Personalized Medicine Test of Multi-Drug Protocols Ex Vivo for Hematological Malignancies.
Authors: Bennett Teresa A , Sapia Sandra , Primo Daniel , Suarez Lilia , Lago Santiago , Matoses Maria , Espinosa Ana , Tudela Consuelo , Arroyo Elena , Gorrochategui Julian , Jackson William C , Okun Alex , Lopez Horacio Hector , Gornemann Isolde , Melcon Soledad Gallego , de Heredia Cristina Diaz , Dapena Jose Luis , Roma Josep M , Delgado Regina Garcia , Queipodellano Mari Paz , Bailen Alicia , Barrios Manuel , Diez Eva , Del Castillo Santiago , Heiniger Ana Isabel , de Toledo Jose Sanchez , Ramirez Manuel , Ortiz Macarena , Gonzáalez Marcos , San-Miguel Jesús F. , Ocio Enrique M , Bosanquet Andrew G. , Orfao Alberto , Ballesteros Joan 
Published: October 2019

Abstract
Abstract 2655
Poster Board II-631

Introduction:
The predictive power of measuring the effect of anticancer treatments on whole living tumor cells freshly removed from cancer patients, called Individualized Tumor Response Testing (ITRT), has been recently further validated in a clinical trial, the UK's LRF CLL4 trial (Bosanquet ASH 2007). It predicts resistance better than sensitivity. We present a novel approach to ITRT based on measuring drug induced apoptosis of tumor cells in whole blood ex vivo (in vitro using freshly extracted samples). It uses a novel automated flow cytometry platform (ExviTech) capable of evaluating hundreds of drugs and drug combinations used in current treatment protocols, and can address the significant scaling of potential future protocols induced by a number of new drug approvals in each indication.

Patients and Methods:
We evaluated 47 samples of peripheral blood or bone marrow from patients diagnosed with hematological malignancies: 20 chronic Lymphocytic Leukemia (CLL), 14 Acute Lymphoblastic Leukemia (ALL), 7 Multiple Myeloma (MM), and 6 Acute Myeloblastic Leukemia (AML). After informed consent, samples, collected into heparin, were processed the same or the next day. Whole blood was diluted and incubated with drugs for 24 and 48 hours. Whole blood was used to retain erythrocytes and serum proteins enabling more clinically relevant physiological conditions. Three types of drugs were tested: 1) Approved drugs for each indication, including all possible pair wise combinations, and combinations administered within current and experimental protocols as advised by the PETHEMA groups in Spain. 2) Concomitant medicines (Con-Meds), including alternative drugs within the same class of antacids, antiemetics, etc… to test whether they may also induce apoptosis 3) Drugs in clinical trials, preferentially Phase III drugs, alone and in combination with approved drugs, which may form the basis of future treatment protocols. Drugs were plated at a final concentration equivalent to their reported plasma Cmax concentration. Synergistic drug combinations were identified as one drug potentiating the effect of the other.

Results:
The efficacy of each drug and combination tested was categorized as highly resistant, intermediate or highly sensitive. Highly resistant drug results were contraindicated. Among the highly sensitive treatments ex vivo, often those that effectively killed all malignant cells, we selected those whose drugs were significantly less toxic as treatment guidelines, highlighting those treatment protocols that act faster ex vivo (24 vs 48 hours) and/or show synergistic combinations. The final result was a set of multiple reasonable ex vivo options for hematologists. The efficacy of individual drugs varied notably from patient to patient, , as reported earlier by other methods. Drug-drug combinations show surprising results. Some combinations, effective at high doses, kill 80% of malignant cells when combined in low concentrations at which the individual drugs kill only 10%20% of these cells. On the contrary, many drug combinations were antagonistic, effectively turning them into cytoprotectors and the patient into potential resistance. Specific combinations that show consistent efficacy across samples are indicative of potential new protocols. Surprisingly, for a proportion of patients, some of the Con-Meds were highly efficient in killing malignant cells selectively. For example, in a particular CLL patient an antacid and an antiviral drug had similar efficacies as the best approved cytotoxic drugs. In other patients, drugs still in clinical trials showed high sensitivity and highly selective apoptosis – suggesting that those patients could be referred for inclusion into these trials, which could represent new alternatives especially for refractory patients with few therapeutic options available.

Conclusions:
We have developed a Personalized Medicine Multi-Drug ex vivo test, evaluating the efficacy of hundreds of drugs and drug combinations in whole blood. This scale could address the predictable expansion of multi-drug potential treatments as the existing extensive drug pipeline delivers new drug approvals, exploring hundreds of new protocols ex vivo. Promising results obtained ex vivo (in vitro using freshly extracted samples) need to be verified in clinical trials.

Disclosures:
Bennett: Vivia Biotech: Employment. Sapia:Vivia Biotech SL: Employment. Primo:Vivia Biotech SL: Employment. Suarez:Vivia Biotech SL: Employment. Lago:Vivia Biotech SL: Employment. Matoses:Vivia Biotech SL: Employment. Espinosa:Vivia Biotech SL: Employment. Tudela:Vivia Biotech SL: Employment. Arroyo:Vivia Biotech SL: Employment. Gorrochategui:Vivia Biotech SL: Employment. Jackson:Vivia Biotech SL: Employment. Okun:Vivia Biotech SL: Research Funding. Lopez:Vivia Biotech SL: Employment. Gornemann:Vivia Biotech SL: Employment. Diez:Vivia Biotech SL: Employment. Gonzáalez:Vivia Biotech SL: Consultancy. Bosanquet:Vivia Biotech SL: Consultancy. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.