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Original Investigation |

Clinical and Histologic Analysis of the Efficacy of Topical Rapamycin Therapy Against Hypomelanotic Macules in Tuberous Sclerosis Complex FREE

Mari Wataya-Kaneda, MD, PhD1; Mari Tanaka, MD, PhD1; Lingli Yang, MD, PhD1; Fei Yang, BS1; Daisuke Tsuruta, MD, PhD2; Ayumi Nakamura, BPharm3; Shoji Matsumoto, PhD3; Toshimitsu Hamasaki, PhD4; Atushi Tanemura, MD, PhD1; Ichiro Katayama, MD, PhD1
[+] Author Affiliations
1Department of Dermatology, Graduate School of Medicine, Osaka University, Osaka, Japan
2Department of Dermatology, Graduate School of Medicine, Osaka City University, Osaka, Japan
3Department of Pharmacy, Osaka University Hospital, Osaka, Japan
4Department of Biomedical Statistics, Graduate School of Medicine, Osaka University, Osaka, Japan
JAMA Dermatol. 2015;151(7):722-730. doi:10.1001/jamadermatol.2014.4298.
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Published online

Importance  Tuberous sclerosis complex (TSC) is an autosomal dominant disorder leading to the aberrant activation of the mammalian target of rapamycin complex 1. Although the efficacy of mammalian target of rapamycin complex 1 inhibitors against tumors in patients with TSC, including facial angiofibroma, has been well investigated, their efficacy against hypomelanotic macules in patients with TSC is unknown.

Objectives  To evaluate objectively the efficacy of topical rapamycin treatment of hypomelanotic macules in patients with TSC and to elucidate the mechanisms of how rapamycin improves the macules.

Design, Setting, and Participants  We performed a prospective, baseline-controlled trial of 6 patients with TSC and hypomelanotic macules in non–sun-exposed and sun-exposed skin at the Department of Dermatology, Osaka University, from August 4, 2011, through September 27, 2012. Rapamycin gel, 0.2%, was applied to the lesions twice a day for 12 weeks. Histologic examinations and blood tests were conducted at the start and completion of treatment. Blood rapamycin levels were analyzed at completion.

Exposures  Topical rapamycin treatment for hypomelanotic macules.

Main Outcomes and Measures  Objective evaluation of rapamycin treatment of hypomelanotic macules in TSC with δ-L (L indicates the brightness of the color) levels on spectrophotometry at the start and completion (12 weeks) of treatment and at 4 and 12 weeks after discontinuation of treatment (16 and 24 weeks, respectively).

Results  Improvement of hypomelanotic macules (in δ-L values) was significant at 12 weeks (mean [SD], 2.501 [1.694]; P < .05), 16 weeks (1.956 [1.567]; P < .01), and 24 weeks (1.836 [1.638]; P < .001). Although efficacy tended to be prominent in sun-exposed skin, we did not observe significant differences (in δ-L values) between sun-exposed and non–sun-exposed skin at 12 weeks (mean [SD], 1.859 [0.629] and 3.142 [2.221], respectively), 16 weeks ( 1.372 [0.660] and 2.539 [2.037], respectively), and 24 weeks (1.201 [0.821] and 2.471 [2.064], respectively). No adverse events were observed, and rapamycin was not detected in the blood of any patient. Electron microscopic analysis of hypomelanotic macules revealed that topical rapamycin treatment significantly improved the uniformity of the melanosome numbers in the TSC melanocytes (pretreatment macules: mean [SD], 25.71 [21.90] [range, 5-63]; posttreatment macules: 42.43 [3.60] [range, 38-49]; P < .001). Moreover, rapamycin treatment induced the recovery of melanosomes in TSC–knocked-down melanocytes from depleted amounts (mean [SD], 16.43 [11.84]) to normal levels (42.83 [14.39]; P < .001).

Conclusions and Relevance  Topical rapamycin treatment was effective and safe against hypomelanotic macules arising from TSC. This efficacy of rapamycin was corroborated as stemming from the improvement of impaired melanogenesis in TSC melanocytes.

Figures in this Article

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder that causes multiple hamartomas, epilepsy, autism, and hypopigmented macules. Tuberous sclerosis complex is caused by mutations in the TSC1 gene (OMIM 605284)1 or the TSC2 gene (OMIM 191092)2; the genes encode hamartin and tuberin, respectively. The hamartin-tuberin complex downregulates the mammalian target of rapamycin complex 1 (mTORC1).3,4 The constitutive activation of mTORC1 that results from the abnormality of the TSC1 or TSC2 gene is associated with abnormal cellular proliferation, which causes TSC-related hamartomas. Recent reports suggest that mTORC1 inhibitors, such as rapamycin, may be effective for the treatment of TSC-related tumorigenesis, including facial angiofibromas.5 The efficacy of topical rapamycin treatment for facial angiofibroma in patients with TSC also has been reported.614 With regard to hypomelanotic macules, which are resistant to effective treatment, the mechanism by which mTORC1 affects melanogenesis is also still unknown, although hypomelanotic macules are one of the major diagnostic criteria for TSC.15,16 Hypomelanotic macules in TSC are very important because they arise at birth or in early infancy, resulting in the early diagnosis of TSC. In addition, hypomelanotic macules appearing on the face are cosmetically displeasing and require treatment. Ohguchi et al17 and Hah et al18 reported that rapamycin upregulated microphthalmia-associated transcription factor, a master regulator of melanogenesis, in B16 and MNT-1 melanoma cells. Jóźwiak and Galus19 reported the inhibition of microphthalmia-associated transcription factor by mTOR in TSC. Ho et al20 reported that TORC1 is related to melanogenesis through the activation of the melanogenic enzyme and the formation of mature melanosomes. In addition, Murase et al21 reported that autophagy, which is regulated by mTORC1, is involved in the degradation of melanosomes in keratinocytes. Recently, Wataya-Kaneda et al22 reported 2 cases of hypomelanotic macules in patients with TSC who recovered after the topical administration of rapamycin. The mTORC1 inhibitors, such as rapamycin, might be effective not only for tumors but also for hypomelanotic macules in patients with TSC.

The aims of this study were to evaluate objectively and precisely the efficacy of topical rapamycin treatment and to investigate the histologic and cytologic effects of topical rapamycin on TSC-related hypomelanotic macules. To assess the findings objectively, we evaluated the efficacy of rapamycin with the use of a spectrophotometer (CM-700d; Konica Minolta). In addition, the specimens of hypomelanotic macules before and after rapamycin treatment were examined by electron microscopy. To corroborate the effect of rapamycin on the melanogenesis that was observed in the rapamycin-treated hypomelanotic macules in TSC, the effect of rapamycin on TSC2–knocked-down melanocytes (TSC-model melanocytes) was examined. We also compared the efficacy of topical rapamycin treatment on hypomelanotic macules in sun-exposed and non–sun-exposed skin in patients with TSC to investigate the effect of sun exposure.

Ethical Consideration

This study was approved by the ethics committee of the Osaka University Faculty of Medicine (approval 10339) and was disclosed to the University Hospital Medical Information Network (report number UMIN000006108). All the patients volunteered for this trial and signed written informed consent agreements. On behalf of the children, written informed consent was obtained from each legal representative.

Study Design

This study was a prospective, baseline-controlled trial. Six patients with definitive TSC and hypomelanotic macules in non–sun-exposed and sun-exposed skin who wanted to participate in this trial were enrolled from among 250 outpatients at the Department of Dermatology, Osaka University. Rapamycin gel, 0.2%, was applied to hypomelanotic macules on non–sun-exposed and sun-exposed (on the face in 5 patients and above the knee in 1 patient) skin on each patient twice a day for 12 weeks. Assessment of each patient was performed once a month during the 12 weeks of rapamycin treatment and at 4 and 12 weeks after discontinuation of the treatment (16 and 24 weeks, respectively). Serum rapamycin levels were analyzed at the end of treatment using liquid chromatographyelectrospray ionization mass spectrometry (detection limit, 0.6 ng/mL).

Powder prepared from 2-mg rapamycin tablets (sirolimus [Rapamune]) was mixed with a 100-mg gel to a concentration of 0.2% rapamycin. We enrolled 6 patients aged 3 to 33 (mean age, 11.7) years who were diagnosed as having definitive TSC according to the diagnostic criteria update.16 Four of the 6 patients were male; 2 were female. All non–sun-exposed skin areas were on the trunk except for 1 thigh, and all the sun-exposed skin areas were on the face except for 1 knee (Table).

Table Graphic Jump LocationTable.  Characteristics of the Patients and the Treated Lesions

To determine the treatment efficacy objectively, assessment was performed using a spectrophotometer to measure the δ-L value of each hypomelanotic macule before and after treatment.2326 Decreased δ-L values reflected improved hypomelanotic macules.

Evaluation of Rapamycin Efficacy
Immunohistochemical Examinations

Skin specimens of hypomelanotic macules, obtained before treatment from 6 participants and after treatment from 1 participant, were fixed in buffered 10% formalin and embedded in paraffin. The paraffin-embedded tissue slides were stained with hematoxylin-eosin and Fontana-Masson. After the antigen was retrieved by boiling each specimen in an oil bath for 15 minutes in a 10mM TRIS/1mM EDTA buffer (pH, 9.0), the slides were also incubated with a goat anti–melan A monoclonal antibody (1:50) (Dako) at 4°C overnight and with a biotinylated-link universal antibody, conjugated streptavidin–alkaline phosphatase, and a fuchsin substrate–chromogen buffer. These slides were observed under a microscope.

Electron Microscopic Examination of the Skin Tissue

Punched skin samples (1 mm each) from the hypomelanotic macules in patients with TSC before and after treatment were fixed in 2.5% glutaraldehyde in a 0.1M phosphate buffer (pH, 7.4) at 4°C. Postfixation took place in 1% osmium tetroxide in a 0.1M phosphate buffer (pH, 7.4) for 1 hour, after which the samples were dehydrated in an ethanol dilution series and embedded in epoxy resin (Quetol-812; Nisshin EM). Semithin sections (0.5 μm) were stained with toluidine blue O, and ultrathin sections (0.1 μm) were stained with saturated uranyl acetate and lead citrate. Sections on copper mesh were examined using a commercially available electron microscope (JEM-1200EX; Jeol).

Melanin Counts in Melanocytes

Seven melanocytes were randomly selected in each sample for counting. The number of melanosomes in each melanocyte was counted directly at a magnification ×8000 and then summarized in a graph.

RNA Interference

For the short interfering RNA (siRNA) experiments, double-stranded RNA duplexes composed of 21-nucleotide sense and antisense oligonucleotides (Cosmo Bio Ltd) were synthesized. The RNA oligonucleotides used for targeting human TSC2 in this study were 5′-CGAACGAGGUGGUGUCCUATT-3′ for TSC2 sense and 5′-UAGGACACCACCUCGUUCGTT-3′ for TSC-2 antisense.

Transfection of Short Interfering SC2 and Rapamycin Stimulation With siRNA-Transfected Melanocytes

Human neonatal epidermal melanocytes from a moderately pigmented donor (HEMn-MP) were purchased from Invitrogen and cultured in medium 254 (M-254-500; Gibco) with the addition of human melanocyte growth supplement (Invitrogen) at 37°C in an atmosphere of 5% carbon dioxide. The melanocytes were used in passages 6 through 8. We seeded HEMn-MP cells onto 6-well plates at a density of 5 × 105 cells/well 12 hours before transfection. Transfection reagent (Lipofectamine 2000; Invitrogen) was used with 30nM siRNA according to the manufacturer’s instructions. The efficacy of the transfection was verified with reverse transcription–polymerase chain reaction at 48 hours after siRNA transfection; HEMn-MP cells were stimulated with 30nM rapamycin (Calbiochem) for 72 hours.

Electron Microscopic Examination of Cultured Cells

After being washed 3 times with phosphate-buffered solution, cultured cells were fixed with 0.5% glutaraldehyde in a 0.1M phosphate buffer (pH, 7.4) at 4°C for 15 minutes at room temperature. After being washed 3 times with phosphate-buffered solution, cells were fixed with 1% osmium tetroxide in phosphate-buffered solution for 3 hours at 4°C. After they were rinsed with water to remove the osmium tetroxide, the cells were dehydrated in a sequential ethanol dilution series. After infiltration in a 1:1 mixture of epoxy resin and ethanol, the cells were embedded in epoxy resin, drained for 60 minutes by inverting the culture dishes on a paper towel, and polymerized overnight at 60°C in a vacuum oven. Ultrathin sections prepared in the same manner as described for the tissue sample were examined with an electron microscope, and the number of melanosomes in each randomly selected melanocyte was counted directly.

Statistical Analysis

We used a nonparametric approach because the data were not normally distributed. To compare the homogeneity and the mean number of melanosomes before and after rapamycin treatments, we used a Levene test27 and Wilcoxon rank sum test, respectively. To compare the improvements in the hypomelanotic macules before and after the topical rapamycin treatment, we used simulation-based multiple comparisons based on generalized estimating equations. P < .05 was considered significant.

Clinical Improvement of Hypomelanotic Macules

The patient information, treated lesions, and histologic characteristics are summarized in the Table. We have included representative photographs of treatment in sun-exposed and non–sun-exposed skin before and after treatment. The photographs demonstrate that topical rapamycin treatment improved the hypomelanotic macules and that the sun-exposed skin improved more than the non–sun-exposed areas (Figure 1). In this trial, blood tests were performed on all participants before and after treatment to examine the systemic influence. Blood rapamycin concentration was also analyzed. No adverse effects were observed in any participant, and the rapamycin concentration in the blood was lower than the detection limit (0.6 ng/mL).

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Figure 1.
Clinical Features of Hypomelanotic Macules in Patient 1

Hypomelanotic macules in sun-exposed and non–sun-exposed skin before and after 12 weeks of topical rapamycin treatment. Color squares are used to correct the difference in color balance of each machine and to keep coherence of real color.

Graphic Jump Location
Improvement of δ-L Spectrophotometry Values

Five of 6 patients demonstrated lower δ-L values at the end of treatment than at initiation, with no significant difference in non–sun-exposed skin at the end of treatment (mean [SD] at 12 weeks, 3.142 [2.221]). However, at 4 weeks after discontinuation of treatment (16 weeks), δ-L values for the non–sun-exposed skin decreased significantly (mean [SD], 2.539 [2.037]; P = .046). The significant decline in δ-L values continued for 12 weeks after discontinuation of treatment (mean [SD] at 24 weeks, 2.471 [2.064]; P < .01) (Figure 2A). Unlike the non–sun-exposed skin, the δ-L values for the sun-exposed skin indicated significant differences at the completion of treatment (mean [SD], 1.859 [0.629]; P = .046) and 4 and 12 weeks after discontinuation of treatment (mean [SD], 1.372 [0.660] and 1.201 [0.821], respectively; P < .01 for both) (Figure 2B).

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Figure 2.
Improvement of Skin Color Measured by Spectrophotometer

A, Change in δ-L values for each patient in non–sun-exposed skin. Decline was significant at 16 and 24 weeks. B, Change in δ-L values for each patient in the sun-exposed skin. Change was significant at 12, 16, and 24 weeks. C, Changes in the mean δ-L values in the non–sun-exposed and sun-exposed skin. Differences between areas were not significant. Whiskers indicate SD. P < .05 was considered significant. Zero weeks indicates initiation of rapamycin treatment; 12 weeks, discontinuation of treatment; and 16 and 24 weeks, posttreatment observations.

aP < .05.

bP < .01.

Graphic Jump Location

To assess the effect of sun exposure on rapamycin treatment, we compared the mean δ-L values for the 6 patients between the non–sun-exposed and sun-exposed skin. Although the mean δ-L values in the sun-exposed skin were lower than those for the non–sun-exposed skin at all points, including at treatment initiation (mean [SD] at initiation, 3.049 [1.912] vs 3.589 [2.580]; at 12 weeks, 1.859 [0.629] vs 3.142 [2.221]; at 16 weeks, 1.372 [0.661] vs 2.539 [2.037]; and at 24 weeks, 1.201 [0.822] vs 2.471 [2.064]), and although both graphs were approximately parallel, we found no significant difference between the δ-L values for the non–sun-exposed and sun-exposed skin (Figure 2C). This result indicated that sun exposure did not affect the efficacy of the topical rapamycin therapy.

Histochemical Examinations of Hypomelanotic Macules

To corroborate the efficacy of rapamycin treatment, we undertook histologic investigations of the TSC-related hypomelanotic macules before and after treatment. Specimens of hypomelanotic macules before treatment were obtained from all 6 participants voluntarily, but an after-treatment specimen was obtained from only patient 6 because the remaining 5 patients refused biopsy of their healed lesions. Six pretreatment skin specimens, 1 posttreatment specimen, and 1 control specimen were stained with Fontana-Masson and anti–melan A antibody. The pretreatment and posttreatment specimens of hypomelanotic macules in patients with TSC demonstrated melan A–positive cells, as did the control skin specimen (eFigure 1A, C, and E in the Supplement). However, in the pretreatment TSC specimens, we observed dispersed faint Fontana-Masson staining. By contrast, as with the control samples, higher and darker Fontana-Masson–stained granules were detected in the posttreatment TSC specimens than in pretreatment specimens (eFigure 1B, D, and F in the Supplement). These results indicated that melanocytes were present but melanin granule levels were decreased in the TSC-related hypomelanotic macules, and topical rapamycin therapy increased the volume of melanin granules observed by microscope in the basal layers.

Electron Microscopic Examinations of Hypomelanotic Macules

Electron microscopic examinations suggested that in TSC hypomelanotic macules, abnormal melanogenesis might occur. To examine the precise abnormality of the melanogenesis, we assessed the numbers and morphologic features of the melanocytic melanosomes in the TSC hypomelanotic macules using an electron microscope. We examined 5 pretreatment samples and 1 posttreatment sample. Before rapamycin treatment, all the melanosomes observed in the samples were well matured at stage III or stage IV. The number of stages III and IV melanosomes varied between melanocytes. Even within the same hypomelanotic lesion from the same patient, some areas had extreme contrasts of few and many melanosomes (Figure 3A and B). By contrast, in posttreatment TSC hypomelanotic macules, the numbers of melanosomes in the melanocytes became uniform and were equal to those in the control melanocytes (Figure 3C).

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Figure 3.
Electron Microscopic Examinations of Melanosome Numbers in Hypomelanotic Macules From Patients With Tuberous Sclerosis Complex Before and After Rapamycin Treatment

A, Melanocytes with many melanosomes before treatment. B, Melanocytes with few melanosomes before treatment. C, Melanocytes after treatment (derived from 1 patient). A-C, Original magnification ×4000. D, Statistical analysis of homogeneity of melanosome numbers in the melanocytes before and after rapamycin treatment and in the control skin specimen. Data markers indicate melanosome numbers in each melanocyte; bold horizontal lines, mean value of the numbers; and limit lines, SD.

aP = .049, compared with control.

bP = .02, compared with pretreatment.

Graphic Jump Location

To confirm these findings, 7 melanocytes were selected at random from each sample, and we counted the numbers of pretreatment and posttreatment melanosomes in each melanocyte from patient 6 in microscopic images at a magnification of ×8000. The numbers of melanosomes in the melanocytes in the pretreatment TSC hypomelanotic macules ranged from 5 to 63. By contrast, the numbers of melanosomes in the posttreatment melanocytes ranged from 38 to 49. The melanosomes in the normal control skin sample ranged from 29 to 58. The range of melanosomes in the melanocytes from the pretreatment TSC hypomelanotic macules was significantly broader than the range from the control specimen (P = .02). By contrast, the range of melanosomes in the melanocytes from the posttreatment TSC hypomelanotic macules was narrower than that for the pretreatment macules (P = .049) (Figure 3D). The numbers of melanosomes in each melanocyte from the pretreatment hypomelanotic macules obtained from the remaining 4 participants were also examined. Three of the 5 patients’ pretreatment specimens exhibited significant variation in the numbers of melanosomes. One specimen (patient 2) that did not show a difference in variability reflected the significantly small number of melanosomes in each melanocyte (range, 1-65; P = .17) (Table and eFigure 2 in the Supplement). Given that the keratinocytes had abnormalities in the number and morphologic features of melanosomes similar to those of their adjacent melanocytes (Figure 3A), we believe that melanin had been transferred to the adjacent keratinocytes.

These results indicate that in hypomelanotic macules in patients with TSC, melanocytes existed, melanin transfer from melanocyte to keratinocyte was normal, and melanogenesis in melanocytes was impaired. As a result, maturation of the melanosomes was completed, but the numbers of melanosomes ranged widely between melanocytes, and topical rapamycin treatment reduced this range.

Electron Microscopic Examination of Cultured TSC–Knocked-Down Melanocytes

To corroborate the characteristics of TSC melanocytes and the efficacy of rapamycin, TSC2–knocked-down melanocytes (ie, TSC-model melanocytes) were prepared using short interfering TSC2, and the effects of rapamycin were examined. Because the effectiveness rate of the TSC2-model melanocytes was approximately 70%, we found verification of the variability to be difficult. However, many melanocytes with only a small number of melanosomes were observed in the TSC-model melanocytes. After rapamycin treatment, the number of melanosomes in the TSC-model melanocytes increased (Figure 4A and B), although no change occurred in the control melanocytes (Figure 4C and D). To confirm the effect of the rapamycin, the numbers of melanosomes in the TSC-model and control melanocytes were counted. The TSC-model melanocytes had significantly fewer melanosomes (mean [SD], 16.43 [11.84]) than did the control melanocytes (mean [SD], 42.5 [20.1]; P < .001). However, after rapamycin treatment, the number of melanosomes in the TSC-model melanocytes significantly increased (mean [SD], 42.83 [14.39]; P < .001) and approached the numbers in the control melanocytes (Figure 4E). These results corroborated that abnormal melanogenesis had occurred in the TSC-model melanocytes and that rapamycin reduced the abnormalities without affecting normal cells.

Place holder to copy figure label and caption
Figure 4.
Electron Microscopic Examination of Melanosome Numbers in Tuberous Sclerosis Complex (TSC) Model and Control Melanocytes Before and After Rapamycin Treatment

A, TSC-model melanocytes before treatment. B, TSC-model melanocytes after treatment. C, Control melanocytes before treatment. D, Control melanocytes after treatment. A-D, Original magnification ×8000. E, Statistical analysis of the mean numbers of melanosomes in each melanocyte. TSC2 and tsc2 indicate short interfering (si) TSC2 knocked-down melanocyte (TSC model melanocyte) before rapamycin treatment. TSC2-rapa indicates si-TSC2 knocked-down melanocyte after rapamycin treatment; scramble, control melanocyte before rapamycin treatment; and scr-rapa, control melanocyte after rapamycin treatment. Data markers indicate melanosome numbers in each melanocyte; bold horizontal lines, mean value of the numbers; and limit lines, SD.

aP < .001 compared with scramble.

bP < .001 compared with TSC2.

Graphic Jump Location

Tuberous sclerosis complex is an autosomal dominant disorder caused by the inactivation of 1 of 2 tumor suppressor genes, TSC11 or TSC2,2 leading to the aberrant activation of mTORC1.3,4 Thus, mTORC1 inhibitors, such as rapamycin, have been used to treat patients with TSC. The mechanism of tumorigenesis via mTORC1 is well investigated, and the efficacy of rapamycin for treating tumors in TSC has been already reported.5,2830 By contrast, only 1 study of hypomelanotic macules has been reported,22 and the mechanisms and therapeutic approaches to hypomelanotic macules in TSC remain unknown, although these macules are a major feature of TSC.15,16 In this report, we treated hypomelanotic macules in 6 patients with definitive TSC and objectively verified the efficacy of a topical rapamycin treatment using a spectrophotometer.

At the completion of 12 weeks of treatment, hypomelanotic macules on non–sun-exposed and sun-exposed skin improved clinically (Figure 1), but significant differences in their conditions at the initiation of treatment were demonstrated only on sun-exposed skin. However, after the discontinuation of treatment, the hypomelanotic macules were more improved than they had been at the completion of treatment, and significant differences were observed at 4 weeks after discontinuation of treatment for non–sun-exposed and sun-exposed skin (P < .05 and P < .01, respectively) and 12 weeks (P < .01) after discontinuation of the treatment on both areas (Figure 2). These results suggest that topical rapamycin therapy was as effective for the hypomelanotic macules in TSC as it is for TSC angiofibromas. However, recovery of the skin color took longer than recovery after angiofibromas, and once skin color was recovered, the effect lasted for several weeks after the discontinuation of treatment. Although topical rapamycin was effective in hypomelanotic macules and angiofibromas, the mechanisms by which the rapamycin improved the hypopigmented macules compared with the angiofibromas might be different.

To examine how rapamycin was involved in the melanogenesis, histochemical and electron microscopic examinations of the hypomelanotic macules in patients with TSC were conducted. Skin color is related to the number of melanin granules in keratinocytes that are transferred from melanocytes.31 Our observations revealed that the melanosomal transfer from melanocytes to keratinocytes was normal (Figure 3A). Therefore, hypomelanotic macules in patients with TSC could be attributed to abnormal melanization in melanocytes but not to the abnormal transfer of melanosomes from melanocytes to keratinocytes or to the increased degradation of melanosomes in keratinocytes, as reported by Murase et al.21 We demonstrated that maturation of melanosomes was observed in the melanocytes of hypomelanotic macules in the patients with TSC (Figure 3A), but the numbers of matured melanosomes in the melanocytes varied (range, 5-63) between melanocytes (Table and eFigure 2 in the Supplement). Jimbow32 reported that TSC-related hypomelanotic macules are associated with a decrease in melanization and melanosome size and an increase in aggregated melanosomes in keratinocytes. Our observations confirmed those findings. The abnormal melanization that resulted in variable numbers of melanosomes between melanocytes might be characteristic of the hypomelanotic macules in TSC. Rapamycin treatment reduced these abnormalities significantly, and the numbers of matured melanosomes in melanocytes after rapamycin treatment became uniform (range, 38-49) (Figure 3D). Because these posttreatment data are derived from a single patient, further studies will be necessary.

Recently, some studies indicated the involvement of mTOR in melanization.1720 If we consider these results, mTORC1 might be involved in melanogenesis, and rapamycin regulated the constitutively activated mTORC1 and improved abnormal melanogenesis in the TSC melanocytes. Then, the gradual improvement of skin color was likely owing to the rapamycin treatment. Skin color further improved after discontinuation of the treatment.

To corroborate the effects of rapamycin on the melanocytes in the patients with TSC, TSC-model melanocytes before and after rapamycin treatment were observed with an electron microscope (Figure 4). The TSC-model melanocytes had significantly small numbers of melanosomes. The clinical findings in the pretreatment biopsy samples showed a variable number of melanosomes. However, TSC model melanocytes showed reduced numbers of melanosomes. We attribute this discrepancy to influence on melanocytes in vivo by many other surrounding cells and the intricate effect of the microenvironment, in contrast to cultured cells. In addition, TSC2 may be inactivated by a mutation or a deletion, and these modes may not be functionally identical.33 Some abnormality might induce the variability and another might induce the decrease of melanosomes. In patient 2, TSC2 abnormality may have induced the decrease of melanosomes rather than the variability (eFigure 2 in the Supplement). Rapamycin treatment significantly increased the numbers of melanosomes in the TSC-model melanocytes, although no influence appeared in the control melanocytes (Figure 4). These results were consistent with the clinical symptoms after topical rapamycin treatment; namely, rapamycin treatment affected only the hypomelanotic macule but did not affect normal skin.

Although clinical improvement of hypomelanotic macules in sun-exposed skin was remarkable compared with the non–sun-exposed skin throughout the study period, including at initiation (Figure 1), the difference in δ-L values between the sun-exposed skin and the non–sun-exposed skin was not statistically significant (Figure 2C). Recently, Kalie et al34 reported that ULK1 regulates melanin levels in MNT-1 melanoma cells independently of mTORC1. Sun exposure might improve melanogenesis in an mTOR-independent manner.

In this trial, no adverse effects were observed, and the rapamycin blood concentrations were lower than the detection limit (0.6 ng/mL) in all participants. Because mTORC1 is a conserved and ubiquitous protein, adverse effects are a concern with the systemic administration of rapamycin. Topical rapamycin treatment is recommended not only for facial angiofibromas but also for hypomelanotic macules attributable to TSC.

In this report, we clarified that topical rapamycin therapy was effective and safe for treating TSC-related hypomelanotic macules. We also revealed that the small or varied numbers of melanosomes in melanocytes might be characteristic of TSC-related hypomelanotic macules and that rapamycin improved the abnormalities. Sun exposure might improve melanogenesis in an mTOR-independent manner. Further studies with more samples are expected.

Accepted for Publication: October 5, 2014.

Corresponding Author: Mari Wataya-Kaneda, MD, PhD, Department of Dermatology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan (mkaneda@derma.med.osaka-u.ac.jp).

Published Online: February 18, 2015. doi:10.1001/jamadermatol.2014.4298.

Author Contributions: Dr Wataya-Kaneda had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Wataya-Kaneda, Tanaka, L. Yang, F. Yang, Tanemura.

Acquisition, analysis, or interpretation of data: Wataya-Kaneda, Tanaka, L. Yang, F. Yang, Tsuruta, Nakamura, Matsumoto, Hamasaki, Katayama.

Drafting of the manuscript: Wataya-Kaneda, Tanaka, Hamasaki, Tanemura.

Critical revision of the manuscript for important intellectual content: Wataya-Kaneda, L. Yang, F. Yang, Tsuruta, Nakamura, Matsumoto, Katayama.

Statistical analysis: Tanaka, L. Yang, F. Yang, Hamasaki.

Obtained funding: Wataya-Kaneda.

Administrative, technical, or material support: Wataya-Kaneda, L. Yang, F. Yang, Tsuruta, Nakamura, Matsumoto, Tanemura.

Study supervision: Wataya-Kaneda, Tanemura, Katayama.

Conflict of Interest Disclosures: None reported.

Funding/Support: This study was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by a grant from the Ministry of Health, Labor and Welfare of Japan.

Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

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Inoki  K, Li  Y, Zhu  T, Wu  J, Guan  KL.  TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002;4(9):648-657.
PubMed   |  Link to Article
Inoki  K, Li  Y, Xu  T, Guan  KL.  Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 2003;17(15):1829-1834.
PubMed   |  Link to Article
Hofbauer  GF, Marcollo-Pini  A, Corsenca  A,  et al.  The mTOR inhibitor rapamycin significantly improves facial angiofibroma lesions in a patient with tuberous sclerosis. Br J Dermatol. 2008;159(2):473-475.
PubMed   |  Link to Article
Haemel  AK, O’Brian  AL, Teng  JM.  Topical rapamycin: a novel approach to facial angiofibromas in tuberous sclerosis. Arch Dermatol. 2010;146(7):715-718.
PubMed   |  Link to Article
Kaufman McNamara  E, Curtis  AR, Fleischer  AB  Jr.  Successful treatment of angiofibromata of tuberous sclerosis complex with rapamycin. J Dermatolog Treat. 2012;23(1):46-48.
PubMed   |  Link to Article
Wataya-Kaneda  M, Tanaka  M, Nakamura  A, Matsumoto  S, Katayama  I.  A topical combination of rapamycin and tacrolimus for the treatment of angiofibroma due to tuberous sclerosis complex (TSC): a pilot study of nine Japanese patients with TSC of different disease severity. Br J Dermatol. 2011;165(4):912-916.
PubMed   |  Link to Article
Tanaka  M, Wataya-Kaneda  M, Nakamura  A, Matsumoto  S, Katayama  I.  First left-right comparative study of topical rapamycin vs vehicle for facial angiofibromas in patients with tuberous sclerosis complex. Br J Dermatol. 2013;169(6):1314-1318.
PubMed   |  Link to Article
Mutizwa  MM, Berk  DR, Anadkat  MJ.  Treatment of facial angiofibromas with topical application of oral rapamycin solution (1mgmL(-1) ) in two patients with tuberous sclerosis. Br J Dermatol. 2011;165(4):922-923.
PubMed   |  Link to Article
Salido  R, Garnacho-Saucedo  G, Cuevas-Asencio  I,  et al.  Sustained clinical effectiveness and favorable safety profile of topical sirolimus for tuberous sclerosis: associated facial angiofibroma. J Eur Acad Dermatol Venereol. 2012;26(10):1315-1318.
PubMed   |  Link to Article
DeKlotz  CM, Ogram  AE, Singh  S, Dronavalli  S, MacGregor  JL.  Dramatic improvement of facial angiofibromas in tuberous sclerosis with topical rapamycin: optimizing a treatment protocol. Arch Dermatol. 2011;147(9):1116-1117.
PubMed   |  Link to Article
Koenig  MK, Hebert  AA, Roberson  J,  et al.  Topical rapamycin therapy to alleviate the cutaneous manifestations of tuberous sclerosis complex: a double-blind, randomized, controlled trial to evaluate the safety and efficacy of topically applied rapamycin. Drugs R D. 2012;12(3):121-126.
PubMed   |  Link to Article
Wheless  JW, Almoazen  H.  A novel topical rapamycin cream for the treatment of facial angiofibromas in tuberous sclerosis complex. J Child Neurol. 2013;28(7):933-936.
PubMed   |  Link to Article
Roach  ES, Gomez  MR, Northrup  H.  Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J Child Neurol. 1998;13(12):624-628.
PubMed   |  Link to Article
Northrup  H, Krueger  DA; International Tuberous Sclerosis Complex Consensus Group.  Tuberous sclerosis complex diagnostic criteria update: recommendations of the 2012 international tuberous sclerosis complex consensus conference. Pediatr Neurol. 2013;49(4):243-254.
PubMed   |  Link to Article
Ohguchi  K, Banno  Y, Nakagawa  Y, Akao  Y, Nozawa  Y.  Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells. J Cell Physiol. 2005;205(3):444-451.
PubMed   |  Link to Article
Hah  YS, Cho  HY, Lim  TY,  et al.  Induction of melanogenesis by rapamycin in human MNT-1 melanoma cells. Ann Dermatol. 2012;24(2):151-157.
PubMed   |  Link to Article
Jóźwiak  J, Galus  R.  Molecular implications of skin lesions in tuberous sclerosis. Am J Dermatopathol. 2008;30(3):256-261.
PubMed   |  Link to Article
Ho  H, Kapadia  R, Al-Tahan  S, Ahmad  S, Ganesan  AK.  WIPI1 coordinates melanogenic gene transcription and melanosome formation via TORC1 inhibition. J Biol Chem. 2011;286(14):12509-12523.
PubMed   |  Link to Article
Murase  D, Hachiya  A, Takano  K,  et al.  Autophagy has a significant role in determining skin color by regulating melanosome degradation in keratinocytes. J Invest Dermatol. 2013;133(10):2416-2424.
PubMed   |  Link to Article
Wataya-Kaneda  M, Tanaka  M, Nakamura  A, Matsumoto  S, Katayama  I.  A novel application of topical rapamycin formulation, an inhibitor of mTOR, for patients with hypomelanotic macules in tuberous sclerosis complex. Arch Dermatol. 2012;148(1):138-139.
PubMed   |  Link to Article
Alaluf  S, Atkins  D, Barrett  K, Blount  M, Carter  N, Heath  A.  The impact of epidermal melanin on objective measurements of human skin colour. Pigment Cell Res. 2002;15(2):119-126.
PubMed   |  Link to Article
Brazzelli  V, Muzio  F, Antoninetti  M,  et al.  The perilesional skin in vitiligo: a colorimetric in vivo study of 25 patients. Photodermatol Photoimmunol Photomed. 2008;24(6):314-317.
PubMed   |  Link to Article
Choi  H, Ahn  S, Lee  BG, Chang  I, Hwang  JS.  Inhibition of skin pigmentation by an extract of Lepidium apetalum and its possible implication in IL-6 mediated signaling. Pigment Cell Res. 2005;18(6):439-446.
PubMed
Fongers  A, Wolkerstorfer  A, Nieuweboer-Krobotova  L, Krawczyk  P, Tóth  GG, van der Veen  JP.  Long-term results of 2-mm punch grafting in patients with vitiligo vulgaris and segmental vitiligo: effect of disease activity. Br J Dermatol. 2009;161(5):1105-1111.
PubMed   |  Link to Article
Levene  H. Robust tests for equality of variances. In: Olkin  I, Ghurye  SG, Hoeffding  W, Madow  WG, Mannet  HB, eds. Contributions to Probability and Statistics: Essays in Honor of Harold Hotelling. Menlo Park, CA: Stanford University Press; 1960:278-292.
Franz  DN, Leonard  J, Tudor  C,  et al.  Rapamycin causes regression of astrocytomas in tuberous sclerosis complex. Ann Neurol. 2006;59(3):490-498.
PubMed   |  Link to Article
Taillé  C, Debray  MP, Crestani  B.  Sirolimus treatment for pulmonary lymphangioleiomyomatosis. Ann Intern Med. 2007;146(9):687-688.
PubMed   |  Link to Article
Herry  I, Neukirch  C, Debray  MP, Mignon  F, Crestani  B.  Dramatic effect of sirolimus on renal angiomyolipomas in a patient with tuberous sclerosis complex. Eur J Intern Med. 2007;18(1):76-77.
PubMed   |  Link to Article
Costin  GE, Hearing  VJ.  Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J. 2007;21(4):976-994.
PubMed   |  Link to Article
Jimbow  K.  Tuberous sclerosis and guttate leukodermas. Semin Cutan Med Surg. 1997;16(1):30-35.
PubMed   |  Link to Article
Govindarajan  B, Brat  DJ, Csete  M,  et al.  Transgenic expression of dominant negative tuberin through a strong constitutive promoter results in a tissue-specific tuberous sclerosis phenotype in the skin and brain. J Biol Chem. 2005;280(7):5870-5874.
PubMed   |  Link to Article
Kalie  E, Razi  M, Tooze  SA.  ULK1 regulates melanin levels in MNT-1 cells independently of mTORC1. PLoS One. 2013;8(9):e75313.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.
Clinical Features of Hypomelanotic Macules in Patient 1

Hypomelanotic macules in sun-exposed and non–sun-exposed skin before and after 12 weeks of topical rapamycin treatment. Color squares are used to correct the difference in color balance of each machine and to keep coherence of real color.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Improvement of Skin Color Measured by Spectrophotometer

A, Change in δ-L values for each patient in non–sun-exposed skin. Decline was significant at 16 and 24 weeks. B, Change in δ-L values for each patient in the sun-exposed skin. Change was significant at 12, 16, and 24 weeks. C, Changes in the mean δ-L values in the non–sun-exposed and sun-exposed skin. Differences between areas were not significant. Whiskers indicate SD. P < .05 was considered significant. Zero weeks indicates initiation of rapamycin treatment; 12 weeks, discontinuation of treatment; and 16 and 24 weeks, posttreatment observations.

aP < .05.

bP < .01.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 3.
Electron Microscopic Examinations of Melanosome Numbers in Hypomelanotic Macules From Patients With Tuberous Sclerosis Complex Before and After Rapamycin Treatment

A, Melanocytes with many melanosomes before treatment. B, Melanocytes with few melanosomes before treatment. C, Melanocytes after treatment (derived from 1 patient). A-C, Original magnification ×4000. D, Statistical analysis of homogeneity of melanosome numbers in the melanocytes before and after rapamycin treatment and in the control skin specimen. Data markers indicate melanosome numbers in each melanocyte; bold horizontal lines, mean value of the numbers; and limit lines, SD.

aP = .049, compared with control.

bP = .02, compared with pretreatment.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 4.
Electron Microscopic Examination of Melanosome Numbers in Tuberous Sclerosis Complex (TSC) Model and Control Melanocytes Before and After Rapamycin Treatment

A, TSC-model melanocytes before treatment. B, TSC-model melanocytes after treatment. C, Control melanocytes before treatment. D, Control melanocytes after treatment. A-D, Original magnification ×8000. E, Statistical analysis of the mean numbers of melanosomes in each melanocyte. TSC2 and tsc2 indicate short interfering (si) TSC2 knocked-down melanocyte (TSC model melanocyte) before rapamycin treatment. TSC2-rapa indicates si-TSC2 knocked-down melanocyte after rapamycin treatment; scramble, control melanocyte before rapamycin treatment; and scr-rapa, control melanocyte after rapamycin treatment. Data markers indicate melanosome numbers in each melanocyte; bold horizontal lines, mean value of the numbers; and limit lines, SD.

aP < .001 compared with scramble.

bP < .001 compared with TSC2.

Graphic Jump Location

Tables

Table Graphic Jump LocationTable.  Characteristics of the Patients and the Treated Lesions

References

van Slegtenhorst  M, de Hoogt  R, Hermans  C,  et al.  Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science. 1997;277(5327):805-808.
PubMed   |  Link to Article
European Chromosome 16 Tuberous Sclerosis Consortium.  Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell. 1993;75(7):1305-1315.
PubMed   |  Link to Article
Inoki  K, Li  Y, Zhu  T, Wu  J, Guan  KL.  TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nat Cell Biol. 2002;4(9):648-657.
PubMed   |  Link to Article
Inoki  K, Li  Y, Xu  T, Guan  KL.  Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 2003;17(15):1829-1834.
PubMed   |  Link to Article
Hofbauer  GF, Marcollo-Pini  A, Corsenca  A,  et al.  The mTOR inhibitor rapamycin significantly improves facial angiofibroma lesions in a patient with tuberous sclerosis. Br J Dermatol. 2008;159(2):473-475.
PubMed   |  Link to Article
Haemel  AK, O’Brian  AL, Teng  JM.  Topical rapamycin: a novel approach to facial angiofibromas in tuberous sclerosis. Arch Dermatol. 2010;146(7):715-718.
PubMed   |  Link to Article
Kaufman McNamara  E, Curtis  AR, Fleischer  AB  Jr.  Successful treatment of angiofibromata of tuberous sclerosis complex with rapamycin. J Dermatolog Treat. 2012;23(1):46-48.
PubMed   |  Link to Article
Wataya-Kaneda  M, Tanaka  M, Nakamura  A, Matsumoto  S, Katayama  I.  A topical combination of rapamycin and tacrolimus for the treatment of angiofibroma due to tuberous sclerosis complex (TSC): a pilot study of nine Japanese patients with TSC of different disease severity. Br J Dermatol. 2011;165(4):912-916.
PubMed   |  Link to Article
Tanaka  M, Wataya-Kaneda  M, Nakamura  A, Matsumoto  S, Katayama  I.  First left-right comparative study of topical rapamycin vs vehicle for facial angiofibromas in patients with tuberous sclerosis complex. Br J Dermatol. 2013;169(6):1314-1318.
PubMed   |  Link to Article
Mutizwa  MM, Berk  DR, Anadkat  MJ.  Treatment of facial angiofibromas with topical application of oral rapamycin solution (1mgmL(-1) ) in two patients with tuberous sclerosis. Br J Dermatol. 2011;165(4):922-923.
PubMed   |  Link to Article
Salido  R, Garnacho-Saucedo  G, Cuevas-Asencio  I,  et al.  Sustained clinical effectiveness and favorable safety profile of topical sirolimus for tuberous sclerosis: associated facial angiofibroma. J Eur Acad Dermatol Venereol. 2012;26(10):1315-1318.
PubMed   |  Link to Article
DeKlotz  CM, Ogram  AE, Singh  S, Dronavalli  S, MacGregor  JL.  Dramatic improvement of facial angiofibromas in tuberous sclerosis with topical rapamycin: optimizing a treatment protocol. Arch Dermatol. 2011;147(9):1116-1117.
PubMed   |  Link to Article
Koenig  MK, Hebert  AA, Roberson  J,  et al.  Topical rapamycin therapy to alleviate the cutaneous manifestations of tuberous sclerosis complex: a double-blind, randomized, controlled trial to evaluate the safety and efficacy of topically applied rapamycin. Drugs R D. 2012;12(3):121-126.
PubMed   |  Link to Article
Wheless  JW, Almoazen  H.  A novel topical rapamycin cream for the treatment of facial angiofibromas in tuberous sclerosis complex. J Child Neurol. 2013;28(7):933-936.
PubMed   |  Link to Article
Roach  ES, Gomez  MR, Northrup  H.  Tuberous sclerosis complex consensus conference: revised clinical diagnostic criteria. J Child Neurol. 1998;13(12):624-628.
PubMed   |  Link to Article
Northrup  H, Krueger  DA; International Tuberous Sclerosis Complex Consensus Group.  Tuberous sclerosis complex diagnostic criteria update: recommendations of the 2012 international tuberous sclerosis complex consensus conference. Pediatr Neurol. 2013;49(4):243-254.
PubMed   |  Link to Article
Ohguchi  K, Banno  Y, Nakagawa  Y, Akao  Y, Nozawa  Y.  Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells. J Cell Physiol. 2005;205(3):444-451.
PubMed   |  Link to Article
Hah  YS, Cho  HY, Lim  TY,  et al.  Induction of melanogenesis by rapamycin in human MNT-1 melanoma cells. Ann Dermatol. 2012;24(2):151-157.
PubMed   |  Link to Article
Jóźwiak  J, Galus  R.  Molecular implications of skin lesions in tuberous sclerosis. Am J Dermatopathol. 2008;30(3):256-261.
PubMed   |  Link to Article
Ho  H, Kapadia  R, Al-Tahan  S, Ahmad  S, Ganesan  AK.  WIPI1 coordinates melanogenic gene transcription and melanosome formation via TORC1 inhibition. J Biol Chem. 2011;286(14):12509-12523.
PubMed   |  Link to Article
Murase  D, Hachiya  A, Takano  K,  et al.  Autophagy has a significant role in determining skin color by regulating melanosome degradation in keratinocytes. J Invest Dermatol. 2013;133(10):2416-2424.
PubMed   |  Link to Article
Wataya-Kaneda  M, Tanaka  M, Nakamura  A, Matsumoto  S, Katayama  I.  A novel application of topical rapamycin formulation, an inhibitor of mTOR, for patients with hypomelanotic macules in tuberous sclerosis complex. Arch Dermatol. 2012;148(1):138-139.
PubMed   |  Link to Article
Alaluf  S, Atkins  D, Barrett  K, Blount  M, Carter  N, Heath  A.  The impact of epidermal melanin on objective measurements of human skin colour. Pigment Cell Res. 2002;15(2):119-126.
PubMed   |  Link to Article
Brazzelli  V, Muzio  F, Antoninetti  M,  et al.  The perilesional skin in vitiligo: a colorimetric in vivo study of 25 patients. Photodermatol Photoimmunol Photomed. 2008;24(6):314-317.
PubMed   |  Link to Article
Choi  H, Ahn  S, Lee  BG, Chang  I, Hwang  JS.  Inhibition of skin pigmentation by an extract of Lepidium apetalum and its possible implication in IL-6 mediated signaling. Pigment Cell Res. 2005;18(6):439-446.
PubMed
Fongers  A, Wolkerstorfer  A, Nieuweboer-Krobotova  L, Krawczyk  P, Tóth  GG, van der Veen  JP.  Long-term results of 2-mm punch grafting in patients with vitiligo vulgaris and segmental vitiligo: effect of disease activity. Br J Dermatol. 2009;161(5):1105-1111.
PubMed   |  Link to Article
Levene  H. Robust tests for equality of variances. In: Olkin  I, Ghurye  SG, Hoeffding  W, Madow  WG, Mannet  HB, eds. Contributions to Probability and Statistics: Essays in Honor of Harold Hotelling. Menlo Park, CA: Stanford University Press; 1960:278-292.
Franz  DN, Leonard  J, Tudor  C,  et al.  Rapamycin causes regression of astrocytomas in tuberous sclerosis complex. Ann Neurol. 2006;59(3):490-498.
PubMed   |  Link to Article
Taillé  C, Debray  MP, Crestani  B.  Sirolimus treatment for pulmonary lymphangioleiomyomatosis. Ann Intern Med. 2007;146(9):687-688.
PubMed   |  Link to Article
Herry  I, Neukirch  C, Debray  MP, Mignon  F, Crestani  B.  Dramatic effect of sirolimus on renal angiomyolipomas in a patient with tuberous sclerosis complex. Eur J Intern Med. 2007;18(1):76-77.
PubMed   |  Link to Article
Costin  GE, Hearing  VJ.  Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J. 2007;21(4):976-994.
PubMed   |  Link to Article
Jimbow  K.  Tuberous sclerosis and guttate leukodermas. Semin Cutan Med Surg. 1997;16(1):30-35.
PubMed   |  Link to Article
Govindarajan  B, Brat  DJ, Csete  M,  et al.  Transgenic expression of dominant negative tuberin through a strong constitutive promoter results in a tissue-specific tuberous sclerosis phenotype in the skin and brain. J Biol Chem. 2005;280(7):5870-5874.
PubMed   |  Link to Article
Kalie  E, Razi  M, Tooze  SA.  ULK1 regulates melanin levels in MNT-1 cells independently of mTORC1. PLoS One. 2013;8(9):e75313.
PubMed   |  Link to Article

Correspondence

CME


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Multimedia

Supplement.

eFigure 1. Histologic Examinations of Hypomelanotic Macules in Tuberous Sclerosis Complex Patients Before and After Rapamycin Treatment

eFigure 2. Numbers of Melanosomes in the Hypomelanotic Macules of Tuberous Sclerosis Complex in Patients 1 to 3, 5, and 6

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