Basal cell carcinoma (BCC) is the most common of all types of skin cancer; approximately three of 10 Caucasians develop basal cell cancer within their lifetime. In 80% of all cases, basal cell cancers are found on the head and neck, but in recent years, there is an increase in the incidence of basal cell cancer in the trunk (Rosai, 2004). Although it is considered to be a malignancy, it rarely metastasizes but it can invade locally (Kirkham, 2005). The major drawback of BCC is its local destructive and disfiguring ability (Wong et al., 2008).
Cutaneous squamous cell carcinoma (SCC) is the second most common form of skin cancer and accounts for 20% of cutaneous malignancies (Fan et al., 2009) and frequently arises on the sun-exposed skin of middle-aged and elderly individuals.
Approximately 60% of nonmelanoma skin cancers are BCCs, and 20% are SCCs. According to Cancer Pathology Registry 2003–2004, in Egypt, BCC and SCC constitute 45.5 and 37% of malignant skin tumours, respectively (Mokhtar et al., 2007).
Despite the increasing incidence of both BCC and SCC, its pathogenesis has remained largely unknown. Recently, it was reported that genes involved in tissue morphogenesis, such as sonic hedgehog or patched, were found to be mutated in BCC and SCC, suggesting the involvement of those molecules in the pathogenesis of these tumours. Furthermore, there is evidence that the Wnt-mediated signalling pathway may be one of the downstream targets of sonic hedgehog-mediated signalling, which has led us to focus on molecular events on the Wnt pathway in BCC. One of the important signal transducers involved in the Wnt pathway is β-catenin (Willert and Nusse, 2008).
Beta-catenin is a member of the E-cadherin/catenin complex, which plays a major role in cell–cell adhesion. Beta-catenin is also known to be involved in signal transduction pathways. Many studies have demonstrated changes in the expression of β-catenin in colorectal carcinomas, suggesting a role for β-catenin in carcinogenesis. In other words β-catenin shows at least two functions in mammalian cells; cell adhesion by interaction with E-cadherin and cytoskeleton or signal transduction by interaction with adenomatous polyposis coli protein in Wnt-signalling pathway, which is involved in carcinogenesis Hulsken et al. (2004).
In the presence of a Wnt signal, the cytoplasmatic pool of β-catenin rises and translocates to the nucleus in which it transactivates transcription factors of the T-cell factor/lymphocyte enhancement factor family. In the absence of Wnt signals, a multiprotein complex promotes the degradation of β-catenin; thus, no activation of T-cell factor/lymphocyte enhancement factor target genes can take place (Piedra et al., 2007).
In the study carried out by Doglioni et al. (2008) the role of β-catenin pathway in human skin carcinogenesis was determined, 135 nonmelanoma skin tumours were analyzed for β-catenin expression and gene mutations. Intense nucleocytoplasmic immunoreactivity for C terminus β-catenin antibodies was observed in all pilomatricomas and in single cases of trichoepithelioma and SCC showing peculiar signs of matrical differentiation. Moderate increase of β-catenin nuclear staining was detected in a significant proportion of BCCs, Bowen disease, spiroadenomas, and occasionally also in SCCs. The aim of this study was to determine the immunohistochemical expression and role of β-catenin in the pathogenesis of BCC and SCC.
Materials and methods
This study included 40 cases, divided as 20 cases of BCC, 15 cases of SCC and five cases of normal skin as a control group. The cases were diagnosed at the Department of Pathology, Faculty of Medicine, Menoufiya University. Clinical and pathological data were obtained from their files.
Histological examination of haematoxylin and eosin-stained sections was performed to confirm the clinical diagnosis of the cases and verify the histological types and grades. BCC types were diagnosed according to the criteria detailed by Kirkham (2005). Grading of SCC was performed in the most aggressive area of the tumour. Cases were divided into well differentiated, moderately differentiated or poorly differentiated. From each representative block, three contiguous 4-μm thick sections were cut and mounted on glass slides, one for routine haematoxylin and eosin staining and two on poly-L-lysine – precoated slides for immunostaining (one as a test slide and the other as a negative control).
Immunohistochemical staining of β-catenin
After deparaffinization and rehydration, the sections were incubated in hydrogen peroxide (3% H2O2 in absolute methyl alcohol) for 10–15 min in a humidity chamber. For antigen retrieval, the heat-induced epitope retrieval procedure was used (New Marker immune histopathology catalogue, 2000). Ultra V Block was applied for 5 min. The sections were incubated overnight with polyclonal primary antibody of β-catenin (0.1 ml) (Lab Vision Corp., Westinghouse, California, USA). Washing with phosphate-buffered saline (PBS), biotinylated secondary anti-immunoglobulin (LSAB 2 system-HRP, akocytomation, DAKO, Copenhagen, Denmark) was applied for 40 min at room temperature. The specimens were washed in PBS and incubated with streptavidin peroxidase for 10 min. The diaminobenzidine chromogen substrate was prepared while the slides were in PBS, and then, controls were applied on slides for 3 min. Finally, the specimens were counterstained with Mayer's haematoxylin.
Interpretation of β-catenin expression
Beta-catenin expression was assessed according to:
(1) Intensity into: moderate and strong.
(2) Location: nuclear or nucleocytoplasmic expression. Positive β-catenin staining was identified as brown colour.
Data were collected, tabulated and statistically analyzed using a personal computer with Statistical Package for the Social Sciences, version 11. The Fisher exact test was used for comparison between qualitative variables. The Mann–Whitney U-test was used to compare between quantitative variables. Differences were considered to be statistically significant when P value was 0.05 or less and highly significant when P value was 0.01 or less.
This study included 15 cases of SCC, their ages ranged from 26–69 years, with median 40 and mean±standard deviation (SD) (42.8±12.4 years). Most of the SCC cases were men (66.6%). The size of the lesions ranged from 2–17 cm, mean±SD (7.36±5.25 cm). The ulcer margin was positive in two thirds of the cases. Histoplathologic examination revealed three cases (20%) with poorly differentiated SCC, seven cases (46.6%) with moderately differentiated and well-differentiated SCC in five cases (33.3%). Three cases (20%) of SCC were present in the early stage, whereas 12 cases (80%) were present in the late stage. The mitotic index ranged between 1 and 11 with mean±SD 6.26±3.8. Only four cases (26.6%) of SCC showed recurrence.
Our study also included 20 cases with BCC. Their ages ranged from 35–77 years, with median 55.5 years and mean±SD 55.6±9.8 years. Fifteen cases (75%) were men and five were women (25%). The size of the lesions ranged from 1.8 to 10 cm (mean±SD 4.6±2.1). The ulcer margin was positive in 14 (70%) cases. Of the patients, 24% presented with multiple masses and the rest (76%) showed solitary lesions. Histopathological examination revealed that 17 (81%) cases showed the solid type. The other four cases were divided into two morphea type and two adenoid basal types. Eleven cases of BCC were present in the early stage, whereas nine cases were present in the late stage. The mitotic index ranges from 1 to 12 with mean±SD 5.91±3.16.
Comparing both tumour groups revealed that our SCC cases showed more aggressive clinicopathological features than BCC cases as the age in SCC was significantly less than that of BCC cases and the tumour grade, stage, mitotic figures and recurrence were higher in the SCC cases (Table 1).
Immunostaining showed that β-catenin was mainly localized in the cytoplasm of epithelial cells of epidermis, eccrine, apocrine and sebaceous glands of normal human skin. With regard to the expression of β-catenin in both tumour groups, it was found that in SCC, 10 (66.6%) cases showed strong nuclear expression of β-catenin and five (33.3%) cases showed weak nucleocytoplasmic expression of β-catenin. However, in BCC, 10 (50%) cases showed strong nuclear expression of β-catenin and 10 (50%) cases showed weak nucleocytoplasmic expression of β-catenin. The difference between SCC and BCC was not statistically significant. (P>0.05). In both tumour groups, there was a highly statistically significant correlation between the intensity of β-catenin expression and its localization in which strong expression showed nuclear localization whereas weak expression showed nucleocytoplasmic localization. (P<0.01).
With regard to the BCC group, the expression of β-catenin varied among different types of BCC, in which nuclear localization was most notable in the infiltrative and morphoeic variants and less notable in superficial and nodular variants. The expression was prominent at tumour margins (Fig. 1).
A statistical significant correlation was observed between intensity of β-catenin expression and mitotic index as well as grade of the tumour in which the tumour of high grade and mitotic index showed strong intensity of β-catenin (P<0.01) (Table 2) (Figs 2 and 3).
A significant correlation was found between intensity of β-catenin expression and size as well as stage of the tumour in which the tumour with higher size and stage showed strong expression. In squamous cell carcinoma the intensity of β-catenin expression showed statistically significant correlation with the tumour grade where tumours of high grade showed strong intensity of β-catenin. Again the localization of β-catenin in squamous cell carcinoma is significantly correlated with tumour grade, thus nuclear localization was present in high grade tumour (Table 1) (Figs 4 and 5).
BCC is the most common of all types of skin cancer and represents 60% of nonmelanoma skin cancers. Cutaneous SCC is the second most common form of skin cancer and accounts for 20% of cutaneous malignancies Wong et al. (2008). According to Cancer Pathology Registry 2003–2004, in Egypt, BCC and SCC constitute 45.5 and 37% of malignant skin tumours, respectively (Mokhtar et al., 2007).
Skin neoplasms represent a heterogeneous and complex group of tumours deriving from the various structures that constitute the human skin. These proliferative lesions are a composite puzzle of entities that remain vague to many pathologists and dermatologists. Difficulties in correlation of these lesions with their normal counterparts, combined with gaps in knowledge about molecular aspects of normal and neoplastic transformation of the different components of human skin, are the major reasons of different interpretations and classifications of cutaneous proliferations. A deeper knowledge of the genetics of human skin tumours would provide important support for a better classification and management of these particular forms of neoplasia (Tsao, 2009).
Beta-catenin is a crucial member of the E-cadherin/catenin complex, which plays a major role in cell–cell adhesion (Jamora and Fuchs, 2002). Beta-catenin is also known to be involved in signal transduction pathways. Many studies have demonstrated changes in the expression of β-catenin in colorectal carcinomas, suggesting a role for β-catenin in neoplastic development (Nelson and Nusse, 2004 and Tetsu and McCormick, 2009).
Recently, the involvement of β-catenin in human cutaneous neoplastic transformation has been suggested by the finding of β-catenin gene mutations in pilomatrixoma, a tumour arising from hair follicle matrix cells (Van Noort et al., 2006). However, the role of Wnt/β-catenin pathway in the development of the different types of skin neoplasm has not been fully examined (Seto and Bellen, 2004).
In this study, immunohistochemical analysis with β-catenin antibodies revealed that β-catenin was localized in the cytoplasm of epithelial cells of epidermis, eccrine, apocrine and sebaceous glands of normal human skin. With regard to the expression of β-catenin in both tumour groups, it was found that in SCC, 10 (66.6%) cases showed strong nuclear expression of β-catenin and five (33.3%) cases showed weak nucleocytoplasmic expression of β-catenin, whereas in BCC, 10 (50%) cases showed strong nuclear expression of β-catenin and 10 (50%) cases showed weak nucleocytoplasmic expression of β-catenin. The difference between SCC and BCC was not statistically significant. In both groups, the strong intensity of β-catenin was correlated with nuclear expression, whereas weak intensity of β-catenin was correlated with nucleocytoplasmic expression and this results agree with the one reported by Doglioni et al. (2008) and indicate that β-catenin is present mainly in the nucleus in the cases of the tumour that refers to the involvement of Wnt-pathway and β-catenin in pathogenesis in both tumours.
The expression of β-catenin in normal skin was mainly cytoplasmic whereas in both BCC and SCC the expression was mainly nuclear and nucleocytoplasmic and this result matched with that reported by Saldanga et al. (2009) and Xia et al. (2009) and indicate the higher nuclear concentration of β-catenin in tumour that means it acts as signal transducer in tumerogenesis.
There was a highly statistically significant correlation between the intensity of β-catenin expression and mitotic index, grade, size and stage of the tumour in which the tumour of high grade, size, stage and mitotic index showed strong intensity of β-catenin and this refers to that the nuclear localization of β-catenin correlate with the aggressiveness of the tumour. This result agrees with that obtained from the study by Xia et al. (2001) and Drees et al. (2005) and means that β-catenin may be implicated in late carcinogenesis of both BCCs and SCCs.
The expression of β-catenin varied between different types of BCC. Nuclear localization was most notable in the infiltrative and morphoeic variants and was less notable in superficial and nodular variants. The expression was prominent at tumour margins, and this result is similar to the result obtained in the study by EL-Bahrawy et al. (2003), and gives evidence that nuclear β-catenin expression correlates with increased proliferation and aggressiveness of the tumour.
In SCC, the grade of the tumour statistically significantly correlated with the intensity of β-catenin expression in which the tumour of high grade showed strong expression of β-catenin. This result matched that obtained by Fan et al. (2009) and indicated that β-catenin may play an important role in progression and may be involved in aggressive biological behaviour of SCC.
From our results we suggest that
(1) Beta-catenin may play a role in the pathogenesis of both BCC and SCC.
(2) Nuclear localization of β-catenin correlates with the aggressiveness of BCC.
(3) Beta-catenin may play an important role in the progression and may be involved in aggressive biological behaviour of SCC.
1. Doglioni C, Piccinin S, Demontis S, Cangi MG, Pecciarini L, Chiarelli C, et al. Alterations of β-catenin pathway in non-melanoma skin tumors. Cell. 2008;84:345–357
2. Drees F, Pokutta S, Yamada S, Nelson WJ, Weis W. Alpha-catenin is a molecular switch that binds E-cadherin/beta-catenin and regulates actin filament assembly. Cell. 2005;123:769–772
3. EL-Bahrawy M, EL-Masry N, Alison M, Poulsom R, Fallowfield M. Expression of β-catenin in basal cell carcinoma. Br J Dermatol. 2003;148:964–970
4. Fan M, Duan Y, XU Q (2009). Beta-catenin role in the carcinogenesis of oral squamous cell carcinoma. In: IADR/AADR/CADR 87th General Session and Exhibition; 1-4 April 2009; Miami. p. 879. pp. 879.
5. Hulsken J, Behrens J, Birchmeier W. Tumor-suppressor gene products in cell contacts: the cadherin-APC-armadillo connection. Curr Opin Cell Biol. 2004;6:711–716
6. Jamora C, Fuchs E. Intercellular adhesion, signalling and the cytoskeleton. Nat Cell Biol. 2002;4:E101–E108
7. Kirkham NDavid EE. Tumors and cysts of the epidermis. Lever's histopathology of the skin. 2005 USA Lippincott Williams & Wilkins:805–865
8. Mokhtar N, Gouda I, Adel IMokhtar N, Gouda I, Adel I. Malignant skin tumours. Cancer pathology registry, 2003–2004 and time trend analysis. 2007 Cairo NCI, El Sheraa Press:83–85
9. Nelson W, Nusse R. Convergence of Wnt, beta-catenin and cadherin pathways. Science. 2004;303:1483–1487
10. Piedra J, Martinez D, Castano J, Miravet S, Dunach M, Herreros AG. Regulation of beta-catenin structure and activity by tyrosine phosphorylation. J Biol Chem. 2007;276:20436–20443
11. Rosai JJuan Rosai and Ackerman. . Basal cell carcinoma, General features. Rosai and Ackerman's surgical pathology. 20049th ed New York, USA Mosby-Year Book Co.:247–277
12. Saldanga G, Ghura V, Potter L, Fletcher A. Nuclear beta-catenin in basal cell carcinoma correlates with increased proliferation. Br J Dermatol. 2009;151:157–164
13. Seto ES, Bellen HJ. The ins and outs of wingless signaling. Trends Cell Biol. 2004;14:45–53
14. Tetsu O, McCormick F. β-catenin regulates expression of cyclin D1 in colon carcinoma cells. Nature. 2009;398:422–426
15. Tsao H. Genetics of non-melanoma skin cancer. Arch Dermatol. 2009;137:1486–1492
16. Van Noort M, Meeldijk J, Van der Zee R, Destree O, Clevers H. Wnt signaling controls the phosphorylation status of β-catenin. J Biol Chem. 2006;277:17901–17905
17. Willert K, Nusse R. Beta-catenin: a key mediator of Wnt signaling. Curr Opin Genet Dev. 2008;8:95–102
18. Wong CS, Strange RC, Lear JT. Basal cell carcinoma. BMJ. 2008;327:794–798
19. Xia X, Qian S, Soriano S, Wu Y, Fletcher AM, Wang XJ, et al. Loss of presenilin 1 is associated with enhanced β-catenin signaling and skin tumorigenesis. Proc Natl Acad Sci USA. 2001;98:10863–10868
©2011Egyptian Journal of Pathology
20. Xia X, Qian S, Soriano S, Wu Y, Fletcher AM, Wang XJ, et al. Loss of presenilin 1 is associated with enhanced β-catenin signaling and skin tumorigenesis. Proc Natl Acad Sci USA. 2009;98:10863–10868