ALBERT

Description: Monocyte-derived dendritic cells (DCs) and macrophages (Mϕs) generated in vitro from the same individual blood donors were exposed to 5 different pathogens, and gene expression profiles were assessed by microarray analysis. Responses to Mycobacterium tuberculosis and to phylogenetically distinct protozoan (Leishmania major, Leishmania donovani, Toxoplasma gondii) and helminth (Brugia malayi) parasites were examined, each of which produces chronic infections in humans yet vary considerably in the nature of the immune responses they trigger. In the absence of microbial stimulation, DCs and Mϕs constitutively expressed approximately 4000 genes, 96% of which were shared between the 2 cell types. In contrast, the genes altered transcriptionally in DCs and Mϕs following pathogen exposure were largely cell specific. Profiling of the gene expression data led to the identification of sets of tightly coregulated genes across all experimental conditions tested. A newly devised literature-based clustering algorithm enabled the identification of functionally and transcriptionally homogenous groups of genes. A comparison of the responses induced by the individual pathogens by means of this strategy revealed major differences in the functionally related gene profiles associated with each infectious agent. Although the intracellular pathogens induced responses clearly distinct from the extracellular B malayi, they each displayed a unique pattern of gene expression that would not necessarily be predicted on the basis of their phylogenetic relationship. The association of characteristic functional clusters with each infectious agent is consistent with the concept that antigen-presenting cells have prewired signaling patterns for use in the response to different pathogens.

Description: Hypereosinophilic syndrome is a rare disorder characterized by hypereosinophilia and eosinophil-mediated tissue injury. An imatinib sensitive myeloproliferative variant (MHES) has been described which has a male predominance, and is associated with elevated serum tryptase levels, tissue fibrosis, increased atypical mast cells, and the presence of the fusion oncogene FIP1L1-PDGFRα which has tyrosine kinase activity. The FIP1L1-PDGFRα mutation has been detected in peripheral blood mononuclear cells, however, the hypercellular bone marrow and elevated serum tryptase levels suggest that multiple lineages might be involved in the clonal process. We analyzed peripheral blood from eight patients with the FIP1L1-PDGFRα mutation. Individual patient samples were sorted by flow cytometry to collect greater than 95% pure populations of CD3, CD14, and CD19 cells. Density gradient centrifugation followed by negative selection for CD16, CD3, CD14, and CD19 using an immunomagnetic bead column was used to purify eosinophils to 〉 99% purity. Bone marrow from one patient was obtained, and mast cells were cultured from CD34 positive cells. Three techniques were used to assay for the presence of the FIPL1-PDGFRα fusion gene: nested RT-PCR, TaqMan quantitative PCR, and FISH. Eosinophils were positive for the fusion gene in all patient samples that were analyzed. Monocytes were also positive in all but one instance. Surprisingly some patients showed positivity in lymphoid lineages as well. The bone marrow derived pure mast cell culture was positive for the mutation, consistent with the elevation of serum tryptase and atypical appearance of mast cells in MHES. In conclusion, although MHES seems to have a multilineage predilection, specific lineages involved may vary between patients. This may reflect differences in the progenitor stage at which the mutation occurs. Whether the pattern of lineage involvement has any relation to the phenotypic expression of the disease remains to be elucidated.

Description: Introduction: Burkitt Lymphoma (BL) is an aggressive B-cell lymphoma with a translocation involving MYC and immunoglobulin(Ig) loci. It is most common in children, but also affects adults, and occurs in sporadic, endemic and HIV-associated forms. The Epstein-Barr virus (EBV)-associated endemic subtype is the most common pediatric cancer in equatorial Africa, but also occurs in other parts of the world, for example in the rain forest of Brazil. Intensive chemotherapy is effective, but the associated toxicity requires supportive care that is not readily available in resource-poor regions. Previously published molecular characterization of small numbers of tumors indicated that the mutation profiles of endemic and sporadic cases are similar, but not identical. One goal of the BLGSP is to conduct comprehensive molecular characterization of BL by sequencing DNA and RNA from a large BL cohort - including endemic, sporadic, pediatric and adult cases - in order to define the genetic and phenotypic features that drive these cancers. These data will be analyzed with an intent toward developing new therapeutic strategies that can be deployed worldwide. Methods: The goal is to collect 160 BL cases, of which 50% will be endemic, 38% sporadic (pediatric and adult) and 12% from HIV+ patients. For the discovery phase, each tumor requires case-matching normal DNA as well as treatment, outcome and other clinical information. The optimal source of tumor DNA and RNA is from frozen tissue with at least 50% tumor nuclei, but FFPE immobilization is also accepted. Accrual locations include Africa, Brazil, Europe and the US. The BLGSP has developed extensive standard operating procedures for tissue collection, pathology review and tissue processing to reduce the variation associated with these parameters in the interpretation of the results (see https://ocg.cancer.gov/programs/cgci/projects/burkitt-lymphoma). The project also established procedures that allow sharing of all clinical and sample information through the National Cancer Institute Genomic Data Commons (https://gdc.cancer.gov). Molecular characterization includes whole genome sequencing of tumor and normal DNA (80X and 40X coverage, respectively), RNA-sequencing (RNA-seq) and micro-RNA sequencing. These data will enable the BLGSP to identify chromosomal rearrangements, chromosomal copy number alternations, somatic mutations (single nucleotide, insertions, deletions), viral insertions, expression signatures, viral expressions and miRNA regulation of transcripts. Results: To date we have accrued 80 cases of BL of which 75% passed diagnostic pathology review. There was an additional 25% attrition at the tissue processing stage, either due to low quality nucleic acids or low percent tumor nuclei. We have completed sequencing for 45 cases, all but one of which have a MYC translocation involving one of the 3 Ig loci; one case has a MYC rearrangement by FISH analysis that is being characterized further. We have identified recurrent mutations in ID3, DDX3X, ARID1A, FOXO1, TP53, SMARCA4 and other genes. Most mutations are supported by the RNA-seq data, which is also useful in defining the pattern of EBV genome transcription. Preliminary unsupervised hierarchical clustering and principal component analysis of gene expression data defined sample clusters that do not correspond to mutation status or EBV infection, warranting further investigation. Some genes accumulated somatic mutations in a BL subtype-specific fashion. Discussion: BLGSP is an ongoing international collaborative project that will provide a comprehensive molecular portrait of BL subtypes when completed, with the potential to suggest new molecular targets for therapy that can eventually lead to effective treatments that are less toxic than the current regimens. Disclosures Casper: Janssen: Consultancy, Research Funding; Roche: Consultancy, Other: Travel, Accommodation, Expenses; TempTime: Consultancy, Other: Travel, Accommodation, Expenses; Up to Date: Patents & Royalties; GSK: Other: Travel, Accommodation, Expenses. Abramson:Kite Pharma: Consultancy; Abbvie: Consultancy; Seattle Genetics: Consultancy; Gilead: Consultancy. Noy:Pharmacyclics, LLC, an AbbVie Company: Other: travel, accommodations, expenses, Research Funding.

Description: Background: Mixed phenotype acute leukemia (MPAL) is a high risk leukemia with features of acute myeloid (AML) and acute lymphoblastic leukemia (ALL), either due to co-expression of antigens of multiple lineages, or the presence of multiple immunophenotypically distinct populations. WHO 2008 classifies MPAL as T/myeloid (T/M), B/myeloid (B/M), MLL rearranged (MLL) MPAL, BCR-ABL1 (Ph+) MPAL, and MPAL not otherwise specified (NOS). Patients are managed with divergent chemotherapeutic approaches with survival estimates of 50-70%. Apart from Ph+ and MLL rearrangement, the genetic basis of MPAL is poorly defined. Our goal was to define the molecular basis of MPAL, and to compare with potentially related forms of leukemia (AML, T-ALL and early T-cell precursor (ETP) ALL) as a rational foundation for future trials. Furthermore, we examined whether multi-lineal cases harbor genetically distinct subclones, or arise from the acquisition of founding alterations in a multi-lineage hematopoietic progenitor. Methods: 155 cases of pediatric leukemia initially diagnosed as MPAL were studied by central pathology review and/or central flow cytometry (134 cases), confirming the diagnosis according to WHO criteria in 115 cases (fig. 1). Median age was 7 years (0-18) with 52 T/M, 37 B/M, 15 MLL, 8 NOS, and 2 Ph+ (fig. 2). Samples were studied by whole genome and/or exome, RNA sequencing, and SNP array analysis. 44 multi-lineal samples were flow sorted into 2-4 lymphoid, myeloid, and ambiguous subpopulations (15 T/M, 19 B/M, 7 MLL, 1 Ph+, 2 NOS) and subjected to exome sequencing and SNP array. Mutational data were compared to data from 196 AML, 39 ETP-ALL, and 245 T-ALL cases. Results: We identified 35 recurrently mutated genes, the most common of which were WT1 (21%), FLT3 (18%), NRAS (16%), JAK3 (11%), RUNX1 (11%), KMT2D (9%), PTPN11 (9%), ASXL1 (7%), and CREBBP (7%). T/M and B/M subtypes are characterized by distinct patterns of genomic alteration. 48% of T/M cases harbored in-frame chimeric fusion, several of which are described in T-ALL, including ETV6-NCOA2 and ZEB2-BCL11B, NUP214-ABL1 and PICALM-MLLT10, and novel fusions involving hematopoietic regulators (e.g. ETV6-MAML and MNX1-IKZF1). 42% of B/M cases had in-frame fusions of ZNF384 with CREBBP, EP300, and TCF3, while we also identified isolated fusions involving ERG and NF1. Mutations of Ras signaling genes were present in 50% of B/M cases, in contrast to 10% of T/M cases. Epigenetic modifying genes, including CREBBP, SETD2, KMT2D, EZH2 and SUZ12 were mutated in 45% of the combined T/M and B/M cohorts. Cases with MLL gene rearrangements had few sequence alterations. In comparison to other subtypes of leukemia, the mutational spectrum of T/M MPAL, with alterations in transcription factors (60% cases), epigenetic genes (50%) and JAK-STAT signaling (35%) was more similar to ETP-ALL (64%, 72%, 44%) and T-ALL (49%, 60%, 21%) than to AML (19%, 21%, 11%). Similarly, B/M cases have increased alterations in these pathways (42%, 42%, 25%) compared to AML. Sequencing of MPAL subpopulations revealed that 27% of cases had the same SNVs/indels in each subpopulation, and 47% of cases had at least two-thirds of mutations present in each subpopulation. All multi-lineal cases with alterations of regulators WT1 and RUNX1 showed similar allele frequencies of these mutations in all populations. Alternatively, cases with mutations in signaling (FLT3, NRAS, KRAS, PTPN11) or epigenetic regulatory genes (CREBBP, KMT2D, SETD2) only showed consistent presence of alterations across each subpopulation in 60% of the cases. Conclusions: Our analysis has shown that T/M and B/M MPAL are distinct subtypes of leukemia. B/M MPAL is characterized by frequent RAS pathway mutations and ZNF384 fusions with multiple different fusion partners, suggesting that this gene plays a critical role in hematopoietic development for progenitor cells with B lymphoid and myeloid potential. The findings of mutational similarity to ETP ALL, and sharing of genomic lesions between subclones in the majority of cases strongly suggests that MPAL represents part of a spectrum of immature leukemias that arise in a hematopoietic progenitors that may propagate multiple immunophenotypic populations. These results will guide the design of therapeutic strategies for each subtype of MPAL and ETP ALL, and xenografts representative of each subtype are being used to examine sensitivity to therapeutic agents. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zwaan:Pfizer: Research Funding; Pfizer: Consultancy. Reinhardt:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation. Inaba:Arog: Research Funding. Mullighan:Loxo Oncology: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau.

Description: Acute leukemia (AL) of ambiguous lineage (AMBI-L) comprises up to 5% of AL cases in both children and adults. Although several definitions exist, a general treatment guideline has been missing. Single country studies usually report fewer than 50 cases of children or adults. Accordingly, the international iBFM AMBI2012 Study/Registry collected 275 AMBI-L cases in patients

Description: Since serum tryptase levels are elevated in some patients with myeloproliferative disorders, we examined their utility in identifying a subset of patients with hypereosinophilic syndrome (HES) and an underlying myeloproliferative disorder. Elevated serum tryptase levels (〉 11.5 ng/mL) were present in 9 of 15 patients with HES and were associated with other markers of myeloproliferation, including elevated B12 levels and splenomegaly. Although bone marrow biopsies in these patients showed increased numbers of CD25+ mast cells and atypical spindle-shaped mast cells, patients with HES and elevated serum tryptase could be distinguished from patients with systemic mastocytosis and eosinophilia by their clinical manifestations, the absence of mast cell aggregates, the lack of a somatic KIT mutation, and the presence of the recently described fusion of the Fip1–like 1 (FIP1L1) gene to the platelet-derived growth factor receptor α gene (PDGFRA). Patients with HES and elevated serum tryptase were more likely to develop fibroproliferative end organ damage, and 3 of 9 died within 5 years of diagnosis in contrast to 0 of 6 patients with normal serum tryptase levels. All 6 patients with HES and elevated tryptase treated with imatinib demonstrated a clinical and hematologic response. In summary, elevated serum tryptase appears to be a sensitive marker of a myeloproliferative variant of HES that is characterized by tissue fibrosis, poor prognosis, and imatinib responsiveness.

Description: Four patients with hypereosinophilic syndrome (HES) refractory to or intolerant of treatment with conventional therapy were treated with a single 1 mg/kg dose of SCH55700. SCH55700 was extremely well tolerated. Two of the 4 patients responded with a fall in eosinophil counts to within the normal range within 48 hours of receiving the drug, accompanied by marked improvement in clinical signs and symptoms. Response was not predicted by serum interleukin-5 (IL-5) levels or presence of the FIP1L1/PDGFRA mutation. Eosinophil counts remained suppressed for up to 12 weeks after treatment; however, exacerbation of symptoms and eosinophilia above baseline levels occurred as drug levels waned. Reinstitution of treatment with monthly SCH55700 led to decreased eosinophilia and symptomatic improvement, albeit to a lesser degree than that seen after the initial dose. These data suggest that anti–IL-5 therapy may be useful in the treatment of HES irrespective of the underlying etiology, although the observed rebound eosinophilia and attenuation of response require further study. (Blood. 2004;103:2939-2941)

Description: Key Points miR-155 promotes myeloproliferation in the bone marrow, spleen, and blood of mice carrying the FLT3-ITD mutation. miR-155 suppresses the IFN response in FLT3-ITD+ mouse hematopoietic stem and progenitor cells, as well as FLT3-ITD+ human AML cells.

Description: Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

Description: Key Points The largest cohort of ambiguous leukemias to date reveals a better prognosis of children who started on lymphoid-directed treatment. Myeloid-type primary treatment correlated with dismal outcomes in CD19+ leukemias.