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

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?

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

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

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

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

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

Glucocorticosteroids affect disease progression

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 16.21) 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)

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

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

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)

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

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 +27.37 (-95 to 175) >7 years (N=111) 7.1 to 15 -37.25 (-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 12-14. Pane, et al. 2014.

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

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

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

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

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

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.

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

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:10.1371/journal.pone.0141240 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 (rs28357094 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 rs28357094 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.

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

(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)

(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)

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

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

Thank you! DMD Italian Group