Direct comparison of 68Ga-DOTA-TOC and 18F-FDG PET/CT in the follow-up of patients with neuroendocrine tumour treated with the first full peptide receptor radionuclide therapy cycle

Purpose To determine the value of 68Ga-DOTA-TOC and 18F-FDG PET/CT for initial and follow-up evaluation of patients with neuroendocrine tumour (NET) treated with peptide receptor radionuclide therapy (PRRT). Methods We evaluated 66 patients who had histologically proven NET and underwent both PRRT and three combined 68Ga-DOTA-TOC and 18F-FDG PET/CT studies. 68Ga-DOTA-TOC PET/CT was performed before PRRT, 3 months after completion of PRRT and after a further 6 – 9 months. 18F-FDG PET/CT was done within 2 months of 68Ga-DOTA-TOC PET/CT. Follow-up ranged from 11.8 to 80.0 months (mean 34.5 months). Results All patients were 68Ga-DOTA-TOC PET-positive initially and at follow-up after the first full PRRT cycle. Overall, 62 of the 198 18F-FDG PET studies (31 %) were true-positive in 38 of the 66 patients (58 %). Of the 66 patients, 28 (5 grade 1, 23 grade 2) were 18F-FDG-negative initially and during follow-up (group 1), 24 (5 grade 1, 13 grade 2, 6 grade 3) were 18F-FDG-positive initially and during follow-up (group 2), 9 patients (2 grade 1, 6 grade 2, 1 grade 3) were 18F-FDG-negative initially but 18F-FDG-positive during follow-up (group 3), and 5 patients (all grade 2) were 18F-FDG-positive initially but 18F-FDG-negative during follow-up (group 4).18F-FDG PET showed more and/or larger metastases than 68Ga-DOTA-TOC PET in five patients of group 2 and four patients of group 3, all with progressive disease. In three patients with progressive disease who died during follow-up tumour SUVmax increased by 41 – 82 % from the first to the last follow-up investigation. Conclusion In NET patients, the presence of 18F-FDG-positive tumours correlates strongly with a higher risk of progression. Initially, patients with 18F-FDG-negative NET may show 18F-FDG-positive tumours during follow-up. Also patients with grade 1 and grade 2 NET may have 18F-FDG-positive tumours. Therefore, 18F-FDG PET/CT is a complementary tool to 68Ga-DOTA-TOC PET/CT with clinical relevance for molecular investigation.

NET typically have a wide range of cellular differentiation. WHO guidelines classify NET into three grades based on cell proliferation, the number of mitoses and the expression of the nuclear antigen Ki-67. Gastroenteropancreatic NET are classified as low grade (grade 1, Ki-67 <2 %), intermediate grade (grade 2, Ki-67 3 -20 %) and high grade (grade 3, Ki-67 >20 %) [12]. Both proliferation index and grade strongly correlate with tumour behaviour and prognosis [10,13,14]. High-grade, poorly differentiated NET often have limited expression of SSTR [10], what can lead to false-negative SSTR imaging results and make the molecular investigation difficult. 18 F-FDG PET/CT is used to assess glycolytic metabolism, and higher uptake of 18 F-FDG has been found to be associated with tumour aggressiveness [15]. 18 F-FDG PET/CT has thus been used increasingly in the recent years for the evaluation of high-grade NET [15,16]. To date, however, only a few studies have investigated the correlation between 18 F-FDG and SSTR imaging and NET grade. A dichotomous behaviour has been found between these approaches in well-differentiated and poorly differentiated NET, where the former was more positive on SSTR imaging [16,17] and the latter on 18 F-FDG PET [18][19][20]. Hence, adopting a dual-tracer approach encompassing SSTR and 18 F-FDG PET imaging, assessing SSTR expression and glycolytic metabolism, respectively, could support better individualization of therapy selection in patients with NET. High 18 F-FDG uptake would suggest an aggressive behaviour and the possibility of treatment refractoriness of the cells at the site, whereas low uptake would indicate a biologically indolent lesion.
We performed an intrapatient comparison of the results of 68 Ga-DOTA-TOC and 18 F-FDG PET/CT in the initial and follow-up evaluation of NET patients who had received the first full treatment cycle with PRRT. We also evaluated whether possible changes in tumour 18 F-FDG uptake correlate with disease course.

Patients
We retrospectively evaluated a cohort of 66 patients with histological confirmation of NET (according to the ENETS criteria) [13] who underwent PRRT (after confirmation of SSTR-positive lesions with 68 Ga-DOTA-TOC PET/CT) and underwent three combined studies with 68 Ga-DOTA-TOC and 18 F-FDG PET/CT at our institution between 2005 and 2013. The methods of tissue collection were resection of the primary tumour in 27 patients, surgical excision of a metastatic lesion in 3 patients and biopsy of a metastatic lesion in the other 36 patients. The Ki-67 index was evaluated with immunohistochemistry. Patient demographics are shown in Table 1.
All patients included in the study were in advanced stages requiring systemic antitumour therapy in a palliative setting. In particular, more than 65 % had metastases in more than one location, and most of them showed widespread metastases. Before undergoing PRRT, 43 patients were treated with other modalities including resection of the primary tumour (27 patients), chemotherapy (7 patients), and radiofrequency ablation or embolization of liver metastases (9 patients). The remaining 23 patients with widespread metastases were referred to our department for PRRT without previous therapy. Three patients were syndromic (two with insulinoma, one with gastrinoma). Fifteen patients had tumour progression at study entry. The average time from initial diagnosis to PRRT was 3.8 years (range 1 -11 years, standard deviation ±1.4 years). The data presented are number of patients, except age in years 68 Ga-DOTA-TOC PET/CT was performed at baseline (i.e. before PRRT), 3 months after completion of the first full PRRT cycle and every 6 -9 months thereafter. All patients proceeding to PRRT underwent a 18 F-FDG PET/CT scan as part of routine work-up. 68 Ga-DOTA-TOC and 18 F-FDG PET/CT were performed within 2 months of each other. Between these two scans no PRRT was given. A total of 198 combined 68 Ga-DOTA-TOC and 18 F-FDG PET/CT studies (baseline and after the first PRRT) were evaluated. Followup ranged from 11.8 to 80.0 months (mean 34.5 months). The periods between baseline evaluation and the first PRRT administration, and between RECIST 1.1 assessment and PRRT administration were <4 weeks. Three to four therapy cycles with 3.7 GBq 90 Y-DOTA-TOC or 7.4 GBq 177 Lu-DOTA-TATE were administered at an interval of 10 -14 weeks. The treatment protocol used in our department [6] included the additional administration of Bcold^long-acting SST analogues. These were administered after PRRT and repeated 4 weeks later. Retreatment with PRRT was performed at least 6 weeks after the use of Bcold^long-acting SST analogues.

Peptide receptor radionuclide therapy
In 23 patients treatment was solely with 90 Y-DOTA-TOC, in 22 patients solely with 177 Lu-DOTA-TATE and in 21 patients with both agents sequentially. Thirty-five patients were retreated with PRRT. The number of therapy cycles with 90 Y-DOTA-TOC ranged from 4 to 11 (cumulative activity 10.6 -27 GBq) and with 177 Lu-DOTA-TATE ranged from 4 to 9 (cumulative activity 16.3 -37.5 GBq).

68
Ga-DOTA-TOC Preparation of 68 Ga-DOTA-TOC was based on a fully automated synthesis, as described previously [21]. The patients received 100 -150 MBq of 68 Ga-DOTA-TOC (20 -30 μg) intravenously. The radiation exposure related to 68 Ga-DOTA-TOC was 2.3 -3.45 mSv [22]. PET acquisition was started 60 -90 min (median 75 min) after injection. Imaging was performed with a dedicated PET scanner (MS-Advance or MS-Discovery 450; GE Healthcare). Images were acquired from the head to the mid-thigh. Attenuation correction was performed using transmission data obtained with a 67 Ge pin source at 3 min per bed position (MS-Advance) or a CT scan (MS-Discovery 450). Ordered-subsets expectation maximization was used for image reconstruction. 18

F-FDG
Patients received 200 -300 MBq of 18 F-FDG intravenously after fasting for at least 8 hours. The radiation exposure related to 18 F-FDG was 2.4 -3.6 mSv [23]. PET acquisition was started 52 -80 min (median 65 min) after injection. The settings and protocol were as described for 68 Ga-DOTA-TOC.
CT A 2.5-mm helical CT scan was performed on a HiSpeed CT/I Advantage scanner (GE Healthcare). Approximately 1.5 mL/ kg body weight of Visipaque 320 contrast medium (GE Healthcare) was administered. The radiation exposure related to CT was 2 -12 mSv [24].

Image review
68 Ga-DOTA-TOC and 18 F-FDG PET images were assessed by two experienced board-certified nuclear medicine physicians. Criteria for a positive finding on PET studies were focal area(s) of increased tracer uptake or diffusely increased uptake, excluding physiological uptake, in comparison with adjacent tissue on axial, coronal and sagittal images. When the PET results corresponded with those of conventional imaging or histopathology, or when a corresponding lesion appeared on conventional imaging during follow-up, the PET results were rated as true-positive. Lesions not detected on PET but seen on conventional imaging and showing progression during follow-up or confirmed by histopathology were rated as false-negative. PET results suggestive of tumour lesions without corresponding lesions found on conventional imaging during follow-up or verification by histopathology were rated as false-positive.
All PET/CT images were analysed using commercially available software (eNTEGRA; GE Healthcare), which allowed review of PET, CT and fused imaging data. Semiquantitative analysis of all pathological lesions on 18 F-FDG PET, calculating the maximum standardized uptake value (SUV max ), was performed. For calculation of the SUV, regions of interest were drawn around areas with focally increased uptake on transaxial slices and automatically adapted to a 3-D volume of interest at a 70 % isocontour. The lesion with the highest SUV max was chosen for data analysis. No SUV max cut-off value was applied to differentiate benign from malignant lesions. RECIST 1.1 was used for determining tumour response to treatment. Based on all imaging and histological findings as appropriated, tumour response was categorized as complete response (CR), partial remission (PR), stable disease (SD) or progressive disease (PD) [25,26].

Statistical analysis
SPSS software (version 18.0 for Windows SPSS Inc., Chicago, IL, and LEAD Technologies, Charlotte, NC) was used for statistical evaluation of the results. Continuous variables are expressed as mean values with standard deviations. The chi-squared test and t test were used. A p value <0.05 was considered statistically significant.

Results
The disease course at last follow-up was CR in 3 patients (4.5 %), PR in 4 (6.1 %), SD in 35 (53.0 %) and PD in 24 (36.4 %). Figure 1 shows the disease course according to primary site. Figure 2 shows the influence of the primary tumour on disease course.

Analysis on a per-patient basis
Patients were classified into four groups according to the 18 F-FDG PET results as defined in the following sections. The 18 F-FDG PET findings and disease course in each patient group are shown in Fig. 3.

Group 2
Patients 18 F-FDG-positive initially and during follow-up (24 patients, 36.4 %) Fig. 1 Disease course according to primary site (CR complete response, PR partial remission, SD stable disease, PD progressive disease) *p < 0.05 as compared with SD and PR; Δ p < 0.05 as compared with PD Fig. 2 Influence of the primary tumour on disease course (CR complete response, PR partial remission, SD stable disease, PD progressive disease) *p < 0.05 as compared with group of inoperable or unknown primaries Ga-DOTA-TOC PET was positive in 9 primary tumours (9 patients) and 55 metastatic sites (24 patients). 68 Ga-DOTA-TOC PET showed more metastases than 18 F-FDG PET in 11 patients (3 patients with SD and 8 patients with PD during follow-up).
The tumour was grade 1 in 5 patients, grade 2 in 13 patients and grade 3 in 6 patients. Five patients died during follow-up (SD in 1 patient with grade 1, PD in 1 patient with grade 1, 2 patients with grade 2 and 1 patient with grade 3 tumour).
Positive 68 Ga-DOTA-TOC PET was seen in 3 primary tumours (three patients) and 13 metastatic sites (nine patients). The tumour was grade 1 in two patients, grade 2 in six patients and grade 3 in one patient.

Group 4
Patients 18 F-FDG-positive initially but 18 F-FDG-negative during follow-up (five patients, 7.6 %) Positive 18 F-FDG PET was found in three primary tumours (three patients) and eight metastatic sites (five patients). Positive 68 Ga-DOTA-TOC PET was seen in three primary tumours (three patients) and eight metastatic sites (five patients). The tumour was grade 2 in all patients. The disease course was CR in one patient (who was re-treated with PRRT and had radiofrequency ablation of liver metastases), PR in one patient (who was re-treated with PRRT; Fig. 7), and SD in three patients (one with surgical resection of primary tumour and LN metastases).

SUV max in 18 F-FDG PET
In patients with 18 F-FDG-positive lesions the SUV max ranged from 3.0 to 13.0. No significant differences in SUV max were found among the tumour grades or the various disease course groups. In patients with a more favourable disease course, the SUV max in the primary tumour and metastases remained unchanged or showed a slight variation (5 % to 13 %) from first to last follow-up. However, in 3 patients with PD who died during follow-up, the SUV max of the primary tumour (1 patient) and in various metastases (3 patients) showed an increase of 41 % to 82 % from first to last follow-up (SUV max ranging from 11.2 to 11.9).

Discussion
In the past few years, SSTR imaging with 68 Ga-labelled peptide PET/CT has been shown to provide excellent sensitivity and specificity for diagnosing and staging NET [2,3]. 18 F-FDG PET is widely used in oncology, but its use in NET is a matter of controversy. Initial studies [20,27], performed in a small number of patients only, cast doubt on the use of 18 F-FDG PET in NET patients. The loss of SSTR expression was found to coincide with a gain in glucose utilization in tumours [28]. Consequently, it was suggested that the use of 18 F-FDG PET be limited to SSTR-negative NET. More recent studies including a larger number of patients evaluated the xsensitivity of 18 F-FDG PET in NET patients in comparison with that of 68 Ga-DOTA-TATE PET/CT [16] and in relation to survival [29]. These studies showed that the higher the grade of NET, the higher is the prevalence of glucose hypermetabolic tumours [16], which have been linked with more aggressive tumour features including a higher risk of death [29]. This study confirmed that 18 F-FDG positivity is strongly correlated with a higher risk of progression, in agreement with the findings of Garin et al. [30] showing that 18 F-FDG PET has a prognostic value for early tumour progression. Furthermore, an important finding of our investigation is the evidence that patients may develop 18  High 18 F-FDG SUVs seem to be strongly correlated with short survival in NET patients. In accordance with Binderup et al. [29] who found that patients with a SUV max higher than 9 are more likely to have a shorter overall survival, in 3 patients with progression who died during follow-up we found SUV max in the range 11.2 to 11.9 in various tumours at the last follow-up, and importantly the values had increased 41 % to 82 % from the first follow-up. We did not perform a correlation study between 18 F-FDG SUVs and Ki-67 index. Further prospective ongoing studies are necessary to establish the value of SUV in NET patients, in particular for assessing survival and progression-free survival.
The ENETS guidelines recommend that in patients with fast progression of NET classified as grade 1 or grade 2 a rebiopsy should be considered, but do not recommend 18 F-FDG PET routinely during follow-up [31]. Guidelines now consider a place for 18 F-FDG PET in a patient with grade 2 tumour in whom liver transplantation is planned to ascertain the absence of extrahepatic tumour lesions or other malignancies [10]. In this study, we confirm that patients with grade 1 or grade 2 NET may also have 18 F-FDG-positive tumours initially and may develop 18 F-FDG-positive lesions during follow-up. These findings must be taken into account, especially in individualized and optimized therapy planning.
The limitations of this retrospective study were the small number of patients in the subgroups, due in part to the heterogeneity of NET and the tumour primary sites, and the different treatments performed before and during follow-up. The treatment protocol used in our department included 90 Y-DOTA-TOC as the radiopharmaceutical of first choice for PRRT. Although the possible superiority of 90 Y for larger tumours has not been shown by direct comparison of 90 Y-DOTA-TOC and 177 Lu-DOTA-TATE, the rationale for using 90 Y-DOTA-TOC in patients with tumours larger than 2 cm in diameter was based on data using a mathematical modelling approach based on the physical characteristics of therapeutic radionuclides [32]. A previous study has indicated that PRRT may lead to a reduction in glucose metabolism in NET lesions, depending on the amount of SSTR uptake as demonstrated by 68 Ga-DOTA-NOC PET [9]. We evaluated mainly patients with grade 1 and grade 2 NET. Therefore, we were not able to further investigate the behaviour of grade 3 NET during the course of the disease with regard to 18 F-FDG positivity of the tumour lesions.
In conclusion, our results show that investigation only of SSTR status by 68 Ga-DOTA-TOC PET/CT may not reflect progression in certain NET lesions. Therefore, the decision on a change in therapeutic strategy in a patient with a poor prognosis cannot be based on this one modality alone. Hence, we recommend performing 18 F-FDG PET in the initial evaluation and during follow-up of NET patients, especially when SSTR PET/CT shows progression. 18 F-FDG PET/CT is a clinically relevant, complementary tool to 68 Ga-DOTA-TOC PET and its use represents a step towards personalized medicine in the management of NET patients.