THE 6-MINUTE WALK TEST IN DUCHENNE/BECKER MUSCULAR DYSTROPHY: LONGITUDINAL OBSERVATIONS

CRAIG M. MCDONALD, MD,1 ERIK K. HENRICSON, MPH,1 JAY J. HAN, MD,1 R. TED ABRESCH, MS,1 ALINA NICORICI, BS,1 LEONE ATKINSON, MD, PhD,2 GARY L. ELFRING, MS,2 ALLEN REHA, BA,2 and LANGDON L. MILLER, MD2
1 2

Department of Physical Medicine and Rehabilitation, University of California Davis School of Medicine, Sacramento, California 95817, USA PTC Therapeutics, South Plaineld, New Jersey, USA

Accepted 3 June 2010 ABSTRACT: In this study we used the 6-minute walk distance (6MWD) to characterize ambulation over time in Duchenne/ Becker muscular dystrophy (DBMD). The 6MWD was assessed in 18 boys with DBMD and 22 healthy boys, ages 412 years, over mean [range] intervals of 58 [3987] and 69 [52113] weeks, respectively. Height and weight increased similarly in both groups. At 52 weeks, 6MWD decreased in 12 of 18 (67%) DBMD subjects (overall mean [range]: 357 [125481] to 300 [0510] meters; D 57 meters, 15.9%), but increased in 14 of 22 (64%) healthy subjects (overall mean [range]: 623 [479754] to 636 [547717] meters; D 13 meters, 2.1%). Two DBMD subjects lost ambulation. Changes in 6MWD depended on stride length and age; improvements usually occurred by 78 years of age; older DBMD subjects worsened, whereas older healthy subjects were stable. The 6MWD changes at 1 year confirm the validity of this endpoint and emphasize that preserving ambulation must remain a major goal of DBMD therapy.
Muscle Nerve 42: 966974, 2010

Duchenne muscular dystrophy (DMD) is a disabling disorder caused by mutations in the gene for dystrophin, a protein that stabilizes muscle cell membranes.1 From birth to $2.5 years of age, boys with DMD are usually presymptomatic.2 Thereafter, progressive proximal muscle weakness leads to impairment of ambulation as one of the major complications of DMD, and most boys with DMD become nonambulatory by early adolescence.37 A small subset of boys may be classied as having Becker muscular dystrophy (BMD), a phenotypically milder form in the continuum of the disease that is associated with later manifestation of symptoms.8 Although systemic corticosteroid administration has been the standard of care for Duchenne/Becker muscular dystrophy (DBMD),9,10 such treatment has not been universally employed due to apprehension regarding the potential for substantial adverse effects. Given that several novel approaches to the treatment of DBMD have shown promise in preclinical and/or proof-of-concept clinical studies,11 the research community has faced the need to identify and develop clinically meaningful outcome measures for use in pivotal therapeutic trials. In boys with DBMD, walking abnormalities are a
Abbreviations: 6MWD, 6-minute walk distance; 6MWT, 6-minute walk test; ATS, American Thoracic Society; BMD, Becker muscular dystrophy; DBMD, Duchenne/Becker muscular dystrophy; DMD, Duchenne muscular dystrophy Key words: casecontrol study, child, disabled children, exercise test, gait disorders, neurologic Correspondence to: C. M. MCDonald; e-mail: cmmcdonald@ucdavis. edu
C 2010 Wiley Periodicals, Inc. V

Published online 15 November 2010 in Wiley (wileyonlinelibrary.com). DOI 10.1002/mus.21808

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major disease manifestation with great importance to patients and families. Clinically meaningful endpoints focused on ambulation should characterize changes in walking function over time as an expression of changes in disease state. The 6-minute walk test (6MWT) was originally developed as an integrated assessment of cardiac, respiratory, circulatory, and muscular capacity.12 More recently, the 6MWT has been used to evaluate functional capacity in neuromuscular diseases13,14 and has served as the basis for regulatory registration of several drugs.1417 In a short-term study, we previously reported that a modied 6MWT is feasible, safe, and reliable in boys with DBMD who have not yet transitioned to fulltime wheelchair use.18 We also documented that such boys have markedly compromised ambulation relative to healthy boys and correlated 6-minute walk distance (6MWD) with age, anthropometric characteristics, stride length, and cadence. Based on our observations in that short-term trial, 6MWD was adopted as the primary outcome measure in a randomized, controlled study of an investigational new drug (ataluren, PTC124)19 in ambulatory boys with DBMD.20 Our short-term study offered important crosssectional information. However, it could not provide longitudinal data regarding how 6MWD might change over time as boys mature and those with DBMD experience the effects of disease progression. For this reason, it was important for us to extend our initial short-term observations to evaluate the performance of the modied 6MWT in assessing changes in ambulatory capacity over a longer period. We elected to contact the same boys with DBMD and healthy boys who had participated in our short-term study to determine whether they would be willing to undergo repeat evaluation $1 year later. We selected this follow-up period because it extended past the 612-month treatment durations for trials that evaluated corticosteroids in DBMD,10 was similar to the period of evaluation in the ataluren registration study in DBMD,20 was consistent with durations of observation ranging from 6 to 18 months in registration trials that evaluated change in walking distance in other genetic disorders,14,16,17,21 and acknowledged the challenge and expense of maintaining a longitudinal drug intervention project for !1 year.
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6MWT in DBMD: Longitudinal Data

The aims of the study were to assess longitudinal changes in ambulatory capacity by measuring 6MWD, stride length, and cadence in boys with DBMD and healthy boys; to correlate changes in 6MWD with patient characteristics and other outcome measures; and to determine the long-term safety of the modied 6MWT.
METHODS Study Subjects.

The institutional review board approved the study protocol. Informed consent/ assent was obtained for each participant prior to conduct of any study procedures. Eligible participants were boys with DBMD and healthy boys who had participated in the prior short-term study.18 At baseline, both DBMD and healthy boys were required to be 412 years of age and capable of walking !10 meters without assistive devices (e.g., leg braces). For boys with DBMD, the diagnosis of DBMD was established based on typical clinical presentation and one or more of the following: documentation of a disease-causing mutation in the dystrophin gene; complete dystrophin deciency as shown by immunostaining on muscle biopsy; or elevated serum creatinine kinase levels and a family history of an affected relative with either a disease-causing mutation in the dystrophin gene and/or complete dystrophin deciency by immunostaining on muscle biopsy. Healthy boys could have no known neuromuscular, cardiovascular, or respiratory restrictions on test performance. Boys included in the long-term assessment were subjects from the short-term study who remained residents of the referral area for the study site, had not been lost to followup, and were willing to undergo repeat testing at an interval $52 weeks from the initial test. At both timepoints, participants had to be able to understand and follow simple instructions and be free of acute illness or other known contraindications to exercise. Systemic corticosteroid use was permitted in boys with DBMD, but other medications (e.g., amphetamines) that might alter test performance were not allowed in either DBMD or healthy subjects.
Test Methods. The 6MWT for this study was identical to that described in the prior study18 and included modications to the method recommended by the American Thoracic Society (ATS) for use in adults.12 These modications were incorporated to permit testing in the pediatric setting and included review of an orientation video prior to testing, continuous verbal encouragement from the testing staff to maintain attention to the task, and a safety chaser to walk behind the participant during testing. During testing, each participant was 6MWT in DBMD: Longitudinal Data

asked to wear an activity monitor (StepWatch; Orthocare Innovations, Seattle, Washington) on one ankle. The device was calibrated to each subjects height and gait characteristics according to previously described methods22,23 and was used to digitally count the number of strides taken during the 6MWT. A stride was dened as the number of times the leg wearing the device touched the oor; that is, if the monitor was worn on the right leg, one stride was equivalent to two sequential steps (the rst step taken with the left leg and the second step taken with the right leg). Because all subjects demonstrated symmetrical right and left step patterns during linear walking, the total step count was effectively twice the number of recorded strides. For some DBMD subjects who received regular medical care at the investigational clinic, evaluations were performed at routine clinic visits. Other DBMD subjects and healthy subjects were called to return to the clinic for testing at times that were mutually convenient for both subjects and testing staff. Testing was videotaped.
Statistical Methods. Because this study comprised a longitudinal evaluation of subjects enrolled in the original short-term observational study and did not test a predened hypothesis, sample sizes were not based on a formal calculation. All subjects with evaluable data at baseline and at follow-up were included in each analysis. A calculated stride length (m) was determined for each subject by dividing 6MWD by the number of strides recorded. Because cadence is usually dened in steps/minute, this variable was calculated by rst multiplying the total stride count during the 6MWT by 2 (i.e., 2 steps per stride) and then dividing this number by the duration of the test (6 minutes in the case of a complete test). Subjects who were unable to perform a follow-up 6MWT due to loss of ambulation were assigned values of 0 for 6MWD, stride length, and cadence on the date of the follow-up visit. To standardize summaries of the data, linear interpolation/extrapolation was used over the observation period to obtain followup values at 52 weeks. Mean percentage changes were calculated as the average of individual percentage changes. Differences between the DBMD and healthy populations were analyzed using Wilcoxons rank-sum tests, with P 0.05 considered statistically signicant. Standard regression techniques were used to evaluate correlations among variables of interest. Fall numbers, incidence, timing, and duration were described. RESULTS

Subjects who participated Subject Characteristics. both at baseline and during long-term follow-up included 18 boys with DBMD and 22 healthy boys
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FIGURE 1. (A) Individual and mean baseline ages, (B) individual and mean follow-up times, (C) individual heights, (D) mean heights, (E) individual weights, and (F) mean weights. In (C) and (E), the ! symbol denotes individual DBMD subjects who were not receiving corticosteroids at the designated beginning or ending timepoints. *Wilcoxon rank-sum test comparing groups; Wilcoxon rank-sum test comparing changes between groups. DBMD, Duchenne/Becker muscular dystrophy.

(Fig. 1). Mean baseline age was lower in DBMD subjects than in healthy subjects, but the distribution of baseline ages was similar (412 years) (Fig. 1A). Although the median [range] inter-test interval was shorter in boys with DBMD, all but 1 of the boys across both groups had !52 weeks of followup (Fig. 1B). The groups were not statistically different in height and weight at baseline, and there were no changes in these parameters during the inter-test period (Fig. 1C and 1D); all DBMD and healthy subjects had increases in height and weight over the observation period except for 1 control boy who showed a decline in weight despite an
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increase in height. Among boys with DBMD, 13 of 18 (72%) were taking corticosteroids at baseline. These boys were all receiving prednisone, although there were variations in the regimen used (e.g., daily, alternate days, 10 day alternating, or weekends only). For the 13 DBMD subjects taking corticosteroids at baseline, 12 continued on corticosteroids through follow-up; at the request of his mother, 1 DBMD subject (age 5 years) discontinued corticosteroids at 6 months after the baseline evaluation. For the 5 DBMD subjects who were not taking corticosteroids at baseline, the physicians of 3 of the subjects (ages 5, 6, and 8 years) initiated
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FIGURE 2. (A) Individual 6MWDs, (B) mean 6MWDs, (C) individual stride lengths, (D) mean stride lengths, (E) individual cadences, and (F) mean cadences. The ! symbol indicates individual DBMD subjects who were not receiving corticosteroids at the designated timepoints (A, C, E). *Wilcoxon rank-sum test comparing changes between groups: : values same or increased; ; values decreased. 6MWD, 6-minute walk distance; DBMD, Duchenne/Becker muscular dystrophy.

such therapy during the inter-test period but !3 months prior to the follow-up assessment. Three DBMD subjects participated in the initial short-term study but did not return for long-term follow-up; the family of 1 boy had moved away from the investigational site area, and the families of the other 2 boys did not wish to travel to the investigational site. Baseline ages for these boys were 7, 8, and 10 years; 2 of these boys were taking corticosteroids, and 1 was not. Twelve healthy subjects who were involved in the initial short-term study did not return for long-term follow-up; these subjects indicated time constraints as the primary reason for not returning.
6MWT in DBMD: Longitudinal Data

Ambulatory Capacity. All participants in the longterm follow-up were considered evaluable for 6MWD at baseline and follow-up. Three ambulatory boys with DBMD and 1 healthy control did not wear the StepWatch activity monitor during initial testing and/or follow-up testing and were therefore excluded from the analyses of stride length and cadence. Boys with DBMD had markedly lower individual and mean baseline 6MWD values relative to healthy boys (Fig. 2A and 2B). The 6MWD decreased in 12 of 18 (67%) boys with DBMD but increased in 14 of 22 (64%) healthy boys (Fig. 2A). Two boys with DBMD lost independent MUSCLE & NERVE December 2010 969

FIGURE 3. 6MWDs by age. The ! symbol denotes individual DBMD subjects who were not receiving corticosteroids at the designated timepoints. 6MWD, 6-minute walk distance; DBMD, Duchenne/Becker muscular dystrophy.

ambulation during the inter-test interval and were assigned a 6MWD of zero at the follow-up visit. One of these boys was a 10-year-old who walked only 125 meters at baseline and was not receiving corticosteroids, making his loss of ambulation within the follow-up period anticipated. However, the other subject was a 9-year-old who began the study with a 6MWD of 350 meters and received corticosteroids throughout the inter-test interval, making his rapid decline in ambulation unexpected. When considering changes for all participants at 52 weeks, the mean 6MWD values showed a decrement of 57 meters (15.9%) in DBMD subjects compared with an increment of 13 meters (2.1%) in healthy subjects; the difference in these changes was statistically signicant (P < 0.037) (Fig. 2B). The pattern of individual and overall changes in stride length mirrored those observed in 6MWD (Fig. 2C and D). Boys with DBMD had lower individual and mean baseline stride length values relative to healthy boys. Stride length decreased in the majority of DBMD subjects but increased in the majority of healthy subjects (Fig. 2C). At 52 weeks, mean stride length values had changed by similar percentages as had 6MWD values. This led to a statistically signicant difference (P < 0.027) in the changes between groups (Fig. 2D). The pattern for cadence was different from that for stride length (Fig. 2E and F). Cadence at baseline was only slightly lower in boys with DBMD than in healthy boys, but it was much more variable. Cadence decreased in most boys with DBMD (primarily in the 2 boys who lost ambulation) but also decreased minimally in most healthy boys. The differences in the changes between the two groups showed a trend toward statistical signicance (P 0.069) (Fig. 2F).
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Relationships among Variables. The relationships among changes in age, height, stride length, cadence, and 6MWD were explored. The pattern of changes in 6MWD showed a biphasic age dependency (Fig. 3). Improvements in 6MWD among DBMD subjects occurred most commonly in those who were 7 years of age by the end of the observation period. Most subjects older than this age had declines in 6MWD; for those DBMD subjects >7 years of age at baseline, the mean change over 52 weeks was 115 meters (36.0%), with 9 of 10 (90%) showing a decreased 6MWD (including the 2 boys with complete loss of ambulation). An agedependent pattern was also observed in healthy subjects. The majority of the acute improvement in 6MWD occurred in healthy boys who were 8 years of age at baseline. Beyond this age, there appeared to be a attening in 6MWD changes. An evaluation of stride length by age (data not shown) showed the same pattern as that for 6MWD shown in Figure 3. An assessment of cadence by age conrmed that, except for the 2 DBMD subjects who lost ambulation, there was generally a modest magnitude of declines across both groups and all ages (data not shown). Over the course of 1 year, changes in 6MWD, stride length, and cadence were not meaningfully correlated with changes in height for DBMD or healthy subjects (r < 0.36, P > 0.11 for all correlations; data not shown). Changes in 6MWD were positively correlated with changes in stride length in both DBMD boys (r 0.94, R2 0.89, P < 0.001) and healthy boys (r 0.81, R2 0.66, P < 0.001; Fig. 4A). Changes in 6MWD were correlated with changes in cadence in boys with DBMD (r 0.85, R2 0.72, P < 0.001) but not in healthy boys (r 0.02, R2 0.0003, P 0.94; Fig. 4B). The small sample, difference in treatment regimens, and uncontrolled nature of corticosteroid treatment in boys with DBMD precluded formal evaluation of the inuence of corticosteroid use on 6MWD. However, among boys with DMD, subjects who were receiving corticosteroids and subjects who were not receiving corticosteroids showed marked declines in 6MWD over the observation period. Safety.

Falls tended to occur more often in boys with DBMD than in healthy control boys at both initial and follow-up timepoints (Table 1). Falls were less frequent at the follow-up visit in both DBMD and healthy subjects. Among the 3 boys with DBMD who fell during the follow-up 6MWT, 1 fell at both the baseline and follow-up visits, and 2 fell only at the followup visit. Among DBMD and healthy subjects who fell, recovery from falls was rapid, and falls consumed little of the overall
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FIGURE 4. Correlation of change in 6MWD with (A) change in stride length, and (B) change in cadence. 6MWD, 6-minute walk distance; DBMD, Duchenne/Becker muscular dystrophy

testing time. No boys sustained injury due to falling, and all boys who fell were able to resume ambulation promptly and complete testing.
DISCUSSION

Having established the short-term feasibility and reliability of the 6MWT as an outcome measure in DBMD,18 we sought to determine the ability of the test to detect changes in 6MWD over a period of 1 year, both in boys with DBMD and in healthy boys of similar age, height, and weight. We also sought to determine any differences in longitudinal changes between these two populations. Our ndings conrm that boys with DBMD already have profoundly compromised ambulatory functioning by an early age relative to normally developing boys. Despite age-appropriate increases in height and weight and prevalent corticosteroid use, most boys with DBMD experienced loss of ambulatory capacity that was readily discernible over 1 year. The data also show that maturational improvements in walking ability in younger boys with DBMD were overtaken by progressive diseaserelated loss of ambulatory capacity as the boys grew older. Change in stride length is also a major determinant of change in 6MWD over time. Boys with DBMD lose stride length despite increasing height, whereas healthy boys show initial growth-

related increases in stride length that then stabilize. The ability to detect differences between these two distinct populations of boys supports the validity of change in 6MWD as an endpoint. The results from this study also support the safety of the 6MWT over time. Of importance, we believe these results provide a representative estimate of changes in 6MWD in ambulatory boys with DBMD. We included subjects with a typical range of ages observed in the ambulatory DBMD population.37,23 The prevalence of initial corticosteroid use was similar to that (71%) recently documented at baseline in a large sample of 174 boys with DBMD who participated in an international ataluren clinical trial.24 The results are supported by the fact that changes in 6MWD among the healthy control boys in our study appear to be similar to age-related trends observed in cross-sectional evaluations of the 6MWT in normally developing children.18,2527 Clinometric considerations relating to test reproducibility also support the validity of the changes observed in boys with DBMD28; the marked drop in mean 6MWD (57 meters) for DBMD subjects in this long-term study lies outside of the spontaneous uctuations encompassed by the 95% condence interval (7 to 22 meters) for the mean testretest changes in these same boys in our prior short-term study.18

Table 1. Falls and recovery from falls. DBMD subjects (N 18) Parameter 6MWTs (n) Total falls (n) 6MWTs with at least one fall (n) [%] Median duration from fall to fall recovery (s) [range] Median time per 6MWT taken for falls (s) [range] Baseline 18 14 5 [28] 1.5 [14] 5 [110] Follow-up 16* 6 3 [19] 4.5 [39] 9 [318] Healthy subjects (N 22) Baseline 22 4 3 [14] 2 [15] 2 [26] Follow-up 22 0 0 [0]

6MWT, 6-minute walk test; DBMD, Duchenne muscular dystrophy. *Excludes 2 boys who lost ambulation and were unable to perform a follow-up 6MWT. Includes only 6MWTs with at least one fall.

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Two limitations of our study were that we did not have frequently repeated measurements of 6MWD over time and that corticosteroid use was not controlled. We do not believe that having only two timepoints materially biased our results, particularly in patients who remained ambulatory throughout the follow-up period. For the 2 boys with DBMD who became totally wheelchair-dependent during the inter-test interval, it is possible that closer follow-up would have identied an early point at which independent mobility was lost (resulting in an even more acute slope in the line marking the loss of ambulation). However, by assigning a value of 0 meters for 6MWD at the follow-up visits, we provided an estimate of decline that is highly conservative and would not alter our conclusions. Changes in corticosteroid use during the inter-test interval occurred in 4 of the subjects, comprising cessation in 1 boy and initiation of such therapy in 3 boys. The 2 younger boys (each age 5 years), 1 of whom stopped steroids and 1 of whom started steroids, both showed an increase in 6MWD. By contrast, 2 older boys (ages 6 and 8 years), both of whom started steroids, had a decrease in 6MWD. Thus, although changes in corticosteroid use may have altered outcomes in these boys, the results in these individual subjects remained consistent with the overall age-related ndings of the study. The validity of 6MWD change as a measure of disease progression is supported by the age-related pattern of changes over time. As the study shows, even young boys with DMD already have markedly impaired ambulation at baseline, with essentially no overlap in performance with typically developing boys of similar ages. Our experience indicates that young boys with or without disease may show improvements in 6MWD, presumably due to increasing stride lengths in association with greater height and greater strength-to-body-weight ratios. However, by $7 years of age, these gains appear to be eclipsed by disease progression in boys with DBMD. Such ndings are consistent with prior observations that showed early functional improvements in young patients followed by waning performance with advancing age.3,4 Taken together, the magnitude of the ambulatory compromise at an early age and the short period during which maturational inuence can compensate for disease progression emphasize the continued need for early diagnosis and intervention in order to augment and/or maintain ambulation in boys with DBMD.2 Our previous natural history study in DBMD showed that all corticosteroid-naive DBMD subjects had lost ambulation by 13 years of age.5 In this study, 2 subjects categorized as having DBMD,
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both of whom were receiving corticosteroids, continued to ambulate past 13 years of age; over the inter-test interval, 1 boy showed an increase in 6MWD and 1 had a modest reduction in 6MWD (Fig. 3). These subjects may manifest what has been termed outlier DMD or intermediateseverity dystrophinopathy.29 These descriptions have previously characterized patients who, even if corticosteroid-naive, did not lose ambulation until middle adolescence. Because our study had only 2 such boys, both of whom were receiving corticosteroids, we could not fully evaluate 6MWD for DBMD patients with a mild phenotype. Ongoing efforts to collect natural history data in populations of DBMD patients with a range of ages and who are corticosteroid-treated or corticosteroid-naive will likely provide an evolving understanding of ambulatory characteristics across the entire DBMD disease continuum. Our previous cross-sectional study of the 6MWT in DBMD showed stride length to be the best predictor of ambulatory capacity, explaining 89% of the variance in 6MWD.18 The longitudinal experience in this study also documented that change in stride length is a major predictor of change in 6MWD over time, both in boys with DBMD and in healthy subjects. Consistent with prior reports,30,31 our results indicate that stride length and change in stride length in boys with DBMD are primarily dependent upon age-related disease progression, and not height. In this regard, heights increased by a similar magnitude in both DBMD and healthy groups; however, stride lengths commonly shortened in boys with DBMD yet lengthened in healthy controls. When considered together with the changes in walking capacity by age, these data have important implications for future randomized, controlled clinical trials in DBMD; the ndings indicate that stratifying for balance by age (particularly considering those 7 years of age) is critical, but that stratifying for height is likely to have little value. Including only patients !7 years of age might increase study power, because most DBMD patients in this age group would be predicted to have a decline in 6MWD over 1 year. However, excluding patients 7 years of age may be problematic, because early intervention in younger patients might be important to maximize treatment effects before extensive muscle loss has occurred. The effects of change in cadence on change in 6MWD were also notable. In healthy boys, minimal decreases in cadence were counterbalanced by increases in stride length. In boys with DBMD, cadence reductions and stride length decreases over 1 year combined to produce shorter 6MWDs. The data suggest that intervening to improve either
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stride length or cadence may offer salutary effects on ambulatory capacity in boys with DBMD. In view of the logical pattern of the changes observed relative to disease status, age, and anthropomorphic characteristics,18 the longitudinal data bolster the utility and validity of change in 6MWD as a clinically meaningful endpoint for use in natural history studies and therapeutic clinical trials in patients with DBMD. Our results document that, for most ambulatory boys with DBMD, 6MWD worsened demonstrably in 1 year; in this experience, boys with DBMD showed an average deterioration of 57 meters (15.9%). This level of decrement exceeds the changes in 6MWD of $10 15% that have been shown to be consistent with improving or worsening patient-reported wellbeing.32 This decrement also exceeds the mean treatment effects of 2844 meters (813%) achieved in registration-directed, randomized studies that employed change in 6MWD as the basis for approval of drugs for several different indications.1417 Collectively, the data suggest that improving ambulatory capacity in boys with DBMD should continue to be a major treatment objective, but minimizing loss of ambulation would also remain a laudable therapeutic goal. In the healthy control subjects (n 22), the variability (standard deviation) of the change in 6MWD over 52 weeks was 40 meters. In boys with DBMD, disease progression affected each boy differently and led to greater variability in the change in 6MWD over time; for this population, the standard deviation for change in 6MWD of 36 meters, observed over a 1-week interval in our prior shortterm study,18 increased to 83 meters (n 16) among those who maintained ambulation and to 104 meters (n 18) over 52 weeks when the 2 boys who lost independent ambulation were included. This variability needs to be considered in sample size calculation for any future controlled therapeutic trial using 6MWD as the primary outcome measure. We used a t-test to estimate sample sizes for a two-arm study with a two-sided signicance level of 0.05 and a standard deviation for 6MWD change of 90 meters, considering power values of 0.80, 0.85, or 0.90 (Fig. 5). As Figure 5 exemplies, targeting 50-, 40-, or 30-meter 6MWD improvements in a treated group relative to a placebo group with 0.80 power would require sample sizes of 52, 81, and 143 patients/arm, respectively. Rank-based or permutation-based analytical methods may be necessary to account for non-normal distribution of 6MWD data; in this situation, a modest upward adjustment of the sample size ($10%) might be appropriate. Change in 6MWD over $1 year has been used in a recently enrolled, registration-directed,
6MWT in DBMD: Longitudinal Data

FIGURE 5. Sample sizes for comparisons of change in 6MWD between control and treatment groups assuming t-test analysis, a two-sided significance level of 0.05, and standard deviation for change of 90 meters. 6MWD, 6-minute walk distance; SD, standard deviation.

randomized, placebo-controlled study that evaluated the investigational drug, ataluren, in 174 patients with DBMD.20 Although our observational study has the advantage of inclusion of a healthy control population, the randomized trial collects 6MWT data at preplanned 6-week intervals and thus may provide more detailed insights about 6MWD changes over time. The ataluren trial also offers the prospect of conrming the relationships between patient characteristics and 6MWD we have observed and of exploring the correlations between 6MWD data and other functional endpoints. Together, both studies provide complementary information that may support additional drug development efforts in DBMD.
Further Information. An equipment list and an instruction manual that describe the conduct of the 6MWT are available in English. In addition, a professionally produced instructional video for staff training and for patient orientation to the test is available in Belgian Dutch, Belgian French, English, French, German, Hebrew, Italian, Spanish, and Swedish. These materials can be licensed through PTC Therapeutics by contacting medicalinfo@ ptcbio.com. This study was sponsored by PTC Therapeutics and funded in part by a grant from the Parent Project Muscular Dystrophy. The authors thank the patients and volunteers who committed their time and effort; Greg Elfring and James Dancy of Innovative Analytics for their expertise in data management; and Peter Riebling of PTC Therapeutics for manuscript support.
REFERENCES 1. Lapidos KA, Kakkar R, McNally EM. The dystrophin glycoprotein complex: signaling strength and integrity for the sarcolemma. Circ Res 2004;94:10231031. 2. Ciafaloni E, Fox DJ, Pandya S, Westeld CP, Puzhankara S, Romitti PA, et al. Delayed diagnosis in Duchenne muscular dystrophy: data from the Muscular Dystrophy Surveillance, Tracking, and Research Network (MD STARnet). J Pediatr 2009;155:380385.

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