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4337 Clonal Dynamics of Leukemic and Clonal Hematopoiesis Mutations Predict Relapse in Single Cell MRD Analysis of AML in First Complete Remission

Program: Oral and Poster Abstracts
Session: 619. Acute Myeloid Leukemias: Disease Burden and Minimal Residual Disease in Prognosis and Treatment: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, AML, Diseases, Myeloid Malignancies, Technology and Procedures, Measurable Residual Disease , Omics technologies
Monday, December 9, 2024, 6:00 PM-8:00 PM

Enise Ceran, MD1*, Anne Kathrin Merbach, PhD1,2*, Christian Rohde, PhD1,2*, Robert Durruthy-Durruthy, PhD3*, Adam Sciambi, PhD3*, Michelle Lotze1*, Gema Fuerte, PhD3*, Matteo Cattaneo, PhD3*, Ivan Lukic, PhD3*, Maxi Wass, MD4*, Judith Schaffrath, MD4*, Christoph Röllig, MD, MSc5*, Richard F. Schlenk, MD1*, Marina Scheller, PhD1*, Simone Kowoll6*, Jörg Steighardt, PhD6*, Bayram Edemir, MD4*, Beatrice Ludwig-Kraus, MD7*, Lutz P. Mueller, MD4*, Sabine Edemir4*, Abi Vainstein-Haras, MD8*, Ella Sorani, PhD8*, Irit Gliko Kabir, PhD8*, Shaul Kadosh, PhD8*, Cora Gromann6*, Andreas Wienke, PhD9*, Claudia Wickenhauser, MD10*, Claudia D Baldus, MD11*, Uwe Platzbecker, MD12, Hubert Serve, MD13, Martin Bornhäuser, MD14* and Carsten Müller-Tidow, MD1,2*

1Department of Internal Medicine V, Heidelberg University Hospital, Heidelberg, Germany
2European Molecular Biology Lab (EMBL), Molecular Medicine Partnership Unit, Heidelberg, Germany
3Mission Bio, South San Francisco, CA
4Department of Internal Medicine IV, Hematology and Oncology, Martin Luther University Halle-Wittenberg, Halle, Germany
5Department of Internal Medicine I, University Hospital Carl Gustav Carus, Dresden, Germany
6Coordination Centre for Clinical Trials Halle, Martin-Luther University Halle-Wittenberg, Halle, Germany
7Department of Laboratory Medicine, Martin Luther University Halle-Wittenberg, Halle, Germany
8BiolineRx, Modi'in, Israel
9Institute of Medical Epidemiology Biometrics and Informatics, Martin Luther University Halle-Wittenberg, Halle, Germany
10Department of Pathology, Martin Luther University Halle-Wittenberg, Halle, Germany
11Department of Hematology and Oncology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany
12Department of Hematology, Cellular Therapy, Hemostaseology and Infectious Diseases, University Leipzig Medical Center, Leipzig, Germany
13Department of Medicine, Hematology/Oncology, Goethe-University Frankfurt, University Hospital, Frankfurt am Main, Germany
14National Center for Tumor Diseases (NCT/UCC) Dresden, Technical University Dresden, Dresden, Germany

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.

Disclosures: Ceran: IPSEN: Other: Funding for Congress attendance (DGHO). Sciambi: Mission Bio, Inc: Current Employment. Fuerte: MissionBio: Current Employment. Lukic: Mission Bio: Current Employment. Schlenk: Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Receipt of equipment, materials, drugs, medical writing, gifts or other services, Research Funding, Speakers Bureau; RECORDATI: Other: Receipt of equipment, materials, drugs, medical writing, gifts or other services, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Receipt of equipment, materials, drugs, medical writing, gifts or other services, Research Funding, Speakers Bureau; BerGenBio: Membership on an entity's Board of Directors or advisory committees, Other: Receipt of equipment, materials, drugs, medical writing, gifts or other services; Roche: Other: Receipt of equipment, materials, drugs, medical writing, gifts or other services, Research Funding; PharmaMar: Other: Receipt of equipment, materials, drugs, medical writing, gifts or other services, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: Receipt of equipment, materials, drugs, medical writing, gifts or other services, Research Funding; AstraZeneca: Other: Receipt of equipment, materials, drugs, medical writing, gifts or other services, Research Funding, Speakers Bureau; Boehringer Ingelheim: Research Funding. Vainstein-Haras: BiolineRX: Consultancy. Sorani: BioLineRx, Ltd.: Current Employment. Gliko Kabir: BiolineRX: Current Employment, Current equity holder in publicly-traded company. Kadosh: BiolineRX: Consultancy. Baldus: Janssen, Astellas, Pfizer, Astrazeneca, Servier, BMS: Consultancy, Honoraria. Platzbecker: Amgen: Consultancy, Research Funding; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; MDS Foundation: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Research Funding; Curis: Consultancy, Honoraria, Research Funding; Geron: Consultancy; Janssen: Consultancy, Honoraria, Research Funding; Merck: Research Funding; Novartis: Consultancy, Research Funding. Serve: IKP Stuttgart: Consultancy, Honoraria, Other: advisory role; Gilead Sciences: Consultancy, Honoraria; Novartis: Honoraria.

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