The Research Of Mutations In The Abl Kinase Domain In Patients With Chronic Myeloid Leukemia Resistant To Tyrosine Kinase Inhibitors

Objective:The development of tyrosine kinase inhibitor（TKI）has enabled patients with chronic myeloid leukemia（CML）to be almost the same as normal life expectancy,but there are still about 20%-30%of patients due to poor drug resistance,ABL kinase domain mutation is currently recognized as the main mechanism of TKI resistance.The objective of this study was to explore the characteristics of mutations in the ABL kinase domain in CML patients,as well as the influencing factors and prognosis.Method:A retrospective analysis was made of 198 patients with chronic myeloid leukemia who received first-line imatinib treatment and developed drug resistance from January 2012 to October 2021,and the basic clinical data such as sex,age at the time of initial diagnosis,blood routine,spleen size,disease stage,bone marrow cell morphology,chromosomes,and BCR-ABL fusion gene qualitative or quantitative were collected,as well as regular monitoring of bone marrow morphology and/or BCR-ABL fusion gene quantitative PCR results every 3-6 months.ABL kinase region mutation detection:In the molecular biology laboratory of our center,the TRIzol Reagent instrument was used to isolate and extract the total RNA in single nuclear cells in the bone marrow or peripheral blood,reverse transcriptase to c DNA,and expand the BCR-ABL kinase region by two rounds.The purified PCR products were sequenced using the ABI3730 sequencer,and the obtained c DNA sequences were compared with the American gene pool（NM-005157.6）to determine the mutation type.Statistical processing:SPSS 26.0 software was used for statistical analysis,and17 factors such as sex,age at the time of initial diagnosis,blood routine,spleen size,and disease staging were analyzed univariately,and then the variables of P<0.1 in the univariate analysis were included in the logistic regression model for multi-factor analysis,and the main factors affecting mutation were obtained,and the Kaplan-Meier method was used for survival analysis.P<0.05 for the difference was statistically significant.Results:1.A total of 198 CML patients with TKI resistance were included,and a total of215 cases of resistance occurred,including 67 cases in the mutant group and 131cases in the non-mutant group,and the mutation detection rate was 38.14%（82/215）.Among the 67 patients in the mutant group,54 patients were detected with first-line imatinib,9 were replaced by second-line nilotinib after first-line IM resistance,and 4cases were replaced by second-line dasatinib.2.The top five mutation types with higher mutation detection rates in TKI-resistant patients were:T315I（8.37%）,E255K/V（6.05%）,F317L（4.65%）,G250E（4.65%）,M244V（3.72%）.3.Mutation detection was higher in patients with advanced disease,high-risk group of ELTS,secondary resistance,6-month BCR-ABL^IS≤1%,and a history of TKI reduction.4.Factors that are meaningful for the occurrence of drug resistance mutations（P<0.1）in univariate analysis include:platelet count,disease staging at the time of initial diagnosis,ELTS score,whether the interval between diagnosis and imatinib is greater than 6 months,3months of BCR-ABL^IS≤10%,6 months of BCR-ABL^IS≤1%,type of resistance,total TKI duration at treatment failure,and history of discontinuation of drugs.5.Multifactorial analysis suggests that patients with advanced disease,high-risk group of ELTS,patients with a history of drug reduction and discontinuation,who have not reached 6 months of BCR-ABL^IS≤1%,and secondary resistance have a higher mutation detection rate.6.Patients with TKI-resistant CML mutations had lower PFS rates and OS rates（89.4%vs 99.2%;56.2%vs 76.9%）than patients without mutations.Multiple mutations had a lower 5-year OS rate（72%vs 95%）than single mutants.Among them,patients with T315I mutations are still ineffective even if they receive allogeneic hematopoietic stem cell transplantation.Conclusion:1.ABL kinase domain mutation is one of the main mechanisms of TKI resistance,and 82 cases（38.14%）of TKI resistant patients can detect ABL kinase region mutations.According to the different domains of the ABL kinase region,the incidence of P-loop mutation is the highest,followed by the IM binding site region.2.The top five mutation types with high mutation detection rate in TKI-resistant patients were T315I（8.37%）,E255K/V（6.05%）,F317L（4.65%）,G250E（4.65%）,M244V（3.72%）,except for T315I and F317L,the remaining mutations were located in the p-loop region.3.Patients with advanced disease,high-risk group of ELTS,secondary drug resistance,6 months bcr-ABLIS≤1%,and a history of TKI reduction and discontinuation of drugs have a higher mutation detection rate.4.Patients with TKI-resistant CML mutation group had shorter OS and PFS than patients without mutation（89.4%vs 99.2%;56.2%vs 76.9%）.T315 mutation group compared with the non-T315I mutation group of 5 years OS and PFS significantly decreased,receiving allogeneic hematopoietic stem cell transplantation can change the prognosis to a certain extent,the current China independent research and development of three generations of TKI Olverembatinib,as well as the US FDA through the approval of the marketing of THE AMP inhibitor asciminib,may be able to break through the T315I mutation.