Dysembryoplastic Neuroepithelial Tumors Share with Pleomorphic Xanthoastrocytomas and Gangliogliomas BRAFV600E Mutation and Expression

Pediatric cortical glioneuronal benign tumors mainly include gangliogliomas (GG) [differential diagnoses pilocytic astrocytomas (PA) and pleomorphic xanthoastrocytomas (PXA)] and dysembryoplastic neuroepithelial tumor (DNT). DNT include the specific form and the controversial non‐specific form that lack the specific glioneuronal element. Our aims were to search for BRAF^V600E mutation and CD34 expression in DNT, PXA, GG and PA to correlate BRAF^V600E mutation with BRAF^V600E expression and to evaluate their diagnostic and prognostic values. Ninety‐six children were included. BRAF^V600E mutation was studied by sequencing and immunohistochemistry; CD34 expression was analyzed by immunohistochemistry. BRAF^V600E mutation was detected in PXA (60%), GG (38.7%), DNT (30%, including 3/11 specific and 3/9 non‐specific forms) and PA (12.5%). BRAF^V600E expression was recorded in PXA (60%), GG (45.2%) and DNT (30%). CD34 expression was recorded in PXA (60%), GG (58.1%), DNT (25%) and PA (12.5%). Neither CD34 expression nor BRAF^V600E status was predictive of prognosis, except for PA tumors where CD34 expression was associated with a shorter overall survival. In conclusion, DNT shared with PXA and GG, BRAF^V600E mutation and/or CD34 expression, which represent molecular markers for these tumors, and we recommend searching for CD34 expression and BRAF^V600E mutation in all DNT, especially the non‐specific forms.     

Frequent <Emphasis Type="Italic">BRAF</Emphasis><Superscript><Emphasis Type="Italic">V600E</Emphasis></Superscript> and Absence of <Emphasis Type="Italic">TERT</Emphasis> Promoter Mutations Characterize Sporadic Pediatric Papillary Thyroid Carcinomas in Japan

Pediatric papillary thyroid carcinoma (PTC) has unique features but requires further genetic investigation. Moreover, there has been increasing concern about the risk for pediatric PTC in Japan after the Fukushima accident. This study aims to evaluate the frequencies of BRAF and TERT promoter mutations and to examine their significance in non-radiation-associated pediatric PTCs in Japan. We enrolled 81 pediatric PTC patients aged ≤20 years. The control group included 91 adult PTCs from patients >20 years old. BRAF and TERT mutations were analyzed by allele-specific-PCR and/or Sanger sequencing. Compared with adult PTCs, pediatric PTCs exhibited larger tumor size, more frequent lymph node metastasis, and less classical histology. The prevalence of BRAF ^V600E in pediatric PTCs was 54% and significantly lower than that in adults of 85%. In the pediatric PTCs, BRAF ^V600E was positively associated with older age, classical histology, and the lymph node metastasis but independent from other clinicopathological factors. TERT mutations were identified in 13% of adults and in none of the pediatric PTCs. In conclusion, pediatric PTCs are characterized by more advanced clinicopathological features, lower BRAF ^V600E frequency, and absence of TERT mutation. The BRAF ^V600E frequency in this study is similar to the reported BRAF ^V600E frequency in the ultrasonographically screened pediatric PTCs in Fukushima.     

Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma

The activating BRAF ^V600 mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF ^V600E mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF ^V600E mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF ^V600E mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7 %) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5 %) and 15 IHC-positive cases (15/39; 38.5 %) showed a BRAF wild-type by SaS. UDS detected a BRAF ^V600E mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100 % and 63 % and specificities of 95 and 100 % for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAF^V600E (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF ^V600E mutations in colorectal carcinoma.     

<Emphasis Type="Italic">BRAF</Emphasis><Superscript><Emphasis Type="Italic">V600E</Emphasis></Superscript> Mutation Analysis from May-Grünwald Giemsa-Stained Cytological Samples as an Adjunct in Identification of High-Risk Papillary Thyroid Carcinoma

The BRAF ^V600E mutation is specific for thyroid papillary cancer (PTC) and correlates with PTCs invasiveness. This study investigated whether detection of BRAF ^V600E mutation can be performed on routinely stained FNABs. We also examined if establishment of the BRAF ^V600E mutation could help in identification of patients at higher risk for metastatic disease. DNA was isolated from 134 FNABs samples (20 follicular neoplasm, ten suspicious for malignancy, and 104 malignant) using Pinpoint Slide DNA Isolation System. BRAF ^V600E mutation was detected by PCR followed by sequencing. DNA was successfully extracted from all examined FNABs samples. In follicular neoplasm, suspicious for malignancy and malignant FNABs, BRAF ^V600E mutation was found in 0/20 (0%), 2/10 (20%), and 47/104 (45.2%) of cases, respectively. Extra-thyroidal extension was detected in 35/47 (74.4%) BRAF ^V600E positive and in 24/57 (42.1%) wild-type BRAF cases (p = 0.001). Metastases were detected in 37/47 (78.7%) BRAF ^V600E positive and in 28/57 (49.1%) wild-type BRAF cases (p = 0.002). Our results showed that stained FNAB specimens can be used for DNA extraction and assessment of BRAF ^V600E mutation. Detection of BRAF ^V600E mutation had limited value in diagnoses of malignancy in follicular neoplasms but can ascertain malignancy in subset of suspicious for malignancy FNABs. In malignant FNABs, BRAF ^V600E mutation was significantly associated with presence of extra-thyroidal extension and metastases after surgery.     

An immunohistochemical and genetic study of BRAFV600E mutation in Japanese patients with ameloblastoma

Ameloblastoma is an odontogenic tumor of the jaw. It most frequently occurs in the mandible, and less often in the maxilla. Mandibular ameloblastoma harbors a BRAF mutation that causes a valine (V) to glutamic acid (E) substitution at codon 600 (BRAF^V600E). We examined specimens from 32 Japanese patients to detect the prevalence of the BRAF^V600E mutation, and to evaluate the relationship between immunohistochemical (IHC) expression and genetic results, of BRAF^V600E+ ameloblastoma. Among the 32 cases, 22 (69%) were IHC positive for BRAF^V600E protein, and 10 (31%) were IHC negative; and polymerase chain reaction showed 16 of 21 tested cases (76%) carried the BRAF^V600E mutation. Our findings indicate that that samples that stain IHC positive for BRAF^V600E protein are more likely to carry the BRAF^V600E mutation. These results support assessments for BRAF mutations, and the use of BRAF inhibitors as targeted therapy for ameloblastoma in Japanese patients.     

<Emphasis Type="Italic">BRAF</Emphasis><Superscript>V600E</Superscript> Mutation in Papillary Thyroid Carcinoma: Significant Association with Node Metastases and Extra Thyroidal Invasion

B-Raf (BRAF) is the strongest activator in the downstream of MAP kinase signaling. The somatic point mutation of BRAF gene (V600E) is the most common and specific event in papillary thyroid carcinoma (PTC). However, its prevalence is variable among different studies and its association with clinico-pathological features is controversial. This study tests the prevalence of BRAF ^V600E mutation in thyroid cancer patients in Indian subcontinental population. We analyzed 140 thyroid tumor specimens for BRAF gene mutation at codon 600 using mutant-allele-specific amplification, single-strand conformation polymorphism, Mutector assay, and DNA sequencing of the PCR-amplified exon 15. BRAF mutation at codon 600 was detected in 46 of 86 PTC patients (53.4%) from Indian subcontinental cohort. Frequency of mutation varied across the subtypes of PTCs. BRAF ^V600E mutation was more common in the conventional PTC (38 out of 62; 61%) than in the follicular variant of PTC (2 out of 17; 11.7%). None of the 8 follicular thyroid adenomas, 14 follicular thyroid carcinomas, 16 medullary thyroid carcinomas, and 16 benign hyperplasia patients showed any exon 15 mutation. We found significant correlation between BRAF mutation status and extra-thyroidal invasion, lymph node metastasis, and tumor stage. However no correlation was observed with gender, age, and tumor size of the patients. Thus our findings suggest that BRAF ^V600E is a prevalent genetic alteration in adult sporadic PTCs in Indian cohort and it may be responsible for the progression of classic variant of PTC to metastatic and poorly differentiated subtype and likely to have significant impact on its diagnostic and prognostic management.     

Mutations in genes encoding PI3K‐AKT and MAPK signaling define anogenital papillary hidradenoma

Papillary hidradenoma (a.k.a. hidradenoma papilliferum) is a benign tumor of the anogenital region that almost exclusively arises in middle‐aged Caucasian women. These tumors may recur and rare cases of malignant development have been reported. The genetic basis of papillary hidradenoma is currently unknown. Hence, we employed targeted high‐coverage next generation sequencing interrogating 50 cancer‐related genes and conventional Sanger sequencing to investigate the mutational landscape in a cohort of 15 cases. Additionally, we analyzed the HPV status of these tumors. Thirteen cases (87%) harbored mutations in cancer‐related genes. Recurrent mutations in PIK3CA and AKT1 were present in 10 of the cases (67%). One PIK3CA mutated case had a concomitant STK11 mutation. Three cases harbored mutually exclusive mutations in BRAF, APC and ERBB4. The remaining two cases showed no mutations. None of the cases harbored DNA of human papilloma virus. Our results also provide evidence that –just as BRAF V600E mutations in hyperplastic polyps and benign nevi‐ a mutated driver gene does not imply malignant behavior per se but may set the basis for malignant transformation. The latter point may explain why rare cases of papillary hidradenoma have been reported to take a malignant course. Lastly, our genetic data may suggest treatment avenues beyond conventional surgery for some of these tumors. © 2015 Wiley Periodicals, Inc.     

Type and prevalence of BRAF mutations are closely associated with papillary thyroid carcinoma histotype and patients’ age but not with tumour aggressiveness

A high prevalence of the BRAF^V600E somatic mutation was recently reported in several series of papillary thyroid carcinomas (PTC). This mutation appears to be particularly prevalent in PTC with a predominantly papillary architecture. Another BRAF mutation (K601E) was detected in a follicular adenoma and in some cases of the follicular variant of PTC. The few studies on record provided controversial data on the relationship between the occurrence of BRAF mutations and clinicopathologic parameters such as gender, age and tumour staging. In an attempt to clarify such controversies we decided to enlarge our previous series to 315 tumours or tumour-like lesions diagnosed in 280 patients, including a thorough analysis of several clinicopathologic features. The BRAF^V600E mutation was exclusively detected in PTC with a papillary or mixed follicular/papillary architecture both of the conventional type (46%) and of other histotypes, such as microcarcinoma (43%), Warthin-like PTC (75%) and oncocytic variant of PTC (55%). The BRAF^K601E mutation was detected in four of the 54 cases of the follicular variant of PTC (7%). The mean age of patients with conventional PTC harbouring BRAF^V600E (46.7 years) was significantly higher (P<0.0001) than that of patients with conventional PTC without BRAF^V600E (29.5 years). The BRAF (BRAF^V600E) mutated PTC did not exhibit signs of higher aggressiveness (size, vascular invasion, extra-thyroid extension and nodal metastasis) and were in fact less often multicentric than PTC without the mutation.V. Trovisco and P. Soares contributed equally to this workFundação para a Ciência e Tecnologia POCTI/FEDER (POCTI/NSE/48171/2002)     

Aberrant BRAF splicing as an alternative mechanism for oncogenic B‐Raf activation in thyroid carcinoma

Activating BRAF mutations have recently been reported in 28–83% of papillary thyroid carcinomas (PTCs). However, it is not known whether aberrant BRAF splicing occurs in thyroid carcinoma. To investigate aberrant BRAF splicing and its association with BRAF mutation in thyroid tumours, we studied aberrant BRAF splicing and BRAF mutation from 68 thyroid tumours. BRAF^V600E mutation was detected in 20 of 43 PTCs and all three anaplastic thyroid carcinomas (ATCs). There is a higher frequency of BRAF mutation in PTC patients with stage III and IV tumours compared with stage I and II. Novel BRAF splicing variants were detected in 12 PTCs, three follicular variants of PTC (FVPTCs), and one ATC, as well as in two thyroid carcinoma cell lines, ARO and NPA. These variants did not have the N‐terminal auto‐inhibitory domain of wild‐type B‐Raf, resulting in an in‐frame truncated protein that contained only the C‐terminal kinase domain and caused constitutive activation of B‐Raf. These variants were significantly associated with advanced disease stage and BRAF^V600E mutation (p < 0.001, Fisher exact test). Furthermore, expression of these variants in NIH3T3 and CHO cells could activate the MAP kinase signalling pathway, transform them in vitro, and induce tumours in nude mice. These data suggest that BRAF splicing variants may function as an alternative mechanism for oncogenic B‐Raf activation. Combination of the BRAF^V600E mutation and its splicing variants may contribute towards disease progression to poorly differentiated thyroid carcinoma. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Association of BRAFV600E mutation with clinicopathological features of papillary thyroid carcinoma: a study on a Chinese population

Background: The new finding of the heterogeneous distribution of BRAF^V600E mutation in primary papillary thyroid carcinoma suggested the percentage of BRAF^V600E alleles should be taken into consideration when evaluating its association with clinicopathological features of papillary thyroid carcinoma. The aim of this study was to detect both the presence and the percentage of BRAF^V600E alleles in fine-needle aspiration biopsy samples and to assess its association with clinicopathological characteristics of papillary thyroid carcinoma in a Chinese population. Materials and methods: Fine needle aspiration samples were collected in a total of 182 patients (132 conventional papillary thyroid carcinomas and 50 goiters). The associations of the presence and percentage of BRAF^V600E alleles genotyped by pyrosequencing with clinicopathological characteristics were evaluated in papillary thyroid carcinomas. Results: 80 (60.61%) of papillary thyroid carcinomas exhibited BRAF^V600E mutation in a range of 7.7% to 46.3% of the total BRAF alleles. The presence of BRAF^V600E mutation was significantly associated with extrathyroidal invasion. There was no significant difference between the presence of BRAF^V600E mutation and other clinicopathological features. It was not found that the significant relationship between percentage of BRAF^V600E alleles and clinicopathological characteristics. Conclusion: We concluded that the presence of BRAF^V600E could be preoperatively predictive of extrathyroidal invasion in a Chinese population.     

DHPLC is a highly sensitive and rapid screening method to detect BRAF mutation in papillary thyroid carcinoma

The BRAF^V600E mutation has been reported to occur in 30% to 80% of papillary thyroid carcinomas (PTCs). Although direct sequencing is the method most commonly used to identify mutations, this technique is not sensitive enough to accurately detect low level mutation. To determine the optimal diagnostic method for detecting the BRAF^V600E mutation in PTC, we compared the diagnostic efficacy of four representative detection methods in formalin-fixed paraffin-embedded thyroid tissues obtained from 40 patients diagnosed with PTC. To detect the BRAF^V600E mutation, we amplified exon 15 of the BRAF gene and performed mutational analysis with direct sequencing, denaturing high-performance liquid chromatography (DHPLC), pyrosequencing and colorimetric assay. The BRAF mutation was detected in 33 cases (82.5%) by DHPLC, 23 cases (57.5%) by direct sequencing, 22 cases (55.0%) by pyrosequencing, and 37 cases (92.5%) by colorimetric assay. The sensitivity, negative predictive value and accuracy of DHPLC were 100%. The specificity and positive predictive values for DHPLC, direct sequencing and pyrosequencing were 100%, and for colorimetric assay they were 14.3% and 83.8%, respectively. The kappa value for DHPLC was a perfect 1.0, which was superior to the other methods. In conclusion, DHPLC is a sensitive, specific and accurate method for detecting the BRAF^V600E mutation, especially low level mutation, in PTC.     

Detection of Plasma BRAFV600E Mutation Is Associated with Lung Metastasis in Papillary Thyroid Carcinomas

Purpose

The BRAF^V600E mutation represents a novel indicator of the progression and aggressiveness of papillary thyroid carcinoma (PTC). The purpose of this study was to determine the clinical significance of free circulating mutant BRAF^V600E in predicting the advanced disease of PTC.

Materials and Methods

Seventy seven matched tumor and plasma samples obtained from patients with both benign and PTC were analyzed for BRAF^V600E mutation using a peptide nucleic acid (PNA) clamp real-time polymerase chain reaction (PCR).

Results

The BRAF^V600E mutation was absent in tumor DNA samples obtained from patients with benign follicular adenomas or adenomatous goiter. In contrast, 49 of 72 (68.1%) PTC tumors were positive for the BRAF^V600E mutation. Among them, 3 (6.1%) patients with PTC were positive for BRAF^V600E mutation in plasma and tumor. However, all 3 patients (100%) had lateral lymph node and lung metastasis.

Conclusion

These findings suggest that the BRAF^V600E mutation can be detected using a PNA clamp real-time PCR in the blood of PTC patients with lung metastasis. Future studies are warranted to determine clinical significance of serum BRAF^V600E mutation in large prospective studies.     

BRAF‐Mutated Pleomorphic Xanthoastrocytoma is Associated with Temporal Location,Reticulin Fiber Deposition and CD34 Expression

BRAF V600E mutation and homozygous deletion of CDKN2A (p16) are frequent molecular alterations in pleomorphic xanthoastrocytomas (PXAs). We investigated 49 PXAs for clinical, histological and immunohistochemical characteristics related to BRAF mutation status. BRAF mutation was detected by immunohistochemical assay and DNA sequencing in 38/49 (78%) tumors. All but one PXA located in the temporal lobe harbored a BRAF V600E mutation (23/24; 96%) compared with 10/19 nontemporal PXAs (53%; P = 0.0009). Histological and immunohistochemical analysis demonstrated increased reticulin deposition (76% vs. 27%; P = 0.003) and a more frequent expression of CD34 in BRAF‐mutant PXAs (76% vs. 27%; P = 0.003). We further investigated the utility of combined BRAF V600E (VE1) and p16 analysis by immunohistochemistry to distinguish PXAs from relevant histological mimics like giant‐cell glioblastoma. Among PXAs, 38/49 (78%) were VE1‐positive, and 30/49 (61%) had a loss of p16 expression. The combined features (VE1 positivity/p16 loss) were observed in 25/49 PXAs (51%) but were not observed in giant‐cell glioblastoma (VE1 0/28, p16 loss 14/28). We demonstrate that temporal location, reticulin deposition and CD34 expression are associated with BRAF mutation in PXA. Combined VE1 positivity and p16 loss represents a frequent immunoprofile of PXA and may therefore constitute an additional diagnostic tool for its differential diagnosis.     

Can Ultrasound Be as a Surrogate Marker for Diagnosing a Papillary Thyroid Cancer? Comparison with BRAF Mutation Analysis

Purpose

We investigated the merit of ultrasound (US) features and BRAF^V600E mutation as an additional study of cytology and compared the diagnostic performances of cytology alone, cytology with US correlation, cytology with BRAF^V600E mutation, and a combination of cytology, US, and BRAF^V600E mutation all together.

Materials and Methods

This study included 185 patients (mean age, 48.4 years; range 20-77 years) with 191 thyroid nodules who underwent US-guided fine-needle aspiration (FNA) with an additional BRAF^V600E mutation test. Three radiologists highly experienced in thyroid imaging retrospectively reviewed US images and classified each nodule into two categories (positive for malignancy or negative for malignancy). Interobserver variability (IOV) of US assessment between the three readers was estimated using the generalized kappa statistic of Landis and Koch. We also calculated the diagnostic performances of these studies.

Results

There were 131 cases of malignancy (131/191, 68.6%) and 60 cases of benign nodules (60/191, 31.4%). In terms of IOV of US assessment, the generalized kappa value was 0.242, indicating fair agreement was reached. The combination of cytology with BRAF^V600E showed higher specificity (100%) and positive predictive value (PPV) (100%) compared to the combination of cytology, BRAF^V600E, and US (specificity 28.3%, 66.7%, 68.3%; PPV 74.6%, 86.6%, 86.8%, respectively; p<0.001). However, cytology with BRAF^V600E showed lower sensitivity (84.7%) than cytology with BRAF^V600E and US (96.2%, 98.5%, 95.4%, respectively; p<0.001).

Conclusion

Considering the diagnostic performance and low reproducibility of US, the combination of FNA with BRAF^V600E is the most reliable and objective method for diagnosing thyroid malignancy.     

BRAF V600E Mutation Is Associated with mTOR Signaling Activation in Glioneuronal Tumors

BRAF V600E mutations have been recently reported in glioneuronal tumors (GNTs). To evaluate the expression of the BRAF V600E mutated protein and its association with activation of the mammalian target of rapamycin (mTOR) pathway, immunophenotype and clinical characteristics in GNTs, we investigated a cohort of 174 GNTs. The presence of BRAF V600E mutations was detected by direct DNA sequencing and BRAF V600E immunohistochemical detection. Expression of BRAF‐mutated protein was detected in 38/93 (40.8%) gangliogliomas (GGs), 2/4 (50%) desmoplastic infantile gangliogliomas (DIGs) and 23/77 (29.8%) dysembryoplastic neuroepithelial tumors (DNTs) by immunohistochemistry. In both GGs and DNTs, the presence of BRAF V600E mutation was significantly associated with the expression of CD34, phosphorylated ribosomal S6 protein (pS6; marker of mTOR pathway activation) in dysplastic neurons and synaptophysin (P < 0.05). In GGs, the presence of lymphocytic cuffs was more frequent in BRAF‐mutated cases (31 vs. 15.8%; P = 0.001). The expression of both BRAF V600E and pS6 was associated with a worse postoperative seizure outcome in GNT (P < 0.001). Immunohistochemical detection of BRAF V600E‐mutated protein may be valuable in the diagnostic evaluation of these glioneuronal lesions and the observed association with mTOR activation may aid in the development of targeted treatment involving specific pathogenic pathways.     

BRAFV600E hot spot mutation in thyroid carcinomas: first Moroccan experience from a single-institution retrospective study

BackgroundThe incidence of thyroid cancer is increasing worldwide at an alarming rate. BRAF^V600E mutation is described to be associated with a worse prognostic of thyroid carcinomas, as well as extrathyroidal invasion and increased mortality.ObjectiveTo our knowledge, there are no reported studies neither from Morocco nor from other Maghreb countries regarding the prevalence of BRAF^V600E mutation in thyroid carcinomas. Here we aim to evaluate the frequency of BRAF^V600E oncogene in Moroccan thyroid carcinomas.MethodsIn this Single-Institution retrospective study realized in the Anatomic Pathology and Histology Service in the Military Hospital of Instruction Mohammed V ‘HMIMV’ in Rabat, we report, using direct genomic sequencing, the assessment of BRAF^V600E in 37 thyroid tumors.ResultsWe detected BRAF^V600E mutation exclusively in Papillary Thyroid Carcinomas ‘PTC’ with a prevalence of 28% (8 PTC out 29 PTC). Like international trends, Papillary Thyroid Carcinomas ‘PTC’ is more frequent than Follicular Thyroid Carcinomas ‘FTC’ and Anaplastic Thyroid Carcinomas ‘ATC’ (29 PTC, 7 FTC and 1 ATC).ConclusionOur finding gives to the international community the first estimated incidence of this oncogene in Morocco showing that this prevalence falls within the range of international trends (30% to 90%) reported in distinct worldwide geographic regions.     

Up-regulation of urinary-type plasminogen activator correlates with high-risk papillary thyroid carcinoma with BRAFV600E mutation and its possible molecular mechanism

The aim of the present study is to investigate the relationship between urinary-type plasminogen activator (uPA) expression and clinicopathological features in papillary thyroid carcinoma (PTC) and to determine the signal transduction of PTC cells in vitro.PTC tissues from 42 patients were analyzed for the expression of uPA and the BRAF^V600E mutation. BCPAP, a PTC cell line harboring the BRAF^V600E mutation, was used to study MAPK signaling. PCR and direct sequencing were applied to analyze BRAF^V600E mutation status. uPA mRNA expression was measured using a quantitative RT-PCR method, and uPA protein was localized using an immunohistochemical method. The ERK protein status was detected by Western blot analysis.uPA gene expression was significantly increased in PTC tissues as compared to the corresponding non-tumor tissues. Furthermore, the up-regulation of uPA mRNAs was correlated with high-risk clinicopathological features, including extrathyroid invasion, loss of cellular polarity/cohesiveness, and the BRAF^V600E mutation. Marked dephosphorylation of ERK1/2 and down-regulation of uPA expression were detected when BCPAP was treated with a MEK inhibitor, U0126.MEK inhibitors might be a potential treatment strategy for aggressive PTC with BRAF^V600E through inhibition of uPA expression.     

Comparison between two widely used laboratory methods in BRAF V600 mutation detection in a large cohort of clinical samples of cutaneous melanoma metastases to the lymph nodes

Aims: The study compares detection rates of oncogenic BRAF mutations in a homogenous group of 236 FFPE cutaneous melanoma lymph node metastases, collected in one cancer center. BRAF mutational status was verified by two independent in-house PCR/Sanger sequencing tests, and the Cobas® 4800 BRAF V600 Mutation Test. Results: The best of two sequencing approaches returned results for 230/236 samples. In 140 (60.9%), the mutation in codon 600 of BRAF was found. 91.4% of all mutated cases (128 samples) represented p.V600E. Both Sanger-based tests gave reproducible results although they differed significantly in the percentage of amplifiable samples: 230/236 to 109/143. Cobas generated results in all 236 cases, mutations changing codon V600 were detected in 144 of them (61.0%), including 5 not amplifiable and 5 negative in the standard sequencing. However, 6 cases positive in sequencing turned out to be negative in Cobas. Both tests provided us with the same BRAF V600 mutational status in 219 out of 230 cases with valid results (95.2%). Conclusions: The total BRAF V600 mutation detection rate didn’t differ significantly between the two methodological approaches (60.9% vs. 61.0%). Sequencing was a reproducible method of V600 mutation detection and more powerful to detect mutations other than p.V600E, while Cobas test proved to be less susceptible to the poor DNA quality or investigator’s bias. The study underlined an important role of pathologists in quality assurance of molecular diagnostics.