Reflectance confocal microscopy features of BRAF V600E mutated thin melanomas detected by immunohistochemistry.

The classification of melanoma into four histological subtypes has been questioned regarding its clinical validity in providing relevant information for treatment for metastatic tumors. Specific genetic alterations are associated with particular clinical and histopathological features, suggesting that these could be helpful in refining existing melanoma classification schemes. We analyzed BRAF V600E mutated melanomas to explore the Reflectance confocal microscopy (RCM) utility as a screening aid in the evaluation of the most appropriate patients for genetic testing. Thus, 32 melanomas were assessed regarding their BRAF V600E mutational status. Experts blinded to dermoscopic images and V600E immunohistochemistry results evaluated RCM images regarding previously described melanoma features. BRAF positive melanomas were related to younger age (p = 0.035), invasive melanomas (p = 0.03) and to the presence of hiporreflective cells (p = 0.02), epidermal nests (p = 0.02), dermal-epidermal junction nests (p = 0.05), edged papillae (p = 0.05), and bright dots (p = 0.05), and to absence of junctional thickening due to isolated cells (p = 0.01) and meshwork (p = 0.02). This study can not characterize other mutations in the BRAF, because the immunohistochemistry is specific to the type V600E. The findings should encourage the genetic evaluation of BRAF mutation. This study highlights the potential of RCM as a supplementary tool in the screening of BRAF-mutated melanomas.

Epidermal growth factor receptor as an adverse survival predictor in squamous cell carcinoma of the penis.

Penile carcinoma (PC) is more frequent in underdeveloped countries, generally is diagnosed at an advanced stage when therapeutic options are restricted, and thus is associated with high morbidity/mortality rates. Recent studies have demonstrated clinical benefits with epidermal growth factor receptor (EGFR)-targeted therapy in patients with PC, although there is no test that provides accurate patient selection. The aim of the present study was to evaluate the prognostic value of EGFR gene and protein status in tumor samples from patients with primary penile squamous cell carcinoma. We assessed the expression of wild-type and 2 mutant EGFR isoforms (delA746-E750 and mL858R) by immunohistochemistry in 139 samples, of which 49 were also evaluated for EGFR copy number by fluorescence in situ hybridization (FISH). Positive immunohistochemical staining of wild-type and mutant EGFR was evidenced by complete and strong membranous staining. For FISH analysis, cases were considered unaltered, polysomic, or amplified, as determined by signals of the EGFR gene and chromosome 7. An independent cohort of 107 PC samples was evaluated for mutations in EGFR, KRAS, and BRAF. Protein overexpression was noted in nearly half of the cases and was associated with cancer recurrence (P=.004) and perineural invasion (P=.005). Expression of the 2 mutated EGFR isoforms was not observed. The FISH status was not associated with protein expression. Altered FISH (polysomy and gene amplification) was an independent risk factor for dying of cancer. Only 1 patient of 107 presented KRAS mutations, and no mutations of EGFR or BRAF were observed.

Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCß showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

HER2 e carcinomas gástricos: padrão de expressão e metodologias para detecção / HER2 and gastric carcinomas: expression pattern and methodologies for detection

O carcinoma gástrico (CG) representa um sério problema de saúde, sendo a segunda causa de morte relacionada ao câncer no mundo. No Brasil, é o quarto tipo de câncer mais comum entre os homens e o quinto mais comum entre as mulheres. O gene HER2 é um proto-oncogene localizado no cromossomo 17, codifica uma proteína transmembrana que pertence a família dos receptores HER e possue atividade tirosina quinase envolvida em vias de sinalizações relacionadas ao crescimento celular e diferenciação. Alterações no gene HER2 têm sido associadas com o desenvolvimento e progressões de várias neoplasias humanas. Em câncer de mama, a amplificação do HER2 é observada em cerca de 20% dos casos e o tratamento desses casos com o trastuzumabe, um anticorpo monoclonal, tem sido bastante efetivo. Em CG, a amplificação do HER2 tem sido observada em uma freqüência de 7% a 34% dos casos. Esta variabilidade é associada as diferentes metodologias utilizadas para a avaliação da expressão e/ou amplificação do HER2. Os resultados do estudo clinico ToGA mostrararm um aumento considerável nas taxas de sobrevida dos pacientes com CG avançados tratados com trastuzumabe e quiomioterapia comparado aqueles que foram tratados somente com quimioterapia. A correta identificação e seleção dos casos que irão se beneficiar desta terapia é o primeiro e mais importante passo para o sucesso do tratamento. O objetivo deste estudo foi avaliar a expressão proteica e o número de cópias do gene HER2 em 762 CG de pacientes submetidos a ressecção cirúrgica no A.C. Camargo Câncer Center...

Gastric carcinoma (GC) is a serious health problem and is the second leading cause of cancer-related death in the world. In Brazil, it is the fourth most common cancer among men and the fifth most common among women. The HER2 gene is a protooncogene located on chromosome 17, encodes a transmembrane protein which belongs to the HER family of receptors and has tyrosine kinase activity involved in signaling pathways related to cell growth and differentiation. Alterations in HER2 gene have been associated with the development and progression of many human cancers. In breast cancer, amplification of HER2 is observed in approximately 20% of cases, and these treatment of these cases with trastuzumab, a monoclonal antibody, has been quite effective. In GC, the amplification of HER2 has been observed at a frequency of 7% to 34% of cases. This variability is associated with the different methodologies used to evaluate the expression and / or amplification of HER2. The results of the clinical study ToGA showed a considerable increase in patient survival rates with advanced CG treated with trastuzumab and chemotherapy compared those treated with chemotherapy alone. The correct identification and selection of the cases that will benefit from this therapy is the first and most important step to successful treatment. The aim of this study was to evaluate the protein expression and the number of copies of the HER2 gene in 762 GC patients undergoing surgical resection at AC Camargo Cancer Center (São Paulo, Brazil) using the methods of immunohistochemistry and in situ hybridization (FISH and DDISH ) compared: different clones and manufacturers of anti-HER2 primary antibodies (4B5-Ventana clone, SP3-Neomarkers...

Use of a New Fibrin Sealant and Laser Irradiation in the Repair of Skull Defects in Rats

This study evaluated the osteogenic capacity of a new fibrin sealant (FS) combined with bone graft and laser irradiation in the bone repair. Defects were created in the skull of 30 rats and filled with autogenous graft and FS derived from snake venom. Immediately after implantation, low-power laser was applied on the surgical site. The animals were divided in: control group with autogenous graft (G1), autogenous graft and laser 5 J/cm2 (G2), autogenous graft and laser 7 J/cm2 (G3), autogenous graft and FS (G4), autogenous graft, FS and laser 5 J/cm2 (G5), autogenous graft, FS and laser 7 J/cm2 (G6). The animals were sacrificed 6 weeks after implant. Results showed absence of inflammatory infiltrate in the bone defect. New bone formation occurred in all groups, but it was most intense in G6. Thus, the FS and laser 7 J/cm2 showed osteoconductive capacity and can be an interesting resource to be applied in surgery of bone reconstruction.

Este estudo avaliou a capacidade osteogênica de um novo selante de fibrina (FS) associado com enxerto ósseo e irradiação laser no reparo ósseo. Defeitos foram criados no crânio de 30 ratos e preenchidos com enxerto autógeno e FS derivado do veneno de cobra. Imediatamente após implantação, foi aplicado laser de baixa potência na área cirúrgica. Os animais foram divididos em grupo controle com autógeno (G1), autógeno e laser 5 J/cm2 (G2), autógeno e laser 7J/cm2 (G3), autógeno e FS (G4), autógeno, FS e laser 5J/cm2 (G5), autógeno, FS e laser 7J/cm2 (G6). Os animais foram sacrificados 6 semanas após implante. Resultados mostraram ausência de infiltrado inflamatório no defeito ósseo. Neoformação óssea ocorreu em todos os grupos, entretanto, foi mais intenso em G6. Desta maneira, o FS e laser 7J/cm2 mostraram capacidade osteocondutiva e podem ser um interessante recurso a ser aplicado nas cirurgias de reconstrução óssea.

Use of a new fibrin sealant and laser irradiation in the repair of skull defects in rats.

This study evaluated the osteogenic capacity of a new fibrin sealant (FS) combined with bone graft and laser irradiation in the bone repair. Defects were created in the skull of 30 rats and filled with autogenous graft and FS derived from snake venom. Immediately after implantation, low-power laser was applied on the surgical site. The animals were divided in: control group with autogenous graft (G1), autogenous graft and laser 5 J/cm2 (G2), autogenous graft and laser 7 J/cm2 (G3), autogenous graft and FS (G4), autogenous graft, FS and laser 5 J/cm2 (G5), autogenous graft, FS and laser 7 J/cm2 (G6). The animals were sacrificed 6 weeks after implant. Results showed absence of inflammatory infiltrate in the bone defect. New bone formation occurred in all groups, but it was most intense in G6. Thus, the FS and laser 7 J/cm2 showed osteoconductive capacity and can be an interesting resource to be applied in surgery of bone reconstruction.

Hepatoblastomas and liver development: a study of cytokeratin immunoexpression in twenty-nine hepatoblastomas.

Hepatoblastomas (HBs) recapitulate liver development. It is possible that HBs result from malignant transformation of hepatic precursor cells, and they may reflect a blockage in normal development. Here we study the expression of cytokeratins (CKs) in order to delineate the immunoprofile and relationship with liver development, as well as vimentin and alphafetoprotein (AFP), of HBs. Immunohistochemistry was performed in a tissue microarray (TMA) containing representative areas of 18 HBs (fetal and/or embryonal and/or mesenchymal); we also reviewed 11 cases not included in the TMA. No cases stained for CKs 1, 5/6, 7, 10, 13, 15, 16, 20, and 34betaE12. CK8 stained 73.07% of fetal, 50% of embryonal, and 18% of mesenchymal areas. CK18 stained 100% of epithelial areas. CK19 staining was intense and diffuse in 100% of embryonal samples, but it was weaker in fetal areas (66.66%). AE1 stained epithelial areas in all cases, and it stained 29.41% of mesenchymal areas. AE3 stained 84.61% of embryonal and 60% of fetal components. AE1/AE3 showed stronger staining in embryonal (100%) than in fetal areas (76.92%). Vimentin staining was strong in embryonal (66.66%) and mesenchymal (84.61%) components but weak in fetal areas (8%). Alphafetoprotein was positive in only 20% of fetal and 70% of embryonal areas. Our results support the hypothesis that immunoexpression of HBs follows the stages of normal liver development. Embryonal areas look less differentiated, expressing vimentin and biliary epithelium CKs, whereas fetal areas display a more developed phenotype, similar to that of mature hepatocytes. These data aid in understanding the ontogenesis of HBs and may be used in histopathological diagnosis.

Pan-cytokeratin immunoexpression in Wilms' tumors: a simple approach for understanding tumor epithelial differentiation.

Wilms' tumor is one of the most common solid tumors in children and is an interesting model for understanding the pathogenesis of embryonal tumors. Cytokeratins are intracellular fibrous proteins present in tissue of epithelial origin. The immunoexpression of the pan-cytokeratin AE1AE3 was studied in paraffin-embedded tissue sections from 24 Wilms' tumors (12 with nephrogenic rests) and also tissue samples from 15 corresponding normal kidneys, to evaluate epithelial differentiation in the genesis of Wilms' tumor. We observed that the intensity of the expression of AE1AE3 in the epithelial component of Wilms' tumors directly correlated with the degree of maturity of the epithelial structures correspondent to the collecting ducts.

Pan-cytokeratin immunoexpression in Wilms' tumors: a simple approach for understanding tumor epithelial differentiation

Os tumores de Wilms são alguns dos mais freqüentes tumores sólidos da infância e reconhecidamente um modelo para a compreensão da patogênese dos tumores embrionários. As citoqueratinas são proteínas intracelulares presentes em tecidos de origem epitelial. Estudamos a imunoexpressão da pan-citoqueratina AE1AE3 em 24 tumores de Wilms, dentre ao quais 15 continham também tecidos renais não-neoplásicos e 12 apresentavam restos nefrogênicos em blocos de parafina, para avaliar a diferenciação epitelial no desenvolvimento dos tumores de Wilms. Observamos aumento na intensidade de expressão de AE1AE3 no componente epitelial dos tumores de Wilms diretamente relacionado ao grau de maturação das estruturas epiteliais correspondentes aos ductos coletores.