microRNA, immunophenotypic, and somatic alterations in hematologic malignancies

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Abstract

1. Methylation study on bone marrow of GATA2 deficiency vs. healthy control marrow. In order to identify the potential role of epigenetics in the bone marrow pathology of GATA2 deficiency and the development of myelodysplastic syndrome/acute myeloid leukemia (MDS/AML) we performed methylation studies. DNA methylation microarray (Illumina EPICv2) experiments were performed with genomic DNA from bone marrow mononuclear cells of 15 GATA2 deficiency patients and 8 healthy controls (HC). Data analysis was conducted in RStudio using DMRcate, Limma, minfi, and other Bioconductor packages. There were significant differences in the methylation signatures in the GATA2 group versus the HC group. Differential methylation was found in genes related to DNA damage repair (TP53, RAD50, RAD23B, ATM, and DDB1), methylation (DNMT3A, DNMT1, and MTRR), and apoptosis (CASP9, BCL2L14, BCL2L13, and BCL2L11). Similarly, genes involved in hematopoiesis including RUNX1 and ETV6 as well as those involved in myeloid malignancy (FLT3 and IDH2) were significantly differentially methylated. Pathway analysis via Gene Ontology revealed a substantial change in methylation between the two groups in pathways involved in leukocyte migration (adjusted p-value = 0.00075), lymphocyte differentiation (adjusted p-value = 0.0081), chemokine production (adjusted p-value = 0.00075), and T-cell differentiation (adjusted p-value = 0.0073). This work is on going. RNAseq studies are planned to evaluate expression of genes differentially methylated in the methylation microarray studies . This work was done in collaboration with the NCI Laboratory of Pathology Methylation Laboratory, Dr. Steven Holland, and Dr. Dennis Hickstein. 2. Myelodysplastic Syndrome (MDS) in VEXAS: Comprehensive bone marrow evaluation by morphology, cytogenetics and next generation sequencing (NGS), was conducted leading to subclassification of bone marrow features in 50 VEXAS patients with UBA1 mutation. Classification was based on World Health Organization (WHO) and International Consensus Classification (ICC) criteria for MDS vs an independent classification comprising: 4.2% VEXAS without cytopenia (3/71) and no dysplasia (ND), 49.3% with cytopenia (35/71) and ND, 38% with cytopenia and dysplasia (27/71), 7.4% with MDS based on dysplasia and MDS-defining genetics (5/71) and 1.4% with MDS/AML (1/71). Cytogenetic aberrations were only found in VEXAS with morphologic dysplasia or MDS including del(5q), del(13q), del(20q), and t(3;12). In addition to UBA1 mutations, 61.1% cases showed coexisting mutations in DNMT3A, TET2, EZH2, single-hit TP53, ASXL1 and SF3B1. Follow-up bone marrow evaluation typically showed disease progression within 10 months to 8 years (13/15), demonstrating acquired genetic aberrations over time. Platelet count and hemoglobin levels showed a declining tendency as patients progressed from cytopenia without dysplasia to dysplasia/ MDS. Dysplastic features were commonly trilineage. Increased p53 protein staining by immunohistochemistry was positively correlated with bone marrow disease severity in the absence of TP53 mutations. Overall bone marrows in VEXAS demonstrated a high degree of morphologic dysplasia at baseline without cytogenetic or genetic aberrations in myeloid driver genes suggesting that diagnosis of MDS based solely on morphology may be inappropriate and result in over diagnosis of MDS in VEXAS. Specialized diagnostic criteria for MDS in VEXAS are proposed to include presence of supporting molecular/genetic aberrations and/or increased blasts as criteria required for a diagnosis of MDS in VEXAS. This study was done in collaboration with Dr. Peter Grayson, Dr. Neal Young, and their clinical teams. 3. Multiple additional collaborative research studies were supported involving investigation of hematologic malignancies and other bone marrow and blood diseases.