Abstract 5828: The genomic architecture of ibrutinib resistance in CLL: Oligoclonal progression with variable anatomical distribution
Authors: Underbayev Chingiz , Chen Jonathan , Harris Hailey M. , Vaughn Lauren T. , Itsara Andy , Wiestner Adrian 
Published: March 2024

Abstract
Progressive disease (PD) in patients with chronic lymphocytic leukemia (CLL) on ibrutinib often presents with acquired mutations in BTK and/or PLCG2. Reported variant allele frequencies (VAF) are often low, leading some to question the role of these mutations. Further, several co-existing mutations are often identified. Here we investigated the clonal composition of PD on single cell level, across time, and different anatomic compartments. 84 CLL patients (52 treatment-naïve; 32 relapsed/refractory) with either a TP53 aberration or age ≥65 years received single-agent ibrutinib 420 mg once daily until PD or limiting side effects on a phase 2 study (NCT01500733). The median progression-free survival was 86.6 months. 39 (46.4%) patients progressed, 31 with CLL (PD-CLL), and 8 with Richter’s transformation. Median time to PD-CLL was 57.2 months. In PD-CLL patients, peripheral blood (PB) was sent for genotyping of known hotspot mutations in BTK/PLCG2 (Neogenomics). We then designed digital droplet PCR probes to test for BTK C481S/R/Y and PLCG2 R665W/L845/S707F mutations in samples collected at baseline, during response to ibrutinib and at PD in 29 patients. 26 (89.7%) patients had detectable BTK/PLCG2 mutations (VAF range 0.04 - 0.96). 12 (41%) patients carried multiple BTK/PLCG2 mutations. In 11 evaluable cases single-cell DNA sequencing (Mission Bio) showed individual mutations present in separate cells consistent with branching evolution. Having confirmed oligoclonality, we calculated the cumulative cell fraction (cCF) of BTK/PLCG2 mutations in the 29 PD-CLL patients. 16 (55%) patients had cCF &gt;0.5 (range 0.59-0.96), 10 patients (35%) had cCF &lt; 0.5 (range 0.04-0.43) and 3 patients (10%) had no detectable mutations. BTK/PLCG2 mutations were detected in samples collected at median 21.6 months (range 0 - 75) prior to PD. In 11 cases, we tested lymph node (LN) biopsies at PD. In 6 cases with cCF &lt;0.5 in PB, 4 corresponding LN samples had higher cCF (0.01/0.92; 0/0.85; 0.06/0.69; 0.46/0.99 for PB/LN, respectively). In 5 cases, PB contained 2-6 distinct subclones with cCF &gt;0.5, in 3 cases the corresponding LN harbored fewer subclones than PB (6/3; 3/2; 3/0 for PB/LN, respectively). To characterize the genomic background of PD-CLL, we performed whole-exome sequencing of longitudinal samples from 26 patients. In addition to BTK/PLCG2, the most frequently mutated genes were TP53, SF3B1, NOTCH1, and POT1. Furthermore, copy number variations (CNA), such as del17p, del13q, del14q, del3p, and del8p, were common. In summary, in our cohort BTK/PLCG2 mutations were identified in 89.7% of PD-CLL patients. These mutations arise in oligoclonal populations with variable distribution across anatomical sites. In rare patients without BTK/PLCG2 mutations, the co-evolution of putative driver mutations and multiple CNAs generated genomic complexity reminiscent of lymphoma transformation.
Citation Format: Chingiz Underbayev, Jonathan Chen, Hailey M. Harris, Lauren T. Vaughn, Andy Itsara, Adrian Wiestner. The genomic architecture of ibrutinib resistance in CLL: Oligoclonal progression with variable anatomical distribution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5828.