Background: MicroRNAs (miRNAs) have gained significant attention as potential biomarkers in childhood leukemia, offering insights into diagnosis, prognosis, and therapeutic targeting. However, the clinical translation of miRNA biomarkers faces several challenges, including inconsistencies in results caused by methodological differences, sample processing variability, and a lack of standardized normalization techniques. In addition, miRNA profiles are highly cell-specific, with unique signatures for different blood cell types and leukemic blasts, reflecting their distinct biological roles and disease states. Pre-analytical factors are critical in ensuring the accuracy and reproducibility of miRNA quantification. The selectively enriched and highly stable exosomal miRNAs have shown great promise for studying intercellular communication and disease-specific miRNAs. The selection of the analytical matrix should align with the specific objectives of the research or diagnostic application. Addressing technical challenges and recording potential confounding variables (e.g., age, gender, ethnicity, body mass index, menstrual cycle, fasting, circadian rhythm, comorbidities, medications, smoking, and physical activity) are essential to enhancing the reproducibility and reliability of miRNA biomarkers. Objective: This review aims to highlight the challenges facing miRNA quantification in childhood leukemias, scrutinizing all relevant pre-analytical issues, including the preferred specimen source, leukemic blast type and count, the selection of suitable blood components, the impact of time and freeze–thaw cycles, collection, processing, and storage variables. Conclusion: Further research is needed to standardize methodologies and expand our understanding of miRNA interaction networks, ultimately advancing the application of miRNAs in childhood leukemia research.

Although advances in the management of pediatric neoplasms have profoundly improved infectious disease outcomes, invasive fungal diseases (IFDs) remain a major cause of morbidity and mortality in children and adolescents with high-risk hematological malignancies. A retrospective study was conducted in the Pediatric Hematology–Oncology Department of the University General Hospital of Heraklion for 2013–2022 to estimate the prevalence and describe the clinical and epidemiological characteristics of IFDs for pediatric and adolescent patients with neoplasia. Demographic, clinical, and laboratory parameters were analyzed to identify risk factors for the development of IFD. The overall prevalence of IFDs was estimated to be 7.8% (12/154 patients) throughout the study. The mean age at IFD diagnosis was 9.8 years (SD 6.4 years). The most common IFD was possible/probable invasive pulmonary aspergillosis (IPA; in ≈50%), followed by candidemia/invasive candidiasis (in 44%). Candida parapsilosis was the most prevalent Candida species (4/6 events). Of interest, the majority (75%) of IFDs were breakthrough infections. Patients with increased risk for IFDs were those who were colonized by fungi in sites other than the oral cavity, hospitalized in the intensive care unit for >7 days, received >7 different antimicrobials in the last 3 months, or had severe neutropenia for >44 days. Two children out of a total of 12 with IFD died due to refractory disease or relapse (16.7%). More detailed and prospective epidemiological studies on fungal infections in pediatric patients with hematological or solid neoplasms can contribute to the optimization of prevention and treatment.

Diamond–Blackfan anemia (DBA) is a ribosomopathy characterized by bone marrow erythroid hypoplasia, which typically presents with severe anemia within the first months of life. DBA is typically attributed to a heterozygous mutation in a ribosomal protein (RP) gene along with a defect in the ribosomal RNA (rRNA) maturation or levels. Besides classic DBA, DBA-like disease has been described with variations in 16 genes (primarily in GATA1, followed by ADA2 alias CECR1, HEATR3, and TSR2). To date, more than a thousand variants have been reported in RP genes. Splice variants represent 6% of identifiable genetic defects in DBA, while their prevalence is 14.3% when focusing on pathogenic and likely pathogenic (P/LP) variants, thus highlighting the impact of such alterations in RP translation and, subsequently, in ribosome levels. We hereby present two cases with novel pathogenic splice variants in RPS17 and RPS26. Associations of DBA-related variants with specific phenotypic features and malignancies and the molecular consequences of pathogenic variations for each DBA-related gene are discussed. The determinants of the spontaneous remission, cancer development, variable expression of the same variants between families, and selectivity of RP defects towards the erythroid lineage remain to be elucidated.