Superficial bladder cancer is characterized by frequent recurrences with a relatively low rate of progression to muscle invasive disease. Cystoscopy provides excellent visualization of the bladder and is routinely used for tumor surveillance. This visual bladder assessment is often supplemented by urine cytology, a test with high specificity but low sensitivity for low-grade cancers. In the past several years, new urine assays have been added to the diagnostic armamentarium. Assays that measure soluble analytes tend to be more sensitive but less specific than cytology. Assays that measure cellular-based analytes seem to have a high specificity but have been less studied than the soluble analyte assays. A number of new markers show promise and are in development. Furthermore, new strategies such as microsatellite analysis and proteomics offer hope that novel markers for bladder cancer will be defined that have both high sensitivity and specificity.
Point of care (POC) devices to measure drugs of abuse (DOA) in urine represent a rapidly growing portion of the drug screening market. These types of devices are based on sophisticated ligand/receptor technology and are rapid and accurate. They are popular in workplace drug screening and for home use, along with clinical settings. In this paper we focus on the technology currently in use, the effect of adulterants on these assays and the possible future directions in POC drug testing.
Current driving forces in proteomics are mass spectrometry and bioinformatics; however, the separation and display of highly complex protein mixtures have equal importance. Two-dimensional gel electrophoresis remains the most powerful method to resolve, purify, and analyze the components of complex protein mixtures, particularly when difference gel electrophoresis (DIGE) is employed. Sample preparation methodology greatly influences the quality of the separation. Recent improvements in both of these areas have made the separation and display process more convenient and robust.Automation has contributed greatly to the throughput of post-separation analysis. Fully automated image analysis saves time and removes human interference. Spot-picking robots are capable of automatically excising interesting spots. The entire spot handling process, including in-gel digestion and spotting peptides and matrix on targets for mass spectrometric analysis, can be fully automated. New hardware and software for mass spectrometry using the matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) method allow automated spot identification via peptide mass profiling and provide amino acid sequence data when used in conjunction with chemically assisted fragmentation. The entire proteomics workflow may be monitored and controlled by laboratory workflow system software. Thus, in the span of a few years, proteomics has developed from a few labor-intensive procedures to an automated, industrial, high-throughput endeavor.
A semi-automated system was developed that processes and analyzes 7.5 mL of blood for the presence of epithelial-derived tumor cells. Cells of epithelial cell origin are immunomagnetically labeled and separated from blood. The magnetically captured cells are differentially fluorescent labeled and placed in an analysis chamber. Four-color fluorescent imaging is used to differentiate between debris, hematopoeitic cells, and epithelial-derived circulating tumor cells (CTC). An algorithm is applied to the captured images to enumerate an internal control and identify all objects that meet predetermined criteria for tumor cells based on a size-intensity algorithm and immunophenotype. Thumbnail images of each object are presented in a user interface from which the user can determine the presence of tumor cells. In processing the blood of normal donors the internal control showed consistent and reproducible results between systems and operators. In 15 of 22 patients with metastatic breast cancer, CTC exceeded the 99% upper confidence interval of normals.
Despite extensive research efforts to identify unique molecular alterations in cancer, to date, no characteristic has emerged that correlates exclusively with malignancy. Recently, it has been demonstrated that the multiprotein DNA replication complex (DNA synthesome) from human breast cancer cells has significantly decreased replication fidelity compared to that of non-malignant breast cells. Proliferating cell nuclear antigen (PCNA) functions in both DNA replication and DNA repair, and is a component of the synthesome. Using two dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis, a novel form of PCNA has been identified in malignant breast cells. This cancer specific form of the protein (csPCNA) is not the result of a genetic alteration, as demonstrated by DNA sequence analysis of the PCNA gene from malignant and non-malignant breast cells. The csPCNA is most likely the result of an alteration in the post-translational modification of the protein in malignant cells. These findings are significant in that it is now possible to link changes in the fidelity of DNA replication with a specific alteration of a component of the DNA synthetic apparatus of breast cancer cells. The csPCNA may prove to be a new signature for cancer cells and has the potential to serve as a powerful biomarker for malignant disease.
The increasing amount of data arising from improvements in the technology of drug screening and related disciplines is succeeding only in moving the bottlenecks elsewhere. Technological developments in target validation, sample handling, data analysis, and lead verification are constantly being sought to maintain speed and reduce costs. The convergent evolution of cellular assay technologies and compatible bio-imaging systems promises to have significant impact on drug development by enabling earlier generation of physiologically relevant information. This is expected to facilitate drug development at many levels, thereby reducing both drug development costs and time to market. This article describes which types of targets are likely to benefit from cell-based assay formats, what imaging platforms are currently available to address this increasingly important aspect of drug development, and some of the instrumentation challenges that must be met in order to make cell-based screening a reality.
Cardiovascular disease continues to represent the primary cause of morbidity and mortality globally. There has been a growing awareness of the need to address cardiovascular diseases, especially coronary artery disease (CAD). Recently, a group of emerging lipid markers has become available. These markers are beginning to significantly contribute to identifying risk for CAD as well as for predicting cardiac events. This review is devoted to two of these emerging markers, lipoprotein (a) [Lp(a)]) and remnant lipoprotein (RLP) cholesterol (RLP-C). Lp(a) is a carbohydrate rich macromolecule of which plasma concentrations are primarily genetically controlled and have been implicated as a significant risk factor for premature atherosclerosis and coronary heart disease. RLPs are the products of lipolytic degradation of triglyceride rich lipoproteins. RLPs of both intestinal (CH) and hepatic (VLDL) origin have been thought to be a subspecies of TRL, which may potentiate cardiovascular disease risk and cause premature atherosclerosis.
Fibronectin (FN) is a multi-functional adhesion glycoprotein encoded by a single gene. Its pre-mRNA can be alternately spliced at three different sites, and as many as 20 spliced FN protein isoforms can be generated. Alternately spliced FN isoforms may be expressed in a tissue-specific manner, and the splicing pattern may be regulated during ontogeny, aging, and cellular transformation. Plasma FN (PFN), synthesized and secreted by hepatocytes, is well-characterized, while mesenchymal, endothelial, and epithelial cells synthesize and secrete alternately spliced cellular FN (CFN) into the extracellular matrix. Because both androgen-responsive and androgen-independent prostate cancer cell lines secreted abundant amount of FN, we determined whether higher than normal FN levels could be observed in the sera of prostate cancer patients. Using an FN immunoassay kit (which measures both CFN and PFN), we found a significantly higher than normal levels of FN in the sera of prostate cancer patients. Serum FN levels, however, did not correlate with levels of prostate-specific antigen. Molecular characterization of the splicing profile of CFN in prostate cancer cells, and the development of specific reagents for measuring the prostate cancer-associated spliced variants of FN will be needed in order to further explore the tumor marker potential of FN.
Human cardiac troponin I (HcTnI) is one of the most specific biochemical markers for acute myocardial infarction (AMI). Since the molecular forms of HcTnI are not clearly defined in human circulation, their reliable detection becomes crucial for rapid diagnosis and monitoring of AMI patients. To formulate immunoassay kits with consistent assay results, monoclonal antibodies were generated against HcTnI. Lack of cross-reactivity of these antibodies to other troponin isoforms were initially screened and their distinct epitopes mapped. Finally, four antibodies designated as TPC-6, TPC-102, TPC-110, and TPC-302 were shown to recognize epitopes in the N-terminal half of HcTnI and were selected to formulate an enzyme immunoassay (EIA) kit and a rapid immunochromatographic dipstick test. The EIA kit was well correlated with the Abbott AxSYM method in a retrospective clinical study (n = 180, correlation = 0.924). The results of this study seem to confirm that immunoassays based on the use of antibodies recognizing the N-terminal half of HcTnI would yield reliable detection of this cardiac marker for monitoring AMI and other related heart diseases.
The abundance of well characterized single nucleotide polymorphism (SNP) markers and a complete draft of the DNA sequence of the human genome have opened up a new era for systematic characterization of the estimated 30,000 human genes, and have enabled analysis of gene functions and their associations with diseases of genetic origin. Together with recent innovations in genetic analysis, these factors greatly enhance the potential for epidemiological studies to identify polymorphic genes that predict susceptibility to cancer, cardiovascular disease, mental illness, autoimmune disease, diabetes, and various environmentally influenced diseases. In this review, we discuss currently available technologies and platforms for SNP genotyping that are useful for population-based epidemiology studies, as well as the genotyping principles underlying each method and solutions to commonly encountered analytical problems in this rapidly evolving field.
Prostate cancer is a clinically significant health care problem in the United States accounting for 29% of all male cancers. Prostate specific antigen (PSA) has revolutionized the detection of prostate cancer resulting in a dramatic stage migration manifested by a significant increase in the incidence of organ-confined disease at the time of diagnosis. The limitations of this tumor marker, namely its low specificity and positive predictive value have also become apparent. Numerous attempts to enhance PSA performance based on prostate gland volume, age, and velocity have demonstrated little clinical improvement. Percent free PSA has shown some improvement but has limited applicability. The role of other markers such as complexed PSA and human kallikrein 2 (hK2) both currently under investigation is discussed. Additional information pertaining to other potential markers as well as more recent technologic advances in computer-assisted diagnostics is also presented.
The majority of cancer patients do not die from their primary tumor but from distant metastases. Understanding the biology underlying these processes is of essential importance to specifically target the cause of malignancy. Proteases degrade the extracellular matrix surrounding the primary tumor, thereby facilitating tumor invasion and metastasis. Cathepsins, matrix metalloproteinases, and serine proteases such as the urokinase plasminogen activator and plasmin, in concert with their respective inhibitors and receptors, initiate tissue degradation and remodeling. Furthermore, components of these proteolytic systems exert additional functions, not related to their proteolytic activity, effecting cell adhesion, migration, proliferation, and vessel growth. In breast cancer, elevated antigen content of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 is an indicator of poor prognosis and therefore helps to identify the patients likely to experience disease recurrence (metastasis) and/or early death. Such high-risk patients do benefit from adjuvant systemic chemotherapy. uPA and PAI-1 antigen is assessed in primary tumor tissue extracts by ELISA. We evaluated common macro-methodologies for disintegration of the primary tumor tissue and preparation of the tumor tissue extract, starting from fresh tumor tissue blocks. In addition, we present a new micro-extraction procedure using cryostat sections as the source of tumor tissue. Such techniques allow rapid and reproducible quantitative determination of uPA and PAI-1, even in small tumor specimens.
Heart failure is the only cardiovascular disease with a prevalence that is increasing, and the leading cause of Medicare hospitalizations. It is extremely difficult to diagnose accurately on clinical grounds alone. Natriuretic peptides are hormones secreted by the failing heart in response to wall stretch and volume overload. Diagnostic assays for the natriuretic peptides represent a laboratory-based tool that hospital staff including emergency department physicians and cardiologists could use for optimal diagnosis and management of this critical and growing patient population. While numerous publications have appeared concerning BNP (brain natriuretic peptide), this review focuses primarily on NT-proBNP (N-terminal pro brain natriuretic peptide). Studies focusing on the clinical utility of NT-proBNP as an objective diagnostic aid, a prognostic indicator of future risk, and a tool for the selection and monitoring of heart failure therapy are discussed, and practical considerations for laboratory implementation of natriuretic peptide testing are also discussed.
Recently, several different immunoassays have been described for the measurement of tissue inhibitor of metalloproteinases-1 (TIMP-1); however, large discrepancies in plasma TIMP-1 levels have been reported among the various assays. Since TIMP-1 exists in several molecular forms, it is obvious that inconsistent results are obtained using TIMP-1 immunoassays constructed with antibodies of differing specificities for these forms of TIMP-1. To clarify this dilemma, we characterized a panel of commercially and non-commercially available monoclonal anti-TIMP-1 antibodies and determined their affinities and specificities for free TIMP-1 and TIMP-1:MMP-9 complex. Our results show that TIMP-1 monoclonal antibodies can be categorized into two groups: those binding to both free TIMP-1 and TIMP-1:MMP-9 complex, and those binding selectively to free TIMP-1. Different molecular forms of TIMP-1 may therefore be unequally recognized by different assays and thus it is recommended that total TIMP-1 assays should be developed employing carefully characterized monoclonal antibodies detecting all molecular forms of TIMP-1 at equimolar levels.
The galactose oxidase Schiff's (GOS) reaction detects D-galactose-beta [1-3]- N-acetyl-D-galactosamine or Thomsen Friedenreich antigen. GOS-reactive material has been associated with a range of cancers including colorectal and lung cancer but has seen limited utility as a tumor marker in part because of the subjective interpretation of the test outcome. We developed a method of quantifying the GOS reaction based on the determination of the color parameters hue or chroma. In a prospective study of 669 individuals scheduled for colonoscopy, we found GOS reactivity in rectal mucus samples to be significantly and independently correlated with cancer and adenomas. In a pilot study of individuals with lung cancer, we detected 77% of lung cancer cases by measuring GOS reactivity in sputum samples. By quantifying the GOS reaction, we have introduced a means to reassess its role as a tumor marker, which may be applicable to a variety of cancers.
