46 results on '"Bjørn-Tore Gjertsen"'
Search Results
2. Prospective Validation of CD34+CD38- Leukemic Stem Cell Frequency in the HOVON-SAKK 132 Trial: Perspectives for Future Improvements
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Lok Lam Ngai, Diana Hanekamp, Fleur Janssen, Jannemieke Carbaat-Ham, Maaike A.M. Hofland, Mona M.H.E Fayed, Angèle Kelder, Laura Oudshoorn-van Marsbergen, Willemijn J. Scholten, Alexander N. Snel, Costa Bachas, Jesse M. Tettero, Dimitri A. Breems, Thomas Fischer, Bjørn Tore Gjertsen, Laimonas Griškevičius, Gunnar Juliusson, Johan A. Maertens, Markus G Manz, Thomas Pabst, Jakob R. Passweg, Kimmo Porkka, Patrycja Gradowska, Bob Lowenberg, David C. de Leeuw, Jeroen J.W.M. Janssen, Gert J. Ossenkoppele, and Jacqueline Cloos
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
3. Measurable Residual Disease Guided Therapy in Intermediate-Risk AML Patients Compared to an Unguided Cohort Using Propensity Score Matching
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Jesse M. Tettero, Lok Lam Ngai, Costa Bachas, Dimitri A. Breems, Thomas Fischer, Bjørn Tore Gjertsen, Patrycja Gradowska, Laimonas Griskevicius, Jeroen J.W.M. Janssen, Gunnar Juliusson, Johan A. Maertens, Markus G Manz, Thomas Pabst, Jakob Passweg, Kimmo Porkka, Peter JM Valk, Bob Lowenberg, Gert J. Ossenkoppele, and Jacqueline Cloos
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
4. Bemcentinib (Oral AXL Inhibitor) in Combination with Low-Dose Cytarabine Is Well Tolerated and Efficacious in Older Relapsed AML Patients.Updates from the Ongoing Phase II Trial (NCT02488408) and Preliminary Translational Results Indicating Bemcentinib Elicits Anti-AML Immune Responses
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Isabel Ben Batalla, Lisa-Marie Rieckmann, Walter Fiedler, Michael Heuser, Melanie Janning, Monica Hellesøy, Claudia Gorcea-Carson, Daniele Mattei, Grerk Sutamtewagul, Hani Gabra, Yesid Alvarado, Nicola Di Renzo, Charles D. Imbusch, Monica Crugnola, Bjørn Tore Gjertsen, Silke Kapp-Schwoerer, Jonas S. Waizenegger, Niklas Beumer, Jörg Chromik, Jaya Nautiyal, Sonja Loges, David Micklem, Roberto M. Lemoli, Austin Rayford, Tzivia Berkman-Gottlieb, and James B. Lorens
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Oncology ,medicine.medical_specialty ,Immune system ,business.industry ,Internal medicine ,Immunology ,medicine ,Low dose cytarabine ,Cell Biology ,Hematology ,business ,Biochemistry - Abstract
Background: Relapsed (REL) & refractory (REF) r/r AML pts unsuitable for intensive therapy (IT) due to age or co-morbidities, have limited treatment options. Low-dose cytarabine (LDAC) demonstrated limited survival benefit (mOS ≤6 months), highlighting the significant unmet need for new treatments in this patient population. Bemcentinib (BEM) is an orally bioavailable, highly selective AXL-inhibitor. AXL is a receptor tyrosine kinase conferring poor prognosis, resistance to chemotherapy and decreased antitumor immune response. AXL is overexpressed on leukemic cells, especially in the stem cell compartment, thus representing an important novel target in AML. Aims: The ongoing BGBC003 PhII trial cohorts receiving BEM+LDAC (B+L) include newly diagnosed (ND) and r/r AML pts unfit for IT. Based on the initial efficacy signal observed, the r/r AML patient sub-group was selected for an expansion cohort, to further explore safety and efficacy and to pursue translational biomarker analysis. Here, we present preliminary efficacy data in r/r pts, with a safety overview for all pts treated with B+L. We additionally include translational plasma biomarker and multiomics data from bone marrow mononuclear cells (BMMNC). Methods: Pts received the combination of BEM at the RP2D (200mg PO/d) and LDAC SoC schedule. Efficacy endpoints were objective response (OR) and clinical benefit (OR+unchanged [UC]+stable disease [SD=UC for at least 3 BEM cycles]). Secondary objectives looked at overall survival (OS) and exploratory biomarker analyses. Longitudinal BMMNC samples (n=36) from 15 patients were subjected to scRNA-seq and CiteSeq (Chromium 10x genomics; TotalSeq, Biolegend). For scRNA-seq data analyses, Cell Ranger (v3.1.0) and the Seurat (v.4.0.1) in R (v.4.0) were used. Pts were stratified by best response: CR, CRi, PR for Responders; SD, UC, PD for Non-Responders. Cell type annotations were based on the identified clusters and inferred from the expression of known markers at both RNA and protein level. Results: As of 15 July 2021, the B+L cohorts comprised 27 r/r (20 REL, 7 REF) AML pts. Median prior lines of therapy: 1 [1-8] in REL, 3 [1-4] in REF. Median age: 75.5yrs [66-86] for REL, 75yrs [71-81] for REF. Adverse cytogenetic risk profile: 6/18 (33%) in REL; 2/7 (29%) in REF. 17/20 REL pts were evaluable for efficacy (BM assessment post-baseline). 4/17 (24%) achieved remission (4 CR/CRi) between wk13(C5)-wk19(C7); 4 pts had SD, 6 pts were UC; observed clinical benefit rate was 82%. Late onset responses may reflect immunological mechanism of action targeting AXL + innate immune cells in REL pts and may also contribute to a longer time-on-treatment (ToT) and survival. Median ToT was 36.9 wks for CR/CRi pts; mDOR 33wks [12.0-69.9]; 4 pts remain on treatment. Median OS currently 13.3 months (historical controls suggest 4.5 months mOS in this population) continues to mature. In contrast, REF pts showed no response (0/7), with 4/7 (57%) demonstrating clinical benefit; mToT 12.0wks for benefitting patients and mOS 5.3 months; no pts ongoing on treatment. Overall, the safety of B+L (compared with previously published BEM monotherapy) is comparable with the known safety profile of LDAC. TRAEs of ≥G3 observed in ≥10% of pts were anaemia (21% B+L; 4% BEM), and ECG QT prolonged (12% B+L; 7% BEM). No G5 TRAEs reported. scRNA and multiomic analysis of longitudinal samples reflect differences in the immune compartment, underscoring the clinical impression of an immune-mediated MOA associated with response to BEM. CD8+ effector T-cells of responding patients demonstrated enhanced pro-inflammatory signatures involving TNF-alpha and IFN-gamma as compared with non-responders. Furthermore, increased activation of B plasma cells was observed in correlation with response to BEM confirming that BEM mediates an anti-AML immune response through activation of the two major adaptive immune cell populations. Conclusion: B+L is well tolerated and offers promising survival benefit to older unfit REL AML patients. Translational research including scRNA and multiomics, identified specific activation of CD8+ T cells and B plasma cells associated with response to treatment, indicating that BEM elicits activation of the two major adaptive immune cell populations responsible for anti-AML immune responses. B+L warrants evaluation in a randomized pivotal trial in this population. Disclosures Loges: BerGenBio ASA: Honoraria, Research Funding, Speakers Bureau; BMS: Research Funding; Eli Lilly: Research Funding; Roche Pharma: Research Funding; ADC Therapeutics: Research Funding; BMS: Honoraria, Speakers Bureau; Boehringer Ingelheim: Honoraria, Speakers Bureau; Eli Lilly: Honoraria, Speakers Bureau; Roche Pharma: Honoraria, Speakers Bureau; Medac GmbH and Sanofi Aventis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; AstraZeneca: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Bayer: Honoraria, Speakers Bureau. Heuser: Astellas: Research Funding; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharma AG: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; BergenBio: Research Funding; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees. Kapp-Schwoerer: BerGenBio ASA: Research Funding. Lemoli: Celgene: Other: Support for attending meetings and/or travel; Jazz, Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie, Daiichi Sankyo, Servier: Honoraria, Speakers Bureau. Ben Batalla: BerGenBio ASA: Research Funding. Hellesøy: BerGenBio ASA: Research Funding. Rayford: BerGenBio ASA: Current Employment. Nautiyal: BerGenBio ASA: Current Employment. Berkman-Gottlieb: BerGenBio ASA: Consultancy, Ended employment in the past 24 months. Micklem: BerGenBio ASA: Current Employment, Current equity holder in publicly-traded company. Gabra: BerGenBio ASA: Current Employment, Current equity holder in publicly-traded company. Gorcea-Carson: BerGenBio ASA: Current Employment. Lorens: BerGenBio ASA: Current equity holder in publicly-traded company, Ended employment in the past 24 months, Patents & Royalties. Fiedler: Servier: Consultancy, Other: support for meeting attendance; Amgen: Consultancy, Other: support for meeting attendance, Patents & Royalties, Research Funding; ARIAD/Incyte: Consultancy; Daiichi Sankyo: Consultancy, Other: support for meeting attendance; Stemline: Consultancy; Abbvie: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Other: support for meeting attendance; Novartis: Consultancy; Pfizer: Consultancy, Research Funding; Celgene: Consultancy; Morphosys: Consultancy. Alvarado: Jazz Pharmaceuticals: Research Funding; BerGenBio: Research Funding; Daiichi-Sankyo: Research Funding; FibroGen: Research Funding; CytomX Therapeutics: Consultancy; Astex Pharmaceuticals: Research Funding; MEI Pharma: Research Funding; Sun Pharma: Consultancy, Research Funding. Gjertsen: BerGenBio: Consultancy; Pfizer Inc.: Consultancy; Alden Cancer Therapy: Current holder of stock options in a privately-held company; KinN Therapeutics: Current holder of stock options in a privately-held company; Novartis: Consultancy.
- Published
- 2021
5. The Transmembrane Protein CD37 Is Not a Promising Therapeutic Target in Acute Myeloid Leukaemia
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Bjørn Tore Gjertsen, Geir Bredholt, Stein-Erik Gullaksen, Øystein Bruserud, Tara Helen Dowling Dowling Steinsland, Emmet McCormack, Mihaela Popa, Calum Leitch, Jostein Dahle, Mireia Mayoral Safont, Helen Heyerdahl, and Astri Maaland
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education.field_of_study ,business.industry ,medicine.medical_treatment ,Immunology ,Population ,Myeloid leukemia ,Cancer ,Cell Biology ,Hematology ,Immunotherapy ,medicine.disease ,Biochemistry ,Targeted therapy ,Haematopoiesis ,chemistry.chemical_compound ,Monomethyl auristatin E ,chemistry ,Cancer research ,Medicine ,Progenitor cell ,business ,education - Abstract
Acute myeloid leukaemia is a disease of the haematopoietic stem and progenitor cells distinguished by an impairment of proliferation, propagation and differentiation of the haematopoietic lineage[1]. AML is a heterogenous disease, where patient stratification and personalised therapeutic strategies are of importance[2]. The transmembrane protein CD37 has been associated with regulation of cell to cell interaction and proliferation in both B and T cells. Mice knockout studies have suggested that CD37 is not crucial for B cell development, however, it is highly expressed in malignant and suboptimal conditions and thus represent a good target for B-cell malignancies[3]. Pereira et al. reported that their anti-CD37 antibody-drug conjugate was a potential therapeutic in AML[4]. Their study revealed anti-leukemic effect in primary AML material and cell lines bothin vivoandin vitro. An investigation of CD37 expression in AML patients is needed to understand the relevance of CD37 immunotherapy. To investigate the relevance of targeted therapy of CD37 the chimeric mAb NNV003 anti-CD37 antibody was evaluated in AML samples. Single-cell time-of-flight mass cytometry (CyTOF), an established tool to analyse the immune signalling in bone marrow and peripheral blood, was used to investigate CD37 expression in 59 AML patient samples. A panel was designed to categorise hierarchical landscape of AML and the leukemic stem cell population (LSCs). CyTOF identified CD37 expression exclusively in the B-cell population of our 59 patients, whereas the AML blast population was negative for CD37 expression. Flow cytometry, however; identified 5/59 patients with CD37 expression in the leukemic blast population. Although previous studies show that an anti-CD37 drug conjugate showed anti-leukemic effectin vivoandin vitro, our results do not support that it is a promising therapeutic biomarker. We have uncovered that malignant expression of CD37 is not a feature of the AML patients investigated. We do not consider CD37 to be a promising and good target for precision therapy of AML. Kayser, S. and M.J. Levis, Advances in targeted therapy for acute myeloid leukaemia. Br J Haematol, 2018. 180(4): p. 484-500.Patel, J.P., et al., Prognostic relevance of integrated genetic profiling in acute myeloid leukemia. N Engl J Med, 2012. 366(12): p. 1079-89.Payandeh, Z., et al., Anti-CD37 targeted immunotherapy of B-Cell malignancies. Biotechnol Lett, 2018. 40(11-12): p. 1459-1466.Pereira, D.S., et al., AGS67E, an Anti-CD37 Monomethyl Auristatin E Antibody-Drug Conjugate as a Potential Therapeutic for B/T-Cell Malignancies and AML: A New Role for CD37 in AML. Mol Cancer Ther, 2015. 14(7): p. 1650-60. Disclosures Heyerdahl: Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Dahle:Nordic Nanovector ASA:Current Employment, Current equity holder in publicly-traded company.Gjertsen:Novartis:Consultancy;KinN Therapeutics AS:Current equity holder in private company;Pfizer Inc:Consultancy;BerGenBio AS:Consultancy, Research Funding;Alden Cancer Therapy AS:Current equity holder in private company.Mccormack:KinN Therapeutics AS:Current equity holder in private company.
- Published
- 2020
6. Durable Responses Observed in Elderly AML Patients Unfit for Intensive Chemotherapy with First-in Class Selective AXL Inhibitor Bemcentinib (BGB324) in Combination with LDAC: Phase II Open-Label Study
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James B. Lorens, Francesca Re, Monica Hellesøy, Wouter Hanekom, Robert J Holt, Jorge E. Cortes, Carlos E. Vigil, Bjørn Tore Gjertsen, Daniele Mattei, Nicola Di Renzo, Michael Heuser, Isabel Ben Batalla, Peter Paschka, Hassan Aly, Sonja Loges, Walter Fiedler, Muhammad Shoaib, Jörg Chromik, Roberto M. Lemoli, and David Micklem
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Oncology ,medicine.medical_specialty ,business.industry ,Surrogate endpoint ,Immunology ,Cancer ,Cell Biology ,Hematology ,Intensive chemotherapy ,medicine.disease ,Biochemistry ,Chemotherapy regimen ,Cytokine release syndrome ,Open label study ,Internal medicine ,Cytarabine ,Medicine ,business ,Febrile neutropenia ,medicine.drug - Abstract
Background There is an unmet need in treating patients (pts) with AML who are unfit for intense chemotherapy due to advanced age and/or presence of co-morbidities. This population often has poor risk disease features. Low-dose cytarabine (LDAC) monotherapy is a SOC therapy for elderly, unfit pts. The CR rate with LDAC is 18% as reported in the MRC AML14 study, with a median OS of approximately 4-5 months (Burnett et al, 2007). To improve the OS of this challenging patient population, an increase in the CR rate and longer response duration is essential. The receptor tyrosine kinase AXL represents a novel therapeutic target promoting AML cell proliferation and survival and is a negative regulator of anti-tumor immunity. Bemcentinib is a first-in-class, highly selective, potent, oral AXL kinase inhibitor with a favorable safety profile. We have previously reported bemcentinib monotherapy anti-leukemic activity in r/r AML and MDS pts. Bemcentinib treatment was also reported to induce T- and B-cell receptor diversification as well as clonal stabilization have also been reported. We report on cohort B2 of an open-label Phase II study (BGBC003), designed to explore the safety and efficacy of bemcentinib given in combinations with LDAC to pts with AML unfit for intensive chemotherapy. Secondary objectives are OS and exploratory biomarker analyses. Methods In Part B2, AML pts unfit for intensive chemotherapy received bemcentinib at the RP2D (200mg PO/d) + SOC LDAC SQ for 10 days in 21-day cycles (dosing according to standard practice). Primary endpoints include identifying safety and tolerability of combination, and secondary endpoints include OR, RFS and OS. Plasma protein biomarker levels including soluble AXL (sAXL) were measured using the DiscoveryMap v3.3 panel (Myriad RBM) at baseline and following treatment. Results A total of 16 pts were enrolled. Median age was 76 years (range 66-83). Of the 13 pts with available cytogenetic results, 5 pts (38%) had a poor risk cytogenetic profile, 7 (54%) were intermediate, 1 (8%) favorable. Bone marrow results showed a median of 35% myeloblasts at screen (range 3-96%). As of July 2019, 15 pts were evaluable for response by BM aspirate on or after C2D1. Six of the evaluable pts had objective responses (ORR 40%, 4 CR/CRis + 2 PRs). Five of the responses (4 CR/CRi + 1 PR) were durable, ranging from 3.4 to 11.7 mos, all still ongoing. All 5 pts with durable responses were >75 years old (range: 76-83). Survival of these 5 pts ranged from 8.8-12.4 months at cut-off, with no relapse or significant events yet reported. Of these 5 pts, 1 had relapsed disease, 3 had secondary AML, and 2 had unfavorable cytogenetics. Additionally, 3 pts (aged 71-78 years old) had durable stable disease lasting approximately 4 mos, with 1 remaining on study. Of the 3 pts, 1 had newly-diagnosed and 2 refractory AML, and at least 1 had secondary AML. The RFS and OS data is not yet mature and will be reported at a later stage. The combination was generally well tolerated with expected and manageable AE profile, with pts continuing the combination up to >19 cycles at cut-off. All pts experienced at least one AE regardless of relation to study treatment, the most common being diarrhea (7/16, 44%) and anaemia (6/16, 38%). The most common treatment-related AEs (TRAEs) were diarrhea (4/16, 25%), electrocardiogram QTc prolongation (3/16, 19%) and decreased platelet count (3/16, 19%). Of these TRAEs, 1 drug interruption and 1 withdrawal were reported; none of these events were reported as SAEs. SAEs occurring in >1 patient were febrile neutropenia (4/16, 25%; 1 related to study drug), and pyrexia (2/16, 12.5%; not related to study drug). These SAEs are typically observed in AML. Conclusions The combination of bemcentinib + LDAC induced a response rate of 40% in pts with newly-diagnosed and relapsed AML. CRs were durable, lasting more than 11 mos in some pts. Durable responses were seen in elderly AML pts including those with adverse disease features. AEs reported were as expected in this population. The DOR of the combination has the potential to improve the OS in this population. Further biomarker exploration will be reported at the conference. Encouraging results in this elderly pt population will lead to further clinical development with this treatment combination. Disclosures Loges: BerGenBio ASA: Honoraria, Other: Advisory Board, Travel Support, Research Funding. Heuser:Synimmune: Research Funding; Bayer Pharma AG, Berlin: Research Funding. Paschka:Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Takeda: Other: Travel expenses; Agios: Membership on an entity's Board of Directors or advisory committees; BMS: Other: Travel expenses, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sunesis: Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Travel expenses; Otsuka: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees, Travel expenses; Amgen: Other: Travel expenses; Abbvie: Other: Travel expenses. Micklem:BerGenBio ASA: Employment, Equity Ownership. Holt:BerGenBio ASA: Employment. Lorens:BerGenBio ASA: Employment, Equity Ownership. Shoaib:BerGenBio ASA: Consultancy. Aly:BerGenBio ASA: Consultancy. Hanekom:BerGenBio ASA: Consultancy. Fiedler:Amgen, Pfizer, Novartis, Jazz Pharmaceuticals, Ariad/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen, Pfizer, Abbvie: Other: Support in medical writing; Amgen, Jazz Pharmaceuticals, Daiichi Sanchyo Oncology, Servier: Other: Support for meeting attendance; Amgen: Research Funding. Cortes:Biopath Holdings: Consultancy, Honoraria; BiolineRx: Consultancy; Immunogen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Sun Pharma: Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding. Gjertsen:Research Council of Norway: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; ACTII AS: Equity Ownership; Seattle Genetics: Consultancy; BerGenBio AS: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy; Helse Vest Health Trust: Research Funding; ERA PerMed: Research Funding; Haukeland University Hospital / University of Bergen: Employment; KinN Therapeutics AS: Equity Ownership; EU Horizon 2020: Research Funding; Daiichi Sankyo: Consultancy; BerGenBio: Consultancy; The Norwegian Cancer Society: Research Funding.
- Published
- 2019
7. Pharmacodynamic Responses to CC-90009, a Novel Cereblon E3 Ligase Modulator, in a Phase I Dose-Escalation Study in Relapsed or Refractory Acute Myeloid Leukemia (R/R AML)
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Daniel J. DeAngelo, Mark D. Minden, Pau Montesinos, Yngvar Fløisand, Suzana Couto, Bjørn Tore Gjertsen, María Belén Vidriales, Geoffrey L. Uy, Michael Pourdehnad, Jessica K. Altman, Jinhong Fan, Amer M. Zeidan, Daniel W. Pierce, Paresh Vyas, Tonia J. Buchholz, Jamie Koprivnikar, Hongbin Wang, and Tsun-Wen Sheena Yao
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medicine.diagnostic_test ,business.industry ,Cereblon ,education ,Immunology ,Myeloid leukemia ,Cancer ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Flow cytometry ,medicine.anatomical_structure ,Refractory ,Pharmacodynamics ,medicine ,Cancer research ,Immunohistochemistry ,Bone marrow ,business ,health care economics and organizations - Abstract
Background: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD), which is currently under investigation in a first-in-human, phase I study (CC-90009-AML-001; NCT02848001) in patients with R/R AML. In preclinical models, CC-90009 drives the binding of the target protein, translation termination factor G1 to S phase transition 1 (GSPT1), to cereblon and induces its ubiquitination and proteasome-dependent degradation. Loss of GSPT1 results in activation of the integrated stress response (ISR), inhibition of nonsense-mediated decay (NMD), and induction of apoptosis. Deep degradation of GSPT1, mediated by CC-90009, led to AML cell death in vitro and potent antitumor activity in patient-derived AML xenograft models. In the ongoing phase I study, CC-90009 has demonstrated antileukemic activity. Here, we characterize the pharmacodynamic responses using a suite of novel assays to support CC-90009 dose and schedule optimization. Methods: Adult patients with R/R AML received intravenous CC-90009 daily on Days 1-5 (D1-5 schedule) or on Days 1-3 and 8-10 (D1-3/8-10 schedule) of a 28-day cycle. Peripheral blood samples taken before, during, and after dosing in the first treatment cycle were analyzed. Levels of intracellular GSPT1 in blasts and normal blood cell types were quantitated by flow cytometry analysis. Transcript levels of ISR and NMD variants in peripheral blood mononuclear cells (PBMC) were measured by qPCR. Bone marrow (BM) core biopsies at screening, Cycle 1 Day 5 and 28, and Cycle 2 and 4 Day 28, were analyzed for GSPT1, cleaved caspase 3, and CD34 protein expression by immunohistochemistry. ATF3 and DDIT3 mRNA levels were assessed in BM samples by RNA in situ hybridization. Results: The rate and depth of GSPT1 loss in T cells and in circulating AML blasts increased with dose. A marked reduction in GSPT1 was observed in T cells and blast cells of most patients after the first dose of CC-90009 at all dose levels, and GSPT1 levels approached the assay floor between Days 2 and 5 at doses of 1.2 mg and higher on the D1-5 schedule. At 2.4 mg and higher on the D1-5 schedule, a reduction in GSPT1 levels of > 90% was observed in T cells (19 of 29 patients) and in blast cells (11 of 29 patients), with stronger GSPT1 reductions detected in AML blasts and normal T cells compared with B cells or granulocytes. In the 3 mg D1-5 cohort, patients with sustained GSPT1 reduction in peripheral blasts in the days following treatment had more persistent blast suppression compared with patients showing an earlier rebound of GSPT1. At 3 mg and 3.6 mg dose levels, continuous treatment (D1-5) resulted in slower kinetics of GSPT1 rebound and conferred superior antileukemic activity compared with the intermittent dosing schedule (D1-3/D8-10). In addition to measuring the direct target of CC-90009, GSPT1, we also investigated markers downstream of GSPT1 degradation. Several patients with deep and sustained GSPT1 loss in the high-dose cohorts (2.4 mg and above) showed increased levels of ISR-related transcripts (ATF3 and DDIT3) and NMD-associated splice variants (SRSF3 and SRSF6) in on-treatment PBMC samples. Similarly, in BM, deep GSPT1 loss coincided with induction of ATF3 and DDIT3 mRNA, increased cleaved caspase 3 expression, and reduced CD34+ blasts. These clinical findings are consistent with our preclinical studies in which GSPT1 loss culminated in apoptosis, which may be mediated through activation of ISR and inhibition of NMD pathways. Conclusions: CC-90009 is a novel CELMoD and a first-in-class GSPT1 degrader. A suite of novel pharmacodynamic assays performed on patient-derived peripheral blood cells and BM demonstrated a dose-dependent modulation of GSPT1, and showed that the preclinical mechanisms of ISR induction, NMD inhibition, and apoptosis can be confirmed in AML cells in patients. Deeper and more rapid GSPT1 degradation as well as delayed rebound were associated with more rapid, deeper, and more persistent blast reductions. Characterization of these pharmacodynamic responses in ongoing dose-schedule explorations will help identify the optimal scheme for the expansion phase and provide further insight into the mechanism of clinical response. Disclosures Fan: Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Yao:Celgene Corporation: Employment. Uy:Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Zeidan:Pfizer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Otsuka: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding. Montesinos:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees. DeAngelo:Jazz Pharmaceuticals, Inc.: Consultancy; Takeda Pharmaceuticals: Consultancy; Shire: Consultancy; GlycoMimetics: Research Funding; Pfizer, Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Incyte Corporation: Consultancy; Celgene Corporation: Consultancy; AbbVie, Inc.: Research Funding; Blue print Medicines: Consultancy, Research Funding; Amgen: Consultancy. Altman:Novartis: Consultancy; Cancer Expert Now: Consultancy; Biosight: Other: US Lead; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; France Foundation: Speakers Bureau; prIME Oncology: Speakers Bureau; PeerView: Speakers Bureau; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy, Honoraria, Other: Data Safety and Monitoring Committee. Koprivnikar:Amgen: Speakers Bureau; Abbvie: Speakers Bureau; Pfizer: Honoraria; Novartis: Speakers Bureau. Vyas:Astellas: Speakers Bureau; Pfizer: Speakers Bureau; Abbvie: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding. Fløisand:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Novartis: Honoraria. Gjertsen:Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; EU Horizon 2020: Research Funding; KinN Therapeutics AS: Equity Ownership; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees; ERA PerMed: Research Funding; The Norwegian Cancer Society: Research Funding; Helse Vest Health Trust: Research Funding; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Research Council of Norway: Research Funding; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; ACTII AS: Equity Ownership. Buchholz:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership.
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- 2019
8. Clinical Activity of CC-90009, a Cereblon E3 Ligase Modulator and First-in-Class GSPT1 Degrader, As a Single Agent in Patients with Relapsed or Refractory Acute Myeloid Leukemia (R/R AML): First Results from a Phase I Dose-Finding Study
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Tonia J. Buchholz, Jamie Koprivnikar, Bjørn Tore Gjertsen, Kristen Hege, Suzana Couto, Michael Pourdehnad, Jinhong Fan, Geoffrey L. Uy, Bishoy Hanna, Yngvar Fløisand, Paresh Vyas, Daniel J. DeAngelo, Mark D. Minden, Li Li, Jessica K. Altman, Jordi Esteve, Daniel W. Pierce, Amer M. Zeidan, Pau Montesinos, and María Belén Vidriales
- Subjects
business.industry ,Cereblon ,medicine.medical_treatment ,Immunology ,Myeloid leukemia ,Cancer ,Cell Biology ,Hematology ,Immunotherapy ,medicine.disease ,Biochemistry ,Tumor lysis syndrome ,Leukemia ,medicine.anatomical_structure ,Refractory ,medicine ,Cancer research ,Bone marrow ,business - Abstract
Background: CC-90009 is a cereblon (CRBN) E3 ligase modulator (CELMoD) and a first-in-class small molecule that drives the binding of a novel target protein, G1 to S phase transition 1 (GSPT1), to CRBN, resulting in the proteasome-dependent degradation of GSPT1. GSPT1 plays a central role in mRNA translation, and loss of GSPT1 activates an integrated stress response that leads to AML cell death (Matyskiela ME, et al. Nature. 2016;535:252-7; Zhouravleva G, et al. EBMO J. 1995;14:4065-72). In preclinical testing, CC-90009 is active across a range of AML cell lines and primary AML patient (pt) samples in vitro and in vivo and exerts its GSPT1- and CRBN-dependent effects through rapid induction of apoptosis. Here we share the first clinical results in pts with R/R AML. Methods: Adult pts with R/R AML enrolled in the dose-finding phase of this first-in-human, multicenter, open-label phase 1 study to evaluate tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of CC-90009; and to establish the recommended phase 2 dose and schedule (RP2D) (CC-90009-AML-001; NCT02848001). Dose escalation proceeded via a modified 3 + 3 design. Treatment was by daily intravenous administration on either Days 1-5 (D1-5) or Days 1-3 and 8-10 (D1-3/8-10) of a 28-day cycle. Treatment response was assessed after Cycles 1, 2, and 4 by modified International Working Group 2003 criteria. Safety and preliminary response data are presented for all treated pts. PK and PD were analyzed for evaluable pts. Results: As of May 15, 2019, 45 pts with R/R AML had been treated, including 35 pts on the D1-5 and 10 pts on the D1-3/D8-10 schedule. Median age was 66 years (range 27-81); 73% were male. Most pts (n = 36; 80%) were refractory to their last therapy and 17 pts (38%) were refractory to all prior therapy; 14 pts (31%) had secondary AML. Pts were treated at dose levels from 0.3 to 3.6 mg. Dose-limiting toxicities (DLTs) reported (only in dose levels from 2.4 to 3.6 mg) included hypotension, systemic inflammatory response syndrome (SIRS), hyperbilirubinemia, pneumonitis, and pericarditis with tamponade. Exploration of the 3.6 mg dose level is ongoing; the RP2D has not yet been determined. CC-90009-related grade 3/4 treatment-emergent adverse events (TEAEs) occurred in 23 pts (51%); those occurring in >1 pt were hypocalcemia (22%); hypotension (13%); and hyperbilirubinemia, hyperglycemia, hypophosphatemia, pneumonitis, sepsis, thrombocytopenia, and tumor lysis syndrome (4%). Preclinically identified hypocalcemia was confirmed as a CC-90009 on-target toxicity in the clinic; it was reversible, manageable and did not lead to any treatment discontinuations. The majority of treated pts experienced ≥1 serious TEAE (80%); most were infections (47%). Two (4%) pts experienced TEAEs leading to permanent discontinuation of the study drug. Dose interruptions due to TEAEs occurred in 12 pts (27%) and dose reductions in 2 pts (4%). Of 40 pts who discontinued treatment, 24 (60%) discontinued due to progressive disease or lack of efficacy. Seven pts discontinued treatment due to death; 4 deaths were secondary to progression from AML, 2 due to sepsis and 1 due to hyperglycemic hyperosmolar nonketotic syndrome. Responses to single-agent treatment were observed in pts treated at 3.0 or 3.6 mg on the D1-5 schedule, with a best response of complete remission (CR; n = 1), morphologic CR with incomplete blood count recovery (CRi; n = 1) and morphologic leukemia-free state (MLFS; n = 1). A dose-dependent decrease in GSPT1 levels in peripheral blood blasts and T cells was observed, with a >90% decrease observed for higher dose levels. Evidence of antileukemic activity (decreases in bone marrow and/or peripheral blasts) was seen in pts treated with CC-90009 at 1.2 mg and above with a trend to more sustained reductions at the highest dose levels. Plasma PK analysis demonstrated dose-dependent exposure. Conclusions: In this phase 1 study of CC-90009, a first-in-class agent, evidence of deep GSPT1 degradation, on-target activity and promising antileukemic activity was observed. The observed TEAEs, in addition to those expected in this heavily pretreated R/R AML pt population, were generally well manageable. The study is ongoing with further optimization of dose, schedule and toxicity mitigation. Expansion cohorts in R/R AML and higher-risk myelodysplastic syndromes are planned. Disclosures Uy: GlycoMimetics: Consultancy; Curis: Consultancy; Astellas: Consultancy; Pfizer: Consultancy. Montesinos:Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding. DeAngelo:Blue print Medicines: Consultancy, Research Funding; Celgene Corporation: Consultancy; Shire: Consultancy; Pfizer, Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Incyte Corporation: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; GlycoMimetics: Research Funding; AbbVie, Inc.: Research Funding; Takeda Pharmaceuticals: Consultancy; Amgen: Consultancy. Altman:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Biosight: Other: US Lead; France Foundation: Speakers Bureau; PeerView: Speakers Bureau; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy, Honoraria, Other: Data Safety and Monitoring Committee; Cancer Expert Now: Consultancy; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; prIME Oncology: Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Koprivnikar:Amgen: Speakers Bureau; Pfizer: Honoraria; Abbvie: Speakers Bureau; Novartis: Speakers Bureau. Vyas:Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding; Daiichi Sankyo: Speakers Bureau; Pfizer: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Fløisand:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Novartis: Honoraria. Gjertsen:BerGenBio: 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; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; EU Horizon 2020: Research Funding; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; The Norwegian Cancer Society: Research Funding; KinN Therapeutics AS: Equity Ownership; ACTII AS: Equity Ownership; ERA PerMed: Research Funding; Helse Vest Health Trust: Research Funding; Research Council of Norway: Research Funding. Esteve:Astellas: Consultancy, Speakers Bureau; Amgen: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Roche: Consultancy; Pfizer: Consultancy. Buchholz:Celgene Corporation: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Fan:Celgene Corporation: Employment, Equity Ownership. Hanna:Celgene Corporation: Employment, Equity Ownership. Li:Celgene Corporation: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership. Hege:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties; Mersana Therapuetics: Membership on an entity's Board of Directors or advisory committees; Society for Immunotherapy of Cancer: Membership on an entity's Board of Directors or advisory committees; Arcus Biosciences: Membership on an entity's Board of Directors or advisory committees. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Zeidan:Pfizer: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Cardinal Health: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Honoraria; Otsuka: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Astellas: Honoraria.
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- 2019
9. Efficacy and Safety of Bosutinib By Charlson Comorbidity Index in Previously Treated Patients with Chronic Myeloid Leukemia: Results from the Phase 4 BYOND Study
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Tim H. Brümmendorf, Bjørn Tore Gjertsen, Valentín García Gutiérrez, Gianantonio Rosti, Frank Giles, Eric Leip, Camille N. Abboud, Andrea Viqueira, Andreas Hochhaus, Carlo Gambacorti-Passerini, and Elisabetta Abruzzese
- Subjects
medicine.medical_specialty ,business.industry ,Immunology ,Cell Biology ,Hematology ,Baseline data ,Increased alanine aminotransferase ,University hospital ,Biochemistry ,Discontinuation ,Family medicine ,Charlson comorbidity index ,medicine ,In patient ,Previously treated ,business ,Bosutinib ,medicine.drug - Abstract
Background: Bosutinib is a tyrosine kinase inhibitor (TKI) approved for the treatment of Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) resistant/intolerant to prior therapy and newly diagnosed Ph+ chronic phase (CP) CML. The efficacy and safety of bosutinib by baseline comorbidities in patients with CML resistant/intolerant to prior TKIs were investigated. Methods: The phase 4 BYOND trial (NCT02228382) is further examining efficacy and safety of bosutinib (starting dose 500 mg/day) in patients with CML resistant/intolerant to prior TKIs. Comorbidities were analyzed using the Charlson Comorbidity Index (CCI), a validated measure of the influence of relevant comorbid conditions on mortality. Scores were derived from baseline data and patients grouped by CCI score 2-3, 4-5, and ≥6. This analysis was based on ≥1 year of follow-up and included patients with Ph+ CP CML. Results: 156 patients with Ph+ CP CML (n=42 [26.9%], 48 [30.8%], and 66 [42.3%] for CCI 2-3, 4-5, and ≥6) received bosutinib. Median treatment duration was 28.9, 23.9, and 20.1 months for CCI 2-3, 4-5, and ≥6; median dose intensity was 366.7, 385.3, and 291.5 mg/day. Across CCI groups, a substantial proportion of patients attained/maintained cytogenetic and molecular responses (Table). Grade 3/4 treatment-emergent adverse events (TEAEs) rates were 64.3%, 72.9%, and 80.3% for CCI 2-3, 4-5, and ≥6. Emerging grade 3/4 TEAEs differed between groups; diarrhea and increased alanine aminotransferase (ALT) were more common for CCI 2-3 (21.4% and 23.8%, respectively) and 4-5 (18.8% and 16.7%) and pleural effusion more common for CCI ≥6 (12.1%). For CCI 2-3, 4-5, and ≥6, 19.0%, 20.8%, and 31.8% of patients discontinued treatment due to AEs and 2.4%, 2.1%, and 9.1% due to insufficient response. Most common AEs leading to discontinuation in the respective groups were increased ALT and aspartate aminotransferase (7.1% each), increased ALT (8.3%), and pleural effusion (3.0%). 10 deaths occurred (n=0 [0%], 1 [2.1%], and 9 [13.6%] for CCI 2-3, 4-5, and ≥6), 8 due to AEs (none bosutinib-related), 1 CML-related, and 1 of unknown cause. No on-treatment transformations to advanced CML occurred. Conclusions: Across CCI groups, a majority of patients with Ph+ CP CML achieved/maintained cytogenetic and molecular responses, and only 1 CML-related death was reported. Patients with CCI ≥6 showed a trend toward higher rates of TEAEs, discontinuations due to AEs, and death. Results demonstrate efficacy of bosutinib across CCI scores, including patients with important comorbidities. However, CCI stratification may enable identification of patients at higher risk of developing TEAEs who require more careful monitoring. Disclosures Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding. Brümmendorf:Janssen: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Merck: Consultancy; University Hospital of the RWTH Aachen: Employment; Ariad: Consultancy. Gjertsen:Astellas: Consultancy; The Norwegian Cancer Society: Research Funding; BerGenBio: Consultancy; ERA PerMed: Research Funding; ACTII AS: Equity Ownership; Daiichi Sankyo: Consultancy; Seattle Genetics: Consultancy; Haukeland University Hospital / University of Bergen: Employment; KinN Therapeutics AS: Equity Ownership; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Helse Vest Health Trust: Research Funding; Research Council of Norway: Research Funding; EU Horizon 2020: Research Funding; BerGenBio AS: Membership on an entity's Board of Directors or advisory committees. Abboud:Jazz Pharma: Speakers Bureau; Novartis: Other: Member on an entity's Board of Directors or advisory committees (Ended 12/30/2017), Research Funding; Agios: Other: Member on an entity's Board of Directors or advisory committees (Ended 12/30/2017); Tetraphase Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; NKarta: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bayer: Consultancy, Honoraria. Rosti:BMS: Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Abruzzese:BMS: Consultancy; Incyte: Consultancy; Novartis: Consultancy; Pfizer: Consultancy. Leip:Pfizer: Employment, Equity Ownership. Viqueira:Pfizer Inc: Employment, Equity Ownership. García Gutiérrez:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Incyte: Honoraria, Research Funding. Giles:Epigene Therapeutics Inc: Consultancy, Other: leadership, stock/other ownership ; Novartis: Consultancy; Actuate Therapeutics Inc: Employment. Hochhaus:Pfizer: Research Funding; Novartis: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding.
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- 2019
10. Cross-Intolerance with Bosutinib after Prior Tyrosine Kinase Inhibitors in Patients with Chronic Phase Chronic Myeloid Leukemia: BYOND Phase 4 Study
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Philipp le Coutre, Gianantonio Rosti, Justin M. Watts, Eric Leip, Jorge E. Cortes, Guillermo Ortí, Bjørn Tore Gjertsen, Frank Giles, Carlo Gambacorti-Passerini, Andrea Viqueira, and Andreas Hochhaus
- Subjects
medicine.medical_specialty ,business.industry ,Immunology ,Drug intolerance ,Imatinib ,Cell Biology ,Hematology ,Biochemistry ,Discontinuation ,Dasatinib ,Imatinib mesylate ,Nilotinib ,Internal medicine ,Medicine ,business ,Adverse effect ,Bosutinib ,health care economics and organizations ,medicine.drug - Abstract
Background: Some patients receiving a tyrosine kinase inhibitor (TKI) for chronic phase (CP) Philadelphia chromosome-positive (Ph+) chronic myeloid leukemia (CML) experience drug intolerance and require switching to an alternative TKI. Bosutinib, a TKI approved for newly diagnosed CP Ph+ CML and Ph+ CML resistant/intolerant to prior therapy, has a distinct adverse event (AE) profile vs other TKIs used to treat Ph+ CML. Methods: The ongoing phase 4 BYOND study (NCT02228382) is further evaluating efficacy and safety of bosutinib for CML resistant/intolerant to prior TKIs. Patients with Ph+ CP CML (n=156) previously treated with imatinib, dasatinib, and/or nilotinib received bosutinib (starting dose 500 mg once daily). Cross-intolerance (AEs leading to permanent discontinuation of both prior TKI and bosutinib), recurrent AEs (grades 1/2 or 3/4), and bosutinib dose modifications due to recurrent AEs were assessed across AEs and AE clusters. This analysis was based on ≥1 year after the last enrolled patient (median treatment duration 23.7 months [range 0.2-42.2]). Results: Of 141, 95, and 79 patients who received prior imatinib, dasatinib, or nilotinib, respectively, 63 (45%), 70 (74%), and 60 (76%) were intolerant and discontinued treatment due to an AE as the primary reason. 1 (2%) imatinib-intolerant and 5 (7%) dasatinib-intolerant patients had cross-intolerance with bosutinib; no cross-intolerance with bosutinib was reported for the 60 nilotinib-intolerant patients (Table). 15 patients discontinued >1 TKI due to the same AE. Of these, only 1 with prior imatinib and dasatinib intolerance due to anemia (below), was cross-intolerant to bosutinib. No deaths occurred due to cross-intolerance to bosutinib. Imatinib-intolerant: The most common cause of imatinib intolerance was musculoskeletal pain in 19 patients. Of these, 9 patients experienced grade 1/2 recurrence and 1 had a grade 3/4 AE. There was no cross-intolerance with bosutinib due to musculoskeletal pain. 6 patients were intolerant due to edema, which recurred as grade 1/2 in 3 bosutinib-treated patients. In 2 patients with intolerance due to diarrhea, both had grade 1/2 diarrhea with bosutinib. 2 patients were imatinib-intolerant due to neutropenia or thrombocytopenia (n=1 each) and experienced grade 3/4 recurrence with bosutinib. 1 patient was cross-intolerant to bosutinib due to anemia. Dasatinib-intolerant: The most common reason for prior dasatinib intolerance was pleural effusion. Of 36 patients, 10 (28%) had recurrence of grade 1/2 AE and 4 (11%) had grade 3/4 AE with bosutinib that caused dose delay and reduction in 6 (17%) and 4 (11%) patients, respectively. 1 patient had cross-intolerance with bosutinib due to pleural effusion. Of 8 patients dasatinib-intolerant due to dyspnea, 1 had cross-intolerance with bosutinib (this patient also developed pleural effusion with dasatinib). Of 2 patients with dasatinib intolerance due to pulmonary hypertension, 1 had grade 1/2 and 1 had grade 3/4 recurrence with bosutinib, leading to cross-intolerance in 1 patient. 2 other dasatinib-intolerant patients experienced cross-intolerance due to anemia and nausea (n=1 each). Grade 3/4 recurrence of thrombocytopenia was experienced in 1 of 3 patients with prior dasatinib-intolerance. 1 patient with dasatinib intolerance due to diarrhea had grade 3/4 diarrhea with bosutinib that was managed with dose delay/reduction. Nilotinib-intolerant: 6 patients discontinued nilotinib due to peripheral ischemia, of whom 1 experienced grade 3/4 recurrence with bosutinib. None of the 4 patients with nilotinib intolerance due to acute coronary syndrome had recurrence with bosutinib. Of 3 patients with prior intolerance due to pancreatitis, 1 had grade 3/4 recurrence with bosutinib. 2 patients had recurrent diarrhea with bosutinib, both grade 1/2. There were no bosutinib dose reductions, delays, or recurrence of AEs in patients with prior nilotinib intolerance due to rash, hematologic AEs, hepatotoxicity, or metabolic disorders. Conclusions: Incidence of cross-intolerance, dose delay, or dose reduction with bosutinib in patients intolerant to prior TKIs was low. Despite recurrence of certain same grade 1/2 or grade 3/4 AEs that caused prior TKI intolerance, these were manageable and patients were able to remain on bosutinib. These findings support the use of bosutinib in patients with CP Ph+ CML intolerant to prior TKI treatment. Disclosures Gjertsen: Haukeland University Hospital / University of Bergen: Employment; ERA PerMed: Research Funding; BerGenBio: Consultancy; Astellas: Consultancy; BerGenBio AS: Membership on an entity's Board of Directors or advisory committees; KinN Therapeutics AS: Equity Ownership; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy; ACTII AS: Equity Ownership; Seattle Genetics: Consultancy; Research Council of Norway: Research Funding; EU Horizon 2020: Research Funding; The Norwegian Cancer Society: Research Funding; Helse Vest Health Trust: Research Funding. Hochhaus:Pfizer: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding; Novartis: Research Funding. Rosti:BMS: Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Watts:Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees. Ortí:Bristol-Myers Squibb: Consultancy, Other: Travel Expenses, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy, Other: Travel Expenses, Speakers Bureau. le Coutre:Bristol-Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Leip:Pfizer: Employment, Equity Ownership. Viqueira:Pfizer Inc: Employment, Equity Ownership. Cortes:Biopath Holdings: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Sun Pharma: Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; BiolineRx: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding. Giles:Novartis: Consultancy; Epigene Therapeutics Inc: Consultancy, Other: leadership, stock/other ownership ; Actuate Therapeutics Inc: Employment. Gambacorti-Passerini:Pfizer: Honoraria, Research Funding; Bristol-Meyers Squibb: Consultancy.
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- 2019
11. Moxetumomab Pasudotox-Tdfk in Heavily Pretreated Patients with Relapsed/Refractory Hairy Cell Leukemia (HCL): Long-Term Follow-up from the Pivotal Phase 3 Trial
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Bjørn Tore Gjertsen, Philipp le Coutre, Wyndham H. Wilson, Ira Pastan, Tadeusz Robak, Claire Dearden, Priti Patel, Xavier Troussard, Douglas E. Gladstone, Lionel Karlin, Pier Luigi Zinzani, Julio Delgado, Robert J. Kreitman, Nataliya Kuptsova-Clarkson, Gail J. Roboz, Francis J. Giles, and Shiyao Liu
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Oncology ,medicine.medical_specialty ,business.industry ,Long term follow up ,Immunology ,Disease progression ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Moxetumomab pasudotox ,Internal medicine ,Reference values ,Relapsed refractory ,Absolute neutrophil count ,Medicine ,Hairy cell leukemia ,Rituximab ,business ,medicine.drug - Abstract
Moxetumomab pasudotox-tdfk is a first-in-class recombinant CD22-directed cytotoxin approved in the US for the treatment of adult patients with relapsed/refractory hairy cell leukemia (HCL). The pivotal, multicenter, open-label, single-arm trial (Study 1053; NCT01829711) evaluated the efficacy, safety, immunogenicity, and pharmacokinetics of moxetumomab pasudotox-tdfk monotherapy in patients with relapsed/refractory HCL. Here we present the final analysis, describing the long-term follow-up of patients in Study 1053, with a median follow-up of 24.6 months (range 1.2-71.7). Eligible patients had received ≥ 2 prior systemic therapies, including ≥ 2 courses of a purine nucleoside analog (PNA) or 1 course of a PNA followed by ≥ 1 course of rituximab or a BRAF inhibitor. Patients received moxetumomab pasudotox-tdfk 40 µg/kg intravenously on days 1, 3, and 5 of each 28-day cycle for 6 cycles, or until minimal residual disease (MRD)-negative complete response (CR), disease progression, initiation of alternate therapy, or unacceptable toxicity. Disease response and immunohistochemistry MRD status were determined by blinded independent central review. The primary endpoint was durable CR (achieving CR and maintaining hematologic remission [HR] for > 180 days). Durable CR with HR ≥ 360 days was measured in this final analysis. HR was defined as hemoglobin ≥ 11.0 g/dL, absolute neutrophil count ≥ 1.5 × 103/µL, and platelet count ≥ 100 × 103/µL without receiving transfusions or growth factors within the 4 preceding weeks of assessment. Eighty patients (63 males; median age 60 years [range 34-84]) were enrolled and treated with moxetumomab pasudotox-tdfk. The median number of prior systemic therapies was 3 (range 2-11); 48.8% of patients were PNA-refractory and 37.5% were unfit for PNA retreatment. The median number of treatment cycles administered was 6 (range 1-7). The durable CR rate was 36.3% (29/80 patients; 95% confidence interval [CI]: 25.8-47.8%), durable CR rate with HR ≥ 360 days was 32.5% (26/80 patients; 95% CI: 22.4-43.9%), and the overall CR rate was 41.3% (33/80 patients; 95% CI: 30.4-52.8%) (Table). Overall, 27/33 (81.6%) patients who achieved a CR also achieved MRD-negative status (33.8% of all patients). HR was achieved by 64/80 patients (80.0%). The median duration of HR from CR was 62.8 months (Figure) and median progression-free survival was 41.5 months (range 0.0+ to 71.7). Per the primary analysis, the most frequent treatment-related adverse events (AEs) were nausea (28%), peripheral edema (26%), headache (21%), and pyrexia (20%). Treatment-related Grade 3/4 AEs were reported in 24 patients (30.0%) and serious AEs in 28 (35.0%). Treatment-related AEs led to study drug discontinuation in 8 patients (10.0%): hemolytic uremic syndrome (HUS), n = 4 (5.0%); capillary leak syndrome (CLS), n = 2 (2.5%); and increased blood creatinine, n = 2 (2.5%). Grade ≥ 3 CLS events occurred in 2 patients (2.5%) and Grade ≥ 3 HUS occurred in 5 patients (6.3%). In general, CLS and HUS events were manageable and reversible with appropriate supportive care and monitoring. Based on primary and follow-up analyses of serum creatinine, there was no decline in renal function over time; 12 months post-treatment all mean (SD) serum creatinine laboratory values stayed within normal limits, which suggests current management strategies for HUS (oral hydration, i.v. fluid supplementation on the day of infusion, and the use of dexamethasone) are adequate. With 24.6 months of follow-up, 4 deaths were reported: 2 due to study disease progression and 2 due to an AE (1 each of pneumonia and septic shock). Moxetumomab pasudotox-tdfk treatment was associated with a manageable safety profile. Moxetumomab pasudotox-tdfk achieved a high rate of durable responses, demonstrating the ability to achieve MRD negativity in heavily pretreated patients with HCL. Disclosures Kreitman: Genentech: Research Funding. Dearden:Abbvie: Honoraria; Genentech: Honoraria; Janssen: Honoraria; Sanofi: Honoraria. Zinzani:MSD: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; VERASTEM: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELLTRION: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; GILEAD: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; JANSSEN-CILAG: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; IMMUNE DESIGN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSAPHARMA: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ROCHE: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SERVIER: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANDOZ: Membership on an entity's Board of Directors or advisory committees; KYOWA KIRIN: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; SANOFI: Consultancy; PORTOLA: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Robak:Amgen: Consultancy, Other: Travel grant; UCB: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grant, Research Funding; Roche: Consultancy, Other: Travel grant, Research Funding; Abbvie: Consultancy, Honoraria, Other: Travel grant, Research Funding; Gilead: Consultancy, Research Funding; BeiGene: Consultancy, Research Funding; Acerta: Research Funding; Morphosys AG: Research Funding; Takeda: Consultancy, Research Funding. le Coutre:Novartis: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Gjertsen:Research Council of Norway: Research Funding; Daiichi Sankyo: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; BerGenBio: Consultancy; KinN Therapeutics AS: Equity Ownership; Seattle Genetics: Consultancy; ERA PerMed: Research Funding; Haukeland University Hospital / University of Bergen: Employment; ACTII AS: Equity Ownership; Astellas: Consultancy; BerGenBio AS: Membership on an entity's Board of Directors or advisory committees; The Norwegian Cancer Society: Research Funding; Helse Vest Health Trust: Research Funding; EU Horizon 2020: Research Funding. Troussard:Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Other: Research Support; Sysmex: Other: Research Support. Roboz:Argenx: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Actinium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amphivena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celltrion: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orsenix: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka: 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; Sandoz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trovagene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Karlin:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Support; Celgene: 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, Other: Travel Support. Kuptsova-Clarkson:AstraZeneca: Employment, Equity Ownership. Liu:Acerta Pharma: Employment. Patel:Acerta Pharma: Employment, Equity Ownership; AstraZeneca: Equity Ownership.
- Published
- 2019
12. Final Analysis of the Dose Escalation, Expansion and Biomarker Correlations in the Ph I/II Trial BGBC003 with the Selective Oral AXL Inhibitor Bemcentinib (BGB324) in Relapsed/Refractory AML and MDS
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Jorge E. Cortes, Joerg Chromik, Walter Fiedler, Robert A. Holt, Carlos E. Vigil, Bjørn Tore Gjertsen, Nuray Akyüz, Sonja Loges, Maxim Kebenko, James B. Lorens, Melanie Janning, Michael Heuser, Anthony Brown, David Micklem, Peter Paschka, Mascha Binder, Murray Yule, and Isabel Ben-Batalla
- Subjects
Oncology ,medicine.medical_specialty ,Myeloid ,business.industry ,Concordance ,Immunology ,Attendance ,Phases of clinical research ,Cell Biology ,Hematology ,Biochemistry ,Discontinuation ,Clinical trial ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Internal medicine ,Cohort ,Medicine ,business ,Adverse effect ,030215 immunology - Abstract
Background AXL has been established as an independent prognostic factor in AML. AXL also represents a novel immune checkpoint because it directly inhibits NK cells and suppresses antigen presentation by antigen-presenting cells. Bemcentinib is a first-in-class, selective, orally bioavailable, AXL kinase inhibitor which is being evaluated as a therapy for solid tumours and myeloid malignancies in multiple phase II clinical trials. Increased levels of the plasma soluble, shed form of the AXL receptor (sAXL) upon bemcentinib exposure have been found across the clinical trial programme. The Phase I/II trial BGBC003 (NCT02488408) evaluates the anti-leukaemic effect of bemcentinib in patients with relapsed / refractory AML and int-2 and high-risk MDS. Methods Part A of the BGBC003 consisted of a dose escalation part per standard 3+3 design, followed by cohort expansion at the recommended phase 2 dose. Plasma protein biomarker levels were measured using the DiscoveryMap v3.3 panel (Myriad RBM) at pre-dose and after one cycle of treatment. Gene expression analysis was carried out on RNA extracted from BM-MNCs by qPCR using TaqMan. The presence of phosphorylated AXL was measured using Western blotting. The TCRß repertoire was quantified by NGS of DNA isolated from PBMNCs using an Illumina MiSeq sequencer. TCRß genes and the IGH repertoire were analysed with BIOMED2-TCRß-A and -B and BIOMED2-FR1/-FR3 primer pools, respectively. Using genomic DNA as template, the amplicons were tagged with Illumina adapters and indices in two consecutive PCR reactions. Demultiplexing and FastQ formated data output was generated by the MiSeq reporter. Analysis of TCRß and IGH data was performed on a Microsoft Cloud using our in-house analysis pipeline Pippa. Results In total, 36 patients were enrolled in part A of the study: 27 male (75%), median age of 72 years old (51 - 85), 3 patients with MDS (8%), median of 2 lines of prior therapy (0 - 6). A loading dose of 400 mg on days one to three followed by 200 mg daily thereafter has been established as safe and recommended phase 2 dose. In patients who received treatment for at least 21 days (n=28), an ORR (CR/Cri/PR) of 25% (7/28) was observed. Median DoR was 63 days. In the subset of patients with available plasma measurements at screening (n=19), 12 showed low levels of plasma soluble shed AXL and an ORR of 58% (sAXL-low; 7 of 12 patients) was observed. No responses occurred in patients with high levels of sAXL at screening (sAXL-high; 0 out of 7). sAXL levels were correlated with exposure and one cycle of treatment with bemcentinib significantly increased sAXL protein levels, particularly in responders to treatment indicating on-target activity of the drug. Median PFS from the day of randomisation until evidence of disease progression (PD) per bone marrow assessment or discontinuation due to PD whichever occurred earlier, was 62 days overall (95% CI: 18 - 106 d), 124 days in sAXL-low patients and 63 days in sAXL-high patients, respectively. Adverse events were mostly gastrointestinal and mild or moderate in severity congruent with bemcentinib monotherapy being well-tolerated in this patient population. There were no grade 4 or 5 events. We observed a diversification of the TCR repertoire in the bone marrow in 75% of evaluable patients (6/8). In the peripheral blood we detected increased TCR diversification in 44% of evaluable patients (4/9). These data indicate that the contribution of the Gas6-AXL axis to immunosuppression might be particularly pronounced in the bone marrow microenvironment. This notion is in concordance with earlier data indicating a specific upregulation of Gas6 by bone marrow stroma cells upon paracrine interaction with AML cells leading to locally increased Gas6 levels. We also observed diversification of the BCR, however less frequently (bone marrow 42%, peripheral blood 11%). Conclusions Bemcentinib is well tolerated in relapsed / refractory MDS and AML patients and exhibits anti-leukaemic activity. Furthermore, we observed a diversification of the TCR repertoire particularly in the bone marrow thereby supporting a role of AXL as immune checkpoint. Patient benefit was correlated with the level of the plasma marker soluble AXL at screening. This predictive biomarker candidate warrants further exploration in larger cohorts and has potential for development as companion diagnostics to select patient populations more likely to respond to bemcentinib treatment. Disclosures Loges: BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Paschka:Agios: Membership on an entity's Board of Directors or advisory committees; Astex: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Takeda: Other: Travel support; Jazz: Speakers Bureau; Sunesis: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Travel support; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Travel support. Micklem:BerGenBio ASA: Employment, Equity Ownership, Patents & Royalties. Holt:BerGenBio ASA: Employment, Patents & Royalties. Brown:BerGenBio ASA: Employment, Equity Ownership, Patents & Royalties. Lorens:BerGenBio ASA: Employment, Equity Ownership, Patents & Royalties. Yule:BerGenBio ASA: Employment, Equity Ownership, Patents & Royalties. Fiedler:Amgen: Research Funding; GSO: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Daiichi Sankyo: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance; Pfizer: Research Funding; Teva: Other: support for meeting attendance; Amgen: Patents & Royalties; Gilead: Other: support for meeting attendance; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Cortes:novartis: Research Funding. Gjertsen:Alden Cancer Therapy 2: Equity Ownership; Alden Cancer Therapy 2: Patents & Royalties: Alden Cancer Therapy II patent application in relation to CryoIT trial.; Kinn Therapeutics: Equity Ownership; Seattle Genetics: Consultancy; Boehringer Ingelheim: Research Funding; BerGenBio: Consultancy; Alden Cancer Therapy 2: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Kinn Therapeutics: Membership on an entity's Board of Directors or advisory committees.
- Published
- 2018
13. Multi-Parametric Single Cell Profiling Defines Distinct Drug Responses in Healthy Hematological Cell Lineages That Are Retained in Corresponding Malignant Cell Types
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Satu Mustjoki, Monica Hellesøy, Caroline A. Heckman, Muntasir Mamun Majumder, Olli-Pekka Kallioniemi, Peter O'Gorman, Kimmo Porkka, Aino-Maija Leppä, Emma I. Andersson, Jing Tang, Bjørn-Tore Gjertsen, Alun Parsons, Alina Malyutina, Despina Bazou, Paul Dowling, and Johan Krister Wennerberg
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Drug ,Multi parametric ,medicine.diagnostic_test ,010405 organic chemistry ,Bortezomib ,media_common.quotation_subject ,Immunology ,Cell ,Cell Biology ,Hematology ,Biology ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Peripheral blood mononuclear cell ,3. Good health ,0104 chemical sciences ,Flow cytometry ,medicine.anatomical_structure ,medicine ,Cancer research ,Malignant cells ,Stem cell ,medicine.drug ,media_common - Abstract
Background During hematopoiesis, multipotent stem cells and pluripotent precursors undergo a complex differentiation program to generate a diverse set of blood cell types with wide-ranging phenotypes and functions that may shape their initial response to therapies. Therefore, innate drug sensitivity in healthy cells provides insight into cell specific responses, aids identification of lineage specific anticancer therapies and reveals off-target effects. To characterize the diversity in drug response and intracellular signal transduction patterns in the major hematopoietic cell types, we simultaneously assessed their sensitivity to 71 small molecules utilizing a multi-parametric flow cytometry assay and mapped onto their proteomic and basal signaling profiles. Methods Human BM aspirates and PB samples were collected after written informed consent in compliance with the Declaration of Helsinki. Mononuclear cells from 331 BM aspirates or PB samples were isolated by density gradient centrifugation (Ficoll-Paque Premium; GE Healthcare). Cytometric analyses of drug responses were performed assay using a high throughput flow cytometer (iQue®Screener PLUS) in both 384 well (in 3 healthy samples, 71 drugs) and 96 well plate formats (in 26 samples, 6 drugs) to study drug effects in 5 concentrations (1-10,000 nM). Effect of bortezomib, clofarabine, dexamethasone, omipalisib, venetoclax and navitoclax were assessed on 10 cell populations, namely hematopoietic stem cells (HSCs) (CD34+CD38-), common progenitor cells (CPCs) (CD34+CD38+), monocytes (CD14+), B cells (CD45+CD19+), cytotoxic T cells (CD45+CD3+CD8+), T helper cells (CD45+CD3+CD4+), NK-T cells (CD45+CD3+CD56+), NK cells (CD45+CD56+CD3-), plasma cells (CD138+CD38) and granulocytes (CD45+, SSC++). Mass cytometry (CyTOF) was applied to investigate basal signaling activity of 9 proteins involved in MAPK, JAK-STAT, NF-κB and PI3K-mTOR signaling. Protein abundances in CD3+, CD14+ and CD19+ cells were investigated in six samples using mass spectrometry. To evaluate whether the distinct drug sensitivities detected in the cells of origin could be exploited in the malignant cell counterpart cell, ex vivo drug responses detected in healthy cells were compared to a cohort of 281 primary samples derived from multiple hematological malignancies. Results Unsupervised hierarchical clustering of drug response to 71 small molecules identified distinct drug responses in healthy cell subsets based on their cellular lineage. Compared to other cell types, B and NK cells were more sensitive to dexamethasone, venetoclax and midostaurin. Monocytes were more sensitive to trametinib which did not correlate to resting ERK1/2 phosphorylation. Venetoclax exhibited dose dependent cell selectivity to lymphocytes that inversely correlated to STAT3 phosphorylation. Elevated expression of catalase (CAT) and calprotectin (S100A8/S100A9) in monocytes corresponded to their intrinsic resistance to dexamethasone and venetoclax. Comparison of drug responses for six aforementioned drugs in healthy and neoplastic cells across 281 patient samples showed that healthy cell responses are predictive of the corresponding malignant cell response. Conclusion Applying a high content, multi-parametric single-cell assay, we could assess the diversity in drug effects on 10 different cell populations in individual donor samples. Our results demonstrate that cell subtypes are drastically different from each other with respect to protein abundance, signaling profiles and drug-response patterns against a diverse collection of anticancer drugs. Importantly, cell subset specific sensitivity and resistance mechanisms were clearly reflected in their malignant state. Taken together, understanding drug sensitivity in the healthy cell-of-origin provides opportunities to obtain a new level of therapy precision and avoid off-target toxicity. Disclosures Mustjoki: Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding. Wennerberg:Novartis: Research Funding. Porkka:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Gjertsen:Seattle Genetics: Consultancy; BerGenBio: Consultancy; Kinn Therapeutics: Membership on an entity's Board of Directors or advisory committees; Alden Cancer Therapy 2: Equity Ownership; Boehringer Ingelheim: Research Funding; Alden Cancer Therapy 2: Membership on an entity's Board of Directors or advisory committees; Alden Cancer Therapy 2: Patents & Royalties: Alden Cancer Therapy II patent application in relation to CryoIT trial.; Kinn Therapeutics: Equity Ownership; Novartis: Consultancy. Heckman:Novartis: Research Funding; Orion Pharma: Research Funding; Celgene: Research Funding.
- Published
- 2018
14. A 36-Dimensional Cytometry by Time of Flight (CyTOF) Analysis of De Novo Acute Myeloid Leukemia (AML) Patients Eligible for Intensive Chemotherapy
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Pilar Ayuda Duran, Sonia Gavasso, Stein-Erik Gullaksen, Jørn Skavland, Benedicte Sjo Tislevoll, Jorrit M. Enserink, Yngvar Fløisand, Dagim Shiferaw Tadele, Oda Helen Eck Fagerholt, Monica Hellesøy, Bjørn Tore Gjertsen, Nello Blaser, and Laure Piechaczyk
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,De novo acute ,Myeloid leukemia ,Cell Biology ,Hematology ,Intensive chemotherapy ,Biochemistry ,Internal medicine ,medicine ,business ,Cytometry - Abstract
Introduction Chemotherapy induced clonal selection and development are major contributors to disease relapse and resistance to therapy in AML. An immediate response to chemotherapy in intracellular signalling networks can be detected within minutes in vivo. (Irish J. et al 2004) We hypothesize that by analyzing the immediate intracellular chemotherapy response we might reveal which cancer clones are resistant to therapy and how they might be targeted. (Anensen N. et al Clin Cancer Res 2006), (Øyan M. et al BMC Cancer 2009) CyTOF combines the principles of conventional flow cytometry with time-of-flight mass spectrometry to allow analyses of multiple parameters simultaneously in single cells. This dramatically increases the numbers of antibodies that can be applied and permits a simultaneous analysis of both intracellular signaling networks and phenotypic characterization of cells. Material and methods We have performed a 36- dimensional mass cytometry analysis (CyTOF), containing 21 surface markers (CD66b, CD16, CD45, CD11b, CD25, CD3, CD4, CD8, CD56, CD20, CD38, CD34, CD117, CD33, CD64, CD14, HLA-DR, Axl-1H12, CD90, CD7, CD123) and 15 intracellular markers (cCaspase-3, p-4EB-P1, p-Stat 5, p-Stat 3, p-Stat 1, p-p38, p- ERK ½, p-Akt, p-NFkB, p-CREB, p-S6, p-Axl, p-Rb, Cyclin B1, p-Histone H3) of over 40 de novo AML patients eligible for intensive chemotherapy. Peripheral blood was sampled at 4 hours and at 18- 24 hours after start of standard "3+7" induction therapy (Lowenberg B. et al Blood 2017). Bone marrow and peripheral blood from healthy donors were also included in the analysis. Results Well-characterized surface markers were used to perform an unsupervised clustering of the high dimensional single cell data from each AML patient before start of induction therapy (Figure 1A). This clustering resulted in 15 unique cell clusters throughout the patient cohort, illustrated by viSNE in Figure 2A. The heatmap in Figure 1A show the expression of surface markers in each cluster. Healthy cells in AML patients clustered together and were recognized by their characteristic phenotype, for example granulocytes in cluster 13 (CD66b+ and CD11b+), CD8 T-cells in cluster 1 (CD3+, CD7+ and CD8+) and monocytes in cluster 11 (CD45+, CD64+, CD38+ and HLA-DR+). Cluster 3 and 4 had high expression of the immature marker CD34 and thereby likely to represent blast populations. Intracellular signaling at time of diagnosis was then compared to 24 hours after start of induction therapy in the same populations. A lasso regression analysis identified intracellular markers that significantly correlated to patient survival, based on their change in expression between pre-treatment and at 24 hours. Figure 1C show a Kaplan-Meier curve of patient survival (24 months) between patients that had high or low expression ratio of an intracellular phosphor-specific antibody in cluster 3 and 15. Both clusters have expression of CD34 and CD117 that are frequently expressed on leukemic cells. The expression ratio of this intracellular marker was significantly correlated to survival (p Conclusions By applying unsupervised clustering analysis of over 40 de novo AML patients we identified leukemic cell subpopulations that had unique patterns of intracellular phospho-specific antibodies during start of induction therapy. Changes in intracellular markers during the first 24 hours of induction therapy could significantly be related to patient survival. By analyzing molecular alterations short time after chemotherapy, we aim to reveal the sub-clonal diversity and the heterogeneous responses to treatment. Identification of cancer cell subsets that are resistant to conventional chemotherapy may in the future be used to guide therapy in AML. Disclosures Gjertsen: Alden Cancer Therapy 2: Patents & Royalties: Alden Cancer Therapy II patent application in relation to CryoIT trial.; BerGenBio: Consultancy; Novartis: Consultancy; Alden Cancer Therapy 2: Membership on an entity's Board of Directors or advisory committees; Alden Cancer Therapy 2: Equity Ownership; Kinn Therapeutics: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; Kinn Therapeutics: Equity Ownership; Boehringer Ingelheim: Research Funding.
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- 2018
15. JAK1/2 and BCL2 inhibitors synergize to counteract bone marrow stromal cell-induced protection of AML
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Caroline A. Heckman, Bjørn Tore Gjertsen, Riikka Karjalainen, Alun Parsons, Jing Tang, Olli Kallioniemi, Tea Pemovska, Minna Suvela, Tero Aittokallio, Mihaela Popa, Mireia Mayoral Safont, David Tamborero, Komal Kumar Javarappa, Krister Wennerberg, Mika Kontro, Dmitrii Bychkov, Kimmo Porkka, Muntasir Mamun Majumder, Jonathan Knowles, Minxia Liu, Bhagwan Yadav, and Emmet McCormack
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0301 basic medicine ,Myeloid ,Chronic lymphocytic leukemia ,Pharmacology ,Biochemistry ,Tyrosine-kinase inhibitor ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,hemic and lymphatic diseases ,Tumor Cells, Cultured ,Sulfonamides ,Drug Synergism ,Hematology ,3. Good health ,Leukemia ,Leukemia, Myeloid, Acute ,STAT Transcription Factors ,medicine.anatomical_structure ,Proto-Oncogene Proteins c-bcl-2 ,030220 oncology & carcinogenesis ,Female ,Signal Transduction ,Stromal cell ,medicine.drug_class ,Immunology ,Antineoplastic Agents ,Bone Marrow Cells ,Cell Line ,03 medical and health sciences ,Nitriles ,medicine ,Animals ,Humans ,Protein Kinase Inhibitors ,Venetoclax ,business.industry ,Cell Biology ,Janus Kinase 1 ,Janus Kinase 2 ,medicine.disease ,Bridged Bicyclo Compounds, Heterocyclic ,030104 developmental biology ,Pyrimidines ,chemistry ,Drug Resistance, Neoplasm ,Pyrazoles ,Bone marrow ,Stromal Cells ,business ,Ex vivo - Abstract
The bone marrow (BM) provides a protective microenvironment to support the survival of leukemic cells and influence their response to therapeutic agents. In acute myeloid leukemia (AML), the high rate of relapse may in part be a result of the inability of current treatment to effectively overcome the protective influence of the BM niche. To better understand the effect of the BM microenvironment on drug responses in AML, we conducted a comprehensive evaluation of 304 inhibitors, including approved and investigational agents, comparing ex vivo responses of primary AML cells in BM stroma-derived and standard culture conditions. In the stroma-based conditions, the AML patient cells exhibited significantly reduced sensitivity to 12% of the tested compounds, including topoisomerase II, B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), and many tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity was most pronounced in patient samples harboring FLT3 or PDGFRB alterations. In contrast, the stroma-derived conditions enhanced sensitivity to Janus kinase (JAK) inhibitors. Increased cell viability and resistance to specific drug classes in the BM stroma-derived conditions was a result of activation of alternative signaling pathways mediated by factors secreted by BM stromal cells and involved a switch from BCL2 to BCLXL-dependent cell survival. Moreover, the JAK1/2 inhibitor ruxolitinib restored sensitivity to the BCL2 inhibitor venetoclax in AML patient cells ex vivo in different model systems and in vivo in an AML xenograft mouse model. These findings highlight the potential of JAK inhibitors to counteract stroma-induced resistance to BCL2 inhibitors in AML.
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- 2016
16. Identification and Clinical Exploration of Individualized Targeted Therapeutic Approaches in Acute Myeloid Leukemia Patients By Integrating Drug Response and Deep Molecular Profiles
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Tero Aittokallio, Kimmo Porkka, Krister Wennerberg, Simon Anders, Caroline A. Heckman, Samuli Eldfors, Bjørn Tore Gjertsen, Jani Saarela, Astrid Murumägi, Riikka Karjalainen, Bhagwan Yadav, Mika Kontro, Maija Wolf, Olli Kallioniemi, Disha Malani, Swapnil Potdar, and Ashwini Kumar
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Oncology ,medicine.medical_specialty ,Ruxolitinib ,Immunology ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Internal medicine ,medicine ,030304 developmental biology ,Trametinib ,0303 health sciences ,Sunitinib ,business.industry ,Venetoclax ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,Temsirolimus ,3. Good health ,Dasatinib ,Leukemia ,chemistry ,030220 oncology & carcinogenesis ,business ,medicine.drug - Abstract
Introduction Most patients with acute myeloid leukemia (AML) are still missing effective options for targeted treatments. Here, we applied individualized systems medicine (ISM) concept1 by integrating deep molecular profiles (genomics, transcriptomics) and ex vivo drug response profiles with 521 oncology drugs in 154 AML patient samples. The aim was to identify new treatment opportunities for molecular subsets of AML patients. When feasible, ISM guided treatment opportunities were applied clinically for AML patient treatment. Serial samples were available to identify molecular alterations in response to targeted drug treatment and to monitor therapeutic success or failure. We also aimed at testing the impact of bone marrow stromal cell conditioned media on drug response profiles in AML patients2. Methods Samples from bone marrow or blood of 122 AML patients and 17 healthy donors were obtained with written consent and ethical approval (239/13/03/00/2010 and 303/13/03/01/2011) from the Hematology Clinic, Comprehensive Cancer Center, Helsinki University Hospital. The ex vivo drug sensitivity and resistance testing (DSRT) assay was performed with 521 approved oncology drugs and investigational oncology compounds as described earlier1. In this study, freshly isolated mononuclear cells were randomly resuspended either in standard mononuclear cell medium (MCM, PromoCell) or in human bone marrow stroma derived conditioned medium (CM) for drug testing. DNA samples from same mononuclear cells were subjected to whole exome and transcriptome sequencing and data were analyzed as described previsously2. Hierarchical clustering and non-parametric rank correlation were performed with drugs and samples. Wilcoxon sign ranked test was applied between wild type and mutated samples to identify significant mutation-drug associations. Results Hierarchical clustering was largely independent of clinical features such as disease status or risk class. A strong drug sub-cluster with a unique response profile was composed of that of the MDM2 antagonist idasanutlin along with BCL-2 inhibitors navitoclax and venetoclax (Figure). BET inhibitors (JQ1, I-BET151, birabresib) and MEK inhibitors (trametinib, selumetinib) were positively correlated with each other suggesting an association between bromodomain mediated epigenetic deregulation and up-regulation of the MEK pathway in a subset of patients. Comparison between patient samples profiled in CM (n=77) vs MCM medium (n=77) indicated higher efficacy of MDM2 modulator idasanutlin in MCM while BET inhibitors responded more strongly in CM. Other differences observed earlier by Karjalainen et al1 between the two media types were also validated. Furthermore, 16 chemorefractory and one diagnostic stage patients were treated with the targeted drugs suggested by this ISM approach. We observed complete remission or leukemia free state in 35% (6/17) of the AML patients given tailored treatment in an observational study. The targeted drugs used for clinical translation included ruxolitinib (in n=4 patients), temsirolimus (n=5), trametinib (n=4), sunitinib (n=7), dasatinib (n=7), sorafeninb (n=4), omacetaxine (n=3) and dexamethasone (n=5). Summary This study highlights the potential of individualized systems medicine (ISM) approach in the identification of effective treatment opportunities for individual patients with AML. Identifying molecular markers for ex vivo drug responses can help to assign treatments to the patient subgroups most likely to respond in clinical trials. Figure Figure. Disclosures Heckman: Orion Pharma: Research Funding; Novartis: Research Funding; IMI2 project HARMONY: Research Funding; Pfizer: Research Funding; Celgene: Research Funding. Porkka: Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.
- Published
- 2017
17. Flt3 Y591 duplication and Bcl-2 overexpression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53
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Øystein Bruserud, Nina Anensen, Anne-Lise Borresen-Dale, Garry P. Nolan, Bjørn Tore Gjertsen, Jørn Skavland, Jonathan M. Irish, and Randi Hovland
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Myeloid ,Tumor suppressor gene ,Immunology ,Gene Expression ,Biology ,medicine.disease_cause ,Biochemistry ,hemic and lymphatic diseases ,medicine ,Humans ,Protein Isoforms ,Electrophoresis, Gel, Two-Dimensional ,Phosphorylation ,Cells, Cultured ,Regulation of gene expression ,Mutation ,Gene Expression Regulation, Leukemic ,Gene Expression Profiling ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,Molecular biology ,Leukemia, Myeloid, Acute ,Leukemia ,medicine.anatomical_structure ,Proto-Oncogene Proteins c-bcl-2 ,fms-Like Tyrosine Kinase 3 ,Cancer research ,Tyrosine ,Tumor Suppressor Protein p53 ,Signal transduction ,DNA Damage ,Signal Transduction - Abstract
Loss or mutation of the TP53 tumor suppressor gene is not commonly observed in acute myeloid leukemia (AML), suggesting that there is an alternate route for cell transformation. We investigated the hypothesis that previously observed Bcl-2 family member overexpression suppresses wild-type p53 activity in AML. We demonstrate that wild-type p53 protein is expressed in primary leukemic blasts from patients with de novo AML using 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and phospho-specific flow cytometry. We found that p53 was heterogeneously expressed and phosphorylated in AML patient samples and could accumulate following DNA damage. Overexpression of antiapoptosis protein Bcl-2 in AML cells was directly correlated with p53 expression and phosphorylation on serine residues 15, 46, and 392. Within those patients with the highest levels of Bcl-2 expression, we identified a mutation in FLT3 that duplicated phosphorylation site Y591. The presence of this mutation correlated with greater than normal Bcl-2 expression and with previously observed profiles of potentiated STAT and MAPK signaling. These results support the hypothesis that Flt3-mediated signaling in AML enables accumulation of Bcl-2 and maintains a downstream block to p53 pathway apoptosis. Bcl-2 inhibition might therefore improve the efficacy of existing AML therapies by inactivating this suppression of wild-type p53 activity.
- Published
- 2006
18. Single Cell Signaling Pharmacodynamics in a Phase 1b Trial of the Axl Inhibitor BGB324 in Acute Myeloid Leukemia
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Monica Hellesøy, Håkon Reikvam, Jorge E. Cortes, Øystein Bruserud, Oda Helen Eck Fagerholt, Stein-Erik Gullaksen, André Sulen, J. Lorens, Jörg Chromik, Benedicte Sjo Tislevoll, Bjørn Tore Gjertsen, David Micklem, Michael Heuser, Sonja Loges, Jørn Skavland, and Gro Gausdal
- Subjects
0301 basic medicine ,Oncology ,AXL Inhibitor BGB324 ,medicine.medical_specialty ,medicine.medical_treatment ,Immunology ,Population ,CD38 ,Pharmacology ,Biochemistry ,Loading dose ,03 medical and health sciences ,Internal medicine ,medicine ,education ,education.field_of_study ,Chemotherapy ,business.industry ,Myeloid leukemia ,Cell Biology ,Hematology ,Clinical trial ,030104 developmental biology ,medicine.anatomical_structure ,Bone marrow ,business - Abstract
Axl is a receptor tyrosine kinase that has been shown to have a strong oncogenic potential in many cancer types. Overexpression and activation of Axl is found in many cancers, and is linked to increased proliferation, migration/invasion and resistance to apoptosis. Axl overexpression has been shown to be a poor prognostic marker, and recently overexpression of Axl has also been linked to the acquired resistance to chemotherapy and other anticancer therapies in many malignancies, including AML. BGB324 (BerGenBio AS) is a first-in-class highly specific small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in a clinical safety trial in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in a phase 1b clinical trial in patients with refractory/relapsed AML and MDS (BGBC003, ClinicalTrials.gov Identifier:NCT02488408; Loges S et al. J Clin Oncol 34, 2016 suppl; abstr 2561). 20 AML and 4 MDS patients have been treated at the following dose levels (loading dose/continuation dose): 400/100mg, 600/200mg and 900/300mg. Objective responses were observed in 2/4 MDS patients and 2/20 AML patients including one CR (AML). Enrollment continues to define MTD. The effect of BGB324 on intracellular signaling and the immune profile of leukemic blasts in patients treated in the clinical study was investigated using phospho-flow cytometry. Blasts were identified using surface markers (CD45low, CD66b-, CD38-, and CD117+ and/or CD34+), and the following direct and indirect downstream targets of Axl were explored: phosphorylated (p)-Akt(S473 and T308), pErk(T202/Y204), pp38(T180/Y182), pPLCγ1(Y783), pNFκB(S529), pCREB(S113) and pSTAT1(Y701), 3(Y705), 5(Y694)and 6(Y641). Preliminary analyses of blood samples from six patients show very rapid responses in signaling pathways downstream of Axl (including Akt, Erk, NFκB and PLCγ1) within hours or days of ingestion of the first dose, although the response patterns varies from patient to patient (Figure 1A). Two distinct blast populations were identified: one CD117+/CD34- and one CD117+/CD34+. In most patients the CD117+/CD34- population displayed the most extensive signaling changes during treatment, and this population also decreased during treatment with BGB324. In contrast, the CD117+/CD34+ population expanded during the course of the treatment (Figure 1B). White cell differential counts of peripheral blood from two patients treated with BGB324 for a prolonged period of time (15 weeks or more) showed a decrease in peripheral blast count, and a corresponding increase in granulocyte and monocyte counts, suggesting that Axl inhibition may push the blasts towards differentiation. The clinical trial is ongoing, and the signaling profile of leukemic blasts in blood and bone marrow of treated patients will be further examined by conventional phosphoflow cytometry and mass cytometry searching for signaling profiles with prognostic information. In conclusion, BGB324 has unique pharmacodynamic properties and molecular responses to exposure can be observed in peripheral blood leukemic blasts by phospho-flow cytometry within hours of ingestion of the first treatment dose. Further studies may establish whether single cell signal profiling can discriminate responders from non-responders and provide information about dose-response in a clinically meaningful way. Disclosures Cortes: Astellas: Research Funding; Arog: Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Ambit: Research Funding. Heuser:Novartis: Consultancy, Research Funding; Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Pfizer: Research Funding. Lorens:BerGenBio AS: Employment, Equity Ownership, Research Funding. Gausdal:BerGenBio AS: Employment. Micklem:BerGenBio AS: Employment, Equity Ownership. Gjertsen:BerGenBio AS: Consultancy, Research Funding.
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- 2016
19. BGB324, an Orally Available Selective Axl Inhibitor Exerts Anti-Leukemic Activity in the First-in-Patient Trial BGBC003 and Induces Unique Changes in Biomarker Profiles
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J. Lorens, Nuray Akyüz, Maxim Kebenko, Isabel Ben Batalla, Jorge E. Cortes, Loges Sonja, Jörg Chromik, Walter Fiedler, Mascha Binder, Melanie Janning, Michael Heuser, Bjørn Tore Gjertsen, Anthony Brown, and David Micklem
- Subjects
0301 basic medicine ,Oncology ,medicine.medical_specialty ,T cell ,Immunology ,Biochemistry ,Loading dose ,03 medical and health sciences ,Internal medicine ,medicine ,Osteopontin ,biology ,Maintenance dose ,business.industry ,T-cell receptor ,Cell Biology ,Hematology ,medicine.disease ,030104 developmental biology ,medicine.anatomical_structure ,biology.protein ,Biomarker (medicine) ,Bone marrow ,business ,Progressive disease - Abstract
Introduction: The RTK Axl represents a therapeutic target promoting AML cell proliferation and survival by pleiotropic mechanisms. Signalling through Axl is also known to suppress immune reactions mainly by inhibiting pro-inflammatory responses of antigen presenting cells. BGB324 is an orally available selective potent inhibitor of Axl, which is currently being investigated in a Phase 1 a/b trial in AML and MDS. We report here the clinical data and the impact of BGB324 on the Axl signalling pathway. Furthermore, we evaluated the T cell receptor (TCR) repertoire in order to assess potential effects of BGB324 on the immune response in AML patients. Methods: Twenty-five patients (twenty-one with relapsed/refractory AML and four with MDS) were treated in a classical 3+3 dose escalation design. Three dose levels were explored 400/100 mg (loading dose d1-2/maintenance dose), 600/200 mg and 900/300 mg. Serial bone marrow samples (BM) were available from n=5 patients. By means of immunoblotting and ELISA we analyzed phosphorylation of Axl and its downstream targets Erk/MapK and Akt as well as the levels of soluble Axl (sAxl) and osteopontin in plasma. The TCRß repertoire was quantified by Next Generation Sequencing of DNA isolated from peripheral blood MNCs using an Illumina MiSeq sequencer. TCRß gene containing the entire Vß, Dß and Jß segments were amplified with BIOMED2-TCRß-A and -B primer pools. Using genomic DNA as template, the amplicons were tagged with Illumina adapters and indices in two consecutive PCR reactions. Demultiplexing and FastQ formated data output was generated by the MiSeq reporter. Analysis of TCRß data was performed on a Microsoft Cloud using our in-house analysis pipeline Pippa, which relies on MiXCR analysis tools. Results: Treatment was generally well-tolerated. Diarrhea and fatigue represented the most frequent adverse events, which were mostly CTC Grade 1 or 2. One DLT (QTc prolongation) occurred and the MTD has not yet been reached. Steady-state levels of BGB324 were reached between three and six days after initiation of treatment. One AML patient achieved a CRi of 5 months, a second achieved a PR and another achieved clearance of circulating blasts accompanied by peripheral blood count recovery > 3 months (ORR 10%). Four additional AML patients (25%) experienced disease stabilisation for more than four months. Two MDS patients experienced a PR. From the five patients in whom BM samples were available three experienced either an objective response or disease stabilization for more than four months and two had progressive disease. We could detect phosphorylated Axl (pAxl) before treatment and decreased pAxl level after 21 days of treatment in 3 out of 3 responders/patients with SD while could not detect pAxl before treatment in the patients with PD (n=5; p=0.025). The patients with response or SD showed inhibition of pErk and pAxl (2 out of 3) or no change of the activity of these Axl downstream pathways (1 out of 3). The patients with PD showed increased pErk and pAkt level after treatment (n=5; p=0.082). Treatment with BGB324 led to an increase in the levels of sAxl after 21 days of treatment in the plasma of 67% of analyzed patients (n=12). SAxl levels were positively correlated to compound exposure (n=12; r=0.829), indicating that sAxl might be used as a biomarker of target engagement. The highest level of sAxl was seen in the patient who achieved a PR. Osteopontin levels decreased in 67% of analyzed patients after three weeks of treatment with BGB324 indicating modulation of this protein by BGB324. However, there was no correlation between increases of sAxl and decreases of osteopontin in this patient cohort. Interestingly, we could demonstrate diversification of the TCR repertoire by higher abundance of small T cell clones after three weeks of therapy with BGB324 compared to pre-treatment levels in two out of four analysed patients. Conclusion: BGB324 is well tolerated in AML patients and exhibits anti-leukemic activity. BGB324 has on-target activity in AML patients and blocks the phosphorylation of Axl and its downstream pathways. Furthermore, BGB324 can induce a diversification of the TCR repertoire in AML patients and might hold potential as an immune-activating drug. Disclosures Sonja: BergenBio: Honoraria, Other: Travel Support, Research Funding. Gjertsen:BerGenBio AS: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Micklem:BerGenBio AS: Employment, Equity Ownership. Janning:Teva: Honoraria. Fiedler:Pfizer: Research Funding; GSO: Other: Travel; Amgen: Consultancy, Other: Travel, Patents & Royalties, Research Funding; Kolltan: Research Funding; Ariad/Incyte: Consultancy; Novartis: Consultancy; Gilead: Other: Travel; Teva: Other: Travel. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.
- Published
- 2016
20. Targeting of JAK/STAT Signaling to Reverse Stroma-Induced Cytoprotection Against BCL2 Antagonist Venetoclax in Acute Myeloid Leukemia
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Bjørn Tore Gjertsen, Alun Parsons, Krister Wennerberg, Mihaela Popa, Mika Kontro, Kimmo Porkka, Emmet McCormack, Mireia Mayoral Safont, Riikka Karjalainen, Caroline A. Heckman, and Minxia Liu
- Subjects
Ruxolitinib ,Stromal cell ,medicine.medical_treatment ,Immunology ,CD34 ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,030304 developmental biology ,0303 health sciences ,Venetoclax ,business.industry ,Cell Biology ,Hematology ,medicine.disease ,3. Good health ,Leukemia ,Cytokine ,medicine.anatomical_structure ,chemistry ,Cancer research ,Bone marrow ,business ,Ex vivo ,030215 immunology ,medicine.drug - Abstract
Several promising new, targeted agents are being developed for the treatment of AML. The BH3 mimetic venetoclax (ABT-199) is a specific inhibitor of BCL2, with results from a phase 2 study showing transient activity of venetoclax in relapsed/refractory AML (Konopleva et al, 2014). The bone marrow (BM) microenvironment is known to protect AML cells from drug therapy and we showed earlier that conditioned medium (CM) from BM stromal cells applied to AML patient cells conferred resistance to venetoclax, which could be reversed by the addition of the JAK1/2 inhibitor ruxolitinib (Karjalainen et al, 2015). Here, we investigated the mechanisms mediating the BM stromal cell induced resistance to venetoclax and its reversal by ruxolitinib. To identify the soluble factor(s) contributing to stroma-induced protection of BCL2 inhibition, we analyzed the cytokine content of 1) CM from the human BM stromal cell line HS-5, 2) CM from BM mesenchymal stromal cells (MSCs) isolated from AML patients, 3) supernatants from BM aspirates collected from AML patients, and 4) supernatants from BM aspirates collected from healthy donors. Although expression levels varied, the cytokines detected were similar among the different samples. In HS-5 CM, IL-6, IL-8 and MIP-3α were among the most abundant cytokines. In addition, gene expression analysis showed the receptors for these cytokines were expressed in AML patient samples. IL-6, IL-8 and MIP-3α were added individually to mononuclear cells collected from AML patients, which were then treated with venetoclax. However, none of the cytokines alone could mimic the reduced sensitivity to venetoclax conferred by the HS-5 CM suggesting that stromal cell induced cytoprotection is likely multi-factorial. Next we tested the effect of AML-derived BM MSCs on the ex vivo response of AML patient samples (n=8) to ruxolitinib or venetoclax alone or in combination in a co-culture setting. Apoptosis assays showed negligible effects of ruxolitinib at a concentration of 300 nM, while venetoclax at a dose of 100 nM induced reduction in the percentage of CD34+ AML cells. Co-treatment with venetoclax and ruxolitinib demonstrated synergistic effects in 6 out of 8 samples and significantly reduced the number of CD34+ AML cells. Mechanistic studies showed that ruxolitinib treatment inhibited the BM stromal medium-induced expression of BCL-XL mRNA on AML cells and the drugs in combination down-regulated BCL2, MCL1 and BCL-XL protein expression, which was in correlation with sensitivity to the drugs. To further evaluate the ability of the venetoclax and ruxolitinib combination to eradicate leukemic cells in vivo we used an orthotopic xenograft model of AML. NSG mice were injected with genetically engineered MOLM-13luc cells and after engraftment treated with venetoclax (25 mg/kg, i.p.), ruxolitinib (50 mg/kg BID, p.o) or both and imaged once per week for 4 weeks. At the end of the treatment period bioluminescent imaging showed significantly reduced leukemia burden in the ruxolitinib and venetoclax co-treated mice compared to controls demonstrating superior anti-tumor efficacy than either agent alone (Figure 1). In summary, our data demonstrate that the combined blockade of JAK/STAT and BCL2 pathways with ruxolitinib and ventoclax is synergistic in ex vivo co-culture models and in vivo in an AML mouse model. The addition of ruxolitinib was able to overcome intrinsic resistance to venetoclax by reducing expression of MCL1, a known escape mechanism of BCL2 inhibition. These results support further clinical investigation of this combination, particularly for relapsed/refractory AML. Disclosures Porkka: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Wennerberg:Pfizer: Research Funding. Gjertsen:BerGenBio AS: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Heckman:Celgene: Research Funding; Pfizer: Research Funding.
- Published
- 2016
21. Effects of Dasatinib and Interferon-α Combination Treatment on the Immune System in CML
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Franz Gruber, Henrik Hjorth-Hansen, Mette Ilander, Bjørn Tore Gjertsen, Perttu Koskenvesa, Hanne Vestergaard, Ulla Olsson-Strömberg, Satu Mustjoki, Tobias Gedde-Dahl, Berit Markevärn, Hanna Lähteenmäki, Ole Weis Bjerrum, Johan Richter, Arta Dreimane, Leif Stenke, Jesper Stentoft, Lene Udby, Kristina Myhr-Eriksson, Kimmo Porkka, Martin Höglund, and Anna Lubking
- Subjects
medicine.medical_treatment ,T cell ,Immunology ,Population ,Pharmacology ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Immunophenotyping ,Medicine ,education ,030304 developmental biology ,0303 health sciences ,education.field_of_study ,business.industry ,Cell Biology ,Hematology ,3. Good health ,Dasatinib ,Cytokine ,medicine.anatomical_structure ,Cytokine secretion ,Interleukin 18 ,business ,CD8 ,030215 immunology ,medicine.drug - Abstract
Background: In chronic myeloid leukemia (CML) the combination treatment of tyrosine kinase inhibitors (TKIs) with interferon-α (IFN-α) has proved to be effective and well-tolerated. IFN-α has long-term immunomodulatory effects, and when combined to TKI therapy, it may increase the success rates for treatment free remission. In our recent clinical trial NordCML007, a low-dose pegylated IFN-α was combined with dasatinib therapy. As dasatinib is also known to have immunostimulatory effects (activation of T and NK cells and downregulation of regulatory T cells), we aimed to monitor the immune effects of dasatinib and IFN-α combination treatment. Methods: 40 newly diagnosed CML patients participated in the NordCML007 clinical trial (NCT01725204). Patients were treated with 100 mg dasatinib QD and after 3 months IFN-α treatment was added (first 3 months 15 μg/week, then 25 μg/week of pegylated IFN-α). After 12 months of combination treatment, patients resumed to dasatinib monotherapy. In this immunological substudy, peripheral blood samples were collected at the diagnosis, 3, 12, and 24 months after the start of therapy. T- and NK-cells were phenotyped with multicolor flow cytometry, and their function (degranulation and cytokine secretion) was studied. In addition, a multiplexed cytokine and growth factor panel was performed (Proseek Multiplex Inflammation I96×96, Olink). Results: Dasatinib monotherapy led to an increase of NK-cell frequencies when compared to pre-treatment values (median diagnosis 6.8% vs. 3 months 12.8%, p The more detailed immunophenotyping showed that at 3 months NK cells were more mature when compared to pre-treatment values. This could be observed as the increased proportion of CD16+ (86% vs. 77%, p=0.008) and CD57+ (79% vs. 67%, p=0.0001) NK cells. Correspondingly, the frequencies of CD56bright (median 3 months 3.9% vs. dg 6.1%, p Similar effects of dasatinib treatment were also observed within the T cell population, and at 3 months patients had more CD8+ terminally differentiated effector memory CD45RA+ (Temra) cells (22% vs. 32%, p=0.03). Correspondingly, both CD8+ and CD4+ effector memory (EM) populations decreased (CD8+ EM 34% vs. 24%, p=0.007; CD4+ EM 21 % vs. 14 %, p=0.0003). As observed with the NK cells, the degranulation and Th1 type cytokine secretion of T cells decreased during dasatinib treatment (CD107a/b+ 19.4% vs. 18.4% of CD8+ T cells, p=0.02; CD107a/b+ 12.7% vs. 8.8% of CD4+ T cells, p=0.005). Interestingly, 3 out of 4 patients who suffered from pleural effusion (PE) had exceptionally high CD57+CD8+ T cell frequencies (>70% of CD8+ T cells, median 38% in all patients) at diagnosis and during the treatment. The combination of IFN-α did not significantly affect the number of NK cells. Interestingly however, the addition of IFN-α treatment seemed to have an opposite effect on the NK cell phenotype than dasatinib treatment. The proportion of CD56bright NK cells increased (median 3 months 3.9% vs. 12 months 5.2%, p=0.03), and a decreasing trend was observed among CD16+ and CD57+ NK cells at 12 months. Similarly, the T cell surface markers shifted towards more immature phenotype (CD4+ EM 14% vs. 19%, p=0.002; CD4+ CM 36% vs. 28%, p=0.002; CD8+ Temra 32% vs. 17%, p=0.003) and the degranulation of CD4+ T cells and NK cells returned to the diagnosis level. The initiation of dasatinib treatment was associated with a decrease in several plasma protein concentrations (IL-18, IL-8, CXCL5, MCP-2, MCP-3, VEGF-A, CD244, LAP TGFβ1, TGFA, 4E-BP1, ADA, STAMPB, Casp8, OSM) and an increase in others (SCF, MCP-1). After IFN-α addition, the concentration of MCP-1 was further increased. Conclusions: These results show that dasatinib and IFN-α modulate both innate and adaptive immune systems. Interestingly, where dasatinib seems to induce a more mature immunophenotype, IFN-α counteracts this by driving a shift towards a more immature phenotype. Comparative analysis of the changes in the immunophenotype and function with the clinical outcome of the patients is ongoing. Disclosures Stentoft: Ariad: Research Funding; Pfizer: Research Funding; Novartis: Research Funding; Bristol-Myers-Squibb: Research Funding. Richter:Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Höglund:Akinion Pharmaceuticals: Consultancy; Janssen-Cilag: Honoraria. Mustjoki:Ariad: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.
- Published
- 2016
22. Multiplexed mAbs: a new strategy in preclinical time-domain imaging of acute myeloid leukemia
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Øystein Bruserud, Maja Mujic, Emmet McCormack, Tereza Osdal, and Bjørn Tore Gjertsen
- Subjects
Acute promyelocytic leukemia ,Pathology ,medicine.medical_specialty ,Myeloid ,Immunology ,Green Fluorescent Proteins ,HL-60 Cells ,Tretinoin ,Mice, SCID ,Biochemistry ,Immunophenotyping ,Mice ,Leukemia, Promyelocytic, Acute ,Differentiation therapy ,Mice, Inbred NOD ,hemic and lymphatic diseases ,Cell Line, Tumor ,Antineoplastic Combined Chemotherapy Protocols ,medicine ,Animals ,Humans ,Anthracyclines ,Fluorescent Dyes ,Mice, Knockout ,CD11b Antigen ,business.industry ,Valproic Acid ,Optical Imaging ,Cytarabine ,Myeloid leukemia ,Antibodies, Monoclonal ,Cell Biology ,Hematology ,medicine.disease ,Xenograft Model Antitumor Assays ,CD11c Antigen ,Leukemia ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Luminescent Measurements ,business ,medicine.drug - Abstract
Antibodies play a fundamental role in diagnostic immunophenotyping of leukemias and in cell-targeting therapy. However, this versatility is not reflected in imaging diagnostics. In the present study, we labeled anti–human mAbs monochromatically against selected human myeloid markers expressed on acute myeloid leukemia (AML) cells, all with the same near-infrared fluorochrome. In a novel “multiplexing” strategy, we then combined these mAbs to overcome the limiting target-to-background ratio to image multiple xenografts of AML. Time-domain imaging was used to discriminate autofluorescence from the distinct fluorophore-conjugated antibodies. Imaging with multiplexed mAbs demonstrated superior imaging of AML to green fluorescent protein or bioluminescence and permitted evaluation of therapeutic efficacy with the standard combination of anthracycline and cytarabine in primary patient xenografts. Multiplexing mAbs against CD11b and CD11c provided surrogate imaging biomarkers of differentiation therapy in an acute promyelocytic leukemia model treated with all-trans retinoic acid combined with the histone-deacetylase inhibitor valproic acid. We present herein an optimizedapplication of multiplexed immunolabeling in vivo for optical imaging of AML cellxenografts that provides reproducible, highly accurate disease staging and monitoring of therapeutic effects.
- Published
- 2012
23. Increased TACE (Tumor necrosis factor-alpha±-converting enzyme; ADAM17) Activity Associates with Decreased CD62L Expression, Increased Soluble CD62L Plasma Levels and Predicts Molecular Response to Nilotinib Therapy in Patients with Early Chronic Phase Chronic Myelogenous Leukemia (CML-CP): Results from an ENEST1st Substudy
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Frank Giles, Guenther Gastl, Satu Mustjoki, Bjørn Tore Gjertsen, Kimmo Porkka, Gert J. Ossenkoppele, Angelica Loskog, Dominik Wolf, Sieghart Sopper, and Andreas Hochhaus
- Subjects
medicine.medical_specialty ,Immunology ,Biochemistry ,Chronic phase chronic myelogenous leukemia ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,medicine ,030304 developmental biology ,0303 health sciences ,Metalloproteinase ,Hematology ,business.industry ,Cancer ,Cell Biology ,medicine.disease ,3. Good health ,Dasatinib ,Endocrinology ,Nilotinib ,Molecular Response ,Cancer research ,Tumor necrosis factor alpha ,business ,030215 immunology ,medicine.drug - Abstract
Background and Aim: Tyrosine kinase inhibitors (TKI) imatinib and dasatinib modulate immune responses in vitro and in vivo. Immunological surveillance in the MRD-situation might be of particular relevance for long-term control or even elimination of CML-repopulating stem cells. Moreover, baseline immunological characteristics may be associated with response to TKI therapy. Little is known about potential immune-modulatory effects of nilotinib in vivo. The ENEST1st study (NCT01061177) evaluated the role of first-line nilotinib therapy in CML-CP. The primary endpoint was the MR4 rate at 18 months. A comprehensive immunological monitoring program within this ENEST1st substudy characterized baseline and therapy-induced immunological variables to correlate them with biological disease characteritics and clinical response parameters. Methods: Peripheral blood was taken prior to treatment initiation and after 6 and12 months (mo) from 52 patients. Samples were analyzed by nine colour flow cytometry employing six panels of optimized antibodies to determine various leukocyte populations (e.g. T cell subpopulations including Treg and NKT cells, NK cells, B cells, monocytes, MDSC, dendritic cell subsets). Plasma concentrations of soluble CD62L (sCD62L) and TACE (tumor necrosis factor-α-converting enzyme; ADAM17, CD156b), the metalloproteinase inducing proteolytic cleavage of CD62L from the cell surface, were either measured by ELISA or (in case of the enzymatic activity of TACE) using a fluorogenic assay. Changes in immune cell parameters were correlated to biological disease features and clinical endpoints. Results: The most striking finding of this study is the drastic loss of the lymph-node homing marker CD62L on immune cells (T cell subsets and granulocytes) at baseline (basCD62L), which increased back to normal levels during nilotinib therapy. The proportion of basCD62L+ cells among both CD4+ and CD8+ T cell subsets significantly correlated with Sokal score (both as continous and categorial variable, i.e. high vs. low/int). Low basCD62L expression levels on both T cell subsets correlate with increased spleen size, higher BM and PB blast and WBC counts as well as it correlates to higher BCR-ABL copy numbers at almost all time points during treatment. Similarly, lower basCD62L on either CD4+ or CD8+ T cells is linked to a longer duration to reach the respective molecular endpoint. Patients reaching MR4 at 18 months (primary study endpoint) had significantly higher levels of basCD62L on both CD4+ (p=0.02) and CD8+ (p=0.008) T cells. Consequently, MR4 at 18 months was attained in a significantly higher percentage of patients in the basCD62hi compared to the CD62lo patients (63% vs. 13.0%). Vice versa, patients who reached MR4 at 18 months had significantly higher proportions of basCD62L expressing cells among both CD4+ and CD8+ T cells. Moreover, as depicted by a cumulative response rate, patients with high proportions of basCD62Lhi T cells, achieved MMR and MR4 significantly earlier and in a higher proportion throughout the observation period. A detailed characterization of other T cell differentiation marker (CD45RA, CD45R0, CD28, CD27, and CD95) did not reveal significant baseline T cell subset alterations as explanation for altered CD62L expression. In contrast to low basCD62L surface expression levels, its shed form sCD62L is significantly increased at diagnosis but subsequently drops back during nilotinib therapy. Similar to surface CD62L expression, also sCD62L associates with biological disease features and molecular response to nilotinib. Finally, low CD62L surface expression was associated with elevated sCD62L levels and increased proteolytic activity but not total amount of TACE. Conclusion: At baseline, increased proteolytic activity of TACE sheds CD62L from the immune cell surface. During nilotinib therapy, TACE activity gets normalized leading to re-expression of CD62L on T cells and vice versa a drop of sCD62L. Low baseline T cell expression levels of CD62L and increased sCD62L levels correlate to a more aggressive CML phenotype and are linked to inferior molecular response to nilotinib in early CML-CP. Larger prospective studies including also other TKIs are needed to confirm the prognostic relevance of sCD62L/CD62L expression as response-prediction marker, as this marker is easy to measure by ELISA in plasma samples or flow-cytometry. Disclosures Mustjoki: Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding. Loskog:RePos Pharma AB: Membership on an entity's Board of Directors or advisory committees; Vivolux AB: Membership on an entity's Board of Directors or advisory committees; Lokon Pharma AB: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; NEXTTOBE AB: Membership on an entity's Board of Directors or advisory committees; Alligator Bioscience AB: Patents & Royalties. Gjertsen:Haukeland University Hospital: Research Funding. Giles:Novartis: Consultancy, Honoraria, Research Funding. Ossenkoppele:Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria.
- Published
- 2015
24. Single Cell-Level Signaling Profiling of Acute Myeloid Leukemia Following Treatment with Axl Kinase Inhibitor BGB324
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Håkon Reikvam, Bjørn Tore Gjertsen, Jørn Skavland, Katarzyna Wnuk-Lipinska, J. Lorens, A. Boniecka, Jorge E. Cortes, David Micklem, Gro Gausdal, Monica Hellesøy, Øystein Bruserud, Sonja Loges, and Eline Milde Nævdal
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Oncology ,medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,Immunology ,CD33 ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Flow cytometry ,Metastasis ,medicine.anatomical_structure ,Internal medicine ,medicine ,Mass cytometry ,Bone marrow ,business ,Protein kinase B ,TYRO3 - Abstract
Axl is a member of the Tyro3, Axl, Mer (TAM) receptor tyrosine kinase family that regulate a wide range of cellular functions, including cell survival, proliferation, migration/invasion and adhesion. Axl has been shown to play a key role in the survival and metastasis of many tumors, and has also been found to be upregulated and constitutively active in human AML. Indeed, Axl has been reported as an independent prognostic marker and a potential novel therapeutic target in AML. BGB324 is a first-in-class highly selective small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in clinical safety studies in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in phase I b clinical trials for AML and non-small cell lung cancer. In this study, we use phosphoflow cytometry to measure changes in signal transduction nodes in single AML cells treated with BGB324. We are applying this approach to monitor signaling profiles in primary AML cells harvested from patients undergoing BGB324 treatment. Results: The human AML cell line MOLM13 was treated in vitro with BGB324 (0.5 and 1µM for 1 hour) and analyzed for signal transduction changes by phosphoflow cytometry. We found a significant reduction in phosphorylation of Axl (pY779), Akt(pS473), Erk1/2(pT202/Y204) and PLCɣ1(pY783). Next we established a systemic MOLM13 preclinical AML model in NOD/SCID mice. The mice were treated with 25 or 50 mg/kg BGB324 until moribund (up to 16 days). We found a dose-dependent and significant increase in overall survival in BGB324-treated mice. We further investigated intracellular signaling in BGB324-treated cells in vivo. Mice carrying systemic AML disease (MOLM13) were treated with BGB324 at 50mg/kg for 4 days, and we monitored CD33/45-positive MOLM13 cells harvested from spleen and bone marrow by flow cytometry. BGB324-treated mice showed a significant reduction in pErk and pPLCɣ1 relative to mice in the control group. PBMCs from peripheral blood of AML patients treated with BGB324 400 mg x1 at day 1 and 2, and thereafter 100 mg daily were collected for single cell signal profiling of signal transduction changes by conventional flow cytometry (phospho-flow) and mass cytometry (CyTOF). Preliminary phopho-flow analyses show decrease of pAkt(T308) and pPLCgamma1(Y783) in one patient. Further analyses are ongoing and will be presented. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Disclosures Hellesøy: BerGenBio AS: Other: Previous employee. Stock option holder. Wnuk-Lipinska:BerGenBio AS: Employment. Boniecka:BerGenBio AS: Employment. Nævdal:BerGenBio AS: Employment. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding. Cortes:Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Lorens:BerGenBio AS: Employment, Equity Ownership. Micklem:BerGenBio AS: Employment, Equity Ownership. Gausdal:BerGenBio AS: Employment. Gjertsen:Haukeland University Hospital: Research Funding.
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- 2015
25. Safety and Efficacy of Addition of Pegylated Interferon alpha2b to Standard Dose Dasatinib in Newly Diagnosed Chronic Phase CML Patients
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Ole Weis Bjerrum, Leif Stenke, Perttu Koskenvesa, Arta Dreimane, Anna Lübking, Henrik Hjorth-Hansen, Hanne Vestergaard, Satu Mustjoki, Franz Gruber, Kimmo Porkka, Tobias Gedde-Dahl, Bjørn Tore Gjertsen, Johan Richter, Martin Höglund, Berit Markevärn, Kristina Myhr-Eriksson, Jesper Stentoft, and Ulla Olsson Strömberg
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Oncology ,medicine.medical_specialty ,Hematology ,business.industry ,Pleural effusion ,Immunology ,Cell Biology ,medicine.disease ,Biochemistry ,3. Good health ,Dasatinib ,Imatinib mesylate ,Nilotinib ,Pegylated interferon ,Internal medicine ,medicine ,Chronic phase CML ,business ,Adverse effect ,medicine.drug - Abstract
Rationale: Dasatinib (DAS) and interferon have different modes of action and may have synergistic activity in CML, due to both antineoplastic and immunostimulatory mechanisms. Addition of pegylated interferon (PegIFN) to imatinib therapy in CP-CML has in previous clinical trials (French SPIRIT and NordCML002) resulted in deeper molecular responses. Thus, an optimal combination of DAS and PegIFN may increase the proportion of patients who reach deep molecular response with potential for treatment-free remission (TFR). Design: Newly diagnosed CP-CML patients were treated with DAS (Sprycel, BMS) 100 mg OD as single drug for three months. Thereafter weekly subcutaneous injections of Peg-IFN α2b (PegIntron, MSD) were added to DAS; from end of month 3 (M3) to M6, 15µg/week, thereafter 25µg/week until M15. Primary end points were safety and the rate of MMR at M12. The doses of PegIFN were lower than in the SPIRIT and NordCML002 studies to increase adherence. Population: Forty patients were included at 14 university centers. One patient was lost to follow-up after M6. All patients were included in analysis up to M12. Mean and median age was 48 years (range 19-71). The proportions of high risk patients were 25% (Sokal), 15% (Hasford), and 15% (EUTOS). Safety and dosing: Treatment was well tolerated with expected DAS and PegIFN related side effects. Six patients had seven serious adverse events (AEs), all hospitalizations. 1 episode each of bradycardia/atrial fibrillation (possibly PegIFN-related), headache (DAS), fever (PegIFN), anaphylaxis-like reaction (PegIFN), fever/malaise/headache (PegIFN), pneumonia and a knee effusion (both unrelated). One pleural effusion occurred (grade 2, 3%). Grade 3-4 neutropenia and thrombocytopenia occurred in 6 and 9 patients respectively. Prolonged hematological toxicity (>2 months) occurred in 8 patients, causing dosing problems in 5. One patient suffered grade 3 depression. Grade 3 flu-like symptoms occurred in 2 patients. One patient had lipase elevation grade 3 and one patient developed hypothyroidism attributed to PegIFN. Grade 2 dermal AEs like rash and acne occurred in about 20%, attributable to both drugs. 94% (DAS) and 76% (PegIFN) of assigned dose was given. Dose reductions occurred in 19 patients for DAS and 20 patients for PegIFN. Two patients discontinued DAS and switched to nilotinib, 1 for headache at M3 and 1 at M12 for lack of efficacy/hematological toxicity. Two patients could not start PegIFN for hematological toxicity (one lost to follow-up after M6). PegIFN was discontinued because of bradycardia/atrial fibrillation (1 patient), anaphylaxis (1 patient), flu-like syndrome (2 patients) and long-term hematological toxicity (2 patients). At 12 months 31/38 pats (82%) were still on PegIFN, a higher proportion than in the French Spirit or NordCML002 studies. Efficacy: We have used the DAS arm of the Dasision study (Kantarjian NEJM 2010) as a historical control. Early response at M3 was very similar between studies. In the present and the Dasision cohorts respectively, 18% vs 16% missed the 10% BCR-ABLIS landmark, 66% vs 56% achieved a CCyR and 8% vs 8% achieved MMR. At M6, three months after introduction of PegIFN, a steep increase in MMR rate was observed compared with Dasision. This was also reflected in deep responses, MR4.0 (see tables) and MR4.5 at M12, 18% vs 5%. The primary efficacy endpoint was MMR at M12, 82% vs 46%. Table 1.MMRDAS+PegIFN (%)DAS (Dasision)(%)Difference (%)M3880M6532726M9663927M12824636Table 2.MR4.0DAS+PegIFN (%)DAS (Dasision) (%)Difference (%)M3303M620614M938830M12481236 Progressions and treatment failure defined by ELN 2013: Failures: No progression was noted. At M3, 2 patients still had >95% Ph+ metaphases (MF). At M6, four patients (11%) had > 35% Ph+MF or >10% BCR-ABL levels. At M12, one patient failed CCgR and two more patients failed Conclusion: The combination of DAS and low dose PegIFN could be safely administered in newly diagnosed CP CML. No unexpected autoimmune phenomena were observed, and pleural effusions were rare. Efficacy appears very promising with high early MMR rates and deep molecular responses. A randomized comparison DAS +/- PegIFN is warranted. Support: Study drug from BMS and MSD. Grant from BMS. Figure 1. Figure 1. Disclosures Hjorth-Hansen: Ariad: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Off Label Use: Dasatinib and Pegylated IFN combination in CML. Richter:Ariad: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding.
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- 2015
26. Single-Cell Immune Signatures in Patients with Chronic Phase Chronic Myeloid Leukemia (CML) Treated with Nilotinib: An ENEST1st Sub Study
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Stein-Erik Gullaksen, Jaroslava Voglová, Waleed Majeed, Boris Labar, Henrik Hjorth-Hansen, Jeroen Janssen, Kimmo Porkka, Alois Lang, Georgi Mihaylov, Gregor Verhoef, Jørn Skavland, Sonia Gavasso, Gert J. Ossenkoppele, Krzysztof Warzocha, Noortje Thielen, Leif Stenke, Werner Linkesch, Andreas Hochhaus, Augustin Ferrant, Dominik Wolf, Sieghart Sopper, Tobias Gedde-Dahl, Vinko Tosevski, Josef Thaler, Bjørn Tore Gjertsen, Frank Giles, Jesper Stentoft, Claudia Dumrese, Satu Mustjoki, and Andrzej Hellmann
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0303 health sciences ,Myeloid ,Cluster of differentiation ,business.industry ,Immunology ,Context (language use) ,Cell Biology ,Hematology ,CD38 ,Biochemistry ,3. Good health ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,Immunophenotyping ,Nilotinib ,030220 oncology & carcinogenesis ,medicine ,Stem cell ,Progenitor cell ,business ,030304 developmental biology ,medicine.drug - Abstract
Earlier evaluation of therapy effect in patients with CML would assist in optimal use of available tyrosine kinase inhibitors (TKI). Single cell analysis by mass cytometry has enabled the quantification of up to 46 antibody epitopes, making it ideally suited for exhaustive immunophenotyping of the haematological hierarchy, and evaluation of associated dynamic signal transduction events, in a clinical setting. By integrating time resolved single cell signalling data with clinical parameters, we searched for prognostic and efficacy-response mass cytometry biomarkers within a month of TKI therapy. We report data from experiments used to validate the custom panels of antibodies, highlighting the power of mass cytometry in the analysis of primary patient material obtained on clinical studies. Peripheral Blood (PB) samples were collected before, 3 hours, 7 days and 28 days, after start of nilotinib (300 mg BID) treatment in a subset of patients (n=55) enrolled in the ENEST1st trial. PB cells were stained with two panels of antibodies, allowing a comprehensive immunophenotyping of numerous cellular subsets, and also the evaluation of intracellular phosphorylation status of several epitopes. Moreover, using a straightforward barcoding scheme, the time-resolved samples from each individual patient were pooled after barcoding and stained with the antibody panels to minimize sample variation. In a pilot study, 7 and 10 cell subsets were identified in PB samples from 4 untreated healthy donors and 2 complete sets of 4 patients enrolled in this sub study, respectively. Furthermore, a robust signal was measured for pCrkL, pStat5, pStat3, pCreb, pAbl Y412 and pAbl Y245. The two sets of samples from study patients showed substantial changes in activation status over the course of therapy. Some changes, such as pStat3 alterations are only detectable in neutrophils and monocytes, while the activity of others i.e. pCreb was found to be ubiquitous. CD34+ cells indicated decreased phosphorylation of CrkL, Stat5, and Abl Y412/245. To increase the immunophenotyping resolution of the myeloid lineage, 3 additional cell surface markers were incorporated into the cell surface panel. In 1 healthy donor, and in diagnostic samples from three patients enrolled in this sub study, this allowed the identification of 13 cell subsets: CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-) (Figure 1 A,B). With respect to the relative number of cells identified for each cell type, the three diagnosis samples differed from the single healthy control. In the patients, we observed an expansion of the granulocytic compartment, as well as the emergence of CD34+ progenitor and stem cells in the peripheral blood. In conclusion, the here presented developed assay is able to resolve most of the cell subpopulations found in the hematopoietic tree, and also robustly measure the activity of central signalling substrates known to be involved in CML pathogenesis. With the addition of new phospho-specific antibodies, the methodology may facilitate the detailed characterization of CML in an immunological context, and may shed new light on both the disease and therapeutic mechanism. Analysis of variation in signal responses and immune profile are now in progress in the subset of patients (n=55) in the ENEST1st trial. Figure 1. Manually annotated SPADE tree from healthy donor and patient (3581_0002). With the incorporation of additional cell surface markers, the protocol was able to identify 13 cellular subsets in healthy donors (A) and a typical CML patient (B): CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-). Figure 1. Manually annotated SPADE tree from healthy donor and patient (3581_0002). With the incorporation of additional cell surface markers, the protocol was able to identify 13 cellular subsets in healthy donors (A) and a typical CML patient (B): CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), monocytes, dendritic cells (DCs), plasmacytoid dendritic cells (pDC's), neutrophils, basophils, B cells, hematopoietic stem cells (Lin- CD34+ CD38-) and progenitor cells (Lin- CD34+ CD38-). Disclosures Thaler: AOP Orphan: Research Funding. Lang:Celgene: Consultancy. Hjorth-Hansen:Bristol-Myers Squibb: Research Funding; Ariad: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding. Hellmann:Novartis: Consultancy, Other: funding of travel, accomodations or expenses, Research Funding, Speakers Bureau; BMS: Consultancy, Other: funding of travel, accomodations or expenses, Speakers Bureau. Giles:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Janssen:ARIAD: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer: Consultancy; Novartis: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Pfizer: Honoraria. Ossenkoppele:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Mustjoki:Signe and Ane Gyllenberg Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Pfizer: Honoraria, Research Funding; the Finnish Cancer Societies: Research Funding; Academy of Finland: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Gjertsen:Bergen University Hospital: Research Funding.
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- 2015
27. Reduced Expression of CD62L Is Associated with Increased ADAM17 Activity and Predicts Molecular Response to Nilotinib Therapy in Patients with Early Chronic Phase Chronic Myelogenous Leukemia (CML-CP)
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Satu Mustjoki, Ute Mark, Andreas Hochhaus, Angelica Loskog, Dominik Wolf, Sieghart Sopper, Nidas Jurjonas, Bjørn Tore Gjertsen, Jens Haenig, Günther Gastl, Frank Giles, Gert J. Ossenkoppele, and Kimmo Porkka
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medicine.medical_specialty ,Hematology ,biology ,business.industry ,Immunology ,Cell Biology ,Biochemistry ,Chronic phase chronic myelogenous leukemia ,Nilotinib ,T cell differentiation ,Internal medicine ,Molecular Response ,medicine ,biology.protein ,Cancer research ,L-selectin ,Stem cell ,business ,medicine.drug ,Whole blood - Abstract
Background: In CML, immunological surveillance in the MRD-situation might be of particular relevance for long-term control or even elimination of CML-repopulating stem cells. Little is known about potential immune-modulatory effects of nilotinib in vivo. A comprehensive monitoring program to characterize nilotinib-induced immunological changes was set up as a substudy to the ENEST1st study (NCT01061177), which is focused on examining the role of first-line nilotinib therapy in CML-CP. Aims: The identification of immunological changes induced by nilotinib and definition of immunological surrogates for response prediction in newly diagnosed CML-CP patients. Methods: Peripheral blood was taken prior to treatment initiation and after 6 and12 months (mo) from 50 patients treated on the ENEST1st study. Whole blood was phenotyped by 9-color flow cytometry employing 6 panels of antibodies to determine various leukocyte populations and expression of differentiation and activation markers. Soluble factors in plasma were measured by ELISA. Changes in immune parameters were correlated to clinical endpoints. Results: 55% of the patients included in this substudy achieved MMR at 6 mo, 75% at 12 mo and 79% at 24 mo of therapy. MR4.5 was achieved by 5%, 18%, 24% of patients at 6, 12 and 24 mo of therapy, respectively. Expression of L-selectin (CD62L), a known lymph node homing marker, was very low on T cells at baseline (median 5.7%) and the proportion of CD62L-expressing cells among CD4+ and CD8+ T cells negatively correlated with SOKAL score and spleen size. During treatment, CD62L expression on both T cell subsets significantly increased (median 72.3%) to normal levels at mo 6. Moreover, higher proportions of CD62L+ cells among both CD4+ and CD8+ T cells at baseline were negatively associated with lower BCR-ABL1 mRNA burden at 3, 6, 9, 15 and 18 mo. Cumulative response rates for MMR and MR4 were significantly higher in patients with high proportions (cutoff: >25%) of CD62L expressing T cells when compared to patients with low proportions of CD62L+ cells. A detailed characterization of other T cell differentiation marker, such as CD45RA, CD45R0, CD28, CD27 and CD95, did not reveal a reduction of cells usually expressing high CD62L levels, such as naïve T cells. CD62L expression levels on granulocytes were also low at baseline and increased to normal levels during nilotinib therapy. Low levels of CD62L expression were associated with elevated plasma levels of soluble CD62L before treatment initiation. Washing whole blood from treatment naïve patient several times before staining partially restored immunoreactivity for CD62L, and plasma taken at baseline but not after treatment initiation reduced CD62L signal on normal T cells, indicating that interference by increased levels of soluble CD62L with assessment of surface CD62L at least in part contributes to low CD62L expression. Importantly, ADAM17 (TACE, CD156b), the metalloproteinase responsible for CD62L shedding was increased on granulocytes and monocytes but not on T cells of CML patients when compared to normal controls suggesting that reduced levels on CD62L on T cells were mediated by ADAM17 on myeloid cells. Conclusion: We here show for the first time the prognostic impact of reduced CD62L expression levels at baseline for later molecular response to nilotinib in early CML-CP. Decreased expression of CD62L is at least in part due to increased CD62L-cleavage, probably by ADAM17, which is aberrantly expressed on the clonal myeloid cells. One may speculate that decreasing CD62L expression on T cells may interfere with immune surveillance of CML cells and that ADAM17 might be a target for adjunctive therapy. Larger prospective studies are needed to confirm the prognostic relevance of increased soluble CD62L and reduced T cell CD62L expression levels for molecular response in TKI-treated CML-CP. Disclosures Sopper: Novartis: Travel Grants Other. Mustjoki:Novartis: Research Funding. Gjertsen:Novartis: Research Funding. Mark:Novartis: Employment. Haenig:Novartis: Employment. Jurjonas:Novartis: Employment. Giles:Novartis: Honoraria, Research Funding. Hochhaus:Novartis: Research Funding; BMS: Research Funding; MSD: Research Funding; Ariad: Research Funding; Pfizer: Research Funding. Ossenkoppele:Novartis: Research Funding. Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Wolf:Nocartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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- 2014
28. Rituximab for primary chronic cold agglutinin disease: a prospective study of 37 courses of therapy in 27 patients
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Fuad Victor Shammas, Elling Ulvestad, Sigbjørn Berentsen, Håvar Knutsen, Henrik Hjorth-Hansen, Ruth Langholm, Waleed Ghanima, Geir E. Tjønnfjord, and Bjørn Tore Gjertsen
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medicine.medical_specialty ,Cold agglutinin disease ,medicine.medical_treatment ,Immunology ,Alpha interferon ,Biochemistry ,Gastroenterology ,Antibodies, Monoclonal, Murine-Derived ,Hemoglobins ,Immunopathology ,Internal medicine ,medicine ,Humans ,Paroxysmal cold hemoglobinuria ,Prospective Studies ,Prospective cohort study ,Interferon alfa ,business.industry ,Antibodies, Monoclonal ,Cell Biology ,Hematology ,Immunotherapy ,Drug Tolerance ,medicine.disease ,Recombinant Proteins ,Surgery ,Immunoglobulin M ,Interferon Type I ,Rituximab ,Anemia, Hemolytic, Autoimmune ,business ,medicine.drug - Abstract
Conventional therapies for primary chronic cold agglutinin disease (CAD) are ineffective, but remissions after treatment with the anti-CD20 antibody rituximab have been described in a small, prospective trial and in some case reports. In this study we report on 37 courses of rituximab administered prospectively to 27 patients. Fourteen of 27 patients responded to their first course of rituximab, and 6 of 10 responded to re-treatment. In both groups combined, responses were achieved after 20 of 37 courses, giving an overall response rate of 54%. We observed 1 complete and 19 partial responses. Two nonresponders and 3 patients who experienced relapse received second-line therapy with interferon-α combined with a new course of rituximab, and 1 nonresponder and 2 patients who experienced relapse achieved partial responses. Responders achieved a median increase in hemoglobin levels of 40 g/L (4 g/dL). Median time to response was 1.5 months, and median observed response duration was 11 months. We conclude that rituximab is an effective and well-tolerated therapy for CAD. Histologic and flow cytometric findings suggest that some of the effect may be mediated by mechanisms other than the elimination of clonal lymphocytes. We were unable to predict responses from the hematologic, immunologic, or histologic parameters before therapy. (Blood. 2004;103:2925-2928)
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- 2004
29. Single Cell Analysis Of Protein Phosphorylation In Chronic Myeloid Leukemia Treated With Dasatinib, Eltrombopag, and Pegfilgrastim
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Roald Lindås, Mihaela Popa, Henrik Hjorth-Hansen, Bjørn Tore Gjertsen, Jørn Skavland, Emmet McCormack, Randi Hovland, Klaus Beiske, and Stein Erik Gullaksen
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medicine.medical_specialty ,Myeloid ,business.industry ,Immunology ,Eltrombopag ,Myeloid leukemia ,Cell Biology ,Hematology ,Neutropenia ,medicine.disease ,Biochemistry ,Dasatinib ,chemistry.chemical_compound ,Endocrinology ,medicine.anatomical_structure ,Nilotinib ,chemistry ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Cancer research ,Bone marrow ,business ,Pegfilgrastim ,medicine.drug - Abstract
Introduction Tyrosine kinase inhibitor (TKI) induced neutropenia and thrombocytopenia may be relieved with G-CSF and trombopoietin analogs. To characterize the signaling response in a CML patient treated with hematopoietic growth factors and TKI, we employed single cell analysis of phosphoproteins in serial samples. Methods We characterized the single cell phosphoprotein responses to treatment with dasatinib, eltrombopag, and pegfilgrastim in CML-like cell lines K562 and KCL-22, normal peripheral blood leukocytes and leukocytes from a patient starting on nilotinib. These data formed a basis for comparison with serial samples (Figure 1) from a CML patients after extensive wash-out before restarted on third-choice TKI, combined with eltrombopag and pegfilgrastim. For characterization of in vivo transformational potential CML patient bone marrow cells were engrafted in duplicate in NOG mice by femoral injection. Basal phosphorylation was determined using 17 phosphorylation specific antibodies: All staining panels contain the same 4 surface antibodies and a live-dead discriminator; CD33(P67.6) PerCP-Cy5.5, CD38(HB7) PE-Cy7, CD34(581) PE, CD45(MEM-28) PE-Dynamics590 and phospho c-PARP(Asp214) AlexaFlour-700. Two phosphospecific antibodies were added to each panel with the respective direct conjugated dye Alexa Flour 488 and Alexa Flour 647. The lowest median signal for each phosphoprotein in CML cells and lymphocytes, respectively, were used as reference value for calculation of basal phosphorylation. Results A 56 y old male was diagnosed with CML (Sokal RR 0.75, Hasford RR 929, karyotype 46,XY,t(9;22)(q34;q11)[20], FISH 9q34 ABL/22q11.2 BCR dual fusion 62% of cells, PCR positive BCR-ABL e13e2/e14a2), developed prolonged thrombocytopenia and neutropenia on various dose levels of imatinib and nilotinib (neutrophile The CML bone marrow engrafted without symptomatic disease in NOG mice over 6 months monitoring, in contrast to engraftment with disseminated disease in an accelerated phase CML patient sample and the KCL-22 cell line. Phosphorylation of patient myeloid cells indicated STAT3(pY705) and STAT5(pY694) phosphorylation 2 and 3 hours after intake of eltrombopag. Dasatinib modestly inhibited SRC(pY418) and STAT3(pY705), but only transiently blunted the effect of eltrombopag. Pegfilgrastim strongly induced STAT3(pY705), STAT3(pS727), and SRC(pY418). Further analyses of cellular subsets and phospho-Abl will be presented. Conclusion Eltrombopag kept stable levels of platelets on dasatinib treatment, and cell phosphorylation reflected persisting signaling induced by eltrombopag after seven days on combination therapy. Pegfilgrastim induced strong phosphorylation of STAT3(pY705) during dasatinib co-therapy. Phosphoprotein signal analysis of patient leukemic cells reflected the hematological response of dasatinib, eltrombopag, and pegfilgrastim and may be used to monitor therapy responses of and growth factors and targeted therapy. Phosphoprotein modulated early after start of treatment. (A) Each day 1-23 is analysed separately with its pretreatment sample as control and signal baseline. (B) Various treatment days are compared, using pretreatment control at day 1 used as baseline. (A) and (B) were analysed by flow cytometry at the same day, using bar code labelling, sample processing and analysis as previously described (Skavland et al. Blood Cancer J 2012). On day 16 dasatinib and eltrombopag is administrated concomitantly. Signalling responses in lymphocytes were distinct different from myeloid cells. Further determination of cellular subsets and their signalling responses will be presented. Disclosures: Popa: KinN Therapeutics AS: Employment.
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- 2013
30. Identification Of AML Subtype-Selective Drugs By Functional Ex Vivo Drug Sensitivity and Resistance Testing and Genomic Profiling
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Riikka Karjalainen, Maija Lepistö, Tero Pirttinen, Jonathan Knowles, Maxim M. Bespalov, Samuli Eldfors, Sonja Lagström, Erkki Elonen, Anna Lehto, Bjørn Tore Gjertsen, Jesus M. Lopez Marti, Aino Palva, Alun Parsons, Tea Pemovska, Satu Mustjoki, Maria E. Rämet, Kimmo Porkka, Agnieszka Szwajda, Laura Turunen, Mika Kontro, Krister Wennerberg, Astrid Murumägi, Evgeny Kulesskiy, Pekka Ellonen, Pirkko Mattila, Caroline A. Heckman, Olli Kallioniemi, Minna Suvela, Imre Vastrik, Tero Aittokallio, Maija Wolf, Tuija Lundán, Henrik Edgren, Muntasir Mamun Majumder, Bhagwan Yadav, and Henrikki Almusa
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Trametinib ,0303 health sciences ,Ruxolitinib ,Sunitinib ,business.industry ,Immunology ,Cell Biology ,Hematology ,Drug resistance ,Bioinformatics ,Biochemistry ,Temsirolimus ,3. Good health ,Dasatinib ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,chemistry ,medicine ,Personalized medicine ,business ,030304 developmental biology ,030215 immunology ,medicine.drug ,Quizartinib - Abstract
Introduction Adult acute myeloid leukemia (AML) exemplifies the challenges of modern cancer drug discovery and development in that molecularly targeted therapies are yet to be translated into clinical use. No effective second-line therapy exists once standard chemotherapy fails. While many genetic events have been linked with the onset and progression of AML, the fundamental disease mechanisms remain poorly understood. There is significant genomic and molecular heterogeneity among patients. Several targeted therapies have been investigated for improved second-line AML therapy but none has been approved for clinical use to date. It would be critically important to identify patient subgroups that would benefit from such therapies and to identify combinations of drugs that are likely to be effective. Methods To identify and optimize novel therapies for AML, we studied 28 samples from 18 AML patients with an individualized systems medicine (ISM) approach. The ISM platform includes functional profiling of AML patient cells ex vivo with drug sensitivity and resistance testing (DSRT), comprehensive molecular profiling as well as clinical background information. Data integration was done to identify disease- and patient-specific molecular vulnerabilities for translation in the clinic. The DSRT platform comprises 306 anti-cancer agents, each tested in a dose response series. We calculated differential drug sensitivity scores by comparing AML responses to those of control cells in order to distinguish cancer-specific drug effects. Next generation RNA- and exome-sequencing was used to identify fusion transcripts and mutations that link to drug sensitivities. Results Individual AML patient samples had a distinct drug sensitivity pattern, but unsupervised hierarchical clustering of the drug sensitivity profiles of the 28 AML patient samples identified 5 functional AML drug response subtypes. Each subtype was characterized by distinct combinations of sensitivities: Bcl-2 inhibitors (e.g. navitoclax; Group 1), JAK inhibitors (e.g. ruxolitinib) (Group 2) and MEK inhibitors (e.g. trametinib) (Groups 2 and 4), PI3K/mTOR inhibitors (e.g. temsirolimus; Groups 4 and 5), broad spectrum receptor tyrosine kinase inhibitors (e.g. dasatinib) (Groups 3, 4 and 5) and FLT3 inhibitors (e.g. quizartinib, sunitinib) (Group 5). Correlation of overall drug responses with genomic profiles revealed that RAS and FLT3 mutations were significantly linked with the drug response subgroups 4 and 5, respectively. Activating FLT3 mutations contributed to sensitivity to FLT3 inhibitors, as expected, but also to tyrosine kinase inhibitors not targeting FLT3, such as dasatinib. Hence, these data point to the potential synergistic combinatorial effects of FLT3 inhibitors with dasatinib for improved therapy outcome (Figure). Early clinical translational results based on compassionate use support this hypothesis. Therefore, by combinations of drugs we expect to see synergistic drug responses that can be translated into efficacious and safe therapies for relapsed AML cases in the clinic. Clinical application of DSRT results in the treatment of eight recurrent chemorefractory patients led to objective responses in three cases according to ELN criteria, whereas four of the remaining five patients had meaningful responses not meeting ELN criteria. After disease progression, AML patient cells showed ex vivo resistance to the drugs administered to the patients, as well as significant changes in clonal architecture during treatment response. Furthermore, we saw genomic alterations potentially explaining drug resistance, such as appearance of novel fusion genes. Summary The ISM approach represents an opportunity for improving therapies for cancer patients, one patient at the time. We show that the platform can be used to identify functional groups of AML linking to vulnerabilities to single targeted drugs and, importantly, unexpected drug combinations. This information can in turn be used for personalized medicine strategies and for creating hypotheses to be explored in systematic clinical trials, both for approved and investigational drugs. Disclosures: Off Label Use: Many of the compounds included in our DSRT platform are not indicated for AML therapy. Mustjoki:BMS: Honoraria, Research Funding; Novartis: Honoraria. Porkka:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Kallioniemi:Medisapiens: Membership on an entity’s Board of Directors or advisory committees; Roche: Research Funding.
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- 2013
31. Dasatinib Treatment Induces Fast and Deep Responses In Newly Diagnosed Chronic Myeloid Leukemia (CML) Patients In Chronic Phase: Clinical Results From a Randomized Phase 2 Study (NordCML006)
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Henrik Hjorth-Hansen, Perttu Koskenvesa, Claes Malm, Satu Mustjoki, Kari Remes, Bjørn Tore Gjertsen, Hans Ehrencrona, Waleed Majeed, Martin Höglund, Ulla Olsson-Strömberg, Johan Richter, Tobias Gedde-Dahl, Bengt Simonsson, Lotta Ohm, Merja Suominen, Berit Markevärn, Stina Söderlund, Leif Stenke, and Kimmo Porkka
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,Phases of clinical research ,Imatinib ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Rash ,Surgery ,Discontinuation ,Dasatinib ,Leukemia ,Imatinib mesylate ,hemic and lymphatic diseases ,Internal medicine ,medicine ,Clinical endpoint ,medicine.symptom ,business ,medicine.drug - Abstract
Background Dasatinib is a potent BCR-ABL1 and SRC tyrosine kinase inhibitor, which in vitro is more effective against progenitor and putative leukemia stem cells than imatinib. This may translate into deeper molecular responses in vivo. Methods We randomized (1:1) 46 newly diagnosed CML patients to receive dasatinib 100 mg or imatinib 400 mg once daily. The primary endpoint of our study was treatment response in stem and progenitor cell fractions (Mustjoki et al, Leukemia 2013). We here summarize the clinical results of the study after a 24-month follow-up focusing on toxicity and standard response evaluation by quantitative BCR-ABL1 PCR and cytogenetics (NCT00852566 www.ClinicalTrials.gov). Results Both imatinib and dasatinib treated patients fared well with deeper and faster treatment responses than what has been reported in the registration studies. By karyotyping, dasatinib induced a faster response by 3 months (median of 5% of Ph+ cells in imatinib group vs. 0% in dasatinib group, p=0.01, n=21 in each group), but already by 12 months the difference disappeared, as all evaluable patients were in complete cytogenetic remission. The rate of molecular response MR3.0 was already at the 3 months time-point better in the dasatinib group (36% vs 8%, p=0.02; see Table), but within 18 months imatinib patients caught up the difference. In contrast, the achievement of deeper therapy responses, MR4.0 and MR4.5, was clearly different between the groups and increased over 24 months. After 18 months 64% and 71% of imatinib- and dasatinib-treated patients had achieved MR3.0 (p=0.59), while the MR4.0 rates were 23% and 62% (p=0.009) and MR4.5 rates 4% and 41% (0.003) (see Table below). The difference in median transcript levels was approximately 1 log (>10-fold difference) in all time-points after 3 months of therapy (see Table below). A total of 7 patients (30%) in both groups discontinued assigned treatment. Main drug-related toxicities were as expected. Dasatinib-induced serosal inflammation (pleural/pericardial effusions) was more frequent than in registration studies (6 patients, 27%). In 4 patients (18%) this led to therapy discontinuation, despite of drug interruption and dose reductions. In the imatinib group 3 patients discontinued due to drug-related toxicity (liver toxicity, rash and severe hypogammaglobulinemia with recurrent infections). Disease progression occurred in one dasatinib-treated patient (cytogenetic progression with the appearance of V299L mutation at month 9) and two imatinib-treated patients (blastic transformation at month 2 and molecular progression at month 18). The patient in blast phase has been transplanted and is currently in molecular remission. No CML-related deaths occurred, but one patient died from lung cancer. Interpretation Dasatinib induced faster and deeper molecular responses than imatinib and overall responses were better in both groups than in the registration studies. Relatively high rate of serosal toxicity was observed among the dasatinib-treated patients, but this had no adverse effect on response. Upcoming studies will show if the deeper treatment responses induced by dasatinib therapy translate into increased probability of successful therapy discontinuation. Disclosures: Hjorth-Hansen: Pfizer: Honoraria, Travel, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Novartis: Honoraria, Travel Other; Merck: Research Funding. Richter:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; Bristol-Myers Squibb: Consultancy, Honoraria, Travel, Travel Other. Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Mustjoki:Novartis: Honoraria; BMS: Honoraria, Research Funding.
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- 2013
32. Leukemic Stem Cell Quantification Is Of Prognostic Value In Newly Diagnosed Patients In Chronic Phase Chronic Myeloid Leukemia (CML-CP) Receiving Nilotinib Therapy: Results From The ENEST1st Stem Cell Substudy
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Jeroen Janssen, Johan Richter, Thoas Fioretos, Gert J. Ossenkoppele, Leif Stenke, Sieghart Sopper, Kimmo Porkka, Bjørn Tore Gjertsen, Noortje Thielen, Gisela Barbany, Andreas Hochhaus, Satu Mustjoki, Dominik Wolf, Sara Thunberg, Gerrit Jan Schuurhuis, and Frank Giles
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,CD34 ,Imatinib ,Cell Biology ,Hematology ,Biochemistry ,Dasatinib ,Haematopoiesis ,medicine.anatomical_structure ,Nilotinib ,Internal medicine ,medicine ,Bone marrow ,Stem cell ,Progenitor cell ,business ,medicine.drug - Abstract
Background Leukemic stem cells (LSCs) are considered to be very important therapeutic targets in CML, as it has been shown both in vitro and in vivo that tyrosine kinase inhibitor (TKI) therapy is not able to eradicate these entirely. We previously reported that LSC burden at diagnosis has significant prognostic impact on therapy outcome in imatinib and dasatinib treated CML patients. The ENEST1st study (NCT01061177) is focused on examining the role of first-line nilotinib therapy in patients in CML-CP. This translational, multinational, multicenter ENEST1st substudy addresses the predictive value of the stem cell profile in these patients. Patients and methods Bone marrow (BM) and/or peripheral blood (PB) samples were collected from newly diagnosed CML-CP patients before and after 1 and 3 months of nilotinib treatment. The LSC burden was analyzed by FISH and flow cytometry (FC). With the FISH method, CD34+ cells were pre-selected with paramagnetic beads after which they were fractionated into CD38+ (upper 80%, “progenitor”) and CD38- (lowest 5%, “stem cell”) pools using flow sorting. The proportion of Ph+ cells was assayed by counting 1,000 cells with interphase FISH using specific BCR-ABL1 probes. In the FC method, the stem cell compartment was defined as the lowest 1% of CD34+CD38- cells. LSCs were distinguished from normal hematopoietic stem cells (nHSCs) by either higher light scatter properties, and/or aberrant expression of CD7, CD11b and CD56, and/or higher CD45 and CD90 expression. Patient samples were assigned to as “residual nHSCs present” or “no residual nHSC present” at diagnosis and as “residual LSCs present” or “no residual LSCs present” at 1 and 3 months. Altogether, 48 patients from 6 European countries were investigated. Results By FISH analysis at diagnosis, the proportion of BCR-ABL+ cells in the stem cell compartment varied markedly in individual patients (1%-100%) and was lower (85%) than in progenitor (96%) or whole BM fractions (96%, p80% of BCR-ABL+ cells in the stem cell fraction at diagnosis. When LSCs were analyzed by the FC method, patients with residual nHSCs at diagnosis had significantly lower BCR-ABL levels at 3 (0.27 vs 1.74, p=0.025) and 6 (0.13 vs 1.21, p=0.03) months, but this was not reflected in lower BCR-ABL levels at 12 or 18 months. During nilotinib therapy, the proportion of BCR-ABL+ cells by FISH decreased rapidly both the in progenitor and stem cell compartments. At 3 months the median LSC percentage was 0.28%, and it did not differ significantly from the values detected either in the progenitor fraction (0.34%) or whole BM (0.29%). Similarly, only a few patients had detectable LSCs left during follow-up time-points with the FC method. Conclusions LSC burden at diagnosis reflects the biology of the disease in newly diagnosed CML-CP patients. It also carries a prognostic value in first-line treated nilotinib patients, and a substantial number of patients not meeting the optimal response criteria at the 12-month time-point have high LSC burden at diagnosis. Furthermore, the presence of residual nHSCs at diagnosis is predictive for molecular response at 3 and 6 months. Nilotinib therapy markedly decreases LSC pool during the first 3 months of treatment. Noteworthy, the reduction is as potent in the LSC compartment as it is in more mature progenitor cell or whole BM fractions. Disclosures: Richter: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Fioretos:Novartis: unrestricted research grant Other. Giles:Novartis: Consultancy, Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Ossenkoppele:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Porkka:Novartis: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau. Wolf:Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Meyers Squibb: Honoraria. Janssen:Novartis: Consultancy, Research Funding. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.
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33. Pharmacological Inhibition Of The SIRT1 Deacetylase With The Small Molecule Inhibitor Tenovin-6 Enhances Ablation Of FLT3-ITD+ LSC In Combination With TKI Treatment
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Tereza Osdal, Sonia Lain, Su Chu, Tinisha McDonald, Emmet McCormack, YinWei Ho, Sookhee Chun, Bjørn Tore Gjertsen, Ling Li, WenYong Chen, Cedric Dos Santos, Ravi Bhatia, Mihaela Popa, and Allen Lin
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Chemotherapy ,medicine.medical_treatment ,Immunology ,CD34 ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Transplantation ,Leukemia ,Apoptosis ,hemic and lymphatic diseases ,Cancer research ,medicine ,Progenitor cell ,Stem cell - Abstract
Acute myeloid leukemia (AML) is propagated by small populations of leukemia stem cells (LSC). Internal tandem duplication (ITD) in the FMS-like tyrosine kinase-3 (FLT3) is the most frequent mutation seen in AML patients, and is associated with poor prognosis. However, FLT3 tyrosine kinase inhibitors (TKI) demonstrate only moderate clinical activity in FLT3-ITD AML patients. Persistent FLT3-ITD+ AML LSC may represent a source of relapse after treatment. Additional therapeutic strategies are required to target LSC and improve outcomes for FLT3-ITD+ AML patients. We have shown that the stress-related deacetylase SIRT1 is expressed at high levels in FLT3-ITD+ AML stem/progenitor cells and in cord blood CD34+ cells ectopically expressing FLT3-ITD. Inhibition of SIRT1 using RNAi or a potent small molecule inhibitor Tenovin-6 (TV-6) induces apoptosis in FLT3-ITD+ AML progenitors via p53-dependent mechanisms while sparing normal CD34+ cells suggesting a potential role for SIRT1 in FLT3-ITD+ AML LSC maintenance (Blood 2012,120: A1368). We now show that treatment of FLT3-ITD+ AML CD34+ cells with the TKI AC220 (20 nM) did not significantly reduce SIRT1 levels, increase p53 acetylation, or increase p53 target gene expression (p21, Bax, Puma, DR5, Noxa), suggesting that kinase independent mechanisms maintain SIRT1 expression and activity in TKI-treated cells. The combination of TV-6 (1μM) with AC220 (20nM) significantly reduced survival of primary human FLT3-ITD+ AML CD34+ cells compared with AC220 or TV-6 alone (combination 47±8% inhibition, AC220 alone 21±7%, TV-6 alone 25±5%, p=0.03, n=7), and significantly increased inhibition of AML CD34+ cell growth compared to AC220 and TV-6 alone (TV-6 44±7% inhibition; AC220 30±6%, TV+AC220 59±8%, p=0.04, n=7). TV-6 treatment increased p53 expression and activity in AC220-treated FLT3-ITD+ AML CD34+ cells, but did not affect FLT3 phosphorylation or STAT5 activation. Interestingly, TV-6 treatment also reduced survival of FLT3-ITD+ AML CD34+ cells expressing the D835Y mutation associated with TKI resistance (AC220 5±5% inhibition, TV 33±8%, p=0.04, n=3], suggesting SIRT1 inhibition can target TKI-resistant FLT3-ITD+ AML cells. We next tested the effect of TV-6, AC220 and the combination on primary cells from one untreated cytogenetically normal and FLT3-ITD+ patient engrafted in NSG mice (2×106 cells/mouse).12 weeks after transplantation, mice were treated with vehicle, AC220 (10mg/kg, gavage), TV-6 (100mg/kg, intraperitoneal), or the combination (n=7 per group) for 4 weeks. Treatment with TV reduced human cell engrafted in NSG mice (TV-6 2.9*107±1.2*107 human CD45+ cells/2 femurs vs. Control 5.3*107±2.6*107, after 4 weeks treatment, p=0.04). Importantly, the combination of TV and AC220 significantly reduced AML cell engraftment compared with AC220 alone (AC220 2.4*107±0.9*107 human CD45+ cells. Combination 1.2*107±0.6*107, p=0.01). Residual functional LSC were assessed by transplantation into secondary recipients. BM cells from the combination treatment group demonstrated significantly reduced engraftment of leukemia cells after secondary transplantation compared to AC220 treatment alone (16 weeks after secondary transplantation, AC220 1.8*106±0.6*106 human CD45+ cells/2 femurs, Combination 0.4*106±0.1*106, p=0.02, n=6). To determine the potential of this combination for FLT3-ITD+ AML patients refractory to standard chemotherapy, AML xenografts established using cells from a chemotherapy-resistant patient (n = 36) were treated with TV-6, AC220, or the combination vs. Standard of care (SOC, Ara-C + DNR), and vehicle control. Molecular imaging of primary patient xenografts with fluorescently labeled monoclonal antibodies after 4 weeks of therapy revealed significant differences between combination and single arm groups (vs. TV-6, p=0.0005, n=8; vs. AC220, p=0.003, n=7). Furthermore, TV-6 alone increased survival over controls (TV-6 vs. Control, p=0.02, n=6), and the combination demonstrated significantly improved survival over AC220 (p=0.02) and SOC (p=0.006. n=6). In conclusion, our studies indicate that elevated SIRT1 expression in FLT3-ITD AML cells is maintained after FLT3 TKI treatment. Inhibition of SIRT1 with TV-6 can enhance ablation of FLT3-ITD+ AML LSC in combination with FLT3 TKI treatment. Our results support further evaluation of inhibition of SIRT1 as a therapeutic strategy in FLT3-ITD+ AML. Disclosures: Popa: KinN Therapeutics AS: Employment.
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- 2013
34. Survival Stratification In Acute Myeloid Leukemia By Single Cell Signal Profiling
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Håkon Reikvam, Bjørn Tore Gjertsen, Jørn Skavland, and Øystein Bruserud
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NPM1 ,Myeloid ,Immunology ,CD33 ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,CD38 ,Biochemistry ,medicine.anatomical_structure ,Ribosomal protein s6 ,Cancer cell ,Cytarabine ,medicine ,Cancer research ,medicine.drug - Abstract
Introduction Dysregulation and mutations in signaling genes of cancer cells characterize more than half of the acute myeloid leukemia (AML) patients, and contribute to chemoresistance through regulation of cellular processes including apoptosis and DNA repair. We investigated if determination of single cell basal phosphorylation of signaling proteins reflected mutation of FLT3 and NPM1, cytogenetics, response to first course of chemotherapy or overall survival. Methods We employed flow cytometric single cell analysis of phosphoproteins central to signal transduction pathways in myeloid cancer cells and analyzed peripheral blood leukocytes from 93 acute myeloid leukemia (AML) patients. Blood samples from consecutively diagnosed AML patients with high peripheral blood blast counts (>7x109/L, >70% blasts) were collected after informed consent was given and biobanked by cryo-preservation. AML samples was thawed and equilibrated for one hour in a defined serum-free medium (StemSpan SFEM medium, Stem Cell Technologies) which include insulin and transferrin. All samples were viability controlled and validated for growth factor response. Basal phosphorylation was determined using 17 phosphorylation specific antibodies: All staining panels contain the same 4 surface antibodies and a live dead discriminator; CD33(P67.6) PerCP-Cy5.5, CD38(HB7) PE-Cy7, CD34(581) PE, CD45(MEM-28) PE-Dynamics590 and phospho c-PARP(Asp214) Alexa Flour 700. Two phosphospecific antibodies were added to each panel with the respective direct conjugated dye Alexa Flour 488 and Alexa Flour 647; p38(pT180/pY182) and ERK2(pT202/pY204), SRC(pY418) and Akt(pT308), PDK1(pS241) and Akt(pS473), STAT1(pY701) and ribosomal protein S6(pS235/36), STAT3(pY705) and STAT5(pY694), CREB(pS133) and STAT3(pS727), ribosomal protein S6(pS240) and NFkB(pS529), 4EBP(pT37/pT45) and STAT6(Y641), without p-antibody and JNK(pT183/pY185). The lowest median signal for each phosphoprotein in AML cells and lymphocytes, respectively, were used as reference value for calculation of basal phosphorylation. Hierarchical clustering with the use of complete linkage were created using TM4, and Principal Component Analysis was carried out using Unscrambler X (CAMO Software). Results Unsupervised clustering revealed two distinct signature clusters based on low or elevated phosphorylation level among AML cells. A similar cluster signature was absent in endogenous non-leukemic lymphocytes from the same patients. No correlations between basal phosphorylation and prognostic mutations (FLT3 or NPM1), cytogenetics or response to first course of chemotherapy were found. In AML patients treated with intensive chemotherapy (n=45) the cluster with low phosphorylation level (n=19) correlated with significant (p=0.007) shorter overall survival. Principal component analysis verified the cluster analysis and guided a reduction to only three phosphoproteins (STAT3, 4EBP1, ribosomal protein S6) with statistically significant (p=0.014) stratification of survival. Conclusion Leukemic cells demonstrated a phosphorylation profile that reflected survival of the intensively treated patients, but surprisingly not correlated with mutational status of FLT3, NPM1, cytogenetics or first course remission status. This suggests that phosphoprotein determination in leukemic cells provide prognostic information so far not available with current diagnostics. The robust and relatively simple method of single cell signal profiling should be tested in clinical trials to examine its feasibility in therapy response prediction. More extensive mutational and epigenetic analyses are needed in search for the molecular origin of the low/high signal profiles. (A) Unsupervised hierarchical cluster analysis based on three phospho protein analysis stratified in two distinct clusters of patients receiving standard intensive induction chemotherapy (n=45; daunorubicin + cytarabine or idarubicin + cytarabine (3+7) similar to HOVON AML protocols 103 and 102, respectively). (B) Kaplan-Meier plot was performed calculating P value with the use mantel-Cox log-rank test. Disclosures: No relevant conflicts of interest to declare.
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35. Elacytarabine Is More Effective Preclinically Than Cytarabine In Combination With Daunorubicin In Primary Patient Xenografts Of AML
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Øystein Bruserud, Marit Liland Sandvold, Emmet McCormack, Mihaela Popa, Birgitte Booij, André Sulen, Bjørn Tore Gjertsen, Siv Lise Bendringaas, Lene Mari Vikebø, and Tereza Osdal
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Oncology ,medicine.medical_specialty ,Severe combined immunodeficiency ,Anthracycline ,Elacytarabine ,business.industry ,Daunorubicin ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,medicine.anatomical_structure ,Refractory ,Internal medicine ,medicine ,Cytarabine ,Bone marrow ,business ,medicine.drug - Abstract
Acute myeloid leukemia (AML) is a heterogeneous disease with poor prognosis. The overall survival rate in AML patients has changed little in the last 20 years with overall survival rates for young patients (< 60 years) remaining at 35%. However, for elderly patients (> 60 years), survival is less than 10%. Despite the fact that AML represents one of the most molecularly characterized malignancies, the standard therapeutic option is unaltered in the last 15 years i.e. we still solely rely upon a combination of anthracycline and Cytarabine (Ara-C) as the cornerstone of induction therapy. Recently many potentially drugable targets have been discovered and while responses have been observed, these are generally amongst sub-sets of patients with good to intermediate prognostic factors and most often in combination with conventional therapy. Elacytarabine (ELA; CP-4055) is a fatty acid derivative (elaidic acid ester) of Ara- C, currently in undergoing phase II clinical trial as second-course remission-induction therapy in combination with anthracycline. In this study we determined the maximum tolerated dose for the combination of ELA with Daunorubicin (DAN) in immunodeficient NOD-scid-IL2Rγcnull (NSG) mice. Subsequently, we compared the preclinical efficacy of the combination of ELA (30 mg/kg; q.d.x5) or Ara-C (500 mg/kg; q.d.x2) with DAN (2.5 mg/kg; q.d.x3) in a primary patient xenograft model of refractory AML following relapse after Ara-C+DAN therapy (n = 24). Efficacy was evaluated employing a novel time-domain imaging strategy utilizing targeted monoclonal antibodies conjugated to near infrared dye that specifically recognize and bind to epitopes on AML cells enabling non-invasive monitoring of disease progression following therapy. Similarly, bone marrow aspirates were assayed for AML content by flow cytometry (CD45+/34+/33+) and IHC analysis. The combination of ELA and DAN was well tolerated in NSG mice xenografted with primary patient AML cells. While longitudinal near infrared optical imaging of AML disease dissemination illustrated lower disease burden in Ara-C + DAN treated mice than controls (p ELA is more effective preclinically than Ara-C in combination with DAN in primary patient xenografts of refractory AML. ELA was found to be well tolerated and significantly increased survival in combination with Daunorubicin. Despite withdrawal of Elacytarabine as a single agent (following Phase III clinical trial earlier this year in relapsed and refractory AML versus investigators choice of treatment) our preclinical results demonstrate combined therapy of ELA+DAN may promise enhanced survival advantages over current standard therapy for AML patients. Disclosures: Popa: KinN Therapeutics AS: Employment. Gjertsen:KinN Therapeutics AS: Membership on an entity’s Board of Directors or advisory committees. McCormack:KinN Therapeutics AS: Membership on an entity’s Board of Directors or advisory committees.
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36. Immune Monitoring In Patients With Early Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Treated With Frontline Nilotinib
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Richard Greil, Ward Posthuma, Gregor Verhoef, Edo Vellenga, Angelica Loskog, Frank Giles, Henrik Hjorth-Jansen, Tobias Gedde-Dahl, Ulla Olsson-Stroemberg, Jesper Stentoft, Andreas Hochhaus, Waleed Majeed, Kimmo Porkka, Augustin Ferrant, Gert J. Ossenkoppele, Alois Lang, Leif Stenke, Jan Van Droogenbroeck, Willem M. Smit, Werner Linkesch, Günther Gastl, Kourosh Lofti, Sieghart Sopper, Satu Mustjoki, Soeren Lehman, Dominik Wolf, Ute Mark, Nidas Jurjonas, Bjørn Tore Gjertsen, Jens Haenig, Josef Thaler, Johan Richter, Guided Treatment in Optimal Selected Cancer Patients (GUTS), and Stem Cell Aging Leukemia and Lymphoma (SALL)
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education.field_of_study ,Myeloid ,business.industry ,T cell ,Immunology ,Population ,Imatinib ,Cell Biology ,Hematology ,Natural killer T cell ,Biochemistry ,Monitoring program ,Dasatinib ,medicine.anatomical_structure ,Nilotinib ,Medicine ,business ,education ,medicine.drug - Abstract
Imatinib and dasatinib modulate immune responses in vitro and in vivo . Immunological surveillance in the MRD-situation might be of particular relevance for long-term control or even elimination of CML-repopulating stem cells. Little is known about potential immune-modulatory effects of nilotinib in vivo . The ENEST1st study ([NCT01061177][1]) is focused on examining the role of firstline nilotinib therapy in CML-CP - this ENEST1st substudy involves a comprehensive immunological monitoring program. To evaluate and characterize the immunomodulatory effects of nilotinib therapy in newly diagnosed CML-CP patients. Peripheral blood was taken prior to treatment initiation and after 6 and12 months (mo) from 50 patients treated on the ENEST1st study. Samples were analyzed by nine color flow cytometry employing six panels of antibodies to determine various leukocyte populations including lymphocyte subsets (e.g. T cell subpopulations including Treg and NKT cells, NK cells, B cells), myeloid populations (e.g. monocytes, MDSC) and dendritic cell subsets (e.g. mDC, pDC), respectively. Changes in immune cell parameters were correlated to clinical endpoints and PK data. 55% of the patients included into this substudy achieved MMR at 6mo, 75% at 12mo and 79% at 12mo of therapy. MR4 was achieved by 21%, 31%, 50% of patients at 6, 12 and 24 mo of therapy, respectively. A high proportion of CD14+ monocytes with aberrant expression of CD56, present at baseline, dropped to undetectable levels at 6 and 12 mo of treatment. The most prominent immunological change from baseline to 12 mo was a dramatic increase of CD19+ B cells. The abundance of CD19+ B cells at baseline was negatively correlated with SOKAL score. Among T-cell subpopulations, the proportion of CD56+ NKT-cells decreased. Similarly, the proportion of CD8+ T cells significantly decreased concomitant with increasing CD4+ T cells during therapy. In parallel to a decrease of CD45RA+ expressing T cells among both CD8+ and CD4+ T cell subsets, the proportion of CD45R0+ memory cells increased. This increase was mainly due to an increase of the CD95+CD28+ central memory population. In contrast to our expectation, CD25high FoxP3+ Treg cells were even increased transiently 6 months after treatment initiation but at 12 months dropped back to levels seen at diagnosis. None of the immunological changes were associated with plasma levels of nilotinib or response to therapy. Various immunological changes were detected during nilotinib therapy. However, most changes after therapy are most likely due to normalization of the peripheral blood compartment. Correlation of a specific immunological signature with response to nilotinib could not be identified. However the increased proportion of memory T cells during therapy suggests a potential contribution of the cellular immune response to the eradication of malignant cells. Molecular analyses of NKT-cells will show whether the increased proportion before therapy is due to aberrant expression of CD56 as in monocytes or a specific expansion of this subset. Mustjoki: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Greil: Roche: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Thaler: Roche: Honoraria, Research Funding. Richter: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Verhoef: MedImmune: Research Funding. Mark: Novartis: Employment. Haenig: Novarts: Employment. Jurjonas: Novartis: Employment. Gastl: Novartis: Honoraria, Research Funding. Giles: Novartis: Consultancy, Research Funding. Hochhaus: Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Ossenkoppele: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Porkka: BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Wolf: Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Meyers Squibb: Honoraria. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01061177&atom=%2Fbloodjournal%2F122%2F21%2F2731.atom
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- 2013
37. A Phase II Study of Elacytarabine/Idarubicin As Second Course Remission-Induction in Patients with Acute Myeloid Leukemia Who Failed Cytarabine/Anthracycline
- Author
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Jianyu Rao, Athos Gianella-Borradori, David A. Rizzieri, Xavier Thomas, Norbert Vey, Bjørn Tore Gjertsen, Jürgen Krauter, Malin Johansen, Françoise Huguet, Thomas Kindler, Trygve Bergeland, Igor Wolfgang Blau, Utz Krug, Tove Flem Jacobsen, and Richard F. Schlenk
- Subjects
Oncology ,medicine.medical_specialty ,education.field_of_study ,Combination therapy ,Anthracycline ,Elacytarabine ,business.industry ,Immunology ,Population ,Phases of clinical research ,Cell Biology ,Hematology ,Pharmacology ,Biochemistry ,Regimen ,Internal medicine ,Cytarabine ,Medicine ,Idarubicin ,business ,education ,medicine.drug - Abstract
Abstract 46 Background Elacytarabine, a fatty acid derivative (elaidic acid ester) of cytarabine, has pharmacokinetic and pharmacodynamic properties that may lead to improved clinical outcomes (Adema et al., 2011) compared to cytarabine. A phase I study had established 1000 mg/m2/d continuous infusion on d 1 – 5 as a safe and effective dose of elacytarabine given in combination with idarubicin at 12 mg/m2/d IV d 1 – 3 (Giles et al., 2012). Mechanism of Action The mechanism of action is similar to cytarabine, but unlike cytarabine, plasma elimination half-life is long, intracellular distribution is prolonged and activity is independent of membrane nucleoside transporters. Resistance to cytarabine has been associated with decreased expression of the human equilibrative nucleoside transporter 1 (hENT1) (Hubeek et al., 2005). Aims The aim of the study is to determine the efficacy and safety of elacytarabine given in combination with idarubicin; to explore the relationship between the hENT1 status in AML cells and response to cytarabine and to elacytarabine; to evaluate the safety and toxicity of the combination therapy. Methods Adult patients with AML who have not attained blast clearance after the first induction course with a standard dose cytarabine based regimen, assessed from day 12 on, were enrolled in the study. Patients received a combination of elacytarabine and idarubicin scheduled as described above, as second induction course. Patients treated with further courses received either the combination therapy or elacytarabine monotherapy at 2000 mg/m2/d on d 1 – 5, at the investigator‘s discretion. hENT1 expression level was analysed retrospectively by immunocytochemistry at time of initial AML diagnosis (pre-therapeutic) and/or before elacytarabine/idarubicin treatment (baseline). Results In the on-going study which will recruit up to 50 evaluable patients, 47 patients [27 male, 20 female, median age 60 years (range 18–78), ECOG 0–2] have currently been treated. 40 patients have been evaluated for response, of whom 35% suffered from secondary leukemia. Median time from start of first induction course to start of elacytarabine treatment was 36 days (range 13 – 189). After treatment with elacytarabine and idarubicin, 18/40 evaluable patients attained a CR/CRi. Median time to remission was 36 days (range 25 – 60). Incidence of low hENT1 expression was approximately 50%. Preliminary data shows a higher response rate of elacytarabine compared to cytarabine in this low hENT1 population (47% versus 37%). The most frequently reported non haematological adverse events grades 3 and 4 were fatigue, infections/sepsis, hypoalbuminaemia, hypokalaemia and hypoxia. Three deaths occurred within 30 days after start of treatment, all due to sepsis. Additional two patients died from progressive AML within 60 days after start of treatment. All these five patients suffered from secondary leukaemia. Summary/Conclusion At the time of this abstract (August 2012) elacytarabine in combination with idarubicin shows a promising clinical activity with a CR/CRi rate of approximately 45% in patients failing a first induction course. Low expression of the hENT1 transporter is associated with a lower probability of achieving remissions following cytarabine treatment. Preliminary data indicates that assessment of hENT1 expression in blast cells could be used to identify patients less likely to benefit from cytarabine and for whom elacytarabine could be an effective therapy. The emerging safety profile is as expected for these groups of cytotoxic therapies. Results of the completed study will be presented at the meeting. Disclosures: Kindler: Novartis: Honoraria. Johansen:Clavis Pharma: Employment. Bergeland:Clavis Pharma: Employment. Gianella-Borradori:Clavis Pharma: Employment. Flem Jacobsen:Clavis Pharma: Employment. Rao:Clavis Pharma: Honoraria.
- Published
- 2012
38. A Phase II Study of Elacytarabine/Idarubicin As Second Course Remission-Induction in Patients with Acute Myeloid Leukemia Who Failed Cytarabine/Anthracycline, and Evaluation of the Impact of the Nucleoside Transporter hENT1 on Response
- Author
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Jianyu Rao, Richard F. Schlenk, David A. Rizzieri, Athos Gianelli-Borradori, Jürgen Krauter, Norbert Vey, Françoise Huguet, Igor Wolfgang Blau, Tove Flem Jacobsen, Utz Krug, Malin Johansen, Bjørn Tore Gjertsen, and Xavier Thomas
- Subjects
Oncology ,medicine.medical_specialty ,Elacytarabine ,Combination therapy ,Anthracycline ,business.industry ,Immunology ,Phases of clinical research ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Regimen ,Internal medicine ,medicine ,Cytarabine ,Idarubicin ,business ,Febrile neutropenia ,medicine.drug - Abstract
Abstract 1533 Background: Elacytarabine is a fatty acid derivative (elaidic acid ester) of cytarabine. The mechanism of action is similar to cytarabine, but unlike cytarabine, cellular uptake and activity of elacytarabine are independent of nucleoside transporters. Resistance to cytarabine has been associated with decreased expression of the human equilibrative nucleoside transporter 1 (hENT1) (Hubeek et al., 2005). An agent such as elacytarabine, active in low hENT1 expressing, cytarabine resistant acute myeloid leukemia (AML), could therefore improve clinical outcome in patients. Aims: To determine the efficacy and safety of elacytarabine given in combination with idarubicin to patients who have failed the first ”standard” induction course with a cytarabine based regimen, as well as to explore the relationship between the hENT1 status in AML cells and response. Methods: Study therapy consisted of one course elacytarabine 1000 mg/m2/d CIV on d1-5 administered in combination with idarubicin 12 mg/m2/d IV d1-3 in adult patients with AML who after a first cytarabine based induction course have not attained blast clearance (bone marrow (BM) >5 % blasts, circulating blasts, or chloroma etc). Assessment of response was at least 12d after induction start. Responding patients could receive the same course or elacytarabine 2000 mg/m2/d CIV on d1-5 d and then consolidation therapy with two courses of either elacytarabine monotherapy or combination with idarubicin as described above or could proceed to allogeneic SCT at any time point. The hENT1 expression level of BM blasts was analyzed by immunocytochemistry at time of initial AML diagnosis (pre-cytarabine course) and/or before elacytarabine treatment. The planned sample size is 50 evaluable patients, and with a target of 40% CR/CRi rate, the lower limit of the 90% confidence interval for the CR/CRi rate will be greater than 22% with at least 80% probability. The CR/CRi rate will be estimated and its corresponding two-sided 90% confidence interval will be provided. The significance of the association between the hENT1 expression level and response status will be assessed through a Chi-square test of hENT1 expression level (high, low) versus CR/CRi (yes, no). Results: In the ongoing study, 26 patients [16 male, 10 female, median age 61 years (range 18–71), ECOG PS 0–2] have been treated with elacytarabine and idarubicin. 23 patients have currently been response evaluated, and 11 attained a CR/CRi and 3 a PR (post one elacytarabine course). The most frequently reported related non-hematologic adverse events (AEs) CTCAE grade ≥ 3 were febrile neutropenia, infections/sepsis and increased liver function tests. 30 patients have been scored for hENT1 expression level at time of diagnosis. Preliminary results indicate that approximately 50 % of the patients hENT1 expression is low (defined as less than 10% of blasts stained). As to response, only approximately 1/3 of patients with low hENT1 blasts respond to cytarabine while for the high hENT1 2/3 respond. Three deaths occurred within 30 days after start of treatment and were all due to sepsis. All 3 patients had secondary leukemia. Summary/Conclusion: Elacytarabine administered at 1000 mg/m2/d CIV d1-5 in combination with idarubicin 12mg/m2/d IV d1-3 showed promising clinical activity with a CR/CRi rate of approximately 45 %. The adverse event profile, is as expected for cytarabine combination therapy. Preliminary data indicate that the assessment of hENT1 transporter expression in blasts could be used to select patients less likely to benefit from cytarabine and for whom elacytarabine could be an effective therapy. Disclosures: Gianelli-Borradori: Clavis Pharma: Employment. Flem Jacobsen:Clavis Pharma: Employment. Krug:MedA Pharma: Honoraria; Novartis: Honoraria; Alexion: Honoraria; Boehringer Ingelheim: Research Funding; Sunesis: Honoraria.
- Published
- 2011
39. Serum Heat Shock Protein Levels and Cytokine Profiles Are Altered In AML Patients Before Treatment and Following Disease Stabilizing Therapy
- Author
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Øystein Bruserud, Håkon Reikvam, Bjørn Tore Gjertsen, and Hanne Fredly
- Subjects
medicine.medical_treatment ,Immunology ,Cellular homeostasis ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Proinflammatory cytokine ,Hsp70 ,Cytokine ,Granulocyte macrophage colony-stimulating factor ,Heat shock protein ,medicine ,biology.protein ,Interleukin 6 ,Interleukin 4 ,medicine.drug - Abstract
Abstract 3599 Heat shock proteins (HSPs) maintain cellular homeostasis and function as molecular chaperones assisting protein folding and translocation. Their intracellular levels are up-regulated in response to a wide variety of insults allowing the cell to survive otherwise lethal conditions. Heat shock proteins are also released to the extracellular space and lead to a more efficient cross-presentation to specific T cells and may also initiate release of proinflammatory cytokines, stimulate NK cells and facilitate DC maturation. Extracellular Heat shock protein (HSP) 70 and HSP90 are characteristics of immunogenic apoptosis, and primary human acute myeloid leukemia (AML) cells release these mediators during spontaneous in vitro apoptosis. Primary AML cells also show constitutive release of several cytokines; these heterogeneous mediators interact through local cytokine networks that regulate growth and differentiation of normal as well as malignant hematopoietic cells. In the present study we investigated serum levels of HSP70/HSP90 and the serum cytokine profiles of patients with untreated AML and patients receiving disease-stabilizing treatment based on all-trans retinoic acid (ATRA) plus valproic acid. Serum samples from 82 untreated AML patients, including 42 patients receiving AML-stabilizing therapy, and 20 healthy controls were analysed. HSP and cytokine levels were determined by ELISA and multiplex analyses. Patients with untreated AML showed significantly increased HSP90 levels compared to healthy controls (Mann Whitney U-test, p AML patients showed an altered serum cytokine profile compared to healthy controls with significantly altered levels of several chemokines, interleukins, growth factors and immunomodulatory cytokines (Tab.1). These cytokine levels were usually increased compared to the healthy controls. Hierarchical cluster analysis of the untreated patients showed a close association between HSP70, HSP90 and Hepatocyte growth factor (HGF) levels, these mediators also showed a significant correlation (Spearman, p We conclude that both HSP levels and serum cytokine profiles are altered in untreated AML, and the modulation of these serum levels by AML-stabilizing chemotherapy may reflect a possible mechanism for the clinical effect of this treatment strategy. The table shows serum levels for 82 AML patients and 20 healthy controls where significant differences were found (Mann-Whitney U-test, p Disclosures: No relevant conflicts of interest to declare.
- Published
- 2011
40. The Proportion of Ph+ CD34+CD38neg Leukemic Stem Cells In the Bone Marrow of Newly Diagnosed Patients with Chronic Myeloid Leukemia (CML) In Chronic Phase (CP) Is Variable and Correlates with High Sokal Risk, High Leukocyte Count, Low Hemoglobin Concentration, Splenomegaly and Increased Hematological Toxicity During Initial TKI-Therapy. Data From a Randomized Phase II NordCML006 Study
- Author
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Ole Weiss Bjerrum, Leif Stenke, Claes Malm, Tobias Gedde Dahl, Perttu Koskenvesa, Ali Moshfegh, Waleed Majeed, Randi Hovland, Ingunn Dybedal, Kimmo Porkka, Ulla Olsson Strömberg, Dag Josefsen, Henrik Hjorth Hansen, Sari E. Jalkanen, Lotta Ohm, Kirsi Latvala, Satu Mustjoki, Bengt Simonsson, Kari Remes, Johan Richter, Jesper Stentoft, Merja Suominen, Gisela Barbany, Tor Olofsson, Berit Markevärn, Thoas Fioretos, Carin Lassen, Katrin Rapakko, Bjørn Tore Gjertsen, and Sara Thunberg
- Subjects
medicine.medical_specialty ,Hematology ,business.industry ,Immunology ,CD34 ,Myeloid leukemia ,Cell Biology ,Neutropenia ,medicine.disease ,Biochemistry ,Gastroenterology ,Dasatinib ,medicine.anatomical_structure ,Imatinib mesylate ,Internal medicine ,medicine ,Bone marrow ,Stem cell ,business ,medicine.drug - Abstract
Abstract 667 Background: Targeted tyrosine kinase inhibitor (TKI) therapy efficiently induces rapid hematologic and cytogenetic responses in most CML patients. In vitro studies have suggested that CML stem cells are resistant to TKIs and therefore the treatment may need to be life-long. However, the in vivo effects of TKIs on the leukemic stem cell pool in a patient population have not been prospectively assessed. In addition, the biological impact and prognostic value of leukemic stem cell burden at diagnosis is unknown. Aim: To analyze the proportions of Ph+ cells in the stem cell compartment in newly diagnosed CP CML patients at diagnosis, and correlate the initial leukemic stem cell burden to biological variables and hematological toxicity during first 3 months of TKI therapy. Patients and Methods: 42 newly diagnosed CP CML patients within the Nordic countries were randomized to receive either dasatinib 100 mg (n=21) or imatinib 400 mg (n=21) once daily. Stem cell assays were performed at diagnosis and at 1, 3, and 6 months from start of TKI therapy. After pre-selection of CD34+ cells from large volume bone marrow (BM) aspirates with paramagnetic beads, the CD34+ cells were fractionated into CD38 positive and negative pools using a sorting flow cytometer. The proportion of Ph+ cells in the stem cell fractions was assayed by counting 1000 cells with interphase FISH for BCR-ABL1. Results: Measurement of Ph+ stem cells was feasible in most patients at diagnosis and results from 36 evaluable patients will be presented. The median volume of BM aspirate was 28, 37, 40 and 36 ml at diagnosis, 1, 3, and 6 months after therapy start, respectively. The median yield of BM mononuclear cells was 1000, 110, 93 and 79 ×106, respectively. The median proportion of Ph+ cells was significantly lower in the more primitive CD34+CD38neg fraction when compared to the CD34+CD38+ fraction or to unfractionated BM (79%, range 0.6–100%; 96%, 50–100%; and 96%, 57–100%, respectively, p=0.0001). The proportion of Ph+ cells in the CD34+CD38neg fraction at diagnosis correlated with high leukocyte count (r=0.59, p Conclusions: The proportion of Ph+ stem cells at the time of diagnosis varied from 1 to 100% between individual CML patients. It was correlated with hemoglobin concentration, leukocyte count, blast percentage and spleen size at diagnosis and with hematological toxicity during early course of treatment, mirroring paucity of healthy hematopoietic stem cell reservoir. The size of the leukemic stem cell pool at diagnosis may be a powerful prognostic marker and a major biological determinant for the high Sokal risk group. The effect of TKI therapy on the malignant stem cell pool size and correlation to therapy responses will be evaluated when all patients have reached the primary study endpoint of 6 months. Disclosures: Mustjoki: BMS, Novartis: Honoraria. Richter:BMS, Novartis: Consultancy, Honoraria. Simonsson:BMS, Novartis: Consultancy, Honoraria, Research Funding. Porkka:BMS, Novartis: Consultancy, Honoraria, Research Funding. Hjorth-Hansen:BMS, Novartis: Consultancy, Honoraria, Research Funding.
- Published
- 2010
41. Identification of Molecular Targets of AML by Phosphoproteomic Screening of Valproic Acid Treated BNML and C. Elegans RNAi Validation
- Author
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Emmet McCormack, Bjørn Tore Gjertsen, Hilde Nilsen, Gry Sjøholt, Rakel Brendsdal Forthun, Tanima SenGupta, Øystein Bruserud, and Siv Lise Bedringaas
- Subjects
Gene knockdown ,Programmed cell death ,Reporter gene ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Synthetic lethality ,Biology ,Biochemistry ,Molecular biology ,Transplantation ,Apoptosis ,RNA interference ,Cancer research - Abstract
Abstract 4151 Disease stabilisation, instead of cure, is proposed as the therapeutic strategy of choice in elderly or chemoresistant acute myeloid leukemia (AML). This approach may also be of particular benefit to patients for whom allogeneic bone marrow (re)transplantation is not an option. Previously, we have clinically investigated the addition of valproic acid (VPA) to various combination chemotherapies with initial results in AML indicating prolonged survival, with follow-up on a predominantly out-patient basis. Consequently, we aimed to identify further molecular targets of VPA, which may enhance its therapeutic efficacy through screening for VPA-modulated phosphoproteins in relevant preclinical models of AML, and validation of these targets in RNAi screen of Caenorhabditis elegans (C. elegans). Brown Norwegian Myeloid Leukemia (BNML) has previously been described as a particularly relevant preclinical rat model of AML. Indeed, leukemic rats treated with 170 mg/kg VPA twice-daily achieved therapeutic serum levels of VPA and demonstrated significant increases in survival in comparison to controls (p = 0.004). To screen for molecular targets of VPA effect in this responsive model, we investigated the differences in control and VPA treated BNML phosphoproteomes by difference gel electrophoresis (DIGE) separation and subsequent differential gel software analysis. This was achieved through harvest of phosphoproteins from leukemic blasts, isolated from the spleens of treated and control BNML rats by immobilized metal ion affinity chromatography (IMAC) and subsequent protein identification via Orbitrap mass-spectrometry. Significant differential expression of 9 phosphoproteins was found in VPA treated BNML rats compared to controls, including Tubulin α-1B chain (TBA1B) and Actin β (ACTB), indicating these genes as possible targets of VPA therapy. To validate the functionality of 7 of these genes, RNAi was performed in wild type Bristol N2 strain of C. elegans at larval stage L1, 24 hours prior to exposure to 15 mM VPA for 72 hours. Knockdown of 4 of 7 genes resulted in larval developmental arrest, defined as synthetic lethality. In order to ascertain if synthetic lethality induced by these 4 genes was resultant of apoptosis, we employed the CED-1::GFP transgenic reporter assay to quantify germline cell death following RNAi depletion and subsequent exposure to VPA (15 mM, 24 hours). Increased numbers of apoptotic corpses in the germline was determined for all genes examined. To further examine the role of p53 in the observed apoptotic induction we used the transgenic strain cep-1::CED-1::GFP, which expresses the C. elegans ortholog of p53, CEP-1. Successive RNAi knockdown of our 4 candidate genes, again effected increased basal number of apoptotic corpses independently of CEP-1. These results suggest that similar combinational treatment of AML may be beneficial, irrespective of p53 status. To further investigate this thesis in a human AML cell line, MOLM-13 cells were co-treated with VPA and small molecule inhibitors of prospective targets TBA1B and ACTB, namely paclitaxel, and cytochalasin B. Inhibition of actin polymerization or stabilisation of tubulin polymerization resulted in increased apoptosis when supplemented with VPA, as determined by DNA specific staining with Hoechst 33342. These results suggest that use of these combinations may be beneficial in the treatment of AML. In conclusion, this study indicates that phosphoproteomic screening of BNML and subsequent target verification in C. elegans worms has the potential to identify future drugable targets for effective combinatorial therapy with valproic acid in acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2009
42. Evaluation of Combinational Therapy of MDM2-Antagonist Nutlin-3 and HDAC-Inhibitor Valproic Acid in Acute Myeloid Leukemia in Vitro and in Vivo
- Author
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Øystein Bruserud, Gurid Venås, Bjørn Tore Gjertsen, Rakel Brendsdal Forthun, Ingvild Haaland, and Emmet McCormack
- Subjects
Myeloid ,Immunology ,Myeloid leukemia ,Cell Biology ,Hematology ,Nutlin ,Biology ,Pharmacology ,Biochemistry ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Apoptosis ,Cell culture ,In vivo ,Toxicity ,medicine ,biology.protein ,Mdm2 - Abstract
Nutlin-3 is a small-molecule antagonist of MDM2 that induces non-genotoxic stabilization and activation of the tumor suppressor protein p53, resulting in therapeutic effects in tumor models comprising wild type TP53. Valproic acid (VPA) is an anti-convulsive drug with histone deacetylase (HDAC) inhibitor activity that induces differentiation and apoptosis in acute myeloid leukemia (AML) cells. Both therapeutic compounds indirectly affect the regulation of p53; nutlin-3 by inhibiting MDM2, the main negative regulator of p53, and VPA by inhibiting HDACs that participate in p53 deacetylation and destabilization. While mutations in TP53 occur in less than 10% of AML, over-expression of MDM2 is frequently observed. Furthermore, aberrant recruitment of histone deacetylases (HDACs) is seen in AML, leading to block of myeloid differentiation. We therefore hypothesized that concomitant inhibition of MDM2 and HDACs would synergistically induce p53-mediated apoptosis and inhibit tumor growth. We examined the anti-leukemic effects of nutlin-3 in combination with low doses valproic acid in AML cell lines, primary AML cells, and in an in vivo xenograft model using optical imaging. To evaluate the efficacy of the combination of nutlin-3 and VPA in AML cells expressing wild type TP53, the AML cell line MOLM-13 was treated with various concentrations of the two drugs both alone and in combinations (nutlin-3; 0.5–10 μM, VPA; 50–1000 μM, nutlin-3:VPA; 1:100) over different time periods (24–72h, nutlin-3 only for the 24 last hours). Synergistic or additive effects were detected in three distinct viability assays; 3H-thymidine incorporation was used to examine effect on proliferation, WST-1 was used to determine number of metabolic active cells in culture, and DNA specific staining with Hoechst 33342 was used to determine apoptosis after drug-treatment. In addition, MOLM-13 cells treated with the combination showed super additive induction of p53 and target genes. The optimal combination and time period found in MOLM-13 cells were tested in 40 different primary AML samples using the three different viability assays. Toxicity of the combination treatment was tested in normal peripheral blood lymphocytes, while preliminary toxicity of nutlin-3 and VPA alone and in combination on healthy NOD/SCID IL2γnull mice permitted determination of treatment regime. We developed an optical imagable model for in vivo evaluation of the combinational therapy by injecting NOD/SCID IL2γnull mice with MOLM-13 cells transfected with a tetracycline activated luciferase expressing construct (termed L192). Bioluminescent imaging was performed using a TD-SAMI (Time-domain small animal molecular imager). The efficacy of the combinational therapy was tested in 20 NOD/SCID IL2γnull mice injected with MOLM-13 L192 cells and divided into four groups; control, VPA (50 mg/kg b.i.d), nutlin-3 (200 mg/kg b.i.d) and nutlin-3 + VPA (200 mg/kg + 50 mg/kg) b.i.d. Treatment was scheduled for three weeks. The combination of nutlin-3 and VPA significantly inhibited disease development after one week, as monitored by in vivo imaging. Limitations due to nutlin related toxicity prevented further evaluation of continuous combinational therapy after 14 days. However, all treatment groups showed a significant increase in survival compared to the control group, with the combination group demonstrating decreased leukaemic burden as visualized by optical imaging and longer mean average survival time. VPA effect on survival was also tested in a BNML rat leukemia model, in which VPA-treatment (170 mg/kg) resulted in significant longer mean survival compared to the control. Together, the results suggest combined targeting of MDM2 and HDACs as a promising therapeutic approach in AML. Future studies will apply the established bioluminescent MOLM-13 AML xenograft model for further evaluation of the combinational therapy, using a different dosing regimen and scheduling. In addition, we will evaluate combinations of differing classes of HDAC inhibitors and MDM2 antagonists.
- Published
- 2008
43. Bcl-2 Protects against p53-Induced Apoptosis through Hdm2
- Author
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Eystein Oveland, Line Wergeland, Guillermina Lozano, Kevin B. Spurgers, Tsuyoshi Honda, Bjørn Tore Gjertsen, James B. Lorens, Manel Cascallo, Torill Høiby, and Timothy J. McDonnell
- Subjects
Programmed cell death ,Immunology ,Wild type ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Ubiquitin ligase ,Proteasome ,Ubiquitin ,Apoptosis ,biology.protein ,Cancer research ,Mdm2 - Abstract
Hdm2 is up-regulated in several malignancies including sarcomas and acute myeloid leukemia, where it counteracts the anti-proliferative and pro-apoptotic effect of wild type p53. The anti-apoptotic protein Bcl-2 is often elevated in many tumors with wild type p53 and serves to block p53-induced apoptosis. We demonstrate that the protein level of Hdm2 positively correlates with the level of Bcl-2 and follows the Bcl-2 level in different cell systems. Over-expression of Bcl-2 protects Hdm2 from DNA-damage induced degradation in a dose dependant manner. In addition, modulation of Bcl-2 by shRNA knockdown reduced the Hdm2 protein level in parallel. Consequently, treatment of AML cells with the Bcl-2 small inhibitory molecule HA14-1 attenuated the level of Hdm2. The Bcl-2 level, but not the DNA damage induced Hdm2 degradation, was affected by disruption of the E3 ubiquitin ligase activity of Hdm2. In addition, the DNA-damage induced Hdm2 down-regulation was blocked by disrupted E1 ubiquitin-activation, defect polyubiquitination and by proteasome inhibitors. Finally, we show that Bcl-2 protection from p53-induced cell death requires co-expression of Hdm2 in double null p53/mdm2 mouse embryonic fibroblasts. Our results indicate that Bcl-2 regulates the Hdm2 level and that Hdm2 is a key mediator in Bcl-2 inhibition of p53-induced apoptosis. This is of particular therapeutic interest for cancers displaying elevated Hdm2 and Bcl-2, like sarcoma and acute myeloid leukemia.
- Published
- 2008
44. Synergistic Anti-Leukemic Effect of Protein Kinase A Activator and PPARγ Agonist Is Independent of Bcl-2 Status
- Author
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Bjørn Tore Gjertsen, Bjarte Skoe Erikstein, Frank Schwede, Karl Johan Tronstad, Rolf Bergen, and Emmet McCormack
- Subjects
Immunology ,Cell ,Tetradecylthioacetic acid ,Cell Biology ,Hematology ,Mitochondrion ,Pharmacology ,Biology ,Biochemistry ,Molecular biology ,In vitro ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Cell culture ,In vivo ,Apoptosis ,medicine ,Protein kinase A - Abstract
The fatty acid derivate tetradecylthioacetic acid (TTA) and 8-CPT-cAMP have previously been shown to have anti-proliferative activities through activation of PPARγ and effects on the mitochondrial metabolism, respectively. Based on our previous work, we hypothesized that these two agents have a converging point of action. The combined effect of these two compounds was investigated in acute myelogenous cell lines (IPC-81 and HL-60), including IPC-81 clones over expressing either Bcl-2 or inducible cAMP early transcription repressors (ICER). Brown Norwegian rat leukaemia (BNML) was used as an in vivo model system. 8-CPT-cAMP- and TTAs anti-leukemic activity was assessed in vivo and in vitro. The compounds were able to synergistically induce a decrease in viability in promyelocytic IPC-81 cells when measured by combinational index. Enforced expression of ICER could partially protect the cell from TTA induced apoptosis. Bcl-2 over expression did not prevent additive apoptosis induction of the two compounds. An additive proliferation inhibition of the two compounds was detected in HL-60 cells at a 8-CPT-cAMP concentration that did not affect viability in itself. TTA and 8-CPT-cAMP had a spleen anti-leukaemic activity in vivo but the schedule of administration will have to be further investigated. In conclusion there seems to be a converging point in the action of 8-CPT-cAMP and TTA since they synergistically decrease in vitro viability of acute leukemic cells.
- Published
- 2007
45. Discrimination and Quantification of Spectrally Similar Near-Infrared Probes by Time-Domain (TD) Optical Imaging in Acute Myeloid Leukemia Mouse Models
- Author
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Alexandre Belankov, Pierre Couture, Bjørn Tore Gjertsen, Emmet McCormack, and Maja Mujic
- Subjects
chemistry.chemical_classification ,medicine.medical_specialty ,Biomolecule ,Immunology ,Near-infrared spectroscopy ,Cell Biology ,Hematology ,Biochemistry ,Fluorescence ,Spectral imaging ,Functional imaging ,Autofluorescence ,chemistry ,In vivo ,medicine ,Biophysics ,Time domain - Abstract
The use of whole-body optical imaging in the near-infrared (NIR) spectrum (650–1100 nm) employing fluorescently labelled reagents recognising cell-specific biomarkers of leukemia has become a standard modality in preclinical models of the human disease. A particular challenge is represented by leukemic infiltrates in liver and spleen, organs with high optical absorbance. While there are increasingly impressive arrays of fluorescently labelled biomolecules available for exploitation via optical imaging, the number of commmercially availible fluorophores for NIR imaging remain limited. In particular, simultaneous imaging of disease progression and functional imaging of more specific biological processes within the same sample is complicated by the requiste for multiple filtersets for fluorophores with similar spectral properties. Subsequent “bleeding” fluorescence through filtersets is unavoidable precluding ones ability to quantify specific fluorophores based on fluorescence. Similarly, descrimination of in vivo autofluorescence of similar spectral properties to commonly employed NIR dyes, consequent of ingested food comlicates contrast even further. More recently spectral imaging techniques have aided discrimination of fluorophores of similar spectral profiles however, these techniques attenuate much of the light reaching the detector. Time-domain (TD) optical imaging through the use of pulsed laser diodes and time resolved detector system, typically a photo-multiplier tube (PMT), has previously been demonstrated to distinguish between changes in physiological processes such as; tissue pH or calcuim concentration, based on changes in fluorescence lifetime of a fluorescently labelled probe. Here we demonstrate employing a single wave lenght TD optical imaging (eXplore Optix™, ART Inc) the potential to discriminate and quantify combinations of diverse NIR probes of spectrally similar properties but differing fluorescence lifetime on the basis of fluorescence lifetime in appropriate in vitro phantoms. Similarly, we illustrate the ability of this technique to discriminate between endogenous autofluorescence from administered fluorophores in vivo of leukemic cells in liver and spleen, and subsequent distinction of mixtures these fluorophores via their inherent fluorescent lifetimes in vivo.
- Published
- 2007
46. The Role of Bcl-2 in Apoptosis Induced by khat (Catha Edulis) in Acute Myeloid Leukemia Cell Lines
- Author
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George W. Francis, Bjørn Tore Gjertsen, Anne Christine Johannessen, Therese Bredholt, Elisabeth Anne Okumo Dimba, Olav Karsten Vintermyr, and Daniela Elena Costea
- Subjects
Programmed cell death ,Small interfering RNA ,Immunology ,Myeloid leukemia ,Endogeny ,Cell Biology ,Hematology ,Transfection ,Biology ,Pharmacology ,biology.organism_classification ,Biochemistry ,Cell culture ,Apoptosis ,Khat - Abstract
The herbal drug khat (Catha edulis Forsk.) is habitually used in Eastern Africa and the Middle East. The pharmacological activity of khat is due to its alkaloid fraction which initiates a rapid release of monoamine neurotransmitters from nerve terminals when ingested. Extracts from khat have been shown to induce cell death caspase-dependent apoptosis in human leukaemia cells. In this study, apoptotic sensitivity is correlated with endogenous protein expression of the anti-apoptogen Bcl-2, as well as transfection enforced over expression of Bcl-2. Exposure of a panel of human leukaemia cell lines to khat extract resulted in the development of similar apoptotic morphotypes. The sensitivity of the cell lines to khat induced apoptosis revealed a trend of inverse correlation with endogenous levels of Bcl-2. Stable or transient transfection of Bcl-2 inhibited khat-induced apoptosis, and the threshold for khat-induced apoptosis was lowered by blocking Bcl-2 synthesis with small interfering RNA. Together, these data suggest Bcl-2 modulation as a possible mechanistic pathway through which khat-induced apoptosis may occur.
- Published
- 2005
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