Longitudinal Mutation Profiling in CLL Patients during Acalabrutinib Therapy and at Progression

Introduction Progressive disease (PD) is driven by clonal evolution in patients with chronic lymphocytic leukemia (CLL) treated with the Bruton tyrosine kinase inhibitor (BTKi) ibrutinib. Specifically, emergence of BTK mutant subclones is frequently observed at relapse. Mutations in BTK have also been reported in a small number of patients who developed PD during treatment with acalabrutinib, a second generation BTKi (Black, et al., Blood. 2022). To gain further insight into genetic mechanisms of acalabrutinib resistance, we performed targeted next generation sequencing (NGS) on longitudinal samples from a single-center phase 2 study for patients with high-risk CLL (NCT02337829). Methods The clinical trial results were previously reported (Sun, et al., Blood. 2020). Eligible patients had treatment naive (TN) CLL or small lymphocytic lymphoma (SLL) with deletion (del) 17p, TP53 mutation, or NOTCH1 mutation, or had relapsed/refractory (R/R) disease. Forty-eight participants were randomized 1:1 to receive acalabrutinib 100 mg twice daily (BID) or 200 mg once daily (QD). Treatment response and PD were assessed according to the International Workshop on CLL guidelines. The Kaplan-Meier method was used to estimate progression-free survival (PFS). Peripheral blood mononuclear cells were obtained at baseline then longitudinally on treatment until PD. A targeted NGS panel comprised of 600 recurrently mutated genes in hematologic malignancies was performed using DNA isolated from CD19+ enriched cells (Collins, et al., Leuk Lymphoma. 2021). Putative CLL driver genes (Knisbacher, et al., Nat Genet. 2022) with variant allele frequencies of ≥5% were included in downstream analysis. In PD patients, Sanger and high-sensitivity Sanger sequencing for BTK and PLCG2 hotspot mutations were also done. Results After a median follow-up of 5.4 years, 17/48 (35%) patients developed PD (14 CLL, 3 Richter transformation). The estimated PFS rate at 5 years was 62% (95% CI 44-75) and median PFS was reached at 6.0 years. All 14 patients with CLL PD had achieved partial remission or better before developing secondary resistance to acalabrutinib. PD occurred in 13/32 (41%) R/R patients compared to 4/16 (25%) TN patients (P=.4) and 8/24 (33%) patients on BID dosing compared to 9/24 (38%) patients on QD dosing (P>.9). IGHV was unmutated in all but one PD patient. This patient had bi-clonal disease with discordant IGHV mutation status. Targeted NGS data are available for 212 samples: 48 samples at baseline, 45 samples at 6 months, 40 samples at 12 months, 34 samples at 24 months, 24 samples at 36 months, 11 samples at 48 months, 2 samples at 60 months, and 8 samples at PD (various timepoints). At baseline, 25 CLL driver genes were mutated in 47/48 (98%) patients with a median of 3 (IQR 2-4) mutated genes per patient. The most commonly mutated (≥10% of patients) CLL driver genes at baseline were NOTCH1, ZNF292, TP53, SF3B1, ATM, and NFKBIE. Baseline mutations in BCOR and SPEN were observed exclusively in PD patients (BCOR: 4/17, P=.012 and SPEN: 3/17, P=.039 vs. non-PD patients). Of note, del 17p, TP53 mutation, and NOTCH1 mutation were overrepresented in TN patients likely as a reflection of clinical trial eligibility. Serial monitoring identified treatment-emergent mutations in 10 CLL driver genes across 21/48 (44%) patients. PD was not associated with the acquisition of CLL driver mutations (P=.8). BTK C481 mutations were detected in 5/13 (39%) patients and PLCG2 mutations in 4/13 (31%) patients at the time of CLL PD. BTK and PLCG2 mutations could not be identified in 4/13 (31%) patients; one patient did not have BTK and PLCG2 sequencing at relapse. CLL PD patients without BTK or PLCG2 mutations acquired mutations in NOTCH1, ZNF292, and BIRC3 during treatment. Conclusions Baseline mutations in BCOR and SPEN were associated with PD during treatment with acalabrutinib. Acquired mutations in NOTCH1, ZNF292, and BIRC3 were detected in PD patients without BTK or PLCG2 mutations. Longitudinal mutation profiling could deepen understanding of acalabrutinib resistance mechanisms in CLL.