Clonal Dynamics of Leukemic and Clonal Hematopoiesis Mutations Predict Relapse in Single Cell MRD Analysis of AML in First Complete Remission

Abstract

Measurable residual disease (MRD) assessment is important in acute myeloid leukemia (AML), as post-remission MRD indicates higher relapse risk. Current techniques, however, lack precision in individual outcome prediction, complicated by AML's clonal heterogeneity. Novel single cell MRD (scMRD) workflows based on DNA mutations offer a potential breakthrough, simultaneously capturing genotypic and immunophenotypic changes at a single-cell level. The Tapestri® platform (Mission Bio) incorporates a microfluidic droplet technology with cell barcoding beads and gene-specific primers that enables amplification and comprehensive identification of low-frequency subclones that may lead to overt disease or resistance. With this approach, complex heterogeneity and differentiation between leukemic clones and clonal hematopoiesis can be deconstructed. Objective: This study represents the first large-scale scMRD analysis in AML using this novel scMRD technology to deconvolute clonal heterogeneity to identify potential relapse markers and to differentiate between leukemic clones and clonal hematopoiesis. Methods: We analyzed a cohort of 69 AML patients in first complete remission (CR) as assessed by bone marrow morphology from the BLAST trial (NCT02502968). For 11 patients, we analyzed paired samples from both first CR and relapse. We performed single-cell DNA + protein sequencing on a total of 250,000 cells across 23 multiplexed pools. Our methodology included 40 selected mutation hotspot genes and a 19-plex antibody oligonucleotide cocktail for AML-specific surface markers as well as patient-specific hash tag antibodies for enhanced demultiplexing. We used magnetic activated cell sorting (MACS) to enrich CD34/CD117-positive blasts to ensure high-quality samples for subsequent analysis. Results: Our analyses revealed a diverse genetic landscape with 25 different mutated genes. DNMT3A and NPM1:p.W228Cfs mutations were the most common alterations. The genetic distribution included a predominance of preleukemic genes (51.5%), chromatin remodelers (12.1%), splice factors (9.1%), transcription factors (6.1%), cell cycle regulators (6.1%), and other functional categories (15.2%). Lower MRD (and clonal hematopoiesis) levels were consistently associated with improved overall survival (OS) and relapse-free survival (RFS) across different thresholds (<5%, <10%, <20%, <30%, <40% and <50% MRD burden per patient), with patients in the <5% MRD group showing a significant difference in RFS (p = 0.0013). The median overall survival in this group was 18.0 months, while the median relapse-free survival was 11,7 months. OS and RFS remained significantly different in all MRD groups even after exclusion of DNMT3A and TET2 mutations, which most likely represented clonal hematopoiesis. In paired samples, TP53 mutations were notably prominent during follow-up, remaining the dominant clone, indicating a mixed composition, and the persistence of treatment resistant clones. Overall, our study highlighted the dynamic nature of clonal evolution under therapeutic pressure by demonstrating continuous clonal diversity during clinical remission and notable changes in clonal composition during relapse. Conclusion: In this AML cohort, our study provides insights into the genetic landscape and clonal dynamics of AML in remission. Thus, our findings emphasize the importance and the potential of single-cell DNA + protein sequencing in refining MRD assessment compared to current techniques.