Lloyd J. Edwards

A Controlled Study of Adenoviral-Vector–Mediated Gene Transfer in the Nasal Epithelium of Patients with Cystic Fibrosis

BACKGROUND Cystic fibrosis is a monogenic disease that deranges multiple systems of ion transport in the airways, culminating in chronic infection and destruction of the lung. The introduction of a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium through gene transfer is an attractive approach to correcting the underlying defects in patients with cystic fibrosis. We tested the feasibility of gene therapy using adenoviral vectors in the nasal epithelium of such patients. METHODS An adenoviral vector containing the normal CFTR complementary DNA in four logarithmically increasing doses (estimated multiplicity of infection, 1, 10, 100, and 1000), or vehicle alone, was administered in a randomized, blinded fashion to the nasal epithelium of 12 patients with cystic fibrosis. Gene transfer was quantitated by molecular techniques that detected the expression of CFTR messenger RNA and by functional measurements of transepithelial potential differences (PDs) to assess abnormalities of ion transport specific to cystic fibrosis. The safety of this treatment was monitored by nasal lavage and biopsy to assess inflammation and vector replication. RESULTS The adenoviral vector was detected in nasal-lavage fluid by culture, the polymerase chain reaction (PCR), or both in a dose-dependent fashion for up to eight days after vector administration. There was molecular evidence of gene transfer by reverse-transcriptase PCR assays or in situ hybridization in five of six patients treated at the two highest doses. However, the percentage of epithelial cells transfected by the vector was very low (< 1 percent), and measurement of PD across the epithelium revealed no significant restoration of chloride transport or normalization of sodium transport. At the lower doses of vector, there were no toxic effects. However, at the highest dose there was mucosal inflammation in two of three patients. CONCLUSIONS In patients with cystic fibrosis, adenoviral-vector-mediated transfer of the CFTR gene did not correct functional defects in nasal epithelium, and local inflammatory responses limited the dose of adenovirus that could be administered to overcome the inefficiency of gene transfer.

Longitudinal analysis of pulmonary function decline in patients with cystic fibrosis

BACKGROUND Chronic progressive lung disease is the most prominent cause of morbidity and death in patients with cystic fibrosis (CF), but severity of lung disease and rate of lung function decline are widely variable. Accurate estimates of decline have been difficult to define and compare because the timing of measurements and duration of follow-up differ in various patient groups. PATIENTS Three hundred sixty-six patients with CF, born from 1960 to 1974, were selected from a CF database birth cohort if they had two or more measurements of pulmonary function, at least one of which was performed before the age of 10 years. METHODS Mixed model regression analysis provided estimates of the average rate of decline of spirometry measurements in subgroups on the basis of survival age, sex, pancreatic status, and genotype. RESULTS Patients who died before the age of 15 years had significantly poorer pulmonary function when first tested and a more rapid decline in pulmonary function thereafter than patients who survived beyond the age of 15 years. In the latter, functional levels at the age of 5 years were normal, but average rates of decline were significantly related to survival age. Female patients had significantly steeper decline than male patients, and those with pancreatic insufficiency had much steeper decline than those with pancreatic sufficiency. In the subset of 197 who survived to 1990 and were subsequently genotyped, rate of decline was greater in those homozygous for the delta F508 mutation, compared with those who were heterozygous for delta F508 or those, who had two other mutations. DISCUSSION All but the most severely affected patients, who died before age 15, appear to have had normal pulmonary function when first tested in early childhood. Pancreatic sufficiency, male gender, and some non-delta F508 mutations are associated with a slower rate of pulmonary function decline. Mixed model analysis is a valuable tool for describing and comparing pulmonary function decline in groups of patients with CF.

A Pilot Study of Aerosolized Amiloride for the Treatment of Lung Disease in Cystic Fibrosis

Excessive active absorption of sodium is a unique abnormality of the airway epithelium in patients with cystic fibrosis. This defect is associated with thickened mucus and poor clearance of airway secretions and may contribute to the pulmonary disease in these patients. To study whether the inhibition of excessive absorption of sodium might affect the course of lung disease in cystic fibrosis, we performed a double-blind, crossover trial comparing aerosolized amiloride (5 mmol per liter; 3.5 ml four times daily), a sodium-channel blocker, with vehicle alone. Fourteen of the 18 adult patients initially enrolled in the study completed the one-year trial (25 weeks for each treatment). The mean (+/- SEM) loss of forced vital capacity (FVC) was reduced from 3.39 +/- 1.13 ml per day during treatment with vehicle alone to 1.44 +/- 0.67 ml per day during treatment with amiloride (P less than 0.04). A measured index of sputum viscosity and elasticity was abnormal during treatment with vehicle alone and improved during treatment with amiloride. Calculated indexes of mucociliary and cough clearance also improved during amiloride treatment. No systemic, respiratory, or subjective toxic effects of amiloride were noted. We conclude from this preliminary study that aerosolized amiloride can be safely administered to adults with cystic fibrosis. The slowing of the loss of FVC and the improvement in sputum viscosity and elasticity suggest a beneficial clinical effect. Aerosolized amiloride deserves further evaluation in the treatment of lung disease in patients with cystic fibrosis.

Improving the Reliability of Stroke Subgroup Classification Using the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) Criteria

Background and Purpose We sought to improve the reliability of the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) classification of stroke subtype for retrospective use in clinical, health services, and quality of care outcome studies. The TOAST investigators devised a series of 11 definitions to classify patients with ischemic stroke into 5 major etiologic/pathophysiological groupings. Interrater agreement was reported to be substantial in a series of patients who were independently assessed by pairs of physicians. However, the investigators cautioned that disagreements in subtype assignment remain despite the use of these explicit criteria and that trials should include measures to ensure the most uniform diagnosis possible. Methods In preparation for a study of outcomes and management practices for patients with ischemic stroke within Department of Veterans Affairs hospitals, 2 neurologists and 2 internists first retrospectively classified a series of 14 randomly selected stroke patients on the basis of the TOAST definitions to provide a baseline assessment of interrater agreement. A 2-phase process was then used to improve the reliability of subtype assignment. In the first phase, a computerized algorithm was developed to assign the TOAST diagnostic category. The reliability of the computerized algorithm was tested with a series of synthetic cases designed to provide data fitting each of the 11 definitions. In the second phase, critical disagreements in the data abstraction process were identified and remaining variability was reduced by the development of standardized procedures for retrieving relevant information from the medical record. Results The 4 physicians agreed in subtype diagnosis for only 2 of the 14 baseline cases (14%) using all 11 TOAST definitions and for 4 of the 14 cases (29%) when the classifications were collapsed into the 5 major etiologic/pathophysiological groupings (&kgr;=0.42; 95% CI, 0.32 to 0.53). There was 100% agreement between classifications generated by the computerized algorithm and the intended diagnostic groups for the 11 synthetic cases. The algorithm was then applied to the original 14 cases, and the diagnostic categorization was compared with each of the 4 physicians’ baseline assignments. For the 5 collapsed subtypes, the algorithm-based and physician-assigned diagnoses disagreed for 29% to 50% of the cases, reflecting variation in the abstracted data and/or its interpretation. The use of an operations manual designed to guide data abstraction improved the reliability subtype assignment (&kgr;=0.54; 95% CI, 0.26 to 0.82). Critical disagreements in the abstracted data were identified, and the manual was revised accordingly. Reliability with the use of the 5 collapsed groupings then improved for both interrater (&kgr;=0.68; 95% CI, 0.44 to 0.91) and intrarater (&kgr;=0.74; 95% CI, 0.61 to 0.87) agreement. Examining each remaining disagreement revealed that half were due to ambiguities in the medical record and half were related to otherwise unexplained errors in data abstraction. Conclusions Ischemic stroke subtype based on published TOAST classification criteria can be reliably assigned with the use of a computerized algorithm with data obtained through standardized medical record abstraction procedures. Some variability in stroke subtype classification will remain because of inconsistencies in the medical record and errors in data abstraction. This residual variability can be addressed by having 2 raters classify each case and then identifying and resolving the reason(s) for the disagreement.

An R2 statistic for fixed effects in the linear mixed model

Statisticians most often use the linear mixed model to analyze Gaussian longitudinal data. The value and familiarity of the R(2) statistic in the linear univariate model naturally creates great interest in extending it to the linear mixed model. We define and describe how to compute a model R(2) statistic for the linear mixed model by using only a single model. The proposed R(2) statistic measures multivariate association between the repeated outcomes and the fixed effects in the linear mixed model. The R(2) statistic arises as a 1-1 function of an appropriate F statistic for testing all fixed effects (except typically the intercept) in a full model. The statistic compares the full model with a null model with all fixed effects deleted (except typically the intercept) while retaining exactly the same covariance structure. Furthermore, the R(2) statistic leads immediately to a natural definition of a partial R(2) statistic. A mixed model in which ethnicity gives a very small p-value as a longitudinal predictor of blood pressure (BP) compellingly illustrates the value of the statistic. In sharp contrast to the extreme p-value, a very small R(2) , a measure of statistical and scientific importance, indicates that ethnicity has an almost negligible association with the repeated BP outcomes for the study.

Factors Associated With Decisions to Undergo Surgery Among Patients With Newly Diagnosed Early-Stage Lung Cancer

CONTEXT Lung cancer is the leading cause of cancer death in the United States. Surgical resection for stage I or II non-small cell cancer remains the only reliable treatment for cure. Patients who do not undergo surgery have a median survival of less than 1 year. Despite the survival disadvantage, many patients with early-stage disease do not receive surgical care and rates are even lower for black patients. OBJECTIVES To identify potentially modifiable factors regarding surgery in patients newly diagnosed with early-stage lung cancer and to explore why blacks undergo surgery less often than whites. DESIGN, SETTING, AND PATIENTS Prospective cohort study with patients identified by pulmonary, oncology, thoracic surgery, and generalist practices in 5 communities through study referral or computerized tomography review protocol. A total of 437 patients with biopsy-proven or probable early-stage lung cancer were enrolled between December 2005 and December 2008. Before establishment of treatment plans, patients were administered a survey including questions about trust, patient-physician communication, attitudes toward cancer, and functional status. Information about comorbid illnesses was obtained through chart audits. MAIN OUTCOME MEASURE Lung cancer surgery within 4 months of diagnosis. RESULTS A total of 386 patients met full eligibility criteria for lung resection surgery. The median age was 66 years (range, 26-90 years) and 29% of patients were black. The surgical rate was 66% for white patients (n = 179/273) compared with 55% for black patients (n = 62/113; P = .05). Negative perceptions of patient-physician communication manifested by a 5-point decrement on a 25-point communication scale (odds ratio [OR], 0.42; 95% confidence interval [CI], 0.32-0.74) and negative perception of 1-year prognosis postsurgery (OR, 0.27; 95% CI, 0.14-0.50; absolute risk, 34%) were associated with decisions against surgery. Surgical rates for blacks were particularly low when they had 2 or more comorbid illnesses (13% vs 62% for <2 comorbidities; OR, 0.04 [95% CI, 0.01-0.25]; absolute risk, 49%) and when blacks lacked a regular source of care (42% with no regular care vs 57% with regular care; OR, 0.20 [95% CI, 0.10-0.43]; absolute risk, 15%). CONCLUSIONS A decision not to undergo surgery by patients with newly diagnosed lung cancer was independently associated with perceptions of communication and prognosis, older age, multiple comorbidities, and black race. Interventions to optimize surgery should consider these factors.

A randomized trial of 3,4-diaminopyridine in Lambert-Eaton myasthenic syndrome

Objectives: The authors report the results of a prospective, placebo-controlled, randomized study to evaluate the effectiveness of 3,4-diaminopyridine (DAP) in patients with Lambert-Eaton myasthenic syndrome (LEMS) and to determine the acute and long-term side effects of DAP. Methods: Twenty-six patients with LEMS completed a two-arm parallel treatment protocol in which DAP, 20 mg three times daily, or placebo was given blindly for 6 days, and a quantitative examination of muscle strength (the quantitative myasthenia gravis [QMG] score) was used as the primary measure of efficacy. After the blinded study, patients were given open-label DAP and monitored for side effects as long as there was symptomatic improvement. Results: Twelve patients took DAP, and 14 took placebo. There was no difference in the age of LEMS onset, gender distribution, incidence of lung cancer, or baseline muscle strength between the patients who were randomly assigned to receive placebo and those randomly assigned to DAP. Statistical analysis using the Wilcoxon’s rank sum test demonstrated that patients who received DAP had a significantly greater improvement in the QMG score and in the summated amplitude of compound muscle action potentials recorded from three sentinel limb muscles. All but one LEMS patient had significant symptomatic improvement from subsequent open-label DAP. Side effects of DAP were negligible, consisting of perioral and digital paresthesia. Laboratory measurements demonstrated no evidence of toxicity affecting liver, renal, hematologic, endocrinologic, encephalographic, or electrocardiologic function acutely or after 6 months of open-label DAP. Conclusions: This study corroborates previous studies and many years of clinical experience showing that DAP is an effective and safe treatment for LEMS.

Modern statistical techniques for the analysis of longitudinal data in biomedical research

Longitudinal study designs in biomedical research are motivated by the need or desire of a researcher to assess the change over time of an outcome and what risk factors may be associated with the outcome. The outcome is measured repeatedly over time for every individual in the study, and risk factors may be measured repeatedly over time or they may be static. For example, many clinical studies involving chronic obstructive pulmonary disease (COPD) use pulmonary function as a primary outcome and measure it repeatedly over time for each individual. There are many issues, both practical and theoretical, which make the analysis of longitudinal data complicated. Fortunately, advances in statistical theory and computer technology over the past two decades have made techniques for the analysis of longitudinal data more readily available for data analysts.

Racial Variation in Initial Stroke Severity

BACKGROUND AND PURPOSE Blacks experience greater morbidity and mortality from stroke than do whites. The degree to which this is due to the severity of the initial stroke is not known. The objective of this study is to determine whether there is a racial difference in initial stroke severity. METHODS A secondary analysis of a prospective cohort of 984 veterans (29.7% black) admitted to any of 9 geographically diverse Veterans Administration Hospitals for acute stroke between April 1995 and March 1997 was performed. Initial stroke severity was ascertained by using the modified Canadian Neurological Scale (CNS) applied retrospectively to medical record data. Stroke severity, unadjusted and adjusted for covariates, was compared between black and white patients. RESULTS Blacks had greater initial stroke severity than did whites (mean CNS score 7.96 versus 8.32, respectively; P=0.039), with a 0.5-point difference on the scale corresponding to a single-level decrement in either speech or strength of half of an extremity. This difference persisted with adjustment for other important predictors of stroke severity (P=0. 035). However, there was no significant racial difference in severity when CNS scores were collapsed into a priori clinically relevant categories. CONCLUSIONS Compared with whites, blacks show greater severity of stroke at hospital admission. It remains uncertain whether the relatively small but significant difference at presentation fully explains the striking racial differences in morbidity and mortality from stroke.

Phosphatidylethanolamine N-methyltransferase (PEMT) knockout mice have hepatic steatosis and abnormal hepatic choline metabolite concentrations despite ingesting a recommended dietary intake of choline

Choline is an essential nutrient for humans and is derived from the diet as well as from de novo synthesis involving methylation of phosphatidylethanolamine catalysed by the enzyme phosphatidylethanolamine N -methyltransferase (PEMT). This is the only known pathway that produces new choline molecules. We used mice with a disrupted Pemt-2 gene (which encodes PEMT; Pemt (-/-)) that have previously been shown to possess no hepatic PEMT enzyme. Male, female and pregnant Pemt (-/-) and wild-type mice ( n =5-6 per diet group) were fed diets of different choline content (deficient, control, and supplemented). Livers were collected and analysed for choline metabolites, steatosis, and apoptotic [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL)] positive cells. We found that, in livers of Pemt (-/-) mice fed any of the diets, there was hepatic steatosis and significantly higher occurrence of TUNEL positive cells compared with wild-type controls. In male, female and pregnant mice, liver phosphatidylcholine concentrations were significantly decreased in Pemt (-/-) choline deficient and in Pemt (-/-) choline control groups but returned to normal in Pemt (-/-) choline supplemented groups. Phosphocholine concentrations in liver were significantly diminished in knockout mice even when choline was supplemented to above dietary requirements. These results show that PEMT normally supplies a significant portion of the daily choline requirement in the mouse and, when this pathway is knocked out, mice are unable to attain normal concentrations of all choline metabolites even with a supplemental source of dietary choline.