P602: single-cell rna-sequencing enables tracking and characterization of rare cll cells with the potential to cause refractoriness.

Background: Chronic lymphocytic leukemia (CLL) is considered an incurable disease due to the limited ability of current treatment options to completely eradicate leukemic cells. The therapy selection pressure favors the preferential proliferation of rare resistant cells, leading to recurrent relapses. Early detection of these rare cells holds the potential to assist in selecting appropriate therapy. Aims: To explore the possibility to identify and characterize rare populations of CLL cells resistant to treatment and causing relapses, and trace the presence of such cells during the disease course by single-cell RNA sequencing (scRNA-seq). Methods: Peripheral blood CLL cells of a single patient from the time of diagnosis (DG), progression (PR), the first relapse (RL), and the refractory phase of terminal massive lymphadenopathy after several therapy lines (RF) were analyzed using 3‘single-cell RNA-seq. The estimation of copy number aberrations (CNA) was performed via the InferCNV tool. CNA results were verified using SNParray together with bulk NGS sequencing using the LYNX panel (PMID: 34082072), providing additional genomic data. In addition, ultra-sensitive TP53 amplicon sequencing was performed. Results: scRNA-seq revealed a striking change in clonal dominance at the refractory disease phase (RF). Refractory cells showed upregulation of negative apoptosis regulators (CFLAR, BIRC3, BCL2, YBX3) and downregulation of MS4A1 (encoding therapy target CD20), immunoglobulin genes, CD19, CD79A, and transcription factors PAX5 and TCF3, indicating a loss of original B-cell phenotype. Furthermore, the RF sample consisted of two cell populations differing by the presence of a complex karyotype as revealed by InferCNV (Fig. 1A, 1B). A complex karyotype comprised a number of chromosomal aberrations, namely deletion in 1p, monosomy of chromosome 9, and deletions of 12q, 13q and 17p. Unsupervised hierarchical clustering assigned 0.6% of PR cells and 1.3 % RL cells into the cluster with complex karyotype of RF cells. SNP arrays and the LYNX NGS panel confirmed the presence of multiple copy number aberrations in RF and revealed the presence of 11q and 13q deletions in all timepoints analyzed. However, the 11q deletion initially present in 96% of DG cells was present in only 20% of cells in RF, supporting the hypothesis of rare resistant cells without 11q deletion at PR and RL selected by subsequent therapies. LYNX also detected mutations in the NOTCH1 and ZMYM3 genes with 50% variant allele frequency (VAF) at DG and an increase to 80% VAF at RF. In addition, mutations in the XPO1, TP53, BIRC3, and RB1 genes arose over time. TP53 amplicon sequencing confirmed the presence of the c.582delT variant at PR and RL with an allele frequency of 0.2 % and 3.0 %, respectively. The frequency of the same variant at the terminal progression (RF) was 64.0 %. Finally, the mutational composition of RF cell populations analyzed by scRNA-seq was reconstructed by combining the information with bulk DNA analysis (SNParray, LYNX panel). Both cell populations contained 13q deletion and mutations in ZMYM3 and NOTCH1. Population with the complex karyotype bore a mutation in the TP53 gene, while the other harbored an 11q deletion and mutations in either XPO1 or BIRC3 (Fig. 1C). Summary/Conclusion: This study highlights scRNA-seq as an efficient method for tracking rare cells potent for causing CLL refractoriness, thus enabling their subsequent detailed characterization. Presented study was funded by the following grants: MH-CZ_AZV_NU20-08-00314, MEYS-CZ_MUNI/A/1224/2022, MH-CZ_RVO_65269705009, NPO_NUVR_LX22NPO5102, TACR_TN02000109.Keywords: TP53, RNA-seq, B-CLL