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Objective To develop specific monoclonal antibodies (mAbs) against human germ cell tumours. Materials and methods A single-cell suspension obtained from tumour tissue fragments (consisting of both tumour and normal compartments) from a patient with seminoma was used as an immunogen. Spleen cells from immunized mice were used to develop mAbs. Tissue specificity, biochemical characteristics and competitive studies were analysed using immunocytochemical staining, dot blots and a Western blot analysis, to identify target antigen(s). Results The immunization protocol led to the development of 107 hybridomas, 90 of which were negative against the original tissue biopsies. The remaining 17 showed positivity against various tissue compartments. One selected mAb (ATC2) showed specific staining on germ cell tumours but not on normal tissues, and positive staining with some human tumour cell lines. The target antigen for ATC2 was confirmed to be placental alkaline phosphatase (PLAP) based on: Western blot analysis compared with commercially available PLAP; comparison of the data with another well-known anti-PLAP mAb (H17E2, although the two mAbs recognized different antigenic epitopes); heat resistance characteristics; high-performance liquid chromatography of the ATC2 target antigen and purified PLAP. Conclusion The selected mAb ATC2 has high specificity for human germ cell tumours, the target antigen for ATC2 being PLAP, although the antigenic epitope(s) differ from those recognized by H17E2. Thus ATC2 may be useful for monitoring serum levels of PLAP in patients with testis cancer and may be relevant for detecting cancer cells in the semen of individuals with suspected testis cancer, particularly in those with equivocal findings on ultrasonography.

In this investigation the profile of p53 and epidermal growth factor receptor (EGFR) expression in tumour tissue biopsies of transitional cell carcinoma of bladder (TCC) and of oral-pharyngeal carcinoma (OP) were compared using an immunocytochemical staining method. In addition, various techniques including sodium dodecyl sulphate-polyacrylamide gel elecrophoresis (SDS-PAGE), colorimetric assay and gene transfection were used to investigate the influence of p53 on the behaviour of human tumour cell lines in vitro. The results showed that: (a) p53 was detectable in more than 45% of cases in both tumour types, although the profile and intensity of expression differed. (b) Concomitant strong expression of EGFR and p53 for TCC and OP was 21% and 38% (P>0.05%), respectively. (c) Treatment of tumour cells by either gamma radiation or by cisplatin resulted in the induction of p53 independent of the origin of the tumour. (d) Susceptibility of two cell lines, one with and one without constitutive expression of p53 showed that the expressing cells were more sensitive to gamma radiation (the percentage inhibition at 250 cGy was 57% versus −15%, P

Cellular alkaline phosphatases (ALP) are increasingly recognised as important markers for monitoring tumour cell behaviour in human malignancies. Colorimetric, flow-cytometric, and immunocytochemical assays were employed to assess the influence of activation on expression of cellular ALP in human tumour cell lines. The results showed the following: (1) Testis tumour biopsies (16/16) unlike bladder (0/14) and head and neck (0/16) tumours showed positive staining for ALP, particularly the placental type, i.e. PLAP, although this was not always present on all the cells of non-seminoma biopsies. (2) The intensity of ALP expression differed widely in tumour cell lines. Based on biochemical analysis, the profile of ALP fell into two categories: (a) low expressing (MW 70 kD, placental type ALP) like Hep2 and KB lines, and (b) those expressing both low and high molecular (MW 95 kD) bands like testis lines Tera II and Ep2102. In all cases treatment of tumour cell lysates with heat prior to biochemical analysis showed the disappearance of the higher and sharpening of the lower molecular weight ALP band. (3) Exposure of tumour cells to epidermal growth factor (EGF) expressing EGF receptor led to a decreased ALP expression by as much as 54% as assessed by biochemical or flow-cytometric techniques. These data demonstrated that testis tumour tissues and cell lines expressed ALP which were different from others. The data also showed that exposure of tumour cell lines expressing EGFr to EGF resulted in suppression of ALP expression. These observations are consistent with the notion that EGFr and PLAP expression may be taken as a marker of proliferation and differentiation in human malignancies, respectively.

Objective To establish the specificity and sensitivity of alkaline phosphatase activity in testicular tumours for the early detection of testicular germ cell tumours (GCTs). Materials and methods Samples from patients with GCTs of the testis were sectioned and stained using the nitroblue tetrazolium salt (NBT)/5 bromo-4-chloro-3-indolylphosphate (BCIP) Sigma Fast substrate technique (referred to as MEDC) and peroxidase anti-peroxidase (PAP) assay. Additionally, several cell lines from GCTs and other tumours were cultured using standard techniques and stained using both methods, and lysates from all samples were stained after gel electrophoresis. Results MEDC showed strong and specific staining of testicular tumours only. The activity on gel electrophoresis appeared as two bands, one at 75 and one at 95 kDa, which was a unique combination for testicular tumours and testis cell lines. Conclusion The MEDC method has extreme sensitivity and intensityfor testicular tumour tissue and has potential use as a powerful tool for the early detection of testicular GCTs by analysing semen samples, particularly in patients with equivocal findings on testicular ultrasonography.