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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Published on: August 4, 2016
An All-In-One Transcriptome-Based Assay to Identify Therapy-Guiding Genomic Aberrations in Nonsmall Cell Lung Cancer
Jiacong Wei1,2, Anna A Rybczynska1, Pei Meng3,4
1Department of Genetics, University Medical Centre Groningen, University of Groningen, 9700RB Groningen, The Netherlands.
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.
Insights
A new transcriptome-based next-generation sequencing assay accurately detects multiple genomic aberrations in non-small cell lung cancer. This single test optimizes molecular profiling for targeted therapy decisions, even with limited tissue.
Area of Science:
- Genomics and Molecular Biology
- Oncology
- Next-Generation Sequencing (NGS)
Background:
- Therapeutic decisions for non-small cell lung cancer (NSCLC) increasingly rely on identifying genomic aberrations.
- Current molecular testing often requires multiple assays, posing challenges with limited patient tissue specimens.
- There is a need for optimized diagnostic approaches for comprehensive genomic profiling in NSCLC.
Purpose of the Study:
- To develop and evaluate a novel transcriptome-based next-generation sequencing (NGS) assay for NSCLC.
- To enable simultaneous detection of multiple clinically relevant genomic alterations from limited clinical samples.
- To assess the sensitivity and accuracy of the assay in diverse sample types.
Main Methods:
- Development of a single primed enrichment transcriptome-based NGS assay.
- Interrogation of various sample types including cell lines, frozen biopsies, pleural effusions, and FFPE samples.
- Analysis of RNA quality (DV200) and unique read counts for successful assay performance.
Main Results:
- The assay successfully detected all 19 single nucleotide variants (SNVs)/small insertions and deletions (INDELs), 3 MET exon 14 skipping events, and 13 fusion gene transcripts.
- In samples with adequate RNA quality (DV200 >30) and sufficient unique reads (>50 K), the test achieved 100% accuracy.
- The assay demonstrated high sensitivity across different sample types, including challenging formalin-fixed paraffin-embedded (FFPE) specimens.
Conclusions:
- The developed lung-cancer-specific, all-in-one transcriptome-based NGS assay is highly sensitive for simultaneous detection of mutations and fusion genes.
- This assay offers an optimized solution for molecular profiling in NSCLC, addressing limitations of small tissue specimens.
- The assay facilitates comprehensive genomic assessment for informed therapeutic decisions in NSCLC patients.
