Novel Ex Vivo Assay Measures Drug-Induced Depletion of Hematopoietic Progenitors As an Estimate of Hematotoxicity
Authors: Primo Daniel , Ballesteros Joan , Jimenez Antonio , Gonzalez Ataulfo , Hernandez Miguel , Cordoba Raul , González Yolanda , Moreno Hernan Jose , Bailen Alicia , Martin Jesus , Chinea Anabelle , Etxeveste Asuncion , Hernandez Jose Mariano , Oriol Albert , Perez de Oteiza Jaime , Palomera Luis , Encinas Cristina , Prosper Felipe , Iglesias Rebeca , Rosiñol Laura , Robles Alicia , Gorrochategui Julian , Garcia Veronica , Hernandez Pilar , Ocio Enrique , Martinez Joaquin 
Published: October 2019

Abstract
Background and Objectives: Recently, knowledge of the specific genetic markers responsible for cancer malignancies and their associated signaling pathways have generated many new targets that promise to increase drug efficacy while reducing side effects such as hematotoxicity. Although hematotoxicity is widely assumed to be the result of depleting hematopoietic progenitors, in particular myeloid progenitors, there are no assays to test new compounds in bone marrow samples to investigate this effect. Because these effects are patient-specific, an assay that identifies those patients risk of severe hematotoxicity by certain treatments could help personalizing patient treatment. Here, we show the ability of our flow cytometry-based automated Exvitech© platform to measure depletion analysis of different subsets of CD34+ progenitors and other cell subsets to potentially establish a new assay to screen drug candidates and combinations for hematotoxicity, as well as personalizing therapy to the individual sensitive patient at risk. So that in multiple myeloma (MM) we can identify at the same time CD34+ cells and pathological plasma cells; we could actually measure depletion of both malignant cells and progenitor cells on the same patient sample.
Patients and methods: 16 normal bone marrow (NBM) and 4 MM samples were studied. For a proof of concept to test the hypothesis, we have selected two known cytotoxic drugs (cytarabine and clofarabine: N=10NBM) and two novel drugs with low expected cytotoxicity (ruxolotinib and volasertib: N=6NBM). The whole sample was plated into 96-well assay plates containing 8 concentrations of each drug and incubated for 48-hours for NBM and 12h with Bortezomib for MM samples. A multiple staining (CD45v450/Anexin-FITC/CD117-PE/CD34PerCP/CD38APC/CD19APCya7) was used to distinguish between both populations. Drug response was evaluated as a depletion survival index of each cell population relative to the average of 6 control wells in each plate.
Results: As expected, nucleoside induces hematotoxicity in most of the studied NBM samples, but not all. Results reflect that cytarabine has similar activity than clofarabine in terms of efficacy (Ymax: 30% vs 23%) but with less potency (EC50: 6µM vs 0.2µM) in the immature population (N=10; Figure 1). This reflects a lower hematological toxicity which is consistent with clinical practice. Interestingly, for both drugs there is a large range of inter-patient variability inside this population in terms of efficacy (cytarabine, range Ymax: 4%-75% and clofarabine, range Ymax: 10%-37%) and potency (cytarabine, range EC50: 1µM-14µM and clofarabine, range EC50: 0.01µM-2µM) suggesting that in a subsets of vulnerable patients, drug doses could be tailored. 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 even at very high concentrations never achieve in vivo (Figure 1).
Figure 2 shows bortezomib activity in MM bone marrow samples measuring both the dose response on malignant and myeloid progenitor cells. For Patient 1 the drug depletes myeloid precursor at lower doses than malignant cells, suggestive of severe hematotoxicity before therapeutic benefit can be achieved. Patient 2 shows the opposite case, where bortezomib depletes malignant cells completely without depleting myeloid precursors, suggestive of a good therapeutic index for this individual patient.
Conclusions: These preliminary results show that Vivia Exvitech© platform is able to measure hematopoietic progenitors depletion in addition to other cell populations for novel drugs or before patientxs treatment that could contribute to a more selective drug development or a better clinical management of patients. This approach enables screening for hematotoxicty potential new discovery compounds, new drug candidates, and their synergistic combinations, thus supporting drug discovery and development. This assay could be helpful to both hematological and solid tumor drugs. The platform can measure both malignant and progenitor cells in bone marrow samples of MM and Non Hodgkin's Lymphoma. This simultaneous analysis shown for bortezomib could help guiding the clinical response and possible hematological toxicities associated to drug treatments.
Figure 1: Figure 1:.
Figure 2: Figure 2:.

Disclosures
Primo: Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Garcia:Vivia Biotech: Employment. Hernandez:Vivia Biotech: Employment. Martinez:Vivia Biotech: Membership on an entity's Board of Directors or advisory committees.