Outcomes of Allogeneic Stem Cell Transplantation for AML and MDS Based on Pre-Transplant MRD Status By Next-Generation Sequencing
Authors: Manning Benjamin M , Sussman Robyn T , Deihimi Safoora , Frey Noelle V. , Hexner Elizabeth O. , Loren Alison W. , Luger Selina , Mangan James K. , Martin Mary Ellen , McCurdy Shannon R. , Morrissette Jennifer J.D. , Perl Alexander E. , Stadtmauer Edward A. , Porter David L. , Gill Saar I. 
Published: October 2019

Abstract
Background
After induction therapy for acute myelogenous leukemia (AML), the presence of minimal residual disease (MRD) by targeted next-generation sequencing (NGS) during complete remission (CR) predicts relapse and survival, particularly after exclusion of pre-leukemic mutations. MRD assessment is not routinely performed for AML prior to transplant, partly because consensus regarding assay methodology, appropriate timing, interpretation of results, and therapeutic value prior to SCT is lacking. We therefore sought to describe the rates of mutational clearance and correlate these with relapse rates post-transplant.
Methods
We conducted a retrospective review of sequential AML or myelodysplastic syndrome (MDS) patients undergoing allogeneic hematopoietic cell transplant (alloHCT) at our institution between 2014 and 2017. There were 119 patients with AML/MDS who were treated with either myeloablative or reduced intensity conditioning regimens. Of the 119 patients transplanted, 60 had both pre- and post-treatment NGS results and were included in the analysis. 56 patients had somatic mutations on initial NGS and were therefore eligible for mutational clearance analysis. Twelve patients were in active disease and excluded from further analyses. The remaining patients (n=44) represent the core dataset.
Blood and/or marrow specimens were analyzed via a clinical NGS panel targeting 68 leukemia-associated genes. Median coverage (across 88 samples) was 2817 reads. Mutations were considered persistent if present at variant allele frequencies (VAF) ≥ 1% for single nucleotide variants (SNV) or ≥ 2 copies for insertions and deletions (indels). Validated laboratory reporting practice at our institution reports VAF > 4% for SNVs and ≥ 1% for indels with a minimum of 250 total reads. We therefore defined three levels of mutational clearance on the basis of the VAF of residual mutations: VAF for SNV <1% (and/or indels ≤1 copy), between 1-4% (and/or indels <1% and ≥ 2 copies), and >4% (and/or indels > 1%). Patients with ≥ 1 mutation meeting these thresholds were designated NGS(-), NGS-low and NGS(+), respectively. The median follow-up was 332 days.
Results
On review of NGS data, 120 mutations were present in initial sequencing, with 64 mutations persistent in pre-transplant samples from 26 patients. The most commonly mutated genes from initial samples were FLT3 (18), ASXL1 (11), TET2 (10), NPM1 (9), RUNX1 (8), SRSF2 (8), and DNMT3A (7) (Figure 1A). Mutational clearance varied widely, with the putative pre-leukemic genes DNMT3A, TET2, and ASXL1 (DTA) demonstrating low rates of mutational clearance (Figure 1A). Mutations persisting below the validated reporting threshold were present in 20 patients, including 10 patients otherwise negative by NGS.
There were 16 patients categorized as NGS(+), 10 NGS-low, and 18 NGS(-), with relapse rates of 31%, 22%, and 30%, respectively. No difference in relapse risk was observed between NGS(-) and NGS-low subgroups (p = 0.72), and no RFS benefit was observed for patients without persistent mutations > 4% relative to the NGS(+) subgroup (p = 0.56, Figure 1B). Recent work has shown a survival benefit in AML patients in CR without persistent mutations that is enhanced when DTA genes were excluded from the analysis (Jongen-Lavrencic, NEJM 2018). In our cohort, after exclusion of DTA mutations, 6 patients were reclassified by mutational clearance status, and 2 were excluded from the analysis as they had only DTA mutations in pre-treatment samples. Similar to the more comprehensive cohort, no RFS benefit based on NGS status was observed in the post-transplant period (p = 0.42, Figure 1C).
Conclusions
There were similar outcomes regardless of molecular MRD findings by NGS for patients with advanced myeloid malignancies who were in morphologic CR prior to alloHCT. These results contrast with those in the published literature that address a more uniform patient population of clinical trial participants, not all of whom went on to transplant. Further detailed analyses from larger more homogeneous populations will be useful to determine the prognostic significance of MRD by NGS prior to allogeneic HCT.
Figure 1 Figure 1.

Disclosures
Frey: Servier Consultancy: Consultancy; Novartis: Consultancy. Perl:Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Daiichi Sankyo: Consultancy. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; AbbVie, Inc: Research Funding; Amgen: Consultancy; Janssen: Consultancy. Porter:Genentech: Other: Spouse employment; Kite Pharma: Other: Advisory board; Novartis: Other: Advisory board, Patents & Royalties, Research Funding. Gill:Extellia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Carisma Therapeutics: Equity Ownership; Novartis: Research Funding.