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

Abstract

Abstract.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, antinucleolin 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 reexamined with immunohistochemical methods, but the intensity of its immunoabsorption with the nucleolin peptide did not exceed that observed in the other anti-hTERT antibodies.

Localization of telomerase hTERT protein in frozen sections of basal cell carcinomas (BCC) and tumor margin tissues

“…In these tissues, the score values with the antibodies EST21-A (Ab 2, Table II) and code 582000 (Ab 6) were the lowest. This result is in accordance with our earlier examination of tissues from squamous cell carcinomas (52). The other antibodies, Ab 3, Ab 4 and Ab 7-Ab 11, showed clear nuclear (or nucleolar) marking of carcinoma cells and squamous epithelial cells.…”

“…To visualize the outcome we performed the alkaline phosphatase anti-alkaline phosphatase (APAAP, 56) for the current study, as it had proved successful in our earlier immunohistochemical examinations (57)(58)(59). The APAAP was more suitable in our study of hTERT evidence than EnVision™ (DakoCytomation, Denmark) (52). After methanol and acetone fixation, the slides with the frozen sections were pretreated for 30 min in a steamer (95-99˚C) in TRS buffer (target retrieval solution) S1699 (Dako Cytomation).…”

“…Methodical procedure and assessment of nuclear demonstration of hTERT on frozen sections from basal cell carcinomas: As already reported (52) we optimized the immunohistochemical nuclear hTERT proof in the basal cell carcinoma and tumor margin tissues with antibodies Ab 1-Ab 4 (Table II) and applied the same method of pretreatment and detection for further anti-hTERT antibodies Ab 5 to Ab 11 and also for the control antibodies (Ab 12 to Ab 15, Table II). We used the same immunohistochemical method, the same Figure 5.…”

“…pretreatments of the frozen slides and the same anti-hTERT antibodies as when we investigated squamous cell carcinoma tissues, Fabricius et al (52). Conforming to Yan et al (73) no unambiguous nuclear staining was produced with the polyclonal antibody code PC563 by Oncogene/Calbiochem, an antibody against the hTERT amino acid sequence 348-358 (our Ab 1).…”

“…As for the in situ proof of hTERT in frozen sections of squamous cell carcinoma (SCC) tissues in the head-neck region (52), hTERT evidence in cryo sections of BCCs was presented using 11 commercial antibodies to hTERT for appropriate nuclear demonstration of hTERT in BCC tissues.…”