Immunohistochemical study of cyclooxygenase-2 and p53 expression in skin tumors

Overexpression of cyclooxygenase-2 (COX-2) has been demonstrated in various cancers, including experimentally promoted tumors, gastrointestinal cancers, breast tumors and skin tumors. The mechanism that controls COX-2 expression is not yet clear. Currently, it is reported that COX-2 expression is frequently associated with mutated p53 genes. The goal of this study was to evaluate the expression patterns of COX-2 and p53 in several skin tumors and their correlation. An immunohistochemical method was used to investigate the expression of COX-2 and p53 proteins on formalin-fixed, paraffin-embedded tissue specimens of squamous cell carcinomas (SCC), basal cell carcinomas (BCC), Bowen's disease (BD), actinic keratosis (AK) and porokeratosis. The expression of COX-2 increased in 50% (5/10) of SCC, 80% (8/10) of BCC, 40% (4/10) of BD, 50% (5/10) of AK, and 20% (2/10) of porokeratosis cases. The expression of p53 increased in 90% (9/10) of SCC, 70% (7/10) of BCC, 70% (7/10) of BD, 50% (5/10) of AK, and 40% (4/10) of porokeratosis cases. COX-2 positivity rates of the p53-positive skin tumors were 56%, 100%, 57%, 80% and 25% in SCC, BCC, BD, AK and porokeratosis, respectively. However, the correlation between p53 and COX-2 expression in skin tumors was not statistically significant ( P  > 0.05). Our results indicate that skin COX-2 and p53 may play roles in skin tumors, but that there is no apparent correlation between the two markers.     

p63 expression in normal skin and usual cutaneous carcinomas

BACKGROUND: p63 is a p53 homologue that is mapped to chromosome 3q27. This gene encodes six different isoforms, which have either transactivating or dominant negative effects on p53-reporter genes. It has been described that in contrast to p53, p63 seems not to be associated with tumor predisposition, as neither p63 knockout mouse models nor germline p63 mutations are related to an increased risk of tumorigenesis. It has been demonstrated that p63 is a reliable keratinocyte stem cell marker and that it is involved in the maintenance of the stem cell population. Scant data on p63 expression in normal skin, basal cell carcinomas (BCCs), keratoacanthomas and squamous cell carcinomas (SCCs) have been reported. We herein evaluated p63 expression in 16 BCCs, one keratoacanthoma and 13 SCCs. METHODS: Immunohistochemistry according to the streptavidin-biotin-peroxidase technique, using the antibody 4A4 raised against all p63 isoforms, was performed. p63 expression was evaluated in epidermal cells and skin appendages. Semi-quantitative evaluation (-, +, ++, +++) of p63 expression in BCCs, keratoacanthoma and SCCs was carried out. Only nuclear expression was considered as specific. RESULTS: p63 was expressed in the nuclei of epidermal basal and suprabasal cells, in the cells of the germinative hair matrix and the external root sheath of hair follicles, in the basal cells of the sebaceous gland and in the myoepithelial/basal cells of the sweat glands. All terminally differentiated cells were negative for p63. All BCCs showed ++ to +++ immunoreactivity. At variance, keratoacanthomas and grade I and II SCCs showed variable p63 reactivity in a basal layer-like distribution, whereas undifferentiated cells of grade III SCCs showed ++ to +++ positivity. A grade IV spindle SCC showed + immunoreactivity. The SCCs in situ showed remarkable expression of p63 in all cell layers. Terminally differentiated squamous cells were either negative or showed only focal immunoreactivity in the carcinomas. CONCLUSIONS: p63 is consistently expressed in the basal cells of epidermis and cutaneous appendages, including the basal/myoepithelial cells of sweat glands. Based on our findings, the balance of probabilities favors that p63 might play a role in the pattern of differentiation and in the oncogenesis of usual carcinomas of the skin.     

Immunophenotypic analysis of the p53 gene in non-melanoma skin cancer and correlation with apoptosis and cell proliferation

BACKGROUND: Sunlight precipitates a series of genetic events that lead to the development of skin cancers such as basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). The p53 tumour suppressor gene, which plays a pivotal role in cell division and apoptosis, is frequently found mutated in sunlight-induced skin tumours. OBJECTIVE: To investigate the immunoreactivity of the p53 gene in non-melanoma skin cancers and to correlate its expression with apoptotic and cell proliferation markers. METHODS: We analysed 35 non-melanoma tumours including 19 BCCs and 16 SCCs from sun-exposed skin areas. p53 protein expression was studied immunohistochemically using the DO7 monoclonal antibody against wild-type and mutant p53 forms. The percentage of p53-immunopositive nuclei was measured by image analysis. Cell proliferation and apoptosis were also assessed by image analysis following Ki-67 immunostaining and application of the TUNEL method on paraffin sections, respectively. RESULTS: The percentage of p53-expressing cells varied from 3.5 to 90 in BCCs (median value 54.4%) and from 3.7 to 94 in SCCs (median value 40.3%). The mean value of Ki-67-positive cells was comparable in both groups of tumours with a mean value of 40.6% in BCCs and 34.6% in SCCs. Conversely, the TUNEL assay showed sporadic staining of apoptotic cells within the tumours with a mean value of 1.12% in BCCs and 1.8% in SCCs. p53 protein expression was correlated positively with cell proliferation (r = 0.75, P = 0.000001) and negatively with apoptosis (r = -0.23, P = 0.05). CONCLUSION: p53 immunoreactivity was high in the majority of the skin carcinomas examined and correlated positively with cell proliferation and negatively with apoptosis. The p53 protein overexpression appears to be related to an inactivated protein resulting from mutations of the p53 gene or other unclear molecular mechanisms.     

生存素和端粒酶逆转录酶在皮肤肿瘤中的表达

目的 研究皮肤肿瘤凋亡抑制基因生存素与人端粒酶逆转录酶(hTERT)的表达及相互关系.方法 免疫组化SP法检测17例鳞状细胞癌(鳞癌)、21例基底细胞癌(基癌)、19例鲍恩病、25例脂溢性角化病和13例正常人皮肤组织中生存素表达水平;原位杂交法检测上述组织中hTERT mRNA的表达水平;分析皮肤肿瘤中生存素与hTERT mRNA表达的相关性.结果 ①在鳞癌、基癌、鲍恩病中生存素及hTERT mRNA表达的阳性率均较正常人皮肤中显著增加;②脂溢性角化病中生存素表达的阳性率与正常人皮肤组织中比较差异有统计学意义,但hTERT mRNA表达的阳性率与正常人皮肤组织中比较差异无统计学意义;③鳞癌、基癌、鲍恩病中生存素阳性率及hTERT mRNA阳性率之间均呈显著正相关,脂溢性角化病中两者阳性率之间无显著相关性.结论 生存素与皮肤肿瘤发病关系密切,在鳞癌、基癌、鲍恩病中生存素与hTERT mRNA的表达具有一定的相关性.     

Overexpression of p2lWaf1/Cip1 immunohistochemical staining in Bowen's disease, but not in disseminated superficial porokeratosis

Disseminated superficial porokeratosis (DSP) consists of multiple small lesions of porokeratosis. Although the pathogenesis of DSP remains unclear, localized cloning of abnormal epidermis has been hypothesized. Malignant cutaneous neoplasms, especially Bowen's disease, have been frequently reported in DSP. Immunopositive p53 has been demonstrated in a variety of human malignant tumours, and its role in oncogenesis and tumour progression is thought to be important. p21Waf1/Cip1 is thought to mediate the signal of p53 induced by DNA damaging agents to arrest the cell cycle. To clarify the role of p53 and p21Waf1/Cip1 in Bowen's disease and DSP, we analysed 12 cases of Bowen's disease and eight cases of DSP by immunohistochemistry. In five of the 12 Bowen's disease patients and two of the eight DSP patients, positive p53 staining was detected. In contrast, whereas p21Waf1/Cip1 overexpression was detected in all Bowen's disease patients, it was not seen in DSP. The present data suggest that p53 immunostaining provides relevant information concerning the pathogenesis of Bowen's disease and DSP. Furthermore, high p21Waf1/Cip1 expression appears to be a useful indicator of tumour activity in Bowen's disease.     

Immunohistochemical prognostic criteria in xeroderma pigmentosum

Xeroderma pigmentosum (XP) is a rare inheritable disease characterized by severe sun sensitivity and early development of skin cancers. We compared the expression of cell proliferation markers and cell cycle checkpoint regulators in squamous cell carcinomas (SCC) and basal cell carcinomas (BCC) from patients with and without XP. Immunostaining for p53, Ki-67, and proliferating cell nuclear antigen (PCNA) was determined in SCCs and BCCs from 18 XP patients and 30 controls. Nine of the 18 XP patients had SCC and BCC, and the other nine had only SCC. In the control group, 15 moderately differentiated SCCs and 15 BCCs were evaluated. Expressions of p53, Ki-67 and PCNA in XP and non-XP patients were assessed statistically by using the Chi-square method. Expression of Ki-67 and PCNA was found to be greater in SCC from XP patients than controls (P = 0.021 and P = 0.033, respectively). Expression of PCNA and p53 by BCCs was greater in XP patients (P < 0.001 and P = 0.027, respectively). There was a significant difference in Ki-67 (P < 0.001) and PCNA (P = 0.001) expression between the lesions of the XP patients who died during the follow up and XP patients who survived. In XP patients, SCCs with more than 10% Ki-67 expression and %50 PCNA expression have a poor prognosis. Our results suggest that increased Ki-67 and PCNA expression may be a predictor for recurrence of nonmelanocytic skin cancer and a poor prognosis.     

Immunohistological analysis of P53 expression in human skin tumors.

The p53 expression in various skin tumors was immunohistologically evaluated using two mouse monoclonal anti-p53 antibodies, PAb421 and PAb1801. The p53 expression was not detected in the normal epidermal cells. Nuclear staining suggested that the p53 expression was observed in 10 of 26 squamous cell carcinomas (SCCs) from 24 patients, in one undifferentiated carcinoma, one proliferating trichilemmal cyst, one malignant proliferating trichilemmal tumor and in one metastatic carcinoma of breast cancer. None off four cases of Bowen's disease (SCC in situ) showed nuclear staining. In the SCCs, five of 20 primary lesions, three of four recurrent lesions and both of two metastatic lesions had positive nuclei. There was one case of SCC in which a primary lesion was negative but a recurrent lesion was positive. Thus, p53 expression was more frequently observed in SCCs at more clinically advanced stages. This may suggest that p53 has some relevance to progression of SCC. Nuclear staining was not detected in any of the following cases: two cases of seborrheic keratosis, one eccrine poroma, one keratoacanthoma, 11 basal cell epitheliomas, two mammary Paget's disease, three genital Paget's disease, one sebaceous carcinoma, four malignant melanomas, six lymphomas, two leukemia cutis and two angiosarcomas.     

The expression of p63 in basal cell carcinomas and association with histological differentiation

BACKGROUND: We aim to examine p63 expression in basal cell carcinomas (BCCs) and to investigate association with their histopathological differentiation subtypes. METHODS: Eighty-four BCCs were classified according to the histopathologic differentiation subtypes. Immunohistochemistry using monoclonal antibody against p63 was performed. RESULTS: In nontumoral skin, p63 expression was consistently seen in basal/suprabasal cells of epidermis, hair matrix cells, and outer root sheath of the hair follicle. In BCCs, the cases were distributed as 47 undifferentiated, 28 differentiated (16 adenoid and 12 keratotic), and nine superficial. The nuclear p63 expression was negative in two cases, whereas 64 BCCs (76.2%) showed homogeneous p63 immunostaining. There was no statistically significant difference between p63 expression and histological differentiation subtypes (p > 0.05). The expression of p63 was found strongly and diffuse in 72.3% of solid undifferentiated and 82.1% differentiated and in 77.8% of superficial type BCCs. CONCLUSIONS: p63 is consistently expressed in epidermal basal/suprabasal and adnexal basal cells. Most BCCs have higher homogeneous p63 expression than nontumoral epidermis, which is not changed according to histological differentiation subtypes. Thus, overexpression of p63 in all histological subtypes may confirm that basaloid progenitor cells are linked tumor-cell lineage and have a role in the tumorigenesis of BCC.     

Expression of apoptotic, cell proliferation regulatory, and structural proteins in actinic keratosis and their association with dermal elastosis

BACKGROUND: Actinic keratosis (AK) is a premalignant lesion caused by ultraviolet (UV) radiation and characterized by epithelial and connective tissue alterations. However, little is known about the link between connective and UV-damaged epithelial tissues in AK. OBJECTIVE AND METHODS: To examine the potential relationship between connective tissue degeneration and molecular alterations in epithelial cells without evident morphologic changes, 30 cases of AK (8, grade I; 10, grade II; 12, grade III), divided into three grades according to the proportion of dermal elastosis (in grade I, up to 30% of collagen degeneration; in grade II, 30-60%; in grade III, more than 60%), were immunohistochemically analyzed for the expression of Ki67, p53, p63, bcl-2, E-cadherin, 34-betaE12, and CD99. RESULTS: The increase in the solar elastosis grade was associated with an increase in positive cell numbers for all analyzed markers. Basal expression predominated in the lesions with low and moderate levels of connective tissue degeneration, while a basal and suprabasal expression pattern was prevalent in the lesions with high degeneration. In grade I and II lesions, proliferation marker, Ki67, expression was found to be significantly associated with the proapoptotic marker p53, while in grade III lesions, its expression was correlated with the anti-apoptotic marker, bcl-2. CONCLUSIONS: These results demonstrate that the epithelial expression of apoptotic, cell proliferation, and structural proteins is augmented with the increase of the solar elastosis grade. Thus, the grade of solar elastosis could be a helpful morphologic marker in the assessment of neoplastic changes in sun-damaged skin.     

Immunohistochemical staining of palisading basal cells in Bowen's disease and basal involvement in actinic keratosis: contrasting staining patterns suggest different cells of origin

Actinic keratosis (AK) and Bowen's disease (BD) are common patterns of in situ squamous cell carcinoma of the epidermis. In AK, atypical keratinocytes proliferate in the lower portion of the epidermis including the basal layer. In contrast, BD features atypical squamous cells in all portions of the epidermis but initially leaves basal cells in palisades along the basement membrane. To characterize immunohistochemically keratocyte proliferation in AK and Palisading Basal Cells (PBC) in BD, we stained microarray samples of 45 AK and 25 BD with Molecular Immunology Borstel (MIB-1). Subsequent immunostaining of full mounted sections examined 11 BD, 7 AK, and 4 examples of psoriasis for MIB-1 (as a proliferative marker) and p53 (as a cell cycle regulatory marker). AK stained for MIB-1 and p53 antibodies only in lower portion of epidermis and included the basal layer. BD with typical PBCs stained positive for both markers throughout the epidermis, except for the basal layer. Psoriatic biopsies stained positively for the 2 markers only in the basal and parabasal layers. Normal epidermis adjacent to the lesions in AK and BD biopsies stained sparsely in the basal layers. The correlation of different histologic patterns of epidermal involvement with different immunohistochemical patterns of stains argues for different cells of origin for BD versus AK. Lack of expression of proliferative antigens in palisading basal cells in BD provides evidence that PBCs are not the cell of origin for BD. Conversely in AK, expression of MIB-1 and p53 in basal cells argues that these cells play a role in histogenesis of AK.     

Aberrant expression of p53 tumour-suppressor protein in non-melanoma skin cancer

Expression of the cellular p53 tumour-suppressor protein was examined in 78 epidermal tumours, including basal and squamous cell carcinomas, keratoacanthomas, solar keratoses, Bowen's disease and viral warts. An immunohistochemical study was employed using the antibody CM-1, raised against recombinant human p53 protein. Positive staining for p53, not detectable in normal cells because wild-type p53 is rapidly degraded, reflects abnormal stabilization of p53 protein, and in many cases suggests p53 gene mutation. p53 immunoreactivity was not observed in normal skin or in viral warts. In contrast, positive staining for CM-1 was seen throughout the tumour in the majority of basal and squamous cell carcinomas and in Bowen's disease. Immunoreactivity to p53 was also observed in the majority of keratoacanthomas and solar keratoses, but was confirmed to areas of dysplastic basal epithelium. This study demonstrates that accumulation of p53 protein, suggestive in many cases of p53 gene mutation and hence loss of tumour-suppressor function, may occur as an important early step in the development of diverse epidermal cancers.     

Lamin expression in normal human skin,actinic keratosis,squamous cell carcinoma and basal cell carcinoma

BACKGROUND: Aberrant expression patterns of nuclear lamins have been described in various types of cancer depending on the subtype of cancer, its aggressiveness, proliferative capacity and degree of differentiation. In general, the expression of A-type lamins (lamins A and C) has been correlated with a non-proliferating, differentiated state of cells and tissues. OBJECTIVES: To establish and compare the expression patterns of lamins in normal human skin, actinic keratosis (AK), squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). METHODS: Expression patterns of the individual lamin subtypes were studied immunohistochemically. The proliferation capacity of the tumour cells was detected using a specific antibody to Ki-67, and was related to the A-type lamin expression patterns. RESULTS: In normal skin, lamin A was expressed in the suprabasal cell compartment of the epidermis, whereas the basal cells were mostly unstained. BCCs and SCCs stained positive in most cells, while the epidermis overlying BCC and SCC and the epidermis in AK stained homogeneously and strongly in the basal cells in addition to the suprabasal cells. Lamin C was expressed in some basal cells of normal epidermis while the suprabasal cells stained strongly positive. Both BCCs and SCCs stained strongly positive for lamin C, with the difference that in BCC the staining was predominantly present in nucleolar structures with occasional staining of the nuclear envelope. The epidermis overlying SCC showed strong positivity in the lamina of virtually all cells. The expression of lamin C in the basal cells of AK resembled the expression pattern seen in the epidermis overlying BCC, i.e. a nucleolar staining next to nuclear envelope staining. Lamin B1 and B2 were found in virtually all cells in normal epidermis, AK, BCC, SCC and the epidermis overlying cancer. The percentage of Ki-67-expressing cells was highest in BCC (45%), and gradually decreased via epidermis overlying BCC, AK, SCC, and epidermis overlying SCC, to normal skin (11%). Simultaneous expression of A-type lamins and Ki-67 occurred in approximately 50% of the proliferating (Ki-67 positive) cells in BCC and SCC. CONCLUSIONS: Significant changes occur in the expression patterns of A-type lamins in both premalignant and malignant lesions of the skin. The profound overlap of lamin A and Ki-67 staining patterns indicates that the proliferating tumour cells may obtain a certain degree of differentiation. Finally, lamin A expression in the basal cell layer of the apparently normal epidermis overlying BCC may suggest its involvement in the primary process.     

Curettage-cryosurgery for non-melanoma skin cancer of the external ear: excellent 5-year results

In this 5-year follow-up study, 60 auricular non-melanoma skin cancers, selected at a skin tumour clinic, were treated by a thorough curettage with different sized curettes followed by cryosurgery in a double freeze-thaw cycle. Tumours of the external auditory canal were excluded. Forty-seven basal cell carcinomas (BCCs), nine squamous cell carcinomas (SCCs), three Bowen's disease and one basisquamous cancer were included. The mean diameter of the malignancies was 18 mm (range 5-70). Morphoeiform BCCs, recurrent BCCs with fibrotic component and most of the SCCs were selected for Mohs' micrographic surgery. Forty-two patients with 47 tumours were followed-up for at least 5 years with only one recurrence. Thirteen patients, followed-up for 2-4 years, died from other causes with no sign of recurrence at their last visit. No major problems after treatment were registered and the cosmetic result was good or acceptable in all patients.     

p73蛋白在正常人皮肤和表皮肿瘤中的表达

目的 探讨p73蛋白在正常人皮肤和不同表皮肿瘤皮损中的表达及意义。方法 应用免疫组化方法检测19例脂溢性角化病、16例基底细胞癌、11例Bowen病、5例鳞状细胞癌及10例正常人皮肤p73、p53、Ki67的表达。结果 在正常人表皮基底层、毛囊外毛根鞘最外层基底样细胞和皮脂腺生发细胞有p73的表达;在基底细胞癌和脂溢性角化病的基底样细胞、Bowen病中异形性明显的瘤细胞p73呈高表达,鳞状细胞癌和脂溢性角化病中的鳞状细胞呈弱阳性或不表达。脂溢性角化病、Bowen病、基底细胞癌、鳞状细胞癌之间p73蛋白表达差异有统计学意义(H=12.71,P<0.01),其中基底细胞癌的表达最强。Ki67在皮肤肿瘤之间差异也有统计学意义(H=14.12,P<0.01),但p53差异无统计学意义(H=2.058,P>0.05)。在各组样本中,p73的表达与p53、Ki67无显著相关性(P>0.05)。结论 p73蛋白可能在皮肤分化中起重要作用。     

Expression of bcl-2, p53 and Ki-67 in arsenical skin cancers

To investigate the regulation of apoptosis and proliferation in arsenic-induced skin cancers, we examined the expression of bcl-2. p53, and Ki-67 using immunohistochemical staining. Thirty patients with Bowen's disease (BD), ten with basal cell carcinoma (BCC), eight with squamous cell carcinoma (SCC) and eleven of perilesional normal skin (PLN) of the non-sun exposure sites from endemic area were examined. The results showed that: 1) bcl-2 was expressed in all of the BCC homogeneously, in none of the SCC, and in 12/30 of the BD focally or homogeneously; 2) p53 was expressed in all of the arsenical skin cancers with a labelling index of 75±14% of BD, 50±17% of BCC. 61±15% of SCC, and also in all of the perilesional normal skin with a labelling index of 55±24%; 3) Ki-67 was expressed in all of the skin cancers with labelling index of 58±17% of BD. 12±7% of BCC, 47±21% of SCC, and in 9/11 of PLN with a labelling index of 41±24%. Expression of bcl-2 in BCC or BD is related to the phenotype of germinative basal cell. The constant expression of bcl-2 i early dysplastic cells of BD and the earliest expression of P53 in the basal cells of perilesional normal skin indicate that the initial step of arsenic-induced carcinogenesis is from the basal germinative cells. There is no mutual relationship between bcl-2, p53 or Ki-67 expression in any type of the arsenical skin cancers, but there is a positive correlation between p53 and Ki-67 expression identified in perilesional normal skin. BD had the highest labelling index of p53 and Ki-67.     

An assessment of the malignant potential of actinic keratoses and Bowen's disease: p53 and PCNA expression pattern correlate with the number of desmosomes

Actinic keratoses (AK) and Bowen's disease (BD), both intraepidermal skin tumors, have a potential progression to squamous cell carcinoma (SCC). To evaluate the malignant potential of AK and BD, the expression pattern of p53 protein and proliferating cell nuclear antigen (PCNA) were examined in five types of AK and BD by immunohistochemistry. The ultrastructural difference of epidermal cells between AK and BD lesions was investigated. In the study of p53 and PCNA expression, the atrophic and acantholytic types of AK showed lower positive rates compared to others. These two types did not demonstrate all layers expression pattern. The number of desmosomes of the epidermal cells was significantly reduced in BD, and in the bowenoid and hypertrophic types of AK compared with other types of AK The number of hemi-desmosomes showed greatest reduction in BD and the bowenoid type of AK On the basis of our findings, it is hypothesized that atrophic and acantholytic types of AK may have the lowest, and the bowenoid type of AK and BD may have the highest, malignant potential.