SPECIAL SECTION AACE/ACE Disease State Commentary AACE/ACE DISEASE STATE COMMENTARY: MOLECULAR DIAGNOSTIC TESTING OF THYROID NODULES WITH INDETERMINATE CYTOPATHOLOGY Victor Bernet, MD; Kenneth H. Hupart, MD; Sareh Parangi, MD; Kenneth A. Woeber MD, FRCPE for the AACE Thyroid Scientific Committee Summary Highlights • Approximately 10 to 25% of fine-needle aspiration (FNA) biopsies yield an indeterminate result often labeled as atypia of undetermined significance or follicular lesion of undetermined significance (AUS/ FLUS) or follicular neoplasm/suspicious for follicular neoplasm (FN/SFN). The risk of malignancy typically varies between 15 and 30% for these categories. • Although many markers are in development and have been studied in a research setting, 2 principal tests are currently marketed for use to improve the malignancy risk assessment of “indeterminate” thyroid nodules. “Rule In” and “Rule Out” tests attempt to confirm or exclude the presence of cancer within a thyroid nodule

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• Submitted for publication February 13, 2014 Accepted for publication March 24, 2014 Address correspondence to Victor Bernet, MD; Mayo Clinic Florida; Division of Endocrinology, 4500 San Pablo Road; Jacksonville, FL 32224. E-mail: [email protected]. Published as a Rapid Electronic Article in Press at http://www.endocrine practice.org. DOI:10.4158/EP14066.PS To purchase reprints of this article, please visit: www.aace.com/reprints. Copyright © 2014 AACE.

The opinions represented in the AACE/ACE Disease State Commentary: Diagnostic Testing of Thyroid Nodules with Indeterminate Cytopathology are the expressed opinions of the Thyroid Scientific Committee of the American Association of Clinical Endocrinologists. AACE/ACE Disease State Commentaries are systematically developed documents written to assist health care professionals in medical decision making for specific clinical conditions, but are in no way a substitute for a medical professional's independent judgment and should not be considered medical advice. Most of the content herein is based on literature reviews. In areas of uncertainty, professional judgment of the authors was applied. This commentary is a working document that reflects the state of the field at the time of publication. Because rapid changes in this area are expected, periodic revisions are inevitable. We encourage medical professionals to use this information in conjunction with, and not a replacement for, their best clinical judgment. The presented recommendations may not be appropriate in all situations. Any decision by practitioners to apply these guidelines must be made in light of local resources and individual patient circumstances. Copyright © 2014 AACE.

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by means of robust positive (PPV) or negative predictive values (NPV), respectively. The Rule In tests determine the presence of single gene point mutations (BRAFV600E or RAS) or gene rearrangements (RET/PTC, PAX8/PPARγ) that have been shown to increase the ability to predict cancer, while the Rule Out test (Afirma® gene expression classifier, GEC) utilizes a proprietary gene expression classifier (RNA expression) specifically designed to maximize the ability to define a process as benign. Among the presently available tests, only the BRAFV600E and RET/PTC rearrangement are associated with a PPV that approaches 100%. The category of cytologically “indeterminate” nodule (AUS/FLUS, FN/SFN),  cytopathology practice patterns, and the prevalence of malignancy within the population being tested all impact the NPVs and PPVs for the tests in question. At present, molecular testing is meant to complement and not replace clinical judgment, sonographic assessment, and visual cytopathology interpretation. As molecular testing is new and advances in the field are regularly occurring, clinicians need to stay informed, as recommendations for use within practice are expected to evolve. (Endocr Pract. 2014;20: 360-363)

Abbreviations: AUS/FLUS = atypia of undetermined significance or follicular lesion of undetermined significance; FN/ SFN = follicular neoplasm/suspicious for follicular neoplasm; FNA = fine-needle aspiration; GEC = gene expression classifier; NPV = negative predicative value; PPV = positive predictive value INTRODUCTION Thyroid nodules occur in 20 to 75% of adults, the frequency rising with age, with ~5 to 15% proving to be malignant (1). Fine-needle aspiration (FNA) biopsy has


emerged as the most reliable clinical tool for determining whether a nodule is benign or malignant. However, approximately 10 to 25% of FNA biopsies yield an indeterminate result with the risk of malignancy varying from 5 to 75% (typically 15-30%) depending on the indeterminate category according to the Bethesda System for Reporting Thyroid Cytopathology (2). This review focuses on nodules that display atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) and follicular neoplasm/suspicious for follicular neoplasm (FN/SFN), Bethesda categories III and IV, respectively, and not category V, often labeled “suspicious for malignancy.” Recent advances in thyroid and cancer biology have led to the development and marketing of several tests to assist in determining whether a nodule with indeterminate cytology (AUS/FLUS or FN/SFN) is benign or malignant. Our goal is to provide a concise review of the commercially available “molecular tests” for thyroid nodule assessment and to provide the clinician with a practical summary of the power and limitations of these tests in the clinical setting. Available Tests At present, 2 main types of molecular tests are marketed for the assessment of thyroid nodules for the presence of cancer. Veracyte’s Afirma® gene expression classifier (GEC) and mutation analysis panels, such as Asuragen’s miRInform®, Quest Diagnostic’s panel, and other similar panels, utilize different methods to assess the risk of malignancy. A third type of test from the Cleveland Clinic utilizing serum thyroid-stimulating hormone (TSH) receptor mRNA levels is also available commercially but has received less scrutiny in the medical literature. Technical Aspects The Afirma® GEC is based on the gene expression profiles of surgically proven benign and malignant thyroid nodules and evaluates for the presence of a benign gene expression profile. Based on validation studies demonstrating high negative predictive value (NPV) among nodules with cytology of AUS/FLUS or FN/SFN, it has been employed as a “Rule Out” test to identify nodules that are benign. However, because a substantial number of benign nodules do not have a gene expression profile classified as benign, this test cannot always “Rule Out” the presence of malignancy. The mutation analysis panel developed by Nikiforov et al and available from Asuragen miRInform®, Quest Diagnostics, and other facilities assesses for BRAFV600E and RAS point mutations, as well as common rearrangements of RET/PTC and PAX8/PPARγ. It is estimated that 1 of these mutations or rearrangements is present in approximately 60% of well-differentiated thyroid cancers. The mutation analysis panel is considered to represent a “Rule In” test, as nodules harboring these mutations or rearrangements have a high likelihood of cancer given the high

positive predictive value (PPV) of this panel. However, as a significant number (~40%) of malignant nodules with indeterminate cytology do not harbor one of these genetic markers, this panel cannot reliably “Rule Out” cancer. Additionally, RAS mutations may be found in some benign adenomas. Of note, the components of the mutation analysis panel are available as stand-alone assays or in various combinations by other laboratories. The mutation analysis panels and Afirma® GEC test are performed using samples from needle passes collected during FNA biopsy. The TSH receptor mRNA test is a serum assay that utilizes quantitative reverse-transcription polymerase chain reaction (RT-PCR) to detect circulating thyroid cancer cells by measuring thyrotropin receptor mRNA in peripheral blood. A large, prospective, blinded, multicenter study was undertaken to assess the clinical validity of the Afirma® GEC (3). It found that a negative result (i.e., a benign gene expression profile) has a NPV ranging from 94 to 95% for cytologically AUS/FLUS and FN/SFN lesions. Accordingly, because of its high NPV for AUS/FLUS and FN/SFN nodules, the Afirma® GEC has been marketed as a “Rule Out” test with sufficient reliability to defer surgery. It is not recommended for use in nodules with FNA cytology labeled “suspicious for malignancy” given a lower 85% NPV reported in this subgroup. The analytical performance of this test has been validated by Veracyte with respect to sensitivity, specificity, and reproducibility (4). However, as the false-negative rate for indeterminate nodules is 5 to 6%, nodules classified as benign by the GEC should be followed closely with repeat ultrasound because malignancy is not absolutely excluded. Selective FNA re-assessment or consideration for resection should occur for any significant nodule growth or suspicious change in appearance. In the case of the mutation analysis panels, results of validation studies have not been reported or published by the laboratories. However, several large, prospective, multicenter studies have independently validated the utility of testing for markers similar to those used in the miRInform® panel in FNA samples with indeterminate cytology (5-7). These studies found that a positive result carries a PPV of approximately 90% for predicting cancer. However, a literature review of articles in which mutation analysis was undertaken for indeterminate cytology revealed that a negative result was associated with an overall risk of 23% for malignancy (8). Accordingly, this test is considered to be a “Rule In” test. Clinical Use Molecular testing is just 1 of many factors that must be considered in the evaluation of a thyroid nodule. Patient characteristics, physical examination, thyroid ultrasound and FNA biopsy, as well as sound clinical judgment should continue to be applied in a balanced manner. Molecular testing should only be used to complement and not to


replace cytopathologic evaluation or clinical and imaging assessment. Molecular testing may be considered in adult patients with thyroid nodules >1 cm in the presence of a cytological diagnosis of AUS/FLUS or FN/SFN. Clinical validation has not been performed for thyroid nodules 4-5 cm); or in patients with compressive manifestations, a high suspicion of malignancy based on clinical or ultrasonographic features, or a preference for or against surgery. As a “Rule Out” test, the Afirma® GEC has been reported to obviate the need for surgical excision in ~50% of cases (9). Preliminary cost analysis has suggested that such an approach may be cost-effective based on the stated study parameters (10). However, the validity of these findings is tempered by the fact that the statistical estimates used for this cost-effectiveness analysis were based upon preliminary data and not data specifically derived from the validation study. Moreover, categorization of a nodule as “suspicious” by the Afirma® GEC carries a reported cancer risk of only 38%, so clinicians and patients need to understand that this result does not establish a diagnosis of cancer (3). A cost analysis has suggested that use of the mutation analysis panel alone might also be cost effective by reducing the need for complete thyroidectomy in comparison to lobectomy ± isthmusectomy (11). At present, there are no data assessing the cost-effectiveness of the combined use of the Afirma® GEC and mutation analysis tests. The importance of obtaining an adequate sample for both FNA cytology and molecular testing cannot be overemphasized. Collecting samples for cytopathology and molecular testing during the same procedure may help ensure that these tests are performed on cells derived from the same nodule. Alternatively, a second FNA for molecular analysis can be performed at a later date after an “indeterminate” cytology diagnosis is received. One should follow the procedures recommended by the testing laboratory regarding the number of needle passes required and the appropriate processing and packaging of the sample. The Veracyte Corporation markets the Afirma® GEC with a protocol that specifies the diagnostic paradigm that many clinicians must follow. Outside of designated healthcare organizations that have been allowed to utilize their own internal cytology assessment given their high volume and internal expertise, 4 FNA passes are required for each

nodule: 2 for cytological examination to be performed by Thyroid Cytopathology Partners (TCP) and the other 2 for molecular testing, with the Afirma® GEC test being reserved for instances where the cytological diagnosis falls within the indeterminate categories of AUS/FLUS or FN/ SFN as determined by TCP. The miRInform® test is designed to be applied to patients harboring thyroid nodules with “indeterminate” classification of malignant risk by cytologic assessment. Therefore, the specimen for molecular analysis can be collected at the time of initial FNA for cytology and sent for analysis only if the cytology reading is AUS/FLUS or FN/SFN. The miRInform® protocol specifies 1 dedicated FNA pass for molecular analysis. Alternatively, the second FNA for molecular analysis can be performed later if the first FNA yields an “indeterminate” cytologic diagnosis. Presently, no consensus exists for use of mutation testing results as a guide for surgical decision making. However, when the cytologic interpretation falls into the AUS/FLUS or FN/SFN category, the presence of a mutation such as BRAFV600E, which has a PPV approaching 100% for papillary thyroid cancer, might well justify consideration of total thyroidectomy over hemithyroidectomy. Based on available data, it is still to be determined whether mutational results should play any role in determining the extent of lymph node dissection. The need for molecular diagnosis only occurs in the minority of cases in which the cytological diagnosis is “indeterminate.” Therefore, one can consider obtaining the additional sample for molecular diagnosis only in the ~10 to 25% of cases where this is pertinent. However, a repeat FNA procedure is associated with extra time and effort by both the physician and patient plus additional cost and anxiety to the patient. Use of material from separate FNA passes, especially from procedures performed at different times, raises the possibility of specimens not originating from the same lesion examined by the cytopathologist, although the risk of this occurring might be low with ultrasound-guided aspiration. Automatically sending all FNA samples for this type of molecular testing is not recommended because the cytological result will provide actionable diagnostic guidance (benign or malignant) in the majority of cases, and molecular testing is not required. Afirma® GEC testing is not validated for the assessment of benign nodules and should be reserved for cases of AUS/FLUS and FN/SFN. With a NPV of 85%, Afirma® GEC testing is also not recommended for evaluating lesions suspicious for malignancy or nodules with cytology consistent with cancer. In the case of mutation testing, the false-positive rate ranges between 5 and 13%, primarily due to the presence of mutated RAS genes in some follicular adenomas (5). The molecular mutation markers in BRAF and RAS genes, as well as rearrangements in PAX8/PPARγ and RET/PTC are available from several laboratories – both


independent and those affiliated with research laboratories and universities. Quest Diagnostics also offers “thyroid FNA cytomorphology with molecular reflex” testing. In their diagnostic paradigm, nodules with indeterminate cytology are subjected to analysis for a selection of common mutations and rearrangements from the list above and are applied selectively based on the diagnostic cytology category. Limitations The ultimate goal of molecular testing is the accurate assessment of a thyroid nodule as being benign or malignant prior to surgery. At present, molecular testing should not supplant clinical and ultrasound assessment because none of the available molecular tests can achieve this goal. When assessing molecular testing clinical usefulness, it must be appreciated that even the gold standard of thyroid histopathology is limited by variations in interpretation. Cibas et al reported a 9% disagreement between pathologists when categorizing excised thyroid nodules, as benign or malignant under blinded conditions (12). Available markers that utilize a “Rule-In” cancer method report that between 6 and 28% of cases of malignancy lack a mutation of one of the included markers, thereby leading to a false-negative result. Because testing for the BRAFV600E mutation appears to be associated with near 100% specificity, the occurrence of a false-positive result does not appear to be a concern. However, RAS mutations have been reported to be present in both follicular cancer and some benign follicular adenomas, although there is ongoing debate as to whether or not the latter represents a premalignant lesion that should be removed anyway. Also, as the analytic sensitivities for mutation testing offered by various facilities could well vary, analytic validity studies are needed to provide proof of comparability of results with validated panels. The strength of cytology support directly impacts the reliability of molecular testing because it determines the pretest probability of malignancy. This in turn will inversely influence the NPV of both types of molecular testing. At least 1 published study with a 33% cancer rate for indeterminate nodules found a lower NPV (89.6%) for Afirma® GEC then previously reported (13). Accordingly, the potential variance of NPV might well impact physician reliance on test results for determining the need for surgery. Finally, both the physician and patient need to be aware that as molecular testing is in its infancy, continued research and advances may change our understanding of how best to utilize molecular markers in the clinical setting. In addition, this statement has not covered other markers and techniques (galectin-3 immunohistochemical staining, HBME-1, microRNAs, etc.) that hold a potential role in this field (14,15). Prospective multicenter studies

are required to validate all of these tests used either singly or in tandem. DISCLOSURE

The authors have no multiplicity of interest to disclose.

REFERENCES 1. Popoveniuc G, Jonklaas J. Thyroid nodules. Med Clin North Am. 2012;96:329-349. 2. Bongiovanni M, Spitale A, Faquin WC, Baloch ZW. The Bethesda system for reporting thyroid cytopathology: a meta-analysis. Acta Cytol. 2012;56:333-339. 3. Alexander EK, Kennedy GC, Baloch ZW, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med. 2012;367:705-715. 4. Walsh PS, Wilde JI, Tom EY, et al. Analytical performance verification of a molecular diagnostic for cytologyindeterminate thyroid nodules. J Clin Endocrinol Metab. 2012;97:E2297-E2306. 5. Nikiforov YE, Steward DL, Robinson-Smith, et al., Molecular testing for mutations in improving the fineneedle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab. 2009;94:2092-2098. 6. Cantara S, Capezzone M, Marchisotta S, et al. Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules improves the diagnostic accuracy of cytology. J Clin Endocrinol Metab. 2010;95:1365-1369. 7. 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;96:3390-3397. 8. 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: 1085-1091. 9. Duick DS, Klopper JP, Diggans JC, et al. The impact of benign gene expression classifier test results on the endocrinologist-patient decision to operate on patients with thyroid nodules with indeterminate fine-needle aspiration cytopathology. Thyroid. 2012;22:996-1001. 10. Li H, Robinson KA, Anton B, Saldanha IJ, Ladenson PW. Cost-effectiveness of a novel molecular test for cytologically indeterminate thyroid nodules. J Clin Endocrinol Metab. 2011;96:E1719-E1726. 11. Yip L, Farris C, Kabaker AS, et al. Cost impact of molecular testing for indeterminate thyroid nodule fine-needle aspiration biopsies. J Clin Endocrinol Metab. 2012;97: 1905-1912. 12. Cibas ES, Baloch ZW, Fellegara G, et al. A prospective assessment defining the limitations of thyroid nodule pathologic evaluation. Ann Intern Med. 2013;159:325-332. 13. 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;20:364-369. 14. Kim MI, Alexander EK. Diagnostic use of molecular markers in the evaluation of thyroid nodules. Endocr Pract. 2012;18:796-802. 15. Ross DS. Diagnostic approach to and treatment of thyroid nodules. In: Cooper DS, ed. UpToDate. Waltham, MA: UpToDate. Available at: http://www.uptodate.com/ contents/diagnostic-approach-to-and-treatment-of-thyroidnodules. Accessed March 22, 2014.

ACE disease state commentary: molecular diagnostic testing of thyroid nodules with indeterminate cytopathology.

• Approximately 10 to 25% of fine-needle aspiration (FNA) biopsies yield an indeterminate result often labeled as atypia of undetermined significance ...
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