Ultrasensitive multifunctional biosensor integrating ECL quenching and DPV enhancement for early classification of thyroid cancer via BRAF V600E and microRNA-221 detection.

Objective To explore the correlation of ultrasound-guided fine-needle aspiration(US-FNA) combined with BRAF V600E mutation detection and ultrasound features and central cervical lymph nodes metastasis of classic papillary thyroid cancer(PTC) for providing a reliable molecular basis for clinical preoperative evaluation of patients. Methods Ninty-three cases of patients collected from October 2017 to November 2018 in Gansu Province Hospital were enrolled, who underwent general ultrasonic examination TI-RADS ≥4a, the US-FNA highly suspicious of PTC, thyroid surgery including total thyroidectomy and central cervical lymph node dissection, with the postoperative pathologic results of classical PTC and whether the central cervical lymph node metastasis happened in the patients. Part of the specimen applied HE staining for cytological diagnosis, the other part of specimen was used real-time for detection of BRAF V600E gene mutation by fluorescent quantitative polymerase chain reaction (PCR) method. Results Univariate analysis showed that the occurrence of cervical lymph node metastasis for classic PTC were significantly correlated with gender(χ2=10.303, P=0.002), BRAF V600E mutation(χ2=31.204, P=0.000) and extrathyroidal invasion(χ2=12.848, P=0.000). Multi-logistic regression analysis showed that BRAF V600E mutation(OR=13.324, 95%CI=4.058-43.744, P=0.000) and extrathyroidal invasion(OR=5.738, 95%CI=1.766-18.643, P=0.004) were the risk predictors of cervical lymph node metastasis of classic PTC. Gender(OR=0.385, 95%CI=0.112-1.324, P=0.130) was not the risk predictor. Conclusions US-FNA combined with BRAF V600E mutation and extrathyroidal invasion are the risk factors in predicting central cervical lymph node metastasis in classic PTC. Patients with these two risk factors should be elected to undergo prophylactic central cervical lymph node dissection. Key words: Ultrasound-guided fine-needle aspiration cytology; Papillary thyroid cancer, classic; Central cervical lymph node; Metastasis; BRAF V600E

Objective To investigate the clinical value of detection of the BRAF V600E combined with ultrasound-guided fine needle aspiration cytology in the diagnosis of indeterminate thyroid nodules in elderly patients. Methods A total of 60 elderly patients with thyroid nodules admitted to our hospital from October 2014 to December 2015 underwent ultrasound-guided fine needle aspirations. The samples were sent for cytology examination and detection of the BRAF V600E mutation. Diagnoses based on cytology alone and those in combination with detection of the BRAF V600E mutation were retrospectively analyzed and compared with clinical follow-up results. Results The fine needle aspiration procedure was successfully performed in 95% (57/60) of the patients. Of the 57 patients, cytological diagnoses in 48 cases were consistent with the follow-up results, with an agreement rate of 84.2% (48/57). The 48 cases included 6 patients diagnosed with benign nodules and 42 patients with thyroid papillary carcinoma. The BRAF V600E mutation was found in 32 cases with thyroid papillary carcinoma, with a detection rate of 76.2%. Of the 9 cases with indeterminate thyroid nodules, 7 had the BRAF V600E mutation. In diagnoses based on the combination of fine needle aspiration biopsy with detection of the BRAF V600E mutation, 55 out of 57 cases were consistent with the follow-up results, achieving an agreement rate of 96.5%. The clinical diagnostic accuracy of the cytology procedure combined with BRAF V600E mutation detection was better than that of the cytology procedure alone (P<0.05). Conclusions Ultrasound-guided fine needle aspiration cytology combined with detection of the BRAF V600E mutation can increase the clinical diagnostic accuracy for thyroid nodules and is highly valuable in the diagnosis of cytologically indeterminate thyroid nodules in elderly patients. Key words: Ultrasonography; Lymph nodes; Immunohistochemistry

Investigation of BRAF V600E detection approaches in papillary thyroid carcinoma

Electrogenerated chemiluminescence biosensor for microRNA detection incorporating enzyme-free dual DNA cyclic amplification and Ru(bpy)32+-functionalized metal-organic framework

Radiofrequency Ablation of Recurrent Metastatic Papillary Thyroid Cancer to a Lymph Node

BackgroundBRAF mutations occur in approximately 8% of all human cancers and approach 50% in melanoma and papillary carcinoma of thyroid. These mutations provide potentially valuable diagnostic, prognostic and treatment response prediction markers. A sensitive, specific, low-cost assay to detect these mutations is needed.ResultsTo detect BRAF V600E mutation in formalin-fixed, paraffin-embedded (FFPE) tissue, we developed a method using Amplification Refractory Mutation System (ARMS)-PCR. This method was designed to amplify three products in a single reaction tube: a 200 bp common product serving as an amplification control, a 144 bp BRAF V600E specific product, and a 97 bp wild-type (wt) specific product. The sensitivity of this method was determined to be as low as 0.5% for the BRAF V600E allele in a wild-type background. This method was successfully validated in 72 thyroid tumors. It detected V600E mutation in 22 out of 33 (67%) of the conventional papillary thyroid carcinoma (PTC), 8 out of 12 (75%) of the tall-cell variant of PTC, whereas none of the 10 follicular variant of PTC showed BRAF V600E mutation. In addition, none of the 14 follicular adenomas and 3 follicular carcinomas had BRAF V600E mutation. As a comparison method, direct dideoxy sequencing found only 27 out of 30 (90%) mutations detected by ARMS-PCR method, suggesting that this ARMS-PCR method has higher sensitivity.ConclusionsOur ARMS-PCR method provides a new tool for rapid detection of BRAF V600E mutation. Our results indicate that ARMS-PCR is more sensitive than automated dideoxy sequencing in detecting low BRAF V600E allele burdens in FFPE tumor specimen. The strategy of this ARMS-PCR design may be adapted for early detection of point mutations of a variety of biomarker genes.

&lt;p&gt;&lt;strong&gt;Background:&lt;/strong&gt;&lt;strong&gt; &lt;/strong&gt;Molecular markers are gaining increasing importance as diagnostic and prognostic tools in patients with well differentiated thyroid cancers and BRAF V600E mutation has received wide attention in this regard &lt;strong&gt;Aim: &lt;/strong&gt;To evaluate the clinical value of immunohistochemistry (IHC) using anti-BRAF V600E antibody (clone RM8) for detection of the BRAF V600E mutant protein in formalin-fixed and paraffin-embedded tissues of patients with papillary thyroid carcinomas. &lt;strong&gt;Materials and Methods: &lt;/strong&gt;Patients who were managed for well differentiated thyroid cancers (&lt;em&gt;n &lt;/em&gt;= 79) during the years 2005 and 2006 were included in the study. We evaluated the fidelity of the RM8 antibody specific for the BRAF V600E and compared its detection accuracy to real time polymerase chain reaction (PCR), which was taken as the gold standard. &lt;strong&gt;Results: &lt;/strong&gt;Mutant BRAF V600E antibody was studied in 79 tissue sections, out of which 21 (26.5%) had staining for BRAF V600E in &amp;gt;20% of the tumour cells and these were considered positive. The BRAF staining was moderate in 10 (47.6%), strong in 9 (42.5%) and very strong in 2 (9.5%) of sections stained. There was a statistically significant concordance (&lt;em&gt;P &lt;/em&gt;= 0.000) with quantitative PCR (qPCR) for BRAF mutant taken as standard. (Kappa agreement: 0.881) Further, the receiver operating characteristics (ROC) curve showed that IHC can be used as a comparable standard to the qPCR. The highest possible sensitivity of 92% and specificity of 92.6% could be achieved by considering the cytoplasmic positivity of &amp;gt;20% of cells with moderate to strong intensity (AUC = 0.923) &lt;strong&gt;Conclusion: &lt;/strong&gt;Our study has shown that BRAF V600E IHC can be done in a conventional manner using rabbit monoclonal antibody RM8 on formalin-fixed and paraffin-embedded tissues of patients with papillary thyroid carcinomas. With a comparable diagnostic accuracy to the gold standard qPCR testing and with an added advantage of being cost effective, this technology can be considered for use as a first-line method for detection of BRAF V600E mutations, especially in resource constrained settings.&lt;/p&gt;

Immunohistochemical detection of the BRAF V600E mutation in papillary thyroid carcinoma. Evaluation against real-time polymerase chain reaction

Evaluation of the Real-Q BRAF V600E Detection Assay in Fine-Needle Aspiration Samples of Thyroid Nodules

Background: PTC is the most common type of thyroid cancer and offers excellent prognosis. However, recurrence PTC remains relatively common, especially PTC with BRAF V600E mutation. PTC is often characterized by RET, RAS or BRAFmutations. RAS, RET and BRAF V600E mutations tend to be mutually exclusive. In this study, we investigated whether these oncogenic driver mutations could propose a molecular classification of PTC into distinct tumor immune microenvironment. Methods: Surgical tissue samples from 252 patients with stage I-III PTC consecutively diagnosed between 07/2020 to 08/2021 at Sun Yat-sen Memorial Hospital (SYMH) were sequenced using a panel targeting 18 cancer-related genes. Clinical and genomic data from SYMH cohort was compared with 499 stage I-III PTC patients (PTCs) from TCGA. Results: Based on the analysis of the genetic alteration profile from SYMH cohort, 252 PTCs can be classed into three major subgroups, BRAFV600E-(83.7%), RAS-(6.0%), and RET-(3.6%) driven PTCs. These three subgroups covered 93.2% of SYMH cohort and 77.6% in TCGA cohort. (Thyroid peroxidase) TPO status was strongly correlated with BRAF V600E (p=0.003), RET (p=0.005) mutations. Median age of RET- driven PTCs was lower than that of BRAFV600E-, RAS- driven PTCs (p=0.034), which was in concordance with TCGA cohort (p=0.0021). In both cohorts, prevalence of lymph node metastasis in RET-driven PTCs (SYMH, 88%; TCGA, 76%) was higher than that in RAS-(SYMH, 20%; TCGA, 21%) driven PTCs (SYMH, RET vs. RAS, P=0.018; TCGA RET vs. RAS, p &amp;lt;0.001). The TPO positive rates in RET- (p=0.034) and RAS- (p &amp;lt;0.01) driven PTCs were higher than BRAF V600E- driven PTCs in SYMH cohort. In TCGA cohort, RET-driven PTCs had longer disease-free survival (DFS) than RAS- driven PTCs, but not statistically significant. Tumor immune microenvironment study in TCGA cohort revealed a more anti-inflammatory tumor state for RAS-driven PTCs, with decreased levels of pro-inflammatory mediators DC cells, Eosinophils, M1 Macrophages, B cells, and CD4 memory T cells, and increased level of anti-inflammatory M2 macrophages. RAS-driven PTCs also had the decreased level of pro-inflammatory cytokines IL-1, IL-2, IL-12, IL-17, IL-18, IFN-γ, and TNF-α. In contrast, significantly increased level of tumor-infiltrating Tregs was observed in BRAFV600E -driven PTCs (p&amp;lt;0.005). BRAF V600E - driven PTCs had higher degree of NKT cells infiltration than RAS-driven PTCs (p&amp;lt;0.05). Conclusion: PTCs can be classified into BRAF V600E-, or RAS-, or RET-driven subgroups with district clinicopathological features and tumor immune microenvironmental differences, which implication for clinical treatment decision. Citation Format: Miaoyun Long, Yunfang Yu, Lili Lin, Yue Zhu, Langping Tan, Yisikandaer Yalikun, Wenhao Ouyang, Shuai Ni, Xi Li, Ting Hou, Wenjie Sun, Zhange Chen. Novelmolecular subtypes of papillary thyroid carcinoma (PTC) driven by BRAFV600E-,RAS- and RET-mutations linked to distinct clinicopathological features and tumor immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5265.

The aim of this study was to explore the correlation between ultrasound-guided fine-needle aspiration cytology(US-FNAC) combined with BRAF V600E mutation analysis and central neck lymph node metastasis in cN0 papillary thyroid cancer, so as to provide reliable molecular evidence to use it for preoperative evaluation, operation procedure design, and postoperative follow-up planning in clinic. Specimens were obtained from 250 patients with cN0 thyroid cancer (TI-RADS≥4a, highly suspected of PTC by US-FNAC) after bilateral thyroidectomy and central neck lymph node dissection with accessible postoperative pathologic results of PTC and central neck lymph nodes and used for cytological diagnosis by H&E stain and BRAF V600E mutation detection. Single-factor analysis showed that differences between the central neck lymph node metastasis and nonmetastasis groups were statistically significant in gender, BRAF V600E mutation, and extracapsular extension. Logistic multivariate regression analysis showed significant differences in gender, BRAF V600E mutation, and extracapsular extension. Positive BRAF V600E mutations by US-FNAC, extracapsular extension, and male gender are risk factors of central neck lymph node metastasis in cN0 PTC metastatic PTC to central neck lymph node. Patients with those factors should undergo prophylactic central neck lymph node dissection.

Objective Telomerase reverse transcriptase (TERT) promoter mutations have recently been described in thyroid carcinoma. The purpose of this study was to investigate the clinical significance of (v-raf murine sarcoma viral oncogene homolog B1) BRAF V600E and TERT promoter mutations in differentiated thyroid carcinoma (DTC). The relationship between the two mutations and NIS/TSHR expression was also analyzed. Methods We have detected BRAF V600E and TERT promoter mutations by direct sequencing and NIS/TSHR expression by immunohistochemistry in 229 cases of DTC, 52 cases of benign nodular goiter, and 31 cases of normal thyroid tissue. Results The BRAF V600E mutation was detected in 142 (62.0%) of 229 cases of DTC [141 cases of papillary thyroid carcinoma (PTC) and 1 case of follicular thyroid carcinoma (FTC)]. TERT promoter mutations were detected in 18 (7.9%) of 229 cases of DTC (14 cases of PTC and 4 cases of FTC), including the mutations C228T (0.9%) and C250T (7.0%), which were mutually exclusive. Moreover, 11 (61.1%) cases also harbored the BRAF V600E mutation, which was not associated with gender, age, tumor size, lymph node metastasis, and recurrence risk stratification (P &gt;0.05). The rate of TERT promoter mutation was higher in males, age ≥45, and in the middle/high-risk group (P &lt;0.05), and the rate of simultaneous BRAF V600E and TERT promoter mutations were higher in the middle/high-risk group (P &lt;0.05). In addition, NIS positive rate in the concurrent BRAF V600E and TERT promoter mutation group (45.5 %) was lower than in other groups (that is, the DTC group with BRAF V600E or TERT promoter mutations (55.1%), the DTC group with no BRAF V600E or TERT promoter mutation (57.5%), the nodules and normal group (75.9%); | r | = 0.171, P = 0.002). Conclusion TERT promoter mutations were lower in patients with DTC, with the C250T mutation being the most common. The detection of BRAF V600E mutation combined with TERT promoter mutations was instructive for the prognosis assessment and treatment of DTC.

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer with high incidence in endocrine tumors, which emphasizes the significance of accurate diagnostics. Still, the commonly used cytological method (fine-needle aspiration (FNA) cytology) and molecular diagnostic methods (such as PCR and sequencing) are limited in terms of diagnostic time, sensitivity, and user-friendliness. In this study, we introduce a novel Zip recombinase polymerase amplification (Z-RPA) strategy to efficiently detect rare mutant alleles in PTC fine-needle aspiration samples, which is sensitive, fast, and simple to manipulate. Using Zip nucleic acid (ZNA) probes to clamp the mutation region, the phi 29 polymerase could selectively displace mismatched ZNA probes and start amplification, while leaving complementary ZNA probes untouched and blocking amplification according to genotype. We demonstrated the good sensitivity and specificity of this strategy with optimized conditions and design, which enabled detection of BRAF V600E mutation in a total 4 ng of genomic DNA within 40 min (≈1 copy). Robust behavior in clinical specimen analysis was also demonstrated. The Z-RPA strategy provides a pragmatic approach to rapidly, sensitively, and easily detect BRAF V600E mutation in clinical fine-needle aspiration samples, which is a promising method for early cancer diagnosis and treatment guideline.

We are pleased to receive a letter from readers regarding our publication entitled "MRI-Based Texture Analysis for Preoperative Prediction of BRAF V600E Mutation in Papillary Thyroid Carcinoma". (ID: 405040). The responses to the readers' comments are as following: 1) Comment: It was clearly stated and done in the experimental works of Zheng T el al. that the BRAF gene mutation was confirmed by sequencing which was performed before MRI-based texture analysis. The inclusion criteria of the samples used in this study regarding the detection of BRAF V600E mutation detected by AB Diagnostic kit, which is based on the amplification-resisted mutation Information Classification: General system (ARMS) real-time polymerase chain reaction (PCR) technology, then confirmed by sequencing was questionable. Response: I apologize for the misunderstanding caused by the unclear presentation. Our institution sent specimens out to companies for testing until October 2021, using the Sanger sequencing method. After that, the amplification-resisted mutation system (ARMS) real-time polymerase chain reaction (PCR) technology has been used. Fifty-nine of our 80 patients were Sanger sequencing method and 21 were tested with ARMS real-time PCR technology. 2) Comment: In this study, the samples used were originally grouped based on their characteristics, BRAF V600E mutant (72.5%) and wild-type (27.5%). The question addressed here is regarding the conclusion taken in the study. It was mentioned that MRI-based texture analysis could be a potential method for predicting BRAF V600E mutation in PTC preoperatively in the conclusion section. However, based on the data, the mutation in BRAF gene was clearly not predicted by MRI. Response: Thank you for your comments. We used the results of genetic testing as a criterion to explore MRI-based texture with statistically significant differences between the mutant and wild groups and build the prediction model. The area under the ROC curves (AUCs) for the T2WI model, CE-T1WI model, and combined model were 0.83 (95% CI: 0.75-0.91), 0.83 (95% CI: 0.73-0.90), and 0.88 (95% CI: 0.81-0.94), respectively. The ICCs of the features we used for modeling were all greater than 0.75 with good agreement. So we think MRI-based texture analysis could be a potential method for predicting BRAF V600E mutation in PTC.

Fine-needle aspiration (FNA) of thyroid lesions may result in infarction and diagnostic difficulties on subsequent thyroidectomy specimens. Next-generation sequencing (NGS) methods for detection of hallmark driver BRAF V600E mutations may help characterize such tumors in which histologic alterations preclude definitive tissue diagnosis. Thyroidectomy specimens with both malignant FNA diagnoses and resultant infarction were identified from our institutional database. NGS methods were used to detect BRAF V600E mutations in the infarcted thyroid carcinomas. Nine thyroid carcinomas with infarction were characterized as BRAF-like papillary thyroid carcinoma based on molecular driver categorization and histologic diagnosis. BRAF V600E mutations were detected in the infarcted tissue in four (67%) of six lesions. We demonstrate detection of hallmark BRAF V600E mutations by NGS within infarcted tissue of thyroid carcinomas after FNA. This suggests a potential ancillary method of characterizing infarcted thyroid carcinomas whose altered histology may be nondiagnostic.