scholarly journals Spatial Genomic Heterogeneity in Chronic Lymphocytic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3017-3017
Author(s):  
Clare Sun ◽  
Yun-Ching Chen ◽  
Aina Zurita Martinez ◽  
Delong Liu ◽  
Daniel Rosebrock ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Research Funding ◽  
Clonal Evolution ◽  
Lymphocytic Leukemia ◽  
Checkpoint Blockade ◽  
Rna Seq ◽  
Stable Group ◽  
Single Nucleotide Variants ◽  
Activation Markers

Activation and proliferation of chronic lymphocytic leukemia (CLL) cells depend on signals from the lymph node (LN) tumor microenvironment (TME). Separately, the genetic makeup of CLL has been closely linked to disease aggressiveness and its capacity to evolve under the selective pressures of treatment. Here, we investigated the intersection between the TME and molecular events in CLL pathogenesis. Whole exome and RNA sequencing (RNA-seq) were performed on CD19+ cells of paired peripheral blood (PB) and LN samples and matched germline DNA from 14 patients with treatment-naïve CLL. RNA-seq was also done on unsorted LN samples containing tumor and non-tumor cells from the same patients. A median of 27 (range 11-69) somatic single nucleotide variants (sSNVs) and 3 (0-10) insertions and deletions (sIndels) were detected per exome. All but one patient had copy number alterations (CNAs), most commonly del 11q and del 13q. Cancer cell fractions (CCFs) of sSNVs, sIndels, and CNAs were inferred from variant allele frequencies then clustered over the two anatomic compartments for each patient. Genetic compartmentalization (ΔCCF > 0.25, false discovery rate [FDR] < 0.1) was observed in 7 patients (50%), of whom 6 demonstrated subclonal expansion in LN. To understand factors contributing to spatial heterogeneity, we compared the tumor transcriptome based on the presence (shifted group) or absence (stable group) of an expanded subclone in LN. Most differentially expressed genes between PB and LN were shared by all patients and reflected the activation of CLL cells in the LN TME as previously shown. However, cell cycle genes (e.g. E2F2, CDC25A) were more upregulated (log2FC > 0.5, FDR < 0.05) in LN of the shifted group, while lymphocyte activation markers (e.g. CD83, CD69) were more upregulated in LN of the stable group. We hypothesized the latter finding could indicate immune-mediated control of clonal outgrowth. We therefore evaluated the expression of an 18-gene T-cell associated inflammatory signature in unsorted LN samples. This signature was originally developed as a predictive biomarker for response to immune checkpoint blockade in multiple cancer types. Unsupervised hierarchical clustering of signature genes revealed an inflamed TME in the stable group relative to the shifted group. In summary, genetic compartmentalization is a common phenomenon in CLL. Clonal equilibrium is maintained by a T-cell inflamed TME. When immune surveillance is inactivated, subclones with a competitive advantage may expand in response to support signals provided by the TME. An immunotherapy-based clinical study using checkpoint blockade to restrict clonal evolution is currently in progress (NCT03204188). This research was supported by the Intramural Research Program of the NIH, NHLBI. Disclosures Getz: Pharmacyclics: Research Funding; IBM: Research Funding; MuTect, ABSOLTUE, MutSig and POLYSOLVER: Patents & Royalties: MuTect, ABSOLTUE, MutSig and POLYSOLVER. Wu:Neon Therapeutics: Other: Member, Advisory Board; Pharmacyclics: Research Funding. Wiestner:Merck: Research Funding; Pharmayclics: Research Funding; Acerta: Research Funding; Nurix: Research Funding.

scholarly journals Mechanisms of Adaptation to Ibrutinib in High Risk Chronic Lymphocytic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 585-585 ◽  
Author(s):  
Valeria Spina ◽  
Gabriela Forestieri ◽  
Antonella Zucchetto ◽  
Alessio Bruscaggin ◽  
Tamara Bittolo ◽  
...  
Keyword(s):  
High Risk ◽  
Chronic Lymphocytic Leukemia ◽  
Nuclear Localization ◽  
Peripheral Blood ◽  
Research Funding ◽  
Clonal Evolution ◽  
Lymphocytic Leukemia ◽  
Adaptation Process ◽  
Over Time ◽  
Stimulation Of

Abstract Introduction. Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a minimal residual disease (MRD) positive partial remission (PR) which is maintained until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. The study aims at characterizing the adaptation process allowing residual CLL cells to persist despite BTK inhibition. Methods. The IOSI-EMA-001 study (NCT02827617) is an observational study consisting in the prospective and longitudinal collection of peripheral blood samples and clinical data from high risk CLL patients treated with ibrutinib. Peripheral blood CLL cells longitudinally drawn from patients before treatment start and at fixed timepoints under ibrutinib were monitored by: i) next generation flow cytometry approaches for changes in proliferation rate, surfaceome, and pathway activation; and ii) CAPP-seq targeted deep next generation (sensitivity ~10-3) for clonal evolution. Results. The study cohort comprised 31 high risk CLL patients, including 15 treatment naïve, 16 relapsed, 80% IGHV unmutated, 42% 17p deleted and 55% TP53 mutated. Median duration of ibrutinib treatment was 45 weeks (24-72 weeks). All patients obtained a MRD positive PR that was maintained in all but one who progressed with a PLCG2 mutation (VAF 3%). Compared to baseline, under ibrutinib therapy CLL cells slowed down their proliferation, as suggested by the decreased expression of Ki-67, the reduction of the proliferating fraction (CXCR4dimCD5bright), and the increase of the resting fraction (CXCR4brightCD5dim). Compared to baseline, under ibrutinib therapy CLL cells also upregulated BCR and adhesion/homing proteins, and decreased the expression of BCR inhibitor proteins. Upon stimulation of the BCR with anti-IgM, the downstream path through pBTK and pPLCG2 was inhibited by ibrutinib, while conversely the downstream path through pAKT and pERK was still inducible throughout all the assessed timepoints. The proportion of CLL cells harboring nuclear localization of NF-kB progressively increased over time under ibrutinib. NF-kB nuclear localization was inducible throughout all the assessed timepoints by CD40L stimulation of the non-canonical NF-kB pathway, but not by anti-IgM stimulation of the BCR/canonical NF-kB pathway. Overall, 880 individual mutations were longitudinally discovered and monitored across a total of 121 sequential timepoints collected during ibrutinib treatment. Clonal evolution was observed in (67.7%) cases, a proportion rate previously documented in CLL treated with chemoimmunotherapy. Clonal evolution appeared to be heterogeneous involving different genes without a stereotypic targeting. Consistently, none of the main driver gene mutations was homogeneously selected or suppressed by ibrutinib suggesting that the biological adaptation of CLL cells under ibrutinib is not genetically driven. Clonal evolution propensity was not associated with any of the biomarkers of the disease, and it did not decrease over time under ibrutinib. Conclusions. Taken together these results suggest that residual CLL cells persisting under ibrutinib therapy adapt their phenotype by upregulating adhesion molecules, chemokine receptors and BCR molecules, and by maintaining a competence of BCR signaling through the PI3K/AKT/ERK pathway. The progressive selection of CLL cells having NF-kB in the nucleus, likely due to the BTK independent non-canonical NF-kB pathway, might explain their survival despite ibrutinib therapy. Finally, clonal evolution is not suppressed by ibrutinib chemotherapy, and despite does not seem to be directly involved in such adaptation process, may ultimately favor the acquisition of BTK and PLCG2 ibrutinib resistance mutations. Disclosures Zucca: Celltrion: Consultancy; AstraZeneca: Consultancy. Ghia:Sunesis: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding. Montillo:Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding. Tedeschi:Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy; AbbVie: Consultancy. Gaidano:AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Morphosys: Honoraria; Roche: Consultancy, Honoraria.

Superagonistic Anti-CD28 Antibody TGN1412 as a Potential Immunotherapeutic for the Treatment of B Cell Chronic Lymphocytic Leukemia.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2519-2519 ◽  
Author(s):  
Chia-Huey Lin ◽  
Thomas Kerkau ◽  
Christine Guntermann ◽  
Martin Trischler ◽  
Niklas Beyersdorf ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mhc Class Ii ◽  
T Cell Activation ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Activation Markers ◽  
Cell Chronic Lymphocytic Leukemia

Abstract B cell chronic lymphocytic leukemia (B-CLL) is characterised by an accumulation of malignant B cells, and impaired humoral and cellular immune responses. Evasion strategies of leukemic cells appear to involve down-regulation of co-stimulatory molecules as well as increased resistance to apoptosis. Here we provide data supporting a novel concept to treat B-CLL with a humanized, superagonistic monoclonal antibody specific for CD28 (TGN1412). Superagonistic anti-CD28 antibodies have been shown to activate human T cells in vitro without requirement for engagement of the T cell antigen receptor (Luhder et al., J. Exp. Med. 2003. 197(8):955–66). Indicative of their activation, TGN1412-triggered T cells from healthy donors upregulate, among other activation markers, CD40L, that has been reported to promote anti-leukemic effects when ectopically expressed on B-CLL cells (Wierda et al., Blood. 2000. 96 (9): 2917–2924). In this report, the responses of PBMCs from B-CLL patients to soluble TGN1412 were examined. We show that in a dose-dependent fashion, polyclonal T cell activation was induced by TGN1412 including proliferation, cytokine production and induction of activation markers such as CD25, CD71, CD134 (Ox40), CTLA-4 (CD152) and CD154 (CD40L). Significantly, modulation of malignant B-CLL cells was also observed. MHC class II molecules (HLA-DR), CD95 and the co-stimulatory molecules CD80 and CD86, but not the proliferation marker Ki-67, were strongly up-regulated upon TGN1412 stimulation. These data suggested that improved antigen-presenting functions of B-CLL cells were induced by TGN1412. Accordingly, preliminary data indicate that B-CLL cells isolated from TGN1412 stimulated cultures induced enhanced proliferation of both allogeneic and autologous T cells, and importantly, TGN1412 activated T cells exhibited enhanced CTL-activity against B-CLL cells. In conclusion, our data suggest that TGN1412 induces polyclonal T cell expansion and activation as well as increased APC function of B-CLL cells. They imply that TGN1412 may have future therapeutic benefit for B-CLL patients.

Prior Treatment with Alemtuzumab Interferes with T-Cell Engraftment After Allogeneic Stem Cell Transplantation in Patients with Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3351-3351
Author(s):  
Johannes Schetelig ◽  
Christian Thiede ◽  
Alexander Kiani ◽  
Uwe Platzbecker ◽  
Uta Oelschlaegel ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Washout Period ◽  
Research Funding ◽  
Limit Of Detection ◽  
Lymphocytic Leukemia ◽  
Cell Engraftment ◽  
Median Level ◽  
Antibody Levels ◽  
Donor Lymphocyte Infusions

Abstract Abstract 3351 Poster Board III-239 Objectives: The majority of patients with chronic lymphocytic leukemia (CLL) who receive allogeneic hematopoietic cell transplantation (HCT) have fludarabine-refractory disease. The most active single agent in this disease stage is alemtuzumab. Alemtuzumab has a long half-life and induces profound T-cell depletion (TCD). Since TCD may mitigate graft-versus leukemia effects we evaluated „pre-conditioning“ with alemtuzumab followed by a washout period in order to minimize in vivo T-cell depletion of the graft in a phase II study (NCT 00337519). Methods: Patients received cytoreductive treatment with 3 × 30 mg alemtuzumab weekly prior to HCT. The scheduled interval between last dose of alemtuzumab and HCT was increased from two weeks to one month during the study. The antibody level at the day of HCT was measured with an ELISA with a lower limit of detection of 31.25 ng/mL (BioAnaLab lim., Oxford, UK). The conditioning regimen contained fludarabine (150 mg/m2) and busulfan (8 mg/kg). Cyclosporine (CSA) and methotrexate (MTX) were applied as GVHD-prophylaxis. Medically fit patients with relapsed CLL were elible. Results: 62 patients with a median age of 57 years were included between April, 2004 and October, 2008. A median of 3 prior regimens had been given. 55% of the patients had fludarabine-resistant disease. Two patients failed to reach HCT due to progressive disease during alemtuzumab therapy. Donors were matched siblings for 26 and matched unrelated donors for 34 patients. The median level of alemtuzumab in peripheral blood after a washout period of two weeks was 62 ng/mL (interquartile range, 45 to 196 ng/mL; minimum below the limit of detection; maximum 490 ng/mL) compared to a median level below the limit of detection after a delay of four weeks (interquartile range, between the limit of detection and 77 ng/mL, maximum 256 ng/mL) (p=0.005). Despite one month time between the last dose of alemtuzumab and HCT 4 out of 30 patients (13%) had alemtuzumab levels greater than 200 ng/mL. No primary or secondary graft failure occurred. A linear relationship between the alemtuzumab level at HCT and the time to complete CD4-T-cell chimerism (TCC) was observed (p=0.003). At day +100 a CD4 positive T-cell-chimerism (TCC) >95% had been achieved by 84% of patients with alemtuzumab levels <100 ng/mL, 83% of patients with antibody levels between 100 and 200 ng/mL and 25% of patients with antibody levels >200 ng/mL (p=0.006). All patients had a complete neutrophil-chimerism at day +100. After early taper of immunosuppression (N=2) or the application of donor lymphocyte infusions in incremental doses (N=5) mixed TCC has been converted to complete TCC in all patients. The median follow-up is 17 months (1 to 61 months). Day +100 non-relapse mortality was 2%. At two years non-relapse mortality and relapse incidence were 21% and 29%, respectively. Two-year overall survival and progression-free survival were 67% (95% CI, 51% to 83%) and 50% (95% CI, 31% to 69%). Conclusions: In patients who received alemtuzumab prior to HCT, residual drug levels may interfere with T-cell engraftment. Lineage specific T-cell chimerism should therefore be assessed prospectively in this group of patients. Persistent mixed T-cell chimerism can be converted by an early taper of immunosuppression and incremental doses of donor lymphocyte infusions. Disclosures: Schetelig: Bayer Schering: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Subcutaneous Epcoritamab in Patients with Relapsed/Refractory Chronic Lymphocytic Leukemia: Preliminary Results from the Epcore CLL-1 Trial

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2627-2627
Author(s):  
Arnon P. Kater ◽  
Jacob Haaber Christensen ◽  
Hans Herluf Bentzen ◽  
Carsten Utoft Niemann ◽  
Martin Hutchings ◽  
...  
Keyword(s):  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Dose Escalation ◽  
Research Funding ◽  
Injection Site Reaction ◽  
Steering Committee ◽  
Lymphocytic Leukemia ◽  
Site Reaction ◽  
Phase 1B ◽  
Dose Levels

Abstract Background: Small molecules such as Bruton tyrosine kinase (BTK) and B-cell lymphoma 2 (BCL2) inhibitors have transformed the management of chronic lymphocytic leukemia (CLL). However, such treatments are not curative, and patients (pts) with relapsed or refractory (R/R) CLL following multiple targeted treatments present an emergent challenge with very limited therapeutic options. In addition, success rates of autologous T-cell-based therapies in CLL have been disappointing. In vitro data in CLL cells suggest potentially high efficacy of bispecific T-cell engagers (Martens et al, J Immunother Cancer 2020), but clinical data are extremely limited. Epcoritamab (GEN3013; DuoBody ®-CD3×CD20) is a bispecific antibody that can induce potent activation and cytotoxic activity of CD4+ and CD8+ T cells to specifically eliminate CD20-expressing cells (van der Horst et al, Blood Cancer J 2021). In the first-in-human trial in R/R B-cell non-Hodgkin lymphoma (B-NHL; EPCORE NHL-1; NCT03625037), epcoritamab showed manageable safety and meaningful antitumor activity across a range of aggressive and indolent B-NHLs. The most common treatment-emergent adverse events (AEs) were pyrexia (69%), cytokine release syndrome (CRS; 59%), and injection-site reaction (47%); CRS events were all grade 1-2 and most occurred in cycle 1 (Clausen et al, J Clin Oncol 2021). However, as CLL is characterized by presence of (high numbers of) circulating tumor cells, acquired T-cell dysfunction, and variable expression of CD20, data obtained in B-NHL are difficult to extrapolate to CLL. Herein we present the first results from the dose-escalation part of a phase 1b/2 trial evaluating epcoritamab in pts with R/R CLL. Methods: In this open-label, multicenter, phase 1b/2 trial, toxicity and efficacy of epcoritamab are investigated in adults with R/R CLL (EPCORE CLL-1; NCT04623541). Eligible pts previously received ≥2 lines of systemic antineoplastic therapy, including treatment with (or intolerance to) a BTK inhibitor. Epcoritamab is subcutaneously administered via 1-mL injections in 4-week cycles as follows: once weekly in cycles 1-3, every 2 weeks in cycles 4-9, and monthly in cycles ≥10 until progression or unacceptable toxicity. Step-up dosing during cycle 1 (ie, priming dose followed by an intermediate dose, then full doses) is used in combination with steroid prophylaxis to reduce the risk of CRS. In the dose-escalation part, pts with R/R CLL received epcoritamab at 2 full-dose levels (24 and 48 mg) according to a modified 3+3 design. Primary end points of the dose-escalation part included dose-limiting toxicities (DLTs) during the first 28-day treatment cycle and the incidence and severity of AEs, CRS, immune effector cell-associated neurotoxicity syndrome (ICANS), and tumor lysis syndrome (TLS). Results: The first pt was enrolled on November 30, 2020. As of July 12, 2021, 7 pts with R/R CLL received epcoritamab subcutaneously administered at 2 full-dose levels: 24 mg (n=3) and 48 mg (n=4). Six pts completed the DLT evaluation period, and 5 pts had a full response assessment. Pts had received a median of 4 lines of prior therapy (range, 2-5). Six of 7 pts had poor-risk features of del(17p), TP53 mutations, or both. Three of 7 pts had bulky disease. No DLTs occurred at 24 or 48 mg. The most common treatment-emergent AEs (&gt;30%) were CRS (100%), fatigue (71%), injection-site reaction (43%), and nausea (43%). All pts experienced CRS in the first cycle, but no CRS events were higher than grade 2. No cases of ICANS were observed. TLS was not observed. Antileukemic activity has been observed at both dose levels, with partial responses in 3 of 5 pts. Updated clinical and pharmacokinetic data, including data for additional pts treated with epcoritamab, will be presented. Conclusions: These data suggest that epcoritamab is well tolerated in pts with R/R CLL at dose levels up to 48 mg and has clinical activity in pts with high-risk features. The expansion part of this study in pts with R/R CLL and Richter syndrome will open later this year. Disclosures Kater: Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding. Niemann: CSL Behring, Genmab, Takeda, Octapharma: Consultancy; Abbvie, AstraZeneca, Janssen: Consultancy, Research Funding; Novo Nordisk Foundation: Research Funding. Hutchings: Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Incyte: Research Funding; Janssen: Honoraria, Research Funding; Genmab: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Genentech: Honoraria, Research Funding. Chen: Genmab: Current Employment. Rios: Genmab: Current Employment. Palenski: AbbVie: Current Employment. Li: Genmab: Current Employment. Mato: AbbVie: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; MSKCC: Current Employment; Genmab: Research Funding; Johnson and Johnson: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; LOXO: Consultancy, Research Funding; Nurix: Research Funding; DTRM BioPharma: Consultancy, Research Funding; TG Therapeutics: Consultancy, Other: DSMB, Research Funding; BeiGene: Consultancy, Research Funding; Acerta/AstraZeneca: Consultancy, Research Funding; AstraZeneca: Consultancy; Sunesis: Consultancy, Research Funding.

scholarly journals Role of Histone Deacetylase-Mediated Gene Silencing in Chronic Lymphocytic Leukemia Progression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2705-2705 ◽  
Author(s):  
Lara Rizzotto ◽  
Arianna Bottoni ◽  
Tzung-Huei Lai ◽  
Chaomei Liu ◽  
Pearlly S Yan ◽  
...  
Keyword(s):  
High Risk ◽  
Research Funding ◽  
Clonal Evolution ◽  
Lymphocytic Leukemia ◽  
Low Risk ◽  
Gene Promoters ◽  
Rna Seq ◽  
Hdac Inhibition ◽  
Protein Coding ◽  
Protein Coding Genes

Abstract Chronic lymphocytic leukemia (CLL) follows a variable clinical course mostly dependent upon genomic factors, with a subset of patients having low risk disease and others displaying rapid progression associated with clonal evolution. Epigenetic mechanisms such as DNA promoter hypermethylation were shown to have a role in CLL evolution where the acquisition of increasingly heterogeneous DNA methylation patters occurred in conjunction with clonal evolution of genetic aberrations and was associated with disease progression. However the role of epigenetic mechanisms regulated by the histone deacetylase group of transcriptional repressors in the progression of CLL has not been well characterized. The histone deacetylases (HDACs) 1 and 2 are recruited onto gene promoters and form a complex with the histone demethylase KDM1. Once recruited, the complex mediate the removal of acetyl groups from specific lysines on histones (H3K9 and H3K14) thus triggering the demethylation of lysine 4 (H3K4me3) and the silencing of gene expression. CLL is characterized by the dysregulation of numerous coding and non coding genes, many of which have key roles in regulating the survival or progression of CLL. For instance, our group showed that the levels of HDAC1 were elevated in high risk as compared to low risk CLL or normal lymphocytes and this over-expression was responsible for the silencing of miR-106b, mR-15, miR-16, and miR-29b which affected CLL survival by modulating the expression of key anti-apoptotic proteins Bcl-2 and Mcl-1. To characterize the HDAC-repressed gene signature in high risk CLL, we conducted chromatin immunoprecipitation (ChIP) of the nuclear lysates from 3 high risk and 3 low risk CLL patients using antibodies against HDAC1, HDAC2 and KDM1 or non-specific IgG, sequenced and aligned the eluted DNA to a reference genome and determined the binding of HDAC1, HDAC2 and KDM1 at the promoters for all protein coding and microRNA genes. Preliminary results from this ChIP-seq showed a strong recruitment of HDAC1, HDAC2 and KDM1 to the promoters of several microRNA as well as protein coding genes in high risk CLL. To further corroborate these data we performed ChIP-Seq in the same 6 CLL samples to analyze the levels of H3K4me2 and H3K4me3 around gene promoters before and after 6h exposure to the HDACi panobinostat. Our goal was to demonstrate that HDAC inhibition elicited an increase in the levels of acetylation on histones and triggered the accrual of H3K4me2 at the repressed promoter, events likely to facilitate the recruitment of RNA polymerase II to this promoter. Initial analysis confirmed a robust accumulation of H3K4me2 and H3K4me3 marks at the gene promoters of representative genes that recruited HDAC1 and its co-repressors in the previous ChIP-Seq analysis in high risk CLL patients. Finally, 5 aggressive CLL samples were treated with the HDACi abexinostat for 48h and RNA before and after treatment was subjected to RNA-seq for small and large RNA to confirm that the regions of chromatin uncoiled by HDACi treatment were actively transcribed. HDAC inhibition induced the expression of a large number of miRNA genes as well as key protein coding genes, such as miR-29b, miR-210, miR-182, miR-183, miR-95, miR-940, FOXO3, EBF1 and BCL2L11. Of note, some of the predicted or validated targets of the induced miRNAs were key facilitators in the progression of CLL, such as BTK, SYK, MCL-1, BCL-2, TCL1, and ROR1. Moreover, RNA-seq showed that the expression of these protein coding genes was reduced by 2-33 folds upon HDAC inhibition. We plan to extend the RNA-seq to 5 CLL samples with indolent disease and combine all the data to identify a common signature of protein coding and miRNA genes that recruited the HDAC1 complex, accumulated activating histone modifications upon treatment with HDACi and altered gene and miRNA expression after HDAC inhibition in high risk CLL versus low risk CLL. The signature will be than validated on a large cohort of indolent and aggressive CLL patients. Our final goal is to define a signature of coding and non coding genes silenced by HDACs in high risk CLL and its role in facilitating disease progression. Disclosures Woyach: Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

scholarly journals Lysis of malignant B cells from patients with B-chronic lymphocytic leukemia by autologous T cells activated with CD3 x CD19 bispecific antibodies in combination with bivalent CD28 antibodies

Blood ◽  
1993 ◽  
Vol 82 (6) ◽  
pp. 1803-1812 ◽  
Author(s):  
H Bohlen ◽  
T Hopff ◽  
O Manzke ◽  
A Engert ◽  
D Kube ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
T Cell Activation ◽  
Cell Activation ◽  
Lymphocytic Leukemia ◽  
Bispecific Antibodies ◽  
Autologous Tumor ◽  
Activation Markers

Abstract Bispecific antibodies (bi-MABs) can be used to target T cells to autologous tumor cells. It has been shown that the activation of resting human T cells requires two independent signals, namely the cross-linking of the T-cell receptor (TCR)-CD3 complex together with the CD28 homodimer. In the present study, we demonstrate the activation of T cells from patients with chronic lymphocytic leukemia (CLL) using bi-MABs against the CD3 and CD19 antigens (CD3 x CD19) in combination with monospecific, bivalent antibodies against the CD28 antigen. Mononuclear cells from patients with CLL were cultured with the bi-MAB CD3 x CD19 and monospecific CD28 antibodies. The CD3 x CD19 bi-MABs were isolated by the hybridoma-hybridoma fusion technique and purified by hydrophobic interaction chromatography. T-Cell activation as demonstrated by increased proliferation, upregulation of T-cell activation markers (CD25, CD38), and cytotoxicity against autologous CLL cells and allogeneic B cells was shown in seven of eight CLL specimens. The stimulation with CD3 x CD19 bi-MABs with CD28 antibodies preferentially induced proliferation of CD4+ T cells. The effective dose of purified antibodies required for optimal T-cell activation was 100 ng/mL in vitro, which suggests that this antibody combination may be useful for immunotherapy of patients with B-CLL.

Clonal Evolution In Patients With Previously Untreated Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1643-1643
Author(s):  
Sameer A. Parikh ◽  
Kari G. Rabe ◽  
Stephanie A. Smoley ◽  
Anne E. Wiktor ◽  
Curtis A. Hanson ◽  
...  
Keyword(s):  
Overall Survival ◽  
Chronic Lymphocytic Leukemia ◽  
Mayo Clinic ◽  
Research Funding ◽  
Clonal Evolution ◽  
Clinical Importance ◽  
Lymphocytic Leukemia ◽  
Risk Category ◽  
Time Interval ◽  
Trisomy 12

Abstract Background Although the clinical importance of del 17p13 in patients with chronic lymphocytic leukemia (CLL) is well recognized, the implications of when this defect is identified are less clearly defined. In particular, the distinction between the identification of del 17p13 at the time of diagnosis and secondary acquisition of del 17p13 during the course of the disease are poorly characterized. Methods We identified all patients with CLL cared for at the Mayo Clinic between 1/1/2000 and 12/31/2012 who underwent baseline fluorescence in-situ hybridization (FISH) testing prior to receiving treatment. The Results of repeat FISH testing were reviewed to identify cases with clinically ascertained clonal evolution. Results A total of 1757 patients with newly diagnosed CLL were seen at Mayo Clinic prior to receiving treatment. Among these, 1243 had FISH testing performed prior to treatment and within 36 months of diagnosis. The median time from diagnosis to initial FISH was 0.8 months (11.5 to 35.4 months). The Results of baseline FISH testing among these patients is as follows: 486 (39%) had del 13q14, 234 (19%) had trisomy 12, 115 (9%) had del 11q23, 59 (5%) had del 17p13, 15 (1%) had other (e.g., del6q) and 334 (37%) had no abnormalities detected. Among these patients, 344 (28%) underwent repeat FISH testing during the course of their disease. Repeat FISH testing was typically performed for clinical suspicion of karyotype evolution or prior to therapy selection in patients with a long time interval between first prognostic FISH and initiation of treatment. Among these 344 patients, 41 (12%) acquired new cytogenetic abnormalities on repeat FISH. Classification of these 344 patients by the Dohner classification at diagnosis and time of follow-up FISH is shown in Table 1. Among the 41 pts who acquired a new FISH detectable genetic abnormality, the newly acquired defect resulted in a change in Dohner FISH risk category for 39 patients including 15/41 (37%) who acquired a del 17p13. Baseline clinical and prognostic characteristics of patients who developed clonal evolution to those who did not are shown in Table 2. Patients with unmutated immunoglobulin heavy chain (IGHV) gene mutation status (p<0.0001) and CD49d (p=0.003) expression were more likely to experience clonal evolution. Among the 319 patients who did not have del 17p13 on baseline FISH, 15 (5%) acquired del 17p13 during the course of their disease. The overall survival from the date del 17p13 was identified is shown for those with del 17p13 at diagnosis (n=59) compared to those who acquired del 17p13 later in the course of the disease (n=15) in Figure 1A. Similarly, among 339 patients who did not have del 6q23 on baseline FISH, 9 (3%) acquired del 6q23 during the course of their disease. The overall survival from the date del 6q23 was identified is shown for those with del 6q23 at diagnosis (n=11) and those who acquired del 6q23 later in the course of the disease (n=9) in Figure 1B. Conclusion Acquired cytogenetic evolution is clinically ascertained by FISH during the course of the disease in approximately 12% of patients. The newly acquired defects in these patients result in a change in Dohner classification for in >90% of these patients including ∼37% who acquire del 17p13. The clinical implications of del 17p13 is influenced by the timing of ascertainment with markedly shorter survival for those who acquire del 17p13 during the course of the disease relative to those with this defect at diagnosis. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.

scholarly journals Interleukin-10 suppression enhances T-cell antitumor immunity and responses to checkpoint blockade in chronic lymphocytic leukemia

2020 ◽  
Author(s):  
J.R. Rivas ◽  
S.S. Alhakeem ◽  
Y. Liu ◽  
J.M. Eckenrode ◽  
F. Marti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Function ◽  
Cell Activity ◽  
Interleukin 10 ◽  
Interferon Γ ◽  
Immune Checkpoint Blockade ◽  
Lymphocytic Leukemia ◽  
Checkpoint Blockade

AbstractT-cell dysfunction is a hallmark of B-cell Chronic Lymphocytic Leukemia (CLL). CLL cells downregulate T-cell responses by expressing regulatory molecules including programmed death ligand-1 (PD-L1) and Interleukin-10 (IL-10). Immune checkpoint blockade (ICB) aims to restore T-cell function by preventing the ligation of inhibitory receptors like PD-1, however most CLL patients do not respond well to this therapy. Thus, we investigated whether IL-10 suppression could enhance antitumor T-cell activity and improve responses to ICB. Since CLL IL-10 expression depends on Sp1, we utilized a novel, better tolerated mithramycin analogue, MTMox32E, to suppress CLL IL-10. We found MTMox32E inhibited mouse and human CLL IL-10 production and maintained T-cell effector function. In the Eμ-Tcl1 mouse model, treatment reduced plasma IL-10 and CLL burden while it increased CD8+ T-cell proliferation, effector and memory cell prevalence, and interferon-γ production. When combined with ICB, suppression of IL-10 improved responses to anti-PD-L1 as shown by a 4.5-fold decrease in CLL cell burden compared with anti-PD-L1 alone. Combination therapy also produced more interferon-γ+, cytotoxic effector KLRG1+, and memory CD8+ T-cells, with fewer exhausted T-cells than ICB alone. Since current therapies for CLL do not target IL-10, this provides a novel strategy to increase the efficacy of T-cell-based immunotherapies.

scholarly journals Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib Show Expansion of T-Cell Clonotypes Composed of Antitumor Cytotoxic CD8+ T-Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3030-3030
Author(s):  
Maria Joao Baptista ◽  
Sivasubramanian Baskar ◽  
Keyvan Keyvanfar ◽  
Erika M Gaglione ◽  
Inhye E Ahn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Chronic Lymphocytic Leukemia ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Tumor Antigens ◽  
Ex Vivo ◽  
Cd8 T Cell ◽  
Fold Change ◽  
Lymphocytic Leukemia

We recently showed that ibrutinib induced a more oligoclonal T-cell receptor (TCR) repertoire in patients with chronic lymphocytic leukemia (CLL). Using next generation sequencing (NGS), we found that overrepresented TCRβ clonotypes were mostly patient-specific and that the degree of oligoclonal expansion correlated with CD8+ T-cell counts. Here, we tested the hypothesis that overrepresented clonotypes are CD8+ T-cells that respond to tumor antigens. Flow cytometric profiling of TCR Vβ (TRBV) chain usage, T-cell subset distribution, and granzyme B (GrB) expression was performed on cryopreserved peripheral blood mononuclear cells obtained at baseline and during response to single-agent ibrutinib (n = 5). Antibodies against TRBV were selected to investigate the ten most abundant TCRβ clonotypes identified by NGS. The cumulative frequency of these clonotypes evaluable by flow cytometry increased at the time of response (mean 31.2% ± SEM 3.9% of all T-cells) compared to baseline (mean 24.1% ± SEM 2.3% of all T-cells). Within each TRBV subpopulation, the proportion of CD4+ T-cells decreased (mean fold-change 0.78 ± SEM 0.04), while the proportion of CD8+ T-cells increased (mean fold-change 1.79 ± SEM 0.36). We further observed that the TRBV clonotypes expanding the most during treatment were comprised exclusively of CD8+ T-cells in every patient. These highly expanded CD8+ clonotypes expressed GrB, consistent with a cytotoxic phenotype. To evaluate whether expanding CD8+ clonotypes are tumor-specific, T-cells were expanded ex vivo in two conditions: (1) co-culture with autologous CLL cells, CD40L, IL-2, and IL-7 or (2) with anti-CD3/CD28/CD137 beads, IL-2, and IL-7. Overall, T-cells expanded with autologous CLL cells had a higher proportion of CD8+ GrB+ cells than T-cells expanded with anti-CD3/CD28/CD137 beads. The dominant CD8+ clonotypes detected in each patient at the time of response were preferentially expanded by co-culture with autologous CLL cells compared to expansion with beads. The biased expansion of cytotoxic T-cell clonotypes in the CLL-primed T-cell product supports a TCR mediated response to tumor antigens. Next, cytotoxicity assays were performed by mixing the expanded T-cell products described above with autologous CLL cells at an effector to target ratio of 3:1, 1:1 and 1:3. CLL-primed T-cells killed CLL cells in a dose-dependent fashion. In contrast, bead-expanded T-cells were ineffective at killing CLL cells or even protected tumor cells from spontaneous apoptosis. Thus, tumor-specific cytotoxic T-cells expand during treatment with ibrutinib and are further enriched ex vivo by co-culture with autologous CLL cells. In summary, increased TCR oligoclonality in patients responding to ibrutinib is driven by the expansion of specific CD8+ clonotypes. These clonotypes expand in co-culture with, and are cytotoxic against, autologous CLL cells. Tumor-specific CD8+ T-cell clonotypes may contribute to the overall treatment response with ibrutinib and supports the investigation of therapeutic strategies combining ibrutinib with T-cell directed immunotherapy. Disclosures Baskar: NIH: Patents & Royalties: ROR1 mAb 2A2. Wiestner:Nurix: Research Funding; Acerta: Research Funding; Merck: Research Funding; Pharmayclics: Research Funding.

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