Bone marrow transplantation for recessive dystrophic epidermolysis bullosa

Abstract

Background: Recessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7 −/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans.

Methods: Between October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography.

Results: One patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal-epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies.

Conclusions: Increased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)

Figure 1. Clinical Photographs and Skin-Biopsy Specimens before and after Transplantation in Six Patients with Recessive Dystrophic Epidermolysis Bullosa

Photographs and skin-biopsy specimens were taken immediately before treatment and at scheduled time points after transplantation. The clinical photographs show specific areas that were consistently blistered before treatment. More rapid wound healing and reepithelialization were noted after transplantation in all six patients, with variable reductions in skin blistering. In Patient 7, marked cutaneous toxicity developed (presumably as a result of the chemotherapy) and was most pronounced on day 17 after transplantation but showed marked improvement by day 25. Immunofluorescence staining for C7 was carried out with skin-biopsy specimens obtained from all six patients, with the use of anti-C7 antibodies provided by Drs. D.T. Woodley and M. Chen (Keck School of Medicine, University of Southern California) for Patient 1 and purchased from BD Biosciences for the other five patients. For Patient 1, immunofluorescence staining before transplantation revealed faint, stippled labeling of C7; continuous, bright, linear C7 labeling was observed at all time points after bone marrow infusion. For Patient 3, faint, stippled C7 labeling was also observed at the dermal–epidermal junction before transplantation and through day 60 after transplantation, with continuous, bright, linear C7 labeling noted on day 145. For Patient 4, a bright, continuous band was observed at the dermal–epidermal junction before transplantation, with the use of anti-C7 antibody from BD Biosciences, whereas a thicker, brighter band was noted on day 198 after transplantation; this observation was confirmed with the use of LH7.2 anti-C7 antibody from Sigma-Aldrich, with no labeling before transplantation but bright labeling on day 198. For Patient 5, faint, stippled C7 labeling was observed at the dermal–epidermal junction with loss of demonstrable C7 labeling early after transplantation (day 28); however, continuous, bright linear, C7 labeling was observed on day 180. For Patient 6, no immunofluorescence staining was detectable before transplantation or at any time after transplantation, through day 102. For Patient 7, immunofluorescence staining before transplantation revealed stippled C7 labeling and continuous, bright, linear C7 labeling at the dermal–epidermal junction as early as day 30 after transplantation. For Patients 3 through 7, keratinocytes (green) were visualized with the use of anti–cytokeratin 5 antibody.

Figure 2. Mean (±SE) Quantification of C7 Fluorescence Intensity at Specific Time Points after Transplantation

Panel A shows the relative fluorescence intensity with the use of an anti-C7 antibody from BD Biosciences. Panel B shows the results with the use of additional anti-C7 antibodies, which in some cases failed to bind mutant C7 before transplantation but did bind C7 after transplantation. An anti-C7 antibody provided by Drs. D.T. Woodley and M. Chen (Keck School of Medicine, University of Southern California) was used for Patients 1, 3, 6, and 7), and an anti-C7 antibody (LH7.2) made by Sigma-Aldrich was used for Patients 4 and 5. (Each antibody was applied to tissue from Patients 3 through 7; the data presented are for samples showing the most pronounced changes in C7 expression.) Species-specific and isotype-specific control antibodies, which were used in assays performed on the same day as those carried out with anti-C7 antibodies, yielded negative results (data not shown). Note that 100% is the maximal measurable fluorescence intensity on the gray scale.

Figure 3. Transmission Electron Micrographs Showing Fibril Formation in Skin Specimens from Patients 4 and 7

Photomicrographs show thin fibrillar structures beneath the lamina densa before transplantation and on day 60 after transplantation in Patient 4 (Panel A, arrowheads) and before transplantation and on day 100 in Patient 7 (Panel B, arrowheads), with an increase in the number of fibrils after transplantation. Although some of these fibrils appear somewhat thicker in Patient 4 than in Patient 7 (Panel A, arrow), none bear the ultrastructural hallmarks of normal anchoring fibrils. The bars represent 250 nm.