Characterization of MET Exon 14 Skipping Alterations (in NSCLC) and Identification of Potential Therapeutic Targets Using Whole Transcriptome Sequencing

Abstract

Introduction: Genomic alterations in the juxtamembrane exon 14 splice sites in NSCLC lead to increased MET stability and oncogenesis. We present the largest cohort study of MET Exon 14 (METex14) using whole transcriptome sequencing.

Methods: A total of 21,582 NSCLC tumor samples underwent complete genomic profiling with next-generation sequencing of DNA (592 Gene Panel, NextSeq, whole exome sequencing, NovaSeq) and RNA (NovaSeq, whole transcriptome sequencing). Clinicopathologic information including programmed death-ligand 1 and tumor mutational burden were collected and RNA expression for mutation subtypes and MET amplification were quantified. Immunogenic signatures and potential pathways of invasion were characterized using single-sample gene set enrichment analysis and mRNA gene signatures.

Results: A total of 533tumors (2.47%) with METex14 were identified. The most common alterations were point mutations (49.5%) at donor splice sites. Most alterations translated to increased MET expression, with MET co-amplification resulting in synergistic increase in expression (q < 0.05). Common coalterations were amplifications of MDM2 (19.0% versus 1.8% wild-type [WT]), HMGA2 (13.2% versus 0.98% WT), and CDK4 (10.0% versus 1.5% WT) (q < 0.05). High programmed death-ligand 1 > 50% (52.5% versus 27.3% WT, q < 0.0001) and lower proportion of high tumor mutational burden (>10 mutations per megabase, 8.3% versus 36.7% WT, p < 0.0001) were associated with METex14, which were also enriched in both immunogenic signatures and immunosuppressive checkpoints. Pathways associated with METex14 included angiogenesis and apical junction pathways (q < 0.05).

Conclusions: METex14 splicing alterations and MET co-amplification translated to higher and synergistic MET expression at the transcriptomic level. High frequencies of MDM2 and CDK4 co-amplifications and association with multiple immunosuppressive checkpoints and angiogenic pathways provide insight into potential actionable targets for combination strategies in METex14 NSCLC.

Keywords: Immune signatures; MDM2; METex14; Non–small cell lung cancer; RNA expression; Whole transcriptome sequencing.

Figure 1

(A) Spatial chromosomal representation of METex14 mutation subtypes. (B) Distribution of METex14 alteration on the basis of mutation subtype. (C) Frequency of often identified DNA alterations. (D) Frequency of co-alterations (left) or co-amplifications (right) of METex14 and MET WT. (E) Oncoprint of METex14 and co-alterations in TP53, MDM2, CDK4, and HMGA2 (green: mutation detected; red: amplification detected; gray: wild type; white: data not available). CNA > 6. CNA, copy number alteration; METex14, MET exon 14; NGS, next-generation sequencing; WT, wild type.

Figure 2

(A) mRNA expression of MDM2, CDK4, and HMGA2 on the basis of MDM2 co-amplification in METex14 and MET WT (y axis, log₂-transformed TPM; error bars represented interquartile range and median presented). (B) Oncoprint of MDM2, CDK4, and HMGA2 represented with mRNA expression levels. Red indicates higher expression and blue lower expression. (C) MET expression on the basis of METex14 mutation subtype (left) and MET co-amplification (right). (D) Ratios of METex14 mutation junction reads to WT junction reads in METex14/Amp+ versus METex14/Amp- (left) and Spearman correlation (right) of MET expression and ratio of METex14 junction reads to WT junction reads (gray dots: METex14/Amp-; red dots: METex14/Amp+). (E) Oncoprint of METex14 and MET co-amplification represented against MET expression and METex14 junction reads. Amp, amplification; METex14, MET exon 14; TPM, Trusted Platform Module; WT, wild type.

Figure 3

(A) Kaplan-Meier plot of overall survival (date of tissue collection to last contact) of METex14 patients with and without amplification. (B) Kaplan-Meier plot of overall survival (date of tissue collection to last contact) of MET WT patients with and without amplification. (C) Kaplan-Meier plot of overall survival (date of tissue collection to last contact) of MET-amplified patients with and without METex14. (D) Kaplan-Meier plot of overall survival (date of tissue collection to last contact) of non–MET-amplified patients with and without METex14. HR, hazard ratio; CI, confidence interval; METex14, MET exon 14; WT, wild type.

Figure 4

(A) METex14 and MET WT patients stratified by PD-L1 less than 1%, 1% to 49%, and greater than or equal to 50% (left). Percentage of patients with high TMB (>10 mut/Mb) in METex14 and MET WT cohorts (right). (B) Oncoprint of METex14 with smoking history against TMB, PD-L1, IFN-γ signature, and T-cell inflammation signature. (C) IFN-γ signature (left) and T-cell inflammation signature (right) in METex14 and MET WT. (D) mRNA expression of immune checkpoints in METex14, MET WT, KRAS mutant, and EGFR mutant cohorts. IFN-γ, interferon-γ; METex14, MET exon 14; mut/Mb, mutations per megabase; PD-L1, programmed death-ligand 1; WT, wild type.

Figure 5

(A) ssGSEA pathway analysis of METex14 patients. (B) mRNA expression of genes previously found to be up-regulated in METex14 in in vitro models in METex14 and MET WT cohorts. METex14, MET exon 14; mut/Mb, mutations per megabase; FC, fold change; ssGSEA, single-sample gene enrichment analysis; WT, wild type.