Methods
Previous reports have detailed the study design and outcomes of the phase I/2, 2, and 3 galsulfase trials in subjects with MPS VI (Harmatz et al.
2004,
2005,
2006,
2008,
2010); details of the clinical trials are outlined in Table
1. Cardiac ultrasound was performed as part of the clinical evaluation of subjects in the phase 1/2, 2, and 3 clinical trials at baseline, before ERT was begun, and at intervals of 24–48 weeks and 72–96 weeks after initiation of ERT (Table
2). An Institutional Review Board (IRB) or Ethics Committee (EC) at each participating clinical site approved each study. All adult patients and parent/guardians gave written consent; patients younger than 18 years old gave written assent according to local IRB regulations.
Table 1
Summary of study populations
Phase 1/2 | Double-blinded, randomized, dose comparison/open-label extension | Jan 2001–Dec 2005 | 240 | 7/5 | 0.2 | 12.0 ± 3.8 (7–16) | 4/3 |
Phase 2 | Open-label, nonrandomized | 2002–2006 | 144 | 10/10 | 1.0 | 12.1 ± 5.3 (6–21) | 7/3 |
Phase 3 | Double-blinded, placebo-controlled, randomized/open-label extension | 2003–2006 | 96 | 39/38 | 1.0 | 13.7 ± 6.5 (rhASB); 10.7 ± 4.4 (placebo); (6–29) | 13/26 |
Table 2
Schedule of cardiac ultrasound evaluations
Phase 1/2 | X | X | X | X | X |
Phase 2 | X | | X | | X |
Phase 3 rhASB/rhASB | X | | X | | X |
Phase 3 placebo/rhASB | X | X | | X | |
Archived ultrasound data, collected from subjects who had participated in phase 1/2, 2, or 3 clinical trials, were reviewed and tabulated for this study. The original echoes were obtained and analyzed at the individual sites; original echo tapes were unavailable for further review. Each archived measurement was reviewed for reliability by a single person (E.B.). Discrepancies were resolved by discussion with individual sites. The entire data set was then subjected to statistical evaluation. Height, weight, and urinary GAG content at study entry were analyzed for the 54 subjects who participated in phase 1/2, 2, or 3 galsulfase trials.
Cardiac ultrasound investigations were performed and interpreted by study protocol at the participating sites during each study point as indicated in Table
2. Measurements included M-mode determination of left ventricular chamber dimension in diastole (LVED) and systole (LVES), as well as diastolic left ventricular posterior wall (LVPWd) and intraventricular septal (IVSd) thicknesses. Body surface areas were calculated by the method of duBois for each subject, and z-scores were determined from the measured value for each chamber dimension and wall thickness at each evaluation (Dyar
2012). The individual z-scores from each subject for each parameter at each time point were summed and averaged to obtain the mean z-score displayed in the tables. By use of the z-score, chamber dimensions and wall thicknesses from subjects of differing body surface areas can be compared, and the changes within a particular subject tracked over time (Kampmann et al.
2000). With this system, a z-score of 0 represents the expected normal value for a given body surface area, a positive z-score is a standard deviation value greater than expected normal, and a negative z-score is a value less than expected normal. Left ventricular shortening fraction (SF) was calculated by standard methods (Lopez et al.
2010).
Doppler interrogation of flow acceleration across, and regurgitation from, all cardiac valves was measured by pulsed, continuous, and color flow Doppler methods. Peak systolic gradient was recorded across aortic valves; mean diastolic gradient was measured across mitral valves; and both were compared to normal values (Lopez et al.
2010; Hatle and Angelsen
1985; Baumgartner et al.
2009; Sohn and Kim
2001). Valvular regurgitation was assigned the following scores: 0 (none), 1 (trace), 2 (mild), 3 (moderate), and 4 (severe). Mitral and aortic valve gradients and regurgitation scores for the cohort were recorded at baseline, 48 and 96 weeks. Three subjects had undergone mitral valve repair or replacement (two before and one during the trials) or aortic valvuloplasty (one before the trials). For purposes of this review, peak systolic aortic gradient was the only variable analyzed after mitral valve replacement in these three subjects. Additional cardiac anomalies were noted when found.
Ultrasound reports were obtained and reviewed retrospectively. Data from the phase I/2 study and phase 3 study were analyzed for differences between combined reduced dose (0.2 mg/kg, phase 1/2 low-dose group) or delayed dose (phase 3, placebo group) and standard dose (1 mg/kg) treatment. Data were pooled from all three treatment trials (phase 1/2, phase 2, and phase 3) and then analyzed for baseline values and for differences between baseline and 24–48 and 72–96 weeks of treatment with galsulfase.
Data were analyzed using all available data and then re-analyzed using only those subjects for whom data were available at all three study points (baseline, weeks 48 and 96) of galsulfase treatment.
Statistical analysis
Initial analyses were based on analysis of variance models or Student’s
t-test to assess differences in demographic variables at baseline between the various subgroups compared. Analysis of differences by gender and by age (<12 years versus ≥12 years) were made both at baseline and between weeks 24–48 and 72–96. Finally, the major hypotheses were evaluated using general linear models and performed for all subjects for whom data were available and again for subjects for whom data were available at all three study points of galsulfase treatment (Pettersen et al.
2008; Diggle et al.
2012). The models investigated differences over the three time points between and within each of the two treatment groups (reduced or delayed vs. standard dose). To estimate more efficient and unbiased regression parameters of data collected as repeated measures over time, the generalized estimating equation approach of Zeger and Liang was used (Diggle et al.
2012). This allowed specification of a working correlation matrix that accounts for the within-subject correlations. A significance level of 0.05 was used for all statistical tests. Data were analyzed using SAS version 9.2.
Discussion
The cardiovascular system is progressively and unambiguously affected in individuals with MPS VI. Left ventricular hypertrophy, as well as anatomic and functional abnormalities of the mitral and aortic valves, have previously been well described by others (Lael et al.
2010; Dangel
1998; Wippermann et al.
1995; Azevedo et al.
2004; Scarpa et al.
2009; Fesslova et al.
2009). Our data obtained prior to the initiation of ERT from a large number of severely affected subjects with MPS VI support these observations but, more importantly, describe the cardiac effects of long-term treatment with galsulfase ERT.
Prior to ERT, left ventricular hypertrophy and mitral valve stenosis were the most prominent cardiac features found by this study. Left ventricular hypertrophy was severe, with mean z-scores approaching 2 SD greater than normal, and was found in subjects of all ages. The mean gradient across the mitral valve was elevated at baseline and increased significantly when those <12 years of age were compared to those ≥12. Mitral valve replacement, performed in three subjects before, or during, these studies confirmed the severity of this finding. By contrast, mitral valve regurgitation, usually a more common finding in most MPS VI pediatric reports (Lael et al.
2010; Dangel
1998; Wippermann et al.
1995; Azevedo et al.
2004; Scarpa et al.
2009; Fesslova et al.
2009) was only mild in our subjects. The presence of mitral stenosis, rather than regurgitation, has been found in older individuals with MPS VI (Diggle et al.
2012; Marwick et al.
1992; Tan et al.
1992) and is consistent with the older age of the subjects in this study.
Aortic valve obstruction at baseline was significantly greater in those 12 years of age or older but, when compared to mitral obstruction, was milder. Only one subject underwent aortic valvuloplasty prior to initiation of enzyme treatment. Only trace aortic regurgitation was present at baseline, a finding considered not physiologically significant.
Long-term enzyme replacement therapy with galsulfase was associated with maintenance of normal left ventricular function in all subjects in this study and regression of left ventricular septal hypertrophy in those who initiated treatment before 12 years of age. This remained true when we evaluated the entire cohort as well as when we analyzed only those in whom all three measurement points were available. During the course of this study, cardiac valve stenosis neither worsened nor improved, regardless of age. Aortic valve regurgitation increased significantly—but not physiologically—after 96 weeks of enzyme replacement in subjects ≥12 years of age. Although this finding had little physiologic consequence to these individuals, it may imply that cardiac valve pathology, once begun, may not be reversible. This progression of aortic regurgitation in the ≥12 years of age group is most likely due to underlying disease. It is difficult to assess causal relationship with ERT treatment given that the treatment group was followed for 96 weeks, placebo for 24 weeks, and echocardiography was not assessed after 24 weeks of treatment or placebo.
Valve obstruction is identified by the measurement of increased Doppler flow velocities across cardiac valves. With a normal cardiac output, the maximum normal Doppler velocity across the mitral valve is 1.3 m/s or 6.7 mmHg in children (Hatle and Angelsen
1985). The mean mitral gradient, a more accurate measure of obstruction, is obtained by averaging the instantaneous mitral flow velocities throughout diastole, resulting in a lower value. Mean mitral valve gradients <5 mmHg are consistent with mild mitral obstruction in adults; no values have been established for children (Baumgartner et al.
2009). The maximum normal Doppler velocities across the aortic valve in adults and children are 1.7–1.8 m/s, respectively, corresponding to peak aortic gradients of 11–13 mmHg (Hatle and Angelsen
1985). At baseline the mean mitral and aortic valve gradients in our subjects exceeded normal values and were greater in older subjects, consistent with the progressive nature of MPS VI.
The lack of response of the cardiac valves to ERT is similar to that reported in a small series of MPS VI patients who underwent hematopoietic stem cell transplantation (HSCT) and were studied an average of 5 (range 1.8–9) years after the procedure (Herskhovitz et al.
1999). The relatively avascular nature of cardiac valve tissue (Dow and Harper
1932) may, in part, explain the lack of improvement in valve morphology and function with either HSCT or ERT. Irreversible valve damage accruing over years may also explain the lack of response in older children and young adults (such as the subjects of this study) to any type of intervention. In support of early intervention, galsulfase, given from 8 weeks of life, has been shown to prevent cardiac abnormalities altogether (McGill et al.
2010).
Although this study had a large number of subjects with MPS VI who were studied over a lengthy time period, there are limitations to the study. The placebo group was followed for only the first 24 weeks of the phase 3 study. If follow-up had been extended for a longer time period, other differences between the placebo and treated groups may have been identified. The second limitation of this study is that ultrasounds were performed and analyzed at local sites. The concept of a central echocardiographic facility to provide reliable and reproducible data for multicenter pediatric cardiac studies emerged during the course of these trials (Lipschultz et al.
2001). Cardiac ultrasound in subjects with MPS can be difficult due to abnormalities of the thorax, poor lung expansion from hepatomegaly and restrictive lung disease, and inability to extend the neck. Comparison of M-mode measurements made in the field versus the central location in two different pediatric studies (Lipschultz et al.
2001; Dai et al.
1999) suggests that repeated measurements from the field may be more reliable than a single measurement. Thus although inter-institutional differences may have affected the absolute values obtained in this study, it is likely that trends (repeated values from the same sites) were less affected.
Acknowledgments
We acknowledge the participation of study patients and their families and the expert assistance of all study site coordinators and study site personnel. This study was an investigator-initiated study sponsored by BioMarin Pharmaceutical Inc., and supported, in part, with funds provided by the National Center for Research Resources, 5 M01 RR-01271 (Dr. Harmatz), 5 M01 RR-00400 (Dr. Whitley), M01 RR-00334 (Dr. Steiner), and UL1-RR-024134 (Dr. Kaplan). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. The support of the European Consortium for Lysosomal Diseases (EUCLYD, 7th Framework program, European Union) is acknowledged (Dr. Beck).
BioMarin reviewed the manuscript to insure the accuracy of all statements regarding enzyme replacement therapy with galsulfase. All authors participated in the galsulfase clinical trials, collection of data, development and writing of the manuscript and are fully responsible for its content.
*The MPS VI Study Group co-investigators are John Waterson, MD, PhD and Elio Gizzi, MD, Children’s Hospital & Research Center Oakland, Oakland, CA; Yasmina Amraoui, MD, Children’s Hospital, University of Mainz, Germany; Bonito Victor, MD, Unidade de Doenças Metabólicas, Departamento Pediatria, Hospital de Sao João, Porto, Portugal; Javier Arroyo, MD, Hospital San Pedro de Alcantara, Hospital de día de Pediatría, Caceres, Spain; D.N. Bennett-Jones, MD, Consultant General and Renal Physician, Whitehaven, UK; Philippe Bernard, MD, Centre Hospitalier d’Arras, Arras, France; Prof. Billette de Villemeur, Hôpital Trousseau, Paris, France; Raquel Boy, MD, Hospital Universitário Pedro Ernesto, Rio de Janeiro, Brazil; Eduardo Coopman, MD, Hospital del Cobre De. Salvador, Calama, Chile; Prof. Rudolf Korinthenberg, Universitätsklinikum Freiburg, Zentrum für Kinderheilkunde und Jugendmedizin, Klinik II Neuropädiatrie und Muskelerkrankungen, Freiburg, Germany; Michel Kretz, MD, Hôpital Civil de Colmar, Le Parc Centre de la Mère et de l’Enfant, Colmar, France; Shuan-Pei Lin, MD, MacKay Memorial Hospital, Department of Genetics, Taipei, Taiwan; Ana Maria Martins, MD, UNIFESP, Instituto de Oncologia Pediátrica, GRAACC/UNIFESP, Departamento de Pediatria, São Paulo, Brazil; Anne O’Meara, MD, Our Lady’s Hospital for Sick Children, Dublin, Ireland; Gregory Pastores, MD, PhD, NYU Medical Center, Rusk Institute, New York, NY; Lorenzo Pavone, MD, Rita Barone, MD, Agata Fiumara, MD, and Prof. Giovanni Sorge, Department of Pediatrics, University of Catania, Catania, Italy; Silvio Pozzi, MD, Ospedale Vito Fazzi, UO Pediatria, Lecce, Italy; Uwe Preiss, MD, Universitätsklinik und Poliklinik fűr Kinder, Halle, Germany; Emerson Santana Santos, MD, Fundação Universidade de Ciências da Saúde de Alagoas Governador, Departamento de Pediatria, Maceió, Brazil; Isabel Cristina Neves de Souza, MD, and Luiz Carlos Santana da Silva, PhD, Universidade Federal do Pará, Centro de Ciências Biológicas, Hospital Universitário João de Barros Barreto, Belém, Brazil; Eugênia Ribeiro Valadares, MD, PhD, Hospital das Clínicas, Faculdade de Medicina da Universidade Federal de Minas Gerais-UFMG, Avenida Professor Alfredo Balena, Belo Horizonte-Minas Gerais, Brazil; Laura Keppen, MD, Department of Pediatrics, University of South Dakota School of Medicine, Sioux Falls, SD; David Sillence, MD, Children’s Hospital, Westmead, Australia; Lionel Lubitz, MD, Royal Children’s Hospital, Melbourne, Australia; William Frischman, MD, The Townsville Hospital, Townsville, Australia; Julie Simon, RN, Children’s Hospital & Research Center Oakland, Oakland, CA; Claudia Lee, MPH, Children’s Hospital & Research Center Oakland, Oakland, CA; Stephanie Oates, RN, Metabolic Unit, SA Pathology at Women’s and Children’s Hospital Adelaide, North Adelaide, Australia; Lewis Waber, MD, PhD, Pediatric Genetics and Metabolism, University of Texas Southwest Medical Center, Dallas, TX; Ray Pais, MD, Pediatric Hematology/Oncology, East Tennessee Children’s Hospital, Knoxville, TN; Laila Arash, MD, Children's Hospital, University of Mainz, Germany; Robert Steiner MD, Departments of Pediatrics and Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR; Chester B Whitley, PhD, MD, University of Minnesota Medical School, Minneapolis, MN; Paige Kaplan, MD, Children’s Hospital of Philadelphia, Philadelphia, PA; Barbara Plecko, MD, Univ. Klinik für Kinder und Jugendheilkunde, Graz, Austria.