RESUMO
BACKGROUND: There is a clear need for assays that can predict the risk of metastatic prostate cancer following curative procedures. Importantly these assays must be analytically robust in order to provide quality data for important clinical decisions. DNA microarray based gene expression assays measure several analytes simultaneously and can present specific challenges to analytical validation. This study describes the analytical validation of one such assay designed to predict metastatic recurrence in prostate cancer using primary formalin fixed paraffin embedded tumour material. METHODS: Accuracy was evaluated with a method comparison study between the assay development platform (Prostate Disease Specific Array) and an alternative platform (Xcel™ microarray) using 50 formalin-fixed, paraffin-embedded prostate cancer patient samples. An additional 70 samples were used to establish the assay reportable range. Determination of assay precision and sensitivity was performed on multiple technical replicates of three prostate cancer samples across multiple variables (operators, days, runs, reagent lots, and equipment) and RNA/cDNA inputs respectively using the appropriate linear mixed model. RESULTS: The overall agreement between the development and alternative platform was 94.7% (95% confidence interval, 86.9-98.5%). The reportable range was determined to be 0.150 to 1.107 for core needle biopsy samples and - 0.214 to 0.844 for radical prostatectomy samples. From the precision study, the standard deviations for assay repeatability and reproducibility were 0.032 and 0.040 respectively. The sensitivity study demonstrated that a total RNA input and cDNA input of 50 ng and 3.5 µg respectively was conservative. CONCLUSION: The Metastatic Assay was found to be highly reproducible and precise. In conclusion the studies demonstrated an acceptable analytical performance for the assay and support its potential use in the clinic.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/patologia , Perfilação da Expressão Gênica/normas , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/normas , Inclusão em Parafina , Prognóstico , Prostatectomia , Neoplasias da Próstata/genética , Controle de Qualidade , Reprodutibilidade dos TestesAssuntos
Pesquisa Biomédica/organização & administração , Prestação Integrada de Cuidados de Saúde/organização & administração , Sistema de Registros , Centros Médicos Acadêmicos , Comportamento Cooperativo , Humanos , Irlanda do Norte , Patologia Molecular/organização & administração , Avaliação de Resultados da Assistência ao Paciente , Assistência Centrada no Paciente , Medicina de Precisão , Parcerias Público-Privadas/organização & administraçãoRESUMO
Breast cancer-associated 1 (BRCA1) plays an important role in breast cancer initiation and progression through its functions in the cell cycle and DNA repair processes; however, its role in metastatic development in human breast cancer is still poorly understood. We have previously shown that osteopontin (OPN) expression was suppressed by wild-type BRCA1 (Wt.BRCA1) and that a natural mutant allele of BRCA1 (Mut.BRCA1) diminished the effect of Wt.BRCA1 on OPN in vitro. In this study, we show that while Wt.BRCA1 suppresses OPN-induced metastasis in a rat syngeneic system, Mut.BRCA1 enhances the development of metastasis through OPN, suggesting that OPN and BRCA1 work closely to regulate metastatic development in the rat. To test whether these findings are relevant to human breast cancer, we have investigated the relationship between BRCA1, OPN, and metastatic properties in human breast cancer-related cells. Using western blot analysis, we show that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN protein expression; and in parallel that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN-mediated in vitro properties associated with the metastatic state in both MCF-7 and MDA MB435s cells. Overall, these results suggest that Mut.BRCA1 can elicit some of the changes involved in metastatic progression in human breast cancer via the overexpression of OPN.
Assuntos
Neoplasias da Mama/patologia , Transformação Celular Neoplásica , Genes BRCA1 , Mutação em Linhagem Germinativa , Osteopontina/fisiologia , Animais , Neoplasias da Mama/genética , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Invasividade Neoplásica , Osteopontina/genética , Ratos , Ratos Wistar , TransfecçãoRESUMO
BRCA1 is a well described breast cancer susceptibility gene thought to be involved primarily in DNA repair. However, mutation within the BRCA1 transcriptional domain is also implicated in neoplastic transformation of mammary epithelium, but responsible mechanisms are unclear. Here we show in a rat mammary model system that wild type (WT) BRCA1 specifically represses the expression of osteopontin (OPN), a multifunctional estrogen-responsive gene implicated in oncogenic transformation, particularly that of the breast. WT.BRCA1 selectively binds OPN-activating transcription factors estrogen receptor alpha, AP-1, and PEA3, inhibits OPN promoter transactivation, and suppresses OPN mRNA and protein both from an endogenous gene and a relevant model inducible gene. WT.BRCA1 also inhibits OPN-mediated neoplastic transformation characterized by morphology change, anchorage-independent growth, adhesion to fibronectin, and invasion through Matrigel. A mutant BRCA1 allele (Mut.BRCA1) associated with familial breast cancer lacks OPN suppressor effects, binds to WT.BRCA1, and impedes WT.BRCA1 suppression of OPN. Stable transfection of rat breast tumor cell lines with Mut.BRCA1 dramatically up-regulates OPN protein and induces anchorage independent growth. In human primary breast cancer, BRCA1 mutation is significantly associated with OPN overexpression. Taken together, these data suggest that BRCA1 mutation may confer increased tissue-specific cancer risk, in part by disruption of BRCA1 suppression of OPN gene transcription.
