Coverage Policy Manual
Policy #: 2012003
Category: Laboratory
Initiated: January 2012
Last Review: July 2018
  Genetic Test: Molecular Markers in Fine Needle Aspirates of the Thyroid

Description:
Fine needle aspiration (FNA) of a thyroid lesion to identify which patients need to undergo surgery has diagnostic limitations and has led to the development of molecular markers in an attempt to improve the accuracy.
 
Background
FNA of the thyroid
Thyroid nodules are common, present in 5% to 7% of the U.S. adult population. Most are benign, and most cases of thyroid cancer are curable by surgery when detected early. FNA of the thyroid is currently the most accurate procedure to distinguish benign thyroid lesions and malignant ones, reducing the rate of unnecessary thyroid surgery for patients with benign nodules and triaging patients with thyroid cancer to appropriate surgery.
 
About 60% to 70% of thyroid nodules are classified cytologically as benign, and 4% to 10% of nodules are cytologically deemed malignant.1 However, the remaining 20% to 30% have equivocal findings, usually due to overlapping cytologic features between benign and malignant nodules; these nodules usually require surgery for a final diagnosis. Thyroid FNA cytology is classified by Bethesda System criteria into the following categories: Category I nondiagnostic; Category II benign; Category III follicular lesion of undetermined significance (FLUS) or atypia of undetermined significance (AUS); Category IV  follicular neoplasm (or suspicious for follicular neoplasm); Category V suspicious for malignancy; and  Category VI malignant. Lesions with FNA cytology in the AUS or FLUS or follicular neoplasm categories are often considered indeterminate.
 
There is some individualization of management for patients with FNA-indeterminate nodules, but many patients will require a surgical biopsy, typically thyroid lobectomy, with intraoperative pathology. Consultation would typically be the next step in diagnosis. Approximately 80% of patients with indeterminate cytology undergo surgical resection; postoperative evaluation reveals a malignancy rate ranging from 6% to 30%, making this a clinical process with very low specificity (Chudova, 2010).
 
Preoperative planning of optimal surgical management in patients with equivocal cytologic results is challenging, as different thyroid malignancies may require different surgical procedures (eg, unilateral lobectomy versus total or subtotal thyroidectomy with or without lymph node dissection) depending on several factors, including histologic subtype and risk-stratification strategies (tumor size, patient age) If a diagnosis cannot be made intraoperatively, a lobectomy is typically performed, and if on postoperative histology the lesion is malignant, a second surgical intervention may be necessary for completion thyroidectomy.
 
Thyroid cancer
Most thyroid cancers originate from thyroid follicular cells and include well-differentiated papillary thyroid carcinoma (PTC) (80% of all thyroid cancers) and follicular carcinoma (15%). Poorly differentiated and anaplastic thyroid carcinomas are uncommon and can arise de novo or from preexisting welldifferentiated papillary or follicular carcinomas. Medullary thyroid carcinoma originates from parafollicular or C cells and accounts for ~3% of all thyroid cancers.
 
The diagnosis of malignancy in the case of PTC is primarily based on cytologic features. If an FNA in a case of PTC is indeterminate, intraoperative consultation is most often diagnostic, although its efficacy and therefore use will vary between institutions, surgeons, and pathologists.  
 
For follicular carcinoma, the presence of invasion of the tumor capsule or of blood vessels is diagnostic and cannot be determined by cytology, as tissue sampling is necessary to observe these histologic characteristics. Intraoperative diagnosis of follicular carcinoma is challenging and often not feasible, because extensive sampling of the tumor and capsule is usually necessary and performed on postoperative permanent sections.
 
New approaches for improving the diagnostic accuracy of thyroid FNA include mutation analysis for somatic genetic alterations, to more accurately classify which patients need to proceed to surgery (and may include the extent of surgery necessary) and a gene expression classifier to identify patients who do not need surgery and can be safely followed.
 
Mutations associated with thyroid cancer
Various mutations have been discovered in thyroid cancer. The 4 gene mutations that are the most common and carry the highest impact on tumor diagnosis and prognosis are BRAF and RAS point mutations and RET/PTC and PAX8/PPARγ rearrangements.  
 
Papillary carcinomas carry point mutations of the BRAF and RAS genes, as well as RET/PTC and TRK rearrangements, all of which are able to activate the mitogen-activated protein kinase (MAPK) pathway (Nikiforov, 2011). These mutually exclusive mutations are found in more than 70% of papillary carcinomas(Nikiforov, 2011). BRAF mutations are highly specific for PTC. Follicular carcinomas harbor either RAS mutations or PAX8/PPARγ rearrangement. These mutations are also mutually exclusive and identified in 70% to 75% of follicular carcinomas(Nikiforov, 2011). Genetic alterations involving the PI3K/AKT signaling pathway also occur in thyroid tumors, although they are rare in well-differentiated thyroid cancer and have higher prevalence in less differentiated thyroid carcinomas.(4) Additional mutations known to occur in poorly differentiated and anaplastic carcinomas involve the TP53 and CTNNB1 genes. Medullary carcinomas, which can be familial or sporadic, frequently possess point mutations located in the RET gene.
 
Regulatory Status
Commercially available panels of molecular markers utilizing FNA specimens from the thyroid include miRInform™ (Asuragen) and Veracyte® (Afirma).
 
miRInform is a panel of 7 analytically validated molecular markers [mutations] (KRAS, BRAF, HRAS, NRAS, RET/PTC 1, RET/PTC3, and PAX8/PPARγ).
 
The Afirma “gene expression classifier” (GEC) is a proprietary diagnostic test offered by Veracyte, which claims to classify a thyroid nodule with indeterminate cytology as benign (with >95% negative predictive value) or as suspicious for malignancy (>50% risk of malignancy). The GEC measures the gene expression of 142 genes and applies a multidimensional algorithm to classify whether a nodule with an indeterminate cytologic diagnosis is benign or suspicious.
 
These commercially available, laboratory-developed tests are regulated under the Clinical Laboratory Improvement Amendments (CLIA). Premarket approval from the U.S. Food and Drug Administration is not required when the assay is performed in a laboratory that is licensed by CLIA for high-complexity testing.
 
Coding
 
There is a specific CPT code for the Afirma® Gene Expression Classifier test:
 
81545 Oncology (thyroid), gene expression analysis of 142 genes, utilizing fine needle aspirate, algorithm
reported as a categorical result (eg, benign or suspicious).

Policy/
Coverage:
Meets Primary Coverage Criteria Or Is Covered For Contracts Without Primary Coverage Criteria
 
Effective July 2018
 
The use of the Afirma Gene Expression Classifier in fine needle aspirates of the thyroid that are cytologically considered to be Bethesda category III (atypia of undetermined significance/follicular lesion of undetermined significance) or category IV (follicular neoplasm) meets member benefit certificate primary coverage criteria in patients who have the following characteristics:
 
· Thyroid nodules without strong clinical or radiologic findings suggestive of malignancy.
· In whom surgical decision making would be affected by test results.
 
The use of any of the following types of molecular marker testing or gene variant analysis in fine needle aspirates of thyroid nodules with indeterminate findings (Bethesda diagnostic category III [atypia/follicular lesion of undetermined significance] or Bethesda diagnostic category IV [follicular neoplasm/suspicion for a follicular neoplasm]) or suspicious findings (Bethesda diagnostic category V [suspicious for malignancy]) to rule in malignancy to guide surgical planning for initial resection rather than a 2-stage surgical biopsy followed by definitive surgery meets member benefit certificate primary coverage criteria:
 
· ThyroSeq v2;
· ThyraMIR microRNA/ThyGenX;
· Afirma BRAF after Afirma Gene Expression Classifier; or
· Afirma MTC after Afirma Gene Expression Classifier.
 
Does Not Meet Primary Coverage Criteria Or Is Investigational For Contracts Without Primary Coverage Criteria
The use of gene expression classifiers, genetic variant analysis, and molecular marker testing in fine needle aspirates of the thyroid not meeting criteria outlined above including but not limited to use of RosettaGX Reveal and single-gene TERT testing, do not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.
 
The use of gene expression classifiers, genetic variant analysis, and molecular marker testing in fine needle aspirates of the thyroid not meeting criteria outlined above including but not limited to use of RosettaGX Reveal and single-gene TERT testing, are considered to be investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
 
Effective Prior to July 2018
The use of the Afirma Gene Expression Classifier in fine needle aspirates of the thyroid that are cytologically considered to be Bethesda category III (atypia of undetermined significance/follicular lesion of undetermined significance) or category IV (follicular neoplasm) meets member benefit certificate primary coverage criteria in patients who have the following characteristics:
 
    • Thyroid nodules without strong clinical or radiologic findings suggestive of malignancy.
    • In whom surgical decision making would be affected by test results.
 
The use of gene expression classifiers in fine needle aspirates of the thyroid not meeting criteria outlined above do not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.
 
The use of gene expression classifiers in fine needle aspirates of the thyroid not meeting criteria outlined above is considered to be investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective October 2014 to October 2017
Mutation analysis in fine-needle aspirates of the thyroid does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.
 
For contracts without primary coverage criteria, mutation analysis in fine-needle aspirates of the thyroid is considered to be investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
The use of a gene expression classifier in fine-needle aspirates of the thyroid that are cytologically considered to be indeterminate, atypical or suspicious for malignancy does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.
 
The use of a gene expression classifier in fine-needle aspirates of the thyroid that are cytologically considered to be indeterminate, atypical or suspicious for malignancy is considered to be investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 
Effective prior to October 2014
Mutation analysis in fine-needle aspirates of the thyroid that are cytologically considered to be indeterminate, atypical or suspicious for malignancy does not meet member benefit certificate primary coverage criteria that there be scientific evidence of effectiveness in improving health outcomes.
 
For contracts without primary coverage criteria, mutation analysis in fine-needle aspirates of the thyroid that are cytologically considered to be indeterminate, atypical or suspicious for malignancy is considered to be investigational. Investigational services are specific contract exclusions in most member benefit certificates of coverage.
 

Rationale:
This policy was created in 2012, with a MEDLINE literature search performed through December 2011.
 
The literature on the use of molecular markers for thyroid nodules diagnosed by FNA as indeterminate, atypical or suspicious consists of approximately 20 publications. These studies have analyzed either panels of mutations or a single mutation in these fine-needle aspirates and compared the preoperative cytologic diagnosis and mutation status to postoperative final histologic diagnosis to determine diagnostic accuracy of the presence of a mutation. However, neither prospective nor comparative studies to determine how the preoperative result of the presence of a mutation in a thyroid nodule with equivocal cytology results would impact patient management have been performed.
 
Ferraz and colleagues evaluated 20 publications that reported on the type and number of mutations in cases of FNA of the thyroid diagnosed as indeterminate and compared the results to final histology after surgical resection (Ferraz, 2011). Sixteen studies analyzed one mutation (e.g. BRAF or RET/PTC) and 4 studies analyzed a panel of several mutations (BRAF, RAS, RET/PTC, and PAX8/PPARγ). The detection of a mutation in a histologically (surgically resected) benign thyroid lesion was categorized as a false positive (FP) case, detecting no mutation in an FNA sample from a histologically benign surgical sample was considered a true negative (TN), and finding no mutation in a histologically malignant lesion was categorized as a false negative (FN). Based on 4 studies that examined a panel of mutations, there was a broad sensitivity range of 38-85.7% (mean 63.7%), a mean specificity of 98% (range 95-100%), mean false positive rate of 1.25% (0-4%) and mean false negative rate of 9% (1-21%). Based on 2 studies that examined RET/PTC rearrangements, mean sensitivity was 55% (50-60%), specificity 100%, false positive rate of 0% and mean false negative rate 3.5% (91-6%). Based on 3 studies that examined BRAF mutations, mean sensitivity was 13% (0-37.5%), mean specificity 92.3% (75-100%), mean false positive rate 0.5% (0-1%) and mean false negative rate of 6% (3-12%). The authors concluded that testing for a panel of mutations leads to an improvement in the sensitivity and specificity for indeterminate FNA of the thyroid, but that further standardizations and further molecular markers are needed before broad application of molecular FNA cytology for the diagnosis of thyroid nodules.
 
Nikiforov and colleagues prospectively tested a panel of mutations (BRAF, RAS, RET/PTC and PAX8/PPARγ) in 470 FNA samples of thyroid nodules from 328 consecutive patients (Nikiforov, 2009).  Mutational status was correlated with cytology, and either surgical pathology diagnosis or follow-up (mean 34 months). 40 patients were excluded for poor quality of specimen or loss to followup. Sixty-nine patients (with 86 thyroid FNA samples) underwent surgery soon after completion of the cytologic evaluation; preoperative cytologic diagnosis was: positive for malignancy in 22 samples, indeterminate (including atypical and suspicious for malignancy) in 52 samples, and negative for malignancy in 12 samples. By FNA, 32 mutations were found (18 BRAF, 8 RAS, 5 RET/PTC, and 1 PAX8/PPARγ); after surgery, 31 mutation positive nodules (97%) were diagnosed as malignant on pathological examination and one was a benign tumor (3%). Thirteen of the 32 mutation-positive FNA samples had a definitive cytologic diagnosis of malignancy, whereas the rest were either indeterminate or negative for malignancy.
 
Of the remaining 219 patients, 147 (229 FNAs) who did not undergo surgery were followed by serial ultrasound with no change in the nodule status (124 patients) or by repeated FNA with cytology negative for malignancy (23 patients) and no mutation found in the FNA material. These nodules were considered as negative for malignancy.
 
The remaining 72 patients that were initially in the follow-up group underwent subsequent surgery. Combining all 3 groups, the specificity for malignancy was high (99.7%), but the sensitivity of the molecular test alone was 62%.
 
Moses and colleagues prospectively tested FNA samples from 417 patients with 455 thyroid nodules for BRAF, NRAS, KRAS and RET/PTC 1 and 3 and TRK1 mutations (Moses, 2010). Overall, 50 mutations (23 BRAF V600E, 21NRAS and 4 RET/PTC1 and 2 RET/PTC3 rearrangements) were detected. There were significantly more mutations detected in malignant nodules than in benign (p=0.0001). For thyroid FNA biopsies that were indeterminate or suspicious (n=137), genetic testing had a sensitivity of 12%, specificity of 98%, positive predictive value of 38% and negative predictive value of 65%.
 
Ohori and colleagues performed mutation screening in 117 FNA samples classified as a follicular lesion of indeterminate significance/atypia of indeterminate significance (Ohori, 2010). BRAF, RAS, RET/PTC, or PAX8/PPARγ mutations were detected in 10% of this category. They demonstrated that the probability of having a malignancy in this cytology category together with a detection of one of the somatic mutations investigated was 100%, whereas the probability of having a thyroid malignancy without a mutation detected was 7.6%.
 
Cantara and colleagues analyzed a panel of mutations in samples of 174 patients undergoing thyroid surgery for indeterminate/inadequate/benign FNA results (Cantara, 2010). The most prevalent mutation was BRAF (49.3% of the positive samples), followed by RAS (34.3%) and RET/PTC (16.4%). The combination of cytology and mutation analysis improved the accuracy for diagnosing cancer from 83% to 93.2% when compared to cytologic analysis alone. Molecular analysis detected 8 thyroid cancers that were missed on cytology from a total of 32 cancers that were diagnosed as indetermate/inadequate/benign. When the FNA mutation analysis was compared with the mutation analysis of the corresponding histologic material from the surgical sample, in 88.2% of cases, the mutation found in the FNA material was also detected in the histologic samples. The 11.8% discrepant results were due to the presence of a mutation in the tissue sample that was not found in the cytology sample.
 
Mathur and colleagues collected thyroid FNA samples, thyroid tissue, clinical and histopathology data, and tumor genotyping for mutations BRAF V600E, NRAS, KRAS, RET/PTC1, RET/PTC3 and NTRK1 for 341 patients with 423 dominant thyroid nodules (Mathur, 2010). A cytologic examination of the samples showed that 51% were benign (one-quarter of these were surgically resected), 21% were malignant, 11% were atypical lesions, 12% were follicular or Hurthle cell neoplasms, and 4% were suspicious for malignancy. On final analysis, 165 nodules were benign and 123 were malignant. Of the 423 FNA samples, 24 BRAF V600E mutations, 7 KRAS, 21 NRAS 4 PAX8-PPARγ rearrangements, 3 RET/PTC1 and 2 RET/PTC3 rearrangements were detected. In all, 17 of 165 (10.3%) benign thyroid nodules had a mutation compared with 26% (32 of 123) malignant tumors (p<.05).
 
BRAF
Adeniran and colleagues conducted a study of 157 cases with equivocal thyroid FNA readings (indeterminate and suspicious for PTC) or a positive diagnosis for PTC, and concomitant BRAF mutation analysis (Adeniran, 2011). The results of histopathologic follow-up were correlated with the cytologic interpretations and BRAF status. Based on the follow-up diagnosis after surgical resection, the sensitivity for diagnosing PTC was 63.3% with cytology alone and 80.0% with the combination of cytology and BRAF testing. No false positives were noted with either cytology or BRAF mutation analysis. All PTCs with extrathyroidal extension or aggressive histologic features were positive for BRAF mutation. The authors concluded that patients with an equivocal cytologic diagnosis and BRAF V600E mutation could be candidates for total thyroidectomy and central lymph node dissection.
 
Xing and colleagues investigated the utility of BRAF mutation testing of thyroid FNA specimens for preoperative risk stratification in PTC in 190 patients (Xing, 2009). A BRAF mutation in preoperative FNA specimens was associated with poorer clinicopathologic outcomes of PTC. In comparison with the wild-type allele, a BRAF mutation strongly predicted extrathyroidal extension (23% v 11%; P = .039), thyroid capsular invasion (29% v 16%; P = .045), and lymph node metastasis (38% v 18%; P = .002). During a median follow-up of 3 years (range, 0.6 to 10 years), PTC persistence/recurrence was seen in 36% of BRAF mutation-positive patients versus 12% of BRAF mutation-negative patients, with an odds ratio of 4.16 (95% CI, 1.70 to 10.17; P = .002). The positive and negative predictive values for preoperative FNA-detected BRAF mutation to predict PTC persistence/recurrence were 36% and 88% for all histologic subtypes of PTC. The authors concluded that preoperative BRAF mutation testing of FNA specimens may provide a novel tool to preoperatively identify PTC patients at higher risk for extensive disease (extrathyroidal extension and lymph node metastases) and those who are more likely to manifest disease persistence/recurrence.
 
National Cancer Institute Clinical Trials
No phase 3 trials analyzing mutation analysis in fine-needle aspirates of the thyroid were identified.
 
Practice Guidelines and Position Statements
The American Thyroid Association (ATA) guidelines suggest consideration of the use of molecular markers like BRAF, RAS, RET/PTC, PAX8-PPARγ or galectin-3 for patients with indeterminate cytology on FNA and that if detectable, these markers can help guide thyroid nodule management by assisting in estimating thyroid cancer risk (Paschke, 2011).  
 
National Comprehensive Cancer Network (NCCN) Guidelines on the treatment of thyroid cancer state that “molecular diagnostics to detect individual mutations in BRAF, RET, or RAS or pattern recognition approaches using molecular classifiers may be useful in the evaluation of FNA samples that are indeterminate (i.e. follicular lesion of undetermined significance”) (V.3.2011).
 
Summary
Mutation analysis of fine needle aspirates (FNA) of the thyroid that are cytologically indeterminate has a high predictive value of malignancy. However, patients with an equivocal FNA result would likely proceed to surgery regardless of mutation status, with intraoperative consultation to guide the necessity and extent of surgery. Mutation analysis does not achieve a high enough negative predictive value to identify which patients can undergo watchful waiting over thyroid surgery, and there are no known markers that can identify benign thyroid lesions.
 
Therefore, the incremental added value of a molecular test to an equivocal FNA result is not known.
Although mutation analysis has 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 patient management or surgical decision making.
 
2014 Update
This policy is being updated with a literature search through September 2014. The following is a summary of the key identified literature.
 
In 2014, Alexander et al reported results from a retrospective analysis of 339 thyroid nodules which underwent Afirma GEC testing for indeterminate cytology on FNA (follicular lesion of undetermined significance/atypia of undetermined significance, follicular neoplasm, or suspicious for malignancy) at 5 academic medical centers (Alexander, 2014). Most of the nodules sent for GEC testing were follicular lesions of undetermined significance/atypia of undetermined significance or follicular neoplasm.
 
A subset of patients whose nodules underwent GEC testing underwent a subsequent thyroid resection. Among 148 cases with suspicious Afirma GEC findings, surgery (thyroid resection) was recommended for 141 (95%). For the 174 cases with benign Afirma GEC findings, surgery was recommended for 4 (2%; p<0.01). Using the assumption that, in the absence of the GEC results, thyroid surgery would be recommended for patients with cytologically indeterminate FNA results, the authors report that the GEC results altered management in 50% of patients.
 
Seventeen patients who had indeterminate cytology, benign Afirma GEC results, and did not undergo surgery had follow-up beyond 1 year. Of those, 3 patients underwent surgical removal of the nodule because of compressive symptoms (n=2) or nodule growth (n=1); all nodules were benign on final histology. The remaining 14 patients had ongoing follow up with ultrasound with no ongoing evidence of malignancy. The study demonstrated site-to-site variation in the proportion of samples that were GEC benign.
 
This study suggests that the Afirma GEC may alter clinical management of patients with indeterminate thyroid nodules. While the treating physicians presumably elected to obtain the GEC testing with the intent of altering management recommendations, the magnitude of the difference in surgical recommendations for patients with GEC suspicious or benign results was large. A limitation of this study is its retrospective, unblinded nature; thus, factors other than GEC testing may have contributed to either the recommendation for surgery or patients’ decisions to undergo surgery. A benign GEC result did not completely rule out malignant pathology. Long-term follow up was available for only a small proportion of patients with benign GEC findings who did not undergo surgery.
 
In a single-center study, Aragon Han et al reported surgical management decision making outcomes among 114 patients with thyroid nodules who underwent molecular testing (Aragon, 2014). Of 114 patients, 87 underwent thyroid surgery. Testing included a combination of the Afirma gene-expression classifier (n=37), a DNA-based somatic mutation panel (n=21), and testing for BRAF mutations (n=29), BRAF/NRAS (n=1), and BRAF/RET/PTC (n=1). A surgical decision-making algorithm that did not include mutation testing was developed by consensus among 4 thyroid surgeons. If the surgeon performed the same surgery as anticipated by the management algorithm, then the molecular test was considered to have no impact. If the surgeon performed a different surgery than anticipated by the management
algorithm, the molecular test was considered to effect a change in management. The authors report that surgical management was not changed by molecular testing in 89.7% of cases. This study is limited by its use of multiple types of molecular testing, along with a nonstandardized incorporation of molecular genetic testing results into the surgical decision making. As such, the study has limited implications for the clinical utility of molecular diagnostics for thyroid cancer.
 
The AACE/ACE published a commentary on molecular diagnostic testing of thyroid nodules with Indeterminate ytopathology  which outlined the following points (Bernet, 2014):
 
• Approximately 10 to 25% of fine-needle aspiration (FNA) biopsies yield an indeterminate result often labeled as atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS) or follicular neoplasm/suspicious for follicular neoplasm (FN/SFN). The risk of malignancy typically varies between 15 and 30% for these categories.
• Although many markers are in development and have been studied in a research setting, 2 principal tests are currently marketed for use to improve the malignancy risk assessment of "indeterminate" thyroid nodules. "Rule In" and "Rule Out" tests attempt to confirm or exclude the presence of cancer within a thyroid nodule by means of robust positive (PPV) or negative predictive values (NPV), respectively.
• The Rule In tests determine the presence of single gene point mutations (BRAFV600E or RAS) or gene rearrangements (RET/PTC, PAX8/PPARγ) that have been shown to increase the ability to predict cancer, while the Rule Out test (Afirma® gene expression classifier, GEC) utilizes a proprietary gene expression classifier (RNA expression) specifically designed to maximize the ability to define a process as benign.
• Among the presently available tests, only the BRAFV600E and RET/PTC rearrangement are associated with a PPV that approaches 100%.• The category of cytologically "indeterminate" nodule (AUS/FLUS, FN/SFN), cytopathology practice patterns, and the prevalence of malignancy within the population being tested all impact the NPVs and PPVs for the tests in question.
•At present, molecular testing is meant to complement and not replace clinical judgment, sonographic assessment, and visual cytopathology interpretation.
• As molecular testing is new and advances in the field are regularly occurring, clinicians need to stay informed, as recommendations for use within practice are expected to evolve. “ (Bernet, 2014)
 
Ongoing and Unpublished Clinical Trials
A search of online database ClinicalTrials.gov identified 1 study currently enrolling patients that is investigating the role of gene expression and thyroid cancer:
 
· Genetic Analysis in Diagnosing Thyroid Cancer in Patients With Thyroid Nodules (NCT00316823). This is an observational study to evaluate the diagnostic accuracy of biomarkers and mRNA expression analysis using FNA biopsy samples from patients with thyroid nodules and correlate the level of gene expression with the aggressiveness of differentiated thyroid cancer in FNA biopsy samples. Enrollment is planned for 400 subjects; the planned study completion date was December 2009. The study was verified on February 21, 2014, but no publications were identified.
 
Summary
 
Mutation analysis
Mutation analysis of fine needle aspirates (FNA) of the thyroid that are cytologically indeterminate has a high positive predictive value for malignancy. However, patients with an equivocal FNA result would likely proceed to surgery regardless of mutation status, with intraoperative consultation to guide the necessity and extent of surgery. Mutation analysis does not achieve a high enough negative predictive value to identify which patients can undergo watchful waiting over thyroid surgery. Although the presence of certain mutations may predict more aggressive malignancies, the clinical utility of identifying these mutations preoperatively has not been established.
  
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 patient management or surgical decision making.
 
Gene expression classifier
 
The reported negative predictive value of the gene expression classifier (GEC) in predicting which thyroid nodules with indeterminate cytology are benign is high. Two relevant retrospective studies on the clinical utility of the GEC have been published and suggest that treatment recommendations for patients with indeterminate cytology are affected by the results of the GEC test. For patients who avoided surgery based on GEC results, limited longer term follow up data are available. Although the available evidence suggests that this group of patients does well, longer term follow up has been reported for only a small number of patients.
  
2018 Update
Annual policy review completed with a literature search using the MEDLINE database through June 2018. The key identified literature is summarized below.
 
Molecular Markers to rule out malignancy
 
Clinical Context and Test Purpose
The purpose of molecular testing in individuals with indeterminate findings on fine needle aspirate(s) (FNA) of thyroid nodules is to rule out malignancy and eliminate the need for surgical biopsy or resection.
 
The relevant question addressed in this evidence review is: Does molecular testing appropriately eliminate the need for surgical biopsy or resection and lead to improved health outcomes?
 
The following PICOTS were used to select literature to inform this review.
 
Patients
The relevant population of interest is individuals with indeterminate findings on FNAs of thyroid nodules who would be willing to undergo watchful waiting, depending on results of their molecular testing. Patients with indeterminate findings after FNA of thyroid nodule presently proceed to surgical biopsy or resection.
 
Interventions
The test being considered is molecular testing, which includes either Afirma Gene Expression Classifier (GEC), ThyroSeq v2, or RosettaGX Reveal.
 
Comparators
The following practice is currently being used: standard surgical management through surgical biopsy or resection for biopsy.
 
Outcomes
The potential beneficial outcome of primary interest would be avoiding an unneeded surgical biopsy or resection (eg, lobectomy or hemithyroidectomy) in a true-negative thyroid nodule that is benign.
 
Potential harmful outcomes are those resulting from false-negative test results, which may delay diagnosis and surgical resection for thyroid cancer. For small, slow-growing tumors, it is uncertain that a delay in diagnosis would necessarily a worsen health outcomes.
 
Timing
The time frame for evaluating the performance of the test varies from the initial FNA to surgical resection to weeks to months following an indeterminate result to years. Papillary thyroid cancer (PTC) is indolent, and a nodule could be observed for many years to ensure no clinical change.
 
Setting
The primary setting would be in endocrinology.
 
Retrospective Clinical Validation
Santhanam et al conducted a meta-analysis of studies reporting on the performance of the Afirma GEC in cytologically indeterminate nodules (Santhanam, 2016). Seven studies met inclusion criteria, which required that studies reported on the use of the Afirma GEC in nodules found indeterminate on FNA (including atypia of uncertain significance [AUS] or follicular lesion of undetermined significance [FLUS]; suspicious for follicular or Hürthle cell neoplasm; suspicious for malignancy), and thyroidectomy was performed as a reference standard in at least the cases where the index test was suspicious. All studies were judged to be at low risk of bias for patient selection and most for GEC test selection, whereas the risk of bias in the final histopathology was low in 3 studies, unclear in 3 studies, and high in 1 study. Although the authors reported pooled results, these results (particularly specificity) were likely biased given the lack of reference standard diagnosis for most lesions in the included studies.
 
Retrospective multicenter and single-center studies, including Harrell and Bimston, Lastra et al, McIver et al, Yang et al, Witt, Baca et al, Harrison et al, Kay-Rivest et al, Hang et al, and Samulski et al have reported on the diagnostic accuracy of the Afirma GEC (Harrell, 2014; Lastra, 2014; McIver, 2014; Yang, 2016; Witt, 2016; Baca, 2017; Harrison, 2017; Kay-Rivest, 2017; Hang, 2017; Samulski, 2016). All studies were subject to ascertainment bias because a large proportion of individuals, with Afirma benign reports did not undergo surgery, which made determining the sensitivity and specificity of the GEC assay impossible. However, the rates of malignancy among patients with Afirma benign results who did undergo surgery were consistently low. One exception is the study by Harrell and Bimston; it may be reflective of a higher-than-usual overall rate of malignancy in patients with indeterminate FNA results (Harrell, 2014). An additional publication reported on Afirma GEC testing; however, included in this publication were individuals with benign and suspicious cytology on FNA¾and those individuals are not necessarily considered to be part of the “target population (Celik, 2015).”
 
There are limited data on the true-negative rates of individuals with indeterminate FNA cytology and Afirma GEC benign results. Supportive information on the accuracy Afirma GEC benign results can be obtained from studies that have reported on long-term follow-up of individuals with indeterminate FNA cytology and Afirma GEC benign results. Angell et al retrospectively compared clinical outcomes for individuals who had indeterminate FNA cytology and Afirma GEC benign results with individuals who had cytologically benign nodule results (Angell, 2015). A total of 95 cytologically indeterminate and Afirma GEC benign nodules in 90 patients were compared with 1224 cytologically benign nodules identified from a single-center, prospectively collected database. Five nodules in the cytologically indeterminate were resected; of the remaining 90 nodules, 58 (64.4%) had follow-up ultrasound available at a median of 13 months postdiagnosis. When nodule growth was defined by a volume increase of 50% or more, 17.2% cytologically indeterminate/Afirma GEC benign were considered to have grown compared with 13.8% of cytologically benign nodules (p=0.44). Surgical resection was more common in cytologically indeterminate and Afirma GEC benign nodules (13.8% vs 0.9%, p<0.001).
 
Clinically Useful
A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, or more effective therapy, or avoid unnecessary therapy, or avoid unnecessary testing.
 
Direct Evidence
Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from randomized controlled trials.
 
No evidence directly demonstrating improved outcomes in patients managed with the Afirma GEC was identified.
 
Chain of Evidence
Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about clinical utility.
 
Because no direct evidence of utility was identified, a chain of evidence was developed, which addresses 2 key questions:
    1. Does use of the Afirma GEC in individuals with cytologically indeterminate thyroid nodules change clinical management (in this case, reduced thyroid resections)?
    2. Do those management changes improve outcomes?
 
Changes in Management
The clinical setting in which the Afirma GEC is meant to be used is well-defined: individuals with AUS or FLUS or follicular neoplasm or who are suspicious for follicular neoplasm (SFN) on FNA, who do not have other indications for thyroid resection (ie, in whom the GEC results would play a role in surgical decision making).
 
Decision impact studies, most often reporting on clinical management changes but not on outcomes after surgical decisions were made, have suggested that, in at least some cases, surgical decision making changed. These studies are described briefly.
 
Duick et al reported on the impact of Afirma GEC test results on physician and patient decision making to resect thyroid nodules with indeterminate cytology and Afirma GEC benign results in a sample of 395 nodules from 368 patients (Duick, 2012). Surgery was performed in 7.6% of the patients with indeterminate cytology and a benign GEC result, less than the historical rate of thyroid resection (74%) in patients with indeterminate cytology.
 
Sipos et al retrospectively analyzed nonacademic medical practices using the Afirma GEC to determine the long-term nonoperative rate of thyroid nodules with benign results (Sipos, 2016). Of the patients with Afirma benign results during 36 months of follow-up, 17.3% underwent surgery. Eighty-eight percent of all surgeries were performed within the first 2 years after a benign Afirma GEC result.
 
The study by Alexander et al provided evidence on clinical management changes for patients with indeterminate thyroid nodules tested with Afirma GEC (Alexander, 2014). While the treating physicians presumably elected to obtain the GEC testing with the intent of altering management recommendations, the magnitude of the difference in surgical recommendations for patients with GEC suspicious or benign results was large.
 
Two studies evaluated the potential for the Afirma GEC test to change surgical decision making by comparing actual surgical decision making when Afirma GEC was used to predict surgical decision making based on a management algorithm (Aragon Han, 2014; Noureldine, 2015). In both, surgical decision making was estimated to change in at least some proportion of patients (10%-15%).
 
Abeykoon et al studied the impact of implementing Afirma GEC at a single center (Abeykoon, 2016). Surgical recommendations for patients with indeterminate thyroid nodules decreased from 81.5% pre-Afirma GEC to 50% post-Afirma GEC. The rate of malignant surgical pathology diagnosis increased from 20% pre-Afirma GEC to 85.7% post-Afirma. The implementation of Afirma GEC decreased the number of surgical recommendations and increased the rate of malignancy detected for patients who received a surgical biopsy.
 
Chaudhary et al studied the impact on surgical outcomes pre- and postimplementation of Afirma GEC (Chaudhary, 2016). A total of 158 FNAs were sent for Afirma GEC with 73 suspicious and 8 benign Afirma cases going for surgeries. Compared with before implementation of Afirma GEC, the rate for surgical biopsy decreased from 61% to 54% but was not statistically significant. In the SFN, the rate of surgical biopsy significantly decreased from 76% to 52%.
 
Dhingra studied the effects of an FNA protocol combining expert thyroid cytopathology plus Afirma GEC in community practice (Dhingra, 2016). Historical data were compared with data after implementation of the FNA protocol. Prior to protocol implementation, the rates of indeterminate cytology and diagnostic surgeries were 26% and 24%. After protocol implementation, the rates of indeterminate cytology and diagnostic surgeries decreased to 10% and 6%. The effect of Afirma GEC implementation could not be ascertained given the FNA protocol combined expert thyroid cytopathology and Afirma GEC.
 
Improved Outcomes
A simplified decision model was developed for use with Afirma GEC in individuals with cytologically indeterminate FNA samples. It is assumed that when Afirma GEC is not used, patients with cytologically indeterminate FNA results undergo thyroid resection. When Afirma GEC is used, those with Afirma suspicious lesions undergo resection, while those who have Afirma benign lesions do not. In this case, compared with the standard care plan, some patients without cancer will have avoided a biopsy, which is weighed against the small increase in missed cancers, in patients who had cancer but tested as Afirma benign.
 
Assuming that the rate of cancer in cytologically indeterminate thyroid nodules is approximately 20%,42 in the standard care plan, 80% of patients with cytologically indeterminate FNA samples will undergo an unnecessary biopsy. Applying the test characteristic values from Alexander et al, it is estimated that approximately 1.6% of individuals with true cancer would be missed, but approximately 38%, instead of 80%, would undergo unneeded surgery (Alexander, 2012).
 
Whether the tradeoff between avoiding unneeded surgeries and the potential for missed cancer is worthwhile depends, in part, on patient and physician preferences. However, some general statements may be made by considering the consequences of a missed malignancy and the consequences of unnecessary surgery. Most missed malignancies will be PTCs, which have an indolent course. Thyroid nodules are amenable to ongoing surveillance (clinical, ultrasound, and with repeat FNAs), with minimal morbidity.
 
Thyroid resection is a relatively low-risk surgery. However, consequences of surgery can be profound. Patients who undergo a hemi- or subtotal thyroidectomy have a risk of recurrent laryngeal nerve damage and parathyroid gland loss.
 
At present, the existing standard of care for thyroid nodules is based on intervention that is stratified by FNA cytology results, which are grouped into categories with differing prognosis. Avoiding an invasive surgery in situations where patients are at very low likelihood of having an invasive tumor is likely beneficial, given the small but potentially significant adverse events associated with thyroidectomy or hemithyroidectomy. Among the low-risk population, the alternative to surgical biopsy is ongoing active surveillance.
 
RosettaGX Reveal
 
Technically Reliable
Assessment of technical reliability focuses on specific tests and operators and requires review of unpublished and often proprietary information. Review of specific tests, operators, and unpublished data are outside the scope of this evidence review, and alternative sources exist. This evidence review focuses on the clinical validity and clinical utility.
 
Clinically Valid
A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).
 
Lithwick-Yanai et al described the development and initial clinical validation of the RosettaGX Reveal quantitative real-time PCR assay for 24 microRNA samples in a multicenter, retrospective cohort study using 201 FNA smears (Lithwick-Yanai, 2017).
 
Clinically Useful
 
Direct Evidence
No evidence directly demonstrating improved outcomes in patients managed with the RosettaGX Reveal was identified.
 
Chain of Evidence
Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about clinical utility.
 
Section Summary: Molecular Markers to Rule Out Malignancy
In a single multicenter validation study, the Afirma GEC test has been reported to have a high NPV (range, 90%-95%). These results are supported by an earlier development and clinical validation study (Chudova et al [2010]), but the classifiers used in the 2 studies do not appear to be identical. In an additional multicenter and multiple single-center studies, there is suggestive evidence that rates of malignancy are low in Afirma benign patients, but the exact NPV is unknown. The available evidence has suggested that physician decision making about surgery is altered by GEC results, although long-term follow-up of patients with thyroid nodules who avoided surgery based on GEC results is limited. A chain of evidence can be constructed to establish the potential for clinical utility with GEC testing in cytologically indeterminate lesions, but with only a single study with the marketed test reporting a true NPV, the clinical validity is uncertain. For the RosettaGX Reveal test, a retrospective clinical validation has been reported. No prospective studies for patients managed with the RosettaGX Reveal were identified, so the clinical validity remains uncertain.
 
Molecular Markers to rule in MALIGNANCY
 
Clinical Context and Test Purpose
The purpose of testing for molecular markers (eg, single nucleotide variants [SNVs] and gene rearrangements) in individuals with indeterminate findings on FNA of thyroid nodules is to rule in malignancy and to guide surgical approach or management.
 
The relevant question addressed in this evidence review is: Does testing for molecular markers predict malignancy and alter surgical approach or management and lead to improved health outcomes?
 
The following PICOTS were used to select literature to inform this review.
 
Patients
The relevant population of interest is individuals with indeterminate findings on FNA(s) of thyroid nodules. Patients with indeterminate findings would presently proceed to surgical biopsy perhaps with intraoperative pathology consultation (ie, intraoperative frozen section) if available.
 
Interventions
The test being considered is testing for molecular markers (eg, SNVs and gene rearrangements) to rule in malignancy and to use molecular marker results that are positive for variants associated with malignancy to guide surgical planning to ensure the capability for intraoperative pathologic confirmation of malignancy to adjust to definitive surgery for initial resection if appropriate.
 
Comparators
The following practices are currently being used: standard surgical management through surgical resection, including a 2-stage surgical biopsy (ie, lobectomy) followed by definitive surgery (ie, hemithyroidectomy or thyroidectomy).
 
Outcomes
The potential beneficial outcome of primary interest is appropriate surgical planning in the preoperative period (eg, hemithyroidectomy or thyroidectomy when malignancy is predicted). This has the potential benefit of reducing the likelihood of having the patient repeating surgery if a diagnosis is not made on frozen pathology section during the initial surgery if lobectomy is done as a first procedure.
 
Potential harmful outcomes are those resulting from false-positive results. However, the use of intraoperative confirmation of malignancy through frozen pathology section in patients with positive molecular marker testing would mitigate any risk of inappropriately performing more extensive thyroidectomy in the absence of malignancy.
 
Timing
The time frame for evaluating the performance of the test varies from the initial FNA to surgical resection to weeks to months following an indeterminate result.
 
Setting
The primary setting would be in endocrinology.
 
Gene Expression Classifiers to Predict Malignancy
 
Technically Reliable
Assessment of technical reliability focuses on specific tests and operators and requires review of unpublished and often proprietary information. Review of specific tests, operators, and unpublished data are outside the scope of this evidence review, and alternative sources exist. This evidence review focuses on the clinical validity and clinical utility.
 
Clinically Valid
A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).
 
Less evidence exists on the validity of gene expression profiling to rule in malignancy (specifically, the Afirma BRAF and Afirma MTC tests, and TERT single-gene testing). Genetic variants can be used to improve the sensitivity and specificity for diagnosing indeterminate FNA of the thyroid, with the goal of identifying variants that predict malignancy in FNA samples.
 
Afirma BRAF and Afirma MTC
In the study by Diggans et al, describing the development and validation of the Afirma BRAF test (previously described), for a subset of 213 thyroid nodule FNA samples for which histopathology was available, Afirma BRAF test results were compared with pathologic findings (Diggans, 2015). Afirma BRAF classified all histopathologically benign samples as BRAF V600E‒negative (specificity, 100%; 95% CI, 97.4% to 100%). Of the 73 histopathologically malignant samples, the Afirma BRAF test identified 32 as BRAF-positive (sensitivity, 43.8%; 95% CI, 32.2% to 55.9%).
 
In a study describing the development and validation of the Afirma MTC classifier, Kloos et al evaluated the MTC classifier in a sample of 10,488 thyroid nodule FNA samples referred for GEC testing (Kloos, 2016). In this sample, 43 cases were Afirma MTC-positive, of which 42 were considered to be clinically consistent with MTC on pathology or biochemical testing, for a positive predictive value (PPV) of 97.7% (95% CI, 86.2% to 99.9%).
 
TERT  Single-Gene Testing
Published literature describing performance characteristics of the marketed version (Interpace Diagnostics) of a TERT single-gene test was not identified. Three studies have reported performance characteristics of a TERT test; all 3 studies evaluated TERT promoter variants C228T and C250T.
 
Nikiforov et al evaluated the accuracy of the ThyroSeq v2 NGS panel that included tests for SNVs in 13 genes (including TERT) and for 42 types of gene fusions in a series of 143 consecutive thyroid FNA samples with a cytologic diagnosis of follicular or Hürthle cell neoplasm or suspicious for follicular or Hürthle cell neoplasm (Nikiforov, 2014). Molecular testing was retrospectively performed for 91 samples and prospectively performed for the remaining 52. Results for performance characteristics of the TERT variant alone were reported. Four of 39 total cancers were identified as TERT-positive (2 were unique diagnostic events); there were no TERT-positive results in the benign samples.
 
Liu and Xing described the performance of TERT as a single-gene test. FNA biopsy specimens were obtained preoperatively from thyroid nodules in 308 patients who underwent thyroidectomy (Liu, 2014). The percentage of samples that showed indeterminate cytologic findings on FNA biopsy was not described. The disposition of samples meeting eligibility criteria and a number of samples that did not produce results, were not described. Standard PCR was performed for direct genomic DNA sequencing to identify TERT promoter variants (C228T, C250T). One hundred twenty-nine (42%) of the samples were positive for thyroid cancer by pathology following surgery (111 PTC, 18 follicular thyroid carcinomas). TERT promoter variants C228T and C250T were found in 9 cases of thyroid cancer and no TERT variants were found in the 179 benign samples.
 
Decaussin-Petrucci et al described molecular testing for BRAF, RAS, and TERT variants in a prospective cohort of 326 cases, including 61 AUS, 124 follicular neoplasms, 72 suspicious for malignancy, and 69 malignant cases (Decaussin-Petrucci, 2017).file:///C:\Users\kxmitchell\AppData\Local\Microsoft\Windows\Temporary%20Internet%20Files\Content.IE5\OJSXZXWQ\20478%20Mutation%20FNA%20thyroid.docx Diagnosis of malignancy was confirmed by histology on paired surgical specimens in 163 samples. The flow of samples meeting eligibility criteria and number of samples that did not produce results were not described. The results here focus on the analysis of TERT single-gene tests. Nine TERT variants were detected, and all were confirmed to be malignant.
 
In summary, no studies of the validity of the marketed version of the TERT single-gene test were identified. Three studies reported information on TERT testing sufficient to calculate performance characteristics. The sample sizes of the included studies are approximately 150 to 350, with the prevalence of TERT variants between 3% and 7% and prevalence of cancer between 27% and 42%. Specificity was 100% in all studies (ie, there were no false-positives); however, the confidence intervals for PPV were extremely wide.
 
As mentioned, none of the 3 studies reported on the marketed version of the test, although one reported on TERT performance within the existing ThyroSeq marketed test. Liu and Xing was not limited to nodules that were indeterminate, and neither Liu nor Decaussin-Petrucci reported on the disposition of all eligible samples.
 
Clinically Useful
A test is clinically useful if the use of the results informs management decisions that improve the net health outcome of care. The net health outcome can be improved if patients receive correct therapy, or more effective therapy, or avoid unnecessary therapy, or avoid unnecessary testing.
 
Direct Evidence
Direct evidence of clinical utility is provided by studies that have compared health outcomes for patients managed with and without the test. Because these are intervention studies, the preferred evidence would be from randomized controlled trials.
 
Testing for specific variants associated with thyroid cancer (eg, BRAF V600E, TERT, and RET variants, RET/PTC and PAX8/PPARγ rearrangements) is generally designed to “rule in” cancer in nodules with indeterminate cytology on FNA (Bernet, 2014). (Of note, some gene panels, such as the ThyroSeq panel, may have a high enough NPV that their clinical use could also be considered as a molecular marker to predict benignancy; see next section.) A potential area for clinical utility for this type of variant testing would be in informing preoperative planning for thyroid surgery following initial thyroid FNA, such as planning for a hemi- vs a total thyroidectomy or performance of a central neck dissection.
 
In a retrospective analysis, Yip et al reported on outcomes after implementation of an algorithm incorporating molecular testing of thyroid FNA samples to guide the extent of initial thyroid resection (Yip, 2014). The study included a cohort of patients treated at a single academic center at which molecular testing (BRAF V600E, BRAF K601E, NRAS codon 61, HRAS codon 61, and KRAS codon 12 and 13 SNVs; RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements) was prospectively obtained for all FNAs with indeterminate cytology (FLUS, follicular neoplasm, suspicious for malignancy), and for selective FNAs at the request of the managing physician for selected nodules with benign or nondiagnostic cytology. The study also included a second cohort of patients who did not have molecular testing results available. For patients treated with molecular diagnosis, a positive molecular diagnostic test was considered an indication for an initial total thyroidectomy. Patients with FLUS and negative molecular diagnostic results were followed with repeat FNA, followed by lobectomy or total thyroidectomy if indeterminate pathology persisted. Patients with follicular neoplasm or suspicious for malignancy results on cytology and a negative molecular diagnostic result were managed with lobectomy or total thyroidectomy.
 
The sample included 671 patients, 322 managed with and 349 without molecular diagnostics. Positive molecular testing results were obtained in 56 (17% of those managed with molecular diagnostics) patients, most commonly RAS variants (42/56 [75%]), followed by BRAF V600E (10/56 [18%]) and BRAF K601E (2/56 [4%]) variants, and PAX8/PPARγ rearrangements (2/56 [4%]). Compared with those managed without molecular diagnostics (63%), patients managed with molecular diagnostics (69%) were nonsignificantly less likely to undergo total thyroidectomy as an initial procedure (p=0.08). However, they had nonsignificantly higher rates of central compartment lymph node dissection (21% vs 15%, p=0.06). Across both cohorts, 25% (170/671) of patients had clinically significant thyroid cancer, with no difference in thyroid cancer rates based on the type of initial surgery (26% for total thyroidectomy vs 22% for lobectomy, p=0.3). The incidence of clinically significant thyroid cancer after initial lobectomy (ie, requiring a 2-stage surgery) was significantly lower for patients managed with molecular diagnostics (17% vs 43%, p<0.001). An indeterminate FNA result had a sensitivity and specificity for the diagnostic of thyroid cancer of 89% and 27%, respectively, with a PPV of 29% and an NPV of 88%. The addition of molecular diagnostics to FNA results increased the specificity for a cancer diagnosis to 95% and the PPV to 82%.
 
Chain of Evidence
Indirect evidence on clinical utility rests on clinical validity. If the evidence is insufficient to demonstrate test performance, no inferences can be made about clinical utility.
 
In 2015, a task force from the American Thyroid Association published a review with recommendations for the surgical management of FNA-indeterminate nodules using various molecular genetic tests (ATA, 2015). This review reported on the estimated likelihood of malignancy in an FNA-indeterminate nodule depending on results of the Afirma GEC test (described above) and other panels designed to rule in malignancy. Depending on the estimated prebiopsy likelihood of malignancy, recommendations for surgery included observation, active surveillance, repeat FNA, diagnostic lobectomy, or oncologic thyroidectomy.
 
Section Summary: Molecular Markers to Predict Malignancy
The available evidence has suggested that use of variant testing in thyroid FNA samples is generally associated with high specificity and PPV for clinically significant thyroid cancer. The most direct evidence related to the clinical utility of variant testing for genes associated with malignancy in thyroid cancer comes from a single-center retrospective study that reported surgical decisions and pathology findings in patients managed with and without molecular diagnostics. There is potential clinical utility for identifying malignancy with higher certainty on FNA if such testing permits better preoperative planning at the time of thyroid biopsy, potentially avoiding the need for a separate surgery. A statement from the American Thyroid Association provides some guidelines for surgeons managing patients with indeterminate nodules. However, adoption of these guidelines in practice and outcomes associated with them are uncertain.
 
Molecular Markers to rule OUT and RULE in MALIGNANCY
 
Clinical Context and Test Purpose
The purpose of the ThyroSeq v2 test and the combined ThyGenX Thyroid Oncogene Panel plus ThyraMIR microRNA classifier in individuals with indeterminate findings on FNA(s) of thyroid nodules is to predict malignancy and inform surgical planning decisions with positive results using ThyroSeq v2 or the ThyGenX, and if negative, to predict benignancy using ThyraMIR microRNA classifier to eliminate or necessitate the need for surgical biopsy and guide surgical planning.
 
The relevant question addressed in this evidence review is: Does the ThyroSeq v2 test or the combined use of ThyGenX and ThyraMIR appropriately eliminate or necessitate the need for surgical resection or biopsy and lead to improved health outcomes?
 
The following PICOTS were used to select literature to inform this review.
 
Patients
The relevant population of interest includes individuals with indeterminate findings on FNA(s) of thyroid nodules. Patients with indeterminate findings presently proceed to surgical resection.
 
Interventions
The tests being considered are either: (a) the ThyroSeq v2 test or (b) the combined ThyGenX Thyroid Oncogene Panel and ThyraMIR microRNA classifier testing.
 
Comparators
The following practices are currently being used: surgical biopsy and/or standard surgical management through surgical resection.
 
Outcomes
The potential beneficial outcomes of primary interest are using a true-negative result to avoid an unneeded surgical biopsy or using a true-positive result to guide surgical resection (eg, hemithyroidectomy or thyroidectomy).
 
Potential harmful outcomes are those resulting from false-positive or false-negative test results. False-positive test results can lead to unnecessary surgical biopsy or resection and procedure-related complications. False-negative test results can lead to lack of surgical biopsy or resection for thyroid cancer and delay in diagnosis.
 
Timing
The time frame for evaluating the performance of the test varies from the initial FNA to surgical resection to weeks to months following an indeterminate result.
 
Setting
The primary setting would be in outpatient endocrinology.
 
ThyroSeq v2 Test
 
Technically Reliable
Assessment of technical reliability focuses on specific tests and operators and requires review of unpublished and often proprietary information. Review of specific tests, operators, and unpublished data are outside the scope of this evidence review, and alternative sources exist. This evidence review focuses on the clinical validity and clinical utility.
 
Clinically Valid
A test must detect the presence or absence of a condition, the risk of developing a condition in the future, or treatment response (beneficial or adverse).
 
A number of studies have evaluated whether testing for SNVs or gene fusions (either SNVs or panels) can be used to improve the sensitivity and specificity for diagnosing indeterminate FNA of the thyroid, with the goal of identifying variants that predict malignancy in FNA samples.
 
Variants Association With Malignancy
Fnais et al conducted a systematic review and meta-analysis of studies reporting on the test accuracy of BRAF variant testing in the diagnosis of PTC (Finais, 2015). Reviewers included 47 studies with 9924 FNA samples. For all cytologically indeterminate nodules, the pooled sensitivity estimate for BRAF variant testing was 31% (95% CI, 6% to 56%). Among nodules suspicious for malignancy on FNA, the pooled sensitivity estimate for BRAF variant testing was 52% (95% CI, 39% to 64%; I2=77%).
 
Ferraz et al evaluated 20 publications that reported on the type and number of variants in cases of FNA of the thyroid diagnosed as indeterminate and compared the results with final histology after surgical resection (Ferraz, 2011). Sixteen studies analyzed a single variant (eg, BRAF variant or RET/PTC rearrangement) and four analyzed a panel of variants (BRAF and RAS variants, RET/PTC and PAX8/PPARγ rearrangements). The detection of a variant in a histologically (surgically resected) benign thyroid lesion was categorized as a false-positive case, detecting no variant in an FNA sample from a histologically benign surgical sample was considered a true-negative, and finding no variant in a histologically malignant lesion was categorized as a false-negative. Based on 4 studies that examined a panel of variants, there was a broad sensitivity range (38%-85.7%; mean, 63.7%), a mean specificity of 98% (range, 95%-100%), a mean false-positive rate of 1.25% (range, 0%-4%), and a mean false-negative rate of 9% (range, 1%-21%). Based on 2 studies that examined RET/PTC rearrangements, mean sensitivity was 55% (range, 50%-60%), specificity 100%, a false-positive rate of 0%, and mean false-negative rate 3.5% (1%-6%). Based on 3 studies that examined BRAF variants, mean sensitivity was 13% (range, 0%-37.5%), mean specificity was 92.3% (range, 75%-100%), the mean false-positive rate was 0.5% (0%-1%), and the mean false-negative rate was 6% (range, 3%-12%). Authors concluded that testing for a panel of variants improved the sensitivity and specificity for indeterminate FNA of the thyroid but that further standardizations and further molecular markers would be needed before broad application of molecular FNA cytology for the diagnosis of thyroid nodules.
 
Additional studies describing the clinical validity of the genes that comprise the ThyroSeq panel or other individual variants and combinations of variants to predict malignancy in thyroid nodules that are indeterminate on FNA have been reported. (In some cases, measures of agreement were calculated from data provided in the published article.)
 
Additional studies have reported on differences in variant frequency in malignant vs benign tumors, and reported on the sensitivity and specificity of gene testing in unselected populations (ie, all patients with nodules, rather than just those with indeterminate cytology) (Mathur, 2010; Eszlinger, 2014; Park, 2015).
 
Genetic Variants Association With Tumor Behavior
As noted, the presence of BRAF or TERT variants is strongly associated with malignancy in thyroid nodule FNA samples. BRAF or TERT variants have also been associated with more aggressive clinicopathologic features in individuals diagnosed with PTC.
 
Adeniran et al assessed 157 cases with equivocal thyroid FNA readings (indeterminate and suspicious for PTC) or with a positive diagnosis for PTC and concomitant BRAF variant analysis (Adeniran, 2011). The results of histopathologic follow-up correlated with the cytologic interpretations and BRAF status. Based on the follow-up diagnosis after surgical resection, the sensitivity for diagnosing PTC was 63.3% with cytology alone and 80.0% with the combination of cytology and BRAF testing. No false-positives were noted with either cytology or BRAF variant analysis. All PTCs with an extrathyroidal extension or aggressive histologic features were positive for a BRAF variant. The authors concluded that patients with an equivocal cytologic diagnosis and a BRAF V600E variant could be candidates for total thyroidectomy and central lymph node dissection.
 
Xing et al investigated the utility of BRAF variant testing of thyroid FNA specimens for preoperative risk stratification of PTC in 190 patients (Xing, 2009). A BRAF variant in preoperative FNA specimens was associated with poorer clinicopathologic outcomes for PTC. Compared with the wild-type allele, a BRAF variant strongly predicted extrathyroidal extension (23% vs 11%; p=0.039), thyroid capsular invasion (29% vs 16%; p=0.045), and lymph node metastasis (38% vs 18%; p=0.002). During a median follow-up of 3 years (range, 0.6-10 years), PTC persistence or recurrence was seen in 36% of BRAF variant‒positive patients and 12% of BRAF variant‒negative patients, with an odds ratio (OR) of 4.16 (95% CI, 1.70 to 10.17; p=0.002). The PPV and NPV for preoperative FNA-detected BRAF variant to predict PTC persistence or recurrence were 36% and 88%, respectively, for all histologic subtypes of PTC. The authors concluded that preoperative BRAF variant testing of FNA specimens might provide a novel tool to preoperatively identify PTC patients at higher risk for extensive disease (extrathyroidal extension and lymph node metastases) and those more likely to manifest disease persistence or recurrence.
 
Yin et al reported on a systematic review and meta-analysis evaluating TERT promoter variants and aggressive clinical behaviors in PTC (Yin, 2016). Eight eligible studies (total N=2035 patients; range, 30-507) were included. Compared with wild-type, TERT promoter variant status was associated with lymph node metastasis (OR=1.8; 95% CI, 1.3 to 2.5; p=0.001), extrathyroidal extension (OR=2.6; 95% CI, 1.1 to 5.9; p=0.03), distant metastasis (OR=6.1; 95% CI, 3.6 to 10.3; p<0.001), advanced TNM stages III or IV (OR=3.2; 95% CI, 2.3 to 4.5; p<0.001), poor clinical outcome (persistence or recurrence; OR=5.7; 95% CI, 3.6 to 9.3; p<0.001), and mortality (OR=8.3; 95% CI, 3.8 to 18.2; p<0.001).

CPT/HCPCS:
0018UOncology (thyroid), microRNA profiling by RT-PCR of 10 microRNA sequences, utilizing fine needle aspirate, algorithm reported as positive or negative result for moderate to high risk of malignancy
0026UOncology (thyroid), DNA and mRNA of 112 genes, next-generation sequencing, fine needle aspirate of thyroid nodule, algorithmic analysis reported as a categorical result (&quot;Positive, high probability of malignancy&quot; or &quot;Negative, low probability of malignancy&quot;)
81210BRAF (B-Raf proto-oncogene, serine/threonine kinase) (eg, colon cancer, melanoma), gene analysis, V600 variant(s)
81406MOLECULAR PATHOLOGY PROCEDURE LEVEL 7
81545Oncology (thyroid), gene expression analysis of 142 genes, utilizing fine needle aspirate, algorithm reported as a categorical result (eg, benign or suspicious)
81599Unlisted multianalyte assay with algorithmic analysis

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