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Acute lymphoblastic leukemia (ALL) is a prevalent hematopoietic malignancy that requires urgent medical intervention to prevent inferior outcome. ALL is characterized by recurrent structural rearrangements, whole chromosome copy number changes and microdeletions of prognostic value. Currently an extensive panel of tests is required, including karyotype, FISH, whole genome arrays, Multiple Ligation-dependent Probe Amplification (MLPA) and PCR-based diagnostic methods. Testing is thus costly, laborious and time-consuming and due to the large number of tests required cascade testing is often performed resulting in lengthy turn-around-times (TAT). This does not conform to treatment protocols that require rapid results to stratify patients into different treatment arms and to identify those who may benefit from "targeted" therapies, such as the Ph-like ALLs. It is therefore interesting to examine new technologies which can simultaneously detect numerical and structural abnormalities in one assay. Such approaches have not yet been extensively introduced into routine diagnostics but implementation would not only be beneficial in reducing TAT but would simplify the analytical pathways and theoretically increase the diagnostic yield. One such novel technology is genome imaging of ultra-long linear molecules, enzymatically labeled at specific sequence motifs. The Saphyr genome imaging device (Bionano Genomics) detects numerical and structural variants (>500bp) with a TAT of less than one week. The aim of the current study is to assess whether implementation of this technology could (partially) replace current analytical strategies. Ten diagnostic T- and 10 diagnostic B-ALL cases were included in this series. All samples were analyzed with the Rare Variant Pipeline (RVP) and filters were set to detect chromosomal aberrations of greater than 5 Mb. For deletions, the limit of detection was set at 500 bp but only regions encompassing clinically relevant loci were investigated. There was an overall excellent concordance between the results: Bionano results were concordant with MLPA, a technique routinely used for rapid detection of key CNAs.All but one recurrent translocations identified by routine strategies were correctly identified by Bionano which failed to detect a STIL-TAL1 rearrangement in one of the two cases with this abnormality. Bionano further identified: A t(5;11) TCF7-SPI1 in a case with a failed karyotype (subsequently confirmed by another method) (Figure 1).A t(12;22) ZNF384-EP300 fusion in a case with a normal karyotype. This recurrent rare fusion was confirmed by FISH. In general there was an excellent concordance of whole/partial chromosome gains and losses and for hyperdiploidy (observed in 3 B-ALLs in this series). Moreover, Bionano identified also hyperdiploidy in a case with a normal karyotype. Interestingly, FISH analysis of the same case suggested the presence of a hypodiploid clone. This demonstrates the limitation of DNA based techniques to accurately determine ploidy when allele information is not available. There were some discrepancies between the results of cytogenetics and Bionano: Bionano could not identify the presence of multiple clones, a common limitation for DNA based techniques.Bionano did not confirm some abnormalities identified by karyotype. Chromosome analysis of ALL samples is notoriously difficult due to low chromosome resolution explaining some of these differences. Some of the discrepant abnormalities were often marked as uncertain or were present only in a subclone ( Overall our data demonstrate that genome imaging is a promising technique to identify structural and numerical abnormalities in ALL. Bionano provided an informative result in each case and results were concordant with other results in the majority of cases. However this study also highlighted a few limitations. Further studies are underway to understand why the STIL-TAL1 was overlooked and to determine whether some of the additional variants identified by Bionano represent true rearrangements or technical artefacts. Disclosures No relevant conflicts of interest to declare.

Purpose Vincristine (VCR) is frequently used for the treatment of pediatric cancer. However, it can lead to dose-limiting vincristine-induced peripheral neuropathy (VIPN). This study aimed to investigate if prolonging the duration of VCR administration (1-hour infusions instead of push injections) reduces VIPN in children with cancer during the first year of treatment. Methods The VINCA trial is an international multicenter randomized controlled trial. Participants were randomized to receive all VCR administrations through push injections or 1-hour infusions. Dose of VCR was 1.5-2 mg/m2 with a maximum of 2 mg. VIPN measurements were performed at baseline and 1-3 times during treatment, depending on the number of VCR administrations and the total treatment time, using 4 items of the common toxicity criteria of adverse events (CTCAE version 4.03): constipation, peripheral sensory neuropathy, peripheral motor neuropathy and neuralgia. Individual item scores range from zero (no complaints) to five (death). The primary outcome of this trial was total sum CTCAE score during first year of treatment. For the current analysis, patients treated for acute lymphoblastic leukemia (ALL) or Hodgkin's lymphoma were included. All included patients were analyzed according to the intention-to-treat principle. Besides VIPN measurements, data on all relevant co-medication during treatment were collected, including data of concurrent azole therapy (as azole treatment is known to interact with VCR treatment). Descriptive data were analyzed using either chi-square tests or t-tests. Longitudinal data were analyzed using repeated measures mixed model analysis for continuous outcomes (total CTCAE sum score) and generalized estimating equations for dichotomous outcomes (having VIPN yes or no, with VIPN defined as a CTCAE score of ≥ 2 on any of the 4 CTCAE items). Patients were considered to have been treated with concurrent azole therapy when azoles were used during the week before or following VCR administration and if ≥ 50% of VCR administrations between two succeeding measurements were given with concurrent azole therapy. Results were corrected for concurrent azole therapy, cumulative VCR dose, disease, age, gender, ethnicity and time since diagnosis. Results In total 90 children (n=45 one hour infusions group, n=45 push injections group) participated in the study, 58 (64%) with ALL and 18 (20%) with HL. Participants in the two randomization groups did not significantly differ regarding gender, age, ethnicity, diagnosis, or cumulative VCR dose. Overall results showed no effect of randomization on total CTCAE score (β=0.07, 95% confidence interval (CI) -0.42-0.56, p=0.78). However, concurrent azole treatment appeared to be an effect modifier in this analysis and therefore results are reported separately for measurements with (n=24) and without concurrent azole therapy (n=226). Among patients who received concurrent azole therapy, total CTCAE sum score was significantly higher in the push group compared to the 1-hour group (β=1.95, 95% CI 0.49-3.41, p=0.01), while among those without concurrent azole therapy, these CTCAE sum scores did not differ between the two randomization groups (β=-0.17, 95% CI: -0.67-0.34, p=0.52). The risk of developing VIPN (no/yes) did not significantly differ between both randomization groups, irrespective whether concurrent azole treatment was given or not (with azole: OR (95% CI)=4.92 (0.60-40.37), p=0.14; without azole: OR (95% CI)=0.97 (0.51-1.82, p=0.92). Conclusions Overall, administration method of VCR given as push injection or 1-hour infusion did not seem to affect the risk of developing VIPN in children treated for ALL or HL when using the current dosing regimen. However, when concurrent azole treatment is given, total CTCAE scores are significantly lower in children in the 1-hour infusion group compared to the push injection group, demonstrating less VIPN. These results indicate that for children treated with VCR and concurrent azole therapy for the prevention or treatment of fungal infections, administration of VCR by 1-hour infusions instead of push injections is recommended. Figure Disclosures Kaspers: Helsinn Healthcare: Consultancy; Boehringer Ingelheim Pharma: Other: Member of a DSMC. van der Sluis:medac: Consultancy; jazz farmaceuticals: Consultancy.

Acute lymphoblastic leukemia (ALL), which is the most common cancer in children, shows extensive genetic intra-tumoral heterogeneity. This heterogeneity might be the underlying reason for an incomplete response to treatment and for the development of relapse. In order to envision the clinical implementation of a refined risk-category strategy based on ALL subclonal composition, it is essential to first generate a reference single-cell map and accumulate evidence on how the subclonal composition affects the response to treatment. For that, we performed large-scale and integrative single-cell genome and transcriptome profiling of pediatric samples at diagnosis, during drug treatment and in case of relapse. We used the 10x Genomics platform for single-cell RNA-sequencing analysis (around 4000 cells per sample) and the Tapestri Platform (Mission Bio) for targeted single-cell DNA-sequencing (around 5000 cells per sample) of the most mutated genomic regions in ALL. For the later, we developed a custom panel that covers 305 ALL mutational hotspots across 110 genes. We have determined a reference single-cell map of the cellular (based on the gene expression profile) and the clonal composition (based on the co-occurrence of mutations at each individual cell) for pediatric ALL at diagnosis (8 T-ALL and 10 B-ALL patients). We have also reconstructed the tumor phylogeny highlighting the order of mutational acquisition and the most likely pattern of evolution. Moreover, we have studied how T-ALL evolves during drug treatment at single-cell resolution in 4 patients, unraveling which are the most sensitive clones to the therapy, which are the most resistant ones and when relapse clones originated. Single-cell RNA-sequencing also provided information on how normal hematopoiesis recovers during chemotherapy treatment. The results show that ALL is typically composed by a major clone and 5-10 smaller clones that have different sensitivities to the therapy. We have been able to detect minor clones ( Disclosures Maertens: Gilead Sciences: Other: Grants, personal fees and non-financial support; Cidara: Other: Personal fees and non-financial support; Amplyx: Other: Personal fees and non-financial support; F2G: Other: Personal fees and non-financial support; Merck: Other: Personal fees and non-financial support; Pfizer: Other: Grant and personal fees; Astellas Pharma: Other: Personal fees and non-financial support.

Purpose Vincristine (VCR) is a commonly used drug in the treatment of several pediatric cancers. Unfortunately, children suffer from dose-limiting vincristine-induced peripheral neuropathy (VIPN). We aimed to assess whether the administration of VCR by means of one-hour infusions, resulting in lower peak levels, leads to less VIPN than bolus injections in children having completed the induction phase of their treatment for acute lymphoblastic leukemia (ALL) or Hodgkin's lymphoma (HL). Methods The study is part of the VINCA trial, an international randomized controlled trial studying the effect of administration method of VCR on the development and severity of VIPN during treatment of several types of childhood cancer. Participants were measured 3-6 times (depending on the number of VCR administrations and the total treatment period) and one final measurement 6 months after cessation of therapy. VIPN was assessed using the pediatric modified total neuropathy scale (ped-mTNS) and four items of the common toxicity criteria of adverse events (CTCAE version 4.03) items: constipation, peripheral sensory neuropathy, peripheral motor neuropathy and neuralgia. For the current analysis two measurements were taken into account. The first measurement was performed before onset of VCR therapy and the second after induction at day 33 (after 4 VCR administrations) in case of ALL or in week 7 (after 6 VCR administrations) in case of HL. VIPN was defined as a CTCAE sum score of ≥ 2 and/or a total ped-mTNS score of ≥ 5, whereas severe PNP was defined as an individual CTCAE item score of ≥ 3 or a total ped-mTNS score of ≥ 10. Analysis were done using logistic regression or cox-regression. Results were corrected for age, gender and ethnicity. Results In total, 91 children participated in the study, 58 (64%) of whom were treated for ALL and 18 (20%) for HL. 15 (20%) dropped out of the trial or could not be included in the current analysis due to insufficient data (n=6 drop-out or no measurements available after induction, n=9 time between measurement was larger than 64 days). Of the remaining 61, 35 (57%) were randomized in the bolus group and 26 (43%) in the one-hour group. Overall, there was a mean increase of 2.77 for the CTCAE sum score and 7.81 for the ped-mTNS sum score in the bolus group, compared to 2.12 and 6.38, respectively, in the one-hour group (CTCAE: β=-0.71, CI: -1.7-0.44, ped-mTNS: β=-0.84, CI=-3.65-1.97). 27 (77%) children developed neuropathy in the bolus group and 19 (73%) in the one-hour group. Children in the bolus group had a slightly increased, but not significant, risk of developing VIPN compared to children in the one-hour group (hazard ratio=1.21; 95% CI: 0.66 - 2.20). The proportion of children with severe neuropathy was equal in both groups (42%). Conclusions Children treated for ALL or HL who receive VCR by means of one-hour infusions or bolus injections seem to be at equal or diminished risk of developing VIPN during induction therapy. Results of longer follow-up that includes the total treatment period should demonstrate whether VIPN occurs less frequently and/or is less severe in case VCR is administered through one-hour infusions compared to administrations by bolus injections. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

On behalf of the Hokusai VTE Cancer Investigators The treatment of cancer-associated venous thromboembolism (VTE) is challenging because these patients are at increased risk of both recurrent VTE and major bleeding. Low-molecular-weight heparin (LMWH) treatment is standard care for these patients, but requires daily subcutaneous injections. Guidelines recommend LMWH treatment for 6 months, but the risk-benefit beyond this time is uncertain. Direct oral anticoagulants are used for the treatment of VTE in patients without cancer, but their role in patients with cancer- associated VTE is uncertain. In this randomized, open-label non-inferiority trial, cancer patients with acute symptomatic or incidental VTE were assigned to receive LMWH for a minimum of 5 days followed by the oral factor Xa inhibitor edoxaban at a dose of 60 mg once daily (or 30 mg once daily in patients with a creatinine clearance of 30 to 50 ml per minute or a body weight below 60 kg), or subcutaneous dalteparin 200 units per kg once daily for one month followed by 150 units per kg thereafter. Patients received these regimens for up to 12 months. The primary outcome was the composite of the first recurrent VTE or major bleeding event during follow-up for 12 months. Secondary outcomes included recurrent VTE and major bleeding analyzed separately, and survival free of recurrent VTE or major bleeding. The study hypothesis was that edoxaban would be noninferior to dalteparin for the primary outcome with an upper 95% confidence interval [CI] for the hazard ratio below 1.5, and a two-sided alpha of 0.05. All outcomes were independently adjudicated by a committee without knowledge of treatment allocation. This committee also assessed the clinical severity of major bleeding events using categorical criteria defined a priori (categories 1 to 4). From July 2015 through December 2016 a total of 1050 patients were enrolled at 114 centers in 13 countries; 525 were randomized to edoxaban and 525 to dalteparin. At entry, pulmonary embolism with or without deep-vein thrombosis was present in 657 patients (63%) while the remainder had isolated deep-vein thrombosis. Of the 1050 patents, 706 (67%) had symptomatic VTE and the rest were incidental. Active cancer at entry was present in 97% of the patients and 53% had metastatic disease. 1046 patients were included in the modified-intention-to-treat analysis. The primary outcome occurred in 67 of 522 patients (12.8%) in the edoxaban group compared with 71 of 524 patients (13.5%) in the dalteparin group (hazard ratio with edoxaban, 0.97; 95% CI, 0.70 to 1.36; P = 0.0056 for noninferiority) for a risk difference (edoxaban minus dalteparin) of - 0.7% (95% CI, - 4.8 to 3.4). The difference in risk for recurrent VTE was -3.8 % (95% CI, -7.1 to -0.4), whereas the corresponding difference in risk for major bleeding was 3.1% (95% CI, 0.5 to 5.7). The frequencies of severe major bleeding events (categories 3 and 4) were similar during treatment with edoxaban or dalteparin (12 patients in each group respectively). Survival at 12 months free of recurrent VTE and major bleeding in the edoxaban and dalteparin groups was similar (55.0% and 56.5% respectively). Oral edoxaban for up to 12 months is noninferior to subcutaneous dalteparin for the treatment of cancer-associated VTE. Disclosures Raskob: BMS: Consultancy, Honoraria; Eli Lilly: Consultancy; Janssen: Consultancy; Johnson and Johnson: Consultancy; Pfizer: Consultancy, Honoraria; Portola: Consultancy; Boehringer-Ingelheim: Consultancy; Medscape: Honoraria; Bayer Healthcare: Consultancy; Daiichi Sankyo: Consultancy, Honoraria. Van Es:Daiichi Sankyo: Honoraria; Pfizer: Honoraria. Verhamme:Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Bayer Healthcare: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Portola: Consultancy; Medscape: Honoraria; Leo: Honoraria, Research Funding; Sanofi Aventis: Research Funding; Medtronic: Honoraria, Membership on an entity's Board of Directors or advisory committees. Carrier:Daiichi Sankyo: Consultancy, Honoraria; BMS: Consultancy, Research Funding; Leo: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria. Di Nisio:Daiichi: Consultancy, Honoraria. Garcia:Daiichi Sankyo: Honoraria, Research Funding; BMS: Consultancy; Boehringer Ingelheim: Consultancy; Janssen: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria; Medscape: Honoraria; Incyte: Consultancy, Honoraria, Research Funding. Grosso:Daiichi Sankyo: Employment. Kakkar:Daiichi Sankyo: Consultancy, Honoraria; Bayer Healthcare: Consultancy, Research Funding; Boehringer Ingelheim: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi SA: Consultancy, Honoraria; Verseon: Consultancy, Honoraria. Kovacs:Daiichi Sankyo: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bayer: Honoraria; Bristol Myers Squibb: Research Funding. Mercuri:Daiichi Sankyo: Employment, Patents & Royalties: pending properties of edoxaban . Meyer:BMS Pfizer: Research Funding; Leo: Other: travel support; Stago: Other: travel support. Segers:Ionis: Research Funding; Daiichi Sankyo: Research Funding; Janssen: Research Funding. Shi:Daiichi Sankyo: Employment. Wang:Daiichi Sankyo: Honoraria. Yeo:Daiichi Sankyo: Honoraria, Research Funding; Bayer Healthcare: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Leo: Consultancy, Honoraria. Zhang:Daiichi Sankyo: Employment. Zwicker:Daiichi Sankyo: Honoraria; Quercegen Pharma: Research Funding; Parexel: Consultancy. Weitz:Daiichi-Sankyo: Consultancy, Honoraria; Ionis Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis Pharmaceuticals: Consultancy, Honoraria; Merck & Co., Inc.: Consultancy, Honoraria; Pfizer, Inc.: Consultancy, Honoraria; Portola Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Medscape: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy; Bayer HealthCare Pharmaceuticals: Consultancy, Honoraria. Büller:Daiichi Sankyo: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Boehringer Ingelheim: Consultancy, Honoraria; Portola: Consultancy; Medscape: Honoraria; Eli Lilly: Consultancy; Sanofi Aventis: Consultancy; Ionis: Consultancy.

The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma cells were located in a unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38. The identity of the sorted cell populations was confirmed by microscopic examination of Wright's stained slides and slides stained for cytoplasmic immunoglobulin using polyclonal antibodies reactive with light chains; ie, anti-kappa fluorescein isothiocyanate and anti lambda phycoerythrin (PE). The purity of the sorted plasma cells was greater than 97% (n = 4). The average frequency of plasma cells in normal bone marrow aspirates was low--0.25% of the nucleated cells (n = 7)--but surprisingly consistent between individuals (SD = .05; range 0.14% to 0.30%). A detailed analysis showed two distinct populations of plasma cells: (1) A population relatively smaller by forward light scattering expressed CD22, CD35, and sigE and was identified as early plasma cells (ie, lymphoplasmacytoid), and (2) a population larger by forward light scattering lacked these markers and was identified as mature plasma cells. The antigenic profile of the normal plasma cells was determined in two-color immunofluorescence studies. The expression of cell surface immunoglobulin G (IgG), IgA, IgE, IgD, IgM, and the cell surface antigens CD10, CD11b, CD13, CD11c, CD14, CD15, CD16, CD19, CD22, CD20, CD33, CD35, CD45, and HLA-DR was determined on the plasma cells. A significant heterogeneity in cell surface antigen expression was observed within the plasma cell population. Unexpectedly, myeloid- specific cell surface antigens such as CD33 and CD13 and the early B- cell antigen identified by CD10 were expressed on a proportion of plasma cells. These observations imply that the association of myeloid and early B-cell markers described in multiple myeloma may not be associated with the neoplasia but is a normal phenomenon.

The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma cells were located in a unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38. The identity of the sorted cell populations was confirmed by microscopic examination of Wright's stained slides and slides stained for cytoplasmic immunoglobulin using polyclonal antibodies reactive with light chains; ie, anti-kappa fluorescein isothiocyanate and anti lambda phycoerythrin (PE). The purity of the sorted plasma cells was greater than 97% (n = 4). The average frequency of plasma cells in normal bone marrow aspirates was low--0.25% of the nucleated cells (n = 7)--but surprisingly consistent between individuals (SD = .05; range 0.14% to 0.30%). A detailed analysis showed two distinct populations of plasma cells: (1) A population relatively smaller by forward light scattering expressed CD22, CD35, and sigE and was identified as early plasma cells (ie, lymphoplasmacytoid), and (2) a population larger by forward light scattering lacked these markers and was identified as mature plasma cells. The antigenic profile of the normal plasma cells was determined in two-color immunofluorescence studies. The expression of cell surface immunoglobulin G (IgG), IgA, IgE, IgD, IgM, and the cell surface antigens CD10, CD11b, CD13, CD11c, CD14, CD15, CD16, CD19, CD22, CD20, CD33, CD35, CD45, and HLA-DR was determined on the plasma cells. A significant heterogeneity in cell surface antigen expression was observed within the plasma cell population. Unexpectedly, myeloid- specific cell surface antigens such as CD33 and CD13 and the early B- cell antigen identified by CD10 were expressed on a proportion of plasma cells. These observations imply that the association of myeloid and early B-cell markers described in multiple myeloma may not be associated with the neoplasia but is a normal phenomenon.

Xenoantibody production directed at a wide variety of T lymphocyte-dependent and T lymphocyte-independent xenoantigens remains the major immunologic obstacle for successful xenotransplantation. The B lymphocyte subpopulations and their helper factors, involved in T-cell-independent xenoantibody production are only partially understood, and their identification will contribute to the clinical applicability of xenotransplantation. Here we show, using models involving T-cell-deficient athymic recipient mice, that rapidly induced, T-cell-independent xenoantibody production is mediated by marginal zone B lymphocytes and requires help from natural killer (NK) cells. This collaboration neither required NK-cell-mediated IFN-gamma production, nor NK-cell-mediated cytolytic killing of xenogeneic target cells. The T-cell-independent IgM xenoantibody response could be partially suppressed by CD40L blockade. ispartof: BLOOD vol:110 issue:12 pages:3926-3935 ispartof: location:United States status: published

Abstract 3529 Polo-like Kinase 1 (PLK1) is an essential regulator of mitosis. It is often overexpressed and a predictor of poor prognosis in different types of solid tumours and adult hematopoietic malignancies, making PLK1 an interesting therapeutic target in these cancer types. No PLK1 inhibitor has entered clinical trials in pediatric malignancies yet. This study therefore aimed to determine the potential of PLK1 as a new target in the treatment of childhood acute lymphoblastic leukemia (ALL), the most common type of childhood cancer. Reverse phase protein arrays were performed to analyze levels of PLK1 protein expression and phosphorylation at T210, the major activating phosphorylation site, in ALL patient samples (n=174) and normal bone marrow mononuclear cells (nBM) (n=11). Both PLK1 expression and T210 phosphorylation were elevated (2.6-fold and 1.8-fold, respectively) in patients compared to nBM (p Taken together, these results show that PLK1 is overexpressed in pediatric ALL and plays a pivotal role in the proliferation and survival of pediatric ALL cells. Moreover, they underline the potency of PLK1-inhibiting drugs as a valuable addition to current ALL treatment strategies, especially for cases expressing high levels of PLK1 protein. Disclosures: No relevant conflicts of interest to declare.

Abstract 1476 AIM: Aurora kinases (AURK) A and B are known regulators of mitosis and are overexpressed in a large number of human cancers, including leukemia. Several AURK-inhibitors have shown anti-tumor activity in vitro and in vivo. However, the efficacy of AURK inhibition in the treatment of childhood acute leukemia is unexplored. We therefore investigated the effect of targeting AURKA and AURKB in leukemic cells of children with newly diagnosed acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Materials & Methods: Affymetrix gene expression data of 297 ALL, 237 AML and 8 normal bone marrow (nBM) samples were analyzed for AURKA and B mRNA expression levels. Protein expression levels in 172 pediatric ALL and 10 nBM samples were determined with a reverse phase protein array. Functional studies were performed in ALL and AML cell lines, in which AURKA and B were silenced using a short hairpin RNA with a lentiviral delivery system or LNA-containing oligonucleotides. Sensitivity of leukemic cell lines to the AURKB-selective inhibitor Barasertib-hQPA (AZD1152-hQPA) was tested in vitro with an MTS assay. Results: AURKA and B mRNA levels were low in ALL and AML patients. In contrast, Aurora A and B proteins were expressed to a greater extent in patients (p Conclusion: These data show that inhibition of AURKB but not AURKA has an anti-proliferative and pro-apoptotic effect on acute leukemic cells. Thus, targeting Aurora Kinase B may offer a new strategy to treat pediatric ALL and AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 3978 Poster Board III-914 A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific kinases as cancer targets has been a slow process. Inhibition of cancer-causing tyrosine kinases offers a promising avenue of therapy, however this strategy of targeted therapy will require a detailed understanding of the oncogenic targets in each cancer patient. Here, we present an RNAi-assisted protein target identification (RAPID) assay by which cells from leukemia patients are functionally screened with siRNA to determine tyrosine kinases that constitute amenable targets for therapeutic intervention. Combination of the RAPID screen with gene-specific therapeutic approaches promises to yield a powerful synthesis of methodologies by which cancer patients can be specifically treated on the basis of functionally diagnosed gene targets. Methods To detect gene targets necessary for viability of malignant cells, we screened primary cells from 150 patients with hematologic malignancies by electroporating siRNAs individually targeting each member of the tyrosine kinase gene family. Four days later, we measured cell viability and tabulated sensitivity to silencing of specific genes. Samples were also screened for sensitivity to small-molecule kinase inhibitors. The mechanism of oncogenesis was investigated for each positive result. Results In total, we have identified 40 patient-specific gene targets in primary leukemia samples. We demonstrate that siRNA screening can identify known oncogenic lesions such as K-RasG13D and JAK2V617F in primary cells from leukemia patients. The RAPID screen has also directed us towards a novel insertional mutation in the thrombopoietin receptor, MPL (1886InsGG). Additionally, we have detected FLT3 sensitivity in patients with FLT3-ITD and loss of heterozygosity, although not in FLT3-ITD heterozygous patients. Agreement between siRNA-sensitive gene targets and small-molecule inhibitor sensitivity profiles has been high. The mechanism of oncogenesis and its relation to the gene target has been established in select other samples with abnormalities including gene overexpression and patient-specific mis-splicing events. Conclusions We demonstrate that RNAi functional screening can determine sensitivity to individual genes in cells obtained directly from cancer patients. Thus, this technique offers the potential to match targeted therapies with patients in a personalized manner. Application of these technologies will enable efficient discovery of the genetic etiology of cancer as well as a means for gene-specific therapeutic intervention. Disclosures: Deininger: Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Calistoga: Research Funding; Genzyme: Research Funding. Druker:OHSU patent #843 - Mutate ABL Kinase Domains: Patents & Royalties; MolecularMD: Equity Ownership; Roche: Consultancy; Cylene Pharmaceuticals: Consultancy; Calistoga Pharmaceuticals: Consultancy; Avalon Pharmaceuticals: Consultancy; Ambit Biosciences: Consultancy; Millipore via Dana-Farber Cancer Institute: Patents & Royalties; Novartis, ARIAD, Bristol-Myers Squibb: Research Funding.

A large percentage of cancer cases present without knowledge of the causative genetic events. Tyrosine kinases are frequently implicated in the pathogenesis of cancer, but identification of specific tyrosine kinases as cancer targets has been a slow process. In the near future, whole-genome sequencing will enable vast amounts of sequence data to be collected, however clinical application of this information will require a detailed understanding of the functional consequences of each sequence change. Here, we present an RNAi-assisted protein target identification (RAPID) assay by which cells from leukemia patients are functionally screened with siRNA to determine tyrosine kinases that constitute amenable targets for therapeutic intervention. These data have led to identification of novel oncogenic anomalies in cancer patients. Combination of the RAPID screen with whole-genome sequencing promises to yield a powerful synthesis of methodologies by which both functional targets and genetic lesions can be rapidly determined. Methods: To detect targets necessary for viability of malignant cells, we screened primary cells from 75 patients with AML, ALL, CMML, and other MPD as well as white blood cells from healthy individuals by electroporating siRNAs individually targeting each member of the tyrosine kinase family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. Where possible, results were confirmed by treating samples with small-molecule inhibitors with activity against the genes identified by the assay. In addition, the mechanism of oncogenesis was investigated for each positive result. Results: We demonstrate that siRNA screening can identify known oncogenic lesions such as K-RasG13D and JAK2V617F in primary cells from leukemia patients. The RAPID screen has also directed us towards a novel insertional mutation in the thrombopoietin receptor, MPL (1886InsGG). Additionally, we have detected FLT3 sensitivity in patients with FLT3-ITD and loss of heterozygosity, although not in FLT3-ITD heterozygous patients. In total, of 75 patients screened, this assay has yielded 25 cases that exhibit sensitivity to one or more tyrosine kinases. The mechanism of oncogenesis and its relation to the gene target has been established in select other samples with genetic abnormalities including evidence of chromosomal rearrangements as well as gene overexpression and mis-spicing events. Conclusions: We demonstrate that RNAi functional screening can determine sensitivity to individual tyrosine kinases in primary samples. Thus, this technique offers the potential to match specific therapies for targeted intervention with individual patients based on a functional assay. Additionally, in many cases, combination of the RAPID screen with whole-genome sequencing will enable efficient discovery of the genetic etiology of cancer.

The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma cells were located in a unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38. The identity of the sorted cell populations was confirmed by microscopic examination of Wright's stained slides and slides stained for cytoplasmic immunoglobulin using polyclonal antibodies reactive with light chains; ie, anti-kappa fluorescein isothiocyanate and anti lambda phycoerythrin (PE). The purity of the sorted plasma cells was greater than 97% (n = 4). The average frequency of plasma cells in normal bone marrow aspirates was low--0.25% of the nucleated cells (n = 7)--but surprisingly consistent between individuals (SD = .05; range 0.14% to 0.30%). A detailed analysis showed two distinct populations of plasma cells: (1) A population relatively smaller by forward light scattering expressed CD22, CD35, and sigE and was identified as early plasma cells (ie, lymphoplasmacytoid), and (2) a population larger by forward light scattering lacked these markers and was identified as mature plasma cells. The antigenic profile of the normal plasma cells was determined in two-color immunofluorescence studies. The expression of cell surface immunoglobulin G (IgG), IgA, IgE, IgD, IgM, and the cell surface antigens CD10, CD11b, CD13, CD11c, CD14, CD15, CD16, CD19, CD22, CD20, CD33, CD35, CD45, and HLA-DR was determined on the plasma cells. A significant heterogeneity in cell surface antigen expression was observed within the plasma cell population. Unexpectedly, myeloid-specific cell surface antigens such as CD33 and CD13 and the early B-cell antigen identified by CD10 were expressed on a proportion of plasma cells. These observations imply that the association of myeloid and early B-cell markers described in multiple myeloma may not be associated with the neoplasia but is a normal phenomenon.

Platelets and endothelial cells store the polymeric factor V(a) binding protein, multimerin 1 (MMRN1), for release upon agonist stimulation. In human megakaryocytes, factor V binding to MMRN1 follows plasma factor V endocytosis, resulting in stored complexes of MMRN1 and factor V in platelet α-granules. The C2 domain of the factor V light chain contains a MMRN1 binding site; however, the affinity and stoichiometry of factor V-MMRN1 binding have not been determined, direct comparisons of factor V and Va binding to MMRN1 have not been done, and potential homologous roles of C1 and C2 domain structures in MMRN1 binding have not been studied. To further explore the mechanism of factor V and Va binding to MMRN1, and the roles of B domain release and C1 domain residues in MMRN1 binding, we used surface plasmon resonance and solid-phase binding studies. Functional consequences of factor V-MMRN1 binding were tested in competitive binding assays with the soluble phospholipid 1,2-Dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), and calibrated automated thrombinography (CAT). Factor V bound to MMRN1 with a higher affinity than factor Va (approximately 2 nM versus 12 nM), and a stoichiometry consistent with binding to MMRN1 trimers. The higher affinity of factor V for MMRN1 was mainly due to differences in rates of formation of a more stable, secondary complex with MMRN1. Factor V activation by thrombin dissociated bound factor V from MMRN1, consistent with the reduced affinity of factor Va for MMRN1. A panel of point mutated, B domain deleted factor V constructs were used to identify MMRN1 binding residues in the C1 domain of factor V and Va. On a three dimensional model of factor Va, these residues mapped to a large, predominantly contiguous region between the C1 and C2 domains, that overlapped residues critical for factor Va phospholipid binding and procoagulant function. Consistent with the lowered affinity of factor Va for MMRN1, C6PS significantly inhibited factor Va-MMRN1, but not factor V-MMRN1 binding (p