Tochigi Cancer Center Research Institute

Tochigi, Japan

Tochigi Cancer Center Research Institute

Tochigi, Japan
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PubMed | National Cancer Center Hospital, Clinical Pathology Laboratories and Tochigi Cancer Center Research Institute
Type: Journal Article | Journal: Cancer science | Year: 2016

Germline PMS2 gene mutations were detected by RT-PCR/direct sequencing of total RNA extracted from puromycin-treated peripheral blood lymphocytes (PBL) and multiplex ligation-dependent probe amplification (MLPA) analyses of Japanese patients with colorectal cancer (CRC) fulfilling either the revised Bethesda Guidelines or being an age at disease onset of younger than 70years, and screened by mismatch repair protein immunohistochemistry of formalin-fixed paraffin embedded sections. Of the 501 subjects examined, 7 (1.40%) showed the downregulated expression of the PMS2 protein alone and were referred to the genetic counseling clinic. Germline PMS2 mutations were detected in 6 (85.7%), including 3 nonsense and 1 frameshift mutations by RT-PCR/direct sequencing and 2 genomic deletions by MLPA. No mutations were identified in the other MMR genes (i.e. MSH2, MLH1 and MSH6). The prevalence of the downregulated expression of the PMS2 protein alone was 1.40% among the subjects examined and IHC results predicted the presence of PMS2 germline mutations. RT-PCR from puromycin-treated PBL and MLPA may be employed as the first screening step to detect PMS2 mutations without pseudogene interference, followed by the long-range PCR/nested PCR validation using genomic DNA.


PubMed | National Cancer Center Research Institute, Nagoya University, National Cancer Center Hospital, Clinical Pathology Laboratories and Tochigi Cancer Center Research Institute
Type: Journal Article | Journal: Histopathology | Year: 2016

The aim of this study was to examine the expression of mismatch repair (MMR) proteins in Lynch syndrome (LS)-associated colorectal adenomas and to evaluate their relationship with clinicopathological variables and potential utility in LS screening.We performed immunohistochemistry for MLH1, PMS2, MSH2 and MSH6 in 134 adenomas obtained from 26 genetically confirmed LS patients. MMR deficiency, as determined by loss of any MMR protein, was observed in 113 adenomas (84%). All the MMR-deficient adenomas exhibited homogeneous loss of MMR proteins, which reflected underlying germline mutations. MMR deficiency was more frequent in adenomas obtained from older patients (aged 60 years; 81 of 86, 94%), with larger tumour size (>5 mm; 71 of 73, 97%) and with high-grade dysplasia (50 of 51, 98%). Multivariate analyses indicated that increased age and larger tumour size were associated independently with MMR deficiency.This study shows that MMR deficiency is associated significantly with increased age, in addition to two previously reported factors-larger size and high-grade dysplasia. When adenomas are analysed during LS screening, high sensitivity is expected if the adenomas are associated with any of these three factors.


Tahara M.,Tochigi Cancer Center Research Institute | Tahara M.,Jichi Medical University | Inoue T.,Tochigi Cancer Center Research Institute | Sato F.,Tochigi Cancer Center Research Institute | And 6 more authors.
Molecular Cancer Therapeutics | Year: 2014

Potent application of topoisomerase I inhibitor plus PARP inhibitor has been suggested to be an effective strategy for cancer therapy. Reportedly, mismatch repair (MMR)-deficient colon cancer cells are sensitive to topoisomerase I inhibitor, presumably due to microsatellite instability (MSI) of the MRE11 locus.Weexamined the synergy of SN-38, an active metabolite of irinotecan, in combination with the PARP inhibitor olaparib in colon cancer cells showing different MMR status, such as MSI or microsatellite stable (MSS) phenotype. Treatment with SN-38 and olaparib in combination almost halved the IC50 of SN-38 for a broad spectrum of colon cancer cells independent of theMMRstatus. Furthermore, olaparib potentiated S-phase-specific doublestrand DNA breaks (DSB) induced by SN-38, which is followed by Rad51 recruitment. siRNA-mediated knockdown of Rad51, but not Mre11 or Rad50, increased the sensitivity to olaparib and/or SN-38 treatment in colon cancer cells. In vivo study using mouse xenograft demonstrated that olaparib was effective to potentiate the antitumor effect of irinotecan. In conclusion, olaparib shows a synergistic effect in colon cancer cells in combination with SN-38 or irinotecan, potentiated by the Rad51-mediated HR pathway, irrespective of the Mre11-mediated failure of theMRNcomplex. These results may contribute to future clinical trials using PARP inhibitor plus topoisomerase I inhibitor in combination. Furthermore, the synergistic effect comprising topoisomerase I-mediatedDNAbreakage-reunion reaction,PARPand Rad51-mediatedHRpathway suggests the triple synthetic lethal pathways contribute to this event and are applicable as a potential target for future chemotherapy. © 2014 American Association for Cancer Research.


Kotake K.,Tochigi Cancer Center | Mizuguchi T.,Tochigi Cancer Center | Moritani K.,Tochigi Cancer Center | Wada O.,Tochigi Cancer Center | And 3 more authors.
International Journal of Colorectal Disease | Year: 2014

Purpose: The clinical significance of D3 lymph node dissection for patients with colon cancer remains controversial. This study aims to clarify the impact of D3 lymph node dissection on survival in patients with colon cancer. Methods: This is a retrospective cohort study from a prospectively registered multi-institutional database of colorectal cancer in Japan. Propensity score matching method was applied to balance potential confounders of the treatment. A cohort of 10,098 patients who underwent radical colectomy for pT3 and pT4 colon cancer between 1985 and 1994 were identified. A total of 3,425 propensity score matched pairs were extracted from the entire cohort. The primary outcome measure was overall survival (OS). Results: In the entire cohort, there was a statistically significant difference in overall survival (OS) between the patients who had D3 and D2 lymph node dissection (p=0.00003). The estimated hazard ratio (HR) for OS of patients who had D3 versus D2 lymph node dissection was 0.827 (95 % confidence interval, 0.757 to 0.904). In the matched cohort, there was also a significant difference in OS between the two groups (p=0.0001), and the estimated HR for OS was 0.814 (95 % confidence interval, 0.734 to 0.904). Conclusions: We found D3 lymph node dissection for pT3 and pT4 colon cancer to be associated with a significant survival advantage in a large-scale database, even after adjusting potential confounders of lymph node dissection. This finding may provide a rationale for D3 lymph node dissection in radical surgery for pT3 and pT4 colon cancer. © 2014 Springer-Verlag.


Sugano K.,Tochigi Cancer Center Research Institute
Biotherapy | Year: 2011

Various cancer predisposing genes were reported between the late 1980s and early 1990s and gene testing was commercially available in clinical settings of the United States by the mid-1990s. So far, in Japan, most of the hereditary cancer-related gene testing has not been covered by public insurance, and are performed in the research setting. In some cases, the tests have been performed at the patient's expense. The current status in Japan concerning preventive medicine for hereditary cancers will be discussed in this article.


Miyakura Y.,Jichi Medical University | Sugano K.,Tochigi Cancer Center Research Institute | Nomizu T.,Hoshi General Hospital | Lefor A.,Jichi Medical University | Yasuda Y.,Jichi Medical University
Japanese Journal of Clinical Oncology | Year: 2012

Lynch syndrome is caused by germline mutations of the DNA mismatch repair genes. Missense mutations are often difficult to evaluate as pathogenic. Previously, we reported a missense mutation in exon 12 at codon 600 of the MSH2 gene, causing a substitution of GTT (Val) for GCT (Ala) in a 35-year-old-man with rectal cancer, while the pathogenicity of this mutation is still unclear. In this report, we confirm the same mutation in his 66-year-old mother who had cecal cancer. PCR/direct sequencing analysis of peripheral blood lymphocytes revealed the same missense mutation in exon 12 at codon 600 of the MSH2 gene. The wave height of the capillary sequencer from the wild-type allele was decreased in tumor tissue, indicating loss of heterozygosity in the wild-type allele. Analysis of the tumor showed microsatellite instability high and loss of MSH2 protein expression. This sequence variant has not been reported in another family. This mutation is considered to play a significant and causative role in Lynch syndrome. © The Author 2011. Published by Oxford University Press. All rights reserved.


Wolff E.M.,University of Southern California | Chihara Y.,University of Southern California | Pan F.,University of Southern California | Weisenberger D.J.,University of Southern California | And 6 more authors.
Cancer Research | Year: 2010

Urothelial cancer (UC) develops along two different genetic pathways, resulting in noninvasive or invasive tumors. However, it is unknown whether there are also different epigenetic pathways in UC. UC is also characterized by a high rate of recurrence, and the presence of a field defect has been postulated. In this study, we compared the DNA methylation patterns between noninvasive and invasive UC and the DNA methylation patterns between normal-appearing urothelium from bladders with cancer and urothelium from cancer-free bladders. We used the Illumina GoldenGate methylation assay at 1,370 loci in 49 noninvasive urothelial tumors, 38 invasive tumors with matched normal-appearing urothelium, and urothelium from 12 age-matched UC-free patients. We found distinct patterns of hypomethylation in the noninvasive tumors and widespread hypermethylation in the invasive tumors, confirming that the two pathways differ epigenetically in addition to genetically. We also found that 12% of the loci were hypermethylated in apparently normal urothelium from bladders with cancer, indicating an epigenetic field defect. X-chromosome inactivation analysis indicated that this field defect did not result in clonal expansion but occurred independently across the urothelium of bladders with cancer. The hypomethylation present in noninvasive tumors may counterintuitively provide a biological explanation for the failure of these tumors to become invasive. In addition, an epithelium-wide epigenetic defect in bladders with cancer might contribute to a loss of epithelial integrity and create a permissible environment for tumors to arise. ©2010 AACR.


Miyake M.,Tochigi Cancer Center Research Institute | Miyake M.,Nara Medical University | Ishii M.,Tochigi Cancer Center Research Institute | Koyama N.,Tochigi Cancer Center Research Institute | And 6 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2010

Activating mutation of the fibroblast growth factor receptor-3 (FGFR3) gene is known as a key molecular event in both oncogenesis and cell proliferation of low-grade noninvasive human bladder urothelial carcinoma (UC), which is characterized by frequent intravesical recurrence. In this study, we investigated the antitumor potentiality of 1-tert-butyl-3-[6-(3,5-dimethoxy- phenyl)-2-(4-diethylamino-butylamino)-pyrido[2,3-d]pyrimidin-7-yl]-urea (PD173074), a small-molecule FGFR3-selective tyrosine kinase inhibitor (TKI), as a therapeutic modality using eight UC cell lines. In our in vitro cell proliferation assay, PD173074 suppressed cell proliferation remarkably in two cell lines, namely, UM-UC-14 and MGHU3, which expressed mutated FGFR3 protein. In contrast, the other six cell lines expressing wild-type FGFR3 or without FGFR3 expression were resistant to PD173074 treatment. Cell cycle analysis revealed the growth inhibitory effect of PD173074 was associated with arrest at G1-S transition in a dose-depending manner. Furthermore, we observed an inverse relationship between Ki-67 and p27/Kip1 expression after PD173074 treatment, suggesting that up-regulation of p27 recruited UC cells harboring activating FGFR3 mutations in G1 that was analogous with the other receptor TKIs acting on the epidermal growth factor receptors. In the mouse xenograft models using subcutaneously transplanted UM-UC-14 and MGHU3, orally administered PD173074 suppressed tumor growth and induced apoptotic changes comparable with the results of our in vitro assay. These findings elucidated the effectiveness of molecular targeted approach for bladder UC harboring FGFR3 mutations and the potential utility to decrease the intravesical recurrence of nonmuscle invasive bladder UC after transurethral surgical resection. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.


BACKGROUND: Methylation of the MLH1 promoter region has been suggested to be a major mechanism of gene inactivation in sporadic microsatellite instability-positive (MSI-H) colorectal cancers (CRCs). Recently, single-nucleotide polymorphism (SNP) in the MLH1 promoter region (MLH1-93G/A; rs1800734) has been proposed to be associated with MLH1 promoter methylation, loss of MLH1 protein expression and MSI-H tumors. We examined the association of MLH1-93G/A and six other SNPs surrounding MLH1-93G/A with the methylation status in 210 consecutive sporadic CRCs in Japanese patients.METHODS: Methylation of the MLH1 promoter region was evaluated by Na-bisulfite polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) analysis. The genotype frequencies of SNPs located in the 54-kb region surrounding the MLH1-93G/A SNP were examined by SSCP analysis.RESULTS: Methylation of the MLH1 promoter region was observed in 28.6% (60/210) of sporadic CRCs. The proportions of MLH1-93G/A genotypes A/A, A/G and G/G were 26% (n=54), 51% (n=108) and 23% (n=48), respectively, and they were significantly associated with the methylation status (p=0.01). There were no significant associations between genotype frequency of the six other SNPs and methylation status. The A-allele of MLH1-93G/A was more common in cases with methylation than the G-allele (p=0.0094), especially in females (p=0.0067). In logistic regression, the A/A genotype of the MLH1-93G/A SNP was shown to be the most significant risk factor for methylation of the MLH1 promoter region (odds ratio 2.82, p=0.003). Furthermore, a haplotype of the A-allele of rs2276807 located -47 kb upstream from the MLH1-93G/A SNP and the A-allele of MLH1-93G/A SNP was significantly associated with MLH1 promoter methylation.CONCLUSIONS: These results indicate that individuals, and particularly females, carrying the A-allele at the MLH1-93G/A SNP, especially in association with the A-allele of rs2276807, may harbor an increased risk of methylation of the MLH1 promoter region.


PubMed | Tochigi Cancer Center Research Institute, Nikko Memorial Hospital and Sapporo Medical University
Type: | Journal: European journal of dermatology : EJD | Year: 2017

Muir-Torre syndrome (MTS) is characterized by sebaceous neoplasms with internal malignancies and regarded as a variant of hereditary nonpolyposis colorectal cancer (HNPCC). Pathogenic variations of MTS have been identified in the MSH2, MLH1, and MSH6 genes, with the majority of variations located in MSH2.To present an MTS patient who was the only individual with skin malignancies within a cancer-prone pedigree and to show the usefulness of RNA-based genetic analysis in the investigation of MTS.A 77-year-old man who had operated X-ray equipment at his workplace in his twenties was clinically diagnosed with MTS and investigated by RNA-based analysis, multiplex ligation-dependent probe amplification, and genomic DNA sequencing.The patient had suffered from sebaceous tumours, squamous cell carcinomas of the skin, and colon cancer. The patients family history was remarkable for visceral malignant diseases. Genetic analysis revealed homologous recombination between two Alu elements within intron 4 and 5 of the MLH1 gene. The rearrangement caused a 1,222-bp deletion, including the entire exon 5. Deletion of exon 5 has previously been reported only in two patients with HNPCC, and not in patients with MTS.For the genetic analysis of MTS, the possibility of rare copy number variations of MLH1, as well as MSH2 variations, should be considered. RNA-based screening using puromycin is recommended in order to identify such variations. It remains unclear why only the proband among the pedigree had skin malignancies, however, the skin carcinogenesis might have been related to occupational radiation exposure.

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