Edward L. Gillette

Effect of heat and radiation on synchronous Chinese hamster cells: killing and repair.

GERWECK, L. E., GILLETTE, E. L., AND DEWEY, W. C. Effect of Heat and Radiation on Synchronous Chinese Hamster Cells: Killing and Repair. Radiat. Res. 64, 611-623 (1975). Synchronous Chinese hamster cells were heated to 45.500C before, during, or after Xirradiation. The lethal effects of heat and radiation treatment were determined by the capacity of single cells to form colonies. Heat treatment prior to irradiation during G, or S was more effective, in terms of cell killing than heat treatment during or after irradiation. The time interval between irradiation and subsequent heat treatment influenced survival. The half-time for maximal survival when heat followed irradiation was 20-30 min in both G1 and S phase cells. Survival of cells which were heated, and then irradiated after heat treatment, indicated an independent, additive or synergistic interaction between heat and radiation, depending on the time of heat treatment and the phase of the cell cycle treated. Nine minutes of heat treatment during G, or 7 min during S decreased the shoulder region and increased the slope of the radiation survival curves. But most important, 7 min of heat prior to irradiation radiosensitized the relatively radioresistant S phase cells more than the relatively radiosensitive G, cells, and thus virtually eliminated the differences in radiosensitivity normally observed between G1 and S. Repair of heat damage as determined by survival to subsequent irradiation, began approximately 6 hr after 9 min heat treatment during Gx, and approximately 12 hr after 7 min heat treatment during S. In both cases, heat damage was repaired in the absence of cell division; for heating in Gi, repair occurred during Gi, but for heating during S, repair may have been associated with movement into G2. Cells exposed to split heat treatments during S repaired heat damage while they were still in S phase, and maximum survival was attained when 6 hr elapsed between the heat treatments.

Peripheral neuropathies following experimental intraoperative radiation therapy (IORT)

Injury to peripheral nerves in the lumbar para-aortic region was evaluated in beagle dogs 2 years following fractionated irradiation (EBRT), intraoperative irradiation (IORT), or a combination of IORT and EBRT. Time to onset of peripheral neuropathy was determined by means of serially completed neurological and electrophysiological examinations. Peripheral neuropathies were seen beginning as early as 6 months following 35 Gy (or greater) IORT only and 35 Gy plus 50 Gy EBRT. The incidence of peripheral neuropathies increased with increasing IORT doses beginning at 15 Gy. Onsets of peripheral neuropathies following IORT alone were clustered between 6 and 18 months, with onset in some dogs occurring as late as 24 months. The combination of IORT and EBRT resulted in an incidence and latency to onset of neuropathies similar to that seen with IORT alone. Neuropathies were not seen with EBRT alone at doses from 50 Gy to 80 Gy. Recovery of nerve function did not occur in affected dogs. Histological studies of nerves 2 years following irradiation demonstrated loss of axons and myelin, with a corresponding increase in endoneurial, perineurial, and epineurial connective tissue. Percentage of axon and myelin decreased to about 60% of normal at 15 Gy IORT, and additionally at higher doses. An insignificant decrease in percentage of axon and myelin was seen following EBRT alone. A significant lesion occurring in and around nerves at most IORT doses was necrosis and hyalinization of the media of small arteries and arterioles. The dose for a 50% probability for causing severe vessel lesions in the 2-year study was 19.5 Gy IORT only and 18.7 Gy when IORT was combined with EBRT. These lesions were not seen with any EBRT only dose. These studies suggest that peripheral nerve is a dose limiting normal tissue in IORT. Neuropathies appear to result from direct effects of irradiation on nerve and secondary effects to nerve resulting from damage to regional vasculature.

Morphometric analyses of the microvasculature of tumors during growth and after x-irradiation.

Morphometric methods were used to investigate changes in the microvasculature of C3H/Bi mammary carcinomas during tumor growth and following single‐dose x‐irradiation of 6 mm average diameter tumors. Estimates included per cent vascular volume, average vessel diameter, and mean vessel length and surface area per unit volume of viable tumor tissue. Necrotic tissue volume was quantitated also. Changes in vascular parameter estimates, particularly a decrease in vascular surface area, indicated that the microvasculature may become less capable of handling the exchange of essential nutrients at an early stage in tumor growth. Following irradiation, there was a transient improvement in colloidal‐carbon‐filling of tumor microvasculature, and a change in morphological character of vessels toward that seen in smaller tumors, which may improve capability for exchange of essential nutrients. The results support the conclusion that these quantitative methods are of value in studying time and dose relationships in radiotherapy or chemotherapy, particularly in conjunction with other radiobiological methods.

Response of aorta and branch arteries to experimental intraoperative irradiation

Injury to the aorta was evaluated in dogs 2 and 5 years after fractionated irradiation (EBRT), intraoperative irradiation (IORT) or a combination. Doses greater than 20 Gy IORT combined with 50 Gy EBRT given in 2 Gy fractions or 30 Gy IORT alone were accompanied by a significant risk of aneurysms or large thrombi as determined at necropsy 4 to 5 years following irradiation. Narrowing of the aorta as detected by aortography occurred at 5 years but was not detected earlier. The ED50 for aortic narrowing was 38.8 Gy IORT and 31 Gy IORT plus 50 Gy EBRT. The ED50 for branch artery injury was 24.8 Gy IORT alone and 19.4 Gy IORT plus 50 Gy EBRT. The difference in ED50s for IORT alone and IORT plus EBRT indicates that the contribution of the EBRT dose in terms of an IORT dose for aortic narrowing was 7.8 Gy and for branch artery injury was 5.4 Gy. The ED50 for incidence of small thrombi in the aorta was about 29 Gy for IORT alone and 23.5 Gy for IORT combined with EBRT. Fibrous thickening of the adventitia was measured and the effect of the 50 Gy EBRT component of a combination of EBRT and IORT was determined to be equivalent to 10 to 12 Gy IORT. Based on the various estimates, IORT doses of 10-15 Gy have an effect of 5 times or greater the amount given in 2 Gy fractions. At all EBRT doses and at lower IORT doses the intima was greatly thickened. At IORT doses of 20 Gy or above there was a dose related decrease in intimal thickness to near normal values. This was probably due to cell killing or inhibition of intimal proliferation that predominated at higher doses. Although the risk of serious vascular complications appears low following IORT of humans, this may be due to short observation times and the fact that IORT doses currently used are usually 20 Gy or less; this may be near the tolerance for late response of larger arteries. Only one dog in this study had complete rupture of the aorta causing death. Five other dogs at high IORT doses had near ruptures of the aorta but were clinically normal.

Effect of heat and ionizing radiation on normal and neoplastic tissue of the C3H mouse.

THRALL, D. E., GILLETTE, E. L., DEWEY, W. C. Effect of Heat and Ionizing Radiation on Normal and Neoplastic Tissue of the C3H Mouse. Radiat. Res. 63, 363-377 (1975). The radiation response of the skin of the C3H mouse was evaluated in terms of the dose of radiation required to produce moist desquamation completely surrounding the lower aspect of the hind leg by 21 days following irradiation (DD50-21). Irradiation of the leg under various conditions of local tissue oxygenation indicated that when the animals were breathing air (ambient conditions), the cells in the skin were not fully oxygenated. Heat was administered by immersing the leg for 15 min in 44.50C water either immediately prior to or immediately following irradiation under various conditions of local tissue oxygenation. Heat administered following irradiation reduced the DD50-21 values by 724 rad for hyperbaric 02, 1210 rad for ambient, and 1656 rad for hypoxic conditions. Approximately these same rad equivalents were observed when heat was administered prior to irradiation, under hyperbaric 02 and hypoxic conditions. However, administration of heat prior to irradiation under ambient conditions sensitized the cells to the effects of ionizing radiation. This sensitization was assumed to result from heat causing an increase in local tissue oxygenation prior to and at the time of irradiation. The effect of the heat dose administered under acute hypoxic conditions immediately prior to acute hypoxic irradiation was not significantly different from the protocol where heat was administered under ambient conditions immediately prior to acute hypoxic irradiation. This indicates an independence of the magnitude of the heat effect on the tissue oxygenation status at the time of heating. The response of the C3H mouse mammary adenocarcinoma to combined wet heat (A) and X-radiation (X) administered under either hypoxic, ambient or hyperbaric 02 conditions of local tissue oxygenation was studied.

Intra-arterial cisplatin with or without radiation in limb-sparing for canine osteosarcoma

Methods. Forty‐nine dogs with spontaneously occurring osteosarcoma underwent limb‐sparing surgery after preoperative therapy consisting of intra‐arterial cisplatin alone or intra‐arterial cisplatin in combination with doses of radiation from 20–40 Gy in 10 fractions. All resections were marginal, and the defect was repaired with a cortical allograft.

Hyperthermia and radiation—A selective thermal effect on chronically hypoxic tumor cells in vivo

Abstract Thermal radiosensitization was evaluated by constructing survival curves for radiation doses required to control C3H mouse tumors (tumor control dose for 50% of the animals (TCD 50 )) and for doses required for moist desquamation in 50% of the legs (DD 50 ). A 15 min heat treatment at a water bath temperature of 44.5°C was delivered under ambient conditions (mice breathing air, and the hind leg with the tumor not clamped) immediately after irradiation under ambient, hypoxic, or hyperbaric OZ conditions. The analysis from survival curves suggested that thermal treatment after irradiation had a similar effect on oxygenated and acutely hypoxic skin and tumor cells. However, the chronically hypoxic tumor cells, i.e. those not oxygenated under hyperbaric O 2 , either were selectively killed or selectively radiosensitized by the thermal treatment.

Intraoperative irradiation of the canine abdominal aorta and vena cava

The canine abdominal aorta and vena cava were examined 6 months after single doses of intraoperatively delivered electrons (IORT), fractionated external beam X rays, or a combination. The predominant pathologic change in aortas given fractionated doses was a segmental thickening of the subendothelial region of the tunica intima which was due to fibroelastic proliferation. In severe cases, the intimal proliferation caused significant narrowing of the aortic lumen. The greatest proliferation and lumen narrowing resulted from 80 Gy given in 30 fractions, whereas 60 Gy produced little response. In contrast, IORT alone or combined with fractionated doses resulted in mild subendothelial intimal proliferation at all doses. In some aortas there was focal aortic wall thinning after IORT alone or combined with fractionated doses. This response may be explained by increased intimal cell death and lost or delayed proliferative capability caused by large single doses. These studies suggest that large single doses produce structural alterations in the walls of large blood vessels that are clinically undetectable at early post-irradiation times. If these changes progress in severity they could lead to late effects such as rupture, fissure, or aneurysm that are clinically more significant than the marked intimal proliferation and lumen narrowing changes seen after fractionated doses. The aortic cell responsible for intimal fibroelastic proliferation appears to be a pluripotential stem cell capable of producing fibrous, elastic, and possibly smooth muscle tissue. There were no significant alterations in any of the irradiated vena cavas.

Percent tumor necrosis as a predictor of treatment response in canine osteosarcoma

The percent tumor necrosis was determined in 200 dogs with spontaneously occurring osteosarcoma. One hundred dogs had no treatment before amputation or death. One hundred other dogs were treated with either radiation therapy alone (n = 23), intraarterial (IA) cisplatin alone (n = 16), intravenous (IV) cisplatin alone (n = 6), radiation therapy plus IA cisplatin (n = 47), or radiation therapy plus IV cisplatin (n = 8). Eighty‐nine of these 100 dogs had their tumors resected 3 weeks after the end of therapy (6 weeks after the initiation of therapy) and replaced with a cortical bone allograft. Dogs with preoperative treatment were evaluated for local tumor control and time to metastasis. The mean percent tumor necrosis in untreated osteosarcoma was 26.8%. The mean percent tumor necrosis for dogs receiving radiation only, IA cisplatin only, and IV cisplatin only was 81.6%, 49.1% and 23.8%, respectively. The mean percent tumor necrosis for dogs receiving radiation therapy plus IA cisplatin or radiation therapy plus IV cisplatin was 83.7% and 78.2%, respectively. There was no significant difference between percent tumor necrosis in untreated osteosarcoma compared with those receiving IV cisplatin, but there was a significant increase in percent tumor necrosis with all other treatments. A mathematic model for the effect of cisplatin and radiation dose was developed using multiple regression analysis. The radiation dose calculated to cause at least 80% tumor necrosis was 42.2 Gy (95% confidence interval [CI], 38.0 to 47.6 Gy) when radiation was given alone and 28.1 Gy (95% CI, 21.3 to 36.6 Gy) when radiation was combined with IA cisplatin. Areas of viable tumor tended to be most frequent adjacent to the articular cartilage and in the joint capsule. Percent tumor necrosis was strongly predictive for local tumor control; 28 of 32 dogs with greater than 80% tumor necrosis had local control, and only eight of 29 dogs with less than 79% tumor necrosis had local control (P = 0.0047). There was no correlation between percent tumor necrosis and time to metastasis.

Bone necrosis and tumor induction following experimental intraoperative irradiation

The bone of the lumbar vertebrae of 153 dogs was examined 2 and 5 years after intraoperative irradiation (IORT), fractionated external beam irradiation (EBRT), or the combination. Groups of dogs received 15 to 55 Gy IORT only, 10 to 47.5 Gy IORT combined with 50 Gy EBRT in 2 Gy fractions or 60 to 80 Gy EBRT in 30 fractions. Six MeV electrons were used for IORT, and EBRT was done using photons from a 6 MV linear accelerator. The paraaortic region was irradiated and the ventral part of the lumbar vertebrae was in the 90% isodose level. Two years after irradiation, the dose causing significant bone necrosis as determined by at least 50% empty lacunae in the vertebral cortex was 38.2 Gy IORT alone and 32.5 Gy IORT combined with EBRT. Five years after irradiation, the dose causing 50% empty lacunae was 28.5 Gy IORT only and 14.4 Gy IORT combined with EBRT. The ED50 for lesions of the ventral vertebral artery was 21.7 Gy IORT only and 20.1 Gy IORT combined with 50 Gy EBRT 2 years after irradiation and 27.0 Gy IORT only and 20.0 Gy IORT combined with 50 Gy EBRT 5 years after irradiation. All lesions after EBRT only were mild. Eight dogs developed osteosarcomas 4 to 5 years after irradiation, one at 47.5 Gy IORT only and the remainder at 25.0 Gy IORT and above combined with 50 Gy EBRT. In conclusion, the extent of empty lacunae, indicating bone necrosis, was more severe 5 years after irradiation than after 2 years. The effect of 50 Gy EBRT in 2 Gy fractions was equivalent to about 6 Gy IORT 2 years after irradiation and to about 14 Gy 5 years after irradiation. Based on these estimates, IORT doses of 10 to 15 Gy have an effect 5 times or greater than the amount given in 2 Gy fractions. Osteosarcomas occurred in 21% of dogs which received doses greater than 25 Gy IORT. Doses of 15 to 20 Gy IORT in combination with 50 Gy EBRT in 2 Gy fractions may be near the tolerance level for late developing bone injury.