DOI:10.1111/cyt.12199
Role of BRAF molecular analysis in the management of papillary thyroid carcinoma: analysis of cytological and histological samples L. Capelli*, C. Marfisi†, M. Puccetti‡, L. Saragoni§, F. De Paola§, A. Zaccaroni†, E. Chiadini*, L. Gagliardi¶, G. Ferretti*, W. Zoli* and P. Ulivi* *Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, †
Endocrine Surgery Unit, Morgagni-Piernatoni Hospital, Forlı, ‡Pathology Unit, S. Maria delle Croci Hospital, Ravenna,
§
Pathology Unit and ¶Endocrinology Unit, Morgagni-Piernatoni Hospital, Forlı, Italy
Accepted for publication 29 June 2014
L. Capelli, C. Marfisi, M. Puccetti, L. Saragoni, F. De Paola, A. Zaccaroni, E. Chiadini, L. Gagliardi, G. Ferretti, W. Zoli and P. Ulivi Role of BRAF molecular analysis in the management of papillary thyroid carcinoma: analysis of cytological and histological samples Background: Although fine needle aspiration (FNA) is the standard diagnostic test for the characterization of a suspicious thyroid nodule, in some cases cytological evaluation is inconclusive. The aim of this study was to determine the role of BRAF mutation in aiding diagnosis and to verify whether archival cytological samples could be suitable for molecular analysis. Methods: Eighty-five patients with suspicious (Thy4) or follicular (Thy3) lesions on cytology were resubmitted to a second FNA for BRAF mutation analysis. Of these, 56 subsequently underwent surgery. The usefulness of archival samples for molecular analysis was also studied in a second cohort of 42 patients with a confirmed diagnosis of papillary thyroid carcinoma for whom both archived paraffin-embedded histological samples and cytological smears were available. A further 15 patients with paired fresh FNA and archived cytological and histological samples were recruited. Results: BRAF mutation was found in the fresh FNA samples from 10 of 56 patients who had surgery with previous inconclusive cytology (4/45, 9%, Thy3 and 6/11, 55%, Thy4). The BRAF test showed a specificity and positive predictive value of 100% (26/26 and 10/10, respectively), sensitivity of 33% (10/30) and negative predictive value of 57% (26/46). There was absolute concordance between the BRAF results obtained with 42 histological and cytological archived samples. BRAF analysis on 15 archived cytological samples showed absolute concordance with histology, whereas there was one false negative on the matched fresh FNA. Conclusion: BRAF analysis is a highly specific test that can facilitate cytological diagnosis in some cases and can also be performed on archived cytological samples. Keywords: thyroid nodules, BRAF, mutation, papillary thyroid carcinoma, fine needle aspiration, cytological samples Introduction Papillary thyroid carcinoma (PTC) is the commonest thyroid cancer, accounting for 80% or more of all thyroid malignancies.1 From a clinical point of view, Correspondence: P. Ulivi, Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via Maroncelli 40, 47014 Meldola (FC), Italy Tel.: +39-0543-739277; Fax: +39-0543-739221; E-mail: [email protected] © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
thyroid nodules are fairly common, and only 5% turn out to be malignant.2 Fine needle aspiration (FNA) is currently considered to be the most effective technique for the diagnosis of thyroid nodules.3,4 Its interpretation, based on The British Thyroid Association (BTA) guidelines, which were used to define the Italian (Tir1–5) terminology (SIAPEC: Societ a Italiana di Anatomia Patologica e Citologia Diagnostica), includes non-diagnostic (Thy1), non-neoplastic (Thy2), follicular lesions (Thy3), suspicious for malignancy (Thy4) and malignant
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(Thy5).5,6 Each category is associated with a specific risk of malignancy: less than 1%, 5–30%, 50–75% and 100% for Thy2, Thy3, Thy4 and Thy5, respectively.7 Patients with follicular lesions, suspicious for malignancy nodules and malignant FNAs are commonly referred for surgery. However, pre-operative FNA results may not always represent an accurate guide to optimal surgical management. Recent advances in the molecular genetics of thyroid cancer are being used to develop new diagnostic markers of FNA samples. One such marker, BRAF, a serine–threonine kinase belonging to the RAF family of proteins,8 is mutated in about half of PTCs.9,10 Previous studies have established an association between BRAF mutations and aggressive clinicopathological characteristics of primary PTCs, e.g. extrathyroidal extension, lymph node metastasis, histological subtypes with a poorer prognosis and advanced disease stages.11–13 A number of studies have demonstrated that BRAF mutation testing in FNA samples, alone or in combination with other alterations, represents an innovative approach to the pre-operative identification of patients with PTC at higher risk for extensive disease.14–24 In the majority of these studies, BRAF molecular analysis was performed on the FNA material left over after preparing the cytological smears16,18–20 or collected by washing the syringe needle.14,17 Alternatively, after an inconclusive cytological diagnosis, patients underwent a second FNA procedure to collect biological material for BRAF molecular analysis.25 All three methods of collection have their drawbacks: leftover material may contain an insufficient number of tumour cells, which can lead to false negative results, whereas two consecutive invasive procedures can cause substantial discomfort to the patient. The present study aimed to verify the role of BRAF mutation in the clinical management of suspicious thyroid nodules or inconclusive diagnostic cytology, to evaluate the real clinical benefit of the molecular characterization, and to assess whether archived cytological smears could also be used/are suitable for molecular analysis. Methods Case series From November 2010 to December 2012, 1321 patients were seen at the Endocrinology Unit of Morgagni-Piernatoni Hospital for the evaluation of a
thyroid nodule. FNA were carried out under ultrasound guidance with 23 gauge needles. FNA samples were smeared directly onto glass slides, fixed in alcohol and stained by the Papanicolaou technique. The following cytological categories were used, in accordance with the BTA guidelines: non-diagnostic (Thy1), non-neoplastic (Thy2), follicular lesions (Thy3), suspicious for malignancy (Thy4) and malignant (Thy5). All specimens were assessed by four experienced cytologists and reviewed by the Chief of the Pathology Unit (L.S.). The FNAs from 110 patients had an inconclusive cytological diagnosis (85 Thy3 and 25 Thy4). Of these, 85 (65 female and 20 male) were submitted to a second FNA for BRAF mutation analysis. Fifty-six patients underwent surgery and BRAF analysis was also performed on tumour paraffin-embedded histological samples. A second cohort of 42 patients (37 females, 5 males), with a confirmed diagnosis of PTC after Thy5 cytology and available archived paraffinembedded histological samples and cytological smears, was also included in this study to evaluate the usefulness of archived material for molecular analysis. A third cohort of 15 patients (11 females, 4 males) was selected to compare the molecular analyses performed on fresh FNA, archived cytological smears and paraffin-embedded histological samples. Of these, seven had a Thy3 cytological diagnosis and eight a Thy4 diagnosis. All patients were informed about the nature of the study and signed a written consent form. DNA extraction and detection of BRAF mutation Genomic DNA was extracted from fresh FNA specimens and from archived cytological and histological samples. For the former, samples were centrifuged at 500 g and the pellet was washed in 500 ll of normal saline and frozen. DNA extraction was performed using a QIAamp DNA micro kit (Qiagen, Spa, Milan, Italy), according to the manufacturer’s instructions. For the latter, specimens were accurately selected before DNA extraction. Non-tumour cells were removed from cytological smears by a scalpel under an optical microscope (2.59 magnification) or a magnifying glass, and the remaining material, comprising almost 70% tumour cells, was scraped off the slides and placed in a test-tube. Cells were lysed in 50 mM KCl, 10 mM Tris-HCl, pH 8.0, 2.5 mM MgCl2 and Tween-20, 0.45%, and supplemented with Proteinase K at a concentration of 1.25 mg/ml overnight at 56 °C. Proteinase K was inactivated at © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
BRAF in cytological samples of PTC
95 °C for 10 minutes, after which samples were centrifuged twice to eliminate debris. The supernatant was assessed for DNA quality and quantity by a Nanodrop spectrophotometer (Celbio, Milan, Italy) and submitted to PCR amplification. In paraffinembedded samples, areas containing at least 70% of tumour cells were identified on haematoxylin and eosin-stained tissue sections, and 5-lm sections of the corresponding areas were macrodissected and collected in specific tubes. DNA extraction was performed as for the cytological smears. DNA quantity and quality were assessed using a Nanodrop spectrophotometer (Celbio). Mutation analysis of exon 15 of the BRAF gene was performed using an anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR MoAb) response (BRAF status) (Diatech Pharmacogenetics, Ancona, Italy) kit and pyrosequencing methodology. The kit was designed to identify mutations at codons 599, 600 and 601 of the BRAF gene. Statistical analysis Statistical analyses were performed using SPSS software (version 20.o; SPSS Inc,, Chicago, IL, USA). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were calculated. Results BRAF mutation in fresh FNA samples Eighty-five patients with a suspicious nodule or inconclusive cytology (73 Thy3 and 12 Thy4) underwent a second FNA. BRAF analysis was performed on a new FNA sample destined for molecular analysis. Ten patients showed a V600E BRAF mutation, four of whom had a Thy3 diagnosis and six a Thy4 diagnosis. Forty-five of the 73 patients with Thy3 underwent surgery (Table 1). BRAF was wild-type in all the benign lesions and in both cases of follicular carcinoma (FC), but mutated in four of 18 (22%) of the PTCs (two were classical and two follicular variant). These last four cases were also mutated in tumour tissue. BRAF analysis performed on paraffinembedded histological sections from the 14 remaining PTCs that were wild type on FNA cytology revealed two BRAF-mutated cases. Of the 12 patients with a previous Thy4 diagnosis, 11 underwent surgery: 10 were diagnosed with PTC (seven classical © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
Table 1. Pathological/morphological diagnosis and presence of BRAF mutations in 56 nodules with inconclusive fine needle aspiration (FNA) cytology Initial FNA Cytological diagnosis
BRAF status
Thy3 (45)
4 mut 41 wt
Thy4 (11)
6 mut 5 wt
Surgical samples Histological diagnosis 4 PTC 12 PTC 2 micro-PTC 2 follicular carcinoma 25 benign lesions 6 PTC 3 PTC 1 micro-PTC 1 benign lesion
BRAF status 4 mut 10 wt; 2 mut 2 wt 2 wt – 6 mut 2 wt 1 mut 1 wt –
Thy3, follicular lesion; Thy4, suspicious for malignancy; mut, mutated; wt, wild-type; PTC, papillary thyroid carcinoma.
variant, one each of follicular and sclerosing variant, and one micro-PTC) and one with adenomatous hyperplasia. BRAF was wild-type in the adenomatous hyperplasia and in the sclerosing variant, but mutated in six of 10 (60%) of the PTCs. BRAF analysis performed on histological tumour samples revealed a mutation in one case that was BRAF wild-type on FNA, indicating a false-negative FNA result (Table 1). Thyroid cancer was diagnosed in all patients with BRAF mutations on FNA (100% PPV), but also in 20 patients with wild-type BRAF (57% NPV). The sensitivity and specificity were 33% and 100%, respectively, with an overall diagnostic accuracy of 67% (Table 2). A partial lobectomy is usually performed in cases of uninodular disease (including follicular proliferation and suspicious nodules). For the latter, intraoperative frozen section analysis is sometimes needed to confirm a diagnosis of malignancy and, when this is inconclusive (or in cases of proliferative lesions), it is necessary to wait for the final histological diagnosis. In the event of nodules of more than 1 cm, aggressive phenotype, specific histological types or multifocal neoplasms, a contralateral lobectomy is also performed. The presence of BRAF mutations may modify surgical options. Patient management in the fresh FNA group. In our study, the presence of a BRAF mutation in three patients with a diagnosis of uninodular proliferation (Thy3) indicated the need for total thyroidectomy.
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Table 2. Diagnostic accuracy of BRAF mutation status in 56 patients with suspicious nodules or inconclusive cytology
Sensitivity, % (n)
Specificity, % (n)
Diagnostic accuracy, %
PPV, % (n)
NPV, % (n)
33 (10/30)
100 (26/26)
67
100 (10/10)
57 (26/46)
NPV, negative predictive value; PPV, positive predictive value.
The cases were discussed among clinical staff and it was decided to perform the surgical procedure. Histological examination confirmed multifocal disease in one case and single tumours of 13 and 8 mm in the other two patients. BRAF analysis eliminated the need to recall patients for a completion (contralateral) lobectomy. Although the patient with subcentimetre PTC was probably overtreated, he suffered from chronic thyroiditis, thus justifying the total thyroidectomy performed. The fourth patient with Thy3 with mutated BRAF had multinodular disease, making total thyroidectomy mandatory. After a clinical group discussion, total thyroidectomy without intra-operative frozen section analysis was performed in six patients with a BRAF-mutated Thy4 nodule. In two patients, the tumour was subcentimetre in size, but multifocal and with invasive characteristics, justifying a total thyroidectomy, and, in three cases, the nodule was either more than 1 cm or had features of infiltrating carcinoma. In the remaining patient, the lesion, measuring 12 mm at the pre-operative ultrasound scan, was actually much smaller (7 mm) at the histological examination. BRAF analysis in 42 archival cytological and histological samples In view of the fact that a second FNA was performed specifically for molecular analysis, we decided to evaluate the feasibility of performing the analysis on archival cytological smears to avoid the need for two FNA procedures. For this purpose, we selected 42 patients with a Thy5 diagnosis for whom both paraffin-embedded histological samples and archival cytological smears were available. BRAF mutation (V600E) was detected in 25 patients (60%), all but two of whom were female; 100% concordance between histological and cytological samples was observed.
first FNA procedure and surgery-derived histological samples were available for BRAF mutation analysis in 15 patients (Table 3). In particular, absolute concordance was observed between the results from archived cytological smears and paraffin-embedded histological samples. Conversely, BRAF analysis performed on fresh FNA samples produced one false-negative result, i.e. a wild-type BRAF, whereas the gene was mutated in the other two sample types (archival cytology and histological sample). Discussion Ultrasound-guided FNA represents the most reliable diagnostic tool for the pre-surgical diagnosis of clinically suspicious thyroid nodules. Indeterminate cytology categories, such as follicular neoplasm and suspicious for malignancy, constitute about 10% of all cytological diagnoses and are characterized by a 5–30% and 50–75% risk of cancer, respectively. Over the last few decades, significant advances have been made in our understanding of the molecular pathogenesis of thyroid cancer. Changes in the mitogen-activated protein kinase (MAPK) pathway are among the most frequent and important alterations seen in this tumour. In particular, mutation of the BRAF gene (V600E) occurs in about 50–60% of PTCs, and there is evidence to suggest that mutated Table 3. Comparative analysis of BRAF status in fresh fine needle aspiration (FNA) samples, archival cytological material and histological specimens BRAF analysis Archival material
Case
Cytological diagnosis
Fresh FNA
Comparison of BRAF analysis in 15 matched fresh and archived FNA samples
1–5 6–7 8 9 10–15
Thy3 Thy3 Thy4 Thy4 Thy4
wt V600E wt wt V600E
Fresh FNA samples destined specifically for molecular analysis, archival cytological smears derived from the
Thy3, follicular lesion; Thy4, suspicious for malignancy; wt, wild-type.
Cytological
Histological
wt V600E wt V600E V600E
wt V600E wt V600E V600E
© 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
BRAF in cytological samples of PTC
BRAF is of prognostic value.26,27 However, the frequency of BRAF mutation is lower in the follicular variant of PTC (10–15%) and in follicular thyroid carcinoma (less than 1%).28 The high specificity of BRAF mutation for PTC could facilitate the pre-operative diagnosis of thyroid cancer, especially in thyroid nodules that are suspicious for PTC, whereas its usefulness in the diagnosis of Thy3 nodules is more limited because of the low prevalence of mutation in the follicular forms of the disease.29 We aimed to evaluate whether BRAF determination on cytological material could facilitate the clinical management of patients. Ten BRAF mutated samples were found in our case series, four with a previous Thy3 cytology and six with a previous suspicious for malignancy diagnosis. No BRAF mutations were found in 16 cases without PTC (100% specificity) and all 10 patients with BRAF mutation were found to have cancer at surgery (100% PPV). This indicates that direct thyroidectomy could be the best approach for such patients, thus bypassing the routine ‘two-step’ protocol or eliminating the need for intra-operative frozen section analysis before total thyroidectomy, and reducing healthcare costs and patient discomfort. However, considering that no guidelines are currently available for the clinical management of patients with an indeterminate FNA diagnosis and BRAF mutation, it is advisable to discuss such cases among clinical staff to decide on the best strategy to adopt. The main limitation of the BRAF test is its low sensitivity, as not all PTCs are BRAF mutated and as the majority of Thy3 cases are diagnosed as the follicular variant, or as FC, the latter only being mutated in about 10% of cases. Other authors have attempted to increase the sensitivity of the molecular test on FNA material by performing analyses of other alterations that are more specific for FC, e.g. PAX8/PPARc (paired box gene 8/peroxisome proliferator-activated receptor c) translocations and mutations of RAS genes.19,20 Although RAS mutation analysis is very straightforward, the evaluation of PAX8/PPARc translocation is a more complex procedure to perform on a routine basis as it requires RNA as starting material. FNA-derived samples destined for BRAF determination are normally collected by washing the needle used for FNA in normal saline solution to collect residual cells or by performing a second FNA procedure for patients with inconclusive cytology. The main problem of the former is related to the possibil© 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
ity of false-negative results as washing the needle does not guarantee that there will be a sufficient number of cells on which to perform the molecular analysis. The main disadvantage of the latter concerns the discomfort of patients who are subjected to two FNA procedures. In the present study, we aimed to verify whether archived cytological samples derived from the first FNA could be used for molecular analysis. The results confirmed that this was possible, showing absolute concordance with the data obtained on histological samples. Moreover, the use of a cytological slide permits an appropriate selection of cells to analyse for BRAF mutation under a microscope. Conversely, the use of a fresh sample derived from fine needle washing or from new FNA material can lead to false-negative results because of the evaluation of unselected cells. The possibility of using archival material from the first FNA is of particular relevance in clinical practice, as it reduces the amount of time required for molecular analysis and also spares patients from having to undergo a second FNA procedure. In conclusion, we confirmed that BRAF mutation analysis is a specific, albeit not highly sensitive, test that was capable of identifying cancer in a proportion of inconclusive cytological samples, thus facilitating the cytological diagnosis. We also verified the possibility of using archived cytological samples for molecular analysis, eliminating the need to resubmit patients to two consecutive FNA procedures. Acknowledgments The authors would like to thank Ursula Elbling for editing the manuscript. References 1. Aschebrook-Kilfoy B, Ward MH, Sabra MM, Devesa SS. Thyroid cancer incidence patterns in the United States by histologic type, 1992–2006. Thyroid 2011; 21:125–34. 2. Mazzaferri EL. Management of a solitary thyroid nodule. N Engl J Med 1993;328:553–9. 3. Baloch Z, LiVolsi VA, Jain P et al. Role of repeat fineneedle aspiration biopsy (FNAB) in the management of thyroid nodules. Diagn Cytopathol 2003;29:203–6. 4. Sidoti M, Marino G, Resmini E et al. The rational use of fine needle aspiration biopsy (FNAB) in diagnosing thyroid nodules. Minerva Endocrinol 2006;31:159–72. 5. The British Thyroid Association and the Royal College of Physicians. Guidelines for the Management of Thyroid
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Cancer, 2nd edn 2007. www.british-thyroid-association. org. Fadda G, Basolo F, Bondi A et al. Cytological classification of thyroid nodules: proposal of the SIAPEC-IAP Italian Consensus Working Group. Pathologica 2010;102:405–8. Layfield LJ, Cibas ES, Gharib H, Mandel SJ. Thyroid aspiration cytology: current status. CA Cancer J Clin 2009;59:99–110. Fagin JA, Mitsiades N. Molecular pathology of thyroid cancer: diagnostic and clinical implications. Best Pract Res Clin Endocrinol Metab 2008;22:955–69. Xing M, Tufano RP, Tufaro AP et al. Detection of BRAF mutation on fine needle aspiration biopsy specimens: a new diagnostic tool for papillary thyroid cancer. Clin Endocrinol Metab 2004;89:2867–72. Giordano TJ, Kuick R, Thomas DG et al. Molecular classification of papillary thyroid carcinoma: distinct BRAF, RAS, and RET/PTC mutation-specific gene expression profiles discovered by DNA microarray analysis. Oncogene 2005;24:6646–56. Vasko V, Hu S, Wu G et al. High prevalence and possible de novo formation of BRAF mutation in metastasized papillary thyroid cancer in lymph nodes. J Clin Endocrinol Metab 2005;90:5265–9. Xing M, Westra WH, Tufano RP et al. BRAF mutation predicts a poorer clinical prognosis for papillary thyroid cancer. J Clin Endocrinol Metab 2005;90:6373–9. Xing M, Alzahrani AS, Carson KA et al. Association between BRAF V600E mutation and mortality in patients with papillary thyroid cancer. J Am Med Assoc 2013;309:1493–501. Sapio MR, Posca D, Raggioli A et al. Detection of RET/ PTC, TRK and BRAF mutations in preoperative diagnosis of thyroid nodules with indeterminate cytological findings. Clin Endocrinol (Oxf) 2007;66:678–83. Xing M, Clark D, Guan H et al. BRAF mutation testing of thyroid fine-needle aspiration biopsy specimens for preoperative risk stratification in papillary thyroid cancer. J Clin Oncol 2007;27:2977–82. Kim SW, Lee JI, Kim JW et al. BRAFV600E mutation analysis in fine-needle aspiration cytology specimens for evaluation of thyroid nodule: a large series in a BRAFV600E-prevalent population. J Clin Endocrinol Metab 2010;95:3693–700. Zatelli MC, Trasforini G, Leoni S et al. BRAF V600E mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma in fine-needle aspiration biopsies. Eur J Endocrinol 2009;161:467–73. Troncone G, Cozzolino I, Fedele M et al. Preparation of thyroid FNA material for routine cytology and BRAF
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testing: a validation study. Diagn Cytopathol 2010;38:172–6. Ohori NP, Nikiforova MN, Schoedel KE et al. Contribution of molecular testing to thyroid fine-needle aspiration cytology of “follicular lesion of undetermined significance/atypia of undetermined significance”. Cancer Cytopathol 2010;118:17–23. 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: 1365–9. Kang G, Cho EY, Shin JH et al. Role of BRAFV600E mutation analysis and second cytologic review of fineneedle aspiration for evaluating thyroid nodule. Cancer Cytopathol 2012;120:44–51. Ohori NP, Singhal R, Nikiforova MN et al. BRAF mutation detection in indeterminate thyroid cytology specimens: underlying cytologic, molecular, and pathologic characteristics of papillary thyroid carcinoma. Cancer Cytopathol 2013;121:197–205. Moon HJ, Kwak JY, Kim EK et al. The role of BRAFV600E mutation and ultrasonography for the surgical management of a thyroid nodule suspicious for papillary thyroid carcinoma on cytology. Ann Surg Oncol 2009;16:3125–31. Poller DN, Glaysher S. BRAF V600 co-testing is technically feasible in conventional thyroid fine needle aspiration (FNA) cytology smears and can reduce the need for completion thyroidectomy. Cytopathology 2014;25:155–9. Salvatore G, Giannini R, Faviana P et al. Analysis of BRAF point mutation and RET/PTC rearrangement refines the fine-needle aspiration diagnosis of papillary thyroid carcinoma. J Clin Endocrinol Metab 2004;89:5175–80. Kim TH, Park YJ, Lim JA et al. The association of the BRAF(V600E) mutation with prognostic factors and poor clinical outcome in papillary thyroid cancer: a meta-analysis. Cancer 2012;118:1764–73. Xing M. Prognostic utility of BRAF mutation in papillary thyroid cancer. Mol Cell Endocrinol 2010;321:86–93. Kebebew E, Weng J, Bauer J et al. The prevalence and prognostic value of BRAF mutation in thyroid cancer. Ann Surg 2007;246:466–70. Johnson SJ, Hardy SA, Roberts C et al. Pilot of BRAF mutation analysis in indeterminate, suspicious and malignant thyroid FNA cytology. Cytopathology 2014;25:146–54.
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Role of BRAF molecular analysis in the management of papillary thyroid carcinoma: analysis of cytological and histological samples L. Capelli*, C. Marfisi†, M. Puccetti‡, L. Saragoni§, F. De Paola§, A. Zaccaroni†, E. Chiadini*, L. Gagliardi¶, G. Ferretti*, W. Zoli* and P. Ulivi* *Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, †
Endocrine Surgery Unit, Morgagni-Piernatoni Hospital, Forlı, ‡Pathology Unit, S. Maria delle Croci Hospital, Ravenna,
§
Pathology Unit and ¶Endocrinology Unit, Morgagni-Piernatoni Hospital, Forlı, Italy
Accepted for publication 29 June 2014
L. Capelli, C. Marfisi, M. Puccetti, L. Saragoni, F. De Paola, A. Zaccaroni, E. Chiadini, L. Gagliardi, G. Ferretti, W. Zoli and P. Ulivi Role of BRAF molecular analysis in the management of papillary thyroid carcinoma: analysis of cytological and histological samples Background: Although fine needle aspiration (FNA) is the standard diagnostic test for the characterization of a suspicious thyroid nodule, in some cases cytological evaluation is inconclusive. The aim of this study was to determine the role of BRAF mutation in aiding diagnosis and to verify whether archival cytological samples could be suitable for molecular analysis. Methods: Eighty-five patients with suspicious (Thy4) or follicular (Thy3) lesions on cytology were resubmitted to a second FNA for BRAF mutation analysis. Of these, 56 subsequently underwent surgery. The usefulness of archival samples for molecular analysis was also studied in a second cohort of 42 patients with a confirmed diagnosis of papillary thyroid carcinoma for whom both archived paraffin-embedded histological samples and cytological smears were available. A further 15 patients with paired fresh FNA and archived cytological and histological samples were recruited. Results: BRAF mutation was found in the fresh FNA samples from 10 of 56 patients who had surgery with previous inconclusive cytology (4/45, 9%, Thy3 and 6/11, 55%, Thy4). The BRAF test showed a specificity and positive predictive value of 100% (26/26 and 10/10, respectively), sensitivity of 33% (10/30) and negative predictive value of 57% (26/46). There was absolute concordance between the BRAF results obtained with 42 histological and cytological archived samples. BRAF analysis on 15 archived cytological samples showed absolute concordance with histology, whereas there was one false negative on the matched fresh FNA. Conclusion: BRAF analysis is a highly specific test that can facilitate cytological diagnosis in some cases and can also be performed on archived cytological samples. Keywords: thyroid nodules, BRAF, mutation, papillary thyroid carcinoma, fine needle aspiration, cytological samples Introduction Papillary thyroid carcinoma (PTC) is the commonest thyroid cancer, accounting for 80% or more of all thyroid malignancies.1 From a clinical point of view, Correspondence: P. Ulivi, Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via Maroncelli 40, 47014 Meldola (FC), Italy Tel.: +39-0543-739277; Fax: +39-0543-739221; E-mail: [email protected] © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
thyroid nodules are fairly common, and only 5% turn out to be malignant.2 Fine needle aspiration (FNA) is currently considered to be the most effective technique for the diagnosis of thyroid nodules.3,4 Its interpretation, based on The British Thyroid Association (BTA) guidelines, which were used to define the Italian (Tir1–5) terminology (SIAPEC: Societ a Italiana di Anatomia Patologica e Citologia Diagnostica), includes non-diagnostic (Thy1), non-neoplastic (Thy2), follicular lesions (Thy3), suspicious for malignancy (Thy4) and malignant
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(Thy5).5,6 Each category is associated with a specific risk of malignancy: less than 1%, 5–30%, 50–75% and 100% for Thy2, Thy3, Thy4 and Thy5, respectively.7 Patients with follicular lesions, suspicious for malignancy nodules and malignant FNAs are commonly referred for surgery. However, pre-operative FNA results may not always represent an accurate guide to optimal surgical management. Recent advances in the molecular genetics of thyroid cancer are being used to develop new diagnostic markers of FNA samples. One such marker, BRAF, a serine–threonine kinase belonging to the RAF family of proteins,8 is mutated in about half of PTCs.9,10 Previous studies have established an association between BRAF mutations and aggressive clinicopathological characteristics of primary PTCs, e.g. extrathyroidal extension, lymph node metastasis, histological subtypes with a poorer prognosis and advanced disease stages.11–13 A number of studies have demonstrated that BRAF mutation testing in FNA samples, alone or in combination with other alterations, represents an innovative approach to the pre-operative identification of patients with PTC at higher risk for extensive disease.14–24 In the majority of these studies, BRAF molecular analysis was performed on the FNA material left over after preparing the cytological smears16,18–20 or collected by washing the syringe needle.14,17 Alternatively, after an inconclusive cytological diagnosis, patients underwent a second FNA procedure to collect biological material for BRAF molecular analysis.25 All three methods of collection have their drawbacks: leftover material may contain an insufficient number of tumour cells, which can lead to false negative results, whereas two consecutive invasive procedures can cause substantial discomfort to the patient. The present study aimed to verify the role of BRAF mutation in the clinical management of suspicious thyroid nodules or inconclusive diagnostic cytology, to evaluate the real clinical benefit of the molecular characterization, and to assess whether archived cytological smears could also be used/are suitable for molecular analysis. Methods Case series From November 2010 to December 2012, 1321 patients were seen at the Endocrinology Unit of Morgagni-Piernatoni Hospital for the evaluation of a
thyroid nodule. FNA were carried out under ultrasound guidance with 23 gauge needles. FNA samples were smeared directly onto glass slides, fixed in alcohol and stained by the Papanicolaou technique. The following cytological categories were used, in accordance with the BTA guidelines: non-diagnostic (Thy1), non-neoplastic (Thy2), follicular lesions (Thy3), suspicious for malignancy (Thy4) and malignant (Thy5). All specimens were assessed by four experienced cytologists and reviewed by the Chief of the Pathology Unit (L.S.). The FNAs from 110 patients had an inconclusive cytological diagnosis (85 Thy3 and 25 Thy4). Of these, 85 (65 female and 20 male) were submitted to a second FNA for BRAF mutation analysis. Fifty-six patients underwent surgery and BRAF analysis was also performed on tumour paraffin-embedded histological samples. A second cohort of 42 patients (37 females, 5 males), with a confirmed diagnosis of PTC after Thy5 cytology and available archived paraffinembedded histological samples and cytological smears, was also included in this study to evaluate the usefulness of archived material for molecular analysis. A third cohort of 15 patients (11 females, 4 males) was selected to compare the molecular analyses performed on fresh FNA, archived cytological smears and paraffin-embedded histological samples. Of these, seven had a Thy3 cytological diagnosis and eight a Thy4 diagnosis. All patients were informed about the nature of the study and signed a written consent form. DNA extraction and detection of BRAF mutation Genomic DNA was extracted from fresh FNA specimens and from archived cytological and histological samples. For the former, samples were centrifuged at 500 g and the pellet was washed in 500 ll of normal saline and frozen. DNA extraction was performed using a QIAamp DNA micro kit (Qiagen, Spa, Milan, Italy), according to the manufacturer’s instructions. For the latter, specimens were accurately selected before DNA extraction. Non-tumour cells were removed from cytological smears by a scalpel under an optical microscope (2.59 magnification) or a magnifying glass, and the remaining material, comprising almost 70% tumour cells, was scraped off the slides and placed in a test-tube. Cells were lysed in 50 mM KCl, 10 mM Tris-HCl, pH 8.0, 2.5 mM MgCl2 and Tween-20, 0.45%, and supplemented with Proteinase K at a concentration of 1.25 mg/ml overnight at 56 °C. Proteinase K was inactivated at © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
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95 °C for 10 minutes, after which samples were centrifuged twice to eliminate debris. The supernatant was assessed for DNA quality and quantity by a Nanodrop spectrophotometer (Celbio, Milan, Italy) and submitted to PCR amplification. In paraffinembedded samples, areas containing at least 70% of tumour cells were identified on haematoxylin and eosin-stained tissue sections, and 5-lm sections of the corresponding areas were macrodissected and collected in specific tubes. DNA extraction was performed as for the cytological smears. DNA quantity and quality were assessed using a Nanodrop spectrophotometer (Celbio). Mutation analysis of exon 15 of the BRAF gene was performed using an anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR MoAb) response (BRAF status) (Diatech Pharmacogenetics, Ancona, Italy) kit and pyrosequencing methodology. The kit was designed to identify mutations at codons 599, 600 and 601 of the BRAF gene. Statistical analysis Statistical analyses were performed using SPSS software (version 20.o; SPSS Inc,, Chicago, IL, USA). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were calculated. Results BRAF mutation in fresh FNA samples Eighty-five patients with a suspicious nodule or inconclusive cytology (73 Thy3 and 12 Thy4) underwent a second FNA. BRAF analysis was performed on a new FNA sample destined for molecular analysis. Ten patients showed a V600E BRAF mutation, four of whom had a Thy3 diagnosis and six a Thy4 diagnosis. Forty-five of the 73 patients with Thy3 underwent surgery (Table 1). BRAF was wild-type in all the benign lesions and in both cases of follicular carcinoma (FC), but mutated in four of 18 (22%) of the PTCs (two were classical and two follicular variant). These last four cases were also mutated in tumour tissue. BRAF analysis performed on paraffinembedded histological sections from the 14 remaining PTCs that were wild type on FNA cytology revealed two BRAF-mutated cases. Of the 12 patients with a previous Thy4 diagnosis, 11 underwent surgery: 10 were diagnosed with PTC (seven classical © 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
Table 1. Pathological/morphological diagnosis and presence of BRAF mutations in 56 nodules with inconclusive fine needle aspiration (FNA) cytology Initial FNA Cytological diagnosis
BRAF status
Thy3 (45)
4 mut 41 wt
Thy4 (11)
6 mut 5 wt
Surgical samples Histological diagnosis 4 PTC 12 PTC 2 micro-PTC 2 follicular carcinoma 25 benign lesions 6 PTC 3 PTC 1 micro-PTC 1 benign lesion
BRAF status 4 mut 10 wt; 2 mut 2 wt 2 wt – 6 mut 2 wt 1 mut 1 wt –
Thy3, follicular lesion; Thy4, suspicious for malignancy; mut, mutated; wt, wild-type; PTC, papillary thyroid carcinoma.
variant, one each of follicular and sclerosing variant, and one micro-PTC) and one with adenomatous hyperplasia. BRAF was wild-type in the adenomatous hyperplasia and in the sclerosing variant, but mutated in six of 10 (60%) of the PTCs. BRAF analysis performed on histological tumour samples revealed a mutation in one case that was BRAF wild-type on FNA, indicating a false-negative FNA result (Table 1). Thyroid cancer was diagnosed in all patients with BRAF mutations on FNA (100% PPV), but also in 20 patients with wild-type BRAF (57% NPV). The sensitivity and specificity were 33% and 100%, respectively, with an overall diagnostic accuracy of 67% (Table 2). A partial lobectomy is usually performed in cases of uninodular disease (including follicular proliferation and suspicious nodules). For the latter, intraoperative frozen section analysis is sometimes needed to confirm a diagnosis of malignancy and, when this is inconclusive (or in cases of proliferative lesions), it is necessary to wait for the final histological diagnosis. In the event of nodules of more than 1 cm, aggressive phenotype, specific histological types or multifocal neoplasms, a contralateral lobectomy is also performed. The presence of BRAF mutations may modify surgical options. Patient management in the fresh FNA group. In our study, the presence of a BRAF mutation in three patients with a diagnosis of uninodular proliferation (Thy3) indicated the need for total thyroidectomy.
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Table 2. Diagnostic accuracy of BRAF mutation status in 56 patients with suspicious nodules or inconclusive cytology
Sensitivity, % (n)
Specificity, % (n)
Diagnostic accuracy, %
PPV, % (n)
NPV, % (n)
33 (10/30)
100 (26/26)
67
100 (10/10)
57 (26/46)
NPV, negative predictive value; PPV, positive predictive value.
The cases were discussed among clinical staff and it was decided to perform the surgical procedure. Histological examination confirmed multifocal disease in one case and single tumours of 13 and 8 mm in the other two patients. BRAF analysis eliminated the need to recall patients for a completion (contralateral) lobectomy. Although the patient with subcentimetre PTC was probably overtreated, he suffered from chronic thyroiditis, thus justifying the total thyroidectomy performed. The fourth patient with Thy3 with mutated BRAF had multinodular disease, making total thyroidectomy mandatory. After a clinical group discussion, total thyroidectomy without intra-operative frozen section analysis was performed in six patients with a BRAF-mutated Thy4 nodule. In two patients, the tumour was subcentimetre in size, but multifocal and with invasive characteristics, justifying a total thyroidectomy, and, in three cases, the nodule was either more than 1 cm or had features of infiltrating carcinoma. In the remaining patient, the lesion, measuring 12 mm at the pre-operative ultrasound scan, was actually much smaller (7 mm) at the histological examination. BRAF analysis in 42 archival cytological and histological samples In view of the fact that a second FNA was performed specifically for molecular analysis, we decided to evaluate the feasibility of performing the analysis on archival cytological smears to avoid the need for two FNA procedures. For this purpose, we selected 42 patients with a Thy5 diagnosis for whom both paraffin-embedded histological samples and archival cytological smears were available. BRAF mutation (V600E) was detected in 25 patients (60%), all but two of whom were female; 100% concordance between histological and cytological samples was observed.
first FNA procedure and surgery-derived histological samples were available for BRAF mutation analysis in 15 patients (Table 3). In particular, absolute concordance was observed between the results from archived cytological smears and paraffin-embedded histological samples. Conversely, BRAF analysis performed on fresh FNA samples produced one false-negative result, i.e. a wild-type BRAF, whereas the gene was mutated in the other two sample types (archival cytology and histological sample). Discussion Ultrasound-guided FNA represents the most reliable diagnostic tool for the pre-surgical diagnosis of clinically suspicious thyroid nodules. Indeterminate cytology categories, such as follicular neoplasm and suspicious for malignancy, constitute about 10% of all cytological diagnoses and are characterized by a 5–30% and 50–75% risk of cancer, respectively. Over the last few decades, significant advances have been made in our understanding of the molecular pathogenesis of thyroid cancer. Changes in the mitogen-activated protein kinase (MAPK) pathway are among the most frequent and important alterations seen in this tumour. In particular, mutation of the BRAF gene (V600E) occurs in about 50–60% of PTCs, and there is evidence to suggest that mutated Table 3. Comparative analysis of BRAF status in fresh fine needle aspiration (FNA) samples, archival cytological material and histological specimens BRAF analysis Archival material
Case
Cytological diagnosis
Fresh FNA
Comparison of BRAF analysis in 15 matched fresh and archived FNA samples
1–5 6–7 8 9 10–15
Thy3 Thy3 Thy4 Thy4 Thy4
wt V600E wt wt V600E
Fresh FNA samples destined specifically for molecular analysis, archival cytological smears derived from the
Thy3, follicular lesion; Thy4, suspicious for malignancy; wt, wild-type.
Cytological
Histological
wt V600E wt V600E V600E
wt V600E wt V600E V600E
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BRAF is of prognostic value.26,27 However, the frequency of BRAF mutation is lower in the follicular variant of PTC (10–15%) and in follicular thyroid carcinoma (less than 1%).28 The high specificity of BRAF mutation for PTC could facilitate the pre-operative diagnosis of thyroid cancer, especially in thyroid nodules that are suspicious for PTC, whereas its usefulness in the diagnosis of Thy3 nodules is more limited because of the low prevalence of mutation in the follicular forms of the disease.29 We aimed to evaluate whether BRAF determination on cytological material could facilitate the clinical management of patients. Ten BRAF mutated samples were found in our case series, four with a previous Thy3 cytology and six with a previous suspicious for malignancy diagnosis. No BRAF mutations were found in 16 cases without PTC (100% specificity) and all 10 patients with BRAF mutation were found to have cancer at surgery (100% PPV). This indicates that direct thyroidectomy could be the best approach for such patients, thus bypassing the routine ‘two-step’ protocol or eliminating the need for intra-operative frozen section analysis before total thyroidectomy, and reducing healthcare costs and patient discomfort. However, considering that no guidelines are currently available for the clinical management of patients with an indeterminate FNA diagnosis and BRAF mutation, it is advisable to discuss such cases among clinical staff to decide on the best strategy to adopt. The main limitation of the BRAF test is its low sensitivity, as not all PTCs are BRAF mutated and as the majority of Thy3 cases are diagnosed as the follicular variant, or as FC, the latter only being mutated in about 10% of cases. Other authors have attempted to increase the sensitivity of the molecular test on FNA material by performing analyses of other alterations that are more specific for FC, e.g. PAX8/PPARc (paired box gene 8/peroxisome proliferator-activated receptor c) translocations and mutations of RAS genes.19,20 Although RAS mutation analysis is very straightforward, the evaluation of PAX8/PPARc translocation is a more complex procedure to perform on a routine basis as it requires RNA as starting material. FNA-derived samples destined for BRAF determination are normally collected by washing the needle used for FNA in normal saline solution to collect residual cells or by performing a second FNA procedure for patients with inconclusive cytology. The main problem of the former is related to the possibil© 2014 John Wiley & Sons Ltd Cytopathology 2015, 26, 297–302
ity of false-negative results as washing the needle does not guarantee that there will be a sufficient number of cells on which to perform the molecular analysis. The main disadvantage of the latter concerns the discomfort of patients who are subjected to two FNA procedures. In the present study, we aimed to verify whether archived cytological samples derived from the first FNA could be used for molecular analysis. The results confirmed that this was possible, showing absolute concordance with the data obtained on histological samples. Moreover, the use of a cytological slide permits an appropriate selection of cells to analyse for BRAF mutation under a microscope. Conversely, the use of a fresh sample derived from fine needle washing or from new FNA material can lead to false-negative results because of the evaluation of unselected cells. The possibility of using archival material from the first FNA is of particular relevance in clinical practice, as it reduces the amount of time required for molecular analysis and also spares patients from having to undergo a second FNA procedure. In conclusion, we confirmed that BRAF mutation analysis is a specific, albeit not highly sensitive, test that was capable of identifying cancer in a proportion of inconclusive cytological samples, thus facilitating the cytological diagnosis. We also verified the possibility of using archived cytological samples for molecular analysis, eliminating the need to resubmit patients to two consecutive FNA procedures. Acknowledgments The authors would like to thank Ursula Elbling for editing the manuscript. References 1. Aschebrook-Kilfoy B, Ward MH, Sabra MM, Devesa SS. Thyroid cancer incidence patterns in the United States by histologic type, 1992–2006. Thyroid 2011; 21:125–34. 2. Mazzaferri EL. Management of a solitary thyroid nodule. N Engl J Med 1993;328:553–9. 3. Baloch Z, LiVolsi VA, Jain P et al. Role of repeat fineneedle aspiration biopsy (FNAB) in the management of thyroid nodules. Diagn Cytopathol 2003;29:203–6. 4. Sidoti M, Marino G, Resmini E et al. The rational use of fine needle aspiration biopsy (FNAB) in diagnosing thyroid nodules. Minerva Endocrinol 2006;31:159–72. 5. The British Thyroid Association and the Royal College of Physicians. Guidelines for the Management of Thyroid
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