Treatment with obinutuzumab and venetoclax reshapes the TRB repertoire of CLL patients
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ABSTRACT: Anti-leukemic treatment in chronic lymphocytic leukemia (CLL) impacts autologous T cells through various direct and indirect mechanisms. However, how first-line treatment with venetoclax and obinutuzumab impacts the TRB repertoire has not been studied. Here, we have used next-generation sequencing to longitudinally characterize the TRB repertoire of previously untreated CLL patients who received treatment with obinutuzumab and venetoclax in the HOVON-139/ GIVE trial. Following treatment, due to the emergence of a polyclonal population of small-sized, diverse clonotypes, TRB repertoire diversity increased (Shannon’s H; trial entry 5.5 [95%CI 5.2-6.2] vs end of induction treatment [EOiT] 6.2 [95%CI 5.7- 6.7], P=0.001). In contrast, treatment with obinutuzumab and venetoclax did not ameliorate TRB oligoclonality (EOiT, cumulative frequency of top 10 clonotypes 32.5% [95%CI 25.3-39.9], P=0.10; R+12 CF10 31.2% [95%CI 24.3-38.1], P=0.17), due to the persistence of large pre-treatment clonotypes. Patients with IGLV3-21R110 CLL had weaker recovery of TRB repertoire diversity (mean Shannon’s H 5.2 [95%CI 4.0-6.3] vs 6.8 [95%CI 6.3-7.3], P=0.01). TRB clonotypes that were shared among patients could be identified, a minority of which had known pathogenic or auto-antigenic affinity. The concurrent presence or future resurgence of MRD did not impact the TRB repertoire, but low TRB diversity (Poisson regression coefficient -0.32±0.15, P=0.046) and high oligoclonality (Poisson regression coefficient 0.02±0.009, P=0.02) were associated with an increased risk of infection. These data provide the groundwork for the future investigation of the fitness of autologous T cells for T cell-mediated immunotherapy, following treatment with obinutuzumab and venetoclax.
Project description:In chronic lymphocytic leukemia, measurable residual disease (MRD) depth following fixed-duration treatment is associated with progression-free survival (PFS). Often, MRD is reported as a static parameter, but successive MRD measurements may more accurately capture MRD dynamics and reveal valuable prognostic information. We used our recently developed IGHV leader-based NGS assay to measure MRD at four timepoints in 60 patients treated in the HOVON-139/GIVE trial, in which previously untreated patients received one year induction treatment with obinutuzumab and venetoclax, followed by randomization to consolidation treatment with venetoclax or MRD-conditional consolidation. Induction treatment resulted in MRD undetectability (<10-5) in 93% of assessed patients, with 85% and 74% of patients retaining MRD undetectability at six and twelve months after randomization. Uniquely, we measured MRD at a very early timepoint, during the fourth week of venetoclax ramp-up after obinutuzumab pre-induction. Here, MRD levels showed large interindividual variation, and, importantly, were associated with the probability of achieving MRD undetectability after induction treatment, MRD conversion after randomization and PFS. Additionally, the IGHV leader-based NGS assay allows characterization of the healthy background IGHV repertoire. Of note, venetoclax consolidation treatment, compared to no consolidation treatment, significantly impaired polyclonal IGHV repertoire reconstitution after induction treatment with venetoclax and obinituzumab.
Project description:We examined the CLL cells transcriptome data of six patients who fail to clear their leukemia cells and develop progressive disease on venetoclax therapy. Methods: CLL cells were collected prior to venetoclax therapy (Sample Collection 1 (SC1)) and then more than 1 year later (Sample Collection 2 (SC2)). Negative isolation of CLL cells to ≥95% purity was performed prior to RNA isolation.
Project description:Immunogenetic studies in CLL revealed clonal expansions of T cells and shared T cell clonotypes between different patients, strongly implying clonal selection by antigens. Recurrent genomic aberrations associated with distinct abnormal expression profiles could represent an alternative source of potent immunogenic neoepitopes that might shape the T cell receptor (TR) gene repertoire in CLL. On these grounds, here we interrogated the TR gene repertoire of CLL patients with different genomic aberration profiles with the aim to identify unique signatures that would allude to distinct antigen selection pressures.The study group included 44 untreated CLL patients with who were categorized in 5 subgroups defined by a unique genomic aberration, as follows: +12, n=17; del(11q), n=10; del(13q), n=7; TP53mut, n=5; NOTCH1mut, n=5. Starting material was RNA extracted from blood mononuclear cells. TRBV-TRBD-TRBJ gene rearrangements were RT-PCR amplified and subjected to paired-end next generation sequencing (NGS). Bioinformatics analysis with a purpose-build pipeline and in silico prediction models were used for the profiling of the TR gene repertoire and the identification of neoepitope-specific TR clonotypes, respectively.
Project description:Inhibition of the B-cell receptor pathway, and specifically of Bruton tyrosine kinase (BTK), is a leading therapeutic strategy in chronic lymphocytic leukemia (CLL). Target occupancy has been measured as a pharmacodynamic parameter in clinical studies of covalent BTK inhibitors. However, the kinetics of BTK turnover, which determines occupancy, and the relationship between occupancy, pathway inhibition and clinical outcomes remains undefined. This randomized phase 2 study investigated the safety, efficacy, and pharmacodynamics of a selective BTK inhibitor acalabrutinib at 100 mg twice daily or 200 mg once daily in 48 patients with relapsed/refractory or high-risk treatment naïve CLL. Acalabrutinib was well tolerated and yielded an overall response rate (ORR) of partial response or better of 95.8% (95% CI 78.9%, 99.9%) and an estimated progression-free survival (PFS) rate at 24 months of 91.5% (95% CI 70.0%, 97.8%) with twice daily dosing and an ORR of 79.2% (95% CI 57.9%, 92.9%) and an estimated PFS rate at 24 months of 87.2% (95% CI 57.2%, 96.7%) with once daily dosing. BTK resynthesis was faster in CLL than in healthy volunteers. Twice daily dosing maintained higher BTK occupancy and achieved more potent pathway inhibition compared to once daily dosing. Additional follow-up is required to address the impact of dosing schedule and BTK occupancy on long-term clinical outcomes.
Project description:The sensitivity of conventional techniques for the quantification of minimal/measurable residual disease (MRD) in chronic lymphocytic leukemia (CLL) is limited to MRD 10-4. Measuring MRD <10-4 could help to further distinguish between CLL patients with durable remission and those at risk of early relapse. We here present a novel, academically developed IGHV leader-based next-generation sequencing (NGS) assay for the quantification of MRD in CLL. We demonstrate, based on measurements in contrived MRD samples, that the linear range of detection and quantification of our novel assay reaches beyond MRD 10-5. If provided with sufficient DNA input, MRD can even be detected down to MRD 10-6. There was high inter-assay concordance between measurements of the IGHV leader-based NGS assay and allele-specific oligonucleotide quantitative PCR (r=0.92, [95%CI 0.86-0.96]) and droplet digital PCR (r=0.93, [95%CI 0.88-0.96]) on contrived MRD samples. In a cohort of 37 patients from the CLL11 trial, using MRD 10-5 as a cut-off, MRD undetectability was associated with superior progression-free survival (PFS) and time to next treatment. Importantly, deeper MRD measurement allowed for additional stratification of patients with MRD <10-4 but ≥10‑5. Whereas the PFS of these patients was significantly shorter, compared to patients with MRD <10-5 (HR 4.9, [95%CI 1.3-17.9], P=0.017), their PFS was superior to those with MRD ≥10‑4 (HR 0.18, [95%CI 0.06-0.46], P<0.001). These results clearly demonstrate the clinical utility of the novel IGHV-leader based NGS assay.
Project description:CLL14 (NCT02242942) is a multinational, open-label Phase III study designed to compare venetoclax-obinutuzumab (Ven-Obi) versus chlorambucil-obinutuzumab (Clb-Obi) in previously untreated patients with CLL and co-existing conditions. Between Aug 7, 2015, and Aug 4, 2016, 432 patients were enrolled and randomly assigned to receive either venetoclax plus obinutuzumab (Ven-Obi, n=216) or chlorambucil plus obinutuzumab (Clb-Obi, n=216). In both study arms, patients received fixed-duration treatment of overall 12 cycles, each cycle 28 days long, and entered subsequent post-treatment surveillance. Peripheral blood samples were collected before the start of therapy, as well as every 3-6 months during and after therapy, including at the time of disease progression. The primary endpoint of the study was progression-free survival, secondary endpoints included overall response rate, minimal residual disease response, and overall survival. The primary read-out was conducted after a median follow-up of 28.1 months, when the primary endpoint was met with 30 primary end-point events (disease progression or death) in the Ven-Obi arm and 77 in the Clb-Obi arm (hazard ratio, 0.35; 95% confidence interval [CI], 0.23 to 0.53;P<0.001). Follow-up is ongoing until 9 years after the last patient enrollment. In an exploratory analysis, bulk RNAseq was conducted with CD19-enriched blood from patients before start of therapy (418 patients) and at first relapse (44 patients).
Project description:Diversity of the T-cell receptor (TCR) repertoire is central to adaptive immunity. The TCR is composed of α and β chains, encoded by the TRA and TRB genes, of which the variable regions determine antigen specificity. To generate novel biological insights into the complex functioning of immune cells, combined capture of variable regions and single-cell transcriptomes provides a compelling approach. Recent developments enable the enrichment of TRA and TRB variable regions from widely used technologies for 3’-biased single-cell RNA-sequencing (scRNA-seq). However, a comprehensive computational pipeline to process TCR-enriched data from 3’ scRNA-seq is not available. Here we present an analysis pipeline to process TCR variable regions enriched from 3’ scRNA-seq cDNA. The tool reports TRA and TRB nucleotide and amino acid sequences linked to cell barcodes, enabling the reconstruction of T-cell clonotypes with associated transcriptomes. We demonstrate the software using peripheral blood mononuclear cells (PBMCs) from a healthy donor and detect TCR sequences in a high proportion of single T-cells. Detection of TCR sequences is negligible in non-T-cell populations, demonstrating specificity. Finally, we show that TCR clones are larger in CD8 Memory T-cells than other T-cell types, indicating an association between T-cell clonotypes and differentiation states.
Project description:Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B-cell lymphocytosis (MBL), a CLL precursor state with a prevalence of up to 12% in aged individuals. However, the duration of MBL and the mechanisms of its evolution to CLL remain largely unknown. In this study, we sequenced the B-cell receptor immunoglobulin heavy chain (BcR IGH) gene repertoire of 124 CLL patients and 118 matched controls in blood samples taken up to 22 years prior to diagnosis. Significant skewing in the BcR IGH gene repertoire was detected in the majority of patients, even before the occurrence of lymphocytosis and irrespective of the clonotypic IGH variable gene somatic hypermutation status. Furthermore, in 14 CLL patients we identified dominant clonotypes belonging to major stereotyped subsets associated with poor prognosis up to 16 years before diagnosis. In 22 patients with longitudinal samples, the skewing of the BcR IGH gene repertoire increased significantly over time to diagnosis or remained stable at high levels. For 14/16 patients with available samples at diagnosis, the CLL clonotype was already present in the pre-diagnostic samples. Overall, our data indicate that the pre-clinical phase of CLL could be longer than previously thought, even in adverse-prognostic cases.
Project description:Chemoradiation therapy (CRT) is a treatment for muscle invasive bladder cancer (MIBC). Using a novel transcriptomic profiling panel, we validated prognostic immune biomarkers for response to CRT on 70 pre-treatment tumor samples from NRG/RTOG 0524 and 0712, two prospective trials of MIBC. Disease-free survival (DFS) and overall survival (OS) was estimated by Kaplan-Meier method and stratified by genes correlated with immune cell proportions and activation. Cox proportional hazards models were used to assess group differences. Sample clustering based on gene expression profiles driven by immune cell proportions demonstrated cluster 2 with a high percentage of immune cell content with significantly longer DFS (Hazard Ratio (HR): 0.53 (95% CI: 0.26 – 1.10), p=0.071) and OS (HR: 0.48 (95% CI: 0.24 – 0.97), p=0.040) than cluster 1 with a low percentage of immune cell content. Higher expression of T cell infiltration genes (CD8A and ICOS ) showed longer DFS (HR: 0.40 (95% CI: 0.21 – 0.75), p=0.005) and OS (HR: 0.49 (95% CI: 0.25 – 0.94), p=0.033). Higher expression of interferon gamma signaling (IDO1) also showed longer DFS (HR: 0.44 (95% CI: 0.24 – 0.88), p=0.021) and OS (HR: 0.49 (95% CI: 0.24 – 0.99), p=0.048). These findings have treatment implications that should be validated in CRT MIBC trials particularly those that integrate immunotherapy.