Next-generation sequencing and integrated transcriptomics in the molecular characterization of haematological and melanocytic neoplasms

High-throughput sequencing (HTS)-based technologies range from targeted sequencing of a limited number of genes (gene panels) to whole-exome sequencing (WES) for the assessment of coding regions of genes or whole-genome sequencing (WGS). In the field of lymphoproliferative neoplasms, NGS has assumed a particularly significant role. Indeed, lymphomas are characterized by marked biological and clinical heterogeneity. Moreover, they demonstrate a genetic complexity that often escapes traditional diagnostic methods. Many lymphomas, although morphologically similar, present substantial genetic differences that influence prognosis, biological behavior, and treatment response. However, HGBCL, NOS, remains a rare category defined by morphology and lack of defining genomic rearrangements. Indeed, its molecular landscape is very diverse and, where available, an extensive molecular investigation is recommended to determine whether individual tumors can be aligned or reassigned to defined entities. Additionally, over the past two decades, through the high sensitivity of NGS it has become apparent that somatic mutations in genes traditionally associated with myeloid malignancies can be detected in the peripheral blood of individuals without any clinical or laboratory signs of hematologic disease. Indeed, Haematopoietic Stem Cells (HSC) can acquire somatic mutations leading to a clonal sub-population sharing the mutational landscape of the progenitors. This event is known as Clonal Haematopoiesis (CH). A variant allele frequency (VAF) ≥ 2% in the CH-related mutated genes has been proposed as a criterion for Clonal Haematopoiesis of Indeterminate Potential (CHIP), regarded as a premalignant state, and risk factors for clonal expansion and progression, mostly, to myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) or myeloproliferative neoplasms (MPNs). NGS it enables the detection of rare mutant clones, the reconstruction of clonal architecture, and the monitoring of clonal dynamics over time. However, for pathologists and clinicians, CH/CHIP etc present both opportunities and challenges. One of the most relevant diagnostic considerations is the potential for CH to confound molecular analysis. Finally, is important to note that the routine application of mutation analyses using highthroughput sequencing via next-generation sequencing (NGS) has not yet been widely established in haematopathology. Continuous research into innovative diagnostic tools and therapeutic strategies will be critical in unlocking the full potential of precision medicine for patients with hematological malignancies.Melanocytic neoplasms represent a heterogeneous group of lesions ranging from benign nevi to malignant melanomas, encompassing a broad spectrum of intermediate histological and biological variants. Despite significant advances in histopathological and molecular diagnostics, the precise classification of many of these entities remains a major challenge in pathological practice. The advent of RNA sequencing (RNA-seq) technologies and transcriptomics in general have able research to to study the sum of all the RNA transcripts and their expression level in an organism or a single cell thus revolutionizing the understanding of the cellular and molecular processes underlying various neoplastic lesions.8  Indeed, these approaches overcome the limitations of traditional gene analysis and to explore the functional complexity of gene expression in a quantitative and spatially resolved manner.  This transition from morphology-based to expression-based diagnostics represents a paradigm shift in dermatopathology, placing the transcriptome at the center of modern molecular classification systems.