of CK19 expression compared to CK19-positive group (p = 0.0002, p Conclusions: Our data report that CK19 expression is an important prognostic biomarker for HCC. Furthermore, since CK19s expression was strongly associated with the methylation of its promoter region, our study provides novel mechanistic insights for the participation of this epigenetic alteration in the development of HCC. Disclosure: All authors have declared no conflicts of interest.