Medical Policy: 02.04.65
Original Effective Date: January 2017
Reviewed: January 2017
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This Medical Policy document describes the status of medical technology at the time the document was developed. Since that time, new technology may have emerged or new medical literature may have been published. This Medical Policy will be reviewed regularly and be updated as scientific and medical literature becomes available.
Fine needle aspiration (FNA) of a thyroid lesion to identify which patients needs to undergo surgery has diagnostic limitations and has led to the development of molecular makers in an attempt to improve the accuracy.
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. Fine needle aspiration (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. However, the remaining 20% to 30% have equivocal findings (inconclusive, indeterminate, atypical or suspicious), usually due to overlapping cytologic features between benign and malignant nodules; these nodules usually require surgery for final diagnosis.
Thyroid FNA cytology is classified according to Bethesda System for Reporting Cytopathology: Recommended Diagnostic Categories, see below:
|I||Non-diagnostic or Unsatisfactory||
|III||Atypia of Undetermined Significance or Follicular Lesion of Undetermined Significance||-|
|IV||Follicular Neoplasm or Suspicious for a Follicular Neoplasm||Specify if Hurthle cell (oncocytic) type|
|V||Suspicious for malignancy||
The current guidelines recommend repeat FNA for patients with a diagnosis of “atypia of undetermined significance” and lobectomy with or without intraoperative pathology consultation for those with a suspicious diagnosis, see below table for Bethesda System for Reporting Cytopathology: Implied Risk of Malignancy and Recommended Clinical Management.
There is some individualization of management for patients with FNA-indeterminate nodules, but many patients will ultimately require a surgical biopsy, typically thyroid lobectomy, with intraoperative pathology consultation to provide diagnosis. Approximately 80% of patients with indeterminate cytology undergo surgical resection; postoperatively evaluation reveals a malignancy rate ranging from 6% to 30%, making this a clinical process with very low specificity. Thus, if an analysis of FNA samples could reliably identify the risk of malignancy as low, there is potential for patients to avoid surgical biopsy.
Preoperative planning of optimal surgical management in patients with equivocal cytologic results is challenging, as different thyroid malignancies may require different surgical procedures (e.g., unilateral lobectomy vs 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.
|Diagnostic Category||Risk of Malignancy (%)||Usual Management (actual management may depend on other factors (e.g. clinical, sonographic) besides FNA interpretation)|
|Non-diagnostic or Unsatisfactory||1-4||Repeat FNA with ultrasound guidance|
|Benign||0-3||Clinical follow up|
|Atypia of Undetermined Significance or Follicular Lesion of Undetermined Significance||5-15
(estimate extrapolated from histopathologic data from patients with “repeated atypicals”
|Follicular Neoplasm or Suspicious for a Follicular Neoplasm||15-30||Surgical lobectomy|
|Suspicious for Malignancy||60-75||Near-total thyroidectomy or surgical lobectomy (in the case of “suspicious for metastatic tumor” or a “malignant” interpretation indicating metastatic tumor rather than a primary thyroid malignancy, surgery may not be indicated).|
|Malignant||97-99||Near-total thyroidectomy(in the case of “suspicious for metastatic tumor” or a “malignant” interpretation indicating metastatic tumor rather than a primary thyroid malignancy, surgery may not be indicated).|
FNA: fine needle aspiration
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 pre-existing well-differentiated papillary or follicular carcinomas. Medullary thyroid carcinoma originates from parafollicular or C cells, and accounts for about 3% of all thyroid cancers.
The diagnosis of malignancy in the case of PTC is primarily based on cytologic features. If an FNA in case of PTC is indeterminate, surgical biopsy with intraoperative pathology consultation is most often diagnostics, although the efficacy and therefore use will vary across 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, because 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.
Various mutations have been discovered in thyroid cancer. The 4 gene mutations that are most common and carry the highest impact on tumor diagnosis and prognosis are BRAF and RAS point mutations and RET/PTC and PAX8/PPARy 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. These mutually exclusive mutations are found in more than 70% of papillary carcinomas. BRAF mutations are highly specific for PTC. Follicular carcinomas harbor either RAS mutations or PAX8/PPARy rearrangement. These mutations are also mutually exclusive and identified in 70% to 75% of follicular carcinomas. Genetic alterations involving PI3K/AKT signaling pathway also occur in thyroid tumors, although rare in well differentiated thyroid cancers and have higher prevalence in less differentiated thyroid carcinomas. 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.
Point mutations in specific genes, including BRAF, RAS and RET, and evaluation for rearrangements associated with thyroid cancers can be accomplished by gene sequencing with Sanger sequencing or pyrosequencing or by real-time polymerase chain reaction (rtPCR) of single or multiple genes or by next generation sequencing (NGS) panels. Panels of tests for mutations associated with thyroid cancer, with varying compositions, are also available.
The ThyroSeq v.2 Next Generation Sequencing Panel (CBLPath, Ocala, FL) is a NGS sequencing panel of more than 60 genes. According to the CBLPath’s website, the test is indicated when FNA cytology indicates atypia of uncertain significance or follicular lesion of undetermined significance, follicular neoplasm or suspicious for follicular neoplasm, or suspicious for malignancy. In particular, it has been evaluated in patients with follicular neoplasm/suspicious for follicular neoplasm on FNA as a test to increase both sensitivity and specificity for cancer diagnosis.
The ThyGenX Thyroid Oncogene Panel (formerly miRInform Thyroid; Interpace Diagnostics, Parsippany, NJ; testing done at Asuragen Clinical Laboratory) is another NGS panel designed to be used in patients with indeterminate thyroid FNA results. It includes sequencing of 8 genes associated with papillary thyroid carcinoma and follicular carcinomas.
Thyroid Cancer Mutation Panel (Quest Diagnostics) is a mutational panel to assist in an accurate diagnosis to avoid unnecessary thyroid surgery for benign disease and select the most appropriate treatment option for cancerous nodules. Molecular tests are relatively new tool for further assessment nodules with atypical, suspicious or indeterminate find needle aspirates (FNA) cytology results. BRAF, RAS (KRAS, HRAS, NRAS), RET/PTC and PAX8/PPARy.
Genetic alterations associated with thyroid cancer can be assessed using gene expression profiling, which refers to analysis of messenger RNA (mRNA) expression levels of many genes simultaneously. Several gene expression profiling tests are now available to biologically stratify tissue from thyroid nodules.
The Afirma Gene Expression Classifier (Afirma GEC; Veracyte, South San Francisco, CA) analyzes the expression of 142 different genes to determine patterns associated with benign findings on surgical biopsy. It is designed to be used for thyroid nodules that have an “indeterminate” classification on FNA as a method to select patients who are at low risk for cancer (“rule out”).
Veracyte also markets 2 “malignancy classifiers” that use mRNA expression-based classification to evaluate for BRAF mutations or mutations associated with medullary thyroid carcinoma (Afirma BRAF and Afirma MTC, respectively). In a description of the generation of the Afirma BRAF test, the following have been proposed as benefits of the mRNA-based expression test for BRAF mutations:
The Afirma MTC is an option when Afirma GEC is ordered for thyroid nodules with an “indeterminate” classification on FNA, and can also be used for thyroid nodules with “malignant” or “suspicious” results on Afirma GEC. The Afirma BRAF is designed to be used for nodules with “suspicious” results on Afirma GEC.
ThyraMIR (Interpace Diagnostics, Parisippany, N.J.) is a microRNA expression based classifier that is intended for use in thyroid nodules with indeterminate cytology on FNA. Based on evaluation of expression of 10 miRNAs.
Molecular markers to predict benignancy are tests designed to have a high negative predictive value (NPV). The focus of this section is the Afirma Gene Expression Classifier (GEC), which is proposed as a risk stratifying test for patients who have determinant findings on fine needle aspirate (FNA). These patients presently proceed to surgical resection. The purpose of the test is to select patients at low risk of malignancy who could avoid unnecessary surgery.
Walsh et. al. verified the analytic performance of the Afirma GEC in the classification of cytologically indeterminate FNAs from thyroid nodules. The analytic 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 intranodule, intraassay, and interlaboratory reproducibility. Concordance of the GEC calls was 100% for samples tested under different shipping conditions, 97.2% across different RNA input amounts, 100% under different dilutions with normal tissue, 96% across different genomic DNA contamination amounts. The intra-assay, interassay, interlaboratory, and intranodule concordances were 93.9%, 97%, 100%, and 95%, respectively. The authors concluded that the analytic sensitivity and specificity, robustness, and quality control of the GEC were successfully verified.
Chudova et. al. described the development and initial clinical validation of a version of the Afirma GEC. The classifier was trained on 178 retrospectively-identified surgical thyroid specimens, which represented a variety of malignant and benign disorders, and separately on a set of 137 FNA samples with known surgical pathology. The classifier was developed with the objective of achieving a NPV (negative predictive value) specificity of 95% and specificity of 70%. The tissue-trained classifier was tested on an independent sample of 48 FNAs (24 with indeterminate cytopathology, 24 with a mix of malignant and benign cytopathology). The FNA trained classifier was separately tested on the same sample of 48 FNAs. In the 24 samples with indeterminate cytopathology, sensitivity and specificity were 100% (95 confidence interval (CI), 64% to 100%) and 73.3% (95% CI, 49% to 89%), respectively.
Alexander et. al. reported on a 19-month, prospective, multicenter (49 academic and community) sites, study of the Afirma GEC. A total of 4812 nodules were screened for inclusion with centralized cytopathology. Local pathology reports of the cytologic diagnosis were collected for all patients, and reports without a definitive benign or malignant diagnosis at the local site were reviewed by 3 expert cytopathologists, who reclassified them as atypical, follicular neoplasm, or suspicious for a follicular neoplasm, or suspicious for malignancy. OF all nodules screened, 577 (12%) were considered indeterminate after central review, and 413 of those had tissue pathology available.
The GEC used in the Alexander et. al. study were retrained on a set of 468 samples, comprised of 220 banked tissue samples, 14 ex vivo operative FNA samples, and 234 prospective clinical FNA samples described above.
After exclusion of the 25 used for test validation and those that did not have a valid GEC result, 265 FNA samples were evaluated with Afirma GEC. Of the 265 samples, 85 were malignant; the GEC correctly identified 78 of the 85 as suspicious (92% sensitivity; 95% CI, 84% to 97%). Specificity was 52% (95% CI, 44% to 59%). NPV (negative predictive value) ranged from 85% for “suspicious cytologic findings” to 95% for “atypia of undetermined clinical significance.” There were 7 FNAs with false-negative results, 6 of which were thought to be due to hypocellular aspirate specimens.
In 2014, Alexander et. al. reported results from a multicenter retrospective analysis of 339 thyroid nodules that 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. Most nodules sent for GEC testing were follicular lesions of undetermined significance/atypia of undetermined significance or follicular neoplasm. The distribution of GEC testing results for each cytologic classification is shown below.
|Cytologic Classification||Total, n||GEC Testing Results, n(%)|
|Follicular lesion of undetermined significance/atypia of undetermined significance||165||Benign 91 (55%), Suspicious 66 (40%), Non-diagnostic 8 (5%)|
|Follicular neoplasm||161||Benign 79 (49%), Suspicious 73 (45%), Non-diagnostic 9 (6%)|
|Suspicious for malignancy||13||Benign 4 (31%), Suspicious 9 (69%), Non-diagnostic 0|
|Total||339||Benign 174, Suspicious 148, Non-diagnostic 17|
A subset of patients whose nodules underwent GEC testing had 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, absent the GEC results, thyroid surgery would be recommended for patients with cytologically indeterminate FNA results, the authors reported that the GEC results altered management in 50% of patients. The below table shows thyroidectomy biopsy results for the subset of patients shown in the table above who underwent surgery.
|GEC Results||Total, n||Surgery recommended, n||Surgery Completed, n||Pathology Malignant, n (%)|
|Suspicious||148||141||121||53 (44% of those with completed surgery)|
|Benign||174||4||11||1 (9% of those with completed sugery)|
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. 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 2016, Santhanam et. al. reported results of a meta-analysis of studies reporting on the performance of the Afirma GEC in cytologically indeterminate nodules. Seven studies met the inclusion criteria, which required that studies reported on the use of the Afirma GEC in nodules that were indeterminate on FNA (including atypia of undetermined significance or follicular lesion of undetermined significance; suspicious for follicular/Hurthle 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. In the pooled cohort, the prevalence of malignancy was 37.1%. The main results of the analysis are summarized below.
|Outcomes||Point Estimate||95% Confidence Interval||I2|
|Sensitivity||95.7%||92.2% to 97.9%||45.4%|
|Specificity||30.5%||26.0% to 35.3%||92.1%|
|Positive likelihood ratio||1.20||0.996 to 1.44||-|
|Negative likelihood ratio||0.2||0.11 to 0.36||-|
|Diagnostic odds ratio||7.9||4.1 to 15.1||-|
Retrospective single center studies, including Harrell and Bimston (2014), Lastra et. al. (2014), McIver et. al. (2014), Yang et. al. (2016) have reported the diagnostic accuracy of the Afirma GEC, are summarized in the table below. These studies are subject to ascertainment bias, because a large proportion of individuals with Afirma benign reports did not undergo surgery, which makes determining the sensitivity and specificity of the GEC assays impossible. However, the rates of malignancy among patients with Afirma benign results who did undergo surgery are consistently low.
|Study (Year)||Population of Indeterminate FNA Samples||Afirma Test Result||N||N with Thyroidectomy||N with Malignancy on Thyroidectomy|
|Harrell and Bimston (2014)||58 FLUS/AUS or FN||Suspicious
|Lastra et. al. (2014)||69 (51.5%) FLUS/AUS
39 (29.5%) FN
25 (19%) FNOF
|McIver et. al. (2014)||12 (11.4%) FLUS/AUS
93 (88.6%) FN/HCN
|Yang et. al. (2016)||165 (76%) FLUS/AUS
24 (11%) SFN/FN
|Witt et. al. (2016)||47 FLUS/AUS or SFN/FN
(32 with GEC attemptedc)
Not applicable: followed clinically
AUS: atypia of undetermined significance; FLUS: follicular lesion of undetermined significance; FN: follicular neoplasm; FNA: fine needle aspirates; FNOF: follicular neoplasm with oncocytic features; HCN: Hurthle cell neoplasm; SFN: suspicious for follicular neoplasm.
There are limited data on the true negative rate 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 report on long-term follow up of individuals with indeterminate FNA cytology and Afirma GEC benign results.
No evidence directly demonstrating improved outcomes in patients managed with the Afirma GEC was identified. Therefore, a chain of indirect evidence was developed, which addresses 2 key questions:
The clinical setting in which the Afirma GEC is meant to be used is well-defined: individuals with atypia of undetermined significance/follicular lesion of undetermined significance or follicular neoplasm/suspicious for follicular neoplasm on FNA, who do not have other indications for thyroid resection (i.e. 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, suggest that in a least some cases, surgical decision-making is 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 operate on thyroid nodules with indeterminate cytology and Afirma GEC benign results in a sample of 395 nodules from 368 patients. Data were collected on 368 patients with 395 nodules. Surgery was performed in 7.6% of the patients with indeterminate cytology.
The 2014 study by Alexander et. al. provides some evidence about clinical management changes for patients with indeterminate thyroid nodules with the use of the Afirma GEC. 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 (Aragon Han et. al. (2014), Noureldine et. al. (2015)) were identified that evaluated the potential for the Afirma GEC to change surgical decision making by comparing actual surgical decision making when the Afirma GEC was used to predict surgical decision making based on a management algorithm. In both, surgical decision making was estimated to change in at least some proportion of patients (approximately 10%-15%).
A simplified decision model was developed for use with the Afirma GEC in individuals with cytologically indeterminate FNA samples, it is shown below. It is assumed that when the Afirma GEC is not used, patients with cytologically indeterminate FNA results undergo thyroid resection. When the Afirma benign lesions do not. In this case, compared to the standard care plan, some patients without cancer will have avoided biopsy, which is weighed against the small increase in missed cancers in patients who had cancer but tested as Afirma benign.
|Indeterminate Nodule (FLUS/AUS, SFN/FN)|
|GEC Test Used||GEC Suspicious||GEC Benign||Relative Effect on Health Outcomes: Compared with “Treat All with Surgery”|
|Yes||Yes à resection done||-||Cancer – no net effect on outcome from GEC testing >No Cancer – no net effect on outcome from GEC testing|
|Yes||-||Yes||Cancer – FALSE NEGATIVES: net harm from delayed cancer diagnosis No cancer – TRUE NEGATIVES: net benefit from avoiding surgery|
|No à resection done||-||-||Cancer – baseline outcomes: all get surgery No cancer – baseline outcomes: all get surgery|
FLUS/AUS: follicular lesion of undetermined significance/atypia of undetermined significance; GEC gene expression classifier; SFN/FN: suspicious for follicular neoplasm/follicular neoplasm
Assuming that the rate of cancer in cytologically indeterminate thyroid nodule is approximately 20%, 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. (2012), it is estimated that approximately 1.6% of individuals with a true cancer would be missed, but approximately 38%, instead of 80%,would undergo unneeded surgery.
Whether the tradeoff between voiding 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 papillary thyroid carcinomas (PTCs), which have an indolent course. Thyroid nodules are amendable 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 effects associated with thyroidectomy or hemi-thyroidectomy. The alternative to surgical biopsy in the low-risk population is ongoing active surveillance.
For individuals with thyroid nodule(s) and indeterminate findings on fine needle aspiration (FNA) who receive FNA sample testing with the Afirma Gene Expression Classifier (GEC), the evidence includes 1 prospective clinical validity study with the marketed test, and indirect chain to support clinical utility. In 1 multicenter validation study, the Afirma GEC has been reported to have a high negative predictive value (NPV) range 90%-95%. These results are supported by an earlier development and clinical validation study (Chudova et. al.), 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 suggests 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. An indirect chain of evidence can be constructed to establish the potential for clinical utility with GEC testing in cytologically indeterminate lesions. Despite there only being 1 study with the marketed test reporting a true NPV and the clinical validity uncertain, there appears to be sufficient evidence that this test can be considered standard in the evaluation of some indeterminate cases of FNA.
In response to requests from BCBSA, input was received from 2 physician specialty societies, 1 of which provided 3 responses, and 1 academic medical center while this policy was under review in 2016. Input focused on the use of gene expression classifiers (GEC) designed to with a high negative predictive value (NPV) in nodules indeterminate on fine needle aspirate (FNA). Although individual use of GEC with NPV in these situations varied, there was general agreement that these tests are considered standard in the evaluation of some indeterminate cases of FNA.
Molecular markers associated with malignancy in thyroid nodules are generally used as “rule-in” tests to identify cancer or tumors with more aggressive behavior. The purpose of the test is in patients with cytologically indeterminate FNA results when knowing the presence of certain mutation or having a high enough pretest probability of cancer would change the surgical approach or some other aspect of management.
For thyroid nodules that have indeterminate findings on FNA cytology, a surgical biopsy with intraoperative pathology consultation would typically be the next step. Following a diagnosis of a thyroid malignancy, preoperative surgical planning with regard to the extent of thyroid resection and lymph node dissection is an important consideration. Conventional factors determining biopsy strategy and surgical resection strategy include histological subtype and risk stratification based on factors such as tumor size and patient age.
Point mutations is specific genes associated with thyroid cancer, such as the BRAF V600E gene, and the detection of genetic rearrangements associated with thyroid cancer, such as the RET/PTC rearrangement, are typically detected with Sanger sequencing or next generation sequencing (NGS) methods. In the case of mutation testing for genes associated with thyroid cancer malignancy, analytic validity refers to a test’s technical accuracy in detecting a mutation that is present or in excluding a mutation that is absent. The rtPCR-based methods are generally considered to have high accuracy.
Next generation sequencing (NGS) is expected to have high accuracy for detecting a mutation that is present. However, with increasing numbers of tested mutations there is increased risk of detection of variants of uncertain significance (VOUS). The VOUS rate for currently available NGS panels for thyroid cancer is not well characterized. Nikiforova et. al. described the development and validation of a multigene NGS panel for thyroid cancer, the ThyroSeq panel. The authors developed a custom library of gene sequence variants based on mutations previously reported in the literature. The assay demonstrated 100% accuracy in evaluating samples of 15 thyroid tumors and 3 cells of known genetic alterations and 15 DNA samples with no mutations. In analysis of 229 DNA samples from frozen tissues (n=105), formalin-fixed, paraffin-embedded (FFPE) tissues (n=72), and FNAs (n=52), the panel indentified mutations in 19 (70%) of 27 of classic PTCs, 25 (83%) of 30 follicular variant PTCs, 14 (78%) of 18 conventional and 7 (39%) of 18 Hurthle cell carcinomas, 3 (30%) of 10 poorly differentiated carcinomas, 20 (74%) of 27 anaplastic thyroid carcinomas, and 11 (73%) of 15 medullary thyroid carcinomas. Of 83 benign nodules, 5 (6%) were positive for mutations.
A number of studies have evaluated whether testing for post mutations or gene fusions (either single mutation or panels of mutations) can be used to improve the sensitivity and specificity for diagnosing indeterminate FNA of the thyroid, with the goal of identifying mutations that predict malignancy in FNA samples.
The largest body of literature on mutation testing for prediction of malignancy in indeterminate thyroid nodules is related to the development an evaluation of next generation sequencing (NGS) panel that includes BRAF, RAS, RET/PTC, or PAX8/PPARy, The ThryoSeq.
Nikiforov et. al. prospectively tested a panel of muations (BRAF, RAS, RET/PTC, PAX8/PPARy) in 470 FNA samples of thyroid nodules from 328 consecutive patients. Mutational status was correlated with cytology and either surgical pathology diagnosis or follow up (mean, 34 months). Forty patients were excluded for poor quality specimens or loss to follow up. 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, 1 PAX8/PPARy); after surgery, 31 (97%) mutation positive nodules were diagnosed as malignant on pathologic examination, and 1 (3%) as benign tumor. 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 using serial ultrasound with no change in the nodule status (124 patients) or using repeated FNA with cytology negative for malignancy (23 patients) and no mutation found in the FNA material. These nodules were considered negative for malignancy. The remaining 72 patients who 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 not (62%).
Ohori et. al. performed mutation screenings in 117 FNA samples classified as a follicular lesion of indeterminate significance/atypia of indeterminate significance. BRAF, RAS, RET/PTC, or PAX8/PPARy mutations were detected in 10% of this category. The screening 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%.
In 2011, Nikiforov et. al. reported results of a prospective study that assessed the clinical validity of a panel of mutations to predict the likelihood of malignancy in thyroid nodules that were indeterminate of FNA. The authors included 1056 consecutive samples with indeterminate cytology on FNA that underwent mutation testing, with 967 of those adequate for molecular analysis (653 follicular lesion of undetermined significance/atypia of undetermined significance; 247 follicular or Hurthle cell neoplasm or suspicious for follicular neoplasm; 67 suspicious for malignant cells). One hundred seventeen of the samples were included in the Ohori et. al. study described above and summarized in the table below. Eighty-seven BRAF, RAS, RET/PTC, or PAX8/PPARy mutations were detected. At the time of analysis, 479 patients had undergone thyroidectomy for further evaluation, providing a histopathologic diagnosis for 513 samples. The presence of a mutation had a low sensitivity for predicting malignant histology (63%, 57%, 68% for samples with follicular lesion of undetermined significance/atypia of undetermined significance, follicular or Hurthle cell neoplasm/suspicious for follicular neoplasm, and suspicious for malignant cells on cytology, respectively), but a high specificity (99%, 97%, 96%, respectively). The negative predictive value (NPV) for the mutation analysis results was 94%, 86% and 72% for samples wth follicular lesion of undetermined significance/atypia of undetermined significance, follicular or Hurthle cell neoplasm/suspicious for follicular neoplasm, and suspicious for malignant cells on cytology, respectively. The authors concluded that mutation analysis might be useful in surgical planning, such as determining whether patients should undergo a thyroid lobectomy or a total thyroidectomy as a first surgery.
In a subsequent study, Nikiforov et. al. evaluated the accuracy of an NGS panel that included tests for point mutations in 13 genes and 42 types of gene fusions (ThyroSeq v2 NGS panel) in a series of 143 consecutive thyroid FNA samples with a cytologic diagnosis of follicular or Hurthle cell neoplasm/suspicious for follicular or Hurthle cell neoplasm. Molecular testing was retrospectively performed for 91 samples and prospectively performed for the remaining 52. The prevalence of cancer on histology was 27.5% and 26.9% in the retrospective and prospective cohorts. In the retrospective cohort, of the 25 malignant nodules, 22 were PTCs, and 3 were follicular thyroid carcinomas (FTCs). In the prospective cohort, of the 14 malignant nodules, 11 were PTCs and 3 were FTCs. The performance of the ThyroSec in both cohorts is shown below.
Performance of ThyroSeq Panel in Nikiforov et. al. (2014) and Nikiforov et. al. (2015)
|Mutation Testing Outcomes||Retrospective (n=91)||Nikiforov et. la. 2014|
|>Overall (N=143)||Nikiforov et. al. (2015)
Patients with Unknown Outcome (N=98
64 (2 cancer; 62 benign)
37 (2 cancer; 35 benign)
73 (2 cancer; 71 benign)
27 (23 cancer; 5 benign)
15 (12 cancer; 3 benign)
26 (20 cancer; 6 benign)
90% (80% to 99%)
90.9% (78.8% to 100%)
93% (88 to 98%)
92.1% (86.0% to 98.2%)
PPV (95% CI)
83% (72% to 95%)
76.9% (60.7% to 93.1%)
NPV (95% CI)
96% (92% to 95%)
97.2% (78.8% to 100%)
The authors noted that, compared with the mutation panel used in their 2011 study, the NGS panel was associated with marked increase in NPV, with a similar positive predictive value (PPV). In this case, the authors proposed that the panel could be used to both “rule in” and “rule out” invasive cancers.
The same group (Nikiforov et. al) reported the performance of a subsequent generation ThyroSeq panel (ThyroSeq v2.1) with an expanded gene panel in a series of 465 thyroid FNA samples with a diagnosis of atypia of undetermined significance/follicular lesion of undetermined significance. Molecular analysis was performed prospectively in all patients. Ninety patients (96 nodules) eventually underwent thyroid surgery, based on either patient preference, the presence of another nodule with a diagnosis of suspicious for malignancy or malignant on FNA, or positive molecular testing. An additional 2 patients were considered to have a definitive nonsurgical diagnosis of primary hyperparathyroidism based on biochemical testing. The performance of the test in patients with a known clinical outcome is summarized in the above table.
In addition to studies that describe the clinical validity of the mutations that compromise the ThyroSeq panel, studies have reported on the diagnostic performance of indivual muations and combinations of mutations to predict malignancy in thyroid nodules that are indeterminate on FNA. The results that pertain to the use of mutation testing in indeterminate thyroid nodules are summarized below.
|Study (year)||Population||Genes Tested||Insufficient or |
Inadequate for Analysis
|Measures of Agreement|
|Moses et. al. (2010)||110 indeterminate thyroid nodules||BRAF, KRAS, NRAS, RET/PTC1, RET/PTC3, NTRK1||2||38||95||67||79||77|
|Ohori et. al. (2010)||100 patients with 117 follicular lesions of undetermined significance/atypia of undetermined significance||BRAF, NRAS, HRAS, KRAS, RET/PTC1, RET/PTC3, PAX8/PPARy||NR||60||100||100||92||93|
|Cantara et. al. (2010)||41 indeterminate and
||BRAF,H-K NRAS, RET/PTC, TRK, PAX8/PPARy||53||86||97||86||97||95|
|54 suspicious thyroid nodules||BRAF,H-K NRAS, RET/PTC, TRK, PAX8/PPARy||53||80||100||100||47||82|
|Xing et. al. (2004)||25 indeterminate
|Jara et. al. (2015)||66 nodules suspicious for PTC||BRAF||NR||46||88||88||44||61|
|Rossi et. al. (2015)||140 indeterminate or||BRAF||NR||90||100||100||93||96|
|suspicious for malignancy
Huibregtset et. al.
|53 nodules with indeterminate/non-diagnostic FNA||BRAF, HRAS, KRAS, NRAS, PAX8/PPARy, RET/PTC1, RET/PTC3||-||48||89||81||64||-|
Sen: sensitivity; Spec: specificity; PPV: positive predictive value; NPV: negative predictive value; Acc: accuracy; FNA: fine needle aspiration; PTC: papillary thyroid carcinoma
ThyroSeq v.2 (CBLPath, Ocala, FL) for indeterminate thyroid FNAs next generation sequencing reports actual gene mutations and their corresponding risk of malignancy to provide diagnostic, prognostic and therapeutic information allowing the patient to either avoid surgery altogether or ensure that they get the right surgery at the right time.
|Point Mutations||Gene Fusions|
|AKT1, HRAS, RET, BRAF, KRAS, TERT, CTNNB1, NRAS, TP53, EIF1AX, PIK3CA, TSHR, GNAS, PTEN||ALK fusions (multiple), ETV6-NTRK3 (2), RET/PTC3, BRAF/AKAPg, NTRK1 fusions (6), RET fusions – other (7), BRAF fusions – other (2), PAX8/PPARg (8), Other fusions (12), CREB3L2/PPARg, RET/PTC1|
|Thyroid FNA Cytology||Diagnostic Information|
|Bethesda III Atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FUS)
|Bethesda IV Follicular neoplasm (FN) or Suspicious for a follicular neoplasm (SFN)
|Bethesda V Suspicious for malignancy (SM)
RoM: risk of malignancy
Additional studies report on differences in mutation frequency in malignant versus benign tumors, and may report on the sensitivity and specificity of mutation testing in unselected populations (i.e. all patients with nodules, rather than just those with indeterminate cytology). These studies are summarized next.
Mathur et. al. collected thyroid FNA samples, thyroid tissue, clinical and histopathology data, and tumor genotyping for BRAF V600E, NRAS, and KRAS mutations, and RET/PTC1, RET/PTC3, and NTRK1 rearrangements for 341 patients with 423 dominant thyroid nodules. A cytologic examination of the samples showed that 51% were benign (one quarter of them 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, 7 KRAS, and 21 NRAS mutations, and 4 PAX8-PPARy, 3 RET/PTC1, and 2 RET/PTC3 rearrangement detections were detected. In all, 17 (10.3%) of 165 benign thyroid nodules had a mutation compared with 26% (32/123) malignant tumors (p<0.05).
Eszlinger et. al. retrospectively analyzed a panel of mutations (BRAF and RAS point mutations and PAX8/PPARy and RET/PTC rearrangements) in a sample of 310 thyroid air-dried FNA specimens with available corresponding FFPE thyroid biopsy samples (164 indeterminate, 57 malignant, and 89 benign on FNA). A total of 47 mutations were detected on FNA: 22 BRAF, 13 NRAS, 3 HRAS mutations, and 8
PAX8/PPARy and 1 RET/PTC rearrangements. The addition of mutation analysis to cytology results associated with a sensitivity of 75.3% and specificity of 90.4% for the detection of malignancy, with a PPV of 77.2% and NPV of 89.4%. The presence of BRAF mutation or a RET/PTC rearrangement was associated with cancer in 100% of samples.
The association between BRAF mutations and PTC is supported by a report by Park et. al. (2015) on 294 patients with thyroid nodules whose FNA samples were evaluated with BRAF mutation testing by 2 methods, real-time PCR with Taq-Man minor groove-binding probes and allele-specific PCR using dual-priming oligonucleotides. The detection rate of PTC by BRAF mutation testing by real time PCR and allele-specific PCR was 80.2% (95% CI, 71.9% to 86.9%) and 76.9% (95% CI, 68.3% to 84.0%).
As reported in studies previously described, the presence of BRAF mutations is strongly associated with malignancy in thyroid nodule FNA samples. BRAF mutations have also been associated with more aggressive clinicopathologic features in individuals who are diagnosed with PTC (papillary thyroid carcinoma).
Adeniran et. al. conducted a study of 157 cases with equivocal thyroid FNA readings (indeterminate and suspicious for PTC) or with a positive diagnosis for PTC and concomitant BRAF mutation analysis. The results of histopathologic follow-up correlated with 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 BRAD 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 et. al. investigated the utility of BRAF mutation testing of thyroid FNA specimens for preoperative risk stratification of PTC in 190 patients. A BRAF mutation in preoperative FNA specimens was associated with poorer clinicopathologic outcomes for PTC. Compared with wild type allele, a BRAF mutation 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/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=0.002). The PPV and NPV 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.
Veracyte also markets two Afirma malignancy classifiers, Afirma MTC which are mutations associated with medullary thyroid carcinoma and Afirma BRAF. Malignancy classifiers are performed only on samples where the cytopathology or Afirma GEC results suggests the patient should be considered for surgery (Afirma GEC Suspicious and cytopathology suspicious and malignant samples). Malignancy classifiers may help guide treatment decisions such as the choice of surgery, enabling them to perform a more appropriate procedure the first time potentially reducing the need for additional surgeries and the risks and costs that accompany them.
Afirma MTC is included when Afirma GEC is run on thyroid nodules with indeterminate cytopathology.
In 2015, Diggans et. al. described the development and validation Afirma BRAF malignancy classifier. The study included FNA biopsies from 716 thyroid nodules. Biopsies were evaluated with quantitative PCR (qPCR) for BRAF V600E gene, with 181 used as a training sample and 535 used as validation sample. The Afirma BRAF malignancy classifier was generated using robust multichip average-normalized gene expression summaries, and the classifier were evaluated for positive percent agreement (PPA) and negative percent agreement (NPA) with the PCR-derived gene classification. The highest scoring classification method and gene set were then used in a final round of model building. The maximum PPA and NPA for all cytology categories were observed when the threshold for BRAF-positive status was 5% or more BRAF mutations. At 5% analytic sensitivity, Afirma BRAF demonstrated a PPA with PCR results of 90.4% (95 exact binomial CI, 83.5% to 95.1%) and an NPA of 99.0% (95% CI, 97.6% to 99.7%). Two samples in the training set and 4 samples in the validation set that were Afirma BRAF-positive but negative (0% mutation) on PCR, which the authors attributed to either technical variability in either assay or to mutations other than the BRAF V600E mutation that cause similar gene expression changes.
Less evidence exists on the validity of gene expression profiling specifically the Afirma BRAF and Afirma MTC tests. Mutations can be used to improve the sensitivity and specificity for diagnosing indeterminate FNA of the thyroid, with eh goal of identifying mutations that predict malignancy in FNA samples.
In the Diggans et. al. study, 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. The 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 the Kloos study describing the development and validation of the Afirma MTC classifier, the MTC classifier was evaluated in a sample of 10,488 thyroid nodule FNA samples referred for GEC testing. In this sample, 43 cases were Afirma MTC – positive, of which 42 were considered to be clinically consistent with medullary thyroid carcinoma on pathology or biochemical testing, for a PPV of 97.7% (95% CI, 96.2% to 99.9%)
Labourier et. al. reported on the sensitivity and specificity of test algorithm combining micro-RNA measurements from 17 genes (miRInform; Asuragen Laboratory, Austin, TX) with a 10 gene GEC in 109 samples with atypia of undetermined significance/follicular lesion of undetermined significance or follicular neoplasm/suspicious for follicular neoplasm on cytology evaluated at Asuragen Laboratory with known final pathology. Seventy-four nodules were diagnosed as benign and 35 as malignant.
Testing for specific mutations associated with thyroid cancer (e.g. BRAF V600E and RET mutations, RET/PTC and PAX8/PPARy rearrangements) are generally designed to “rule in” cancer in nodules that have indeterminate cytology on FNA. Of note, some mutation panels, such as ThryoSeq panel may have a high enough NPV that their clinical use could also be considered as a molecular marker to predict benignancy. A potential area for clinical utility for this type of mutation testing would be informing preoperative planning for thyroid surgery following initial thyroid FNA, such as planning for hemi- versus a total thyroidectomy or performance of a central neck dissection.
In a retrospective analysis, Yip et. al. reported outcomes after implementation of an algorithm incorporating molecular testing of thyroid FNA samples to guide the extent initial thyroid resection. 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 point mutations; RET/PTC1, RET/PTC3, and PAX8/PPARy rearrangements) was prospectively obtained for all FNAs with indeterminate cytology (follicular lesion of undetermined significance, 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 to be an indication for an initial total thyroidectomy. Patients with follicular lesion of undetermined significance and negative molecular diagnostic results were followed with repeat FNA, followed by a 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 mutations (42/56 [75%]), followed by BRAAF V600E (10/56 [18%]) and BRAF K601E (2/56 [4%]) mutations, and PAX8/PPARy 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 (i.e. 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 sensitivity and specificity for the diagnostic of thyroid cancer of 89% and 27%, respectively, with PPV and NPV of 29% and 88%. The addition of molecular diagnostics to FNA results increased the specificity for a cancer diagnosis to 95% and the PPV to 82%.
In 2015, a task force from the American Thyroid Association (ATA) reported on a review with recommendations for the surgical management of FNA-indeterminate nodules with various molecular genetic tests. This review reported on the estimated likelihood of malignancy in an FNA-indeterminate nodule depending on results of the Afirma GEC (described above) and other panels designed to rule in malignancy. Depending on the estimated pre-biopsy likelihood of malignancy, recommendations for surgery include observation, active surveillance, repeat FNA, diagnostic lobectomy, or oncologic thyroidectomy.
For individuals with thyroid nodule(s) and indeterminate findings on FNA who receive FNA sample testing with molecular markers to predict malignancy, the evidence includes prospective and retrospective studies of clinical validity. 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. Single-center studies suggest that testing for a panel of mutations associated with thyroid cancer may allow the appropriate selection of patients for surgical management with an initial total thyroidectomy. Prospective studies in additional populations are needed to validate these results. Mutation analysis does not achieve enough NPV to identify which patients can undergo watchful waiting over thyroid surgery. Although the presence of certain mutations may predict more aggressive malignancies, the management changes that would occur as a result of identifying higher risk tumors are not well-established. The evidence is insufficient to determine the effects of the technology on health outcomes.
In 2015, the American Thyroid Association (ATA) issued updated guidelines on the management of adult patients with thyroid nodules and differentiated thyroid cancer. These guidelines make the following recommendations regarding molecular diagnostic testing:
The National Comprehensive Cancer Network (NCCN) guideline on the treatment of thyroid cancer (Version 1.2016) make the following recommendations on the use of molecular diagnostics in thyroid cancer.
Molecular diagnostic testing to detect individual mutations (e.g. BRAF V600E, RET/PTC, RAS, PAX8/PPAR (peroxisome proliferator-activated receptors gamma) or pattern recognition approaches using molecular classifiers may be useful in the evaluation of FNA samples that are indeterminate to assist in management decisions. The BRAF V600E mutation occurs in about 45% of patients with papillary carcinoma and is the most common mutation. Although controversial, data suggest that BRAF V600E mutations may predict for increased recurrence of papillary carcinoma. The choice of the precise molecular test depends on the cytology and the clinical question being asked. Indeterminate groups include: 1) follicular or Hurthle cell neoplasms and 2) AUS/FLUS.
The NCCN Panel recommends (category 2B) molecular diagnostic testing for evaluating FNA results that are suspicious for: 1) follicular or Hurthle cell neoplasms; or 2) AUS/FLUS. The panel noted that molecular testing (both the Gene Expression Classifier and individual mutational analysis) was available in the majority of NCCN Member Institutions (>75%). About 70% of the panelists would recommend using a gene expression classifier in the evaluation of follicular lesions. The gene expression classifier measures the expression of at least 140 genes. BRAF V600E mutation analysis was recommended by 50% of the panelists in the evaluation of thyroid nodules (not restricted to follicular lesions). Furthermore about 60% of the panelists would recommend BRAF V600E testing in the evaluation of follicular lesions.
Rather than proceeding to immediate surgical resection to obtain definitive diagnosis for these indeterminate FNA cytology groups (follicular lesions), patients can be followed with observation if the application of a specific molecular diagnostic test (in conjunction with clinical and ultrasound features) results in a predicted risk of malignancy that is comparable to the rate seen in cytologically benign thyroid FNAs (approximately < 5%). It is important to note that the predictive value of molecular diagnostics may be significantly influenced by the pre-test probability of disease associated with the various FNA cytology groups. Furthermore, the cytologically indeterminate groups, the risk of malignancy for FNA can vary widely between institutions.
Published studies have focused primarily on adult patients with thyroid nodules, the diagnostic utility of molecular diagnostics in pediatric patient’s remains to be defined. Therefore, proper implementation of molecular diagnostics into clinical care requires an understanding of both the performance characteristics of the specific molecular test and its clinical meaning across a range of pre-test disease probabilities.
Follicular and Hurthle cell carcinomas are rarely diagnosed by FNA, because of the diagnostic criterion for these malignancies requires demonstration of vascular or capsular invasion. Nodules that yield an abundance of follicular cells with little or no colloid are nearly impossible to categorize as benign or malignant on the basis of FNA. Approximately 20% of these lesions are malignant. Repeat FNA will not resolve the diagnostic dilemma. However, molecular diagnostic testing may be useful.
Clinical laboratories may develop and validate tests in-house and market them as a laboratory service’ laboratory developed tests (LDTs) must meet general regulatory standards of the Clinical Laboratory Improvement Amendments (CLIA). Thyroid mutation testing and gene expression classifiers are available under the auspices of CLIA. Laboratories that offer LDTs must be licensed by CLIA for high-complexity testing. To date, the U.S. Food and Drug Administration (FDA) has chosen not to require any regulatory review of this test.
See also Medical Policy 02.01.20 Serum Tumor Markers in the Management of Malignancies
Afirma gene expression classifier (GEC) to assess fine needle aspirates of thyroid nodules may be considered medically necessary when all of the following criteria are met:
Afirma gene expression classifier (GEC) testing will be considered not medically necessary, including but not limited to the following:
For individuals less than 18 years of age, published studies have focused primarily on adult patients with thyroid nodules, the diagnostic utility of gene expression profiling (GEC) and molecular diagnostics in pediatric patient’s remains to be defined. Further studies are needed to determine the clinical utility of this testing in this patient population and therefore is considered investigational.
Mutation analysis in fine needle aspirates (FNA) of the thyroid including but not limited to the following will be considered investigational:
For individuals with thyroid nodule(s) and indeterminate findings on FNA who receive FNA sample testing with molecular markers to predict malignancy, the evidence includes prospective and retrospective studies of clinical validity. 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. Single-center studies suggest that testing for a panel of mutations associated with thyroid cancer may allow the appropriate selection of patients for surgical management with an initial total thyroidectomy. Prospective studies in additional populations are needed to validate these results. Mutation analysis does not achieve enough NPV to identify which patients can undergo watchful waiting over thyroid surgery. Although the presence of certain mutations may predict more aggressive malignancies, the management changes that would occur as a result of identifying higher risk tumors are not well-established. The evidence is insufficient to determine the effects of the technology on health outcomes
To report provider services, use appropriate CPT* codes, Alpha Numeric (HCPCS level 2) codes, Revenue codes and / or diagnosis codes.
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Yip L, Wharry LI, Armstrong MJ, et al. A clinical algorithm for fine-needle aspiration molecular testing effectively guides the appropriate extent of initial thyroidectomy. Ann Surg. Jul 2014;260(1):163-168. PMID 24901361
Ferris RL, Baloch Z, Bernet V, et. al. American Thyroid Association statement on surgical application of molecular profiling for thyroid nodules: current impact on perioperative decision making. Thyroid Jul 2015;25(7):760-768. PMID 26058403
Haugen BR, Alexander EK, Bible KC, et. al. American Thyroid Association management guidelines for adult patients with thyroid nodules and differentiated thyroid cancer. Thyroid Jan 2016;26(1):1-133. PMID 26462967
National Comprehensive Cancer Network (NCCN) Thyroid Carcinoma Version 1.2016.
UpToDate. Diagnostic Approach to and Treatment of Thyroid Nodules. Douglas S. Ross, M.D., Topic last updated January 22, 2016.
UpToDate. Oncogenes and Tumor Suppressor Genes in Thyroid Nodules and Nonmedullary Thyroid Cancer. Carl D. Malchoff M.D., Topic last updated September 17, 2015.
UpToDate. Thyroid Nodules and Cancer in Children. Stephen LaFranchi, M.D., Topic last updated November 4, 2015. UpToDate. Tools for Genetics and Genomics: Gene Expression Profiling. Avrum Spira M.D., MSc and Katrina Steiling M.D., MPH. Topic last updated January 14, 2016.
Veracyte. Afirma Thyroid FNA Analysis.
Interpace Diagnsotics. ThyGenX and ThyraMIR.
Quest Diagnostics. Thyroid Cancer Mutation Panel.
Palmetto GBA. MoIDX: Afirma Assay by Veracyte Coding and Billing Guidelines. Last updated December 4, 2015.
January 2017 - New Policy
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