INTRODUCTION. Original Article. Jiyeon Hyeon, MD 1 ; Soomin Ahn, MD 1 ; Jung Hee Shin, MD, PhD 2 ; and Young Lyun Oh, MD, PhD 1

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1 The Prediction of Malignant Risk in the Category Atypia of Undetermined Significance/Follicular Lesion of Undetermined Significance of the Bethesda System for Reporting Thyroid Cytopathology Using Subcategorization and BRAF Mutation Results Jiyeon Hyeon, MD 1 ; Soomin Ahn, MD 1 ; Jung Hee Shin, MD, PhD 2 ; and Young Lyun Oh, MD, PhD 1 BACKGROUND: The atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) category in the Bethesda System for Reporting Thyroid Cytopathology is a heterogeneous category of cases that are not clearly benign or malignant. METHODS: We conducted an analysis of cytologic and histologic evaluations of thyroid nodules that had been interpreted as AUS/FLUS on fine-needle aspiration (FNA) at a single institution from April 2011 to April Those cases were classified into 2 subgroups according to the predominance of nuclear atypia (AUS) or microfollicular architecture (FLUS). In addition, for a number of these cases, BRAF gene mutation analyses were performed. RESULTS: Of 6402 thyroid FNAs performed, 431 cases were diagnosed as AUS and 120 as FLUS. Follow-up cytologic or histologic outcome data were available for 315 AUS cases and 73 FLUS cases. Among AUS cases, 52.7% were malignant on repeat FNA or histologic diagnosis. In contrast, for FLUS, 6.8% were malignant on repeat FNA or histologic diagnosis. Among AUS/FLUS cases, 147 had adequate BRAF mutation analysis, which accompanied the histologic diagnosis. BRAF mutations were found in 87 AUS cases, 86 of which were papillary carcinoma. In contrast, there was only 1 case of BRAF mutation in FLUS. Correlating molecular results with histologic outcome revealed a 98.9% cancer probability for AUS cases with BRAF mutation. CONCLUSIONS: The AUS subcategory indicates a higher risk of malignancy than the FLUS subcategory. Furthermore, BRAF molecular testing is helpful in stratifying the malignant risk of AUS cases into high-risk and low-risk groups. Cancer (Cancer Cytopathol) 2014;122: VC 2014 American Cancer Society. KEY WORDS: fine needle aspiration; Bethesda System for Reporting Thyroid Cytopathology; atypia of undetermined significance; follicular lesion of undetermined significance; BRAF mutation. INTRODUCTION The worldwide incidence of papillary thyroid cancer is rapidly growing, and it is already a common malignancy of the endocrine system. 1-4 When a thyroid nodule is detected on routine examination, patients are advised to undergo fine-needle aspiration (FNA). FNA cytology is the most useful screening test to evaluate thyroid nodules. 5 It is a reliable and cost-effective method for the differential diagnosis of a thyroid nodule and for reducing the unnecessary risk of undergoing surgery for a benign nodule. 6 Accordingly, it is essential that cytopathologists and endocrinologists use a standardized reporting system for thyroid FNAs to communicate findings more accurately and clearly. To satisfy this need, the 2007 National Cancer Institute Thyroid Fine Needle Aspiration State of the Science Conference and the subsequent Bethesda System for Reporting Thyroid Cytopathology Corresponding author: Young Lyun Oh, MD, Department of Pathology, Samsung Medical Center, Sungkyunkwan, University School of Medicine, 50 Irwon-dong, Gangnam-gu, Seoul, , Korea; Fax: (011) ; bijou@skku.edu 1 Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea; 2 Department of Radiology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea Received: November 18, 2013; Revised: December 26, 2013; Accepted: January 1, 2014 Published online March 3, 2014 in Wiley Online Library (wileyonlinelibrary.com) DOI: /cncy.21396, wileyonlinelibrary.com 368 Cancer Cytopathology May 2014

2 AUS/FLUS With BRAF Mutation Results/Hyeon et al (BSRTC) recommend that each thyroid FNA report begin with 6 distinct diagnostic categories. 7 Each category has an implied cancer risk, which ranges from 0% to 3% for the benign category and up to 100% for the malignant category. Linked to each risk category are evidence-based clinical management guidelines. 8 Among the categories in the BSRTC, atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) has been heavily debated. 9 This is a heterogeneous category of cases that are neither clearly benign nor sufficiently atypical for a category IV diagnosis (ie, follicular neoplasm or suspicious for follicular neoplasm) or a category V diagnosis (ie, suspicious for malignancy). 10 AUS/FLUS was intended to be an exclusion category for cases with features that cannot be clearly benign; however, it also includes cases with nonspecific patterns affected by compromising factors such as air-drying artifact, low cellularity, or obscuring elements. 11,12 Therefore, many controversies have arisen regarding the AUS/FLUS category since the adaptation of the BSRTC, and there has been significant variability in how individual cytopathologists as well as institutions use these categories Furthermore, this uncertainty surrounding the risk of malignancy in AUS/FLUS cases leads to confusion regarding patient management. 16 To resolve some of these problems, recent studies of the malignancy risk in AUS/FLUS suggest a 2-tier subclassification of the AUS/FLUS category: 1) low cellularity with predominant microfollicular architecture and absence of colloid and 2) nuclear features not characteristic of benign lesions (nuclear atypia) attributable to strict criteria for the evaluation of cancer probability. 10,17 In this study, we classified AUS/FLUS cases into 2 subgroups on the basis of the predominance of architectural atypia or nuclear atypia and compared malignancy rates in the 2 groups. In addition, we investigated the usefulness of BRAF mutation analysis to predict malignancy risk in AUS/FLUS cases by comparing rates of BRAF mutation in the 2 subgroups. MATERIALS AND METHODS Patients and Specimen Preparation All cases of thyroid aspirates between April 2011 and April 2012 were retrospectively analyzed from the computerized record system at our institution. These cases included both in-house patient and outside-hospital samples. All patients provided written informed consent. In-house cases were obtained from fine-needle aspirations (FNAs), which were performed by skilled interventional radiologists using a 22- or 23-gauge needle attached to a 10-mL syringe under ultrasound (US) guidance. On average, 2-4 passes were performed for each nodule. For patients with multiple nodules, the specimen was obtained from the lesion that was the most suspicious for malignancy on ultrasonographic examination. For all cases, the size, location, composition, and vascularity of the thyroid nodules were evaluated. Nodules that seemed malignant had 1 or more of the following ultrasonographic characteristics: 1) taller than wide shape, 2) spiculated margins, 3) marked hypoechogenicity, and 4) calcification. 18 Aspirates were smeared onto a glass slide, immediately fixed in 95% alcohol for Papanicolaou and hematoxylin and eosin staining. The remaining material was collected for BRAF V600E mutation analysis. Outside cases included in this study were prepared at other institutions by methods similar to that describe above and transferred to our institution for analysis and recommendations on clinical management. Repeat FNA was performed for a proportion of referral cases in our institution. The BRAF mutation test was followed for selective cases. Cytologic Diagnosis and Follow-up In April 2011, our institution adopted a 6-tier diagnostic scheme according to BSRTC recommendations. Based on aspiration cytology, each specimen was classified into 1 of 6 categories: 1) nondiagnostic, 2) benign, 3) AUS/ FLUS, 4) follicular neoplasm or suspicious for a follicular neoplasm, 5) suspicious for malignancy, and 6) malignant. We further subdivided the AUS/FLUS category into 2 subcategories. One subcategory (AUS) includes cases that show nuclear atypia, such as the presence of occasional nuclear pseudoinclusion, grooves, abnormal chromatin pattern, and nuclear overlapping or crowding, but which are not significant enough to be considered suspicious for malignancy (Fig. 1). The other subcategory (FLUS) contains cases with architectural atypia such as the presence of a prominent population of microfollicles or Hurthle cells with scant colloid, but not enough to be diagnosed as a follicular neoplasm or suspicious for a follicular neoplasm (Fig. 2). In a fraction of AUS/FLUS cases, surgical resection was performed, and histologic diagnoses for those cases were correlated with cytologic interpretations and BRAF mutation analysis. Cancer Cytopathology May

3 Figure 1. A case of atypia of undetermined significance with cytologic atypia (AUS). (A) There is mild cytologic atypia with vague microfollicular structures (Papanicolaou stain, 3400). (B,C) The aspirates focally demonstrate nuclear enlargement, grooves, and nuclear membrane thickening with pale chromatin (Papanicolaou stain, 31000). (D) The histology of the surgical specimen reveals a follicular variant papillary thyroid carcinoma (hematoxylin and eosin staining, 340 and 3400 [inset]). Detection of BRAF V600E Mutation BRAF mutations were detected using both allele-specific polymerase chain reaction (AS-PCR) and direct sequencing. The results were considered positive when a mutation was detected by either method. Genomic DNA was extracted using a QIAamp DNA mini kit (Qiagen, Chatsworth, CA) according to the manufacturer s instructions. PCR amplification of exon 15 of BRAF was performed using dual oligonucleotide primers: 5 0 -TTCATGAAGACCTCACAGTAAAAA-3 0 (forward) and 5 0 -CCACAAAATGGATCCAGACA-3 0 (reverse). AS-PCR analysis was performed on a GeneAmp 9700 PCR machine (Applied Biosystems, Foster City, CA) using a Seeplex BRAF ACE detection system (Seegene, Seoul, Korea) designed to detect the V600E variant according to the manufacturer s instructions. Each PCR reaction mixture contained 4 ll of 53 BRAF primer, 3 ll of extracted DNA (10 ng/ll), 3 ll of 8-methoxypsoralen solution, and 10 ll of 23 multiplex master mix (Seegene) for a total volume of 20 ll. After 15 minutes of incubation at 94 C, amplification was performed in a Thermo cycler (Applied Biosystems, Foster City, CA) with 35 cycles of denaturation at 94 Cfor 30 seconds, annealing at 62 C for 30 seconds, extension at 72 C for 60 seconds, and a final extension at 72 Cfor 10 minutes. The amplified PCR products were visualized 370 Cancer Cytopathology May 2014

4 AUS/FLUS With BRAF Mutation Results/Hyeon et al Figure 2. Cytologic and histologic features of follicular lesion of undetermined significance with architectural atypia (FLUS). (A) The aspirates demonstrate moderately cellular clusters with lack of colloid (Papanicolaou stain, 3400). (B,C) Although there is no cytologic atypia, some population of microfollicles is characteristic. However, the overall proportion of microfollicles is not sufficient for the diagnosis of follicular neoplasm/suspicious for follicular neoplasm (Papanicolaou stain, 3400 [B], [C]). (D) This case was confirmed as a follicular adenoma on histologic examination (hematoxylin and eosin staining, 340 and 3400 [inset]). and analyzed on a screentape system (Lab901 Ltd., Edinburgh, Scotland, UK) with Seegene viewer software. The level of internal control/v600e amplicon band intensity was recorded by a mutation ratio >10%. After performing PCR, bidirectional Sanger sequencing was done on an ABI PRISM 3730xl DNA analyzer using a BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems). Sequences were compared with those of normal BRAF exon15 in the GenBank database with Sequencer 4.8 (Gene Codes Corporation, Ann Arbor, MI). Statistical Analysis The sensitivity, specificity, positive predictive value, and negative predictive value of the BRAF mutation as a marker for papillary carcinoma in AUS/FLUS were calculated and compared in both subcategories of AUS/FLUS. Only cases with histologic confirmation were included in the calculation. The chi-square test and Fisher s exact test were used to compare categorical variables. P <.05 was considered statistically significant. Statistical analyses were performed using SPSS software (SPSS Inc., Chicago, IL). RESULTS The general distribution of cytologic diagnoses for 6402 specimens over the 13-month period is presented in Figure 3. Of the 6402 thyroid FNAs, 551 (8.6%) were diagnosed as AUS/FLUS. The overall rate of category III Cancer Cytopathology May

5 Figure 3. General distribution of cytologic diagnoses for 6402 specimens over a 13-month period. of BSRTC was within the recommended range described in the literature. This group included 97 men and 454 women (mean age, 49.7 years; range, years). Of the 551 AUS/FLUS cases, 431 (6.7% of 6402) were diagnosed as AUS, and 120 (1.9% of 6402) were diagnosed as FLUS. Follow-up cytologic or histologic outcomes data were available for 388 cases, which consisted of 315 AUS cases and 73 FLUS cases. Of these 388 cases, repeated FNA without histologic evaluation was performed in 157 cases, and the cytologic diagnoses provided were: nondiagnostic, 1.9%; benign, 59.9%; AUS/FLUS, 21.7%; suspicious for a follicular neoplasm, 2.5%; suspicious for malignancy, 5.1%; and malignancy, 8.9%. Those in the AUS group were diagnosed as nondiagnostic, 2.5% (3 of 122); benign, 57.4% (70 of 122); AUS, 20.5% (25 of 122); FLUS, 0% (0 of 122); suspicious for a follicular neoplasm, 1.6% (2 of 122); suspicious for malignancy, 6.6% (8 of 122); and malignancy, 11.5% (14 of 122). In the FLUS group, they were diagnosed as nondiagnostic, 0% (0/35); benign, 68.6% (24 of 35); AUS, 0% (0 of 35); FLUS, 25.7% (9 of 35); suspicious for a follicular neoplasm, 5.7% (2 of 35); and suspicious for malignancy and malignancy, 0% (0 of 35). To sum up, in the AUS group, 14 of 122 cases were diagnosed as malignant. No cases were diagnosed as malignant or suspicious for malignancy on repeated FNA in the FLUS group (P <.001). Histologic outcomes with or without repeated FNA data were obtained in 231 cases, of which 74 (32%) were benign and 157 (68%) were malignant. In the AUS group, 21.2% (41 of 193) were benign and 78.8% (152 of 193) were malignant based on histologic outcomes. Among the nodules diagnosed as AUS on FNA cytology, histologic confirmation of malignancy revealed the classic variant of papillary carcinoma as the most common final malignant diagnosis (classic variant of papillary thyroid carcinoma [PTC], 138 cases; follicular variant of PTC, 9 cases; oncocytic variant of PTC, 1 case). Furthermore, follicular and medullary carcinomas were the second most 372 Cancer Cytopathology May 2014

6 AUS/FLUS With BRAF Mutation Results/Hyeon et al Table 1. Relationship Between AUS/FLUS Diagnosis at Follow-Up FNA With BRAF Mutation Analysis Diagnosis of Follow-Up FNA FNA diagnosis BRAF Mutation Status Nondiagnostic Benign AUS/FLUS Follicular Neoplasm Suspicious for Malignancy Malignancy Total AUS (n 5 74) Positive 0 (0.0%) 0 (0.0%) 1 (8.3%) 0 (0.0%) 4 (33.3%) 7 (58.3%) 12 (100.0%) Negative 1 (1.6%) 48 (77.4%) 10 (16.1%) 1 (1.6%) 1 (1.6%) 1 (1.6%) 62 (100.0%) FLUS (n 5 16) Positive 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 0 (0.0%) Negative 0 (0.0%) 11 (68.8%) 5 (31.3%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 16 (100.0%) Table 2. Relationship Between AUS/FLUS Diagnosis Specified by histologic outcomes and BRAF Mutation Analysis Histologic Diagnosis FNA Diagnosis BRAF Mutation Status Benign/ Nonneoplastic Papillary Carcinoma Follicular Carcinoma Benign/Neoplastic (Adenoma, etc.) Total P AUS (n 5 135) Negative 12 (25.0%) 33 (68.8%) 1 (2.1%) 2 a (4.2%) 48 (100.0%) <.001 Positive 1 (1.1%) 86 (98.9%) 0 (0.0%) 0 (0.0%) 87 (100.0%) FLUS (n 5 12) Negative 4 (36.4%) 1 (9.1%) 2 (18.2%) 4 b (36.4%) 11 (100.0%) Positive 0 (0.0%) 1 (100.0%) 0 (0.0%) 0 (0.0%) 1 (100.0%) a Hyalinizing trabecular tumor, n 5 1. b Follicular adenoma, n 5 4. P value derived by v 2 and Fisher s exact test. common malignancies (follicular carcinoma, 3 cases; medullary carcinoma, 1 case). Among histologic outcomes of AUS, 4 follicular adenomas and a hyalinizing trabecular tumor were also identified; even though they were not malignant, they were neoplastic disease. The remainder were confirmed as nonneoplastic benign diseases such as nodular hyperplasia or chronic lymphocytic thyroiditis. In contrast, in the FLUS group, 86.8% of cases (33 of 38) were benign and 13.2% (5 of 38) were malignant based on histology. Among those nodules diagnosed as FLUS on FNA cytology, only 3 classic variants of papillary carcinoma and 2 follicular carcinomas were diagnosed on histologic examination. Eight follicular adenomas, a Hurthle cell adenoma, and a parathyroid adenoma were also identified on histologic confirmation. The remainder were confirmed as nonneoplastic benign diseases such as nodular hyperplasia on histologic examination. Compared with the AUS subgroup, malignancy rates of FLUS were significantly low (P <.001). Overall, among AUS cases, 35.2% (111 of 315) were benign and 52.7% (166 of 315) were malignant on repeat FNA or histologic confirmation. In contrast, for FLUS, 78.1% (57 of 73) were benign and 6.8% (5 of 73) were malignant on repeat FNA or histologic confirmation. Among the AUS/FLUS cases, 237 samples had adequate BRAF mutation analyses with or without histologic outcomes. Of the 237 cases, 209 (88.2%) were diagnosed as AUS, and 99 of these 209 cases (47.4%) had BRAF mutations. In contrast, only 1 of 28 FLUS cases (3.6%) had BRAF mutations (P <.001). Of the 237 cases, 90 had follow-up FNA results only. For these cases, the cytologic diagnoses with BRAF mutation analysis are presented in Table 1. BRAF mutations were found in 12 AUS cases, 7 of which were diagnosed as malignancy and 4 as suspicious for malignancy. In contrast, all FLUS cases were negative for BRAF mutation, and there were no malignancies on follow-up FNA. This difference between AUS and FLUS was statistically significant (P <.001). Among 237 cases, 147 had adequate BRAF mutation analysis and were accompanied by histologic diagnosis (Table 2). BRAF mutations were found in 87 AUS cases, 86 of which were papillary carcinoma. In contrast, there was a single case of BRAF mutation in the FLUS group, for which final histology confirmed papillary carcinoma. In correlating molecular results with histologic outcomes, we found that there was a 98.9% risk of cancer for AUS cases with a BRAF mutation, whereas those without BRAF mutation were associated with a 70.8% cancer risk (P <.001; Table 3). In terms of US findings, nodule size ranged from 0.2 to 9.4 cm (mean size, 1.3 cm). The mean diameter of the nodules diagnosed as AUS was mm and Cancer Cytopathology May

7 Table 3. Sensitivity, Specificity, Positive Predictive Value, and Negative Predictive Value of BRAF V600E Mutation in the Prediction of Malignancy significantly smaller than that of the FLUS nodules mm (P <.001). A majority of AUS cases had predominantly ill-defined, spiculated margins on ultrasonography, whereas a majority of FLUS cases had welldefined smooth margins (62.2% vs. 19%, respectively; P <.001). Similarly, initial impressions of nodules on US were significantly different for each group. A majority of AUS cases demonstrated radiographic evidence of malignancy or lesions suspicious for malignancy, whereas FLUS cases did not have these features on US (31.5% vs. 3.4%, respectively; P <.001). DISCUSSION AUS FLUS AUS/FLUS Sensitivity 71.7% 25% 70.2% Specificity 93.3% 100% 95.7% Positive predictive value 98.9% 100% 98.9% Negative predictive value 29.2% 72.7% 37.3% It is important that pathologists inform clinicians of the possible clinical implications of a pathologic diagnosis. After the adoption of BSRTC, it has been crucial that communication between clinicians and cytopathologists becomes more clear and precise. However, lots of controversies were raised on the AUS/FLUS category after adoption of the BSRTC. The category of AUS/FLUS was originally proposed for a group of lesions that contained cells with too great a degree of architectural and/or nuclear atypia to be definitively diagnosed as benign but insufficient atypical features to be classified as suspicious for a follicular neoplasm, suspicious for a malignancy, or malignant. The frequency of AUS/FLUS interpretation should be in the range of approximately 7% of all thyroid FNA interpretations, and it is emphasized that AUS/FLUS is a final report and should not be used indiscriminately. 19 It is a heterogeneous category that includes a variety of abnormal features but is assumed to have low risk for malignancy. Furthermore, it also includes specimens with borderline cellularity/compromised quality. Therefore, there have been differences in its utilization, with significant variability in rates of AUS/FLUS among institutions. Moreover, rates of malignancy among this category have varied widely, ranging from 6% to 48% among selected subsets of the patients undergoing surgery. 20 Given the heterogeneity of AUS/FLUS cases, it is expected that different patterns of AUS/FLUS may carry different risks of malignancy. 9 Studies on this subject are ongoing. Some studies have shown that the presence of cytologic atypia resembling nuclear characteristics of papillary carcinoma confers a higher risk of malignancy than other patterns. On the other hand, architectural atypia alone is about half as likely to be malignant as cytologic atypia or both cytologic and architectural atypia and is more likely to signify a follicular adenoma. 21 Olson et al suggested that the subcategorization of AUS/FLUS is necessary to more accurately estimate the malignancy risk of a lesion. The subcategory of nuclear atypia within AUS/ FLUS indicates a higher malignancy risk than other subcategories of AUS/FLUS, but has a lower malignancy risk than the suspicious for papillary thyroid carcinoma category does. 9 Horne et al suggested a similar concept regarding the subcategorization for AUS/FLUS. They reported that among AUS/FLUS cases, low cellularity with predominant microfollicular pattern in the absence of colloid does not carry the same implication as the nuclear features not diagnostic of benign lesions subcategory. The latter clearly has a higher malignancy risk and requires more aggressive follow-up than the other subcategory. 10 Kelman et al also reported similar findings. They noticed that up to 60% of indeterminate thyroid nodules showing nuclear atypia on cytologic examination went on to develop malignancy, whereas only 7% of nodules without nuclear atypia were malignant. 22 Other studies also suggest that further subcategorization of AUS/FLUS cases based on nuclear atypia may provide an added predictive value regarding malignancy risk. 23 At our institution, we also routinely classified AUS/ FLUS into 2 subcategories as mentioned above. One subcategory (AUS) included cases with nuclear atypia including the presence of occasional nuclear pseudoinclusion, grooves, an abnormal chromatin pattern, and nuclear overlapping or crowding, but with features not significant enough to be considered suspicious for malignancy. The other subcategory (FLUS) contained cases that showed architectural atypia, such as the presence of a prominent population of microfollicles or Hurthle cells with scant colloid, not significant enough to be considered a follicular neoplasm. We assumed that the AUS cases had higher malignancy potential than the FLUS cases. Because the incidence of PTC is higher than that of follicular neoplasm or other thyroid malignancy, the presence of 374 Cancer Cytopathology May 2014

8 AUS/FLUS With BRAF Mutation Results/Hyeon et al nuclear atypia, which is crucial for diagnosis of PTC, is considered the most reliable cytologic abnormality in predicting malignancy. Follow-up cytologic and histologic results for these 2 subgroups confirmed our expectations. Thyroid molecular analysis has also risen as a powerful method for predicting malignancy risk, because 60% to 70% of thyroid cancers harbor at least 1 known genetic mutation. 24,25 A proportion of thyroid papillary carcinomas may carry point mutations of the BRAF gene that are able to activate the mitogen-activated protein kinase (MAPK) pathway Adeniran et al subclassified AUS/ FLUS cases into 2 categories: 1) cases with borderline/low cellularity and a microfollicular pattern eliciting concern for follicular neoplasm and 2) cases with nuclear atypia. The latter referred to the presence of occasional nuclear grooves, nuclear enlargement, mild nuclear overlapping and crowding, and abnormal chromatin pattern (ie, anomalous findings eliciting concern for a papillary carcinoma). They examined AUS/FLUS cases with BRAF mutation analysis and surgical pathology outcomes. 31 According to their results, the sensitivity and specificity of the BRAF mutation in detecting PTC in FNA specimens with indeterminate diagnosis were 59.3% and 100%, respectively. These findings were quite similar to our results, which showed a sensitivity of 71.6% and a specificity of 92.3% for the BRAF mutation in detecting PTC in FNA specimens in the AUS subgroup. Many other studies have also suggested that preoperative BRAF mutation testing can significantly help to increase the sensitivity, accuracy, and negative predictive value for detecting papillary thyroid carcinomas compared with using cytology alone. 30,32-35 Our results also support the use of BRAF mutation analysis to predict malignancy risk in patients with indeterminate FNA diagnoses, especially those diagnosed as AUS on the basis of nuclear atypia. Thus, BRAF mutation testing is a helpful tool to stratify cases of category III BRSTC, thus allowing better clinical management and surgical treatment. 36,37 The radiologic findings in AUS more closely resemble characteristics of a malignant nodule compared with FLUS. Moon et al reported US criteria that distinguish malignant thyroid nodules, including taller than wide shape, spiculated margins, marked hypoechogenicity, and micro- or macrocalcifications. 18 In our study, a majority of AUS cases had spiculated margins with taller than wide shape; the radiologists commented that these specimens had features more consistent with a malignant or indeterminate nodule rather than a benign nodule. In contrast, a majority of FLUS cases were more similar to benign nodules on US examination. Furthermore, a majority of AUS cases were smaller than FLUS cases on US examination. The results of this study demonstrated an estimate of AUS/FLUS diagnoses for our institutional practice. Thus, there is the possibility for bias given that a proportion of AUS/FLUS cases were derived from patients coming to our institution for a second opinion and consultation for proper management. Although follow-up for these 2 patient groups did not differ significantly, there is still the possibility that confounding factors, such as the quality of smear slides or ultrasonographic findings, were responsible for bias in our results. However, our study included a large number of cases and was the first report to follow up BSRTC results in Korea. With increased recognition that a proportion of cases with predominant nuclear atypia in category III specimens have a higher risk of malignancy, there is an increasing need for original research and clinical recommendations within TBSRTC. The risk of malignancy for AUS is so high (52.7% in either the repeated FNA group or the histologically confirmed group, 78.8% in the histologically confirmed group) that this diagnostic category may need to be reconsidered and managed differently from FLUS. Conclusions The AUS subcategory indicates a higher risk of malignancy than the FLUS subcategory. Therefore, it is important to report the subcategorized group of AUS/FLUS given its potential clinical implications. Furthermore, BRAF molecular testing is helpful in predicting malignancy risk in AUS cases and can stratify cases into highrisk and low-risk categories. When it is difficult to decide the malignant potential of a thyroid nodule on FNA examination with BRAF mutation analysis, ultrasonographic findings are also worth considering. FUNDING SOURCES No specific funding was disclosed. CONFLICT OF INTEREST DISCLOSURES The authors made no disclosures. REFERENCES 1. Davies L, Welch HG. Increasing incidence of thyroid cancer in the United States, JAMA. 2006;295: Cancer Cytopathology May

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