High Prevalence of BTK Mutations on Ibrutinib Therapy after 3 Years of Treatment in a Real-Life Cohort of CLL Patients: A Study from the French Innovative Leukemia Organization (FILO) Group
Authors: Quinquenel Anne , Fornecker Luc Matthieu , Letestu Remi , Fleury Carole , Ysebaert Loic , Dilhuydy Marie-Sarah , Guieze Romain , Feugier Pierre , Roos-Weil Damien , Willems Lise , Michallet Anne-Sophie , Delmer Alain Jacques , Taverna Pietro , Fox Judith A. , Levy Vincent , Cymbalista Florence , Baran-Marszak Fanny 
Published: October 2019

Abstract
Background
Long-term outcome of chronic lymphocytic leukemia (CLL) patients (pts) under ibrutinib therapy has been evaluated within clinical trials, and CLL progression seems to be strongly linked with the onset of BTK and PLCg2 mutations. These mutations have largely been studied in resistant pts. Limited data are available regarding the BTK and/or PLCγ2 mutations in pts on ibrutinib without evidence of disease progression. Ibrutinib was available in France during 2014 as an early access program (EAP) thus allowing the evaluation of prolonged use of Ibrutinib in a real-life setting. The objective of this study was to analyze the genetic profile of pts still on Ibrutinib after at least 3 years of treatment. We also explored minimal residual disease (MRD) and T-lymphocyte subsets.
Results
Data were collected from all pts who received ibrutinib via the EAP in 29 centers of the FILO group. Between February 2014 and April 2015, 204 pts began ibrutinib treatment. Only 63 (31%) remained on therapy 3 years later and blood samples were collected for 57 of them. For these 57 pts, median age at ibrutinib initiation was 67.7 years (46.9 to 84.1). Forty-five percent and 42.5% pts presented with 11q or 17p deletion respectively, and 17/18 pts tested harbored unmutated IGVH genes. Pts received a median number of 2 (0-5) previous therapies, consisting mainly of fludarabine-based regimens (73%), bendamustine + rituximab (48%), chlorambucil + anti-CD20 antibody (25%) or alemtuzumab (16%). The best response on ibrutinib therapy was clinical complete response, partial response and partial response with lymphocytosis in 31%, 56% and 13% of cases respectively.
Median time between ibrutinib initiation and blood sample collection was 3.5 years (2.75 to 4.2). Median lymphocyte count was 2.62 G/L (0.37 to 121), and median CLL cell count was 0.52 G/L (0-117). No patient achieved MRD < 0.01% in the blood (by FCM). Thirty pts had CLL cell count higher than 0.5 G/L, allowing mutational analysis by next-generation sequencing. At least one BTK mutation was found in 17/30 (57%) pts, (C481S N=13, C481Y N=2, C481G N=1 and C481R N=2) (VAF 0.2-73%), and 4/30 (13%) pts harbored PLCγ2 mutations (L845G N=3, D993H N=1, R665W N=2 and C849R N=1) (VAF 1-11%). TP53 mutations were found in 14/30 (47%) cases, SF3B1 and NOTCH1 mutations in 6/30 (20%) cases, XPO1 and RPS15 mutations in 4/30 (13%) cases, ATM mutations in 3/30 (10%) cases, BIRC3 and POT1 mutations in 1/30 (3%) case. All pts with PLCγ2 mutations presented with both BTK and TP53 mutations. Neither the incidence of TP53 mutation nor the other CLL frequently mutated genes were different between BTK mutated and unmutated pts (p=NS). Moreover, the number of previous therapies (0-1 vs > 1) was not different between pts harboring BTK mutations or not (p=0.2). Study of T lymphocyte subsets showed a highly variable CD4/CD8 T- cell ratio (median=1, range 0.41-3.42), but, interestingly, the median number of CD4 T-cells was 584/mm3 (141-2481), with 92% of pts presenting with CD4 T-cells >250/mm3. This result suggests that ibrutinib treatment may not affect CD4 T-cell counts.
With a median follow-up of 3.5 months from sample collection, 12/57 pts (21%) discontinued ibrutinib because of CLL progression (N=8), Richter syndrome (N=1), adverse event (N=1), septic shock (N=1) and unknown reason (N=1). Twelve pts progressed within 6 months after sample collection. Among them, BTK mutations were present in 11/12 cases and PLCγ2 in 2 cases. Only one patient experienced progression without BTK or PLCγ2 mutations. BTK mutations were found in 6 pts without evidence of progression at the time of last follow-up and longitudinal analysis is ongoing. Variant allele frequencies of BTK mutations were highly variable between pts (range 0.2%-73%) and were not different between progressing and non-progressing pts (p=0.2).
Conclusion and perspectives
In this real-life setting, only one third of pts remained on ibrutinib after 3 years. Even after such a long treatment exposure, ibrutinib as a monotherapy did not appear to induce complete immunophenotypic response. Fifty-seven percent of pts with CLL cell count > 0.5 G/L had BTK C481 mutations, and BTK mutations were found in all but one pt who progressed. This study also highlighted the onset of BTK and PLCγ2 mutations in long-term responders on therapy, before any evidence of disease progression.

Disclosures
Quinquenel: Jansen Cilag: Honoraria, Research Funding; Abbvie: Honoraria. Fornecker:Takeda: Honoraria; Servier: Honoraria. Ysebaert:Janssen: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding. Guieze:abbvie: Honoraria; janssen: Honoraria; gilead: Honoraria. Feugier:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Taverna:Sunesis Pharmaceuticals: Employment. Fox:Sunesis Pharmaceuticals: Employment; Amphivena Therapeutics: Employment. Cymbalista:AbbVie, Inc: Honoraria; Sunesis: Research Funding; Janssen: Honoraria; Gilead: Honoraria. Baran-Marszak:Sunesis: Research Funding.