Detection of BRAF c.1799t >A (p.v600e) Mutation Using Residual Routine Fine-Needle Aspiration Specimens of Papillary Thyroid Carcinoma Huan Zhao, 1 Zhi-hui Zhang, 1 Bin Zhou, 1 Ting Xiao, 2 Qin-jing Pan, 1 and Hui-qin Guo 1 * Background: mutation is the most common genetic alteration in papillary thyroid cancer (PTC) and has been used as a diagnostic and prognostic marker in PTC. The aim of this study was to investigate the utility of preoperative mutation analysis as an adjunctive diagnostic and prognostic tool to routine fine-needle aspiration (FNA). Methods: Specimens were collected from thyroid nodules by FNA. Cytology diagnosis and testing were performed on these specimens. Molecular and cytological results were correlated with histology outcomes. Results: A total of 195 patients with thyroid nodules were enrolled, including 25 benign lesions and 170 PTCs. BRAF p.v600e testing was successfully performed in all specimens. The combination of testing and cytology improved the sensitivity of cytology from 70% to 85.3% (P 5 0.001). This significant increase in sensitivity was due to the detection of PTC by testing in the nodules with atypical or suspicious PTC cytology results. Patients with -positive tumors were significantly older than those who did not harbor mutations (45.6 years vs. 39.8 years, P 5 0.002). No correlations between mutation and other clinical-pathology parameters were observed. 1 Department of Pathology, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, People s Republic of China 2 State Key Laboratory of Molecular Oncology, Department of Etiology and Carcinogenesis, Cancer Institute/Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, People s Republic of China Contract grant sponsor: Youth Fund of Peking Union Medical College. Conflict of interest: The authors have no conflicts of interest to declare. *Correspondence to: H.-Q. Guo, Associate Prof., Department of Pathology, Cancer Institute/Hospital Chinese Academy of Medical Sciences, 17 Nanli Panjiayuan Lane, Chaoyang District, Beijing 100021, People s Republic of China. E-mail: ghqin2006@163.com Received 25 June 2014; Revised 10 April 2015; Accepted 19 June 2015 DOI: 10.1002/dc.23302 Published online 7 July 2015 in Wiley Online Library (wileyonlinelibrary.com). Conclusions: Detection of mutation can be successfully carried out using residual liquid-based materials. It can be performed as a diagnostic tool to supplement traditional thyroid FNA, especially in cases with atypical or suspicious PTC. However, the role of in guidance of the extent of thyroidectomy and nodal clearance requires further study. Diagn. Cytopathol. 2015;43:786 790. VC 2015 Wiley Periodicals, Inc. Key Words: papillary thyroid carcinoma; mutation; fine-needle aspiration; diagnosis; prognosis Fine-needle aspiration (FNA) is now the most successful diagnostic test in the management of thyroid nodules. 1 3 However, FNA may also be nondiagnostic or may demonstrate indeterminate or suspicious cytologic features in 20 30% of all biopsies. 4 7 Hence, an ancillary test for suspicious FNA results is required. Moreover, the detection rate of microcarcinoma is increasing with the widespread use of ultrasound. However, the optimal management of microcarcinoma remains controversial, and often focuses on the extent of thyroidectomy and lymph node clearance. Thus, additional prognostic information provided by preoperative FNA may be helpful for surgeons in making a decision on initial treatment. BRAF c.1799t > A (p.v600e) mutation is the most common genetic alteration in papillary thyroid cancer (PTC) and is found in 15 80% of these tumors and confined to malignant nodules. 8 10 It is possible to speculate a diagnostic role of testing in PTC. The prognostic significance of the mutation has been explored in several studies, in which -positive PTC was found to have a more aggressive behavior. 8,11 18 In this study, we investigated the usefulness of preoperative mutation analysis as an adjunctive diagnostic and prognostic tool to routine FNA. 786 Diagnostic Cytopathology, Vol. 43, No 10 VC 2015 WILEY PERIODICALS, INC.
BRAF P.V600E TESTING IN THYROID FNA Materials and Methods The materials were obtained from the Cancer Institute/ Hospital, Chinese Academy of Medical Sciences (CAMS) between November 2010 and July 2012. The FNA samples were collected preoperatively and intraoperatively. Of the 195 cases, 95 samples were obtained in the outpatient clinic before surgery. The remaining 100 samples were obtained at the time of frozen examination. Intraoperative FNA biopsies were performed on surgically resected thyroid nodules by one of the two cytopathologists (Dr. Guo and Dr. Zhao). The thyroid nodules were cut in half, one half was sent for frozen pathologic diagnosis and the other half was used for FNA. FNA was performed under direct visualization. At least two passes were performed on each tumor. One pass aspirate was expressed onto a glass slide for cytologic diagnosis. The other pass was rinsed in PreservCyt solution (Hologic, Marlborough, MA, USA) for DNA extraction. FNA was repeated one or more times until suspended white particles were observed in the PreservCyt solution macroscopically. Preoperative FNA samples were retrospectively collected after obtaining clinical cytologic diagnoses. FNA biopsies were performed with one to two passes. Palpable lesions were aspirated by a cytopathologist in the FNA clinic; nonpalpable lesions using ultrasound-guided FNA were aspirated by radiologists. Aspirates were rinsed in PreservCyt solution. One ThinPrep (Hologic, Marlborough, MA, USA) slide was prepared from each case for assessment of both morphology and cellularity. Residues were collected for DNA extraction. In this way, we only selected the specimens which were diagnosed as PTC or suspicious PTC by cytology. Residue selection criteria were defined as 10 groups of cells on the slide in 10 ml of PreservCyt solution. Previous studies suggested selecting 6 10 groups of cells on the slide to perform PCR detection. 19 In order to guarantee enough DNA, our residue selection was based on both cellularity and volume. Approximately 50% of routine FNA specimens met the selection criteria and were used for molecular testing. The liquid-based materials were stored at 48C and submitted for molecular testing within 3 months. This study was reviewed and approved by the ethics committee of the Cancer Institute/Hospital, CAMS. All patients gave informed consent. Cytology Diagnosis According to the The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), cytological diagnoses were categorized as benign, atypia of indeterminate significance (AUS), follicular neoplasm (FN), suspicious malignant/suspicious papillary thyroid carcinoma (SM/ SPTC), and malignant/ptc. DNA Extraction Cells in PreservCyt solution were first centrifuged, then incubated in 500 ll of DNA lysis solution [1 mg/ml proteinase K, 10 mmol/l Tris HCl (ph 8.0), 0.1 mol/l EDTA (ph 8.0), 0.5% (w/v) SDS] at 558C for approximately 12 hours, and the DNA was then extracted by phenol and chloroform, and stored at 2208C until use. The nucleic acids were quantified using a NanoDrop spectrophotometer. Detection of BRAF P.V600E Mutation The primer sets for PCR amplification were designed as follows: (codon 600), 5 0 -TCATAATGCT TGCTCTGATAGGA-3 0 (forward), and 5 0 -GGCCAAAAA TTTAATCAGTGGA-3 0 (reverse). PCR was carried out using TaKaRa Taq DNA polymerase (TaKaRa, in a final volume of 50 ml, with 13 TaKaRa Taq Buffer with Mg 21, 200 lm of each dntp, 200 nm of forward and reverse primers, 1.25 U of TaKaRa Taq, and 50 100 ng of DNA template). PCR cycling parameters were as follows: an initial denaturation at 958C for 5 min followed by 35 cycles at 958C for 30 seconds, 55 618C for 45 seconds, 728C for 30 seconds; and a final extension at 728C for 10 min. Five microliters of PCR product was run on a 1.5% agarose gel to check the specificity of amplification, and 20 ml was sent for direct sequencing with forward and reverse primers for each gene (SinoGenoMax Co., Ltd., China). Statistical Analysis The histological diagnosis in each patient was obtained from their medical records. The results of histopathology were taken as the gold standard. Cytological and molecular results were compared with histopathology. Group comparisons of categorical variables were performed using the v 2 test. The association between clinicopathologic factors and presence of the mutation was validated by the Pearson v 2 and independent t tests. Statistical analysis was carried out using SPSS 13.0 and P < 0.05 was considered statistically significant. Results A total of 195 FNA samples from 44 male and 151 female patients (mean age: 45.1 years), were included in this study. All FNA specimens were sufficient to successfully perform DNA extraction and PCR analysis. On surgical follow-up, 170 cases were confirmed as PTC, and the lesions in the other 25 cases were benign. Using cytology alone, the diagnostic sensitivity, specificity, and accuracy for the detection of PTC were 70% (119/170), 100% (25/25), and 73.8% (144/195), respectively. testing showed similar sensitivity (67.1%), specificity (100%), and accuracy (71.3%) to that Diagnostic Cytopathology, Vol. 43, No 10 787
ZHAO ET AL. Table I. Diagnostic Values of FNA Cytology, Testing or a Combination of Both for Detecting PTC Diagnostic modality SN (%) SP (%) PPV (%) NPV (%) AC (%) Cytology a 70.0 100.0 100.0 32.9 73.8 Cytology b 91.8 84.0 97.5 60.0 90.8 67.1 100.0 100.0 30.9 71.3 testing Combined a 85.3 100.0 100.0 50.0 87.2 Combined b 92.9 84.0 97.5 63.6 91.8 a Cytology diagnosis of malignant was taken as the diagnostic threshold. b Cytology diagnosis of suspicious malignant was taken as the diagnostic threshold. Abbreviations: SN, sensitivity; SP, specificity; PPV, positive predictive value; NPV, negative predictive value; AC, accuracy. Table II. Correlation Between Cytologic Diagnoses and Mutation Status Cytologic diagnoses (n) (1) (n) (%) PTC (n 5 170) PTC 119 88 73.9 SPTC 37 24 64.9 FN 2 0 0 AUS 7 2 28.6 Benign 5 0 0 Total 170 114 67.1 Benign (n 5 25) SPTC 4 0 0 FN 2 0 0 AUS 1 0 0 Benign 18 0 0 Total 25 0 0 Abbreviations: AUS, atypia of undetermined significance; FN, follicular neoplasm; SPTC, suspicious papillary thyroid carcinoma; PTC: papillary thyroid carcinoma. of cytology. When FNA cytology was combined with testing, improved sensitivity (85.3% vs 70.0%, P 5 0.001) and accuracy (87.2% vs 73.8%, P 5 0.001) were obtained for PTC diagnosis (Table I). Table II shows the correlation between mutations and cytological diagnoses. In terms of the two cytology categories of SPTC and AUS, testing contributed to cytology diagnosis. In the group with SPTC (n 5 41), 24 samples had mutations. At final histology, 37 were confirmed as PTC by histopathology and all mutated cases were histologically confirmed as PTC. testing confirmed 64.9% (24/37) of PTC with SPTC cytology diagnosis. In the group with AUS cytology (n 5 8), 7 were PTC. Two samples had mutations, and both were confirmed as PTC. BRAF p.v600e testing detected 28.6% (2/7) of PTC in AUS cytology samples. The clinical pathologic features of PTC according to mutation status are shown in Table III. Of 170 PTCs, 13 recurrent patients (patients who already had a diagnosis of papillary carcinoma) were excluded from this Table III. Univariate Analysis of the Association Between BRAF p.v600e Mutation and Clinicopathologic Factors Characteristic n % n % P Age at diagnosis (years) Mean 6 SD 45.60 6 10.89 39.8 6 10.80 0.002* Range 21 76 18 67 Gender Male 23 21.9 11 21.2 0.914 Female 82 41 Capsular invasion Present 83 79 41 78.8 0.977 Absent 22 11 Extra-thyroid extension Present 49 46.7 22 42.3 0.606 Absent 56 30 Lymph node metastasis Present 54 51.5 30 57.7 0.459 Absent 51 22 Tumor size 1 cm 34 32.4 16 30.8 0.838 >1 cm 71 36 Multifocality Yes 41 39.0 18 34.6 0.589 No 64 34 AJCC stages I/II 78 74.3 42 80.8 0.368 III/IV 27 10 study. Of the other 157 cases, 123 were female and 34 were male aged 18 76 years, with an average age of 44 years. Patients with positive tumors were significantly older than those who did not harbor the mutation (45.6 years vs. 39.8 years, P 5 0.002). However, BRAF p.v600e mutation was not associated with patient gender, capsular invasion, extra-thyroid extension, lymph node metastasis, tumor size, multifocality, and TNM stage. In addition, no significant relationships between mutation and clinicopathologic characteristics were observed in the PTC group with tumor size 1 cm(tableiv). Discussion Positive (n 5 105) Negative (n 5 52) It has been reported that gene mutations can be successfully identified using liquid-based material with high reproducibility. 20 Therefore, it is necessary to establish criteria regarding adequate cell number for molecular testing and to avoid false-negative findings. Previous studies suggested selecting 6 10 groups of cells on the slide to perform PCR detection. 19 In order to guarantee enough DNA for analysis, we chose 10 groups of cells as the criterion in this study. With regard to the correct volume of tumor sample, most studies do not mention this. In this study, we were able to extract 300 ng to [mt]110,000 ng DNA from 10 ml of residual routine FNA specimens. This was sufficient to perform PCR detection. As this was an initial study on liquid-based residual specimens, further studies should be performed to 788 Diagnostic Cytopathology, Vol. 43, No 10
BRAF P.V600E TESTING IN THYROID FNA Table IV. Univariate Analysis of the Association Between BRAF p.v600e Mutation and Clinicopathologic Factors of PTCs with Tumor Size 1 cm Positive (n 5 34) Negative (n 5 16) Characteristic n % n % P Age at diagnosis (years) Mean 6 SD 47.44 6 10.56 41.69 6 9.36 0.069 Range 25 65 28 56 Gender Male 6 17.6 3 18.8 0.925 Female 28 13 Capsular invasion Present 15 44.1 7 43.8 0.981 Absent 19 9 Extra-thyroid extension Present 11 32.4 3 18.8 0.318 Absent 23 13 Lymph node metastasis Present 12 35.3 5 31.3 0.778 Absent 22 11 Multifocality Yes 8 23.5 5 31.3 0.562 No 26 11 AJCC stages I/II 26 76.5 14 87.5 0.363 III/IV 8 2 determine the smallest residual sample which can be used for molecular detection. mutation is known to be highly specific to PTC and has not been reported in follicular carcinoma, adenoma, or benign thyroid nodules. 21 Due to its high specificity, this genetic alteration is now considered a useful diagnostic marker for thyroid FNA. Zatelli et al. reported that mutation analysis increased the sensitivity of cytology for PTC from 77% to 87%. 22 Another author observed that the rate of PTC diagnosis using cytology alone was 62.3% (56/90), while the rate of PTC diagnosis using a morphomolecular method was 82.2% (74/90), with an increase of 20% in diagnostic accuracy due to mutation analysis. 23 In our study, we found that mutation testing increased the sensitivity of the FNA procedure from 70% to 85.3%. Therefore, the addition of molecular analysis resulted in an increase in sensitivity of 15.3% compared to cytology alone. Diagnostic accuracy also increased from 73.8% to 87.2%. These findings are further evidence of the efficacy of BRAF p.v600e as a diagnostic marker in thyroid FNA. In the study by Xing, the authors believed that if BRAF p.v600e testing was performed before cytology examination, nearly half of the patients with PTC did not need to undergo cytology examination, which could result in a substantial cost saving. 24 However, in clinical practice, the majority (60 80%) of FNA specimens are benign lesions and the risk of malignancy in cytology benign lesions is estimated to be <1%. 25 27 This means that if testing is used for screening FNA specimens, the majority of FNA specimens require an additional cytology examination. Therefore, is unsuitable as a screening tool for PTC. In this study, a significant increase in sensitivity was due to the detection of PTC by testing in nodules with suspicious FNA, which included lesions with cellular atypia and those which were suspicious malignant. In this study, testing detected 28.6% of PTC in AUS cytology samples and 64.9% of PTC in SPTC samples. Therefore, we suggest that BRAF p.v600e testing is an additional method for suspicious FNA biopsy samples. For suspicious FNA results, BRAF p.v600e mutation analysis would allow the formulation of an unambiguous surgical plan, while forgoing the need for other less specific diagnostic tests such as repeat FNA and intraoperative frozen section evaluation. The relationship between mutation and the clinicopathological outcomes of PTC has been investigated in several studies. Some authors believe that BRAF p.v600e mutation is associated with poor prognosis. 8,11 18 Li et al. conducted a meta-analysis which included 32 studies and6372patients.theyfoundthat mutation was associated with lymph node metastasis, stage, extrathyroidal extension, tumor size, male gender, multifocality, absence of capsule, classic, and tall-cell variant PTC in patients with PTC. 28 However, some studies showed different results. A report from Korea indicated that the BRAF p.v600e mutation was found in 335 of 424 cases (79%) and was associated with extra-thyroidal extension and variants of PTC, and a high-risk of metastasis, patient age, local invasion, tumor size, and lymph node metastasis were not significantly associated with mutation. 29 Astudy of 214 consecutive Italian patients with classic PTC found that 41% of tumors harbored the mutation, and noted that no significant correlation with status, with the exception of patient age (52.7 years vs. 33.4 years). 30 These findings seem to indicate that it is unclear whether the presence of the mutation is associated with poor prognosis of PTC. In this study, mutations showed a strong association with older patients (45.6 years vs. 39.8 years); however, there was no relationship between BRAF p.v600e mutation and other clinicopathologic parameters including patient gender, capsular invasion, extra-thyroid extension, lymph node metastasis, tumor size, multifocality, and TNM stage. Similar results were found in our groupof50ptcswithtumorsize1 cm. Patient age was an indicator of poor prognosis in PTC. However, age is a known factor in patients waiting for a surgical decision on the extent of thyroidectomy and nodal clearance. Our results show that did not provide more information on the aggressive behavior of PTC, except in older patients. Therefore, there is no evidence to show that mutation can guide the surgical decision on the extent of thyroidectomy and nodal clearance. Diagnostic Cytopathology, Vol. 43, No 10 789
ZHAO ET AL. In conclusion, detection of mutation can be used successfully in liquid-based residues. mutation analysis has significant potential as a diagnostic tool to supplement traditional thyroid FNA cytology, especially in cases with atypical or suspicious PTC diagnosis. The role of in guidance of the extent of thyroidectomy and nodal clearance requires further study. Acknowledgments We thank radiologists Li-juan Niu, Yong Wang, Li Zhu, Qing Chang, and Yu Chen for their help in ultrasound guided sampling. References 1. 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;90:357 363. 2. Werga P, Wallin G, Skoog L, Hamberger B. Expanding role of fine-needle aspiration cytology in thyroid diagnosis and management. World J Surg. 2000;24:907 912. 3. Frates MC, Doubilet PM, Gawande AA, et al. Long-term assessment of a multidisciplinary approach to thyroid nodule diagnostic evaluation. Cancer. 2007;111:508 516. 4. F uhrer D, M ugge C, Paschke R. Questionnaire on management of nodular thyroid disease (Annual Meeting of the Thyroid Section of the German Society of Endocrinology 2003). Exp Clin Endocrinol Diabetes. 2005;113:152 159. 5. Filetti S, Durante C, Torlontano M. Nonsurgical approaches to the management of thyroid nodules. Nat Clin Pract Endocrinol Metab. 2006;2:384 294. 6. Gharib H, Papini E. Thyroid nodules: clinical importance, assessment, and treatment. Endocrinol Metab Clin North Am. 2007;36: 707 735. 7. Mazeh H, Beglaibter N, Prus D, Ariel I, Freund HR. Cytohistologic correlation of thyroid nodules. Am J Surg. 2007;194:161 163. 8. Xing M. BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev. 2007; 28:742 762. 9. Guan H, Ji M, Bao R, et al. Association of high iodine intake with the T1799A BRAF mutation in papillary thyroid cancer. J Clin Endocrinol Metab. 2009;94:1612 1617. 10. Melck AL, Yip L, Carty SE. The utility of BRAF testing in the management of papillary thyroid cancer. Oncologist. 2010;15:1285 1293. 11. Elisei R, Ugolini C, Viola D, et al. BRAF(V600E) mutation and outcome of patients with papillary thyroid carcinoma: A 15-year median follow-up study. J Clin Endocrinol Metab. 2008;93:3943 3949. 12. 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;97:4390 4398. 13. Kebebew E, Weng J, Bauer J, et al. The prevalence and prognostic value of BRAFV600E mutation in thyroid cancer. Ann Surg. 2007; 246:466 470. 14. Lupi C, Giannini R, Ugolini C, et al. Association of BRAFV600E mutation with poor clinicopathological outcomes in 500 consecutive cases of papillary thyroid carcinoma. J Clin Endocrinol Metab. 2007;92:4085 4090. 15. Prescott JD, Sadow PM, Hodin RA, et al. BRAFV600E status adds incremental value to current risk classification systems in predicting papillary thyroid carcinoma recurrence. Surgery. 2012;152:984 990. 16. Xing M, Clark D, Guan H, et al. BRAFV600E mutation testing of thyroid fine-needle aspiration biopsy specimens for preoperative risk stratification in papillary thyroid cancer. J Clin Oncol. 2009;27: 2977 2982. 17. Xing M, Alzahrani AS, Carson KA, et al. Association between BRAFV600E mutation and mortality in patients with papillary thyroid cancer. Jama. 2013;309:1493 1501. 18. Yip L, Nikiforova MN, Carty SE, et al. Optimizing surgical treatment of papillary thyroid carcinoma associated with BRAF mutation. Surgery. 2009;146:1215 1223. 19. Dyhdalo K, MacNamara S, Brainard J, et al. Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples. Cancer Cytopathol. 2014;122:114 122. 20. Rossi ED, Martini M, Capodimonti S, et al. BRAF(V600E) mutation analysis on liquid-based cytology-processed aspiration biopsies predicts bilaterality and lymph node involvement in papillary thyroid microcarcinoma. Cancer Cytopathol. 2013;121:291 297. 21. Nikiforova MN, Nikiforov YE. Molecular diagnostics and predictors in thyroid cancer. Thyroid. 2009;19:1351 1361. 22. Zatelli MC, Trasforini G, Leoni S, et al. Mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma in fine-needle aspiration biopsies. Eur J Endocrinol. 2009;161:467 473. BRAFV600E ;:-. 23. Marchetti I, Lessi F, Mazzanti CM, et al. A morpho-molecular diagnosis of papillary thyroid carcinoma: BRAFV600E detection as an important tool in preoperative evaluation of fine-needle aspirates. Thyroid. 2009;19:837 842. 24. Xing M. BRAFV600E mutation in thyroid cancer. Endocr Relat Cancer. 2005;12:245 262. 25. Yang J, Schnadig V, Logrono R, Wasserman PG. Fine-needle aspiration of thyroid nodules: A study of 4703 patients with histologic and clinical correlations. Cancer. 2007;111:306 315. 26. Yang GC, Liebeskind D, Messina AV. Ultrasound-guided fineneedle aspiration of the thyroid assessed by Ultrafast Papanicolaou stain: Data from 1135 biopsies with a two- to six-year follow-up. Thyroid. 2001;11:581 589. 27. Baloch ZW, LiVolsi VA, Asa SL, et al. Diagnostic terminology and morphologic criteria for cytologic diagnosis of thyroid lesions: A synopsis of the National Cancer Institute Thyroid Fine-Needle Aspiration State of the Science Conference. Diagn Cytopathol. 2008;36:425 437. 28. Li C, Lee KC, Schneider EB, Zeiger MA. BRAFV600E mutation and its association with clinicopathological features of papillary thyroid cancer: A meta-analysis. J Clin Endocrinol Metab. 2012;97: 4559 4570. 29. Nam JK, Jung CK, Song BJ, et al. Is the BRAF(V600E) mutation useful as a predictor of preoperative risk in papillary thyroid cancer?. Am J Surg. 2012;203:436 441. 30. Rodolico V, Cabibi D, Pizzolanti G, et al. BRAFV600E Mutation and p27 kip1 expression in papillary carcinomas of the thyroid <or=1 cm and their paired lymph node metastases. Cancer. 2007; 110:1218 1226. 790 Diagnostic Cytopathology, Vol. 43, No 10