4004 - Tracking CLL cells with mutation in the TP53 gene using scRNAseq in relapsed/refractory patients

Abstract Text:

Background: Aberrations of the TP53 gene (mutation and/or deletion 17p) are the most important adverse prognostic markers in chronic lymphocytic leukemia (CLL). Low-burden TP53 mutations are often detectable in CLL cells prior to the therapy and expand upon the selective pressure of chemoimmunotherapy, thus contributing to disease aggressiveness. Other genomic alterations accompany aberrations in the TP53 gene. Bulk analysis, such as whole genome/exome sequencing or genomic array, cannot precisely determine the co-occurrence of abnormalities in individual cells. The engagement of scRNAseq technology could contribute to identifying expression profiles of malignant cells from patients with relapsed/refractory chronic lymphocytic leukemia (CLL) with high-burden TP53 mutations in relapse and tried to track them back in the cell populations sampled before therapy intervention with confirmed low-burden TP53 mutation using ultra-deep next-generation sequencing (NGS).

Aims: To explore the possibility of identifying and characterize populations of CLL cells resistant to treatment and causing refractoriness in samples from patients with relapse and trace back the presence of such cells in samples prior to the therapy using scRNAseq in patients with a focus on patients with TP53 mutations.

Methods: In our previous study (PMID: 33945616), the TP53 status was assessed by targeted NGS in 511 patients entering first-line treatment with chemo- and/or immunotherapy. 16% of patients carried low-burden TP53 mutations (0.1% to 10% VAF). For detailed scRNAseq combined with bulk DNA analysis, we selected five patients with TP53 aberration that was detected as low-burden before the first therapy (0.14 – 0.9 % VAF) and expanded in relapse. The transcriptomes of CLL cells separated from peripheral blood were analyzed using scRNAseq (Chromium system, 10x Genomics). Data analysis was carried out with the Seurat R package. Copy number aberrations (CNA) were estimated via the InferCNV tool. CNA results in relapse samples were verified using SNParray (Termo Fisher Sci) together with bulk NGS sequencing using the LYNX panel (PMID: 34082072), providing additional genomic data. Patients were tested prior to the chemoimmunotherapy and during the relapse.

Results: Using scRNA-seq, we identified the dominant clone at the refractory disease phase in patients with mutated TP53. Among others, 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 in advanced disease phase. Furthermore, the expansion of cells with TP53 mutation was connected with a complex karyotype, as revealed by InferCNV and confirmed by SNParray and NGS. A complex karyotype comprised of structural as well as numerous chromosomal aberrations, including deletion 17p-. The InferCNV tool enabled the identification of subclonal cell populations bearing the same chromosomal changes. The mutational composition of refractory cell populations analyzed by scRNA-seq was reconstructed in all patients by combining the information from bulk DNA analysis using SNParray and NGS. Besides TP53 mutations, NGS panel LYNX detected mutations in other recurrently mutated genes in CLL, like NOTCH1, ATM, BIRC3, RB1, XPO1, and CD79B. In addition, we detected either an increase in VAF of genes affected by prior therapy or newly occurred aberrations (e.g., RPS15, ZMYM3, BIRC3).
Summary/

Conclusion: Our study highlights scRNA-seq as an efficient method for tracking rare cells potent for causing CLL refractoriness, confirming their presence as a subclone. The therapy selection pressure favors the preferential proliferation of rare resistant cells, leading to recurrent relapses. Expression profiles of cells bearing TP53 mutation are altered and can be distinguished from expression profiles of unaffected cells using advanced techniques like single-cell RNA sequencing.
Grants: MH-CZ_AZV_NU20-08-00314, MEYS-CZ_MUNI/A/1224/2022, MH-CZ_RVO_65269705, NPO_NUVR_LX22NPO5102.

Keywords : TP53 defect, single-cell analysis, relapsed/refractory

Please indicate how this research was funded. : Grants: MH-CZ_AZV_NU20-08-00314, MEYS-CZ_MUNI/A/1224/2022, MH-CZ_RVO_65269705, NPO_NUVR_LX22NPO5102.

Please indicate the name of the funding organization.: The Ministry of Health of the Czech Republic; Ministry of Education, Youth and Sports of the Czech Republic; Masaryk University Brno.