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.     

Expression of hypoxia-inducible factor 1 alpha in papillary thyroid carcinoma is associated with desmoplastic stromal reaction and lymph node metastasis

Papillary thyroid carcinoma (PTC) is the most common thyroid cancer, and it early metastasizes into regional lymph nodes. We evaluated immunohistochemically the expression of the hypoxia marker hypoxia-inducible factor 1α (HIF-1α) as well as extracellular matrix protein tenascin C as a marker of stroma remodelling in a cohort of 160 PTCs. Expression of HIF-1α was seen focally accentuated in 100 of the 160 tumours (62.5 %) including 16 cases with equivocal staining (faint staining or only single-cell staining) and 84 cases with unequivocal staining. HIF-1α expression correlated with the degree of desmoplastic stromal reaction as well as with the expression of tenascin C (p?<?0.001, Kruskal–Wallis test, respectively). Moreover, expression of HIF-1α was significantly associated with the presence of lymph node metastases (p?<?0.001, Mann–Whitney test) as well as signs of invasion, namely, peritumoural and extrathyroidal invasion as well as angioinvasion (p?=?0.024, p?<?0.001, p?=?0.017, Mann–Whitney test, respectively). Additionally, PTC with unequivocal HIF-1α nuclear staining was a larger tumour than PTC with negative (?) or equivocal HIF-1α expression (p?<?0.001, Kruskal–Wallis test). Interestingly, the expression of HIF-1α was not significantly associated with BRAF V600E status (p?>?0.05, Mann–Whitney test). Our data show that the expression of HIF-1α is associated with stroma remodelling processes within the tumour and, thus, may play an important role in an invasive behaviour of these tumours independent of BRAF mutation status.     

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.     

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.     

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.     

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)     

<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.     

Thyroid stimulating hormone levels and BRAFV600E mutation contribute to pathophysiology of papillary thyroid carcinoma: Relation to outcomes?

AimsThe aim of this study was to evaluate the relation between the level of thyroid stimulating hormone (TSH) and progression of papillary thyroid carcinoma (PTC) with or without BRAF^V600E mutation.MethodsThe medical records and laboratory data of 547 patients with PTC and 94 patients with follicular adenoma (FA) were collected. The relationship between hormones levels and such end-points as extrathyroid extension (ETE), lymphovascular invasion (LVI) and lymph node metastasis (LNM) was assessed. In addition, age, gender, BRAF^V600E mutation status, histological type and Hashimoto’s thyroiditis (HT) were considered.Key findingsMost of the patients with PTC had hormones levels within the normal range, however, serum TSH concentration was significantly higher in PTC comparing with FA (P = 0.022). High levels of TSH in PTC were more frequent among women rather than men (P = 0.03) due to the gender differences in coexisting HT rate (P = 0.003). In contrast, LNM rate was higher in men (P = 0.0014). Coexisting HT significantly decreased the risk of ETE (OR = 0.67; 95%CI 0.44–1.00; P = 0.05) and LNM (OR = 0.59; 95%CI 0.37?0.94; P = 0.028) among males with PTC. However, there was no significant relationship between HT and PTC-related ETE and LNM in females. BRAF^V600E mutation was associated with presence of lymphocytic infiltration (P < 0.001) but not with HT (P = 0.08) and violation of thyroid function.ConclusionThe present study showed the lack of significant relationship between TSH levels and PTC aggressiveness (LNM, TNM stage, BRAF^V600E mutation). Higher TSH levels were found in patients with coexisting HT that was associated with female sex and multifocality of PTC.     

<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.     

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.     

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.     

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.     

Investigation of BRAF mutation in a series of papillary thyroid carcinoma and matched-lymph node metastasis with ARMS PCR

Objectives

To figure out that if there is a consistency relationship of the BRAF^V600E mutation in matched-lymph node metastasis and original papillary thyroid carcinoma (PTC) specimen for the same patient.

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.     

Synchronous Occurrence of Medullary and Papillary Carcinoma of the Thyroid in a Patient with Cutaneous Melanoma: Determination of BRAFV600E in Peripheral Blood and Tissues. Report of a Case and Review of the Literature

The purpose of this study is to describe a case of concurrent medullary and papillary thyroid carcinoma (MTC and PTC) and cutaneous melanoma and to analyze BRAF^V600E mutation in plasma and tissues. We report the clinical history and the laboratory, imaging, and histopathological findings of a 47-year-old man affected by multinodular goiter. BRAF^V600E-mutated DNA was quantified in plasma samples and in cancer sections by quantitative real-time polymerase chain reaction (qPCR). At ultrasound examination, the dominant right nodule of the thyroid was weakly hyperechoic and hypervascularized, while the left one was hypoechoic without internal vascularization. Regional lymphadenomegalia was not detected. Basal plasma calcitonin was elevated, and the patient underwent total thyroidectomy and resection of central cervical lymph nodes. Histopathological examination identified two distinct foci of MTC and PTC and micrometastasis of well-differentiated carcinoma in one of the six resected lymph nodes. RET proto-oncogene germline mutations were not detected. Cutaneous melanoma of the thorax was subsequently diagnosed. BRAF^V600E tissue DNA was detected in PTC and melanoma but not in MTC. The cell-free plasma percentage of BRAF^V600E DNA was detected in pre-thyroidectomy peripheral blood and was drastically reduced after cancer treatments. This study confirms the occurrence of synchronous MTC and PTC and is the first evidence of the co-existence of melanoma and distinct thyroid cancers of different origin. BRAF^V600E allele was detected in PTC and melanoma but not in MTC tissues. BRAF^V600E molecular quantification in pre- and post-treatment blood supports our previous data, suggesting its possible role in diagnosis and follow-up of BRAF-positive tumors.     

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.     

Expression of cell surface transmembrane carbonic anhydrase genes CA9 and CA12 in the human eye: overexpression of CA12 (CAXII) in glaucoma

Purpose: Carbonic anhydrase enzymes (CAs) are universally involved in many fundamental physiological processes, including acid base regulation and fluid formation and movement. In glaucoma patients, CA inhibitors are very effective in lowering intraocular pressure by reducing the rate of aqueous humour secretion mediated by the CAs in the ciliary epithelium. In this work, we investigated the expression and tissue distribution of two recently discovered CA genes CA9 (CAIX) and CA12 (CAXII) in fetal, neonatal, and adult human eyes with and without glaucoma.

Methods: CAIX and CAXII expression in 16 normal and 10 glaucomatous eyes, and in cultured non-pigmented ciliary epithelial cells (NPE) from normal and glaucoma eye donors was assessed by immunostaining. In addition, northern blot hybridisation was performed to assess expression of CA4, CA9, and CA12 mRNA in cultured NPE cells from normal and glaucoma donors.

Results: CAXII was localised primarily to the NPE with its expression prominent during embryonic eye development but which decreased significantly in adults. CAIX expression in the NPE was very low. The epithelium of cornea and lens occasionally expressed both enzymes at low levels during development and in adult eye, and no expression was detected in the retina. The NPE from glaucoma eyes expressed higher levels of CAXII, but not CAIX, in comparison with normal eyes. This expression pattern was retained in cultured NPE cell lines. NPE cells from a glaucoma patient showed a five-fold increase in the CA12 mRNA level with no detectable expression of CA9 mRNA. Also, no expression of the CA4 gene encoding a GPI anchored plasma membrane protein was detected on these northern blots.

Conclusions: Transmembrane CAIX and CAXII enzymes are expressed in the ciliary cells and, thus, may be involved in aqueous humour production. CA12 may be a targeted gene in glaucoma.

     

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.