Relationship Of Clinicopathological Features And KRAS Gene Mutation In Left And Right-sided Colon Cancers

Research background and purpose:Colon cancer is one of the most common malignant tumors of the digestive tract.Worldwide,the incidence and mortality rate is third in all malignant tumors.In recent years,the incidence of colon cancer has increased significantly in Asian countries.The latest cancer epidemiology data in China show that the incidence rate is fourth,and the mortality rate is fifth.The biological behavior of left and right colon cancer has long been the focus of clinical attention.In 1990,De Groot et al.proposed dividing the colon into left-sided colon and right-sided colon.Also,he argued that LCC and RCC were two different cancers by elucidating the difference between LCC and RCC for the first time,involving difference in epidemiology and pathological and molecular characteristics,this finding shed light on the exploration of LCC and RCC for many scholars.It has clinically proven that epidermal growth factor(EGFR)is a therapeutic target closely associated with colon cancer and is used for colorectal cancer treatment.As a targeted therapeutic agent,EGFR inhibitor has shown obvious inhibition on colon cancer tumor growth.KRAS gene is an important regulatory gene in EGFR signaling pathway,while mutant-type KRAS gene reduces the function of EGFR antibody agent and leads to chemoresistance.2016 NCCN(National Comprehensive Cancer Network)has provided strict guidelines for molecular pathological diagnosis of mutant-type KRAS gene:1.It is necessary to detect the phenotype of KRAS gene of all CC patients.2.Only CC patients with wide-type KRAS gene were suggested for EGFR inhibitor(cetuximab and panitumumab)therapy.Therefore,KRAS gene mutation detection is the most direct and effective method for screening of anti-EGFR targeted drug.The clinicopathological features of left and right colon cancer and the correlation analysis with KRAS gene are not consistent in domestic reports.Most of them come from non random,small sample retrospective studies.This study aims to reveal tumor biological differences in different parts of colon cancer,especially molecular level,to provide a theory for the realization of individualized treatment and precise treatment of swelling tumors.Accurate treatment is based on accurate detection results.At present,there are still some laboratories in China that have not been tested in the laboratory,and the methods that have been carried out in the laboratory have not been completely unified.The different detection methods lead to the inconsistent results,which affect the clinical treatment and the future research.This article will also analyze and compare the two methods of Sanger DNA sequencing and ARMS,in order to provide a theoretical basis for the standardized detection of KRAS gene mutation in clinical laboratories.Methods:The clinicopathological data of 236 CC patients having surgery in general surgical department of Nanfang hospital were collected:site of tumor;clinicopathological diagnosis;sex;age;with or without abdominal pain;with or without crissum hemorrhage/fecal occult blood;tumor size;tumor differentiation degree,TNM stage;lymph node metastases is grouped by N0 and N1+N2;distant metastases is grouped by M0 and M1);198 CC patients were chosen for statistical analysis of clinicopathological data,aiming to find the difference of clinic-pathological features between LCC and RCC;198 chosen samples were made into formalin-fixed and parrffin-embedded specimens.113 samples were randomly selected to detect the mutation of KRAS gene by sequencing,and the correlation of KRAS gene mutation type,KRAS gene mutation and clinicopathological features in left and right colon cancer was analyzed.Then the ARMS method was used to detect the KRAS gene mutation in the 113 samples,and the two methodological differences were statistically analyzed.The statistical software is SPSS Statitics 20.0,and the analysis methods are chi square test and Kappa consistency test.The difference was statistically significant for P<0.05;the results of the two methods were Kappa consistency test and Kappa=0.679(0.4<Kappa<0.75),indicating consistency.Results:1.The correlation between clinic-pathological features and KRAS gene mutation in LCC and RCC.1)Clinic-pathological features of LCC and RCC.Among the 198 colon cancer cases,statistical analysis of the relationship between primary tumor site of colon cancer patients and pathological parameters showed that colon cancer tumor site is closely correlated with sex,age,tumor size,differentiation degree and distant metastases(P<0.05).Results of analysis showed no relevance between colon cancer primary tumor site and abdominal pain,T stage and N stage,the difference was not statistically significant(P?0.05).2)KRAS mutation type in LCC and RCC.Among the randomly divided 113 colon cancer cases,the total mutation rate of KRAS gene was 34.5%(39/113),among which the single base mutation rate of RCC was 52.6(20/38)and LCC was 25.3%(19/75).RCC patients had higher single base mutation rate compared with LCC,and the difference was statistically significant(P<0.05).Further analysis showed that the mutation rate of codon 12 of KRAS gene GLY12ASP(GGT>GAT)in RCC was 26.3%(10/38)and LCC was 26.3%(10/38)lower than the rate in RCC(P<0.05).The difference was statistically significant(P<0.05).However,we observed no relevance when analyzing the mutation rate of GLY12 VAL(GGT>GTT),GLY12ALA(GGT>GCT),GLY12CYS(GGT>TGT)and GLY13ASP(GGC>GAC),the difference was not statistically significant(P? 0.05).In our study,we found no single base mutation type other than the five mentioned types,but we found double mutation of GLY12ASP(GGT>GAT)and GLY13ASP(GGC>GAC)in a specific specimen.3)The relevance between KRAS gene mutation and clinic-pathological features.After analyzing the relationship between KRAS gene mutation and colon cancer clinic-pathological features in 113 specimens,we found KRAS mutation rate is closely correlated with tumor primary site,tumor differentiation degree and lymph node metastases(N stage),which was statistically significant(P<0.05).Analysis of 113 specimens on the relationship between KRAS mutation and clinic-pathological features displayed that KRAS mutation rates were various in CC patients with different sex,age(<60 years or ?60 years),tumor size(<5cm or ?5cm),tumor invasion depth(T1+T2,T3+T4)and with or without distant metastases(M0,M1).but none of them was statistically significant(P>0.05).2.Comparison of ARMS method and Sanger DNA sequencing method on detection of KRAS mutation in CC.1)Detect KRAS gene overall mutation rate using the two methods.KRAS mutation rate of 113 specimens were determined applying ARMS method and Sanger DNA sequencing method,and the positive rare were 47.8%(54/113)and 34.5%(39/113)respectively.The positive coincidence rate of the two methods was 72.2%(39/54).Obviously,KRAS mutation rate detected by ARMS method were higher than Sanger DNA sequencing method,the difference was statistically significant(P<0.05).2)Detect single base mutation types of KRAS gene utilizing the two methods.In our study,we detected 5 types of single base mutation types of KRAS gene using both methods,they were GLY12ASP(GGT>GAT),GLY12ALA(GGT>GCT),GLY12VAL(GGT>GTT),GLY12CYS(GGT>TGT),GLY13ASP(GGC>GAC).Among the above types,GLY12ASP(GGT>GAT),GLY12ALA(GGT>GCT),GLY12VAL(GGT>GTT),GLY13ASP(GGC>GAC)can be determined by ARMS method.GLY12ASP(GGT>GAT),GLY12ALA(GGT>GCT),GLY12VAL(GGT>GTT),GLY12CYS(GGT>TGT),GLY13ASP(GGC>GAC)can be detected by Sanger DNA sequencing method.Detection of Sanger sequencing to above 4 GLY12CYS and a total of 5 types,ARMS detected 4 single base mutation rate of Sanger DNA sequencing method of the 4 kinds of single nucleotide mutation rate had no correlation(P>0.05).Moreover,we successfully detected the mutation site of GLY12CYS(GGT>TGT)using Sanger DNA sequencing method with 4 positive cases,whereas ARMS method failed to detect this mutation site.Meanwhile,19 positive specimens diagnosed by ARMS method were negative when using Sanger DNA sequencing method.Among them,9 cases were GLY12ASP(GGT>GAT),4 cases were GLY12VAL(GGT>GTT),1 case was GLY12ALA(GGT>GCT)and 5 cases were GLY13ASP(GGC>GAC).Intriguingly,one specimen was diagnosed with two mutation of GLY12ASP(GGT>GAT)and GLY13ASP(GGC>GAC),which can be detected by both ARMS method and Sanger DNA sequencing method.The results of the two methods were consistent with Kappa consistency test and Kappa=0.679.Conclusion:1.The relevance between KRAS gene mutation and clinic-pathological features in LCC and RCC.The clinic-pathological features of LCC and RCC are quite different,and are closely correlated with KRAS gene mutation.Thus,there are great differences on the treatment strategies and therapeutic sensitivity between LCC and RCC.Based on this,we need to make a distinction between LCC and RCC during diagnosis and treatment.2.Both ARMS method and Sanger DNA sequencing method have advantages and disadvantages.ARMS method yields more positive outcomes and distinct results,while Sanger DNA sequencing method can detect the mutation site which is undetectable for ARMS kit.The consistency of the two detection methods is satisfactory.Based on FDA approval scheme,ARMS is the first choice for clinical detection of KRAS gene in CC surgical specimens,although this method fails to detect some mutation sites.Departments with sequencing facility can supplement the detection via direct sequencing.Taken together,combining these two complementary methods will improve and perfect the detection rate of KRAS gene mutation.