scholarly journals CD200 Is a Stem Cell-Specific Immunosuppressive Target in AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2768-2768
Author(s):  
Shelley Herbrich ◽  
Keith Baggerly ◽  
Gheath Alatrash ◽  
R. Eric Davis ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Cd200 Receptor ◽  
Blast Cells ◽  
Equity Ownership

Abstract Acute myeloid leukemia (AML) stem cells (LSC) are an extremely rare fraction of the overall disease (likely <0.3%), largely quiescent, and capable of both long-term self-renewal and production of more differentiated leukemic blasts. Besides their role in disease initiation, they are also hypothesized as the likely source of deadly, relapsed leukemia. Due to the quiescent nature of the LSCs, they are capable of evading the majority of chemotherapeutic agents that rely on active cell-cycling for cytotoxicity. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression data to identify genes significantly over-expressed in LSCs when compared to their normal hematopoietic stem cell (HSC) counterparts (Herbrich et al Blood 2017 130:3962). These datasets contain gene expression arrays on human AML patient samples sorted by leukemia stem, progenitor, and blast cells (with normal hematopoietic cell subsets for comparison). We have since expanded our statistical model to identify targets that are both significantly overexpressed in AML LSCs when compared to HSC as well as LSCs compared to their corresponding, more differentiated blast cells. Instead of traditional methods for multiple testing corrections, we looked at the intersection of genes that met the above criteria in 3 independently generated datasets. This resulted in a list of 30 genes, 28 of which appear to be novel markers of AML LSCs. From this list, we first chose to focus on CD200, a type-1 transmembrane glycoprotein. CD200 is broadly expressed on myeloid, lymphoid, and epithelial cells, while the CD200 receptor (CD200R) expression is strictly confined to myeloid and a subset of T cells. CD200 has been shown to have an immunosuppressive effect on macrophages and NK cells and correlates with a high prevalence FOXP3+ regulatory T cells (Coles et al Leukemia 2012; 26:2146-2148). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget 2015; 6:30212-30221). To date, we have screened 20 primary AML patient samples by flow cytometry, 90% of which are positive for CD200. Expression is significantly enriched in the CD34+/CD123+ stem cell compartment. To examine the role of CD200 in AML, we established two in vitro model systems. First, we used CRISPR/Cas9 to knockout the endogenous CD200 protein in Kasumi-1. Further, we induced CD200 in the OCI-AML3 cell line that had no expression at baseline. Both cell lines did not express the CD200 receptor before or after manipulation, negating any autocrine signaling. In both systems, CD200 manipulation did not affect the proliferation rate or viability of the cells. To examine the immune function of CD200 in AML, we performed a series of mixed lymphocyte reactions. We cultured normal human peripheral blood mononuclear cells (PBMCs) with the CD200+ or CD200- cells from each line both. Cells were incubated in the culture media for 4-48 hours before being harvested and measured by flow cytometry for apoptosis or intracellular cytokine production. The presence of CD200 on the cell surface reduced the rate of immune-specific apoptosis among these leukemia cells. The difference in cell killing was most likely attributable to a CD200-specific suppression of CD107a, a surrogate marker or cytotoxic activity. In the OCI-AML3 model, PBMCs co-cultured with CD200+ cells produced approximately 40% less CD107a when compared to the CD200- co-culture. Additionally, we characterized our new cell lines using RNA sequencing. By comparing the CD200+ to the CD200- cells within each line, we observed that CD200+ cells significantly downregulate genes involved in defining an inflammatory response as well as genes regulated by NF-κB in response to TNFα. This indicates that CD200 may have an undiscovered intrinsic role in suppressing the immune microenvironment of AML LSCs. In conclusion, we have expanded our novel bioinformatics approach for robustly identifying AML LSC-specific targets. Additionally, we have shown that one of these markers, CD200, has a potential role as a stem cell-specific immunosuppressive target by reducing immune-mediated apoptosis and transcriptionally suppressing inflammatory cell processes. We are extending our study to explore CD200 in primary patient samples using a CD200-blocking antibody. Disclosures Andreeff: SentiBio: Equity Ownership; Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; Reata: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Jazz Pharma: Consultancy; Astra Zeneca: Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy. Konopleva:Stemline Therapeutics: Research Funding.

scholarly journals High-Parameter Mass Cytometry (CyTOF) Evaluation of Relapsed/Refractory Multiple Myeloma (MM) Pts (Pts) Treated with Daratumumab Supports Immune Modulation As a Novel Mechanism of Action

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4521-4521 ◽  
Author(s):  
Homer Adams ◽  
Frederik Stevenaert ◽  
Jakub Krejcik ◽  
Koen Van der Borght ◽  
Tineke Casneuf ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Granzyme B ◽  
Advisory Committees ◽  
Activation Markers ◽  
Equity Ownership

Abstract Introduction: Daratumumab (DARA) is a human CD38-targeting monoclonal antibody that induces deep clinical responses in MM pts through multifaceted mechanisms of action (MOA) including complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis and induction of apoptosis. Flow cytometry analysis revealed a previously unknown immunomodulatory role of DARA, via T-cell induction expansion, T-cell activity enhancement, and reduction of immune suppressive cell populations including CD38+ myeloid-derived suppressor cells, CD38+ regulatory T cells (TRegs), and CD38+ regulatory B cells (BRegs). Next-generation mass cytometry (CyTOF), which allows high parameter evaluation of the immune system, was used to assess the effects of DARA alone or in combination on a more comprehensive profile of immune cell subpopulations. Methods: Relapsed/refractory MM pt samples from a subset of single agent studies; SIRIUS (32 pts; whole blood [WB] only; Lonial S et al. The Lancet, 2016) and GEN501 (5 pts; WB and bone marrow [BM], Lokhorst HM et al. NEJM, 2015) along with GEN503, a study of DARA plus lenalidomide and dexamethasone (9 pts; WB and BM; Plesner T et al. ASH 2015) were analyzed. Fluorochrome or metal-conjugated antibody panel stained samples were evaluated by flow cytometry or cytometry by time-of-flight (CyTOF®) platforms, respectively. FACS analyses were performed and analyzed by FACS Canto II flow cytometers and FACSDiva software. For CyTOF analysis, events were clustered by phenotype by a spanning tree progression of density normalized events (SPADE) algorithm, and each cluster was associated with an immune population via Cytobank® software. Differential analysis of population fractions and marker intensity, over time and between response groups, derived raw P values from t-tests and single cell level bootstrap adjusted P values corrected for multiple dependent hypothesis testing. Results were visualized using SPADE trees (Figure) and Radviz projections, a new method that allows for the comparison of populations and conditions while preserving the relation to original dimensions. Results: Flow cytometry and high-dimensional CyTOF analyses confirmed previous findings including higher CD38 expression on plasma cells compared with other immune populations of natural killer (NK), monocytes, B and T cells, and depletion of both plasma cells and NK cells upon DARA treatment. Interestingly, while NK cells were significantly reduced with DARA treatment, remaining active NK cells (CD16+CD56dim) demonstrated increased expression of activation markers CD69, CD25 and CD137 while also decreasing granzyme B and increasing naive marker CD27. Though functionality tests weren't performed, the ability to evaluate several markers simultaneously suggests these cells possess limited cytotoxicity. Additionally, these studies indicated depletion of CD38 positive immune suppressive subsets of Tregs and Bregs. CD38+ basophil reductions occurred independent of response and may provide insight to short-lived infusion related reactions. Several observations within the T-cell compartment were indicative of a DARA-mediated adaptive response in both WB and BM samples. T cells displayed increases in total numbers and shifted towards higher CD8:CD4 and effector:naïve ratios after 2 months of DARA treatment. Responders had higher expression levels of several activation markers including CD69 and HLA-DR along with increased production of cytolytic enzyme granzyme B in CD8+ T cells following DARA treatment. Interestingly, in the GEN503 sample set, pts who achieved a complete response presented with a distinct BM CD4 T-cell phenotype of high granzyme B positivity versus those that achieved a partial response or very good partial response. This observation suggests pts with an active immune phenotype may achieve deeper responses to DARA in combination with standard of care agents lenalidomide and dexamethasone. Conclusion: CyTOF analysis of pt samples from both single agent and combination DARA studies agree with flow cytometry and support the pharmacodynamics and immune modulatory MOA of DARA while providing additional insight into changes in T-cell subtypes and activation status. Future CyTOF analyses of clinical samples from phase 3 combination studies aim to confirm these observations and expand the understanding of the MOA of DARA. Disclosures Adams: Janssen Research & Development, LLC: Employment. Stevenaert:Janssen: Employment. Van der Borght:Janssen: Employment. Casneuf:Janssen R&D, Beerse, Belgium: Employment; Johnson & Johnson: Equity Ownership. Smets:Janssen: Employment. Bald:Janssen: Employment. Abraham:Janssen: Employment. Ceulemans:Janssen: Employment. Vanhoof:Janssen: Employment; Johnson & Johnson: Equity Ownership. Ahmadi:Janssen: Employment. Usmani:Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Array: Research Funding; BioPharma: Research Funding; Pharmacyclics: Research Funding; Takeda: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Millenium: Membership on an entity's Board of Directors or advisory committees; Skyline: Membership on an entity's Board of Directors or advisory committees. Plesner:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lonial:Janssen: Consultancy; BMS: Consultancy; Merck: Consultancy; Novartis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Onyx: Consultancy; Millenium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Lokhorst:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Mutis:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding; Celgene: Research Funding. van de Donk:Janssen: Research Funding; BMS: Research Funding; Amgen: Research Funding; Celgene: Research Funding. Sasser:Janssen Pharmaceuticals R&D: Employment; Johnson & Johnson: Equity Ownership.

scholarly journals Immunotherapy with T-Cells Engineered with a Chimeric Antigen Receptor Bearing a Human CD19-Binding Single Chain Variable Fragment for Relapsed or Refractory Acute Lymphoblastic Leukemia and B-Cell Non-Hodgkin Lymphoma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1415-1415 ◽  
Author(s):  
Jordan Gauthier ◽  
Alexandre V. Hirayama ◽  
Kevin A. Hay ◽  
Alyssa Sheih ◽  
Barbara S. Pender ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership ◽  
Grade 3

Abstract Background We previously reported high response rates and durable remissions in patients (pts) with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL; Turtle, JCI 2016) and non-Hodgkin lymphoma (NHL; Turtle, Sci Transl Med 2016) treated with CD19-specific chimeric antigen receptor T (CD19 CAR-T) cells. In a subset of pts, we identified CD8+ T cell responses to epitopes in the murine CD19-binding single chain variable fragment (scFv) of the CAR that could limit CAR-T cell persistence and responses to subsequent infusions. In an effort to reduce the potential for immune CAR-T cell rejection, the murine CD19-binding scFv of the CAR was replaced with a fully human scFv linked to 4-1BB and CD3z signaling domains (JCAR021; Sommermeyer, Leukemia 2017). Here we report the initial clinical results of immunotherapy with JCAR021. Methods We initiated a phase I trial investigating lymphodepletion with cyclophosphamide 300 mg/m2/d and fludarabine 30 mg/m2/d for 3 days (Cy/Flu) followed by infusion of JCAR021 in pts with R/R ALL and aggressive NHL (NCT03103971). Pts were enrolled into 1 of 3 cohorts: high marrow burden ALL (HMB; > 5% blasts in bone marrow [BM] before lymphodepletion); low marrow burden ALL (LMB; ≤ 5% blasts in BM before lymphodepletion); and NHL. The starting dose was 7x104 JCAR021 cells/kg for the HMB ALL cohort, and 7x105 JCAR021 cells/kg in both the LMB ALL and NHL cohorts. Dose escalation/de-escalation follows a modified toxicity probability interval algorithm (Guo, Contemp Clin Trials 2017). Responses in the NHL cohort and in the HMB/LMB ALL cohorts were determined by the Lugano criteria (Cheson, JCO 2014) and the 2018 NCCN guidelines, respectively. Cytokine release syndrome (CRS) was graded according to consensus criteria (Lee, Blood 2014) and neurotoxicity was graded according to CTCAE v4.03. Results Pt characteristics are detailed in Table 1. As of June 15, 2018, 9 pts were enrolled on the trial. Two pts did not receive JCAR021: one pt was excluded after aggressive NHL was reclassified as indolent after pathology review and one pt had no detectable disease upon pre-treatment restaging. The 7 pts who received JCAR021 had a median age of 63 years (range: 29 - 69). Both pts in the LMB ALL cohort had bulky extramedullary disease (> 5 cm diameter). One patient (LMB ALL cohort) had failed two allogeneic transplants and one patient (HMB ALL cohort) had failed an allogeneic transplant prior to treatment with JCAR021. Four of 4 pts in the NHL cohort and 2 of 2 pts in the LMB ALL cohort received 7x105 JCAR021 cells/kg. The pt treated in the HMB ALL cohort received 7x104 JCAR021 cells/kg. No pt in any cohort developed grade ≥ 3 CRS. All ALL pts developed grade 2 CRS. No pts with NHL developed CRS; one pt in the NHL cohort who had CNS disease prior to CAR-T cell immunotherapy developed grade 3 neurotoxicity in the absence of CRS. We did not observe other neurologic events. No other grade ≥ 3 non-hematopoietic organ toxicity was observed and all 7 treated pts have completed response evaluation. Four weeks after infusion of a low dose of JCAR021, both patients in the LMB ALL cohort had undetectable marrow disease by high resolution flow cytometry and regression of bulky extramedullary disease (1 complete response [CR] and 1 partial response [PR] by PET-CT). One pt treated with a low dose (7x104 cells/kg) of JCAR021 in the HMB ALL cohort did not achieve CR (decrease in BM blasts from 79.8% to 29.5%) but CNS disease was cleared by flow cytometry. In the NHL cohort, we observed objective responses in 2 of 4 patients (1 CR, 1 PR). JCAR021 was detected in blood by flow cytometry and/or quantitative PCR for up to 112 days after infusion. Conclusion JCAR021 appears to have a favorable toxicity profile in R/R ALL and NHL pts. JCAR021 cells expanded in vivo and have persisted in all pts. We observed responses at very low doses of CAR-T cells in ALL pts with bulky disease. This trial continues to enroll to define optimal dosing and determine the safety and efficacy of JCAR021. Disclosures Hirayama: DAVA Oncology: Honoraria. Hay:DAVA Oncology: Honoraria. Till:Mustang Bio: Patents & Royalties, Research Funding. Kiem:Homology Medicine: Consultancy; Magenta: Consultancy; Rocket Pharmaceuticals: Consultancy. Shadman:TG Therapeutics: Research Funding; Mustang: Research Funding; Gilead: Research Funding; Pharmacyclics: Research Funding; AstraZeneca: Consultancy; Qilu Puget Sound Biotherapeutics: Consultancy; Acerta: Research Funding; Abbvie: Consultancy; Verastem: Consultancy; Genentech: Consultancy, Research Funding; Beigene: Research Funding; Celgene: Research Funding. Cassaday:Amgen: Consultancy, Research Funding; Seattle Genetics: Other: Spouse Employment, Research Funding; Adaptive Biotechnologies: Consultancy; Incyte: Research Funding; Pfizer: Consultancy, Research Funding; Merck: Research Funding; Kite Pharma: Research Funding; Jazz Pharmaceuticals: Consultancy. Acharya:Teva: Honoraria; Juno Therapeutics: Research Funding. Riddell:NOHLA: Consultancy; Adaptive Biotechnologies: Consultancy; Cell Medica: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Maloney:Juno Therapeutics: Research Funding; Seattle Genetics: Honoraria; Janssen Scientific Affairs: Honoraria; GlaxoSmithKline: Research Funding; Roche/Genentech: Honoraria. Turtle:Aptevo: Consultancy; Nektar Therapeutics: Consultancy, Research Funding; Caribou Biosciences: Consultancy; Gilead: Consultancy; Juno Therapeutics / Celgene: Consultancy, Patents & Royalties, Research Funding; Bluebird Bio: Consultancy; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Consultancy; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Increased Early Mortality after Fludarabine and Melphalan Conditioning with Peripheral Blood Grafts in Haploidentical SCT with Post-Transplant Cyclophosphamide

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4496-4496 ◽  
Author(s):  
Luke Eastburg ◽  
David A. Russler-Germain ◽  
Ramzi Abboud ◽  
Peter Westervelt ◽  
John F. DiPersio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Early Mortality ◽  
Cell Transplant ◽  
Advisory Committees ◽  
Post Transplant ◽  
Equity Ownership

The use of post-transplant cyclophosphamide (PTCy) in the context of haploidentical stem cell transplant (haplo-SCT) has led to drastically reduced rates of Graft-vs-Host (GvH) disease through selective depletion of highly allo-reactive donor T-cells. Early trials utilized a reduced-intensity Flu/Cy/TBI preparative regimen and bone marrow grafts; however, relapse rates remained relatively high (Luznik et al. BBMT. 2008). This led to the increased use of myeloablative (MA) regimens for haplo-SCT, which have been associated with decreased relapse rates (Bashey et al. J Clin Oncol. 2013). Most studies have used a MA total body irradiation (TBI) based regimen for haplo-SCT. Preparative regimens using fludarabine and melphalan (FluMel), with or without thiotepa, ATG, and/or low dose TBI have also been reported using bone marrow grafts. Reports on the safety and toxicity of FluMel in the haplo-SCT setting with PTCy and peripheral blood stem cell (PBSC) grafts are lacking. In this two-center retrospective analysis, the safety/toxicity of FluMel as conditioning for haplo-SCT was evaluated. We report increased early mortality and toxicity using standard FluMel conditioning and PBSC grafts for patients undergoing haplo-SCT with PTCy. 38 patients at the University of Rochester Medical Center and the Washington University School of Medicine underwent haplo-SCT with FluMel conditioning and PBSC grafts between 2015-2019. Outcomes were measured by retrospective chart review through July 2019. 34 patients (89.5%) received FluMel(140 mg/m2). Two patients received FluMel(100 mg/m2) and two patients received FluMel(140 mg/m2) + ATG. The median age at time of haplo-SCT was 60 years (range 21-73). 20 patients were transplanted for AML, eight for MDS, two for PMF, two for NHL, and five for other malignancies. The median Hematopoietic Cell Transplantation-specific Comorbidity Index (HCT-CI) score was 4 (≥3 indicates high risk). 11 patients had a history of prior stem cell transplant, and 16 patients had active disease prior to their haplo-SCT. Seven patients had sex mismatch with their stem cell donor. Median donor age was 42 (range 21-71). 20 patient deaths occurred by July 2019 with a median follow up of 244 days for surviving patients. Nine patients died before day +100 (D100, "early mortality"), with a D100 non-relapse mortality (NRM) rate of 24%. Median overall and relapse free survival (OS and RFS, respectively) were 197 days (95% CI 142-not reached) and 180 days (95% CI 141-not reached), respectively, for the entire cohort. The 1 year OS and NRM were 29% and 50%. The incidence of grades 2-4cytokine release syndrome (CRS) was 66%, and 52% of these patients were treated with tocilizumab. CRS was strongly associated with early mortality, with D100 NRM of 36% in patients with grade 2-4 CRS compared to 0% in those with grade 0-1. The incidence of acute kidney injury (AKI) was 64% in patients with grade 2-4 CRS, and 8% in those without (p < 0.001). 28% of patients with AKI required dialysis. Grade 2-4 CRS was seen in 54% of patients in remission prior to haplo-SCT and in 92% of those with active disease (p = 0.02). Of the 9 patients with early mortality, 89% had AKI, 44% needed dialysis, and 100% had grade 2-4 CRS, compared to 31%, 10%, and 55% in those without early mortality (p = 0.002, p = 0.02, p = 0.01). Early mortality was not significantly associated with age, HCT-CI score, second transplant, disease status at transplant, total dose of melphalan, volume overload/diuretic use, or post-transplant infection. In conclusion, we observed a very high rate of NRM with FluMel conditioning and PBSC grafts for haplo-SCT with PTCy. The pattern of toxicity was strongly associated with grade 2-4 CRS, AKI, and need for dialysis. These complications may be mediated by excessive inflammation in the context of allo-reactive donor T-cell over-activation. Consistent with this, multiple groups have shown that FluMel conditioning in haplo-SCT is safe when using bone marrow or T-cell depleted grafts. Based on our institutional experiences, we would discourage the use of FluMel as conditioning for haplo-SCT with PTCy with T-cell replete PBSC grafts. Alternative regimens or variations on melphalan-based regimens, such as fractionated melphalan dosing or inclusion of TBI may improve outcomes but further study and randomized controlled trials are needed. This study is limited in its retrospective design and sample size. Figure Disclosures DiPersio: WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Karyopharm Therapeutics: Consultancy; Magenta Therapeutics: Equity Ownership; Celgene: Consultancy; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Amphivena Therapeutics: Consultancy, Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liesveld:Onconova: Other: Data safety monitoring board; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Plerixafor (Mozobil ®)Plus G-CSF Is Effective in Mobilizing Hematopoietic Stem Cells in Patients with Concurrent Thrombocytopenia Undergoing Autologous Hematopoietic Stem Cell Transplantation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3229-3229 ◽  
Author(s):  
Ivana N Micallef ◽  
Eric Jacobsen ◽  
Paul Shaughnessy ◽  
Sachin Marulkar ◽  
Purvi Mody ◽  
...  
Keyword(s):  
Stem Cell ◽  
Platelet Count ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Platelet Counts ◽  
Cd34 Cells ◽  
Low Platelet Count ◽  
Equity Ownership

Abstract Abstract 3229 Poster Board III-166 Introduction Low platelet count prior to mobilization is a significant predictive factor for mobilization failure in patients with non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD) undergoing autologous hematopoietic stem cell (HSC) transplantation (auto-HSCT; Hosing C, et al, Am J Hematol. 2009). The purpose of this study is to assess the efficacy of HSC mobilization with plerixafor plus G-CSF in patients with concomitant thrombocytopenia undergoing auto-HSCT. Methods Patients who had failed successful HSC collection with any mobilization regimen were remobilized with plerixafor plus G-CSF as part of a compassionate use program (CUP). Mobilization failure was defined as the inability to collect 2 ×106 CD34+ cells/kg or inability to achieve a peripheral blood count of ≥10 CD34+ cells/μl without having undergone apheresis. As part of the CUP, G-CSF (10μg/kg) was administered subcutaneously (SC) every morning for 4 days. Plerixafor (0.24 mg/kg SC) was administered in the evening on Day 4, approximately 11 hours prior to the initiation of apheresis the following day. On Day 5, G-CSF was administered and apheresis was initiated. Plerixafor, G-CSF and apheresis were repeated daily until patients collected the minimum of 2 × 106 CD34+ cells/kg for auto-HSCT. Patients in the CUP with available data on pre-mobilization platelet counts were included in this analysis. While patients with a platelet count <85 × 109/L were excluded from the CUP, some patients received waivers and were included in this analysis. Efficacy of remobilization with plerixafor + G-CSF was evaluated in patients with platelet counts ≤ 100 × 109/L or ≤ 150 × 109/L. Results Of the 833 patients in the plerixafor CUP database, pre-mobilization platelet counts were available for 219 patients (NHL=115, MM=66, HD=20 and other=18.). Of these, 92 patients (NHL=49, MM=25, HD=8 and other=10) had pre-mobilization platelet counts ≤ 150 × 109/L; the median platelet count was 115 × 109/L (range, 50-150). The median age was 60 years (range 20-76) and 60.4% of the patients were male. Fifty-nine patients (64.1%) collected ≥2 × 109 CD34+ cells/kg and 13 patients (14.1%) achieved ≥5 × 106 CD34+ cells/kg. The median CD34+ cell yield was 2.56 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 68.5%. The median time to neutrophil and platelet engraftment was 12 days and 22 days, respectively. Similar results were obtained when efficacy of plerixafor + G-CSF was evaluated in 29 patients with platelet counts ≤ 100 × 109/L (NHL=12, MM=10, HD=3 and other=4). The median platelet count in these patients was 83 × 109/L (range, 50-100). The median age was 59 years (range 23-73) and 60.4% of the patients were male. The minimal and optimal cell dose was achieved in 19(65.5%) and 3(10.3%) patients, respectively. The median CD34+ cell yield was 2.92 × 106 CD34+ cells/kg. The proportion of patients proceeding to transplant was 62.1%. The median time to neutrophil and platelet engraftment was 12 days and 23 days, respectively. Conclusions For patients mobilized with G-CSF alone or chemotherapy ±G-CSF, a low platelet count prior to mobilization is a significant predictor of mobilization failure. These data demonstrate that in patients with thrombocytopenia who have failed prior mobilization attempts, remobilization with plerixafor plus G-CSF allows ∼65% of the patients to collect the minimal cell dose to proceed to transplantation. Thus, in patients predicted or proven to be poor mobilizers, addition of plerixafor may increase stem cell yields. Future studies should investigate the efficacy of plerixafor + G-CSF in front line mobilization in patients with low platelet counts prior to mobilization. Disclosures Micallef: Genzyme Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding. Jacobsen:Genzyme Corporation: Research Funding. Shaughnessy:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Marulkar:Genzyme Corporation: Employment, Equity Ownership. Mody:Genzyme Corporation: Employment, Equity Ownership. van Rhee:Genzyme Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Deregulation of TNFRSF18 (GITR) Through Promoter CpG Island Methylation Induces Tumor Proliferation in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2424-2424
Author(s):  
Yang Liu ◽  
Yong Zhang ◽  
Phong Quang ◽  
Hai T Ngo ◽  
Feda Azab ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Research Funding ◽  
Plasma Cells ◽  
Cpg Island ◽  
Advisory Committees ◽  
Brdu Assay

Abstract Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Blockade of PD-1 in Combination with Dendritic Cell/Myeloma Fusion Cell Vaccination Following Autologous Stem Cell Transplantation Is Well Tolerated, Induces Anti-Tumor Immunity and May Lead to Eradication of Measureable Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4218-4218 ◽  
Author(s):  
Jacalyn Rosenblatt ◽  
Irit Avivi ◽  
Noam Binyamini ◽  
Lynne Uhl ◽  
Poorvi Somaiya ◽  
...  
Keyword(s):  
Stem Cell ◽  
Dendritic Cell ◽  
Stem Cell Transplantation ◽  
Board Of Directors ◽  
Research Funding ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Post Transplant ◽  
Fusion Cell ◽  
Equity Ownership

Abstract Autologous stem cell transplantation (ASCT) for multiple myeloma (MM) offers a unique setting to incorporate immunotherapy in an effort to target residual disease. Our group has developed a cancer vaccine in which dendritic cells (DCs) are fused to autologous tumor cells resulting in the presentation of multiple tumor antigens with the capacity to elicit a broad anti-tumor response. A fundamental challenge to developing a more effective tumor vaccine is overcoming the immunosuppressive milieu by which tumor cells evade host immunity. Up-regulation of the PD-1/PDL1 pathway represents a key element contributing to tumor-mediated tolerance, and potentially muting response to vaccination. We are conducting a clinical trial in which patients with MM are treated with an anti-PD1 antibody (Pidilizumab, MDV9300) in combination with a dendritic cell/myeloma fusion cell vaccine following autologous transplantation. 22 patients have been treated with post-transplant immunotherapy. Mean age was 64. MM cells were isolated from bone marrow and were identified by expression of CD38 or CD138. Mean tumor cell yield was 118x106 cells. Adherent mononuclear cells were isolated from leukapheresis collections and cultured with GM-CSF and IL-4 for 5-7 days, then exposed to TNFα for 48-72 hours to generate mature DCs. DCs expressed co-stimulatory (mean CD86 75%) and maturation markers (mean CD83 50%). DC and MM cells were co-cultured with PEG and fusion cells were quantified by determining the percentage of cells that co-express unique DC and myeloma antigens. Mean fusion efficiency was 41% and the mean cell dose generated was 4 x 106 fusion cells. Mean viability of the DC, myeloma, and fusion preparations was 92%, 89%, and 85%, respectively. As a measure of their potency as antigen presenting cells, DC/MM fusions potently stimulate allogeneic T cell proliferation ex-vivo (Mean stimulation index of 1.9, 9.2 and 7.1 for tumor, DC and DC/myeloma fusions respectively, n=21) Post-transplant immunotherapy was initiated after recovery from transplant-related toxicities. Median time from transplant to initiation of post-transplant immunotherapy was 80 days. Patients received 3 doses of Pidilizumab at 6-week intervals. DC/myeloma fusion cells vaccination is administered 1 week before each dose of Pidilizumab. To date, 22 patients have completed vaccinations and Pidilizumab. Adverse events judged to be potentially treatment related included grade 1-2 diarrhea, arthralgias, myalgias, fatigue, headache, nausea, chills, transaminitis, cytopenia, elevated TSH, and vaccine site reactions. A significant increase in circulatingtumor reactive lymphocytes was noted following post-transplant immunotherapy, as determined by T cell expressionof IFN-γ by CD8 cells following ex-vivo co-culture withautologous myeloma cell lysate. Mean percentage of tumor reactiveCD8 cells increased from 1.8% post-transplant to a peak of 9.16% following immunotherapy. In the post-transplant period, regulatory T cells fell to minimal levels and remained low throughout the period of immunotherapy. 6 patients achieved a best response of VGPR, 6 patients have achieved a nCR/CR, including 3 who converted to CR following immunotherapy. Median PFS from transplant is 19 months with ongoing follow up. In summary, DC/MM fusion cell vaccination in conjunction with PD1 blockade following ASCT was well tolerated, potently induced anti-tumor immunity, and in a subset of patients, resulted in the eradication of post-transplant measurable disease. Disclosures Richardson: Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Laubach:Novartis: Research Funding; Onyx: Research Funding; Celgene: Research Funding; Millennium: Research Funding. Anderson:Celgene: Consultancy; Millennium: Consultancy; BMS: Consultancy; Gilead: Consultancy; Oncopep: Equity Ownership; Acetylon: Equity Ownership. Rowe:BioSight Ltd.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BioLineRx Ltd.: Consultancy. Kufe:Genus Oncology: Consultancy, Equity Ownership.

scholarly journals Characterization of Residual CLL Following Long-Term Bruton Tyrosine Kinase Inhibitor Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2620-2620
Author(s):  
Shanmugapriya Thangavadivel ◽  
Alexander Pan ◽  
Xi Chen ◽  
Chen Song ◽  
Claire Snyder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Immune Cell ◽  
Advisory Committees ◽  
Time Points ◽  
Bruton Tyrosine Kinase ◽  
Over Time

Abstract Introduction: The development of Bruton tyrosine kinase inhibitors (BTKi) and their introduction into clinical practice represent a major advance in the treatment of chronic lymphocytic leukemia (CLL). Ibrutinib and other second generation BTKi as monotherapies generally do not produce minimal residual disease negative (MRD-) complete remissions even with extended therapy. The reason for lack of continued elimination of CLL to a MRD- status over time is unknown, and we hypothesized that biological differences in the CLL cells or immune microenvironment might make them resistant to elimination. Methods: Samples were obtained from patients on continuous ibrutinib who hadn't relapsed at time points of 3 years on treatment and 5 years on treatment; and pre-ibrutinib. Isolated CLL cells were subject to B-cell receptor (BCR) sequencing using NEBNext Immune Sequencing Kit by New England Biolabs (NEB, Inc., USA). In a separate cohort, 10X VDJ+5'-sequencing was performed on peripheral blood mononuclear cells. Flow cytometry and ELISA were used to identify alterations in immune cell subtype and identify immune profiles associated with MRD positive (MRD+) status. Results: To identify the clonal pattern in MRD+, we performed deep sequencing of the BCR repertoire on samples from 13 patients with 3 time points each. We found that dominant clones tended to remain constant, but new clones appeared in later time points (Figure 1). MiXCR (v3.0.5) was used with default parameters to identify preprocessed reads containing CDR3 regions from B-cell heavy, kappa, and lambda chains, generating a list of unique productive and nonproductive CDR3 sequences associated with their relative abundances and specific V(D)J gene usage. Two out of three patients (patients 1 and 3) showed significant change in the clone over time. In patients 1 and 2, we saw that heavy chain clones emerge at later time points. In patient 3 alone, we observed that at 5 years there are two dominant clones. Our findings suggest that each patient shows a diverse repertoire of CLL clones and that the dominant clone does not change significantly across time points. To identify cell populations based on gene expression patterns, we performed 10X VDJ+5'-seq. Based on the expression of known markers, we identified CLL cells and other immune cell subtypes. We identified differentially expressed genes (DEGs) for CLL cells in each time points. Over time, we observed upregulation of CD79a, LTB, TAGLN2, and LGALS, genes typically associated with leukemic cell survival. Suggesting differential expression of pro-survival genes contribute to continued presence of MRD over time. T cells are known to be dysfunctional in CLL and have not previously been extensively studied in the setting of long term BTKi. We performed flow cytometry to determine the repertoire and function of T cells at 3 and 5 years of ibrutinib therapy. We found that the percentage of CD3+ T cells increases at later time points in all the 8 patients (p&lt;0.05). Although T cell numbers increase, we do see skewing of these cells towards a terminally differentiated phenotype (p&lt;0.05). We also observed significant increases in NK cells across time points (p&lt;0.05), albeit non-functional due to high expression of inhibitory receptor KLRG1 in 7 out of 8 patients (p&lt;0.05). Although overall the number of immune cells increase in long time ibrutinib therapy, they exhibit exhausted or non-functional phenotypes. Conclusion: Extended ibrutinib treatment yields a subset of patients who become MRD- whereas a large majority remain MRD+. Our findings suggest that BCR repertoire in CLL MRD might change in long term ibrutinib therapy and induce necessary genes for its survival in the microenvironment. Although T cells and NK cells are non-functional at later time points, better understanding of these subtypes may lead to new strategies and to improve antitumor function of these cells. Differentiating the biology of why certain patients attain MRD- status on BTK inhibitor is of high interest as it could provide rationale for therapy discontinuation or add on approaches. Figure 1 Figure 1. Disclosures Rogers: AbbVie Inc.: Consultancy, Research Funding; Acerta Pharma: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy, Research Funding; Innate Pharma: Consultancy; Pharmacyclics LLC: Consultancy; Janssen Pharmaceuticals, Inc: Research Funding; ovartis Pharmaceuticals Corporation: Research Funding. Bhat: Beigene: Consultancy; AstraZeneca: Consultancy; Aptitude Health: Honoraria; Onclive: Honoraria. Kittai: Bristol-Meyers Squibb: Consultancy; Abbvie: Consultancy; Janssen: Consultancy. Blachly: INNATE: Consultancy, Honoraria; KITE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Newave: Membership on an entity's Board of Directors or advisory committees. Woyach: AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy; AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; Gilead Sciences Inc: Other: Data & Safety.

scholarly journals Comparative Analysis of Independent Ex Vivo functional Drug Screens Identifies Predictive Biomarkers of BCL-2 Inhibitor Response in AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2763-2763 ◽  
Author(s):  
Brian S. White ◽  
Suleiman A. Khan ◽  
Muhammad Ammad-ud-din ◽  
Swapnil Potdar ◽  
Mike J Mason ◽  
...  
Keyword(s):  
Gene Expression ◽  
Board Of Directors ◽  
Research Funding ◽  
Drug Response ◽  
Ex Vivo ◽  
Predictive Performance ◽  
Data Sets ◽  
Advisory Committees ◽  
Data Set ◽  
Equity Ownership

Abstract Introduction: Therapeutic options for patients with AML were recently expanded with FDA approval of four drugs in 2017. As their efficacy is limited in some patient subpopulations and relapse ultimately ensues, there remains an urgent need for additional treatment options tailored to well-defined patient subpopulations to achieve durable responses. Two comprehensive profiling efforts were launched to address this need-the multi-center Beat AML initiative, led by the Oregon Health & Science University (OHSU) and the AML Individualized Systems Medicine program at the Institute for Molecular Medicine Finland (FIMM). Methods: We performed a comparative analysis of the two large-scale data sets in which patient samples were subjected to whole-exome sequencing, RNA-seq, and ex vivo functional drug sensitivity screens: OHSU (121 patients and 160 drugs) and FIMM (39 patients and 480 drugs). We predicted ex vivo drug response [quantified as area under the dose-response curve (AUC)] using gene expression signatures selected with standard regression and a novel Bayesian model designed to analyze multiple data sets simultaneously. We restricted analysis to the 95 drugs in common between the two data sets. Results: The ex vivo responses (AUCs) of most drugs were positively correlated (OHSU: median Pearson correlation r across all pairwise drug comparisons=0.27; FIMM: median r=0.33). Consistently, a samples's ex vivo response to an individual drug was often correlated with the patient's Average ex vivo Drug Sensitivity (ADS), i.e., the average response across the 95 drugs (OHSU: median r across 95 drugs=0.41; FIMM: median r=0.58). Patients with a complete response to standard induction therapy had a higher ADS than those that were refractory (p=0.01). Further, patients whose ADS was in the top quartile had improved overall survival relative to those having an ADS in the bottom quartile (p<0.05). Standard regression models (LASSO and Ridge) trained on ADS and gene expression in the OHSU data set had improved ex vivo response prediction performance as assessed in the independent FIMM validation data set relative to those trained on gene expression alone (LASSO: p=2.9x10-4; Ridge: p=4.4x10-3). Overall, ex vivo drug response was relatively well predicted (LASSO: mean r across 95 drugs=0.62; Ridge: mean r=0.62). The BCL-2 inhibitor venetoclax was the only drug whose response was negatively correlated with ADS in both data sets. We hypothesized that, whereas the predictive performance of many other drugs was likely dependent on ADS, the predictive performance of venetoclax (LASSO: r=0.53, p=0.01; Ridge: r=0.63, p=1.3x10-3) reflected specific gene expression biomarkers. To identify biomarkers associated with venetoclax sensitivity, we developed an integrative Bayesian machine learning method that jointly modeled both data sets, revealing several candidate biomarkers positively (BCL2 and FLT3) or negatively (CD14, MAFB, and LRP1) correlated with venetoclax response. We assessed these biomarkers in an independent data set that profiled ex vivo response to the BCL-2/BCL-XL inhibitor navitoclax in 29 AML patients (Lee et al.). All five biomarkers were validated in the Lee data set (Fig 1). Conclusions: The two independent ex vivo functional screens were highly concordant, demonstrating the reproducibility of the assays and the opportunity for their use in the clinic. Joint analysis of the two data sets robustly identified biomarkers of drug response for BCL-2 inhibitors. Two of these biomarkers, BCL2 and the previously-reported CD14, serve as positive controls credentialing our approach. CD14, MAFB, and LRP1 are involved in monocyte differentiation. The inverse correlation of their expression with venetoclax and navitoclax response is consistent with prior reports showing that monocytic cells are resistant to BCL-2 inhibition (Kuusanmäki et al.). These biomarker panels may enable better selection of patient populations likely to respond to BCL-2 inhibition than would any one biomarker in isolation. References: Kuusanmäki et al. (2017) Single-Cell Drug Profiling Reveals Maturation Stage-Dependent Drug Responses in AML, Blood 130:3821 Lee et al. (2018) A machine learning approach to integrate big data for precision medicine in acute myeloid leukemia, Nat Commun 9:42 Disclosures Druker: Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Fred Hutchinson Cancer Research Center: Research Funding; Celgene: Consultancy; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Patient True Talk: Consultancy; Millipore: Patents & Royalties; Monojul: Consultancy; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Bristol-Meyers Squibb: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Novartis Pharmaceuticals: Research Funding. Heckman:Orion Pharma: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Tyner:AstraZeneca: Research Funding; Incyte: Research Funding; Janssen: Research Funding; Leap Oncology: Equity Ownership; Seattle Genetics: Research Funding; Syros: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Aptose: Research Funding; Agios: Research Funding. Aittokallio:Novartis: Research Funding. Wennerberg:Novartis: Research Funding.

scholarly journals Biomarkers of Overall Survival and Response to Glasdegib and Intensive or Non-Intensive Chemotherapy in Patients with Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2733-2733 ◽  
Author(s):  
Jorge E. Cortes ◽  
Akil Merchant ◽  
Catriona Jamieson ◽  
Daniel A Pollyea ◽  
Michael Heuser ◽  
...  
Keyword(s):  
Gene Expression ◽  
Board Of Directors ◽  
Research Funding ◽  
Intensive Therapy ◽  
Employment Equity ◽  
Advisory Committees ◽  
Expression Levels ◽  
Biomarker Analysis ◽  
Cytokine Levels ◽  
Equity Ownership

Abstract Background: In a previously reported Phase 2 randomized study of patients with acute myeloid leukemia (AML), addition of the investigational agent glasdegib (PF-04449913) to low-dose cytarabine (LDAC) improved overall survival (OS) when compared with LDAC alone. In a non-randomized study arm, glasdegib together with 7+3 chemotherapy was well tolerated and associated with clinical activity. We used a comprehensive biomarker analysis, evaluating gene expression, circulating cytokine levels, and gene mutations, to identify molecular drivers that predict overall response (OR) and OS. Methods: In this Phase 2 multicenter study (NCT01546038), patients with AML who were suitable for non-intensive therapy were randomized (2:1) to LDAC + glasdegib 100 mg QD or LDAC alone, and patients suitable for intensive therapy were assigned 7+3 plus glasdegib 100 mg QD. Whole blood, serum, and bone marrow aspirate samples were collected at baseline, and used to assess 19 genes for expression analysis, 38 analytes for circulating cytokine levels, and 109 genes for mutation analysis. Gene expression was analyzed using TaqMan Low Density Array Cards (TLDCs), cytokine levels were analyzed using quantitative, multiplexed immunoassays (Myriad RBM), and mutation analysis was performed using the Illumina® MiSeq instrument (San Diego, CA). All correlations were performed either for OS or for OR. For gene expression and cytokine analysis, a cut-off value above or below the median expression level for each treatment arm was used to separate samples into two subgroups (< or ≥ the median value) to explore the relationship of expression levels with OS data. Criteria for significance in the non-intensive cohort required one subgroup to have a p-value of <0.05 in the between-treatment arms comparison and the HR difference between the two subgroups to be ≥2 fold. Responses were defined as patients with a complete remission (CR), CR with incomplete blood count recovery (CRi), morphologic leukemia-free state, partial remission (PR), or PRi. For response correlations, genes or cytokines were considered to be differentially expressed if they had a p-value <0.05 and were differentially expressed by ≥2-fold. Results: Within the non-intensive arm (LDAC + glasdegib, n=68; LDAC alone, n=30), expression levels of several genes correlated with improved OS with glasdegib plus LDAC. Lower levels of expression of FOXM1 and MSI2, and higher expression levels of BCL2 and CCND2 correlated with improved OS with the combination. Additionally, lower levels of the cytokines 6CKINE (CCL21), ICAM-1, MIP-1α, and MMP-3 correlated with improved OS. An analysis of correlations of gene expression and cytokine levels with OR could not be completed due to the low number of responders in the LDAC only group (n=2). In the intensive treatment arm (glasdegib and 7+3, n=59), higher PTCH1 expression correlated with improved OS (p=0.0219, median OS 10.8 versus 39.5 months). In this cohort, lower levels of IL-8 (p=0.0225) and MIP-3β (p=0.0403) correlated with lower OS. Expression levels of no genes or cytokines significantly correlated with OR in this arm. We also examined correlations between gene mutation status and OS in both study arms. In the non-intensive arm (LDAC + glasdegib, n=58; LDAC alone, n=25), no genes mutated in at least 5 patients correlated with OS. In the intensive treatment arm (n=47), mutations in FLT3, TP53, CEP170, NPM1, and ANKRD26 correlated with OS (all p<0.05). Patients in this arm with FLT3 mutations responded better than patients with wild type FLT3 (p=0.0336, median OS of 13.1 months versus unreached for FLT3 mutant). Conclusions: In this biomarker analysis, we found that expression levels of a select number of genes and circulating cytokines implicated in AML correlated with OS in the non-intensive and the intensive arms. The improved response for patients with FLT3 mutations and high PTCH1 expression levels in the intensive arm deserves further investigation. These findings need to be verified in larger controlled studies, which are ongoing. Disclosures Cortes: Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Astellas Pharma: Consultancy, Research Funding; Arog: Research Funding. Pollyea:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy; Celyad: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding; Curis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Heuser:Astellas: Research Funding; Daiichi Sankyo: Research Funding; Sunesis: Research Funding; Tetralogic: Research Funding; Bayer Pharma AG: Consultancy, Research Funding; StemLine Therapeutics: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; BergenBio: Research Funding; Karyopharm: Research Funding. Chan:Pfizer: Employment, Equity Ownership. Wang:Pfizer: Employment, Equity Ownership. Ching:Pfizer Inc: Employment, Equity Ownership. Johnson:Pfizer Inc: Employment, Equity Ownership. O'Brien:Pfizer Inc: Employment, Equity Ownership.

scholarly journals Development and Evaluation of a Human Single Chain Variable Fragment (scFv) Derived Bcma Targeted CAR T Cell Vector Leads to a High Objective Response Rate in Patients with Advanced MM

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 742-742 ◽  
Author(s):  
Eric L Smith ◽  
Sham Mailankody ◽  
Arnab Ghosh ◽  
Reed Masakayan ◽  
Mette Staehr ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Employment Equity ◽  
Advisory Committees ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract Patients with relapsed/refractory MM (RRMM) rarely obtain durable remissions with available therapies. Clinical use of BCMA targeted CAR T cell therapy was first reported in 12/2015 for RRMM, and based on small numbers, preliminary results appear promising. Given that host immune anti-murine CAR responses have limited the efficacy of repeat dosing (Turtle C. Sci Trans Med 2016), our goal was to develop a human BCMA targeted CAR T cell vector for clinical translation. We screened a human B cell derived scFv phage display library containing 6x1010 scFvs with BCMA expressing NIH 3T3 cells, and validated results on human MM cell lines. 57 unique and diverse BCMA specific scFvs were identified containing light and heavy chain CDR's each covering 6 subfamilies, with HCDR3 length ranges from 5-18 amino acids. 17 scFvs met stringent specificity criteria, and a diverse set was cloned into CAR vectors with either a CD28 or a 4-1BB co-stimulatory domain. Donor T cells transduced with BCMA targeted CAR vectors that conveyed particularly desirable properties over multiple in vitro assays, including: cytotoxicity on human MM cell lines at low E:T ratios (&gt;90% lysis, 1:1, 16h), robust proliferation after repeat antigen stimulation (up to 700 fold, stimulation q3-4d for 14d), and active cytokine profiling, were selected for in vivo studies using a marrow predominant human MM cell line model in NSG mice. A single IV injection of CAR T cells, either early (4d) or late (21d) after MM engraftment was evaluated. In both cases survival was increased when treated with BCMA targeted CAR T cells vs CD19 targeted CAR T cells (median OS at 60d NR vs 35d p&lt;0.05). Tumor and CAR T cells were imaged in vivo by taking advantage of luciferase constructs with different substrates. Results show rapid tumor clearance, peak (&gt;10,000 fold) CAR T expansion at day 6, followed by contraction of CAR T cells after MM clearance, confirming the efficacy of the anti-BCMA scFv/4-1BB containing construct. Co-culture with primary cells from a range of normal tissues did not activate CAR T cells as noted by a lack of IFN release. Co-culture of 293 cells expressing this scFv with those expressing a library of other TNFRSF or Ig receptor members demonstrated specific binding to BCMA. GLP toxicity studies in mice showed no unexpected adverse events. We generated a retroviral construct for clinical use including a truncated epithelial growth factor receptor (EGFRt) elimination gene: EGFRt/hBCMA-41BBz. Clinical investigation of this construct is underway in a dose escalation, single institution trial. Enrollment is completed on 2/4 planned dose levels (DL). On DL1 pts received cyclophosphamide conditioning (3g/m2 x1) and 72x106 mean CAR+ T cells. On DL2 pts received lower dose cyclophosphamide/fludarabine (300/30 mg/m2 x3) and 137x106 mean CAR+ T cells. All pts screened for BCMA expression by IHC were eligible. High risk cytogenetics were present in 4/6 pts. Median prior lines of therapy was 7; all pts had IMiD, PI, high dose melphalan, and CD38 directed therapies. With a data cut off of 7/20/17, 6 pts are evaluable for safety. There were no DLT's. At DL1, grade 1 CRS, not requiring intervention, occurred in 1/3 pts. At DL2, grade 1/2 CRS occurred in 2/3 pts; both received IL6R directed Tocilizumab (Toci) with near immediate resolution. In these 2 pts time to onset of fever was a mean 2d, Tmax was 39.4-41.1 C, peak CRP was 25-27mg/dl, peak IL6 level pre and post Toci were 558-632 and 3375-9071 pg/ml, respectively. Additional serum cytokines increased &gt;10 fold from baseline in both pts include: IFNg, GM CSF, Fractalkine, IL5, IL8, and IP10. Increases in ferritin were limited, and there were no cases of hypofibrinogenemia. There were no grade 3-5 CRS and no neurotoxicities or cerebral edema. No pts received steroids or Cetuximab. Median time to count recovery after neutropenia was 10d (range 6-15d). Objective responses by IMWG criteria after a single dose of CAR T cells were observed across both DLs. At DL1, of 3 pts, responses were 1 VGPR, 1 SD, and 1 pt treated with baseline Mspike 0.46, thus not evaluable by IMWG criteria, had &gt;50% reduction in Mspike, and normalization of K/L ratio. At DL2, 2/2 pts had objective responses with 1 PR and 1 VGPR (baseline 95% marrow involvement); 1 pt is too early to evaluate. As we are employing a human CAR, the study was designed to allow for an optional second dose in pts that do not reach CR. We have treated 2 pts with a second dose, and longer follow up data is pending. Figure 1 Figure 1. Disclosures Smith: Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: BCMA targeted CAR T cells, Research Funding. Almo: Cue Biopharma: Other: Founder, head of SABequity holder; Institute for Protein Innovation: Consultancy; AKIN GUMP STRAUSS HAUER & FELD LLP: Consultancy. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xu: Eureka Therapeutics, Inc: Employment, Equity Ownership. Park: Amgen: Consultancy. Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Dogan: Celgene: Consultancy; Peer Review Institute: Consultancy; Roche Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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