An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer Patients

Abstract

The number of genomic aberrations known to be relevant in making therapeutic decisions for non-small cell lung cancer patients has increased in the past decade. Multiple molecular tests are required to reliably establish the presence of these aberrations, which is challenging because available tissue specimens are generally small. To optimize diagnostic testing, we developed a transcriptome-based next-generation sequencing (NGS) assay based on single primed enrichment technology. We interrogated 11 cell lines, two patient-derived frozen biopsies, nine pleural effusion, and 29 formalin-fixed paraffin-embedded (FFPE) samples. All clinical samples were selected based on previously identified mutations at the DNA level in EGFR, KRAS, ALK, PIK3CA, BRAF, AKT1, MET, NRAS, or ROS1 at the DNA level, or fusion genes at the chromosome level, or by aberrant protein expression of ALK, ROS1, RET, and NTRK1. A successful analysis is dependent on the number of unique reads and the RNA quality, as indicated by the DV200 value. In 27 out of 51 samples with >50 K unique reads and a DV200 >30, all 19 single nucleotide variants (SNVs)/small insertions and deletions (INDELs), three MET exon 14 skipping events, and 13 fusion gene transcripts were detected at the RNA level, giving a test accuracy of 100%. In summary, this lung-cancer-specific all-in-one transcriptome-based assay for the simultaneous detection of mutations and fusion genes is highly sensitive.

Keywords: RNA sequencing; exon skipping; gene fusion; mutation; non-small cell lung cancer.

Figure 1

Schematic representation of the 51 samples included in our all-in-one transcriptome assay and the 60 known mutations. Shown are the number of samples for each source of tumor material. Lower boxes indicate the expected number of single nucleotide variants (SNVs)/insertions or deletions (INDELs); MET exon skipping mutations and fusion genes are indicated. WT: wild-type, samples without known mutations; PE, pleural effusions; FFPE, formalin-fixed paraffin-embedded tissue.

Figure 2

Integrated Genomics Viewer (IGV) screenshot of reads mapping to MET exons 13 to 15 for a randomly selected control sample (P34_S2) without MET exon 14 skipping, two cell lines H596 and Hs746, and one patient (P21) with known MET exon 14 skipping mutations. Numbers indicate the average coverage per exon.

Figure 3

Validation by droplet digital (dd)PCR and threshold estimation for FFPE samples. (A) Comparison of the variant allele frequencies (VAFs) as detected by the all-in-one transcriptome-based assay and ddPCR. The Y-axis represents VAFs of the mutations, as assessed by our all-in-one next-generation sequencing (NGS) assay. The X-axis represents the fraction abundance calculated from ddPCR. (B) Overview of unique read counts (Y-axis) in samples for which we did and did not observe the genomic aberrations with our all-in-one transcriptome-based assay. •Blue dots indicate samples with DV200 above 30. •Red dots indicate samples with DV200 below 30. •Black dots indicate samples for which the DV200 value was not measured. Dashed line indicates the cut-off level of 50,000 unique reads.