Comparison of the genomic background of MET-altered carcinomas of the lung: biological differences and analogies

Abstract

Although non-small-cell lung cancer is a leading cause of cancer-related deaths, the molecular characterization and classification of its genetic alterations has drastically changed treatment options and overall survival within the last few decades. In particular, tyrosine kinase inhibitors targeting specific molecular alterations, among other MET, have greatly improved the prognosis of non-small-cell lung cancer patients. Here, we compare the genomic background of a subset of non-small-cell lung cancer cases harboring either a MET high-level amplification (n = 24) or a MET exon 14 skipping mutation (n = 26), using next-generatison sequencing, fluorescence in situ hybridization, immunohistochemistry, and Nanostring nCounter® technology. We demonstrate that the MET-amplified cohort shows a higher genetic instability, compared with the mutant cohort (p < 0.001). Furthermore, MET mutations occur at high allele frequency and in the presence of co-occurring TP53 mutations (n = 7), as well as MDM2 (n = 7), CDK4 (n = 6), and HMGA2 (n = 5) co-amplifications. No other potential driver mutation has been detected. Conversely, in the MET-amplified group, we identify co-occurring pathogenic NRAS and KRAS mutations (n = 5) and a significantly higher number of TP53 mutations, compared with the MET-mutant cohort (p = 0.048). Of note, MET amplifications occur more frequently as subclonal events. Interestingly, despite the significantly (p = 0.00103) older age at diagnosis of stage IIIb/IV of MET-mutant patients (median 77 years), compared with MET high-level amplified patients (median 69 years), MET-mutant patients with advanced-stage tumors showed a significantly better prognosis at 12 months (p = 0.04). In conclusion, the two groups of MET genetic alterations differ, both clinically and genetically: our data strongly suggest that MET exon 14 skipping mutations represent an early driver mutation. In opposition, MET amplifications occur usually in the background of other strong genetic events and therefore MET amplifications should be interpreted in the context of each tumor's genetic background, rather than as an isolated driver event, especially when considering MET-specific treatment options.

Comparison of in situ and extraction-based methods for the detection of MET amplifications in solid tumors

“…A technical comparison on MET expression analysis by immunohistochemistry also showed a high interlaboratory variability and highlighted the need for harmonization of MET IHC [21]. Nevertheless, in other studies MET IHC positivity was significantly (P < 0.001, χ 2 test) associated with MET high-level amplification and MET high-level amplified samples can be selected by high MET IHC scores, which is in concordance with our study [13], [22].…”

“…In the extraction-based technologies, however, intratumoral heterogeneity in the signal pattern of the MET FISH can result in a lower MET copy numbers and thus in false negative results. Partly, this has been reported previously [13]. It was also shown in gastric-/esophageal adenocarcinoma that MET amplification was markedly higher in tumor specimens with a heterogeneous signal distribution [26], which might be problematic for the detection by extraction-based technologies.…”

“…It was also shown in gastric-/esophageal adenocarcinoma that MET amplification was markedly higher in tumor specimens with a heterogeneous signal distribution [26], which might be problematic for the detection by extraction-based technologies. Further, in our study FFPE material with varying DNA quality was used, therefore after extensive validation, literature research [13], [14] and manufactures instruction the cut-offs were set quite high to a gene copy number ≥2.5 and a log2 ≥0.5 to decrease the number of false positives by elevated background noise.…”

“…MET IHC was performed on 1–2 µm thick FFPE slides cut and mounted on X-tra Adhesive Slides (Leica, Wetzlar, Germany) using a Ventana Benchmark Ultra automated immunostainer and the clone SP44 (Ventana Medical Systems, Tucson, AZ) as previously described [13]. Each sample was assessed using the following scoring criteria for staining intensity: 0 = no staining or <50% of tumor cells with any intensity; 1+ = ≥50% of tumor cells with weak staining but <50% with moderate or higher intensity, 2+ = ≥50% of tumor cells with moderate or higher staining but <50% with strong intensity and 3+ = ≥50% of tumor cells were stained with strong intensity.…”

“…All samples were analyzed for copy-number alterations of MET using the NanoString nCounter platform (NanoString Technologies, Seattle, WA, USA). Copy number analysis was performed as previously described [13], [14] using 200–600 ng of genomic DNA. Based on the manufacturer’s protocol, published literature [13], [14] and validation studies the gene was considered to be a single copy if the average copy number was below 1.4, two copies if between 1.5 and 2.4, three copies if between 2.5 and 3.4 and continued.…”