Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replacement therapy: Data from the Fabry Registry

YMGME-06068; No. of pages: 9; 4C: Molecular Genetics and Metabolism xxx (2016) xxx–xxx

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Molecular Genetics and Metabolism journal homepage: www.elsevier.com/locate/ymgme

Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replacement therapy: Data from the Fabry Registry Robert J. Hopkin a,b,⁎, Gustavo Cabrera c, Joel Charrow d, Roberta Lemay e, Ana Maria Martins f, Michael Mauer g, Alberto Ortiz h, Manesh R. Patel i, Katherine Sims j, Stephen Waldek k, David G. Warnock l, William R. Wilcox m a

Cincinnati Children's Hospital Medical Center, Cincinnati, USA Department of Pediatrics, University of Cincinnati College Medicine, Cincinnati, USA c Centro Cardiovascular Bolivar, Buenos Aires, Argentina d Division of Genetics, Birth Defects and Metabolism, Ann and Robert H. Lurie Children's Hospital of Chicago, Chicago, USA e Strategic Epidemiology & Biostatistics, Rare Diseases, Sanofi Genzyme, Cambridge, USA f Reference Center for Inborn Errors of Metabolism, Federal University of São Paulo, São Paulo, Brazil g Departments of Pediatrics and Medicine, University of Minnesota, Minneapolis, USA h Unidad de Dialisis, IIS-Fundacion Jimenez Diaz, School of Medicine, UAM, IRSIN, REDINREN, Madrid, Spain i Division of Cardiovascular Medicine, Duke University School of Medicine, Durham, USA j Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, USA k University of Sunderland, Sunderland, United Kingdom l Division of Nephrology, University of Alabama at Birmingham, Birmingham, USA m Division of Medical Genetics, Department of Human Genetics, Emory University School of Medicine, Atlanta, USA b

a r t i c l e

i n f o

Article history: Received 26 April 2016 Received in revised form 10 June 2016 Accepted 10 June 2016 Available online xxxx Keywords: Fabry disease Severe clinical events Risk factors Agalsidase beta

a b s t r a c t Background: Fabry disease, an X-linked lysosomal storage disorder, causes intracellular accumulation of glycosphingolipids leading to progressive renal, cardiovascular, and cerebrovascular disease, and premature death. Methods: This longitudinal Fabry Registry study analyzed data from patients with Fabry disease to determine the incidence and type of severe clinical events following initiation of enzyme replacement therapy (ERT) with agalsidase beta, as well as risk factors associated with occurrence of these events. Severe events assessed included chronic dialysis, renal transplantation, cardiac events, stroke, and death. Results: The analyses included 969 male and 442 female Fabry patients. The mean age at first agalsidase beta infusion was 35 and 44, and median treatment follow-up 4.3 years and 3.2 years, respectively. Among males, cardiac events were the most common on-ERT events, followed by renal, stroke, and non-cardiac death. Among females, cardiac events were also most common followed by stroke and renal events. Patients with on-ERT events had significantly more advanced cardiac and renal disease at baseline as compared with patients without on-ERT events. Severe events were also associated with older age at ERT initiation (males and females), a history of pre-ERT events (females; approaching statistical significance in males), and a higher urinary protein/creatinine ratio (females). Approximately 65% of patients with pre-ERT events did not experience subsequent on-ERT events. Of patients without pre-ERT events, most (84% of males, 92% of females) remained event-free. Conclusions: Patients with on-ERT severe events had more advanced Fabry organ involvement at baseline than those without such events and patients who initiated ERT at a younger age had less residual risk of on-ERT events. The observed patterns of residual risk may aid clinicians in multidisciplinary monitoring of male and female patients with Fabry disease receiving ERT, and in determining the need for administration of adjunctive therapies. © 2016 Published by Elsevier Inc.

Abbreviations: eGFR, estimated glomerular filtration rate; ERT, enzyme replacement therapy; ESRD, end-stage renal disease; GL-3, globotriaosylceramide; IEC, independent ethics committee; IRB, institutional review board; LVH, left ventricular hypertrophy; UP/Cr, urinary protein/creatinine ratio. ⁎ Corresponding author at: Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229, USA. E-mail addresses: [email protected] (R.J. Hopkin), [email protected] (G. Cabrera), [email protected] (J. Charrow), [email protected] (R. Lemay), [email protected] (A.M. Martins), [email protected] (M. Mauer), [email protected] (A. Ortiz), [email protected] (M.R. Patel), [email protected] (K. Sims), [email protected] (S. Waldek), [email protected] (D.G. Warnock), [email protected] (W.R. Wilcox).

http://dx.doi.org/10.1016/j.ymgme.2016.06.007 1096-7192/© 2016 Published by Elsevier Inc.

Please cite this article as: R.J. Hopkin, et al., Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replace..., Mol. Genet. Metab. (2016), http://dx.doi.org/10.1016/j.ymgme.2016.06.007

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1. Introduction Fabry disease is a rare, X-linked, systemic, multi-organ disorder in which globotriaosylceramide (GL-3) and other glycosphingolipids accumulate within cells owing to insufficient activity of the enzyme αgalactosidase A. Progressive accumulation of GL-3 in multiple cell types can lead to irreversible end-organ damage and is associated with severe cardiovascular, renal, and cerebrovascular complications, as well as premature death [1,2]. Hemizygous males with little or no residual enzyme — most commonly seen in those with non-sense mutations or large scale deletions — typically manifest the most severe consequences of Fabry disease [1,2]. Heterozygous females are not just carriers; they also frequently develop severe complications [3,4], including cardiac events [5], renal failure [6], cerebrovascular events [7], and premature death [8,9]. Fabry disease is currently treated with enzyme replacement therapy (ERT). The phase 3 clinical trial of agalsidase beta demonstrated near complete clearance of GL-3 from some cell types including endothelial cells, but incomplete clearance of others [10,11]. Subsequent studies of agalsidase beta have reported long-term stabilization of renal function, improvements in cardiac hypertrophy, and, in patients with advanced Fabry disease, slowing of the progression to the composite clinical outcome of renal, cardiac, and cerebrovascular complications and death [12–16]. In this study, we analyzed Fabry Registry data from male and female patients with Fabry disease with the objective of determining the incidence and type of severe clinical events, and factors predicting the occurrence of these events after the initiation of ERT with agalsidase beta. 2. Patients and methods

designees and submitted for central processing. Patients with nonclassic, later-onset variant mutations (p.Asn215Ser [N215S], p.Arg112His [R112H], p.Ala97Val [A97V], p.Gly35Arg [G35R], p.Ile317Thr [I317T], p.Arg301Gln [R301Q]; n = 61) [17–22], as reported in the “fabry-database.org” Fabry mutation database [23,24], were excluded from the analysis. The “fabry-database.org” database of clinical phenotypes, genotypes, and structures of mutant GLAs has been created and maintained by an independent research group led by Dr. H. Sakuraba to help researchers and clinicians who study Fabry disease. The database currently contains N640 different mutations [23]. Individuals with non-pathogenic Fabry gene polymorphisms (p.Asp313Tyr [D313Y], p.Arg118Cys [R118C]; n = 3) [25–27] were also excluded. Thus, the analysis population included: 1. patients with mutations categorized in the “fabry-database.org” Fabry mutation database as being associated with “classic” Fabry disease; 2. patients with mutations not entered or classified in this database; 3. patients for whom mutations were not reported in the Fabry Registry. 2.3. Severe clinical events The following four severe, potentially life-threatening, clinical events were included in the analyses: 1) renal events: first occurrence of either the initiation of chronic dialysis (N 40 days) or renal transplantation; 2) cardiac events: cardiac-related death, myocardial infarction, first-time congestive heart failure, atrial fibrillation, ventricular tachycardia, evidence of progressive heart disease severe enough to require pacemaker placement, bypass surgery, coronary artery dilatation, or implantation of a cardioverter/defibrillator; 3) cerebrovascular events: hemorrhagic or ischemic stroke, or stroke of unknown type; and, 4) death: non-cardiac-related death. Hereafter, for brevity, “severe clinical events” are also named “severe events” or “events”.

2.1. Fabry Registry 2.4. Characterization of patients at ERT baseline The Fabry Registry was initiated in 2001 as part of an effort to help healthcare professionals involved in the diagnosis and/or treatment of Fabry disease to better understand the disease and its management and to help develop Fabry disease treatment monitoring guidelines. It is a multi-center, international, longitudinal, observational program designed to track the natural history and outcomes of patients with Fabry disease. Patient and investigator participation is voluntary. Each independent site is responsible for obtaining a patient's informed written consent to submit their health information to the Registry, and to use and disclose this information in subsequent aggregate analyses. The Registry protocol, informed consent form, and any locally required authorization documents to send patient information to the Registry are reviewed and approved by the local fully constituted Institutional Review Board (IRB) or Independent Ethics Committee (IEC) unless the site provides the Registry with documentation that approval is not required or has been waived by a particular IRB/IEC. The Fabry Registry is registered at www.ClinicalTrials.gov under the identifier NCT00196742 and is sponsored by Sanofi Genzyme. 2.2. Patients Fabry Registry patients of any age who received agalsidase beta (Fabrazyme; Sanofi Genzyme, Cambridge, MA, USA) as their initial source of ERT given at the recommended licensed dose of 1.0 mg/kg every other week [qow; averaged; dose range: ≥ 0.9 to b1.1 mg/kg qow] were included in this analysis. The observation interval extended from the time each patient began treatment until the earliest of: 1) their most recently reported data to the Fabry Registry, through June 25, 2009, when many began temporary agalsidase beta dose reductions owing to manufacturing issues; 2) agalsidase beta treatment was discontinued; or 3) agalsidase beta was switched to another treatment. Data in the Fabry Registry were collected by physicians or their

In addition to demographic data and severe clinical events that had occurred before initiation of ERT, several disease factors were evaluated to characterize patients at the time of ERT initiation. Baseline cardiac characteristics evaluated included left ventricular hypertrophy (LVH), arrhythmias (other than atrial fibrillation or ventricular tachycardia), and hypertension. Baseline renal function evaluation included estimated glomerular filtration rate (eGFR) and the urinary protein/creatinine (UP/Cr) ratio. eGFR was calculated from serum creatinine values using the Chronic Kidney Disease Epidemiology Collaboration equation (adults) [28] or the Bedside Schwartz equation (patients aged b18 years) [29]. 2.5. Statistical analysis Patients who received agalsidase beta as defined by the inclusion criteria were analyzed. Patients were stratified by whether they had a severe event pre-ERT and by whether they had a severe event on-ERT. Baseline refers to the time of initiation of first agalsidase beta treatment. Continuous data were analyzed via the t-test (or Wilcoxon, as appropriate). The Chi-square test (or Fisher's Exact test, as appropriate) was used for categorical data. Covariate selection (based on available data and model-fit statistics) for multivariate Poisson regression was performed to identify factors associated with the occurrence of on-ERT events. These factors included: event pre-ERT, age at initiation of ERT, and UP/Cr ratio (females). A Kaplan–Meier time-to-event model depicting event-free time on ERT was performed stratified by pre-ERT event status (event vs. no event). Incidence rates were calculated and are expressed as the number of events per 1000 person-years of follow-up, following initiation of ERT (first dose to first event). The small number of excluded individuals with later-onset variant mutations or Fabry gene polymorphisms did not allow for a meaningful

Please cite this article as: R.J. Hopkin, et al., Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replace..., Mol. Genet. Metab. (2016), http://dx.doi.org/10.1016/j.ymgme.2016.06.007

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separate analysis of these groups. For all analyses, an alpha level of 0.05 was used as the criterion for determining statistical significance. Statistical analyses were performed using SAS statistical software version 9.1 (SAS Institute Inc., Cary, NC, USA). 3. Results 3.1. Demographic characteristics of patients Of the 3930 patients (1924 males, 2006 females) enrolled in the Fabry Registry, 1411 patients (969 males, 442 females) met the inclusion criteria for these analyses. The baseline demographic and clinical characteristics of patients are reported by gender and by presence or absence of events on-ERT in Table 1. The mean (SD) age at first agalsidase beta infusion for males and females was 35 (14.2) years and 44 (14.3) years, respectively. The median agalsidase beta treatment follow-up was 4.3 years (range 0–11.1 years) and 3.2 years (range 0–10 years) in males and females, respectively. Of the 1411 patients, 446 (32%) had mutations categorized in the “fabry-database.org” database as being associated with classic Fabry disease, 426 (30%) had mutations not entered or classified in this database [23], and 539 (38%) did not have genotype data available. 3.2. Demographic and clinical characteristics by on-ERT event status Male patients without an on-ERT event were started on agalsidase beta treatment on average 13 years earlier than those with an on-ERT event (mean age 32 vs. 45 years, respectively; p b 0.001; Table 1). They were also younger at diagnosis (mean age 27 vs. 34 years, respectively; p b 0.001), and had shorter time between symptom onset and treatment (mean time 18 vs. 30 years, respectively; p b 0.001). Female patients were older than male patients at symptom onset, diagnosis and ERT initiation. Females without an on-ERT event were started on ERT a decade earlier than those who had an on-ERT event

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(mean age 43 vs. 53 years, respectively; p b 0.001). These females were also younger at diagnosis (mean age 36 vs. 45 years, respectively; p b 0.001). The mean time between symptom onset and ERT initiation was similar for females who had no on-ERT event or had an event (20 vs. 22 years, respectively; p = 0.521). Male patients without an on-ERT event had a much higher baseline eGFR than males with an on-ERT event (mean eGFR 89 vs. 53 ml/min/1.73 m2, respectively; p b 0.001), and a significantly lower baseline UP/Cr ratio (mean UP/Cr ratio: 0.7 vs. 1.6 g/g; p b 0.001). Females without an on-ERT event also had a higher baseline eGFR (mean eGFR: 88 vs. 64 ml/min/1.73 m2, respectively; p b 0.001) and lower baseline UP/Cr ratio compared with those with an event (mean UP/Cr ratio: 0.8 vs. 1.6 g/g; p = 0.01). Pre-ERT LVH, arrhythmias and hypertension were significantly less frequent in both males and females without events on-ERT compared with those with events. 3.3. Incidence rates of on-ERT events by pre-ERT event status On-ERT events were recorded in 202 males (21%) and 56 females (13%), whereas pre-ERT events had been recorded in 249 males (26%) and 74 females (17%) (Fig. 1). On-ERT events were cardiac (males n = 85 [9% of all males], females n = 31 [7% of all females]), renal (males n = 65 [7%], females n = 11 [2%]), cerebrovascular (males n = 47 [5%], females n = 14 [3%]), and non-cardiac death (males n = 41 [4%], females n = 5 [1%]). Pre-ERT events were cardiac (males n = 83 [9% of all males], females n = 38 [9% of all females]), renal (males n = 143 [15%], females n = 13 [3%]), and cerebrovascular (males n = 82 [8%], females n = 30 [7%]) (Fig. 1). Pre-ERT events were nearly twice as common in males with on-ERT events than in males without on-ERT events. Among the males with events on-ERT (n = 202), 89 (44%) had experienced pre-ERT events, whereas among males who did not have an on-ERT event (n = 767), 160 (21%) had a pre-ERT event (p b 0.001). Of the males without a

Table 1 Baseline characteristics by event status on-treatment. Males (n = 969)

Age at first ERTa, n Mean (SD) ERT follow-up timea, n Mean (SD) Age at diagnosisa, n Mean (SD) Age at symptom onseta, n Mean (SD) Symptom onset to first ERTa, n Mean (SD) eGFR at ERT baselineb, n Mean (SD) eGFR b 60b, n (%) UP/Cr ratio at ERT baselinec, n Mean (SD) Cardiac echography prior to ERT, n LVH prior to ERT, n (%) Electrocardiogram prior to ERT, n Arrhythmia prior to ERT, n (%) Blood pressure at ERT baseline, n Hypertension at ERT baseline, n (%)

No event on-ERT

Event on-ERT

(n = 767, 79.2%)

(n = 202, 20.8%)

767 32.3 (13.9) 767 3.9 (2.5) 759 27.0 (15.3) 647 14.1 (12.3) 647 17.6 (12.6) 465 88.5 (35.6) 102 (22) 248 0.7 (1.1) 405 221 (28.8) 418 120 (29) 507 240 (47)

202 45.2 (10.0) 202 5.0 (2.5) 201 33.6 (15.4) 166 15.6 (13.5) 166 29.7 (13.7) 144 52.6 (34.9) 92 (64) 82 1.6 (1.5) 115 100 (49.5) 122 66 (54) 149 99 (66)

Females (n = 442) p

b0.001 b0.001 b0.001 0.171 b0.001 b0.001

b0.001 b0.001 b0.001 b0.001

No event on-ERT

Event on-ERT

(n = 386, 87.3%)

(n = 56, 12.7%)

386 42.8 (14.4) 386 3.1 (2.1) 381 36.3 (16.3) 281 21.6 (16.1) 280 20.3 (15.7) 224 88.2 (27.0) 34 (15) 172 0.8 (1.3) 258 141 (36.5) 250 79 (32) 249 147 (57)

56 52.5 (10.4) 56 4.4 (1.8) 55 45.3 (14.2) 43 29.2 (19.0) 43 22.0 (17.3) 34 64.0 (31.6) 14 (41) 22 1.6 (2.0) 40 33 (58.9) 34 23 (68) 41 31 (76)

p

b0.001 b0.001 b0.001 0.006 0.521 b0.001

0.010 b0.001 b0.001 0.022

LVH is defined as ≥12 mm from values in the range 5–35 mm and/or a ‘Yes’ checkbox response. Arrhythmia is defined as other than atrial fibrillation or ventricular tachycardia from electrocardiogram values and/or ‘Yes’ checkbox response. Hypertension is defined as ≥130/80 mmHg and/or a ‘Yes’ checkbox response. eGFR, estimated glomerular filtration rate; ERT, enzyme replacement therapy (agalsidase beta); LVH, left ventricular hypertrophy; SD, standard deviation; UP/Cr, urinary protein/creatinine ratio. a All times and ages are in years. n represents numbers of patients reporting data. b Baseline eGFR (ml/min/1.73 m2) is the value closest to and within 3 months of first agalsidase beta infusion. c Baseline UP/Cr ratio (g/g) is the value closest to and within −6/+3 months of first agalsidase beta infusion.

Please cite this article as: R.J. Hopkin, et al., Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replace..., Mol. Genet. Metab. (2016), http://dx.doi.org/10.1016/j.ymgme.2016.06.007

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pre-ERT event, 607 of 720 males (84%) did not experience an on-ERT event (Fig. 1). Pre-ERT events were also far more common among females with onERT events than in those without on-ERT events. Among the females with on-ERT events (n = 56), 26 (46%) had experienced pre-ERT events, whereas among females who did not have an on-ERT event (n = 402), 48 (12%) had a pre-ERT event (p b 0.001). Of the females without a preERT event, 338 of 368 females (92%) had not experienced an on-ERT event (Fig. 1). Fig. 2 shows the results of the analysis of the incidence rates of onERT events per 1000 person-years of treatment stratified by the preERT event status (pre-ERT events vs. no events). Incidence rates were higher for patients who had a pre-ERT event of cardiac, stroke and/or non-cardiac death as compared with those without pre-ERT events. More male than female patients had a pre-ERT renal event. By definition, renal events were no longer counted once a patient reached endstage renal disease (ESRD) as indicated by dialysis or transplant (thus, a patient could not have multiple renal events). Particularly for females, the number of events per event type on-ERT were low. For example, 14 out of 442 females had a cerebrovascular event of whom 10 had experienced a pre-ERT event. The 95% confidence intervals are relatively wide. A Kaplan–Meier time-to-event analysis was performed for both male and female patients (Fig. 3). After 6 years of ERT, approximately 80% of males without pre-ERT events had not developed any on-ERT event, compared with approximately 55% of males who had a pre-ERT event. Approximately 85% of females without a pre-ERT event had not developed an on-ERT event, compared with just over 50% of females who did have a pre-ERT event. 3.4. Type of on-ERT events by pre-ERT event type Fig. 4 shows the frequency and type of severe events occurring while on ERT by pre-ERT event type (renal, cardiac, stroke, and none) for males (Fig. 4A) and for females (Fig. 4B), respectively. For males with pre-ERT renal or cardiac events, cardiac events on-ERT were the most common events. Those with pre-ERT stroke events were most likely to experience non-cardiac death on-ERT. For males who did not have any pre-ERT events, a renal event was the most common on-ERT event.

For female patients with pre-ERT cardiac events, cardiac events were the most common on-ERT events. For those who had pre-ERT stroke events, stroke events were the most common on-ERT events. Those with renal events pre-ERT were most likely to experience non-cardiac death on-ERT. For females without pre-ERT events, cardiac events were the most common on-ERT events. 3.5. Regression modeling of risk factors for on-ERT events Independent risk factors for events after starting ERT were assessed by regression modeling. The final models are shown in Table 2. The final model for male patients (n = 969) suggested that the risk for an on-ERT event increased when ERT was initiated at an older age. Males aged ≥40 years at the time of first ERT had a 7.8-fold higher risk of on-ERT events than those aged b 30 years (p b 0.001). Males aged between 30 and 40 years had a 3.7-fold higher risk than those aged b30 years (p b 0.001). A trend for an increased risk for an on-ERT event was found for males who had a pre-ERT event (1.3-fold higher risk, p = 0.058). Of note, pre-ERT renal events (dialysis or transplantation) occurred in 15% of the males and, by definition, these males could not have another renal event on-ERT. For male patients UP/Cr was not included in the final model due to missing values. Risk for females (n = 282) also increased with age. The risk for an on-ERT event for females starting ERT at an age higher than the median age at start of ERT was 3.6-fold higher than that of females starting ERT at an age lower than the median age (p = 0.006). In females, having a pre-ERT event increased the risk for an on-ERT event by 2.8-fold (p = 0.004). Females with a UP/Cr ratio ≥ 0.5 g/g at baseline had a 2.1-fold increased risk of having an on-ERT event versus those who had a UP/Cr ratio b 0.5 g/g (p = 0.038; Table 2). 4. Discussion These analyses represent the first time that baseline risk factors for severe clinical events after ERT initiation have been assessed, and the incidence rates and pattern of such events evaluated in a long-term follow-up of a large cohort of male and female patients with Fabry

Fig. 1. Severe clinical events pre- and on-ERT. ERT = enzyme replacement therapy (agalsidase beta).

Please cite this article as: R.J. Hopkin, et al., Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replace..., Mol. Genet. Metab. (2016), http://dx.doi.org/10.1016/j.ymgme.2016.06.007

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Fig. 2. Overall incidence rates for on-ERT events, stratified by pre-ERT event status: (A) male and (B) female patients. ERT = enzyme replacement therapy (agalsidase beta). *Incidence rate is the number of events per 1000 person-years of follow-up, calculated as the time from first ERT to the date of the first on-ERT severe event. The figure includes the 95% confidence intervals.

disease treated with agalsidase beta. The results show that the most common type of reported severe events on-ERT among male patients was cardiac, followed by renal, and less frequently, stroke, and noncardiac death; in females it was cardiac, followed by stroke and renal events. Patients with on-ERT events had significantly more advanced cardiac and renal disease at baseline than patients without events onERT. Moreover, the occurrence of severe events was associated with an older age at initiation of ERT, whereas a pre-ERT history of events appeared to have a greater impact on residual risk in females than in males. Of patients with pre-ERT events, approximately 65% did not experience subsequent on-ERT events. Of patients without pre-ERT events, most (84% of males, 92% of females) remained event-free. Cardiac events were the most common on-ERT events. They were also the most common events in patients who were not receiving ERT in previous studies evaluating multiple events per patient [3–5], and are the most frequently reported cause of death in patients with Fabry disease [8,9]. The extent of myocardial fibrosis (not evaluated in the current study) has been recognized as a crucial determinant of cardiac outcome with or without ERT [30]. Pre-ERT renal events occurred in 15% of the males and were more prevalent in patients with on-ERT events. These renal events may have increased the risk of cardiac and CNS events while on ERT. Chronic

renal failure itself is associated with ~10 to 20-fold greater cardiovascular risk than that of the general population [31,32]. In this study, we cannot determine if some of these renal disease-associated risks are due to ESRD rather than directly due to Fabry disease. Stroke risk is significantly increased in patients with Fabry disease. The pathogenesis of cerebrovascular disease is not well understood, but is undoubtedly complex [7,33]. Fabry-related cardiac and renal disease may contribute to cerebrovascular complications. The frequencies of occurrence of cerebrovascular events (pre-ERT: 8% of males, 7% of females; on-ERT: 5% of males, 3% of females) and incidence rates reported here cannot be directly compared with data from another Fabry Registry study (6.9% of males, 4.3% of females) [7] as the definitions of cerebrovascular events and inclusion criteria were different. The latter study analyzed natural history data from 2446 patients (1243 males) [7], whereas only patients who were eventually initiated on ERT (i.e. disease severity criteria for treatment initiation had been met) and had been followed during treatment were included in the current analysis. A recent study reported that Fabry patients with stable eGFR suffered fewer strokes than those with more rapidly progressing renal disease [34]. A study in young stroke patients without Fabry disease demonstrated that lower eGFR rates, although still within the normal

Please cite this article as: R.J. Hopkin, et al., Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replace..., Mol. Genet. Metab. (2016), http://dx.doi.org/10.1016/j.ymgme.2016.06.007

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Fig. 3. Time to first on-ERT event: (A) male and (B) female patients. ERT = enzyme replacement therapy (agalsidase beta).

range, were associated with the presence of white matter hyperintensity lesions in the brain [35]. Stroke has been observed in young patients with Fabry disease who had no evidence of renal or cardiac disease at the time of the stroke (unpublished personal observations of authors). Therefore, the risk of a stroke event may not precisely correlate with measured renal or heart function, suggesting that other factors may play a causal role. Aging of the present cohort during natural history follow-up would be expected to result in an ever-increasing rate of events. Moreover, no therapy is expected to decrease the risk of events driven by advanced pre-existing permanent fibrotic organ damage such as ESRD, i.e. Fabry patients with advanced disease who receive ERT may still be at-risk of events [12,15]. This is supported by our observation that patients without severe pre-ERT events were more likely to remain free of events onERT than those who already had a severe pre-ERT event. It is encouraging that, of patients who started agalsidase beta therapy relatively late, most did not have severe events over the first 6 years of treatment with agalsidase beta. Nevertheless, the rate of severe events on-ERT is higher than in the general population without Fabry disease and residual risk remains above optimal levels [36,37]. The current analyses were not designed to draw conclusions that agalsidase beta reduces the incidence of severe events in comparison with the natural disease course of Fabry disease. This question has already been directly addressed in a placebo-controlled clinical trial of patients with advanced Fabry disease. That study showed that treatment with agalsidase beta significantly reduced the incidence of major cardiac, renal, and cerebrovascular events, and death, as compared with patients receiving placebo (baseline

proteinuria adjusted analysis) [12]. Another study analyzed the occurrence of severe clinical events during 10 years (median) of agalsidase beta treatment in 52 patients (2 females) with classic Fabry disease. Eighty-one percent of the patients remained free of severe clinical events and 94% of patients were alive at the end of the observation interval [15]. Another Fabry Registry study found that the occurrence of severe events is highest in the first 6 months of agalsidase beta treatment, and decreases thereafter, suggesting that disease burden at the time that ERT is begun influences early on-ERT events [38]. Agalsidase beta has not been shown to cross the blood–brain barrier. It is, therefore, of interest that a recent post-hoc analysis of data from a small cohort of Fabry patients aged younger than 50 years suggested that white matter lesion burden was more likely to remain stable in patients receiving agalsidase beta as compared with patients on placebo [39]. This is the first time the relative burden of each event category has been quantified in a way that allowed comparison of the pattern of risk for each gender. The analyses demonstrate that the clinical course of the disease differs somewhat between genders. Males starting ERT more often have greater renal impairment than females. Conversely, pre-ERT cardiac events made up slightly more than half of the severe events in females, followed by stroke and renal events. It is important to describe such differences in the patterns of risk between the genders and these should be taken into account when designing monitoring and treatment protocols for Fabry disease. It might also be important in designing future clinical trials because it appears unlikely that the results of studies focused predominantly on males with Fabry disease will be readily generalizable to both genders.

Please cite this article as: R.J. Hopkin, et al., Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replace..., Mol. Genet. Metab. (2016), http://dx.doi.org/10.1016/j.ymgme.2016.06.007

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Fig. 4. On-ERT events by pre-ERT event type: (A) male and (B) female patients. ERT = enzyme replacement therapy (agalsidase beta).

Several management guidelines have recommended that early initiation of ERT should be considered in females with Fabry disease at the first sign of organ involvement [40–43]. The present study suggests

that waiting for clinical evidence of vital organ injury in order to initiate treatment in female patients exposes them to residual risk of severe events while on ERT. It is particularly worrisome that females with

Table 2 Regression models: independent risk factors for on-ERT events in male and female Fabry Registry patients.a Parameter Male model: 3 factors, n = 969 Pre-ERT event (vs. no event) Age at ERT initiation 30 to b40 years (vs. age b 30 years) Age at ERT initiation ≥40 years (vs. age b 30 years) ERT follow-up time ≥ median (vs. ERT follow-up time b median) Female model: 3 factors, n = 282 Pre-ERT event (vs. no event) Age at ERT initiation ≥ median (vs. age at ERT initiation bmedian) Median UP/Cr ratio ≥ 0.5 g/g (vs. b0.5 g/g)

Odds ratio

95% CIb

p

1.32 3.73 7.79 0.48

0.99–1.77 2.1–6.61 4.56–13.29 0.36–0.64

0.058 b0.001 b0.001 b0.001

2.83 3.56 2.09

1.40–5.73 1.44–8.79 1.04–4.19

0.004 0.006 0.038

Only patients who had data available for each factor were included. a Results from a generalized linear model regression analysis using the Poisson distribution. b Lower and upper CIs for the estimates were calculated using the Wald test. CI, confidence interval; UP/Cr, urinary protein/creatinine ratio.

Please cite this article as: R.J. Hopkin, et al., Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replace..., Mol. Genet. Metab. (2016), http://dx.doi.org/10.1016/j.ymgme.2016.06.007

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R.J. Hopkin et al. / Molecular Genetics and Metabolism xxx (2016) xxx–xxx

on-ERT events were older at symptom onset than those who were event-free (mean age 29 vs. 22 years). Thus, the absence of reported symptoms at an early age may convey a false sense of security to the physician deciding on initiation of ERT in females. Contrary to the expectation that nearly all males with classical Fabry disease should receive ERT [40–42], it cannot be assumed that all female patients will require intervention. The goal of early treatment for those who do need intervention must be balanced by the goal of minimizing exposure to unnecessary interventions and expense for females. Clearly, additional biomarkers and predictors of risk in females who have not yet manifested overt evidence of end-organ injury are needed. For all patients, close monitoring and, if indicated, administration of adjunctive therapies such as angiotensin-converting enzyme/angiotensin receptor blockers, lipid control, and antiplatelet agents are essential to optimize their management. Fabry disease is a clinically heterogeneous disease and little is known about the natural progression of Fabry disease in patients with later-onset variant mutations. Therefore, patients with such mutations were excluded from the analysis. The main analysis population was restricted to patients with mutations categorized as being associated with classic Fabry disease, patients with mutations not entered or classified in the “fabry-database.org” database [23], and patients for whom mutations were not reported in the Fabry Registry. It is reasonable to presume that some of the patients in the latter three subgroups have later-onset disease; however, all received ERT, suggesting that disease severity criteria for treatment initiation, as per published general guidelines or local/national protocols, had been met during the course of their disease. Nevertheless, the impact of the inclusion of these patients would be expected to reduce the strength of the observations of this study. Other limitations associated with using data from the Fabry Registry must be considered when interpreting the results of these analyses. Patient and investigator participation in the Fabry Registry is voluntary and not all diagnosed Fabry patients have been enrolled. The total number of patients who participate in the Fabry Registry and the associated person-years of follow-up are not pre-defined. The Fabry Registry contains observational data and it is not a prospective clinical trial. Furthermore, data interpretation is limited by the lack of an appropriately matched control group. Although the Fabry Registry provides a recommended schedule of assessments, patients and treating physicians determine what assessments patients receive and the time intervals at which they are done. Thus, the clinical data presented may be incomplete for some patients, and some assessments or events may not have been reported. Additionally, patients may have an incompletely reported medical history or can be lost to follow-up. For these analyses, the types of clinical events evaluated were operationally defined; different event definitions may result in slightly different findings and conclusions. Moreover, the present study did not address several medically important complications of Fabry disease such as pain, gastrointestinal symptoms, hearing impairment, and lymphedema, and adjunctive treatment of modifiable risk factors, e.g. hypertension, likely changed over the observation interval. Incompleteness of the data set for the temporary dose reduction period precluded a meaningful analysis of clinical events occurring after dose reductions. Finally, regression modeling did not identify a significant association between a history of pre-ERT events and increased risk of on-ERT events in males. Nonsignificance of this finding (i.e., p = 0.058) may be related to the fact that, by definition, males with pre-ERT renal events (dialysis or transplantation; 15% of the males) could not have had another renal event on-ERT. This analysis was restricted to patients in the Fabry Registry. Further studies are needed to better understand how to recognize the risk of progressive disease and to establish guidelines for the initiation of treatment before organ damage occurs. To further characterize clinical issues related to Fabry disease outcomes, additional longitudinal data as well as subset analyses will help identify other important risk

factors to assist clinicians in optimizing the management of their patients with Fabry disease. 5. Conclusions This analysis of severe clinical events in patients with Fabry disease treated with agalsidase beta represents the largest sample size reported to date with this type of longitudinal follow-up data. The rate of progression and burden of disease appeared to be different between males and females included in the analysis, as judged by the type of events for each gender. The data suggest that both male and female patients with Fabry disease are at risk of severe events while on agalsidase beta treatment, especially when treatment is initiated at an older age. Patients with on-ERT events had significantly more advanced cardiac and renal disease at ERT baseline. Of the patients without a pre-ERT event, the vast majority remained event-free on ERT. The observed patterns of residual risk may aid clinicians in multidisciplinary monitoring of male and female patients with Fabry disease receiving ERT, and in determining the need for administration of adjunctive therapies. Funding This research was funded (statistical programming and medical writing support, and manuscript submission assistance; see “Acknowledgments” section) by Sanofi Genzyme, the sponsor of the Fabry Registry. The funder had a role in data collection and analysis. Competing interests R.J.H. consults with Sanofi Genzyme and Shire HGT, and has been an investigator in clinical trials sponsored by Sanofi Genzyme, Shire HGT, and Amicus Therapeutics. These activities have been monitored and found to be in compliance with the conflict of interest policies at Cincinnati Children's Hospital Medical Center. G.C. has active research support and consulting arrangements with Sanofi Genzyme. J.C. is a consultant for Sanofi Genzyme, Protalix Corporation, and Shire HGT, and has been an investigator in clinical trials sponsored by Sanofi Genzyme, Shire HGT, BioMarin Pharmaceuticals, and Amicus Therapeutics. These activities have been reviewed and managed by the Ann and Robert H. Lurie Children's Hospital of Chicago and Northwestern University's Feinberg School of Medicine, in accordance with their conflict of interest policies. R.L. is an employee of Sanofi Genzyme. A.M.M. consults for and has been an investigator in clinical trials sponsored by Sanofi Genzyme. M.M. consults for Sanofi Genzyme and has research funding for an investigator-initiated grant from Sanofi Genzyme. These interests have been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policy. M.M. also consults for and performs renal structural studies for Amicus Therapeutics and has received grants from Shire HGT. A.O. consults for Sanofi Genzyme and has received honoraria from Sanofi Genzyme and Shire HGT. M.R.P. is a consultant for Sanofi Genzyme, Janssen Pharmaceuticals, Bayer AG, and AstraZeneca; and has received research grants from Johnson & Johnson and AstraZeneca. K.S. is an investigator for clinical trials sponsored by Amicus Therapeutics and receives research funding from Sanofi Genzyme. These interests have been reviewed and managed by the Massachusetts General Hospital in accordance with its conflict of interest policy. S.W. has consulted for Sanofi Genzyme and Shire HGT. In the past, he has received research funding from Sanofi Genzyme, Shire HGT, Amicus Therapeutics, and BioMarin Pharmaceuticals. He has also received funding for travel and presenting at meetings.

Please cite this article as: R.J. Hopkin, et al., Risk factors for severe clinical events in male and female patients with Fabry disease treated with agalsidase beta enzyme replace..., Mol. Genet. Metab. (2016), http://dx.doi.org/10.1016/j.ymgme.2016.06.007

R.J. Hopkin et al. / Molecular Genetics and Metabolism xxx (2016) xxx–xxx

D.G.W. consults with Sanofi Genzyme, Amicus Therapeutics, and Shire HGT; and has been an investigator in clinical trials sponsored by Sanofi Genzyme and Amicus Therapeutics. These activities have been monitored and found to be in compliance with the conflict of interest policies at the University of Alabama at Birmingham. W.R.W. consults for Sanofi Genzyme and Shire HGT, and is an investigator in clinical trials sponsored by Sanofi Genzyme and Amicus Therapeutics. These activities are monitored and are in compliance with the conflict of interest policies at Emory University School of Medicine. Acknowledgments The authors would like to thank the many patients who have agreed to participate in the Fabry Registry as well as the physicians and research coordinators who have entered clinical data on behalf of these patients. We also acknowledge Pr. Dr. Jörg Strotmann, for input on the methodology and analysis plans, and Badari Gudivada (Sanofi Genzyme), for statistical programming support. We thank Cheryl Lathrop and Hans Ebels for providing medical writing/editing services on behalf of Sanofi Genzyme. A.O. was supported by ISCIII intensificación de la actividad investigadora. The authors received submission assistance provided by Alessia Piazza of Excerpta Medica, funded by Sanofi Genzyme.

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