Longitudinal High-Throughput T Cell Repertoire Profiling of Chronic Lymphocytic Leukemia Patients Under Different Types of Treatment: Implications for Combination Strategies

Abstract Using next-generation sequencing (NGS), we recently documented the clonal architecture of the T cell repertoire in treatment-naive chronic lymphocytic leukemia (CLL), with ample immunogenetic evidence indicating selection by restricted antigens. Our preliminary NGS study in 16 patients pre- and 3-month post-treatment indicated a differential impact of standard chemoimmunotherapy (FCR) versus B cell receptor signaling inhibitors (BcRi) on CLL T cells. Prompted by these observations, here we sought to comprehensively assess CLL T cell repertoire changes over treatment in relation to both treatment type and clinical response by combining NGS immunoprofiling, flow cytometry and functional assays. NGS profiling of the T cell receptor (TR) gene repertoire was performed in 28 CLL patients who received FCR (n=9), ibrutinib (IB, n=15) and/or rituximab-idelalisib (R-ID, n=10) at successive timepoints (pre, +3mo, +9mo and at deepest clinical response, total samples: n=113). TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end NGS. Raw reads were processed through a purpose-built, validated bioinformatics pipeline, culminating to 20,347,768 productive, filtered-in TRB sequences (median 155,479/sample). For repertoire analysis, clonotypes (i.e. rearrangements with identical TRBV gene usage and amino acid complementarity-determining region 3 sequence) were considered (median 11,420 distinct clonotypes/sample). All cases displayed significant clonal T cell expansions both pre- and post-treatment [median clonality, measured as the cumulative frequency of the 10 most expanded (major) clonotypes/sample: 30.3% and 39.6%, respectively]. Median clonality significantly increased at +3mo in the FCR (29.0% to 46.9%, p .05). Overtime analysis revealed a gradual increase of clonality over deepening clinical response (pre-, +3mo, +9mo, deepest response) in the R-ID group (33.0% to 39.1% to 46.0% to 46.1%, respectively; p .05). Considering that FCR resulted in T cell repertoire reconstitution whereas BcRis retained pre-treatment clones, we then focused on major clones persisting over treatment and found that they significantly expanded in the R-ID group, peaking at +3mo (p .05). Taken together, NGS immunoprofiling suggests that BcRis retain T cell clones that may have developed in response to CLL-related antigens, which in the case of R-ID expand and peak at +3mo. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response. Overall, this data provides rationale for designing combination strategies, e.g. of R-ID with immunomodulating drugs, aiming to boost cytotoxic anti-tumor responses. Moreover, identifying the relevant neoepitopes may eventually pave the way for stratified treatments by means of engineered T cells or peptide vaccines, especially if these epitopes are conserved among CLL. Disclosures Vardi: Janssen: Honoraria; Gilead: Research Funding. Gemenetzi: Gilead: Research Funding. Ramsay: MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding. Stamatopoulos: Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding. Hadzidimitriou: Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding.