RESUMO
Interest is increasing in biological scaffolds for tissue regeneration such as extracellular matrix membranes, developed through soft tissue decellularization. Extracellular matrix membranes were developed to heal different tendon and soft tissue lesions that are very frequent in the general population with high health-care costs and patient morbidity. The aim of this research was to evaluate a human dermal matrix (HDM) decellularized by a chemico-physical method. A primary culture of rat tenocytes was performed: tenocytes were seeded on HDM samples and on polystyrene wells as controls (CTR). Cell viability and synthetic activity were evaluated at 3 and 7 days. An in vitro microwound model was used to evaluate HDM bioactivity: after tenocyte expansion, artificial wounds were created, HDM extracts were added, and closure time and decorin synthesis were monitored histomorphometrically at 1, 4, 24, and 72 hr. A significant higher amount of collagen I was observed when cells were cultured on HDM in comparison with that on CTR (3 days: p < 0.0001; 7 days: p < 0.05). In HDM group, fibronectin synthesis was significantly higher at both experimental times (p < 0.0001). At 3 days, proteoglycans and transforming growth factor-ß1 releases were significantly higher on HDM (p < 0.0001 and p < 0.005, respectively). The artificial microwound closure time and decorin expression were significantly enhanced by the addition of 50% HDM extract (p < 0.05). In vitro data showed that the decellularization technique enabled the development of a matrix with adequate biological and biomechanical properties.
Assuntos
Derme/citologia , Estudos de Avaliação como Assunto , Lacerações/patologia , Lacerações/terapia , Lesões do Manguito Rotador , Animais , Sobrevivência Celular , Células Cultivadas , Decorina/metabolismo , Humanos , Ratos , Ratos Sprague-Dawley , Manguito Rotador/patologia , Suturas , CicatrizaçãoRESUMO
Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.
Assuntos
Neoplasias Encefálicas/secundário , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Ativação Enzimática , Receptores ErbB/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/genética , Taxa de SobrevidaRESUMO
PURPOSE: Activating mutations within the tyrosine kinase domain of epidermal growth factor receptor (EGFR) are found in approximately 10% to 20% of non-small-cell lung cancer (NSCLC) patients and are associated with response to EGFR inhibitors. The most common NSCLC-associated EGFR mutations are deletions in exon 19 and L858R mutation in exon 21, together accounting for 90% of EGFR mutations. To develop a simple, sensitive, and reliable clinical assay for the identification of EGFR mutations in NSCLC patients, we generated mutation-specific rabbit monoclonal antibodies against each of these two most common EGFR mutations and aimed to evaluate the detection of EGFR mutations in NSCLC patients by immunohistochemistry. EXPERIMENTAL DESIGN: We tested mutation-specific antibodies by Western blot, immunofluorescence, and immunohistochemistry. In addition, we stained 40 EGFR genotyped NSCLC tumor samples by immunohistochemistry with these antibodies. Finally, with a panel of four antibodies, we screened a large set of NSCLC patient samples with unknown genotype and confirmed the immunohistochemistry results by DNA sequencing. RESULTS: These two antibodies specifically detect the corresponding mutant form of EGFR by Western blotting, immunofluorescence, and immunohistochemistry. Screening a panel of 340 paraffin-embedded NSCLC tumor samples with these antibodies showed that the sensitivity of the immunohistochemistry assay is 92%, with a specificity of 99% as compared with direct and mass spectrometry-based DNA sequencing. CONCLUSIONS: This simple assay for detection of EGFR mutations in diagnostic human tissues provides a rapid, sensitive, specific, and cost-effective method to identify lung cancer patients responsive to EGFR-based therapies.
Assuntos
Anticorpos Monoclonais , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutação/imunologia , Animais , Bioensaio , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/secundário , Análise Mutacional de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Imunoglobulina G/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Coelhos , Sensibilidade e Especificidade , Deleção de Sequência , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
To analyze the potential diagnostic relevance of free plasma DNA (FPDNA), we enrolled 64 patients with localized prostate cancer (CaP). FPDNA was quantified by real-time polymerase chain reaction assessment of the HTERT gene in blood samples from 64 patients with CaP and 45 healthy males. Methylation of the GSTP1 gene was used to confirm the neoplastic origin of FPDNA in selected cases. The mean +/- SD levels of FPDNA were higher in patients with CaP (15.4 +/- 10.9 ng/mL) than in control subjects (5.5 +/- 3.5 ng/mL; P <.001). By using the best cutoff value, the sensitivity of the test was 80%, the specificity was 82%, the area under the receiver operating characteristic curve, 0.881. High FPDNA values were significantly associated with pathologic T3 stage (P = . 035). Methylation of the GSTP1 gene was found in 4 (25%) of 16 FPDNA samples and 15 (94%) of 16 tissue samples. Quantification of FPDNA discriminates between patients with CaP and healthy subjects and correlates with pathologic tumor stage. FPDNA is a candidate biomarker for early diagnosis and monitoring of CaP.
Assuntos
Biomarcadores Tumorais/sangue , DNA de Neoplasias/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnóstico , Idoso , Área Sob a Curva , Metilação de DNA , Glutationa S-Transferase pi/genética , Humanos , Masculino , Pessoa de Meia-Idade , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Telomerase/sangue , Telomerase/genéticaRESUMO
Gene expression profiling has identified several potentially useful gene signatures for predicting outcome or for selecting targeted therapy. However, these signatures have been developed in fresh or frozen tissue, and there is a need to apply them to routinely processed samples. Here, we demonstrate the feasibility of a potentially high-throughput methodology combining automated in situ hybridization with quantum dot-labeled oligonucleotide probes followed by spectral imaging for the detection and subsequent deconvolution of multiple signals. This method is semiautomated and quantitative and can be applied to formalin-fixed, paraffin-embedded tissues. We have combined dual in situ hybridization with immunohistochemistry, enabling simultaneous measurement of gene expression and cell lineage determination. The technique achieves levels of sensitivity and specificity sufficient for the potential application of known expression signatures to biopsy specimens in a semiquantitative way, and the semiautomated nature of the method enables application to high-throughput studies.
Assuntos
Linhagem da Célula , Perfilação da Expressão Gênica/métodos , Hibridização In Situ/métodos , Técnicas de Diagnóstico Molecular/métodos , Pontos Quânticos , Animais , DNA Complementar/genética , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica/métodos , Camundongos , Sondas de Oligonucleotídeos , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Uveal melanoma (UM) has a high propensity to develop hepatic metastases. We sought to define the mechanisms required for preferential liver homing and to understand further the biologic behavior of this disease. The Met tyrosine kinase receptor and its ligand hepatocyte growth factor are expressed in hepatocytes. We therefore considered Met/hepatocyte growth factor signaling as a candidate migration/growth factor for UM cells. We further explored the relationship between c-Met and other growth factor receptors prevalent in the liver and their roles in UM metastatic potential. UM cell lines were evaluated for c-Met, epidermal growth factor receptor (EGFR), and insulin-like growth factor-1R (IGF-1R) expression by immunoblotting, and gene amplification by comparative genomic hybridization and fluorescence in-situ hybridization. High c-Met, phosphorylated c-Met, and EGFR expression were noted in two of nine cell lines, independent of IGF-1R levels. Knockdown of c-Met decreased proliferation of high c-Met-expressing UM cells but did not induce apoptosis. Selective inhibitors of EGFR and IGF-1R decreased proliferation and induced apoptosis in UM cells regardless of the expression levels of c-Met, EGFR, and IGF-1R. Although c-Met, EGFR, and IGF-1R play proliferative roles, EGFR and IGF-1R are also critical for UM cell survival. High c-Met/EGFR-expressing cell lines possessed the greatest migration potential. c-Met knockdown and selective inhibitors of c-Met, EGFR, and IGF-1R revealed independent contribution of these receptors to migration. UM can be categorized by levels of c-Met and EGFR expression which are associated with migratory/invasiveness responses to soluble factors present at high levels in the liver. This provides biologic relevance for UM clinical behavior with potential therapeutic implications.
Assuntos
Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento Insulin-Like I/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Hibridização Genômica Comparativa , Hepatócitos/citologia , Humanos , Hibridização in Situ Fluorescente , Neoplasias Hepáticas/patologia , Modelos Biológicos , Invasividade Neoplásica , Metástase NeoplásicaRESUMO
In a subset of lung adenocarcinomas, the epidermal growth factor receptor (EGFR) is activated by kinase domain mutations and/or gene amplification, but the interaction between the two types of abnormalities is complex and unclear. For this study, we selected 99 consecutive never-smoking women of East Asian origin with lung adenocarcinomas that were characterized by histologic subtype. We analyzed EGFR mutations by PCR-capillary sequencing, EGFR copy number abnormalities by fluorescence and chromogenic in situ hybridization and quantitative PCR, and EGFR expression by immunohistochemistry with both specific antibodies against exon 19 deletion-mutated EGFR and total EGFR. We compared molecular and clinicopathologic features with disease-free survival. Lung adenocarcinomas with EGFR amplification had significantly more EGFR exon 19 deletion mutations than adenocarcinomas with disomy, and low and high polysomy (100% versus 54%, P = 0.009). EGFR amplification occurred invariably on the mutated and not the wild-type allele (median mutated/wild-type ratios 14.0 versus 0.33, P = 0.003), was associated with solid histology (P = 0.008), and advanced clinical stage (P = 0.009). EGFR amplification was focally distributed in lung cancer specimens, mostly in regions with solid histology. Patients with EGFR amplification had a significantly worse outcome in univariate analysis (median disease-free survival, 16 versus 31 months, P = 0.01) and when adjusted for stage (P = 0.027). Lung adenocarcinomas with EGFR amplification have a unique association with exon 19 deletion mutations and show distinct clinicopathologic features associated with a significantly worsened prognosis. In these cases, EGFR amplification is heterogeneously distributed, mostly in areas with a solid histology.
Assuntos
Adenocarcinoma/genética , Receptores ErbB/genética , Amplificação de Genes , Neoplasias Pulmonares/genética , Mutação/genética , Fumar/genética , Adenocarcinoma/patologia , Adenocarcinoma Bronquioloalveolar/genética , Adenocarcinoma Bronquioloalveolar/patologia , Adulto , Idoso , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Feminino , Dosagem de Genes , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Taxa de Sobrevida , Proteínas ras/genéticaRESUMO
Prostate cancer (PCa) is a heterogeneous disease with regard to molecular alterations and clinical course. The investigation of genetic alterations associated with PCa pathogenesis is highly challenging. Genome-wide analyses and epidemiological studies have identified only a handful of candidate genes possibly associated with hereditary or sporadic PCa. Cancer cells often rely for survival on common biochemical pathways such as enhanced anaerobic glycolysis and lipogenesis. The lipogenic enzyme fatty acid synthase seems to play a crucial part in PCa by conferring growth and survival advantages to cancer cells. We summarize the current understanding of the molecular events in PCa, and highlight the importance of altered lipid metabolism in the development and progression of prostate malignancy.