Basal cell carcinoma (BCC) is the most common skin neoplasm accounting for 75% of non-melanoma skin tumors. In addition to fair skin and chronic sun exposure which are the most predisposing factors in the pathogenesis of BCC, genetic syndromes (xeroderma pigmentosum, Rombo syndrome, Bazex syndrome), immune suppression, chemical exposure (arsenic, tar), and ionizing radiation contribute to its pathogenesis (1-3). Although metastasis is rarely seen secondary to BCC, local destruction and recurrences have been reported. Factors such as site and size of the lesion, histo-pathological subtypes, perineural and intravascular invasion are associated with recurrence and local aggressiveness of BCC lesions (4-7).
BCC is classified into BCC1 or low risk (nodular, superficial type) and BCC2 or high risk (micronodular, morpheaform, infiltrative, and basosquamous types) based on clinical behavior. BCC1 is characterized by cellular nests with peripheral palisading and cleft artifact. BCC2 is characterized by cellular nests infiltrated into collagenous stroma with high mitotic activity, necrotic cells, and deeper invasion within dermis and subcutaneous tissue without peripheral palisading and cleft artifact (8). This study, for the first time in Iran, attempted to evaluate immunehistochemical findings (IHC) and clinical features associated with local aggressiveness and recurrence in BCC lesions.
Materials and Methods
This is a cross-sectional descriptive study carried out on 42 paraffin blocks (22 BCC1, 20 in BCC2) at Pathology Department of Afzalipour Teaching Hospital. Demographic features of the patients including age, sex, location, and size of the lesions were recorded. Pathology blocks were revised by two pathologists with light microscope (Olympus, Bx53 series) and then classified into two groups of BCC1 and BCC2 based on their histopathological types. BCC1 included nodular and superficial types and BCC2 included micronodular, morpheaform, and basosquamous types.
For IHC study, histologic sections of paraffin blocks (3 mm thick) were deparaffinized with xylene and rehydrated with ethanol. Antigen retrieval was performed with Tris-EDTA (PH=9) buffer and microwaved for 10 minutes. Tissue peroxidase activity was deactivated by methanol 0.5% and H2O2. Then, primary monoclonal antibodies including CD10 (Dako 56C6, dilution factor (df) 1:30, Denmark), CD1a (Dako M 3571, df 1: 50, Denmark), SMA (Dako 1A4, df 1:1500, Denmark), Ki67 (Dako MIB1, df 1:50, Denmark) and P53 (Dako DO7, df 1:50, Denmark) were incubated for 30 minutes in room temperature. Secondary antibodies were added by biotin and streptavidin peroxidase technique. For demonstration of antigen-antibody bands, diaminobenzidine tetrahy-drochloride (DAB) chromogen was utilized and background was stained with hematoxylin.
To avoid bias, each slide was coded with a number and evaluated blindly by two pathologists. Counting of Langerhans cells (LCs) was done at 5 different fields of 0.32 mm2 and the mean number of cells was recorded. Only LCs with obvious nucleus were counted in order to avoid recounting. For counting of blood vessels, firstly all the slides (stained with SMA) were observed by 10 high power fields (HPFs) to select fields with higher density of vessels. Then, counting was done by 200 HPFs in 3 fields; and the mean number, minimum and maximum of blood vessels were recorded. Only post-capillary venules, capillaries, and endothelial cells without lumen (single or clustered) were counted (8-10).
For evaluation of SMA and CD10, modified quick score was used (7). Intensity and distribution of these markers were classified as follows: 0 (no staining), 1 (weak staining; only observed by high HPFs), 2 (moderate, easily observed by low HPFs), and 3 (strong; prominently observed by low HPFs).
Ki67 and P53 were recorded as percentage of positive staining cells among 100 tumoral cells. Finally, the 2 types of BCCs were compared according to IHC markers and demographic features of cases.
In 42 skin biopsy samples (22 BCC1 and 20 BCC2) were evaluated. The number of samples belonging to male patients was higher (52.4%) and the mean age of the patients was 67.69±12.29 (min=38, max=87) years old. Table (1) illustrates clinical features of BCC lesions.
The mean number of LCs within epidermis above tumor mass was 14+1.92 in BCC1 and 4.7±1.23 in BCC2, which indicated a significant difference between the two groups (P=0.001).
P53 was positive in 41.13±6.39% and 74.5 ±6.26% of the tumor cells in BCC1 and BCC2, respectively (statistically significant: P=0.001). Ki67 was positive in 28.54±28.54% and 45.8±7.74% in BCC1 and BCC2, respectively (P=0.081).
The mean number of blood vessels was 14.40±1.30 and 21.40±1.97 in BCC1 and BCC2 (statistically significant: P=0.005). SMA was positive in 72.7% and 90% in BCC1 and BCC2 groups, respectively (P=0.083). Meanwhile, CD10 was positive in 46.3% and 55% in BCC1 and BCC2, respectively (P=0.655) (Table2).