Pre-operative role of BRAF in the guidance of the surgical approach and prognosis of differentiated thyroid carcinoma

in European Journal of Endocrinology
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  • 1 Unidade de Tireoide, Serviço de Endocrinologia, Serviço de Anatomia Patológica, Serviço de Cirurgia de Cabeça e Pescoço, Laboratório de Endocrinologia Celular e Molecular (LIM25), Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Arnaldo, 455, 4 ° Andar, Sala 4305, CEP 01246-903, São Paulo, Brazil

Objective

The p.V600E BRAF and RAS mutations are found in 30–80% of differentiated thyroid carcinoma (DTC). BRAF mutation has been associated with poor prognosis. This study investigated the role of molecular studies in preoperative diagnosis of DTC and the association of p.V600E mutation with prognostic factors.

Design

Prospective study.

Methods

A total of 202 patients with cytological diagnosis of Bethesda III–VI underwent preoperative molecular studies and subsequent thyroidectomy. p.V600E and RAS mutations were studied in the cytology smears, using real-time PCR genotyping technique. The BRAF mutation (BRAF+ or BRAF) was correlated with histological and clinical findings.

Results

Molecular study of 172 nodules with Bethesda III–V cytology improved negative predictive value and accuracy of Bethesda III and IV diagnosis. BRAF mutation was present in 65% of 94 DTC and p.Q61R NRAS in one. Except for age, BRAF+ and BRAF did not differ in sex, tumor size, histological subtype, multifocality, vascular invasion, extrathyroidal extension, or prognostic staging. Among papillary carcinomas, lymph node (LN) metastasis was diagnosed in 23% BRAF+ and 37% BRAF. Distant metastasis occurred in four BRAF. Recurrent or persistent disease was more frequent in BRAF (26.7 vs 3.3% BRAF+, P=0.002) along follow-up of 29.8±10 months. BRAF+ patients without LN metastasis by pre-operative evaluation submitted to thyroidectomy with central neck dissection (CND) had more frequent LN metastasis (45 vs 5% no CND, P=0.002), but no difference in clinical outcome was observed.

Conclusions

Pre-operative identification of BRAF mutation improved cytological diagnosis of DTC, but it was not associated with poor prognostic factors. Prophylactic CND did not guarantee better outcome in BRAF+ patients.

Abstract

Objective

The p.V600E BRAF and RAS mutations are found in 30–80% of differentiated thyroid carcinoma (DTC). BRAF mutation has been associated with poor prognosis. This study investigated the role of molecular studies in preoperative diagnosis of DTC and the association of p.V600E mutation with prognostic factors.

Design

Prospective study.

Methods

A total of 202 patients with cytological diagnosis of Bethesda III–VI underwent preoperative molecular studies and subsequent thyroidectomy. p.V600E and RAS mutations were studied in the cytology smears, using real-time PCR genotyping technique. The BRAF mutation (BRAF+ or BRAF) was correlated with histological and clinical findings.

Results

Molecular study of 172 nodules with Bethesda III–V cytology improved negative predictive value and accuracy of Bethesda III and IV diagnosis. BRAF mutation was present in 65% of 94 DTC and p.Q61R NRAS in one. Except for age, BRAF+ and BRAF did not differ in sex, tumor size, histological subtype, multifocality, vascular invasion, extrathyroidal extension, or prognostic staging. Among papillary carcinomas, lymph node (LN) metastasis was diagnosed in 23% BRAF+ and 37% BRAF. Distant metastasis occurred in four BRAF. Recurrent or persistent disease was more frequent in BRAF (26.7 vs 3.3% BRAF+, P=0.002) along follow-up of 29.8±10 months. BRAF+ patients without LN metastasis by pre-operative evaluation submitted to thyroidectomy with central neck dissection (CND) had more frequent LN metastasis (45 vs 5% no CND, P=0.002), but no difference in clinical outcome was observed.

Conclusions

Pre-operative identification of BRAF mutation improved cytological diagnosis of DTC, but it was not associated with poor prognostic factors. Prophylactic CND did not guarantee better outcome in BRAF+ patients.

Introduction

Thyroid nodular disease is a common condition, particularly with the advent of higher resolution ultrasound scanners, reaching up to 69% of patients that undergo US examination (1, 2). A proper preoperative diagnosis would restrict most thyroid surgeries to thyroid carcinoma, which represent no more than 5–15% of the cases (1).

Fine-needle aspiration biopsy (FNAB) guided by ultrasound is considered the gold standard technique to pre-operative diagnosis of thyroid carcinomas. However, indeterminate cytological diagnosis still occurs in 15–30%, requiring unnecessary surgeries in up to 80% of them (3, 4).

Papillary thyroid carcinoma (PTC) corresponds to about 85% of differentiated thyroid carcinomas (DTC) (1). In an effort to improve pre-operative DTC diagnosis, molecular studies are proposed. PTC is associated with the p.V600E mutation of the BRAF gene in 30–80% and less frequent to RAS mutations (5, 6, 7, 8, 9). Therefore, it has been proposed that their identification in cytological material would improve sensitivity and accuracy of FNAB, especially in indeterminate or suspicious for thyroid cancer (Bethesda III, IV, and V) (10). Besides, it has been suggested that the p.V600E mutation is associated with poor prognosis (11). Recently, a meta-analysis concluded that p.V600E mutation was correlated with male gender, larger tumor, extra-thyroid extension, multifocality, and greater incidence of cervical lymph nodes (LNs) metastasis, resulting in more frequent advanced clinical stages (odds ratio 1.82) (12). Presumably, pre-operative molecular evaluation of the BRAF p.V600E mutation in FNAB material could not only improve cytological diagnosis accuracy but also guide to a selective neck dissection of the central compartment, a more aggressive surgical therapy, to prevent poor outcome.

This study aims to analyze the benefits of molecular studies as a tool to improve pre-operative diagnosis in indeterminate or suspicious nodules in FNAB. In addition, we analyzed the correlation of p.V600E mutation with poor prognostic factors.

Subjects and methods

Patients

We prospectively recruited consecutive patients with thyroid nodules submitted to ultrasound-guided FNAB from 2009 to 2011 in the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo and Instituto do Câncer do Estado de São Paulo. Subjects with cytological diagnosis of atypia of undetermined significance (Bethesda III), follicular, or Hürthle cell neoplasm (Bethesda IV), suspicious for malignancy (Bethesda V) or PTC (Bethesda VI), were selected for molecular studies. BRAF and RAS genes mutations were investigated in cytological material and confirmed in tumor tissue. All FNAB diagnoses were revised by the same pathologist. Thyroid nodules with Bethesda III, IV, and V cytology were selected for analysis of the performance of cytological and molecular diagnosis.

Patients with cytological diagnosis of Bethesda III and IV were submitted to partial thyroidectomy (PT) or total thyroidectomy (TT). All patients with cytological diagnosis of Bethesda V and VI were submitted to total or near TT. Patients with pre-operative evidence of LNs metastasis underwent therapeutic neck dissection. A subgroup of patients without clinical or ultra sonographic evidence of cervical LN metastasis was submitted to prophylactic central neck dissection (CND), as a part of another institutional protocol (13). Dutenhefner et al. (13) selected prospectively 45 patients without known LN metastasis from a group of 52 consecutive patients with cytological diagnosis of Bethesda V and VI to be submitted to TT and CND. Twenty-three of them had been included in our study. Both patients' physicians and surgeons were blinded to the result of the molecular study.

Postoperatively, patients with thyroid carcinoma were evaluated with serial cervical ultrasound and serum thyroglobulin. Functional sensitivity for the thyroglobulin assay was 0.2 ng/ml (Access, Beckman Coulter, Fullerton, CA, USA). Recurrent or persistent disease was defined in the presence of distant metastasis, loco-regional recurrence, and suppressed or stimulated serum thyroglobulin >2 ng/ml after >1 year of follow-up. The sixth edition of the tumor, LN, and metastasis classification was used for staging (TNM, American Joint Committee on Cancer classification) (14), as well as American Thyroid Association (ATA) classification for the risk of recurrence (1). Clinical characteristics, molecular analysis, cytological and histological patterns as well as initial surgical approach and recurrence of disease were analyzed.

The study was approved by the Institutional Research Ethics Committee and all subjects gave their informed written consent to participate.

BRAF and RAS mutations

DNA extraction from thyroid FNAB stained slides followed the standard phenol/chloroform/isoamyl alcohol protocol (15). Briefly, slides were placed in the bath with histological xylene for 24 h to remove the coverslip and the material was then scraped from the slide and transferred to a clean microtube for extraction using phenol/chloroform/isoamyl alcohol. DNA extraction from material conserved in paraffin was performed as previously described (13). One milliliter of xylene preheated in an incubator at 95 °C was added in a microtube of 1.5 ml with three sections equivalent to 10 μm of conserved tumor. They were agitated and placed in an incubator at 37 °C for 30 min, and then centrifuged for 5 min at 15 000 g, after which the supernatant was discarded. For effective elimination of the paraffin, this step was repeated another two times. The samples were then submitted to two washings with 500 μl absolute ethanol to remove the organic solvent. After each addition of absolute ethanol, the microtubes were centrifuged at 13 000 g for 5 min at 4 °C and the supernatant was discarded. The samples were dried by inverting the microtubes on absorbent paper.

For DNA extraction: 20 μl of proteinase K (10 mg/ml) and 480 μl of a solution of 2.5 ml of Tris–HCl 1 M (pH 8.0); 500 μl EDTA 0.5 M (pH 8.0); 250 μl of Tween 20; and 46.75 ml of deionized water were added to each microtube. The samples were incubated for 18 h at 37 °C. Two extractions were carried out with 500 μl of phenol–chloroform. The tubes were carefully inverted and centrifuged at 13 000 g for 2 min at 4 °C. The supernatants were transferred to new microtubes and a new extraction was carried out with phenol–chloroform. DNA extracted from NPA cells (a melanoma cell line containing a biallelic BRAF p.V600E mutation) was used as positive control (kindly provided by E T Kimura). As negative control, we used DNA extracted from normal thyroid tissue, which showed the WT sequence of exon 15 of the BRAF gene.

The pellets were resuspended in sterile water. Total nucleic acids were extracted and sample adequacy for molecular analysis was assessed based on the general quantity and quality of isolated nucleic acids.

Searching for p.V600E mutation in cytology smears was performed by real-time PCR genotyping with primers defined by Benlloch et al. (16). Samples were genotyped using TaqMan according to the manufacturer's instruction (Applied Biosystems). The fluorescence data were analyzed with the allelic discrimination software of the StepOne version 2.0 Software (Applied Biosystems) and amplification plots produced in the PCR were also checked. In order to confirm genotyping findings, all samples were automatic sequenced using BigDye Terminator Kit on the ABI3100xl (Applied Biosystems).

Codons 12–13 and 61 of K-RAS, N-RAS, and H-RAS genes were directly sequenced only in samples without BRAF mutation as previously described (17).

Statistical analysis

Data were processed using PASW Statistics Software, version 17.0 (SPSS, Inc.) and MedCalc for Windows (version 12.0.3.0; MedCalc Software). Two-tailed P values were used and P values <0.05 were considered statistically significant.

Categorical variables are presented as absolute and relative (percentages) frequencies. Differences were evaluated by Pearson's χ2-test and Fisher's exact test when appropriate. Continuous variables are presented as mean±s.d. Differences among studied subgroups were determined using Student's t-test if presenting normal distribution, and Mann–Whitney U test for non-normal distributions.

Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for the diagnosis of malignancy in the subgroups of Bethesda III, IV, and V biopsies were calculated for cytological and molecular diagnosis of p.V600E mutation, either alone or in combination. We defined the true-positive, true-negative, false-positive, and false-negative results based on final histological diagnosis (benign and malignant).

Results

Clinical characteristics and pre-operative diagnosis

Two hundred and two patients were submitted to FNAB to investigate 208 thyroid nodules. A 116 nodules had the diagnosis of Bethesda III (55.8%), 20 Bethesda IV (9.6%), 36 Bethesda V (17.3%), and 36 Bethesda VI (17.3%). Female patients were more frequent than males in both benign thyroid nodular diseases and thyroid carcinomas (88 vs 84%, P=0.42); however, patients were older in the first group (53.6±13.7 vs 46.9±15.8 years old, P=0.002). We selected 172 thyroid nodules with Bethesda III, IV, and V cytology for pre-operative molecular studies and analysis of the performance of cytological and molecular diagnosis.

According to cytological diagnosis, DTC was diagnosed in 19.8% of Bethesda III, 35% of Bethesda IV, and 80.6% of Bethesda V. Classical PTC were more frequent among Bethesda V and VI diagnoses. Forty-three out of 64 Bethesda V and VI patients (67%) were classical papillary carcinoma compared with 13 of 30 Bethesda III and IV (43%) (P=0.028). The frequency of p.V600E mutation was 6.9% in Bethesda III, 20% in Bethesda IV, and 52.8% in Bethesda V. The p.Q61R NRAS mutation was identified in only one patient. All data were confirmed with the molecular analysis of surgically removed tumor.

Among benign nodules, 69.3% were adenomatous nodules, 21.9% were follicular adenomas, and 8.8% were thyroiditis. Size was statistically different between benign lesions and DTCs (26.2±16.8 vs 21±16.5 mm, P=0.01). None of the benign nodules had a p.V600E or RAS mutations in cytological material and tissue analysis.

The diagnostic performance of cytology and molecular results are described in Table 1. The presence of the p.V600E BRAF mutation provided a specificity and PPV for diagnosis of DTC of 100%. The combination of cytological diagnosis and pre-operative molecular study improved the NPV and accuracy of both Bethesda III and IV specimens. On the other hand, in Bethesda V and VI cytology, the BRAF study was associated with a worse NPV and accuracy than the cytological diagnosis alone.

Table 1

Diagnostic performance of cytology and molecular test in identification of 94 differentiated thyroid carcinomas.

nSensitivity (%)Specificity (%)Positive predictive value (%)Negative predictive value (%)Accuracy (%)
Bethesda III2324.518.419.822.821.2
Bethesda IV77.588.63553.751.9
Bethesda V2930.993.980.662.265.4
Bethesda VI3537.299.197.265.771.2
BRAF6164.910010077.684.1
Bethesda III+BRAF1534.810010086.187.1
Bethesda IV+BRAF457.110010081.285
Bethesda V+BRAF1965.510010041.272.2
Bethesda VI+BRAF2365.71001007.766.7

n, number of cases with histological diagnosis of differentiated thyroid carcinoma; BRAF, presence of p.V600E BRAF gene mutation.

Molecular analysis and prognosis

From 94 patients with final histological diagnosis of DTC, 61 (65%) had pre-operative cytological identification of the p.V600E mutation. There was no significant difference in clinical and histological features between individuals with or without BRAF mutation, except for age (BRAF+49.6±15 vs BRAF 41.8±16.3 years old, P=0.021; Table 2).

Table 2

Clinical and histological characteristics of 94 differentiated thyroid carcinomas according to the presence or absence of p.V600E BRAF mutation.

BRAF+ (n=61)BRAF (n=33)P
Sex (female/male)53/826/70.306
Age (years)49.6±1541.8±16.30.021
Bethesda0.977
 III15 (24.6%)8 (24.2%)
 IV4 (6.6%)3 (9.1%)
 V19 (31.1%)10 (30.3%)
 VI23 (37.7%)12 (36.4%)
Surgery0.093
 TT41 (67.2%)15 (45.5%)
 TT+CND11 (18%)12 (36.4%)
 TT+CND+LND7 (11.5%)6 (18.2%)
 PT2 (3.3%)0
RIT37 (68.5%)17 (31.5%)0.374
Dose147±34170±530.243
Size (mm)20.4±13.922.2±20.60.565
Histological diagnosis0.255
Papillary carcinoma variants
 Classical38 (62.3%)18 (54.5%)
 Follicular17 (27.9%)11 (33.3%)
 Oncocytic2 (3.3%)0
 Solid3 (4.9%)1 (3%)
 Warthin-like1 (1.6%)0
Follicular carcinoma02 (6.1%)
Follicular carcinoma oncocytic variant01 (3%)
Multifocality31 (50.8%)14 (42.4%)0.437
Vascular invasion4 (6.6%)3 (9.1%)0.693
Extrathyroidal extension18 (29.5%)5 (15.2%)0.140
Lymph nodes metastasis14 (23%)11 (33%)0.277
Distant metastasis04 (12.1%)0.014
TNM staging0.301a
 I40 (65.6%)24 (72.7%)
 II5 (8.2%)4 (12.1%)
 III12 (19.7%)3 (9.1%)
 IV4 (6.6%)2 (6.1%)
ATA staging0.985b
 Low risk35 (57.4%)19 (57.6%)
 Intermediate risk24 (39.3%)8 (24.2%)
 High risk2 (3.3%)6 (18.2%)
Follow-up (months)27±1030±100.076
Recurrent or persistent disease2 (3.3%)8 (24.2%)0.003

BRAF+ and BRAF, presence or absence of p.V600E BRAF gene mutation; TT, total thyroidectomy; CND, central neck dissection; LND, lateral neck dissection; PT, partial thyroidectomy; RAI, radioiodine ablation; TNM, American Joint Committee on Cancer classification (13); ATA, American Thyroid Association classification (1).

TNM stages I+II vs III+IV.

ATA low risk vs intermediate+high risk.

Among all PTC cases, LN metastasis was diagnosed in 23% BRAF+ and 37% BRAF (P=0.168). Distant metastasis occurred in only four BRAF patients. Recurrent or persistent disease was more frequent in BRAF patients (26.7 vs 3.3% BRAF+, P=0.002) during follow-up of 29.8±10 and 28.5±6.6 months respectively.

Eighty-four (89%) DTCs corresponded to classical and follicular variants of papillary carcinoma. Subgroup analysis confirmed anterior findings. The only prognostic factor associated with BRAF mutation was age (Table 3). The frequency of LN metastasis did not significantly differ between BRAF+ (20%) and BRAF (31%) patients. However, distant metastasis, diagnosed only in BRAF, and recurrent or persistent disease (BRAF+ 3.6% vs BRAF 24.1%) were also more frequent in BRAF patients.

Table 3

Clinical and histological characteristics of 84 classical and follicular variants of papillary thyroid carcinomas according to the presence or absence of p.V600E BRAF mutation.

BRAF+ (n=55)BRAF (n=29)P
Sex (female/male)47/823/60.473
Age (years)49.5±15.141.1±15.80.020
Size (mm)20.5±14.019.9±20.10.159
Multifocality28 (50.9%)13 (44.8%)0.596
Vascular invasion3 (5.5%)2 (6.9%)1.0
Extrathyroidal extension16 (29.1%)4 (13.8%)0.178
Lymph nodes metastasis11 (20%)9 (31%)0.259
Distant metastasis03 (10.3%)0.040
TNM staging0.153a
 I36 (65.5%)23 (79.3%)
 II5 (9.1%)3 (10.3%)
 III10 (18.2%)2 (6.9%)
 IV4 (7.3%)1 (3.4%)
ATA staging0.775b
 Low risk34 (61.8%)17 (58.6%)
 Intermediate risk19 (34.5%)7 (24.1%)
 High risk2 (3.6%)5 (17.2%)
Follow-up (months)27±1030±100.076
Recurrent or persistent disease2 (3.6%)7 (24.1%)0.008

BRAF+ and BRAF, presence or absence of p.V600E BRAF gene mutation.

TNM stages I+II vs III+IV.

ATA low risk vs intermediate+high risk.

The N-RAS p.Q61R mutation was identified in a 30-year-old female patient, whose thyroid nodule was diagnosed as Bethesda V at FNAB. She presented with a multifocal follicular variant of PTC with the largest tumor measuring 1.9 cm. She was classified as TNM stage I and low risk of recurrence. She had no recurrence over a follow-up of 32 months.

Prophylactic CND and BRAF mutation

Thirteen patients had prior diagnosis of LNs metastasis, so they were excluded from subsequent analyses. Fifty-four patients BRAF+ without preoperative diagnosis of LNs metastasis were submitted to either TT or PT (n=43) or TT and prophylactic CND (TT+CND, n=11). LNs metastasis was significantly more frequent in subjects submitted to TT+CND (45 vs 5% TT/PT, P=0.002), resulting in more frequent TNM stages III and IV (45.5 vs 14% TT/PT, P=0.035) and ATA stages of intermediate and high risk of recurrences (63.6 vs 27.9% TT/PT, P=0.038). Despite a larger extent of local disease in patients submitted to prophylactic CND, no difference in clinical outcome was observed between subgroups, as no patient had recurrent or persistent disease even with a longer mean follow-up of patients submitted to TT+CND (33.5±7.2 vs 26.1±9.3 months in TT/PT, P=0.013).

Similar results were found in BRAF patients. LNs metastasis was present in 6.7% of patients submitted to TT and 50% of patients submitted to TT+CD (P=0.024). Only one patient in the second subgroup had persistent distant metastatic disease (P=0.44) at final evaluation.

Discussion

It has been proposed that molecular studies would improve the accuracy of cancer detection in FNAB of thyroid nodules. Recent studies have demonstrated the feasibility of mutations detection in clinical FNAB samples, extensively BRAF and RAS mutations, and the improvement of accuracy of FNAB especially in indeterminate cytology. Detection of other molecular markers such as as RET/PTC and PAX8/PPARγ mutations does not contribute substantially to cancer diagnosis and in addition, they are technically more difficult to study (18).

Although only one case presented a mutation of RAS gene, the p.V600E BRAF mutation was detected in 65% of thyroid carcinomas in our institution. The molecular study of BRAF gene showed 100% specificity and PPV to diagnosis of DTC and an accuracy of 84.1%.

In our study, we could observe that the combination of BRAF and cytology analysis was especially useful in patients with a cytological diagnosis of Bethesda III and IV (Table 1), while in Bethesda V and VI specimens, the cytological diagnosis alone was associated with a high specificity (93.9 and 99.1% respectively) and high PPV (80.6 and 97.2% respectively) and the combination with BRAF analysis added no benefit but, in fact, was associated with a worse NPV (62.2–41.2% and 65.7–7.7% respectively). The BRAF study increased the NPV and accuracy of combined pre-operative diagnosis of Bethesda III and IV cytology possibly because of their low sensitivity for the diagnosis of DTC attributed to low prevalence of malignant disease in these groups (19.8 and 35% respectively).

In clinical practice, the precise pre-operative diagnosis of thyroid nodule must be done to avoid unnecessary surgery in benign disorders. NPV around 84% in Bethesda III and IV (86.1 and 81.2% respectively) indicate that 16% of negative results in molecular study of these groups would still represent malignant cases. Therefore, the absence of BRAF mutation in Bethesda III and IV is not sufficient to defer surgery. As an alternative, Alexander et al. (19) proposed a panel of benign gene-expression classifier with 92% sensitivity and NPV of 95% to improve diagnosis and avoid surgery in most benign thyroid nodules. While this panel is not available worldwide, BRAF and RAS studies remain feasible methods that, despite not useful to indicate benign disease and defer surgery, can allow detection of malignant disease with indication for surgery. It has been suggested that the p.V600E BRAF mutation is associated with poor prognosis. Except for older age, we found no association of the p.V600E mutation with poor prognostic risk factors or with unfavorable outcome. The frequency of LN metastasis was not different between BRAF+ and BRAF cases. In addition, distant metastasis was observed only in BRAF tumors (Table 2) and recurrent and persistent disease were also observed more frequently in BRAF patients (P=0.002), despite a short period of follow-up. Li et al. suggested the association of BRAF mutation with poor prognostic factors, but they recognized that most of the studies analysed were retrospective and there was no consensus on performance of prophylactic CND; different histological subtypes were also studied altogether, even unfavorable ones such as tall cell and diffuse sclerosing variants (12). In this study, only four out of 91 PTC had unfavorable solid variant and three PTCs had variants of undefined prognosis (oncocytic and Warthin-like). There was no tall cell variant frequently associated with p.V600E and known as more aggressive. The most prevalent subgroup of classical and follicular variants of PTC showed no association of BRAF mutation with poor prognostic factors or poor outcome. Our findings corroborated recent studies (20, 21, 22, 23) and also discarded the association of p.V600E with higher frequency of cervical LN and distant metastases (13, 24, 25, 26). In order to justify the lack of association, it has also been proposed that it is not the intratumoral presence of BRAF mutation that determines prognosis but the higher frequency of mutant BRAF alleles, identified in an innovatory analysis of pyrosequencing technique, that favors more frequent recurrence (27).

Nevertheless, it has been suggested that the identification of a possible unfavorable risk factor, such as the presence of BRAF mutation, before surgery would allow an aggressive initial approach, such as central LN dissection (28). We evaluated BRAF+ patients submitted either to thyroidectomy or thyroidectomy plus prophylactic CND and found no difference in outcome, despite more frequent LNs metastasis in subjects submitted to TT+CND (P=0.002). A recent study that investigated the prognostic significance of microscopic LNs metastasis detected with prophylactic neck dissection has proved they had no impact in the low loco regional recurrence rates (29). Dutenhefner et al. (13) studied PTC submitted to prophylactic CND and found no difference in the LNs metastasis presence between BRAF+ and BRAF. Our prospective study confirmed the high frequency of microscopic LNs metastasis diagnosed in prophylactic CND, despite the presence of BRAF mutation. However, as it was not associated with a better outcome, we question its benefits, especially considering the higher risk of surgical comorbidities, such as permanent hypoparathyroidism.

This study has some limitations as we analyzed only 94 patients with DTC and partly submitted to thyroidectomy plus central node dissection (11/54 BRAF+). Besides, a short period of follow-up was analyzed (28.1±9.9 months) and a longer period would be more revealing regarding DTC.

In summary, the identification of p.V600E BRAF mutation in cytology smears improved accuracy to preoperative diagnosis of DTC in Bethesda III and IV in which cytology alone is associated with low sensitivity. In accordance, the molecular study does not avoid surgery, but permits identification of BRAF+ PTC that requires surgery.

In this study, BRAF mutation was not associated with poor prognostic factors or unfavorable outcome. We conclude that the role of pre-operative molecular analysis is restricted to improving the diagnosis of indeterminate cytological specimens and is still controversial in guiding surgical extent and postoperative adjuvant treatment of PTC.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This study had the financial support of Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP), grant numbers 09/07544-0 and 10/12883-6.

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    • Export Citation
  • 12

    Li C, Lee KC, Schneider EB, Zeiger MA. BRAF V600E mutation and its association with clinicopathological features of papillary thyroid cancer: a meta-analysis. Journal of Clinical Endocrinology and Metabolism 2012 97 45594570. (doi:10.1210/jc.2012-2104).

    • Search Google Scholar
    • Export Citation
  • 13

    Dutenhefner SE, Marui S, Santos AB, Lima EU, Inoue M, Neto JD, Shiang C, Fukushima J, Cernea CR, Friguglietti C. Braf, a tool in the decision to perform elective neck dissection? Thyroid 2013 23 15411546. (doi:10.1089/thy.2012.0304).

    • Search Google Scholar
    • Export Citation
  • 14

    Greene FL. In AJCC Cancer Staging Manual, 6th edn, pp 77–87. New York: Springer-Verlag, 2002

  • 15

    Vince A, Poljak M, Seme K. DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods. British Journal of Haematology 1998 101 349351. (doi:10.1046/j.1365-2141.1998.00702.x).

    • Search Google Scholar
    • Export Citation
  • 16

    Benlloch S, Paya A, Alenda C, Bessa X, Andreu M, Jover R, Castells A, Llor X, Aranda FI, Massuti B. Detection of BRAF V600E mutation in colorectal cancer: comparison of automatic sequencing and real-time chemistry methodology. Journal of Molecular Diagnostics 2006 8 540543. (doi:10.2353/jmoldx.2006.060070).

    • Search Google Scholar
    • Export Citation
  • 17

    Nikiforov YE, Steward DL, Robinson-Smith TM, Haugen BR, Klopper JP, Zhu Z, Fagin JA, Falciglia M, Weber K, Nikiforova MN. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules. Journal of Clinical Endocrinology and Metabolism 2009 94 20922098. (doi:10.1210/jc.2009-0247).

    • Search Google Scholar
    • Export Citation
  • 18

    Cantara S, Capezzone M, Marchisotta S, Capuano S, Busonero G, Toti P, Di Santo A, Caruso G, Carli AF, Brilli L. Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules improves the diagnostic accuracy of cytology. Journal of Clinical Endocrinology and Metabolism 2010 95 13651369. (doi:10.1210/jc.2009-2103).

    • Search Google Scholar
    • Export Citation
  • 19

    Alexander EK, Kennedy GC, Baloch ZW, Cibas ES, Chudova D, Diggans J, Friedman L, Kloos RT, LiVolsi VA, Mandel SJ. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. New England Journal of Medicine 2012 367 705715. (doi:10.1056/NEJMoa1203208).

    • Search Google Scholar
    • Export Citation
  • 20

    Liu RT, Chen YJ, Chou FF, Li CL, Wu WL, Tsai PC, Huang CC, Cheng JT. No correlation between BRAFV600E mutation and clinicopathological features of papillary thyroid carcinomas in Taiwan. Clinical Endocrinology 2005 63 461466. (doi:10.1111/j.1365-2265.2005.02367.x).

    • Search Google Scholar
    • Export Citation
  • 21

    Fugazzola L, Puxeddu E, Avenia N, Romei C, Cirello V, Cavaliere A, Faviana P, Mannavola D, Moretti S, Rossi S. Correlation between B-RAFV600E mutation and clinico-pathologic parameters in papillary thyroid carcinoma: data from a multicentric Italian study and review of the literature. Endocrine-Related Cancer 2006 13 455464. (doi:10.1677/erc.1.01086).

    • Search Google Scholar
    • Export Citation
  • 22

    Ito Y, Yoshida H, Maruo R, Morita S, Takano T, Hirokawa M, Yabuta T, Fukushima M, Inoue H, Tomoda C. BRAF mutation in papillary thyroid carcinoma in a Japanese population: its lack of correlation with high-risk clinicopathological features and disease-free survival of patients. Endocrine Journal 2009 56 8997. (doi:10.1507/endocrj.K08E-208).

    • Search Google Scholar
    • Export Citation
  • 23

    Nam JK, Jung CK, Song BJ, Lim DJ, Chae BJ, Lee NS, Park WC, Kim JS, Jung SS, Bae JS. Is the BRAF(V600E) mutation useful as a predictor of preoperative risk in papillary thyroid cancer? American Journal of Surgery 2012 203 436441. (doi:10.1016/j.amjsurg.2011.02.013).

    • Search Google Scholar
    • Export Citation
  • 24

    Trovisco V, Couto JP, Cameselle-Teijeiro J, de Castro IV, Fonseca E, Soares P, Sobrinho-Simoes M. Acquisition of BRAF gene mutations is not a requirement for nodal metastasis of papillary thyroid carcinoma. Clinical Endocrinology 2008 69 683685. (doi:10.1111/j.1365-2265.2008.03243.x).

    • Search Google Scholar
    • Export Citation
  • 25

    Lee KC, Li C, Schneider EB, Wang Y, Somervell H, Krafft M, Umbricht CB, Zeiger MA. Is BRAF mutation associated with lymph node metastasis in patients with papillary thyroid cancer? Surgery 2012 152 977983. (doi:10.1016/j.surg.2012.08.019).

    • Search Google Scholar
    • Export Citation
  • 26

    Sancisi V, Nicoli D, Ragazzi M, Piana S, Ciarrocchi A. BRAFV600E mutation does not mean distant metastasis in thyroid papillary carcinomas. Journal of Clinical Endocrinology and Metabolism 2012 97 E1745E1749. (doi:10.1210/jc.2012-1526).

    • Search Google Scholar
    • Export Citation
  • 27

    Guerra A, Fugazzola L, Marotta V, Cirillo M, Rossi S, Cirello V, Forno I, Moccia T, Budillon A, Vitale M. A high percentage of BRAFV600E alleles in papillary thyroid carcinoma predicts a poorer outcome. Journal of Clinical Endocrinology and Metabolism 2012 97 23332340. (doi:10.1210/jc.2011-3106).

    • Search Google Scholar
    • Export Citation
  • 28

    Joo JY, Park JY, Yoon YH, Choi B, Kim JM, Jo YS, Shong M, Koo BS. Prediction of occult central lymph node metastasis in papillary thyroid carcinoma by preoperative BRAF analysis using fine-needle aspiration biopsy: a prospective study. Journal of Clinical Endocrinology and Metabolism 2012 97 39964003. (doi:10.1210/jc.2012-2444).

    • Search Google Scholar
    • Export Citation
  • 29

    Randolph GW, Duh QY, Heller KS, LiVolsi VA, Mandel SJ, Steward DL, Tufano RP, Tuttle RM. The prognostic significance of nodal metastases from papillary thyroid carcinoma can be stratified based on the size and number of metastatic lymph nodes, as well as the presence of extranodal extension. Thyroid 2012 22 11441152. (doi:10.1089/thy.2012.0043).

    • Search Google Scholar
    • Export Citation

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     European Society of Endocrinology

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  • 1

    Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, Mazzaferri EL, McIver B, Pacini F, Schlumberger M. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid 2009 19 11671214. (doi:10.1089/thy.2009.0110).

    • Search Google Scholar
    • Export Citation
  • 2

    Guth S, Theune U, Aberle J, Galach A, Bamberger CM. Very high prevalence of thyroid nodules detected by high frequency (13 MHz) ultrasound examination. European Journal of Clinical Investigation 2009 39 699706. (doi:10.1111/j.1365-2362.2009.02162.x).

    • Search Google Scholar
    • Export Citation
  • 3

    Hegedus L. Clinical practice. The thyroid nodule. New England Journal of Medicine 2004 351 17641771. (doi:10.1056/NEJMcp031436).

  • 4

    Baloch ZW, LiVolsi VA, Asa SL, Rosai J, Merino MJ, Randolph G, Vielh P, DeMay RM, Sidawy MK, Frable WJ. 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. Diagnostic Cytopathology 2008 36 425437. (doi:10.1002/dc.20830).

    • Search Google Scholar
    • Export Citation
  • 5

    Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA. High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC–RAS–BRAF signaling pathway in papillary thyroid carcinoma. Cancer Research 2003 63 14541457.

    • Search Google Scholar
    • Export Citation
  • 6

    Soares P, Trovisco V, Rocha AS, Lima J, Castro P, Preto A, Maximo V, Botelho T, Seruca R, Sobrinho-Simoes M. BRAF mutations and RET/PTC rearrangements are alternative events in the etiopathogenesis of PTC. Oncogene 2003 22 45784580. (doi:10.1038/sj.onc.1206706).

    • Search Google Scholar
    • Export Citation
  • 7

    Puxeddu E, Moretti S, Elisei R, Romei C, Pascucci R, Martinelli M, Marino C, Avenia N, Rossi ED, Fadda G. BRAF(V599E) mutation is the leading genetic event in adult sporadic papillary thyroid carcinomas. Journal of Clinical Endocrinology and Metabolism 2004 89 24142420. (doi:10.1210/jc.2003-031425).

    • Search Google Scholar
    • Export Citation
  • 8

    Xing M, Vasko V, Tallini G, Larin A, Wu G, Udelsman R, Ringel MD, Ladenson PW, Sidransky D. BRAF T1796A transversion mutation in various thyroid neoplasms. Journal of Clinical Endocrinology and Metabolism 2004 89 13651368. (doi:10.1210/jc.2003-031488).

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    • Export Citation
  • 9

    Chung KW, Yang SK, Lee GK, Kim EY, Kwon S, Lee SH, Park do J, Lee HS, Cho BY, Lee ES. Detection of BRAFV600E mutation on fine needle aspiration specimens of thyroid nodule refines cyto-pathology diagnosis, especially in BRAF600E mutation-prevalent area. Clinical Endocrinology 2006 65 660666. (doi:10.1111/j.1365-2265.2006.02646.x).

    • Search Google Scholar
    • Export Citation
  • 10

    Zatelli MC, Trasforini G, Leoni S, Frigato G, Buratto M, Tagliati F, Rossi R, Cavazzini L, Roti E, degli Uberti EC. BRAF V600E mutation analysis increases diagnostic accuracy for papillary thyroid carcinoma in fine-needle aspiration biopsies. European Journal of Endocrinology 2009 161 467473. (doi:10.1530/EJE-09-0353).

    • Search Google Scholar
    • Export Citation
  • 11

    Xing M, Haugen BR, Schlumberger M. Progress in molecular-based management of differentiated thyroid cancer. Lancet 2013 381 10581069. (doi:10.1016/S0140-6736(13)60109-9).

    • Search Google Scholar
    • Export Citation
  • 12

    Li C, Lee KC, Schneider EB, Zeiger MA. BRAF V600E mutation and its association with clinicopathological features of papillary thyroid cancer: a meta-analysis. Journal of Clinical Endocrinology and Metabolism 2012 97 45594570. (doi:10.1210/jc.2012-2104).

    • Search Google Scholar
    • Export Citation
  • 13

    Dutenhefner SE, Marui S, Santos AB, Lima EU, Inoue M, Neto JD, Shiang C, Fukushima J, Cernea CR, Friguglietti C. Braf, a tool in the decision to perform elective neck dissection? Thyroid 2013 23 15411546. (doi:10.1089/thy.2012.0304).

    • Search Google Scholar
    • Export Citation
  • 14

    Greene FL. In AJCC Cancer Staging Manual, 6th edn, pp 77–87. New York: Springer-Verlag, 2002

  • 15

    Vince A, Poljak M, Seme K. DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods. British Journal of Haematology 1998 101 349351. (doi:10.1046/j.1365-2141.1998.00702.x).

    • Search Google Scholar
    • Export Citation
  • 16

    Benlloch S, Paya A, Alenda C, Bessa X, Andreu M, Jover R, Castells A, Llor X, Aranda FI, Massuti B. Detection of BRAF V600E mutation in colorectal cancer: comparison of automatic sequencing and real-time chemistry methodology. Journal of Molecular Diagnostics 2006 8 540543. (doi:10.2353/jmoldx.2006.060070).

    • Search Google Scholar
    • Export Citation
  • 17

    Nikiforov YE, Steward DL, Robinson-Smith TM, Haugen BR, Klopper JP, Zhu Z, Fagin JA, Falciglia M, Weber K, Nikiforova MN. Molecular testing for mutations in improving the fine-needle aspiration diagnosis of thyroid nodules. Journal of Clinical Endocrinology and Metabolism 2009 94 20922098. (doi:10.1210/jc.2009-0247).

    • Search Google Scholar
    • Export Citation
  • 18

    Cantara S, Capezzone M, Marchisotta S, Capuano S, Busonero G, Toti P, Di Santo A, Caruso G, Carli AF, Brilli L. Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules improves the diagnostic accuracy of cytology. Journal of Clinical Endocrinology and Metabolism 2010 95 13651369. (doi:10.1210/jc.2009-2103).

    • Search Google Scholar
    • Export Citation
  • 19

    Alexander EK, Kennedy GC, Baloch ZW, Cibas ES, Chudova D, Diggans J, Friedman L, Kloos RT, LiVolsi VA, Mandel SJ. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. New England Journal of Medicine 2012 367 705715. (doi:10.1056/NEJMoa1203208).

    • Search Google Scholar
    • Export Citation
  • 20

    Liu RT, Chen YJ, Chou FF, Li CL, Wu WL, Tsai PC, Huang CC, Cheng JT. No correlation between BRAFV600E mutation and clinicopathological features of papillary thyroid carcinomas in Taiwan. Clinical Endocrinology 2005 63 461466. (doi:10.1111/j.1365-2265.2005.02367.x).

    • Search Google Scholar
    • Export Citation
  • 21

    Fugazzola L, Puxeddu E, Avenia N, Romei C, Cirello V, Cavaliere A, Faviana P, Mannavola D, Moretti S, Rossi S. Correlation between B-RAFV600E mutation and clinico-pathologic parameters in papillary thyroid carcinoma: data from a multicentric Italian study and review of the literature. Endocrine-Related Cancer 2006 13 455464. (doi:10.1677/erc.1.01086).

    • Search Google Scholar
    • Export Citation
  • 22

    Ito Y, Yoshida H, Maruo R, Morita S, Takano T, Hirokawa M, Yabuta T, Fukushima M, Inoue H, Tomoda C. BRAF mutation in papillary thyroid carcinoma in a Japanese population: its lack of correlation with high-risk clinicopathological features and disease-free survival of patients. Endocrine Journal 2009 56 8997. (doi:10.1507/endocrj.K08E-208).

    • Search Google Scholar
    • Export Citation
  • 23

    Nam JK, Jung CK, Song BJ, Lim DJ, Chae BJ, Lee NS, Park WC, Kim JS, Jung SS, Bae JS. Is the BRAF(V600E) mutation useful as a predictor of preoperative risk in papillary thyroid cancer? American Journal of Surgery 2012 203 436441. (doi:10.1016/j.amjsurg.2011.02.013).

    • Search Google Scholar
    • Export Citation
  • 24

    Trovisco V, Couto JP, Cameselle-Teijeiro J, de Castro IV, Fonseca E, Soares P, Sobrinho-Simoes M. Acquisition of BRAF gene mutations is not a requirement for nodal metastasis of papillary thyroid carcinoma. Clinical Endocrinology 2008 69 683685. (doi:10.1111/j.1365-2265.2008.03243.x).

    • Search Google Scholar
    • Export Citation
  • 25

    Lee KC, Li C, Schneider EB, Wang Y, Somervell H, Krafft M, Umbricht CB, Zeiger MA. Is BRAF mutation associated with lymph node metastasis in patients with papillary thyroid cancer? Surgery 2012 152 977983. (doi:10.1016/j.surg.2012.08.019).

    • Search Google Scholar
    • Export Citation
  • 26

    Sancisi V, Nicoli D, Ragazzi M, Piana S, Ciarrocchi A. BRAFV600E mutation does not mean distant metastasis in thyroid papillary carcinomas. Journal of Clinical Endocrinology and Metabolism 2012 97 E1745E1749. (doi:10.1210/jc.2012-1526).

    • Search Google Scholar
    • Export Citation
  • 27

    Guerra A, Fugazzola L, Marotta V, Cirillo M, Rossi S, Cirello V, Forno I, Moccia T, Budillon A, Vitale M. A high percentage of BRAFV600E alleles in papillary thyroid carcinoma predicts a poorer outcome. Journal of Clinical Endocrinology and Metabolism 2012 97 23332340. (doi:10.1210/jc.2011-3106).

    • Search Google Scholar
    • Export Citation
  • 28

    Joo JY, Park JY, Yoon YH, Choi B, Kim JM, Jo YS, Shong M, Koo BS. Prediction of occult central lymph node metastasis in papillary thyroid carcinoma by preoperative BRAF analysis using fine-needle aspiration biopsy: a prospective study. Journal of Clinical Endocrinology and Metabolism 2012 97 39964003. (doi:10.1210/jc.2012-2444).

    • Search Google Scholar
    • Export Citation
  • 29

    Randolph GW, Duh QY, Heller KS, LiVolsi VA, Mandel SJ, Steward DL, Tufano RP, Tuttle RM. The prognostic significance of nodal metastases from papillary thyroid carcinoma can be stratified based on the size and number of metastatic lymph nodes, as well as the presence of extranodal extension. Thyroid 2012 22 11441152. (doi:10.1089/thy.2012.0043).

    • Search Google Scholar
    • Export Citation