Expression of cyclin D1a and D1b as predictive factors for treatment response in colorectal cancer.

BACKGROUND: The aim of this study was to investigate the value of the cyclin D1 isoforms D1a and D1b as prognostic factors and their relevance as predictors of response to adjuvant chemotherapy with 5-fluorouracil and levamisole (5-FU/LEV) in colorectal cancer (CRC). METHODS: Protein expression of nuclear cyclin D1a and D1b was assessed by immunohistochemistry in 335 CRC patients treated with surgery alone or with adjuvant therapy using 5-FU/LEV. The prognostic and predictive value of these two molecular markers and clinicopathological factors were evaluated statistically in univariate and multivariate survival analyses. RESULTS: Neither cyclin D1a nor D1b showed any prognostic value in CRC or colon cancer patients. However, high cyclin D1a predicted benefit from adjuvant therapy measured in 5-year relapse-free survival (RFS) and CRC-specific survival (CSS) compared to surgery alone in colon cancer (P=0.012 and P=0.038, respectively) and especially in colon cancer stage III patients (P=0.005 and P=0.019, respectively) in univariate analyses. An interaction between treatment group and cyclin D1a could be shown for RFS (P=0.004) and CSS (P=0.025) in multivariate analysis. CONCLUSION: Our study identifies high cyclin D1a protein expression as a positive predictive factor for the benefit of adjuvant 5-FU/LEV treatment in colon cancer, particularly in stage III colon cancer.

Hyperthermia exhibits anti-vascular activity in the s.c. BT4An rat glioma: lack of interaction with the angiogenesis inhibitor batimastat.

We investigated the anti-vascular activity of local hyperthermia (44 degrees C, 60 min) in s.c. BT4An rat gliomas, and the influence on tumour growth of hyperthermia and the anti-angiogenic compound batimastat (30 mg/kg i.p.). Heat-induced vascular damage was assessed in small (82 mm3) and large (171 mm3) tumours using confocal microscopy and immunostaining for von Willebrand factor. The influence of hyperthermia on tumour blood flow was measured using the 86RbCl method. The anti-tumour activity of hyperthermia (one or two fractions a week apart) and batimastat (7 or 14 daily injections) and various combination schedules were examined. Hyperthermia disrupted 25-50% of the vasculature in the BT4An tumours, and the vascular damage was most extensive in the central part of the large tumours. The heated tumours exhibited a 40-60% blood flow reduction, which persisted until the last measurement after 24 h. One fraction of hyperthermia caused a significant growth delay of 4 days, compared with the control group (p = 0.01), but no additional tumour response was produced by a second heating session. Batimastat had no influence on tumour growth and the combination of drug and local heating did not enhance the tumour response, compared with heating alone. It was concluded that hyperthermia at 44 degrees C for 60min exhibits anti-vascular activity and inhibits tumour growth in the BT4An tumour model. Batimastat had no effect on tumour growth.

Referee: hyperthermia alone or combined with cisplatin in addition to radiotherapy for advanced uterine cervical cancer.

There are two evidence based therapeutic options for locally advanced cervical cancer: Radiotherapy and concurrent chemotherapy (cisplatin alone or combined with other drugs) or radiation and hyperthermia, documented in randomised trials. The weight of evidence is less for the most advanced stages. Combination of all three options are currently tested in several centres with good clinical response and acceptable toxicity. Based on a pragmatic approach we propose to proceed with a trial selecting cisplatin concurrent with radiation therapy as the standard arm to be compared with the same regimen with the addition of hyperthermia once a week.

Tumor vasculature is targeted by the combination of combretastatin A-4 and hyperthermia.

BACKGROUND AND PURPOSE: Combretastatin A-4 disodium phosphate (CA-4) enhances thermal damage in s.c. BT(4)An rat gliomas. We currently investigated how CA-4 and hyperthermia affect the tumor microenvironment and neovasculature to disclose how the two treatment modalities interact to produce tumor response. METHODS: By confocal microscopy and immunostaining for von Willebrand factor, we examined the extent of vascular damage subsequent to CA-4 (50 mg/kg) and hyperthermia (waterbath 44 degrees C, 60 min). The influence on tumor oxygenation was assessed using interstitial pO(2)-probes (Licox system) and by immunostaining for pimonidazole. We examined the direct effect of CA-4 on the tumor cell population by flow cytometry (cell cycle distribution) and immunostaining for beta-tubulin (cytoskeletal damage). RESULTS: Whereas slight vascular damage was produced by CA-4 in the BT(4)An tumors, local hyperthermia exhibited moderate anti-vascular activity. In tumors exposed to CA-4 3 h before hyperthermia, massive vascular damage ensued. CA-4 reduced the pO(2) from 36.1 to 17.6 mmHg (P=0.01) in the tumor base, and tumor hypoxia increased slightly in the tumor center (pimonidazole staining). Extensive tumor hypoxia developed subsequent to hyperthermia or combination therapy. Despite a profound influence on beta-tubulin organization in vitro, CA-4 had no significant effect on the cell cycle distribution in vivo. CONCLUSION: Our results indicate that the anti-vascular activity exhibited by local hyperthermia can be augmented by previous exposure to CA-4.

Vinblastine and hyperthermia target the neovasculature in BT(4)AN rat gliomas: therapeutic implications of the vascular phenotype.

PURPOSE: The antivascular and antitumor activity of vinblastine and hyperthermia at different tumor volumes were examined in the subcutaneous (s.c.) BT(4)An rat glioma model. METHODS AND MATERIALS: The influence of vinblastine (3 mg/kg) and hyperthermia (44 degrees C/60 min) on tumor growth was assessed in small (100 mm(3)) and large (200 mm(3)) BT(4)An tumors. To disclose how vinblastine and hyperthermia interacted in the neoplasms, tumor blood flow and the extent of vascular damage, hypoxia, cell proliferation, and apoptosis were assessed after treatment. The content of smooth muscle cells/pericytes in the tumor vasculature was examined in small and large tumors to assess how the vascular phenotype changed during tumor growth. RESULTS: In the large tumors, vinblastine reduced the blood flow, but the tumor growth was not affected. The combination of drug and local heating yielded massive vascular damage and a significant tumor response. The small neoplasms had a higher content of smooth muscle cells/pericytes in the vessel walls (host vasculature), and the tumor vasculature displayed a higher resistance to vascular damage than the large neoplasms. Yet, vinblastine alone exhibited a potent antiproliferative activity and induced massive apoptosis in the small tumors, and the drug significantly inhibited tumor growth. The addition of hyperthermia yielded no additional growth delay in the small tumors. CONCLUSION: The antivascular properties of vinblastine and hyperthermia can be exploited to facilitate vascular damage in BT(4)An solid tumors with a low content of host vasculature.

Combretastatin A-4 and hyperthermia;a potent combination for the treatment of solid tumors.

BACKGROUND AND PURPOSE: Attacking tumor vasculature is a promising approach for the treatment of solid tumors. The tubulin inhibitor combretastatin A-4 disodium phosphate (CA-4) is a new vascular targeting drug which displays a low toxicity profile. We wanted to investigate how CA-4 influences tumor perfusion in the BT4An rat glioma and how the vascular targeting properties of CA-4 could be exploited to augment hyperthermic damage towards tumor vasculature. MATERIAL AND METHODS: We used the (86)RbCl extraction technique to assess how CA-4 influences tumor perfusion, and the tumor endothelium was examined for morphological changes induced by the drug. We combined CA-4 (50 mg/kg i.p.) with hyperthermia (44 degrees C, 60 min) at different time intervals to evaluate how therapy should be designed to affect tumor growth, and we studied the tumors histologically to assess tissue viability. RESULTS: We found that CA-4 induced a profound, but transient reduction in tumor perfusion 3-6 h postinjection. If hyperthermia was administered 3-6 h after injecting CA-4, massive hemorrhagic necrosis developed, and tumor response was significantly enhanced compared to simultaneous administration of the two treatment modalities (P<0.005). CA-4 alone had no influence on tumor growth and failed to disrupt the vasculature of the BT4An solid tumors. Interestingly though, a mild endothelial edema was observed in some tumor areas 3 h after injecting CA-4. CONCLUSIONS: We conclude that the combination of CA-4 and hyperthermia is a potent therapeutic option for BT4An tumors, but the selection of adequate time intervals between CA-4 and hyperthermia are imperative to obtain tumor response.

The new tubulin-inhibitor combretastatin A-4 enhances thermal damage in the BT4An rat glioma.

PURPOSE: To investigate the toxicity of combretastatin A-4 disodium phosphate (CA-4) and its vascular effects in the subcutaneous (s.c.) BT4An rat glioma, and additionally, to determine the tumor response of CA-4 combined with hyperthermia. METHODS AND MATERIALS: For assessment of drug toxicity, rats were given 50, 75, or 100 mg/kg CA-4 and followed by daily registration of weight and side effects. Interstitial tumor blood flow was determined by laser Doppler flowmetry in rats injected with 50 mg/kg CA-4. In the tumor response study we administered CA-4 50 mg/kg alone or combined with hyperthermia (waterbath 44 degrees C for 60 min) 0 or 3 h later. RESULTS: We found that CA-4, at a well-tolerated dose of 50 mg/kg, induced a considerable time-dependent decrease in the tumor blood flow. Tumor blood flow was reduced by 47-55% during the first 110 min after injecting CA-4, and thereafter remained decreased until the measurements were terminated. Administering CA-4 3 h before hyperthermia yielded the best tumor response and increased tumor growth time significantly compared with simultaneous administration of CA-4 and hyperthermia (p = 0.03). Interestingly, CA-4 alone did not influence tumor growth. CONCLUSION: CA-4 induces a gradual reduction in tumor blood flow which can be exploited to sensitize the BT4An tumor for hyperthermia.

Double duplex invasion by peptide nucleic acid: a general principle for sequence-specific targeting of double-stranded DNA.

Pseudocomplementary PNAs containing diaminopurine.thiouracil base pairs have been prepared and are shown to bind with high specificity and efficiency to complementary targets in double-stranded DNA by a mechanism termed "double duplex invasion" in which the duplex is unwound and both DNA strands are targeted simultaneously, each by one of the two pseudocomplementary peptide nucleic acids (PNAs). On the basis of our results we predict that (for decameric targets) more than 80% of all sequences can be targeted by straightforward Watson-Crick base pairing by using this approach in its present form. Targeting of pseudocomplementary PNAs to the promoter of the T7 phage RNA polymerase effectively inhibits transcription initiation. These results have important implications in the development of gene therapeutic agents as well as for genetic diagnostic and molecular biology applications.

Quality assurance for radiofrequency regional hyperthermia.

Today most treatments with regional hyperthermia are applied using radiofrequency systems with 'focus' steering by amplitude and phase control. This paper deals with quality assurance procedures developed to ensure controlled and safe treatments in such systems. Our results show how the deviations between requested and observed phase and amplitude vary with frequency, and how these deviations depend on both the geometry of the object (phantom) inside the system and the power level applied. The results also indicate that the investigated systems' internal quality assurance procedures were inadequate and that additional procedures should be applied. Since the system parameters depend on patient and treatment specific conditions it is concluded that there is a need for QA measurements before or during treatment. This paper deals specifically with the commercial BSD-2000 system from BSD Medical Corp. in Salt Lake City, Utah, as installed in Bergen, but the procedure outlined can be applied to other phase and amplitude-controlled RF-RHT systems with only minimal adjustments.

Increased DNA binding and sequence discrimination of PNA oligomers containing 2,6-diaminopurine.

The synthesis of a diaminopurine PNA monomer, N-[N6-(benzyloxycarbonyl)-2,6-diaminopurine-9-yl] acetyl-N-(2-t-butyloxycarbonylaminoethyl)glycine, and the incorporation of this monomer into PNA oligomers are described. Substitution of adenine by diaminopurine in PNA oligomers increased the T m of duplexes formed with complementary DNA, RNA or PNA by 2.5-6.5 degrees C per diaminopurine. Furthermore, discrimination against mismatches facing the diaminopurine in the hybridizing oligomer is improved. Finally, a homopurine decamer PNA containing six diaminopurines is shown to form a (gel shift) stable strand displacement complex with a target in a 246 bp double-stranded DNA fragment.

Use of the vasodilator sodium nitroprusside during local hyperthermia: effects on tumor temperature and tumor response in a rat tumor model.

PURPOSE: The effect of a decrease in the mean arterial blood pressure (MAP) induced by sodium nitroprusside (SNP) on the tumor temperature during hyperthermia (HT), and on the cytotoxic effect of HT, was studied in the BT4An tumor transplanted to the hind limb of BD IX rats. Experiments with two different anesthetics, pentobarbital and the midazolam/fentanyl/fluanisone combination (MFF), were performed to secure reliable conclusions. METHODS AND MATERIALS: In the tumor response experiments local waterbath HT at 44.0 degrees C was given for 60 min. Sodium nitroprusside was administered as a continuous intravenous infusion to lower the MAP to 60 or 80 mmHg during HT. In two other experiments the temperature at the base of the tumor during HT was measured before and during SNP infusion. In animals without tumor the temperature was measured subcutaneously on the foot during HT with or without SNP-induced hypotension. RESULTS: When SNP was given to lower the MAP to 60 mmHg during HT in MFF anesthetized animals, the median tumor growth time (TGT) was 70 days, compared to 14.5 days in the HT alone group. The corresponding figures were 127 and 12.1 days with pentobarbital anesthesia. In the HT + SNP group, more than 40% cure was observed in both experiments. No cures were seen in any of the other groups. Hyperthermia alone prolonged the TGT slightly, whereas SNP given alone had no effect, compared to controls. When the MAP was lowered to 80 mmHg by SNP infusion during HT (MFF anesthesia), the median TGT was 19.9 days, which was significantly longer than that in the HT alone group (10.9 days). In the MAP range from 60 to 120 mmHg, a nearly linear relationship between the MAP and the tumor temperature was found during HT in MFF anesthetized animals. With both anesthetics, the median temperature at the base of the tumor was about 0.8 degrees C higher during HT when the MAP was lowered to 60 mmHg by SNP. In animals without tumors, the temperature subcutaneously on the foot was 0.3 and 0.4 degrees C higher during SNP infusion in the MFF and pentobarbital group, respectively. CONCLUSION: We have developed a small animal model in inbred rats feasible for exploring the influence of a stable blood pressure reduction induced by SNP, on the effect of HT given alone or in combination with other treatment modalities to a transplantable tumor. The greatly increased cytotoxic effect of local waterbath HT in the present tumor response experiments is probably a consequence of increased tumor temperature during SNP infusion.

Interaction between microwave-induced brain hyperthermia and high dose rate radiation in the BT4 An brain glioma in rats.

The cerebral BT4An glioma model in BD IX rats was used to study the effect of hyperthermia given in combination with radiotherapy (thermoradiotherapy) in the treatment of brain tumours. A single treatment with high dose rate radiation was given to a local brain field. Local brain hyperthermia was given at 42.4 degrees C for 45 min by externally applied microwaves (700 MHz), immediately before radiotherapy (10 Gy). In a pilot study, thermoradiotherapy increased the median life span with 20 days compared to controls, which was significantly better than that observed after radiotherapy alone (7 days). In an extended experiment the corresponding figures for thermoradiotherapy, hyperthermia alone and radiotherapy alone were 12.5, 3.5, and 3.5 days, respectively. Thermoradiotherapy was significantly better than radiotherapy and hyperthermia alone. There was no acute mortality in these experiments. Neurological side-effects were infrequent, of slight degree and reversible. The present study shows that a survival benefit of adding hyperthermia to radiotherapy can be achieved without unacceptable neurological side-effects in an animal glioma model.

Effect of hypertonic glucose in thermotolerant rat BT4An gliomas treated with combined hyperthermia and BCNU in vivo.

The influence of hypertonic glucose i.p. on development of thermotolerance and thermochemosensitivity with BCNU in thermotolerant tumours, and on intratumoural pH alterations in previously unheated and thermotolerant tumours, was investigated in BT4An tumours grown on the hind foot of BD IX rats. Thermotolerance was induced with local waterbath hyperthermia (44 degrees C/45 min) 24 h before start of test treatment. Hypertonic glucose immediately after priming hyperthermia did not inhibit development of thermotolerance, despite a significant reduction of pH by glucose. The pH reduction was less in thermotolerant tumours. Glucose administered before treatment of thermotolerant tumours did not change the growth rate of tumours treated with hyperthermia (44 degrees C/45 min), BCNU (20 or 25 mg/kg i.p.) or thermochemotherapy with a low or high dose BCNU (10 or 20 mg/kg), in contrast to previous results, where glucose enhanced thermochemosensitivity of non-thermotolerant tumours.

Hyperthermia as an adjuvant to radiation therapy of recurrent or metastatic malignant melanoma. A multicentre randomized trial by the European Society for Hyperthermic Oncology.

The ESHO protocol 3-85 is a multicentre randomized trial investigating the value of hyperthermia as an adjuvant to radiotherapy in treatment of malignant melanoma. A total of 134 metastatic of recurrent malignant melanoma lesions in 70 patients were randomized to receive radiotherapy alone (3 fractions in 8 days) or each fraction followed by hyperthermia (aimed for 43 degrees C for 60 min). Radiation was given with high voltage photons or electrons. Tumours were stratified according to institution and size (above or below 4 cm) and randomly assigned to a total radiation dose of either 24 or 27 Gy to be given with or without hyperthermia. The endpoint was persistent complete response in the treated area. A number of 128 tumours in 68 patients were evaluable, with an observation time between 3 and 72 months. Sixty-five tumours were randomized to radiation alone and 63 to radiation + heat. Sixty received 24 Gy and 68 tumours received 27 Gy, respectively. Size was < or = 4 cm in 81 and > 4 cm in 47 tumours. Overall the 2-year actuarial local tumour control was 37%. Univariate analysis showed prognostic influence of hyperthermia (rad alone 28% versus rad + heat 46%, p = 0.008) and radiation dose (24 Gy 25% versus 27 Gy 56%, p = 0.02), but not of tumour size (small 42% versus large 29%, p = 0.21). A Cox multivariate regression analysis showed the most important prognostic parameters to be: hyperthermia (odds ratio: 1.73 (1.07-2.78), p = 0.02), tumour size (odds ratio: 0.91 (0.85-0.99), p = 0.05) and radiation dose (odds ratio: 1.17 (1.01-1.36), p = 0.05). Analysis of the heating quality showed a significant relationship between the extent of heating and local tumour response. Addition of heat did not significantly increase the acute or late radiation reactions. The overall 5-year survival rate of the patients was 19%, but 38% in patients if all known disease was controlled, compared to 8% in the patients with persistent active disease.

[Chemotherapy in colorectal cancer. Recommendations of the Norwegian Gastrointestinal Cancer Group]. / Kjemoterapi ved kolorektalcancer. Anbefalinger fra Norsk Gastrointestinal Cancer Gruppe.

The scientific documentation for fluorouracil-based chemotherapy in patients with colorectal cancer has increased during recent years, both for the adjuvant primary situation and in recurrent and metastatic disease. In the case of operable colorectal cancer (stage Dukes B and C), the patients should be included in the ongoing randomized trial on use of fluorouracil and levamisol. In advanced disease, fluorouracil combined with calcium folinate may have palliative effects in terms of relief of symptoms, improved quality of life, delay of onset of symptoms and probably also prolonged survival in 40-50% of the patients, without serious toxicity. In many cases, however, the patient should not be given chemotherapy.

The use of non-proven therapy among patients treated in Norwegian oncological departments. A cross-sectional national multicentre study.

A national multicentre study was performed to investigate the prevalent use of "alternative medicine", here called "non-proven therapies (NPT)", applied among Norwegian cancer patients. Of 911 patients invited to take part in the study, 642 were included in the analysis. Demographic characteristics were collected for all patients. The participating physicians gave information about the patients' clinical characteristics. Among 630 evaluable patients, 20% had been or were present users of NPTs for their oncological disease. The preferred methods were healing by hand and faith healing. Herbs, vitamins, diets and Iscador were other popular methods. As many as 40% of the users of NPTs had used NPTs earlier for non-malignant diseases. Elderly patients were less likely to use NPTs. Use was high in the northern part of Norway.

Randomised trial of hyperthermia as adjuvant to radiotherapy for recurrent or metastatic malignant melanoma. European Society for Hyperthermic Oncology.

The value of hyperthermia as an adjuvant to radiotherapy in patients with malignant melanoma was studied in a European multicentre trial. 134 metastatic or recurrent lesions of malignant melanoma in 70 patients were randomly assigned to receive radiotherapy (three fractions of 8 Gy or 9 Gy in 8 days) alone or followed by hyperthermia (43 degrees C for 60 min). Overall, the 2-year actuarial local tumour control was 37 (SE 5)%. Univariate analysis showed a beneficial effect of hyperthermia (radiation alone 28% vs combined treatment 46%, p = 0.008) and radiation dose (24 Gy 25% vs 27 Gy 56%, p = 0.02), but no effect of tumour size (< or = 4 cm 42% vs > 4 cm 29%, p = 0.21). Cox multivariate regression analysis showed the most important prognostic variables to be hyperthermia (odds ratio for 2-year local control 1.73 [95% CI 1.07-2.78], p = 0.023), tumour size (0.91 [0.85-0.99], p = 0.05), and radiation dose (1.17 [1.01-1.36], p = 0.05). Addition of heat did not significantly increase acute or late radiation reactions. Heating was well tolerated, but because of difficulties with equipment only 14% of treatments achieved the protocol objective. The overall 5-year survival rate was 19%, but 38% of the patients for whom all known disease was controlled survived 5 years. Adjuvant hyperthermia significantly improved local tumour control when applied in association with radiation in treatment of malignant melanoma. Successful local treatment of patients with a single or a few metastatic malignant melanoma lesions has significant curative potential.