Characteristics And Clinical Impact Of TET2 Mutations In Adult Cytogenetically Normal Primary Acute Myeloid Leukemia Based On Targeted Next-generation Sequencing

Objective:Based on targeted next-generation sequencing data,the mutation status,clinical characteristics and impact of TET2 gene mutation in adult primary cytogenetically normal acute myeloid leukemia（CN-AML）were further investigated deeply.Methods:1.Case collection:Retrospective analysis of 157 patients with primary cytogenetically normal AML who were diagnosed and treated in the Second Hospital Center of Shanxi Medical University from February 2017 to December 2020,and collected clinical data（including blood routine,biochemistry,next-generation sequencing）.2.Targeted next-generation sequencing of gene mutations:The samples were all newly diagnosed bone marrow,and all patients were detected by 34 kinds of gene-targeted next-generation sequencing methods.3.Treatment plan:In this study,125 patients were treated with standard induction chemotherapy regimen（3+7 regimen or containing decitabine）;32 patients were treated with low-intensity induction chemotherapy regimen（CAG regimen or containing decitabine）.Patients received high-dose or moderate-dose cytarabine-based chemotherapy after remission.Chemotherapy consolidation in elderly patients is selected on an individual basis.Twenty-one patients with intermediate or poor risk underwent allogeneic hematopoietic stem cell transplantation（allo-HSCT）after CR.4.Evaluation indicators and follow-up:In this analysis,the CR,OS,and RFS were used to evaluate patients;in this study,patients were followed up by case review and telephone and the follow-up of AML patients has been started since the initial diagnosis,and the deadline is January 1,2021.5.Statistical analysis:SPSS25.0 software was used for analysis,and some data statistics and graphs were drawn by Graph Pad Prism^TM8.01.Results:1.Analysis of TET2 gene mutation characteristics in adult patients with cytogenetically normal primary AML（1）A total of 34 patients（22%）with TET2 gene mutation were detected in 157 patients,and the mutation types included 42 types,of which 8 patients had TET2 double-site mutation,and 26 patients had single-site mutation;TET2 mutation types specifically included missense9 cases of mutation,20 cases of frameshift mutation,9 cases of nonsense mutation,2 cases of in-frame insertion and 2 cases of abnormal splicing;all detected mutations were located in the range between exon 3 and exon 11 of TET2 gene,among which the hot spot The regions were mainly concentrated in the exon 3（Cys domain）and exon 11（DSBH domain）regions.Co-mutation analysis showed that TET2 mutation in this group of patients had higher co-occurrence frequency with NPM1 gene mutation（P=0.031）and ASXL1 gene mutation（P=0.048）,and there was no significant difference in other co-mutations.（2）TET2 Variant allele frequency and clonal dominance analysis showed that the median VAF of TET2 mutations was 44.88%（7.58%-96.58%）,and TET2 mutations were mainly in the dominate clone.Among them,there were 28 patients with TET2 mutations as the dominate clone,and patients with subclonal TET2 mutations were 6 cases.2.Overall assessment of the clinical impact of TET2 mutation on adult patients with cytogenetically normal primary AML（1）Comparing the basic clinical characteristics of patients in the TET2mut group and the TET2wt group at the time of initial diagnosis showed that there were more elderly patients in the TET2mut group,with a statistically significant difference（P<0.001）.There were no statistically significant differences in other indicators between the two groups.（2）Comparing the treatment response and survival time of the total TET2mut group（n=34）and TET2wt group（n=123）,the results showed that the CR rate（68%）of the TET2mut group was significantly lower than that of the TET2wt group（89%）and had Significant statistical difference（P=0.003）.The median time of OS and RFS in the TET2mut group was significantly shorter than that in the TET2wt group（OS:15.0 vs 15.0,P=0.002;RFS:10.0 vs12.0,P=0.032）and there was a statistical difference.（3）In the group of age≤60 years old,the CR rate of TET2mut patients was 73%,and the OS（P=0.018）and RFS（P=0.035）of TET2mut patients were still significantly shorter than those of TET2wt patients with statistical difference3.Evaluation of the clinical impact of TET2 gene mutation on adult patients with cytogenetically normal primary AML in different genetic backgrounds（1）The TET2mut/NPM1mut patient group（n=13）compared with the TET2mut/NPM1wt patient group（n=21）showed that the TET2mut/NPM1wt patient group had fewer elderly patients（P=0.002）and higher platelet count（P=0.003）.There was no significant difference in basic clinical characteristics between the two groups;there was no significant difference in the CR rate（84%vs 57%,P=0.096）between the two groups;however,the median time of OS and RFS in the TET2mut/NPM1mut patient group was significant shorter than that of patients in TET2mut/NPM1wt group（12.0vs18.0 months,P=0.050;7.0vs12.0 months,P=0.048）.The comparison of TET2mut/ASXL1 mut patient group（n=7）and TET2mut/ASXL1wt patient group（n=27）showed that:TET2mut/ASXL1wt patient group had a higher proportion of elderly patients（≥60 years old）（P=0.008）,and other basic clinical characteristics There was no significant difference between the two groups;CR rate（57%vs 84%,P=0.096）,OS（15.0 vs 17.0 months,P=0.443）and RFS（8.0 vs 11.0 months,P=0.267）between the two groups)was not statistically different.The comparison between the TET2mut/CEBPAdou-mut patient group（n=7）and the TET2mut/CEBPAwt patient group（n=22）showed that there was no significant difference in the basic clinical characteristics between the two groups;the CR rate between the two groups（57%vs.73%,P=0.668,and the median OS time（14 vs 19 months,P=0.299）was not statistically different.（3）TET2mut VAF≥50%was the high-load group（n=11）,and TET2mut VAF<50%was the low-load group（n=23）.The comparison between the two groups showed that there was no significant difference in basic clinical characteristics between the two groups;the CR rate（70%vs 64%,P=1.000）and RFS（7.0 vs 12.0 months,P=0.327）between the two groups There was also no significant difference;however,the median OS time in the high-load group was significantly shorter than that in the low-load group（10.0 vs 19.0 months,P=0.009）.（4）TET2mutation patients were divided into TET2mut main clone group（n=26）and TET2mut subclone group（n=8）according to clonal grade.The comparison between the two groups showed that the WBC of the main clone group was significantly higher than that of the subclone group（25.22 vs 6.10×10⁹/L,P=0.034）,but the remaining basic clinical features,CR rate（65%vs 75%,P=0.611）,OS（2.0 vs19.0个月,P=0.757）and RFS（11.0vs12.0个月,P=0.867）were not significantly different.（5）TET2mut patients were divided into missense mutation group（n=8）,frameshift mutation group（n=12）and nonsense mutation group（n=6）,according to the type of TET2 mutation.There were no statistically significant differences in clinical basic characteristics,CR rate（50%vs 83%vs 67%,P=0.260）and among the three groups.However,the median time of OS and RFS in the nonsense mutation group was significantly shorter than that in the missense mutation group（OS:11.5 vs 18.5,P=0.027;RFS;5.5 vs 12,P=0.034）and the frameshift mutation group（OS:11.5 vs 18.5,P=0.027;RFS;5.5 vs 12,P=0.034）11.5 vs 21.5,P=0.017;RFS:5.5 vs 10,P=0.022）.（6）There were 26 patients in the TET2 single mutation group and 8 patients in the TET2double mutation group.Comparing the two groups,the results showed that the basic clinical characteristics,CR rate（69%vs 62%,P=1.000）,median OS time（19 vs 9,P=0.673）and median RFS time（10 vs 5,P=0.867）,there was no statistical difference between the two groups.4.Multivariate analysis of the prognostic effect of TET2 gene mutation on adult patients with cytogenetically normal primary AMLCox proportional hazards model was used for multivariate analysis,and the results showed that the HR（95%CI）of OS in the TET2mut VAF≥50%group was 5.203（1.240-21.827）,which was statistically significant compared with the TET2mut VAF<50%group（P=0.024）;the HR（95%CI）of RFS in the TET2mut VAF≥50%group was 5.011（1.335-18.804）,which was also statistically significant compared with the TET2mut VAF<50%group（P=0.017）.Conclusion:1.Adult primary CN-AML patients with TET2 mutations have unique mutational characteristics and are prone to co-occurrence of NPM1 and ASXL1 mutations.2.TET2 mutation significantly reduces the CR rate,OS and RFS for adult primary CN-AML,mainly in patients aged<60 years.3.In the ELN favorable group,TET2 mutation patients showed shorter OS;in the ELN intermediate group,TET2 mutation significantly reduced the CR rate of patients;in the ELN adverse group,TET2 mutation patients had a shorter RFS.4.TET2 mutation co-occurred with NPM1 mutation had worse OS and RFS.5.TET2 nonsense mutations had significantly worse OS and RFS relative to missense mutations and frameshift mutations.6.TET2 mutation clonal rank analyzed that single and double mutations had no significant effect on CR rate,OS and RFS.7.Multivariate analysis showed that TET2mut VAF≥50%was an independent risk factor for OS and RFS in patients with primary CN-AML.