FR ¿El uso de fotoprotectores elimina el riesgo de desarrollar melanoma? / Do Sunscreens Eliminate the Risk of Melanoma?

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Assessment of BRAF V600E Status in Colorectal Carcinoma: Tissue-Specific Discordances between Immunohistochemistry and Sequencing.

Although sequencing provides the gold standard for identifying colorectal carcinoma with BRAF V600E mutation, immunohistochemistry (IHC) with the recently developed mouse monoclonal antibody VE1 for BRAF V600E protein has shown promise as a more widely available and rapid method. However, we identified anecdotal discordance between VE1 IHC and sequencing results and therefore analyzed VE1 staining by two different IHC methods (Leica Bond and Ventana BenchMark) in whole tissue sections from 480 colorectal carcinomas (323 BRAF wild-type, 142 BRAF V600E mutation, and 15 BRAF non-V600E mutation). We also compared the results with melanomas and papillary thyroid carcinomas (PTC). With the Bond method, among 142 BRAF V600E-mutated colorectal carcinomas, 77 (54%) had diffuse VE1 staining and 48 (33%) had heterogeneous staining, but 17 (12%) were negative. Among 323 BRAF wild-type colorectal carcinomas, 196 (61%) were negative, but 127 (39%) had staining, including 7 with diffuse staining. When positivity was defined as staining in ≥ 20% of tumor cells, VE1 IHC had sensitivity of 75% and specificity of 93% for BRAF V600E mutation. With the Ventana method, among 57 BRAF V600E-mutated colorectal carcinomas, 36 (63%) had diffuse VE1 staining, whereas 6 (11%) had no or weak (<20% of tumor cells) staining. Among 33 BRAF wild-type colorectal carcinomas, 16 (48%) had no or weak staining, whereas 15 (45%) had heterogeneous staining. In contrast with colorectal carcinoma, Bond and Ventana VE1 IHC in melanoma and PTC were highly concordant with sequencing results. We conclude that VE1 IHC produces suboptimal results in colorectal carcinoma and should not be used to guide patient management.

Elution profile analysis of SDS-induced subcomplexes by quantitative mass spectrometry.

Analyzing the molecular architecture of native multiprotein complexes via biochemical methods has so far been difficult and error prone. Protein complex isolation by affinity purification can define the protein repertoire of a given complex, yet, it remains difficult to gain knowledge of its substructure or modular composition. Here, we introduce SDS concentration gradient induced decomposition of protein complexes coupled to quantitative mass spectrometry and in silico elution profile distance analysis. By applying this new method to a cellular transport module, the IFT/lebercilin complex, we demonstrate its ability to determine modular composition as well as sensitively detect known and novel complex components. We show that the IFT/lebercilin complex can be separated into at least five submodules, the IFT complex A, the IFT complex B, the 14-3-3 protein complex and the CTLH complex, as well as the dynein light chain complex. Furthermore, we identify the protein TULP3 as a potential new member of the IFT complex A and showed that several proteins, classified as IFT complex B-associated, are integral parts of this complex. To further demonstrate EPASIS general applicability, we analyzed the modular substructure of two additional complexes, that of B-RAF and of 14-3-3-ε. The results show, that EPASIS provides a robust as well as sensitive strategy to dissect the substructure of large multiprotein complexes in a highly time- as well as cost-effective manner.

BRAF V600E mutation detection by immunohistochemistry in colorectal carcinoma.

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

Identification of BRAF inhibitors through in silico screening.

The BRAF protein kinase, a molecule in the RAS-RAF-MEK-ERK signaling pathway, is mutated to harbor elevated kinase activity in approximately 7% of human cancers, which makes it an important therapeutic target for inhibition. Several BRAF protein-kinase inhibitors have been developed through high-throughout screening in vitro; however, many of these compounds suffer from a lack of suitable kinase specificity and other chemotherapeutic properties. In silico screening has evolved as a powerful complimentary approach to protein-kinase inhibitor identification. Here we describe an in silico screen for BRAF inhibitors that leads to the identification of a series of purine-2,6-dione analogues with IC50 values in the single-digit micromolar range and with significant selectivity for BRAF over other representative protein kinases. The binding modes of these inhibitors to BRAF are analyzed through molecular docking to derive structure-activity relationships and to assist in the future development of more potent and more specific BRAF inhibitors.