Clonal Dynamics of ASM-AHN with Avapritinib Treatment

INTRODUCTION. Aggressive systemic mastocytosis (ASM) is characterized by the expansion of clonal mast cells (MCs) that cause organ damage. Some ASM cases involve only the mast cell lineage, but most are associated with another myeloid hematologic neoplasm (ASM - AHN), most commonly chronic myelomonocytic leukemia (CMML). The D816V mutation of KIT (KIT^D816V) is present in almost all ASM cases and thought to be the key disease driver. ASM-AHN patients (pts) have few MCs in the peripheral blood, yet KIT^D816V can be detectable at high variant allele frequency, consistent with the presence of KIT^D816V in non-MC lineage cells. To understand how MC and Non-MC compartments of ASM-AHN respond to disruption of KIT^D816V signaling, we combined single cell RNA sequencing (scRNAseq) with whole genome sequencing (WGS) to map clonal dynamics in ASM-AHN pts treated with avapritinib, a selective KIT^D816V inhibitor.

PATIENTS AND METHODS. White blood cells (WBCs) from 4 pts with ASM-AHN (Table 1) collected prior to and after initiation of avapritinib therapy were subjected to deep (120X) WGS, with skin fibroblasts as controls. WBCs from the same and one additional timepoint were analyzed by scRNAseq (10x Genomics). Mononuclear cells from CMML pts without MC component (N=3) and age-matched healthy donors (N=3) served as controls. scRNAseq data were analyzed using Seurat and scBayes, a Bayesian-statistical approach developed by us for clonal attribution of single cells (Yi et al. Genome Research, in review). MC burden in bone marrow biopsies (BmBx) was analyzed with anti-tryptase antibody.

RESULTS. WGS detected an average of 1891 mutations/genome. Pt1 had a focal homozygous deletion in chromosome (Chr) 21 (including RUNX1), and pts2 and 3 had subclonal Chr 7q and 4q LOH, respectively, all of which did not change on therapy. Pt4 had a subclonal Chr 18 deletion (no change on treatment), a subclonal Chr 7q LOH (increased), and a subclonal Chr 21 deletion (became undetectable). Subclone analysis revealed stable clonal structure in pts 1 and 2. In contrast, in pts 3 and 4, the KIT^D816V containing subclone shrank/disappeared on avapritinib, while founder clones with mutations in TET2, SRSF2, ETNK1, or ASXL1 persisted (Figure 1). BmBx showed reduced MC burden in all 4 pts. scRNAseq revealed abnormal monocytes in pts 1-4 that clustered distinct from each other, CMML monocytes and healthy controls. On avapritinib, monocytes from pts 1 and 2 gradually shifted toward normal controls, while monocytes from pts 3 and 4 continued to cluster closer to normal than CMML monocytes. Pseudo bulking revealed that transcriptomes from ASM-AHN monocytes are distinct from CMML and normal monocytes. Other features distinguishing pt cells from controls included the presence of immature neutrophils and higher levels of CXCR4 in both CD4⁺ and CD8⁺ T cells. Pt 4 (ASM-CEL) showed a prominent population of CD34⁺ cells expressing markers of mature eosinophils (LAIR1, ITGA4, IL3RA) and mast cells (TPSAB1, CPA3), suggesting origin from a transformed granulocyte-macrophage progenitor (GMP) with restricted MC/eosinophil potential (Drissen et al. PMID: 31126997).

CONCLUSIONS. (i) Clonal dynamics of ASM-AHN on avapritinib are dependent on the localization of KIT^D816V in the hierarchy, with truncal upstream mutations predicting AHN responses. (ii) ASM-AHN monocytes are distinct from CMML monocytes. (iii) Eosinophils in ASM-CEL may be derived from a GMP whose lineage potential is limited to eosinophil/MC differentiation. Validation using targeted single cell and colony sequencing is underway and will be presented.

Ahmann:Recursion: Current Employment. George:Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cogent Biosciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARUP Laboratories: Other: Associated. Deininger:Novartis: Consultancy, Honoraria, Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Galena Biopharma: Consultancy, Honoraria; Pfizer Inc: Consultancy, Honoraria, Research Funding.