Despite the immense genetic heterogeneity of B-lymphoblastic leukemia (B-ALL), RNA-Seq could comprehensively interrogate its genetic drivers, assigning a specific molecular subtype in >90% of patients. However, study groups have only started to use RNA-Seq. For broader clinical use, technical, quality control and appropriate performance validation are needed.
Researchers at the National University of Singapore describe the development and validation of an RNA-Seq workflow for subtype classification, TPMT/NUDT15/TP53 variant discovery and IGH disease clone identification for Malaysia-Singapore ALL-Seq 2020 (MS2020). The researchers validated this workflow in 377 patients in their preceding MS2003/MS2010 studies and proposed the quality control measures for RNA quality, library size, sequencing, and data analysis using the International Organization for Standardization ISO 15189 quality and competence standard for medical laboratories. Compared to conventional methods, the researchers achieved >95% accuracy in oncogene fusion identification, digital karyotyping as well as TPMT and NUDT15 variant discovery. They found 7 pathogenic TP53 mutations, confirmed with Sanger sequencing, which conferred a poorer outcome. Applying this workflow prospectively to the first 21 patients in MS2020, they identified the genetic drivers and IGH disease clones in >90% of patients with concordant TPMT, NUDT15 and TP53 variants using PCR-based methods. The median turnaround time was 12 days which was clinically actionable. In conclusion, RNA-Seq workflow could be used clinically in management of B-ALL patients.