Added Convenience of Classifying Nodules With ThyroSeq Test?

Miriam E. Tucker

April 28, 2019

LOS ANGELES — New findings bring the advantage of convenience to the latest version of ThyroSeq (University of Pittsburgh/CBLPath), the thyroid nodule multigene classifier test, as it can potentially be performed using routinely prepared cytology smears without the need for biopsy samples to be collected into preservative solution.

Findings from a study of the ThyroSeq Version 3 Genomic Classifier were presented April 27 here at the American Association of Clinical Endocrinologists (AACE) 2019 Annual Scientific & Clinical Congress by the test's creator Yuri E. Nikiforov, MD, PhD, professor of pathology and director, Division of Molecular and Genomic Pathology, University of Pittsburgh School of Medicine, Pennsylvania.

ThyroSeq is a relatively new molecular test used to help determine which of the 20-30% of thyroid nodules deemed "indeterminate" by cytology are malignant and which patients therefore need to undergo surgery based on assessing samples against 112 known thyroid cancer genes. The test calculates the probability of cancer and assigns the results as positive or negative.

The standard method for collecting a sample for molecular testing is to take it while performing the ultrasound-guided fine-needle aspiration (FNA) biopsy used to collect cytology samples that are prepared as slides. Cells collected during the procedure can be placed directly into a dedicated tube containing solution (ThyroSeqPreserve) that conserves the sample for molecular testing, if needed.

But if that additional sample isn't collected during initial biopsy, the patient has to return for a second FNA biopsy to collect the sample for molecular testing. Now, new data suggest cytology smears can be used to perform ThyroSeq testing, as long as sufficient cells are collected.   

"The presented study demonstrates that the ThyroSeq test can be performed with high accuracy in cytology smears, which expands the samples acceptable for testing. This means that patients with ambiguous biopsy results who have not previously had a sample collected for [molecular] testing can avoid a second visit for sample collection because the cytology smears from the original biopsy can be sent for ThyroSeq testing," Nikiforov told Medscape Medical News.

As a result, he added, "these patients will also avoid the associated frustrations, discomfort [needle in the neck], and delays associated with a second visit for a molecular sample collection. Although the cytology material is not specifically collected for molecular testing, the performance of this complex molecular test remains highly accurate in this newly acceptable sample type."

Asked to comment, session moderator Matthew Levine, MD, an endocrinologist at Scripps Clinic, La Jolla, California, and the University of California, San Diego, said, "With these results, the practicality goes up significantly because now you don't need to prepare a preservative solution with needle washout...From a convenience standpoint, it's an enormous deal."

It may also "possibly" represent an advantage over the competitor product, the Afirma test (Veracyte), Levine said.

Results Nearly as Good, Albeit a Bit Slower  

In the current study, ThyroSeq v3 GC analysis was performed on 33 routinely prepared Diff-Quik or Papanicolaou clinically stained slides, with about 200-1000 cells per slide from 14 indeterminate thyroid nodules.

In the cytology smears, adequate DNA results and copy number alterations were obtained in 93% (13/14) of samples, and adequate RNA results for gene fusions and gene expression in 79% (11/14) of samples.

Two smears with fewer than 200-300 cells failed in RNA analysis, as did one cellular smear that was 6 years old because of degraded nucleic acids. 

Used with unstained reference cytology slides with KRAS, NRAS, BRAF, and PIK3CA mutations in allele frequencies of 10%, 5%, 1%, and 0%, ThyroSeq detected all mutations down to 5% and BRAF and PIK3CA mutations down to 1% allele frequency.

No mutations were detected in the negative control slide.

"Based on this analysis, we concluded that the test is sensitive down to about 5% of allele frequency. This means you need to be sure your smear contains at least 10% of cells with mutation. The other 90% can be lymphocytes, contaminants, or normal cells, but as long as there are 10% of cells positive for mutation on the slide, you can expect to have an accurate result," Nikiforov commented during his presentation.

The 79%-93% efficacy of ThyroSeq for providing an informative result was slightly less than the 96% obtained when using the samples from the preserved solution. Also, the turnaround time was slower — about 8-9 days compared to 5-7 days — as removal of the coverslip and preparation from the slide take extra time.

But, Nikiforov pointed out, those issues may be worth it to the patient in terms of not having to return and endure another needle stuck in their neck. 

Still, he told Medscape Medical News, "We still strongly recommend to collect a portion of FNA into a dedicated preservative solution vial because it provides the highest success rate of testing and shortest turnaround time...However, when cytology smears have adequate cellularity, the success rate of testing is sufficiently high and the test remains highly accurate."

He and his colleagues did caution, nevertheless, that "additional studies are needed to provide more extensive validation of cytology smears adequacy for ThyroSeq testing."

Does This Give ThyroSeq an Advantage Over Afirma? "Possibly"

When asked whether this suggests an advantage for ThyroSeq over its competitor, the Afirma test, Levine pointed out that although both tests are used to assess which patients with indeterminate thyroid nodules need to be sent for surgery, they're different in that, traditionally, ThyroSeq has been viewed as a test to "rule-in" cancer and Afirma to "rule-out" cancer (although with newer versions the two have moved closer in those capacities).    

"Possibly this is in favor of ThyroSeq, but you have two tests that are looked at a little bit differently, so that would have to be reconciled…It would be up to clinical practitioners, [based on] what their level of suspicion was based on ultrasound characteristics, cytology results, and other factors, which test they would want to use, but conceivably there could be a benefit of practicality here."

Nikiforov owns intellectual property related to ThyroSeq. Levine is a speaker for Novo Nordisk, Janssen, Merck, Amgen, and Boehringer Ingelheim.   

AACE 2019. Presented April 27, 2019.

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