BRAFV600E Expression Research Articles

The Correlation of BRAF V600E Expressions with Histopathological Variant and Lymphocyte Infiltration in Papillary Thyroid Carcinoma

BackgroundPapillary thyroid carcinoma is the most common endocrine gland malignancy that has the highest incidence rate of 60-80%. In papillarythyroid carcinoma BRAF V600E mutation plays an important role in carcinogenesis. BRAF V600E is a RAS-regulated-serine-threoninekinase and activator of the MAPK pathway cascade that functions to regulate cell proliferation, differentiation and survival. BRAFV600E mutations in PTC is associated with a high incidence of recurrence and reduced survival. The purpose of this study is to lookat the relationship of BRAF V600E expression with histopathological variants and lymphocyte infiltration in PTC.MethodsThis research was a retrospective observational cross sectional study with 40 cases of papillary thyroid carcinoma which werediagnosed in 4 Pathology Anatomy Laboratory in West Sumatera. Samples were obtained from paraffin blocks of tissue from surgeryand were reevaluated for histopathological variant and lymphocyte infiltration. Expression of BRAF V600E in tumor cell were analyzedusing immunohistochemistry staining. Bivariate statistical analysis of Chi-Square were used and p value <0,05 were consideredsignificance.ResultsPositive BRAF V600E expression was found in 24 case (60%). Positive BRAF V600E expression was mostly found in classic PTC(50%). Papillary thyroid carcinoma with lymphocyte infiltration mostly found with negative BRAF V600E expression (79.2%). Statisticalanalysis showed no significant correlation between BRAF V600E with histopathological variant (p=0.74) and no significancecorrelation between BRAF V600E expression with lymphocyte infiltration (p=0.12).ConclusionThe conclusion is BRAF V600E expression has no correlation with histological variants in papillary thyroid carcinoma and no significantcorrelation of BRAF V600E expression with lymphocyte infiltration.

Lactate Dehydrogenase A as a Potential New Biomarker for Thyroid Cancer.

BackgroundSeveral cancers show increased levels of lactate dehydrogenase A (LDHA), which are associated with cancer progression. However, it remains unclear whether LDHA levels are associated with papillary thyroid cancer (PTC) aggressiveness or with the presence of the PTC prognostic marker, the BRAFV600E mutation. This study aimed to evaluate the potential of LDHA as a PTC prognostic marker.MethodsLDHA expression was examined in 83 PTC tissue specimens by immunohistochemistry. Human thyroid cell lines were genetically manipulated to overexpress BRAFV600E or were treated with a BRAF-specific short hairpin RNA (shBRAF), whose effects on LDHA expression were evaluated by Western blotting. Data from 465 PTC patients were obtained from The Cancer Genome Atlas (TCGA) database and analyzed to validate the in vitro results.ResultsLDHA was aberrantly overexpressed in PTC. Intense immunostaining for LDHA was observed in PTC specimens carrying mutated BRAF, whereas the intensity was less in wild-type BRAF samples. Overexpression of BRAFV600E resulted in LDHA upregulation, whereas treatment with shBRAF downregulated LDHA in human thyroid cell lines. Furthermore, LDHA mRNA expression was significantly elevated and associated with BRAFV600E expression in thyroid cancer tissues from TCGA database. Additionally, LDHA overexpression was found to be correlated with aggressive clinical features of PTC, such as lymph node metastases and advanced tumor stages.ConclusionLDHA overexpression is associated with the BRAFV600E mutation and an aggressive PTC behavior. Therefore, LDHA may serve as a biomarker and therapeutic target in PTC.

Expression of B-type RAF V600E in Thyroid Carcinoma and its Association with Histological Type

Introduction: Thyroid cancer is one of the most common cancers amongst all endocrine cancers. Incidence of thyroid malignancy is about 3-4% of all malignancy in India and 80% of thyroid malignancy belongs to Papillary Thyroid Carcinoma (PTC). Factors affecting the prognosis of PTC include patient’s gender, age, histological findings, tumour size, lymph node metastasis, extrathyroidal extension, and remote metastasis. Presence of B-type RAF V600E (BRAF V600E) mutation in thyroid carcinoma patients tends to present with more aggressive clinicopathological behaviours of PTC, prompting more aggressive radioiodine treatment. Aim: To find out the frequency of occurrence and expression of BRAF V600E mutation by Immunohistochemistry (IHC) in thyroid cancer and its association with histological type and Tumour Nodes Metastases (TNM) Staging. Materials and Methods: The present observational retrospective study included patients treated for thyroid carcinoma between January 2014 to February 2019 at RL Jalappa hospital and Research centre, Kolar, Karnataka, India. The IHC was done with rabbit monoclonal anti-BRAF V600E antibody IgG Clone RM8 (VE1). Clinical records, Fine Needle Aspiration Cytology (FNAC) diagnosis were analysed for 45 thyroid carcinoma cases. Immunopositivity was scored positive when unambiguous clear cytoplasmic staining for the antibody was observed in tumour cells. Categorical data was presented in the form of frequencies and proportions and continuous data was presented as mean and standard deviation. The t-test were applied to find out the difference in means among the groups. The p-value <0.05 was considered statistically significant. Results: Out of total 45 cases, 18 were classical PTC, 18 were Follicular Variant of Papillary Thyroid Cancer (FV-PTC), 4 were micropapillary carcinomas, 3 were Follicular carcinomas, 1 was Oncocytic variant of PTC and 1 undifferentiated thyroid carcinoma. Out of total 45 cases, 33 cases (73%) were found to be BRAF V600E positive and 12 (26%) were negative for BRAF V600E. Out of the total 33 BRAF V600E positive cases, 19 cases showed strong staining, 14 cases showed moderately positive staining. Conclusion: The PTC is the most frequent type of thyroid carcinoma amongst all the sub-types. BRAF V600E expression is commonly seen in higher tumour size (T3, T4) and classical PTC. Tumours with extrathyroidal extension and capsular invasion showed strong positivity.

Immunohistochemical Examination is Highly Sensitive and Specific for Detection of the V600E BRAF Mutation in Colorectal Serrated Lesions.

Mutations in BRAF are important events in colorectal serrated lesions and specific genetic markers for the serrated pathway. However, examination of BRAF mutations is not easy in routine histopathologic analyses. Here, the authors examined 73 colorectal serrated lesions, including 21 hyperplastic polyps, 32 traditional serrated adenomas, and 30 sessile serrated lesions, for comparison of BRAF mutation status with immunopositive expression of the anti-BRAF V600E mutation-specific antibody VE1. Thirty-two tubular adenomas (TAs) were examined as controls. In addition, 5 examples of sessile serrated lesion with dysplasia were included. Mutations in BRAF (exon 15; V600E) and KRAS (exon 2) were analyzed in serrated lesions and TAs using pyrosequencing. Finally, the authors compared BRAF mutations with immunohistochemical expression of VE1 antibodies against the BRAF V600E mutation, which was examined based on quantitative analyses and correlations between semiquantitative (0, 1+, or 2+) and quantitative results in colorectal serrated lesions. The cut-off value of VE1 expression (32%) was set based on receiver operating characteristic curve analysis. In the current study, mutations in BRAF were well correlated with VE1 expression in serrated lesions, although no TAs without BRAF mutations were immunopositive. In contrast, serrated lesions and TAs with mutations in KRAS were not stained for VE1 expression. In serrated lesions, although the sensitivity was 96.2% to 100%, the specificity was 90.0% to 100%. In addition, there was also good correlation between semiquantitative and quantitative results. Analysis of BRAF V600E expression may be pathologically useful, particularly in routine histopathologic diagnosis.

PATH-10. PROGNOSTIC RELEVANT IMMUNOPHENOTYPES OF PEDIATRIC HIGH-GRADE NON-BRAINSTEM GLIOMAS

Pediatric diffuse astrocytomas comprise a wide range of malignancies with variable prognosis. The 4th grading system used now not always correctly characterizes the biological behavior of these tumors. We collected 24 pediatric supratentorial non-brainstem high grade glioma cases. Patient age ranged from 1 to 18 years old (median 11y). Main tumor locations were as follows: parietal lobe 8 cases; temporal lobe, 10 cases; frontal lobe, 3 cases; occipital lobe 3 cases. Eight of them were totally removed. All patients were treated with standard CT and RT. The main objective was to assess the prognostic impact of histopathological and molecular criteria on progression‐free(PFS) and overall survival (OS) of high grade gliomas. The following criteria were analyzed: IDH1 R132H, BRAF V600E expression, ALT-phenotype, CDKN2A deletion, 1p/19q co-deletion, glial and neuronal markers expression. RESULTS: IDHR132H mutation was identified in 3 cases. 4 cases carried BRAFV600E mutation with CDKN2A deletion and displayed PXA phenotype. 5 cases showed undifferentiated glial morphology and ALT–phenotype. Also there was a group of tumors without any of the above mentioned genetic changes. Interestingly 3 of them were post radiation tumors. Statistical analysis showed that low OS correlated with ALT-phenotype(p-0.015), absence of neuronal markers expression and absence of molecular changes (p-0.03). Mutation of IDH1R132H was a favorable prognostic factor as in the adult population. PFS was affected only by the presence of neuronal expression (p-0.015). Employing immunohistochemical analysis with surrogate molecular markers in complex with FISH can provide additional prognostic information in case of pediatric high grade gliomas.

BRAF V600E mutation-specific immunohistochemical analysis in ameloblastomas: a 44-patient cohort study from a single institution.

The aim of this study is to investigate the presence and prevalence of BRAF V600E mutation in ameloblastomas using anti-BRAF V600E monoclonal antibody (VE1 clone) and to identify any clinicopathologic correlation with BRAF V600E mutation in ameloblastoma. The pathology files of the Department of Oral Pathology and Oral Medicine, Faculty of Dentistry, Lagos State University College of Medicine, Lagos, Nigeria, were searched for the diagnosis of ameloblastoma from 2016 to 2020. Archived non-decalcified formalin-fixed paraffin-embedded tissue underwent immunohistochemistry using anti-BRAF V600E antibody at the University of Pittsburgh, Pennsylvania. Clinicopathologic data such as age at diagnosis, gender, jaw bone involved (mandible or maxilla), tumor location (anterior or posterior) and histologic subtype were collected. The clinicopathologic parameters were analyzed using Chi-square test and Fisher's exact test according to the BRAF status. Forty-four cases of ameloblastoma were retrieved. The male to female ratio was 1.32:1. The average age of patients at diagnosis was 33.3years. Thirty-nine cases were located in the mandible and 5 cases in the maxilla. Only cases in the mandible were positive for anti-BRAF V600E antibody (n = 15/39; 38.5%). There was a significant correlation between BRAF V600E expression in mandibular tumors and histologic subtype (p = 0.02); however, no significance was observed for gender, age and tumor location. BRAF V600E mutation preferentially occurs in mandibular ameloblastomas, especially in non-plexiform ameloblastomas. These patients may benefit therapeutically from the use of BRAF inhibitors.

TMOD-23. A NOVEL BRAF V600E LOW-GRADE GLIOMA MOUSE MODEL HIGHLIGHTS EXOMIC AND TUMOR IMMUNE ALTERATIONS AND DIFFERING THERAPEUTIC RESPONSES IN LOW- AND HIGH-GRADE GLIOMAS

Abstract Pediatric low-grade glioma (pLGG), the most common brain cancer in children, is difficult to treat especially at recurrence. The BRAF V600E mutation is the second most common mutation in pLGG, and in a high-risk group for progression is associated with deletion of the tumor suppressor CDKN2A. A better understanding of the factors contributing to progression, in particular the role of the immune infiltrate is needed, but studies have been hindered by the lack of low-grade glioma mouse models. We utilized transgenic mice with a cre-activatable (CA) allele of BRAF V600E to generate endogenous models for low-grade gliomas. We found that BRAF V600E expression cooperates with hemizygous CDKN2A deletion to induce low-grade gliomas, with tumors forming at a greater latency than by homozygous deletion. Cell line derivatives from low-grade lesions continue to grow slower upon orthotopic injection than those we previously derived from high-grade tumors. Murine LGG can progress to higher grade tumors within the mouse lifespan and we observe exomic changes and alterations in the tumor immune infiltrate associated with progression, the details of which will be discussed at the meeting. High-grade cells’ phenotypic changes within in vivo passage are accompanied by exomic changes. The high-grade glioma immune infiltrate is altered by dual MAPK pathway inhibition with dabrafenib and trametinib. Adding dual immune checkpoint inhibition by anti-PD-L1 and anti-CTLA-4 antibodies significantly extends survival of dabrafenib-trametinib dual treatment. Human BRAF V600E mutant tumors reportedly have a higher tumor immune infiltrate than that of BRAF wildtype gliomas, consistent with our murine RESULTS: Here we present a novel model for BRAF V600E mutant gliomas in mice that has a frequent rate of progression, similar to human BRAF V600E mutant gliomas, and an active immune infiltrate in high grade tumors which makes them susceptible to the immunostimulatory effects of dual checkpoint inhibition.

Abstract PR12: Regulation of the tumor-suppressive miR-29 family by oncogenic MAPK signaling in melanoma

Abstract MicroRNAs are a group of noncoding RNAs that can regulate gene expression by targeting the mRNA of a gene. Many microRNAs respond to different stimuli such as growth signals or cellular stress. The tumor-suppressive miR-29 family (miR-29a, b and c), for example, is induced during aging and senescence as a p53-dependent response to DNA damage. Given the tumor-suppressive potential of miR-29, we sought to examine if its expression is induced by oncogenic stress in a p53-dependent manner. Ectopic overexpression of oncogenic Braf-V600E and Kras-G12D in mouse embryonic fibroblasts (MEFs) using retroviral transduction resulted in increased expression of pri-miR-29a-b1 and, to a lesser extent, pri-miR-29b2~c. While ectopic Kras-G12D overexpression stimulated p53 activity, Braf-V600E failed to elicit a p53 response, suggesting that this oncogene provokes miR-29 expression in a p53-independent manner. Moreover, expression of Braf-V600E at physiologic levels induced miR-29 in the absence of a p53 response. Accordingly, expression of endogenous Braf-V600E stimulated miR-29 in MEFs even when p53 was silenced by RNAi. When Braf-V600E MEFs were treated with a MEK inhibitor, miR-29 levels decreased in both the presence and absence of p53, suggesting that the MAPK pathway downstream of Braf-V600E directly regulates miR-29 transcription. Importantly, TPA-mediated stimulation of the MAPK pathway increased miR-29 levels in human melanocytes. Similarly, expression of Braf-V600E induced miR-29 in human melanocytes and MEK inhibition rescued this effect. These results strongly support the notion that miR-29 transcription is induced by MAPK signaling and suggest that oncogenic BRAF provokes a tumor-suppressive miR-29 checkpoint that needs to be inactivated for further tumor progression. Indeed, human melanoma cells lines express lower levels of miR-29 compared to human melanocytes, and inactivation of miR-29 by a lentiviral sponge in Braf-V600E mutant melanocytes and melanoma cells promotes cell transformation. We are now utilizing our ESC-GEMM platform to generate Braf-V600E; Pten-deficient mice expressing the miR-29 sponge and will assess if counteracting the induction of miR-29 by Braf-V600E promotes melanoma formation in vivo. Our results suggest a biphasic role of miR-29 during melanomagenesis where Braf-V600E-induced miR-29 upregulation prevents the progression of early lesions until miR-29 is downregulated, likely by inactivation of the ARF-p53 axis. This abstract is also being presented as Poster B24. Citation Format: Olga Vera, Ilah Bok, Neel Jasani, Oluwashanu Balogun, Koji Nakamura, Florian Karreth. Regulation of the tumor-suppressive miR-29 family by oncogenic MAPK signaling in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR12.

HOME / ARCHIVES / VOL. 10 NO. 4 (2020): VOLUME 10 ISSUE 4 / Original Articles Expression of BRAF V600E in Tissue Samples of Colorectal Carcinoma and Its Correlation with Various Clinico-Pathological Parameters

Objective: To determine the expression of BRAF V600E in tissue samples of colorectal carcinoma and to correlate it with various clinico-pathological parameters. Study design and setting: Cross-sectional study was conducted at department of Pathology, Pakistan Navy Station Shifa hospital Karachi from 1st March 2016 to 28th February 2019 Methodology: Total of 51 cases of colorectal cancer were analyzed for immunohistochemical staining using BRAF antibodies on representative tissue blocks. Clinical and pathological records were retrieved for data collection. The results of immunohistochemical analysis were correlated with the recorded clinico-pathological parameters. Results: In this study 51 cases of colorectal cancer were analyzed for immune expression of BRAF V600E. The age of the patients ranged from 14 to 85 years with the mean age of 60.96 years. Among the 51 cases, 37(72.5%) cases were males and 14(27.4%) were females. 37(72.5%) were localized to left side colon and 14(27.4%) were found in the right colon. For BRAF V600E, positive expression was seen in 20(39.2%) cases, whereas 31(60.7%) cases showed negative expression of BRAFV600E. No significant association was seen between BRAF V600E expression and histological variants like age, gender, tumor location and glandular carcinomas. Conclusion: BRAF V600E immunosuppression was seen in 39.2% of colorectal carcinoma in this study. No significant association was seen in BRAF V600E expression and histological variants

CORRIGENDUM.

CorrigendumCORRIGENDUMPublished Online:14 Sep 2020https://doi.org/10.1152/z9k-5591-corr.2020Original articleMoreSectionsPDF (17 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmail Goz RU, Akgül G, LoTurco JJ. BRAFV600E expression in neural progenitors results in a hyperexcitable phenotype in neocortical pyramidal neurons. J Neurophysiol 123: 2449–2464, 2020. First published May 13, 2020; doi:10.1152/jn.00523.2019.In the third paragraph of the Introduction to this article, the third sentence should begin “In a related finding, Yeh et al. (2018) in a CFC patient with another BRAF activating mutation (Q257R) in induced pluripotent stem cells (iPSCs)-derived neuronal cultures….”This article has no references to display. Previous Back to Top FiguresReferencesRelatedInformationRelated articlesBRAFV600E expression in neural progenitors results in a hyperexcitable phenotype in neocortical pyramidal neurons 18 Jun 2020Journal of Neurophysiology More from this issue > Volume 124Issue 3September 2020Pages 1005-1005 Copyright & PermissionsCopyright © 2020 the American Physiological Societyhttps://doi.org/10.1152/z9k-5591-corr.2020PubMed32924791History Received 31 August 2020 Published online 14 September 2020 Published in print 1 September 2020 Metrics Downloaded 158 times

Different expression of BRAFV600E, ALK and PD-L1 in melanoma in children and adolescents: a nationwide retrospective study in Finland in 1990–2014

Background Pediatric melanoma may have a different biological background and more favorable prognosis compared with melanoma in adults. The aim of this study was to investigate melanoma in children and adolescents in the Finnish population in terms of incidence, clinical course, treatment, prognosis and BRAFV600E-, ALK- and PD-L1-positivity of the primary tumors. Materials and Methods Primary tumor samples and clinical records of all patients aged 0-19 years diagnosed with cutaneous melanoma in Finland in 1990–2014 were collected using the Finnish Cancer Registry database, Finnish hospitals and private pathology laboratories. BRAFV600E, ALK and PD-L1 were analyzed from 54 primary tumors and BRAFV600E from six metastasis samples. Results A total of 122 patients diagnosed with cutaneous melanoma were retrieved from the Cancer Registry database. The primary tumor samples of 73 patients were obtained for the review, and 56 cases were included in the study. The incidence of pediatric melanoma increased from 0.02 to 0.1/100 000 during the period 1990–2014. Spitzoid melanoma was the most common subtype (66%). The 10-year cancer-specific survival (CSS) was 88.7% in all patients. The 10-year-CSS did not differ in SLNB-positive or -negative groups. BRAFV600E was positive in 48%, ALK in 9% and PD-L1 in 2% of the tumors. BRAFV600E mutation was associated with 83% of melanoma deaths. Conclusions Young melanoma patients had more favorable prognosis and a different staining profile for BRAFV600E, ALK, and PD-L1 in primary tumor than reported in adults. SLNB status was not an indicator for survival. BRAFV600E-positive patients have worse prognosis and could benefit from surveillance and treatment similarly to adults.

Heme oxygenase promotes B-Raf-dependent melanosphere formation.

Biosynthesis and degradation of heme, an iron-bound protoporphyrin molecule utilized by a wide variety of metabolic processes, are tightly regulated. Two closely related enzymes, heme oxygenase 1 (HMOX1) and heme oxygenase 2 (HMOX2), degrade free heme to produce carbon monoxide, Fe2+ , and biliverdin. HMOX1 expression is controlled via the transcriptional activator, NFE2L2, and the transcriptional repressor, Bach1. Transcription of HMOX1 and other NFE2L2-dependent genes is increased in response to electrophilic and reactive oxygen species. Many tumor-derived cell lines have elevated levels of NFE2L2. Elevated expression of NFE2L2-dependent genes contributes to tumor growth and acquired resistance to therapies. Here, we report a novel role for heme oxygenase activity in melanosphere formation by human melanoma-derived cell lines. Transcriptional induction of HMOX1 through derepression of Bach1 or transcriptional activation of HMOX2 by oncogenic B-RafV600E results in increased melanosphere formation. Genetic ablation of HMOX1 diminishes melanosphere formation. Further, inhibition of heme oxygenase activity with tin protoporphyrin markedly reduces melanosphere formation driven by either Bach1 derepression or B-RafV600E expression. Global transcriptome analyses implicate genes involved in focal adhesion and extracellular matrix interactions in melanosphere formation.

Expression and clinical significance of BRAF V600E in children with Langerhans cell histiocytosis

目的： 探讨儿童朗格汉斯细胞组织细胞增生症（Langerhans cell histiocytosis，LCH）中BRAF V600E蛋白和基因的表达与临床病理特征的关系。 方法： 收集儿童LCH病例75例，分别用免疫组织化学法（Ventana）和ARMS荧光PCR/Sanger测序法检测BRAF V600E蛋白和基因情况，比较两种方法的一致性，并分析BRAF V600E蛋白/基因的表达与临床病理特征的关系。 结果： 免疫组织化学法（Ventana）检测BRAF V600E阳性率为53.3%（40/75），ARMS荧光PCR/Sanger测序法检测BRAF V600E基因突变阳性率为53.3%（40/75），两种方法的一致性高（Kappa=0.893，P<0.001）。BRAF V600E蛋白表达/基因突变率在男性和女性中分别为53.8%和58.3%，差异无统计学意义（P>0.05）；年龄范围为出生1 d至13岁，在3岁以下组突变率高于3岁及以上组（63.2%∶48.6%），但差异无统计学意义（P>0.05）；皮肤病变者突变率为58.3%，高于骨病变55.6%，但差异无统计学意义（P>0.05）；单发者突变率55.6%，多发者突变率57.1%，差异无统计学意义（P>0.05）。 结论： 儿童LCH中BRAF V600E蛋白和基因采用免疫组织化学法（Ventana）与ARMS荧光PCR/Sanger测序法有较好的一致性。3岁以下低龄儿童、累及皮肤的病变中BRAF V600E突变率有增高的趋势，但尚需扩大样本进一步研究。.

BRAFV600E expression in neural progenitors results in a hyperexcitable phenotype in neocortical pyramidal neurons.

Somatic mutations have emerged as the likely cause of focal epilepsies associated with developmental malformations and epilepsy-associated glioneuronal tumors (GNT). Somatic BRAFV600E mutations in particular have been detected in the majority of low-grade neuroepithelial tumors (LNETS) and in neurons in focal cortical dysplasias adjacent to epilepsy-associated tumors. Furthermore, conditional expression of an activating BRAF mutation in neocortex causes seizures in mice. In this study we characterized the cellular electrophysiology of layer 2/3 neocortical pyramidal neurons induced to express BRAFV600E from neural progenitor stages. In utero electroporation of a piggyBac transposase plasmid system was used to introduce transgenes expressing BRAF wild type (BRAFwt), BRAFV600E, and/or enhanced green fluorescent protein (eGFP) and monomeric red fluorescent protein (mRFP) into radial glia progenitors in mouse embryonic cortex. Whole cell patch-clamp recordings of pyramidal neurons in slices prepared from both juvenile and adult mice showed that BRAFV600E resulted in neurons with a distinct hyperexcitable phenotype characterized by depolarized resting membrane potentials, increased input resistances, lowered action potential (AP) thresholds, and increased AP firing frequencies. Some of the BRAFV600E-expressing neurons normally destined for upper cortical layers by their birthdate were stalled in their migration and occupied lower cortical layers. BRAFV600E-expressing neurons also displayed increased hyperpolarization-induced inward currents (Ih) and decreased sustained potassium currents. Neurons adjacent to BRAFV600E transgene-expressing neurons, and neurons with TSC1 genetically deleted by CRISPR or those induced to carry PIK3CAE545K transgenes, did not show an excitability phenotype similar to that of BRAFV600E-expressing neurons. Together, these results indicate that BRAFV600E leads to a distinct hyperexcitable neuronal phenotype.NEW & NOTEWORTHY This study is the first to report the cell autonomous effects of BRAFV600E mutations on the intrinsic neuronal excitability. We show that BRAFV600E alters multiple electrophysiological parameters in neocortical neurons. Similar excitability changes did not occur in cells neighboring BRAFV600E-expressing neurons, after overexpression of wild-type BRAF transgenes, or after introduction of mutations affecting the mammalian target of rapamycin (mTOR) or the catalytic subunit of phosphoinositide 3-kinase (PIK3CA). We conclude that BRAFV600E causes a distinct, cell autonomous, highly excitable neuronal phenotype when introduced somatically into neocortical neuronal progenitors.

Protein Expression in Metastatic Melanoma and the Link to Disease Presentation in a Range of Tumor Phenotypes.

Malignant melanoma is among the most aggressive skin cancers and it has among the highest metastatic potentials. Although surgery to remove the primary tumor is the gold standard treatment, once melanoma progresses and metastasizes to the lymph nodes and distal organs, i.e., metastatic melanoma (MM), the usual outcome is decreased survival. To improve survival rates and life span, advanced treatments have focused on the success of targeted therapies in the MAPK pathway that are based on BRAF (BRAF V600E) and MEK. The majority of patients with tumors that have higher expression of BRAF V600E show poorer prognosis than patients with a lower level of the mutated protein. Based on the molecular basis of melanoma, these findings are supported by distinct tumor phenotypes determined from differences in tumor heterogeneity and protein expression profiles. With these aspects in mind, continued challenges are to: (1) deconvolute the complexity and heterogeneity of MM; (2) identify the signaling pathways involved; and (3) determine protein expression to develop targeted therapies. Here, we provide an overview of the results from protein expression in MM and the link to disease presentation in a variety of tumor phenotypes and how these will overcome the challenges of clinical problems and suggest new promising approaches in metastatic melanoma and cancer therapy.

High frequency of BRAF V600E mutation in Iranian population ameloblastomas.

Background Ameloblastoma is a common locally invasive but slow-growing neoplasm of the jaws with an odontogenic origin. Association between BRAF V600E mutation and clinicopathologic features and behavior of ameloblastoma remains controversial. This study aimed to evaluate BRAF V600E gene mutation and expression of its related proteins with clinicopathologic parameters in conventional ameloblastoma. Material and Methods 50 Formalin-fixed paraffin-embedded blocks were included in this study. Immunohistochemistry was done using rabbit monoclonal BRAF V600E mutation-specific antibody VE1. Quantitative real-time polymerase chain reaction assay was used for evaluating of BRAF V600E mutation. Results Expression of BRAF V600E antibody was Positive in 42 out of 50 cases (84%). 46 (92%) out of 50 specimens showed BRAF V600E mutation. There were 13 cases of recurrence (26%). 3 out of 4 cases with negative mutations did not show recurrence. Conclusions We report the highest frequency (92%) of BRAF V600E mutation in ameloblastomas in the Iranian population. Although there was not a significant association between BRAF V600E‑positive immunoexpression and recurrence and clinicopathologic parameters, its high frequency could emphasize its role as a therapeutic marker in the future. Key words:Conventional ameloblastoma, BRAF V600E, recurrence.

PTPN11 Knockdown Prevents Changes in the Expression of Genes Controlling Cell Cycle, Chemotherapy Resistance, and Oncogene-Induced Senescence in Human Thyroid Cells Overexpressing BRAF V600E Oncogenic Protein.

The MAPK (RAS/BRAF/MEK/ERK) signaling pathway is a kinase cascade involved in the regulation of cell proliferation, differentiation, and survival in response to external stimuli. The V600E mutation in the BRAF gene has been detected in various tumors, resulting in a 500-fold increase in BRAF kinase activity. However, monotherapy with selective BRAF V600E inhibitors often leads to reactivation of MAPK signaling cascade and emergence of drug resistance. Therefore, new targets are being developed for the inhibition of components of the aberrantly activated cascade. It was recently discovered that resistance to BRAF V600E inhibitors may be associated with the activity of the tyrosine phosphatase SHP-2 encoded by the PTPN11 gene. In this paper, we analyzed transcriptional effects of PTPN11 gene knockdown and selective suppression of BRAF V600E in a model of thyroid follicular epithelium. We found that the siRNA-mediated knockdown of PTPN11 after vemurafenib treatment prevented an increase in the expression CCNA1 and NOTCH4 genes involved in the formation of drug resistance of tumors. On the other hand, downregulation of PTPN11 expression blocked the transcriptional activation of genes (p21, p15, p16, RB1, and IGFBP7) involved in cell cycle regulation and oncogene-induced senescence in response to BRAF V600E expression. Therefore, it can be assumed that SHP-2 participates not only in emergence of drug resistance in cancer cells, but also in oncogene-induced cell senescence.

1O - Suppression of KRAS-G12D and BRAF-V600E oncogene transcription with PNA-conjugates

BackgroundWe have developed a strategy to block transcription of oncogenes such as BRAF V600E and KRAS G12D directly using modified complementary peptide nucleic acid (PNA) oligomers that target oncogenesis specifically causing inhibition of tumour growth. This proof of principle strategy against BRAF V600E and KRAS G12D in vitro and in vivo should provide a new means to develop PNA-delivery peptide conjugates as targeted drug therapeutics across a broad range of oncogenes that drive cancer cell growth. MethodsObstruction of KRAS G12D and BRAF V600E expression was evaluated through suppression of cell proliferation and specific mRNA transcription. Tumour reduction was assessed through Xenograft mouse models. ResultsExposure of KRAS G12D-dependent cell line to modified PNA-peptide conjugate complementary to KRAS G12D mutation sequence also results in concentration-dependent and time-dependent inhibition of cell growth and specific and complete suppression in mRNA transcription. Cell lines expressing KRAS WT and KRAS G12C, both differing by a single nucleobase, show no suppression. Exposure of the melanoma cell lines to a modified PNA-peptide conjugate complementary to BRAF V600E mutation sequence results in a concentration-dependent and time-dependent inhibition of cell growth that is specific for the BRAF V600E mutant melanoma cell lines with inhibition of mRNA and protein expression. Xenograft mouse trials show tumour growth delay and necrosis compared to PNA controls. This 50mg/kg dose was well tolerated without associated weight loss. By H&E staining, tumor tissue from trials shows ablation and extensive scaring upon exposure to BRAF V600E-complementary PNA-peptide conjugate whereas saline and scramble PNA sequence controls do not. Similarly quantitative measurement shows a 2.5-fold decrease in Ki67 and a 3-fold increase in TUNEL expression. ConclusionsOur results indicate that these PNA-peptide derivatives could represent a novel and promising new therapy for patients with genes specific for and causative of tumorigenesis. This strategy could be applied to a multitude of cancers either with specific translocations or mutations differing from wild-type cells even by only a single base pair. Legal entity responsible for the studyThe authors. FundingCelgene, Fortress Biotech. DisclosureAll authors have declared no conflicts of interest.

COCCIDIOMYCOSIS MASQUERADING AS PULMONARY HISTIOCYTOSIS X

SESSION TITLE: Interesting Presentations of Infectious Diseases SESSION TYPE: Fellow Case Reports PRESENTED ON: 10/20/2019 1:00 PM - 2:00 PM INTRODUCTION: Pulmonary Langerhans Cell Histiocytosis X (PLCH) and coccidiomycosis are two distinct disease entities with significant overlap in presenting symptoms and imaging findings; however, are rarely mentioned as differentials of one another. PLCH is a non-neoplastic smoking related condition that causes dysregulation of dendritic cells(1), while coccidiomycosis is an infection by the dimorphic fungi, Coccidioides sp., found mostly in the subtropics of North America(2). Here we describe a case of a young man who presented with symptoms consistent with both diseases (cough, fevers, chills, malaise, weight loss) and had an initial workup including a biopsy that was consistent with PLCH, but was ultimately diagnosed with Coccidiomycosis. CASE PRESENTATION: 29-year-old male smoker with no past medical history presented to our hospital with fatigue, weight loss, dyspnea, cough, and fevers for 1 month. Two weeks prior to presentation started to develop cyclical nighttime fevers and went to urgent care who gave him diagnosis of pneumonia. His symptoms worsened over the following 2 weeks despite finishing a course of antibiotics thus he presented to the hospital for further evaluation. CT showed multiple stellate pulmonary nodules with a cavitary lesion in the left lung. Extensive workup was completed including testing for fungal, bacterial, mycobacterium, and vasculitis sources which came back negative. Bronchoscopy was performed and transbronchial biopsies showed abundant proliferation of langerhans cells with strong CD1a staining (figure 1), in the absence of foreign organisms (negative methenamine silver stain) (figure 2), most consistent with a diagnosis of pulmonary histiocytosis X. He was discharged on steroids, however in one week returned with worsening cough and development of night sweats. Repeat CT showed interval increase in lymphadenopathy. Mediastinoscopy was performed revealing 5 small dense lymph nodes. An excisional biopsy was positive for coccidioides (figure 3). The patient was subsequently started on fluconazole and experienced an improvement in symptoms. DISCUSSION: This patient had risk factors, symptoms, imaging, laboratory studies, and biopsies consistent with a diagnosis PLCH; however, was ultimately diagnosed with coccidiomycosis. This case emphasizes the insidious nature that this fungal disease may take (3). Treatment of PLCH and coccidioidomycosis are diametrically opposed in that one requires immunosuppressives and the other antifungals, therefore its is crucial that one make a proper diagnosis prior to initiation of treatment (1,2). CONCLUSIONS: This case illustrates the importance of recognizing coccidiomycosis as an important differential to Pulmonary Langerhans Cell Histiocytosis X, even in a patient with with significant data supporting the diagnosis. Reference #1: BRAF V600E expression in Langerhans cell histiocytosis: clinical and immunohistochemical study on 25 pulmonary and 54 extrapulmonary cases. Roden AC Et al; Am J Surg Pathol. 2014 Apr;38(4):548-51. Reference #2: Valley Fever: Timely Diagnosis, Early Assessment, and Proper Management | Fungal Diseases | CDC.” Centers for Disease Control and Prevention, Centers for Disease Control and Prevention, 17 May 2017. Reference #3: Saubolle MA, Et al. Epidemiologic, clinical, and diagnostic aspects of coccidioidomycosis. Journal of Clinical Microbiology 2007; 45:26. DISCLOSURES: No relevant relationships by Kristyn Galbraith, source=Web Response No relevant relationships by Anil Ghimire, source=Web Response No relevant relationships by Isaac Huml, source=Web Response

BRAF V600E and Pten deletion in mice produces a histiocytic disorder with features of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by the accumulation of Langerin (CD207)-expressing histiocytes. Mutational activation of mitogen-activated protein kinase pathway genes, in particular BRAF, drives most cases. To test whether activated BRAF is sufficient for the development of LCH, we engineered mice to express BRAF V600E under the control of the human Langerin promoter. These mice have shortened survivals, smaller lymphoid organs, absent Leydig cells, and fewer epidermal LCs than controls, but do not accumulate histiocytes. To test whether the absence of histiocyte proliferation could be due to oncogene-induced senescence, we engineered homozygous Pten loss in the same cells that expressed BRAF V600E. Like mice with intact Pten, these mice have shortened survivals, smaller thymi, and absent Leydig cells. However, loss of Pten also leads to the accumulation of CD207+ histiocytes in spleen, thymus, and some lymph nodes. While many CD207+ histiocytes in the thymus are CD8-, reminiscent of LCH cells, the CD207+ histiocytes in the spleen and lymph nodes are CD8+. These mice also accumulate large numbers of CD207- cells in the lamina propria (LP) of the small intestine. Both the lymphoid and LP phenotypes are likely due to human Langerin promoter-driven BRAF V600E expression in resident CD8+ dendritic cells in the former and LP dendritic cells in the latter and confirm that Pten loss is required to overcome inhibitory pathways induced by BRAF V600E expression. The complex phenotype of these mice is a consequence of the multiple murine cell types in which the human Langerin promoter is active.