Genomic events associated with poor outcome in chronic myeloid leukemia (CML) are poorly understood. We performed whole-exome sequencing, copy-number variation, and/or RNA sequencing for 65 patients to discover mutations at diagnosis and blast crisis (BC). Forty-six patients with chronic-phase disease with the extremes of outcome were studied at diagnosis. Cancer gene variants were detected in 15 (56%) of 27 patients with subsequent BC or poor outcome and in 3 (16%) of 19 optimal responders ( = .007). Frequently mutated genes at diagnosis were ,, and The methyltransferase was a novel recurrently mutated gene. A novel class of variant associated with the Philadelphia (Ph) translocation was detected at diagnosis in 11 (24%) of 46 patients comprising fusions and/or rearrangement of genes on the translocated chromosomes, with evidence of fragmentation, inversion, and imperfect sequence reassembly. These were more frequent at diagnosis in patients with poor outcome: 9 (33%) of 27 vs 2 (11%) of 19 optimal responders ( = .07). Thirty-nine patients were tested at BC, and all had cancer gene variants, including kinase domain mutations in 58%. However, mutations cooccurred with other mutated cancer genes in 89% of cases, and these predated mutations in 62% of evaluable patients. Gene fusions not associated with the Ph translocation occurred in 42% of patients at BC and commonly involved fusion partners that were known cancer genes (78%). Genomic analysis revealed numerous relevant variants at diagnosis in patients with poor outcome and all patients at BC. Future refined biomarker testing of specific variants will likely provide prognostic information to facilitate a risk-adapted therapeutic approach.

GATA2 deficiency syndrome (G2DS) is a rare autosomal dominant genetic disease predisposing to a range of symptoms of which myeloid malignancy and immunodeficiency including recurrent infections are most common. In the last decade since it was first reported, there have been over 465 individuals identified carrying a pathogenic or likely pathogenic germline GATA2 variant with symptoms of G2DS, with 231 of these confirmed to be familial and 22 de novo. For those that develop myeloid malignancy (75% of all carriers with G2DS disease symptoms), the median age of onset is 17 years (range 0-78 years) and myelodysplastic syndrome (MDS) is the first diagnosis in 75% of these cases with acute myeloid leukemia (AML) in a further 9%. All variant types appear to predispose to myeloid malignancy and immunodeficiency. Apart from lymphedema in which haploinsufficiency seems necessary, the mutational requirements of the other less common G2DS phenotypes is still unclear. These predominantly loss-of-function variants impact GATA2 expression and function in numerous ways including perturbations to DNA binding, protein structure, protein:protein interactions, and gene transcription, splicing and expression. In this review, we provide the first expert curated ACMG/AMP classification with codes of published variants compatible for use in clinical or diagnostic settings.

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GATA2 deficiency syndrome (G2DS) is a rare autosomal dominant genetic disease predisposing to a range of symptoms, of which myeloid malignancy and immunodeficiency including recurrent infections are most common. In the last decade since it was first reported, there have been over 480 individuals identified carrying a pathogenic or likely pathogenic germline GATA2 variant with symptoms of G2DS, with 240 of these confirmed to be familial and 24 de novo. For those that develop myeloid malignancy (75% of all carriers with G2DS disease symptoms), the median age of onset is 17 years (range 0–78 years) and myelodysplastic syndrome is the first diagnosis in 75% of these cases with acute myeloid leukemia in a further 9%. All variant types appear to predispose to myeloid malignancy and immunodeficiency. Apart from lymphedema in which haploinsufficiency seems necessary, the mutational requirements of the other less common G2DS phenotypes is still unclear. These predominantly loss‐of‐function variants impact GATA2 expression and function in numerous ways including perturbations to DNA binding, protein structure, protein:protein interactions, and gene transcription, splicing, and expression. In this review, we provide the first expert‐curated ACMG/AMP classification with codes of published variants compatible for use in clinical or diagnostic settings.

Key Points Klf5 functions in hematopoiesis to regulate HSC and progenitor proliferation and localization in the bone marrow. Klf5 is required in the granulocyte lineage and positively affects neutrophil output at the expense of eosinophil production.

The apoptotic cysteine protease caspase-2 has been shown to suppress tumourigenesis in mice and its reduced expression correlates with poor prognosis in some human malignancies. Caspase-2-deficient mice develop normally but show ageing-related traits and, when challenged by oncogenic stimuli or certain stress, show enhanced tumour development, often accompanied by extensive aneuploidy. As stem cells are susceptible to acquiring age-related functional defects because of their self-renewal and proliferative capacity, we examined whether loss of caspase-2 promotes such defects with age. Using young and aged Casp2−/− mice, we demonstrate that deficiency of caspase-2 results in enhanced aneuploidy and DNA damage in bone marrow (BM) cells with ageing. Furthermore, we demonstrate for the first time that caspase-2 loss results in significant increase in immunophenotypically defined short-term haematopoietic stem cells (HSCs) and multipotent progenitors fractions in BM with a skewed differentiation towards myeloid progenitors with ageing. Caspase-2 deficiency leads to enhanced granulocyte macrophage and erythroid progenitors in aged mice. Colony-forming assays and long-term culture-initiating assay further recapitulated these results. Our results provide the first evidence of caspase-2 in regulating HSC and progenitor differentiation, as well as aneuploidy, in vivo.

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DEAD-Box helicase 41 (DDX41) is one of the most commonly reported familial hematological malignancy (HM) genes, first reported in 2015. Mutated, it predisposes to both MDS/AML and lymphoma with an age of diagnosis similar to that of sporadic cases. Consequently, unrecognized DDX41 mutated cases are present in 'sporadic' cohorts with families often identified this way. Individuals with DDX41 mutation or deficiency have generally poor outcome with no effective targeted therapies available. The biological mechanism by which mutant DDX41 predisposes to HM is poorly understood making risk assessment for asymptomatic carriers difficult. In over 50% of HM affected individuals with germline DDX41 mutations, an acquired somatic mutation is identified on the other DDX41 allele with R525H being most common. Analysis of other somatic mutations in germline DDX41 mutation carriers reveals a minimal fingerprint of acquired somatic alterations in known myeloid malignancy driver genes with an absence of NPM1 and FLT3 mutations and an under-representation of mutations in DNMT3A, TET2, IDH1/2 and RUNX1. This may indicate a mechanism of leukemia development unique to germline DDX41 cases. Through an integrative combinatorial genomics approach of whole exome sequencing, leukemic panel sequencing and RNA-sequencing, we are in the process of comprehensively assessing six independent DDX41 germline mutated families. We and others have identified that DDX41 mutations are recurrently associated with MDS with erythroid dysplasia and erythroid subtype leukemia consistent with functional in vitro experiments showing that DDX41 mutations including loss-of-function can impact erythroid differentiation. Our preliminary RNA-Seq analysis on peripheral blood mononuclear cells of DDX41 carriers with HM reveals altered expression of genes involved in hemoglobin complex and innate immunity. DDX41 is reported to bind RNA splicing components such as SF3B1 and PRPF8 which are recurrently mutated in MDS/AML and intriguingly, the R525H mutation disrupts this interaction. Further analysis of our RNA-Seq data interrogating alternative splicing, gene expression analysis and biological pathways to uncover mechanisms for mutant DDX41 oncogenicity is currently ongoing. We have used CRISPR-Cas9 technology to generate Ddx41M1I mice as a model for one of the most common germline mutations in familial HM to investigate the direct effect of DDX41 mutation on hematopoiesis. Preliminary results indicate a sub-clinical reduction in hemoglobin levels in mice carrying the M1I mutation in heterozygosity when compared to their wildtype littermates. We are further investigating these mice with particular emphasis on characterizing hematopoiesis longitudinally to better understand mechanisms of disease onset and progression. This integrative genomic and functional approach to evaluate both mutations and biological pathways affected in DDX41 mutated malignancies will provide biological insight facilitating advancement in diagnosis, risk assessment, monitoring and personalized treatment. Disclosures Scott: Celgene: Honoraria.