Imaging oxygenation of human tumours
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- Padhani, A.R., Krohn, K.A., Lewis, J.S. et al. Eur Radiol (2007) 17: 861. doi:10.1007/s00330-006-0431-y
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Tumour hypoxia represents a significant challenge to the curability of human tumours leading to treatment resistance and enhanced tumour progression. Tumour hypoxia can be detected by non-invasive and invasive techniques but the inter-relationships between these remains largely undefined. 18F-MISO and Cu-ATSM-PET, and BOLD-MRI are the lead contenders for human application based on their non-invasive nature, ease of use and robustness, measurement of hypoxia status, validity, ability to demonstrate heterogeneity and general availability, these techniques are the primary focus of this review. We discuss where developments are required for hypoxia imaging to become clinically useful and explore potential new uses for hypoxia imaging techniques including biological conformal radiotherapy.
The suspicion that tumour hypoxia increased resistance to radiotherapy was first considered in the 1930’s but it was not until 1955 that Tomlinson and Gray showed that chronic hypoxia occurred in human bronchial carcinomas with necrosis occurring approximately 150 μm from blood vessels , which is a little larger than the currently known diffusion distance of soluble oxygen in tissues (approximately 70 μm). Decades of research in radiation therapy then followed, much of which focused on attempts to circumvent hypoxia-mediated radio-resistance but these efforts were only moderately successful. Over the last decade, it has become evident that hypoxia changes the patterns of gene expression in several ways that alters the malignant potential of tumours, leading to more aggressive survival traits. As a result, hypoxic cancers are difficult to treat, particularly by radiation and photodynamic therapy , but also by cytotoxic chemotherapy. Attempts at circumventing the cure-limiting impact of hypoxia have included the use of hyperbaric oxygen and radiation sensitizer drugs but these have, in general, not proved widely advantageous. However, attempts to take advantage of the presence of tumour hypoxia, such as hypoxia-specific cytotoxins, are more promising. As hypoxia-directed therapies enter into clinical trials, it has become important to non-invasively assess for the presence of hypoxia and to be able to follow how it is modulated by new therapies. Hypoxia imaging may help select the most appropriate population that would benefit from novel hypoxia-directed therapies. In this review we describe the causes for and the effects of tumour hypoxia, as well as summarise the lead contenders for human tumour imaging. We also assess where developments are required for them to become clinically useful imaging tests and explore potential new uses for hypoxia imaging techniques including biologically-directed conformal radiotherapy.
Overview of tumour hypoxia & its importance
For the majority of solid tumours hypoxia develops because of the inability of the vascular system to supply the growing tumour mass with adequate amounts of oxygen. Consequently, both low oxygen tensions and nutrient deprivation contribute to impaired tumour growth such that growth beyond 2 mm requires tumour neovascularisation. The major factors that play roles in the development of tumour cell hypoxia are the known abnormalities in structure and functioning of tumour microvessels , the increased diffusion distances between blood vessels (many of which may not even carry oxygenated red blood cells), the expanding tumour cell mass competing for oxygen and the reduced oxygen carrying capacity of blood due to disease- or treatment related anaemia. Thus, there are three distinct types of tumour hypoxia : (1) Perfusion related (acute) hypoxia that results from inadequate blood flow in tumours that is generally the consequence of recognised structural and functional abnormalities of the tumour neovasculature. Such acute hypoxia is often transient, caused by temporary occlusions and temporary rises in interstitial pressure and can affect all cells right up to the vessel wall; (2) Diffusion related (chronic) hypoxia is caused by increased oxygen diffusion distances due to tumour expansion and affects cells greater than 70–100 μm from the nearest capillary, depending on where tumour cells lie in relation to the arterial or venous end of a capillary; (3) Anaemic hypoxia, which relates to reduced O2-carrying capacity of the blood and may be tumour associated or treatment related.
The presence of hypoxia within human tumours before starting treatment has been observed in a variety of tumour types including squamous cell carcinomas, gliomas, adenocarcinomas (breast & pancreas) and in sarcomas. For example, in the normal cervix the pO2 is a median of 42 mmHg compared to a median of 10 mmHg in squamous carcinomas, and for cervix cancer the oxygenation status is independent of size, stage, histopathological type, and grade of malignancy . Oxygen probes, that is, electrodes implanted directly into tumours to measure oxygen concentration by a polarographic technique [6–8] have shown (1) heterogeneity within and between the same tumour types of oxygen concentration and, (2) that hypoxia contributes to poor prognosis; pO2 < 10 mmHg results in poor local tumour control, disease-free survival and overall survival in squamous carcinomas of the head and neck and of the cervix [9, 10].
There is debate about whether there is a critical intratumoural pO2 below which detrimental changes begin to occur that is common across cell types. This occurs because experiments performed in cell cultures may not be applicable to in-vivo environments and some of the literature variation can be attributed to the tumour cell type chosen for experiments and the demands of host tissues. With these caveats in mind, the critical pO2 tensions below which cellular functions progressively cease or anticancer treatments are impaired are approximately as follows : Effectiveness of immunotherapy becomes impaired (30–35 mmHg); Photodynamic therapy (15–35 mmHg); Cell death on exposure to radiation (25–30 mmHg); Binding of hypoxia immunohistochemical markers (10–20 mmHg); Proteome changes (1–15 mmHg) and Genome changes (0.2–1 mmHg). The differences in these numbers are smaller than the similarities so that, from a practical perspective, for solid tissue tumors in vivo, a value of between 5–15 mmHg is a good number to remember because of its impact on therapy. This number is in contrast to ischaemic hypoxia in the myocardium or stroke where detrimental effects are experienced at higher O2 . In all these instances the critical oxygen level in tissues reflects the drive to match delivery with metabolic demand.
As noted above, the presence of tumour hypoxia appears to impair the effectiveness of radiotherapy and radiosensitivity is progressively limited as tumour pO2 levels fall. Hypoxia-induced radioresistance is multifactorial with the presence of oxygen mediating DNA damage through the formation of oxygen free radicals which occurs after the interaction of radiation with intracellular water. The ratio of doses administered under well-oxygenated to hypoxic conditions needed to achieve the same biological effect (i.e., cell kill) is called the oxygen enhancement ratio (OER). For sparsely ionising radiations such as x- and gamma rays, the OER at therapeutic doses is between 2.5–3.5 . That is, well oxygenated cells are about three times more sensitive to x- and gamma radiation than the same cells when they are hypoxic. Half maximal sensitivity to x- and gamma rays occurs at oxygen tensions of approximately 2–5 mmHg; above pO2 values of approximately 10–15 mmHg near maximal oxygen effects are seen. However, it should be recognised that sensitivity of cells to radiation is dependent on the phase of the cell cycle, with cells in the G1 phase having a lower OER (i.e., more radiosensitive) than cells in S-phase. As noted above, the oxygen effect is not the only mechanism for radioresistance in hypoxic tumour cells. Evidence is accumulating that the hypoxia-mediated proteomic and genomic changes may also contribute to radioresistance by increasing the levels of heat shock proteins (heat shock proteins (HSPs), are induced in response to environmental stresses like heat, cold and oxygen deprivation ) or by increasing the number of tumour cells that can resist apoptosis by mutating p53 (the slowing of cell division is dependent on a protein brake known as p53; the disruption of the functioning of this protein is associated with approximately 50–55% of human cancers).
Clinical imaging of hypoxia
As tumour hypoxia is an important biological characteristic and there is no good or easy clinical way to predict its presence, it has been suggested that imaging may be a good way of non-invasively selecting cancer patients who would benefit from treatments that overcome, circumvent or take advantage of the presence of hypoxia. Since tumour hypoxia is a key mechanism that leads to radioresistance, it has been repeatedly suggested that a hypoxia mapping technique could be integrated with conformal radiotherapy techniques to improve target delineation and dose delivery; this is discussed in more detail below. Imaging could also be used to document whether or not and the extent to which reoxygenation of tumours occurs during radiotherapy. Key requirements of any method that evaluates tumour hypoxia include non-invasive assessments that allow serial changes during treatment to be monitored and evaluation of heterogeneity between and within tumours.
There are a number of ways in which tissue oxygenation status can be assessed in vivo (both invasive and non-invasive) or in vitro using material from biopsy. Non-imaging methods of assessing for the presence of hypoxia in tissues include histological appearance, immunohistochemical staining for intrinsic markers of hypoxia (e.g., carbonic anhydrase IX (CA-IX) and hypoxia inducible factor-1 (HIF-1)) and for the binding of externally administered nitroimidazoles [17, 18].
Comparison of techniques for evaluating human tumour hypoxia
Technique and key references
Clinically Validated in RT
General availability (1–5; poor-wide)
Monitors changes in pO2
18F-AZA PET 
18F-EF5 PET 
[dHb] in RBCs
Hypoxia can be imaged with 18F-MISO PET in a procedure that is well-tolerated by the patients. Imaging requires 20–30 min and starts anywhere from 75 to 150 min after injection, making it similar to the bone scan with which most cancer patients are familiar. Useful and well-validated images can be achieved with a modest dose of radiation, typically 250 MBq. No arterial sampling or metabolite analysis is required and synthesis is achieved through relatively simple modifications of nucleophilic displacement / deprotection synthesis boxes such as are used for fluoro-deoxyglucose (18F-FDG). In the USA, F-MISO has investigational new drug (IND) authorisation from the Food and Drug Administration (FDA) as an investigational product for use in humans. Unlike Eppendorf pO2 histography, 18F-MISO is only sensitive to the presence of hypoxia in viable cells; 18F-MISO is not retained in necrosis because the electron transport chain that reduces the nitroimidazole to a bioreductive alkylating agent is no longer active (Fig. 3). Limitations of 18F-MISO PET include the modest signal-to-noise ratio of raw 18F-MISO PET images but if a venous blood sample is acquired during the mid-course of the imaging procedure and used to calculate a Tumour:Blood (T/B) ratio image, then normoxic uptake (T/B < 1) can be electronically subtracted to increase image contrast. Several studies in a range of hypoxic tumours, stroke and hypoxic myocardium  have shown that a T/B of >1.2 reliably identifies the presence of hypoxia. The presence of high normal liver uptake impairs complete assessment of liver lesions and urinary excretion interferes with imaging near the bladder.
18F-MISO PET is able to monitor the changing hypoxia status of lung tumours during radiotherapy . Studies in sarcoma  and head and neck cancer [22–24] have demonstrated a correlation of 18F-FMISO uptake with poor outcome to radiation and chemotherapy.
In human studies of lung  and cervix cervical cancers , encouraging evidence has emerged that 60Cu-ATSM can act as a prognostic indicator for response to therapy. In the prospective study of 14 humans with non-small cell lung cancer, a semi-quantitative analysis of the 60Cu-ATSM muscle-to-tumour ratio was able to discriminate those likely to respond to therapy from non-responders . A similar study in 14 women with cervical cancer demonstrated a similar predictive value in the tumour response to therapy .
The observations made in the previous paragraph imply two intuitive inferences. (1) BOLD-MRI images are more likely to reflect on acute (perfusion-related) tissue hypoxia which, as stated above, occurs because of transient occlusions of vessels, simply because hypoxic areas extend to the level of the blood vessels. In contradistinction, chronic hypoxia is less likely to be reflected by BOLD-MRI because the red blood cells in vessels are too distant from the area of hypoxia. (2) For BOLD-MRI to be able to inform on tissue oxygenation status, it is important for red blood cells to be delivered to the tissue in question. Human and xenograft studies have shown that tumour perfusion varies widely and that red blood cell perfusion is not simply related to the absence/presence of vessels; plenty of tumour vessels maybe present but perfusion by red blood cells may not occur . This observation probably explains in part why no direct correlations between baseline R2* and tissue pO2 have been observed (that is, R2* does NOT measure tissue pO2). So it is necessary to know or to determine the distribution of blood volume in tissue in order to be able to correctly interpret R2* images in order to infer oxygenation status. Thus, if a tissue is perfused but has a high baseline R2* in one area/region compared to another area/region in the same tissue (i.e. the statistic components are the same), then one can infer that the high R2* region is relatively more hypoxic; this hypothesis is supported by recent preclinical and clinical data [38, 39].
As stated above, the use of BOLD-MRI for assessment of tissue hypoxia is predicated on the assumption that the oxygenation of haemoglobin is proportional to blood arterial pO2 which is in equilibrium with oxygenation of surrounding tissues. Many studies have shown that changes in R2* in response to vasomodulation with Carbogen (95% CO2:5% O2) inhalation, for example, are temporally correlated with changes in tissue pO2. Tumours differ in their responses to carbogen inhalation with only 50–60% of human tumours showing changes in R2* [40, 41]. The reasons for these limited and heterogeneous responses are complex but undoubtedly include the fact that tumours have adapted to widely different perfusion and that, even when vessels are present, red blood cell transport along these vessels may not be effective as demonstrated by Robinson et al. . Thus, hypoxic tumours with high blood volume (due to high microvessel density coupled with large vessels) will not only have raised baseline R2* values but are more likely to respond to Carbogen. This will be reflected by large changes in R2*; and it is these hypoxic tumours that show positive radiosensitisation with Carbogen. On the other hand, hypoxic tumours with low blood volume (due to lower microvessel density, or due to small vessels) will have lower baseline R2* values and are thus less likely to respond to Carbogen. In this situation, there will be negligible changes in R2* and such hypoxic tumours do not show radiosensitisation with Carbogen . Readers should also note that the BOLD response to Carbogen is also dependent on the ability of the underlying maturity of the vasculature with mature vessels able to respond actively to vasoconstrictory and vasodilatory stimuli .
The primary advantages of BOLD-MRI are that there is no need to administer exogenous radioactive contrast material and images at high temporal and with high spatial resolution can be obtained and repeated as needed. It is possible to decouple the effects of flow and deoxyhaemoglobin which are seen in native BOLD images and so to demonstrate changes in oxygenation independent of changes in blood flow. Major limitations of BOLD-MRI include the fact that they do not measure tissue pO2 directly (either in blood or tissues because of a non-linear relationship of R2* and tissue pO2), the images obtained have low signal to noise ratio and clinical studies with Carbogen vasomodulation are technically challenging (approximately 25–35% of patient examinations fail due to respiratory distress caused by an increased respiratory drive induced by Carbogen [40, 41]. BOLD-MRI appears most sensitive to oxygen levels adjacent to perfused vessels (that is, perfusion related or acute hypoxia) and BOLD-MRI sensitivity to more distant diffusion related or chronic hypoxia is an unknown.
Hypoxia guided radiotherapy
Recently introduced, technological improvements in radiotherapy delivery systems, including intensity-modulated radiotherapy (IMRT), have provided a means for shaping the dose distribution not only to the geometry of target volumes, and also to the differences in radiobiology across tumours . Thus, it is now possible to define an additional “target within the target” as 3D pixel maps of the prescribed dose incorporating biological information derived from functional images; sometimes called dose painting by numbers [45, 46]. This allows treatment to the desired dose with escalation based on biologically relevant data, such as hypoxia which was discussed above, that is mechanistically related to therapeutic outcome. As an example of this, the spatial distribution of 60/64Cu-ATSM uptake on PET was successfully fused with CT radiotherapy planning images to show a proof of concept. This theoretical treatment planning would deliver higher doses of radiation via intensity modulated radiotherapy techniques (IMRT) to the most hypoxic regions of head and neck tumours .
Challenges for hypoxia imaging techniques
As hypoxia imaging techniques move from academic research environments to routine clinical usage, it becomes important to recognise the unique challenges of clinical translation. For example, it is important that patient examination times are short to improve patient compliance particularly for repeated examinations. Thus, the need for doing dynamic scanning followed by several static scans over a prolonged period of time with PET techniques could prove a disincentive for patients. However, as noted above, not all patients undergoing hypoxia studies by PET need dynamic imaging although it may be useful in selected patients where the aim is to demonstrate both perfusion and hypoxia (for example to demonstrate reperfusion-reoxygenation). In-contradistinction, the interpretation of BOLD-MRI does require that the distribution of blood flow/volume is known and this can be done in clinical studies using dynamic contrast enhanced MRI (DCE-MRI) . Whatever the chosen technique for clinical translation, there needs to be standardisation of imaging procedures and analysis methods in order to allow techniques to become more completely validated, for use in clinical trials. Amongst other issues that require addressing when clinical trials are being designed include the need for quantification, test-retest variability and data collection in body parts where there is a large degree of physiological movement such as the lungs and liver.
A practical question often asked is whether it is necessary to quantify imaging data to answer important clinical questions. Subjective assessments work well enough in the clinic; however it is important to realise that subjective criteria cannot be applied simply from one centre to another particularly, when different equipment and imaging routines and human observers are used. Quantification techniques aim to minimise errors that can result from the use of different equipment and imaging protocols. Quantification techniques also enable the derivation of parameters that are based on some understanding of physiological processes and so can provide insights into tumour biology, for example the simple T/B and T/M ratios described for the two PET procedures. Quantification techniques are preferred when serial imaging studies are anticipated, for example when evaluating response to novel anticancer therapeutics.
The reproducibility of the imaging technique should also be known in order to estimate the sample size required to evaluate therapy efficacy. Variation between measurements of the same quantity on the same individual can be caused either by measurement error or by physiological changes between measurements. Whilst it is possible (in theory) to reduce measurement error, physiological variation is inherent, and can cause difficulty in attempts to characterise disease or to monitor the effects of therapy. An estimate of measurement error enables us to decide whether a change in observation represents a real change. Data addressing the precision and measurement variability of hypoxia imaging techniques are urgently needed and should be an integral part of any prospective study that evaluates functional response to therapy to allow assessments of individual patients and group changes.
It is intuitive that analysis and presentation of imaging data needs to take into account the heterogeneity of tumour hypoxia. The presence of motion can invalidate functional parameter estimates particularly for pixel-by-pixel analyses and this is especially true for high spatial resolution techniques such as BOLD-MRI. Motion is averaged in PET imaging because imaging times are long. In that case, pixel-analyses of the data and the issues of heterogeneity assessment can become less meaningful. The first step in heterogeneity analysis includes ROI definition which should be performed independent on the hypoxia imaging being assessed. For BOLD-MRI this could be done by anatomic MRI images and for PET studies could include the CT component of CT-PET studies although some groups have used ROIs defined in 18FDG-PET images. However, ROIs defined on 18FDG-PET images are know to be prone to error as far as tumour boundaries are concerned and are highly dependent on the level of threshold chosen. Whole tumour ROIs yield outputs with good signal-to-noise ratio, but lack spatial resolution and are prone to partial volume averaging errors and thus are unable to evaluate tumour heterogeneity. Pixel mapping has the advantages of an improved appreciation of heterogeneity of hypoxia and the risk of missing important diagnostic information and of creating ROIs that contain more than one tissue type is reduced. An important advantage of pixel mapping is being able to spatially map tumour characteristics such as hypoxia, glucose metabolism and blood flow and to be able to probe the spatial correlations between different kinetic parameters, providing unique insights into tumour structure, function and response to treatment.
To summarise, tumour hypoxia is common and its effects represents a significant challenge to the curability of human tumours, leading to treatment resistance and enhanced tumour progression. Tumour hypoxia can be detected by non-invasive and invasive techniques but the inter-relationship between these techniques needs to be better defined; human validation of the utility of hypoxia imaging is sparse at best. Anti-hypoxia therapies exist in the clinic and more are on their way. Either they don’t work very well or we don’t know how to use them optimally. Hypoxia imaging may allow better definition of a sub-population of cancer patients that would benefit for novel anti-hypoxia directed therapies.
The hypoxia imaging studies in Seattle were initiated in collaboration with Janet Rasey. The work described in this review was by Joseph Rajendran and Janet Eary, whom the authors gratefully acknowledge, along with funding from NIH, CA-42045. The BOLD-MRI work was done in collaboration with Jane Taylor & Peter Hoskin (Mount Vernon Hospital, London) and Simon Robinson (Institute of Cancer Research, London). The Cu-ATSM studies described in this review was undertaken with Farrokh Dehdashti and her collaborators, Perry W. Grigsby, Jeffrey Bradley, Ramaswamy Govindan, Michael J. Welch. Mark Mintun and Barry A. Siegel. The development of Cu-ATSM as a hypoxia imaging agent was supported by the US Department of Energy (DE-FG02-87ER60512). The Image guided radiotherapy work in Tuebingen is done in collaboration with Daniella Thorwarth and SM Eschmann.