Medical Policy: 02.04.65
Original Effective Date: January 2017
Reviewed: January 2018
Revised: January 2018
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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.
To determine which patients need thyroid resection, many physicians will perform cytological examination of fine needle aspiration (FNA) samples from a thyroid lesion; however, this method has diagnostic limitations. As a result, assays using molecular makers have been developed to improve the accuracy of thyroid FNA biopsies.
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. Sampling thyroid cells by fine needle aspiration (FNA) 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||
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 would typically be the next step in the diagnosis. Approximately 80% of patients with indeterminate cytology undergo surgical resection; postoperative evaluation has revealed a malignancy rate ranging from 6% to 30%, making this a clinical process with very low specificity. Thus, if 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 fine needle aspiration interpretation)|
|Non-diagnostic or Unsatisfactory||1-4||Repeat fine needle aspiration|
|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”
|Repeat fine needle aspiration|
|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).|
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 FNA in a case of PTC is indeterminate, surgical biopsy with intraoperative pathology consultation is most often diagnostic, 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 variant 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 genetic variants 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 single-nucleotide variants (SNVs) and RET/PTC and PAX8/PPARy rearrangements.
Papillary carcinomas carry single-nucleotide variants (SNVs) 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 SNVs are highly specific for PTC. Follicular carcinomas harbor either RAS SNVs or PAX8/PPARy rearrangement. These mutations identified in 70% to 75% of follicular carcinomas. Genetic alterations involving PI3K/AKT signaling pathway also occur in thyroid tumors, although they are rare in well differentiated thyroid cancers and have higher prevalence in less differentiated thyroid carcinomas. Additional variants 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 SNVs located in the RET gene.
Studies have evaluated the association between various genes and cancer phenotype in individuals with diagnosed thyroid cancer.
Single-nucleotide variants (SNVs) in specific genes, including BRAF, RAS and RET, and evaluation for rearrangements associated with thyroid cancers can be accomplished with Sanger sequencing or pyrosequencing or with real-time polymerase chain reaction (PCR) of single or multiple genes or by next generation sequencing (NGS) panels. Panel tests for genes associated with thyroid cancer, with varying compositions, are also available. For example, Quest Diagnostics offers a Thyroid Cancer Mutation Panel, which includes BRAF and RAS variant analysis and testing for RET/PTC and PAX8/PPARy rearrangements.
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 and/or 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 is a next-generation sequencing panel that sequences 8 genes and identifies specific gene variants and translocations associated with thyroid cancer. ThyGenX is intended to be used in conjunction with the ThyraMIR microRNA expression test when the initial ThyGenX test is negative.
TERT (telomerase reverse transcriptase) promoter mutations (Interpace Diagnostics, Parsippany, NJ) is a molecular marker predictor of aggressiveness of thyroid cancer. Currently, the ThyGenX mutation panel includes the following markers that are predictive of thyroid cancer from cytologically indeterminate thyroid nodules: BRAF, HRAS, KRAS, NRAS, RET/PTC, PAX8/PPARy and PIK3CA. By adding TERT, the ThyGenX panel will be a strong predictor of thyroid cancer, but will also provide evidence that a positive result indicates that the cancer is likely to be more aggressive in nature.
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 (“rule out”) who are at low risk for cancer.
ThyraMIR (Interpace Diagnostics, Parisippany, N.J.) is a microRNA expression based classifier intended for use in thyroid nodules with indeterminate cytology on FNA following a negative result from the ThyGenX Thyroid Oncogene Panel. There are 10 microRNAs evaluated.
RosettaGX Reveal (Rosetta Genomics, Philadelphia, PA) is a microRNA expression based classifier to differentiate indeterminate thyroid nodules as benign, suspicious for malignancy or as having high risk for medullary carcinoma (an aggressive form of thyroid cancer). RosettaGX Reveal can be performed using the existing FNA smears from routinely prepared cytology slides from the patient’s initial biopsy. There are 24 microRNAs evaluated.
Algorithmic testing involves the use of 2 or more tests in a prespecified sequence, with a subsequent test automatically obtained depending on results of an earlier test.
In addition to Afirma GEC, Veracyte also markets 2 “malignancy classifiers” that use mRNA expression-based classification to evaluate for BRAF variants (Afirma BRAF) or variants associated with medullary thyroid carcinoma (Afirma MTC).
|Test 1||Test 1 Result||Reflex to Test 2|
|Thyroid nodule on fine needle aspirate||“Intermediate”||Afirma MTC|
|Afirma GEC||“Malignant” or “Suspicious”||Afirma MTC|
|Afirma GEC||“Suspicious”||Afirma BRAF|
In a description of the Afirma BRAF test, the following have been proposed as benefits of the mRNA-based expression test for BRAF variants:
The testing strategy for both Afirma MTC and Afirma BRAF is to predict malignancy from a FNA sample with increased pretest probability for malignancy. A positive result with Afirma MTC or Afirma BRAF would inform preoperative planning such as planning for hemi vs a total thyroidectomy or performance of a central neck dissection.
The testing strategy for combined ThyGenX and ThyraMIR testing is to first predict malignancy. A positive result on ThyGenX would “rule in” patients for surgical resection. The specific testing results from a ThyGenX positive test would be used to inform preoperative planning when positive. For a ThyGenX negative result, the reflex testing involves the ThyraMIR microRNA expression test to “rule out” for a surgical biopsy procedure given the high NPV (negative predictive value) of the second test. Patients with a negative result from the ThyraMIR test would be followed with active surveillance and avoid a surgical biopsy.
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 resection. The relevant population of interest includes 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 resection. The potential beneficial outcomes of primary interest would result from avoiding an unneeded surgical resection (e.g. lobectomy or hemithyroidecrtomy) in a true-negative thyroid nodule that is benign. A potential harmful outcome are those resulting from a false-negative testing result, 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 result in worsening of health outcomes.
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. (2010) 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. (2012) 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(%)|
|Atypia or Follicular lesion 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. One exception is the study by Harrell and Bimston (2014); it may be reflective of a higher-than-usual overall rate of malignancy in patients with indeterminate FNA results.
|Study (Year)||Population of Indeterminate fine needle aspiration Samples||Afirma Test Result||N||N with Thyroidectomy||N with Malignancy on Thyroidectomy|
|Harrell and Bimston (2014)||58 follicular lesion of undetermined significance/atypia of undetermined significance or follicular neoplasm||Suspicious||36Two samples inadequate due to low mRNA content||30||21|
|Lastra et. al. (2014)||69 (51.5%) follicular lesion of undetermined significance/atypia of undetermined significance
39 (29.5%) follicular neoplasm
25 (19%) follicular neoplasm with oncocytic features
|McIver et. al. (2014)||12 (11.4%) follicular lesion of undetermined significance/atypia of undetermined significance
93 (88.6%) follicular neoplasm/Hurthle cell neoplasm
|Suspicious||44GEC results were available for 60 subjects||32||5|
|Yang et. al. (2016)||165 (76%) follicular lesion of undetermined significance/atypia of undetermined significance
24 (11%) suspicious for follicular neoplasm/follicular neoplasm
|Witt et. al. (2016)||47 follicular lesion of undetermined significance/atypia of undetermined significanceor suspicious for follicular neoplasm/follicular neoplasm
(32 with GEC attemptedthree samples were adequate)
|Benign||14||0||Not applicable: followed clinically|
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. Angell et. al. (2015), retrospectively compared clinical outcomes for individuals with indeterminate FNA cytology and Afirma GEC benign results with individuals to cytologically benign nodules. A total of 95 cytologically indeterminate/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 post-diagnosis. 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/Afirma GEC benign nodules (13.8% vs 0.9%, p<0.001).
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 (AUS)/follicular lesion of undetermined significance (FLUS) 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. (2012) 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. 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.
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%).
Sipos et. al. (2016) performed a retrospective study of nonacademic medical practices using the Afirma GEC and determine the long-term nonoperative rate of thyroid nodules with benign results. Of the patients with Afirma “benign” results during 36 months (+ 3 months) of follow-up, 17.3% underwent surgery. 88% of all surgeries were performed within the first 2 years after a “benign” Afirma GEC result.
Abeykoon et. al. (2016) studied the impact of implementing Afirma GEC at a single center. 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. (2016) studied the impact on surgical outcomes pre- and post implementation of Afirma GEC. A total of 158 FNAs were sent for Afirma GEC with 73 “suspicious” and 8 “benign” Afirma cases going through 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 surgival biopsy significantly decreased from 76% to 52%.
Dhingra et. al. (2016) studied the effects of a FNA protocol combining the expert thyroid cytopathology and Afirma GEC in a community practice. Historical data was compared with data after implementation of the FNA protocol. Prior to implementation of the FNA protocol, the rates of indeterminate cytology and diagnostic surgery were 26% and 24%. After implementation of the FNA protocol, the rates of indeterminate cytology and diagnostic surgery decreased to 10% and 6%. The effect of Afirma GEC implementation could not be ascertained given the FNA protocol combining expert thyroid cytopathology and Afirma GEC used in the study.
Benjamin et. al. (2016) reported on the analytic performance of the RosettaGX Reveal in the classification of cytologically indeterminate thyroid FNA smears from routinely prepared cytology slides. The analytic sensitivity of the assay was determined to be 1.28 x 10-2 ng. The assay was reported to be able to classify FNA smears with low amounts of thyroid cells. The interlaboratory agreement for samples extracted in single laboratory and processed at 2 different laboratories was 92.68%. For samples that were extracted and cDNA synthesis run in a single laboratory with subsequent quantitative real-time PCR (TR-PCR) run in 2 different laboratories, the interlaboratory agreement was 90.9%.
Lithwick-Yanai et. al. (2017) described the development and initial clinical validation of using the RosettaGX Reveal quantitative RT-PCR assay for 24 microRNAs in a multicenter, retrospective cohort study using 201 FNA smears. The results of the clinical validation study are reported in the table below:
|Outcomes||Value||95% Confidence Interval|
|Samples passing QC (n=189)|
|Sensitiviy %||85||74 to 93|
|Specificity %||72||63 to 79|
|Negative Predictive Value||91||84 to 96|
|Samples with 3 Pathologists agreeing on final diagnosis (n=150, subset of samples passing QC)|
|Sensitiviy %||98||87 to 100|
|Specificity %||78||69 to 85|
|Negative Predictive Value||99||94to 100|
No prospective clinical studies for RosettaGX Reveal were identified.
No evidence directly demonstrating improved outcomes in patients managed with RosettaX Reveal was identified.
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 to 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%, 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 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 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 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 hemithyroidectomy. The alternative to surgical biopsy in the low-risk population is ongoing active surveillance.
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.), 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 Afirma GEC testing in cytologically indeterminate lesions, but with only 1 study marketed test reporting a true NPV, the clinical validity is uncertain. For the RosettaGX Reveal test, 1 analytic validation study and 1 retrospective clinical validation have been reported. No prospective studies for patients managed with the RosettaGX Reveal were identified, so the clinical utility remains uncertain.
The purpose of testing for molecular markers (e.g. single nucleotide variants (SNVs) and gene rearrangement) in individuals with indeterminate findings on FNA of thyroid nodules is to rule in malignancy and to guide surgical approach or management. The relevant population of interest includes individuals with indeterminate findings on fine needle aspirate(s) of thyroid nodules. Patients with indeterminate findings would presently proceed to surgical biopsy perhaps with intraoperative pathology consultation (i.e. intra-operative frozen section) if available. The relevant intervention of interest is testing for molecular markers single nucleotide variants (SNVs) and gene rearrangements to rule in malignancy and to use molecular marker results that are positive for variant associated with malignancy to guide surgical planning to ensure the capability for intraoperative pathologic confirmation of malignancy in order to be able to adjust to definitive surgery for initial resection if appropriate.
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 for the BRAF V600E gene, with 181 used as training sample and 535 used as a validation sample. The Afirma BRAF malignancy classifier was generated using robust multichip average-normalized gene expression summaries, and the classifiers were evaluated for positive percent agreement (PPA) and negative percent agreement (NPA) with 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 variants. At 5% analytic sensitivity, Afirma BRAF demonstrated a PPA with PCR results of 90.4% (95% 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 were Afirma BRAF positive but negative (0% variant) on PCR, which the authors attributed to technical variability in either assay or to variants other than then BRAF V600E variant that cause similar gene expression changes.
Intra and interrun reproducibility of the classifier were evaluated using 9 FNA biopsies and 3 tissue controls selected from training samples with high (BRAF-positive) or low (BRAF-negative) classifier scores and scores near classifier decision boundary. Each FNA biopsy and tissue was processed from total RNA in triplicate in each of 3 different runs across days, operators and reagent lots. The intra-assay standard devision (SD) of Afirma BRAF scores was 0.171 (95% CI, 0.146 to 0.204). Of the 106 Afirma BRAF calls produced (2 arrays failed quality control requirements), 106 resulted in concordant calls across all 3 runs (100% concordance). The inter-assay standard deviation (SD) of scores was 0.204 (95% CI, 01.78 to 0.237) for scores measured on a 6-point scale. These results suggest low intra and interrun variability.
In 2016, Kloos et. al. described the development of the Afirma MTC classifier in a study that also described the clinical validity of the MTC classifier.
Pankratz et. al. (2016) studied the analytic performance of Afirma MTC classifier from fresh-frozen tissue specimens with a confirmed medullary thyroid carcinoma (MTC) diagnosis. Twenty-seven MTC tissue specimens were compared with 20 deidentified FNA samples with normal donors. The reported clinical sensitivity of the Afirma MTC classifier was 96.3% (95% CI, 81.0% to 99.9%).
Less evidence exists on the validity of gene expression profiling (specifically, the Afirma BRAF and Afirma MTC tests). 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.
In the Diggans 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. 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 (2016) 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 to GEC testing (the Afirma GEC described above). 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 PPV of 97.7% (95% CI, 86.2% to 99.9%).
Testing for specific variants associated with thyroid cancer (e.g. BRAF V600E and RET variants, RET/PTC and PAX8/PPARy rearrangements) is generally designed to “rule in” cancer in nodules that have indeterminate cytology on FNA. A potential areas 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. (2014) 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 single nucleotide variants; 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 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 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/PPARy rearrangements (2/56 [4%]). Compared with those managed without molecular diagnostics (63%), patients managed with molecular diagnostics (69%) were nonsignifcantly 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 a sensitivity and specificity for the diagnosis of thyroid cancer of 89% and 27%, retrospectively, with a PPV and NPV of 29% and 88% respectively. 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) published a review of 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 the results of the Afirma GEC test (described above) and other molecular testing designed to rule in malignancy. Depending on the estimated pre-biopsy likelihood of malignancy, recommendations for surgery included observation, active surveillance, repeat FNA, diagnostic lobectomy or oncologic thyroidectomy.
The available evidence has suggested that use of variant testing in thyroid FNA samples is generally associated with a 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 high certainty on FNA if such testing permits better pre-operative planning at the time of thyroid biopsy, potentially avoiding the need for a separate surgery. An American Thyroid Association (ATA) statement provides some guidelines for surgeons managing patients with indeterminate nodules. However, adoption of these guidelines in practice and outcomes associated with them are uncertain.
The purpose of the ThyroSeq V2 test and the combined ThyGenX Thyroid Oncogene Panel and ThyraMIR microRNA classifier in individuals with indeterminate findings on FNAs of thyroid nodules is to predict malignancy and inform surgical planning decisions with positive results using ThyroSeq v2 or the ThyGenX, and if negative, predict benignancy using ThyraMIR microRNA classifier to eliminate or necessitate the need for surgical biopsy and guide surgical planning.
Single nucleotide variants (SNVs) in specific genes associated with thyroid cancer (e.g. the BRAF V600E gene) and the detection of genetic rearrangements associated with thyroid cancer (e.g. the RET/PTC rearrangement) are typically detected with Sanger sequencing or next-generation sequencing (NGS) methods. In the case of testing for gene variants associated with thyroid cancer malignancy, analytic validity refers to a test’s technical accuracy in detecting a variant that is present or in excluding a variant that is absent. The real-time polymerase chain reaction (PCR) based methods are generally considered to have high accuracy. For example, Smith et. al. (2014) reported on the technical performance characteristics for BRAF variant detection by qualitative PCR in thyroid FNA samples with high within and between-run reproducibility.
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 (VUS). The VUS rate for currently available NGS panels for thyroid cancer is not well characterized. Nikiforova et. al. (2013) 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 variants. 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 identified variants 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 variants.
A number of studies have evaluated whether testing for single nucleotide variants (SNVs) (either single variantsor panels of variants) 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.
In 2015, 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. The review 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. (2011) evaluated 20 publications that reported on the type and number of variants in cases of FNA of the thyroid diagnosed as indeterminate and compared with results with final histology after surgical resection. Sixteen studies analyzed 1 variant (e.g. BRAF variant or RET/PTC rearrangement) and 4 studies analyzed a panel of several variants (BRAF and RAS variants, RET/PTC and PAX8/PPARy 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%), mean false-positive rate of 1.25% (range, 0%-4%), and mean false-negative rate of 9% (range, 1%-21%). Based on 2 studies the examined RET/PTC rearrangement, mean sensitivity was 55% (range, 50%-60%), specificity 100%, a false-positive rate of 0% and mean false-negative rate 3.5% (91%-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%), mean false-positive rate was 0.5% (0%-1%), and mean false-negative rate was 6% (range, 3%-12%). Authors concluded that testing for a panel of variants 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.
The largest body of literature on variant testing for prediction of malignancy in indeterminate thyroid nodules is related to the development a NGS panel (ThyroSeq) that includes BRAF, RAS, RET/PTC, or PAX8/PPARy. Studies that address these panels are described in more detail; studies that include subsets of these variants or additional variants are summarized in the following section.
Nikiforov et. al. (2009) prospectively tested a panel of variants (BRAF, RAS, RET/PTC, PAX8/PPARy) in 470 FNA samples of thyroid nodules from 328 consecutive patients. Variant 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 variants were found (18 BRAF, 8 RAS, 5 RET/PTC, 1 PAX8/PPARy); after surgery, 31 (97%) variant positive nodules were diagnosed as malignant on pathologic examination, and 1 (3%) as benign tumor. Thirteen of the 32 variant-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 variant 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. (2010) 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 variants 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 variants 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 variants to predict the likelihood of malignancy in thyroid nodules found indeterminate on FNA. The authors included 1056 consecutive samples with indeterminate cytology on FNA that underwent variant testing, with 967 of those adequate for molecular analysis (653 follicular lesion of undetermined significance (FLUS)/atypia of undetermined significance (AUS); 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 variants 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 variant had a low sensitivity for predicting malignant histology (63%, 57%, 68% for samples with follicular lesion of undetermined significance (FLUS)/atypia of undetermined significance (AUS), 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 variants analysis results was 94%, 86% and 72% for samples with follicular lesion of undetermined significance (FLUS)/atypia of undetermined significance (AUS), follicular or Hurthle cell neoplasm/suspicious for follicular neoplasm, and suspicious for malignant cells on cytology, respectively. The authors concluded that variants 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. (2014) evaluated the accuracy of an NGS panel that included tests for single nucleotide variants 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 in the table 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)
|Negative||64 (2 cancer; 62 benign)||37 (2 cancer; 35 benign)||-||73 (2 cancer; 71 benign)|
|Positive||27 (23 cancer; 5 benign)||15 (12 cancer; 3 benign)||-||26 (20 cancer; 6 benign)|
(95% confidence interval)
|92%||86%||90% (80% to 99%)||90.9% (78.8% to 100%)|
(95% confidence interval)
|94%||92%||93% (88 to 98%)||92.1% (86.0% to 98.2%)|
|positive predictive value (95% confidence interval)||85%||80%||83% (72% to 95%)||76.9% (60.7% to 93.1%)|
|NPVnegative predictive value (95% confidence interval)||97%||95%||96% (92% to 95%)||97.2% (78.8% to 100%)|
The authors noted that, compared with the gene 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 (AUS)/follicular lesion of undetermined significance (FLUS). Molecular analysis was performed prospectively in all patients. Ninety patients (96 nodules) 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.
In addition to studies that describe the clinical validity of the genes that comprise the ThyroSeq panel, studies have reported on the diagnostic performance of individual variants and combinations of variants 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 in the table below.
|Study (year)||Population||Genes Tested||Insufficient or |
Inadequate for Analysis
|Measures of Agreement|
|sensitivity||specificity||positive predictive value||negative predictive value||accuracy|
|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 papillary thyroid carcinoma||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 fine needle aspiration||BRAF, HRAS, KRAS, NRAS, PAX8/PPARy, RET/PTC1||-||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
Additional studies report on differences in variant frequency in malignant versus benign tumors, and may report on the sensitivity and specificity of gene 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 variants, 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 (25% 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 rearrangements were detected. In all, 17 (10.3%) of 165 benign thyroid nodules had a variant compared with 26% (32/123) malignant tumors (p<0.05).
Eszlinger et. al. (2014) retrospectively analyzed a panel of variants (BRAF and RAS sinlgle nucleotide variants 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 variants were detected on FNA: 22 BRAF, 13 NRAS, 3 HRAS mutations, and 8 PAX8/PPARy and 1 RET/PTC rearrangements. The addition of variant analysis to cytology results was 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 variants 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 variants using 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 variant 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%) respectively.
As reported in studies previously described, the presence of BRAF mutations is strongly associated with malignancy in thyroid nodule FNA samples. BRAF variants have also been associated with more aggressive clinicopathologic features in individuals who are diagnosed with PTC (papillary thyroid carcinoma).
Adeniran et. al. (2011) 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. 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 variant analysis. All PTCs with extrathyroidal extension or aggressive histologic features were positive for BRAF variant. 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. (2009) investigated the utility of BRAF variant testing of thyroid FNA specimens for preoperative risk stratification of PTC in 190 patients. A BRAF variant 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 variant-positive patients versus 12% of BRAF variant-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 variant to predict PTC persistence/recurrence were 36% and 88% for all histologic subtypes of PTC. The authors concluded that preoperative BRAF variant 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 more likely to manifest disease persistence or recurrence.
Hadd et. al. (2013) reported on targeted next-generation sequencing (NGS) of cancer genes in 38 formalin-fixed paraffin-embedded and 10 FNA tumor specimens. The results show an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing; Sanger sequencing has analytic sensitivity of approximately 15% to 20%. When NGS was compared with a multiplex detection system with a 1% variant detection rate, the accuracy was reported to be 99.6% (95% CI, 97.9%-99.9%).
Wylie et. al. (2016) reported on the development of the ThyraMIR microRNA classifier, along with a 17-variant oncogene panel including BRAF, RAS, RET, or PAX. A miRNA classifier was originally developed using rtPCR methodology in a sample of 257 surgical specimens, and validated in an independent set of 42 nodules with indeterminate cytology. A 17-variant panel covering validated oncogenic gene alterations for BRAF, RAS, RET or PAX8 genes was tested on preoperative FNA and surgical specimens. Optimization of microRNA classifiers A and B resulted in the commercial ThyraMIR Classifier. ThyraMIR was used on a subset of thyroid tissues negative 17-variant panel and resulted in a sensitivity of 85% and specificity of 95%.
The analytic validity of targeted NGS of cancer genes is expected to be high. Concordance rates between Sanger sequencing and NGS are high but limited lower analytic sensitivity of Sanger sequencing. Concordance rates increased with NGS is compared with an orthogonal technology with a 1% variant detection rate. One study describing the development of a miRNA classifier was identified.
Labourier et. al. (2015) evaluated the diagnostic algorithm combining a 17-variant panel with ThyraMIR on a cross-sectional cohort of thyroid nodules comprised of 109 FNA samples with atypia of undetermined significance (AUS)/follicular lesion of undetermined significance (FLUS) or follicular neoplasm (follicular neoplasm)/suspicious for a follicular neoplasm (SNF) across 12 endocrinology centers across the United States. A qualitative molecular results were compared with surgical histopathology to determine diagnostic performance and model clinical effect. Mutations were detected in 69% of nodules with malignant outcome. Among mutation negative specimens, miRNA testing correctly identified 64% of malignant cases and 98% of benign cases. The diagnostic sensitivity and specificity of the combined algorithm was 89% (95% confidence interval [CI], 73-97%) and 85% (95% CI, 75-92%), respectively. At 32% cancer prevalence, 61% of the molecular results were benign with a negative predictive value of 94% (95% CI, 85-98%). Independently of variations in cancer prevalence, the test increased the yield of true benign results by 65% relative mRNA-based gene expression classification and decreased the rate of avoidable diagnostic surgeries by 69%. The authors concluded that a diagnostic algorithm combining miRNA expression and gene mutation detection yields clinically actionable molecular information in thyroid nodules with AUS/FLUS or follicular neoplasm/SFN cytology. Based on the high PPV and NPV of the MPT (multiplatform mutation test), it is reasonable to propose that patients with positive (malignant) MPT results may be sent to surgery while patients with negative (benign) MPT results may benefit from a more conservative management, i.e. active follow-up without surgery.
Evidence for clinical validity of combined testing for miRNA gene expression using ThyraMIR and a targeted 17-variant panel comes from two retrospective studies using archived surgical specimens and FNA samples. One study combined a 17-variant panel with ThyraMIR testing on arvhiced surgical specimens and resulted in a sensitivity of 85% and specificity of 95%. The second study combined a 17-variant panel (miRInform) with ThyraMIR testing on FNA samples and resulted in a sensitivity of 89%, specificity of 85%, PPV of 74% and NPV of 94%. No studies identified were identified that demonstrated the clinical validity of a combined ThyGenX and ThyraMIR test on FNA samples.
Direct evidence for the clinical utility for the ThyroSeq v2 test and the combined ThyGenX and ThyraMIR diagnostic testing algorithm are lacking. In the absence of direct evidence for the clinical utility of the combined testing, an indirect chain of evidence may be constructed to infer potential clinical utility of the combined diagnostic testing algorithm. No studies using ThyGenX NGS panel in FNA samples was indentified. However, available evidence was suggested that use of variant testing using NGS in thyroid FNA samples is generally associated with a high specificity and PPV for clinically significant thyroid cancer. 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. However, variant analysis does not achieve a high enough NPV to identify which patients can undergo active surveillance over thyroid surgery. In the diagnostic algorithm that reflexes to the ThyraMIR after a negative ThyGenX result, patients receiving reflex testing could identify who may under active surveillance over thyroid surgery. A single study using a 17-variant panel with ThyraMIR showed a NPV of 94%. Therefore, the high NPV of ThyraMIR has the potential to accurately predict benignancy and triage patients to active surveillance.
Direct evidence for the clinical utility for the ThyroSeq v2 test and the combined ThyGenX and ThyraMIR reflex testing is lacking. However, available evidence suggests that testing for gene variants and rearrangements can predict malignancy and inform surgical planning decisions when the test is positive. Pooled retrospective and prospective clinical validation studies of ThyroSeq v2 have reported a combined NPV of 96% (95% CI, 92% to 95%) and PPV of 83% (95% CI, 72% to 95%) and may potentially assist in selecting patients to avoid surgical biopsy in negative and guide surgical planning in positive. The NPV or the ThyGenX to identify patients who should undergo active surveillance over thyroid surgery is unknown. In a reflex testing setting, the high NPV for microRNA gene expression test used on the subset of patients with a negative result from a variant and gene rearrangement test has the potential clinical utility in identifying patients appropriately for active surveillance.
TERT (telomerase reverse transcriptase) promoter mutations has been proposed for risk stratification and predicting patient outcomes. TERT can be performed as part of the ThyGenX mutation panel or on an individual basis. Published data suggests that TERT mutations can extend the life span of the tumor cell and allow time for other mutations to develop. Mutations in the TERT promoter region are found in thyroid cancers and seem to act synergistically when they occur with the BRAF V600 mutation. The coexistence of mutations in TERT and BRAF genes have shown to dramatically increase the risk of thyroid cancer aggressiveness. By adding TERT, the panel (ThyGenX) not only acts as a strong predictor of thyroid cancer, but also provides evidence that a positive result indicates the cancer is more likely to be more aggressive, which enables the physician to make the most informed surgical choice for the patient.
In 2016, Liu et. al. evaluated TERT promoter mutations in thyroid cancer which concluded, it has been less than three years since the initial report on TERT promoter mutations in thyroid cancer, while substantial progress has occurred in this exciting new field. Much has been known about the biological and clinical relevance of these mutations in thyroid cancer in this short time. Studies from various populations and regions in the world uniformly found TERT promoter mutations to be present in thyroid cancers, but not benign thyroid tumors, and be more common in aggressive types of thyroid cancers. These mutations are also more commonly associated with aggressiveness tumor behaviors and poor clinical outcomes, including tumor recurrence and patient mortality. A particular interesting and important aspect of TERT promoter mutations in papillary thyroid carcinoma (PTC) is their association with the BRAF V600E mutation and the robust synergistic impact of the coexisting two mutations on aggressive clinicopathological outcomes of PTC, particularly tumor recurrence and patient mortality. These results are consistent with the proposed model in which TERT promoter mutations create consensus binding sites for ETS transcriptional factors for the later to activate the expression of TERT, a process that can be upregulated by the BRAF V600E/MAP kinase signaling pathway. A similar synergistic effect between TERT promoter mutations and RAS mutations, likely through activating the PI3K pathway, may also exist in thyroid cancer. These clinicopathological data strongly support a prominent role of TERT promoter mutations in the tumorigenesis and progression of thyroid cancer, which is well corroborated by previous results on similar differential expression patterns of TERT in benign and malignant thyroid tumors. As such, TERT promoter mutations are promising diagnostic and prognostic genetic makers for thyroid cancer, which, in combination with BRAF V600E mutation or other genetic markers (e.g. RAS mutations), are proving to be clinically useful for the management of thyroid cancer. Future studies will specifically define such clinical utilities, clarify the biological mechanisms, and explore the potential therapeutic targets of TERT promoter mutations in thyroid cancer.
Based on review of the peer reviewed medical literature the TERT (telomerase reverse transcriptase) promoter mutations may be performed as part of the ThyGenX mutation panel or on an individual basis, and TERT promoter mutations are commonly associated with aggressive tumor behaviors and poor clinical outcomes including tumor recurrence and patient mortality. While the studies may be promising as a diagnostic and prognostic genetic markers for thyroid cancer, future studies are needed to more specifically define clinical utility, clarify the biological mechanisms of the role of TERT promoter mutations in thyroid cancer, and explore and establish therapeutic utilities of targeting TERT for thyroid cancer. The evidence is insufficient to determine the effects of the technology on net health outcomes.
For individuals with thyroid nodule(s) and indeterminate findings on FNA who receive FNA sample testing with molecular markers to predict malignancy to avoid surgical biopsy, the evidence includes one prospective study of clinical validity with the Afirma Gene Expression Classifier (GEC)., and an indirect chain of evidence to support clinical utility. In a multicenter validation study, the Afirma GEC was reported to have 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 two studies do not appear to be identical. In other 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. 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 of the marketed test reporting a turn NPV, the clinical validity is uncertain. Based on clinical input received in 2017 from physician specialty societies and academic medical centers, the clinical input supports that this testing for certain indications provides a clinically meaningful improvement in net health outcome and are consistent with generally accepted medical practice (see below information on Clinical Input).
For the RosettaGX Reval test, no prospective clinical studies were identified and there is no evidence directly demonstrating improved outcomes in patients managed with RosettaGX Reveal. The evidence is insufficient to determine the effects of technology on health outcomes. Based on clinical input received in 2017 from physician specialty societies and academic medical centers, based on the evidence the clinical input provided does not support this testing as this testing does not provide a clinically meaningful improvement in net health outcome or is consistent with generally accepted medical practice.
For individuals with thyroid nodule(s) and indeterminate findings on FNA who receive FNA sample testing with molecular markers to rule in malignancy to guide surgical planning, the evidence includes prospective and retrospective studies of clinical validity. Variant 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 have suggested that testing for a panel of genetic variants associated with thyroid cancer may allow for the appropriate selection of patients for surgical management with an initial complete thyroidectomy. Prospective studies in additional populations are needed to validate these results. Variant analysis does not achieve enough NPV to identify which patients can undergo active surveillance over thyroid surgery. Although the presence of certain variants 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. However, based on clinical input received in 2017 from physician specialty societies and academic medical centers, the clinical input supports that this testing for certain indications provides a clinically meaningful improvement in net health outcome and are consistent with generally accepted medical practice (see below information on Clinical Input).
For individuals with thyroid nodule(s) and indeterminate findings on FNA who receive FNA sample testing with molecular markers to rule out malignancy and to avoid surgical biopsy and rule in to surgical planning, the evidence includes multiple retrospective and prospective clinical validation studies for the ThyroSeq v2 and two retrospective clinical validation studies that utilized a predicate test 17-variant panel (miRInform) test to the current ThyGenX and ThyraMIR. In a retrospective validation study on FNA samples, the 17-variant panel (miRInform) test and ThyraMIR had a sensitivity of 89% and a NPV of 94%. Pooled retrospective and prospective clinical validation studies of ThyroSeq v2 have reported a combined negative predictive value (NPV) of 96% and a positive predictive value (PPV) of 83%. No studies were identified demonstrating the diagnostic characteristics of the marketed ThyGenX. No studies were identified demonstrating evidence of direct outcome improvements. A chain of evidence for the ThyroSeq v2 test and the combined ThyGenX and ThyraMIR testing would rely on establishing clinical validity. The evidence is insufficient to determine the effects of the technology on health outcomes. However, based on clinical input received in 2017 from physician specialty societies and academic medical centers, the clinical input supports that this testing for certain indications provides a clinically meaningful improvement in net health outcome and are consistent with generally accepted medical practice (see below information on Clinical Input).
In 2017, clinical input was sought by Blue Cross Blue Shield Association (BCBSA) to help determine whether the evidence and clinical experience supports a clinical benefit of testing for molecular markers in fine needle aspirates (FNA) of the thyroid for management of individuals with thyroid nodule(s) with an indeterminate finding on the fine needle aspirate. In response to requests, clinical input on 7 tests for molecular markers was received from 9 respondents, including 1 specialty society-level response, 1 physician from academic center and 7 physicians from 2 health systems. Based on the evidence and independent clinical input, the clinical input supports that the following indications provide a clinically meaningful improvement in net health outcome and are consistent with generally accepted medical practice.
Use of the following types of molecular marker testing in fine needle aspirate of thyroid nodules with indeterminate cytologic findings (i.e. Bethesda diagnostic category III – atypia/follicular lesion of undetermined significance or Bethesda diagnostic category IV – follicular neoplasm/suspicion for a follicular neoplasm) to rule out malignancy and to avoid surgical biopsy:
Use of the following type of molecular markers testing in FNA of thyroid nodules with indeterminate cytological findings or Bethesda diagnostic category V – suspicious for malignancy to rule in the presence of malignancy to guide surgical planning for the initial resection rather than a 2-stage surgical biopsy followed by definitive surgery:
Based on the evidence and independent clinical input, the clinical input does not support whether the following indications provides a clinically meaningful improvement in net health outcome or is consistent with generally accepted medical practice:
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 thyroid carcinoma (version 2.2017) make the following recommendations on the use of molecular diagnostics in thyroid cancer:
The NCCN Panel recommends molecular diagnostic testing for evaluating FNA results that are suspicious for follicular cell neoplasms or AUS/FLUS. Molecular diagnostic testing is not recommended for suspected Hurthle cell neoplasms. Molecular diagnostic testing may include multigene assays (e.g. the gene expression classifier) or individual mutational analysis. The gene expression classifier measures the expression of at least 140 genes. BRAF V600E mutation analysis was recommended by some panelists for the evaluation of thyroid nodules (not restricted to the follicular lesions). Furthermore, a majority of the panelists would recommend BRAF V600E testing in the evaluation of follicular lesions. A minority of panelists expressed concern regarding observation of follicular lesions because they were perceived as potentially pre-malignant lesions with a very low, but unknown, malignant potential if not surgically resected (leading to recommendations for either observation or considering lobectomy in lesions classified as benign by molecular testing). Clinical risk factors, sonographic patterns, and patient preference can help determine whether observation or lobectomy is appropriate for these patients.
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.
Current NCCN Panel recommendations regarding molecular diagnostic testing for evaluating FNA results for indeterminate cytopathology does not include or indicate the use of TERT promoter mutations.
In 2016, the American Association of Clinical Endocrinologists (AACE), American College of Endocrinology (ACE) and Associazone Medici Endocrinology (AME) updated its joint guideline and made the following statements:
Patient specific characteristics, the prevalence of cancer within a given population, as well as the distribution and diagnostic accuracy for each cytologic classification have substantial impacts on assessing the odds of malignancy. This was highlighted in a 2012 meta-analysis showing that the malignancy rates across studies for AUS and FLUS ranged from 6 to 48% and 14 to 34%, respectively (154 [EL 2]). While molecular analysis of FNA genetic material from thyroid nodules shows great promise in refining the diagnosis, prognosis, and treatment of thyroid cancer, there are currently insufficient data to support a universal recommendation for molecular testing in the further categorization of “indeterminate” thyroid nodules.
Molecular testing for cytologically indeterminate thyroid nodules:
Role of molecular testing for deciding the extent of surgery:
How should patients with nodules that are negative at mutation testing be monitored:
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.
|Afirma GEC||mRNA gene expression||167 genes||Benign/suspicious|
|Afirma BRAF||mRNA gene expression||1 gene||Negative/positive|
|Afirma MTC||mRNA gene expression||-||Negative/positive|
|ThyroSeq v2||Next-generation sequencing||60+ genes||Specific gene variant/translocation|
|ThyGenX||Next-generation sequencing||8 genes||Specific gene variant/translocation|
|ThyraMIR||microRNA expression||10 miroRNAs||Negative/positive|
|RosettaGX Reveal||microRNA expression||24 microRNAs||Benign/suspicious for malignancy/high risk for medullary carcinoma|
*TERT currently Interpace Diagnostics website does not include any specific information related to this test to include methodology, analyte(s) or how this test is reported.
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 (FNA) 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:
Afirma Malignancy Classifiers (Afirma MTC) and/or Afirma BRAF may be considered medically necessary when the following criteria are met:
The use of ThyroSeq v2, ThyraMIR microRNA and ThyGenX to assess fine needle aspirates (FNA) of thyroid nodules may be considered medically necessary when all of the following criteria are met:
The RosettaGX Reveal test to assess fine needle aspirates (FNA) of thyroid nodules is considered investigational.
Based on the review of the peer review medical literature no prospective clinical studies were identified and the evidence is insufficient in demonstrating improved outcomes in patients managed with RosettaGX reveal.
Gene expression classifiers (GEC), genetic variant analysis and molecular marker testing in fine needle aspirates of the thyroid not meeting the criteria outlined above, including but not limited to the following are considered investigational as there is insufficient evidence to support a conclusion concerning net health outcomes:
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