Tumor DNA mismatch repair (MMR) deficiency testing is important to the identification of Lynch syndrome and decision making regarding adjuvant chemotherapy in stage II colorectal cancer (CRC) and has become an indispensable test in metastatic tumors due to the high efficacy of immune checkpoint inhibitor (ICI) in deficient MMR (dMMR) tumors. CRCs greatly benefit from this testing as approximately 15% of them are dMMR but only 3% to 5% are at a metastatic stage. MMR status can be determined by two different methods, microsatellite instability (MSI) testing on tumor DNA, and immunohistochemistry of the MMR proteins on tumor tissue. Recent studies have reported a rate of 3% to 10% of discordance between these two tests. Moreover, some reports suggest possible intra- and inter-tumoral heterogeneity of MMR and MSI status. These issues are important to know and to clarify in order to define therapeutic strategy in CRC. This review aims to detail the standard techniques used for the determination of MMR and MSI status, along with their advantages and limits. We review the discordances that may arise between these two tests, tumor heterogeneity of MMR and MSI status, and possible explanations. We also discuss the strategies designed to distinguish sporadic versus germline dMMR/MSI CRC. Finally, we present new and accurate methods aimed at determining MMR/MSI status.

Approximately 30% of patients with wild type RAS metastatic colorectal cancer are non-responders to anti-epidermal growth factor receptor monoclonal antibodies (anti-EGFR mAbs), possibly due to undetected tumoral subclones harboring RAS mutations. The aim of this study was to analyze the distribution of RAS mutations in different areas of the primary tumor, metastatic lymph nodes and distant metastasis. A retrospective cohort of 18 patients with a colorectal cancer (CRC) was included in the study. Multiregion analysis was performed in 60 spatially separated tumor areas according to the pathological tumor node metastasis (pTNM) staging and KRAS, NRAS and BRAF mutations were tested using pyrosequencing. In primary tumors, intra-tumoral heterogeneity for RAS mutation was found in 33% of cases. Inter-tumoral heterogeneity for RAS mutation between primary tumors and metastatic lymph nodes or distant metastasis was found in 36% of cases. Moreover, 28% of tumors had multiple RAS mutated subclones in the same tumor. A high proportion of CRCs presented intra- and/or inter-tumoral heterogeneity, which has relevant clinical implications for anti-EGFR mAbs prescription. These results suggest the need for multiple RAS testing in different parts of the same tumor and/or more sensitive techniques.

“Glioma Stem Cells” (GSCs) are known to play a role in glioblastoma (GBM) recurrence. Homologous recombination (HR) defects and cell cycle checkpoint abnormalities can contribute concurrently to the radioresistance of GSCs. DNA repair protein RAD51 homolog 1 (RAD51) is a crucial protein for HR and its inhibition has been shown to sensitize GSCs to irradiation. The aim of this study was to examine the consequences of ionizing radiation (IR) for cell cycle progression in GSCs. In addition, we intended to assess the potential effect of RAD51 inhibition on cell cycle progression. Five radiosensitive GSC lines and five GSC lines that were previously characterized as radioresistant were exposed to 4Gy IR, and cell cycle analysis was done by fluorescence-activated cell sorting (FACS) at 24, 48, 72, and 96 h with or without RAD51 inhibitor. Following 4Gy IR, all GSC lines presented a significant increase in G2 phase at 24 h, which was maintained over 72 h. In the presence of RAD51 inhibitor, radioresistant GSCs showed delayed G2 arrest post-irradiation for up to 48 h. This study demonstrates that all GSCs can promote G2 arrest in response to radiation-induced DNA damage. However, following RAD51 inhibition, the cell cycle checkpoint response differed. This study contributes to the characterization of the radioresistance mechanisms of GSCs, thereby supporting the rationale of targeting RAD51-dependent repair pathways in view of radiosensitizing GSCs.

Background: In most countries, participation in colorectal cancer (CRC) screening programs with the immunological fecal occult blood test (iFOBT) is low. Mutations of RAS and BRAF occur early in colorectal carcinogenesis and “liquid biopsy” allows detection of mutated circulating tumor DNA (ctDNA). This prospective study aims to evaluate the performance of RAS and BRAF-mutated ctDNA in detecting CRC and advanced adenomas (AA). Methods: One hundred and thirty patients who underwent colonoscopy for suspicion of colorectal lesion were included and divided into four groups: 20 CRC, 39 AA, 31 non-advanced adenoma and/or hyperplastic polyp(s) (NAA) and 40 with no lesion. Mutated ctDNA was analyzed by droplet digital PCR. Results: ctDNA was detected in 45.0% of CRC, in 2.6% of AA and none of the NAA and “no-lesion” groups. All patients with stage II to IV mutated CRC had detectable ctDNA (n = 8/8). Among the mutated AA, only one patient had detectable ctDNA (4.3%), maybe due to limited technical sensitivity or to a low rate of ctDNA or even the absence ctDNA in plasma. Specificity and sensitivity of KRAS- and BRAF-mutated ctDNA for the detection of all CRC and AA were 100% and 16.9%, respectively. Conclusions: ctDNA had high sensitivity in detection of advanced mutated CRC but was unable to sensitively detect AA. ctDNA analysis was easy to perform and readily accepted by the population but requires combination with other circulating biomarkers before replacing iFOBT.