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Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder diagnosed by thrombocytopenia and hemolytic anemia, associated with a deficiency in von Willebrand factor (VWF)–cleaving protease ADAMTS13. Current treatment is based on plasma infusion for congenital TTP, or plasma exchange, often in combination with immunosuppressive agents, for acquired TTP. These treatment methods are not always effective; therefore, new treatment methods are highly necessary. N-acetylcysteine (NAC), an FDA-approved anti-mucolytic agent, is a possible new treatment strategy for TTP, as it was demonstrated to reduce disulfide bonds in VWF, thereby decreasing VWF multimers size and hence their prothrombotic potential. We investigated whether NAC, without concurrent plasma exchange and immunosuppressive therapy, is effective in preventing and resolving TTP signs, using well-established murine and baboon models for TTP. In mice, prophylactic administration of NAC was effective in preventing severe TTP signs. This in vivo finding was supported by in vitro data demonstrating the VWF multimer–reducing properties of NAC in solution. Nonetheless, in both mice and baboons, administration of NAC was not effective in resolving preexisting TTP signs; thrombocytopenia, hemolytic anemia, and organ damage could not be reversed, as thrombus resolution was not achieved. Failure to improve clinical outcome occurred even though a reduction in VWF multimers was observed, demonstrating that NAC was efficient in reducing disulfide bonds in circulating VWF multimers. In conclusion, prophylactic administration of NAC, without concurrent plasma exchange, was effective in preventing severe TTP signs in mice, but NAC was not effective in resolving preexistent acute TTP signs in mice and baboons.

Acute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.

ALX-0681 is a therapeutic Nanobody targeting the A1-domain of VWF. It inhibits the interaction between ultra-large VWF and platelet GpIb-IX-V, which plays a crucial role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In the present study, we report the efficacy and safety profile of ALX-0681 in a baboon model of acquired TTP. In this model, acute episodes of TTP are induced by administration of an ADAMTS13-inhibiting mAb. ALX-0681 completely prevented the rapid onset of severe thrombocytopenia and schistocytic hemolytic anemia. After induction of TTP, platelet counts also rapidly recovered on administration of ALX-0681. This effect was corroborated by the full neutralization of VWF activity. The schistocytic hemolytic anemia was also halted and partially reversed by ALX-0681 treatment. Brain CT scans and post mortem analysis did not reveal any sign of bleeding, suggesting that complete neutralization of VWF by ALX-0681 under conditions of thrombocytopenia was not linked with an excessive bleeding risk. The results obtained in this study demonstrate that ALX-0681 can successfully treat and prevent the most important hallmarks of acquired TTP without evidence of a severe bleeding risk. Therefore, ALX-0681 offers an attractive new therapeutic option for acquired TTP in the clinical setting.

The pathophysiology of thrombotic thrombocytopenic purpura (TTP) can be explained by the absence of active ADAMTS13, leading to ultra-large von Willebrand factor (UL-VWF) multimers spontaneously interacting with platelets. Preventing the formation of UL-VWF–platelet aggregates therefore is an attractive new treatment strategy. Here, we demonstrate that simultaneous administration of the inhibitory anti-VWF monoclonal antibody GBR600 and the inhibitory anti-ADAMTS13 antibody 3H9 to baboons (prevention group) precluded TTP onset as severe thrombocytopenia and hemolytic anemia were absent in these animals. In addition, partial VWF inhibition was not enough to prevent thrombocytopenia, demonstrating the specificity of this therapeutic strategy. GBR600 treatment of baboons during acute TTP (treatment group) resulted in a rapid recovery of severe thrombocytopenia similar to the platelet count increases observed in TTP patients treated by plasma exchange. Baboons in the control group only injected with 3H9 developed early stages of TTP as previously described. Hence, inhibiting VWF-GPIb interactions is an effective way to prevent and treat the early symptoms of acquired TTP in baboons.

Thrombotic thrombocytopenic purpura (TTP) is the prototypical microangiopathy characterized by disseminated microthromboses, hemolytic anemia, and ultimately organ dysfunction. A link with deficiency of the von Willebrand factor–cleaving protease (ADAMTS13) has been demonstrated, but additional genetic and/or environmental triggers are thought to be required to incite acute illness. Here we report that 4 days of ADAMTS13 functional inhibition is sufficient to induce TTP in the baboon (Papio ursinus), in the absence of inciting triggers because injections with an inhibitory monoclonal antibody (mAb) consistently (n = 6) induced severe thrombocytopenia (< 12 × 109/L), microangiopathic hemolytic anemia, and a rapid rise in serum lactate dehydrogenase. Immunohistochemical staining revealed the characteristic disseminated platelet- and von Willebrand factor–rich thrombi in kidney, heart, brain, and spleen but not lungs. Prolonged inhibition (14 days, n = 1) caused myocardial ischemic damage and asplenia but not death. Control animals (n = 5) receiving equal doses of a noninhibitory anti-ADAMTS13 mAb remained unaffected. Our results provide evidence for a direct link between TTP and ADAMTS13 inhibition and for a mild disease onset. Furthermore, we present a reliable animal model of this disease as an opportunity for the development and validation of novel treatment strategies.

The malignant Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (HL) are derived from mature B cells, but have lost a considerable part of the B cell–specific gene expression pattern. Consequences of such a lineage infidelity for lymphoma pathogenesis are currently not defined. Here, we report that HRS cells aberrantly express the common cytokine-receptor γ-chain (γc) cytokine IL-21, which is usually restricted to a subset of CD4+ T cells, and the corresponding IL-21 receptor. We demonstrate that IL-21 activates STAT3 in HRS cells, up-regulates STAT3 target genes, and protects HRS cells from CD95 death receptor–induced apoptosis. Furthermore, IL-21 is involved in up-regulation of the CC chemokine macrophage-inflammatory protein-3α (MIP-3α) in HRS cells. MIP-3α in turn attracts CCR6+CD4+CD25+FoxP3+CD127lo regulatory T cells toward HRS cells, which might favor their immune escape. Together, these data support the concept that aberrant expression of B lineage–inappropriate genes plays an important role for the biology of HL tumor cells.

The malignant Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (HL) are derived from mature B cells, but have lost a considerable part of the B cell–specific gene expression pattern. Consequences of such a lineage infidelity for lymphoma pathogenesis are currently not defined. Here, we report that HRS cells aberrantly express the common cytokine-receptor γ-chain (γc) cytokine IL-21, which is usually restricted to a subset of CD4+T cells, and the corresponding IL-21 receptor. We demonstrate that IL-21 activates STAT3 in HRS cells, up-regulates STAT3 target genes, and protects HRS cells from CD95 death receptor–induced apoptosis. Furthermore, IL-21 is involved in up-regulation of the CC chemokine macrophage-inflammatory protein-3α (MIP-3α) in HRS cells. MIP-3α in turn attracts CCR6+CD4+CD25+FoxP3+CD127loregulatory T cells toward HRS cells, which might favor their immune escape. Together, these data support the concept that aberrant expression of B lineage–inappropriate genes plays an important role for the biology of HL tumor cells.

Autoantibodies to proteinase 3 (PR3) are involved in the pathogenesis of autoimmune-mediated vasculitis in Wegener granulomatosis (WG). To address the question how the autoantigen PR3 becomes a target of adaptive immunity, we investigated the effect of PR3 on immature dendritic cells (iDCs) in patients with WG, healthy blood donors, and patients with Crohn disease (CD), another granulomatous disease. PR3 induces phenotypic and functional maturation of a fraction of blood monocyte-derived iDCs. PR3-treated DCs express high levels of CD83, a DC-restricted marker of maturation, CD80 and CD86, and HLA-DR. Furthermore, the DCs become fully competent antigen-presenting cells and can induce stimulation of PR3-specific CD4+T cells, which produce IFN-γ. PR3-maturated DCs derived from WG patients induce a higher IFN-γ response of PR3-specific CD4+T cells compared with patients with CD and healthy controls. The maturation of DCs mediated through PR3 was inhibited by a serine protease inhibitor, by antibodies directed against the protease-activated receptor-2 (PAR-2), and by inhibition of phospholipase C, suggesting that the interactions of PR3 with PAR-2 are involved in the induction of DC maturation. Wegener autoantigen interacts with a “gateway” receptor (PAR-2) on iDCs in vitro triggering their maturation and licenses them for a T helper 1 (Th1)–type response potentially favoring granuloma formation in WG.

T(11;18)(q21;q21) is the most common structural abnormality in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) leading to the fusion of the apoptosis inhibitor-2 (API2) gene and the MALT lymphoma-associated translocation (MALT1) gene. In 2 patients with MALT lymphoma of the liver and skin, respectively, t(14;18)(q32;q21) was observed by cytogenetic analysis. Subsequent fluorescence in situ hybridization (FISH) studies disclosed that the immunoglobulin heavy-chain locus (IGH) and the MALT1 gene were rearranged by this translocation. In order to screen a large series of MALT lymphomas for this aberration, a 2-color interphase FISH assay was established. Among a total of 66 cases, t(14;18)(q32;q21) involving IGH and MALT1 was detected in MALT lymphomas of the liver (4 of 4), skin (3 of 11), ocular adnexa (3 of 8), and salivary gland (2 of 11), but did not occur in MALT lymphomas of the stomach (n = 10), intestine (n = 9), lung (n = 7), thyroid (n = 4), or breast (n = 2). In total, 12 of 66 (18%) MALT lymphomas harbored t(14;18)(q32;q21); 7 additional cases of splenic marginal zone lymphoma tested negative. All of the 12 MALT lymphomas featuring the t(14;18)(q32;q21) were negative for t(11;18)(q21;q21) by reverse transcriptase–polymerase chain reaction (RT-PCR). However, trisomy 3 and/or 18 was found in 4 of 12 cases, suggesting that the t(14;18)(q32;q21) does not occur as the sole genetic abnormality. This study identifies IGH as a new translocation partner of MALT1 in MALT lymphomas, which tend to arise frequently at sites other than the gastrointestinal tract and lung. In contrast to t(11;18)(q21;q21)+ MALT lymphomas, those with t(14;18)(q32;q21) may harbor additional genetic abnormalities.

T(11;18)(q21;q21) is the most common structural abnormality in extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) leading to the fusion of the apoptosis inhibitor-2 (API2) gene and the MALT lymphoma-associated translocation (MALT1) gene. In 2 patients with MALT lymphoma of the liver and skin, respectively, t(14;18)(q32;q21) was observed by cytogenetic analysis. Subsequent fluorescence in situ hybridization (FISH) studies disclosed that the immunoglobulin heavy-chain locus (IGH) and the MALT1gene were rearranged by this translocation. In order to screen a large series of MALT lymphomas for this aberration, a 2-color interphase FISH assay was established. Among a total of 66 cases, t(14;18)(q32;q21) involving IGHand MALT1was detected in MALT lymphomas of the liver (4 of 4), skin (3 of 11), ocular adnexa (3 of 8), and salivary gland (2 of 11), but did not occur in MALT lymphomas of the stomach (n = 10), intestine (n = 9), lung (n = 7), thyroid (n = 4), or breast (n = 2). In total, 12 of 66 (18%) MALT lymphomas harbored t(14;18)(q32;q21); 7 additional cases of splenic marginal zone lymphoma tested negative. All of the 12 MALT lymphomas featuring the t(14;18)(q32;q21) were negative for t(11;18)(q21;q21) by reverse transcriptase–polymerase chain reaction (RT-PCR). However, trisomy 3 and/or 18 was found in 4 of 12 cases, suggesting that the t(14;18)(q32;q21) does not occur as the sole genetic abnormality. This study identifies IGHas a new translocation partner of MALT1in MALT lymphomas, which tend to arise frequently at sites other than the gastrointestinal tract and lung. In contrast to t(11;18)(q21;q21)+MALT lymphomas, those with t(14;18)(q32;q21) may harbor additional genetic abnormalities.

Intravenous recombinant (r)-hirudin has a potent antithrombotic effect in aspirin- and heparin-resistant platelet-dependent thrombus formation in baboon models. However, these thrombi reform when therapy is stopped after 60 minutes. To determine if 4 hours of therapy can produce a lasting antithrombotic effect, we investigated the extent of deposition of 111In-labeled platelets onto 0.5-cm2 segments of Dacron vascular grafts for 53 hours. These grafts had been incorporated into exteriorized permanent femoral arteriovenous shunts in baboons. Platelet deposition in eight untreated animals was generally sigmoidal. Maximum platelet deposition, 1.7% +/- 0.9% of injected labeled platelets, was reached after approximately 4 hours. Deposition then gradually decreased to 0.4% +/- 0.2% of injected labeled platelets after 53 hours. After a thrombus was allowed to form for 15 minutes in six animals, intravenous treatment with r-hirudin at a dose of 20 nmol (0.14 mg)/kg-min-1 (aPTT > 300 seconds) was started and maintained for 4 hours. Platelet deposition was interrupted during treatment. After infusion was stopped, platelets accumulated again, but not as much as in the untreated animals. Maximum platelet deposition, 0.7% +/- 0.2% of injected labeled platelets, was significantly less (P < .01), and was reached after approximately 23 hours. Thereafter, deposition decreased to 0.4% +/- 0.2% at 53 hours. The shunts in all of the untreated animals occluded at some stage during the study, while only one shunt occluded in the treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Mullighan: Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding.

Chromosomal translocations are common in acute myeloid leukemia (AML), causing gene fusions that encode oncogenic proteins. The NUP98gene participates in chromosomal rearrangements with over 30 fusion partners and comprises 6-10% of de novocases of pediatric AML. These fusions are associated with poor prognosis in children and adults. Among them, NUP98-KDM5Ais further enriched in specific subpopulations, found in 15% of non-Down syndrome acute megakaryoblastic leukemia cases and 20% of pediatric acute erythroleukemia. Despite these associations, the direct impact of NUP98-KDM5Aon the growth and differentiation of human hematopoietic cells has not been systemically studied.

Therapy-related myeloid neoplasms (tMN) occur in children secondary to cytotoxic therapies used to treat pediatric malignancies, are typically resistant to conventional chemotherapy, require hematopoietic cell transplantation as the only curative option, and have a dismal prognosis. The genomic alterations that drive tMN in children have yet to be comprehensively described, and it is unclear if particular genomic lesions hold prognostic value. We have characterized the genomic profile of 62 pediatric tMN cases (tMDS: n=23, tAML: n=39) obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1987 and 2018. These cases arose following treatment for a variety of primary tumors (hematological (74%), bone and soft tissue (23%), and brain (3%)). Acute lymphoblastic leukemia was the most frequent primary tumor (n=39, 63%). Conventional cytogenetics (n=60) showed a complex karyotype (≥3 structural alterations) in 19 (32%) cases, and 7 of these cases contained a deletion involving chromosome 7 (del(7)). Eleven (18%) other cases without complex karyotypes had del(7). Deletions of chromosome 5 were present in 9 (15%) cases, but only in the context of a complex karyotype. We hypothesized that the patients' younger age and the different spectrum of primary tumor types and chemotherapy would give rise to a mutational spectrum distinct from adult tMN.

Gruber: Bristol-Myers Squibb: Consultancy.

Gruber: Bristol-Myers Squibb: Consultancy. Rubnitz:AbbVie: Research Funding. Reinhardt:Jazz: Other: Participation in Advisory Boards, Research Funding; CSL Behring: Research Funding; Novartis: Other: Participation in Advisory Boards; Roche: Research Funding. Locatelli:bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Zwaan:Roche: Consultancy; Servier: Consultancy; Daiichi Sankyo: Consultancy; Novartis: Consultancy; Sanofi: Consultancy; Pfizer: Consultancy, Research Funding; BMS: Research Funding; Celgene: Consultancy, Research Funding; Jazz pharmaceuticals: Other: Travel support; Janssen: Consultancy; Incyte: Consultancy.

We and others recently showed that the mutational spectrum of de novopediatric myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is different than those in adults. MDS and AML also occur in children as a consequence of cytotoxic therapies used to treat childhood malignancies and are collectively referred to as therapy-related myeloid neoplasms (tMN). The incidence of pediatric tMN is ~1% in the pediatric cancer population. These secondary malignancies are usually resistant to conventional chemotherapy and managed with hematopoietic cell transplantation (HCT). These patients have a dismal prognosis. TP53mutations and somatic alterations in chromatin modifiers predominate in adults with tMN, yet whether children with tMN have a similar constellation of genetic alterations remains unclear since comprehensive genomic profiling has not been completed in a large pediatric tMN cohort. We hypothesize that the mutational profile of pediatric tMN will be different than adult tMN given the patients' younger age and the different spectrum of primary tumor types and chemotherapies.

Background: Clostridium difficile(CD) is an important cause of infectious diarrhea in patients with hematologic malignancies (HM) admitted to the hospital for chemotherapy or hematopoietic cell transplantation (HM-HCT patients). These patients are at increased risk of developing Clostridium difficileinfection (CDI) compared to non-oncology patients, and they are more likely to suffer adverse outcomes from the infection. Asymptomatic colonization of patients with CD is known to precede infection. However, the reported prevalence of asymptomatic colonization varies widely between studies. The primary objective of this study was to define the prevalence of toxigenic CD colonization in patients with hematologic malignancies who were admitted to the hospital for scheduled chemotherapy or hematopoietic cell transplantation (HCT). The secondary objective was to determine what microbiology tests are accurate in detecting CD colonization compared to the gold standard of toxigenic culture.

No relevant conflicts of interest to declare.

Kasenda: Riemser: Other: Travel Support. Schmidt-Wolf:Janssen: Research Funding; Novartis: Research Funding. Röth:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Stilgenbauer:Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding. Illerhaus:Riemser: Honoraria; Amgen: Honoraria.

Introduction:For fit multiple myeloma (MM) patients, autologous hematopoietic stem cell transplant (HSCT) is standard of care as part of first line therapy, demonstrating longer progression-free survival when compared to upfront bortezomib, lenalidomide, and dexamethasone (IFM/DFCI 2009, ASH 2015). The use of granulocyte colony stimulating factor (G-CSF) after HSCT accelerates time to neutrophil recovery by 1 - 6 days when compared with control (Klumpp TR et al, JCO, 1995 & Schmitz N et al, BMT2004). The American Society of Clinical Oncology guidelines recommend that G-CSF should be initiated 1-5 days after administration of high-dose chemotherapy and should be continued until the absolute neutrophil count (ANC) is 2000-3000/mm3(Smith TJ, JCO, 2015). We have evaluated the role of G-CSF starting day +1, day +5, and day +7 post-transplant in three sequential cohorts of MM patients focusing on the duration of severe neutropenia (rather than the time to neutrophil engraftment), infections, and mucositis.

Spencer: Cofactor Genomics: Consultancy.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Hofmeister: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Honoraria, Research Funding; ARNO Therapeutics: Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees.

Illerhaus: Riemser: Honoraria. Wolf:Bayer: Honoraria; Geo Pharma: Honoraria. Stilgenbauer:Pharmacyclics, Janssen: Honoraria, Research Funding.

Hofmeister: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Honoraria, Research Funding; ARNO therapeutics: Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Mutations in the de novoDNA methyltransferase DNMT3Aare found in ~25% of patients with acute myeloid leukemia (AML) and most commonly affect codon 882 within the catalytic domain of the protein. We have previously shown that this mutation has dominant negative activity in vitroand is associated with hypomethylation at specific CpG dinucleotides in primary AML samples using array-based methylation data. However, the genome-wide extent and patterns of DNA methylation associated with this hypomethylation are currently unknown. In addition, it is unclear if the methylation differences caused by this mutation result in RNA expression changes at specific targets across the genome, or whether they are associated with altered chromatin structure.

Mutations in DNMT3A(encoding one of two mammalian de novoDNA methyltransferases) are found in >30% of normal karyotype AML cases and correlate with poor clinical outcomes. Most DNMT3Amutations occur at position R882 within the catalytic domain (most commonly R882H) and are virtually always heterozygous. This over-representation suggests that mutations at R882 may result in gain-of-function or dominant-negative activity that contributes to leukemogenesis. However, how DNA methylation might be altered in DNMT3A-mutant cases of AML remains unclear, and no published study to date has addressed the effects of mixing wild-type (WT) and R882H DNMT3A. Importantly, mouse HSPCs deficient in Dnmt3adramatically expand over time and have a concurrent defect in differentiation (Challen, GA et al. Nat Genet, 2011). Mice haploinsufficient for Dnmt3a, on the other hand, do not have a measurable defect in hematopoiesis. Collectively, these data suggest that the heterozygous R882 mutations probably cause more than a simple loss-of-function phenotype.

No relevant conflicts of interest to declare.

Welch: Eisai: Research Funding.

Most recurrent somatic mutations in acute myeloid leukemia (AML) have been identified. However, it is now clear that many mutations are not always present in all the cells within an AML sample. Our previous studies have shown that all AML samples are comprised of a founding clone and usually one or more subclones that are derived from the founding clone. The clonal architecture of an AML sample can be identified using single nucleotide variants (SNVs) that cluster according to discrete variant allele fractions (VAFs); the accurate identification of these clusters in AML samples generally requires deep digital sequencing of all the variants identified by whole genome sequencing (WGS), since AML samples have so few mutations in coding sequences. However, it is not yet clear whether human AML samples engrafted in immunodeficient mice accurately recapitulate the subclonal architecture of the injected sample. Similarly, it not yet clear whether the presence of human cytokines, such as those expressed in the NSG-SGM3 strain (NSG mice with transgenes expressing human IL-3, SCF, and GM-CSF) would alter the engrafting potential of individual subclones within an injected sample.

Primary central nervous system lymphoma (PCNSL) is an aggressive non-Hodgkin′s lymphoma with poor prognosis. Addition of methotrexate (MTX) to whole brain radiotherapy (WBRT) has improved the prognosis of patients (pts) with PCNSL, but a significant proportion are still not cured. Preliminary reports suggested that dose-intensified chemotherapy (HDT) and autologous stem-cell transplantation (ASCT) are highly effective in the treatment of newly-diagnosed PCNSL in younger pts. To strengthen the evidence of this approach, we initiated a prospective multicenter phase II study with early HDT and ASCT to investigate efficacy, safety and survival. This trial is registered at ClinicalTrials.gov (NCT 00647049).Immunocompetent pts <65 years with untreated biopsy proven PCNSL were eligible. Induction treatment consisted of 4 repetitive cycles of MTX (8g/m2) and 2 cycles cytarabine (2×3g/m2) and thiotepa (TT, 40mg/m2). Rituximab (375mg/m2) was added by amendmend after 2 included patients; it was given on day −7 before induction treatment and before each chemotherapy cycle. After the 2nd cycle cytarabine/TT stem-cells were collected after mobilisation with rG-CSF. The HDT regimen included carmustine (400mg/m2) and TT (4×5mg/kgBW) prior ASCT. Primary endpoint was complete remission (CR) 30 days after ASCT. Secondary end-points were overall-survival (OS), duration of response and toxicity. Patients not in complete remission after HDT and ASCT received WBRT.From 2007 to 2011 79 pts (44 female, 35 male) were enrolled from 18 German centers and evaluable for analysis (median age 55 years, range 20–66). Seventy eight pts had aggressive B-cell lymphomas and one T-cell-lymphoma. Median Karnofsky performance status at diagnosis was 90% (range 30–100). After induction treatment, 73 of 76 (96%) evaluable pts responded, (26,9% CR, 55,7 PR). Seventy-three pts (96%) received HDT and ASCT according to protocol. Six pts were treated off-protocol due to low performance status (n=1), progressive disease (n=1) and infectious complications (n=4). Regarding the primary endpoint, CR was achieved in 77% and partial remission (PR) in 14% of patients (overall response rate 91%) after HDT and ASCT. Ten pts in PR after HDT and ASCT received consolidating WBRT. After a median follow-up of 28.8 months (range 1–63 mo) 1 and 2 years OS was 92% and 87%, respectively. Myelotoxicity was the most frequent CTC grade 3–4 toxicity with grade 3–4 infections in 41/73 pts (56,2%) during the transplant-phase. Two patients had lethal infectious complications during induction treatment with cytarabine/TT, three further pts died after HDT and ASCT due to severe infection (n=1), renal failure (n=1) and pneumonitis (n=1). Further results will be presented.Sequential MTX-based immuno-chemotherapy followed by carmustine/TT containing HDT and ASCT is highly effective and feasible in younger patients. Treatment related toxicity is of concern and comparable to non-high-dose protocols. Further randomised trials to compare HDT with conventional CT are needed.Illerhaus: Riemser: Honoraria.

No relevant conflicts of interest to declare.

Ley: Washington University: Patents & Royalties.

Ley: Washington University: Patents & Royalties.

Abstract 1329

Ley: Washington University: Patents & Royalties.

Abstract 2527

Abstract 407

Westervelt: Novartis: Speakers Bureau.

Abstract 99

Westervelt: Novartis: Honoraria; Celgene: Honoraria, Speakers Bureau. DiPersio:Genzyme: Honoraria.

Objectives: A pilot phase II trial was performed to evaluate response rate, response duration, overall survival, and toxicity in primary central nervous system lymphoma (PCNSL) after systemic and intraventricular chemotherapy with deferred radiotherapy. Patients and Methods: From 09/1995 to 12/2002, 65 patients with PCNSL (median age 62 years) were enrolled into a pilot/phase II study evaluating chemotherapy without radiotherapy. A high-dose methotrexate (MTX) (cycles 1,2,4,5) and cytarabine (ara-C) (cycles 3,6) based systemic therapy (including dexamethasone, vinca-alkaloids, ifosfamide and cyclophosphamide) was combined with intraventricular MTX, prednisolone and ara-C. Primary endpoint was time to treatment failure (TTF), secondary endpoints were response, overall survival, response duration, 5-year-survival fraction and (neuro)toxicity. Results: 34 patients were male, 31 female. Sixty-one of 65 patients were evaluable for response. Of these, 37 (61%) achieved a complete response (CR), 6 (10%) complete response/unconfirmed, and 12 (20%) progressed under therapy. Overall response rate was 71% for all patients and 86% for patients younger than 61 years. Six (9%) out of 65 patients died due to treatment-related complications. Follow-up time is 78 to 151 months in surviving patients (median 100 months). Kaplan Meier estimates for median time to treatment failure (TTF), median overall survival and median response duration are 22 months, 54 months and 37 months, respectively. For patients aged 60 years or older, the respective numbers were 7 months, 34 months and 30 months; in patients younger than 60 years, the Kaplan Meier estimate for TTF is 49 months, median overall survival and median response duration have not yet been reached. The 5-year survival fraction is 72% in patients < 60 years and 24% in older patients. Systemic toxicity was mainly hematologic. Ommaya reservoir infection occurred in 19% of the patients. At present, 17/30 (57%) of younger and 4/35 (9%) elderly patients are still alive. Only 5/30 (17%) of younger, but 19/35 (54%) of elderly patients received radiation salvage therapy at relapse. In 2/65 (3%), secondary cancers developed. In the subgroup of patients with long-term survival (n=17/30 under 61 years), 12 had an ongoing response, 1 an isolated CNS relapse (resolved by radiation), 1 an isolated ocular relapse (resolved by ocular radiation) and 3 a pure systemic relapse without CNS involvement (all resolved by systemic chemotherapy). Eleven of these 17 patients could be investigated by comprehensive neuropsychological testing, which revealed normal cognitive function in all of them. Conclusions: Primary chemotherapy based on high-dose MTX and ara-C is highly efficient in PCNSL. A substantial fraction of patients < 60 years can obviously be cured with this regimen.

Background: Data on relapsed central nervous system lymphoma (CNSL) is limited. Therefore, we have evaluated the clinical characteristics and outcome of relapsed CNSL patients at our centers. A central nervous system relapse is a serious complication of aggressive lymphomas. The prognosis is generally regarded as poor and standard therapies of relapsed CNSL have not yet been established. Patients and Methods: All pts with primary and secondary CNSL who were treated at Bonn University Hospital and Cologne University Hospital from 09/1995 – 12/2007 were included into the study. 125 pts could be identified; 102 with newly diagnosed primary CNSL (PCNSL) and 23 pts with secondary CNSL (SCNSL). First-line- treatment for pts with PCNSL consisted of high-dose methotrexate (MTX) in combination with vincristine, ifosfamide, prednisolone and cytarabine (Bonn protocol). Patients with SCNSL received a CHOP-like combination chemotherapy first-line. At cerebral relapse, for 17/23 pts SCNSL-treatment was Bonn polychemotherapy, 4/23 pts received an acute leukaemia regimen (GMALL-B-ALL protocol) at relapse and 2/23 received radiotherapy only. 81/125 pts (64%) were male with a median age of 62 yrs (range 27–77) and 72/125 pts (57%) were older > 60 years. Results: Overall response rate (CR+PR) was 63% (49CR +19PR) for PCNSL and 65% (12CR+3PR) for SCNSL. Median overall survival was 15.8 months for all 125 pts and 23 vs. 7.1 months for PCNSL and SCNSL. There was a difference in OS of 15 months between pts >60 yrs (median 13 months) vs. pts <60 yrs (median 28 months). After a median follow-up of 12 months (range 1–119) 80/125 (64%) pts with PCNSL and SCNSL relapsed (37) or progressed (43). All 80 patients relapsed/progressed again within the CNS. 65/80 pts initially suffered from PCNSL, 15/80 pts from SCNSL. Prognosis of relapsed/progressed PCNSL and SCNSL was dismal. 56/80 patients died. Median OS was short with only 8.5 months (range 1–124) survival time after relapse. Treatment at relapse was radiotherapy alone (16), radiotherapy in combination with temozolomide (2), radiotherapy in combination with MTX, dexamethasone and cytarabine (1), PVC-Chemotherapy (1), BEAM-Polychemothapy (1), Bonn polychemotherapy (7), cytarabine and etoposide (CYVE) with autologous stem cell transplantation (5) and “wait and see” (4). Response to relapse treatment was in 12 cases CR, in 9 cases PR and in 16 cases PD. Treatment at progress was radiotherapy alone (16), radiotherapy in combination with temozolomide (3), radiotherapy in combination with MTX, dexamethasone and cytarabine (1), BEAM-polychemotherapy (1), Bonn polychemotherapy (1), cytarabine and etoposide (CYVE) with autologous stem cell transplantation (2) and “wait and see”/no therapy (16). Response to progress treatment was in 3 cases CR, in 3 cases PR and in 37 cases PD. Conclusions: HD-MTX chemotherapy according to the Bonn Protocol is equally effective in PCNSL and SCNSL pts although the prognosis of pts with SCNSL seems to be worse. Prognosis of relapsed and refractory CNSL pts is dismal. Median OS was only 8.5 months. Therefore, new treatment strategies are urgently needed.

Background: A systemic and intraventricular polychemotherapy regimen (“Bonn protocol”) with deferred radiotherapy had resulted in durable responses in 75% of patients < 60 years with primary CNS lymphoma (PCNSL), but had been complicated by a high rate of Ommaya reservoir infections. Purpose: Here, efficacy and toxicity of this regimen but without intraventricular treatment was evaluated in PCNSL. Patients and Methods : From 08/03 to 11/05, 18 patients with PCNSL < 60 years (median age 53 years) were treated within a phase II trial with a high-dose methotrexate (MTX; cycles 1,2,4 and 5) and cytarabine (Ara-C; cycles 3 and 6) based systemic therapy including dexamethasone, vinca-alkaloids, ifosfamide and cyclophosphamide. Results: Study accrual was prematurely stopped in 11/05 due to a high rate of early relapses. Seventeen/18 patients were assessable for response: Nine (53%) achieved complete response (CR), two (12%) complete response/unconfirmed (CRu), two (12%) partial response (PR), four (24%) showed progressive disease (PD); in one treatment was stopped due to toxicity. Median follow-up is 23 months; Kaplan-Meier estimates for median response duration were ten months only in responding patients and for median time to treatment failure (TTF) eight months in the whole group; median overall survival (OS) has not yet been reached. Systemic toxicity was mainly hematologic. Conclusions: In patients < 60 years with PCNSL polychemotherapy without intraventricular treatment results in a high response rate, but is associated with early relapses in the majority of cases. This is in contrast to the results achieved with the same protocol but with inclusion of intraventricular treatment.

Background: Treatment of primary CNS lymphoma (PCNSL) with a combined systemic and intraventricular chemotherapy (Bonn protocol) has achieved an overall response rate (ORR) of 84% and long term complete remissions in a substantial fraction of patients younger than 60 years. Purpose: Due to a high infection rate of the Ommaya reservoir the question was addressed if intraventricular treatment is dispensable in this polychemotherapy protocol. Patients and Methods: Fifty patients with histologically confirmed PCNSL were enrolled onto a phase II-study evaluating chemotherapy without radiotherapy and without intraventricular treatment. A high-dose methotrexate (MTX) (cycles 1,2,4,5) and cytarabine (ara-C) (cycles 3,6) based systemic therapy (including dexamethasone, vinca-alkaloids, ifosfamide and cyclophosphamide) was administered. Results: In an ongoing trial thirty-five of 50 patients (18 pat. < 60 years, 17 pat. over 60 years) are yet assessable for response after a median follow up of nine months (range: 1 to 26 months). In 18 patients < 60 years, the ORR was 78%. However, median time to treatment failure (TTF) was eight months, and median progression free survival (PFS) only 7 months according to frequent early relapses. Conclusions: Early relapses are frequent in younger patients treated with the modified Bonn protocol without intraventricular treatment despite a high ORR. These preliminary results support the assumption that intraventricular treatment is essential to achieve sustained remissions after successful treatment of PCNSL.

In patients with plaque rupture, platelets adhere, aggregate and form a thrombus. Current strategies to prevent thrombus formation consist of the use of Aspirin®, Plavix® and integrin αIIbβ3 blockers (e.g. Reopro®) in combination with Heparin®. These drugs are associated with high bleeding risk. Several in vivo experiments have shown that neutralizing the collagen von Willebrand Factor (vWF) platelet glycoprotein (GP)Ib-IX-V axis strongly inhibits arterial thrombosis without bleeding complications, therefore, these targets are of high interest to develop new anti-thrombotic drugs. Nanobodies are antibody-derived therapeutic proteins with the structural and functional properties of naturally occurring single-chain antibodies derived from camelids. ALX-0081 is a bivalent humanized Nanobody targeting the GPIb-IX-V binding site at the A1 domain of vWF. The precursor molecule was isolated from a llama immunized with the recombinant A1 domain of vWF and then humanized and engineered into a bivalent format to maximally benefit from the avid binding to vWF. In vitro, ALX-0081 can completely inhibit platelet adhesion to collagen at nanomolar concentrations. This inhibition is specific for the high shear rates relevant for coronary and carotid arteries whilst platelet adhesion and aggregation under low shear conditions is unaltered. The Nanobody also inhibits platelet adhesion to ultra large vWF (ULvWF) whilst it does not inhibit cleavage of ULvWF by ADAMTS-13. In a modified Folt’s model in baboons ALX-0081 inhibits thrombus formation more efficiently than a combination of Aspirin, Heparin and Plavix. Inhibition of thrombus formation is sustained in the presence of epinephrine and upon a new injury confirming the strong anti-thrombotic effect of ALX-0081. The Nanobody is effective at doses approximately 10–20 times lower than the dose required for Reopro. Ex vivo analysis of plasma samples after ALX-0081 administration in baboons in the ristocetin induced platelet aggregation (RIPA) assay reflects the efficacy seen in the Folt’s model. Therefore, this assay seems to be suited to predict effective ALX-0081 concentrations in vitro. In comparison to Reopro and Plavix, ALX-0081 is associated with less bleeding complications, even at doses exceeding the effective dose by a factor of 10 probably because of its selective inhibition of platelet aggregation under high shear but not under low shear conditions. After treatment with ALX-0081 no effect on other hematological parameters such as PT, aPTT, platelet count, VWF concentration and FVIII levels is seen and no immunogenicity is detected in baboons after repeated administration of ALX-0081. The terminal half-life of ALX-0081 in baboons is 8 hours, indicating that the molecule adopts the half-life of vWF. This high efficacy combined with an improved safety compared to the currently marketed drugs suggests that Ablynx’ drug development candidate ALX-0081 can become a powerful drug to treat acute thrombotic events in indications such as ACS, stroke, and TTP.

In 1999 the German Multicenter Study Group for Adult ALL (GMALL) activated a pilot study (GMALL 06/99). One major aim was to develop a new, shortened and intensified induction regimen based on the following new principles compared to previous GMALL trials: 1) Dexamethasone (DEXA) instead of prednisone to improve antileukemic activity and prophylaxis of CNS relapse 2) prephase with cyclophosphamide (CYCLO) 3) G-CSF parallel to chemo 4) intensified daunorubicin with two 2day cycles (DNR) vs 4 wkly applications 5)1 dose PEG-L-Asparaginase (ASP) instead of 14 d conventional ASP Induction I was followed by GMALL induction phase II as previously reported and a uniform consolidation I. Remission control took place on d24 and d44. Thereafter treatment was risk adapted. Induction I consisted of DEXA, CYCLO and G-CSF. In addition pts received PEG-ASP 1000 U/m2 (d13), vincristin 2 mg (d4,11,18) and DNR 45 mg/m2 (d4+5,11+12). The regimen was modified by 3 amendments which separated the study to 4 pilot phases. The major modifications referred to reduction of DEXA/CYCLO and earlier application of G-CSF. Table 1: Major modifications of induction phase I Drug Pilot 1 Pilot 2 Pilot 3 DEXA 40 mg/m2 (d1–3) 10 mg/m2 (d4–17) 10 mg/m2 (d 1–6,11–16) 10 mg/m2 (d 1–5,11–14) CYCLO 200 mg/m2 (d1–3) none none G-CSF from d13 from d4 from d4 Overall 843 pts were included between 4/99 and 10/03. The median age was 36 (15–65) yrs. Subtypes distribution was c-/pre B 65%, pro B 8%, early T 8%, thymic 14%, mature T 6%. 23% had Ph/BCR-ABL+ ALL. The overall CR rate was 83%, with 12% failure/PR and 7% early death (ED). Significant differences were detected for the pilot phases (p=.0008). The high mortality in pilot I was mainly due to infections. With lower doses of DEXA the rate of ED (p=.0002) and severe infections decreased significantly whereas the failure rate increased slightly. The earlier application of G-CSF contributed to a significant decrease of grade III/IV granulocytopenias and probably also mucositis. Table 2: Results and major toxicities (grade III/IV) of induction therapy Pilot 1 Pilot 2 Pilot3 P Evaluable 103 100 605 CR 76% 83% 82% .0008 PR/Failure 9% 9% 14% ED 16% 8% 5% Survival (3y) 45% 47% 47% >.05 Granulopenia 84% 72% 69% .008 Median duration 17d 15d 12d <.0001 Infections 30% 33% 14% <.0001 Mucositis 23% 14% 6% <.0001 We conclude that dose and schedule of DEXA in induction of ALL has a significant effect on rate of infections and ED. A dose reduced schedule with interruption to allow for early detection of infections led to a favourable CR rate of 82% with ED of 5% after only 6 wks of induction therapy. The parallel application of chemo and G-CSF rendered the regimen more feasible and contributed to the lower rate of infections and mucositis. The optimised regimen which includes again prephase with CYCLO is now used as induction therapy of the ongoing GMALL study 07/2003. Interim results confirmed the high antileukemic activity with CR rates of 89% and the feasibility with 4% ED. Beside the optimised induction the use of Imatinib in Ph+ ALL parallel to induction contributed to the improvement. Further progress is now attempted by additional use of rituximab in CD20 positive pts.