370 Immediate Interest
Authors
A. Crescenzi1, L. Guidobaldi1, N. Nasrollah2, S. Taccogna3, D. D. Cicciarella Modica1, L. Turrini3, G. Nigri4, F. Romanelli5, S. Valabrega4, L. Giovanella6, A. Onetti Muda7, P. Trimboli8
Affiliations
Affiliation addresses are listed at the end of the article
Key words
Abstract
▶ thyroid ● ▶ cancer ● ▶ BRAF(V600E) ● ▶ VE1 ● ▶ core needle biopsy (CNB) ●
received 21.10.2013 accepted 21.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368700 Published online: February 25, 2014 Horm Metab Res 2014; 46: 370–374 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Dr. A. Crescenzi Anatomia Patologica Ospedale Israelitico di Roma Via Fulda, 14 00148 Rome Italy Tel.: + 39/06/655 891 Fax: + 39/06/655 89329 [email protected]
▼
BRAF(V600E) is the most frequent genetic mutation in papillary thyroid cancer (PTC) and has been reported as an independent predictor of poor prognosis of these patients. Current guidelines do not recommend the use of BRAF(V600E) mutational analysis on cytologic specimens from fine needle aspiration due to several reasons. Recently, immunohistochemistry using VE1, a mouse anti-human BRAF(V600E) antibody, has been reported as a highly reliable technique in detecting BRAF-mutated thyroid and nonthyroid cancers. The aim of this study was to test the reliability of VE1 immunohistochemistry on microhistologic samples from core needle biopsy (CNB) in identifying BRAF-mutated PTC. A series of 30 nodules (size ranging from 7 to 22 mm) from 30 patients who underwent surgery fol-
Introduction
▼
In the last decade, the risk stratification of thyroid nodules has been improved by several immunohistochemical and molecular markers [1]. While galectin-3, HBME-1 and cytokeratin-19 have been recognized as a reliable panel to predict thyroid malignancy [2, 3], molecular analyses, such as of BRAF, RAS, RET/PTC, and PAX8/PPAR have been associated with higher cancer aggressiveness and their use has been proposed for clinical evaluation [4–6]. In particular, great relevance has been ascribed to the V600E mutation of the BRAF gene [BRAF(V600E)]. The latter, which involves substitution of glutamate (E) for valine (V) at codon 600, is found in 30–70 % of papillary thyroid carcinomas (PTC) and has been reported as an independent predictor of poor prognosis, also in patients with intrathyroid PTC clinically at low-risk of recurrence [7]. Furthermore, in a large retrospective multicenter study, BRAF(V600E) mutation seemed to be associated with increased cancer-
Crescenzi A et al. VE1 Expression on Thyroid Microhistology… Horm Metab Res 2014; 46: 370–374
lowing CNB were included in the study. All these lesions had had inconclusive cytology. In all cases, both VE1 and BRAF(V600E) genotypes were evaluated. After surgery, final histology demonstrated 21 cancers and 9 benign lesions. CNB correctly diagnosed 20/20 PTC and 5/5 adenomatous nodules. One follicular thyroid cancer and 4 benign lesions were assessed at CNB as uncertain follicular neoplasm. VE1 immunohistochemistry revealed 8 mutated PTC and 22 negative cases. A 100 % agreement was found when positive and negative VE1 results were compared with BRAF mutational status. These data are the first demonstration that VE1 immunohistochemistry performed on thyroid CNB samples perfectly matches with genetic analysis of BRAF status. Thus, VE1 antibody can be used on thyroid microhistologic specimens to detect BRAF(V600E)-mutated PTC before surgery.
related mortality among PTC patients [8]. Unfortunately, current guidelines do not recommend the use of BRAF(V600E) mutational analysis on cytologic specimens from fine needle aspiration (FNA) owing to several reasons [9–11]. Currently, the relationship between immunohistochemistry for VE1, a mouse anti-human BRAF(V600E) antibody, and BRAF genotyping is under investigation, and a strong correlation with BRAF-mutated nonthyroid cancers has been reported [12–14]. The microhistologic evaluation of samples obtained by core needle biopsy (CNB) has been proposed as a complementary test for thyroid nodules with inconclusive FNA. By CNB, a very large percentage of nodules that are read as inadequate or indeterminate at cytologic examination may be re-assessed as diagnostic, with high tolerability and good comfort for patients [15– 23]. To date, immunohistochemistry using VE1 has not been investigated in thyroid CNB specimens.
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Immunohistochemistry for BRAF(V600E) Antibody VE1 Performed in Core Needle Biopsy Samples Identifies Mutated Papillary Thyroid Cancers
Fig. 1 Gross image of a core sample.
The aim of the present study was to test the reliability of VE1 immunohistochemistry performed on thyroid CNB samples in detecting BRAF(V600E)-mutated PTC. Accordingly, both VE1 and BRAF genotypes were evaluated in a series of thyroid nodules from patients who had undergone CNB and surgery, and the results obtained from the 2 tests in histologically proven malignant and benign lesions were compared.
Patients and Methods
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Patients The series included 30 nodules from 30 patients (24 females and 6 males, age ranging from 16 to 67 years) who underwent surgery following CNB. These 30 lesions (size from 7 to 22 mm) were subjected to CNB because of inconclusive cytologic report of indeterminate (i. e., Thy/Tir 3) or not adequate (Thy/Tir 1) diagnosis. In these patients, thyroidectomy was indicated due to several reasons: PTC demonstrated at microhistology (n = 20), adenomatous nodule in a goiter with compressive symptoms (n = 5), or uncertain follicular neoplasm at CNB diagnosis (n = 5). Informed consent was obtained from all patients.
Biopsy procedure As we have previously described [21, 22], CNB was performed using a modified 21-G Menghini cutting needle (Biomol, Hospital Service, Rome, IT) in freehand fashion under ultrasound guidance. The procedure was performed in the outpatient surgery unit of Ospedale Israelitico of Rome by an experienced surgeon ▶ Fig. 1) were fixed in 10 % buffered for(NN). The core samples (● malin immediately following the biopsy.
Microhistology, VE1 immunohistochemistry, and BRAF mutational analysis Formalin-fixed tissue cores were automatically processed and embedded in paraffin. Serial 4-micron sections were collected on polarized slides and stained with hematoxylin-eosin for morphologic evaluation. Microscopic diagnosis was reported as PTC when the typical features were present, as follicular hyperplasia when microfollicular pattern was seen next to non neoplastic parenchyma, and as follicular neoplasm when microfollicular pattern was separated from non-neoplastic parenchyma by fibrous septa (probably fibrous capsule). Following microscopic diagnosis, additional paraffin sections were cut from the blocks, collected on polarized slides and submitted for immunohistochemical evaluation using an Autostainer 480S (Thermo Fisher Scientific Inc, Fremont, CA, USA): sections were pretreated with EDTA, pH 8, for 30 min and incu-
bated with diluted 1:50 mouse anti-human BRAF(V600E) antibody, clone VE1 (Spring Bioscience, Pleasanton, CA, USA) for 30 min. The reaction product was revealed by peroxidase using the biotin-free method (Thermo Fisher Scientific Inc, Fremont, CA, USA). Cases were reported as positive when a clear brown granular reaction product was seen within the cytoplasm of neoplastic cells, in the absence of background staining. A control study was performed by omitting the primary antibody. All immunostained slides were evaluated twice by 2 expert pathologists (AC, ST), blinded to the clinico-pathologic and genetic data. Eight 5-micron thick sections were cut from each block, dewaxed, hydrated and subjected to DNA extraction using the Qiagen mini kit. Mutational analysis was performed by pyrosequencing. Briefly, 5 μl of genomic DNA at 20 ng/μl, was amplified and sequenced using Anti-EGFR Moab Response® (BRAF status) for both CE-IVD marked kits (DIATECH Pharmacogenetics, Italy) on Rotor-Gene™ 6000 (Corbett Research, Australia), according to the manufacturer’s instructions. After amplification, the presence of PCR products was detected by melting analysis with a denaturation step from 65 °C upto 95 °C. For pyrosequencing analysis, single-strand biotinylated DNA was purified using the PyroMark Vacuum Prep Workstation (Biotage-Diatech, Iesi, An, Italy). A volume of 20 μl of the PCR biotinylated product was captured using streptavidin-Sepharose beads (Streptavidin Sepharose™ High Performance, GE Healthcare Bio-Science AB, Uppsala, Sweden). The primed single-stranded DNA templates were then transferred to the microtiter plate-based PSQ HS 96 (Biotage, Sweden), where real-time sequencing of the sequence surrounding codon 600 of BRAF was performed by using PyroMark Gold reagents (Qiagen) on a PyroMark™ Q96 ID instrument (Biotage, Sweden).
Final histology After surgery, thyroid samples were formalin fixed and paraffin embedded for routine histology. Thyroid tumors and lesions were classified according to the most recent criteria of the World Health Organization [24].
Statistical analysis The BRAF(V600E) genotype mutation analysis performed on microhistologic samples was considered the gold standard to verify the reliability of VE1 immunohistochemistry in detecting the BRAF mutation.
Results
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After surgery, final histology demonstrated 21 thyroid cancers. Of these, 20 PTC diagnosed at CNB were confirmed at final examination, while 1 minimally invasive follicular thyroid cancer was previously assessed at CNB as uncertain follicular neoplasm. The remaining 9/30 nodules had benign final histology following benign (n = 5) or uncertain (n = 4) microhistology by CNB. All 30 biopsies provided adequate core samples with the exception of 5 uncertain follicular neoplasms at CNB, and the malignant and benign microhistologic diagnoses were confirmed in 25/25 cases. Of importance, one cystic PTC had had inadequate FNA ▶ Table 1 summarizes the and was correctly detected by CNB. ● characteristics of the series. VE1 immunohistochemistry showed 8 BRAF(V600E)-mutated PTC (8/20 PTC, 40 %) and 22 negative cases. In particular, positive
Crescenzi A et al. VE1 Expression on Thyroid Microhistology… Horm Metab Res 2014; 46: 370–374
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Immediate Interest 371
372 Immediate Interest
Id 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
FNA class* 3 3 3 3 3 1 3 1 1 3 3 3 1 3 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
Microhistology PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC FN FN FN FN FN AN AN AN AN AN
BRAF (V600E)
Final Histology
VE1 immunohistochemistry
Mutational Status
– + + – + – – + – – – + – – + – – – + + – – – – – – – – – –
Wild type V600E + V600E + Wild type V600E + Wild type Wild type V600E + Wild type Wild type Wild type V600E + Wild type Wild type V600E + Wild type Wild type Wild type V600E + V600E + Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type
Table 1 Cytologic, CNB, and histologic characteristics of the series.
FV-PTC mixed C- and FV-PTC SV-PTC FV-PTC mixed C- and FV-PTC cystic PTC mixed C- and FV-PTC SV-PTC SV-PTC mixed C- and FV-PTC FV-PTC C-PTC SV-PTC mixed C- and FV-PTC SV-PTC FV-PTC mixed C- and FV-PTC mixed C- and FV-PTC mixed C- and FV-PTC C-PTC MI-FTC FA FA FA FA AN HCAN AN HCAN AN
PTC: Papillary thyroid cancer; FN: Follicular neoplasm; AN: Adenomatous nodule; FV-PTC: Follicular variant of PTC; SV-PTC: Sclerosing variant of PTC; C-PTC: Conventional PTC; MI-FTC: Minimally invasive follicular thyroid cancer; FA: Follicular adenoma; HCAN: Hürthle cell adenomatous nodule *Italian consensus for thyroid cytology 2010 [30]
a
b
c
Fig. 2 a Low power VE1 immunohistochemistry in a BRAF mutated PTC. b High power field from previous case: note the fine granular cytoplasmatic evidence of brown reaction product within neoplastic cells. c Pyrogram from the same sample show mutated allele of BRAF V600E.
cytoplasmic granular VE1 expression was recorded in 3 mixed conventional and follicular variant PTC, 3 sclerosing variant PTC, ▶ Fig. 2a, b). Five of these were multifoand 2 conventional PTC (● cal cancers at final histology. The remaining 22 cases with negative VE1 immunohistochemistry were histologically classified as PTC (n = 12), minimally invasive follicular cancer (n = 1), follicular adenoma (n = 4), adenomatous nodule (n = 3), and Hürthle cell adenomatous nodule (n = 2). No reaction product was detected in immunohistochemistry when primary antibody was omitted.
After extraction, mean DNA concentration in the eluate was 40 ng/μl with a A260/A280 ratio of 1.6:1.9. Eight cases resulted ▶ Fig. 2c), matching with in mutated V600E on pyrosequencing (● positive VE1 immunohistochemistry with 100 % concordance. Among the BRAF-mutated cases the mutation rate at pyrosequencing ranged from 14.2–39.1 %. In all 22 negative VE1 cases, the BRAF(V600E) mutational status resulted in wild type with 100 % agreement.
Crescenzi A et al. VE1 Expression on Thyroid Microhistology… Horm Metab Res 2014; 46: 370–374
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Core Needle Biopsy
Discussion
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Generally, differentiated thyroid cancers have an indolent behavior. However, a non-negligible percentage of these malignancies can manifest as more aggressive tumors with local invasion, neck lymph node involvement and distant metastases. These situations account for the majority of deaths from PTC. As a consequence, identifying those cancers with high aggressiveness could allow a more tailored treatment of patients. The latter is one of the most relevant challenges for thyroidologists. In this regard, the detection of BRAF mutation is recognized to be associated with aggressive PTC types [7, 8]. Indeed, BRAFmutated PTC account for about 45 % of cases, and this rate increases in more aggressive PTC subtypes (i. e., tall cell variant PTC). Also, its independent oncogenic role was demonstrated in transgenic mouse studies [25]. Thus, the identification of BRAF mutation could be useful in the stratification of patients for less or more extended treatments. Despite these data, BRAF mutation analysis is not yet widely performed due to its high cost and poor reproducibility in FNA samples. In fact, this test requires DNA extraction from at least 50 % of malignant cells obtained from well-targeted lesions. Thus, current guidelines do not recommend its use in clinical practice [10, 11]. Very recently, the approach by CNB has been proposed to better assess thyroid lesions. The cutting needles used for CNB sampling allow to obtain a tissue section from both large- and smallsize nodules, with consequent high diagnostic accuracy of the microhistologic examination. The main results of these studies show that a large proportion of lesions with prior indeterminate or inadequate cytology (Thy/Tir 3 and Thy/Tir 1, respectively) can be correctly diagnosed by CNB alone or combined with a second FNA [15–23]. Nevertheless, no studies evaluating the potential assessment of BRAF mutation in thyroid CNB samples had been performed. Here, we aimed to investigate the reliability of VE1 immunohistochemical staining in assessing BRAF mutational status in thyroid microhistology. The results demonstrated the perfect agreement between immunophenotypical VE1 expression and BRAF(V600E) mutational status. In particular, all 8 positive and 22 negative VE1 cases were confirmed in genetic analysis. This finding is highly important to better tailor the surgical approach of thyroid cancer patients [10, 11]. Also, according to previous reports, 40 % of PTC was BRAF-mutated [8]. The immunohistochemical use of the monoclonal antibody VE1, which specifically recognizes BRAF(V600E) protein, was described in several nonthyroid cancers, such as melanoma, colon adenocarcinoma, ovarian carcinoma, lung carcinoma, and hairy cell leukemia [12– 14]. These studies showed strong agreement between immunohistochemistry and genotype analysis. More recently, Zimmermann and colleagues studied VE1 antibody on cell block slides from PTC with some equivocal results [26]. Our findings corroborate and extend these interesting data, showing that BRAF(V600E) mutation of PTC can be detected by VE1 immunohistochemistry performed on CNB samples with no false results. This allows to stratify better the aggressiveness of PTC before surgery. Patients with conventional PTC type represent the main cohort that can benefit from the VE1 test, and other patients with encapsulated, mixed or follicular variant PTC, could be better identified and treated [27]. Also, the present data should prompt the development of specific immunochemical kits [28]. To date, several attempts have been made to improve the preoperative diagnosis of thyroid neoplasms by molecular tests on
cytologic samples, with discordant results [29]. The samples obtained by CNB should be the optimal material for extensive studies and ancillary techniques [23]. Certainly, microhistology can assess nuclear changes, architectural alterations in the follicular structures as well as relations with adjacent tissues. Of high relevance, CNB can be used to determine whether the nodule capsule is present or lacking [21]. Furthermore, the paraffin core sections permit automated immunostaining with high reproducibility and low cost. Here, we have shown that CNB samples should be the actual specimens for VE1 immunohistochemical profiling. The feasibility of this technique is maintained with poor cellular or fibrous samples, as demonstrated in small cell aggregates from BRAF-mutated cases with inconclusive genetic results [14]. The difference in the cost of VE1 immunohistochemistry and BRAF(V600E) mutation analysis is not a minor point. Remarkably, while the genetic analysis of BRAF has a significant expense in working time and cost of reagents, VE1 immunohistochemistry can be performed routinely, especially on paraffin core biopsy sections. The latter should greatly promote widespread BRAF testing. Some limitations of these findings have to be briefly discussed. The present study was conducted on a small sample of 30 PTC cases. Future studies including larger series and a prospective design are necessary to confirm our results for clinical practice use of VE1. In conclusion, we have shown here that VE1 immunohistochemistry performed on thyroid CNB samples perfectly matched with genetic analysis of BRAF(V600E) mutational status. These data encourage the use of VE1 immunostaining of thyroid CNB to detect BRAF(V600E)-mutated PTC before surgery.
Conflicts of Interest
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The authors declare that they have no conflicts of interest in the authorship or publication of this contribution. Affiliations Section of Pathology, Ospedale Israelitico, Rome, Italy 2 Section of Surgery, Ospedale Israelitico, Rome, Italy 3 Section of Pathology, Ospedale Regina Apostolorum/IHG2, Albano Laziale (Rome), Italy 4 Department of Surgical and Medical Sciences, Sapienza University, Ospedale S. Andrea, Rome, Italy 5 Department of Experimental Medicine, Sapienza University, Rome, Italy 6 Department of Nuclear Medicine and Thyroid Centre, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland 7 Integrated Research Center (CIR), Campus Bio-Medico University, Rome, Italy 8 Section of Endocrinology and Diabetology, Ospedale Israelitico, Rome, Italy 1
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374 Immediate Interest
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Authors
A. Crescenzi1, L. Guidobaldi1, N. Nasrollah2, S. Taccogna3, D. D. Cicciarella Modica1, L. Turrini3, G. Nigri4, F. Romanelli5, S. Valabrega4, L. Giovanella6, A. Onetti Muda7, P. Trimboli8
Affiliations
Affiliation addresses are listed at the end of the article
Key words
Abstract
▶ thyroid ● ▶ cancer ● ▶ BRAF(V600E) ● ▶ VE1 ● ▶ core needle biopsy (CNB) ●
received 21.10.2013 accepted 21.01.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1368700 Published online: February 25, 2014 Horm Metab Res 2014; 46: 370–374 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Dr. A. Crescenzi Anatomia Patologica Ospedale Israelitico di Roma Via Fulda, 14 00148 Rome Italy Tel.: + 39/06/655 891 Fax: + 39/06/655 89329 [email protected]
▼
BRAF(V600E) is the most frequent genetic mutation in papillary thyroid cancer (PTC) and has been reported as an independent predictor of poor prognosis of these patients. Current guidelines do not recommend the use of BRAF(V600E) mutational analysis on cytologic specimens from fine needle aspiration due to several reasons. Recently, immunohistochemistry using VE1, a mouse anti-human BRAF(V600E) antibody, has been reported as a highly reliable technique in detecting BRAF-mutated thyroid and nonthyroid cancers. The aim of this study was to test the reliability of VE1 immunohistochemistry on microhistologic samples from core needle biopsy (CNB) in identifying BRAF-mutated PTC. A series of 30 nodules (size ranging from 7 to 22 mm) from 30 patients who underwent surgery fol-
Introduction
▼
In the last decade, the risk stratification of thyroid nodules has been improved by several immunohistochemical and molecular markers [1]. While galectin-3, HBME-1 and cytokeratin-19 have been recognized as a reliable panel to predict thyroid malignancy [2, 3], molecular analyses, such as of BRAF, RAS, RET/PTC, and PAX8/PPAR have been associated with higher cancer aggressiveness and their use has been proposed for clinical evaluation [4–6]. In particular, great relevance has been ascribed to the V600E mutation of the BRAF gene [BRAF(V600E)]. The latter, which involves substitution of glutamate (E) for valine (V) at codon 600, is found in 30–70 % of papillary thyroid carcinomas (PTC) and has been reported as an independent predictor of poor prognosis, also in patients with intrathyroid PTC clinically at low-risk of recurrence [7]. Furthermore, in a large retrospective multicenter study, BRAF(V600E) mutation seemed to be associated with increased cancer-
Crescenzi A et al. VE1 Expression on Thyroid Microhistology… Horm Metab Res 2014; 46: 370–374
lowing CNB were included in the study. All these lesions had had inconclusive cytology. In all cases, both VE1 and BRAF(V600E) genotypes were evaluated. After surgery, final histology demonstrated 21 cancers and 9 benign lesions. CNB correctly diagnosed 20/20 PTC and 5/5 adenomatous nodules. One follicular thyroid cancer and 4 benign lesions were assessed at CNB as uncertain follicular neoplasm. VE1 immunohistochemistry revealed 8 mutated PTC and 22 negative cases. A 100 % agreement was found when positive and negative VE1 results were compared with BRAF mutational status. These data are the first demonstration that VE1 immunohistochemistry performed on thyroid CNB samples perfectly matches with genetic analysis of BRAF status. Thus, VE1 antibody can be used on thyroid microhistologic specimens to detect BRAF(V600E)-mutated PTC before surgery.
related mortality among PTC patients [8]. Unfortunately, current guidelines do not recommend the use of BRAF(V600E) mutational analysis on cytologic specimens from fine needle aspiration (FNA) owing to several reasons [9–11]. Currently, the relationship between immunohistochemistry for VE1, a mouse anti-human BRAF(V600E) antibody, and BRAF genotyping is under investigation, and a strong correlation with BRAF-mutated nonthyroid cancers has been reported [12–14]. The microhistologic evaluation of samples obtained by core needle biopsy (CNB) has been proposed as a complementary test for thyroid nodules with inconclusive FNA. By CNB, a very large percentage of nodules that are read as inadequate or indeterminate at cytologic examination may be re-assessed as diagnostic, with high tolerability and good comfort for patients [15– 23]. To date, immunohistochemistry using VE1 has not been investigated in thyroid CNB specimens.
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Immunohistochemistry for BRAF(V600E) Antibody VE1 Performed in Core Needle Biopsy Samples Identifies Mutated Papillary Thyroid Cancers
Fig. 1 Gross image of a core sample.
The aim of the present study was to test the reliability of VE1 immunohistochemistry performed on thyroid CNB samples in detecting BRAF(V600E)-mutated PTC. Accordingly, both VE1 and BRAF genotypes were evaluated in a series of thyroid nodules from patients who had undergone CNB and surgery, and the results obtained from the 2 tests in histologically proven malignant and benign lesions were compared.
Patients and Methods
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Patients The series included 30 nodules from 30 patients (24 females and 6 males, age ranging from 16 to 67 years) who underwent surgery following CNB. These 30 lesions (size from 7 to 22 mm) were subjected to CNB because of inconclusive cytologic report of indeterminate (i. e., Thy/Tir 3) or not adequate (Thy/Tir 1) diagnosis. In these patients, thyroidectomy was indicated due to several reasons: PTC demonstrated at microhistology (n = 20), adenomatous nodule in a goiter with compressive symptoms (n = 5), or uncertain follicular neoplasm at CNB diagnosis (n = 5). Informed consent was obtained from all patients.
Biopsy procedure As we have previously described [21, 22], CNB was performed using a modified 21-G Menghini cutting needle (Biomol, Hospital Service, Rome, IT) in freehand fashion under ultrasound guidance. The procedure was performed in the outpatient surgery unit of Ospedale Israelitico of Rome by an experienced surgeon ▶ Fig. 1) were fixed in 10 % buffered for(NN). The core samples (● malin immediately following the biopsy.
Microhistology, VE1 immunohistochemistry, and BRAF mutational analysis Formalin-fixed tissue cores were automatically processed and embedded in paraffin. Serial 4-micron sections were collected on polarized slides and stained with hematoxylin-eosin for morphologic evaluation. Microscopic diagnosis was reported as PTC when the typical features were present, as follicular hyperplasia when microfollicular pattern was seen next to non neoplastic parenchyma, and as follicular neoplasm when microfollicular pattern was separated from non-neoplastic parenchyma by fibrous septa (probably fibrous capsule). Following microscopic diagnosis, additional paraffin sections were cut from the blocks, collected on polarized slides and submitted for immunohistochemical evaluation using an Autostainer 480S (Thermo Fisher Scientific Inc, Fremont, CA, USA): sections were pretreated with EDTA, pH 8, for 30 min and incu-
bated with diluted 1:50 mouse anti-human BRAF(V600E) antibody, clone VE1 (Spring Bioscience, Pleasanton, CA, USA) for 30 min. The reaction product was revealed by peroxidase using the biotin-free method (Thermo Fisher Scientific Inc, Fremont, CA, USA). Cases were reported as positive when a clear brown granular reaction product was seen within the cytoplasm of neoplastic cells, in the absence of background staining. A control study was performed by omitting the primary antibody. All immunostained slides were evaluated twice by 2 expert pathologists (AC, ST), blinded to the clinico-pathologic and genetic data. Eight 5-micron thick sections were cut from each block, dewaxed, hydrated and subjected to DNA extraction using the Qiagen mini kit. Mutational analysis was performed by pyrosequencing. Briefly, 5 μl of genomic DNA at 20 ng/μl, was amplified and sequenced using Anti-EGFR Moab Response® (BRAF status) for both CE-IVD marked kits (DIATECH Pharmacogenetics, Italy) on Rotor-Gene™ 6000 (Corbett Research, Australia), according to the manufacturer’s instructions. After amplification, the presence of PCR products was detected by melting analysis with a denaturation step from 65 °C upto 95 °C. For pyrosequencing analysis, single-strand biotinylated DNA was purified using the PyroMark Vacuum Prep Workstation (Biotage-Diatech, Iesi, An, Italy). A volume of 20 μl of the PCR biotinylated product was captured using streptavidin-Sepharose beads (Streptavidin Sepharose™ High Performance, GE Healthcare Bio-Science AB, Uppsala, Sweden). The primed single-stranded DNA templates were then transferred to the microtiter plate-based PSQ HS 96 (Biotage, Sweden), where real-time sequencing of the sequence surrounding codon 600 of BRAF was performed by using PyroMark Gold reagents (Qiagen) on a PyroMark™ Q96 ID instrument (Biotage, Sweden).
Final histology After surgery, thyroid samples were formalin fixed and paraffin embedded for routine histology. Thyroid tumors and lesions were classified according to the most recent criteria of the World Health Organization [24].
Statistical analysis The BRAF(V600E) genotype mutation analysis performed on microhistologic samples was considered the gold standard to verify the reliability of VE1 immunohistochemistry in detecting the BRAF mutation.
Results
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After surgery, final histology demonstrated 21 thyroid cancers. Of these, 20 PTC diagnosed at CNB were confirmed at final examination, while 1 minimally invasive follicular thyroid cancer was previously assessed at CNB as uncertain follicular neoplasm. The remaining 9/30 nodules had benign final histology following benign (n = 5) or uncertain (n = 4) microhistology by CNB. All 30 biopsies provided adequate core samples with the exception of 5 uncertain follicular neoplasms at CNB, and the malignant and benign microhistologic diagnoses were confirmed in 25/25 cases. Of importance, one cystic PTC had had inadequate FNA ▶ Table 1 summarizes the and was correctly detected by CNB. ● characteristics of the series. VE1 immunohistochemistry showed 8 BRAF(V600E)-mutated PTC (8/20 PTC, 40 %) and 22 negative cases. In particular, positive
Crescenzi A et al. VE1 Expression on Thyroid Microhistology… Horm Metab Res 2014; 46: 370–374
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Immediate Interest 371
372 Immediate Interest
Id 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
FNA class* 3 3 3 3 3 1 3 1 1 3 3 3 1 3 1 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
Microhistology PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC PTC FN FN FN FN FN AN AN AN AN AN
BRAF (V600E)
Final Histology
VE1 immunohistochemistry
Mutational Status
– + + – + – – + – – – + – – + – – – + + – – – – – – – – – –
Wild type V600E + V600E + Wild type V600E + Wild type Wild type V600E + Wild type Wild type Wild type V600E + Wild type Wild type V600E + Wild type Wild type Wild type V600E + V600E + Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type Wild type
Table 1 Cytologic, CNB, and histologic characteristics of the series.
FV-PTC mixed C- and FV-PTC SV-PTC FV-PTC mixed C- and FV-PTC cystic PTC mixed C- and FV-PTC SV-PTC SV-PTC mixed C- and FV-PTC FV-PTC C-PTC SV-PTC mixed C- and FV-PTC SV-PTC FV-PTC mixed C- and FV-PTC mixed C- and FV-PTC mixed C- and FV-PTC C-PTC MI-FTC FA FA FA FA AN HCAN AN HCAN AN
PTC: Papillary thyroid cancer; FN: Follicular neoplasm; AN: Adenomatous nodule; FV-PTC: Follicular variant of PTC; SV-PTC: Sclerosing variant of PTC; C-PTC: Conventional PTC; MI-FTC: Minimally invasive follicular thyroid cancer; FA: Follicular adenoma; HCAN: Hürthle cell adenomatous nodule *Italian consensus for thyroid cytology 2010 [30]
a
b
c
Fig. 2 a Low power VE1 immunohistochemistry in a BRAF mutated PTC. b High power field from previous case: note the fine granular cytoplasmatic evidence of brown reaction product within neoplastic cells. c Pyrogram from the same sample show mutated allele of BRAF V600E.
cytoplasmic granular VE1 expression was recorded in 3 mixed conventional and follicular variant PTC, 3 sclerosing variant PTC, ▶ Fig. 2a, b). Five of these were multifoand 2 conventional PTC (● cal cancers at final histology. The remaining 22 cases with negative VE1 immunohistochemistry were histologically classified as PTC (n = 12), minimally invasive follicular cancer (n = 1), follicular adenoma (n = 4), adenomatous nodule (n = 3), and Hürthle cell adenomatous nodule (n = 2). No reaction product was detected in immunohistochemistry when primary antibody was omitted.
After extraction, mean DNA concentration in the eluate was 40 ng/μl with a A260/A280 ratio of 1.6:1.9. Eight cases resulted ▶ Fig. 2c), matching with in mutated V600E on pyrosequencing (● positive VE1 immunohistochemistry with 100 % concordance. Among the BRAF-mutated cases the mutation rate at pyrosequencing ranged from 14.2–39.1 %. In all 22 negative VE1 cases, the BRAF(V600E) mutational status resulted in wild type with 100 % agreement.
Crescenzi A et al. VE1 Expression on Thyroid Microhistology… Horm Metab Res 2014; 46: 370–374
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Core Needle Biopsy
Discussion
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Generally, differentiated thyroid cancers have an indolent behavior. However, a non-negligible percentage of these malignancies can manifest as more aggressive tumors with local invasion, neck lymph node involvement and distant metastases. These situations account for the majority of deaths from PTC. As a consequence, identifying those cancers with high aggressiveness could allow a more tailored treatment of patients. The latter is one of the most relevant challenges for thyroidologists. In this regard, the detection of BRAF mutation is recognized to be associated with aggressive PTC types [7, 8]. Indeed, BRAFmutated PTC account for about 45 % of cases, and this rate increases in more aggressive PTC subtypes (i. e., tall cell variant PTC). Also, its independent oncogenic role was demonstrated in transgenic mouse studies [25]. Thus, the identification of BRAF mutation could be useful in the stratification of patients for less or more extended treatments. Despite these data, BRAF mutation analysis is not yet widely performed due to its high cost and poor reproducibility in FNA samples. In fact, this test requires DNA extraction from at least 50 % of malignant cells obtained from well-targeted lesions. Thus, current guidelines do not recommend its use in clinical practice [10, 11]. Very recently, the approach by CNB has been proposed to better assess thyroid lesions. The cutting needles used for CNB sampling allow to obtain a tissue section from both large- and smallsize nodules, with consequent high diagnostic accuracy of the microhistologic examination. The main results of these studies show that a large proportion of lesions with prior indeterminate or inadequate cytology (Thy/Tir 3 and Thy/Tir 1, respectively) can be correctly diagnosed by CNB alone or combined with a second FNA [15–23]. Nevertheless, no studies evaluating the potential assessment of BRAF mutation in thyroid CNB samples had been performed. Here, we aimed to investigate the reliability of VE1 immunohistochemical staining in assessing BRAF mutational status in thyroid microhistology. The results demonstrated the perfect agreement between immunophenotypical VE1 expression and BRAF(V600E) mutational status. In particular, all 8 positive and 22 negative VE1 cases were confirmed in genetic analysis. This finding is highly important to better tailor the surgical approach of thyroid cancer patients [10, 11]. Also, according to previous reports, 40 % of PTC was BRAF-mutated [8]. The immunohistochemical use of the monoclonal antibody VE1, which specifically recognizes BRAF(V600E) protein, was described in several nonthyroid cancers, such as melanoma, colon adenocarcinoma, ovarian carcinoma, lung carcinoma, and hairy cell leukemia [12– 14]. These studies showed strong agreement between immunohistochemistry and genotype analysis. More recently, Zimmermann and colleagues studied VE1 antibody on cell block slides from PTC with some equivocal results [26]. Our findings corroborate and extend these interesting data, showing that BRAF(V600E) mutation of PTC can be detected by VE1 immunohistochemistry performed on CNB samples with no false results. This allows to stratify better the aggressiveness of PTC before surgery. Patients with conventional PTC type represent the main cohort that can benefit from the VE1 test, and other patients with encapsulated, mixed or follicular variant PTC, could be better identified and treated [27]. Also, the present data should prompt the development of specific immunochemical kits [28]. To date, several attempts have been made to improve the preoperative diagnosis of thyroid neoplasms by molecular tests on
cytologic samples, with discordant results [29]. The samples obtained by CNB should be the optimal material for extensive studies and ancillary techniques [23]. Certainly, microhistology can assess nuclear changes, architectural alterations in the follicular structures as well as relations with adjacent tissues. Of high relevance, CNB can be used to determine whether the nodule capsule is present or lacking [21]. Furthermore, the paraffin core sections permit automated immunostaining with high reproducibility and low cost. Here, we have shown that CNB samples should be the actual specimens for VE1 immunohistochemical profiling. The feasibility of this technique is maintained with poor cellular or fibrous samples, as demonstrated in small cell aggregates from BRAF-mutated cases with inconclusive genetic results [14]. The difference in the cost of VE1 immunohistochemistry and BRAF(V600E) mutation analysis is not a minor point. Remarkably, while the genetic analysis of BRAF has a significant expense in working time and cost of reagents, VE1 immunohistochemistry can be performed routinely, especially on paraffin core biopsy sections. The latter should greatly promote widespread BRAF testing. Some limitations of these findings have to be briefly discussed. The present study was conducted on a small sample of 30 PTC cases. Future studies including larger series and a prospective design are necessary to confirm our results for clinical practice use of VE1. In conclusion, we have shown here that VE1 immunohistochemistry performed on thyroid CNB samples perfectly matched with genetic analysis of BRAF(V600E) mutational status. These data encourage the use of VE1 immunostaining of thyroid CNB to detect BRAF(V600E)-mutated PTC before surgery.
Conflicts of Interest
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The authors declare that they have no conflicts of interest in the authorship or publication of this contribution. Affiliations Section of Pathology, Ospedale Israelitico, Rome, Italy 2 Section of Surgery, Ospedale Israelitico, Rome, Italy 3 Section of Pathology, Ospedale Regina Apostolorum/IHG2, Albano Laziale (Rome), Italy 4 Department of Surgical and Medical Sciences, Sapienza University, Ospedale S. Andrea, Rome, Italy 5 Department of Experimental Medicine, Sapienza University, Rome, Italy 6 Department of Nuclear Medicine and Thyroid Centre, Oncology Institute of Southern Switzerland, Bellinzona, Switzerland 7 Integrated Research Center (CIR), Campus Bio-Medico University, Rome, Italy 8 Section of Endocrinology and Diabetology, Ospedale Israelitico, Rome, Italy 1
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