Abstract 1130P
Background
The increasing number of drugs targeting genomic rearrangements make their detection an important step for cancer management. Fluorescence in-situ hybridization (FISH) is the gold standard for the detection of ALK and ROS1 fusions but multiplexed technologies are needed to allow analysis of several potential targets simultaneously. We have evaluated a targeted RNAseq panel based on Anchored Multiplex PCR (AMP) for the detection of rearrangements in NSCLC patients.
Methods
92 patients with late stage NSCLC were evaluated. Total nucleic acids were extracted from tissue sections using the Maxwell RSC RNA FFPE kit (Promega). ALK and ROS1 status were determined by immunohistochemistry (IHC) and confirmed by FISH. Libraries were prepared using the QIAseq Targeted RNAscan Lung Cancer Panel (Qiagen) with 200ng of RNA and sequenced on an Illumina MiSeq (300 cycle MiSeq Reagent Kit V2). Discordant cases were confirmed by RT-PCR.
Results
Analysis of tumor samples with the QIAseq panel allowed us to detect 20 ALK rearrangements (with 3 different partners, EML4, KLC1, STRN) and 6 ROS1 fusions (with CD74, CTNND1, SLC34A2). Five analyses were not contributive. The remaining samples were negative for ALK and ROS1. 15 of the 20 ALK RNAseq+ were FISH+. The remaining 5 samples (1 FISH-, 1 FISH not contributive and 3 results not available) were further analyzed by RT-PCR. 4 were positive, and 1 was negative (STRN-ALK not covered by the assay). 4 of the 6 ROS1 RNAseq+ were FISH+. One was FISH- and 1 result was not available. RT-PCR confirmed the ROS1 rearrangement for the first one but not for the second despite a high NGS coverage (2000 reads, fusion fraction 36%). In the RNAseq ALK and ROS1 negative tumors, 1 was ALK FISH+ and confirmed by RT-PCR. In addition, 3 RET fusions (with CCDC6 and KIF5B) and 1 TPM3-NTRK1 fusion were detected. Altogether, using FISH as a reference, the overall agreement of RNA sequencing for ALK and ROS1 fusions detection was 96% (Sensitivity: 95%; Specificity: 96%), and additional gene rearrangements were identified.
Conclusions
Using an AMP based library preparation, RNA sequencing on FFPE samples allowed us to identify multiple gene rearrangements with a turn around time compatible with clinical requirement.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
Marc G Denis.
Funding
Has not received any funding.
Disclosure
All authors have declared no conflicts of interest.