Immunohistochemical Changes in Acquired Melanocytic Nevi Following Narrow Band Ultraviolet-B Therapy

Transcription

1 Med. J. Cairo Univ., Vol. 84, No. 2, March: , Immunohistochemical Changes in Acquired Melanocytic Nevi Following Narrow Band Ultraviolet-B Therapy HESHAM ZAHER, M.D.*; SAFINAZ SAYED, M.D.**; DALIA BASSIOUNY, M.D.*; RANIA ABD EL-HAY, M.D*; NESREEN SAMIR, M.D.* and NANEES RAGAB, M.Sc.* The Departments of Dermatology* and Histology**, Faculty of Medicine, Cairo University 373 Studies of the risk of melanoma following UV therapy revealed controversial results [2]. Distinction between benign and malignant melanocytic lesions may be difficult even for highly skilled dermatopathologists emphasizing the importance of advanced diagnostic tools such as immunohistochemistry [3]. Ki67 which is a nuclear protein and a cellular marker of proliferation is the most important marker in distinguishing benign from malignant melanocytic tumors [4]. MART 1 (melanoma antigen recognized by T cells 1) also known as Melan A is a protein antigen found on the surface of melanocytes. It is another marker highly specific for melanoma. Ki67/MART 1 stains are valuable diagnostic tools to distinguish melanomas and nevi with a large degree of certainty [5]. Aim of the study: To detect the immunohistochemical changes in acquired melanocytic nevi following average therapeutic doses of narrow band UVB therapy in our Phototherapy Department. To detect whether narrow band UVB therapy increases the potential risk of melanoma. Patients and Methods This analytical, case control study was conducted in the Phototherapy Unit of the Dermatology Department in Cairo University. 40 patients with different dermatological conditions in which NB- UVB was planned for treatment were included in the study. The study was approved by the research ethical committee of the Dermatology Department, Cairo University from July 2012 April Each patient signed a written informed consent before participation in the study.

2 374 Immunohistochemical Changes in AMN Following Narrow Band Ultraviolet-B Therapy Inclusion criteria: For each patient at least 2 melanocytic nevi were selected in non sun exposed area and located on healthy skin. All lesions had both benign clinical and dermoscopic features. Exclusion criteria: Age <18 years, family history of melanoma or other skin cancers, photosensitivity, atypical mole syndrome and history of recieving phototherapy within a period of 6 months before participation in the study. Irradiation protocol: A Waldmann W UV 100L cabinet with 40 narrow band UVB fluorescent tubes installed (Philips TL 100 W/01, Philips Company, Eindhoven, The Netherlands) and an emission spectrum of nm was used. NB-UVB was given thrice weekly on non consecutive days. Suberythemogenic doses were used. The initial dose was 0.3J/cm 2 in cases of vitiligo, 0.5J/cm 2 in cases of psoriasis and 0.7J/cm 2 in cases of mycosis fungoids and parapsoriasis with increments of 0.3J/cm 2 every other dose, until very faint erythema occurred up to a maximum of 5J/cm 2. Eyes and genitilia were covered during treatment. One nevus was surgically removed for each patient by a suitable sized punch prior to the sessions as a control. The other nevus from the same patient was removed after the irradiation cycle (30 sessions or clearance of disease). Histological and immunohistochemical analysis: Specimens were fixed in formol saline 10% and processed to obtain paraffin blocks. Paraffin sections 5-7µm thick were cut and stained by: 1- Hematoxylin and eosin: For histopathological confirmation of the clinical diagnosis. 2- Immunohistochemical staining for: A- MART- 1/Melan-A (Melanoma Marker). B- Ki67 a marker for proliferating cells. Immunohistochemical staining: MART- 1/Melan-A primary was a mouse monoclonal antibody Cat. #MS-799-R7. Ki67 was a rabbit polyclonal antibody Cat. #RB R7. Staining of formalin-fixed tissues requires boiling tissue sections in 10mM citrate buffer, ph 6.0, (NEOMARKERS' Cat.#AP-9003), for minutes followed by cooling at RT for 20 minutes. Immunostaining was completed by the use of Ul- travision Detection System Anti-Polyvalent, HRP/AEC (Cat.#TP-015- HA). Counterstaining was done using Mayer's hematoxylin catalogue number (TA-060-MH). MART- 1 and Ki67 primary antibodies, ultravision detection system and Mayer's hematoxylin were purchased from lab vision coporation Vermont, CA, USA and Thermo Fischer scientific, Cheshire, UK. Positive immunostaining for MART-1/Melan- A appeared as red cytoplasmic deposits while positivity for Ki67 appeared as red nuclear reaction. Morphometric study: Counting the number of cells showing positive immunostaining for ki67 in the epidermis and dermis of all specimens from all subjects of the study. This was done in five non overlapping fields at X 400 magnification in a field area of µm 2 for every subject. Measuring the area percent of Melan-A positive immune staining in the epidermis and dermis of all specimens from all subjects of the study. This was done in five non overlapping fields at X 400 magnification in a field area of µm 2 for every subject using Leica Qwin 500C image analyzer computer system (England) present in Histology Department, Faculty of Medicine, Cairo University. Images were captured live on the screen from sections under a light microscope (Olympus BX- 40, Olympus Optical Co. Ltd., Japan) with affixed video camera (Panasonic Color CCTV camera, Matsushita Communication Industrial Co. Ltd., Japan). The video images were digitized using Lecia Qwin which is a Lecia's windows based image analysis tool kit fitted to an IBM compatible personal computer with a color monitor. The positive immunoreactivity for Melan A appeared as red deposits. Binary images were generated by color thresholding for the red color then the area of these binaries is measured by the Lecia Qwin 500 software. This is done for every field of the five fields for each subject to obtain a mean area for each. Mean area percent is the relation between the areas of positivity marked by the binary images to the field area which is µm 2. Results obtained were subjected for statistical analysis. Statistical methods: Data were statistically described in terms of mean ± standard deviation (±SD), median and

3 Hesham Zaher, et al. 375 range, or frequencies (number of cases) and percentages when appropriate. Comparison of numerical variables between the study groups was done using Mann Whitney U-test for independent samples. For comparing more than 2 groups Kruskal Wallis test was used. Within group comparison between pre-and post-treatment values was done using paired t-test for paired (matched) samples. For comparing categorical data, Chi square ( χ 2 ) test was performed. Exact test was used instead when the expected frequency is less than 5. Correlation between various variables was done using Pearson moment correlation equation for linear relation in normally distributed variables and Spearman rank correlation equation for non-normal variables/non-linear monotonic relation. p-values less than 0.05 was considered statistically significant. All statistical calculations were done using computer program SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) release 15 for Microsoft Windows (2006). Results Clinical data and cumulative dose: This analytical case control study included 40 patients with clinically acquired melanocytic nevi. However 10 patients dropped out due to personal circumstances. The enrolled patients were 19 (63%) females and 11 (36%) males. As regards the clinical diagnosis 3 (10%) were diagnosed with Mycosis Fungoids, 2 (6%) with Parapsoriasis, 16 (53%) with vitiligo and 9 (30%) with Psoriasis. Age of patients ranged from 18 to 62 years with a mean age of 36.42± As regards skin type, 22 (73%) patients were skin type IV and 8 (26%) patients were skin type III. All patients received NB-UVB sessions as a treatment modality for their skin conditions with a cumulative dose of NB- UVB that ranged from 15 joules to 55.8 joules; with mean value 30.33±10.24 joules. Ki 67 and MA immunohistochemical changes after NB-UVB sessions: The mean value of Ki67 has increased as well as the mean value of MA but it was not statistically significant. Mean Ki67 change was 0.13± 1.42 ( p= 0.621). Mean MA change was 0.29±1.68 (p=0.350) [(Table 1) & Fig. (1)]. No statistically significant association was found between different skin phototypes and immunohistochemical changes in nevi following NB-UVB therapy (p=0.707). No statistically significant association was found between immunohistochemical changes in nevi and the site of nevus (p=0.771). No statistically significant association was found between immunohistochemical changes in nevi and the total cumulative dose received (p=0.871) Before NB-UVB After NB-UVB Ki67 (Mean count) MA (Mean area %) Fig. (1): Ki67 and Melan A expression before and after NB- UVB. Table (1): Ki 67 and MA immunohistochemical expression before and after NB-UVB sessions. Range Mean±SD Range Mean±SD p- before before after after value Ki67 mean count ± ± MA mean area % ± ± Discussion In this study repeated irradiation of AMN with suberythemogenic doses of NB-UVB showed an increase in Ki67 (marker of proliferation) as well as Melan A (marker of melanogenesis) expression but this increase was not statistically significant and this agreed with Managoni et al., (2011) who did not demonstrate a statistically significant vari- ation of Ki67 and Melan A expression among irradiated and non irradiated nevi after repeated suberythemogenic doses of NB-UVB and Tronnier et al., (1997) who didn't detect a statistically significant increase in Ki67 expression in AMN irradiated with suberythemogenic doses of UVB. On the other hand, previous investigations in which AMN were exposed to a single erythemo-

4 376 Immunohistochemical Changes in AMN Following Narrow Band Ultraviolet-B Therapy genic UVB exposure showed a statistically significant increase in melanogenesis related proteins including Melan A [6] and a statistically significant increase in Ki67 expression suggesting melanocyte proliferation [5-7]. This disagreement could be attributed to the irradiation protocol in our study using repeated suberythemogenic doses of NB-UVB. Suberythemogenic doses probably increase the melanin content or cause redistribution of the melanin within nevi while erythemogenic doses increase the proliferative capacity of nevi. Morphological changes and enhanced proliferative and reparative activity in melanocytes were much more frequent in nevi irradiated with a single erythemogenic UV dose than in those given repeated suberythemogenic doses [5]. In addition, keratinocytes showed an increased labelling for PCNA and p53 after a single erythemogenic irradiation. These data might support the safety of suberythemogenic phototherapy [5], and reinforce the importance of sunburns in the development of both benign and malignant melanocytic lesions [8]. This was supported by our study which did not demonstrate any high Ki67 expression following repeated suberythemogenic exposures. Conclusion: Suberythemogenic doses of NB-UVB did not induce a statistically significant change in Ki67 (proliferation marker) and Melan A (melanogenesis related protein). These findings support the safety of suberythemogenic NB-UVB phototherapy and reinforce the importance of sunburns in the development of both benign and malignant melanocytic lesions. Future studies utilizing new in vivo imaging methods such as confocal laser scanning microscope and multiphoton laser scanning tomography could be used to detect subcellular skin structures and find cytological and subcellular changes in UV-exposed nevi. Further studies comparing erythemogenic versus non erythemogenic doses of NB-UVB could con- firm the safety of suberythemogenic doses. The safety of suberythemogenic doses should be further investigated by studies on larger cumulative doses and larger number of patients. References 1- MANGANONI A.M., ROSSI M.T., SALA R., VENTURI- NI M., SERENI E., UNGARI M., MAROCOLO D., LONARDI S. and CALZAVARA-PINTON P.: Dermoscopic, histological and immunohistochemical evaluation of cancerous features in acquired melanocytic nevi that have been repeatedly exposed to UVA or UVB. Experimental Dermatology, 21: 86-90, ARCHIER E., DEVAUX S., CASTELA E., GALLINI A., AUBIN F., Le MAÎTRE M., ARACTINGI S., BACHELEZ H., CRIBIER B., JOLY P., JULLIEN D., MISERY L., PAUL C., ORTONNE J.P. and RICHARD M.A.: Carcinogenic risks of psoralen UV-A therapy and narrowband UV-B therapy in chronic plaque psoriasis: A systematic literature review. J. Eur. Acad. Dermatol. Venereol., 3: 22-31, NIELSEN P.S., RIBER-HANSEN R. and STEINICHE T.: Immunohistochemical double stains against Ki67/ MART1 and HMB45/MITF: Promising diagnostic tools in melanocytic lesions. Am. J. Dermatopathol., 33: , OHSIE S.J., SARANTOPOULOS G.P., COCHRAN A.J. and BINDER S.W.: Immunohistochemical characteristics of melanoma. J. Cut. Pathol., 35: , TRONNIER M., RUDOLPH P., KOSER T., RAASCH B. and BRINCKMANN J.: One single erythemagenic UV irradiation is more effective in increasing the proliferative activity of melanocytes in melanocytic naevi compared with fractionally applied high doses. Br. J. Dermatol., 137: 534-9, CARRERA C., PUIG S., LLAMBRICH A., PALOU J., LECHA M., MASSI D. and MALVEHY J.: Development of a Human in vivo Method to Study the Effect of Ultraviolet Radiation and Sunscreens in Melanocytic Nevi. Dermatology, 217: , RUDOLPH P., TRONNIER M. and MENZEL R.: Enhanced expression of Ki-67, topoisomerase IIalpha, PCNA, p53 and p21 WAF 1/Cip 1 reflecting proliferation and repair activity in UV-irradiated melanocytic nevi. Hum. Pathol., 29: , LIN C.Y., OAKLEY A., RADEMAKER M., HILL S. and YUNG A.: Effect of narrow band ultraviolet-b phototherapy on melanocytic nevi. Br. J. Dermatol., 168: 815-9, 2013.

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