RNA sequencing fails to detect many genetic variants in thyroid tumor, tissue samples
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RNA sequencing detected less than half of identifiable genetic mutations found in DNA sequencing of thyroid and brain tumor, thyroid tissue and fine-needle aspiration samples, according to findings published in Thyroid.
“Even though it sounds plausible to detect both mutations and fusions from RNA sequencing data, a significant number of coding and noncoding mutations will be missed,” Cihan Kaya, PhD, lead bioinformatics scientist in the division of molecular and genomic pathology at the University of Pittsburgh Medical Center, told Healio. “This limitation should be accounted for in clinical practice for diagnosis, prognostication and selection of targeted therapeutics.”
Kaya and colleagues analyzed 35 randomly selected tissue specimens from de-identified DNA and RNA samples using whole-exome DNA sequencing and whole-transcriptome RNA sequencing. There were 18 brain tumors analyzed at the Englander Institute for Precision Medicine at Weill Cornell Medicine in New York and 17 thyroid tumors analyzed at the University of Pittsburgh Medical Center. DNA and RNA from 44 thyroid fine-needle aspiration samples and 47 thyroid tissue samples were also studied at the University of Pittsburgh using targeted DNA sequencing and RNA sequencing.
During analysis of the 35 tumor samples, DNA sequencing identified 162 gene variants. RNA sequencing detected 77 of the 162 variants. When broken down by site, RNA sequencing identified 15 of 32 pathogenic variants in 18 brain tumors and 64 of 130 variants in 17 thyroid tumors.
Analysis of 44 thyroid fine-needle aspiration and 47 thyroid tissue samples revealed 118 gene mutations with targeted DNA sequencing. Of that group, 57 mutations were identified through RNA sequencing.
The most common mutations detected in thyroid samples through targeted DNA sequencing were BRAF (n = 27) and RAS (n = 25). RNA sequencing detected 48% of BRAF mutations and 75% of RAS mutations. Of tumor suppressor genes, TP53 was detected 75% of the time by RNA sequencing and PTEN 50%. RNA sequencing detected none of the 23 TERT promoter mutations revealed in DNA sequencing.
Of the 118 mutations detected in the tissue and fine-needle aspiration samples, 89 had a high allelic frequency of 10% or greater, whereas 29 had a low allelic frequency of 5% to 10%. RNA sequencing detected 62% of high allelic-frequency mutations, but only 7% of the low allelic-frequency mutations (P < .0001). Among BRAF and RAS oncogenes, RNA sequencing detected 94% of high allelic mutations and 11% of low allelic mutations.
RNA sequencing detection in thyroid fine-needle aspiration samples was lower compared with tissue samples (36% vs. 49%; P = .02). It remained lower after TERT mutations were excluded.
“DNA sequencing is extremely powerful in detecting mutations with a low allelic frequency, which will provide high-quality information that makes the clinical decision-making process easier for both clinicians and patients,” Kaya said. “RNA sequencing is relying heavily on the expression of the gene, which could be problematic for low-level coding mutations and, as expected, non-coding TERT promoter mutations, which emerged as a valuable prognostic marker that defines an aggressive class of thyroid cancer.”
Kaya said new methods need to be introduced for calling mutations through RNA sequencing data to make it more accurate. In absence of new methods, he said providers should take the possibility of missing relevant mutations into account.
Perspective
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Genetic alterations in thyroid nodules and cancers are increasingly recognized as having prognostic significance and therapeutic implications. Understanding the current ability to accurately identify these meaningful gene changes is therefore crucial to improving care. The recent study by Kaya and colleagues succinctly provides direct comparisons between DNA and RNA methods for assessing these alterations on a common set of thyroid samples. This strong work consistently suggests that measuring RNA expression fails to detect relevant mutations, particularly when mutated allele frequency is low. The inability to detect TERT promotor mutations in RNA was particularly meaningful given the negative prognostic implications of this finding.
How to translate these findings into practice will be an ongoing process. It is noteworthy that the samples were chosen to provide broad inclusion of mutations found in thyroid cancers rather than reflecting a representative clinical population, so the implications of these results in clinical practice is less clear. Though enticing to draw inferences to diagnostic testing, data evaluating selected individual genes provide little information on which kind of test would be superior for the assessment of indeterminate thyroid nodules given the complexity of current commercially available platforms. Advances in RNA interrogation may reveal profiles of RNA expression that predict the presence of a mutation without direct measurement of that genetic sequence. Nevertheless, these valuable data support the robustness of current DNA based testing for assessing individual genes.
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