Background/Objectives: Treatment with tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) has revolutionized disease management and has transformed CML from a life-threatening disease to a chronic condition for many patients. However, overcoming resistance, particularly related to leukemic stem cells (LSC) that can persist even when the bulk of the leukemic cells are eliminated, remains a significant challenge. Methods: K562 and KU812 cell lines were treated in vitro with the TKI Imatinib (IM). Gene expression, protein analysis, and metabolomic screening were conducted to investigate the ability of the drug to enhance stem cell (SC) features. Moreover, a gene ontology analysis was performed on different available datasets, to further consolidate our data. Results: 48 h of IM treatment can significantly increase the expression of genes related to SC self-renewal, particularly SOX2 and OCT 3/4. Interestingly, these modulations occur in cells that remain alive after drug treatment and that displayed features consistent with leukemia stem-like CML cells, suggesting that SC genes levels are crucial even in cell population survived upon TKI treatment. Moreover, after in silico analysis of available data, we observed an enrichment of SOX2/NANOG and OCT 3/4 signatures after TKI treatment, thus strengthening our results. Conclusions: Our results confirmed the relevance of LSC features after TKI treatment, highlighting the need for more effective and potentially curative strategies targeting LSCs to overcome resistance in CML.

The clinical management of myeloid neoplasms increasingly relies on the accurate detection and longitudinal monitoring of disease-defining genetic alterations. Many clinically relevant mutations are often present at very low variant allele frequencies, below the detection limits of conventional approaches routinely used in diagnostic workflows. In recent years, a growing number of ultra-sensitive molecular technologies have been developed to overcome these limitations, enabling the detection of rare variants with unprecedented precision, offering complementary strengths in terms of sensitivity, quantification, throughput, and clinical applicability. This review provides a comprehensive overview of established and emerging ultra-sensitive technologies for the diagnosis and molecular monitoring of myeloid neoplasms, discussing their technical principles, advantages, and limitations.

R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid duplex and a displaced single-stranded DNA loop. Unscheduled or persistent R-loops drive genome instability by creating conflicts with transcription and replication. Up to 75% of the human genome comprises repetitive DNA elements that are prone to R-loop formation. We show that the RNA binding protein SFPQ suppresses R-loop mediated replication stress and DNA damage at repeat elements such as telomeres, (peri)-centromeres, LINE-1 and SINE elements. SFPQ exhibits in-vitro R-loop binding activity, associates with chromatin containing R-loops, and recruits the histone H3.3 specific chaperon DAXX to preserve a correct nucleosome template that counteracts R-loop accumulation. Loss of SFPQ results in DAXX displacement from repeat elements, reduced histone H3.3 incorporation, replication stress-mediated genome instability and the emergence of cytoplasmatic DNA. This leads to activation of innate immune signaling via the cGAS/STING pathway, ultimately correlating with improved survival of sarcoma patients.

The FLT3-ITD mutation is a critical prognostic marker in acute myeloid leukemia (AML) and recent clinical trials demonstrate that FLT3-based measurable residual disease (MRD) is both prognostic and predictive, guiding therapeutic interventions in intensive and post-transplant settings. Conventional detection methods lack the sensitivity required for effective MRD monitoring. We developed a patient-specific droplet digital PCR (ddPCR) approach achieving analytical sensitivity of 10⁻⁵ (0.001%) for FLT3-ITD quantification. In our cohort, ddPCR enabled longitudinal monitoring of clonal dynamics, allowing the detection of re-emerging FLT3-ITD clones months before hematologic relapse and earlier than standard capillary electrophoresis. Notably, 25% of patients who relapsed as FLT3-ITD positive despite being classified as FLT3-negative at diagnosis harbored detectable microclones when retrospectively analyzed by ddPCR, suggesting that FLT3-ITD-positive relapse frequently originates from pre-existing subclones below conventional detection thresholds. These findings challenge current diagnostic classification and may influence risk stratification and treatment decisions, particularly regarding FLT3 inhibitor eligibility. While ddPCR is limited to tracking known dominant clones, it represents a practical, cost-effective solution for high-sensitivity MRD surveillance. In the era of targeted FLT3 therapies, integrating sensitive molecular monitoring into routine AML management may enable timely therapeutic adjustments and improve patient outcomes.