Neuroendocrine tumors (NET) are neoplasms characterized by tissue immunoreactivity for neuroendocrine differentiation markers, appearance as a small mass that can be off white to yellow often in submucosa and prevalence throughout the body but typically found in the intestine or lungs (Oberg2011). The tumors can be malignant and are typically detected through hormone markers in a first diagnosis (Arnold 2003). Because of the diffuse nature of this type of tumor, detecting and imaging NETs requires scanning and typically requires a contrast agent to detect the location of tumors in tissue (Tan 2011). Many NET cells possess an overexpression of somatostatin receptors on their surface (Kwekkeboom 2005, VenEssen 2007). Somatostatin receptors are cell surface proteins that bind to somatostatin, a growth hormone inhibiting hormone that regulates the endocrine system and affects neurotransmission and cell proliferation (Florio 2002). The overexpressed somatostatin receptors can be used to both detect these tumors with image contrast agents in scanning and for targeting the tumors with chemotherapeutic agents (Forrer 2006). Somatostatin has a 3-5-minute half life in circulation which limits the application of this molecule for detection and treatment of NET cells; synthetic analogues have been developed to target the overexpressed receptors (Atunes 2007).
Imaging with a radionucleotide requires a high degree of specificity for the target molecule at the receptor site or non-targeted tissue will exhibit uptake. The synthetic somatostatin analogue and chelating agent complexed to short peptide, DOTA-TOC [N-(4,7,10-(tris (carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl) acetyl-D-Phe-c[Cys-D-Tyr-Trp-Lys- Thr-Cys]-Thr (ol))] has been developed as a molecule that both targets the overexpressed receptors and allows rapid and strong chelation to the radio nucleotide 68Ga. DOTA or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid is a cyclic polydentate chelating ligand with four secondary amine groups and four acetic acid pendants. The molecule can be linked to phenylalanine-tyrosine and then a short peptide where it behaves as a strong somatostatin analogue showing
: DOTATOC. DOTA is the cyclic molecule containing four secondary nitrogen atomspreferential binding to somatostatin receptors. The resulting molecule has a strong binding site for several transition metals including Gallium (Ga3+). The radionucleotide, 68Ga, has been shown to act as a high contrast agent in positron emission tomography (PET) scans while allowing formation with a conventional nuclear formation rather than more expensive cyclotron formation (Fani 2008).
Preliminary research showed that the DOTA-TOC somatostatin analogue showed preferential binding for somatostatin receptors and the potential to target the overexpressed somatostatin receptors found in NET cells with these molecules coordinated to a 68Ga3+ atom could provide a high contrast agent for PET imaging with high specificity for tumor cells within tissue (Gabriel 2007). Biokinetics and preclinical animal imaging demonstrated a 3-fold affinity for multiple somatostatin receptor types with superior specificity and imaging under PET scanning (Antunes 2007). PET has been shown to offer higher resolution and improved pharmacokinetics as compared to somatostatin scintography (Chiti 2000).
Preclinical animal studies have been performed on multiple species for biocompatibility, biodistribution, and small animal PET studies. Biodistribution studies were performed on mice implanted with murine melanoma cells that were grown for 10 days and control groups (Velikyan 2012). Similar animals were studied for PET scanning. Testing the tumor specificity with a control group that involved pre-administration of a blocking dose of unlabeled DOTA-TOC significantly reduced uptake of the labeled imaging agent coupled to 68Ga3+. Fast clearance was shown for the molecule with tissue in the blood, liver, heart, lung and muscle tissue clearing the background radioactivity to very low levels 2 hours post injection (Velikyan 2012). Some non-specific kidney uptake has been shown which can hinder the specificity in this organ, however the injection of lysine with the 68 GaDOTA-TOC allowed tumor uptake without significant renal accumulation (Froidveaux 2003). Small animal PET imaging studies showed significant accumulation of 68Ga within implanted B16 melanoma tumors indicating that 68 GaDOTA-TOC is a selective target for imaging melanoma in vivo. Standard uptake values for the target followed trends in bioavailability that were determined in an acute study demonstrating that the uptake in liver, kidney, and muscle were lower than that of the tumor even at a non-acute dose (Hofmann, 2001).
Preclinical animal studies and toxicology studies on 68 GaDOTA-TOC showed promise toward the application of this conjugate for PET imaging of NET cells in human tissue since the specificity and biocompatibility showed efficacy in tumor detection and corresponding low toxicity. The first human studies of 68 GaDOTA-TOC compared the results with 111In-DTPAOC SPECT were performed on patients diagnosed with NET and a comparison was made between the two molecules (Kowalski 2003). In this study, twenty seven patients were imaged with both agents. Findings showed that both agents had similar SUV's for non-tumor tissue, however, the 68 GaDOTA-TOC showed a higher specificity and the findings led one case to have a change in surgical intervention plan for subsequent treatment (Kowalski 2003).
The use of 68 GaDOTA-TOC as an effective PET diagnosis system has been tested along with subsequent treatment with receptor-mediated Radionucleotide therapy. A study on 84 patients showed that 68 GaDOTA-TOC had better efficacy than the diagnosis obtained with scintography and diagnostic CT scans (Gabriel 2007). Seven of the patients scanned within this study were selected to receive 90 YDOTA-TOC or 177 LuDOTA-TOCate on the basis of very strong uptake of the 68 GaDOTA-TOC tracer as observed in the imaging scan.
A larger study of 68 GaDOTA-TOC evaluated the specificity for the tracer in PET scanning observing the specificity again comparing to the tracer, 111 InDOTA-TOCate (Buchmann 2007). The study showed high specificity for tumors with a high expression of receptors:
Gastro enteropancreatic tumors
Sympatho adrenal system tumors
Medullary thyroid carcinoma
Merkel cell carcinoma
Small cell lung cancer
Tumors with low expression of receptors that did not respond well to 68 GaDOTA-TOC imaging included:
Non-small cell carcinoma
The conjugate peptides could be used to determine the location of primary tumors and secondary metastases, determine SST receptor status level in tumors to allow for secondary therapy decisions, and monitor response to therapy. The 68 GaDOTA-TOC were rapidly cleared from the blood and no radioactive metabolites were detected in serum or urine within 4 hours of administration. Maximum tumor uptake was observed between 50 and 90 minutes (Buchmann 2007).
GaDOTA-TOC has been used in the detection of bone metastases in patients with NET's (Putzer 2009). In this study, fifty one patients with verified NETs were scanned with diphosphonate bone scans and 68 GaDOTA-TOC PET scans. The results showed the 68 GaDOTA-TOC had a sensitivity of 97% and a specificity of 92% which was a significantly higher rate compared to the phosphate bone scan.
Clinical administration of 68 GaDOTA-TOC should be performed using an indwelling catheter and the activity administered should ranger from 100 to 300 MBQ and the amount of 68 GaDOTA-TOC injected should be below 50 micrograms. Generation of the 68Ga is performed in strong HCl solutions typically with anion exchange with testing of purity and sterility used with the preparation kit (Decristoforo 2007). Image acquisition is performed using a dedicated PET/CT scanner with the timing for imaging between 60 and 90 minutes post administration (Pettinato 2008).
Normal biodistribution and abnormal accumulation should be evaluated by a physician trained in diagnosing medical images in nuclear medicine. Accumulation higher than background is considered to be pathological while non-focal intestinal uptake is non-pathological. 68 GaDOTA-TOC may show variable sensitivity in different tumor types and uptake can occur with high SST receptors rather than malignant disease such as activated lymphocytes at locales of inflammation.
Continued investigation of 68 GaDOTA-TOC as a diagnostic for the evaluation of NETs is of critical importance to the development of new therapies. The somatostatin receptor specificity for the agent combined with new therapies in delivering targeted agents for radiation or conventional chemotherapies of molecules bound to DOTA-TOC allows for strong potential in treating NET metastases.
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