The Research Of The Association Between BCR-ABL1 Fusion Gene Mutation And TKIs Resistance Based On Next-generation Sequencing

Background :Tyrosine kinase inhibitors(TKIs),including Imatinib(IM)could specifically inhibit the activity of BCR-ABL1 gene product which can inhibit the proliferation of tumor.However,about 20%~30% of the patients occured TKI resistance,the main reason was the point mutation in the kinase domain(KD)of BCR-ABL1.Sanger sequencing(SS)is the most widely used method for routine ABL1 KD mutation screening,but it has several disadvantages,including low sensitivity,failure to identify 15%~20% of low-level mutation abundance and inability of analyzing multiple clonal structures in mutation population.By increasing throughput sequence,the sensitivity of next-generation sequencing(NGS)is more than 1%?NGS could uncover complex clonal textures in many cases.Objectives:1.This study aims to verify NGS to identity whether it can detect the low-level mutation of BCR-ABL1 KD with certain accuracy in chronic myeloid leukemia(CML)patients,and to establish a novel method of high sensitivity for testing BCR-ABL1 fusion gene mutation.2.We also intend to further reveal the multiple clonal structures and analysis the association between this high-sensitive method and clinical work for the purposes of this study.Materials and methods:We collected 40 samples of peripheral blood for analysis from 22 of diagnosed CML patients who lost major molecular reaction(MMR: BCR-ABL1?1%IS)after TKIs sequential therapy.Firstly,2ml from a blood sample was drawn and total RNA extracted via Trizol method.And after reverse transcription,c DNA was obtained and BCR-ABL1 KD amplified through PCR.DNA was recycled and purified afterwards.Then,we adopted SS and NGS these two different sequencing methods for detection of BCR-ABL1 KD gene mutation,compared their differences and also analyzed the correlation between clonal evolution of mutated gene and TKIs resistance.In the last part of the experiment,we directed sequencing those special samples with TA clonal technique in order to verify the complexity of clonal population(complex mutation or multiple clonal mutation).All the data was proceeded and analyzed by the statistic software,SPSS 19.0.And we set P<0.05 as the standard for statistic differnces.Results1.12 kinds of resistance-related mutation was detected by NGS,including M244 V,Y253H,E255 K,E292V,V299 L,T315I,F359C/I,F317 L,H396R,E450A/K and C475 Y,and SS were detected mutation: E450 A / K,F359 C / I,T315 I,V299L,Y253 H,M244V.2.7 samples were negative by SS whereas NGS detected 10 point mutations,with the abundance within the range of 1.20% to 33.18%.And 8 out of these 10 mutation abundance were already lower than the lowest threshold of SS,which meant that the sensitivity of NGS was apparently higher than that of SS(P<0.05).3.At least 8 cases were accounted for complex mutation in 12 multiple mutation samples,in which 2 were never been reported,F359C/E450 A and T315I/E450 A.Conclusions:1.Classification errors and underestimation may occur when analyzing the samples via SS,in which 1%~15% mutation abundance could be detected by NGS.2.Early prediction for therapeutic responses of TKIs could be done by adopting NGS to monitor the mutation changes of ABL1 kinase domain and their clonal evolution.Thus,basic theory of early drug switching for individualized therapy is provided.