ALBERT

Description: Acute myeloid leukemia expressing mutated NPM1 gene and cytoplasmic nucleophosmin (NPMc+ AML) [Falini B et al, NEJM2005;352:254–266] is a new entity of WHO classification that shows distinctive biological and clinical features, including a unique molecular signature characterized by downregulation of CD34 and upregulation of most HOX genes [Falini B et al, Blood2007;109:874–885]. Involvement of HOX genes in the maintenance of the stem-cell phenotype strongly suggest that AML with mutated NPM1 originates from a multipotent hematopoietic progenitor (HSC). This view is also supported by immunohistological findings showing that AML with mutated NPM1 frequently displays multilineage involvement [Pasqualucci L et al, Blood2006;108:4146–4155]. On the other hand, the frequent negativity of NPMc+ AML for the HSC-associated antigen CD34 raises the question of whether the mutation event occurs in a CD34-negative HSC (these cells have been identified in mice) or whether a minimal pool of CD34-positive NPM1-mutated leukemic cells does exist. Currently, the hierarchical level of stem cell involvement in NPMc+ AML is unknown. To address this issue, we purified CD34+ cells from NPMc+ AML patients and detected NPM1 mutant protein in the sorted population by Western blot with anti-NPM mutant specific antibodies [Martelli MP et al, Leukemia 2008] (Figure 1A). We investigated 6 NPMc+ AML patients presenting at diagnosis with 0.12%, 0.14%, 0.38%, 5%, 22%, and 28% of CD34+ cells in the peripheral blood. In all cases, CD34+ fractions (purity 〉90%) harboured NPM1 mutant protein, indicating they belong to the leukemic clone (Figure 1B). The percentage of most undifferentiated CD34+/CD38− cells in the CD34+ fractions ranged from 5 to 97%. Notably, in at least one case, all CD34+ NPM1-mutated leukemic cells were CD38−negative. Moreover in all cases, CD34+ NPM1-mutated leukemic cells appeared to express CD123 (IL-3 receptor), considered a marker of the leukemic stem cell and target of potential therapy. Double staining of bone marrow biopsies with anti-CD34 and anti-NPM antibodies revealed that the rare CD34+ cells expressed NPM1 aberrantly in the cytoplasm. Inoculation of CD34+ NPM1-mutated AML cells into sublethally irradiated NOD/SCID mice resulted into leukemia engrafment in various body sites, especially bone marrow, spleen, lung and liver. Preliminary results showed that CD34+ leukemic cells reacquired the same leukemic phenotype as the original patient’s, including CD34-negativity of the leukemic bulk in spite of any lack of differentiation. This finding suggests that NPM1 mutant protein may be involved in downregulation of CD34 antigen, while keeping a gene expression profile typical of the hematopoietic stem cell. These findings suggest the CD34+ fraction contains the SCID-leukemia initiating cells (SL-IC) and point to CD34+/CD38− HSC as the cell of origin of AML with mutated NPM1. Figure Figure

Description: Acute myeloid leukemia (AML) with mutated NPM1 shows distinctive biologic and clinical features, including absent/low CD34 expression, the significance of which remains unclear. Therefore, we analyzed CD34+ cells from 41 NPM1-mutated AML. At flow cytometry, 31 of 41 samples contained less than 10% cells showing low intensity CD34 positivity and variable expression of CD38. Mutational analysis and/or Western blotting of purified CD34+ cells from 17 patients revealed NPM1-mutated gene and/or protein in all. Immunohistochemistry of trephine bone marrow biopsies and/or flow cytometry proved CD34+ leukemia cells from NPM1-mutated AML had aberrant nucleophosmin expression in cytoplasm. NPM1-mutated gene and/or protein was also confirmed in a CD34+ subfraction exhibiting the phenotype (CD34+/CD38−/CD123+/CD33+/CD90−) of leukemic stem cells. When transplanted into immunocompromised mice, CD34+ cells generated a leukemia recapitulating, both morphologically and immunohistochemically (aberrant cytoplasmic nucleophosmin, CD34 negativity), the original patient's disease. These results indicate that the CD34+ fraction in NPM1-mutated AML belongs to the leukemic clone and contains NPM1-mutated cells exhibiting properties typical of leukemia-initiating cells. CD34− cells from few cases (2/15) also showed significant leukemia-initiating cell potential in immunocompromised mice. This study provides further evidence that NPM1 mutation is a founder genetic lesion and has potential implications for the cell-of-origin and targeted therapy of NPM1-mutated AML.

Description: Key Points We develop and validate Karyogene, a comprehensive one-stop diagnostic platform for the genomic analysis of myeloid malignancies. Karyogene simultaneously detects substitutions, insertions/deletions, translocations, copy number and zygosity changes in a single assay.

Description: Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by dysplastic hematopoiesis and peripheral blood cytopenias. Recently, somatic mutations affecting components of the spliceosomal machinery have been discovered in the majority of MDS patients. SF3B1 mutations are most frequent and strongly correlate with the presence of bone marrow ring sideroblasts and a favorable prognosis. SF3B1 mutations, including the K700E substitution which accounts for more than 50% of all mutations, are missense, heterozygous and cluster in a hotspot within the heat domain of the protein suggesting that they are gain-of-function variants. The molecular effects of SF3B1 mutations and the mechanisms through which they drive clonal expansion and dyserythropoiesis remain obscure. Therefore, to assess their molecular and phenotypic consequences, we generated a mouse model carrying a conditional floxed knock-in allele (Sf3b1flox-K700E/+) by homologous recombination of JM8 murine embryonic stem cells. To induce expression of Sf3b1 K700E in adult hematopoietic stem and progenitor cells, Sf3b1flox-K700E/+/Mx1-Cre+ were injected with pIpC from 4-8 weeks of age. Here we report the initial characterization of these animals. Monthly peripheral blood counts from mutants and wild-type (WT) littermates starting one month post-pIpC injection showed a reduction in hemoglobin levels (at 8 weeks WT=17g/dl mut=14.5g/dl, p

Description: Background. Age-dependent clonal expansion of somatic mutations in the hematopoietic system is associated with an increased risk of hematological cancers (including myelodysplastic syndromes, MDS) and other illnesses (coronary heart disease and stroke). However, the presence of clonal hematopoiesis per se in a given individual has only limited predictive power. We hypothesized that the study of oldest-old population can define more specifically the relationship between mutations in the hematopoietic system and risk for MDS, inflammation and vascular diseases. Methods. We analyzed 1004 oldest-old subjects (median age 84.2y, range 80-105) included in the "Health and Anemia" population-based study [Haematologica 2010;95:1849]. Using peripheral blood DNA, we looked for somatic mutations in 47 genes recurrently mutated in hematologic cancers. Results. Clonal mutations were observed in 32.8% of individuals (range 1-5). The majority of variants occurred in 3 genes: DNMT3A (36.4%), TET2 (24.3%) and ASXL1 (6.5%). Mutations in splicing genes, PPM1D and TP53 were found in 7.4%, 5% and 2% of cases, respectively. The mutation frequencies increased with age, up to 50% in individuals aged over 90 years (P=.011). Clonal hematopoiesis was associated with a lower 5-y probability of survival (P=.03), and prognosis was even poorer in patients carrying ≥2 mutations (P=.002) We first focused on the relationship between clonal hematopoiesis and MDS phenotype. Carrying a somatic mutation with a variant allele frequency (VAF) ≥.10, carrying ≥2 mutations, spliceosome gene mutations and co-mutation patterns involving TET2, DNMT3A had a positive predictive value for MDS (from .85 to 1.0). The most frequent early phenotypic changes in patients who developed MDS included an increasing red blood cell distribution width (RDW) and mean corpuscular volume (MCV). Preliminary analyses suggested that the combination of mutations and non-mutational factors (RDW, MCV, after excluding iron/vitamin depletion and thalassemia) may improve the capability to capture individual risk of developing MDS with respect to molecular data alone (P=.01) We studied clonal evolution in 72 patients with multiple samples available over a period of 5y. Clonal hematopoiesis was found at baseline in 22 cases: 2 individuals acquired additional mutations during follow-up, and 5 displayed significant increase in VAF. In 9 subjects without clonal hematopoiesis, mutations were acquired during follow-up. RDW and MCV changes, induction of unexplained cytopenia and overt MDS phenotype were significantly restricted to subjects displaying clonal evolution. We hypothesized that in oldest-old populations MDS could be underdiagnosed (many patients are not considered for bone marrow aspiration because of age). Cytopenia was a common finding in our cohort (20%) the underlying cause remaining unexplained in 27% of cases. In patients with unexplained anemia, carrying a somatic mutation had a positive predictive value for persistent, progressive, multilineage cytopenia (findings consistent with a MDS phenotype) and shorter survival (from .8 to .94). On this basis, 8% of all cytopenias might be undiagnosed MDS. Finally, we investigated the association between clonal hematopoiesis with inflammatory and vascular diseases. Mutations in DNMT3A, TET2, and ASXL1 were each individually associated with risk of coronary heart disease and death, and preliminary analyses suggest that clonal hematopoiesis is also associated with increased risk of rheumatological diseases (P from .03 to.009). We identified mutations in macrophages isolated from synovial fluid of 4/17 patients with rheumatoid arthritis and from atherosclerotic plaques of 3/25 patients with carotid stenosis. Functional studies of macrophages (expression of specific chemokine and cytokine gene patterns) are ongoing. All these findings are under validation in an independent cohort of 800 subjects enrolled in the "Monzino 80-plus" study [Alzheimers Dement 2015;11:258]]. Conclusion. Clonal hematopoiesis was associated with reduced survival in an oldest-old population. Specific mutational profiles define different risks of developing MDS and inflammatory/vascular diseases. Non mutational factors, such as early changes in red blood cell indices, may improve the capability to identify patients at increased risk of developing myeloid cancers. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Bolli:Celgene: Honoraria. Vassiliou:KYMAB: Consultancy, Equity Ownership; Celgene: Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.