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Importance: Thyroid cancer, predominantly papillary thyroid carcinoma (PTC), is common, but an estimated 30% of ultrasonography-guided fine-needle aspiration (FNA) biopsies of thyroid nodules are indeterminate. BRAF variation, associated with poor clinicopathological characteristics, is a useful molecular marker for diagnostics., Objective: To develop a sensitive molecular assay for BRAF V600E detection in remaining tissue of thyroid FNA biopsies to identify patients with cancer carrying a BRAF variation., Design, Setting, and Participants: This diagnostic study used tumor tissue from surgical formalin-fixed, paraffin-embedded (FFPE) specimens and residual tissue from thyroid FNA biopsies for genomic DNA extraction. FFPE specimens served as the validation set, and residual tissue from FNA biopsies served as the test set. A molecular assay was developed for accurate detection of BRAF V600E variation using locked nucleic acid (LNA) probe-based droplet digital polymerase chain reaction (dPCR), and the assay was validated by BRAF V600E immunohistochemical staining (IHC). The study was conducted between February 2019 and May 2021., Results: A total of 271 specimens, including 77 FFPE specimens (with a follow-up of 48 matched surgical specimens) and 146 residual FNA samples, were collected from 223 patients (mean [SD] age, 53.8 [15.3] years; 174 [78.0%] women; 49 [22.0%] men). The molecular assay by dPCR was first established to specifically and accurately detect and quantify wild-type BRAF and variant BRAF in DNA from human follicular thyroid carcinoma-derived FTC-133 and papillary thyroid carcinoma-derived BCPAP cells. The linearity of quantification of BRAF V600E was calculated (y = 0.7339x; R2 = 0.9996) with sensitivity at 0.02 copies/μL and reproducibility in detecting variant DNA at various dilutions(coefficient of variance in 0.3% DNA, 9.63%; coefficient of variance in 1.0% DNA, 7.41%). In validation testing, the dPCR assay and IHC staining exhibited 100% specificity in concordantly identifying BRAF V600E in PTCs (κ = 0.873; P < .001) and sensitivity of 32.0% (95% CI, 19.1% to 44.9%) in dPCR and 26.0% (95% CI, 13.1% to 38.9%) in IHC staining, with an improvement by 23.08% in dPCR compared with the IHC staining. The dPCR assay further detected BRAF V600E in 39 of 146 residual FNA specimens (26.7%). At short-term follow-up, 48 patients, including 14 of 39 patients with BRAF variation and 34 of 107 patients without BRAF variation on residual FNA specimens, underwent resection. The dPCR assay of BRAF status in the matched surgical specimens showed BRAF V600E variations in 12 patients and wild-type BRAF in 36 patients, with a high agreement to that in residual tissue of FNA specimens (κ = 0.789; P < .001). Among 14 patients with BRAF variations on residual FNA, 13 were diagnosed with PTC and 1 was diagnosed with anaplastic thyroid cancer at the thyroidectomy., Conclusions and Relevance: This diagnostic study developed a sensitive molecular assay for detection and quantification of BRAF V600E variation in residual tissue from thyroid FNA biopsies to identify patients with cancer harboring BRAF V600E in a cost-effective manner, highlighting the clinical value of molecular assay of the remaining FNA tissue in the management of thyroid nodules.

Somatic mutations in cancer driver genes can help diagnosis, prognosis and treatment decisions. Formalin-fixed paraffin-embedded (FFPE) specimen is the main source of DNA for somatic mutation detection. To overcome constraints of DNA isolated from FFPE, we compared pyrosequencing and ddPCR analysis for absolute quantification of BRAF V600E mutation in the DNA extracted from FFPE specimens and compared the results to the qualitative detection information obtained by Sanger Sequencing. Sanger sequencing was able to detect BRAF V600E mutation only when it was present in more than 15% total alleles. Although the sensitivity of ddPCR is higher than that observed for Sanger, it was less consistent than pyrosequencing, likely due to droplet classification bias of FFPE-derived DNA. To address the droplet allocation bias in ddPCR analysis, we have compared different algorithms for automated droplet classification and next correlated these findings with those obtained from pyrosequencing. By examining the addition of non-classifiable droplets (rain) in ddPCR, it was possible to obtain better qualitative classification of droplets and better quantitative classification compared to no rain droplets, when considering pyrosequencing results. Notable, only the Machine learning k-NN algorithm was able to automatically classify the samples, surpassing manual classification based on no-template controls, which shows promise in clinical practice.

Abstract: This study investigated the feasibility of using immunohistochemistry (IHC) instead of PCR to detect BRAF V600E mutant protein in papillary thyroid carcinoma (PTC), and to determine the value of using preoperative BRAF V600E mutant protein by IHC to assist in the diagnosis of thyroid nodule patients with Hashimoto's thyroiditis (HT).The expression of BRAFV600E mutant protein was measured in 23 cases of HT+PTC, 31 cases of PTC, and 28 cases of HT by IHC, followed by PCR in the same samples for validation. SPSS 19.0 software was used for statistical analysis.The sensitivity and specificity of IHC to detect BRAF V600E mutation were 100% and 42.86%, respectively. In addition, the mutation rate of BRAF V600E protein in the HT+PTC group (34.78%, 8/23) was lower than that in the PTC group (80.65%, 25/31).The application of IHC to detect BRAF V600E mutant protein has good sensitivity but not specificity to diagnose PTC. IHC can be used as a preliminary screening method to detect BRAF V600E mutation. The strongly positive (+++) staining of IHC potently indicated BRAF V600E gene mutation. For suspicious thyroid nodules combined with HT, the detection of BRAF V600E mutant protein with IHC alone is not of great significance for differentiating benign and malignant nodules., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Abstract: Colorectal cancer is a heterogeneous disease with multiple epigenetic alterations and different molecular features. The molecular classification is based on 2 major distinct pathways: microsatellite stable pathway and the microsatellite instability pathway. Molecular profiling of colorectal cancer provides important information regarding treatment and prognosis. Aim of the study was to assess the frequency of microsatellite instability in colon cancer and the clinicopathological characteristics of the tumors with high level of microsatellite instability (MSI-H) in our region. The secondary outcome was to assess the frequency of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations in colon cancer.The study included 129 patients with colon cancer fit for surgery. Demographic data, clinical and pathological data, immunohistochemistry staining pattern (4 mismatch repair proteins were investigated), and BRAF gene mutations were assessed. According to microsatellite instability status by polymerase chain reaction, patients were divided into 3 groups: microsatellite stable (MSS) = 108 patients, high level of microsatellite instability (MSI-H) = 15 patients and low level of microsatellite instability (MSI-L) = 6 patients. Different clinicopathological comparisons between MSS and MSI-H patients, and between MSS and MSI-L patients were performed.Microsatellite instability was found in 16.3% patients: 11.6% had MSI-H and 4.7% had MSI-L. Significantly more patients in the MSI-H group than in the MSS group were female (P = .01) and had a family history of colon cancer (P < .001). MSI-H and MSI-L groups were associated with the ascending colon location of the tumors, were mostly type G3, T2, and stage I whereas MSS tumors were mostly G2, pT3, and stage III. Overall, BRAF mutations were identified in 18/129 patients (13.9%). BRAF mutant tumors were predominantly associated with MSI-H and MSI-L tumors. Immunohistochemistry had a sensitivity of 76% and a specificity of 89% in detecting MSI tumors and an accuracy of 87.6%.The frequency of microsatellite instability in our study was 16.3%. MSI-H is a distinct molecular phenotype of colon cancer with particular features: female gender, family history of colorectal cancer, a predilection for the ascending colon, poorly differentiated, predominantly T2, and stage I. The frequency of BRAF mutations was 13.9% and mutations were more often present in the MSI tumors., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Introduction: Molecular profiling has led to significantly longer survival in metastatic colorectal cancer (mCRC) patients. Clinical guidelines recommend testing for KRAS/NRAS, BRAF and MSI status, and new biomarkers such as HER2 amplification and NTRK fusions have emerged more recently in refractory CRC, supported by overwhelming clinical relevance. These biomarkers can guide treatment management to improve clinical outcomes in these patients., Areas Covered: Preclinical and clinical data over the last decade were reviewed for known and novel biomarkers with clinical implications in refractory CRC. Molecular alterations are described for classic and novel biomarkers, and data for completed and ongoing studies with targeted and immunotherapies are presented., Expert Opinion: Use of targeted therapies based on biomarker testing in CRC has enabled impressive improvements in clinical outcomes in refractory patients. BRAF, MSI, NRAS and KRAS should be tested upfront in all patients given their indisputable therapeutic implications. Other molecular alterations such as HER2 and NTRK are emerging. Testing for these alterations may further improve outcomes for refractory CRC patients. Nonetheless, many key aspects remain to be defined including the optimal timing and technique for testing, the most adequate panel, and whether all patients should be tested for all alterations.

Melanoma is widely known as the most lethal skin cancer. Specific tumor-related mortality can be significantly reduced if diagnosis and treatment are properly performed during initial phases of the disease. The current search for biomarkers in early-stage melanomas is a high-priority challenge for physicians and researchers. We aimed to assess the immunoexpression of BRAFV600E and KIT in a case series consisting of 44 early-stage melanomas. Formalin-fixed paraffin-embedded samples were systematically evaluated using a semi-quantitative method based on scores of percentage and intensity for immunostained tumor cells. We observed significant concordance between BRAFV600E and KIT immunoexpression in thin invasive melanomas. Our findings corroborate previous evidence showing abnormal expression of proteins associated with MAPK intracellular signaling pathway in early-stage melanomas.

This study utilized a microfluidic mixer for the sample pretreatment of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation and protein enzymatic digestion. The time of sample pretreatment was reduced and thus the throughput of quantitative mutant proteins was increased by using the proposed method. Whole cell lysates of the cancer cell line HT-29 with gene mutations were used as the sample. The target protein BRAF was immunoprecipitated using magnetic beads in a pneumatic micromixer. Purified protein was then eluted and digested by trypsin in another two micromixers to yield peptide fragments in the solution. Using stable isotope-labeled standard as the internal control, wild-type and mutant BRAF proteins were quantified using mass spectrometry, which could be used for cancer screening. Compared with conventional methods in which protein immunoprecipitation lasts overnight, the micromixer procedure takes only 1 h, likely improving the throughput of mutant BRAF protein quantification by mass spectrometry. Graphical abstract Three micromixers were used to reduce the sample pretreatment time of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation, protein elution, and protein enzymatic digestion.

Objective: To investigate the association of the BRAFV600E mutation with papillary thyroid carcinoma using clinical, morphological and prognostic parameters. We also intend to assess the utility of the BRAFV600E immunohistochemistry and compare it with BRAF polymerase chain reaction (RT-PCR)., Material and Method: We applied BRAFV600E immunohistochemistry in a cohort of 107 papillary carcinomas, 19 adenomas and 13 normal thyroid tissues that was chosen retrospectively between 2011 and 2015. Statistical analysis was based on semiquantitative immunohistochemistry findings. We also applied BRAF RT-PCR in a subgroup of 14 papillary carcinomas, 13 metastatic lymph nodes and 4 adenomas that was chosen randomly., Results: In regard to the comparison of BRAFV600E immunohistochemistry and BRAF RT-PCR, a 3+ nuclear and cytoplasmic immunoexpression was considered 'positive'. The BRAFV600E mutation was most frequently observed in classic variant cases. No mutation was detected in follicular variant cases. The mutational status of the primary tumour and the lymph node metastasis was consistent. A significant relationship of the BRAFV600E mutation was found with prognostic factors such as higher pT stage, classic variant, lymphatic invasion, perineural invasion, lower mitotic index, lack of tumour capsule, intrathyroidal spread and extrathyroidal extension., Conclusion: Immunohistochemistry, using the VE1 clone, is a reliable technique for detection of the BRAFV600E mutation. Our results with immunohistochemistry are consistent with a previous effort. In our study, despite the correlation between some pathological prognostic parameters and the BRAFV600E mutation; poor prognosis was found to be irrelevant overall. Morphological parameters seem to be keener than the BRAFV600E mutation. Nevertheless, different series display different results, possibly due to environmental factors. Considering this and the proven success of targeted therapies against the BRAFV600E mutation a thorough assessment would be important.

Protein detection and quantification play critical roles in both basic research and clinical practice. Current detection platforms range from the widely used ELISA to more sophisticated, and more expensive, approaches such as digital ELISA. Despite advances, there remains a need for a method that combines the simplicity and cost-effectiveness of ELISA with the sensitivity and speed of modern approaches in a format suitable for both laboratory and rapid, point-of-care applications. Building on recent developments in DNA structural nanotechnology, we introduce the nanoswitch-linked immunosorbent assay (NLISA), a detection platform based on easily constructed DNA nanodevices that change conformation upon binding to a target protein with the results read out by gel electrophoresis. NLISA is surface-free and includes a kinetic-proofreading step for purification, enabling both enhanced sensitivity and reduced cross-reactivity. We demonstrate femtomolar-level detection of prostate-specific antigen in biological fluids, as well as reduced cross-reactivity between different serotypes of dengue and also between a single-mutation and wild-type protein. NLISA is less expensive, uses less sample volume, is more rapid, and, with no washes, includes fewer hands-on steps than ELISA, while also achieving superior sensitivity. Our approach also has the potential to enable rapid point-of-care assays, as we demonstrate by performing NLISA with an iPad/iPhone camera for imaging., Competing Interests: Conflict of interest statement: Patent applications have been filed for various aspects of this work by all authors. M.A.K. and W.P.W. are founders of and have equity interest in Confer Health, Inc., a company that is developing point-of-care diagnostics.

Background: Lymph nodal involvement is very common in differentiated thyroid cancer, and in addition, cervical lymph node micrometastases are observed in up to 80 % of papillary thyroid cancers. During the last decades, the role of routine central lymph node dissection (RCLD) in the treatment of papillary thyroid cancer (PTC) has been an object of research, and it is now still controversial. Nevertheless, many scientific societies and referral authors have definitely stated that even if in expert hands, RCLD is not associated to higher morbidity; it should be indicated only in selected cases., Main Body: In order to better analyze the current role of prophylactic neck dissection in the surgical treatment of papillary thyroid cancers, an analysis of the most recent literature data was performed. Prophylactic or therapeutic lymph node dissection, selective, lateral or central lymph node dissection, modified radical neck dissection, and papillary thyroid cancer were used by the authors as keywords performing a PubMed database research. Literature reviews, PTCs large clinical series and the most recent guidelines of different referral endocrine societies, inhering neck dissection for papillary thyroid cancers, were also specifically evaluated. A higher PTC incidence was nowadays reported in differentiated thyroid cancer (DTC) clinical series. In addition, ultrasound guided fine-needle aspiration citology allowed a more precocious diagnosis in the early phases of disease. The role of prophylactic neck dissection in papillary thyroid cancer management remains controversial especially regarding indications, approach, and surgical extension. Even if morbidity rates seem to be similar to those reported after total thyroidectomy alone, RCLD impact on local recurrence and long-term survival is still a matter of research. Nevertheless, only a selective use in high-risk cases is supported by more and more scientific data., Conclusions: In the last years, higher papillary thyroid cancer incidence and more precocious diagnoses were worldwide reported. Among endocrine and neck surgeons, there is agreement about indications to prophylactic treatment of node-negative "high-risk" patients. A recent trend toward RCLD avoiding radioactive treatment is still debated, but nevertheless, prophylactic dissections in low-risk cases should be avoided. Prospective randomized trials are needed to evaluate the benefits of different approaches and allow to drawn definitive conclusions.

We investigated whether sessile serrated adenomas/polyps (SSA/Ps) are direct precursors of colorectal carcinomas. We identified colon carcinomas that arose from SSA/Ps among 2646 colorectal cancers included in the surgical pathology database at the Mayo Clinic (2006-2012). Molecular features of the serrated neoplasia pathway were analyzed in these tumors by immunohistochemical analyses of mutant BRAF (V600E) and MLH1 proteins. Among the 33 identified SSA/P-associated colonic adenocarcinomas (median patient age, 75 y), 24 developed in women (73%), 31 were located in the proximal colon (94%), and 23 (69%) were TNM stage I or II. Thirty-one of the tumors (94%) expressed mutant BRAF; of these, 26 also had loss of MLH1 (79%), indicating deficient DNA mismatch repair of sporadic origin. Twenty-two of the tumors (67%) were interval cancers that were more common in women and did not differ significantly in TNM stage, BRAF mutation, or loss of MLH1. By histopathology, SSA/Ps that were associated with colon carcinomas contained frequent dysplasia (48%). Most cancers that arose from SSA/Ps were located on the right side of the colon and had mutant BRAF and loss of MLH1. These findings indicate that SSA/Ps are precursors of most sporadic colon carcinomas with deficient DNA mismatch repair., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Purpose: V600E is the most common activating BRAF mutation in colorectal carcinomas (CRCs). It is a crucial biomarker for patient selection and response to targeted therapy with BRAF V600E inhibitors. Previous studies using immunohistochemistry (IHC) have shown different results. In this study, we evaluated the IHC expression of the mutated BRAF protein in archival material from CRC specimens and correlated it with DNA sequence analysis., Methods: 51 cases of primary colon adenocarcinoma were stained with BRAF V600E-specific clone VE1 antibody against mutated BRAF protein. DNA sequence analysis was performed and the results were compared., Results: BRAF V600E protein was detected in the cytoplasm of neoplastic cells in 15 of the 51 examined cases (29.4%). The correlation between IHC staining and DNA sequence analysis showed 93.75% sensitivity and 100% specificity., Conclusions: Our data show that IHC could be used in routine clinical practice as a screening method for BRAF V600E mutant protein detection in CRC patients.

Purpose: We performed this retrospective study to have a comprehensive investigation of the clinicopathological characteristics of ALK fusion-positive lung adenocarcinoma in Chinese populations., Methods: We screened 1407 patients with primary lung adenocarcinoma from October 2007 to May 2013. Quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (RT-PCR), and fluorescence in situ hybridization were performed to detect ALK fusion genes, with validation of positive results using immunohistochemistry. Clinicopathological characteristics were collected to assess prognosis in ALK fusion-positive patients., Results: Of 1407 patients with lung adenocarcinoma, there were 74 (5.3 %) ALK fusion-positive patients. Patients harboring ALK fusion were significantly younger (56.0 years vs. 59.8 years p = 0.002) and were more likely to have advanced stages (stage III or stage IV) (OR 1.761; 95 % CI 1.10-2.82, p = 0.017). Lepidic predominant adenocarcinoma was rarely found in ALK fusion patients (2.7 vs. 13.5 % p = 0.025), while IMA (invasive mucinous adenocarcinoma) predominant adenocarcinoma was more frequently found (21.6 vs. 5.0 % p < 0.001). ALK fusion was neither a risk factor nor protective factor in relapse-free survival and overall survival. Male, current smoker, and EML4-ALK variant 3 indicated poor prognosis among ALK fusion-positive lung adenocarcinomas., Conclusions: ALK fusion was detected in 5.3 % (74/1407) of the Chinese patients with lung adenocarcinoma. ALK fusion defines a molecular subset of lung adenocarcinoma with unique clinicopathological characteristics. Different ALK fusion variants determine distinct prognoses.

Research and development of multi-target inhibitors has attracted increasing attention as anticancer therapeutics. B-RafV600E synergistically works with vascular endothelial growth factor receptor 2 (KDR) to promote the occurrence and progression of cancers, and the development of dual-target drugs simultaneously against these two kinds of kinase may offer a better treatment advantage. In this paper, docking and three-dimensional quantitative structure activity relationship (3D-QSAR) studies were performed on a series of dual B-Raf/KDR inhibitors with a novel hinge-binding group, [5,6]-fused bicyclic scaffold. Docking studies revealed optimal binding conformations of these compounds interacting with both B-Raf and KDR. Based on these conformations, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) 3D-QSAR models were constructed, and the best CoMFA (q²=0.542, r²=0.989 for B-Raf; q²=0.768, r²=0.991 for KDR) and CoMSIA models (q²=0.519, r²=0.992 for B-Raf; q²=0.849, r²=0.993 for KDR) were generated. Further external validations confirmed their predictability, yielding satisfactory correlation coefficients (r²pred=0.764 (CoMFA), r²pred=0.841 (CoMSIA) for B-Raf, r²pred=0.912 (CoMFA), r²pred=0.846 (CoMSIA) for KDR, respectively). Through graphical analysis and comparison on docking results and 3D-QSAR contour maps, key amino acids that affect the ligand-receptor interactions were identified and structural features influencing the activities were discussed. New potent derivatives were designed, and subjected to preliminary pharmacological evaluation. The study may offer useful references for the modification and development of novel dual B-Raf/KDR inhibitors.

Vemurafenib is approved for the treatment of metastatic melanoma in patients with BRAF V600 mutation. In pivotal clinical trials, BRAF testing has always been done with the approved cobas 4800 BRAF test. In routine practice, several methods are available and are used according to the laboratories usual procedures. A national, multicenter, non-interventional study was conducted with prospective and consecutive collection of tumor samples. A parallel evaluation was performed in routine practice between the cobas 4800 BRAF V600 mutation test and home brew methods (HBMs) of 12 national laboratories, labelled and funded by the French National Cancer Institute (INCa). For 420 melanoma samples tested, the cobas method versus HBM showed a high concordance (93.3%; kappa = 0.86) in BRAF V600 genotyping with similar mutation rates (34.0% versus 35.7%, respectively). Overall, 97.4% and 98.6% of samples gave valid results using the cobas and HBM, respectively. Of the 185 samples strictly fulfilling the cobas guidelines, the concordance rate was even higher (95.7%; kappa = 0.91; 95%CI [0.85; 0.97]). Out of the 420 samples tested, 28 (6.7%) showed discordance between HBM and cobas. This prospective study shows a high concordance rate between the cobas 4800 BRAF V600 test and home brew methods in the routine detection of BRAF V600E mutations.

BRAF mutations can be found in various solid tumors. But accurate and reliable screening for BRAF mutation that is compatible for clinical application is not yet available. In this study, we used an automated immunohistochemistry (IHC) staining coupled with mouse monoclonal anti-BRAF V600E (VE1) primary antibody to screen the BRAF V600E mutation in 779 tumor cases, including 611 colorectal carcinomas (CRC), 127 papillary thyroid carcinomas (PTC) and 41 malignant melanomas. Among the 779 cases, 150 cases were positive for BRAF (V600E) staining, including 38 (of 611, 6%) CRCs, 102 (of 127, 80%) PTCs and 10 (of 41, 24%) malignant melanomas. Sanger sequencing and real-time PCR confirmed the sensitivity and specificity of IHC staining for the V600E mutation are 100% and 99%, respectively. Therefore, our study demonstrates that the fully automated IHC is a reliable tool to determine BRAF mutation status in CRC, PTC and melanoma and can be used for routine clinical screen.

Advances in next-generation sequencing have provided new insights and new therapeutic options for patients with cancer. However, assessment of genomic aberrations, which is required for precision medicine, has been challenging because of the difficulties in capturing intratumoral heterogeneity and in real-time assessment of tumors. Recent advances in technology have enabled detection and analysis of cell-free DNA in cancer patients, which provides real-time assessment of tumor evolution. Here, we review the recent advances in our understanding of the clinical utility of cell-free DNA and the future directions for its use in cancer management.

Background: The prognosis for gliomas is still dismal when treated with traditional approaches. Molecular targeted therapy has become a trend in treating tumors, including gliomas. However, molecular characteristics vary among different kinds of tumors and ethnic groups., Objectives: The aim of the research was to study the expression characteristics of key components in the EGFR pathway of gliomas in order to contribute new data for the molecular targeted treatment of gliomas., Material and Methods: EGFR, KRAS, BRAF, PI3K, phospho-EGFR and Ki67 expression were detected with immunohistochemistry in 82 glioma specimens., Results: The expression of EGFR was positively correlated with the patients' age. There were no significant differences in clinicopathological characteristics between gliomas with and without phospho-EGFR expression. EGFR overexpression was significantly correlated with phospho-EGFR expression. In gliomas with EGFR activation, overexpressions of EGFR, BRAF and PI3K were significantly correlated with proliferation. However, in gliomas without phospho-EGFR expression, only BRAF overexpression was significantly related to the proliferation of tumor cells., Conclusions: BRAF overexpression could be an independent factor causing tumorigenesis in gliomas regardless of phospho-EGFR expression. The molecular characteristics can vary with the increasing age of glioma patients.

Aim: to compare the immunoexpression of BRAF V600E among stage of colorectal cancer., Methods: this was a cross sectional, and retrospective study involving Dukes' stage A, B, and C colorectal carcinoma, each with 15 cases. Immunohistochemistry was performed in paraffin-embedded specimens of tumor mass for the assessment of BRAF V600E. The proportion differences of immunoexpression of BRAF V600E among Dukes' stage A, B, and C were tested using Chi-Square test., Results: the result of positive BRAF V600E immunoexpression (moderately to strongly positive) in Dukes' stage A, B, and C were found in 1 of 15 cases, 4 of 15 cases and 13 of 15 cases respectively. BRAF V600E immunoexpression was statistically significant more frequent in Dukes' stage C (p<0,001, Chi-Square test)., Conclusion: positive BRAF V600E immunoexpression could be used as a marker of aggresive colorectal carcinoma.

Purpose: Cetuximab improves survival in patients with K-ras wild-type advanced colorectal cancer. We examined the predictive and prognostic significance of additional biomarkers in this setting, in particular BRAF, PIK3CA, and PTEN., Experimental Design: Available colorectal tumor samples were analyzed from the CO.17 study. BRAF mutations were identified in tumor-derived DNA by direct sequencing and PIK3CA mutations were identified using a high-resolution melting screen with confirmation by sequencing. PTEN expression by immunohistochemistry (IHC) was performed on tissue microarrays. For each biomarker, prognostic and predictive effects were examined using a Cox model with tests for treatment-biomarker interaction., Results: A total of 572 patients with pretreated colorectal cancer were randomly assigned to receive cetuximab or best supportive care (BSC). Of 401 patients assessed for BRAF status, 13 (3.2%) had mutations. Of 407 patients assessed for PIK3CA status, 61 (15%) had mutations. Of 205 patients assessed for PTEN, 148 (72%) were negative for IHC expression. None of BRAF, PIK3CA, or PTEN was prognostic for overall or progression-free survival in the BSC arm. None was predictive of benefit from cetuximab, either in the whole study population or the K-ras wild-type subset. In the K-ras wild-type subgroup, the overall survival adjusted HR according to BRAF mutation status was 1.39 (interaction P = 0.69), PIK3CA mutation status HR = 0.79 (interaction P = 0.63), and PTEN expression HR = 0.75 (interaction P = 0.61)., Conclusions: In chemotherapy-refractory colorectal cancer, neither PIK3CA mutation status nor PTEN expression were prognostic, nor were they predictive of benefit from cetuximab. Evaluation of predictive significance of BRAF mutations requires a larger sample size., (©2013 AACR.)

Background: The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations., Methods: Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively)., Results: There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM., Conclusion: Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

BRAF(V600E) mutations are frequent in melanomas originating from intermittently sun-exposed skin and also in common acquired melanocytic nevi, suggesting that BRAF mutation is an early event in melanocytic neoplasia. All neoplastic melanocytes within such a nevus would be expected to carry the BRAF mutation, and thus we evaluated the frequency of cells with BRAF(V600E) mutations within acquired nevi by droplet digital polymerase chain reaction. In BRAF-mutant nevi the number of BRAF mutant alleles equaled the number of wild-type (WT) alleles in the neoplastic cell population, consistent with a fully clonal heterozygous BRAF mutation. The allelic ratio of BRAF(V600E) to BRAF(WT) in the eight VE1-positive nevi, adjusted for degree of stromal contamination, ranged from 0.84 to 1.12 with an average ratio of 1.01. This was confirmed by immunohistochemistry with an antibody specific for BRAF(V600E), which uniformly labeled the neoplastic cells without any evidence of heterogeneity. We found BRAF(V600E) mutations in the melanocytic nevi to be fully clonal, strongly suggesting that BRAF-activating mutations typically are early initiating events in melanocytic neoplasia.

Despite thousands of studies about colorectal cancer (CRC) as much as extensively usage of prognostic antibodies÷genes and clinical trials that include the newest targeted drugs, this tumor still remains in the top of both incidence and cancer-related mortality. In this review, we intended to correlate our experience in field of colorectal cancer with the literature data and to present our vision about the prognostic and predictive role of some of the most used molecular and immunohistochemical examinations in the field. The prognostic and predictive values of parameters such as microsatellite instability, angiogenesis, Maspin gene/protein, K-ras and BRAF mutations are discussed in relationship to the classical antibodies such as Keratin 7/20, p53 or HER2. At the end, we correlated these informations and tried to realize a molecular classification of colorectal cancer, similar to breast carcinomas, in order to establish targeted groups of patients for targeted therapy.

For some, glioma biomarkers have been expected to solve common diagnostic problems in routine neuropathology service caused by insufficient material, technical shortcomings or lack of experience. Further, biomarkers should predict patient outcome and direct optimal therapy for the individual patient. Unfortunately, current biomarkers still fall somewhat short of these grand expectations. While there has been some progress, it has generally been slow and in small steps. In this review, the newest set of glioma biomarkers: O(6) -methylguanine-DNA methyltransferase (MGMT) methylation, BRAF fusion and IDH1 mutation are discussed. MGMT methylation is well established as a prognostic/predictive marker for glioblastoma; however, technical questions regarding testing remain, it is not currently utilized widely in guiding patient management, and it has proven to be of no assistance in diagnostics. In contrast, BRAF fusion and IDH1 mutation analyses promise to be very helpful for classifying and grading gliomas, while their potential predictive value has yet to be established., (© 2010 The Authors; Brain Pathology © 2010 International Society of Neuropathology.)

This study presents an ultra-sensitive technique for the electrochemical detection of the mutated BRAF gene associated with papillary thyroid carcinomas (PTC). In the proposed approach, a biotinylated 30-nucleotides probe DNA was immobilized in a streptavidin-modified 96-well microtiter plate and the free active sites of the streptavidin were blocked using biotinylated bovine serum albumin (BSA). The biotinylated target DNA was then added and allowed to hybridize with the immobilized probe DNA for 30min. Subsequently, streptavidin-labeled gold nanoparticles were added, and a nanoparticle enlargement process was performed using gold ion solution and formaldehyde reductant. The gold particles were then dissolved in bromide and DNA hybridization detection process was performed using a square wave stripping voltammetry (SWSV) technique. The results indicated a stable SWSV response in differential detection between blank solution and target DNA solution with a concentration of 130aM. Moreover, the coefficient of determination (R(2)) of the semi-log plot of the SWSV response current against the target DNA concentration (0.52-1300aM) was found to be 0.9982. The detection limit was estimated to be 0.35aM (based on a signal-to-noise ratio of 3:1). This value was approximately three orders of magnitude lower than that obtained using the same method but without gold amplification process. Finally, the proposed approach is successful in differentiating between the mutant and wildtype BRAF sequences that are present in genuine 224-nucleotides DNA.

IQGAP1 modulates several cellular functions, including cell-cell adhesion, transcription, cytoskeletal architecture, and selected signaling pathways. We previously documented that IQGAP1 binds ERK and MAPK kinase (MEK) and regulates EGF-stimulated MEK and ERK activity. Here we characterize the interaction between IQGAP1 and B-Raf, the molecule immediately upstream of MEK in the Ras/MAPK signaling cascade. B-Raf binds directly to IQGAP1 in vitro and coimmunoprecipitates with IQGAP1 from cell lysates. Importantly, IQGAP1 modulates B-Raf function. EGF is unable to stimulate B-Raf activity in IQGAP1-null cells and in cells transfected with an IQGAP1 mutant construct that is unable to bind B-Raf. Interestingly, binding to IQGAP1 significantly enhances B-Raf activity in vitro. Our data identify a previously unrecognized interaction between IQGAP1 and B-Raf and suggest that IQGAP1 is a scaffold necessary for activation of B-Raf by EGF.

The activating point mutation of the BRAF gene, BRAF(T1799A), is the most common and specific genetic alteration in adult papillary thyroid carcinoma (PTC) and a possible marker of malignant potential of PTC. We have applied the PCR-RFLP method using fine-needle aspiration biopsy samples not only to our clinical practice but also to the international medical assistance effort around the Semipalatinsk Nuclear Testing Site in Kazakhstan. Seventy-seven cases (100 nodules) from Japan and 131 cases (137 nodules) from Kazakhstan were examined. There were 14 Japanese and 76 Kazakhstani cases of cytological malignant tumors from the examined samples. We detected 12 (85.7% of PTC) and 19 (25% of PTC) cases with BRAF(T1799A) among the Japanese and Kazakhstani cases, respectively. Of these cases, we found mutations in one cytologically "suspicious" case and even in two pathologically "benign" cases (after surgery in Kazakhstan). All of the BRAF mutation-positive cases, including those three, were confirmed as PTC by careful pathological examination, including immunohistochemical analysis. In summary, our PCR-RFLP method for BRAF(T1799A) detection using FNAB samples is useful not only for preoperative diagnosis of PTC but also as a complementary diagnostic tool for accurate pathological diagnosis, even after surgery.

Mutationally activated BRAF(V600E) (BRAF(VE)) is detected in approximately 6% of human malignancies and promotes sustained MEK1/2-ERK1/2 pathway activation. We have designed BRaf(CA) mice to express normal BRaf prior to Cre-mediated recombination after which BRaf(VE) is expressed at physiological levels. BRaf(CA) mice infected with an Adenovirus expressing Cre recombinase developed benign lung tumors that only rarely progressed to adenocarcinoma. Moreover, BRaf(VE)-induced lung tumors were prevented by pharmacological inhibition of MEK1/2. BRaf(VE) expression initially induced proliferation that was followed by growth arrest bearing certain hallmarks of senescence. Consistent with Ink4a/Arf and TP53 tumor suppressor function, BRaf(VE) expression combined with mutation of either locus led to cancer progression.

Purpose: Recently, activating mutations of the epidermal growth factor receptor (EGFR) gene were discovered in non-small cell lung cancers sensitive to gefitinib (ZD1839, an EGFR tyrosine kinase inhibitor) but not in gefitinib-resistant cancers. Abnormalities of EGFR and related pathways may have an effect on responsiveness of advanced colorectal cancer to combination chemotherapy with gefitinib., Experimental Design: We examined patients with previously untreated metastatic colorectal cancer, who were enrolled into two phase I/II trials of combination chemotherapy (irinotecan, leucovorin, and 5-fluorouracil) and daily oral gefitinib. We obtained paraffin tissue blocks of primary tumors from 31 patients, sequenced the EGFR, KRAS, and BRAF genes, and did immunohistochemistry for EGFR, phosphorylated AKT1, p53, p21, and p27., Results: Twelve (39%) of the 31 patients experienced a partial objective response to the therapy. A novel EGFR mutation in exon 18 (c.2170G>A, p.Gly724Ser) was identified in only one patient who did not experience an objective tumor response. EGFR immunohistochemistry was not predictive of responsiveness. In contrast, loss of p21 was associated with a higher response rate to therapy (P = 0.05). Moreover, the response rate among patients whose tumors maintained p21 expression and possessed a mutation in p53 was only 9% (1 of 11, P = 0.005). Overexpression of phosphorylated AKT1 also seemed to predict a trend towards resistance to the therapy., Conclusions: p21 expression in colorectal cancer, especially in combination with p53 mutation, is a predictor of resistance to the combination chemotherapy with gefitinib. Activating EGFR mutations are rare in colorectal cancer and do not seem to confer sensitivity to gefitinib and chemotherapy.

Cardiovascular complications are the leading cause of morbidity and mortality in autosomal dominant polycystic kidney disease. Pkd2+/- vascular smooth muscle cells (VSMCs) have an abnormal phenotype and defective intracellular Ca2+ ([Ca2+]i) regulation. We examined cAMP content in vascular smooth muscles from Pkd2+/- mice because cAMP is elevated in cystic renal epithelial cells. We found cAMP concentration was significantly increased in Pkd2+/- vessels compared with wild-type vessels. Furthermore, reducing the wild-type VSMC [Ca2+]i by Verapamil or BAPTA-AM significantly increased cellular cAMP concentration (mainly by phosphodiesterase [PDE] inhibition), the rate of VSMC proliferation (determined by direct cell counting, 3H-incorporation, FACS analysis of cells entering S phase, and quantitative Western PCNA and ERK1/2 analyses), and the rate of apoptosis (by Hoechst staining, FACS analysis of the Annexin-V positive cells, and quantitative Western Bax, cytochrome c, and activated caspase 9 and 3 analyses). The low [Ca2+]i induced VSMC proliferation was independent of cAMP/B-Raf signaling, while that of apoptosis was promoted by cAMP. In summary, Pkd2+/- VSMCs have elevated cAMP levels. This elevation can also be induced by reducing [Ca2+]i in wild-type VSMCs. The [Ca2+]i reduction and cAMP accumulation can cause an increase in both cellular proliferation and apoptosis, resembling Pkd mutant phenotype.