Sridhar Nimmagadda • Anthony F. Shields
Uncontrolled proliferation is a hallmark of cancer. Monitoring and quantification of this uncontrolled growth for diagnostic and therapeutic purposes has long been a primary interest of the imaging community.1 Proliferation can be monitored in several ways based on identifying the active biologic process required for cell growth that is, carbohydrate metabolism, DNA synthesis, amino acid metabolism, lipid metabolism to name a few. Glucose metabolism, as monitored using 18FDG PET, is the mainstay of nuclear medicine PET imaging.2 However, high 18FDG uptake is nonspecific because of inflammation or immune cell proliferation in the tumors. High intense glucose metabolism in tissues like the brain and radioactivity in the bladder caused by excretion of tracer may also interfere with the interpretation in the brain or prostate, respectively. Importantly, cells tend to maintain glucose metabolism even after cessation of replication therefore complicating assessment of the effectiveness of therapeutic agents.
Amino acid and lipid precursors can also be used to quantify cell growth. Both of these precursors, however, tend to have more than one metabolic pathway.3,4 Therefore, it is sometimes difficult to assess the uptake observed in the tumors as a measure of proliferation. Despite these limitations, recent developments in this category such as utilization of [18F]FACBC and [11C/18F]choline, respectively have gained attention for imaging of prostate and other cancers.5,6 Recently, [11C]choline-PET received FDA approval to detect recurrent prostate cancer underscoring the importance of the availability of an armamentarium of complementary imaging agents for a thorough cancer diagnosis.
RADIOLABELED THYMIDINE (Tdr) AS A PROLIFERATION IMAGING AGENT
One of the first biologic processes to be halted in response to a cellular insult is DNA synthesis. An unambiguous way to assess proliferation, therefore, would be to monitor the DNA synthesis. In addition, nucleosides, particularly thymidine (Tdr) and its analogs, are unique because of their involvement in the DNA salvage pathway. Of all the nucleic acid bases, Tdr is the only one that is selectively incorporated into DNA and not into RNA. Hence, retention of this radiolabeled base reflects the level of DNA synthesis and cell replication. Cytosine, adenosine, and guanosine are used in the synthesis of RNA after replacement of the deoxyribose. Accordingly, for more than five decades Tdr labeled with either 3H or 14C and more recently other nonradioactive click chemistry–based analogs such as 5-ethynyl-2′-deoxyuridine (EdU) have become a standard to characterize proliferation.7 Interestingly, Tdr is not essential for DNA synthesis. It is introduced into DNA synthesis by an endogenous process called the de novo pathway. It is synthesized by methylation of dUMP and converted to dTMP by an enzyme called thymidylate synthase (TS) (Fig. 38.1). TS is a key enzyme for the de novo synthesis of pyrimidines. TS catalyzes the reductive methylation of dUMP, derived from either the deamination of dCMP (catalyzed by dCMP deaminase), or from the hydrolysis of dUTP (catalyzed by dUTPase), by N5, N10-methylene tetrahydrofolate to result in 2′-deoxyTdr-5′-O-monophosphate (dTMP).8 dTMP is further di- and triphosphorylated to dDTP and dTTP by Tdr monophosphate kinase (TMPK) and nucleotide diphosphate kinase (NDPK), respectively. All the Tdr kinase family members require ATP and magnesium for the catalytic addition of phosphate groups. The formed dTTP is incorporated into DNA by DNA polymerase. This de novo pathway is tightly autoregulated by different anabolic and catabolic processes including the end product dTTP. The formed dTTP inhibits several enzymes involved in its synthesis process including (1) aspartate transcarbamylase involved in the synthesis of carbamoyl aspartic acid, a precursor for all pyrimidine nucleotides; (2) deoxyuridine kinase involved in the synthesis of dUMP; (3) deoxycytidine monophosphate deaminase involved in the synthesis of dUMP; (4) cytidine diphosphate reductase involved in the synthesis of dCDP (Fig. 38.1).9 Our understanding of these processes played an important role in the development of imaging agents and how therapeutic agents modulate these processes is further exploited for proliferation-based therapeutic monitoring.
When Tdr is introduced exogenously, it is rapidly transported across the cell membrane by both equilibrative (ENT1 and ENT2) and concentrative (CNT1 and CNT3) transporters that produce equilibration within seconds.10 Once intracellular, the Tdr DNA incorporation time course is determined by the nature of the system with dTMP formation as the rate-limiting step and dTTP as the largest fraction of metabolite composition. In the case of exogenous TdR, TK1 catalyzes the transfer the γ-phosphate group of ATP to the 5′-hydroxyl group of Tdr resulting in dTMP. Expression of TK1 is strictly cell cycle regulated and increases markedly during S-phase followed by a rapid decline as cells enter G2-phase.11 Increased human TK1 activity is observed and proposed as a prognostic marker in several cancers.12 Because of a high proliferation rate, cancer cells often express high levels of TK1, which can be used for the detection of tumors and metastases by means of positron-labeled tracers.13 However, considering only 8% to 20% of the cells are in S-phase but every cell has active glucose metabolism, the absolute accumulation of TK1-catalyzed radioactivity, though highly specific, is relatively low compared to 18FDG accumulation within the tumors.
FIGURE 38.1. Thymidine synthesis and metabolism. Biochemical de novo pathways leading to the DNA synthesis. Double arrows indicate reversible processes, catalyzed not necessarily by the same enzyme. The sites of dTTP inhibition pathways are shown in X. Enzymes discussed in this chapter that play a role in proliferation imaging are shown in blue. CA, carbamyl aspartic acid. (Modified from Loffler M, Zameitat E. Pyrimidine Biosynthesis. In: William JL, Lane MD, eds. Encyclopedia of Biological Chemistry. New York, NY: Elsevier; 2004:600–605.)
FIGURE 38.2. [11C]Thymidine catabolism leading to radioactive metabolites. BAIB, β-aminoisobutyric acid.
One of the issues encountered with labeled thymidine is that, exogenously introduced Tdr could be metabolized before it gets into the cell by thymidine phosphorylase (TP). TP is involved in catalyzing the phosphorolysis of the nucleosidic linkage of pyrimidine 2′-deoxynucleosides with the formation of the pyrimidine and 2-deoxy-α-D-ribofuranose-1-phosphate.14 Because this phosphorolysis reaction is reversible, the catabolic function of TP is inhibited by excess thymine and enhanced by low Tdr levels. The formed thymine undergoes further degradation to dihydrothymine, β-ureiodoisobutyric acid (BUIB), β-aminoisobutyric acid (BAIB), carbon dioxide, and water (Fig. 38.2). Although the steps involving TP occur in many tissues where TP is expressed, including intestines, liver, bone marrow, kidney, brain, etc., the steps involving the formation of BAIB, CO2, NH3, and water preferentially occur in liver, spleen, and kidneys. By regulating nucleotide pools, TP plays a critical role in maintaining nucleic acid homeostasis by ensuring the correct supply of deoxyribonucleoside triphosphates (dNTPs) for DNA replication and repair.15,16To prevent the TP-associated catabolism, nucleoside analogs with greater stability are developed by substituting the 2′ or 3′ hydroxyl group of the sugar with a fluoride as in the case of 3′-fluoro-3′-deoxy-thymidine (FLT) (Fig. 38.3). Clearly, knowledge of TP levels in tumors will be useful for therapeutic dosing and better interpretation of nucleoside-PET images. Grierson et al., developed 5′-deoxy-5′-[18F]fluorothymidine (18F-DFT) as a probe for intracellular TP expression imaging. The tracer uptake did not show a correlative uptake with TP expression levels in A459 and U937 cell lines, reason being that initial metabolite formed resulted in a diffusible secondary metabolite allowing efflux of radioactivity from the cells.17 Recent developments include the synthesis of 5-fluoro analog of (5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]uracil) (TPI), a known inhibitor of TP, was shown to have an IC50 value of 9 nM for [3−H]TdR cleavage inhibition by human TP. The presence of a fluoride functional group suggests a potential radiopharmaceutical for TP expression imaging.18
For detailed molecular characterization of Tdr metabolism, many of the early studies, both in vitro and in vivo, measured the retention of the radiolabel, either 3H- or 14C-Tdr, after the extraction of macromolecules. These extraction processes were done by acid precipitation of the DNA from mixtures of disrupted cellular contents and usually removed small molecules, including Tdr itself and its metabolic breakdown products. Studies were also performed using fixed tissues, where fixed cells retained labeled DNA. Such detailed characterization is not possible in vivo with PET, because PET cannot distinguish the molecular form of the radiotracer detected. In PET, all of the radioactivity present in a given volume element is measured irrespective of the associated molecular entity. These issues were encountered when 11C-Tdr was used to measure DNA synthesis. The initial synthesis of 11C-Tdr was achieved by introducing the radiolabel at the methyl position by enzymatic conversion of deoxyuridine-5′-phosphate to [11C]thymidylate. Further alkaline phosphatase treatment of [11C]thymidylate resulted in [11C]-Tdr.19 Evaluation in a VX2 rabbit tumor model showed clear tumor uptake of radioactivity. However, longer synthesis time and poor yields limit routine use of this method. Later in the 1980s, improved synthesis and evaluation of 11C-Tdr was achieved through the production and introduction of a radiolabel using 11C-CH3I in the methyl position.20 Comparison of in vivo studies of 11C-Tdr with other labeled Tdr analogs (3H and 14C) demonstrated similar results that is, a minor fraction of radioactivity incorporated into DNA with a major fraction present in metabolites. Further HPLC analysis of metabolites demonstrated that within 2 minutes post injection, 11C-Tdr is degraded into thymine and later into other smaller metabolites BAIB, CO2, and NH3, which are excreted (Fig. 38.2). Extensive metabolism of 11C-thymidine demands complicated image analysis and this detailed analysis of metabolites by HPLC certainly improved the modeling of imaging kinetics.21–23 In the 1990s, developments in radiosynthesis allowed the synthesis of [2-11C]Tdr radiolabeled at the 2′-position of the pyrimidine ring.24 The advantage of this radiolabeled analog is that, the principal metabolite, [11C]CO2, is readily transported into and washed out of tissues. It can be quantified and modeled more readily than BAIB.25–27 Initial studies with this agent identified 11 of 14 untreated brain tumors with an average uptake approximately twice that of normal brain.28 However, no correlation was seen between tumor grade and radioactivity uptake. In another study, Eary et al.29 compared [11C]Tdr-PET imaging, FDG, and MRI in 13 patients. Comparison of PET scans with MRI showed different uptake patterns in about half of the patients, indicating that different information was being obtained. Even though proliferation images would be expected to match clinical progression, it was not observed in all cases. This preliminary application of a kinetic model of brain tumor proliferation showed promising results and suggested an improved ability to delineate active tumor from treatment effects. Nevertheless, both [11C] radiolabeled analogs of thymidine required quantification of labeled metabolites for comprehensive interpretation of time–activity curves and DNA incorporation. Several factors including (1) fast metabolism of Tdr limiting the amount of radiolabel available for DNA incorporation; (2) complex metabolite analysis and extensive modeling effort required for interpretation of images and (3) the short half-life of 11C limited the routine clinical use of 11C-Tdr. These studies not only demonstrated the potential of proliferation imaging with [11C]Tdr-PET but also illustrated the need for metabolically stable Tdr analogs, preferably labeled with 18F, for successful in vivo proliferation imaging applications.
FIGURE 38.3. Thymidine analogs. Molecular structures of thymidine-based agents used for proliferation imaging.
DEVELOPMENT AND EVALUATION OF 18F-LABELED NUCLEOSIDE ANALOGS AS PROLIFERATION IMAGING AGENTS
Nucleoside analogs with nonalcohol functional groups in the 2′- or 3′-position are generally stable from degradation by TP.30 Initial screening of several analogs with 2′-fluorine substitution of the sugar in dog plasma demonstrated greater stability than Tdr. However, most of these agents are also substrates for mitochondrial thymidine kinase TK2.31–33 FLT demonstrated high substrate specificity to TK1 and is a poor substrate for TK2.31–33 FLT is taken up by tumor cells similar to thymidine,34 phosphorylated by TK1 and trapped intracellularly. Unlike thymidine, the presence of 3′-fluorine in FLT makes it not only resistant to degradation by TP but also less susceptible to incorporation into DNA by DNA polymerases. Therefore, the majority of the intracellularly trapped radioactivity represents the sum of the activities of its specific transport and phosphorylation. Based on these observations, initial radiosynthesis of 18F-FLT was achieved through a displacement of 3′-O-nosyl group on the precursor protected with dimethoxy benzoyl and dimethoxy trityl groups.35 Other improved syntheses were later developed using 5′-O-(4,4′-dimethoxy-trityl)-2,3′-anhydro-Tdr or 5′-O-benzoyl-2,3′-anhydro-Tdr as precursors.36,37 FLT was initially developed as an antiviral agent; however, its use was discontinued because of hematologic and hepatic toxicity and peripheral neuropathy.38 One of the advantages of PET and SPECT imaging agents is that, at tracer doses, toxicity is not an issue. Nonetheless, an NCI sponsored safety trial did not show any detectable toxicity symptoms at a maximum injected dose of 6.1 μg.39
The suitability of 18FLT as a proliferation imaging agent was demonstrated in an elegant study by Rasey et al.,40 in which 18FLT uptake was correlated with TK1 activity in A549 lung carcinoma cells. Using growth arrested and actively proliferating cells, the authors demonstrated that growth arrested cells take up little 18FLT whereas actively growing cells show increased 18FLT uptake. Increased 18FLT uptake is correlated with increased TK1 activity and S-phase cell fraction. In comparative studies, [14C]deoxyglucose showed that 18FLT uptake is better correlated with growth changes than glucose utilization suggesting that 18FLT is perhaps a better measure of proliferation than 18FDG. However, correlation of 18FLT uptake in vivo in rodents with TK1 activity is not always straightforward. Rodents have a 10-fold higher serum level (1 μM) of Tdr compared to humans (0.1 μM).41 Therefore, in addition to Tdr being a better substrate for TK1 injected 18FLT has 10-fold higher competition from endogenous Tdr for TK1 activity to be phosphorylated and trapped intracellularly.
Interestingly, dogs have similar serum Tdr levels as humans. Initial studies of 18FLT in dogs demonstrated a clear uptake of radioactivity in bone marrow of the spine, which is a highly proliferative tissue. Confirming the previous in vitro results, very little uptake was observed in the heart which has very high levels of TK2. An initial report of 18FLT in humans clearly showed uptake in the non– small-lung cancer lesion and marrow.13 High uptake was also observed in the liver in humans that was not observed in dog studies. This could be attributed to the intraspecies differences in glucuronidation of 18FLT. Further metabolite analysis performed showed that 18FLT was conjugated in the liver with glucuronide and rapidly excreted in urine. In spite of glucuronidation, in humans, nearly 60% to 70% of the extracted 18FLT is intact. Based on these observations, a robust mechanism-based 4-compartment kinetic model has been developed and validated in human.42,43 Sampling and metabolite analysis at five time points is sufficient to derive a 18FLT flux parameter with compartmental analysis.44 Analysis of 18FLT metabolites from in vitro studies showed that 18FLT and 18FLT-MP are primary metabolic species. The di- and triphosphate products were also reported in smaller fractions.45 As mentioned previously, one important aspect of 18FLT is that it is not incorporated into DNA and is therefore only a surrogate marker of DNA synthesis based on TK1 activity. Factors that may confound the 18FLT uptake are changes in nucleoside transporter expression and the contributions from the salvage and de novo pathways including the removal of the phosphate by nucleotidases. Because most 18FLT is trapped inside cells as 18FLT-MP, dephosphorylation of 18FLT-MP is a possibility and needs to be accounted for during kinetic analysis. In addition, many parameters such as the plasma thymidine levels, degradation of the tracer by plasma proteins, and uncoupling of TK1 activity from the cell cycle in P53 mutated tumors46 may affect tracer uptake.
18FLT PET and Pathology Comparisons
Increased tumor proliferation has been assessed by many methods using pathologic specimens. These began using simple measurement of the mitotic index in tumors, but this simply determines the fraction of cells in M-phase and does not take into account the rate of proliferation. With the production of thymidine labeled with long-lived tracers (e.g., 3H and 14C) autoradiography was used to measure the fraction of cells in S-phase, or the labeling index (LI). This approach, although frequently used in preclinical studies, has never been routinely used clinically, because the tracers need to be injected intravenously followed by biopsy and laborious tissue processing and measurements. The approach that has gained the most use is the assessment of the nuclear antigen Ki-67 in tumors using immunohistochemical methods.47 Ki-67 is absent in cells in G0 but it is present in proliferative cells, in other cell cycle phases. This method has now become routine at many medical centers. When tumor proliferation with PET tracers, such as 18FLT, was being evaluated, among the first studies was its comparison with Ki-67 to determine the association between these approaches. Both of these approaches have been found to be highly correlated, in many cancers, such as lung cancer, but not in all tumors (Fig. 38.4).48 It is important to understand what is being measured, and the limitations of each method.
The measurement of Ki-67 is done using fixed tissues stained for immunochemistry and detects the presence of the antigen, which is associated with cell proliferation. There are technical issues associated with staining for Ki-67.49Although a number of antibodies have been developed over the years to assess Ki-67, MIB-1 has become the standard (Dako, Glosstrup, Denmark). It is routinely used by most investigators in clinical services and has been the best validated. The tissue must be fixed, stored, and sectioned in a consistent manner and then stained using an approach such as avidin-biotin with immunoperoxidase. Although the fraction of stained cells should be quantitated. The choice of areas for measurement, method of interpretation, and use of a cut-point for staining intensity all require consideration. Comparisons of Ki-67 measurements have been performed with multiple other approaches, such as histologic grade, mitotic index, and gene expression profiles (genomic grade index, [GGI]).50 Overall, in patients with node-positive breast cancer GGI appeared to provide the best marker of disease-free survival. A recent meta-analysis has compared 18FLT uptake and Ki-67 from 27 studies.51 Overall, they found the average Ki-67 expression using surgical or biopsy specimens, has high correlation with 18FLT uptake (r = 0.70, p < 0.001). The maximum Ki-67 value from biopsies did not correlate with 18FLT uptake, presumably because of sampling issues. The maximum Ki-67 from surgical specimens, however, did correlate with 18FLT uptake. They reported that in brain, lung, and breast cancer there was enough data to validate 18FLT imaging to predict Ki-67 levels.
FIGURE 38.4. Comparison of 3′-fluoro-3′-deoxy-thymidine (FLT) retention and Ki-67 measurement in patients with lung cancer. (Based on graph from Buck AK, Halter G, Schirrmeister H, et al. Imaging proliferation in lung tumors with PET: 18F-FLT versus 18F-FDG. J Nucl Med. 2003;44:1426–1431.)
ROLE OF 18FLT IN THERAPEUTIC MONITORING
The value of 18FLT-PET as a predictor of anticancer treatment response has been demonstrated in various cell culture and tumor models. Often, a decrease in 18FLT uptake in response to therapy is reported both in cell culture systems and mouse models. The sensitivity and superiority of 18FLT over 18FDG in detecting early therapeutic changes induced by cytotoxic chemotherapy, radiotherapy, or chemoradiotherapy has been well characterized.52,53Similar results were also observed with specifically targeted therapeutic agents such as Aurora kinase inhibitors that fall into the general category of antimitotic agents. Pharmacodynamic effects of antimitotic agents are somewhat difficult to assess because the traditional biomarker, the mitotic index, is different from one cell line to another in the duration of mitotic arrest as well as the mitotic slippage-based mechanism.54,55 Aurora A and B kinases are frequently expressed in human tumors. They belong to the serine/threonine kinase family and are essential for the formation of normal mitotic spindles and function of centrosomes. Inhibition or depletion of Aurora Kinase A has been shown to result in inappropriate chromosomal segregation and G2-M arrest resulting apoptosis.56 Similarly, Aurora Kinase B plays an important role in mitosis and functions in both chromosome attachment and orientation. Because of these important roles, Aurora kinases are considered important therapeutic targets and several agents are in clinical trials.56 Alisertib (MLN8237), an Aurora Kinase A inhibitor, is a potent inhibitor of cell division in vitro and shows growth inhibition in various tumor xenografts.57 In an effort to identify pharmacodynamic imaging biomarkers for alisertib therapy, Manfredi et al., assessed 18FLT-PET uptake compared to mitotic index, spindle bipolarity, and chromosome alignment assays in HCT116 colon cancer xenografts. Although there was no change in tumor volumes over the course of 3 weeks of treatment, the authors demonstrate that 18FLT uptake and, therefore, cell proliferation, significantly decreased within 7 days of treatment.57 These observations also show potential of alisertib in clinical trials. Similar observations have been made in preclinical studies with therapeutics targeting HSP90, histone deacetylase, epidermal growth factor receptor, and mitogen-activated protein kinase as summarized in Table 38.1.58–62,64–80,82–86
TABULATED SUMMARY OF FLT-PET FOR THERAPEUTIC MONITORING IN PRECLINICAL AND CLINICAL STUDIES
Although in most cases, decrease in 18FLT uptake is observed following treatment, when thymidine synthesis is blocked by antimetabolites such as 5-fluorouracil (5-FU) and methotrexate, an increase in 18FLT uptake can be observed. Though TK1-based phosphorylation is the primary trapping mechanism, it is postulated that the role of transport predominates resulting in an increase, rather than decrease, in 18FLT uptake is referred to as “flare effect.”81In response to de novo DNA synthesis inhibition such as TS inhibition that results in short thymidine supplies, tumor cells respond through a temporary compensatory mechanism by increasing the TK1 activity that may result in flare response. However, this posttherapeutic flare response window is rather short, generally 14 to 24 hours, until the thymidine pool requirement by the salvage pathway could not be met and results in DNA synthesis arrest. Indeed, significant flare response of 18FLT uptake was observed in response to 5-FU and methotrexate treatment in esophageal squamous cell carcinoma cell lines.87 A similar increase in 18FLT uptake was also observed in human breast cancers as early as 1 hour after administration of capecitabine.88
Endogenous plasma and tumor thymidine levels could also influence the tracer uptake as there is a considerable competition from the natural TK1 substrate, thymidine. This was elegantly demonstrated by Wang et al. in a series of imaging experiments correlated with metabolite measurements and ex vivo histology. These studies show that, even in rapidly growing tumors, 18FLT uptake is not proportional to TK1 expression. This discordance was caused by high intrinsic plasma or tumor thymidine levels suggesting that careful consideration should be given to plasma and tumor thymidine levels while interpreting PET images. Because most therapeutic assessment studies often use baseline scans, these interpatient differences might influence imaging results.
Another important aspect that could confound 18FLT uptake is the deregulation of TK1 from cell cycle in P53 mutated tumors. P53 is considered a guardian of the genome and many cancers harbor P53 mutations. Under wild-type p53 conditions, DNA damage leads to p53-mediated negative regulation of TK1 followed by cell cycle arrest at the G1/S-phase leading to cell death or senescence and reduced FLT uptake. However, in p53-deficient tumors, DNA damage results in halting of the cell cycle progression at the G2/S checkpoint, rather than G1/S checkpoint, allowing accumulation of TK1 in S-phase resulting in an increase in FLT uptake. These changes may result in dramatic differences in FLT uptake in p53 wild type and null tumors as illustrated by Schwartz et al. and others.46,89 These studies suggest factors confounding 18FLT uptake in tumors are not fully understood and require further investigation.
ALTERNATIVE Tdr ANALOGS FOR PROLIFERATION IMAGING
Although 18FLT is very promising as a proliferation imaging agent, it is poorly incorporated into DNA and therefore does not reflect DNA synthesis. Several 2′-substituted analogs that could be incorporated into DNA exist. 11C90,91and 18F-labeled 1-(2′-deoxy-2′-fluoro-beta-D-arabinofuranosyl)thymine (FMAU)92,93 have been studied in humans. FMAU was initially developed as an antiviral agent and is a slightly better substrate for TK2 than TK1. Similar to Tdr, FMAU is phosphorylated by Tdr kinases, and incorporated into DNA by DNA polymerases because of the presence of 3′-hydroxyl group but it is not a substrate for TP. In vitro studies have shown that the amount of FMAU incorporation into DNA was proportional to DNA synthesis.94 FMAU was initially labeled in good yield with 11C in the 5-methyl position of the pyrimidine ring using the 11CH3I chemistry. Studies in beagle dogs showed clear tumor accumulation of radioactivity and correlation with BrdU staining demonstrating potential for imaging cellular proliferation. The 18F-labeled version of FMAU was achieved through a complicated 4-step synthesis involving initial fluorination of the sugar followed by condensation with the thymine precursor. In vivo studies in normal dogs using 18F-FMAU showed substantial quantities of radioactivity in the acid precipitable large molecular fraction suggesting FMAU incorporation into DNA.92 In addition, 18F-FMAU studies in humans provide a clear visualization and uptake of activity in tumors of the brain, thorax, prostate, and bone indicating that it might be useful as a direct marker to monitor DNA synthesis.93 Although FMAU uptake is seen within the marrow of dogs, uptake within the normal human marrow has not been seen.92,93 Studies using human cell lines in culture demonstrate that FMAU retention increases as cells are stressed, whereas 18FLT retention declines in the same setting.95 This reflects the phosphorylation of FMAU by mitochondrial TK2 and suggests that FMAU may be more relevant application to measure oxidative stress rather than proliferation. Further studies are warranted.
In an effort to develop imaging agents that can accurately reflect DNA synthesis, 76Br- or 18F-labeled 1-(2′-deoxy-2′-fluoro-1-β-D-arabinofuranosyl)-5-bromouracil (FBAU) was synthesized and studied for use as a proliferation marker96–101 in mice, rats, and dogs. The rationale for studying FBAU is that, similar to Tdr and its analogs, it will be phosphorylated by cytosolic Tdr kinases and subsequently incorporated into newly synthesized DNA. Such cellular trapping of the tracer combined with labeling with a long-lived isotope (76Br; 16.7 hours) would be a good indicator of DNA synthesis for protracted studies that is not possible with currently available imaging agents. However, 18FBAU is relatively a poor substrate for TK1 in comparison to 18FLT or Tdr which results in low tumor-to-background ratios in in vivo studies.101
Recently, Toyohara et al. described the evaluation of a thymidine analog 4′-[methyl-11C]thiothymidine ([11C]4DST). In [11C]4DST, replacement of the 4′-position oxygen with sulfur was shown to prevent cleavage of deoxyribose from the nucleoside by TP conferring resistance to degradation. Initial stability studies in mice showed the molecule to be stable with >97% as the parent molecule at 60 minutes post injection. In vivo biodistribution in the EMT-6 tumor model showed the majority of activity in the spleen, duodenum, thymus, and tumor with little activity in nonproliferative tissue. Further tissue extraction studies showed 50% to 80% of the radioactivity in the rapidly proliferating tissues was present in the DNA fractions. In response to a DNA synthesis inhibitor treatment, reduced uptake in the tumor was also observed confirming the specificity of the tracer for DNA synthesis.102 11C-labeled analog was synthesized using 11C-CH3I chemistry and a tributyltin precursor. Initial clinical studies in brain tumor patients showed high tumor, bone-marrow, and liver uptake. Very little uptake was observed in the heart suggesting that 4DST is not a good substrate for TK2.103 In contrast to mouse studies, glucuronidation and high liver uptake was observed in humans with only 30% as intact molecule and several hydrophilic metabolites. In another study of 18 non–small-cell lung cancer (NSCLC) patients, correlation coefficient between SUVmax and Ki-67 index was higher with 11C-4DST than that of FDG.104 Though promising, the metabolism observed indicates that a thorough kinetic and compartmental analysis is necessary and the role of 4DST in monitoring antiproliferative therapy is yet to be established.
The TK1-based assessment of proliferative status of the tumors primarily works in cells that are in S-phase and use salvage pathway but does not account for cells that exclusively rely on de novo pathway, uncoupled cell cycle-TK1 activity or the presence of quiescence cells. Solid tumors, as they increase in size, can outgrow their blood supply and become hypoxic and nutrient deprived. During the hypoxic state, proliferative cells (P cells) can exit the cell cycle and enter a prolonged quiescence (Q) state. A quiescent tumor cell can remain undifferentiated for a long time and can be recruited back into a P-cell state once the conditions of hypoxia and/or nutrient deprivation are eliminated. Quiescence cells play a significant role in tumor resistance to therapy. Accumulating evidence suggests that σ2 receptor may serve as an imaging biomarker of cell proliferation based on its expression in proliferating cells. This receptor is a 21.5-kDa protein that has not been sequenced or cloned. Its role in proliferation is not yet clearly established. Its natural ligand is yet to be determined. The initial characterization and overexpression of σ2 receptor in cancer cell lines was demonstrated by differential binding observed with two antagonists to its sister σ1 receptor.105 The σ2 receptor is reported to be expressed 10-fold higher in proliferating cells compared to quiescent cells and is regulated in a manner similar to other proliferation markers such as Ki-67. Because tumor cells cycle between P and Q states, low radioactivity uptake in tumors as determined by σ2 receptor quantitation may provide a window into the quiescent status of the tumor cells suggesting that its expression could be used as a proliferation marker.106–108 In vitro receptor-binding studies on brain and liver tissues suggest that receptor is localized in the endoplasmic reticulum, mitochondria, and plasma membrane. Studies with σ2 receptor binding fluorescent probes in combination with cell organelle compartmentalizing tracker dyes in cancer cells show that this receptor is localized in mitochondria, lysosomes, endoplasmic reticulum, and the cytoplasmic membrane.109 Time lapsed confocal microscopy studies revealed a rapid internalization of the bound probe with nearly 40% of the receptors internalized by receptor-mediated endocytosis with T1/2 of 16 seconds which could have therapeutic implications.
FIGURE 38.5. Molecular structures of various σ2 receptor ligands and their Ki values. Where applicable, radiolabelled position on the analogs is shown as an *.
Based on the high correlation observed between σ2 receptors and proliferative status of solid tumors significant efforts have been invested into the developing imaging agents. Currently available agents fall into two broad categories namely the 9-azabucyclo[3.3.1]nonanes (granatane) and the benzamides (Fig. 38.5). Of these two, the benzamides have been more amenable to modifications and several 11C-labeled derivatives were synthesized. Initial evaluation of these analogs (Fig. 38.5B) showed high affinity for σ2 receptors. The short half-life of 11C and reduced tumor-to-background ratios observed because of high lipophilicity of the compounds underscored the need for the development of more hydrophilic 18F-labeled agents.110 Further structure–activity modifications resulted in the development of 2-fluoroethoxy analog (Fig. 38.5C), with a Ki of 6.95 ± 1.63 nM for the σ2 receptor and a σ1/σ2 selectivity ratio of 48. Biodistribution studies of this analog in female Balb/c mice-bearing EMT-6 tumor allografts demonstrated 1.14 ± 0.10% ID/g in the tumor and acceptable tumor/normal tissue ratios at 1 and 2 hours post i.v. injection. Blocking studies further confirmed that uptake was indeed σ2 receptor specific.111 This agent is currently in clinical investigation in cancer patients (NCT00968656). In addition to the analogs mentioned above, several analogs labeled with either 76Br, 123I, 125I, or 99mTc have also been synthesized and evaluated as potential PET or SPECT imaging agents for proliferative index of tumors.112–114 Fluorescent probes developed have already contributed to the understanding of the σ2 receptor localization and intracellular processing.108,109
Although uncertainties remain about the functional role of σ2 receptor in cell cycle and tumor biology, accumulating evidence suggests that its expression correlates well with proliferation status of the tumors and may provide complementary information to that of thymidine analogs.
ASSESSMENT OF TREATMENT RESPONSE WITH PROLIFERATIVE IMAGING AGENTS
18FLT PET has been utilized to help detect and assess the prognosis in patients with high-grade brain tumors.115 Grade III and IV lesions are usually visualized whereas grade II lesions may not show increased tracer retention. Measurement of the proliferative volume (PV), using an adaptive approach taking into account the signal-to-background ratio, has demonstrated information predictive of survival in patients with gliomas.116 Receiver operating characteristic curve (ROC) analysis resulted in a cut-off volume of 11.4 mL whereas patients with a PV of less than 7.4 mL had prolonged survival and those with a volume of over 24 mL had short survival and none over 2 years.
18FLT PET imaging of patients with gliomas has also been utilized to assess the response to treatment in 31 patients treated with bevacizumab usually given with irinotecan (Fig. 38.6).117,118 Imaging was done at baseline and after 2 and 6 weeks of treatment. Using an ROC analysis, the optimal cut-off was 25% to define responders based on the change in SUVpeak (1 cm circular region of interest). Both follow-up scans predicted the overall survival ( p < 0.001) although the baseline to 6-week scans appear to be slightly better at predicting overall survival (OS) (HR: 7.869 and 5.416, respectively) and improved progression-free survival (PFS). The OS was 3.3 times longer in those with a PET response (12.5 versus 3.8 months, p = 0.001) whereas comparison with MRI response, also done around 6 weeks, was significant but not as useful in predicting OS (12.9 versus 9, p = 0.05). 18FLT PET imaging at 6 weeks provided the best predictor of OS and PFS in a multivariate analysis. Although this early study is intriguing and provided a useful predictive marker, its limitations must also be considered. 18FLT does not cross the intact blood–brain barrier, hence uptake in tumors depends on breakdown of this barrier and tracer delivery, as well as trapping by TK1 within the tumor. To overcome this issue, one needs to obtain dynamic images and use kinetic modeling to fully assess the flux of 18FLT into brain tumors.43,119 Delivery of 18FLT to the tumor might be further altered because the antivascular agent bevacizumab also alters blood flow and/or vessel permeability, further altering delivery of 18FLT unrelated to proliferation. No kinetic modeling was done as part of this study to take these issues into account but the changes in simple measurement of SUV were predictive nonetheless of OS and PFS.
FIGURE 38.6. Images obtained from two patients with glioblastoma multiforme demonstrating decreased 3′-fluoro-3′-deoxy-thymidine (FLT) uptake in a responding patient when comparing baseline (A)with scans at 2 weeks (B)and 6 weeks (C). The nonresponding patient (baseline D) demonstrated no significant change in SUV on the scan at 2 weeks (E) or 6 weeks (F), although the uptake is clearly increased in the final scan. (Reprinted by permission of the Society of Nuclear Medicine from Schwarzenberg J, Czernin J, Cloughesy TF, et al. 3′-Deoxy-3′-18F-Fluorothymidine PET and MRI for Early Survival Predictions in Patients with Recurrent Malignant Glioma Treated with Bevacizumab. J Nucl Med. 2012; 53(1): 29–36.)
EGFR inhibition using gefitinib was evaluated by Sohn et al.63 using 18FLT PET for the assessment of patients with lung cancer with scans before and 7 days after the start of therapy (Fig. 38.7). In the 28 patients analyzed, they compared the results of PET to CT obtained 6 weeks after the start of therapy. SUVmax declined by a mean 36% in responders (mean SUVmax 3.2 decreased to 2) compared to a 10.1% increase in nonresponders (Fig. 38.8). Using ROC analysis, they found that a decline of 10.9% in 18FLT SUVmax, resulted in 92.9% positive and negative predictive values. 18FLT PET responders had a median time to progression of 7.9 months, compared to 1.2 months in nonresponders (p = 0.0041).
FIGURE 38.7. 3′-Fluoro-3′-deoxy-thymidine (FLT) of patients with lung cancer treated with gefitinib comparing FLT assessment 7 days after the start of therapy to CT done at 6 weeks. A: A responding patient with a decline in SUVmax from 4.8 to 2.3, and (B) a nonresponder with SUVmax increasing from 7.2 to 8. (Reprinted from Sohn HJ, Yang YJ, Ryu JS, et al. [18F]Fluorothymidine positron emission tomography before and 7 days after gefitinib treatment predicts response in patients with advanced adenocarcinoma of the lung. Clin Cancer Res. 2008, 14:7423–7429, with permission of the American Association for Cancer Research [AACR].)
FIGURE 38.8. Percent change in SUVmax by FLT PET in patients with lung cancer treated with gefitinib. Imaging was done prior to and 7 days after the start of treatment. Patients had disease status, disease progression (DP), stable disease (SD), and partial response (PR), determine on CT at 6 weeks. (Based on a graph from Sohn HJ, Yang YJ, Ryu JS, et al. [18F]Fluorothymidine positron emission tomography before and 7 days after gefitinib treatment predicts response in patients with advanced adenocarcinoma of the lung. Clin Cancer Res. 2008;14:7423–7429, with permission of the American Association for Cancer Research [AACR].)
Although 18FLT use appears promising for evaluation of EGFR inhibition, it should also be noted that 18FDG can also be used to assess such treatment.120 Imaging was done at baseline and again around days 14 and 56 after the start of erlotinib in the study by Mileshkin et al. The scans at both time points were quantitated using the SUVmax and a partial metabolic response (PMR) was defined as a decrease of equal or greater than 15%. PMR for 18FLT at days 14 and 56 was associated with improved PFS; hazard ratio for responders compared with nonresponders 0.41, p: 0.02; HR: 0.38, p: 0.02, respectively. The trend was slightly better for 18FDG PET done at the same time points (HR: 0.28, p < 0.001; HR: 0.32, p: 0.01, respectively). Furthermore, the HR for OS was significant for 18FDG scans done at day 14 (HR: 0.44, p: 0.03), but not for 18FDG at day 56 (HR: 0.40, p: 0.13) or 18FLT at either time point (HR: 0.87, p: 0.74; HR: 0.80, p: 0.62).120 Zander et al.121 also demonstrated that early 18FDG PET predicted improved PFS and OS, whereas 18FLT only predicted improved PFS.
Total lesion glycolysis (TLG) or total lesion proliferation (TLP) analyses use a combination of tumor volume and tracer accumulation to measure activity. Using a 20% decrease in TLG or 30% decrease in TLP 1 week into therapy with erlotinib for advanced NSCLC, investigators could separate those with improved PFS.122 Imaging at 6 weeks of therapy also demonstrated improved PFS for those with lower TLG and TLP, whereas baseline values were not predictive. Early SUV measurements showing low activity at 1 week with FDG and FLT were found to correlate with PFS.123 At this time, further study is needed to determine the optimal method to assess such therapies.
Head and Neck Cancer
The use of 18FLT has been studied for the detection and assessment of treatment response in patients with head and neck cancers to detect cancer and its metastases as well as the response to therapy. 18FDG PET has been regularly used in the staging and assessment of head and neck cancers and the two tracers have been compared in some studies. Cobben et al.,124 studied 21 patients evaluated for such cancers, and similar to what has been demonstrated in lung cancer, 18FDG showed an increased uptake (mean SUVmax: 3.3, range: 1.9 to 8.5). Although 18FLT could readily detect the tumors there was lower contrast (mean SUVmax: 1.6, range: 1 to 5.7). Even though 18FLT has somewhat lower background the tumor-to-normal tissue ratio was still better with FDG, mean 1.9 and 1.5 respectively). Overall both tracers were able to correctly detect the cancers in 15 of 17 patients. 18FLT had two false-positive patients, one of which was found to be associated with inflammation. In the initial development of 18FLT, it was thought that one advantage would be that there would be minimal tracer uptake in inflammatory lesions, which certainly had been a problem with 18FDG. In patients with head and neck cancer FDG uptake in inflammatory nodes in follicular dendritic cells in secondary lymphoid follicles and was associated with GLUT1 expression.125 In a study of FLT imaging prior to neck dissection increased 18FLT uptake was seen in 9 of 10 patients, but six of these were false positives based on pathology.126 In addition to PET imaging, these patients had the thymidine analog iododeoxyuridine (IUdR) injected just before surgery. Lymph nodes were evaluated by staining for IUdR and Ki-67 and they found increases in both markers in the germinal centers caused by B cells. Hence, proliferation of immune cells can also be detected with FLT imaging, as it is with 18FDG, and this limitation needs to be considered.
To be able to utilize 18FLT PET imaging to assess treatment response, the quantitative measurements must be reproducible. In general, the simple measurement of SUV has been utilized to measure treatment response, although dynamic imaging may be particularly useful if there are differences in metabolism, delivery, or clearance of the tracer. Most commonly SUV measurements are obtained about 60 minutes after injection for convenience, as is routinely done in for clinical 18FDG measurements. Fortunately, the exact timing for 18FLT imaging is not at issue, because a study has shown that the measurement of SUV at 55 to 60 minutes, obtained as part of a dynamic scan, produced uptake that was highly correlated with results obtained at around 100 minutes as part of a whole-body scan (SUVmax Spearman’s ρ:0.97, p < 0.0001).127 Furthermore, SUV measurements were found to correlate with both kinetic analysis using compartmental models and graphical (Patlak) approaches (correlation coefficient: 0.91 and 0.99, respectively).128 In this study they also compared baseline scans to those obtained after 1,000 cGy along with chemotherapy in seven patients. They found that the SUV declined from a mean of 2.47 to 1.66 (p< 0.01) and the results correlated with those obtained with Patlak analysis (R2 = 0.99). Kishino et al.129 obtained 18FLT and 18FDG assessment prior to therapy in patients with head and neck cancer and after 4 weeks of chemoradiotherapy and then again 5 weeks after treatment was completed. Imaging results were compared to routine clinical follow-up and pathology, when biopsies were done. Midtreatment 18FLT had improved specificity (72%) and accuracy (74%), whereas the specificity of 18FDG was 19% and accuracy 30%. On the other hand, both 18FLT and 18FDG imaging proved to be predictive of 3-year control when comparing those lesions with and without tracer uptake at the end of treatment. The recurrence rates were slightly higher in those visible with 18FDG (68.7%) compared to 18FLT (45.8%). Further study is needed to demonstrate if PET, with either tracer, can be used to modify the planned treatment course.
The ability to predict the response to neoadjuvant therapy with rectal cancer would be helpful in determining subsequent therapy, including the need for further surgery and chemotherapy. 18FLT PET done before and two cycles into chemoradiotherapy was not found to be predictive of pathologic response in a study of 10 patients.130 In another study of 14 patients imaged before and about 2 weeks after the start of chemoradiotherapy the change in tracer retention did not predict those who had complete or partial pathologic response compared to nonresponders (Fig. 38.9A).131 On the other hand, patients with ≥60% decline in 18FLT retention had improved disease-free survival (p < 0.05), as did those that had SUVmax <2.2 during therapy (p < 0.05) (Fig. 38.9B). The ability to predict long-term outcome in such patients needs to be verified, but could provide useful information to help direct treatment.
FIGURE 38.9. Assessment of rectal cancer with 3′-fluoro-3′-deoxy-thymidine (FLT) PET obtained before and after about 2 weeks of therapy. A: Change in FLT retention did not predict pathologic response. B: Disease-free survival was significantly improved in patients with ≥60% decrease in FLT retention. (Based on Dehdashti F, Grigsby PW, Myerson RJ et al. Positron Emission Tomography with [(18)F]-3′-Deoxy-3′fluorothymidine (FLT) as a Predictor of Outcome in Patients with Locally Advanced Resectable Rectal Cancer: A Pilot Study. Mol Imaging Biol. 2013;15:106–113.)
18FLT PET has primarily been studied for use in the assessment of treatment response in patients with both metastatic and locally advanced breast cancer undergoing neoadjuvant chemotherapy. The latter setting has the advantage of allowing one to compare imaging results to the “gold” standard of pathologic response in a relatively short period of time. Given the low 18FLT uptake in normal breast and chest wall tissues, imaging and analysis becomes relatively straightforward (Fig. 38.10).132 On the other hand, there are a number of approaches that can be used to quantitate the changes with chemotherapy. Lubberink et al.133 compared SUV measurements, tumor-to-whole blood ratio (TBR), and influx rate using a three-compartment model and Patlak analysis. Fifteen patients were imaged before and after the first cycle of therapy. The uptake rate by nonlinear regression (NLR) from the compartmental model was highly correlated with Patlak, SUV, and TBR measurements (Spearman’s ρ ≥ 0.96). They also compared the relative change in tracer retention using each approach and found that change in NLR was highly correlated with SUV (0.96), TBR (0.93), and Patlak (0.81). Changes in SUV, however, demonstrated a slight bias and slope of less than one (0.69, CI: 0.57 to 0.88) compared to TBR (0.82, CI: 0.56 to 1.13). Although TBR may have some advantages, the slight bias with SUV is not likely to cause a problem in assessment of response.
Pio et al.134 studied 14 patients before and after the first cycle of treatment in patients receiving chemotherapy or hormonal therapy primarily for metastatic disease. They demonstrated that the change in 18FLT uptake correlated well with the percent change in the tumor marker CA 27.29 (r = 0.79, p = 0.001) and in tumor size (r = 0.74, p = 0.01). In a study of 17 patients the reproducibility of 18FLT imaging was obtained with two scans prior to therapy and then again about 1 week after the start of treatment.135 18FLT uptake was quite reproducible when measured by SUV (mean percent difference SD: 10.5%) and 18FLT flux (15.1%) with a high correlation (≥0.97). Both approaches found that 18FLT assessment could predict response (p = 0.022). In another study FLT PET done before and mostly after one cycle of docetaxel demonstrated that the decline in SUV correlated with change in size after three cycles of treatment.136
Another use of 18FLT imaging is to assess pharmacodynamic changes in patients undergoing therapy that will interfere with thymidylate synthase (TS) activity.88 Six patients with breast cancer were imaged before and then 1 hour after the first dose of capecitabine, a TS inhibitor. Because TS inhibition leads to decreased thymidine production, they found increased 18FLT SUV and flux parameters in most lesions consistent with tumor working to compensate by increasing the use of exogenous thymidine via the salvage pathway. Further studies of this approach are needed to determine if this will be predictive of treatment response.
FDG has been the radiopharmaceutical of choice for PET for more than three decades. At this juncture in time, it is safe to say that 18FLT is gaining ground in oncology as an alternate choice for diagnosis and therapeutic response monitoring. This is because of the importance of proliferation in assessing the tumor metabolic activity, 18FLT uptake sensitivity to proliferative state of the tumor and the commercial availability of the agent. Even in the era of targeted therapeutics, it is clear that proliferation imaging has a significant role for therapeutic monitoring. This is more so in monitoring a combination therapy response where it may not always be feasible to follow one or both of the targeted pathways using respective pathway specific imaging agents. Studies described earlier in this chapter also underscore the importance of serum or tumor endogenous thymidine levels and other factors that need to be taken in to account in the interpretation of tumor 18FLT or any other thymidine analog uptake. As our understanding of the effectors of 18FLT uptake improves, so will its effective use in oncologic imaging.
FIGURE 38.10. 3′-Fluoro-3′-deoxy-thymidine (FLT) PET of a patient with locally advanced breast cancer image before, after one cycle of chemotherapy, and 5 months after the start of treatment and just prior to resection. Pathology demonstrated persistent cancer. Courtesy of Dr. Anthony Shields.
Although a number of proliferative tracers have been developed, none is yet available for routine clinical use. This reflects the common gulf in moving a new imaging agent from the laboratory, preclinical work, and limited clinical studies to the larger trials needed for government approval, commercial distribution, and routine clinical adoption. Only 18FLT is widely available and studied at many PET centers worldwide as part of research studies. In the United States, 18FLT is supplied commercially by vendors and it has been utilized in a cooperative group sponsored trial in the evaluation of neoadjuvant treatment for breast cancer. There have been ongoing discussions with the Food and Drug Administration on the studies and steps needed to file a successful new drug application. One essential component of such an application is a study or studies that demonstrate the utility of such an imaging approach in the selection and monitoring of patient therapy. Further work is now being done to provide the needed data. Many investigators and pharmaceutical companies are presently utilizing proliferative imaging to understand cancer and develop new treatments, hopefully demonstrate the best approaches to incorporating these imaging approaches into routine care.
This work was partially supported by the National Cancer Institute grants CA148722 (AFS) and CA22453 (AFS) and by the Elsa U. Pardee Foundation and CA166131 (SN).
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