METHODS
Fifty-nine biopsies were taken from 49 Japanese female patients (4 males and 45 females, age ranged from 16 to 60 (ave. = 36.16)) with hyperpigmented skin lesions after informed consent using an IRB-approved protocol. The lesions were clinically diagnosed as follows: ADM (n =12); solar (senile) lentigines (n = 8); lichen pilaris (n = 4); ripple/reticulate hyperpigmentation in atopic dermatitis (RHAD) (n = 7); nevus spilus (cafe au lait macules) (n = 4); pigmented contact dermatitis (n = 4); pigmented nipple areolar complex (PNAC) (n = 3); pigmentatio petaloides actinica (PPA) (n = 4); ephelides (n = 2); friction melanosis (n = 2); periorbital hyperpigmentation (n = 2); nevus of Ota (n = 2); postinflammatory hyperpigmentation resulted from a single inflammatory event (PIH-S) (n = 1); seborrheic keratosis (n = 1); melasma (ML) (n = 1); pigmented external genitalia (n = 1); and erythromelanosis follicularis faciei et colli (EFFC) (n = 1). In addition, 10 samples were taken from 10 patients at baseline and during RA-HQ therapy; these lesions included diagnoses of solar lentigines (n = 1), lichen pilaris (n = 2), nevus spilus (n = 1), pigmented contact dermatitis (n = 2), PNAC (n = 1), RHAD (n = 1), ADM (n = 1), and PPA (n = 1).
Biopsied tissue was fixed in 4% buffered neutral formaldehyde solution and embedded in paraffin for sectioning. The sections were stained with hematoxylin-eosin and Fontana-Masson stains. The pathological examination focused on the existence of hyperkeratosis and the location (layers) of melanin deposits or melanocytes, since these two factors determined our therapeutic strategy. Comparing samples obtained before and during treatment, morphological alterations due to the RA-HQ therapy were observed.
Details of the RA-HQ therapy protocol were described previously.4-6 Briefly, the protocol consisted of a bleaching phase and a healing phase. We originally prepared 0.1, 0.2, and 0.4% tretinoin aqueous gels and an ointment with 5% hydroquinone. For bleaching, tretinoin was first applied only to the pigmented area with a cotton tip applicator, and then hydroquinone was applied to the larger area surrounding the lesion (the whole face, for example). Initially, the application was done twice a day, depending on skin reactions such as erythema and scaling. When sufficient reduction in pigmentation was obtained, the application of tretinoin was discontinued. The duration of this phase was 2 to 6 weeks. For healing, hydroquinone alone was applied twice a day until the erythema disappeared completely; the duration of this phase was usually 4 weeks.

RESULTS
Biopsies of pigmented lesions
The histological characteristics and diagnoses of the biopsied samples are summarized in Table 1. Clinical appearance and histology of representative cases of each morbidity are shown in Figures 1 and 2, respectively.

Horny layers
In general, dermatoses located on the trunk and extremities rather than on the face have a physiological tendency toward hyperkeratosis. In agreement with this, in pigmented lesions located on the body, i.e. on the nipples/areola or on the external genitalia, the horny layer was relatively thicker than for pigmented dermatoses on the face. Solar lentigines and relatively flat seborrheic keratoses resembled each other histologically. Both were characterized by distinctive hyperkeratosis, which was more obvious in lesions located on areas other than the face. Note that in solar lentigines, mild hyperkeratosis was observed even in lesions that appeared flat. Lichen pilaris and EFFC also showed mild hyperkeratosis, but the hyperkeratosis was more distinctive in lichen pilaris. This difference in the degree of hyperkeratosis was greater than expected. In EFFC, the epidermis was acanthotic and hyperkeratotic without parakeratosis, and follicular hyperplasia with follicular plugging was also observed. No other dermatoses investigated in this study showed hyperkeratosis.

Epidermal pigmentation
Epidermal pigmentation was enhanced in all the morbidities in this study except for the nevus of Ota case. The degree of epidermal melanin pigmentation varied among the morbidities, and substantial differences were found between cases.

Dermal pigmentation
Dermal melanosis (melanin incontinence) was observed in some of the morbidities. Lesions resulting from chronic or repeated inflammation, such as pigmented contact dermatitis, friction melanosis, RHAD, and PPA, showed breakdown of the dermo-epidermal junction and severe dermal melanosis (deposits of melanophages) in the upper dermis. Some melanophage deposits in the upper dermis were also seen in cases of melasma and solar lentigines, although degenerative changes of the dermo-epidermal junction were not detected. ADM and periorbital hyperpigmentation (a subtype of ADM) had dermal melanocytes with a highly pigmented, elongated dendritic appearance in the upper dermis, while dermal melanocytes were scattered throughout the dermis in nevus of Ota. In several morbidities such as PIH-S, ephelides, and nevus spilus, dermal pigmentation, either melanosis or melanocytosis, was not detected irrespective of the degree of epidermal pigmentation.

Samples taken during RA-HQ treatment
The histological and clinical manifestations of representative cases of hyperpigmentation are shown in Figures 3 and 4. In sections obtained during RA-HQ therapy, substantial epidermal hyperplasia, dramatic reduction of melanin deposits around the basal layer, and temporal enhancement of parakeratosis were consistently observed (Figure 3B and D). These changes were most likely a result of enhanced basal keratinocyte mitosis and accelerated turnover of the epidermis. Epidermal pigmentation was significantly reduced, while dermal melanophages or melanocytes, if any, appeared unchanged (Figure 4B and D). The clinical observations agreed with the histological findings. In lesions without dermal pigmentation involvement, the pigmented color was clinically improved (Figure 3E), while in lesions with dermal pigmentation, the color change was usually moderate (Figure 4C) until laser therapy was also used (Figure 4E).

DISCUSSION
Hyperpigmentation in the epidermis and dermis
Although the degree of pigmentation differed among both morbidities and cases, the mechanisms underlying melanin deposits in the epidermis can be understood as follows. Epidermal melanin is continuously provided to keratinocytes by melanocytes located in the basal layer, transported to the stratum corneum by keratinocyte turnover, and finally removed by stratum corneum sloughing. In healthy skin, the total amount of epidermal melanin, the main determinant of skin color, is the result of the balance between production and discharge of melanin. Production of epidermal melanin is regulated by the genetic makeup and gene expression profile of the melanocytes located in the basal layer, but production can be enhanced by external factors such as UV irradiation10 and inflammation. Skin phototype is determined by the genetic makeup of melanocytes, which is modified by genetic transformation of melanocytes in morbidities such as solar lentigines11 and PPA.12 Melanin production can be suppressed by agents such as hydroquinone that are toxic to melanocytes or that inhibit melanocytes. On the other hand, the discharge of epidermal melanin is determined mainly by epidermal turnover, which can be increased by topical retinoids. On the contrary, external factors such as topical corticosteroids and aging can attenuate melanin discharge. The regulatory mechanism of epidermal pigmentation (skin color) is a key concept for establishing our bleaching strategy to epidermal hyperpigmentation; that is a combined use of tretinoin (a discharge enhancer) and hydroquinone (a production suppressor).
In morbidities such as ephelides,11 nevus spilus,13 melasma,14,15 lichen pilaris, and EFFC,16,17 melanin production is pathologically enhanced. In addition to the underlying genetic factors, PNAC and external genitalia are also influenced by hormones and topical inflammation, such as that induced by the friction caused by clothing.18 In solar lentigines and PPA, UV irradiation induces genetic alterations in melanocytes, though other internal and external factors can also be pathogenetic.10,19 On the other hand, PIH-S is the result of an external factor: in this condition, melanogenesis is temporally enhanced due to an inflammatory event. PIH-S usually involves only epidermal hyperpigmentation without degenerative changes in the dermo-epidermal junction, and tends to gradually disappear, but occasionally it can persist for a long time.
Dermal pigmentation is influenced by another mechanism. Although melanophages are occasionally present in the normal dermis of Asians,20 they are mainly observed in pigmented lesions with a grayish appearance known as areas of “pigmentary incontinence.”21,22 Histologically, pigmentary incontinence is caused by melanosome translocation from the epidermis to the dermis, usually following dermo-epidermal junction damage. Pigmented contact dermatitis,23 friction melanosis,24 and RHAD25,26 are caused by repeated inflammatory events, which induce repeated hypermelanogenesis and degeneration of the dermo-epidermal junction, and result in scattered melanophages located in the upper dermis. Our results confirmed the histological features of these lesions, and also revealed that some lesions classified as solar lentigines and melasma also have melanophages in the upper dermis without apparent dermo-epidermal damage; the pigmentary incontinence of these lesions may result from inflammatory events or excessive UV irradiation encountered after the onset of the lesion.
In acquired dermal melanosis and periorbital hyperpigmentation, there are ectopic and aberrant melanocytes in the upper dermis (dermal melanocytosis); these conditions can be distinguished from the pigmented lesions accompanying dermal melanosis (pigmented incontinence) described above.27 Nevus of Ota,28 nevus of Ito,29 and Mongolian spots29 are known to have aberrant melanocytes throughout the dermis.

Treatment-oriented histological classification of hyperpigmented lesions
Based on the features of hyperkeratosis and epidermal and dermal pigmentation discussed above, we propose a new classification system for pigmented dermatoses; diagnosis and histological features of each Category are summarized in Table 2.
Morbidities with apparent hyperkeratosis are classified in Category I; our strategy suggests that these lesions should be treated with CO2 lasers. Based on the location of the pigmentation, the character of the pigmentation (melanosis or melanocytosis), and the melanocyte activity (genetically upregulated or temporally upregulated), lesions can be placed into Categories II to VII. Category II and III include only epidermal hyperpigmentation and Category VII includes only dermal hyperpigmentation, while Categories IV to VI includes lesions with both epidermal and dermal hyperpigmentation. Melanocytes may be temporally upregulated by local inflammatory events such as dermatitis, scratching, and UV irradiation (Categories II and IV), while melanocytes have undergone genetic changes leading to upregulation (Categories III and V). The epidermal hypermelanosis observed in lesions in Category VI may be due to stimulatory effects of melanocytes localized in the upper dermis.4,6

Therapeutic strategy for pigmented lesions based on their histological characteristics
Based on our classification system for pigmented skin lesions, we have broadened the indications for our therapeutic strategy. This therapeutic strategy involves CO2 laser therapy, QSR laser irradiation, and topical bleaching treatment (RA-HQ therapy), and is schematically summarized in Figure 5. RA-HQ treatment accelerates the removal of accumulated melanosomes in the epidermis and replaces them with much less pigmented cells. QSR laser irradiation not only reduces dermal melanosis and melanocytosis, but also removes the pigmented and hyperkeratotic epidermis associated with solar lentigines. The former can also be accomplished using a Q-switched Alexandrite laser, and the latter can be accomplished with other lasers, such as the Q-switched ND:Yag laser. A CO2 laser was used to treat lesions with excessive hyperkeratosis (Category I lesions) which could not be effectively treated with a QSR laser. Hyperkeratotic lesions need to be treated with lasers: because the thickened horny layer prevents percutaneous absorption of topical ointment, RA-HQ therapy does not work.
Lesions in Categories II and III are treated only with topical RA-HQ therapy, except for solar lentigines with hyperkeratosis. The treatment usually needs to be performed repeatedly for melasma.4 PIH-S does not recur after treatment, but ephelides, lentigo simplex, and nevus spilus (cafe au lait macules) tend to reappear within a few months unless topical hydroquinone is used for post-treatment maintenance. Although the RA-HQ therapy cannot treat dermal pigmentation and hyperkeratotic lesions, it can be used as a pre-treatment for QSR laser irradiation in treating Categories IV to VI lesions. It can also be used after QSR laser treatment, because it is quite effective for treating the PIH frequently seen after QSR laser treatment of darker-colored skin.
A critical difference between lesions in Category IV to VI and those in Category VII is the extent of epidermal pigmentation, which determines the beneficial and adverse effects of QSR laser on dermal pigmentation. If the lesion has only dermal melanocytosis (Category VII), it can be effectively treated with repeated sessions of QSR laser irradiation alone.30,31 However, the sole use of Q-switched laser irradiation failed in the treatment of morbidities with both epidermal and dermal pigmentation (Categories IV to VI), ,resulting in high frequency of PIH and/or depigmentation.32-34 We believe that, in these morbidities, epidermal melanin deposits obstruct laser irradiations to dermal pigmentation as competing chromophores; laser irradiation thus induces considerable inflammation in the epidermis and, consequently, severe PIH results especially in patients with darker-colored skin.6 To overcome these problems, we propose that topical bleaching be performed prior to QSR laser therapy. The pretreatment can be applied not only to ADM, but also to other skin conditions with both epidermal and dermal pigmentation, such as friction melanosis, pigmented contact (cosmetic) dermatitis, and RHAD (lesions in Categories IV to VI; Figure 4). For Category VI lesions (dermal melanocytosis), the combination of RA-HQ treatment and QSR laser must usually be performed 2 or 3 times, while only one QSR session is usually needed for treating lesions in Categories VI and V (dermal melanosis).

CONCLUSONS
We refined a strategy for treating hyperpigmentation by studying lesion histology and by reviewing our extensive clinical experience in treating Asian skin. Here is our proposed standard strategy: first, treat epidermal pigmentation with RA-HQ, unless the lesion has hyperkeratosis; second, treat dermal pigmentation without epidermal hypermelanosis with repeated QSR laser irradiation; third, treat dermal pigmentation with substantial epidermal pigmentation with a combined RA?HQ treatment and QSR irradiation; fourth, repeat each session as needed. We believe that the classification system and the histology-based treatment principles proposed in this study may be helpful for establishing a standardized treatment algorithm for hyperpigmented skin lesions, especially in Asians.



Table 1.　A summary of the histological characteristics of samples stained with Fontana-Masson stain.
Clinical diagnosis Epidermis Dermis
Hyperkeratosis Melanin deposits Melanophages (Pigmentary incontinence) Dermal melanocytes


Table 2. 　Proposed 7-category classification system for hyperpigmented lesions
* Morbidities classified into two Categories.

Figure legends


Figure 1. Hyperpigmented lesions in Asians: clinical manifestations.
(A) Postinflammatory hyperpigmentation resulting from a single inflammatory event. (B) Pigmented nipple/areola. (C) Ephelides. (D) Nevus spilus. (E) Lichen pilaris. (F) Seborrheic keratoses. (G) Solar lentigines. H) Melasma. (I) Pigmented external genitalia. (J) Pigmented contact dermatitis. (K) Friction melanosis. (L) Ripple hyperpigmentation in atopic dermatitis. (M) Pigmentatio petaloides actinica. (N) Erythromelanosis follicularis faciei. (O) Acquired dermal melanosis. (P) Periorbital hyperpigmentation. (Q) Nevus of Ota.

Figure 2. Hyperpigmented lesions in Asians: histological findings.
Specimens were obtained from the lesions shown in Figure 1. Photos A through Q: original magnification ×100. Photos O’, P’, and Q’: corresponding ×200 magnification photos of specimens O, P, and Q.

Figure 3. A 26-year-old-woman with pigmented nipple-areola complex. (A) At baseline, the nipple-areola complex showed dark brown hyperpigmentation. (B) Histological examination revealed epidermal pigmentation. (C) Two weeks after starting bleaching treatment with 0.2% tretinoin and 5% hydroquinone, the hyperpigmentation had improved. Some tretinoin-associated erythema was observed. (D) Histological examination revealed hyperplasia of the epidermis. Epidermal pigmentation was highly reduced. (E) Eight weeks after starting treatment (final result). For 4 weeks, 0.2% tretinoin was used together with 5% hydroquinone, followed by treatment with 5% hydroquinone alone for 4 weeks. Pigmentation improved and no post-inflammatory hyperpigmentation was observed.

Figure 4. A 56-year-old woman with pigmented contact (cosmetic) dermatitis. (A) At baseline, the patient showed dark brown or dark grey macules distributed symmetrically on a wide area of the face. (B) Histology at baseline. The dermo-epidermal junction was severely damaged and a number of melanosomes were found in the upper dermis (pigmentary incontinence). (C) At 8 weeks, just after the topical bleaching treatment with 0.1 and 0.4 % tretinoin and 5% hydroquinone, the pigmentation was reduced with slight erythema, but the macules still had a grayish color. (D) Histology at 8 weeks. Epidermal pigmentation was significantly improved, while the dermal melanocytosis appeared not to change at all. (E) Clinical appearance at 20 weeks. QSR irradiation was performed to reduce dermal melanosis at 8 weeks. Four weeks after the QSR irradiation, RA-HQ therapy was performed again: 4 weeks of the bleaching phase and 4 weeks of the healing phase.

Figure 5. Schematic summary of our therapeutic strategy for pigmented lesions.
A treatment protocol is suggested for lesions in each Category (I to VII) except for Category II, which was divided into three subclasses: solar lentigines, melasma and others. Solar lentigines frequently have a thicker horny layer, so that it is initially treated with a Q-switch laser followed by RA-HQ therapy for post-laser hyperpigmentation. Melasma sometimes requires two or three sessions of the RA-HA therapy. Category IV to VI lesions require combination protocols using QSR laser and RA-HQ therapy, while Category VII can be treated with QSR laser treatment alone.