<div>AbstractPurpose:<p>Using next-generation sequencing (NGS), we recently documented T-cell oligoclonality in treatment-naïve chronic lymphocytic leukemia (CLL), with evidence indicating T-cell selection by restricted antigens.</p>Experimental Design:<p>Here, we sought to comprehensively assess T-cell repertoire changes during treatment in relation to (i) treatment type [fludarabine-cyclophosphamide-rituximab (FCR) versus ibrutinib (IB) versus rituximab-idelalisib (R-ID)], and (ii) clinical response, by combining NGS immunoprofiling, flow cytometry, and functional bioassays.</p>Results:<p>T-cell clonality significantly increased at (i) 3 months in the FCR and R-ID treatment groups, and (ii) over deepening clinical response in the R-ID group, with a similar trend detected in the IB group. Notably, in constrast to FCR that induced T-cell repertoire reconstitution, B-cell receptor signaling inhibitors (BcRi) preserved pretreatment clones. Extensive comparisons both within CLL as well as against T-cell receptor sequence databases showed little similarity with other entities, but instead revealed major clonotypes shared exclusively by patients with CLL, alluding to selection by conserved CLL-associated antigens. We then evaluated the functional effect of treatments on T cells and found that (i) R-ID upregulated the expression of activation markers in effector memory T cells, and (ii) both BcRi improved antitumor T-cell immune synapse formation, in marked contrast to FCR.</p>Conclusions:<p>Taken together, our NGS immunoprofiling data suggest that BcRi retain T-cell clones that may have developed against CLL-associated antigens. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response.</p></div>
<div>AbstractPurpose:<p>Using next-generation sequencing (NGS), we recently documented T-cell oligoclonality in treatment-naïve chronic lymphocytic leukemia (CLL), with evidence indicating T-cell selection by restricted antigens.</p>Experimental Design:<p>Here, we sought to comprehensively assess T-cell repertoire changes during treatment in relation to (i) treatment type [fludarabine-cyclophosphamide-rituximab (FCR) versus ibrutinib (IB) versus rituximab-idelalisib (R-ID)], and (ii) clinical response, by combining NGS immunoprofiling, flow cytometry, and functional bioassays.</p>Results:<p>T-cell clonality significantly increased at (i) 3 months in the FCR and R-ID treatment groups, and (ii) over deepening clinical response in the R-ID group, with a similar trend detected in the IB group. Notably, in constrast to FCR that induced T-cell repertoire reconstitution, B-cell receptor signaling inhibitors (BcRi) preserved pretreatment clones. Extensive comparisons both within CLL as well as against T-cell receptor sequence databases showed little similarity with other entities, but instead revealed major clonotypes shared exclusively by patients with CLL, alluding to selection by conserved CLL-associated antigens. We then evaluated the functional effect of treatments on T cells and found that (i) R-ID upregulated the expression of activation markers in effector memory T cells, and (ii) both BcRi improved antitumor T-cell immune synapse formation, in marked contrast to FCR.</p>Conclusions:<p>Taken together, our NGS immunoprofiling data suggest that BcRi retain T-cell clones that may have developed against CLL-associated antigens. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response.</p></div>
Background: Chronic lymphocytic leukemia (CLL) is divided into two broad prognostic categories, namely mutated (M) and unmutated (U) CLL, according to the somatic hypermutation (SHM) status of the clonotypic heavy chain immunoglobulin (IGHV) gene. This is perceived to remain stable over time, as evidenced by low‐throughput studies, which however precluded investigation of subclonal architecture and evolution overtime. Aims: Here, we sought to comprehensively assess the B cell receptor (BcR) IG gene repertoire at CLL diagnosis and 1 st relapse after chemoimmunotherapy (FCR) by next‐generation sequencing (NGS). Methods: We studied 5 patients, of whom 1 each expressed BcR IG typical of stereotyped subset #1 (U‐CLL), subset #4 (M‐CLL) or subset #6 (U‐CLL), whereas the remaining 2 concerned non‐stereotyped U‐CLL. Genomic DNA was amplified by multiplex PCR and products were subjected to paired‐end NGS. Sequence data were processed by a validated bioinformatics pipeline performing strict quality filtering. Rearrangements with identical IGHV gene and CDR3 amino acid (aa) sequence were defined as clonotypes. The dominant clonotype was defined as major, and clonotypes with the same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3 were considered as its satellite subclones. Results: In total, we analyzed 1,682,728 filtered‐in, productive IGHV‐IGHD‐IGHJ gene rearrangements (median 195,460 rearrangements/sample). In all cases, the major clonotype (IGHV and CDR3 aa sequence) was identical to that determined by Sanger sequencing and remained the same at diagnosis and relapse. However, it consisted of multiple distinct nucleotide (nt) sequences; while most nt sequences displayed the same SHM load as with Sanger (median 73.9% of all nt sequences of the major clonotype), there were other nt sequences with higher or lower SHM load. In all cases, nt sequences with discordant SHM status were practically negligible (median frequency 0.01%). Notably, the relative frequency of the dominant nt sequence increased at relapse for all cases (median increase by 3.7%, p < 0.01). A particular note for subset #4, which by definition displays high SHM, was the emergence of major clonotype nt sequences with lower or even absent SHM at relapse. The median frequency of the major clonotype at diagnosis was 92.5% (range 86.1–93.3%) and remained stable at relapse (92.6%, range 89.9–93.8%). In all cases, the major clonotype came along with numerous satellite subclones (same IGHV gene, CDR3 length, and ≤2 aa differences within the CDR3) at both diagnosis and relapse (median n = 685 vs n = 604 respectively, p>0.05). When considering both the major clonotype and its satellite subclones, the respective cumulative frequency corresponded to nearly the entire B cell repertoire/sample (median 99.6%, range 97.6–99.8%). Subset #4 satellite sublones displayed recurrent SHM patterns within the CDR3 at relapse, which actually diverged from the major clonotype and converged towards the consensus CDR3 sequence for this subset as defined by 176 different subset #4 patients. Summary/Conclusion: Overall, our study provides insight into the evolving architecture of the BcR IG repertoire of relapsing CLL, revealing that, similar to genomic subclones, there is a universe of BcR IG subclones as well. While the possibility of technical error cannot be excluded, the identification of recurrent SHM patterns and trends at relapse strongly points towards functional selection.
T cell large granular lymphocyte (T-LGL) lymphoproliferations constitute a disease spectrum ranging from poly/oligo to monoclonal. Boundaries within this spectrum of proliferations are not well established. T-LGL lymphoproliferations co-occur with a wide variety of other diseases ranging from autoimmune disorders, solid tumors, hematological malignancies, post solid organ, and hematopoietic stem cell transplantation, and can therefore arise as a consequence of a wide variety of antigenic triggers. Persistence of a dominant malignant T-LGL clone is established through continuous STAT3 activation. Using next-generation sequencing, we profiled a cohort of 27 well-established patients with T-LGL lymphoproliferations, aiming to identify the subclonal architecture of the T-cell receptor beta (TRB) chain gene repertoire. Moreover, we searched for associations between TRB gene repertoire patterns and clinical manifestations, with the ultimate objective of discriminating between T-LGL lymphoproliferations developing in different clinical contexts and/or displaying distinct clinical presentation. Altogether, our data demonstrates that the TRB gene repertoire of patients with T-LGL lymphoproliferations is context-dependent, displaying distinct clonal architectures in different settings. Our results also highlight that there are monoclonal T-LGL cells with or without STAT3 mutations that cause symptoms such as neutropenia on one end of a spectrum and reactive oligoclonal T-LGL lymphoproliferations on the other. Longitudinal analysis revealed temporal clonal dynamics and showed that T-LGL cells might arise as an epiphenomenon when co-occurring with other malignancies, possibly reactive toward tumor antigens.
Abstract Purpose: Using next-generation sequencing (NGS), we recently documented T-cell oligoclonality in treatment-naïve chronic lymphocytic leukemia (CLL), with evidence indicating T-cell selection by restricted antigens. Experimental Design: Here, we sought to comprehensively assess T-cell repertoire changes during treatment in relation to (i) treatment type [fludarabine-cyclophosphamide-rituximab (FCR) versus ibrutinib (IB) versus rituximab-idelalisib (R-ID)], and (ii) clinical response, by combining NGS immunoprofiling, flow cytometry, and functional bioassays. Results: T-cell clonality significantly increased at (i) 3 months in the FCR and R-ID treatment groups, and (ii) over deepening clinical response in the R-ID group, with a similar trend detected in the IB group. Notably, in constrast to FCR that induced T-cell repertoire reconstitution, B-cell receptor signaling inhibitors (BcRi) preserved pretreatment clones. Extensive comparisons both within CLL as well as against T-cell receptor sequence databases showed little similarity with other entities, but instead revealed major clonotypes shared exclusively by patients with CLL, alluding to selection by conserved CLL-associated antigens. We then evaluated the functional effect of treatments on T cells and found that (i) R-ID upregulated the expression of activation markers in effector memory T cells, and (ii) both BcRi improved antitumor T-cell immune synapse formation, in marked contrast to FCR. Conclusions: Taken together, our NGS immunoprofiling data suggest that BcRi retain T-cell clones that may have developed against CLL-associated antigens. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response.
