Clonal architecture and growth dynamics of ibrutinib-resistant CLL: Oligoclonal BTK/PLCG2 mutations and emerging non-BTK/PLCG2 drivers

Abstract In patients with chronic lymphocytic leukemia (CLL) undergoing treatment with ibrutinib, progressive disease (PD) frequently manifests with acquired mutations in BTK and/or PLCG2. The reported variant allele frequencies (VAF) are often low, prompting some to question the significance of these mutations. Additionally, multiple co-existing mutations are commonly observed in these patients. This study examined the clonal structure and dynamics of PD in the peripheral blood (PB) of patients with CLL. In a phase 2 study (NCT01500733), 84 CLL patients (52 who had not received prior treatment and 32 with relapsed/refractory disease) with either a TP53 aberration or aged ≥ 65 years were administered ibrutinib 420 mg daily until PD or intolerable side effects. The median progression-free survival was 86.6 months. Of 84 patients, 39 (46.4%) experienced disease progression, 31 (36.9%) had CLL progression (PD-CLL) and 8 (9.5%) developed transformation. The median time to PD-CLL was 57.2 months. PB samples were sent for genotyping of known hotspot mutations in BTK and PLCG2 (Neogenomics). Digital droplet PCR probes were designed to detect BTK C481S/R/Y and PLCG2 R665W/L845/S707F mutations in samples collected at baseline, during response to ibrutinib, and at PD in 28 PD-CLL patients. BTK/PLCG2 mutations were detectable in 25 (89.3%) patients, with VAF ranging from 0.04 to 0.96. Twelve (41.4%) patients exhibited multiple BTK/PLCG2 mutations, and in 11 evaluable cases, single-cell DNA sequencing (Mission Bio) revealed individual mutations in separate cells, indicating a branching evolution. After confirming oligoclonality, the cumulative cell fraction (cCF) of BTK/PLCG2 mutations was calculated in 28 patients with PD-CLL. Sixteen (57%) patients had a cCF greater than 0.5 (range 0.59-0.96), 9(32%) had a cCF less than 0.5 (range 0.04-0.43), and three (11%) had no detectable mutations. To evaluate the clonal architecture and dynamics of PD-CLL in peripheral blood, we performed whole-exome sequencing of longitudinal samples from 26 patients, followed by clonal decomposition analysis. The most frequently mutated ancestral driver genes identified were TP53, SF3B1, NOTCH1, and POT1. The growth kinetics of resistant clones were assessed by calculating the lymphocyte doubling time (DT) using the cellular fraction (CF) of the identified emerging clones and CD19+ counts in two or more sequential samples. In 11 evaluable cases, the median DT of BTK/PLCG2 clones was 79 days (n=10, IQR: 35-155); no significant difference in DT was observed between BTK and PLCG2 clones when analyzed individually (Student's t-test, p=0.5). Next, we estimated the DT of co-evolving non-BTK/PLCG2 clones in patients with low BTK/PLCG2 CF, and in patients without detectable BTK/PLCG2 mutations. The median DT for these clones was 98 days (n=6, IQR: 54-170). There was no significant difference in DT of BTK/PLCG2 and non-BTK/PLCG2 clones across all patients analyzed (Wilcoxon Rank-Sum Test, W=32, p=0.44). In patients without BTK/PLCG2 mutations and copy number variations (CNV) in IKBKB, SETD2, TRAF3, and CDKN1B emerged as potential alternative drivers of resistance. In conclusion, BTK/PLCG2 mutations were detected in 89.7% of patients with PD-CLL. These mutations occur in oligoclonal populations that have variable growth rates commonly showing fast doubling times of median of <3 months. In emerging clones without BTK/PLCG2 mutations, we identified putative driver mutations, while for some clones multiple CNVs contributed to genomic complexity. Growth rates of clones with and without BTK/PLCG2 mutations were comparable. The putative drivers of BTK inhibitor resistance warrant further investigation and validation in independent studies.