P051 | BEYOND THE DROP CURTAIN: DECODING LEUKEMIA'S MORPHOLOGICAL MASQUERADE

Background: Despite advances in the molecular characterization of hematologic malignancies, morphological evaluation of peripheral blood (PB) and bone marrow (BM) remains critical for diagnosis and prognostic assessment, particularly in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), juvenile myelomonocytic leukemia/chronic myelomonocytic leukemia (JMML/CMML), and acute leukemias of ambiguous lineage (ALAL). The blast percentage serves as a key diagnostic parameter, but its assessment can be complicated by cells with ambiguous morphology, such as immature promonocytes. These "blast equivalents," introduced in the 2001 World Health Organization (WHO) classification, may exhibit immunophenotypic similarities to blasts while lacking their typical morphology, thereby complicating disease classification. Current protocols for targeted cell isolation via Laser Microdissection (LMD) exist primarily for solid tissue specimens. Aim: This study aims to validate LMD as a reliable diagnostic tool for isolating morphologically ambiguous cells in hematologic malignancies, characterize their molecular profiles via next-generation sequencing (NGS). Methods: We applied LMD either to PB and BM smears or to PB and BM mononuclear cells (PBMCs) separated via Ficoll gradient, subsequently stained with May-Grünwald Giemsa for morphological identification. Isolated cells undergo NGS for mutational profiling. Results: We demonstrated efficient isolation and preservation of cell morphology, with high-quality nucleic acid yield suitable for downstream molecular analyses. First, we confirmed the preservation of cellular morphology on polyethylene naphthalate (PEN) membrane slides. Second, we evaluated the quality and quantity of genetic material extracted from approximately 80 laser-microdissected cells harboring inv(16)(p13.1q22) (CBFB::MYH11), t(15;17)(q24;q21) (PML::RARA), and t(9;22)(q34;q11.2) (BCR::ABL1) rearrangements. The extracted cDNA was analyzed by qualitative polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR) using fusion transcript-specific primers, confirming suitability for downstream molecular applications. Conclusions: We developed an optimized LMD protocol specifically for liquid samples, enabling precise microdissection and molecular characterization of rare or atypical hematopoietic cells. Our liquid sample–adapted LMD protocol represents a novel strategy for enhancing integrated morpho-molecular diagnostics in hematology.