Joan M. C. Bull

Interorgan glutamine metabolism in the tumor-bearing rat

The effects of progressive malignant disease on gut/liver glutamine metabolism were studied in order to gain further insight into the altered glutamine metabolism that characterizes the host with cancer and to further elucidate the causes and consequences of glutamine depletion in tumor-bearing rats. Rats were inoculated on Day 0 with 2 X 10(6) viable fibrosarcoma cells and blood glutamine was measured every 6 days. On Day 24 the animals underwent laparotomy and sampling of arterial, portal venous, and hepatic venous blood. Arterial glutamine fell by more than one-third in tumor-bearing rats and the arterial-portal venous concentration difference for glutamine across the intestinal tract was diminished by 50% (P less than 0.01). Simultaneously the fractional extraction of glutamine by the gut was reduced from 21 to 15% (P less than 0.05). The liver switched from an organ of near glutamine balance in control rats to one of marked glutamine output in tumor-bearing rats (P less than 0.01). The wet weight of the small intestine was diminished by 15% in tumor-bearing rats and villous height was uniformly decreased in tumor-bearing rats with an average reduction in villous height of 26% (P less than 0.05). The causes of glutamine depletion in this tumor-bearing rat model remain unclear. The growing tumor may behave as a glutamine trap but also appears to alter glutamine metabolism in vital metabolic processing centers such as the gut and liver. Malignant cells may compete with gut mucosal cells for glutamine resulting in a diminished gut glutamine utilization and detrimental changes in mucosal architecture.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of coagulation during therapeutic whole body hyperthermia.

Coagulation parameters were initially monitored in 8 patients receiving whole body hyperthermia (WBH). Patients were heated by the warm water blanket technique to 41.8 degrees C (Tmax), maintained at this temperature for 2 hours, then allowed to cool. A fall in platelets was apparent by the time Tmax was achieved and continued during the 18 hours after WBH. Levels of beta-thromboglobulin (BTG) and platelet factor 4 rose by 56% and 191% by the end of treatment but returned to baseline 18 hours later. Fibrinogen, plasminogen and alpha 2-antiplasmin levels declined and FDP and fibrinopeptide A (FPA) levels increased during WBH. Factor XII and Factor VIII:C fell moderately during WBH while Factors VIII R:Ag, VIII:RC and V did not change or showed a late rise. Factor VII levels fell in 7 of 8 patients, reaching levels of 30% of normal in four. To better define the sequence of these coagulations perturbations, earlier and more frequent timepoints were studied in an additional 3 patients. This revealed that decreases in fibrinogen and plasminogen and increases in FPA and BTG occur very early (by the time the patient reaches 39 degrees C). On the other hand, a decrease in Factor VII activity was not apparent until patients had reached Tmax. WBH is therefore associated with a consumption coagulopathy. Possible mechanisms are discussed and extrapolations to the situation seen in heat stroke are suggested.

Thermal enhancement of drug uptake and DNA adducts as a possible mechanism for the effect of sequencing hyperthermia on cisplatin-induced cytotoxicity in L1210 cells

An optimal scheduling of hyperthermia andcis-diammine-dichloroplatinum(II) (cisplatin) may increase the therapeutic gain of the combination of these two modalities. In this study, intracellular platinum accumulation, total platinum binding to DNA, and DNA interstrand crooslinks (ISC) were assayed to investigate the molecular mechanisms responsible for the effect of sequencing hyperthermia on the thermal enhancement of cisplatin-induced cytotoxicity in mouse leukemia L1210 cells. Simultaneous treatment with heat (41.5°C, 60 min) and cisplatin produced maximal cell killing with a 4-fold decrease in the 50% growth-inhibitory concentration (IC50) of the platinum complex. Super-additive cell killing was also shown when cells were exposed to heat before cisplatin treatment, whereas no thermal enhancement in cisplatin-mediated cytotoxicity was observed in cells given heat after exposure to cisplatin. These results correlated with the degree of formation of ISC observed in cells following various treatments. A 2-to 3-fold increase in ISC formation was observed in cells given heat before or during cisplatin exposure, whereas heat after cisplatin treatment did not alter either the formation or the reversal of ICC as compared with cisplatin alone. The increased ISC formation was associated with an increase in intracellular platinum accumulation and total platinum binding to DNA in cells given heat before or during cisplatin exposure. These data, showing that hyperthermia potentiates cisplatin cytotoxicity by increasing drug uptake and the formation of DNA adducts without inhibiting the repair of DNA lesions, demonstrate the potential utility of sequencing hyperthermia combined with cisplatin as a clinical anticancer therapy.

The effects of anaesthetics on cisplatinum-induced toxicity at normal temperatures and during whole-body hyperthermia: the influence of NaCl concentration of the vehicle

The effects of various anaesthetics, including a balanced combination of different anaesthetics (consisting of ketamine, xylazine, and acepromazine), Nembutal, and halothane anaesthesia on DDP-induced renal and intestinal toxicities at 37 degrees C and at 41.5 degrees C were studied using a F344 rat model. The combination anaesthesia decreased the DDP-induced lethality (LD50) and toxic side-effects as evidenced by decreased in BUN and diarrhoea at day 5, whereas nembutal anaesthesia increased DDP-induced toxic side-effects at 37 degrees C. The inhalation anaesthetic halothane had only minor influence on these DDP-induced toxicities. Increasing the NaCl concentration of the DDP vehicle from 0.9 to 1.8 per cent decreased the DDP-induced toxicity both in non-anaesthetized and anaesthetized animals. When applied simultaneously with DDP administration, whole-body hyperthermia (WBH; 120 min at 41.5 degrees C) increased the DDP-induced toxicity as indicated by the thermal enhancement ratio of between 2.1 and 2.7 for the LD50 values. With combined WBH + DDP treatment the effect of anaesthetics on DDP-induced toxicities was generally similar to that observed at 37 degrees C. The protective effects of the high NaCl (1.8 vs 0.9 per cent) concentration of the DDP vehicle, however, were minimal under hyperthermic conditions. The data suggest the need for caution in the use of DDP in combination with WBH.

Fever-range whole-body thermal therapy combined with cisplatin, gemcitabine, and daily interferon-α: A description of a phase I-II protocol

Purpose: The purpose of the Phase I component of this study was to find the maximally tolerated dose (MTD) of cisplatin administered within a regimen of fever-range whole body thermal therapy (FR-WB-TT), cisplatin, gemcitabine, and low-dose interferon-alpha (IFN-α). The Phase II component aimed to assess which cancer diagnoses responded to the regimen, the response rate, and response duration. Materials and methods: The protocol design derived from a schedule-optimized preclinical regimen. Drugs were administered together, and also with thermal therapy in a schedule that optimized the therapeutic index. Eligible patients were those with therapy-resistant, metastatic or advanced solid malignancies. Beginning at 40 mg/m2, the cisplatin dose was escalated by 10 mg/m2 to the maximally tolerated dose (MTD) in successive cohorts of 3 patients. A treatment cycle consisted of cisplatin on day one, followed by thermal therapy and simultaneous gemcitabine 36 hours later; then a second dose of gemcitabine one week later; and daily IFN- α. Results: Thirty-seven patients were treated on protocol. The MTD of cisplatin in the thermochemotherapy regimen was established to be 60 mg/m2. The dose limiting toxicities (DLT) were peripheral neuropathy and ototoxicity. Complete and partial responses combined were 43%. The therapy improved the quality of life of responding patients. Conclusion: The protocol was well tolerated and was associated with antitumor activity in patients with a variety of advanced metastatic solid tumors. Tumor response occurred with the thermochemotherapy treatment despite treating malignancies that had progressed on the same chemotherapy drugs administered as standard treatment. Notably, good responses were observed in patients with high-grade neuroendocrine and pancreas cancers. This regimen will be tested in a phase II study.

Long duration-mild whole body hyperthermia of up to 12 hours in rats: feasibility, and efficacy on primary tumour and axillary lymph node metastases of a mammary adenocarcinoma: implications for adjuvant therapy.

The feasibility and efficacy of low temperature (40 degrees C) long duration whole body hyperthermia (LL-WBH) was investigated in rats bearing a highly metastatic mammary adenocarcinoma (MTLn3). We compared the treatment effects of various durations of LL-WBH (40 degrees C for 2-12 h) to that of conventional short duration-high temperature WBH (SH-WBH, 41.5 degrees C for 2 h). SH-WBH, 2 h LL-WBH, and 4 h LL-WBH resulted in only modest primary tumour growth delays (TGDs) of 0.9, 1.1 and 1.8 d (days) respectively. In contrast, significantly increased TGDs of 2.8, 3.2, 2.6, and 3.1 d were achieved with 6, 8, 10 and 12 h LL-WBH, respectively (p < 0.05 compared to SH-WBH, 2 h-LL-WBH, and 4 h-LL-WBH). Notably, LL-WBH reduced the incidence of axillary lymph node metastasis at 14 days post-treatment, from 100% in normothermic controls and 92% after SH-WBH, to 33, 40, 50, and 60% following 4, 6, 8 and 10 h LL-WBH respectively. When the duration of LL-WBH was extended to 12 h, no reduction in axillary lymph node metastasis was observed. Normal tissue toxicity of LL-WBH appeared to be minimal and LL-WBH durations of up to 12 h were well tolerated. These data show that LL-WBH for durations of from 4 to 10 h has greater antitumour activity than SH-WBH, against mammary adenocarcinoma, suggesting that LL-WBH may have therapeutic potential in the treatment of malignant disease.

Therapeutic efficacy and apoptosis and necrosis kinetics of doxorubicin compared with cisplatin, combined with whole-body hyperthermia in a rat mammary adenocarcinoma.

We have compared the therapeutic efficacy as well as the kinetics of treatment‐induced apoptosis and necrosis of the maximum tolerated dose (MTD) of doxorubicin (DOX) or cisplatin (CDDP) combined with long‐duration, low‐temperature whole‐body hyperthermia (LL‐WBH, at 40.0°C for 6 hr), with the combination of the MTDs of either DOX or CDDP with short‐duration, high‐temperature WBH (SH‐WBH, at 41.5°C for 2 hr), in a rat mammary adenocarcinoma (MTLn3). The MTD of LL‐WBH + DOX resulted in increased therapeutic efficacy, compared with the MTD of DOX alone and SH‐WBH + DOX. The MTD of LL‐WBH + CDDP, however, did not increase therapeutic efficacy, when compared with the MTD of CDDP alone or SH‐WBH + CDDP. The MTD of LL‐WBH + DOX caused a significant delay in the development of spontaneous axillary lymph node (ALN) metastasis and tended to cause longer mean survival, compared with SH‐WBH + DOX. The peak of treatment‐induced apoptosis was higher for the MTD of DOX + LL‐WBH, compared with SH‐WBH + DOX, whereas the apoptosis peak of the MTD of SH‐WBH + CDDP was higher than that of LL‐WBH + CDDP. The most extensive levels of tumor necrosis appeared to occur earlier with SH‐WBH alone and the MTD of SH‐WBH + DOX or CDDP than with other groups. Our results suggest that LL‐WBH + DOX may be a promising therapy for breast cancer, and the extent of treatment‐induced tumor apoptosis appears to correlate with antitumor response for MTDs of LL‐WBH + DOX and SH‐WBH + DOX, but not for the MTDs of CDDP with SH‐WBH or LL‐WBH. Int. J. Cancer 76:499–505, 1998.© 1998 Wiley‐Liss, Inc.

The effect of whole-body hyperthermia combined with 'metronomic' chemotherapy on rat mammary adenocarcinoma metastases

Many women diagnosed with invasive breast cancer have undetected occult metastases at the time of their primary tumour diagnosis. The development and growth of these micro-metastases relies heavily on angiogenesis. Therefore, administering an angiogenesis-blocking treatment from the time of diagnosis could reduce the incidence of metastasis and, ultimately, increase patient survival. It is hypothesized that an antiangiogenesis strategy combining fever-range whole-body hyperthermia (FR-WBH) and metronomic chemotherapy could inhibit the development of metastatic disease with minimal toxicity. To test this theory, a low, daily dose of the topoisomerase-I inhibitor irinotecan hydrochloride (CPT-11) was administered over a prolonged period of time to rats bearing the highly metastatic MTLn3 mammary adenocarcinoma primary tumour surgically excised on day 12 after implantation. The metronomic CPT-11 was combined with long-duration, low-temperature, fever-range whole body hyperthermia (FR-WBH). This systemic hyperthermia enhances chemotherapy-induced cytotoxicity as well as immunological activity. Both the group treated with FR-WBH alone and the combined FR-WBH+CPT-11 group had delayed onset and reduced incidence of axillary lymph node metastases compared to control ( p < 0.05). Combination therapy of FR-WBH+CPT-11 resulted in a significantly greater inhibition of axillary lymph node metastasis volume compared to both control and CPT-11 alone ( p < 0.02) at day 16. Interestingly, none of the therapies significantly affected inguinal lymph node metastases. Lung metastases were decreased by 36% at the time of death in rats treated with FR-WBH+CPT-11, by 25% in the CPT-11 alone group and by 14% in the FR-WBH alone group. Rats treated with FR-WBH, + CPT-11 survived significantly longer (35%) than control animals ( p < 0.04). Neither significant body weight loss nor gastrointestinal toxicity was observed in any group. These data suggest that, after excision of the primary tumour, FR-WBH and metronomic CPT-11 can be safely combined to reduce distant lymph node and lung metastases and, thus, to increase survival.

Long-duration, mild whole body hyperthermia with cisplatin : tumour response and kinetics of apoptosis and necrosis in a metastatic rat mammary adenocarcinoma

This study examines antitumour effect and induction of apoptosis and necrosis after treatment with long-duration, mild whole body hyperthermia (LL-WBH, 40.0 degrees C, for 6 h) in simultaneous combination with cisplatin (CDDP) on primary and metastatic tumour growth in a rat mammary adenocarcinoma. A significantly greater delay in primary mammary tumour growth was observed after treatment with LL-WBH + CDDP, compared to either modality alone (p < 0.05). LL-WBH alone caused a significant delay in spontaneous metastasis to the axillary lymph node (ALN) and LL-WBH + CDDP tended to further increase the delay in ALN metastasis. Survival was longest in rats receiving LL-WBH + CDDP, compared to other groups (p < 0.05). CDDP induced a peak of tumour apoptosis at 24 h after treatment beginning that was significantly greater than LL-WBH alone (p < 0.05). The peak of tumour apoptosis induced by LL-WBH + CDDP from 12 to 24 h was significantly greater than any other group (p < 0.01). These results suggest that the extent of treatment-induced apoptosis seems to correlate positively with antitumour response and the combination or LL-WBH with CDDP may lead to a promising adjuvant therapy for breast cancer.

Chemotherapy resistant sarcoma treated with whole body hyperthermia (WBH) combined with 1-3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

Seventeen patients with chemotherapy-resistant metastatic sarcoma were treated with whole body hyperthermia (WBH) combined simultaneously with 1-3-Bis(2-chloroethyl)-1-nitrosourea (BCNU). All of the patients had chemotherapy resistant metastases to major organ sites. Patients were heated to 41.8-42.0 degrees C for 2 h using an insulated blanket heating technique. Two patients (12%) experienced partial responses (PR). In addition, four objective tumour responses (OR) lasting more than 4 months were documented. One patient with previously rapidly growing chondrosarcoma pulmonary metastases experienced stable disease (SD) for 38 months from the onset of treatment. Median survival of seven patients with responding tumours (PR, OR and SD) compared with 10 patients with progressive disease was 15 versus 2 months, respectively. Cumulative thrombocytopenia was a therapy-limiting toxicity of the combined treatment, and occurred in six of seven patients. Acute toxicities attributable to WBH alone included transient thrombocytopenia in all patients, non-cardiogenic pulmonary oedema in two patients, and mild hypotension in five patients. Acute granulocytosis was observed in all patients. No treatment related deaths occurred. These data suggest that WBH combined with chemotherapy is associated with disease response in patients with chemotherapy-resistant, widely disseminated sarcoma metastases.