GMP-conform production of [ 68Ga ]Ga-NeoB for positron emission tomography imaging of patients with gastrointestinal stromal tumor

Background: [ 68 Ga]Ga-NeoB is a novel DOTA-coupled Gastrin Releasing Peptide Receptor (GRPR) antagonist with high anity for GRPR and good in vivo stability. This study aimed at (1) the translation of preclinical results to the clinics and establish the preparation of [ 68 Ga]Ga-NeoB using a GMP conform kit approach and a licensed 68 Ge/ 68 Ga generator and (2) to explore the application of [ 68 Ga]Ga-NeoB in patients with gastrointestinal stromal tumors (GIST) before and/or after interventional treatment (selective internal radiotherapy, irreversible electroporation, microwave ablation). Results: Validation of the production and quality control of [ 68 Ga]Ga-NeoB for patient use had to be performed before starting the GMP production. Six independent batches of [ 68 Ga]Ga-NeoB were produced, all met the quality and sterility criteria and yielded 712 ± 73 MBq of the radiotracer in a radiochemical yield of >50%, a radiochemical purity of >95% and a molar activity of 14.2 ± 1.5 GBq/µmol within 20 min synthesis time and additional 20 min quality control. Three patients (2 females, 1 male, 51–77 yrs of age) with progressive gastrointestinal stromal tumor metastases in the liver or peritoneum not responsive to standard tyrosine kinase inhibitor therapy underwent both [ 68 Ga]Ga-NeoB scans prior and after interventional therapy. Radiosynthesis of 68 Ga-NeoB was performed using a kit approach under GMP conditions. No specic patient preparation such as fasting or hydration was required for [ 68 Ga]Ga-NeoB PET/CT imaging. Contrast-enhanced PET/CT studies were performed. A delayed, second abdominal image was acquired at 120 minutes p.i. of admistration of the rst dose of [ 68 Ga]Ga-NeoB. Conclusions: A fully GMP compliant kit preparation of [ 68 Ga]Ga-NeoB enabling the routine production of the tracer under GMP conditions was established for clinical routine PET/CT imaging of patients with metastatic GIST and proved to adequately visualize tumor deposits in the abdomen expressing GRPR.


Introduction
Gastrointestinal stromal tumors (GIST) are rare soft tissue mesenchymal tumors occurring in the gastrointestinal tract and are thought to be derived from the cells of Cajal, which drive peristalsis in the intestine [1,2]. In GIST with high risk of developing metastases, early detection of metastases detection of GIST with high sensitivity and by non-invasive methods would be an important improvement to allow for immediate treatment and to monitor or predict of the e cacy of therapy which currently is mainly in uenced by assessing the type of mutations in KIT or PDGFRA gene [3].
In this regard, morphological and functional imaging methods may be important for detection, staging and to follow-up of GIST patients undergoing therapy. Computed tomography (CT) is the most frequently used morphological imaging procedure, although it lacks sensitivity and/or speci city. Functional imaging with positron emission tomography (PET) using 2-deoxy-2-[ 18 F] uoro-d-glucose combined with computed tomography ([ 18 F]FDG PET/CT) is the most commonly used nuclear medicine functional imaging modality in clinic routine and has shown to be advantageous over morphological imaging procedures alone when assessing therapy response. However, earlier studies using [ 18 F]FDG PET/CT for GIST detection reported only the low/moderate sensitivity [4]. Hence, more accurate, speci c and sensitive non-invasive diagnostic tools visualizing GIST are needed.
The purpose of our study was to explore the applicability of [ 68 Ga]Ga-NeoB for the determination of the status of GIST in patients with different GRPR expression levels con rmed by previous biopsies of those lesions. Furthermore, the GMP compliant production of [ 68 Ga]Ga-NeoB was established in our good manufacturing practice (GMP) environment including risk management, installation quali cation (IQ), operation quali cation (OQ) and validation of the process in six independent productions of [ 68 Ga]Ga-NeoB following most recent guidelines [30].
In addition, the applicability of [ 68 Ga]Ga-NeoB for visualization of GIST metastases before and/or after selective and patient-oriented speci c interventional therapy (selective internal radiotherapy (SIRT), irreversible electroporation (IRE), microwave ablation (MWA)) was assessed.

Radiochemistry
The 68 Ge/ 68 Ga-generator used holds a license for patient application according to the European pharmacopeia and was purchased from Eckert&Ziegler (1.85 GBq, GalliaPharm, Eckert & Ziegler, Berlin, Germany). The NeoB radiolabeling kit was received from Advanced Accelerator Applications S.A. (AAA).

Quality control under GMP conditions for the patient use
The quality control (QC) of the injectable radiotracer solution was performed on an Elysia-Raytest QC-Cubicle compact unit within a cleanroom class D environment. The in-house production of radiopharmaceuticals is regulated in the German Pharmaceuticals Act and the European Pharmacopeia.
There are monographs for example for [ 68 Ga]Ga-Octreotide [31] on which the quality control (QC) was based on. The following criteria were tested: the pH value has to be between 3.0-4.0. A bubble-point test of the sterile lter was performed. The radiochemical purity as determined by radio-TLC and radio-HPLC have to exceed > 97% and > 95%, respectively. The half-life of the product has to be 1.133 ± 0.1 h and was determined using an activimeter (Elysia Raytest). The nuclide purity has to exceed 99.999% and was determined by gamma spectroscopy at an energy of the γ-line to be 511 ± 70 keV. The product was also tested for endotoxins using an Endosafe unit (Charles River, Wilmington, MA, USA), and the endotoxin level had to be below 35 EU/mL in a maximum application volume of 5 mL. Finally, the product was externally tested for sterility.

Validation of production under GMP conditions
The validation of the production process was performed on six independent [ 68 Ga]Ga-NeoB syntheses which met the above mentioned criteria.
The generator was eluted automatically using the mentioned GRP module equipped with an 20 mL syringe by pushing 3 mL of 0.1 M HCl through the generator (2 mL/min) and a sterile lter into the reaction vial containing the precursor for [ 68 Ga]Ga-NeoB [32]. 68 Ga-NeoB PET/CT Imaging Protocol

Validation runs for [ 68 Ga]Ga-NeoB
The results of the validation runs are summarized in Table 1 (Fig. 3). Additionally, an abdominal wall metastasis in the left lower abdomen was found, which was not previously known from a three-month preliminary [ 18 F]FDG PET/CT and was later proven histologically by surgical resection. In addition, physiological distribution of [ 68 Ga]Ga-NeoB was visible in the study area. The single metastasis in liver segment VII was treated by IRE and no lesions were found by magnetic resonance imaging (MRI) follow-up 11 months later. Therefore, the patient was considered to have experienced a complete response.
Patient #3 (female, 51 years old, multiple progressive hepatic metastases of GIST of the stomach carrying a D842V mutation in PDGFRa and being pretreated as treated by SIRT and microwave ablation at previously known hepatic metastases. Three months later the patient received [ 68 Ga]Ga-NeoB (135 MBq) PET/CT for staging (Fig. 4). An inhomogeneous, at tracer accumulation within the uterine cavity with decrease of uptake in the temporal course (SUV max 60 min p.i. of 26.5, SUV max 120 min p.i. of 7.1, SUV max 180 min p.i. of 6) was found, most likely a physiological enrichment. In addition, physiological radiopharmaceutical distribution in the study area was observed. However, there was no pathologically increased uptake of [ 68 Ga]Ga-NeoB in the known hepatic metastatic lesions which had an unchanged morphology in comparison to the three-month previously performed [ 18 F]FDG PET/CT. No tumor uptake and no new metastases were found in the region of interest also at later time-points indicating a stable disease. In the 120 min p.i. images, an increased nuclide uptake was found in the region of the gall bladder neck (Fig. 4b). However, after fatty eating, there was no correlation in 180 min p.i. images (Fig. 4c). An additional MRI follow-up examination six months after therapy was performed, which con rmed a stable disease.  [29].
It is well known that GIST have a wide spectrum of mutations, some of it with very low incidence [33].

CONSENT FOR PUBLICATION
All data is consent for publication.

AVAILABILITY OF DATA AND MATERIAL
The data used and analysed during the current study are available from the corresponding author on reasonable request.

COMPETING INTERESTS
The authors declare that they have no competing interests

AUTHORS' CONTRIBUTIONS
MP, LR and BW were responsible for the radiopharmaceutical translation to clinical routine production und GMP conditions. MP, CW and PH analyzed and interpreted the patient data. MP, CW and PH were major contributor in writing the manuscript. SD, SOS and BW were responsible for funding of the project. SD, SOS and PH were responsible for selection of patients and study design. All authors read and approved the nal manuscript. Study concept and design: