PET imaging of tumour hypoxia.

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]Fluoromisonidazole-3-fluoro-1-(2'-nitro-1'-imidazolyl)-2-propanol ([18F]MISO) and Cu-diacetyl-bis(N4-methylthiosemicarbazone (Cu-ATSM)-positron emission tomography (PET), and blood oxygen level-dependent (BOLD)-magnetic resonance imaging (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; PET techniques are the primary focus of this review. (c) International Cancer Imaging Society.

Introduction

The fact that hypoxia increases tumour resistance to the effects of radiotherapy has been known for over 70 years. Forty-five years ago, Tomlinson and Gray showed that hypoxia exists in human tumours and that necrosis occurs about 100 μm from the nearest blood vessel which is also the diffusion distance of soluble oxygen [1]. Decades of research in radiation therapy have focused on attempts to circumvent hypoxia mediated radio-resistance with moderate success. Over the last decade, it has become known that hypoxia changes the pattern of gene expression that alters the malignant potential of tumours leading to more aggressive survival traits, as a result of which cancer cells become difficult to treat by radiation and chemotherapy [2].

Tumour hypoxia overview

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. Tumour growth beyond 2 mm requires tumour neovascularisation to both supply oxygen and nutrients. The major factors in the development of tumour cell hypoxia are the known abnormalities in structure and functioning of tumour microvessels [3], the increased diffusion distances between blood vessels (many of which may not even carry oxygenated red blood cells), the expanding tumour cell mass with increased metabolic needs and the reduced oxygen carrying capacity of blood due to disease- or treatment-related anaemia. Thus, there are three distinct types of tumour hypoxia [4]:

(1) Perfusion related (acute) hypoxia which results from inadequate blood flow in tumours; it 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. Such hypoxia affects tumour 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 tumour 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 capillaries.

(3)Anaemic hypoxia, which is related to the reduced O 2-carrying capacity of the blood.

As noted above, the presence of tumour hypoxia appears to impair the effectiveness of radiotherapy and radiosensitivity is progressively limited as tumour pO 2 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 and 3.5 [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 pO 2 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 G 1 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 (HSPs) (HSPs are induced in response to environmental stresses like heat, cold and oxygen deprivation [6]) 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).

The presence of hypoxia within human tumours before starting treatment has been observed in squamous cell carcinomas, gliomas, adenocarcinomas (breast and pancreas) and in sarcomas. Oxygen probes, that is, electrodes implanted directly into tissues have shown that the normal cervix pO 2 is a median of 42 mmHg compared to a median of 10 mmHg in squamous carcinomas [7-9]. Furthermore, in cervix cancer, for example, the oxygenation status is independent of size, stage, histopathological type, grade of malignancy, and heterogeneity within and between the same tumour types. Hypoxia contributes to poor prognosis; pO 2<10 mmHg results in poor local tumour control, disease-free survival and overall survival in squamous carcinomas of the head and neck and of cervix cancers [10, 11].

There is debate about whether there is a critical intratumoural pO 2 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 pO 2 tensions below which cellular functions progressively cease or anticancer treatments are impaired are approximately as follows [12]: 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 and 15 mmHg is a good number to remember because of its impact on therapy.

Clinical imaging of hypoxia

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 [13]. From an imaging perspective, an ideal test would: (1) distinguish normoxia/hypoxia/anoxia/necrosis; (2) distinguish between perfusion-related (acute) and diffusion-related (chronic) hypoxia if possible; (3) reflect cellular in preference to vascular pO 2; (4) be applicable to any tumour site with complete loco-regional evaluation; (5) be simple to perform, non-toxic and allow repeated measurements, and (6) be sensitive at pO 2 levels relevant to tumour therapy. Therefore, the challenge for hypoxia imaging is to measure low levels of tissue pO 2 on a spatial scale similar to the O 2 diffusion distance (70–100 μm), a much smaller dimension than can be achieved with human imaging techniques. [18F]Fluoromisonidazole-3-fluoro-1-(2 ′-nitro-1 ′-imidazolyl)-2-propanol ([18F]MISO) and [60/64Cu]diacetyl-bis(N4-methylthiosemicarbazone (Cu-ATSM)-positron emission tomography (PET), the leading imaging contenders, are compared with other techniques in Table 1 and discussed in greater detail below.

Table 1

Comparison of techniques for evaluating human tumour hypoxia

Technique and	Invasive	Requires	Measures	Monitors changes	General availability
key references	investigation	injection		in pO 2	(1–5; poor-wide)
Polorographic electrode [7–9]	Yes	No	pO 2	Yes	2
[64Cu]ATSM [21, 22, 24, 30, 31]	No	Yes	Hypoxia	No	3
[18F]MISO PET [14, 20, 32–34]	No	Yes	Hypoxia	No	3
[18F]AZA PET [35]	No	Yes	Hypoxia	No	2
[18F]EF5 PET [36]	No	Yes	Hypoxia	No	2
BOLD-MRI [37]	No	No	[dHb] in RBCs a	Yes	4

a[dHb] in RBCs, deoxyhaemoglobin concentration in red blood cells.

[18F]MISO

[18F]MISO is the prototype hypoxia imaging agent. Uptake is homogeneous in most normal tissues, reflecting its high partition coefficient (near unity), and delivery to tumours is not limited by perfusion [14]. The initial distribution of [18F]MISO is flow dependent, as with any freely diffusible tracer, but local oxygen tension is the major determinant of its retention above normal background in tissues after 2 h. [18F]MISO accumulates in tissues by binding to intracellular macromolecules when pO 2<10 mmHg. Retention within tissues is dependent on nitroreductase activity (i.e. on reduction status of a NO 2 group on the imidazole ring) and accumulation in hypoxic tissues over a range of blood flows has been noted, including within the intestinal lumen where it is retained in anaerobes.

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 fluorodeoxyglucose ([18F]FDG). In the United States, F-MISO has Investigational New Drug (IND) authorization from the Food and Drug Administration (FDA) as an investigational product for use in humans. Unlike Eppendorf pO 2 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. 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 [15] 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 [16]. Studies in sarcoma [17] and head and neck cancer [18-20] have demonstrated a correlation of [18F]FMISO uptake with poor outcome to radiation and chemotherapy.

Cu-ATSM

Cu-ATSM holds exceptional promise as an agent for delineating the extent of hypoxia within tumours with PET. Numerous pre-clinical studies have evaluated and validated its use for imaging of hypoxia in tumours and other tissues [21-28]. The mechanism of retention of the reagent in hypoxic tissues is largely attributed to the low oxygen tensions and the subsequent altered redox environment of hypoxic tumours (increased NADH levels). Clinical studies, well tolerated by patients, involved [60Cu]ATSM imaging sessions of about 60 min with analysis of 30–60 min summed images. This time frame yields excellent data with good image quality in a very short time frame which opens up the opportunity with the shorter-lived 60Cu to perform multiple imaging sessions. A number of radioactive copper isotopes with longer half lives are available, e.g. 64Cu (t1/2=12.74 h) [29], enabling wide geographic distribution.

In human studies of lung [30] and cervical cancers [31], encouraging evidence has emerged that [60Cu]ATSM can act as a prognostic indicator for response to therapy. In a 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 [30]. A similar study in 14 women with cervical cancer demonstrated a similar predictive value in the tumour response to therapy [31].

Conclusions

To summarize, 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 imaging finding is sparse at best. Anti-hypoxia therapies exist in the clinic (but do not work very well or we do not know how to use them optimally) and more are on their way. Hypoxia imaging may allow better definition of a population that would benefit from novel anti-hypoxia directed therapies.