P349: CLONAL ARCHITECTURE DISSECTING AT SINGLE-CELL RESOLUTION REVEALS MECHANISMS OF CHEMOTHERAPY RESISTANCE AND RELAPSE IN T CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Abstract

Background: T-lineage acute lymphoblastic leukemia (T-ALL) comprises approximately 10-15% of pediatric ALL cases with distinct feature in biology and largely inferior outcome compared to B-ALL. Growing evidence has reflected pivotal roles of clonal evolution in T-ALL recurrence, but bulk sequencing may not serve as the perfect model to reliably infer clonal heterogeneities and their immunomodulatory milieu during leukemia development. In this study, single-cell sequencing was applied to uncover leukemic clonal relationships with relapse throughout chemotherapy in T-ALL at a more accurate resolution. Aims: We performed bulk and single cell multi omics analysis for diagnostic and relapse T-ALL, with the aim to dissect mechanisms of chemotherapy resistance and relapse in T cell acute lymphoblastic leukemia. Methods: We performed bulk whole-exome sequencing for sorted CD7+ BMMCs from 5 pairs of diagnosis-relapse (Dx_Rel) samples, revealing a series of well-reported hotspot mutations in T-ALL. To dissect clonal diversities within and across the 5 Dx_Rel T-ALL pairs, we carried out high-throughput droplet-based 5’-single-cell RNA-seq (scRNA-seq) and paired T cell receptor sequencing (scTCR-seq). Results: By performing unsupervised clustering of scRNA- seq profiles encompassing 10 samples, we identified 23 distinct T-lineage clusters (Cluster 0-22) based on the two-dimensional UMAP visualization. In 2 out of 5 patients, diffusion map of T-lineage sub-clusters between diagnostic and relapsed samples appeared to be almost identical, while distinct shifts from diagnosis to relapse in the compositions have been observed in the other 3 out of 5 patients. We sought to further deconvolute the clonal architecture trajectory for T-ALL Dx_Rel pairs. We observed that dominant diagnostic clones of 4 patients diminished or vanished at relapse, sparing newly emerged subclones predominantly substituted at relapse. We clearly depicted two distinct patterns of evolutionary trajectories in these 4 Dx_Rel pairs by comprehensively mapping hierarchical TCR clonotypes onto leukemic clonotypes at single cell levels. Specifically, in T956 and T723, we observed significant outgrowth of incidental diagnostic sub-clones at relapse, whereby surrogate TCR repertoires correspondingly enumerated, suggestive of dynamic shifts in dominant clone over continuous chemo-exposure. Whereas in T593 and T856, expanding clones at relapse were showed up with completely different gene signatures from the diagnostic ones, but dominant clones at diagnosis and relapse were surprisingly presented with identical TCR repertoires. This was undoubtedly informative of leukemic “clonal drift” within which hypothetical intrinsic transformation happened to the same subclones over persistent chemotherapy. Image:Summary/Conclusion: Collectively, our presented study accurately distinguished leukemic cells from normal T cells in T-ALL at a single-cell resolution. By tracking transcriptomic profiles within and across Dx_Rel T-ALL pairs, we further identified distinct clonal evolutionary patterns, which may determine diversified fates of leukemic clones in response to therapeutic pressures, extending significant implications for future precise chemotherapies.