RNA-Based Detection of Gene Fusions in Formalin-Fixed and Paraffin-Embedded Solid Cancer Samples

Abstract

Oncogenic gene fusions are important drivers in many cancer types, including carcinomas, with diagnostic and therapeutic implications. Hence, sensitive and rapid methods for parallel profiling in formalin-fixed and paraffin-embedded (FFPE) specimens are needed. In this study we analyzed gene fusions in a cohort of 517 cases where standard treatment options were exhausted. To this end the Archer® DX Solid tumor panel (AMP; 285 cases) and the Oncomine Comprehensive Assay v3 (OCA; 232 cases) were employed. Findings were validated by Sanger sequencing, fluorescence in situ hybridization (FISH) or immunohistochemistry. Both assays demonstrated minimal dropout rates (AMP: 2.4%; n = 7/292, OCA: 2.1%; n = 5/237) with turnaround times of 6–9 working days (median, OCA and AMP, respectively). Hands-on-time for library preparation was 6 h (AMP) and 2 h (OCA). We detected n = 40 fusion-positive cases (7.7%) with TMPRSS2::ERG in prostate cancer being most prevalent (n = 9/40; 22.5%), followed by other gene fusions identified in cancers of unknown primary (n = 6/40; 15.0%), adenoid cystic carcinoma (n = 7/40; 17.5%), and pancreatic cancer (n = 7/40; 17.5%). Our results demonstrate that targeted RNA-sequencing of FFPE samples is feasible, and a well-suited approach for the detection of gene fusions in a routine clinical setting.

A gene expression signature associated with B cells predicts benefit from immune checkpoint blockade in lung adenocarcinoma

“…In a growing number of clinical laboratories, mRNA analysis is performed in parallel to DNA analysis as part of the routine NGS diagnostic work-up of lung adenocarcinoma for the detection of oncogenic gene fusions. 20 Gene expression based immune profiling could be easily integrated in this workflow without the need for additional tissue and would represent an excellent opportunity to incorporate comprehensive TME characterization into the clinical work-up.…”

NTRKtesting: First results of theQuiP‐EQAscheme and a comprehensive map ofNTRKfusion variants and their diagnostic coverage by targetedRNA‐basedNGSassays

“…While we are aware that this diagnostic coverage map only represents a snapshot of a dynamic and emerging landscape of fusion variants and assays, the data emphasize that knowledge of both, wet‐lab performance data 23 and technical dry‐lab specifications support the informed use of RNA‐based tNGS assays in routine diagnostics.…”

Predictive molecular pathology of lung cancer in Germany with focus on gene fusion testing: Methods and quality assurance

“…[65][66][67] In the past few years many attempts were underway to replace the conventional techniques by methods like the nCounter technology 68 or parallel sequencing. 11,12 Several algorithms for fusion diagnostics in a clinical setting have been suggested, all of which include parallel sequencing as a last resort. 15,42,69…”

“…[8][9][10] Technical advances and improved protocols for the extraction of RNA from formalin-fixed, paraffin-embedded Cancer Cytopathology September 2020 (FFPE) tissue also facilitate the detection of gene fusions involving, for instance, ALK, ROS1, NTRK, and RET using targeted parallel sequencing approaches. [11][12][13] All molecular methods used for the identification of targetable biomarkers can be applied on cytological material as well. 14 Especially in the context of a low amount of tumor cells, parallel sequencing has the advantage of analyzing multiple parameters in a single approach.…”