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Summary The majority of Myeloproliferative Neoplasms (MPNs) are characterised by mutations in genes encoding molecules or receptors involved in cell signalling, the most common being the JAK2 V617F mutation. This mutation leads to ligand‐independent activation of downstream signalling pathways by constitutive phosphorylation. The signalling pathways affected include the Janus kinase‐signal transducers and activators of transcription (JAK‐STAT) and phosphotidylinositide‐3 kinase (PI3K) pathways, which regulate cell survival and apoptosis respectively. Monoclonal antibodies to phospho‐STAT5 and phospho‐Akt were generated and assessed by immunocytochemistry on bone marrow biopsies of MPN patients with JAK2 V617F, JAK2 exon 12, MPL exon 10 and KIT D816V mutations. JAK2 V617F mutation was associated with significantly increased levels of phosphorylated STAT5 and Akt in haemopoietic cells, most marked in megakaryocytes. In contrast, JAK2 exon 12 and MPL exon 10 mutations did not affect the level of phosphorylation. In systemic mastocytosis with KIT D618V mutation there was significantly increased expression of phosphorylated STAT5 and Akt in neoplastic mast cells although there was no change in the expression in other haemopoietic cells. JAK2 V617F is associated with upregulated phosphorylation of STAT5 and Akt in megakaryocytes, and to a lesser extent in other haemopoietic cells. Immunocytochemistry of bone marrow trephines for these phospho‐proteins can be used as a supplementary diagnostic test with a high negative predictive value.

Acute promyelocytic leukaemia (APML) can be promptly diagnosed by detecting abnormal diffuse staining patterns of PML bodies in abnormal promyelocytes using immunofluorescence microscopy. However, this technique is subjective, with low sensitivity. Using samples from 18 patients with acute myeloid leukaemia (AML) (including four with APML), the authors investigated whether imaging flow cytometry could be a viable alternative to this current technique and improve sensitivity levels. Bone marrow/peripheral blood cells were stained with an antibody to PML, and data were acquired on an ImageStream (Amnis Corporation, Seattle, Washington, USA). Using the modulation feature for data analysis, the authors demonstrated that this technique could successfully identify cases of APML. Imaging flow cytometry, by analysing greater numbers of cells and with the potential to include disease-specific antigens, increases the sensitivity of the current immunofluorescence technique. Imaging flow cytometry is an exciting technology that has many possible applications in the diagnosis of haematological malignancies, including the potential to integrate modalities.

IntroductionApproximately one-third of cases of de novo acute myeloid leukemia (AML) carry mutations in the C-terminal region of the nucleophosmin (NPM1) gene.1-2 AML with mutated NPM1 shows distinctive biological and clinical features, including female gender, monocytoid morphology, CD34-negativity and a unique gene expression profile.3 These features are irrespective of whether NPM1-mutated AML carries a normal karyotype (about 85% of cases) or secondary chromosomal aberrations (about 15%), thus reinforcing the view that NPM1 mutation is a founder genetic lesion. 4 In the absence of FLT3-ITD mutations, AML with a normal karyotype and NPM1 mutation confers a significantly better prognosis than other AML cases with a normal karyotype.5-8 A recent study indicates that these prognostic considerations also apply to NPM1-mutated AML patients carrying secondary chromosomal aberrations.4 Frame-shift NPM1 mutations create a new leucine-rich sequence at the protein's C-terminus which serves as a nuclear export signal. This, accompanied by loss of tryptophan residues, is responsible for the increased nuclear export and aberrant cytoplasmic accumulation of the NPM1 leukemic mutants. 9NPM1 mutations were initially detected following the observation that in B5-fixed/EDTA decalcified bone marrow trephine (BMT) biopsies, NPM protein was aberrantly expressed in the cytoplasm of leukemic cells of about onethird of AML patients.1 This anomalous staining pattern is closely correlated with the presence of mutated NPM1.9 As BMT biopsies are not always performed at diagnosis of AML, the ability to detect cNPM on cell smears would be advantageous. We have established a technique to assess NPM expression in routine hematologic smear samples and assessed its correlation with NPM1 mutation status. Design and Methods SamplesCell lines FL18 and L428 were cultured with recommended media and OCI-AML3 (DSMZ, Braunschweig, Germany) as described else-