ABSTRACT
Knowledge of estrogen receptor (ER) and progesterone receptor (PR) status has been critical in the evolution of modern targeted therapy of breast cancer and remains essential for making informed therapeutic decisions. Recently, growth factor receptor HER2/neu (ERBB2) status has made it possible to provide another form of targeted therapy linked to the overexpression of this protein. Presently, pathologists determine the receptor status in formalin-fixed, paraffin-embedded sections using subjective, semiquantitative immunohistochemistry (IHC) assays and quantitative fluorescence in situ hybridization for HER2. We developed a single-tube multiplex TaqMan (mERPR+HER2) assay to quantitate mRNA levels of ER, PR, HER2, and two housekeeping genes for breast cancer formalin-fixed, paraffin-embedded sections. Using data from the discovery sample sets, we evaluated IHC-status-dependent cutoff-point and IHC-status-independent clustering methods for the classification of receptor status and then validated these results with independent sample sets. Compared with IHC-status, the accuracies of the mERPR+HER2 assay with the cutoff-point classification method were 0.98 (95% CI: 0.97-1.00), 0.92 (95% CI: 0.88-0.95), and 0.97 (95% CI: 0.95-0.99) for ER, PR, and HER2, respectively, for the validation sets. Furthermore, the areas under the receiver operating-characteristic curves were 0.997 (95% CI: 0.994-1.000), 0.967 (95% CI: 0.949-0.985), and 0.968 (95% CI: 0.915-1.000) for ER, PR, and HER2, respectively. This multiplex assay provides a sensitive and reliable method to quantitate hormonal and growth factor receptors.
Subject(s)
Breast Neoplasms/diagnosis , Molecular Diagnostic Techniques/methods , RNA, Messenger/analysis , Receptor, ErbB-2/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/genetics , Female , Formaldehyde/chemistry , Humans , Paraffin Embedding , RNA, Messenger/genetics , Reproducibility of Results , Sensitivity and Specificity , Tissue Fixation , Up-RegulationABSTRACT
UNLABELLED: Responsiveness to hepatitis C virus (HCV) therapy depends on viral and host factors. Our aim was to assess sustained virologic response (SVR)-associated early gene expression in patients with HCV receiving pegylated interferon-alpha2a (PEG-IFN-alpha2a) or PEG-IFN-alpha2b and ribavirin with the duration based on genotypes. Blood samples were collected into PAXgene tubes prior to treatment as well as 1, 7, 28, and 56 days after treatment. From the peripheral blood cells, total RNA was extracted, quantified, and used for one-step reverse transcription polymerase chain reaction to profile 154 messenger RNAs. Expression levels of messenger RNAs were normalized with six "housekeeping" genes and a reference RNA. Multiple regression and stepwise selection were performed to assess differences in gene expression at different time points, and predictive performance was evaluated for each model. A total of 68 patients were enrolled in the study and treated with combination therapy. The results of gene expression showed that SVR could be predicted by the gene expression of signal transducer and activator of transcription-6 (STAT-6) and suppressor of cytokine signaling-1 in the pretreatment samples. After 24 hours, SVR was predicted by the expression of interferon-dependent genes, and this dependence continued to be prominent throughout the treatment. CONCLUSION: Early gene expression during anti-HCV therapy may elucidate important molecular pathways that may be influencing the probability of achieving virologic response.
