Initial Clinical Experience with 68Ga-DOTA-NOC Prepared Using 68Ga from Nanoceria-polyacrylonitrile Composite Sorbent-based 68Ge/68Ga Generator and Freeze-dried DOTA-NOC Kits.

Somatostatin receptor positron emission tomography-computed tomography (PET/CT) with 68Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) peptides have become an indispensable part of disease assessment in patients with neuroendocrine tumors and forms the basis of personalized therapy with peptide receptor-based radionuclide therapy. With growing utilization of PET/CT in developing countries, availability of the indigenous GMP-certified 68Ge/68Ga generators is expected to further promote cost-effective molecular imaging service to the cancer patients. We present our initial clinical experience in 32 patients injected with 68Ga-DOTA-NOC prepared using 68Ga eluted from Bhabha Atomic Research Centre nanoceria-polyacrylonitrile sorbent-based 68Ge/68Ga generator and freeze-dried DOTA-NOC cold kits.

Introduction

Widespread availability of positron emission tomography–computed tomography (PET/CT) along with the development of many radiolabeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated peptides for imaging and therapy of neuroendocrine tumors (NETs) has led to significant progress in the rapidly evolving field of theranostics.[1] In a major step toward providing a cost-effective molecular imaging service to the increasing demands of many nuclear medicine centers in India, Bhabha Atomic Research Centre (BARC), Mumbai, has developed a nanoceria-polyacrylonitrile (CeO2-PAN) composite sorbent-based 68Ge/68Ga Generator and single vial freeze-dried cold kits of DOTA-NOC for instantaneous labeling with 68Ga from the generator without the need for automated systems.[23]

Compared to the other available commercial generators, this novel generator is reported to have consistent 68Ga yield, minimal metal impurities, and very low 68Ge breakthrough when long-term elution profiles were studied.[4] Another advantage is that it provides 68Ga of requisite quality without the need for postelution processing. Hence, such cost-effective, GMP-compliant generator could be easily used in clinical settings in hospital radio-pharmacy like the 99Mo-99mTc generators.[123]

We present our initial clinical experience in 32 patients injected with 68Ga-DOTA-NOC prepared using 68Ga eluted from BARC CeO2-PAN sorbent-based 68Ge/68Ga generator and freeze-dried DOTA-NOC cold kits for various clinical indications.

Materials and Methods

Materials

DOTA-NOC was procured from ABX, Germany. Sodium acetate and Suprapure HCl were purchased from Fluka, USA, and MERCK, Darmstadt, Germany, respectively. All reagents were prepared using sterile high-performance liquid chromatography (HPLC) grade water (Merck, India). Sterility test kits were obtained from Himedia Laboratories, India, and Endosafe PTS equipment and cartridges were from Charles River Laboratories Pvt. Ltd., India. ITLC SG paper was from Agilent Technologies, USA, whereas Whatman 3 mm chromatography strips were from Whatman, UK.

Radioactivity measurements were made using a NaI (Tl) counter (ECIL, India). An HPLC system (JASCO, Japan) equipped with a C-18 reversed phase column coupled to an ultraviolet/visible detector and a NaI (Tl) radioactivity detector (Raytest, Germany) was used for characterization of radiolabeled peptides. Alpha 1–2 LD plus freeze dryer was purchased from Martin Christ, GmBH while 0.22 μm membrane filters (33 mm) were from M/s. Millipore Corporation, Bedford, Massachusetts, USA. PET/CT studies were acquired on Philips Medical Systems, Ohio, USA GEMINI TOF 64.

Methods

Development of 68Ge/68Ga generator

The CeO2-PAN sorbent was prepared by the decomposition of the cerium oxalate precursor to cerium oxide followed by incorporation in the PAN matrix, as reported earlier.[2] To fabricate a 68Ge/68Ga generator, a borosilicate glass column of dimension 7 cm × 0.8 cm (id) with a sintered disk (G2) at the bottom was packed with 0.5 g of the sorbent, in a lead shield. The column matrix was conditioned at pH 3 by passing 100 mL of 0.001 M HCl solution, at a flow rate of ~2 mL/min. The loading solution (100 mL) containing 740 MBq (20 mCi) of 68Ge maintained at pH ~3 was allowed to percolate into the column at a flow rate of 0.5–1 mL/min. The column was then washed with 200 mL of 0.1 M HCl (pH 1) solution.

Formulation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-NOC kits and radiolabeling with 68Ga

Single vial kits of DOTA-NOC were formulated in 0.5 M sodium acetate, freeze dried, vacuum sealed under sterile conditions and stored at −20°C, as reported elsewhere.[35] Aseptically prepared 0.1 N HCl was provided with the kits for elution of 68Ga from the generator. Quality control of the kits was carried out by evaluating their sterility and apyrogenicity, as per the guidelines of Indian Pharmacopeia. Radiochemical yield (RCY) and radiochemical purity of 68Ga-DOTA-NOC were determined by paper chromatography (PC) as per the reported procedure.[35] For preparation of patient dose of 68Ga-labeled DOTA-NOC, 68Ga Cl3 from the nanoceria-PAN 68Ge/68Ga generator was eluted using 1 mL of sterile 0.1 N HCl directly into DOTA-NOC cold kit vial. Reaction was carried out at 90°C for 10 min. RCY of 68Ga-DOTA-NOC was estimated by PC. 68Ga-DOTA-NOC was then filtered through 0.22 μ sterile filter, diluted with sterile saline (2 mL), and injected into the patients.

Clinical studies

We retrospectively studied 32 patients from June to July 2013 (age 16–73, mean 52) wherein 68Ga-DOTA-NOC PET/CT was done as part of their treatment management. After obtaining a scout image, breath hold CT was acquired followed by whole body CT and then PET acquisition. PET acquisition time was 2 min/per bed position. CT parameters for breath-hold CT includes slice thickness 3 mm, pitch 1.08, field of view – 356 mm, voltage – 120 kV with automated mA correction, image matrix – 512 mL × 512. 80 mL of low osmolar nonionic intravenous contrast was administered in all eligible patients at a rate of 1.8 mL/s and scan delay was 50 s. Images were reconstructed iteratively using RAMLA algorithm. Images were viewed on display system having extended brilliance workspace software (EBW) version 4.5.3.40140 (Netherlands), Philips Healthcare. In addition to the PET/CT, all patients underwent 99mTc-hydrazinonicotinyl-Tyr3-octreotide (99mTc-HYNIC-TOC) whole body planar scan with single photon emission CT (SPECT) acquisition on Infinia, Hawkeye, GE Healthcare, as standard of care. Relative performances of PET and SPECT studies were analyzed. All studies were acquired with prior informed consent from patients.

Results

The generator was able to provide 68Ga activity (in the form of 68GaCl3) in consistent yields and acceptable radionuclidic purity (<10 − 4% of 68Ge breakthrough). Schematic of 68Ge/68Ga Generator is depicted in Figure 1. DOTA-NOC cold kits each consisting of 50 μg of the peptide conjugate in 0.5 M sodium acetate (13 mg) could be successfully formulated. The formulated kits were found to be of pharmaceutical grade as per sterility tests and BET tests. Greater than 95% radiolabeling yields could be obtained on radiolabeling with 68Ga, as ascertained by the chromatography techniques. Specifications of DOTA-NOC kit and 68Ga-DOTA-NOC are given in Table 1.

Figure 1

Schematic diagram of Bhabha Atomic Research Centre 68Ge-68Ga generator

Table 1

Generator and cold kit performance parameters

68Ga-DOTA-NOC PET/CT was positive in 22/32 patients (12 – initial characterization/staging/diagnosis, 10 – restaging) [Figures 2 and 3]. PET/CT was negative in 10/32 patients (4 – medullary Ca thyroid, 1 – suspected Insulinoma, 3 metastatic NET with MiB index, 1 – Ectopic Cushing's syndrome, 1 – thymoma) [Tables 2a and b]. Number of lesions identified on 68Ga-DOTA-NOC PET/CT was 142 and on the whole body 99mTc-HYNIC-TOC scintigraphy was 85 [Table 3].

Figure 2

68Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-NOC positron emission tomography–computed tomography of a 54-year-old patient, showing maximum intensity projection (a), transaxial positron emission tomography (b) and fused positron emission tomography–computed tomography (c) showing focal increased tracer uptake in the left glomus jugulare (white arrow)

Figure 3

Case of solitary liver metastasis from an operated case of duodenal neuroendocrine tumor showing fused images of negative 99mTc-hydrazinonicotinyl-Tyr3-octreotide single photon emission computed tomography/computed tomography (a) study and positive 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-NOC positron emission tomography–computed tomography (b, white arrow) study

Table 2a

Indications and results of 68Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-NOC positron emission tomography–computed tomography for initial characterization

Table 2b

Indications and results of 68Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-NOC positron emission tomography–computed tomography for follow-up evaluation

Table 3

Comparison of relative performance of 99mTc-hydrazinonicotinyl-Tyr3-octreotide TOC single photon emission computed tomography and 68Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-NOC positron emission tomography–computed tomography for lesions detection

Discussion

Molecular imaging with 68Ga radiopharmaceuticals is rapidly evolving. Somatostatin receptor (SSTR) targeting 68Ga-DOTA peptides have been successfully investigated in multiple clinical indications such as neuro-endocrine tumors, para-gangliomas, neuroblastomas, meningiomas, and oncogenic oseteomalacia.[6] Beyond SSTR imaging, promising clinical studies have emerged using 68Ga with biomolecules such as PSMA, bombesin, and macroaggregated albumin.[789]

The first and foremost important clinical indication for 68Ga-DOTA peptides have been NETs. NET represents a subset of tumors originating from enterochromaffin cells of the foregut, midgut, or hindgut. Survivor, Epidemiology and End results database has shown rise in the incidence of NET for the past few decades, probably related to the increased detection rate.[10] Prognosis of this disease is highly dependent on stage with 5-year survival in localized disease above 905 and 5-year survival about < 30% in patients with distant metastasis.[11] Knowing the tumor biology, its location and extent through imaging has been to improve patient's treatment outcomes.[12] SSTR PET/CT is proven to be diagnostically superior to conventional scinitigraphy and CT and used extensively for staging, restaging and assessing response to treatment in patients with NET.[13] 68Ga-DOTA-NOC PET/CT impacts management of NET patients by modifying treatment in over 50% of referred patients.[14] 68Ga-SSTR-PET/CT forms the basis of selecting patients for PRRT, a new and promising treatment for NET patients. PRRT has shown response rates of 25–30% with comparable progression-free survival and overall survival with other available medical treatments.[15]

The increasing need of 68Ga-labeled molecules for the above-mentioned oncological and nononcological indications has propelled the development of many GMP-certified 68Ga-Generators. Despite excellent advances of present-day 68Ga-radiopharmacy, it is pertinent to point out that majority of the commercially available 68Ge/68Ga generators have several shortcomings and are not directly amenable for use in clinical context.[1617] To circumvent these limitations, “state-of-the-art” automated modules have been developed over the last few years for postelution processing of 68Ga and subsequent radiopharmaceutical preparation.[1819] However, these automated modules are highly expensive and beyond the reach of most nuclear medicine departments, especially in developing countries. Therefore, we have indigenously developed a 68Ge/68Ga generator using CeO2-PAN sorbent which could directly be used for the preparation of radiopharmaceuticals. Recently, Isotope Technologies Garching GmBH has launched a new GMP-certified 68Ge/68Ga generator in the clinical market that utilizes pyrogallol-derivatized SiO2 as an adsorbent and is directly amenable for clinical use. A detailed performance evaluation of both these generators was recently carried out by our group, and the performances of these two generators were found to be comparable.[20] Thus, we could ensure that our indigenously developed generator is suitable for clinical use.

To achieve the goal of making 68Ga as the “work-horse” of PET, just as 99mTc is for SPECT, it is prudent to develop cold kits containing precursors in freeze-dried form for extemporaneous preparation of 68Ga radiopharmaceuticals. Our group has demonstrated feasibly of preparation of 68Ga radiopharmaceuticals utilizing cold kits of peptide conjugates.[35] Herein, we report our first clinical experience with 68Ga-DOTA-NOC prepared using 68Ga from the indigenous generator and freeze-dried cold kits of DOTA-NOC. 68Ga-DOTA-NOC in consistently high yields could be prepared in 10 min and within 35 min, radiolabeled DOTA-NOC was ready for injection into the patients after quality control analysis. Moreover, availability of cold kits independent of 68Ga generator provides more control over the reaction parameters compared to automated modules for 68Ga radiopharmaceutical preparation.

We analyzed the application of 68Ga-DOTA-NOC imaging for a wide variety of indications, which included pulmonary/gastro-enteropancreatic NETs, paragangliomas, ectopic ACTH syndrome, suspected meningioma, and tumor-induced osteomalacia. PET/CT showed intense uptake in two patients with paraganglioma and two patients with suspected meningioma, supporting the increasing use of PET/CT for these indications. PET/CT was successful in localizing the primary tumor in four patients (1 – tumor induced osteomalacia, 3 – metastatic NET of unknown primary). PET/CT was negative in 10 patients (4 – medullary Ca thyroid, 1 – suspected insulinoma, 3 metastatic NET with high proliferation index, 1 – ectopic Cushing's syndrome, 1 – thymoma). Compared to the standard imaging for NET, 99mTc-HYNIC TOC, significantly more number of lesions was identified on 68Ga-DOTA-NOC PET-CT. However a change of management was noted in only one patient, which could be explained by small number of patients and heterogeneous clinical indications in our study.

Conclusion

The first clinical experience with BARC's CeO2-PAN sorbent-based 68Ge/68Ga Generator and freeze-dried DOTA-NOC kits was found to be satisfactory in various clinical indications. Larger studies can be considered, to promote its widespread use as a cost-effective molecular imaging agent for the assessment of NETs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.