Radioimmunotherapy of breast cancer xenografts with monoclonal antibody ICR12 against c-erbB2 p185: comparison of iodogen and N-succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate radioiodination methods.

C-erbB2 p185 is a proto-oncogene product expressed in 25-30% of human invasive breast cancers that is associated with poor prognosis and resistance to endocrine therapy and chemotherapy. It is minimally expressed in normal adult tissues (M. F. Press et al., Oncogene, 5: 953-962, 1990). For this reason, it is an attractive target for radioimmunotherapy and other antibody-directed therapies. ICR12 is a rat IgG2a monoclonal antibody directed against a protein epitope of the external domain of the c-erbB2 p185. We performed experiments to optimize the direct iodination of ICR12 with 131I using the IodoGen method, and we found impairment of immunoreactive fraction with increasing specific activity. N-Succinimidyl 4-methyl-3-(tri-n-butylstannyl)benzoate (MATE) is a tin ester that can be radioiodinated easily and then coupled to the epsilon-amino group of lysine residues. This method has been shown to have improved uptake in tumors compared with antibody labeled by direct iodination (P. K. Garg et al., Nucl. Med. Biol., 20: 379-387, 1993). ICR12 could be labeled up to 16 mCi/mg by this technique without loss of immunoreactive fraction. Whole-body retention of MATE-labeled ICR12 was less than IodoGen (P < 0.0001). Radioimmunotherapy experiments in athymic mice bearing established MDA MB 361 human breast cancer xenografts showed growth inhibition for > 24 days at a dose of 600 microCi/mouse (P < 0.0001) when labeled by the IodoGen technique, and 12 days using the MATE method (P < 0.0001).

Correlation between tumour blood flow and fluorouracil distribution in a hypovascular liver metastasis model.

The poor response of colorectal liver metastases to fluorinated pyrimidine chemotherapy may be due to poor drug penetration into the tumour. Chemotherapy delivered by the blood to well perfused areas of tumour must reach less well perfused areas by diffusion. This study examined the relationship between intratumoural blood flow and drug uptake in a hypovascular liver metastasis animal model. We used a double isotope technique to examine the microdistribution of the blood flow tracer [125I]-iodoantipyrine (IAP) and fluorinated pyrimidine 5-[6-3H]-fluorouracil (5-FU) within intrahepatic, hypovascular HSN tumours. There was a significant fall (P < 10(-6)) in both IAP and 5-FU uptake between the liver/tumour edge and tumour centre which resulted in a significant covariation (P < 10(-5)) in tracer uptake with distance. The finding of a close covariation between blood flow and drug uptake in liver metastases suggested that 5-FU diffusion did not compensate for low 5-FU delivery in areas of poor tumour blood flow. The lower 5-FU levels in low compared with high areas of tumour blood flow could reduce the cytotoxic effect and increase the potential for development of drug resistance.

Effect of regional angiotensin II infusion on the relationship between tumour blood flow and fluorouracil uptake in a liver metastasis animal model.

The aim of this study was to assess the relationship between tumour:liver blood flow and 5-fluorouracil (5-FU) uptake ratios in a hypovascular liver metastasis animal model, and examine whether they were similarly affected by a 5 min infusion of angiotension II via the hepatic artery. Tumour:liver blood flow ratio was measured using the isotope tracer 64Copper (II)-pyruvaldehyde bis(n-4 methyl thiosemicarbazone, and 5-FU was tritiated. There was a wide variation in tumour:liver blood flow and 5-FU uptake ratios which could only partly be explained by between animal variation, and was not related either to individual tumour size or overall tumour burden within the liver. There was a close correlation (r = 0.957, P < 0.0001) between tumour:liver blood flow and 5-FU uptake ratios. Angiotensin II infusion significantly increased tumour:liver blood flow (nested analysis of variance, P= 0.05) but not 5-FU uptake (P = 0.29) ratios. There was a poor correlation (r = 0.51, P = 0.13) between tumour:liver blood flow and 5-FU uptake ratios with angiotensin II infusion. Thus, despite an increased 5-FU blood concentration arising from angiotensin-induced reduction in blood flow at constant 5-FU infusion dose, tumour:liver 5-FU uptake ratio did not increase as expected, and there ceased to be a significant correlation between tumour:liver blood flow and 5-FU uptake ratios. We conclude that the vasoactive changes within the hypovascular tumour circulation produced by a 5 min angiotensin II infusion did not significantly increase tumour 5-FU uptake.

Quantitative in vivo measurements of tumor perfusion using rubidium-81 and positron emission tomography.

Rubidium-81 (t1/2 = 4.58 hr) was investigated as a tumor perfusion tracer in the VX2 carcinoma implanted into rabbit thigh muscle using a large-area, multiwire proportional chamber positron emission tomography (PET) system. Perfusion was determined using the arterial reference sample method, and the results from PET imaging were compared with postmortem tissue sampling. Absolute quantitation of tumor perfusion was achieved using external probes to estimate local extraction fraction. Redistribution of rubidium-81 (81Rb) was investigated using a dual-tracer technique. Average perfusion was found to be 13.5 and 3.7 ml/min/100 g in tumor and normal muscle, respectively. The extraction fraction as estimated from a two-compartment model ranged from 0.94 to 1.00. No significant redistribution of 81Rb was observed in these tissues. Nine patients with malignancies were studied using 81Rb and PET. Tumor perfusion in four patients with carcinoma of the breast was elevated by a factor of 1.8 (range 1.2-2.3) compared to contralateral normal breast.

Thermal enhancement of radiation response: a growth delay study on superficial human tumour metastases.

In a quantitative study of 96 nodules in 10 patients, an enhancement of radiation response has been demonstrated following the addition of a single heat treatment 3-4 h after a single dose of radiation. Thermal enhancement ratios ranging from greater than 1.6 to 4.2 have been derived from growth-delay curves obtained by ultrasonic and caliper tumour volume measurements. Nodules subjected to heat and radiation regress more rapidly than those receiving radiation alone, with median tumour volume halving times of 15.5 and 70 days, respectively. The total volume reduction was greater in heated nodules and the nadir values were observed later compared with nodules receiving radiation alone.

Radiolabelled 5'-iodo-2'-deoxyuridine: a promising alternative to [18F]-2-fluoro-2-deoxy-D-glucose for PET studies of early response to anticancer treatment.

We investigated the potential of radiolabelled 5-iodo-2'-deoxyuridine (IUdR) as a pharmacodynamic probe for use with positron emission tomography (PET) in studies of early proliferative response to anticancer treatment. Using the hormone-responsive rat mammary carcinoma OES.HR1, we used a multiple radiotracer method to examine treatment-induced changes in 24 h tumour retention of [131I]IUdR, uptake of [3H]2-deoxy-D-glucose ([3H]DG) together with [99mTc]hexylmethylpropylene amineoxine ([99mTc]HMPAO) uptake as a measure of blood flow. Radiotracer data were compared with macroscopic changes in tumour growth, and cell proliferation as determined by DNA histogram flow cytometry. From 4 days after tumour growth arrest induced by oestrogen ablation, a sustained fall in tumour cell proliferation was demonstrated, which was associated with reduced tumour uptake of each tracer. Whereas reduced levels of tumour [3H]DG could be accounted for by changes in blood flow, this was not the case for [131I]IUdR, which was found to be closely related to percentage S-phase cells within tumour (r = 0.73, p < 0.002). It was also estimated that residual levels of radioiodide may contribute significantly, to the low levels of retained radioactivity associated with responding tumours at 24 h following IUdR administration, suggesting that metabolite correction methods should be implemented as part of IUdR PET imaging protocols. We conclude that [124I]IUdR is a promising alternative to [18F]fluorodeoxyglucose ([18F]FDG) for the early assessment by PET of tumour response to treatments directed at targets associated with cell proliferation.

Pharmacokinetics and biodistribution of radiolabelled idoxifene: prospects for the use of PET in the evaluation of a novel antioestrogen for cancer therapy.

With a view to evaluating the role of PET imaging in the development of new anticancer drugs, we are investigating the novel antioestrogen pyrrolidino-4-iodotamoxifen (idoxifene). [125I]idoxifene and [131I]idoxifene have been produced in no-carrier-added form using a tributyl stannylated precursor, and the bio-distribution and dynamic behaviour of the compound investigated using syngeneic transplantable mammary tumours in the rat. Our findings support the use of PET imaging with 124I to study the clinical pharmacology of idoxifene. Factors other than hormone receptor levels appear to influence tumour uptake and therefore, possibly the biological effects of this compound.

Metal complexes of bleomycin: evaluation of [Rh-105]-bleomycin for use in targeted radiotherapy.

Bleomycin has been used as a carrier for several radioisotopes; however, its potential for clinical use has been limited either by the in vivo stability of the complexes or the half-life of the isotope used. The chemical, biological, and radiological properties of 105Rhodium appear to make it an ideal choice for targeted radiotherapy. The synthesis and purification of a hereto unreported 105Rhodium-bleomycin (105Rh-BLM) complex is described. The stability of this complex in plasma is sufficient to allow targeted delivery of the radioisotope. 57Cobalt-bleomycin was studied under identical conditions for comparative purposes. The suitability of 105Rh-BLM for targeted therapy, which appears to be limited by the renal clearance of this agent, is discussed.

Effect of blood perfusion on the ablation of liver parenchyma with high-intensity focused ultrasound.

This paper discusses the effect of blood perfusion on the ablation of rat liver tissue with high-intensity focused ultrasound (HIFU). For this study a practical method has been developed, in which the liver blood flow can be reduced by ligation of the hepatic artery and portal vein. During the treatment the rat liver was mobilized out of the abdomen and the blood flow was measured using both the radioactive microsphere method and a laser Doppler blood-flow monitor. The results show that the hepatic blood flow was about 23 ml/100 g min-1 via the hepatic artery and about 227 ml/100 g min-1 via the portal vein. The total liver blood flow was reduced by 98% when both the hepatic artery and portal vein were ligated. Comparative lesions were made on the same liver lobes of rats with both normal and reduced blood flow using a focused ultrasound beam of 1.7 MHz, 67-425 W cm-2 spatially averaged focal intensity ISAL and 2-20 s exposure duration. A marked difference has been found between the lesion dimensions obtained with normal blood flow and that with reduced blood flow. For exposures at 169 W cm-2 the lesion diameter with normal blood flow was reduced by 14% for 3 s exposure duration compared to that obtained with both hepatic artery and portal vein ligated, while the reduction was more than 20% for longer durations.

The rabbit as an experimental model for regional chemotherapy. 1. Intra-arterial hindlimb infusion.

Regional delivery of chemotherapy to a tumour or tumour-bearing region has pharmacokinetic advantages over the systemic route. The applications of an animal model for regional drug delivery are outlined. The technique for intra-arterial infusion in the rabbit hindlimb is described. The use of the implantable VX2 rabbit carcinoma as a model for solid human tumours may be studied by this method. Pharmacokinetic data obtained with the model allow comparison between systemic and regional routes of delivery. The distribution of the cancerostatic plant toxin ricin following regional delivery has been investigated using this experimental model.

Nickel-57-doxorubicin, a potential radiotracer for pharmacokinetic studies using PET: production and radiolabelling.

The clinical use of anthracyclines, such as doxorubicin (DXR), is hampered by tumour development of multidrug resistance (MDR). The drug efflux associated with MDR could be characterised in vivo using Positron Emission Tomography (PET) in conjunction with a suitable radiolabelled drug. We are investigating DXR labelled with the positron emitter 57Ni as a potential analogue of the parent drug. Essential to this work is the production of a high purity radionuclide in a suitable chemical form for the preparation of radiolabelled DXR. To optimise production parameters, excitation functions (reaction cross section as a function of beam energy) for proton induced reactions in cobalt were measured up to 60 MeV. The excitation function for the 59Co(p,3n)57Ni reaction shows a maximum cross section of 13.8 +/- 1.5 mb at 38 MeV. The optimum energy range for production of 57Ni was found to be 41-->26 MeV resulting in an experimental thick target yield of 17.8 MBq/muAh. The level of the 56Ni impurity is only 0.21% at the end of bombardment. A radiochemical procedure, based on cation-exchange chromatography, has been developed for the separation of radionickel from the cobalt target and other radiochemical and chemical impurities. The 57Ni activity was eluted, using 2M HCl, from a Dowex-50Wx8(H+) column, in a 95% radiochemical yield. Optimum labelling of DXR has been investigated in terms of pH, reaction time and temperature, achieving radiochemical yields > 94%. DXR labelled with 57Ni therefore shows promise as a radiotracer for pharmacokinetic studies using PET.

The practical use of thermocouples for temperature measurement in clinical hyperthermia.

The use of thermocouples as invasive thermometers in clinical hyperthermia is comprehensively and critically reviewed. The ability to construct thermocouple probes as small-bore, multiple junction assemblies is a major reason for their popularity and full constructional details are given. The potential sources of measurement error when using thermocouples both in temperature gradients and in electromagnetic or ultrasonic heating fields are discussed. Emphasis is placed upon simple practical solutions to these problems and a combination of good measurement practice and electrical filtering can reduce errors to an insignificant level. Techniques are suggested for the assessment of thermocouple performance during clinical measurement. With careful use, thermocouples can be reliable and convenient thermometers.

Uptake and distribution of L-3-[I-125] iodo-alpha-methyl tyrosine in experimental rat tumours: comparison with blood flow and growth rate.

Radiolabelled amino acids combined with positron emission tomography (PET) show promise for the accurate delineation of viable tumour extent and may also provide a rapid and sensitive indicator of response to therapy. We have investigated the potential use of the radioiodinated amino acid analogue L-3-iodo-alpha-methyl tyrosine (IMT) for these purposes using experimental tumours in hooded rats. Preliminary studies using HSN tumours and IMT labelled with iodine-125 demonstrated maximum tumour uptake at 15 min post injection although an improved tumour-to-brain ratio was seen at 24 h due to the relatively poor retention of IMT in normal brain. Brain uptake of IMT was also found to be substantially reduced by competition with another large neutral amino acid phenylalanine; however, relatively less effect was seen in tumour, and in skeletal muscle no change in IMT uptake was observed. Quantitative autoradiography revealed no sign of heterogeneity in tumour IMT uptake: good penetration was seen even in poorly vascularised regions as confirmed by endothelial immunohistochemistry. Similar levels of IMT uptake were found in the OES.HR1 tumour during growth supplemented by exogenous oestrogen. Following arrest of tumour growth by removal of the oestrogen stimulus, IMT uptake was seen to fall from 1.7% to 1.0% of the injected dose per gram: this was matched by a fall in tumour blood flow as estimated by technetium-99m hexamethylpropylene amine oxime distribution. It appears that IMT uptake is more strongly influenced by blood flow than cell proliferation and that intratumoural distribution of IMT is principally determined by diffusion.

Nitric oxide inhibition sustains vasopressin-induced vasoconstriction.

Hepatic parenchymal vasoconstriction increases cytotoxic drug uptake into hepatic metastases by increasing the tumour to liver blood flow ratio. Prolonged infusion of the vasoconstrictor vasopressin does not result in sustained vasoconstriction, and this may limit the benefit of vasopressin in infusional chemotherapy. We have assessed whether loss of vasopressin-induced vasoconstriction is mediated by nitric oxide. Hepatic and tumour blood flow were continuously monitored, in an animal hepatic tumour model, by laser Doppler flowmetry. The response to regionally infused vasopressin and the nitric oxide inhibitor N-nitro-L-arginine methyl ester (L-NAME) were assessed over a 30 min infusion period. The vasopressin-induced vasoconstrictor effect diminished after 15 min despite continued infusion. Vasoconstriction was significantly prolonged when L-NAME was infused in addition to vasopressin. The increase in tumour to normal blood flow ratio was greater over the infusion period when L-NAME was co-administered with vasopressin. Our results suggest that the loss of vasopressin-induced vasoconstriction seen in liver parenchyma after regional infusion is prevented by the nitric oxide synthase inhibitor L-name and may be mediated by nitric oxide.

Effect of continuous regional vasoactive agent infusion on liver metastasis blood flow.

Regionally administered vasopressors might increase tumour chemotherapy uptake by differentially constricting normal and tumour blood vessels, leading to a selective increase in blood flow to the tumour. In this study, we compared the effects of the vasopressors angiotensin II, vasopressin and endothelin I and the vasodilator calcitonin gene-related peptide (CGRP) by continuously measuring liver parenchymal and tumour blood flow during a 30-min regional vasoactive infusion in a rat HSN liver metastasis model. Vasopressin and angiotensin II produced a vasoconstriction that decreased despite continued infusion, while endothelin I infusion led to prolonged vasoconstriction with a more gradual onset. CGRP infusion resulted in increased vessel conductance but a reduction in blood flow due to systemic hypotension. The tumour to normal flow ratio (TNR) was transiently increased during infusion of all pressors, but only endothelin I produced sufficient change to result in a rise in average TNR throughout pressor infusion. Continuous liver and tumour blood flow measurement throughout vasoactive infusion demonstrated that the extent and the duration of blood flow change varied with the agents assessed. No vasoactive agent increased tumour blood flow, but endothelin I had the most suitable vasoactive properties for enhancing tumour uptake of continuously infused chemotherapy.

Tissue blood flow estimation with copper(II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) and PET.

Copper (II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) (Cu-PTSM) labelled with 62Cu or 64Cu is currently under investigation as a radiotracer for imaging the distribution of blood flow with positron emission tomography (PET). The application of a simple trapped tracer model in conjunction with tissue uptake and continuous arterial sampling to estimate blood flow has been compared with the 57Co-microsphere method in the rat. After intraventricular injection the cumulative arterial function for 64Cu increased progressively due to the presence of circulating non lipophilic complexes. The cumulative function for lipophilic 64Cu-PTSM extracted in n-octanol plateaued at levels corresponding to those reached by 57Co-microspheres. No consistent disagreement was found between cardiac output and blood flow estimated by 64Cu-PTSM and 57Co-microspheres in low to moderate flow tissues: muscle (0.08, 0.07 mL/min/g; 64Cu mean, 57Co mean), brain (0.52, 0.43 mL/min/g) and kidney (2.29, 2.45 mL/min/g). However, 64Cu-PTSM underestimated blood flow measured by 57Co-microspheres in myocardium (4.09, 6.55 mL/min/g). A simple tissue trapping model may therefore be suitable for the derivation of blood flow estimates in low to moderate flow tissues using 62,64Cu-PTSM, PET imaging and continuous arterial sampling with n-octanol extraction.

Uptake of radiolabelled tyrosine and iodo-methyl tyrosine in experimental rat tumours: influence of blood flow and tumour growth rate.

Radiolabelled amino acids combined with Positron Emission Tomography (PET) may be useful for delineation of the extent of viable tumour and may also provide a rapid and sensitive indicator of response to therapy. Promising early clinical reports led us to investigate the potential use of the amino acid analogue L-3-iodo-alpha-methyl tyrosine (IMT), which may be radioiodinated with isotopes suitable for PET or conventional single photon imaging. We have studied the biodistribution and kinetics of [125I]IMT using two transplantable tumour systems in hooded rats, and have compared the findings with those using the natural amino acid L-tyrosine (TYR) radiolabelled with tritium. Similar levels of IMT and TYR uptake were found in HSN and OES.HR1 tumours during tumour growth. Following arrest of OES.HR1 tumour growth by oestrogen ablation, reduced IMT and TYR uptake was found to be closely matched by a fall in tumour blood flow. Unlike IMT, a substantial proportion of TYR uptake in tumours was found to be protein incorporated, even following tumour growth arrest. Quantitative autoradiography revealed sharp delineation of tumour boundary using either radiotracer. We conclude that IMT and TYR kinetics are strongly influenced by blood flow and diffusion, and that tumour growth status may not be closely associated with amino acid uptake.

Biodistribution and kinetics of radiolabelled pyrrolidino-4-iodo-tamoxifen: prospects for pharmacokinetic studies using PET.

With a view to evaluating the role of PET imaging in early clinical studies of new anticancer drugs, we are investigating the recently developed antiestrogen compound pyrrolidino-4-iodo-tamoxifen (idoxifene). Preliminary experimental studies have been undertaken using [125,131I]idoxifene, following synthesis of a tributyl-stannyl-idoxifene precursor to facilitate radioiodination. We have investigated the tissue biodistribution and kinetics of [125I]idoxifene following i.v. infusion in hooded rats bearing the hormone-dependent transplantable mammary tumour OES.HR1. Clearance of idoxifene from the circulation is accompanied by an increase in uptake by tumour and uterus, to peak levels after 24 hours (0.33 +/- 0.037% dose/g (mean +/- 1 SD) and 0.40 +/- 0.033% dose/g, respectively). Highest uptake of idoxifene was found in the liver (11.0 +/- 0.8% dose/g), with a progressive fall after 24 hours consistent with hepatobiliary excretion of the radiotracer. No evidence of idoxifene metabolism was found in tissue extracts taken up to 48 hours. Whole body clearance of [131I]idoxifene was characterised by a single exponential decay (t1/2 = 140 hours) up to 350 hours post administration. We conclude that 124I-labelled idoxifene combined with PET imaging would facilitate human in vivo pharmacokinetic studies of this new anticancer drug and provide an opportunity to investigate relationships between drug uptake and tumour response.

Evaluation of copper(II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) for tissue blood flow measurement using a trapped tracer model.

Copper(II)-pyruvaldehyde bis (N-4-methylthiosemicarbazone) (Cu-PTSM) labelled with 62,64Cu is a promising radiotracer for the study of blood flow using positron emission tomography (PET). We have investigated the application of a simple trapped tracer model to measurements of tissue 64Cu-PTSM uptake combined with continuous arterial sampling. A dual-tracer method was used to compare blood flow estimated by 64Cu-PTSM with values derived from measurements using cobalt-57 microspheres in the rat. Prolonged retention of 64Cu-PTSM following intravenous administration was initially confirmed in both normal tissues and tumours. After intraventricular 64Cu-PTSM infusion, cumulative arterial 64Cu activity increased progressively, and after extraction in n-octanol was found to plateau to levels corresponding with those reached following administration of 57Co microspheres. Rapid and species-dependent rates of 64Cu-PTSM decomposition to non-extractable 64Cu complexes were found in rat and human blood in vitro (70% +/- 6% and 43 +/- 5% respectively at 16 min), demonstrating the need for immediate processing of arterial samples. Close agreement was found between blood flow estimated by 64Cu-PTSM and 57Co microsphere methods in tissues of low to moderate flow: muscle (0.01, 0.08, 0.07 ml/min per gram; mean difference, mean 64Cu, mean 57Co), brain (0.09, 0.52, 0.43 ml/min per gram) and kidney (-0.16, 2.29, 2.45 ml/min per gram). Estimates of cardiac output also compared favourably between the two methods (5.7, 59.8, 54.1 ml/min). We conclude that a simple tissue trapping model may be suitable for the derivation of blood flow estimates using 62,64Cu-PTSM, PET imaging and continuous arterial blood sampling.

The vascularity of cutaneous melanoma: a quantitative histological study of lesions 0.85-1.25 mm in thickness.

The vascularity of 107 primary cutaneous melanomas has been characterized by morphometric histological analysis. The lesions selected for study were of thickness 0.85-1.25 mm and the aim was to evaluate the prognostic significance of tumour vascularity. Two groups of patients were identified; 86 with no evidence of recurrence after a minimum follow-up period of 5 years and 21 with locoregional recurrence and/or metastasis. The lectin Ulex europaeus type I was used for endothelial cell staining of tissue sections and morphometric analysis was performed to derive the vascular length, surface and volume density from independent measurements of tumour, adjacent dermis and the junctional zone between tumour and underlying tissue. A wide range of values was obtained for each parameter with increased vascularity always found at the tumour base compared with the tumour as a whole. In relation to the adjacent normal dermis, vascularity was generally found to be higher at the tumour base but either higher or lower in the tumour overall. Tumour recurrence could not be predicted by any of the derived vascular parameters either independently or together with other histological and clinical features. This study suggests that tumour vascularity is of no prognostic significance in melanoma of the above thickness range. The highly variable extent of tumour vascularity was not correlated with other clinical or histological parameters, but may have implications for the delivery of pharmaceutical agents used for diagnosis or therapy.