RESUMEN
BACKGROUND: The importance of definitive histological subclassification has increased as drug trials have shown benefit associated with histology in nonsmall-cell lung cancer (NSCLC). The acuity of this problem is further exacerbated by the use of minimally invasive cytology samples. Here we describe the development and validation of a 4-protein classifier that differentiates primary lung adenocarcinomas (AC) from squamous cell carcinomas (SCC). METHODS: Quantitative immunofluorescence (AQUA) was employed to measure proteins differentially expressed between AC and SCC followed by logistic regression analysis. An objective 4-protein classifier was generated to define likelihood of AC in a training set of 343 patients followed by validation in 2 independent cohorts (n = 197 and n = 235). The assay was then tested on 11 cytology specimens. RESULTS: Statistical modeling selected thyroid transcription factor 1 (TTF1), CK5, CK13, and epidermal growth factor receptor (EGFR) to generate a weighted classifier and to identify the optimal cutpoint for differentiating AC from SCC. Using the pathologist's final diagnosis as the criterion standard, the molecular test showed a sensitivity of 96% and specificity of 93%. Blinded analysis of the validation sets yielded sensitivity and specificity of 96% and 97%, respectively. Our assay classified the cytology specimens with a specificity of 100% and sensitivity of 87.5%. CONCLUSIONS: Molecular classification of NSCLC using an objective quantitative test can be highly accurate and could be translated into a diagnostic platform for broad clinical application.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/clasificación , Neoplasias Pulmonares/clasificación , Proteínas/análisis , Adenocarcinoma/química , Adenocarcinoma/clasificación , Adenocarcinoma del Pulmón , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/clasificación , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Modelos Logísticos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Análisis de Matrices TisularesRESUMEN
Detachment of epithelial cells from matrix or attachment to an inappropriate matrix engages an apoptotic response known as anoikis, which prevents metastasis. Cellular sensitivity to anoikis is compromised during the oncogenic epithelial-to-mesenchymal transition (EMT), through unknown mechanisms. We report here a pathway through which EMT confers anoikis resistance. NRAGE (neurotrophin receptor-interacting melanoma antigen) interacted with a component of the E-cadherin complex, ankyrin-G, maintaining NRAGE in the cytoplasm. Oncogenic EMT downregulated ankyrin-G, enhancing the nuclear localization of NRAGE. The oncogenic transcriptional repressor protein TBX2 interacted with NRAGE, repressing the tumor suppressor gene p14ARF. P14ARF sensitized cells to anoikis; conversely, the TBX2/NRAGE complex protected cells against anoikis by downregulating this gene. This represents a novel pathway for the regulation of anoikis by EMT and E-cadherin.
Asunto(s)
Anoicis/fisiología , Cadherinas/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Transducción de Señal/fisiología , Animales , Ancirinas/genética , Ancirinas/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Cadherinas/genética , Línea Celular Tumoral , Células Epiteliales/citología , Células Epiteliales/fisiología , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Proteína p14ARF Supresora de Tumor/genética , Proteína p14ARF Supresora de Tumor/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence.
