High Yield of RNA Sequencing for Targetable Kinase Fusions in Lung Adenocarcinomas with No Mitogenic Driver Alteration Detected by DNA Sequencing and Low Tumor Mutation Burden

Abstract

Purpose: Targeted next-generation sequencing of DNA has become more widely used in the management of patients with lung adenocarcinoma; however, no clear mitogenic driver alteration is found in some cases. We evaluated the incremental benefit of targeted RNA sequencing (RNAseq) in the identification of gene fusions and MET exon 14 (METex14) alterations in DNA sequencing (DNAseq) driver-negative lung cancers.Experimental Design: Lung cancers driver negative by MSK-IMPACT underwent further analysis using a custom RNAseq panel (MSK-Fusion). Tumor mutation burden (TMB) was assessed as a potential prioritization criterion for targeted RNAseq.Results: As part of prospective clinical genomic testing, we profiled 2,522 lung adenocarcinomas using MSK-IMPACT, which identified 195 (7.7%) fusions and 119 (4.7%) METex14 alterations. Among 275 driver-negative cases with available tissue, 254 (92%) had sufficient material for RNAseq. A previously undetected alteration was identified in 14% (36/254) of cases, 33 of which were actionable (27 in-frame fusions, 6 METex14). Of these 33 patients, 10 then received matched targeted therapy, which achieved clinical benefit in 8 (80%). In the 32% (81/254) of DNAseq driver-negative cases with low TMB [0-5 mutations/Megabase (mut/Mb)], 25 (31%) were positive for previously undetected gene fusions on RNAseq, whereas, in 151 cases with TMB >5 mut/Mb, only 7% were positive for fusions (P < 0.0001).Conclusions: Targeted RNAseq assays should be used in all cases that appear driver negative by DNAseq assays to ensure comprehensive detection of actionable gene rearrangements. Furthermore, we observed a significant enrichment for fusions in DNAseq driver-negative samples with low TMB, supporting the prioritization of such cases for additional RNAseq.See related commentary by Davies and Aisner, p. 4586

Assessment of a New ROS1 Immunohistochemistry Clone (SP384) for the Identification of ROS1 Rearrangements in Patients with Non–Small Cell Lung Carcinoma: the ROSING Study

“…38,44 The choice of RNA-based NGS can reduce the risk of false negatives, and using another sample or a third technology (i.e., FISH) when the initial NGS approach fails is mandatory to confirm those positive IHC results. 39,47 In conclusion, the new SP384 clone showed high sensitivity without compromising specificity, so it is another excellent analytical option for the proposed CAP/IASLC/AMP molecular testing algorithm. Consideration of the clinical problem of NSCLC highlights the need to be aware of how the methods that we use perform in the real-world setting.…”

“…24,37,45 The percentage of cases in which the suboptimal RNA quality and/or quantity resulted in low sequencing coverage highlights the need for an evidence-based algorithmic approach. 39,46,47 The fusion partner can influence both the IHC staining and the FISH pattern, with the EZR variant usually being associated with a membranous accentuation and isolated 3 0 signals, respectively. 13,14,25,45 This latter association could explain some of the cases with false-negative FISH results that were found to contain the EZR-ROS1 transcript, as this atypical pattern is in fact the most difficult to score because the isolated 3 0 signals can sometimes be absent or barely visible.…”

TRK Fusions Are Enriched in Cancers with Uncommon Histologies and the Absence of Canonical Driver Mutations

“…All pediatric and adult patients with TRK fusion-positive cancers identified at Memorial Sloan Kettering (MSK) between April 7, 2015 and August 15, 2018 were included for analysis under an IRB-approved retrospective research protocol. Patients were characterized as harboring TRK fusions on the basis of DNA-based hybrid-capture next-generation sequencing (MSK-IMPACT) 14 or targeted RNA sequencing using anchored multiplex PCR technology (MSK-Fusion) 15 . Three MSK-IMPACT assay versions were used (version 1, 310 genes; version 2, 410 genes; version 3, 468 genes).…”

Updated guidelines for predictive biomarker testing in advanced non-small-cell lung cancer: a National Consensus of the Spanish Society of Pathology and the Spanish Society of Medical Oncology

“…All these procedures urgently require quality control on the pre-test fixation parameters, as well as control of tumour cellularity and quality control for the nucleic acids [94]. Although there are advantages and limitations associated with amplicon-based and hybrid capture solutions, the most important thing to keep in mind is the need to use RNA when looking for druggable fusions to avoid a significant risk of false negatives [95].…”