The in vivo proliferation kinetics of human solid tumours

Abstract

The study of tissue and tumour cell kinetics is one approach to better understanding of human tumour biology and to the formulation of better treatment strategies. Past efforts to measure in vivo the duration of cell cycle phases has been severely handicapped by the need to use radioisotopes and to take sequential biopsies. The drug 5-bromo-2-deoxyuridine (BRdU) is a safe, ethical alternative to tritiated thymidine for in vivo labelling of DNA during the S phase of the cell cycle. The label can be detected by conventional histochemistry, and by multiparameter flow cytometry. The latter provides measurements of ploidy, phase related DNA labelling, and the duration of the S phase and of the potential doubling time (Tpot) of the tumour or tissue. These parameters have not been characterised in many types of tumour, nor have their relationships to prognosis or to treatment strategies been explored. A series of 100 colorectal adenocarcinomas, 53 invasive ductal carcinomas of the breast, 3 5 gastrooesophageal carcinomas and a small number of lymphomas, melanomas, sarcomas and squamous carcinomas were studied prospectively. Hospital ethical committee approval and informed patient consent was obtained for a single intravenous injection of 250mg BRdU prior to conventional surgery for malignant disease. The DNA index, total and aneuploid labelling indices, S phase duration and Tpot were measured. No correlations were found to exist between these parameters and current prognostic indicators. Long term follow-up will be undertaken to relate the data to clinical outcome. The Tpot of gastrointestinal tumours was typically less than ten days, whereas the actual volume doubling time from clinical experience is known to be of the order of 100 days or more. This discrepancy is largely produced by a high rate of cell loss by exfoliation and necrosis in these tumours. A validation study of the Tpot concept was undertaken using the MC28 rat sarcoma grown subcutaneously in nude mice. There was a good correlation between the measured Tpot and the volume doubling time during the exponential phase of tumour growth. A study was undertaken of mucosal kinetics using tissue from resection specimens throughout the gastrointestinal tract. An original method is described for the calculation of the crypt labelling index and crypt cell turnover rate combining histological and flow cytometric data. A good correlation was obtained with previously published data. The method offers advantages over previous methods of studying mucosal kinetics in normal and disease states. The counting of confluent features of interest such as labelled nuclei in stained tissue sections is laborious and prone to observer errors. An original application of the automated planar image analyser is described which allows automation of the measurement of the labelling index in peroxidase stained tissues. This has a number of uses in clinical research in histopathology. A reliable intrinsic marker of tumour proliferation would offer substantial practical advantages over BRdU labelling. The "p62" protein product of the putative oncogene c-myc was measured by multiparameter flow cytometry in colorectal tumours and tissues. Results were compared with BRdU labelling in the same speccimens. p62c-myc levels were found to be unexpectedly high in normal and polyposis coli mucosa, raising new questions about the function of the c-myc gene. The combination of in vivo BRdU labelling and multiparameter flow cytometry has been demonstrated to be valid and practical in the study of tumour and tissue kinetics. The possible of use of the technique in devising better chemotherapy and radiotherapy regimes are discussed. Future lines of research are considered.

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ORCID for David Rew: orcid.org/0000-0002-4518-2667

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