Abstract 3960: BCL-2 G101V mutations develop in one-third of patients on continuous venetoclax
Authors: Kuang Yanan , Fernandes Stacey M. , Fardoun Rayan , Vasquez Kevin , Mogili Abhishek , Paweletz Cloud P. , Brown Jennifer R. 
Published: June 2022

Abstract
The development of targeted therapies has revolutionized the treatment of chronic lymphocytic leukemia (CLL). To date, these therapies are generally given continuously, indefinitely, leading to the development of resistance, which is often on target. Venetoclax is the first-in-class BCL-2 inhibitor which was initially approved for continuous therapy in relapsed high-risk CLL. In that context the BCL-2 G101V mutation (mut) was identified in post-progression samples and shown to reduce venetoclax binding to BCL-2, limiting its efficacy. The mut can be identified at low variant allele frequency (VAF) prior to clinical progression. We were therefore interested to identify the frequency of this mut in our cohort of relapsed refractory CLL patients (pts) on continuous venetoclax, and to assess the sensitivity of measurements in blood vs bone marrow. To this end we utilized a ddPCR assay which has LNA probes that specifically bind to either the BCL2 G101wt or G101V sequences, to screen for G101V muts in DNA extracted from patient samples. We also started to investigate additional co-occurring BCL2 muts in G101V positive samples by Sanger sequencing.
Our patient cohort included 28 pts, of whom 20 had serial samples collected during venetoclax therapy. The median age of the pts was 66, and they had a median of 3 prior therapies before venetoclax, including chemoimmunotherapy in 67.9% and a BTK inhibitor in 60.7%. Deletion of 17p was present in 43%, with five additional pts having isolated TP53 mut (total with known TP53 aberrancy, 61%). 75% (21/28) of those evaluated had an unmutated IGHV. The median duration of venetoclax treatment was 43.5 months (mos). The timing of the first sample tested was a median of 23.3 mos after venetoclax initiation.
We detected the G101V allele in peripheral blood mononuclear cells (PBMCs) in 9 out of 28 pts, at a median allele frequency (AF) of 1.38% (range 0.04%-22.31%), at a median of 44.6 mos on venetoclax. Out of the three pts who had G101V detected at multiple timepoints, two had AF increased with time (7.8 fold increase over 6 mos and 7.7 fold increase over 5 mos, respectively), one had similar AF with time (4.68% at 18.9 mos, 3.43% at 23.8 mos on treatment). Six of these pts also had bone marrow evaluated and all were also positive (at a median AF of 0.21%; range 0.2%-18.66%); one additional patient without a PBMC sample at that timepoint was positive in bone marrow. In order to screen for any co-occurring acquired resistance muts in BCL2 G101V positive samples, we performed Sanger sequencing across the BCL2 open-reading frame. We have identified a duplication mut (R107-R110dup) in one of the samples.
In conclusion, this study shows that approximately one-third of pts on continuous venetoclax for 2+ years develop evidence of low-level BCL-2 G101V mut. Further work is underway to identify additional co-existing muts in BCL2 or other genes, and to characterize the additional genetic events at the time of clear clinical progression.
Citation Format: Yanan Kuang, Stacey M. Fernandes, Rayan Fardoun, Kevin Vasquez, Abhishek Mogili, Cloud P. Paweletz, Jennifer R. Brown. BCL-2 G101V mutations develop in one-third of patients on continuous venetoclax [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3960.