**4. Choline-based probes**

In recent years, choline metabolism has received a growing interest in cancer research. Choline is incorporated into membrane phospholipid in the form of phosphatidylcholine through the multistep Kennedy pathway. Phosphatidylcholine is one of the major lipid components of plasma membranes in mammalian cells and is essential for membrane structural stability and cell proliferation. Following its transport into the cell, choline undergoes ATP-dependent phosphorylation to form phosphocholine, a reaction catalyzed by choline kinase. High levels of choline uptake and increased choline kinase activity relative to normal tissues have been reported in various cancers including brain tumors (Fulham et al. 1992). This has motivated the development of choline based PET imaging for noninvasive evaluation of gliomas.

Shinoura et al. (1997) were the first to use choline as a PET tracer of brain tumor imaging. They evaluated 20 patients with brain tumors using [11C]choline PET. Progressive uptake of [11C]choline was observed in brain tumors, while uptake by surrounding normal cerebral cortex was 10-fold lower. Later, Ohtani et al. (2001) compared [11C]choline-PET with [18F]FDG-PET in 22 patients with histopathologically confirmed benign lesions and brain tumors from grade I-IV. Higher uptake of [11C]choline relative to [18F]FDG was observed in high-grade grade III and grade IV gliomas. Furthermore, [11C]choline was able to detect the extent of tumor better than MRI and could differentiate high-grade from low-grade lesions, but could not differentiate low-grade lesions from benign lesions.

The short half-life of [11C]choline limits the use of this tracer to facilities having an on-site cyclotron. In view of this, a choline radiotracer with a longer half-life is highly desirable. [18F]Fluorinated analogs of choline [18F]FCH are promising options for choline based PET tumor imaging.

DeGrado et al. (2001) first reported brain tumor imaging with [18F]FCH in a patient with previously resected anaplastic astrocytoma. The maximal T/N ratio of 10:1 was attained within 5 min after injection. [18F]FDG -PET revealed a corresponding area of increased [18F]FDG uptake; however, the tumor boundaries were difficult to assess with FDG because of high uptake by normal cortex. Hara et al. (2003) performed studies with [18F]fluoroethylcholine (FECH) in 12 glioma patients. The T/N ratio of [18F]FECH was 10.5- 12 in anaplastic astrocytoma and 13.2-21 in glioblastoma. These ratios were slightly higher

PET Imaging of Gliomas 169

with tumor progression and invasiveness of gliomas (Bello et al. 2001). Therefore, this

The most common integrin-targeting radiopharmaceuticals are radiolabeled RGD containing peptides. Cyclic RGD peptides (cRGD peptides) are preferred over linear RGD peptides due to their higher metabolic stability (Bogdanowich-Knipp et al. 1999). However, radiolabeled cRGD peptides (18F-FB-cRGDs (Chen et al. 2004) and 64Cu-DOTA-cRGDs (Chen et al. 2004)) commonly suffer the drawback of poor tumor retention and high renal and/or hepatic uptake. To improve the tumor imaging properties of these tracers, dimeric (Chen et al. 2004) and tetrameric (Wu et al. 2005) cyclic RGD congeners have been developed. The multimeric cyclic RGD probes showed higher tumor uptake but nevertheless exhibited rapid uptake by liver and kidneys. To decrease uptake in liver and kidneys, modifications such as glycosylation (Schnell et al. 2009) or PEGylation (Chen et al. 2004) have helped to achieve higher uptake in tumors along with decreased uptake in liver and kidney in U87MG glioblastoma models (Chen et al. 2004; Chen et al. 2004; Wu et al. 2005) and in patients with

New developments to integrin imaging are in progress. Recently, a new generation of RGD containing probes have been designed that show better potential for imaging gliomas. The new approach is based on cystine knot proteins or knottins that are relatively stable in physical, chemical and biological environments due to presence of scaffold of disulfidebonded framework and a triple-stranded ß-sheet fold (Kimura et al. 2009). The Knottin family members also possess one or more surface-exposed loops that can tolerate sequence diversity. RGD peptides have been grafted into these surface-exposed loops, while radiolabeling accomplished with conjugation of PET radionuclide, 18F-FB or 64Cu-DOTA. In a U87MG glioblastoma model, a knottin based probe demonstrated rapid and high tumor accumulation, fast clearance from blood and normal organs, and low uptake in the kidney

Increased cellular proliferation is an integral part of the cancer phenotype (Bading and Shields 2008). The primary requirement for cell proliferation is replication of nuclear DNA. Of the 4 nucleotides (adenine, guanine, cytosine and thymidine) required for DNA synthesis, thymidine is the only one that is specific for DNA. In cells, thymidine is derived either *de novo* or through the salvage pathway (Bading and Shields 2008). The *de novo* pathway is not a viable alternative for monitoring DNA synthesis in proliferating cells because the relevant precursors (deoxyuridine, uridine, and uracil) are routed into both DNA and RNA (Bading and Shields 2008). Thus, thymidine salvage pathway is a better choice for indication of proliferation. PET radiotracers of thymidine have been developed, including [11C]thymidine ([11C]TdR) (De Reuck et al. 1999) and 3'-deoxy-3'-fluorothymidine ([18F]FLT). These probes are currently being evaluated for their potential for imaging cellular proliferation in gliomas. The thymidine-based tracers are transported into cells and subsequently phosphorylated by thymidine kinase-1 (TK-1), thereby rendering them trapped within the cell. Since TK-1 activity correlates to a significant extent with the cellular proliferation rate, the PET-measurement of tissue retention of radioactivity is a noninvasive indicator of proliferation. The advantage of using the fluorinated thymidine tracer [18F]FLT over [11C]TdR is twofold. First, replacement of the hydroxy group of

integrin has received attention as a target for imaging probe development.

*de novo* or recurrent GBM (Schnell et al. 2009).

and liver (Miao et al. 2009).

**6. Cellular proliferation probes** 

than those obtained with [11C]choline in the same patients. A preliminary study by Kwee et al. (2004) in 2 patients suggested that [18F]FCH uptake was significantly higher in glioblastoma multiforme (GBM) than benign demyelinating disease. Subsequently, Kwee et al. (2007) performed a more extensive [18F]FCH PET study on 30 consecutive patients (14 women, 16 men; age range, 26-79 years) with solitary brain lesions defined by MRI. In this study, the order of SUV and T/N ratios were benign lesions < high-grade gliomas < metastases from distant tumors with appreciable separation of these classes. [18F]FCH is also useful in detecting recurrent GBMs (**Figure 5**).

Pre-clinical studies on the *in vivo* kinetics and metabolism of [18F]FCH and choline in a 9L glioma allograft tumor model showed marked washout from the tumor possibly resulting from the hypoxic nature of these tumors. However, a strong association of [18F]FCH uptake and angiogenesis was found in the C6 glioma xenograft model (Wyss et al. 2007) and increased [18F]FCH uptake was observed in a multidrug resistant U87MG glioma tumor model (Vanpouille et al. 2009).

Fig. 5. [18F]FCH uptake in recurrent GBM. PET/CT (left) and [18F] FCH-PET (right) image shows a 6 mm focus of increased [18F]FCH uptake along a right frontal lobe resection cavity. This lesion was noted to increase in size on serial brain MRI consistent with the diagnosis of recurrent tumor. A post-craniotomy defect is evident on the PET/CT image. [18F] FCH-PET is potentially advantageous for imaging brain tumors such as GBM given the low amounts of physiologic cerebral [18F]FCH uptake. *Image Courtesy of Dr. Sandi A. Kwee, MD, Nuclear Medicine Department, Queen's Medical Center, Honolulu, HI, USA.* 
