ALBERT

Description: Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P = .01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n = 3), glucocorticoid receptor NR3C1 (n = 4), and components of the mismatch repair pathways (n = 3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P = .02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies.

Description: Abstract 1214 Immune thrombocytopenia (ITP) is the most common acquired thrombocytopenia in children. Typically, external triggers as infections or vaccinations cause the rise of antibodies that crossreact with antigens expressed on the platelet surface. These anti-platelet antibodies are mostly directed against glycoprotein complexes GPIIb/IIIa or GPIb/IX/V, resulting in an increased turnover of antibody-decorated platelets which are then sequestered by the reticuloendothelial system. Recently, it has been suggested that thrombocytopenia might also be due to an insufficient platelet production as serum of some patients with ITP can impair the maturation of CD34+ hematopoietic stem cells to bone marrow megakaryocytes (MKs) in vitro or abrogate the formation of proplatelets in an in vitro culture system. The accelerated platelet turnover demands the generation of platelets de novo. Bone marrow smears often reveal normal or slightly increased MKs, although they seem to be smaller and of altered morphology. However, very little is known about the consequences of anti-platelet antibodies on bone marrow MKs in vivo and in situ. Here, we took advantage of a simple animal model of passive ITP by single or multiple intraperitoneal injections of an anti-GPIb antibody into mice. MKs were evaluated by multi-color immunofluorescence histology on whole femur sections in a modified staining procedure that bypasses decalcification. MK numbers on day 3 were doubled in response to a single injection and tripled on day 8 when mice were injected additionally on day 3 and 7. In these mice platelet counts were up to 2000/nL on day 10, indicating the power to produce platelets. MK area per section was transiently upregulated on day 3 in single injected mice and quadrupled after multiple injections on day 8 before shrinking below norm on day 14. Staining with an anti-rat IgG antibody showed that the antibody was present on MKs within the bone marrow several hours to days after injection. The signal was present for 5 days and no antibody was detected on day 7. MKs had an overall normal morphology and showed no signs of apoptosis or DNA blebbing. All MKs analyzed were negative for TdT in a classical TUNEL assay, indicating that there were no single strand breaks. As platelet counts rose markedly while the antibody was still present on the MK surface, we sought to identify whether the pool of MKs is expanded or formed de novo. To address this, mice where fed with nucleotide analogue EdU for up to 12 days and femur sections stained with Click-It-647 reagent to stain for newly incorporated DNA while mice were treated with anti-platelet antibody or isotype control. We found EdU-positive MKs after 12 days in control isotype-injected mice indicating the de novo formation from hematopoietic stem cells. In antibody-injected mice, newly formed MKs were negative or stained weakly for EdU on day 12, suggesting that they arise partially from an existing pool of progenitors. Finally, we analyzed platelet formation in vivo by imaging of the cranial bone marrow of GPIIb-eYFP-heterozygous mice. The depletion antibody was labeled with Atto-590-fluorophore and injected hours before imaging. Vasculature was counterstained by Quantum dots. We found that MKs residing at the bone marrow were decorated with the antibody and released pre- and proplatelets into the vasculature, indicating that platelet biogenesis can occur in the presence of anti-platelet antibodies on MKs. Our data thus provide novel insight into the pathomechanism of platelet production in patients with ITP. Disclosures: No relevant conflicts of interest to declare.

Description: Acute lymphoblastic leukemia (ALL) is the most common malignancy in childhood. While improved multi-agent chemotherapy regimens with individualized risk stratification have led to increased survival rates of approximately 80 percent, 20 percent of patients respond poorly to therapy or relapse. Therefore, novel therapeutic avenues are urgently needed to improve treatment outcome, overcome resistance and reduce side effects. Failure to undergo cell death represents a key survival mechanism of cancer cells and results in drug resistance and clonal escape. Since inhibitor of apoptosis proteins (IAPs) are often overexpressed in malignant cells and their overexpression correlates with inferior survival rates, they provide an attractive molecular target for therapeutic intervention. Small molecule inhibitors have been developed that act as SMAC mimetics (SMs) to counteract the cell death inhibitory function of IAPs. SMs can activate and/or modulate cell death pathways, and are currently being evaluated in clinical trials. Their successful therapeutic implementation requires identification of patients who could benefit from a SM-based treatment regimen ideally before start of therapy. Here, we analyzed the intrinsic activity of two monovalent (AT406 and LCL161) and two bivalent (Birinapant or BV6) SMs on 29 unselected patient-derived pediatric precursor B-cell (BCP)-ALL samples and identified a subset of BCP-ALL primografts to be sensitive to SM treatment (n=8). When we compared gene expression of SM-sensitive (n=8) and SM-insensitive (n=6) patient-derived BCP-ALL samples, we identified a characteristic gene expression signature with 127 differentially regulated genes, amongst them upregulation of TNFRSF1A (TNFR1) in the SM-sensitive subset. In line with previous reports, we confirmed a critical role of the TNF/TNFR1-axis for SM-induced cell death in BCP-ALL by functional analysis. Expression of TNFRSF1A alone, however, did not correlate with sensitivity to SM-induced cell death indicating that TNFR1 is not the only factor regulating cell fate decisions in response to SM treatment. To identify potential biomarker genes for prediction of patient response to SM monotherapy in BCP-ALL, we compared differentially regulated genes of SM responders and non-responders from our cohort with data from a published cohort. Interestingly, we found 4 genes to overlap between these two cohorts. Of these 4 genes TSPAN7, FAM69C, and TNFRSF1A were upregulated whereas MTX2 was downregulated in SM-sensitive samples. The signature identified may reflect a particular TNF network. Analysis of expression levels of these 4 genes in BCP-ALL cell lines (Nalm6, Reh, UoCB6 and RS4;11) revealed that Reh cells, sensitive to SM-induced cell death, exhibited the biomarker profile of primograft sensitivity, i.e. upregulation of TSPAN7, FAM69C, TNFRSF1A and downregulation of MTX2. Nalm6 cells resembled the expression pattern of SM-insensitive samples with a downregulation of TSPAN7, FAM69C, TNFRSF1A and an upregulation of MTX2 and were resistant to SM-induced cell death. RS4;11 and UoCB6 cells showed no pattern. Based on these findings we hypothesized that the respective expression patterns of TSPAN7, FAM69C, TNFRSF1A and MTX2 could predict sensitivity to SMs. An extended screen of additional primary BCP-ALL samples for their expression levels of TSPAN7, FAM69C, TNFRSF1A and MTX2 and response to SMs substantiated this hypothesis. In summary, the subset of primary BCP-ALL samples with sensitivity to SMs is characterized by a gene signature with MTX2 low and TSPAN7, FAM69C and TNFRSF1A high. By using this expression profile, sensitivity to SMs in BCP-ALL could be identified in cell lines and additional primografts. Based on these results, we suggest the identified gene expression pattern as a biomarker for selecting patients to be treated by SM monotherapy in clinical trials. Disclosures No relevant conflicts of interest to declare.

Description: The Southeastern Cancer Study Group conducted a post-remission induction randomized trial in adult acute myelogenous leukemia to assess the efficacy of alternate drug therapy during consolidation and of immunotherapy during maintenance. Of 508 evaluable patients entered into the study, 335 (66%) achieved a complete remission treated with a 7-day infusion of cytosine arabinoside at a dose of 100 mg/sq m/day and 3 days of daunorubicin at a dose of 45 mg/sq m/day. Those in remission were randomized to receive 3 courses of 1 of 3 consolidation regimens: (A) a continuous infusion of 5-azacytidine, 150 mg/sq m/day for 5 days; (B) 5-azacytidine plus beta-deoxythioguanosine, 300 mg/sq m/day for 5 days; or (C) cytosine arabinoside, 100 mg/sq m/day intravenously, and thioguanine, 100 mg/sq m orally every 12 hr, plus daunorubicin, 10 mg/sq m every 24 hr daily for 5 days. There was no difference in relapse rate among the 3 arms. Those completing consolidation and remaining in remission were randomized to 1 of 3 maintenance regimens: (D) chemotherapy, 5-day infusion of cytosine arabinoside and 2 days of daunorubicin (same doses as induction) given every 13 wk for 1 yr; (E) BCG given twice weekly for 1 mo and then monthly for 1 yr; or (F) the combination of regimens D and E. The median duration of remission was significantly better on regimen D (17.4 versus 9.4 and 9.5 mo), and median survival was 29 mo compared to 21 mo for the other regimens. Those given different drugs during consolidation than used for induction (regimens A and B) and subsequent chemotherapy for maintenance (regimen D) had the longest remission durations and survival. Immunotherapy was not as good as intensive chemotherapy for maintenance.

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: Childhood Leukemia treatment is one of the most common malignancies seen across the globe in the pediatric age group. Severe Hemophilia A is still considered a rare bleeding disorder. Only 13 patients with hemophilia A or B have been reported in literature to also be diagnosed with acute leukemia in childhood . This rarity appeared again in a 13 year boy with severe hemophilia A who presented with worsening bone pain and joint swelling, weight loss and leukocytosis. Morphology and molecular diagnostics confirmed Acute Pre B Lymphoblastic Leukemia . The patient happens to be the first case of Acute Lymphoblastic Leukemia in a patient with severe Hemophilia with Inhibitors. Severe hemophilia with inhibitors pose challenge in clinical management given their propensity of bleeding and poor response to traditional therapies due to a neutralizing antibody as is. Treatment of acute lymphoblastic leukemia requires an intensive treatment with systemic and intrathecal chemotherapy. Medications are commonly administered through a central line, in many cases an implantable catheter. Coexistence of both these life threatening disorders posed a unique practical challenge in providing standard care and our discussion aims at highlighting strategies in management of Severe Hemophilia in challenging clinical scenarios. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Platelet β1 integrin−mediated signals control granule secretion and hemostasis β1 integrin−mediated outside-in signaling is independent of direct kindlin-integrin interaction

Description: Key Points The incidence of venous thromboembolism is high in patients with a solid tumor and implanted port in the real-life practice setting. The risk factors for catheter-related thrombosis differ from those for venous thromboembolism unrelated to the catheter.

Description: Recently, we showed that during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP), ADAMTS13 circulates in an open conformation. Although the cause of this conformational change in acute iTTP remains elusive, ADAMTS13 is mainly closed in iTTP patients (i) in remission with an ADAMTS13 activity 〉50% and undetectable anti-ADAMTS13 autoantibodies, and (ii) after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, IgGs from 18 acute iTTP patients were purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14/18 (78%) samples, proving that indeed anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n=197) that also included plasma samples of iTTP patients in remission where ADAMTS13 activity was