Diagnostic perspectives – WGS and WTS

The diagnosis and classification of hematological neoplasia is a complex process. Various diagnostic technologies (cytomorphology, histology, cytogenetics, immunophenotyping and molecular genetics) are needed to meet the clinical challenges for a rapid, accurate and therapeutically relevant diagnosis for each patient. Genetic testing in particular has changed considerably in recent years, making it a quick and comprehensive option for a variety of issues.

Conventional molecular tests have so far been mainly based on the study of genes or gene regions known to be associated with hematological neoplasia. However, for many patients, these tests do not yet provide a conclusive, molecularly justifiable diagnosis. However, recent improvements in sequencing technology and bioinformatics have made time and cost-effective sequencing of the individual genome (WGS) and transcriptome (WTS) a reality. This allows for an equally wide range of molecular information to be collected with a single approach and without prior knowledge and any previously necessary limitation to known genetic aberrations. This can, for example, improve the determination of clonal markers, which can be helpful in the diagnosis of cytopenia of unclear origin.

The WGS opens up the possibility of creating a complete and personalized molecular profile for each patient by simultaneously analyzing somatic changes such as individual nucleotide variants (SNV), small insertions and deletions (indels), structural variants (SV) and variations in the number of copies (CNV). In particular, this advantage can be seen in cases with limited material, such as with multiple myeloma, where the isolation of a sufficient number of plasma cells is often difficult, limiting the number of possible tests. However, assessing the clinical relevance of SNVs and indels is also a major challenge for which there is neither a gold standard nor a standardized pipeline. 

The WTS has been established in diagnostics for about 20 years, mostly in the form of microarray experiments, and is used to determine gene expression and detect fusion transcripts. With the introduction of RNA-Seq, the analysis is performed without prior knowledge for known biomarkers or fusions. RNA-Seq can be used by determining specific expression profiles for the classification of hematological neoplasia, as well as for prognosis assessment. Gene expression is used in acute lymphocytic leukemia (ALL), for example, to identify cases that are similar to BCR-ABL1-positive ALL despite the lack of t(9;22) in its expression pattern and potentially benefit from treatment with kinase inhibitors. In addition, SNVs, indels and CNVs can be determined to a limited extent; however, accuracy is not (yet) comparable to DNA-based analyses.

As part of the 5,000 genome project, more than 4,500 genomes and their transcriptomes have already been sequenced and analyzed at the MLL to gain better research insight into the molecular genetic profile of the various subgroups of hematological neoplasia. The data generated in this way already contributes to the understanding and knowledge of the accuracy of WGS and WTS data. One goal is also to test a practical and efficient use of WGS and WTS for routine diagnostics.