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.

Detecting therapy-guiding RNA aberrations in platelets of non-small cell lung cancer patients

“…The DV200 value of the platelet-derived RNA samples ranged from 49-76 and fulfilled the cut-off criterium of 30 as set for our assay [30]. The number of unique on-target reads ranged from 1.2⨯10 5 to 1.4⨯10 6 (mean 8.4⨯10 5 ), again well above 5⨯10 4 which is the threshold determined in our previous study (Figure 2).…”

“…As a quality marker for purity of the platelet samples, landing-probes for the platelet marker GP9 (platelet glycoprotein IX/CD42a) and the white blood cell marker PTPRC (CD45) were included in our targeted NGS design. Results obtained for the platelet samples were compared to our previous results for the same targeted NGS design on cell lines, pleural effusion, fresh-frozen and FFPE tissue samples [30]. In the current study we have applied the criteria for good quality data as determined in our previous study, i.e.…”

“…The DV200 value, as determined by fragment analyzer (Agilent), was used as RNA-quality measurement. Libraries were prepared according to the user guide of the ovation fusion panel target enrichment system and ovation cDNA module for target enrichment (NuGEN) using the custom designed gene panel described elsewhere [30] with a total RNA input of 200-400 ng. Libraries were mixed in equimolar amounts and sequenced on the MiSeq platform (Illumina, San Diego, USA) with 150 bp paired-end reads according to the protocol of the manufacturer.…”

“…If so, platelets would be an additional source for minimally invasive molecular testing for tumor specific abbreviations. We tested the most common therapy-driving variants in platelet RNA from NSCLC patients using our customized RNA-based NGS assay as described previously [30]. This assay showed high sensitivity to detect these variants even in FFPE tissue biopsies.…”

New Therapeutic Strategies for Lung Cancer

“…However, since the available tissue samples are generally small and several molecular tests are required to reliably establish the presence of genomic aberrations, this determination can be difficult. Jiacong Wei et al [ 12 ] present a novel method that can detect genomic alterations in a single RNA-based, assay. This test has a high accuracy even on formalin-fixed-paraffin-embedded tissue samples.…”