Histopathologic Features in Vitiligo You Chan Kim, MD,* Yun Jeon Kim, MD,* Hee Young Kang, MD,* Seonghyang Sohn, Phd,† and Eun-So Lee, MD*

ORIGINAL ARTICLE

Histopathologic Features in Vitiligo You Chan Kim, MD,* Yun Jeon Kim, MD,* Hee Young Kang, MD,* Seonghyang Sohn, PhD,† and Eun-So Lee, MD*

characterized by melanocyte loss in the epidermis.8–10 Le Abstract: Nevus depigmentosus is a congenital disorder charac- Poole et al8 reported that melanocytes were lost in vitiligo in terized by a nonprogressive hypopigmented lesion, which may not be their study, which used 18 antibodies against melanocytes. In apparent at birth. Thus, it is sometimes difficult to differentiate contrast, Husain et al11 discovered dihydroxyphenylacetic acid vitiligo from nevus depigmentosus only by clinical features. We pos- (DOPA)-positive melanocytes in vitiligo. Lesional skin in tulated that the histologic changes in lesional and perilesional skin vitiligo may not always be devoid of melanocytes, and melanin might be different in the 2 conditions. We took biopsies from both pigmentation may remain for a period of time after the lesional and perilesional skin of 100 cases of vitiligo to assess the development of vitiligo.12 Also, melanocytes and melanin number of melanocytes, the amount of melanin, dermal inflammatory pigment may be observed in the border areas around lesions of infiltrate, and other changes. We compared them with 30 cases of nevus vitiligo.13 Therefore, differential diagnosis of vitiligo from depigmentosus. Histologically, lesions of vitiligo showed more basal other depigmented disorders can be difficult even when hypopigmentation and dermal inflammation than perilesional normal a biopsy is performed. For example, the lesions of nevus skin. With Fontana–Masson staining, 16% of cases of vitiligo showed depigmentosus still contain some melanocytes. Thus, there is the presence of melanin. The ratio of pigmented area to epidermal a need for accurate histological investigation. Nevus depig- area was 0.06% in vitiligo, whereas 17% in perilesional normal skin mentosus is generally known to be a congenital disorder and 8.9% in nevus depigmentosus. In NKI/beteb staining, 12% of characterized by a hypopigmented macule or patch that vitiligo showed the presence of melanocytes, and their average num- remains stable over time. It may develop at various ages, which ber was 7.68 per square millimeter. The number of melanocytes was often makes the differential diagnosis of vitiligo challeng- also decreased in nevus depigmentosus but not as much as in vitiligo. ing.14,15 It is important to distinguish vitiligo from nevus We also confirmed the presence of melanocytes in 1 of 3 cases of depigmentosus because the prognosis and treatment of these 2 vitiligo by electron microscopy. In conclusion, there are a few mela- entities differ. Vitiligo is progressive and requires treatment, nocytes and melanin in some cases of vitiligo. Therefore, the diag- whereas nevus depigmentosus is stable and generally does not nosis of vitiligo should be made considering these points. require treatment. The misdiagnosis of nevus depigmentosus Key Words: immunohistochemistry, melanocytes, vitiligo as vitiligo may lead to tremendous psychologic trauma and economic loss, whereas the misdiagnosis of vitiligo as nevus (Am J Dermatopathol 2008;30:112–116) depigmentosus may also lead to the failure of treatment at the appropriate time.14 We investigated the number of melanocytes, the amount of melanin pigment, the dermal inflammatory infiltrate, and other changes between lesional skin in vitiligo, adjacent normal- INTRODUCTION appearing skin, and nevus depigmentosus skin to delineate the Vitiligo is a common acquired disorder characterized by histopathologic features that are important in differentiating patches of depigmentation. Its etiological factors include the vitiligo from nevus depigmentosus. contribution of a genetic predisposition, the nervous system, autoimmunity, and oxidative stress.1–4 A convergence theory combining all these pathogenetic hypotheses has also been suggested.5 In the majority of patients with vitiligo, the diag- MATERIALS AND METHODS nosis is established by direct clinical inspection followed by Subjects examination of the depigmented areas under Wood light. We examined 100 patients with vitiligo who presented Therefore, there are only a few reports on histopathological to the Department of Dermatology, Ajou University Hospital, changes in vitiligo.6,7 Vitiligo is generally known to be Suwon, Korea, between January 2003 and December 2004. There were 43 men and 57 women. Their mean age was 24.8 From the *Department of Dermatology; and †Laboratory of Cell Biology, years, and the mean duration of vitiligo was 13.3 months. We Ajou University School of Medicine, Suwon, Korea. also included 30 patients with nevus depigmentosus patients Supported by 2004 grant from Department of Medical Sciences, The Graduate (12 men and 18 women) for comparison. The mean age of the School, Ajou University. patients was 15.4 years, and the mean duration was 12.5 Reprints: You Chan Kim, MD, Department of Dermatology, Ajou University School of Medicine, 5 Wonchon-dong, Yeongtong-ku, Suwon, 443-721, months. Two-millimeter punch biopsies from lesional and Korea (South) (e-mail: maychan@ ajou.ac.kr). perilesional normal-appearing skin were obtained from all Copyright Ó 2008 by Lippincott Williams & Wilkins patients.

112 Am J Dermatopathol Volume 30, Number 2, April 2008 Am J Dermatopathol Volume 30, Number 2, April 2008 Histopathologic Features in Vitiligo

Stains RESULTS A hematoxylin and eosin stain was used to study the general histopathological changes such as hyperkeratosis, General Histopathologic Features of Vitiligo acanthosis, exocytosis, spongiosis, basal hypopigmentation, In 78 of 100 (78%) cases, vitiligo skin showed more basal dermal melanophages, dermal inflammatory infiltrate, rete hypopigmentation compared with perilesional normal skin, ridge elongation, and telangiectasia in the vitiligo skin. Mel- whereas only 7 of 30 (23%) cases of nevus depigmentosus skin anin pigment was visualized with the Fontana–Masson stain, did (P , 0.05). The vitiligo skin showing mild dermal inflam- performed by the usual methods. mation accounted for 41% of the cases, which was more frequent than the 23% of cases of the perilesional normal skin (P , 0.05). Other features such as hyperkeratosis, acanthosis, Immunohistochemistry exocytosis, spongiosis, melanophages, rete ridge elongation, The melanocytes and inflammatory cells were stained and telangiectasia were observed in the vitiligo skin but were by immunohistochemistry using the standard technique.16 We not statistically significant in comparison to the perilesional used the following antibodies: NKI/beteb (1:20, Monosan, Uden, normal skin (P = 0.28) or the nevus depigmentosus skin (P = the Netherlands), MART-1 (1:100, Neomarker, Fremont, CA) to 0.74) (Table 1). visualize melanocytes, and CD1a (1:100, Novocastra, New castle upon Tyne, Tyne and Wear) to visualize Langerhans Quantitative Analysis of Melanin Pigment and cells. We also used CD3 (Novocastra), CD20 (Dako), and the Number of Melanocytes CD68 (Dako) to analyze dermal inflammation. In the stains for With the Fontana–Masson staining, 16 of 100 (16%) inflammatory cells (CD3, CD20, CD68, and CD1a), each com- cases of vitiligo skin showed remaining melanin pigment. ponent of the inflammatory cells was expressed as a percentage PA/EA was significantly decreased in the epidermal layers of of all inflammatory cells. vitiligo skin (0.06 6 0.00) compared with perilesional normal skin (17.25 6 5.56) or nevus depigmentosus skin (8.94 6 1.17, P , 0.05). The PA/EA of nevus depigmentosus skin was Image Analysis also decreased in lesional skin as compared with perilesional Quantification was performed with a computerized normal skin (14.11 6 4.79), but the amount of decrease was image analyzer (Image Pro Plus Version 4.5, Media Cybertics much smaller than in vitiligo skin (Fig. 1). Co., Silver Spring, MD) as described previously.16 The ratio of Remaining melanocytes were found in 12 of 100 (12%) pigmented area to epidermal area (PA/EA) was measured in vitiligo skin cases. The MC/EA was 7.68 6 24.43, and MC/1R lesional and control skin with Fontana–Masson staining. With was 0.35 6 1.16, whereas MC/EA and MC/1R were 384.36 6 the immunohistochemical stains for melanocytes (NKI/beteb 161.71 and 72.62 6 43.42, respectively, in perilesional normal and MART-1), the number of melanocytes was estimated using skin with NKI/beteb staining (P , 0.001). MART-1 staining 2 methods: the number of melanocytes per millimeter rete showed findings similar to the NKI/beteb staining (P = 0.01). ridge length (MC/1R) and the ratio of melanocytes to Nevus depigmentosus lesional skin showed a decreased MC/ epidermal area (MC/EA). Each melanocyte was counted as EA and MC/1R both for NKI/beteb and MART-1 staining com- 1 cell when its nucleus was confirmed. All morphometric pro- pared with that of the perilesional normal skin. But the amount cedures were performed by manually tracing the borders of the of decrease was greater in vitiligo than in nevus depigmentosus epidermis and rete ridges, and the epidermal area that con- (P , 0.05) (Table 2, Fig. 2). We then examined the rela- tained hair follicles was excluded from this tracing. tionship between duration of vitiligo and the number of

Electron Microscopic Evaluation TABLE 1. Comparison of Histologic Features of Lesional and For electron microscopic evaluation, 3 pairs of lesional Perilesional Normal Skin in Vitiligo and Nevus Depigmentosus and normal skin specimens were processed as previously Nevus described.16 Depigmentosus Vitiligo (N = 100) (N = 30) Normal Lesional Normal Lesional Statistical Analysis (%) (%) (%) (%) The data were expressed as mean 6 standard deviation. Hyperkeratosis 2 (2) 5 (5) 1 (3) 2 (7) These were analyzed with the Statistical Package for Social Acanthosis 1 (1) 2 (2) 0 1 (3) Science Version 10.0 (SPSS, Chicago, IL). Comparison of Exocytosis 1 (1) 2 (2) 0 0 general histologic features, melanin, and the number of mela- Spongiosis 1 (1) 2 (2) 0 0 nocytes in the lesional skin of vitiligo; the perilesional normal- Basal hypopigmentation 0 78 (78) 0 7 (23) appearing skin; and the lesional skin of nevus depigmentosus Dermal melanophage 5 (5) 11 (11) 1 (3) 1 (3) was made using the Kruskal–Wallis test. Comparison of the Dermal inflammation 23 (23) 41 (41) 4 (13) 4 (13) number of melanocytes and Langerhans cells between areas of Rete ridge elongation 1 (1) 1 (1) 0 0 inflammatory vitiligo and noninflammatory vitiligo was made Telangiectasia 4 (4) 5 (5) 2 (7) 3 (10) using the Mann–Whitney test. A probability value of less than The number of specimen that exhibited each feature was demonstrated. 0.05 was considered as statistically significant. q 2008 Lippincott Williams & Wilkins 113 Kim et al Am J Dermatopathol Volume 30, Number 2, April 2008

FIGURE 1. Melanin pigment in the vitiligo lesional, perilesional normal skin, and nevus depigmentosus. A, Perilesional normal skin of vitiligo patient; B, vitiligo lesional skin of same subject; and C, nevus depigmentosus lesional skin. Vitiligo skin showed decreased pigment compared with normal or nevus depigmentosus skin. Fontana–Masson stain, 3200. remaining melanocytes. Of the 100 vitiligo skin specimens, Analysis of the Components of the Dermal 12 cases had a history of 5 or more years. The melanocytes Inflammatory Infiltrate were present in 3 of 12 vitiligo skin lesions with a duration of Dermal inflammation was found in 41% of vitiliginous 5 or more years. NKI/beteb staining revealed an MC/EA of skin specimens and 17.63% of perilesional normal skin spec- 5.58 6 16.43 in vitiligo skin lesions with a duration of 5 or imens. In vitiligo skin, CD3-positive cells accounted for more years, which is not significantly different from vitiligo 65.38% of the inflammatory cells. Other cells such as CD68- skin lesions with a duration of less than 5 years. There was no positive cells, CD20-positive cells, and CD1a-positive cells definite relationship between the duration of vitiligo and the accounted for 13.5%, 5.67%, and 1.89%, respectively. Similar number of melanocytes (correlation coefficient = 0.04). The findings were observed in normal skin, which were 69.87%, melanin pigment was present in 4 of 12 vitiligo skin lesions 9.32%, 2.74%, and 1.75%, respectively. There was no signif- with a duration of 5 or more years. Fontana–Masson staining icant difference in the components of the dermal inflammatory revealed a PA/EA of 0.05 6 0.01. Also, there was no definite infiltrate between lesional and perilesional normal skin. relationship between the duration of vitiligo and the amount of the pigment. To investigate the relationship between inflammation and the number of remaining melanocytes, we divided Ultrastructural Characteristics of Vitiligo the vitiligo skin into vitiligo skin with inflammation and with- Each of the 3 perilesional normal skin specimens dem- out inflammation. There was no significant difference in the onstrated melanocytes with dilated endoplasmic reticulum, number of NKI/beteb-positive melanocytes between these granular deposits, prominent Golgi apparatus, and vacuoliza- 2 subgroups. MART-1 staining also did not reveal significant tion. The mitochondria were also damaged. There were no differences (Table 3). intact melanosomes in the melanocytes or in the keratinocytes. Of the 3 vitiligo lesional skin specimens, 1 specimen showed residual melanocytes with melanosomes. Although Quantitative Analysis of the Number of the intact melanosomes were difficult to discern, poorly mel- Langerhans Cells anized melanosome organelles remained. They also had With CD1a staining, there was no significant difference dilated, rough endoplasmic reticulum and vacuolization. Lang- in the number of Langerhans cells in the epidermis between vit- erhans cells with Birbeck granules were also noted, and their iligo lesional skin and perilesional normal skin. However, the changes included vacuolization, cellular swelling, irregular number of Langerhans cells in the epidermis of vitiligo skin nuclei, and damaged mitochondria. In the keratinocytes of with inflammation was increased (24.66 6 19.69 per square vitiligo lesional skin, irregular nuclei were observed. Dilated millimeter) when compared with that of vitiligo without Golgi complex and intracytoplasmic vacuoles were also noted, inflammation (15.70 6 1.98 per square millimeter, P , 0.05). and tonofilaments were fragmented.

TABLE 2. Quantitative Analysis of the Number of Melanocytes in Vitiligo Lesional, Perilesional Normal Skin, and Nevus Depigmentosus Skin Vitiligo Nevus Depigmentosus Antibodies Normal Lesional P Value Normal Lesional P Value NKI/beteb MC/EA 384.36 6 161.71 7.68 6 24.43 ,0.001 412.65 6 218.12 243.85 6 149.08 ,0.001 MC/1R 72.62 6 43.42 0.35 6 1.16 ,0.01 77.16 6 32.45 68.49 6 43.16 ,0.001 MART-1 MC/EA 411.35 6 203.06 12.45 6 41.72 0.01 361.73 6 203.06 209.58 6 107.36 ,0.001 MC/1R 61.98 6 6.27 0.52 6 1.88 ,0.01 54.79 6 28.46 35.88 6 6.27 ,0.01

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FIGURE 2. Melanocytes in vitiligo lesional, perilesional normal skin, and nevus depigmentosus skin. A, Perilesional normal skin of vitiligo patient; B, vitiligo lesional skin; and C, nevus depigmentosus lesional skin; a, NKI/beteb staining and b, MART-1 staining. The number of positive cells was decreased in vitiligo lesional skin compared with nevus depigmentosus or perilesional normal skin in NKI/beteb staining and MART-1 staining. Original magniﬁcation 3200.

DISCUSSION against melanocytes and concluded that melanocytes are The diagnosis of vitiligo may be challenging, as absent within vitiligo lesions. However, there are some spo- histopathologic findings have not been confirmed. Indeed, radic reports indicating that vitiligo lesions are not fully devoid 12 there have been few studies specific to the histopathological of melanocytes. Tobin et al also showed that melanocytes characteristics of vitiligo and none comparing histopatholog- could be isolated and established in vitro from all samples of ical findings in vitiligo, perilesional normal skin, and other lesional and normal skin, independent of disease duration and 11 depigmented lesions, such as nevus depigmentosus. The pres- treatment. Husain et al also found epidermal melanocytes in ent investigation of the histopathology of vitiligo, in com- vitiligo and showed enzymatic hydroxylation of tyrosinase to parison to perilesional normal skin or nevus depigmentosus DOPA. And nonnegligible amounts of melanin were detected skin, is a prerequisite for diagnosis or differential diagnosis of in basal keratinocytes in 1- to 3-year-old vitiligo lesions. vitiligo. Therefore, the possibility of intact melanogenesis or melanin 13 The general histologic features of vitiligo revealed more transfer in vitiligo was suggested. In our study, we found basal hypopigmentation and dermal inflammation in viti- 64.01 6 37.04 NKI/beteb-positive cells per square millimeter ligo lesional skin than in perilesional normal skin. Basal in 12 of 100 vitiligo lesional skin specimens indicating that, hypopigmentation in nevus depigmentosus compared with whereas the level is much lower than normally pigmented or perilesional normal skin was found occasionally, but the incidence was much less than in vitiligo. There was no difference in dermal inflammation in nevus depigmentosus TABLE 3. Comparative Analysis of the Number of between lesional skin and perilesional normal skin. Therefore, Melanocytes Between in Vitiligo With Inflammation and basal hypopigmentation and mild dermal inflammation with Vitiligo Without Inflammation hematoxylin and eosin stain may sometimes provide a hint Vitiligo With Vitiligo Without in the diagnosis of vitiligo, especially when a melanocyte- Antibodies Inflammation Inflammation P Value specific immunohistochemical stain is not available. NKI/beteb MC/EA 5.84 6 18.22 7.65 6 8.13 NS Almost all authors of the earlier studies unanimously MC/1R 0.37 6 1.16 0.32 6 1.17 NS concluded that long-standing vitiligo patches show a com- MART-1 MC/EA 7.72 6 78.48 7.41 6 65.91 NS plete loss of melanin and absence of melanocytes from the MC/1R 0.77 6 1.75 0.37 6 1.03 NS 8–10 8 epidermis. Le Poole et al reported a comprehensive NS, not significant. immunohistochemical study using a panel of 18 antibodies q 2008 Lippincott Williams & Wilkins 115 Kim et al Am J Dermatopathol Volume 30, Number 2, April 2008 nevus depigmentosus skin, melanocytes may exist in some repeatedly indicated that skin lesions show an absence of vitiliginous skin. We also confirmed the presence of mela- melanin and melanocytes, along with degenerative changes nocytes in 1 of 3 vitiligo skin specimens on electron micros- affecting both melanocytes and basal/suprabasal keratino- copy. Even in vitiligo skin of 5 or more years duration, cytes, more recent investigations including our study dem- melanocytes have been confirmed to be occasionally present in onstrated that melanocytes may never be completely absent in lesional skin. Because NKI/beteb targets gp100, which is a the depigmented epidermis and that these melanocytes main- premelanosomal protein, we suggest that the remaining mela- tain the capability of recovering their functionality. Further nocytes produce premelanosomes. The amount of melanin was studies are therefore needed to clarify this highly debated issue 0.06 per square millimeter in the epidermal layers in vitiligo that has obvious therapeutic implications. skin compared with 17.25 in normal skin or 8.94 in nevus depigmentosus skin with Fontana–Masson staining. There- fore, the possibility that the remaining melanocytes may be REFERENCES 1. Casp CB, She JX, McCormack WT. Genetic association of the catalase functioning is suggested. gene (CAT) with vitiligo susceptibility. Pigment Cell Res. 2002;15:62–66. To make differential diagnosis of vitiligo histologically, 2. Bystryn JC. Neural pathogenesis. In: Hann SK, Nordlund J, eds. 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