Abstract 1517: The integration of bulk DNA sequencing and single-cell analysis reveals diverse clonal evolution in CLL patients treated with BTKi
Authors: Huang Xiaomeng , Qiao Yi , Moos Philip , Tarapcsák Szabolcs , Woyach Jennifer A. , Byrd John C. , Stephens Deborah M. , Marth Gabor T. 
Published: September 2020

Abstract
Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia and is considered incurable. Hyperactivity of B-cell receptor signaling is a central driver of CLL's molecular pathogenesis. Targeting this pathway with Bruton's tyrosine kinase inhibitors (BTKi) has improved clinical outcomes for CLL patients when compared to standard chemo-immunotherapies (Woyach, NEJM 2018; Shanafelt, NEJM 2019). To characterize cancer evolution in CLL patients who received BTKi treatment, we collected blood samples from 4 CLL patients at 3 timepoints: pre-treatment, 1 year, and 2 years after initiation of treatment. Sorted B and T cells (normal control) were subjected to 200X WES and 60X WGS. Using somatic mutations and their allele frequencies from WGS/WES data across the 3 timepoints, we reconstructed the subclonal evolution of the malignant B cell population using our subclone deconvolution algorithm SubcloneSeeker (Qiao, Genome Biol. 2014). Our analysis revealed diverse patterns of subclonal genomic evolution across patients in terms of (1) presence or absence of subclonal evolution between time points; (2) emergence of new subclones across time points; and (3) clonal replacement across time points.To elucidate subclone-specific phenotypic behavior in each patient, we collected single-cell RNA sequencing (sc-RNA-seq) and IgG VDJ sequencing (sc-VDJ-seq) from unsorted blood samples at the same time points at which we had the genomic data. Using our new scBayes algorithm (manuscript in preparation), we assigned individual cells in the scRNA-seq data to genomic subclones based on the presence or absence of subclone-defining DNA mutations. This allowed us to isolate and examine the expression phenotype corresponding to subclonal populations. We observed three patterns: 1) no/minimal genomic and transcriptomic evolution across time points; 2) no/minimal genomic subclonal evolution but transcriptomic shift of the same subclones across time points; 3) clonal/subclonal replacement with distinct transcriptomic and VDJ profiles. We found that the two patients in our study with no/minimal genomic and transcriptomic evolution had progressive disease. In contrast, the two other patients whose tumor showed transcriptomic shift and/or clonal replacement had clinically complete remission. Although larger sample sizes are needed to draw further biological conclusions, this study highlights the power of multi-omic, and especially single-cell transcriptional profiling of BTKi treatment impact on CLL patients.
Citation Format: Xiaomeng Huang, Yi Qiao, Philip Moos, Szabolcs Tarapcsák, Jennifer A. Woyach, John C. Byrd, Deborah M. Stephens, Gabor T. Marth. The integration of bulk DNA sequencing and single-cell analysis reveals diverse clonal evolution in CLL patients treated with BTKi [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1517.