Immunohistochemical Expression of CCND1 and p16 Genes in Tissue Microarray a Sima Ataollahi Eshkoor, a Patimah Ismail, b Sabariah Abdul Rahman, a Mirsaed Mirinargesi c Soraya Ataollahi Oshkour a Department of Biomedical, b Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400UPM, Serdang, Selangor, Malaysia, c Shafa st. Hasan Makiabadi clinic, Sirjan, Iran. ABSTRACT in the skin tissue. It is a reliable method, which assesses a large number of samples in a research setting. This study illustrated Background and objectives: Immunohistochemistry using a significant protein expression of CCND1 and p16 genes in Tissue Microarrays (IHC-TMA) is a useful method to identify TMA samples of 25 patients of BCC compared to normal skin the alteration of CCND1 and p16 genes. This study determined tissue (p<0.05). The findings of this study demonstrated the the sensitivity of this analysis to find the protein over-expression over-expression of CCND1 and p16 proteins in BCC tissue of CCND1 and p16 in Basal Cell Carcinoma (BCC). samples using IHC-TMA method as compared to normal human skin tissue. Method: Twenty-five spot samples obtained from different patients who were diagnosed with BCC and four spot samples Interpretation and Conclusion: Alteration of both CCND1 of normal skin tissue. The slides were assessed by IHC-TMA and p16 genes could lead to the abnormal prolifration activity technique. in the cells and resulting in BCC. Results: The study revealed this technique is a feasible and INTRODUCTION efficient method to diagnose the minute numbers of BCC cells Tissue microarray (TMA) is a method to exhibit tissues from multiple blocks in a single histological section. It allows examination of the expression of several molecular markers such as protein, mRNA, DNA for improving the discovery process and accelerating its efficiency.1-4 A large number of tissue samples are accommodated in a single conventional Key words: Basal cell carcinoma (BCC), CCND1, paraffin block without significant damage to the donor Immunohistochemistry (IHC), p16, skin cancer, Tissue block. This method allows evaluation of antibody Microarray (TMA) performance in the IHC analysis.5 Correspondence to: Prof. Dr. Patimah Ismail PhD, Department of Biomedical, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia 43400 UPM, Serdang, Selangor, Malaysia. 103 Tel: +60389472314, E-mail: [email protected] Patimah Ismail This method permits application of high-dimensional humans over-expression of CCND1 is seen in many tumours biology techniques in the pathology and translational such as endometrial,30 thyroid,31 urothelial bladder,32 researches. The TMA improved efficient use of material, breast,33 brain gliomas,34 esophageal35 and colorectal personnel, and reagents.3, 4 One of the main advantages of cancers36 and also Squemous Cell Carcinoma (SCC).35 the TMA is the histological studies such as IHC, fluorescence in situ hybridization, chromogenic in situ p16 protein plays a key role in cell cycle control at the G1- hybridization and RNA in situ hybridization with standard S transition.37 p16 gene is one of the most altered genes protocols.1, 3-4, 6-8 It has been widely used in cancer research observed in different human neoplasms.38, 39, 40, 41 It is identifying molecular biomarkers with diagnostic and believed to encode a negative regulatory protein which prognostic values.1, 9-12 The TMA and the conventional prevents cell cycle progression from G1 to S phase by whole-section tissue have same validity for histology and inhibiting the CDK4 or CDK6/cyclin D complex and IHC approaches.9, 10, 13-18 The TMAs containing two to subsequently Rb protein phosphorylation.38, 42 It reveals the four tissue cores provide a degree of concordance ranging p16 gene may play a critical role in the pathogenesis of from 95% to 97% in immunoreactivity.10, 13, 14, 17, 18 Therefore, neoplasms.43 it is a tool for biomarker discovery.18 Pathologic examination of the BCC suggests important The BCC is the most common type of skin information about the potential mechanisms of disease cancer.19, 20, 21, 22 The incidence of the BCC in the white associated with the genes. Thus far, TMAs have not been populations, who are at higher risk, is between 18% and used for the systematic studies of the BCC and for biomarker 40% with higher occurrence in men. Before the age of 20 discovery in research study. The point of this study was to years the BCC is rarely seen but the incidence of it grows assess the feasibility and efficiency of TMAs with BCC up with increased age. The BCCs are slow growing tissue using IHC analysis in the finding of the role of malignancies but without the treatment can become CCND1 and p16 genes alterations in the formation of BCC. dangerous. The metastatic rate ranges from 0.0028% to 0.55%.23 MATERIAL AND METHODS The principal cause of BCC is chronic sun exposure over Twenty-five spot samples of formalin-fixed, paraffin a number of years as the result of chronic sunlight exposure embedded AccuMax® TMA slides obtained from twenty- as a child. Mostly the patients with BCC are over 40 years five different patients from Korea (CAT#: A216; LOT#: old24 and the greatest incidence is in people older than 55 122120310121, Location: 73 and 74) who were diagnosed years.25 with BCC and four spot samples of normal skin tissue. Each size of single spot is 1.0 mm in diameter. IHC studies were BCCs occur most frequently on parts of the body that are performed on the slides using commercially available exposed to the sun, such as the face, ears, neck, scalp, monoclonal anti- CCND1 antibody obtained from Research shoulders, and back. Some people whose occupations Biolabs (Danvers, USA) and anti-p16 polyclonal antibody require a significant amount of time outdoors or spend as named anti-p16INK4a antibody from Lab vision extensive leisure time in the sun are at higher risk of taking (Fremont, CA, USA). the BCC of skin.26 Immunohistochemical staining was conducted using CCND1 (Cyclin D1)/(CDK)4-6 complexes initiate the DAKO Envision TM system + HRP DAB + Rb / Mo Kit phosphorylation of pRb and cyclin E/CDK2 complex (DAKO Co., Carpinteria, CA, USA) according to the completes the procedure in late G1 phase. Alterations in manufacturer’s instructions. Briefly, the slides were Cyclin and Cyclin-Dependent Kinase (CDK) expression dewaxed by heating on hot plate at 600C and then result in increased cell proliferation and contribute to deparaffinised. The slides were heated in a microwave oven malignancy.27 CCND1 protein is a member of cell-cycle- for 20 min in 10 mM citrate-Na (pH 6.0). After incubation associated nuclear proteins and is encoded by the CCND1 with dual endogenous blocking enzyme for 10 min, sections gene. CCND1 gene induces G1 to S-phase transition, were incubated with primary antibodies described above promotes cell proliferation and plays a major role in for over night at 40C with an antibody dilution of 1: 400 for oncogenesis.28 anti-p16 antibody and 1: 300 for anti-CCND1 antibody. After washing by TBS the slides incubated with polymer CCND1 gene is disrupted in the cancer cell genome in the kit for 30 min and for labeling incubated with usually by the process of gene amplification or chromosome DAB+substrate buffer in the kit for 10 min. After translocation what may lead to cancer development.29 In counterstaining with hematoxylin and mounting the slides Austral - Asian Journal of Cancer ISSN-0972-2556, Vol. 8, No. 2, April 2009 pp 103-108 104 Immunohistochemistry analysis of CCND1 and p16 genes were ready to view by light microscope (BX 51, New York, p=0.032). USA). Depending on the gene the staining reaction in IHC is SCORING SYSTEM seen in the nucleus, cytoplasm or both. Protein expression of CCND1 and p16 in the BCC is higher than normal skin IHC interpretation is qualitative and semi-quantitative tissue. CCND1 (Fig1) and p16 (Fig 3) protein are visible in study which minimized the many known inconsistencies normal skin tissue. Figure 2 shows the protein expression of among laboratories with regard to reagents and methods. CCND1 and figure 4 expresses the p16 protein expression This assay may improve intra and inter-laboratories in the malignant tissue. The brown staining in the involved standardization and reproducibility of the study.44 cells demonstrated the protein expression of the genes. The number of stained cells in the malignant tissue for both of As the strength of reaction was variable we graded the these genes is more than that noted in the normal skin intensity of reaction on a numerical scale from + to +++, tissue. reflecting weak, moderate, and strong reactions. We also recorded the extent of reactivity within target cell population on 1 to 4 scales indicating convincingly positive nucleus reaction in 1-25, 26-50, 51- 75, and 76-100% of the cells. The evaluation of IHC staining45, 46, 47 on the slides was based on signal intensity which was carried out using scoring system. The total scoring for every single spot in the slides counted based on the score for percentage of positive cells and staining intensity. Statistical Analysis The data of IHC was stored and analyzed by means of SPSS.12 version software (SPSS Inc, Chicago, IL, USA). The protein expression data of CCND1 and p16 genes were evaluated non-parametrically using Mann-Whitney test. Tests were considered significant when their p value was less than 0.05. Fig 1: Immunostaining analysis of CCND1 in normal skin tissue illustrates the mild staining intensity (+1) in nuclei of normal epidermal cells (arrow). (Magnification 40X) RESULTS Site of reaction product Twenty-five paraffin embedded tissue samples obtained from the patients diagnosed with BCC.