Imaging Flow Cytometry Detection of Cytogenetic Abnormalities in Circulating <scp>CD34</scp> + Cells Predicts Leukemic Transformation in Myelofibrosis

Myelofibrosis is a myeloproliferative neoplasm with potential to transform to acute myeloid leukemia. This evolution is unpredictable and current assays lack the sensitivity and applicability needed to predict this transformation. While population-level data utilizing comprehensive genomic profiling can identify subgroups at higher risk of progression, they do not provide individualized information on the likelihood or timing of leukemia. Cytogenetic alterations are typically present in secondary leukemia. We aimed to determine whether these changes could be detected at an early stage. To achieve this we established and tested a single-cell imaging flow cytometric method for chromosomal aberrations using fluorescence in situ hybridization (FISH) probes to analyze circulating CD34/CD45-positive cells. Peripheral blood samples from 14 patients, collected at up to eight timepoints over a 34-month period, were analyzed for defects involving chromosomes 1, 5, and 17. Following cell immunophenotyping and FISH probe hybridization, a mean of 174,216 mononuclear cells was assessed per sample. Chromosomal abnormalities including gain(1q), del(5q), idic (5), monosomy 17, and/or del(17p) were identified in eight patients, at frequencies down to 0.2% of mononuclear cells. Serial analyses revealed emergence of new chromosomal lesions, clonal evolution, dominance, and multi-hit abnormalities. In three patients, acquired chromosome 17 abnormalities preceded progression to secondary leukemia by up to 7 months. This pilot study demonstrates that imaging flow cytometry-based FISH of circulating CD34/CD45-positive cells enables real-time, blood-based surveillance for cytogenetic evolution in myelofibrosis. The ability to dynamically track clone size and hierarchy highlights its potential as an early predictor of leukemic transformation in myelofibrosis.