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PDF file - 269K, Significance of methylation in prostate cancer tissue samples from 4 microarrays with the batch effect

Supplementary Methods Additional methodology of experimental methods.

Supplementary Figure 2. A) In vitro comparison of LNCaP and LNCaP-AR and DNA damage post-radiotherapy. B) In vivo correlation of baseline AR-output using bioluminescence to tumor progression.

Supplementary Figure legends for Supplementary Figure 1 and 2

PDF file - 70K, Significance of PTGS2 methylation in different groups of prostate cancer tissues

PDF file - 172K, Prostate cancer tissues used for methylation analysis by pyrosequencing

Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival. Cancer Res; 75(22); 4688–96. ©2015 AACR.

PDF file - 419K, Significantly methylated genes in prostate cancer tissues from the binary group comparisons

Supplementary Figure 1. In vitro experiments demonstrating increase AR protein level post-radiotherapy; A) Nuclear AR staining, B) 18F-FDHT uptake. C) Isodose distribution of multibeam animal EBRT.

XLS file - 34K, Most significantly methylated genes associated with prostate cancer recurrence from the Cox univariate analysis

XLS file - 43K, Most significantly methylated genes associated with prostate cancer recurrence from the Cox multivariate model

Objectives: To prospectively evaluate the performance of a pre-specified statistical model based on four kallikrein markers in blood (total prostate-specific antigen [PSA], free PSA, intact PSA, and human kallikrein-related peptidase 2), commercially available as the 4Kscore, in predicting Gleason Grade Group (GG) ≥2 prostate cancer at biopsy in an international multicentre study at three academic medical centres, and whether microseminoprotein-β (MSP) adds predictive value. Patients and Methods: A total of 984 men were prospectively enrolled at three academic centres. The primary outcome was GG ≥2 on prostate biopsy. Three pre-specified statistical models were used: a base model including PSA, age, digital rectal examination and prior negative biopsy; a model that added free PSA to the base model; and the 4Kscore. Results: A total of 947 men were included in the final analysis and 273 (29%) had GG ≥2 on prostate biopsy. The base model area under the receiver operating characteristic curve of 0.775 increased to 0.802 with the addition of free PSA, and to 0.824 for the 4Kscore. Adding MSP to the 4Kscore model yielded an increase (0.014–0.019) in discrimination. In decision-curve analysis of clinical utility, the 4Kscore showed a benefit starting at a 7.5% threshold. Conclusion: A prospective multicentre evaluation of a pre-specified model based on four kallikrein markers (4Kscore) with the addition of MSP improves the predictive discrimination for GG ≥2 prostate cancer on biopsy and could be used to inform biopsy decision-making. (Less)

Introduction Heterogeneity of nephrotic diseases and a lack of validated biomarkers limits interventions and reduces the ability to examine outcomes. Urinary CD80 is a potential biomarker for minimal change disease (MCD) steroid-sensitive nephrotic syndrome (NS). We investigated and validated a CD80 enzyme-linked immunosorbent assay (ELISA) in urine in a large cohort with a variety of nephrotic diseases. Methods A commercial CD80 ELISA was enhanced and analytically validated for urine. Patients were from Mayo Clinic (307) and Nephrotic Syndrome Study Network Consortium (NEPTUNE; 104) as follows: minimal change disease (MCD, 56), focal segmental glomerulosclerosis (FSGS, 92), lupus nephritis (LN, 25), IgA nephropathy (IgAN, 20), membranous nephropathy (MN, 49), autosomal dominant polycystic kidney disease (ADPKD, 10), diabetic nephropathy (DN; 106), pyuria (19), and controls (34). Analysis was by Kruskal−Wallis test, generalized estimating equation (GEE) models, and receiver operating characteristic (AUC) curve. Results Urinary CD80/creatinine values were highest in MCD compared to other glomerular diseases and were increased in DN with proteinuria >2 compared to controls (control = 36 ng/g; MCD = 139 ng/g, P < 0.01; LN = 90 ng/g, P < 0.12; FSGS = 66 ng/g, P = 0.18; DN = 63, P = 0.03; MN = 69 ng/g, P = 0.33; ng/g, P = 0.07; IgA = 19 ng/g, P = 0.09; ADPKD = 42, P = 0.36; and pyuria 31, P = 0.20; GEE, median, P vs. control). In proteinuric patients, CD80 concentration appears to be independent of proteinuria levels, suggesting that it is unrelated to nonspecific passage across the glomeruli. CD80/creatinine values were higher in paired relapse versus remission cases of MCD and FSGS (P < 0.0001, GEE). Conclusion Using a validated ELISA, urinary CD80 levels discriminate MCD from other forms of NS (FSGS, DN, IgA, MN) and primary from secondary FSGS., Graphical abstract

Background: To assess whether a prespecified statistical model based on the four kallikrein markers measured in blood—total, free, and intact prostate-specific antigen (PSA), together with human kallikrein-related peptidase 2 (hK2)—or any individual marker measured in pretreatment serum were associated with biochemical recurrence-free (BCR) or metastasis-free survival after radical prostatectomy (RP) in a subgroup of men with very high-risk disease. Methods: We identified 106 men treated at Mayo Clinic from 2004 to 2008 with pathological Gleason grade group 4 to 5 or seminal vesicle invasion at RP. Univariable and multivariable Cox models were used to test the association between standard predictors (Kattan nomogram and GPSM [Gleason, PSA, seminal vesicle and margin status] score), kallikrein panel, and individual kallikrein markers with the outcomes. Results: BCR and metastasis occurred in 67 and 30 patients, respectively. The median follow-up for patients who did not develop a BCR was 10.3 years (interquartile range = 8.2-11.8). In this high-risk group, neither Kattan risk, GPSM score, or the kallikrein panel model was associated with either outcome. However, after adjusting for Kattan risk and GPSM score, separately, preoperative intact PSA was associated with both outcomes while hK2 was associated with metastasis-free survival. Conclusions: Conventional risk prediction tools were poor discriminators for risk of adverse outcomes after RP (Kattan risk and GPSM risk) in patients with very high-risk disease. Further studies are needed to define the role of individual kallikrein marker forms in the blood to predict adverse prostate cancer outcomes after RP in this high-risk setting. (Less)

Objectives To analyze consistency of reference limits and widths of reference intervals (RIs) calculated by six procedures and evaluate a protocol for merging intrainstitutional reference data. Methods The differences between reference limits were compared with "optimal" bias goals. Also, widths of the RIs were compared. RIs were calculated using Mayo-SAS quantile, EP Evaluator, and four International Federation of Clinical Chemistry and Laboratory Medicine methods: parametric and nonparametric (NP) with and without latent abnormal values exclusion (LAVE). Regression parameters from cotested samples were evaluated for harmonizing intrainstitutional reference data. Results Mayo-SAS quintile, LAVE(-)NP, and EP Evaluator generated similar RIs, but these RIs often were wider than RIs from parametric procedures. LAVE procedures generated narrower RIs for nutritional and inflammatory markers. Transformation with regression parameters did not ensure homogeneity of merged data. Conclusions Parametric methods are recommended when inappropriate values cannot be excluded. The nonparametric procedures may generate wider RIs. Data sets larger than 200 are recommended for robust estimates. Caution should be exercised when merging intrainstitutional data.

In vitro diagnostic (IVD) reagent stability is typically evaluated using regression analysis of measurand drift across time following CLSI guideline EP25-A. The corresponding stability duration est...

L’entretien entre Madeleine de Scudéry et Johann Christoph Wagenseil qui fait l’objet de notre présente étude, n’est pas absolument inconnu. Pourtant il n’a jamais fait l’objet d’une recherche scientifique ni chez les romanistes, ni chez les germanistes. Dans la mesure où ce document constitue un témoignage extraordinaire des temps modernes, cette négligence ne peut qu’étonner. En effet, dans la seconde moitié du XVIIe siècle, Madeleine de Scudéry était l’auteur français le plus connu en Alle...

ObjectiveTo examine the use of the Prostate Health Index (PHI) as a continuous variable in multivariable risk assessment for aggressive prostate cancer in a large multicentre US study.Materials and methodsThe study population included 728 men, with prostate-specific antigen (PSA) levels of 2-10 ng/mL and a negative digital rectal examination, enrolled in a prospective, multi-site early detection trial. The primary endpoint was aggressive prostate cancer, defined as biopsy Gleason score ≥7. First, we evaluated whether the addition of PHI improves the performance of currently available risk calculators (the Prostate Cancer Prevention Trial [PCPT] and European Randomised Study of Screening for Prostate Cancer [ERSPC] risk calculators). We also designed and internally validated a new PHI-based multivariable predictive model, and created a nomogram.ResultsOf 728 men undergoing biopsy, 118 (16.2%) had aggressive prostate cancer. The PHI predicted the risk of aggressive prostate cancer across the spectrum of values. Adding PHI significantly improved the predictive accuracy of the PCPT and ERSPC risk calculators for aggressive disease. A new model was created using age, previous biopsy, prostate volume, PSA and PHI, with an area under the curve of 0.746. The bootstrap-corrected model showed good calibration with observed risk for aggressive prostate cancer and had net benefit on decision-curve analysis.ConclusionUsing PHI as part of multivariable risk assessment leads to a significant improvement in the detection of aggressive prostate cancer, potentially reducing harms from unnecessary prostate biopsy and overdiagnosis.

A woman in her 70s presented to the department of dermatology with an acute, non-painful, pruritic rash on the forehead, which had been present for about three days. The rash had worsened after treatment with topical corticosteroids. Her medical history included breast cancer, which had been treated with surgery, chemotherapy, and radiotherapy eight years previously. Subsequent oncological follow-up had been unremarkable. The patient had no risk factors for HIV infection, was a lifelong non-smoker, and had no respiratory symptoms. Examination showed multiple monomorphic erosions with surrounding erythema, extending across the midline of the forehead, in a bilateral V1 dermatomal distribution (fig 1). Hutchinson’s sign was negative—that is, no vesicles were seen on the tip or either side of the nose. In addition, isolated urticated plaques, some with blistering, involving several thoracic dermatomes were seen. Fig 1 Monomorphic erosions with surrounding erythema, extending across the midline of the forehead in a bilateral V1 dermatomal distribution Routine physical examination was otherwise unremarkable. Table 1 shows the results of routine blood tests. An autoimmune blistering skin disorder was excluded …

To analyze consistency of reference limits and widths of reference intervals (RIs) calculated by six procedures and evaluate a protocol for merging intrainstitutional reference data.The differences between reference limits were compared with "optimal" bias goals. Also, widths of the RIs were compared. RIs were calculated using Mayo-SAS quantile, EP Evaluator, and four International Federation of Clinical Chemistry and Laboratory Medicine methods: parametric and nonparametric (NP) with and without latent abnormal values exclusion (LAVE). Regression parameters from cotested samples were evaluated for harmonizing intrainstitutional reference data.Mayo-SAS quintile, LAVE(-)NP, and EP Evaluator generated similar RIs, but these RIs often were wider than RIs from parametric procedures. LAVE procedures generated narrower RIs for nutritional and inflammatory markers. Transformation with regression parameters did not ensure homogeneity of merged data.Parametric methods are recommended when inappropriate values cannot be excluded. The nonparametric procedures may generate wider RIs. Data sets larger than 200 are recommended for robust estimates. Caution should be exercised when merging intrainstitutional data.

Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival. Cancer Res; 75(22); 4688–96. ©2015 AACR.

Clinical laboratory tests play a vital role in the medical decision making process, including diagnosis, prognostic assessment and drug dosage prescription. Drift or degradation in the performance of the analytic instrument over time can have a significant effect on the uncertainty of the clinical laboratory measurement test result. In this paper, we model the drift in the analytic instrumentation used to perform the laboratory tests, and estimate its effect on the uncertainty of the measurement result. This is accomplished developing a physics-based mathematical model of the total bilirubin laboratory test that describes the measurement result as a function of various sources of uncertainty operating within the total bilirubin measurement process. The Monte Carlo method is used to estimate the uncertainty associated with this model. Drift in the instrument is modeled as affecting both the mean (inaccuracy) and the standard deviation (imprecision) of each source of uncertainty. Further, recalibrating the ...

Spliced variant forms of androgen receptor were recently identified in castration resistant prostate cancer cell lines and clinical samples. We identified the cistrome and gene signature of androgen receptor splice variants in castration resistant prostate cancer cell lines and determined the clinical significance of androgen receptor splice variant regulated genes.The castration resistant prostate cancer cell line 22Rv1, which expresses full-length androgen receptor and androgen receptor splice variants endogenously, was used as the research model. We established 22Rv1-ARFL(-)/ARV(+) and 22Rv1-ARFL(-)/ARV(-) through RNA interference. Chromatin immunoprecipitation coupled with next generation sequencing and microarray techniques were used to identify the cistrome and gene expression profiles of androgen receptor splice variants in the absence of androgen.Androgen receptor splice variant binding sites were identified in 22Rv1-ARFL(-)/ARV(+). A gene set was regulated uniquely by androgen receptor splice variants but not by full-length androgen receptor in the absence of androgen. Integrated analysis revealed that some genes were directly modulated by androgen receptor splice variants. Unsupervised clustering analysis showed that the androgen receptor splice variant gene signature differentiated benign from malignant prostate tissue as well as localized prostate cancer from metastatic castration resistant prostate cancer specimens. Some genes that were modulated uniquely by androgen receptor splice variants also correlated with histological grade and biochemical failure.Androgen receptor splice variants can bind to DNA independent of full-length androgen receptor in the absence of androgen and modulate a unique set of genes that is not regulated by full-length androgen receptor. The androgen receptor splice variant gene signature correlates with disease progression. It distinguishes primary cancer from castration resistant prostate cancer specimens and benign from malignant prostate specimens.

Purpose: The Prostate Health Index (phi) is a new test combining total, free and [-2] proPSA into a single score. It was recently approved by the FDA and is now commercially available in the U.S., Europe and Australia. We investigate whether phi improves specificity for detecting clinically significant prostate cancer and can help reduce prostate cancer over diagnosis. Materials and Methods: From a multicenter prospective trial we identified 658 men age 50 years or older with prostate specific antigen 4 to 10 ng/ml and normal digital rectal examination who underwent prostate biopsy. In this population we compared the performance of prostate specific antigen, % free prostate specific antigen, [-2] proPSA and phi to predict biopsy results and, specifically, the presence of clinically significant prostate cancer using multiple criteria. Results: The Prostate Health Index was significantly higher in men with Gleason 7 or greater and "Epstein significant" cancer. On receiver operating characteristic analysis phi had the highest AUC for overall prostate cancer (AUCs phi 0.708, percent free prostate specific antigen 0.648, [-2] proPSA 0.550 and prostate specific antigen 0.516), Gleason 7 or greater (AUCs phi 0.707, percent free prostate specific antigen 0.661, [-2] proPSA 0.558, prostate specific antigen 0.551) and significant prostate cancer (AUCs phi 0.698, percent free prostate specific antigen 0.654, [-2] proPSA 0.550, prostate specific antigen 0.549). At the 90% sensitivity cut point for phi (a score less than 28.6) 30.1% of patients could have been spared an unnecessary biopsy for benign disease or insignificant prostate cancer compared to 21.7% using percent free prostate specific antigen. Conclusions: The new phi test outperforms its individual components of total, free and [-2] proPSA for the identification of clinically significant prostate cancer. Phi may be useful as part of a multivariable approach to reduce prostate biopsies and over diagnosis.

Uncertainty in the calibration of a clinical laboratory measurement process has a significant effect on the uncertainty of the measurement result. We develop a mathematical model of the analytical stage of the measurement of serum triglyceride concentration in the clinical laboratory, and use the Monte Carlo method to estimate the net uncertainty associated with this model. We then use the model to study the effect of instrument calibration on the uncertainty of the laboratory measurement result. The effect of the correlation between the parameters of the linear calibration function on the measurement result is quantified using the model. In addition, the effect of the choice of calibrator concentration levels on the measurement result distribution is studied using the model, by studying the effect of the value or the position of the calibrator concentration, and the difference or the distance between calibrator concentrations, on the uncertainty of the measurement result.

Diagnostic decisions based on decision limits according to medical guidelines are different from the majority of clinical decisions due to the strict dichotomization of patients into diseased and non-diseased. Consequently, the influence of analytical performance is more critical than for other diagnostic decisions where much other information is included. The aim of this opinion paper is to investigate consequences of analytical quality and other circumstances for the outcome of "Guideline-Driven Medical Decision Limits".Effects of analytical bias and imprecision should be investigated separately and analytical quality specifications should be estimated accordingly.Use of sharp decision limits doesn't consider biological variation and effects of this variation are closely connected with the effects of analytical performance. Such relationships are investigated for the guidelines for HbA1c in diagnosis of diabetes and in risk of coronary heart disease based on serum cholesterol. The effects of a second sampling in diagnosis give dramatic reduction in the effects of analytical quality showing minimal influence of imprecision up to 3 to 5% for two independent samplings, whereas the reduction in bias is more moderate and a 2% increase in concentration doubles the percentage of false positive diagnoses, both for HbA1c and cholesterol.An alternative approach comes from the current application of guidelines for follow-up laboratory tests according to clinical procedure orders, e.g. frequency of parathyroid hormone requests as a function of serum calcium concentrations. Here, the specifications for bias can be evaluated from the functional increase in requests for increasing serum calcium concentrations.In consequence of the difficulties with biological variation and the practical utilization of concentration dependence of frequency of follow-up laboratory tests already in use, a kind of probability function for diagnosis as function of the key-analyte is proposed.

SHBG concentrations correlate inconsistently with metabolic parameters.SHBG assay platforms contribute to nonuniformities according to the literature.The design of the study was a noninterventional quantification of SHBG by two immuno- and two mass spectrometric assays and abdominal visceral fat by computed tomography scan.The study was conducted at the Center for Translational Science Activities.Healthy men (n=120) aged 18-80 years with a body mass index of 20-43 kg/m2 participated I the study.Outcomes of the study included a correlation of log SHBG with age, metabolic surrogates [body mass index, albumin, glucose, insulin, abdominal (total and visceral) fat, homeostasis model assessment insulin resistance index], sex steroids (estrone, 17β-estradiol, T, and dihydrotestosterone by mass spectrometry), and adipocytokines (IL-1β, IL-6, IL-8, IL-10 and IL-12, TNF-α, and adiponectin).By univariate regression, age (P10(-4)), dihydrotestosterone (P10(-4)), T (P≤.00022), and adiponectin (P≤.0084) were positive correlates, and insulin and homeostasis model assessment insulin resistance index were negative correlates (P≤.0060) of SHBG in all four assays. Stepwise multivariate analysis unveiled that age and T together could explain 38.1%-52.5% of the statistical variance in SHBG in all assays (P10(-11)). Multivariate regression without sex steroids unveiled that age (P10(-5)) and insulin (P10(-3)) are jointly associated with SHBG levels in the four assays with overall R2=0.215-0.293 and P10(-6). In one immunological SHBG assay each, abdominal visceral fat and adiponectin were weak multivariates also.Immunological and mass spectrometric SHBG assays yield both consistent and inconsistent correlations with key metabolic variables in healthy men, thereby potentially explaining earlier inconsistencies in the literature.

Objective To determine the distribution of, and racial differences in, changes in prostate-specific antigen (PSA) from a population-based sample of men. Materials and Methods Data from 2 prospective cohort studies of a random sample of white men, aged 40-79 years in 1990, followed biennially through 2007, and African American men, aged 40-79 years in 1996, followed through 2000, were examined to assess the longitudinal changes in PSA concentrations. Serum PSA levels were determined at each examination for both cohorts and observations after a diagnosis of prostate cancer or treatment of benign prostatic hyperplasia were censored. The observed and estimated annual percentage of change in the serum PSA levels were examined by race. Results At baseline, the median PSA level in the white men did not differ from the median level observed in the African American men (white men 0.9 ng/mL; African American men 0.9 ng/mL; P = .48). However, African American men had a much more rapid increase in the PSA level over time compared with the white men (median annual percent change in PSA for white men 3.6%/y, African American men 7.9%/y; P Conclusion These data suggest that African American men have more rapid rates of change in the PSA levels over time. If the difference in the rate of changes between African American and white men is an early indicator of future prostate cancer diagnosis, earlier detection in African American men could help to alleviate the racial disparities in prostate cancer diagnosis and mortality.

Background: A statement of measurement uncertainty describes the quality of a clinical assay analysis result, and uncertainty models of clinical assays can be used to evaluate and optimize laboratory protocols designed to minimize the measurement uncertainty associated with an assay. In this study, we propose a methodology to lend systematic structure to the uncertainty modeling process. Methods: Clinical laboratory assays are typically classified based on the chemical reaction involved, and therefore, based on the assay analysis methodology. We use this fact to demonstrate that uncertainty models for assays within the same category are structurally identical in all respects except for the values of certain model parameters. This is accomplished by building uncertainty models for assays belonging to two categories – substrate assays based on optical absorbance analysis of endpoint reactions, and ion selective electrode (ISE) assays based on potentiometric measurements of electromotive force. Results: Uncertainty models for the substrate assays and the ISE assays are built, and for each category, a general mathematical framework for the uncertainty model is developed. The parameters of the general framework that vary from assay to assay for each category are identified and listed. Conclusions: Estimates of measurement uncertainty from the models were compared with estimates of uncertainty from quality control data from the clinical laboratory. We demonstrate that building a general modeling framework for each assay category and plugging in parameter values for each assay is sufficient to generate uncertainty models for an assay within a given category.

This article proposes analytic performance goals for five quality indicators: precision, trueness, linearity, detection limits, and consistency across instruments and time. We defined our goals using methods linked to clinical practice data. Goals for desirable precision and trueness are based on biological variation. Linearity goals are related to total error recommendations. Detection limit goals are derived from 0.1 percentile of patient values. Goals for consistency are derived from the variability of distributions of patient test values. Data were collected and evaluated for each of these quality indicators for 46 chemistry tests measured on the Roche cobas 8000 analyzer.

Available data associate lipids concentrations in men with body mass index, anabolic steroids, age, and certain cytokines. Data were less clear in women, especially across the full adult lifespan, and when segmented by premenopausal and postmenopausal status. Subjects: 120 healthy women (60 premenopausal and 60 postmenopausal) in Olmsted County, MN, USA, a stable well studied clinical population. Dependent variables: measurements of 10 h fasting high-density lipoprotein cholesterol, total cholesterol, low-density lipoprotein cholesterol, and triglycerides. Independent variables: testosterone, estrone, estradiol, 5-alpha-dihydrotestosterone, and sex-hormone binding globulin (by mass spectrometry); insulin, glucose, and albumin; abdominal visceral, subcutaneous, and total abdominal fat [abdominal visceral fat, subcutaneous fat, total abdominal fat by computerized tomography scan]; and a panel of cytokines (by enzyme-linked immunosorbent assay). Multivariate forward-selection linear-regression analysis was applied constrained to P

Objectives: The IFCC Committee on Reference Intervals and Decision Limits coordinated a global multicenter study on reference values (RVs) to explore rational and harmonizable procedures for derivation of reference intervals (Rls) and investigate the feasibility of sharing RIs through evaluation of sources of variation of RVs on a global scale. Methods: For the common protocol, rather lenient criteria for reference individuals were adopted to facilitate harmonized recruitment with planned use of the latent abnormal values exclusion (LAVE) method. As of July 2015, 12 countries had completed their study with total recruitment of 13,386 healthy adults. 25 analytes were measured chemically and 25 immunologically. A serum panel with assigned values was measured by all laboratories. Rls were derived by parametric and nonparametric methods. Results: The effect of LAVE methods is prominent in analytes which reflect nutritional status, inflammation and muscular exertion, indicating that inappropriate results are frequent in any country. The validity of the parametric method was confirmed by the presence of analyte-specific distribution patterns and successful Gaussian transformation using the modified Box-Cox formula in all countries. After successful alignment of RVs based on the panel test results, nearly half the analytes showed variable degrees of between-country differences. This finding, however, requires confirmation after adjusting for BMI and other sources of variation. The results are reported in the second part of this paper. Conclusion: The collaborative study enabled us to evaluate rational methods for deriving Rls and comparing the RVs based on real-world datasets obtained in a harmonized manner. Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) Japan Society for the Promotion of Science - 24256003

Objectives: The intent of this study, based on a global multicenter study of reference values (RVs) for serum analytes was to explore biological sources of variation (SVs) of the RVs among 12 countries around the world. Methods: As described in the first part of this paper, RVs of 50 major serum analytes from 13,396 healthy individuals living in 12 countries were obtained. Analyzed in this study were 23 clinical chemistry analytes and 8 analytes measured by immunoturbidimetry. Multiple regression analysis was performed for each gender, country by country, analyte by analyte, by setting four major SVs (age, BMI, and levels of drinking and smoking) as a fixed set of explanatory variables. For analytes with skewed distributions, log-transformation was applied. The association of each source of variation with RVs was expressed as the partial correlation coefficient (rp). Results: Obvious gender and age-related changes in the RVs were observed in many analytes, almost consistently between countries. Compilation of age-related variations of RVs after adjusting for between-country differences revealed peculiar patterns specific to each analyte. Judged from the rp, BMI related changes were observed for many nutritional and inflammatory markers in almost all countries. However, the slope of linear regression of BMI vs. RV differed greatly among countries for some analytes. Alcohol and smoking-related changes were observed less conspicuously in a limited number of analytes. Conclusion: The features of sex, age, alcohol, and smoking-related changes in RVs of the analytes were largely comparable worldwide. The finding of differences in BMI-related changes among countries in some analytes is quite relevant to understanding ethnic differences in susceptibility to nutritionally related diseases. Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) Japan Society for the Promotion of Science - 24256003

Background: Updated National Academy of Clinical Biochemistry (NACB) Laboratory Medicine Practice Guidelines for the use of tumor markers in the clinic have been developed.Methods: Published reports relevant to use of tumor markers for 5 cancer sites—testicular, prostate, colorectal, breast, and ovarian—were critically reviewed.Results: For testicular cancer, α-fetoprotein, human chorionic gonadotropin, and lactate dehydrogenase are recommended for diagnosis/case finding, staging, prognosis determination, recurrence detection, and therapy monitoring. α-Fetoprotein is also recommended for differential diagnosis of nonseminomatous and seminomatous germ cell tumors. Prostate-specific antigen (PSA) is not recommended for prostate cancer screening, but may be used for detecting disease recurrence and monitoring therapy. Free PSA measurement data are useful for distinguishing malignant from benign prostatic disease when total PSA is Conclusions: Implementation of these recommendations should encourage optimal use of tumor markers.

Free human kallikrein 2 (free-hK2) and hK2 pro-form (pro-hK2) have been found to be increased in tumor tissues and serum from patients with prostate cancer. We established semiautomatic assays for free-hK2 and pro-hK2 using a research version of the Beckman Coulter ACCESS2 system. Serum samples from a cohort of 189 men undergoing radical prostatectomy for known high-risk disease were assayed for Prostate-Specific Antigen (PSA), free-PSA, free-hK2, and pro-hK2. Univariate Cox regression and multivariate models were used to predict both Gleason scores and progression-free survival (PFS). Free-hk2 levels ≥80 ng/L were predictive of both Gleason scores ≥7 ( p = 0.04) and PFS ( p = 0.03). PSA ≥8.0 µg/L also was predictive of PFS ( p = 0.02). However, neither % free-PSA nor pro-hK2, when treated as continuous or cutoff variables were associated with Gleason score or PFS. Multivariable models showed that clinical stage T1c versus T2/T3, Gleason score ≥7, and PSA ≥8.0 µg/L or clinical stage T1c versus T2/T3, Gleason scores ≥7, and free-hK2 ≥80 ng/L were among the best models predicting PFS. Both free-hK2 and PSA in conjunction with clinical stage and Gleason score are good predictors of PFS in prostate cancer.

This paper, prepared by the EFLM Task and Finish Group on Allocation of laboratory tests to different models for performance specifications (TFG-DM), is dealing with criteria for allocating measurands to the different models for analytical performance specifications (APS) recognized in the 1st EFLM Strategic Conference Consensus Statement. Model 1, based on the effect of APS on clinical outcome, is the model of choice for measurands that have a central role in the decision-making of a specific disease or clinical situation and where cut-off/decision limits are established for either diagnosing, screening or monitoring. Total cholesterol, glucose, HbA1c, serum albumin and cardiac troponins represent practical examples. Model 2 is based on components of biological variation and should be applied to measurands that do not have a central role in a specific disease or clinical situation, but where the concentration of the measurand is in a steady state. This is best achieved for measurands under strict homeostatic control in order to preserve their concentrations in the body fluid of interest, but it can also be applied to other measurands that are in a steady state in biological fluids. In this case, it is expected that the “noise” produced by the measurement procedure will not significantly alter the signal provided by the concentration of the measurand. This model especially applies to electrolytes and minerals in blood plasma (sodium, potassium, chloride, bicarbonate, calcium, magnesium, inorganic phosphate) and to creatinine, cystatin C, uric acid and total protein in plasma. Model 3, based on state-of-the-art of the measurement, should be used for all the measurands that cannot be included in models 1 or 2.

Background: Laboratory protocols used to calibrate commercial clinical chemistry systems affect test result quality. Mathematical models of system uncertainty can be developed using performance parameters provided by the manufacturer for various subsystems. These models can be used to evaluate protocols for specific laboratory operations. Methods: A mathematical model was developed to estimate the uncertainty inherent in the Roche Diagnostics P-Modular system, and included uncertainties associated with the sample and reagent pipettes, spectrometer and the calibration process. The model was then used to evaluate various alternate calibration protocols: calibration based on mean of replicate measurements (n=1–6) and calibration based on conditional acceptance when the following quality control specimen was within one standard deviation of target. The effect of calibrator concentrations on assay measurement uncertainty was also studied, and calibrator concentrations that minimize uncertainty at a specific concentration were identified. Results: The simulation model produced uncertainty estimates of 3.5% for the serum cholesterol assay and identified sample pipette (40%) and spectrometer (21%) as the largest contributors to measurement uncertainty. Each additional replicate calibrator measurements result in diminishing reductions in measurement uncertainty, with maximum reductions (19%) achieved with five replicate measurements. The conditional acceptance of calibration only when the control was within 1s resulted in an 18% reduction. Conclusions: The model can be used to evaluate the utility of laboratory protocols and establish realistic assay performance targets. The model also can help instrument manufacturers and laboratorians identify major contributors to assay measurement uncertainty, which helps improve performance in future assay systems.

We describe cross-sectional associations of benign prostate specific antigen with clinical urological measures and examined the risk of future urological outcomes in 2 population based cohorts of black and white men, respectively.Two population based cohort studies were established to characterize the natural history of and risk factors for prostate disease progression in white and black male residents of Olmsted County, Minnesota, and Genesee County, Michigan, respectively.The benign prostate specific antigen distribution was similar in black men at a median of 32.9 pg/ml (25th, 75th percentiles 17.3, 68.0) and white men at a median of 32.2 pg/ml (25th, 75th percentiles 16.6, 68.9, respectively). However, it was much lower than in previous reports. For Olmsted County men in the upper quartile of benign prostate specific antigen there was a fifteenfold increased risk of prostate cancer (HR 14.6, 95% CI 3.1-68.6) and a twofold higher risk of treatment for benign prostatic hyperplasia (HR 2.2, 95% CI 1.2-4.2) after adjusting for age. After additional adjustment for baseline prostate specific antigen the association between benign prostate specific antigen and prostate cancer risk was attenuated but remained almost ninefold higher for men in the upper quartile of benign prostate specific antigen (HR 8.7, 95% CI 1.8-42.4). The twofold higher risk of treatment for benign prostatic hyperplasia also remained after adjusting for baseline prostate specific antigen for men in the upper benign prostate specific antigen quartile (HR 1.9, 95% CI 0.9-4.0).Results suggest that increased benign prostate specific antigen may help identify men with prostate cancer and those at risk for benign prostatic hyperplasia treatment.

We provide cross-sectional normative data on [-2]proenzyme-prostate specific antigen from the Olmsted County Study of Urinary Symptoms and Health Status among Men, and the Flint Men's Health Study. We also describe associations with clinical urological measures and the risk of prostate cancer diagnosis.Measurements of [-2]proenzyme-prostate specific antigen were obtained from 420 white men from Olmsted County, Minnesota, and 328 black men from Genesee County, Michigan. Cross-sectional associations between [-2]proenzyme-prostate specific antigen and prostate enlargement/elevated prostate specific antigen were assessed. Cox proportional hazard models were used to assess associations between [-2]proenzyme-prostate specific antigen and the incident diagnosis of prostate cancer.Baseline [-2]proenzyme-prostate specific antigen was slightly higher in black men at a median of 6.3 pg/ml (25th, 75th percentiles 4.1, 8.9) than in white men at a median of 5.6 pg/ml (25th, 75th percentiles 3.9, 7.7, respectively, p = 0.01). Baseline [-2]proenzyme-prostate specific antigen was highly predictive of biopsy confirmed prostate cancer in the Olmsted County Study cohort. Relative to men in the [-2]proenzyme-prostate specific antigen lower quartile those in the upper quartile were at almost eightfold increased risk for prostate cancer (HR 7.8, 95% CI 2.2-27.8) after adjusting for age and baseline prostate specific antigen.In these cohorts of community dwelling black and white men [-2]proenzyme-prostate specific antigen was much lower than in previous studies. These data suggest that [-2]proenzyme-prostate specific antigen may help identify prostate cancer in men with serum prostate specific antigen in an indeterminate range, although the reference ranges for white and black men may differ slightly.

Clinical laboratory testing is a vital component of many stages of the medical decision making process, and therefore information about the quality of the measurement process is critical to the medical decision-making process. A statement of uncertainty of the result of a laboratory test provides this information. To obtain this information, the clinical laboratory measurement process is conceptualized as a self-contained system, the concept of process phases is introduced, and a broadly applicable algorithm describing the modeling and estimation of uncertainty of such processes is developed. The article discusses how performance specifications for individual components can be used to characterize their uncertainty, and uses Monte Carlo simulation to integrate these individual component uncertainties into a net system uncertainty. The proposed approach is illustrated by developing a mathematical model of the serum cholesterol assay analysis procedure. The uses of the model are to: 1) simulate, evaluate an...

Objective To determine the distribution of longitudinal changes in serum prostate-specific antigen (PSA) levels from a population-based sample of men.

Purpose Hereditary hyperferritinemia cataract syndrome (HHCS), an autosomal-dominant disorder characterized by hyperferritinemia and bilateral cataracts, is caused by mutations in the iron-responsive element of the ferritin light chain ( FTL ) gene. The purpose of this study is to describe the genotypic and phenotypic manifestations of HHCS observed in 2 large sets of unrelated American families. Methods Forty-five patients were recruited from 2 unrelated families. Each underwent ophthalmological and general physical evaluation as well as laboratory testing of serum ferritin, iron, transferrin saturation, and total iron binding capacity. Serum DNA was evaluated for mutations by DNA amplification and sequencing of the FTL gene. Results Numerous cortical and nuclear white opacities in a stellate pattern occurred in 22 affected individuals and were the only clinical manifestation of HHCS. Of the 22, 16 (73%) demonstrated >1.00 D of astigmatism. Genetic analysis revealed mutation G32A in Pedigree 1 and mutation G32T in Pedigree 2, both heterozygous and located in the iron-responsive element of the ferritin light chain mRNA. Serum ferritin levels of affected subjects ranged from 555 to 2,453 μg/L (normal range, 24–336 μg/L male, 11–307 μg/L female), with greater ferritin levels and more severe cataracts associated with mutation G32A. Conclusions Most clinical and genetic findings from these families are consistent with previous reports of HHCS. Astigmatism, previously not associated with HHCS, was present in the majority. Ferritin levels and age of cataract surgery varied among subjects with both FTL gene mutations, suggesting that phenotypic variability is modulated by other genetic or environmental factors.

The development of Hot-Dry Rock (HDR) geothermal energy in Australia with drillings to some kilometres depth yields an impetus for deep stress logging. For the Olympic Dam HDR-project, borehole Blanche-1 was drilled to almost 2 km depth and provided the possibility to estimate the in situ stresses within the granitic borehole section by the analysis of borehole breakouts and core discing, as well as by hydraulic fracturing combined with acoustic borehole televiewer logging for fracture orientation determination. Although the stress magnitudes derived by the different methods deviate significantly, they clearly indicate for the depth range between 800 and 1,740 m a compressional stress regime of S v ≤ S h

Purpose: Prostate specific antigen and free prostate specific antigen have limited specificity to detect clinically significant, curable prostate cancer, leading to unnecessary biopsy, and detection and treatment of some indolent tumors. Specificity to detect clinically significant prostate cancer may be improved by [-2]pro-prostate specific antigen. We evaluated [-2]pro-prostate specific antigen, free prostate specific antigen and prostate specific antigen using the formula, ([-2]pro-prostate specific antigen/free prostate specific antigen X prostate specific antigen(1/2)) to enhance specificity to detect overall and high grade prostate cancer. Materials and Methods: We enrolled 892 men with no history of prostate cancer, normal rectal examination, prostate specific antigen 2 to 10 ng/ml and 6-core or greater prostate biopsy in a prospective multi-institutional trial. We examined the relationship of serum prostate specific antigen, free-to-total prostate specific antigen and the prostate health index with biopsy results. Primary end points were specificity and AUC using the prostate health index to detect overall and Gleason 7 or greater prostate cancer on biopsy compared with those of free-to-total prostate specific antigen. Results: In the 2 to 10 ng/ml prostate specific antigen range at 80% to 95% sensitivity the specificity and AUC (0.703) of the prostate health index exceeded those of prostate specific antigen and free-to-total prostate specific antigen. An increasing prostate health index was associated with a 4.7-fold increased risk of prostate cancer and a 1.61-fold increased risk of Gleason score greater than or equal to 4 + 3 = 7 disease on biopsy. The AUC of the index exceeded that of free-to-total prostate specific antigen (0.724 vs 0.670) to discriminate prostate cancer with Gleason 4 or greater + 3 from lower grade disease or negative biopsy. Prostate health index results were not associated with age and prostate volume. Conclusions: The prostate health index may be useful in prostate cancer screening to decrease unnecessary biopsy in men 50 years old or older with prostate specific antigen 2 to 10 ng/ml and negative digital rectal examination with minimal loss in sensitivity.