The retrospective study included specimens from 145 randomly selected patients having primary renal pelvis (n
30) or bladder (n
115) urothelial carcinoma retrieved from the files of the Reina Sofia University Hospital, Cordoba, Spain. Treatment included complete transurethral resection of the bladder or cystectomy. Clinical information was obtained from the patient's record. Patient follow‐up (mean (SD)), calculated as the number of months from the date of surgery to the date of the most recent cystoscopy (or the last visit or death) was 65 (28) (range 6–120) months. Tumour recurrence in stage Ta/T1 bladder tumours was defined as reappearance of tumour after treatment with at least one tumour‐free cystoscopy interval.
Specimens were fixed in formalin and embedded in paraffin. Available H&E stained slides of tumours were reassessed without knowledge of the clinical status. The resulting grade and stage were in accordance with the criteria of the 1973 World Health Organization and the 2002 Tumour, Node and Metastasis, respectively.2,13
The presence of intercellular bridges or keratinisation was indicative of SqD.1,2,3,4,5
For immunohistochemistry, 4‐μm‐thick sections were deparaffinised in xylene and rehydrated in graded alcohol. Immunohistochemical analysis was carried out using streptavidin–biotin peroxidase. Briefly, sections were incubated overnight with the primary mouse monoclonal antibody detecting myelomonocytic L1 antigen at 4°C (Mac387, Dakopatts, Glostrup, Denmark) diluted at 1:400. Biotinylated secondary antibody and the streptavidin–biotin peroxidase complex were sequentially applied (LSAB2 kit, Dako, Glostrup, Denmark). Diaminobenzidine was used as chromogen (Dakopatts). Endogenous peroxidase activity was blocked by incubating the slides in 1% hydrogen peroxide in methanol for 10 min. For epitope retieval, the slides were boiled in 10 mM citrate buffer (pH 6.0). Negative and positive controls were included in every procedure. A control group for the immunoreactivity included normal urothelium (n
5), flat urothelial hyperplasia (n
5) and normal trigone mucosa (n
Bivariate and multivariate statistical analyses included the χ2
test with the SPSS 8.0 for Windows Software. All were two sided tests. A p value
0.05 was considered significant.