Key points
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Distinct genetic alterations involving the mitogen-activated protein kinase and phosphatidylinositol 3 kinase pathways characterize thyroid cancer subtypes, and can be detected in preoperative fine-needle aspiration biopsy specimens.
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Identification of these gene mutations and rearrangements can improve the diagnostic utility of preoperative testing, particularly for nodules that are cytologically indeterminate.
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Mutation testing also adds preoperative prognostic information including the identification of thyroid cancers that have a high risk of aggressive histopathologic features, which may further guide the extent of initial thyroidectomy and lymphadenectomy.
Introduction
Thyroid cancer is the most common endocrine malignancy, its incidence is steadily growing, and it is currently the fifth most common cancer diagnosed in women. Thyroid cancer is typically diagnosed during the evaluation of thyroid nodules, which are highly prevalent in the general population. The incidence of nodule detection has been increasing as the population ages, and as high-resolution diagnostic imaging is increasingly being used. Although most thyroid nodules are benign, the challenge is to accurately and effectively identify malignant nodules. As a standard diagnostic approach, current evidence-based guidelines recommend ultrasonography evaluation with ultrasonography-guided fine-needle aspiration biopsy (FNAB) of thyroid nodules for cytologic examination. However in approximately 25% of nodules, the cytology is indeterminate, which limits the clinical management. Several molecular testing techniques have been investigated in an attempt to improve diagnostic accuracy. This article focuses on the diagnostic utility of testing for somatic mutations and rearrangements commonly found in thyroid cancer, discusses how preoperative testing can affect operative management, and examines how recently introduced technologies such as next-generation sequencing (NGS) can further expand the diagnostic capability of preoperative FNAB.
Before the routine use of thyroid nodule FNAB, malignancy was found in only 14% of resected thyroid glands. In a recent meta-review, ultrasonography guidance improved diagnostic sensitivity of FNAB to 95%, but the specificity remained low at 47%. When FNAB cytology has adequate cellularity, the results are classified into one of 3 categories: benign, malignant, and indeterminate. FNAB results that are benign or positive for malignancy are often accurate, with false-negative and false-positive rates of 3% to 4% and 1% to 2%, respectively. However, 13% to 40% can be classified as indeterminate, which represents the limitations of cytologic analysis. Indeterminate FNAB results have been further subdivided by the National Cancer Institute in the widely accepted 2007 Bethesda Classification System ( Table 1 ) into the following 3 categories: (1) suspicious for malignancy, (2) follicular neoplasm (FN), and (3) follicular lesion of undetermined significance (FLUS)/atypia of undetermined significance (AUS). The suspicious category represents 3% of all FNAB results, and up to 75% prove to be malignant. Morphologic criteria for an FN result predominantly include a hypercellular aspirate of follicular cells indicating follicular proliferation with or without microfollicles, and the differential diagnosis encompasses follicular adenoma (FA), follicular thyroid carcinoma (FTC), or follicular variant of papillary thyroid carcinoma (fvPTC). The malignancy rate is lower (up to 26%), but cytology results indicating FN currently require the patient to undergo at least thyroid lobectomy for definitive diagnosis. The FLUS/AUS category is the most heterogeneous and represents those cytology specimens that are neither benign nor malignant, but may have a degree of cellular and/or architectural atypia that does not meet morphologic criteria for being FN or suspicious. A repeat FNAB may lead to a benign cytology result in up to 40% of nodules ; however, the malignancy risk is as high as 16% and diagnostic surgery may still be necessary. Thus, many patients undergo surgical resection of benign disease resulting in potentially avoidable morbidity as well as suboptimal use of limited health care resources. Improved diagnostic accuracy of FNAB cytology evaluation ideally obviates diagnostic surgery and guides the appropriate extent of initial surgery.
| FNA Result | Alternate Accepted Nomenclature | Risk of Malignancy (%) |
|---|---|---|
| Benign | — | <1 |
| Atypia of undetermined significance | Atypical lesion of undetermined significance, follicular lesion of undetermined significance, indeterminate follicular lesion, atypical follicular lesion | 5–15 |
| Neoplasm | Suspicious for neoplasm, follicular neoplasm | 20–30 |
| Suspicious for malignancy | — | 50–75 |
| Malignant | — | 100 |
| Nondiagnostic | Unsatisfactory | — |
Introduction
Thyroid cancer is the most common endocrine malignancy, its incidence is steadily growing, and it is currently the fifth most common cancer diagnosed in women. Thyroid cancer is typically diagnosed during the evaluation of thyroid nodules, which are highly prevalent in the general population. The incidence of nodule detection has been increasing as the population ages, and as high-resolution diagnostic imaging is increasingly being used. Although most thyroid nodules are benign, the challenge is to accurately and effectively identify malignant nodules. As a standard diagnostic approach, current evidence-based guidelines recommend ultrasonography evaluation with ultrasonography-guided fine-needle aspiration biopsy (FNAB) of thyroid nodules for cytologic examination. However in approximately 25% of nodules, the cytology is indeterminate, which limits the clinical management. Several molecular testing techniques have been investigated in an attempt to improve diagnostic accuracy. This article focuses on the diagnostic utility of testing for somatic mutations and rearrangements commonly found in thyroid cancer, discusses how preoperative testing can affect operative management, and examines how recently introduced technologies such as next-generation sequencing (NGS) can further expand the diagnostic capability of preoperative FNAB.
Before the routine use of thyroid nodule FNAB, malignancy was found in only 14% of resected thyroid glands. In a recent meta-review, ultrasonography guidance improved diagnostic sensitivity of FNAB to 95%, but the specificity remained low at 47%. When FNAB cytology has adequate cellularity, the results are classified into one of 3 categories: benign, malignant, and indeterminate. FNAB results that are benign or positive for malignancy are often accurate, with false-negative and false-positive rates of 3% to 4% and 1% to 2%, respectively. However, 13% to 40% can be classified as indeterminate, which represents the limitations of cytologic analysis. Indeterminate FNAB results have been further subdivided by the National Cancer Institute in the widely accepted 2007 Bethesda Classification System ( Table 1 ) into the following 3 categories: (1) suspicious for malignancy, (2) follicular neoplasm (FN), and (3) follicular lesion of undetermined significance (FLUS)/atypia of undetermined significance (AUS). The suspicious category represents 3% of all FNAB results, and up to 75% prove to be malignant. Morphologic criteria for an FN result predominantly include a hypercellular aspirate of follicular cells indicating follicular proliferation with or without microfollicles, and the differential diagnosis encompasses follicular adenoma (FA), follicular thyroid carcinoma (FTC), or follicular variant of papillary thyroid carcinoma (fvPTC). The malignancy rate is lower (up to 26%), but cytology results indicating FN currently require the patient to undergo at least thyroid lobectomy for definitive diagnosis. The FLUS/AUS category is the most heterogeneous and represents those cytology specimens that are neither benign nor malignant, but may have a degree of cellular and/or architectural atypia that does not meet morphologic criteria for being FN or suspicious. A repeat FNAB may lead to a benign cytology result in up to 40% of nodules ; however, the malignancy risk is as high as 16% and diagnostic surgery may still be necessary. Thus, many patients undergo surgical resection of benign disease resulting in potentially avoidable morbidity as well as suboptimal use of limited health care resources. Improved diagnostic accuracy of FNAB cytology evaluation ideally obviates diagnostic surgery and guides the appropriate extent of initial surgery.
| FNA Result | Alternate Accepted Nomenclature | Risk of Malignancy (%) |
|---|---|---|
| Benign | — | <1 |
| Atypia of undetermined significance | Atypical lesion of undetermined significance, follicular lesion of undetermined significance, indeterminate follicular lesion, atypical follicular lesion | 5–15 |
| Neoplasm | Suspicious for neoplasm, follicular neoplasm | 20–30 |
| Suspicious for malignancy | — | 50–75 |
| Malignant | — | 100 |
| Nondiagnostic | Unsatisfactory | — |
Molecular causes of thyroid malignancies
The identification of molecular pathways known to be implicated in thyroid carcinogenesis has significantly expanded options for developing diagnostic adjuncts. The identification of these somatic mutations and rearrangements has helped to further elucidate the specific genetic alterations that occur during the progression from follicular hyperplasia to well-differentiated carcinoma, and much attention has been directed toward elucidating the molecular signature of the 3 most commonly encountered histologic subtypes, which are difficult to differentiate based on FNAB alone: fvPTC, FTC, and FA. Although the diagnosis of histologic variants such as poorly differentiated and anaplastic thyroid cancers is typically straightforward, identification of markers that may herald development of aggressive biological behavior also contributes to improved preoperative risk stratification.
Papillary Thyroid Carcinoma
The mitogen-activated protein kinase (MAPK) pathway alters signaling pathways, induces cell cycle progression, and is typically activated by receptor tyrosine kinases. However, MAPK-mediated tumorigenesis can also be modulated by alternate oncogenic mechanisms such as methylation, chemokine activation, and alterations in components of the tumor microenvironment. Several mutations comprising components of the MAPK pathway have been documented in the development of papillary thyroid carcinoma (PTC) including RAS and BRAF mutations, and RET/PTC rearrangements. Alterations in this oncogenic pathway have been shown in many different malignancies and targeted therapies inhibiting this pathway have been used effectively for a variety of tumor types (eg, Hodgkin lymphoma, glioblastoma multiforme, squamous cell carcinoma, non–small cell lung cancer, and melanoma).
The most common genetic mutation for PTC is an activating point mutation in the BRAF gene, occurring in approximately 45% of all PTC. The mutation that leads to substitution of valine for glutamate at residue 600 ( BRAF V600E) has been implicated in other tumors including melanoma and, less frequently, colorectal adenocarcinoma. Transgenic mice with thyroid-specific BRAF V600E expression develop aggressive and radioiodine-resistant PTC. The mutation is more commonly seen in the classic version of PTC and the tall-cell variant, but can also be identified in anaplastic carcinoma, poorly differentiated thyroid cancer, and primary thyroid lymphomas. More importantly, FTC and benign lesions do not carry this mutation, thereby making it a specific diagnostic marker for PTC. In a study of more than 4500 cytology samples, all BRAF V600E–positive FNAB specimens were histologic malignancies. BRAF K601E is the second most common BRAF mutation identified in thyroid cancer and is more likely to be associated with fvPTC.
RAS mutations are found in approximately 10% to 20% of PTC tumors, and are more commonly encountered in fvPTC and FTC. However, RAS mutations can be found in the full spectrum of thyroid neoplasms ranging from benign follicular hyperplasia and FA to anaplastic carcinomas. The RAS gene encodes for G proteins that are bound to cell membrane receptors and, when stimulated by extracellular signals, result in cellular dysregulation. Activated RAS is bound to guanosine triphosphate (GTP), which is tightly mediated by intrinsic RAS -regulated GTPase, leading to the inactive GDP-bound RAS . Mutations within RAS cause constitutive activation either by inactivating the autocatalytic GTPase function (codon 12 or 13) or increasing the binding affinity for GTP (codon 61). The 3 RAS gene isoforms ( NRAS , KRAS , and HRAS ) can activate both the MAPK and phosphatidylinositol 3 kinase (PI3K) pathways. Mutations resulting in RAS activation have been shown in 20% to 25% of all human tumors.
RET proto-oncogene point mutations are classically identified in medullary thyroid carcinoma (MTC) and, when genomic, are associated with MEN2 and familial MTC. However, the role of RET rearrangements in PTC has been well documented with more than 10 different types of translocations described that are identified in 10% to 20% of PTC. The 3′ tyrosine kinase portion of the RET gene fuses with the 5′ of a different gene resulting in ligand-independent dimerization and constitutive activation of effector genes in the MAPK and PI3-AKT pathways. The 2 most common fusion proteins are RET/PTC1 and RET/PTC3 , which are paracentric versions with the 5′ domain of 2 genes on chromosome 10: CCDC6 and NCOA4 , respectively. A higher incidence of RET/PTC rearrangements are seen in patients with PTC with a previous history of radiation exposure (50%–80%) or in young patients (40%–70%). RET/PTC1 -positive tumors show either classic papillary architecture or diffuse sclerosing features, whereas RET/PTC3 is associated with the solid variant. All of the RET/PTC tumor subtypes possess a high rate of lymph node metastases. Using highly sensitive detection methods, nonclonal RET/PTC rearrangements have also been identified in up to 45% of benign nodules.
TRK rearrangements are gene fusions of the NTRK1 receptor tyrosine kinase gene on chromosome 1q22, which is one of at least 3 different genes described to date. These rearrangements can also be found in less than 5% of PTC.
FTC and FA
Genetic alterations in the PI3K-AKT pathway are common in FTC and FA. Germline mutations in the PTEN gene are responsible for Cowden syndrome, which is characterized by benign and malignant follicular thyroid tumors. Increased expression and activation of AKT was reported in FTC and poorly differentiated thyroid cancer, but has also been observed in all subtypes. Somatic point mutations in PTEN are identified in ∼10% of FTC, but gene methylation resulting in reduced expression levels may be a more frequent mechanism of PTEN loss in follicular thyroid lesions. As previously discussed, RAS mutations can also activate the PI3K-AKT pathway, and are found in 40% to 50% of FTC and 20% to 40% of FA. Although benign follicular lesions carrying the RAS mutations may be precancerous lesions, the identification of RAS is nonspecific for histologic malignancy.
A gene rearrangement leading to the fusion of the thyroid-specific paired domain transcription factor PAX8 and the peroxisome proliferator-activated receptor gene PPARγ , which plays an important role in lipid metabolism, was discovered in FTC in 2000. The PAX8/PPARγ rearrangement results in overexpression of the fusion protein, but the carcinogenic mechanism of action is unclear. PAX8 plays an essential role in thyrocyte development, as well as in the gene expression of the sodium-iodide symporter, thyroglobulin, and the thyroid-stimulating hormone receptor. The fusion protein antagonizes the action of PPARγ via a dominant-negative inhibition that has been shown to be a causative agent in FTC in vitro and in vivo tumorigenesis. However, the exact carcinogenic consequence of the PAX8/PPARγ fusion protein remains theoretic. PAX8/PPARγ translocation is found in 30% to 40% of classic FTC, 2% to 10% of FA, and rarely in fvPTC. PAX8/PPARγ – positive FA tends to have positive immunohistochemical staining for markers more consistent with FTC, but does not meet histologic criteria for malignancy. Much like RAS -positive FAs, PAX8/PPARγ -positive FA may represent carcinomas in situ. PAX8/PPARγ -positive FTCs tend to occur in young patients with tumor characteristics that have solid patterns and vascular invasion.
Poorly Differentiated Thyroid Carcinomas
The progression from differentiated thyroid cancer to poorly differentiated or anaplastic thyroid cancer is less well explicated, and could theoretically be caused by either accumulation of multiple genetic alterations resulting in oncogenic amplification, or coexistent MAPK and PI3K-AKT pathway dysregulation that accelerates the genetic alterations that promote tumor growth. Poorly differentiated thyroid carcinomas (PDTC), although uncommon, display more aggressive behavior than well-differentiated thyroid carcinomas, but are less aggressive than the undifferentiated or anaplastic forms. Histology, immunohistochemistry, and molecular genetic tests reinforce the diagnosis of PDTC. Given the aggressiveness of PDTC and the poor survival rates in patients who undergo surgery alone, a multimodality treatment approach is required.
Although rearrangements are rarely identified in PDTC, point mutations found in differentiated thyroid cancer are well-represented. BRAF V600E is present in ∼15% to 25% of patients with PDTCs but is rarely found in PDTCs arising from FTC. RAS gene alterations (commonly point mutations at HRAS codon 12 or 61 and NRAS at codon 13) are present in 25% to 35% of PDTCs. RAS -induced chromosomal instability may predispose to tumor dedifferentiation, perhaps explaining the increased prevalence of mutant RAS in anaplastic thyroid carcinomas. However, mutant RAS is unlikely to be solely capable of driving tumor dedifferentiation, given its high prevalence (45%) in patients with differentiated thyroid cancer and in those with benign thyroid adenomas. In contrast, Garcia-Rostan and colleagues stated that histologic dedifferentiation is not necessarily driven by BRAF or RAS mutations individually, but rather represents the cooperation of multiple genetic alterations that likely stimulate dedifferentiation.
Inactivating point mutations of TP53 are rarely associated with differentiated thyroid cancers; however, they are highly prevalent (25%–30%) in patients with PDTC and ATC. Because p53 is known as the guardian of the genome, unlike RAS and BRAF gene alterations, which regulate proliferative signals, p53 mutations possess an important function in triggering tumor dedifferentiation and evolution to PDTC and ATC. Other gene mutations that are associated with tumor dedifferentiation include point mutations in PIK3CA (10%–20%), CTNNB1 encoding beta-catenin (10%–20%), and AKT1 (5%–10%).
Biomarkers used for tumor detection and prognostication
Several prospective and retrospective studies have shown that the diagnostic accuracy of FNAB can be significantly improved using modern molecular detection techniques to identify genetic alterations. In addition, although most patients with differentiated thyroid cancer fare well, biomarkers have also been shown to provide prognostic information that can be used to guide further management.
Molecular Testing of FNAB Specimens
BRAF
Most studies of biomarker detection in FNAB specimens have focused on the BRAF V600E mutation. In a 2009 review, Nikiforova and Nikiforov reported on testing for BRAF in 2766 samples from 9 prospective fine-needle aspiration (FNA) studies, 7 retrospective FNAB studies, and 2 studies of research FNAB performed on postoperative thyroid specimens ( Table 2 ). In this meta-analysis, all 580 BRAF -positive clinical FNAB samples studied prospectively and retrospectively were positive for papillary carcinoma, and there was only 1 reported false-positive sample, obtained by research aspiration of the nodule in a surgically removed thyroid gland, resulting in a false-positive rate of 0.2%. BRAF V600E mutation can occasionally be detected in false-negative benign FNAB results from histologic malignancies; however, BRAF V600E is less common overall than other gene alterations in cytologically indeterminate FNAB specimens. Regardless of cytology category, when preoperative BRAF V600E is detected in FNAB testing, a diagnosis of thyroid cancer should be suspected. BRAF K601E is less frequently detected, but is associated with indeterminate cytology and indolent histology. In a series of 29 indeterminate FNAB specimens with positive BRAF testing, 8 (28%) were BRAF K601E with histologic malignancies confirmed in all (100%). Only 1 of the 8 malignancies was a solid variant PTC, whereas the remaining 7 were histologic fvPTC.
