Yoji Nishimura

Three DNA Methylation Epigenotypes in Human Colorectal Cancer

Purpose: Whereas the CpG island methylator phenotype (CIMP) in colorectal cancer associates with microsatellite instability (MSI)-high and BRAF-mutation(+), the existence of an intermediate-methylation subgroup associated with KRAS-mutation(+) is controversial, and suitable markers for the subgroup have yet to be developed. Our aim is to clarify DNA methylation epigenotypes of colorectal cancer more comprehensively. Experimental Design: To select new methylation markers on a genome-wide scale, we did methylated DNA immunoprecipitation-on-chip analysis of colorectal cancer cell lines and re-expression array analysis by 5-aza-2′-deoxycytidine/Trichostatin A treatment. Methylation levels were analyzed quantitatively in 149 colorectal cancer samples using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. Colorectal cancer was epigenotyped by unsupervised two-way hierarchical clustering method. Results: Among 1,311 candidate silencing genes, 44 new markers were selected and underwent quantitative methylation analysis in colorectal cancer samples together with 16 previously reported markers. Colorectal cancer was clustered into high-, intermediate-, and low-methylation epigenotypes. Methylation markers were clustered into two major groups: group 1 showing methylation in high-methylation epigenotype, and group 2 showing methylation in high- and intermediate-methylation epigenotypes. A two-step marker panel deciding epigenotypes was developed with 95% accuracy: the 1st panel consisting of three group-1 markers (CACNA1G, LOX, SLC30A10) to extract high-methylation epigenotype, and the 2nd panel consisting of four group-2 markers (ELMO1, FBN2, THBD, HAND1) and SLC30A10 again to divide the remains into intermediate- and low-methylation epigenotypes. The high-methylation epigenotype correlated significantly with MSI-high and BRAF-mutation(+) in concordance with reported CIMP. Intermediate-epigenotype significantly correlated with KRAS-mutation(+). KRAS-mutation(+) colorectal cancer with intermediate-methylation epigenotype showed significantly worse prognosis. Conclusions: Three methylation epigenotypes exist in colorectal cancer, and suitable classification markers have been developed. Intermediate-methylation epigenotype with KRAS-mutation(+) correlated with worse prognosis. Clin Cancer Res; 16(1); 21–33

Identification of chromosomal aberrations of metastatic potential in colorectal carcinoma.

In colorectal cancer (CRC) care, treatment decisions depend on the efforts to estimate the metastatic potential of tumors. The liver is one of the most common metastatic sites of CRC and the prognosis of CRC patients often reflects metastases to distant sites. To identify chromosomal aberrations associated with liver metastasis, we performed allelic copy number analysis for CRC with or without synchronous liver metastasis using genotyping arrays. By allelic copy number analysis of CRC samples, we observed common aberrations in 14 chromosomal arms in two groups, that is, gains on 7p22.3‐p11.2, 8q22.3‐q24.3, 13q12.12‐q34, and 20q11.22‐q13.33 and loss of heterozygosity (LOH) on 4q12‐q35.1, 5q11.2‐q35.3, 8p23.3‐p12, 15q11.2‐q26.3, 17p13.3‐p11.2, 17q11.2‐q25.1, 18p11.32‐p11.21, 18q11.2‐q23, 20p13‐p12.1, and 22q11.1‐q13.32. We found that gains on 20p13‐p12.1 and 20q11.21‐q13.33 and LOH on 6q14.1‐q25.1 were more frequent in CRC with liver metastasis. We also compared chromosomal aberrations in primary CRC lesions with those of the corresponding liver metastasis and found that the allelic genome imbalance status of a metastatic lesion is similar to that of the primary cancer, which suggests that chromosomal aberrations are largely maintained on hematogenous spread. Intriguingly, several chromosomal aberrations in CRC were found in the primary cancer but not in the corresponding liver metastasis, thus suggesting heterogeneity of cancer cells within solid tumors or the presence of events uniquely developed in primary tumors. Consequently, CRC with and without liver metastasis harbor similar chromosomal aberrations, and chromosomal aberration at 6q, 20p, and 20q may be involved in the process of liver metastasis of CRC.

Prognostic value of KRAS and BRAF mutations in curatively resected colorectal cancer.

AIMnTo investigate the prognostic role of KRAS and BRAF mutations after adjustment for microsatellite instability (MSI) status in Japanese colorectal cancer (CRC) population.nnnMETHODSnWe assessed KRAS and BRAF mutations and MSI status in 813 Japanese patients with curatively resected, stageu2005I-III CRC and examined associations of these mutations with disease-free survival (DFS) and overall survival (OS) using uni- and multivariate Cox proportional hazards models.nnnRESULTSnKRAS and BRAF mutations were detected in 312 (38%) of 812 and 40 (5%) of 811 tumors, respectively. KRAS mutations occurred more frequently in females than in males (P=0.02), while the presence of BRAF mutations was significantly associated with the female gender (P=0.006), proximal tumor location (P<0.001), mucinous or poorly differentiated histology (P<0.001), and MSI-high tumors (P<0.001). After adjusting for relevant variables, including MSI status, KRAS mutations were associated with poorer DFS (HR=1.35; 95%CI: 1.03-1.75) and OS (HR=1.46; 95%CI: 1.09-1.97). BRAF mutations were poor prognostic factors for DFS (HR=2.20; 95%CI: 1.19-4.06) and OS (HR=2.30; 95%CI: 1.15-4.71). Neither the BRAF by MSI interaction test nor the KRAS by MSI interaction test yielded statistically significant results for DFS and OS.nnnCONCLUSIONnKRAS and BRAF mutations are associated with inferior survival, independent of MSI status, in Japanese patients with curatively resected CRC.

Indicators for surgical resection and intraoperative radiation therapy for pelvic recurrence of colorectal cancer.

AbstractPURPOSE: We retrospectively analyzed prognostic factors for surgical resection and intraoperative radiation therapy to identify indicators for this treatment strategy. nMETHODS: Thirty-nine consecutive patients with locally recurrent colorectal cancer who underwent surgical resection with intraoperative radiation therapy from January 1, 1987, to June 30, 1999, were analyzed. The mean electron energy was 10.5 MeV and the mean intraoperative radiation dose was 22.6 Gy. Kaplan-Meier survival estimates were obtained for the 37 patients who recovered postoperatively. Prognostic factors were analyzed univariately by log-rank test and multivariately by Cox’s proportional hazards model. nRESULTS: Three-year cumulative survival was 44 percent (standard error = 11) for 26 patients free of unresectable distant metastasis who underwent surgical resection and intraoperative radiation therapy for pelvic recurrence of colorectal cancer, but none of the 11 patients with unresectable distant metastasis survived 3 years. Preoperative prognostic factors which were significant on univariate and multivariate analysis were unresectable distant metastasis (P = 0.001) and elevated preoperative serum CA 19–9 (P = 0.02). Patients with synchronous resection of local recurrence and distant metastasis had a significant survival advantage over those without resection of metastases (P = 0.02). Univariate analysis in a subgroup of 26 patients without unresectable distant metastasis revealed pain (P = 0.0003) to be a useful preoperative prognostic indicator, whereas tumor fixation (P = 0.01) and amount of residual tumor after surgical resection (P = 0.01) were significant intraoperative and postoperative factors, respectively. Fluorouracil-based postoperative systemic chemotherapy produced a significant survival benefit (P = 0.04). nCONCLUSIONS: Patients with unresectable distant metastasis are not suitable candidates for surgical resection and intraoperative radiation therapy, whereas those with resectable metastasis are potential candidates. Intraoperative radiation therapy may be less useful for patients with pain, elevated preoperative CA19–9, fixed tumors, or gross residual tumor after surgical resection. Multimodal treatment strategies combining preoperative and/or postoperative external beam radiation therapy and intraoperative radiation therapy with fluorouracil-based systemic chemotherapy are recommended for patients with these indicators.

High concordance rate of KRAS/BRAF mutations and MSI-H between primary colorectal cancer and corresponding metastases

Genetic testing is needed for the treatment of colorectal cancer (CRC), especially molecular-targeted therapy. The effects of anti-EGFR therapy and prognosis are affected by the presence of KRAS mutations. However, whether primary CRC or metastatic tissues are appropriate in the analysis is still unclear. In the present study, we assessed the concordance of KRAS/BRAF mutation status and microsatellite instability (MSI) in primary CRC and corresponding metastases. This study enrolled 457 patients with surgically resected primary and corresponding metastatic CRC (499xa0synchronous metastases and 57xa0metachronous metastases) and seven local recurrences, and KRAS/BRAF mutation and MSI status were analysed for these tumours. The concordance rates of KRAS mutation, BRAF mutation, wild-type, MSI-H and MSS between primary CRC and corresponding metastases were 93.9% (214/228), 100% (30/30), 99.3% (304/306), 87.5% (21/24) and 100% (137/137), respectively. These high concordance rates were not different between synchronous and metachronous metastases. In conclusion, a high concordance of KRAS/BRAF mutation status and MSI status was observed between primary CRC and corresponding metastases in this study. Either primary CRC or metastatic tissues can be used for testing KRAS/BRAF mutation status and MSI status.

Laparoscopic Versus Open Lateral Lymph Node Dissection for Locally Advanced Low Rectal Cancer: A Subgroup Analysis of a Large Multicenter Cohort Study in Japan

BACKGROUND: Mesorectal excision with lateral lymph node dissection is the standard treatment for locally advanced low rectal cancer in Japan. However, the safety and feasibility of laparoscopic lateral lymph node dissection remain to be determined. OBJECTIVE: The purpose of this study was to evaluate the safety and feasibility of laparoscopic versus open lateral lymph node dissection for locally advanced low rectal cancer. DESIGN: This was a retrospective cohort study using an exact matching method. SETTING: We conducted a multicenter study of 69 specialized centers in Japan. PATIENTS: Patients with consecutive midrectal or low rectal adenocarcinoma cancer stage II to III who underwent mesorectal excision with curative intent between 2010 and 2011 were recruited. MAIN OUTCOME MEASURES: Short-term and oncological outcomes were compared between the laparoscopic and open-surgery groups. RESULTS: Of the 1500 eligible patients, 676 patients who underwent lateral lymph node dissection were analyzed, including 137 patients who were treated laparoscopically and 539 patients who were treated with open surgery. After matching, the patients were stratified into laparoscopic (n = 118) and open-surgery (n = 118) groups. Operative times in the overall cohort were significantly longer (461 vs 372u2009min) in the laparoscopic versus the open-surgery group. In the laparoscopic group, the blood loss volume was significantly smaller (193 vs 722u2009mL), with fewer instances of blood transfusion (7.3% vs 25.5%) compared with the open-surgery group. The postoperative complication rates were 35.8% and 43.6% for the laparoscopic and open-surgery groups (p = 0.10). The 3-year relapse-free survival rates were 80.3% and 72.6% for the laparoscopic and open-surgery groups (p = 0.07). LIMITATIONS: The study was limited by its retrospective design and potential selection bias. CONCLUSIONS: Laparoscopic lateral lymph node dissection is safe and feasible for cancer stage II to III low rectal cancer and is associated with similar oncological outcomes as open lateral lymph node dissection. See Video Abstract at http://links.lww.com/DCR/A334.

Molecular and clinical characteristics of MSH6 germline variants detected in colorectal cancer patients

The MSH6 gene is one of the mismatch repair genes involved in Lynch syndrome and its mutations account for 10-20% of Lynch syndrome. Although previous studies suggested that the difference of the geographical region affects the clinical phenotype of Lynch syndrome, there has been no report on the detailed features of Japanese Lynch syndrome patients carrying an MSH6 mutation. The aim of the present study was to investigate the clinical and molecular features of MSH6 mutation carriers in Japan. Surgically resected 1720 colorectal carcinoma specimens were screened by microsatellite instability (MSI) testing and the MSI-high cases were subjected to a germline mutation analysis of the mismatch repair genes MLH1, MSH2 and MSH6. We investigated the clinical and molecular features of the MSH6 variants, such as the family cancer history, pathological findings, immunohistochemistry, methylation status of the MLH1 promoter and BRAF mutation in the colorectal tumor. Furthermore, the impact of the missense variants on MSH6 protein was predicted by using in silico tools. We identified nine novel pathogenic mutations and eight unclassified missense variants. Among the eight missense variants, three were suspected pathogenic by in silico analysis. We also found that most colorectal cancers in the MSH6 mutation carrier were diagnosed after the age of 50 and were localized distally. Furthermore, the mean age at diagnosis of endometrial cancer in Japanese MSH6 mutation carriers (49.2 years) was earlier than previous reports from Western countries (56.5 years). These results may improve the surveillance program for Japanese MSH6 mutation carriers.

Predictive model for high-frequency microsatellite instability in colorectal cancer patients over 50 years of age

Microsatellite instability (MSI) is an important biomarker for screening for Lynch syndrome, and also of response to immune checkpoint inhibitors. The aim of this study is to create a predictive model to determine which elderly patients with colorectal cancer (CRC) should undergo MSI and/or immunohistochemistry testing on the basis of clinicopathological data. We analyzed a test cohort of CRC patients aged ≥50 years (n = 2219) by multivariate logistic regression analyses to identify predictors of high‐frequency MSI (MSI‐H). The created prediction model was validated in an external cohort (n = 992). The frequency of MSI‐H was 5.5% among CRC patients aged ≥ 50 years. The following five predictors of MSI‐H were identified in the test cohort: female (1 point), mucinous component (2 points), tumor size ≥ 60 mm (2 points), location in proximal colon (3 points), and BRAF mutation (6 points). The area under curve (AUC) in the receiver‐operating characteristic (ROC) analysis of this prediction model was 0.832 (95% confidence interval: 0.790–0.874). The sensitivity and specificity were 74.4% and 77.7%, respectively, for a cut‐off score of 4 points. The receiver‐operating characteristic curve of the validation cohort also showed an AUC of 0.856 (95% CI: 0.806–0.905). This prediction model is useful to select elderly CRC patients who should undergo MSI testing, and who may benefit from treatment with 5‐FU‐based adjuvant chemotherapy and cancer immunotherapy.