ALBERT

Description: Here we report that T-cell lymphomas characterized by the expression of anaplastic lymphoma kinase (ALK+ TCL) fail to express the TNFα and frequently display DNA methylation of the TNFα gene promoter. While only a subset of the ALK+ TCL-derived cell lines showed a high degree of the promoter methylation, all 6 showed low to nondetectable expression of the TNFα mRNA, and none expressed the TNFα protein. All 14 ALK+ TCL tissue samples examined displayed some degree of the TNFα promoter methylation, which was the most prominent in the distal portion of the the promoter. Treatment with a DNA methyltransferase inhibitor, 5′-aza-2′-deoxy-cytidine (5-ADC), reversed the promoter methylation and led to the expression of TNFα mRNA and protein. Furthermore, in vitro DNA methylation of the promoter impaired its transcriptional activity in the luciferase reporter assay. This impairment was seen even if only either distal or proximal portion were methylated, with methylation of the former exerting a more profound inhibitory effect. Notably, the ALK+ TCL cell lines uniformly expressed the type 1 TNFα receptor (TNF-R1) protein known to transduce the TNFα-induced pro-apoptotic signals. Moreover, exogeneous TNFα inhibited growth of the ALK+ TCL cell lines in a dose-dependent manner and induced activation of the members of the cell apoptotic pathway: Caspase 8 and caspase 3. These findings provide additional rationale for the therapeutic inhibition of DNA methyltransferases in ALK+ TCL. They also suggest that treatment with TNFα may be highly effective in this type of lymphoma.

Description: Bone marrow failure syndromes (BMFS) are a diverse group of rare life-threatening blood disorders characterized by inadequate hematopoiesis, clonal evolution, and increased risk of hematologic malignancies. Despite recent advances in the understanding of the molecular pathogenesis of BMFS, the ability to diagnose, risk-stratify, and treat patients with these rare disorders remains limited. In both the acquired and the inherited BMFS, the major contributors to mortality are complications of progressive cytopenias, and, albeit to a lesser extent—transformation to myelodysplastic syndrome (MDS) and acute myeloid leukemia. The main predictor of malignant transformation is acquisition of clonal cytogenetic abnormalities. Recently, single nucleotide polymorphism arrays (SNP-A) were proposed as a promising tool for high resolution cytogenetic analysis and surveillance of early clonal changes in BMFS, however, their clinical utility still remains to be established. In 2009, the Comprehensive Bone Marrow Failure Center at the Children’s Hospital of Philadelphia and the Hospital of the University of Pennsylvania incorporated high-density SNP-A as an adjunct to conventional cytogenetics in evaluation of BMFS patients. Here we present a comprehensive analysis of genetic changes in BMFS using 124 SNP-A from 91 patients, who were referred for evaluation of bone marrow failure. SNP-A genotyping was correlated with medical histories, hematopathology, cytogenetic, and molecular data. To assess the potential role of SNP-A in screening for early clonal evolution, longitudinal analysis of SNP-A was performed in 25 patients. We found that acquired copy number-neutral loss of heterozygosity (CN-LOH) was significantly more frequent in acquired aplastic anemia (aAA) than in other BMFS (OR 12.240, 95% CI 1.333-573.696, p

Description: Immune Thrombocytopenia (ITP) usually presents with isolated, severe thrombocytopenia with very low platelet count (generally less than

Description: Introduction: Castleman disease (CD) is an uncommon lymphoproliferative disorder of unclear etiology. CD is broadly subclassified into unicentric (UCD) and multicentric (MCD) disease based on based on clinical, radiological, laboratory and histomorphological findings. UCD presents with minimal symptoms, is restricted to a single lymph node station, and excision is usually curative. Multicentric CD, in contrast, presents with systemic inflammatory symptoms, multicentric lymphadenopathy, and vital organ dysfunction. MCD is further subclassified into Human Herpesvirus-8(HHV-8)-associated MCD and HHV-8-negative/idiopathic MCD (iMCD), the etiology of which is poorly understood. Histologically, UCD frequently shows a hyaline vascular pattern with atretic follicles, thickened mantle zones and increased interfollicular vascularity with hyalinization. iMCD frequently shows increased interfollicular plasma cells and hyperplastic follicles. iMCD is also characterized by a systemic hypercytokinemia that includes IL-6, VEGF, IL-2, TNF-α, IL-10 and CXCL13. However, the cell(s) involved in initiating and amplifying the cytokine network are unclear. We combined histomorphology and cytokine in situ hybridization to identify the hypercytokine-producing cells in lymph nodes from CD patients. In addition, T and B lymphocytes play important roles in initiating and amplifying the cytokine response and their clonality has not been well-studied in CD. Here, we performed deep sequencing of the immunoglobulin heavy chain and T cell receptor gene loci. Methods: Lymph node biopsies from patients with UCD and iMCD were identified from the pathology archives of the Children's Hospital of Philadelphia. 17 UCD cases and 8 HHV8-negative iMCD cases were examined. Reactive lymph nodes (N=10) with plasmacytosis and/or other CD-like features served as controls. Cytokine expression was determined by RNA in situ hybridization (RNAscope) on formalin fixed paraffin embedded tissue. IL-6, IL-6R, VEGF, IL-10, TNF-α, IL-1β, IL-2, and IL-8 RNA expression patterns were analyzed in conjunction with histomorphological features. Expression was manually quantified with a semi-quantitative grading scale (0-4) per manufacturer recommendations and statistical analysis was performed using the Chi-square test. Fresh frozen lymph node tissue was utilized for deep sequencing of the TCR Vβ and IgH gene loci. VDJ usage, clonal frequency and CDR3 sequence was determined and compared between subtypes of CD and reactive lymph node controls. Results: Lymph nodes from patients with iMCD express significantly higher levels of VEGF compared to patients with UCD and controls (75% vs. 29% vs. 0%; p=0.014). Atretic follicles and interfollicular regions were the source of increased VEGF expression. Potential cell types responsible for the increased VEGF production in these regions are follicular dendritic cells in the atretic follicles and plasma cells in the interfollicular areas. IL-6 expression was also significantly higher in iMCD cases compared to UCD and controls (75% vs. 25% vs. 20%, p=0.026) in a subset of cells within the interfollicular regions. This cellular source of the excess IL-6 in the interfollicular region may be endothelial cells. Thus, follicular dendritic cells in the germinal centers and endothelial cells, T cells, and plasma cells in the interfollicular spaces are potential sources for increased VEGF and IL-6. IL-6R, IL-10, TNF-α, IL-1β, IL-2, and IL-8 showed no significant differences between the various subtypes of CD. Deep sequencing of the TCRα gene loci revealed mildly expanded clonal T-cell populations (5% of total sequences) in a subset of iMCD cases (2/6) and UCD cases (1/9) compared to controls (0/15). B cell populations were polyclonal in both subtypes of CD and in reactive lymph nodes. Conclusion: The findings suggest that cells in the interfollicular region and atretic follicles in the lymph nodes are a potential source of the systemic hypercytokinemia in iMCD. The locations and patterns of cytokine expression implicate follicular dendritic cells, endothelial cells, and plasma cells specifically as potential hypercytokine-producing cells. Additionally, T cells in CD show oligoclonality in some cases and may play an important role in initiating or amplifying the immune response in CD. Disclosures Fajgenbaum: Janssen Pharmaceuticals, Inc.: Research Funding.

Description: Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare hematological malignancy of early childhood with characteristics of both myeloproliferative neoplasms and myelodysplastic syndromes. JMML shares pathological features and diagnostic criteria with chronic myelomonocytic leukemia (CMML), a malignancy predominantly affecting the elderly. While 85% of patients with JMML have somatic or germline mutations in RAS pathway genes (NF1, NRAS, KRAS, PTPN11, and CBL), the most frequently mutated genes in CMML include TET2, SRSF2, ASXL1, and RAS and are generally somatic-only. The extent to which histone modification genes (ASXL1, EZH2) or spliceosome machinery genes (SF3B1, SRSF2, U2AF1, ZRSR2) play a role in JMML pathogenesis is unclear. Despite mutational differences, both JMML and CMML manifest as myelomonocytic proliferation with varying amounts of dysplasia in the bone marrow. Clusters of clonally-related CD123+ plasmacytoid dendritic cells (PDCs) have been observed in the bone marrow of patients with CMML but have not been investigated in JMML. Here, we report the mutation profiles and immunophenotypic characteristics of JMML specimens from children treated at our institution. Methods: The pathology archives (1987-2017) at the Children's Hospital of Philadelphia (CHOP) were searched to identify JMML cases (n=21) and included formalin fixed paraffin-embedded diagnostic bone marrow biopsies and splenectomy tissue obtained prior to hematopoietic stem cell transplant. JMML diagnosis was confirmed in all cases by clinicopathological review. Cytogenetic analysis and whole genome SNP array were performed at initial clinical presentation. Genomic DNA and RNA were extracted from JMML patients' bone marrow (n=8) and spleen tissue (n=10) for next-generation sequencing analysis of 118 cancer genes for sequence and copy number variants and 110 genes for known and novel fusions via our custom CHOP Hematologic Cancer Panel. CD123 immunohistochemical (IHC) staining was performed on bone marrow and spleen tissues from children with JMML. Presence of CD123+ PDC clusters was evaluated manually and by digital image analysis. CD123 staining was enumerated using the Aperio Image Scope quantitation of membranous staining v9 with the analysis parameters set such that normal endothelial staining was quantified as 1+, and true CD123 staining cells were quantified as 2+ or 3+. The percentage of CD123+ cells (out of total cellularity) was calculated. Bone marrow from patients with non-JMML myeloid malignancies (n=6) and splenectomy tissue from patients with sickle cell anemia (n=8) were used as controls for the CD123 IHC analysis. Results: We confirmed canonical JMML-associated somatic or germline NF1 (n=3), NRAS (n=4), KRAS (n=2), PTPN11 (n=6), or CBL (n=2) mutations in 16 of the 17 (94%) patients with sequencing data. Interestingly, both PTPN11A72T and NF1R2637* mutations were detected in one patient. In addition, we found potential variants in genes affecting histone modifications (ASXL1, DNMT3A, KDM6A, SETD2), spliceosomal processes (SF3B1, U2AF1), transcription (BCOR, RUNX1, ETV6), or cellular growth (SETBP1, BRAF) in 8/17 patients (47%). While mutations in these genes have been well-characterized in other myeloid disorders, many of these alterations have not been reported to date in children with JMML or are currently of unclear biologic and prognostic significance. We also observed increased clustering of CD123+ PDCs in bone marrow and spleens from patients with JMML compared to IHC staining of control tissues. 2.2 ± 0.42% and 1.8 ± 0.74% of cells expressed CD123 in the spleen and bone marrow specimens, respectively. Control bone marrow and spleen samples did not show significant CD123+ staining. Conclusions: Our study demonstrates frequent variants in histone modification, splicing, and transcription-associated genes in JMML specimens in addition to known pathogenic RAS pathway mutations. We further report histopathologic CD123+ PDC clustering in JMML specimens analogous to that observed in CMML, which may aid in the workup of this often difficult-to-diagnose disease. Our findings of genetic and immunophenotypic overlap between JMML and CMML suggest similarities in pathogenesis despite typical presentation at extremes of age. Disclosures Tasian: Aleta Biopharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding; Incyte Corporation: Research Funding.

Description: Abstract 3445 Associations of chemotherapeutic TOP2 poisons with secondary leukemia have implicated TOP2-mediated DNA damage in balanced chromosomal translocations underlying many common forms of leukemia. “TOP2 poisons” convert native TOP2 into a cellular toxin by disrupting the cleavage/re-ligation equilibrium, either by decreasing the reverse rate of re-ligation or increasing the forward rate of cleavage, thus increasing cleavage complexes and causing DNA strand breaks that can promote recombination or initiate apoptosis. Besides anticancer chemotherapy, several dietary substances and the benzene metabolite p-benzoquinone are TOP2 poisons. Population and molecular epidemiology, translocation breakpoint junction sequences, temporal origins of translocations, and correlations of TOP2 in vitro cleavage sites with translocation breakpoints have pointed to a model in which TOP2 is the DNA damage mediator and resolution of TOP2 cleavage complexes, whether induced by chemotherapy, dietary substances, environmental toxins or ROS mediated damage, forms translocation breakpoint junctions. Still, the DNA damage mechanism(s) remain controversial. Investigation of cleavage complexes at the DNA sequence level in a human hematopoietic progenitor cell model that approximates target cells for translocations is the critical next step in testing this model. We invented a high-throughput sequencing-based method (Provisional Patent Filed) to address cause-and-effect relationships between TOP2 cleavage complexes and translocation breakpoints in the context of DNA topological structure in the chromatin of human hematopoietic cells. TOP2 relaxes supercoiled DNA by transiently cleaving and re-ligating both strands of the double helix. Each subunit of the TOP2 homodimer forms a phosphodiester bond with the base 3' to the cleavage. This creates a fleeting covalent TOP2-DNA intermediate called the cleavage complex with 4-base staggered DNA ends tethered by the enzyme. We implemented, refined, optimized and validated a novel assay system for immunodepletion of the alpha isoform of TOP2 (TOP2A) including DNA-bound TOP2A, with concomitant isolation and purification of the DNA from cleavage complexes in human hematopoietic cells. By taking advantage of the covalent phosphodiester bonds between TOP2A and DNA, the activity of calf intestinal phosphatase (CIP) (i.e. hydrolysis of phosphodiester bonds via removal of 5' phosphates) was used for a purpose never used before: to release DNA from cleavage complexes at exact sites of cleavage. These steps were performed in CEM cells and, to better mimic target cells for translocations, fresh cord blood mononuclear cells (MNCs) from three newborn infants. Western blot and Q-PCR analyses proved that we achieved: 1) isolation and immunodepletion of TOP2A and TOP2A-bound DNA, 2) CIP release of TOP2A-bound DNA from the cleavage complexes, and 3) quantitative enrichment of DNA amplicons near known MLL translocation breakpoint hotspots over that obtained using a negative control antibody for immunodepletion. By morphology and immunophenotype, the cord blood MNCs contained lymphocyte, monocyte, and minor CMP and GMP populations, and they mainly were in G0/G1 by cell cycle analysis. Even though TOP2A cleavage complex enrichment was evident, on Western blot analysis the TOP2A in the cord blood MNCs appeared predominantly degraded, which is consistent with known TOP2A cell cycle dependence and downregulation in quiescent cells. This validation forms the basis for the next steps in the assay to localize cleavage complexes at single base resolution genome-wide through high-throughput sequencing of DNA ends created by TOP2 and mapping them to the genome. This strategy comprises an entirely novel application of high-throughput sequencing with many possible future uses to define TOP2 cleavage complexes, as well as other adaptations to identify covalent DNA modifications with exact base precision. Secondary leukemias are a growing problem, and the incidence of infant leukemia where TOP2 poisons also have been implicated is increasing. Solving how DNA breaks arise and form chromosomal translocations would have profound implications for anticancer treatment and leukemia prevention. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points An international panel established the first ever diagnostic criteria for iMCD based on review of 244 clinical cases and 88 tissue samples. The criteria require multicentric lymphadenopathy with defined histopathology, ≥2 clinical/laboratory changes, and exclusion of iMCD mimics.