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On-line version ISSN 1806-4841
An. Bras. Dermatol. vol.84 no.5 Rio de Janeiro Sept./Oct. 2009
Marilho Tadeu DornelasI; Michele Fernandes RodriguesII; Dequitier Carvalho MachadoIII; Ângela Maria GollnerIV; Ana Paula FerreiraV
IFull Member, Brazilian Society of
Plastic Surgery; Professor, Discipline of Specialized Surgery, Medical School,
Universidade Federal de Juiz de Fora. Master in Brazilian Public Health, focused
on Immunology, Medical School Universidade Federal de Juiz de Fora (FM/UFJF)
Juiz de Fora (MG), Brazil
IIBiologist, Master in Brazilian Health, focused on Immunology, Medical School, Universidade Federal de Juiz de Fora (FM/UFJF) Juiz de Fora (MG), Brazil
IIIUndergraduate, Medical School, Universidade Federal de Juiz de Fora. Intern, Service of Plastic Surgery, University Hospital, Universidade Federal de Juiz de Fora (HU/UFJF) Juiz de Fora (MG), Brazil
IVStudy Coordinator. Ph.D. in Pathology, Universidade Federal Fluminense. Associate Professor, Department of Pathology, Medical School, Universidade Federal de Juiz de Fora (FM/UFJF) Juiz de Fora (MG), Brazil
VStudy Coordinator. Ph.D. in Immunology, Universidade de São Paulo (USP), Associate Professor, Department of Parasitology, Microbiology and Immunology, Universidade Federal de Juiz de Fora (UFJF) Juiz de Fora (MG), Brazil
Skin cancer is the most frequent type of human cancer and has shown an increase
in its incidence. In many cases, before the onset of the carcinoma, there might
be a precursor lesion - actinic keratosis, which can develop into squamous cell
carcinoma. Studies have been carried out in order to determine the parameters
that have prognostic significance in predicting those tumors which have more
OBJECTIVE: To evaluate the expression of markers of cell proliferation (PCNA, Ki-67) and apoptosis (p53,Bcl-2) in patients with squamous cell carcinoma and actinic keratosis.
METHOD: We studied samples from 30 patients, ten patients of squamous cell carcinoma, ten with actinic keratosis and ten lesion-free samples from blepharoplasty.
RESULTS: p53 protein was expressed in all cases with different quantitative patterns. Bcl-2 was expressed at low intensity in six cases of actinic keratosis in the skin from blepharoplasty and negative in cases of squamous cell carcinoma. PCNA showed intense expression in all samples. Ki-67 showed variable expression in cases of keratosis and carcinoma and negative in the skin from the eyelid.
CONCLUSION: The high expression of Ki-67 associated with low expression of Bcl-2 indicates proliferation in the carcinoma group. Thus, expression of p53 and Bcl-2 in patients with actinic keratosis indicates cell immortalization.
Keywords: Apoptosis; Carcinoma, squamous cell; Cell proliferation; Immunohistochemistry; Keratosis
Skin cancer is the most frequent type of human cancer and it has shown increase in recent years, which transforms it into a growing problem to world public health 1. The number of new cases of skin cancer estimated to Brazil in 2008 was 55,890 in men and 59,120 in women, with estimated risk of 62 new cases for each 100,000 men and 60 to each 100,000 women 2.
Squamous cell carcinoma (SCC) represents 20% of cutaneous malignant neoplasms. It is originated from the proliferation of atypical squamous cells, with invasive characteristics and producing metastases. In general, primary skin SCC originate from regions exposed to sun and may arise from non-invasive lesions, such as actinic keratosis (AK), actinic keilitis, oral leukoplasia, and chronic radiodermitis 3.
Molecular and epidemiological data suggest the existence of a close association between excessive exposure to ultraviolet radiation (UV) from the sun and development of non-melanoma skin cancer. UV radiation causes a specific damage to DNA, with mutations of suppressor gene p53, in people with squamous cell carcinoma that developed in areas exposed to the sun 4. The decrease in p53 function (control of cell cycle and apoptosis) may lead to uncontrolled cell proliferation, accumulation of mutated DNA and finally, cancer 5,6.
The expression of protein p53, apoptosis marker, and KI-67, cell proliferation marker, was studied in skin neoplastic and non-neoplastic disease and the results suggested that the expression rate of p53 and Ki-67 reflected the level of malignancy of the examined skin neoplasms 7,8. Therefore, the study by Balassiano was carried out to validate the expression of markers Bcl-2, p53, mutated p53, caspase-3 and Ki-67 as prognostic factors in inflammatory fibrous hyperplasia, actinic keilitis and lower lip squamous cell carcinoma. The author found higher expression of p53 and mutated p53 in SCC, high expression of Ki-67 in all lesions and increasing expression of Bcl-2 in relation to lesion severity 9.
Considering the increase in number of new cases and frequent recurrences, studies have been intensified to determine the parameters with prognostic meaning in predicting those tumors that will have a more aggressive behavior, which may suffer recurrence and progress with metastases 3.
Therefore, the purpose of the present study was to assess the expression of apoptosis markers (p53, Bcl-e) and cell proliferation (PCNA, KI-67) in patients with SCC and AK using immunohistochemistry technique and to compare the results obtained between these groups and those represented by healthy subjects undergoing blepharoplasty (eye lid surgery). Moreover, we intended to check whether there was correlation between apoptosis and cell proliferation markers in cases of AK, SCC and in those free from the disease.
MATERIAL AND METHOD
We studied samples from 30 patients undergoing reconstructive surgery between August 2000 and July 2008. Among them, ten had histopathological diagnosis of actinic keratosis, ten had squamous cell carcinoma and ten were skin samples from the eyelid collected in the blepharoplasty.
The study was approved by the Research Ethics Committee of UFJF.
The expression of markers p53, PCNA, Ki-67 and BCl-2 (Chart 1) was assessed in histological sections from skin lesion, using immunohistochemistry technique with streptoavidine-biotine peroxidase method. For markers of PNCA, KI-67 and Bcl-2, we used sections of palatine tonsil as positive controls and for p53, we used sections of positive breast carcinoma.
The immunohistochemical profile of the lesions was considered as positive when the cells had brownish color only at the nucleus for markers p53, PCNA and Ki-67. For Bcl-2, we considered cytoplasmatic marking as being positive.
We decided for the descriptive analysis of the expression of all markers, using the criteria of crosses proposed by Sannino and Shousha 10, which takes into account staining intensity and number of stained cells. Staining intensity was classified as mild, moderate and strong. Concerning the number of stained cells, cases were considered negative when there were no stained cells; positive, marked as one cross (+) when there was occasional presence of positive cell, two crosses (++) in cases with up to 1/3 of moderate intensity stained cells, three crosses (+++) in those in which 2/3 were stained from moderate to strong intensity or moderate intensity in over 2/3, and four crosses (++++) in those that had strong intensity staining in more than 2/3 of the cells. The quantification of the expression of markers was made by observing the three microscopic fields under 100x magnification.
Non-parametric tests were used to analyze quantified variables of the system of crosses. To test the difference in mean values of the same marker among the diagnoses, we used Mann-Whitney test. To check the difference in mean values among all markers of the same diagnosis, we applied Friedman test and then for each pair of markers, we used Wilcoxon test.
The distribution of studied cases concerning gender, age, location, dimension, histopathological diagnosis and expression of immunohistochemical markers of the lesions is shown in Table 1.
Concerning gender, there was a greater trend of occurrence of keratosis among women and SCC among men, but there was no statistically significant difference (p value given by Fisher exact test = 0.378).
The expression of p53 was shown in all cases of AK, with strong intensity (4+) in five cases and 3+ in four cases. Only one case had 2+. Concerning SCC, four cases had 4+ and four cases had 2+, and only two cases had 1+.
The samples of SCC patients did not express marker Bcl-2 and in six samples of AK the expression was weak, ranging from 1 to 2+ and, in the others, it was negative.
In 19 samples, cell proliferation marker PCNA was expressed with great intensity (3 to 4+). Only one case had weak marker level (2+).
Concerning marker Ki-67 (cell proliferation), there is great variability in intensity of marking. Two AK samples (cases 6 and 9) and two of SCC (cases 12 and 17) did not express Ki67. Chart 1 shows the mean value observed for each marker in relation to the diagnosis. The highest values were observed for PCMA (3, 7) for both diagnoses. Next, there is marker p53, with mean values of 3. 4 and 2.6 for diagnoses of AK and SCC, respectively. Markers of lower score were: Ki-67 (1.6 to AK and 1.7 to SCC) and Bcl-2 (0.8 to AK and 0.0 to SCC).
Figure 1 illustrates immune expression of cell proliferation and apoptosis markers in samples of tissue from case eight, representing the group of AK. Figure 2 represents the immune expression of cell proliferation and apoptosis markers in case 20 of SCC, with 100x magnification. Using 400x magnification, the expression of PCNA and Ki-67 in characteristically malignant cells is shown in Figures 2B and 2D, respectively.
Mann-Whitney test for the difference of mean values of scores between Figure 1 diagnoses was statistically significant for the marker that presented the lowest scores (Bcl-2).
Another analysis with protein pairs Bcl-2 - Ki-67 and p53 - Ki-67 (apoptosis x proliferation) showed that in the group SCC the relation between proliferation (Ki-67) x apoptosis (Bcl-2) was significant, with p< 0.05, whereas in group AK the relation p53 x Ki-67 showed apoptosis marker greater than the proliferation one, with p< 0.05.
The negative control group free from AK and SCC showed that the eyelid samples from the blepharoplasty presented atrophy and elastosis owing to aging and sun exposure, without epithelial atypia. In the immunohistochemical study, there was marked diffuse expression of PCNA (basal and parabasal). Marker Ki-67 was not expressed in the samples. Markers of apoptosis p53 and Bcl-2 showed weak expression (1+), with basal location in some cells.
Skin tumors comprise the most common group of neoplasms in human beings and among them the ones induced or related with excessive exposure to sun ultraviolet radiation have been detected with increasing incidence in the population groups with fair skin that live in tropical and temperate climate areas of the world, even though people are highly compliant with use of sun blocking lotions.
SCC is caused by the atypical proliferation of keratinocytes that stem from the basal layer of the epidermis. Keratinocytes in SCC are larger than those in the normal basal layer and they lose polarity. The nucleus is vesicular or hyperchromatic and the ratio nucleus: cytoplasm is abnormal if compared to normal keratinocytes. There is rupture in the natural sequence of maturation as the lesion becomes advanced, with keratinocyte atypia extending above the basal layer 11.
When keratinocyte atypia involves the whole epidermis the lesion is named in situ squamous cell carcinoma, whereas when atypia is less extensive, more evident on the basal and parabasal regions, the name used is actinic keratosis 11,12.
The diagnosis in histological basis is made by analyzing fragments from skin lesion biopsy.
Even though skin SCC has a relative good prognosis, information such as lesion dimension, level of differentiation, depth of invasion, neural and vascular sheath invasion, and distance from the resected margins are essential 12. In many cases, only histology analysis does not reveal the real behavior of these lesions, which may lead to clinical underestimation concerning management and follow-up 13. It is important that the pathologist can contribute with additional information of prognostic value for the clinician. It is not possible to identify the exact moment in which the neoplasm contained in the epidermis acquires the capacity to invade the basal membrane, get into the dermis and becomes potentially capable of having a metastasis, which can be done only in histological basis.
The expansion of knowledge about molecular genetics and the creation of molecular markers with the use of paraffin sections have enabled different studies to be performed to identify possible oncogenes, suppressor genes and their products with significant prognostic meaning for skin cancer 3,4,7,13.
Therefore, we compared the expression of immune markers involved in the process of carcinogenesis, p53 and Bcl-2 (apoptosis), Ki-67 and PCN (cell proliferation) in subjects with AK and SCC with negative controls represented by skin free from tumor from the upper eyelid in patients undergoing blepharoplasty.
Gene p53, tumor suppressor, is essential to regulate cell proliferation, has critical role in maintaining the integrity of human genome and its inactivation is considered the key event in carcinogenesis 7.
The expression of protein p53 in AK and SCC has varied in different publications. In our study, p53 was expressed in all assessed cases with tendency to show greater intensity in AK. Nagano et al. 4, using polyclonal p53, found 48% positivity in 23 cases of AK and 54% positivity in ten cases of SCC; Kim et al. 5 using monoclonal p53, found 50% positivity in 10 cases of AK and 90% positivity in ten cases of SCC, whereas Park et al. 14 found 50% positivity in ten cases of AK and 100% positivity in 10 cases of SCC. The expression of p53 in pre-cancer lesions such as AK suggests that it may be used as early marker of malignancy, according to Kim et al. 5. It is important to emphasize that it was poorly positive in some cells of healthy subjects in the study and in 39% of normal skins studied by Batinac et al 7.
The overexpression of Bcl-2 protein in a genetically abnormal cell contributes to its clonal expansion because it interrupts apoptosis, leading the cell to immortalization 8.
Differently from the expression of p53, protein Bcl-2 was expressed at low intensity in six cases of AK and negative in cases of SCC. Balassiano8 identified in his study the expression of Bcl-2 in 90% of the 20 cases of lip squamous cell carcinoma and in 75% of the four cases of actinic keilitis with dysplasia, and Tomkova et al. 15 also demonstrated that there was no expression of Bcl-2 in patients with SCC.
Many markers for cell proliferation have been studied to predict the behavior and the progression of pre-neoplastic lesions and cancer.
PCNA, one marker of cell proliferation, was uniformly expressed on the basal layer of all samples.
Monoclonal antibody DVB-2, marker of cell proliferation, recognizes the epitope of antigen Ki-67 in tissues fixed in formol and included in paraffin block 16. Its expression occurred in all stages of tissue cycle, except for stage G0 in which cells are at rest. In the present study, only two cases of AK and two of SCC did not express Ki-67, even though we had observed varied marking intensity.
Despite the promoting effect of proliferative activity, the way through which p53 is related with cell proliferation markers (Ki-67 and PCNA) seems to be independent and variable.
Expression of Ki-67 proved to be important evidence of more marked cell proliferation in SCC. Batinac et al. 7 described a similar pattern of expression of Ki-67 and p53, suggesting an association between cell proliferation and expression of p53.
The expression of Ki-67 and absence of expression of Bcl-2 in SCC group seem to indicate that in SCC there is intensification of proliferative activity. However, concerning AK and greater expression of p53 in relation to Ki-67, it seems to indicate that in this type of lesion there is reduction of apoptosis, facilitating the perpetuation of cells with damage in non-repaired DNA.
Therefore, pre-invasive lesions (AK) overexpress p53 with consequent blocking of apoptosis, resulting from continuous exposure to UV radiation. These findings suggest that Ki-67 rates and expression of p53 and Bcl-2 reflect the grade of malignancy in target skin lesions in the study.
Ruksha et al 17 studied the peripheral benzodiazepam receptor (PBR), PCNA and caspase-3 involved in the synthesis of steroid hormones, regulation of cell proliferation and apoptosis, considered that cells of squamous cell carcinoma and melanoma comprise models for the study of PBR function during the development of UV light-induced skin tumors.
The modulation of cell proliferation and apoptosis induced by ultraviolet light radiation may be a potential therapeutic target in skin malignant tumors.
New studies should be carried out trying to identify other molecular components involved in the development of UV light-induced skin cancer and possible novel therapeutic agents.
Expression of Ki-67 and absence of expression of Bcl-2 in the group of SCC indicate intensification of proliferative activity, whereas greater expression of p53 and Bcl-2 in group AK suggests apoptotic abnormalities and consequence cell immortalization. Marker PCNA did not prove to be effective in differentiating lesions because they had uniform staining in all samples.
The intensity of expression of PCNA, Ki-67, p53 and Bcl-2 associated with other indicators, such as histopathological analysis and clinical presentation, provides information about the likely progression of neoplasm, enabling safer diagnosis. Thus, the adoption of directed therapy provides a likely prognosis concerning the relation with cosmetic-functional outcomes and survival of the patient with neoplasm.
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Marilho Tadeu Dornelas
Rua Dom Viçoso nº20 - Alto dos Passos
36026 390 Juiz de Fora MG
Tel./Fax: 32 3239 8282
Conflict of interest:
Financial Funding: None
How to cite this article: Gelatti LC, Bonamigo RR, Becker AP, dAzevedo PA.
Staphylococcus aureus resistentes à meticilina: disseminação emergente na comunidade. An Bras Dermatol. 2009;84(5):501-6.