Novel NPM1 exon 5 mutations and gene fusions leading to aberrant cytoplasmic nucleophosmin in AML

Nucleophosmin (NPM1) mutations in acute myeloid leukemia (AML) affect exon 12, but also sporadically affect exons 9 and 11, causing changes at the protein C-terminal end (tryptophan loss, nuclear export signal [NES] motif creation) that lead to aberrant cytoplasmic NPM1 (NPM1c+), detectable by immunohistochemistry. Combining immunohistochemistry and molecular analyses in 929 patients with AML, we found non–exon 12 NPM1 mutations in 5 (1.3%) of 387 NPM1c+ cases. Besides mutations in exons 9 (n = 1) and 11 (n = 1), novel exon 5 mutations were discovered (n = 3). Another exon 5 mutation was identified in an additional 141 patients with AML selected for wild-type NPM1 exon 12. Three NPM1 rearrangements (NPM1/RPP30, NPM1/SETBP1, NPM1/CCDC28A) were detected and characterized among 13 979 AML samples screened by cytogenetic/fluorescence in situ hybridization and RNA sequencing. Functional studies demonstrated that in AML cases, new NPM1 proteins harbored an efficient extra NES, either newly created or already present in the fusion partner, ensuring its cytoplasmic accumulation. Our findings support NPM1 cytoplasmic relocation as critical for leukemogenesis and reinforce the role of immunohistochemistry in predicting AML-associated NPM1 genetic lesions. This study highlights the need to develop new assays for molecular diagnosis and monitoring of NPM1-mutated AML

Platelet size for distinguishing between inherited thrombocytopenias and immune thrombocytopenia: a multicentric, real life study.

The most frequent forms of inherited thrombocytopenia (IT) are characterized by platelet size abnormalities and it has been suggested that this parameter is useful for their differentiation from immune thrombocytopenia (ITP). Recently, a monocentric study identified cut-off values for mean platelet volume (MPV) and mean platelet diameter (MPD) with good diagnostic accuracy in this respect. To validate these cut-off values in a different and larger case series of patients, we enrolled 130 subjects with ITP and 113 with IT in six different centres. The platelet count and MPV was each measured by the instrument routinely used in each institution. In some centres, platelet count was also measured by optical microscopy. MPD was evaluated centrally by image analysis of peripheral blood films. The previously identified cut-off value for MPV had 91% specificity in distinguishing ITP from inherited macrothrombocytopenias (mono and biallelic Bernard-Soulier, MYH9-related disease), while its sensitivity was greatly variable depending on the instrument used. With an appropriate instrument, specificity was 83%. The diagnostic accuracy of MPD was lower than that obtained with MPV. We concluded that MPV is a useful parameter for differentiating ITP from IT provided that it is measured by appropriate cell counters

Analysis of Clonal Type-Specific Antibody Reactions in Toxoplasma gondii Seropositive Humans from Germany by Peptide-Microarray

BACKGROUND: Different clonal types of Toxoplasma gondii are thought to be associated with distinct clinical manifestations of infections. Serotyping is a novel technique which may allow to determine the clonal type of T. gondii humans are infected with and to extend typing studies to larger populations which include infected but non-diseased individuals. METHODOLOGY: A peptide-microarray test for T. gondii serotyping was established with 54 previously published synthetic peptides, which mimic clonal type-specific epitopes. The test was applied to human sera (n = 174) collected from individuals with an acute T. gondii infection (n = 21), a latent T. gondii infection (n = 53) and from T. gondii-seropositive forest workers (n = 100). FINDINGS: The majority (n = 124; 71%) of all T. gondii seropositive human sera showed reactions against synthetic peptides with sequences specific for clonal type II (type II peptides). Type I and type III peptides were recognized by 42% (n = 73) or 16% (n = 28) of the human sera, respectively, while type II-III, type I-III or type I-II peptides were recognized by 49% (n = 85), 36% (n = 62) or 14% (n = 25) of the sera, respectively. Highest reaction intensities were observed with synthetic peptides mimicking type II-specific epitopes. A proportion of the sera (n = 22; 13%) showed no reaction with type-specific peptides. Individuals with acute toxoplasmosis reacted with a statistically significantly higher number of peptides as compared to individuals with latent T. gondii infection or seropositive forest workers. CONCLUSIONS: Type II-specific reactions were overrepresented and higher in intensity in the study population, which was in accord with genotyping studies on T. gondii oocysts previously conducted in the same area. There were also individuals with type I- or type III-specific reactions. Well-characterized reference sera and further specific peptide markers are needed to establish and to perform future serotyping approaches with higher resolution

Eltrombopag for the treatment of the inherited thrombocytopenia deriving from MYH9 mutations.

Platelet transfusion is currently the primary medical treatment for reducing thrombocytopenia in patients with inherited thrombocytopenias. To evaluate whether stimulating megakaryopoiesis could increase platelet count in these conditions, we treated patients with a severe thrombocytopenia induced by MYH9 mutations (MYH9-related disease) with a nonpeptide thrombopoietin receptor agonist, eltrombopag. Twelve adult patients with MYH9-RD and platelet counts of less than 50 7 10(9)/L received 50 mg of eltrombopag orally per day for 3 weeks. Patients who achieved a platelet count higher than 150 7 10(9)/L stopped therapy, those with 100 to 150 platelets 7 10(9)/L continued treatment at the same eltrombopag dose for 3 additional weeks, while those with less than 100 platelets 7 10(9)/L increased the eltrombopag dose to 75 mg for 3 weeks. Major responses (platelet count of at least 100 7 10(9)/L or 3 times the baseline value) were obtained in 8 patients, minor responses (platelet counts at least twice the baseline value) in 3. One patient did not respond. Bleeding tendency disappeared in 8 of 10 patients with bleeding symptoms at baseline. Mild adverse events were reported in 2 patients. The availability of thrombopoietin mimetics opened new prospects in the treatment of inherited thrombocytopenias. This study is registered at www.clinicaltrials.gov as NCT01133860 (European Union Drug Regulating Authorities Clinical Trials number 2008-001903-42)