1975, British Journal of Radiology, 48, 872 Correspondence THE EDITOR—SIR, ARE NEGATIVE PIMESONS REALLY NECESSARY?

There are three reasons for developing the use of negative pimesons and heavy ions for the treatment of cancer. These are: (1) The high RBE of these radiations at a depth, in the region of the peak dose. (2) The low OER. (3) The improved dose distribution in a phantom. Unless the desired dose distribution in the patient can be made to coincide with the position of the cancer the advantages of the first two of these reasons is lost. Indeed, it is possible that errors in positioning could result in a high RBE seriously damaging a sensitive organ such as the kidney. On the other hand it must be assumed that the facilities and expertise available in connection with such costly installations will be such as to ensure avoiding vital and sensitive normal structures even if the exact position of the malignant process cannot be known. Some disadvantages of such installations are: (1) The high cost resulting in too few centres to deal with the patients requiring treatment. (2) Deterioration of dose distribution with increasing area of single beams. (3) Diminished dose-rate if all the beams in a multiple beam machine cannot be used {e.g. in the Stanford multiple beam machine). (4) The difficulties always inherent in radiation therapy, of knowing the precise position and size of the malignant process. (5) The production of other particles which can reach and damage other tissues. On the other hand, the use of 4TT curietherapy with suitable radioactive sources and afterloading techniques has much to recommend it. (The term 4TT is more logical than brachytherapy as a distinction from beam therapy.) 1. The dose distribution can be limited to the region of the target volume with a rapid fall-off outside it. 2. The shape of the treated volume can be varied, e.g. a curved plane on the chest wall or an ellipsoid in the prostate gland. 3. The radiopaque active sources used for treatment can be used as markers for the position of the tumour so that supplementary external radiation can be used. 4. Such sources can be inserted at a surgical operation. What is needed to exploit the use of active sources is more ingenuity in devising methods of applying them so that the geometry is good, and in producing the necessary isotopes in suitable form and quantity. For temporary implants they should be available for afterloading at a suitable time after an operation. For permanent implants they should be available immediately. In addition the training of radiation therapists to think in terms of such treatment and of surgeons to become awake to the possible advantages is most desirable. It may be that the local application of heat could be facilitated by the presence in microwave beams of the radioactive sources and their metal jackets. If primary tumours in difficult situations such as the lung, pancreas and prostate can be dealt with consistently well by local methods, the further cooperation of chemical oncologists and immunological oncologists in dealing with systemic metastases may have greater success. AT: curietherapy can be used more widely, less expensively and more immediately than negative pimesons. It would seem to be worthwhile to put a larger slice of the financial pie into such methods and into the appropriate training than has so far been the case. Yours, etc., F.

Memorial Hospital for Cancer and Allied Diseases, New York, 10021, U.S.A.



We have read with interest the letter by G. M. Ardran, W. A. Langmead and H. E. Crooks in your March issue discussing a new screen/film combination. In this they mention a reduction in radiation exposure in mammography obtained by using Trimax XD film in conjunction with an Alpha 8 back screen and compared it with Medichrome film and a high definition calcium tungstate back screen. Recently Agfa-Gevaert Ltd. have provided us with new rare earth intensifying screens which we have used as a vacuum packed back screen with Medichrome film for mammography. The screen has the advantage of emitting blue light and thus normal blue-sensitive black and white and Medichrome film can be used and handled under conventional darkroom conditions. Tests with a standard Kodak Pathe mammogram phantom showed that a 50 per cent reduction of exposure time could be achieved when compared with Medichrome and an Ilford high definition screen. Photomicrographs of the 50 /xm mesh on this phantom showed that resolution and sharpness were identical, and similar to that of Cronex "Lo dose" film. White fused bauxilite (white fused alumina) particles embedded in paraffin wax of a mean size of 145 fim could be identified readily on all these films with a hand lens. (On an industrial film, Structurix D4 used without an intensifying screen, 105 yu,m particles were identifiable.) This system has been used on 50 patients and we have found that mammograms taken with Medichrome and the rare earth screen are indistinguishable from those taken with an Ilford high definition screen and the radiation exposure is cut by a half. The exposure conditions are 100 mAs, 30 kVp and 60 cm FFD; the skin dose is in the range of 0-15-0-3 rad per exposure, measured by thermoluminescent dosimetry. Yours etc., J. L. PRICE, P. D. BUTLER.

Royal Surrey County Hospital, Guildford.


There is abundant evidence that cancer cells grown in vivo or in vitro are sensitive to elevated temperature (Cavaliere, 1967; Overgaard and Overgaard, 1972). Clinically, Coley (1893), near the beginning of this century, achieved a considerable success in the regression of tumours by inducing high temperatures in cancer patients with bacterial toxins. Since then, the literature contains numerous cases of the arrest or total disappearance of cancer after prolonged fever (Cavaliere, 1967). Recently, renewed interest in the combined use of hyperthermia and radiotherapy or chemotherapy has been generated at both the laboratory and the clinical level (Suit and Shwayder, 1974). In the case of radiotherapy, a single most important radiobiological concept relevant to therapy is the fact that large solid tumours contain a population of hypoxic cells which are still clonogenic. These cells constitute the most radioresistant fraction, and require two to three times more radiation dose compared with well-oxygenated cells. It is generally thought that this radioresistant tumour cell fraction results in failure of local tumour control by radiation therapy. Various means have been employed to overcome this problem: hyperbaric oxygen chambers have been used to increase the oxygenation of tumours and high LET radiations are being explored because of their relative independence from an oxygen effect on cell killing.












Hours at elevated temperature FIG. 1. Survival curves of HeLa cells plated immediately after heat treatment at various temperatures for various periods of time. Hypoxia was induced by incubating at 37°C for two hours with 6 X 108/ml. heavily irradiated feeder cells. The plating efficiency of corresponding unheated controls was 50-60 per cent. The points at 42°C are the average values obtained from four separate experiments, and the points at 41 °C and 43°C are from one experiment each. The standard error of the mean is indicated by bar where greater than symbol representation.

The present study was designed to investigate the differential thermal sensitivity of oxic and hypoxic cells, using in vitro culture of HeLa cells as the model system. In this system, hypoxia was induced in the culture by the oxygen scavenging ability of an excess of heavily irradiated metabolizing cells admixed with assay cells (Djordjevic, Anderson and Kim, 1973). HeLa S-3 cells were grown and maintained in Eagle's minimum essential medium supplemented with 10 per cent fetal calf serum. Details of tissue culture procedures and preparation of hypoxic cells are described elsewhere (Djordjevic 5et al., 1973; Kim, Kim and Eidinoff, 1965). Briefly, 1 X 10 assay cells and a 30- or 60fold excess of heavily irradiated feeder cells were suspended per ml. of calcium-free Eagle's minimum essential medium containing 25 mM Hepes buffer. From this mixture 0-6 ml. fractions of the cell suspension were sealed in 1 0 ml. plastic pipettes and incubated at 37°C for 2-8 hours to deplete oxygen by the respiratory activity of feeder cells. For heat application, the sealed pipettes were immersed in a constant temperature water bath for the desired time period. Immediately following heat treatment, the cells were diluted in regular medium and an appropriate number of cells was plated in 60 mm diameter plastic Petri dishes for colony formation. To determine the effect of heat on the cells, the colony-forming ability of plated single cells was enumerated throughout the study. The chronicity and degree of hypoxia attained was studied in relation to the thermal sensitivity of cells sub-


Cell state

No. of feeder cells per ml.

Hours incubated at 37°C to deplete oxygen 2

% survival of Oxic 0 cells at 42°C for 2 hours Hypoxic 3xlO 66 6xlO




40% 42% 39% 40% 45% 25% 11% 5% 15% 3% 0-45% —

*The percentage survivals of heat-treated cells were calculated from corresponding unheated controls which were treated similarly except for heating. The plating efficiency of the unheated oxic and hypoxic controls: 50-60 per cent for oxic cells, and 40-60 per cent for hypoxic cells. sequently incubated for two hours at 42°C. The results, summarized in Table I, clearly indicate that survival of hypoxic cells is dependent upon the degree of hypoxia, i.e., the higher the number of feeder cells and the longer the



48, No. 574 Correspondence

incubation period at 37°C, the lower the survival. Survival of oxygenated cells remained constant at approximately 40 per cent. Subsequent experiments were carried out to study the temperature dependency of the previously observed killing of hypoxic cells. The survival curves of HeLa cells heattreated at different temperatures for different periods of time in both oxic and hypoxic state are shown in Fig. 1. A differential thermal sensitivity between oxic and hypoxic state of cells became evident when the temperature was increased from 41 °C to 43 °C and the incubation time proceeded from half an hour to three hours at the elevated temperatures. To investigate whether the reduction in survival of hypoxic cells was caused by the production of toxic metabolites from the irradiated feeder cells during the induction of hypoxia, two different methods of inducing hypoxia were employed, e.g., by crowding large numbers of viable (unirradiated) cells in a small volume so that the metabolic process would bring about oxygen depletion, as occurs in a tumour, or by placing the cells in a glass ampoule sealed after bubbling through nitrogen (Zeitz et al., 1974). Similar results were obtained in both cases, indicating that reduction in the survival of hypoxic cells in heat treatment is not caused by the feeder cells in hypoxic cell suspension. Furthermore, when cells were kept in a hypoxic state for six hours and then heated (42°C for two hours) in the oxic state by releasing from hypoxia, survival was equal to that of oxic cells (Kim, Kim and Hahn, 1975a). This further reduces the possibility of toxic effects from the feeder cells during the induction of hypoxia. Thus, it appears that the lack of oxygen in the HeLa cells was responsible for the enhanced cell killing in hyperthermia. Nonetheless, unidentifiable metabolites produced as a result of crowding or from irradiated feeder cells might also have contributed to the enhancement of hyperthermic cell killing. This hypoxic system gives an oxygen enhancement ratio of 3-0 (Kim, Kim and Hahn, 1975b). Since cellular hypoxia usually develops only in malignant solid tumours, particularly large ones, and it is currently believed that radiotherapy fails locally mainly because of the presence of this hypoxic cell fraction, any means to overcome this resistant cell population would have important clinical significance. Our studies of combined use of heat and radiation on mice bearing a Ridgway osteogenic sarcoma suggest that the cure rate following the combined treatment appeared to be independent of tumour size (0-7 cm to 1-2 cm average diameter) (Hahn, Alfieri and Kim, 1974). This may be of clinical relevance because the radiation doses employed in tumour therapy and the success rate in achieving local tumour control are, generally, related to the initial size of the tumour. This relationship may, in part, be due to the hypoxic cells contained in solid tumour. Several factors are known to be related to the thermal sensitivity of cells, such as the metabolic state of cells (protein and DNA synthesis) and the stage of cells during the division cycle (Palzer and Heidelberger, 1973 a; Palzer and Heidelberger, 1973b; Westra and Dewey, 1971). Hypoxia appears to be another modifying factor of thermal sensitivity. It is interesting to note that cells in a hypoxic state and in late S phase are the most radioresistant, while such cells in similar state are the most heat sensitive. We reported recently that when HeLa cells were heated for two hours at 42°C immediately after irradiation, the oxygen enhancement ratio was reduced by a factor of approximately 2 and this led us to suggest that heating irradiated hypoxic cells could be an effective means of reducing the oxygen dependence factor in cell killing with low LET radiations (KAm et al., \975b). The mechanisms involved in hyperthermic killing of both oxic and hypoxic mammalian cells are not understood. However, this observation, along with the finding we reported recently (Kim et al., 1975b), could be of significant importance to clinical radiotherapy because the radioresistant hypoxic cells could be overcome by hyperthermia and radiation. This

work was supported in part by NCI grant CA-08748 and NCI grant RO 1 CA-16178-01. Yours, etc., S. H. KIM, J.H.KIM, E. W. HAHN.

Division of Radiotherapy Research, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, U.S.A. ACKNOWLEDGMENTS

The authors gratefully acknowledge Dr. G. J. D'Angio, Chairman, Department of Radiation Therapy, for his support and advice in carrying out these studies and Miss C. Browne for her secretarial assistance in the preparation of this manuscript. REFERENCES CAVALIERE, R., CIOCATTO, E. C , GIOVANELLA, B. C , HEIDELBERGER, C , JOHNSON, R. O., MARGOTTINI, M., MONDOVI, B., MORICCA, G., and Rossi-FANELLI, A., 1967.

Selective heat sensitivity of cancer cells. Biochemical and clinical studies. Cancer, 20, 1351-1381. COLEY, W. B., 1893. The treatment of malignant tumours by repeated inoculations of erysipelas—With a report of ten original cases. American Journal of Medical Science, 105,487-511. DJORDJEVIC, B., ANDERSON, L. L., and KIM, S. H., 1973.

Oxygen enhancement ratios in HeLa cells irradiated with californium and radium sources. Radiology, 107, 429-434. HAHN, E. W., ALFIERI, A. A., and KIM, J. H., 1974.

Increased cures using fractionated exposures of X-irradiation and hyperthermia in the local treatment of the Ridgway osteogenic sarcoma in mice. Radiology, 113, 199-202. KIM, J. H., KIM, S. H., and EIDINOFF, M. L., 1965. Cell

viability and nucleic acid metabolism after exposure of HeLa cells to excess thymidine and deoxyadenosine. Biochemical Pharmacology, 14, 1821-1829. KIM, S. H., KIM, J. H., and HAHN, E. W., 1975a. Unpublished data. 1975b. The radiosensitization of hypoxic tumour cells by hyperthermia. Radiology. (In press). OVERGAARD, K., and OVERGAARD, J., 1972. Investigations on

the possibility of a thermic tumour therapy—I. European Journal of Cancer, 8, 65-78. PALZER, R. J., and HEIDELBERGER, C , 1973a. Studies on

the quantitative biology of hyperthermic killing of HeLa cells. Cancer Research, 33, 415—421. 1973b. Influence of drugs and synchrony on the hyperthermic killing of HeLa cells. Cancer Research, 33, 422427. SUIT, H., and SHWAYDER, M., 1974. Hyperthermia: Po-

tential as an anti-tumour agent. Cancer, 34, 122-129. WESTRA, A., and D E W E Y , W . C , 1971. Variation in sensitivity

to heat shock during the cell-cycle of Chinese hamster cells in vitro. International Journal of Radiation Biologv, 19, 467-477. ZEITZ, L., CANADA, T. R., DJORDJEVIC, B., DYMORT, G., FREEMAN, R., MACDONALD, J. C , and LAUGHLIN, J. S.,

1974. Biological determination of variation of fast neutron field quality with depth, RBE and OER. Radiation Research. (In press). Editor's note: This letter was originally submitted for publication in November 1974. We apologize to the authors for the delay. THE EDITOR—SIR, THE RADIOLOGICAL DIAGNOSIS OF ADRENAL TUMOURS

Whilst offering congratulations on the review article by Dr. D. Sutton in the April issue of the Journal, I must


Letter: Enhanced killing of hypoxic tumour cells by hyperthemia.

1975, British Journal of Radiology, 48, 872 Correspondence THE EDITOR—SIR, ARE NEGATIVE PIMESONS REALLY NECESSARY? There are three reasons for develo...
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