Millennium Pharmaceuticals Research Articles

TAK-243, a Small Molecule Inhibitor of Ubiquitin-Activating Enzyme (UAE), Induces ER Stress and Apoptosis in Diffuse Large B-Cell Lymphoma (DLBCL) Cells

Despite the promise of targeting B-cell receptor-associated signaling kinases in indolent lymphomas, new agents in this class show limited activity in DLBCL, which remains an unmet clinical need. Ubiquitin-proteasome systems (UPS) are altered in cancer, leading to destabilization of tumor suppressors (e.g., TP53), overexpression of proto-oncogenes (e.g., MYC), and impaired DNA repair. We and others have shown that neoplastic B-cells exhibit a state of heightened cellular stress and thereby are susceptible to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Proteasome inhibitors (e.g., bortezomib) have demonstrated clinical activity in B-cell neoplasia. Our group has previously targeted proximal components of the UPS in B-cell neoplasia by disrupting the Nedd8 pathway with pevonedistat, which inhibits the function of a subset of E3 ubiquitin ligases known as cullin-ring ligases. Here we investigated an alternative approach to targeting the UPS in DLBCL using TAK-243, a first-in-class small molecule UAE inhibitor. By inhibiting UAE, TAK-243 blocks ubiquitin conjugation, disrupting monoubiquitin signaling as well as global protein ubiquitination. Here we demonstrate that UAE inhibition effectively kills DLBCL cells via induction of ER stress.Experiments were conducted in activated B-cell (ABC)-type (OCI-LY3, U2932) and germinal center (GC)-type (OCI-LY19, SUDHL4/10/16, VAL) DLBCL cell lines, and primary neoplastic B-cells derived from peripheral blood and lymph nodes of patients with CLL and DLBCL, respectively. CD40L-expressing stromal cells were used to induce NFκB activation in primary cells. TAK-243 was obtained from Millennium Pharmaceuticals, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA).TAK-243 induced ER stress and the UPR , followed by rapid apoptosis, as evidenced by PARP cleavage within 2 h, in both DLBCL cell lines and primary neoplastic B-cells. 24 h drug treatment yielded an IC50 of ~50 nM, as estimated by Annexin V expression. TAK-243 induced apoptosis of DLBCL cell lines independent of cell-of-origin (ABC- vs. GC). By contrast, primary T cells were significantly less sensitive to TAK-243.Treatment with TAK-243 rapidly disrupted ubiquitin conjugation and subsequent degradation of proteins controlled by the UPS in neoplastic B-cells. TAK-243-induced UPR occurred within 2 h, as evidenced by oligomerization and autophosphorylation of PERK, induction of BiP/GRP78 and activation of eIF2α. After 4 h of treatment, cells exhibited late apoptotic phase of the UPR: transcriptional induction of CHOP, GADD34 (10-fold) and NOXA (4-fold), accompanied by upregulation of pro-apoptotic BH3-only proteins, stabilization of MCL-1, activation of BAK, and ultimately cleavage of caspase-3 and PARP.TAK-243 inhibited both canonical and non-canonical NFκB pathways in neoplastic B-cells, as evidenced by accumulation of IκBα, a negative pathway regulator, and diminished p100/p52 processing, respectively. We and others have shown that CD40L induces NFκB and promotes resistance to spontaneous and drug-induced apoptosis in primary neoplastic B-cells. By contrast, CD40L-stimulated cells were fully sensitive to TAK-243 (IC50=50 nM). Furthermore, TAK-243 showed lower IC50 compared to bortezomib in DLBCL cell lines and primary samples, and induced accelerated and more pronounced ER stress and NFκB inhibition.We observed increased MYC mRNA transcript and protein expression in neoplastic B-cells following treatment with TAK-243. DLBCL cells expressing high baseline MYC levels (by protein expression) showed enhanced susceptibility to TAK-243-mediated apoptosis compared with MYC-low cells. Engineered expression of MYC sensitized OCI-LY3 cells to TAK-243, while siRNA-mediated downregulation of MYC resulted in decreased apoptosis. It has been suggested that MYC may sensitize cells to apoptosis through the extrinsic pathway. Indeed, we found that TRAIL ligand sensitized DLBCL cell lines to TAK-243.Thus, the UAE inhibitor TAK-243 induces ER stress, abrogates NFκB pathway activation and promotes apoptosis in DLBCL cell lines and primary neoplastic B-cells. Its in vitro activity compares favorably with bortezomib. Targeting UAE is a novel approach to disrupt the ubiquitin-proteasome system which may hold promise in therapy of B-cell malignancies. DisclosuresBerger:Takeda Pharmaceuticals International Co.: Employment.

Adar-Mediated Aberrant a-to-I RNA Editing Is Driven By 1q Amplification and Contributes to Proteasome Inhibitor Resistance in Multiple Myeloma

Multiple Myeloma (MM) is an incurable malignancy of plasma cells characterized by wide and remarkable genetic heterogeneity. The amplification of 1q identifies a class of patients characterized by a more aggressive disease course and poor prognosis. While previous research has identified several oncogenes with prognostic relevance located in 1q, the biological mechanisms underlying the poor clinical outcome associated with this alteration is still unclear.ADAR (Adenosine Deaminase Acting on RNA) is an enzyme responsible for A-to-I editing, a post-transcriptional process which contributes to transcriptome and proteome diversity by conversion of specific adenosines into inosines in both coding and non-coding transcripts. Such changes may affect RNA stability and regulation and introduce mutations in the corresponding proteins.Recent research has reported dysregulation of RNA editing in cancer, revealing its oncogenicity and clinical relevance. Since ADAR is located in 1q21, the critical amplified region in MM, we sought to investigate the implications of ADAR activity in MM.We have previously shown increased ADAR expression and A-to-I RNA editing in a small group of newly diagnosed patients with 1q21 amplification enrolled in the MMRF CoMMpass study (Lagana et al, ASH 2016), and that ADAR expression could differentiate prognosis. To further investigate and characterize the role of ADAR and RNA Editing in MM, we have analyzed 590 RNAseq samples from newly diagnosed patients in CoMMpass. ADAR copy number gain was observed in 111 samples (19%).ADAR expression was significantly higher in patients with 1q21 gain (p < 0.0001), supporting the role of copy number gain as a driver of ADAR expression. Moreover, patients with high expression of ADAR and no CN alterations, showed significant activation of Interferon pathways (Fig. 1A). This can be explained by the fact that one isoform of ADAR (p150) is regulated by an interferon-inducible promoter. Multivariate regression of ADAR expression against ADAR CN and STAT1 expression, as a measure of interferon signaling activation, showed that both ADAR CN and STAT1 expression were significant independent predictors of ADAR expression.To investigate the functional impact of ADAR activity, we implemented a computational pipeline to calculate sample-wise Alu Editing Index (AEI). A-to-I RNA editing targets predominantly Alu elements, repetitive sequences abundantly interspersed throughout the human genome, mostly within introns and untranslated regions (UTRs). AEI has been developed and validated as a global measure of RNA editing activity at the sample level (Bazak et al, Genome Res 2014). Our analysis in MM revealed that AEI could discriminate between good and poor prognosis in terms of both PFS and OS (p < 0.0001). Moreover, AEI could critically stratify patients with 1q21 gain in terms of PFS (p < 0.01) (Fig. 1B). This finding suggests that RNA editing can be an adverse prognostic factor independently of 1q21 gain.Patients with high AEI were characterized by down-regulation of immunoglobulin transcripts and of the Unfolded Protein Response (UPR) pathway. In particular, patients with high AEI exhibited down-regulation of key activators of UPR such as NFYC, ATF4 and XBP1, and of transcriptional targets of XBP1s, the active spliced form of XBP1. Decreased UPR and low XBP1s expression in pre-plasmablasts have been shown to induce resistance to proteasome inhibitors (PI) (e.g. Bortezomib) through de-differentiation of plasma cells (Leung-Hagesteijn et al, Cancer Cell 2013). Among patients who received Bortezomib-based first line therapy, we found that AEI was significantly higher (p<0.01) in patients whose best response was stable disease (SD) or progressive disease (PD) (23; 4.5%) compared to patients who had complete (CR) or stringent complete response (sCR) (118; 23%) (Fig. 1C). siRNA meditated knockdown of ADAR increased sensitivity to Bortezomib in MM cell lines with 1q amplification and high ADAR expression (OPM2), consistently with our in-silico findings.In conclusion, we propose a model of MM where ADAR-mediated RNA editing is driven by gain of 1q21 and interferon signaling and contributes to disease aggressiveness and resistance to PI-based therapy through down-regulation of UPR (Fig. 1D). [Display omitted] DisclosuresMadduri:Foundation Medicine, Inc.: Consultancy. Chari:Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Janssen: Consultancy, Research Funding. Cho:Agenus, Inc.: Research Funding; Genentech: Other: advisory board, Research Funding; Ludwig Institute for Cancer Research: Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Multiple Myeloma Research Foundation: Research Funding. Barlogie:Millenium Pharmaceuticals: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding. Jagannath:Merck: Consultancy; Medicom: Speakers Bureau; Celgene: Consultancy; Bristol-Meyers Squibb: Consultancy; Novartis: Consultancy; MMRF: Speakers Bureau. Dudley:Ecoeos, Inc.: Equity Ownership; Ayasdi, Inc.: Equity Ownership; Ontomics, Inc.: Equity Ownership; Personalis: Patents & Royalties; AstraZeneca: Consultancy; NuMedii, Inc.: Equity Ownership, Patents & Royalties; Janssen Pharmaceuticals, Inc.: Consultancy; GlaxoSmithKline: Consultancy.

Is Durability of Response Reported in Refractory CTCL: A Systematic Review

Introduction: Primary cutaneous T-cell lymphomas (CTCL) are a group of non-Hodgkin lymphomas derived from T-lymphocytes that infiltrate the skin. In refractory CTCL, treatments generally include systemic therapies in conjunction with skin-directed therapies. Numerous assessment criteria are used to evaluate the efficacy of different therapies administered to patients with CTCL. Robust treatment comparisons in CTCL are currently complicated by differences between studies in the efficacy outcomes reported. A systematic review (SR) was performed to summarise published evidence on the efficacy of treatments in patients with relapsed/refractory CTCL.Methods: A SR was performed in January 2017 to identify clinical studies published in the last 10 years enrolling >20 patients treated for refractory CTCL.Results: In total, 63 publications reporting on 54 unique studies and 21 different therapies for refractory CTCL were included in the SR. The majority of included studies (n=49) were single arm investigations. Five of the studies included were randomised controlled trials (RCT) comparing CTCL therapies. Two RCTs compared multiple doses of a single therapy (bexarotene or denileukin diftitox), two compared multiple therapies (interferon and methotrexate vs interferon and retinoids or brentuximab vs methotrexate or bexarotene), and one was a placebo-controlled trial of denileukin diftitox. Patient cohorts ranged in size from 21 to 216 patients and all patients had recurrent or refractory disease following ≥1 therapy for CTCL prior to the therapy for which data were extracted. The most frequently reported outcomes were objective response rate (ORR) reported in 82% of studies, complete response rate (80%), progression-free survival (52%), duration of response (48%), overall survival (24%) and time to tumour progression (20%) (Table 1). However, not all outcomes were reported for all therapies, outcome assessment criteria were not reported in all studies, and durable responses were rarely reported. In addition, there was substantial heterogeneity between studies in terms of patient characteristics, outcome assessment criteria, study design, and follow-up time. As such, a robust comparison of relative efficacy is not feasible. ORR was the most consistently reported outcome across studies and was reported for 19 different therapies in 44 different studies. The assessment criteria for ORR was reported in 15 studies. ORR is a useful measure of efficacy as it is directly attributable to drug effect and can be assessed in single-arm studies; however, as the time-point for assessment of ORR or the definition of ORR differed between studies or was not reported, the durability of response was difficult to interpret. The novel outcomes, objective global responses lasting at least 4 (ORR4) and 6 months (ORR6), were reported in three and two trials investigating the efficacy of brentuximab vedotin, respectively. ORR4 and ORR6 capture both the proportion of patients achieving a response and response duration as a single measurement and were 51-56% and 34%, respectively, following treatment with brentuximab vedotin. Health-related quality of life was investigated in only one study in which key endpoints, such as Skindex-29 symptom domains, were reported. Adverse events were reported following treatment with all therapies for refractory CTCL. However, there was a lack of consistency in the definition of the severity of adverse events reported.Conclusion: Although progress has been made towards the standardisation of response criteria in CTCL, the evaluation of durability of response to treatment remains inconsistent. The routine use of clearly defined efficacy outcomes in clinical trials is required to enable robust comparisons of therapeutic efficacy. [Display omitted] DisclosuresDalal:Takeda Pharmaceuticals International Co: Employment, Equity Ownership. Mitchell:DRG Abacus: Employment. McCloskey:DRG Abacus: Employment. Zagadailov:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Gautam:Takeda Pharmaceuticals International Co: Employment, Equity Ownership.

T Cell Versus T Cell; A Study of the Immune Checkpoint Landscape in Cutaneous T Cell Lymphoma

Introduction.Tumor infiltrating T cells (TILs) are capable of recognising tumor-specific mutations presented as peptides on MHC class I. Overexpression of immune checkpoint ligands on cancer cells, and the corresponding expression of the receptors on T cells, appears to be an important mechanism of immune evasion, exploited with success in immune checkpoint therapy. However, when the cancer cell is itself a T cell, the microenvironment interaction is likely to be more complex. To investigate this further we utilized the clonal nature of mycosis fungoides (MF), a primarily CD4 T cell cancer, by targeting the TCR receptor, differentiating between TIL and tumor in the microenvironment.Methods.43 patients with mycosis fungoides (MF) were prospectively consented to the study, and a skin biopsy and blood sample were obtained. The skin biopsy was macerated and digested with collagenase D, and a TCR V-beta clonogram was used to identify the tumor population. Flow cytometry was used to isolate five cell populations: peripheral blood CD4 and CD8, TIL CD4 and CD8, and tumor cells. The expression of a panel of immune checkpoint receptors (PD-1, TIM-3, LAG-3, Fas & TIGIT) and ligands (PD-L1, PD-L2, Galectin 9, Fas ligand) were measured using conjugated antibodies.Results.46 biopsies from MF patients with stage IA(n=6), IB(n=18), IIA(n=1), IIB(N=14), IIA(n=1) & IVA(n=3) were analysed with paired blood. The technique was effective in distinguishing tumour from TIL in 20 cases, isolating a population with CD7 downregulation, high forward and side scatter (indicative of larger and more complex cells) and a unique immune checkpoint profile (figure 1a & 1b). Tumour infiltrating lymphocytes of both CD4 and CD8 phenotype demonstrated significant overexpression of immune checkpoint receptors PD-1 (p < 0.0001), TIGIT (p < 0.0001) & fas (p < 0.0001), with an increased population of T regulatory cells in the CD4 cells (p < 0.001) and CD8 expressing high TIM-3 (p < 0.0001) compared to peripheral blood. While the TIL population was similar across the demographic, the tumour population demonstrated a significantly varied phenotype, with up and down-regulation of immune checkpoints and HLA-DR (figure 1c). Principal component analysis (figure 1d) revealed three tumour subtypes:1. High PD-L1, Fas, HLA-DR (n=4)2. High PD-1. Low PD-L1 (n=12)3. Low PD-1, PD-L1, Fas (n=3)These subtypes did not relate to disease type, stage, presence of large cell transformation or folliculotropic disease, though those with advanced disease had tumor phenotypes which differed more from the TILs. Tumor cells not only expressed MHC class I and HLA-DR, but upregulated these compared to peripheral blood (p < 0.001).Discussion.The TIL phenotype in MF is surprisingly homogeneous across the demographic, demonstrating upregulation of immune checkpoint receptors and an exhaustion phenotype seen in other cancers. However, the tumor cells did not show consistent upregulation of immune checkpoint ligands, instead their phenotype across patients was heterogeneous with both up and down-regulation. Expression of immune checkpoint ligands by the tumor cells may have negative growth consequences for both tumor cells as well as the immune system, particular since tumor T cells often express immune checkpoint receptors. We did not see concurrent high expression of PD-L1 and PD-1 on tumor cells, which would be expected to be detrimental to tumor survival. Immune checkpoint ligand over-expression is unlikely to be the main method of immune evasion in mycosis fungoides, and as a result, immune checkpoint inhibition may have unexpected responses in this disease.Legends.Figure 1a. TCR V-beta antibody used to separate three CD4 populations (red: peripheral blood CD4, green: CD4 TIL, blue: tumor) and compare HLA-DR expression.Figure 1b. tSNE clustering of cells in the tumour microenvironment, demonstrating clear separation of three populations - tumor, CD4 TIL and CD8 TIL. Coloring by expression of PD-L1 (red: high, blue: low).Figure 1c. Heatmap of immune checkpoint markers (red: high, blue: low) in the tumor cell population only. This demonstrates the heterogeneity, particularly in PD-1, Fas, PD-L1 and HLA-DR expression.Figure 1d. Principal component analysis demonstrating the observation that TIL expression of immune checkpoints are similar between patients (green and blue), whereas tumor expression of these markers (red) can vary markedly and cluster. [Display omitted] DisclosuresScarisbrick:NHS: Employment; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Mallinckrodt: Consultancy, Honoraria; Innate Pharma: Consultancy; Actelion: Consultancy.

Incremental Financial and Health-Related Burden of Novel Drugs Used in the Treatment of Relapsed and/or Refractory Multiple Myeloma (RRMM): A Meta-Analysis of Phase III Randomized Controlled Trials (RCTs)

Background:The remarkable progress in the outcomes of RRMM in the last 2 decades with the introduction of novel therapies have come with significant additional cost and adverse events (AEs). We quantified the incremental efficacy, AEs, and cost of newly approved anti-RRMM drugs by conducting a meta-analysis of pivotal phase III Randomized Controlled Trials (RCTs) used for registration.Methods: A systematic search of MEDLINE, SCOPUS and Cochrane databases was performed to identify RCTs leading to approvals of new agents for the treatment of RRMM from January 1, 1999 to July 1, 2017. Abstracts presented within the last four years at major hematology and oncology meetings were also identified. Hazard Ratios (HR) for Progression free Survival (PFS), Overall survival (OS), and Odds Ratios (OR) for all grade III/ IV AEs along with the respective 95% Confidence Intervals (CI) reported in the included studies were weighed using a generic inverse variance approach and were pooled using random effects model meta-analyses using RevMan 5.3 software. Incremental costs were calculated from 1) drug prices listed in Micromedex Redbook and 2) costs of management of AEs and health services costs based on published literature (adjusted for inflation to match 2017-US dollar value) for experimental compared to control arms.Results: Sixteen RCTs (n=8,683) involving 14 different treatment options qualified for inclusion (Table 1). Pooled analyses demonstrated a significant improvement in OS (HR 0.74; 95% CI: 0.62-0.89;p=0.002) and PFS (HR 0.55; 95% CI 0.48-0.64; p<0.00001) with the use of newly approved drugs or combinations compared to control arm. However, there was substantial increase in the risk of adverse events with the use of new-targeted agents and combination compared to the controls (OR for Grade III-IV toxicities 1.56, 95% CI: 1.29-1.88, p <0.00001). (Fig 1). There was no difference in treatment discontinuation and toxic deaths between the two groups. The median incremental drug price for experimental agents was $ 13,588/patient/month compared to the control arm. The new agents or combinations led to incremental cost of care by a median of $ 133,914/patient/month.Conclusion: Despite improvements in efficacy, newly approved drugs for RRMM are associated with increased rates of AEs, and add exponentially to financial burden, exceeding by far the acceptable willingness-to-pay threshold of most developed countries. Toxicities and costs are highest for immunomodulators and are likely to increase when the agents are used in unselected populations after the approvals leading to heavier health-related and financial burden to society. Strategies to lower cost of novel therapies, and better predictive tools to select patients are imperative for sustainability of RRMM treatments. [Display omitted] DisclosuresDhakal:Celgene: Honoraria. Chhabra:Cardinal Health: Honoraria; Incyte: Honoraria. Hari:Janssen: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Honoraria, Research Funding; Takeda Pharmaceuticals: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy; Sanofi: Honoraria, Research Funding.

Single-Cell RNA Sequencing Reveals Distinct Transcriptomic Profiles of Multiple Myeloma with Implications for Personalized Medicine

Multiple Myeloma (MM) is a heterogeneous malignancy with genomic translocations and mutations conferring varying levels of risk of recurrence and confounding treatment options across patients. Intra-tumor clonal heterogeneity has also been observed which further complicates treatment. We hypothesize that the use of Single-Cell RNA sequencing (scRNA-seq) can elucidate the complex clonal transcriptomic structure of individual patients' disease, and can be used in concert with other genomic techniques to better characterize and treat patients on a personalized level.Methods:Individual CD138+ tumor cells were isolated and barcoded from bone marrow aspirates from eight relapsed myeloma patients using the 10x Genomics Chromium microfluidics platform and V2 chemistry for deep sequencing using an Illumina HiSeq 2500 machine. The 10x Genomics Chromium platform allows for large-scale simultaneous cellular sequencing which facilitates a more robust analysis of cellular populations than previous technologies. The number of cells analyzed ranged from 393 to 3,359 per sample, depending on sample cell suspension density. We used 10x Genomics Loupe software to visualize scRNA-seq data via TSNE dimensional reduction plots. Cells were then clustered via Graph-Based, K-Means, or custom gating methods.Results:Analysis of bulk CD138+ whole-exome sequencing data has identified previously known mutational drivers and allowed estimation of cellular sub-populations (Fig. 1a) using copy-number alteration and mutational data via Phylo-WGS (Deshwar et al, Genome Biol 2015). Integrative analysis of bulk RNA-seq data revealed concordant activation of relevant pathways (e.g. MAP kinase) in patients with corresponding somatic mutations. On an individual patient level, we were able to process scRNA-seq data to visualize clusters of single cells with transcriptional similarity using t-distributed stochastic neighbor embedding (t-SNE) plots. We identified pathway activation in individual scRNA-seq clones via differential expression analysis and Signaling Pathway Impact Analysis (SPIA), in concordance with known somatic mutations (Fig. 1b). Genomic drivers and stages of differentiation in cell subsets (plasma cell vs immature B cell-like plasma cells) within a given patient were also revealed by distinct expression profiles (Fig. 1c). We utilized open-source drug repurposing tools with the RNA signature of individual clones to interrogate transcriptional signatures of drugs in public databases such as LINCS. The use of such drug repurposing methods will be effective in generating more targeted personalized therapies, utilizing cluster specific expression profiles. This is especially useful for cellular populations which do not contain a known target based on DNA mutational analysis. In a pooled analysis of all samples, each patient sample remarkably formed its own spatially distinct cluster of cells (Fig. 1d). These data suggest that relapsed myeloma patients form transcriptionally distinct clones, and myeloma therapy may need to be highly individualized to overcome clonal heterogeneity. scRNA-seq is a powerful tool which can be utilized alone, or in harmony with other sequencing technologies and genomic techniques, to improve characterization and treatment of myeloma patients in a clone specific manner. [Display omitted] DisclosuresMadduri:Foundation Medicine, Inc.: Consultancy. Chari:Novartis: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc.: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Amgen: Honoraria, Research Funding. Cho:Genentech: Other: advisory board, Research Funding; Bristol Myers-Squibb: Other: advisory board, Research Funding; Ludwig Institute for Cancer Research: Research Funding; Multiple Myeloma Research Foundation: Research Funding; Agenus, Inc.: Research Funding. Barlogie:Celgene Corporation: Consultancy, Research Funding; Millenium Pharmaceuticals: Consultancy, Research Funding. Jagannath:MMRF: Speakers Bureau; Medicom: Speakers Bureau; Merck: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Bristol-Meyers Squibb: Consultancy. Dudley:NuMedii, Inc.: Equity Ownership, Patents & Royalties; Janssen Pharmaceuticals, Inc.: Consultancy; Ayasdi, Inc.: Equity Ownership; Ecoeos, Inc.: Equity Ownership; Ontomics, Inc.: Equity Ownership; Personalis: Patents & Royalties; AstraZeneca: Consultancy; GlaxoSmithKline: Consultancy.

Immunological Correlates of Treatment with the CTLA-4 Inhibitor Ipilimumab in Patients with Refractory Myelodysplastic Syndromes (MDS)

Introduction: Failure of hypomethylating agents (HMAs) in patients with myelodysplastic syndromes (MDS) is associated with dismal survival and there are no currently approved therapies in this setting. We recently presented the results of a multi-center phase 1b study of the immune checkpoint blocker (ICB) ipilimumab given as monotherapy after HMA failure in 29 MDS patients. Eight doses of 3mg/kg administered in induction and maintenance phases were safe and well tolerated. Very limited data has been reported on immunologic correlates of response after ICB therapy among patients with hematologic malignancies. Here we present a detailed analysis of the immunological correlates of achieving clinical benefit with ipilimumab.Methods: We defined patients who achieved marrow complete response (mCR) or prolonged stable disease (PSD) for ≥46 weeks as having achieved meaningful clinical benefit (MCB) for analyses of immunologic studies. 11 patients were treated during the dose escalation phase (6 patients at 3mg/kg dose and 5 patients at 10 mg/kg), while another 18 patients were treated in the dose expansion part at the 3 mg/kg dose. Correlative immunologic studies were performed on mononuclear cells from both peripheral blood (PBMC) and bone marrow (BMMC) samples that were collected at baseline and at pre-determined timepoints after ipilimumab treatment (after induction cycle 2 and 4; after each maintenance cycle 1-4, and end of treatment or study discontinuation). Correlative assays included immunophenotyping of PBMCs and BMMCs by multicolor flow cytometry, as well as high throughput sequencing of the T cell antigen receptor (TCR) repertoire. T cell percentages, absolute numbers, and ratios were summarized by time point for patients and overall for healthy controls using descriptive statistics (mean, SD). Differences in log-transformed T cell values were compared between healthy controls and patients at pre-treatment using two-sample t tests. Differences from pre-treatment to cycle 2 and cycle 4 were estimated for patients and tested for positive or negative changes with paired t tests. Analyses were completed using R version 3.3.2.Results: MCB was observed in 28% of patients, 2 (7%) patients with mCR and 6 (21%) patients with PSD, respectively. Immunologic studies were performed on 16 patients who had samples available from at least two time points. Compared to solid tumor literature, we did not observe a correlation between early dynamic changes in serial absolute lymphocyte count and probability of achieving MCB. Expression of co-stimulatory molecules (ICOS - Inducible T Cell CO-Stimulator) and co-inhibitory molecules (PD-1 and CTLA-4) showed that a higher fraction of CD4+ and CD8+ T-cells in PB expressed ICOS in patients who achieved MCB following ipilimumab treatment (P=0.05 and P=0.01, respectively, Figure 1]. Similarly, expression of the immune co-inhibitory receptor CTLA-4 followed a similar pattern showing a trend of increased expression in both CD4+ and CD8+ T-cells (average increase of 6% and 2.4% respectively, P=ns.) over the first 4 cycles of ipilimumab treatment among patients who achieved MCB, followed by a gradual decrease. In contrast to CTLA-4, there was a wide range in frequencies of CD4+ and CD8+ T-cells expressing PD-1 in both patients and HC and PD-1 levels remained stable throughout ipilimumab treatment suggesting that the lack of T-cell activation in non-responders was not due to increased expression of other co-inhibitory molecules. There was a significantly higher frequency of CD3+CD4+Foxp3+ and activated effector Tregs (eTregs) prior to treatment which persisted throughout the examined time points and neither their levels, nor the CD8 T-cell to Treg (CD8/Treg) ratio were impacted by ipilimumab treatment.Conclusions: These data represent, to our knowledge, the first detailed analysis of immunologic correlates of response to ICB therapy in MDS patients. Our data suggest that ICOS expression is positively correlated with achievement of MCB with single agent ipilimumab in patients with MDS and should be further explored as a biomarker for response. The results of the high throughput sequencing of the T cell antigen receptor (TCR) repertoire are currently being analyzed and will be presented at the meeting. [Display omitted] DisclosuresSmith:Celgene: Other: DSMB member; Jazz: Membership on an entity's Board of Directors or advisory committees. Zeidner:Tolero Pharmaceuticals: Honoraria; Agios: Honoraria; Tolero Pharmaceuticals: Research Funding; Takeda: Research Funding; Merck: Research Funding; Celgene: Honoraria. Frattini:Astellas: Honoraria; Lin Bioscience: Consultancy. Levy:Actinium Pharmaceuticals: Equity Ownership; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; Takeda: Consultancy, Speakers Bureau. Zeidan:AbbVie, Otsuka, Pfizer, Gilead, Celgene, Ariad, Incyte: Consultancy, Honoraria; Takeda: Speakers Bureau; Otsuka: Consultancy.

Phase II Trial of Natalizumab (Tysabri®) with Corticosteroids As Initial Treatment of Gastrointestinal Acute Graft Versus Host Disease

Background: Graft-vs.-host disease (GVHD) is caused by allorecognition of host antigens by tissue-specific donor lymphocytes. We have previously demonstrated that α4ß7 integrin is upregulated on both naïve and memory T cell subsets in patients who subsequently developed intestinal acute GVHD (Chen, BMT 2013). This integrin interacts with endothelial cell adhesion molecules and mediates homing of lymphocytes to the gastrointestinal (GI) tract. Natalizumab (Tysabri®, Biogen Inc.) is a selective adhesion molecule inhibitor, acting directly against the α4 subunit of α4 integrins. Given the poor outcomes of GI GVHD when treated with corticosteroids alone, we initiated a phase II study to determine the safety and efficacy of natalizumab in combination with corticosteroids for the initial treatment of acute GI GVHD.Methods: Subjects with new-onset, acute GVHD of the lower GI tract were eligible to participate. Other organ involvement was permitted but not required. Biopsy confirmation of GI GVHD and exclusion of infectious causes of diarrhea were required. Natalizumab was given at a dose of 300 mg IV, with a second dose permitted 4 weeks later in the event of an incomplete response. An initial corticosteroid dose of 2 mg/kg of methylprednisolone was suggested, but dosing and dose reductions were at the discretion of the treating physician. Steroids could be initiated up to 7 days prior to the initiation of natalizumab while biopsy and infectious disease specimens were being analyzed. The primary endpoint was GVHD-free survival rate 56 days from enrollment. Secondary endpoints included response rates at day 28 and 56, steroid dose at days 28, 56, 100, 180 and 365, incidence of systemic infections at day 56 and 180, and overall survival.Results: 18 subjects were enrolled and received at least one dose of natalizumab. Subjects had a median age of 63.5 years, and 12 were male. Fifteen had a non-myeloablative transplant, 12 had a matched unrelated donor, and all had received PBSCs. All subjects had newly diagnosed acute GI GVHD at time of enrollment; 4 had concomitant cutaneous and 1 had concomitant hepatic involvement. Stage 1, 2, 3, and 4 GI involvement was noted in 7, 4, 4, and 3 subjects respectively, corresponding to overall grade II GVHD in 7 and grade III in 11 subjects. Sixteen patients had data for response assessment. The day 56 GVHD-free survival rate was 37.5%. The overall response rate (PR+CR) for GI GVHD at day 28 and day 56 was 75% and 62.5% respectively. All surviving subjects except one achieved steroid dose reduction by day 56, with a median reduction of 61.6%. Three patients experienced treatment-related toxicity possibly related to natalizumab; one subject had grade 2 hepatic toxicity, one had grade 4 encephalopathy, and one developed hepatic failure. Three subjects have developed chronic GVHD. Four subjects had documented infections with bacteria (2), yeast (1), and viral pathogens (1- polyomavirus). Six subjects had measurable JC viremia prior to initiation of natalizumab and 8 subjects had measurable JC viremia at day 56, although only 1 subject had a viral load greater than 1000 copies/ml. No subject has developed JC-associated disease. Seven subjects have died. Causes of death include GVHD (2), disease relapse (2), and organ failure (3). With a median follow-up of 180 days, the 6 month overall survival was 52% (95% CI 25-74%).Conclusion: Natalizumab in combination with corticosteroids as initial treatment of acute gastrointestinal GVHD is feasible and well tolerated. These early results suggest a higher rate of response at day 56 than is typically observed with steroids alone. Inhibition of T-cell homing to the GI tract may improve GI GVHD response and potentially mitigate progression of acute GVHD. DisclosuresKoreth:Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding. Armand:Bristol-Myers Squibb: Consultancy, Research Funding; Tensha: Research Funding; Affimed: Research Funding; Sequenta/Adaptive: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Roche: Research Funding; Genmab: Consultancy; Pfizer: Consultancy, Research Funding; Infinity: Consultancy; Otsuka: Research Funding; Sigma Tau: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Cutler:Pfizer: Consultancy; Pharmacyclics: Consultancy; Kite: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

T Follicular Regulatory (TFR) Cell Deficiency during Chronic Graft-Versus-Host Disease Is Improved By Low-Dose IL-2 Therapy

Background: Chronic Graft versus Host Disease (cGVHD) reflects the inability to achieve immune tolerance after allogeneic stem cell transplantation (allo-SCT) resulting in the persistence of allo and auto-reactive T cells and B cells. We previously showed that regulatory T cells (Treg) are decreased in patients with cGVHD and may be rescued by low dose IL-2 therapy resulting in improved Treg number and survival and clinical improvement of cGVHD symptoms. More recently, we showed that circulating T follicular helper cells (cTFH) have increased function during cGVHD, thus stimulating B cells to produce auto and allo-antibodies involved in cGVHD. T follicular regulatory cells (TFR) regulate the germinal center (GC) reaction and suppress B cell differentiation and immunoglobulin secretion. TFR reside primarily in lymphoid tissues but also circulate in small numbers. In the present study we characterized the numbers and function of blood CD3+CD4+CD45RA-CD25hiFoxP3+CXCR5+ TFR in healthy donors (HD; N=18) and allo-SCT patients (N=64).Results: Patients undergoing allo-SCT showed a significant reduction of circulating TFR (cTFR) compared to HD (median: 0.08% vs 0.34% of CD4+T respectively, p<10-4). Moreover, patients with active cGVHD (N=37) had significantly lower cTFR frequency compared to patients with no cGVHD (N=15) (median: 0.034 vs 0.18% of CD4+, p=0.0012) and resolved cGVHD (N=12) (median: 0.034 vs 0.14, p=0.02). Interestingly, cTFR values tended to be negatively associated with cGVHD grade (median cTFR %: 0.11 vs 0.029, p=0.004 for mild vs moderate; 0.11 vs 0.012, p=0.005 for mild vs severe; 0.029 vs 0.012, p=0.2 for moderate vs severe). The numeric reduction of cTFR in cGVHD patients appears to be a consequence of decreased survival as attested by lower BCL-2 expression (median: 1831 vs 1939, p=0.037 in active cGVHD and without cGVHD, respectively). This favors the survival of TFH effector cells (CD4+CD45RA-CXCR5+ excluding CD25hiFoxP3+) as measured by the ratio of expression of BCL-2 in effectors/regulators in active compared to no cGVHD (BCL-2 E/R ratio: 1.26 vs 1.14, p=0.0013 respectively). Similar to tonsil TFR, blood TFR exhibit suppressive functions in in vitro assays. Purified TFR were co-cultured with TFH and memory B cells at different ratios. The addition of TFR was associated with a significant reduction of plasmablast (PB) generation (CD27hiCD38hiCD19+ cells) (Figure 1A, t-test: p=0.0087) and IgG production (Figure 1B, t-test: p=0.0087). Human cTFR in vivo display similar suppressive function toward B cells, as analysis of active cGVHD patients revealed an inverse correlation between TFR and PB frequency in the peripheral blood (Spearman test: r = -0.49, p=0.003) (Figure 1C). As IL-2 has been shown to enhance regulatory T cells, we assessed its impact on the GC reaction in vitro . Purified TFH and memory B cells from HD were co-cultured at different doses of IL-2 from 0 to 100 IU/mL, simulating a germinal center reaction in vitro . Low dose IL-2 starting at 10 IU/mL was associated with a suppressive effect on PB generation and IgG production that peaked at 50 IU/mL reflecting the high expression level of IL-2 high affinity receptor (CD25). We also evaluated the capacity of IL-2 to restore in vivo cTFR:cTFH ratio using mass cytometry (CyTOF) in a cohort of patients receiving IL-2 (1x106 IU/M2/day) for 12 weeks as previously described (Koreth et al, Blood 2016). We observed an improvement of blood cTFR:cTFH ratio over the course of IL-2 therapy, with a maximum ratio observed at 2 weeks, and subsequently decreasing to reach baseline values after stopping IL-2 (Figure 2).Conclusion: Our results confirm that blood TFR exert a suppressive function as evidence by an inverse correlation with PB and in vitro suppression of memory B cell Ig production. This suppressive subset is deficient in patients with cGVHD due to altered TFR survival, thus favoring imbalance toward TFH effectors which may promote a GC reaction in vivo resulting in PB and memory B cell pathogenic Ig production. By enhancing TFR, low-dose IL-2 therapy promotes effective control of the GC reaction. [Display omitted] DisclosuresForcade:Neovii: Other: Travel grant. Koreth:Millennium Pharmaceuticals: Research Funding; Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Nikiforow:Kite Therapeutics: Membership on an entity's Board of Directors or advisory committees. Armand:Roche: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Affimed: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sigma Tau: Research Funding; Otsuka: Research Funding; Pfizer: Consultancy, Research Funding; Infinity: Consultancy; Tensha: Research Funding; Genmab: Consultancy. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Characteristics and Outcome of Patients with Acute Myeloid Leukemia and t(6;9)(p22;q34)

Background: Acute myeloid leukemia (AML) with t(6;9)(p22;q34) is a distinct entity in the 2016 World Health Organization classification of myeloid neoplasms and accounts for a small subgroup (1-2%) of AML patients. A substantial proportion of AML patients with t(6;9) have a concomitant FLT3 -ITD. While outcomes are dismal with intensive chemotherapy, limited evidence suggests allogeneic hematopoietic stem cell transplantation (allo-HSCT) may improve survival if applied early during first complete remission (CR). However, neither prospective nor larger retrospective cohort studies are available to support these results which are derived from small series.Aims: To characterize AML patients with t(6;9)(p22;q34) and compare outcomes with different treatment strategies.Methods: We retrospectively studied 123 AML patients with t(6;9)(p22;q34) (median age at diagnosis, 46 years; range, 16-76 years) treated between 1989 and 2016 within fourteen study groups/institutions of the US and Europe. Standard statistical methods were applied.Results: Median white blood cell count at diagnosis was 20/nl (range, 0.5-274/nl) and platelets 52.5/nl (range, 9-332/nl). The type of AML was de novo in 111 (90%), secondary after myelodysplastic syndrome/myeloproliferative neoplasm in 9 (8%), and therapy-related in 3 (2%) of the patients.Fifty-four patients (44%) were female. Cytogenetic analysis revealed additional abnormalities in 25 (20%) patients, most frequently ≥3 (n=15) abnormalities including +8 (n=2), +13 (n=1) or combined +8/+13 (n=4). A total of 76 patients (62%) had FLT3 -ITD mutation testing, and 48 (63%) of those patients harbored FLT3 -ITD mutations.Data on response to intensive anthracycline-based induction therapy were available in 121 patients. CR was achieved in 79% (n=95) with 16/95 (17%) requiring an intensive salvage treatment cycle with HiDAC. The CR rate in FLT3 -ITD positive patients was 88% (n=42/48) as compared to 68% (n=19/28) in FLT3 -ITD negative patients (p=0.07). An allo-HSCT was performed in 75 (61%) patients, of whom 51 patients were transplanted in first CR after induction therapy. Additionally 6 patients achieved first CR after salvage chemotherapy and went on to allo-HSCT. Overall, the majority of patients underwent allo-HSCT in first (n=57) or second (n=9) CR; another 9 patients received allo-HSCT with active disease. Type of donor was matched-related in 31, matched-unrelated in 31, haplo-identical in 8, cord blood in 4, and unknown in 1 of the 75 patients, respectively. The majority of patients (72%) received myeloablative conditioning, including TBI in half of them. The proportion of patients receiving an allo-HSCT increased over the time.Median follow-up for the entire cohort was 61 months (95%-CI, 47-83%). Five-year overall survival (OS) was 36% (95%-CI, 28-47%). Five-year relapse-free survival (RFS) and OS were 50% (95%-CI, 36-68%) and 55% (95%-CI, 41-74%) in patients proceeding to allo-HSCT in first CR after induction therapy (n=51), as compared to 8% (95%-CI, 3-24%; p<0.001) and 18% (95-CI, 9-36%; p<0.001), respectively, in those who received consolidation chemotherapy (n=44). Of note, OS rates after allo-HSCT were comparable if performed in CR1 (56%; 95%-CI, 43-73%; n=57), CR2 (58%; 95%-CI, 30-100%; n=9) or active disease (53%; 95%-CI, 28-100%; n=9; p=0.91). In subgroup analysis FLT3 -ITD positivity had no prognostic impact on OS, either in the total analyzed cohort (p=0.11), or in those patients proceeding to allo-HSCT (p=0.37). Similarly, additional chromosomal abnormalities had no prognostic impact on OS in the afore mentioned cohorts (p=0.92; p=0.83; respectively).Conclusions: Our cohort of AML patients with t(6;9)(p22;q34) showed a high CR rate after intensive induction therapy; however, treatment with chemotherapy alone resulted in dismal survival outcomes. Those patients who proceeded to allo-HSCT achieved very encouraging OS rates, regardless of FLT3- ITD status, additional cytogenetic abnormalities or timing of transplant, suggesting that transplant should be standard of care when possible for these patients. DisclosuresKayser:Novartis: Honoraria, Speakers Bureau; TEVA: Honoraria. Perl:Pfizer: Other: Advisory Board; Novartis: Other: Advisory Board; Actinium Pharmaceuticals: Other: Scientific Advisory Board; Arog Pharmaceuticals: Consultancy; Seattle Genetics: Other: Advisory board; Daiichi Sankyo: Consultancy; Asana Biosciences: Other: Scientific advisory board; Astellas: Consultancy. Mayer:Eisai: Research Funding; Novartis: Research Funding. Walter:ADC Therapeutics: Research Funding; Aptevo Therapeutics: Research Funding. Stone:Ono: Consultancy; Celgene: Consultancy; Fuji Film: Consultancy; Arog: Consultancy; Pfizer: Consultancy; Novartis: Consultancy; Abbvie: Consultancy; Amgen: Consultancy; Agios: Consultancy; Astellas: Consultancy; Jazz: Consultancy; Sumitomo: Consultancy. Levis:Daiichi Sankyo, Inc.: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; FujiFilm: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Astellas Pharma Us: Consultancy, Research Funding.

Utility of Clinical-Grade Sequencing of Relapsed Multiple Myeloma Patients; Interim Analysis of the Multiple Myeloma Research Foundation (MMRF) Molecular Profiling Protocol

Utility of Clinical-Grade Sequencing of Relapsed Multiple Myeloma Patients; Interim Analysis of the Multiple Myeloma Research Foundation (MMRF) Molecular Profiling Protocol

Treatment Patterns of Hypomethylating Agents in Older (≥65 Years) Acute Myeloid Leukemia Patients Who Did Not Receive Intensive Chemotherapy: A Retrospective Claims Database Analysis

BACKGROUND: Acute myeloid leukemia (AML) and therapy for patients with AML represent a striking example of age-related outcome disparity in oncology. Older patients experience shorter overall survival, have usually more comorbidities, and are at greater risk for treatment-related mortality. Consequently, a substantial portion of older patients are considered “unfit” for standard intensive chemotherapy and are offered less intensive regimens or best supportive care. In recent years, hypomethylating agents (HMAs), namely azacitidine (AZA) and decitabine (DEC), have emerged as standards of care for patients with AML unfit for intensive chemotherapy. The objective of this study was to identify and describe HMA utilization in older patients with AML (≥65 years) not receiving intensive chemotherapy in a real-world claims database.METHODS: AML patients were retrospectively identified in the MarketScan® Research Database (Truven Health) including both Commercial and Medicare Supplemental claims databases between 01/2011 to 07/2016. Patients ≥ 65 years old with a minimum of two claims with an AML diagnosis, ≥ 1 bone marrow procedure (the first AML diagnosis following the bone marrow diagnosis procedure was defined as the index date), and ≥ 12 months of continuous eligibility (baseline period) prior to the index date were included. Exclusion criteria were the following: ≥ 1 diagnosis for AML relapse or remission on or before the bone marrow diagnosis procedure, ≥ 2 diagnoses for another blood cancer, ≥ 1 allogeneic hematopoietic stem cell transplantation before the bone marrow diagnosis procedure, or ≥ 1 claim for intensive chemotherapy at any time. We recorded the percentages of HMA usage (AZA and DEC) among patients treated with ≥ 1 antineoplastic agent (excluding hormonal therapy agents) overall and in first and second lines of therapy, and treatment duration (i.e., cumulative duration of AZA- or DEC-based lines of therapy) along with the corresponding number of 28-day treatment cycles obtained by dividing the treatment duration by 28. In patients using HMAs in first- or second-line therapy, the percentages of monotherapy and blood transfusion use were tabulated and the proportion of patients who initiated a subsequent line of therapy was recorded.RESULTS: After applying the selection criteria, 1,492 patients were identified for inclusion in our study population. Mean (standard deviation [SD]) patient age was 76.8 (7.0) years, 36.6% were ≥ 80 years old, and 61.0% were male. The mean (SD) Quan-Charlson Comorbidity Index (CCI) was 3.4 (2.4), with 33.5% of patients having a prior diagnosis of myelodysplastic syndrome. Among 610 (40.9%) patients treated with an antineoplastic agent, 449 (73.6%) patients received HMAs at any time during the observation period: 250 (55.7%; Table 1) of them used AZA (mean [SD] age = 77.3 [6.4] years; 59.6% male; mean [SD] CCI = 3.2 [2.3]) and 230 (51.2%; Table 1) used DEC (mean [SD] age = 76.5 [6.3] years; 60.0% male; mean [SD] CCI = 3.4 [2.4]). Mean (SD) total treatment duration was 134.8 (150.6) days with AZA and 139.8 (128.6) days with DEC, which is equivalent to a mean (SD) number of 28-day treatment cycles of 4.8 (5.5) for AZA and 4.9 (4.6) for DEC (Table 1). Among the 436 (71.5%) patients that received HMAs in first line, 232 (53.2%) used AZA, 204 (46.8%) used DEC, 395 (90.6%) used them in monotherapy, 379 (86.9%) had ≥ 1 blood transfusion during the first line of therapy, and 91 (20.9%) initiated a second line of therapy (Table 1). Of 120 (19.7%) patients with a second-line therapy, 65 (54.2%) received HMAs. Among them, 28 (43.1%) used AZA, 39 (60.0%) used DEC, 38 (58.5%) used them as monotherapy, 54 (83.1%) had ≥ 1 blood transfusion during the second line of therapy, and 10 (15.4%) patients proceeded to a third line of therapy (Table 1).CONCLUSION: This study confirms that HMAs are commonly used in older (≥65 years) patients with AML who did not receive intensive chemotherapy, following National Comprehensive Cancer Network guideline recommendations. Utilization and treatment patterns were similar between both HMAs. A large proportion of patients received blood supportive products during their therapy and few of them initiated a subsequent line of therapy suggesting the need for additional treatment options. Additional studies with detailed evaluation of HMA use (including sequencing) are needed to further evaluate the important unmet need in the management of older patients with AML. [Display omitted] DisclosuresSundaram:Johnson & Johnson, LLC: Equity Ownership; Janssen: Employment. Lafeuille:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd: Research Funding; Janssen Scientific Affairs, LLC: Research Funding. Hoehn:Johnson & Johnson, LLC: Equity Ownership; Janssen: Employment. Emond:Janssen Scientific Affairs, LLC: Research Funding. Romdhani:Janssen Scientific Affairs, LLC: Research Funding. Lefebvre:Janssen Scientific Affairs, LLC: Research Funding.

Optimizing Checkpoint Blockade As a Treatment for Relapsed Hematologic Malignancies after Allogeneic Hematopoietic Cell Transplantation

Optimizing Checkpoint Blockade As a Treatment for Relapsed Hematologic Malignancies after Allogeneic Hematopoietic Cell Transplantation

Managing Acute Myeloid Leukemia in Older Patients: Are We Making Progress? An Analysis of Treatment Patterns and Healthcare Resource Utilization

▪BACKGROUND: Despite advances in the understanding of disease pathogenesis, improved treatment strategies, and evidence of better survival with treatment, a large proportion of older patients (pts) with acute myeloid leukemia (AML) remains untreated and has poor outcomes. The aim of this study is to describe current treatment patterns in pts with AML aged <60, 60-64, and ≥65 years (yrs), and assess healthcare resource utilization (HRU) in pts with AML aged <65 and ≥65 yrs in a real-world claims database.METHODS: AML pts were retrospectively identified in the MarketScan® Research Database (Truven Health) including both Commercial and Medicare Supplemental claims databases between 01/2011 to 07/2016. Pts with ≥2 AML diagnoses, ≥1 bone marrow procedure (the first AML diagnosis following the procedure was defined as the index date), and ≥12 months of continuous eligibility (baseline period) prior to the index date were included. Exclusion criteria were ≥1 diagnosis for AML relapse or remission on or before the bone marrow procedure, ≥2 diagnoses for another blood cancer, and ≥1 allogeneic hematopoietic stem cell transplantation before the bone marrow diagnosis procedure. Furthermore, pts aged ≥65 yrs at the index date were excluded if they had ≥1 claim for intensive chemotherapy at any time. The observation period spanned from index date up to the first of health plan disenrollment or the end of data availability. To accommodate different age cut-offs in accordance with National Comprehensive Cancer Network treatment guidelines, top treatments (excluding hormonal therapy agents) received post-AML diagnosis were reported in pts aged <60, 60-64, and ≥65 yrs. Listings that lacked codes for specific chemotherapy agents, but offered adequate International Classification of Diseases codes were included and reported as “unknown antineoplastic agents”. HRU, including days of hospital stay, days with outpatient services, days with emergency room visits, days with hospice care, and days with durable medical equipment use, was evaluated in pts <65 and ≥65 yrs old. To account for varying length of follow-up among pts, HRU was reported per patient per month.RESULTS: After applying the selection criteria, 2,954 AML pts were identified: 1,492 pts ≥65 yrs old (mean [SD] age: 76.8 [7.0]; 36.6% were ≥80 yrs old; mean [SD] follow-up: 212 [255] days) and 1,462 pts <65 yrs old (mean [SD] age: 51.8 [11.5]; mean [SD] follow-up: 350 [350] days), including 468 (32.0%) aged 60-64 yrs and 994 aged <60 yrs. Pts <60 yrs old received the most therapeutic interventions (i.e., ≥1 antineoplastic agent, ≥1 blood transfusion, or ≥1 hematopoietic stem cell transplant), followed by pts 60-64 and pts ≥65 yrs old (Fig. 1). Type of antineoplastic agents (excluding hormonal therapy agents) received varied by age group with “other antineoplastic agent” (other than HMAs and excluding hormonal therapy agents) and “unknown antineoplastic agent” more commonly used in younger pts. Hypomethylating agents (HMAs) were more commonly used in older pts (Fig. 1). In pts aged <60, 60-64, and ≥65 yrs treated with ≥1 HMA, azacitidine was given in 56.2%, 59.8%, and 55.7% of pts and decitabine was given in 50.5%, 47.1%, and 51.2%, respectively. In pts <60 yrs who received “other antineoplastic agents”, tretinoin (31.2%), cytarabine (30.6%), and arsenic trioxide (22.4%) were most commonly used. Pts 60-64 yrs old received cytarabine (30.7%), hydroxyurea (20.5%), and tretinoin (17.3%) as most common agents. Pts ≥65 yrs old received most commonly hydroxyurea (53.4%) and cytarabine (14.3%). Post-AML diagnosis, pts ≥65 yrs old had half the monthly all-cause average number of inpatient days compared to pts <65 yrs old (mean [SD]: 3.74 [5.28] vs. 7.74 [7.14]). Differences in the number of days with outpatient services (mean [SD]: 5.64 [4.62] vs. 5.27 [4.25]) or emergency room visits (mean [SD]: 0.22 [0.52] vs. 0.10 [0.30]; Fig. 2) were smaller.CONCLUSION: This study revealed that >40% of older AML pts (≥65 yrs) did not receive any antineoplastic therapy. This group also had fewer inpatient days compared to younger AML pts (<65 yrs old). These results show that despite medical advances, a large proportion of older AML pts remains either untreated or undertreated, revealing important unmet needs and suggesting that AML treatment has to face a much-needed expansion to improve disease management in this population. [Display omitted] DisclosuresLafeuille:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Ltd: Research Funding; Janssen Scientific Affairs, LLC: Research Funding. Sundaram:Johnson & Johnson, LLC: Equity Ownership; Janssen: Employment. Hoehn:Janssen: Employment; Johnson & Johnson, LLC: Equity Ownership. Emond:Janssen Scientific Affairs, LLC: Research Funding. Romdhani:Janssen Scientific Affairs, LLC: Research Funding. Lefebvre:Janssen Scientific Affairs, LLC: Research Funding.

Patient and Physician Preferences for Front-Line Treatment of Advanced Stage Hodgkin Lymphoma in Germany, France and the United Kingdom

Introduction: The standard treatment regimens for advanced-stage Hodgkin lymphoma (HL) are adriamycin/bleomycin/vinblastine/dacarbazine (ABVD) and escalated dose bleomycin/etoposide/ adriamycin/cyclophosphamide/vincristine/procarbazine/prednisone (BEACOPPesc) in patients under age 60. The objective of this study was to understand drivers of treatment preferences among patients and physicians for choice of front-line treatments in advanced stage HL.Methods: A discrete choice experiment (DCE) was administered to hematologists/oncologists who manage patients with HL and individuals diagnosed with HL in the previous 2 years located in France, Germany and the United Kingdom. The attributes and levels were informed from a targeted literature review, physician interviews, and patient qualitative interviews. A total of six attributes were included: 5-year overall survival (OS; levels: 84%, 89%, 94%); 5-year progression free survival (PFS; levels: 68%, 77%, 85%); risk of side effects resulting in hospitalization (levels: 40%, 60%, 80%); risk of peripheral neuropathy following treatment (levels: low risk, high risk); risk of infertility (levels: low risk, high risk); and risk of permanent pulmonary toxicity (levels: no risk, approximately 20%). Based on patient interviews, the PFS attribute was altered slightly for the patient DCE, described as the risk of HL coming back or getting worse within 5 years (levels: 26%, 15%, 4%). Participants reviewed 12 unique DCE scenarios, developed using a d-efficient design, and selected their preference between two hypothetical unnamed treatments. Patients considered themselves when selecting their preference whereas physicians considered five different advanced HL patient types that differed in gender, age (30 vs 65 years), smoking status, and desire to have children. The DCE data were analyzed using a mixed logit model, using effects coding, which produces coefficients for the mean and standard deviation of each attribute level. Separate models were run for each of the patient types for the physician DCE. The relative importance of each attribute was calculated by dividing the difference between the coefficients of the minimum and maximum of each attribute by the sum of the differences for all the attributes. The least important attribute was assigned a value of 1.00 and the remaining attributes' importance level were reported relative to 1.00.Results: A total of 281 physicians and 289 patients provided logical responses and were included in the analysis. Physicians were a mean of 47 years of age, 73% were male and they had a mean of 15 years' experience in treating HL. Patients were a mean of 37 years of age (8% > 55 years of age), 63% were male, and nearly half with known disease stage were in advanced stages of disease (43% in stage III, IV, intermediate or advanced stage).OS was considered the most important attribute to physicians in 3 of 5 patient profiles (Table 1). For smokers (profile 4), physicians ranked pulmonary toxicity as the most important attribute, over 1.5 times as important than OS, over twice as important as PFS and over 5 times as important as short-term side effects. Among older patients (profile 5), short term side effects was most important to physicians' preferences.From the patients' perspective, PFS was the most important attribute, followed by OS, both of which were over twice as important as short-term side effects. Patients considered long-term side effects (peripheral neuropathy, infertility, and pulmonary toxicity) almost twice as important as short-term side effects. OS was most important to male patients followed by PFS, whereas PFS was most important to female patients and OS was less important than long-term side effects (Table 2). Among older patients, PFS was most and infertility least important. Short-term side effects were the least important attribute to younger patients.Conclusion: Generally, patients are willing to endure treatments with worse short and long-term effects if the treatment provided better efficacy outcomes. Physicians' treatment preferences are patient-specific, with age and potential comorbidities impacting the importance of pulmonary toxicity and short-term side effect attributes. This study demonstrates the complexity of the decision making process for front-line HL treatments. Novel therapies would provide treatment options to address patient-specific needs. [Display omitted] DisclosuresBroeckelmann:Takeda: Consultancy, Honoraria, Research Funding; BMS: Honoraria, Research Funding. Stamatoullas:Celgene Corporation: Honoraria; Takeda: Consultancy. Zagadailov:Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Gautam:Takeda Pharmaceuticals International Co: Employment, Equity Ownership. Huebner:Takeda Pharmaceuticals Co.: Employment, Equity Ownership. Dalal:Takeda Pharmaceuticals International Co: Employment, Equity Ownership. Illidge:Nordic Nanovector: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Research Funding; Takeda: Consultancy, Honoraria; University of Manchester: Consultancy, Employment.

Evaluation of the Impact of Minimal Residual Disease, FLT3 Allelic Ratio, and FLT3 Mutation Status on Overall Survival in FLT3 Mutation-Positive Patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) in the Chrysalis Phase 1/2 Study

Evaluation of the Impact of Minimal Residual Disease, FLT3 Allelic Ratio, and FLT3 Mutation Status on Overall Survival in FLT3 Mutation-Positive Patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) in the Chrysalis Phase 1/2 Study

Dasatinib Discontinuation in Patients (Pts) with Chronic-Phase Chronic Myeloid Leukemia (CML-CP) and Stable Deep Molecular Response (DASFREE)

Dasatinib Discontinuation in Patients (Pts) with Chronic-Phase Chronic Myeloid Leukemia (CML-CP) and Stable Deep Molecular Response (DASFREE)

Extra-Corporeal Photopheresis Plus Low-Dose Interleukin-2 for Steroid-Refractory Chronic Graft-Vs.-Host Disease: Efficacy, Safety and Immune Correlates

CD4+CD25+FOXP3+ regulatory T cells (Treg) are dominant suppressors of innate and adaptive immune effector cells that enhance tolerance. Treg augmentation can control immune activation underlying chronic graft-versus-host disease (cGVHD). In prior studies, daily subcutaneous (SC) low-dose interleukin-2 (IL-2) therapy for 8-12 weeks induced preferential Treg expansion in-vivo, with objective clinical responses in over half of evaluable participants (NEJM 2011, Blood 2016). Alternative immunomodulatory approaches like extra-corporeal photopheresis (ECP) also have efficacy in cGVHD and may function via Treg augmentation. Combining ECP plus IL-2 could offer therapeutic synergy.We report on a prospective single arm phase 2 trial of twice-weekly ECP (weeks 1-16) plus sequential low-dose IL-2 (1x106 IU/m2/d SC, weeks 9-16) for treatment of steroid-refractory cGVHD. The study enrolled 25 participants (48% male, 88% HLA-matched, 100% PBSC grafts, 52% prior aGVHD). Median participant age was 62 years (range, 34-76). Median time from HSCT and from cGVHD onset to start of treatment was 919 days (range, 177-2842) and 524 days (range, 58-2697) respectively. Participants had a median of 3 cGVHD organ sites (range, 2-5). The median follow-up time in survivors is 16 months (range, 2.1-26).ECP plus IL-2 was well tolerated: 1 participant withdrew due to IL-2 adverse effects (AE) (myalgia, fatigue), and 3 were additionally unevaluable (1 cardiac arrest; 1 parainfluenza infection, 1 still undergoing treatment). None required IL-2 dose reduction. There were four grade 3 AEs during the 16-week study (1 participant had dehydration/hypotension/syncope, possibly IL-2-related;1 had ECP-related anemia). None experienced relapse. At week 16, cGVHD objective partial responses (PR) were documented in 14 of 21 evaluable participants (67%). None had cGVHD progression. Response sites included skin (n=9/18), joint/fascia/muscle (n=7/16), liver (n=1/1), lung (n=2/9), and GI tract (n=3/5). 18 participants with clinical benefit (PR, and stable disease with minor response) proceeded to extended ECP plus IL-2 therapy.Immunologically, 8 weeks of ECP alone did not induce significant changes in buffy coat WBC or absolute lymphocyte count (ALC). Peripheral blood CD3 T cell counts/µL fell, with a median CD8 and CD4 conventional T cell (Tcon) decrement of -109 (Q1, Q3: -220, -13; p<0.001) and -68 (Q1, Q3: -244, -19; p=0.023), respectively, at week 8 (Figure 1A). There was an early fall in Treg cell counts/µL, with a median decrement of -4 (Q1, Q3: -9, 0; p=0.033) at week 4, without change in NK or B cell counts or Treg:Tcon ratio. The addition of low-dose IL-2 thereafter induced a >5-fold increase in median Treg count/µL (p<0.001), from a baseline of 9 (Q1, Q3: 5, 17) at week 8 to a peak of 72 (Q1, Q3: 22, 186) at week 10, plateauing at 50 (Q1, Q3: 33, 71) at week 16 (Figure 1A). No significant change in CD4 Tcon or B cell count was noted. NK cell count increased 1.7-fold (p=0.009). The median Treg:Tcon ratio increased 4-fold (p<0.001): from a baseline of 0.06 (Q1, Q3: 0.03, 0.08) at week 8 to a peak of 0.26 (Q1, Q3: 0.12, 0.42) at week 12, and remained elevated at 0.17 (Q1, Q3: 0.14, 0.34) at week 16 (Figure 1B). Treg count and Treg:Tcon ratio remained elevated in responders during extended ECP plus IL-2 therapy.Immunologically, clinical responders had a higher median Treg count/µL vs. non-responders after 1 and 6 weeks of IL-2 therapy (21 vs. 11, p<0.001; 73 vs. 29, p=0.016 respectively). Treg:Tcon ratio at week 2 and 6 after IL-2 therapy was also higher in responders (0.29 vs. 0.15, p=0.038; 0.28 vs. 0.13, p=0.016 respectively). Applying our previous cutoffs (Blood 2016), Treg:Tcon ratio ≥0.2 after 1 week of IL-2 was also predictive of clinical response (p=0.05).In summary, in steroid-refractory cGVHD, ECP was associated with a fall in peripheral CD8, CD4 Tcon, and Treg counts. The addition of low-dose IL-2 thereafter induced profound Treg and NK cell augmentation. Clinical responses (PR) to combination ECP plus IL-2 therapy were documented in 67% of the evaluable participants. Those with early rise in Treg count and Treg:Tcon ratio after the addition of IL-2 were likelier to benefit. However, the overall clinical efficacy of ECP plus IL-2 did not appear markedly higher than low-dose IL-2 alone. [Display omitted] DisclosuresKoreth:Prometheus Labs: Research Funding; Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy. Armand:Bristol-Myers Squibb: Consultancy, Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Sequenta/Adaptive: Research Funding; Sigma Tau: Research Funding; Tensha: Research Funding; Affimed: Research Funding; Roche: Research Funding; Pfizer: Consultancy, Research Funding; Genmab: Consultancy; Infinity: Consultancy; Otsuka: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Haploidentical Hematopoietic Cell Transplantation Followed By Donor Natural Killer Cell Infusion and Sirolimus Maintenance Can Successfully Treat Patients with High-Risk Ewing Sarcoma

Background: Approximately 30-40% of patients with newly diagnosed localized Ewing Sarcoma (EWS), and 60-80% with disseminated EWS, will eventually relapse. Patients with late relapse have a 5 year overall survival (OS) of 30%, while patients with early relapse (<2 years from initial diagnosis) have a more dismal 5 year OS of <10% (Stahl Ped Blood Cancer 2011). Unfortunately, conventional treatments including chemotherapy, autologous transplantation, surgery, and radiation therapy often lead to additional morbidities without substantial gains in disease remissions or survival. Allogeneic hematopoietic cell transplantation (HCT) using human leukocyte antigen (HLA)-matched related donors has shown some success for patients with high-risk EWS (Baird BBMT 2012). Additionally, EWS cells are exquisitely sensitive to natural killer (NK) cell cytotoxic activity (Cho CCR 2010), and many EWS tumors have upregulated the mammalian target of rapamycin (mTOR) pathway making them susceptible to anti-mTOR agents (Wan et al The Oncologist 2007). With growing literature supporting the safety and efficacy of HLA-haploidentical HCT with post-transplant cyclophosphamide (CY) for hematological malignancies, we developed a novel treatment regimen adding a single infusion of donor NK cells on day +7 after HLA-haploidentical HCT, followed by sirolimus as part of a GVHD prophylaxis strategy. We hypothesized that this multi-faceted immunotherapy approach, in combination with an mTOR-inhibitor post-transplant, would be well-tolerated and efficacious in patients with relapsed EWS.Methods: Patients received reduced-intensity conditioning using fludarabine (150 mg/m2), CY (29 mg/kg) and 3 Gy total body irradiation, followed by infusion of HLA-haploidentical related marrow (n=4) or peripheral blood stem cells (n=2). Post-transplant immune suppression consisted of CY (50 mg/kg, days +3 and +4), tacrolimus (beginning day +5, and transitioned to sirolimus from day +28 to 6 months), and mycophenolate mofetil (MMF) (day +9 to +84). MMF initiation was delayed until day +9 to decrease concerns of inhibiting NK activity. For NK cell therapy, non-mobilized peripheral blood mononuclear cells were collected from the same haplo donors on day +6 using apheresis. NK cells were selected on day +7 using the Miltenyi CliniMACS system (CD3 depletion followed by CD56 selection) and were administered as a single, fresh infusion that same day without prior culturing, expansion, or exogenous cytokine exposure.Results: Four pediatric and 2 adult patients with median age of 18.5 (10.5-34.0) years having relapsed EWS were enrolled on this Phase II study. All patients had stable or undetectable gross disease at HCT. Patients had 1 (n=3), 2 (n=2), or 5 (n=1) relapses prior to HCT. Median time from EWS diagnosis to first relapse was 22.5 (15-56) months. Two patients had prior autologous HCTs. Median HCT-CI score at time of transplant was 4 (2-5), demonstrating increased risk for transplant-related mortality (TRM). Patients received a median CD34 dose of 5.24 (1.3-8.3) x 106/kg. NK cell products had a median log T cell depletion of 6.25 (5.18-6.42), median NK recovery of 54% (47-71%), and median NK purity of 91% (78-96%). Excellent viability (>99%) was seen in all NK products. Donor NK infusions were well-tolerated without fevers or other adverse reactions. Sustained full donor chimerism (>95% CD3) was achieved in all 6 patients. One patient developed stage 1 (grade II) gut graft-versus-host disease (GVHD) at day +52 which was treated with budesonide. To date, no patients have developed chronic GVHD nor have died from TRM. With a median follow-up of 1.3 years (range, 180 days - 2.7 years), 1 year overall survival is estimated at 75% (see figure). Three patients have relapsed or progressed at 187, 284, and 455 days after HCT, of which 1 patient (who had 5 prior relapses), died of progressive disease at day +350. The other 2 patients have received salvage treatments. They remain alive with full-donor chimerism and low-level/absent disease at 1.7 and 2.7 years post-HCT.Summary: This dual immunotherapy approach, using HLA-haploidentical HCT with donor NK cell infusion, followed by sirolimus maintenance, was well-tolerated by all patients. While longer follow-up is needed, preliminary results of this ongoing novel approach demonstrate feasibility in EWS patients, with better than expected 1 year OS and promising disease control. [Display omitted] DisclosuresThakar:Miltenyi Biotec: Other: funding of partial costs - disposable agents. Hari:Janssen: Honoraria; Sanofi: Honoraria, Research Funding; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy; Takeda Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Spectrum: Consultancy, Research Funding; Amgen: Consultancy, Honoraria, Research Funding.

Bone Marrow Stroma Protects FLT3 Acute Myeloid Leukemia (AML) through CYP3A4-Mediated Drug Metabolization of FLT3 Tyrosine Kinase Inhibitors (TKIs)

IntroductionFms-like tyrosine kinase 3 internal tandem duplications (FLT3/ITD) are among the most common mutations found in AML patients. Interestingly, although initial remission rates of FLT3/ITD AML are similar to standard-risk AMLs, these AML have a high relapse rate and poor overall survival. Moreover, FLT3 TKIs have been much more successful in eliminating peripheral FLT/ITD blasts, than eradicating marrow disease and generating durable responses clinically. Studies have addressed several mechanisms underlying the FLT3 TKI resistance, including a protective role for the bone marrow (BM) microenvironment. However, the mechanisms underlying this protection remain unknown. We previously showed that human bone marrow stromal cells (BMSCs) highly express most cytochrome P450 (CYP) enzymes, and these enzymes are a major means of chemoprotection within the BM niche (Alonso et al. Oncotarget 2015). Among them, CYP3A4 is a key enzyme responsible for hepatic inactivation of many chemotherapy agents, including many FLT3 TKIs used clinically. Therefore, we hypothesized that BM stromal CYP3A4 contributes to BM microenvironment mediated FLT3 TKI resistance by FLT3/ITD AML.Methods and ResultsTo test our hypothesis in vitro, FLT3/ITD AML cell lines (MV4-11 and Molm14) were co-cultured with FLT3 TKIs with or without BMSCs (F/STRO), and their clonogenic activity was assessed. We found that 3 different clinically active FLT3 TKIs (sorafenib, quizartinib, and gilteritinib), while active against the FLT3/ITD AML in liquid culture, had little activity when co-cultured with BMSCs (Figure 1A). Moreover, when FLT3/ITD AMLs were co-cultured with BMSCs with short hairpin (sh) RNA knockdown of CYP3A4 (shCYP3A4), the activity of the FLT3 TKIs was restored (Figure 1A). To test the hypothesis in vivo, we investigated the growth of FLT3/ITD AML tumors containing control or shCYP3A4 human primary BMSCs upon FLT3 TKI treatments in a xenograft mouse model. We observed that the tumors with shCYP3A4 BMSCs were significantly more sensitive to FLT3 TKI treatments compared to those with control BMSCs.To further examine the role of CYP3A4 in BMSCs, we examined the inhibitory effects of FLT3 TKIs in the absence of stromal cellular contact with FLT3/ITD AML cells. FLT3 TKI-containing media was incubated with and without control BMSCs and shCYP3A4 BMSCs, and then incubated with FLT3/ITD AML cells. Western blot performed on the FLT3/ITD AML cells showed that FLT3 TKI media conditioned with BMSCs blocked the inhibition of FLT3 phosphorylation, and this was reversed by CYP3A4 knockdown in the BMSCs. These results demonstrated that stromal CYP3A4 protected against FLT3 TKIs even in the absence of stromal cell contact.We next studied if combining a clinically active CYP3A4 inhibitor with FLT3 TKIs would increase the FLT3/ITD AML cells' sensitivities to FLT3 TKIs in the presence of BMSCs. Clarithromycin, a clinically active CYP3A4 inhibitor, significantly reversed the protective effects of BMSCs to 3 different FLT3 TKIs (Figure 1B).ConclusionsThese data confirm the protective effects of stroma for FLT3/ITD AML against FLT3 TKIs. Further, we found for the first time that CYP3A4 expressed in BMSCs contributes to BMSC mediated FLT3/ITD AML protection against FLT3 TKIs both in vitro and in vivo mouse model. Our results also demonstrated that combining FLT3 TKIs with clinically available CYP3A4 inhibitors may become a promising strategy to overcome drug resistance in FLT3/ITD AML treatment. [Display omitted] DisclosuresLevis:Novartis: Consultancy, Honoraria, Research Funding; Daiichi Sankyo, Inc.: Consultancy, Honoraria; Astellas Pharma Us: Consultancy, Research Funding; Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Research Funding; FujiFilm: Research Funding.