Opposite Effects of Bruton Tyrosine Kinase (BTK) Inhibitor Therapy with Ibrutinib and FCR Chemo-Immunotherapy on the Normal B Lymphocyte Repertoire in Patients with Chronic Lymphocytic Leukemia
Authors: Schliffke Simon , Sivina Mariela , Kim Ekaterina , Thiele Benjamin , Akyüz Nuray , Falker-Gieske Clemens , Thenhausen Toni , Krohn-Grimberghe Artus , Bokemeyer Carsten , Jain Nitin , Estrov Zeev , Ferrajoli Alessandra , Wierda William G. , Keating Michael J. , Burger Jan A. , Binder Mascha 
Published: October 2019

Abstract
Disease-inherent and treatment-related immune dysfunction remain leading causes for morbidity and mortality in patients with chronic lymphocytic leukemia (CLL). The advent of kinase inhibitors that target B cell receptor (BCR) signaling, which lack myelo- and T lymphocyte toxicity, raised hopes that these new agents may be less immunosuppressive and allow for better immune reconstitution when compared to chemo-immunotherapy (CIT). The effects of the BTK inhibitor ibrutinib or CIT with fludarabine, cyclophosphamide and rituximab (FCR) on the normal B cell repertoire have not been well characterized. Here, we used state-of-the-art immunosequencing technology to investigate how ibrutinib treatment affects the regeneration of non-malignant B-cells when compared to patients treated with FCR.
Clinical data on infection rates and immunoglobulin levels was analyzed from 40 CLL patients treated with ibrutinib (median number of two pre-treatments) or frontline CIT with FCR at MD Anderson Cancer Center. In a representative subset of 20 patients, flow cytometry and next generation sequencing (NGS) of the immunoglobulin heavy chain (IGH) gene locus was used to monitor non-malignant B-cell immune reconstitution for 24 months after start of treatment with ibrutinib or FCR.
Comparison of ibrutinib treatment with CIT revealed that immunoglobulin levels remained stable and relatively low in both cohorts, except for an increase in IgA during ibrutinib treatment, as previously reported. NGS results showed that ibrutinib treatment significantly decreased the non-malignant B-cells count after 24 months of treatment, while the counts were quantitatively stable in the FCR cohort. Next, we determined the dynamics of non-malignant B-cell immune repertoire composition over treatment. Based on the mutational status of the V gene, non-malignant B-cells were classified as IGH hypermutated (<98% identity to the corresponding germline V gene, corresponding to antigen-experienced B-cells) or IGH unmutated (≥98% identity to the corresponding germline V gene, corresponding to antigen-naïve B-cells). Before treatment initiation, the mean percentage of antigen-experienced B-cells did not significantly differ between the groups (ibrutinib 39%, FCR 48%). After 24 months, a significant decrease of antigen-experienced B-cells was observed in the FCR cohort, while the ratio of antigen-experienced and antigen-naïve B-cells remained unchanged in ibrutinib treated patients (ibrutinib 39%, FCR 22%, p=0.01). Analysis of the IGH clonotype repertoire using the Shannon-Wiener and the inverse Simpson diversity indices confirmed these results, showing that the non-malignant IGH repertoire was composed of balanced numbers of antigen-experienced and antigen-naïve medium sized clones before treatment initiation in both cohorts. In line with the IGH repertoire shift towards antigen-naïve B-cells in FCR treated patients, the medium-sized clones disappeared after treatment, with large numbers of small-sized unmutated clones dominating after 24 months (p<0.0001). In ibrutinib treated patients, the repertoire diversity remained stable throughout the course of treatment.
Taken together, our data indicate that continuous treatment with ibrutinib preserves preexisting (partially antigen-experienced) B-cells but impairs de-novo generation of naive B-cells. In contrast, FCR leads to a deletion of memory B-cells but also a subsequent substantial renewal of the B-cell repertoire. Both patterns may differentially affect immune-competence towards infections.

Disclosures
Bokemeyer: Karyopharm: Research Funding. Jain:Pfizer: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Genentech: Research Funding; Abbvie: Research Funding; Pharmacyclics: Consultancy, Honoraria, Research Funding; Infinity: Research Funding; Novartis: Consultancy, Honoraria; Servier: Consultancy, Honoraria; Novimmune: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Honoraria, Research Funding; BMS: Research Funding; Celgene: Research Funding; Seattle Genetics: Research Funding. Wierda:Gilead: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding; Genentech: Research Funding. Burger:Pharmacyclics: Research Funding.