George M. Hahn

Treatment of superficial human neoplasms by local hyperthermia induced by ultrasound

This study was designed to test the safety and efficacy of local heating of superficial tumors by ultrasound. Twenty‐five patients were treated who had recurrent or metastatic tumor in superficial locations refractory to conventional treatment modalities. Heating was done with high intensity ultrasound fields produced by a 2 cm or 4 cm piezoelectric transducer at frequencies of 1‐3 MHz. Tumors were held at 43, 44, or 45 C center temperature for 30 minutes each treatment. Analysis of response was done after 6 treatments. Ultrasound and thermal fields were carefully mapped. Some nonuniformity of tumor heating was noted despite relatively uniform output of ultrasound energy by the transducer. Twenty‐one of the 25 patients were evaluable after 26 courses of local hyperthermia. Fourteen of 26 courses of treatment resulted in objective tumor response (54%) However, most responses were partial and median follow‐up time was short (5 weeks) because many patients had systemic disease and died or were put on chemotherapy after treatment. Eight of 12 lesions in patients with squamous cell carcinoma of the head and neck regressed. Response rate did not correlate with central tumor temperature. Treatment was, in general, well tolerated although 10/26 courses of treatment caused significant local pain. Pain always subsided after treatment. Five of 26 courses of treatment resulted in small (0.5 cm) cutaneous burns. Previous irradiation did not limit heating. We conclude that superficial tumors can be safely and effectively heated by ultrasound. Objective tumor responses were seen with hyperthermia alone, although we stress that, because of the short follow‐up time and, because most responses were partial, it is likely that the effects noted were transitory.

Repair of potentially lethal radiation damage in vitro and in vivo.

The repair of potentially lethal damage was studied in plateauphase cultures of human LICH cells in vitro and in NCTC-2472 mouse fibrosarcoma grown in vivo in both the ascites and solid forms. Solid tumors and old, slowly-growing ascites tumors repaired potentially lethal damage; the kinetics and amount of repair were similar to those found in plateau-phase cultures. Repair in vitro was associated with a change in the slope of the survival curve without an accompanying increase in the shoulder of the curve. The effects of repair of sublethal and potentially lethal damage were additive. Thus this repair is another factor which influences the response of tumors to fractionated irradiation.

Thermotolerance and Heat Shock Proteins in Mammalian Cells

One of the more intriguing aspects of thermal biology is the response of heated cells to subsequent exposures at elevated temperatures. It has been clearly demonstrated by Gerner and Schneider (1) and Henle and Leeper (2) that an initial heat dose can induce a temporary state of heat resistance. This state has been termed thermotolerance. If the initial heat dose is at temperatures greater than about 43?C, the transient resistance manifests itself only if an intervening period at 37?C is permitted. Maximum thermotolerance is reached around 6 hr after transfer to 37?C; tolerance maintains itself for some time, decays slowly, and disappears by perhaps 100 hr. Alternatively, if the initial heating is at temperatures lower than 43?C, thermotolerance can develop during the heating period. The crucial role of protein synthesis in the development of thermotolerance has been recognized for some time (3). Protein synthesis is inhibited at temperatures of 43?C or higher, but goes on at lower temperatures. Thermotolerance also cannot develop if protein synthesis is inhibited by means other than heat itself, as, for example, during incubation in medium in which water has been replaced by deuterium oxide (4) or if cells are at low temperature (1, 2). It has been known for a considerable time that heat induces synthesis of a family of proteins, usually referred to as heat shock proteins (hsp) (5). These proteins, perhaps as many as 20 or more, range in molecular weight from less than 20,000 to more than 100,000. Because of the important role that protein synthesis appears to play in the development of thermotolerance, as discussed in the previous paragraph, it is not surprising that experiments were performed to determine whether or not hsp may play a role in conferring heat resistance on cells. Indeed, several studies have now shown that in mammalian cells there is a good temporal correlation between the induction of heat shock proteins and the development of thermotolerance (6, 7). Furthermore, it has been demonstrated that thermotolerance can be

Chromosome aberrations induced by thymidine

Chromosome aberrations were observed in several lines of Chinese hamster cells treated with high concentrations (1 mM or greater) of thymidine. Gaps, constrictions, chromatid exchanges and breaks were most frequent, although chromosome breaks and exchanges were also seen. The effect of thymidine was concentration dependent, at least in the range tested (1 to 20 mM). Cells incubated in medium containing 2 mM thymidine did not grow; in medium containing 2 mM thymidine and 10−4 M deoxycytidine, growth was normal. Chromosome aberrations were still observed although at a reduced rate. Chromosome damage appears to be related to interruption of DNA synthesis as well as to DNA synthesis in the presence of an unbalanced pool of nucleosides.

Two or six hyperthermia treatments as an adjunct to radiation therapy yield similar tumor responses: results of a randomized trial.

From March 1984 to February 1988, 70 patients with 179 separate treatment fields containing superficially located (less than 3 cm from surface) recurrent or metastatic malignancies were stratified based on tumor size, histology, and prior radiation therapy and enrolled in prospective randomized trials comparing two versus six hyperthermia treatments as an adjunct to standardized courses of radiation therapy. A total of 165 fields completed the combined hyperthermia-radiation therapy protocols and were evaluable for response. No statistically significant differences were observed between the two treatment arms with respect to tumor location; histology; initial tumor volume; patient age and pretreatment performance status; extent of prior radiation therapy, chemotherapy, hormonal therapy, or immunotherapy; or concurrent radiation therapy. The means for all fields of the averaged minimum, maximum, and average measured intratumoral temperatures were 40.2 degrees C, 44.8 degrees C, 42.5 degrees C, respectively, and did not differ significantly between the fields randomized to two or six hyperthermia treatments. The treatment was well tolerated with an acceptable level of complications. At 3 weeks after completion of therapy, complete disappearance of all measurable tumor was noted in 52% of the fields, greater than or equal to 50% tumor reduction was noted in 7% of the fields, less than 50% tumor reduction was noted in 21% of the fields, and continuing regression (monotonic regression to less than 50% of initial volume) was noted in 20% of the fields. No significant differences were noted in tumor responses at 3 weeks for fields randomized to two versus six hyperthermia treatments (p = 0.89). Cox regression analyses were performed to identify pretreatment or treatment parameters that correlated with duration of local control. Tumor histology, concurrent radiation doses, and tumor volume all correlated with duration of local control. The mean of the minimum intratumoral temperatures (less than 41 degrees C vs. greater than or equal to 41 degrees C) was of borderline prognostic significance in the univariate analysis, and added to the power of the best three covariate model. Neither the actual number of hyperthermia treatments administered nor the hyperthermia protocol group (two versus six treatments) correlated with duration of local control. The development of thermotolerance is postulated to be, at least in part, responsible for limiting the effectiveness of multiple closely spaced hyperthermia treatments.

Rapid increases in inositol trisphosphate and intracellular Ca++ after heat shock

Heat shock (45 degrees C) caused a rapid (less than 1 min) release of inositol trisphosphate from the membranes of HA-1 CHO fibroblasts. The rise in inositol trisphosphate concentration was followed by an increase in intracellular free Ca++. In addition to the heat induced rise in intracellular free Ca++, we observed an increase in 45Ca++ influx following nonlethal heat shock (45 degrees C/10 min). The heat-induced increase in 45Ca++ influx was linearly related to membrane accumulation of phosphatidic acid, phosphoinositide metabolite that may be involved in Ca++ gating. These results suggest that the membrane may be the proximal target of heat shock; stimulation of rapid breakdown of polyphosphoinositides and subsequent increases in intracellular free Ca++ may provide a mechanistic insight into the pleiotropic effects of heat. In addition, the large increases in Ca++ influx could initiate a Ca++ dependent mechanism of thermal cell killing.

Effects of hyperthermia in a malignant tumor

The mechanisms of immediate and delayed tumor cell killing by hyperthermia were investigated in EMT‐6 neoplasms implanted in BALB/cKa mice. Radiofrequency electromagnetic fields were used to achieve a curative local dose of 44°C for 30 minutes. The tumors were sampled sequentially, during and after heat therapy, and studied by light and electron microscopy. Assays for cell survival, including cell cultures, were performed at various times after completion of therapy. Focal cytoplasmic swelling, rupture of the plasma membrane and peripheral migration of heterochromatin were observed 5 minutes after initiation of therapy and led to cytoplasmic fragmentation by the end of the treatment period (30 minutes). Necrosis of most cells occurred 2–6 hours after the end of treatment. At 48 hours, there were no recognizable tumor cells. A scar replaced the tumor bed 14 days later. Viable (clonogenic) tumor cells were still 2% of control levels at the end of therapy and then progressively decreased to 0.0003% at 48 hours, confirming the morphologic observations and indicating that factors other than the direct effect of heat on tumor cells contributed to complete tumor eradication. Our findings, coupled with previous studies, suggest that the immediate heat induced necrosis in this tumor occurs through the mechanisms of physical changes in the plasma membrane. The delayed (post‐therapy) cell death is likely due to modifications in the environment of the tumor bed.

Thermal sensitivity and resistance of insulin-receptor binding

Membrane proteins may be key targets in thermal cell killing. In the present study, insulin binding to cellular receptors has been used as a probe for membrane protein behavior after heat shock (42-45 degrees C). Heating and binding studies were carried out on monolayers of HA-1 Chinese hamster ovary cells. Binding was unaffected by temperatures below 43 degrees C, but above this temperature (43-45 degrees C) it was inhibited in a time-temperature dependent manner. The kinetics of inhibition of insulin binding were similar to those for cell inactivation. Scatchard analysis indicated a decrease in receptor number rather than affinity for insulin in heated cells. In cells made resistant to further heat treatment (thermotolerant cells) by a mild pretreatment dose of hyperthermia (45 degrees C/10 min) insulin binding was also made heat resistant. Heating appeared to act directly on the insulin receptor rather than indirectly on subsequent energy dependent processes such as internalization. The data thus indicate that the fate of at least one membrane protein is closely tied up with the ultimate destiny of the cell per se after heating.

Effects of Heat on Cell Calcium and Inositol Lipid Metabolism

Hyperthermia causes a large (three-to fivefold) increase in intracellular free calcium ([Ca2+]i) in HA-1 fibroblasts. Increased [Ca2+]i appears initially to be due to release of Ca2+ from an internal store, probably located in the endoplasmic reticulum. A subsequent influx of Ca2+ from the extracellular medium is then observed. These heat-induced changes in Ca2+ homeostasis are correlated with turnover of the phosphoinositides (PI), a class of phospholipids whose metabolism has been shown to regulate Ca2+ in a wide variety of cells (M. J. Berridge and R. F. Irvine, Nature 312, 315 (1984]. Hyperthermia induces rapid release of inositol 1,4,5-trisphosphate (IP3) within 1 min at 45 degrees C; IP3 release precedes the heat-induced rise in [Ca2+]i. IP3 release, a result of phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C, is the initial step in PI turnover. Later accumulation of phosphatidic acid, another metabolite in the PI pathway, is correlated with the delayed, heat-induced influx of 45Ca2+ from the extracellular environment. The data thus indicate that heat-induced changes in Ca2+ homeostasis are correlated with activation of PI turnover. They indicate that this class of lipids may be closely involved in heat-induced changes in cellular Ca2+ homeostasis. Cell Ca2+ appears to be important in some aspects of the cellular response to heat.

Chinese hamster cell monolayer cultures: I. Changes in cell dynamics and modifications of the cell cycle with the period of growth☆☆☆

Abstract An investigation was carried out to contrast cellular kinetics of Chinese hamster cells in exponential and in plateau periods of growth, because of the possibility that cells in the plateau period may constitute a better model of tumor cell populations than do logarithmically growing cultures. The rates of synthesis (per cell) of RNA and protein were independent of cell number. On the other hand, the rate of DNA synthesis was closely coupled to the growth period of the culture. DNA synthesis (per cell) was constant during exponential growth but dropped to 10 per cent of its former value just before the cell number reached its maximum, indicating that a large proportion of the plateau period cells were in a G1-like part of the cell cycle. Those cells in the plateau period which continued to synthesize DNA went on to divide. The newly born cells replaced dead cells that had lysed. Thus the cell number remained stationary. The cell cycle of the reproducing cells in the plateau period was found to be characterized by an extended G2 and an extended and highly variable S phase. M did not appear to be changed greatly and G1 could not be measured. The similarities between the plateau period cell renewal system and populations of tumor cells are pointed out.