Radiobiology

In1 J. Rudrurron &,CdOgJ~ Blol. Phw Vol. Printed in the U.S.A. All rights reserved.

14. pp. S25-S28 Copyright

0360-3016/88 $3.00 + .X3 0 1988 Pergamon Press plc

0 Special Priorities RADIOBIOLOGY

E. J, HALL,

D.PHIL.,

D.Sc.’

AND J. F. FOWLER,

PH.D.,

M.D. (HoN)~

‘Radiological Research Laboratory, College of Physicians and Surgeons, Columbia University New York, N.Y.; and ‘Gray Laboratory, Northwood, U.K.

RATIONALE

FOR RADIOBIOLOGY

CLINICAL PARAMETERS OF TREATMENT OUTCOME UNDERSTANDABLE IN RADIOBIOLOGICAL TERMS

INPUT

The function of a radiobiology committee in a cooperative trials group is two-fold: First to provide input, counsel and advice to existing and functioning modality committees, particularly those based on radiobiological principles perceived to offer a potential advantage over conventional therapy. These include Hyperthermia, High LET Radiation, Chemical Modifiers, and Altered Fractionation Patterns. Second to keep abreast of new developments, identify promising trends that may impact on cooperative clinical trials 1 to 10 years in the future, and make this information available to the group. In the latter category, there are a number of headings under which possible progress can be discussed.

PREDICTIVE

ASSAYS OF TUMOR TO RADIATION

Predictive parameters, other than the usual ones of tumor size, histology, stage and the patients sex, age and hormone status include: (1) Hemoglobin level (Bush et al., 1978;2 Dische et al., 1983;5 Taskiren, 1969;” Overgaard, 1986).* Despite convincing evidence of the predictive value of hemoglobin levels, this appears to be relatively neglected. (2) Measurements of tumor physiology.

HYPOXIC

RESPONSE

Accepted for publication

Reprint requests to: E. J. Hall, D.Phil. Acknowledgments-The manuscript was read, and constructive criticism offered by Drs. H. Rodney Withers, J. D. Chapman, and Norman

INDICATORS

The presence of foci of viable hypoxic cells which limit tumor curability by X rays delivered in a single or small number of fractions appears to be the rule, rather than the exception, in transplanted tumors in laboratory rodents (Moulder and Rockwell 1982).6 It is difficult to say what the position is in the human (Chapman et al., 1983).3,4 A great deal of effort has gone into the development of strategies to overcome the perceived problems of hypoxic cells, including the development of neutrons, hypoxic cell sensitizers, and hyperbaric oxygen. These approaches have shown some limited success, but clearly do not represent an across the board gain. A pressing need is to perfect ways to select individual tumors that contain hypoxic cells. Methods have been devised to tag misonidazole with a radioactive label, such that the radioactivity is deposited in hypoxic cells when the misonidazole is metabolized and broken down under anaerobic conditions. Although feasible, exploitation of this technology has been slow even

Relative simple observations, or short-term assays of complexity that may give some information on the relative sensitivity of individual tumors based on observations of biopsy material include the following: (1) Ploidy, Streffer et al., 1986;” (2) Micronuclei Production, Streffer et al., 1986;” Peters et al., 1986;9 (3) Clonogenic Potential, Peters et al., 1986;9 (4) Capillary Density, Revesz, 1986;” (5) Kinetic parameters (by flow cytometry). None of the above have been developed, tested or used to the point where they can give routinely reliable and meaningful indications of the sensitivity of individual tumors to X rays or to a new modality. But this is clearly the goal.

Todd Wasserman,

CELL

Bleehen. s25

4 January 1988.

S26

I. J. Radiation

Oncology

0 Biology 0 Physics

to demonstrate the presence or absence of hypoxic cells in representative tumors, much less to use it routinely as a diagnostic test in individual patients. To date, 4 of 9 human tumors have shown uptake of tritium labeled Miso indicative of chronic hypoxia. The need is to tag the sensitizer with a gamma or positron emitter, so that it may be detected outside of the body by conventional nuclear medicine techniques; this has proved difficult to achieve in practice. Routine realization of the potential benefits of this idea have been slow to come. Because these early results indicate that less than half the human tumors studied contain a significant proportion of overtly hypoxic cells, this further emphasizes and highlights the need for patient selection in sensitizer studies. An alternative approach first suggested by Olive (1983) is to use nitro-aromatic compounds which undergo nitroreduction to fluorescent forms which can then be detected either by the microscope or by flow cytometry. The latter method has the advantages ofspeed and quantitation and, in principal, could be used to estimate hypoxic fractions in human tumors before and during therapy. To date a number of compounds have been tested in vitro (Olive and Durand, 1983;’ Begg et al., 1985)’ several of which have fluoresced well in hypoxic cells. However, suitable compounds for in vivo use have not yet been developed. Compounds that worked well in vitro were either too toxic for in vivo use or underwent reductive metabolism in the liver (as well as in tumors) to such an extent that considerable non-specific fluorescent labelling of cells resulted. In the long run, perhaps the most promising approach to the detection of the presence of hypoxic cells is the development of Magnetic Resonance Spectroscopy (MRS) using magnets of high field strength to image phosphorus, or magnetic spin labelled drugs which can localize in hypoxic cells. A technique of this kind enjoys the enormous advantage of being non invasive.

DEVELOPMENT OF MORE EFFECTIVE HYPOXIC CELL RADIOSENSITIZERS (I) Dual function bioreductive drugs The bifunctional compound RSU 1069, which has radiosensitizing properties because it is a 2-nitroimidazole, and an alkylating function because of the aziridine ring on the side-chain, has shown greatly enhanced activity compared to misonidazole. Phase I clinical trials have shown the toxicity of the new compound to be considerably higher than misonidazole, but there are prospects for analogues that retain much of the enhanced sensitizing activity, without unacceptable additional toxicity. The mechanism of action of these drugs is not clear and merits detailed investigation. The hypoxic-specific bioreduction of this class of compound offers considerable promise. (2) Non-nitro compounds Selective radiosensitization of hypoxic cells has been demonstrated in several classes of oxidants. Although nitro

1988 Volume

14, Supplement

I

compounds have received the most attention, relatively little effort has been directed towards optimizing the properties of other possible candidates, either as selective radiosensitizers of hypoxic cells or as selective cytotoxic agents of hypoxic cells. There is plenty of scope for further work in this area. PROLIFERATION

IN TUMORS

Fractionation in radiotherapy confers the benefits of possibly increasing tumor response due to reoxygenation and redistribution (towards asynchrony) within the division cycle between dose fractions, while decreasing normal tissue damage due to repair of sublethal damage and to proliferation. In recent years, hyperfractionation has been proposed and used, with a view to further separate early and late radiation effects in normal tissues, based on the premise that kute responding tissues are more sensitive to fractionation because they are characterized by a “curvier” dose response relationship. Another modification of conventional fractionation is indicated for fast growing tumors, where tumor cell proliferation during the several weeks required in conventional (or hyper-fractionated) regimens may more than offset the benefits of more fractions. In this situation “accelerated treatment” may be the method of choice, where the total number of fractions of a conventional regimen is maintained, while shortening the overall treatment time, by the expedient of using multiple treatments per day. It has been postulated that a cell cycle time of about 5 days represents the transition between selecting hyperfractionation and accelerated treatment as the method of choice. Before such choices can be made, however, methods must be available to allow rapid estimates of the cell cycle of tumor cells in individual patients. Potential methods of varying complexity include: 1. Labelling index from biopsy specimens. 2. Fast flow cytometry using fluorescent dyes and BUdR. The labelling index provides some information about repopulation rates in tumors, but the potential doubling time is a much better parameter to use. This requires knowledge of both the labelling index and the duration of S-phase. Begg et al., (1985)’ have described a method for measuring these two parameters from a single biopsy sample following in vivo labelling with bromodeoxyuridine (BUdR), then using an anti-BUdR monoclonal antibody and flow cytometry. This has the advantage that the information can be obtained within a day of obtaining the biopsy. Comparison of this technique have already been made with conventional autoradiography using tritiated thymidine, and similar results were obtained (Wilson et al., 1985).i3 Wilson et al., (1985)13 have reported the first in vivo measurement of the labelling index of human tumors using BUdR and flow cytometry. Bromo-, or iododeoxyuridine can be used as the label. Both have been used extensively as radiosensitizers in man. It is likely that a 30 to 60 minute i.v. infusion of either agent to a total dose of 0.5 to 1 gm will be non-toxic and provide

Radiobiology

sufficient incorporation into tumor cell DNA for analysis by flow cytometry. It should then be possible to measure the in vivo labelling index and probably the potential doubling time, routinely and rapidly to provide predictive information on human tumor cell kinetics. Cell cycle parameters may one day be ordered for each patient as readily as a differential blood count.

INTRINSIC

CELLULAR

RADIORESISTANCE

Further from the clinic, but of much basic interest are studies of inherent resistance to radiation and other cytotoxic agents at the cellular level. This will be discussed under 3 headings.

(1) Identification of repair genes The existence of mutants exquisitely sensitive to some agents (e.g. mitomycin C) has made it possible to identify and clone repair genes specific for that type of DNA damage. This has not yet been done for ionizing radiations. The search is in progress in many laboratories for the appropriate radiation mutants, but repair genes have not yet been identified-the problem may turn out to be much more difficult for ionizing radiation because the DNA damage produced is less specific than those produced by UV or mitomycin C. Certainly, sensitivity to DNA damaging agents is dominated by the efficiency of repair processes, the manipulation of which would be a major step forward. A study of the sensitivity of fresh explants of human tumors, as well as repair-deficient cells, such as those from patients with Ataxia Telangiectasia, are also likely to provide useful insight. (2) Inhibition of “repair” As pointed out above, the molecular mechanisms involved in radiation repair have not yet been elucidated, so that “sublethal damage repair” and “potentially lethal damage repair” remain as purely operational terms. Nevertheless, a large body of empirical data are available concerning drugs that tend to inhibit repair. For example, actinomycin D has been shown to inhibit SLDR, whereas cordycepin and ARA-A have been shown to inhibit PLDR. This may not be a very promising approach, and clinical trials on the inhibition of the repair of radiation damage would appear to be premature at the present time. It must be proved conclusively that in a given tumor type a differential exists between normal and tumor tissues that would lead to an increase in tumor control. This has not yet been demonstrated, and given the greater repair occurring in late responding normal tissues, may never be.

s21

0 E. J. HALL

(3) Biochemical manipulation of cells Because X ray damage is mediated by the production of free radical species, the role of sullhydryls such as glutathione (which can, in various ways, scavenge free radicals) has received much attention. The development of a specific inhibitor of GSH synthesis, namely buthionine sulfoximine (BSO) has enabled researchers to investigate the role of GSH in radiation response. Other less specific thiol depleting or oxidizing agents have been used in the past. These “non-specific” depleters often exhibit more radiosensitization than specific GSH depletion mediated through a specific agent such as BSO. This single observation indicates the potential importance of identification of other biochemical systems, in addition to GSH, that are affected by these various non-specific thiol depleting or oxidizing agents. The free radical scavenging hypothesis suggests that thiols such as GSH would not significantly contribute to the aerobic radiation response due to the greater efficiency of damage fixation by oxygen. However, GSH depletion does sensitize aerobic cells to radiation, and recently it has been shown that addition of extracellular GSH to GSH depleted cells reverses the aerobic sensitization. Since GSH is thought not to traverse the cell membrane, these recent studies suggest a possible involvement of the cell membrane in detoxification by either the shuttling of reducing equivalents to the inside of cells via disulfide/sulfide coupling or possibly by direct detoxification of hydrogen peroxide or organoperoxides through GSH peroxidase. The role of GSH in the radiation response has been studied intensely during the past 2 years, yet contribution of other sullhydryl containing compounds, namely protein thiols, which are present in cells at high equivalent concentrations, has not been assessed. It would seem that a quantitative non-protein sullhydryls (NPSH) profile of cells within individual tumors might relate to the inherent tumor cell sensitivities to radiation and several chemotherapeutic drugs.

GENES

FOR

METASTASIS

Whereas radiation is predominantly considered to be a local treatment, the long-term results are always modified by the problem of distant metastases. The recent discovery that the process of metastases may be governed by a specific gene, holds the promise that, at the very least, the potential for metastases of a given tumor may one day be assessed and in the long run may be amenable to genetic manipulation. It is to be hoped that, like other exciting advances in molecular biology, this finding may soon be translated into a practical gain, although at present it is clearly far removed from the clinic.

REFERENCES 1. Begg, A.C., McNally,

N.J., Shrieve, D.C., Karcher, H.: A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6: 620-626, 1985.

2. Bush, R.S., Jenkin, R.D., Allt, W.E.C., Beale, F.A., Bean, H., Dembo, A.J., Pringle, J.F.: Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br. J. Cancer 37(Suppl III): 302-306, 1978.

S28

I. J. Radiation Oncology 0 Biology 0 Physics

3. Chapman, J.D., Franko, A.J., Koch, C.J.: The fraction of hypoxic clonogenic cells in tumor populations. In Biological

Bases and Clinical Implications of Tumor Radioresistance, G.H. Fletcher, C. Neri, H.R. Withers (Eds.). NY, Mason Pub. 1983, pp. 61-73. 4. Chapman, J.D., Franko, A.J., Sharplin, J.: A marker for hypoxic cells in tumors with potential clinical applicability. Br. J. Cancer 43: 546-550, 198 1. 5. Dische, S., Anderson, P.J., Sealy, R., Watson, E.R.: Carcinoma of the cervix-anaemia, radiotherapy and hyperbaric oxygen. Br. J. Radiol. 56: 251-255, 1983. 6. Moulder, J.E., Rockwell, S.: Hypoxic fractions of solid tumors; experimental techniques, methods of analysis and a survey of existing data. Int. J. Radiat. Oncol. Biol. Phys.

10: 695-7 12, 1984. 7. Olive, P.L., Durand, R.E.: Fluorescent nitroheterocyles for identifying hypoxic cells. Cancer Res. 43: 3276-3280, 1983. 8. Overgaard, J.: Misonidazole combined with split-course radiotherapy in the treatment of invasive carcinoma larynx and pharynx. In Progress in Radio-Oncology III, K.H. Karcher, H.D. Kogelnik, T. Szepesi (Eds.). New York, Raven Press. 1987 (In press).

1988 Volume 14, Supplement I

9. Peters, L.J., Brock, W.A., Johnson,

T., Meyn, R.E., Tofilon, P.J., Milas, L.: Potential methods for predicting tumor radiocurability. Int. J. Radiat. Oncol. Biol. Phys. 12: 459-

467, 1986. S., Sharipova, M., Kolycheva, 10 Revesz, L., Balmukhanov, N.: Vascularity as a predictor of radiation response: Its possible practical usefulness in selecting cases for treatment with radiosensitizers. In Progress in Radio-Oncology III, K.H. Karcher, H.D. Kogelnik, T. Szepesi (Eds.). New York, Raven Press. 1987 (In press). 11 Streffer, C., van Beuningen, D., Gross, E., Schabronath, J., Eigler, F.W., Rebmann, A.: Predictive assays for the therapy of rectum carcinoma. In Progress in Radio-Oncology III, K.H. Karcher, H.D. Kogelnik, T. Szepesi (Eds.). New York, Raven Press. 1987 (In press). and TNM classification of 12. Taskinen, P.J.: Radiotherapy cancer of the larynx. Acta Radiol. 287, (Suppl.): I- 12 1, 1969. 13. Wilson, G.D., McNally, N.J., Dunphy, E., Pfragner, R., Karcher, H.: The labelling index of human and mouse tumors assessed by bromodeoxyuridine staining in vivo and in vitro and flow cytometry. Cytometry 6: 641-647, 1985.