Summary Marrow fibrosis is a significant complication of myeloproliferative neoplasms (MPN) that affects up to 20% of patients and is associated with a poor prognosis. The pathological processes that lead to fibrotic progression are not well understood, but megakaryocytes have been implicated in the process. The aim of this study was to determine whether platelets, derived from megakaryocytes, have transcriptomic alterations associated with fibrosis. Platelets from MPN patients with and without fibrosis and non‐malignant control individuals were assessed using next generation sequencing. Results from the initial training cohort showed discrete changes in platelet transcripts in the presence of marrow fibrosis. We identified more than 1000 differentially expressed transcripts from which a putative 3‐gene fibrotic platelet signature (CCND1, H2AX [previously termed H2AFX] and CEP55) could be identified. This fibrosis‐associated signature was assessed blinded on platelets from an independent test MPN patient cohort. The 3‐gene signature was able to discriminate between patients with and without marrow fibrosis with a positive predictive value of 71% (93% specificity, 71% sensitivity). This demonstrates that assessment of dysregulated transcripts in platelets may be a useful monitoring tool in MPN to identify progression to marrow fibrosis. Further, sequential monitoring could have clinical applications for early prediction of progression to fibrosis.

Imaging flow cytometry is emerging as a diagnostic tool for the assessment of leukemia. It has the functionality of standard flow cytometry and generates high‐resolution digital images of each cell with quantifiable numerical data. We demonstrate the use of an automated high‐throughput method for performing fluorescence in situ hybridization (FISH) on immunophenotyped whole cells in suspension and analyzed by imaging flow cytometry, a technique called “Immuno‐flowFISH”. The aim of this study was to demonstrate the application of immuno‐flowFISH for the detection of chromosomal abnormalities in CLL, specifically trisomy 12 and del(17p). Mononuclear cells were isolated and immunophenotyped with fluorescently conjugated CD3, CD5, and CD19 monoclonal antibodies. Following fixation, cells were permeabilized, dsDNA denatured and hybridized with chromosome 12 or 17 enumeration (CEP 12 and CEP17) and 17p12 locus‐specific FISH probes. Cells were analyzed on the Amnis ImageStream®X Mark II to assess the number and percent FISH‐positive CLL cells and the ratio of FISH spot counts for CD5/CD19‐positive CLL cells to CD3/CD5‐positive T cells (FISH “mean spot ratio”). Deletion of 17p was detected in about 8% of cases to date, with del(17p) ranged from 3.5–22.8% and the FISH “mean spot ratio” 0.86–0.96. Immuno‐flowFISH also detected a minimal residual disease case with +12 with a limit of detection of 0.13% and a rare case that presented with atypical phenotype and cytogenetics. Immuno‐flowFISH could detect del(17p) in phenotypically identified CD5/CD19‐positive B‐cells. The 100‐fold increase in analyzed cells, as well as the addition of cell phenotype increased the sensitivity and specificity over current clinical FISH testing. Furthermore, immuno‐flowFISH analysis demonstrated specific utility in unique clinical scenarios such as residual disease and atypical biology cases which may be of significant benefit with regards to prognostication and MRD analysis. The method will assist in therapeutic decision making and disease monitoring for many hematological malignancies. © 2019 International Society for Advancement of Cytometry

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