Marker For Papillary Thyroid Carcinoma Research Articles

Angiogenesis and CK19 expression in papillary thyroid carcinoma

Purpose: Aim of the present study is to evaluate immunohistochemically the diagnostic value of CK19 in papillary thyroid carcinoma (PC) and to determine the microvascular density (MDV) in tumor tissue by CD34. Methods: We studied immunohistochemical expression of CK19 in both tumor tissue and adjacent non-tumor tissue of ten patients that had undergone surgical resection for PC. We evaluated the MVD by CD34, an endothelial marker, in the same zones. Results: All neoplasms were CK19 positive and the tumor tissue showed a diffuse and strong cytoplasmic expression. In most of the cases, the adjacent non-tumor tissue was negative for CK19. However, a focal modarate staining occured in oncocytic cells and in areas of degeneration. The MVD in tumor tissue was 29.1±6.05. The microvessels were unevenly distributed as opposed to those in the adjacent tissue. The MVD in non-tumor tissue was 36.4±6.38. Conclusions: CK19 is a highly specific marker for PC, which means that it may be used as an additive tool in diagnosis of this carcinoma.. Angiogenesis is reduced and this may affect the biological behavior of the PC.

Morphological parameters able to predict BRAF(V600E) -mutated malignancies on thyroid fine-needle aspiration cytology: Our institutional experience.

The BRAF(V600E) mutation is the most common diagnostic/prognostic marker in papillary thyroid carcinoma (PTC). Its evaluation is typically performed with DNA-based techniques; nonetheless, a few articles have recently proposed the morphological prediction of BRAF(V600E) in histological PTCs. We investigated this morphological parameter in our cytological series. We re-analyzed all 72 cytohistological samples diagnosed as positive for malignancy (favoring PTC) on fine-needle aspiration cytology from January 2012 to December 2013. We included 22 male patients and 50 female patients. The cytological cases were processed with liquid-based cytology. We performed molecular analysis and immunocytochemistry for the VE1 BRAF(V600E) antibody. We reported 47 mutated cases and 25 wild-type (WT) cases with 100% cytohistological concordance. The cytological evaluations revealed plump cells (abundant eosinophilic cytoplasm and PTC nuclei) in all 47 mutated cases, with only 6 having a focal plump cell component (≤20% cells). Furthermore, 5 of the 25 WT cases showed focal plump cells. A distinctive sickle nuclear shape was found only in the mutated cases. VE1 yielded 100% positivity for all 24 mutated cases that were tested, including 3 cases with focal plump cells. We demonstrated that the BRAF(V600E) mutation might be predicted in cytological samples on the basis of some specific morphological features. Although the detection of plump cells (mainly focal) was also observed in WT cases, the detection of sickle-shaped nuclei provided the highest specificity and sensitivity as a predictive mutational parameter. These morphological features might be a valid tool for selecting cases for molecular analysis.

Anterior gradient protein 2 promotes survival, migration and invasion of papillary thyroid carcinoma cells.

BackgroundThrough a transcriptome microarray analysis, we have isolated Anterior gradient protein 2 (AGR2) as a gene up-regulated in papillary thyroid carcinoma (PTC). AGR2 is a disulfide isomerase over-expressed in several human carcinomas and recently linked to endoplasmic reticulum (ER) stress. Here, we analyzed the expression of AGR2 in PTC and its functional role.MethodsExpression of AGR2 was studied by immunohistochemistry and real time PCR in normal thyroids and in PTC samples. The function of AGR2 was studied by knockdown in PTC cells and by ectopic expression in non-transformed thyroid cells. The role of AGR2 in the ER stress was analyzed upon treatment of cells, expressing or not AGR2, with Bortezomib and analyzing by Western blot the expression levels of GADD153.ResultsPTC over-expressed AGR2 at mRNA and protein levels. Knockdown of AGR2 in PTC cells induced apoptosis and decreased migration and invasion. Ectopic expression of AGR2 in non-transformed human thyroid cells increased migration and invasion and protected cells from ER stress induced by Bortezomib.ConclusionsAGR2 is a novel marker of PTC and plays a role in thyroid cancer cell survival, migration, invasion and protection from ER stress.

CD117: a novel ancillary marker for papillary thyroid carcinoma in fine-needle aspiration biopsies.

c-KIT/CD117 down-regulation has been described in papillary thyroid carcinoma (PTC). In this study, the authors investigated CD117 as an ancillary immunocytochemical test for PTC in fine-needle aspiration biopsy (FNAB). The expression of CD117 was assessed in cytologic samples of histologically confirmed classic PTC (n = 35) and in a control group of benign thyroid nodules (BTNs) (n = 30) using immunocytochemistry. The corresponding PTCs and BTNs from surgical resection specimens also were evaluated immunohistochemically for CD117. The expression of CD117 was assessed semiquantitatively using a scoring system from 0 to 5 that combined the proportion and the intensity of staining. The difference in immunoreactivity for CD117 between PTC and BTNs was highly significant both in FNAB samples and in the corresponding histology samples (P < .0001). On cytology, most PTCs (74%; n = 26) lacked immunoreactivity for CD117, and 9 samples (26%) had faint reactivity in <2% of tumor cells (score, 0). In contrast, all BTNs (100%; n = 30) were positive for CD117, and at least 50% of the cells (score, 2-4) were positive in 79% (n = 24) of BTNs. Results with corresponding histology were similar to the cytology results; most PTCs (89%; n = 31) lacked immunoreactivity for CD117, and 4 samples (11%) had faint reactivity in <2% of tumor cells. In contrast, all BTNs (n = 30) were variably positive for CD117 (score, 2-4). Normal follicular epithelium also was positive for CD117 (score, 2-4). CD117 is expressed in both normal follicular epithelium and in BTNs. In contrast, expression of CD117 is absent or weak in PTC. These results suggest that CD117 may be useful as an ancillary marker for PTC.

Detection of serum p53 antibodies from Chinese patients with papillary thyroid carcinoma using phage-SP-ELISA: correlation with clinical parameters

The goal of the present study was to investigate whether p53 antibodies (Abs) could be a relevant marker for papillary thyroid carcinoma (PTC). Three types of enzyme-linked immunosorbent assay (ELISA) methods were developed for the detection of p53 Abs, including p53-ELISA, phage-SS-ELISA, and phage-SP-ELISA. A total of 304 patients, including 117 cases with thyroid adenoma and 187 PTC patients, were enrolled in this study. Expression of p53 protein and mutation in BRAF gene were evaluated in paraffin-embedded tissue from 44 patients with PTC, in order to elucidate their correlations with the presence of p53 Abs. Compared with p53-ELISA and phage-SS-ELISA, phage-SP-ELISA presented the highest detection efficiency of p53 Abs in patients with PTC, and a combination of these three ELISA systems could make the detection of p53 Abs more sensitive than using each of the individual ELISA methods. Furthermore, p53 Abs was positively associated with clinical stage (P = 0.044), node metastasis (P = 0.010), and p53 protein accumulation (P = 0.019). These results indicate that serum p53 Abs could be a useful marker for PTC.

A testing algorithm for detection of the B-type Raf kinase V600E mutation in papillary thyroid carcinoma.

The B-type Raf kinase (BRAF) V600E mutation is a useful diagnostic marker for papillary thyroid carcinoma (PTC). We developed a testing algorithm for the BRAF mutation using BRAF immunohistochemistry (IHC) in PTC. Formalin-fixed, paraffin-embedded PTC tissues from 91 patients were immunostained with a BRAF V600E mutation-specific antibody. The immunostaining results were interpreted semiquantitatively by adding the scores of the proportion of positive cells (scored 0 to 5) and staining intensity (score 0 to 3). To validate BRAF IHC, 4 molecular methods were used: direct sequencing, pyrosequencing, peptide nucleic acid clamping polymerase chain reaction, and real-time polymerase chain reaction. The cut-off score of BRAF IHC for predicting the BRAF mutation was 5.5. In 68 cases, where the BRAF IHC score was 6 or more, 89.7% were positive for the BRAF mutation by 2 or more molecular methods and 97.1% by one or more methods. In 13 cases, where the IHC score was between 4 or more and less than 6, 61.5% were positive for the BRAF mutation by 2 or more methods and 69.2% by at least one method. In 10 cases, where the IHC score was less than 4, BRAF mutation was noted in one case only by one molecular method. BRAF V600E IHC scores of less than 4 or 6 or more should not require confirmation by additional tests. However, BRAF V600E IHC scores of 4 or 5 may require confirmation of the BRAF V600E mutation. In addition, BRAF IHC scores 6 or more were more sensitive than any single molecular method; therefore, BRAF IHC may overcome the limitations of using a single type of molecular method.

Thyroglobulin Antibodies Could be a Potential Predictive Marker for Papillary Thyroid Carcinoma

Hashimoto thyroiditis (HT) is associated with an increased risk of developing papillary thyroid carcinoma (PTC). The relationship between thyroid autoimmunity and cancer remains controversial. The purpose of this study was to investigate whether the preoperative TgAb could be a potential predictor of PTC in patients with thyroid nodules and to assess whether there is an association of preoperative TgAb with lymph node metastases. This retrospective, nonrandomised study included 854 patients who underwent standard total thyroidectomy. Benign thyroid nodules were diagnosed in 447 patients, and 407 presented with malignant nodules. The examined parameters included the clinical characteristics, preoperative TSH and TgAb levels, and the histopathological characteristics of the tumour. Tumour size >10mm (p=0.01), the presence of PTC (p<0.001), elevated TSH levels (2.64±1.28 μU/ml vs. 2.09±0.98μU/ml, p=0.001), HT (p<0.001), and lymph node metastasis (p=0.005) were significantly associated with positive TgAb. Additionally, tumour size >10mm (p<0.001), preoperative TgAb positivity (p=0.003), and elevated TSH levels (TSH>3.4μU/ml, p=0.038) were independent risk factors for PTC based on the multivariate logistic regression analysis. This study showed that TgAb positivity was an independent risk factor for PTC. A positive correlation between TgAb and PTC in patients with indeterminate nodules was existed. Additionally, a positive correlation existed between TgAb and lymph node metastases in patients with PTC. Prospective studies with a larger number of patients and long-term follow-up are needed clarify the potential role of positive serum TgAb in the prediction of PTC.

The prognostic significance of the BRAFV600E mutation in papillary thyroid carcinoma detected by mutation-specific immunohistochemistry

BRAF mutation has been shown in a large meta-analysis to be an independent prognostic marker for papillary thyroid carcinoma (PTC) with poorer survival and higher recurrence rates. We studied prevalence of BRAF mutation in 77 patients with PTC from an Australian cohort using competitive polymerase chain reaction (C-PCR) and immunohistochemistry (IHC) with BRAF-specific antibody, VE1. Clinicopathological parameters, recurrence and mortality were analysed according to BRAF mutation status. Median follow-up was 84.5 months. BRAF mutation was demonstrated in 65% of cases combining both C-PCR and IHC; in 71% (37/77) of tumours >1 cm and 52% (13/25) of microcarcinomas (<1 cm). IHC was positive in 69% (49/71) and C-PCR in 53% (41/77); 87% (67/77) of our patients were treated with total thyroidectomy and 65% (50/77) also had radioactive ablation. BRAF positive tumours had a significantly higher rate of subsequent lymph node metastases (p=0.035). Significant association was found between BRAF mutation and male sex (p=0.034), but not between age >45 years at diagnosis, size of primary tumour, extrathyroidal extension, lymph node or distant metastases or clinical stage at diagnosis. BRAF mutation in PTC determined by IHC is associated with significantly increased risk of lymph node recurrence.

Quantitative assessment of RASSF1A methylation as a putative molecular marker in papillary thyroid carcinoma

Epigenetic alterations such as DNA methylation are widespread cancer, contributing to tumorigenesis and acting as markers for prognostication. Papillary thyroid cancer (PTC) demonstrates tumor-specific methylation of numerous genes, including RASSF1A. Although the function of RASSF1A in PTC tumorigenesis is still being defined, quantitative evaluation of RASSF1A methylation and its correlation with tumor characteristics has not been performed. Analysis of RASSF1A methylation was performed using quantitative polymerase chain reaction after methylation-dependent and -sensitive restriction enzyme digestion in PTC (n = 41) and normal (n = 18) thyroid tissue. Methylation was then evaluated for correlation with tumor size, stage, and multiple histopathologic characteristics. RASSF1A promoter hypermethylation was observed in nearly all PTC cases versus normal thyroid tissue, with mean hypermethylation 4.2 times greater in PTC (P < .05). Hypermethylation was greater in multifocal than unifocal PTC (P < .05). Furthermore, tumor methylation was inversely correlated with extracapsular invasion (P < .05). RASSF1A methylation differs in PTC compared with normal thyroid, is associated with multifocality, and is inversely correlated with extracapsular invasion. The ease of evaluating methylation status with minute amounts of DNA suggests a potential role for RASSF1A as a molecular marker for characterization of PTC histopathology.

Comprehensive MicroRNA Expression Profiling Identifies Novel Markers in Follicular Variant of Papillary Thyroid Carcinoma

Follicular variant of papillary thyroid carcinoma (FVPTC) shares features of papillary (PTC) and follicular (FTC) thyroid carcinomas on a clinical, morphological, and genetic level. MicroRNA (miRNA) deregulation was extensively studied in PTCs and FTCs. However, very limited information is available for FVPTC. The aim of this study was to assess miRNA expression in FVPTC with the most comprehensive miRNA array panel and to correlate it with the clinicopathological data. Forty-four papillary thyroid carcinomas (17 FVPTC, 27 classic PTC) and eight normal thyroid tissue samples were analyzed for expression of 748 miRNAs using Human Microarray Assays on the ABI 7900 platform (Life Technologies, Carlsbad, CA). In addition, an independent set of 61 tumor and normal samples was studied for expression of novel miRNA markers detected in this study. Overall, the miRNA expression profile demonstrated similar trends between FVPTC and classic PTC. Fourteen miRNAs were deregulated in FVPTC with a fold change of more than five (up/down), including miRNAs known to be upregulated in PTC (miR-146b-3p, -146-5p, -221, -222 and miR-222-5p) and novel miRNAs (miR-375, -551b, 181-2-3p, 99b-3p). However, the levels of miRNA expression were different between these tumor types and some miRNAs were uniquely dysregulated in FVPTC allowing separation of these tumors on the unsupervised hierarchical clustering analysis. Upregulation of novel miR-375 was confirmed in a large independent set of follicular cell derived neoplasms and benign nodules and demonstrated specific upregulation for PTC. Two miRNAs (miR-181a-2-3p, miR-99b-3p) were associated with an adverse outcome in FVPTC patients by a Kaplan-Meier (p < 0.05) and multivariate Cox regression analysis (p < 0.05). Despite high similarity in miRNA expression between FVPTC and classic PTC, several miRNAs were uniquely expressed in each tumor type, supporting their histopathologic differences. Highly upregulated miRNA identified in this study (miR-375) can serve as a novel marker of papillary thyroid carcinoma, and miR-181a-2-3p and miR-99b-3p can predict relapse-free survival in patients with FVPTC thus potentially providing important diagnostic and predictive value.

CXC motif ligand 12 as a novel diagnostic marker for papillary thyroid carcinoma

We subjected chemokine (C-X-C motif) ligand 12 (CXCL12) to immunohistochemistry to determine its utility as a novel diagnostic marker for papillary thyroid carcinoma (PTC) in comparison to cytokeratin 19 (CK19), Hector Battifora mesothelial epitope-1 (HBME-1), and galectin-3. We analyzed the expressions of CXCL12, CK19, HBME-1, and galectin-3 using immunohistochemical staining in 258 cases of thyroid lesions (196 PTCs and 62 thyroid lesions excluding PTC). Remarkably, CXCL12 expression was exclusively found in PTC compared to other thyroid lesions (90.8% vs 3.2%; p < .001). In total, 91.1% (62 of 68) of the variant PTCs and 90.6% of conventional type (116 of 128) were associated with CXCL12 immunohistochemical expression, irrespective of the histological subtype. In contrast, very few of the thyroid lesions, excluding PTC, were positive for CXCL12 (2 of 62; 3.2%). Diagnostic performances for PTCs were as follows: sensitivity, 90.8%; specificity, 96.8%; positive predictive value, 98.9%; negative predictive value, 76.9%; and diagnostic accuracy, 92.2%. Our findings indicate that CXCL12 might serve as an effective novel supplementary diagnostic marker for PTC.

Application of Chemokine CXC Motif Ligand 12 as a Novel Diagnostic Marker in Preoperative Fine-Needle Aspiration Biopsy for Papillary Thyroid Carcinoma

Objectives: A panel composed of three immunomarkers, cytokeratin 19, HBME-1 and galectin-3, is recommended for improving the accuracy of diagnosing papillary thyroid carcinoma (PTC), but the results are sometimes inconsistent and difficult to interpret. We used immunocytochemistry to assess the utility of chemokine CXC ligand 12 (CXCL12) as a novel diagnostic marker for PTC employing paraffin-embedded cell blocks. Methods: We analyzed the expression of CXCL12 using immunocytochemical staining in 82 cases of thyroid lesions (47 PTCs and 35 thyroid lesions other than PTC). To determine the optimal cut-off value for the assessment of CXCL12 positivity, we used receiver operating characteristics (ROC). Results: ROC curves showed that the optimum diagnostic cut-off was 10% (area under the curve 0.950, 95% confidence interval 0.891-1.008), with exclusive CXCL12 expression in PTC compared to other thyroid lesions (p < 0.001). In total, more than 90% of the PTCs were associated with CXCL12 immunohistochemical staining, while only up to 11.4% of thyroid lesions other than PTC were positive for CXCL12. The follicular variant PTC showed 90% CXCL12 expression compared to 10.5% positivity in follicular neoplasm. Conclusion: Our findings indicate that CXCL12 may be an effective supplementary diagnostic marker for PTC in preoperative fine-needle aspiration cytology using the cell block method.

CirculatingBRAFV600Ein the Diagnosis and Follow-Up of Differentiated Papillary Thyroid Carcinoma

Cell-free nucleic acids circulating in plasma are considered a promising noninvasive tool for cancer monitoring. BRAF(V600E) mutation in cell-free DNA (cfDNA) could represent an appropriate marker for papillary thyroid carcinoma (PTC). Our aim is to investigate the role of BRAF(V600E)-mutated allele in cfDNA as a marker for the diagnosis and follow-up of PTC. BRAF(V600E) allele was detected and quantified by an allele-specific real-time quantitative PCR assay in plasma from 103 patients affected by nodular goiter. As control populations, we enrolled 49 healthy subjects and 16 patients with non-nodular thyroid diseases. The percentage of circulating BRAF(V600E) was significantly different between patients and controls and throughout different cytological categories of ultrasound-assisted fine-needle aspiration. Patients with a histopathological diagnosis of PTC showed a higher percentage of circulating BRAF(V600E) (P = .035) compared to those with benign histology. In 19 patients, a second blood draw, taken 3-6 months after surgery, showed a lower percentage of BRAF(V600E) in cfDNA than the presurgical sample (P < .001). The diagnostic performance of circulating BRAF(V600E) was assessed by receiver operating characteristic curve analysis resulting in an area under the curve of 0.797. A cutoff value was chosen corresponding to maximum specificity (65%) and sensitivity (80%). On this basis, we evaluated the predictive value of BRAF(V600E) in Thy 3 patients with a resulting positive predictive value of 33% and a negative predictive value of 80%. The results of the present study provide encouraging data supporting the possibility to take advantage of circulating BRAF(V600E) in the management of PTC.

Allele Percentage of theBRAFV600E Mutation in Papillary Thyroid Carcinomas and Corresponding Lymph Node Metastases: No Evidence for a Role in Tumor Progression

The relevance of the BRAF V600E mutation in papillary thyroid carcinoma (PTC) as a negative prognostic factor is a subject of intense debate. This mutation has been associated with several clinicopathological features, but the lack of consistency among data does not support its usefulness as marker of tumor aggressiveness and poorer outcome. Due to the genetic heterogeneity of the tumor, both the occurrence and the allele percentage of the BRAF mutation should be considered to unravel this controversy. We aimed to evaluate the impact of the BRAF V600E mutation occurrence and the allele percentage on the metastatic process in PTCs. The presence and allele percentage of the BRAF mutation were determined by pyrosequencing in 132 cases of well-differentiated PTCs with (n = 37) or without lymph node metastases (LNMs) (n = 95) and in 40 LNMs matched with 35 PTCs. No significant differences were observed in either the occurrence or the allele percentage of V600E mutation between the 2 groups of PTCs with or without LNMs. The LNMs were heterogeneous for the V600E mutation as the primary lesions. In this study, the occurrence and percentage of the BRAF V600E mutated allele was not preferentially associated with the development of metastases and the average mutated allele percentage decreased as the tumor progresses from the primary site to the lymph node metastatic sites. These observations support the need to reevaluate the role of the BRAF V600E mutation as a negative prognostic marker in PTCs.

Sorting nexin 5 of a new diagnostic marker of papillary thyroid carcinoma regulates Caspase‐2

Papillary thyroid carcinoma (PTC) is a well-differentiated endocrine malignant tumor that develops from thyroid follicular epithelium. The tumor represents the most common type of endocrine malignancy; however, its tumorigenesis is not fully elucidated. The aim of this study was to address the functional role of the sorting nexin (SNX) family in PTC because of recent experimental evidence suggesting that the SNX family members actively control endocytotic transportation as well as cell fate. Expression profiles of SNX family members of PTC showed a significant quantity of transcripts of SNX5. Further immunohistochemical analysis with an SNX5-specific monoclonal antibody established in this study consistently demonstrated the preferential expression of SNX5 in PTC (94.2%, 113/120 cases) as indicated by studies on 440 cases of various tumors. In contrast, other major carcinomas originating from the lung (2.6%, 1/38 cases), breast (5.1%, 2/39 cases), and intestine (4.2%, 1/24 cases) scarcely expressed SNX5. When we investigated models of murine thyroid tumors induced by the administration of carcinogens, high expression of Snx5 was also observed in well-differentiated thyroid tumors, further implying that the tumorigenesis of the thyroid gland was tightly associated with the abundance of SNX5/Snx5. Moreover epithelial cells expressing excess SNX5 showed high levels of Caspase-2 of an initiator caspase. Collectively these findings suggest that the evaluation of SNX5 expression would support pathological diagnosis of primary and secondary PTC.

Role of Chronic Lymphocytic Thyroiditis in Central Node Metastasis of Papillary Thyroid Carcinoma

(1) To investigate the role of chronic lymphocytic thyroiditis (CLT) in central node metastasis of papillary thyroid carcinoma (PTC) and (2) to evaluate the presence of chronic lymphocytic thyroiditis according to PTC-specific molecular markers. Historical cohort study. Academic medical center. All patients who underwent total thyroidectomy with central neck dissection for PTC at Oregon Health & Science University between 2005 and 2010 were screened for the presence of CLT and reviewed for clinical prognostic factors. Patients with inadequate central neck dissections were excluded. Molecular markers for PTC were analyzed on archived tumor samples. A total of 139 patients met selection criteria. The rate of CLT was 43.8%. The rate of central node positivity was 63%. Presence of CLT was associated with a significantly lower proportion of central node metastases (49% vs 74%, P = .003) and angiolymphatic invasion (31% vs 15%, P = .03). There was no significant difference in mean age, tumor size, and extracapsular extension. Molecular genotyping did not reveal a significant difference in the types of mutations found in both groups. The data indicate a lower incidence of central compartment lymph node metastasis in those with CLT in this patient population, suggesting a potential protective role in tumor spread. The equal distribution of tumor mutations between the carcinomas with and without evidence of CLT argues against a mutation-specific antigen as the immunologic stimulus. Further research is needed to characterize the role of autoimmunity in thyroid cancer.

A High Percentage of BRAFV600E Alleles in Papillary Thyroid Carcinoma Predicts a Poorer Outcome

BRAF(V600E) is considered a negative prognostic marker in papillary thyroid carcinoma (PTC), but unexplained conflicting results are present in the literature. In light of the new finding that most PTC consist of a mixture of tumor cells with wild-type and mutant BRAF, we examined the associations between the percentage of BRAF(V600E) alleles and both the clinicopathological parameters at time of diagnosis and the disease outcome in a large series of PTCs. Tumors from 168 patients with PTC were genotyped for BRAF(V600E) using BigDye Terminator sequencing and pyrosequencing, and the clinical parameters were analyzed. The associations between clinicopathological characteristics, including disease recurrence at follow-up (median 5.1 yr) and the percentage of mutant BRAF alleles were assessed. The observed prevalence of BRAF(V600E) was higher when using pyrosequencing then when using BigDye Terminator sequencing (53.6 vs. 36.9%). In the PTC positive for BRAF(V600E), the percentage of mutant alleles ranged from 5.1 to 44.7% of the total BRAF alleles, with a median of 20.6%. The presence or the percentage of BRAF(V600E) alleles did not correlate significantly with sex, multicentricity, lymph node metastasis, or tumor stage. The percentage of BRAF(V600E) alleles directly correlated with age at diagnosis and tumor volume (R(2) = 0.223, P = 0.039, and R(2) = 0.166, P < 0.001, respectively). The percentage of BRAF(V600E) alleles (P = 0.014), tumor volume (P = 0.012), and lymph node metastasis (P = 0.008) predicted the disease outcome. The odds ratio of recurrence for PTC with BRAF(V600E) alleles of 30% or greater, compared with that for PTC with BRAF(V600E) alleles of less than 30%, was 5.31 (P = 0.002). A high percentage of BRAF(V600E) alleles defines a PTC molecular subtype and predicts a poorer disease outcome. The analysis of BRAF mutations by pyrosequencing is useful to refine the risk stratification of patients with PTC.

Cytokeratin 19 immunoreactivity in the diagnosis of papillary thyroid carcinoma

Context:The diagnosis of papillary thyroid carcinoma (PTC) is based on nuclear features. These features may be present in focal areas in benign thyroid diseases and follicular adenoma (FA), leading to diagnostic difficulty.Aims:To evaluate the expression and pattern of the distribution of cytokeratin 19 (CK19) in PTC and compare its reactivity with other neoplastic and non-neoplastic conditions to assess its potential as a useful marker for PTC.Materials and Methods:Twenty two cases of papillary carcinoma (usual type, follicular and diffuse sclerosing variant), eight follicular adenomas, eight multinodular goiters (MNG) were collected for a period of two years and six months. Sections were taken from thyroidectomy specimens fixed in 10% buffered neutral formalin. Hematoxylin and eosin staining and immunohistochemical staining for CK19 were done using standard protocol. Results were semiquantitatively scored as follows: 1+ (<5% positively stained cells), 2+ (5-25%), 3+ (25-75%) and 4+ (>75%), and then analyzed.Statistical Analysis and Results:All 22 (100%) papillary carcinomas showed diffuse and strong (3+ and 4+) CK19 expression. Six out of eight (75%) FAs and four out of eight (50%) MNG were positive for CK19, but it was of weaker intensity (1+ and 2+) and focal in distribution.Conclusion:Focal CK19 staining may be found in benign disease, but diffuse and strong positivity is characteristic of PTC, which can be used in the diagnosis of PTC in lesions of equivocal morphological appearances.

BRAF mutation testing in clinical practice

Serine/threonine-protein kinase BRAF, a downstream effector of the RAS oncogene along the MEK/ERK signaling pathway, has emerged as an important biological marker for diagnosis, prognosis and therapeutic guidance for human cancers. The high prevalence of BRAFV600E activating mutation in papillary thyroid carcinoma, cutaneous malignant melanoma and hairy cell leukemia implies that the mutation is an important ’driver‘ or ’codriver‘ in the development of a subset of these cancers. Diagnostically, the BRAFV600E mutation is a powerful molecular marker for papillary thyroid carcinoma and, quite possibly, hairy cell leukemia as well. Cancers with a BRAF mutation are generally more aggressive than their counterparts without the mutation. Importantly, mutant BRAF has been a highly attractive target for precision cancer therapy. Indeed, recent studies in the clinical trials of BRAF inhibitors in patients with malignant melanoma are changing the treatment paradigm of this highly lethal disease. BRAF mutation testing using highly sensitive and specific methodology in a molecular diagnostic laboratory is essential in the current clinical practice of oncology.

The prognostic significance of the V600E B-Raf mutation in papillary thyroid carcinoma: data from an Australian population

Aims B-Raf V600E mutation in the thyroid is a specific marker of papillary thyroid carcinoma (PTC) and correlates with poorer survival and rates of recurrence.1,2 We assessed the prevalence of this mutation in a series of PTCs from an Australian population, its association with aggressive clinicopathological features, recurrence and mortality. Methods 82 patients with a median follow up of 6 years were studied. V600E mutation status was determined by PCR on DNA extracted from paraffin blocks. Clinicopathological parameters, recurrence and mortality were analysed according to V600E mutation status. Results The V600E mutation was demonstrated in 44 of 82 (54%) tumours. Up to 58% of patients with V600E positive PTCs suffered a recurrence compared to 12% of patients with V600E negative PTCs (p = 0.005). Up to 15% of patients with V600E positive PTCs died during the follow-up period compared to 5% of patients without the mutation (p = 0.19). The V600E mutation was associated with male sex (p = 0.0002) and the classic subtype (p = 0.005). Discussion The prevalence of B-Raf V600E mutation in PTCs in this study is similar to other series. We confirmed that this mutation was associated with a poorer prognosis, specifically higher rates of recurrence. B-Raf V600E mutation in the thyroid is a specific marker of papillary thyroid carcinoma (PTC) and correlates with poorer survival and rates of recurrence.1,2 We assessed the prevalence of this mutation in a series of PTCs from an Australian population, its association with aggressive clinicopathological features, recurrence and mortality. 82 patients with a median follow up of 6 years were studied. V600E mutation status was determined by PCR on DNA extracted from paraffin blocks. Clinicopathological parameters, recurrence and mortality were analysed according to V600E mutation status. The V600E mutation was demonstrated in 44 of 82 (54%) tumours. Up to 58% of patients with V600E positive PTCs suffered a recurrence compared to 12% of patients with V600E negative PTCs (p = 0.005). Up to 15% of patients with V600E positive PTCs died during the follow-up period compared to 5% of patients without the mutation (p = 0.19). The V600E mutation was associated with male sex (p = 0.0002) and the classic subtype (p = 0.005). The prevalence of B-Raf V600E mutation in PTCs in this study is similar to other series. We confirmed that this mutation was associated with a poorer prognosis, specifically higher rates of recurrence.