Clinical UM Guideline
Subject: Molecular Marker Evaluation of Thyroid Nodules
Guideline #: CG-GENE-04 Publish Date: 07/06/2022
Status: Reviewed Last Review Date: 05/12/2022

This document addresses the use of molecular markers for the evaluation of thyroid nodules to identify genetic mutations (mutation analysis) and to identify benign thyroid nodules preoperatively. Examples of these tests include, but are not limited to:

Note: Please see the following related document for additional information:

Clinical Indications

Medically Necessary:

Molecular marker evaluation of a thyroid nodule is considered medically necessary for use with fine needle aspirates when the following criteria are met:

  1. Initial cytopathology is indeterminate (that is, atypia of undetermined significance [AUS], follicular lesion of undetermined significance [FLUS], suspicious for follicular neoplasm [SFN], follicular neoplasm [FN], or suspicious for malignancy [SUS]); and
  2. One of the following gene expression classifiers will be used:
    1. Afirma Genomic Sequencing Classifier; or
    2. Afirma Medullary Thyroid Carcinoma (MTC) Classifier when results from the Genomic Sequencing Classifier are indeterminate; or
    3. ThyGeNEXT/ThyraMIR; or
    4. ThyroSeq Genomic Classifier.

Not Medically Necessary:

The use of molecular marker evaluation of thyroid nodules is considered not medically necessary for repeat testing of the same nodule and all other indications not listed above as medically necessary.

The use of other molecular marker evaluations of thyroid nodules (for example, Afirma Xpression Atlas) is considered not medically necessary.


The following codes for treatments and procedures applicable to this guideline are included below for informational purposes. Inclusion or exclusion of a procedure, diagnosis or device code(s) does not constitute or imply member coverage or provider reimbursement policy. Please refer to the member's contract benefits in effect at the time of service to determine coverage or non-coverage of these services as it applies to an individual member.

When services may be Medically Necessary when criteria are met:




Oncology (thyroid), mRNA, gene expression analysis of 10,196 genes, utilizing fine needle aspirate, algorithm reported as a categorical result (eg, benign or suspicious)
Afirma® Genomic Sequencing Classifier, Veracyte, Inc


Oncology (thyroid), microRNA profiling by RT-PCR of 10 microRNA sequences, utilizing fine needle aspirate, algorithm reported as a positive or negative result for moderate to high risk of malignancy
ThyraMIR, Interpace Diagnostics, Interpace Diagnostics


Oncology (thyroid), DNA and mRNA of 112 genes, next-generation sequencing, fine needle aspirate of thyroid nodule, algorithmic analysis reported as a categorical result ("Positive, high probabilityof malignancy" or "Negative, low probability of malignancy")
Thyroseq Genomic Classifier, CBLPath, Inc, University of Pittsburgh Medical Center


Oncology (thyroid), mutation analysis of 10 genes and 37 RNA fusions and expression of 4 mRNA markers using next-generation sequencing, fine needle aspirate, report includes associated risk of malignancy expressed as a percentage
ThyGeNEXT® Thyroid Oncogene Panel, Interpace Diagnostics, Interpace Diagnostics


Oncology (thyroid), DNA and mRNA, next-generation sequencing analysis of 112 genes, fine needle aspirate or formalin-fixed paraffin-embedded (FFPE) tissue, algorithmic prediction of cancer recurrence, reported as a categorical risk result (low, intermediate, high)
ThyroSeq® CRC, CBLPath, Inc, University of Pittsburgh Medical Center



ICD-10 Diagnosis



All diagnoses

When services are Not Medically Necessary:
For the procedure codes listed above when criteria are not met or when the code describes a situation designated in the Clinical Indications section as not medically necessary.

When services are also Not Medically Necessary:
For the following procedure codes; or when the code describes a procedure designated in the Clinical Indications section as not medically necessary.




Unlisted multianalyte assay with algorithmic analysis [when specified as testing for thyroid molecular markers by other gene expression classifiers]


Oncology (thyroid), mRNA, gene expression analysis of 593 genes (including BRAF, RAS, RET, PAX8, and NTRK) for sequence variants and rearrangements, utilizing fine needle aspirate, reported as detected or not detected
Afirma Xpression Atlas, Veracyte, Inc, Veracyte, Inc



ICD-10 Diagnosis



All diagnoses

Discussion/General Information

According to the National Cancer Institute (NCI), there were an estimated 52, 890 new cases of thyroid cancer and 2180 deaths in 2020. Thyroid cancer is more common in women and typically affects people between 25 and 65 years old. Thyroid cancer usually starts as a nodule; however, thyroid nodules are common and most never become cancerous. While some nodules may be visible or palpable, most are found during imaging of the head and neck for unrelated reasons. Suspicious nodules, such as those that are large or have calcifications, are usually evaluated for cancer by ultrasound and fine needle aspiration (FNA) biopsy with cytological evaluation. For up to 30% of nodules, the results of the FNA are classified as indeterminate by the Bethesda system (see Appendix): atypia of undetermined significance (AUS), follicular lesion of undetermined significance (FLUS), follicular neoplasm (FN), suspicious for follicular neoplasm (SFN), or suspicious for malignancy (SUS). When nodules are considered indeterminate, a cancer diagnosis cannot be established. To make a diagnosis, indeterminate nodules are excised surgically (lobectomy or partial thyroidectomy) for histopathological evaluation, and if the result is positive, a second surgery may be needed to completely remove the thyroid (total thyroidectomy). The majority of surgically resected indeterminate nodules are found to be benign, resulting in many unnecessary surgeries. Molecular marker tests are proposed as an option to help individuals avoid unnecessary surgery by ruling out cancer preoperatively. In addition, molecular marker tests are used for mutation analysis to help determine the appropriate intervention, such as a total thyroidectomy instead of partial thyroidectomy for individuals with an aggressive cancer type.

Various genetic mutations have been discovered in thyroid cancer. The four gene mutations that are most common and carry the highest impact on tumor diagnosis and prognosis are BRAF, RAS, RET/PTC, and PAX8/PPARγ. rearrangements (Bellevicine, 2020; Cantara 2010; Ferraz 2011; Mathur 2010; Moses 2010; Nikiforov, 2011; Ohori 2010; Xing 2009). These non-overlapping genetic alterations are found in more than 70% of papillary and follicular thyroid carcinomas (Nikiforov, 2011).

Studies on the use of molecular markers for thyroid nodules have analyzed single mutations or panels of mutations and compared the preoperative cytologic findings with postoperative histologic diagnosis to determine the diagnostic accuracy of mutation presence and to predict the presence and potential aggressiveness of a malignancy. Additionally, gene expression classifiers have been developed to predict the likelihood that a thyroid lesion with indeterminate cytology is benign, allowing an individual to potentially avoid unnecessary surgical excision.


The ThyroSeq test, developed at the University of Pittsburgh, uses next-generation sequencing (NGS) of DNA and RNA. The test started out as a 7-gene panel in 2007 and expanded to 15 genes in 2013 (version 1), 56 genes in 2014 (version 2), and 112 genes in 2017 (version 3). The test is primarily marketed to rule out cancer for nodules with indeterminate FNA cytology. In addition, ThyroSeq is marketed for cancer prognostication to determine the type of surgery needed for malignant nodules (for example, lobectomy for indolent cancer versus total thyroidectomy for aggressive cancer). The latest version of the test provides information on > 12,000 mutation hotspots and > 120 gene fusion types, while detecting mutations, gene fusions, gene expression alterations, and copy number variations. A proprietary genomic classifier is used to report positive or negative results.

Nikiforova and colleagues (2013) examined the use of targeted NGS for simultaneous testing of multiple mutations in thyroid cancer. The aim of the study was to create an NGS approach to allow for the detection of most point mutations and small insertions or deletions known to occur in thyroid cancer. A custom panel, ThyroSeq, was designed to target 12 thyroid cancer genes with 284 mutational hotspots. DNA from 228 thyroid neoplastic and non-neoplastic samples, including 105 frozen, 72 formalin-fixed, and 51 FNA samples representing all major types of thyroid cancer, was analyzed. The analytical accuracy for mutation detection was reported to be 100% with the sensitivity of 3–5% of mutant allele. ThyroSeq DNA assay identified mutations in 19/27 (70%) of classic PTC, 25/30 (83%) of follicular variant PTC, 14/18 (78%) of conventional and 7/18 (39%) of oncocytic follicular carcinomas, 3/10 (30%) of poorly differentiated carcinomas, 20/27 (74%) of anaplastic (ATC), and 11/15 (73%) medullary carcinomas. In contrast, 5/83 (6%) of benign nodules were positive for mutations. Most tumors had a single mutation; however, several ATC and PTC demonstrated two or three mutations. The most common mutations detected were BRAF and RAS.

Additional research by Nikiforov and others (2014, 2015) further studied the use of NGS testing using ThryoSeq. In the 2014 study, the authors evaluated 143 consecutive FNA samples with a cytologic diagnosis of follicular neoplasm or suspicious for follicular neoplasm (FN/SFN) from individuals with known surgical outcomes. Included were 91 retrospective samples and 52 prospective samples. Analyses were performed on a proprietary sequencer using the targeted ThyroSeq v2 NGS panel, simultaneously testing for point mutations in 13 genes and for 42 types of gene fusions that occur in thyroid cancer. The expression of 8 genes was used to assess the cellular composition of FNA samples. Histologic analysis revealed 104 benign nodules and 39 malignant nodules. The most common point mutations involved the neuroblastoma RAS viral oncogene homolog (NRAS), followed by the Kirsten rat sarcoma viral oncogene homolog (KRAS), the telomerase reverse transcriptase (TERT) gene, and the thyroid-stimulating hormone receptor (TSHR) gene. The identified fusions involved the thyroid adenoma associated (THADA) gene; the peroxisome proliferator-activated receptor gamma (PPARG) gene; and the neurotrophic tyrosine kinase, receptor, type 3 (NTRK3) gene. Performance characteristics were similar in the retrospective and prospective groups. Among all FN/SFN nodules, preoperative ThyroSeq v2.0 performed with 90% sensitivity (95% confidence interval [CI], 80% to 99%), 93% specificity (95% CI, 88% to 98%), a PPV of 83% (95% CI, 72% to 95%), a NPV of 96% (95% CI, 92% to 100%), and 92% accuracy (95% CI, 88% to 97%).

In 2015, the authors similarly tested the ThryoSeq v2.1 (v2.1 has an additional gene for analysis when compared to v2.0) for AUS/FLUS. A total of 465 consecutive FNA samples with the cytologic diagnosis of AUS/FLUS underwent prospective molecular testing. A total of 98 (21%) of these nodules had definitive surgical (n = 96) or nonsurgical (n = 2) follow-up and were used to determine the assay performance. Among 465 AUS/FLUS nodules, 3 were found to be composed of parathyroid cells and 462 of thyroid follicular cells. Of the latter, 31 (6.7%) were positive for mutations. The most frequently mutated genes were NRAS and HRAS, and overall point mutations in seven different genes and five types of gene fusions were identified in these nodules. Among 98 nodules with known outcome, histologic analysis revealed 22 (22.5%) cancers. ThyroSeq v2.1 was able to classify 20/22 cancers correctly, showing a sensitivity of 90.9% (95% CI, 78.8 to 100), specificity of 92.1% (95% CI, 86.0 to 98.2), PPV of 76.9% (95% CI, 60.7 to 93.1), and NPV of 97.2% (95% CI, 78.8 to 100), with an overall accuracy of 91.8% (95% CI, 86.4 to 97.3).

Early investigation of the ThyroSeq and ThyroSeq v2 largely included retrospective studies with inconsistent conclusions regarding the tests’ validity and clinical utility (Shrestha, 2016; Taye, 2018; Valderrabano, 2017).

In 2018, Nikiforova and colleagues reported on the analytical performance of newly formulated ThyroSeq v3 (expanded to analyze 112 genes, providing information on > 12,000 mutation hotspots and > 150 gene fusion types). Study samples included 238 surgically removed tissue samples, 175 FNA indeterminate cytology samples, 16 cell lines, and 4 reference controls. For the surgically removed tissue samples, the test detected over 100 alterations and accurately classified the majority of papillary carcinomas (91.2%), follicular carcinomas (90.9%), Hürthle cell lesions (93.1%), medullary thyroid carcinomas (100%), and parathyroid lesions (100%). The genomic classifier cutoffs for differentiating cancerous and benign nodules had a 93.9% sensitivity, 89.4% specificity, and 92.1% accuracy. For the FNA samples, the validation set had a 98.0% sensitivity, 81.8% specificity, and 90.9% accuracy. The authors concluded that the test is valid for clinical use, and further studies will be done to determine clinical utility.

Steward and colleagues (2018) published a prospective, double-blinded, multicenter study that evaluated the diagnostic accuracy of ThyroSeq v3. Inclusion criteria included 18 years of age and older, 1 or more thyroid nodule, and an FNA procedure to collect samples for cytological examination and molecular analysis. Those who had a cytologic diagnosis of Bethesda III, IV, or V underwent thyroid surgery. The study was conducted across 10 sites (9 in the United States and 1 in Singapore) between January 2015 and December 2016. The primary outcome was the sensitivity, specificity, NPV, and PPV of the test to predict the histopathologic diagnosis of benign nodule versus cancer/NIFTP in indeterminate nodules with a Bethesda III and IV cytology. The secondary outcome was the prediction of cancer/NIFTP by specific genetic alterations in Bethesda III, IV, and V cytology nodules. A total of 286 FNA samples met inclusion criteria. After some samples were found inadequate for molecular analysis, a total of 257 samples (90%) from 232 subjects were in the final study set (Bethesda III [n=154], Bethesda IV [n=93], and Bethesda V [n=10]). For Bethesda III and IV combined, the sensitivity was 94% (95% CI, 86 to 98%), and the specificity was 82% (95% CI, 75 to 87%). Considering a cancer/NIFTP prevalence of 28%, the NPV was 97% (95% CI, 93 to 99%) and the PPV was 66% (95% CI, 56-75%). For Bethesda III and IV nodules, the negative benign call rate was 61%. Of 152 test-negative samples, 5 (3%) were found to be false-negative. For the nodules that tested positive, specific groups of genetic alterations had cancer probabilities from 59 to 100%. The authors concluded:

The study documents a high sensitivity and correspondingly high NPV of the ThyroSeq GC test for Bethesda III and IV indeterminate cytology nodules, which together with high specificity may prevent diagnostic surgeries in the majority of such patients. The availability of detailed genetic information in test-positive cases may help to further inform individualized treatment for these patients after integration with imaging and other clinical information.

Several additional recently published cohort studies and retrospective reviews also indicate that ThyroSeq v3 testing may contribute to avoidance of surgery for initially indeterminate thyroid nodules (Marcadis, 2019; Ohori, 2019).

Afirma Thyroid FNA Analysis

The Afirma Thyroid FNA Analysis combines specialized cytopathology with the Afirma Gene Expression Classifier (GEC). The GEC analyzes the mRNA expression of 167 genes in aspiration material and reclassifies FNAs with ambiguous cytopathology diagnoses as either benign or suspicious for cancer. Approximately 10% of FNA samples have inadequate RNA yield or quality and are reported by the Afirma GEC as “no result” (Duick 2012; Ali 2013). The Afirma is intended to rule out thyroid cancer (Ward, 2013).

In the initial development of the GEC, Chudova and colleagues (2010) had set out to develop a molecular test to distinguish between benign and malignant thyroid nodules using FNA. More than 247,000 transcripts in 315 thyroid nodules were measured by mRNA expression analysis. The data set consisted of 178 retrospective surgical specimens, representing the most common benign and malignant histologic subtypes, and 137 prospectively collected aspirate specimens. The performance of the resulting classifier was markedly lower in the FNAs than in tissue, likely due to differences in cellular heterogeneity between the two types of specimens. On the test set of this early version of the GEC, NPV and specificity were estimated to be 96% and 84%, respectively. Subsequently, Walsh and colleagues (2012) sought to verify the analytical performance of the Afirma GEC in the classification of cytologically indeterminate thyroid nodule FNAs. The analytical performance studies were designed to characterize the stability of the RNA in the aspirates during collection and shipment, analytical sensitivity and specificity, and assay performance studies including intra-nodule, intra-assay, inter-assay, and inter-laboratory reproducibility. Analytical sensitivity, specificity, robustness and quality control of the GEC were all verified in the study. Based on these results, the authors concluded that routine testing of FNA specimens is feasible.

Much of the early investigation of the GEC’s performance was conducted in small, retrospective studies with limited follow-up and inconsistent reporting of the test’s validity and clinical utility (Alexander, 2014; Angell, 2015; Duick, 2012; Harrell, 2014; McIver, 2014; Witt, 2015).

In 2012, Alexander and colleagues performed a 19-month, prospective, multicenter validation study of the Afirma GEC, involving 49 clinical sites (both academic and community centers), 3789 individuals and 4812 FNAs of thyroid nodules that were at least 1 cm in size. Histopathologic reports of the cytologic diagnosis were collected for all cases, and reports without a definitive benign or malignant diagnosis at the local site were reviewed by three expert cytopathologists, who reclassified them as atypical, follicular neoplasm or suspicious for a follicular neoplasm, or suspicious for malignancy. Corresponding histopathologic diagnoses from excised specimens were available (excisions were performed without knowledge of the results of the GEC). After inclusion criteria were met, 265 FNA samples considered to be cytologically indeterminate were tested with the GEC assay at Veracyte Laboratory. Of the 265 indeterminate samples, 85 were reported as malignant. The GEC correctly identified 78 of the 85 as suspicious (92% sensitivity; 95% CI, 84 to 97%), with a specificity of 52% (95% CI, 44 to 59%). NPVs ranged from 85% for “suspicious cytologic findings” to 95% for “atypia of undetermined clinical significance” with a PPV of 47%. Limitations of this study, as reported by the authors, included imperfect interobserver agreement possibly affecting the sensitivity or specificity of the classifier, since pathological assessment of benign versus malignant disease is not always absolute.

In 2016, Santhanam and colleagues performed a meta-analysis that combined individual data from seven studies that examined the GEC test for indeterminate thyroid nodules. The pooled sensitivity of the GEC was reported to be 95% and the specificity was 30.5%. Individuals with benign GEC results were not followed long enough to determine reliable false negative rates. The authors indicated that GEC is useful to rule out malignancy in thyroid nodules that have indeterminate cytology although the long-term benefits are unclear. Specifically, while the GEC “might prevent some unnecessary thyroid surgeries…for many persons it might represent an additional layer of testing prior to diagnostic thyroidectomy.” A significant limitation of the meta-analysis was the heterogeneity of the studies.

A 2016 population-based, retrospective cohort study by Singer and colleagues assessed long-term management patterns and rates of thyroid surgery for individuals with benign GEC results as compared to a control group with cytopathology benign results. Individuals who underwent FNA biopsy between January 1, 2011 and July 31, 2013 were included in the study. Study outcomes included rates of thyroid-related follow-up clinic visits, ultrasound examinations, and thyroid-related surgeries. A total of 159 of the 2059 participants who met study inclusion criteria had their thyroid nodule FNA biopsy diagnosed as indeterminate, and only a molecular sample was sent for GEC testing. Remaining were 1900 participants whose paired cytopathology and GEC samples were sent for evaluation. Nodule cytology pathology results for this group were: non-diagnostic for 157 subjects (8.3%), benign for 1308 subjects (68.8%), indeterminate for 357 subjects (18.8%), suspicious of malignancy in 19 subjects (1.0%), and malignant for 59 subjects (3.1%). Altogether, 532 subjects had samples that underwent GEC testing and 35 of those molecular samples did not produce a result. Of the remaining 497, results were GEC benign for 218 (43.9%) subjects, and GEC suspicious for 279 (56.1%) subjects. Of the 218 GEC-benign subjects, 201 were successfully matched 1:3 to 603 subjects with a cytopathology benign diagnosis. The number of GEC-benign and cytopathology-benign subjects that underwent thyroid surgery (11.4% versus 10.1%, p=0.594), and received a follow-up ultrasound exam (60.2% versus 61.7%, p=0.706), respectively, were not significantly different. The majority of subjects in both groups did not require surgery and were managed with routine care consisting of ultrasounds and clinical follow-up. The authors concluded their data suggested a GEC diagnosis of “benign” could be clinically managed in the manner as an initially cytopathologic “benign” thyroid nodule.

A retrospective analysis by Yang and colleagues (2016) was completed at a single institution that performed the Afirma GEC test between August 24, 2012 (when GEC testing began in the institution) and April 1, 2014. Cases of indeterminate cytology that also had GEC testing were selected and GEC results were compared to the histopathology findings. A total of 1693 thyroid FNAs were performed and of these, 789 (46.7%) had GEC samples collected for potential testing and 217 (13% of the total number of FNAs, rate of indeterminate cytology results in the study institution) had GEC completed. Among the 217 cases, 189 were of indeterminate cytology. Of the indeterminate cytology cases, 42% were benign and 50% were suspicious by GEC. The rate of excision of atypia of undetermined significance-follicular lesion of undetermined significance in the pre-GEC category was 63% with the rate decreasing to 47% in the post-GEC category. The malignancy rate of excised thyroids increased from 35% in the pre-GEC group to 47% in the post-GEC group. Findings were similar for lesions suspicious for a follicular neoplasm-follicular neoplasm lesion. The authors concluded that GEC testing contributed to an avoidance of surgery for initially indeterminate thyroid nodules.

Sipos and colleagues (2016) conducted a retrospective study of non-academic medical practices utilizing the GEC between September 2010 and June 2014.The primary study objective was to evaluate the rate of surgical intervention in subjects with a benign Afirma GEC result during long-term follow-up. The secondary study objective was to determine the opinion of treating physicians regarding the safety of GEC use compared to the hypothetical situation of providing care without the GEC. During the 36-month follow-up period, 17 of 98 subjects (17.3%) with a benign GEC result had surgery. After a benign GEC, 88% of surgeries occurred during the first 2 years. Additionally, a survey was administered to treating physicians to assess their perception of safety in using the GEC. Reports from treating physicians indicated that patient safety was improved by using the GEC compared to not using the GEC in 78 of 91 (86%) cases.

Abeykoon and colleagues (2016) conducted a retrospective cohort study at a single institution comparing the rate ofsurgical recommendations for all cytologically indeterminate thyroid nodules pre-and post-Afirma introduction and found a statistically significant reduction from 81.5% to 50% (p=0.01) after Afirma GEC implementation. Of the individuals who underwent surgery, 85.7% in the post-Afirma cohort showed evidence of malignancy, as compared with 20% of those in the pre-Afirma cohort (p<0.01).

Several additional recently published case series and retrospective reviews also indicate that Afirma GEC testing may contribute to avoidance of surgery for initially indeterminate thyroid nodules (Chaudhary, 2016; Dhingra, 2016; Jug, 2019; Parajuli, 2019).

A more recent systematic review and meta-analysis by Valderrabano (2019) sought to compare post-marketing findings to the initial clinical validation findings of GEC testing. A total of 19 studies were included, comprising a total of 2568 thyroid nodules. Based on a simulation using the sensitivity and specificity reported in the initial validation study, the observed benign call rate and PPV values in post-marketing studies would have to be explained by enormously different underlying prevalence rates of cancer (15% vs 30%); an impossibility. The findings suggest that the initial validation study cohort was not representative of the populations in whom the GEC has been used, raising doubt regarding the accuracy of its reported diagnostic performance, including the negative predictive value.

In 2017, a second-generation assay (Afirma Genomic Sequence Classifier [GSC]) was made available for clinical use based on early research by Patel and colleagues (2018) and a confirmatory clinical study published by Harrell and colleagues (2019). Clinical outcomes after 11 months of use with the GSC (n=146) were compared with 6.5 years of experience with the GEC (n=509). When compared to GEC, GSC identified less indeterminate cytology nodules as suspicious (38.8%; 54/139 vs. 58.4%; 281/481, respectively). A total of 82.7% of oncocytic FNAB subjects were classified as suspicious in the GEC group (86 of 104 oncocytic indeterminates) whereas only 35.3% were classified as suspicious by GSC (12 of 34). There was a 45% reduction in the rate of subsequent surgery in individuals with oncocytic aspirates (56% in the GEC group vs. 31% in the GSC group). Pathology analysis demonstrated a false-negative percentage for an incomplete surgical group of 9.5% for GEC and 1.2% for GSC. Authors conclude that the GSC further reduces surgery in indeterminate thyroid nodules by improving the specificity without compromising sensitivity, especially considering the significant improvement in the specificity of the GSC test in oncocytic FNAB aspirate.

In 2020, Vora and colleagues published a retrospective review and clinical study (median follow-up = 27.8 months) from a single institution of 416 indeterminate thyroid nodules (n=368) analyzed by the AFIRMA GEC. Nodules classified as ‘suspicious’ by the GEC were resected 85% of the time with a positive predictive value (PPV) of 37%, whereas nodules classified as ‘benign’ were resected 24% of the time with a negative predictive value (NPV) of 90% (prevalence of malignant nodules was 41%). The resection rate in nodules classified as ‘no result’ was 57% with a malignancy rate of 42%. Authors recommend that a ‘suspicious’ result be coupled with other indications of malignancy before resection is recommended as classification of ‘suspicious’ alone was a poor predictor of a malignant nodule. Limitations of this trial include the single-institution, retrospective design.

Afirma Medullary Thyroid Cancer (MTC) Classifier

The Afirma MTC classifier was developed to identify the presence of MTC from the same FNA sample used for Afirma GSC in lieu of traditional serum calcitonin testing which has historically had a high false positive rate (low specificity). Based on the preliminary work by Kloos and colleagues (2016), Pankratz and colleagues (2016) sought to demonstrate the analytic validity of the Afirma MTC using 27 fresh-frozen tissue specimens with histologically confirmed MTC diagnoses. FNA biopsies and whole blood from normal donors were obtained. Gene expression data from the tissue and FNA samples were used to model classifier response to the following: mixtures of MTC samples with normal thyroid tissue, a benign thyroid nodule, a Hürthle cell adenoma, and whole blood. The resulting MTC classifier sensitivity was 96.3% [confidence interval 81.0–99.9%]. No false positives were detected, suggesting the specificity is near 100%. The tests reproducibility was 100% concordant. The MTC classifier demonstrated robust sensitivity, specificity, accuracy, and reproducibility. When included as part of the routine molecular testing of indeterminate thyroid nodules following Afirma GSC testing, the Afirma MTC can distinguish MTC with a specificity that exceeds that of traditional serum calcitonin testing.

Afirma Xpression Atlas

The Afirma Xpression Atlas (XA), originally launched in 2018, is designed to provide genomic alteration information from the same FNA sample used for Afirma GSC testing with the end-goal of providing clinicians a more detailed genetic profile to make a more informed decision, enabling tailored surgery and therapeutic treatment options for thyroid nodules that are cancerous or suspicious. The RNA sequencing-based test originally measured 761 DNA variants and 130 RNA fusions in over 500 genes. In 2020, based on analysis of over 37,000 samples (surgical histology unknown), the Afirma XA was expanded to include 593 genes informing on 905 variants and 235 fusions (Expanded Afirma XA) (published abstract; [Wirth, 2020]).

In 2019, Angell and colleagues first reported on the analytical and clinical validity of the original Afirma XA. DNA and RNA from 943 blinded FNA samples were analyzed by whole-transcriptome RNA-seq, targeted RNA-seq, and targeted DNA-seq. Additionally, 695 blinded FNAs were used to define performance for fusions between whole-transcriptome RNA-seq and targeted RNA-seq. The reproducibility of the whole-transcriptome RNA-seq assay was verified and the variants and fusions were compared to histopathology results. The intra-plate reproducibility ranged from 89% to 94%, inter-plate reproducibility from 86–91%, and inter-lab accuracy 90%. As a standalone test, the sensitivity of Afirma XA was 49%. The study investigators concluded that when the Afirma GSC is used as a rule-out test for Bethesda III/IV nodules the Afirma XA can provide supplemental genomic insight among those that are classified as suspicious by the GSC. This retrospective study does not report clinical outcomes from the adjunct use of the Afirma XA, thus its value as an add-on to the Afirma GSC remains to be established.

ThyGenX, ThyGeNEXT and ThyraMIR

ThyGenX (based on the predicate 17-gene alteration panel miRInform test) is a mutational panel for the detection of 8 genes associated with thyroid papillary carcinoma and follicular carcinoma. ThyraMIR is a micro RNA (miRNA) gene expression classifier that is based on the evaluation and expression of 10 miRNAs. The tests are marketed to use in combination, in that ThyraMIR can identify malignancy when ThyGenX has a negative result. ThyGenX and ThyraMIR are designed to minimize the need for surgery when nodules are indeterminate or to assist in making surgical decisions when nodules are malignant (lobectomy versus total thyroidectomy).

Beaudenon-Huibregtse and colleagues (2014) performed a prospective, double-blind study on the performance of FNA cytology combined with the molecular analysis of a 17-gene alteration panel. The researchers collected 806 FNA specimens from 618 subjects at 5 U.S. clinical sites. A total of 737 nodules from 581 subjects met inclusion criteria, and at the end of the study there were 109 specimens that had post-surgical histopathology. For those 109 specimens, oncogenic mutations were present in 50% of malignant nodules missed by FNA cytology. A total of 14 nodules that were indeterminate after FNA were negative by molecular testing but positive after surgical resection and histopathology (false-negative detection rate of 25%). There were 6 false-positive molecular test results. The researchers concluded that molecular testing compliments cytopathological testing, but “not all malignant tumors carry one of the 17 genetic alterations evaluated, and it is important to emphasize that, unless the local pretest probability of cancer is known and sufficiently low, a negative molecular result alone should not be used to rule out surgical therapy for nodules with an indeterminate or nondiagnostic cytologic diagnosis.”

Labourier and colleagues (2015) evaluated a diagnostic algorithm that combined mutation detection and miRNA expression. A total of 638 surgical specimens and preoperative FNAs were tested for 17 validated gene alterations using the miRInform Thyroid test and a 10-miRNA gene expression classifier that generates positive (malignant) or negative (benign) results. A cross-sectional sampling of 109 thyroid nodules with AUS/FLUS or FN/SFN cytology was performed at 12 endocrinology centers. Mutations were detected in 24 of 35 (69%) nodules with malignant outcome. Of the mutation-negative specimens, miRNA testing identified 64% of malignant cases and 98% of benign cases. The diagnostic sensitivity and specificity of the combined algorithm was 89% and 85%, respectively. At 32% cancer prevalence, 61% of the molecular results were benign with an NPV of 94%. The authors concluded that the diagnostic algorithm “combining miRNA expression and gene mutation detection yields clinically actionable molecular information in thyroid nodules with AUS/FLUS or FN/SFN cytology.”

Wylie and colleagues (2016) performed an analysis on the diagnostic potential of miRNA for the development of ThyraMIR. The researchers analyzed 534 archived nodule remnants from FNAs, including 257 nodules that underwent surgical resection. The samples, which were from 14 centers across the United States, were histologically reviewed by a single pathologist and classified according to current World Health Organization schemes. Expression profiling was done by reverse transcription-quantitative polymerase chain reaction (PCR). The researchers extracted total nucleic acids for 17 gene alterations (BRAF, RAS, RET, or PAX8), selected 31 miRNA candidates based on literature review and differential expression analyses, and genotyped over 1500 unique gene alterations using a custom sequencing assay. Eight supervised machine learning algorithms were used to distinguish benign and malignant samples. The AUROC was invariant in cross-validation (0.89) and was optimal for 235 preoperative aspirates (0.94). The models also classified 92% of benign lesions as low risk/negative and 92% of malignant lesions as high risk/positive. miRNA significantly increased the diagnostic performance of the 17-mutation panel (p<0.001). For a subset of resected tissue samples (n=54) and an independent indeterminate set of nodules (n=42), miRNA increased sensitivity by 30-39% and was able to classify all the benign nodules as negative. When the researchers compared miRNA and NGS testing, they found that both methods increased sensitivity of the 17-mutation panel; however, NGS decreased the specificity. For 30 nodules reported benign by the 17-mutation panel, miRNA was able to correctly classify 67% of malignant cases and 100% of benign cases. The combination of miRNA expression combined with the 17-gene panel resulted in an 85% sensitivity and 95% specificity. The authors concluded that “a molecular test combining an optimized miRNA classification algorithm with a validated panel of somatic driver mutations displays high diagnostic sensitivity and specificity.”

Based on the preliminary work of Banizs (2019) and Ablordeppey (2019), the latter of which pioneered an expanded version of the ThyGenX, called the ThyGeNEXT®, Lupo and colleagues (2020) sought to validate this expanded mutation panel. A blinded, retrospective, multicenter study was conducted using consensus histopathology diagnosis among three pathologists to validate the test along with the microRNA risk classifier, ThyraMIR (combination test is known as the ancillary multiplatform test [MPTX]). A total of 243 subjects with indeterminate thyroid nodules that later underwent surgical resection, were enrolled. Consensus diagnosis was unanimous in 197 (81%) subjects with indeterminate thyroid nodules; 36% confirmed malignancy. Overall, the MPTX had a 95% sensitivity (95% CI, 86%-99%) and 90% specificity (95% CI, 84%-95%) for disease with a 97% NPV (95% CI, 91%-99%) at a 30% disease prevalence, and a 75% PPV (95% CI, 60%-86%).

Sistrunk (2020) evaluated the clinical performance of the MPTX for the management of 140 indeterminate thyroid nodules. MPTX test results were determined with clinical outcomes blinded. In total, 73% (102 of 140) of the study cohort had MPTX low-risk results, while 20% (28 of 140) had MPTX moderate-risk results and 7% (10 of 140) had MPTX high-risk results. In this study population, 13% of the nodules were malignant. When MPTX resulted in a negative test status or low-risk status, there was a high probability (94%) that study participants would remain cancer-free at median follow-up of 9 months. The probability of malignancy was 53% when an MPTX positive test status resulted (Hazard Ratio [HR]=11.2, p<0.001) and also 53% when an MPTX moderate-risk test status resulted (HR=8.5, p=0.001). The probability of malignancy was 70% when MPTX high-risk category resulted (HR=38.5, p<0.001). Authors concluded that the MPTX accurately stratifies individuals by risk for development of thyroid nodule malignancy.

Other Considerations

The American Thyroid Association (ATA) Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer (Haugen, 2015) indicate that there is currently no single optimal molecular test that can definitively rule in or rule out malignancy in all cases of indeterminate cytology. However, the ATA did offer the following specific recommendations for molecular testing in adults:

The corresponding ATA pediatric guidelines (2015) indicate that although studies have shown molecular testing aids in the management of thyroid nodules with indeterminate cytopathology in adults, there are no studies determining its usefulness in the evaluation of indeterminate pediatric thyroid nodules. As a result, the ATA was unable to offer a recommendation for the use of molecular diagnostics in children, and reported that additional studies are required prior to a recommendation being made. The ATA also indicated that their pediatric guidelines applied to ages18 years and below.

The ATA (Ferris, 2015) also published a Statement on Surgical Application of Molecular Profiling for Thyroid Nodules: Current Impact on Perioperative Decision Making and reported:

Techniques for molecular profiling of thyroid cytology specimens have evolved as adjuncts to guide the appropriate management of cytologically indeterminate nodules. However, it must be stressed that the utility of any molecular test is only applicable clinically when combined with clinical and sonographic risk factors for malignancy and with understanding of the prevalence of malignancy for the Bethesda cytologic categories at the reporting institution. For example, a ‘‘rule out’’ test such as the GEC will perform better in a setting of lower cancer frequency, as well as in a cytologic category of low cancer frequency such as AUS/FLUS or FN, than it will in a setting of higher cancer frequency such as SMC or a site with a high prevalence of malignancy in a given cytologic category.

In 2017, the ATA released a recommendation (Haugen, 2017) on the proposed renaming of non-invasive EFVPTC to NIFTP:

The histopathologic nomenclature for Encapsulated Follicular Variant Papillary Thyroid Carcinoma (eFVPTC) without invasion may be re-classified as a NIFTP given the excellent prognosis of this neoplastic variant. Prospective studies are needed to validate the observed patient outcomes (and test performance in predicting thyroid cancer outcomes), as well as implications on patients’ psychosocial health and economics (Weak Recommendation, Moderate-quality evidence).

The ATA further states:

The proposed name change will also affect the performance of molecular tests when applied to patients with indeterminate cytology. For example, neoplasms harboring RAS mutations, will likely have a lower positive predictive value for malignancy, while nodules with no genetic mutation or a negative gene expression classifier will likely have a slightly higher negative predictive value. These effects will be dependent on the prevalence of NIFTP in a given population. Since NIFTP, like follicular adenoma, requires surgery for a definitive diagnosis, the changes in PPV and NPV of the molecular tests will not alter the requirement of surgical intervention for these patients. Potential frameworks for addressing cytology and molecular reporting are still evolving.

In 2021 the ATA published Guidelines for Management of Patients with Anaplastic Thyroid Cancer; the following recommendations are made related to molecular testing:

FNA cytology can play an important diagnostic role in the initial evaluation of ATC, but parallel core biopsy may be necessary for definitive diagnosis and to obtain sufficient material for molecular interrogation. (Strength of Recommendation: Strong; Quality of Evidence: Low)

The American Association of Clinical Endocrinologists, American College of Endocrinology, and Associazione Medici Endocrinologi Medical Guidelines for Clinical Practice for the Diagnosis and Management of Thyroid Nodules – 2016 Update (Gharib, 2016) states:

National Comprehensive Cancer Network (NCCN) Guidelines on the treatment of thyroid carcinoma (V1.2022) report that molecular diagnostics to detect individual mutations in BRAF, V600E, RET/PTC, RAS, PAX8/PPAR or pattern recognition approaches using molecular classifiers may be useful in the evaluation of FNA samples that are indeterminate (that is, follicular lesion of undetermined significance). NCCN issued 2A recommendations that molecular diagnostics may be used for reclassification of follicular lesions (FN, AUS, and FLUS) as either more or less likely to be benign or malignant based on the genetic profile. NCCN stated that historically studies have not shown that molecular diagnostics perform well for Hürthle cell neoplasms. NCCN discusses the encouraging evidence surrounding Afirma and ThyroSeq specifically for molecular testing of cytologically indeterminate thyroid nodules; other proprietary tests are not mentioned by name at this time.

The strength of the cytology report impacts the reliability of molecular testing. Cibas and colleagues (2013) assessed inter- and intraobserver variability of preoperative cytopathologic and postoperative histopathologic thyroid diagnoses. A total of 653 subjects with 776 surgically resected thyroid nodules of 1 cm or greater were evaluated at 14 academic centers and 35 clinical sites. Study samples were collected in a prospective, multicenter trial validating a GEC between June 2009 and December 2010. Intraobserver concordance among two or more central histopathologists who independently read histopathology slides was calculated. Interobserver concordance between the diagnoses made by the central histopathologists and those made by local pathologists was also calculated. Intra- and interobserver concordance for cytopathology was similarly calculated by comparing diagnoses made by local pathologists with those made by a central panel of three cytopathologists. Concordance on the histopathologic distinction between benign and malignant diagnoses was 91% comparing local with central histopathologists and 90% comparing two central histopathologists. Sixty four percent of diagnoses made by central and local cytopathologists and 74.7% of intraobserver diagnoses were concordant using the six category Bethesda System for reporting thyroid cytopathology. Central cytopathologists made fewer indeterminate diagnoses than local pathologists (41.2% vs. 55.0%). A significant limitation of this study was that many local pathologists did not use the Bethesda System, and their reports were translated to allow for comparison.


The incremental added value of mutation analysis to an equivocal FNA result is not known, and although mutation analysis has the potential to improve the accuracy of an equivocal FNA of the thyroid and may play a role in preoperative risk stratification and surgical planning, at this time, it is not clear how it will impact clinical management of an individual or surgical decision making.

Published data provides sufficient evidence demonstrating that the Afirma GSC, Afirma MTC, ThyGeNEXT+ ThyraMIR and the ThyroSeq genomic classifier contribute to the avoidance of surgery for those with indeterminate initial cytopathology of a thyroid nodule. As such, an improvement in net health outcome may occur as a result of molecular marker testing.

The commercially available, laboratory-developed molecular marker tests are regulated under the Clinical Laboratory Improvement Amendments (CLIA). At this time, premarket approval from the U.S. Food and Drug Administration (FDA) is not enforced when the assay is performed in a laboratory that is licensed by CLIA.


Bethesda System for Reporting Thyroid Cytopathology (TBSRTC): A standardized reporting system used by pathologists to classify FNA specimens (see Appendix below).

BRAF: A protein which influences the regulation of the MAP kinase/ERKs signaling pathway, which affects cell division, differentiation, and secretion. BRAF is also known as serine/threonine-protein kinase B-Raf, v-raf murine sarcoma viral oncogene homolog B1.

mRNA: Messenger RNA conveys genetic information from DNA to the ribosome, where they specify the amino acid sequence of the protein products of gene expression.

miRNA: Micro RNA is a small non-coding RNA molecule containing about 22 nucleotides found in plants, animals and some viruses, that functions in RNA silencing and post-transcriptional regulation of gene expression.

Mutation: A permanent, transmissible change in genetic material.

Next-generation sequencing (NGS): Any of the technologies that allow rapid sequencing of large numbers of segments of DNA, up to and including entire genomes.


Peer Reviewed Publications:

  1. Abeykoon JP, Mueller L, Dong F, et al. The effect of implementing gene expression classifier on outcomes of thyroid nodules with indeterminate cytology. Horm Cancer. 2016; 7(4):272-278.
  2. Ablordeppey KK, Timmaraju VA, Song-Yang JW, et al. Development and analytical validation of an expanded mutation detection panel for next-generation sequencing of thyroid nodule aspirates. J Mol Diagn. 2020; 22(3):355-367.
  3. Adeniran AJ, Theoharis C, Hui P, et al. Reflex BRAF testing in thyroid fine-needle aspiration biopsy with equivocal and positive interpretation: a prospective study. Thyroid. 2011; 21(7):717-723.
  4. Alexander EK, Kennedy GC, Baloch ZW, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med. 2012; 367(8):705-715.
  5. Alexander EK, Schorr M, Klopper J, et al. Multicenter clinical experience with the Afirma gene expression classifier. J Clin Endocrinol Metab. 2014; 99(1):119-125.
  6. Ali SZ, Fish SA, Lanman R, et al. Use of the Afirma® gene expression classifier for preoperative identification of benign thyroid nodules with indeterminate fine needle aspiration cytopathology. PLoS Curr. 2013; 5.
  7. Angell TE, Frates MC, Medici M, et al. Afirma benign thyroid nodules show similar growth to cytologically benign nodules during follow-up. J Clin Endocrinol Metab. 2015; 100(11):E1477-E1483.
  8. Angell TE, Wirth LJ, Cabanillas et al. Analytical and clinical validation of expressed variants and fusions from the whole transcriptome of thyroid FNA samples. Front Endocrinol (Lausanne). 2019; 10:612.
  9. Banizs AB, Silverman JF. The utility of combined mutation analysis and microRNA classification in reclassifying cancer risk of cytologically indeterminate thyroid nodules. Diagn Cytopathol. 2019; 47(4):268-274.
  10. Beaudenon-Huibregtse S, Alexander EK, Guttler RB, et al. Centralized molecular testing for oncogenic gene mutations complements the local cytopathologic diagnosis of thyroid nodules. Thyroid. 2014; (10):1479-87.
  11. Bellevicine C, Migliatico I, Sgariglia R, et al. Evaluation of BRAF, RAS, RET/PTC, and PAX8/PPARg alterations in different Bethesda diagnostic categories: A multicentric prospective study on the validity of the 7-gene panel test in 1172 thyroid FNAs deriving from different hospitals in South Italy. Cancer Cytopathol. 2020; 128(2):107-118.
  12. Benjamin H, Schnitzer-Perlman T, Shtabsky A, et al. Analytical validity of a microRNA-based assay for diagnosing indeterminate thyroid FNA smears from routinely prepared cytology slides. Cancer Cytopathol. 2016; 124(10):711-721.
  13. Borowczyk M, Szczepanek-Parulska E, Olejarz M, et al. Evaluation of 167 Gene Expression Classifier (GEC) and ThyroSeq v2 diagnostic accuracy in the preoperative assessment of indeterminate thyroid nodules: bivariate/HROC meta-analysis. Endocr Pathol. 2019; 30(1):8-15.
  14. Cantara S, Capezzone M, Marchisotta S, et al. Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules improves the diagnostic accuracy of cytology. J Clin Endocrinol Metab. 2010; 95(3):1365-1369.
  15. Chaudhary S, Hou Y, Shen R, et al. Impact of the Afirma gene expression classifier result on the surgical management of thyroid nodules with category III/IV cytology and its correlation with surgical outcome. Acta Cytol. 2016; 60(3):205-210.
  16. Chudova D, Wilde JI, Wang ET, et al. Molecular classification of thyroid nodules using high-dimensionality genomic data. J Clin Endocrin Metab. 2010; 95(12):5296-5304.
  17. Cibas ES, Baloch ZW, Fellegara G, et al. A prospective assessment defining the limitations of thyroid nodule pathologic evaluation. Ann Intern Med. 2013; 159(5):325-332.
  18. Dhingra JK. Office-based ultrasound-guided FNA with molecular testing for thyroid nodules. Otolaryngol Head Neck Surg. 2016; 155(4):564-567.
  19. Duick DS, Klopper JP, Diggans JC et al. The impact of benign gene expression classifier test results on the endocrinologist-patient decision to operate on patients with thyroid nodules with indeterminate fine-needle aspiration cytopathology. Thyroid. 2012; 22(10):996-1001.
  20. Ferraz C, Eszlinger M, Paschke R. Current state and future perspective of molecular diagnosis of fine-needle aspiration biopsy of thyroid nodules. J Clin Endocrinol Metab. 2011; 96(7):2016-2026.
  21. Harrell RM, Bimston DN. Surgical utility of Afirma: effects of high cancer prevalence and oncocytic cell types in patients with indeterminate thyroid cytology. Endocr Pract. 2014; 20(4):364-369.
  22. Harrell RM, Eyerly-Webb SA, Golding AC, et al. Statistical comparison of Afirma GSC and AFIRMA GSC outocomes in a community endocrine surgical practice: early findings. Endocr Pract. 2019; 25(2):161-164.
  23. Jug R, Parajuli S, Ahmadi S, Jiang XS. Negative results on thyroid molecular testing decrease rates of surgery for indeterminate thyroid nodules. Endocr Pathol. 2019; 30(2):134-137.
  24. Kloos RT, Monroe RJ, Traweek ST, et al. A genomic alternative to identify medullary thyroid cancer preoperatively in thyroid nodules with indeterminate cytology. Thyroid. 2016; 26(6):785-793.
  25. Labourier E, Shifrin A, Busseniers AE, et al. Molecular testing for miRNA, mRNA, and DNA on fine-needle aspiration improves the preoperative diagnosis of thyroid nodules with indeterminate cytology. J Clin Endocrinol Metab. 2015; 100(7):2743-2750.
  26. Lithwick-Yanai G, Dromi N, Shtabsky A, et al. Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilising fine needle aspirate smears. J Clin Pathol. 2017; 70(6):500-507.
  27. Livhits MJ, Zhu CY, Kuo EJ, et al. Effectiveness of molecular testing techniques for diagnosis of indeterminate thyroid nodules: A randomized clinical trial. JAMA Oncol. 2021 Jan 1;7(1):70-77.
  28. Lupo MA, Walts AE, Sistrunk JW, et al. Multiplatform molecular test performance in indeterminate thyroid nodules. Diagn Cytopathol. 2020; 48(12):1254-1264.
  29. Marcadis AR, Valderrabano P, Ho AS, et al. Interinstitutional variation in predictive value of the ThyroSeq v2 genomic classifier for cytologically indeterminate thyroid nodules. Surgery. 2019;165(1):17-24.
  30. Mathur A, Weng J, Moses W, et al. A prospective study evaluating the accuracy of using combined clinical factors and candidate diagnostic markers to refine the accuracy of thyroid fine needle aspiration biopsy. Surgery. 2010; 148(6):1170-1177.
  31. McIver B, Castro MR, Morris JC, et al. An independent study of a gene expression classifier (Afirma) in the evaluation of cytologically indeterminate thyroid nodules. J Clin Endocrinol Metab. 2014; 99(11):4069-4077.
  32. Moses W, Weng J, Sansano I, et al. Molecular testing for somatic mutations improves accuracy of thyroid fine-needle aspiration biopsy. World J Surg. 2010; 34(11):2589-2594.
  33. Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med. 2011; 135(5):569-577.
  34. Nikiforov YE, Carty SE, Chiosea SI, et al. Highly accurate diagnosis of cancer in thyroid nodules with follicular neoplasm/suspicious for a follicular neoplasm cytology by ThyroSeq v2 next-generation sequencing assay. Cancer. 2014; 120(23):3627-3634.
  35. Nikiforov YE, Carty SE, Chiosea SI, et al. Impact of the multi-gene ThyroSeq next-generation sequencing assay on cancer diagnosis in thyroid nodules with atypia of undetermined significance/follicular lesion of undetermined significance cytology. Thyroid. 2015; 25(11):1217-1223.
  36. Nikiforov YE, Steward DL, Robinson-Smith TM, et al. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab. 2009; 94(6):2092-2098.
  37. Nikiforova MN, Mercurio S, Walk AI, et al. Analytical performance of the ThyroSeq v3 genomic classifier for cancer diagnosis in thyroid nodules. Cancer. 2018; 124(8):1682-1690.
  38. Nikiforova MN, Wald AI, Roy S, et al. Targeted next-generation sequencing panel (ThyroSeq) for detection of mutations in thyroid cancer. J Clin Endocrinol Metab. 2013; 98(11):E1852-1860.
  39. Ohori NP, Landau MS, Carty SE, et al. Benign call rate and molecular test result distribution of ThyroSeq v3. Cancer Cytopathol. 2019; 127(3):161-168.
  40. Ohori NP, Nikiforova MN, Schoedel KE, et al. Contribution of molecular testing to thyroid fine-needle aspiration cytology of  “follicular lesion of undetermined significance/atypia of undetermined significance.” Cancer Cytopathol. 2010; 118(1):17-23.
  41. Pankratz DG, Hu Z, Kim SY, et al. Analytical performance of a gene expression classifier for medullary thyroid carcinoma. Thyroid. 2016; 26(11):1573-1580.
  42. Parajuli S, Jug R, Ahmadi S, Jiang XS. Hurthle cell predominance impacts results of Afirma gene expression classifier and ThyroSeq molecular panel performance in indeterminate thyroid nodules. Diagn Cytopathol. 2019; 47(11):1177-1183.
  43. Patel KN, Angell TE, Babiarz J, et al. Performance of a genomic sequencing classifier for the preoperative diagnosis of cytologically indeterminate thyroid nodules. JAMA Surg. 2018; 153(9):817-824.
  44. Santhanam P, Khthir R, Gress T, et al. Gene expression classifier for the diagnosis of indeterminate thyroid nodules: a meta-analysis. Med Oncol. 2016; 33(2):14.
  45. Shrestha RT, Evasovich MR, Amin K, et al. Correlation between histological diagnosis and mutational panel testing of thyroid nodules: a two-year institutional experience. Thyroid. 2016; 26(8):1068-1076.
  46. Silaghi CA, Lozovanu V, Georgescu CE, et al. Thyroseq v3, Afirma GSC, and microRNA panels versus previous molecular tests in the preoperative diagnosis of indeterminate thyroid nodules: A systematic review and meta-snalysis. Front Endocrinol (Lausanne). 2021. Available at: Accessed on April 14, 2022.
  47. Singer J, Hanna JW, Visaria J, Gu T, et al. Impact of a gene expression classifier on the long-term management of patients with cytologically indeterminate thyroid nodules. Curr Med Res Opin. 2016; 32(7):1225-1232.
  48. Sipos JA, Blevins TC, Shea HC, et al. Long-term nonoperative rate of thyroid nodules with benign results on the Afirma gene expression classifier. Endocr Pract. 2016; 22(6):666-672.
  49. Sistrunk JW, Shifrin A, Frager M, et al. Clinical performance of multiplatform mutation panel and microRNA risk classifier in indeterminate thyroid nodules. J Am Soc Cytopathol. 2020; 9(4):232-241.
  50. Steward DL, Carty SE, Sippel RS, et al. Performance of a multigene genomic classifier in thyroid nodules with indeterminate cytology: a prospective blinded multicenter study. JAMA Oncol. 2019; 5(2):204-212.
  51. Taye A, Gurciullo D, Miles BA, et al. Clinical performance of a next-generation sequencing assay (ThyroSeq v2) in the evaluation of indeterminate thyroid nodules. Surgery. 2018; 163(1):97-103.
  52. Valderrabano P, Khazai L, Leon ME, et al. Evaluation of ThyroSeq v2 performance in thyroid nodules with indeterminate cytology. Endocr Relat Cancer. 2017; 24(3):127-136.
  53. Valderrabano P, Hallanger-Johnson JE, et al. Comparison of postmarketing findings vs the initial clinical validation findings of a thyroid nodule gene expression classifier: a systematic review and meta-analysis. JAMA Otolaryngol Head Neck Surg. 2019;e191449.
  54. Vargas-Salas, Martínez JR, Urra S, et al. Genetic testing for indeterminate thyroid cytology: review and meta-analysis. Endocr Relat Cancer. 2018; 25(3):R163-R177.
  55. Vora A, Holt S, Haque W, Lingvay I. Long-term outcomes of thyroid nodule AFIRMA GEC testing and literature review: An institutional experience. Otolaryngol Head Neck Surg. 2020; 162(5):634-640.
  56. Walsh PS, Wilde JI, Tom EY et al. Analytical performance verification of a molecular diagnostic for cytology-indeterminate thyroid nodules. J Clin Endocrinol Metab. 2012; 97(12):E2297-2306.
  57. Ward LS, Kloos RT. Molecular markers in the diagnosis of thyroid nodules. Arq Bras Endocrinol Metabol. 2013; 57(2):89-97.
  58. Wirth L, Grubbs E, Livhits M, et al. Identification of novel and rare receptor tyrosine kindase fusions in thyroid fine needle aspirates. J Endocr Soc. A1102.
  59. Witt RL. Outcome of thyroid gene expression classifier testing in clinical practice. Laryngoscope. 2016; 126(2):524-527.
  60. Wylie D, Beaudenon-Huibregtse S, Haynes BC, et al. Molecular classification of thyroid lesions by combined testing for miRNA gene expression and somatic gene alterations. J Pathol Clin Res. 2016; 2(2):93-103.
  61. Xing M, Clark D, Guan H et al. BRAF mutation testing of thyroid fine-needle aspiration biopsy specimens for preoperative risk stratification in papillary thyroid cancer. J Clin Oncol. 2009; 27(18):2977-2982.
  62. Yang SE, Sullivan PS, Zhang J, et al. Has Afirma gene expression classifier testing refined the indeterminate thyroid category in cytology? Cancer Cytopathol. 2016; 124(2):100-109.

Government Agency, Medical Society, and Other Authoritative Publications:

  1. Bible K, Kebebew E, Brierley J, et al. American Thyroid Association guidelines for management of patients with anaplastic thyroid cancer Force. 2021. Available at: Accessed on April 14, 2022..
  2. Cibas ES, Ali SZ. The 2017 Bethesda System for Reporting Thyroid Cytopathology. Thyroid. 2017; 27(11):1341-1346.
  3. Ferris RL, Baloch Z, Bernet V, et al. American Thyroid Association Surgical Affairs Committee. American Thyroid Association statement on surgical application of molecular profiling for thyroid nodules: current impact on perioperative decision making. Thyroid. 2015; 25(7):760-768.
  4. Francis GL, Waguespack SG, Bauer AJ, et al; American Thyroid Association Guidelines Task Force. Management guidelines for children with thyroid nodules and differentiated thyroid cancer. Thyroid. 2015; 25(7):716-759.
  5. Gharib H, Papini E, Garber JR, et al. American Association of Clinical Endocrinologists, American College of Endocrinology, and Associazione Medici Endocrinologi medical guidelines for clinical practice for the diagnosis and management of thyroid nodules–2016 Update. Endocr Pract. 2016; 22(5):622-639.
  6. Haugen BR, Alexander EK, Bible KC, et al. 2015 American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid. 2016; 26(1):1-133.
  7. Haugen BR, Sawka AM, Alexander EK, et al. American Thyroid Association guidelines on the management of thyroid nodules and differentiated thyroid cancer task force review and recommendation on the proposed renaming of encapsulated follicular variant papillary thyroid carcinoma without invasion to noninvasive follicular thyroid neoplasm with papillary-like nuclear features. Thyroid. 2017; 27(4):481-483.
  8. NCCN Clinical Practice Guidelines in Oncology®. © 2022 National Comprehensive Cancer Network, Inc. Thyroid cancer (V.1.2022). Updated April 05, 2022. For additional information visit the NCCN website: Accessed on April 14, 2022.
Websites for Additional Information
  1. National Cancer Institute. Thyroid Cancer. Available at:‌types/thyroid/hp. Accessed on April 14, 2022.

Afirma Thyroid FNA Analysis

The use of specific product names is illustrative only. It is not intended to be a recommendation of one product over another, and is not intended to represent a complete listing of all products available.







Medical Policy & Technology Assessment Committee (MPTAC) review. Updated Discussion/General Information, References and Websites sections.



Updated Coding section with 01/01/2022 CPT changes; added 0287U effective 01/01/2022, removed 0208U deleted 12/31/2021.



MPTAC review. ThyGeNEXT and ThyraMIR added to the MN statement. ThyGenX and ThyraMIR removed from NMN statement. RosettaGX removed from the position statement. Updated Coding, Discussion/General Information, References and Websites sections.



Updated Coding section with 04/01/2021 CPT changes; added 0245U.



MPTAC review. Added Afirma MTC to MN statement and Afirma Xpression Atlas to NMN statement. Updated Background/Overview, References and Websites sections. Reformatted Coding section, updated to add CPT PLA codes 0204U, 0208U and 01/01/2021 CPT changes 81546 replacing 81545 deleted 12/31/2020.



MPTAC review. Revised MN statement to reflect updated second generation Affirma test, Genomic Sequencing Classifier (GSC). Updated Description/Scope, Background/Overview, References and Websites sections.



MPTAC review.



Hematology/Oncology Subcommittee review. Added ThyroSeq to the MN statement. Coding, References and Websites sections updated.



MPTAC review.



Hematology/Oncology Subcommittee review. Updated acronym in Clinical Indications section. Description, Discussion/General Information, and References sections updated. Added Websites for Additional Information section.



MPTAC review.



Hematology/Oncology Subcommittee review. Initial document development. Moved content of GENE.00032 Molecular Marker Evaluation of Thyroid Nodules to new clinical utilization management guideline document with the same title. Addition of Afirma, ThyGenX, ThyraMIR, Thryoseq, and Rosetta to Clinical Indications section. Updated Coding section with 01/01/2018 CPT changes; added code 0026U.


Bethesda System for Reporting Thyroid Cytopathology: Recommended Diagnostic Categories


(Cibas and Ali, 2017)

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