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Noninvasive tests for liver fibrosis.

The field of noninvasive markers for the assessment of hepatic fibrosis has seen tremendous growth in the last two decades. Surrogate markers are gradually being substituted for biomarkers that reflect the complex balance between synthesis and degradation of the extracellular matrix. Coupled with these promising tests are artificial intelligence networks devised by sophisticated statistical instruments that incorporate a battery of laboratory tests and biomarkers with imaging modalities. Recently, gene identification and protein analysis have shown promise in identifying selected patients with mild or advanced fibrosis. Despite such progress, an important limitation of these tests is the frequent inability to detect mild fibrosis although patients at the end of the histological spectrum (i.e., advanced fibrosis or cirrhosis) are readily identified. With time, it is anticipated a combination of serological and radiological tests together with genetic and proteomic investigations will provide accurate assessment of fibrosis in a cost-effective and timely manner.

Biomarkers↗

Identification of a chemoprevention cohort from a population of women at high risk for breast cancer.

In a prospective pilot study, we performed breast fine needle aspirations (FNAs) on 213 high-risk and 30 low-risk women and analyzed these aspirates for cytologic changes and biomarker abnormalities of aneuploidy and overexpressed estrogen receptor (ER), epidermal growth factor receptor (EGFR), p53 and HER-2/neu. High-risk women were those with a first degree relative with breast cancer (73%), prior biopsy indicating premalignant breast disease (26%), a history of breast cancer (13%), or some multiple of these risk factors (11%). Median ages of the high-risk and low-risk groups were 44 and 42, respectively. Sixty-three percent of the high-risk and 73% of the low-risk group were premenopausal. Sixty-eight percent of the high-risk and 17% of low-risk women had cytologic evidence of hyperplasia with or without atypia (P < .0001). Aneuploidy and overexpression of EGFR and p53 occurred in 25%, 36%, and 28% of high-risk subjects but in less than 4% of low-risk subjects (P < .0002). Overexpression of ER and HER-2/neu occurred in 8% and 19%, respectively of high-risk women; nc low-risk women had these abnormalities. Sixty-eight percent of high-risk women and 7% of low-risk women had abnormalities of one or more of these biomarkers exclusive of cytology. Thirty-one percent of high-risk women, but no low-risk women had abnormalities of two or more biomarkers (P = .0004). Biomarker abnormalities were more frequent with increasing cytologic abnormality. Eighteen percent of women with normal cytology, 29% of women with epithelial hyperplasia and 60% of women with hyperplasia with atypia had abnormalities of two or more biomarkers (P = .048 and < .0001, respectively). Restricting the analysis to those three biomarkers most frequently overexpressed in the high-risk group (ploidy, EGFR, p53), 13% of high-risk women with normal cytology, 20% of high-risk women with epithelial hyperplasia and 51% of high-risk women with atypical hyperplasia had abnormalities of 2 or more of these 3 biomarkers. At a median follow up of two years, 8 of 213 women have been diagnosed with in situ (n = 5) or invasive (n = 3) cancer. Later detection of neoplasia was associated with prior FNA evidence of atypical hyperplasia (P < .0001) and multiple biomarker abnormalities in the 5 test battery (P = .006) by univariate analysis. By multivariate analysis, development and/or detection of cancer was primarily predicted by atypical hyperplasia (P = .0047) and secondarily by multiple biomarker abnormalities (P = 0.021). Atypical hyperplasia, EGFR, and p53 in breast FNAs have promise as risk markers and as surrogate endpoint biomarkers for breast cancer chemoprevention trials.

Adult↗

Sulfadimethoxine and rhodamine B as oral biomarkers for European badgers (Meles meles).

A field study was carried out on Little Island (County Waterford, Ireland) in June 2000 to evaluate the potential of a bait-marking system for use in European badgers (Meles meles). Two oral biomarkers, sulfadimethoxine (SDM) and rhodamine B, were incorporated into fishmeal baits and distributed by hand at main sets in five test territories for 3 consecutive days. In parallel, non-biomarked baits were distributed at a single control territory. The objectives of the study were to: (1) assess the effects of SDM and rhodamine B on palatability and thus bait acceptance, and (2) investigate the marking capacity of SDM and rhodamine B in serum and hair samples taken from badgers. Trapping was carried out in each territory for 5 consecutive days immediately after bait distribution. Analysis of data revealed that 90-100% of baits were removed in four of the test territories and from the control territory. In the fifth test territory, 61% of baits were removed. Of the badgers (n = 26) trapped in the test territories, 18 (69%) were positive when tested for both biomarkers. In contrast, the remaining eight animals and those captured in the control territory (n = 6 badgers) were negative. In the marked animals, the highest levels of SDM were recorded in serum samples taken soon after bait distribution. Thereafter, the levels declined in each badger over the course of the study. In contrast, rhodamine B was readily detectable by fluorescence microscopy of hair samples throughout the period of study. The results indicate that SDM and rhodamine B act as systemic markers in badgers and have potential future applications for monitoring of oral vaccine uptake.

Administration, Oral↗

Biodegradability of the X-ray contrast compound diatrizoic acid, identification of aerobic degradation products and effects against sewage sludge micro-organisms.

Pharmaceuticals and contrast media have been detected in hospital effluents, sewage treatment plants, surface water, and ground water. Only little is known about their elimination during sewage treatment and effects of possible biotransformation products against bacteria. The modified Zahn-Wellens test (ZWT, OECD 302 B) and a test simulating biological sewage treatment (modified OECD 303 A test) were used to assess the biodegradability of the widely used ionic iodinated contrast agent diatrizoic acid (diatrizoate). Effects against sewage sludge bacteria were studied in the two test systems by monitoring the biomarkers quinones, polyamines, phospholipids and adenosine triphosphate. Diatrizoate was biotransformed into 2,4,6-triiodo-3,5-diamino-benzoic acid in the ZWT. 2,4,6-Triiodo-3,5-diamino-benzoic acid was stable under the test conditions of the ZWT. Diatrizoate was not eliminated in the OECD 303 A simulation test. It was not adsorbed by the sewage sludge. No effects of the test compound or its aerobic transformation products against the bacteria present in the sewage sludge were detected using phospholipids, quinones, polyamines, and adenosine triphosphate as biomarkers.

Aerobiosis↗

Adoption of array technologies into the clinical laboratory.

Array-based methods are making substantial contributions to the discovery of disease biomarkers and are fueling the growth of multianalyte testing for disease diagnosis and treatment. The distillation of high-density array results into sets of signature markers promises to improve disease staging, risk stratification and treatment decisions. To accommodate the growing requirement for multiplex testing, clinical laboratories are converting several single-analyte tests into array-based formats. However, adoption of array technologies provides several challenges to the laboratory, which must evaluate these new formats, train laboratory personnel, market the new services and obtain reimbursement for new analytes. Liquid-bead arrays are an attractive format for routine clinical diagnostics due to a combination of appropriate analyte density, simultaneous array decoding and detection, and flexibility for rapid customization. In this review, the suitability of several array platforms to diagnostic testing and applications of liquid-bead arrays for cystic fibrosis testing, multidisease carrier status assays and leukemia subtyping are discussed. As our understanding of the clinical utility of new or established biomarkers and recommendations for testing change, flexibility and adaptability of array platforms will be imperative. Future development of novel assay formats and improved quantitation will expand the number of diseases tested and lead to further integration into the diagnostic laboratory.

Genetic Testing↗

In search of secreted protein biomarkers for the anti-inflammatory effect of beta2-adrenergic receptor agonists: application of DIGE technology in combination with multivariate and univariate data analysis tools.

Two-dimensional difference gel electrophoresis (DIGE) in combination with univariate (Student's t-test) and multivariate data analysis, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to study the anti-inflammatory effects of the beta(2)-adrenergic receptor (beta(2)-AR) agonist zilpaterol. U937 macrophages were exposed to the endotoxin lipopolysaccharide (LPS) to induce an inflammatory reaction, which was inhibited by the addition of zilpaterol (LZ). This inhibition was counteracted by addition of the beta(2)-AR antagonist propranolol (LZP). The extracellular proteome of the U937 cells induced by the three treatments were examined by DIGE. PCA was used as an explorative tool to investigate the clustering of the proteome dataset. Using this tool, the dataset obtained from cells treated with LPS and LZP were separated from those obtained from LZ treated cells. PLS-DA, a multivariate data analysis tool that also takes correlations between protein spots and class assignment into account, correctly classified the different extracellular proteomes and showed that many proteins were differentially expressed between the proteome of inflamed cells (LPS and LZP) and cells in which the inflammatory response was inhibited (LZ). The Student's t-test revealed 8 potential protein biomarkers, each of which was expressed at a similar level in the LPS and LZP treated cells, but differently expressed in the LZ treated cells. Two of the identified proteins, macrophage inflammatory protein-1beta (MIP-1beta) and macrophage inflammatory protein-1alpha (MIP-1alpha) are known secreted proteins. The inhibition of MIP-1beta by zilpaterol and the involvement of the beta(2)-AR and cAMP were confirmed using a specific immunoassay.

Adrenergic Agonists↗

Serologic assessment of type 1 and type 2 immunity in healthy Japanese adults.

We assessed the informativeness of several serologic biomarkers of immune function using serum specimens collected in the Miyazaki Cohort Study from subjects who were seronegative for anti-human T-cell lymphotrophic virus I and anti-hepatitis C virus. To broadly characterize type 1 immune status, we measured EBV antibody titers, because titer profiles associated with cellular immune suppression are well described. We also tested for three type 2 biomarkers: total serum IgE, soluble CD23, and soluble CD30. Nonreactivity to a tuberculin purified protein derivative (PPD) skin test is indicative of diminished delayed-type hypersensitivity (type 1) responsiveness in the study population due to a history of tuberculosis exposure or Bacillus Calmette-Guérin vaccination. We therefore evaluated the serologic markers as predictors of PPD nonreactivity using logistic regression. Subjects whose EBV antibody profiles were consistent with deficient type 1 immunity were more than thrice as likely to be PPD nonreactive as persons with "normal" antibody titers. Elevated total IgE was also strongly associated with PPD nonreactivity (odds ratio 3.4, 95% confidence interval 1.2-9.9); elevated soluble CD23 had a weaker, but positive, odds ratio, whereas soluble CD30 levels were not predictive of PPD status. Therefore, PPD nonreactivity is associated, in this population, with a pattern of serum biomarkers that is indicative of diminished type 1 and elevated type 2 immunity. We conclude that, with the exception of soluble CD30, the serologic markers are informative for the characterization of type 1/type 2 immune status using archived sera from study populations of healthy adults.

Adult↗

Combining several screening tests: optimality of the risk score.

The development of biomarkers for cancer screening is an active area of research. While several biomarkers exist, none is sufficiently sensitive and specific on its own for population screening. It is likely that successful screening programs will require combinations of multiple markers. We consider how to combine multiple disease markers for optimal performance of a screening program. We show that the risk score, defined as the probability of disease given data on multiple markers, is the optimal function in the sense that the receiver operating characteristic (ROC) curve is maximized at every point. Arguments draw on the Neyman-Pearson lemma. This contrasts with the corresponding optimality result of classic decision theory, which is set in a Bayesian framework and is based on minimizing an expected loss function associated with decision errors. Ours is an optimality result defined from a strictly frequentist point of view and does not rely on the notion of associating costs with misclassifications. The implication for data analysis is that binary regression methods can be used to yield appropriate relative weightings of different biomarkers, at least in large samples. We propose some modifications to standard binary regression methods for application to the disease screening problem. A flexible biologically motivated simulation model for cancer biomarkers is presented and we evaluate our methods by application to it. An application to real data concerning two ovarian cancer biomarkers is also presented. Our results are equally relevant to the more general medical diagnostic testing problem, where results of multiple tests or predictors are combined to yield a composite diagnostic test. Moreover, our methods justify the development of clinical prediction scores based on binary regression.

Bayes Theorem↗

Alternatives to cytology in the management of non-muscle invasive bladder cancer.

The natural history of non-muscle invasive bladder cancer is characterized by a high probability of recurrence and in the case of high-grade tumors, progression to muscle invasive cancer. This mandates a follow-up strategy designed to identify recurrences in the bladder early in their evolution in order to facilitate early intervention and ablation. Urine cytology is considered the gold standard urine biomarker. Although specificity exceeds 90% to 95%, its overall sensitivity ranges from 40% to 60% in expert hands and is both tumor grade and operator dependent. While cytology is an excellent test for detection of high-grade disease, the sensitivity is particularly weak for the detection of low grade tumors. This has spawned an entire field of research of in vitro diagnostic tests and cell-based assays in order to improve the diagnostic accuracy for detection of incident or recurrent disease. To date, the US Food and Drug Administration approved dipstick and immunoassays marketed as point-of-care tests. The point-of-care tests are intended for use as an adjunct to cystoscopy and cytology, and may have a role in the office evaluation of hematuria patients. Monoclonal antibody-based tests combined with cytology may improve the diagnostic accuracy and are superior to cytology alone. A recently approved cell-based assay, utilizing fluorescent in situ hybridization technology, may help resolve suspicious cytologies, and provide early and additional information about the biology of the bladder urothelium beyond that provided by cytology, a marker of disease relatively late in evolution. Novel promising markers are in various stages of clinical testing, and a panel of biomarkers may serve in the future as a feasible alternative to urine cytology and cystoscopy for the screening, detection, and follow-up of non-muscle invasive bladder cancer.

Antibodies, Monoclonal↗

Mediating effects of inflammatory biomarkers on insulin resistance associated with obesity.

OBJECTIVE: Obesity is associated with elevated levels of biomarkers of inflammation and endothelial dysfunction [including C-reactive protein (CRP), E-selectin, and intercellular adhesion molecule-1], as well as insulin resistance (IR) and type 2 diabetes. We tested the hypothesis that these biomarkers mediate associations among obesity, IR, and risk of diabetes. RESEARCH METHODS AND PROCEDURES: We stratified 510 initially non-diabetic women in the Nurses' Health Study cohort into four phenotypes above/below median BMI (27 kg/m2) and waist circumference (81 cm): low BMI-low waist (LBLW; N = 190), low BMI-high waist (LBHW; N = 74), high BMI-low waist (HBLW; N = 27), and high BMI-high waist (HBHW; N = 219). RESULTS: In models assessing associations of weight phenotype with IR [fasting insulin (FI)], adjusted for age and diabetes risk factors, mean FI was higher comparing HBHW women (13.6 microU/mL, p < 0.0001) and LBHW (11.5 microU/mL, p = 0.02) with LBLW women (8.6 microU/mL); HBLW and LBLW women were not significantly different. Differences in FI levels were most strongly attenuated after adjustment for E-selectin comparing LBHW with LBLW women (11.7 vs. 9.7 microU/mL, p = 0.2). DISCUSSION: In logistic regression models, LBHW predicted diabetes (risk factor-adjusted relative risk 2.06, 1.05 to 6.40), compared with LBLW, but was no longer significant after adjustment for E-selectin or CRP. After adjusting for CRP and E-selectin, only HBHW and E-selectin were significantly associated with risk of diabetes. In women with central adiposity and low BMI, endothelial dysfunction and inflammation may mediate the relationship among central fat, IR, and incident diabetes.

Adiposity↗

Negative correlation between poly-ADP-ribosylation of spleen cell histone proteins and initial duration of dimethylnitrosamine exposure to mice in vivo measured by Western blot immunoprobe assay: a possible biomarker for cancer detection.

Improved cancer detection involving suitable biomarkers with easy applicability is a challenge to our fight against cancer. Poly-ADP-ribosylation (PAR) of proteins is a likely candidate biomarker for this purpose because it meets the criterion well. This report is a step towards testing suitability of PAR as a biomarker for cancer detection. Swiss albino mice were exposed to hepatocarcinogen, dimethylnitrosamine (DMN), at a chronic dose, which is known to induce carcinogenesis in liver. PAR was monitored by a Western blot immunoprobe assay in spleen, a lymphoid organ, to find a correlation between PAR of spleen histone proteins and duration of DMN exposure. A negative, non-linear correlation was found for most histone proteins. The inhibition of PAR of histones was significant from 4 weeks onwards until the end of the observation. The inhibition was potentiated when 3-aminobenzamide was simultaneously administered. The results open up the possibility of PAR of cellular proteins being used as biomarker for cancer detection.

Animals↗

[LE cell test].

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Antibodies, Antinuclear↗

Identification of parotid salivary biomarkers in Sjogren's syndrome by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry and two-dimensional difference gel electrophoresis.

OBJECTIVES: To identify the most significant salivary biomarkers in Sjögren's syndrome (SS) using proteomic methods. METHODS: Parotid saliva from 20 non-SS subjects and 41 primary SS patients was analysed. Protein expression profiles for each sample were generated by surface-enhanced laser desorption/ionization time-of-flight-mass spectrometry (SELDI-TOF-MS). Mean peak intensities of SS patients and non-SS subjects were compared by univariate analyses. Samples pooled by diagnosis (SS and non-SS) and labelled with different Cy dyes were compared by two-dimensional difference gel electrophoresis (2D-DIGE). Two protein levels that were most significantly different by SELDI-TOF-MS and 2D-DIGE were validated by enzyme-linked immunosorbent assay in individual samples. RESULTS: SELDI-TOF-MS of 10-200 kDa peaks revealed eight peaks with >2-fold changes in the SS group that differed from non-SS at P < 0.005. Peaks of 11.8, 12.0, 14.3, 80.6 and 83.7 kDa were increased, while 17.3, 25.4, and 35.4 kDa peaks were decreased in SS samples. 2D-DIGE identified significant increases of beta-2-microglobulin, lactoferrin, immunoglobulin (Ig) kappa light chain, polymeric Ig receptor, lysozyme C and cystatin C in all stages of SS. Two presumed proline-rich proteins, amylase and carbonic anhydrase VI, were reduced in the patient group. Three of these ten biomarkers have not been associated previously with SS. CONCLUSIONS: The salivary proteomic profile of SS is a mixture of increased inflammatory proteins and decreased acinar proteins when compared with non-SS. Future studies will test the ability of these biomarker levels, alone and in combination, to diagnose the salivary component of SS.

Amylases↗