Yasuyuki Miyakura

Mutations of BRAF are associated with extensive hMLH1 promoter methylation in sporadic colorectal carcinomas

Activating mutations of BRAF have been frequently observed in microsatellite unstable (MSI+) colorectal carcinomas (CRCs), in which mutations of BRAF and KRAS are mutually exclusive. Previously, we reported that hypermethylation of hMLH1 might play an important role in the tumorigenesis of right‐sided sporadic CRCs with MSI showing less frequency of KRAS/TP53 alteration. Therefore, we have assumed that BRAF mutations might be highly associated with hMLH1 methylation status rather than MSI status. In this study, mutations of BRAF and KRAS and their relationship with MSI and hMLH1 methylation status were examined in 140 resected specimens of CRC. The methylation status was classified into 3 types: full methylation (FM), partial methylation (PM) and nonmethylation (NM). Only FM closely linked to reduced expression of hMLH1 protein. BRAF mutations were found in 16 cases (11%), all leading to the production of BRAFV599E. As for MSI status, BRAF mutations were found in 43% of MSI+ and 4% of MSI− cases (p < 0.0001). Among the MSI+ individuals, BRAF mutations were more frequent in cases with hMLH1 deficiency (58%) than those with hMSH2 deficiency (0%; p = 0.02). Moreover, they were found in 69% of FM, 4% of PM and 4% of NM, revealing a striking difference between FM and the other 2 groups (FM vs. PM or NM; p < 0.0001). These findings suggest that BRAF activation may participate in the carcinogenesis of sporadic CRCs with hMLH1 hypermethylation in the proximal colon, independently of KRAS activation.

Extensive but hemiallelic methylation of the hMLH1 promoter region in early-onset sporadic colon cancers with microsatellite instability

BACKGROUND AND AIMS Methylation of the hMLH1 promoter region is frequently observed in microsatellite instability (MSI)-positive sporadic colorectal carcinomas. We studied hMLH1 promoter methylation in peripheral blood lymphocytes of 87 index patients representing 29 cases of hereditary nonpolyposis colorectal cancers (HNPCCs), 28 cases of atypical HNPCCs, and 30 sporadic cases of the development of early-onset colorectal carcinomas or multiple primary cancers. METHODS Methylation of the hMLH1 promoter region was analyzed by Na-bisulfite polymerase chain reaction/single-strand conformation polymorphism analysis or methylation-specific polymerase chain reaction. MSI, allelic status of the hMLH1 locus, and loss of hMLH1 protein expression were examined in cases for which tumor tissues were available. RESULTS Extensive methylation of the hMLH1 promoter was detected in peripheral blood lymphocytes of 4 of 30 patients with sporadic early-onset colon cancer, among whom multiple primary cancers (1 colon and 1 endometrial cancer) developed in 2 cases. This methylation was not detected in analyses of HNPCC or atypical HNPCC groups or healthy control subjects. MSI was positive, and extensive methylation was detected in both cancers (colon and endometrial cancer) and normal tissues (colon, gastric mucosa, endometrium, and bone marrow) in all of the examined cases (3 of 3). Analysis of a polymorphic site in the hMLH1 promoter in 2 informative cases showed that methylation was hemiallelic. In 1 case, the unmethylated allele was lost in the colon cancer but not in the metachronous endometrial cancer. CONCLUSIONS Constitutive, hemiallelic methylation of the hMLH1 promoter region was shown to be associated with carcinogenesis in sporadic, early-onset MSI-positive colon cancers.

Evaluation of venous invasion by Elastica van Gieson stain and tumor budding predicts local and distant metastases in patients with T1 stage colorectal cancer.

Evaluation of pathologic predictors of metastases in T1 stage colorectal cancer may be difficult with hematoxylin and eosin (HE) staining alone. The aim of this study was to clarify the role of pathologic predictors by using immunohistochemical staining and Elastica van Gieson (EVG) staining. One hundred and twenty-four patients who underwent bowel resection for single T1 stage colorectal cancer from 1990 to 2004 in 1 institution were studied. D2-40, EVG staining, and CAM5.2 were used to detect lymphatic invasion, venous invasion, and tumor budding, respectively. These 3 factors were separately evaluated based on HE staining. Histology was reviewed by 1 pathologist. Lymph node metastases in the surgical specimen were the standard reference, and distant metastases were identified by periodic computed tomography for 2 years or more after surgery. A logistic regression model was applied to analyze risk factors for lymph node metastases and a Cox regression model for distant metastases. In predicting lymph node metastases, univariate analysis demonstrated significance for all predictors except venous invasion by HE staining. Multivariate analysis showed that venous invasion by EVG and tumor budding by HE showed significance as predictors. In predicting distant metastases, univariate analysis showed significance for lymphatic invasion shown by D2-40, tumor budding shown by CAM5.2 and HE, and lymph node metastases. Multivariate analysis showed only venous invasion by EVG stain as being significantly associated with distant metastases (P=0.001). In conclusion, venous invasion evaluated shown by EVG staining is a useful pathologic predictor for metastases in T1 stage colorectal cancer.

Chromosome copy number analysis in screening for prognosis-related genomic regions in colorectal carcinoma

Colorectal carcinoma (CRC) remains the major cause of cancer death in humans. Although chromosomal structural anomaly is presumed to play an important role in the carcinogenesis of CRC, chromosomal copy number alterations (CNA) and loss of heterozygosity (LOH) have not yet been analyzed extensively at high resolution in CRC. Here we aim to identify recurrent CNA and LOH in human CRC with the use of single nucleotide polymorphism‐typing microarrays, and to reveal their relevance to clinical outcome. Surgically resected CRC specimens and paired normal mucosa were obtained from a consecutive series of 94 patients with CRC, and both of them were subjected to genotyping with Affymetrix Mapping 50K arrays. CNA and LOH were inferred computationally on every single nucleotide polymorphism site by integrating the array data for paired specimens. Our large dataset reveals recurrent CNA in CRC at chromosomes 7, 8, 13, 18, and 20, and recurrent LOH at chromosomes 1p, 4q, 5q, 8p, 11q, 14q, 15q, 17p, 18, and 22. Frequent uniparental disomy was also identified in chromosomes 8p, 17p, and 18q. Very common CNA and LOH were present at narrow loci of <1 Mbp containing only a few genes. In addition, we revealed a number of novel CNA and LOH that were linked statistically to the prognosis of the patients. The precise and large‐scale measurement of CNA and LOH in the CRC genome is efficient for pinpointing prognosis‐related genome regions as well as providing a list of unknown genes that are likely to be involved in CRC development. (Cancer Sci 2008; 99: 1835–1840)

Microsatellite instability as a marker in predicting metachronous multiple colorectal carcinomas after surgery. A cohort-like study

AbstractPURPOSE: In case-control studies, it was reported that microsatellite instability might be helpful in predicting the development of metachronous multiple colorectal cancers. The purpose of this cohort-like study was to determine whether microsatellite instability is a novel independent marker in predicting metachronous colorectal carcinomas after colorectal cancer surgery. METHODS: Three hundred twenty-eight colorectal carcinoma patients were surveyed by periodic colonoscopy for at least three years after surgery. Among these, DNA from paraffin-embedded sections was available for 272 cases. DNA of these cases was studied for six microsatellite markers (five dinucleotide repeats, one mononucleotide repeat). Microsatellite instability phenotype was defined as alterations in one or more loci. RESULTS: Median follow-up period was 74 months, and the median number of colonoscopies was 4.6. The percentage of microsatellite instability-positive cases was 26.4 percent (72/272). Seventeen metachronous colorectal carcinomas were detected during the follow-up period. Incidences of metachronous colorectal carcinomas in microsatellite instability-positive and microsatellite instability-negative cases were 15.3 and 3 percent, respectively (P < 0.001). The cumulative five-year incidence of metachronous colorectal carcinomas was significantly higher in microsatellite instability-positive cases than in microsatellite instability-negative cases (12.5 vs. 2.5 percent, P < 0.0001). Logistic regression analysis of the relationship between incidence of metachronous colorectal carcinomas and possible risk factors (namely, coexistence of adenoma at the time of surgery, family history of colorectal carcinoma , history of extracolonic malignancy, and microsatellite instability status) showed that microsatellite instability and coexistence of adenoma were significant independent risk factors for the occurrence of metachronous colorectal carcinomas, with values of P = 0.001 and 0.02, respectively. CONCLUSION: These data indicate that microsatellite instability can be regarded as a novel independent and important marker for predicting the development of metachronous colorectal carcinoma after surgery.

Methylation profile of the MLH1 promoter region and their relationship to colorectal carcinogenesis.

Methylation of the MLH1 promoter region has been suggested to be a principal mechanism of gene inactivation in sporadic microsatellite instability (MSI)–positive colorectal carcinoma. Recently, we have shown a novel methylation profile of the MLH1 promoter region (i.e., full, partial, and no methylation), among which full methylation was strongly associated with MSI. In this study, to confirm whether methylation requires the involvement of both alleles, we studied the MLH1 promoter region concerning the methylation profile and allelic loss. Furthermore, we studied correlations of methylation profiles with genetic alternations such as loss of heterozygosity (LOH) of the TP53 locus and KRAS mutation. Eighty‐eight tumors were classified as full (n = 14), partial (n = 26), and no methylation (n = 48). Full methylation was observed in 78% (14/18) of high‐frequency MSI, in which all CpG sites in the promoter region were methylated. Full methylation differed significantly from partial methylation regarding absence of TP53 LOH (0/12) and KRAS mutation (0/14). In cases with full methylation, we could show biallelic methylation by use of a single‐base nucleotide polymorphism in the promoter. However, this did not accompany LOH of the MLH1 locus. In contrast, there were no significant differences in molecular features between partial and no methylation, except for low frequencies of LOH of the MLH1 locus (P = 0.02). In conclusion, biallelic extensive methylation of the MLH1 promoter region plays a significant role in gene inactivation and is independent of KRAS mutation and TP53 LOH.

The Use of Olaparib (AZD2281) Potentiates SN-38 Cytotoxicity in Colon Cancer Cells by Indirect Inhibition of Rad51-Mediated Repair of DNA Double-Strand Breaks

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. We examined 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 the MMR status. Furthermore, olaparib potentiated S-phase–specific double-strand 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 the MRN complex. These results may contribute to future clinical trials using PARP inhibitor plus topoisomerase I inhibitor in combination. Furthermore, the synergistic effect comprising topoisomerase I-mediated DNA breakage–reunion reaction, PARP and Rad51-mediated HR pathway suggests the triple synthetic lethal pathways contribute to this event and are applicable as a potential target for future chemotherapy. Mol Cancer Ther; 13(5); 1170–80. ©2014 AACR.

Cell diameter measurements obtained with a handheld cell counter could be used as a surrogate marker of G2/M arrest and apoptosis in colon cancer cell lines exposed to SN-38

In vitro assessment of chemosensitivity are important for experiments evaluating cancer therapies. The Scepter 2.0 cell counter, an automated handheld device based on the Coulter principle of impedance-based particle detection, enables the accurate discrimination of cell populations according to cell size and volume. In this study, the effects of SN-38, the active metabolite of irinotecan, on the colon cancer cell lines HCT116 and HT29 were evaluated using this device. The cell count data obtained with the Scepter counter were compared with those obtained with the (3)H-thymidine uptake assay, which has been used to measure cell proliferation in many previous studies. In addition, we examined whether the changes in the size distributions of these cells reflected alterations in the frequency of cell cycle arrest and/or apoptosis induced by SN-38 treatment. In our experiments using the Scepter 2.0 cell counter, the cell counts were demonstrated to be accurate and reproducible measure and alterations of cell diameter reflected G2/M cell cycle arrest and apoptosis. Our data show that easy-to-use cell counting tools can be utilized to evaluate the cell-killing effects of novel treatments on cancer cells in vitro.

Colocutaneous fistula after left inguinal hernia repair using the mesh plug technique.

A 34-YEAR-OLD MAN was admitted with purulent discharge from a left inguinal hernia scar. Three years before, a hernioplasty using the mesh plug technique was performed at another hospital, with an uneventful postoperative course. Two years later, he noted purulent discharge from the operative site that was treated unsuccessfully with oral antibiotics, and he was referred to our hospital. On admission, his temperature was 36.2 C. He had a yellow-green foul-smelling discharge at the lateral edge of the operative scar. Induration was palpable under the scar, and the surrounding skin was erythematous (Fig 1). Laboratory findings were normal (WBC 9000/mm). Fistulography (Fig 2) demonstrated contrast flowing past the mesh plug and into the sigmoid colon; a colocutaneous fistula caused by migration of the mesh plug was diagnosed. After partial resection of the

cDNA array analysis for prediction of hepatic metastasis of colorectal carcinoma

PurposeDistant metastasis is a significant prognostic factor of colon carcinoma. Adjuvant chemotherapy has been shown to decrease its recurrence. However, there are no definitive methods for the diagnosis of hepatic recurrence after potentially curative surgery. The aim of this study was to evaluate the accuracy of mRNA expression profiling using samples obtained from primary tumors to predict hepatic recurrence.MethodsPatients with stage III colorectal carcinoma without any recurrence for at least 5 years (group A: n = 9) and patients with stage IV carcinoma with hepatic metastasis (group B: n = 10) were included in this study. Tissue samples were collected from the primary tumor and adjacent normal colonic mucosa at the time of surgery in each patient. Total RNA was extracted and the mRNA expression profile was examined using a cDNA macroarray.ResultsA hierarchical clustering analysis revealed a dendrogram in which the patients were divided into two clusters. One cluster consisted of seven patients in group A and two in group B. The other consisted of two patients in group A and eight in group B. Therefore, the positive and negative predictive value of hierarchical clustering analysis for hepatic metastasis was 80.0% and 78.8%, respectively. Fifteen genes were revealed to be upregulated and 12 were downregulated in group B. The upregulated genes included CCNA2, TP53, and MDM2, while the downregulated genes included CDH1, GADD45A, and BCL2L2.ConclusionsmRNA expression profiling by a cDNA array analysis of specimens obtained from primary tumors was found to be useful for distinguishing patients with and without hepatic metastasis. This method is expected to contribute to the identification of patients at high risk for hepatic recurrence, while also helping in the administration of intensive adjuvant chemotherapy for such high risk patients.