Aggiornamenti di Diagnostica Molecolare in Ematologia

[Autom. eng. transl.] Nowadays, advanced diagnostics of hematological neoplasms cannot ignore the integration of the clinical, morphological and immunophenotypic aspects, with the genetic-molecular alterations characterized at the level of cytogenetics, FISH and molecular biology methods For some neoplasms the mutation of a single gene is predominant and defines the disease, however in the vast majority of cases there is a complex pattern of genetic alterations with the greater frequency of some compared to others with the distinction of early driver mutations, late alterations and passing mutations. In the hematological field, clinically relevant genomic aberrations have been identified with diagnostic, prognostic, predictive impact on response to a given treatment and as markers of measurable residual disease (MRD), which need to be identified and reported for each individual patient. Technological advances in recent years, especially thanks to high-throughput sequencing methods, have changed molecular diagnostics, moving it from studies on single markers to more comprehensive approaches with analysis of several genes simultaneously at the DNA and RNA level. This made it possible to characterize the genetic-molecular panorama of neoplasms of the lymphoid and haematopoietic tissues in a more in-depth manner and contributed to the revision of their classification as well as to the better definition and distinction of new and old nosographic entities. High-throughput sequencing methods have joined traditional PCR-based techniques and vary from target-sequencing of a limited number of genes to whole exome sequencing (WES), which analyzes the coding regions of genes, or whole-genome sequencing ( WGS), which analyzes the entire genome. These methods have different abilities to detect somatic aberrations, as the targeted approaches have a higher sequence depth and greater sensitivity in detecting subclonal alterations compared to genome-wide technologies capable of identifying mutations characterized by a greater allele frequency. Target sequencing methods can study DNA or RNA and have panels based either on amplicons or on the hybridization capture technique. The latter panels are able to simultaneously study multiple single nucleotide variations (SNVs), insertions/deletions (indels), copy number aberrations (CNAs) and structural variants such as rearrangements. “All-in-one” capture-based panels have been developed that can identify the most frequent genomic alterations in lymphoid and myeloid neoplasms, which are easier to routinely apply than molecular diagnostics. Gene Expression Profiling (microarray) methods have contributed to identifying biologically and prognostically distinct subtypes, especially in lymphomas, and to studying the tumor microenvironment. Target approaches that are easier to apply in routine have subsequently been developed, such as Nanostring, Reverse Transcriptase Multiplex Ligation–Dependent Probe Amplification (RT-MLPA) and Whole Transcriptome Sequencing.