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Multiple myeloma (MM) is characterized by an immunosuppressive microenvironment that enables tumor development. One of the mechanisms of immune evasion used by MM cells is the inhibition of natural killer (NK) cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal to increase tumor cell recognition, avoid tumor escape and potentially enhancing the effect of other drugs. In this study, we evaluated the ability of the investigational medicine NKTR-255, an IL-15 receptor agonist, to engage the IL-15 pathway and stimulate NK cells against MM cells. We observed that incubation with NKTR-255 was able to tilt the balance toward an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of patients with MM, with increased expression of activating receptors on the surface of treated NK cells. This resulted in an enhanced degranulation, cytokine release, and anti-tumor cytotoxicity when the NK cells were exposed to both MM cell lines and primary MM cells. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared with placebo, weekly injection of the mice with NKTR-255 increased the number of circulating NK cells in peripheral blood and delayed tumor growth. Finally, we observed that combination of NKTR-255 with the anti-CD38 antibody, daratumumab, was effective against MM cells in vitro and in vivo. Taken together, our data suggest a significant impact of NKTR-255 in inducing NK cell function against MM cells with important translational implications.

170 Background: CRC is the 4th leading cause of global cancer-related deaths, and novel therapeutic strategies for advanced CRC are urgently needed. Adoptive cell therapy (ACT) is effective in treating hematological malignancies; however, ACT in solid tumors is hindered by target antigen identification, restricted migration into tumors, and survival in the tumor microenvironment (TME) due to immunosuppressive signals and scarcity of nutrients. NK cells are central to anti-tumor immunity and can directly eliminate tumor cells without prior sensitization. Through cytokine reprogramming, NK cells can also gain memory-like features that augment their anti-tumor potential. WU-NK-101 is a cytokine-reprogrammed, expanded, cryopreserved, off-the-shelf NK cell product derived from peripheral blood mononuclear cells, with no additional engineering. Methods: WU-NK-101 ± Ctx was evaluated in vitro in 2D cytotoxicity assays in complete (N) and TME-aligned medias. WU-NK-101 cytotoxicity was further assessed against primary CRC surgical samples in native-TME-aligned 3D assays. Proteomic analysis was performed using tandem-mass spectrometry. In vivo efficacy of WU-NK-101 ± Ctx was evaluated in NSG mice bearing LoVo xenograft CRC tumors. Cell trafficking/penetration to TME was measured by tracking labeled WU-NK-101 ± trastuzumab in NSG mice bearing subcutaneous SKOV-3 xenografts. Results: Compared to conventional NK cells (cNK), WU-NK-101 had a unique phenotype consistent with rapid activation and proliferation (higher expression of activating receptors, Ki67, and GZMB), and showed enhanced in vitro cytotoxicity. WU-NK-101 also exhibited potent cytotoxicity against LoVo CRC tumors in vivo, which was further enhanced in combination with Ctx. Antibody combination improved WU-NK-101 penetration, and persistence in TME. WU-NK-101’s metabolic profile was consistent with aerobic (Warburg) glycolysis, potentially facilitating effector functions in the TME. WU-NK-101 also showed enhanced metabolic fitness and flexibility, as proteins in several metabolic pathways were upregulated in TME vs N media. Consistent with this, WU-NK-101 had increased cell-surface expression of nutrient transporters. While cNK and T cells cytotoxicity was significantly suppressed in TME-aligned media, WU-NK-101’s function was not impacted. Conclusions: We show that WU-NK-101 exerted potent activity against CRC, and in combination with Ctx showed improved intra-tumor infiltration/persistence and anti-tumor activity. Also, WU-NK-101 cells had enhanced metabolic fitness/flexibility and decreased susceptibility to immunosuppression, overcoming limitations encountered by ACT for solid tumors. A Phase 1b clinical trial is in development, which may reshape ACT in CRC and other EGFR-expressing tumors. [Table: see text]

Background Multiple myeloma (MM) is characterized by an immunosuppressive microenvironment that enables tumor development. One of the mechanisms of immune evasion used by MM cells is the inhibition of NK cell effector functions; thus, the restoration of NK cell antitumor activity represents a key goal for new immunotherapeutic approaches, increasing tumor cell recognition, avoiding tumor escape and potentially enhancing the effect of other drugs. Methods Here we investigate the potential of NKTR-255, a novel polymer-conjugated human IL-15 to engage the IL-15 pathway and overcome the inhibitory status observed in NK cells from MM patients. For this purpose, we have analyzed ex vivo and in vivo effects of NKTR-255 on phenotypic features, effector functions and cytotoxicity of NK cells against MM cells. Results We observed that incubation with NKTR-255 was able to tilt the balance towards an activated phenotype in NK cells isolated from peripheral blood mononuclear cells of MM patients, with increased expression of activating receptors (NKG2D, NKp46, NKp30, DNAM-1, CD69, TRAIL) on the surface of treated NK cells. This resulted in an enhanced degranulation, cytokine release and anti-tumor cytotoxicity when the NK cells were exposed to both MM cell lines and primary MM cells. For a more accurate assessment of the effect of NKTR-255 on NK cell activity in an autologous setting in the presence of the bone marrow (BM) milieu, we cultured whole BM samples from non-treated newly-diagnosed MM patients with increasing doses of NKTR-255 for 5 days. NK cells experienced a dose-dependent induction of proliferation and activation (as shown by increased expression of CD69 and NKG2D), which translated in a reduced viability of CD138+ MM cells in the presence of NKTR-255. We further evaluated the in vivo effect of NKTR-255 in fully humanized immunocompetent mice subcutaneously engrafted with H929 MM cells. Compared to placebo, weekly injection of the mice with NKTR-255 increased the number of circulating NK cells in peripheral blood and delayed tumor growth. Finally, we also tested in vitro and in vivo efficacy of a combination of NKTR-255 with daratumumab. We observed a more efficient antibody-dependent cellular cytotoxicity against MM cells in vitro and decreased tumor growth in vivo, where NKTR-255 rescued CD38+ NK cell levels from depletion by daratumumab. Conclusions Taken together, these results support the restoration and expansion of NK cell activity in MM with NKTR-255, providing rationale for its clinical use as a novel immunotherapeutic approach for MM patients alone or in combination with monoclonal antibodies or other immunomodulatory drugs.

Introduction: Precision Medicine (PM) by measuring the pharmacological activity of drug combination treatments in patient samples by flow cytometry is a new field. This approach has been validated in an observational study of 236 Acute Myeloid Leukemia (AML) patients from Spain and China. Patients were treated with commonly administered chemotherapeutic regimens in AML, and the PM test generated a ranking of the activity of alternative treatments in each patient sample to support personalizing treatments in clinical practice. Material and Methods: Results were processed from an observational study collecting 236 bone marrow samples from AML adult patients from hospitals in China (113) and Spain (123). Most were de novo patients (223) with 13 relapse-refractory patients. The PM test was carried out in the laboratories of Hosea Precision Medical (China) or Vivia Biotech (Spain). Whole bone marrow samples maintaining their Native Environment were incubated for 48h or 72h in well plates containing 23 treatments (Tx) representing the most common drugs and drug combinations used in induction treatments with AML patients. The PM test activity was calculated by the percentage of the maximum Area Under the Curve (AUC) from curve functions models fitted to dose-response experiments results. Besides that, the test includes a measurement of synergy in drug combinations estimated by the application of surface interaction models. The predicted sensitivities of alternative treatments for each individual patient are ranked in five 20% categories following a traffic lights color code, from most sensitive 80-100% green to most resistant 0-20% red. Statistical analysis by response frequency on the predicted green (most sensitive) patients with a target frequency of 85% responders in the green group and a target error of 10% required N=236 total samples. Results: The clinical % CR (Complete Remission) correlates with the predicted responses, decreasing from green to orange to red categories (Figure). PM Test predicts well 85.5% CR for green Tx. 70% of all patient samples have ≥1 green Tx among these 23 Tx. For these 70% patients, the analysis and projection of these observed prediction frequencies suggest that the use of the PM Test might achieve 85.5% CR vs the clinical 62.0%, a 23.5% increase. Within the remaining 30% samples without any green Tx, 17% have at least 1 dark orange Tx (60-80%), with a good prediction CR rate of 68.4%. An additional 8% have ≥1 orange Tx, without any Green or Dark Orange Txs, and these patients have a low clinical 22.2% CR, consistent with being multiresistant to most 23 drug treatments in PM Test. PM Test might increase CR rates from 22.2 to 40%, an increase of 17.8% CR in very difficult patients. If we include all patient samples, PM Test might increase CR from 54.9% to 71.8%, 16.9% more patients achieving CR. Results from Chinese or Spanish patients were consistent with each other. Conclusions: Ranking each treatment across all patient samples in a database, and ranking the alternative treatments that can be administered to a new patient, generates a new Precision Medicine test that might help selecting the optimal treatment for each individual patient. This should be validated by a prospective, two-arms, study. Disclosures Wang: Hosea Precision Medical Technology Co., Ltd: Current Employment. Ge:Hosea Precision Medical Technology Co., Ltd: Current Employment. Zhou:Hosea Precision Medical Technology Co., Ltd: Current Employment. Gorrochategui:VIVIA BIOTECH: Current Employment. Primo:Vivia Biotech: Current Employment. Ballesteros:Vivia Biotech: Current Employment. Martinez-López:Janssen, BMS, Sanofi, Novartis, Incyte, F. Hoffmann-La Roche and Amgen: Honoraria, Other: Advisory boards; Hosea and Altum: Membership on an entity's Board of Directors or advisory committees; Janssen, Novartis, BMS, Incyte: Consultancy.

Background: Phase I clinical trial for oncology drugs have the highest clinical failure rate. Drug candidates may be discarded if they don´t show activity. Yet these drugs may have valuable activity in patient subgroups. Biomarkers attempt to identify these sensitive subpopulations, but are normally evaluated and validated in Phase II when good drugs may have been discarded. OPB-111077 is a novel, oral, low-molecular-weight compound that inhibits STAT3 and mitochondrial electron transport. A prior Phase I trial on 145 patients from all tumor types gave only 1 partial response (PR). A strong anti-proliferative activity was identified in Acute Myeloid Leukemia (AML) samples ex vivo. This new biomarker was used for patient selection in this trial. We present a novel design of a Phase Ib trial directly selecting patients with a potential biomarker, that can benefit the patient without increasing toxicity risks, in Relapsed/Refractory (RR) AML patients. Method: This is an open-label, phase Ib dose-escalation clinical trial to evaluate the safety profile, maximum tolerated dose (MTD) and preliminary efficacy of oral OPB-111077 in AML relapsed or refractory to chemotherapy patients. Patients > 18 years old with high risk AML, ECOG ≤2 and adequate organ functions were selected for biomarker screening. Biomarker consisted on a dose response curve for antiproliferative activity of agent in a bone marrow sample of patients incubated 72-96-120h (Figure), quantified as the Area Under the Curve (AUC). Patient samples whose ex vivo biomarker showed low sensitivity (70% worst) we excluded, since they were potentially predicted as resistant to the agent. The remaining 30% with higher sensitivity were included. OPB-111077 was administered orally on a once daily dose schedule in 28-day cycles until intolerable toxicity or disease progression. Two dose schemas were evaluated: 200 (DL1) and 250 mg/day (DL2). Dose limiting toxicities were any Grade ≥ 3 non-hematologic toxicity and any unexpected non-tolerable grade II that requires delay beyond 1 week until recovery and evaluated during the first 28 days of treatment. Adverse events were graded according to NCI-CTCAE vs 4.03 and response criteria was based on those given by Cheson et al. IC50 and area under the curve (AUC) determined by the Vivia ex vivo biomarker were correlated with clinical response. Results: We screened 47 RR AML patients, twelve patients were included and selected by personalized medicine test (5 in DL1 and 7 in DL2), median age 76 years, 92% men, 42% ECOG 0 were included in the study. AML patients were refractory in 41.7% and patients were in median of relapses 2 (range 1-6). No DLT was reported. Adverse events related to study treatment were reported in three patients, all G 1-2: in DL1 one patient had vomiting; in DL2 one patient had extrasystoles and other had anorexia, diarrhoea, epigastric discomfort, nausea and vomiting. Two patients died during study treatment due to disease progression and respiratory infection. Half of the patients discontinued study treatment due to disease progression and 25% due to adverse events (respiratory failure G5, respiratory infection G5 and extrasystoles G2). Over the 6 patients evaluable for efficacy, 3 of them achieved PR and 3 treatment failure as best response. Progression free survival and overall survival were 57 days (95% CI: 37 - 77) and 95 days (95% CI: 27 - 163) respectively. The biomarker AUC and IC50 values stratified patients consistent with their clinical response, although without enough sampling potency for statistical significance. Figure shows the relative position of patients included in the study in the context of the overall population of samples considered to build up the mixed effect population pharmacodynamic model used to analyze samples results and define inclusion criteria. In green those that correspond to patients who showed a positive response; in orange those who failed. Conclusions: OPB-111077 is generally well tolerated and has a manageable toxicity profile. The MTD was not reached. The 50% PR rate achieved by OPB-111077, albeit in a few patients, is substantially higher that the 0.7% (1/145) PR rate achieved in a prior phase I trial on all tumor types1. This innovative Phase Ib design selecting patients using a biomarker may enable to rescue drug failures in the future. References: 1. Tolcher A, Flaherty K, Shapiro GI et al. Oncologist. 2018, 23(6):658-e72 Figure Disclosures Martinez-Lopez: Hosea: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BMS: Research Funding, Speakers Bureau; Amgen: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Vivia Biotech: Honoraria; Altum: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Novartis: Research Funding; Takeda: Speakers Bureau; Incyte: Research Funding, Speakers Bureau. Gorrochategui:VIVIA BIOTECH: Current Employment. Rojas:VIVIA BIOTECH, S.L.: Current Employment. Primo:Vivia Biotech: Current Employment. Perez De Oteyza:MACROGENICS: Research Funding. Ballesteros:Vivia Biotech: Current Employment.

Ruxolitinib is the front-line non-palliative treatment for myelofibrosis (MF). However, a significant number of patients lose or present suboptimal response, are resistant or have unacceptable toxicity. In an attempt to improve response and avoid the adverse effects of this drug, we evaluated the combination of 17 drugs with ruxolitinib in ex vivo models of peripheral blood mononuclear cells from MF patients and cell lines. We found that the combination ruxolitinib and nilotinib had a synergistic effect against MF cells (ΔEC50 nilotinib, -21.6%). Moreover, the addition of prednisone to combined ruxolitinib/nilotinib improved the synergistic effect in all MF samples studied. We evaluated the molecular mechanisms of combined ruxolitinib/nilotinib/prednisone and observed inhibition of JAK/STAT (STAT5, 69.2+11.8% inhibition) and MAPK (ERK, 29.4+4.5% inhibition) signaling pathways. Furthermore, we found that the triple therapy combination inhibited collagen protein and COL1A1 gene expression in human bone marrow mesenchymal cells. Taken together, we provide evidence that combined ruxolitinib/nilotinib/prednisone is a potential therapy for MF, possibly through the anti-fibrotic effect of nilotinib, the immunomodulatory effect of ruxolitinib and prednisone, and the anti-proliferative effect of ruxolitinib. This combination will be further investigated in a phase Ib/II clinical trial in MF. This study was supported by the Subdireccion General de Investigacion Sanitaria (Instituto de Salud Carlos III, Spain) grants PI13/02387 and PI16/01530, and the CRIS against Cancer foundation grant 2014/0120. M.L. holds a postdoctoral fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409). Sí

PI3Kδ (idelalisib) and BTK (ibrutinib) inhibitors have demonstrated significant clinical activity in chronic lymphocytic leukemia (CLL) interfering with the cross-talk between CLL cells and the lymph node microenviroment, yet their mechanism of action remains to be fully elucidated. Here, we developed an ex vivo model with the aim of reproducing the effects of the microenvironment that would help shed light on the in vivo mechanism of action of idelalisib and ibrutinib and predict their clinical efficacy in individual patients. First we explored the effects of various cell-extrinsic elements on CLL apoptosis and proliferation and found that the combination of CpG+IL2+HS5 stromal cell line + human serum +CLL plasma and erythrocyte fractions represented the best co-culture conditions to test the effects of the novel inhibitors. Then, using this assay, we investigated the impact of idelalisib and ibrutinib on both survival and proliferation in 30 CLL patients. While both drugs had a limited direct pro-apoptotic activity, a potent inhibition of proliferation was achieved at clinically achievable concentrations. Notably, up to 10% of CLL cells still proliferated even at the highest concentrations, likely mirroring the known difficulty to achieve complete responses in vivo. Altogether, this novel assay represents an appropriate ex vivo drug testing system to potentially predict the clinical response to novel inhibitors in particular by quantifying the antiproliferative effect., This work was supported in part by H2020 “MEDGENET, Medical Genomics and Epigenomics Network” (Grant 692298), funded by the European Union; the Swedish Cancer Society, the Swedish Research Council, and Lion’s Cancer Research Foundation; Associazione Italiana per la Ricerca sul Cancro AIRC (Investigator Grant #20246 to PG and Special Program Molecular Clinical Oncology - 5 per mille #9965), Milano, Italy, Ricerca; ERA-NET TRANSCAN-2 JTC 2016, GCH-CLL. Vivia has received unrestricted research support from Gilead.

[Background]: Despite recent advances in the treatment of multiple myeloma (MM), the prognosis of most patients remains poor, and resistance to traditional and new drugs frequently occurs. EDO-S101 is a novel therapeutic agent conceived as the fusion of a histone deacetylase inhibitor radical to bendamustine, with the aim of potentiating its alkylating activity. [Methods]: The efficacy of EDO-S101 was evaluated in vitro, ex vivo and in vivo, alone, and in combination with standard anti-myeloma agents. The underlying mechanisms of action were also evaluated on MM cell lines, patient samples, and different murine models. [Results]: EDO-S101 displayed potent activity in vitro in MM cell lines (IC 1.6-4.8 μM) and ex vivo in cells isolated from MM patients, which was higher than that of bendamustine and independent of the p53 status and previous melphalan resistance. This activity was confirmed in vivo, in a CB17-SCID murine plasmacytoma model and in de novo Vk∗MYC mice, leading to a significant survival improvement in both models. In addition, EDO-S101 was the only drug with single-agent activity in the multidrug resistant Vk12653 murine model. Attending to its mechanism of action, the molecule showed both, a HDACi effect (demonstrated by α-tubulin and histone hyperacetylation) and a DNA-damaging effect (shown by an increase in γH2AX); the latter being again clearly more potent than that of bendamustine. Using a reporter plasmid integrated into the genome of some MM cell lines, we demonstrate that, apart from inducing a potent DNA damage, EDO-S101 specifically inhibited the double strand break repair by the homologous recombination pathway. Moreover, EDO-S101 treatment reduced the recruitment of repair proteins such as RAD51 to DNA-damage sites identified as γH2AX foci. Finally, EDO-S101 preclinically synergized with bortezomib, both in vitro and in vivo. [Conclusion]: These findings provide rationale for the clinical investigation of EDO-S101 in MM, either as a single agent or in combination with other anti-MM drugs, particularly proteasome inhibitors., This work was in part funded by Mundipharma-EDO GmbH, the Spanish Instituto de Salud Carlos III (ISCIII-FIS), (PI 15/0067 and PI 15/2156) and FEDER, the Spanish RTICC (RD12/0036/0058), Spanish Association Against Cancer (AECC, GCB120981SAN), and the Regional Council of Castilla y León (GRS 1175/A/15 and FIC335U14). All this funding was employed for the execution of experiments and the analysis of data. AALI was supported by a grant from the Spanish Society of Hematology and Hemotherapy. MMS is supported by the Network of Centers for Regenerative Medicine and Cellular Therapy from Castilla y León, Spain.

Background: Acute myeloid leukemia (AML) is a hematological malignancy with a very low overall survival. Among the new treatment modalities, chimeric antigen receptor (CAR) therapy is showing promising results in other hematological malignancies. Since AML exhibits high heterogeneity and does not have specific differential antigens of the hematopoietic stem cell, using NKG2D-CAR cells could be an appropriate therapeutic strategy against AML. NKG2D receptor has a wide range of specific tumor cell ligands (MICA, MICB, ULBP-1, ULBP-2 and ULBP-3) which are expressed in more than 80% of all tumors. For this reason, the objective of this work was to evaluate the anti-tumor activity of activated and expanded natural killer cells (NKAE) and T cells expressing an NKG2D CAR. Methods: T cells and NK cells were isolated from the healthy donor´s peripheral blood mononuclear cells ring (n = 5) by immunomagnetic depletion. NKAE cells were obtained by co-culture with subletally irradiated CSTX002 cells. The purified NKAEs and T cells were transduced with an NKG2D CAR with 4-1BB and CD3z signaling domains. The efficiency of transduction was evaluated by flow cytometry detecting NKG2D expression. Also, the immunoprofiling of surface molecules and the cytotoxicity against primary blasts of AML, as well as the expression of NKG2D ligands in tumor cells (AML cell lines and primary blasts) were analyzed by flow cytometry. The cytotoxicity of untransduced NKAE, CAR-NKAE cells, untransduced T cells and CAR-T cells was evaluated by 4 hour europium release assay. Toxicity on healthy tissue (healthy lung cells and PBMCs from third party) was analyzed in the same way. The safety of NKG2D-CAR transduced cells was evaluated using CGH arrays to detect chromosomal abnormalities. Results: Both the AML cell lines and primary blasts from AML patients showed expression of the MICA/B and ULBPs-1 to 3 ligands. NKG2D ligands expression was highly variable from a cell line to another. However, all the cell lines and samples showed high expression of at least 1 of the 5 analyzed ligands. Four hour europium release assays revealed that untransduced T cells had higher cytotoxicity than untransduced NKAE cells at the same ratio (32: 1) against both OCI-AML-3 cytarabine-resistant cell line (52.7% ± 14.2% vs. 32.5% ± 7.2%) as to the cytarabine-sensitive cell line (56.42% ± 5.6% vs. 50.14 % ± 5.9%). T cells showed a better transduction efficiency than NKAE cells at a multiplicity of infection (MOI) of 5. It was observed that both the CAR-NKAE cells and the CAR-T cells had higher cytotoxicity against these two lines (OCI-AML-3R and OCI-AML-3S) after being transduced with our NKG2D-CAR. However, the antitumor activity of CAR-T cells always remained superior to that of the CAR-NKAE for both OCI-AML-3R (54.26% ± 3.8% vs. 35.3% ± 6.7%) and for OCI-AML-3S (63.36% ± 3.5% vs. 54.3% ± 3.7%) cell lines, with a greater difference compared to drug resistant cells. The antitumor activity of CAR-T cells and CAR-NKAE cells was always superior on the sensitive cell line. The antitumor activity of NKG2D CAR-T cells was evaluated against primary blasts from AML patients (n = 4), observing a nearly complete destruction of the blasts after 24 hours, at a very low target: effector ratio of 4:1. CAR-T cells populations were analyzed by flow cytometry and we found that CAR-T cells were highly positive to CD45RO and reduced expression of CD45RA, on the other hand, they showed high IL-2 production exhibiting a central memory phenotype. Slight toxicity was observed against lung cells in both cell types. However, CAR-T cells exhibited some toxicity on third party PBMCs being null in the case of CAR-NKAE. CGH arrays studies showed no variation in the copy number, resulting from the introduction of the CAR, of genes which their variation has been associated with chromosomal instability. No significant changes associated with transduction process were observed in the surface phenotype of CAR-NKAE cells or CAR-T cells. Conclusions: We have demonstrated that AML cells could be target with an NKG2D-CAR. Primary NKAE cells and T cells can be transduced with an NKG2D-CAR at a very low MOI to enhance their antileukemic activity. CD3+ CAR-T cells are more effective than CAR-NKAE cells. Moreover, CAR-T cells were able to completely destroy AML blasts. Although further studies are needed, these results show the potential of NKG2D-CAR T and NK cell therapy in AML. Disclosures Rio: Rocket Pharmaceuticals: Equity Ownership, Patents & Royalties, Research Funding. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Functional ex vivo assays that predict a patient's clinical response to anticancer drugs for guiding cancer treatment have long been a goal, but few have yet proved to be reliable. To address this, we have developed an automated flow cytometry platform for drug screening that evaluates multiple endpoints with a robust data analysis system that can capture the complex mechanisms of action across different compounds. This system, called PharmaFlow, is used to test peripheral blood or bone marrow samples from patients diagnosed with hematological malignancies. Functional assays that use the whole sample, retaining all the microenvironmental components contained in the sample, offer an approach to ex vivo testing that may give results that are clinically relevant. This new approach can help to predict the patients' response to existing treatments or to drugs under development, for hematological malignancies or other tumors. In addition, relevant biomarkers can be identified that determine the patient's sensitivity, resistance, or toxicity to a given treatment. We propose that this approach, which better recapitulates the human microenvironment, constitutes a more predictive assay for personalized medicine and preclinical drug discovery.

Cytogenetic analysis is still an important and mandatory component of Acute Myeloid Leukemia (AML) diagnosis and prognosis. Pretreatment cytogenetic and molecular genetic findings are one of the major independent prognostic markers in AML, and they determine chemotherapy response and outcome. However, cytogenetic does not provide alternative treatments when a patient have a high cytogenetic risk, and requires relatively long time until obtaining the results despite the treatment of these patients should begin as soon as possible. The aim of this study is providing data about the utility of a new AML Precision Medicine (PM) Test as a complementary tool to conventional cytogenetic to overcome the main obstacles this later has. For this purpose, AML bone marrow from 111 patients were received at the laboratory 24h from extraction and incubated for 48h in 96-well plates containing single drugs or combinations, representing up to 31 different treatments that are currently given in the clinical practice. The analyses were performed in the automated flow cytometry PharmaFlow platform and the test results can be sent to the hematologists 72h after the extraction of the sample. Pharmacological responses were calculated using pharmacokinetic population models. Induction response was assessed according to the Cheson criteria (2003). Patients attaining a complete remission (CR) or CR with incomplete blood count recovery (CRi) were classified as responders and the remaining as resistant, excluding early deaths. The probability of being resistant or non-responder was modeled using binary logistic generalized additive models (GAM) with Cytarabine (CYT) and Idarubicin (IDA) area under the curve (AUC) data and over the cytogenetic risk (favorable/intermediate/adverse). The empirical ROC curves were calculated for the probabilities of being non-responder from each GAM. Final scores and treatments ranking are based on a therapeutic algorithm that integrates ex vivo activity of single drugs, quantified by the AUC and synergism, referred as α parameter, using a surface interaction model. Clinical and cytogenetic risk data of the patients were monitored and collected. A simple logistic model of the probability of being non-responder over the cytogenetic risk (favorable/intermediate/adverse) explained less variability (29.4%) than the GAM over the AUC values (40.8%) in the subset of 111 patients in whom the cytogenetic risk was informed. Figure 1 shows the results of the clinical correlation of cytogenetics vs PM Test in the cohort of 111 patients analyzed. In both approaches prediction of sensitive patients (Negative Prediction Value, NPV) is better than resistant patients (Positive Predictive Value, PPV), being the PM Test slightly better in predicting the sensitive patients (NPV=93% vs 88%), while the cytogenetics shows a 20% improvement in the prediction of resistant patients (PPV= 76% vs 56% with PM Test). The correlation achieved by the PharmaFlow PM test was 80% that is almost similar than the correlation obtained with the cytogenetic data using the same cut off point (86%). Figure 1 (right) also shows an example of the classification of AML treatments with the PharmaFlow PM Test in a patient sample according to a color scale from higher (green) to lower (red) ex vivo activity. In summary, despite the PharmaFlow PM Test and cytogenetics provide similar information, results from cytogenetic risk are available typically in 10-14 days, and thus after patient treatment, while results from this novel PM Test are available in 48-72h, prior to treatment. Hence, this novel approach provides information to hematologist with higher predictive value than risk factor (deviance explained 40.8% vs 29.4%) and ahead of treatment, and thus represent a valuable in-time prior to treatment decision making. In addition, the PM Test can provide alternative treatments to AML patient in a basis of their ex vivo activity. Disclosures Ballesteros: Vivia Biotech: Employment. Martinez Lopez:Novartis: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau. Hernandez:Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Montesinos:Novartis: Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Speakers Bureau.

Despite the effects of BCR ABL on cell proliferation, no study has compared the proliferative rate of different haematopoietic cell compartments from chronic myeloid leukaemia (CML) with those of normal bone marrow (NBM). We comparatively analysed the cell cycle distribution and BCR/ABL expression in different compartments of BM cells from 15 CML and 11 NBM. Overall, our results showed similar proliferative indices in CML patients and NBM. However, CD34+ myeloid precursors from CML patients displayed an increased proportion of S + G2/M-phase cells (P = 0.04), while no significant differences were found between CML and NBM for other BM cell subsets analysed. In BM cells separated by fluorescence-activated cell sorting, decreasing levels of BCR/ABL mRNA were found from CD34+/CD38+ myeloid precursors to myeloblasts; BCR/ABL expression increased afterwards with a peak at the myelocyte/metamyelocyte stage, decreasing in the more mature band/neutrophil compartment. Unexpectedly, BCR/ABL gene expression showed an inverse correlation with the proportion of S + G2/M-phase cells (R = -0.33; P = 0.04). These results suggest that in CML, BCR/ABL expression is associated with an increased proliferation of CD34+ myeloid haematopoietic progenitor cells but not of other more mature myeloid precursors, as confirmed by the observation of an inverse correlation between the amount of BCR/ABL transcripts and the proportion of S + G2/M-phase cells.

Philadelphia-positive (Ph(+)) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous disease with a very poor prognosis. In this study, we analyzed the frequency of supernumerary Ph, trisomy 8, monosomy 7, and del(9p21) by FISH and its relationship with the characteristics of the disease, in 46 BCR/ABL(+) adult BCP-ALL patients. The frequency of supernumerary Ph, trisomy 8, monosomy 7 and del(9p21) was 30%, 20%, 15%, and 24%, respectively. Although all patients displayed a BII/common phenotype, supernumerary Ph and trisomy 8 were associated with higher expression of CD19 and CD22 and of CD19, CD34, CD45, and HLA-DR, respectively; in turn, cases with monosomy 7 showed lower CD19, CD22, CD34, and cCD79a and del(9p21)(+) blasts were CD13(-) and CD33(-). Overall, similar clinical and hematological features were observed at presentation, independently of the underlying genetic abnormalities. However, relapse-free survival (RFS) was significantly shorter in cases with supernumerary Ph, trisomy 8, and del(9p21), the latter being the most powerful independent prognostic factor for RFS.

Interphase fluorescence in situ hybridization (iFISH) is increasingly used for the identification of BCR/ABL gene rearrangements in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). In the present study, we have explored the incidence of both typical and atypical iFISH patterns of BCR/ABL gene rearrangements in a series of 168 consecutive BCR/ABL+ patients--135 CML, 31 precursor B-ALL and two acute myeloblastic leukemia (AML) cases--and established their underlying genetic alterations through further molecular and chromosome analyses. Two different FISH probes (Vysis Inc., Downers Grove, IL, USA) were used: the LSI BCR/ABL dual color extra signal (ES) and the dual color dual fusion BCR/ABL probe (D-FISH). Our results show that most BCR/ABL+ patients (83%, including 88% of all CML, 61% of ALL and one of two AML) displayed typical iFISH patterns of either Major (M) BCR/ABL (87% of CML, 13% of ALL and one of the two AML) or minor (m) BCR/ABL gene rearrangements (1% of all CML and 48% of ALL cases) with the two probes. Further molecular and cytogenetic studies confirmed the presence of such typical rearrangements in all except one of these ALL cases who had coexistence of an MBCR/ABL and an mBCR/ABL gene rearrangement together with monosomy 9. In the remaining 29 cases (17%), up to five different atypical iFISH patterns were detected with the ES probe. Atypical iFISH patterns were most frequently due to additional numerical changes--most often supernumerary Philadelphia (Ph) chromosome (7%) but also gain or loss of chromosome 9 (1%) or 22 (1%). Deletion of 9q sequences proximal to the breakpoint were also frequently observed with the ES probe (8%). Application of the D-FISH probe showed that in most of these latter cases (5%) deletion of 22q sequences distal to the breakpoint also occurred. The remaining cases with atypical iFISH had cryptic insertion of BCR in 9q34 (1%). Exact interpretation of each iFISH pattern was supported by FISH on metaphases and molecular determination of the BCR breakpoint. In summary, our results indicate that despite the high incidence of typical iFISH patterns of BCR/ABL gene rearrangements, atypical patterns are also found in BCR/ABL+ acute leukemias; the precise definition of the alteration present in individual cases is dependent on metaphase studies and molecular definition of the breakpoint.

Objectives: Bispecific antibodies (BsAbs) act through the formation of an immunologic synapse between T-cells (CD3) and a tumor-associated surface antigen (TAA) leading to T-cell activation and serial lysis of tumor cells. The aim of the present study is to explore the mechanism of action (MOA) and the in vitro effect of BsAbs on hematologic samples with the PharmaFlow platform. Methods: Fresh whole bone marrow (BM) and peripheral blood (PB) from 41 samples from 3 different hematologic diseases (31 AML, 3 ALL, and 7 CLL) and two AML cell lines were tested with CD3-CD123 (for AML patients and cell lines) or CD3-CD19 (for ALL and CLL patients) BsAbs in the PharmaFlow platform, an innovative proprietary method that uses flow cytometry (FCM) to efficiently count the number of tumor cells killed by activated T-cells. We analyzed the populations of leukemic cells, activated T-cells, and residual normal cells. Additional key parameters were also used to explore the MOA after BsAb exposure at different time incubations (24h-144h), such as the effective E:T ratio (the number of T cells that kill a number of leukemic cells), real basal E:T ratio, tumor antigen expression, T-cell expansion, and expression of immune checkpoint proteins on target and effector cells before and after cell culture. For some experiments, fluorescence-activated cell sorting (FACS) was performed to evaluate T-cell cytotoxicity after BsAb exposure. Results: Most of the samples demonstrated T-cell activation and effective lysis of tumor cells after BsAb exposure independent of TAA expression and in a dose-response manner. Once sorted, these T cells could kill tumor cells in the absence of BsAb, as well as tumor cells that did not express the TAA target. Interestingly, these activated T cells selectively killed tumor cells with low cytotoxicity in residual normal cells from the same patients. Moreover, differential T-cell cytotoxicity was observed between samples. We observed samples with leukemic resistance or no T-cell activity (especially in CLL with CD3-CD19), as well as others with higher T-cell cytotoxicity and minimal number of activated T cells (especially in AML with CD3-CD123). The integration of all the predictive parameters (E:T ratios, Tumor-Specific Antigen (TSA) expression, etc.) allowed us to generate an in vitro response model and select samples with higher T-cell cytotoxicity after the BsAb exposure. Conclusion: Our findings are consistent with a model where, in addition to the standard MOA inducing tumor cells lysis by proximity, BsAbs can highly enrich cytotoxic clonal T-cell subsets with TSA and induce strong activation and proliferation of T cells capable of killing tumor cells in an effective and selective manner. This differential in vitro T-cell cytotoxicity effect between patients could allow us to select better candidates for adoptive antitumor immunotherapy with BsAbs. Citation Format: Daniel Primo, Pilar Hernandez, Julian Gorrochategui, Maria L. Vicente, David Martinez-Cuadron, Pau Montesinos, Joaquin Mártinez-López, Joan Ballesteros. A novel in vitro approach elucidates a new mode of cytotoxic action of bispecific antibodies on hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B012.

We have estimated the pharmacological sensitivity and synergism of 125 individual patient samples for all drugs and combination treatments for acute myeloid leukemia in the context of the overall patient population. Each ex vivo pharmacological profile identifies drugs and treatments for which the patient's malignant cells are particularly sensitive or resistant, assisting in the selection of individualized treatments. Background: We have evaluated the ex vivo pharmacology of single drugs and drug combinations in malignant cells of bone marrow samples from 125 patients with acute myeloid leukemia using a novel automated flow cytometry-based platform (ExviTech). We have improved previous ex vivo drug testing with 4 innovations: identifying individual leukemic cells, using intact whole blood during the incubation, using an automated platform that escalates reliably data, and performing analyses pharmacodynamic population models. Patients and Methods: Samples were sent from 24 hospitals to a central laboratory and incubated for 48 hours in whole blood, after which drug activity was measured in terms of depletion of leukemic cells. Results: The sensitivity of single drugs is assessed for standard efficacy (E-MAX) and potency (EC50) variables, ranked as percentiles within the population. The sensitivity of drug-combination treatments is assessed for the synergism achieved in each patient sample. We found a large variability among patient samples in the dose-response curves to a single drug or combination treatment. Conclusion: We hypothesize that the use of the individual patient ex vivo pharmacological profiles may help to guide a personalized treatment selection.

The present paper describes for the first time an intrinsic fluorescent square-planar platinum(II) complex carrying two ursodeoxycholate ligands ([Pt(UDC)2(en)], where UDC(-) = ursodeoxycholate), that emits at room temperature once free in solution. Kinetic studies were carried out in aqueous solution and in the presence of different NaCl concentrations: 4 mM (similar to cytoplasmic concentration) and 150 mM (similar to plasmatic concentration). This novel compound was synthesized from a [PtCl2(en)] complex and shows increased cytotoxic activity against both resting and cycling HeLa cells, with no toxicity for cell lines derived from neoplastic haematopoietic cells.

The microenvironment (ME) critically promotes progression of chronic lymphocytic leukemia (CLL), favoring leukemic cell survival and proliferation as well as inducing drug resistance. We aimed at reproducing the effects of ME stimuli for the development and optimization of an ex vivo assay that would enable to predict in vivo drug efficacy for agents interfering with ME protective effects e.g. the novel BCR inhibitors. To this purpose, we exploited the following 2 new approaches: 1) the Exvitech® proprietary automated flow cytometry-based platform by Vivia Biotech that enables evaluation of up to 20.000 wells and conditions per sample, and 2) Viviaxs Precision Medicine Native Environment approach that utilizes the whole blood sample rather than isolated leukocytes. We present this assay optimization using primary CLL samples and its validation when exposing CLL cells to the registered PI3Kd inhibitor, Idelalisib, for which only a weak pro-apoptotic effect has been reported, besides the known tissue mobilization activity in vivo. Cryopreserved peripheral blood (PB) mononuclear cells were provided from CLL patients in need of treatment. In order to more closely reproduce the complexity of the in vivo ME, the following elements were evaluated in different combinations: (i) 3 backbone stimulations, previously reported to improve, to different extent, CLL viability and proliferation: CD40L+CpG, CD40L+IL21, CpG+IL2; (ii) "Native Environment", defined as the plasma & erythrocyte/granulocyte fraction of a Ficoll gradient, already shown to improve ex vivo drug testing (Bennet et al. Clin Lymphoma Myeloma Leuk. 2014;14:305-18): the two fractions were added to thawed CLL samples and were obtained from fresh samples of normal donor PB or bone marrow as well as from CLL patients at different stages of disease; (iii) the stroma cell line H5S, added at different ratios (1:10 or 1:100); (iv) both human and bovine fetal serum (at 10 or 20% total volume); (v) stimulatory B cell factors, including IL-21, soluble CD40L, BAFF, and B cell receptor stimulation (anti-IG). CLL cell viability and proliferation was then tested and, although CLL cells from PB are notorious for a low proliferative index and tend to die quickly ex vivo by apoptosis, we achieved a median of 30±3% proliferation (assessed by the CFDA dye) and 60±5% viability (assessed by Annexin V staining) in cryopreserved progressive CLL samples. These results were obtained with the combination of the following assay conditions: CpG+IL2, HS5 (at 1:100 ratio), human serum 10%, and "native environment" from PB of CLL samples (pooled samples to prevent interpatient variability). We then tested the dose responses of Idelalisib in 16 cryopreserved progressive CLL samples and found little effect on the non-proliferative CLL fraction (Fig, 1A), suggesting a limited direct pro-apoptotic activity of the drug. In contrast, potent inhibition of proliferation with median potency (EC50) of 14 nM was observed (Fig 1B). The efficacy was nearly complete leaving a median of 5% resistant CLL cells that proliferated at the highest doses of Idelalisib. In conclusion, we report a novel ex vivo assay that enables high-throughput pharmacological characterization of compounds and combinations, optimized for CLL cells by incorporating ME stimuli and thereby more accurately simulating in vivo interactions. The increased cell viability and proliferation achieved with this innovative assay offers improved opportunities for ex vivo pharmacology, in particular unraveling a hitherto unknown anti-proliferative mode of action for Idelalisib, a drug interfering with the interaction of CLL cells with the ME. Figure 1. Dose response curves of Idelalisib incubated for 96 h with 16 CLL samples in the new Microenvironment Native Environment assay. The effect on non-proliferative (A) and proliferative (B) CLL cells identified using flow cytometry as subpopulations with different CFDA staining is shown. Figure 1. Dose response curves of Idelalisib incubated for 96 h with 16 CLL samples in the new Microenvironment Native Environment assay. The effect on non-proliferative (A) and proliferative (B) CLL cells identified using flow cytometry as subpopulations with different CFDA staining is shown. Disclosures Ballesteros: Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Robles:Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Munugalavadla:Gilead Sciences: Employment. Stamatopoulos:Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding. Quéva:Gilead Sciences: Employment, Equity Ownership. Ghia:AbbVie: Consultancy; Pharmacyclics: Consultancy; Gilead: Consultancy, Research Funding, Speakers Bureau; Adaptive: Consultancy; Acerta Pharma BV: Research Funding; GSK: Research Funding; Roche: Consultancy, Research Funding; Janssen: Consultancy.

e218 were substantially minimized after the heat treatment. The heat treatment also suppressed the clonogenic or self-renewal capacity of these MM cells as determined by in vitro colony formation and in vivo tumor formation in SCID mice, suggesting targeting MM progenitors. Further, the Pim inhibitor SMI16a also reduced the SP sizes and the ability of colony formation in RPMI8226 and KMS11 cells. Interestingly, the Pim inhibition in combination with heat treatment enhanced the induction of CHOP, a suicide mediator, while further reducing the protein levels of IRF4 and c-Myc to facilitate MM cell death. These results collectively demonstrated that hyperthermia is able to impair clonogenic drug-resistant fractions of MM cells, which may be augmented in combination with ER stress inducers, such as bortezomib, as well as Pim inhibition. We are now developing superparamagnetic mesoporous nanoparticles, which are able to deliver tumor-selective hyperthermia and drug release. A new strategy with tumor-selective hyperthermia and drug release warrants further study especially in the setting of drugresistant extramedullary plasmacytomas.

The relationship between different Abelson/breakpoint cluster region (BCR/ ABL + ) gene rearrangements and the involvement of different haematopoietic cell lineages were investigated in 15 chronic myeloid leukaemia patients. Analysis of purified cell populations confirmed the involvement of the neutrophil (89%), monocytic (89%), eosinophil (88%), erythroid (100%), and CD34 + cells (100%) in virtually all patients, without differences between minor BCR/ABL + and major BCR/ABL + cases; BCR/ABL + B- and natural killer (NK)-cells were detected in 43% and 31% of cases, respectively, whereas BCR/ABL + T-cells were rare (7%). All three minor BCR/ABL + patients showed involvement of both B- and NK-cells, which was infrequent (27%, P = 0·06 and 10%, P = 0·01) among major BCR/ABL + cases.

We compared the incidence of Her-2/ neu amplification in patients with and without a family history of breast cancer and correlated gene status with clinicobiologic and prognostic features in sporadic and familial cases. Of 108 patients, 28.7% had gene amplification. Among 96 cases with family history information available, 28 had an affected first-degree relative. The gene was amplified more frequently in familial than in sporadic cases (13/28 [46%] vs 14/68 [21%]; P = .01). Among familial cases, amplification was associated with adverse clinicobiologic features (poorly differentiated tumors [ P = .05], larger tumors [ P = .05], more lymph nodes involved [ P = .04], and DNA aneuploid [ P = .02] and highly proliferative tumors [ P = .005]), and the relapse ( P = .02) and disease-related death ( P = .05) rates were higher than in cases without amplification. Among sporadic cases, amplification was not associated with significantly different disease features, except for a higher incidence of DNA aneuploid tumors ( P = .01), percentage of S-phase tumor cells ( P = .006), and lower proportion of estrogen ( P = .001) and progesterone ( P = .002) receptors. Her-2/ neu amplification was observed more frequently among patients with a family history of breast cancer, in whom it was associated with adverse clinicobiologic features and a worse clinical outcome.

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.

Background Alkylating histone deacetylase inhibitors (HDACi) enhance the anticancer efficacy of alkylators by increasing chromatin accessibility and also down regulating DNA repair. EDO-S101 is a first-in-class fusion molecule that combines DNA damaging effect of bendamustine with the pan-HDACi vorinostat. Objectives To study the bi-functional properties of EDO-S101 as an alkylating agent and a pan-HDACi in various in vitro and in vivo xenograft models of hematological malignancies. Methods In vitro inhibition of HDAC Class I and II enzymes by EDO-S101 and vorinostat was tested using an recombinant human enzymatic assay (BPS Bioscience, Enzo Life Science) and in vivo in rat peripheral blood mononuclear cells (PBMCs). The degree of inhibition was measured 1 hour following a single dose of 10–50 mg/kg i.v. and duration of inhibition over 24 hours after a single i.v. dose of EDO-S101 of 25 mg/kg. HDAC inhibition, alkylation and apoptotic activity were evaluated in vitro in myeloid (HL60 AML cell line) and lymphoid cell lines, including Daudi Burkitt’s lymphoma (BL) and a panel of 6 MM cell lines (MM1S, MM1R, RPMI-8226, RPMI-LR5, U266, U266-LR7). In vivo intra-tumor effects were analyzed after short courses of treatment with EDO-S101 in MM1S human plasmacytoma (PC) and BL xenograft models. Changes in pathway activation, protein expression and activities influencing the cell cycle were measured by Western blot and immunohistochemistry. Anti-tumor activity in vitro was measured by MTT and in vivo using a caliper to assess tumor size at regular intervals. Results In vitro, EDO-S101’s pan-HDACi activity, at nanomolar concentrations in Class I and II recombinant enzymes, was similar to vorinostat. In vivo, in intact rat PBMCs, HDAC inhibition was maximal at 1 hour after a single dose of 10 mg/kg i.v.–the dose where antitumor activity starts. HDAC inhibition did not increase with doses up to 50 mg/kg, recovery began within 3 hours and was nearly complete at 16 hours. In the AML HL60 cell line in vitro, hyperacetylation of lysine residues K9, K14, K23 and K56 on histone 3 was found after exposure to 2–4 µM of EDO-S101. Histone 3 and 4 hyperacetylation was also demonstrated in MM cell lines at 1–5 µM concentrations. In xenograft models of human plasmacytoma and BL, EDO-S101 induced histone 3 hyperacetylation, indicating an HDACi effect in vivo. Alkylating activity was demonstrated in vitro in HL60 and MM cell lines by DNA cross-linking and double strand break formation in the comet assay by immunofluorescence. In vivo, in xenograft models of human plasmacytoma (60 mg/kg d 1, 8, 15) and BL (40 and 80mg/kg d1) exposure to EDO-S101 caused a strong DNA-repair response shown by activation of pH2AX and p53 (PC and BL) followed by an increase of DNA damage check point proteins pCHK1 (PC) and even more prominent pCHK2 (PC and BL). The kinetics of this effect, studied in vivo in BL tumors, showed that the pH2AX response fell at Day 8 after dosing while the p53 response lasted, particularly in the group treated with 80mg/kg. In Daudi-bearing mice tumors, p-ATR was completely suppressed at Day 8 after treatment, which was not clear in the PC tumors. EDO-S101 triggered apoptosis in vitro and in vivo, resulting in strong antitumor activity in HL60, Daudi and the panel of six MM cell lines. Initial in vitro experiments in HL60 cells showed an activation of the intrinsic pathway of apoptosis with cleavage of caspases 3, 9 and PARP and a marked reduction of anti-apoptotic proteins XIAP and Mcl-1. In the MM cell line, MM1S activation of the intrinsic and extrinsic pathways of apoptosis (C 8, 9, 3, 7 and PARP cleavage) was seen with a loss of mitochondrial membrane potential by DiOC6. Tumors of human plasmacytoma and BL in vivo were rapidly shrinking or completely eradicated after i.v. administration of EDO-S101. A decrease in proliferation (Ki67) and slight PARP cleavage was found in the tumor tissue (PC), and evidence of activation of apoptosis by cleavage of caspases 7 and 9 at Day 4 and caspase 8 and PARP at Day 8 after treatment in BL tumors. The level of caspase 3, different to MM, remained unchanged. Importantly, EDO-S101 induced a rapid and dose-dependent strong decrease of XIAP and Mcl-1 which lasted until Day 8. Conclusions This study demonstrates the bi-functional mechanism of ED0-S101 in both myeloid and lymphoid hematological malignancies. The data support the clinical investigation of EDO-S101 in treating hematological malignancies. Disclosures Ocio: Mundipharma: Honoraria, Research Funding. Mehrling:Mundipharma: Employment.

Background: Finding the best therapy option to treat myelofibrosis (MF) represents a huge challenge. Ruxolitinib (R) is a potent JAK1/2 inhibitor that has demonstrated improved survival and symptomatology in MF patients. There are a lot researches looking for the best combination of JAK inhibitor to improve its efficacy. In a previous work, we screened different drugs to evaluate if they were synergistic with ruxolitinib, we found that ruxolitinib was synergistic with nilotinib (N) and Prednisolone (P) as well as other drugs including bortezomib and HSP-90 inhibitors. Aim: To study the synergistic behavior and mechanism of actions of ruxolitinib in combination with nilotinib and prednisolone both in patient samples and cell lines. Methods: We have studied 20 secondary or primary MF patients and cell line: BA/F3 transfected with mutated JAK2 V617F (BA/F3 JAK2V617F). Classical assay was performed for patient samples: mononuclear cells isolated from peripheral blood were cultured during 2 weeks in Methocult TM GF_H4535 with 20 ng/ml IL-3, and 50 ng/ml SCF, in presence of increasing concentrations of R, N or P or their combinations. Also, mononuclear cells were cultured as described above, during 2 weeks but without drugs. Then, cells were washed with PBS and cultured 72 hours in RPMI 10% FBS and plated at 15,000 per well in 96-well plates with increasing concentrations of drugs. After both assay, cells were labeled with Annexin V and CD13 and analyzed in the Exvitech platform, an automated multiparametric flow cytometry platform. For cell lines, these were cultured in RPMI 10% FBS in the presence of increasing concentration of drugs. After 48 h of incubation, we performed a wst-8 assay to evaluate cell viability. To analyzed effect of treatment on survival and proliferation signaling pathways, cell lines were treated for 30 min with R 0.032 µM, N 1.6 µM and P 0.8 µM or combinations and total lysates were collected. To perform western blot, we use phospho-STAT5 (Tyr 964) and STAT5 after stripping as primary antibody and β-actin as load control. Graphpad Prism or XLFit was used to analyze dose-response curves. Synergism will be evaluated by the Median Effect methods described by T-C Chou and P. Talalay. Results: In patient samples when performed the classical assay, we found synergistic interaction in all combination in most of the patients (Table 1). Briefly, all combinations showed synergetic behavior (C Figure 1 Figure 1. However, when pathologic cells were amplified in a methylcellulose culture and then analyzed, we found a strong potentiation, representing an important dose reduction of R in the presence of N (Dose-Reduction Index (DRI) = 6.10, (n = 4) and a DRI = 110.17 when R is combined with P (n =2). There was not a synergistic interaction between R with N or P, because R was not efficient enough in this model. Interestingly, R had more effect in the classical assay, always less than 0.500 µM, than in the amplification assay even on less sensitive patient samples (Table 2). R seems inhibit growth and development of hematopoietic stem cells but not differentiated cells. N had a wide range of action in classical assay, but in amplification assay the effect was homogeneous, around 9 µM. P had similar heterogeneous effect on both assays from high nM to low µM. Figure 2 Figure 2. The 3 drugs were more potent in cell line BA/F3V617F than in patients: IC50 R = 15 nM; IC50 N = 3.35 µM; IC50 P = 4 nM. All drug combination tested were synergistic (CI Conclusions: we have showed that ruxolitinib have a synergistic effect with nilotinib and prednisone in MF patient samples. This effect is in part mediated through a greatest inhibition of STAT5 pathway. This preclinical study warranties a clinical trial with this combination in patients with MF. Disclosures Hernandez-Campo: Vivia Biotech: Employment. Gorrochategui:Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment, Equity Ownership. Martinez-Lopez:Vivia Biotech: Honoraria.

Background and Objectives: B-cell chronic lymphoid leukemia (CLL) is a lymphoproliferative disorder where specific microenvironment between B-cells and nurse-like Cells (NLC) seem to be involved in disease progression providing cell survival, proliferation and drug resistance. Consequently, functional screening platforms that can assess drug candidates within this microenvironment are needed. Our aim is to show the ability of the Exvitech® automated flow cytometry platform to screen agents that interfere with the microenvironmentxs protective scenario such as ibrutinib or idelalisib or standard CLL drugs such as fludarabine or prednisolone. This approach will allow us to select candidates in an in vitro assay and could personalize the treatment according the response to the drugs. Patients and methods: We have adapted Jan Burger's published assay1. Peripheral blood mononuclear cells (PBMCs) from not previously treated CLL patients were isolated by density gradient centrifugation over Ficoll-Pacque and were used fresh (N=14) or cryopreserved (N=6). B-cells were assessed to be >90% viable by flow cytometry. NLC co-cultures were stablished by suspending PBMCs from CLL patients in complete RPMI medium with 10% FBS to a concentration of 2x107/ml. Cells were incubated for 14 days in 96-well plates and presence of NLC was confirmed by microscopy. After that, viability was investigated in B-cells treated with 8 concentrations of ibrutinib, idelalisib, fludarabine and prednisolone after 72h of incubation with annexin-V and the appropriate CLL flow cytometry markers. Drug response was evaluated as a depletion survival index of the B-cell population relative to the average of the control wells with NLC but without drug. Results: As expected, depletion of B-cells cultured without NLCs were significant greater than with NLC after the 72h incubation supporting the assay where NLCs protect B-CLL cells from in vitro spontaneous apoptosis. In a similar way, viability of fresh samples with NLC was higher than the corresponding frozen samples (84% vs 25%), though both could be used. Our results show a lower pharmacological median potency, measured as a higher EC50, when we work with NLC versus without NLC for ibrutinib (10µM vs 4µM), idelalisib (17µM vs 0.4µM) and prednisolone (3.5µM vs 1.5µM). However, the effect of fludarabine seems to be independent of the presence of the NLC in the cell culture (7µM vs 6µM). This is consistent with the protective role of microenvironment; more pronounced for ibrutinib and idelalisib. Interestingly with NLC, for each drug there is a significant interpatient variability (Figure 1); each line correspond to a different patient, reflecting the possibility that patients might be more sensitive or resistant to a certain drug in this particular scenario. There is a higher degree of patient sample stratification for idelalisib, fludarabine or prednisolone, where there are still an important % of B-cells alive for some patients after drug exposure at high concentration, supporting the notion of drug resistance. Synergism between some of these drugs was evaluated in 3 samples, with some samples being more synergistic than other, requiring a larger number of samples. Conclusions: Cellular and molecular interactions between B-cells and the microenvironment represented here with the NLC, have become an attractive target for CLL therapy. Because novel drugs such as ibrutinib or idelalisib are transforming CLL therapy targeting the microenvironment, novel technologies that could predict its effect are necessary. Here we have adapted a Nurse-Like Cell assay mimicking the microenvironment published by Burger1 to our ExviTech platform. The automated platform enables scaling of the data points acquired with this assay supporting characterization of drug activity by pharmacological dose response curves, as well as exploring synergistic interactions. As showed in the results and illustrated in Figure 1, there is a interpatient variability of the pharmacological profile for the studied drugs, if clinically validated, could help guiding a personalized treatment selection; measuring the drug activity inside this particular microenvironment responsible of drug resistance. 1.- Burger JA et al. Blood. 2000 Oct 15;96(8):2655-63. Figure 1: Figure 1:. Disclosures Primo: Vivia Biotech: Employment. Martinez:Vivia Biotech: Membership on an entity's Board of Directors or advisory committees. Gorrochategui:Vivia Biotech: Employment. Espinosa:Vivia Biotech: Employment. Arroyo:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Employment. Hernandez:Vivia Biotech: Employment.

Background Identifying the most promising synergistic drugs combinations for a drug is a challenge for researchers. Identification of optimal combinations are key and should be translated in better designed clinical trials with fewer patients and ultimately more effective treatments. Ruxolitinib is a potent JAK1/JAK2 inhibitor that has demonstrated improved survival, rapid and durable improvements in splenomegaly, in patients with myelofibrosis (MF), however although improve survival in most of patients it does not change the natural history of the disease. There is however always a drive to improve outcomes and we hypothesize that treatment with a synergistic drug could enhance the activity in MF. Aim To design an ex-vivo model, based on flow cytometry, to identify the most synergistic drugs with Ruxolitinib in cell lines and primary samples from MF patients. Methods We have studied five secondary or primary MF patients (n = 5) and one cell line, BA/F3 transfected with mutated JAK2 V617F (BA/F3 JAK2V617F). We combined Ruxolitinib with a panel of 30 drugs whose mechanism of action is implicated in proliferation, differentiation and survival, cell-cycle inhibition, protein stabilisation, epigenetic, immune response. Briefly, mononuclear cells from peripheral blood, isolated, was cultured in Methocult TM GF_H4535 supplemented with 20 ng/ml interleukin (IL)-3, and 50 ng/ml stem-cell factor (SCF). After 2 weeks incubation, viable cells were plated at 15.000 per well in 96-well plates in increasing concentrations of each drug, alone or in combination with Ruxolitinib, in 8 or 5 point dose response curve. After 72 hr incubation, we performed a multiparametric flow cytometry, using Annexin V-fluorescein isothiocyanate (FITC) and CD13 to monitoring drug sensibility of myeloid lineage, in the ExviTech platform for screening by flow cytometry. Synergism will be evaluated by the Median Effect methods described by T-C Chou and P. Talalay. Regarding the cell line model, it confirms the results obtained in patients samples: Panobinostat was the most potent drugs tested in the assay with an IC50 of 86 nM and the most synergistic drugs with Ruxolitinib was Everolimus (CI = 0.613 when Ruxolitinib and Everolimus were 370 nM and 7.41 μM respectively). Conclusions This Vivia Ex vivo platform is highly efficient to study multiple synergisms of drugs in myeloproliferative diseases. We can test 30 drugs, inhibitors of multiple signaling pathways, epigenetics and immune response, alone or in combination with Ruxolitinib and test its activity and its potential synergy with Ruxolitinib. Based in these results, clinical trials combination Ruxolitinib with BKM120 (ongoing), Everolimus and LDE225 (ongoing) could potentially be explored in phase I clinical trials. Disclosures: Hernandez-Campo: Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Equity Ownership. Martínez-López:Vivia Biotech: Honoraria; Novartis: Research Funding.

Background and objectives Complete remission (CR) after induction therapy is the first treatment goal in acute myeloid leukemia (AML) patients. The aim of this study is to determine the ability of the Vivia’s novel ex vivo drug sensitivity platform Exvitech analyzing leukemic cell death to predict the CR rates after induction chemotherapy with cytarabine (Ara-C) and idarubicin (Ida) in 1st line AML. Patients and Methods This non-interventional and prospective study included samples from adult patients over 18 years of age diagnosed with de novo AML in Spanish centers from the PETHEMA group. Marrow samples were collected at diagnosis, sent to the Vivia laboratories, and incubated for 48 hours in whole samples in well plates containing Ara-C, Ida, or the combination Ara-C+Ida, each at 8 different concentrations to calculate dose responses. Annexin V-FITC was used to quantify the drug-induced apoptosis. Pharmacological responses are calculated using pharmacokinetic population models. Induction response was assessed according to the Cheson criteria (2003). Patients attaining a CR/CRi were classified as responders. The remaining patients were considered as resistant. Patients dying during induction response assessment were non-evaluable. The correlation was modeled using a generalized additive model with a logit link and a binomial distribution for residuals. Kernel density estimates were then used to plot empirical probability density functions for both groups. Their separation was quantified as the area under the ROC curve and a cut point was selected using the Youden’s criteria to optimize the classification probabilities (sensitivity, specificity). 95% confidence intervals for sampling errors were calculated for all these quantifiers. Results 125 patient samples were used to calculate the dose response curves for Ara-C alone, Ida alone, and the synergism of the Ara-C plus Ida combination. For clinical correlation we used 64 patients with a median age of 55 years (range 31 to 72). Dose responses for Ara-C alone are shown in Figure 1.A; note that for many samples there is a significant number (>20%) of resistant cells to Ara-C (bracket). This is a strong clinical predictor of resistance because in the patient the drug will never be present at these high doses for 48 h. The second variable that is a good predictor of response is the synergism between these 2 drugs. The generalized additive model identified an algebraic combination of these 2 variables that yielded the best marker to separate both groups of patients. The probability density functions had minimal overlap. The area under the corresponding ROC curve was 0.965 (0.928, 1.000), and the classification probabilities for the optimal cut point (set at 0.414 for the marker), expressed as percentages, were 85% (62.1% to 96.8%) and 86.4% (72.6% to 94.8%) for sensitivity and specificity, respectively. Results are shown in Figure 1.B; Forty-four patients (68.8%) achieved CR after Ida+Ara-C, and the remaining 20 (31.3%) were resistant. Correlations of the PM test are shown in Figure 1.B. Seventeen of the 20 (85%) patients who fail to achieve CR were predicted as resistance in the ex vivo test. Thirty-eight of the 44 patients (86.4%) who achieved CR showed good ex vivo sensitivity to Ida+Ara-C predicting for CR. When the ex vivo test predicted a patient as sensitive it was correct in 38/39 cases (93%), and when it predicted resistant it was correct 17/23 cases (74%). Overall, 45 patients (86%) had an accurate prediction of their response to treatment. Conclusions This study shows that this novel ex vivo pharmacological profile test is able to predict the clinical response to Ida+Ara-C induction. We are increasing the number of patients in this ongoing study, and we are planning a PM Test-adapted Clinical Trial. Disclosures: Martínez: Vivia Biotech: Employment. Ortega:Vivia Biotech: Employment. Primo:Vivia Biotech: Employment. Hernandez-Campo:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Bennett:Vivia Biotech: Employment. Liebana:Vivia Biotech: Employment. Lopez:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Equity Ownership.

Background and objectives Protocols for acute myeloid leukemia (AML) 1st line patients are centered on the combination of Cytarabine and an anthracycline; Idarubicin (IDA), Daunorubicin (DNR), or Mitoxantrone (MIT). Patients may be treated with IDA, DNR, or MIT depending on the country of residence, because multiple clinical trials have not found significant differences among them. A new Personalized Medicine (PM) test developed by Vivia Biotech based on pharmacological responses in patient samples (ex vivo) is uncovering individual responses to these treatments. Our objective is to explore whether a significant % of individual patients may respond differently to IDA vs DNR vs MIT treatments, in spite that of their “on average” similar response shown by clinical trials. Patients and Methods Multicenter, prospective, non-interventional study of the PETHEMA group for treatment of AML. Bone Marrow (BM) samples were collected at diagnosis for 160 AML patients. Samples were incubated for 48 hours in 96-well plates, each well containing different drugs or drug combinations, each at 8 different concentrations, enabling calculation of dose response curves for each single drug (CYT, IDA, DNR, MIT) and combination used in treatments (CYT-IDA, CYT-DNR, CYT-MIT). Drug response was evaluated as depletion of AML malignant cells in each well after 48 hours incubations. Annexin V-FITC was used to quantify the ability of the drugs to induce apoptosis. Malignant cells were identified with monoclonal antibodies and light scatter properties. 1) We use the whole bone marrow sample, retaining the erythrocyte population and serum proteins, during the entire incubation period; and after 48 h leukocytes are isolated prior to evaluation by flow cytometry. 2) We have pioneered development of a proprietary automated flow cytometry platform called ExviTech. 3) Pharmacological responses are calculated using pharmacokinetic population models. Results Figure left panel shows dose responses for both IDA (red) and DNR (blue) in 125 AML patient samples. Although their average curves (thick red & blue) are similar, the interpatient variability of either drug is quite large. We hypothesized that some patients could show very differential sensitivities to both drugs, as illustrated by the green arrow where a patient sample is resistant to DNR (right shifted dose response curve) but sensitive to IDA (left shifted dose response curve). To identify these cases Figure right panel shows a comparison of the potency IDA vs DNR. Potency is represented by their EC50 (concentration that kills 50% of the cells). Most dots tend to line up, but red dots represent patient samples with a difference in potency between these drugs >30%. Repeating this exercise for IDA-MIT and DNR-MIT to cover all alternatives among the 3 anthracyclines identifies 40% of patients samples with >30% different potency among IDA-DNR-MIT. Repeating this exercise with the combination treatments CYT-IDA, CYT-DNR, CYT-MIT increases to 58% the population of patients whose samples have a differential sensitivity to these anthracyclines. A fraction of this 57% of patients may benefit in if treatment selection among these 3 treatments were to be aided by this ex vivo testing sensitivities. To identify which fraction would benefit we would need a trial specifically designed. Conclusions This preliminary results show that Vivia's PM test seems able to identify a subset of AML patients who's ex vivo pharmacological response to anthracycline drugs is significantly different. Because this ex vivo test accurately predicts the clinical response to CYT-IDA, if these selective anthracycline ex vivo responses translate to clinical responses, a fraction of this 57% subpopulation could benefit significantly from receiving 1st or 2nd line treatments based on either IDA, DNR, MIT, and their combinations. Hence this approach stands for European integration of treatment protocols, based on ex vivo individual responses data rather than nationality. Disclosures: Primo: Vivia Biotech: Employment. Hernandez-Campo:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Bennett:Vivia Biotech: Employment. Liebana:Vivia Biotech: Employment. Lopez:Vivia Biotech: Employment. Ballesteros:Vivia Biotech: Equity Ownership.

Background To aid in the identification of effective treatments for individual patients, ex vivo assays for detecting cell death inducible by drugs for hematological malignancies have been in development for over 20 years. We have developed a novel approach incorporating 4 key innovations; incubating drugs in whole bone marrow sample without isolating leukocytes, using flow cytometry enables identification of the malignant cells selectively, an automated flow cytometry-based platform (ExviTech) decreases errors and enables full pharmacological characterization, and analyzing the data using pharmacodynamic population models. Aim The purpose of this study is to derive the ex vivo pharmacological profiles across the AML patient population of single drugs and combination treatments as a tool for individualized treatment selection. Patients and Methods Bone-marrow samples from 160 patients diagnosed with AML were sent to Vivia from 24 hospitals across Spain within 24 hrs. The plates were incubated for 48-hours prior to analysis with ExviTech, The percentage of leukemic cell death was determined via labeling with monoclonal antibodies and AnnexinV-FITC. A survival index is computed for each drug, the lower the survival index, the more effective the drug. Dose-response curves of cytarabine, idarubicin, daunorubicine, etoposide, mitoxantrone, fludarabine, clofarabine, and 6-thioguanine were measured in 160 patient samples. The added benefit of combining these drugs into 12 combination treatments was assessed by measuring their synergy in each individual patient. In 39 patients treated with CYT IDA we had clinical data of response, and then we performed a blinded interpretation of this in vitro test by an expert hematologist, to predict the clinical response based in this test result. Results There was a large range of interpatient variability in the response to a single drug and even larger in the synergism between drugs. The Population Pharmacological Profiles for an individual patient is shown on the figure below. The relative drug potency in terms of their percentile ranking within the population is shown in the left panel from 0 (weakest) to 100 (most potent). Green lines represent the individual patient potency relative to the population ranking, with confidence intervals. Third column lists when a drug leaves a significant % of leukemic cells alive, potential resistant clones. The panel on the right side shows the synergism of the drug combinations treatments shown as box-plots at 10-25-75-90% to highlight their distribution. The synergism value for an individual patient in each combination is shown in green, with confidence interval as parallel dotted green lines. This representation of the Pharmacological Profile of an individual patient sample quickly identifies extreme values, when a drug or combination is very sensitive (rightward shift green lines, green boxes) or very resistant (leftward shift green lines, red boxes). This patient showed average sensitivities for most drugs though highly resistant to Clofarabine (red box) that leaves 45% alive. However this patient showed lack of synergism in multiple treatments (right, red boxes). CYT and IDA show average potencies but lack of synergism, suggesting CYT-DAU might be a more efficient treatment. These representations lead to clear guidelines in >90% samples, and based on hematologist's interpretation of these guidelines show a clinical correlation with clinical responses to CYT-IDA of 84%. Conclusion We have developed an improved a methodology to measure the pharmacological activity of drugs and drug combinations in AML patient samples as well as modeling their pharmacological behavior. This information may be useful in selecting the optimal treatment for the individual patient, especially relapse/refractory patients in need of therapeutic alternatives. By testing the drugs used in the treatment protocols for AML directly on patient samples, a pharmacological based model has been developed to infer drug resistance or sensitivity, patient by patient. Disclosures: Ballesteros: Vivia Biotech: Equity Ownership. Primo:Vivia Biotech: Employment. Hernandez-Campo:Vivia Biotech: Employment. Rojas:Vivia Biotech: Employment. Liebana:Vivia Biotech: Employment. Lopez:Vivia Biotech: Employment. Iñaki:Vivia Biotech: Consultancy. Bennett:Vivia Biotech: Employment.

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.

Abstract 4414 Introduction Discovery of novel non cytotoxic drugs for cancer focuses on targets selectively expressed in malignant cells, only testing at the end if they are toxic to patients. We have developed a novel approach to discover these drugs starting at the end; we screen 2.000 approved drugs with proven safety, directly on freshly extracted (ex vivo) blood samples of patients with Chronic Lymphocytic Leukemia (CLL). These screens are enabled by a novel technology platform based on automated flow cytometry we call ExviTech for ex vivo technology. Patients and Methods All screening studies were performed directly on either peripheral blood or bone marrow samples from 44 patients diagnosed with various subtypes of B-cell malignancies, after informed consent. Patient samples were diluted and plated with each of the 2.000 drugs individually, retaining the erythrocyte population and serum proteins to enable clinically relevant concentrations. The experimental assay was setup the same or a day after sample extraction. Each sample was diluted to achieve a leukemic cell concentration of approximately 3,000 cells/μl; then 45μl of the suspension is added to each well of 96-well plates that contain the pharmacological agents (final concentration of 30μM). The compound plates were then sequentially incubated for 24 hours at 37°C with 5% CO2 for screening (sterile conditions). After incubation, the erythrocytes were lysed and the leucocytes incubated with Annexin V-FITC, anti-CD45-APC and anti-CD19-PE added to each well. The plates were then transferred to an automated flow cytometry system where the contents of each well were aspirated and analyzed by a CyAn flow cytometer. Candidates from the primary screens were validated in additional samples with dose-responses, combinations with approved drugs, multiple incubation times, etc… Results Analyzing primary screens from 24 CLL patients, three related compounds (Vivia007, Vivia008 and Vivia009) were found to consistently induce apoptosis of nearly all leukemic B-cells from most of the patient samples diagnosed with B-cell chronic lymphocytic leukemia at levels equal to or greater than known CLL active cytotoxic agents. Notably, these candidates are equally effective against samples of p53 mutated patients. These 3 drugs are pharmacologically me-too drugs sharing the same target and mechanism of action, and are non cytotoxic drugs with a known and good safety profile, administered to millions of patients over many years. Validation experiments were done on 20 additional CLL patients and Vivia009 emerged as the most effective agent with an average EC50 of 18.2μM. The mechanism of action is different than the known mechanism of Vivia009 and its class members for their approved indications. Consistent with this observation, only 3 of 15 members of the same pharmacological drug class were efficacious against CLL malignant cells. All 3 Vivia′s candidates were equally efficacious against other B-Cell Malignancies such as B-ALL (pediatric and adult), and Multiple Myeloma. These drugs are not effective in their current oral formulation and require a novel intravenous formulation. Interestingly, kinetics of induction of apoptosis were faster for Vivia009 than for fludarabine, cyclophosphamide and mitoxantrone. Vivia009 requires only 1 hour of incubation with fresh cells to induce maximal apoptosis. This timeline is less than the 3 hours in which Vivia009 was found present at high concentrations in bone marrow of rats using a single intravenous bolus. Thus, Vivia009 seems to fulfill the pharmacokinetic criteria to eliminate all leukemic cells with a single intravenous bolus, which would be a major advantage over current treatments (5-days fludarabine or 3 days FCR). Animal models are ongoing to confirm the non cytotoxic nature of the candidates in the novel IV formulation and the fewer days needed to reach remission, both compared with fludarabine monotherapy. Conclusions In summary, our results demonstrate the potential of the ExviTech technology platform as a successful model for the systematic search of new uses for already existing approved drugs directly on patient samples of hematological malignancies. A new drug candidate with excellent safety profile has been identified with similar efficacy ex vivo as the best approved cytotoxic drugs, which is a non-cytotoxic drug with fast kinetics that might enable significantly safer and shorter treatments. 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: Employment. Espinosa:Vivia Biotech: Ana Espinosa, Employment. Tudela:Vivia Biotech SL: Employment. Arroyo: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. González:Vivia Biotech SL: Consultancy. Dominguez-Gil:Vivia Biotech SL: Consultancy. Troconiz:Vivia Biotech SL: Consultancy. Rodriguez de Fonseca:Vivia Biotech SL: Consultancy. Saunders:Vivia Biotech: Consultancy. Montejo:Vivia Biotech SL: Consultancy. Caveda:Vivia Biotech SL: Employment. Orfao:Vivia Biotech SL: Research Funding. Ballesteros:Vivia Biotech SL: Equity Ownership.