Impact of Rare and Multiple Concurrent Gene Fusions on Diagnostic DNA Methylation Classifier in Brain Tumors.

DNA methylation is an essential molecular assay for central nervous system (CNS) tumor diagnostics. While some fusions define specific brain tumors, others occur across many different diagnoses. We performed a retrospective analysis of 219 primary CNS tumors with whole genome DNA methylation and RNA next-generation sequencing. DNA methylation profiling results were compared with RNAseq detected gene fusions. We detected 105 rare fusions involving 31 driver genes, including 23 fusions previously not implicated in brain tumors. In addition, we identified 6 multi-fusion tumors. Rare fusions and multi-fusion events can impact the diagnostic accuracy of DNA methylation by decreasing confidence in the result, such as BRAF, RAF, or FGFR1 fusions, or result in a complete mismatch, such as NTRK, EWSR1, FGFR, and ALK fusions. IMPLICATIONS: DNA methylation signatures need to be interpreted in the context of pathology and discordant results warrant testing for novel and rare gene fusions.

Towards a single-assay approach: a combined DNA/RNA sequencing panel eliminates diagnostic redundancy and detects clinically-relevant fusions in neuropathology.

Since the introduction of integrated histological and molecular diagnoses by the 2016 World Health Organization (WHO) Classification of Tumors of the Nervous System, an increasing number of molecular markers have been found to have prognostic significance in infiltrating gliomas, many of which have now become incorporated as diagnostic criteria in the 2021 WHO Classification. This has increased the applicability of targeted-next generation sequencing in the diagnostic work-up of neuropathology specimens and in addition, raises the question of whether targeted sequencing can, in practice, reliably replace older, more traditional diagnostic methods such as immunohistochemistry and fluorescence in-situ hybridization. Here, we demonstrate that the Oncomine Cancer Gene Mutation Panel v2 assay targeted-next generation sequencing panel for solid tumors is not only superior to IHC in detecting mutation in IDH1/2 and TP53 but can also predict 1p/19q co-deletion with high sensitivity and specificity relative to fluorescence in-situ hybridization by looking at average copy number of genes sequenced on 1p, 1q, 19p, and 19q. Along with detecting the same molecular data obtained from older methods, targeted-next generation sequencing with an RNA sequencing component provides additional information regarding the presence of RNA based alterations that have diagnostic significance and possible therapeutic implications. From this work, we advocate for expanded use of targeted-next generation sequencing over more traditional methods for the detection of important molecular alterations as a part of the standard diagnostic work up for CNS neoplasms.