Clinical Effect Observation And Analysis Of ABL Kinase Domain Mutations Of Ph-positive Leukemia In The Era Of Tyrosine Kinase Inhibitors

Objective:The most common types of Ph+ leukemia are chronic myeloid leukaemia(CML)and Ph chromosome-Positive acute lym Phocytic leukemia(Ph+ ALL).The adventment of tyrosine protein kinase inhibitors(TKI)has completely changed the treatment of CML patients.Now the life expectancy is close to that of the general population,but some patients obtain TKI resistance at some point during the course of treatment.For Ph+ ALL patients,TKI has improved from a cure rate of less than 5%with standard chemotherapy alone to a complete response rate(CR)of more than 90%and a 5-year overall survival rate(OS)of 40-60%.However,TKIs resistance and disease progression,especially the high cumulative recurrence rate of Ph+ALL patients,are still important factors influencing the poor prognosis of these two types of leukemia patients.This study aims to explore and analyze the basic clinical characteristics of these two types of patients,the efficacy of TKI medication,the most important cause of drug resistance,ABL kinase region mutations,and the discovery of new ABL gene abnormal spliceosomes.Methods:1.The basic information,follow-up information and bone marrow blood samples of patients with CML and Ph+ALL were collected,and RNA was extracted for preservation.The clinical characteristics and medication information were recorded,and the medication efficacy,molecular reaction and survival of such patients were analyzed.2.Abl kinase domain mutations in CML and Ph +ALL patients were detected by Sanger generation sequencing.PCR products were screened by PAGE gel electrophoresis.3.The expression level of BCR-ABL was detected by real-time fluorescent quantitative PCR(q-PCR)and droplet digital PCR(dd-PCR).4.T-A clone sequencing to analyze the composition of double mutations in the kinase region of ABL.Results:1.82.7% of CML patients used first-generation TKI as the initial treatment agent,19.6% of CML patients tolerated TKI,and the rest of the patients had different adverse reactions,with face swelling and eye swelling(24.8%)as the main adverse reaction.Among the 14 patients who discontinued the drug after reaching MR4.5,2patients(14.3%)were still in treatment-free remission(TFR)at 36 months of discontinuation.2.At 3 months of treatment,the early molecular responses(EMR)rate of the second generation TKI was higher than that of the first generation TKI(P=0.04).There was no statistical difference in major molecular responses(MMR)rate(P=0.15).At 12 months of treatment,the second-generation TKI had a higher rate of deep molecular responses(DMR)than the first-generation TKI(P=0.03).There was no statistical difference in MMR rate(P=0.097).Compared with the MMR group,the non-MMR group was more likely to take domestic TKI(P=0.015),higher white blood cell count(P=0.031),more abnormal chromosomes(P=0.01),fewer people reached MR4.5 by the end of follow-up(P=0.004).3.The incidence of ABL kinase mutations in resistant CML patients was 44.2%.The main regions of mutation concentration were the P-loop region and the direct binding region with TKI.D391 G,p.Pro230Alafs*14,p.Val304Argfs*13,p.Asp363＿386Argdel24Aa,p.Gly372＿447Glndel76Aa were identified for the first time.4.The incidence of ABL kinase mutations in resistant Ph +ALL patients was63.9%.The main regions of mutation concentration were the P-loop region and the direct binding region with TKI.V338 M,G426E,p.Val304Argfs*13,p.Asp363＿386Argdel24aa,p.Val371Hisfs*29 were identified for the first time.5.There is up to 54% of the co-expression rate of P190 and P210 in patients with relapsed Ph+ALL.The ABL kinase domain mutation load shows a difference between two transcripts in relapsed Ph+ ALL patients co-expressing p190 and p210 transcripts,and were positively correlated with their respective BCR-ABL levels.6.The incidence of p.Asp363＿386 Argdel24 aa spliceosome in healthy control group,incipient CML group,relapsed CML group,incipient Ph+ ALL and relapsed Ph+ ALL were 0%(0/60),3.3%(1/30),3.3%(1/30),0%(0/20),0%(0/20),respectively.Conclusion:1.In CML patients,the use of second-generation TKIs is more likely to achieve EMR and DMR than first-generation TKIs;patients who achieve the treatment goals early are more likely to achieve MR4.5.2.ABL kinase mutation has a higher incidence in CML resistant and Ph+ALL resistant patients with relapse,and the type of mutation is mainly point mutation.Abnormal spliceosome may participate in this process.New spliceosome p.Asp363＿386 Argdel24 aa is reproducible in patients with drug-resistant Ph+leukemia,which may be related to TKI resistance.3.In Ph+ALL co-expressed with P210 and P190,the mutation load of the ABL kinase region presents different proportions in two different transcripts,and is positively correlated with the level of their respective BCR-ABL fusion genes.