Deficient Mismatch Repair And RAS Mutation In Colorectal Carcinoma Patients In Shandong Province

Background and ObjectivesColorectal cancer(CRC)is one of the most common cancers.The morbidity has increased steadily due to the growth of an aging population and the change of lifestyle in recent years.Microsatellite instability(MSI),caused by deficient mismatch repair(dMMR)system,is molecular phenotype of dMMR CRC.Compared with proficient MMR(pMMR),patients with dMMR CRCs are reported to have unique clinicopathological characteristics and better clinical outcome.Studies in China showed a lower frequency of dMMR compared with that in western populations,and the clinicopathological characteristics were also inconsistent.The RAS gene family,included KRAS and NRAS,is the other significant biomarker.Although several studies reported the frequency of KRAS mutation in Chinese CRC patients,the number of samples was limited in most of these studies.Moreover,information about NRAS mutation in Chinese CRC patients was limited.Little has been studied on the association between status of dMMR and RAS mutation.BRAF mutation has been proved to be responsible for poor response to epidermal growth factor receptor(EGFR)monoclonal antibody Cetuximab,and lead to poor prognosis in patients with metastatic colorectal carcinomas(CRCs).Moreover,it has been used to distinguish sporadic microsatellite instability(MSI)CRCs from Lynch syndrome.In papillary thyroid carcinomas(PTCs),more than 50% tumors harbor BRAF mutation,and the presence of BRAF mutation is associated with worse prognosis.Detection of BRAF mutation is useful to make a reliable diagnosis and treatment strategy.The routine methods for screening BRAF V600 E status are direct sequencing and amplification refractory mutation system(ARMS)-PCR study,which are time-consuming and expensive.Recently,a BRAF V600 E mutation-specific monoclonal antibody has been developed,which has been used to evaluate the V600 E status in several cancers.However,the results including assay conditions and scoring criteria were controversial indifferent cancer tissues.Therefore,813 formalin-fixed,paraffin-embedded tumor specimens from CRC patients who underwent primary surgical resection were selected.We analyzed the dMMR and RAS mutation status of CRC patients to evaluate possible associations between dMMR,RAS mutation and the clinicopathological characteristics in primary colorectal carcinoma and we also attempted to explore the prognostic roles of dMMR and RAS mutation.Moreover,we developed a fully automated IHC assay for BRAF V600 E mutation in formalin-fixed paraffin-embedded(FFPE)samples of CRC and PTC to explore the optimum detection conditions and investigate whether IHC could be used clinically to screen BRAF mutation status.Methods1.1 Clinical and pathological information from 813 patients were reviewed and recorded.1.2 Expression of mismatch repair proteins was tested by immunohistochemistry.1.3 Mutation analyses for RAS gene were performed by real-time polymerase chain reaction.2.1 Eight surgical resected samples with known BRAF V600 E status which was tested by Sanger sequencing,including 2 CRCs with mutated BRAF V600 E,2 PTCs with mutated BRAF V600 E,2 CRCs with wild BRAF V600 E,and 2 PTCs with wild BRAF V600 E,were selected to explore the optimal IHC conditions for the BRAF V600 E immunostaining.2.2 A total of 255 samples(123 CRCs and 132 PTCs)underwent IHC screening for BRAF V600 E using the optimal condition.2.3 Thereafter,ARMS-PCR was performed to determine the BRAF V600 E mutation in these 255 samples.The cases with discordant result were further validated by Sanger sequencing.Results1.The overall percentage of dMMR was 15.18%(121/797).The proportion of dMMR was higher in patients <50 years old and in the right side of the colon.Deficient mismatch repair was also associated with mucinous production,poor differentiation,early tumor stage and bowel wall invasion.2.The overall RAS mutation rate was 45.88%,including 42.56%(346/813)KRAS mutation and 3.69%(30/813)NRAS mutation.KRAS mutation was significantly associated with mucinous production,early tumor stage and was higher in non-smokers and patients with a family history of colorectal carcinoma.3.Moreover,dMMR,KRAS and NRAS mutation were not prognostic factors for stage I~III colorectal carcinoma.4.Pretreatment for 32 min and 64 min using the VENTANA Medical System showed satisfactory intensity and homogeneity of VE1 staining in CRC and PTC samples,respectively.5.The concordance between IHC and ARMS/Sanger sequencing was 99.2%(122/123)in CRCs and 96.2%(127/132)in PTCs.Conclusions1.The status of dMMR reflects the specific clinicopathological characteristics of colorectal carcinoma.The proportion of dMMR was higher in patients <50 years old and in the right side of the colon.Deficient mismatch repair was also associated with mucinous production.2.KRAS mutation was significantly associated with mucinous production and was higher in non-smokers and patients with a family history of colorectal carcinoma.3.dMMR,KRAS and NRAS mutation were not prognostic factors for stage I~III colorectal carcinoma.4.IHC is a suitable method to screen BRAF V600 Emutation in FFPE samples of CRCs and PTCs.