Kazuhiro Matsunaga

Influence of HRH2 promoter polymorphism on aberrant DNA methylation of DAPK and CDH1 in the gastric epithelium

BackgroundAberrant methylation patterns in CpG island are known to be influential in gene silencing. Histamine plays important physiological roles in the upper gastrointestinal tract and acts via the H2 receptor. We report an investigation into the effect of HRH2 promoter polymorphism (rs2607474 G > A) on the methylation of DAPK and CDH1.MethodsNon cancerous gastric mucosa samples were obtained from 115 subjects with gastric cancer (GC) and 412 non-cancer subjects (non-GC). Methylation status of genes was determined by MSP. The genotyping of rs2607474 was performed by PCR-SSCP.ResultsMethylation of DAPK and CDH1 was observed in 296 and 246 subjects, respectively. The frequency of CDH1 methylation in the subjects with GC was significantly lower in cancer lesion than in non cancerous mucosa, whereas that of DAPK methylation was not different. The allelic distribution of rs2607474 was 401GG, 119GA and 7AA. The GG homozygote was associated with a significantly increased risk for methylation of both DAPK and CDH1 (p < 0.0001 and p = 0.0009, respectively). In the non-GC subjects or more than 60 years of age, GG homozygote was more closely associated with both DAPK and CDH1 methylation. However, this genotype did not show an increased risk for the development of methylation of both genes in patients with GC. In H. pylori negative subjects, GG homozygote showed an increased risk for the methylation of both DAPK and CDH1 (p = 0.0074 and p = 0.0016, respectively), whereas this genotype was associated with an increased risk for the development of DAPK methylation in H. pylori positive subjects (p = 0.0018). In addition, in subjects older than 60 years of age, atrophy and metaplasia scores were significantly higher in the GG homozygote (p = 0.011 and p = 0.039, respectively) and a significant correlation was observed between age and atrophy or metaplasia.ConclusionsOur results suggest that rs2607474 GG homozygote confers a significantly increased risk for age- and inflammation-related DAPK and CDH1 methylation.

Genetic polymorphisms of IL17A and pri-microRNA-938, targeting IL17A 3'-UTR, influence susceptibility to gastric cancer.

We report an association between gastric cancer (GC) and polymorphisms in IL17A, rs2275913 (-197 G > A), rs3748067 (*1249 C > T), and pri-miR-938, rs2505901 (T > C). We employed the multiplex PCR-SSCP method to detect gene polymorphisms in 337 GC cases and 587 controls. The minor allele frequency of rs2275913 was significantly higher, and those of rs3748067 and rs2505901 significantly lower, in GC cases than controls. The rs2275913 AA homozygote was associated with an increased risk (OR, 2.38; 95%CI, 1.63-3.46; p < 0.0001) for the development of both intestinal and diffuse types of GC. The rs3748067 T polymorphism was associated with a decreased risk for intestinal GC (OR, 0.511; 95%CI, 0.272-0.962; p = 0.037), whereas rs2505901 C locus carried a decreased risk overall for GC (OR, 0.733; 95%CI, 0.545-0.985; p = 0.039). In addition, rs3748067 T allele was inversely correlated with lymph node metastasis. Our results suggest that polymorphisms in both IL17A and pri-miR-938 contribute to cancer risk susceptibility and therefore can affect the development of gastric cancer.

Functional promoter polymorphisms of NFKB1 influence susceptibility to the diffuse type of gastric cancer

In the present study, we report an association between gastric cancer and polymorphisms in NFKB1 (rs28362941 and rs78696119). We employed the PCR-SSCP method to detect gene polymorphisms in 479 gastric cancer cases and 880 controls. The rs28362941 del/del homozygote was significantly associated with gastric cancer development; in particular it was closely associated with diffuse type gastric cancer. The rs78696119 GG homozygote was also associated with the diffuse type of gastric cancer. In young subjects, both polymorphisms were significantly associated with the development of gastric cancer. In addition, both polymorphisms were related to tumor progression such as tumor invasion and lymph node metastasis. The inflammatory cell infiltration into non-cancerous gastric mucosa was greater in the subjects with the rs28362491 del/del or rs78696119 GG genotype when compared to those with the other genotypes. In conclusion, functional polymorphisms of NFKB1 are associated with an increased risk of gastric cancer; in particular they are closely associated with the development of diffuse type of gastric cancer via severe gastric inflammation. These polymorphisms also appear to be associated with gastric cancer progression.

White globe appearance is a novel specific endoscopic marker for gastric cancer: A prospective study

White globe appearance (WGA) is a small white lesion with a globular shape that can be identified by magnifying endoscopy with narrow‐band imaging (M‐NBI). WGA was recently reported as a novel endoscopic marker that can differentiate between gastric cancer (GC) and low‐grade adenoma. However, the usefulness of WGA for differentiating GC from non‐cancerous lesions (NC), including those of gastritis, is unknown.

Importance of colonoscopy in patients undergoing endoscopic resection for superficial esophageal squamous cell carcinoma

Background The aim of the study was to clarify the frequency of colorectal neoplasm (CRN) complicating superficial esophageal squamous cell carcinoma (ESCC) and the need for colonoscopy. Methods We retrospectively reviewed 101 patients who had undergone initial endoscopic resection (ER) for superficial ESCC. Control group participants were age- and sex-matched asymptomatic subjects screened at our hospital over the same period of time. Advanced adenoma was defined as an adenoma ≥10 mm, with villous features, or high-grade dysplasia. Advanced CRN referred to advanced adenoma or cancer. We measured the incidence of advanced CRN in superficial ESCC and controls, and we compared the characteristics of superficial ESCC patients with and without advanced CRN. Results In the superficial ESCC group, advanced CRNs were found in 17 patients (16.8%). A history of smoking alone was found to be a significant risk factor of advanced CRN [odds ratio 6.02 (95% CI 1.30-27.8), P=0.005]. Conclusion The frequency of synchronous advanced CRN is high in superficial ESCC patients subjected to ER. Colonoscopy should be highly considered for most patients who undergo ER for superficial ESCC with a history of smoking, and is recommended even in superficial ESCC patients.

Su1501 Morphological Changes of Intraductal Papillary Mucinous Neoplasm of the Pancreas Are Not Enough for Indicators of the Development of Pancreatic Carcinoma During Follow Up

G A A b st ra ct s receiver operator characteristics curve. The factors associated with malignant IPMNs were determined by logistic regression analysis for BD-IPMN and MD-IPMN separately. Diagnostic accuracy of the determined predictors of malignancy was calculated. Results: Total 241 patients consisted of 202 BD-IPMN and 39 MD-IPMN were included. Cutoff values of cyst size, MPD diameter, and nodule size were determined for BD-IPMN as 30 mm, 6 mm, and 10 mm, respectively, whereas for MD-IPMN as 24 mm, 12 mm and 10 mm, respectively. For BD-IPMN, nodule size ≥10 mm (OR=176.97, p<0.0001) and positive cytology (OR127.14, p<0.0001) were significantly associated with malignant IPMN (Table 1). For MD-IPMN, nodule size ≥10 mm (OR=16.07, p=0.002) and positive cytology (OR=160.00, p<0.0001) were significantly associated with malignant IPMN. Sensitivity, specificity, and overall diagnostic accuracy of nodule size ≥10 mm alone were 59%, 98%, 90%, respectively, and positive cytology alone were 83%, 96%, 94%, respectively (Table 2). In combination of nodule size ≥10 mm and/or positive cytology, sensitivity, specificity, and overall diagnostic accuracy were 96%, 95%, and 95%, respectively. Conclusions: The common predictors, nodule size ≥10 mm and positive cytology, were demonstrated to be appropriate for both BD-IPMN and MD-IPMN. In combination of nodule size ≥10 mm and/or positive cytology as predictors of malignancy, diagnostic accuracy was shown to be excellent. Table 1. Factors associated with malignant IPMN determined by logistic regression analysis.

Mo1366 Genetic Polymorphisms of MAFK, Encoding Small Maf Protein MAFK, Are Associated With the Susceptibility to Ulcerative Colitis

[Background] The small Maf proteins have emerged as crucial regulators of mammalian gene expression. Small Mafs do not contain an obvious transcriptional activation domain. However, it has been clear that small Maf proteins are regulated transcription factors, such as Nrf2 or Bach1. MafK, one of the small Maf proteins, is encoded by MAFK gene. Within 20kbp around MAFK gene, there are two linkage disequilibrium blocks, with above 0.05 of HWE p value and above 0.05 of minor allele frequency. In this study, we investigated an association between ulcerative colitis (UC) and each tag polymorphism, rs4268033G.A and rs3735656 (*910T.C), and the other one rs10226620 (*1506T.C). [Material and Methods] The studied population comprised 922 subjects, including patients with UC (UC cases, n=174), who were enrolled Fujita Health University Hospital, and the subjects without UC (controls, n=748). We employed the PCR-SSCP method to detect a gene polymorphism. [Results] In controls, mean age was 57.1 years old and male/female ratio was 438/310. In UC cases, mean age was 40.3 years old, mean age of onset was 33.0 years old and male/ female ratio was 98/76. The distributions of rs4268033, rs3735656 and rs10226620 in controls were 366GG, 324GA and 58AA (HWE p=0.25), 329TT, 346TC and 73CC (HWE p=0.21) and 345TT, 328TC and 75CC (HWE p=0.87) respectively, whereas the distributions in UC cases was 80GG, 66GA and 28AA, 80TT, 69TC and 25CC, 80TT, 72TC and 22CC, respectively. The genotype frequency of rs4268033 AA and allelic frequency of rs4268033A allele were significantly higher in UC cases than in controls (p=0.0013 and 0.045 by Fisher). By logistic regression analysis after adjustment for age and gender, rs4268033 AA and rs3735656 CC genotypes were significantly associated with the susceptibility to UC development (OR, 2.62; 95%CI, 1.54-4.48; p=0.0004 and OR, 1.79; 95%CI, 1.05-3.04; p=0.031, respectively). In addition, rs4268033 AA genotype was significantly associated with all phenotypes of UC (i.e. total colitis phenotype: OR, 2.58; 95%CI, 1.29-5.17; p=0.0073, not total colitis: OR, 2.39; 95%CI, 1.22-4.72; p=0.012). The rs3735656CC genotype was significantly associated with steroid-independent or not required phenotype of UC (OR, 1.87; 95%CI, 1.08-3.26; p=0.026). There was no significant association between rs10226620 and UC. [Conclusion] Our results provided the first evidence that MAFK gene polymorphisms were significantly associated with the susceptibility to UC development. In particular, rs4268033 was closely associated with an increased risk for the development of UC.

Mo1131 Genetic Polymorphism of MAFK (Rs4268033) Is Associated With the Susceptibility to Gastric Cancer in Japan

[Background] The small Maf proteins have emerged as crucial regulators of mammalian gene expression. Small Mafs do not contain an obvious transcriptional activation domain. However, it has been clear that small Maf proteins are regulated transcription factors, such as Nrf2 or Bach1. MafK, one of the small Maf proteins, is encoded by MAFK gene. Within 20kbp around MAFK gene, there are two linkage disequilibrium blocks, with above 0.05 of HWE p value and above 0.05 of minor allele frequency. In this study, we investigated an association between gastric cancer (UC) and each tag polymorphism, rs4268033G .A and rs3735656 (*910T.C), and the other one rs10226620 (*1506T.C). [Material and Methods] The study was performed in 334 patients with gastric cancer (GC cases) and 661 subjects with no evidence of gastric malignancies (controls) on upper gastro-duodenal endoscopy. We employed the PCR-SSCP method to detect the gene polymorphism. [Results] The mean age, male/female ratio and Helicobacter pylori (HP) positive ratio in GC cases were significantly higher than those in controls. The distributions of rs4268033, rs3735656 and rs10226620 in controls were 326GG, 284GA and 51AA (HWE p=0.35), 291TT, 304TC and 66CC (HWE p=0.33) and 304TT, 288TC and 69CC (HWE p=0.93) respectively, whereas the distributions in GC cases was 127GG, 163GA and 44AA, 142TT, 153TC and 39CC, 144TT, 154TC and 36CC, respectively. The allelic frequency of rs4268033 Awas significantly higher in GC cases than in controls ‘(37.6% vs. 29.2%, p=0.0002). Overall, rs4268033 minor variant had a significantly increased risk for the development of gastric cancer by logistic regression analysis after adjustment for gender, age and HP infection status (recessive genetic model: OR, 1.72 95%CI, 1.10-2.69; p=0.016 and dominant genetic model: OR, 1.61; 95%CI, 1.21-2.14; p=0.0009), whereas no significant association was found between gastric cancer susceptibility and rs3735656 or rs10226620. The rs4268033 minor allele was more strongly associated with intestinal type of gastric cancer (recessive genetic model: OR, 2.12; 95%CI, 1.27-3.54; p=0.0040 and dominant genetic model: OR, 1.66; 95%CI, 1.17-2.35; p=0.0042), and slightly associated with diffuse type of gastric cancer (dominant genetic model: OR, 1.50; 95%CI, 1.02-2.21; p=0.040). In addition, rs4268033 minor allele was significantly associated with non-cardiac gastric cancer (recessive genetic model: OR, 1.76; 95%CI, 1.11-2.77; p=0.015 and dominant genetic model: OR, 1.68; 95%CI, 1.252.25; p=0.0005). [Conclusion] Our results provided the first evidence that MAFK gene polymorphism is significantly associated with the susceptibility to gastric cancer development. The rs4268033 minor allele is closely associated with an increased risk for the development of gastric cancer.

Mo1129 Genetic Polymorphism of NFKB1 (rs28362491, -94 ATTG ins/del) Is Associated With Age-Related Gene Methylation in Helicobacter pylori Infected Japanese Subjects

[Background] CpG island aberrant methylation is shown to be an important mechanism in gene silencing. The important role of NF-κB in inflammatory response to H. pylori colonization has been indicated. We investigated the influence of NFKB1 polymorphism (rs28362491, -94 ATTG ins/del) on the aberrant gene methylations under H. pylori infection. [Materials and Methods] Gastric mucosal samples were obtained from 243 H. pylori infected subjects without malignancies. Methylation status of genes (p14ARF, p16INK4a, CDH1and DAPK) was determined by methylation-specific polymerase chain reaction. Two or more gene methylations were defined as CIHM. The genotyping of NFKB1 was performed by PCRSSCP. [Results] Mean age of the subjects was 60.5 years old and male/female ratio was 154/ 89. Methylation of p14ARF, p16INK4a, CDH1and DAPK was seen in 90, 91, 109 and 139 of 243 subjects, respectively. The ratio of CIHMwas 136/243. The distribution of rs28362491 genotype was 90ins/ins, 122ins/del and 31del/del (HWE p=0.34). The -94 del/del homozygote was significantly associated with the risk for development of CIHM (OR, 4.16; 95%CI, 1.14-15.3; p=0.031), especially DAPK (OR, 5.35; 95%CI, 1.17-24.5; p=0.031) and CDH1 (OR, 2.91; 95%CI, 1.02-8.30; p=0.046) methylations, in the subjects older than 60 years old by logistic regression analysis using recessive genetic model after adjustment for gender and age. On the other hand, there was no association between this polymorphism and CDKN2A (p14ARF and/or p16INK4a) methylation. The number of methylated genes was significantly higher in -94 del/del homozygote than ins/del + ins/ins genotype (ins carrier) in older subjects (p=0.040 by ANOVA). In addition, this methylated gene number was significantly increased with age in del/del homozygote (p=0.027 by ANCOVA), but not in ins carrier. Furthermore, inflammation score was significantly higher in del/del homozygote than ins carrier. [Conclusions] NFKB1 rs28362491 (-94 ATTG ins/del) polymorphism is significantly associated with the increased risk for the development of age-related gene mathylations in non-cancerous gastric mucosa under H. pylori-induced inflammation.

Tu1097 Application of the New WHO Classification and the Possibility of Molecular-Targeted Therapies Against Colorectal Neuroendocrine Tumors

[Background] Colorectal neuroendocrine tumors (NETs) may cause distant unresectable metastases, for which no effective chemotherapy has been established. Trastuzumab, which targets HER2/neu, has been effective in the treatment of advanced tumors that overexpress the target molecule. Bevacizumab, which targets VEGF, is moderately effective against advanced tumors, but the predictor of efficacy for this drug is unknown. Specifically, it is unclear whether these target molecules are involved in colorectal NETs. Furthermore, the expression patterns of EGFR and SSTR have not been fully characterized in NETs. [Objective] To assess the possibility of molecular-targeted therapies against colorectal NETs, we classified NET specimens according to the new WHO 2010 classification criteria and examined the expression of HER2/neu, VEGF, EGFR, and SSTR. [Specimens] Thirty-six paraffin-embedded specimens of colorectal NET excised from 2000 to 2010. [Methods] Each specimen was classified according to the new WHO 2010 classification criteria and examined for the expression of HER2/neu, VEGF, EGFR, and SSTR2A (a subtype of SSTR) by immunohistochemistry. [Results] 1. WHO classification: Of the 36 specimens, 31 were classified into NET G1, 1 into NET G2, and 4 into NEC. 2. Immunohistochemical staining: The overall proportions of the stained specimens (and proportion by WHO class) were as follows: 5% (G1: 0%, G2: 0%, NEC: 50%) for HER2/neu, 78% (G1: 80%, G2: 100%, NEC: 50%) for VEGF, 0% for EGFR, and 16.6% (G1: 19%, G2: 0%, NEC: 0%) for SSTR2A. [Discussion] Among colorectal NETs, HER2/neu was expressed in highly proliferative tumors.While trastuzumab is expected to be effective against these unresectable advanced tumors, its efficacy may be limited due to low HER2/neu expression levels (rated as 1+). VEGF was expressed in 78% of the tested specimens, indicating that bevacizumab monotherapy is effective in these tumors. However, more basic studies are required to clarify the role of VEGF in tumor proliferation. Sunitinib, a VEGFR-1,2,3-TKI drug, has been reported to be effective against PNET, and its potential efficacy against colorectal NEC should be assessed in future studies. Because EGFR was not detected in any specimen, cetuximab should not be effective. SSTR2A expressionwas inversely correlated with malignancy, suggesting that SSTR degrades as the proliferative capacity increases. Thus, SSTR will not serve as a target molecule for highly malignant colorectal NETs.