OPTIMAL CONDITIONS FOR IMMUNOHISTOCHEMICAL DETERMINATION OF THE INVITRO DNA-SYNTHESIS LABELING INDEX WITH BROMODEOXYURIDINE IN HEAD AND NECK-CANCER

The in vitro DNA synthesis labelling index was assessed immunohistochemically in 24 freshly obtained specimens of head and neck cancer using bromodeoxyuridine (BrdUrd) as the DNA precursor to determine the influence of BrdUrd concentration on labelling index (LI). Initially, tumour fragments were incubated in varying concentrations of BrdUrd from 2 to 100-mu-M for 2 h, and BrdUrd was detected with an anti-BrdUrd monoclonal antibody using immunoperoxidase labelling. There was a dose-response gradient with mean LI varying from 1.6% at 2-mu-M BrdUrd to 8.8% at 100-mu-M. The concentration-response gradient best fit a quadratic model when LI was plotted against log BrdUrd concentration (r = 0.65, P < 0.0001). Eleven additional tumours were then studied to determine whether LI increased for BrdUrd concentrations above 100-mu-M. The mean LI at 125-mu-M and at 150-mu-M in these 11 tumours did not differ from the value at 100-mu-M, suggesting a plateau at this level. The gradient effect accounted for 17% of the variance in LI, while 60% of the variance was explained by between tumour differences. Within individual tumours, three response patterns were observed: (i) LI rose at a constant rate to the highest concentration tested (n = 8), (ii) the LI plateaued or declined at high BrdUrd concentrations (n = 6); and (iii) there was a biphasic slope slope in which the rate of rise in the LI increased at the higher BrdUrd concentrations (n = 2). The data show that BrdUrd concentration is an important variable in the immunohistochemical assessment of the in vitro LI in head and neck cancer.