Digital droplet PCR and liquid biopsy in acute myeloid Leukemia: bridging translational research and clinical...

Acute myeloid leukemia (AML) is a genetically heterogeneous and dynamically evolving malignancy in which morphologic remission does not necessarily indicate eradication of disease. Bone marrow examination remains central to diagnosis and response assessment, yet repeated aspiration is invasive, may be affected by hemodilution and spatial sampling, and provides only intermittent snapshots of an evolving clonal process. Liquid biopsy offers a complementary strategy by interrogating circulating cell-free DNA (cfDNA), the leukemia-derived fraction termed circulating tumor DNA (ctDNA), extracellular vesicles, exosomal nucleic acids, and circulating leukemic cells. Among the analytical platforms applicable to these materials, droplet digital polymerase chain reaction (ddPCR) is attractive because it partitions a specimen into thousands of reactions and enables highly precise absolute quantification without a calibration curve. This narrative review critically examines the biological rationale, analytical performance, and clinical evidence supporting ddPCR-based liquid biopsy in AML. The strongest current evidence concerns molecular measurable residual disease (MRD), particularly for NPM1 mutations and selected patient-specific variants, with additional applications in IDH1/2, FLT3-TKD, KIT, CEBPA, DNMT3A, and fusion-transcript monitoring. We discuss the roles of peripheral blood and plasma cfDNA at diagnosis, during induction and consolidation, before and after allogeneic hematopoietic stem-cell transplantation, and at suspected molecular relapse. We also compare ddPCR with multiparameter flow cytometry, reverse-transcription quantitative PCR, and error-corrected next-generation sequencing, emphasizing that analytical sensitivity alone does not establish clinical validity. Major barriers include pre-analytical variation, low and fluctuating ctDNA abundance, assay-specific false-positive droplets, incomplete target coverage, clonal hematopoiesis, and the absence of harmonized thresholds. Emerging multi-analyte strategies that combine mutation tracking, methylation, exosomal RNA, chimerism, and computational modeling may improve robustness. At present, ddPCR is best positioned as a rapid, targeted, and complementary tool embedded in a multimodal MRD framework rather than as a universal replacement for bone marrow or broad genomic profiling.