Next-Generation Molecular Tests for Thyroid Nodules: Which to Use

A closer look at the validity and efficacy of four molecular tests, including an expert review and assessment of their recommended use in clinical practice.

With Kepal Patel, MD, Marina N. Nikiforova, MD, and Yuri E. Nikiforov MD, PhD

Recent advances in molecular testing may enhance clinical management of patients with thyroid nodules of indeterminate cytology.1,2  

Currently, up to one-third of thyroid nodules receive this designation based on interpretation of fine needle aspiration (FNA) biopsy.3 Of this subset of nodules, at least two-thirds are benign. Too many of these patients have undergone invasive surgical procedures before clinicians are comfortable arriving at this diagnosis but enhanced technology finally may remove this diagnostic uncertainty.

Four molecular tests are now commercially available to provide a clearer cytological assessment of thyroid tumors: Afirma (Veracyte), ThyroSeq (CBLPath), ThyGenX/ThyraMIR (Interpace Diagnostics), and RosettaGX (Rosetta Genomics).1    

Evaluating the differences in validity and efficacy of molecular tests for thyroid nodules.

“Familiarity with data from the validation studies as well as the emerging literature about test performance in the post-validation setting can help users to select and interpret these tests in a clinically meaningfully way,”1 according to Michiya Nishino, MD, PhD, and Marina Nikiforova, MD, in a review published in the Archives of Pathology & Laboratory Medicine.   Here is a closer look at the data and functionality for each of the four tests:

Afirma Gene Sequencing Classifier More Specific, Comparably Accurate to Older Test

Independent testing revealed a 36% increase in specificity of Afirma Gene Sequencing Classifier (GSC) as compared to its predecessor,2 said Darren McGuire, MD, MHSc, professor of medicine at the University of Texas Southwestern Medical Center in Dallas.

Afirma Gene Expression Classifier (GEC), and test sensitivity has been maintained, according to Kepal N. Patel, MD, of New York University Langone Medical Center, and colleagues,2 who published these findings JAMA Surgery.

The Afirma GSC is a next-generation genomic test that relies on RNA sequencing and advanced machine learning methodology to categorize tissue from cytologically indeterminate FNA biopsy as either benign or suspicious.2    

The researchers conducted a blinded validation study of the Afirma GSC using FNA biopsy data collected from 49 clinical sites across the United States. One hundred ninety-one samples with Bethesda III or IV indeterminate cytopathology were assigned to a panel of experts from which a consensus diagnosis was derived.2  

The results supported a sensitivity of 91% (95% CI, 79-98), specificity of 68% (95% CI, 60-76), negative predictive value of 96% (NPV; 95% CI, 90-99), and positive predictive value of 47% (PPV; 95% CI, 36-58), based on a cancer prevalence of 24%.2  

“We were happy with these findings as the improved classifier identified more benign thyroid nodules than in the past, demonstrating improved specificity of the test,” Dr. Patel told EndocrineWeb. “The GSC reliably identified benign thyroid nodules when the cytology was indeterminate.”

ThyroSeq v3 Genomic Classifier Also Well-Validated as Clinical Tool

According to a blinded validation study conducted by Marina Nikiforova, MD, medical director of the Molecular & Genomic Pathology Laboratory at the University of Pittsburgh School of Medicine in Pennsylvania, and colleagues, the ThyroSeq v3 Genomic Classifier (GC) is highly sensitive and specific, and therefore robust for clinical use.3  

ThyroSeq v3 GC is a genomic test using DNA- and RNA-based sequencing for analyzing five different classes of molecular alterations and to categorize lesions as either benign or suspicious.3  

To test the efficacy, a set of 238 tissues samples and 175 FNA samples with known surgical follow-up were analyzed.3 Among the tissue samples, ThyroSeq v3 GC had a sensitivity of 93.9% and a specificity of 89.4% with benign nodules distinguished from cancerous cells with 92.1% accuracy. In the FNA sample set, the test had a sensitivity of 98.0%, a specificity of 81.18%, and an accuracy of 90.9%.3  

In discussing the results with EndocrineWeb, Dr. Nikiforova said: “the test’s advantage of not only denoting whether a nodule is negative or positive but also providing information on individual mutations of concern will be particularly helpful in informing surgical decisions.”   

RosettaGX MicroRNA Classifier Comparable to ThyGenX/ThyraMIR Combination Testing

Dr. Kristen Partyka of Indiana University School of Medicine, Indianapolis, and colleagues conducted a retrospective blinded validation study on RosettaGX and ThyGenX/ThyraMIR,4 results of which were published in Diagnostic Cytopathology.    

RosettaGX microRNA classifier and the ThyGenX/ThyraMIR combination test analyze direct cytology smears for classification of lesions as benign versus malignant. The ThyGenX/ThyraMIR combination test is comprised of an oncogene panel and microRNA classifier respectively. Both tests differ from Afirma GSC and ThyroSeq v3 GC in that aspirate material from an initial FNA is not required.4  

Ten thyroid FNAs with known surgical follow-up were included in this analysis.4  Each smear contained at least 60-100 lesioned cells and was stained with Diff-Quik and Papanicolaou before application of genomic testing.  NPV was 100% for both molecular tests. PPV was 75% in RosettaGX vs 60% in ThyGenX/ThyraMIR.4  

“This study demonstrated that two molecular testing platforms performed equally well using our stained direct smears,” wrote the authors, who did not respond to requests for comment. “The data was undoubtedly affected by our small sample size, but it shows promising results,” reported Dr. Partyka et al.

Expert Assessment of the Clinical Utility of the Available Molecular Tests

“To use these tests for clinical decision-making, they must be well-validated and the clinician needs to be able to evaluate its performance in all types of thyroid cancers and for all types of nodules. As such, of the commercially available options reviewed, Afirma and ThyroSeq best fit these criteria,” Yuri E. Nikiforov, MD, PhD, professor of pathology and director of the Division of Molecular & Genomic Pathology at the University of Pittsburgh School of Medicine in Pennsylvania, told EndocrineWeb.

When discussing the advantages of RosettaGX and ThyGenX/ThyraMIR, Dr. Partyka and colleagues wrote, “The ability to employ direct smears holds great promise for personalized medicine. Molecular assays can potentially be performed on prior cases in the archives to further aid treatment plans. It creates additional options, more flexibility, and a larger window of opportunity for its application in the clinical setting.”

“This is a rapidly evolving field,” Dr. Patel told EndocrineWeb. “The next step would be to assist in prognostication.”

Yuri Nikiforov MD, PhD, is the lead developer of ThyroSeq v3 GC; none of the other authors had any conflicts to disclose.

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Evolving Care of Thyroid Nodules: Improving Cancer Detection, Determining Need for Active Surveillance
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