Negative reactions of BRAF mutation‐specific immunohistochemistry to non‐V600E mutations of BRAF

BRAF mutations are rare driver mutations in non‐small cell lung cancer (NSCLC), accounting for 1%–2% of the driver mutations, and the mutation spectrum has a wide range in contrast to other tumors. While V600E is a dominant mutation in melanoma, more than half of the mutations in NSCLCs are non‐V600E. However, treatment with dabrafenib plus trametinib targets the BRAF V600E mutation exclusively. Therefore, distinguishing between V600E and non‐V600E mutations is crucial for biomarker testing in NSCLC in order to determine treatment of choice. Immunohistochemistry (IHC) using the BRAF V600E mutation‐specific antibody is clinically used in melanoma patients, but little is known about its application in NSCLC, particularly with regard to the assay performance for non‐V600E mutations. In the present study, we examined 117 tumors with BRAF mutations, including 30 with non‐V600E mutations, using BRAF mutation‐specific IHC. None of the tumors with non‐V600E mutations, including two compound mutations, showed a positive reaction. Furthermore, all V600E mutations were positive except for one case with combined BRAF V600E and K601_W604 deletion. Our findings confirmed that the BRAF V600E mutation‐specific IHC is specific without any cross‐reactions to non‐V600E mutations, suggesting that this assay can be a useful screening tool in clinical practice.     

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.     

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.     

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)     

Pediatric Brainstem Gangliogliomas Show BRAFV600E Mutation in a High Percentage of Cases

Brainstem gangliogliomas (GGs), often cannot be resected, have a much poorer prognosis than those located in more common supratentorial sites and may benefit from novel therapeutic approaches. Therapeutically targetable BRAF c.1799T>A (p.V600E) (BRAF^V600E) mutations are harbored in roughly 50% of collective GGs taken from all anatomical sites. Large numbers of pediatric brainstem GGs, however, have not been specifically assessed and anatomic—and age‐restricted assessment of genetic and biological factors are becoming increasingly important. Pediatric brainstem GGs (n = 13), non‐brainstem GGs (n = 11) and brainstem pilocytic astrocytomas (PAs) (n = 8) were screened by standard Sanger DNA sequencing of BRAF exon 15. Five of 13 (38%) pediatric GG harbored a definitive BRAF^V600E mutation, with two others exhibiting an equivocal result by this method. BRAF^V600E was also seen in five of 11 (45%) non‐brainstem GGs and one of eight (13%) brainstem PAs. VE1 immunostaining for BRAF^V600E showed concordance with sequencing in nine of nine brainstem GGs including the two cases equivocal by Sanger. The equivocal brainstem GGs were subsequently shown to harbor BRAF^V600E using a novel, more sensitive, RNA‐sequencing approach, yielding a final BRAF^V600E mutation frequency of 54% (seven of 13) in brainstem GGs. BRAF^V600E‐targeted therapeutics should be a consideration for the high percentage of pediatric brainstem GGs refractory to conventional therapies.     

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.     

<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.     

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.     

<Emphasis Type="Italic">BRAF</Emphasis> mutations typical of papillary thyroid carcinoma are more frequently detected in undifferentiated than in insular and insular-like poorly differentiated carcinomas

Somatic mutations of the BRAF gene (BRAF^V599E and BRAF^K600E) were found to be closely associated with different histotypes of papillary thyroid carcinoma (PTC). The V599E mutation is highly prevalent in PTC with a papillary or mixed papillary follicular growth pattern, and the K600E mutation is apparently restricted to the follicular variant of PTC. It is usually accepted that thyroid malignancies may follow a progression path from well-differentiated to poorly differentiated (PDC) and undifferentiated (UC) carcinomas. One would expect that at least some of the less differentiated carcinomas would harbour the genetic alterations of pre-existing well-differentiated tumours. In order to find the prevalence of BRAF mutations in PDC and UC, we screened a series of 19 PDCs and 17 UCs, as well as 3 UC-derived cell lines, for both mutation types. The group of PDCs was restricted to the so-called insular and insular-like PDCs, thus excluding PTCs with solid, insular or trabecular foci of growth and PDCs displaying typical PTC nuclei. No BRAF mutations were detected in any of the 19 cases of PDC, whereas 6 of the UCs (35%) and one UC-derived cell line presented the BRAF^V599E mutation. The BRAF^K600E mutation was not detected in any case. We conclude that UC may progress from BRAF^V599E-mutated PTC. The absence of BRAF mutations in our series of PDC supports the assumption that pure insular and insular-like PDCs are more closely related to follicular carcinoma than to PTC.Paula Soares and Vítor Trovisco contributed equally to this work.     

The correlation of sodium iodide symporter and BRAFV600E mutation in classical variant papillary thyroid carcinoma

BRAF^V600E mutation was analyzed by real-time polymerase chain reaction in 96 consecutive cases with classical variant papillary thyroid cancer, and immunohistochemical staining of Na +/I − symporter (NIS) protein was evaluated. Localization (intracellular or membranous), density, and the intensity of cytoplasmic staining were characterized semiquantitatively. Extrathyroidal invasion, surgical margin positivity, and lymph node metastasis were compared with BRAF^V600E mutation and NIS expression. Eighty-eight patients who had at least 24-month follow-up were also included in survival analysis. BRAF^V600E mutation was determined in 78.1% (75/96) and functional NIS activity in 74% (71/96) of the cases. There were statistically significant differences in mean ages between BRAF^V600E mutation–positive (48.6) and BRAF^V600E mutation–negative cases (37.3; Levene test, P = .419; Student t test, P = .001). The surgical margin positivity (46.7%) and extrathyroidal extension percentage (54.7%) in the BRAF^V600E mutation–positive group were higher than the negative (28.6% and 33.3%, respectively) group, without statistical significance (P = .138 and P = .084, respectively). Functional NIS activity was higher in BRAF^V600E mutation–positive cases (78.1%) than mutation-negative ones (57.1%; P = .047). The possibility of moderate and intense cytoplasmic staining in BRAF^V600E mutation–positive cases (72%) was 6.3 times higher than the possibility of weak staining (28%) in the mutation-positive cases (95% confidence interval, 2.2-18.8; P = .001). Functional NIS expression is higher in patients with classical variant papillary thyroid cancer with BRAF^V600E mutation. However, the clinical features were not found to be associated with NIS expression. There may be different mechanisms determining the outcome of therapy.     

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.     

BRAF mutation in sporadic colorectal cancer and Lynch syndrome

The aim of the study was to detect mutations of BRAF oncogene in colorectal cancer and to use this information to identify Lynch syndrome patients. Consecutive cases of primary colorectal cancer (n?=?137) were analyzed for MLH1 protein expression using immunohistochemistry (IHC). BRAF V600E mutation was detected by IHC using a specific monoclonal antibody (VE1) and by qPCR. All MLH1 protein-negative cases were subjected to microsatellite instability analysis and MLH1 promoter methylation assay. MLH1 protein expression deficiency and high microsatellite instability (MSI-H) were detected in 18 of the 137 (13.1 %) consecutive colorectal cancer specimens. Detection of the BRAF V600E mutation by IHC was 100 % sensitive and specific as compared to qPCR, and this mutation was frequently present in the MSI-H group (77.8 %; 14/18) and less frequently in the microsatellite-stable group (7.6 %; 9/118). All BRAF V600E mutated cases of the MSI-H group presented with a MLH1 promoter methylation (14/14) as detected by methylation-specific multiplex ligation-dependent probe amplification. When BRAF was wild type in the MSI-H group, only one MLH1 promoter methylation was detected (1/4), and of the remaining three cases without MLH1 methylation, two were identified to harbor an MLH1 mutation consistent with Lynch syndrome. Finally, 11 previously confirmed Lynch syndrome cases were analyzed for BRAF V600E mutation, and all of them were wild type. In conclusion, detection of BRAF V600E in colorectal cancer specimens by IHC is sensitive and specific and may help to identify Lynch syndrome patients.     

Morphology predicts BRAF V600E mutation in papillary thyroid carcinoma: an interobserver reproducibility study

Papillary thyroid carcinomas (PTC) with BRAF ^V600E mutation are morphologically distinctive. They are typically classic or tall cell variants, show infiltrative borders, and are associated with desmoplasia/fibrosis, psammoma bodies, and well-developed nuclear features of papillary carcinoma. We hypothesize that morphologic features of PTC can help in the prediction of BRAF ^V600E mutation, and we evaluate the accuracy and the interobserver reproducibility of such prediction. Hematoxylin and eosin-stained sections from 50 PTCs comprising of 26 mutation-positive and 24 mutation-negative tumors were examined. BRAF ^V600E mutation was predicted correctly in 42/50 tumors (accuracy, 84 %) with 96 % sensitivity, 71 % specificity, and 78 % positive and 94 % negative predictive values (NPV). Subtle nuclear features of PTC (n?=?10) had the highest (100 %) negative predictive value followed by well-circumscribed non-infiltrative tumor borders (17/22 mutation-negative tumors, 95 % NPV). The positive predictive value of infiltrative tumor borders (21/28 [75 %] mutation-positive), desmoplasia/fibrosis (23/31 [74 %] mutation-positive), and psammoma bodies (13/20 [65 %] mutation-positive) increased to 100 % when all three features were present (n?=?8/8 mutation-positive). To assess interobserver reproducibility, two pathologists blinded to the mutational status evaluated 30 PTCs (15 mutation-positive and 15 mutation-negative) after self-training on 10 PTCs with known BRAF ^V600E mutational status (five mutation-positive and five mutation-negative). The prediction of the mutation was achieved with substantial agreement (κ value, 0.79) and accuracy (25/30, 83 %). This study demonstrates that BRAF ^V600E mutation in papillary thyroid carcinoma can be predicted on morphology with accuracy and with substantial interobserver agreement.     

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.     

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.     

BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma

The serine/threonine‐protein kinase B‐raf (BRAF) is an oncogene mutated in various neoplasms, including 5–15% of colorectal carcinomas. The T1799A point mutation, responsible for a large majority of these alterations, results in an amino acid substitution (V600E) causing the constitutive activation of a protein kinase cascade. BRAF V600E in MLH1 deficient tumors implicates somatic tumor‐only methylation of the MLH1 promoter region instead of a germline MLH1 mutation. BRAF V600E also predicts poor prognosis in microsatellite stable colorectal cancers and may be a marker of resistance to anti‐EGFR therapy in metastatic disease. Currently, only molecular methods are available for assessing BRAF mutational status. An immunohistochemical approach is evaluated here. Colon cancers from 2008 to 2012 tested by pyrosequencing for BRAF V600E mutation were selected. A total of 31 tumors with (n = 14) and without (n = 17) the BRAF V600E mutation were analyzed by immunohistochemistry using a commercially available antibody specific to the V600E‐mutated protein. All 14 colorectal carcinomas with the BRAF V600E mutation demonstrated cytoplasmic positivity in tumor cells with the anti‐BRAF antibody. In a minority of cases, staining intensity for the mutated tumor samples was weak (n = 2) or heterogeneous (n = 4); however, the majority of cases showed diffuse, strong cytoplasmic positivity (8 of 14 cases). None of the 17 BRAF wild‐type colorectal cancers showed immunoreactivity to the antibody. The overall sensitivity and specificity of the immunohistochemical BRAF V600E assay was 100%. Detection of the BRAF V600E mutation in colorectal cancer by immunohistochemistry is a viable alternative to molecular methods. © 2013 Wiley Periodicals, Inc.     

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.     

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.     

Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is rare,benign lesion using modified stringent diagnostic criteria: Reclassification and outcome study

BackgroundRigid diagnostic criteria for NIFTP have been recently proposed. The frequency of NIFTP using the new criteria is unknown, and whether abortive papillae are associated with BRAF^V600E mutation has not been studied. The aim of this study is to identify NIFTP by a retrospective review of Follicular Variant of Papillary Thyroid Carcinoma (FVPTC), and to study its incidence as well as the association between immunohistochemical BRAF^V600E expression and abortive papillae in NIFTP.DesignThyroid tumors diagnosed as FVPTC or NIFTP over a period of 18 years (2000–2017) were identified using the laboratory information system. The final pathology reports were reviewed and potential NIFTP were retrieved. The archived slides for these cases were independently reviewed by 2 pathologists. BRAF^V600E (clone: VE1) immunostain was performed on representative tumor blocks. Clinical information including follow-up data was obtained from the electronic medical records.ResultsAmong the 1918 cases with the diagnosis of papillary thyroid carcinoma (PTC), 589 (30.7%) of FVPTC and 136 cases of potential NIFTP were identified. After the review of the archived pathology slides, 29 lesions were morphologically reclassified as NIFTP. Four (13.7%) of these were positive for BRAF^V600E; no association was found between the presence of abortive papillae and BRAF^V600Eexpression (p=0.3). Exclusion of the 4 cases with BRAF^V600Eexpression resulted in 25 lesions of final NIFTP, representing 4.2% of the FVPTC and 1.3% of the PTC. The mean age of the NIFTP patients was 50 years, 87.5% were females. The mean size of the lesions was 1.4 cm (0.1–4.0 cm). Intranuclear pseudoinclusions were not identified, and abortive papillae were identified in 60% of NIFTP. The average follow-up was 70 (28–166) months. There were no adverse events (recurrence or metastasis) in the NIFTP group.ConclusionWhen strictly defined, NIFTP comprises 1.3% of cases perviously classified as PTC. In morphological NIFTP, no correlation is found between the presence of abortive papillae and the BRAF^V600E expression. Intranuclear pseudo-inclusions are not observed in NIFTP. Modification of current morphological criteria to include BRAF^V600E immunohistochemistry test may stratify NIFTP with benign outcome.