**4. Discussion**

combination with tacalcitol treatment was adopted. A typical case, a 17-year-old female patient

**Figure 10.** CALM on the nose of a 20-year-old male NF1 patient treated with OCT in combination with Q-switched Ruby laser irradiation. A) Before treatment, (B) after 10 months of topical OCT application twice a day, and (C) after 6 months of Q-switched Ruby laser irradiation twice every 3 months in combination with topical OCT treatment.

**Figure 11.** Side-by-side treatment of hyperpigmentation and small pigmented spots using Nd:YAG laser toning with or without topical tacalcitol ointment. Pigmented lesions of a 17-year-old female patient were treated seven times with laser toning only (right side) or seven times with laser toning in combination with topical tacalcitol ointment (left side).

Her facial pigmented spots were treated with either laser toning alone (right side) or laser toning plus tacalcitol treatment (left side, **Figure 11**). Hyperpigmentation around her mouth and small pigmented spots around her neck became lighter on the left side compared with those on the right. Since laser toning with an Nd:YAG laser exerts effects mainly on epidermal melanocytes, this treatment was thought to have little beneficial effect on CALMs. However,

Pigmentation of her left side was more improved compared with the right side.

(B)

with NF1, is shown in **Figure 11**.

172 A Critical Evaluation of Vitamin D - Clinical Overview

(C)

(A)

NF1 is one of the major neurocutaneous syndromes. Patients with this disease experience decreased quality of life caused by cutaneous morbidities such as CALMs, skin-fold freckling, and NFs. Moreover, if malignant peripheral nerve sheath tumors should arise from plexiform NFs, it is potentially lethal to patients with NF1. Our understanding of the etiopathogenesis of NF formation in NF1 has progressed with use of mouse models and various cells isolated from NFs. Tumors are comprised of Schwann cells, mast cells, fibroblasts, and perineurial cells; however, Schwann cells are thought to be the primary tumor cell-type as they possess both germline and second-hit mutations in the NF1 gene (NF1−/−) [18]. Other components, such as mast cells and fibroblasts with haploinsufficient NF1 gene mutations (NF1+/−) are essential to sustain the formation of NFs. In a mouse model, Nf1−/− diploinsufficient Schwann cells rapidly proliferated and secreted KIT ligand at approximately sixfold higher levels compared with wild-type controls [19]. Mast cells haploinsufficient for the NF1 gene infiltrated into NFs in response to KIT ligand and exhibited potency to proliferate. Precise etiopathogenesis of CALMs remains obscure, but it has been suggested that melanocyte density is increased within CALMs [20]. Also, somatic mutation analysis yielded two NF1 hits in melanocytes isolated from CALMs [21]. A one-hit mutation in melanocytes has been thought to cause skin-fold freckling and global hyperpigmentation.

VD3 and its analogs have been found to inhibit in vitro growth of primarily isolated fibroblasts and mast cells, but not Schwann cells. We also have found that growth of commercially available human epidermal melanocytes is efficiently inhibited by VD3 and its analogs. We used these human epidermal melanocytes in our study because it was difficult to obtain informed consent from patients to excise a large enough sample of a CALM to get the required number of melanocytes. Thus, whether the growth inhibition rate of primarily isolated melanocytes (NF1+/−) from NF1 patient CALMs is the same as that of the human epidermal melanocytes (NF1+/+) remains to be examined.

Given that we observed that the growth of Schwann cells was not affected by VD3 and its analogs, we extended our study further to whether an mTOR inhibitor (rapamycin) or Ras-MEK pathway inhibitor (lovastatin) could inhibit the growth of primary Schwann cells and fibroblasts isolated from NFs. It was found that these agents could inhibit growth of both Schwann cells and fibroblasts isolated from NFs. A combination of VD3 with rapamycin and/ or lovastatin did not increase the suppressive effects of either of these drugs on the growth of Schwann cells, but it did cause additive suppression of fibroblast growth. With regard to clinical use of VD3 or its analogs for NF1 patient skin lesions, our in vitro experimental results indicate a combination of rapamycin and/or lovastatin with VD3 should be more effective at suppressing NF growth in vivo. Although the tumorigenic cells in NFs are considered to be NF1−/− Schwann cells, supporting NF1+/− mast cells and/or fibroblasts are considered to be essential for NF formation.

We found definite inhibitory effects of 308-nm UVB irradiation (excimer light) on growth of all cells comprising the NF1 phenotype in vitro. We then studied whether NB-UVB irradiation brought about beneficial effects on skin lesions of patients with NF1. In addition, we measured changes in serum VD3 levels of these patients after long-term whole body NB-UVB irradiation. We observed that at least 6 months of irradiation significantly increased serum VD3 levels, which were accompanied by a lightening of generalized hyperpigmentation of the skin of most patients examined. Hyperpigmentation commonly resulting from UV or sun exposure, caused by induction of endothelin-1, was not detected with NB-UVB irradiation at doses of 0.2– 0.5 J/cm2 once weekly or biweekly, even if NB-UVB irradiation was continued for more than 3 years.

Increases in NF1 patient serum VD3 levels by NB-UVB irradiation are in accord with a previous report suggesting NB-UVB irradiation for patients with either atopic dermatitis or psoriasis causes upregulation of serum 25-hydroxyvitamin D (calcidiol) [22]. Other reports suggest serum calcidiol levels in patients with NF1 are significantly lower than those of control subjects [23], and low levels of calcidiol have a negative correlation with severity of NF formation [24]. Also, reduced bone density and increased incidence of calcidiol deficiency in adults with NF1 have been reported [25]. Therefore, oral supply of VD3 and long-term NB-UVB irradiation could bring about benefits for both skin and internal lesions of NF1 patients; although, care should be taken to identify and minimize any adverse events caused by longterm NB-UVB irradiation.

To date, no evidence supports use of laser therapy for removal of CALMs. However, we experienced the virtual disappearance of a CALM on the nose of a young male patient with NF1 after two treatments with Q-switched Ruby laser irradiation in combination with continuous topical OCT application during a 16-month period. We also observed multiple small pigmented spots on the torso of a female patient with NF1 virtually disappeared after treatment with IPL-RF in combination with topical OCT application. Our recent studies using laser toning with an Nd:YAG laser in combination with topical tacalcitol application showed fairly good results with regard to multiple facial small pigmented spots. So, we recommend adopting this irradiation with topical VD3 ointment for pigmented spots associated with severe cosmetic problems, especially those on the face, in patients with NF1.

Our clinical investigative results with VD3 have been obtained from a small number of patients with NF1, and no double-blind studies have been performed. Therefore, further studies of a larger scale are needed to clarify to what extent pigmented lesions such as CALMs, small pigmented spots, and skin-fold freckling can be improved by irradiation with laser or IPL-RF in combination with VD3. Additional studies examining suppression of new NF formation with long-term whole body NB-UVB irradiation, which significantly enhances serum VD3 levels in patients with NF1, would also be of great value. It is generally considered that either topical application or internal ingestion of VD3 should exert the same biological mechanism of action. On the one hand, direct topical application of VD3 or its analogs onto skin lesions may be more effective than internal ingestion because of locally higher concentrations of VD3. On the other hand, skin lesions related with NF1, such as CALMs or NFs, are generally scattered across the whole body, ingestion, or injection of VD3 may be more useful for practical therapeutic applications. In conclusion, use of VD3 or its analogs is encouraging for either improving pigmented skin lesions or suppressing new NF formation in patients with NF1.
