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1.
J Immunol Methods ; 442: 35-41, 2017 03.
Article in English | MEDLINE | ID: mdl-28093271

ABSTRACT

BACKGROUND: The Papanicolaou (Pap) screen has been successful in reducing cervical cancer; but exhibits low sensitivity when detecting cervical dysplasia. Use of molecular biomarkers in Pap tests may improve diagnostic accuracy. DESIGN: Monoclonal antibodies to Minichromosome Maintenance Protein 2 (MCM2) and DNA Topoisomerase II α (TOP2A) were selected for use in IHC based on their ability to differentiate normal from diseased cervical tissues in tissue microarrays. Enhanced Green Fluorescent Protein Western blot analysis was used to help identify binding epitopes specific to MCM2 and TOP2A antibody clones. Antibody affinity was determined by solution phase affinity measurement and immunohistochemistry was performed using high affinity MCM2 or TOP2A antibodies on serial histological sections. RESULTS: Antibody clones to MCM2 and TOP2A clones were selected based on their ability to detect over expression in abnormal cervical epithelia. In IHC, MCM2-27C5.6 and MCM2-26H6.19 demonstrated superior staining in abnormal cervical tissue over the MCM2-CRCT2.1 antibody. A combination of MCM2 and TOP2A antibodies showed greater staining when compared to staining with any of the antibodies alone on serial histological sections. Distinct linear epitopes were elucidated for each of the MCM2 and TOP2A clones. Affinity values (Kd) for MCM2 or TOP2A antibodies had a similar range. In a research study, the MCM2 and TOP2A (BD ProEx™ C) antibody cocktail showed increased epithelia staining with increasing dysplasia. The use of BD ProEx™ C in combination with H&E staining enhanced immunohistochemical discrimination of dysplastic and non-dysplastic FFPE cervical tissue specimens. CONCLUSIONS: BD ProEx™ C containing MCM2 and TOP2A antibodies showed strong specific nuclear staining that correlated with increased dysplasia and lesion severity. Enhanced performance of the antibodies was linked to their unique topography recognition. BD ProEx™ C incorporates antibodies that enhance detection of CIN2+ cervical disease.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens, Neoplasm/immunology , Cervix Uteri/immunology , DNA Topoisomerases, Type II/immunology , DNA-Binding Proteins/immunology , Immunohistochemistry , Minichromosome Maintenance Complex Component 2/immunology , S Phase , Tissue Array Analysis/methods , Uterine Cervical Dysplasia/diagnosis , Uterine Cervical Neoplasms/diagnosis , Biopsy , Blotting, Western , Cell Nucleus/enzymology , Cell Nucleus/immunology , Cell Nucleus/pathology , Cervix Uteri/enzymology , Cervix Uteri/pathology , Epitope Mapping/methods , Epitopes , Female , Humans , Poly-ADP-Ribose Binding Proteins , Predictive Value of Tests , Severity of Illness Index , Uterine Cervical Dysplasia/enzymology , Uterine Cervical Dysplasia/immunology , Uterine Cervical Dysplasia/pathology , Uterine Cervical Neoplasms/enzymology , Uterine Cervical Neoplasms/immunology , Uterine Cervical Neoplasms/pathology
2.
J Mol Diagn ; 11(2): 117-30, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19225135

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-Regulation
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