Utility of BRAF mutation detection in fine-needle aspiration biopsy samples read as suspicious for papillary thyroid carcinoma

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1 ORIGINAL ARTICLE Utility of BRAF mutation detection in fine-needle aspiration biopsy samples read as suspicious for papillary thyroid carcinoma Sebastian M. Jara, MD, 1 Ramneesh Bhatnagar, MD, 2,4 Hui Guan, MD, PhD, 2,5 Christopher D. Gocke, MD, 3 Syed Z. Ali, MD, 2 Ralph P. Tufano, MD, MBA 1 * 1 Division of Head and Neck Endocrine Surgery, Department of Otolaryngology Head and Neck Surgery, Johns Hopkins University, School of Medicine, Baltimore, Maryland, 2 Division of Cytopathology, Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, 3 Division of Molecular Pathology, Department of Pathology, Johns Hopkins University, School of Medicine, Baltimore, Maryland, 4 Department of Pathology, University of Maryland, School of Medicine, Baltimore, Maryland, 5 Department of Pathology, Wayne State University, School of Medicine, Detroit, Michigan. Accepted 29 June 2014 Published online 25 September 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI /hed ABSTRACT: Background. The purpose of this study was to evaluate the diagnostic utility of BRAF mutation testing on thyroid nodules suspicious for papillary thyroid carcinoma (PTC) cytology. Methods. A chart review of patients with fine-needle aspiration (FNA) results suspicious for PTC with subsequent thyroidectomy was performed. Corresponding archived FNA slides underwent BRAF mutation testing. Results. Sixty-six patients with FNA suspicious for PTC underwent thyroidectomy. Forty-two (63.6%) had PTC diagnosed on final histopathology, whereas 21 (31.8%) had benign findings. Thirty-five patients (83%) with histologically proven PTC underwent total thyroidectomy, whereas 7 (17%) underwent hemithyroidectomy. BRAF mutation was detected in 17 of 49 samples (34.6%) available for testing and had 45.5% sensitivity, 87.5% specificity, 88.2% positive predictive value (PPV), and 43.8% negative predictive value (NPV) for diagnosing PTC. Two of 4 patients (50%) who underwent hemithyroidectomy with subsequent completion thyroidectomy had mutated BRAF detected. Conclusion. BRAF testing is a useful adjunct to improve PPV for patients with suspicious for PTC cytology. VC 2014 Wiley Periodicals, Inc. Head Neck 37: , 2015 KEY WORDS: BRAF mutation, fine-needle aspiration biopsy, papillary thyroid carcinoma, suspicious for papillary carcinoma, molecular marker testing *Corresponding author: R. P. Tufano, Division of Head and Neck Endocrine Surgery, Department of Otolaryngology Head and Neck Surgery, Johns Hopkins Medical Institute, 601 N. Caroline St., Rm. 6242, Baltimore, MD rtufano@jhmi.edu INTRODUCTION Fine-needle aspiration (FNA) biopsy has been shown to be a cost-effective and efficacious diagnostic test for the detection of malignancy in thyroid nodules. However, an estimated 20% to 30% of FNA samples have equivocal findings and continue to pose a therapeutic dilemma. 1 5 Indeed, FNA samples read as atypia of undetermined significance/follicular lesion of undetermined significance, follicular or oncocytic (H urthle cell) neoplasm/ suspicious for follicular or oncocytic (H urthle cell) neoplasm, and suspicious for malignancy by the Bethesda system have been shown to have 5% to 15%, 15% to 30%, and 60% to 75% risk of malignancy, respectively. 6,7 In particular, FNA findings suspicious for papillary thyroid carcinoma (PTC) have been shown to have a 60% to 75% risk of malignancy and thus warrant surgical intervention to establish a histopathological diagnosis. 8 Most patients undergo total thyroidectomy, with the hindsight of needless surgical risk and financial expenditures in the patients ultimately found to have a benign diagnosis who require a lifetime of thyroid hormone replacement. Conversely, for patients with malignancy confirmed by diagnostic lobectomy, it is common to recommend completion thyroidectomy. This results in additional surgical risks and financial expenditures that could have been prevented by up-front total thyroidectomy if the diagnosis could have been predicted more accurately. Thus, adjunctive preoperative tests that can better predict PTC may be helpful for guiding surgical management in these patients. Numerous studies have examined the role of molecular marker testing as an adjunct to FNA in patients with indeterminate cytology It is estimated that over 80% of PTCs exhibit a genetic alteration, with the BRAF V600E mutation being the most common genetic alteration identified BRAF mutation is estimated to be present in approximately 50% to 80% of PTCs and has been shown to be highly specific for PTC compared to other thyroid malignancies Furthermore, very few cases demonstrating the BRAF mutation in benign thyroid tissue have been identified. 27,28 Additionally, prognostic information gleaned from the presence of BRAF may be useful preoperatively and impact the decision for extent of surgery It has been shown that BRAF testing is feasible, easy, and inexpensive to assay from thyroid FNAs. 27 A considerable number of studies have examined the role of BRAF testing, as well as that of molecular marker panel testing, in patients with indeterminate FNA cytology However, data on its efficacy specifically for FNA 1788 HEAD & NECK DOI /HED DECEMBER 2015

2 BRAF MUTATION AND FINE NEEDLE ASPIRATION FOR SUSPICIOUS BIOPSY SAMPLES cytology suspicious for PTC are limited, with many studies providing a secondary analysis of a limited number of samples within a much larger cohort of patients with indeterminate findings. In this retrospective study, we report the prevalence of the BRAF mutation among FNA read as suspicious for PTC, correlate BRAF results with the likelihood of the presence of malignancy on final histopathology, and investigate the feasibility of testing suspicious for PTC FNA samples for BRAF. Using Medicare reimbursement data, we show that BRAF testing is cost-effective and may impact surgical decision-making for patients with FNA cytology suspicious for PTC. MATERIALS AND METHODS Patient selection and chart review Institutional review board (IRB) approval was obtained through both the Johns Hopkins Medicine IRB and the University of Maryland Medical Center IRB. Patients were initially identified by a query of the cytopathology database. All patients from both institutions who underwent FNA for a thyroid nodule with cytology findings suspicious for PTC between January 2001 and July 2011 were identified. Patients who then underwent subsequent thyroidectomy at the same institution as the FNA were selected for this study. We excluded all patients with thyroidectomy performed at an outside hospital. We also excluded all patients identified as consult cases that underwent FNA at an outside hospital without archived FNA samples physically available for testing. Cytopathology reports were reviewed for the site, size, and cytopathologic diagnosis of each nodule biopsied. Patients were excluded if any additional nodule with a cytopathologic diagnosis of PTC was identified on the report. Surgical pathology reports were reviewed for final histopathologic diagnosis and for papillary carcinoma subtype, site, size, focality, number of lymph nodes excised, number of lymph nodes positive for cancer, pathologic TNM staging according to the American Joint Committee on Cancer staging system of 2010, positive surgical margins, and lymphovascular invasion if the final histopathologic diagnosis showed malignancy. Patients were only included if the nodule biopsied by FNA could be directly correlated (by site and size) to the final histopathologic diagnosis on the surgical pathology report. Patients with cytopathologic and histopathologic findings that could not be correlated were excluded from this study. Operative reports were reviewed to determine the type and extent of thyroidectomy performed. Patient charts were reviewed for demographic and clinical data. Fine-needle aspiration samples diagnostic terminology and interpretation Slides of archived FNA samples from the patients selected for our study were identified. The selected slides were cytology smears that were either air-dried slides stained with the Diff-Quik method or were alcohol-fixed slides stained with Papanicolaou method. One representative slide was selected from each case. The selected cytology slides underwent repeat review by a cytopathologist at our institution to identify cells on the slide consistent with suspicious for PTC cytology (ie, cytomorphologic features suggestive, but not diagnostic of, PTC quantitatively or qualitatively). These cytomorphologic features included oval-shaped nuclear enlargement, pale finely granular chromatin, nuclear grooves, membrane thickening and irregularity, or nuclear pseudoinclusions. 34 A slide was considered to have adequate cellularity for BRAF mutation testing if it contained 100 atypical cells making up >10% of all cells in the selected area of the slide. Slides of archived FNA samples that did not meet these criteria could not be subjected to BRAF mutation testing and were excluded. Selected areas of each slide were marked with a diamond pen on the back of the glass slide to identify the suspicious cells to target for BRAF mutation testing. BRAF mutational analysis Microscopically identified and marked areas enriched for suspicious cells were removed from the slide using the Pinpoint DNA Isolation System (Zymo Research) in accord with the manufacturer s directions. 35 In brief, a thin layer of Pinpoint solution (Zymo #D3001-1) was painted on the targeted areas and dried for 30 to 40 minutes. The dried solution and cells were scraped off the slide and extracted with a Proteinase K solution for 8 hours. DNA was recovered using the BioRobot EZ1 and the DNA Blood Kit (Qiagen) in accord with the manufacturer s directions. The DNA was amplified by polymerase chain reaction using primers (5 0 -GAAGACCTCACAGTAAAAATAG-3 0 (forward) and 5 0 -biotin-atagcctcaattcttaccatcc-3 0 (reverse)) and HotStar Taq (Qiagen) with conditions of initial denaturation 95 C for 15 minutes and 42 cycles of 95 C for 20 seconds, 53 C for 30 seconds, and 72 Cfor 20 seconds with a final extension of 72 Cfor5minutes. Pyrosequencing was performed with the PyroMark Q24 KRAS v2.0 Kit (Qiagen) and sequencing primer 5 0 AGGTGATTTTGGTCTAGCTACAG-3 0 in accord with the manufacturer s directions. Cost and statistical analysis Medicare part A and part B data from 2011 were collected to determine the net costs for hemithyroidectomy, total thyroidectomy, and completion thyroidectomy at our institution. Data on the commercial cost of BRAF mutation testing and Quest Diagnostics Thyroid Cancer Mutation Panel (for BRAF V600E, RAS, RET/PTC, and PAX8/ PPARg) were obtained from Quest Diagnostics. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated using the BRAF test results for the histopathologic outcome for the nodule that had been targeted by FNA. For calculations, a true-positive was taken as a case with a BRAF mutation on the cytology specimen and PTC on final histology; true-negative as negative for BRAF mutation (wild-type) on cytology and non-ptc (either nonneoplastic, a benign neoplasm, or other malignancy) on final histology; false-positive as BRAF mutation on cytology and non-ptc histology; and false-negative as negative for BRAF on cytology and PTC on histology. RESULTS Clinical characteristics of the study population are summarized in Table 1. Overall, 66 patients with FNA HEAD & NECK DOI /HED DECEMBER

3 JARA ET AL. TABLE 1. (n 5 66). Demographic and clinical characteristics for all patients TABLE 2. Histopathologic characteristics of patients with papillary thyroid carcinoma on final histopathology (n 5 42). Characteristic Patients (%) Sex Female 50 (75.8) Male 16 (24.2) Median age at diagnosis, y 49.5 (10 73)* <45 21 (31.8) (68.2) Race/ethnicity White 46 (69.7) African American 13 (19.7) Asian/Pacific Islander 4 (6.1) Hispanic 1 (1.5) Other 2 (3.0) Type of thyroidectomy Total thyroidectomy 53 (80.3) Hemithyroidectomy 13 (19.7) Final surgical pathology Papillary carcinoma 42 (63.6) Follicular carcinoma 2 (3.0) Mucoepidermoid carcinoma 1 (1.5) Benign diagnosis 21 (31.8) BRAF testing BRAF1 17 (25.8) BRAF- 32 (48.4) Not tested 17 (25.8) * Median (range) for age. Characteristic Patients with PTC (%) Mean tumor size, cm 2.0 Histopathologic subtype Conventional 23 (54.8) Follicular variant 17 (40.4) Tall cell variant 1 (2.4) Trabecular variant 1 (2.4) pt classification pt1a 7 (16.7) pt1b 18 (42.9) pt2 10 (23.8) pt3 3 (7.1) Missing 4 (9.5) pn classification pn0 15 (35.7) pn1a 5 (11.9) pnx 17 (40.4) Missing 5 (11.9) Focality Unifocal 21 (50.0) Multifocal 14 (33.3) Missing 7 (16.7) Margins involved No 39 (92.8) Yes 1 (2.4) Missing 2 (4.8) Lymphovascular invasion Absent 31 (73.8) Present 7 (16.7) Indeterminate 1 (2.4) Missing 3 (7.1) Abbreviation: PTC, papillary thyroid carcinoma. findings for 66 nodules suspicious for PTC who subsequently underwent thyroidectomy were identified. Of these, 50 of 66 were women (76%) and 16 were men (14%). The median age at diagnosis was 49.5 years (range, years). White people comprised the majority of the cohort, who accounted for 46 patients (70%). Fifty-three patients (80%) underwent up-front total thyroidectomy, whereas 13 patients (20%) underwent initial hemithyroidectomy. Of the 66 patients identified with cytology suspicious for PTC, only 42 (64%) were found to have PTC on final surgical pathology (Table 1). Twenty-one patients (32%) were found to have a benign diagnosis, whereas 2 patients (3%) had follicular thyroid carcinoma and 1 patient (1.5%) had mucoepidermoid carcinoma. Of the patients with PTC on final surgical pathology, 23 (55%) had conventional PTC, 17 (40%) had follicular variant PTC, and 1 patient (2.4%) each had tall cell variant and trabecular variant PTC, respectively (Table 2). The final surgical pathologic findings of the 21 patients with benign diagnoses are listed in Table 3. Forty-nine of the 66 patients (74%) had sufficient cytopathologic samples to undergo BRAF testing. Of these, 17 (35%) tested positive for the BRAF V600E mutation, whereas 32 (65%) tested negative for the mutation (Table 1). Data illustrating BRAF mutation status stratified by final surgical pathologic diagnosis and type of thyroidectomy performed are shown in Table 4. The sensitivity, specificity, PPV, NPV, and accuracy of BRAF mutation testing for PTC were 45.5%, 87.5%, 88.2%, 43.8%, and 59.2%, respectively. Of note, 2 of the patients (10%) with a benign diagnosis on final surgical pathology tested positive for BRAF. The benign diagnoses with positive BRAF test results included chronic lymphocytic thyroiditis and multinodular hyperplasia. Four of the 33 patients (12%) who underwent BRAF testing and had PTC on final surgical pathology underwent an initial hemithyroidectomy and required subsequent completion thyroidectomy. Of those 4 patients, 2 (50%) tested positive for the BRAF mutation. DISCUSSION We present one of the largest retrospective studies evaluating the prevalence of the BRAF mutation in patients with cytology suspicious for PTC. Our results show that BRAF mutation positivity is highly associated with PTC on final surgical pathology. Thus, BRAF mutation TABLE 3. Patients with benign histopathology (n 5 21). Benign diagnoses No. of patients (%) Multinodular hyperplasia 7 (33.3) Adenomatoid nodule 5 (23.8) Follicular adenoma 5 (23.8) Chronic lymphocytic thyroiditis 2 (9.5) H urthle cell adenoma 1 (4.8) Hyalinizing trabecular adenoma 1 (4.8) 1790 HEAD & NECK DOI /HED DECEMBER 2015

4 BRAF MUTATION AND FINE NEEDLE ASPIRATION FOR SUSPICIOUS BIOPSY SAMPLES TABLE 4. BRAF mutation status of patients by final histopathology and surgery type. BRAF status Papillary carcinoma Benign diagnosis FTC/ mucoepidermoid Total BRAF Total thyroidectomy Hemithyroidectomy BRAF Total thyroidectomy Hemithyroidectomy Not tested Total thyroidectomy Hemithyroidectomy Total Abbreviation: FTC, follicular thyroid carcinoma. detection may help guide cost-effective surgical decisionmaking in these patients. The overall prevalence (Table 4) of BRAF mutation among suspicious for PTC FNA samples was 35% (17 of 49), whereas the prevalence among those with PTC on final histopathology was 45% (15 of 33). The prevalence was highest among tall cell variant PTC (1 of 1) and lowest among follicular variant PTC (4 of 13), with no mutations identified in the sole trabecular cell variant PTC, follicular carcinoma, or mucoepidermoid carcinoma. This is consistent with previous studies showing follicular variant PTC is more often found to have mutations in RAS than in BRAF The high specificity and PPV, with lower sensitivity and NPV, is also consistent with previous studies examining BRAF testing in cytology suspicious for malignancy We report 2 cases in which BRAF mutation was positively identified from the FNA sample but correlated to a benign final histopathologic diagnosis. Very few previous studies have reported cases of BRAF mutation detection in benign thyroid tissue. Reported false-positive cases have occurred through the use of highly sensitive BRAF detection methods (such as dual-priming oligonucleotidebased multiplex polymerase chain reaction) and in the setting of patients with chronic lymphocytic thyroiditis (CLT). 27,28 One of our 2 false-positive cases occurred in a patient with CLT on final histopathology. This provides evidence that BRAF testing may be confounded in the evaluation of a suspicious thyroid nodule in a patient with CLT. The other case occurred in a patient with multinodular goiter on final histopathology. To date, there are no reported cases showing that benign pathology, aside from CLT, can produce a false-positive BRAF mutation. In addition to the risk of false positivity, these cases also highlight the importance of the clinical circumstances under which molecular testing is considered. For example, a patient with CLT may already require thyroid hormone replacement or may be counseled on the risk of cooccurrence of PTC with CLT, leading them to a lower threshold to consider undergoing a total thyroidectomy, independent of molecular testing. The patient with a multinodular goiter was 63 years old and found to have an FNA with suspicious for PTC cytology upon serial assessment of her thyroid nodules in the setting of a history of multiple, large (>1 cm), bilateral thyroid nodules. Because of her age and the presence of multiple bilateral thyroid nodules, she elected to undergo total thyroidectomy. In contrast, a young woman with a single thyroid nodule who is otherwise euthyroid and asymptomatic may be more reluctant to undergo a total thyroidectomy without a high level of confidence that malignancy is present. Hence, BRAF mutation testing may have the greatest utility in young, euthyroid patients with a single suspicious for PTC thyroid nodule looking for further evidence to justify initial total thyroidectomy and may be best reserved for the clinical circumstances in which the decision to undergo diagnostic lobectomy versus initial total thyroidectomy is equivocal. We report a 64% accuracy of suspicious for PTC FNAs for diagnosing PTC on final surgical pathology. This fits within the range of widely cited concordance between suspicious for malignancy cytology and malignancy on final histopathology at most academic centers, which generally ranges from 60% to 75% but has been reported as low as 54% and as high as 94%. 40 In contrast, the concordance between cytology and final histopathology in community hospitals has been cited by 2 studies at 56% and 57%, respectively. 41,42 Molecular marker testing may have a higher utility in helping to guide extent of thyroidectomy in community hospitals with lower thyroid cytopathology volume. The variability observed across these studies also reinforces the idea that cytologic diagnosis from a thyroid FNA sample is inherently subjective. Further prospective studies are needed to assess the role of adjunct molecular marker testing in patients with FNAs read as suspicious for PTC. Although the majority of the cohort underwent up-front total thyroidectomy, 20% (13/66) underwent initial diagnostic hemithyroidectomy with 54% (7 of 13) of those undergoing completion thyroidectomy after identification of PTC on final histopathology. Table 5 illustrates the overall cost of several thyroid surgeries. A hemithyroidectomy followed by completion thyroidectomy costs over $6000 more than a total thyroidectomy. The 4 patients in our study who underwent initial hemithyroidectomy and required completion thyroidectomy for PTC incurred more than an additional $24,000 in costs than if they had received an up-front total thyroidectomy. Two of these 4 patients were positively identified to have the BRAF mutation. If BRAF mutation status had been known preoperatively in these patients, the decision to undergo up-front total thyroidectomy may have resulted in $12,280 in cost savings, not including savings attributable from the reduction of recovery time from surgery and time away from work. The commercial cost of BRAF mutation testing is about $670 per sample (Quest Diagnostics), and we were able to test samples for $100 per sample. Thus, the cost incurred to test our entire cohort for BRAF at our TABLE 5. Procedure 2011 Medicare cost data for thyroid surgeries. Cost Hemithyroidectomy $ Completion thyroidectomy $ Hemithyroidectomy 1 completion $14, Total thyroidectomy $ HEAD & NECK DOI /HED DECEMBER

5 JARA ET AL. FIGURE 1. Proposed clinical algorithm for management of patients with fine-needle aspiration (FNA) cytology suspicious for papillary thyroid carcinoma (PTC) applying the results of BRAF mutation testing. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com] institution would have been $6600, still resulting in a net savings of $5680. This demonstrates that BRAF mutation testing may be cost-effective in patients with FNA cytology suspicious for PTC deliberating the extent of initial thyroidectomy. The commercial cost of the Quest Diagnostics Thyroid Cancer Mutation Panel (BRAF V600E, RAS, RET/PTC, and PAX8/PPARg) is $ per sample. This panel has been shown to provide a high sensitivity, specificity, PPV, and NPV for thyroid nodules with indeterminate cytology. 20 Data from the Nikiforov et al 20 large prospective study demonstrated a PPV of 95% after panel testing a prospective series of 52 FNA samples with cytology suspicious for malignancy, resulting in an improvement from a PPV of 54% predicted by cytology alone. An analysis of their data shows that BRAF testing was a large driver for the high reported PPV, with the positive RAS mutation test providing a false-positive result in a follicular adenoma. In our study, BRAF mutation testing demonstrated a PPV of 88%. This is an increase from 64% predicted by the suspicious for PTC cytology finding. Although a more modest increase compared to the 4- molecular marker panel test, the increase in PPV from BRAF testing alone is sufficient to strongly consider a total thyroidectomy after a positive BRAF test in a suspicious for PTC FNA sample. Because BRAF testing alone provides a comparable PPV at a lower price, it may be cost-effective to offer BRAF testing as a first test for patients with suspicious cytology, followed by testing for the remaining 3 molecular markers in the subset of patients not identified to have mutations in BRAF (see Figure 1). There were several limitations to this study. Although this is one of the largest studies assessing patients with FNA cytology suspicious for PTC, our study was limited to only 66 matched samples and records. In addition, not all patients identified through the cytology database as suspicious for PTC had an archived FNA sample available for testing or sufficient identifiable suspicious cells to undergo BRAF mutation testing. Another limitation was the method used to test samples for BRAF. Specifically, suspicious cells were microscopically identified and removed from archived slides. In contrast, BRAF testing in clinical practice would involve DNA extraction and amplification directly from the aspirate collected by FNA. The testing method used in clinical practice would allow more suspicious cells to be tested while reducing the signal-to-noise ratio. We hypothesize that testing performed in clinical practice might yield even more accurate results than those reported here. Last, the retrospective design of this study imposed inherent limitations on the content and availability of clinical records. In summary, although numerous studies have explored the use of molecular marker testing in patients with indeterminate cytology, few have also explored its use in patients with cytology suspicious for malignancy Our large series of BRAF mutation testing in a cohort of patients with cytology suspicious for PTC only suggest that BRAF mutation testing is an easy, helpful, and cost-effective adjunct to FNA that may help guide the need for up-front total thyroidectomy before testing for additional molecular markers. The results may be most helpful in community hospitals with a low volume of thyroid cytopathology review. Future prospective studies are needed to validate the effectiveness, utility, and cost-effectiveness of BRAF mutation testing in this patient population. REFERENCES 1. Castro MR, Gharib H. Thyroid fine-needle aspiration biopsy: progress, practice, and pitfalls. Endocr Prac 2003;9: Blansfield JA, Sack MJ, Kukora JS. Recent experience with preoperative fine-needle aspiration biopsy of thyroid nodules in a community hospital. Arch Surg 2002;137: Renshaw AA. Accuracy of thyroid fine-needle aspiration using receiver operator characteristic curves. Am J Clin Pathol 2001;116: Ravetto C, Colombo L, Dottorini ME. Usefulness of fine-needle aspiration in the diagnosis of thyroid carcinoma: a retrospective study in 37,895 patients. Cancer 2000;90: HEAD & NECK DOI /HED DECEMBER 2015

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