Abstract Introduction: Although therapies targeting Bruton Tyrosine Kinase (BTK), either as monotherapy or in combination have revolutionized the treatment in chronic lymphocytic leukemia (CLL), the disease remains incurable. Continuous treatment with these agents frequently leads to the development of resistance and disease progression, mostly associated with mutations in BTK or PLCG2. However, a significant fraction of BTKi-resistant patients does not harbor mutations in these genes, revealing that multiple distinct mechanisms of resistance can independently develop in these patient cohorts. Identifying novel molecular pathways associated with drug resistance in the relapsed/refractory (RR) setting of CLL requires more exploration. Materials and Methods: Using the Mayo Clinic CLL Database and tissue bank, we identified 30 RR patients, who had had disease progressionon a BTKi. The median lines of prior therapies were four. The median age at start of treatment was 57.9 (range, 36-78), 77% were male. Rai stage at diagnosis was 0 in 10% of patients, stage I in 37%, stage II in 20%, stage III in 6%, and stage IV in 27%. IGHV unmutated was observed in 87% of cases. Five patients subsequently evolved to Richter's Transformation (RT). A total of 64 blood samples were collected at different timepoints: baseline (before BTKi therapy), during treatment and at disease progression from BTKi (Ibrutinib/Acalabrutinib/Pirtobrutinib/Nemtabrutinib). The CD5+/CD19+ tumor population was enriched by flow sorting. We performed whole genome sequencing (WGS) in tumor/germline pairs with an average sequencing depth of 60X. Copy number variants (CNVpytor), structural variants – SV (Manta and DELLY2), single nucleotide variants (SNV) and indels (MuTect2, Strelka) were calculated. Mutational signature analysis was conducted independently on each disease timepoint with sigProfiler. De novo signatures were extracted with non-negative matrix factorization, then decomposed and mapped to known signatures from the COSMIC Human Cancer Signatures database. Results: Prior to start of BTKi treatment, the most common abnormality was del13q (65%), followed by abnormalities in TP53 (mutation and/or del17p) in 59% of patients, del8p, del11q (23% each), and mutations in SF3B1 (35%), NFKIE, NOTCH1 and MAPK signaling pathway (BRAF, KRAS, NRAS, PTPN11) in 23% of cases, each. Complex karyotype, defined as 3 copy number abnormalities was found in 59% of cases. Disease progression was observed 4.4 years (median; range 1.9-10.8 yrs) after starting BTKi therapy. At disease progression, there was an overall increase in genomic complexity. Overall, 17 cases (56%) showed mutations in BTK (p.C481S, p.C481Y, p.L528W, p.A428D) and/or PLCG2 (p.P844H). Four cases showed mutations in both genes or two or more independent mutations in BTK. Del17p and/or TP53 mutations were found in 20 (67%) cases, 9 of them with biallelic abnormalities. A third of patients (N=10) were found to have MAPK mutations, and 9 (30%) cases had mutations in the epigenetic regulators CREBBP, SETD2, SETD1A andKMT2D. Loss of heterozygosity, including 11q and 17p was found in 13% of cases. Chromothripsis was found in 23% of cases. In patients without BTK or PLCG2 mutations (N=13), TP53 abnormalities were present in 92% of cases and mutations in the MAPK pathway in 46% of these patients. Finally, we identified several SVs involving driver genes at disease progression, including inversions in RB1 (N=1), NOTCH1 (N=1) and translocations in MYC (N=1) and IGH (N=2). Analyzing the mutational signatures, baseline timepoints were dominated heavily by aging signatures (SBS5, SBS1). However, in cases that progressed after therapy, we found enrichment of mutational signatures associated with defective DNA mismatch repair (SBS44, 6%), chemotherapy treatment (SBS25, 14%), homologous and nucleotide excision repair deficiency (SBS8, 48%), and indirect effects of AID-induced somatic mutagenesis (SBS85, 7%). Additionally, the Melphalan signature (SBS99, 10%) was found in patients treated alkylating agents. Conclusions: Disease progression of CLL on BTKi confirmed the existence of multiple driver genes beyond BTK, PLCG2 andTP53, including the high prevalence of mutations in MAPK pathway and epigenetic regulators, and increased genomic complexity. Ongoing efforts are focused on integrating these findings with clinical demographics, epigenetic modifications, and preclinical drug sensitivity profiles.
Loaiza et al. (Mon,) studied this question.