173 Radiolabeled Amino Acids

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PET and SPECT in IMRT: Future Prospects

Christophe Van de Wiele

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Contents

2.1 Introduction . . . 171

2.2 Basic Technology: SPECT and PET Imaging . . . 171

2.3 PET and SPECT Tracers of Interest for 3D-image Characterization of Tumors for IMRT Planning . . . . 172

2.3.1 FDG . . . 172

2.3.2 Proliferation Markers . . . 173

Radiolabeled Thymidines and Derivatives . . . 173

Radiolabeled Amino Acids . . . 173

2.3.3 Hypoxia Markers . . . 174

2.3.4 Apoptosis Markers . . . 174

2.3.5 Others . . . 174

2.4 Discussion and Future Prospects . . . 175

References . . . 175

2.1 Introduction

Intensity modulated radiotherapy is characterized by the high-precision application of radiation to an ex- actly deﬁned target and by very rapid dose falloff to spare normal tissue. Improvement in the physical dose distribution obtained by IMRT has raised the issue of accuracy of target volume selection and delineation on a 3D basis. For both technical and logistic reasons, computed tomography (CT scan) has become the reference imaging modality for 3D tumor delineation in IMRT.

CT does not suffer from geometric distortion and in- herently contains information on the density mapping which is used by the algorithms for dose calculation.

As compared to CT, MRI allows better target volume definition compared with CT in some specific sites and provides multi-plane images, facilitating the assessment of tumor extension. On the other hand, MRI images may be degraded by geometric distortion at the edge of the field of view, do not allow precise delineation of the ex- ternal contour of the body and the bony structures and lack information on tissue density. Whilst CT-MRI image fusion is feasible and may overcome some of the

abovementioned limitations, both techniques have dif- ﬁculties in detecting lymph node metastases when they show a normal appearance. Additionally, primary tumor boundaries on CT and MRI images may be vague when there are inﬂammatory changes around the tumor or when metal artifacts hamper image interpretation.

PET (positron emission tomography) and SPECT (single photon emission tomography) may offer a solution to solve these problems for certain clinical situations.

As opposed to CT and MRI, SPECT and PET imaging provides biological information. Using this information, a more specific, biological target volume rather than morphological target may be delineated which may help to guide customized dose delivery. For instance, the use of specific markers to visualize biological path- ways known to influence response to ionizing radiation (e.g. tumor hypoxia and proliferation) could lead to delineation of sub-target volumes for delivering an extra boost dose.

2.2 Basic Technology: SPECT and PET Imaging

The gamma camera is an imaging device that is able to detect scintillations (ﬂashes of light) produced when gamma rays, resulting from radioactive decay, interact with a thallium doped sodium iodide crystal at the front of the camera [1]. The scintillations are detected by photomultiplier tubes (PMTs), and whilst the areas of crystal seen by tubes overlap, the location of each scintillation can be computed from the relative response in each tube. The energy of each scintillation is also meas- ured from the response of the tubes, and the electrical signal to the imaging computer consists of the location and photon energy. In front of the crystal resides a collimator, which is made of lead and usually man- ufactured with multiple elongated holes (parallel-hole collimator). The holes allow only gamma rays that are traveling perpendicularly to the crystal face to enter. In conventional planar gamma camera imaging the gamma photons absorbed by the crystal therefore form a pro- jection of the distribution of the radiopharmaceutical

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in front of the camera. In SPECT imaging the camera is rotated around the patient and several projections at different angles are acquired, tomographic images can be generated through the use of speciﬁc reconstruction algorithms [2].

As with SPECT, positron emission tomography (PET) relies on computerized reconstruction procedures to produce tomographic images, however, by means of indirectly detecting positron-emission [3]. Positrons when emitted by radioactive nuclei will combine with an electron from the surroundings and annihilate. Upon annihilation both the positron and the electron are then converted to electromagnetic radiation in the form of two high-energy photons which are emitted 180^◦away from each other. It is this annihilation radiation that can be detected externally and is used to measure both the quantity and the location of the positron emitter. Simul- taneous detection of two of these photons by detectors on opposite sides of an object places the site of the annihilation on or about a line connecting the centers of the two opposing detectors. At this point mapping the distribution of annihilations by computer is allowed.

If the annihilation originates outside the volume between the two detectors, only one of the photons can be detected, and since the detection of a single photon does not satisfy the coincidence condition, the event is rejected.

Since radioisotopes suitable for PET have a short half-life (e.g., 110 min for ¹⁸F, an on-site cyclotron is needed for production of such isotopes [4]. Also, special radiosynthesis facilities are required restricting the availability of non-commercially available PET- radiopharmaceuticals to specialized centers. Opposed to PET, the synthesis of SPECT radiopharmaceuticals is mostly less expensive. As the half-lives of the isotopes used in SPECT are longer than those of isotopes used in PET (hours vs minutes), longer acquisition times are also possible in SPECT. This may, for instance, allow receptor imaging at equilibrium, a prerequisite in order to obtain reliable information with respect to relative receptor density measurements. On the other hand, the resolution of a conventional PET camera is twice as good as that of a conventional gamma camera and PET allows for more accurate quantiﬁcation when compared to SPECT.

2.3 PET and SPECT Tracers of Interest for 3D-image Characterization of Tumors for IMRT Planning

2.3.1 FDG

The most widely used PET tracer in oncology imaging is 2-¹⁸ﬂuoro-2-deoxy-glucose (FDG) [5]. The rationale

behind its use is the ﬁnding of an increased rate of glucose consumption in malignant tissues, due to an increase of glycolytic enzymes and of the number of glucose transporters expressed on malignant cells [6–8].

After injection, FDG is transported by facilitated dif- fusion into neoplastic cells where it is phosphorylated by hexokinase and subsequently trapped as it is not a substrate for the subsequent enzymatic driven path- ways for glucose metabolism. As the neoplastic cells accrue larger amounts of FDG due to their increased metabolism, increased activity is detected that delin- eates the hypermetabolic tumor from the surrounding normal tissues. Since its ﬁrst application in the detection of primary brain tumors, FDG PET has been increasingly used for its ability to detect primary malignant tumors, but also for its ability to detect both regional and distant metastases, distinguish benign from malignant tissue or recurrent cancer from treatment- related scarring, and document response to therapy [9].

A major limitation of FDG PET is the limited spatial resolution, approximately 5–8 mm for ¹⁸F with cur- rent PET machines [10]. Below a threshold of twice this resolution, due to partial volume effect, tracer activity will be underestimated eventually leading to false-negative results. On the other hand, as leucocytes and macrophages also accumulate FDG, when select- ing for FDG PET, data obtained in patients presenting with inﬂammatory conditions should be closely corre- lated to conventional imaging as to avoid false-positive ﬁndings [11].

Several, mainly retrospective studies, have provided evidence that the detection of hypermetabolic tumor tissue by means of FDG PET may lead to a better definition of the clinical target volume, i.e. the local and regional extension of the neoplastic disease. Available data on FDG PET and IMRT are scarce and limited to patients suffering from cervical cancer, non- small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN).

Mutic et al. evaluated a treatment planning method for dose escalation to the para-aortic lymph nodes (PALNs) based on FDG PET with IMRT in four cervical cancer patients with PALN involvement [12]. The treatment plans for the four patients revealed that escalated prescription doses could be delivered to target volumes while maintaining acceptable doses to the surrounding critical structures. More specifically, radiation doses could be escalated from the conventional 45 Gy to 59.4 Gy for the gross target volume (positive PALNs defined on FDG PET) and 50.4 Gy for the clinical target volume (para-aortic bed). The data indicate that PET-guided IMRT could be used in a clinical trial in an attempt to escalate doses delivered to patients with cervical cancer who have positive PALNs. The guidelines regarding the selection of the appropriate treatment parameters (e.g., number of beams, beam geometry) and organ specific parameters (e.g., importance weighting

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and tolerance dose) for IMRT planning when aiming for a goal dose of 50.4 Gy to the clinical target volume and of 59.4 Gy to the gross volume where described in a separate paper.

Grills et al. evaluated four different techniques of radiation therapy, respectively IMRT, limited and opti- mized three-dimensional conformal radiotherapy (3D- CRT) and traditional radiotherapy, used to treat non- small cell lung cancer and also determined their efficacy in meeting multiple normal-tissue constraints while maximizing tumor coverage and achieving dose escalation [13]. In this series the target volume was delineated using information from both the treatment planning CT and the treatment planning PET scan to create a composite tumor/nodal volume. The primary tumor volume (GTVprimary) was defined on the PET scan using a previously defined formula, respectively [(0. 3069× mean standardized uptake value) + 0. 583]. GTVnodalin- cluded all lymph nodes larger than 1 cm on CT scan and all lesions smaller than 1 cm on CT scan that were positive on FDG PET. The clinical target volume (CTV) was defined as 0.5 cm 3D expansion of the GTVprimary+ the GTVnodal. The planning target volume was defined as the CTV with appropriate margin to com- pensate for variability in internal target motion due to respiration or other internal motion, as well as variability in patient set-up. Their data show that whereas IMRT is of limited additional value (compared to 3D-CRT) in node-negative cases, it is beneficial in node-positive patients and in patients with target volumes close to the esophagus. When meeting all normal- tissue constraints in node-positive patients, IMRT can deliver RT doses 25–30% greater than 3D-CRT and 130–140% greater than traditional radiotherapy.

Scarfone et al. defined conventional GTVs, FDG PET GTVs and final FDG PET|CT GTVs, based on co-registered images, in six patients suffering from SC- CHN [14]. The resulting PET|CT GTV was larger than the original CT GTV volume by an average of 15% with the CT GTV being modified in five out of six patients.

2.3.2 Proliferation Markers

A non-invasive, reliable and repeatable technique allowing assessment of tumor proliferation would constitute a useful tool to the radiotherapist to estimate the potential of repopulation of clonogens during radiotherapy.

Information obtained by such techniques would allow for a customized “dynamic” dose delivery to “clono- genic” subvolumes when performing IMRT.

Radiolabeled Thymidines and Derivatives

Tumor cell proliferation has been studied extensively using autoradiography to detect uptake of tritiated thymidine into cellular DNA. The proportion of labeled cells at a short interval after administration

of tritiated thymidine (the labeling index) is a measure of the proportion of cells that were in S-phase and thus actively dividing [15, 16]. Unfortunately, assessment of the labeling index requires invasive biopsy and is invariably subject to sampling er- rors. Accordingly, radiolabeled thymidines, respectively 2-[¹¹C]-thymidine, [methyl-¹¹C]thymidine and ¹⁸F- fluorothymine, as well as halogenated deoxyuridines, respectively ⁷⁶Br-deoxyuridine, 123,131,124I-deoxyuridine and¹⁸F-fluorodeoxyuridine, were developed for imaging tumor proliferation [17]. Out of these, both 2-[¹¹C]- thymidine and [methyl-¹¹C]thymidine as well as⁷⁶Br- deoxyuridine and 123,131,124I-deoxyuridine are rapidly metabolized, resulting in high background activity and low tumor uptake. In contrast,¹⁸F-fluorothymidine is much more resistant to in vivo degradation and is more avidly taken up by tumor tissues. As initial clinical results suggest¹⁸F-fluorothymidine tumor uptake values significantly correlate with tumor proliferative status, this tracer may prove of interest for IMRT planning.

Radiolabeled Amino Acids

Amino acids uptake by cells is largely mediated by carriers that are either sodium dependent, e.g. system A, ASC and Gly that transport amino acids with short, polar or linear side chains such as alanine, serine and glycine, or sodium independent, e.g. system L, Bô,+and system y⁺that are transporters of branched chain and aromatic amino acids, such as leucine, valine, tyrosine and phenylalanine [18–21]. Tumors generally show increased pooling of amino acids, amongst others by up- regulation of carriers, e.g. system A [22, 23]. Al- though part of the pooled amino acids in tumor tissue is shuttled into protein synthesis, a fraction will be used for other purposes, e.g. metabolic fuel. In general, the fraction of radiolabeled amino acids that is incorporated into proteins is small as compared to the total amount that is taken up by the cell [24, 25]. Neverthe- less, despite the fact that imaging shows the sum of both fractions, usually the total amino acid signal generated relates to tumor proliferation. Furthermore, as inflammatory cells have a low protein metabolism as compared to glucose metabolism, radiolabeled amino acid uptake in tumor tissue is less obscured by in- terfering uptake in concomitant inflammatory tissues than is the case for FDG [26–28]. As a result, radiolabeled amino acid imaging could allow for a better discrimination between tumor tissue and inflammatory tissue and thus also in a more appropriate tumor volume delineation when considering radiation treatment planning.

To date, virtually all amino acids have been radiolabeled for tumor imaging by means of PET. However, given their ease of synthesis and limited metabolite formation following intravenous injection in man,¹¹C- methyl-methionine (MET) and¹¹C-tyrosine (TYR) have

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been most extensively studied. Evidence that their uptake reﬂects tumor proliferation in vivo in humans has so far only been provided for¹¹C-methyl-methionine.

Changes in¹¹C-methyl-methionine uptake were shown to reﬂect response to radiotherapy treatment in patients suffering from a wide variety of tumors. Studies imple- menting¹¹C-methione for IMRT planning may prove worthwhile.

2.3.3 Hypoxia Markers

For a number of tumors, radiotherapeutic treatment may fail due to the presence of tumor hypoxia (pO2< 5 mm). For instance, approximately 80% of head and neck squamous cell carcinoma have signiﬁcant hypoxic fractions (pO2< 2.5 mm Hg) while the ﬁgure for carcinoma of the uterine cervix is around 50% [29–31].

One approach to overcome hypoxic tumor resistance is to escalate radiation dose. However, increasing radiation indiscriminately may increase normal tissue complica- tion rates in areas where critical structures are in close proximity of gross tumor or a high-risk surgical bed, e.g. head and neck cancer. PET or SPECT-guided imaging of hypoxia could provide a novel avenue to escalate radiation doses, without compromising normal tissue function.

Several investigators have focused on the develop- ment of radiolabeled compounds that are selectively retained in hypoxic areas and can be visualized non- invasively and repetitively by means of SPECT or PET. The chemical basis for most of these compounds has been to incorporate a 2-nitroimidazole moiety to act as a bioreductive molecule accepting a single electron and producing a free radical anion which, after further reduction, is then incorporated into cell constituents under hypoxic conditions. To date, four of these radiopharmaceuticals have been injected in oncological patients, respectively the N-1 substitute 2-nitroimidazole derivatives ¹8F-misonidazole (¹⁸F- MISO) and¹²³I-iodoazomycin arabinoside (123-IAZA) and the bioreductive non imidazole moiety con- taining^99mTc-2,2-(1,4-diaminobutane)bis(2-methyl-3- butanone)dioxime (^99mTc HL91) and ⁶⁰Cu-diacetyl- bis(N-4-methylthiosemicarbazone) (⁶⁰Cu-ATSM) [32–

40]. Although the mechanisms of tumor accumulation of both ^99mTc HL91 and ⁶⁰Cu ASTM have yet to be clariﬁed, it is believed that the^99mTc and⁶⁰Cu com- plexes are bio-reducible groups by themselves [40, 41].

Out of these four tracers, ⁶⁰Cu ASTM has the best tracer kinetics, allowing imaging as early as 10 min following tracer injection with high enough contrast to identify hypoxic tumor sub-volumes. Importantly,

60Cu-ASTM retention occurs only in cells with intact mitochondria, allowing straightforward discrimination of necrotic from hypoxic cells. The feasibility of

60Cu-ASTM guided IMRT was recently demonstrated

in a patient suffering from head and neck carcinoma [42].

2.3.4 Apoptosis Markers

Since its recognition as a major form of cell death after radiation, apoptosis is being increasingly studied as a marker of cellular radiosensitivity and progno- sis for radiotherapy treatment outcome. The positive correlation of tumor response to radiation and the background level of apoptotic cells seen in murine systems raises the possibility of developing the in-vivo visual- ization of apoptosis as an assay for deﬁning subvolumes for IMRT planning. If the spontaneous level of apoptosis plays a similar role in tumor responsiveness to radiation in humans, then patient tumor subvolumes whose pretreatment biopsy specimens exhibit low levels of apoptosis may beneﬁt from higher local treatment doses.

Annexin V binds to membrane-bound phosphatidyl serine (PS), a constitutive anionic membrane phospho- lipid that is normally restricted to the inner leaﬂet of the plasma membrane lipid bilayer but is selectively exposed on the surfaces of cells as they undergo apoptosis (programmed cell death) [43]. To date, Annexin V has been ﬂuorinated for PET and radioiodinated and coupled to a wide variety of linker molecules such as di- amide dimercaptide (N2S2) or hydrazino nicotinamide for complexation with ^99mTc for SPECT. In particular in vivo uptake of^99mTc-radiolabeled annexin V as assessed by means of SPECT imaging was shown to allow for non-invasive monitoring of cell death dynam- ics and effectiveness of therapies aimed at reducing cell death in patients suffering from myocardial infarction and reperfusion injury as well as in viral and auto-immune myocarditis and nonischemic cardiomy- opathies [44–47]. Studies assessing in vivo quantitative

99mTc-Annexin V uptake in human tumors and their relationship to radiotherapy outcome as well as its potential to modulate radiation treatment planning are underway.

2.3.5 Others

Advances in fundamental radiobiology suggest that im- provements in tumor control can be achieved through strategies that combine radiation and molecular targeting. One approach which is currently being clinically evaluated is to target speciﬁc molecules involved in tumor cell survival after irradiation, using inhibitors of EGFR or Ras [48]. Because of tumor heterogeneity and the existence of multiple tumor radio-resistance path- ways, an extension of this approach being investigated at the pre-clinical level is to use Hsp90 inhibitors as a means of reducing the levels of multiple radioresponse

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regulatory proteins. In addition, it may also be possible to target normal tissue processes, such as angiogenesis, to enhance the radioresponse of tumors. Finally, an alternative approach to combining radiation and molecular targeting is to exploit radiation-induced gene expression to induce targets for other modalities or to increase their effectiveness. Several studies have demonstrated that radiosensitivity of cells may be inﬂuenced by the addition of a wide variety of exogenous growth factors or hormones in receptor positive cells before or after irradiation. The tissue radiation interactions resulting in the increase of radiosensitivity are complex and still poorly understood. PET and SPECT tracers allowing in vivo assessment of the local tumor distribution of these molecular targets may help to delineate subvolumes of interest for dose-increase. For instance, when administering EGFR-blocking antibodies as radiotherapeutic adjuvant, areas of low or absent EGFR expression may beneﬁt from higher dose-delivery in IMRT planning.

2.4 Discussion and Future Prospects

Till recently, traditional radiation treatment planning relied solely on density imaging such as chest radiogra- phies and CT in order to obtain anatomic information of structures of interest for treatment, including target and normal tissue. The advent of FDG PET has now made it possible to exclude or include particular areas based on their level of glucose metabolism. A limited number of studies, respectively in NSCLC-, cervix- and head and neck carcinoma suggest that integration of information obtained by means of FDG PET in intensity modulated radiotherapy is feasible. In these patient populations, re- currences following radiotherapy are mainly located in the high- dose-prescription regions, suggesting the need for even higher doses in these areas. Inclusion of areas of increased FDG tumor uptake in the target definition process for these malignancies may provide information that is complementary to conventional CT and may result in target volumes that contain proliferating tumor burden. If these volumes are small, focused dose escalation of large magnitude can be attempted which might result in improved local control by IMRT. The medical significance of including these additional data in the original treatment plan on final patient outcome will than need to be determined prospectively.

Aside from FDG, in the future new and more speciﬁc radiopharmaceuticals are likely to become routinely available that may permit a more accurate imaging of tumor clonogen density to complement the information gained by FDG and CT. Of special interest are SPECT and PET radiopharmaceuticals for the evaluation of tumor hypoxia, angiogenesis, apoptosis and receptor status, variables that play an important role in determining

the outcome of radiation therapy. In this regard, the study by Chao et al. demonstrating the feasibility of

60Cu-ATSM-guided IMRT following co-registration of hypoxia⁶⁰Cu-ATSM PET images to the corresponding CT images for IMRT planning is worth mentioning.

To the degree that PET provides physiologic data not available on CT, hybrid PET/CT treatment volumes may reduce the risk of geographic misses, particularly when using IMRT to constrict treatment volumes. When re- viewing differences between CT and PET target volumes, however, careful consideration will need to be given to the quality of the co-registration and its potential role in these differences.

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