Self-Reported Functional Vision in USH2A-Associated Retinal Degeneration as Measured by the Michigan Retinal Degeneration Questionnaire.

Purpose: The purpose of this study was to evaluate self-reported functional vision (FV) and the impact of vision loss in patients with USH2A-associated retinal degeneration using a patient-reported outcome (PRO) measure, the Michigan Retinal Degeneration Questionnaire (MRDQ), to correlate MRDQ scores with well-established visual function measurements. Design: An observational cross-sectional study (n = 93) of participants who had Usher Syndrome Type 2 (USH2, n = 55) or autosomal recessive non-syndromic retinitis pigmentosa (ARRP; n = 38) associated with biallelic variants in the USH2A gene. Methods: The study protocol was approved by all ethics boards and informed consent was obtained from each participant. Participants completed the MRDQ at the 48-month study follow-up visit. Disease duration was self-reported by participants. One-way ANOVA was used to compare subgroups (clinical diagnosis, age, disease duration, and full-field stimulus threshold [FST] Blue-Red mediation) on mean scores per domain. Spearman correlation coefficients were used to assess associations between MRDQ domains and visual/retinal function assessments. Results: Of the study sample, 58% were female participants and the median disease duration was 13 years. MRDQ domains were sensitive to differences between subgroups of clinical diagnosis, age, disease duration, and FST Blue-Red mediation. MRDQ domains correlated with static perimetry, microperimetry, full-field stimulus testing, and best-corrected visual acuity (BCVA). Conclusions: Self-reported FV measured by the MRDQ, when applied to USH2 and ARRP participants, had good distributional characteristics and correlated well with visual function tests. MRDQ adds a new dimension of understanding on vision-related functioning and establishes this PRO tool as an informative measure in evaluating USH2A outcomes.

Functional Vision in Patients With Biallelic USH2A Variants.

PURPOSE: To describe functional vision (FV) and investigate the relationship between FV, visual acuity (VA), and hill of vision (VTOT) at baseline in patients with biallelic USH2A variants. DESIGN: Multicenter, international, cross-sectional study. METHODS: In individuals with biallelic disease-causing variants in USH2A, clinical diagnosis of Usher syndrome type 2 (USH2) or autosomal recessive nonsyndromic retinitis pigmentosa (ARRP) was based on history of hearing loss and audiology examinations. The VALVVFQ-48 was administered verbally to participants ≥18 years old. VA was measured in both eyes; VTOT was determined from static perimetry in the study eye (better VA). FV scores were calculated using Rasch analysis. RESULTS: Median age of 121 participants (76 with USH2, 45 with ARRP) was 41 years (range: 19-80); 54% were female. FV scores varied from -2.0 to 7.6 logits (median [interquartile range (IQR)]: 2.8 [1.5-3.8]). ARRP and USH2 participants had similar FV scores, both before [mean (95% CI): 2.8 (2.3-3.4) and 2.7 (2.3-3.2), respectively], and after [mean (95% CI): 2.5 (2.1-3.0) and 2.9 (2.6-3.3), respectively; P = .24] adjusting for age, VA, disease duration, and VTOT. VA and VTOT accounted for 29% and 26% of the variance in FV scores, respectively (P < .001 for each). Together, they accounted for 36% of variance observed. CONCLUSIONS: Biallelic USH2A variants were associated with a large range of FV, yet similar in ARRP and USH2, despite hearing loss in USH2. The modified VALVVFQ-48 we evaluated is not ideal for detecting the impact of USH2A-associated retinal degenerations on activities of daily living.

Static Perimetry in the Rate of Progression in USH2A-related Retinal Degeneration (RUSH2A) Study: Assessment Through 2 Years.

PURPOSE: To evaluate disease progression using static perimetry (SP) in patients with USH2A-related retinal degeneration, including Usher syndrome type 2 (USH2) and nonsyndromic autosomal recessive retinitis pigmentosa. DESIGN: Prospective, observational cohort study. METHODS: A total of 102 patients with biallelic disease-causing sequence variants in USH2A with baseline best-corrected visual acuity (BCVA) letter score ≥54 were recruited from 16 clinical sites in Europe and North America. SP, BCVA, full-field stimulus thresholds, spectral domain optical coherence tomography macular scans, and fundus-guided mesopic microperimetry were performed at baseline and annually. The main outcome measures were total hill of vision (VTOT), hill of vision in the central 30° (V30), VTOT minus V30 (VPERIPH), and mean sensitivity. RESULTS: The average decline (95% CI) was 2.05 (1.40, 2.70) decibel-steradian (dB-sr)/y for VTOT, 0.48 (0.32, 0.65) dB-sr/y for V30, 1.53 (0.97, 2.08) dB-sr/y for VPERIPH, and 0.55 (0.40, 0.71) dB/y for mean sensitivity. Average percentage decline per year was 8.3 (5.5, 11.1) for VTOT, 5.2 (3.0, 7.4) for V30, 16.0 (9.5, 22.0) for VPERIPH, and 5.1 (3.5, 6.7) for mean sensitivity. Changes from baseline to year 2 in all SP measures were highly correlated (r's ranging from 0.52 [V30 vs VPERIPH] to 0.98 [VTOT vs VPERIPH]). CONCLUSIONS: Quantitative measures of SP declined significantly over 2 years in USH2A-related retinal degeneration. The annual percentage rate of change was greatest for VTOT and VPERIPH, whereas V30 and mean sensitivity changed least, reflecting earlier and more severe peripheral degeneration compared with central loss.

Baseline Microperimetry and OCT in the RUSH2A Study: Structure-Function Association and Correlation With Disease Severity.

PURPOSE: To investigate baseline mesopic microperimetry (MP) and spectral domain optical coherence tomography (OCT) in the Rate of Progression in USH2A-related Retinal Degeneration (RUSH2A) study. DESIGN: Natural history study METHODS: Setting: 16 clinical sites in Europe and North AmericaStudy Population: Participants with Usher syndrome type 2 (USH2) (N = 80) or autosomal recessive nonsyndromic RP (ARRP) (N = 47) associated with biallelic disease-causing sequence variants in USH2AObservation Procedures: General linear models were used to assess characteristics including disease duration, MP mean sensitivity and OCT intact ellipsoid zone (EZ) area. The associations between mean sensitivity and EZ area with other measures, including best corrected visual acuity (BCVA) and central subfield thickness (CST) within the central 1 mm, were assessed using Spearman correlation coefficients. MAIN OUTCOME MEASURES: Mean sensitivity on MP; EZ area and CST on OCT. RESULTS: All participants (N = 127) had OCT, while MP was obtained at selected sites (N = 93). Participants with Usher syndrome type 2 (USH2, N = 80) and nonsyndromic autosomal recessive Retinitis Pigmentosa (ARRP, N = 47) had the following similar measurements: EZ area (median (interquartile range [IQR]): 1.4 (0.4, 3.1) mm2 vs 2.3 (0.7, 5.7) mm2) and CST (median (IQR): 247 (223, 280) µm vs 261 (246, 288), and mean sensitivity (median (IQR): 3.5 (2.1, 8.4) dB vs 5.1 (2.9, 9.0) dB). Longer disease duration was associated with smaller EZ area (P < 0.001) and lower mean sensitivity (P = 0.01). Better BCVA, larger EZ area, and larger CST were correlated with greater mean sensitivity (r > 0.3 and P < 0.01). Better BCVA and larger CST were associated with larger EZ area (r > 0.6 and P < 0.001). CONCLUSIONS: Longer disease duration correlated with more severe retinal structure and function abnormalities, and there were associations between MP and OCT metrics. Monitoring changes in retinal structure-function relationships during disease progression will provide important insights into disease mechanism in USH2A-related retinal degeneration.

Tissue-specific genotype-phenotype correlations among USH2A-related disorders in the RUSH2A study.

We assessed genotype-phenotype correlations among the visual, auditory, and olfactory phenotypes of 127 participants with Usher syndrome (USH2) (n =80) or nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) (n = 47) due to USH2A variants, using clinical data and molecular diagnostics from the Rate of Progression in USH2A Related Retinal Degeneration (RUSH2A) study. USH2A truncating alleles were associated with USH2 and had a dose-dependent effect on hearing loss severity with no effect on visual loss severity within the USH2 subgroup. A group of missense alleles in an interfibronectin domain appeared to be hypomorphic in ARRP. These alleles were associated with later age of onset, larger visual field area, better sensitivity thresholds, and better electroretinographic responses. No effect of genotype on the severity of olfactory deficits was observed. This study unveils a unique, tissue-specific USH2A allelic hierarchy with important prognostic implications for patient counseling and treatment trial endpoints. These findings may inform clinical care or research approaches in others with allelic disorders or pleiotropic phenotypes.

The RUSH2A Study: Dark-Adapted Visual Fields in Patients With Retinal Degeneration Associated With Biallelic Variants in the USH2A Gene.

Purpose: To measure visual fields using two-color dark-adapted chromatic perimetry in a subset of participants in the Rate of Progression of USH2A-related Retinal Degeneration (RUSH2A), a study of USH2A-mediated syndromic (USH2) and autosomal recessive nonsyndromic retinitis pigmentosa, determine percentage retaining rod function, and explore relationships between dark-adapted visual fields (DAVF) and rod function from ERG and full-field stimulus thresholds (FST). Methods: Full-field rod mean sensitivity, number of rod loci, maximum sensitivity, DAVF full-field hill of vision (DAVF VTOT), and 30° hill of vision (DAVF V30) were measured in one eye for DAVF ancillary study participants (n = 49). Loci where cyan relative to red sensitivity was more than 5 dB on dark-adapted chromatic perimetry were considered rod mediated. Correlation coefficients between the DAVF measures and standard clinical measures were estimated, as were kappa statistics (κ) for agreement between DAVF and other measures of rod function. Results: Of 49 participants tested with DAVF, 38 (78%) had evidence of rod function, whereas 15 (31%) had measurable rod ERGs. DAVF maximum sensitivity was highly correlated with FST white thresholds (r = -0.80; P < .001). Although not statistically significant, the number of rod loci and DAVF VTOT were lower in eyes with longer disease duration by 0.82 (95% confidence interval, -1.76, 0.12) loci/year and 0.59 (95% confidence interval, -1.82, 0.64) dB-steradians/year, respectively. Conclusions: Rod-mediated function on FST and DAVF is present in many patients with symptomatic USH2A-related retinal degeneration, including some without measurable rod ERGs. RUSH2A longitudinal data will determine how these measures change with disease progression and whether they are useful for longitudinal studies in inherited retinal degenerations.

Auditory and olfactory findings in patients with USH2A-related retinal degeneration-Findings at baseline from the rate of progression in USH2A-related retinal degeneration natural history study (RUSH2A).

Sensorineural hearing loss (SNHL) is characteristic of Usher syndrome type 2 (USH2), but less is known about SNHL in nonsyndromic autosomal recessive retinitis pigmentosa (ARRP) and olfaction in USH2A-associated retinal degeneration. The Rate of Progression of USH2A-related Retinal Degeneration (RUSH2A) is a natural history study that enrolled 127 participants, 80 with USH2 and 47 with ARRP. Hearing was measured by pure-tone thresholds and word recognition scores, and olfaction by the University of Pennsylvania Smell Identification Test (UPSIT). SNHL was moderate in 72% of USH2 participants and severe or profound in 25%, while 9% of ARRP participants had moderate adult-onset SNHL. Pure-tone thresholds worsened with age in ARRP but not in USH2 participants. The degree of SNHL was not associated with other participant characteristics in either USH2 or ARRP. Median pure-tone thresholds in ARRP participants were significantly higher than the normative population (p < 0.001). Among 14 USH2 participants reporting newborn hearing screening results, 7 reported passing. Among RUSH2A participants, 7% had mild microsmia and 5% had moderate or severe microsmia. Their mean (±SD) UPSIT score was 35 (±3), similar to healthy controls (34 [±3]; p = 0.39). Olfaction differed by country (p = 0.02), but was not significantly associated with clinical diagnosis, age, gender, race/ethnicity, smoking status, visual measures, or hearing. Hearing loss in USH2A-related USH2 did not progress with age. ARRP patients had higher pure-tone thresholds than normal. Newborn hearing screening did not identify all USH2A-related hearing loss. Olfaction was not significantly worse than normal in participants with USH2A-related retinal degeneration.

Tackling the Challenges of Product Development Through a Collaborative Rare Disease Network: The Foundation Fighting Blindness Consortium.

The Foundation Fighting Blindness, a 501(c)(3) nonprofit organization, established an international consortium of inherited retinal disease specialists in 2016, with a mission to accelerate the development of treatments for rare, inherited retinal degenerations, such as retinitis pigmentosa, Stargardt disease, Leber congenital amaurosis, Usher syndrome, choroideremia, and achromatopsia. The Consortium accomplishes its mission by evaluating novel outcome measures, sharing standardized study protocols and datasets, and disseminating findings. Having established research infrastructure in the first 3 years, including 39 global research sites, the network is now poised to expand its infrastructure for trials of new therapies in partnership with industry. This model represents an innovative approach to overcome challenges of therapeutic development for rare diseases.

The RUSH2A Study: Best-Corrected Visual Acuity, Full-Field Electroretinography Amplitudes, and Full-Field Stimulus Thresholds at Baseline.

Purpose: The purpose of this study was to evaluate baseline best corrected visual acuity (BCVA), full-field electroretinography (ERG), full-field stimulus thresholds (FST), and their relationship with baseline demographic and clinical characteristics in the Rate of Progression in Usher syndrome type 2 (USH2A)-related Retinal Degeneration (RUSH2A) multicenter study. Methods: Participants had Usher syndrome type 2 (USH2, N = 80) or autosomal recessive nonsyndromic retinitis pigmentosa (ARRP, N = 47) associated with biallelic variants in the USH2A gene. Associations of demographic and clinical characteristics with BCVA, ERG, and FST were assessed with regression models. Results: In comparison to ARRP, USH2 had worse BCVA (median 79 vs. 82 letters; P < 0.001 adjusted for age), lower rod-mediated ERG b-wave amplitudes (median 0.0 vs. 6.6 µV; P < 0.001) and 30 Hz flicker cone-mediated ERG amplitudes (median 1.5 vs. 3.1 µV; P = 0.001), and higher (white, blue, and red) FST thresholds (means [-26, -31, -23 dB] vs. [-39, -45, -28 dB]; P < 0.001 for all stimuli). After adjusting for age, gender, and duration of vision loss, the difference in BCVA between diagnosis groups was attenuated (P = 0.09). Only diagnosis was associated with rod- and cone-mediated ERG parameters, whereas both genders (P = 0.04) and duration of visual loss (P < 0.001) also were associated with FST white stimulus. Conclusions: USH2 participants had worse BCVA, ERG, and FST than ARRP participants. FST was strongly associated with duration of disease; it remains to be determined whether it will be a sensitive measure of progression. Translational Relevance: Using standardized research protocols in RUSH2A, measures have been identified to monitor disease progression and treatment response and differentiate features of prognostic relevance between USH2 and ARRP participants with USH2A mutations.

Baseline Visual Field Findings in the RUSH2A Study: Associated Factors and Correlation With Other Measures of Disease Severity.

PURPOSE: To report baseline visual fields in the Rate of Progression in USH2A-related Retinal Degeneration (RUSH2A) study. DESIGN: Cross-sectional study within a natural history study. METHODS: Setting: multicenter, international. STUDY POPULATION: Usher syndrome type 2 (USH2) (n = 80) or autosomal recessive nonsyndromic retinitis pigmentosa (ARRP) (n = 47) associated with biallelic disease-causing sequence variants in USH2A. OBSERVATION PROCEDURES: Repeatability of full-field static perimetry (SP) and between-eye symmetry of kinetic perimetry (KP) were evaluated with intraclass correlation coefficients (ICCs). The association of demographic and clinical characteristics with total hill of vision (VTOT) was assessed with general linear models. Associations between VTOT and other functional and morphologic measures were assessed using Spearman correlation coefficients and t tests. MAIN OUTCOME MEASURES: VTOT (SP) and III4e isopter area (KP). RESULTS: USH2 participants had more severe visual field loss than ARRP participants (P < .001, adjusting for disease duration, age of enrollment). Mean VTOT measures among 3 repeat tests were 32.7 ± 24.1, 31.2 ± 23.4, and 31.7 ± 23.9 decibel-steradians (intraclass correlation coefficient [ICC] = 0.96). Better VA, greater photopic ERG 30-Hz flicker amplitudes, higher mean microperimetry sensitivity, higher central subfield thickness, absence of macular cysts, and higher III4e seeing area were associated with higher VTOT (all r > .48; P < .05). Mean III4e isopter areas for left (4561 ± 4426 squared degrees) and right eyes (4215 ± 4300 squared degrees) were concordant (ICC = 0.94). CONCLUSIONS: USH2 participants had more visual field loss than participants with USH2A-related ARRP, adjusting for duration of disease and age of enrollment. VTOT was repeatable and correlated with other functional and structural metrics, suggesting it may be a good summary measure of disease severity in patients with USH2A-related retinal degeneration.

How Did These Data Get Here? Recommendations for the Analysis of Data From ClinicalTrials.gov.

A paper by Drs Okada and Sengoku that appears in this issue of TIRS shows how data from Clinicaltrials.gov can be used for research on the pharmaceutical industry. This commentary identifies several challenges associated with using these data for research and concludes with 3 recommendations from a statistical perspective.

Utilization of Standard and Target-Specific Oral Anticoagulants Among Adults in the United Kingdom With Incident Atrial Fibrillation.

New oral anticoagulants (OACs) and updated risk stratification have the potential to improve the quality of care for patients with atrial fibrillation (AF). To describe the time from AF diagnosis to the initiation of an OAC, characteristics associated with treatment, and the incidence of switching OACs, we conducted this retrospective cohort study of 23,018 adults with incident AF receiving care between 2010 and 2014 in 647 primary care practices participating in the United Kingdom Clinical Practice Research Datalink. In patients with moderate to high stroke risk (CHA2DS2-VASc ≥ 2), the median time from diagnosis to OAC initiation decreased from 10 to 2 months. Among 980 at very low stroke risk (CHA2DS2-VASc = 0), 29% received OAC prescriptions after 90 days. Being prescribed an OAC was associated with a history of stroke or transient ischemic attack (relative risk 1.3); severe dementia or psychosis was most associated with not being prescribed an OAC (relative risk 0.3). After 1 year, the risk of OAC switching was higher for patients initiating dabigatran (19%) than warfarin (6%), rivaroxaban (8%), or apixaban (9%). The prescribing of OACs in moderate-to-high-risk patients in the United Kingdom increased annually; 1/3 of very low-risk patients were prescribed OACs contrary to guidance. In conclusion, future research should refine decision-making tools to minimize the unwanted effects of underutilization and overutilization of OACs.

Cost-Effectiveness Analysis of Four Simulated Colorectal Cancer Screening Interventions, North Carolina.

INTRODUCTION: Colorectal cancer (CRC) screening rates are suboptimal, particularly among the uninsured and the under-insured and among rural and African American populations. Little guidance is available for state-level decision makers to use to prioritize investment in evidence-based interventions to improve their population's health. The objective of this study was to demonstrate use of a simulation model that incorporates synthetic census data and claims-based statistical models to project screening behavior in North Carolina. METHODS: We used individual-based modeling to simulate and compare intervention costs and results under 4 evidence-based and stakeholder-informed intervention scenarios for a 10-year intervention window, from January 1, 2014, through December 31, 2023. We compared the proportion of people living in North Carolina who were aged 50 to 75 years at some point during the window (that is, age-eligible for screening) who were up to date with CRC screening recommendations across intervention scenarios, both overall and among groups with documented disparities in receipt of screening. RESULTS: We estimated that the costs of the 4 intervention scenarios considered would range from $1.6 million to $3.75 million. Our model showed that mailed reminders for Medicaid enrollees, mass media campaigns targeting African Americans, and colonoscopy vouchers for the uninsured reduced disparities in receipt of screening by 2023, but produced only small increases in overall screening rates (0.2-0.5 percentage-point increases in the percentage of age-eligible adults who were up to date with CRC screening recommendations). Increased screenings ranged from 41,709 additional life-years up to date with screening for the voucher intervention to 145,821 for the mass media intervention. Reminders mailed to Medicaid enrollees and the mass media campaign for African Americans were the most cost-effective interventions, with costs per additional life-year up to date with screening of $25 or less. The intervention expanding the number of endoscopy facilities cost more than the other 3 interventions and was less effective in increasing CRC screening. CONCLUSION: Cost-effective CRC screening interventions targeting observed disparities are available, but substantial investment (more than $3.75 million) and additional approaches beyond those considered here are required to realize greater increases population-wide.

Multicenter Study of a Novel Topical Interleukin-1 Receptor Inhibitor, Isunakinra, in Subjects With Moderate to Severe Dry Eye Disease.

OBJECTIVES: Isunakinra, formerly known as EBI-005, is a novel interleukin (IL)-1 receptor inhibitor developed for topical treatment of patients with dry eye disease (DED). This phase 1b/2a multicenter, double-masked, randomized, vehicle controlled environmental trial assessed the safety and biological activity of isunakinra in patients with moderate to severe DED. METHODS: Subjects (N=74) were randomized to vehicle (placebo) or isunakinra (5 or 20 mg/mL) 3×/daily for 6 weeks. Evaluations included safety, tolerability, biological activity for signs (corneal fluorescein staining [CFS]), symptoms (pain or sore eyes and total Ocular Surface Disease Index [OSDI]), and reduction in rescue artificial tear use. RESULTS: Topical administration of isunakinra (5 and 20 mg/mL) was safe and well tolerated and resulted in clinically relevant improvements in symptoms (OSDI score, painful/sore eye component of OSDI) and signs (total CFS) compared with baseline with no dose response. OSDI scores improved from baseline by 38% (18.9 points) at 6 weeks and CFS scores improved by 33% (3 points) in the isunakinra groups. These changes were not statistically significant compared with the vehicle. Use of artificial rescue tears was significantly reduced in the isunakinra treatment groups (mean=9 vials) compared with vehicle (mean=31 vials). The differences between isunakinra and vehicle treatments were more pronounced in subjects with OSDI scores less than 50 at baseline. CONCLUSIONS: Isunakinra was safe, well tolerated and showed clinically meaningful improvements in signs and symptoms of DED. These results encouraged the design of an adequately powered study to characterize the safety and efficacy of isunakinra in ocular surface diseases.

Denufosol tetrasodium in patients with cystic fibrosis and normal to mildly impaired lung function.

RATIONALE: Intervention for cystic fibrosis lung disease early in its course has the potential to delay or prevent progressive changes that lead to irreversible airflow obstruction. Denufosol is a novel ion channel regulator designed to correct the ion transport defect and increase the overall mucociliary clearance in cystic fibrosis lung disease by increasing chloride secretion, inhibiting sodium absorption, and increasing ciliary beat frequency in the airway epithelium independently of cystic fibrosis transmembrane conductance regulator genotype. OBJECTIVES: To evaluate the efficacy and safety of denufosol in patients with cystic fibrosis who had normal to mildly impaired lung function characteristic of early cystic fibrosis. METHODS: A total of 352 patients greater than or equal to 5 years old with cystic fibrosis who had FEV(1) greater than or equal to 75% of predicted normal were randomized to receive inhaled denufosol, 60 mg, or placebo three times daily in a Phase 3, randomized, double-blind, placebo-controlled, 24-week trial. MEASUREMENTS AND MAIN RESULTS: Main outcome measures included change in FEV(1) from baseline to Week 24 endpoint and adverse events. Mean change from baseline to Week 24 endpoint in FEV(1) (primary efficacy endpoint) was 0.048 L for denufosol (n = 178) and 0.003 L for placebo (n = 174; P = 0.047). No significant differences between groups were observed for secondary endpoints including exacerbation rate and other measures of lung function. Denufosol was well tolerated with adverse event and growth profiles similar to placebo. CONCLUSIONS: Denufosol improved lung function relative to placebo in cystic fibrosis patients with normal to mildly impaired lung function. Clinical trial registered with www.clinicaltrials.gov (NCT00357279).

Randomization-based nonparametric methods for the analysis of multicentre trials.

Multicentre trials offer several advantages over single centre trials in clinical research, including the ability to recruit patients at a faster rate over the course of the study, increased generalizability through the use of a broader patient population, and the ability to shed light on the replication of findings at multiple centres in a single study. A nonparametric approach to the analysis of multicentre trial data provides a convenient way for addressing the role of centres as well as baseline covariables during data analysis. With the use of randomization-based nonparametric methods, the strategy for evaluating the null hypothesis of no treatment effect can be prespecified during study planning without requiring a specific structure for the relationship of response criteria (or endpoints) to centres, covariables, or potential interaction terms. Further, the basis of inference for the application of these methods is the randomization mechanism, and the population to which inference can be directly made is the study population itself. No assumptions about underlying distributions, data structures, likelihood functions, or samples from super populations of inference are required. A three-step approach is proposed for handling centres via randomization-based nonparametric methods. In Step 1, a test of overall treatment effect is carried out using data from all centres simultaneously, without any assumption about treatment by centre interaction. In Step 2, the question of treatment by centre interaction is addressed, usually through the use of parametric multiple regression methods. In cases with suggestion of such interaction, Step 3 is conducted to evaluate different weighting schemes in forming pairwise treatment comparisons averaged across centres to assess the robustness of treatment effects observed in Step 1. An attractive inferential feature of this three-step approach is that the Type I error for the test of treatment effect is controlled by requiring statistical significance at each step to proceed to the next step. Extended Mantel-Haenszel methods with stratification adjustment for centre can be used to provide a nonparametric assessment of treatment effect. When adjustment for other covariates, such as baseline values, is desired, the more recent nonparametric analysis of covariance methods are available. Both methods are easy to use, require no assumptions beyond that of a valid randomization mechanism, and can be applied in a similar manner to dichotomous, ordinal, failure time, or continuous response criteria (endpoints). The methods are illustrated using data from a confirmatory clinical trial of a therapeutic agent for the treatment of dry eye disease.