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Prognostic significance of the hair follicle stem cell marker nestin in patients with malignant melanoma |
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European Journal of Dermatology. Volume 20, Number 3, 283-8, May-June 2010, Investigative report |
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DOI : 10.1684/ejd.2010.0909 |
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Author(s) : Kenichi Tanabe, Yasuyuki Amoh, Maho Kanoh, Hiroshi Takasu, Naohiko Sakai, Yuichi Sato, Kensei Katsuoka , Department of Dermatology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan, Department of Plastic Surgery, Kitasato University School of Medicine, Department of Molecular Diagnostics, Kitasato University School of Allied Health Sciences, Sagamihara, Kanagawa, Japan. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Summary : Nestin is an intermediate filament protein, and serves as a hair follicle stem cell and neural stem cell marker. Recent studies have suggested that nestin expression is also important for tumorigenesis. Previous reports from our laboratory have revealed that nestin is a marker of HMB-45-negative melanoma cells in dermal invasive lesions of nodular malignant melanoma. The present study examines nestin expression in malignant melanoma and investigates the relationship between nestin expression and prognosis in patients. We immunohistochemically stained 78 formalin-fixed and paraffin-embedded malignant melanomas for nestin, HMB-45 and S100 reactivity. We found that nestin, HMB-45 and S100 protein were detected in 56.5%, 88.4% and 100% of malignant melanomas, respectively. The 5-year survival rate of stage I and II nestin-positive cases was significantly decreased compared to the nestin-negative cases (p <\; 0.05). In addition, the 5-year survival rate exceeded 80% in nestin-negative malignant melanomas at all stages of tumor development. We conclude that nestin expression may be a predictor of poor prognosis in patients with malignant melanoma. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Keywords : malignant melanoma, hair follicle stem cells, nestin, HMB-45, prognosis | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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ARTICLE
Auteur(s) : Kenichi Tanabe1, Yasuyuki Amoh1, Maho Kanoh1, Hiroshi Takasu1, Naohiko Sakai2, Yuichi Sato3, Kensei Katsuoka1 1Department of Dermatology, Kitasato University
School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa
228-8555, Japan accepté le 9 Decembre 2009 Nestin is a neural stem cell marker protein that is also expressed in the bulge area stem cell compartment of the hair follicle. Nestin-expressing hair follicle stem cells give rise to the outer root sheath and the nestin-expressing interfollicular vascular network. Nestin-expressing stem cells isolated from the hair follicle bulge region are negative for the keratinocyte marker keratin 15 (K15), and are able to differentiate into numerous cell types in vitro, including neurons, glia, keratinocytes, smooth muscle cells and melanocytes. This primitive state demonstrated by the nestin-positive stem cells is compatible with their pluripotency [1-9]. Moreover, as various types of cutaneous tumors, including melanoma, originate from the hair follicle and/or epidermal stem cells [10], nestin expression is thought to be important in tumorigenesis. In the present study, we examined nestin expression in malignant melanoma and investigated the relationship between nestin expression levels and the prognosis of patients with malignant melanomas. Materials and methodsPatients and tumor collectionFormalin-fixed and paraffin-embedded tissues were obtained from 78 (35 male and 43 female) malignant melanoma patients who were observed over a period of at least 5 years and who were surgically resected at the Department of Dermatology or Plastic Surgery of Kitasato University Hospital in Japan. The patients (4 to 85 years old; median, 56.6 years old) between 1988 and 2004 were retrospectively reviewed. And we observed the five year or more passages (the longest is 21 years) of all the cases, excluding the patients who died within five years. All patients underwent subsequent tumor excision. The tumor samples analyzed were obtained from central areas of the primary tumor. Paraffin blocks representing the most typical tumor tissue (mainly central portions of primary tumors) were selected, and 4-μm thick sections were prepared for immunohistochemistry. The samples were histologically analyzed according to the pathological staging criteria of the American Joint Committee on Cancer (AJCC) and subdivided into 6 cases of stage 0, 18 cases of stage I, 33 cases of stage II, 13 cases of stage III and 8 cases of stage IV. The majority of patients in stages II to IV, 51 out of 54 (without three patients in Stage IV), received chemotherapy (DAV-F: intravenous (i.v.) dacarbazine, 160 mg/m2/day on days 1-5; nimustine, 80 mg/m2/day i.v. on day 1; vincristine, 0.8 mg/m2/day i.v. on day 1; and β-feron, 3 million Units s.c. on days 1-5). The survival time was calculated as the date of surgery to the date of death. Informed consent was obtained from all patients prior to admission into the study. This study was approved by the Ethics Committee of Kitasato University School of Medicine.Immunohistochemical analysisIndirect immunohistochemical staining was performed using the following primary antibodies: polyclonal anti-nestin (1:20, IBL, Gunma, Japan), monoclonal anti-melanosome (clone HMB-45, undiluted, Dako, Glostrup, Denmark) and polyclonal anti-S100 (1:400, Dako). Bound primary antibodies were detected using the dextran polymer reagent (ChemMate Envision, Dako). Following deparaffinization, rehydration and elimination of endogenous peroxidase activity by treatment with 3% hydrogen peroxide for 5 min at room temperature (RT), sections were incubated with 5% fetal calf serum in PBS for 5 min at RT to block non-specific protein binding. The sections were incubated with each antibody for 60 min and then with ChemMate Envision for 30 min at RT. Finally, the color reaction was developed with 8-amino-9-ethylcarbazole solution (Dako) and the sections counterstained with Mayer's hematoxylin (Wako Pure Chemical, Osaka, Japan). S100 protein staining was considered positive when definite expression was observed in the nucleus and/or cytoplasm of > 5% of tumor cells, while HMB-45 and nestin staining was considered positive when definite expression was observed in the cytoplasm of tumor cells. The slide of Nestin staining was evaluated according to staining extent and intensity. Staining extension was assessed by the percentage of stained cells and scored semi-quantitatively, using a 0 to 4 scale for expression: 0 = no expression; 1+ = 1-25%, 2+ = 26-50%, 3+ = 51-75%, 4+ = 76-100%. Staining intensity was categorized into three groups by comparing the staining intensity of tumor cells with vascular endothelial cells: 1+ = weaker than endothelial cells; 2+ = same as endothelial cells; and 3+ = stronger than endothelial cells. By adding the staining extensity and intensity scores, the combined scores were calculated. The combined scores were then divided into 4 groups: Negative = combined score 0; weak staining = combined score 2, moderate staining = combined scores 3-4; strong staining = combine scores 5-7 [10].Analysis of clinical parametersThe overall survival of the patients among the 6 groups was analyzed using the Kaplan-Meier method, Z-test and Fisher's exact test. Values < 0.05 were considered statistically significant.ResultsImmunohistochemical analysisThe expression of S100 protein was detected in the nucleus and cytoplasm of tumor cells in all cases. The expression of HMB-45 protein was also observed in the cytoplasm of tumor cells. The expression of HMB-45 was detected in 5 out of 6 (83.3%) stage 0 tumors, 17 out of 18 (94.4%) stage I tumors, 29 out of 33 (87.9%) stage II tumors, 10 out of 13 (76.9%) stage III tumors, and 8 out of 8 (100%) stage IV tumors. Nestin expression was observed in the cytoplasm of tumor cells in 59.0% of malignant melanomas (n = 78). Positivity rates were 0 out of 6 (0%) stage 0 tumors, 6 out of 18 (33.3%) stage I tumors, 23 out of 33 (69.7%) stage II tumors, 10 out of 13 (76.9%) stage III tumors and 7 of 8 (87.5%) stage IV tumors. The frequency and combined scores of nestin expression of groups are shown in table 1. Average combined score was 0 in stage 0 tumors, 1.5 in stage I tumors, 3.18 in stage II tumors, 3.46 in stage III tumors and 4.57 in stage IV tumors. Average combined score rose gradually as the stage progressed. In addition, some vascular endothelial cells, fibroblasts and peripheral nerve cells were also nestin positive. Figure 1 indicates the nestin- and HMB-45-positive melanoma cells in the junctional area (figure 1A), dermis (figure 1B), and deep dermis (figure 1C).Table 1 The frequency and combined scores (all patients)
Prognostic relevanceFollow-up of patients ranged from 5 to 180 months. During this time, 29.5% of the patients died. The survival curves for each of the different tumor stages are presented in figure 2, and the 5-year survival rates of nestin-positive and nestin-negative patients are shown in figure 3. The 5-year survival rates were 6 out of 6 (100%) for stage 0 tumors, 16 out of 18 (88.9%) for stage I tumors, 28 out of 33 (84.8%) for stage II tumors, 8 out of 13 (61.5%) for stage III tumors and 1 out of 8 (12.5%) for stage IV tumors. The difference in survival rate among the stages was significant (figure 2). Comparison of the survival rate between nestin-positive patients and nestin-negative patients for each stage also showed significant differences in all cases (P < 0.001 Fisher's exact test). That is, the 5-year survival rate was 100% in all patients exhibiting nestin-negative tumors (stage 0: 6 cases, stage I: 12 cases, stage II: 10 cases, stage III: 3 cases and stage IV: 1 case). In stage I tumors, nestin-positive patients demonstrated a decreased 5-year survival rate (66.7%) compared to the nestin-negative patients (100%, P < 0.05, Z-test). Nestin-positive patients also exhibited a decreased 5-year survival rate (78.3%) in patients with stage II tumors compared to the nestin-negative patients with stage II tumors (100%, P < 0.05 Z-test) (figure 3). And the frequency and combined scores of nestin expression of groups, divided 5-year survival groups and death groups, are shown in table 2. In each stage, average combined scores in 5-year survival groups were higher than in death groups.Table 2 The frequency and combined scores (divided 5 year survival groups and death groups)
DiscussionNestin is a neural stem cell marker protein that is also expressed in hair follicle stem cells in the bulge region [5-9]. The nestin-expressing hair follicle stem cells give rise to the outer root sheath and the nestin-expressing interfollicular vascular network in nestin-GFP transgenic mice [6]. We have recently demonstrated that nestin-expressing stem cells isolated from the hair follicle stem cell region in mice that were negative for the keratinocyte marker K15 were able to differentiate into neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro [5-9]. In addition to being K15-negative, the pluripotent nestin-expressing stem cells are also positive for the stem cell marker CD34, demonstrating their relatively undifferentiated state. This primitive state of the nestin-expressing stem cells appears to be compatible with their pluripotency [6-9]. Recently, numerous studies have suggested that various forms of cutaneous tumors, including melanoma, originate from the hair follicle and epidermal stem cells. Brychtova et al. [11] demonstrated via immunohistochemical analysis that nestin is expressed in malignant melanoma and melanocytic nevi. They also demonstrated that nestin immunoreactivity was significantly increased in malignant melanomas, and that the precise levels of nestin correlated with the clinical stage of the tumor. Moreover, immunohistochemical analysis demonstrated nestin-positive cells in 35 of 42 (83.3%) nodular melanomas, 10 of 32 (31.3%) superficial spreading melanomas, 10 of 12 (83.3%) metastatic melanomas, 2 of 10 (20.0%) dysplastic nevi and 20 of 43 (46.5%) nevomelanocytic nevi [12]. Thus, the expression levels of nestin significantly correlate with the aggressiveness of malignant melanoma [13]. In addition, a significantly greater percentage of CD166-, CD133- and nestin-positive tumor cells were identified in malignant melanomas compared to nevomelanocytic nevi, while all cases of metastatic melanoma expressed at least one stem cell marker. However, statistical significance for nestin expression was only detected between the primary and metastatic melanomas [13]. Moreover, nestin expression has not been observed in HMB-45-negative melanotic and amelanotic malignant melanomas [12].As the precise diagnosis of HMB-45-negative malignant melanoma is clinically most important, we investigated the relationship between nestin expression and melanoma class in the present study. We found that nestin was a useful marker for the diagnosis of HMB-45-negative malignant melanoma. Tumor cells in epidermal lesions that also expressed melanin failed to express nestin. This result correlated with previous studies demonstrating that tumor cells proliferating into the dermis, especially within the invasive front, lacked melanin and expressed nestin [12]. Xu et al. evaluated the reactivity of a panel of antibodies against markers associated with melanoma and melanocytic differentiation in HMB-45-negative, non-desmoplastic melanomas. They concluded that melanoma antigen 1 (MAGE-1), melanocyte-specific transcription factor (MITF), tyrosinase and Melan-A served as useful markers for the diagnosis of malignant melanotic lesions when HMB-45 is not present [14]. In contrast, we reported the presence of Melan-A in only 6 out of 10 cases of dermal lesions in nodular malignant melanoma, and only 2 out of 5 cases of dermal lesions of amelanotic malignant melanoma. In addition, MITF immunoreactivity was only observed in 5 out of 10 cases of dermal lesions of nodular malignant melanoma and 4 out of 5 cases of dermal lesions of amelanotic malignant melanoma, while MAGE-1 immunoreactivity was only observed in 1 case of dermal lesion, 10 cases of nodular malignant melanoma and 5 cases of amelanotic malignant melanoma. Thus, nestin may be a useful marker of HMB-45-negative melanoma cells in dermal lesions of melanotic and amelanotic nodular malignant melanomas [12]. Recently, Flammiger et al. described that the intermediate filament protein and stem cell marker nestin, as well as the lineage restricted transcription factors BRN2, SOX9, and SOX10, are expressed in melanoma cell lines of all progression stages as well as in melanoma tissues, and that SOX9 and SOX10 but not BRN2 can alter nestin expression in melanoma [15]. And Bakos et al. demonstrated that nestin and SOX9 expression are increased, respectively, in ulcerated melanomas and advanced-stage melanoma, and may be markers of tumor aggressiveness [16]. In addition, Yang et al. suggested that the expression of nestin may play an important role in the development of some neoplasms, such as GIST and angiosarcoma [17]. In the present study, we investigated the relationship between nestin expression and patient outcome in malignant melanomas. We found that nestin, HMB- 45 and S100 protein were detected in 56.5%, 88.4% and 100% of malignant melanomas, respectively. The 5-year survival rates of stages I and II nestin-positive cases was significantly decreased compared to the nestin-negative cases (p < 0.05). In addition, the 5-year survival rates were 100% in nestin-negative malignant melanomas at all stages of tumor development. We conclude that nestin expression may be a predictor of poor prognosis in patients with malignant melanoma. AcknowledgementsThis work was partially supported by Grants-in-Aid for the All Kitasato Project Study 2007 and 2008, and the Parents’ Association Grand of Kitasato University, School of Medicine. We are also grateful to Ms Masako Ishii for providing excellent technical assistance. Conflict of interest: none.References1 Hoffman RM. The hair follicle as a gene therapy target. Nat Biotechnol 2000; 18: 20-1.2 Oshima H, Rochat A, Kedzia C, Kobayashi K, Barrandon Y. Morphogenesis and Renewal of Hair Follicles from Adult Multipotent Stem Cells. Cell 2001; 104: 233-45. 3 Cotsarelis G, Sun T-T, Lavker RM. Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 1990; 61: 1329-37. 4 Taylor G, Lehrer MS, Jensen PJ, Sun TT, Lavker RM. Involvement of Follicular Stem Cells in Forming Not Only the Follicle but Also the Epidermis. Cell 2000; 102: 451-61. 5 Li L, Mignone J, Yang M, et al. Nestin expression in hair follicle sheath progenitor cells. Proc Natl Acad Sci USA 2003; 100: 9958-61. 6 Amoh Y, Li L, Yang M, et al. Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells. Proc Natl Acad Sci USA 2004; 101: 13291-5. 7 Amoh Y, Li L, Yang M, et al. Multipotent nestin-positive, keratin-negative hair-follicle bulge stem cells can form neurons. Proc Natl Acad Sci USA 2005; 102: 5530-4. 8 Amoh Y, Li L, Campillo R, et al. Implanted hair follicle stem cells form Schwann cells which support repair of severed peripheral nerves. Proc Natl Acad Sci USA 2005; 102: 17734-8. 9 Amoh Y, Li L, Katsuoka K, Hoffman RM. Chemotherapy targets the hair-follicle vascular network but not the stem cells. J Invest Dermatol 2007; 127: 11-5. 10 Levent Y, Mehmet K, Oguz A, Bedri K. Fascin expression in melanocytic lesions of the skin. Eur J Dermatol 2009; 19: 445-50. 11 Brychtova S, Fiuraskova M, Hlobilková A, Brychta T, Hirnak J. Nestin expression in cutaneous melanomas and melanocytic nevi. J Cutan Pathol 2007; 34: 370-5. 12 Kanoh M, Amoh Y, Tanabe K, Maejima H, Takasu H, Katsuoka K. Nestin is expressed in HMB-45 negative melanoma cells in dermal lesion of nodular malignant melanoma. Eur J Derm 2008; 18: 518-23. 13 Klein WM, Wu BP, Zhao S, Wu H, Klein-Szanto AJ, Tahan SR. Increased expression of stem cell markers in malignant melanoma. Mod Pathol 2007; 20: 102-7. 14 Xu X, Chu AY, Pasha TL, Elder DE, Zhang PJ. Immunoprofile of MITF, Tyrosinase, Melan-A, and MAGE-1 in HMB-45 negative Melanomas. Am J Surg Pathol 2002; 26: 82-7. 15 Flammiger A, Besch R, Cook AL, Maier T, Sturm RA, Berking C. SOX9 and SOX10 but Bot BRN2 Are Required for Nestin Expression in Human Melanoma Cells. J Invest Dermatol 2009; 129: 945-53. 16 Bakos RM, Maier T, Besch R, Mestel DS, Ruzicka T, Sturm RA, Berking C. Sturm and Carola Berking. Nestin and SOX9 and SOX10 transcription factors are coexpressed in melanoma. Exp Dermatol 2009 Oct 21 (Epub ahead of print). 17 Yang XH, Wu QL, Yu XB, et al. Nestin expression and malignant grade for different tumors. J Clin Pathol 2008; 61: 467-73. |
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