Perspective from Trevor Angell, MD
Disclosures: Kaya reports no relevant financial disclosures. Please see the study for all other authors’ relevant financial disclosures.
November 18, 2020
3 min read

RNA sequencing fails to detect many genetic variants in thyroid tumor, tissue samples

Perspective from Trevor Angell, MD
Disclosures: Kaya reports no relevant financial disclosures. Please see the study for all other authors’ relevant financial disclosures.
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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.

Cihan Kaya

“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.”

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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.