Letters to the Editor / Journal of Dermatological Science 74 (2014) 159–182

Fig. 2. Relative miRNA concentrations determined by real-time PCR as described in Table S3 are shown on the ordinate.

Acknowledgements This study was supported in part by a grant for scientific research from the Japanese Ministry of Education, Science, Sports and Culture, by Shiseido Research Grant, and by grants from Rohto Award. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jdermsci. 2014.01. 005. References [1] Kroh EM, Parkin RK, Mitchell PS, Tewari M. Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR). Methods 2010;50(4):298–301.

Photodynamic diagnosis of metastatic lymph nodes using 5-aminolevulinic acid in mouse squamous cell carcinoma

Keywords: Squamous cell carcinoma; Photodynamic diagnosis; 5-Aminolevulinic acid; Protoporphyrin IX

To the Editor Squamous cell carcinoma (SCCs) is one of the most frequent types of non-melanoma skin cancer. In certain cases, SCCs spread to the regional lymph nodes (LNs), which results in poor prognosis. It is essential to accurately evaluate LN metastasis in order to select the appropriate therapeutic strategy and predict the outcomes of the patients with SCCs. In addition to using sentinel lymph node biopsy (SLNB), which is a potentially promising procedure for assessing LN metastasis in patients with SCCs [1], achieving accurate and rapid intraoperative diagnosis is vital to ensure the use of a less invasive surgery. The photosensitizer 5-aminolevulinic acid (5-ALA) has recently been clinically used for detecting the primary lesion in certain

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[2] Kanemaru H, Fukushima S, Yamashita J, Honda N, Oyama R, Kakimoto A, et al. The circulating microRNA-221 level in patients with malignant melanoma as a new tumor marker. J Dermatol Sci 2011;61(3):187–93. [3] Sonkoly E, Wei T, Janson PC, Sa¨a¨f A, Lundeberg L, Tengvall-Linder M, et al. microRNAs: novel regulators involved in the pathogenesis of psoriasis? PLoS One 2007;2(7):e610. [4] Sonkoly E, Sta˚hle M, Pivarcsi A. microRNAs: novel regulators in skin inflammation. Clin Exp Dermatol 2008;33(3):312–5. [5] Zibert JR, Løvendorf MB, Litman T, Olsen J, Kaczkowski B, Skov L. microRNAs and potential target interactions in psoriasis. J Dermatol Sci 2010;58(3): 177–85. [6] Joyce CE, Zhou X, Xia J, Ryan C, Thrash B, Menter A, et al. Deep sequencing of small RNAs from human skin reveals major alterations in the psoriasis miRNAome. Hum Mol Genet 2011;20(20):4025–40. [7] Xu N, Brodin P, Wei T, Meisgen F, Eidsmo L, Nagy N, et al. MiR-125b: a microRNA downregulated in psoriasis, modulates keratinocyte proliferation by targeting FGFR2. J Invest Dermatol 2011;131(7):1521–9. [8] Gu X, Nylander E, Coates PJ, Nylander K. Effect of narrow-band ultraviolet B phototherapy on p63 and microRNA (miR-21 and miR-125b) expression in psoriatic epidermis. Acta Derm Venereol 2011;91(4):392–7. [9] Ralfkiaer U, Hagedorn PH, Bangsgaard N, Løvendorf MB, Ahler CB, Svensson L, et al. Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL). Blood 2011;118(22):5891–900. [10] Bhandari A, Gordon W, Dizon D, Hopkin AS, Gordon E, Yu Z, Andersen B. The Grainyhead transcription factor Grhl3/Get1 suppresses miR-21 expression and tumorigenesis in skin: modulation of the miR-21 target MSH2 by RNAbinding protein DND1. Oncogene 2012;32(12):1497–507.

Yusaku Koga, Masatoshi Jinnin*, Asako Ichihara, Akihiko Fujisawa, Chikako Moriya, Keisuke Sakai, Satoshi Fukushima, Yuji Inoue, Hironobu Ihn Department of Dermatology and Plastic Surgery, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan *Corresponding author. Tel.: +81 96 373 5233; fax: +81 96 373 5235 E-mail addresses: [email protected], [email protected] (M. Jinnin). Received 28 July 2013 http://dx.doi.org/10.1016/j.jdermsci.2014.01.005

types of non-melanoma skin cancers—this process is termed as photodynamic diagnosis. The exogenous administration of 5-ALA causes selective accumulation of the heme precursor protoporphyrin IX (PpIX) in cancer cells [2]. PpIX is a fluorescent substance that emits a strong red fluorescence at approximately 635 nm on blue light excitation [3,4]. In the present study, we aimed to evaluate the feasibility of using 5-ALA-induced PpIX fluorescence as a rapid intraoperative diagnostic tool for LN metastasis in mouse SCCs. A mouse model of SCCs with LN metastasis was established, as previously described by Matsumoto et al. [5] with minor modifications. In brief, NR-S1M cells (6  106 cells) were intracutaneously injected into the back area—adjacent to the tail—of 6–8-week-old C3H/He mice (SHIMIZU Laboratory Supplies, Kyoto, Japan). Three to four weeks after tumor implantation, the mice were administered 5-ALA hydrochloride (Wako Pure Chemical Industries, Kyoto, Japan) at a dose of 250 mg/kg body weight through a tail vein injection [6]. Six to nine hours after the injection, the inguinal, axiliary, and para-aortic LNs were excised and examined using fluorescence stereomicroscopy, as described previously [6,7]. Briefly, light emitting from a mercury lamp was filtered through a 405  10 nm band-pass filter and used for excitation. The fluorescent emission at a wavelength longer than 430 nm was transmitted through a long-pass filter and detected by charge-coupled device camera. We performed intensity analysis by

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Fig. 1. Representative images of metastatic lymph nodes (A) and non-metastatic inguinal (B) and para-aortic (C) lymph nodes. White-light and fluorescence images (left panels, white images; middle panels, fluorescence images, scale bar: 1 mm) and hematoxylin and eosin-stained sections (right panels, original magnification: 12.5) of each lymph node are shown. The yellow arrows indicate metastatic foci. Red fluorescence, representative of metastatic foci, is observed in the metastatic lymph node (A). Microscopic fluorescence images: excitation, 395–415 nm; emission, >430 nm. (For interpretation of the references to color in this legend, the reader is referred to the web version of the article.)

using Image-J software (National Institutes of Health, Bethesda, MD), as described previously [7]. The absolute mean R value and the signal intensity ratio of red to the sum of red, green, and blue (R/[R + G + B]) were used as indices corresponding to the red fluorescence of PpIX. All procedures were performed in accordance with the guidelines of the Animal Care and Use Committees of Kyoto Prefectural University of Medicine. In total, 33 LNs obtained from 7 mice were examined, 12 of which were metastatic LNs. Strong accumulation of PpIX fluorescence was detected within the metastatic lesions in metastatic LNs (Fig. 1A). Dimly weak to moderate fluorescence in the entire LN was detected in most of the non-metastatic inguinal or axillary LNs (Fig. 1B). However, para-aortic LNs without metastasis showed no fluorescence or imperceptible fluorescence (Fig. 1C). Compared to that in non-metastatic LNs, the R value (Fig. 2A) and R/(R + G + B) ratio (Fig. 2B) indicated significant increments in metastatic LNs (R value: metastatic LNs, 69.92  38.29 vs. non-metastatic LNs, 23.73  19.07, P < 0.001; R/(R + G + B) ratio: metastatic LNs, 0.782  0.153 vs. non-metastatic LNs, 0.501  0.154, P < 0.001). Thus, we observed that the evaluation of 5-ALA-induced PpIX fluorescence can be a useful method for detecting metastatic LNs in a mouse SCC model. To our knowledge, this is the first report that applied photodynamic diagnosis using 5-ALA for the detection of LN metastasis in SCCs. Histological analysis is commonly performed for detecting LN metastasis through rapid intraoperative diagnosis. However, several studies have reported that rapid

Fig. 2. The absolute mean R value (A) and the signal intensity ratio of red to the sum of red, green, and blue (R/[R + G + B]) (B) between metastatic lesions of metastatic lymph nodes and non-metastatic lymph nodes (*, P < 0.001).

intraoperative pathological diagnosis is associated with a significant risk of overlooking metastasis, as only a few hematoxylin and eosin-stained slides from whole LNs are examined during routine histological examinations [8,9]. In addition to SCCs, photodynamic diagnosis or therapy of the primary lesions is widely used for non-melanoma skin cancers as well, including extramammary Paget disease, wherein LN metastasis would result in a poor prognosis. As fluorescence imaging methods are simple, rapid, and easily applicable, we believe that the 5-ALA-induced PpIX fluorescence method for rapid intraoperative diagnosis can be a novel and promising tool for patients with various types of non-melanoma skin cancers. Acknowledgements This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (J.A., N.K.) and project of the Japan Science and Technology Agency (JST), Japan (J.A.). We thank Mr. Shin-ichiro Kotani for assistance with fluorescence stereomicroscopy. References [1] Matthey-Gie ML, Boubaker A, Letovanec I, Demartines N, Matter M. Sentinel lymph node biopsy in nonmelanoma skin cancer patients. J Skin Cancer 2013;2013. 8 pp. [Article ID 267474]. [2] Peng Q, Berg K, Moan J, Kongshaug M, Nesland JM. 5-Aminolevulinic acid-based photodynamic therapy: principles and experimental research. Photochem Photobiol 1997;65:235–51. [3] Leunig A, Rick K, Stepp H, Gutmann R, Alwin G, Baumgartner R, et al. Fluorescence imaging and spectroscopy of 5-aminolevulinic acid induced protoporphyrin IX for the detection of neoplastic lesions in the oral cavity. Am J Surg 1996;172:674–7.

Letters to the Editor / Journal of Dermatological Science 74 (2014) 159–182 [4] Harada K, Harada Y, Beika M, Koizumi N, Inoue K, Murayama Y, et al. Detection of lymph node metastases in human colorectal cancer by using 5-aminolevulinic acid-induced protoporphyrin IX fluorescence with spectral unmixing. Int J Mol Sci 2013;14:23140–52. [5] Matsumoto G, Yajima N, Saito H, Nakagami H, Omi Y, Lee U, et al. Cold shock domain protein A (CSDA) overexpression inhibits tumor growth and lymph node metastasis in a mouse model of squamous cell carcinoma. Clin Exp Metastasis 2010;27:539–47. [6] Murayama Y, Harada Y, Imaizumi K, Dai P, Nakano K, Okamoto K, et al. Precise detection of lymph node metastases in mouse rectal cancer by using 5-aminolevulinic acid. Int J Cancer 2009;125:2256–63. [7] Koizumi N, Harada Y, Murayama Y, Harada K, Beika M, Yamaoka Y, et al. Detection of metastatic lymph nodes using 5-aminolevulinic acid in patients with gastric cancer. Ann Surg Oncol 2013;20:3541–8. [8] Ghossein RA, Rosai J. Polymerase chain reaction in the detection of micrometastases and circulating tumor cells. Cancer 1996;78(10–16). [9] Natsugoe S, Aikou T, Shimada M, Yoshinaka H, Takao S, Shimazu H, et al. Occult lymph node metastasis in gastric cancer with submucosal invasion. Surg Today 1994;24:870–5. a,1,

b,1

b

Jun Asai *, Yoshinori Harada , Masatomo Beika , Hideya Takenakaa, Norito Katoha, Tetsuro Takamatsub a Department of Dermatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan;

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Department of Pathology and Cell Regulation, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan *Corresponding author at: Department of Dermatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Hirokoji, Kawaramachi, Kamigyo-Ku, Kyoto, 602-8566, Japan. Tel.: +81 75 251 5586; fax: +81 75 251 5586 E-mail address: [email protected] (J. Asai). 1

These authors equally contributed to this work.

Received 2 December 2013 Accepted 25 December 2013

http://dx.doi.org/10.1016/j.jdermsci.2013.12.009

Letter to the Editor Oculocutaneous albinism (OCA) in Japanese patients: Five novel mutations

Keywords: Pigment disorder; Melanin; Gene diagnosis

Oculocutaneous albinism (OCA) is a group of autosomal recessive disorders characterized by a reduction or deficiency of melanin in the eyes, skin, and hair [1]. The lack of pigment in the skin results in severe photosensitivity and a high risk of skin cancer, while the lack of pigment in the eyes results in photophobia, nystagmus, and greatly decreased visual acuity. OCA is classified into two groups: non-syndromic and syndromic. The former is caused by a reduction in melanogenesis, resulting in hypopigmentation and amblyopia, whereas the latter has a number of additional features, such as bleeding diathesis, immunodeficiency, and neurological dysfunction [2]. To date, there are seven types of non-syndromic OCA: OCA type 1 (OCA1, MIM 203100) that is caused by mutations in tyrosinase gene(TYR) [1], OCA type 2 (OCA2, MIM 203200) that is caused by mutations in P gene [3], OCA type 3 (OCA3, MIM 203290) that is caused by mutations in tyrosinase-related protein 1 gene (TYRP1) [4], OCA type 4 (OCA4, MIM 611409) that is caused by mutations in solute carrier family 45 member 2 gene(SLC45A2) [5], and OCA types 5, 6, and 7 [6]. The latter three types were reported very recently and are rare. Hermansky-Pudlak syndrome (HPS) is a well-known syndromic OCA characterized by OCA, mild to severe bleeding diathesis, and ceroid storage disease [1]. Among Japanese OCA patients, 10% have been diagnosed with HPS type 1(HPS1, MIM 203300), which is caused by mutations in HPS1 [2,7]. We examined five Japanese patients who were clinically diagnosed with OCA and screened for mutations in TYR (OCA1), P (OCA2), TYRP1 (OCA3), SLC45A2 (OCA4), and HPS1. Informed consent and blood samples were obtained following protocols approved by the Ethics Committee of Yamagata University School of Medicine. After the amplification of each gene by polymerase chain reaction, the products were tested using single strand

conformational polymorphism analysis and direct sequencing in order to identify sequence variations. This analysis allowed us to genetically diagnose the type of OCA, and resulted in the detection of five novel mutations (Table 1). These mutations included four missense mutations; two in TYR (c.131G > A, p.S44 N; c.283T > C, p.F95L), one in P (IVS17-3C > A), one in SLC45A2 (c.233C > T, p.P78L), and one deletion mutation in SLC45A2 (c.192delT, p.G64fsX112). We confirmed that all the amino acids altered by these novel mutations were conserved among species, including chimpanzee, horse, dog, mouse, chicken, fish, and frog. In addition, the newly identified mutations were not found in 100 unrelated, normally pigmented Japanese adults, indicating that the mutations are likely not polymorphisms and are probably pathological. Severe or relatively severe phenotypes were observed in patients 1, 2, and 4, who had the novel p.G64fsX112(SLC45A2), p.S44N(TYR), p.F95L(TYR), p.P78L(SLC45A2) and the reported p.D157N(SLC45A2) mutations, suggesting that these newly identified mutations may result in the loss of melanogenesis activity. Patient 5 had two heterozygous mutations in P. The p.A481T mutation is associated with a mild phenotype, occasionally with no distinctive skin manifestations when present with a null mutation [8]. He had another novel splicing mutation, IVS17-3C > A, and he displayed a mild phenotype, suggesting that this novel mutation may be a null mutation resulting in no melanogenesis activity. Patient 3 was diagnosed with OCA3. This type is rare worldwide, especially in the East Asian area [2]. To date, only two Chinese and one Japanese patient have been reported [9,10]. This is the fourth case from East Asia. The patient was a 5-year-old Japanese boy. Physical examination showed blond hair and eyebrows, dark brown eyelashes and irises, lighter skin than that of the parents, and several pigmented nevi on the face (Fig. 1). He had normal visual acuity with no nystagmus but had mild photophobia. His elder brother was normal, and his parents were cousins with an inbreeding coefficient of 1/16, leading us to predict a homozygous causative mutation (i.e., autozygous). Mutation screening revealed a homozygous mutation at c.1100delG,p.G367fsX384 in TYRP1 for OCA3. The patient had an apparent clinically tyrosinase-positive OCA despite the homozygous frameshift mutation. This fact might support the results of Yamada et al., who reported that TYRP1 protein is not the sole rate-limiting factor in melanogenesis [10].