Retraction Notice The Mutation and the Clinicopathological Features of the Papillary Thyroid Carcinoma

There has been a dramatic increase in the morbidity of papillary thyroid carcinoma (PTC), which accounts for more than 85% to 90% of all thyroid ma-lignancies. A somatic BRAF V600E oncogenic mutation has been deemed as the most frequent genetic alteration that occurs in approximately 32% - 83% of papillary thyroid cancer cases, associated with more aggressive clinical be-haviors and worse prognosis. Nevertheless, the prognostic value of the BRAF V600E mutation remains to be confirmed. Herein, we examined BRAF V600E mutation by performing qPCR as well as immunohistochemistry (IHC), and analyzed the relationship between BRAF V600E mutation status and multiple clinicopathological features of a total of 188 patients with PTC. There was no significant difference in the prevalence of BRAF V600E mutation, with regard to patient gender (χ 2 = 1.252, P = 0.263), ethnicity (χ 2 = 0.756, P = 0.384) and age (younger than 40 years versus 40 years or older, χ 2 = 0.002, P = 0.957). Also, our study found no association between the frequency of V600E mutation and various pathological characteristics of PTC, including calcification (χ 2 = 0.2186, P = 0.640), hashimoto (χ 2 = 0.072, P = 0.789), tumor size (χ 2 = 1.453,

There has been a dramatic increase in the morbidity of papillary thyroid carcinoma (PTC), which accounts for more than 85% to 90% of all thyroid malignancies. A somatic BRAF V600E oncogenic mutation has been deemed as the most frequent genetic alteration that occurs in approximately 32% -83% of papillary thyroid cancer cases, associated with more aggressive clinical behaviors and worse prognosis. Nevertheless, the prognostic value of the BRAF V600E mutation remains to be confirmed. Herein, we examined BRAF V600E mutation by performing qPCR as well as immunohistochemistry (IHC), and analyzed the relationship between BRAF V600E mutation status and multiple clinicopathological features of a total of 188 patients with PTC. There was no significant difference in the prevalence of BRAF V600E mutation, with regard to patient gender (χ 2 = 1.252, P = 0.263), ethnicity (χ 2 = 0.756, P = 0.384) and age (younger than 40 years versus 40 years or older, χ 2 = 0.002, P = 0.957).

Introduction
According to papers published during the last decades, there has been a rapid and continuous increase worldwide in the incidence rate of thyroid cancer (TC), the most common endocrine malignancy [1] [2]. Papillary thyroid cancer (PTC) accounts for the vast majority (more than 85% to 90%) of all thyroid malignancies [3]. Despite the steadily raising morbidity, mortality related to PTC appears to be stationary [1] [4]. Indeed, due to the intrinsic indolent behavior of the disease and the effective initial management, the prognosis of patients affected with PTC is generally promising with a 10-year overall survival rate of approximately 85% [5] [6]. However, a relapse from complete remission has been reported in about 5% to 25% of the patients [7] [8]. In case a tumor metastasis occurs, the 10-year survival rate of PTC can be reduced to 40%.
Interestingly, the incidence rates of different TC histotypes vary considerably: the rates of PTC have exclusively increased for last decades, particularly the follicular variant of PTC [9], whereas an extremely modest increase in the morbidity of follicular thyroid cancers (FTC) has been observed [10] and the rates of anaplastic thyroid cancer are stable or even have shrunk [11]. This suggests that some carcinogenetic factors are involved in PTC onset by dysregulating specific molecular signaling.
BRAF is a Ser/Thr specific protein kinase gene, which is a member of the Raf kinase family of growth signal transduction protein kinases. This protein plays a role in regulating the MAP kinase/ERKs signaling pathway, which affects cell division, differentiation, and secretion. The BRAF V600E mutation results in substitution of valine to glutamate in codon 600 of a serine-or threonine-specific protein kinase named BRAF, consequently leading to constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway [12]. It occurs in approximately 36% -83% of PTC cases [13] and plays an essential role in maintaining and progression of thyroid cancer [14]. Actually, recent studies reported that there has been an increasing trend of patients with PTC carrying the BRAF V600E oncogenic mutation [15] [16]. BRAF V600E mutation is the most frequent genetic aberration correlated with more aggressive tumors based upon conventional staging, and patients harboring BRAF V600E mutation are more susceptible to an increased risk for recurrence and lymph node metastasis in comparison to those patients with PTC devoid of the mutation [17]. However, the value of the BRAF V600E mutation as a prognostic hallmark in PTC was controversial and unclear. To this end, the present study aimed to define the correlation between the BRAF V600E mutation and PTC-related clinicopatho-logical characteristics. In addition, the study evaluated the clinical values of qPCR and immunohistochemical strategies for the identification of V600E mutation.

Patients
A total of 188 patients with primary papillary thyroid carcinoma diagnosed at the First Affiliated Hospital of Xinjiang Medical University (Urumqi, Xinjiang, China) from June 2015 to December 2015 were recruited in this study. Tumor tissues were obtained by surgical resection during the surgery. And we record and statistical analysis the gender, ethnicity, age, calcification, tumor diameter, hashimoto, thyroid focality, thyroid capsular invasion, vascular invasion, lymph node metastasis, BRAF immunity and BRAF V600E PCR results in these cases.
Written informed consents were given by all included patients and the sample collection as well as investigation were approved by the local ethics committee of the First Affiliated Hospital of Xinjiang Medical University.

Quantitative PCR Assay
Total DNA was extracted from paraffin-embedded tissues. The resuspended DNA samples were subjected for PCR analysis with the human BRAF V600E detection kit (ACCB Biotech Ltd., Beijing, China) according to the manufacturer's protocol. The amplification was performed in an ABI 7300 plus machine (Applied Biosystems, Foster City, CA, USA) ( Figure 1). The amplification profile was started with an initial denaturation at 95˚C for 10 min, followed by 40 cycles of denaturation 95˚C for 15 s, annealing at 60˚C for 30 s, and extension at 72˚C for 1 min.

Immunohistochemistry
Immunohistochemistry was performed as previously described [18]. Briefly, formalin-fixed, paraffin-embedded tissue sections (4 μm-thick) were deparaffinized and rehydrated, and were incubated in retrieval solution at 60˚C and overnight to retrieve antigens. After inhibition of the preprimary peroxidase and blocking in 1% BSA in TBS for 2 hrs at room temperature, slides were incubated with the anti-BRAF V600E (VE1) (1:50, Roche, Ventana Medical Systems, Tucson, AZ, USA) mouse monoclonal primary antibody at 37˚C for 30 minutes. Afterwards, the primary antibodies were detected using an OptiView DAB IHC Detection kit (Ventana Medical Systems) following incubation with hematoxylin and a bluing reagent (for 4 minutes each).
Statistical analysis was performed using SPSS software (version 11.0; SPSS Inc., Chicago, IL). Chi square test was used to compare frequencies of BRAF V600E mutation between groups. Categorical variables were expressed as numbers and percentages. Statistical significance was assumed when the P value was < 0.05. International Journal of Clinical Medicine

Clinicopathological Features of Patients with PTC
With the extensive application of ultrasound-guided fine needle aspiration and imaging studies, there have been an increasing number of PTC cases over the past several decades in our hospital. During the study period, a total of 188 patients were diagnosed with PTC, of which 160 patients carried BRAF V600E mutation (85.1%) and 28 were mutation-negative (14.9%) patients. The basic demographics of PTC patients was retrospectively analyzed and summarized in Table 1. The majority of the patients were women (69.7%), Han people (74.5%), and older than 40 years (63.8%). Calcification was detected in 60 cases (31.9%) and hashimoto was present in 63 cases (33.5%).Multifocal tumors were observed in 114 patients (60.6%). The frequencies of thyroid capsular invasion, vascular invasion and the lymph node metastasis were 36.2%, 4.8% and 30.9% respectively. 62 patients (33.0%) had thyroid tumors with a diameter more than 1 cm.
To assess the correlation between BRAF V600E mutation and age, 188 patients were divided into two groups: the younger segment aged less than 40, the older segment at least 40 years of age. BRAF V600E mutation was detected in 102 (85%) of the 120 patients at least 40 years of age, and 58 (85.29%) of the 68 patients younger than 40. There was neither a direct association between BRAF V600E and patient age (χ 2 = 0.002, P = 0.957).
Also, PTC patients were divided into two groups according to whether they were Han people or not. However, there was no significant difference existed between the groups when considering the incidence of BRAF V600E (86.43% vs 81.25%, χ 2 = 0.756, P = 0.384).
The associations between BRAF V600E mutation and focal number as well as tumor size were also evaluated. Multifocality was seen in 60.64% of all the involved patients with PTC. For the 74 patients with monofocal PTC, 64 (86.49%) International Journal of Clinical Medicine whereas that in the latter was 57 (83.82%). There was no direct correlation of the mutant frequency and the incidence of thyroid capsular invasion (χ 2 = 0.138, P = 0.710). In 9 patients with vascular invasion, the BRAF mutation rate was 77.78%, while the non-invasive patient, the mutation rate was 85.47%. There was no statistical difference between the two groups (χ 2 = 1.132, P = 0.860). Of the 58 patients with lymph node metastasis, the frequency of BRAF V600E mutation was 86.21% (50/58) and in patients who had no lymph node metastasis, the mutation frequency was 84.62% (110/130). No significant differences in the mutation frequency were noticed between the metastatic and non-metastatic PTCs (χ 2 = 0.080, P = 0.777).
In aggregate, we analyzed the relationship between the incidence of BRAF V600E mutation and diverse clinicopathological factors including age, gender, ethnicity, tumor size, and the presence of calcification, hashimoto, multifocality, thyroid capsular invasion, vascular invasion and lymph node metastasis. However, Univariate analysis showed that the presence of mutation was not apparently influenced by these factors.

Comparison of the BRAF V600E Mutation by Using IHC and PCR
We also performed the mutation detection by using IHC, todetermine if the PCR method can be used as a reliable substitute for BRAF V600E detection. Using IHC, the mutation was found in 153 cases ( Figure 2). The positive rate was 81.38%, relatively lower compared with that using PCR molecular test (85.11%).
In the 153 V600E mutation-positive cases which were detected by IHC, 147 (96.08%) could also betested by PCR, whereas in the 35 mutation-immuno-negative cases, 13 was detected by PCR. Giving the superior performance of PCR strategy in detecting barely detectable cells carrying a V600E mutation, probably due to International Journal of Clinical Medicine its amplification effect on the mutant sequences froma fraction of BRAF mutated tumor cells in tissues, we can conclude that the PCR presents a superior sensitivity compared to IHC method.

Discussion
A worldwide increase in the PTC incidence results in a relevant public health concern. Nowadays, clinical decision-making remains controversial in absence of specific assessment of the malignancy [19]. Despite the importance of prognostic implications of other tumor-specific genetic alterations is increasingly apparent [20], their clinical predictive power is limited. The RAS-ERK-MAPK signaling pathway is involved in cell responses to environmental stimuli and plays a crucial part in human carcinomas [19]. This pathway comprises RAS, MEK, ERK and the protein kinases RAF, which includes three protein kinases with nonredundant functions. On incitement of cytokines or hormones, active RAS recruits RAF to themembrane and the latter phosphorylates and activates the scaffold protein MEK, which afterwards activates ERK. BRAF is one of the RAF kinase family members and acts as an important transduction factor in the RAS-ERK-MAPK signaling pathway that can regulate a variety of biological events such as cell survival, division, differentiation, senescence, apoptosis, and secretionin normal cells [20].
BRAF V600E mutation is the most frequent genetic event in PTC, and it has been found in approximately 32% -83% of PTC cases, whereas this alterations carcely appears in other thyroid tumors [13]. The V600E mutation has been considered to simulate constitutive phosphorylation on T599 and S602 residues, thereby abnormal activation of BRAF protein kinase [21]. Indeed, it was reported that BRAF V600E was 500-fold activated, leading to constitutively acti-vated ERK-MAPK signaling and inducting proliferation and survival of cancer cells. This activating mutation BRAF V600E has been extensively observed in PTC, colorectal cancer, melanoma, and non-small-cell lung cancer [22] [23] [24]. The clinical researchers using the BRAF inhibitors vemurafenib, dabrafenib improved the survival rate among patients with advanced melanoma [25] and initiated an objective response in refractory hairy-cell leukemia (HCL) [26]. And the BRAF-selective inhibitor vemurafenib restrained growth of BRAF mutated anaplastic thyroid cancerin mice [27]. These data validated BRAF V600E mutation is a therapeutic target in many cancers and, furthermore, BRAF-selective drugs have been applied in the clinic and display excellent effects in patients with BRAF mutant melanomas [28].
Although results from some individual studies suggested that BRAF V600E mutation was associated with multiple aggressive clinicopathological characteristics of PTCs, including extrathyroid extension, multifocality, advanced tumor stage, and lymph node metastasis [29] [30], it is still controversial and indeterminate. We herein carried out an additional study involving a larger number of patients with PTC, to determine whether the frequency of BRAF V600E mutation is correlated with the aforementioned aggressive clinicopathological features as well as other characteristics such as patients age, gender, ethnicity, tumor size, the presence of hashimoto and calcification. The features of a total of 188 patients with PTC were retrospectively studied, and qPCR analysis was used to identify if the BRAF V600E mutation were existed. By chi-square analysis, we found that all variables showed no correlation to BRAF V600E mutation, indicating that each of these factors cannot function alone. Thus we speculated that two or more factors might be collectively involved in the presence of V600E mutation. However, the amount of BRAF V600E negative specimens was not enough for us to accurately evaluate the phenotypic differences between mutation-positive and negative tumors. On the other hand, we assumed that the mutation was preferably contributed to the tumorigenesis rather than progression of PTC. And this study has potential limitations that we did not analysis subtypes (the classical type, the follicular variant, the oncocytic variant and the tall cell variant) of PTC.
Recent studies have demonstrated that the BRAF V600E mutation has emerged as a useful diagnostic hallmark and a specific factor for poor clinical outcomes of PTCs [13] [31] [32]. An efficient and sensitive method with higher specificity therefore needs to be developed to detect the presence of BRAF V600E mutation.
Of the 188 patients with PTCs in the present study, the mutation was identified in 153 cases by immunohistochemistry (IHC), while it was detected in 160 patients by using PCR assay. The detection rate for the mutation by IHC was 81.38%, slightly lower compared with PCR method (85.11%). In the 153 V600E mutation-positive cases which were detected by IHC, 147 (96.08%) could be also tested by PCR, whereas in the 35 mutation-immunonegative cases, 13 was detected by PCR. Giving the preferable performance of PCR strategy in detecting barely detectable cells carrying a V600E mutation, we can conclude that it presents a superior sensitivity compared to IHC method. Therefore, it is recommendable to combine the results obtained from PCR molecularstrategy together with conventional IHC method for clinical evaluation of BRAF V600E mutation.
The differential diagnostic value of the BRAF V600E mutation in thyroid tumors has been widely recognized and applied. The results of this study indicate that the sensitivity of the ARMS PCR assay to the detection of the BRAF V600E mutation to diagnose PTC is as high as 85%comparingwith an average rate of 44% (36% -83%), [33]. This is similar to Li et al. [34] (83.1%), Wang et al. [35] (86.8%). This shows that the ARMS PCR method has a better clinical application prospect than the direct sequencing method [36].