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Glucose tetrasaccharide as a biomarker for monitoring the therapeutic response to enzyme replacement therapy for Pompe disease
Molecular Genetics and Metabolism 85 (2005) 247–254 www.elsevier.com/locate/ymgme
Glucose tetrasaccharide as a biomarker for monitoring the therapeutic response to enzyme replacement therapy for Pompe disease Yan An a,1, Sarah P. Young a,¤,1, Priya S. Kishnani a, David S. Millington a, Andrea AmalWtano a, Deyanira Corzo b, Yuan-Tsong Chen a,c a
Division of Medical Genetics, Department of Pediatrics, Duke University Medical Center, Durham and RTP, North Carolina, USA b Genzyme Corporation, Cambridge, MA, USA c Institute of Biochemical Sciences, Academica Sinica, Taipei, Taiwan Received 21 December 2004; received in revised form 17 March 2005; accepted 23 March 2005 Available online 10 May 2005
Abstract A tetraglucose oligomer, Glc1-6Glc1-4Glc1-4Glc, designated Glc4, has been shown to be a putative biomarker for the diagnosis of Pompe disease. The purpose of this study was to assess whether Glc4 could be used to monitor the therapeutic response to recombinant human acid glucosidase (rhGAA) enzyme replacement therapy (ERT) in patients with Pompe disease. Urinary Glc4 levels in 11 patients receiving rhGAA therapy was determined by both HPLC-UV and stable isotope dilution ESI-MS/MS. Combined Glc4 and maltotetraose, Glc1-4Glc1-4Glc1-4Glc, (M4) concentrations, designated Hex4, in plasma from these patients were measured by HPLC-UV only. Baseline urinary Glc4 and plasma Hex4 in these patients (mean § SD: 34.2 § 11.3 mmol/mol creatinine and 1.7 § 0.8 M, respectively) were higher than age-matched control values (mean § SD, 6.1 § 5.1 mmol/mol creatinine and 0.22 § 0.15 M, respectively). Both urinary Glc4 and plasma Hex4 levels decreased after initiation of ERT for all patients. In the four patients with the best overall clinical response in both skeletal and cardiac muscle, levels decreased to within, or near, normal levels during the Wrst year of treatment. In contrast, levels Xuctuated and were persistently elevated above the control ranges in those patients with a less favorable clinical response (good cardiac response but limited motor improvement). These results suggest that urinary Glc4 and plasma Hex4 could serve as a valuable adjunct to clinical endpoints for monitoring the eYcacy of therapeutic interventions such as rhGAA ERT in Pompe disease. 2005 Elsevier Inc. All rights reserved. Keywords: Glycogen storage disease type II; GSD-II; Pompe disease; Lysosome storage disease; Glc4; Oligosaccharide; HPLC; LC-MS/MS; Enzyme replacement therapy; Acid -glucosidase
Introduction Pompe disease, also known as glycogen storage disease type II (GSD-II), is an inherited muscle disorder of glycogen metabolism caused by deWciency of lysosomal acid -glucosidase (GAA), a glycogen-degrading enzyme *
1
Corresponding author. Fax: +1 919 549 0709. E-mail address: [email protected] (S.P. Young). An Y. and Young S.P. made equal contributions to this manuscript.
1096-7192/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.03.010
[1]. In its most severe form, the disease is characterized by massive cardiomegaly, macroglossia, progressive muscle weakness, and marked hypotonia in early infancy. Most infantile patients are diagnosed between 3 and 6 months of age and patients with the most severe presentation die around the Wrst year of life. Although at present there is no proven eVective treatment for Pompe disease, therapies are currently being developed and evaluated. Recent eVorts have focused on producing human recombinant GAA (hrGAA) for
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enzyme replacement therapy (ERT), in a manner similar to that utilized for other lysosomal disorders [2–4]. rhGAA has been produced in Chinese hamster ovary (CHO) cell cultures [5,6], and in transgenic mouse and rabbit milk [7,8], and its therapeutic eVects have been demonstrated in animal models [8,9]. Results from human clinical trials utilizing two diVerent forms of the recombinant enzyme have shown that rhGAA is capable of improving both cardiac and skeletal muscle function in patients with infantile Pompe disease [10–15]. Multiple gene therapy studies utilizing hGAA gene transfer to foster in vivo expression of hGAA have also shown high levels of eYcacy in several animal models of Pompe disease [16–20]. A tetraglucose oligomer, Glc1-6Glc1-4Glc1-4Glc, designated Glc4, has been shown to be elevated in both urine [21–24] and plasma [24,25] of patients with Pompe disease. Recently, we developed HPLC-UV [24] and stable isotope dilution ESI-MS/MS [26] methods for the analysis of Glc4 in biological Xuids as the butyl-p-aminobenzoate (BAB) derivative. These assays have been utilized for the determination of Glc4 levels in both normal individuals and patients with Pompe disease. Patients with Pompe disease were shown to have elevated Glc4 levels in urine and plasma by the HPLC-UV method [24]. Further analysis of plasma samples by hydrophilic interaction LC-MS/MS demonstrated a combined elevation of Glc4 and maltotetraose (Glc1-4Glc14Glc1-4Glc) in Pompe disease patients (unpublished observations). The objective of this study was to apply both the HPLC-UV and ESI-MS/MS methods to determine the utility of Glc4 as a non-invasive biomarker to monitor the therapeutic response in patients with Pompe disease receiving ERT with CHO cell-derived rhGAA.
Subjects and methods Materials and instrumentation Glc1-6Glc1-4Glc1-4Glc ((Glc)4), maltotetraose (M4), maltopentaose (M5), maltohexaose (M6), maltoheptaose (M7), cellopentaose (C5), sodium cyanoborohydride (NaBH3CN), butyl-4-aminobenzoate (BAB) and 1-phenyl-3-methyl-5-pyrazolone (PMP) were purchased from Sigma–Aldrich (St. Louis, MO). A [13C6]-labeled Hex4-internal standard was synthesized as previously described [17]. All other reagents were of analytical grade and commercially available. Sep-Pak Vac C18 cartridges (100 mg) and YMC-Pack Pro C18 column (250 £ 4.6 mm ID, 5 m) were purchased from Waters (Franklin, MA). Liquid chromatography–mass spectrometric analysis was performed using a Quattro-LC tandem mass spectrometer with electrospray ionization (ESI-MS/MS) (Waters, Franklin, MA) with a HP1100 quaternary pump (Agilent Technologies, Pal Alto, CA) and a HTS PAL sample injector (Leap Technologies, Carborro, NC). HPLC-UV
analysis was performed on a Alliance 2690 with a 2487 UV detector (Waters, Franklin, MA). Subjects A total of 11 patients with infantile-onset Pompe disease who participated in clinical trials of rhGAA enzyme replacement therapy were enrolled in this study. This includes three patients (1A–1C) from a pilot study and eight patients (2A–2H) from a subsequent study [12,13]. All patients received rhGAA puriWed from genetically engineered CHO cells expressing GAA [6]. The initial dosage for the patients in the Wrst study was 5 mg/kg biweekly. For the two patients (1A and 1B) who showed clinical decline after the Wrst 4 months of treatment, there were periods of therapy that included rhGAA administered in a dose of 10 mg/kg 2–5 times per week. All patients involved in the second study were given a weekly dose of 10 mg/kg. The urine and plasma oligosaccharide samples were collected at baseline and at regular intervals (every 1 or 2 weeks for the Wrst 2 months and then every 1–3 months) during the Wrst year’s course of the ERT. Both studies were approved at all participating sites and parental written informed consent was obtained. Glc4 measurements Urinary Glc4 was measured by HPLC-UV and ESIMS/MS methods as previously described [24,26], whereas Hex4, the combined concentration of plasma Glc4 and maltotetraose (M4), was measured by HPLC-UV only. Measurement of the glycogen content and Hex4 level in Pompe disease quail The glycogen content in muscle tissue from Pompe disease and control quail was determined by treatment of tissue extracts with amyloglucosidase and measurement of glucose released as previously described [6]. The Hex4 level in the same tissue extracts was measured using the HPLC-UV method. Digestion of glycogen by -amylase Glycogen (20 mg/mL) (type III, from rabbit liver, Sigma G8876) in PBS and 400 U/mL -amylase (type XIII-A, from human saliva, Sigma, A1031) in 1 mM CaCl2 were incubated at 37 °C for up to 96 h. Reaction products were determined using HPLC-UV analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives [27]. Statistical analysis Comparisons of urinary Glc4 and plasma Hex4 levels between groups of patients and at diVerent time points were performed using a two-tailed homoscedastic
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Student’s t test analysis. SigniWcance was deWned as a p value < 0.05.
Results Oligosaccharides as breakdown products of glycogen
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Glycogen and Hex4 levels in muscle from an animal model for Pompe disease Glycogen and Hex4 concentrations were determined in muscle from an -glucosidase-deWcient quail, an animal model resembling human late-onset Pompe disease and compared to that of a control quail. The Hex4 level in Pompe quail muscle ranged from 0.01 to 0.02 g/mg, whereas it was undetectable in normal quail. The ratio of Hex4 to total glycogen content in Pompe quail muscle was 2%. This was comparable to the ratio of Glc4 produced from amylolytic degradation of glycogen relative to the initial glycogen concentration, which ranged from 0.5 to 3.6% over 1–96 h.
Incubation of glycogen with -amylase produced oligosaccharides (Fig. 1). The production of glucose oligosaccharides from 3 to 7 residues (G3 to G7) with unconWrmed linkage was analyzed using the PMP derivative HPLC-UV method [24]. This method allowed the separation of Glc4 from M4, which were identiWed by comparison of retention times with those of standards. The separation of other oligosaccharide isomers by this method was not evaluated. All oligosaccharides except G3, tentatively identiWed as maltotriose, M3, showed a rapid rise in the Wrst 6 h of the incubation. Overtime, however, only Glc4, and M4 continued to rise, suggesting that, under these conditions, both compounds were stable products of amylolytic glycogen degradation (limit dextrins) and/or that the rate of production was greater than the rate of degradation. On the contrary, neither Glc4 nor M4 was detectable in digests of glycogen by GAA, the main product being glucose (data are not shown). Levels of glucose hexasaccharide (G6) and glucose heptasaccharide (G7), decreased with time suggesting that these oligosaccharides undergo further degradation. Glucose pentasaccharide (G5) initially increased and then remained fairly constant over the incubation period, suggesting production and digestion were balanced. The instability of M3 in the reaction mixture was demonstrated by its rapid disappearance (Fig. 1).
The direct isotope dilution ESI-MS/MS method used in this study is speciWc for hexose tetrasaccharides but cannot distinguish between Glc4 and M4. Similarly the BAB-derivative HPLC-UV method does not separate them. Hence measurements of Glc4 by these methods actually represent the combined glucose tetrasaccharide (Hex4) concentration in the samples. Using a hydrophilic interaction LC-MS/MS, which enabled the separation of diVerent hexose tetrasaccharide isomers, we have recently demonstrated that in urine, Glc4 is by far the major component of the total glucose tetrasaccharide fraction (>92%), whereas plasma contains variable amounts of Glc4 and M4 (manuscript in preparation). In the remainder of this manuscript, we therefore represent our urine assay as an assay for Glc4 and the plasma assay as an assay for Hex4. Fig. 2 shows a comparison of
Fig. 1. Time course of oligosaccharide production from glycogen incubated with -amylase. Key: pink square for Glc4, diamond for maltotetraose (M4), triangle for glucose pentasaccharide (G5), blue square for glucose hexasaccharide (G6), star for glucose heptasaccharide (G7), and circle for maltotriose (M3).
Fig. 2. Comparison of glucose tetrasaccharide levels in urine from a Pompe disease patient during 3 years of rhGAA enzyme replacement therapy measured by ESI-MS/MS and HPLC-UV methods. Squares represent HPLC-UV values and stars represent the ESI-MS/MS values.
Measurement of urinary Glc4 and plasma Hex4 from patients with Pompe disease
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urinary Glc4 levels determined by ESI-MS/MS and HPLC-UV for patient 1A during 3 years of ERT. The correlation between these methods is good (r2 > 0.8). Our results showed that absolute urinary Glc4 levels are 1–3 orders of magnitude greater than the plasma Hex4 levels. Because of its accuracy and speed, we applied the ESIMS/MS method to measure Glc4 levels in the urine from all patients receiving ERT. The HPLC-UV method was used to measure combined Hex4 levels in plasma, primarily because the rapid ESI-MS/MS method was not validated for plasma. Glc4 as a biomarker for monitoring therapeutic response The patients’ baseline urinary Glc4 (mean § SD, 34.2 § 11.3 mmol/mol creatinine) and plasma Hex4 levels (1.7 § 0.8 M) were higher than the mean of agematched controls (6.1 § 5.1 mmol/mol creatinine (n D 56), and 0.22 § 0.15 M (n D 19), respectively). The urinary Glc4 and plasma Hex4 levels from these patients during the Wrst year of ERT are shown in Fig. 3 and Table 1. All patients, except 2H, had elevated urinary Glc4 and plasma Hex4 levels at baseline (Table 1). The urinary Glc4 and plasma Hex4 levels for most patients decreased after initiation of the therapy, as early as 2 weeks after the Wrst infusion (Table 1) and may reXect an initial decrease in glycogen storage in various tissues (see below). It is noteworthy that the urinary Glc4 and plasma Hex4 levels in the patients with the best-maintained overall clinical response at 1 year of treatment (1C, 2A, 2E, and 2F) remained near or within the control range during this Wrst year of treatment (Fig. 3). Their clinical improvements included most signiWcantly, amelioration of the cardiomyopathy (as measured by left ventricular mass index (LVMI) by echocardiogram), improvement in growth parameters, and achievement of independent ambulation [10,12,13]. The decrease in the mean urinary Glc4 level compared with baseline at 2–7 and at 52 weeks was signiWcant for this group. The decrease in the mean plasma Hex4 level was signiWcant at 52 weeks (p < 0.05) but not at 2–7 weeks (p D 0.08). The mean urinary Glc4 level, but not plasma Hex4, in another group of patients (1A, 1B, 2B, 2C, 2D, and 2G) also showed a signiWcant initial decrease at 2–7 weeks of treatment. However, the overall trend for these patients was a persistent and Xuctuating elevation during the 1 year course of the therapy, with a signiWcant increase in the mean urinary Glc4 level at 52 weeks compared with baseline values (Fig. 3). Although these patients also showed improvement of the cardiomyopathy and growth gains, they had a less favorable motor response, ranging from a need for non-invasive respiratory support to development of respiratory insuYciency, frequently secondary to concurrent pneumonia. Comparing the group of patients that showed improvement in motor function and those that did not, there was no
Fig. 3. Urinary and plasma glucose tetrasaccharide levels in 11 Pompe disease patients during the Wrst year of the enzyme therapy. x-axis: days on treatment. Right y-axis: (¤) hexose tetrasaccharide (Hex4) levels in plasma (mol/L) measured by HPLC-UV. Left x-axis (䊏) glucose tetrasaccharide (Glc4) levels in urine (mmol/mol creatinine) measured by ESI-MS/MS. The age-matched cutoV values are shown in plain (for urine) and dotted (for plasma) lines. The urinary cutoV values are 16.4 and 5.0 mmol/mol creatinine for age groups of <1 and 1–5 years, respectively. The plasma cutoV values are 0.5 and 0.46 mol/L for age groups of <1 and 1–5 years, respectively.
signiWcant diVerence in the mean urinary Glc4 or plasma Hex4 at baseline. However, at 2–7 and 52 weeks, the mean urinary Glc4 level was signiWcantly lower in the patients with improved motor function. The mean
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Table 1 Summary of urinary Glc4 and plasma Hex4 levels in 11 GSD-II patients on ERT Patient ID
Age at start of therapy (months)
Urine Glc4 (mmol/mol Cn) Baseline
2–7 Weeks ERT
Plasma Hex4 (mol/L) 1 Year ERT
Baseline
2–7 Weeks ERT
Patients with improved cardiac (decreased LVM) and motor (independent ambulation) response 1C 0.4 20 20 17 1.07 0.83 20 6 1.91a 0.92 2A 4.7 38a 2E 3.1 47 18 8 1.13 0.48 2F 2.9 37 19 15 2.65 1.15 Mean (SD)
2.8 (1.8)
36 (11)
19 (1.0)
11.5 (5.3)
Patients with improved cardiac (decreased LVM), but limited motor response 1A 4.2 37 30 93 1B 1.7 23 30 42 2B 4.8 41 25 79b 2C 8.1 36 24 51 2D 8.5 42 19 95 2G 14.4 46 33 46c 2H 3.7 12 6.2 33d Mean (SD)
6.5 (4.2)
34 (12)
24 (9.1)
63 (26)
1 Year ERT
Urine Glc4 vs Plasma Hex4 (r2)
0.21 0.84 0.69 0.29
0.26 0.84 0.62 0.92
1.7 (0.7)
0.8 (0.3)
0.5 (0.3)
0.66 (0.29)
3.20 ND 1.22 1.86 0.84 2.10 0.39
2.19 1.71 1.26 1.09 0.54 0.95 0.37
1.38 0.50 ND 1.02 1.26 0.90c 0.96d
0.26 0.08 0.07 0.01 0.44 0.06 0.70
1.6 (1.0)
1.2 (0.6)
1.0 (0.3)
0.23 (0.26)
Control ranges, mean § SD (range). Urine Glc4 (mmol/mol Cn): <1 year, 6.1 § 5.1 (1.0–21.8) n D 56; 1–5 years, 1.9 § 1.5 (0.05–6.5) n D 25. Plasma Hex4 (mol/L): <1 year, 0.22 § 0.15, n D 19. LVM, left ventricular mass index; ERT, enzyme replacement therapy; ND, not determined. a Taken at 6 days on ERT. b After 10 months on ERT. c After 3 months on ERT. d After 11 months on ERT.
plasma Hex4 level was also lower at 52 weeks, but not at the level of signiWcance (p D 0.06). For patients 1A and 1B, there appeared to be an association between rising urinary Glc4 and plasma Hex4 levels and the development of respiratory insuYciency. Patient 2B died approximately 10 months into therapy following progressive respiratory insuYciency. The Glc4 level in the last urine specimen (79 mmol/mol Cn) collected from this patient was approximately 2.5 times greater than the average Glc4 value observed during therapy (mean § SD: 30 § 7.7 mmol/mol Cn, n D 9 time points). Patients 2C, 2D, and 2G were older and already in an advanced stage of the disease when the therapy was started. One of these patients (patient 2G) died approximately 3 months into therapy. All three patients had persistently high levels of urinary Glc4 and plasma Hex4 while on ERT. Another patient (patient 2H) had an initial good clinical response (cardiac and motor improvement) but developed respiratory insuYciency after approximately 8 months of ERT, following an episode of pneumonia. The urinary Glc4 and plasma Hex4 levels remained at or below the upper control limit for the Wrst 8 months of treatment, but were found to be increased at about 11 months, correlating with the clinical events. Fig. 2 shows the urinary Glc4 levels for patient 1A over a period of 3 years of treatment. There was an initial slight decrease or stabilization over the Wrst 3–4 months. This may be explained by an initial decrease in glycogen storage in some tissues during this time. Evi-
dence for this includes a decrease in the left ventricular mass index and motor improvement during the initial phase of treatment [10]. It was also reported by Winkel et al. [28] that in Pompe patients receiving rhGAA, the vascular endothelium and peripheral nerves showed a decrease in glycogen content within the Wrst 12 weeks. This occurred regardless of whether further treatment resulted in signiWcant glycogen clearance from muscles. The increase in urinary Glc4 levels in patient 1A after about 4 months likely reXects increasing glycogen storage in skeletal muscle. At 4 months there was no change in muscle glycogen content in this patient compared with baseline [10]. The LVMI continued to decrease during this time, but the initial gains in motor function observed over the Wrst 3–4 months were subsequently lost [10]. The Wnal decrease in urinary Glc4 levels during the last year on treatment probably reXects the overall slow clinical decline of the patient that was also noted simultaneous with these measurements. This slowly progressive and worsening clinical course reXects the multiple manifestations of the advancing infantile-onset Pompe disease phenotype. A comparison of date-matched urinary Glc4 and plasma Hex4 levels showed a high correlation (r2 > 0.5, Table 1) for some patients, while for others the correlation was low. The mean correlation was higher for the group of patients with the best-maintained overall clinical response and lowest oligosaccharide levels (1C, 2A, 2E, and 2F).
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Discussion Our group and others have previously reported that Glc4 is a putative biomarker for the diagnosis of Pompe disease, being elevated in the urine from patients with infantile-onset Pompe disease [21–26]. Two earlier studies by Lundblad, Zopf, and co-workers provided evidence that Glc4 is formed through intravascular degradation of glycogen released into the circulation [29,30]. This is supported by the study described here on Pompe quail muscle that was found to have increased Hex4 levels compared with a control. It is therefore hypothesized that urinary Glc4 should correlate with the degree of glycogen accumulation in cardiac and skeletal muscle of patients with Pompe disease. It is thus a potential biomarker for monitoring rhGAA ERT eYcacy, which aims to reduce glycogen accumulation in patients’ tissues. A similar approach has been used for monitoring the eYcacy of enzyme replacement therapy in Fabry disease, using plasma globotriaosylceramide (GL-3) [31,32] and in the mucopolysaccharidoses, using urinary glycosaminoglycans [33–36] as biomarkers. To date, there are no blood or urine biomarkers that can be utilized for monitoring ERT with rhGAA. Blood CK was invariantly elevated in these patients. However, the Xuctuation of CK values limited its use in monitoring the therapeutic response (unpublished results). In our 1-year study of measuring urinary Glc4 in patients with Pompe disease receiving ERT with rhGAA, we found that changes in urinary Glc4 were associated with the clinical response. Four out of 11 patients had the best clinical outcomes after 1 year of treatment, including the achievement of independent ambulation. The urinary Glc4 levels of these patients was initially elevated at baseline and then decreased to within or slightly above the normal control range over the Wrst 2 months on treatment and remained low during the Wrst year on treatment. In those patients with a more limited motor response to treatment, the urinary Glc4 levels never normalized, but remained elevated. One patient who had an initial good clinical response, but developed respiratory insuYciency after 1 year, had normal levels of urinary Glc4 for most of the treatment period, with levels rising in conjunction with the decline in clinical response. Plasma Hex4 was also evaluated as a biomarker. In general, the same trends observed for urinary Glc4 were also seen for the plasma Hex4 levels. The two major hexose tetrasaccharide isomers in plasma, Glc4 and M4, are both glycogen amylolytic degradation products as shown in this study. Thus, plasma M4 in addition to plasma Glc4 may also reXect the degree of glycogen storage and turnover. Interestingly, although the same trends were observed for urinary Glc4 and plasma Hex4, date-matched urine and plasma values were not corre-
lated for all patients. The reason for this is unclear, but is probably related to the diVerence in hexose isomer composition in the two sample types. It is not necessary to determine hexose tetrasaccharide levels in both urine and plasma. Urine is the sample of choice as the analysis by tandem mass spectrometry is rapid (<3 min) and concentrations are 1–3 orders of magnitude higher than in plasma. Furthermore, we have previously demonstrated that urinary Hex4 can be determined from a dried urine spot [26]. Hence, urine can be collected at home, soaked onto Wlter paper, dried, and mailed directly to the laboratory. In conclusion, urinary Glc4 and plasma Hex 4 levels in the 11 patients receiving rhGAA ERT correlated well with their clinical response and course. Most patients demonstrated an initial decrease in the Wrst few weeks of treatment regardless of outcome. Therefore, it is important to continue monitoring levels beyond 3 months of treatment. The tetrasaccharide levels did not decrease to normal levels in those patients with a limited motor response and this was evident by 6 months of treatment. Hence, a decrease of Glc4 levels to within or close to the normal range between 3 and 6 months of treatment may be an important predictor of the clinical response. Hex4 is a valuable adjunct to clinical endpoints in that it would provide an indication of the degree of glycogen clearance from skeletal muscles. Assessment of the glycogen content of skeletal muscle is important for the understanding of the clinical response [10,28]. For this study, one biopsy from the quadriceps was taken at 0, 12, and 52 weeks [12]. This is an invasive procedure and only provides information in the response from that particular muscle. Hex4 levels, on the other hand, would provide a more general indication of glycogen storage and can be monitored at many time points. Our results suggest that urinary Glc4 and plasma Hex4 can be used as a noninvasive biomarker to monitor the therapeutic response in Pompe disease.
Acknowledgments This work was supported by a grants from Synpac of N.C. Inc., N.C. Biotechnology Center, RTP, NC, Kenan Institute, RTP, NC, and Genzyme Corporation, Cambridge, MA Genzyme Corp. and by M01-RR30, National Center for Research Sources, General Clinical Research Centers Program. P.S.K. is a recipient of 1 K23 RR16060-01 (National Center for Research Resources, General Clinical Research Centers Program, NIH). The authors thank Drs. Joel Charrow, George Tiller, John Philips, BradleySchaefer, R. Curtis Rogers, C.H. Tsai, Gail Herman, John Waterson, Marc Nicolino, and Thomas Voit for their participation in the clinical trials and patient care.
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[14] J.M. Van den Hout, J.H. Kamphoven, L.P. Winkel, W.F. Arts, J.B. De Klerk, M.C. Loonen, A.G. Vulto, A. Cromme-Dijkhuis, N. Weisglas-Kuperus, W. Hop, H. Van Hirtum, O.P. Van Diggelen, M. Boer, M.A. Kroos, P.A. Van Doorn, E. Van der Voort, B. Sibbles, E.J. Van Corven, J.P. BrakenhoV, J. Van Hove, J.A. Smeitink, G. de Jong, A.J. Reuser, A.T. Van der Ploeg, Long-term intravenous treatment of Pompe disease with recombinant human alphaglucosidase from milk, Pediatrics 113 (2004) e448–e457. [15] L.P. Winkel, J.M. Van den Hout, J.H. Kamphoven, J.A. Disseldorp, M. Remmerswaal, W.F. Arts, M.C. Loonen, A.G. Vulto, P.A. Van Doorn, G. De Jong, W. Hop, G.P. Smit, S.K. Shapira, M.A. Boer, O.P. van Diggelen, A.J. Reuser, A.T. Van der Ploeg, Enzyme replacement therapy in late-onset Pompe’s disease: a three-year follow-up, Ann. Neurol. 55 (2004) 495–502. [16] A. AmalWtano, A.J. McVie-Wylie, H. Hu, T.L. Dawson, N. Raben, P. Plotz, Y.T. Chen, Systemic correction of the muscle disorder glycogen storage disease-type II after hepatic targeting of a modiWed adenovirus vector encoding human acid-glucosidase, Proc. Natl. Acad. Sci. USA 96 (1999) 8861–8866. [17] E.Y. Ding, H. Hu, B.L. Hodges, F. Migone, D. Serra, F. Xu, Y.T. Chen, A. AmalWtano, EYcacy of gene therapy for a prototypical lysosomal storage disease (GSD-II) is critically dependent upon vector dose, transgene promoter, and the tissues targeted for vector transduction, Mol. Ther. 5 (2002) 436–446. [18] A.J. McVie-Wylie, E.Y. Ding, T. Lawson, D. Serra, F.K. Migone, D. Pressley, M. Mizutani, T. Kikuchi, Y.T. Chen, A. AmalWtano, Multiple muscles in the AMD quail can be “cross-corrected” of pathologic glycogen accumulation after intravenous injection of an [E1-,polymerase-] adenovirus vector encoding human acidalpha-glucosidase, J. Gene Med. 5 (2003) 399–406. [19] F. Xu, E. Ding, S. Liao, F. Migone, A. Schneider, D. Serra, Y. Chen, A. AmalWtano, Improved eYcacy of gene therapy approaches for Pompe disease using a new, immune deWcient GSD-II mouse model, Gene Ther. 11 (2004) 1590–1598. [20] B. Sun, H. Zhang, L.M. Franco, S.P. Young, A. Schneider, A. Bird, A. AmalWtano, Y.-T. Chen, D. Koeberl, EYcacy of an adeno-associated virus 8 pseudotyped (AAV2/8) vector in glycogen storage disease type II (GSD-II), Mol. Ther. 11 (2005) 57–65. [21] P. Hallgren, G. Hansson, K.G. Henriksson, A. Häger, A. Lundblad, S. Svensson, Increased excretion of a glucose-containing tetrasaccharide in the urine of a patient with glycogen storage disease type II (Pompe’s disease), Eur. J. Clin. Invest . 4 (1974) 429–433. [22] M.A. Chester, A. Häger, S. Sjöblad, C. Loonen, J.M. Tager, D. Zopf, Increased urinary excretion of a glycogen-derived tetrasaccharide in heterozygotes with glycogen storage diseases type II and III, Lancet 1 (1983) 994–995. [23] G.O.H. Peelen, J.G.N. de Jong, R.A. Wevers, HPLC analysis of oligosaccharides in urine from oligosaccharidosis patients, Clin. Chem. 40 (1994) 914–921. [24] Y. An, S.P. Young, S.L. Hillman, J.L.K. van Hove, Y.-T. Chen, D.S. Millington, Liquid chromatographic assay for a glucose tetrasaccharide, a putative biomarker for the diagnosis of Pompe disease, Anal. Biochem. 87 (2000) 136–143. [25] T. Rozaklis, S.L. Ramsay, P.D. WhitWeld, E. Ranieri, J.J. Hopwood, P.J. Meikle, Determination of oligosaccharides in Pompe disease by electrospray ionization tandem mass spectrometry, Clin. Chem. 48 (2002) 131–139. [26] S.Y. Young, R.D. Stevens, Y. An, Y.-T. Chen, E.S. Millington, Analysis of a glucose tetrasaccharide elevated in Pompe disease by stable isotope dilution-electrospray ionization tandem mass spectrometry, Anal. Biochem. 316 (2003) 175–180. [27] D. Fu, D. Zopf, Analysis of sialyllactoses in blood and urine by high-performance liquid chromatography, Anal. Biochem. 269 (1999) 113–123. [28] L.P.F. Winkel, J.H.J. Kamphoven, H.J.M.P. Van Den Hout, L.A. Severijnen, P.A. Van Doorn, A.J.J. Reuser, A.T. Vand Der Ploeg,
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[33] E.D. Kakkis, J. Muenzer, G.E. Tiller, L. Warber, J. Belmont, M. Passage, B. Izkowski, J. Phillips, R. Doroshow, I. Walot, R. Hoft, E.F. Neufeld, Enzyme-replacement therapy in mucopolysaccharidosis I, New Engl. J. Med. 344 (2001) 182–188. [34] J.E. Wraith, L.A. Clarke, M. Beck, E.H. Kolodny, G.M. Pastores, J. Muenzer, D.M. Rapoport, K.I. Berger, S.J. Swiedler, E.D. Kakkis, T. Braakman, E. Chadbourne, K. Walton-Bowen, G.F. Cox, Enzyme replacement therapy for mucopolysaccharidosis I: a randomized, double-blinded, placebo-controlled, multinational study of recombinant human alpha-L-iduronidase (laronidase), J. Pediatr. 144 (2004) 581–588. [35] A. Crawley, S.L. Ramsay, S. Byers, J. Hopwood, P.J. Meikle, Monitoring dose response of enzyme replacement therapy in feline mucopolysaccharidosis type VI by tandem mass spectrometry, Pediatr. Res. 55 (2004) 585–591. [36] P. Harmatz, C.B. Whitley, L. Waber, R. Pais, R. Steiner, B. Plecko, P. Kaplan, J. Simon, E. Butensky, J.J. Hopwood, Enzyme replacement therapy in mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), J. Pediatr. 144 (2004) 574–580.
Glucose tetrasaccharide as a biomarker for monitoring the therapeutic response to enzyme replacement therapy for Pompe disease Yan An a,1, Sarah P. Young a,¤,1, Priya S. Kishnani a, David S. Millington a, Andrea AmalWtano a, Deyanira Corzo b, Yuan-Tsong Chen a,c a
Division of Medical Genetics, Department of Pediatrics, Duke University Medical Center, Durham and RTP, North Carolina, USA b Genzyme Corporation, Cambridge, MA, USA c Institute of Biochemical Sciences, Academica Sinica, Taipei, Taiwan Received 21 December 2004; received in revised form 17 March 2005; accepted 23 March 2005 Available online 10 May 2005
Abstract A tetraglucose oligomer, Glc1-6Glc1-4Glc1-4Glc, designated Glc4, has been shown to be a putative biomarker for the diagnosis of Pompe disease. The purpose of this study was to assess whether Glc4 could be used to monitor the therapeutic response to recombinant human acid glucosidase (rhGAA) enzyme replacement therapy (ERT) in patients with Pompe disease. Urinary Glc4 levels in 11 patients receiving rhGAA therapy was determined by both HPLC-UV and stable isotope dilution ESI-MS/MS. Combined Glc4 and maltotetraose, Glc1-4Glc1-4Glc1-4Glc, (M4) concentrations, designated Hex4, in plasma from these patients were measured by HPLC-UV only. Baseline urinary Glc4 and plasma Hex4 in these patients (mean § SD: 34.2 § 11.3 mmol/mol creatinine and 1.7 § 0.8 M, respectively) were higher than age-matched control values (mean § SD, 6.1 § 5.1 mmol/mol creatinine and 0.22 § 0.15 M, respectively). Both urinary Glc4 and plasma Hex4 levels decreased after initiation of ERT for all patients. In the four patients with the best overall clinical response in both skeletal and cardiac muscle, levels decreased to within, or near, normal levels during the Wrst year of treatment. In contrast, levels Xuctuated and were persistently elevated above the control ranges in those patients with a less favorable clinical response (good cardiac response but limited motor improvement). These results suggest that urinary Glc4 and plasma Hex4 could serve as a valuable adjunct to clinical endpoints for monitoring the eYcacy of therapeutic interventions such as rhGAA ERT in Pompe disease. 2005 Elsevier Inc. All rights reserved. Keywords: Glycogen storage disease type II; GSD-II; Pompe disease; Lysosome storage disease; Glc4; Oligosaccharide; HPLC; LC-MS/MS; Enzyme replacement therapy; Acid -glucosidase
Introduction Pompe disease, also known as glycogen storage disease type II (GSD-II), is an inherited muscle disorder of glycogen metabolism caused by deWciency of lysosomal acid -glucosidase (GAA), a glycogen-degrading enzyme *
1
Corresponding author. Fax: +1 919 549 0709. E-mail address: [email protected] (S.P. Young). An Y. and Young S.P. made equal contributions to this manuscript.
1096-7192/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.03.010
[1]. In its most severe form, the disease is characterized by massive cardiomegaly, macroglossia, progressive muscle weakness, and marked hypotonia in early infancy. Most infantile patients are diagnosed between 3 and 6 months of age and patients with the most severe presentation die around the Wrst year of life. Although at present there is no proven eVective treatment for Pompe disease, therapies are currently being developed and evaluated. Recent eVorts have focused on producing human recombinant GAA (hrGAA) for
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enzyme replacement therapy (ERT), in a manner similar to that utilized for other lysosomal disorders [2–4]. rhGAA has been produced in Chinese hamster ovary (CHO) cell cultures [5,6], and in transgenic mouse and rabbit milk [7,8], and its therapeutic eVects have been demonstrated in animal models [8,9]. Results from human clinical trials utilizing two diVerent forms of the recombinant enzyme have shown that rhGAA is capable of improving both cardiac and skeletal muscle function in patients with infantile Pompe disease [10–15]. Multiple gene therapy studies utilizing hGAA gene transfer to foster in vivo expression of hGAA have also shown high levels of eYcacy in several animal models of Pompe disease [16–20]. A tetraglucose oligomer, Glc1-6Glc1-4Glc1-4Glc, designated Glc4, has been shown to be elevated in both urine [21–24] and plasma [24,25] of patients with Pompe disease. Recently, we developed HPLC-UV [24] and stable isotope dilution ESI-MS/MS [26] methods for the analysis of Glc4 in biological Xuids as the butyl-p-aminobenzoate (BAB) derivative. These assays have been utilized for the determination of Glc4 levels in both normal individuals and patients with Pompe disease. Patients with Pompe disease were shown to have elevated Glc4 levels in urine and plasma by the HPLC-UV method [24]. Further analysis of plasma samples by hydrophilic interaction LC-MS/MS demonstrated a combined elevation of Glc4 and maltotetraose (Glc1-4Glc14Glc1-4Glc) in Pompe disease patients (unpublished observations). The objective of this study was to apply both the HPLC-UV and ESI-MS/MS methods to determine the utility of Glc4 as a non-invasive biomarker to monitor the therapeutic response in patients with Pompe disease receiving ERT with CHO cell-derived rhGAA.
Subjects and methods Materials and instrumentation Glc1-6Glc1-4Glc1-4Glc ((Glc)4), maltotetraose (M4), maltopentaose (M5), maltohexaose (M6), maltoheptaose (M7), cellopentaose (C5), sodium cyanoborohydride (NaBH3CN), butyl-4-aminobenzoate (BAB) and 1-phenyl-3-methyl-5-pyrazolone (PMP) were purchased from Sigma–Aldrich (St. Louis, MO). A [13C6]-labeled Hex4-internal standard was synthesized as previously described [17]. All other reagents were of analytical grade and commercially available. Sep-Pak Vac C18 cartridges (100 mg) and YMC-Pack Pro C18 column (250 £ 4.6 mm ID, 5 m) were purchased from Waters (Franklin, MA). Liquid chromatography–mass spectrometric analysis was performed using a Quattro-LC tandem mass spectrometer with electrospray ionization (ESI-MS/MS) (Waters, Franklin, MA) with a HP1100 quaternary pump (Agilent Technologies, Pal Alto, CA) and a HTS PAL sample injector (Leap Technologies, Carborro, NC). HPLC-UV
analysis was performed on a Alliance 2690 with a 2487 UV detector (Waters, Franklin, MA). Subjects A total of 11 patients with infantile-onset Pompe disease who participated in clinical trials of rhGAA enzyme replacement therapy were enrolled in this study. This includes three patients (1A–1C) from a pilot study and eight patients (2A–2H) from a subsequent study [12,13]. All patients received rhGAA puriWed from genetically engineered CHO cells expressing GAA [6]. The initial dosage for the patients in the Wrst study was 5 mg/kg biweekly. For the two patients (1A and 1B) who showed clinical decline after the Wrst 4 months of treatment, there were periods of therapy that included rhGAA administered in a dose of 10 mg/kg 2–5 times per week. All patients involved in the second study were given a weekly dose of 10 mg/kg. The urine and plasma oligosaccharide samples were collected at baseline and at regular intervals (every 1 or 2 weeks for the Wrst 2 months and then every 1–3 months) during the Wrst year’s course of the ERT. Both studies were approved at all participating sites and parental written informed consent was obtained. Glc4 measurements Urinary Glc4 was measured by HPLC-UV and ESIMS/MS methods as previously described [24,26], whereas Hex4, the combined concentration of plasma Glc4 and maltotetraose (M4), was measured by HPLC-UV only. Measurement of the glycogen content and Hex4 level in Pompe disease quail The glycogen content in muscle tissue from Pompe disease and control quail was determined by treatment of tissue extracts with amyloglucosidase and measurement of glucose released as previously described [6]. The Hex4 level in the same tissue extracts was measured using the HPLC-UV method. Digestion of glycogen by -amylase Glycogen (20 mg/mL) (type III, from rabbit liver, Sigma G8876) in PBS and 400 U/mL -amylase (type XIII-A, from human saliva, Sigma, A1031) in 1 mM CaCl2 were incubated at 37 °C for up to 96 h. Reaction products were determined using HPLC-UV analysis of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives [27]. Statistical analysis Comparisons of urinary Glc4 and plasma Hex4 levels between groups of patients and at diVerent time points were performed using a two-tailed homoscedastic
Y. An et al. / Molecular Genetics and Metabolism 85 (2005) 247–254
Student’s t test analysis. SigniWcance was deWned as a p value < 0.05.
Results Oligosaccharides as breakdown products of glycogen
249
Glycogen and Hex4 levels in muscle from an animal model for Pompe disease Glycogen and Hex4 concentrations were determined in muscle from an -glucosidase-deWcient quail, an animal model resembling human late-onset Pompe disease and compared to that of a control quail. The Hex4 level in Pompe quail muscle ranged from 0.01 to 0.02 g/mg, whereas it was undetectable in normal quail. The ratio of Hex4 to total glycogen content in Pompe quail muscle was 2%. This was comparable to the ratio of Glc4 produced from amylolytic degradation of glycogen relative to the initial glycogen concentration, which ranged from 0.5 to 3.6% over 1–96 h.
Incubation of glycogen with -amylase produced oligosaccharides (Fig. 1). The production of glucose oligosaccharides from 3 to 7 residues (G3 to G7) with unconWrmed linkage was analyzed using the PMP derivative HPLC-UV method [24]. This method allowed the separation of Glc4 from M4, which were identiWed by comparison of retention times with those of standards. The separation of other oligosaccharide isomers by this method was not evaluated. All oligosaccharides except G3, tentatively identiWed as maltotriose, M3, showed a rapid rise in the Wrst 6 h of the incubation. Overtime, however, only Glc4, and M4 continued to rise, suggesting that, under these conditions, both compounds were stable products of amylolytic glycogen degradation (limit dextrins) and/or that the rate of production was greater than the rate of degradation. On the contrary, neither Glc4 nor M4 was detectable in digests of glycogen by GAA, the main product being glucose (data are not shown). Levels of glucose hexasaccharide (G6) and glucose heptasaccharide (G7), decreased with time suggesting that these oligosaccharides undergo further degradation. Glucose pentasaccharide (G5) initially increased and then remained fairly constant over the incubation period, suggesting production and digestion were balanced. The instability of M3 in the reaction mixture was demonstrated by its rapid disappearance (Fig. 1).
The direct isotope dilution ESI-MS/MS method used in this study is speciWc for hexose tetrasaccharides but cannot distinguish between Glc4 and M4. Similarly the BAB-derivative HPLC-UV method does not separate them. Hence measurements of Glc4 by these methods actually represent the combined glucose tetrasaccharide (Hex4) concentration in the samples. Using a hydrophilic interaction LC-MS/MS, which enabled the separation of diVerent hexose tetrasaccharide isomers, we have recently demonstrated that in urine, Glc4 is by far the major component of the total glucose tetrasaccharide fraction (>92%), whereas plasma contains variable amounts of Glc4 and M4 (manuscript in preparation). In the remainder of this manuscript, we therefore represent our urine assay as an assay for Glc4 and the plasma assay as an assay for Hex4. Fig. 2 shows a comparison of
Fig. 1. Time course of oligosaccharide production from glycogen incubated with -amylase. Key: pink square for Glc4, diamond for maltotetraose (M4), triangle for glucose pentasaccharide (G5), blue square for glucose hexasaccharide (G6), star for glucose heptasaccharide (G7), and circle for maltotriose (M3).
Fig. 2. Comparison of glucose tetrasaccharide levels in urine from a Pompe disease patient during 3 years of rhGAA enzyme replacement therapy measured by ESI-MS/MS and HPLC-UV methods. Squares represent HPLC-UV values and stars represent the ESI-MS/MS values.
Measurement of urinary Glc4 and plasma Hex4 from patients with Pompe disease
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urinary Glc4 levels determined by ESI-MS/MS and HPLC-UV for patient 1A during 3 years of ERT. The correlation between these methods is good (r2 > 0.8). Our results showed that absolute urinary Glc4 levels are 1–3 orders of magnitude greater than the plasma Hex4 levels. Because of its accuracy and speed, we applied the ESIMS/MS method to measure Glc4 levels in the urine from all patients receiving ERT. The HPLC-UV method was used to measure combined Hex4 levels in plasma, primarily because the rapid ESI-MS/MS method was not validated for plasma. Glc4 as a biomarker for monitoring therapeutic response The patients’ baseline urinary Glc4 (mean § SD, 34.2 § 11.3 mmol/mol creatinine) and plasma Hex4 levels (1.7 § 0.8 M) were higher than the mean of agematched controls (6.1 § 5.1 mmol/mol creatinine (n D 56), and 0.22 § 0.15 M (n D 19), respectively). The urinary Glc4 and plasma Hex4 levels from these patients during the Wrst year of ERT are shown in Fig. 3 and Table 1. All patients, except 2H, had elevated urinary Glc4 and plasma Hex4 levels at baseline (Table 1). The urinary Glc4 and plasma Hex4 levels for most patients decreased after initiation of the therapy, as early as 2 weeks after the Wrst infusion (Table 1) and may reXect an initial decrease in glycogen storage in various tissues (see below). It is noteworthy that the urinary Glc4 and plasma Hex4 levels in the patients with the best-maintained overall clinical response at 1 year of treatment (1C, 2A, 2E, and 2F) remained near or within the control range during this Wrst year of treatment (Fig. 3). Their clinical improvements included most signiWcantly, amelioration of the cardiomyopathy (as measured by left ventricular mass index (LVMI) by echocardiogram), improvement in growth parameters, and achievement of independent ambulation [10,12,13]. The decrease in the mean urinary Glc4 level compared with baseline at 2–7 and at 52 weeks was signiWcant for this group. The decrease in the mean plasma Hex4 level was signiWcant at 52 weeks (p < 0.05) but not at 2–7 weeks (p D 0.08). The mean urinary Glc4 level, but not plasma Hex4, in another group of patients (1A, 1B, 2B, 2C, 2D, and 2G) also showed a signiWcant initial decrease at 2–7 weeks of treatment. However, the overall trend for these patients was a persistent and Xuctuating elevation during the 1 year course of the therapy, with a signiWcant increase in the mean urinary Glc4 level at 52 weeks compared with baseline values (Fig. 3). Although these patients also showed improvement of the cardiomyopathy and growth gains, they had a less favorable motor response, ranging from a need for non-invasive respiratory support to development of respiratory insuYciency, frequently secondary to concurrent pneumonia. Comparing the group of patients that showed improvement in motor function and those that did not, there was no
Fig. 3. Urinary and plasma glucose tetrasaccharide levels in 11 Pompe disease patients during the Wrst year of the enzyme therapy. x-axis: days on treatment. Right y-axis: (¤) hexose tetrasaccharide (Hex4) levels in plasma (mol/L) measured by HPLC-UV. Left x-axis (䊏) glucose tetrasaccharide (Glc4) levels in urine (mmol/mol creatinine) measured by ESI-MS/MS. The age-matched cutoV values are shown in plain (for urine) and dotted (for plasma) lines. The urinary cutoV values are 16.4 and 5.0 mmol/mol creatinine for age groups of <1 and 1–5 years, respectively. The plasma cutoV values are 0.5 and 0.46 mol/L for age groups of <1 and 1–5 years, respectively.
signiWcant diVerence in the mean urinary Glc4 or plasma Hex4 at baseline. However, at 2–7 and 52 weeks, the mean urinary Glc4 level was signiWcantly lower in the patients with improved motor function. The mean
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Table 1 Summary of urinary Glc4 and plasma Hex4 levels in 11 GSD-II patients on ERT Patient ID
Age at start of therapy (months)
Urine Glc4 (mmol/mol Cn) Baseline
2–7 Weeks ERT
Plasma Hex4 (mol/L) 1 Year ERT
Baseline
2–7 Weeks ERT
Patients with improved cardiac (decreased LVM) and motor (independent ambulation) response 1C 0.4 20 20 17 1.07 0.83 20 6 1.91a 0.92 2A 4.7 38a 2E 3.1 47 18 8 1.13 0.48 2F 2.9 37 19 15 2.65 1.15 Mean (SD)
2.8 (1.8)
36 (11)
19 (1.0)
11.5 (5.3)
Patients with improved cardiac (decreased LVM), but limited motor response 1A 4.2 37 30 93 1B 1.7 23 30 42 2B 4.8 41 25 79b 2C 8.1 36 24 51 2D 8.5 42 19 95 2G 14.4 46 33 46c 2H 3.7 12 6.2 33d Mean (SD)
6.5 (4.2)
34 (12)
24 (9.1)
63 (26)
1 Year ERT
Urine Glc4 vs Plasma Hex4 (r2)
0.21 0.84 0.69 0.29
0.26 0.84 0.62 0.92
1.7 (0.7)
0.8 (0.3)
0.5 (0.3)
0.66 (0.29)
3.20 ND 1.22 1.86 0.84 2.10 0.39
2.19 1.71 1.26 1.09 0.54 0.95 0.37
1.38 0.50 ND 1.02 1.26 0.90c 0.96d
0.26 0.08 0.07 0.01 0.44 0.06 0.70
1.6 (1.0)
1.2 (0.6)
1.0 (0.3)
0.23 (0.26)
Control ranges, mean § SD (range). Urine Glc4 (mmol/mol Cn): <1 year, 6.1 § 5.1 (1.0–21.8) n D 56; 1–5 years, 1.9 § 1.5 (0.05–6.5) n D 25. Plasma Hex4 (mol/L): <1 year, 0.22 § 0.15, n D 19. LVM, left ventricular mass index; ERT, enzyme replacement therapy; ND, not determined. a Taken at 6 days on ERT. b After 10 months on ERT. c After 3 months on ERT. d After 11 months on ERT.
plasma Hex4 level was also lower at 52 weeks, but not at the level of signiWcance (p D 0.06). For patients 1A and 1B, there appeared to be an association between rising urinary Glc4 and plasma Hex4 levels and the development of respiratory insuYciency. Patient 2B died approximately 10 months into therapy following progressive respiratory insuYciency. The Glc4 level in the last urine specimen (79 mmol/mol Cn) collected from this patient was approximately 2.5 times greater than the average Glc4 value observed during therapy (mean § SD: 30 § 7.7 mmol/mol Cn, n D 9 time points). Patients 2C, 2D, and 2G were older and already in an advanced stage of the disease when the therapy was started. One of these patients (patient 2G) died approximately 3 months into therapy. All three patients had persistently high levels of urinary Glc4 and plasma Hex4 while on ERT. Another patient (patient 2H) had an initial good clinical response (cardiac and motor improvement) but developed respiratory insuYciency after approximately 8 months of ERT, following an episode of pneumonia. The urinary Glc4 and plasma Hex4 levels remained at or below the upper control limit for the Wrst 8 months of treatment, but were found to be increased at about 11 months, correlating with the clinical events. Fig. 2 shows the urinary Glc4 levels for patient 1A over a period of 3 years of treatment. There was an initial slight decrease or stabilization over the Wrst 3–4 months. This may be explained by an initial decrease in glycogen storage in some tissues during this time. Evi-
dence for this includes a decrease in the left ventricular mass index and motor improvement during the initial phase of treatment [10]. It was also reported by Winkel et al. [28] that in Pompe patients receiving rhGAA, the vascular endothelium and peripheral nerves showed a decrease in glycogen content within the Wrst 12 weeks. This occurred regardless of whether further treatment resulted in signiWcant glycogen clearance from muscles. The increase in urinary Glc4 levels in patient 1A after about 4 months likely reXects increasing glycogen storage in skeletal muscle. At 4 months there was no change in muscle glycogen content in this patient compared with baseline [10]. The LVMI continued to decrease during this time, but the initial gains in motor function observed over the Wrst 3–4 months were subsequently lost [10]. The Wnal decrease in urinary Glc4 levels during the last year on treatment probably reXects the overall slow clinical decline of the patient that was also noted simultaneous with these measurements. This slowly progressive and worsening clinical course reXects the multiple manifestations of the advancing infantile-onset Pompe disease phenotype. A comparison of date-matched urinary Glc4 and plasma Hex4 levels showed a high correlation (r2 > 0.5, Table 1) for some patients, while for others the correlation was low. The mean correlation was higher for the group of patients with the best-maintained overall clinical response and lowest oligosaccharide levels (1C, 2A, 2E, and 2F).
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Discussion Our group and others have previously reported that Glc4 is a putative biomarker for the diagnosis of Pompe disease, being elevated in the urine from patients with infantile-onset Pompe disease [21–26]. Two earlier studies by Lundblad, Zopf, and co-workers provided evidence that Glc4 is formed through intravascular degradation of glycogen released into the circulation [29,30]. This is supported by the study described here on Pompe quail muscle that was found to have increased Hex4 levels compared with a control. It is therefore hypothesized that urinary Glc4 should correlate with the degree of glycogen accumulation in cardiac and skeletal muscle of patients with Pompe disease. It is thus a potential biomarker for monitoring rhGAA ERT eYcacy, which aims to reduce glycogen accumulation in patients’ tissues. A similar approach has been used for monitoring the eYcacy of enzyme replacement therapy in Fabry disease, using plasma globotriaosylceramide (GL-3) [31,32] and in the mucopolysaccharidoses, using urinary glycosaminoglycans [33–36] as biomarkers. To date, there are no blood or urine biomarkers that can be utilized for monitoring ERT with rhGAA. Blood CK was invariantly elevated in these patients. However, the Xuctuation of CK values limited its use in monitoring the therapeutic response (unpublished results). In our 1-year study of measuring urinary Glc4 in patients with Pompe disease receiving ERT with rhGAA, we found that changes in urinary Glc4 were associated with the clinical response. Four out of 11 patients had the best clinical outcomes after 1 year of treatment, including the achievement of independent ambulation. The urinary Glc4 levels of these patients was initially elevated at baseline and then decreased to within or slightly above the normal control range over the Wrst 2 months on treatment and remained low during the Wrst year on treatment. In those patients with a more limited motor response to treatment, the urinary Glc4 levels never normalized, but remained elevated. One patient who had an initial good clinical response, but developed respiratory insuYciency after 1 year, had normal levels of urinary Glc4 for most of the treatment period, with levels rising in conjunction with the decline in clinical response. Plasma Hex4 was also evaluated as a biomarker. In general, the same trends observed for urinary Glc4 were also seen for the plasma Hex4 levels. The two major hexose tetrasaccharide isomers in plasma, Glc4 and M4, are both glycogen amylolytic degradation products as shown in this study. Thus, plasma M4 in addition to plasma Glc4 may also reXect the degree of glycogen storage and turnover. Interestingly, although the same trends were observed for urinary Glc4 and plasma Hex4, date-matched urine and plasma values were not corre-
lated for all patients. The reason for this is unclear, but is probably related to the diVerence in hexose isomer composition in the two sample types. It is not necessary to determine hexose tetrasaccharide levels in both urine and plasma. Urine is the sample of choice as the analysis by tandem mass spectrometry is rapid (<3 min) and concentrations are 1–3 orders of magnitude higher than in plasma. Furthermore, we have previously demonstrated that urinary Hex4 can be determined from a dried urine spot [26]. Hence, urine can be collected at home, soaked onto Wlter paper, dried, and mailed directly to the laboratory. In conclusion, urinary Glc4 and plasma Hex 4 levels in the 11 patients receiving rhGAA ERT correlated well with their clinical response and course. Most patients demonstrated an initial decrease in the Wrst few weeks of treatment regardless of outcome. Therefore, it is important to continue monitoring levels beyond 3 months of treatment. The tetrasaccharide levels did not decrease to normal levels in those patients with a limited motor response and this was evident by 6 months of treatment. Hence, a decrease of Glc4 levels to within or close to the normal range between 3 and 6 months of treatment may be an important predictor of the clinical response. Hex4 is a valuable adjunct to clinical endpoints in that it would provide an indication of the degree of glycogen clearance from skeletal muscles. Assessment of the glycogen content of skeletal muscle is important for the understanding of the clinical response [10,28]. For this study, one biopsy from the quadriceps was taken at 0, 12, and 52 weeks [12]. This is an invasive procedure and only provides information in the response from that particular muscle. Hex4 levels, on the other hand, would provide a more general indication of glycogen storage and can be monitored at many time points. Our results suggest that urinary Glc4 and plasma Hex4 can be used as a noninvasive biomarker to monitor the therapeutic response in Pompe disease.
Acknowledgments This work was supported by a grants from Synpac of N.C. Inc., N.C. Biotechnology Center, RTP, NC, Kenan Institute, RTP, NC, and Genzyme Corporation, Cambridge, MA Genzyme Corp. and by M01-RR30, National Center for Research Sources, General Clinical Research Centers Program. P.S.K. is a recipient of 1 K23 RR16060-01 (National Center for Research Resources, General Clinical Research Centers Program, NIH). The authors thank Drs. Joel Charrow, George Tiller, John Philips, BradleySchaefer, R. Curtis Rogers, C.H. Tsai, Gail Herman, John Waterson, Marc Nicolino, and Thomas Voit for their participation in the clinical trials and patient care.
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