Next Generation Sequencing (NGS)-Based Post-Transplant Monitoring in Acute Myeloid Leukemia Patients Receiving Allogeneic Hematopoietic Stem Cell Transplantation (HCT)

Abstract

▪ObjectiveTo assess applicability of serial next generation sequencing (NGS) in monitoring acute myeloid leukemia (AML) patients after allogeneic hematopoietic stem cell transplantation (HCT).IntroductionClinical application of NGS in allogeneic HCT is a topic of interest. Post-HCT mutation dynamics using longitudinal NGS have not been thoroughly examined. We hypothesized that serial sequencing of pre- and post-HCT using NGS could provide a clinically relevant value in AML patients receiving HCT. The present study aimed to evaluate mutation dynamics in AML using serial samples from pre- and post-HCT with respect to transplant outcomes.Patients and Methods104 AML patients were enrolled and sequenced using an Illumina HiSeq 2000 sequencer (84 myeloid custom gene panel) on 529 bone marrow/peripheral blood samples at diagnosis (n=104), pre-HCT (n=104), 21 days post-HCT (n=104), at 6 (n= 38), 12 (n= 20), beyond 12 months (n=4) and at relapse (n=20). Two patients relapsed by day 21. T-cell (n=80) and donor samples (n=57) were also sequenced. All computational and statistical analyses were performed using Python and R.ResultsThe average on-target coverage in 529 samples was 1725.6x. In total, we detected 256 mutations at diagnosis in 90/104 patients (86.5%, median 3 mutations per patient). DNMT3A ( n=26, 25.0%), NPM1 ( n=23, 22.1%), CEBPA ( n=15, 14.4%), IDH2 ( n=15, 14.4%), FLT3 (n=14, 13.5%), PTPN11 (n=11, 10.6%) , and NRAS (n=11, 10.6%) were commonly mutated at diagnosis (Figure A). When grouped by 8 defined biological pathways, activated signaling (n=45, 43.2%), DNA methylation (n=44, 42.3%), NPM1 (n=23, 22.1%), and myeloid transcription factors (n=21, 20.2%) were mutated in more than 20% of the cohort (Figure B). Assessment of mutation dynamics in serial samples revealed that mutations from non-relapsed patients show rapid clearance after HCT without acquisition of somatic mutations and that allelic burden is maintained at very low level in longer follow-up period (less than 0.1%) (Figure C). On the other hand, relapsed patients tend to carry higher allelic burden even at day 21 after HCT (2.9% as opposed to 0.04% in non-relapsed patients), which suggests that allelic burden throughout the course of the disease and treatment can possess predictive power for detecting early relapse and predicting long-term overall survival (Figure D). We then assessed whether the mutation status at pre- and post-HCT has any impact on overall survival (OS) and relapse after HCT. With median follow-up of 6.3 years (range 0.7 - 12.3 years), patients with overall variant allele frequency (VAF) ≥ 0.2% at day 21 after HCT showed worse OS (Hazard Ratio (HR) 2.62 [1.28-5.35], p= 0.008) as well as increased risk of relapse (HR 6.16 [2.57-14.73], p < 0.001) (Figure E and F). Multivariate analyses verified that VAF ≥ 0.2% at day 21 was an adverse prognostic factor independent of age and cytogenetics risk group for OS (HR 3.06, [1.46-6.39], p = 0.003). Non-relapse mortality did not show significant difference regardless of VAFs ≥ 0.2% at day 21 (p = 0.21). Overall, 23 mutations from 16 patients were detected at day 21 after HCT (VAFs ≥ 0.2%) where 22 mutations were detected either at diagnosis or relapse, or both. In addition, mutations in DNMT3A (n=10) and NPM1 (n=3) were found in one than one patients at day 21 after HCT (13/23 mutations, 56%).Conclusion and summaryThis study revealed that the allelic burden immediately after HCT, within 21 days, with low VAF (0.2%) can be used to predict relapse after HCT as well as maintenance of nearly cleared allelic burden was observed in samples taken at longer follow-up period (mostly 6 and 12 months) from non-relapsed patients (less than 0.1%). Altogether, our data and analyses illustrated the value of longitudinal NGS-based monitoring strategies for AML patients after allogeneic HCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.