David G. Hirst

Anemia: A problem or an opportunity in radiotherapy?

Anemia may often become a problem in the treatment of the cancer patient. There are insufficient clinical data to assess the overall importance of anemia in radiotherapy, but there is clear evidence that uncorrected anemia is detrimental to local tumor control in some sites. There may be situations, however, when the transfused, previously anemic patient is at an advantage. These patients have shown a dramatically better response than non-anemic patients when radiotherapy for cancer of the cervix was given in hyperbaric oxygen. Animal experiments suggest that adaptive processes may be responsible for this effect. There is an important difference between acute and chronic anemia in their influence on the radiosensitivity of mouse tumors; while acute anemia consistently causes radioresistance, this effect is lost as the duration of the anemia prior to irradiation is prolonged. This would suggest that anemia per se should not cause tumor radioresistance in the chronically anemic patient. Blood transfusion in previously anemic animals has been shown to produce a markedly increased tumor radiosensitivity, but again this is only transient and sensitivity returns to normal when the interval between transfusion and irradiation is extended to 24 hrs. The mechanisms responsible for tumor adaptation to anemia and blood transfusion are not known, but there is evidence that changes in diffusion distances occur within tumors in response to alterations in oxygen availability and that changes in blood chemistry through the 2,3-DPG system may alter the release of oxygen to the tissues. These are complex processes and it remains to be determined what influence they have in the treatment of human cancer. However, the animal data suggest a clear benefit of blood transfusion to restore the hemoglobin level in radiotherapy, but they also emphasize the need to irradiate immediately so that adaptive mechanisms cannot erode the effect.

Changes in misonidazole binding with hypoxic fraction in mouse tumors

Binding of misonidazole (MISO) or a derivative to hypoxic cells in tumors has been proposed as a method for identifying tumors and measuring their level of hypoxia. We have recently shown that the hypoxic fraction of tumor cells can be altered over a wide range in vivo by acutely changing the hematocrit of the host animal by transfusion. The present study aimed to investigate the changes in binding by 14C MISO that accompanied this procedure. Tumor bearing mice were injected with 14C MISO, irradiated with a single dose of X rays (20 Gy) and their tumor excised and bisected. One half of each tumor was used to determine cell survival in vitro, the other was used for 14C scintillation counting. As previously described, tumor cell survival was dramatically increased in acutely anemic mice and this was accompanied by an increase in 14C MISO binding to the tumors. The relationship between clonogenic cell survival and binding was found to be linear on a log-log plot for each of the tumor lines studied, but the slopes of the lines were different tumor lines and generally steeper than the value of 1.0 expected for a 1:1 correspondence between cells binding radioactivity and radiobiological resistance. We attribute these differences to MISO binding to cells in the tumor which were not clonogenic.

The Effect of Alterations in Haematocrit on Tumour Sensitivity to X-rays

Hypoxic cells in human tumours probably contribute to the failure of radiotherapy in some sites. Changes in the oxygen carrying capacity of the blood, such as in anaemia, have been shown to influence tumour response. The effect of acute and chronic changes in haematocrit on the radiosensitivity of three mouse tumours (EMT6, KHT and RIF-1) were studied. Alterations in haematocrit were achieved by bleeding followed by retransfusion. When radiation was preceded immediately by an acute reduction in haematocrit (anaemia), radiosensitivity was markedly reduced in each tumour. An acute rise in haematocrit (polycythaemia) increased or decreased X-ray sensitivity depending on its severity. The optimum haematocrit for maximum sensitivity was always found to be at a level 5-10 per cent above normal. When the time between induction of anaemia and irradiation was increased, simulating a progressively longer duration of anaemia, marked changes in radiosensitivity of all the tumours were observed. A short duration of anaemia resulted in a resistant tumour with each cell line, but the resistance was gradually lost as the anaemia was prolonged, even though no recovery in haematocrit occurred. The rate of recovery to normal radiosensitivity varied from 24 to 72 hours in the different tumours. Therefore, only haematocrit changes which occurred within 1-3 days of a dose of radiation affect the radiosensitivity of these tumours.

Oxygen delivery to tumors

Sensitization of hypoxic cells in tumors, by increasing their oxygen supply, has been attempted for at least 30 years. Only the use of hyperbaric oxygen has been shown unequivocally as a beneficial adjunct to radiotherapy; and even then, the number of sites sensitized is limited to head and neck and cervix. It is not clear whether this implies that all other tumors reoxygenate fully during treatment, or whether a better method would sensitize other sites. Nevertheless, the elimination of hypoxic cells is viewed by many as a worthy goal in radiobiology and many strategies have been tested in animal systems. These include: oxygen releasing chemicals, artificial oxygen carriers, inhibitors of oxygen consumption, blood flow modifiers, or the exploitation of tumor adaptation to altered oxygen availability. We must be aware that any procedure which improves tumor oxygenation will not only increase radiosensitivity, but will induce an adaptive response in the tumor such that, sensitization will be of limited duration. It is likely that in the apparent failure of measures to improve substantially the oxygen delivery to tumors, the elimination of most of the hypoxic cells, of the type accessible to them, may have been achieved. If, as has been suggested, there are two distinct types of hypoxic cells, a combination of more than one strategy may be necessary to achieve more substantial gains.

Calcium antagonists as radiation modifiers: Site specificity in relation to tumor response pauline

The calcium antagonists, verapamil, nifedipine, and flunarizine, were studied for their effects on blood flow and radiation response in SCCVII/St intradermal back tumors, over a dose range of 0.05-50 mg/kg. Verapamil, at low doses, increased tumor blood flow, as measured by relative fluorescence intensity of Ho33342 stain, and increased tumor radiosensitivity. However, at doses of 20 mg/kg and above, verapamil reduced Ho33342 fluorescence intensity, and increased tumor radioresistance. Nifedipine reduced tumor radiosensitivity and Ho33342 fluroscence intensity at doses above 1 mg/kg, but below this dose increased Ho33342 intensity was observed and a small radiosensitization was apparent. Flunarizine sensitized tumors to X rays at all doses tested, although increased Ho33342 intensity was seen only at 5 mg/kg. The relative affinities of these compounds for different sites within the host may explain the variations in blood flow and radiation sensitivity in this tumor system.

The Adaptive Response of Mouse Tumours to Anaemia and Retransfusion

We have developed exchange transfusion methods to alter the hematocrit of tumour-bearing mice. The effects of anaemia and its correction by blood transfusion on the radiosensitivity of two mouse tumours (SCCVII/St and RIF-1) were studied using excision, in vivo/in vitro assay. An acute reduction in haematocrit caused a high degree of radioresistance equivalent to an increase in the hypoxic fractions by factors of 10 (SCCVII/St) and 30 (RIF-1). As the duration of the anaemia was prolonged, radioresistance was lost until within about 6 h normal radiosensitivity was observed even though the anaemia persisted. The restoration of the normal haematocrit by red blood cell transfusion after 24 h of anaemia caused increased radiosensitivity equivalent to a reduction in the hypoxic fraction by factors of 5 (SCCVII/St) and 10 (RIF-1), but again the effect was transient and normal radiosensitivity was re-established within 24-48 h of retransfusion. Measurements of 14C misonidazole (MISO) binding to RIF-1 tumours after these procedures indicated changes in the number of hypoxic cells which were qualitatively almost identical to those using the cell survival endpoint, leading us to believe that changes in oxygenation were responsible for the altered radiosensitivity. We feel that transfusion procedures could be used to advantage in the radiotherapy of some cancers.

Chlorophenoxy acetic acid derivatives as hemoglobin modifiers and tumor radiosensitizers

We have studied the influence of the antilipidemia drug, clofibrate, and several structurally related analogs on the binding affinity of hemoglobin for oxygen and the radiation sensitivity of the SCCVII/St carcinoma in the mouse. Several compounds in this class reduced hemoglobin affinity in vivo; and two of these, ML1024 (etophyline clofibrate) and ML1037, were at least as effective as clofibrate at reducing hemoglobin affinity and much less toxic. When given orally at a dose of 4.1 m mole/Kg, 1/2-2 hrs before 20 Gy X rays, clofibrate gave radiosensitization in the SCCVII/St tumor equivalent to a 40-fold reduction in hypoxic fraction. ML1024 and ML1037 at a dose (3.0 m mole/Kg), which had a similar effect on hemoglobin, gave much less sensitization of the tumor. Only ML1024 produced a statistically significant effect, equivalent to a four-fold reduction in hypoxic fraction. We conclude that there are several clofibrate analogs which in relation to their toxicity are much better hemoglobin modifiers than the parent compound. They do not, however, show the same radiosensitizing effects, leading us to believe that mechanisms other than changes in hemoglobin/oxygen binding must also be involved.

The therapeutic potential of misonidazole enhancement of alkylating agent cytotoxicity.

The effect of prolonged exposure to low misonidazole (MISO) levels on the cytotoxicity of three alkylating agents was studied in mouse tumors. A concentration of 100 micrograms/ml was maintained in the plasma for 7 hr by multiple injections of MISO. Cyclophosphamide (CYC). Melphalan (L-PAM), or CCNU were given after 4 hrs of MISO exposure. In each case, prolonged low level MISO exposure enhanced tumor response as measured by regrowth delay or a cloning assay. The effect of this treatment was also studied in several normal tissues: bone marrow, white blood cell counts, and spermatogonia. In none of these was any enhancement seen after prolonged MISO exposure. These encouraging results show that clinically relevant exposures to MISO can greatly improve tumor response to alkylating agents without increased normal tissue toxicity.

The effect of timing on chemosensitization by clinical levels of SR-2508

The nitroimidazole SR-2508 is currently being tested clinically as a radiosensitizer. Its relatively low toxicity allows it to be used at higher doses than misonidazole so that its potential as a chemosensitizer is also of considerable interest. Multiple injections of SR-2508 were given to SCC VII/St tumor-bearing mice to achieve a clinically realistic plasma concentration of approximately 300 micrograms/ml over 8 hrs. Single doses of melphalan (L-PAM) or cyclophosphamide (CY) were given at different times after the first SR 2508 injection. With L-PAM, a delay of at least 2 hr was necessary before enhancement of L-PAM cytotoxicity was observed. A similar result was obtained when a simulation was carried out with SCC VII/St tumor cells in vitro. Results with CY were less clear, although the most consistent enhancement was observed when a 4 to 8 hr interval elapsed between the beginning of SR 2508 exposure and the CY injection. In general, although precise timing was not essential for enhancement, an interval of at least 4 hr is recommended between the administration of SR 2508 and either alkylating agent. This is particularly important for L-PAM where no enhancement would be expected if the drugs were given simultaneously.

Enhancement of melphalan cytotoxicity in vivo and in vitro by inhibitors of poly (ADP-ribose) polymerase

In these preliminary experiments, we have found enhanced cell killing by the bifunctional alkylating agent L-phenylalanine mustard (L-PAM) in the presence of inhibitors of poly (ADP-ribose) polymerase (ADPRP) in vitro. In vivo enhancement of the tumoricidal effects of L-PAM was observed with the ADPRP inhibitor nicotinamide (1000 mg/kg), although enhanced myelosuppression was also demonstrated. Nicotinamide also increased the plasma elimination half-life of L-PAM by a factor of at least 2. This alteration of L-PAm pharmacokinetics makes it difficult to assess the role that ADPRP inhibition plays in the enhancement of L-PAM tumor cell killing in vivo.