ALBERT

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: MicroRNA (miR)-mediated gene regulation plays critical roles in B-cell development and dysregulated expression of miRs has been implicated in the pathogenesis of various types of B-cell malignancies. Somatic deletions of chromosome 13q14, harboring miR-15a/16-1, occurs frequently in B-cell lymphomas suggesting that members of this miR family are tumor suppressors. Consistently, mice with CD19-Cre-induced deletion of miR-15a/16-1 in early B-cells and follicular B-cells develop chronic lymphocytic leukemia (CLL). Since the 13q14 deletion is observed in a broader range of B-cell malignancies, we hypothesized that the type of B-cell malignancy resulting from miR-15a/16-1 down-regulation may depend on the stage of B-cell development at which this deletion occurs. Therefore, we generated a transgenic mouse model in which conditional deletion of miR-15a/16-1 takes place at later stages of B-cell development. To delete miR-15a/16-1 in activated B-cells, miR-15a/16-1fl/fl mice were mated with AID-Cre+/+ mice to obtain AID-Cre+/-; miR-15a/16-1fl/fl compound mice that expressed Cre recombinase from the Activation-induced Cytidine Deaminase (AID promoter) gene - a gene needed for generation of somatic hypermutations in the immunoglobulin (Ig) variable region (V) genes that is highly expressed in activated B-cells and is a well-known marker for germinal center (GC) B-cells. Expression levels of both miR-15a and miR-16-1, but not miR-15b were decreased in GC B-cells of AID-Cre+/-; miR-15a/16-1fl/fl mice as compared with control AID-Cre+/- mice when evaluated by In Situ Hybridization (ISH) analysis. Given that in humans miR-15a, b and 16 are also expressed in GC B-cells, these results demonstrate the validity of this mouse model in which the biological consequences of miR-15a/16-1 deletion can be studied. Next we assessed whether miR-15a/16-1 deletion could affect proliferation and/or survival of GC B-cells. GCs in the spleens of AID-Cre+/-; miR-15a/16-1fl/fl mice at 10 weeks of age were significantly increased in both number and size, and contained a larger number of Ki-67-positive B-cells as compared with spleens of AID-Cre+/- mice. No significant differences in the number of apoptotic cells, neither in the expression of the miR-15a/16-1 putative target BCL2 were detected, indicating that miR-15a/16-1 may play important roles in the proliferation, but not survival of GC B-cells. Apart from mild splenic enlargement and increased number and size of GCs, AID-Cre+/-, miR-15a/16-1fl/fl mice where indistinguishable from AID-Cre+/- mice between 8 and 40 weeks of age as assessed by weight and posture. However, after 48 weeks of age and at variable times thereafter, 80% (32/40) of AID-Cre+/-, miR-15a/16-1fl/fl mice but none from control cohorts (0/30) showed signs of disease. Gross pathologic examination of euthanized AID-Cre+/-; miR-15a/16-1fl/fl mice revealed enlargement of the spleen and lymph nodes. Detailed histological examination revealed in most instances an effacement of normal tissue architecture by a nodular or diffuse population of atypical lymphoid cells, or less commonly by sheets of plasma cells in interfollicular areas. Two distinct patterns of B220+BCL6+BCL2- B-cell lymphomas were identified after detailed analysis. The most common (47%) resembled human follicular lymphoma (FL) and the next in frequency (28%) resembled human diffuse large B-cell lymphoma (DLBCL). The other group of tumors (25%) resembled human plasmacytoma (PC). All three tumor subtypes were clonal, hypermutated and associated with different degrees of preservation of the dendritic meshwork in the lymph nodes. The comparison of lymphomas arising in AID-Cre+/-; miR-15a/16-1fl/fl mice and CD19-Cre+/-; miR-15a/16-1fl/fl mice corroborated that deletion of miR-15a/16-1 at different stages of B-cell development leads to distinct subtypes of B-cell malignancies. Finally, we investigated miR-15a/16-1 expression in human FL and PC and showed that miR-15a/16-1 abundance is significantly decreased in those malignancies when compared with nodal B-cells in reactive GCs and normal plasma cells in interfollicular areas respectively, suggesting that miR-15a/16-1 may play important roles in normal GC B-cell development as well as in the pathogenesis of FL and PC in humans. Disclosures Ghobrial: BMS: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Noxxon: Honoraria; Amgen: Honoraria. Anderson:Oncoprep: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Millennuim: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Equity Ownership; C4 Therapeutics: Equity Ownership; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees.

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: Chromosome 13q deletion (del(13q)), harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B-cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without the del(13q), suggesting an important tumor suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B-cells induces moderate but widespread molecular and functional changes including increased number of GC B-cells, percentage of dark zone B-cells, and maturation into plasma cells. With time this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM which has progressed to extramedullary disease. We corroborate that human primary EP have low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor suppression function in plasma cell and B-cell malignancies.