Myelosuppression is a major side effect of chemotherapy in cancer patients and can result in infections, bleeding complications, and increased risk of morbidity and mortality, as well as limit the drug dose and frequency of administration. Chemotherapy-induced myelosuppression is caused by the disruption of normal hematopoiesis. Thus, prior understanding of the adverse effects of chemotherapies on hematopoietic cells is essential to minimize the side effects of cancer treatment. Traditional methods such as colony-forming assays for studying chemotherapy-induced myelosuppression are time-consuming and labor intensive. High-throughput flow cytometry technologies and methods to detect rare hematopoietic cell populations are critical in advancing our understanding of how different blood cell types in complex biological samples respond to chemotherapeutic drugs. In the present study, hematopoietic progenitor cells were induced to differentiate into megakaryocytes and myeloid lineage cells. The expanded cells were then used in a multiplexed assay to monitor the dose-response effects of multiple chemotherapies on different stages of megakaryocyte differentiation and myeloid cell populations in a 96-well plate format. The assay offers an alternative method to evaluate the myelosuppressive potential of novel chemotherapeutic drugs compared to traditional lower throughput and labor-intensive assays.

Introduction: The median progression free survival (PFS) and overall survival (OS) of multiple myeloma (MM) patients have been prolonged due to novel agents combined with ASCT but the median OS in MM is still 7-8 years. Thus, the feasibility of new combinations and dosing of available agents must be investigated. The first proteasome inhibitor (PI), bortezomib (B), combined with elotuzumab and dexamethasone (d) showed superiority to Bd with PFS of 9.7 vs. 6.9 months, respectively, without excessive toxicity (Jakubowiak et al. Blood 2016;127:2833-40). In our study we investigate the safety, feasibility and initial efficacy of a second generation PI carfilzomib (K), SLAMF7 antibody elotuzumab (E) and dexamethasone (D) combination (KED) in relapsed or refractory MM (RRMM) patients. Patients and methods: Forty RRMM patients after 1-3 prior lines will be included in this phase 2 study after written informed consent. The primary endpoint is overall response rate (ORR). In patients achieving at least very good partial remission (VGPR) the quality of response will be assessed with high-sensitivity multicolour flow cytometry (MFC) according to the 8-colour EuroFlow protocol. Carfilzomib is given once weekly 20 mg/m2 on D1C1 and thereafter 70 mg/m2 in 28 day cycles on days 1, 8 and 15 in cycles 1-8 and on days 1 and 15 thereafter combined with weekly elotuzumab 10 mg/kg on days 1, 8 and 15 in cycles 1-2, thereafter on days 1 and 15; dexamethasone 40 mg on days 1, 8, 15 and 22 on cycles 1-8, thereafter on days 1 and 15. Treatment will continue until progression or excessive toxicity. Carfilzomib dose was 20/56 mg/m2 for the first two cycles for the first five patients to evaluate the safety. Additionally, patient samples collected prior to treatment will be comprehensively profiled by whole exome and RNA sequencing and evaluated for ex vivo response to the agents. Together, the study addresses clinical response, ex vivo-in vivo translation, identifies molecular biomarkers for the KED combination and facilitates precision guided clinical trials for RRMM. Results: By the end of July 2018, 11 patients have been enrolled. Median number of prior lines was 2 (1-3). Seven IgG-κ, two IgA-κ and one kappa and lambda light-chain patient are included. After a median of 6 (2-12) cycles ORR is 91% with 3 patients in VGPR (median MFC-MRD of 0,002%), 7 patients in PR, one in MR. Initial molecular characterization highlighted diverse subclonal backgrounds among the treated patients (Figure 1), but interestingly, the best responding VGPR patients displayed mutations to RAS genes in the dominant clones (NRAS 828, 2662; KRAS 733). At least PR was achieved after a median of 1 (1-4) cycle. Three patients have progressed and one patient withdrawn due to suspected thrombotic microangiopathy with manifestation of convulsions and pulmonary embolism. We noticed only one grade 2 infusion reaction after premedication. One patient developed autoimmune hemolytic anemia (AIHA), without red cell antibodies, suspected to be related to elotuzumab. She recovered with steroids and elotuzumab discontinuation and continued Kd without reappearance of AIHA. Another patient had grade 3 liver transaminase elevation but was able to continue treatment with dose reduction of carfilzomib and dexamethasone. Conclusion: To the best of our knowledge this is the first study evaluating the carfilzomib, elotuzumab and dexamethasone combination in RRMM with comprehensive molecular annotations. Among this small group of patients we noticed two unexpected severe adverse events; atypical AIHA and suspected thrombotic microangiopathy. AIHA should be excluded if unexpected anemia will appear during elotuzumab treatment. Preliminary results of this KED combination showed efficacy in patients with clonal RAS mutations and ORR of 91% after the median of two prior treatment lines using weekly carfilzomib dose of 70 mg/m2. Figure 1: Subclonal diversity in patient derived plasma cells. Clonal and subclonal fractions were evaluated by assessing peaks of variant allele frequencies of somatic mutations present in the myeloma plasma cells using kernel density estimation. For each plot, the fraction of cells carrying the mutation is represented on the x axis (1 = 100% of cells), and the probability density on the y axis. The mutated cell fraction (x axis) was calculated by adjusting mutant allele burden by copy number of the mutated loci. Disclosures Silvennoinen: Takeda: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Tsallos:Novartis: Research Funding. Anttila:Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees. Räsänen:Amgen: Honoraria. Luoma:Amgen: Honoraria. Jantunen:Amgen: Honoraria; Genzyme/Sanofi: Honoraria; Takeda: Honoraria. Heckman:Celgene: Research Funding; Orion Pharma: Research Funding; Novartis: Research Funding.

BACKGROUND: Despite mounting knowledge, the management of patients with acute myeloid leukemia (AML) can be challenging, particularly for relapsed/refractory patients who lack innovative, effective treatments. Recent preclinical studies of AML have demonstrated promissory therapeutic activity of the novel class of inhibitors of bromodomain and extra terminal (BET) family proteins. To identify AML patients that may benefit from BET inhibitor treatment, we sought to discover and evaluate predictive molecular biomarkers for sensitivity towards BET inhibition, focusing on the prototype BET inhibitor JQ1. METHODS: Following written informed consent and according to the Declaration of Helsinki, bone marrow, peripheral blood, and skin biopsy samples were obtained prospectively from AML patients (n=139) and analyzed for their whole exome profiles and ex vivo responses to JQ1 exposure, as part of an Individualized Systems Medicine (ISM) study of AML (Pemovska et al., 2013). Consequently, associations between molecular features and drug responses were investigated using logistic regression models for data integration. Novel signals emerging from our cohort of patients were carried forward for reproducibility assessment in publicly available cell line datasets (Cancer Cell Line Encyclopedia and Catalogue of Somatic Mutations in Cancer for genomic data, and Cancer Therapeutics Response Portal and Genomics of Drug Sensitivity in Cancer for drug response data). In addition, literature-reported indicators of sensitivity were examined in the AML cohort in order to evaluate their clinical translatability. RESULTS: Enabling a genome-wide search for predictive biomarkers for BET inhibitor response, our bioinformatics pipeline returned a set of signals significantly associated with JQ1 treatment. Frequently mutated in AML, isocitrate dehydrogenases IDH1 and IDH2, tet methylcytosine dioxygenase TET2, and Wilms' tumor WT1 each appeared to confer resistance upon mutation to the samples harboring them. Participating in a common biochemical pathway, the 4 genes have been shown to be mutated following a mutual exclusivity pattern in AML (Scourzic et al., 2015), which was successfully replicated in our cohort. Accordingly, grouping of samples with mutation in any of the genes yielded a significantly more resistant group to JQ1 treatment in comparison to wild-type samples. Incorporation of TET2 and WT1 deletions from the samples' copy number variation data supported this hypothesis. Importantly, this observation was also successfully validated in cell line drug sensitivity data (Figure 1A). Moreover, FLT3-mutated samples in our cohort returned significantly higher sensitivity scores to JQ1 compared to wild-type samples, irrespective of their NPM1 mutation status. The superior response trend observed within this group was maintained independently of whether the mutation was an internal tandem duplication (FLT3-ITD) or in the tyrosine kinase domain (FLT3-TKD) of the gene (Figure 1B). Finally, and in contrast to other studies, AML samples harboring mutation to NPM1 were not significantly more sensitive to BET inhibition with respect to NPM1 wild-type samples. CONCLUSION: Taken together, the results suggest novel insights for the stratification of AML patients for treatment using BET inhibition therapy, taking this malignancy's heterogeneity into account. Particularly, while FLT3 mutations are to be considered as sensitizing factors to BET inhibitors, samples with mutations in IDH1, IDH2, TET2, or WT1 were resistant, and thus, patients harboring these gene mutations may not benefit. These observations also propose drug partners that are worth investigating in combination with BET inhibitors in the defined patient subpopulations, namely FLT3 inhibitors and hypomethylating agents (or IDH1/2 inhibitors), respectively. Furthermore, the variability of responses observed among samples harboring NPM1 mutations highlights the molecular heterogeneity existing within this group of patients, stressing the need for identifying additional companion diagnostics for a more effective stratification of this population. Although our findings require additional validation in a larger cohort and through clinical investigations, this study provides evidence of molecular indicators that could help identify AML patients that would benefit from BET inhibitor therapy. Disclosures Tsallos: Novartis: Research Funding. Wennerberg:Novartis: Research Funding. Porkka:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Novartis: Research Funding; Orion Pharma: Research Funding; Celgene: Research Funding.