Maturity State and MCL-1 Dependence Predetermines Response to NOTCH1 Inhibition in T-ALL

Abstract Introduction: Acute T cell lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy in children and young adults that frequently becomes treatment-refractory and relapses. The Notch1 pathway is a key oncogenic driver in T-ALL and is aberrantly activated in more than 50% of the cases. Despite promising pre-clinical data using gamma secretase inhibitors such as DBZ to target NOTCH1, resistance is rapidly occurring in vivo. As molecular heterogeneity has been linked to treatment escape, we focused our study on defining transcriptional cell states driving resistance to NOTCH inhibition and understanding their relation to mitochondrial priming. Methods: 5 primary T-ALLs harboring NOTCH activating mutations were engrafted in NSG (NOD-scidIL2Rgnull) mice. Upon reaching ~ 10% of human CD45+ positive leukemic blasts in the peripheral blood, randomized groups of 8 mice per primary T-ALL were treated with DBZ (Dipenzazepine; 10 μM/kg every other day through tail vein) or vehicle (VEH). 3 mice per group were sacrificed after one week of treatment to assess short-term effect of DBZ, while the remaining 5 mice were weekly monitored for disease progression, leukemic blasts were collected from lymphoid organs and overall survival was determined. Full-length transcriptome analysis of 3188 blasts present in the blood of 20 sensitive and 22 refractory mice was performed by Smart-Seq2. Based on scRNA features, 'scVelo' and 'CytoTRACE' were used to identify developmental potential and differentiation trajectories. Cell fate and transcriptional regulatory networks were defined and reconstructed using 'SCENIC'. Assessment of mitochondrial priming as measured by BH3 profiling was used to identify anti-apoptotic vulnerabilities present in these PDX models. Results: Upon DBZ, short or long-term disease control was observed in two strains, while rapid resistance occurred in three strains, thus establishing two sensitive and three refractories to NOTCH inhibition PDX models. Immunohistochemical analysis showed decreased expression of active NOTCH1 in spleen biopsies of all strains, validating the efficacy of DBZ and suggesting a mechanism of resistance independent of ICN1. Single cell transcriptional profiling showed enrichment of immature hematopoietic signatures and co-expression of lymphoid and myeloid progenitor programs in refractory models. Interestingly, pre-existing cells harboring refractory-like transcriptional circuits within the untreated sensitive population were identified. Upon treatment, despite increased differentiation in all models, lineage promiscuity was maintained in refractory strains, suggesting that cellular plasticity mediates treatment escape. Next, we characterized cell states driving treatment refraction. RNA velocity projections identified two distinct immature states differing in cell cycle and oncogenic signaling. Clustering of untreated, sensitive leukemic cells in immature state imply that aberrant lineage commitment can predict response to NOTCH inhibition in vivo. These observations were further confirmed by differentiation state analysis, where prior to treatment, high developmental potential was correlated to treatment escape. Surprisingly, in addition to early lineage differentiation drivers such as BCL11A, state-specific regulons analysis associated immature states with BCLAF1 a transcriptional regulator of apoptosis. We postulated that these transcriptional circuits lead to differential apoptotic priming, therefore the dependence on individual anti-apoptotic proteins was evaluated. Mitochondrial priming at baseline revealed BCL-2 dependence in sensitive strains whereas MCL1-dependence was observed in refractory ones. Upon DBZ treatment, while dependency profiles in refractory strains remained unchanged, a functional switch from BCL-2 to MCL1-dependency occurred in sensitive models. Conclusion: Our results suggest that response to NOTCH inhibition is predetermined by cell maturity states and their associated transcriptional circuits responsible for differential sensitivity to apoptotic priming via BCL2 and MCL1. These data suggest that combining BH3 and lineage commitment profiling may predict drug responses in vivo. Moreover, our findings highlight the importance of targeting co-existing cell states to overcome transcriptional heterogeneity as a driver of treatment escape. Disclosures Letai: Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board; Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board. Weinstock: Daiichi Sankyo: Consultancy, Research Funding; Verastem: Research Funding; Abcuro: Research Funding; Bantam: Consultancy; ASELL: Consultancy; SecuraBio: Consultancy; AstraZeneca: Consultancy; Travera: Other: Founder/Equity; Ajax: Other: Founder/Equity.

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Dissecting Richter's Syndrome in a Multiplexed CRISPR-Based Mouse Model Reveals Key Changes in MYC, Interferon and BCR Signaling Underlying Transformation

Blood ◽

10.1182/blood-2021-148614 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 251-251

Author(s):

Elisa Ten Hacken ◽

Shanye Yin ◽

Tomasz Sewastianik ◽

Livius Penter ◽

Neil Ruthen ◽

...

Keyword(s):

Mouse Model ◽

Interferon Gamma ◽

Research Funding ◽

Advisory Board ◽

Scientific Advisory Board ◽

Bcr Signaling ◽

Scientific Advisory ◽

Equity Holding ◽

Richter’S Syndrome

Abstract Richter's syndrome (RS) represents one of the foremost challenges in CLL management, and its pathogenesis remains largely undefined. We recently leveraged CRISPR-Cas9 in vivo gene editing to develop mouse models of RS by engineering multiplexed loss-of-function lesions typical of CLL (Atm, Trp53, Chd2, Birc3, Mga, Samhd1) in early stem and progenitor cells [Lineage - Sca-1 + c-kit + (LSK)] from MDR-Cd19Cas9 donor mice. These animals express Cas9-GFP in a B-cell restricted fashion and the leukemogenic MDR lesion, which mimics del(13q) when the sgRNA-transduced LSK cells are transplanted in CD45.1 immunocompetent recipients. Through these methods, we observed not only development of CLL, but also transformation into RS, and even captured a stage where CLL and RS were co-existing in the same animal (CLL/RS). We hypothesized that the molecular events underlying RS development would be markedly distinct from those of CLL and performed transcriptome analysis of FACS-sorted CLL and/or RS cells (5 CLL, 4 CLL/RS, 10 RS) and normal B cell controls from 4 age-matched wild type MDR-Cd19Cas9 mice. We identified a unique transcriptional profile of RS (ANOVA, FDR<0.1), characterized by upregulation of pathways involved in cell survival and proliferation (E2F/MYC targets, G2-M checkpoint, mitotic spindle). In contrast, genes involved in interferon gamma response, JAK-STAT and BCR signaling were predominantly downregulated. We asked whether these oncogenic circuitries would be recapitulated in human RS. By correlating the differentially expressed genes in murine RS with those of 7 human RS cases (compared to matched CLL), we identified similar pathway dysregulations with >100 commonly altered genes including upregulated cell cycle regulators (CDK1, CCNA2) and downregulated signaling adapters (ITPKB, MAP3K9). To further dissect gene regulatory networks driving transformation in the mouse, we profiled one CLL and one RS case by single cell ATAC sequencing (scATAC-seq). Consistent with the RNA-seq profiles, we detected increased chromatin accessibility of MYC-family associated transcription factor motifs (MAX, MYCN), and reduced accessibility of the pro-inflammatory STAT2 motif in RS (-log10adjP>50). Functionally, decreased interferon gamma responses were confirmed by the reduced ability of RS cells to phosphorylate STAT1 and STAT3 at 5' and 15' after IFN-gamma stimulation, compared to CLL and normal B cells (Western Blot). To define the genetic landscape underlying these changes, we performed whole genome sequencing analysis, and identified loss of chr12 and chr16 as recurrent events in RS (6/8 cases) and CLL/RS (2/2), but not in CLL cases (0/5). Among the genes encoded by these chromosomes, we identified several epigenetic drivers (Dnmt3a, Crebbp, Setd3/4), MAP kinase family members (Map4k5, Mapk1), cytoskeletal regulators (Hcls1, Rhoj), and interferon family receptors (Ifnar1/2, Ifngr2), suggesting that broad epigenetic modifications together with loss of BCR and interferon signaling molecules represent key events of transforming disease. RS cases were also characterized by a significantly higher number of full chromosome amplifications or deletions (median=6; range: 2-9), as compared to CLL or CLL/RS (1; 0-5, P=0.0008), consistent with the high degree of genomic instability observed in human disease. Finally, we asked whether the observed changes would impact RS therapeutic vulnerabilities, and exposed 15 primary murine RS splenocyte samples to 20 drugs in vitro for 24 hours, followed by CellTiter-Glo assessment of cellular viability. We observed strong sensitivity to the BRD4 inhibitor JQ1 and the mTOR inhibitor everolimus (both reported to interfere with MYC signaling, P<0.0001), and to CDK inhibitors (e.g. the CDK4/6 inhibitor palbociclib, P=0.0007), modest activity of the JAK1/2 inhibitor ruxolitinib (P=0.05), and minimal, if any, response to ibrutinib, venetoclax and fludarabine. In conclusion, we define the evolutionary trajectories and therapeutic vulnerabilities of RS in a mouse model, with unique transcriptional, genetic, and epigenetic features, indicative of broad changes in MYC, IFN and BCR signaling pathways and remarkable similarities with human disease. In-depth analyses of BCR signaling and in vivo treatment studies are underway and will refine mechanistic insights into the biology of RS. Disclosures Davids: Surface Oncology: Research Funding; Eli Lilly and Company: Consultancy; Genentech: Consultancy, Research Funding; Takeda: Consultancy; MEI Pharma: Consultancy; Janssen: Consultancy; Verastem: Consultancy, Research Funding; Ascentage Pharma: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding; Merck: Consultancy; Adaptive Biotechnologies: Consultancy; Research to Practice: Consultancy; AbbVie: Consultancy; MEI Pharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; BeiGene: Consultancy. Letai: Dialectic Therapeutics: Other: equity holding member of the scientific advisory board; Flash Therapeutics: Other: equity holding member of the scientific advisory board; Zentalis Pharmaceuticals: Other: equity holding member of the scientific advisory board. Neuberg: Madrigal Pharmaceuticals: Other: Stock ownership; Pharmacyclics: Research Funding. Wu: Pharmacyclics: Research Funding; BioNTech: Current equity holder in publicly-traded company.

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The VLA-4 Integrin Is Constitutively Activated in a Fraction of CD49d-Expressing Chronic Lymphocytic Leukemia Via Autonomous BCR-Mediated Signaling

Blood ◽

10.1182/blood-2019-124313 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 849-849

Author(s):

Erika Tissino ◽

Tamara Bittolo ◽

Dania Benedetti ◽

Riccardo Bomben ◽

Andrea Härzschel ◽

...

Keyword(s):

Research Funding ◽

Gene Families ◽

Advisory Board ◽

Lymphocytic Leukemia ◽

Scientific Advisory Board ◽

Ighv Gene ◽

Bcr Signaling ◽

Scientific Advisory ◽

Inside Out

Introduction. In chronic lymphocytic leukemia (CLL), CD49d, the alpha chain of the heterodimer CD49d/CD29 (VLA-4), is a strong negative prognosticator, and a key player of CLL microenvironmental interactions. The adhesive properties of VLA-4 can be rapidly inside-out activated by signals through the B-cell receptor (BCR), thus favoring the capability of the integrin to interact with its ligands. Especially, a continuous BCR signaling, which is either induced by canonical autoantigen-dependent mechanism, or by an autonomous manner, may augment VLA-4 activation. Aim. To investigate the constitutive VLA-4 activation state in CLL cells and to connect this feature with the presence of signals from the BCR. Methods. Constitutive VLA-4 activation was determined in flow cytometry by combining expression of CD49d and activated CD29, the latter using the conformation-sensitive anti-CD29 mAb HUTS21 (Tissino et al, J Exp Med, 2018) in whole blood (WB), as source of VLA-4 ligands, from: i) 1,044 consecutive CLL all with IGHV gene mutations available; ii) sequential samples (0, 14, 30, 90 days) from CLL patients treated in vivo with ibrutinib (IB) in real-world (n=15) and from a clinical trial (NCT02827617, n=15). HUTS21 staining was also performed in the presence of: i) plasma depletion/replacement; ii) soluble (s) VCAM-1; iii) fibronectin (FN); iv) blocking anti-CD49d (HP1/2) mAbs. ELISA assays were used to quantify sVCAM-1 in plasma samples (n=122). Antigene driven BCR signaling and autonomous BCR signaling were investigated by Ca++ influx assay in a 4-hydroxy tamoxifen (4-OHT) inducible manner in murine TKO cells system as described before (Dühren-von Minden M et al, Nature, 2012). VLA-4 activation was assessed by "real-time" flow cytometry measuring the binding of the VLA-4 ligand LDV-FITC and its replacement by unlabeled LDV. The calculated Koff values indicate the VLA-4 affinity state (Koff <0.02 s−1 high affinity; Koff >0.06 s−1 low affinity). BCR engagement was performed using goat F(ab′)2 anti-human IgM. Results. 1) A fraction of CD49d+ CLL shows constitutive VLA-4 activation - Out of 1,044 CLL, 534 (51%) expressed CD49d (cutoff 30%), and HUTS21 (cutoff 20%) was scored positive in 112/534 (21%) cases. HUTS21 staining was: i) impaired by depletion of plasma from WB samples (Fig.A), and reconstituted by specific plasma components (sVCAM1, FN; Fig.B); ii) impaired by pre-incubation with anti-CD49d HP2/1 blocking mAbs before addition of plasma, sVCAM-1 and FN (Fig.C). 2) Plasma levels of sVCAM-1 are dependent on the VLA-4 activation state - sVCAM-1 (n=122 plasma samples) was higher in CD49d+ vs CD49d- cases (p<0.0001); among CD49d+ cases, sVCAM-1 was lower in HUTS21+ cases (p=0.0096), suggesting ligand sequestration by activated surface VLA-4 (Fig.D). 3) Constitutive VLA-4 activation associates to specific BCR - By comparing cases expressing high vs low levels of constitutively activated VLA-4 (HUTS21>50% vs HUTS21<5%), a skewing in IGHV gene family usage was detected (Fig.E). In particular, cases bearing the IGHV gene families 4-34, 3-21, 4-39, 3-7 frequently associated with a high constitutive VLA-4 activation state (12/26 cases, black arrows), while cases bearing the IGHV gene families 1-2, 1-3, 1-18, 1-8 associated with a very low constitutive VLA-4 activation state (21/134 cases, grey arrows). 4) Constitutive VLA-4 activation is hampered by IB exposure in-vivo - A decrease in constitutive VLA-4 activation was observed in CLL cells collected at pre-treatment and at day 14, 30 and 90 from patients undergoing IB treatment (Fig.F), suggesting an IB-dependent impairment of VLA-4 activation via BCR signal. 5) VLA-4 is activated via BCR autonomous signaling - Murine TKO cells express high level of cell surface VLA-4 (Fig.G). TKO cells expressing different BCRs derived from CLL cases (e.g. BCR17, a CLL- subset#2 case) show autonomous Ca++ influx as well as VLA-4 activation as compared to TKO cells expressing BCRs derived from healthy donor (BCR53, mature recirculating B cells). Notably, both BCR17 and BCR53, upon anti-IgM stimulation, induced high VLA-4 affinity state, while only BCR17 induced a significant increase of VLA-4 affinity in the absence of anti-IgM stimulation (Fig.H). Conclusion. The presence of a constitutively activated form of VLA-4 is observed in a significant fraction of CD49d+ CLL, due to a continuous VLA-4 inside-out stimulation derived from an autonomous BCR signaling. Figure Disclosures Di Raimondo: Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board.

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Secreted Factors Determine Resistance to Idelalisib in Marginal Zone Lymphoma Models of Resistance

Blood ◽

10.1182/blood-2019-124299 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2569-2569

Author(s):

Alberto J Arribas ◽

Sara Napoli ◽

Eugenio Gaudio ◽

Luciano Cascione ◽

Alessandra Di Veroli ◽

...

Keyword(s):

B Cell ◽

Cell Lines ◽

Research Funding ◽

Advisory Board ◽

Institutional Research ◽

Rna Seq ◽

Scientific Advisory Board ◽

Scientific Advisory ◽

Erbb Signaling ◽

Travel Grant

Background . PI3Kδ is expressed in B-cells and has a central role in the B-cell receptor signaling in B-cell derived malignancies. Idelalisib was the first-in-class PI3Kδ inhibitors and several second-generation compounds are undergoing clinical investigation as single agents and in combinations. To identify modalities to overcome the resistance that develops to this class of agents, we have developed two idelalisib-resistant models derived from splenic marginal zone lymphoma (SMZL) cell lines. Materials and Methods. Cells were kept under idelalisib (IC90) until acquisition of resistance (RES) or with no drug (parental, PAR). Stable resistance was confirmed by MTT assay after 2-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out. Cells underwent transcriptome and miRNA profiling by RNA-Seq, whole exome sequencing (WES), lipidomics profiling, pharmacological screening (348 compounds), and FACS analysis. Cytokines and growth factor secretion was performed by ELISA. Results. Two RES models were obtained from VL51 and Karpas1718 with 7-10 fold times higher IC50s than PAR counterparts. In both models, conditioned media from RES cells transferred the resistance in the PAR cells. While WES did not identify somatic mutations associated with resistance, RNA-Seq and lipidomics analyses showed that the two cell lines had developed resistance activating different modalities. The VL51 RES model showed an enrichment in BCR-TLR-NFkB (TLR4, CD19, SYK), IL6-STAT3 (IL6, CD44), chemokines (CXCL10, CXCR4, CXCR3) and PDGFR (PDGFRA, PRKCE) signatures, paired with increased p-AKT and p-BTK levels, decreased cardiolipins and sphingomyelins levels, and increased levels of specific triacylglycerols and glycerophosphocholines. In particular, there was an over-expression of surface expression of PDGFRA and secretion of IL6 in the medium. Silencing of both IL6and PDGFRA by siRNAs reverted the resistance, while the silencing of the individual genes had only a partial effect. These data were paired with the acquired sensitivity to the PDGFR inhibitor masitinib, identified in the pharmacologic screening. In the Karpas1718 model, we observed an increased p-AKT activity with an enrichment for B-cell activation signatures (RAG1, RAG2, TCL1A), proliferation (E2F2, MKI67), ERBB signaling (HBEGF, NRG2, ERRB4), increased levels of some triacylglycerols and repressed levels for specific glycerophosphocholines. HBEGF secretion was confirmed by ELISA. The addition of recombinant HBEGF to the medium induced resistance in the PAR cells. Combination with the pan ERBB inhibitor lapatinib was beneficial in the K1718 RES. Recombinant HBEGF also induced resistance to the BTK inhibitor ibrutinib in the PAR cells and in the mantle cell lymphoma SP-53 cell line. Specific members of the let-7 family of miRNAs were repressed in the RES lines derived from both cell lines, indicating the involvement of miRNA deregulation in the mechanism of resistance. Indeed, let-7 members are known to directly target IL6-STAT3 and cytokine signaling cascade, as well PI3K-AKT network. In solid tumors, let-7 members are also expressed at low levels in tumors with constitutive active ERBB signaling, in accordance with the activation of ERBB pathway and p-AKT we observed in our Karpas1718model. Experiments with a LIN28B inhibitor are now on-going. Finally, we validated the findings across a panel of 34 B-cell lymphoma cell lines, in which IL6, PDGFRA, HBEGF and LIN28 expression levels were negatively correlated with idelalisib sensitivity, while the latter was positively correlated with let-7 levels (P <0.05). Conclusions. We developed two distinct models derived from MZL of secondary resistance to the PI3Kδ inhibitor idelalisib. We identified treatments that might overcome resistance to idelalisib and are worth of further investigations. The two models, driven by different biologic processes, will allow the evaluation of further alternative therapeutic approaches. Disclosures Stathis: PharmaMar: Other: Renumeration; ADC Therapeutics: Other: Institutional research funding; Abbvie: Other: Renumeration; Bayer: Other: Institutional research funding; Novartis: Other: Institutional research funding; MEI-Pharma: Other: Institutional research funding; Roche: Other: Institutional research funding; Pfizer: Other: Institutional research funding; Merck: Other: Institutional research funding. Stuessi:Gilead: Speakers Bureau. Zucca:Gilead: Honoraria, Other: travel grant. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Bertoni:Nordic Nanovector ASA: Research Funding; Acerta: Research Funding; Jazz Pharmaceuticals: Other: travel grants; ADC Therapeutics: Research Funding; Bayer AG: Research Funding; Cellestia: Research Funding; CTI Life Sciences: Research Funding; EMD Serono: Research Funding; Helsinn: Consultancy, Research Funding; ImmunoGen: Research Funding; Menarini Ricerche: Consultancy, Research Funding; NEOMED Therapeutics 1: Research Funding; Oncology Therapeutic Development: Research Funding; PIQUR Therapeutics AG: Other: travel grant, Research Funding; HTG: Other: Expert Statements ; Amgen: Other: travel grants; Astra Zeneca: Other: travel grants.

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Conventional Immunochemotherapy (R-CHOEP) Vs High-Dose Immunochemotherapy (R-MegaCHOEP) in Younger Patients with High-Risk Aggressive B-Cell Lymphoma: 10-Year Long-Term Follow-up of a German Lymphoma Alliance (GLA) Study

Blood ◽

10.1182/blood-2019-123637 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1589-1589

Author(s):

Fabian Frontzek ◽

Marita Ziepert ◽

Maike Nickelsen ◽

Bettina Altmann ◽

Bertram Glass ◽

...

Keyword(s):

High Risk ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

High Dose ◽

Advisory Committees ◽

Scientific Advisory Board ◽

Scientific Advisory

Introduction: The R-MegaCHOEP trial showed that dose-escalation of conventional chemotherapy necessitating autologous stem cell transplantation (ASCT) does not confer a survival benefit for younger patients (pts) with high-risk aggressive B-cell lymphoma in the Rituximab era (Schmitz et al., Lancet Oncology 2012; 13, 1250-1259). To describe efficacy and toxicity over time and document the long-term risks of relapse and secondary malignancy we present the 10-year follow-up of this study. Methods: In the randomized, prospective phase 3 trial R-MegaCHOEP younger pts aged 18-60 years with newly diagnosed, high-risk (aaIPI 2-3) aggressive B-cell lymphoma were assigned to 8 cycles of CHOEP (cyclophosphamide, doxorubcine, vincristine, etoposide, prednisone) or 4 cycles of dose-escalated high-dose therapy (HDT) necessitating repetitive ASCT both combined with Rituximab. Both arms were stratified according to aaIPI, bulky disease, and center. Primary endpoint was event-free survival (EFS). All analyses were calculated for the intention-to-treat population. This follow-up report includes molecular data based on immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) for MYC (IHC: 31/92 positive [40-100%], FISH: 14/103 positive), BCL2 (IHC: 65/89 positive [50-100%], FISH: 23/111 positive) and BCL6 (IHC: 52/86 positive [30-100%], FISH: 34/110 positive) and data on cell of origin (COO) classification according to the Lymph2CX assay (GCB: 53/88; ABC: 24/88; unclassified: 11/88). Results: 130 pts had been assigned to R-CHOEP and 132 to R-MegaCHOEP. DLBCL was the most common lymphoma subtype (~80%). 73% of pts scored an aaIPI of 2 and 27% an aaIPI of 3. 60% of pts had an initial lymphoma bulk and in 40% more than 1 extranodal site was involved. After a median observation time of 111 months, EFS at 10 years was 57% (95% CI 47-67%) in the R-CHOEP vs. 51% in the R-MegaCHOEP arm (42-61%) (hazard ratio 1.3, 95% CI 0.9-1.8, p=0.228), overall survival (OS) after 10 years was 72% (63-81%) vs. 66% (57-76%) respectively (p=0.249). With regard to molecular characterization, we were unable to detect a significant benefit for HDT/ASCT in any subgroup analyzed. In total, 16% of pts (30 pts) relapsed after having achieved a complete remission (CR). 23% of all relapses (7 pts) showed an indolent histology (follicular lymphoma grade 1-3a) and 6 of these pts survived long-term. In contrast, of 23 pts (77%) relapsing with aggressive DLBCL or unknown histology 18 pts died due to lymphoma or related therapy. The majority of relapses occurred during the first 3 years after randomization (median time: 22 months) while after 5 years we detected relapses only in 5 pts (3% of all 190 pts prior CR). 11% of pts were initially progressive (28 pts) among whom 71% (20 pts) died rapidly due to lymphoma. Interestingly, the remaining 29% (8 pts) showed a long-term survival after salvage therapy (+/- ASCT); only 1 pt received allogeneic transplantation. The frequency of secondary malignancies was very similar in both treatment arms (9% vs. 8%) despite the very high dose of etoposide (total 4g/m2)in the R-MegaCHOEP arm. We observed 2 cases of AML and 1 case of MDS per arm. In total 70 pts (28%) have died: 30 pts due to lymphoma (12%), 22 pts therapy-related (11 pts due to salvage therapy) (9%), 8 pts of secondary neoplasia (3%), 5 pts due to concomitant disease (2%) and 5 pts for unknown reasons. Conclusions: This 10-year long-term follow-up of the R-MegaCHOEP trial confirms the very encouraging outcome of young high-risk pts following conventional chemotherapy with R-CHOEP. High-dose therapy did not improve outcome in any subgroup analysis including molecular high-risk groups. Relapse rate was generally low. Pts with aggressive relapse showed a very poor long-term outcome while pts with indolent histology at relapse survived long-term. Secondary malignancies occurred; however, they were rare with no excess leukemias/MDS following treatment with very high doses of etoposide and other cytotoxic agents. Supported by Deutsche Krebshilfe. Figure Disclosures Nickelsen: Roche Pharma AG: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grants; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel Grant; Janssen: Membership on an entity's Board of Directors or advisory committees. Hänel:Amgen: Honoraria; Celgene: Other: advisory board; Novartis: Honoraria; Takeda: Other: advisory board; Roche: Honoraria. Truemper:Nordic Nanovector: Consultancy; Roche: Research Funding; Mundipharma: Research Funding; Janssen Oncology: Consultancy; Takeda: Consultancy, Research Funding; Seattle Genetics, Inc.: Research Funding. Held:Roche: Consultancy, Other: Travel support, Research Funding; Amgen: Research Funding; Acrotech: Research Funding; MSD: Consultancy; Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding. Dreyling:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: scientific advisory board, Research Funding, Speakers Bureau; Bayer: Consultancy, Other: scientific advisory board, Speakers Bureau; Celgene: Consultancy, Other: scientific advisory board, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Research Funding; Gilead: Consultancy, Other: scientific advisory board, Speakers Bureau; Novartis: Other: scientific advisory board; Sandoz: Other: scientific advisory board; Janssen: Consultancy, Other: scientific advisory board, Research Funding, Speakers Bureau; Acerta: Other: scientific advisory board. Viardot:Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosenwald:MorphoSys: Consultancy. Lenz:Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding; Agios: Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Employment, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy. Schmitz:Novartis: Honoraria; Gilead: Honoraria; Celgene: Equity Ownership; Riemser: Consultancy, Honoraria.

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Identification of Two CAR T-Cell Populations Associated with Complete Response or Progressive Disease in Adult Lymphoma Patients Treated with Axi-Cel

Blood ◽

10.1182/blood-2019-130815 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 779-779 ◽

Cited By ~ 2

Author(s):

Zinaida Good ◽

Jay Y. Spiegel ◽

Bita Sahaf ◽

Meena B. Malipatlolla ◽

Matthew J. Frank ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Advisory Committees ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory

Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

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Interaction of Mantle Cell Lymphoma with the Microenvironment Induces Phospho Akt-Mediated Resistance Against BTK Inhibition and Can be Overcome By Co-Treatment with a Specific Akt Inhibitor (MK-2206)

Blood ◽

10.1182/blood-2019-128782 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2802-2802

Author(s):

Elisabeth Silkenstedt ◽

Claudia Schwandner ◽

Johanna Deuss ◽

Natalie Mack ◽

Yvonne Zimmermann ◽

...

Keyword(s):

Western Blot ◽

Research Funding ◽

Direct Interaction ◽

Cell Interaction ◽

Advisory Board ◽

Akt Inhibitor ◽

Scientific Advisory Board ◽

Mantle Cell ◽

Scientific Advisory ◽

Btk Inhibitor

Mantle cell lymphoma (MCL) is a distinct lymphoma subtype representing 6-8% of non-Hodgkin's lymphoma (NHL). Although with current standard therapy high initial response rates can be achieved, early relapses and rapid disease progression determine the clinical course of most MCL patients. Recently, Bruton´s tyrosine Kinase (BTK) inhibitors have been introduced with highly promising clinical activity. Nevertheless, interindividual responsiveness is heterogenous and primary and secondary resistance has been reported. However, molecular mechanisms driving resistance to BTK inhibition are not well understood yet. Among other factors, interactions between the tumor and its microenvironment have been proposed to play an important role in response to targeted therapy. In this study, we investigated the influence of tumor cell interaction with its microenvironment on sensitivity to the BTK inhibitor CC292 in vitro. MCL cell lines JeKo-1, Z-138 and Granta-519 were treated with 5 µM of CC292 alone or in co-culture with human bone marrow stromal cells (HS-5) and cell death induction and proliferation were assessed. Expression of proteins involved in BCR signaling and other tumor-promoting pathways was analyzed by Western Blot. Co-cultured MCL cells settled within the stromal cell layer were separated using MACS Feeder removal microbeads prior to Western Blot analysis. In all cell lines, direct interaction with the microenvironment markedly reduced sensitivity towards CC292 treatment (by 22% (JeKo-1), 33% (Granta) and 64 % (Z-138)). Importantly, cell-cell contact was shown to play a crucial role for mediating resistance to CC292 as only those MCL cells settled within the stromal cell layer proved to be significantly less vulnerable to the inhibitor compared to MCL cells co-cultured with HS-5 but separated by a transwell insert. Western Blot analysis showed a reduction of protein levels of phBTK upon treatment with CC292 in both, mono- and co-cultured cells. Interestingly, direct interaction of MCL cells with the microenvironment strongly induced protein expression of phAkt. Accordingly, phosphorylation (inactivation) of the pro-apoptotic FoxO1, a downstream-target of phAkt, was increased and its translocation to the nucleus was decreased in those cells. We could show that the effect of microenvironment interaction on sensitivity towards CC292 is mediated by Akt as knockdown of Akt using siRNA restored sensitivity to the drug. Furthermore, co-treatment of MCL cells with CC292 and the specific Akt inhibitor MK-2206 hampered upregluation of phAkt in co-cultivated cells and prevented Akt-mediated sequestration of FoxO1 in the cytoplasm, resulting in translocation of FoxO1 to the nucleus. Thus, combination with MK-2206 could significantly overcome microenvironment-mediated protection from growth inhibition and apoptosis induction upon CC292 treatment. Moreover, combination of the BTK inhibitor CC292 and the Akt inhibitor MK-2206 proved to act synergistically in MCL cells in all dose combinations tested (Combination index 0,73-0,93 in Z-138; 0,47-0,78 in JeKo-1). Taken together, cell-cell-interaction of MCL cells with their microenvironment protected them from CC292-induced cell death. This effect was mediated by increased phAkt expression resulting in inhibition of pro-apoptotic signaling and could effectively be overcome by combination with the specific Akt inhibitor MK-2206. Furthermore, CC292 and MK-2206 acted synergistically in MCL cells. Our results indicate that co-targeting the PI3K/Akt-pathway might be a promising strategy to overcome resistance to BTK inhibition mediated by interaction with the microenvironment. Disclosures Dreyling: Sandoz: Other: Scientific advisory board; Roche: Other: Scientific advisory board, Research Funding, Speakers Bureau; Novartis: Other: Scientific advisory board; Mundipharma: Other: Scientific advisory board, Research Funding; Janssen: Other: Scientific advisory board, Research Funding, Speakers Bureau; Gilead: Other: Scientific advisory board, Speakers Bureau; Celgene: Other: Scientific advisory board, Research Funding, Speakers Bureau; Bayer: Other: Scientific advisory board, Speakers Bureau; Acerta: Other: Scientific advisory board.

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Central Nervous System Involvement By Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2021-148447 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2426-2426

Author(s):

Nicole McLaughlin ◽

Jonas Paludo ◽

Yucai Wang ◽

David J. Inwards ◽

Nora Bennani ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Data Monitoring Committee ◽

Advisory Board ◽

Control Group ◽

Cns Involvement ◽

Extranodal Involvement ◽

Advisory Committees ◽

Scientific Advisory Board ◽

Scientific Advisory

Abstract Background: While extranodal involvement by mantle cell lymphoma (MCL) is relatively common, involvement of the central nervous system (CNS) is rare (<5% of cases), with limited treatment options. We report the outcomes of 36 patients (pts) with CNS involvement compared to 72 matched control MCL pts without CNS involvement. Methods: MCL pts with CNS involvement seen at Mayo Clinic between 1/1995-9/2020 were identified using the Mayo Data Explorer tool. CNS involvement was defined by tissue biopsy confirmed CNS MCL, CSF analysis demonstrating lymphoma cells, and/or neuroimaging findings compatible with CNS involvement. A 2:1 control group of MCL pts without CNS involvement, matched by age (+/- 2 years) and year of diagnosis (+/- 1 year), was selected among all MCL cases. Medical records were reviewed for baseline characteristics, treatment modalities, and outcomes. Kaplan-Meier method was used for time to event analysis. Wilcoxon test was used to compare continuous variables and Chi square test was used for categorical variables. Results: Out of 1,753 pts with MCL, 36 (2%) had evidence of CNS involvement, including 4 pts with CNS involvement at initial MCL diagnosis. Baseline characteristics of pts with CNS involvement (CNS MCL group) and those without CNS involvement (control group) are shown in Table 1. At MCL diagnosis, non-CNS extranodal involvement was seen in 30 (83%) pts in the CNS MCL group (24 pts with 1 site and 6 pts with ≥ 2 sites), with bone marrow being the most common extranodal site of involvement (n=24, 67%). For the control group, 54 (75%) pts had extranodal involvement (44 pts with 1 site and 10 pts with ≥ 2 sites), and bone marrow was also the most common extranodal site of involvement (n=50, 69%). Notably, advanced stage disease (stage 3-4) was more commonly seen in the CNS MCL group (n=32, 97%) than in the control group (n=59, 83%) (p=0.04) at MCL diagnosis. Blastoid variant was present in a higher proportion of pts in the CNS MCL group (n=11, 31%) compared to the control group (n=8, 11%) (p=0.02). The CNS MCL group also presented with a higher median serum LDH at diagnosis (239 U/L [range 153-1901] vs. 187 U/L [range 124-588], p=0.02), and higher Ki-67 (40% [range 15-100] vs. 30% [range 10-90], p=0.04) compared to the control group. The most common frontline treatment regimen was anthracycline-based therapies (i.e. R-CHOP, Nordic regimen, R-hyperCVAD) for both groups (58% in CNS MCL group and 56% in control group). 14 (39%) pts in the CNS MCL group underwent autologous stem cell transplant in CR1 vs. 31 pts (43%) in the control group. Similar use of rituximab maintenance was seen in both groups (31% in CNS MCL group and 25% in control group). Median total lines of therapy from initial MCL diagnosis was 3 (range 1-9) in CNS MCL group and 2 (range 1-9) in the control group. The median follow-up from MCL diagnosis was 134 months (95% CI:119-163) for the entire cohort. Median OS from MCL diagnosis was 50.3 months (95% CI: 20.9-71.1) for the CNS MCL group compared to 97.1 months (95% CI: 82.6-192.7; p=<0.001) for the control group (Figure 1). Median time from MCL diagnosis to CNS involvement was 25 months (range 0-167). Median OS from CNS involvement was 4.7 months (95% CI: 2.3-6.7). At last follow up, 31 (86%) pts were deceased from the CNS MCL group, compared to 38 (52%) pts in the control group. For the CNS MCL group, the causes of death were CNS lymphoma in 10 (32%) pts, systemic lymphoma in 9 (29%) pts, treatment-related complication in 7 (23%) pts, and other/unknown in 5 (16%) pts. For the control group, the causes of death were systemic lymphoma in 15 (39%) pts, treatment-related in 2 (5%) pts, and other/unknown in 21 (55%) pts. Conclusion: In pts with MCL, CNS involvement is associated with worse outcomes as evident by a shorter median OS from initial MCL diagnosis (50 months vs. 97 months). Involvement of the CNS by lymphoma is an important contributor for the shorter OS as suggested by the median OS of only 5 months from CNS involvement. Advanced stage, blastoid variant, elevated LDH, and elevated Ki67 at MCL diagnosis were features more commonly seen in the CNS MCL cohort. Validation of risk factors at initial MCL diagnosis associated with CNS involvement and exploring the role of CNS prophylaxis are important topics for further investigation. Figure 1 Figure 1. Disclosures Paludo: Karyopharm: Research Funding. Wang: Novartis: Research Funding; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; MorphoSys: Research Funding; InnoCare: Research Funding; Eli Lilly: Membership on an entity's Board of Directors or advisory committees; LOXO Oncology: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding. Bennani: Purdue Pharma: Other: Advisory Board; Daichii Sankyo Inc: Other: Advisory Board; Kyowa Kirin: Other: Advisory Board; Vividion: Other: Advisory Board; Kymera: Other: Advisory Board; Verastem: Other: Advisory Board. Nowakowski: Celgene, MorphoSys, Genentech, Selvita, Debiopharm Group, Kite/Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene, NanoString Technologies, MorphoSys: Research Funding. Witzig: Karyopharm Therapeutics, Celgene/BMS, Incyte, Epizyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS, Acerta Pharma, Kura Oncology, Acrotech Biopharma, Karyopharm Therapeutics: Research Funding. Habermann: Seagen: Other: Data Monitoring Committee; Tess Therapeutics: Other: Data Monitoring Committee; Incyte: Other: Scientific Advisory Board; Morphosys: Other: Scientific Advisory Board; Loxo Oncology: Other: Scientific Advisory Board; Eli Lilly & Co.,: Other: Scientific Advisor. Ansell: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, AI Therapeutics, Pfizer, Trillium and Takeda: Research Funding.

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Phase I Trial Using CD19/CD22 Bispecific CAR T Cells in Pediatric and Adult Acute Lymphoblastic Leukemia (ALL)

Blood ◽

10.1182/blood-2019-129411 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 744-744 ◽

Cited By ~ 10

Author(s):

Liora M Schultz ◽

Lori S Muffly ◽

Jay Y. Spiegel ◽

Sneha Ramakrishna ◽

Nasheed Hossain ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Adult Patients ◽

Advisory Committees ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory

Introduction: Chimeric antigen receptor (CAR) T cells targeting either CD19 or CD22 have yielded striking complete remission (CR) rates of 70%-90% in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL), but CD19 negative and CD22 low relapse limits the curative potential of these single-antigen CAR T cell approaches. We hypothesized that a bivalent CAR-T construct that can target CD19 and/or CD22 would prevent antigen negative/low relapse. Here we present the combined single institution experience to date of pediatric and adult patients with R/R ALL treated with this novel bispecific CAR. Methods: We conducted parallel Phase I clinical trials of CD19/CD22 bispecific CAR T cells in pediatric and adult patients with relapsed/refractory ALL. We utilized lentiviral transduction of a bivalent CAR construct incorporating the fmc63 CD19 and m971 CD22 single chain variable fragments (scFvs) and a 41BB costimulatory endodomain. After lymphodepletion with fludarabine and cyclophosphamide, patients were infused with fresh or cryopreserved CAR T cells manufactured using a 7-11 day process. Two dose levels were tested during dose escalation: Dose level 1 was 1x106 CAR T cells/kg and dose level 2 was 3x106 cells/kg. Primary objectives assessed the ability to successfully manufacture CAR19/22 CAR T cells and safety while response at Day 28 post-infusion was a secondary objective. Blood, bone marrow and cerebrospinal fluid samples were obtained at protocol defined intervals for correlative biology studies. Results: Nineteen patients have been enrolled (10 pediatric; 9 adult) with a median age of 23 years (range, 2-68) and median of 4 (range, 2-11) prior lines of leukemia-directed therapy. Ten patients received prior HCT, 9 were treated with prior Blinatumomab, 3 with prior CD19 directed CAR T cells and 4 with prior Inotuzumab. Fourteen patients (8 pediatric, 6 adult) have been infused to date with CD19/CD22 bispecific CAR T cells; 7 were treated at dose level 1 (DL1) and 7 at dose level 2 (DL2). Successful manufacturing of cells at target dose levels was achieved in all patients. Twelve patients have reached day 28 and are included in the safety and response analysis presented here. Nine of 12 (75%) experienced cytokine release syndrome (CRS) and 2/12 (17%) developed immune-effector cell neurotoxicity syndrome (ICANS). The CRS and ICANS were all grade 1 or 2 across both dose levels and across pediatric and adult patients except for one adult with high disease burden who experienced grade 4 CRS and grade 4 ICANS, both of which were reversible. No differences in toxicities were seen across the patient age spectrum and there were no cases of treatment-related mortality within 28 days following CAR T infusion. Eleven of 12 (92%) patients achieved a CR, 10 of whom achieved CR at day 28 and one with a PR of extramedullary disease at day 28 which improved to CR by day 180 without further leukemia-directed intervention. One patient had primary progressive disease prior to day 28. Peak CAR expansion as detected by peripheral blood flow cytometry reached a median level of 11.13% (DL1) and 29.1% (DL2) CAR T of CD3+ cells with a range of 0.7-22.54% and 3.8-86.96%, respectively. To date, 3 patients (1 pediatric and 2 adult patients) have relapsed, all with retention of CD19. Post-remission practice differed across pediatric and adult patients; Six pediatric patients reaching day 28 underwent consolidative hematopoietic cell transplantation (HCT) whereas no adult patients received subsequent HCT. One patient died from complications post HCT while in remission. Therefore, the overall survival for all infused patients was 92% with a median follow-up of 9.5 months from time of infusion (range, 1-20). Conclusion: The combined pediatric and adult phase I trials of bispecific CD19/CD22 targeting CAR T cells in relapsed/refractory ALL demonstrates safety and tolerability at two dose levels. Expanded accrual at dose level 2 is ongoing and clinical outcomes will be updated. This work additionally demonstrates feasibility of delivering unified B-ALL CAR T cell therapy across age boundaries. Multi-parametric CyTOF studies permitting CAR T cell phenotyping in conjunction with single cell TCR tracking, proteomics, epigenomics and cytokine profiling are ongoing and will be used to further characterize persisting CAR T cells and define inter-product and inter-patient variability. Disclosures Muffly: Pfizer: Consultancy; KITE: Consultancy; Adaptive: Research Funding. Majzner:Xyphos Inc.: Consultancy; Lyell Immunopharma: Consultancy. Feldman:Octane Biotech, Inc.: Employment; Personalized Medicine Initiative Science: Membership on an entity's Board of Directors or advisory committees. Miklos:Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Becton Dickinson: Research Funding; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; AlloGene: Membership on an entity's Board of Directors or advisory committees. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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Evaluation of the International Prognostic Index for Chronic Lymphocytic Leukemia (CLL-IPI) and Validation of a Proposed Novel Risk Model (BALL Score) in Real-World Relapsed/Refractory (R/R) CLL Patients Receiving Idelalisib and Rituximab

Blood ◽

10.1182/blood-2019-126717 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5485-5485

Author(s):

Massimo Gentile ◽

Gianluigi Reda ◽

Francesca Romana Mauro ◽

Paolo Sportoletti ◽

Luca Laurenti ◽

...

Keyword(s):

High Risk ◽

Risk Stratification ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Low Risk ◽

Intermediate Risk ◽

Advisory Committees ◽

Scientific Advisory Board ◽

Scientific Advisory

The CLL-IPI score, which combines genetic, biochemical, and clinical parameters, represents a simple worldwide model able to refine risk stratification for CLL patients. This score, developed in the era of chemo-immunotherapy, has not been gauged extensively in R/R-CLL patients treated with novel targeted agents, such as BCR and BCL2 inhibitors. Soumerai et al (Lancet Hematol 2019) assembled a novel risk model for OS in the setting of R/R-CLL receiving targeted therapies in clinical trials. This model, consisting of four accessible markers (β2M, LDH, Hb, and time from initiation of last therapy; BALL score), is able to cluster 3 groups of CLL patients with significantly different OS. This multicenter, observational retrospective study aimed to validate the proposed Soumerai (BALL) and/or CLL-IPI scores for R/R-CLL real-world patients treated with idelalisib and rituximab (IDELA-R). The primary objectives were to determine whether: i) the CLL-IPI retains its prognostic power also in R/R patients treated with IDELA-R; ii) the BALL score is of prognostic value for IDELA-treated R/R-CLL patients, and iii) the BALL score is predictive of PFS. This study, sponsored by Gilead (ISR#IN-IT-312-5339), included CLL patients collected from 12 Italian centers, who received IDELA-R (idelalisib 150 mg b.i.d. and a total of 8 rituximab infusions intravenously) outside clinical trials as salvage therapy with available data for the calculation of the CLL-IPI and BALL scores at the time of treatment start. OS was estimated for all subgroups of both scores. Additionally, risk-specific PFS was assessed. Kaplan-Meier curve, log-rank test, and Cox regression analyses were performed. The prognostic accuracy of the predictive model was assessed by Harrell's C-index. Overall, 120 CLL patients were included in this analysis. The majority of patients were Binet stage B and C (94.2%). The median age was 75 years and 83 cases (69.2%) were male. The median number of previous therapies was 3 (range 1-9) Baseline patient features are listed in Table 1. After a median follow-up of 1.6 years (1 month to 5.8 years), 33 patients had died and 39 experienced an event (death or progression). CLL-IPI scoring (115/120 evaluable cases) indicated that 6 patients (5.2%) were classified as low-risk, 24 (20.9%) as intermediate-risk, 58 (50.4%) as high-risk, and 27 (23.5%) as very high-risk. Stratification of patients according to the CLL-IPI score did not allow prediction of significant differences in OS. Thus, low-risk patients had a 2-year OS probability of 75% (HR=1), with an intermediate-risk of 68% (HR=2.9, 95%CI 0.37-23.3, P=0.3), high-risk of 83% (HR=1.58, 95%CI 0.2-12.5, P=0.66), and very high-risk of 63% (HR=5.9, 95%CI 0.78-45.2, P=0.86). Next, we tested a modified CLL-IPI by assigning a more balanced score to the original CLL-IPI variables (Soumerai et al, Leukemia Lymphoma 2019), partially overlapping previous results. Specifically, modified CLL-IPI high-risk group showed a significantly different OS as compared with intermediate- and low-risk groups. However, differently from the original report no difference was observed between low- and intermediate-risk). According to the BALL score (120/120 evaluable cases), 33 patients (27.5%) were classified as low-risk, 68 (56.7%) as intermediate-risk, and 19 (15.8%) as high-risk. Stratification of patients according to the BALL score predicted significant differences in terms of OS. Thus, low-risk patients had a 2-year OS probability of 92% (HR=1), intermediate-risk of 76% (HR=5.47, 95%CI 1.3-23.7, P=0.023), and high-risk of 54% (HR=15.1, 95%CI 3.4-67, P<0.0001) (Figure 1). Harrell's C-statistic was 0.68 (P<0.001) for predicting OS. To note, BALL score failed to significantly stratify patients in terms of PFS. As for Soumerai et al (Leukemia Lymphoma 2019), the original CLL-IPI score did not retain discriminative power in term of OS in R/R-CLL patients receiving IDELA-R. The modified CLL-IPI failed to stratify low- and intermediate-risk groups, likely due to the number of cases analysed in the current cohort and the heterogeneous IDELA-containing regimens included in the Soumerai study (Soumerai et al, Leukemia Lymphoma 2019). The CLL-IPI was designed for CLL patients treated with first-line chemo-immunotherapy. Herein, we confirm the prognostic power of the BALL score in this real-world series for OS, while losing the predictive impact of patient outcomes in terms of PFS. Disclosures Mauro: Gilead: Consultancy, Research Funding; Jannsen: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Roche: Consultancy, Research Funding. Coscia:Abbvie: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Varettoni:ABBVIE: Other: travel expenses; Roche: Consultancy; Janssen: Consultancy; Gilead: Other: travel expenses. Rossi:Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Honoraria, Other: Scientific advisory board; Janseen: Honoraria, Other: Scientific advisory board; Roche: Honoraria, Other: Scientific advisory board; Astra Zeneca: Honoraria, Other: Scientific advisory board. Gaidano:AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesys: Consultancy, Honoraria; Astra-Zeneca: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Myeloablative Fractionated Busulfan Conditioning Regimen with Sorafenib in Patients with AML: Results of Phase I Clinical Trial

Blood ◽

10.1182/blood-2021-152450 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 556-556

Author(s):

Uday R. Popat ◽

Roland Bassett ◽

Peter F. Thall ◽

Amin M. Alousi ◽

Gheath Alatrash ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Conditioning Regimen ◽

Advisory Board ◽

License Agreement ◽

Advisory Committees ◽

Post Transplant ◽

Scientific Advisory Board ◽

Scientific Advisory

Abstract Background: Myeloablative conditioning can be given safely to older patients by administering busulfan over a longer period (fractionated busulfan regimen) than the standard four-day regimen. (Popat, et al Lancet Haematology 2018). This longer conditioning regimen duration allows the addition of oral targeted agents like sorafenib, which may be synergistic with conditioning chemotherapy and thus further improve disease control. Therefore, we added sorafenib to fludarabine and fractionated busulfan regimen (f-bu) in a phase 1 dose-finding trial studying 4 different doses of sorafenib with f-bu (NCT03247088). Here we report the results of this trial. Methods: Between 3/2018 and 6/2021, 24 patients with AML aged 18 to 70 years with adequate organ function and 8/8-HLA matched related or unrelated donors were enrolled prospectively. The dose of sorafenib was varied among the four values 200, 400, 600, and 800 mg administered from day -24 to -5. Dose-limiting toxicity (DLT) was defined as grade 3 or higher regimen-related non-hematologic, non-infectious, non-GVHD toxicity occurring between day -24 and day 3. The Bayesian Model Averaging Continual Reassessment Method (BMA-CRM) with target DLT probability 0.30 was used to choose doses for successive cohorts of 3 patients. The first cohort was treated at the lowest sorafenib dose 200, with all successive cohorts' doses chosen adaptively by the BMA-CRM. The doses and schedules of busulfan and fludarabine were fixed, with f-Bu dose targeting an area under the concentration vs time curve (AUC) of 20,000 ± 12% μmol.min given over 3 weeks. The first two doses of busulfan (80 mg/m2 IV each) were administered on days -20 and -13 on an outpatient basis. The last four Bu doses were calculated to give a total course AUC of 20,000 ± 12% μmol.min and were given as inpatient following each dose of Flu 40 mg/m2 on days -6 through -3. GVHD prophylaxis was post-transplant cyclophosphamide (PTCy) 50mg/kg on days 3 and 4 and tacrolimus. Recipients of unrelated donor grafts also received MMF. All patients were eligible to receive post-transplant maintenance sorafenib after engraftment. Results: The median age was 52 years (range, 30-70). Disease status was CR in 16 (66.6%) patients, CRi in 5 (20.8%), and advanced in 3 (12.5%). Adverse risk karyotype was present in 10 (41.7%) patients. MRD was present in 13 (54.2%). 9 (38%) had mutated flt3. The donor was unrelated in 14 (58%), and peripheral blood stem cells were the graft source in 21(87.5%). Due to the absence of DLTs, the BMA-CRM assigned 200mg, 400mg, 600mg, and 800mg of sorafenib, respectively, to the first 4 cohorts, and the next 4 cohorts were given 800mg. Only 2 dose-limiting skin toxicities were seen, one in cohort 3 with 600mg of sorafenib and the second in cohort 6 with 800mg of sorafenib. 800mg was the final recommended phase 2 dose. The median follow-up in 20 surviving patients was 7.6 months and 1-year progression free survival was 89% (95% CI 75-100%). Other outcomes are summarized in Table 1. Conclusion: Sorafenib can be safely added to the fractionated busulfan regimen. Early data on efficacy appear promising, with an 89% PFS at 1 year of follow up. Figure 1 Figure 1. Disclosures Popat: Bayer: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding. Hosing: Nkarta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Rezvani: Bayer: Other: Scientific Advisory Board ; AvengeBio: Other: Scientific Advisory Board ; Navan Technologies: Other: Scientific Advisory Board; GSK: Other: Scientific Advisory Board ; Virogin: Other: Scientific Advisory Board ; Affimed: Other: License agreement and research agreement; education grant, Patents & Royalties, Research Funding; Pharmacyclics: Other: Educational grant, Research Funding; Caribou: Other: Scientific Advisory Board; GemoAb: Other: Scientific Advisory Board ; Takeda: Other: License agreement and research agreement, Patents & Royalties. Qazilbash: Bristol-Myers Squibb: Other: Advisory Board; Biolline: Research Funding; Amgen: Research Funding; Oncopeptides: Other: Advisory Board; NexImmune: Research Funding; Angiocrine: Research Funding; Janssen: Research Funding. Daver: Daiichi Sankyo: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; Abbvie: Consultancy, Research Funding; Hanmi: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Sevier: Consultancy, Research Funding; Novimmune: Research Funding; Trovagene: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Ravandi: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AstraZeneca: Honoraria; Novartis: Honoraria; Xencor: Honoraria, Research Funding; Taiho: Honoraria, Research Funding; Astex: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Prelude: Research Funding; Syros Pharmaceuticals: Consultancy, Honoraria, Research Funding. Shpall: Magenta: Consultancy; Bayer HealthCare Pharmaceuticals: Honoraria; Magenta: Honoraria; Adaptimmune: Consultancy; Novartis: Consultancy; Navan: Consultancy; Novartis: Honoraria; Takeda: Patents & Royalties; Affimed: Patents & Royalties; Axio: Consultancy. Mehta: CSLBehring: Research Funding; Kadmon: Research Funding; Syndax: Research Funding; Incyte: Research Funding.

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