Purpose: The aim of this study was to evaluate the effect of combined 68Ga and 18F-FDG PET/CT on treatment management for patients with pancreatic neuroendocrine tumor (PNET). Methods: Between January 2012 and April 2014, 49 consecutive patients with a cytologically and/or histologically proven diagnosis of PNET underwent combined 68Ga and 18FDG PET/CT on the same day. Results: The study group consisted of 21 males and 28 females with a median age of 59 years. Disease detection was achieved in 48 out of the 49 cases with 68Ga imaging, and in 36 of the 49 cases with 18FDG PET/CT. These results corresponded to sensitivities of 98% for 68Ga versus 73% for 18FDG PET/CT. Patients with NET-G1/NET-G2 had a positive 68Ga and negative 18FDG PET/CT in 13 cases, whereas both 68Ga and 18FDG PET/CT were positive in 27 cases. Patients with NEC-G3 were positive by both 68Ga and 18FDG PET/CT in 7 cases and positive only by 18FDG in 1 case. Another NEC-G3 patient was only positive by 68Ga PET/CT. The median Ki67 was 7% for 68Ga PET/CT-positive tumors and 10% for tumors with both 68Ga and 18FDG PET/CT positivity (p = 0.130). Half of the patients with a prevalent uptake of 18FDG (n = 7) had an NEC-G3 compared with 12% of patients with a prevalent uptake of 68Ga (p = 0.012). There were no significant differences between patients with positive 68Ga and those with positive 18FDG with regards to treatment choice. Conclusions: The association of 18FDG slightly increases sensitivity of 68Ga PET/CT alone in the diagnosis of PNET. A combined dual tracer PET/CT does not influence the choice of treatment strategy.
Pancreatic neuroendocrine tumors (PNETs) comprise a wide and heterogeneous group of neoplasms. Diagnosis can often be challenging due to their rarity. The treatment strategy should be adapted to each single patient according to the tumor characteristics and patient comorbidities . 68Ga PET/CT is becoming the new gold standard for somatostatin receptor imaging of PNET. The sensitivity of this technique varies between 91 and 95% with a specificity of 82-97% . 68Ga-labeled octreotide has several advantages. Firstly, 68Ga is a generator product instead of a cyclotron-produced product and a relatively simple labeling can be performed on an everyday basis . Secondly, 68Ga octreotide is similar to its counterparts that are used for peptide receptor radionuclide therapy (PRRT). Lastly, PET imaging has an accuracy of 2-3 mm with a consequent high sensitivity . On the other hand, 18FDG PET is the most widely used functional imaging for cancer in general. It has been demonstrated in patients affected by NET that 18FDG PET has a high accuracy for poorly differentiated forms despite a relatively low overall sensitivity for these tumors [5, 6]. Dual tracer PET/CT can be performed on a single day owing to the short half-life of 68Ga and can be superior to histopathology by demonstrating tumor heterogeneity, especially for patients with multiple metastases . Only a handful of studies have explored the value of combined 68Ga and 18FDG PET/CT for PNET [7, 8]. Theoretically, a more accurate functional mapping of these lesions with demonstration of different areas of uptake could select tumors to proper therapeutic groups. Although it has been demonstrated that routine execution of 68Ga PET/CT in patients with PNET causes a therapy modification in almost 50% of cases , the clinical impact of combined 68Ga and 18FDG PET/CT and its role in the treatment strategy is scarcely known. The aims of the current study were: (1) to evaluate the influence of combined 68Ga and 18FDG PET/CT on treatment management for patients with cytologically or histologically proven PNET, and (2) to analyze the correlation between uptake of dual tracers and tumor grade.
This was a retrospective, bi-institutional observational study carried out at the University of Marche, Ancona, and University ‘La Sapienza', Rome, Italy. Between March 2012 and April 2014, all consecutive patients with cytologically or histologically proven PNET underwent combined 68Ga and 18FDG PET/CT. The inclusion criteria were the presence of a cytologically or histologically proven PNET and age >18 years. The only exclusion criterion was the inability to provide written informed consent. The routine use of combined 68Ga and 18FDG PET/CT was introduced in order to demonstrate tumor heterogeneity and to possibly change the management strategy according to the available evidence . Ethical committee approval was waived because of the retrospective nature of the study according to the regulations of the involved institutions. Demographics, presenting symptoms and radiological findings were prospectively collected. All patients underwent at least one high-resolution imaging technique (CT or MR). A suspected diagnosis of PNET was made in the presence of a hypervascularized mass of the pancreas. Patients underwent PET/CT imaging after a cytological/histological diagnosis was obtained. A routine evaluation of chromogranin A and/or other tumor markers was not considered.
All patients with a localized pancreatic mass underwent endoscopic ultrasound with fine-needle aspiration. Patients with a pancreatic mass and liver metastases underwent ultrasound-guided liver biopsy. For patients who underwent surgery, a final pathological examination on a surgical specimen was carried out. The diagnosis of PNET was based on cytological or histological findings and immunochemistry, which was performed for all tumors for chromogranin A, synaptophysin and CD56. All cases were classified according to criteria proposed by the World Health Organization  and were assigned to a TNM (tumor-node-metastasis)-based staging system according to Rindi et al. . The Ki67 proliferative index was expressed as a percentage based on the count of Ki67-positive cells in tumor cells in the areas of highest immunostaining using the MIB1 antibody (DBA, Milan, Italy). Tumors were then classified into three categories: PNET-G1 (Ki67 ≤2%), PNET-G2 (Ki67 between 2 and 20%) and PNEC-G3 (Ki67 >20%).
After diagnostic work-up was concluded, all cases were discussed during a multidisciplinary team meeting held at the two involved institutions. A specific treatment protocol based on dual 68Ga and 18F-FDG PET/CT findings was not included. Generally, the treatment strategy was chosen according to the latest European Neuroendocrine Tumor Society (ENETS) guidelines . Briefly, surgical exploration was always indicated in the presence of localized PNET >2 cm or in the presence of symptoms. For PNET with liver metastases, a possible surgical approach was evaluated for each individual patient. Surgery was always contraindicated in the presence of: (i) severe patient comorbidities, (ii) extra-abdominal disease and (iii) diffuse peritoneal carcinomatosis. Somatostatin receptor-targeted therapies were offered only to those patients with a positive 68Ga-DOTANOC ([DOTA, 1-Nal3]-octreotide) PET/CT.
The peptide-chelator conjugate DOTANOC was synthesized by standard Fmoc solid-phase synthesis on 2-chlorotritylchloride resin on a peptide synthesizer (Switch 24; Rink CombiChem Technologies®). 68Ga-DOTANOC was labelled under sterile conditions in an isolator using a modification of the methoddescribed by Zhernosekov et al. . The final product was diluted with 7 ml of saline and then subjected to sterile filtration using a Millex 0.22-μm filter (Millipore®). The labelling yield was analyzed by silica gel instant thin-layer chromatography (Pall Inc.®) and by high-performance liquid chromatography using a Luna 5-μm, C18(2) 50 × 3-mm column (Phenomenex®) and an acetonitrile-water gradient. The labelling yield and radiochemical purity of 68Ga-DOTANOC were greater than 98% at a specific activity of 14.5-34 GBq/μmol.
Combined 68Ga-DOTANOC and 18FDG PET/CT Acquisition
PET/CT images were acquired with an mCT 64-slice scanner (Siemens®). Patients fasted from at least 6 h before imaging and blood glucose levels were checked. Those with a blood glucose level above 150 mg/dl did not undergo scanning. Images from the vertex to the proximal femur were obtained while the patients were in the supine position. The 68Ga-DOTANOC PET/CT was performed at 8.00 a.m. and the 18FDG PET/CT was always subsequently performed at 2.00 p.m. on the same day regardless of the findings of the 68Ga-DOTANOC PET/CT. Both the whole-body 68Ga-DOTANOC (1.5 MBq/kg) PET/CT and whole-body 18FDG PET/CT (2.96 MBq/kg) imaging were performed approximately 1 h after an intravenous injection. During the waiting period, patients rested in a quiet room without taking any muscle relaxants. PET images were acquired for 2 min per bed position. CT images were also obtained from the patient's integrated 68Ga-DOTANOC PET/CT and 18FDG PET/CT with the use of a standardized protocol of 120 kV, care dose of 100 mAs, tube rotation time of 0.5 s per rotation, a pitch of 1.4 and a slice thickness of 5 mm. Patients were allowed to breathe normally during the procedure. Attenuation-corrected PET/CT fusion images were reviewed in three planes (transaxial, coronal and sagittal) with True-D software (Siemens).
The results are presented as frequencies and percentages for categorical variables and as median with interquartile range (IQR) for continuous variables. Patients were divided in two groups according to the PET/CT findings. The first group included those patients who had only a positive 68Ga-DOTANOC PET/CT. The second group included those with tumors that were positive for both 68Ga-DOTANOC and 18FDG PET/CT, or only positive for the latter. When comparing these two groups, normally distributed continuous variables were analyzed using a two-sample Student t test, while the Mann-Whitney U test was used for nonnormally distributed variables. Categorical variables were compared using the χ2 test and Fisher's exact test as appropriate. A Wilcoxon matched-pairs signed-rank test was used to compare uptake of 68Ga-DOTANOC and 18FDG according to tumor grades. All p values were two sided and considered significant when <0.05. Statistical analyses were performed using IBM SPSS 20.0 for Mac software (SPSS Inc., Chicago, Ill., USA).
The demographics and clinical characteristics of the entire cohort are summarized in table 1. All patients had nonfunctioning PNET except for 1 who had a metastatic insulinoma. Of the 49 patients, 68Ga-DOTANOC PET/CT was true positive in 48 and false negative in 1. The overall sensitivity for 68Ga-DOTANOC PET/CT was 98%. 18FDG PET/CT was true positive in 36 and false negative in 14 of the patients. The overall sensitivity for 18FDG PET/CT was therefore 73%. The combined 68Ga-DOTANOC and 18F-FDG PET/CT sensitivity was 100%. Overall, 14 patients (28.5%) had only a positive 68Ga-DOTANOC PET/CT, whereas 34 patients (69%) were positive for both 68Ga-DOTANOC and 18F-FDG PET/CT. Only 1 patient was only positive by 18FDG PET/CT. Overall, 8 patients (16%) had a prevalent 18FDG uptake. In the majority of patients (35/49) a PNET was diagnosed for the first time, whereas 14 patients had a previous diagnosis of PNET. These 14 patients were then restaged after the failure of previous treatment and all but 5 of them were positive on 18FDG PET/CT.
Pathological Characteristics according to 68Ga-DOTANOC and 18FDG PET/CT Findings
A comparison of pathological characteristics is illustrated in table 2. Only 3 patients had a tumor diameter <2 cm (6%). A cytological diagnosis was made in only 8 patients (16%), whereas the remaining 41 (84%) had a histological diagnosis. The radiological tumor stage was concordant with the PET/CT tumor stage in all cases. 68Ga-DOTANOC failed to detect the primary tumor in 1 case, whereas 18FDG PET/CT failed to detect a pancreatic lesion in 2 cases. The radiological number of liver metastases was concordant with the PET/CT number of liver metastases in all cases except 2. In 1 of these cases 68Ga-DOTANOC PET/CT detected 6 liver metastases compared with 2 metastases detected by CT alone. In the other case, both 68Ga-DOTANOC and 18FDG PET/CT detected only two liver metastases compared with multiple lesions observed on the CT scan. A total of 30 metastatic sites were defined in 24 patients. The most frequent site was the liver (n = 24) followed by bone (n = 4), lung (n = 1) and spleen (n = 1). 18FDG was false negative for skeletal metastases in 1 case and liver metastases in another. The pathological data of patients with positive 68Ga-DOTANOC PET/CT were comparable with those who were both 68Ga-DOTANOC and 18FDG PET/CT positive, or only 18FDG positive. The two groups were also similar regarding tumor grading, although all but 1 of the patients with NEC-G3 had a positive 18FDG PET/CT. The median Ki67 was 7% for 68Ga PET/CT-positive tumors and 10% for tumors that were both 68Ga and 18FDG PET/CT positive (p = 0.130). Half of the patients with a prevalent uptake of 18FDG (n = 7) had an NEC-G3 compared with 12% of patients with a prevalent uptake of 68Ga (p = 0.012). The median SUVmax for 68Ga-DOTANOC PET/CT was 43.5 (IQR 18-107) and 8.5 (IQR 5-23) for 18FDG PET/CT. The tracer uptake according to tumor grade is depicted in table 3.
Impact of 68Ga-DOTANOC and 18F-FDG PET/CT on Treatment Choice
Table 4 illustrates a comparison of treatment choice between patients who were positive by 68Ga-DOTANOC and those positive by both 68Ga-DOTANOC and 18FDG PET/CT, or only 18FDG. Overall, 16 patients underwent surgery (5 pancreaticoduodenectomies, 6 distal pancreatectomy, 3 atypical pancreatic resections and 2 pancreatic resection with associated liver resection). Only 8 patients (16%) underwent chemotherapy. All these 8 patients were positive on both 68Ga-DOTANOC and 18FDG PET/CT. Of these, 5 had a PNEC-G3 tumor whereas 3 had a PNET-G2 tumor (table 5). Two patients with a PNET-G2 tumor underwent chemotherapy because of the failure of previous treatment with somatostatin analogue (SSA) and PRRT. One patient with PNET-G2 (Ki67 = 12%) with high uptake for 68Ga-DOTANOC and moderate uptake for 18FDG PET/CT (fig. 1) underwent SSA and chemotherapy with oxaliplatin and capecitabine during hospitalization for severe diarrhea with hepatorenal syndrome. Of 8 patients with a prevalent uptake of 18FDG, 3 underwent surgery for a localized tumor and 4 underwent chemotherapy. The remaining patient was not treated due to chronic leukemia. Only 1 patient was negative by 68Ga-DOTANOC PET/CT and positive only by 18FDG PET/CT (fig. 2). This patient had a localized PNEC-G3 and underwent radical surgery. He then developed multiple liver metastases (positive on 18FDG PET/CT) only 8 months after surgery. Another patient with PNEC-3 underwent PRRT as the high uptake for 68Ga-DOTANOC (SUVmax, 44) whereas the remaining patient with PNEC-G3 was administered everolimus.
The current study demonstrates that a routine use of combined 68Ga-DOTANOC and 18FDG PET/CT does not significantly influence the treatment strategy in patients affected by PNET despite dual tracer functional imaging often revealing tumor heterogeneity. Prior studies have focused mainly on the accuracy of dual tracer PET/CT imaging in the diagnosis and staging of this disease [7, 8]. The sensitivity of 68Ga PET/CT was reported to be between 91 and 95% , which is much higher compared with that reported for 18FDG PET/CT [7, 8, 14]. In the present study we found similar results, as the sensitivity of 68Ga PET/CT was 98%. This sensitivity was higher than that reported by Kayani et al. , possibly related to the profile of tracer DOTANOC which has affinity for SSTR2, SSTR3 and SSTR5 compared with that of DOTATATE . The accuracy could also have been biased by the inclusion of only histologically proven PNET in the study and, similarly, the fact that high pretest probability usually affects the accuracy of functional imaging for PNET. For these reasons, specificity is generally not considered for this imaging and specificity of 18FDG is inevitably low as this represents the most accurate functional imaging for cancer . If we exclude the value of PET/CT in the diagnostic accuracy of PNET, the real significance of this procedure remains mostly related to its potential influence on treatment strategy. The clinical impact of 68Ga-DOTANOC has been previously proven . Ambrosini et al.  demonstrated that 68Ga-DOTANOC PET/CT influenced the management of patients in more than half of the studied population. In particular, the effect of PET on therapy was to initiate or continue PRRT or SSA medical therapy on the basis of the demonstration of somatostatin receptor expression. Data on the clinical impact of combined dual tracer PET/CT imaging are limited to those described by Kayani et al. . In their study they reported that for 4 patients (3 high-grade NET and 1 intermediate-grade NET), 18FDG showed more intense and more widespread uptake than 68Ga PET/CT. In view of this pattern, the patients were assigned to receive chemotherapy instead of PRRT. They concluded that the use of 18FDG led to a change in clinical management from PRRT to chemotherapy in 25% of intermediate- and high-grade NET. In the present study, therapy was not influenced by the routine use of dual tracer PET/CT imaging. In particular, the use of 18FDG did not influence the clinician's choice regarding chemotherapy. Moreover, 1 patient with localized pancreatic NET underwent surgery despite the high uptake of 18FDG. The treatment strategy in the present study was mainly influenced by tumor grade, symptoms and previous clinical history. Two patients with moderately differentiated tumors underwent chemotherapy, in 1 case due to the presence of a severe clinical syndrome and in the other because of the failure of previous receptor-based treatment. Nevertheless, the majority of patients had a higher uptake of 68Ga-DOTANOC, except for 8 patients with a localized tumor. Evaluations on treatment strategy were then more influenced by the expression of somatostatin receptors than by the increased glycolytic activity of tumor cells. The real value of combined 68Ga and 18FDG PET CT consists mainly in the possibility to demonstrate areas of different tumor grading among the same lesions. This was clearly demonstrated by the Kayani et al.  study, in which patients with high-grade tumors had a significantly higher median SUVmax of 18FDG, whereas patients with well-differentiated tumors had a higher uptake for 68Ga-DOTATATE. In the present study, patients who also had positive 18FDG PET/CT imaging had tumors with a trend toward a higher median Ki67, although this was not statistically significant. Moreover, we observed a significantly higher incidence of NEC-G3 lesions among those patients with a prevalent uptake of 18FDG. This result confirms the presence of tumor heterogeneity that seems to be a specific trait of neuroendocrine neoplasms . Tumor heterogeneity cannot be assessed by tumor biopsy and this can represent a distinctive advantage of combined dual tracer PET/CT imaging. On the other hand, in this preliminary experience, it seems that physicians still place greater trust in histology, or even cytology, rather than functional imaging. Long-term survival data will be of paramount importance to definitely assess the value of dual tracer PET/CT and the role of tumor heterogeneity on outcomes.
This study has some limitations. Firstly, it was a retrospective study although a prospective evaluation could potentially bias the physicians' choices on treatment strategy. Secondly, a relatively small sample size could have influenced some results, especially after patients were classified into subgroups. In particular, the relatively small sample size, as well as the wide range of therapeutic options, did not allow a statistical analysis between the different groups to be performed. As a consequence, these results should be taken cautiously and corroborated by larger series.
A routine use of combined 68Ga and 18FDG PET/CT in patients with PNET seems not to be justified in clinical practice and still represents an area of research. In routine use the association of these two tracers could be reasonable in selected patients affected by PNET with a Ki67 >10%. The association of long-term outcomes and findings obtained by dual tracer PET/CT imaging has to be evaluated.
This work was supported by the Italian Association for Neuroendocrine Tumors (It.A.Net).
The authors have no conflict of interest to declare.