1163 - Sensitive monitoring of sequential targeted therapy resistance in relapsed/therapy refractory chronic lymphocytic leukemia

Targeted therapies, including the BTK inhibitor ibrutinib and the Bcl-2 inhibitor venetoclax, have transformed the therapeutic landscape of relapsed/therapy refractory chronic lymphocytic leukemia (R/R CLL) demonstrating previously unmet efficacy and high response rates across heavily pre-treated patient populations. Despite the prolonged progression-free survivals, secondary or acquired resistance and subsequent progressive CLL occur in some patients and are the leading cause of treatment failure. Since eventual loss of efficacy and sequential resistance to both ibrutinib and venetoclax confer dismal outcomes, early detection of secondary resistance could potentially allow for the introduction of alternative treatment strategies with ideal timing over the disease course and the identification of patients who may benefit from change of therapy.

With a series of studies, our specific aims were to develop a droplet digital polymerase chain reaction (ddPCR)-based method for the detection of the most frequent resistance mutations conferring secondary resistance to ibrutinib or venetoclax therapy. We further aimed to dissect the temporal heterogeneity observed in R/R CLL in the context of targeted therapy by the molecular monitoring of the BTK p.Cys481Ser and BCL-2 hotspot mutations relevant in acquired ibrutinib and venetoclax resistance, respectively. Our ultimate goal was to aid the standard-of-care monitoring of the efficacy of ibrutinib and venetoclax therapies in CLL.

Serial peripheral blood (PB) samples were collected from 83 R/R CLL patients treated with ibrutinib. This cohort comprised a subset of 126 patients investigated within the framework of the nationwide Hungarian Ibrutinib Resistance Analysis Initiative. Further PB samples were collected from 67 patients with R/R CLL treated with venetoclax monotherapy or venetoclax in combination with rituximab. Progressive disease and relapse were defined as the emergence of at least one novel symptom based on the iwCLL criteria for active disease and change of therapy was issued at the physician’s discretion in both cohorts. Screening and quantitative assessment of the BTK p.Cys481Ser and selected BCL2 resistance mutations (p.Gly101Val and p.Asp103Tyr) were performed by ddPCR using assays designed for the discriminative analysis of mutant and wild-type alleles. Reactions were carried out on the QX200 Droplet Digital PCR platform (Bio-Rad Laboratories, California, USA) according to the manufacturer’s instructions.

In the ibrutinib- and venetoclax-treated patient groups, median follow up time was 40 and 23 months, respectively, with both cohorts representing the largest real-world patient populations in which resistance mutations have been longitudinally monitored at the date of publishing. In 83 ibrutinib-treated R/R CLL patients, BTK p.Cys481Ser was detected in nearly half of the cases with 80% of the mutation-bearing patients showing signs of disease progression. In the venetoclax-treated patient population, BCL2 p.Gly101Val and p.Asp103Tyr were detected in 10.4% and 11.9% of the cases, respectively, with 4 patients harbouring both alterations. Ten out of eleven (90.9%) patients carrying p.Gly101Val and/or p.Asp103Tyr experienced relapse or disease progression, representing 43.5% (10/23) of all cases developing secondary venetoclax resistance during the follow-up period. Intriguingly, 21 patients receiving ibrutinib have experienced relapse or disease progression during the follow up and were administered subsequent venetoclax therapy. Of this patient subpopulation, 18 cases were found to harbour the BTK p.Cys481Ser resistance mutation with the emergence of the variant predating the first clinical observation of resistance with a median of 11 months. Sequential resistance to ibrutinib and venetoclax occurred in 11 cases, with 3 patients carrying both BTK p.Cys481Ser and either of the two BCL2 hotspot mutations.

In summary, with the ultra-sensitive ddPCR-based analyses of one mutation hotspot associated with ibrutinib resistance and two hotspots linked to venetoclax failure, we successfully identified the underlying molecular mechanisms in two-thirds (32/44 and 10/23 of all ibrutinib- and venetoclax-resistant cases, respectively) of the patients undergoing relapse or disease progression during targeted treatments. Molecular monitoring of sequential resistance to ibrutinib and venetoclax by ddPCR is a feasible method able to capture the temporal heterogeneity observed in R/R CLL. Our results imply the selected gene mutations as promising potential molecular biomarkers for use in the standard-of-care diagnostic work-up of targeted therapy resistance in CLL.