Multiplex RNA‐based detection of clinically relevant MET alterations in advanced non‐small cell lung cancer

Abstract

MET inhibitors have shown activity in non-small cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect and thus response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with Next Generation Sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally co-exist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

Detecting ALK, ROS1, and RET fusions and the METΔex14 splicing variant in liquid biopsies of non‐small‐cell lung cancer patients using RNA‐based techniques

“…DNA or RNA‐based NGS is currently the multiplex method of choice to determine gene fusions and splicing variants in liquid biopsies, while qPCR‐derived techniques are widely used to detect specific alterations. Our results demonstrate that nCounter and dPCR can also be successfully applied for the detection of fusions and splicing transcripts not only in tissue [12,14] but also in blood and other fluids. Remarkably, the combination of nCounter and dPCR analysis enabled the detection of aberrant transcripts in 87.5% of the cfRNA samples from patients positive in tumor (Fig.…”

Performance of an RNA-Based Next-Generation Sequencing Assay for Combined Detection of Clinically Actionable Fusions and Hotspot Mutations in NSCLC

“…This aspect gains in interest as characterization of fusion variants to predict clinical response and concerns for missing MET exon 14 skipping mutations when using DNA-based NGS only is emerging. 14 , 21 In addition, the focused hotpot approach for SNVs/indel calling reduces the burden of interpreting unknown variants. Although counterintuitive for the NGS technology, it responds to recommendations observed in some jurisdiction to filter out variants not relevant to the clinical indication or tumor type, as reflected in some decisions rendered from health technology assessment bodies.…”

RNA-Based Multiplexing Assay for Routine Testing of Fusion and Splicing Variants in Cytological Samples of NSCLC Patients

“…RNA was retrotranscribed with the M-MLV retrotranscriptase (ThermoFisher Scientific, Waltham, MA, USA) and random primers. HotStart Taq polymerase (Qiagen) was used for MET∆ex14 and EML4-ALK amplification using a 20 µL reaction (45 cycles) and visualized in agarose gels, as described [9,11]. Positive samples were confirmed by bidirectional Sanger sequencing of RT-PCR products, using the big-dye 3.1 sequencing kit (Applied Biosystems, Foster City, CA, USA).…”

“…In both cases, a BenchMark ULTRA automated tissue staining system was used (Ventana Medical Systems, Valle del Oro, AZ, USA). Membrane staining was graded as described [9,11].…”

“…We have previously reported the use of nCounter for simultaneous detection of fusions in ALK, ROS1, and RET [9,10] and, more recently, for MET alterations [11]. In this article, we analyze the prospective use of the nCounter technology for the detection of fusions, MET∆ex14 splicing and MET very high expression in NSCLC cytological samples and we compare the results with FFPE biopsies.…”