Molecular markers refine the cytopathological diagnosis of thyroid neoplasms.
Home Is Where the Heart Is. Photograph courtesy of Craig Damlo. www.soapboxrocket.com.
Molecular Assays in Cytopathology for Thyroid Cancer Pablo Valderrabano, MD, Victor E. Zota, MD, Bryan McIver, MD, PhD, Domenico Coppola, MD, and Marino E. Leon, MD Background: Despite lack of adequate, validated, independently performed clinical studies, several molecular tests are commercially available on the market and are being used on indeterminate thyroid nodules to guide patient-care decisions. Methods: We summarize the current evidence on the role and limitations of molecular tests used in combination with thyroid cytopathology to refine the presurgical diagnosis of thyroid nodules. Results: The clinical performance of molecular tests depends on the pretest risk of malignancy within the specific cytological group being assessed. This risk is variable and should be assessed at each institution to optimize the selection of the molecular test and the interpretation of its results. Next-generation sequencing has increased the sensitivity of oncogene panels while maintaining high specificity. Tests assessing the gene expression pattern have shown promising results, with high sensitivity but low specificity. The impacts of molecular markers on clinical practice remains in flux and their effect on health care costs remains poorly understood. Conclusions: Further large, independent, confirmatory, clinical validation studies and real-world, cost-effectiveness studies are necessary before the widespread adoption of these tests can be endorsed as standard of care.
Introduction The incidences of benign thyroid nodules and thyroid cancer have increased in the last several decades.1 An accurate presurgical diagnosis of thyroid nodules is important because of the implications in clinical management. Although fine needle aspiration (FNA) From the Endocrine Tumor Program (PV, BM), Departments of Head and Neck (PV, BM), Endocrine Oncology (PV, BM), and Anatomic Pathology (VEZ, DC, MEL), H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida. Submitted September 4, 2014; accepted March 13, 2015. Address correspondence to Marino E. Leon, MD, Department of Anatomic Pathology, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612. E-mail: [email protected] Dr Valderrabano acknowledges financial support from the Alfonso Martín Escudero Foundation. No other significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. 152 Cancer Control
cytology provides valuable information in the presurgical evaluation of thyroid nodules, approximately 25% of biopsy samples do not render diagnostic information and are classified as indeterminate.2 Repeating FNA on an indeterminate specimen may be helpful at times; however, for many patients, diagnostic surgery is eventually needed to clarify the diagnosis. One-third of these nodules resected for diagnostic purposes will prove to be malignant and, in many cases, will require additional surgery to complete thyroidectomy.3,4 Conversely, two-thirds of such surgeries might have been avoided with a more accurate presurgical diagnosis.3 The Bethesda System for Reporting Thyroid Cytopathology (Bethesda) has standardized reporting terminology and diagnostic criteria, but it has not improved the performance of FNA.5 Indeterminate specimens are stratified by Bethesda in 3 diagnostic April 2015, Vol. 22, No. 2
categories according to risk of malignancy (ROM): • Category III: Atypia/follicular lesion of undetermined significance • Category IV: Follicular neoplasm/suspicious for follicular neoplasm and Hürthle cell neoplasm/suspicious for Hürthle cell neoplasm • Category V: Suspicious for malignancy The estimated ROMs of Bethesda categories III to V are 5% to 15%, 15% to 30%, and 60% to 75%, respectively.5 However, the observed ROMs in the indeterminate categories, particularly Bethesda categories III and IV, are broader, ranging from 0% to 48% and 14% to 49%, respectively, in multiple large institutional studies.2,3 This diagnostic challenge reflects subtle morphological differences and significant morphological overlap in the cytological and histological features exhibited by thyroid follicular pattern lesions, including follicular hyperplasia, adenomatous nodule/ follicular thyroid adenoma, follicular thyroid carcinoma, and the follicular variant of papillary thyroid carcinoma (PTC).6,7 The subjectivity of the pathological diagnosis further complicates this issue. Broad intraobserver and interobserver variabilities have been reported for follicular thyroid lesions in cytological and histological specimens.7-10 Several molecular tests have been developed in an attempt to better and more reliably characterize these lesions to avoid diagnostic surgery. These tests use different approaches to identify molecular signatures specific for thyroid cancer. Some scrutinize the DNA to detect mutations and others characterize the gene expression profiles at the transcriptional (messenger ribonucleic acids [RNAs]) or post-transcriptional level (microRNAs). All these methods have limitations and no single test is 100% accurate. Furthermore, the predictive values of these tests — which represent their true clinical value — depend on the cytological ROM, which represents the pretest probability of malignancy; as the ROM increases, so does the positive predictive value (PPV) at the expense of a reduction in the negative predictive value (NPV), and vice versa.11 Therefore, until further standardization is achieved in the classification of indeterminate cytology, the results obtained at one center may not extrapolate to others. Marketing a test on the basis of a fixed NPV, without first knowing the true pretest ROM, is disingenuous. In addition, some of these molecular markers, such as the point mutation BRAF V600E, are also considered prognostic factors, and their study may be helpful for individualizing clinical management.12-15
During recent years, our understanding and knowledge regarding driver mutations have been expanding. A recent publication by The Cancer Genome Atlas (TCGA) has significantly reduced the proportion of PTCs with unknown driver mutations from 25% to 3.5%.17 Such mutations are almost always mutually Table. — Common Mutations and Rearrangements in Common Thyroid Tumors Tumor Type
Mutation and Rearrangement
Papillary thyroid carcinoma
BRAF V600E (classic, tall cell variant) TERT RET/PTC1 (RET/CCDC6) RET/PTC3 (RET/ELE1) RET/PTC6 (RET/HTIF1)
Papillary thyroid carcinoma (follicular variant)
EIF1AX BRAF K601E NRAS HRAS KRAS PAX8/PPARγ
Follicular thyroid carcinoma
PAX8/PPARγ NRAS HRAS KRAS BRAF K601E TSHR
Follicular thyroid carcinoma (oncocytic variant)/Hürthle cell carcinoma
TP53 HRAS KRAS PTEN
Poorly differentiated thyroid carcinoma
NRAS PIK3CA GNAS RAF V600E
Undifferentiated thyroid carcinoma (anaplastic carcinoma)
TP53 BRAF V600E NRAS PIK3CA PTEN CTNNB1
Medullary thyroid carcinoma
RET HRAS KRAS
Follicular thyroid adenoma
PAX8/PPARγ NRAS KRAS
Benign thyroid “hyperplastic nodule”
TSHR NRAS HRAS KRAS PTEN GNAS
Somatic Alterations Most thyroid cancers exhibit somatic point mutations or rearrangements that activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase/protein kinase B pathways (Table16-21).22 April 2015, Vol. 22, No. 2
Data from references 16 to 21.
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exclusive and homogeneously present within the tumor.17,18 Most well-differentiated thyroid cancers exhibit a single point mutation or chromosomal rearrangement, although 2 or more mutations may be found in more aggressive tumors.17 Some mutations are highly specific for thyroid cancer such as the point mutation BRAF V600E; other mutations, like RAS mutations, are also found in benign lesions, thus limiting their diagnostic specificity. However, this finding could be explained by the evolution hypothesis, which posits that the accumulation of mutations induces progression from a polyclonal proliferation (hyperplasia) to a benign monoclonal proliferation (follicular thyroid adenoma) to a differentiated thyroid cancer (PTC [follicular variant] or follicular thyroid carcinoma) and potentially from there to undifferentiated thyroid cancer. If this concept holds true, then a RAS mutation present in follicular thyroid adenoma would represent an early neoplastic change that might progress to malignancy (precancer) rather than represent a false-positive result. The presurgical identification of the oncogenic driver mutation in thyroid cancer has been explored for several years as a method to segregate thyroid nodules with indeterminate cytology. Initial studies searched for 1 or just a few specific mutations.23-28 If researchers must focus on a single marker, then the most useful mutation is BRAF V600E for several reasons: (1) It is the most prevalent mutation among PTC (45%) and PTC represents 85% of all thyroid malignancies, (2) its specificity for malignancy is nearly 100%, and (3) it can independently predict extrathyroidal extension and lymph node involvement, thus implying a higher risk for disease persistence or recurrence that could influence the extent of the surgery.29-32 However, the sensitivity of BRAF for detecting cancer improves when other frequently occurring mutations are included in the screening panel. Such an observation led to the development of oncogene panels that simultaneously studied several mutations. The most well-studied panel assessed 14 point mutations in BRAF and RAS and for RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements. This panel has been prospectively studied in the characterization of indeterminate thyroid nodules achieving estimated sensitivity and specificity for malignancy around 60% and 98%, respectively.33-35 Results from the commercially available variant of this panel marketed as mIRinform (Asuragen, Austin, Texas) were published in a validation study that indicated the panel had a slightly worse performance than anticipated, perhaps in part because of differences in the technology used in the commercial version of the test.36 In the study, the panel achieved sensitivity and specificity of 48% (29%–68%) and 89% (72%–98%), respectively, for indeterminate thyroid nodules.36 A modified version of this test using next-genera154 Cancer Control
tion sequencing is now commercially available as ThyGenX (Interpace Diagnostics, Parsippany, New Jersey), but the performance of this modified assay has not yet been reported.37 The advent of next-generation sequencing has allowed the rapid translation of new data on additional, low-prevalence, somatic alterations into the clinical field. An additional commercially available panel is based on this technology and is marketed as ThyroSeq V2 (University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania). This panel now includes more than 400 point mutations in 14 genes,38 including AKT1, BRAF, CTNNB1, GNAS, HRAS, KRAS, NRAS, PIK3CA, PTEN, RET, TP53, TSHR, TERT, and EIF1AX, along with 42 gene rearrangements and can be performed on as little as 10 ng of DNA.20,39 In the initial publication on this panel, the test included mutations in 13 genes (rather than the 14 genes now in the commercial offering) and 42 gene rearrangements, and it achieved sensitivity and specificity of 90% (80%–99%) and 93% (88%–98%), respectively, among cytological specimens classified as Bethesda category IV.19 Such performance should improve and simplify the interpretation (NPVs and PPVs) of the results among different institutions with distinct pretest ROM. The NPV and PPV achieved in this study were 96% and 83%, respectively, with an overall ROM of 27%.19 Based on the reported sensitivity and specificity, we calculate that the NPV will remain above 95% at any ROM up to 34%, whereas the PPV will remain above 75% at any ROM above 15%. As more mutations are added to this panel, we anticipate an increase in the test sensitivity, but it is possible that the specificity could decrease, thus negatively impacting the predictive values. Consequently, although these early results are promising, they still require external validation and additional information regarding the performance of the test on Bethesda category III. Data on this category were presented at the annual meeting and exposition of the Endocrine Society in early 2015, suggesting that sensitivity and specificity are well preserved in this cytological group.40
Gene Expression Analysis Somatic mutations directly impact the gene expression profile by acting on signaling pathways. For example, BRAF V600E is associated with high MAPK signaling, whereas RAS mutants have lower MAPK signaling. This observation was used by TCGA to develop a score that quantified the extent to which the gene expression profile of each somatic mutation resembled either BRAF V600E–mutant or RAS-mutant PTCs.17 This score is associated with the degree of thyroid differentiation of the tumor, histological grade, American Thyroid Association risk of recurrence stage,4 and the MACIS score.41 RAS-like tumors were better differentiated and April 2015, Vol. 22, No. 2
BRAF V600E–like tumors were less differentiated; in addition, RAS-mutant tumors were homogeneous in terms of gene expression and degree of thyroid differentiation.17 Conversely, BRAF V600E–mutant tumors had a heterogeneous gene expression profile and included at least 4 different groups, a finding that could explain the uncertainty regarding the prognostic and predictive power of this mutation alone.17 In light of these results, the authors suggested that PTCs may be more appropriately classified according to their genetic profiles rather than to their morphological appearance.17 Although these data have the potential to transform histological classification and clinical practice, their utility in routine clinical practice has yet to be established. However, prior studies have already used gene expression profiles (at the transcriptional or post-transcriptional level) to differentiate thyroid nodules with indeterminate cytology; these are discussed below. Transcriptional Level Several tools exploit the analysis of the gene expression profile at the transcriptional level to differentiate benign from malignant lesions among indeterminate thyroid nodules. Although several RNA-based markers have investigated single genes, a commercially available multigene expression panel has achieved promising results. The Afirma Gene Expression Classifier (Veracyte, South San Francisco, California) assesses the relative mRNA expression of 167 genes processed through a support vector machine trained on a group of histological and FNA material derived from a broad spectrum of samples from benign and malignant thyroid disease. Using a proprietary algorithm, the classifier reports the nodule as benign or suspicious. In a prospective, double-blind, multicenter study, 265 indeterminate thyroid nodules were analyzed.42 The overall performance of the test showed sensitivity and specificity of 92% (84%–97%) and 52% (44%–59%), respectively.42 In this study, the test achieved an NPV of approximately 95% for Bethesda categories III and IV. Such an NPV justifies patient observation in lieu of surgery per the recommendations of the National Comprehensive Cancer Network.43 However, an NPV of 95% can be assumed only if the midpoint sensitivity and specificity achieved in this single study are used and the ROM of the cytology is below 24%.11,42 Because the ROM in the indeterminate categories is higher in many institutions,3 its use should be individualized according to the specific ROM of each center. In an independent study, the sensitivity and specificity rates achieved by Afirma were reported to be 83% and 36%, respectively, and were the best rates of the case scenarios.44 If lower sensitivity and specificity than anticipated are confirmed, then both predictive values would be negatively affected. April 2015, Vol. 22, No. 2
Therefore, further validation is needed before the widespread use of this test. As single-gene markers, HMGA2 and cancer-related E2 ubiquitin-conjugating enzyme have also been studied in the cytology specimens of indeterminate thyroid nodules.45,46 However, neither has demonstrated sufficiently high sensitivity or specificity to justify widespread use. Nonetheless, their combined use, along with other markers, continues to be explored.47 The detection of thyrotropin receptor mRNA in peripheral blood achieved better results in a study on thyroid nodules with cytological diagnosis of follicular neoplasm, reaching sensitivity and specificity of 97% and 88%, respectively, for detecting thyroid cancer.48 However, further studies are necessary to validate these results. Post-Transcriptional Level MicroRNAs are small, noncoding RNA molecules that bind to mRNA to promote or silence their translation into proteins.49-51 These molecules can be analyzed in cytological specimens and those affecting oncogenes or tumor suppressor genes are also being studied. Several studies have reported attempts to identify the specific signatures of thyroid cancer by developing predictive models on excised specimens later investigated as single microRNAs or in microRNA panels on FNA samples of thyroid nodules.52-59 One meta-analysis found that panels had better performance rates compared with single microRNAs.59 The overall sensitivity and specificity of 4 studies using microRNA panels in FNA specimens were 87% (77%–92%) and 85% (78%–90%), respectively.59 Performance was best in a study validating a panel of 4 microRNAs (222, 328, 197, and 21) in which sensitivity and specificity of 100% and 86%, respectively, were achieved.55 Larger prospective studies of indeterminate thyroid nodules must be performed to validate their diagnostic utility. Some microRNAs also appear to have prognostic value. In the study by TCGA, 4 different expression patterns stratified the BRAF-like PTCs into groups with various histological grades of differentiation.17 The overexpression of microRNA-21 and microRNA-146b, as well as the down-regulation of microRNA-204 — which acts as a tumor suppressor — was found in the group with the most aggressive PTCs.17 Conversely, higher expressions of microRNA-182 and microRNA-183 were associated with RAS-like PTCs that had a higher degree of thyroid differentiation.17,60
Follicular Neoplasm (Oncocytic Variant) /Hürthle Cell Neoplasm Most of the molecular markers previously discussed are not helpful for Hürthle cell neoplasms. Hürthle Cancer Control 155
cell carcinomas rarely exhibit one of the commonly studied mutations.61 However, the use of next-generation sequencing might improve the sensitivity of previous oncogene panels. The initial version of ThyroSeq detected a mutation in 39% of cases of Hürthle cell carcinoma analyzed.20 Most of them had a TP53 mutation, typically detected in undifferentiated thyroid cancer but not in well-differentiated carcinomas.20 Moreover, Hürthle cell neoplasms have a distinct profile of genes and transcription factors involved in tumorigenesis.62 Possibly because of this, Afirma classifies 90% of the Hürthle cell neoplasms as “suspicious,” even despite the fact that the ROM in these tumors is typically lower than that for the overall Bethesda category IV group, resulting in a low PPV in these nodules.42,43,63 In part, this likely reflects the fact that the training of the support vector machine that encodes the proprietary algorithm included a number of Hürthle cell carcinomas but almost no benign Hürthle cell adenomas; as a consequence, the gene expression classifier determines all Hürthle cell lesions as suspicious. Similarly, microRNA panels may inadequately classify this group of tumors. In a study, the accuracy of the panel rose from 90% to 97% when Hürthle cell neoplasms were excluded.55 Thus, in our opinion, Hürthle cell neoplasms should not be routinely evaluated with the currently available molecular tests, and, if used, the test results should be interpreted with caution.
Conclusions The rapid translation into the clinical field of new discoveries in the molecular basis of thyroid cancer has led to the development of several molecular tests that address the deficiencies of thyroid cytopathology. The initial results of such tests are promising, and the rapid expansion of our knowledge in this regard promises further advances in the near future. Additional studies that independently validate these results and define the role of these tests in routine clinical practice are needed. Until the cytological classification has been further standardized, each institution interested in using these tests should determine its own risk of malignancy for each of the indeterminate categories to select the most appropriate test and to adequately interpret the results. However, in the future, molecular markers will be instrumental in refining the diagnostic accuracy of cytology and to individualize thyroid cancer management and treatment. References 1. Chen AY, Jemal A, Ward EM. Increasing incidence of differentiated thyroid cancer in the United States, 1988-2005. Cancer. 2009;115(16):3801-3807. 2. Bongiovanni M, Spitale A, Faquin WC, et al. The Bethesda System for reporting thyroid cytopathology: a meta-analysis. Acta Cytol. 2012;56(4):333-339. 3. Wang CC, Friedman L, Kennedy GC, et al. A large multicenter cor-
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Lloyd RV, Erickson LA, Casey MB, et al. Observer variation in the diagnosis of follicular variant of papillary thyroid carcinoma. Am J Surg Pathol. 2004;28(10):1336-1340. 10. Hirokawa M, Carney JA, Goellner JR, et al. Observer variation of encapsulated follicular lesions of the thyroid gland. Am J Surg Pathol. 2002;26(11):1508-1514. 11. McIver B. Evaluation of the thyroid nodule. Oral Oncol. 2013;49(7):645-653. 12. Kim TH, Park YJ, Lim JA, et al. The association of the BRAF(V600E) mutation with prognostic factors and poor clinical outcome in papillary thyroid cancer: a meta-analysis. Cancer. 2012;118(7):1764-1773. 13. Elisei R, Viola D, Torregrossa L, et al. The BRAF V600E mutation is an independent, poor prognostic factor for the outcome of patients with low-risk intrathyroid papillary thyroid carcinoma: single-institution results from a large cohort study. J Clin Endocrinol Metab. 2012;97(12):4390-4398. 14. Howell GM, Carty SE, Armstrong MJ, et al. 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BRAF mutation analysis in fine needle aspiration (FNA) cytology of the thyroid. Diagn Mol Pathol. 2006;15(3):136-143. 29. Joo JY, Park JY, Yoon YH, et al. Prediction of occult central lymph node metastasis in papillary thyroid carcinoma by preoperative BRAF analysis using fine-needle aspiration biopsy: a prospective study. J Clin Endocrinol Metab. 2012;97(11):3996-4003. 30. Xing M, Clark D, Guan H, et al. BRAF mutation testing of thyroid fine-needle aspiration biopsy specimens for preoperative risk stratification in papillary thyroid cancer. J Clin Oncol. 2009;27(18):2977-2982. 31. Howell GM, Nikiforova MN, Carty SE, et al. BRAF V600E mutation independently predicts central compartment lymph node metastasis in patients April 2015, Vol. 22, No. 2
with papillary thyroid cancer. Ann Surg Oncol. 2013;20(1):47-52. 32. Lin KL, Wang OC, Zhang XH, et al. The BRAF mutation is predictive of aggressive clinicopathological characteristics in papillary thyroid microcarcinoma. Ann Surg Oncol. 2010;17(12):3294-3300. 33. Nikiforov YE, Steward DL, Robinson-Smith TM, et al. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab. 2009;94(6):2092-2098. 34. Ohori NP, Nikiforova MN, Schoedel KE, et al. Contribution of molecular testing to thyroid fine-needle aspiration cytology of “follicular lesion of undetermined significance/atypia of undetermined significance”. Cancer Cytopathol. 2010;118(1):17-23. 35. Nikiforov YE, Ohori NP, Hodak SP, et al. Impact of mutational testing on the diagnosis and management of patients with cytologically indeterminate thyroid nodules: a prospective analysis of 1056 FNA samples. J Clin Endocrinol Metab. 2011;96(11):3390-3397. 36. Beaudenon-Huibregtse S, Alexander EK, Guttler RB, et al. Centralized molecular testing for oncogenic gene mutations complements the local cytopathologic diagnosis of thyroid nodules. Thyroid. 2014;24(10):1479-1487. 37. Interpace Diagnostics. Thyroid cancer diagnosis. http://www.interpa cediagnostics.com/hcpw.php. Accessed April 1, 2015. 38. CBLPath, Inc. ThyroSeq v.2 next generation sequencing. http://www. cblpath.com/products-and-services/test-menu-cblpath/item/1628-thyroseq-v2. Accessed April 28, 2015. 39. University of Pittsburgh Medical Center. ThyroSeq - thyroid cancer next-generation sequencing panel. http://mgp.upmc.com/Applications/mgp /Home/Test/ThyroSeq_Details. Accessed April 1, 2015. 40. Nikiforova MN. Next-gen sequencing for diagnosis, prognostication and treatment of thyroid cancer. Presented at: Endocrine Society 97th Annual Meeting & Expo; March 5–8, 2015; San Diego, CA. https://endo.confex.com/ endo/2015endo/webprogram/Paper18101.html. Accessed April 28, 2015. 41. Hay ID, Bergstralh EJ, Goellner JR, et al. Predicting outcome in papillary thyroid carcinoma: development of a reliable prognostic scoring system in a cohort of 1779 patients surgically treated at one institution during 1940 through 1989. Surgery. 1993;114(6):1050-1058. 42. Alexander EK, Kennedy GC, Baloch ZW, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med. 2012;367(8):705-715. 43. National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology. v2.2014. Published August 12, 2014. http://www.nccn.org/professionals/physician_gls/pdf/thyroid.pdf. Accessed April 13, 2015. 44. McIver B, Castro MR, Morris JC, et al. An independent study of a gene expression classifier (Afirma) in the evaluation of cytologically indeterminate thyroid nodules. J Clin Endocrinol Metab. 2014:jc20133584. 45. Jin L, Lloyd RV, Nassar A, et al. HMGA2 expression analysis in cytological and paraffin-embedded tissue specimens of thyroid tumors by relative quantitative RT-PCR. Diagn Mol Pathol. 2011;20(2):71-80. 46. Guerriero E, Ferraro A, Desiderio D, et al. UbcH10 expression on thyroid fine-needle aspirates. Cancer Cytopathol. 2010;118(3):157-165. 47. Prasad NB, Kowalski J, Tsai HL, et al. Three-gene molecular diagnostic model for thyroid cancer. Thyroid. 2012;22(3):275-284. 48. Milas M, Shin J, Gupta M, et al. Circulating thyrotropin receptor mRNA as a novel marker of thyroid cancer: clinical applications learned from 1758 samples. Ann Surg. 2010;252(4):643-651. 49. Calin GA, Dumitru CD, Shimizu M, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proceed Natl Acad Sci U S A. 2002;99(24):15524-15529. 50. Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proceed Natl Acad Sci U S A. 2004;101(32):11755-11760. 51. Ambros V. MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell. 2003;113(6):673-676. 52. Kitano M, Rahbari R, Patterson EE, et al. Expression profiling of difficult-to-diagnose thyroid histologic subtypes shows distinct expression profiles and identify candidate diagnostic microRNAs. Ann Surg Oncol. 2011;18(12):3443-3452. 53. Mazeh H, Mizrahi I, Halle D, et al. Development of a microRNA-based molecular assay for the detection of papillary thyroid carcinoma in aspiration biopsy samples. Thyroid. 2011;21(2):111-118. 54. Kitano M, Rahbari R, Patterson EE, et al. Evaluation of candidate diagnostic microRNAs in thyroid fine-needle aspiration biopsy samples. Thyroid. 2012;22(3):285-291. 55. Keutgen XM, Filicori F, Crowley MJ, et al. A panel of four miRNAs accurately differentiates malignant from benign indeterminate thyroid lesions on fine needle aspiration. Clin Cancer Res. 2012;18(7):2032-2038. 56. 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59. Zhang Y, Zhong Q, Chen X, et al. Diagnostic value of microRNAs in discriminating malignant thyroid nodules from benign ones on fine-needle aspiration samples. Tumour Biol. 2014;35(9):9343-9353. 60. Reddi HV, Driscoll CB, Madde P, et al. Redifferentiation and induction of tumor suppressors miR-122 and miR-375 by the PAX8/PPARγ fusion protein inhibits anaplastic thyroid cancer: a novel therapeutic strategy. Cancer Gene Ther. 2013;20(5):267-275. 61. Filicori F, Keutgen XM, Buitrago D, et al. Risk stratification of indeterminate thyroid fine-needle aspiration biopsy specimens based on mutation analysis. Surgery. 2011;150(6):1085-1091. 62. Máximo V, Lima J, Prazeres H, et al. The biology and the genetics of Hurthle cell tumors of the thyroid. Endoc Relat Cancer. 2012;19(4):R131-R147. 63. Harrell RM, Bimston DN. Surgical utility of afirma: effects of high cancer prevalence and oncocytic cell types in patients with indeterminate thyroid cytology. Endocr Pract. 2014;20(4):364-369.
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Home Is Where the Heart Is. Photograph courtesy of Craig Damlo. www.soapboxrocket.com.
Molecular Assays in Cytopathology for Thyroid Cancer Pablo Valderrabano, MD, Victor E. Zota, MD, Bryan McIver, MD, PhD, Domenico Coppola, MD, and Marino E. Leon, MD Background: Despite lack of adequate, validated, independently performed clinical studies, several molecular tests are commercially available on the market and are being used on indeterminate thyroid nodules to guide patient-care decisions. Methods: We summarize the current evidence on the role and limitations of molecular tests used in combination with thyroid cytopathology to refine the presurgical diagnosis of thyroid nodules. Results: The clinical performance of molecular tests depends on the pretest risk of malignancy within the specific cytological group being assessed. This risk is variable and should be assessed at each institution to optimize the selection of the molecular test and the interpretation of its results. Next-generation sequencing has increased the sensitivity of oncogene panels while maintaining high specificity. Tests assessing the gene expression pattern have shown promising results, with high sensitivity but low specificity. The impacts of molecular markers on clinical practice remains in flux and their effect on health care costs remains poorly understood. Conclusions: Further large, independent, confirmatory, clinical validation studies and real-world, cost-effectiveness studies are necessary before the widespread adoption of these tests can be endorsed as standard of care.
Introduction The incidences of benign thyroid nodules and thyroid cancer have increased in the last several decades.1 An accurate presurgical diagnosis of thyroid nodules is important because of the implications in clinical management. Although fine needle aspiration (FNA) From the Endocrine Tumor Program (PV, BM), Departments of Head and Neck (PV, BM), Endocrine Oncology (PV, BM), and Anatomic Pathology (VEZ, DC, MEL), H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida. Submitted September 4, 2014; accepted March 13, 2015. Address correspondence to Marino E. Leon, MD, Department of Anatomic Pathology, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612. E-mail: [email protected] Dr Valderrabano acknowledges financial support from the Alfonso Martín Escudero Foundation. No other significant relationships exist between the authors and the companies/organizations whose products or services may be referenced in this article. 152 Cancer Control
cytology provides valuable information in the presurgical evaluation of thyroid nodules, approximately 25% of biopsy samples do not render diagnostic information and are classified as indeterminate.2 Repeating FNA on an indeterminate specimen may be helpful at times; however, for many patients, diagnostic surgery is eventually needed to clarify the diagnosis. One-third of these nodules resected for diagnostic purposes will prove to be malignant and, in many cases, will require additional surgery to complete thyroidectomy.3,4 Conversely, two-thirds of such surgeries might have been avoided with a more accurate presurgical diagnosis.3 The Bethesda System for Reporting Thyroid Cytopathology (Bethesda) has standardized reporting terminology and diagnostic criteria, but it has not improved the performance of FNA.5 Indeterminate specimens are stratified by Bethesda in 3 diagnostic April 2015, Vol. 22, No. 2
categories according to risk of malignancy (ROM): • Category III: Atypia/follicular lesion of undetermined significance • Category IV: Follicular neoplasm/suspicious for follicular neoplasm and Hürthle cell neoplasm/suspicious for Hürthle cell neoplasm • Category V: Suspicious for malignancy The estimated ROMs of Bethesda categories III to V are 5% to 15%, 15% to 30%, and 60% to 75%, respectively.5 However, the observed ROMs in the indeterminate categories, particularly Bethesda categories III and IV, are broader, ranging from 0% to 48% and 14% to 49%, respectively, in multiple large institutional studies.2,3 This diagnostic challenge reflects subtle morphological differences and significant morphological overlap in the cytological and histological features exhibited by thyroid follicular pattern lesions, including follicular hyperplasia, adenomatous nodule/ follicular thyroid adenoma, follicular thyroid carcinoma, and the follicular variant of papillary thyroid carcinoma (PTC).6,7 The subjectivity of the pathological diagnosis further complicates this issue. Broad intraobserver and interobserver variabilities have been reported for follicular thyroid lesions in cytological and histological specimens.7-10 Several molecular tests have been developed in an attempt to better and more reliably characterize these lesions to avoid diagnostic surgery. These tests use different approaches to identify molecular signatures specific for thyroid cancer. Some scrutinize the DNA to detect mutations and others characterize the gene expression profiles at the transcriptional (messenger ribonucleic acids [RNAs]) or post-transcriptional level (microRNAs). All these methods have limitations and no single test is 100% accurate. Furthermore, the predictive values of these tests — which represent their true clinical value — depend on the cytological ROM, which represents the pretest probability of malignancy; as the ROM increases, so does the positive predictive value (PPV) at the expense of a reduction in the negative predictive value (NPV), and vice versa.11 Therefore, until further standardization is achieved in the classification of indeterminate cytology, the results obtained at one center may not extrapolate to others. Marketing a test on the basis of a fixed NPV, without first knowing the true pretest ROM, is disingenuous. In addition, some of these molecular markers, such as the point mutation BRAF V600E, are also considered prognostic factors, and their study may be helpful for individualizing clinical management.12-15
During recent years, our understanding and knowledge regarding driver mutations have been expanding. A recent publication by The Cancer Genome Atlas (TCGA) has significantly reduced the proportion of PTCs with unknown driver mutations from 25% to 3.5%.17 Such mutations are almost always mutually Table. — Common Mutations and Rearrangements in Common Thyroid Tumors Tumor Type
Mutation and Rearrangement
Papillary thyroid carcinoma
BRAF V600E (classic, tall cell variant) TERT RET/PTC1 (RET/CCDC6) RET/PTC3 (RET/ELE1) RET/PTC6 (RET/HTIF1)
Papillary thyroid carcinoma (follicular variant)
EIF1AX BRAF K601E NRAS HRAS KRAS PAX8/PPARγ
Follicular thyroid carcinoma
PAX8/PPARγ NRAS HRAS KRAS BRAF K601E TSHR
Follicular thyroid carcinoma (oncocytic variant)/Hürthle cell carcinoma
TP53 HRAS KRAS PTEN
Poorly differentiated thyroid carcinoma
NRAS PIK3CA GNAS RAF V600E
Undifferentiated thyroid carcinoma (anaplastic carcinoma)
TP53 BRAF V600E NRAS PIK3CA PTEN CTNNB1
Medullary thyroid carcinoma
RET HRAS KRAS
Follicular thyroid adenoma
PAX8/PPARγ NRAS KRAS
Benign thyroid “hyperplastic nodule”
TSHR NRAS HRAS KRAS PTEN GNAS
Somatic Alterations Most thyroid cancers exhibit somatic point mutations or rearrangements that activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase/protein kinase B pathways (Table16-21).22 April 2015, Vol. 22, No. 2
Data from references 16 to 21.
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exclusive and homogeneously present within the tumor.17,18 Most well-differentiated thyroid cancers exhibit a single point mutation or chromosomal rearrangement, although 2 or more mutations may be found in more aggressive tumors.17 Some mutations are highly specific for thyroid cancer such as the point mutation BRAF V600E; other mutations, like RAS mutations, are also found in benign lesions, thus limiting their diagnostic specificity. However, this finding could be explained by the evolution hypothesis, which posits that the accumulation of mutations induces progression from a polyclonal proliferation (hyperplasia) to a benign monoclonal proliferation (follicular thyroid adenoma) to a differentiated thyroid cancer (PTC [follicular variant] or follicular thyroid carcinoma) and potentially from there to undifferentiated thyroid cancer. If this concept holds true, then a RAS mutation present in follicular thyroid adenoma would represent an early neoplastic change that might progress to malignancy (precancer) rather than represent a false-positive result. The presurgical identification of the oncogenic driver mutation in thyroid cancer has been explored for several years as a method to segregate thyroid nodules with indeterminate cytology. Initial studies searched for 1 or just a few specific mutations.23-28 If researchers must focus on a single marker, then the most useful mutation is BRAF V600E for several reasons: (1) It is the most prevalent mutation among PTC (45%) and PTC represents 85% of all thyroid malignancies, (2) its specificity for malignancy is nearly 100%, and (3) it can independently predict extrathyroidal extension and lymph node involvement, thus implying a higher risk for disease persistence or recurrence that could influence the extent of the surgery.29-32 However, the sensitivity of BRAF for detecting cancer improves when other frequently occurring mutations are included in the screening panel. Such an observation led to the development of oncogene panels that simultaneously studied several mutations. The most well-studied panel assessed 14 point mutations in BRAF and RAS and for RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements. This panel has been prospectively studied in the characterization of indeterminate thyroid nodules achieving estimated sensitivity and specificity for malignancy around 60% and 98%, respectively.33-35 Results from the commercially available variant of this panel marketed as mIRinform (Asuragen, Austin, Texas) were published in a validation study that indicated the panel had a slightly worse performance than anticipated, perhaps in part because of differences in the technology used in the commercial version of the test.36 In the study, the panel achieved sensitivity and specificity of 48% (29%–68%) and 89% (72%–98%), respectively, for indeterminate thyroid nodules.36 A modified version of this test using next-genera154 Cancer Control
tion sequencing is now commercially available as ThyGenX (Interpace Diagnostics, Parsippany, New Jersey), but the performance of this modified assay has not yet been reported.37 The advent of next-generation sequencing has allowed the rapid translation of new data on additional, low-prevalence, somatic alterations into the clinical field. An additional commercially available panel is based on this technology and is marketed as ThyroSeq V2 (University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania). This panel now includes more than 400 point mutations in 14 genes,38 including AKT1, BRAF, CTNNB1, GNAS, HRAS, KRAS, NRAS, PIK3CA, PTEN, RET, TP53, TSHR, TERT, and EIF1AX, along with 42 gene rearrangements and can be performed on as little as 10 ng of DNA.20,39 In the initial publication on this panel, the test included mutations in 13 genes (rather than the 14 genes now in the commercial offering) and 42 gene rearrangements, and it achieved sensitivity and specificity of 90% (80%–99%) and 93% (88%–98%), respectively, among cytological specimens classified as Bethesda category IV.19 Such performance should improve and simplify the interpretation (NPVs and PPVs) of the results among different institutions with distinct pretest ROM. The NPV and PPV achieved in this study were 96% and 83%, respectively, with an overall ROM of 27%.19 Based on the reported sensitivity and specificity, we calculate that the NPV will remain above 95% at any ROM up to 34%, whereas the PPV will remain above 75% at any ROM above 15%. As more mutations are added to this panel, we anticipate an increase in the test sensitivity, but it is possible that the specificity could decrease, thus negatively impacting the predictive values. Consequently, although these early results are promising, they still require external validation and additional information regarding the performance of the test on Bethesda category III. Data on this category were presented at the annual meeting and exposition of the Endocrine Society in early 2015, suggesting that sensitivity and specificity are well preserved in this cytological group.40
Gene Expression Analysis Somatic mutations directly impact the gene expression profile by acting on signaling pathways. For example, BRAF V600E is associated with high MAPK signaling, whereas RAS mutants have lower MAPK signaling. This observation was used by TCGA to develop a score that quantified the extent to which the gene expression profile of each somatic mutation resembled either BRAF V600E–mutant or RAS-mutant PTCs.17 This score is associated with the degree of thyroid differentiation of the tumor, histological grade, American Thyroid Association risk of recurrence stage,4 and the MACIS score.41 RAS-like tumors were better differentiated and April 2015, Vol. 22, No. 2
BRAF V600E–like tumors were less differentiated; in addition, RAS-mutant tumors were homogeneous in terms of gene expression and degree of thyroid differentiation.17 Conversely, BRAF V600E–mutant tumors had a heterogeneous gene expression profile and included at least 4 different groups, a finding that could explain the uncertainty regarding the prognostic and predictive power of this mutation alone.17 In light of these results, the authors suggested that PTCs may be more appropriately classified according to their genetic profiles rather than to their morphological appearance.17 Although these data have the potential to transform histological classification and clinical practice, their utility in routine clinical practice has yet to be established. However, prior studies have already used gene expression profiles (at the transcriptional or post-transcriptional level) to differentiate thyroid nodules with indeterminate cytology; these are discussed below. Transcriptional Level Several tools exploit the analysis of the gene expression profile at the transcriptional level to differentiate benign from malignant lesions among indeterminate thyroid nodules. Although several RNA-based markers have investigated single genes, a commercially available multigene expression panel has achieved promising results. The Afirma Gene Expression Classifier (Veracyte, South San Francisco, California) assesses the relative mRNA expression of 167 genes processed through a support vector machine trained on a group of histological and FNA material derived from a broad spectrum of samples from benign and malignant thyroid disease. Using a proprietary algorithm, the classifier reports the nodule as benign or suspicious. In a prospective, double-blind, multicenter study, 265 indeterminate thyroid nodules were analyzed.42 The overall performance of the test showed sensitivity and specificity of 92% (84%–97%) and 52% (44%–59%), respectively.42 In this study, the test achieved an NPV of approximately 95% for Bethesda categories III and IV. Such an NPV justifies patient observation in lieu of surgery per the recommendations of the National Comprehensive Cancer Network.43 However, an NPV of 95% can be assumed only if the midpoint sensitivity and specificity achieved in this single study are used and the ROM of the cytology is below 24%.11,42 Because the ROM in the indeterminate categories is higher in many institutions,3 its use should be individualized according to the specific ROM of each center. In an independent study, the sensitivity and specificity rates achieved by Afirma were reported to be 83% and 36%, respectively, and were the best rates of the case scenarios.44 If lower sensitivity and specificity than anticipated are confirmed, then both predictive values would be negatively affected. April 2015, Vol. 22, No. 2
Therefore, further validation is needed before the widespread use of this test. As single-gene markers, HMGA2 and cancer-related E2 ubiquitin-conjugating enzyme have also been studied in the cytology specimens of indeterminate thyroid nodules.45,46 However, neither has demonstrated sufficiently high sensitivity or specificity to justify widespread use. Nonetheless, their combined use, along with other markers, continues to be explored.47 The detection of thyrotropin receptor mRNA in peripheral blood achieved better results in a study on thyroid nodules with cytological diagnosis of follicular neoplasm, reaching sensitivity and specificity of 97% and 88%, respectively, for detecting thyroid cancer.48 However, further studies are necessary to validate these results. Post-Transcriptional Level MicroRNAs are small, noncoding RNA molecules that bind to mRNA to promote or silence their translation into proteins.49-51 These molecules can be analyzed in cytological specimens and those affecting oncogenes or tumor suppressor genes are also being studied. Several studies have reported attempts to identify the specific signatures of thyroid cancer by developing predictive models on excised specimens later investigated as single microRNAs or in microRNA panels on FNA samples of thyroid nodules.52-59 One meta-analysis found that panels had better performance rates compared with single microRNAs.59 The overall sensitivity and specificity of 4 studies using microRNA panels in FNA specimens were 87% (77%–92%) and 85% (78%–90%), respectively.59 Performance was best in a study validating a panel of 4 microRNAs (222, 328, 197, and 21) in which sensitivity and specificity of 100% and 86%, respectively, were achieved.55 Larger prospective studies of indeterminate thyroid nodules must be performed to validate their diagnostic utility. Some microRNAs also appear to have prognostic value. In the study by TCGA, 4 different expression patterns stratified the BRAF-like PTCs into groups with various histological grades of differentiation.17 The overexpression of microRNA-21 and microRNA-146b, as well as the down-regulation of microRNA-204 — which acts as a tumor suppressor — was found in the group with the most aggressive PTCs.17 Conversely, higher expressions of microRNA-182 and microRNA-183 were associated with RAS-like PTCs that had a higher degree of thyroid differentiation.17,60
Follicular Neoplasm (Oncocytic Variant) /Hürthle Cell Neoplasm Most of the molecular markers previously discussed are not helpful for Hürthle cell neoplasms. Hürthle Cancer Control 155
cell carcinomas rarely exhibit one of the commonly studied mutations.61 However, the use of next-generation sequencing might improve the sensitivity of previous oncogene panels. The initial version of ThyroSeq detected a mutation in 39% of cases of Hürthle cell carcinoma analyzed.20 Most of them had a TP53 mutation, typically detected in undifferentiated thyroid cancer but not in well-differentiated carcinomas.20 Moreover, Hürthle cell neoplasms have a distinct profile of genes and transcription factors involved in tumorigenesis.62 Possibly because of this, Afirma classifies 90% of the Hürthle cell neoplasms as “suspicious,” even despite the fact that the ROM in these tumors is typically lower than that for the overall Bethesda category IV group, resulting in a low PPV in these nodules.42,43,63 In part, this likely reflects the fact that the training of the support vector machine that encodes the proprietary algorithm included a number of Hürthle cell carcinomas but almost no benign Hürthle cell adenomas; as a consequence, the gene expression classifier determines all Hürthle cell lesions as suspicious. Similarly, microRNA panels may inadequately classify this group of tumors. In a study, the accuracy of the panel rose from 90% to 97% when Hürthle cell neoplasms were excluded.55 Thus, in our opinion, Hürthle cell neoplasms should not be routinely evaluated with the currently available molecular tests, and, if used, the test results should be interpreted with caution.
Conclusions The rapid translation into the clinical field of new discoveries in the molecular basis of thyroid cancer has led to the development of several molecular tests that address the deficiencies of thyroid cytopathology. The initial results of such tests are promising, and the rapid expansion of our knowledge in this regard promises further advances in the near future. Additional studies that independently validate these results and define the role of these tests in routine clinical practice are needed. Until the cytological classification has been further standardized, each institution interested in using these tests should determine its own risk of malignancy for each of the indeterminate categories to select the most appropriate test and to adequately interpret the results. However, in the future, molecular markers will be instrumental in refining the diagnostic accuracy of cytology and to individualize thyroid cancer management and treatment. References 1. Chen AY, Jemal A, Ward EM. Increasing incidence of differentiated thyroid cancer in the United States, 1988-2005. Cancer. 2009;115(16):3801-3807. 2. Bongiovanni M, Spitale A, Faquin WC, et al. The Bethesda System for reporting thyroid cytopathology: a meta-analysis. Acta Cytol. 2012;56(4):333-339. 3. Wang CC, Friedman L, Kennedy GC, et al. A large multicenter cor-
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