Monitoring the 3-Year Efficacy of Enzyme Replacement Therapy in Fabry Disease by Repeated Skin Biopsies

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1 See related Commentary on page iv Monitoring the 3-Year Efficacy of Enzyme Replacement Therapy in Fabry Disease by Repeated Skin Biopsies Beth L. Thurberg, H. Randolph Byers,w 1 Scott R. Granter,z 1 Robert G. Phelps,y 1 Ronald E. Gordon,y and Michael O Callaghan Departments of Pathology and Preclinical Biology, Genzyme Corporation, Cambridge, Massachusetts, USA; wdepartments of Dermatology and Pathology, Boston University Medical Center, Boston, Massachusetts, USA; zdepartment of Pathology, Brigham and Women s Hospital and Harvard Medical School, Boston, Massachusetts, USA; ydepartment of Pathology, Mount Sinai School of Medicine, New York, New York, USA The earliest clinical signs of Fabry disease often manifest as dermatologic disturbances such as angiokeratomata, hypohidrosis, acroparesthesias, and impaired thermal and vibration detection. These disturbances are caused by cellular globotriaosylceramide accumulation in the skin due to deficient lysosomal a-galactosidase A activity. In this histologic study, we analyzed pre- and post-treatment dermatologic biopsies from 58 Fabry patients enrolled in a 5 mo, Phase 3 double-blind, randomized, placebo-controlled trial followed by a 30 mo open label extension study of recombinant human a-galactosidase A (r-hagala), administered i.v. at 1 mg per kg every 2 wk. Baseline evaluations revealed globotriaosylceramide in multiple dermal cell types (vascular endothelial cells, vascular smooth muscle cells, perineurium). Five months of r-hagala treatment in the Phase 3 trial resulted in complete clearance of globotriaosylceramide from the superficial capillary endothelium in all treatment patients and in only 1 (3%) placebo patient (po0.001). The placebo group achieved similar results after 6 mo of r-hagala in the open label trial. The capillary endothelium remained free of globotriaosylceramide for up to 30 mo into the extension study among 39 of 40 (98%) patients who underwent biopsies. Globotriaosylceramide clearance from deep vascular endothelial cells was similarly robust. Vascular smooth muscle cells and perineurium demonstrated moderate clearance. These findings suggest that long-term treatment with r-hagala may halt the progression of pathology and prevent the dermatologic disturbances in Fabry patients, and that periodic dermal biopsies can serve as a reliable monitor of sustained efficacy. Keywords: a-galactosidase A/angiokeratomas/globotriaosylceramide/lysosomal storage disease/phase 3 trial J Invest Dermatol 122: , 2004 Deficiency of the lysosomal enzyme a-galactosidase A is the pathological basis of Fabry Disease, an X-linked recessive disorder which primarily affects males, but may be symptomatic in female heterozygotes depending on the pattern of Lyonization (Gubler et al, 1978; Farge et al, 1985; Marguery et al, 1993). As a result of enzyme deficiency, neutral glycosphingolipids, mainly globotriaosylceramide (GL-3), progressively accumulate in tissues throughout the body. Males affected with the classical phenotype have absent or very low levels of a-galactosidase A activity, and manifest a number of well-recognized clinical features such as angiokeratomas, hypohidrosis, acroparesthesias, transient ischemic attacks and stroke, congestive heart failure, cardiac conduction abnormalities, myocardial infarction, and progressive renal failure (Desnick et al, 2001). Safety and efficacy of recombinant human a-galactosidase A (r-hagala, Fabrazyme) replacement therapy has been demonstrated in a Phase 1/2 trial (Eng et al, 2001a) and a Phase 3 trial with an open label extension (Eng et al, 2001b). Replacement of the missing enzyme with r-hagala Abbreviations: ERT, enzyme replacement therapy; GL-3, globotriaosylceramide; r-hagala, recombinant human alpha-galactosidase A 1 Contributed equally to this study. was highly effective in clearing GL-3 from the renal peritubular (interstitial) capillary endothelium (the primary endpoint of the Phase 3 trial) and significantly cleared substrate from other cell types of the kidney, based on blinded microscopic examination of biopsy samples (Thurberg et al, 2002). Enzyme replacement therapy (ERT) was equally effective in clearing GL-3 from the capillary endothelium of the heart, skin, and liver. Biochemical analyses also demonstrated effective clearance of GL-3 from the plasma, urinary sediment, and homogenized kidney tissue (Eng et al, 2001a, b). The vascular endothelial endpoint for these Fabry trials was chosen based on the prominent role that vascular pathology plays in Fabry disease. Renal failure due to vascular injury is the most common cause of death in Fabry patients (Sakuraba et al, 1987; Sterzel and Lovett, 1988; DeGraba et al, 2000; Desnick et al, 2001). Skin biopsies were also examined for vascular GL-3 deposition since angiokeratomas are a prominent feature of the disease. In addition, ischemic damage to small vessels has been implicated in the altered sensory threshold and painful crises that characterize the earliest clinical manifestations of Fabry disease (Hilz et al, 2000). Furthermore, GL-3 accumulation in vascular smooth muscle cells has been suggested as a cause for the Raynaud s-like vasospasm Copyright r 2004 by The Society for Investigative Dermatology, Inc. 900

2 122 : 4 APRIL 2004 induced by changes in temperature in Fabry patients (Seino et al, 1983; Hilz et al, 2000). Pain induced by such ischemic lesions, therefore, may be alleviated or prevented by clearance of GL-3 in the skin of Fabry patients. The range of vascular pathology and organ damage to the heart, kidney, liver and CNS in Fabry disease has prompted others to investigate whether impairment of the vascular endothelium, particularly reduction in the anticoagulant properties of the vascular lining, accelerates atherosclerosis and thromboembolism in these patients. Platelet hyper-reactivity capable of accelerating thromboembolic vascular change has been documented in Fabry patients as young as 9 yr of age, and detected in both hemizygous males as well as heterozygous females (Igarashi et al, 1986). Furthermore, organized thrombi have been identified in the dermal vasculature of Fabry patients (Palungwachira and Yaguchi, 2002). Markers of endothelial cell injury and activation (ICAM-1, VCAM-1, P-selectin), leukocyte activation (CD11b) and coagulation (tpa, vwf, PAI) are also elevated suggesting that the vascular endothelial cells of Fabry patients are in a chronic proinflammatory and pro-thrombotic state (Sakuraba et al, 1987; DeGraba et al, 2000). The Phase 3 clinical trial (Eng et al, 2001b) established the significant response of the primary vascular endothelial endpoints and demonstrated the safety of r-hagala treatment for Fabry disease. A detailed analysis of GL-3 clearance from the critical renal cell types has also recently been described (Thurberg et al, 2002). During the conduct of the Phase 3 trial and throughout the subsequent Extension study the histologic response of the various cell types was regularly monitored to demonstrate persistent efficacy of r-hagala treatment (1 mg per kg, biweekly). The main dermatopathologic characteristics of glycolipid accumulation in dermal tissue (vascular endothelium, smooth muscle, fibroblasts, perineurium, and eccrine sweat glands) from Fabry patients have been well described (Van Mullem and Reuter, 1970; van Mullem, 1972; Lao, 1998; Palungwachira and Yaguchi, 2002). Herein, based on the analysis of the most prevalent dermal cell types in 3 mm punch biopsies (vascular endothelium, vascular smooth muscle cells, and perineurium) we report the GL-3 clearance characteristics of each cell type in response to r-hagala at selected time points. The findings demonstrate the persistent benefits of long-term treatment with r-hagala in patients with Fabry disease. Results Baseline biopsies At baseline, GL-3 accumulation in the dermal biopsies appeared as small, dark beaded granules. In endothelial cells, accumulated GL-3 appeared as small, dense, beaded, perinuclear cytoplasmic inclusions. In severely affected cells, the accumulations appeared as larger grouped inclusions in the peripheral cytoplasm. These larger cytoplasmic inclusions often clustered and protruded into the lumens of small capillaries (Figs 1a and 2a). On electron microscopy, many of these granules were dense and laminated myelin figures (Fig 1c). Vascular smooth muscle MONITORING FABRY DISEASE WITH SKIN BIOPSIES 901 cells also accumulated moderate amounts of granular, cytoplasmic GL-3 irregularly distributed throughout the cytoplasm (Fig 3a). GL-3 in the perineurium appeared as dense granules clustered around the nuclei (Fig 4a). Post-treatment biopsies Vascular endothelium Treatment with r-hagala resulted in clearance of lipid from endothelial cells of all vessel types examined: superficial dermal capillaries and deep dermal vascular endothelium. In the patient group receiving r-hagala from baseline to 5 mo, 100% of patients achieved a score of zero after 5 mo of treatment (Fig 1b) for the superficial dermal capillary endothelium (baseline mean ¼ 2.1; 5 mo post-treatment mean ¼ 0.0; Fig 5a). After an additional 30 mo of treatment, 100% of the patients that remained in the study maintained a score of zero. In the placebo group, 0% of patients had a reduction to zero score from baseline to 5 mo, with one patient (3%) maintaining a zero score from baseline (baseline mean ¼ 2.3; 5-mo post-placebo mean ¼ 2.2, Fig 5a). There was a statistically significant difference between r-hagala and placebo-treated patients in the placebocontrolled (5 mo) study (po0.001; based on a w 2 test). After cross-over to treatment, the majority of patients achieved and maintained a score of zero for an additional 30 mo, with 95% of placebo patients achieving a score of zero (Fig 5a and Table I), demonstrating complete clearance. For deep dermal vascular endothelial cells (Fig 2a, b), patients receiving r-hagala from baseline to 5 mo demonstrated 100% reduction in GL-3, with 85% achieving or maintaining a zero score (baseline mean ¼ 2.4; 5 mo posttreatment mean ¼ 0.2; Fig 5b). After an additional 30 mo of treatment, 89% of patients remaining in the trial had achieved a score of zero (36 mo mean ¼ 0.2; Fig 5b). In the placebo group, only 8% of patients achieved or maintained zero scores from baseline to 5 mo (baseline mean ¼ 2.4; 5 mo post-placebo mean ¼ 2.2; Fig 5b). There was a statistically significant difference in the percentage of zero scores at 5 mo between the r-hagala and placebotreated patients in the placebo-controlled (5 mo) study (po0.001). After crossover to treatment, 85% of the former placebo patients achieved deep vessel endothelial scores of zero at month 36 (month 36 mean ¼ 0.2; Fig 5b), demonstrating complete clearance. Vascular smooth muscle cells ERT removed GL-3 from vascular smooth muscle cells (Fig 3a, b), although clearance was less complete than in endothelial cells. In addition, the sample size was small at all time points; due to the superficial nature of the biopsies, not all samples contained arterioles or arteries for evaluation. In the patient group treated with r-hagala from baseline to 5 mo, 33% of patients (1 of 3) with evaluable smooth muscle cells at both timepoints achieved a zero score (Table I). When all available scores were examined, the baseline mean score was 1.5 and the 5 mo post-treatment mean score was 1.2 (Fig 5c). After an additional 30 mo of treatment, 33% of patients with evaluable smooth muscle cells at both baseline and 36 mo showed a reduction in score (Table I). When all available scores were examined, however, the 36 mo mean was 1.0 (Fig 5c). In the placebo group, one

3 902 THURBERG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 1 GL-3 is cleared from superficial capillary endothelial cells after ERT. (a) Baseline biopsy, pre-treatment. GL-3 is present near endothelial cell nuclei and scattered throughout the cytoplasm. The larger granules protrude into the capillary lumen. (b) Post-treatment biopsy, month 36. Capillary endothelium is clear of GL-3. (magnification of a and b, 100 objective, scale bar ¼ 10 mm) (c) Baseline biopsy, pre-treatment, electron microscopy. GL-3 can be seen as dense bodies and myelin figures, causing occlusion of the vascular lumen (magnification 2600, scale bar ¼ 2.5 mm). (d) Posttreatment biopsy, month 36, electron microscopy. Clearance of GL-3 from the vascular endothelium relieves the occlusion observed in (c). Three red blood cells can be observed floating within the lumen (magnification 2600, scale bar ¼ 2.5 mm). patient with evaluable smooth muscle cells at both baseline and month 5 showed no reduction to zero in GL-3 score. When all available scores were examined, the baseline mean score was 1.5 (n ¼ 13), and the 5 mo post-placebo mean score was 1.2 (n ¼ 5; Fig 5c). After an additional 30 mo of treatment, 60% of the patients with evaluable smooth muscle cells at both baseline and 36 mo showed a reduction in score, with 20% (1 of 5) achieving a score of zero (Table I). When all available scores were examined, the 36 mo mean was 1.1 (n ¼ 9; Fig 5c). Perineurium GL-3 accumulation Perineurium responded to r-hagala enzyme therapy with a modest, but progressive reduction in GL-3 inclusions (Table I; Fig 4a, b). In the patient group treated with r-hagala from baseline to 5 mo, 46% demonstrated a reduction in GL-3, with 4% achieving or maintaining a zero (baseline mean ¼ 2; 5 mo post-treatment mean ¼ 1.7; Fig 5d). After an additional 30 mo of treatment with r-hagala, the patients who remained in the study continued to improve: 61% demonstrated a reduction in score and 22% achieved a score of zero from baseline (month 36 mean ¼ 1.2; Fig 5d). In the placebo group, 30% demonstrated a reduction in GL-3 with a single patient (4%) maintaining a score of zero from baseline (baseline mean ¼ 2.0; 5 month post-placebo mean ¼ 1.9; Fig 5d). After cross-over to r-hagala treatment for an additional 30 months however, 75% of the remaining former placebo patients demonstrated a reduction in GL-3 and 15% achieved or maintained a score of zero from baseline, (36 month mean ¼ 1.1; Fig 5d). Discussion In the current study, we have demonstrated sustained clearance of GL-3 from dermal cells during a 36 mo trial of ERT. This represents the longest trial period during which pathology was used to routinely monitor the efficacy of ERT. Furthermore, the sustained clearance observed from multiple cell types suggests that in Fabry disease the skin is highly responsive to ERT and therefore a good surrogate with which to monitor long-term efficacy. The renal biopsies performed during the trial at baseline, 5, and 12 mo provided valuable information about GL-3 clearance, demonstrating that renal capillaries remain clear of GL-3 after 12 mo of ERT (Thurberg et al, 2002). Skin biopsy is less invasive, however, and we were therefore able to obtain samples throughout the entire trial to monitor long-term response to ERT. The clearance of GL-3 from dermal capillaries paralleled that observed in renal capillaries up to 12 mo, and was maintained in subsequent skin biopsies taken over a 36 mo treatment period. The potential use of skin biopsies as a monitor of disease is a critical finding, since dermatologic lesions are often the first indicator of Fabry disease during childhood when it is otherwise clinically silent. Despite this clinical silence, a number of studies have shown that accumulation of GL-3 begins in utero and continues progressively during childhood and into adulthood (Tondeur and Resibois, 1969; Malouf et al, 1976; Breathnach et al, 1980; Tsutsumi et al, 1985). One case report demonstrated the accumulation of GL-3 in capillaries of clinically uninvolved skin from a 1-yr-old boy (Breathnach et al, 1980). The burning pain (acroparesthesias) which accompanies early Fabry disease has been described as early as age seven (Gordon et al, 1995) and is easily overlooked in a pediatric population. Yet this is likely the most opportune period in which to confirm the diagnosis, initiate treatment and prevent the serious pathologic changes, which later lead to the more serious manifestations of Fabry disease. In another illustrative case, a 6-yr-old patient had been referred to a hospital clinic for unexplained distal limb pain, but was never diagnosed with

4 122 : 4 APRIL 2004 MONITORING FABRY DISEASE WITH SKIN BIOPSIES 903 Figure 2 GL-3 is cleared from deep vessel endothelial cells after ERT. (a) Baseline biopsy, pre-treatment. Endothelial cells are heavy with granular GL-3 inclusions (b) Post-treatment biopsy, month 36. Endothelial cells are free of GL-3. (magnification of a and b, 100 objective, scale bar ¼ 10 mm) (c) Baseline biopsy, pre-treatment, electron microscopy. Black arrows indicate capillary endothelial cell GL-3; red arrows indicate pericyte GL-3. (magnification of c, 6000, scale bar ¼ 1.25 mm). (d) Post-treatment biopsy, month 36, electron microscopy. GL-3 has been removed from both endothelial cells (black) and pericytes (red) (magnification of d, 3300, scale bar ¼ 1.93 mm). Figure 3 GL-3 is cleared from endothelial cells (black arrows) and is reduced in vascular smooth muscle cells (red arrows) of small arteries after enzyme replacement therapy. (a) Baseline biopsy, pre-treatment. (b) Post-treatment biopsy, month 36. (magnification of a and b, 100 objective, scale bar ¼ 10 mm). (c) Baseline biopsy, pretreatment, electron microscopy (magnification 3000, scale bar ¼ 2.43 mm). (d) Post-treatment biopsy, month 36, electron microscopy (magnification 2000, scale bar ¼ 2.95 mm). Fabry disease until age 52 when he again presented with neurologic symptoms. At that latter time, characteristic angiokeratomas were noted and biopsied; the diagnosis of Fabry disease was thus made, and confirmed by subsequent enzyme assays (Mohanraj et al, 2002). Biochemical confirmation is critical, since angiokeratomas are also present in other enzyme deficiencies such as a-fucosidase, a-neuraminidase, C-mannosidase, and aspartylglycosaminidase deficiencies (Shelley et al, 1995; Schiller and Itin, 1996). The anhidrosis associated with Fabry disease is present as young as 6 yr of age and is often accompanied by the characteristic telangiectasias of Fabry disease. The dermatologic findings of Fabry disease, therefore, are important to recognize and help establish the early diagnosis and treatment of these patients. In one report, a decrease in

5 904 THURBERG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Figure 4 GL-3 is cleared from the perineurium after enzyme replacement therapy. (a) Baseline biopsy, pre-treatment. (b) Posttreatment biopsy, month 36 (magnification of a and b, 100 objective, scale bar ¼ 10 mm). (c) Baseline biopsy, pretreatment, electron microscopy (magnification 3500, scale bar ¼ 2.09 mm). (d) Post-treatment biopsy, month 36, electron microscopy. Very few GL-3 granules remain (magnification 2500, scale bar ¼ 2.79 mm). sweat gland function was detected in a pair of 6- and 23-yrold affected siblings only after the 23-yr-old brother was diagnosed with Fabry disease following repeated bouts of heat intolerance, collapse, and loss of consciousness (Shelley et al, 1995). The older brother went on to develop renal failure 6 yr later. Again, biopsy and analysis of the skin lesions was instrumental in making the correct diagnosis (Massi et al, 2000). Based on our current knowledge of the cell biology and time course of Fabry disease, it is reasonable to expect that clearance of GL-3 from each of the four cell types examined will afford cell-specific functional benefit. Clearance of GL-3 from superficial capillaries of the papillary dermis is important for the delivery of nutrients to the skin as well as the maintenance of their thermoregulatory role that they share with eccrine sweat glands (Urmacher, 1997). Weakness of the capillary wall due to GL-3 accumulation is believed to be responsible for vascular ectasia and the subsequent development of angiokeratomas present within the epidermis and dermis, lesions which are particularly susceptible to thrombosis (Schiller and Itin, 1996; Silverberg, 1997). In addition, Fabry patients have been shown to have activated platelet function, (Igarashi et al, 1986); this procoagulative state is supported by the observation of organized fibrin thrombi in the dilated vessels present in skin biopsies of Fabry patients (Palungwachira and Yaguchi, 2002), causing occlusion and endothelial cell injury (Nakamura et al, 1981). Clearance from deep vessel endothelium of the reticular dermis is important to prevent the stenosis of the vasa nervorum, which impairs blood supply to superficial tissues and nerves and leads to transient ischemia of small nerve fibers, causing intense pain on cold exposure (Hilz et al, 2000). An ultrastructural study of Fabry skin pathology noted that the small blood vessels around the eccrine sweat glands were narrowed by heavy endothelial cell inclusions and may be responsible for hypohidrosis (Palungwachira and Yaguchi, 2002), a symptom also present in female heterozygotes (Yamamoto et al, 1996). GL-3 deposits have also been observed in eccrine sweat gland epithelium and may also contribute to deficient sweat production (Yamamoto et al, 1996; Lao et al, 1998). Clearance from vascular smooth muscle cells is important for proper control of blood flow to superficial tissues. Deposition of GL-3 in vascular smooth muscle cells has been observed to be associated with muscle cell disarray (Nakamura et al, 1981), a feature which is likely to be associated with altered vasodilatory and vasoconstrictive function. In turn, impaired vasoconstriction of stenotic vessels may lead to ischemia-induced pain in patients exposed to extremes of temperature (Hilz et al, 2000). In a study which examined peripheral hemodynamics, Fabry patients were found to have decreased forearm venous capacitance and increased forearm vascular resistance, compared to controls. In addition, finger and toe pulse volumes, blood flows and temperature were significantly lower than control subjects. These data suggest that accumulation of glycolipid in vessel walls/vascular smooth muscle cells plays a role in the integumentory pathophysiology of Fabry disease. Therefore, clearance of GL-3 from vascular smooth muscle cells would be expected to alleviate some of these symptoms (Seino et al, 1983). Clearance of perineurial GL-3 is a critical element to the restoration of conduction velocity in large nerve fibers and sensory function of small nerve fibers (Dütsch et al, 2002). Recent studies comparing motor and sensory conduction velocities (MCV, SCV) with sensory skin responses (SSR) such as vibratory, cold, and heat-pain detection thresholds, have shown that small fiber dysfunction was more severe than large fiber dysfunction (Dutsch et al, 2002; Luciano

6 122 : 4 APRIL 2004 MONITORING FABRY DISEASE WITH SKIN BIOPSIES 905 Figure 5 The mean GL-3 scores with standard deviations were calculated for each cell type in both the treatment and placebo groups at baseline, 5, 12, 24, and 36 mo. Note the rapid decline in mean GL-3 scores for the placebo group once ERT was initiated after the 5 mo time point. Treatment groups maintained their low GL-3 scores from the pivotal study (baseline to 5 mo) into the extension segment of the study (evaluated at 12, 24, and 36 mo); (a) superficial dermal capillary endothelial cells; (b) deep vessel capillary endothelium; (c) vascular smooth muscle cells; (d) perineurium. a- gal ¼ patient treated with recombinant human enzyme, a-galactosidase A. et al, 2002). These findings suggest that small diameter nerves are more vulnerable in Fabry patients. Similar studies investigating small nerve fiber function after cold challenge, suggest that the GL-3 accumulation in small dermal vessels and small nerve axons cause skin and small fiber malperfusion during cold-induced vasoconstriction, leading to transitory ischemia and burning pain (Hilz et al, 2000). A study which compared the nerve fiber density of large fibers (via sural nerve biopsy) versus small fibers (via cutaneous biopsy) in Fabry and normal subjects also demonstrated a predominance of small fiber neuropathy (Scott et al, 1999). In addition, GL-3 accumulation in the dorsal root ganglia has been reported at autopsy and may also contribute to axonal degeneration and Fabry pain (Steward and Hitchcock, 1968; Machinami, 1972; Kahn, 1973; Gadoth and Sandbank, 1983). This histologic study confirmed the rapid and persistent efficacy of Fabrazyme by removing glycosphingolipid from the lysosomes of affected dermal tissues, and then maintaining the beneficial effects over at least 3 yr. This study also confirmed that not all cell types respond equally to ERT. In the skin, as in the kidney (Thurberg et al, 2002), the vascular endothelium was highly responsive to Fabrazyme therapy. Other cell types such as vascular smooth muscle and perineurium responded in a more gradual manner, but with significant and substantial substrate removal over the period of the study. The accumulation of substrate in many lysosomal storage diseases causes artifactual vacuolization in affected cells when tissues are processed by traditional, routine formalin fixation and paraffin embedding. While this characteristic artifact may be recognized in cells which accumulate large amounts of substrate (such as cardiomyocytes and podocytes in Fabry disease), it may be less apparent when the substrate is present in smaller quantities, as in the cells of the vasculature. This is particularly

7 906 THURBERG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Table I. Percentage of patients achieving zero scores in skin Score change from baseline to 5 mo Score change from baseline to month 36 Skin cells evaluated TX group Zero scores at baseline (%) Zero scores at month 5 (%) Zero score or reduction at 5 mo (%) Total N Zero scores at baseline (%) Zero scores at month 36 (%) a Zero score or reduction at 36 mo (%) Total N Superficial endothelial cells Placebo A-gal Deep endothelial cells Placebo A-gal Smooth muscle Placebo cells b A-gal Perineurium Placebo A-gal The clearance of GL-3 from multiple dermal cell types was determined before and after enzyme replacement therapy. There were two groups of patients: (1) the placebo group received placebo for the first 5 months and then a-galactosidase A for the remaining 30 months and (2) the A-gal group which received a-galactosidase A for the entire 36 mo. The percentage of patients who attained a zero score at each biopsy time point is displayed and the percentage of all patients who demonstrated a reduction in score, including all zeros, is presented in the third column. Values are presented for both the placebo and treatment groups. For statistical purposes, it was necessary for each patient to have both a baseline score and a shift score (5 or 36 mo) for each cell type, in order to be included in this data set. Two baseline columns are presented; the N designates the number of paired scores available for each cell type (paired scores meaning baseline and 5 mo, or baseline and 36 mo). This permitted calculation of the statistical significance of the change in score from either baseline to 5 mo, or from baseline to 36 mo, when comparing the placebo and treated groups. This distinction was necessary because: (1) some patients had a poorly preserved or missed biopsy at one of the time points, and (2) some biopsy specimens did not contain deep skin structures and therefore those cells could not be evaluated at all time points. a Placebo group has crossed over to treatment by this time point. b Represents a small evaluable sample size; few skin biopsies contained smooth muscle cells for evalulation.

8 122 : 4 APRIL 2004 Table II. Scoring system for GL-3 accumulation and clearance in different dermal cell types Cell/tissue Superficial dermal capillary endothelial cells Deep dermal vascular endothelial cells Vascular smooth muscle cells Perineurium true in the cells of small skin biopsies. In the present study, therefore, we chose to proceed with the more laborintensive glutaraldehyde fixation and epon embedding methodology, which preserves GL-3 substrate so that it is easily stained and visualized, quantifiable, and easily confirmed by electron microscopy when necessary. We recognize that it is difficult, if not impossible in a clinical setting, to anticipate which patients may have Fabry disease and utilize this methodology in order to make a primary diagnosis. It is hoped, however, that clinicians will find it useful on occasion to request such special processing in order to follow patients, who have already been diagnosed through family history and genetic testing, as they are treated over time. Materials and Methods Score 0 ¼ none or trace accumulation 1 ¼ mild accumulation 2 ¼ moderate accumulation 3 ¼ severe accumulation Patients and study design Fifty-eight Fabry patients (56 males, 2 females) were enrolled in the 5 mo Phase 3 double-blind, randomized, placebo-controlled trial (29 patients in placebo and treatment arms, mean age SD, and yr, respectively), followed by a 30 mo open label extension study, in which all 58 patients received ERT. Patients were required to be 16 yr of age or older, with native plasma a-galactosidase A levels less than 1.5 nmol per h per ml or leukocyte activity levels less than 4 nmol per hr per mg, and baseline creatinine levels less than, or equal to, 2.2 mg per dl. Patients who had undergone kidney transplantation or who were undergoing dialysis were excluded. Recombinant a-galactosidase A (r-hagala; agalsidase beta; Fabrazyme, Genzyme, Cambridge, Massachusetts) was administered intravenously at 1 mg per kg of body weight every 2 wk at a perfusion rate of 0.25 mg per min. Three-millimeter punch biopsies (taken from the lower back, avoiding angiokeratomas) were taken from all 58 Fabry patients at baseline. Additional biopsies were obtained from the majority of patients at 5, 12, 24, and 36 mo poststudy initiation. There was approximately a 1 mo break in treatment after month 5, followed by an additional 30 mo of continuous treatment. The institutional review boards at all sites approved the double-blind and open label protocols, and all patients gave written informed consent. Light microscopy Dermal biopsies were fixed in 3% glutaraldehyde in 0.2 M sodium cacodylate buffer, ph 7.3, followed by postfixation in 1% osmium tetroxide in 0.2 M sodium cacodylate (Electron Microscopy Sciences, Fort Washington, Pennsylvania). Tissue was infiltrated overnight, then embedded in a 1:1 mixture of Epon 811 A and B (Electron Microscopy Sciences), plus DMP-30/ propylene oxide (Electron Microscopy Sciences). One-micron sections were stained with a 1:1 mixture of methylene blue in 1% sodium borate and 1% Azure II (Fisher Scientific, Fairlawn, New Jersey). Scoring of light microscopy Light microscopic examination of the dermal biopsies was performed by three independent dermatopathologists blinded as to patient identity and pivotal MONITORING FABRY DISEASE WITH SKIN BIOPSIES 907 treatment status. To establish consistent criteria for grading baseline GL-3 content and subsequent clearance from affected tissues, all pathologists underwent a preliminary training session during which criteria for scoring were reviewed. Cell types evaluated included: superficial dermal capillary endothelial cells (one of the secondary endpoints of the Phase 3 trial), deep dermal capillary/arteriolar endothelial cells, vascular smooth muscle cells and perineurium (see Table II for scoring criteria). The majority score (the score common to at least 2 of the 3 evaluating pathologists) for each cell type was derived from the individual scores of the three pathologists. In the cases in which no common score could be determined for the secondary endpoint (superficial dermal endothelial cells), an adjudication meeting of all pathologists at a multi-headed scope was held to reach a consensus score. The median score was selected for other cell types. Figure 5 shows the mean values for all scores available at each time point, while Table I shows the distribution of score differences from baseline. Note that in some biopsies certain cell types were absent and could not be evaluated at all time points, accounting for some of the discrepancies in the n values in the table. Electron microscopy Thin sections cut from the same tissue blocks were stained with 5% uranyl acetate (in a 1:1 mixture, methanol:water) and modified Reynold s Lead Citrate. Photographs were taken at a range of 2000 to 6000 magnification using a JEM 100CX electron microscope (JEOL, Tokyo, Japan). Electron micrographs were not used to score GL-3 content, but were made available to confirm its presence or absence as observed in light microscopic sections. Many thanks to the Genzyme Pathology Department staff for their help in developing a histologic grading system, and to Biometrics for statistical data analysis. We also thank the clinical investigators and their patients at the multiple sites who participated in this trial: C.M. Eng, R.J. Desnick, M.Banikazemi, J. Ibraham, and A.P. Cheng (New York, USA); W.R. Wilcox and L.J. Raffel (Los Angeles, USA); N. Guffon and P. Cochat (Lyons, France); D.P. Germain, M. Azizi, and X. Jeunemaitre (Paris, France); P. Lee and A. Vellodi (London, UK); S. Waldek and J.E. Wraith (Manchester, UK); L. Caplan, C.J. Chaves, K.B. Kanis, I. Linfante, and R. Llinas, (Boston, USA); C.E.M. Hollak, G.E. Linthorst, D.K. Bosman, H.S.A. Heymans, and F.A. Wijburg (Amsterdam, The Netherlands). This work was supported in part by grants from the National Institutes of Health including a research grant (R29 DK Merit Award), a grant (5 MO1 RR00071) for the Mount Sinai General Clinical Research Center Program from the National Center of Research Resources, a grant (5 P30 HD28822) for the Mount Sinai Child Health Research Center, and a research grant from Genzyme Corporation. DOI: /j X x Manuscript received September 12, 2003; revised October 22, 2003; accepted for publication November 11, 2003 Address correspondence to: Beth L. Thurberg, Department of Pathology, Genzyme Corporation, One Mountain Road, Framingham, MA , USA. Beth.Thurberg@genzyme.com References Breathnach SM, Black MM, Wallace HJ: Anderson Fabry disease: Characteristic ultrastructural features in cutaneous blood vessels in a 1-year old boy. Br J Dermatol 103:81 84, 1980 DeGraba T, Azhar S, Dignat-George F, et al: Profile of endothelial and leukocyte activation in Fabry patients. Ann Neurol 47: , 2000 Desnick RJ, Ionnou YA, Eng CM: a-galactosidase A deficiency: Fabry disease. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds). The Metabolic and Molecular Basis of Inherited Disease, Vol. 3, 8th edn. New York: McGraw- Hill, 2001; p Dutsch M, Marthol H, Stemper B, et al: Small fiber dysfunction predominates in Fabry neuropathy. J Clin Neurophysiol 19: , 2002

9 908 THURBERG ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Eng CM, Banikazemi M, Gordon RE, et al: A Phase 1/2 clinical trial of enzyme replacement in Fabry disease: Pharmacokinetic, substrate clearance, and safety studies. Am J Hum Genet 68: , 2001a Eng CM, Guffon N, Wilcox WR, et al: Safety and efficacy of recombinant human a-galactosidase: A replacement therapy in Fabry s disease. N Engl J Med 345:9 16, 2001b Farge D, Nadler S, Wolfe LS, et al: Diagnostic value of kidney biopsy in heterozygous Fabry s disease. Arch Pathol Lab Med 109:85 88, 1985 Gadoth N, Sandbank U: Involvement of dorsal root ganglia in Fabry s disease. J Med Genetics 20: , 1983 Gordon KE, Ludman MD, Finley GA: Successful treatment of painful crises of Fabry disease with low dose morphine. Pediatr Neurol 12: , 1995 Gubler MC, Lenoir G, Grunfeld JP, et al: Early renal changes in hemizygous and heterozygous patients with Fabry s disease. 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