Immunohistochemical determination of the appropriate anti-hTERT antibodies for in situ detection of telomerase activity in frozen sections of head and neck squamous cell carcinomas and tumor margin tissues.

In previous studies we demonstrated telomerase activity in frozen tissue from head and neck squamous cell carcinoma (HNSCC) and their tumor-free tumor margins. In the present study frozen sections from the same tissues were examined for in situ presence of hTERT. In preliminary investigations we established that the most suitable method of tissue preparation was fixation in acetone and methanol followed by steaming and visualization by APAAP. Most of the assays involved eleven anti-hTERT antibodies and were supplemented with the inclusion of antibodies Ki-67, anti-nucleolin and CD45. hTERT expression was investigated in the tissues of 61 patients with HNSCC and 37 patients without tumor. Semi-quantitative immunoreactive scores were correlated with telomerase activity. We examined the prognostic significance of hTERT expression with Kaplan-Meier curves and tested the immunological specificity of the antibodies by immunoabsorption with two hTERT peptides and a nucleolin peptide. Nuclear staining of satisfactory distribution and intensity was achieved in seven anti-hTERT antibodies both in the carcinomas and in the squamous epithelia of the tumor resection margins and in the control tissues. Proof of hTERT did not differ from telomerase activity. The telomerase activity demonstrated in tumor-free resection margins and in control tissues did, however, correlate with lymphocytic-monocytic infiltration (CD45 expression). This telomerase activity might be related to nuclear hTERT expression in the squamous epithelium, given that the hTERT score values in the connective tissue tended to be negative. The prognostic significance of hTERT expression demonstrated on paraffin sections from different tumor localizations was not confirmed for the frozen sections of patients with HNSCC. The hTERT specificity of the monoclonal NCL-L-hTERT, whose use as an antibody against hTERT has been questioned, was re-examined with immunohistochemical methods, but the intensity of its immunoabsorption with the nucleolin peptide did not exceed that observed in the other anti-hTERT antibodies.