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Drug induced depletion of myeloid progenitors in bone marrow samples as an ex vivo estimation of hematotoxicity
Abstracts Research Topics
900 First Report of NRAS Mutations Contributing to Donor Cell Leukemia after allogeneic Bone Marrow Transplantation for Severe Aplastic Anemia Rita Assi,1 Rami Mahfouz,2 Hussein Abou Ghaddara,1 Ali Bazarbachi1 1
Internal Medicine/Hematology Oncology, American University of
Beirut Medical Center; 2Pathology and Laboratory Medicine/Molecular Diagnostics, American University of Beirut Medical Center
Introduction: Donor cell leukemia (DCL) is a rare event following allogeneic bone marrow transplantation (BMT) for severe aplastic anemia (SAA). Context: A 25 year old female underwent BMT for SAA from an HLA-identical brother in April 2012 with no subsequent GVHD. She presented in October 2013 with severe thrombocytopenia and 94% circulating blasts. Bone marrow (BM) studies confirmed the diagnosis of B-acute lymphoblastic leukemia. Cytogenetic evaluation demonstrated that the patient had an abnormal clone 45,XY,-7,del(9)t(7;9)(q11;p13)[11]/46,XY[2] as well as a chimerism with 99% of donor origin. Materials and Methods: BM studies were also performed on the donor. DNA was extracted from the donor and the recipient samples for Next Generation Sequencing (NGS) genetic analysis using the Ion AmpliSeqÔ Comprehensive Cancer Panel of the ION TORRENT platform, which screens for 409 genes involved in leukemia and implicated in cancer research. Results: The donor did not have any mutations other than in PTPN11 with a non-pathogenic polymorphism that was passed on to the recipient. The recipient on the other hand, developed two mutations that were not present prior to the transplant and consisted of two mutations at the same codon of NRAS gene: p.Gly12Ser and pGly12Asp, with mixed allele burdens of 76 and 8%, respectively. We also performed DNA chimerism analysis using Short tandem repeat (STR) markers in order to rule out any residual patient cells remaining prior to transplant (example by inefficient eradication of the bone marrow). We found out that 10 markers (each with two alleles) were informative between pre-transplant and donor and, in addition, the donor and the post-transplant samples were identical and there does not appear to be any residual DNA marker from pretransplant sample. Thus, there was no evidence of chimerism and the source of the leukemia is donor cells. The NRAS results obtained by NGS were confirmed by another technique using realtime PCR and ultra-sensitive hybridization assay and results were also compatible. Conclusions: Our results provide the first report of NRAS mutations contributing to the leukemisation of donor cells. In-depth analysis of the mechanisms of oncogenic transformation of donor-derived cells might provide a unique human model for studying the leukemogenesis in vivo.
S240
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Clinical Lymphoma, Myeloma & Leukemia June 2015
901 Drug induced depletion of myeloid progenitors in bone marrow samples as an ex vivo estimation of hematotoxicity Daniel Primo,1 Joan Ballesteros,1 Antonio Jiménez,2 Ataulfo González Fernández,3 Miguel Hernández,4 Raúl Córdoba,5 Yolanda González,6 M. José Moreno,7 Alicia Bailén,2 Jesús Martín,8 Anabelle Chinea,9 Asunción Etxveste,10 José Mariano Hernández,11 Albert Oriol,12 Jaime Pérez de Oteyza,13 Luis Palomera,14 Cristina Encinas,15 Felipe Prósper,16 Rebeca Iglesias,17 Laura Rosiñol,18 Alicia Robles,1 Julián Gorrochategui,1 Verónica García,1 Pilar Hernández,1 Enrique Ocio,19 Joaquín Martínez20 1
Vivia Biotech, Madrid; 2Hospital Regional Universitario de Málaga
Carlos Haya, Málaga; 3Hospital Universitario Clínico San Carlos, Madrid; 4Hospital Universitario de Canarias, Santa Cruz de Tenerife; 5
Hospital Infanta Sofía, Madrid; 6Hospital Universitari de Girona
Doctor Josep Trueta, Girona; 7Hospital General universitario Morales Meseguer, Murcia; 8Hospital Universitario Virgen del Rocío, Sevilla; 9
Hospital Universitario Ramon y Cajal, Madrid; 10Hospital Universitario
Donostia, Guipúzcoa;
11
Hospital General de Segovia, Segovia;
12
Hospital Universitari Germans Trias i Pujol, Barcelona;
Universitario Madrid Sanchinarro, Madrid; versitario Lozano Blesa, Zaragoza; Gregorio Marañón, Madrid;
15
13
Hospital
14
Hospital Clínico Uni-
Hospital General Universitario
16
Clínica Universidad de Navarra, Nav-
arra; 17MD Anderson Cancer Center, Madrid; 18Hospital Clínic de Barcelona, Barcelona; 19Hospital Clínico Universitario de Salamanca; 20
Hospital Universitario 12 de Octubre, Madrid
Background and Objectives: We show the ability of our flow cytometry-based automated ExvitechÓ platform to measure depletion of different subsets of CD34+ progenitors and other cell subsets to potentially establish a new assay to screen drug candidates and combinations for hematotoxicity. Patients and Methods: 16 Normal Bone Marrow (NBM) samples were included. For a proof of concept, we have selected two known cytotoxic drugs (Cytarabine and Clofarabine: N¼10NBM) and two novel drugs with low expected cytotoxicity (Ruxolotinib and Volasertib: N¼6NBM). A multiple staining (CD45v450/Anexin-FITC/CD117-PE/CD34 PerCP/CD38APC/ CD19APCya7) was used to distinguish between populations. Drug response was evaluated as a depletion survival index of each cell population relative to the average of 6 control wells. Results: As expected, nucleosides induce hematotoxicity in almost all the studied NBM samples. Results reflect that Cytarabine has similar activity than Clofarabine in terms of efficacy (Ymax: 30% vs 23%) but with less potency (EC50: 6mM vs 0.2mM) in the immature population (N¼10; Figure 1). This reflects a lower hematological toxicity which is consistent with clinical practice. Interestingly, for both drugs there is a large range of interpatient variability inside this population in terms of efficacy (Cytarabine, range Ymax: 4%-75% and Clofarabine, range Ymax: 10%-37%) and potency (Cytarabine, range EC50: 1mM-14mM and Clofarabine, range EC50: 0.01mM-2mM) suggesting it may be useful to
Abstracts personalize treatments and doses to patients. By contrast, ruxolitinib and volasertib had little effect (Ymax: 80% vs 73%) in the immature population with minimal interpatient variability confirming the low toxicity expected for these novel drugs. Conclusions: These preliminary results show that Vivia ExvitechÓ platform is able to measure hematopoietic progenitors depletion in addition to other cell populations for novel drugs or before patient’s treatment that could contribute to a better clinical management of patients. This
assay could be helpful to both hematological and solid tumor drugs. The platform can measure the effect of drugs on both malignant (efficacy) and progenitor cells (hematotoxicity) in bone marrow samples of Multiple Myeloma and Non-Hodgkin’s Lymphoma. This approach enables screening for hematotoxicity potential new discovery compounds, new drug candidates, and their synergistic combinations, thus also supporting drug discovery and development.
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S241
900 First Report of NRAS Mutations Contributing to Donor Cell Leukemia after allogeneic Bone Marrow Transplantation for Severe Aplastic Anemia Rita Assi,1 Rami Mahfouz,2 Hussein Abou Ghaddara,1 Ali Bazarbachi1 1
Internal Medicine/Hematology Oncology, American University of
Beirut Medical Center; 2Pathology and Laboratory Medicine/Molecular Diagnostics, American University of Beirut Medical Center
Introduction: Donor cell leukemia (DCL) is a rare event following allogeneic bone marrow transplantation (BMT) for severe aplastic anemia (SAA). Context: A 25 year old female underwent BMT for SAA from an HLA-identical brother in April 2012 with no subsequent GVHD. She presented in October 2013 with severe thrombocytopenia and 94% circulating blasts. Bone marrow (BM) studies confirmed the diagnosis of B-acute lymphoblastic leukemia. Cytogenetic evaluation demonstrated that the patient had an abnormal clone 45,XY,-7,del(9)t(7;9)(q11;p13)[11]/46,XY[2] as well as a chimerism with 99% of donor origin. Materials and Methods: BM studies were also performed on the donor. DNA was extracted from the donor and the recipient samples for Next Generation Sequencing (NGS) genetic analysis using the Ion AmpliSeqÔ Comprehensive Cancer Panel of the ION TORRENT platform, which screens for 409 genes involved in leukemia and implicated in cancer research. Results: The donor did not have any mutations other than in PTPN11 with a non-pathogenic polymorphism that was passed on to the recipient. The recipient on the other hand, developed two mutations that were not present prior to the transplant and consisted of two mutations at the same codon of NRAS gene: p.Gly12Ser and pGly12Asp, with mixed allele burdens of 76 and 8%, respectively. We also performed DNA chimerism analysis using Short tandem repeat (STR) markers in order to rule out any residual patient cells remaining prior to transplant (example by inefficient eradication of the bone marrow). We found out that 10 markers (each with two alleles) were informative between pre-transplant and donor and, in addition, the donor and the post-transplant samples were identical and there does not appear to be any residual DNA marker from pretransplant sample. Thus, there was no evidence of chimerism and the source of the leukemia is donor cells. The NRAS results obtained by NGS were confirmed by another technique using realtime PCR and ultra-sensitive hybridization assay and results were also compatible. Conclusions: Our results provide the first report of NRAS mutations contributing to the leukemisation of donor cells. In-depth analysis of the mechanisms of oncogenic transformation of donor-derived cells might provide a unique human model for studying the leukemogenesis in vivo.
S240
-
Clinical Lymphoma, Myeloma & Leukemia June 2015
901 Drug induced depletion of myeloid progenitors in bone marrow samples as an ex vivo estimation of hematotoxicity Daniel Primo,1 Joan Ballesteros,1 Antonio Jiménez,2 Ataulfo González Fernández,3 Miguel Hernández,4 Raúl Córdoba,5 Yolanda González,6 M. José Moreno,7 Alicia Bailén,2 Jesús Martín,8 Anabelle Chinea,9 Asunción Etxveste,10 José Mariano Hernández,11 Albert Oriol,12 Jaime Pérez de Oteyza,13 Luis Palomera,14 Cristina Encinas,15 Felipe Prósper,16 Rebeca Iglesias,17 Laura Rosiñol,18 Alicia Robles,1 Julián Gorrochategui,1 Verónica García,1 Pilar Hernández,1 Enrique Ocio,19 Joaquín Martínez20 1
Vivia Biotech, Madrid; 2Hospital Regional Universitario de Málaga
Carlos Haya, Málaga; 3Hospital Universitario Clínico San Carlos, Madrid; 4Hospital Universitario de Canarias, Santa Cruz de Tenerife; 5
Hospital Infanta Sofía, Madrid; 6Hospital Universitari de Girona
Doctor Josep Trueta, Girona; 7Hospital General universitario Morales Meseguer, Murcia; 8Hospital Universitario Virgen del Rocío, Sevilla; 9
Hospital Universitario Ramon y Cajal, Madrid; 10Hospital Universitario
Donostia, Guipúzcoa;
11
Hospital General de Segovia, Segovia;
12
Hospital Universitari Germans Trias i Pujol, Barcelona;
Universitario Madrid Sanchinarro, Madrid; versitario Lozano Blesa, Zaragoza; Gregorio Marañón, Madrid;
15
13
Hospital
14
Hospital Clínico Uni-
Hospital General Universitario
16
Clínica Universidad de Navarra, Nav-
arra; 17MD Anderson Cancer Center, Madrid; 18Hospital Clínic de Barcelona, Barcelona; 19Hospital Clínico Universitario de Salamanca; 20
Hospital Universitario 12 de Octubre, Madrid
Background and Objectives: We show the ability of our flow cytometry-based automated ExvitechÓ platform to measure depletion of different subsets of CD34+ progenitors and other cell subsets to potentially establish a new assay to screen drug candidates and combinations for hematotoxicity. Patients and Methods: 16 Normal Bone Marrow (NBM) samples were included. For a proof of concept, we have selected two known cytotoxic drugs (Cytarabine and Clofarabine: N¼10NBM) and two novel drugs with low expected cytotoxicity (Ruxolotinib and Volasertib: N¼6NBM). A multiple staining (CD45v450/Anexin-FITC/CD117-PE/CD34 PerCP/CD38APC/ CD19APCya7) was used to distinguish between populations. Drug response was evaluated as a depletion survival index of each cell population relative to the average of 6 control wells. Results: As expected, nucleosides induce hematotoxicity in almost all the studied NBM samples. Results reflect that Cytarabine has similar activity than Clofarabine in terms of efficacy (Ymax: 30% vs 23%) but with less potency (EC50: 6mM vs 0.2mM) in the immature population (N¼10; Figure 1). This reflects a lower hematological toxicity which is consistent with clinical practice. Interestingly, for both drugs there is a large range of interpatient variability inside this population in terms of efficacy (Cytarabine, range Ymax: 4%-75% and Clofarabine, range Ymax: 10%-37%) and potency (Cytarabine, range EC50: 1mM-14mM and Clofarabine, range EC50: 0.01mM-2mM) suggesting it may be useful to
Abstracts personalize treatments and doses to patients. By contrast, ruxolitinib and volasertib had little effect (Ymax: 80% vs 73%) in the immature population with minimal interpatient variability confirming the low toxicity expected for these novel drugs. Conclusions: These preliminary results show that Vivia ExvitechÓ platform is able to measure hematopoietic progenitors depletion in addition to other cell populations for novel drugs or before patient’s treatment that could contribute to a better clinical management of patients. This
assay could be helpful to both hematological and solid tumor drugs. The platform can measure the effect of drugs on both malignant (efficacy) and progenitor cells (hematotoxicity) in bone marrow samples of Multiple Myeloma and Non-Hodgkin’s Lymphoma. This approach enables screening for hematotoxicity potential new discovery compounds, new drug candidates, and their synergistic combinations, thus also supporting drug discovery and development.
Clinical Lymphoma, Myeloma & Leukemia June 2015
- S241