Cost-Efficient and Easy to Perform PCR-Based Assay to Identify Met Exon 14 Skipping in Formalin-Fixed Paraffin-Embedded (FFPE) Non-Small Cell Lung Cancer (NSCLC) Samples

Abstract

MET is a receptor tyrosine kinase (RTK) that plays important roles in carcinogenesis. Despite being frequently overexpressed in cancer, clinical responses to targeting this receptor have been limited. Recently novel splicing mutations involving the loss of exon 14 (called METex14 skipping) have emerged as potential biomarkers to predict for responsiveness to targeted therapies with Met inhibitors in non-small cell lung cancer (NSCLC). Currently, the diverse genomic alterations responsible for METex14 skipping pose a challenge for routine clinical diagnostic testing. In this report, we examine three different methodologies to detect METex14 and assess their potential utility for use as a diagnostic assay for both the identification of METex14 and intra-tumoural distribution in NSCLC.

Keywords: Met exon 14 skipping; PCR; RNA hybridisation; diagnostic assay; next generation sequencing.

Figure 1

End-point PCR detection of METex14 in three cohorts of non-small cell lung cancer (NSCLC) formalin-fixed paraffin-embedded (FFPE). (A) Amplification of the Wild-Type amplicon is limited primarily to high-quality RNA. Samples from snap-frozen tumour tissues versus a sample isolated from an FFPE embedded specimen show that the integrity/quality of RNA is important for detecting WT MET in FFPE embedded samples. A subset of FFPE samples were subsequently examined for the expression of 18S rRNA. Amplification was observed across all specimens; (B) Confirmation of the specificity of the MetEx14 assay on FFPE extracted RNA from known METex14 skipped NSCLC cases from three hospitals: St James’s Hospital (SJH); St Vincent’s University Hospital (SVUH) and St Gallen (SG); (C) Assessment of a cohort of 20 pulmonary sarcomatoid carcinomas (PSCs) for METex14 skipped samples. * indicates that a given sample exhibits the MetEx14 skipping mutation.

Figure 2

Assay sensitivity for end-point PCR detection of METex14 skipping. Sensitivity to detect MetEx14 was measured using either (A) GBlocks or (B) admixtures of patient FFPE RNA with limiting amounts/percentages of WT/METex14 as indicated. The limit of detection was found to be 10%.

Figure 3

RISH analysis. Probe design for the detection of METex14 using the ACDbio BaseScope™ assay. One probe (blue—(i)), designed to detect the junction between exons 12 and 13, is considered common and can detect both wild-type MET and METex14 mRNA. A second probe (orange) is designed to detect the junction between exons 14 and 15 (ii) and detects only MET mRNA containing exon 14 (wild-type MET). A third probe is designed to detect the junction between exons 13 and 15 (iii) and detects only METex14 skipped mRNA (METex14). The three probes are tested in parallel. An example of RISH on a patient sample using these probes demonstrating the presence of METex14 skipping. 20× magnification of (Panel A) exon 12/13 probe (detects all MET); (Panel B) exon 14/15 probe (detects wild-type MET); (Panel C) exon 13/15 probe (detects (METex14)). In this example, almost the entire sample demonstrates METex14. (Panel D) 40× magnification of the (iii) exon 13/15 probe (METex14), while (Panel E) is a H&E stain from the same sample.