Genomic Determinants of Pirtobrutinib Resistance in Refractory CLL: BRUIN Analysis

New genomic analysis from the phase 1/2 BRUIN trial (NCT03740529) suggests that pirtobrutinib (Jaypirca), a noncovalent Bruton tyrosine kinase inhibitor (BTKi), maintains high efficacy in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) while inducing a distinct shift in the clonal architecture of resistance.¹ The study, published in Blood, demonstrates that while pirtobrutinib effectively clears or reduces common mutations associated with covalent BTKi (cBTKi) resistance, it also selects for novel BTK mutations, primarily in the gatekeeper and kinase domains, that may guide future therapeutic sequencing.

“We report, to our knowledge, the largest study of mechanisms of response and resistance to pirtobrutinib,” wrote Brown et al. “Using a highly sensitive assay, we find that 37% of these mutations can be detected at baseline, likely having emerged on prior cBTKi therapy.”

Study Design and Patient Population

The research focused on 245 patients with R/R CLL enrolled in BRUIN²who had previously been treated with covalent inhibitors such as ibrutinib (Imbruvica; 89%), acalabrutinib (Calquence; 16%), or zanubrutinib (Brukinsa; 3%).¹ This cohort was heavily pretreated, with a median of 4 prior lines of therapy (range, 1–11). Genomic profiling at baseline revealed a high prevalence of high-risk alterations, including TP53 mutations (38%) and 17pdeletions (28%).¹

Notably, 43% of patients entered the trial with baseline BTK mutations, the most common being C481 substitutions (85% of those with BTK mutations) which are known drivers of resistance to covalent inhibitors. The investigators performed targeted next-generation sequencing (NGS) on peripheral blood mononuclear cells at baseline and at the time of progressive disease (PD) to track clonal evolution under the selective pressure of pirtobrutinib.

Efficacy and Clonal Clearance

Pirtobrutinib demonstrated a robust overall response rate (ORR) of 82% among the 245-patient analysis group. The median time on pirtobrutinib treatment was 20.7 months. A key finding of the genomic analysis was the drug’s ability to overcome established resistance; 84% of patients with baseline BTK C481 mutations showed a significant decrease or complete clearance of these clones at the time of progression on pirtobrutinib.

This clearance indicates that pirtobrutinib successfully suppresses the clones that drove failure on prior covalent therapies. However, clinical progression eventually occurred in 57% of the overall population at a median follow-up of 29 months.

Emerging Resistance Mechanisms

The analysis of 88 patients with paired samples at baseline and PD identified several distinct pathways of acquired resistance:

• Acquired BTK Mutations: Approximately 44% of patients acquired new BTK mutations at progression. The most prevalent were gatekeeper mutations at residue T474(26%) and kinase-impaired L528W mutations (16%). Other less frequent mutations included V416L, A428D, and substitutions at the D539 and Y545 residues.
• Non-BTK Genomic Alterations: Nearly 24% of patients acquired mutations in genes other than BTK, including TP53, PLCG2, and PIK3CA.
• Nongenomic Resistance: Interestingly, 32% of patients experienced disease progression without the emergence of any identifiable mutations in the targeted sequencing panel, suggesting that nongenomic mechanisms or off-target pathways may also drive pirtobrutinib resistance.

High-sensitivity NGS (limit of detection 0.5%) revealed that 37% of the acquired BTK mutations were actually present at baseline at very low subclonal frequencies. This suggests that pirtobrutinib may select for these preexisting resistant clones rather than inducing new mutations.

Clinical Implications for Sequencing

The identification of specific resistance mutations like L528W and T474x has significant implications for subsequent therapy. For instance, L528W has been associated with reduced BTK kinase activity but preserved B-cell receptor signaling through noncatalytic scaffolding roles. Furthermore, laboratory assays confirmed that these variants significantly reduce pirtobrutinib’s binding affinity and residence time.

Understanding these genomic determinants allows oncologists to better anticipate the trajectory of CLL after pirtobrutinib failure. As noncovalent inhibitors become more common in the second- and third-line settings, characterizing the mutational landscape will be essential for determining whether a patient should transition to BCL2 inhibitors, CAR T-cell therapy, or other emerging modalities.

REFERENCES

1. Brown JR, Nguyen B, Desikan SP, et al. Genomic determinants of response and resistance to pirtobrutinib in relapsed/refractory chronic lymphocytic leukemia. Blood. 2026;147(1):24-34.

2. A Study of Oral LOXO-305 in Patients With Previously Treated CLL/​SLL or NHL. ClinicalTrials.gov. Updated September 25, 2025. Accessed January 5, 2026. https://clinicaltrials.gov/study/NCT03740529