microRNA, immunophenotypic, and somatic alterations in hematologic malignancies

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Abstract

After the completion of our major miRNA GATA2 project in 2020-2021, which was published in the Journal of Leukocyte Biology (PMID 34270823), we shifted focus on the second objective studying the somatic mutations, disease progression, and bone marrow pathology in GATA2 Deficiency and RUNX1 Familial Platelet Disorder. Project activities are listed below. 1. Somatic mutation analysis of myeloid neoplasms and marrow failure in GATA2 deficiency. GATA2 deficiency patients harbor de novo or inherited germline mutations in the GATA2 transcription factor gene, predisposing them to myeloid malignancies. There is considerable variation in disease progression, even among family members with the same mutation in GATA2. In my laboratory we performed NGS somatic myeloid mutation analysis of the bone marrow of 106 patients with GATA2 deficiency over several years to identify acquired mutations that are associated with disease progression to myeloid malignancy. The peripheral blood of the same patients underwent whole exome sequencing in Dr. Dennis Hicksteins lab in NCI. We combined the NGS data sets to characterize the molecular landscape of MDS, acute myeloid leukemia (AML), chronic myelomonocytic leukemia (CMML), and marrow failure in GATA2 deficiency. Myelodysplastic Syndrome (MDS) was the most common diagnosis (44%), followed by GATA2 bone marrow immunodeficiency disorder (G2BMID) (37%) which had features of marrow failure and did not meet criteria for MDS. Thirteen percent of the cohort had GATA2 mutations but displayed no disease manifestations. There were no correlations between patient age or sex with disease progression or survival. Cytogenetic analyses showed a high incidence of abnormalities (43%)- notably trisomy 8 (23%) and monosomy 7 (12%), but these changes did not correlate with lower survival. Somatic mutations in ASXL1 and STAG2 were detected in 25% of patients, though these mutations were rarely concomitant. Mutations in DNMT3A were found in 10% of patients. These somatic mutations were found similarly in G2BMID and MDS, suggesting clonal hematopoiesis in early stages of disease and that G2BMID may be an early manifestation of MDS. ASXL1 mutations conferred a lower survival probability and were more prevalent in female patients. STAG2 mutations also conferred a lower survival probability, but did not show a statistically significant sex bias. There was a conspicuous absence of many commonly mutated genes associated with myeloid malignancies, including TET2, IDH1/2, and the splicing factor genes. Notably, somatic mutations in chromatin-related genes and cohesin genes characterized disease progression in GATA2 Deficiency. These findings were published in Blood Advances in February 2022 (PMID 34529785). 2. Spectrum of pathologic findings and clonal evolution in RUNX1 familial platelet disorder with associated myeloid malignancy. The RUNX1 Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM) is a rare autosomal dominant cancer predisposition syndrome. The lifetime risk of developing hematopoietic malignancies (HM) is 3040%, often MDS/AML. Understanding the baseline spectrum of bone marrow morphologic and genetic findings as well as temporal disease progression is critical to ensure diagnostic accuracy and develop criteria to recognize the onset of HM. We analyzed the clinicopathologic and genetic findings of 38 patients; 26 adult and 12 pediatric. Of these, 31 patients did not develop HM over a 24-month follow-up period; 7 patients developed or had a history of HM including 1 AML; 1 CMML, 2 MDS, 1 T-ALL, and 2 plasma cell neoplasm. Development of HM was associated with acquired genetic alterations, additional cytopenia, 2 or more dysplastic lineages, and/or increased blasts. Peripheral blood abnormalities including thrombocytopenia (78%), abnormal MPV (39%), anemia (22%), and eosinophilia (25%) were present at initial evaluation. All 38 patients showed baseline bone marrow abnormalities: low-for-age cellularity (49%), high-for-age cellularity (8%), megakaryocytic atypia (89%) ranging from mild to moderate with features of dysmegakaryopoiesis, dysplastic erythroid and myeloid lineages (8%), eosinophilia (39%), low to absent B-cell precursors (14%), and monoclonal B-cell lymphocytosis (2%). Twelve different germline RUNX1 mutations were identified across the gene, most commonly in the RUNT homology domain. In 76% of the patients, RUNX1 mutation was the only genetic finding. Molecular analysis identified clonality in 24% of patients. Mutations in BCOR, NRAS, and TET2 were identified in patients without progression to HM. Patients with HM harbored mutations in genes affecting signaling (JAK2, NRAS), tumor suppressors (PHF6), cellular metabolism (IDH1), chromatin-spliceosome machinery (SF3B1), and epigenetic regulators (BCOR, EZH2). Of the clinically stable patients with isolated thrombocytopenia, normal karyotype, and no acquired clonal genetic alterations, all but one had megakaryocytic atypia. Given the frequency of baseline megakaryocytic atypia in RUNX1 FPD which could be misinterpreted as MDS with single lineage dysplasia, we propose separate MDS diagnostic criteria in RUNX1-FPD requiring unexplained persistent non-platelet cytopenia(s) and multilineage dysplasia, ring sideroblasts, MDS-defining cytogenetic abnormality, and/or increased blasts. This work was presented at the 2022 USCAP Annual Meeting. 3. Pilot study of transcriptomic analysis of dysmegakaryopoiesis in GATA2 deficiency. We performed a pilot study to evaluate the ability to transcriptionally profile abnormal megakaryocytes in bone marrow core biopsies in MDS in GATA2 deficiency in comparison with normal megakaryocytes in the marrow of healthy individuals. This was done using digital spatial profiling technology to assay the expression of over 18,000 genes in megakaryocytes selected by morphology and expression of CD61. The pilot study was successful demonstrating significant differential expression of over 100 genes in dysplastic megakaryocytes vs. normal megakaryocytes. Subsequent studies are planned in 2022-2023. 4. Bone marrow failure, immunodeficiency, and hematologic malignancies. We continued collaborative research support of multiple clinical and translational research protocols in areas of bone marrow failure, immunodeficiency, hematologic malignancies and other rare diseases resulting in multiple collaborative publications.