1. Trial design in DMD, lessons learned from natural history
Eugenio Mercuri

2. In the last decade increasing attention to the use of outcome measures and to natural history studies
Can we use what we learned from natural history studies for clinical trial design?

3. Current Natural History of ambulant DMD is well described
PLoS One | January 2013 | Volume 8 (1) | e52512
PLoS One. 2014 Jan 8;9(1):e83400

4. New natural history 5 years 9 years 14 years 20 years
Contemporary: with steroids and improved cardiac management
Loss of standing
Loss of ambulation Dx
Loss of self-feeding
Need for ventilation
Death
Ambulant DMD
Very young
Non ambulant

5. Natural history of Duchenne muscular dystrophy
Contemporary: with steroids and improved cardiac management
Loss of self-feeding
Need for ventilation Dx
Loss of ambulation
Death
Functional gains
Functional decline of upper limb function
Functional decline of ambulation
Novel
clinical
endpoints
in DMD
6MWT
Northstar ambulatory assessment
9-hole peg test
Patient-reported outcomes: NeuroQOL and neuromuscular module of PedsQL

6. Non linear pattern of disease evolution
6MWD
Goemans et al,
Neuromus Dis 2013
McDonald et al
Muscle &Nerve 2013

7. Corticosteroids Interfere with pathophysiologic cascade of events
Scientific evidence for increase in strength and delay of progression
Preservation of ambulation into mid teens
Reduced need for scoliosis surgery
Preservation of upper limb function and respiratory capacity
Possible cardioprotective effect
Known side effects
So this treatment is indeed standard of treatment as it has been proven to dealy disease progression

8. Glucocorticosteroids affect disease progression

10. EFFECT OF STEROIDS ON NON AMBULANT BOYS/YOUNG ADULTS
91 BOYS AGE 12-25
18 were steroid naive (mean age 17.9 years)
25 stopped steroids at loss of ambulation (mean age )
48 still on steroids after loss of ambulation (mean age 16,98)
Baseline PUL
The percentage decrease at 12 months was
-10% in patients still using GC as compared to -34% and -36% in those who stopped and never used GC respectively (p<0.001).
(Pane et al, 2015)

11. Other Key Prognostic Factors Affecting Rate of Disease Progression
6MWT distance
Age
Type of mutation
While the order of disease progression is very predictable in DMD, the rate at which individual patients experience loss of abilities can vary.
Over the last few years multiple natural history studies have identified key prognostic factors that reliably predict rate of decline in DMD. The role of steroids on disease progression is well known but in recent years we have been able to identify other factors, such as age, type of mutation or the values of 6MWT

12. Baseline walking ability < 350 m predicts the decline in ambulation in patients with DMD
800 20
–20
–40
–60
–80
–100
–120
Time (weeks) 18 24 30 36 42
Baseline 6 12 48
700
Placebo (n = 57)
600
–6 m
≥ 350 m
500
6MWD (m)
400
Change in 6MWD, mean (m)
300
200
< 350 m
Baseline 6MWD ≥ 350 m (n = 34)
100
Baseline 6MWD < 350 m (n = 23)
–101 m 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Age (years)
6MWD, 6-minute walk distance
Left panel, adapted from McDonald CM et al. Muscle Nerve 2013;48:343–56; right panel, PTC Therapeutics data on file, 007 study

13. Baseline 6MWT values can predict decline in ambulation in DMD
36 MONTH CHANGES
IN BOYS
BELOW (ORANGE)
AND ABOVE (GREEN)
350 meters at baseline
Pane et al, PLOS One 2014)

14. Other Key Prognostic Factors Affecting Rate of Disease Progression
6MWT distance
Age
Type of mutation
While the order of disease progression is very predictable in DMD, the rate at which individual patients experience loss of abilities can vary.
Over the last few years multiple natural history studies have identified key prognostic factors that reliably predict rate of decline in DMD. The role of steroids on disease progression is well known but in recent years we have been able to identify other factors, such as age, type of mutation or the values of 6MWT

15. 6MWT 12 Month Change (range)
6MWT Declines After Age 7
Age
Age Range, yrs
6MWT 12 Month Change (range)
≤7 years (N=80)
3.2 to 7
(-95 to 175)
>7 years (N=111)
7.1 to 15
(-325 to 102)
As I mentioned earlier, boys with DMD initially gain in functional ability due to growth and development before experiencing a progressive and irreversible decline. This is demonstrated in natural history studies of 6MWT as shown here.
In this study of 113 patients with DMD assessed at different ages , we see that 6MWT initially improve on 6MWT approximately until age 7. After age 7, patients in the progressive decline phase walk shorter and shorter distances until they lose ambulation, typically between age
Pane, et al

16. Baseline age can also predict decline in ambulation in DMD
36 MONTH CHANGES IN BOYS
BELOW (BLUE)
AND ABOVE (RED)
THE AGE OF 7 YEARS
Pane et al, 2014

17. Other Key Prognostic Factors Affecting Rate of Disease Progression
6MWT distance
Age
Type of mutation
While the order of disease progression is very predictable in DMD, the rate at which individual patients experience loss of abilities can vary.
Over the last few years multiple natural history studies have identified key prognostic factors that reliably predict rate of decline in DMD. The role of steroids on disease progression is well known but in recent years we have been able to identify other factors, such as age, type of mutation or the values of 6MWT

18. Genetic Mutation Impacts 6MWT and Rate of Progression
Baseline mean
Whole cohort (n=191)
Mean 378 m
Duplications (n=15)
Point mutations (n=44)
All deletions (n=132)
Skipping 45 (n=15)
Skipping 51 (n=27)
This graph illustrates how different genetic mutations may impact the performance on the 6MWT. In this study of 191 patients with DMD, some differences in baseline 6MWT were observed for different mutation types.
The line in the middle of the graph represents the mean values for the whole cohort. On average, the entire cohort walked XXX meters at baseline.
Patients with duplications or point mutations had better perormance and on average walked more meters than patients with deletions.
Some variations however were also observed within theboys of deletions depending on which exons are deleted.
Patients with deletions amenable to skipping exon 44 walk further which is consistent with other reports indicating milder phenotype for this patient group which is believed to be due to a higher rate of spontaneous exon skipping and the observation that these patients have more revertant fibers expressing dystrophin.
In contrast, patients with exon deletions amenable to skipping exons 45, 51 or 53 all walked less far, indicating a more severe phenotype.
While these baseline differences were not statistically significant, subsequent, longitudinal 6MWT studies as well as NSAA studies have supported these findings with statistically significant results.
Skipping 53 (n=28)
Skipping 44 (n=18)
Adapted from Pane M, et al. PLOS One 2014

19. Ricotti 2015 – Decline in Total Score Exon Amenable vs. DMD
Exon 51 (p=0.01) and Exon 53 (p<0.001) amenable patients declined on NSAA significantly faster than All DMD patients

20. Baseline 6MWD less than (m)
A 30-m decrease in walking ability is associated with an increased risk of losing ambulation within 2 years 80
–30 m
–30 m 60
–30 m
–30 m
Percent ambulatory over 2 years (%) 40
–30 m
–30 m 20
We have learned that maintaining 6MWT is important because 6MWT distance can predict loss of ambulation.
This graph reports the results of a study performed on 131 boys with DMD followed for over 2 years evaluating the risk of losing ambulation in different subgroups subdivided according to their 6MWT.
It is obvious looking from left to right, that the risk of losing ambulation increases as 6MWT distance decreases.
These results suggest that if we are able to maintain or to slow the deterioration of the 6MWD we therefore also decrease the risk of losing ambulation.
< 410 m (n = 89)
< 380 m (n = 63)
< 350 m (n = 45)
< 320 m (n = 28)
< 290 m (n = 16)
< 260 m (n = 10)
< 230 m (n = 6)
Baseline 6MWD less than (m)
Mazzone ES et al. PLoS ONE 2013;8:e52512

21. Age at Loss of Ambulation Correlates to Age of Subsequent Disabilities
Age at Loss of Ambulation (N=278)
Age at loss of ability to raise hand to mouth (self-feed) Mean (+SD)
Age of severe respiratory insufficiency
Mean (+SD)
< 8 years old
10.4 (1.36)
14.7 (1.89)
8-11 years old
12.1 (2.05)
18.1 (2.62)
>11 years old
14.4 (1.72)
22.1 (5.72)
Correlation
<0.0001
Maintaining ambulation is of course important per se, but it is also important as loss of ambulation is related to the onset of further progression of other aspects of disability. In a recent French study, a cohort of boys with DMD followed for over 20 years was subdivided into three groups based on age of loss of ambulation. This study showed that boys who lost ambulation at a later stage, after the age of 11, also had a significant delay in the need for ventilation or in the time when they lost the ability to self feed.
I would like to stress how important this chain of events is, because if we delay progression in the 6MWT we delay loss of ambulation and we delay the subsequent events of disease progression such as lost of self feeding or need for ventilation.
Humbertclaude, V. et al. Eur J Paediatr Neurol. 2012;16:149–160.

22. MRI and MRS provide further elements to identify patients at risk of sudden deterioration
Imaging data illustrate infiltration of muscle by fat and fibrous tissue
6MWD
300
Data courtesy of H. Lee Sweeney, Ph.D. Myology Institute, University of Florida

23. Modifier genes can predict progression
RESEARCH ARTICLE
Genetic Modifiers of Duchenne Muscular Dystrophy and Dilated Cardiomyopathy
Andrea Barp, Luca Bello, Luisa Politano, Paola Melacini, Chiara Calore, Angela Polo, Sara Vianello, Gianni Sorarù, Claudio Semplicini, Boris Pantic, Antonella Taglia, Ester Picillo, Francesca Magri, Ksenija Gorni, Sonia Messina, Gian Luca Vita, Giuseppe Vita, Giacomo P. Comi, Mario Ermani, Vincenzo Calvo, Corrado Angelini, Eric P. Hoffman, Elena Pegoraro
PLOS ONE | DOI: /journal.pone October 29, 2015
Objective Dilated cardiomyopathy (DCM) is a major complication and leading cause of death in Duchenne muscular dystrophy (DMD). DCM onset is variable, suggesting modifier effects of genetic or environmental factors. We aimed to determine if polymorphisms previously associated with age at loss of independent ambulation (LoA) in DMD (rs in the SPP1 promoter, rs10880 and the VTTT/IAAM haplotype in LTBP4) also modify DCM onset.
Methods A multicentric cohort of 178 DMD patients was genotyped by TaqMan assays. We performed a time-to-event analysis of DCM onset, with age as time variable, and finding of left ventricular ejection fraction < 50% and/or end diastolic volume > 70 mL/m2 as event (confirmed by a previous normal exam < 12 months prior); DCM-free patients were censored at the age of last echocardiographic follow-up.
Results Patients were followed up to an average age of 15.9 ± 6.7 years. Seventy-one/178 patients developed DCM, and median age at onset was 20.0 years. Glucocorticoid corticosteroid treatment (n = 88 untreated; n = 75 treated; n = 15 unknown) did not have a significant independent effect on DCM onset. Cardiological medications were not administered before DCM onset in this population. We observed trends towards a protective effect of the dominant G allele at SPP1 rs and recessive T allele at LTBP4 rs10880, which was statistically significant in steroid-treated patients for LTBP4 rs10880 (< 50% T/T patients developing DCM during follow-up [n = 13]; median DCM onset 17.6 years for C/C-C/T, log-rank p = 0.027)
Conclusions We report a putative protective effect of DMD genetic modifiers on the development of cardiac complications, that might aid in risk stratification if confirmed in independent cohorts.

24. After correcting for age, 6MWT, steroid use, four latent trajectory classes were observed

25. (n= 96 boys, baseline plus 3 years follow-up)
Functional Performance of Telethon Patients grouped by Natural History Cluster
Patients were assigned to cluster class by longitudinal trajectory analysis of annual 6MWD
(n= 96 boys, baseline plus 3 years follow-up)
Mean 6MWD +/- s.e.
(N= 8)
(N= 22)
(N= 44)
Age (years)

26. (n= 96 boys, baseline plus 3 years follow-up)
Similarity of longitudinal trajectories of functional performance on 6MWD and NSAA within a Natural History cluster class
6MWD
NSAA score
Age (years)
Age (years)
Patients were assigned to cluster class by longitudinal trajectory analysis of annual 6MWD
(n= 96 boys, baseline plus 3 years follow-up)

27. What else did we learn?
Demonstrating efficacy of a study is not simple
kids
families
evaluators
Natural history studies have provided important information for inclusion and stratification criteria but these must be integrated with
Mechanism of action
Duration of the study

28. Progressive loss of function highlights the complexities associated with conducting clinical studies in DMD
450m
400m
300m
150m
Ambulatory Function (6MWT)
Optimal window for
1 year clinical trials
6MWD
Timed Function Tests
(10m walk, stair climb, stair descend)
Loss of rise
From floor
Loss of
Stair climb
Loss of
ambulation 11

30. Thank you!
DMD Italian Group