An immunologically relevant rodent model demonstrates safety of therapy using a tumour-specific IgE.

BACKGROUND: Designing biologically informative models for assessing the safety of novel agents, especially for cancer immunotherapy, carries substantial challenges. The choice of an in vivo system for studies on IgE antibodies represents a major impediment to their clinical translation, especially with respect to class-specific immunological functions and safety. Fcε receptor expression and structure are different in humans and mice, so that the murine system is not informative when studying human IgE biology. By contrast, FcεRI expression and cellular distribution in rats mirror that of humans. METHODS: We are developing MOv18 IgE, a human chimeric antibody recognizing the tumour-associated antigen folate receptor alpha. We created an immunologically congruent surrogate rat model likely to recapitulate human IgE-FcεR interactions and engineered a surrogate rat IgE equivalent to MOv18. Employing this model, we examined in vivo safety and efficacy of antitumour IgE antibodies. RESULTS: In immunocompetent rats, rodent IgE restricted growth of syngeneic tumours in the absence of clinical, histopathological or metabolic signs associated with obvious toxicity. No physiological or immunological evidence of a "cytokine storm" or allergic response was seen, even at 50 mg/kg weekly doses. IgE treatment was associated with elevated serum concentrations of TNFα, a mediator previously linked with IgE-mediated antitumour and antiparasitic functions, alongside evidence of substantially elevated tumoural immune cell infiltration and immunological pathway activation in tumour-bearing lungs. CONCLUSION: Our findings indicate safety of MOv18 IgE, in conjunction with efficacy and immune activation, supporting the translation of this therapeutic approach to the clinical arena.

[18F]tetrafluoroborate-PET/CT enables sensitive tumor and metastasis in vivo imaging in a sodium iodide symporter-expressing tumor model.

Cancer cell metastasis is responsible for most cancer deaths. Non-invasive in vivo cancer cell tracking in spontaneously metastasizing tumor models still poses a challenge requiring highest sensitivity and excellent contrast. The goal of this study was to evaluate if the recently introduced PET radiotracer [18F]tetrafluoroborate ([18F]BF4-) is useful for sensitive and specific metastasis detection in an orthotopic xenograft breast cancer model expressing the human sodium iodide symporter (NIS) as a reporter. In vivo imaging was complemented by ex vivo fluorescence microscopy and γ-counting of harvested tissues. Radionuclide imaging with [18F]BF4- (PET/CT) was compared to the conventional tracer [123I]iodide (sequential SPECT/CT). We found that [18F]BF4- was superior due to better pharmacokinetics, i.e. faster tumor uptake and faster and more complete clearance from circulation. [18F]BF4--PET was also highly specific as in all detected tissues cancer cell presence was confirmed microscopically. Undetected comparable tissues were similarly found to be free of metastasis. Metastasis detection by routine metabolic imaging with [18F]FDG-PET failed due to low standard uptake values and low contrast caused by adjacent metabolically active organs in this model. [18F]BF4--PET combined with NIS expressing disease models is particularly useful whenever preclinical in vivo cell tracking is of interest.

Al(OH)3 facilitated synthesis of water-soluble, magnetic, radiolabelled and fluorescent hydroxyapatite nanoparticles.

Magnetic and fluorescent hydroxyapatite nanoparticles were synthesised using Al(OH)3-stabilised MnFe2O4 or Fe3O4 nanoparticles as precursors. They were readily and efficiently radiolabelled with (18)F. Bisphosphonate polyethylene glycol polymers were utilised to endow the nanoparticles with excellent colloidal stability in water and to incorporate cyclam for high affinity labelling with (64)Cu.

Imaging tumour hypoxia with positron emission tomography.

Hypoxia, a hallmark of most solid tumours, is a negative prognostic factor due to its association with an aggressive tumour phenotype and therapeutic resistance. Given its prominent role in oncology, accurate detection of hypoxia is important, as it impacts on prognosis and could influence treatment planning. A variety of approaches have been explored over the years for detecting and monitoring changes in hypoxia in tumours, including biological markers and noninvasive imaging techniques. Positron emission tomography (PET) is the preferred method for imaging tumour hypoxia due to its high specificity and sensitivity to probe physiological processes in vivo, as well as the ability to provide information about intracellular oxygenation levels. This review provides an overview of imaging hypoxia with PET, with an emphasis on the advantages and limitations of the currently available hypoxia radiotracers.

PET and SPECT imaging of a radiolabeled minigastrin analogue conjugated with DOTA, NOTA, and NODAGA and labeled with (64)Cu, (68)Ga, and (111)In.

Cholecystokinin-2 (CCK-2) receptors, overexpressed in cancer types such as small cell lung cancers (SCLC) and medullary thyroid carcinomas (MTC), may serve as targets for peptide receptor radionuclide imaging. A variety of CCK and gastrin analogues has been developed, but a major drawback is metabolic instability or high kidney uptake. The minigastrin analogue PP-F11 has previously been shown to be a promising peptide for imaging of CCK-2 receptor positive tumors and was therefore further evaluated. The peptide was conjugated with one of the macrocyclic chelators DOTA, NOTA, or NODAGA. The peptide conjugates were then radiolabeled with either (68)Ga, (64)Cu, or (111)In. All (radio)labeled compounds were evaluated in vitro (IC50) and in vivo (biodistribution and PET/CT and SPECT/CT imaging). IC50 values were in the low nanomolar range for all compounds (0.79-1.51 nM). In the biodistribution studies, (68)Ga- and (111)In-labeled peptides showed higher tumor-to-background ratios than the (64)Cu-labeled compounds. All tested radiolabeled compounds clearly visualized the CCK2 receptor positive tumor in PET or SPECT imaging. The chelator did not seem to affect in vivo behavior of the peptide for (111)In- and (68)Ga-labeled peptides. In contrast, the biodistribution of the (64)Cu-labeled peptides showed high uptake in the liver and in other organs, most likely caused by high blood levels, probably due to dissociation of (64)Cu from the chelator and subsequent transchelation to proteins. Based on the present study, (68)Ga-DOTA-PP-F11 might be a promising radiopharmaceutical for PET/CT imaging of CCK2 receptor expressing tumors such as MTC and SCLC. Clinical studies are warranted to investigate the potential of this tracer.

PET imaging of cardiac hypoxia: opportunities and challenges.

Myocardial hypoxia is a major factor in the pathology of cardiac ischemia and myocardial infarction. Hypoxia also occurs in microvascular disease and cardiac hypertrophy, and is thought to be a prime determinant of the progression to heart failure, as well as the driving force for compensatory angiogenesis. The non-invasive delineation and quantification of hypoxia in cardiac tissue therefore has the potential to be an invaluable experimental, diagnostic and prognostic biomarker for applications in cardiology. However, at this time there are no validated methodologies sufficiently sensitive or reliable for clinical use. PET imaging provides real-time spatial information on the biodistribution of injected radiolabeled tracer molecules. Its inherent high sensitivity allows quantitative imaging of these tracers, even when injected at sub-pharmacological (≥pM) concentrations, allowing the non-invasive investigation of biological systems without perturbing them. PET is therefore an attractive approach for the delineation and quantification of cardiac hypoxia and ischemia. In this review we discuss the key concepts which must be considered when imaging hypoxia in the heart. We summarize the PET tracers which are currently available, and we look forward to the next generation of hypoxia-specific PET imaging agents currently being developed. We describe their potential advantages and shortcomings compared to existing imaging approaches, and what is needed in terms of validation and characterization before these agents can be exploited clinically.

Harnessing engineered antibodies of the IgE class to combat malignancy: initial assessment of FcɛRI-mediated basophil activation by a tumour-specific IgE antibody to evaluate the risk of type I hypersensitivity.

BACKGROUND: IgE antibodies, sequestered into tissues and retained locally by the high-affinity IgE receptor, FcɛRI, on powerful effector cells such as mast cells, macrophages and eosinophils, may offer improvements in the therapy of solid tumours. The chimeric antibody, MOv18 IgE, against the human ovarian carcinoma antigen, folate receptor α (FRα), is more effective than its IgG1 counterpart in xenograft models of ovarian cancer. Although MOv18 IgE binds to a single epitope on FRα and cannot cross-link IgE receptors on basophils, there remains a risk that components in the circulation of ovarian cancer patients might cross-link FRα-MOv18-IgE-receptor-FcɛRI complexes on basophils to cause type I hypersensitivity. OBJECTIVE: To assess the propensity for MOv18 used in a therapeutic setting to cause FcɛRI-mediated type I hypersensitivity. METHODS: As validated readouts of the potential for MOv18 to cause FcɛRI-mediated type I hypersensitivity we measured release of a granule-stored mediator from a rat basophilic leukaemia cell line RBL SX-38 stably transfected with human tetrameric (αßγ2) FcɛRI, and induction of CD63 on blood basophils from patients with ovarian carcinoma and healthy controls ex vivo. RESULTS: Serum FRα levels were increased in ovarian cancer patients compared with healthy controls. MOv18 IgE alone, or in the presence of its antigen recombinant human FRα, or of healthy volunteer (n=14) or ovarian carcinoma patient (n=32) sera, did not induce RBL SX-38 cell degranulation. Exposure to FRα-expressing ovarian tumour cells at target-to-effector ratios expected within tumours induced degranulation. MOv18 IgE did not induce expression of CD63 in blood basophils from either healthy volunteers (n=6), or cancer patients, despite detectable levels of circulating FRα (n=5). CONCLUSION AND CLINICAL RELEVANCE: These encouraging data are compatible with the hypothesis that, when ovarian carcinoma patients are treated with MOv18, FcɛRI-mediated activation of effector cells occurs within the tumour mass but not in the circulation mandating, with due caution, further pre-clinical studies.

SPECT/CT lymphoscintigraphy of heterotopic cardiac grafts reveals novel sites of lymphatic drainage and T cell priming.

The normal function of lymphatic vessels is to facilitate the trafficking of antigen presenting cells to draining lymph nodes where they evoke an immune response. Donor lymphatic vessels are not connected to that of recipients' during organ transplantation. The pathophysiology of this disruption has received little attention. Murine heterotopic cardiac transplantation has been used extensively in transplantation research. Following vascularized organ transplantation, the main site of allosensitization is thought to be in the spleen of the recipient as a result of migration of donor passenger leukocytes via blood. Here, using Single Photon Emission Computed Tomography/Computerized Tomography (SPECT/CT) lymphoscintigraphy, we studied the pattern of lymphatic flow from mouse heterotopic abdominal cardiac grafts and identified mediastinal lymph nodes as the draining nodes for the donor graft. Staining with HY tetramer after transplantation of HY mismatched heart grafts and ELISPOT following allogeneic grafts to detect donor specific T cells revealed them as important sites for allosensitization. Our data indicates that mediastinal lymph nodes play a crucial role in the alloimmune response in this model, and should be used for ex vivo and adoptive transfer studies after transplantation in addition to the spleen.

Bifunctional bisphosphonate complexes for the diagnosis and therapy of bone metastases.

Easily synthesised and structurally well-defined novel imaging/therapeutic radiopharmaceutical agents for bone metastases are described.

99mTc-SnF2 colloid "LLK": particle size, morphology and leucocyte labelling behaviour.

99mTc-SnF2 colloid (Radpharm LLK) leucocyte labelling agent is used in whole blood, exploiting phagocytosis. The objectives of this work were to optimize leucocyte labelling in leucocyte-enriched plasma, and to investigate: (i) the effect of temperature and other factors on labelling efficiency; (ii) the selectivity for different leucocyte types; (iii) the viability of the labelled cells and efflux of the radiolabel; and (iv) the physical characteristics of the colloid. Density gradient centrifugation was used to investigate the labelling efficiency, cell selectivity and efflux, Trypan blue to study the viability, and laser scattering, electron microscopy and membrane filtration to investigate particle size and morphology. Particles appeared as loose, coiled, chain-like aggregates of much smaller particles (<0.05 microm). The aggregate diameter ranged from <0.1 to >5 microm and increased with time. The distribution of radioactivity amongst the particle sizes varied widely. The labelling efficiency in leucocyte-rich plasma was enhanced at 37 degrees C compared to room temperature, and by centrifuging during labelling. The selectivity for different leucocyte types varied markedly between batches and blood samples, in some cases showing preference for mononuclear cells and in others for granulocytes. Viability was excellent and comparable with 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO)-labelled cells. A significant fraction of radiolabel, comparable to that observed with 99mTc-HMPAO, was lost from leucocytes during incubation in vitro over 4 h. Thus, 99mTc-SnF2 is a convenient, efficient labelling agent for leucocytes, but shows variable cell selectivity which may be linked to particle size variability, and there is significant efflux of radioactivity from labelled cells.

Synthesis and characterization of 111In-DTPA-N-TIMP-2: a radiopharmaceutical for imaging matrix metalloproteinase expression.

The matrix metalloproteinases (MMPs) are enzymes involved in the turnover of the extracellular matrix. Their overexpression in tumors is implicated in the metastatic process and may provide a target for diagnostic tumor imaging by using a radiolabeled inhibitor. MMPs are inhibited by endogenous tissue inhibitors of metalloproteinases (TIMPs). Thus, TIMPs are potential targeting molecules which could be used as vehicles for selective radionuclide delivery by virtue of their binding to MMPs. The aim of this work was to produce a radiopharmaceutical with which to evaluate this potential. The 127 amino acid N-terminal domain of recombinant human TIMP-2 (N-TIMP-2) was conjugated with the bifunctional chelator diethylenetriamine pentaacetic acid (DTPA). Singly modified DTPA-N-TIMP-2 conjugate (identified by electrospray ionization mass spectrometry) was isolated by anion-exchange chromatography. The primary site of DTPA modification on N-TIMP-2 was mapped to lysine-116, which is distant from the site of MMP interaction. The conjugate was radiolabeled with indium-111 to give 111In-DTPA-N-TIMP-2 with a specific activity of at least 4 MBq/microg and a radiochemical yield and purity of >95%, by incubation with 111InCl3, without need for postlabeling purification. The product was sterile, pyrogen-free, and stable in serum over 48 h and retained full inhibitory activity in a fluorimetric binding assay. With these attributes, 111In-DTPA-N-TIMP-2 is a suitable radiopharmaceutical for in vivo biological and clinical investigation of the potential benefits of imaging MMP expression.

In vivo evaluation of 111In-DTPA-N-TIMP-2 in Kaposi sarcoma associated with HIV infection.

Matrix metalloproteinases are the major agents responsible for the degradation of extracellular matrix and are produced at high levels by transformed and tumour cells, where they participate in the metastatic process by allowing local invasion. They are also more active at sites of new normal growth and angiogenesis. In the early stages of Kaposi sarcoma (KS), in vitro studies have demonstrated that vascular invasion can be inhibited by inhibitors of matrix metalloproteinases. Imaging of visceral and cutaneous KS presents a problem and therefore the potential use of a labelled inhibitor of metalloproteinases, N-TIMP-2, with indium-111 was thought to present a possible imaging tool. The biokinetics, dosimetry and potential for imaging with 111In-DTPA-N-TIMP-2 were assessed in five patients with HIV infection and KS. Between 103.1 and 108.0 MBq of this agent was injected into each patient, and the dynamic uptake over the kidneys was assessed, whole body scans were performed and blood samples were obtained. The clearance from the blood was rapid, with a first component half-time of 16.6+/-3.4 min and a second component half-time of 9.68+/-2.68 h. Two out of five patients experienced minor shivering but one of these patients was generally unwell before the study. The last three patients had no such problems. The tracer distributed predominantly to the kidneys and did not localise in other tissues. No KS lesions were clearly identified. 111In-DTPA-N-TIMP-2 can be successfully prepared and administered to patients safely, with a biodistribution and dosimetry which would allow its use as an imaging tracer. It is unlikely to be of use for imaging KS, but may have a role in other tumours that produce matrix metalloproteinases.

Towards new transition metal-based hypoxic selective agents for therapy and imaging.

The greater lability of Co(II) relative to Co(III) can potentially be used to achieve selective delivery of nitrogen mustard type molecules to hypoxic cells. Attempts to improve the stability of the Co(II) state by utilising tripodal tetradentate ligands are described, together with the results of DF calculations. Rhenium has two beta-emitting isotopes (186)Re and (188)Re that have potential for use to treat cancer if the complexes can be targeted with sufficient specificity. We describe some new rapid low temperature routes using hydrazines to labile Re(V) and Re(III) species which provide potential convenient access to a wide range of oxo- and diazenido-complexes. The synthesis of new Re(V) and Re(III) thiosemicarbazone complexes is presented in the context of obtaining hypoxic selective species. Copper(II) bis(thiosemicarbazone) complexes are known to be hypoxic selective and spectroscopic, cyclic voltammetric and computational studies of the mechanism are presented, together with the synthesis of new Cu(II) complexes directed towards the hypoxic selective delivery of nitrogen mustard type molecules.

99mTc(V)DMSA quantitatively predicts 188Re(V)DMSA distribution in patients with prostate cancer metastatic to bone.

Rhenium-188 dimercaptosuccinic acid complex [188Re(V)DMSA], a potential therapeutic analogue of the tumour imaging agent 99mTc(V)DMSA, is selectively taken up in bone metastases in patients with prostate cancer. It would be helpful in planning palliative radionuclide therapy if 99mTc(V)DMSA could be used to predict tumour and kidney retention of 188Re(V)DMSA. The aim of this study was to determine the correlation between tumour-to-normal tissue ratios and kidney-to-soft tissue ratios of 99mTc(V)DMSA and 188Re(V)DMSA. This would determine whether a scan with 99mTc(V) DMSA could be used to identify patients for whom 188Re(V)DMSA treatment would be contra-indicated, and enable prediction of relative kidney and tumour radiation absorbed dose in 188Re(V)DMSA treatment. Ten patients with prostate carcinoma were recruited following observation of disseminated bone metastases on a recent 99mTc-hydroxydiphosphonate bone scan. Whole-body planar scans were obtained at ca. 4 h and 24 h after hydration and injection of 600 MBq 99mTc(V)DMSA, and a week later, at similar times after hydration and injection of 370 MBq 188Re(V)DMSA. A triple-energy window (TEW) scatter correction was applied to the 188Re scans. Counts per pixel were determined in regions of interest drawn over metastatic sites, kidneys and normal soft tissue. Tumour-to-soft tissue ratios were significantly lower (by a factor of approximately 0.8 after the TEW was applied) on 188Re scans than on 99mTc scans, but the two were highly linearly correlated both in all individual patients and in tumours pooled from all patients together both at 4 h and at 24 h. Kidney-to-soft tissue ratios were similarly correlated and were lower for 188Re than for 99mTc by a similar factor. Both tumour- and kidney-to-soft tissue ratios increased between 4 and 24 h but the latter increased more. In conclusion, only minor differences were seen between 99mTc and 188Re scans, and kidney-to-background ratios on 188Re scans were not higher than on 99mTc scans. These differences are insufficient to infer that they are due to a real difference in biodistribution, and they may be due only to different physical imaging characteristics. Thus 99mTc(V)DMSA scans are predictive of 188Re(V)DMSA biodistribution and could be used to estimate tumour and renal dosimetry and assess suitability of patients for 188Re(V)DMSA treatment.

Copper bis(diphosphine) complexes: radiopharmaceuticals for the detection of multi-drug resistance in tumours by PET.

Experience with imaging of the multi-drug resistance (MDR) phenotype in tumours using technetium-99m sestamibi, a substrate of the P-glycoprotein (Pgp) transporter, suggests that better quantification of images and separation of MDR from other variables affecting tracer uptake in tumours are required. One approach to these problems is the development of short half-life positron-emitting tracers which are substrates of Pgp. Several lipophilic cationic copper(I) bis(diphosphine) complexes labelled with copper-64 have been synthesised and evaluated in vitro as substrates for Pgp. The synthesis is rapid and efficient with no need for purification steps. The chemistry is suitable for use with very short half-life radionuclides such as copper-62 (9.7 min) and copper-60 (23.7 min). Incubation of the complexes with human serum in vitro showed that they are sufficiently stable in serum to support clinical imaging, and the more lipophilic members of the series are taken up rapidly by cells (Chinese hamster ovary and human ovarian carcinoma) in vitro with great avidity. Uptake in human ovarian carcinoma cells is significantly reduced after several months of conditioning in the presence of doxorubicin, which induces increased Pgp expression. Uptake in hooded rat sarcoma (HSN) cells, which express Pgp, is significantly increased in the presence of the MDR modulator cyclosporin A. Biodistribution studies in hooded rats show rapid blood clearance, excretion through both kidneys and liver, and low uptake in other tissues. The one complex investigated in HSN tumour-bearing rats showed uptake in tumour increasing up to 30 min p.i. while it was decreasing in other tissues. We conclude that diphosphine ligands offer a good basis for development of radiopharmaceuticals containing copper radionuclides, and that this series of complexes should undergo further evaluation in vivo as positron emission tomography imaging agents for MDR.

Trithiacyclononane as a ligand for potential technetium and rhenium radiopharmaceuticals: synthesis of [M(9S3)(SC2H4SC2H4S)][BF4] (M = 99Tc, Re, 188Re) via C-S bond cleavage.

Chemical or electrochemical reduction of the 1,4,7-trithiacyclononane (9S3) complexes [MII(9S3)2][BF4]2 (M = Re (3a) or Tc (3b)) results in instantaneous C-S bond cleavage to yield ethene and the stable MIII thiolate complexes [MIII(9S3)L][BF4] (M = Re (4a) or Tc (4b), L = SCH2CH2SCH2CH2S). Compounds 4 have been characterized by 1H NMR spectroscopy, and the pseudo-octahedral geometry of 4b has been confirmed by X-ray crystallography. Upon electrochemical reduction 4a loses ethene, while 4b can be reversibly reduced to [TcII(9S3)L], which is then further reduced to Tc(I) with loss of ethene. Successive ethene loss is observed in the mass spectra of compounds 3 and 4. The radiosynthesis of 4a with 188Re can be comfortably completed within 10 min starting with 188ReO4- from a 188W/188Re generator, with a radiochemical yield in excess of 90%, and thus represents a practical approach to the preparation of stable 188Re (and 99mTc) thioether complex derivatives/conjugates for clinical use. Crystal data: 4b, C10H20S6Tc, orthorhombic Pbca, a = 12.233(2) A, b = 14.341(2) A, c = 20.726(3) A, Z = 8.

(99m)Tc(V)DMSA quantitatively predicts (188)Re(V)DMSA distribution in patients with prostate cancer metastatic to bone.

Rhenium-188 dimercaptosuccinic acid complex [(188)Re(V)DMSA], a potential therapeutic analogue of the tumour imaging agent (99m)Tc(V)DMSA, is selectively taken up in bone metastases in patients with prostate cancer. It would be helpful in planning palliative radionuclide therapy if (99m)Tc(V)DMSA could be used to predict tumour and kidney retention of (188)Re(V)DMSA. The aim of this study was to determine the correlation between tumour-to-normal tissue ratios and kidney-to-soft tissue ratios of (99m)Tc(V)DMSA and (188)Re(V)DMSA. This would determine whether a scan with (99m)Tc(V)DMSA could be used to identify patients for whom (188)Re(V)DMSA treatment would be contra-indicated, and enable prediction of relative kidney and tumour radiation absorbed dose in (188)Re(V)DMSA treatment. Ten patients with prostate carcinoma were recruited following observation of disseminated bone metastases on a recent (99m)Tc-hydroxydiphosphonate bone scan. Whole-body planar scans were obtained at ca. 4 h and 24 h after hydration and injection of 600 MBq (99m)Tc(V)DMSA, and a week later, at similar times after hydration and injection of 370 MBq (188)Re(V)DMSA. A triple-energy window (TEW) scatter correction was applied to the (188)Re scans. Counts per pixel were determined in regions of interest drawn over metastatic sites, kidneys and normal soft tissue. Tumour-to-soft tissue ratios were significantly lower (by a factor of approximately 0.8 after the TEW was applied) on (188)Re scans than on (99m)Tc scans, but the two were highly linearly correlated both in all individual patients and in tumours pooled from all patients together both at 4 h and at 24 h. Kidney-to-soft tissue ratios were similarly correlated and were lower for (188)Re than for (99m)Tc by a similar factor. Both tumour- and kidney-to-soft tissue ratios increased between 4 and 24 h but the latter increased more. In conclusion, only minor differences were seen between (99m)Tc and (188)Re scans, and kidney-to-background ratios on (188)Re scans were not higher than on (99m)Tc scans. These differences are insufficient to infer that they are due to a real difference in biodistribution, and they may be due only to different physical imaging characteristics. Thus (99m)Tc(V)DMSA scans are predictive of (188)Re(V)DMSA biodistribution and could be used to estimate tumour and renal dosimetry and assess suitability of patients for (188)Re(V)DMSA treatment.

Low-doses of ionising radiation induce melanoma metastases and trigger the immune system--adrenal axis feedback loop.

Low-doses of ionising radiation are frequently implicated in triggering and/or accelerating the growth of skin and other malignancies. It seemed probable that the radiation at similar dose levels might initiate metastasis from already existing tumours. Highly pigmented human melanoma xenograft that had lost its ability for a spontaneous metastasising and grown subcutaneously in athymic mice was exposed to very low and well-defined doses of ionising radiation to determine whether low linear energy transfer radiation can restore metastatic potential of the tumour. To ensure that all effects derived from radiation-activated neoplastic cells only, I was delivered selectively to the cutaneous melanoma instead of using the external beam. The direct response of these tumours to radiation was monitored by determining the growth rate of the lesions. Histopathological methods were employed to detect metastases. The lowest radiation dose of approximately 6 cGy deposited in the tumours initiated metastatic spread in all animals. Gradual increase of the radiation doses diminished both the frequency of the appearance of metastases and their distance from the primary lesions. There were no metastases from non-irradiated melanomas. The highest dose used (60 cGy) did not affect significantly the growth of cutaneous (primary) tumours, but lower doses that enhanced inflammatory infiltration of the lesions reduced tumour growth. Such radiation-stimulated immune responses were accompanied by increased pigmentation in cutaneous lesions and activation of the adrenal cortex indicating that the immune system-adrenal axis feedback loop had been triggered. The results demonstrate that very low-doses of ionising radiation induce melanoma metastases. The phenomenon is accompanied by the stimulation of the immune system-adrenal axis feedback loop that regulates eicosanoid synthesis, thereby suggesting an involvement of these molecules in the process. Radiation doses approaching the therapeutic level do not initiate melanoma dissemination.

Early detection of melanoma metastases with radioiodinated methylene blue.

Melanin synthesised in melanoma cells presents a unique target to which the treatment can be selectively addressed, provided the pigment is recognised by a suitable drug. Methylene blue (MTB) possesses a high affinity for melanin and, therefore, accumulates preferentially in melanoma cells. Since not directly toxic to the tumour, MTB serves as a carrier for radioisotopes and, once taken up by melanoma cells, acts as a selectively localised source of radiation. Hence, radioderivatives of the compound can be used for both diagnosis and therapy of disseminated melanoma. Eleven patients with confirmed metastatic melanoma and one with a recent local recurrence were studied using radioiodinated (iodine-123 or iodine-131) MTB and a gamma camera. Biopsies of cutaneous lesions were taken to determine directly the compound uptake in tumours. This first clinical investigation concerning the diagnostic potential of radioiodinated MTB in patients with disseminated melanoma confirmed the existence of approximately 80% of internal lesions previously identified by routine methods and, additionally, enabled detection of unknown secondaries in 6 of 12 patients studied. There were no false-positive gamma camera images regardless of whether 123I or 131I was used. 131I proved to be more suitable than 123I for detecting melanoma metastases with radioiodinated MTB. Hazy images of the lesions treated with external beam radiation and/or some drugs suggest that initial radio- and chemotherapy might affect MTB uptake in melanoma metastases and reduce the clarity of the scintigrams obtained from a gamma camera. However, small, untreated internal lesions that cannot be visualised easily with the standard diagnostic methods are revealed with 131I-MTB regardless of their localisation. It is concluded that use of radioiodinated MTB in conjunction with gamma camera or positron emission tomographic imaging might prove to be a useful and accessible tool for the detection of early melanoma dissemination.

Design of hypoxia-targeting radiopharmaceuticals: selective uptake of copper-64 complexes in hypoxic cells in vitro.

The well-known perfusion tracer CuPTSM, labelled with 62Cu or 64Cu, is believed to be trapped in cells non-selectively by a bioreductive mechanism. It is proposed that by modifying the ligand to increase its electron donor strength (for example by adding alkyl functionality or replacing sulphur ligands with oxygen ligands), the copper complexes will become less easily reduced and tracers with selectivity for hypoxic tissues could thus be developed. The aim of this work was to prepare 64Cu-labelled complexes of two series of ligands, based on the bis(thiosemicarbazone) (13 ligands) and bis(salicylaldimine) (3 ligands) skeletons, and to evaluate the hypoxia dependence of their uptake in cells. The complexes were incubated with Chinese hamster ovary cells under normoxic and hypoxic conditions, and the cells isolated by centrifugation to determine radioactivity uptake at various time points up to 90 min. Several members of both series demonstrated significant (P<0.05) or highly significant (P<0.01) hypoxia selectivity, indicating that both series of complexes offer a basis for development of hypoxia-targeting radiopharmaceuticals for positron emission tomography (60Cu, 61Cu, 62Cu, 64Cu) and targeted radiotherapy (64Cu, 67Cu).