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BRAFv600e mutation combined with thyroglobulin and fine-needle aspiration in diagnosis of lymph node metastasis of papillary thyroid carcinoma
Accepted Manuscript Title: BRAFv600e mutation combined with thyroglobulin and fine-needle aspiration in diagnosis of lymph node metastasis of papillary thyroid carcinoma Authors: Jianming Li, Jibin Liu, Xiaomeng Yu, Xiaoli Bao, Linxue Qian PII: DOI: Reference:
S0344-0338(18)30769-6 https://doi.org/10.1016/j.prp.2018.09.003 PRP 52178
To appear in: Received date: Revised date: Accepted date:
20-6-2018 21-8-2018 11-9-2018
Please cite this article as: Li J, Liu J, Yu X, Bao X, Qian L, BRAFv600e mutation combined with thyroglobulin and fine-needle aspiration in diagnosis of lymph node metastasis of papillary thyroid carcinoma, Pathology - Research and Practice (2018), https://doi.org/10.1016/j.prp.2018.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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BRAFv600e mutation combined with thyroglobulin and fine-needle aspiration in diagnosis of lymph node metastasis of papillary thyroid carcinoma Li Jianming1, MD, Liu Jibin2, PhD, Yu Xiaomeng3, BD, Bao Xiaoli4, BD,
Ultrasound Department, Beijing Friendship Hospital, Capital Medical University, Beijing,
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Qian Linxue1 PhD*
China
Ultrasound Department, Thomas Jefferson University, Philadelphia, PA, USA
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Pathology Department, Beijing Friendship Hospital, Capital Medical University, Beijing,
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Laboratory Department, Beijing Friendship Hospital, Capital Medical University, Beijing,
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China
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China
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*Corresponding author: Qian Linxue, MD, Department of Ultrasound, Beijing Friendship Hospital, Capital Medical University, No. 95 Yong’an Road, Xicheng District,
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Beijing, China. Tel: +86-13311100999; Email: [email protected]
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Abstract
Background: Papillary thyroid carcinoma (PTC) is often associated with cervical lymph node metastasis (LNM), which may cause poor prognosis. Both fine-needle aspiration cytology (FNAC) and thyroglobulin measurement with fine-needle aspiration (Tg-FNA)
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have high diagnostic efficacy, but the diagnostic values and relationships among BRAFv600e mutation (BRAFMUT) testing, FNAC and FNA-Tg are unclear. Methods: We enrolled 145 patients with confirmed PTC and lymph nodes (LNs) that were
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suspected to be metastatic based on ultrasound findings, who were treated from May 2017 to April 2018, and underwent FNAC, Tg-FNA and BRAFMUT tests. Diagnostic efficacy was
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calculated by diagnostic and chi-square tests.
Results: Diagnostic values were FNAC—sensitivity: 67%, specificity: 100%, PPV: 100%,
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NPV: 66%, accuracy: 80%; and FNA-Tg (at a cut-off of 2.23 ng/ml)—sensitivity: 97.3%,
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specificity: 87.8%, PPV: 87.8%, NPV: 97.3%, accuracy: 92.3%. In the BRAFMUT+ group,
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sensitivities and specificities were FNAC: 68.6% and 100%, Tg-FNA: 94.4% and 85.7%;
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compared with FNAC: 75% and 90.9%, Tg-FNA: 80% and 100% in the BRAFMUT− group.
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Conclusion: The combination of Tg-FNA + FNAC is useful in diagnosing metastatic PTC.
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BRAF mutational status does not affect the diagnostic performance of FNAC or Tg-FNA. Keywords: Lymph node metastasis, Papillary thyroid carcinoma, BRAFv600e test, FNAC,
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FNA-Tg
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Introduction
In China, the incidence of thyroid carcinoma has increased to 10.16 per 100,000 over the last 10 years [1], of which a large proportion is papillary thyroid carcinoma (PTC). PTC is frequently associated with cervical lymph node metastasis (LNM), which may promote loco-regional recurrence of PTC [2].
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Although ultrasound (US)-guided fine-needle aspiration cytology (FNAC) is a conventional and highly specific diagnostic method for LNM [3], its sensitivity is less robust and surgeons must face higher rates of missed diagnosis. Thyroglobulin
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measurement with fine-needle aspiration (Tg-FNA) was initially proposed in 1992 by Pacini et al. to detect LNM by PTC [4]. Several studies have shown Tg-FNA to be more
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sensitive than FNAC [5-7].
Changes in the V600E amino acid in the BRAF proto‑ oncoprotein (BRAF) cause
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constitutive activation of the mitogen‑ activated protein kinase signaling pathway, which
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induces spontaneously metastasizing tumors [8]. Some studies have confirmed the
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BRAFv600e mutation (BRAFMUT) to be the most common genetic change in PTC [9-11].
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Several scholars have also shown a positive relationship among BRAFMUT, LNM, and high
intriguing possibility.
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TNM stages [12]. Thus, BRAFMUT testing as an indicator of lymph node (LN) status is an
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However, the diagnostic powers of FNAC and FNA-Tg for BRAFMUT are unclear. To our
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knowledge, no reports on the diagnostic efficiency and interrelationships of thyroglobulin levels, FNAC and BRAFMUT are available.
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Our study compared the diagnostic value of performing FNAC, BRAFMUT and FNA-Tg, alone and in combination, to detect LMN from PTC; and evaluated whether the diagnostic accuracy of FNAC and FNA-Tg is affected by BRAFMUT.
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Materials and methods Patient selection This prospective study was approved by the institutional review board at the Beijing
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Friendship Hospital of Capital Medical University, and informed consent was obtained from all patients.
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From May 2017 to April 2018, 312 patients with (collectively) 356 LNs were evaluated by US and FNAC. Of these patients, we excluded 102 patients for whom we had incomplete
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information and 65 patients who did not undergo their surgeries in our hospital. Finally, the
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remaining 145 patients with 154 LNs were enrolled in this study. All these patients
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underwent total thyroidectomy or hemi-thyroidectomy.
All enrolled patients fulfilled the following criteria: (a) their PTC was confirmed by FNAC
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or post-surgical pathology; (b) US examinations of suspicious LNs showed any two of:
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cystic lesions, micro-calcification, rounded shape (long/short axis ratio <1.5), irregular margins, hyperechoic medulla and/or indistinct hilum. Lymph node compartments were
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defined according to the 2012 consensus classification of the American Thyroid Association.[13]
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FNAC
Ultrasound-guided FNA was performed with a 25-gauge needle connected to a 5-ml syringe. Under local anesthesia (1% lidocaine), the needle was inserted perpendicularly into a palpable lesion of a suspicious LN of the thyroid carcinoma (Figure 2). The needle
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contents were expelled onto four slides by double puncture, using a third and fourth slide, smeared and immediately immersed in 95% ethanol. For our purposes, benign cytologies were considered negative and malignant cytologies were considered positive.
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Tg-FNA After collecting FNAC samples, each needle was washed with 1.0 ml saline (0.9 % w/v of
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NaCl). Wash solution from all needles for each FNAC was pooled (final volume:1.0−1.5
ml) and sent to the laboratory within 1 week. Wash-out specimens were stored at −20 °C.
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FNAC needle washes with blood contamination were centrifuged, and clear supernatant
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was removed and tested for thyroglobulin (Tg). Blood-free washes were directly tested. In
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the needle wash solution, Tg was measured using automated immunoassay analyzer
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(Immulite, XPI, 2000, Siemens, Germany). In our laboratory, the detectable Tg
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Tg as 300+ ng/mL.
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concentration range was: 0.2−300 ng/mL. We recorded values greater than the maximum
BRAFMUT analysis
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Genomic DNA isolated from the suspicious LN was extracted from paraffin-embedded tissues. Sections were deparaffinized and collected for DNA extraction. The process was
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performed using the FFPE DNA Kit (Cat No. ADx-FF01, Xiamen, China) according to manufacturer’s instructions. Absorbance of DNA samples was measured with an ultraviolet spectrophotometer; O260/O280 values were all between 1.8 and 2.0. The DNA samples were stored at −20°C
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until real-time polymerase chain reaction (PCR) analysis (within 6 months). The BRAFMUT status of each LN was determined using the AmoyDx BRAF V600E Mutation Detection Kit (Amoy Diagnostics, Xiamen, China). The mutant BRAF gene (encoding BRAF, with
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V600E as the area of interest) was amplified with specific primers and detected with novel probes using the Applied Biosystems 7500 Fast Real-Time PCR System, according to
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manufacturer instructions. The FAM fluorescence signal was used to evaluate the mutation status of the sample. When the sample FAM Ct value was ≥28 or below the detection limit of the kit, the sample was classified as BRAFMUT−. When the sample FAM Ct value was
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< 28, the sample was classified as BRAFMUT+.
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Statistical analysis
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We used SPSS (Version 19; IBM, Armonk, New York) statistical software to analyze the
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data. Comparisons of patient and LN characteristics from the different diagnostic methods were done by the chi-square test or Fisher’s exact test. Quantitative data measurements
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were expressed as mean ± SD and range. Histological results were the diagnostic gold
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standard. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of FNAC, Tg-FNA and BRAFMUT were calculated and
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compared by diagnostic test. Kappa analysis was used to compare inter-rater reliability among the different methods. Receiver operating characteristic (ROC) curve analyses were used to determine the most appropriate threshold value for Tg-FNA, and areas under the
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ROC curves (AUCs). Comparisons of significant differences between groups were done by the chi-square test. P<0.05 was considered significant. Results
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Characteristics of patients, LNs and BRAFMUT All 145 patients (male: female ratio: 1: 3.1) and their collective 154 LNs were initially
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evaluated for metastasis, using US. They then underwent FNAC, Tg-FNA, and/or BRAF
mutation tests to make definitive diagnoses, including 69 patients (male: female 1:2.6) with
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78 LNs tested by Tg-FNA, 90 patients (1:2.6) with 90 LNs tested by BRAF mutation
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analysis, and 82 patients (1:2.3) with thyroid carcinoma tested by BRAF mutation analysis.
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Most patients in the FNAC group (63%, 92/145) underwent central neck dissection, which was similar to rates in the Tg-FNA group (72%, 50/69) and BRAF mutation group (75.6%,
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68/90). The other patients underwent other procedures, including selective neck dissection
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and radical dissection. All LNs were characterized by their compartment locations in the cervical region. Metastatic PTC tumors (per FNAC/Tg-FNA/BRAFv600e tests, respectively)
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were located in Region I (n [by test method]=2/0/1), Region II (n=7/3/5), Region III (n=12/5/8), Region IV (n=15/6/13), Region V (n=9/4/8) and Region VI (n=51/19/37).
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Among metastatic PTC that were histologically verified, US imaging features included cystic lesions (n=21), micro-calcification (n=16), indiscernible LN medulla (n=86), rounded shape (n=82) and irregular margins (n=68). Among non-metastatic PTC that were histologically verified but misdiagnosed by US, US imaging features included cystic
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lesions (n=2), micro-calcification (n=1), indiscernible LN medulla (n=53), rounded shape (n=48) and irregular margins (n=46). Region VI had the highest incidence of LN dissection (Table 1). Patients who were evaluated by FNAC, Tg-FNA or BRAFMUT tests (respectively)
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did not significantly differ in demographic data prior to examination (Table 1). Lymph node metastasis occurred in 89 patients (27 men and 62 women); the remaining 56
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patients (12 men and 44 women) had non-metastatic LNs. Mean patient ages were 40.56±12.53 years in the LNM subgroup, and 43.93±13.15 years in the non-LNM
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subgroup (Table 2). Mean LN sizes were 9.87±4.38 mm (range: 4.5–27 mm) in the LNM
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group and 8.73±4.23 mm (range: 3.5–22.5 mm) in the non-LNM group.
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Tg-FNA measurement in the LNM and non-LNM subgroups The mean values ± SD for Tg-FNA were 241.28±110.05 ng/mL (range: 1.13–300+ ng/mL)
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subgroup (P<.0001).
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in the LNM subgroup and 1.36±3.5 ng/mL (range: 0.2–19.80 ng/mL) in the non-LNM
We used ROC curves to evaluate the diagnostic capacity of Tg-FNA to detect metastatic
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PTC, based on the 78 LNs from 75 patients. The AUC was 0.987 (95% CI: 0.971–1.000), and the optimal cutoff value for FNA-Tg was 2.23 ng/mL (sensitivity: 97.3%, specificity:
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87.8%, PPV: 87.8%, NPV: 97.3%, accuracy: 92.3%; Table 4; Figure 1). The relationship between BRAFMUT and LNM Of the 90 patients with 90 tested LNs, 56 were BRAFMUT+ in the LNM subgroup, 10 were BRAFMUT−in the non-LNM subgroup, 16 were BRAFMUT− in the LNM subgroup and 8
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were BRAFMUT+ non-LNM subgroup. The association between regional LNM and BRAF V600E mutation was statistically significant (P=0.005). The relationship between the severity and extent of papillary thyroid cancer and
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BRAFMUT state LMN was significantly associated with BrafV600E state (P=0.01). Mean tumor size did not
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significantly differ between the BRAFMUT+ group (0.997±0.66 cm) and the the BRAFMUT− group (1.106±0.37 cm; P = 0.089). In addition, BRAFV600E protein expression did not
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significantly differ between male vs female patients (P = 0.779), patients with multiple
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lesions vs single lesions (P = 0.868), smooth vs rough nodule borders (P = 0.23), with vs
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without calcification (P = 0.707), with vs without extrathyroidal extension (P =0.449), with
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vs without flow color in CDFI (P =0.062), or with vs without cystic lesions (P =0.217;
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Table 3).
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Diagnostic ability of FNAC, Tg-FNA and BRAFMUT combinations FNAC found lesions to be malignant in 61 cases, benign in 30 cases, suspicious for
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malignancy in 3 cases, and nondiagnostic in 2 cases, with 67% sensitivity (61/91), 100% specificity (58/58), 100% PPV (61/61), 66% NPV (58/88), and 80% accuracy (119/149;
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Table 4).
We regarded histological results as the gold standard. Kappa values were Tg-FNA: 0.847, FNAC: 0.613, and BRAFMUT: 0.286. The kappa value of Tg-FNA +FNAC was the highest
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at 0.874. However, addition of BRAFMUT to Tg-FNA did not improve diagnostic accuracy (Table 4). Influence of BRAFMUT on the diagnostic efficacy of FNAC and/or Tg-FNA
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The influence of BRAFMUT on the diagnostic performances of FNAC and Tg-FNA is shown in Table 5. Sensitivities were FNAC: 68.6% Tg-FNA: 94.4% in the BRAFMUT+ group; and
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FNAC: 75%, Tg-FNA: 80% in the BRAFMUT−group. Specificities were FNAC: 100%, TgFNA: 85.7% in the BRAFMUT+ group; and FNAC: 90.9%, Tg-FNA: 100% in the
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BRAFMUT−group. These values did not significantly differ for either FNAC (sensitivity:
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P=0.716, specificity: P=0.193) or Tg-FNA (sensitivity: P=0.311; specificity: P=0.423;
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Table 5). Comparison of FNAC and BRAFMUT tests
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Histological results were regarded as the gold standard. For cytology analyses, we
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classified the FNAC diagnostic results as metastatic, suspicious, indeterminate or benign. FNAC confirmed 61 metastatic LNM, of which 42 LNs underwent BRAFMUT analysis (35
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positive, 7 negative). Of the 58 LNs identified as benign by FNAC, 30 were histologically found to be malignant. BRAFMUT analysis identified 17 positive and 10 negative LNs
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correctly; and 17 positive and 2 negative LNs incorrectly. Of 2 metastatic LNs identified as suspicious and 3 metastatic LNs identified as indeterminate by FNAC, 2 were verified to be BRAFMUT+, and 3 patients did not undergo the BRAFMUT test. Details of BRAFMUT analyses are shown in Table 6.
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Discussion Thyroid malignancies are among the most common cancers worldwide. In 2014, the incidence of thyroid carcinoma was 10.16 cases per 100,000 in Beijing [1]. The increased
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reported incidence of thyroid cancer may reflect improved detection methods [14]. In 2017, the age-standardized incidence rate of thyroid cancer was 21.0 cases among women
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and 7.0 cases among men per 100,000 in North America [15]. Despite increasing
incidences, the total mortality is low, as benign nodules account for a large proportion.
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Lymph node metastases increase the risk of recurrence and death in thyroid malignancies.
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Therefore, the use of imaging modalities and FNAC to detect LNM are more widely used
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to define the appropriate extent of surgery necessary to optimize regional control [2].
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Reportedly, preoperative US has low accuracy for thyroid cancer LNM, at only 32.9%
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[16]. Contrast-enhanced ultrasonography (CEUS) is a helpful technique that can identify vascular architectural changes in LNs that are suggestive of malignant infiltration, but
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cannot easily reduce the differential diagnosis of LN status [17]. FNAC is thus the most
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important diagnostic tool, with high specificity for LNM [5]. In our study, its diagnostic values were sensitivity: 67%, specificity: 100%, PPV: 100%, NPV: 66% and accuracy:
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80%—similar to the results of Zhao et al [5]. In 2015, American Thyroid Association (ATA) Management Guidelines recommended TgFNA as a useful ancillary tool for detecting LNM from PTC, with high sensitivity and specificity [2]. We conclude that Tg-FNA of metastatic PTC measurement is
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241.28±110.05 ng/ml, and 1.36±3.5 ng/ml in non-metastatic PTC at preoperative evaluation. The optimal cutoff value we recommend is 2.23 ng/mL (sensitivity: 97.3%, specificity: 87.8%, PPV: 87.8%, NPV: 97.3%, accuracy: 92.3%). FNA-Tg alone was
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superior to FNAC in identifying LNM. The addition of Tg-FNA to FNAC further improved the diagnostic value (sensitivity:100%, specificity: 88.4%, PPV: 87.8%, NPV:
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100%, accuracy: 93.7%). Our results also showed kappa values of Tg-FNA + FNAC:
0.874, Tg-FNA alone: 0.847, and FNAC alone: 0.613. These results were similar to those
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of other reported studies [5, 6].
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Our study confirmed that BRAFMUT in the thyroid nodule is associated with LN metastasis,
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in accordance with other studies [12]. The relationship between BRAFMUT in the thyroid
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nodule and other invasive symptoms (extrathyroidal extension, calcification, cystic lesions
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etc.) is not statistically significant [10]. Furthermore, some studies found no correlation between BRAFMUT and tumor severity [18-20], possibly because this BRAF mutation may
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perform differently in various PTC subtypes [18]. Our study tested for BRAF mutations in
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90 LNs and 82 thyroid carcinomas. The carcinoma of one patient was confirmed positive, whereas his LN was confirmed negative. Immunohistochemical analysis has shown
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BRAFV600E expression to vary in normal thyroid tissue, nodule hyperplasia and PTC [12]. The association between BRAFMUT and LNM has been widely investigated. Our study confirmed BRAFMUT in LNs was significantly associated with metastatic PTC (P=0.005), in agreement with previous studies [10, 11]. Our results also confirmed the kappa value of BRAFMUT to be 0.286 with respect to diagnosis (sensitivity: 77.8%, specificity: 55.6%,
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PPV: 87.5%, NPV: 38.5%, accuracy: 73.3%). These results are in line with the study of Dong et al [18]. Although BRAFMUT alone may not accurately indicate LN status, 5 metastatic LNs in our study that were assessed as suspicious or indeterminate by FNAC
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were identified as positive in BRAFMUT analysis. This result suggests that BRAFMUT tests of LNs could be a supportive diagnostic method for PTC patients. Our findings were
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similar to those of Kim et al [9].
The cutoff value and diagnostic efficiency of Tg-FNA are still controversial [5, 6, 21], and
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can be influenced by many factors, such as the presence or absence of the thyroid gland
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[5]. Therefore, we explored the influence of BRAF mutation on the diagnostic efficiency of
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FNAC and FNA-Tg. We found that the presence or absence of BRAF mutations had little
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effect on FNAC and FNA-Tg diagnostic sensitivities.
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Our study has certain limitations. First, as the sample size was small, and may reflect selection bias. A multi-institutional study with a larger cohort is needed to verify our
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results. Second, whether non-metastatic BRAFMUT+ LNs may deteriorate is unclear, and
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should also be checked in a future large-scale study. Conclusion
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Our study demonstrates that Tg-FNA combined with FNAC is a useful diagnostic test that improves detection of metastatic PTC. BRAF mutation status did not obviously affect the diagnostic performance of FNAC or Tg-FNA.
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Authors’ contributions Li Jianming: research design, acquisition and analysis of data, drafting the paper, approval of the submitted and final versions; Liu Jibin: critical paper revisions; Bao Xiaoli:
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thyroglobulin measurement; Yu Xiaomeng: thyroglobulin measurement; QianLinxue: research design, author coordination, approval of the submitted and final versions.
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Funding
This work was funded by the National Natural Science Foundation of China (041320015)
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Competing interests
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All the authors report no conflicts of interest
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Acknowledgment
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We thank Marla Brunker, from Liwen Bianji, Edanz Group China
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(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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papillary thyroid cancer: Different histological subtypes and preoperative lymph node status should be taken into account, Oncology letters, 14 (2017) 4122-4134.
[19] G.C. Leonardi, S. Candido, M. Carbone, F. Raiti, V. Colaianni, S. Garozzo, D. Cina, J.A.
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McCubrey, M. Libra, BRAF mutations in papillary thyroid carcinoma and emerging targeted therapies (review), Molecular medicine reports, 6 (2012) 687-694.
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[20] D. Chen, W. Qi, P. Zhang, Y. Zhang, Y. Liu, H. Guan, L. Wang, Investigation of BRAF V600E
detection approaches in papillary thyroid carcinoma, Pathology, research and practice, 214 (2018)
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303-307.
N
[21] A. Salmaslioglu, Y. Erbil, G. Citlak, F. Ersoz, S. Sari, A. Olmez, M. Tunaci, D. Yilmazbayhan,
A
N. Colak, S. Ozarmagan, Diagnostic value of thyroglobulin measurement in fine-needle aspiration
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biopsy for detecting metastatic lymph nodes in patients with papillary thyroid carcinoma,
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Figure legend
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Langenbeck's archives of surgery, 396 (2011) 77-81.
Figure 1. Flow chart of recommended management of suspicious lymph nodes in papillary
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thyroid carcinoma, using US. BRAFV600E: V600E amino acid in the BRAF proto‑ oncoprotein; FNAC: Fine-needle aspiration cytology; Tg-FNA: Thyroglobulin measurement with fine-needle aspiration; US: Ultrasound.
19
Figure 2. Receiving operating characteristic (ROC) curve of lymph nodes measured using Tg-FNA. The optimal cutoff value for FNA-Tg was 2.23 ng/mL. Area under ROC curve: 0.987 (95% confidence interval: 0.971–1.000); sensitivity: 97.3%; specificity: 87.8%; PPV:
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87.8%; NPV: 97.3%; accuracy: 92.3%. NPV: Negative predictive value; PPV: Positive predictive value; Tg-FNA: Thyroglobulin measurement with fine-needle aspiration.
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Detection of lymph node in
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nodes in papillary thyroid carcinoma, using US.
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Figure 1. Flow chart of recommended management of suspicious lymph
A
preoperative patients with papillary
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thyroid carcinoma
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At least two of US features: (312 patients and 356 lymph
incomplete information; Cystic lesions, Micro-calcification, 65 patients: did not Rounded shape (long/short axis ratio <1.5), Irregular margins, (145 patients with 154 lymph nodes) undergo their surgeries HyperechoicFNAC medulla and/or indistinct hilum
A
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1. 2. 3. 4. 5.
Excluded 102 patients :
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nodes)
BRAFv600e mutation Test
(90 patients with 90 lymph nodes)
in hospital FNA-Tg Test (69
BRAFv600e mutation and FNA-Tg
patients with 78 lymph
Test (41 patients with 41 lymph
nodes)
nodes)
20
Figure 2. Receiving operating characteristic (ROC) curve of lymph nodes measured on TgFNA. The optimal cutoff value for FNA-Tg was 2.23 ng/mL. Area under the ROC curve: 0.987; 95% confidence interval: 0.971–1.000); sensitivity: 97.3%; specificity: 87.8%; PPV:
A
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ED
M
A
N
U
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87.8%; NPV: 97.3%; accuracy: 92.3%.
21
Table 1. Clinical characteristics of patients with papillary thyroid carcinoma by diagnostic method. FNAC
Tg-FNA
BRAFv600e
35 vs 110
19 vs 50
25 vs 65
Central neck dissection
98
50
68
Selective neck dissection
41
12
13
Radical dissection
15
7
9
I Region
2
0
II Region
7
3
III Region
12
5
IV Region
15
6
V Region
9
4
VI Region
51
19
Total
96
37
Gender Male vs female
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Location of Metastatic PTC
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Surgical procedures
1 5 8
13
N
U
8
37 72
A
CC E
PT
ED
M
A
FNAC= Fine-needle aspiration cytology; Tg-FNA= Thyroglobulin measurement with fineneedle aspiration; BRAFv600e=V600E amino acid in the BRAF proto‑ oncoprotein; PTC= Papillary thyroid carcinoma.
22
Table 2. Characteristics of patients with metastatic PTC or non-metastatic PTC Histology
Metastatic PTC
Non-metastatic PTC
P value
27 vs 62
12 vs 44
0.239
Age
40.56±12.53
43.93±13.15
0.203
Number of nodes
96
58
NA
Size of node (mm)
9.87±4.38
8.73±4.23
0.429
Gender Male vs female
NA 21
2
micro-calcification
16
1
medulla or hilum disappears
86
53
rounded shape
82
48
irregular margins
68
46
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cystic lesions
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US imaging
A
CC E
PT
ED
M
A
N
U
PTC= Papillary thyroid carcinoma; US= Ultrasound.
23
Table 3. Relationships between thyroid carcinoma characteristics and BRAFv600e mutations. Mutation Detected
Mutation Not Detected
Gender
64
18
Male
20
5
Female
44
13
Tumor Size
0.997±0.66
Calcification
0.779
1.106±0.37
No
49
13
Nodule border in the US
15
5
Rough
22
9
Blood flow in the CDFI
42
9
Smooth
38
Capsule evolving
26
3
Multiple lesions
52
M
12
Multiple
No Yes
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No
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Lymph node metastasis
0.230
0.062
0.449
2 16
20
6
44
12
7
4
57
14
46
7
18
11
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Single Yes
A
Yes No
0.707
15
N
No
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Yes
0.089
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Yes
Cystic lesion
P
BRAF V600E status of primary tumor
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Characteristic
0.868
0.217
0.01*
A
BRAFv600: V600E amino acid in the BRAF proto-oncoprotein; CDFI: Color Doppler flow imaging; US: Ultrasound.
24
Table 4. Diagnostic values of FNAC, Tg-FNA and BRAFv600e mutations. Diagnostic modality Sensitivity
Specificity
PPV
NPV
Accuracy
Kappa
BRAFv600e
77.8%(56/72)
55.6%(10/18)
87.5%(56/64)
38.5%(10/26)
73.3%(66/90)
0.286
Tg-FNA
97.3%(36/37)
87.8%(36/41)
87.8%(36/41)
97.3%(36/37)
92.3%(72/78)
0.847
FNAC
67%(61/91)
100%(58/58)
100%(61/61)87. 66%(58/88)
80%(119/149)
0.613
FNAC + Tg-FNA
100%(36/36)
88.4%(38/43)
8%(36/41)
100%(38/38)
93.7%(74/79)
0.874
BRAFv600e+FNAC
96.6%(57/59)
34.5%(10/29)
75%(57/76)
83.3%(10/12)
76.1%(67/88)
0.365
22.2%(4/18)
61.1%(22/36)
80%(4/5)
63.4%(26/41)
0.194
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BRAFv600e+Tg-FNA 95.7%(22/23)
A
CC E
PT
ED
M
A
N
U
SC R
FNAC= Fine-needle aspiration cytology; Tg-FNA= Thyroglobulin measurement with fineneedle aspiration; BRAFv600e= V600E amino acid in the BRAF proto‑oncoprotein; PPV= positive predictive value; NPV= negative predictive value
25
Table 5. Influence of BRAFv600e mutations on the diagnostic efficacy of FNAC and TgFNA BRAF status
Histology
FNAC+
FNAC-
Tg-FNA+
Tg-FNA-
BRAFv600e+
Metastatic PTC
35
16
17
1
Non-metastasis
0
18
2
12
Metastatic PTC
6
2
4
1
Non-metastasis
1
10
0
4
BRAFv600e
-
A
CC E
PT
ED
M
A
N
U
SC R
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BRAFv600e=V600E amino acid in the BRAF proto-oncoprotein; FNAC= Fine-needle aspiration cytology; Tg-FNA= Thyroglobulin measurement with fine-needle aspiration; PTC= Papillary thyroid carcinoma.
26
Table 6. Comparison of FNAC and the BRAFv600e mutation test FNAC
Histology
BRAF mutation analysis
Metastatic PTC
61(Metastatic PTC)
35 positive,7 negative,19 NA
Suspicious
2(Metastatic PTC)
1 positive, 1 NA
Benign
58(Non-metastasis),
17 positive, 10 negative, 31 NA
30(Metastatic PTC)
17 positive, 2 negative, 11 NA
3(Metastatic PTC)
1 positive, 2 NA
Indeterminate
A
CC E
PT
ED
M
A
N
U
SC R
IP T
FNAC= Fine-needle aspiration cytology; BRAFv600e=V600E amino acid in the BRAF proto‑oncoprotein; PTC= Papillary thyroid carcinoma.
S0344-0338(18)30769-6 https://doi.org/10.1016/j.prp.2018.09.003 PRP 52178
To appear in: Received date: Revised date: Accepted date:
20-6-2018 21-8-2018 11-9-2018
Please cite this article as: Li J, Liu J, Yu X, Bao X, Qian L, BRAFv600e mutation combined with thyroglobulin and fine-needle aspiration in diagnosis of lymph node metastasis of papillary thyroid carcinoma, Pathology - Research and Practice (2018), https://doi.org/10.1016/j.prp.2018.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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BRAFv600e mutation combined with thyroglobulin and fine-needle aspiration in diagnosis of lymph node metastasis of papillary thyroid carcinoma Li Jianming1, MD, Liu Jibin2, PhD, Yu Xiaomeng3, BD, Bao Xiaoli4, BD,
Ultrasound Department, Beijing Friendship Hospital, Capital Medical University, Beijing,
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1
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Qian Linxue1 PhD*
China
Ultrasound Department, Thomas Jefferson University, Philadelphia, PA, USA
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Pathology Department, Beijing Friendship Hospital, Capital Medical University, Beijing,
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2
Laboratory Department, Beijing Friendship Hospital, Capital Medical University, Beijing,
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4
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China
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China
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*Corresponding author: Qian Linxue, MD, Department of Ultrasound, Beijing Friendship Hospital, Capital Medical University, No. 95 Yong’an Road, Xicheng District,
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Beijing, China. Tel: +86-13311100999; Email: [email protected]
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Abstract
Background: Papillary thyroid carcinoma (PTC) is often associated with cervical lymph node metastasis (LNM), which may cause poor prognosis. Both fine-needle aspiration cytology (FNAC) and thyroglobulin measurement with fine-needle aspiration (Tg-FNA)
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have high diagnostic efficacy, but the diagnostic values and relationships among BRAFv600e mutation (BRAFMUT) testing, FNAC and FNA-Tg are unclear. Methods: We enrolled 145 patients with confirmed PTC and lymph nodes (LNs) that were
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suspected to be metastatic based on ultrasound findings, who were treated from May 2017 to April 2018, and underwent FNAC, Tg-FNA and BRAFMUT tests. Diagnostic efficacy was
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calculated by diagnostic and chi-square tests.
Results: Diagnostic values were FNAC—sensitivity: 67%, specificity: 100%, PPV: 100%,
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NPV: 66%, accuracy: 80%; and FNA-Tg (at a cut-off of 2.23 ng/ml)—sensitivity: 97.3%,
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specificity: 87.8%, PPV: 87.8%, NPV: 97.3%, accuracy: 92.3%. In the BRAFMUT+ group,
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sensitivities and specificities were FNAC: 68.6% and 100%, Tg-FNA: 94.4% and 85.7%;
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compared with FNAC: 75% and 90.9%, Tg-FNA: 80% and 100% in the BRAFMUT− group.
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Conclusion: The combination of Tg-FNA + FNAC is useful in diagnosing metastatic PTC.
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BRAF mutational status does not affect the diagnostic performance of FNAC or Tg-FNA. Keywords: Lymph node metastasis, Papillary thyroid carcinoma, BRAFv600e test, FNAC,
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FNA-Tg
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Introduction
In China, the incidence of thyroid carcinoma has increased to 10.16 per 100,000 over the last 10 years [1], of which a large proportion is papillary thyroid carcinoma (PTC). PTC is frequently associated with cervical lymph node metastasis (LNM), which may promote loco-regional recurrence of PTC [2].
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Although ultrasound (US)-guided fine-needle aspiration cytology (FNAC) is a conventional and highly specific diagnostic method for LNM [3], its sensitivity is less robust and surgeons must face higher rates of missed diagnosis. Thyroglobulin
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measurement with fine-needle aspiration (Tg-FNA) was initially proposed in 1992 by Pacini et al. to detect LNM by PTC [4]. Several studies have shown Tg-FNA to be more
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sensitive than FNAC [5-7].
Changes in the V600E amino acid in the BRAF proto‑ oncoprotein (BRAF) cause
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constitutive activation of the mitogen‑ activated protein kinase signaling pathway, which
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induces spontaneously metastasizing tumors [8]. Some studies have confirmed the
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BRAFv600e mutation (BRAFMUT) to be the most common genetic change in PTC [9-11].
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Several scholars have also shown a positive relationship among BRAFMUT, LNM, and high
intriguing possibility.
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TNM stages [12]. Thus, BRAFMUT testing as an indicator of lymph node (LN) status is an
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However, the diagnostic powers of FNAC and FNA-Tg for BRAFMUT are unclear. To our
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knowledge, no reports on the diagnostic efficiency and interrelationships of thyroglobulin levels, FNAC and BRAFMUT are available.
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Our study compared the diagnostic value of performing FNAC, BRAFMUT and FNA-Tg, alone and in combination, to detect LMN from PTC; and evaluated whether the diagnostic accuracy of FNAC and FNA-Tg is affected by BRAFMUT.
4
Materials and methods Patient selection This prospective study was approved by the institutional review board at the Beijing
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Friendship Hospital of Capital Medical University, and informed consent was obtained from all patients.
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From May 2017 to April 2018, 312 patients with (collectively) 356 LNs were evaluated by US and FNAC. Of these patients, we excluded 102 patients for whom we had incomplete
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information and 65 patients who did not undergo their surgeries in our hospital. Finally, the
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remaining 145 patients with 154 LNs were enrolled in this study. All these patients
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A
underwent total thyroidectomy or hemi-thyroidectomy.
All enrolled patients fulfilled the following criteria: (a) their PTC was confirmed by FNAC
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or post-surgical pathology; (b) US examinations of suspicious LNs showed any two of:
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cystic lesions, micro-calcification, rounded shape (long/short axis ratio <1.5), irregular margins, hyperechoic medulla and/or indistinct hilum. Lymph node compartments were
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defined according to the 2012 consensus classification of the American Thyroid Association.[13]
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FNAC
Ultrasound-guided FNA was performed with a 25-gauge needle connected to a 5-ml syringe. Under local anesthesia (1% lidocaine), the needle was inserted perpendicularly into a palpable lesion of a suspicious LN of the thyroid carcinoma (Figure 2). The needle
5
contents were expelled onto four slides by double puncture, using a third and fourth slide, smeared and immediately immersed in 95% ethanol. For our purposes, benign cytologies were considered negative and malignant cytologies were considered positive.
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Tg-FNA After collecting FNAC samples, each needle was washed with 1.0 ml saline (0.9 % w/v of
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NaCl). Wash solution from all needles for each FNAC was pooled (final volume:1.0−1.5
ml) and sent to the laboratory within 1 week. Wash-out specimens were stored at −20 °C.
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FNAC needle washes with blood contamination were centrifuged, and clear supernatant
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was removed and tested for thyroglobulin (Tg). Blood-free washes were directly tested. In
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the needle wash solution, Tg was measured using automated immunoassay analyzer
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(Immulite, XPI, 2000, Siemens, Germany). In our laboratory, the detectable Tg
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Tg as 300+ ng/mL.
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concentration range was: 0.2−300 ng/mL. We recorded values greater than the maximum
BRAFMUT analysis
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Genomic DNA isolated from the suspicious LN was extracted from paraffin-embedded tissues. Sections were deparaffinized and collected for DNA extraction. The process was
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performed using the FFPE DNA Kit (Cat No. ADx-FF01, Xiamen, China) according to manufacturer’s instructions. Absorbance of DNA samples was measured with an ultraviolet spectrophotometer; O260/O280 values were all between 1.8 and 2.0. The DNA samples were stored at −20°C
6
until real-time polymerase chain reaction (PCR) analysis (within 6 months). The BRAFMUT status of each LN was determined using the AmoyDx BRAF V600E Mutation Detection Kit (Amoy Diagnostics, Xiamen, China). The mutant BRAF gene (encoding BRAF, with
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V600E as the area of interest) was amplified with specific primers and detected with novel probes using the Applied Biosystems 7500 Fast Real-Time PCR System, according to
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manufacturer instructions. The FAM fluorescence signal was used to evaluate the mutation status of the sample. When the sample FAM Ct value was ≥28 or below the detection limit of the kit, the sample was classified as BRAFMUT−. When the sample FAM Ct value was
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< 28, the sample was classified as BRAFMUT+.
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Statistical analysis
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We used SPSS (Version 19; IBM, Armonk, New York) statistical software to analyze the
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data. Comparisons of patient and LN characteristics from the different diagnostic methods were done by the chi-square test or Fisher’s exact test. Quantitative data measurements
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were expressed as mean ± SD and range. Histological results were the diagnostic gold
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standard. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of FNAC, Tg-FNA and BRAFMUT were calculated and
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compared by diagnostic test. Kappa analysis was used to compare inter-rater reliability among the different methods. Receiver operating characteristic (ROC) curve analyses were used to determine the most appropriate threshold value for Tg-FNA, and areas under the
7
ROC curves (AUCs). Comparisons of significant differences between groups were done by the chi-square test. P<0.05 was considered significant. Results
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Characteristics of patients, LNs and BRAFMUT All 145 patients (male: female ratio: 1: 3.1) and their collective 154 LNs were initially
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evaluated for metastasis, using US. They then underwent FNAC, Tg-FNA, and/or BRAF
mutation tests to make definitive diagnoses, including 69 patients (male: female 1:2.6) with
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78 LNs tested by Tg-FNA, 90 patients (1:2.6) with 90 LNs tested by BRAF mutation
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analysis, and 82 patients (1:2.3) with thyroid carcinoma tested by BRAF mutation analysis.
M
A
Most patients in the FNAC group (63%, 92/145) underwent central neck dissection, which was similar to rates in the Tg-FNA group (72%, 50/69) and BRAF mutation group (75.6%,
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68/90). The other patients underwent other procedures, including selective neck dissection
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and radical dissection. All LNs were characterized by their compartment locations in the cervical region. Metastatic PTC tumors (per FNAC/Tg-FNA/BRAFv600e tests, respectively)
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were located in Region I (n [by test method]=2/0/1), Region II (n=7/3/5), Region III (n=12/5/8), Region IV (n=15/6/13), Region V (n=9/4/8) and Region VI (n=51/19/37).
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Among metastatic PTC that were histologically verified, US imaging features included cystic lesions (n=21), micro-calcification (n=16), indiscernible LN medulla (n=86), rounded shape (n=82) and irregular margins (n=68). Among non-metastatic PTC that were histologically verified but misdiagnosed by US, US imaging features included cystic
8
lesions (n=2), micro-calcification (n=1), indiscernible LN medulla (n=53), rounded shape (n=48) and irregular margins (n=46). Region VI had the highest incidence of LN dissection (Table 1). Patients who were evaluated by FNAC, Tg-FNA or BRAFMUT tests (respectively)
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did not significantly differ in demographic data prior to examination (Table 1). Lymph node metastasis occurred in 89 patients (27 men and 62 women); the remaining 56
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patients (12 men and 44 women) had non-metastatic LNs. Mean patient ages were 40.56±12.53 years in the LNM subgroup, and 43.93±13.15 years in the non-LNM
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subgroup (Table 2). Mean LN sizes were 9.87±4.38 mm (range: 4.5–27 mm) in the LNM
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group and 8.73±4.23 mm (range: 3.5–22.5 mm) in the non-LNM group.
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Tg-FNA measurement in the LNM and non-LNM subgroups The mean values ± SD for Tg-FNA were 241.28±110.05 ng/mL (range: 1.13–300+ ng/mL)
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subgroup (P<.0001).
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in the LNM subgroup and 1.36±3.5 ng/mL (range: 0.2–19.80 ng/mL) in the non-LNM
We used ROC curves to evaluate the diagnostic capacity of Tg-FNA to detect metastatic
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PTC, based on the 78 LNs from 75 patients. The AUC was 0.987 (95% CI: 0.971–1.000), and the optimal cutoff value for FNA-Tg was 2.23 ng/mL (sensitivity: 97.3%, specificity:
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87.8%, PPV: 87.8%, NPV: 97.3%, accuracy: 92.3%; Table 4; Figure 1). The relationship between BRAFMUT and LNM Of the 90 patients with 90 tested LNs, 56 were BRAFMUT+ in the LNM subgroup, 10 were BRAFMUT−in the non-LNM subgroup, 16 were BRAFMUT− in the LNM subgroup and 8
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were BRAFMUT+ non-LNM subgroup. The association between regional LNM and BRAF V600E mutation was statistically significant (P=0.005). The relationship between the severity and extent of papillary thyroid cancer and
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BRAFMUT state LMN was significantly associated with BrafV600E state (P=0.01). Mean tumor size did not
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significantly differ between the BRAFMUT+ group (0.997±0.66 cm) and the the BRAFMUT− group (1.106±0.37 cm; P = 0.089). In addition, BRAFV600E protein expression did not
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significantly differ between male vs female patients (P = 0.779), patients with multiple
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lesions vs single lesions (P = 0.868), smooth vs rough nodule borders (P = 0.23), with vs
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without calcification (P = 0.707), with vs without extrathyroidal extension (P =0.449), with
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vs without flow color in CDFI (P =0.062), or with vs without cystic lesions (P =0.217;
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Table 3).
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Diagnostic ability of FNAC, Tg-FNA and BRAFMUT combinations FNAC found lesions to be malignant in 61 cases, benign in 30 cases, suspicious for
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malignancy in 3 cases, and nondiagnostic in 2 cases, with 67% sensitivity (61/91), 100% specificity (58/58), 100% PPV (61/61), 66% NPV (58/88), and 80% accuracy (119/149;
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Table 4).
We regarded histological results as the gold standard. Kappa values were Tg-FNA: 0.847, FNAC: 0.613, and BRAFMUT: 0.286. The kappa value of Tg-FNA +FNAC was the highest
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at 0.874. However, addition of BRAFMUT to Tg-FNA did not improve diagnostic accuracy (Table 4). Influence of BRAFMUT on the diagnostic efficacy of FNAC and/or Tg-FNA
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The influence of BRAFMUT on the diagnostic performances of FNAC and Tg-FNA is shown in Table 5. Sensitivities were FNAC: 68.6% Tg-FNA: 94.4% in the BRAFMUT+ group; and
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FNAC: 75%, Tg-FNA: 80% in the BRAFMUT−group. Specificities were FNAC: 100%, TgFNA: 85.7% in the BRAFMUT+ group; and FNAC: 90.9%, Tg-FNA: 100% in the
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BRAFMUT−group. These values did not significantly differ for either FNAC (sensitivity:
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P=0.716, specificity: P=0.193) or Tg-FNA (sensitivity: P=0.311; specificity: P=0.423;
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Table 5). Comparison of FNAC and BRAFMUT tests
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Histological results were regarded as the gold standard. For cytology analyses, we
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classified the FNAC diagnostic results as metastatic, suspicious, indeterminate or benign. FNAC confirmed 61 metastatic LNM, of which 42 LNs underwent BRAFMUT analysis (35
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positive, 7 negative). Of the 58 LNs identified as benign by FNAC, 30 were histologically found to be malignant. BRAFMUT analysis identified 17 positive and 10 negative LNs
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correctly; and 17 positive and 2 negative LNs incorrectly. Of 2 metastatic LNs identified as suspicious and 3 metastatic LNs identified as indeterminate by FNAC, 2 were verified to be BRAFMUT+, and 3 patients did not undergo the BRAFMUT test. Details of BRAFMUT analyses are shown in Table 6.
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Discussion Thyroid malignancies are among the most common cancers worldwide. In 2014, the incidence of thyroid carcinoma was 10.16 cases per 100,000 in Beijing [1]. The increased
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reported incidence of thyroid cancer may reflect improved detection methods [14]. In 2017, the age-standardized incidence rate of thyroid cancer was 21.0 cases among women
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and 7.0 cases among men per 100,000 in North America [15]. Despite increasing
incidences, the total mortality is low, as benign nodules account for a large proportion.
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Lymph node metastases increase the risk of recurrence and death in thyroid malignancies.
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Therefore, the use of imaging modalities and FNAC to detect LNM are more widely used
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to define the appropriate extent of surgery necessary to optimize regional control [2].
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Reportedly, preoperative US has low accuracy for thyroid cancer LNM, at only 32.9%
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[16]. Contrast-enhanced ultrasonography (CEUS) is a helpful technique that can identify vascular architectural changes in LNs that are suggestive of malignant infiltration, but
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cannot easily reduce the differential diagnosis of LN status [17]. FNAC is thus the most
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important diagnostic tool, with high specificity for LNM [5]. In our study, its diagnostic values were sensitivity: 67%, specificity: 100%, PPV: 100%, NPV: 66% and accuracy:
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80%—similar to the results of Zhao et al [5]. In 2015, American Thyroid Association (ATA) Management Guidelines recommended TgFNA as a useful ancillary tool for detecting LNM from PTC, with high sensitivity and specificity [2]. We conclude that Tg-FNA of metastatic PTC measurement is
12
241.28±110.05 ng/ml, and 1.36±3.5 ng/ml in non-metastatic PTC at preoperative evaluation. The optimal cutoff value we recommend is 2.23 ng/mL (sensitivity: 97.3%, specificity: 87.8%, PPV: 87.8%, NPV: 97.3%, accuracy: 92.3%). FNA-Tg alone was
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superior to FNAC in identifying LNM. The addition of Tg-FNA to FNAC further improved the diagnostic value (sensitivity:100%, specificity: 88.4%, PPV: 87.8%, NPV:
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100%, accuracy: 93.7%). Our results also showed kappa values of Tg-FNA + FNAC:
0.874, Tg-FNA alone: 0.847, and FNAC alone: 0.613. These results were similar to those
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of other reported studies [5, 6].
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Our study confirmed that BRAFMUT in the thyroid nodule is associated with LN metastasis,
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in accordance with other studies [12]. The relationship between BRAFMUT in the thyroid
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nodule and other invasive symptoms (extrathyroidal extension, calcification, cystic lesions
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etc.) is not statistically significant [10]. Furthermore, some studies found no correlation between BRAFMUT and tumor severity [18-20], possibly because this BRAF mutation may
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perform differently in various PTC subtypes [18]. Our study tested for BRAF mutations in
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90 LNs and 82 thyroid carcinomas. The carcinoma of one patient was confirmed positive, whereas his LN was confirmed negative. Immunohistochemical analysis has shown
A
BRAFV600E expression to vary in normal thyroid tissue, nodule hyperplasia and PTC [12]. The association between BRAFMUT and LNM has been widely investigated. Our study confirmed BRAFMUT in LNs was significantly associated with metastatic PTC (P=0.005), in agreement with previous studies [10, 11]. Our results also confirmed the kappa value of BRAFMUT to be 0.286 with respect to diagnosis (sensitivity: 77.8%, specificity: 55.6%,
13
PPV: 87.5%, NPV: 38.5%, accuracy: 73.3%). These results are in line with the study of Dong et al [18]. Although BRAFMUT alone may not accurately indicate LN status, 5 metastatic LNs in our study that were assessed as suspicious or indeterminate by FNAC
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were identified as positive in BRAFMUT analysis. This result suggests that BRAFMUT tests of LNs could be a supportive diagnostic method for PTC patients. Our findings were
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similar to those of Kim et al [9].
The cutoff value and diagnostic efficiency of Tg-FNA are still controversial [5, 6, 21], and
U
can be influenced by many factors, such as the presence or absence of the thyroid gland
N
[5]. Therefore, we explored the influence of BRAF mutation on the diagnostic efficiency of
A
FNAC and FNA-Tg. We found that the presence or absence of BRAF mutations had little
M
effect on FNAC and FNA-Tg diagnostic sensitivities.
ED
Our study has certain limitations. First, as the sample size was small, and may reflect selection bias. A multi-institutional study with a larger cohort is needed to verify our
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results. Second, whether non-metastatic BRAFMUT+ LNs may deteriorate is unclear, and
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should also be checked in a future large-scale study. Conclusion
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Our study demonstrates that Tg-FNA combined with FNAC is a useful diagnostic test that improves detection of metastatic PTC. BRAF mutation status did not obviously affect the diagnostic performance of FNAC or Tg-FNA.
14
Authors’ contributions Li Jianming: research design, acquisition and analysis of data, drafting the paper, approval of the submitted and final versions; Liu Jibin: critical paper revisions; Bao Xiaoli:
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thyroglobulin measurement; Yu Xiaomeng: thyroglobulin measurement; QianLinxue: research design, author coordination, approval of the submitted and final versions.
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Funding
This work was funded by the National Natural Science Foundation of China (041320015)
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Competing interests
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All the authors report no conflicts of interest
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Acknowledgment
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We thank Marla Brunker, from Liwen Bianji, Edanz Group China
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(www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
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N. Colak, S. Ozarmagan, Diagnostic value of thyroglobulin measurement in fine-needle aspiration
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biopsy for detecting metastatic lymph nodes in patients with papillary thyroid carcinoma,
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Figure legend
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Langenbeck's archives of surgery, 396 (2011) 77-81.
Figure 1. Flow chart of recommended management of suspicious lymph nodes in papillary
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thyroid carcinoma, using US. BRAFV600E: V600E amino acid in the BRAF proto‑ oncoprotein; FNAC: Fine-needle aspiration cytology; Tg-FNA: Thyroglobulin measurement with fine-needle aspiration; US: Ultrasound.
19
Figure 2. Receiving operating characteristic (ROC) curve of lymph nodes measured using Tg-FNA. The optimal cutoff value for FNA-Tg was 2.23 ng/mL. Area under ROC curve: 0.987 (95% confidence interval: 0.971–1.000); sensitivity: 97.3%; specificity: 87.8%; PPV:
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87.8%; NPV: 97.3%; accuracy: 92.3%. NPV: Negative predictive value; PPV: Positive predictive value; Tg-FNA: Thyroglobulin measurement with fine-needle aspiration.
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Detection of lymph node in
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nodes in papillary thyroid carcinoma, using US.
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Figure 1. Flow chart of recommended management of suspicious lymph
A
preoperative patients with papillary
M
thyroid carcinoma
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At least two of US features: (312 patients and 356 lymph
incomplete information; Cystic lesions, Micro-calcification, 65 patients: did not Rounded shape (long/short axis ratio <1.5), Irregular margins, (145 patients with 154 lymph nodes) undergo their surgeries HyperechoicFNAC medulla and/or indistinct hilum
A
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1. 2. 3. 4. 5.
Excluded 102 patients :
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nodes)
BRAFv600e mutation Test
(90 patients with 90 lymph nodes)
in hospital FNA-Tg Test (69
BRAFv600e mutation and FNA-Tg
patients with 78 lymph
Test (41 patients with 41 lymph
nodes)
nodes)
20
Figure 2. Receiving operating characteristic (ROC) curve of lymph nodes measured on TgFNA. The optimal cutoff value for FNA-Tg was 2.23 ng/mL. Area under the ROC curve: 0.987; 95% confidence interval: 0.971–1.000); sensitivity: 97.3%; specificity: 87.8%; PPV:
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ED
M
A
N
U
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87.8%; NPV: 97.3%; accuracy: 92.3%.
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Table 1. Clinical characteristics of patients with papillary thyroid carcinoma by diagnostic method. FNAC
Tg-FNA
BRAFv600e
35 vs 110
19 vs 50
25 vs 65
Central neck dissection
98
50
68
Selective neck dissection
41
12
13
Radical dissection
15
7
9
I Region
2
0
II Region
7
3
III Region
12
5
IV Region
15
6
V Region
9
4
VI Region
51
19
Total
96
37
Gender Male vs female
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Location of Metastatic PTC
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Surgical procedures
1 5 8
13
N
U
8
37 72
A
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M
A
FNAC= Fine-needle aspiration cytology; Tg-FNA= Thyroglobulin measurement with fineneedle aspiration; BRAFv600e=V600E amino acid in the BRAF proto‑ oncoprotein; PTC= Papillary thyroid carcinoma.
22
Table 2. Characteristics of patients with metastatic PTC or non-metastatic PTC Histology
Metastatic PTC
Non-metastatic PTC
P value
27 vs 62
12 vs 44
0.239
Age
40.56±12.53
43.93±13.15
0.203
Number of nodes
96
58
NA
Size of node (mm)
9.87±4.38
8.73±4.23
0.429
Gender Male vs female
NA 21
2
micro-calcification
16
1
medulla or hilum disappears
86
53
rounded shape
82
48
irregular margins
68
46
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cystic lesions
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US imaging
A
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PT
ED
M
A
N
U
PTC= Papillary thyroid carcinoma; US= Ultrasound.
23
Table 3. Relationships between thyroid carcinoma characteristics and BRAFv600e mutations. Mutation Detected
Mutation Not Detected
Gender
64
18
Male
20
5
Female
44
13
Tumor Size
0.997±0.66
Calcification
0.779
1.106±0.37
No
49
13
Nodule border in the US
15
5
Rough
22
9
Blood flow in the CDFI
42
9
Smooth
38
Capsule evolving
26
3
Multiple lesions
52
M
12
Multiple
No Yes
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No
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Lymph node metastasis
0.230
0.062
0.449
2 16
20
6
44
12
7
4
57
14
46
7
18
11
ED
Single Yes
A
Yes No
0.707
15
N
No
U
Yes
0.089
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Yes
Cystic lesion
P
BRAF V600E status of primary tumor
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Characteristic
0.868
0.217
0.01*
A
BRAFv600: V600E amino acid in the BRAF proto-oncoprotein; CDFI: Color Doppler flow imaging; US: Ultrasound.
24
Table 4. Diagnostic values of FNAC, Tg-FNA and BRAFv600e mutations. Diagnostic modality Sensitivity
Specificity
PPV
NPV
Accuracy
Kappa
BRAFv600e
77.8%(56/72)
55.6%(10/18)
87.5%(56/64)
38.5%(10/26)
73.3%(66/90)
0.286
Tg-FNA
97.3%(36/37)
87.8%(36/41)
87.8%(36/41)
97.3%(36/37)
92.3%(72/78)
0.847
FNAC
67%(61/91)
100%(58/58)
100%(61/61)87. 66%(58/88)
80%(119/149)
0.613
FNAC + Tg-FNA
100%(36/36)
88.4%(38/43)
8%(36/41)
100%(38/38)
93.7%(74/79)
0.874
BRAFv600e+FNAC
96.6%(57/59)
34.5%(10/29)
75%(57/76)
83.3%(10/12)
76.1%(67/88)
0.365
22.2%(4/18)
61.1%(22/36)
80%(4/5)
63.4%(26/41)
0.194
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BRAFv600e+Tg-FNA 95.7%(22/23)
A
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ED
M
A
N
U
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FNAC= Fine-needle aspiration cytology; Tg-FNA= Thyroglobulin measurement with fineneedle aspiration; BRAFv600e= V600E amino acid in the BRAF proto‑oncoprotein; PPV= positive predictive value; NPV= negative predictive value
25
Table 5. Influence of BRAFv600e mutations on the diagnostic efficacy of FNAC and TgFNA BRAF status
Histology
FNAC+
FNAC-
Tg-FNA+
Tg-FNA-
BRAFv600e+
Metastatic PTC
35
16
17
1
Non-metastasis
0
18
2
12
Metastatic PTC
6
2
4
1
Non-metastasis
1
10
0
4
BRAFv600e
-
A
CC E
PT
ED
M
A
N
U
SC R
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BRAFv600e=V600E amino acid in the BRAF proto-oncoprotein; FNAC= Fine-needle aspiration cytology; Tg-FNA= Thyroglobulin measurement with fine-needle aspiration; PTC= Papillary thyroid carcinoma.
26
Table 6. Comparison of FNAC and the BRAFv600e mutation test FNAC
Histology
BRAF mutation analysis
Metastatic PTC
61(Metastatic PTC)
35 positive,7 negative,19 NA
Suspicious
2(Metastatic PTC)
1 positive, 1 NA
Benign
58(Non-metastasis),
17 positive, 10 negative, 31 NA
30(Metastatic PTC)
17 positive, 2 negative, 11 NA
3(Metastatic PTC)
1 positive, 2 NA
Indeterminate
A
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PT
ED
M
A
N
U
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FNAC= Fine-needle aspiration cytology; BRAFv600e=V600E amino acid in the BRAF proto‑oncoprotein; PTC= Papillary thyroid carcinoma.