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A 67-year-old woman underwent polypectomy for a tumor at the descending colon. Pathologically, the tumor was diagnosed as adenocarcinoma with an invasion of 2000 μm. Computed tomography showed a swollen paracolic lymph node and a mass lesion in the presacral space. Magnetic resonance imaging revealed a multio-cular cystic lesion. On diagnosis of descending colon cancer and tailgut cyst, she underwent synchronous lapa-roscopic resection. Histopathologically, the colon cancer was diagnosed as pT1bN1M0, pStage IIIa. The pre-sacral cystic lesion was diagnosed as a nonmalignant tailgut cyst with negative surgical margin. The patient is currently doing well without recurrence at 28 months.

Introduction: Gastric cancer is divided into four subtypes by their molecular features linked with genetic alterations, e.g., Epstein-Barr virus (EBV), microsatellite instability-high (MSI-high), chromosomal instability (CIN), and genomically stable (GS), called as TCGA classification. In this study, we tried to clarify the epigenetic features of the four GC subtypes according to aberrant methylation status in 23 loci. Methods: A total of 98 gastric cancers and their normal gastric mucosa samples were included in this study. We divided gastric cancers into TCGA subtypes which were determined in line with MSI-high, EBV, CIN, to GS by their molecular features. The 13 loci of polymorphic microsatellite sequences were used to determine loss of heterogeneity for the detection of CIN. The MSI status was determined by three mononucleotide repeat markers. Infection of EBV was determined by recovering EBV BNRF1 sequence from genomic DNA collected from gastric cancers. Methylation status of 23 loci was investigated by the combined bisulfite restriction analysis. Status of other findings, e.g., KRAS mutations, HER2 expression status, and infection of helicobacter pylori were confirmed. Results: Gastric cancers were divided into MSI (13%), EBV (7%), CIN (53%), and GS (27%). By histological classification, poorly differentiated adenocarcinoma was more in tumors categorized in MSI-high, and GS and signet-ring cell carcinoma (sig) were more in GS. Among the 23 loci investigated their methylation status, 18 loci were significantly hypermethylated in caner tissues. An unsupervised clustering divided gastric cancers into two clusters and revealed that most GS tumors clustered together in a cluster that exhibited lower methylation levels, distinct from the other subtypes. The inter-variable clustering revealed that a cluster contained the three loci (SFRP2-region 1/2 and APC) belonging to the Wnt signal cascade (Wnt-associated loci). The mean methylation score of Wnt-associated loci was the lowest in GS tumors (MSI-high: 2.7 [95% confidence interval, 2.3–2.9]; EBV: 2.1 [1.2–3.1]; CIN: 2.4 [2.2–2.7]; GS: 1.3 [0.8–0.7]). In contrast, the mean methylation score of the other 15 loci was significantly higher in MSI-high, while that in GS was as same as that in EBV or CIN (MSI-high: 10.4 [8.3–12.4]; EBV: 5.7 [1.7–9.7]; CIN: 4.4 [3.6–5.1]; GS: 3.4 [2.2–4.6]). Additionally, the lower methylation score of Wnt-associated loci was observed only in sig tumors. Conclusions: GS subtype tumors have the potential to possess distinct signatures in DNA hypomethylation profiles in Wnt signaling pathway, especially in sig.

Because of the different forms of circulating miRNAs in plasma, Argonaute2 (Ago2)-miRNAs and extracellular vesicles (EV-miRNAs), we examined the two forms of extracellular miRNAs in vitro and developed a unique methodology to detect circulating Ago2-miRNAs in small volumes of plasma. We demonstrated that Ago2-miR-21 could be released into the extracellular fluid by active export from viable cancer cells and cytolysis in vitro. As miR-21 and miR-200c were abundantly expressed in both metastatic liver sites and primary lesions, we evaluated Ago2-miR-21 as a candidate biomarker of both active export and cytolysis while Ago2-miR-200c as a biomarker of cytolysis in plasma obtained from colorectal cancer (CRC) patients before treatment and in a series of plasma obtained from CRC patients with liver metastasis who received systemic chemotherapy. The measurement of Ago2-miR-21 allowed us to distinguish CRC patients from subjects without CRC. The trend in ΔCt values for Ago2-miR-21 and -200c during chemotherapy could predict tumor response to ongoing treatment. Thus, capturing circulating Ago2-miRNAs from active export can screen patients with tumor burdens, while capturing them from passive release by cytolysis can monitor tumor dynamics during chemotherapy treatment.

Aim: To evaluate whether PCR-reverse sequence-specific oligonucleotide can examine the concordance between liquid biopsy and metastatic lesions with acquired resistance. Materials & methods: We examined acquired mutations in chemoresistant lesions and blood obtained from four patients with RAS wild-type metastatic colorectal cancer who underwent treatment with anti-epidermal growth factor receptor antibodies. Results: In one patient, metastatic lesions harbored diverse acquired mutations in KRAS in all seven metastases; the two acquired mutations were detectable in blood collected after the patient acquired resistance. None of the other patients exhibited liquid biopsy mutations, except one, with a BRAF mutation confirmed in primary tumor and peritoneal dissemination. Conclusion: Liquid biopsy based on PCR-reverse sequence-specific oligonucleotide is a successful procedure for capturing acquired mutations with precise information on the RAS mutational spectrum.

Background Mutations in the POLE gene result in an ultra-hypermutated phenotype in colorectal cancer (CRC); however, the molecular characterisation of epigenetic alterations remains unclear. We examined the genetic and epigenetic profiles of POLE-mutant CRC to elucidate the clinicopathological features of the associated genetic and epigenetic alterations. Results Tumour tissues (1,013) obtained from a cohort of patients with CRC were analysed to determine associations between the proofreading domain mutations of POLE with various clinicopathological variables, microsatellite instability (MSI) status, BRAF and KRAS mutations, and the methylation status of key regions of MLH1, MGMT, and SFRP2 promoters by calculating the methylation scores (range 0–6). Only four cases (0.4%) exhibited pathogenic POLE hotspot mutations (two p.P286R [c.857C > G], one p.V411L [c.1231G > C], and p.S459F [c.1376C > T] each), which were mutually exclusive to BRAF and KRAS mutations and MSI. CRC patients were divided into four subgroups: patients with POLE mutations (POLE, 0.4%, n = 4), patients with both MSI and extensive methylation in MLH1 (MSI-M, 2.9%, n = 29), patients with MSI but no extensive methylation in MLH1 (MSI-U, 3.6%, n = 36), and patients without MSI (non-MSI, 93.2%, n = 944). The POLE group was younger at diagnosis (median 52 years, P P P POLE-mutant tumours was small, the number of CD8-positive cells increased in tumours with partial methylation in the MLH1 gene. Conclusions CRC patients with POLE proofreading mutations are rare. Such mutations are observed in younger individuals, and tumours are primarily located in the right colon. Diagnosis occurs at an earlier stage, and distinct epigenetic alterations may be associated with CD8 cell infiltration.

Background: Acquired mutations are detected in plasma. However, still few reports examine the concordance between liquid biopsy and metastatic lesions with acquired resistance. Herein we evaluated whether a polymerase chain reaction-reverse sequence-specific oligonucleotide (PCR-rSSO) method can examine the concordance between liquid biopsy and metastatic lesions with acquired resistance.Methods: Firstly, we examined the presence of acquired mutations in 7 chemoresistant metastatic lesions and blood samples obtained from a metastatic colorectal cancer (mCRC) patient without RAS activating mutations treated with anti-EGFR treatment. The patient (patient 1) displayed initial early tumor shrinkage and finally progressed to disease (PD). Blood samples were collected before the development of PD and after acquiring resistance. Next, we evaluated RAS and BRAF mutational status among blood samples, primary tumors, and metastatic lesions obtained from three additional mCRC patients without RAS activating mutations. Acquired mutations were examined using Sanger sequencing and the PCR-rSSO approach.Results: Of patient 1, metastatic tumor specimens harbored diverse acquired mutations in the KRAS gene in all of the 7 (100%) metastases, and the three acquired mutations were detected in blood specimens collected after acquiring resistance. Next, we analyzed primary tumors, metastatic lesions after chemotherapy, and blood samples from three additional mCRC patients but noted that none of the patients exhibited mutations in liquid biopsy except for one case with BRAF V600E mutation, which was confirmed in both primary tumor and peritoneal dissemination. Of the four cases, acquired mutations of RAS, as well as BRAF V600E mutation, was detected in the blood obtained only after confirmation of acquiring resistance by radiological examinations.Conclusions: Our results suggest liquid biopsy based on the PCR-rSSO is a successful procedure for capturing acquired mutations with precise information of mutational spectrum that may lend us to reach selective target agents for RAS mutations.

We present the case of a Tailgut cyst occurring in the retrorectal space that was curatively resected using a posterior approach. A 40-year-old man presented to the Kochi Health Sciences Center with the chief complaint of perineal incongruity. Pelvic magnetic resonance imaging revealed a multilocular cystic lesion in the retrorectal space, with high signal intensity on T2-weighted imaging. After diagnosing a Tailgut cyst, we performed resection of the tumor using a posterior approach. The lesion was removed en bloc with the coccyx. Histopathologically, the lesion was diagnosed as a non-malignant Tailgut cyst, and the surgical margin was negative. The patient is currently doing well without recurrence at 20 months.

Background: Acquired mutations are detected in circulating cell-free DNA or circulating tumor cells in blood. However, there are still few reports that examine the concordance between liquid biopsy and metastatic lesions with acquired resistance. Methods: We examined the presence of acquired mutations in 7 chemoresistant metastatic lesions and blood samples obtained from a metastatic colorectal cancer (mCRC) patient without RAS activating mutations treated with anti-EGFR treatment. The patient displayed initial early tumor shrinkage and finally progressed to disease (PD). The 7 metastatic lesions showing acquired resistance to chemotherapy were extracted at morbid autopsy. Blood samples were collected before the development of PD during first-line chemotherapy and after acquiring resistance to second- and third-line chemotherapies. Acquired mutations were analyzed using two methodologies: conventional Sanger sequencing and MEBGENTM RASKET KIT based on Luminex® technology.Results: Metastatic tumor specimens harbored diverse acquired mutations in the KRAS gene in all of the 7 (100%) metastases in the patient resistant to EGFR-targeted treatment. Of the 7 diverse acquired mutations, only one acquired mutation observed in a liver metastasis was clearly detected in the blood collected at third-line chemotherapies. Two other acquired mutations were also detected but did not reach meaningful values. Conclusions: Liquid biopsy is a successful procedure for capturing acquired mutations that may arise from progressive metastatic lesions with acquired resistance over time.

// Yoshiko Mori 1, 2 , Akihiro Nyuya 3 , Kazuya Yasui 1 , Toshiaki Toshima 1 , Takashi Kawai 1 , Fumitaka Taniguchi 1 , Keisuke Kimura 1 , Ryo Inada 1 , Masahiko Nishizaki 1 , Junko Haraga 4 , Keiichiro Nakamura 4 , Yuzo Umeda 1 , Hiroyuki Kishimoto 1 , Toshiyoshi Fujiwara 1 , Yosuke Katata 3 , Yoshiyuki Yamaguchi 3 and Takeshi Nagasaka 3 1 Departments of Gastroenterological Surgery, Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan 2 Clinical Genomic Medicine, Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan 3 Department of Clinical Oncology, Kawasaki Medical School, Kurashiki City, Japan 4 Obstetrics and Gynecology, Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan Correspondence to: Takeshi Nagasaka, email: takeshin@med.kawasaki-m.ac.jp Keywords: ovarian metastases; colorectal cancer; ovarian metastatectomy; BRAF; RAS Received: August 15, 2017 Accepted: February 21, 2018 Published: March 27, 2018 ABSTRACT 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.

The aim of this single-institution, retrospective, observational case-control study was to evaluate the safety and feasibility of laparoscopic proctocolectomy (PC) for ulcerative colitis (UC), by comparing it with a case-control series of open PC. Twenty UC patients who underwent laparoscopic PC were retrospectively compared with the open PC group of 12 patients matched for age, sex, and urgency of the operation. In the laparoscopic PC group, the operative time was significantly longer, but the amount of blood loss was significantly smaller. The open PC patients underwent an intraoperative blood transfusion significantly more often, and the serum C-reactive protein level on the first postoperative day was significantly higher in the open PC group. In the laparoscopic PC group, the rate of severe postoperative morbidities, grades 3 and 4 on the Clavien-Dindo classification, was significantly lower, and the median length of hospital stay was significantly shorter. Laparoscopic PC for patients with UC showed superior perioperative outcomes to open PC, except for longer operative time.

A case of advanced rectal cancer treated by aggressive local and systemic treatment who has survived more than 7 years from initial recurrence is presented. A 55-year-old woman was diagnosed with advanced lower rectal cancer and underwent a low anterior resection with complete removal of all regional lymph nodes and total mesorectal excision. The tumor was diagnosed as a moderately differentiated adenocarcinoma, pStage IIIB (T3, N2a, M0). Twenty-six months after the initial surgery, local recurrence in the pelvis was detected by computed tomography, and total pelvic exenteration with distal sacrectomy (TPES) was performed after systemic chemotherapy with a molecular-targeted drug. Six months after the TPES, multiple lung metastases were detected. Consequently, the patient underwent radiofrequency ablation (RFA) and chemotherapy. The disease has since been controlled for 38 months. As volume control is essential for cancer treatment, it may be important to combine appropriate local therapy with systemic therapy to metastatic or recurrent sites in order to achieve much longer disease control.

We report a successful laparoscopic resection of a cecal tumor in a 95-year-old Japanese man. The patient visited an initial hospital with a complaint of constipation in March 2014. Computed tomography scan and colonoscopy showed a stenotic ileocecal cancer with pericolic lymph node metastases, and he was referred to our department for management. Since his general condition was maintained, we performed a laparoscopic ileocecal resection with regional lymph node dissection for the patient. The operation achieved curative resection, and the tumor was diagnosed as a moderately differentiated adenocarcinoma and graded as pStage IIIa (pT3, pN0, pM0) according to the Japanese Classification of Colorectal Carcinoma, eighth edition. He was discharged on the 11th postoperative day without perioperative complications. Several large-scale randomized controlled trials （RCTs） revealed that laparoscopic surgeries for colorectal cancers have some advantages compared to open surgeries, including superior short-term outcomes and comparable long-term outcomes. Unfortunately, since these RCTs did not include enough elderly patients, the safety and feasibility of laparoscopic surgery for extremely elderly patients are still unknown. With respect to less-invasive procedures, these advantages of laparoscopic surgery are also thought to be the advantages for elderly colorectal cancer patients.

A man in his 50s underwent high anterior resection for rectosigmoid cancer in January 2010. The primary tumor was diagnosed as a moderately differentiated adenocarcinoma with KRAS mutation, pStage III a. In May 2011, the patient had a recurrent lung tumor detected by computed tomography(CT); the tumor was resected using video-assisted thoracoscopic surgery. However, additional recurrent lung tumors arose, and radiofrequency ablation (RFA) was performed to treat these in February 2012. After RFA therapy, capecitabine was administered as adjuvant chemotherapy. Unfortunately, 10 months later, positron emission tomography (PET) /CT suggested a new recurrence in a left lateral lymph node. Although the pelvic lymph node was surgically removed immediately, a new lung recurrence was found on CT three months after the surgery. RFA was again used to treat this lung lesion. After the second RFA, the patient is doing well without any evidence of recurrence. We describe a case of recurrent rectal cancer successfully treated with multimodality therapy. The combination of appropriate local therapy with systemic chemotherapy is an essential strategy to treat advanced colorectal cancer, especially in patients with KRAS mutation when anti-EGFR antibodies are not effective.

Background: Classification of colorectal cancer (CRC) according to gene expression profiling remains controversial with regards to their ability to stratify patients for precision therapy. Several studies have suggested that ‘stem cell like’ or ‘epithelial-mesenchymal transition (EMT)’ signature associates with poor prognosis. Meanwhile CRCs with BRAF V600E mutation have shown poor outcomes in several clinical trials, but the biological roles of BRAF mutation have not been fully elucidated. Materials and Methods: To evaluate biological roles of BRAF mutations in CRC, we performed mRNA microarray analysis in the following three subsets; eight BRAF-mutant CRC tissues without MSI, six BRAF-mutant CRCs with MSI and five BRAF-wild type CRCs with MSI. Pathway analyses were performed by Gene Set Enrichment Analysis (GSEA). Following identification of candidate biomarkers that influence poor outcomes and associate with BRAF V600E mutation, we examined these candidate biomarkers in a cohort of 1068 CRC patients who underwent surgical resection of their primary tumor and/or metastatic lesions from 1994 to 2015 at the Okayama University Hospital. Results: By the GSEA, prominent signatures enriched in CRCs with BRAF V600E mutation vs. wild-type were EMT-related processes (EMT and myogenesis). Additionally, CRC with BRAF V600E mutation strongly associated with inactivation of Wnt signaling and intestinal differentiation-related genes. Among the differentially expressed genes between CRCs with BRAF V600E mutation versus wild-type, Wnt-antagonist, Secreted frizzled-related proteins (SFRPs) were significantly upregulated in CRCs with BRAF V600E mutation. As SFRPs are well known to be inactivated in CRCs by promoter hypermethylation, and up to 80% of CRCs are methylated in the promoter region of the SFRP2 gene, we focused and analyzed SFRP2 promoter methylation status in 782 stage II-IV CRCs of our cohort by a modified highly sensitive assay for bisulfite DNA followed by fluorescence-based PCR, as we previously reported (JNCI 2009). SFRP2 methylation was observed in 498 (64.2%) stage II-IV CRCs. Interestingly, SFRP2 unmethylated CRCs with genetic mutation in the KRAS/BRAF genes demonstrated significantly poorer outcome in RFS compared with SFRP2 methylated CRCs with genetic mutations in the KRAS/BRAF genes (5-years RFS rate were 46.8% vs 72.1%; P = 0.015, Hazard ratio 2.06 [95% CI, 1.12 to 3.69]; P = 0.021). Conclusion: Our results suggest that SFRP2 methylation status could be a potential prognostic biomarker for stage II-IV CRCs, especially with genetic mutations in the KRAS/BRAF genes. Citation Format: Kazuya Yasui, Takeshi Nagasaka, Toshiaki Toshima, Takashi Kawai, Kunitoshi Shigeyasu, Yoshiko Mori, Junko Haraga, Keiichiro Nakamura, Yuzo Umeda, Hiroshi Tazawa, Ajay Goel, Toshiyoshi Fujiwara. Gene expression profiles in BRAF V600E mutant colorectal cancer and association with SFRP2 methylation status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5711. doi:10.1158/1538-7445.AM2017-5711

Severe toxicity in patients with a deficiency of dihydropyrimidine dehydrogenase(DPD), an enzyme that reduces fluoropyrimidine, is very rare, and reports on this condition are few. Accordingly, diagnosis is very difficult. The patient was 70-year-old man who was admitted for adjuvant chemotherapy with capecitabine(3,600mg/day)for rectal cancer. He was admitted to our hospital because of severe oral mucositis(grade 3)and hand-foot syndrome(grade 3). After hospitalization, he experienced complications with neutropenia(grade 4)and thrombocytopenia(grade 4). The patient died 25 days after the onset of chemotherapy. Despite the measurement of the DPD value in mononuclear cells of peripheral blood and urophanic uracil and dihydrouracil, we were unable to diagnose DPD deficiency. However, we suspected a partial deficiency of DPD on the basis of the clinical course.

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.

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.

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.

Background: Intrahepatic cholangiocarcinoma (ICC) is a primary liver cancer with poor prognosis and limited therapeutic options. Recently comprehensive genetic profiles have elucidated novel fusion genes and somatic mutations. Although somatic mutations in the isocitrate dehydrogenase (IDH) and KRAS gene were frequently found in ICC, the clinical features of this malignancy with mutations in these genes remain unclear. Materials and Methods: A cohort of 49 patients with ICC who underwent curative resection from 2000 to 2013 at Okayama University Hospital were enrolled and analyzed. KRAS (exon2), IDH1 (codon132), and IDH2 (codon172 and codon140) mutations were confirmed by Sanger sequencing. Associations between the mutational profiles of these genes and clinic-pathological features were investigated. Kaplan-Meier curves were plotted, and survival rates were compared using the log-rank analysis. Results: KRAS mutations were observed in twelve ICC patients (24.5%, KRAS-mutant). IDH mutations were found in eight patients (16.3%); five patients harbored IDH1 mutations and three patients harbored IDH2 mutations (IDH1/2-mutant). Among three patients with IDH2 mutations, a rare IDH2 R140L was identified. One patient harbored both KRAS G12A and IDH2 R172K mutations (classified as KRAS-mutant). The rest of 30 ICCs harbored no mutation in either KRAS or IDH1/2 gene, thus categorized as Wild-type. Infection of Hepatitis B (HBV) or C (HCV) was observed in 10 ICCs (6 ICCs were HBV-positive and 4 ICCs were HCV-positive). Of 10 ICCs with viral infection, only two ICCs harbored KRAS mutations (categorized as virus-infected) and eight ICCs with viral infection harbored no mutation in KRAS and IDH1/2 genes. Finally, ICCs were divided into four subsets; viral-infected, KRAS-mutant, IDH1/2-mutant, and ‘others’). With regard to clinic-pathological features, lymph node metastasis was more frequently observed in KRAS-mutant (p = 0.039) compared with the others. IDH1/2 -mutant were more frequently observed in mass-forming type (p = 0.047). By survival analysis, 3-years survival rate was 85.7% in IDH1/2-mutant, 58.3% in the others, 36.3% in viral-infected, and 22.2% in KRAS-mutant, respectively (p = 0.05). Thus, our cohort suggests that IDH1/2, as well as KRAS mutations, have the potential to serve as prognostic biomarkers in ICC. Conclusion: We conclude that not only viral infections, but also IDH1/2 and KRAS mutations could be potential predictive markers for the identification of good and worse prognosis in ICCs. Hence the mutational profiles of these genes provide an attractive rationale for developing a molecular signature for the development of non-invasive screening for ICCs in future. Citation Format: Kazuya Yasui, Takeshi Nagasaka, Yuzo Umeda, Tomokazu Fuji, Fumitaka Taniguchi, Toshiaki Toshima, Keisuke Kimura, Takashi Kawai, Yoshiko Mori, Hiroshi Tazawa, Takahito Yagi, Ajay Goel, Toshiyoshi Fujiwara. Comprehensive analysis of intrahepatic cholangiocarcinoma based on viral infections and mutational status in the IDH1/2 and KRAS genes. [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 3172.

We report a case of adenocarcinoma of the small intestine responding to XELOX chemotherapy, leading to a partial metabolic response(PMR). The patient was a 58-year-old male with multiple peritoneal dissemination of adenocarcinoma of the small intestine. Chemotherapy with XELOX(L-OHP 130 mg/m² on day 1 , and capecitabine 1,000 mg/m2 on days 1-14)was performed. After 4 courses, a significant tumor reduction was obtained. This case suggests that chemotherapy with XELOX is a potential regimen for small intestinal adenocarcinoma.

A 55-year-old male had complained of melena.Colonoscopy revealed a type 2 tumor at the rectum.CT demonstrated hepatic lymph nodes and multiple liver metastases(stage IV).Low anterior resection was performed(tub2, RsRa, circ, type 2, pSS, pN1, sH3, cHN1, sP0, cM0: fstage IV).The patient was treated with mFOLFOX6 and sLV5FU2 after operation.CT revealed a partial response after 14 courses of systemic chemotherapy.sLV5 FU2 therapy was converted to capecitabine because he experienced bone marrow suppression.CT showed that the liver metastases had enlarged but the hepatic lymph nodes disappeared.Right portal vein embolization was performed.After 4 weeks, right hepatectomy and hepatic lymph node dissection were performed.Preoperative chemotherapy with mFOLFOX6 seems beneficial as a neoadjuvant chemotherapy for hepatic lymph node-positive advanced colorectal cancer.

A 45-year-old male was admitted to our hospital complaining of anemia. Gastric endoscopy showed a type IIa+IIc tumor at the anterior wall of the gastric angle. Based on the pathology of the biopsy specimen, poorly-differentiated adenocarcinoma was diagnosed. Computed tomography scans showed regional lymph node swelling. Distal gastrectomy with a D2 lymph node dissection was performed. On pathology, the tumor was immunohistochemically positive for chromogranin A and synaptophysin. The Ki67 index was 70%. The tumor was diagnosed as poorly-differentiated neuroendocrine carcinoma of the stomach. He was treated with S-1 and CPT-11. Neuroendocrine cell carcinoma of the stomach is rare and usually has a very poor prognosis. Thus, we are reporting this case of early poorly-differentiated neuroendocrine carcinoma of the stomach that was curatively resected and had 12-month survival without recurrence.