Positron Emission Tomography Imaging of Tumor Cell Metabolism and Application to Therapy Response Monitoring

Cancer cells do reprogram their energy metabolism to enable several functions, such as generation of biomass including membrane biosynthesis, and overcoming bioenergetic and redox stress. In this article, we review both established and evolving radioprobes developed in association with positron emission tomography (PET) to detect tumor cell metabolism and effect of treatment. Measurement of enhanced tumor cell glycolysis using 2-deoxy-2-[18F]fluoro-D-glucose is well established in the clinic. Analogs of choline, including [11C]choline and various fluorinated derivatives are being tested in several cancer types clinically with PET. In addition to these, there is an evolving array of metabolic tracers for measuring intracellular transport of glutamine and other amino acids or for measuring glycogenesis, as well as probes used as surrogates for fatty acid synthesis or precursors for fatty acid oxidation. In addition to providing us with opportunities for examining the complex regulation of reprogramed energy metabolism in living subjects, the PET methods open up opportunities for monitoring pharmacological activity of new therapies that directly or indirectly inhibit tumor cell metabolism.

Cancer cells do reprogram their energy metabolism to enable several functions, such as generation of biomass including membrane biosynthesis, and overcoming bioenergetic and redox stress. In this article, we review both established and evolving radioprobes developed in association with positron emission tomography (PET) to detect tumor cell metabolism and effect of treatment. Measurement of enhanced tumor cell glycolysis using 2-deoxy-2-[ 18 F]fluoro-D-glucose is well established in the clinic. Analogs of choline, including [ 11 C]choline and various fluorinated derivatives are being tested in several cancer types clinically with PET. In addition to these, there is an evolving array of metabolic tracers for measuring intracellular transport of glutamine and other amino acids or for measuring glycogenesis, as well as probes used as surrogates for fatty acid synthesis or precursors for fatty acid oxidation. In addition to providing us with opportunities for examining the complex regulation of reprogramed energy metabolism in living subjects, the PET methods open up opportunities for monitoring pharmacological activity of new therapies that directly or indirectly inhibit tumor cell metabolism.
Keywords: tumor metabolism, positron emission tomography, choline, acetate, methionine, glutamine iNTRODUCTiON Mammalian cells possess molecular machineries that regulate their proliferation, differentiation, and death. Malignant transformation is a multistep process involving genetic alterations, disruption of regulatory circuits, and dynamic changes in the genome. It has been suggested that malignant growth is governed by six essential alterations in cell physiology: self-sufficiency in growth signals, insensitivity to growth-inhibitory (antigrowth) signals, evasion of programed cell death (apoptosis), limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis (1). Recent advances led to the notion that progressive evolution of normal cells to a neoplastic state involves not only the successive acquisition of hallmark capabilities but also contributions of recruited normal cells (which form tumor-associated stroma, constituting the "tumor microenvironment") (2).
Metabolic reprograming is an important property of the cancer cells. In the presence of abundant nutrients, oncogenic signaling facilitates assimilation of carbons into macromolecules, such as lipids, proteins, and nucleic acids. The net result of this is to support cell growth and proliferation. Glucose and glutamine are abundant nutrients, and both feed into multiple nodes of central metabolism. Glutamine also provides two nitrogen atoms for synthesis of hexosamines, nucleotides, and amino acids, all of which are also required for growth (3). Among the host of pathways altered in cancer, glucose and glutamine metabolism are consistently reprogramed by mutations in MYC, TP53, the Ras-related oncogenes, and the LKB1-AMP kinase (AMPK) and PI3 kinase (PI3K) signaling pathways. Oncogenic Ras increases both glucose uptake via enhanced expression of glucose transporter (GLUT) 1, and utilization (4), in addition to regulating glutamine metabolism, promoting cell survival and growth (5). Increased MYC also enhances glycolysis, and glutamine catabolism, resulting in cell growth (6).
The hallmarks of cancer are all linked to proliferation of cancer cells, thus making cell proliferation an important capability leading to immortalization and generation of macroscopic tumors. The framework of hallmarks assumes a homogeneous population of cancer cells and considers the hallmarks as distinct entities, with a one-to-one relation between oncogenic events (the inducers), the signaling pathways (transmission), and the hallmarks (the effects). However, one oncogenic event, or one signaling cascade, could induce several hallmarks accounting for the dynamic and spatial heterogeneity of tumors (7). This heterogeneity provides a framework to interpret pathological, diagnostic, and therapeutic observations of tumors and supports the need for non-invasive serial studies on the whole tumor mass and the use of simultaneous, multi-targeted therapies for treating cancer.
Routine clinical evaluation of cancer therapeutics involves assessment of the change in tumor burden (anatomical measurements). Tumor shrinkage (objective response) and time to disease progression are both important endpoints, as these have been linked to an improvement in overall survival or other time to event measures in randomized phase III studies (8). These measures also determine the efficacy of drugs under study. In order to have standardized and widely accepted criteria for measurement of response to allow comparisons to be made across studies, the Response Evaluation Criteria in Solid Tumors (RECIST) criteria were formulated (9). These criteria have been widely adopted for trials where the primary endpoints are objective response or disease progression. Since the introduction of RECIST in 2000, the increasing utilization of imaging technologies, such as MRI, FDG positron emission tomography (PET), and targeted cytostatic therapies, have prompted an update in the guidelines (RECIST v1.1) (10). The definitions of the criteria used to determine objective tumor response for target lesions are as follows: 1) Complete response (CR): disappearance of all target lesions.
Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. 2) Partial response (PR): at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. 3) Progressive disease (PD): at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression.) 4) Stable disease (SD): neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
RECIST has limitations due to its reliance on changes in tumor size with therapy. First, uni-dimensional measurements may be apparent only after three to four cycles of chemotherapy. In non-responders, this means subjecting patients to cumulative toxicity of three to four cycles of treatment with little benefit. Moreover, the change in the tumor diameter may be non-uniform. Second, changes in measurements of smaller lesions are not reliable (11). Third, cytostatic treatments may not necessarily cause a decrease in tumor size or volume. Use of functional imaging overcomes several of these limitations. The use of PET has, for example, resulted in accurate imaging of subtle tumor biologic changes and the detection of early response to anti-cancer therapy (12). Tumors having increased metabolic activity may take up greater amounts of a radioactive tracer as compared to adjacent normal tissues; in that regard, sub-millimeter tumors have been known to show significant radiotracer uptake for certain tracers (13). Similarly, any change in metabolic or signaling pathway activity consequent to successful treatment could result in changes in uptake of the tracer on PET (14). Thus, PET is a useful tool in oncology to image certain metabolic pathways and response to therapy.
This review gives an overview of metabolic processes imaged by PET focusing on both established and evolving radioprobes to detect tumor glycolysis, choline metabolism, intracellular transport of glutamine, and other amino acids, as well as fatty acid metabolism (Figure 1). In particular, we emphasize the role of radiolabeled choline, acetate, and amino acid tracers for monitoring efficacy or predicting response to new therapies that directly or indirectly inhibit tumor cell metabolism. glycolysis (AG) refers to glucose utilization in excess of that needed for oxidative phosphorylation, despite sufficient oxygen to metabolize glucose to carbon dioxide and water. AG plays an important role in the biosynthesis of glycogen, proteins, lipids, and nucleic acids (15). AG, also known as the Warburg effect, supports the biosynthetic requirements of proliferating cancer cells (16). PET using 2-deoxy-2-[ 18 F]fluoro-D-glucose ([ 18 F] FDG) has been widely used in the evaluation of various tumor types on the basis that an increase in AG will be reflected in an increase in the total glucose consumption of the tissue.
In a large pooled review of over 18,000 patient studies, it was shown that [ 18 F]FDG PET has a sensitivity of 84% and a specificity of 88% for tumor detection (17). [ 18 F]FDG PET has also been evaluated in response assessment following treatment with conventional chemotherapeutic agents both in the preclinical (18) and in the clinical setting (19)(20)(21)(22)(23)(24)(25), with the premise that decreases in glycolysis may occur in tumors that are sensitive to the applied cancer therapeutics and that the tumors that are resistant to treatment will show unchanged glucose metabolism. The prediction of treatment response has also been analyzed in many studies following treatment with different targeted therapies, e.g., monoclonal antibodies and small molecules inhibitors (26). However, [ 18 F]FDG PET has the following limitations: (1) False positive uptake in some benign processes, such as infection and inflammatory lesions (27); (2) low sensitivity in well-differentiated/low-grade tumors that have relatively low glucose metabolism such as carcinoid tumors, bronchoalveolar cell carcinoma, and renal cell carcinoma (RCC) (28)(29)(30); (3) low sensitivity in hypocellular cancers, such as desmoplastic or mucinous tumors (31); (4) increased [ 18 F]FDG accumulation in some normal body regions, such as lymphoid tissue and brown fat (32); and (5) lack of clinical utility due to high urinary excretion and low expression of GLUT in prostate cancer (PCa) (30). Thus, newer radiotracers to image tumors accurately are being developed to address these shortcomings, as well as explore other metabolic pathways of tumors that can be imaged using PET. Two evolving imaging strategies somewhat linked to glycolysis will be discussed next.
Further to the Warburg effect, the final rate-limiting step in AG, catalyzed by pyruvate kinase (PK), controls the balance between energy production and metabolic precursor synthesis. The M2 isoform of PK (PKM2) is preferentially expressed in cancer cells and channels glycolytic intermediates into pentose phosphate pathway for nucleotide synthesis (33). PKM2 can be allosterically regulated to assume a high-or low-activity state. In cancer cells, there is downregulation of PK activity favoring a microenvironment that is conducive to cell proliferation. 1-((2,6-difluorophenyl)sulfonyl)-4-((methoxy-11 C)phenoxy)sulfonyl)piperazine ([ 11 C]DASA-23) (Figure 1) has been developed as a non-invasive PET probe to measure activity of PKM2 in preclinical glioblastoma models (34). Witney and co-workers have demonstrated that [ 11 C]DASA-23 improved tumor visualization and predicted response to PKM2 activator, which resulted in loss of PET signal. The clinical translation of these findings is eagerly awaited.
Another glycolysis-linked pathway that has come to the fore is glycogenesis. Glycogen, the principal glucose store in mammalian cells, is synthesized from uridine diphosphate glucose (UDP-glucose) catalyzed by glycogen synthase (GS). Tumor cells originating from epithelial tissues, especially in the quiescent state also accumulate glycogen, in addition to increased glycolytic flux (35,36). In order to gain biological insight into the role of glycogenesis, PET with [ 18 F]-N-(methyl-(2-fluoroethyl)-1H [1,2,3] triazole-4-yl) glucosamine ([ 18 F]NFTG) has been studied (37). The authors showed that glycogen levels, [ 18 F]NFTG, but not [ 18 F] FDG uptake, increased proportionately with cell density and G1/ G0 arrest. This increase in glycogen levels and [ 18 F]NFTG uptake has potential application in the assessment of oncogenic pathways related to glycogenesis and the detection of post-treatment tumor quiescence. However, there have been no studies evaluating response to therapy.

Choline Metabolism: Choline PeT
Choline is a precursor of phosphatidylcholine (PC), an essential component of phospholipids in the cell membrane (38) and is required for structural stability and cell growth. It is also essential for the synthesis of neurotransmitters such as acetylcholine (by reaction of choline with acetyl-CoA), and production of potent lipid mediators, such as platelet-activating factor. Choline kinase (CHK) is the first enzyme in the Kennedy pathway (39), and is responsible for the de novo synthesis of PC. CHK phosphorylates choline to phosphocholine (PCho), the rate-limiting step in the Kennedy pathway. PCho is further phosphorylated to cytidine diphosphate-choline (CDP-choline) by the enzyme cytidylyltransferase and then to other intermediates before being incorporated into cell membrane phospholipids as PC. Malignant transformation is associated with enhanced choline transport and utilization, characterized in a large part by increased CHKα expression, which leads to a phenotype of increased radiolabeled choline uptake (40,41).

Choline Tracers
Choline has been radiolabeled with [ 11 C], [ 18 F] for tracing choline transport and phosphorylation in several tumor types. In one of the first studies, Hara and colleagues showed that phosphorylation led to intracellular retention of the carbon label [ 11 C] in PCa (42), thus enabling imaging of this metabolic pathway. The same group also showed that [ 11 C]choline had good uptake in brain tumors with almost negligible activity in the blood after 5 min (43). This work inspired others to use [ 11 C]choline as a PET radiotracer to image other tumors, including renal (30), esophageal (44)(45)(46)(47)(48), and non-small cell lung cancer (NSCLC) (44). [ (50), the tracer has proven safe for human administration (51) and has been extensively used in patients for diverse pathologies.
[ 11 C]Choline (and fluoro-analog) is, however, readily oxidized to [ 11 C]betaine by choline oxidase mainly in kidney and liver tissues, with metabolites detectable in plasma soon after injection of the radiotracer (52)(53)(54). This makes discrimination of the relative contributions of parent radiotracer from catabolites difficult when a late imaging protocol is used. A more metabolically stable [ 18 (54,56,57). This modification is hypothesized to increase the net availability of the parent tracer for phosphorylation and trapping within cells leading to a better signal-to-background contrast, thus improving tumor detection sensitivity of PET. [ 18 F]D4-FCH has been validated for imaging tumors preclinically (56,57). [ 18 F]D4-FCH injection was also found to be safe and well tolerated in healthy volunteers with a As the large proportion of studies evaluating response with choline radiotracers has been conducted in PCa -a disease that is managed by a plethora of agents, including androgen deprivation therapy (ADT), radiotherapy (RT), and chemotherapy -this will be the main aspect of the review although other malignancies will be briefly discussed.

Preclinical Studies
Radiolabeled choline uptake has been extensively investigated in cells and animal models of cancer to determine factors that affect intrinsic uptake and allow interpretation of clinical findings ( Table 1).
Hara and colleagues demonstrated that androgen depletion markedly suppressed the uptake of [ 3 H]choline in androgendependent LNCaP cells but not in androgen-independent PC3 cells (59). Anti-androgens were subsequently shown to modulate choline uptake in androgen-dependent cell lines and also inhibit proliferation (60,61). Regarding chemotherapy, the effects of docetaxel have been studied by Krause et al. (62), who showed a reduction in the mean [ 11 C]choline uptake in PC3 xenograft mouse model as early as 1 week after initiation of docetaxel. A significant reduction of mean tracer uptake of 45% was associated with a mean  (64). Radiationinduced effects were variable with a transient increase in radiotracer uptake in androgen-independent PC3 cells but a decrease in androgen-dependent LNCaP cells. In both cell lines, modulation of tracer uptake was dose-independent following irradiation with 2-12 Gy with a mean increase to 120% in PC3 cells and a mean decrease to 74% in LNCaP cells. The authors suggested that changes of tumor choline uptake monitored by PET after RT may be due to a combination of factors, including therapeutic efficacy and altered tracer transport in cancer cells as a consequence of irradiation. Photodynamic therapy (PDT) responses have also been evaluated. Fei and co-workers evaluated the potential use of [ 11 C]choline PET to monitor early tumor response to PDT in animal models. For treated tumors, normalized [ 11 C]choline uptake decreased significantly at 24 and 48 h after PDT, associated with decreases in prostate-specific antigen (PSA) levels. The authors concluded that [ 11 C]choline PET has the potential to determine response in a PDT-treated tumor within 48 h after therapy (65).

Clinical Studies
To date, only anecdotal reports (50,66) and two small clinical studies (67,68) have assessed the role of [ 11 C]choline PET as a method to monitor the therapeutic effects of ADT ( Table 2). Fuccio et al. (67) retrospectively evaluated the effect of 6 months of androgen deprivation (Zoladex in 12 and Bicalutamide in 2 patients) in 14 PCa patients with recurrence after radical prostatectomy. They concluded that androgen deprivation significantly decreases [ 11 C] choline uptake in androgen-sensitive patients. In another study in six primary PCa patients having bicalutamide therapy, Giovacchini et al. (68) showed an average reduction of 45% in the [ 11 C]choline uptake (SUVmax from 11.8 to 6.4) corresponding to a 78% decrease in PSA following a median of 4 months of therapy. A similar   (71). Thus, the concern that RT might increase labeled choline transport does not appear to occur in patients at clinically relevant doses. Furthermore, in a proof of concept study, Challapalli and co-workers showed that choline uptake in prostate tumors, determined by [ 11 C] choline PET/CT, is sensitive to ADT and RT, and could be used as an objective quantitative tool for response assessment. ADT decreased tumor-imaging variables -SUV60,ave, SUV60,max, TMRave, and TMRmax -by 26-60%. RT also decreased [ 11 C]choline uptake within primary prostate tumors (though of lesser magnitude: 12-27%), compared to that seen with ADT, except for TMRmax where a significant reduction (40%) was seen (Figure 2)   CT changes (72,73). These studies show the potential of radiolabeled choline to detect response of early PCa to therapies used routinely in the clinic. Chemotherapy, Radium-223, and drugs interfering with androgen receptor (AR) machinery, such as enzalutamide and abiraterone, are the main stay of treatments in metastatic castrate resistant prostate cancer (mCRPC). Currently, there is excessive reliance on changes in serum PSA as an indicator of therapeutic efficacy and there are no predictive diagnostic tools to identify an early objective response in patients with mCRPC treated with abiraterone acetate or enzalutamide, although AR splice variants detectable in circulating tumor cells (CTCs) are evolving (74). The Prostate Cancer Clinical Trials Working Group recommends waiting 12 weeks before the first evaluation of response to ensure adequate drug exposure (75). Therefore, studies investigating new biomarkers for outcome prediction and disease monitoring are urgently needed. To this effect, labeled choline PET is under evaluation to assess therapeutic response.
Kwee and colleagues evaluated effects of docetaxel chemotherapy on circulating cell-free DNA (cfDNA) and [ 18 F]FCH PET/ CT uptake in CRPC. Tumor-derived plasma cfDNA concentrations increased significantly after one and three treatment cycles, possibly due to post-chemotherapy necrotic cell lysis. Lower cfDNA concentrations at baseline were found to be associated with PET responses as measured after the third chemotherapy cycle. They concluded that it is feasible to annotate potential tumor sources of cfDNA using [ 18 (79). Miyazaki and co-workers evaluated acute changes in net metabolically active tumor volume (MATV) and total lesion activity (TLA) detected by [ 18 F]FCH PET/CT imaging midway during treatment with radium-223 dichloride, in two patients. After the third dose of radium-223 dichloride, near-total disappearance of abnormal skeletal activity was observed in one case, while a heterogeneous tumor response was observed in the other (80). It can be summarized that, while being a proliferation-independent phenotype (13), changes in labeled choline uptake reflects response to therapy although the optimal time still needs to be clarified.

Non-Prostate Tumors
In addition to PCa, radiolabeled choline has been utilized in other tumor histotypes. Parashar et al. explored whether [ 18 F]FCH PET could serve as an predictive biomarker for early detection of RT/ chemo-radiotherapy (CRT) response in patients with lesions at the base of the tongue, tonsil, nodes, hypopharynx, maxilla, palate, lung, pancreas, brain, uterus, and rectum. They demonstrated reductions in SUVmax in 88% of lesions (CR: 7/16 and PR: 7/16) and concluded that changes in SUVmax after 3-4 weeks of initiation of RT were predictive of final outcome (81). Panagiotidis and co-workers showed that simultaneous PET/MRI with [ 18 F] FCH in a patient with pineal germ cell tumor demonstrated a reduction in both size and radiotracer activity of the mass after chemotherapy (82).
While the choline phenotype has been reported as being proliferation independent in PCa (13), the phenotype is intrinsically associated with proliferation in breast cancer cells (83). In particular, PCho formation is linked to the activity of mitogenactivated protein kinase (MAPK) signaling function (84). It was, thus, postulated that therapy response in breast cancer might be accompanied by predictable changes in the tumor retention of [ 11 C]choline. In a clinical study involving breast cancer patients receiving trastuzumab, [ 11 C]choline uptake decreased in two patients responding to trastuzumab compared to nonresponders (85). Regarding targeted therapies, Middendorp et al. also evaluated use of [ 18 F]fluoroethylcholine (FEC) PET/CT in staging and monitoring therapy response of advanced RCC before and 10 weeks after two cycles of tyrosine kinase inhibitor (TKI) therapy. FEC PET/CT showed heterogeneous changes, with progression in one patient and a PR in the second patient, which were concordant with the RECIST response (86). Thus, changes in uptake of labeled choline in non-prostate histotypes also appear to reflect therapy response.

Fatty Acid Metabolism
Fatty Acid Synthesis: Acetate PET Cancer cells are dependent on their ability to gain access to energy and substrate precursors, by reprograming the normal metabolic pathways required for the proliferation and survival of tumor cells, to synthesize of proteins, nucleotides, and lipids (87). Cancer cells are also characterized by a lipogenic phenotype (88), and often require that fatty acids be generated de novo to maintain proliferation and viability. As a result, fatty acid biosynthesis has gained significance as a potential therapeutic target in multiple cancers.
Acetate is metabolized in the tricarboxylic acid (TCA) cycle yielding CO2 and water (89). However, in cancer cells, acetate is preferentially utilized for fatty acid synthesis as a component of acetyl-CoA. Intracellularly, acetate is converted to acetyl-CoA by acetyl-CoA synthase (ACeS), and fed into the fatty acid synthesis pathway. [ 11 C]Acetate PET was originally used to assess myocardial function (90). In myocardial tissues, carbons derived from [ 11 C]acetate are incorporated into CO2 during the TCA cycle, allowing for PET visualization of oxygen consumption. However, in tumor cells, [ 11 C]acetate is incorporated into membrane lipids due to over-expression of fatty acid synthase (FASN). This property is exploited for tumor imaging with [ 11 C]acetate (91). The majority of studies analyzing the efficacy of [ 11 C]acetate PET in tumors have focused on the detection of PCa (91). In addition to PCa, there are a number of other tumor types in which [ 11 C]acetate PET shows high contrast, including hepatocellular carcinoma (HCC), thymomas, renal cancers, brain tumors, and bronchioloalveolar carcinoma (92). These studies demonstrate that [ 11 C]acetate is useful in diagnosis, staging, and predicting disease progression in certain cancers, and it is logical that [ 11 C] acetate could also be used to stratify patients for specific therapies, as well as a method to monitor response to therapy. Emonds (96). While these studies are promising (Table 1), it has been noted recently that optimal acquisition of [ 11 C]acetate images may require late imaging (~90 min) to increase sensitivity toward lipid incorporation (97).

Clinical Studies
Yu and co-workers, tested the feasibility of [ 11 C]acetate PET imaging to assess response to therapy in men with bone metastatic PCa. Patients were imaged before and 6-12 weeks after initial  (101). [ 11 C] acetate PET has also been used in assessing response to therapy in multiple myeloma. Bone marrow histology and whole-body (WB) MRI were used as comparators. In 13 patients who had repeat examination after induction therapy, visual and quantitative analysis, suggested a higher detection rate for both diffuse and focal myeloma lesions at diagnosis. After treatment, a 66% reduction in SUVmax was seen in patients with at least a very good PR (≥ 90% reduction in M-protein) versus a 34% reduction in those with a PR (≥50% reduction in M-protein). They concluded that [ 11 C]acetate may be valuable for response assessment (102). In aggregate, the initial data with [ 11 C]acetate for monitoring response is promising ( Table 3) but the short half-life of the tracer may reduce sensitivity for imaging lipid incorporation, and its use is limited to centers with in-house cyclotron. Moreover, acetate is not specific to fatty acid synthesis, it also serves as a substrate for protein acetylation, and is utilized in cholesterol synthesis. [

Fatty Acid Oxidation: [ 18 F]Fluoropivalic Acid PET
In addition to fatty acid synthesis, the critical nature of fatty acid oxidation for cancer cells survival has been recognized (105). Short-chain carboxylates, including acetate, propionate, butyrate, and the non-natural substrate pivalate (trimethylacetate) use the early steps of the fatty acid oxidation pathway involving acyl-CoA and acyl-carnitine synthesis (106). A new radioprobe, 3-18 F-fluoro-2,2-dimethylpropionic acid, also called [ 18 F]fluoropivalic acid ([ 18 F]FPIA), for imaging the early steps of the fatty acid oxidation pathway has been validated and has shown promise for cancer detection (107). Further studies are eagerly awaited.

Amino Acid Metabolism
Amino acids play an important role in the synthesis of a variety of nitrogen-containing compounds, such as proteins and nucleotides during cell growth, and their increased transport and utilization are be associated with early events in carcinogenesis (108). Natural amino acids are transported into cells by specific carrier-mediated transport systems and further incorporated into proteins and intermediary metabolites to different extents. Thus, investigators have studied the utility of several radiolabeled natural amino acids (including methionine, glycine, tyrosine, phenylalanine, and leucine) as tumor-imaging agents with PET (109). Amino acid scanning provides higher contrast between tumor and normal brain compared to [ 18 F]FDG PET, due to the low uptake of amino acids in normal brain. However, of the several amino acid tracers investigated for tumor imaging, only a few have been evaluated beyond the initial feasibility studies in human patients.

Glutamine
Glutamine is the most abundant amino acid in plasma and occupies a unique niche in intermediary metabolism by providing a major inter-organ shuttle for both nitrogen and carbon (110). This makes it essential for cell proliferation by contributing to synthesis of nucleic acids, proteins, and hexosamines. Loss of amino and amido groups in glutamine produces alpha-ketoglutarate that also promotes cell growth, anaplerosis and adenosine-tri-phosphate (ATP) generation (111). Malignant transformation, involving enhanced c-Myc expression, increases glutamine metabolism by increased expression of cell surface transporters (112,113).  Glutamine metabolism lends itself to evaluation by PET imaging, most relevant in non-[ 18 F]FDG avid tumors, such as prostate, bronchoalveolar carcinomas, carcinoid tumors, and low-grade lymphomas. These malignancies may use glutamine as an alternative nutrient source and as such are more easily detected by a glutamine-based tracer. Venneti and co-workers, for example demonstrated that PET imaging in vivo with the glutamine analog 4-[ 18 F]-(2S,4R)-fluoroglutamine ([ 18 F]FGln) facilitates clear tumor delineation due to high tumor-to-background ratio. Chemo/radiation therapy reduced [ 18 F]FGln tumor uptake, which was associated with decreased tumor burden, confirmed on autoradiography. In contrast, there were no anatomical or structural changes seen on T2-weighted MRI sequences, within the same time frame. These findings were translated into humans (six patients) and an increased [ 18 F]FGln uptake was seen in patients with progressive brain tumors, but not in patients with SD (114).

Methionine
Methionine, an essential sulfur amino acid, is necessary for growth and development. It plays an important role in protein synthesis and is a predominant methyl group donor for multiple metabolic pathways. Malignant transformation enhances demand for methionine in cancer cells caused by increased fluxes in the pathways of protein synthesis, transmethylation, and transsulfuration. This forms the basis for higher uptake of labeled methionine in tumors.
Currently, PET using L-[methyl-11 C]-methionine ([ 11 C] methionine) is the most popular amino acid imaging modality for tumors, although its use is restricted to PET centers with an on-site cyclotron facility. [ 11 C]methionine PET has been extensively studied in gliomas. Its role in initial diagnosis, differentiation of tumor recurrence from radiation injury, grading, prognostication, tumor extent delineation, biopsy planning, surgical resection and RT planning has been evaluated (115). A number of oncologic imaging studies have evaluated the role of [ 11 C]methionine in response assessment and have been described in detail in the preclinical setting (116)(117)(118)(119)(120)(121)(122)(123)(124)(125)(126)(127)

Leucine Analogs
Leucine is one of the preferential amino acid required for proliferating tumor cells and is, therefore, of interest in molecular imaging of anabolic cancer processes. 1-Amino-3-[ 18 F]fluorocyclobutane-1-carboxylic acid (anti-[ 18 F]FACBC), a synthetic non-natural leucine analog, has been widely studied in imaging brain (171,172), prostate tumors (173,174), and pulmonary lesions (175). The non-natural amino acids are not metabolized but are taken up through both the L-type transporter and the energy-dependent A-type transporter (176). The tracer accumulation in PCa cells correlates with the expression level of the alanine, serine, and cysteine preferring system-mediated amino acid transport with the large neutral amino acid transporter (LAT1) as an important transport system (177,178

Tryptophan Analogs
Tryptophan is an essential amino acid required for biosynthesis of proteins, serotonin, and niacin in the brain and other tissues (179). The amino acid PET tracer alpha-[ 11 C]methyl-Ltryptophan (AMT) is transported in tumor tissue by LAT1 but is not incorporated into proteins (180). Instead, AMT is utilized by the kynurenine immunomodulatory pathway (181). Under pathological conditions, induction of this pathway's rate-limiting enzyme, indoleamine 2,3-dioxygenase (IDO), leads to increased metabolism of tryptophan and, thus, AMT accumulation (182). Tryptophan analogs have been widely studied in imaging highgrade gliomas (182,183), CRPC (184), and neuroendocrine tumors (185). In a case report, Peng and co-workers suggested that AMT PET may be useful for assessing progression and therapeutic response of optic glioma (186). Further studies are eagerly awaited.

DiSCUSSiON
Several metabolic pathways are deranged in cancer in a proliferation-dependent or proliferation-independent manner. These metabolic pathways, particularly enhanced glycolysis, offer the opportunity to detect cancer often with high contrast. In this review article, we discuss about the role of established and evolving metabolism tracers for prediction/monitoring response to therapy. The effect of drug or radiation therapy on each metabolic phenotype ought to be carefully considered to enable assignment of biological and clinical relevance to the changes seen. Notably, these therapies may directly or indirectly inhibit tumor cell metabolism, or indeed the changes may simply reflect loss of cell viability and influence the timing of post-treatment scanning. For [ 18 F]FDG PET, the effect of the so-called targeted or biologic therapies on response monitoring has been reviewed (26) with the suggestion that the drugs may directly affect GLUT/ hexokinase expression or activity with changes occurring within hours to days after initiating treatment. This type of information is less mature when other metabolism tracers are considered. For example, as discussed above, only a few studies have attempted to directly link the biology of androgen deprivation to changes in the tumor labeled choline signal. Regarding imaging variables, different variables have been used in the assessment of non-FDG tracers (see Tables 1-4). Some of these variables, e.g., TMR, may be considered, for instance, when RT is the choice of therapy to account for the effect of radiation on normal tissues. Whatever the mechanism of signal change, be it direct or via loss of cell viability, it is important to consider the intrinsic variability of the quantitative measure, as well as that magnitude of change (threshold) for response. For [ 18 F]FDG uptake, the intrinsic measurement variability (without treatment) ranges from 10 to 20% in different tumor phenotypes (187,188). Based on pooling together reproducibility data, a consensus for quantifying PET response by EORTC PET study group was reached (189). The tumor responses were graded as follows: 1) Complete metabolic response (CMR): complete resolution of FDG uptake. 2) Partial metabolic response (PMR): a decrease (across all lesions) of minimum of 15% in tumor SUV after one cycle or >25% after more than one cycle of chemotherapy. 3) Stable metabolic disease (SMD): an increase of <25% or a decrease of <15% in SUV, and no visible increase in extent of FDG tumor uptake (20% in longest dimension). 4) Progressive metabolic disease (PMD): an increase in FDG tumor SUV of >25% within tumor region defined on baseline scan; visible increase in extent of FDG tumor uptake (20% in longest dimension) or appearance of new FDG uptake in metastatic lesions.
More recently, PET Response Criteria in Solid Tumors (PERCIST) guidelines have been formulated (190). These are based on the premise that cancer response as assessed by PET is a continuous and time-dependent variable. The tumor responses were graded as follows: 1) CMR: visual disappearance of all metabolically active tumors. 2) PMR: more than a 30% decline and a 0.8-unit decline in SULpeak between the most intense lesion before treatment and the most intense lesion after treatment, although not necessarily the same lesion. 3) SMD: not CMR, PMR, or PMD. 4) PMD: more than a 30% and 0.8-unit increase in SULpeak or new lesions, if confirmed. A >75% increase in total lesion glycolysis is also proposed as another metric of progression.
The PERCIST criteria differ from the EORTC criteria in that the SUV is normalized to the lean body mass and five tumors (up to two per organ) with the most intense [ 18 F]FDG uptake lesions being considered target lesions; SULmean is the recommended imaging variable, as it has better test-retest variability (8-10%), is statistically less susceptible to variance, and is less subjective due to clear definition of target lesions.
Notably, these criteria are specific for [ 18 F]FDG PET and may differ for other tracers. For example, Kenny and co-workers have evaluated the reproducibility of [ 11 C]choline in breast cancer (85). A decrease of 40% for SUV30min, and 24% for SUV60min, was classified statistically as response. However, it is not clear if these criteria could be widely applied across different tumor sites or across different PET tracers, as the intrinsic variability may be isotope, patient, or scanner related.
In the future, further evaluation is required to assess the role of metabolic-PET imaging in assessing response to treatment and follow-up after treatment. These include what the optimal time (early or delayed) for performing the scan after treatment is, what the relevant imaging variables for predicting response are, how often to scan, whether imaging sensitivity and specificity are sufficient to predict response or progression, and whether changes in imaging variables can be used as surrogates for predicting patient outcomes. Future studies will need to be designed to establish the answers to these questions.

CONCLUSiON
In this article, we aimed to give an overview of metabolic processes imaged by PET and focused on both established and evolving radioprobes to detect tumor glycolysis, choline metabolism, intracellular transport of glutamine, and other amino acids, as well as fatty acid metabolism. In particular, we emphasize the role of radiolabeled choline, acetate, and amino acid tracers for monitoring efficacy or predicting response to new therapies that directly or indirectly inhibit tumor cell metabolism. The optimal imaging time point, pertinent imaging variable, and criteria for response will require further interrogation.

AUTHOR CONTRiBUTiONS
AC and EA have contributed to the conception, layout of the review, and reviewed and proof read.

FUNDiNG
EA's laboratory receives core funding from Cancer Research UK and the UK Medical Research Council.