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Summary A promising new treatment for glioma involves Auger electron emitters such as 1251 or 1231 conjugated to deoxyuridine (lUdR). However, the presence in tumour deposits of non-proliferating cells with clonogenic potential poses a major limitation to this cycle-specific therapy. We have used multicellular tumour spheroids derived from the human glioma cell line UVW to study [1251]1UdR-targeted radiotherapy in aggregates containing cells in different proliferative states. Autoradiographic identification of labelled cells indicated that nuclear incorporation of [1251]1UdR decreased markedly with increasing size of spheroid. IUdR incorporation was maximal in the surface layer of cells and decreased with depth within spheroids. Radiopharmaceutical uptake corresponded closely to the regions of cell cycling as indicated by staining for the nuclear antigen Ki67. The uptake of drug was enhanced by increasing the duration of incubation from 52 h to 104 h. These observations suggest that significant sparing of non-cycling malignant cells would result from treatment delivered as a single injection of radiolabelled lUdR. To achieve maximal therapeutic effect, lUdR should be administered by multiple injections, by slow release from biodegradable implants or by slow-pump delivery.

Radioiodinated iododeoxyuridine (IUdR) is a novel, cycle-specific agent that has potential for the treatment of residual malignant glioma after surgery. As only cells in S-phase incorporate IUdR into DNA, a major limitation to this therapy is likely to be proliferative heterogeneity of the tumour cell population. Using a clonogenic end point, we have compared the toxicities of three radioiodoanalogues of IUdR--[123I]IUdR, [125I]IUdR and [131I]IUdR--to the human glioma cell line UVW, cultured as monolayers in the exponential and the plateau phase of growth and as multicellular spheroids. Monolayers treated in the exponential growth phase were most efficiently sterilized by [125I]IUdR (concentration resulting in 37% survival (C37) = 2.36 kBq ml(-1)), while [123I]IUdR and [131I]IUdR were less effective eradicators of clonogens (C37 = 9.75 and 18.9 kBq ml(-1) respectively). Plateau-phase monolayer cultures were marginally more susceptible to treatment with [123I]IUdR and [125I]IUdR (40% clonogenic survival) than [131I]IUdR (60% clonogenic survival). In cells derived from glioma spheroids, both [125I]IUdR and [123I]IUdR were again more effective than [131I]IUdR at concentrations up to and including 20 kBq ml(-1). However, the survival curve for [131I]IUdR crossed the curves for the other agents, resulting in lower survival for [131I]IUdR than [123I]IUdR and [125I]IUdR at concentrations of 40 kBq ml(-1) and higher, the clonogenic survival values at 100 kBq ml(-1) were 13%, 45% and 28% respectively. It was concluded that IUdR incorporating the Auger electron emitters 123I and 125I killed only cells that were in S-phase during the period of incubation with the radiopharmaceutical, whereas the superior toxicity to clonogenic cells in spheroids of [131I]IUdR at higher concentration was due to cross-fire beta-irradiation. These findings suggest that [131I]IUdR or combinations of [131I]IUdR and [123I]IUdR or [125I]IUdR may be more effective than Auger electron emitters alone for the treatment of residual glioma, if proliferative heterogeneity exists.

We developed a sensitive EGF receptor detection method for frozen tissue sections using biotinylated EGF as the primary reagent. The method was directly compared with an immunohistochemical technique based on an anti-EGF receptor monoclonal antibody (MAb EGFR1) in normal human and rat tissues and in human tumors. The method was more sensitive than a previously published biotinylated EG"Sed technique. In normal human tissues and in 37 of the 50 tumors, the binding pattern mirrored that of positive staining with EGFR1. Five further tumors showed weak immunoreactivity, but in these no binding of biotinylated EGF

The relationship between expression of the c-erbB-2 proto-oncogene and the biology of breast cancer has been investigated widely, most studies using immunohistochemistry in formalin-fixed, paraffin-embedded tissues. This technique is at best semiquantitative and there is a high degree of interstudy variability because of its subjective nature and poor methodological standardization. The relationship between the levels of expression and biology can be examined thoroughly only with an accurately quantitative technique. We have developed a radioimmunohistochemical assay to measure p185(erbB-2) in tissue biopsy specimens. The method involves incubating frozen sections with 125I-labeled monoclonal antibody, microautoradiograpy, and grain counting with image analysis. Sections of cell pellets with known c-erbB-2 levels are processed with each batch of samples as internal calibration standards. We have quantified c-erbB-2 expression in 60 breast carcinomas and compared the results with conventional immunohistochemistry. Radioimmunohistochemistry measured receptor levels throughout the range of expression in breast carcinomas, whereas conventional immunohistochemistry detected the protein only in the highest expressing tumors. The quantitative, objective data produced by radioimmunohistochemistry allow a more thorough evaluation of the relationship between c-erbB-2 expression and tumor biology. This technique may have applications in other fields where quantitative data is required and relevant monoclonal antibodies are available.

The Auger electron emitting agent 5-[125I]iodo-2′-deoxyuridine (i.e.[125I]IUdR) holds promise for the treatment of residual glioma after surgery because this thymidine analogue kills only proliferating cells. However, malignant cells which are not synthesizing DNA during exposure to the radiopharmaceutical will be spared. To determine whether tumour incorporation of [125I]IUdR could be enhanced by protracted administration, we used a C6 cell line, growing in the brains of Wistar rats, as a glioma model and compared three methods of intracerebral delivery of [125I]IUdR. Twenty-four hours after administration of drug, autoradiography of brain sections demonstrated nuclear uptake of the radiopharmaceutical in cells throughout tumour while normal brain cells remained free of radioactivity. The [125I]IUdR labelling indices (% ± s.e.m.) achieved were 6.2 (0.4) by single injection, 22.5 (4.1) using a sustained release polymer implant (poly(lactide-co-glycolide)) and 34.3 (2.0) by mini-osmotic pump. These results emphasize the need for a sustained delivery system as a prerequisite for effective treatment. These findings are also encouraging for the development of a sustained release system for radiolabelled IUdR for use in the treatment of intracranial tumours, particularly in the immediate postoperative setting. © 2000 Cancer Research Campaign

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