I. A. MicroRNA profiling of the Bone Marrow Microenvironment, Serum and Plasma of Myeloma and Precursor Diseases. During 2015 our manuscript reporting the significant findings of this study was submitted and accepted for publication in the Journal of Molecular Diagnostics, and is in press, scheduled to be released in the November 2015 (Wang W et.al. Aberrant Level of miRNAs in Bone Marrow Microenvironment and Peripheral Blood of Myeloma Patients and Disease Progression). In brief, our findings showed that the extracellular bone marrow (BM) microenvironment in myeloma contains a unique miRNA signature (involving let-7a, let-7b, let-7i; miR-15a/b, miR-16, and miR-20a) which is partially represented in the peripheral blood. A subset of these miRs showed aberrant expression in the precursor disease MGUS, while aberrant expression of the remaining miRs were associated with disease progression and myeloma. The findings suggest that miR profiles in the serum/plasma may correlate with disease state. I. B. Monoclonal Gammopathy of Undetermined Significance (MGUS) and MicroRNAs in Ranch Hand Veterans. In 2014 we began microRNA isolation from de-identified Air Force Heath Study (AFHS) serum samples received via our collaborators at the CDC/ATSD (Dr. Youn Shim and Dr. Robert Vogt). These samples were previously obtained at multiple timepoints, spanning three decades, from Air Force Ranch Hand Veterans exposed to TCDD (Agent Orange). In parallel, we assisted Dr. Ola Landgren's lab, in the processing of these samples for SPEP, IFE and serum light chain ratios in order to confirm diagnoses, correlate laboratory values with miRNA expression levels, and for additional statistical analyses. In 2015 we completed quantitative PCR on the RNA isolated from the serum samples, assaying a panel of 10-15 miRs associated with MM, MGUS, SMM and/or TCDD exposure. The miRNA data is undergoing analysis by our collaborators at the CDC for correlation with M-protein levels over time, disease status, TCDD exposure, clinical outcome, demographic, and other available data. In 2015, the initial findings related to TCDD exposure and serum protein levels diagnostic of MGUS and myeloma were published in JAMA Oncology (Landgren O et al. Agent Orange Exposure and Monoclonal Gammopathy of Undetermined Significance: An Operation Ranch Hand Veteran Cohort Study). I.C. MicroRNA profiling of GATA2 deficiency associated myelodysplasia (MDS) / acute myeloid leukemia (AML). Somatic and inherited germline mutations in GATA2 have been identified in patients diagnosed with monocytopenia, B-cell and NK-cell lymphopenia, susceptibility to opportunistic infections (e.g. disseminated MAC), and a strong propensity to develop hypocellular MDS/AML or CMML. We previously identified significantly increased levels of miR-181c in EBV transformed cell lines from patients with GATA2 deficiency. CHIP-seq data demonstrates GATA2 binding in the putative promoter region of the miR-181c gene suggesting that GATA2 regulates miR-181c expression. mRNA targets of miR-181c include MCL-1 transcripts involved in the regulation of apoptosis. We found significantly decreased levels of MCL-1 in cell lines derived from patients with GATA2 deficiency and MDS. Interestingly, deletion of Mcl-1 is known to cause apoptosis and loss of hematopoietic stem cells in murine studies. During 2015 we continued functional studies that strongly suggest the down-regulation of MCL-1 in GATA2 deficiency occurs through a mechanism involving aberrant expression miR-181c. These findings suggest a potential mechanism for the progressive cytopenias in GATA2 deficiency involving down regulation of MCL-1 in stem cells, permitting apoptosis and the depletion of hematopoietic progenitors. A manuscript with these findings is being drafted. I.D. MicroRNA changes in chronic lymphocytic leukemia in response to treatment. In 2014 we began a collaboration with Dr. Adrian Wiestner in NHLBI to analyze microRNA levels in lymph node and peripheral blood derived CLL cells from patients pre and post treatment with BTK inhibitors. The goals were to compare miR patterns of CLL cells in the lymph node and peripheral blood compartments with respect to miRs involved in B-cell signaling, neoplasia and activation; and to measure miR expression patterns in response to treatment with targeted inhibitors. In 2015 we found 1) unique miR profiles in CLL cells in the LN related to B-cell activation that become down regulated upon release into the peripheral blood; 2) a subset of 4 miRNA related to BCR activation become significantly down regulated in circulating CLL cells after treatment with ibrutinib, 3) multiple targets of the 4 miRNA include tumor suppressor transcripts that are significantly upregulated after ibrutinib treatment. These findings suggest that response to Ibrutinib involves upregulation of tumor suppressors via downregulation of miRNAs, possibly representing a novel mechanism of treatment response. A manuscript with these findings is being drafted. II. A. Immunophenotypic and morphologic features distinguishing GATA2 deficiency associated bone marrow failure from idiopathic aplastic anemia and hypocellular MDS. Germline GATA2 gene mutations, leading to haploinsufficiency have been identified in patients with familial myelodysplastic syndrome/ acute myeloid leukemia (MDS/AML), monocytopenia and mycobacterial infections (MonoMAC), Emberger syndrome, and dendritic cell, monocyte, B and NK-cell deficiency (DCML). GATA2 deficiency-associated bone marrow failure can present with features that overlap with idiopathic aplastic anemia and hypocellular MDS. Distinguishing GATA2 patients from those with AA or hypocellular MDS is critical for selecting appropriate therapy. In 2015 we published the results of the first phase of this project in Blood (Ganapathi, K et al Blood 2015). In brief, we compared the BM flow cytometric, morphologic and cytogenetic features of 28 GATA2 patients to those of 32 patients being evaluated for idiopathic AA. We identified a unique bone marrow flow cytometry profile that was capable of distinguishing bone marrow failure in the setting of GATA2 deficiency from idiopathic aplastic anemia. These findings show that routine flow cytometric analysis of bone marrow can identify patients for whom GATA2 sequencing is indicated. The next phase of this project is ongoing and involves characterizing the flow cytometry immunophenotypic profiles of de novo hypocellular MDS in comparison to GATA2 deficiency to determine whether flow cytometry is useful in distinguishing these two entities. II.B. Immunophenotypic and morphologic analyses of other rare diseases with bone marrow and peripheral blood pathology. Multiple collaborative research studies continued in 2015 involving immunophenotypic and morphologic characterization of PI3Kinase deficiency, WHIM syndrome, CANDLE syndrome, HIV, RAS-associated autoimmune leukoproliferative disorder (RALD), aplastic anemia, hypocellular MDS and AML. The unique immunophenotypic findings related to RALD were published in Blood in 2015 (Calvo et al. Blood 2015) and presented at the International JMML symposium at the Annual Society of Hematology meeting in December 2014.

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Salem, Dalia Abdel-Raouf; Korde, Neha; Venzon, David J et al. (2018) Expression of the IL-6 receptor alpha-chain (CD126) in normal and abnormal plasma cells in monoclonal gammopathy of undetermined significance and smoldering myeloma. Leuk Lymphoma 59:178-186
Bogaert, Delfien J; Kuehn, Hye Sun; Bonroy, Carolien et al. (2018) A novel IKAROS haploinsufficiency kindred with unexpectedly late and variable B-cell maturation defects. J Allergy Clin Immunol 141:432-435.e7
Giannelou, Angeliki; Wang, Hongying; Zhou, Qing et al. (2018) Aberrant tRNA processing causes an autoinflammatory syndrome responsive to TNF inhibitors. Ann Rheum Dis 77:612-619
Ahn, Inhye E; Farooqui, Mohammed Z H; Tian, Xin et al. (2018) Depth and durability of response to ibrutinib in CLL: 5-year follow-up of a phase 2 study. Blood 131:2357-2366
Sanchez, Gina A Montealegre; Reinhardt, Adam; Ramsey, Suzanne et al. (2018) JAK1/2 inhibition with baricitinib in the treatment of autoinflammatory interferonopathies. J Clin Invest 128:3041-3052
Dulau-Florea, Alina E; Young, Neal S; Maric, Irina et al. (2018) Bone Marrow as a Source of Cells for Paroxysmal Nocturnal Hemoglobinuria Detection. Am J Clin Pathol :
McReynolds, Lisa J; Calvo, Katherine R; Holland, Steven M (2018) Germline GATA2 Mutation and Bone Marrow Failure. Hematol Oncol Clin North Am 32:713-728
Parta, Mark; Shah, Nirali N; Baird, Kristin et al. (2018) Allogeneic Hematopoietic Stem Cell Transplantation for GATA2 Deficiency Using a Busulfan-Based Regimen. Biol Blood Marrow Transplant 24:1250-1259
Boutboul, David; Kuehn, Hye Sun; Van de Wyngaert, ZoƩ et al. (2018) Dominant-negative IKZF1 mutations cause a T, B, and myeloid cell combined immunodeficiency. J Clin Invest 128:3071-3087
Kim, Miriam Y; Yu, Kyung-Rok; Kenderian, Saad S et al. (2018) Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia. Cell 173:1439-1453.e19

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