Limitations of Detecting Genetic Variants from the RNA Sequencing Data in Tissue and Fine-Needle Aspiration Samples
Cihan Kaya1, Princesca Dorsaint2,3,4, Stephanie Mercurio1
1Division of Molecular and Genomic Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA.
Abstract:
Genetic profiling of resected tumor or biopsy samples is increasingly used for cancer diagnosis and therapy selection for thyroid and other cancer types. Although mutations occur in cell DNA and are typically detected using DNA sequencing, recent attempts focused on detecting pathogenic variants from RNA. The aim of this study was to determine the completeness of capturing mutations using RNA sequencing (RNA-Seq) in thyroid tissue and fine-needle aspiration (FNA) samples. To compare the detection rate of mutations between DNA sequencing and RNA-Seq, 35 tissue samples were analyzed in parallel by whole-exome DNA sequencing (WES) and whole-transcriptome RNA-Seq at two study sites. Then, DNA and RNA from 44 thyroid FNA samples and 47 tissue samples were studied using both targeted DNA sequencing and RNA-Seq. Of 162 genetic variants identified by WES of DNA in 35 tissue samples, 77 (48%) were captured by RNA-Seq, with a detection rate of 49% at site 1 and 46% at site 2 and no difference between thyroid and nonthyroid samples. Targeted DNA sequencing of 91 thyroid tissue and FNA samples detected 118 pathogenic variants, of which 57 (48%) were identified by RNA-Seq. For DNA variants present at >10% allelic frequency (AF), the detection rate of RNA-Seq was 62%, and for those at low (5-10%) AF, the detection rate of RNA-Seq was 7% (p < 0.0001). For common oncogenes (BRAF and RAS), 94% of mutations present at >10% AF and 11% of mutations present at 5-10% AF were captured by RNA-Seq. As expected, none of TERT promoter mutations were identified by RNA-Seq. The rate of mutation detection by RNA-Seq was lower in FNA samples than in tissue samples (32% vs. 49%, p = 0.02). In this study, RNA-Seq analysis detected only 46-49% of pathogenic variants identifiable by sequencing of tumor DNA. Detection of mutations by RNA-Seq was more successful for mutations present at a high allelic frequency. Mutations were more often missed by RNA-Seq when present at low frequency or when tested on FNA samples. All TERT mutations were missed by RNA-Seq. These data suggest that RNA-Seq does not detect a significant proportion of clinically relevant mutations and should be used with caution in clinical practice for detecting DNA mutations.
Insights
RNA sequencing (RNA-Seq) captures less than half of cancer DNA mutations, especially those at low allelic frequency or in fine-needle aspiration samples. This method should be used cautiously for clinical detection of DNA mutations.
Area of Science:
- Genomics
- Molecular Biology
- Oncology
Background:
- Genetic profiling of tumors is crucial for cancer diagnosis and treatment selection.
- While DNA sequencing is standard, RNA sequencing (RNA-Seq) is being explored for detecting mutations.
- Thyroid cancer and other cancers rely on accurate mutation detection.
Purpose of the Study:
- To evaluate the completeness of mutation detection using RNA sequencing (RNA-Seq) in thyroid tissue and fine-needle aspiration (FNA) samples.
- To compare the mutation detection rates between DNA sequencing and RNA-Seq.
- To assess the impact of variant allelic frequency (AF) and sample type on RNA-Seq detection rates.
Main Methods:
- Whole-exome DNA sequencing (WES) and whole-transcriptome RNA-Seq were performed on 35 tissue samples.
- Targeted DNA sequencing and RNA-Seq were used on 44 thyroid FNA and 47 tissue samples.
- Detection rates were compared based on variant AF, sample type, and specific genes (BRAF, RAS, TERT).
Main Results:
- RNA-Seq captured 48% of genetic variants identified by WES and 48% of pathogenic variants from targeted DNA sequencing.
- Detection rates were significantly higher for variants with >10% AF (62%) compared to low AF (5-10%, 7%).
- RNA-Seq missed all TERT promoter mutations and showed lower detection rates in FNA samples (32%) versus tissue samples (49%).
Conclusions:
- RNA-Seq detects less than half of the pathogenic variants identifiable by DNA sequencing.
- High allelic frequency is critical for successful mutation detection by RNA-Seq.
- RNA-Seq is unreliable for comprehensive detection of DNA mutations in clinical practice, particularly for low-frequency variants and FNA samples.
