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.

Routine genetic testing of lung cancer specimens derived from surgery, bronchoscopy and fluid aspiration by next generation sequencing.

After the development of EGFR tyrosine kinase inhibitors (TKIs), genetic testing of EGFR became required for effective treatment of lung cancer. Initially, the testing was conducted separately for each mutated region. However, many EGFR mutations have since been identified that determine the efficacy of EGFR-TKIs. Therefore, genetic testing of EGFR by next generation sequencing (NGS) may be a suitable strategy for lung cancer. Here we examined the applicability of the NGS method in regard to sensitivity, time and cost. A total of 939 specimens were obtained from 686 lung cancer patients at our hospital. DNA and RNA were simultaneously extracted from specimens derived from surgery, bronchoscopy, and fluid aspiration. Specimens included cerebrospinal fluid, pleural effusion, abdominal fluid, and pericardial effusion. From RNA, target regions (EGFR, KRAS, ALK fusion and RET fusion) were enriched by RT-PCR and sequenced with MiSeq. From DNA, PCR or PCR-RFLP conventional methods were performed. NGS and conventional methods were carried out routinely per week. Among the total 939 specimens, 38 specimens could not be examined with NGS. Among these, 34 specimens were analyzed by conventional testing with simultaneously extracted DNA. The remaining four specimens could not be tested with either method. Compared with the conventional method, the concordance rate of mutations was 99% (892/901), excluding specimens with NGS failure. The time period required from processing of specimens to results was 4 days, and the cost per sample was sufficiently low. In conclusion, the genetic testing with NGS method was useful for lung cancer treatment. The cost, sensitivity and time were able to tolerate routine examinations.

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 (499 synchronous metastases and 57 metachronous 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.

Metastatic Pattern of Stage IV Colorectal Cancer with High-Frequency Microsatellite Instability as a Prognostic Factor.

BACKGROUND: A recent clinical trial on the immune check-point inhibitor pembrolizumab demonstrated that microsatellite instability (MSI) is a good biomarker for response to this inhibitor. However, clinicopathological features of advanced colorectal cancer (CRC) with high-frequency MSI (MSI-H) are unclear. PATIENTS AND METHODS: A total of 2,439 surgically resected CRC tissues were analyzed for MSI status, and mutational status of V-Ki-Ras2 Kirsten rat sarcoma 2 viral oncogene homolog (KRAS), neuroblastoma RAS viral oncogene homolog (NRAS) and v-Raf murine sarcoma viral oncogene homolog B (BRAF). Stage IV cases were selected, and clinical and molecular features were evaluated. RESULTS: There was no significant survival difference observed between MSI-H CRC and microsatellite-stable (MSS) CRC in patients with stage IV disease (3.92 vs. 2.50 years; p=0.766). However, hematogenous and lymphogenous metastasis-dominant CRC with MSI-H demonstrated poor prognosis, whereas peritoneal metastasis-dominant CRC with MSI-H demonstrated good prognosis, (1.33 vs. 5.2 years; p=0.006). CONCLUSION: Prognosis of stage IV CRC with MSI-H depended on the metastatic pattern. These findings provide useful information for the adaptation of CRC immunotherapy.

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

AIM: To investigate the prognostic role of KRAS and BRAF mutations after adjustment for microsatellite instability (MSI) status in Japanese colorectal cancer (CRC) population. METHODS: We assessed KRAS and BRAF mutations and MSI status in 813 Japanese patients with curatively resected, stage I-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. RESULTS: KRAS 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. CONCLUSION: KRAS and BRAF mutations are associated with inferior survival, independent of MSI status, in Japanese patients with curatively resected CRC.

[Tm analysis method using a quenching probe is a simple and rapid way to simultaneously detect KRAS and BRAF mutations].

Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that, on ligand binding, triggers the RAS-RAF-MAPK signaling pathway which is mainly associated with cell proliferation. Drugs targeting EGFR have recently entered clinical practice and have proven to be effective in providing clinical benefits. However, the presence of mutated KRAS alleles in cancer is a predictive marker of anti-EGFR drug resistance. Similarly, alterations of other members of the RAS-RAF-MAPK signaling pathway may also be such predictive markers, especially the BRAF mutation in colorectal cancer. Therefore, the identification of KRAS and BRAF mutations may be important in predicting resistance to EGFR-targeted therapies. We looked at the Tm analysis for simultaneous detection of KRAS and BRAF mutations using a quenching probe. The oligonucleotide probes modified with certain fluorescent dyes at 5'-end cytosine are quenched by their interaction with a uniquely positioned guanine. When the probe is hybridized with target DNA, its fluorescence is quenched by the guanine in the target DNA. However, as the temperature is raised, perfect match probes and miss match probes dissociate at different temperatures. Dissociated probes generate fluorescence. This simple and rapid method for simultaneous detection of these mutations is useful in clinical practice.

Microsatellite instability-low colorectal cancer acquires a KRAS mutation during the progression from Dukes' A to Dukes' B.

The classification of colorectal cancer (CRC) by microsatellite instability (MSI) status is important for effective clinical management. In fact, microsatellite instability-high (MSI-H) cancer has distinctive clinicopathological and molecular features. However, microsatellite instability-low (MSI-L) cancer is not clearly defined. The objective of this study was to further clarify the characteristics of MSI-L CRC. A consecutive series of 940 primary CRCs were subdivided into three groups according to the level of MSI and analyzed the clinicopathological features and genetic changes in the KRAS, BRAF and p53 mutation and the loss of heterozygosity (LOH) of adenomatous polyposis coli (APC) gene and methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) and MLH1 promoter. Of the 940 CRCs, 5.9% were MSI-H, 7.1% were MSI-L and 87% were microsatellite stable (MSS). KRAS and BRAF mutations were detected in 39.4 and 4.6% of the CRCs, respectively. The frequency of KRAS mutations in MSI-H, MSI-L and MSS cancer was 30, 48 and 39%, respectively. The proportion of KRAS mutations in MSI-L cancer increased from 16 to 63% accompanying the progression from Dukes' A to Dukes' B. While the LOH of D5S346, which is located near the APC gene, and p53 mutation was observed in 75 and 67% of MSI-L CRC at Dukes' A, respectively. These results indicated that the LOH of APC and p53 mutation has already occurred by the Dukes' A lake 'suppressor pathway' but not the KRAS mutation in MSI-L CRCs. The genes involving MSI-L carcinogenesis are similar to MSS but the timing and frequency of the KRAS mutation is different.

The clinical features of rectal cancers with high-frequency microsatellite instability (MSI-H) in Japanese males.

Colorectal cancers with high-frequency microsatellite instability (MSI-H) are known to display striking differences in their clinical and pathological features compared to microsatellite stable (MSS) cancers. Previous studies revealed that MSI-H cancers are more likely to occur in women, irrespective of the higher incidence of colorectal cancer in men. In this study we investigated the gender-specific clinico-pathological features of MSI positive colorectal cancer in Japanese individuals. A series of 478 colorectal carcinomas were collected in an unbiased manner and analyzed for microsatellite instability status. Seven percent, 6% and 87% of tumors showed MSI-H, low-frequency microsatellite instability (MSI-L) and MSS, respectively. A comparison of gender indicated a statistical significance in terms of the distribution of the subsite and age of onset of MSI-H colorectal cancer. In general, MSI-H cancers prone to develop in the proximal colon however, we found that about 36% of MSI-H cancers in men developed in the rectum. These rectal cancers were more likely to have a mucinous component and multiple colorectal cancers including critical lesion. These findings should be useful to find MSI positive rectum cancer.