ALBERT

Description: Temporary or long-lasting decrease of one or more blood cell lineages following allogeneic hematopoietic stem cell transplant (allo-HSCT) can lead to increased morbidity and mortality. Post-transplant cytopenia is often secondary to drug toxicity, viral infections, graft versus host disease (GVHD), or relapse. Less is known about the significance of cytopenia of unknown cause. Here we retrospectively analyzed 91 consecutive adult patients (median age 46 years; range: 19-63) who had received allo-HSCT conditioned with myeloablative fludarabine/ I.V. busulfan at our institution. Diagnoses included: acute myeloid leukemia (AML) or myelodysplatic syndrome (MDS) (n=57), acute lymphoblastic leukemia (ALL) (n=15), chronic myeloid leukemia (CML) (n=9), non-Hodgkin lymphoma (NHL) (n=5), B cell chronic lymphocytic leukemia (B-CLL) (n=2), myelofibrosis (n=1), unclassified myeloproliferative neoplasms (MPN) (n=2). Stem cell source was granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSC) in 81, bone marrow in 8 and cord blood in 2 patients. Median follow up for the entire cohort was 20 months (range 1-162 months). Patients with cytopenia of unknown cause were identified if they had full donor chimerism, negative cytomegalovirus (CMV) by molecular analysis, no active infections, acute GVHD

Description: Deregulated gene expression due to genetic alterations, such as gene fusions affecting transcription and/or epigenetic factors is the hallmark of acute myeloid leukemia and the basis for the differentiation block of hematopoietic progenitors. Acute megakaryoblastic leukemia (AMKL) is a subtype of poor prognosis acute myeloid leukemia (AML) affecting primarily young children. Recently, the ETO2-GLIS2 fusion has been identified in 20-30% of de novo AMKL and associated with the worst prognosis in this subtype of AML. To characterize the transformation induced by ETO2-GLIS2, we first defined the consequences of ETO2-GLIS2 expression on hematopoietic progenitors and the contribution of ETO2 and GLIS2 on differentiation and self-renewal. Using methylcellulose replating assays and phenotype characterization, we show that the GLIS2 moiety drives the megakaryocytic phenotype whereas both the ETO2 and GLIS2 moieties are required for maintaining self-renewal. Global expression profiling and comparison to patients' signature consistently identify ETO2-GLIS2-mediated deregulation of major transcriptional regulators of hematopoiesis and leukemogenesis, including overexpression of the ERG oncogene. ChIP-seq analysis reveals that ETO2-GLIS2 is recruited at normal ETO2 complexes sites and also at GLIS2-specific targets through binding via GLIS2 DNA-binding domain. We demonstrate that ETO2-GLIS2 fusion localize at half of H3K27Ac-dense enhancers, so called super-enhancers, to control transcription of associated genes. We show that interaction of ETO2-GLIS2 with ETO2 complexes is an essential node for the transcriptional control by the fusion at enhancer elements. Indeed, ETO2-GLIS2 dimerizes and interacts with endogenous ETO2 via its NHR2 domains. An NHR2 peptide-interference strategy inhibits oligomerization, reverses the transcriptional activation at enhancers, promotes megakaryocytic differentiation and abrogates human AMKL cells maintenance in vivo. Finally, upregulation of ERG by ETO2-GLIS2 further strengthen enhancers formation as ERG is co-recruited generating a feed forward loop at these elements and its knockdown or genetic inactivation downregulates expression of ETO2-GLIS2 targets required for leukemic cells survival. We propose that the megakaryocytic differentiation arrest and self-renewal controlled by ETO2-GLIS2 results from an imbalance in the expression of master transcription factors imposed by aberrant chromatin structures at enhancers that may be disrupted by targeting the NHR2 interface. Disclosures No relevant conflicts of interest to declare.

Description: Multipotent mesenchymal stromal cells (MSCs) have immunomodulatory properties and have been successfully used for treatment of autoimmune diseases and acute or chronic graft-versus-host disease. Therapy with MSCs is not always effective. It has been shown that MSCs immunomodulatory properties can be improved by means of various agents, such as IFN-g, TNF-a, IL-17. After 4 hours of IFN-g exposure the expression level of immunomodulatory genes increased - IDO1 300, CSF1 - 7, and IL6 - 2.4 times. MSCs typically express low levels of MHC class I, and no MHC class II or co-stimulatory molecules (e.g., B7-1, B7-2, or CD40), making them partially immunoprivileged. However, treatment with IFN-g leads to increased expression of HLA-DR antigens on MSCs. After injection to the patient the characteristics of MSCs differ from those which have been studied in culture due to their interactions with other cells in the bloodstream and tissues. In this study the model of MSCs and MSCs treated with IFN-g (IFN-g-MSC) interactions with allogeneic lymphocytes in vitro was developed. The aim of the study was to identify the changes in MSCs and IFN-g-MSCs characteristics after co-cultivation with lymphocytes in vitro in dynamics. Materials and methods MSCs were isolated from 13 bone marrow (BM) samples used for allogeneic hematopoietic cells transplantation and cultured by a standard method in aMEM with 10% fetal bovine serum (FBS). MSCs on 2-3-d passages were seeded 105 cells per flask with 25 cm2 bottom area and a day later 500 units/mL of IFN-g were added for 4 hours to half of the cultures. Then the media was changed on RPMI-1640 with 10% FBS. Some cultures were seeded with 106 allogeneic lymphocytes, to half of these cultures 5 mg/ml phytohemagglutinin (PHA) was added for lymphocytes activation. All flasks were cultured up to 4 days at 37°C and 5% CO2. After 1, 2, 3 and 4 days lymphocytes were washed from MSCs. MSCs were removed from the flasks with trypsin and the number of viable cells was determined by dye exclusion method (trypan blue). For each of the MSCs cultures the mean fluorescent signal intensity level (MFI) of HLA-DR was determined by direct immunofluorescent staining with anti-HLA-DR APC (BD Pharmingen) antibodies and measured on flow cytometer BD FACS Canto II (BD Biosciences, USA). Data are presented as mean ± standard error. Statistical analysis was performed using Student's t-test (considered reliable p

Description: Mixed Lineage Leukemia gene rearrangements (MLL-r) account for nearly 10% of human acute leukemia cases and are generally associated with poor prognosis. Previous studies have revealed an essential role of the histone H3K79 methyltransferase Disruptor of Telomeric Silencing-1 Like (DOT1L) in MLL-r leukemogenesis. Our recent report (Chen et al. 2015 Nature Medicine) further identified a role for histone acetylation in DOT1L dependent gene expression driven by MLL-fusion proteins including MEIS1 and HOXA cluster genes. A first-in-human Phase I clinical trial demonstrated clinical activity of DOT1L inhibition in MLL-r leukemia patients, thus providing a potential opportunity for treating these malignant diseases. Nevertheless, the incomplete silencing of the leukemic program by only targeting DOT1L motivates the need for additional and perhaps combinational approaches to improve therapies against MLL-r leukemias. To enhance the efficacy of DOT1L inhibition, we sought to identify genes whose suppression would synergize with the DOT1L inhibitors to suppress the proliferation of mouse bone marrow progenitors transformed with MLL-AF9. We conducted a pooled RNAi screen using a customized library composed of 2,252 shRNA targeting 468 epigenetic regulators (i.e. writers, readers, and erasers of chromatin modifications; Fig 1). The integrated shRNA sequences were assessed using high-throughput sequencing. By comparing the change in frequency of each shRNA construct cultured in control vs. an IC50 DOT1L inhibitor EPZ4777, we identified several candidate modulators of DOT1L dependency, which had multiple shRNAs selectivity depleted only in the DOT1L suppressed condition. Notably, using a network correlation study, we found that one of the top candidate genes Plant Homeodomain Finger Protein 20 (PHF20) is highly associated with histone acetylation in the mammalian epigenome. Knockdown of PHF20 drastically increased the sensitivity of MLL-AF9 leukemic blasts to DOT1L inhibitors through enhanced myeloid differentiation and reduced cell proliferation, colony formation, and re-plating capacity. Similar phenotypes were also observed in PHF20-deficient MLL-AF9 cells generated by CRISPR/Cas9-mediated gene knockout. PHF20 is an epigenetic adaptor protein that has no predicted enzymatic activity. To investigate the role of PHF20, we conducted a CRISPR functional domain screen and identified the requirement of the chromatin reader domains in PHF20, including the Tudor domains and the PHD-finger, in supporting the survival of MLL-r leukemic cells upon DOT1L inhibition. We also performed RNA-seq and found that suppression of PHF20 facilitated the silencing of the MLL-AF9 leukemic program induced by DOT1L inhibitor treatment. Chromatin immunoprecipitation and sequencing (ChIP-seq) analyses validated that PHF20 contributes to the maintenance of histone acetylation including H3K9ac and H4K16ac at MLL-AF9 target loci. In line with the profound loss of histone acetylation at MLL-AF9 target loci in PHF20-depleted cells, we found that knockdown of a known PHF20 interacting partner KAT8 (a histone acetyltransferase; also known as MOF or MYST1) phenocopies the effects observed in PHF20-knockdown cells. Finally, we showed that pharmacological inhibition of DOT1L and KAT8 synergistically suppresses the proliferation and survival of MLL-AF9 leukemic cells. These data collectively highlight the involvement of a novel DOT1L-PHF20-KAT8 axis in mammalian gene regulation and MLL-r leukemogenesis. In summary, our studies show that MLL-rearrangements may drive leukemic transformation by coordinating an epigenetic network involving several histone modifications associated with gene transcription (e.g. H3K79 methylation and H3K9/H4K16 acetylation). Our results also suggest that simultaneous targeting of multiple components of this epigenetic feed-forward loop including DOT1L and PHF20/KAT8 may provide a novel and more effective approach against MLL-r leukemia. Disclosures Bradner: Novartis Institutes for BioMedical Research: Employment. Armstrong:Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

Description: Background In younger and fit multiple myeloma (MM) patients (pt), autologous stem cell transplantation (ASCT) remains the gold standard treatment. Mobilization chemotherapy is usually administered in an inpatient regimen and Cyclophosphamide (CY) at different doses is the most used chemoterapy for collecting peripheral blood stem cells (PBSC) in MM. Clinical trials have demonstrated that intermediate dose CY (3 and 4 g/m2, ID-CY) combined with G-CSF, is an efficient mobilizing regimen with less toxicity compared with high dose CY (7 g/m2, HD-CY) in term of neutrophil recovery, thrombocytopenia, need of transfusions and IV antibiotics. (Fitoussi et al, BMT 2001; Goldschmidt et al, BMT 1996). Objective To evaluate the safety of mobilization therapy administered in an outpatient regimen, with the prospect to lower costs and minimize patient inconvenience, maintaining an optimal yield. Methods 92 pt with newly diagnosed MM underwent outpatient stem cell mobilization between 2002 and 2016 with CY 3 g/m2 (82%) or 4 g/m2 (18%) + G-CSF after induction therapy with bortezomib-based (79%) or VAD-like (21%) regimens. No antibiotics prophylaxis was routinely used. Day 0 was defined as the CY infusion day. CY was administered in 2-4 consecutive 1h infusions (depending on total dose). Hyper-hydration (3.5/4 l), antiemetics and the uroprotectant Uromitexan were began IV 1 hour before CY infusion. Subsequently, Uromitexan was continued at home orally in the next 12h. Furthermore, the patient was advised to drink 2.5/3 l of water in the next 24h. G-CSF 10 mcg/Kg was started by day +5 and continued until completion of apheresis. Blood count was monitored at day + 4 and daily from day +7. CD34+ cells were counted on peripheral blood by day 7; apheresis was started at leukocyte rise and with a value of at least 20 CD34+/μl. Number of apheresis depended on the number of CD34+ cells collected to obtain al least 4x106 CD34+/Kg. Results Median age at diagnosis of was 56y (range 34-68). MM isotype was IgA, IgG and micromolecular respectively in 18%, 58% and 24%. Prior MGUS was present in 37 cases (43%). LDH was elevated in 7 pt (11%), whereas ISS was 1/2/3 in 47%/30%/23%. Bone disease was detectable in 74% of pt, with 56% having 3 or more osteolysis. Median bone marrow plasma cell at diagnosis was 60% (range 10-95%). Pt received induction with bortezomib-based regimens (79%) or chemoterapy, mostly VAD (21%). 8 pt (9%) required second line therapy before mobilization. Response prior of mobilization was CR/sCR in 15%, VGPR in 59%, PR in 24%, and SD in 2%. Stem cell collection was successful in 98% of pt, with a median CD34+ harvest of 9.8x106/Kg. Chemotherapy was very well tolerated. Most frequently observed adverse events (AEs) were nausea and vomiting of grade 1-2. 2 pt experienced cystitis (one grade 1, one grade 2), 2 pt infections, 2 pt hyperthermia regressed rapidly without therapy, 1 patient diarrhea. 3 pt had neurological symptoms: in 2 cases they were aspecific (headache, instability); the other case presented a sudden appearance of 7th cranial nerve deficit at the end of mobilization chemotherapy infusion with negative imaging and successively regressed in few hours, interpreted as transient ischemic attack not correlated with Cy. Only 2 patient required hospitalization for AEs: 1 patient for fever grade 3 without microbiological findings, rapidly regressed with IV antibiotics; the second one for 7th cranial nerve deficit. These were the only grade 3 AEs, no grade 4 AEs verified. There were no other significant AEs related to chemotherapy. All pt except 2 proceeded to stem cell harvest and reached CD34+ target, but 5 pt required administration of Plerixafor on demand. The 2 pt not reaching CD34+ target successfully mobilized afterwards, 1 with different chemoterapy and the other with G-CSF and Plerixafor. After mobilization, 88 pt proceeded to single (45%) or double (55%) ASCT. Conclusion In conclusion, outpatient mobilization with ID-CY appears to be an efficient and safe procedure, with minimal and manageable side effects and low rate of hospitalization. Outpatient mobilization could ameliorate the quality of life of pt and reduce costs, avoiding or minimizing the hospitalization rate, without compromising the safety profile and the success of PBSC collect. Disclosures No relevant conflicts of interest to declare.

Description: Background: Histone acetylation plays a key role in regulating gene expression and in control of cellular activities in multiple pathways involved in normal and cancer cell growth.Panobinostat (pano) is a pan histone de-acetylase inhibitor (HDAC-i) approved by the FDA on February 23, 2015 for use withbortezomib (btz) and dexamethasone (dex) for patients with multiple myeloma (MM) who have had at least 2 prior lines of therapy including bothbtz and an immunomodulatory agent (IMiD). The combination ofpano withIMiDs and proteasome inhibitors (PIs) has been found to demonstrate enhanced anti-myeloma activity in clinical trials (Berdeja JG et al, 2015,Haematologica;Mateos M et al, 2010, ASCO Abstract 8030, JCO 28:15s). The goal of this retrospective study is to evaluate the real world experience on efficacy and safety ofpano in combination with a variety of FDA approved agents including a PI, anIMiD or a monoclonal antibody-based regimen in patients with relapsed/refractory MM. Methods: Between February 23, 2015 and July 1, 2016, 34 consecutive patients with relapsed/refractory MM who were treated with commercialpano were identified from the JohnTheurer Cancer Center. Charts were analyzed for response and safety data. The study was approved by the institutional review board. Results: Median age was 63 (range 27-78), with 58% percent men. Thirty-one patients (91.2%) wereDurie-Salmon stage II or III. Ten (30%) had high-risk FISH as defined byt(14;16), t(4;14), del p53, and gain 1q21. Median number of prior lines was 5 (range 2-9). All patients were relapsed/refractory to their last line of therapy, and 18 (53%) werebtz-refractory, 25 (74%) werelenalidomide-refractory, 27 (79%) werepomalidomide-refractory, and 29 (85%) were carfilzomib-refractory. Twenty-five (74%) were refractory to the combination of carfilzomib with anIMiD. Five patients (14.7%) had priordaratumumab, and 4 (12%) had prior HDAC-i therapy. Median number of cycles withpano was 1 (range 1-5). The overall response rate (≥ partial response (PR)) was 23.5% and the clinical benefit rate (≥ minor response (MR)) was 67.6%. The median duration of response (≥ stable disease (SD)) was 3 months. The median progression-free survival (PFS) for all patients was 2.3 months (95% CI: [1.27 - 4.07]). See Figure 1. Median overall survival (OS) from initiation ofpano through 7/27/16 was 5.5 months (95% CI: [3.93, NA]). See Figure 2. Of the 4 patients who were refractory to a prior HDAC-i, 1 achieved PR (4 cycles), 1 achieved MR (5 cycles) and 2 had disease progression. Only 1 patient discontinuedpano due to toxicities. Grade 3 and 4 non-hematologic toxicities were diarrhea (N=1), and hypoxia/respiratory failure (N=1). Grade 3 and 4 hematologic toxicities occurred in 11 (32%) patients, with 5 (15%) anemia, 9 neutropenia (26%), and 8 (24%) thrombocytopenia. Serious adverse events included acute kidney injury, GI bleed, and febrile neutropenia in 3 patients, respectively. Conclusions: These observations demonstrate that real-world use ofpano outside of the FDA indication in combination with PI andIMiD-based regimens has activity and is well tolerated in heavily pretreated patients with relapsed/refractory MM, even those who have exhausted conventional treatments. Further assessment in a larger prospective study is warranted. Figure 1 PFS of all patients receivingpanobinostat-based regimens Figure 1. PFS of all patients receivingpanobinostat-based regimens Figure 2 OS of all patients receivingpanobinostat-based regimens from time of initiatingpanobinostat Figure 2. OS of all patients receivingpanobinostat-based regimens from time of initiatingpanobinostat Disclosures Biran: Takeda: Speakers Bureau; Celgene: Speakers Bureau; Novartis: Speakers Bureau; Amgen: Speakers Bureau. Vesole:Janssen: Speakers Bureau; Novartis: Speakers Bureau; Takeda: Speakers Bureau; Celgene: Speakers Bureau; Amgen: Speakers Bureau. Richter:Celgene: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Jannsen: Speakers Bureau. Siegel:Celgene: Honoraria, Speakers Bureau; Merck: Honoraria; Takeda: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau.

Description: Genomic stability and integrity in Hematopoietic Stem Cells (HSCs) is maintained via DNA damage checkpoints, DNA proofreading and DNA repair (Moehrle et al., 2015; Cell Rep). Despite these mechanisms, recurring and non-recurring mutations accumulate in HSCs upon aging, which correlate with an elevated incidence of myeloproliferative diseases (Rossi, Bryder, and Weissman, Exp. Gerontol. 2007) as well as changes in clonality (Akunuru and Geiger, 2016; Trends in Mol. Med.). The rate at which such mutations accumulate in individual HSCs and the selection advantage/disadvantage that they provide is unclear and is an active area of investigation. Evolutionary theory supports a strong influence of the aged niche on the selection of HSCs clones upon aging (Rozhok, Salstrom, and DeGregori, 2014; Aging). We hypothesized that variant profiling of single HSCs based on RNA transcripts will reveal mutational signatures adapted to the selection pressure of the aging microenvironment. We performed Single cell RNA-seq of daughter cell pairs from young and aged murine HSCs (LSK, CD34-, flk2-). The Genome Analysis Toolkit (GATK; Broad Institute) RNA-seq variant/mutation calling algorithm pipeline was applied with some modifications. Only variants that were observed in both daughter cells of a given pair were selected, which significantly decreased our false discovery rate (tested by a Monte Carlo simulation). First and most interestingly, we observed no significant difference in the overall number of variants/mutations between young and aged HSCs, further supporting our recently published observations on the frequency of DNA mutations in HSCs upon aging (Moehrle et al., 2015; Cell Rep). We then used an approach that takes into account the 3' and 5' bases flanking a variant to generate motifs whose frequencies can be mathematically analyzed to deduce characteristic mutational patterns, termed as mutational signatures (Nik-Zainal et al., 2012; Cell). We employed a non-negative matrix factorization (nmf) and principal component analysis (pca) algorithms to generate 10 mutational signatures that explained 〉 95% of the variance in the dataset. We then analyzed the signatures in a pairwise fashion and selected two signatures with the highest discrimination score between young and aged HSC. Based on this, cells fell into two major groups: group 1 predominantly contained aged single cells (~90% of the cells in this group) whereas, interestingly, group 2 contained a mix of young and aged HSCs. The segregation of young and aged single HSCs counts between groups 1 and 2 was tested using Fisher's exact test and was statistically significant (p-value 0.0029). These data indicate that while the overall mutational load is not elevated, majority of aged HSCs acquire a mutational signature distinct from young HSCs, while a proportion of aged HSCs present with a young-like HSC signature. Furthermore, our results show that even those cells that have acquired an aging signature aren't homogeneous and show sub-clustering tendencies, providing the first hint that they may potentially evolve further into more distinct clones. In conclusion, our results show that individual HSCs reflect a mixed mutational profile reminiscent of a non-uniform accumulation of variants. As such signatures are a reflection of underlying mechanisms by which the mutations accumulate (Nik-Zainal et al., 2012; Cell), the proportion of aged HSCs sharing similar mutational signatures but distinct from the young HSCs reveal an aging signature that indicates specific mutational factors and selection pressure of the aging microenvironment. Disclosures Mulaw: NuGEN: Honoraria.

Description: Autologous stem cell transplant (ASCT) remains the standard of care for Multiple Myeloma (MM) patients younger than 70 years old. The role of induction therapy is crucial within a program of high-dose therapy since deeper is the response before, higher is the outcome of transplant. In this study, we analyzed a real life setting of patients treated with three different induction approaches: VAD (Vincristine-Adriamycin-Dexamethasone), VD (Bortezomib - Dexamethasone), and VTD (Bortezomib-Thalidomide-Dexamethasone) in terms of depth of response, 2 years therapy-free rate and toxicity. One hundred and sixty-three MM patients (pts) were included in the analysis: 62 pts treated with VAD (38%), 44 with VD (27%) and 57 with VTD (35%). In VTD group 49 pts (86%) received Bortezomib subcutaneously. As shown in Table 1, patients of the three groups were similar for D&S stage (p 0.59), a higher rate of ISS stage 3 was observed in VAD group (p=0.019), patients in VTD group were significantly older (p=0.024), median follow-up was significantly lower in VTD pts (p

Description: Introduction: Neutrophil extracellular traps (NETs) are structures composed of DNA, histones, and bactericidal factors that are expelled by neutrophils in order to trap and neutralize bacteria. NETs play a role in host defense by trapping and killing infecting bacteria and inactivating bacterial virulence factors. Activation of the coagulation cascade by these components can lead to "immunothrombosis" and facilitate the containment and destruction of bacteria within a fibrin clot. Although extracellular nucleosomes (structures consisting of DNA wound around a histone protein core) within NETs can contribute to host defense, they can also play a role in disease pathology by leading to inflammation, endothelial damage, and pathological thrombosis. Disseminated intravascular coagulation (DIC) is a condition characterized by systemic activation of the coagulation and fibrinolytic systems that can occur in conjunction with several underlying conditions, including sepsis. Links between infection, host response, and systemic coagulation, extracellular nucleosomes may play a significant role in the pathophysiology of sepsis-associated DIC. The purpose of this study was to quantify extracellular nucleosomes in the plasma of patients with sepsis-associated DIC. Materials and Methods: Citrated, de-identified plasma samples were collected from patients with sepsis and suspected DIC at ICU admission and on ICU days 4 and 8 under an IRB approved protocol. DIC score was evaluated in each sample using the ISTH scoring algorithm incorporating platelet count, PT/INR, fibrinogen (Recombiplastin, Instrumentation Laboratory, Bedford, MA), and D-Dimer (HyphenBioMed,Neuville-Sur-Oise, France). Plasma from healthy individuals was purchased from a commercial laboratory (George King Biomedical, Overland,KS). Nucleosomes in plasma were measured using the Cell Death Detection ELISA (Roche Diagnostics, Indianapolis, IN). The correlation of variation for both intra-assay and inter-assay variation was 0.2, p

Description: Background: Erythropoiesis Stimulating Agents (ESAs) are FDA approved for chemotherapy induced anemia and anemia of chronic kidney disease. These indications are more frequent in patients with multiple myeloma. Use of ESAs has been associated with an increased risk of heart attack, stroke, venous thromboembolism (VTE), and all-cause mortality. In patients with cancer, ESA's have been associated with worse progression free survival (PFS) and overall survival (OS) Previous research regarding ESA use in patients with multiple myeloma has been limited and conflicting. In this study, we evaluate the effects of ESA use in patients with multiple myeloma. Additionally, we examined the frequency of ESA usage after the 2007 FDA safety update revising product labeling for ESAs. Methods: A retrospective chart review was conducted on patients diagnosed with active multiple myeloma between January 1st, 2000 and December 31st, 2015 at Gundersen Health System in La Crosse Wisconsin. Collected data include patient demographics, medications, lab tests, comorbidities, and the dates of any VTE, stroke, or myocardial infarction. Both a logistic regression and a matched case-control analysis were used to compare rates of complications in patients using ESAs to those that did not. ESA effect on median survival time was also calculated for each International Staging System (ISS) stage of disease. Results:There were 278 patients included for demographic analysis (Table 1), of which 268 were included in the logistic regression analysis and 124 (62 pairs) in the matched case-control analysis. A logistic regression model constructed via stepwise selection found that bone lesions at diagnosis (Odds Ratio (OR): 2.5 [1.2-5.1]), antiplatelet drug use (OR: 3.7 [1.2-11.3]) and ESA use (OR: 4.7 [2.2-9.9]) were associated with increased risk of VTE in our patient population. Use of an Angiotensin Converting Enzyme (ACE) inhibitor (OR: 0.4 [0.2-0.9]) was associated with a decreased risk of VTE. Increased odds of VTE with ESA usage (OR: 5.9 [1.9 - 18.8]) were also noted in the matched case-control analysis. There was no association found between rate of stroke and ESA usage in either logistic or matched case-control analysis. No significant association between ESA use and overall survival was noted in either logistic or matched case-control analysis. When comparing outcomes based upon pre and post FDA revised product labeling, we found a 50% reduction in ESA usage within our institution. In this same time period, the percentage of patients with multiple myeloma developing VTEs has been significantly reduced (18.6% vs 12.8% [p

Description: Introduction Multiple myeloma (MM) is a largely incurable plasma cell malignancy characterised by marked genomic heterogeneity, in which chromosome 1q21 amplification (amp1q21) associates with poor prognosis. Genomic analysis using next generation sequencing has identified recurrent mutations, but no universal acquired somatic mutation(s) have emerged in MM, suggesting that understanding pathways of survival will require analysis of individual tumours in distinct disease subsets. To compound complexity of the problem, intraclonal variation (ICV), known as a major driver mechanism in cancer plasticity, in which clonal competitor cells undergo selection during disease evolution and progression by Darwinian principles, will need to be fully mapped at the genome level. Identifying the true level of ICV in a tumour will thus require analysis at the level of whole exome sequencing (WES) in single cells (SCs). In this study, we sought to establish WES methodology able to identify ICV in SCs in an index case of amp1q21 MM. Methods Cell selection and sequencing CD138+ tumour cells and CD3+ T-cells were isolated from a presentation case of amp1q21 MM as bulk populations to high purity (〉97%). Single MM cells and normal T cells were individually isolated and used for single cell (SC) whole exome sequencing (WES). Whole genome amplification (WGA) was performed by multiple displacement amplification (Qiagen REPLI-g Mini kit), and exome capture was performed using Agilent SureSelect. Libraries were then 90 bp paired end sequenced on an Illumina HiSeq2000 (BGI, China). Data analysis Data was produced for bulk (1000 cells) MM and bulk germline T cells, twenty MM SCs and five T cell SCs. Raw data was aligned to hg19 reference sequence using NovoAlignMPI (v3.02.03). Variant calling was performed using SAMtools (v1.2.1) and VarScan (v2.3.6) and variants were annotated using ANNOVAR. High confidence variants were called in the bulk tumour WES by pairwise comparison with bulk germline WES. Variant lists were also cross-searched against various variant databases (CG46, 1000 genomes, dbSNP, esp650 and in-house database) in order to exclude variants that occur in the general population. Multiple quality control measures were employed to reduce the number of false positive calls. Results and Discussion Data and bioinformatics pipelines are of a high quality SC WES generated raw data reads that were similar to bulk WES of 1000 cells, with comparable mapping to Agilent SureSelect target exome (69-76% SC vs. 70% bulk) and mean fold coverage (56.8-59.1x vs. 59.7x bulk). On average, 82% of the exome was covered sufficiently for somatic variant (SV) calling (often considered as ≥ 5x), which was higher than seminal published SC WES studies (70-80%) (Hou et al., Cell, 2012; Xu et al., Cell, 2012). We identified 33 potentially deleterious SVs in the bulk tumour exome with high confidence bioinformatics, 21 of which were also identified in one or more SC exomes. The variants identified include suspected deleterious mutations in genes involved in MAPK pathway, plasma cell differentiation, and those with known roles in B cell malignancies. To confirm SV calls, randomly selected variants were validated by conventional Sanger sequencing, and of 15/15 variants in the bulk WES and of 55/55 variants in SCs, to obtain 100% concordance. Intraclonal variation in MM Significantly, ICV was apparent from the SC exome variant data. Total variant counts varied considerably among SCs and most variant positions had at least several cells where no evidence of the variant existed. Bulk WES lacks crucial information We identified an additional 23 variants that were present in 2+ SC exomes, but absent in the bulk MM tumour exomes. Of these, 30% (7 variants) were examined for validation, and were amplifiable in at least one cell to deliver 100% concordance with variant calls. These variants are of significant interest as they reveal a marked occurrence of subclonal mutations in the MM tumour population that are not identified by bulk exome sequencing. They indicate that the mutational status of the MM genome may be substantially underestimated by analysis at the bulk tumour population level. Conclusion In this work we establish the feasibility of SC WES as a method for defining intraclonal genetic variation in MM. Disclosures No relevant conflicts of interest to declare.

Description: Background: Glucocorticoids has been a backbone of various treatment regimens for multiple myeloma (MM). The repeated and chronic use of high doses of glucocorticoids is associated with development of secondary adrenal insufficiency (AI), and AI could be a major problem in critically-ill patients. However, there has been no specialized data about incidence and clinical significance of secondary AI in hospitalized patients with MM. Purpose: The objective of this retrospective study is to evaluate incidence, predictive factors, and clinical significance of secondary AI in hospitalized patients with MM. Methods: We retrospectively evaluated medical records of MM patients who were hospitalized in Chonnam National University Hwasun Hospital, South Korea from December 2014 to December 2015. The definite AI was diagnosed when the peak cortisol concentration was less than 〈 500 nmol/L (18 mcg/dL) after ACTH administration. Results: Between December 2014 and December 2015, 77 patients were hospitalized, and 58 underwent rapid ACTH stimulation test. The most frequent cause of hospitalization was infection (70.7%), followed by weakness (24.1%), and the others (5.3%). The definite AI was confirmed in 19 patients (32.7%). To evaluate the predictive factors of AI, all variables including clinical characteristics, laboratory results, cumulative steroid dose, and treatment duration at hospitalization were analyzed, but there were no significant predictors for AI. In addition, the patients with AI had a significantly poor survival outcomes compared to those without AI (the median overall survival of 42.3 months vs. 82.7 months; P = 0.037) (Figure 1). Conclusions: This study showed that the secondary AI is not a rare condition among hospitalized patients with MM, and there was no specific predictable symptoms or signs. In addition, development of AI in the treatment period is associated with a poor prognosis. This study suggests that evaluation of AI is routinely needed in hospitalized patients with MM. Disclosures No relevant conflicts of interest to declare.

Description: Background and objective: Multiple Myeloma (MM) is a plasma cell malignancy with a well documented immune dysfunction. Previous reports in murine models and our previous work in MM patients, showed that myeloid cells, both granulocyte-myeloid derived suppressor cells (G-MDSC) and neutrophils sedimenting on top of red cells after density gradients, defined as high-density neutrophils (HDN) are increased and exert an immunosuppressive activity. Moreover, we showed that myeloid precursors could be activate and acquire G-MDSC phenotype if cultures in presence of MM mesenchymal cells. Thus, we characterized functionally HDN to gauge their contribution in immunesuppression, angiogenesis and bone resorption in MM microenvironment. Methods: In HDN freshly-isolated from a series of 60 newly-diagnosed MM, 30 smoldering MM and 30 MGUS we investigated by RT-PCR, flow-cytometry, western-blot and immunofluorescence genes and proteins related to immunesuppression (Arg-1, IDO), angiogenesis (BV8, VEGF, MMP9) and bone resorption (IL1-b, MMP9, CHI3L). Results: First, MM-HDN exhibited an increased expression of immunesuppressive molecules Arg-1 and IDO compared to MGUS and healthy subjects (25.5 vs 6.2 vs 1 fold changes in gene expression, p=0.003), confirmed by functional assay of enzymatic activity, immunostaining and WB, positively correlated with advanced disease. Second, MM-HDN showed increased expression of BV8 and MMP9 (p=0.01, p=0.05) but not VEGF, as confirmed by immunofluorescence. In line with this observation, by using migration assay (CIM plate) MM-HDN induced neo-vasculogenesis in vitro by forming more and aberrant tubes than healthy or MGUS-HDN, thus to disclosure a novel proangiogenic mechanism driven by BV8. Third, MM-HDN showed increased expression of IL1-b, MMP9, CHI3L, associated to increased bone resorption activity, evaluated by dentin-disk assay. The same findings could be obtained treating bone matrix with MM-HDN-conditioned media, suggesting the crucial role of molecules released by MM-HDN but no from MGUS-HDN. Conclusion: taken together, our observations disclosure the contribution of aberrantly activated HDN in MM clinical features, contributing to immunosuppression, angiogenesis and bone resorption. Disclosures No relevant conflicts of interest to declare.

Description: Background:Novel insights into the biology of myeloma cells have led to the identification of relevant prognosis factors.Cytogenetic abnormalities (CA) has become one of the most important prognostic factors, and the presence of t(4;14), t(14;16) or del(17p) are associated with poor prognosis. Although there are some reports indicating that 1q gains may be considered as a poor-risk feature, the information is not uniform. Furthermore, there are important controversies about whether or not novel agents-based combinations are able to overcome the poor prognosis of CA. In the relapse setting, the combinations including proteasome inhibitors and immunomodulatory drugs have shown to improve, and some of them to overcome, the outcome of patients with high-risk CA. Here we report a preplanned analysis, in a series of elderly newly diagnosed myeloma patients included in the Spanish GEM2010 trial and receiving VMP and Rd, in a sequential or alternating approach, in order to evaluate the influence of CA by FISH on the response rate and outcome. Patients and methods: 242 pts were randomized to receive a sequential scheme consisting of 9 cycles of VMP followed by 9 cycles of Rd or the same regimens in an alternating approach (one cycle of VMP alternating with one Rd, up to 18 cycles. VMP included the IV administration of weekly bortezomib (except in the first cycle that was given twice weekly) at 1.3 mg/m2 in combination with oral melphalan 9 mg/m2 and prednisone 60 mg/m2once daily on days 1-4. Rd treatment consisted on lenalidomide 25 mg daily on days 1-21 plus dexamethasone 40 mg weekly. FISH analysis for t(4;14), t(14;16), del(17p) and 1q gains was performed at diagnosis according to standard procedures using purified plasma cells. Results: In 174 out of the 233 patients evaluable for efficacy and safety, FISH analysis at diagnosis were available and two groups were identified: high-risk group (n= 32 patients with t(4;14) and/or t(14;16) and/or del(17p)) and standard-risk group (n=142 patients without high-risk CA). The rates of CA was similar in both treatment arms. Response Rates (RR) were no different in the high-risk vs standard-risk groups, both in the sequential (74% vs 79% RR and 42% vs 39% CR) and alternating arms (69% vs 86% RR and 39% vs 38% CR). After a median follow-up of 51 months, high-risk patients showed shorter PFS as compared to standard risk in the alternating arm (24 versus 33 months, p=0.03) and this also translated into a significantly shorter OS (38.4m vs not reached, p=0.002). However, in the sequential arm, high-risk and standard-risk patients showed similar PFS (29.5 months vs 31.5 months, p=0.9) and OS (46m vs 63m, p=0.1). This beneficial effect observed in the sequential arm applied to both t(4;14) or del(17p). As far as 1q gains is concerned, 151 patients had 1q information and 76 of them had 1q gains (50.3%), defined as the presence of more than 3 copies in at least 10% of plasma cells. The rate of 1q gains was well balanced in both sequential and alternating arms. The ORR was similar in patients with or without 1q gains (83% vs 80%) as well as the CR rate (45% vs 31%), and no differences were observed between sequential and alternating arms. Patients with or without 1q gains had a similar PFS (36 months vs 29 months) and 4-years OS (63% vs 68%) in the whole series and no differences were observed between the sequential and alternating arms. This effect has been observed in patients with 1q gains as isolated CA and the outcome was slightly but not significantly worse when 1q gains were present plus either t(4;14) and/or del17p. Conclusions: The total therapy approach including VMP and Rd administered in a sequential approach is able to overcome the poor prognosis of the presence of high-risk CA in elderly patients with newly diagnosed MM. The presence of 1q gains has no impact in the PFS and OS of elderly patients treated with VMP and Rd. Disclosures Mateos: Janssen, Celgene, Amgen, Takeda, BMS: Honoraria. Martínez-López:Novartis: Honoraria, Speakers Bureau. Oriol:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Paiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Description: Introduction: Idelalisib (IDELA) is a selective, small molecule inhibitor of PI3Kd that has shown significant efficacy in treatment of patients (pts) with relapsed chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). A common adverse event (AE) observed in IDELA studies is diarrhea/colitis (DC): grade ≥3 ~15%. Published preclinical data suggests that PI3Kd plays a critical role in regulating the function and development of regulatory T-cells (T-regs). This biomarker analysis aimed to evaluate possible immune mechanisms that may have contributed to DC in IDELA-treated pts. Methods: Longitudinal absolute peripheral blood T (CD4+ and CD8+), NK (CD16+/CD56+) cell subsets, cytokines, and chemokine levels from patients treated with IDELA were analyzed (Table 1). Since absolute numbers of T-reg cells were not available, we utilized epigenetic qPCR method (Kleen T. et. al. J Immunother Cancer 2015) to assess the status of T-regs by quantifying FOXP3 utilizing banked peripheral blood mononuclear cells (PBMCs). The following cytokines and chemokines were measured: IL-12p40, IL-17A, IFNγ, TNFα, G- CSF, MIP1α (CCL3), CCL5 (RANTES), IL-10, IL-1RA, IL-6, IL-7, IL-8, IL-15, CRP, and IP-10 (CXCL10). We evaluated the association of changes from baseline of these biomarker(s) with the occurrence and severity of DC events during IDELA treatment. Association of cytomegalovirus (CMV) with DC was not addressed in this study and is being presented separately. Results: There were no differences in absolute numbers of T (CD4+ or CD8+) and NK cells between pts treated with IDELA in both trials with grade ≥3 DC vs those with no DC. Consistently, results from epigenetic qPCR analysis also demonstrated no differences in temporal profiles for peripheral T-cell subsets (CD3+, CD8+, or FOXP3+) in CLL pts treated with IDELA with grade ≥3 DC vs no DC. Baseline and on-treatment changes in peripheral T-cell subsets were not predictive of DC. Analysis of T-cell subsets from the visit immediately prior (t-1) to the first occurrence of grade ≥3 DC was not predictive, and revealed no differences compared to pts with no DC. Lower levels of CD3+, CD8+, and FOXP3+ were noted longitudinally as well as at t-1 visits in grade 1/2 DC vs non-DC pts, but these changes were not predictive of grade 1/2 DC. Increased levels of circulating pro-inflammatory cytokines (IL-15, IFN-γ, and CLL5) were noted in both CLL and indolent non-Hodgkin lymphoma (iNHL) pts treated with IDELA. IL-17A level was significantly higher at the t-1 visit in CLL pts with grade ≥3 DC vs no DC. However, Receiver Operating Characteristic analysis deemed that neither individual cytokine/chemokine or in combination was not predictive for DC occurrence. CLL/iNHL pts with grade ≥3 DC vs no DC were noted to have higher on treatment IL-8. CLL pts presented lower baseline IL-6 and G-CSF levels in patients with grade ≥3 DC vs no DC (Table 2). There were no associations between baseline circulating plasma markers and DC in pts with iNHL. Conclusion: With currently available data, no single circulating immune biomarker is associated with or is predictive for the development of DC during treatment with IDELA. Lower levels of CD3+, CD8+, and FOXP3+ were noted longitudinally in grade 1/2 DC vs no DC pts. No differences were observed in temporal profiles for T-cell subsets in pts with grade ≥3 DC vs those with no DC. However, higher on-treatment IL-8 and lower baseline IL-6 and G-CSF were noted in the relapsed CLL pts with grade ≥3 DC when compared with no DC pts. While quantitative analysis of these T-cell subsets was not associated with grade ≥3 DC, the qualitative function of T-cells may play a role in mediating DC. Functional assays for T-cells were not explored in this study. In addition, our concurrent analysis of colonic biopsies and association with CMV in pts with IDELA associated DC will be presented separately. Disclosures Furman: Pharmacyclics: Consultancy, Speakers Bureau; Gilead Sciences: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Abbvie: Consultancy, Honoraria. Hallek:Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding, Speakers Bureau. Sharman:Gilead Sciences, Inc.: Honoraria, Research Funding. Hillmen:Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding. Zelenetz:Gilead Sciences: Research Funding. Flinn:Janssen: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership. Jurczak:Gilead Sciences: Research Funding; Janssen: Research Funding; Celltrion, Inc: Research Funding; Acerta: Research Funding; Bayer: Research Funding. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Xiao:Gilead Sciences: Employment, Equity Ownership. Zheng:Gilead Sciences: Employment, Equity Ownership. Rao:Gilead Sciences: Employment, Equity Ownership. Dreiling:Gilead Sciences: Employment, Equity Ownership. Salles:Roche/Genentech: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Mundipharma: Honoraria. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria.

Description: Background: The amyloidoses comprise a heterogeneous group of diseases characterized by misfolding of amyloidogenic proteins and subsequent deposition as amyloid fibrils. To date, over 30 proteins are known to be amyloidogenic (Sipe Amyloid 2014). Immunoglobulin light chain (AL) amyloidosis, a plasma cell dyscrasia, is the most common subtype. The standard diagnostic algorithm in AL amyloidosis is to obtain a biopsy of a clinically involve organ, and once Congo red positivity is confirmed, perform subtyping analyses with immunohistochemistry or mass spectrometry. Accurate subtyping of amyloidosis is essential to appropriate treatment, as misdiagnosis occurs in up to 10% of patients and may lead to inappropriate administration of chemotherapy (Comenzo Blood 2006; Lachmann NEJM 2002). We sought to determine the patterns of amyloid subtyping among patients with a diagnosis of AL amyloidosis referred to a tertiary referral center for HDM/SCT. Methods: Sequential patients with confirmed amyloidosis, age ≥ 18 years who underwent HDM/SCT between 2001 and 2014 at the Fred Hutchinson Cancer Research Center and University of Washington Medical Center were eligible. Presence of a Congo red-positive biopsy for each patient referred for transplant was confirmed and the pathology reports and medical records were reviewed to determine if subtyping was performed, and which modality was used. Results: Fifty-one patients with AL amyloidosis were referred for transplant; of these, 45 proceeded with HDM/SCT. The organ systems most commonly involved were renal in 34/51, and gastrointestinal in 5/51. Of the biopsies, subtyping was performed in 35 (68.6%), and no subtyping was performed in 16 patients (31.3%). Immunofluorescence was the most common modality used for subtyping in 33 biopsies (94.2%) and laser capture/mass spectrometry (LC/MS) was used in 2 patients (5.7%). All patients had evidence of a clonal plasma cell dyscrasia by bone marrow biopsy and peripheral blood testing. Of the patients without subtyping, 8 (50%) were diagnosed before 2008. Discussion: Misdiagnosis of amyloidosis due to a lack of appropriate subtyping is a well-described and ongoing problem for patients with amyloidosis. These data suggest that definitive subtyping is still not routinely performed in the evaluation of amyloidosis. At our center, efforts to standardize the evaluation of Congo-red positive biopsies using definitive typing are underway. Disclosures Gopal: Seattle Genetics: Research Funding.

Description: Introduction: Network meta-analyses (NMA) are used commonly to allow formal comparisons between drugs that were not compared head-to-head in a clinical trial through a common comparator. Two main assumptions are made to allow such methodology: a) the study samples are drawn from similar populations and b) the common comparator in the trials represents the same intervention. Clinical trials for first-line treatment of chronic lymphocytic leukemia (CLL) offer an opportunity to perform NMA through a common comparator chlorambucil (CHL). In cases where the common comparators differ, the results of the NMA may be impacted; for example, the dose for CHL for most trials was 0.5 to 0.8 mg/kg of body weight given orally on days 1 and 15 of each 28-day cycle (total dose = 70 to 112 mg; denoted as CHL-low arm) in CLL-11 for obinutuzumab (OBI)-CHL and rituximab (RTX)-CHL, in RESONATE for ibrutinib (IBR), and in Knauf 2012 for bendamustine (BEN). The dose for CHL for COMPLEMENT-1 trial for ofatumumab (OFA)-CHL was 10 mg/m2 per day given orally on days 1-7 of each 28 day cycle (total dose = 132 mg; denoted as CHL-high arm). We sought to evaluate the impact of the dose difference, CHL0.5 vs. CHL10 on efficacy outcomes in CLL patients receiving these treatments. Methods: A systematic literature review was conducted in PubMed, Embase, and Cochrane library for the time period of 2010-2016 and in conference proceedings of American Society of Hematology (ASH), the American Society of Clinical Oncology (ASCO), and the European Hematology Association (EHA) for 2014-2016. Search was limited to clinical trials conducted on humans and published in English language. Randomized clinical trials (RCTs) evaluating first-line CLL therapies were included. Bayesian NMA of efficacy outcomes, including progression-free survival (PFS), complete response (CR), and overall response rate (ORR) were conducted without accounting for CHL dose-difference (base-case analyses) and with accounting for CHL dose-difference (secondary analyses) using SAS® (v9.3). For the base case analyses, the CHL-high and CHL-low comparator arms were assumed to have similar effects and treated as the same intervention. For secondary analyses, the two doses of CHL were evaluated as different interventions and trials where fludarabine treatment arms were a common comparator was included. Results: Of the 77 studies screened, four RCTs (n = 1,624) were included in the NMA. In the base-case NMA where CHL dose-difference is not accounted, hazard ratios (HRs) for PFS with OFA-CHL in comparison to OBI-CHL and RTX-CHL were both non-significant (95% CI: 0.01, 〉100), the odds ratios (95% CI) for ORR with OFA-CHL in comparison to OBI-CHL and RTX-CHL were both non-significant, and the odds ratios for CR with OFA-CHL in comparison to OBI-CHL and RTX-CHL were 0.07 (0.01-0.90) and 0.29 (0.01-6.03) respectively. In the secondary NMA where CHL dose difference is taken into account, HRs for PFS and odds ratios for ORR with OFA-CHL in comparison to OBI-CHL and RTX-CHL were both also non-significant, and the odds ratios for CR were 0.03 (0.01- 〉100) and 0.01 (0.01- 〉100) respectively. Conclusion: The differences in estimations and their respective confidence intervals demonstrate the importance of employing appropriate methodology and clinical considerations in NMA. In this case, conclusions arising from the results of two scenarios differed based on accounting for any clinically significant dose differences in "a potential common comparator." When CHL dose was not accounted for, OBI-CHL appeared to be superior to OFA-CHL in CR. After accounting for the dose-difference, OBI-CHL, OFA-CHL and RTX-CHL were similar to each other. Accurate estimation of treatment effects is important as these results are used to inform health policies and treatment guidelines through regulatory or HTA bodies and professional clinical bodies respectively. These data are also used to inform health economic models and budget impact models. Inaccurate results are propagated to future evidence synthesis efforts that further misinform policies and guidelines. Disclosures Mwamburi: Novartis Pharmaceuticals: Consultancy. Bal:Novartis Pharmaceuticals: Employment. Cascella:Novartis Oncology: Employment. Shah:Novartis Pharmaceuticals: Consultancy. Nanavaty:Novartis Pharmaceuticals: Consultancy. Gala:Novartis Pharmaceuticals: Consultancy.

Description: Background: CLL is the most common leukemia in Western World. Hypogammaglobulinemia is the most common immune deficiency detected in CLL. Patients with CLL have increased risk of recurrent infections. Despite hypogammaglobulinemia reported as a cause of both infections and morbidity, controversy exists in its role. Aim: In the present study, we have evaluated the impact of hypogammaglobulinemia at diagnosis on survival and infection risk in CLL. Patients and Mathods: We have included 75 CLL patients diagnosed between 2000 and 2014 with B lymphocyte count 〉5000mm3, CD5, CD19, CD30, CD 23, CD79b positivity and surface Ig light chain restriction in flow cytometry at Ankara University School of Medicine Department of Hematology. The diagnosis and treatment criterias were depended on IWCLL 2008. The patient's performance status was evaluated by physician. If levels were considered decreased when values were below the normal ranges, defined as IgG 6.1-14.9 g/L, IgA 0.8-4.9 g/L and IgM 0.41-2.2 g/L. Overall survival (OS) was calculated by Kaplan-Meier method. Results: The median age at diagnosis was 59 (range, 32-85). 47 patients (63%) were male. The frequencies of RAI staging from 0 to 4 were as follows; 17%, 13%, 25%, 13%, 32%. The ECOG performance status at diagnosis were stage 1 in 81%, stage 2 in %16, stage 3 in %3 of patients. FISH abnormality was detected in 19 patients (25%), del 13q was the most common. 39 patients (52%) (25 fit, 12 unfit, 2 unknown) received treatment. Fit patients got Rituximab-Fludarabine-Cyclophosphomide (R-FC) in first line whereas RFClite was preferred in unfit patients. 37 patients (49%) had any hypogammaglobulinemia at diagnosis; low IgG in 7 (9%), low IgM in 28 (37%), low IgA in 18 (24%) patients. During first year after diagnosis 26 patients (35%) had moderate to severe infections. Twenty five patients (68%) received intravenous immunoglobulin (IVIG) after diagnosis of hypogamaglobulinemia. No significant associations were found with Ig levels and infections. Six patients died during follow-up. 5-year OS in patients with normal and decreased gammaglobulin were 90% vs 86% (P=0.72). There were no survival advantage detected in low IgG, IgM, IgA subgroup analysis. Conclusion: Hypogammaglobulinemia is considered predictive risk of infections. However we have not detected any significant increase of infections or decrease of survival in our cohort. Most of patients with hypogammaglobulinemia received IVIG after diagnosis which would effect our results. Disclosures No relevant conflicts of interest to declare.

Description: Background : TheHevylite assay (HLC Assay) is a novel assay using antibodies that recognize unique conformational epitopes presented by the association of the heavy and light chain constant regions of intact immunoglobulins (Ig) allowing quantitative measurement of each Ig class concentration and generating ratios for each pair (e.g. IgGK/IgGL). Recent studies indicate that HLC Assay can enhance the ability to detect and quantify monoclonal Ig, potentially providing greater sensitivity for detection of minimal residual disease or early relapse after treatment and providing a prognostic indicator of progression free survival (Ludwig, H. et al. Leukemia (2013) 27, 213-219; Kraj, M. et al. Adv Clin Exp Med 2014, 23, 1, 127-133). While HLC Assay could simplify and enhance the assessment of monoclonal protein response in multiple myeloma (MM), its utility as compared to standard assays (SA) (Serum Protein Electrophoresis (SPEP) and Immunofixation (IF)) is not well established. The goal of this retrospective study was to compare the performance of these tests in patients with MM seen at Memorial Sloan Kettering Cancer Center. Methods : We have previously reported on the patterns of relapse and/or progression (R/POD) in 179 patients with MM transplanted between 2001 and 2009 at MSKCC and determined the precise date of R/POD based on IMWG standard clinical criteria using serum and urine PEP and IF, as well as Free Light Chain Assay (FLCA) (Zamarin, Bone Marrow Transplant, 2013). Serum samples from 63 of these patients, collected at the time of R/POD and/or at time points preceding R/POD were analyzed by HLC Assay and compared to results obtained by SA. Results : Among the 63 patients, 22 had IgA and 41 had IgG isotype. Overall, 207 samples were available for all 63 patients, including 72 IgA and 135 IgG samples. Figure 1 shows the concordance of Hevylite ratio (on the Y-axis) with the results obtained by SA (on the X-axis), for IgA and IgG samples, respectively. These graphs reveal an excellent association between HLC Assay and SA results in the IgA samples: all IgA samples revealing an M spike by SPEP had an abnormal HLC ratio even for low M spike levels (between 0 and 0.5 g/dL). Among IgA samples with a monoclonal band detectable by IF, 43 out of 48 samples also had an abnormal ratio by HLC Assay; and among samples with no detectable band by IF, 18 out of 24 had a normal ratio by HLC Assay (sensitivity 90%, specificity 75%, p〈 0.001) (Table 1, Panel 1). Interestingly, when looking at samples taken prior to relapse in IgA patients having achieved CR, HLC Assay was abnormal in 4 out of 7 patients while SA was still reported as normal. In contrast, the lack of association between the SA and HLC Assay is striking for the IgG samples, with poor sensitivity for the HLC Assay to detect monoclonal gammopathies with M spikes below 1 g/dL on SPEP. Among IgG samples with a monoclonal band detectable by IF, only 48 out of 89 samples also had an abnormal ratio by HLC Assay; while among samples with no detectable band by IF, 40 out of 46 had a normal ratio by HLC Assay (sensitivity 53% and specificity of 86% , p 〈 0.001) (Table 1, Panel 2). Conclusions: Although retrospective, this analysis suggests the following: 1) HLC Assay may be more sensitive than IF or SPEP in patients with IgA disease, as it can detect an abnormal HLC ratio at a time prior to relapse by SA, when IF and SPEP are still normal; 2) HLC Assay appears to be less useful in IgG patients, as its sensitivity in these patients appears much lower than IF; 3) There is a need for further detailed analysis on larger prospective cohorts to test the utility of HLC Assay compared to SA in the management of multiple myeloma patients, especially those with IgA disease. Figure 1 Association between HLC Assay and SA (SPEP/IF) results in patients with IgA disease. Figure 1. Association between HLC Assay and SA (SPEP/IF) results in patients with IgA disease. Figure 2 Association between HLC Assay and SA (SPEP/IF) results in patients with IgG disease. Figure 2. Association between HLC Assay and SA (SPEP/IF) results in patients with IgG disease. Disclosures Hassoun: Celgene: Research Funding; Binding Site: Research Funding; Novartis: Consultancy; Takeda: Consultancy, Research Funding. Kazunori:Binding Site: Research Funding. Landau:Prothena: Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx/Amgen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy; Spectrum Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Korde:Medscape: Honoraria. Landgren:Takeda: Honoraria; Amgen: Honoraria, Research Funding; BMS: Honoraria; Medscape Myeloma Program: Honoraria; Merck: Honoraria; Celgene: Honoraria, Research Funding.

Description: Ankyrin repeat and KH domain-containing protein 1, ANKHD1, is highly expressed in myeloma cells and plays an important role in multiple myeloma (MM) progression and growth. ANKHD1 is found to be overexpressed in S phase of cell cycle in MM cells and silencing of ANKHD1 expression leads to accumulation of cells in S phase, suggesting a role in S phase progression (1). Earlier studies by our group reported that ANKHD1 silencing downregulates all replication dependent histones and that this downregulation may be associated with replication stress and DNA damage (2). We observed increased expression of γH2AX protein (phosphorylated histone H2A variant, H2AX, at Serine 139), a marker for DNA double strand breaks (DSBs) and an early sign of DNA damage induced by replication stress, in ANKHD1 silenced MM cells. In the present study we further sought to investigate the mechanisms underlying the induction of DNA damage on ANKHD1 silencing. We first confirmed the increased expression of γH2AX by flow cytometry analysis and observed that both the mean fluorescence intensity as well as percentage of γH2AX positive cells were higher in ANKHD1 silenced MM cells as compared to control cells. Phosphorylation of histone 2AX requires activation of the phosphatidylinositol-3-OH-kinase-like family of protein kinases, DNA-PKcs (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated)andATR (ATM-Rad3-related) that serves as central components of the signaling cascade initiated by DSBs. Hence, we checked for the expression of these kinases and observed increased phosphorylation of both ATM and ATR kinases in ANKHD1 silenced MM cells. There was no difference in the expressions of DNA-PKcs in control and ANKHD1 silenced cells by western blot. We next checked for the expression of CHK1 (checkpoint kinase 1) and CHK2 (checkpoint kinase 2), essential serine threonine kinases downstream of ATM and ATR. We observed a decrease in pCHK2 (phosphorylated CHK2 at Thr 68), with no change in expression of pCHK1 (phosphorylated CHK1 at Ser 345) total CHK1 or total CHK2. We also checked for expression of CDC25a (a member of the CDC25 family of dual-specificity phosphatases), that is specifically degraded in response to DNA damage (DSBs) and delays S phase progression via activation of ATM /ATR-CHK2 signaling pathway. Expression of CDC25a was significantly decreased in ANKHD1 silencing cells, confirming the induction of DSBs, and probably accounting for S phase delay on ANKHD1 silencing. Since there was decrease in active CHK2 (pCHK2) and no change in CHK1 required for degradation of CDC25a, we assume that decrease in CDC25a in ANKHD1 silenced MM cells may be via activation of ATM/ ATR pathway independent of CHK2/CHK1. Expression of several other downstream factors of DSBs induced DNA damage response and repair such as BRCA1, PTEN, DNMT1, SP1, HDAC2 were also found to be modulated in ANKHD1 silenced MM cells. In conclusion, ANKHD1 silencing in MM cells leads to DNA damage and modulates expression of several genes implicated in DNA damage and repair. DNA damage induced after ANKHD1 silencing in MM cells activates ATM/ ATR-CDC25a pathway which may lead to the activation of S phase checkpoint in MM cells. Results however are preliminary and further studies are required to understand the role of ANKHD1 in intra S phase check point. References: 1) ANKHD1 regulates cell cycle progression and proliferation in multiple myeloma cells. Dhyani et al. FEBS letters 2012; 586: 4311-18. 2) ANKHD1 is essential for repair of DNA double strand breaks in multiple myeloma. Dhyani et al. ASH Abstract, Blood 2015; 126:1762. Disclosures No relevant conflicts of interest to declare.

Description: CONTEXT Chronic Lymphocytic Leukemia (CLL) is a clonal proliferation of small mature B-cell lymphocytes diagnosed clinically when the peripheral blood clonal B lymphocyte count is persistently 〉5,000/mcL with distinctive immunological markers defined by co-expression of CD5 and CD23 with additional expression of CD19, CD20 (weak) with weak surface immunoglobulin expression, usually IgM heavy chain. Expression of CD79b and FMC7 is typically negative to weakly positive. With the recent advancement in cancer genetics and the continued understanding of the transforming events in CLL, the importance of the various somatic genomic aberrations has been well documented. Multiple studies have shown the clinical implications of these aberrations in terms of their prognostic and predictive relevance in clinical practice and these genomic aberrations are usually assessed clinically by cytogenetic analysis and FISH. OBJECTIVE We proposed this study to determine the incidence of the genomic aberrations in the CLL patients diagnosed at the Rose Cancer Treatment Center of the William Beaumont Hospital between 2010 and 2015 and to determine their impact on survival among the patients diagnosed during the study period. STUDY DESIGN A retrospective review of all the patients diagnosed with CLL between 2010 and 2015 at the Rose Cancer Treatment Center was conducted with the assistance of the staff in the William Beaumont cancer registry office. We determined the demographic variables and analyzed the incidence of CLL among the subjects diagnosed within the study period. Data analysis was performed using SPSS 21 and Kaplan-Meier curves were plotted for survival analysis and log rank (Mantel-Cox) was used to compare these curves. 12-Month and 36-Month overall survival rates were analyzed by actuarial method. The distribution of the various genomic aberrations was determined using descriptive statistical analysis. RESULTS A total of 151 patients were identified at the Rose Cancer Center of the William Beaumont Hospital during the study period. The median age at diagnosis was 74 years (range 38-101) of which 90 were male (59.6%) and 61 female (40.4%). One-hundred and twenty-four (82.1%) patients were white, six patients (4.0%) were African American, two patients (1.3%) were Asian and nineteen (12.6%) patients declined to identify their race. Analysis of cytogenetic distribution showed that, twenty patients (13.2%) had normal cytogenetic, eleven patients (7.3%) had del(13q) alone, eight (5.3%) had both del(13q) and del(11q), four patients (2.6%) had del(13q) plus trisomy 12 aberrations, ten (6.6%) patients had del(13q) and other karyotypes (TP53, RB1, trisomy 1q, del(6q23), unmutated IGHV). Thirteen patients (8.6%) had trisomy 12 abnormality, five patients (3.3%) had del(11q), three patients (2.0%) had del(17p), twenty-five patients (16.6%) had complex cytogenetic abnormalities and fifty-two (34.4%) patients cytogenetic were not checked (see Fig.1). The median follow-up duration for the cohort was 22.5 months (range 0 to 70 months). The survival rates at 12 months and 36 months for the cohorts based on cytogenetic are described in table 1 and fig.2. CONCLUSION Our study showed that majority of our patients (34.4%) did not have their cytogenetics checked at diagnosis, patients with del(13q) abnormality alone had the most favorable 36-Month overall survival rate and those with del(17p) fared worst with the most unfavorable outcome followed by patients with complex cytogenetic abnormalities. Presence of del(13q) with either del(11q) or trisomy 12 abnormalities appeared to ameliorate their poor and intermediate adverse prognostic effects, respectively. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Background: Mutations of MAP2K1, which encodes MEK1, have been identified in up to half of patients with variant Hairy Cell Leukemia (vHCL).[Waterfall et al., Nat Gen 2014, Mason et al., Leukemia & Lymphoma 2016], and have been associated with vHCL with IGHV4-34 gene usage, which This form of HCL tends to have a worse prognosis than classic HCL or wild type vHCL (Arons et al., Blood 2009), with inferior responses to chemotherapy and shorter durations of remission. Trametinib, an oral inhibitor of MEK1 and MEK2, is FDA approved for treatment of patients with BRAF p.V600E mutant melanoma. We hypothesized that this MEK inhibitor would have activity in MAP2K1 mutant vHCL. Case Report: The patient is a 52 year old man with a history of CD25+, BRAF wildtype, IGHV4-34 usage vHCL diagnosed in 2005. His previous treatments included cladribine, BL22, pentostatin/rituximab, splenectomy, single agent rituximab, ibrutinib, bendamustine/rituximab, and allogeneic transplantation from a matched unrelated donor. The patient experienced disease relapse day +350 post transplant when he developed skin nodules as well as a generalized skin rash. The skin rash appeared clinically consistent with acute GVHD. However, when biopsies of both the skin nodules and skin rash were performed he was found to have relapsed vHCL. He was consented for paired tumor and germline next generation sequencing with a 25-gene amplicon panel which revealed a somatic MAP2K1 K57N mutation that has been shown to constitutively activate MEK [Marks et al., Cancer Res 2008]. As the patient had exhausted the majority of available treatment options, he was prescribed trametinib 2 mg po daily (commercial supply, according to approved melanoma dosing). Within a week of therapy initiation his skin nodules were markedly diminished in size and his generalized rash had resolved. He did develop a new acneiform rash over his face consistent with drug toxicity. This was managed with topical agents with improvement and did not require a dose reduction. Disease restaging following cycle 2 of therapy showed near complete resolution of skin nodules, with disappearance of visible skin rash. Repeat bone marrow biopsy showed unchanged hairy cell index. Skin biopsies were repeated and phospho-ERK (T202/Y204) staining of skin biopsies pre- and post-trametinib were performed (Figure 1). This showed diminished lymphocyte involvement on H&E staining with a decrease in p-ERK expression on immunostaining, indicative of decreased signaling downstream of MEK and consistent with on target trametinib effects. As of this writing, the patient has remained on trametinib for 12 weeks with no recurrence of leukemia cutis rash. Discussion: MEK inhibition with the oral MEKi trametinib is a well tolerated therapy with clinical activity in MAP2K1 mutant vHCL. Additional studies of this agent are warranted. Optimal dose and duration of therapy will need to be explored in prospective clinical trials. Figure 1 Skin biopsies pre- and post-trametinib. (A)(C) H&E staining shows diminished lymphocyte involvement. (B)(D) PhosphoERK immunostaining shows decrease of phosphoERK expression. Bar = 500 μm Figure 1. Skin biopsies pre- and post-trametinib. (A)(C) H&E staining shows diminished lymphocyte involvement. (B)(D) PhosphoERK immunostaining shows decrease of phosphoERK expression. Bar = 500 μm Disclosures Andritsos: Hairy Cell Leukemia Foundation: Research Funding. Anghelina:Hairy Cell Leukemia Foundation: Research Funding. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Genentech: Research Funding; Stemline Therapeutics Inc.: Research Funding. Jones:Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Recently published clinical trials have confirmed the effectiveness of anti-CD38 monoclonal antibody therapy in myeloma. Furthermore, in vitro studies of chronic lymphocytic leukaemia (CLL) cells suggest that CD38 expression can be enhanced by treatment with retinoid derivatives and thus may enhance the cytotoxic effects of anti-CD38 therapy. However, retinoids have been shown to have diverse effects on cellular function and we have previously shown that the retinoid drug acitretin upregulates CD38 expression while also reducing cell homing to the chemokine CXCL12 in primary CLL cells. To investigate possible key mechanisms for these effects, we purified CD20+ B cells from the peripheral blood of 20 CLL patients (9 previously treated, 11 untreated) and, using flow cytometry, measured percentage cell surface expression of CD38 and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4, CD152). We also measured gene expression levels of the key retinoid receptor, stimulated by retinoic acid 6 (STRA6) and it's agonist, retinol-binding protein 4 (RBP4), as well as CTLA-4, cyclin D1 (CCND1) and the transcription factors, lymphoid enhancer factor 1 (LEF1) and signal transducer and activator of transcription 3 (STAT3) using RT-PCR. GAPDH was used as a reference gene. Mean percentage surface expression of CD38 and CTLA-4 was 21.96% and 45.25% respectively. Mean ∆CT gene expression levels of CCND1, CTLA-4, LEF1 and STAT3 were 12.03, 5.57 , 5.99 and 8.98 respectively. RBP4 and STRA6 gene expression levels were undetectable in all 20 patients. Gene expression of LEF1 showed significant correlations with CTLA-4 (rs=0.572, p=0.008), CCND1 (rs=0.61, p=0.004) and STAT3 (rs=0.587, p=0.006). There was also a significant correlation between gene expression of CCND1 and of STAT3 (r =0.499, p=0.025). No significant correlations were found between percentage surface expression of CTLA-4 and gene expression levels of either CTLA-4 or of LEF1. A weak negative correlation between percentage surface expression of CTLA-4 and of CD38 was not statistically significant. Comparing untreated and previously treated patients, there was no significant difference in gene expression levels of CTLA-4 and of LEF1 or in surface expression of CTLA-4. The failure to detect RBP4 and STRA6 gene expression in unstimulated peripheral blood CLL cells is evidence against an autocrine retinoid effect in CLL, although upregulation of STRA6 gene expression following stimulation by retinoids might be anticipated. The Wnt signalling pathway has been shown to be active in CLL, including aggressive disease subtypes, highlighting the potential benefits in targeting this pathway. Intriguingly, CTLA-4 expression, although found to be the most highly induced gene following treatment with recombinant Wnt-3a in melanoma cell lines, is associated with a favourable outcome in CLL, possibly by inhibiting cell proliferation and survival. In contrast, expression of LEF1, which is a direct target of the Wnt signalling pathway, is associated with disease progression in CLL. Our finding that CTLA-4 and LEF1 gene expression levels are strongly correlated suggests that further investigation of the relationship between CTLA-4 and the Wnt/β-Catenin pathway in CLL is required and that targeting of the Wnt/β-catenin pathway may have unwanted consequences on CTLA-4 expression and function. Disclosures Quinn: Celgene: Honoraria; Janssen Cilag: Honoraria.

Description: Background: The established clinical staging systems (Rai/Binet) of chromic lymphoma leukemia (CLL) had limitedly discriminated among prognostic groups, especially for the patients in early stages and without treatment indications. In the past two decades, several prognostic factors have been identified to predict the outcome of patients with CLL, but only a few studies investigated the prognostic improvement of the combination of more markers together. In order to predict the time to first treatment (TTFT) in patients of early stages, we evaluated the prognostic role of conventional bio-markers as well as cytogenetic abnormalities and combined them together in a new prognostic scoring system, the CLL prognostic index. Patients and Methods: Taking advantage of a population of 402 untreated Chinese patients with CLL at early and advanced stage of disease, we identified the strongest prognostic markers of TTFT and, subsequently, in a cohort of 173 patients who had complete data for all 3 variables, we integrated the data of traditional staging system, cytogenetic aberrations and mutational status of immunoglobulin heavy chain variable region (IGHV) in CLL-PI. The median follow-up time was 45 months (ms). Multivariable statistics were applied with software SPSS 17.0, and the end point was TTFT. Results: Based on multivariate Cox regression analysis, three independent factors for TTFT were identified: advanced clinical stage (Rai risk group), 17p deletion and unmutated IGHV. Applying weighted grading of these independent factors, a CLL-PI was constructed based on regression parameters, which could categorize four different risk groups [low risk (score 0), intermediate low (score 1), intermediate high (score 2) and high risk (score 3-6)] with significantly different TTFT (median TTFT of NR, 65.0 ms, 36.0 ms and 19.0 ms, respectively, p

Description: The clinical course of chronic lymphocytic leukemia (CLL) is highly heterogeneous, from stable to a rapidly progressive. The variety of prognostic factors has been already described, nevertheless they are not fully efficient in predicting the course of CLL, especially when the disease is diagnosed at an early stage. Recently, beside well established cytogenetic prognostic factors, novel molecular mutations of predictive value have been identified. Many of them have been thoroughly characterized, including TP53 mutation, which is commonly considered as a strong, negative prognostic factor. The use of next-generation sequencing technology has also revealed previously unknown genomic alterations, such as neurogenic locus notch homolog protein1 (NOTCH1), splicing factor 3B subunit 1 (SF3B1) or myeloid differentiation primary response 88 (MYD88). These new mutations could partly explain the CLL heterogeneity and help in identifying clinically relevant groups of patients. The aim of the study was to characterize CLL patients with NOTCH1, MYD88 and SF3B1 mutations with regard to molecular and immunological prognostic markers in CLL. Peripheral blood mononuclear cells (PBMCs) were obtained from 369 CLL patients at the moment of diagnosis and the median age reached 65 years. Sixty percent of the patients were male. Distribution of disease stages according to the Rai classification was the following: 0 stage=93, I stage=52, II stage=69, III stage=14, IV stage=26. Clinically, the prognostic significance in terms of time to first treatment (TTFT) was assessed for 202 CLL patients. DNA samples were extracted from PBMCs obtained after Ficoll density gradient centrifugation. NOTCH1 c.7544_7545delCT (n=316) in PEST domain (exon 34) and MYD88 L265P (n=323) mutations were investigated by ARMS PCR. Screening for SF3B1 (n=364) mutations K700, E622/R625 and H662/K666 (exons 14 and 15) were performed using HRM analysis and the results were confirmed by Sanger sequencing. The IGHV gene mutations were investigated by Sanger sequencing. NOTCH1 mutations were found in 19/316 (6.0%) patients. Patients harbouring NOTCH1 mutations prevalently have unfavourable prognostic factors including unmutated IGHV gene status, expression of CD38 (〉30%) and expression of ZAP-70 (〉20%). The complete analysis of correlations between NOTCH1, MYD88 and SF3B1 mutations and prognostic markers in CLL are presented in Table 1. Analysis of IGHV subsets in patients with NOTCH1 mutation revealed frequent presence of subset #1 in n=2/19 (10.5%), which is associated with particularly poor prognosis in CLL. Patients belonging to subsets #5, #6, #201 and #202 were also present, each in single NOTCH1 mutated CLL case (5.2%). MYD88 mutation occurred in 12/323 (3.7%), of whom one patient was characterized as subset #2 and another as subset #4. MYD88 mutations were nearly equally distributed in patients with mutated/unmutated IGVH status (5 vs. 7). SF3B1 mutations occurred in 17/364 (4.7%) patients, furthermore two of them carrying negative prognostic features of subsets #2 and one subset #1. Patients belonging to subsets #3 and #6 were also present. Certain negative prognostic factors accompany poor clinical outcome. The assessment of median TTFT revealed the significant differences between patients from various prognostic groups (Fig. 1). Patients with unmutated IGHV gene status were characterized by significantly shorter TTFT than patients harbouring the mutation (p

Description: Chronic lymphocytic leukaemia (CLL) is reported to be an uncommon hematopoietic malignancy in the Indian subcontinent (1.7-8.8%), whereas it is seen in 25-30% of all leukemias in west. Previous reports from India report CLL to occur at younger age, have larger spleen and present in high risk Rai stages. It is possible that this more aggressive disease in India may be due to differences in the biology of CLL in India. Thus, in the present study we aimed to elucidate the prevalence of various known biological and molecular prognostic parameters in CLL patients in India to evaluate the biology of CLL in India. 100 newly diagnosed cases of CLL along with 50 healthy individuals in similar age group, were taken for the study. Flowcytometry was performed for ZAP70 and CD38. Molecular studies were performed to include mutational analysis of IGVH and P53 gene, and PCR RFLP for polymorphism of P2X7 (1513 A-C) and BaxG (-248) A gene. Sandwich ELISA was performed to determine level of serum beta 2 microglobulin and Thymidine kinase. FISH cytogenetics was available in 30 patients. Patients with asymptomatic early stage disease (Rai 0) were monitored without therapy unless they had evidence of disease progression. Patients with intermediate (Rai stage I and II) and high (Rai stage III and IV) risk were treated conventionally with Fludarabine+ Cyclophosphamide + Rituximab (FCR) or Bendamustin+ Rituximab (BR).Response to induction chemotherapy was assessed according to the criteria proposed by the National Cancer Institute (NCI) sponsored Working Group Guidelines for CLL. Outcome analysis was based on Progression free survival (PFS), Treatment free survival (TFS) and Overall survival (OS). Median age of CLL patients at presentation was 60.5 years (range 35 - 82); M:F:: 3.7:1 and haemoglobin 11.3 g/dl (range 4.6 - 15.3g/dl); Rai stage 0 was seen in 26% patients, Rai stage I in 15%, Rai stage II in 28%, Rai stage III in 16% and Rai stage IV in 15% patients. Diffuse involvement of bone marrow biopsy by CLL was seen in 65% patients. Treatment was given to 78% patients during the study period. Response to chemotherapy was observed in 48/78 (61.5%) with complete response (CR) in 19 (24.3%) patients and partial response (PR) in 29(37.2%) patients, while 30/78 (38.5%) patients showed no response. The median follow-up of the study group was 36.11 months (range 1.03 to 71.7 months). High risk cytogenetic abnormalities like 17p- and 11q- were seen in 13.3 % patients .This is thus more common in India than that reported in the West. Expression of Zap70 only was seen in 37% patients, CD38 only expression in 44% of patients and combined expression of Zap70 and CD38 expression was present in 26%.Unmutated IGVH genes were seen in 36% patients, a prevalence similar to that in the West.The highest prevalence was of VH1-69 gene segment (10%) followed by VH3-73; (7%), VH3-48; (6%), VH2-70; (5%), VH3-23; (4%) and VH1-03; (3%). 8/10 of VH1-69 IGVH gene were unmutated and 5/8 patients died. Gene segments VH3-23 and VH3-21, each of which is known to be a bad prognostic marker, were found in 4 and 2 % each. However, VH3-73, VH3-48, and VH2-70 gene segments, which were found to be associated with poor prognosis in our study, showed a higher prevalence in Indians, compared to West. TP53 mutation ,examined in highly conserved region between Exon-5 and Exon-9,was seen in 18/100 (18%) patients,5/18 mutations were seen in Exon-6 and Exon-8. 2 mutations was present each in Exon-7 and Exon-9, and four patients showed mutation in Exon-5. Analysis of P2X7 (1513A-C) gene polymorphism revealed 33/100 (33%) patients to have heterozygous genotype (1513A/C) and 13/100 (13%) patients to have homozygous genotype (1513C/C), while Bax G(-248)A gene polymorphism showed the frequency of GG, GA, and AA genotypes were 78%, 18%, and 4% respectively. P2X7 and Bax gene polymorphism had no prognostic significance. Serum β2-microglobulin level (≥ 4.5 ug/ml) was seen in 36% patients and Thymidine kinase level (≥7.4 U/L) was seen in44% patients. The above results indicate that ,in contrast to reported prevalence from the West, Indian CLL has higher prevalence of bad prognostic markers like CD38 expression, combined Zap70 and CD38expression, 17p-, 11q-, TP53 mutation, β2-microglobulin level and VH3-73, VH3-48, and VH2-70 gene segments of IGVH which may be responsible for a different biology. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: BDNF is a neuronal growth factor that previously showed to exert survival effect in B cells. The role of BDNF in CLL is unknown. Objectives: To study the circulating levels of BDNF in CLL patients and their association with disease prognosis. We also looked for CXCR-4 as a possible mechanisms underlying BDNF effect in CLL. Design and methods: The total BDNF levels and the levels of the BDNF precursor; proBDNF were quantified in the serum of 36 CLL patients and 5 healthy controls by commercial ELISA kits (DY248 and DY3175 DuoSet, respectively, from R&D Systems, Minneapolis, MN, USA). The patients' BDNF levels were correlated to the disease characteristics and clinical course. mRNA and protein expression of the high affinity receptor for BDNF; TrkB were evaluated by real time PCR and flow cytometry respectively. The effect of BDNF on CXCR4 surface expression and migration of CLL cells towards SDF-1 were studied in-vitro. Results: The total serum levels of BDNF in CLL patients was not statistically different compared to healthy controls (mean±SD; 19.9±15.1ng/ml vs. 10.5±9.5ng/ml respectively, p=0.19). Serum proBDNF levels in both CLL patients and healthy controls were under the detection limit of the kit. Within the CLL group we found higher total BDNF levels in patients with mutated immunoglobulin variable heavy-chain (IGHV) status than in patients with unmutated IGHV (25.2±14.6ng/ml vs. 14.1±13.0 in mutated and unmutated status, respectively, p=0.028). We also found higher serum levels of total BDNF in CLL patients in Binet stage A compared to those with a more advance clinical stage (mean±SD ; 24.5±14.6ng/ml vs. 17.0±16.9 and 10.2±9.6 in A,B and C, respectively, p=0.013). Accordantly, higher BDNF levels were detected in patients who had a clinically stable disease compared to patients who had disease progression and required treatment (23.1±14.7ng/ml vs. 13.4±14.5, respectively, p=0.013). The mRNA levels of the BDNF receptor; TrkB, were twofold lower in CLL cells compared to normal B cells. However, the overall TrkB mRNA transcripts were very low in CLL cells and normal B cells compared to the normalized housekeeping genes of Beta-2-microglobulin and CD19. Likewise, surface expression of TrkB in CLL was undetectable using several commercial monoclonal Abs by flow cytometry. In vitro culture of CLL cells in serum free media for 24h resulted in an increased CXCR4 surface expression and greater apoptosis. Culture of CLL cells in the presence of recombinant BDNF (50ng/ml) resulted in downregulation CXCR-4 surface levels and protection from spontaneous apoptosis, irrespectively of the IGHV mutational status. The effect of BDNF on CXCR4 downregulation was also confirmed at 1h culture as it shows to induce similar effect to SDF-1 (50ng/ml) and additive effect when combined with SDF-1. Finally, possible competition between BDNF and SDF-1 was indicated as pretreatment of CLL cells with BDNF inhibit their migration toward SDF-1. Discussion: Our findings show association between high BDNF levels and favorable disease prognosis in CLL. Undetected TrkB expression in CLL cells is compatible with previous reports showing sequestration of TrkB in normal and malignant B cells and suggests TrkB independent effect of BDNF in CLL. The effect of BDNF on the survival and migration of CLL cells via CXCR-4 needs to be further investigated. Disclosures No relevant conflicts of interest to declare.

Description: Introduction : Chronic anemia impairs quality of life and adversely affects survival in patients with MDS. Recombinant erythropoietin (EPO) has been proven in maintaining hemoglobin (Hb) level and reducing red blood cell (RBC) transfusion in this group of patients. However, there were limited reports of EPO-β in MDS patients especially in Asian populations. Method: We have conducted a phase IV, multicenter, prospective, open labelled study in patients with low/intermediate-1 risk MDS to evaluate safety and efficacy of EPO-β 30,000 or 60,000 IU/week up to 12 weeks (NCT02145026). Patients with baseline Hb 〈 10g/dL were eligible if they required RBC transfusion 〈 4 unit/ 8 weeks and serum erythropoietin 〈 500 mU/ml. All enrolled patients will start EPO-β 30,000 IU weekly and their erythroid response will be assessed at week 4 and every 4 weeks thereafter until the end of study. If hemoglobin level reaches ≥ 12 g/dL at any time, EPO-β should be discontinued until Hb levels are ≤ 10 g/dL. While patients with Hb level is less than 12 g/dL and increased less than 1 g/dL from baseline level, a 60,000 IU weekly of EPO-β will be administered subcutaneously until week 12. Up to the clinical cut-off date for this interim analysis, 58 patients were screened, 27 of which were eligible for safety analysis whereas 25 patients were eligible for primary efficacy analysis to evaluate response according to International Working Group (IWG 2006) response criteria in MDS. Results: The median age of patients was 74.6 (range; 53.1-88.5), 18 (67%) were female. Three major subtypes of MDS were refractory cytopenia with multi-lineage dysplasia (RCMD, 44%), refractory cytopenia with uni-lineage dysplasia (RCUD, 32%) and refractory anemia with ring sideroblasts (RARS, 16%). Baseline Hb, hematocrit (Hct) and serum EPO were 8.1±1.3 g/dL, 25.2±3.7% and 89.5±116.6 mU/ml respectively. Twenty-five patients (92%) had serum EPO ≤ 200 mU/ml. There were 16 patients (64.0%) achieved hematologic improvement on erythroid (HI-E), while 6 patients (24.0%) achieved hematologic improvement on platelet (HI-P). Eleven patients (44.0%) (68% of responding patients) achieved Hb≥12 g/dL. Sixteen of 23 patients (69.6%) with serum EPO ≤ 200mU/ml achieved HI-E. Proportion of patients who required at least one RBC transfusion was reduced from 37% (n=10) to 11% (n=3) at the end of the study. By univariate analysis, none of baseline characteristics, including age, gender, comorbidity and MDS subtype, predicted response to EPO-β. From 27 patients eligible for safety analysis, there were 14 adverse events (AEs) and one serious adverse event (SAE) were reported in 13 patients (48.1%). The most frequently reported AEs were increased blood pressure (28.6%; all grade1), infections (21.4%) and gastrointestinal disorders (14.3%). There was no major AEs or SAEs which considered to be related to study drug by the investigators including hypertension, pure red cell aplasia (PRCA), thromboembolism and seizure. Conclusions Preliminary results suggest efficacy and safety of EPO-β in the treatment of anemia in low/intermediate-1 risk MDS patients. Two-third of patients demonstrated HI-E. There were no new unknown AEs found in this study and no SAEs considered related to study drug. Disclosures Charnwiboonsri: Roche (Thailand) Ltd.: Employment.

Description: Myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by a deregulation of blood cell formation with cytopenia in varying degrees and frequently develop into acute myeloid leukemia (AML). Next generation sequencing (NGS) has significantly contributed to diagnosis and prognostication in patients with MDS. To explore the role of acquired mutations in MDS biology and clinical features, we performed mutational profiling of 111 genes in 125 patients with MDS based on target-sequencing. The results showed that 89% of patients had one or more carcinogenic mutations. Mutation frequencies of several genes, including ASXL1 (16.8%), RUNX1 (14.4%) and TET2 (12%), were above 10% in MDS patients. According to univariate analysis in 108 patients with survival data, mutations of GATA1/2, TP53 and DNMT3A were identified as adverse prognostic factors for overall survival (OS), while RUNX1, KRAS/NRAS, SRSF2 and TET2 mutations were determined as unfavorable factors for progression free survival (PFS). By multivariate COX regression analysis, mutations of KRAS/NRAS, GATA1/2 and DNMT3A were independent risk factors for OS, whereas IDH1/2 gene mutations were favorable factors for PFS. By evaluation of the clinical benefit of hypomethylating agents (HMAs), it showed that for patients harboring mutations associated DNA methylation or not and receiving HMAs treatment or not, the order of the outcome from good to bad was geneWT with non-HMAs, geneWT with HMAs, genemutwith HMAs and genemutwith non-HMAs. Therefore, target-sequencing of mutational spectrum is a feasible and highly promising prediction method for prognostic evaluation in patients with MDS, which would contribute to personalized therapeutic decisions. Disclosures No relevant conflicts of interest to declare.

Description: The recent advances in cytogenetic and molecular diagnostic techniques has provided a better understanding of biology of CLL and a better prediction of disease progression and refractoriness but they are not easily accessible to all Institutions. CLL cells depend on microenvironmental interactions for proliferation and survival that are at least partially mediated through B cell receptor (BCR) signaling. An increased number of studies describe the emerging role of neutrophils and monocytes as mediators of the inflammation process and antitumor immunity modulation that support tumorigenesis and tumor progression. The neutrophil to lymphocyte ratio (NLR), has been recently published as new marker of systemic inflammation associated with outcome in different cancer types. In our study we retrospectively evaluated the prognostic significance of peripheral blood neutrophils, monocytes and non-neoplastic lymphocytes in a unicentric, unselected, CLL cohort of 400 CLL patients (160 were treated). The study was approved by the institutional board review;informed consent was obtained from patients. Using the blood count and the Flow cytometric analysis reports, peripheral blood absolute neutrophil count (ANC), absolute monocyte count (AMC) and absolute T-lymphocyte count (ALC-CD3+) were evaluated and the neutrophil to T-lymphocyte ratio (NLR), the monocyte to T-lymphocyte ratio (MLR) and the neutrophil to monocyte ratio (NMR) were calculated. Clinical and biological data from all patients were also retrieved. Serial count and ratio were evaluated at diagnosis, during follow up and at relapse to make a better comparison with the major clinical and prognostic markers commonly used. The median ratio for NLR 2.67 and patients with pre-treatment NLR 〉 3 had a shorter time from diagnosis to treatment initiation. CLL patients showed an increase in the absolute number of monocytes compared to normal controls (788±65 cells/μL vs 469±51 cells/μL, p=0.005) and MLR appeared higher in advanced stage (stage Binet C) and bulky disease (p=0.06). High level of both NLR and MLR (cut off 〉3) correlated with the presence of a complex karyotype detected by FISH (p=0.03, p=0.016). NLR ratio was associated with the absence of serum prognostic markers, such as the expression of CD38, ZAP-70 and CD49d. This result, apparently conflicting, could strengthen the NLR as an independent prognostic factor. NMR was higher in asymptomatic patients (absence vs. presence of B symptoms, p=0.02) and this data resulted independent from disease stage. NMR median value was significantly higher in untreated patients than in patients who received treatment (p=0.01), strengthening the hypothesis that this ratio is associated with a more indolent form of disease. In this contest, the subset of patients with CD49d positive disease showed the lowest NMR value. This data seems of relevance, being CD49d a recently discovered and validated prognostic marker. ANC/ALC and AMC/ALC ratio significantly increased at relapse compare to baseline (NLR average onset 2.3±0.4 vs 3.4±0.6 at relapse). The median NMR value showed conversely the opposite trend: NMR is reduced in first relapse (NMR average onset 7.2±0.4 vs. 5.4±0.5 at relapse). These combined ratios reflect both the inflammatory status, the immune response and the microenviromental network that contribute to aggressive tumor biology and disease progression. They are simple, cheap, easily measured and reproducible and can be integrated into daily clinical practice as new prognostic markers for CLL. Disclosures Chiarenza: Gilead: Consultancy; Janssen: Consultancy; Roche: Consultancy.

Description: Background: 'Myelodysplastic syndrome (MDS) with isolated del(5q),' as defined by the World Health Organization (WHO) criteria (SwederlowSH, et al, 2008) is a unique pathological entity with favorable outcomes. The 2016 revision to the classification expands this entity to include cases that have an additional cytogenetic abnormality, with the exception of monosomy 7 or del(7q) (Arber DA, et al, Blood 2016). The objective of our study was to evaluate the prognostic impact of an additional cytogenetic abnormality, other than monosomy 7 or del(7q), in patients with 'MDS with isolated del(5q)'. Methods: After due IRB approval, the Mayo Clinic MDS database (n=1067) was utilized for this study. All patients had bone marrow (BM) biopsies and cytogenetic studies performed at diagnosis. The International Society for Cytogenetic Nomenclature guidelines were used for cytogenetic nomenclature, while the 2008 and 2016 WHO criteria were used for morphological diagnosis. Results: Patient Characteristics: 72 patients (7.2%) met the 2016 WHO criteria for 'MDS with isolated del(5q)' of which 60% were female and median age was 74 years (28-90). In 61 (85%) cases del(5q) was the only cytogenetic abnormality, while in 11 (15%), del(5q) was present with an 'additional cytogenetic abnormality' (ACA). One additional case within the database had del(5q) accompanied with monosomy 7, which was not included in the analysis. Risk stratification by IPSS-R was as follows; 24 (29%) 'very low', 44 (64%) 'low' and 4 (6%) 'Intermediate' risk, with no patient classified as 'high' or 'very high' risk. At a median follow up of 43 months, 55 (76%) deaths and 5 (7%) leukemic transformations were documented. del(5q) versus del(5q) with an additional cytogenetic abnormality- phenotypic correlates: In the 'del(5q) with ACA' group, the additional abnormalities included trisomy 8 (n=4), del(20q) (n=3), der(9;18) (n=1), inv(3)(p25,q21)(n=1), -Y (n=1), and i(Xp) (n=1) (Table 1). There was no significant difference between the 'del(5q)' and 'del(5q) with ACA' groups in terms of age, gender, hemoglobin, platelet count, white cell count, absolute neutrophil count, bone marrow blast percentage or transfusion requirement. A greater proportion of the 'del(5q) with ACA' group (27%) had IPSS-R risk in the 'intermediate' category compared to the 'del(5q)' group (2%) (p=0.01). 18 of 42 cases diagnosed after 2004 (43%) were treated with lenalidomide, with no difference in the proportions treated between the two groups (p=1.00). del(5q) versus del(5q) with an additional cytogenetic abnormality- impact on overall survival (OS) and leukemia-free survival (LFS): The median survival of the cohort was 54 months. Survival was not significantly different between the 'del(5q)' group (median 55 months) and the 'del(5q) with ACA' group (median 38 months) (p=.75, Figure 1). This finding was consistent when analysis was restricted to patients in both groups treated with lenalidomide (p=0.29). The incidence of leukemic transformation in the del(5q) group was 5%, compared with 18% for the 'del(5q) with ACA' group (p=0.16), however there was no significant difference in LFS between the two groups (p=0.57). Conclusion : In our cohort of primary MDS patients meeting the 2016 WHO definition of 'MDS with isolated del(5q)', we confirm no significant survival difference between cases with del(5q) as the sole cytogenetic abnormality versus cases where del(5q) was accompanied by an additional cytogenetic abnormality. Table 1 Additional Cytogenetic Abnormalities with del(5q): Table 1. Additional Cytogenetic Abnormalities with del(5q): Figure 1 del(5q) vs del(5q) with an additional abnormality (ACA): Overall Survival Figure 1. del(5q) vs del(5q) with an additional abnormality (ACA): Overall Survival Disclosures Al-Kali: Onconova Therapeutics, Inc.: Research Funding; Celgene: Research Funding.

Description: Biochemical aspects of cellular process are well characterized, but more recently, it has been shown that cells dynamically sense and respond to biophysical cues such as substrate stiffness and geometrical constraints; physical cues even direct cell differentiation and stem cell lineage (Discher et al, Science, 2005). In hematology, we know that platelets are shear activated and attenuate force based on substrate stiffness, and that endothelial cells align with flow and are activated by shear stress. Blood cells pass through, and interact with, biological matrices such as fibrin clots and the vascular wall, but the physical and biochemical aspects of these interactions are indistinguishable from one another in vivo. As such, there is a gap in knowledge as to how blood cells respond to matrices as they transit through them. To decouple the physical and biochemical interactions of blood cells and biological matrices, we sought to recreate the physical geometry of a fibrin network in a controlled, non-biological, in vitro microfluidic system. To this end, we designed a two-part microfluidic device comprised of an array of micron sized pillars (~1 µm diameter, 3 µm height, and 2 µm gap between pillars) overlaid with a microfluidic channel (Fig 1). The dimensions of the pillars are on the order of the diameter of fibrin fibers and the mesh size of a fibrin gel (Okada et al, J. Biol. Chem, 1985), while channels of various dimensions can be bonded over the pillar array to represent various biological scenarios. Standard microfluidic processes cannot produce pillars with the feature sizes reported herein, so electron beam lithography was used to create the mold from which the elastomeric pillars are made. The biophysical interaction of platelets flowing through fibrin mesh (absent biological factors) was recreated by a pillar array oriented perpendicular to the direction of flow in a 6 µm tall channel. When washed platelets are perfused through the system, they adhere to the pillars, aggregate, and form an occlusive mass that extends to the edges of the array (Fig 2A). Platelet adhesion initiates exclusively at the pillars and aggregation propagates to the extents of the channel area perpendicular to flow, resulting in channel occlusion and flow cessation. These findings show that in the absence of platelet agonists and biological ligands, platelets are activated by the shear environment afforded by the presence of fibrin fibers. Thus, in addition to the biochemical players in clot formation, the geometry of the fibrin mesh plays a role in platelet adhesion, and clot propagation. As expected, passive adsorption of fibrinogen and collagen to the pillar surfaces enhances platelet aggregation, as evidenced by a decrease in time to channel occlusion from 10 min to 2 min and 6 min, respectively. With thus we see the synergistic effect of biophysical and biochemical factors in clot propagation. This novel microfluidic system both separates biophysical and biochemical aspects of clot formation and allows researchers to specify the precise location and extent of clot formation in vitro. Platelets are not the only blood cells to interact with and react to physical barriers. Red blood cell (RBC) deformation has been historically studied in single cell assays, SEM studies of fixed clots, and more recently after RBCs have passed through a filtration system comprised of either beads or long slits (Deplaine et al, Blood, 2010); however, real time visualization of RBC deformation in geometries representative of biological matrices has remained elusive. The deformation (and possible fragmentation) that RBCs undergo when passing through the physical challenges of a fibrin matrix or the interendothelial slits of the spleen can be visualized in our system: an array of pillars overlaid by a 3 µm channel. Our findings visually suggest that red blood cells are able to deform through the matrix with little effect on their membranes, and that exposure to high shear gradients alone does not cause cell fragmentation (Figure 2B). The ready deformation and transit of healthy RBCs in our system confirms recent computational studies of RBC filtration by the spleen (Pivkin et al, PNAS, 2016). Further studies will give insight into the deformation and transit of sickled cells and malaria infected RBCs in physical matrices. Overall, our microfluidic studies give novel insight into the biophysical aspect of blood cell interactions with biological matrices. Disclosures Lam: Sanguina, LLC: Equity Ownership.

Description: BACKGROUND: Agent Orange (AO), a 1:1 mixture of herbicides + TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), was used during the Vietnam War to destroy dense jungle and enemy crops. In 2002 the Department of Veteran Affairs (VA) determined that chronic lymphocytic leukemia (CLL) was associated with AO exposure. Case-control studies suggest an increased risk of death from CLL in areas where herbicide use was highest. There is also an increased incidence of other cancers (prostate, melanoma) in AO-exposed veterans. Limited data exists as to the specific impact of AO exposure on CLL disease presentation and outcome. METHODS: Patients (pts) diagnosed with CLL from 2009-2013 were identified in the National VAMC Tumor Registry. Baseline demographic and laboratory parameters were obtained, including Rai stage, marrow cytogenetics (when available), and lymphocyte doubling time (LDT). AO exposure was identified according to the medical record. The VA Benefits and Compensation officers determine AO exposure based on whether a person served on land and in the brown waters in Vietnam during the appropriate timeframe. Timing and types of CLL therapies were identified to determine if AO exposure influenced CLL treatment. RESULTS: 2052 CLL pts were identified, of which 418 had AO exposure. AO-exposed pts presented at a younger age (63.2 versus [vs] 70.5 years (yrs), p

Description: Chronic lyhocytic leukemia displays a strong dependency on tumor-stroma interactions. CLL cells were found to rapidly die when cultivated in vitro, whereas co-cultivation with mesenchymal stromal cells promotes cell proliferation and ensures leukemia cell survival. It is therefore of great interest to obtain a thourough picture of the exact molecular players involved in this critical interplay. While a number of protagonists are already well described, there are still unresoved issues concerning the effect of stromal cells on the metabolism of CLL cells. In this study we co-cultivated for one week primary CLL cells obtained from peripheral blood with mesenchymal cells (hMSC). Subsequently, hMSC were lysed and fractionated to obtain cytoplamic, nuclear and secreted proteins. To allow comparative analyses, B-cells from healthy individuals and hMSC were co-cultivated. Mass spectrometry-based proteomic analysis resulted in the identification and relative quantification of more than 3700 proteins. Comparative analyses between co-cultures with CLL and healthy B-cells showed that already at a ratio of 0.25% CLL cells could induce a significant proteomic alteration in hMSC. Furthermore, hMSC were found to undergo a metabolic change upon co-cultivation with CLL cells, an effect which was most pronounced in the secreted protein fraction. Annotation analyses according to gene ontology revealed that proteins related to "generation of precursor metaboites" were significantly up-regulated in hMSC upon co-culture with CLL cells. This indicated that these stromal cells "feed" the leukemia cells by producing and secreting high energy metabolite, satisfying the elevated metabolic demands of the CLL cells. Revealing the metabolic interactions between CLL cells and surrounding stromal cells by mass spetrometry-based methods should contribute to the development of innovative treatment strategies. Disclosures No relevant conflicts of interest to declare.

Description: Background: The accurate detection of cytogenetic abnormalities in Acute Myeloid Leukemias (AML) and Myelodysplastic Syndromes (MDS) play an important role in prognostic value and often assist in therapeutic choice. The most commonly used genetic test for the diagnostics of these conditions is karyotype (KT), which is able to detect frequent non-random chromosomal abnormalities. However, this technique requires metaphases and offers low resolution (5Mb), which may lead to the loss of clinically important smaller genomic rearrangements. Therefore, a complementary technique is sometimes needed. FISH, for example, is a highly reliable technique, it can be performed in interphases, has higher resolution, and can detect balanced translocations (important for prognosis mainly in AML), although it is very expensive. As an alternative method, MLPA is usually less costly, is able to evaluate more genomic regions at once and is performed with genomic DNA, which is convenient for biological material availability. On the other hand, it is not able to detect balanced translocations due to its semi-quantitative PCR-based method. The aim of this study was to evaluate MLPA as a complementary technique to KT, and to compare the outcomes of MLPA and FISH panels for the high resolution detection of common unbalanced genetic alterations in MDS and AML. Methods: A total of 23 samples from patients diagnosed with MDS (n=16)/AML(n=6) were tested for KT and MLPA. 20 metaphases were analyzed in G-banding KT. MLPA (Kits P144 and P145, MRC-Holland) was performed with genomic DNA extracted from bone marrows. A subset of 10 samples was tested for FISH MDS/AML panel (PML/RARA, CBFB/MYH11, RUNX1/RUNX1T1, MLL rearrangement, del5q, del7q, de17p and del20q, Cytocell), and the outcomes were compared to MLPA. Reagent costs were also compared between MLPA and FISH techniques. Results: Outcomes from KT and MLPA were mostly concordant: 91.3% of samples had either concordant or partially concordant results (considering "partially concordant" when MLPA could detect smaller genetic alterations not achieved by KT). Discordance between KT and MLPA occurred in two cases: (i) MLPA detected a deletion of RUNX1 gene (chr 21) in a previously normal KT, and (ii) MLPA showed different abnormalities than those in a complex KT case, with low mitotic index. When comparing regions that are notoriously related to MDS/AML (-5/del5q, -7/del7q, +8, del11q, del13q, del17p, del20q and del21q), all results were concordant between MLPA and KT. Comparison of MLPA and FISH was performed with 10 samples, of which 4 were negative for genetic alterations and 6 were positive. 90% of samples had concordant results, and 10% (one sample) showed extra genetic alterations in MLPA (del7q; del11q and del20q). This is expected since MLPA analyzes more genetic regions then FISH MDS/AML panel at once. Genomic regions accessed both by MLPA and FISH included -7, +8, del17p, del21q. Regarding financial expenses (cost of commercial MDS/AML panels and additional reagents), MLPA costs approximately U$280.00 (considering test sample plus internal controls), while FISH costs U$555.00 (values were converted from quotes provided by regional distributors and converted to dollars). Conclusions: MLPA results are equivalent to FISH for the detection of MDS/AML frequent unbalanced genetic alterations. Both techniques are reliable as complementary molecular biology tests for KT. Moreover, MLPA is a friendly and less expensive technique for the detection of high resolution genomic unbalanced abnormalities, frequently adding extra information to KT results. Nevertheless, it is important to notice that only FISH can detect balanced translocations with significant prognostic value in AML. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Fli1 (Friend leukemia virus integration 1) together with other transcription factors induces the megakaryocytic differentiation of MEP (megakarycytic and erythroid progenitor). Refractory anemia and thrombocythemia is typical for 5q- syndrome. We found increased mRNA level of Fli1 in mononuclear bone marrow cells of 5q- syndrome patients in comparison with healthy controls (Neuwirtova et al., Ann Hematol 2013). The reason of the elevated Fli1 in 5q- syndrome is haploinsufficiency of microRNA-145, which targets Fli1 mRNA (Kumar et al., Blood 2011). Due to haploinsufficiency of RPS14 in 5q- syndrome non-consumed ribosomal proteins cause ribosomal stress and inactivate HDM2 in erythroblasts. E3 ubiquitin ligase HDM2 regulates p53 level by p53 degradation in proteasome. Inactivated HDM2 in erythroid precursors of 5q- syndrome leads to apoptosis of erythroblasts and to anemia. Why ribosomal stress does not cause thrombocytopenia and ineffective megakaryopoiesis as well? Our previous results support significant role of Fli1 in this process. Fli1 binds to promoter of the HDM2 gene and increases its transcription (Truong et al., Oncogene 2005). The increased activity of HDM2 in megakaryocytes inspite of ribosomal stress maintains p53 regulation and its degradation in proteasome. Megakaryopoiesis remains effective. Why it is not the case in erythroid precursors? To answer this question it was necessary to detect Fli1 as the protein and to determine in which cells Fli1 is present. Material and Methods. Twenty-three control representative bone marrow trephine biopsies of patients from controls (8 negative staging biopsies in lymphoma) and of patients with various hematological diagnoses (7 MDS with normal chromosome 5, 4 MPN, 3 AML and 1 RARS-T) and from 15 patients with 5q- syndrome were examined. In 13 patients with 5q- syndrome, samples taken before and 6 months after lenalidomide (Revlimid) therapy were available. The expression of Fli1 protein was investigated by immunohistochemistry (IHC). Expression of Fli1 on erythroid precursors was studied by double staining IHC procedure utilizing antibodies against Fli1 and either glycophorin A or E-cadherin known as reliable markers for erythroid precursors. Results. Nuclear expression of Fli1 was demonstrated in normal as well as in dysplastic megakaryocytes, in most cells of granulocytic series and lymphocytes. No staining for Fli1 was seen in erythroblasts and proerythroblasts visualized by expression of either glycophorin A or E-cadherin both in 5q- syndrome and controls. There were no significant differences in Fli1 expression between samples taken before and after lenalidomide treatment.The used IHC technique does not permit quantitative analysis of Fli1 protein levels. This fact could explain why we did not find any difference in Fli1 protein labeling in megakaryocytes before and after lenalidomide treatment while Fli1 mRNA level was decreased in majority of 5q- syndrome patients after six months of this therapy. Conclusion. Fli1 expression was found in normal as well as in dysplastic megakaryocytes. However, no Fli1 positivity was found in erythroid precursors in both 5q- syndrome and controls. Negativity of Fli1 expression in erythroid precursors in 5q- syndrome support our hypothesis of protective role of Fli1 against apoptosis under ribosomal stress in megakaryocytes in contrast to erythroblasts lacking Fli1. This protective role of Fli1 in megakaryocytes consists in Fli1 potentiation of expression of the E3 ubiquitine ligase HDM2 (Truong et al., Oncogene 2005). The presence of increased Fli1 in megakaryocytes helps to explain effective megakaryopoiesis in 5q- syndrome and is the answer to the question in the title of our abstract. Supported by Ministry of Health, Czech Republic-conceptual development of research organization Institute of Hematology and Blood Transfusion 00023736, RVO-VFN64165 and PRVOUK P-27/LF1/2. Disclosures No relevant conflicts of interest to declare.

Description: Introduction : Oxidative stress is closely related to iron overload in myelodysplastic syndrome (MDS) and induces DNA damage. A recent study has suggested that oxidative stress leads to increased mutation frequency in a murine model of MDS, and that additional mutations lead to the progression of MDS. We have recently reported that oxidative stress marker reflects not only iron overload but also disease progression in a MDS patient (2016, Shimizu et al). So there is a possibility that the increase in oxidative stress markers may have not only caused the iron overload but also resulted in the disease progression of MDS. To clarify this point, we evaluated the association among oxidative stress marker and disease progression in eight patients with MDS. In this study, we first examined the oxidative stress marker, derivatives of reactive oxygen metabolite (dROM), ferritin in serum and Wilms Tumor 1 gene (WT1) in the peripheral blood throughout the course of treatment. Then, we analyzed the correlations of the levels of dROM, ferritin and WT1. Next, we investigated ROS expression in each fraction of WBC by flow cytometry in a healthy donor and some MDS patients. Materials and methods: Levels of serum dROM were measured by colorimetry, using samples of MDS (RA n=3, RAEB-1 n=4, RAEB-2 n=1). Ferritin in serum and WT1 in peripheral blood were also measured. Statistical analyses were performed by Pearsonfs test. The ROS expression was evaluated during the clinical course by flow cytometry. Results: Table 1 shows the relationship between dROM, ferritin and WT1 in MDS patients throughout the treatment. Case 1 to 3 patients showed disease progression with excess blasts in spite of azacitidine treatment. These patients showed a correlation tendency between dROM and ferritin, dROM and WT1. Case 1 showed close relationship between ferritin and WT1. Case 3 shows slight relation between ferritin and WT1. Case 4 to 8 patients showed no disease progression. They were treated with supportive therapy, including blood transfusion or cytokine therapy. These patients showed no relation between dROM and the elevation of ferritin and WT1. In these patients, there was no relation between ferritin and WT1 except Case 6. We also evaluated the score of WPSS, IPSS and dROM at diagnosis. dROM did not correlate with WPSS and IPSS. Figure 1 shows increased ROS expression during disease progression in a MDS patient compared with healthy control. Considering that other MDS patients with excess blasts showed the similar pattern, the present result suggested that the oxidative stress markers were produced by tumor-derived cells. Conclusions: dROM levels were slightly related with the ferritin and WT1 mRNA expression levels in MDS patients with excess blasts. We speculate that the increase in the oxidative stress markers is a cause of not only iron overload but also the disease progression of MDS. Oxidative stress markers may contribute to evaluate the progression of treatment efficacy for MDS patients. Table 1 Patientfs characteristics and correlation between serum dROM and ferritin, WT1 mRNA in MDS patients. Table 1. Patientfs characteristics and correlation between serum dROM and ferritin, WT1 mRNA in MDS patients. Figure 1 The increment of ROS expression during disease progression in a MDS patient with excess blasts. Figure 1. The increment of ROS expression during disease progression in a MDS patient with excess blasts. Disclosures Nakaseko: BMS: Honoraria, Research Funding; PFIZER: Honoraria, Research Funding; NOVARTIS: Honoraria.

Description: BACKGROUND AND AIMS The high incidence of mutations in patients with myelodysplastic syndrome (MDS) strongly suggests an implication of defective DNA repair mechanisms in the MDS pathogenesis. Based on the hypothesis that genetic changes are amplified during evolution of MDS-cell clone and disease progression; we investigated abnormalities in DNA repair gene expressions and monitored their possible development during MDS progression. Further, we focused on sequencing of selected DNA repair genes and searched for their mutations associated with the disease. METHODS First, gene expression of 84 DNA repair genes in bone marrow (BM) CD34+ cells of 18 MDS patients was measured by RT² Profiler PCR Arrays (Qiagen). Validation of expression data in selected genes was performed on a cohort of 100 MDS patients. Moreover, paired samples from 15 patients with disease progression were used for monitoring of RAD51 and XRCC2 gene expressions in the course of disease. Mutational analysis was performed on 84 DNA repair genes in 16 patients by targeting next generation sequencing (NGS) (SeqCap EZ System, NimbleGen). Detected mutation was confirmed by Sanger sequencing. Multivariate analysis using a Cox regression model to determine the independent impact of each variable (BM blasts, hemoglobin, neutrophils, platelet count, karyotype, and gene expressions of RAD51 and XRCC2) examined for overall survival (OS) was done. RESULTS RAD51 (p

Description: Introduction: Myelodysplastic syndromes (MDS) are considered as a "stem cell disorders", in which hematopoietic stem cells and lineage-committed progenitor cells acquire genetic and epigenetic alterations and provide aberrant, clonal hematopoiesis, sometimes resulted in the progression to acute myeloid leukemia (Elias HK et al, Oncogene 2014). We previously reported a rare case of which the patient developed Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) 2.5 years after being diagnosed with MDS (Kohno T et al, Br J Haematol 1996). p190 BCR-ABL1 mRNA was detected in the Ph+ALL cells. Metaphase cytogenetics showed the karyotypes: 46, XY, 20q- in MDS phase and 46, XY, t(9;22)(q34;q11), 20q- in ALL phase, indicating that MDS and Ph+ALL in this patient were of the same clonal origin. To uncover the detail of the clonal evolution, we analyzed bone marrow samples of MDS and Ph+ALL in this patient by targeted massively parallel sequencing with a panel of 154 genes including known driver genes of hematologic malignancies. Methods: Genomic DNAs (gDNAs) were extracted from the bone marrow mononuclear cells of MDS and Ph+ALL in this patient. Targeted sequencing was performed on the Illumina HiSeq2500 platform. Single nucleotide variants (SNVs) and small insertions and deletions (INDELs) were called using HaplotypeCaller of Genome Analysis Toolkit (GATK) version 3.4-46. We also attempted to detect the breakpoint of BCR-ABL1 translocation from the targeted sequencing data using the computational method, BreaKmer (Abo RP et al, Nucleic Acids Research 2015). The candidates of the mutations and structural variations were validated by amplicon-based deep sequencing and Sanger sequencing. Copy number variations were analyzed using Affymetrix CytoScan HD Array. Results: The mutations in ASXL1 and U2AF1 genes were identified in the MDS sample with variant allele frequencies (VAFs) of about 45%. At the progression of Ph+ALL, the mutations in SETBP1, SMC1A, and SLC5A8 genes were newly acquired while the ASXL1 and U2AF1 mutations were also identified with the same level of VAFs (about 50%) as the other mutations. VAFs of all of the mutations were decreased to about 20% after the chemotherapy for Ph+ALL, and then increased to about 40% at the recurrence of the disease. Furthermore, we identified the breakpoint of BCR-ABL1 translocation at intron 1 of ABL1 genes and intron 1 of BCR genes, that is the well-known cluster region, m-bcr, only among the samples of Ph+ALL. Copy number analysis confirmed that both MDS and Ph+ALL samples harbored the deletion of chromosome 20q. And the deletion of IKZF1 gene, which is frequently identified in Ph+ALL cases (Mullighan CG et al, Nature 2008), was not identified during the progression from MDS to Ph+ALL. These results demonstrated that the MDS clone harboring 20q- and ASXL1 and U2AF1 mutations acquired the mutations in SETBP1, SMC1A, and SLC5A8 genes and the p190 BCR-ABL1, resulted in the development of Ph+ALL in this patient. Conclusion: The alterations of SETBP1, SMC1A, and SLC5A8 genes are usually reported in myeloid malignancies (Makishima H et al, Nat Genet 2013, Kon A et al, Nat Genet 2013, Whitman SP et al, Blood 2008). Previous study in transgenic mouse demonstrated the distinct role of p190 BCR-ABL1 in the development of an ALL (Voncken JW et al, Blood 1995). Recapitulating this scenario, p190 BCR-ABL1 may play a critical role in the development of Ph+ALL from the MDS stem cells in this patient. This study may provide a new insight into the stem cell origin of MDS and the role of p190 BCR-ABL1 in the development of Ph+ALL. Disclosures No relevant conflicts of interest to declare.

Description: Background and Aims: Myelodysplastic syndromes (MDS) are mainly diagnosed in the elderly with an increasing burden on healthcare systems. As a consequence, population-based studies are important in order to estimate the evolution of this emerging disease in different countries. Objectives: The objective of this study was to provide trends ofannual case frequency, morphological subtypes, incidence, mortality and survival of patients diagnosed with MDS in Switzerland between 2001 and 2012. Methods: A retrospective, population-based, epidemiological study was carried out on MDS cases reported to the Swiss Cantonal Cancer Registries and aggregated by the National Institute for Epidemiology and Cancer Registration. The Swiss Federal Statistical Office provided mid-year population estimates and cause of death statistics. Due to changes in the WHO classification of MDS, data was stratified for two time periods 2001-2007 and 2008-2012, respectively. 56 million person-years (py) were observed, covering 60%-65% of the Swiss population, during a time period of 12 years. Results: 2138 MDS cases were reported with a median age of 77 years (range of means: 75-78 years). The estimated annual case frequency increased from 263 to 316 (+20%) but the overall age-standardized (adjusted) incidence-rate did not change between the time periods (Table 1). A substantial increase in incidence was only visible for men aged 80-84 (+57.7%), men aged 85+ (+29.3%) and women aged 85+ (+13.4%). With respect to mortality rates, a 10% decline was observed for men aged 85+. Incidence and mortality were very low below the age of 60 years but the rates steeply increased thereafter (Figure 1a). Irrespective of time period, incidence- and mortality-rates were almost twice as high among men compared to women (Figure 1b). Classification in MDS subtypes was poor and improved only modestly from 20% to 39% with a higher awareness for diagnosis of higher-risk diseases. Relative survival at 5 years (RS at 5y) for all patients was 37% in 2008-2012 with better survival for younger patients 〈 65 years (61%) compared to older patients 〉 65 years (24-37%). No differences in survival could be observed between the two time periods (Figure 2). Conclusions: In this study we provide the first population-based, epidemiologic data from MDS patients in Switzerland. The analysis showed a 20% increase of annual incidence mainly due to population aging and not explained by increase in age-specific risk. This observation will impact on the future prevalence of the disease and its burden on healthcare systems.The age-specific incidence-ratesin patients 〉 75 years increased markedlyconsistent with the general increase of cancer-incidence in the elderly population. An increased diagnostic awareness of higher-risk disease seems to shift the population-based data for MDS sub-classification. We observed that younger patients without classified MDS subtypes have a similar survival like lower-risk disease, indicating that lower-risk MDS is underreported. Unsurprisingly, our data showed that younger patients have a better survival than elderly patients. This is most likelyrelated to higher frequency of lower-risk diseases in younger patients and their eligibility for allogeneic HSCT. However,the lack of a survival benefit observed in elderly patients on population-based level, after introduction of hypomethylating agents as standard treatment for transplant ineligible patients, is intriguing. The underlying reasons require further health-service research investigations. Relevance: The currently available data from CCRs in Switzerland is insufficient for detailed health service research on MDS patients, since important data is lacking on treatment, side effects and outcomes. A new cancer registration law with mandatory notification of cancer cases will be implemented in Switzerland by 2018. Moreover, the Swiss MDS Study Group has launched a Swiss MDS Registry that has started recruitment in 2016. Both initiatives will be of great value to improve data collection in order to foster future health service research of MDS patients in Switzerland with international collaborators. Table 1 Incidence and Mortality of MDS in Switzerland in 2001-2007 and 2008-2012 Table 1. Incidence and Mortality of MDS in Switzerland in 2001-2007 and 2008-2012 Figure 1 Incidence and mortality of MDS in Switzerland in 2001-2007 and 2008-2012 Figure 1. Incidence and mortality of MDS in Switzerland in 2001-2007 and 2008-2012 Figure 2 Age-specific relative survival of MDS patients diagnosed in 2001-2007 and 2008-2012 Figure 2. Age-specific relative survival of MDS patients diagnosed in 2001-2007 and 2008-2012 Disclosures Bonadies: Bristol-Myers Squibb: Consultancy; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ruefer:Novartis: Consultancy; Celgene: Consultancy, Research Funding. Gerber:Celgene: Consultancy. Benz:Celgene: Consultancy. Lehmann:Novartis: Consultancy.

Description: Blood glycoprotein von Willebrand Factor (VWF) is constantly circulating in the blood vessel, with its platelet GPIb-IX recognizing unit-the A1 domain-being masked to avoid its adventitious binding and activating platelets. This "self-mask" mechanism, however, remains poorly understood. The impediment in identifying the self-mask ligand is to have the right pair of VWF fragments with opposite binding behavior for comparison. Here we characterize using hydrogen-deuterium exchange mass spec a pair of VWF fragments containing the A1 domain with opposite association to platelets. The results suggest that the short N-terminal region (1238-1260) in front of the A1 domain might involve in protecting the VWF A1 domain by masking a critical region near the binding site for GPIb. We expressed and purified using baby hamster kidney cells two C-terminally His-tagged proteins: both contain the entire A1 domain (1273-1453), with one has more extra N-terminal residues (1238-1472, named lA1) than the other (1261-1472, named sA1). Both proteins seem to be glycosylated and stable without obvious deformation for over one week when stored at 4°C. The analytical ultracentrifugation analysis determined that both proteins are primarily in the monomeric form: the sedimentation coefficient (S) and the derived apparent molecular weight for lA1 are 2.50 S and 36.6 kDa, and those for sA1 are 1.37 S and 27.4 kDa. Interaction of lA1 and sA1 to GPIb, as determined by flow cytometry, is surprisingly opposite: only sA1, but not lA1, efficiently binds washed platelets at the concentration similar to the physiological concentration of vWF in the blood vessel. Increasing lA1 concentration 10 times did not improve its binding. Replacing the platelets with CHO cells that express human GPIb-IX complex exhibited similar result. Implied in these results are that either lA1 adopt a different conformation that disadvantages its binding or the binding site on A1 is masked by the extra amino acids in lA1 compared to sA1. Protein hydrogen-deuterium exchange is a sensitive assay to determine the local conformational change and the existence of the masked region. We acquired hydrogen-deuterium exchange data for both proteins and got nearly 100% sequences coverage from over 150 overlaid peptides for each protein, with on average over 10 redundancy per residue, which allows us to compare the hydrogen-deuterium exchange rate/extent of the A1 domain in lA1 and sA1 in good resolution. The results show that the global hydrogen-deuterium exchange pattern is the same between the two, indicating that the tertiary structures of the two proteins are similar. However, a contiguous region in lA1 is clearly protected from hydrogen-deuterium exchange compared to that in sA1 (indicated by the colorful region in figure 1). The protected region is located at the bottom of the globular A1 domain, including three loop regions (1272-1276; 1304-1310; and 1332-1340) and a part of the a1 helix (1289-1300). Compared to sA1, in this protected region of lA1 the deuterium exchange extents are reduced by 20-60% even after 3-hour incubation. Furthermore, 11 of out of 15 identified "gain-of-function" mutation sites of type 2B VWD are located in this masked region (P1266, H1268, C1272, M1304, R1306, R1308, I1309, S1310, W1313, P1337, R1341); the other 4 are located adjacently (V1314, V1316, L1460, A1461). This good alignment implies that the "gain-of-function" might associate with the detachment of the identified masking ligand in this region. Based on our findings, we propose that this short region in front of the A1 domain may involve in masking the A1 domain from interacting with platelet GPIb-IX. If confirmed, this internal masking ligand might offer new clues on how the mutations on A1 domain affect its role -as "gain-of-function" in type 2B VWD and "loss-of-function" in type 2M VWD. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: The detection of chromosomal abnormalities in myelodysplastic syndromes (MDS) supports the diagnosis, classification, prognostic stratification, therapy option, treatment monitoring and better understanding of the biology of disease. The main chromosomal abnormalities of MDS are losses and gains of genetic material, and these changes are among the most important prognostic parameters in IPSS-R. As a consequence, major advances have been achieved in the treatment and survival of patients. However, nearly half of the patients present a normal karyotype, which prevents their further characterization. Therefore, there is the desire to increase the abnormalities detection rate by other methods. Single nucleotide polymorphisms array (SNPa), also referred to as chromosomal microarray, is a sensitive technology used to perform high-resolution genome-wide DNA losses, gains and copy-neutral loss of heterozygosity (CN-LOH). The genomic DNA is hybridized to polymorphic probes which are SNP markers and non-polymorphic probes which are copy number markers, providing information about CN-LOH and copy number alteration (CNA), respectively. Previous studies have shown that the addition of the SNPa to karyotype (KT) increases by 28% the detection rate of cytogenetic abnormalities. Objective: To substantiate this idea we describe two cases of MDS to whom SNPa added valued information to karyotype. Methods: DNA was extracted from bone marrow cells for genomic clonal evaluation. The DNA was digested, amplified, fragmented, labeled and hybridized to the chip (Affymetrix - CytoScan HD®) containing the probes. The chip was scanned to detect the signals' intensity emitted by the hybridized probes which were further analyzed by a software (ChAS) that allows visualization of CNA and CN-LOH. CNA´s analysis relies on the comparison of the obtained signals to a reference diploid DNA signal, and the difference encountered is characterized as loss or gain. CN-LOH analysis is based on two possible nucleotide signals (A or B), which are evaluated to discriminate three genotypes: AA, AB and BB. CN-LOH occurs when one allele is lost and duplicate another, resulting in genotypes AA or BB. Results: Case 1, 64y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II fibrosis. KT: 46,XY,del(5)(q15q33),del(17)(p11.2)[16] / 46,XY[4]. SNPa: 3p21.31p21.2 CN-LOH; 5q21.1q35.3 loss (CN:1.00); -7; +8; 12p13.33p12.3, 12p12.1p11.22, 12q22q23.3 loss (CN:1.00); -16; 17p13.3p11.2 mosaic loss (CN: 1.50) and -Y. Case 2, 74y-male-patient, classified as MDS-EB (WHO 2016), Bone marrow histology showed grade II/lll fibrosis. KT: 46,XY[15]. SNPa: 21q21.1q22.3 CN-LOH. Discussion and Conclusion: The SNPa has the advantage of detecting genomic alterations regardless of the cell cycle, even when the cell is quiescent or growth is defective. It also enables the identification of CN-LOH (also known as uniparental disomy, UPD), submicroscopic amplifications and deletions that are not detected by KT. On the other hand, SNPa does not allow the identification of balanced translocations and polyploidy. In case 1, after SNPa analysis, some chromosomal abnormalities (−7, +8, −16 and −Y) were found in sporadic metaphases during the KT reanalysis, but had not initially been described because they did not meet the criterion to be considered as a cytogenetic clone. The risk-stratification (IPSS-R) for this patient was intermediate, but the addition of the SNPa results the risk-stratification could be changed to very poor. Seven months after diagnosis the patient developed acute myeloid leukemia and died. In case 2, CN-LOH detected by SNPa could be responsible for homozygosity of mutations in critical genes located in the 21p region, such as RUNX1 that encodes a protein, which is a transcription factor critical in hematopoiesis. Indeed, sequencing of candidate genes in CN-LOH regions should be considered a priority in the search of driver mutations of MDS. Twenty-four months after diagnosis the patient died due to other non-hematologic causes. In Summary, SNPa analysis may add value to KT non-informative results and occasionally reveal cryptic abnormalities not recognized by karyotyping. However, SNPa analysis should be viewed as a complimentary tool. Acknowledgment: The SNPa test was supported by Grupo Fleury Disclosures No relevant conflicts of interest to declare.

Description: Introduction Myelodysplastic syndromes (MDS) are phenotypically and genotypically heterogeneous diseases with several driver mutations, which are closely related to patient prognosis. Dynamic evolution of mutations reflects the selection of subclones during disease evolution until transformation in secondary acute myeloid leukemia. Thanks to increasing knowledge in gene functions, target drugs are now available in therapeutic. However, questions remain on the impact of such treatments on malignant cells. We have previously investigated the effects of lenalidomide on clonal evolution, by monitoring variant allele frequencies (VAF) using next generation sequencing (NGS) in non-del5q MDS patients (Chesnais et al, Blood 2015). Here, we present a rapid and ultra-sensitive method using picoliter-droplet digital PCR for mutation detection in MDS with ring sideroblasts (RS). Materials and Methods Bone marrow aspirates were obtained from MDS patients included at diagnosis in a multicentric observational trial (PHRC MDS-04, NCT02619565). Three cell lines (HL60, OCI-AML3, UKE-1) were also used to establish the specificity and the sensitivity of assays. Both frozen living cells and extracted DNA were used. Selected samples were screened for mutations in 39 genes by an NGS approach using a Personal Genome Machine® (PGM, ThermoFisher Scientific, Waltham, MA, USA). Primers and probes were designed for Taqman assays based on allelic discrimination of recurrent mutations found in DNMT3A, SF3B1, JAK2 and NRAS genes. For the detection of SF3B1 p.K700E mutation, 3 locked nucleic acids were notably added to the probes to improve specificity. Picoliter-droplet digital PCR was performed on RainDrop® Digital PCR System (RainDance™ Technologies). Results Allelic discrimination assays were validated on genomic DNA extracted from cell lines and patient samples harboring or not targeted mutations using the RainDance system. About 5.106 droplets were generated using RainDrop Source. Wild-type (WT) DNA was tested in order to assess false positive signals for each design, characterized by λFP (mean number of false positive signals), limit of blanck (LOB) and limit of detection (LOD) for all experiments. The limit of blanck (LOB) defined here the highest number of droplets corresponding to apparent droplets containing mutated amplicons while testing wild type DNA. The limit of detection (LOD) was the lower number of droplets which can be distinguish from LOB while testing DNA with very low concentration of mutant genome. All the designed assays were also strongly approved for linearity using mixtures of mutated and WT DNA from cell lines (0.01% to 100% mutated allele frequency). Specificity, linearity and sensibility of the selected assays were validated on genomic DNA. Later on, we investigate genomic DNA of 3 MDS patients with RS and harboring JAK2 and SF3B1 mutations. For these patients, we obtained comparable results using both NGS and picoliter-droplet digital PCR in term of mutant allele burden quantification. Moreover, a triplex assay allowing mutant allele discrimination in JAK2 and SF3B1 genes was established on these patients. Further analyses were conducted on living cells harboring JAK2 or NRAS mutations. This approach was first conducted using a "home made" microfluidic system based on the detection of fluorescent probes in living cells encapsulated into agarose beeds. We obtained specific fluorescent signals corresponding to the genotypes. In parallel, an alternative method based on the QX100™Droplet Digital™PCR system (Biorad) also demonstrated the feasibility of allelic discrimination in living cells. Experiments based on frozen cells of MDS patients are currently under investigation. Conclusion This study is the first application of multi-target digital PCR used to detect and quantify somatic mutations recurrently found in MDS. Analyses of the clonal architecture determined on living cells and its evolution upon treatment in MDS patients with RS by this approach will help us to investigate the monitoring of the therapeutic response. Our study supports a proof of principle for further large-scale analyses of MDS patients at diagnosis and follow-up. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Hypereosinophilic syndrome (HES) is a rare hematologic disorder in which the eosinophils proliferate and infiltrate and damage multiple organs. Without treatment, the condition is fatal, usually due to cardiac dysfunction, particularly left ventricular dysfunction. Causes of reactive eosinophilia include allergic and hypersensitivity reactions, drug allergies, cytokine therapy (recombinant human interleukin), cutaneous disorders, connective tissue diseases, collagen vascular disorders, parasitic infections, immunodeficiency disorders, sarcoidosis, and neoplasms, such as Hodgkin's and non-Hodgkin's lymphoma (B- and T-cell), acute lymphoplastic leukemia, chronic myeloproliferative disorders. Jakafi is approved for myelodysplastic syndrome (MDS) and is effective in reducing splenomegaly. The medication is off-label for HES. This report demonstrates the successful treatment of HES with ruxolitinib. Methods: Case report Results: A 77 year-old Asian male presented with sudden onset pruritus. There was no history of drug allergy or scabies. Past medical history included hypertension, benign prostatic hypertrophy, and early glaucoma. He had shingles 3 times in his 50's on his thorax. Past surgical history included vasectomy, laser iridotomies, and pterygectomies. His medications included losartan, doxazosin and silodosin, amlodipine, carvedilol, and multi-vitamins. He was a retired pediatrician who was actively involved in gardening, carpentry, roller-blading, swimming, and walking his dogs. The patient first presented to his primary care physician, who offered a psychosomatic explanation for his symptoms. A CBC with a differential demonstrated elevated eosinophils of 36%. He was referred to an allergist, who started oral prednisone. There was some relief. He was given interferon. This resulted in fatigue and purple fingernails and no improvement in the pruritus, so interferon was discontinued. He was referred to a dermatologist who prescribed hydroxyzine and fluocinolone cream. This provided minimal relief. He saw a 2nd dermatologist who performed a skin biopsy, which was negative. The patient was referred to an oncologist. Bone marrow biopsy revealed a slight hypercellularity of 30-40% and increased eosinophilia of 16%. There was no evidence of leukemia, lymphoma, or MDS. Cytogenetic testing revealed the following: 46XY, del 9 (q13q22). Kappa/Lambda ratio was slightly elevated at 4.15. KIT mutation by PCR, T-cell clonality by PCR, and FISH panel were negative. Immunoglobulin testing revealed: IgA 140, IgG 1317, IgM 65, and IgE 358. The patient also saw an infectious disease specialist. HIV, ANA, serum tryptase, LDH, Vitamin B12,urine protein electrophoresis, parasitic work-up (trichinella, toxocara, strongyloides) was negative. Echocardiogram demonstrated an ejection fraction of 62%. The patient continued to have chronic pruritus with sleeplessness. In desperation, he attempted to discontinue his prostate medications in hopes that his condition was a simple allergy to medications, but this resulted in urinary retention with fever, sepsis, and anasarca, necessitating urgent antibiotic treatment. Although the patient was negative for the FIP1L1-PDGFRA mutation, a trial of imatinib (tyrosine kinase inhibitor) was given. Not surprisingly, there was no improvement in his symptoms or condition. The patient's symptoms worsened, so oral prednisone was increased from 20 to 80 mg. There was no improvement in his symptoms. Additional testing was obtained, revealing a JAK 2 kinase mutation. He was prescribed ruxolitinib 20 mg po bid. By the 3rd day, he could sleep a full night without pruritus. His prednisone was tapered; by one month, he had tapered off of his prednisone. He developed mild neutropenia, so ruxolinitib was decreased. He noted that when he missed even one dose of his ruxolitinib, the pruritus would recur. Most recent labs (4 years after initial presentation) revealed normal eosinophils of 2%. He had mild anemia and thrombocytosis, which were stable. Conclusions: This is the first case of HES treated with ruxolitinib. The patient had side effects from the standard treatments (prednisone, interferon, imatinib). Newer treatments should be available to patients who had documented HES to prevent delay in treatment and increased morbidity. Ruxolitinib is a potential treatment for HES who are intolerant of other treatments. Disclosures No relevant conflicts of interest to declare.

Description: Objective: To investigate the change of mitophagy level of bone marrow nucleated red blood cell in anemia patients with myelodysplastic syndromes (MDS), to explore its significance in the anemic pathogenesis of MDS. Methods: Fifty anemia patients with MDS and twenty-six normal controls were enrolled in this study. The mitophagy were observed by transmission electron microscopy (TEM). The level of autophagy-associated protein LC3B, the number of mitochondria, mitochondrial membrane potential, the level of ROS in GlycoA + nucleated red blood cell were measured by Flow cytometry .The expression of AMPK, ULK1, mTOR mRNA of GlycoA + nucleated red blood cell were measured by real time PCR. The expression of the mitochondrial outer membrane protein TOM20 in GlycoA + nucleated red blood cell were detected by Western blotting. Results: (1) Autophagosome or autolysosome including mitochondria were scarcely observed by TEM. (2) The expression of LC3B in GlycoA + nucleated red blood cell in high-risk MDS patients(0.22±0.12) was significantly lower than that in normal controls (0.43±0.22, P

Description: Low-grade systemic inflammation is often associated with metabolic syndrome, which plays a critical role in the development of the obesity-associated inflammatory diseases, including insulin resistance and atherosclerosis. Here, we investigate how Toll-like receptor-MyD88 signaling in myeloid and endothelial cells coordinately participates in the initiation and progression of high fat diet-induced systemic inflammation and metabolic inflammatory diseases. MyD88 deficiency in myeloid cells inhibits macrophage recruitment to adipose tissue and their switch to an M1-like phenotype. This is accompanied by substantially reduced diet-induced systemic inflammation, insulin resistance, and atherosclerosis. MyD88 deficiency in endothelial cells results in a moderate reduction in diet-induced adipose macrophage infiltration and M1 polarization, selective insulin sensitivity in adipose tissue, and amelioration of spontaneous atherosclerosis. Both in vivo and ex vivo studies suggest that MyD88-dependent GM-CSF production from the endothelial cells might play a critical role in the initiation of obesity-associated inflammation and development of atherosclerosis by priming the monocytes in the adipose and arterial tissues to differentiate into M1-like inflammatory macrophages. Collectively, these results implicate a critical MyD88-dependent interplay between myeloid and endothelial cells in the initiation and progression of obesity-associated inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoietic stem cell (HSC) activation is accompanied by mitochondria activation and a shift in metabolic activity from glycolysis to OXPHOS, which provides energy and increases the production of ROS and other mitochondrial metabolites that can act both as signaling molecules and substrates/co-activators for epigenetic enzymes. Metabolically activated HSCs are poised to undergo lineage priming and produce different lineage-biased multipotent progenitors (MPP). However, activated HSCs must also return to quiescence to maintain the stem cell pool. In this context, autophagy plays an essential role by clearing activated mitochondria to allow OXPHOS-driven HSCs to efficiently revert to a mostly glycolysis-based quiescence metabolism. Without autophagy, HSCs display an overactive OXPHOS-driven metabolism that promotes myeloid-biased differentiation and loss of stemness, likely as a direct consequence of epigenetic reprogramming. At steady state, blood production reflects the differential generation by HSCs of a small number of myeloid-biased MPP2/3 and a large number of lymphoid-biased MPP4, which both contribute to myeloid output. In contrast, during blood regeneration, activated HSCs are induced to overproduce MPP2/3, and MPP4 are reprogrammed towards almost exclusive myeloid output in large part due to cytokine stimulations and the triggering of specific regulatory pathways. Altogether, the metabolic activation of HSCs and the remodeling of the MPP compartment represent emergency myelopoiesis pathways that are transiently activated during regeneration, and are continuously triggered in myeloid disease conditions. Disclosures No relevant conflicts of interest to declare.

Description: My laboratory studies the mechanisms that control the development, homeostasis and function of tissue resident myeloid cells, which mostly consists of macrophages and dendritic cells (DC). Macrophages are hematopoietic cells that perform tissue-specific functions critical for regulating and maintaining organ homeostasis. In contrast to most other hematopoietic cells, macrophages that reside in quiescent tissues originate from early hematopoietic precursors that take residence in tissues prior to birth. In this presentation, I will discuss how tissue cues control macrophage functional diversity to ensure tissue integrity. I will also discuss recent data from our laboratory showing that developmental diversity contributes to shaping macrophage functional specialization and that ontogenically distinct macrophages differently regulate tumor response to radiation therapy. In contrast to macrophages, DC are constantly repopulated by DC restricted precursors that are recruited from the blood circulation. Our laboratory has contributed to the mapping of the regulatory network of DCs, and the identification of a lineage of DC, the CD103+ DC, which are specialized in the induction of CD8+ T-cell immunity. Here I will discuss recent data from the laboratory that revealed the critical role for tumor-associated CD103+ DC in tumor response to checkpoint blockade and argue for the need to study human CD103+ DC equivalent in clinical cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.

Description: In iron overload conditions such as thalassemia major and hereditary hemochromatosis, the iron-carrying capacity of plasma transferrin is exceeded, giving rise to non-transferrin-bound iron (NTBI). NTBI is taken up preferentially by the liver, and to a lesser extent, the kidney, pancreas, and heart. How NTBI is taken up by various tissues has been elusive. We recently demonstrated that the plasma membrane metal-ion transporter SLC39A14 (ZIP14) mediates NTBI uptake and iron loading of the liver and pancreas, but not the kidney, heart or most other tissues¹ Given that the heart is particularly susceptible to iron-related toxicity, we are currently investigating the contribution of other iron transporters to iron loading of this organ. Possible alternative cardiac iron importers include L-type and T-type calcium channels, divalent metal transporter 1 (DMT1), and SLC39A8 (ZIP8). To examine the role of DMT1 and ZIP8 in cardiac iron metabolism, we generated mice with cardiomyocyte-specific disruption of DMT1 (Dmt1heart/heart) or ZIP8 (Zip8heart/heart). The mice were then crossed with hemojuvelin knockout (Hjv-/-) mice, a model of juvenile hemochromatosis characterized by high circulating levels of NTBI. Dmt1heart/heart mice were found to have cardiac non-heme iron concentrations that were 30% lower (P

Description: Introduction Refractory anemia with ring sideroblasts (RARS), as a form of acquired primary sideroblastic anemia, represents about 11% of myelodysplastic syndromes (MDS) and is classified as a low risk MDS. It´s defined by the WHO as a pure dyserythropoietic disorder with presence of 〉15% ringed sideroblasts in the bone marrow. Abnormal expression of several genes of heme synthesis in this MDS subgroup and excessive accumulation of iron especially in mitochondria, the main place of reactive oxygen species (ROS) formation, contributes to the elevated oxidative stress. Oxidative stress is also known as one of the factors involved in the pathogenesis of MDS. High iron concentrations catalyse a Fenton reaction, where a hydroxyl radical is produced from hydrogen peroxide and causes an increase in ROS which may lead to the oxidation of DNA, lipids, and proteins, thereby causing cell damage. The aim of this study was to find a useful method for detection and identification of oxidatively modified proteins in plasma unique for RARS patients. Methods Carbonylated protein levels were determined spectrophotometrically using dinitrophenylhydrazine (DNPH) derivatization. Oxidatively modified proteins of plasma samples were derivatized with biotin hydrazide. The dialyzed biotin hydrazide labeled samples and negative controls were mixed with monomeric avidin resin. Captured carbonylated proteins were digested by trypsin and then identified by MS/MS mass spectrometry coupled to a nano-LC system. Mascot (Matrix Science, London, UK) was used for database searching (Swiss-Prot). Two unique peptides (with a higher Mascot score than the minimum for identification, P

Description: A 77 year-old man initially presented in July 2013 with anemia, splenomegaly, and constitutional symptoms. A bone marrow biopsy revealed a hypercellular marrow with megakaryocytic hyperplasia and atypia and mild reticulin fibrosis, consistent with a diagnosis of primary myelofibrosis (PMF). Cytogenetics revealed a normal karyotype. JAK2 V617F testing was negative. Initiation of treatment with the JAK inhibitor ruxolitinib led to marked symptomatic improvement. The patient was then enrolled in a Phase 2 study with the anti-lysyl oxidase-like-2 (LOXL2) monoclonal antibody simtuzumab, administered via IV infusion every two weeks (while continuing ruxolitinib). He tolerated the simtuzumab infusions well initially, but with the 11th and 12th infusions experienced rigors, hypotension, and hypoxia. This occurred ~8 months after his initial PMF diagnosis. A repeat bone marrow biopsy revealed large aggregates of mast cells comprising 30-40% of the marrow cellularity (with 16% mast cells enumerated on the aspirate). A subset of the mast cells exhibited spindled morphology, and CD25 co-expression was demonstrated by flow cytometry in a subset of CD117-positive cells. Testing for the KITD816V mutation was positive. Tryptase levels were significantly elevated (375 ng/mL). These findings were consistent with a diagnosis of aggressive systemic mastocytosis with an associated hematologic non-mast cell lineage disorder (ASM-AHNMD). Compassionate-use approval for the KIT inhibitor midostaurin was obtained, and treatment with midostaurin (in addition to continuation of ruxolitinib) was initiated. The patient initially reported symptomatic improvement with midostaurin treatment, but after several months his symptoms began to worsen, with a corresponding increase in tryptase (875 ng/mL). A repeat bone marrow biopsy revealed overt evolution to mast cell leukemia (MCL) with 〉 90% mast cell involvement. Based on the presence of an IDH2mutation identified on a clinical next-generation sequencing assay, the patient was evaluated for a clinical trial with the mutant IDH2 inhibitor AG-221. Unfortunately, the patient decompensated and expired before he could enroll in the study. To identify contributing driver mutations and to delineate clonal hierarchy associated with disease initiation and progression in this unique case of PMF with concomitant ASM, exome sequencing was performed on serial samples obtained at the following disease stages:PMF diagnosis (pre-ruxolitinib) (Day 0)PMF on ruxolitinib (before ASM diagnosis) (Day 181)ASM diagnosis (post-anti-LOXL2 antibody, pre-midostaurin) (Day 394)Progression to MCL (on midostaurin) (Day 519)Matched skin (normal) sample Likely driver mutations in IDH2 and SRSF2 were identified at ~40-50% variant allele frequency (VAF) in all samples and were therefore likely present in the founding clone. The KIT D816V mutation was found at 23% VAF at Day 0, then ~40% VAF in all other samples, suggesting it was present in a daughter subclone of the IDH2/SRSF2-containing clone that became dominant over time with disease progression. These findings also suggest that targeting KIT with midostaurin would be unlikely to eradicate the founding clone. Rather, selective targeting of IDH2 and/or SRSF2 could potentially ameliorate both diseases (PMF and ASM/MCL). To provide pre-clinical evidence of the potential utility of targeting IDH2, peripheral blood mononuclear cells obtained at the time of ASM diagnosis were plated in liquid culture in the presence or absence of the mutant IDH2 inhibitor AGI-6780. After 14 days in culture, the differentiation status of the cultured cells was examined by mass cytometry (CyTOF). Treatment with AGI-6780 resulted in a marked enhancement of myeloid differentiation (denoted by CD15 and CD66b expression) along with a corresponding decrease in CD34+ progenitor cells. These effects were not seen in cells cultured in the absence of AGI-6780. These results are consistent with prior studies in acute myeloid leukemia indicating that the beneficial effects of mutant IDH2 inhibition are likely related to inducing differentiation of primitive cells. In summary, this study highlights the capacity of serial genomic analysis to define the clonal architecture that drives disease initiation and evolution, and to distinguish founding vs subclonal mutations to identify the most promising targets for therapeutic intervention. Disclosures Oh: Janssen: Research Funding; CTI: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Natural killer (NK) cells are defined as lymphocytes capable of target cell killing and cytokine production independent of somatic antigen receptor recombination. Challenging the paradigm of continuous production of non-specific cytotoxic NK cells from undifferentiated precursors, a subset of murine NK cells sharing many properties with cytotoxic effector T cells was shown to specifically respond to a viral infection, resulting in expansion and protective immunity upon adoptive transfer, uncovering unexpected "adaptive" capabilities for NK cells. In humans, CD56dimCD16+ NK cells expressing NKG2C and variegated patterns of KIR are associated with Cytomegalovirus (CMV) infection, and may represent human analogs of murine adaptive NK cells. In contrast to CD56bright and canonical CD56dim NK cells, we recently showed that human adaptive NK cells lack expression of specific signaling proteins and transcription factors, and epigenetically resemble CD8+ effector T cells, suggesting shared pathogen-driven developmental pathways (Schulms et al, 2015). NK cell heterogeneity, ontogeny, and mechanisms sustaining NK cell memory are poorly understood, particularly in humans, because in contrast to adaptive T and B cells, clonal tracking of NK cells based on receptor gene structure is not possible. We applied autologous transplantation of genetically barcoded CD34+ rhesus macaque hematopoietic stem and progenitor cells (HSPC) to interrogate the landscape of NK cell production and expansion at a clonal level. We followed 3 monkeys for up to 4 years post-transplantation, and observed a distinct clonal ontogeny for CD56-CD16+ (corresponding to human CD56dimCD16+ NK) versus CD56+CD16- rhesus NK cells. CD56-CD16+ NK were oligoclonal, and dominated by small numbers of highly biased clones, a pattern showing little overlap with highly polyclonal multipotent contributions to T, B, myeloid and CD56+CD16- NK cell lineages. There were clear expansions and contractions of groups of these highly biased CD56-CD16+ NK cell clones over time, with individual clones contributing up to 20% to this lineage, similar to patterns of waxing and waning T cell clones, suggesting responses of both cell types to extrinsic cues such as viruses. In contrast, all other lineages, including CD56+CD16- NK cells, were characterized by very stable contributions from long-term engrafting multipotent HSPC clones over time. The highly biased, greatly expanded, and waxing and waning CD56-CD16+ NK cell clones were specifically enriched in a subpopulation of CD56+CD16- NK with high KIR and low SYK expression,and decreased methylation at IFNG the CNS1 locus, analogous to human CMV-associated adaptive NK cells. This population of cells was increased in CMV+ compared to CMV- macaques; our transplanted macaques were CMV+. Canonical CD56+CD16- NK cells, lacking an adaptive phenotype, had clonal patterns similar to CD56+CD16- NK cells. We profoundly depleted CD56-CD16+ NK cells with an anti-CD16 antibody, and observed rapid regeneration of the same CD56-CD16+ NK clonal pattern, without recruitment of contributions from multipotent HSPC via CD56+CD16- putative precursor NK cells. There was a marked increase in cycling of CD56-CD16dim NK cells just prior to reappearance of CD56-CD16bright NK cells, and no change in cycling of CD56+CD16- cells. Thus, adaptive NK cell clones, both at steady state and under regenerative stress do not appear to require ongoing production from HSPC via CD56+CD16- precursors. These results suggest that adaptive NK cells or their immediate precursors have acquired self-renewal properties and high proliferative potential, with clonal restriction potentially based selection and expansion of cells with specific, epigenetically-controlled receptor expression able to interact with external stimuli such as viruses. In conclusion, via tracking of individual NK cell clonal histories in rhesus macaques, we provide the first direct evidence for persistence of adaptive NK cell clones in any species, offering potential insights into the acquisition of NK cell memory. Disclosures Dunbar: GSK/Novartis: Research Funding.

Description: Backgroud and objective: Myeloproliferative neoplasms (MPNs) are a group of clonal haematological disorders that characteristic with a multipotent haematopoietic stem cell transformation, often associating with the progression of myelofibrosis in the evolution course of disease. Progressive myelofibrosis finally turned out a high risk factor of transformation to leukemia and bone marrow failure. Cancer Associated Fibroblasts (CAFs) are recently thought to be a critical mediator in several hematological malignancies tumor microenvironment and associate with fibrosis. Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that promote the crosslinking of collagens or elastins in the extracellular matrix and mediate epithelial-mesenchymal transition (EMT). Here, we want to verify CAFs mediating myelofibrosis and explore the potential role of LOXL2 association with CAFs in simulated vivo microenvironment. Patients and methods:For bone marrow specimens, normal samples (n=19) and patients with polycythemia vera (PV) (n=21), essential thrombocythemia (ET) (n=32), and primary myelofibrosis (PMF) (n=9) were contrasted. Markers of CAFs including α-smooth actin(α-SMA), fibroblast activation protein(FAP), transforming growth factor-β1(TGF-β1) and LOXL2 were detected by quantitative reverse transcription-PCR(RT-PCR). we also detected α-SMA, FAP, LOXL2 and reticulin protein by western blot and immunohistochemical staining. For cell lines, α-SMA and FAP were measured after cocultured mesenchymal stem cell(MSCs) with recombinant human lysyl oxidase homolog 2 Protein(rhLOXL2) in hypoxic niche for 24, 48, 72 and 96 hours, respectively. Results: Markers of CAFs displayed a differential pattern of expression in MPNs especially in PMF(P

Description: Background: The combination of MPD and amyloidosis is rare, and this patient population's clinical outcome is not well studied. Methods: Pts with a MPD and amyloidosis were identified via an clinical note search engine that searches through the electronic medical records of patients seen at Mayo Clinic Rochester, Jacksonville, and Scottsdale between 1990 and 2016. Terms used included amyloid or amyloidosis, chronic myelogenous leukemia or CML, essential thrombocytopenia or ET, and polycythemia vera or PV. Demographic and clinical data were abstracted from the medical record. Pts with both disorders were analyzed and their mortality rates along with median time to death were calculated. Prevalences at the Mayo Clinic were calculated for the years 2014 and 2015. Results: Twenty-three pts diagnosed with both a MPD and amyloidosis were identified. Thirteen (56.5%) were male, 10 (43%) were female. Eleven (47.8%) were initially diagnosed at the Mayo Clinic. Types of amyloidosis were as follows: Eleven (47%) had immunoglobulin light-chain (AL), four had localized (17%), two (8.7%) had wild-type transthyretin (ATTR), one (4.3%) had mutant ATTR, and five (21.8%) were unknown. Types of MPD were as follows: Seven (30%) had polycythemia vera (PV), seven (30%) had chronic myelogenous leukemia (CML), five (22%) had myelofibrosis, and four (17%) had essential thrombocytosis (ET). Fifteen (65%) were initially diagnosed with a MPD. Median time to last follow-up from second diagnosis was 1.7 years, and median time to death following second diagnosis was 1.4 years. The mortality rate was 87% in the total population. The median time to death for AL and PV was 2.7 years, AL and CML 1.0 years, and AL and ET 1.17 years. Myelofibrosis did not occur with AL. The most common combination was AL and PV, which accounted for five (22%) of the cases. Treatment regimens for the patients with AL were varied. Multiple drugs were combined with dexamethasone including melphalan, velcade, pomalidomide, lenalidomide, doxorubicin, and revlimid. Cyclophosphamide, bortezomib, and dexamethasone (CyBorD) were used in two cases. The mean time to diagnosis of amyloidosis from symptom onset was 10.3 months. Prevalences of AL and a MPD in 2014 and 2015 at the Mayo Clinic were 2.8% and 1.5%, respectively. Conclusions: The mortality rate for the combined diagnoses is high. The most common combination of diagnoses was that of AL and PV, which was associated with a mean time to death of 2.7 years. Disclosures Al-Kali: Celgene: Research Funding; Onconova Therapeutics, Inc.: Research Funding.

Description: BACKGROUND: Symptom burden in essential thrombocythemia (ET) and polycythemia vera (PV) is severe even among individuals with low risk disease (Blood 2012. 12;123(24):3803-10). New therapies exist which alleviate the severe symptom burden and reduce splenomegaly in ET and PV patients (N Engl J Med 2015; 372:426-435). This analysis is the first to date to evaluate thresholds at which symptom-based treatment can be considered for ET and PV patients who are intolerant or resistant to hydroxyurea (HU). METHODS: Patient demographics, symptom burden, and disease traits were collected from ET and PV patients at a single time point during therapy. The MPN-10 total symptom score (TSS, JCO 2012;30(33)4098-103) was utilized to assess symptom burden. Symptom criteria models were determined as previously described among a population of MF patients (Scherber et. al. EHA 2016: a2250). Cutoffs were then evaluated in a cohort of ET and PV patients to assess for utility as a symptom model among this population. RESULTS: Demographics and symptom burden: 838 PV and 867 ET patients with previous hydroxyurea therapy were included in this analysis. Patients were of mean age (54.9 years ET, 64.0 years PV) and gender (69.2% female ET, 55.7% female). Mean disease duration was 6.0 years for ET and 7.3 years for PV.Among ET and PV patients, 15.0% and 24.2% had prior thrombosis respectively. In evaluating prognostic risk, ET patients tended to be low (45.5%) or intermediate risk (42.9%) with only a minority of patient meeting criteria forhigh risk disease (11.6%). Laboratory findings: ET patients had a mean platelet value of 598.7x 109/L(SD=283.4). Among PV patients, mean hematocrit was 45.8% (SD=8.1) and 42.6% of patients had a hematocrit of greater than 45%. White blood cell count was normal between the two groups (ET mean 8.3 x 109/L, PV mean 9.0 x 109/L). Symptoms: Mean worst symptom severity was 6.4 out of 10 (SD=2.7). Among ET patients, worst symptom was most frequently fatigue (32.7%, mean 5.0/10, SD=3.1, overall prevalence 88%) followed by night sweats (13.6%, mean 2.0/10, SD=3.0, overall prevalence 53%) and concentration difficulties (8.6%, mean 3.1/10, SD=3.0, overall prevalence 68%). For PV, worst individual symptom items were most frequently fatigue (29.2%, mean 5.2/10, SD=3.0, overall prevalence 91%), pruritus (14.1%, mean 3.2/10, SD=3.2, overall prevalence 69%), and night sweats (12.8%, mean 2.5/10, SD=3.0, overall prevalence 57%). Cutoff Scoring: 47.0% of ET patients fit criteria for TSSgreater than to equal to 20; 59.0%% had a single itemgreater than 5; and 45.7% had both a TSS greater than or equal to 20 and a single item greater than 5. Among PV participants, 54.5% had aTSS greater than to equal to 20; 66.1% had a single itemgreater than 5; and 51.5% had both a TSS greater than or equal to 20 and a single item greater than 5. Each scoring method was significantly associated with individual item scores (Table 1). Prognostic scoring was not significantly associated with any of the symptom cutoffs evaluated. Correlations: Among ET patients, a prior history of thrombosis was significantly associated with having a worst symptom item greater than 5 (p=0.043). ET patients with lower hemoglobin were significantly more likely to meet criteria for a MPN-10 score greater than or equal to 20 or to meet combined criteria for a MPN-10 greater than or equal to 20 and single worst item greater than 5 (for both p=0.01 or less). For PV, lower hematocrit levels were significantly associated with having an individual worst symptom score of greater than 5 (44.9% versus 46.7%, p=0.0376). CONCLUSION: Assessment of ET and PV symptoms, now measurable through standardized and practical instruments such as the MPN-10, is an integral part of determining therapeutic impact of newer therapies in both clinical practice and trial settings. In our modeling, patients with severe symptom burden profiles are well represented by utilizing cutoff criteria including aworst individual symptom item of greater than 5 out of 10, an MPN-10 score of greater than or equal to 20, or combined criteria of both cutoffs. These cutoffs can be considered when determiningwhich HU intolerant or resistant patients would most benefit fromsymptom orientedtreatment. Disclosures Kiladjian: AOP Orphan: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Research Funding. Schouten:Novartis: Consultancy; Sanofi: Consultancy. Etienne:BMS: Speakers Bureau; ARIAD: Speakers Bureau; Pfizer: Speakers Bureau; novartis: Consultancy, Speakers Bureau. Harrison:Incyte Corporation: Honoraria, Speakers Bureau; Shire: Honoraria, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Baxaltra: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses, Research Funding, Speakers Bureau. Radia:Pfizer: Honoraria; Novartis: Honoraria. Cervantes:AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Mesa:Promedior: Research Funding; Celgene: Research Funding; CTI: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Galena: Consultancy; Ariad: Consultancy; Novartis: Consultancy.

Description: Introduction: Interferon (IFN-α) is frequently used in various hematologic neoplastic disorders either at failure of first-line treatment or as first-line therapy in selected patients. IFN-α has shown significant activity in treatment, but the therapy is handicapped by high dropout rates due to side effects and cumbersome dosing schedules. Pegylated (peg) IFN- α is formulated by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side chain of IFN- α. This modification prolongs serum half-life and patient exposure to medicine, decreases renal excretion, thus allowing for weekly administration, acceptable toxicity, tolerability and activity profiles. The results of peg-IFN in polycythemia vera (PV) and essential thrombocythemia (ET) in phase 2 studies are encouraging, with molecular responses in some patients. There is at least one phase 3 study of hydroxyurea versus peg-IFN as first-line cytoreductive therapy for PV. In spite of ongoing international efforts to evaluate peg-IFN in PV, there is limited experience on peg-IFN use in other hematologic neoplasms. Method: All patients who were put on peg-IFN in our center were found in our institutional electronic database. The patients had been recorded prospectively. All patients who had been treated with peg-IFN had been approved by national health authority and all patients had given informed written consent. Responses were evaluated by ELN (chronic myeloid leukemia), IWG-MRT (PV and ET), EUMNET (myelofibrosis), Nordic proposal criteria (hypereosinophilic syndrome), modified consensus criteria (systemic mastocytosis) (Am. J. Hematol 2009;84:790), Cheson criteria (lymphomatoid granulomatosis) and PET response criteria (Erdheim Chester disease) standardized criteria. Adverse effects were classified according to Common Terminology Criteria for Adverse Events (CTCAE4.0). Categorical and continuous data were expressed as ratio (%) and median (range). Statistical analyses were done by SPSS v17 (Chicago, IL). Results: There were 30 patients with 8 different hematologic neoplasms: 12 PV, 6 ET, 2 chronic myeloid leukemia (CML), 3 systemic mastocytosis (SM), 4 Erdheim-Chester disease (ECD), 1 primary myelofibrosis (PM), 1 hypereosinophilic syndrome (HES), and 1 lymphomatoid granulomatosis (LG). Detailed descriptive data, toxicity and treatment results are presented in table 1. Responses were observed in all disease categories. Toxicities were frequent in PV/Et, including significant autoimmune disorders (nephritic syndrome, autoimmune hepatitis) Conclusion: Peg-IFN is effective in many hematologic neoplastic disorders. Unfortunately, toxicity is a major concern in this treatment. Disclosures No relevant conflicts of interest to declare.

Description: Background: Myelodysplastic syndrome (MDS) represents a heterogenous group of meyloid clonal disorders typically characterized by aberrant myeloid differentiation, dysplasticchanges, ineffective hematopoiesis and high potential to progress into acute meyloid leukemia. In the general population, the incidence of MDS is approximately 5/100000 and the mean age of onset of MDS is 68 years. Most of the patients in middle-2 and high risk were uneligible for intensive therapy. It have been confirmed that epigenetic play important roles in processes of tumorigenesis both in soild tumer and hematopoic malignacies. The aberrant changes of this key epigenetic process contribute significantly to the development of hematologic malignancies.Irregular DNA hypermethylation which lead to the silencing of tumor suppressor genes is a very commonamong MDS. The epigenetic therapy with hypomethylating agents has become a new strategy for the treatment ofpatients with MDS.The decitabine (DAC) is an inhibiter of DNA hypermethylationand act as an anti-tumor by reactivating the tumor suppressor genes. While the treatment dose and schedule of DAC haven't reach a consensus. The CAG chemotherapy regimen is widely used for the treatment of patients with relapsed AML since 1995. The regimen is composed of low-dose cytosine arabinoside (10 mg/m2/12 h, usually day 1~14), low-dose aclarubicin (10-14 mg/m2/day, day 1~4), and concurrent use of G-CSF (200 micrograms/m2/day).But the cardiotoxicity limit its application among elder patients. So we tried to modify the regimen by replacing the aclarubicin with homoharringtonine (HHT) which was reported have the function of arresting leukemic cell cycle, inducing apoptosis and it has been used in the treatment of AML in China for decades. The aim of this study was to provide a retrospective analysis of the safety and performance of ultra low-dose decitabine combined a modified protocol GHA to find an option for middle-2 and high risk MDS patients. Methods: Retrospective analysis of 40 MDS patients who were enrolled in the low-dose decitabine combined with GHA regimen. All the patients were classified in to RAEB1/2(Refractory anemia with excess blasts) or RCMD (Refractory cytopenia with multilineage dysplasia) by 2008 WHO classification of MDS. In IPSS (international prognostic scoring system), all the patients were categoried in intermediate-2 or high risk. The decitabine were used at the dose of 10 mg/d given intravenously over 1 h daily for 5 days (d1~5). The GHA regimen consisted of homoharringtonine 1mg/m2 daily by intravenous continuous infusion d 1~14), cytarabine 10mg/m212hr by subcutaneous injection (day 1~14), and G-CSF200 mg/m2/day by subcutaneousinjection one day prior to chemotherapy and continued until the end of chemotherapy or when the peripheral white blood cell (WBC) 〉20*109/L. The clinical efficiency and side effects were observed. Results: 27 patients achieved CR and 5 patients achieved PR. The CR and PR rate were 67.5% and 12.5%, respectively. Total effective rate was 80%. In the induction therapy, a total of 10/40(25.0 %) patients experienced absolute neutrophol count (ANC) less than 0.5*109/L with a mean 8 days of agranulocytic period, and 28/40 (70%) patients experienced platelets less than 20*109/L with a mean 7 days of recovery over 20*109/L. The nonhematologic toxicities were mild and early death was zero. The median follow up time was 28 months; the median overall survival (OS) and disease free survival (DFS) were 25 and 12 months, respectively. Conclusion: The ultra low-dose decitabine combined with regimen is effective and well tolerated in induction therapy for intermediate-2 and high-risk MDS who were uneligible for intensive therapy and the results are better than the single regimen of decitabine or GHA. Disclosures No relevant conflicts of interest to declare.

Description: Despite advances in understanding of the biology of acute myeloid leukemia (AML), cure remains elusive for the majority of patients. Pro-survival molecules of BCL-2 family play critical roles in leukemia transformation and chemoresistance. The anti-leukemia potency of selective BCL-2 inhibitor venetoclax (ABT-199/GDC-0199) has been demonstrated in AML models (Pan et al. Cancer Discovery 2014). However, venetoclax is often associated with resistance due to its poor inhibition of MCL-1. RAF/MEK/ERK (MAPK) pathway is commonly activated in AML, and can stabilize anti-apoptotic MCL-1 and inactivate the pro-apoptotic BIM. In this study, we evaluated the anti-leukemia effects of concomitant BCL-2 and MAPK blockade by venetoclax in combination with MEK1/2 inhibitor GDC-0973 (cobimetinib). First, anti-leukemia activity of cobimetinib and venetoclax was examined in 18 primary AML samples with diverse genetic alterations. The combination significantly enhanced cell death, as compared to the single agent treatment (Fig 1A). Cobimetinib inhibited cell proliferation in the majority of AML cases (34.2 ± 23.7%) and the cell growth suppression was more profound in the combination group (60.2 ± 28.8%, p

Description: Polycythemia vera (PV) is a myeloid neoplasm characterized by thrombotic risk related to high hematocrit (HCT) and to platelet hyperactivation. According to PVSG, the antithrombotic therapy include low dose aspirin (ASA). However, there are insensitive ASA PV patients. It is debated if enherited thrombophilia is responsible to affect platelet activation and, hence, to cause ASA insensitivity. Therefore, we evaluated gene polymorphism human platelet antigen-1 (HPA-1) as thrombophilic indicator associated with abnormal platelet activity, HCT, platelet count, b-TG and PF4, as markers of platelet activation, the platelet functional activity (PFA) and the maximum clot firmness (MCF), as direct and indirect indicators of ASA sensitivity. We studied 40 patients (28 men, 12 women; mean age 64 years, range 35-85 years) with PV according to WHO criteria. Fifty subjects served as controls. The mean duration of disease was 9 years. All patients were on phlebotomy and ASA (100 mg once daily). Platelets and HCT were measured by automated analyzer. b-TG and PF4 were determined by ELISA. PFA and MCF were measured by Platelet Function Analyzer (PFA-100) and by ROTEM delta. Of 40 patients, 24 were homozygous HPA-1a/a and 16 heterozigous HPA-1a/b. The mean HCT value was 47±3% and platelets were 428±180x109/L. All patients had high b-TG and PF4 (113±47 IU/ml vs 20±11 IU/ml and 45±21 IU/ml vs 6±2 IU/ml, respectively), prolonged C/EPI closure time (CT, unit: s, n.v. 84-160 s) (233±65 s) and normal MCF (MCF, unit: mm, n.v. 50-72 mm) (65±5 mm). These findings suggest that PFA-100 and Thromboelastometric assays may be an useful tool to detect ASA platelet sensitivity in PV patients. Disclosures No relevant conflicts of interest to declare.

Description: Background: The Philadelphia chromosome negative chronic myeloproliferativeneoplasms (MPNs) including essential thrombocythemia (ET), polycythemia vera(PV), and myelofibrosis (MF) are a group of diseases of myeloid clonal lineage with significant symptom burden. MPN patients have also been found to have a high likelihood of mood disturbance related to their MPN, including symptoms of depression and anxiety (BMC Cancer. 201616:167). To date, the relationship between disease factors and depressive symptoms among MPN patients has not been well characterized. Methods: We have previously published the results of a 70-item internet-based survey which evaluated fatigue-relieving strategies among MPN patients (Cancer. 2016 Feb 1;122(3):477-85). Surveyed data included mental health questionnaires including a measure of symptoms of depression, the PHQ-2, (Ann Fam Med. 2010. 8(4): 348-353), a measure of positive and negative moods the POMS-short form (J Nerv Ment Dis. 1979;167(10):612-4) and a measure of symptoms of depression and anxiety, the MHI-5 (J Consult Clin Psychol. 1983. 51;730-742). This abstract is a secondary analysis of data collected among this cohort. Data: Demographics A total of 1788 individuals diagnosed with an MPN participated in the survey. 1389 patients answered questions regarding mood and psychological comorbidities. Many MPN patients reportedthat they had been seen by a health provider for care or diagnosis of depression (32%), anxiety (29.5%), stress (26.2%), or grief(15.0%). Overall, 24.0% of ET, 22.2% of PV, and 22.9% of MF patients endorsed symptoms of depression based on their score of greater than or equal to 3 on the PHQ-2. Demographic and Psychosocial Correlates Younger patients were significantly more likely to have PHQ-2 score of equal to or greater than three (57.0 years vs 59.5 years, p=0.0006). Gender, race, and country of origin did not significantly correlate with screening positive for symptoms of depression. Higher BMI was associated with a higher likelihood of screening positive for symptoms of depression (BMI 26.2 vs 24.6, p=0.005). Not surprisingly, having received treatment for mood problems in the last 6 months (p

Description: Introduction. A generic drug is a pharmaceutical drug considered to be equivalent to a brand-name product. A generic drug has to contain the same active ingredients as those of the original formulation. Regulatory agencies used to require that generics be identical to their brand-name counterparts with regards to pharmacokinetic properties. In most cases, generic products are available after the patent protection given to a drug's original developer expires. In Russia, a patent protection lasts for a 10-year period from registration of the original drug. To this day, twelve Imatinib generics have been registered in Russia. Aim. To assess the safety and efficacy of Imatinib generics for treatment of newly diagnosed Chronic myelogenous leukemia patients that have been in our center since August 2012. Materials and methods. 30 newly diagnosed CML patients were started on generics. The drugs: 1) GenericPh 100 mg, in capsules (Ph-Syntez, Russia); 2) GenericG 100 mg, in tablets (Laboratorio TUTEUR S.A.C.I.F.I.A., Argentina); 3) GenericIm 100 mg, in tablets (Sandoz d.d. (Slovenia). Switching from one generic to another was done due to intolerance. We analyzed the range and frequencies of adverse events (AE), cumulative incidences of complete hematologic (CHR), major cytogenetic (MCyR), complete cytogenetic (CCyR), and early molecular responses (BCR-ABL

Description: Multiple trials now demonstrate the potential for treatment-free remission (TFR) in patients with sustained deep molecular response to tyrosine kinase inhibitors (TKIs). We describe two patients who remain in treatment-free remission after achieving undetectable BCR-ABL with ponatinib on the phase II PACE trial, despite being multiply resistant/intolerant to other agents or carrying the T315I mutation. Patient 1 previously had a suboptimal response to imatinib (BCRABL 〉10% at 15mo) and then was intolerant of nilotinib and dasatinib due to grade 3 liver enzyme abnormalities. He achieved undetectable BCR-ABL (Molecular MD laboratories) by six months on ponatinib. His ponatinib dose was gradually reduced from 45mg/d to 15mg/d due to vascular events (myocardial infarction requiring stenting, carotid stenosis requiring endarterectomy, recurrent ischaemic colitis). At the time of his last episode of colitis, his BCR-ABL had been undetectable for 47 months and hence a decision to attempt TFR was made. He remains in TFR 11 months post-cessation. Patient 2 progressed on imatinib and was found to carry the T315I mutation. He achieved undetectable BCR-ABL by nine months on ponatinib. His ponatinib dose was also gradually reduced from 45mg/d to 15mg/d due to vascular events (popliteal stenosis requiring stenting, embolic stroke, coronary artery disease requiring stenting). At the time of his last event, his BCR-ABL had been undetectable for 30 months and hence a decision to attempt TFR was made. He remains in TFR 17 months post-cessation. This series adds to the one prior case report of ponatinib facilitating treatment-free remission (Engel NW et al. J Oncol Pract 2016. 12(6);592-4) and these remissions are notable because they occur in patients not considered for other TFR studies because of refractoriness to other agents or known T315I mutations. This is an alternative avenue to dose reduction to reduce the risk of vascular events in ponatinib-treated patients. Disclosures Mills: Ariad: Other: This clinical study funded by Ariad.

Description: Erythropoiesis is a highly controlled process partly regulated in erythroblastic islands by the central erythroblastic island macrophage (EI MΦ) which provides iron, growth factors and mediates enucleation of the maturing erythroblasts. As macrophages are key effectors of inflammation, we investigated the effect of bacterial LPS in vivo on erythropoiesis and EI MΦ defined as CD11b+ F4/80+ VCAM1+ CD169+Ly6G+ in mice. C57BL/6 mice were injected i.p. with 2.5 mg/kg/day LPS from E. coli for 2 days and the effect on medullar erythropoiesis examined at various time-points after this. LPS administration caused a marked whitening of the bone marrow (BM) with decreased numbers of basophilic (9-fold), polychromatic (3.7-fold), orthochromatic erythroblasts (2.2-fold) and reticulocytes (2.5-fold) 48hrs after LPS challenge. Those remained significantly reduced up to 6 days post-LPS. Likewise, EI MΦ were suppressed in the BM 24-48hrs after LPS challenge and remained significantly reduced 6 days post-LPS. This loss of medullar erythropoiesis was compensated by increased number of EI MΦ (13.6-fold), pro-erythroblasts (1.5-fold), polychromatic (1.9-fold), orthochromatic (3.2-fold) and reticulocytes (2.3-fold) in the spleen. As this phenotype resembled suppression of medullar erythropoiesis following G-CSF treatment, we examined whether the mechanism could be indirect via endogenous G-CSF release. LPS induced a transient 80-fold increase in G-CSF concentration in the blood from 123pg/mL (n=6) to 10ng/ml (n=6) 2 days post-LPS. LPS was administered to TLR4 KO and G-CSF receptor (GCSFR) KO mice. These LPS-mediated responses were abrogated in TLR4 KO mice demonstrating that erythropoiesis suppression in response to LPS is fully TLR4-dependant. However responses in GCSFR KO mice were more contrasted. EI MΦ numbers did not change in GCSFR KO mice in response to LPS demonstrating that suppression of EI MΦ in response to LPS is an indirect effect of endogenous G-CSF release. In contrast, medullar erythropoiesis was still suppressed in GCSFR KO mice with significantly reduced numbers of basophilic, polychromatic and orthochromatic erythroblasts demonstrating that medullar erythroblast suppression 1) persists despite the presence of EI MΦ, and 2) is not G-CSF-dependent. Unexpectedly, the BM from GCSFR KO mice treated with LPS was not whitened with high numbers of reticulocytes/erythrocytes. To further understand how BM erythrocytes could be increased whilst erythropoiesis is suppressed in LPS-treated GCSFR KO mice, we measured vascular leakage by injecting Evans Blue i.v. Blood plasma volume in the BM of LPS-treated GCSFR KO mice was 2.9-fold higher compared to LPS-treated wild-type mice and untreated WT and KO mice (6.0±2.0 µL vs 2.1±0.9 µL blood plasma/femur, p=0.005) suggesting that GCSFR-mediated signaling is necessary to maintain the integrity of the BM vasculature in response to LPS. In conclusion LPS-mediated medullar erythropoiesis suppression involves at least two different TLR4-dependent mechanisms in regards to their requirement for GCSFR: 1) GCSFR-dependent suppression of EI MΦ, 2) GCSFR-independent and EI MΦ-independent suppression of maturing erythroblasts. We are currently investigating whether the latter mechanism involves hepcidin. Finally we also discovered that GCSFR-mediated signaling is necessary to maintain the BM vasculature integrity following LPS challenge. Disclosures Winkler: GlycoMimetics: Research Funding. Levesque:GlycoMimetics: Equity Ownership.

Description: Introduction Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) associated with the JAK2 V617F mutation. Recombinant interferon alfa-2b (rIFNα-2b) or pegylated rIFN2α (pegrIFNα-2a) treatment in PV is effective in inducing hematologic and molecular responses. Reduction in JAK2V617Fallele burden (%V617F) has been used increasingly as a surrogate marker of disease response to treatment and as a unique criterion for discontinuation of therapy. To date, no studies have evaluated the relationship between changes in %V617F and established indicators of disease activity such as bone marrow cellularity and degree of fibrosis. Having observed persistent marrow hypercellularity or progressive fibrosis in PV patients despite a significant reduction in %V617F, we therefore systematically examined the correlation of %V617F with marrow morphology. We also assessed immunohistochemical expression of pSTAT5 as a marker of JAK2 activation. Methods The diagnosis of PV was based on the WHO criteria which include presence of erythrocytosis and/or an increased Cr51 red cell mass, JAK2V617F mutation and typical bone marrow findings such as hypercellularity due to panmyelosis and megakaryocyte morphology consistent with PV. Patients with two marrow examinations and quantitative JAK2 levels measured at a median of 1.2 months from the biopsy date were eligible for inclusion. The JAK2V617Fallele burden in peripheral blood samples was determined by pyrosequencing. Clinical, hematologic and molecular responses were graded according to ELN criteria. Marrow fibrosis was scored according to the WHO three-tiered semi quantitative grading system. Immunohistochemical staining for p-STAT5 was performed on clot sections in a subset of patients. Results We identified 15 patients for inclusion. The median %V617F at the time of the initial biopsy was 62.2% (18.2-100%), which decreased to a median of 33% (0-93.7%) on subsequent evaluation (p=0.02). Patients were initially treated with peg rIFNα-2a 45-90 mcgm weekly (n=13) or rIFNα-2b (n=2), 1x106units thrice weekly, and gradually increased based upon response and tolerance. The median duration of treatment between biopsy was 4.2 years (0.8 - 6.6). All patients achieved either a complete (CHR) (n=8) or a partial (PHR) (n=7) hematologic response. Of these 15 patients, 3 achieved a complete molecular response (CMR), 4 a partial molecular response (PMR), and 8 patients no molecular response (NMR). We observed no correlation between clinical and molecular response: of the 8 patients who achieved CHR, 2 achieved a CMR and 6 did not. There was no correlation between clinical response and fibrosis or cellularity; of 8 patients who achieved CHR, 5 showed persistent/increased hypercellularity and 6 had persistent/increased fibrosis. We also found no correlation between marrow morphologic changes and molecular response. Of the 3 patients with CMR, all had persistent hypercellularity (range: 60-90%) and increased/persistent fibrosis. Among the 4 patients who achieved a PMR, 2 had increased cellularity and 2 decreased cellularity. All had increased or persistent fibrosis. Among the 8 patients with NMR, 2 had an increase in marrow cellularity, 2 no change, and 4 an insignificant decrease. Fibrosis increased in 2, was unchanged in 3, and decreased in 3. Thus, there were no significant correlations between changes in %V617F and cellularity (κ=0.02) and fibrosis (κ=0.04). Immunohistochemical staining for p-STAT5 expression in megakaryocyte nuclei showed no correlation between degree of positivity and %V617F. Conclusions Although we observed a statistically significant decrease in %V617F after treatment with rIFNα, there were no parallel changes in marrow morphology. In all patients with CMR, we observed persistent or worsening marrow disease, suggesting to us a need for continued therapy. In patients who achieved either CHR or PHR, we observed persistent hypercellularity and persistent/progressive fibrosis. Our results question the use of JAK2V617F allele burden as the sole criterion for discontinuation of rIFNα therapy. The prognostic significance of failing to attain a morphologic response in patients who have achieved a hematologic and/or molecular response remains unresolved. Resolution of these issues with longer follow-up is important, as premature discontinuation of rIFNα may allow the pathogenic mechanisms of the disease to progress unfettered. Disclosures Orazi: Novartis: Honoraria.

Description: Background.- Nilotinib has 30-fold higher potency and increased selectivity against Bcr-Abl.Fast and deep responses can be achieved with nilotinib after failure to imatinib. In this study, we report the response rates and long-term results of using nilotinib after failure to imatinib or beyond. At the Specialty Hospital CMN "La Raza" nilotinibexperience launching this late 2006, has developed institutional experience. Objective.- To evaluate the cytogenetic and molecular response to treatment with nilotinib in the second line treatment of patients with CML-CP and its correlation with early molecular-response. Methods- During the experience with nilotinib as second-line therapy or beyond on CML-CP, 110 CML patients have been treated; established dose of nilotinib for all phase was 400 mg BID. Dose adjustments due toxicity were performed in accordance to previous publications, hematological and no hematological toxicity were scored in conformance to CTCAE v3. Patients were evaluated for hematologic, cytogenetic and molecular responses. Of all patients with CP-CML diagnosed treated with nilotinib, sixty-nine patients were included in the analysis, we selected those who had undergone a molecular evaluation at 3 months. In this study by BCR-ABL mutation status was analyzed. Twenty-seven percent (30 of 110) of the imatinib-resistant patients had BCR-ABL mutation(s) at baseline. One imatinib-resistant patients had the T315I mutation at baseline. The data from 69 patients with CP-CML were analyzed. Distribution of Sokal risk groups were as follows low-16/intermediate-14/high-57/NA-13%. Results.- Overall, 57 of the 69 patients (83%) of them had a transcript of ≤ 10% at 3 months. Forty-five out of 69 patients (65%) with transcript of ≤10% at 3 months achieved MMR at 12 months, while only 2 of 12 patients (17%) with ratio 〉10% had MMR, and 83% did not reach the MMR. The EFS by level of transcripts ≤10% at 3 months in 57 patients with 2 events was 96% while in 〉10% in 12 patients with 4 events was 67%, with an average estimate of time of EFS of 62 vs 43 months respectively. The EFS by MMR at 12 months in 32 patients without events was 100%, while without MMR in 37 patients with 6 events was 84% P=0.022. Nilotinib induced complete cytogenetic responses in 60 percent and 48% molecular responses in the 110 patients treated, 58% of which were major molecular responses, and deep molecular response 42%. To date, 96 of the treated patients remain alive (87%) and continue on treatment. Conclusions.- Nilotinib (Tasigna) is efficacy and well-tolerated treatment for patients with CML as second-line or beyond in our patients, The 48-month follow-up results show that nilotinib therapy continued to be effective, is comparable to diverse results of other international reports in developed countries. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Imatinib mesylate (Glivec, Novartis) is the first tyrosine kinase inhibitor (TKI) targeting the BCR-ABL1 fusion protein responsible for the pathogenesis of chronic myeloid leukemia (CML). Low cost generic alternatives to imatinib are an integral part of cost effective healthcare strategies for developing countries. However, the use of generics has been associated with different clinical outcomes. In this study, we compared outcomes of two groups of patients who received Glivec as first-line therapy (Group 1) to patients who received generic imatinib as first-line therapy (Group 2) in Bosnia and Herzegovina. Material and methods This was a multicenter retrospective cohort study of BCR-ABL1 positive CML patients (n = 53) in the Federation of Bosnia and Herzegovina between 1 June 2005 and 31 March 2016. Glivec was used from 01 June 2005 until 30 September 2013, when all patients had to switch to generics, which was mandated by the Federal Solidarity Fund that allocates targeted cancer therapies. The following generic imatinib was available: Anzovip (Zdravlje, Actavis) from 09/2013 to 09/2014, Meaxin (Krka) from 09/2014 to 12/2015, and Plivatinib (Pliva) from 12/2015. Patient data was collected from the database of the Federal Solidarity Fund, a subsidiary of the Federal Health Insurance Agency. Branded and generic imatinib was administered orally at dosage of 400 mg/day. Patients who were switched to nilotinib received orally 400 mg/day. Patients on Glivec included in this study started therapy from 0-6 months from time of diagnosis, while patients who started with generics did not wait for therapy. Patient variables that were collected included age, gender, town, date of diagnosis, date of start of therapy, monthly TKI dosage, adverse side effects, progression, lethal outcome, prognostic factors and diagnostic parameters, including cytogenetics and molecular testing. In September 2013, Glivec stopped being available in Bosnia and all CML patients were switched to generic therapy Anzovip. Median duration of each therapy is given in Table 1. Results We compared patients on Glivec as first-line therapy (Group 1, n=26) to patients on first-line generic imatinib (Group 2, n=27) with the follow-up period of at least three years for each group. When we compare Groups 1 and 2 using intention to treat analysis, Kaplan-Meier estimated rate of overall survival at 24 months of therapy was 88% vs. 68%, respectively (p=0.14), while 69% vs. 70% achieved CCyR (p=0.12), respectively. In Group 1, 27% (7/26) patients switched to nilotinib (treatment failure in 2 patients and side effects in 5 patients), 54% (14/26) patients switched to generics because Glivec was no longer available, and 19% (5/26) patients stopped therapy (2 patients stopped therapy and 3 patients died). Of the 7 patients who switched to nilotinib, 71% (5/7) achieved CCyR, 29% (2/7) achieved MMR and none died. Of 19 patients who stayed on imatinib, 68% (13/19) achieved CCyR, 63% (12/19) achieved MMR and 3/19 (16%) died. Of the 54% (14/26) patients who were switched from branded imatinib to generic imatinib, one patient (7%) lost complete cytogenetic response. Regarding Group 2, 52% (14/27) of patients switched to nilotinib due to treatment failure (n=8) and side effects (n=6), while 48% (13/27) of patients stayed on generics. Of patients who switched to nilotinib, 43% (6/14) achieved CCyR and 15% (2/14) achieved MMR. Of the patients who stayed on generic imatinib, 100% (13/13) achieved CCyR and 85% (11/13) achieved MMR. Conclusion Our results suggest that there was no obvious difference in the treatment efficacy between generic and branded imatinib. At 3 years, there was no significant difference in the overall suvival and achievement of CCyR between first-line Glivec and first-line generic imatinib (p=0.14, and p=0.12, respectively). * Median duration of therapy on generic imatinib Table Table. Disclosures Radich: Bristol-MyersSquibb: Consultancy; Pfizer: Consultancy; ARIAD: Consultancy; TwinStrand: Consultancy; Novartis: Consultancy, Other: laboratory contract.

Description: Introduction: The Philadelphia chromosome (Ph+) is a hallmark of CML and is also present in a subset of patients with acute lymphoblastic leukemia (ALL). Lymphoid blast phase (CML-BP) and Ph+ ALL almost always affect B cell lineage. In this report, we present the clinical characteristics of the rare subset of patients with Ph+ ALL or CML-BP with T-cell lymphoid phenotype. Methods: We reviewed the institutional database for all patients with blast phase CML (n=498) and de novo T-ALL (n=150) diagnosed since 07/1997. All clinical characteristics at the time of the diagnosis of BP or Ph+ ALL were collected. Seven patients with Ph+ T-cell lymphoid leukemia were identified. Results: Among the total of 498 CML-BP patients, 5 patients had T-cell lymphoid CML-BP (0.01%); in addition 2 patients with de novo Ph+ T-ALL were identified among 150 patients with T-ALL seen during the same time period (1.3%). Median age of the seven patients was 57 years (range 31-72 years); 71% were male. Among patients with CML-BP, all were in BP at the time they were referred to MDACC (one had progressed from an initial diagnosis in CP, 1 from accelerated phase, and 3 were in BP at initial diagnosis). Only 2 patients with CML-BP received prior TKI therapy; the 2 patients with T-ALL were previously untreated at the time of referral. Immunophenotype included 2 cortical, 3 early T-cell and 2 early T-cell precursor; 2 patients were negative and one was dim positive for TdT (summarized in Table-1). Extramedullary disease at initial presentation was seen in all patients [2 with lymphadenopathy alone, 2 with mediastinal involvement (one also with lymphadenopathy and the other with splenomegaly and lymphadenopathy), 2 with pleural effusion (1 with lymphadenopathy and another with splenomegaly) and 1 patient with splenomegaly alone]. None had CNS involvement at initial presentation, but one patient developed CNS involvement at first relapse. One patient had variant Ph+ and 2 had complex karyotype. Three patients had lymphoblastic lymphoma and 4 patients had T-ALL. Five patients died (3 from disease progression, one from complications of SCT, and one from intestinal obstruction and sepsis) and 2 are alive (25 and 49 months from BP diagnosis). The median survival for all patients was 13 months (range 1-49 months). Five patients received hyper-CVAD based therapy as an induction regimen: 3 of them received hyper-CVAD alone - one achieved CR then underwent a SCT but died due to complications of SCT, another achieved PR and was given imatinib followed by dasatinib which he did not tolerate; he was subsequently lost to follow up; and one patient had no response. The other 2 patients received hyper-CVAD with dasatinib - one achieved PR, then underwent allo-SCT and achieved CR but later had a CNS relapse and progressed; the other patient developed nodal progression 9 months after achieving CR with HCVAD-dasatinib which was later found to represent metastatic prostate cancer in the lymph nodes with no T-ALL recurrence and is currently on dasatinib with major molecular response. Two patients never received any TKI therapy and progressed on chemotherapy alone. Two patients received nelarabine as first salvage - one as a single agent (no response) and another with dasatinib (with PR). Conclusions: Ph+ T-cell lymphoid leukemias are very rare and have clinical features similar to those of de novo T-ALL. Induction with a combination of HCVAD and TKIs may achieve prolonged remission in some patients. Disclosures Jabbour: ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. O'Brien:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria. Cortes:ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding.

Description: Background: MPNs including Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF), are clonal hematopoietic diseases in which the discovery of molecular driver mutations (JAK2, CALR, MPL) has deeply modified diagnostic approach in recent years. To date available data on epidemiology of MPNs and perspective analysis are rare. Our aim is to study the incidence of MPN Ph negative in a specific region of Italy named Latium and its variability across five years. Moreover we prospectively report the general features of our population. Method: We present here the prospective epidemiologic analysis of 1116 adult patients affected by MPNs (PV=289, ET=550, PMF=209) diagnosed according to 2008 WHO criteria, from January 2011 to December 2015 in 15 hematological Centers (5 academic and 10 community-based Hospitals) in Latium. A total of 289 PV, 550 ET and 209PMF were identified. The overall incidence rate of 289PV was 1.0/105 in 2011 and 2012, 1.1/105 in 2013, 0.9/105 in 2014 and 2015. The overall incidence rate of 550ET was 2.0/105 in 2011, 2.4/105 in 2012, 2.2/105 in 2013, 1.8/105 in 2014 and 1,2/105 in 2015 and the overall incidence rate of 209PMF was 0.7/105 in 2011 and 2012, 1.0/105 in 2013, 0.7/105 in 2014 and 0.5/105 in 2015. We have observed also 63 cases of MPNu (36M/32F) and the incidence rate was 0.3/105 in 2011 and 2012, 0.14/105 in 2013, 0.24/105 in 2014 and 0.22/105 in 2015. Baseline features of PV, ET and PMF patients are summarized in table 1. We have also analyzed the presence of comorbidities including obesity, arhythmia and neoplasia observed at the diagnosis in 1.6, 6.2 and 4% of all population, respectively; thirty-five percent of 1116 pts presented other comorbidities such as diabetes, inflammatory bowel disease, renal and liver failure. As thrombotic risk factors we considered diabetes, dislipidemia, smoke, essential hypertension and thrombophilia observed in 11,8, 16,2, 13,2, 51,7 and 3% of total pts, respectively. Conclusions: We confirm in our prospective observational protocol the overall incidence of MPN Ph negative, previously reported in the literature and the major incidence of male gender in PV and PMF, female in of ET. The annual incidence from 2011-2015 in Latium is remained substantially the same during the observation period. The decreasing trend observed in 2015 is probably due to the different update of some Centers that was done in October 2015 not including patients diagnosed in the last two months. Disclosures Latagliata: Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Janssen: Consultancy, Honoraria; Shire: Honoraria. Breccia:Pfizer: Honoraria; Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria. Cimino:Celgene: Honoraria; Bristol-Mayer: Honoraria.

Description: Despite the remarkable clinical responses achieved with BCR-ABL tyrosine kinase inhibitors (TKIs) in the treatment of chronic phase-chronic myeloid leukemia (CML), these TKIs have been less effective as single agents in blast phase (BP) CML. Identification of new therapeutic strategies is needed for the better clinical management of BP-CML. It is well known that the mitochondrial metabolic properties of tumor cells are different from those of normal cells, making this as an attractive target for cancer treatment. Previously, we screened a number of antimicrobial drugs with possible mechanisms of action related to mitochondrial metabolism and identified mefloquine as a potential candidate for CML treatment. Mefloquine is a FDA-approved antimalarial drug and has been reported to have anti-cancer activities. In this work, we investigated the effect of mefloquine and its underlying mechanisms in CML. We show that mefloquine induces apoptosis of CML cells in a dose-dependent manner (Fig. 1A). In addition, mefloquine is also effective in targeting BP-CML CD34+ progenitor cells. It induces apoptosis, inhibits colony formation and self-renewal capacity of CD34+ cells derived from a TKI-resistant BP-CML patient (Fig. 2). Mefloquine significantly enhanced anti-proliferative and pro-apoptotic effects of imatinib and dasatinib in CML cell lines as well as BP-CML CD34 cells, suggesting that mefloquine augments the effects of BCR-ABL TKIs (Fig. 1B, 2A and 2B). Mechanistically, we show that mefloquine significantly induces oxidative stress by increasing levels of mitochondrial superoxidase in K562 cells (Fig, 1C). Consistent with this, mefloquine disrupts lysosomal integrity/function in CML cells as measured by LysoTracker labelling (Fig. 1D). Taken together, we demonstrate that mefloquine is active against BP-CML and enhances the efficacy of BCR-ABL TKIs. Our work also highlights the therapeutic value of targeting oxidative stress and lysosome in the treatment of BP-CML. Figure 1 Mefloquine induces apoptosis, ROS, and lysosomal dysfunction in CML cells. (A)Mefloquine induces apoptosis of K562, LAMA84 and KU812 cells in a dose-dependent manner. (B) Combination of mefloquine and imatinib or dasatinib induces more much apoptosis than single drug alone. Cells were treated with drugs for 72 h. (C) Mefloquine increases levels of mitochondrial superoxidase in K562 cells. (D) Less Lysotracker staining in mefloquine-treated K562 cells compared to control. Cells were treated with mefloquine at 15 µM for 24 h. Figure 1. Mefloquine induces apoptosis, ROS, and lysosomal dysfunction in CML cells. (A)Mefloquine induces apoptosis of K562, LAMA84 and KU812 cells in a dose-dependent manner. (B) Combination of mefloquine and imatinib or dasatinib induces more much apoptosis than single drug alone. Cells were treated with drugs for 72 h. (C) Mefloquine increases levels of mitochondrial superoxidase in K562 cells. (D) Less Lysotracker staining in mefloquine-treated K562 cells compared to control. Cells were treated with mefloquine at 15 µM for 24 h. Figure 2 Mefloquine effectively targets BP-CML CD34 progenitor cells. Mefloquine induces apoptosis (A) and colony formation (B) of BP-CML CD34 cells and combination of mefloquine and dasatinib is superior in inducing apoptosis and decreasing colony formation. (C) Mefloquine inhibits self-renewal capacity of BP-CML CD34 cells. Figure 2. Mefloquine effectively targets BP-CML CD34 progenitor cells. Mefloquine induces apoptosis (A) and colony formation (B) of BP-CML CD34 cells and combination of mefloquine and dasatinib is superior in inducing apoptosis and decreasing colony formation. (C) Mefloquine inhibits self-renewal capacity of BP-CML CD34 cells. Disclosures Hwang: Sanofi: Honoraria, Other: Travel support; Janssen: Honoraria, Other: Travel support; BMS: Honoraria, Other: Travel support; Celgene: Honoraria, Other: Travel support; Roche: Honoraria, Other: Travel support; Pfizer: Honoraria, Other: Travel support; Novartis: Honoraria, Other: Travel support; MSD: Honoraria, Other: Travel support. Chuah:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Chiltern: Honoraria.

Description: An understanding of cellular events that are propagated within erythroid progenitor cells upon HGF-R / JAK complex activation is of basic importance for generating new insight into regulated red cell formation, anemia and myeloproliferative disease. Using the EPO/EPOR/JAK2 system as a paradigm, our group is successfully applying post-translational modification-based proteomics to uncover important new mediators of EPO-dependent human erythropoiesis (certain of which may also relate to EPO's untoward effects on hypertension and cancer progression). Here, we report on the discovery of a novel ORF, "C1ORF150", that is strongly tyrosine phosphorylated in response to EPO, possesses several unique features, and modulates EPO- dependent erythroid progenitor cell formation. In human erythroid progenitor UT7epo cells, EPOR ligation leads to C1ORF150 phosphorylation at tandem tyrosine p-Y69, p-Y89 and p-Y110, p-Y129 sites (up to 10-fold within 15 minutes). For each PTM site, EPOR/JAK2 mediated- phosphorylation was validated in independent LC-MS/MS experiments using Hematide as an EPOR agonist. p-Y69 and p-Y89 are predicted SFK sites, while p-Y110 and p-Y129 are predicted RTK sites (including KIT). Notably, C1ORF150 is conserved in H. sapiens and primates, but is not represented in mouse, rat or lower vertebrates. In addition, C1ORF150 has no obvious orthologues, but within EPO-regulated pY regions exhibits sequence homology with HGAL, an important factor for B cell receptor signaling. To assess C1ORF150's functional effects, we used a lentiviral shRNA loss-of-function approach (80% knockdown efficiency). At physiological EPO levels, the knockdown of C1ORF150 substantially compromised UT7epo erythroid progenitor cell (EPC) survival, including 200% increases in apoptosis observed relative to control sh-NT transduced EPCs (p 〈 0.01). The ectopic expression of C1ORF150, in contrast, heightened baseline JAK2 activation, and potentiated STAT5 activation following EPO challenge. C1ORF150's subcellular localization proved to be predominantly membrane associated. With regards to expression profiles, C1ORF150 levels were markedly elevated in bone marrow (among 30 human tissues), and during erythroid development were maximal at a CFUe stage. Furthermore, transcriptome profiles of myelodysplastic syndrome (MDS) CD34pos hematopoietic progenitor cells revealed elevated C1ORF150 expression in MDS refractory anemia (p=0.005) and refractory anemia-blast patients (p=0.05) compared to normal controls. In summary, via PTM-proteomics we have discovered "C1ORF150" as a major new pY- regulated EPOR/JAK2 target and membrane associated phosphoprotein that is proposed to have evolved in human erythroid progenitor cells to support EPO's cytoprotective effects, and red cell formation, in part by reinforcing JAK2 and STAT5 activation. In anemia and pre-leukemic contexts, attention also is brought to possible roles for C1ORF150 in the onset and progression of MDS. Disclosures No relevant conflicts of interest to declare.

Description: Background: Prognosis of diffuse large B-cell lymphoma (DLBCL) and other aggressive lymphoma entities has improved with the advent of Rituximab, and R-CHOP-21 and variants is SOC. Nevertheless, a substantial proportion of patients fail first line treatment. Salvage therapies are often effective. However, no more than 25-50% achieve a long term remission even when consolidative high dose chemotherapy (HDT) followed by hematopoietic stem cell transplantation (SCT) is applied. In case of failure or intolerance to HDT, regimen like Gemcitabine/Oxaliplatin are applied but show limited efficacy, indicating the need for new treatments. Obinutuzumab (GA101) is a type II anti-CD20 antibody. Superiority of Obinutuzumab could be demonstrated in xenograft models of mantle cell lymphoma and DLBCL. Although desirable, cumulative dose-related, progressive cardiotoxicity eliminates anthracyclins from higher treatment lines. With Pixantrone, a drug structurally related to anthracyclines and especially anthracenediones, a re-exposition against this drug class has been shown to be feasible. In 70 heavily pre-treated patients, a best ORR of 40% (20% CR/CRu) was observed (Pettengell et al). Experiences from further antibody drug combinations lead to the assumption that the effects of Pixantrone will be augmented by a monoclonal antibody without increasing toxicity. We thus initiated a trial combining both agents for the first time. The trial has opened in Q4/2015 and recruitment is ongoing. Overall, a total of up 70 patients will be enrolled for a number of 64 evaluable patients. Primary endpoint will be the objective overall response rate, with secondary endpoints being safety, PFS and OS. Methods: this is a multicenter, national, prospective trial. Inclusion criteria: patients were eligible if they had histologically proven DLBCL, FL grade IIIb or transformed indolent lymphoma, CD20 positive disease, no curative option available, relapsed disease, measurable disease, ECOG 〈 3, sufficient bone marrow reserve, no severe concomitant diseases and given informed consent. There was no upper limit or prior treatment lines. Treatment consisted of Pixantrone 50mg/m² day 1, 8 and 15 of each cycle, Obinutuzumab 1000 mg flat dose day 1, 8 and 15 of cycle one and day 1 of each subsequent cycle. A total of 6 cycles was planned with interim staging after 3 cycles. Results: 24 patients (pts) have been included until now. Concerning clinical characteristics, all were caucasian, 12 were female and the other 12 male and median age was 75 years. Most of the patients suffered from DLBCL (18 pts, 82%). Median number of prior therapies was 2 (1 to 6). Until now 55 evaluable cycles of chemotherapy (median 2 cycles (0 to 6)) have been performed. At this time, the treatment seems to be well tolerated, with no unforeseen side effects. Observed toxicity was predominantly hematologic. The following hematologic adverse events of grade 3/4 were noted: leukopenia (4 pts, 17%), neutropenia (6 pts, 25%), granulocytopenia (1 pts, 4%), as well as thrombocytopenia (2 pts). Non-hematologic grade 3/4 adverse events were observed in at least two patients: hypertension (2 pts) and pelvic pain (2 pts). Response: currently, best responses were 4 PR, 1 SD, and 8 PD in 13 patients evaluable so far. Four patients died, all after progression of lymphoma. Summary: the combination of Obinutuzumab and Pixantrone seems to be feasible and safe with early signs of efficacy. Updated results of this trial in progress with a focus on safety will be presented. Disclosures Hess: Janssen: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Celgene: Honoraria; Roche, CTI, Pfizer, Celgene: Research Funding; Roche: Honoraria. Marks:Pfizer: Honoraria. Witzens-Harig:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dreyling:Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau. Viardot:Amgen: Consultancy; Janssen: Consultancy; BMS: Consultancy; Roche: Honoraria; Takeda: Other: travel support; Pfizer: Honoraria. Keller:Spectrum Pharmaceutical: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Description: Background.Treatment of APL in the elderly with conventional ATRA-anthracycline based CT regimens is associated, like in younger patients, with very few relapses, but high death rates (CR rate of 87.3%, 10-year CIR of 9.3%, 21.7% deaths in CR, and a 10-year OS of 58% in patients aged〉 65 years in our previous APL trials ;Blood 2010 115:1690). Recent results have shown that, in standard risk APL, ATRA+ATO combinations (without CT) are at least as effective as classical ATRA + CT regimens while being less myelosuppressive (Lo Coco, NEJM 2014; Burnett, Lancet Oncol, 2015) thus constituting a very appealing approach for elderly patients. However, when our APL 2006 trial started, the feasibility of treatment of APL without CT was unknown. Furthermore, access to ATO still remains limited for frontline treatment of APL in most countries. We present results of APL 2006 trial, where we combined ATO to ATRA and reduced CT in patients aged older than 70 with standard risk APL (baseline WBC 70 years with WBC 1 G/L and platelets〉 50G/l after the first consolidation course was 16.2 and 11.9 days in the original vs 5.6 and 4.0 days in the amended protocol (p

Description: PD-L1/PD-L2 are immunomodulatory molecules that engage with the PD-1 receptor on immune effector T and NK-cells to inhibit anti-lymphoma immunity. PD-1/PD-L1/PD-L2 axis molecules are prognostic in Hodgkin Lymphoma (HL, Roemer et al J Clin Oncol 2016) and Diffuse Large B-cell Lymphoma (DLBCL, Keane et al Lancet Haem 2015). Importantly, blockade of the axis is associated with particularly potent clinical responses in relapsed/refractory HL (Ansell et al NEJM 2015), as well as response in DLBCL (Armand et al J Clin Oncol 2013). Focus has been on the interaction of PD-L1 on malignant B-cells with PD-1 on HLA-class I restricted CD8+ effector T-cells. This is despite considerable evidence that: A) malignant B-cells in HL and DLBCL frequently lack the ability to present HLA-class I due to mutations in b2M and associated antigen presenting molecules (Challa-Malladi et al Cancer Cell 2013). This makes them insensitive to direct lysis by CD8+ T-cells (Zaretsky et al NEJM 2016) but potentially enhances their sensitivity to NK-cells; B) PD-L1/PD-L2 are expressed by inhibitory CD163+ monocytes/macrophages as well as by malignant B-cells (Chen et al CCR 2013). Here, we seek to establish the contribution of NK-cells and inhibitory CD163+ expressing monocytes/macrophages in the setting of HL and DLBCL. CD163/PD-1/PD-L1/PD-L2 gene expression was quantified by nanoString in 194 patients and was elevated in HL relative to DLBCL tissues (P

Description: Introduction: APL, while highly curable in the modern era, remains a therapeutic challenge in the high-risk subset, with high rates of early mortality and relapse compared to non-high-risk APL. Recent evidence has confirmed excellent outcomes in non- high-risk APL with the combination of ATO and ATRA, and a previous pilot study had indicated the efficacy of a combination of ATO + ATRA + GO in high-risk APL. The North American Leukemia Intergroup designed a larger phase 2 study to confirm the efficacy and safety of this combination in high-risk APL. Primary Objectives: 1) assessment of 3-year event-free survival (EFS); 2) assessment of early (6 week) death rate. Methods: Adult patients with newly diagnosed high-risk APL (WBC ≥10k/uL) were eligible. Induction therapy consisted of: ATRA (45 mg/m2/day), beginning on day 1; ATO 0.15 mg/kg/day, beginning on day 10; GO 9 mg/m2on day 1. ATRA and ATO were continued until remission achieved. Patients in remission went on to receive consolidation with ATO x 2 cycles, followed by ATRA + daunorubicin x 2 cycles, followed by GO x 2 cycles. Subsequent maintenance therapy consisted of ATRA + 6-mercaptupurine + methotrexate for up to 1 year. Results: Between 2008 and 2013, 73 registered patients began protocol treatment. Median age was 46.5 years, with 52% females and 48% males. Sixty-two (85%) patients completed induction therapy as planned, and 50 patients (68%) completed all planned consolidation. Sixty-two patients (85%) achieved a documented complete response (CR). Non-responses were attributable to lack of response assessment (n=10), most commonly related to death. One patient had resistant disease. With a median follow-up of 3.3 years, the Kaplan Meier 3-year EFS estimate was 79% (95% CI 68% - 87%), which was significantly improved compared to the protocol-defined historical rate of 50% (p

Description: Background: BCL6 is known as a protooncogene and transcriptional repressor in diffuse large B cell lymphoma, where it is frequently involved in chromosomal rearrangements. We recently identified BCL6 as a novel mediator of drug resistance to tyrosine kinase inhibitors (TKI) in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (Duy et al., Nature 2011; Hurtz et al., J Exp Med 2011). In addition,BCL6 directly competes with the tumor suppressor BACH2 for p53 promotor binding to protecting cells from p53-mediated apoptosis in multiple ALL subgroups (Swaminathan et al., Nature Medicine 2013). Based on this, our current study is focusing on the function of BCL6 in different subtypes of human ALL. Results: Analysis of gene expression data from 207 children with high-risk B cell precursor ALL (COG P9906) showed that high expression levels of BCL6 at the time of diagnosis correlated with a poor overall and relapse-free survival (p=0.007). Furthermore, 49 matched sample pairs from patients at diagnosis and relapse showed increased BCL6 levels at relapse compared to diagnosis (p=0.003). To test whether or not there are specific subtypes of leukemia with high BCL6 expression levels, we studied BCL6 expression via western blot and immunohistochemistry staining in Non-Ph+ cell lines and ALL patient samples (n=76). Interestingly, BCL6 levels are particularly elevated in MLL-rearranged (MLLr) ALL cases. In addition, patients from the clinical trial that had high BCL6 levels and had MLL rearrangements had the worst clinical outcome (p=0.0009). We next tested if MLLr oncogenes drive aberrant BCL6 expression. First, we performed a ChIP-analysis using antibodies against MLL, AF4, and ENL, which provided evidence for direct binding to the BCL6 promoter. We then performed a BCL6 Western blot analysis of inducible MLL-AF4-transgenic and retrovirally transduced MLL-ENL pre-B cells, demonstrating that both oncogenes are sufficient to induce ~10-fold upregulation of BCL6 protein levels. Additionally, we used a newly developed conditional BCL6 knock out/reporter mouse model to decipher the function of BCL6. We transduced B cell progenitor cells from BCL6fl/fl mice with MLL-ENL and either with a control or Cre-expression vector. Using the BCL6 reporter capability of the mouse we found that BCL6 is significantly higher expressed in MLL-ENL transduced cells. To test if MLL is required for BCL6 upregulation, we used a conditional MLL knock out mouse and found that after Cre-mediated deletion of MLL, pro-B, mature-B, and MLL-AF4 transduced ALL cells almost lost the ability to upregulate BCL6. Interestingly, using inducible BCL6 transgenic and knockout as well as retrovirally transduced pre-B and ALL cells showed that overexpression of BCL6 leads to higher expression levels of MLL and deletion of BCL6 results in lower expression levels of MLL. Therefore, we conclude that MLL and BCL6 both cooperate and activate each other's expression in an activating feedback loop. Strikingly, Cre-mediated BCL6- deficiency results in apoptosis of MLL-ENL transduced cells. Clinical relevance: To verify if the high BCL6 expression levels in MLL-AF4 patients are important for the disease progression, we transduced primary human ALL xenografts with a dominant-negative BCL6-mutant (BCL6-DN). Expression of BCL6-DN rapidly induced cell cycle arrest and cell death. To test if pharmacological inhibition of BCL6 is of potential use for patients with MLLr leukemia, we treated multiple MLL-AF4 rearranged human xenograft cases with a RI-BPI a BCL6 peptide inhibitor. Strikingly, treatment with RI-BPI not only compromised colony formation in methylcellulose it also prevents leukemia-initiation in transplant recipient mice. RI-BPI also had a strong synergistic effect when combined with the chemotherapy drug Vincristine, which represents the backbone for most high risk regimen in pediatric ALL. Conclusions: These findings identify BCL6 as a central factor in leukemia initiation and survival and its pharmacological inhibition as a novel strategy to treat MLL-rearranged ALL. Aberrant expression of BCL6 in MLLr ALL is the direct consequence of the MLLr oncogenic activity in these cells. Based on these findings, we propose combinations of BCL6 inhibitors with currently used chemotherapeutics as potential approach to reduce the risk of ALL relapse and improve overall outcome. Disclosures Armstrong: Epizyme, Inc: Consultancy. Ernst:Amgen: Other: stocks. Melnick:Janssen: Research Funding.

Description: Introduction: Allogeneic stem cell transplantation is a potentially curative treatment for patients with high-risk non-Hodgkin lymphoma (NHL). Fludarabine/busulfan based conditioning regimens are widely used in Europe for this purpose. Busulfan dose intensity discriminates between reduced intensity (FB2, 2 days of busulfan at 4 mg/Kg/d per os or 3.2 mg/kg/d iv) and reduced-toxicity myeloablative (FB3/FB4, 3 or 4 days of busulfan at 4 mg/Kg/d per os or 3.2 mg/kg/d iv) conditioning regimens (Bacigalupo, 2009). While some data have been recently published showing some advantages of higher busulfan dose intensity for myeloid malignancies, there is no such data available in the lymphoid setting. Methods: This was a large retrospective study conducted on behalf of the SFGM-TC including all adults allografted in France between January 2004 and December 2014 for NHL (n=378). Clinical data were obtained through ProMISe (Project Manager Internet Server), an internet-based system shared by all French transplantation centers. We aim to compare various outcomes (overall (OS) and lymphoma free (LFS) survivals, relapse incidence (RI), non-relapse mortality (NRM), acute and chronic GVHD) between those who received FB2 (n=277) or FB3/FB4 (n=101) as conditioning regimens. GVHD free relapse free survival (GRFS) was also studied (defined as alive with no previous grade III-IV aGvHD, no moderate or severe chronic GvHD (cGvHD) and no relapse). Results: Both groups were comparable for the following variables: median follow-up (FB2: 24.9 vs FB3/4: 23 months), gender (male 61% vs 53%), disease type (low-grade lymphoma 25% vs 21%, mantle-cell lymphoma 17% vs 13%, high-grade lymphoma 25% vs 21%, T cell lymphoma 32% vs 45%), disease status at transplant (complete remission/very good partial response 64% vs 62%, partial response 28% vs 31%, active disease 8% vs 7%), donor type (sibling 43% vs 49.5%, matched unrelated 56% vs 47), median number of previous courses of treatment (2 vs 2, p=0.44), stem cell source (peripheral blood 96% vs 95%). FB2 patients were significantly older (median 57.3 vs 53.1 years, p=0.07), have been transplanted more recently (median year of transplant: 2011 vs 2010, p=0.001) and have been more previously autografted (69% vs 50.5%, p=0.001). FB3/4 patients have been allotransplanted earlier during the evolution of their disease (median time between diagnosis and allograft 18.2 vs 33.8 months, p=30 months), there were also no significant differences between both groups in terms of OS, LFS, RI or NRM. In multivariate analysis there was a trend for worse outcome using FB3/FB4 regimens (OS: HR 1.46, 95%CI: 0.96-2.23, p=0.07; LFS: HR: 1.43, 95%CI: 0.99-2.06, p=0.05; RI: HR 1.54; 95%CI: 0.95-2.48, p=0.07). These results were also confirmed using a propensity score-matching strategy including 184 FB2 and 98 FB3/4 patients. Conclusion: This large retrospective study showed that reduced toxicity myeloablative fludarabine/busulfan regimens did not improve outcomes of adults allografted for NHL. FB2 conditioning regimen still should be considered as the standard of care conditioning regimen in this setting. To validate these results, prospective studies are needed, like the French prospective trial currently ongoing for myeloid diseases (NCT01985061). Also, new conditioning regimens and post-allograft strategies should be tested to improve outcomes of patients. Disclosures Peffault De Latour: NOVARTIS: Consultancy, Honoraria, Research Funding; PFIZER: Consultancy, Honoraria, Research Funding; ALEXION: Consultancy, Honoraria, Research Funding.

Description: Several groups have shown that leukemic cells create a self-reinforcing bone marrow (BM) niche that functionally impairs normal hematopoietic stem and progenitor cells (HSPC) indirectly through stroma-secreted factors. We recently demonstrated an alternative mechanism whereby extracellular vesicles (EVs) from acute myeloid leukemia (AML) patients and cell lines, but not BM CD34 controls, suppress their clonogenicity through EV trafficking of microRNA that directly downregulate critical transcription factors (c-Myb and HoxA9). Here, we aimed to clarify the fate of residual HSPC in in vivo AML xenografts, as well as ex vivo intrafemural (IF) injection and in vitro exposure of EVs experiments. Among KSL cells we observed a significant increase in the frequency of the long-term hematopoietic stem cell (SLAM, CD150+CD48−) subpopulation, but not the multipotent progenitors even at low levels of AML infiltration or direct IF injection of EVs. The HSPC pool redistribution was accompanied by cell cycle alterations in residual HSPC that showed AML EVs consistently induced quiescence (G0) in KSL (cKit+Sca1+Lin−) HSPC populations. When we assessed their DNA damage, residual HSPC showed a distinct increase in the gH2AX foci relative to control non-engrafted mice as well as the transcriptional upregulation of Rad51 and P21 genes along with gains in phosphorylation of the tumor suppressor p53. Yet, the reprogrammed KSL showed no evidence of apoptosis indicated by the lack of upregulation of the p53 target, Puma, and Annexin V staining, nor evidence of senescence (P16 and Sparc transcripts). To gain additional insight, we performed a tandem mass tag (TMT) proteomic profiling of AML-EV exposed HSPC with or without exposure to EVs derived from AML cells. The results showed significant enrichment of DNA methylation regulatory pathway such as DNMT1, HELLS and UHRF1 as well as inflammatory pathways including IL1b, NOS, CEBPB and NFkB pathway-targets, confirmed by transcriptional profiling of KSL from xeno-transplanted mice. Based on our recent report that miR-1246 is one of the most highly enriched miRNA in AML derived EVs and proceeded to determine its target transcripts using an attenuated RISC complex (RISC-Trap), followed by high-throughput sequencing. Bioinformatics analysis identified a set of 27 miR-1246-specific targets relative to control microRNAs. Strikingly, the target set was selectively enriched for a panel of negative cell-cycle regulator genes (CDK1, CDK7, CDK11, CCNF, HDAC2 and GATA3) as well as the DNA methylation regulators (DNMT1 and HELLS).Collectively, our results demonstrated that residual HSPC in the AML BM are phenotypically reprogrammed and suppressed in their proliferation along with DNA damage accumulation via paracrine EV microRNA trafficking. Our study provides insight into HSPC fates in the AML niche and echoes observations of cell competition, as a mode of non-cell autonomous regulation where p53 activation in the reprogrammed cells leads to a progressive decline in proliferation and fitness. We propose that AML EV trafficking of miR-1246 specifically may contribute to the altered fate of residual HSPC via transcriptional regulation of proliferation-related genes. Disclosures No relevant conflicts of interest to declare.

Description: Recent reports propose high-dose (HD) methotrexate (MTX)-based cytoreduction followed by consolidation with HD chemotherapy (HDC) with or without autologous stem-cell transplantation (ASCT) as first-line treatment for primary central nervous system lymphoma (PCNSL). The main rationale is that HDC-ASCT may improve disease control by achieving higher therapeutic concentrations of cytotoxic chemotherapy in the CNS, thus circumventing the blood-brain barrier (BBB). We have employed an alternative approach to treat PCNSL patients effectively. This first-line treatment enhances delivery of standard dose (SD) MTX-based chemotherapy administered intra-arterially (i.a.) with osmotic BBB opening plus the anti-CD20 monoclonal antibody (mAb) rituximab, followed in complete responders by anti-CD20 mAb maintenance immunotherapy. The main features that distinguish our approach from HDC-ASCT consolidation are to utilize BBB properties to maximize delivery of SD chemotherapy to the CNS sanctuary, and maintenance immunotherapy to delay recurrence. A multi-center study of 149 PCNSL patients (2,079 i.a. BBB treatments) resulted in one death within 48 hrs after treatment, from pulmonary embolism. Prior to routine use of granulocyte colony stimulating factor, granulocytopenic fever occurred in 3% of BBB treatments (Angelov, J Clin Oncol 2009). This contrasts with HDC-ASCT consolidation which carries substantial morbidity and treatment-related mortality (6% to 9%) (Ferrari, Lancet Hemat 2016; Omuro, Blood 2015). Further, HDC-ASCT is used primarily in younger PCNSL patients (〈 60-65 yrs) with good performance status and is not feasible in many PCNSL patients given the median age at diagnosis is 60-65 yrs. First-line enhanced BBB delivery of SD MTX-based chemotherapy in 149 patients provided an overall response rate (ORR) of 82% (58% CR rate) and median overall survival (OS) of 37 mo. In low risk patients (age 〈 60 yrs and KPS ≥ 70) median OS was approximately 14 yrs. Survivors in CR a minimum of 2 yrs after diagnosis (n = 24) were evaluated with standard neuropsychological tests. Median follow-up was 12 yrs (range 2 to 26 yrs) and results indicated long-term preserved or improved cognitive function (Doolittle, J Clin Oncol 2013). Recently we compared efficacy from two first-line MTX-based regimens: HD MTX (intravenous, 4gr/m2) (Thiel, Lancet Oncol 2010), and enhanced BBB delivery of MTX (i.a., 3.5gr/m2). After adjusting for patient characteristics, CR rate and progression free survival were higher in patients 〈 65 yrs treated with enhanced delivery of MTX (p 〈 0.001). When first-line rituximab was added to the i.a. enhanced delivery regimen in 24 patients (median age 64 yrs; median KPS 65), the ORR improved to 92% (75% CR) and OS to 61 mo. To prevent recurrence, Ney (Leuk Lymphoma 2009) treated a small group of patients in CR after standard PCNSL treatment, with maintenance rituximab. The addition of maintenance immunotherapy resulted in average disease control of 22 mo or longer. Consistent with the Ney report, we noted prolonged median CR duration of 38 mo (range: 10 to 85 mo) in a small subset treated with maintenance rituximab (n = 9). Increased survival was also seen in translational laboratory studies of a rodent model of intracerebrally implanted MC116 human B-cell lymphoma cells, using single agent rituximab, whether or not delivered with BBB disruption and whether or not MTX was included (Muldoon, Clin Cancer Res 2011). These results suggest that rituximab slowly leaked into main CNS tumor mass even in absence of BBB opening; and given the long half-life, was trapped by binding to CD20+ on tumor cells, attaining sufficient concentration for antitumor efficacy. Our goal is to maximize first-line enhanced BBB delivery of SD chemotherapy to enable less treatment-related morbidity and mortality than is associated with HDC-ASCT in PCNSL, and increase CR duration. Neurocognitive safety has been demonstrated in long-term survivors treated with BBB delivery. Based on the encouraging results from Ney et al, our pilot data using maintenance rituximab, and translational lab studies of mAb delivery to brain, a new randomized multi-center trial is underway to study the effect of maintenance obinutuzumab on CR duration, neurocognition and quality of life in PCNSL patients who achieve CR following first-line MTX-based chemotherapy (NCT02498951). A trial update will be presented during the ASH 2016 meeting. Disclosures No relevant conflicts of interest to declare.

Description: Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy and is most often of B-cell lineage (Roberts, et al. NEJM 2014). One subtype of B-cell ALL, Philadelphia chromosome-like ALL (Ph-like ALL), is BCR-ABL negative with a gene expression signature similar to that of BCR-ABL positive ALL and is prognostic for poor clinical outcomes (Roberts, et al. NEJM 2014). Ph-like ALL is often associated with rearrangements involving the cytokine receptor-like factor 2 (CRLF2) component of the thymic stromal lymphopoietin receptor (TSLPR) leading to its overexpression (Shochat, et al., JEM 2011). TSLPR is a heterodimer of CRLF2 and IL-7Rα that signals to promote the proliferation and differentiation of B-cell progenitors and acts to promote B-cell transformation in the context of Ph-like ALL (Maude, et al. Blood 2012). Glucocorticoid (GC) therapy plays a central role in the treatment of childhood ALL and resistance to GCs confers a poor prognosis (Piovan, et al. Cancer Cell, 2013). This study examined the role of TSLPR signaling in mediating primary GC resistance and the effects of downstream signal transduction inhibitors to confer GC sensitivity. Methods: Viably cryopreserved splenocytes were obtained from 19 patient-derived xenografts of Ph-like ALL banked in the Children's Oncology Group or Children's Hospital of Philadelphia leukemia biorepositories. Assays were also performed using the Mutz 5 Ph-like ALL cell line. Flow cytometry was used to assess the protein expression of TSLPR and GC receptor (GR). Levels of pERK and pAKT were measured by phosphoflow cytometry at baseline and following TSLP stimulation. Cells were cultured in the presence of 1µM dexamethasone (dex), a GC, with or without 1µM trametinib, a MEK1/2 inhibitor, or 1µM MK2206, a pan-AKT inhibitor, in the presence of TSLP and viability was assessed by Hoechst staining and flow cytometry at 48 hours. Results: Of the 19 Ph-like ALL samples in this study, 11 were CRLF2-rearranged (CRLF2R) and 8 were non-rearranged (CRLF2NR). CRLF2 rearrangements involved P2RY8 or IGH and 9 of 11 samples had concomitant activating mutations in JAK1 or JAK2. CRLF2NRsamples expressed a variety of other translocations involving genes such as JAK2, PDGFR, and ABL1. CRLF2R cells were shown to have significantly greater TSLPR protein expression relative to CRLF2NR cells (p = 0.03). In the presence of TSLP, CRLF2 rearrangement status predicted responsiveness to dex, with CRLF2Rsamples demonstrating significant resistance to dex relative to CRLF2NRsamples (p = 0.004). There was no significant reduction in cell viability following dex treatment in CRLF2R samples (p = 0.5), while dex effectively attenuated cell viability in CRLF2NRsamples (p = 0.008). Importantly, there was no difference in GR expression between these two groups (p = 0.6). CRLF2R samples demonstrated hyperresponsiveness to TSLP stimulation, with a significant induction of pERK and pAKT that exceeded the response of CRLF2NRsamples (p = 0.007 and p = 0.0005, respectively) despite no differences in basal phosphoprotein levels between the two sample groups. Inhibition of MAPK signaling with trametinib or of AKT signaling with MK2206 significantly sensitized CRLF2Rcells to dex in the presence of TSLP when used in combination relative to dex alone (p = 0.0003 and p 〈 0.0001, respectively) and resulted in a significant reduction in cell viability relative to untreated cells (p 〈 0.0001 and p 〈 0.0001, respectively). The Mutz 5 cell line, which expresses both a CRLF2 rearrangement and a JAK2 activating mutation, was used to assess the effect of simultaneous pathway inhibition. Mutz 5 cells were treated with dex alone or dex in combination with trametinib and/or MK2206. While dex alone had no significant effect on cell viability, the addition of trametinib or MK2206 resulted in a 43% and 36% reduction in cell viability, respectively. Furthermore, combined treatment with dex, trametinib, and MK2206 resulted in a 72% reduction in cell viability, demonstrating the efficacy of simultaneous MAPK and PI3K pathway inhibition to confer dex sensitivity. Conclusion: MAPK and PI3K pathway signaling play a role in mediating primary GC resistance in CRLF2R Ph-like ALL, making these pathways potential therapeutic targets for enhancing the efficacy of GC therapy in this patient group. Disclosures Maude: Novartis: Consultancy. Teachey:Novartis: Research Funding.

Description: Background: In standard risk APL, ATRA+ATO combinations (without CT) are at least as effective as classical ATRA + anthracycline based chemotherapy (CT) while being less myelosuppressive (Lo Coco, NEJM 2014, Burnett, Lancet Oncol, 2015). In high risk APL (WBC〉 10G/l), it is still unclear if CT can be avoided or greatly reduced, but addition of ATO to ATRA + CT reduces relapses (Powell, Blood 2010). In a randomized trial (APL2006 trial) in high-risk APL patients (pts) who received ATRA + CT induction treatment, we evaluated the addition of ATO to CT during consolidation. Methods: Between 2006 and 2015, newly diagnosed APL pts 10 G/L, after an induction of ATRA 45mg/m2/d until CR with Idarubicin (Ida) 12 mg/m2/dx3 and AraC 200mg/m2/dx7, were randomized for consolidation between CT and CT+ATO. The CT group (standard group) received a first consolidation with Ida 12 mg/m2/dx3 and AraC 200mg/m2/dx7, a second consolidation with Ida 9 mg/m2/dx3 and AraC 1g/m2/12h x4d, and 2-year maintenance with intermittent ATRA and continuous 6 MP + MTX. The CT+ATO group received the same treatment except that ATO 0.15 mg/Kg/d d1 -25 was added during both consolidation courses. After a first interim analysis in Sept 2010, based on 81 pts, AraC was deleted from consolidation cycles of the CT+ ATO group. The primary endpoint was EFS from CR achievement. Results: 211 pts 10 G/L were included (after the exclusion of 8 diagnostic errors) in 58 centers. 95.7% achieved CR, 3.3% had early death and 1% resistant leukemia. 193 pts were randomized for consolidation, 97 in the CT and 96 the CT+ ATO groups. Pre-treatment characteristics were well balanced between the 2 groups. 7 pts (3 CT vs 4 CT+ATO) had relapsed (5-year cumulative incidence of relapse (CIR) of 2.5% vs 3.9%; p= 0.39) and 9 pts had died in CR :7 (7.8%), 2 (5.1%), 0 (0%) in the CT, CT (with AraC) + ATO, CT (without AraC) + ATO groups respectively (p=0.04). Causes of death in CR were bleeding (n=5), infection (n=2), previous cancer relapse (n=2). One patient in the CT+ATO arm developed AML/MDS. 5-year OS was 93% vs 94% (p=0.56) and 5-year EFS was 89% vs 93% (p=0.47) in the CT and CT+ATO groups, respectively. Omission of AraC (after the amendment) in the CT+ATO group did not increase CIR (5 year CIR 5.3% with and 3.3% without AraC, p=0.57). In the CT, CT (with AraC) + ATO, CT (without AraC) + ATO groups respectively, median time to ANC〉1 G/L after consolidation 2 was 22, 25 and 19 days (p50G/l was 24, 26 and 20 days (p

Description: Introduction: Daratumumab (DARA) is a first-in-class human CD38 IgG1κ monoclonal antibody that has demonstrated activity as monotherapy and in combination with standard of care regimens for multiple myeloma (MM). Population pharmacokinetics (PPK) analyses were conducted to describe the PK characteristics of DARA following its administration in combination therapies, to evaluate the influence of covariates on its disposition in patients with MM who had received ≥1 prior therapy, and to compare its PK in combination therapies with that of monotherapy. Exposure-efficacy/safety analyses were performed to investigate the relationship between DARA exposure and selected efficacy and safety endpoints. Methods: The PPK analysis primarily included data from two phase 3 studies in which DARA was combined with background regimens: MMY3003 (POLLUX; lenalidomide [R]/dexamethasone [d]) and MMY3004 (CASTOR; bortezomib [V]/d). Data from two phase 1/2 studies (GEN503 [Rd] and MMY1001 [pomalidomide/d; Vd; V-thalidomide-d; V-melphalan-d]) were also used. Most patients included in the analysis (684 of 694) received 16 mg/kg DARA intravenously. A PPK model based on previous monotherapy studies was used to fit the concentration-time data from combination studies. Subgroup analyses were conducted to evaluate the influence of patient and disease characteristics on exposure to DARA. Based on data from MMY3003 and MMY3004, the exposure-efficacy analyses investigated the relationship between maximal trough concentrations (Cpre-infusion,max) and progression-free survival (PFS), duration of response (DOR), and overall response rate (ORR), while the exposure-safety relationship was explored for infusion-related reactions (IRRs), thrombocytopenia, anemia, neutropenia, lymphopenia, and infections. Results: Exposure to DARA was similar between the monotherapy and combination therapies. Based on combination therapy data, the effects of the intrinsic and extrinsic factors (age, sex, race, renal and hepatic impairment, baseline albumin, type of MM, region, type of combination therapy, ECOG, refractory status, and number of prior lines of therapy) were similar to or smaller than those in the monotherapy studies. Consistent with the monotherapy studies, none of the investigated intrinsic and extrinsic factors had clinically important effects on the exposure to DARA as all the covariate effects were within 25%. Although the clearance and volume of distribution of DARA increased with increasing body weight, the exposure to DARA was relatively consistent across the range of body weights after administration on a mg/kg-basis. Despite the decreasing concentration of DARA over time due to less frequent dosing, the current dosing schedule was adequate to produce concentration levels that maintained target saturation during the Q4W dosing period in the dosing schedules for MMY3003 (QW for 8 weeks, Q2W for 16 weeks, and Q4W, thereafter) and MMY3004 (QW for 9 weeks, Q3W for 15 weeks, and Q4W, thereafter). The exposure-efficacy analyses on the data from combination therapies suggest that maximum clinical benefit to PFS, DOR, and ORR was attained for the majority of the patients with an acceptable safety profile at the recommended dose of 16 mg/kg. No apparent relationships between drug exposure and IRRs, thrombocytopenia, anemia, neutropenia, and lymphopenia were identified within the studied concentration range. Although the overall rate of infection (any grade) appeared to increase with drug exposure, this trend was not observed for grade ≥3 infections. These findings were consistent with results from the monotherapy studies. Conclusion: The PK of DARA was similar between monotherapy and combination studies. No clinically relevant demographic or clinical characteristics were identified. Therefore, no dose adjustment based on these factors is recommended. Population PK and exposure-response analyses for combination therapies support the recommended body weight-based 16 mg/kg dose and the dosing schedules for the MMY3003 and MMY3004 studies. Disclosures Xu: Janssen: Employment, Equity Ownership. Liao:Pharmax: Employment; Janssen Research & Development: Consultancy; Johnson & Johnson: Equity Ownership. Dimopoulos:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Amgen, Celgene, Janssen, Karyopharm, Takeda: Consultancy, Honoraria; Amgen, Celgene, Janssen, Karyopharm: Research Funding. Ho:Janssen, Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Belch:Amgen, Celgene, Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Capra:Janssen: Speakers Bureau. Gomez:Janssen, Bristol Myers Squibb, Celgene, Amgen: Consultancy. Medvedova:Oregon Health & Science University: Employment. Iida:Janssen Pharmaceuticals, Takeda Pharmaceuticals Co, Celgene, Bristol-Myers Squibb, Ono Pharmaceuticals Co: Honoraria; Janssen Pharmaceuticals, Takeda Pharmaceuticals Co, Celgene, Bristol-Myers Squibb, Chugai Pharmaceuticals, Kyowa Hakko Kirin Co, Eli Lilli Japan, Novartis Pharma, Sanofi, Bayer Yakuhin, Toyama Chemical Co, Teijin Pharma, Astellas Pharma: Research Funding. Qi:Janssen: Employment. Schecter:Janssen: Employment, Equity Ownership. Khokhar:Janssen: Employment. Yan:Janssen: Employment; Johnson & Johnson: Equity Ownership. Zhang:Janssen: Employment, Equity Ownership. Clemens:Johnson & Johnson: Equity Ownership; Janssen: Employment.

Description: Introduction: Histone deacetylase inhibitors (HDACis) have demonstrated clinical efficacy in multiple myeloma, particularly in combination with proteasome inhibitors. CHR-3996 is a class 1 selective HDACi with potent anti-myeloma activity in vitro. Aminopeptidase inhibitors act downstream of the proteasome and prevent breakdown of proteasome generated peptides into amino acids. Synergistic cytotoxicity was observed in vitro when CHR-3996 was combined with the aminopeptidase inhibitor, tosedostat through rapid activation of NFkB followed by increased expression of the repressors IκBα, A20, CYLD, BIRC3. The MUK-three study was designed to translate these pre-clinical findings into a phase 1 clinical trial. This dose escalation study aimed to determine the maximum tolerated dose, safety and preliminary activity of CHR-3996 administered in combination with Tosedostat for patients with relapsed, refractory MM. Here we present the final study results. Methods: MUK-three was an open label multi-centre UK Phase I/IIa trial for patients with relapsed and relapsed/ refractory myeloma who had failed conventional treatments. Patients were permitted to meet the haematological entry criteria using growth factor and/or blood product support. During dose escalation subjects received CHR-3996 (20-40mg days1-28) and Tosedostat (0-60mg days 1-28) (Table 1) every 28 day cycle until disease progression or withdrawal. Dose limiting toxicities (DLTs) were evaluated during cycle 1 and dose escalation followed the 3+3 design. Responses were assessed using modified IMWG uniform response criteria, with the primary endpoint for the expansion phase of stable disease (SD) rate after 4 cycles of therapy. Toxicity was graded by CTCAE V4.0. Results: The trial was open to recruitment from July 2012 to December 2015. 20 patients were treated during dose escalation, including 8 at the recommended dose (RD) and 12 at dose levels (DL) 1-3. Only 1 DLT was observed at DL3 (grade 4 thrombocytopenia); however, this DL was deemed not tolerable due to the high incidence of low grade gastrointestinal toxicities. Hence the RD was determined as DL3b, CHR-3996 20mg and Tosedostat 60mg. A further 2 patients were treated at RD during dose expansion to make the required 10 patients for the protocol defined initial analysis at which point the trial closed. At the RD (n=10) median age was 63 years (range 47-73). 80% of patients had received at least 4 prior lines of therapy (median 4, range 2-9); 50% were ISS II, 30% ISS III; 4/6 patients with evaluable FISH data had 1q gain. The median time from diagnosis to treatment for the overall population was 85.3 months (27.5-198.8). The median number of cycles received was 2.5 (range 2-8) and 2 patients remain on treatment with 8 stopped due to disease progression. The 2 patients ongoing (received 5 & 9 prior lines) had their schedule adjusted to a 5 day a week dosing to further improve tolerability. Both had a clinical response (1MR, 1PR) and remained progression free at 6 months. 3/10 patients had SD after 4 cycles, the overall response rate (≥PR) was 1/10(10%) and the clinical benefit rate (≥MR) 2/10 (20%). Overall outcomes were: PR 10%, MR 10%, and SD 30%. Median time to maximum response was 1.84 months (95% CI [1.09, 8.65]). Toxicities at the RD were manageable, 30% of patients required a dose reduction. 22 serious adverse events were reported in 16 patients across all doses, mainly infections (10/22, 45.5%). The commonest grade 3-4 toxicities reported for all 22 patients were: platelet count decrease (12, 54.5%), white blood cell decreased (6, 27.2%), diarrhoea (5, 22.7%). The most frequent grade 1-2 toxicities were fatigue (15, 68.2%), nausea (14, 63.3 %), anorexia (14, 63.6%), anaemia (13, 59.1%). 1 patient withdrew due to toxicity, and there were no treatment related deaths. Conclusions: This study demonstrated that the novel combination of CHR-3996 and tosedostat was safe and tolerable in multiply relapsed, refractory myeloma patients many of which had poor bone marrow function. The recommended dose of the combination was 20mg and 60mg, respectively. Following further adjustment to an intermittent 5 day/ week dosing schedule, treatment was well tolerated and clinical benefit observed. This suggests that further evaluation of this novel combination is warranted. Acknowledgments: This trial was part of the Myeloma UK Clinical Trial Network, ISRCTN: 24989786. Disclosures Williams: Novartis: Honoraria; Janssen: Honoraria, Other: Travel support, Speakers Bureau; Celgene: Honoraria, Other: Travel support, Speakers Bureau; Takeda: Honoraria, Other: Travel support, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Yong:Autolus Ltd: Equity Ownership, Patents & Royalties: APRIL based chimeric antigen receptor; Janssen: Research Funding. Cook:Takeda Oncology: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Glycomimetics: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding, Speakers Bureau. Jenner:Amgen: Consultancy, Honoraria, Other: Travel support; Takeda: Consultancy, Honoraria, Other: Travel support; Janssen: Consultancy, Honoraria, Other: Travel support, Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding. Morgan:Univ of AR for Medical Sciences: Employment; Bristol Meyers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Davies:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria.

Description: Triplet regimens combining an immunomodulatory agent, a proteasome inhibitor (PI), and a steroid are used to treat newly diagnosed and relapsed multiple myeloma (MM). Although ixazomib (Ix), an oral PI with single agent activity, can be combined with lenalidomide (LEN), patients (pts) with relapsed/refractory (R/R) MM are often LEN-refractory. Pomalidomide (POM) has single agent activity in LEN-refractory disease, and both POM and Ix also show activity in poor cytogenetic risk pts. Methods: Primary objectives: 1) determine the maximum tolerated dose (MTD) of Ix in combination with standard dose POM and dexamethasone (DEX), and 2) evaluate the anti-tumor activity of the triplet. The treatment regimen included two dose levels (3 mg and 4 mg) of Ix on days 1, 8, 15; POM 4 mg days 1-21; and DEX 40 mg days 1, 8, 15, 22, of a 28 day cycle. Eligibility: R/R MM after 〉1 prior therapy, LEN-refractory, and ≤ grade(gr) 1 peripheral neuropathy (PN). Pts were treated until progression or unacceptable toxicity. Design: Phase I study utilizing a standard 3+3 design; dose limiting toxicities (DLTs) defined during cycle 1. Results: 32 pts treated, 31 evaluable for toxicity and response. Pts received a median 4 cycles (range 1-13); median follow-up is 5.5 months (range 1.8-21.1). Six pts treated on DL1, 25 treated on DL2, the MTD/Phase II dose (P2D). Median age: 62 years (range 38-84); median time from diagnosis: 3.7 years (range 1.0-8.9); median number prior therapies: 3 (range 1-5); prior transplant: n = 23 (74%); double (LEN/Bortezomib[BOR]) or triple (LEN/BOR/Carfilzomib[CFZ]) refractory: 19 (61%). Phase I: DL1 expanded to n=6 after 1/3 pts experienced DLT (gr3 lung infection); no further DLT seen on DL 1 or 2. Adverse events (AEs) related to POM and/or Ix: ANC decrease Gr1/2 n=11 (35%), Gr3/4 n=10 (32%), platelet decrease Gr1/2 n=9 (29%), lymphocyte decrease Gr1/2 n=8 (26%), Gr3/4 n=11 (35%), PN Gr1/2 n=9 (29%), no Gr3/4. Response: Phase I and II, n=31 pts treated. ORR: 45% (6 VGPR, 8 PR); Clinical Benefit Rate (CBR): 81% (6 VGPR, 8 PR, 3 MR, 8 SD). In the pts with high risk cytogenetics (7[23%] 1q, 3[10%] 17p, 2[6%] t(4;14)) an ORR of 58% (3 VGPR, 4 PR) was seen, and the CBR was 83%. In the double or triple refractory pts, an ORR of 26% and CBR of 79% (1 VGPR, 4 PR, 3 MR, 7 SD) were observed. Conclusions: Ix/POM/DEX is a well-tolerated oral combination therapy, and responses were seen even at DL1 and in high risk patients, including those with poor-risk cytogenetics or advanced refractory disease. Disclosures Kapoor: Celgene: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Kumar:Millennium: Consultancy, Research Funding; AbbVie: Research Funding; Glycomimetics: Consultancy; Sanofi: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; BMS: Consultancy; Kesios: Consultancy; Onyx: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding. Lonial:Novartis: Consultancy; BMS: Consultancy; Janssen: Consultancy; Merck: Consultancy; Celgene: Consultancy; Onyx: Consultancy; Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Onyx: Consultancy. Nathwani:Carevive Systems, Inc.: Research Funding. Forman:Mustang Therpapeutics: Other: Construct licensed by City of Hope. Berdeja:Abbvie, Acetylon, Amgen, Bluebird, BMS, Calithera, Celgene, Constellation, Curis, Epizyme, Janssen, Karyopharm, Kesios, Novartis, Onyx, Takeda, Tragara: Research Funding.

Description: Introduction: Multiple myeloma is a heterogeneous plasma cell neoplasm that remains all but incurable despite significant advances in treatment. We anticipate that the ability to overcome this hurdle resides in personalized strategies designed to specifically recognize, target, and anticipate dynamic tumor subpopulations with variable drug response profiles within an individual. To this end, we have developed a novel multi-disciplinary approach using organotypic drug screening and mathematical modeling to assess drug sensitivity of the different subpopulations within the tumor burden of individual patients and, in turn, provide accurate predictions of clinical outcome to anti-myeloma therapy. Material and methods: We have used a novel combination of ex vivo drug sensitivity assay and mathematical models to predict clinical response of 48 MM patients (11 newly diagnosed and 37 relapsed, 18 females and 30 males, median age 64.5, range 45-77) treated with a combination of proteasome inhibitors and IMIDs (37), nuclear export and topo2 isomerase inhibitors (10), and high dose melphalan (1). MM cells (CD138+) were extracted from fresh bone marrow aspirates and seeded in an ex vivo co-culture model with human stroma in 384-well plates. These cells were exposed to a number of chemotherapeutic and experimental agents (up to 31) for a period of 4 days, during which viability was assessed continuously using bright field imaging and digital image analysis. A mathematical model was used to interpolate the dose response dynamics to each drug, and combined with drug and regimen-specific pharmacokinetic data, generate predictions of clinical response to each individual drug. We have then validated ex vivo-based predictions with actual outcome 90 days post-biopsy. In patients treated with combinations, the mathematical model combined the effect of each single drug assuming additivity. Results: To examine the accuracy of the predicted in silico responses, we have assessed the model according to three increasingly strict standards of accuracy: (A) The model correctly predicted 32 out of 32 responders (100%) and 14 out of 16 non-responders (88%), with an overall accuracy of 96%; (B) According to IMWG stratification, the model correctly stratified 14 out of 16 patients as stable or progressive disease (PD/SD, 88%, the remaining 2 incorrectly predicted as MR/PR), 15 our of 18 as minimal or partial response (MR/PR, 83%, the remaining 3 incorrectly predicted as VGPR/CR), and 10 out of 14 patients as very good partial response or complete response (71%, the remaining 4 incorrectly classified as MR/PR), with an overall accuracy of 81%; (C) The 48 patients from this study provided a total of 120 measures of tumor burden (M-spike or SFLC) within the 90-day post-biopsy period. The direct correlation between tumor burden measures and model predictions led to a Pearson r=0.5547 (P

Description: Introduction: A recent study revealed an antiproliferative and apoptotic effect of propranolol on multiple myeloma (MM) cells. Our previous small matched case-control study showed longer survival in patients with propranolol and other beta-blockers (BB) intake than those without. This larger scale study was conducted to confirm the positive association of BB and MM survival. Methods: We identified 1971 newly diagnosed pts seen at Mayo Clinic between 1995 and 2010. Cardiac medication usage after diagnosis of MM was extracted from patient records and categorized based on BB intake. Cause of death was collected with death due to MM as the primary interest event and death due to cardiac disease or other reasons as competing risk events. The primary outcomes were MM disease-specific survival (DSS) and overall survival (OS). Cumulative incidence functions and Kaplan-Meier method were used to estimate the 5-year cumulative incidence rate (CIR) of MM death and OS rate, respectively. DSS and OS were compared by Gray's test and log-rank test, respectively. Multivarable Cox proportional hazard models were used to estimate the adjusted cause-specific HR (HRCSadj.) and hazard ratio (HRadj.) for DSS and OS, respectively, adjusting for demographics, disease characteristics, diagnosis year, and various chemotherapies. Results: 930 (47.2%) of MM patients had no intake of any cardiac medications; 260 (13.2%) had BB only; 343 (17.4%) used both BB / non-BB cardiac medications; and 438 patients (22.2%) had non-BB cardiac drugs. Five-year CIR of MM death and OS rate were shown in table. Superior MM DSS was observed for BB only users, compared to patients without any cardiac drugs (HRCSadj., .53, 95% confidence interval [CI], .42-.67, padj.

Description: Dysregulated oncogenic serine/threonine kinases (STKs) play a pathological role in diverse forms of malignancies, including multiple myeloma (MM), and thus represent potential therapeutic targets. Here, we evaluated the biological and functional role of p21-activated kinase 4 (PAK4), and its potential as a new therapeutic target in MM. The PAK4 gene lies within chromosome region 19q13.2 commonly amplified in 30% of MM patients. We confirmed a correlation between copy number amplification and increased expression of PAK4 in two large myeloma patient datasets. High expression of total and phosphorylated PAK4 was also observed in the premalignant MGUS stage, suggesting that its overexpression may be an early event in the pathogenesis of myeloma. Myeloma cells with high PAK4 expression display sensitivity to conditional PAK4 knockdown, implying that an oncogene-addicted state exists in such cells. Moreover, we show that PAK4 promotes myeloma cell proliferation in vitro as well as in vivo in murine models of human myeloma through activation of the MEK/ERK pathway. This is further highlighted by the correlation between the basal levels of ERK activity and sensitivity to a novel small molecule inhibitor of PAK4, KPT-9274, a lead clinical agent. KPT-9274 effectively inhibits MM cell growth and survival via ERK inhibition in a large panel of MM cell lines and primary MM cells, also in the presence of bone marrow microenvironment, with no significant effect on normal PBMCs suggesting a potentially favorable therapeutic index. Inhibition of ERK by KPT-9274 also correlates with decreased DNA binding activity of ERK-dependent transcription factors AP1, ETS, CREB and EGR and decreased expression of ERK target genes such as CCND2, CCR1 and MYC. Finally, using a protein binding array, we have identified FGFR3, a commonly disrupted tyrosine kinase receptor, and Grb2, an adaptor protein involved in RAS activation, as novel PAK4 binding partners in myeloma and report disruption of this binding by KPT-9274. As a results, t(4;14)-positive MM cells expressing FGFR3 show greater sensitivity to PAK4 inhibition compared to MM cell lines without FGFR3 aberrations (e.g. U266 and ANBL6). FGFR3 may therefore serve as a predictive marker of KPT-9274 sensitivity in MM patients, with the potential for broader application to other malignancies associated with dysregulation of FGFR3 such as human bladder and cervical carcinomas. In conclusions, our study sheds light on the oncogenic role of the serine/threonine kinase PAK4 as survival and anti-apoptotic factor in myeloma and its inhibition with a new allosteric modulator, KPT-9274, as a potential novel therapeutic intervention in MM especially in high risk t(4:14)-positive patients. Disclosures Senapedis: Karyopharm Therapeutics Inc: Employment. Oliva:Celgene: Honoraria; Takeda: Honoraria; Amgen: Honoraria. Anderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

Description: Large B-cell lymphomas (LBCL) exhibit multiple genetic alterations, often in specific combinations. However, little is known about the biologic consequences of these concurrent genetic features. We recently characterized the comprehensive molecular signatures of two LBCL subtypes that exclusively involve extranodal disease sites, primary central nervous system lymphoma (PCNSL) and primary testicular lymphoma (PTL). In contrast to systemic diffuse large B-cell lymphomas (DLBCLs) or most activated B-cell (ABC)-type DLBCLs, the majority of PCNSLs and PTLs exhibit oncogenic Toll-like receptor (TLR) signaling (MYD88L265P activating mutations), often with concurrent B-cell receptor (BCR) activation (CD79BY196mut) and/or additional mutations or inactivating rearrangements of TBL1XR1 (Chapuy and Roemer et al, Blood 2016; 127:869-81). TBL1XR1 is a component of the NCoR/SMRT co-repressor complex that modulates TLR/MYD88 signaling by increasing the clearance of NCoR/SMRT transcriptional co-repressors from certain TLR/MYD88 target genes. However, the functional consequences of somatic mutations in TBL1XR1 (TBL1XR1mut) or reduced TBL1XR1wt expression in LBCLs remain to be defined. For these reasons, we performed in silico modeling of the identified TBL1XR1mut and functionally characterized TBL1XR1mut and decreased TBL1XR1wt expression in model LBCL systems. First, we evaluated the frequency and types of TBL1XR1mut in our local series of PCNSLs and 151 primary DLBCLs (102 local and 49 previously published) for which we had whole exome sequenceing data. Thirty-six percent of PCNSLs and 6% of DLBCLs in this series exhibit TBL1XR1mut, over half of which occur in the context of MYD88 and/or CD79B mutations. In these PCNSLs and DLBCLs, we identifed 13 different non-synonymous TBL1XR1 mutations. All identified non-overlapping TBL1XR1 mutations were in the WD40 propellar domains, which are critical for protein-protein interactions. To gain insights into the structural consequences of TBL1XR1mut, we overlayed these alterations onto the TBL1XR1 protein crystal structure, modeled the corresponding mutations in silico and assessed protein stability changes. Eighty percent of the TBL1XR1mut resulted in significant destabilization of the mutant protein (ΔΔGibbsFreeEnergy median: 9.78 kcal/mol, range: 1.91 - 28.31). We found that mutations often perturbed the 3D protein structure by abrogating critical polar interactions (representative example TBL1XR1wt vs. TBL1XR1H390R in Figure 1). These in silico modeling data suggest that genetic perturbations of TBL1XR1mut disrupt the function of the encoded protein. For that reason, we modeled reduced TBL1XR1wt expression in informative DLBCL cell lines with or without concurrent CD79B/MYD88 mutations (TMD8 and OCI-Ly1, respectively) by genetically depleting endogenous TBL1XR1 alleles with CRISPR/Cas9. All of the resulting LBCL cell lines with loss of either one or two TBL1XR1 alleles exhibited significantly increased proliferation in comparison to the parental controls. We next assessed the role of TBL1XR1mut by generating HA-tagged versions of each identified TBL1XR1 mutation with site-directed mutagenesis. Viral particles of TBL1XR1mut constructs and the TBL1XR1wt control were used to reconstitute mutant and wild type protein in the TBL1XR1-/- Ly1 cell line. Reconstitution with each TBL1XR1mut significantly enhanced LBCL proliferation, in comparison to TBL1XR1wt. Taken together, these in silico and experimental data suggest that TBL1XR1mut in PCNSL and DLBCL are inactivating events and establish TBL1XR1 as a tumor suppressor in these diseases. Additional analysis of potential interactions between perturbed TBL1XR1 and MYD88/CD79B are underway. Disclosures Rodig: Bristol-Myers Squibb: Honoraria, Research Funding; Perkin Elmer: Membership on an entity's Board of Directors or advisory committees.

Description: Introduction: Recent studies linking cancer genomics and immunity have reinforced the concepts that some mutations trigger T cell effector responses and that the likelihood of an immunogenic mutation increases with increasing mutation load. Importantly, these data highlight the potential utility of such markers in identifying patient subsets likely to respond to cancer immunotherapies. This study investigated the clinical impact of mutation load and its association with T cell gene expression in newly diagnosed patients with follicular lymphoma (FL). Methods: We used clinical and genomic data from FL patients (n = 249; 216 with follow-up information) with evaluable pre-treatment tumor tissue who were treated in a randomized study of rituximab maintenance vs observation (PRIMA; ClinicalTrials.gov ID: NCT00140582). We estimated mutation load per megabase (Mb) as a proxy for neoantigen formation using FoundationOne Heme (Foundation Medicine, Inc). We quantified expression of T cytotoxic effector genes (GZMA, GZMB, PRF1, IFNG, EOMES, CD8A) as a surrogate for pre-existing immunity (and the inflammatory state of the tumor) using TruSeq (Illumina, Inc) RNA seq (n = 142). We used Cox regression to examine associations between these markers and progression-free survival (PFS), adjusting for the FL International Prognostic Index, age, sex, treatment arm and response to induction therapy. Pvalues were calculated for exploratory purposes. Results: The mutation load estimate among newly diagnosed patients with FL was highly variable (range, 0-33 mutations/Mb; Q1: 4.2; median: 6.6; Q3: 10.0). Patients with 〉 15 mutations/Mb (n = 19) were considered to have a high probability of neoantigen formation, and the remaining patients were stratified into mutation-low (〈 6.6 mutations/Mb; n = 112) or mutation-mid (≥ 6.6 mutations/Mb and ≤ 15 mutations/Mb; n = 85) groups. The 3-year PFS in patients with high mutation load was 83% compared with 66% for mid-mutation load and 68% for low-mutation load groups, but mutation load was not independently prognostic in either the rituximab (P = .13) or observation (P = .66) arms. Of note, 92% of FL patients with high mutation load (n = 12/13) also had high T-effector gene expression compared with 49% of those with midlevel (n = 24/49) and 44% of those with low mutation load (n = 35/80) (P = .001). Mutation load was also associated with benefit from rituximab maintenance: FL patients with low mutation load experienced a significant benefit from rituximab maintenance (HR, 0.29 [95% CI, 0.15-0.54]; P 〈 .001), whereas no statistically significant benefit was seen among FL patients with medium (HR, 0.81 [95% CI, 0.43-1.5]; P = .51) or high mutation load (HR, 0.29 [95% CI, 0.026-3.3]; P = .32). Importantly, the T/NK gene signature was prognostic as a continuous predictor (P = .008) and clearly separated 2 large groups of FL patients into an "inflamed" subset (T-effector signature high; n = 74) and an "uninflamed" subset (T-effector signature low; n = 75), with longer PFS seen in the "inflamed" FL subset (PFS HR, 0.39 [95% CI, 0.21-0.70]; P = .002). T-effector gene expression may be particularly useful for identifying the immunologically primed FL subset among patients with low/mid mutation load: there was a trend in 3-year PFS in 84.4% vs 56.6% for T-effector-high vs T-effector-low among low-mutation load patients (P = .002) and 76.2% vs 58.3% of mid-mutation load patients (P = .17), respectively. The subset of inflamed (T-effector signature high) FL tumors also demonstrated high expression of IDO1, which similarly correlated with longer PFS (HR, 0.25 [95% CI, 0.14-0.45]; P 〈 .001), and a strong correlation was observed between IDO1 and IFNG expression (R2 = 0.61; P〈 .001). This is consistent with an interplay of pro- and anti-inflammatory immunity, wherein pro-inflammatory IFNγ drives the clinical outcome. Conclusions: Collectively, our results suggest that mutation load and T-effector gene expression may help identify immunologically distinct lymphoma subsets appropriate for modern immunotherapies. Disclosures Bolen: Genentech, Inc.: Employment. McCord:Genentech, Inc.: Employment. Frampton:Foundation Medicine: Employment, Equity Ownership. Bourgon:Genentech, Inc.: Employment; F Hoffman-La Roche: Other: Shareholder. Punnoose:Genetech, Inc.: Employment. Szafer-Glusman:Genentech, Inc.: Employment. Xerri:Novartis: Honoraria. Salles:Gilead: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mundipharma: Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Venstrom:Genentech: Employment.

Description: Diffuse large B cell lymphoma (DLBCL) comprises two distinct molecular subtypes: germinal center B cell (GCB) subtype and activated B cell (ABC) subtype. The pathogenesis of ABC-DLBCL is characterized by two processes - the activation of NF-KB and a block in terminal B-cell differentiation. However, in GCB-DLBCL only a few biologically relevant pathways have been identified, which has hampered the development of targeted therapies with specific efficacy in this subtype. Recurrent genetic alterations involved in PI3K-AKT signaling pathway have been identified in the patients with solid cancers, and dramatic responses have been observed to PI3K inhibitors in clinical trials. The prevalence and clinical significances of genetic alterations in PI3K-AKT pathway have not been well studied in DLBCL. Previous studies have reported that loss of PTEN expression was observed in almost half of GCB-DLBCL cases but were largely absent in the ABC-DLBCL. In addition, functional studies have recently shown that the inactivation of Gα13 signaling pathway genes may also activate AKT in germinal center driven lymphoma, raising the possibility that additional genes within these pathways are affected in GCB-DLBCL. Herein, we identified genetic alterations involved in the PI3K-AKT pathway and evaluated their clinical impact. We analyzed biopsies from 347 patients newly diagnosed with de novo DLBCL uniformly treated with R-CHOP at the BC Cancer Agency. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Mutation status was determined using deep targeted re-sequencing of the coding exons of 61 genes with a Truseq Custom Amplicon assay (Illumina) and/or Fluidigm Access Array chips, and RNAseq. Immunohistochemical staining of phospho-AKT (pAKT) was performed on tissue microarrays (n=332). Cell-of-origin (COO) was assigned by gene expression (Lymph2Cx assay) in 323 cases - 183 GCB, 104 ABC and 36 unclassifiable. GISTIC analysis revealed several COO-specific peaks with copy number changes. Among them, focal 10q23.3 deletion including PTEN was detected in GCB-DLBCL (q-value=1.7e-7), but not in ABC-DLBCL. We also identified two focal amplification peaks in GCB-DLBCL containing microRNAs MIR17HG (13q31.1;q=1.01e-24) and MIR21 (17q23.3; q=1.45e-6), which are known to down-regulate PTEN and result in activation of PI3K-AKT signaling. Furthermore, we detected recurrent INPP4B deletion (4q21.23) in GCB-DLBCL (q= 0.004) only. Of note, the lipid phosphatase INPP4B has been shown to play a role as a tumor suppressor that controls the levels of PI3K lipid products leading to AKT activation and metastasis of some solid cancers. This is consistent with the observation that PTEN and INPP4B deletions were individually associated with increased pAKT protein expression in our cohort (p=0.015 and p

Description: Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of hematopoietic stem cell leading to lack of phosphatidylinositol glycoproteins, sensitizing cells to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allo-HCT is the only curative treatment for the disease, although outcomes presented in the past were controversial. Material and methods: We report 41 allo-HCTs: 37 from MUD and 4 from MRD performed for PNH in 2004-2016. Median age of recipients was 29(20-62) years and donors 30(19-53), median time from diagnosis to allo-HCT was 16(2-307) months. Median size of PNH clone was 80% granulocytes (0.5%-100%). Indication for allo-HCT was PNH with aplastic/hypoplastic bone marrow (19 pts), MDS (2 pts), overlapping MDS/aplasia (3 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (17 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts), chronic hepatitis B (1 pt) and C (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (31 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (10 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG in MUD-HCT. 2 pts instead of cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (13 pts) or peripheral blood (28 pts) with median 6.3x108NC/kg, 5.7x106CD34+cells/kg, 24.7x107CD3+cells/kg. Myeloablation was complete in all pts with median 9(1-20) days of absolute agranulocytosis