Assessment of the Clonal Dynamics of Acquired Mutations in Patients (Pts) with Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL) Treated in the Randomized Phase 3 Murano Trial Supports Venetoclax-Rituximab (VenR) Fixed-Duration Combination Treatment (Tx)
Authors: Seymour John F. , Wu Jenny Qun , Popovic Relja , Eichhorst Barbara , Hillmen Peter , Kipps Thomas J. , Langerak Anton W. , Owen Carolyn , Dubois Julie , Mellink Clemens , Van Der Kevie-Kersemaekers Anne-Marie , Dunbar Fengjiao , Jiang Yanwen , Chyla Brenda , Boyer Michelle , Thadani-Mulero Maria , Lefebure Marcus , Harrup Rosemary , Kater Arnon P. 
Published: November 2021

Abstract
Introduction: In the MURANO trial (NCT02005471), fixed-duration VenR (2 yrs Ven + R for the first 6 mo) improved survival and rates of undetectable minimal residual disease (MRD) over bendamustine (Benda)-R in pts with R/R CLL. Novel recurrent mutations in BCL2 and family genes (eg BAX and PMAIP1 [encoding Noxa]) have been linked to Ven resistance. We analyzed MRD+ samples from VenR-treated pts in MURANO to assess the nature and frequency of acquired mutations in BCL2 family genes (marker of emergent Ven resistance) and TP53 (may negatively impact response to re-Tx). We also analyzed the impact of these mutations on time to next CLL Tx (TTNT) and subsequent response.
Methods: Pts included in this analysis received up to 2 yrs of VenR and had, at any time, ≥1 CLL cell per 100 leukocytes (high MRD+) in peripheral blood. Deep next generation sequencing (NGS) was performed without cell selection using a custom targeted NGS panel, encompassing 87 cell death related genes (coding regions and selected promoters). A CD19-enriched pre-Tx sample, analyzed by whole-exome sequencing, was available for all pts for comparison. Data cutoff was May 8, 2020, for the MURANO main study and Oct 13, 2020, for the substudy (where pts received next-line VenR following progression after initial VenR Tx). TTNT was calculated using the main study Ven completion/discontinuation date and the date of the first subsequent CLL Tx.
Results: 107 samples from 42 pts, collected 21-42 mo post-Tx initiation, were evaluable. No BCL2 mutations were identified (limit of detection - variant allele frequency [VAF] 1%). Acquired BAX and PMAIP1 mutations were seen in 4 and 2 pts, respectively. Discordance between VAF and CLL MRD, and lack of proportionality in results with serial testing, suggested presence of BAX mutations in the non-CLL compartment in some pts, with further testing of sorted populations needed to clarify the source. All BAX mutations identified have been predicted to lead to loss of protein function, including 4 frameshift mutations. PMAIP1 mutations included a frameshift mutation (T86fs) and a mutation affecting gene splicing, both suggesting a loss-of-function effect. Nineteen TP53 mutations in 15/42 pts were observed across longitudinal samples (8/19 mutations in 6 pts present at baseline, 11/19 in 11 pts newly acquired, and 2 pts with both baseline and acquired mutations). Co-occurrence of TP53mut with BAXmut or PMAIP1mut was observed in 3/15 and 1/15 pts, respectively; based on VAF and assuming heterozygosity, these were likely in different clones.
After Ven cessation, 28/42 (66.7%) had received an additional anti-CLL Tx by the cutoff; 19 pts received Ven-based regimens (15 in the substudy; 4 received other Ven-containing regimens per investigators' choice), 8 pts received ibrutinib (Ibr) monotherapy and 1 pt received Benda. Of the 19 pts who received Ven-based regimens (median 22.6 mo [range 11.9-38.1] post-main study end of Tx), 8 had TP53mut (5 acquired, 3 baseline), with 5/8 (63%) responding (complete response, 3 pts; partial response [PR], 2 pts). Six/11 pts (55%) with TP53-wild-type (WT) responded, all PRs. Two pts with TP53mut received Ibr as next Tx (one responded) vs 6 TP53-WT pts (all responded). The one pt treated with Benda without response had coexisting TP53mut and BAXmut. One patient with BAXmut received Ven re-Tx without response, while in pts with BAXmut and PMAIP1mut, responses to Ibr were 100% (2/2 pts) and 50% (1/2 pts), respectively (Table). No association was apparent between acquired TP53mut and TTNT, median 784 days (d; range 248-1051) vs 617 d (range 302-1142) in TP53-WT pts. Pts harboring BAXmut (n=5) had a shorter median TTNT (305 d; range 248-874) than BAX-WT pts (678 d; range 302-1142).
Conclusion: No acquired BCL2 mutations were found in this analysis of pts from MURANO who received fixed-duration VenR Tx and subsequently manifest high MRD+. This suggests that fixed-duration Tx may reduce the propensity for selection of BCL2 resistance mutations, although confirmation using orthogonal methodology with higher sensitivity is required. Acquired TP53 mutations, noted in ~40% of pts, did not preclude response to subsequent Tx (primarily Ven-based regimens). Consistent with the TP53-independent mechanism of Ven, Ven re-Tx may be effective for pts with MRD+ disease who have relapsed following initial fixed-duration VenR Tx. More data are needed to better understand the significance of BAX or PMAIP1 mutations.
Figure 1 Figure 1.

Disclosures
Seymour: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Mei Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Morphosys: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sunesis: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Research Funding, Speakers Bureau. Wu: Roche/GNE: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Genentech, Inc.: Current Employment. Popovic: AbbVie: Current Employment, Current equity holder in publicly-traded company. Eichhorst: Consultant Department I for Internal Medicine: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Speakers Bureau; Adaptive Biotechnologies: Speakers Bureau; Hexal: Speakers Bureau; ArQule: Membership on an entity's Board of Directors or advisory committees; Oxford Biomedica (UK): Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; University Hospital of Cologne: Current Employment. Hillmen: University of Leeds: Current Employment; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Research Funding; Pharmacyclics: Honoraria, Research Funding; AstraZeneca: Honoraria; SOBI: Honoraria. Kipps: European Research Initiative on CLL (ERIC): Honoraria; DAVA Pharmaceuticals: Speakers Bureau; Bionest Partner: Other; Celgene: Consultancy, Honoraria, Other, Research Funding; Genetech: Honoraria, Other; Genentech-Roche: Consultancy; Gilead Sciences: Consultancy, Honoraria, Other, Speakers Bureau; Janssen: Consultancy, Honoraria, Other, Research Funding, Speakers Bureau; Roche: Honoraria, Other; MD Anderson Cancer Center: Research Funding; Velos: Research Funding; CRIM: Research Funding; Indy Hematology Review: Other; TG Therapeutics: Other; Verstem: Other, Speakers Bureau; University of California, San Diego: Current Employment; Pharmacyclics/AbbVie: Honoraria, Research Funding; Breast Cancer Research Foundation: Research Funding; SCOR - The Leukemia and Lymphoma Society: Research Funding; National Cancer Institute/NIH: Honoraria, Research Funding; DAVAOncology: Consultancy, Honoraria, Other; AbbVie: Consultancy, Honoraria, Other, Speakers Bureau; Oncternal Therapeutics, Inc.: Current holder of stock options in a privately-held company, Other: Stock or other ownership, Patents & Royalties: Cirmtuzumab was developed by Thomas J. Kipps in the Thomas J. Kipps laboratory and licensed by the University of California to Oncternal Therapeutics, Inc., which provided stock options and research funding to the Thomas J. Kipps laboratory., Research Funding; Genentech/Roche: Honoraria; Moores Cancer Center: Current Employment; MedImmune Inc: Research Funding; GlaxoSmithKline: Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding; Genentech, Inc.: Honoraria, Research Funding, Speakers Bureau; Pharmacyclics LLC, an Abbvie Company: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Research Funding; Abbott Laboratories: Consultancy, Research Funding. Langerak: Janssen: Speakers Bureau; Gilead: Research Funding; F. Hoffmann-La Roche Ltd/Genentech, Inc.: Research Funding; Erasmus MS, University Medical Center: Current Employment. Owen: Incyte: Honoraria; AbbVie: Honoraria, Research Funding; Merck: Honoraria; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Servier: Honoraria; Pharmacyclics: Research Funding; Genentech: Research Funding; Gilead: Honoraria. Dubois: Abbvie: Research Funding; Genentech: Research Funding; Roche: Research Funding. Mellink: Cytogenetic Field: Consultancy; Genome Diagnostics Laboratory, AUMC: Current Employment; Financial support related to microarray analysis of Murano samples: Research Funding. Van Der Kevie-Kersemaekers: Amsterdam University Medical Centers: Current Employment. Dunbar: AbbVie: Current Employment. Jiang: Genentech, Inc./F.Hoffmann-La Roche Ltd: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company. Chyla: AbbVie: Current Employment, Current equity holder in publicly-traded company. Boyer: Roche: Current Employment. Thadani-Mulero: Roche: Current Employment, Current equity holder in publicly-traded company. Lefebure: Roche: Current Employment; F. Hoffmann-La Roche Ltd: Current equity holder in publicly-traded company. Harrup: AstraZeneca: Other: Advisory board. Kater: Genmab, LAVA: Other: Ad Board, Steering Committee; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding.