Leukemia is the most common childhood malignancy, with an incidence of 4.7 per 100,000 in pediatric patients (< 20 years of age). Acute Leukemias (AL) represent a heterogeneous group of diseases that originate from the interaction between genetic and epigenetic alterations in hematopoietic progenitors, leading to the dysregulation of multiple crucial signal transduction pathways, resulting in hematopoietic insufficiency due to a clonal proliferation of immature precursors. Clonal heterogeneity has been implicated as a driving mechanism of leukemogenesis since a high degree of genetic variability is associated with an increased risk of particular subclones having a proliferative advantage leading to clonal expansion. In this context, cytogenetics is still considered one of the most valuable tools for understanding the mechanisms which lead to AL transformation since chromosomal abnormalities are found in 80% of children affected by the disease. In this regard, due to the relative rarity and aggressive potential of childhood AL, the prognostic impact of these abnormalities remains to be elucidated. Thus, by combining cytogenetics and genomic high throughput sequencing (NGS), it may be possible to characterize the genomic profile of pediatric patients with AL, identifying putative genes/gene fusions and genomic variants to help determine the risk stratification and provide appropriate treatment to patients, including adaptations in therapeutic protocols. Herein we describe the cytogenetic and genomic profile of six children with acute leukemia. Firstly, using banding cytogenetics and FISH, patients were sorted for further genomic investigations by NGS.

We performed G-banding and FISH experiments. NGS was performed on a targeted approach.

We characterized six (6) pediatric patients diagnosed with acute leukemias presenting with rare cytogenetic abnormalities. The cytogenetic abnormalities studied were associated with genomic pathogenic variants by NGS analysis. We observed variants in IDH1 (2 cases); multi-hit TP53, Li-Fraumei related (1); novel RUNX1 (1); KIT (1); WT1 (1), besides a CBFA2T3-GLIS2 fusion associated with a Philadelphia chromosome.

The prognostic evaluation of acute leukemias at diagnosis is a critical step for proper clinical management. According to the WHO classification, risk stratification is based on cytogenetic and molecular data, morphological characteristics, and mutational screening. In this regard, NGS can be a powerful tool, allowing the parallel sequencing of multiple targets and detecting variants with prognostic impact. NGS became relevant in discovering genes/variants and characterizing the clonal architecture. Thus, due to its capability to screen a high number of genes and detect different types of molecular aberrations, targeted NGS has been increasingly incorporated into the clinical routine for the management of AL patients.

We highlight the importance of cytogenetic studies combined with NGS in identifying leukemic putative genes and selecting pediatric patients with AL as possible candidates for future targeted therapies. Cytogenetic, molecular, and genomic technologies combined have been shown to help understand the AL mutational landscape and impact clinical practice.