Analytical and Clinical Sample Performance Characteristics of the Onclarity Assay for the Detection of Human Papillomavirus.

The objective of this study was to determine the result reproducibility and performance of the BD Onclarity human papillomavirus (HPV) assay (Onclarity) on the BD Viper LT platform using both contrived and clinical specimens. Reproducibility was assessed in BD SurePath liquid-based cytology (LBC) medium (SurePath) using contrived panels (HPV genotype 16 [HPV16] positive, HPV18 positive, or HPV45 positive) or clinical specimens (HPV16, -18, -31, -33/58, -45, or -52 positive or HPV negative). In addition, specimens from 3,879 individuals from the Onclarity trial were aliquoted prior to or following cytology processing and tested for HPV. Finally, specimens were collected using either the Cervex-Brush or Cytobrush (or Cytobrush/spatula) for comparison of HPV results. Contrived specimens showed >95% concordance with the expected results, and pooled clinical specimens had standard deviations and coefficients of variation ranging from 0.87 to 1.86 and 2.9% to 5.6%, respectively. For precytology and postcytology aliquot analyses, specimens showed >98.0% overall agreement and mean differences in cycle threshold (CT ) scores for HPV ranging from -0.07 to 0.31. Positivity rates were close between the Cervex-Brush and Cytobrush/spatula for all age groups tested. Onclarity results are reproducible and reliable, regardless of sample collection before or after cytology aliquoting. Onclarity performs well regardless of the method of specimen collection (Cervex-Brush or Cytobrush/spatula) for cervical cancer screening.

Fundamentals of pharmacology and applications in pharmacogenetics.

This article provides an introduction to the fundamental principles of pharmacokinetics (PK) and pharmacodynamics (PD) as they apply to understanding the application of pharmacogenetics (PGx) in a clinical setting. PGx establishes connections between the disciplines of pharmacology and genetics. As such, one functional component of PGx involves establishing relationships between phenotypes and genotypes with respect to predicting the response of medications in individual patients. The article begins by describing each of the concepts, followed by discussing the clinical utility of PGx and pharmacodynamics in a laboratory medicine setting; it then makes a link with the evolving field of PGx from the perspective of clinical laboratory medicine. Laboratory medicine serves as a catalyst for transitioning PGx into clinical settings, and as such, the article concludes by describing the future role of clinical laboratories in the application of PGx to patient care.

Comparative proteomic analysis of matched primary and metastatic melanoma cell lines.

Identification of the biochemical pathways involved in the transformation from primary to metastatic melanoma is an area under intense investigation. A 2DE proteomics approach has been applied herein to the matched patient primary and metastatic melanoma cell lines WM-115 and WM-266-4, respectively, to better understand the processes that underlie tumor progression. Image analysis between samples aligned 470 common gel spots. Quantitative gel analysis indicated 115 gel spots of greater intensity in the metastatic line compared with the primary one, leading to the identification of 131 proteins via database searching of nano-LC-ESI-Q-TOF-MS/MS data. This more than tripled the number of proteins previously shown to be of higher abundance during melanoma progression. Also observed were 22 gel spots to be of lesser intensity in the metastatic line with respect to the primary one. Of these gel spots 15 proteins could be identified. Numerous proteins from both groups had not been reported previously to participate in melanoma progression. Further analysis of one protein, cyclophilin A, confirmed that this protein is expressed at higher levels in metastatic melanoma compared with primary melanoma and normal fibroblasts. Overall, this study expands our knowledge of protein modulation during melanoma stages, and suggests new targets for inhibitor development.

Mammalian cardenolides in cancer prevention and therapeutics.

Digoxin-like immunoreactive factor (DLIF) and ouabain-like factor (OLF) are the mammalian counterparts to the plant-derived cardiotonic steroids digoxin and ouabain. Compelling evidence indicates that the cardiotonic steroids may have anticancer properties. Recent evidence indicates that low (nanomolar) concentrations of DLIF selectively induce cell death in transformed cells, while sparing normal cells, and is even more potent than the plant-derived compounds. The discovery that these endogenous molecules may play a role in the regulation of cancer cell proliferation provides a potentially new paradigm for the physiologic role of DLIF and OLF. In addition, the possible use of digoxin itself as a therapeutic agent in cancer has been explored, and evidence suggests that its conversion to dihydrodigoxin may be involved in regulating anticancer activity. The mechanism(s) for the pro-apoptotic property of these compounds is not known. In this brief review, we will discuss the proposed mechanism of action of digoxin, ouabain, DLIF, and OLF as anticancer compounds and discuss the effects that metabolic conversion to their dihydro-derivatives may have on this activity. From the perspective of therapeutic drug monitoring, these findings suggest some potential new challenges in the need to measure concentrations of digoxin and dihydrodigoxin as well as their endogenous counterparts DLIF and OLF in serum.

Proteolytic profiling of the extracellular matrix degradome.

The profiling of protein function is one of the most challenging scientific tasks in the postgenomic age. Traditional protein expression methodologies have focused only on the quantification of proteins under varying conditions or pathologies. Determining the functional differences between protein populations allows for a more accurate view of the outcomes in normal vs diseased proteomes. Because the presence or absence of a protein's function can affect its complex surroundings (consisting of multiple other proteins and substrates), the study of proteome functionality yields information on protein-protein interactions, amplification cascades, signaling pathways, and posttranslational modifications. Of significant interest are proteinases, as proteolysis is responsible for tight regulation of various cellular and tissue processes. Proteinase activities, or lack there of, alter the proteome makeup by regulating other proteins or by generating cleavage products. This chapter describes current proteolytic profiling technologies using activity or target-based formats. In particular, the analysis of collagenolytic matrix metalloproteinase activity using fluorogenic triple-helical substrates is discussed.

Differential modulation of human melanoma cell metalloproteinase expression by alpha2beta1 integrin and CD44 triple-helical ligands derived from type IV collagen.

Tumor cell binding to components of the basement membrane is well known to trigger intracellular signaling pathways. Signaling ultimately results in the modulation of gene expression, facilitating metastasis. Type IV collagen is the major structural component of the basement membrane and is known to be a polyvalent ligand, possessing sequences bound by the alpha1beta1, alpha2beta1, and alpha3beta1 integrins, as well as cell surface proteoglycan receptors, such as CD44/chondroitin sulfate proteoglycan (CSPG). The role of alpha2beta1 integrin and CD44/CSPG receptor binding on human melanoma cell activation has been evaluated herein using triple-helical peptide ligands incorporating the alpha1(IV)382-393 and alpha1(IV)1263-1277 sequences, respectively. Gene expression and protein production of matrix metalloproteinases-1 (MMP-1), -2, -3, -13, and -14 were modulated with the alpha2beta1-specific sequence, whereas the CD44-specific sequence yielded significant stimulation of MMP-8 and lower levels of modulation of MMP-1, -2, -13, and -14. Analysis of enzyme activity confirmed different melanoma cell proteolytic potentials based on engagement of either the alpha2beta1 integrin or CD44/CSPG. These results are indicative of specific activation events that tumor cells undergo upon binding to select regions of basement membrane collagen. Based on the present study, triple-helical peptide ligands provide a general approach for monitoring the regulation of proteolysis in cellular systems.