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This study investigated the impact of temporary vascular collapse on tumour control probability (TCP) in stereotactic body radiotherapy (SBRT), taking into account different radiosensitivities of chronically and acutely hypoxic cells.Three-dimensional tumours with heterogeneous oxygenation were simulated assuming different fractions of collapsed vessels at every treatment fraction. The modelled tumours contained a chronically hypoxic subvolume of 30-60% of the tumour diameter, and a hypoxic fraction ≤5 mm Hg of 30-50%. The rest of the tumours were well-oxygenated at the start of the simulated treatment.For all simulated cases, the largest reduction in TCP from 97% to 2% was found in a tumour with a small chronically hypoxic core treated with 60 Gy in eight fractions and assuming a treatment-induced vascular collapse of 35% in the well-oxygenated region.The timing of SBRT fractions should be considered together with the tumour oxygenation to avoid loss of TCP in SBRT.

Background: Tumour hypoxia is associated with increased radioresistance and poor response to radiotherapy. Pre-treatment assessment of tumour oxygenation could therefore give the possibility to tailor the treatment by calculating the required boost dose needed to overcome the increased radioresistance in hypoxic tumours. This study concerned the derivation of a non-linear conversion function between the uptake of the hypoxia-PET tracer F-18-HX4 and oxygen partial pressure (pO(2)).Material and methods: Building on previous experience with FMISO including experimental data on tracer uptake and pO(2), tracer-specific model parameters were derived for converting the normalised HX4-uptake at the optimal imaging time point to pO(2). The conversion function was implemented in a Python-based computational platform utilising the scripting and the registration modules of the treatment planning system RayStation. Subsequently, the conversion function was applied to determine the pO(2) in eight non-small-cell lung cancer (NSCLC) patients imaged with HX4-PET before the start of radiotherapy. Automatic segmentation of hypoxic target volumes (HTVs) was then performed using thresholds around 10mmHg. The HTVs were compared to sub-volumes segmented based on a tumour-to-blood ratio (TBR) of 1.4 using the aortic arch as the reference oxygenated region. The boost dose required to achieve 95% local control was then calculated based on the calibrated levels of hypoxia, assuming inter-fraction reoxygenation due to changes in acute hypoxia but no overall improvement of the oxygenation status.Results: Using the developed conversion tool, HTVs could be obtained using pO(2) a threshold of 10mmHg which were in agreement with the TBR segmentation. The dose levels required to the HTVs to achieve local control were feasible, being around 70-80Gy in 24 fractions.Conclusions: Non-linear conversion of tracer uptake to pO(2) in NSCLC imaged with HX4-PET allows a quantitative determination of the dose-boost needed to achieve a high probability of local control.

Purpose: In tumour, the imbalance between oxygen supply and demand leads to hypoxia, which represents a negative prognostic factor associated with aggressive tumour phenotype and therapy resistance. This review provides an overview of the use of positron emitter-labelled radiopharmaceutical used to image hypoxia in preclinical models of cancer. Methods: A critical and comprehensive PubMed search was performed identifying articles related to PET imaging for hypoxia assessment in preclinical setting from January 2007 up to January 2017. Results: We have considered and described a total of 54 original articles, exploring tumour-associated hypoxia in preclinical models. Results underlined the potential application together with the advantages and pitfalls of the use of PET in preclinical research. Multi-target imaging allowed to better define the relationship between hypoxia and other biological hallmarks of tumour; imaging of hypoxia was proved as a useful tool for lesions stratification and response prediction to radiotherapy; however, cutoff indexes were identified in few studies. Hypoxia PET showed remarkable tracer delivery limitations in the study of vascular disrupting agents but suggested the potential use of PET as a marker of response or resistance to anti-angiogenics. Finally, the effect of anaesthesia on tracer kinetics and tumour oxygenation as well as perfusion dependency in tracer uptake should be carefully evaluated to avoid artefactual results. Conclusions: Preclinical studies highlight the advantages and the limitations of the available hypoxia-radiotracers and their potential usefulness for the evaluation of treatments outcome and radiotherapy planning.

BACKGROUND/AIM: The aim of this study was to investigate radiation-induced tumour vascular damage and its impact thereof on the outcome of stereotactic body radiotherapy (SBRT). MATERIALS AND METHODS: Vessel densities in animal tumours before and after a single dose of 20 Gy were quantified and used as input for simulations of three-dimensional tumours with heterogeneous oxygenation. SBRT treatments of the modelled tumours in 1–8 fractions were simulated. The impact of vessel collapse on the outcome of SBRT was investigated by calculating tumour control probability (TCP) and the dose required to obtain a TCP of 50% (D(50)). RESULTS: A radiation-induced increase of acute hypoxia in tumours during SBRT treatment could be simulated based on the experimental data. The D(50) values for these tumours were higher than for the simulated tumours without vessel collapse. CONCLUSION: The vascular changes after high doses of radiation could compromise the outcome of SBRT by increasing tumour hypoxia.

Hypoxia is known to be a poor prognostic indicator for nearly all solid tumours and also is predictive of treatment failure for radiotherapy, chemotherapy, surgery and targeted therapies. Imaging has potential to identify, spatially map and quantify tumour hypoxia prior to therapy, as well as track changes in hypoxia on treatment. At present no hypoxia imaging methods are available for routine clinical use. Research has largely focused on positron emission tomography (PET)-based techniques, but there is gathering evidence that MRI techniques may provide a practical and more readily translational alternative. In this review we focus on the potential for imaging hypoxia by measuring changes in longitudinal relaxation [R (1); termed oxygen-enhanced MRI or tumour oxygenation level dependent (TOLD) MRI] and effective transverse relaxation [R (2)*; termed blood oxygenation level dependent (BOLD) MRI], induced by inhalation of either 100% oxygen or the radiosensitising hyperoxic gas carbogen. We explain the scientific principles behind oxygen-enhanced MRI and BOLD and discuss significant studies and their limitations. All imaging biomarkers require rigorous validation in order to translate into clinical use and the steps required to further develop oxygen-enhanced MRI and BOLD MRI into decision-making tools are discussed.

Tumour hypoxia is a well‐established factor of resistance in radiation therapy (RT). Myo‐inositol trispyrophosphate (ITPP) is an allosteric effector that reduces the oxygen‐binding affinity of haemoglobin and facilitates the release of oxygen by red blood cells. We investigated herein the oxygenation effect of ITPP in six tumour models and its radiosensitizing effect in two of these models. The evolution of tumour pO2 upon ITPP administration was monitored on six models using 1.2 GHz Electron Paramagnetic Resonance (EPR) oximetry. The effect of ITPP on tumour perfusion was assessed by Hoechst staining and the oxygen consumption rate (OCR) in vitro was measured using 9.5 GHz EPR. The therapeutic effect of ITPP with and without RT was evaluated on rhabdomyosarcoma and 9L‐glioma rat models. ITPP enhanced tumour oxygenation in six models. The administration of 2 g/kg ITPP once daily for 2 days led to a tumour reoxygenation for at least 4 days. ITPP reduced the OCR in six cell lines but had no effect on tumour perfusion when tested on 9L‐gliomas. ITPP plus RT did not improve the outcome in rhabdomyosarcomas. In 9L‐gliomas, some of tumours receiving the combined treatment were cured while other tumours did not benefit from the treatment. ITPP increased oxygenation in six tumour models. A decrease in OCR could contribute to the decrease in tumour hypoxia. The association of RT with ITPP was beneficial for a few 9L‐gliomas but was absent in the rhabdomyosarcomas.

Hypoxia, or low oxygen partial pressure, is a common feature of many solid tumours, associated with therapy resistance and poor prognosis for cancer patients. This chemical feature of the tumour microenvironment represents an imbalance of blood oxygen supply and tissue oxygen demand. Given the prognostic significance of hypoxia, there is a clinical unmet need to provide validated, non-invasive, imaging biomarkers that can be used to detect and monitor the spatiotemporal distribution of hypoxia at the point of cancer diagnosis and during treatment. In this talk, I will give an overview of how photoacoustic imaging can address this unmet need, providing examples from both preclinical studies in mouse models of cancer and clinical studies in human patients.

Intravital imaging within heterogenic solid tumours is important for understanding blood perfusion profiles responsible for establishment of multiple parameters within the tumour mass, such as hypoxic and nutrition gradients, cell viability, proliferation and drug response potentials. Herein, we developed a method based on a volumetric multispectral optoacoustic tomography (vMSOT) for cancer imaging in preclinical models and explored its capacity for three-dimensional imaging of anatomic, vascular and functional tumour profiles in real time. In contrast to methods based on cross-sectional (2D) image acquisition as a basis for 3D rendering, vMSOT has attained concurrent observations from the entire tumour volume at 10 volumetric frames per second. This truly four dimensional imaging performance has enabled here the simultaneous assessment of blood oxygenation gradients and vascularization in solid breast tumours and revealed different types of blood perfusion profiles in-vivo. The newly introduced capacity for high-resolution three-dimensional tracking of fast tumour perfusion suggests vMSOT as a powerful method in preclinical cancer research and theranostics. As the imaging setup can be equally operated in both stationary and handheld mode, the solution is readily translatable for perfusion monitoring in a clinical setting. • vMSOT visualizes 3D anatomic, vascular and functional tumour profiles in real time. • Three types of blood perfusion profiles are revealed in breast tumour model. • The method is readily adaptable to operate in a handheld clinical mode.

Purpose To estimate the rate constant for pyruvate to lactate conversion in tumours in response to a hypoxic challenge, using hyperpolarised 13C1-pyruvate and magnetic resonance spectroscopy. Methods and materials Hypoxic inspired gas was used to manipulate rat P22 fibrosarcoma oxygen tension (pO2), confirmed by luminescence decay of oxygen-sensitive probes. Hyperpolarised 13C1-pyruvate was injected into the femoral vein of anaesthetised rats and slice-localised 13C magnetic resonance (MR) spectra acquired. Spectral integral versus time curves for pyruvate and lactate were fitted to a precursor-product model to estimate the rate constant for tumour conversion of pyruvate to lactate (kpl). Mean arterial blood pressure (MABP) and oxygen tension (ArtpO2) were monitored. Pyruvate and lactate concentrations were measured in freeze-clamped tumours. Results MABP, ArtpO2 and tumour pO2 decreased significantly during hypoxia. kpl increased significantly (p

Background: Isolated limb perfusion with TNF-alpha and melphalan (TM-ILP) is a highly effective treatment for locally advanced tumours of the extremities. Previous research suggests an almost immediate disintegration of the blood supply of the tumour. The aim of the present study was to verify this hypothesis using non-invasive measurements of microvascular perfusion and tissue oxygenation. Methods: A total of 11 patients were included in the study. TM-ILP was performed under mildly hyperthermic conditions (39 °C) in the extremities via proximal vascular access. Capillary-venous microvascular blood flow, haemoglobin level (Hb) and oxygen saturation (SO2) were determined using laser Doppler and white-light spectroscopy, respectively, before TM-ILP and at 30 min, 4 h, 1 day, 4 days, 1 week, 2 weeks and 6 weeks after TM-ILP from tumour and healthy muscle tissues. Results: Blood flow and Hb were mostly higher, whereas SO2 was lower, in tumour tissue compared with muscle tissue. In both tumour and muscle tissues, blood flow significantly increased immediately after TM-ILP and remained elevated for at least 2 weeks, followed by a return to the initial values 6 weeks after the procedure. Conclusion: No signs were found of early destruction of the tumour vasculature. The observations suggest that an inflammatory reaction is one of the key elements of TM-ILP.

Mild hyperthermia has been known to enhance the response of tumours to radiotherapy or chemotherapy by increasing tumour blood flow, thereby increasing tumour oxygenation or drug delivery. The purpose of this study was to assess the changes in temperature and blood flow in human cervical cancer in response to regional heating with modulated electro-hyperthermia (mEHT).The pelvic area of 20 patients with cervical carcinoma was heated with mEHT. The peri-tumour temperature was measured using an internal organ temperature probe. The tumour blood flow was measured using 3D colour Doppler ultrasound by determining the peak systolic velocity/end-diastolic velocity ratio (S/D ratio) and the resistance index (RI) within blood vessels.The mean peri-tumour temperature was 36.7 ± 0.2 °C before heating and increased to 38.5 ± 0.8 °C at the end of heating for 60 min. The marked declines in RI and S/D values strongly demonstrated that heating significantly increased tumour blood perfusion.Regional heating of the pelvic area with mEHT significantly increased the peri-tumour temperature and improved the blood flow in cervical cancer. This is the first demonstration that the blood flow in cervical cancer is increased by regional hyperthermia. Such increases in temperature and blood flow may account for the clinical observations that hyperthermia improves the response of cervical cancer to radiotherapy or chemotherapy.

Functional imaging of tumour hypoxia has been suggested as a tool for refining target definition and treatment optimization in radiotherapy. The approach, however, has been slow to be adopted clinically as most of the studies on the topic do not take into account the in-treatment changes of hypoxia. The present study aimed to introduce a function that quantifies the changes of oxygen distributions in repeated PET images taken during treatment. The proposed approach for determining the reoxygenation function was tested for feasibility on patients with head and neck cancer, repeatedly imaged with FMISO PET during radiotherapy. Reoxygenation functions were derived by solving the convolution between functions describing the oxygen distributions of successive images. The method was found to be mathematically feasible. The results indicate that the reoxygenation functions describing the change in oxygenation have distinct shapes prompting the hypothesis that oxygenation changes reflected by them might have predictive power for treatment outcome. Future studies on a larger patient population to search for predictive correlations based on the reoxygenation function are planned.

The efficacy of Stereotactic Body Radiation Therapy (SBRT) in early-stage non-small cell lung cancer for severely hypofractionated schedules is clinically proven. Tumour control probability (TCP) modelling might further optimize prescription dose and number of treatment fractions (n). To this end, we will discuss the following controversial questions. Which is the most plausible cell-survival model at doses per fraction (d) as high as 20Gy? Do clinical data support a dose-response relationship with saturation over some threshold-dose? Given the reduced re-oxygenation for severe hypofractionation, is the inclusion of tumour hypoxia in TCP modelling relevant? Can iso-effective schedules be derived by assuming a homogeneous tumour-cell population with α/β≈10Gy, or should distinct cell subpopulations, with different α/β values, be taken into account? Is there scope for patient-specific individualization of n? Despite the difficulty of providing definite answers to the above questions, reasonable suggestions for lung SBRT can be derived from the literature. The LQ model appears to be the best-fitting model of cell-survival even at such large d, and is therefore the preferred choice for TCP modelling. TCP increases with dose, reaching saturation above 90% local control, but there is still uncertainty on the threshold-dose. In silico simulations accounting for variations in tumour oxygenation are consistent with an improved therapeutic ratio at 5-8 fractions instead of the current 3-fraction reference schedules. Tumour hypoxia modelling might also explain how α/β changes with n, identifying the clonogen subpopulation which determines tumour response. Finally, an optimal patient-specific n can be derived from the planned lung dose distribution.

Hyperthermic isolated limb perfusion (ILP) with recombinant tumour necrosis factor alpha (TNF) and melphalan contributes to limb-saving treatment in patients with locally advanced extremity soft tissue sarcoma (STS). This study was conducted to evaluate the dynamic changes of tumour oxygenation and temperature during ILP and their effects on treatment response.Tumour oxygenation (pO(2)) and tumour temperature were measured by intratumourally placed O(2)-sensitive catheter electrodes in 34 patients who underwent ILP for locally advanced or recurrent STS. Tumour response to ILP was assessed by the fraction of tumour necrosis in the resection specimen.Mean tumour pO(2) prior to application of TNF and melphalan was 36 mm Hg (range: 2-116 mm Hg) and dropped significantly to 13 mm Hg (range: 0-67 mm Hg, p = 0.03) during ILP. Mean tumour tissue temperature increased from 34.4°C (range 32.4-36.4) to 38.5°C (range 34.1-40.4, p = 0.0001). The mean fraction of necrosis in the resection specimen was 65% (range 5-99). Only the tumour tissue temperature at the onset of ILP correlated with the extent of tumour necrosis (p = 0.01).ILP with TNF and melphalan induces severe oxygen deprivation in soft tissue sarcoma. However, changes in tumour oxygenation did not correlate with treatment response.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) is a functional imaging technique that assesses the physiology of tumour tissue by exploiting abnormal tumour microvasculature. Advances made through DCE-MRI include improvement in the diagnosis of cancer, optimization of treatment choices, assessment of treatment efficacy and non-invasive identification of prognostic information. DCE-MRI enables quantitative assessment of tissue vessel density, integrity, and permeability, and this information can be applied to study of angiogenesis, hypoxia and the evaluation of various biomarkers. Reproducibility of DCE-MRI results is important in determining the significance of observed changes in the parameters. As improvements are made towards the utility of DCE-MRI and interpreting biologic associations, the technique will be applied more frequently in the study of cancer in animals. Given the importance of tumour perfusion with respect to tumour oxygenation and drug delivery, the use of DCE-MRI is a convenient and powerful way to gain basic information about a tumour.

Contains fulltext : 51464.pdf (Publisher’s version ) (Closed access) Tumour hypoxia has been found to be a characteristic feature in many solid tumours. It has been shown to decrease the therapeutic efficacy of radiation treatment, surgery and some forms of chemotherapy. Successful approaches have been developed to counteract this resistance mechanism, although usually at the cost of increased short- and long-term side-effects. New methods for qualitative and quantitative assessment of tumour oxygenation have made it possible to establish the prognostic significance of tumour hypoxia. The ability to determine the degree and extent of hypoxia in solid tumours is not only important prognostically, but also in the selection of patients for hypoxia-modifying treatments. To provide the best attainable quality of life for individual patients it is of increasing importance that tools be developed that allow a better selection of patients for these intensified treatment strategies. Several genes and proteins involved in the response to hypoxia have been identified as potential candidates for future use in predictive assays. Although some markers and combinations have shown potential benefit and are associated with treatment outcome, their clinical usefulness needs to be validated in prospective trials. A review of published studies was carried out, focusing on the assessment of tumour hypoxia, patient selection and the possibilities to overcome hypoxia during treatment.

Introduction: Radiation plays a major role in treatment of locoregional control of Head and Neck Squamous cell carcinoma (HNSCC). Anaemia is considered a contributor to intra-tumour hypoxia and tumour resistance to ionizing radiation and most evidences are from developed world, we prospectively investigated the exact role of anaemia in treatment outcome of Stage III/IVA HNSCC in our patient population. Aim of the Study: Primary end point: To analyse the PreRadiotherapy haemoglobin level and early response of treatment in stage III/IVA HNSCC and to determine the relationship of Pre-Radiotherapy haemoglobin level with other prognostic factors. Materials and Methods: This non-interventional single blinded randomized study enrolled patients attending the OPD consecutively, who met our eligibility criteria. Inclusion Criteria: HNSCC patients of Stage III/IVA aged ≥18 years and ≤ 70 years with ECOG status of 1or 2 and willing for concurrent chemoradiation and at least 6 weeks of follow up. Exclusion Criteria: 1) Previous history of treatment for malignancy or radiation in head and neck site. 2) Patients with other fatal and non-fatal pre-morbid or co-morbid conditions that can affect the outcome or the overall survival. Patients with Pre-radiotherapy haemoglobin status < 10 g/dl were given haematinic support and/or blood transfusion. All patients received concurrent chemotherapy (weekly cisplatin) and radiation in conventionally fractionated dose of 66Gy. Early treatment responses were evaluated with Revised RECIST version 1.1 and Data analysis using SPSS version 17.0. Results: Ninety one patients enrolled had mean age of 55.63 (range: 32-69), a median of 56 and mode of 60. Seventy one were males (78%) and 20 females (22%) with a performance status of ECOG 1 in 43 (47%) patients and ECOG 2 in 48 (53%); Pre-RT Hb level of 5 days (p = 5 days and nondevelopment of grade III mucositis was found to be significantly associated with good loco-regional control. Haemoglobin level ≥10.7 g/dl was associated with better treatment outcome, higher performance status, fewer treatment interruptions and lesser degree of mucositis. Transfusion did not affect the outcome. Definitive conclusions and recommendations need further expansion of our study for better statistical power.

The microenvironment of a tumour, in particular its hypoxic status, is a crucial factor in its response to radiotherapy. Conventional techniques for measuring hypoxia are either invasive or follow surgical intervention, and thus not ideal. Positron emission tomography allows the non-invasive pre-surgical assessment of oxygen status by measuring the spatiotemporal distribution of hypoxia-specific tracers. However, the relationship between levels of uptake and the underlying oxygen tension are yet to be elucidated. Furthermore, it is not fully understood how changes in the underlying physiology affect the appearance of uptake. This paper presents a modular simulation of the tumour microenvironment, underpinned by a probability density function (PDF) to model the vasculature. The model is solved numerically, to simulate both the steady-state oxygenation of a tumour and the spatiotemporal distribution of the hypoxia-specific tracer, [18F]-fluoromisonidazole (Fmiso), in a 2D environment. The results show that using a PDF to represent the vasculature effectively captures the 'hypoxic island' appearance of oxygen-deficient tissues seen ex vivo. Simulated tissue activity curves (TACs) demonstrate the general two-stage trend of empirical data, with an initial perfusion-dominated uptake, followed by hypoxia-specific binding. In well-perfused tissue, activity follows plasma levels in early stages, with binding of Fmiso only becoming apparent at a later stage. In structurally hypoxic tissue, a more gradual initial increase in activity is observed, followed by the same accumulation slope. We demonstrate the utility of theoretical modelling of tracer uptake, by quantifying the changes in TAC structure that arise as a result of altering key physiological characteristics. For example, by decreasing either the proximity of tissue to the vasculature, or the effective diffusion coefficient of Fmiso, we can observe a shift of TAC structure from corresponding to well-perfused to avascular regions, despite wholly different underlying causes.

Purpose To investigate the relationship between tumour hypoxia and serum and tumour osteopontin (OPN) levels. Materials and methods Experiments were performed in CDF1 or C3H/Km mice implanted with a C3H mammary carcinoma (CDF1) or SCCVII squamous cell carcinoma (C3H/Km), respectively. Mice were either untreated or gassed with 10% oxygen for 1–72 h. Serum and tumour OPN levels were measured with an ELISA and tumour OPN mRNA levels using RT-PCR. Tumour oxygenation was estimated using the Eppendorf histograph with the percentage of pO2 values ⩽5 mm Hg (HF5) as the endpoint. Results OPN levels were 50-fold higher in the serum of non-tumour bearing CDF1 mice compared to C3H/Km mice. A tumour related increase in serum OPN levels was observed in CDF1 but not in C3H/Km mice. Low oxygen breathing increased HF5 in both tumour models and in the C3H mammary carcinoma model both serum and tumour OPN decreased after prolonged hypoxia (24 h and more). When 12 h of hypoxia was followed by 24 h reoxygenation there was a twofold increase in serum OPN levels. No changes were observed in the SCCVII model. No changes in tumour OPN mRNA expression were observed during hypoxia and reoxygenation in these tumour models. Conclusion Clear strain and tumour specific differences in the effect of hypoxia on OPN levels have been observed in two different mouse tumour models. These data emphasize the complexity in the relationship between poor oxygenation (and/or reoxygenation) of tumours and serum levels of OPN.

The polarographic measurement of tissue oxygenation is one of the most widely used methods in clinical practice for the quantification of tumour hypoxia. However, due to the particular features of the electrode measuring process, the results of the measurements do not accurately reflect the tumour oxygenation. This study aimed to find a correlation between the electrode measurements and the tumour oxygenation in an attempt to improve the accuracy of the predictions regarding the response to treatment based on electrode measurements. A previously developed computer model that allows the simulation of tumour tissue and electrode measurements was used. The oxygenation of a large number of tumours with biologically relevant distributions of blood vessels was theoretically calculated. Simulations of electrode measurements allowed the comparison between the real tissue oxygenation and the results obtained with the electrode. A semi-empirical relationship between the hypoxic fraction measured by the electrode and the real hypoxic fraction in the tissue has been found. The impact of the correction of the electrode measurements in terms of predictions for tumour control probability was estimated for a few clinical examples. The range of possible true values corresponding to one measurement has also proven useful for explaining the apparently unexpected response to the treatment of some patients. The corrected hypoxic fraction which is believed to be closer to the real value of tissue hypoxia predicts much smaller control probabilities than the raw electrode measurements. This could provide an explanation for the apparently unexpected failure to respond to the treatment of some of the patients with apparently favourable tumour oxygenation. This also means that the electrode measurements cannot be used directly for the quantitative modelling of tumour response to the treatment. The conversion method proposed in this paper might however strengthen the statistical power of the correlations between the electrode measurements and the treatment outcome.

Darbepoetin alfa (Aranesp®, Amgen) is a novel erythropoiesis-stimulating protein with a serum half-life longer than recombinant human erythropoietin (Epo), used in the treatment of cancer-associated anaemia. Anaemia is known to adversely affect prognosis and response to treatment in cancer patients. Solid tumours contain regions of hypoxia due to poor vascular supply and cellular compaction. Although hypoxic stress usually results in cell death, hypoxia-resistant tumour cells are genetically unstable and often acquire a drug-resistant phenotype. Increasing tumour oxygenation and perfusion during treatment could have the doubly beneficial outcome of reducing the fraction of treatment-resistant cells, while increasing drug delivery to previously hypoxic tissue. In this study, we examined the effect of darbepoetin alfa on chemotherapy sensitivity and delivery in an in vivo model of Lewis lung carcinoma, shown here to express the Epo receptor (EpoR). We identified that weekly darbepoetin alfa treatment, commencing 10 days before chemotherapy, resulted in a significant reduction in tumour volume compared to chemotherapy alone. This was mediated by the prevention of anaemia, a reduction in tumour hypoxia and a concomitant increase in drug delivery. Darbepoetin alfa treatment alone did not modulate the growth of the EpoR-expressing tumour cells. This study identifies an important role for darbepoetin alfa in increasing the therapeutic index of chemotherapy.

It is known that tumour oxygenation levels play a role in the success of cancer treatment, especially radiation therapy. Current research continues to show that tumour hypoxia adversely affects patient outcome and is focused on finding methods of improving tumour oxygen levels or using poor oxygenation as a tumour-specific characteristic in order to develop new therapeutic methods. The radiobiology of oxygen in radiation-induced cell eradication will be discussed, as well as the effects of tumour hypoxia on malignant progression and metastases. There are currently methods to improve poor oxygenation, or drugs can be used that mimic the effect of oxygen in the cell, or target other therapies to the hypoxic cells while sparing normal tissues. Anemia and angiogenesis are related topics that are important to tumour oxygenation and patient outcome. Novel ideas on improving anemia and influencing tumour angiogenesis to increase treatment success have also been included.

Accelerated radiation carbogen nicotinamide (ARCON) therapy is generally well tolerated, but some patients experience intolerance to nicotinamide and carbogen (95% O(2)+5% CO(2)). This study investigated the effect of reducing both the nicotinamide dose and carbogen CO(2) content on radiation response.A C3H mouse mammary carcinoma grown in the right rear foot of female CDF1 was used and treated when at 200 mm(3). Nicotinamide was intraperitoneally injected 20 min prior to irradiation. Carbogen (CO(2) content of either 2 or 5%, balance O(2)) breathing was started 5 min before, and continued during, additional treatments. Radiation was given locally to tissues of restrained non-anaesthetised mice either as a single or fractionated (10 fractions in 12 days) schedule. The endpoints were local tumour control at 90 days, development of moist desquamation 11-23 days after treatment of normal foot skin, or tumour oxygenation measured with the Eppendorf electrode.The TCD50 values in this tumour following single or fractionated radiation treatment were 52 and 71Gy, respectively. Carbogen (5% CO(2) content) breathing with every radiation treatment in the fractionated schedule significantly (Chi-squared test; P0.05) enhanced radiation response (ER 1.25). Significant enhancements were also seen with nicotinamide given either as 10x120 mg/kg (ER 1.25), 6x120 mg/kg (ER 1.11) or 10x90 mg/kg (ER 1.18), although the 6x120 schedule was significantly less effective than 10x120. Combining nicotinamide with carbogen resulted in ERs of 1.39-1.44, and these were independent of the nicotinamide treatments. There was also no significant difference in the enhancement of tumour radiation response or improved tumour oxygenation status if the CO(2) content of the gas breathing was varied from 0% (i.e. 100% O(2)) to 2 or 5% (balance O(2)), although a CO(2) content of 2% did give a smaller enhancement of radiation-induced normal skin damage than 5%.Both the nicotinamide dose, but not the frequency, and carbogen CO(2) content may be reduced in patients experience intolerance without any significant loss of sensitisation.

Haemoglobin concentrations and tumour-pO(2) were evaluated pre-therapy in 30 patients with head and neck cancers. Anterior tibialis muscle-pO(2) was additionally measured in 16 of these patients. Tumour-pO(2) was lower in the most anaemic patients (P=0.032) and correlated with muscle-pO(2) (r=0.809, P0.001). These results suggest that haemoglobin concentration influences tumour-oxygenation.

A number of laboratories are utilising both hypoxia and perfusion markers to spatially quantify tumour oxygenation and vascular distributions, and scientists are increasingly turning to automated image analysis methods to quantify such interrelationships. In these studies, the presence of regions of necrosis in the immunohistochemical sections remains a potentially significant source of error. In the present work, frozen MCa-4 mammary tumour sections were used to obtain a series of corresponding image montages. Total vessels were identified using CD31 staining, perfused vessels by DiOC7 staining, hypoxia by EF5/Cy3 uptake, and necrosis by haematoxylin and eosin staining. Our goal was to utilise image analysis techniques to spatially quantitate hypoxic marker binding as a function of distance from the nearest blood vessel. Several refinements to previous imaging methods are described: (1) hypoxia marker images are quantified in terms of their intensity levels, thus providing an analysis of the gradients in hypoxia with increasing distances from blood vessels, (2) zonal imaging masks are derived, which permit spatial sampling of images at precisely defined distances from blood vessels, as well as the omission of necrotic artifacts, (3) thresholding techniques are applied to omit holes in the tissue sections, and (4) distance mapping is utilised to define vascular spacing. British Journal of Cancer (2002) 86, 1831–1836. doi:10.1038/sj.bjc.6600343 www.bjcancer.com © 2002 Cancer Research UK

Over the last ten years the impact of fundamental radiation biology into daily radiotherapy has been of concern chiefly to fractionation, prediction of radiation response, tumour oxygenation, intrinsic radiosensitivity including genetic approaches, and the determinants of the outcome of chemoradiotherapy combinations. Future goals will rely on sophisticated approaches, based on the progress of molecular and cellular biology and the characterisation of new targets for radiation. Some of these novel advances will be discussed.

Perfusion and oxygenation of experimental tumours were studied during angiotensin II (AT II) administration whereby the rate of the continuous AT II infusion was chosen to increase the mean arterial blood pressure (MABP) by 50–70 mmHg. In subcutaneous DS- sarcomas the red blood cell (RBC) flux was assessed using the laser Doppler technique and the mean tumour oxygen partial pressure (p O 2) was measured polarographically using O 2-sensitive catheter and needle electrodes. Changes in RBC flux with increasing MABP depended mainly on tumour size. In small tumours, RBC flux decreased with rising MABP whereas in larger tumours RBC flux increased parallel to the MABP. As a result of these volume-dependent effects on tumour blood flow, the impact of AT II on tumour p O 2 was also mainly tumour volume-related. In small tumours oxygenation decreased with increasing MABP during AT II infusion, whereas in large tumours a positive relationship between blood pressure and O 2 status was found. This disparate behaviour might be the result of the co-existence of two functionally distinct populations of tumour vessels. In small tumours, perfusion decreases presumably due to vasoconstriction of pre-existing host vessels feeding the tumour. In larger malignancies, newly formed tumour vessels predominate and seem not to have this vasoresponsive capability (lack of smooth muscle cells and/or AT receptors), resulting in an improvement of perfusion which is not tumour-related per se, but is due to the increased perfusion pressure. © 2000 Cancer Research Campaign

This study quantifies the spatial distribution of pO2 in glioma and in the surrounding brain tissue. Both glioma and peritumoural brain contain regions at oxygen tensions less than 2.5 mmHg. Modalities targeting hypoxia to improve the efficacy of therapy may have an important role in the management of this disease.

Tumour hypoxia is increasingly recognized as a major deleterious factor in cancer therapies, as it compromises treatment and drives malignant progression. This review seeks to clarify the oxygen levels that are pertinent to this issue. It is argued that normoxia (20% oxygen) is an extremely poor comparator for “physoxia”, i.e. the much lower levels of oxygen universally found in normal tissues, which averages about 5% oxygen, and ranges from about 3% to 7.4%. Importantly, it should be recognized that the median oxygenation in untreated tumours is significantly much lower, falling between approximately 0.3% and 4.2% oxygen, with most tumours exhibiting median oxygen levels

Hypoxia plays an important role in tumour recurrence among head and neck cancer patients. The identification and quantification of hypoxic regions are therefore an essential aspect of disease management. Several predictive assays for tumour oxygenation status have been developed in the past with varying degrees of success. To date, functional imaging techniques employing positron emission tomography (PET) have been shown to be an important tool for both pretreatment assessment and tumour response evaluation during therapy. Hypoxia-specific PET markers have been implemented in several clinics to quantify hypoxic tumour subvolumes for dose painting and personalized treatment planning and delivery. Several new radiotracers are under investigation. PET-derived functional parameters and tracer pharmacokinetics serve as valuable input data for computational models aiming at simulating or interpreting PET acquired data, for the purposes of input into treatment planning or radio/chemotherapy response prediction programs. The present paper aims to cover the current status of hypoxia imaging in head and neck cancer together with the justification for the need and the role of computer models based on PET parameters in understanding patient-specific tumour behaviour.

Tumour oxygenation is an important determinant of radiotherapy outcome as it could modulate cellular radiation sensitivity. Advanced PET imaging able to characterise this microenvironmental aspect in vivo might be used to devise counteracting therapies as it could provide information on the severity and the spatial distribution of the hypoxic regions. This study reviews the advantages and limitations of PET for imaging and quantifying tumour hypoxia and proposes a novel approach to obtain absolute levels of hypoxia from PET images through the use of EPR oximetry. This would offer a significant advantage over proposals based on empirical conversions of the intensities in the PET images to relative radiosensitivities. Thus, tumour hypoxia must be taken into account at the stage of treatment planning for photons and particle therapy by accounting for its extent and severity through the use of PET imaging combined with absolute EPR measurements.

The purpose of this study was to evaluate the feasibility of polarographic oxygen electrode measurements and phosphorus magnetic resonance spectroscopy (31P-MRS) in extravisceral soft tissue tumours, designated to receive preoperative radiotherapy. Pretreatment tumour oxygenation was determined in 41 cases and 31P-MRS was amenable to lesions in 34 patients. Biopsies were characterized histopathologically as 25 primary soft tissue sarcomas (STS), 2 recurrent STS, 9 benign and 5 other malignancies. Evaluation of phosphorus (31P) spectra was possible in 11 cases. The oxygenation status of normal tissue was higher than that of tumours, whereas no difference was found between oxygenation status of benign lesions and that of STS. There was substantial variation between tumours in the median pO2 and the bioenergetic status (beta-NTP/Pi). No correlation was found between tumour pO2 and volume (n = 25). Moreover, there was no correlation between beta-NTP/Pi and the median tumour pO2, the fraction of pO2 valuesor =2.5 mmHg or tumour volume (n = 10), respectively. In conclusion, oxygen electrode assessment was found to be a clinically applicable and feasible technique for measuring tumour oxygenation status, whereas the success of 31P-MRS in human neoplasms was limited by a very poor resolution in the phosphorus signal that allowed analysis of 31P spectra in 11 tumours out of 34 cases.

The oxygenation status of C3H mammary carcinomas, grown in the feet of CDF1 mice, was measured with an Eppendorf oxygen electrode. Tumours were locally heated in a water bath at temperatures of 38.5-41.5°C for 1 h. Measurements made during heating showed temperature-dependent increases in tumour oxygenation. However, measurements performed after heating showed a rapid return to normal oxygenation status. Mild hyperthermia thus improves tumour oxygenation and this can explain the radiosensitization seen with low heat treatments, but only when the heat and radiation are administered concurrently, which is typically not the way that are given clinically.

There are areas of limited oxygen availability in most solid tumours, including breast cancer. Hypoxia in solid tumours is mainly a consequence of poor perfusion. Structural and functional abnormalities of newly formed tumour vessels cause spatial and temporal heterogeneity of tissue perfusion. The two principal mediators of hypoxia response, HIF-1 and HIF-2, are known to be stabilized at different oxygen levels and to have different temporal responses to hypoxia. Recently, stromal HIF-1 and HIF-2 have been suggested to have opposing roles in breast cancer progression. There is an established link between intralesional, severe hypoxia near areas of necrosis with high levels of HIF-1 and poor prognosis in breast cancer. However, the biological effects of moderate hypoxia and the hypoxic response of stromal cells are currently topics of intense investigation.

Hydralazine can substantially decrease blood flow and increase hypoxia in transplanted tumours. Previous indirect studies have suggested that hydralazine does not induce such effects in spontaneous tumours. We have now directly investigated the ability of hydralazine to increase hypoxia in both transplanted and spontaneous murine tumours by measuring tumour oxygen partial pressure (pO2) distributions using an Eppendorf oxygen electrode. Spontaneous tumours arose at different sites in CDF1 mice, while transplanted tumours were produced by implanting a C3H mouse mammary carcinoma on the backs of the same mouse strain. Measurements of pO2 were made in anaesthetised mice immediately before and 45 min after an intravenous injection of 5 mg kg-1 hydralazine. In the transplanted tumours hydralazine significantly decreased tumour oxygenation, such that the percentage of pO2 values < or = 5 mmHg increased from 45% to 87%, and median pO2 decreased from 5 to 3 mmHg. Similar significant changes were induced by hydralazine in the spontaneous tumours, the percentage of pO2 values < or = 5 mmHg increasing from 60% to 94% while the median pO2 values decreased from 8 to 2 mmHg. These results clearly show that there is no difference in the response of transplanted and spontaneous mouse tumours to hydralazine.

Tumour oxygenation and bioenergetic status were measured in the same tumour and these results related to radiobiological hypoxia. A C3H mouse mammary carcinoma grown in the feet of CDF1 mice was used. Bioenergetic status was assessed by 31P MRS using a SISCO 7 Tesla magnet, oxygen measurements were done by a polarographic electrode and the hypoxic fraction was determined from direct analysis of the radiation dose-response data. During all examinations restrained, non-anaesthetized mice were allowed to breathe either 100% oxygen, carbogen, normal air, carbon monoxide (CO) at 75, 220, or 660 ppm or had blood flow occluded by clamping. Results showed a significant correlation between the radiobiological hypoxic fraction and % pO2

The relationship between tumour tissue oxygenation and necrosis at different tumour sizes was investigated in a C3H mammary carcinoma implanted in the feet of female CDF1 mice. Experiments were performed using tumours that ranged in size from 80 to 800 mm3. Necrosis was estimated histologically. Tumour tissue oxygenation (pO2) was estimated with an Eppendorf electrode. Our results showed that as tumour volume increased there was a corresponding increase in necrotic fraction ranging from 1% in small tumours up to 51% in large tumours. The percentage of pO2 valuesor = 5 mmHg increased from 2% up to 79%, in small and large tumours respectively. After correcting for necrosis, the apparent, significant increase in the % of pO2 valuesor = 5 mmHg was lost. We conclude that correcting for necrotic fraction in this tumour model is necessary when attempting to measure tumour oxygenation using electrodes.

Tumours that are low in oxygen (hypoxic) tend to be more aggressive and respond less well to treatment. Knowing the spatial distribution of oxygen within a tumour could therefore play an important role in treatment planning, enabling treatment to be targeted in such a way that higher doses of radiation are given to the more radioresistant tissue. Mapping the spatial distribution of oxygen in vivo is difficult. Radioactive tracers that are sensitive to different levels of oxygen are under development and in the early stages of clinical use. The concentration of these tracer chemicals can be detected via positron emission tomography resulting in a time dependent concentration profile known as a tissue activity curve (TAC). Pharmaco-kinetic models have then been used to deduce oxygen concentration from TACs. Some such models have included the fact that the spatial distribution of oxygen is often highly inhomogeneous and some have not. We show that the oxygen distribution has little impact on the form of a TAC; it is only the mean oxygen concentration that matters. This has significant consequences both in terms of the computational power needed, and in the amount of information that can be deduced from TACs. ISSN:1932-6203

Hypoxia is one of the key features conferring resistance to oncologic treatment. Modern radiotherapy aims at overcoming hypoxia-induced resistance by escalating or redistributing dose or by modulating the sensitivity of poorly oxygenated but viable tumour cells. To accomplish this, it is necessary to detect hypoxia both spatially and temporally and to recognise limitations in sensitivity to differentiate oxic and hypoxic tumour subvolumes. Currently, PET/CT-based imaging using radiolabelled nitroimidazole or thiosemicarbazone compounds is the preferred technique for biological dose planning targeting hypoxia. Hypoxic tumour cells identified on PET/CT may be treated by giving a graded higher dose in a limited number of hypoxic compartments or by individually prescribing a dose to each volume unit based on 3D mapping of tumour oxygenation status. The latter technique is commonly called dose painting by numbers (DPN) to illustrate the heterogeneous dose received by the hypoxic target. Dose planning requires sophisticated computer algorithms where intensity-modulated radiotherapy (IMRT) is used to deliver irradiation. It is not yet known which strategy for planning and delivering hypoxia-targeted radiotherapy is the most appropriate in the clinical setting and what role chemical and biological modifiers of oxygenation will play given the lack of outcome data. Furthermore, adaptive strategies accounting for the effect of reoxygenation and cyclic hypoxia should be studied as well. This chapter outlines biological, methodological and technical issues associated with hypoxia-directed radiation therapy planning with emphasis on their potential application in clinical practice.

The ability of hyperbaric oxygen (HBO) treatment to modulate the response of a rodent fibrosarcoma to interstitial photodynamic therapy (PDT) using 630 nm light and intravenous polyhaematoporphyrin (PHP) was examined. Application of HBO for 30 min immediately prior to PDT resulted in a light dose-dependent increase in tumour growth retardation, maximum effect (+227%) being observed with 100J light, and no enhancement being seen at or above 400J. Application of HBO during or immediately after PDT had no effect on response. Reducing the interval between giving PHP and administering light from the usual 48 h to just 1 h increased the efficacy of PDT even in the absence of HBO pre-treatment. Under these circumstances, application of HBO did not realise any further increase in tumour response at any light dose. Our findings suggest that manipulation of tumour oxygenation may be able to improve the outcome of PDT under some circumstances, but that careful consideration of other treatment variables, such as light dose and drug-to-light interval, may provide similar, more easily achievable, improvements in efficacy.

There is much interest in positron emission tomography (PET) for measurements of regional tracer concentration in hypoxic tumour-bearing tissue, focusing on the need for accurate radiotherapy treatment planning. Generally, relevant data are taken over multiple time frames in the form of tissue activity curves (TACs), thus providing an indication of vasculature structure and geometry. This is a potential key in providing information on cellular perfusion and limited diffusion. A number of theoretical studies have attempted to describe tracer uptake in tissue cells in an effort to understand such complicated behaviour of cellular uptake and the mechanism of washout. More recently, a novel computerized reaction diffusion equation method was developed by Kelly and Brady (2006 A model to simulate tumour oxygenation and dynamic [18F]-FMISO PET data Phys. Med. Biol. 51 5859-73), where they managed to simulate the realistic dynamic TACs of (18)F-FMISO. The model was developed over a multi-step process. Here we present a refinement to the work of Kelly and Brady, such that the model allows simulation of a realistic tissue activity curve (TAC) of any hypoxia selective PET tracer, in a single step process. In this work we show particular interest in simulating the TAC of perhaps the most promising hypoxia selective tracer, (64)Cu-ATSM. In addition, we demonstrate its potential role in tumour sub-volume delineation for radiotherapy treatment planning. Simulation results have demonstrated the significant high contrast of imaging using ATSM, with a tumour to blood ratio ranging from 2.24 to 4.1.

Hypoxic tissues lack adequate oxygenation and it has been long established that tumours commonly exhibit hypoxia and that hypoxia is a factor contributing towards resistance to radiotherapy. To develop computer models and make predictions about the affects of tumour hypoxia on treatment outcome, quantitative tumour oxygenation and reoxygenation data from in vivo systems is required. The aim of this study was to investigate the timing and degree of reoxygenation during radiotherapy in a human head and neck squamous cell carcinoma xenograft mouse model (FaDu). Mice were immobilised using a novel restraining system and exposed unanaesthetised in 3 or 5 Gy fractions, up to a maximum of 40 Gy. Partial pressures of oxygen (pO2) measurements were recorded at six time points throughout the 2 week course of radiotherapy, using a fibre optic system. Tumours receiving 0–30 Gy did not exhibit an increase in pO2. However, the mean pO2 after 2 weeks of accelerated fractionated radiotherapy (40 Gy) was significantly increased (P < 0.01) compared to the mean pO2 of tumours not receiving the full schedule (0–30 Gy). These results lead to the conclusion of an average reoxygenation onset time of 2 weeks in this group of xenografts. A relatively large range of pO2 values measured at each dose point in the study indicate a large inter-tumour variation in oxygenation among the tumours. Data from this experimental work will be used to define the range of reoxygenation onset times implemented in a Monte Carlo computer model, simulating hypoxic head and neck cancer growth and radiotherapy. Refereed/Peer-reviewed

Tumour oxygenation could be investigated through several methods that use various measuring principles and can therefore highlight its different aspects. The results have to be subsequently correlated, but this might not be straightforward due to intrinsic limitations of the measurement methods. This study describes an analysis of the relationship between vascular and tissue oxygenations that may help the interpretation of results. Simulations have been performed with a mathematical model that calculates the tissue oxygenation for complex vascular arrangements by taking into consideration the oxygen diffusion into the tissue and its consumption at the cells. The results showed that while vascular and tissue oxygenations are deterministically related, the relationship between them is not unequivocal and this could lead to uncertainties when attempting to correlate them. However, theoretical simulation could bridge the gap between the results obtained with various methods.

Information on tumour oxygenation could be obtained from various imaging methods, but the success of incorporating it into treatment planning depends on the accuracy of quantifying it. This study presents a theoretical analysis of the efficiency of measuring tumour hypoxia by PET imaging. Tissue oxygenations were calculated for ranges of biologically relevant physiological parameters and were then used to simulate PET images for markers with different uptake characteristics. The resulting images were used to calculate dose distributions that could lead to predefined tumour control levels. The results have shown that quantification of tumour hypoxia with PET may lead to different values according to the tracer used and the tumour site investigated. This would in turn be reflected into the dose distributions recommended by the optimisation algorithms. However, irrespective of marker-specific differences, focusing the radiation dose to the hypoxic areas appears to reduce the average tumour dose needed to achieve a certain control level.

The expression of the hypoxia-inducible protein erythropoietin in tumour cells correlates with levels of tumour hypoxia. Our aim was to look for an interrelation of directly measured oxygenation levels, the presence of tissue erythropoietin and its receptor. Data of tumour oxygenation status, plasma and tissue erythropoietin and its receptor in a group of spontaneously occurring tumours in 15 dogs were collected. Polarographic tumour oxygen partial pressure measurements were obtained and data were correlated. Significant positive correlations were found between tissue erythropoietin and the percentages of pO2 values < or = 10 mmHg. Multivariate analysis revealed no parameters influencing plasma erythropoietin levels. Our results show that a co-expression of erythropoietin receptor and its ligand in spontaneous canine tumours exists, that the level of hypoxia in tumour cells correlates with the level of tissue erythropoietin and suggest the need to be quantitatively and functionally tested as novel prognostic biological parameters in neoplastic tissues.

Theoretical simulation of tissue oxygenation is a robust method that can be used to quantify the tissue oxygenation for a variety of applications. However, it is necessary that the relevant input parameters are used for the model describing the tumour microenvironment. The results of the simulations presented in this article suggest that the accuracy of the simulations depends very much on the method of calculation of the effects of the temporal change of the hypoxic pattern due to the opening and the closure of blood vessels. Thus, the use of average oxygenations might lead to dangerous overestimations of the treatment response. This indicates that care should be taken when incorporating hypoxia information into the biological modelling of tumour response.

A high level of hypoxia in solid tumours is an adverse prognostic factor for the poor outcome of cancer patients following treatment. This review describes the status of research into finding a practical method for measuring hypoxia and treating hypoxic tumours. The application of such methodology would enable the selection of head and neck cancer treatment based on an individual's tumour oxygenation status. This individualization would include the selection not only of surgery or radiotherapy, but also of novel hypoxia-modification strategies.