Thyroid carcinoma is the most common endocrine neoplasm and is increasing worldwide (in the USA, 8.7 per 100,000 people) [
Between January 2013 and July 2014, 70 Caucasian out-patients living in Liguria were referred to our “Thyroid Clinic” with an indeterminate cytological diagnosis according to the 2009 BTA classification [
Clinical and pathological characteristics.
Patient characteristics |
|
---|---|
Age (years) |
|
Sex: | |
Male | 13/57 (22.8%) |
Female | 44/57 (77.2%) |
US diagnosis: | |
GUN | 35/57 (61.4%) |
GMN | 22/57 (38.6%) |
Cytology diagnosis: | |
Thy 3 | 39/57 (68.4%) |
Thy 4 | 18/57 (31.6%) |
F = female; M = male; GMN = multinodular goiter; GUN = uninodular goiter.
The purpose of the study was to evaluate the effectiveness of a surgical choice based not only on the cytological diagnosis but also on the detection of BRAF mutations, in our Ligurian population.
The following protocol was adopted: If a sample was positive for a BRAF mutation, we suggested total thyroidectomy (with lymphadenectomy if the initial cytological diagnosis was Thy 4; without lymphadenectomy if the initial cytological diagnosis was Thy 3). If a sample was negative for the presence of mutation, we chose a less aggressive approach: if the initial cytological diagnosis was Thy 4, we suggested total thyroidectomy without lymphadenectomy; if the initial cytological diagnosis was Thy 3 and no nodular disease was observed in the contralateral lobe, we suggested only lobe-isthmectomy; if the initial cytological diagnosis was Thy 3 but there were nodules in the contralateral lobe and/or chronic thyroiditis, we suggested total thyroidectomy without lymphadenectomy.
Thyroids were evaluated by ultrasonography (US) using color Doppler equipment (MyLab Five, Esaote Biomedica, Genoa, Italy) equipped with a 7.5 MHz linear probe. Ultrasound-assisted FNAB was performed with the aid of the same machine. In accordance with the current guidelines for ultrasound [
Somatic point mutation in the BRAF V600 gene was determined on cytological material smeared on a slide after the pathologist had verified the adequacy of the sample and had selected areas with the highest number of neoplastic cells. The presence of nonneoplastic cells, that is, normal thyrocytes, stromal cells, and blood-derived leukocytes, was evaluated to determine the ratio of neoplastic/nonneoplastic cellular compartment. Only areas with a neoplastic/nonneoplastic cells ratio of >50% were considered to be suitable for molecular testing. After removal of the coverslip (48–72 hours), DNA was extracted from selected areas by means of a “home-made” buffer (pH 8, 1% tween) and digestion with Proteinase K, as recommended (Qiagen, Hilden, Germany). The optimal number of cells suitable for molecular studies should be 100 or more. Two methods were used to study the mutation: (i) conventional PCR followed by the direct Sanger sequencing method (according to the recommendations of the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytology (SIAPEC)); and (ii) Real Time PCR (RT PCR) with commercial kits approved for clinical use (therascreen BRAF RGQ PCR, Qiagen).
Briefly, 100–200 ng of genomic DNA was amplified by PCR using 1.5 U Platinum Taq DNA polymerase (Thermo Fisher Scientific, TFS, Milan, Italy), 1x buffer, 2 mM MgCl2, 200 nM dNTPs, 30 pmoles of forward and reverse primers in a final volume of 50
The therascreen BRAF RGQ PCR Kit is a molecular diagnostic tool for detection of the 4 different V600 mutations (V600E, V600D, V600K, and V600R, including the V600E complex) and utilizes two technologies: ARMS (Amplification Refractory Mutation System), which allows mutation-specific amplification, and Scorpions probes for the detection of amplification. This combination of techniques provides high sensitivity and high specificity. The Real Time PCR protocol and analysis of the data were performed on a Rotor-Gene Q MDx 5 plex HRM instrument by using the Rotor-Gene Q software v. 2.2.3 according to the manufacturer’s instructions.
The sensitivity of Sanger sequencing was about 12.5% mutated DNA/wt DNA, as determined in home-made experiments described in [
Only PCR assay and Sanger sequencing were able to detect the K601E mutation.
The choice of the method used for detection of BRAF mutations was based on the material and the amount of DNA extracted and assayed. Whenever possible, both procedures were performed. The laboratory was accredited by Bureau Veritas ISO (International Organization for Standardization) 9001: 2008 and the external quality control for the determination of BRAF mutations was promoted by AIOM in 2014 SIAPEC.
Statistical evaluation (Prism 6.0, GraphPad) of the correlations among cytology, histology, and molecular analysis was performed on fully evaluable patients. All data are reported as mean ± standard deviation (SD) unless otherwise specified. Continuous data were compared by means of nonparametric statistical tests. Percentages were compared by means of Fisher’s exact test. Correlations between continuous variables were determined by means of Spearman’s test.
The cytological diagnosis was undetermined in 70 patients; however, cytohistological correlation was available for only 57/70 patients (81.4%). In only one case was the material insufficient for mutations to be sought. A higher percentage of BRAF mutations were found in Thy 4 lesions (8/18 cases, 44%) than in Thy 3 lesions (6/38 cases, 16%) (Table
Correlation of BRAF status and clinicopathological characteristics.
BRAF status | ||
---|---|---|
BRAF wild-type | BRAF-mutated | |
Cytology diagnosis: | ||
Thy 3 | 34/38 (90%) | 6/38 (16%) |
Thy 4 | 10/18 (56%) | 8/18 (44%) |
Final histology: | ||
FTC | 5/42 (12%) | 0/14 (0%) |
PTC | 2/42 (5%) | 11/14 (79%) |
Nodular hyperplasia colloid or follicular adenoma | 28/42 (67%) | 2/14 (14%) |
mPTC | 3/42 (7%) | 0/14 (0%) |
Combined cell carcinoma and PTC | 1/42 (2.25%) | 0/14 (0%) |
MTC | 1/42 (2.25%) | 0/14 (0%) |
Hurtle neoplasia | 1/42 (2.25%) | 0/14 (0%) |
fPTC | 1/42 (2.25%) | 1/14 (7%) |
PTC = papillary thyroid carcinoma; FTC = follicular thyroid carcinoma; MTC = medullary thyroid carcinoma; fPTC = follicular variant of papillary carcinoma; mPTC = papillary microcarcinoma.
The nucleotide sequence of the entire exon 15 region of the BRAF gene was obtained in all 56 cases, while RT PCR was performed in 32/56 cases (57%). Mutations in the BRAF gene were detected in 14/56 cases (25%): 2/14 (14%) males and 12/14 (86%) females. In 12/14 cases (86%), the mutation identified was BRAF V600E, while in 2/14 cases (14%) BRAF K601E was detected. Notably, there was 100% concordance between the two different methods used for the detection of V600E mutations, that is, Sanger sequencing and Real Time PCR (32/32 cases).
A similar rate of V600E BRAF mutations was found on Sanger sequencing and RT PCR (12/56 mutated cases (21.4%) and 5/32 mutated cases (15.6%), resp.). The relative slightly high percentage of the V600E mutation rate among the cases analyzed by means of the sequencing method can be ascribed to the fact that the cases that proved BRAF nonmutated on sequencing were then preferentially selected for RT PCR. Of those cases in which molecular analysis of cytology specimens revealed a BRAF mutation, 2/14 (14%) proved benign on histology, while 12/14 (86%) were malignant neoplasms: 11/12 (92%) PTC and 1/12 (8%) PTC follicular variant;
Of the nonmutated BRAF cases (42/56 cases, 75%) which were later found to be malignant on definitive histology (14 cases), 5 were follicular carcinomas (36%), 3 were incidentally found papillary microcarcinomas (22%), 2 were classic papillary carcinomas (14%), 1 was a follicular variant of papillary carcinomas (7%), 1 was a medullary carcinoma (7%), 1 was a Hurtle cell tumor (7%), and 1 as a combined cell carcinoma and papillary oncocytic carcinoma (7%) (Table
If we consider only the classical variant of papillary carcinoma, a correlation between BRAF V600E mutation and a histological diagnosis of malignancy was found in 11/12 (92%) of cases. No false positive results were recorded in our series.
As a result of BRAF gene analysis, the surgical approach was changed in 17/56 patients (30% of cases). These patients had Thy 3 cytology and nonmutated BRAF gene; instead of thyroidectomy, they underwent lobe-isthmectomy alone: the histological diagnosis was benign in all these cases. The decision to undertake more conservative surgery was based on (i) Thy 3 nodule cytology, (ii) wild-type BRAF gene, and (iii) absence of ultrasound signs of malignancy. There was discordance between the endocrinological indication and the type of surgery performed in 2/56 cases (3.5%). This discrepancy involved only patients with Thy 3 nodules on cytology without the presence of multinodular goiter or thyroiditis; in these cases, a more aggressive surgical approach was adopted. The reasons for adopting a more aggressive approach were the following: in 1 case, the presence at ultrasound of a suspect lymph node which subsequently proved positive for metastasis from a papillary carcinoma and, in the other case, the intraoperative suspicion of malignancy (final histology of this case: Hurtle cell tumor).
The mean size of the nodules that underwent FNAB was
Situated in the northwest of Italy, Liguria is a region with a population of 1,583,628. The region is bounded on the south by the Ligurian Sea, on the west by France (Provence-Alpes-Cote d’Azur), on the north and east by Piedmont and Emilia Romagna, and on the southeast by Tuscany. The region is part of the Mediterranean Alps.
Thyroid nodules with indeterminate cytology have been studied for many years, with the aim of orienting surgery more accurately and reducing the number of total thyroidectomies for benign thyroid nodules (80% of Thy 3 lesions proved benign in our data).
BRAF mutations have been studied in various Italian regions. Research by Guerra et al. evaluated the frequency of the BRAF V600E mutation in thyroid nodules diagnosed in the Naples area and highlighted the importance of such research in that area [
It should be borne in mind that BRAF gene mutation, besides genetic and racial factors, seems to be influenced by the geographic area, as is testified by the percentage of papillary carcinomas in the population studied [
A recent paper by our team [
Several studies [
In 2009 [
In a recent paper, Yip et al. proposed a clinical algorithm based not only on the initial cytology but also on mutational research, as a guide to more or less radical surgery. Moreover, Yip recorded a considerably lower frequency of thyroid carcinomas in patients undergoing lobectomy if presurgical mutational research had been carried out [
A multicenter study conducted in 2013 confirmed the close correlation between the presence of a BRAF mutation and increased mortality in PTC patients [
A recent study by Liu et al. [
Another recent paper demonstrated the possibility of searching for mutations not only on fresh FNAB material but also in air-dried samples [
The latest guidelines also suggest (recommendation rating: C) the use of molecular markers (e.g., BRAF, RAS, RET/PTC, and PAX8-PPAR) in the management of thyroid nodules with indeterminate cytology [
Thus, on the basis of the data reported, it seems that patients with a BRAF V600E mutation on thyroid FNAB cytology should undergo more aggressive surgery, at least if a microcarcinoma is diagnosed [
The data obtained from our population, who had never undergone mutational study, seem to support the suggestion made in the literature, that is, more aggressive initial surgery and closer follow-up. It should also be emphasized that, on considering only the classical variant of papillary carcinoma in our population, we found a correlation between mutations on cytology and a histological diagnosis of malignancy in a high percentage of cases (92%). Another important finding is that no false positive results were recorded in our series.
In conclusion, in agreement with the literature data, the data from our population suggest that the presence of the BRAF V600E mutation should determine a more aggressive surgical approach (currently adopted only in cases of papillary carcinoma on final histology). In our limited number of cases, the BRAF K601E mutation did not correlate with malignancy indexes. A retrospective study to determine whether patients with the BRAF V600E mutation have a worse outcome than those with wild-type BRAF is currently underway.
The limitation of our study is the relatively small number of cases. In a larger population, we might have been able to discern a correlation between the ELX index and both BRAF V600E and RAS mutations.
The authors declare that they have no competing interests.
This study was supported by grants from the Italian Ministry of Health (5x1000 Funds, 2011) and Fondazione Carige 2013.