June Pierce

TRANSVAGINAL ULTRASONOGRAPHY COMPARED WITH ENDOMETRIAL BIOPSY FOR THE DETECTION OF ENDOMETRIAL DISEASE

BACKGROUND Transvaginal ultrasonography is a noninvasive procedure that may be used to detect endometrial disease. However, its usefulness in screening for asymptomatic disease in postmenopausal women before or during treatment with estrogen or estrogen-progesterone replacement is not known. METHODS We compared the sensitivity and specificity of transvaginal ultrasonography and endometrial biopsy for the detection of endometrial disease in 448 postmenopausal women who received estrogen alone, cyclic or continuous estrogen-progesterone, or placebo for three years. RESULTS Concurrent ultrasonographic and biopsy results were available for 577 examinations in the 448 women, 99 percent of whom were undergoing routine annual follow-up. Endometrial thickness was less than 5 mm in 45 percent of the examinations, 5 to 10 mm in 41 percent, more than 10 mm in 12 percent, and not measured in 2 percent, and it was higher in the women receiving estrogen alone than in the other groups. Biopsy detected 11 cases of serious disease: 1 case of adenocarcinoma, 2 cases of atypical simple hyperplasia, and 8 cases of complex hyperplasia. Biopsy also detected simple hyperplasia in 20 cases. At a threshold value of 5 mm for endometrial thickness, transvaginal ultrasonography had a positive predictive value of 9 percent for detecting any abnormality, with 90 percent sensitivity, 48 percent specificity, and a negative predictive value of 99 percent. With this threshold, a biopsy would be indicated in more than half the women, only 4 percent of whom had serious disease. CONCLUSIONS Transvaginal ultrasonography has a poor positive predictive value but a high negative predictive value for detecting serious endometrial disease in asymptomatic postmenopausal women.

HLA genotyping in the international Type 1 Diabetes Genetics Consortium

Background Although human leukocyte antigen (HLA) DQ and DR loci appear to confer the strongest genetic risk for type 1 diabetes, more detailed information is required for other loci within the HLA region to understand causality and stratify additional risk factors. The Type 1 Diabetes Genetics Consortium (T1DGC) study design included high-resolution genotyping of HLA-A, B, C, DRB1, DQ, and DP loci in all affected sibling pair and trio families, and cases and controls, recruited from four networks worldwide, for analysis with clinical phenotypes and immunological markers. Purpose In this article, we present the operational strategy of training, classification, reporting, and quality control of HLA genotyping in four laboratories on three continents over nearly 5 years. Methods Methods to standardize HLA genotyping at eight loci included: central training and initial certification testing; the use of uniform reagents, protocols, instrumentation, and software versions; an automated data transfer; and the use of standardized nomenclature and allele databases. We implemented a rigorous and consistent quality control process, reinforced by repeated workshops, yearly meetings, and telephone conferences. Results A total of 15,246 samples have been HLA genotyped at eight loci to four-digit resolution; an additional 6797 samples have been HLA genotyped at two loci. The genotyping repeat rate decreased significantly over time, with an estimated unresolved Mendelian inconsistency rate of 0.21%. Annual quality control exercises tested 2192 genotypes (4384 alleles) and achieved 99.82% intra-laboratory and 99.68% inter-laboratory concordances. Limitations The chosen genotyping platform was unable to distinguish many allele combinations, which would require further multiple stepwise testing to resolve. For these combinations, a standard allele assignment was agreed upon, allowing further analysis if required. Conclusions High-resolution HLA genotyping can be performed in multiple laboratories using standard equipment, reagents, protocols, software, and communication to produce consistent and reproducible data with minimal systematic error. Many of the strategies used in this study are generally applicable to other large multi-center studies. Clinical Trials 2010; 7: S75—S87. http:// ctj.sagepub.com

Does withdrawal of antihypertensive medication increase the risk of cardiovascular events

The Fifth Report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure recommends that attempts to discontinue antihypertensive drug therapy be considered after blood pressure (BP) has been controlled for 1 year. However, discontinuation of drug therapy could unmask underlying conditions and precipitate clinical cardiovascular events. The Trial of Nonpharmacologic Interventions in the Elderly (TONE) was a clinical trial of the efficacy of weight loss and/or sodium reduction in controlling BP after withdrawal of drug therapy in patients with a BP<145/85 mm Hg on 1 antihypertensive medication. Of 975 participants, 886 entered the drug withdrawal phase of the trial and 774 were successfully withdrawn from their medications. Thirty-three events (stroke, transient ischemic attack, myocardial infarction, arrhythmia, congestive heart failure, angina, other) occurred between randomization and the onset of drug withdrawal (median time 3.6 months), 57 events occurred either during or after drug withdrawal (14.0 months), and 36 events occurred after resumption of antihypertensive therapy (15.9 months). Event rates per 100 person-years were 5.5, 5.5, and 6.8 for the 3 time periods (p = 0.84) in the nonoverweight group and 7.2, 5.2, and 5.6 (p = 0.08) in the overweight group. The study shows that antihypertensive medication can be safely withdrawn in older persons without clinical evidence of cardiovascular disease who do not have diastolic pressure ≥150/90 mm Hg at withdrawal, providing that good BP control can be maintained with nonpharmacologic therapy.The Fifth Report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure recommends that attempts to discontinue antihypertensive drug therapy be considered after blood pressure (BP) has been controlled for 1 year. However, discontinuation of drug therapy could unmask underlying conditions and precipitate clinical cardiovascular events. The Trial of Nonpharmacologic Interventions in the Elderly (TONE) was a clinical trial of the efficacy of weight loss and/or sodium reduction in controlling BP after withdrawal of drug therapy in patients with a BP< 145/85 mm Hg on 1 antihypertensive medication. Of 975 participants, 886 entered the drug withdrawal phase of the trial and 774 were successfully withdrawn from their medications. Thirty-three events (stroke, transient ischemic attack, myocardial infarction, arrhythmia, congestive heart failure, angina, other) occurred between randomization and the onset of drug withdrawal (median time 3.6 months), 57 events occurred either during or after drug withdrawal (14.0 months), and 36 events occurred after resumption of antihypertensive therapy (15.9 months). Event rates per 100 person-years were 5.5, 5.5, and 6.8 for the 3 time periods (p=0.84) in the nonoverweight group and 7.2, 5.2, and 5.6 (p=0.08) in the overweight group. The study shows that antihypertensive medication can be safely withdrawn in older persons without clinical evidence of cardiovascular disease who do not have diastolic pressure > or = 150/90 mm Hg at withdrawal, providing that good BP control can be maintained with nonpharmacologic therapy.

Serial audiometry in a clinical trial of AIED treatment.

Objective: We analyzed pure-tone and speech audiometric results from a prospective trial of anti-inflammatory treatment of subjects with active autoimmune inner ear disease (AIED).We sought to characterize the pattern and size of the treatment effect as reflected in clinical audiometry and to identify audiometric predictors of response to steroid treatment of AIED. Subjects: Adult participants demonstrated clinically established criteria for AIED (n = 116). Eligibility required audiometric evidence of active AIED as indicated by idiopathic sensorineural hearing loss with threshold elevations within 3 months of enrollment. Methods: We evaluated audiometric changes after 4 weeks of treatment with pharmacologic doses (60 mg/day) of prednisone. We examined the relationship between audiometric pure-tone thresholds at baseline and changes in word intelligibility score (WIS) using parametric and nonparametric analyses. Magnitudes of change were assessed using independent or paired t-tests. Separate analyses were performed on subgroups that did or did not show improved WIS score with steroid treatment. Results: Overall mean pure-tone averages improved from baseline to closeout of prednisone treatment in better hearing ears from 52.4 to 48.3 dB (p < .0001). Mean WIS improved in the better ear from 71.4% to 78.1% (p < .0001). Of pure-tone measures, only the six-tone average showed significant correlation with both the absolute improvements in WIS and with the percentage change in WIS after treatment. Individual frequencies at baseline showed no significant relationship with changes in WIS score after treatment. In 69 (59.5%) of 116 subjects, WIS improved (range, 2-80%) in the better ear. In these subjects, the baseline pure-tone thresholds and pure-tone averages correlated significantly and positively with improvement in WIS. Conclusions: Steroid treatment in AIED-mediated hearing loss produce variable but significant hearing gains. Neither a focal, cochleotopic region of greatest vulnerability to AIED nor frequency-specific amenability to treatment were evident. We did observe that analysis of predictors and the degree of treatment effect vary with different approaches to measuring change in the WIS. Depending on the approach adopted, the size of the treatment effect may be greatest across intermediate hearing levels at baseline. These observations offer an audiometric database that may enable greater precision in judging clinically meaningful parameters for future studies of AIED treatment and other interventions for sensorineural hearing loss.

Designing and implementing sample and data collection for an international genetics study: the Type 1 Diabetes Genetics Consortium (T1DGC)

Background and Purpose The Type 1 Diabetes Genetics Consortium (T1DGC) is an international project whose primary aims are to: (a) discover genes that modify type 1 diabetes risk; and (b) expand upon the existing genetic resources for type 1 diabetes research. The initial goal was to collect 2500 affected sibling pair (ASP) families worldwide. Methods T1DGC was organized into four regional networks (Asia-Pacific, Europe, North America, and the United Kingdom) and a Coordinating Center. A Steering Committee, with representatives from each network, the Coordinating Center, and the funding organizations, was responsible for T1DGC operations. The Coordinating Center, with regional network representatives, developed study documents and data systems. Each network established laboratories for: DNA extraction and cell line production; human leukocyte antigen genotyping; and autoantibody measurement. Samples were tracked from the point of collection, processed at network laboratories and stored for deposit at National Institute for Diabetes and Digestive and Kidney Diseases (NIDDK) Central Repositories. Phenotypic data were collected and entered into the study database maintained by the Coordinating Center. Results T1DGC achieved its original ASP recruitment goal. In response to research design changes, the T1DGC infrastructure also recruited trios, cases, and controls. Results of genetic analyses have identified many novel regions that affect susceptibility to type 1 diabetes. T1DGC created a resource of data and samples that is accessible to the research community. Limitations Participation in T1DGC was declined by some countries due to study requirements for the processing of samples at network laboratories and/or final deposition of samples in NIDDK Central Repositories. Re-contact of participants was not included in informed consent templates, preventing collection of additional samples for functional studies. Conclusions T1DGC implemented a distributed, regional network structure to reach ASP recruitment targets. The infrastructure proved robust and flexible enough to accommodate additional recruitment. T1DGC has established significant resources that provide a basis for future discovery in the study of type 1 diabetes genetics. Clinical Trials 2010; 7: S5—S32. http://ctj.sagepub.com

Age-Stratified QTL Genome Scan Analyses for Anthropometric Measures

With the availability of longitudinal data, age-specific (stratified) or age-adjusted genetic analyses have the potential to localize different putative trait influencing loci. If age does not influence the locus-specific penetrance function within the range examined, age-stratified analyses will tend to yield comparable results for an individual trait. However, age-stratified results should vary across age strata when the locus-specific penetrance function is age dependent. In this paper, age-stratified and age-adjusted quantitative trait loci (QTL) linkage analyses were contrasted for height, weight, body mass index (BMI), and systolic blood pressure on a subset of the Framingham Heart Study. The strata comprised individuals with data present in each of three age groups: 31–49, 50–60, 61–79. Genome-wide QTL analyses were performed using SOLAR. Over all ages, a linkage signal for height was detected on chromosome 14q11.2 near marker GATA74E02A (LOD for ages 31–49 = 2.38, LOD for ages 50–60 = 1.84, LOD for ages 61–79 = 2.45). Evidence of linkage to BMI in the 31–49 age group was found on chromosome 3q22 (GATA3C02, LOD = 2.89, p = 0.0003) at the same location as the signal for weight (LOD = 3.10, p = 0.0002). Linkage was also supported on chromosome 1p22.1 for BMI (LOD = 2.21, p = 0.0014) and weight (LOD = 2.47, p = 0.0007) in the 31–49 age group. Our age-stratified results suggest that QTL that are expressed over long periods of time and affecting multiple, correlated traits may be identified using genome scan and variance-component methodology to help detect early and/or late gene expression.

Age at menopause in women participating in the postmenopausal estrogen/progestins interventions (PEPI) trial: An example of bias introduced by selection criteria

Our objective is to illustrate the bias introduced in assessing factors associated with age at menopause when the population sample has been selected using restricted criteria, i.e. number of years since menopause, by using a cross-sectional analysis of baseline data from a population-based randomized clinical trial. The participants were women who participated in the Postmenopausal Estrogen/Progestins Intervention (PEPI) trial, had not had a hysterectomy, were between 45 and 64 years old, and were menopausal for at least 1 but not greater than 10 years. The outcome measures were self-reported age at menopause and factors thought to be associated with it, including smoking, alcohol use, oral contraceptive use, number of pregnancies, education, income, body mass index, waist-hip ratio, thigh girth, and systolic and diastolic blood pressures. At entry, the mean age of the 601 women was 56.2 years. Mean age at menopause was 51.0 years. Chronologic (current) age was strongly correlated with age at menopause (r = 0.74, p = 0.0001). In bivariate analyses, factors associated with younger age at menopause were ever-use of cigarettes, former oral contraceptive use, and higher thigh girth; factors associated with later age at menopause were greater number of pregnancies, higher waist-hip ratio, and higher systolic blood pressure. After stratification by 5-year age intervals, these associations were no longer statistically significant. Because of restricted sampling, an artificial association was observed between chronologic age and age at time of menopause. This artifact made it difficult to distinguish between factors associated with chronologic age and those that may be independently associated with menopause. Failure to recognize this bias could lead to erroneous conclusions.

Measurement of islet cell antibodies in the Type 1 Diabetes Genetics Consortium: efforts to harmonize procedures among the laboratories.

Background and Purpose Three network laboratories measured antibodies to islet autoantigens. Antibodies to glutamic acid decarboxylase (GAD65 [GADA]) and the intracellular portion of protein tyrosine phosphatase (IA-2ic [IA-2A]) were measured by similar, but not identical, methods in samples from participants in the Type 1 Diabetes Genetics Consortium (T1DGC). Methods All laboratories used radiobinding assays to detect antibodies to in vitro transcribed and translated antigen, but with different local standards, calibrated against the World Health Organization (WHO) reference reagent. Using a common method to calculate WHO units/mL, we compared results reported on samples included in the Diabetes Autoantibody Standardization Program (DASP), and developed standard methods for reporting in WHO units/mL. We evaluated intra-assay and inter-assay coefficient of variation (CV) in blind duplicate samples and assay comparability in four DASP workshops. Results Values were linearly related in the three laboratories for both GADA and IA-2A, and intra-assay technical errors for values within the standard curve were below 13% for GADA and below 8.5% for IA-2A. Correlations in samples tested 1—2 years apart were >97%. Over the course of the study, internal CVs were 10—20% with one exception, and the laboratories concordantly called samples GADA or IA-2A positive or negative in 96.7% and 99.6% of duplicates within the standard curve. Despite acceptable CVs and general concordance in ranking samples, the laboratories differed markedly in absolute values for GADA and IA-2A reported in WHO units/mL in DASP over a large range of values. Limitations With three laboratories using different assay methods (including calibrators), consistent values among them could not be attained. Conclusions Modifications in the assays are needed to improve comparability of results expressed as WHO units/mL across laboratories. It will be essential to retain high intra- and inter-assay precision, sensitivity and specificity and to confirm the accuracy of harmonized methods. Clinical Trials 2010; 7: S56—S64. http:// ctj.sagepub.com

Collection and processing of whole blood for transformation of peripheral blood mononuclear cells and extraction of DNA: the Type 1 Diabetes Genetics Consortium

Background and Purpose To yield large amounts of DNA for many genotype analyses and to provide a renewable source of DNA, the Type 1 Diabetes Genetics Consortium (T1DGC) harvested DNA and peripheral blood mononuclear cells (PBMCs) from individuals with type 1 diabetes and their family members in several regions of the world. Methods DNA repositories were established in Asia-Pacific, Europe, North America, and the United Kingdom. To address region-specific needs, different methods and sample processing techniques were used among the laboratories to extract and to quantify DNA and to establish Epstein-Barr virus transformed cell lines. Results More than 98% of the samples of PBMCs were successfully transformed. Approximately 20—25 μg of DNA were extracted per mL of whole blood. Extraction of DNA from the cell pack ranged from 92 to 165 μg per cell pack. In addition, the extracted DNA from whole blood or transformed cells was successfully utilized in each regional human leukocyte antigen genotyping laboratory and by several additional laboratories performing consortium-wide genotyping projects. Limitations Although the isolation of PBMCs was consistent among sites, the measurement of DNA was difficult to harmonize. Conclusions DNA repositories can be established in different regions of the world and produce similar amounts of high-quality DNA for a variety of high-throughput genotyping techniques. Furthermore, even with the distances and time necessary for transportation, highly efficient transformation of PBMCs is possible. For future studies/trials involving several laboratories in different locations, the T1DGC experience includes examples of protocols that may be applicable. In summary, T1DGC has developed protocols that would be of interest to any scientific organization attempting to overcome the logistical problems associated with studies/trials spanning multiple research facilities, located in different regions of the world. Clinical Trials 2010; 7: S65—S74. http://ctj.sagepub.com

The Kidney Outcomes Prediction and Evaluation (KOPE) Study: A Prospective Cohort Investigation of Patients Undergoing Hemodialysis: Study Design and Baseline Characteristics

PURPOSE The purpose of the Kidney Outcomes Prediction and Evaluation (KOPE) study, was to more fully characterize the end-stage renal disease (ESRD) population with respect to social, psychological, and clinical characteristics, and to prospectively study the biomedical, social, and psychological factors that influence a range of ESRD outcomes in a large observational study of black and white patients on hemodialysis. This paper focuses on the KOPE study design as well as characteristics of patients at baseline. METHODS KOPE was a prospective cohort investigation of patients treated at four dialysis centers in Forsyth County, North Carolina. Participants were interviewed at the dialysis centers, semi-annually over a 3 1/2 year period. Prevalent cases who were being treated with hemodialysis at the initiation of the study were enrolled into KOPE. Incident cases were subsequently enrolled as they presented to the participating units for hemodialysis. A total of 304 prevalent and 162 incident cases were enrolled into the study. The baseline health and sociodemographic characteristics of KOPE participants reported in this paper were obtained from medical records and Southeast Kidney Council data. Laboratory values taken within a 30-day interval around the baseline interview are also reported. RESULTS KOPE participants differ from national statistics on race, age, and gender. Differences between KOPE participants and patients living in the region, but who did not participate in the study, can be explained by our recruitment criteria. CONCLUSIONS KOPE will enable the characterization of the ESRD population, identification of factors related to poor outcomes, and identification of opportunities for interventions to prevent death and morbidity.