Mediterranean Journal of Hematology and Infectious Diseases Original Article Differences in ex-vivo Chemosensitivity to Anthracyclines in First Line Acute Myeloid Leukemia Juan Eduardo Megías-Vericat1, David Martínez-Cuadrón1,2, Joaquín Martínez López3, Juan Miguel Bergua4, Mar Tormo5, Josefina Serrano6, Ataulfo González7, Jaime Pérez de Oteyza8, Susana Vives9, Belén Vidriales10, Pilar Herrera11, Juan Antonio Vera12, Aurelio López Martínez13, Adolfo de la Fuente14, Mª Lourdes Amador15, José- Ángel Hernández-Rivas16, Mª Ángeles Fernández17, Carlos Javier Cerveró18, Daniel Morillo19, Pilar Hernández Campo20, Julián Gorrochategui20, Daniel Primo20, José Luis Rojas20, Margarita Guenova21, Joan Ballesteros20, Miguel Sanz1,2 and Pau Montesinos1,2 on behalf of the Spanish PETHEMA group. 1 Hospital Universitari i Politècnic La Fe, Valencia, Spain. 2 CIBERONC, Instituto Carlos III, Madrid, Spain. 3 Hospital Universitario 12 de Octubre, UCM, CNIO, CIBERONC, Madrid, Spain. 4 Hospital San Pedro de Alcántara, Cáceres, Spain. 5 Hospital Clínico Universitario, Valencia, Spain. 6 Hospital Universitario Reina Sofía, Córdoba, Spain. 7 Hospital Universitario Clínico San Carlos, Madrid, Spain. 8 Hospital de Madrid Norte Sanchinarro, Madrid, Spain. 9 ICO-Hospital Germans Trias i Pujol, Josep Carreras Leukemia Research Institute, Universitat Autònoma de Barcelona, Badalona, Spain. 10 Complejo Asistencial Universitario de Salamanca, Salamanca, Spain. 11 Hospital Universitario Ramón y Cajal, Madrid, Spain. 12 Hospital Universitario Virgen Macarena, Sevilla, Spain. 13 Hospital Arnau de Vilanova, Valencia, Spain. 14 MD Anderson Cancer Center, Madrid, Spain. 15 Hospital de Montecelo, Pontevedra, Spain. 16 Hospital Universitario Infanta Leonor, Universidad Complutense de Madrid, Madrid, Spain. 17 Hospital Xeral Cies, Vigo, Spain. 18 Hospital Virgen de la Luz, Cuenca, Spain. 19 Fundación Jiménez Díaz, Madrid, Spain. 20 Vivia Biotech, Tres Cantos, Madrid, Spain. 21 Specialized Hospital for Active Treatment of Hematological Diseases, Sofía, Bulgaria. Competing interests: The authors have declared that no competing interests exist. Abstract. Background: Induction schedules in acute myeloid leukemia (AML) are based on combinations of cytarabine and anthracyclines. The choice of the anthracycline employed has been widely studied in multiple clinical trials showing similar complete remission rates. Materials and Methods: Using an ex vivo test we have analyzed if a subset of AML patients may respond differently to cytarabine combined with idarubicin, daunorubicin or mitoxantrone. Bone marrow (BM) samples of 198 AML patients were incubated for 48 hours in 96 well plates, each well containing different drugs or drug combinations at different concentrations. Ex vivo drug sensitivity analysis was made using the PharmaFlow platform maintaining the BM microenvironment. Drug response was evaluated as depletion of AML blast cells in each well after incubation. Annexin V-FITC was used to quantify the ability of the drugs to induce apoptosis, and pharmacological responses were calculated using pharmacokinetic population models. Results: Similar dose-respond graphs were generated for the three anthracyclines, with a slight decrease in EC50 with idarubicin (p=1.462E-06), whereas the interpatient variability of either drug was large. To identify those cases of selective sensitivity to anthracyclines, potency was www.mjhid.org Mediterr J Hematol Infect Dis 2019; 11; e2019016 Pag. 1 / 11 compared, in terms of area under the curve. Differences in anthracycline monotherapy potency greater than 30% from 3 pairwise comparisons were identified in 28.3% of samples. Furthermore, different sensitivity was detected in 8.2% of patients comparing combinations of cytarabine and anthracyclines. Discussion: A third of the patients could benefit from the use of this test in the first line induction therapy selection, although it should be confirmed in a clinical trial specifically designed. Keywords: Anthracycline; ex-vivo test; Idarubicin; Daunorubicin; Mitoxantrone; Acute myeloid leukemia; Personalized medicine. Citation: Megías-Vericat J.E., Martínez-Cuadrón D., López J.M., Bergua J.M., Tormo M., Serrano J., González A., de Oteyza J.P., Vives S., Vidriales B., Herrera P., Vera J.A., Martínez A.L., de la Fuente A., Amador MªL., Hernández-Rivas J.Á., Fernández MªÁ., Cerveró C.J., Morillo D., Campo P.H., Gorrochategui J., Primo D., Rojas J.L., Guenova M., Ballesteros J., Sanz M., Montesinos P. Differences in ex-vivo chemosensitivity to anthracyclines in first line acute myeloid leukemia. Mediterr J Hematol Infect Dis 2019, 11(1): e2019016, DOI: http://dx.doi.org/10.4084/MJHID.2019.016 Published: March 1, 2019 Received: October 10, 2018 Accepted: January 12, 2019 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Correspondence to: Pau Montesinos. Hospital Universitari i Politècnic La Fe and CIBERONC, Instituto Carlos III, Madrid, Spain. Tel: +34 961411966. E-mail: montesinos_pau@gva.es Introduction. Induction 1st line schedules in de novo Classification (WHO) criteria.26 This study was acute myeloid leukemia (AML) are based in a approved by the Research Ethics Board of each combination of an anthracycline with cytarabine (CYT) participating institution and was conducted according (3+7 schedule), obtaining complete remission (CR) to the Spanish law 14/2007 of biomedical research. rates of 70-80% after 1-2 cycles.1,2 Daunorubicin Informed consent was provided to all patients. (DNR), idarubicin (IDA), mitoxantrone (MIT, an anthracenedione), and less frequently other Vivia’s PharmaFlow PM Test. anthracyclines have been employed in these schemes. • Native environment whole bone marrow sample The choice of the anthracycline employed has been Ex vivo drug sensitivity analysis was made using the widely studied in several randomized clinical trials PharmaFlow platform (previously termed (RCT),3-22 showing similar CR rates, with some ExviTech®)25 maintaining the bone marrow (BM) exceptions in which IDA reported higher CR than microenvironment. A minimum BM sample volume DNR,4,6-8,12 finding reproduced in a Cochrane meta- between 1 and 2 ml was collected by aspiration at analysis.23 AML diagnosis, before starting induction Different ex vivo tests have been employed to select chemotherapy, and was processed by an automated the most effective drug combination from the method in Vivia Biotech laboratories 24 hours after individualized sensitivity and resistance assays, but extraction. Samples were incubated for 48 hours in 96 none of them have been recommended in clinical well plates, each well containing different drugs or practice.24 We are developing a Precision Medicine drug combinations at different concentrations, enabling (PM) test based on an actionable native environment calculation of dose-response curves for every single method (PharmaFlow platform), which showed drug (CYT, IDA, DNR, MIT) and combination used in excellent correlations with clinical responses in AML, treatments (CYT-IDA, CYT-DNR, CYT-MIT). The avoiding some limitations of other ex vivo assays.25 number of BM samples analyzed were 289 with IDA, The objective of this non-interventional study is to 333 with DNR and 274 with MIT. A more detailed explore whether a significant percentage of patients description of the procedure has been published AML samples may show different ex-vivo sensitivity elsewhere.25 The concentrations assayed for each to IDA vs DNR vs MIT combined with CYT. anthracycline were: - Concentrations for IDA (µM): > 0.0002 ; 0.001 ; Patients and Methods. 0.002 ; 0.006 ; 0.01 ; 0.018 ; 0.02 ; 0.04 ; 0.05 ; Patients and study design. A multicenter, prospective, 0.055 ; 0.08 ; 0.13 ; 0.16 ; 0.2 ; 0.26 ; 0.4 ; 0.5 ; non-interventional cohort study was carried out in 33 0.6 ; 1.5. Spanish institutions of the PETHEMA group. The - Concentrations for DNR (µM): > 0.001; 0.05 ; inclusion period lasted five years (2012-2017), 0.075 ; 0.093 ; 0.15 ; 0.18 ; 0.25 ; 0.3 ; 0.37 ; enrolling patients aged 18 years and older with newly 0.45 ; 0.75 ; 0.85 ; 1.25 ; 1.5 ; 2.7 ; 3. diagnosed AML. Diagnosis and classification of AML were performed according to the World Health www.mjhid.org Mediterr J Hematol Infect Dis 2019; 11; e2019016 Pag. 2 / 11 - Concentrations for MIT (µM): > 0.001 ; 0.0016 ; 72h. Therefore, the typical parameter value for the 0.008 ; 0.01 ; 0.04 ; 0.08 ; 0.2 ; 0.38 ; 0.6 ; 0.8 ; 1 ; maximum fractional effect (Emax) was set to 1 for both 2.33 ; 3.5 ; 7. drugs. For γ, the typical parameter value was calculated • Modeling of ex vivo activity of CYT, IDA, DNR, but limited to the range 0-3. IPV for both parameters MIT. could not be determined with this dataset. Evaluation of drug response was done by counting For interaction analysis, a Surface Interaction the number of live pathological cells (LPC) remaining model29 was used to estimate the degree of synergy, after incubation at increasing drug concentrations. referred as α parameter, between both drugs (R Dying cells (apoptosis) were excluded using Annexin environment (v3.3.1) for statistical computing).30 In V-FITC. Pharmacological responses were estimated this analysis, a value equal to 0 is an additive effect, a using pharmacodynamic (PD) population-based value > 0 indicates a synergistic effect, and a value < 0 models27 which essentially perform the fitting of the reflects an antagonistic effect. dependent variable (natural log of LPC) in a non-linear mixed-effects model to derive typical population Study endpoints. The primary end-point was the values (fixed effects) and the magnitude of inter-patient comparison between the selective sensitivities of the and residual variability (random effects). Model different anthracyclines individually using the AUCs in development was performed with the first-order the dose-response curve. For the comparisons between conditional estimation method using interaction option the combinations of anthracyclines with CYT, we with the software NONMEM (v7.2)28, according to the employed the volume under the surface (VUS) of the following equation: dose-response curves. Besides, the differences in either  C  drug potency or synergism ex vivo were also calculated LPC = LPC 0  1 − E max      according to the observed and predicted response after  C + EC 50  induction. Where LPC0 parameter refers to the number of LPC after incubation in the absence of drug, Emax represents Results. the maximum fractional decrease in LPC that the drug Patient Characteristics. Overall, 332 BM samples from can elicit, EC50, is the drug concentration exerting half patients with AML suspicion were received at the of Emax , and γ is the parameter governing the steepness laboratory, from which 261 BM samples were of the LPC vs drug concentration (C) curve. Potency completely monitored at the end of the study. Of them, (EC50) and efficacy (Emax) are PD parameters that 63 (24%) were not evaluable because of the following characterize the pharmacological response and are protocol issues: 1) incorrect informed consent form (32 integrated into a single value corresponding to the patients), 2) no available case report form (23 patients), measurement of the area under the dose-response curve 3) misdiagnosis (3 patients), and 4) other unknown (Area Under the Curve, AUC). reasons (5 patients). Overall, clinical data from 198 For data presentation, the survival index was patient’s samples (60%) were available at the end of computed, with the number of LPC in control wells this study. The main baseline characteristics of these that were not exposed to any drugs being set as 100%. patients are displayed in Table 1. In summary, the The number of live cells in each drug-treated well was median age was 61 years (range, 19 to 91), all patients compared with this control value, and the survival were newly diagnosed AML, and 37 patients (19%) index for each drug at each concentration was were categorized as having high-risk cytogenetics. CR determined as the percentage of LPC at every tested rate was obtained in 93 patients (47%), whereas 65 concentration. patients obtained partial remission or were resistant to Interpatient variability (IPV) associated with all induction. parameters was described using an exponential model of the components of variance. An additive error Ex vivo PharmaFlow Test characterization of IDA, structure was used for the residual variability. DNR and MIT models. Dose-response graphs were Population PD models were built with BM samples generated for the single drugs (IDA, DNR, and MIT) from 227 patients that were incubated with IDA, 271 using PD models (Figure 1). Most of the observations with DNR, and 212 with MIT. Bayesian estimation were contained within the simulation-based 95% methods were then used to retrieve individual patient confidence intervals of the 5-95th population parameters based on their available exposure-response percentiles proving good predictability of the selected measurements in conjunction with the PD population models. Pharmacological population parameters, as parameters. After several trials with different modeling well as variability and error values, are shown in Table strategies, we could conclude that optimal approach, in 2. terms of correlation with clinical output, was achieved The average dose-responses of the three anthracyclines by forcing typical parameters to values obtained in a were similar, with a slight decrease in EC50 values with different model using a dataset from samples tested at IDA (p-value = 1.462E-06; Table 2), reproducing www.mjhid.org Mediterr J Hematol Infect Dis 2019; 11; e2019016 Pag. 3 / 11 Table 1. Baseline characteristics of the 198 analyzed patients. the results of the clinical trials.4,6-8,12 However, the Median Range interpatient variability of either drug is quite large Age (years) 61 19-91 (Table 2, Figure 1), which could explain why some n % patients could show very differential sensitivities to 18-29 7 3.5 these three drugs. As an example, Figure 2 illustrates a 30-39 20 10.1 patient sample that is resistant to IDA and DNR (right 40-49 31 15.7 shifted dose-response curve) but sensitive to MIT (left 50-59 30 15.2 shifted dose-response curve). >60 110 55.6 To identify these cases of selective sensitivity to Gender n % anthracyclines, we compared the potency, regarding Male 113 57 AUC, between IDA vs. DNR, IDA vs. MIT, and DNR Female 85 43 vs. MIT (Figure 3, Table 3). Most dots tend to line up, ECOG n % but red dots represent patient samples with a difference 0 29 in potency between these drugs >30%. Red dots from 3 57 pairwise comparisons identify 28.3% of patient 1 73 37 samples with >30% different potency among IDA- 2 24 12 DNR-MIT (Figure 4). 3-4 8 4 Unknown 36 18 Ex vivo PharmaFlow Test characterization of CYT- FAB subtype n % IDA, CYT-DNR, and CYT-MIT combinations and their M0 14 7 synergism. The pairwise comparison of the M1 46 23 combination treatments CYT-IDA, CYT-DNR, and M2 44 22 CYT-MIT obtained differential sensitivity to these M4 32 16 anthracyclines (red dots of Figure 5). In this case, the M5 27 14 red dots represent patient samples with a difference in M6 2 1 CYT + anthracyclines synergy differences >30%, and Unknown 33 17 Median Range Table 2. Estimates of the ex vivo population pharmacodynamic WBC (count x 109/L) 18.65 0.6 - 270 parameters. Parameters typical and random (variability and residual n % error percentage) are shown together with the corresponding relative standard error calculated as the ratio between the standard 0-10 74 37 error provided by NONMEM and the estimate. Estimates of inter- 10-50 63 32 patient variability (IPV) are expressed as coefficient of variation >50 51 26 (%). Unknown 10 5 Parameter Cytogenetic risk profilea n % Mitoxantrone Idarubicin Daunorubicin (units) Favorable 15 8 LPC0 (cells) 7443 ( 10.04 ) 8384 ( 14.18 ) 7926 ( 10.21 ) Intermediate 111 56 Emax (unitless) 1(-) 1(-) 1(-) Adverse 37 19 EC50 (μM) 0.329* ( 16.84 ) 0.07* ( 14.58 ) 0.458*( 12.08 ) Unknown 35 18 γ (unitless) 0.77 ( - ) 1.04 ( - ) 1.13 ( - ) FLT3-ITD status n % Wild 119 60 Residual Error (log(μM)) 845 ( 10.07 ) 1027 ( 15.61 ) 924 ( 11.79 ) Mutant 22 11 Unknown 57 29 Inter-patient variability NPM1 status n % (IPV) Wild type 92 46 LPC0 86.4 ( 6.56 ) 107.3 ( 6.83 ) 92.9 ( 5.76 ) Mutant 50 25 Unknown 56 28 Emax N/D N/D N/D Response n % EC50 224.2 ( 6 ) 181.8 ( 5.46 ) 168.6 ( 4.6 ) CR/CRi 93 47 γ N/D N/D N/D PR/resistance 65 33 Residual Error Unknown 40 20 83.1 ( 7.63 ) 107.4 ( 7.21 ) 97.4 ( 6.38 ) aBased on the risk groups described by Grimwade et al (2010). Emax: maximum fractional decrease in live pathological cells that ECOG-PS: Eastern Cooperative Oncology Group performance the drug can elicit; EC50: drug concentration exerting half of E max; status; FAB: French-American-British classification; FLT3-ITD: LPC0: Starting live pathological cells in the absence of drug; N/D: fms-like tyrosine kinase 3-internal tandem duplication; NPM1: not determined; γ: parameter governing the steepness of the LPC vs Nucleophosmin 1; WBC: white blood cells; PR: partial remission. drug concentration curve. * p value = 1.462E-06 www.mjhid.org Mediterr J Hematol Infect Dis 2019; 11; e2019016 Pag. 4 / 11 Figure 1. Average and Individual Dose Responses ex vivo for Figure 2. Example of differential individual sensitivities to AML Drugs. Dose-Response Analysis was Completed for 3 anthracyclines. Dotted lines represented individual response to each Anthracyclines in Bone Marrow Samples From Patients With Acute drug and cotinuous lines the median response to each drug. Panel Myeloid Leukemia; 227 with Idarubicin, 212 with Mitoxantrone (A) shows an example of a patient resistant to Idarubicin (right and 271 with Daunorubicin. The Survival Index (y-Axis) Ranges shifted dose response curve) but sensitive to Mitoxantrone (left From 100% to 0%, Displaying the Selective Acute Myeloid shifted dose response curve). Panel (B) shows an example of a Leukemia Cell Depletion Calculated With Population Models. The patient resistant to Idarubicin and Daunorubicin (right shifted dose Gray Lines Display Each Individual Response, With the Median response curve). Panel (C ) shows an example of a patient resistant Response Shown in yellow for Idarubicin, Panel (A); in blue for to Daunorubicin (right shifted dose response curve) but sensitive to Mitoxantrone, Panel (B); and in red for Daunorubicin, Panel (C). Mitoxantrone (left shifted dose response curve). Table 3. Differences in Area Under the Dose-Response Curve between anthracyclines. AUC Over30% Normal Total % DNR_IDA 15 102 117 12.82 DNR_MIT 32 172 204 15.69 IDA_MIT 17 100 117 14.53 AUC: area under the curve; DNR: daunorubicin; IDA: idarubicin; MIT: mitoxantrone. www.mjhid.org Mediterr J Hematol Infect Dis 2019; 11; e2019016 Pag. 5 / 11 Table 4. Differences in Volume Under the Surface (VUS) between the combinations of cytarabine and different anthracyclines. VUS Over30% Normal Total % CYT+DNR_CYT+IDA 2 125 127 1.57 CYT+MIT_CYT+IDA 6 81 87 6.90 CYT+DNR_CYT+MIT 9 153 162 5.56 CYT: cytarabine; DNR: daunorubicin; IDA: idarubicin; MIT: mitoxantrone; VUS: volume under the surface. red dots from 3 pairwise comparisons identified an 8.2% of patient samples (Figure 6, Table 4). Furthermore, the values for the alpha parameters of the interaction models of CYT-IDA, CYT-MIT, CYT- DNR were 0.72, 0.59 and 0.25, indicating synergistic response in the ex vivo combination experiments. Discussion. The findings of this study show that PharmaFlow PM test seems able to identify a subset of AML patients who have a significantly different ex vivo pharmacological response to anthracycline drugs. We can hypothesize that if these selective anthracycline ex vivo responses were translated to in vivo responses, a fraction of this 28.3% subpopulation could benefit significantly from receiving a specific anthracycline-based on the ex vivo test sensitivity results. Furthermore, an 8.2% of patients showed a significant difference in the synergy between CYT and anthracyclines, in which the choice of the anthracycline could be crucial. The first line induction therapy recommended by ELN1 and NCCN2 clinical guidelines includes seven days of a standard dose of CYT plus three days of an anthracycline, especially IDA (12 mg/m2) or DNR (60- 90 mg/m2). The combination of CYT-MIT was not considered standard therapy, although it has been widely employed. The influence of the anthracycline´s selection in the efficacy of induction therapy was analyzed in some RCTs.3-22 The comparison between CYT-DNR and CYT-IDA has been studied in 13 different trials,3-15 but only five studies reported differences in CR rates in favor of CYT-IDA.4,6-8,12 A meta-analysis confirmed the superiority of CYT-IDA against CYT-DNR, obtaining higher overall survival (OS), disease-free survival (DFS), CR, lower relapse rate, although this scheme increased induction death and mucositis.23 Regarding the employment of CYT-DNR or CYT- MIT, a clinical trial reported similar CR, length of duration of CR, OS, and toxicity.16 No evidence of Figure 3. Comparison of the potency between anthracyclines. differences between CYT-IDA and CYT-MIT in CR, Panels A-C represented the pairwise comparisons between Area survival rates, and toxicity was observed in 6 Under (AUC) the Dose-Response Curve of the anthracyclines, with their bisectors, linear regression lines and R2 values. Red dots RCTs9,11,17-20 and one meta-analysis.23 Combinations of represent patient samples with a difference in potency between CYT-doxorubicin showed worse outcomes than CYT- these drugs greater than 30%. Panel (A) comparison between AUCs DNR21 and CYT-IDA.22 According to clinical trials, in of Idarubicin and Mitoxantrone; Panel (B) comparison between our study the average dose-responses of IDA, DNR, AUCs of Daunorubicin and Mitoxantrone; Panel (C) comparison and MIT were similar, with a slight decrease in EC50 between AUCs of Daunorubicin and Idarubicin. www.mjhid.org Mediterr J Hematol Infect Dis 2019; 11; e2019016 Pag. 6 / 11 Figure 4. Differences in Area Under the Dose-Response Curve between anthracyclines. A 28.3% of patients samples showed >30% different potency among Idarubicin-Daunorubicin-Mitoxantrone Area Under the Dose-Response Curve (AUC). with IDA, indicating a probable higher potency with PM test aims to solve technical limitations including IDA than DNR and MIT. However, the anthracycline some novelties25: dosage of induction protocols assumed a cumulative a) the use of whole BM sample, maintaining the doses proportion of 4:1 for DNR: IDA and DNR: native environment, which has been hypothesized that MIT,31 but these proportions are not based in well- it can influence the emergence of resistance;45-48 designed trials. In our cohort, according to this b) the increase of the accuracy obtained modeling proportion and EC50 of DNR (0.458), the estimated ex vivo activity with PD population models in one EC50 of IDA and MIT was 0.115, a proportion 1.6 fold single step;49 higher than IDA EC50 and three fold lower than MIT c) the improvements in the measures performed by EC50 measured with ex vivo test. automated flow cytometry platform (PharmaFlow). Other studies analyzed the role of different The correlation between in vitro and in vivo therapy anthracyclines in the AML induction with CYT and a sensitivity of PharmaFlow PM test has been recently third component, but CR and survival rates were demonstrated in a cohort of 123 AML patients after similar for DNR, MIT, and aclarubicin.32,33 Besides the induction therapy with CYT-IDA (most of these selection of the anthracycline, the dose intensity is patients were also included in this study).50 This study crucial in the therapy success. An RCT34 reported achieved an 81% of overall accuracy in the correlations significant improvements in CR, OS and event-free between test predictions and hematological response, survival (EFS) using DNR doses of 90 mg/m2 identifying with success responders (CR/CR with compared to doses of 45 mg/m2. The response-oriented incomplete recovery) in 93% of cases and non- individualized induction therapy is another approach responders (partial remission/resistance) in 60% of tested with IDA+CYT scheme without any advantage cases. The present study generates a theoretical role of over the standard scheme.35 In addition, some specific PM tests in individual anthracycline selection but does AML characteristics could modify the anthracycline not provide enough data and critical analyses to allow response, such as FLT3-ITD mutated patients which to translate their use in the routine clinical practice. showed higher CR and survival with high-dose DNR Regarding the synergism between anthracyclines compared to standard-dose DNR or IDA.36,37 These and CYT, we observed a synergistic response with the findings were reproduced in vitro in FLT3-ITD- three combinations, especially with CYT-IDA and mutated cell lines.37 Unfortunately, we have not CYT-MIT. In a previous study, we also reported a enough data to analyze the impact of this mutation in higher synergy with CYT-IDA and CYT-MIT our cohort. combination and a trend to an additive effect with Despite the previous experiences of ex vivo drug CYT-DAU.25 Curiously, a novel approach in AML testing with limited sensitivity38-44, the PharmaFlow therapy is the use of the liposomal formulation of CYT and DNR in a molar ratio concentration of 5:1, based www.mjhid.org Mediterr J Hematol Infect Dis 2019; 11; e2019016 Pag. 7 / 11 Figure 5. Comparison of the potency between combinations of cytarabine and anthracyclines. Panels A-C represented the pairwise comparisons between Volume Under the Surface (VUS) of the combinations of cytarabine (CYT) with anthracyclines, with their bisectors, linear regression lines and R2 values. Red dots represent patient samples with a difference in potency between these drugs greater than 30%. Panel (A) comparison between VUS of Cytarabine + Mitoxantrone (CYT+MIT) and Cytarabine + Idarubicin (CYT+IDA); Panel (B) comparison between VUS of CYT+MIT and Cytarabine + Daunorubicin (CYT+DNR); Panel (C) comparison between VUS of CYT+DNR and CYT+IDA. www.mjhid.org Mediterr J Hematol Infect Dis 2019; 11; e2019016 Pag. 8 / 11 Figure 6. Differences in Volume Under the Surface between combinations of cytarabine and different anthracyclines. An 8.2 % of patients samples obtained >30% of different sensitivity in Volume Under the Surface (VUS) of Cytarabine + Idarubicin (CYT+IDA), Cytarabine + Daunorubicin (CYT+DNR) and Cytarabine + Mitoxantrone (CYT+MIT). on a probable higher synergistic effect.51,52 Finally, the findings reported are not yet validated in an Furthermore, the pairwise comparisons between independent cohort. combinations of CYT-IDA, CYT-DNR, and CYT-MIT found in an 8.2% of patients synergy differences Conclusions. The ex vivo PharmaFlow PM test >30%, probably associated to the interpatient obtained in a 28.3% of the BM samples analyzed variability in drug sensibility observed in dose- overall differences in sensitivity to anthracyclines in response graphs. monotherapy. This test could allow designing a trial to Some limitations should be addressed in this study. explore a personalized selection of anthracycline First, this study analyzes the differences between ex therapy in AML patients. A similar approach is being vivo sensitivities to three different anthracyclines tested in a clinical trial by PETHEMA group in combined with CYT in BM samples of AML patients relapsed or refractory AML patients to select the at diagnosis, but the correlation between ex vivo salvage therapy based on the ex vivo sensitivity to responses and clinical response was not analyzed. conventional chemotherapy agents. The role an Second, although the incubation time was relatively adequate selection in this subset of AML patients is short, additional transportation and processing time critical because none of the salvage regimens53 has could lead, in several patients, to a non-affordable achieved outstanding CR rates, long-lasting remissions, delay to start induction chemotherapy while receiving and acceptable OS. the test report. Third, associations of the different in vitro response of each anthracycline and specific Acknowledgments. We are grateful to all participating characteristics of AML (age, WBC, cytogenetic risk, institutions and clinicians in the PETHEMA group, and FLT3-ITD, and NPM1 status, etc.) were not analyzed. all the patients included. References: 1. 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