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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.

Background/aim The objective of this study was to determine the molecular and clinicopathological features, as well as the prognosis of patients with endometrial cancer (EC) having prior malignancy (second primary EC: SPEC) compared with those without a history of prior malignancy (first primary EC: FPEC). Materials and methods We enrolled 294 FPEC patients and 32 SPEC patients who had undergone surgical resection with curative intent. EC was divided into four groups according to Cancer Genome Atlas Research Network (TCGA) classification. Results SPEC patients having greater than a 10-year interval from prior malignancy had risk factors including type II histology, deeper myometrial invasion, cervical invasion, and copy number high (CNH) phenotype compared with patients having less than a 10-year interval (p=0.007, p=0.002, p=0.015 and p=0.001). Conclusion SPEC patients having greater than a 10-year interval from prior malignancy possessed numerous high-risk factors for EC.

Background: The neutrophil-to-lymphocyte ratio (NLR) is a well-known prognostic marker for gastric cancer (GC) patients. However, for GC patients treated with palliative chemotherapy, the prognostic value of NLR remains obscure. Therefore, we evaluated the clinical impact of NLR in GC patients treated with multiple lines of chemotherapy.Methods: We retrospectively evaluated 83 unresectable GC patients who received a series of chemotherapy treatments. NLR in the blood was calculated before the initiation of each line of chemotherapy (before 1st-, 2nd-, and 3rd-line, respectively).Results: Of the 83 patients enrolled, 56 (67%) received 2nd-line chemotherapy and 34 (41%) received 3rd-line treatment. NLR at 1st-line treatment ranged from 0.72 to 48.9 (the median NLR was 3.00). Thus, we used this median NLR value (3.00) as the cut-off for NLR throughout this study. All patients were divided into a NLR-high (> 3.00) or NLR-low group (< 3.00) by NLR values determined at before each line of chemotherapy. The median overall survival (OS) of the NLR-low group was better than that of the NLR-high group throughout 1st- to 3rd-line treatment (1st-line: 18.1 vs. 8.0 months, p = 0.06; 2nd-line: 10.7 vs. 4.5 months, p = 0.0001; 3rd-line: 8.7 vs. 4.7 months, p = 0.003), respectively. Of the 34 patients who received 3rd-line chemotherapy, 24 were treated with nivolumab. Of the 24 patients treated with nivolumab, patients in the NLR-low group showed better OS compared with those in the NLR-high group (8.3 months in NLR-low and 6.6 months in NLR-high, p = 0.06).Conclusions: Estimates of NLR prior to each chemotherapy line may be readily performed in a clinical setting and are useful for predicting outcome in unresectable GC patients, including in patients treated with nivolumab.

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.

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.

Colorectal cancer (CRC) manifests after the accumulation of genetic and epigenetic alterations along with tumor microenvironments. MicroRNA (miRNA/miR) molecules have been revealed to serve in critical roles in the progression various types of cancer, and their expression level is often an important diagnostic, predictive or prognostic biomarker. The aim of the present study was to evaluate the potential of miRNAs as prognostic biomarkers for patients with advanced CRC. miRNA arrays were performed on CRC specimens obtained from tumors with various molecular statuses [e.g. KRAS proto-oncogene, GTPase (KRAS)/B-Raf proto-oncogene, serine/threonine kinase (BRAF)/microsatellite instability (MSI)], and their paired normal mucosal specimens. The miRNA array revealed that miR-31-5p (miR-31) was specifically upregulated in CRCs with the BRAF V600E mutation, the results of which were supported by subsequent analysis of a dataset retrieved from The Cancer Genome Atlas (TCGA) database, which contained information regarding 170 patients with CRC including 51 BRAF-mutant CRCs. Of our cohort of 67 patients with stage IV CRC, 15 (22%) and 4 (6%) showed KRAS and BRAF V600E mutations, respectively. Since the median miR-31 expression was 3.45 (range, 0.004–6330.531), the cut-off value was chosen as 3.5, and all tumors were categorized into two groups accordingly (high-/low-miR-31 expression). The high miR-31 expression group (n=33) was significantly associated with a poorer mortality (univariate hazard ratio=2.12; 95% confidence interval, 0.23–0.95; P=0.03) and exhibited a shorter median survival time (MST; 20.1 months) compared with the low miR-31 expression group (n=34) (MST, 38.3 months; P=0.03), indicating that miR-31 is a promising prognostic biomarker for patients with advanced CRC. Thus, performing a functional analysis of miR-31 expression may lead to the development of new targeted therapies for the various genetic subtypes of CRC.

// 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.

Background: Endometrial cancer accounts for the second percentage of Lynch syndrome related tumors following colorectal cancer. Inactivation of MSH2 was frequently observed in endometrial cancer with microsatellite instability (MSI) or mismatch repair complex deficiency (dMMR). With respect to MSH2 deficiency (dMSH2), not like MLH1 deficiency, most of dMSH2 were caused by germline mutations in the MSH2 gene or germline EpCAM deletions. Meanwhile, heritable germline epimutations in MSH2 have been reported in a few Lynch syndrome families that lacked germline mutations in the MSH2 gene. We previously provided evidence for frequent MSH2 hypermethylation in Lynch syndrome colorectal tumors with dMSH2 and MSH2 methylation may serve as the “second hit” at the wild-type allele. Materials and Methods: In this study, we analyzed MSH2 promoter methylation status, as well as MLH1 methylation status, and expression status of the mismatch repair proteins (MLH1, MSH2, PMS2, and MSH6) by immunohistochemistry in a cohort of 138 endometrial cancer tissues surgically resected at Okayama University Hospital. DNA was extracted from formalin-fixed, paraffin-embedded tissue, analyzed MSI status by four mononucleotide markers and both MLH1 and MSH2 promoter methylation status by a fluorescent quantitative bisulfite PCR assay. Results: Endometrial cancers displaying MSI or dMMR were observed in 40 (29.0%) or 41 cases (29.7%), respectively. Endometrial cancers with MSH2 deficiency were observed in eight (5.8%) of 138 tumors (19.5% of dMMR). MSH2 promoter methylation was present in 7 cases (5.1% in 138 tumors), and significantly correlated with dMSH2 (P=0.0041, Fisher's exact probability test). Then, we also examined the family history of first-degree relatives retrospectively. In this cohort, although patients with MMR deficiency were significantly associated with family history of Lynch syndrome related tumor (P Conclusions: In this study, we demonstrated that MSH2 methylation significantly correlated with dMSH2 and may have strong relation with family history of Lynch syndrome related tumor, taking a role as “second hit” to the MSH2 gene. Citation Format: Junko Haraga, Takeshi Nagasaka, Keiichirou Nakamura, Tomoko Haruma, Takeshi Nishida, Akihiro Nyuya, Kazuya Yasui, Hisashi Masuyama, Toshiyoshi Fujiwara, Yuji Hiramatsu. Clinicopathological significance of endometrial cancer with MSH2 deficiency [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 4277. doi:10.1158/1538-7445.AM2017-4277

Background: Downregulated expression of the netrin-1 dependence receptors, UNC5C and DCC, is a frequent event in significant proportion of colorectal tumors. However, the tumor suppressive role of this important cancer-related pathway in other gastrointestinal cancers remains unclear. We hypothesized that similar to colorectal cancer, down-regulation of UNC5C and DCC may have an important growth-regulatory function in gastric tumorigenesis as well. Methods: We performed a series of genetic (loss of heterozygosity, LOH) and epigenetic analysis (aberrant DNA methylation) for UNC5C and DCC in a cohort of 105 sporadic gastric cancers and the corresponding normal gastric mucosa. Results: In this study, the distribution of gastric cancers as categorized by TNM staging system (UICC 7th) was as follows; 20, 24, 28, and 29 cases in stages I, II, III, and IV, respectively. Overall, 58% (48 out of 83 informative cases) and 76% (65 out of 85 informative cases) of gastric cancers demonstrated either a genetic or epigenetic alteration in the UNC5C and DCC genes, respectively. Interestingly, although epigenetic mechanisms were the primary cause for UNC5C inactivation, methylation-induced transcriptional silencing served also served as the second hit responsible for the DCC gene loss. Taken together, 52% (36 out of 69 gastric cancers with informative data) of gastric cancers showed cumulative defects in these two dependence receptors. The frequency of LOH and aberrant methylation of both dependence receptors gradually increased with progressive tumor stages. Conclusions: This is the first study that unravels the observation that majority of gastric cancers harbor defects in netrin1 receptors. Our observation that both genetic and epigenetic alterations are present early, and continue to escalate in a stepwise manner with the disease progression, highlights the fundamental importance of this growth regulatory pathway in gastric carcinogenesis.