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Keywords:

  • thyroid papillary carcinoma;
  • liquid-based cytology;
  • BRAF mutation;
  • prognostic parameters

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. FUNDING SOURCES
  9. REFERENCES

BACKGROUND

Activating mutations in the valine-to-glutamic acid substitution at position 600 of the v-raf murine sarcoma viral oncogene homolog B1 (BRAF-1) gene are detected frequently in patients with papillary thyroid carcinoma (PTC). These mutations have been identified in approximately 29% to 69% of PTCs and in >80% of PTCs of the tall cell variant, whereas they have not been detected in benign lesions or in the majority of those (80%) with the follicular variant of PTC. The objective of the current study was to evaluate the role of liquid-based cytology (LBC) for the detection of BRAF mutations in the outcome of patients who have thyroid PTC measuring ≤1 cm and, hence, in guiding their clinical and surgical management.

METHODS

From October 2010 through June 2011, 230 consecutive cases were diagnosed as positive for malignancy on fine-needle aspiration cytology processed by LBC. Of these, 73 PTCs ≤1 cm underwent BRAF mutational analysis. The aspirated material was processed using an LBC technique. After DNA extraction of the residual material, BRAF mutation analysis was performed using a direct sequencing method.

RESULTS

Fifty of 73 patients (68.5%) underwent surgery, and 34 of those patients (68%) had tumors that expressed a BRAF mutation (31 PTCs, including 11 tall cell variants and 3 follicular-variant PTCs). A significant association between BRAF mutation and bilaterality of cancer was observed (odds ratio, 0.077; P = .0007). BRAF mutation was associated significantly with lymph node involvement (odds ratio, 19; P = .0007) but not with extracapsular infiltration (odds ratio, 0.298; P = .179).

CONCLUSIONS

The current results indicated that BRAF gene mutations can be identified successfully on LBC material and using other cytologic methods with high reproducibility, feasibility, and informative results. The presence of a BRAF mutation may preoperatively predict the behavior of microscopic PTC, suggesting a more aggressive surgical approach. Cancer (Cancer Cytopathol) 2013;121:291–7. © 2012 American Cancer Society.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. FUNDING SOURCES
  9. REFERENCES

Thyroid nodules are a common finding in the general population. The differential diagnoses of these lesions encompass numerous entities: either non-neoplastic or neoplastic. Among the latter, the distinction between benign and malignant neoplasms represents an area fraught with inconclusive diagnoses, especially in the presence of small tumor.[1, 2]

Fine-needle aspiration cytology (FNAC) is the most important diagnostic tool for evaluating thyroid lesions. It has come into worldwide application because of its simplicity, safety, and cost-effectiveness; and its essential role is focused mainly on discriminating between benign and malignant neoplasms, correctly diagnosing the nature of a thyroid lesion in >70% of cases and leading to a correct clinical approach in >90%.[1] Conventional cytology was the first and most useful technique until the introduction, in 1996, of liquid-based cytology (LBC), which has gained popularity as an alternative method for the collection and preparation of cytologic specimens.[1, 2]

LBC includes the collection of aspirated cells into a methanol-based preservative solution then their processing with a semiautomated device. Although distinguishing slight differences in morphologic features requires only a limited training period, this alternative method has several positive aspects especially in terms of cost-effectiveness, time-sparing, and the possible simple use of many additional techniques. Indeed, a variable amount of aspirated material is usually stored in a permanent preservative solution for the preparation of a second slide or for the useful applications of immunocytochemistry and molecular testing with excellent results.[1]

Recent advances in molecular genetics of thyroid cancer are being applied for the development of new diagnostic markers on FNAC samples. The issue is based on the finding that mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF), rearranged in transformation/papillary thyroid carcinoma (RET/PTC), or rat sarcoma (RAS) genes are identified in >70% of papillary thyroid carcinomas (PTCs), and some of these mutations are associated with more aggressive tumor behavior.[10] Many groups have investigated the role of BRAF mutations in thyroid cancer and have demonstrated correlations with extrathyroid extension, advanced tumor stage, and both lymph node and (less frequently) distant metastases.[15] In particular, the detection of a valine-to-glutamic acid substitution at position 600 (V600E) of the BRAF gene on thyroid FNAC samples appears to be associated with a poorer outcome and, thus, assumes a significant diagnostic role and provides some valuable prognostic information useful for preoperative risk stratification of thyroid lesions.[16, 17]

The role of BRAF mutation identified on FNAC has been assessed by several studies in which the analyses were applied mainly to conventional cytology or using specific solutions, although very few studies analyzed the application of LBC. The objectives of the current study were: 1) to evaluate the feasibility of molecular analysis using LBC and 2) to correlate BRAF mutation in thyroid aspiration cytology of neoplastic nodules measuring ≤1 cm with thyroid tumor aggressiveness and establish a preoperative evaluation of thyroid cancer in patients who are diagnosed using LBC.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. FUNDING SOURCES
  9. REFERENCES

From October 2010 through June 2011, 230 consecutive thyroid FNAC specimens that were diagnosed as positive for malignancy (favor PTC) were recorded in the files of the Division of Anatomic Pathology and Histology of the Catholic University, Agostino Gemelli Hospital of Rome (Rome, Italy). All FNACs were carried out under ultrasound guidance, mostly by surgeons and endocrinologists, and were processed with the LBC cytologic method (Thin Prep 2000; Hologic Cytyc Company, Marlborough, Mass).

Of the 230 specimens that were positive for malignancy (favor papillary carcinoma), all 73 nodules that measured ≤1 cm were studied for the application of detecting BRAF mutations on LBC. All of these subcentimetric lesions were discovered during a routine ultrasound thyroid check-up performed at the Screening Center for Thyroid Disease in the Department of Endocrinology of our hospital. The series included 47 men and 26 women, and the median patient age was 42 years (range, 20-70 years). All aspirations (usually 2 passes for each lesion) were obtained with 25-gauge to 27-gauge needles, and no fast checks for the adequacy of the material were done. All patients had been appropriately informed regarding use of the LBC method for processing their samples, and a written informed consent form was signed by all patients.

The aspirated material was fixed with a hemolytic and preservative solution (Cytolit; Hologic Cytyc Company) after rinsing the needle in this solution. Next, the cells were spun at 1500 revolutions per minute, and the sediment was transferred to Preservcyt solution (Hologic Cytyc Company) to be processed with the T2000 automated processor according to the manufacturer's suggestions. The resulting slide was fixed in 95% ethanol and stained with Papanicolaou, whereas the remaining material was stored in Preservcyt solution to be used for further investigations.

All of our cytologic cases were classified as positive for malignancy (favor papillary carcinoma) according to the morphologic criteria adopted by the Bethesda System, which was identical to the tier-5 (TIR 5) category of the Italian Working Group Italian Society of Anatomical Pathology and Diagnostic Cytopathology-International Academy of Pathology (SIAPEC-IAP) classification.[18, 19] The lower limit for the adequacy of each sample was established, according to Goellner in 6 groups of at least 10 cells.[20] The percentage of disease-specific cells for molecular analysis was at least 50% in all LBC samples.

Fifty of 73 patients underwent surgery (total thyroidectomy) in the Division of Endocrine Surgery at our hospital. The remaining 23 patients were lost to follow-up in our hospital, although all cytologic diagnoses were confirmed, but no further management was reported concerning these 23 patients. All surgical cases included several central perithyroid lymph nodes (range, 2-14 central perithyroid lymph nodes). All surgical specimens were fixed in 10% buffered formaldehyde and embedded in paraffin, and the 5-micron-thick microtomic sections were stained with hematoxylin and eosin.

BRAF Mutational Analysis

DNA extraction was performed on fine-needle cytologic samples using the ThinPrep 2000 system (Hologic Cytyc Company) or on paraffin slides using the QIAamp tissue kit (Qiagen, Hilden, Germany). Exons 11 and 15 of the BRAF gene were amplified using the following primers: for exon 11, the forward primer was 5′-TTA TTG ATG CGA ACA GTG AAT AT-3′, and the reverse primer was 5′-TTA CAG TGG GAC AAA GAA TTG-3′; for exon 15, the forward primer was 5′-TCA TAA TGC TTG CTC TGA TAG GA-3′, and the reverse primer was 5′-GGC CAA AAA TTT AAT CAG TGG A-3′. Briefly, DNA (100-200 ng) was amplified in a mixture containing 1 times polymerase chain reaction (PCR) buffer (20 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl2), deoxyribonucleotide triphosphates (200 mM each), primers (20 pM each), and 0.5 U GoTaq polymerase (Promega, Milan, Italy) in a final volume of 25 μL. PCR conditions were as follows: initial denaturation at 95°C for 8 minutes was followed by 35 cycles at 95°C for 40 seconds, 55°C for 40 seconds, and 72°C for 40 seconds. After visualization on agarose gels, PCR products were treated with ExoSAP-IT (USB Corporation, Cleveland, Ohio) according to the manufacturer's protocol, amplified with a BigDye Terminator cycle sequencing kit (version 3.1; Applied Biosystems, Milan, Italy) using forward and reverse primers, and sequenced with an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). Water was used as negative control. The sensitivity and specificity of this mutation assay in our laboratory were 85% and 97%, respectively.[21]

Statistical Analysis

Statistical analyses were performed using a commercially available statistical software package (SPSS version 10.0; SPSS Inc., Chicago, Ill) for Windows (Microsoft, Redmond, Wash). Comparison of categorical variables was performed by chi-square statistic using the Fisher exact test when appropriate. All P values < .05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. FUNDING SOURCES
  9. REFERENCES

During the period between October 2010 and June 2011, 230 consecutive ultrasound-guided FNAC samples were diagnosed as positive for malignancy with the material processed by LBC. Among these 230 samples, 73 that measured ≤1 cm (median size, 0.2-1.0 cm) were diagnosed as positive for malignancy (PTC), including 11 that were diagnosed as positive for malignancy (favor tall cell variant of papillary carcinoma [TCV]). All 73 cytologic samples were analyzed preoperatively for BRAF mutations.

In this group of 73 PTCs, 50 patients underwent surgery for 24 classic PTCs, 15 follicular variant of PTC (FVPCs), and 11 TCVs (Table 1). The remaining 23 patients were lost to follow-up in our hospital, and we did not have any further information. Concerning those 23 patients who did not undergo surgery, 11 (47.8%) had BRAF mutations, and 12 (52.1%) had wild-type BRAF.

Table 1. Evaluation of Cases With Cytohistologic Control
 No. of Cases (%)
VariableFNACHistologyBilateralityExtracapsular InvasionLymph Node Metastases
  1. Abbreviations: FNAC, fine-needle aspiration cytology; PTC/FVPC, papillary thyroid carcinoma/follicular variant of papillary thyroid carcinoma.

  2. a

    aAll of these tall cell variant cases were included in the cytologic diagnosis of “positive for malignancy.”

Positive for malignancy (PTC/FVPC)62391429
Tall cell varianta1111111111
Overall735025 (50)13 (26)20 (48.8)

In the surgical group, 25 of 50 patients (50%) had a bilateral localization, 13 of 50 had (26%) microscopic extracapsular invasion, and 20 out 50 (48.8%) had lymph node involvement. It is noteworthy that all TCVs were bilateral, with both extracapsular invasion and lymph node involvements (Table 1).

In the surgical series, a V600E BRAF activating mutation was detected in 34 of 50 patients (68%), whereas 16 of 50 patients (32%) had a wild-type sequence (Table 1). All but 1 patient (who had a double mutation involving 2 nucleotides at codon 600; GTG[RIGHTWARDS ARROW]GAA) had a point mutation of GTG to GAG at codon 600 of BRAF exon 15. We did not observe any BRAF exon 11 mutations. In contrast to patients with TCV and PTC, who had BRAF mutations with high frequency (in all patients with TCV and in 21 of 24 patients [87.5%] with PTC), only 3 of 15 patients with FVPC had this mutation (20%) (see Table 2).

Table 2. Distribution of Histologic Diagnosis Based on the BRAF Molecular Testing
DiagnosisBRAF MutationBRAF Wild TypeOverall
  1. Abbreviations: BRAF, v-raf murine sarcoma viral oncogene homolog B1; FVPC, follicular variant of papillary thyroid carcinoma; PTC, papillary thyroid carcinoma.

FVPC31215
PTC21324
Tall cell variant11011

When we analyzed the relation between BRAF mutation status and thyroid cancer parameters of aggressiveness, we observed a significant association between the bilateral localization of thyroid cancer and this molecular alteration (odds ratio [OR], 0.015; 95% confidence interval [CI], 0.016-0.422; P = .0007; Fisher exact test) (Table 3). Table 3 indicates that the presence of a BRAF mutation also was associated significantly with lymph node-metastatic cancer (OR, 19; 95% CI, 2.247-160.7; P = 0.0007; Fisher exact test) (Table 3). Conversely, we did not observe a significant association between BRAF mutation and extracapsular infiltration (OR, 0.298; 95% CI, 0.057-1.551; P = .179; Fisher exact test) (Table 3).

Table 3. Distribution of Parameters of Multifocality, Capsular Involvement, and Lymph Node Metastases Based on BRAF Molecular Pattern
 BRAF Status 
ParameterPositiveNegativeP
  1. Abbreviations: BRAF, v-raf murine sarcoma viral oncogene homolog B1.

Monolateral cancer1214.0007
Bilateral cancer222 
Intrathyroid cancer2314.179
Extrathyroid cancer112 
Lymph node status   
Negative1515.0007
Positive191 

We also performed a BRAF mutational analysis on DNA extracted from surgical specimens that contained ≥70% tumor. The concordance of mutational analysis between surgical specimens and LBC samples was 100%.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. FUNDING SOURCES
  9. REFERENCES

Thyroid nodules are a common finding in the adult population, but only a small proportion of such nodules are malignant. The increased use of ultrasound examination in routine thyroid investigation allows the easier and more frequent identification and the possible cytologic aspiration of very small nodules (up to 1 cm), reducing the overall preoperative risk of malignancy in the general population and defining the correct treatment.[22] Thyroid papillary microcarcinomas (TPMCs) are defined by the World Health Organization Histological Classification of Thyroid Tumors[26, 27] by 1 of these 2 criteria: 1) size ≤1 cm and 2) incidental finding on histopathologic examination after thyroid surgery. Regarding this definition, TPMCs are very common neoplasms and some surgical series have reported that as much as 17% of patients undergo thyroidectomy for benign lesions. The clinical management of patients with TPMCs remains nonstandardized, because these tumors generally are considered clinically indolent and innocuous, although some of them may exhibit aggressive clinical behavior.[17, 26, 27]

A meta-analysis that encompassed >4000 papillary TPMCs indicated that 28% of patients with TPMCs had lymph node metastases, 0.6% had distant metastases, and 3.3% presented with recurrent disease.[26] These data support the idea that TPMCs may include at least 2 biologically distinct subpopulations: a population with indolent tumors that have minimal or no potential of progression and a second population that has tumors with more aggressive behavior. The ability to identify markers capable of distinguishing these 2 different subpopulations, especially in the preoperative phase, is crucial for appropriate clinical management.

For this reason, many recent studies have addressed the adoption of genetic and molecular tests to be carried out on FNAC samples to improve the accuracy of the cytologic diagnosis of these thyroid nodules.[28] Many studies have demonstrated that BRAF mutation is associated significantly with papillary carcinoma and, in particular, with aggressive histopathologic features, such as extrathyroid extension, advanced stage at presentation, lymph node metastases, tumor size >5 mm, and multifocality.[28]

BRAF mutation also predisposes tumors to dedifferentiation and to progression toward poorly differentiated and anaplastic forms, which explain the unfavorable prognosis of these patients.[30] In agreement with data from the literature, we observed that FVPCs had a low rate of BRAF mutations (3 of 15) and, conversely, that this molecular alteration was detected in all 11 TCVs, which constitute an aggressive variant of PTC.[16]

Recently, Niemeier et al proposed a combined molecular-pathologic score for the stratification of TPMCs supported by the evidence that, by adding histologic criteria to BRAF status, sensitivity increased from 77% to 96% and specificity increased from 68% to 80% in a cohort of 403 TPMCs.[26] In this manner, in the current study, we adopted the detection of BRAF mutations in all microcarcinomas that were processed using this “alternative” LBC method to improve FNAC cytologic information. However, with the possible exception of the application of LBC for prognostic purposes, this did not help in the diagnosis, for which morphology alone was satisfactory. Our results clearly indicate that the activating mutation of BRAF was particularly frequent in papillary carcinoma and in its more aggressive variant (TCV). Furthermore, we demonstrated that the presence of a BRAF mutation was associated significantly with 2 parameters of aggressiveness in thyroid tumors: lymph node metastasis (P = .0007) and bilateral localization (P = .0007).

The substantial, innovative point of our current report is the application of molecular detection to LBC material, and we have demonstrated the simplicity and feasible results obtained with this alternative method. The introduction of LBC, which offers the possibility of molecular testing for common somatic mutations in thyroid FNAC material, is emerging as a promising routine and alternative approach, especially considering that approximately 66% of thyroid cancers of follicular cell origin have at least 1 of the common genetic alterations that are absent in benign thyroid neoplasms.[28, 33, 34] From this point of view, and considering our data, the BRAF mutation analysis performed on cytologic material from FNAC of small papillary cancers may help to identify a group of patients with a greater risk of bilateral spread and lymph node involvement who should undergo more extensive surgery and prophylactic central neck dissection.[35]

To our knowledge, this is the first study that addresses the preoperative correlation between BRAF expression and clinical prognostic parameters using exclusively, and with good results, an LBC method from papillary microcarcinomas. Several studies, including a review by Mazzaferri,[36] have examined the role of BRAF in microcarcinomas exclusively in histologic samples and have reported that BRAF expression is correlated with aggressive histologic parameters and with worse prognostic behavior.

The role of BRAF detection also was studied in 38 microcarcinomas by Marchetti et al, who focused only on the role of BRAF in increasing the diagnostic efficacy of the conventional cytologic technique.[37] Those authors used a manual macrodissection technique with good diagnostic results, although, in our opinion, it was not a simple tool for improving diagnostic cytologic accuracy. Lin et al, in their 20 cytologic cases, used a specific aspiration for molecular material, correlating the BRAF mutation with lymph node involvement and extrathyroid extension.[38]

In our experience, the application of molecular analyses on LBC is feasible and highly reproducible, and it also provides high yield with informative molecular results some months after FNAC material is obtained. The quality of the detection carried out on LBC slides also is supported by our observation of complete concordance of results from BRAF mutational analyses between cytology specimens and surgical specimens. In our opinion, the quality of the results on LBC slides is comparable to the quality from conventional smears in terms of morphologic details and purity of background, as demonstrated in several studies, including some from our group.[39]

The use of LBC for molecular procedures is simple and cost-effective, and it also provides material for immunocytochemistry, such as that with HBME-1 (human mesothelial cell 1) and galectin-3, which may represent a possible aid to enhance the efficacy of the morphologic diagnosis, especially in achieving a complete preoperative assessment of a thyroid neoplasm as demonstrated in some other studies from our group in the field of follicular patterned lesions.[2, 38] Furthermore and honestly, we also add that some authors have reported high numbers of false-positive and false-negative results with the application of immunocytochemistry.

Apart from its prognostic role, this molecular application on preoperative FNAC material also assumes the critical significance of a possible focus on which specific, targeted therapies against BRAF inhibitors would be directed, especially in those patients who are not candidates for surgery. In conclusion, the BRAF evaluation described in this report plays a useful role in the preoperative prediction of the behavior of cytologic thyroid microcarcinomas, and it may be used for planning either a more aggressive surgery for the gland or an extensive lymph node dissection.

Acknowledgments

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. FUNDING SOURCES
  9. REFERENCES

We thank Elena Visca, Flavia Barbetti, Laura Cammarota, and Antonella Evangelista for their invaluable technical assistance.

FUNDING SOURCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. FUNDING SOURCES
  9. REFERENCES

No specific funding was disclosed.

CONFLICT OF INTEREST DISCLOSURES

The authors made no disclosures.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgments
  8. FUNDING SOURCES
  9. REFERENCES
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