ABSTRACT
OBJECTIVES: The benefits of liquid-based cytology (LBC) in routine cervical cancer screening are often associated with the availability of instrumented platforms and economic considerations. A low-cost alternative to LBC in low-volume settings remains an unmet need. METHODS: A multisite evaluation of the BD SurePath (SurePath) LBC Direct to Slide (DTS) method was conducted. The DTS preparations were evaluated across 3 sites. Cytology features for DTS preparation included predetermined thresholds for total cellularity, cell distribution, cellular preservation, and stain quality. Rare event detection was evaluated using SiHa cells spiked into pools from negative cytology specimens. Concordance between Bethesda classification results was evaluated for SurePath LBC and DTS methods using routinely collected SurePath specimens in a split-sample study design. RESULTS: The DTS specimens met criteria for total cellularity, cell distribution, cellular preservation, and stain quality in more than 98% of all cases. Rare event detection was observed with an average detection of 5 SiHa cells per 2 mL of specimen. Concordant cervical cytology classifications were observed between SurePath LBC and DTS methods. CONCLUSIONS: The results demonstrate that the DTS process is suitable for routine cervical cytology evaluation. The procedure is reproducible and detected abnormal cervical cells in concordance with standard SurePath LBC preparation.
ABSTRACT
BACKGROUND: Screening efforts using the Papanicolaou test have significantly reduced the incidence of cervical cancer in countries with an active screening program. However, this test does not accurately identify all abnormal cases. Significant effort has been expended investigating molecular markers that could improve the sensitivity and specificity of detection of high-grade disease. In this study, we describe the selection and characterization of a set of antibodies to the minichromosome maintenance proteins MCM6 and MCM7 that highlight cervical disease in an immunoassay. METHODS: Antibodies to MCM6 or MCM7 proteins were identified from hybridoma clones screened against tissue microarrays containing different grades of diseased cervical tissue along with normal controls. We determined epitopes by western blotting against nested truncations of either the MCM6 or MCM7 proteins fused to GFP protein. We also determined specificity by western blotting against a panel of major MCM proteins (MCM2-MCM8). Affinity to recombinant antigen and epitope-only peptides was determined using solution-phase binding and determination of free antibody concentration by ELISA. Optimization studies resulted in the selection of antibodies specific to MCM6 and MCM7 for use in immunocytochemistry (ICC) with cervical cytology samples. RESULTS: Four antibodies were identified that demonstrated strong nuclear staining of abnormal cervical epithelial cells in immunohistochemistry (IHC) of cervical biopsies with minimal background staining of normal cervical tissues. Of these four antibody clones, 2E6.7 (MCM7) and 9D4.3 (MCM6) were chosen for further study. Linear epitopes of at most 12 amino acids were identified and verified by binding to epitope-only peptides. Affinities of at least 4×10(-9) M were determined for these two antibodies and both were found to be specific for their respective antigens by western blotting. Clones 9D4.3 and 2E6.7 were also determined to stain abnormal cells in high-grade squamous intraepithelial lesion cytology samples, with minimal background staining of normal cells. CONCLUSION: In this study, we present a method for selecting antibodies that perform well in IHC and ICC applications and characterize two antibodies generated by this method that effectively stain abnormal cells in cervical cancer tissue and cervical cytology samples.
Subject(s)
Antibodies, Monoclonal/analysis , Blotting, Western/methods , Cell Cycle Proteins/immunology , DNA-Binding Proteins/immunology , Epitope Mapping/methods , Immunohistochemistry/methods , Nuclear Proteins/immunology , Uterine Cervical Dysplasia/chemistry , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Antibody Specificity , Biopsy , Cell Cycle Proteins/analysis , DNA-Binding Proteins/analysis , Enzyme-Linked Immunosorbent Assay , Epitopes/analysis , Epitopes/chemistry , Epitopes/immunology , Female , Humans , Minichromosome Maintenance Complex Component 6 , Minichromosome Maintenance Complex Component 7 , Molecular Sequence Data , Nuclear Proteins/analysis , Uterine Cervical Dysplasia/immunology , Uterine Cervical Dysplasia/pathologyABSTRACT
Molecular diagnostic adjuncts could improve the specificity of cervical cancer screening. Since persistent infection with human papillomavirus (HPV) is found in virtually 100% of cervical cancer cases, testing for markers of HPV integration may have a role in identifying underlying high-grade lesions in patients with low-grade cytologic abnormalities. Several proteins associated with the cell cycle are known to be affected by HPV integration into the host's DNA. Immunocytochemical identification of these upregulated proteins can assist in the identification of small numbers of pre-neoplastic or neoplastic cells in routine cytologic sampling.
