Somatostatin receptor sst1-sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands

Somatostatin receptors are known to be expressed in a large number of human tumours and represent the basis for in vivo tumour targeting. Stable somatostatin derivatives such as octreotide or lanreotide are the most frequently used radiopharmaceuticals acting through specific binding to somatostatin receptors; however, they do not bind with high affinity to all five receptor subtypes. Whereas the mRNAs for most receptor subtypes have been detected in tumours, it is in most cases unclear which of the receptor subtype proteins are expressed. Since in vitro receptor binding methods are close correlates and predictors of in vivo peptide receptor targeting, we took advantage of the recently developed subtype-selective analogues and evaluated approximately 200 tumours for their receptor subtype protein expression in specific binding assays using autoradiography with 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 and displacement by subtype-selective analogues. The majority of the tested neuroblastomas, meningiomas, medulloblastomas, breast carcinomas, lymphomas, renal cell carcinomas, paragangliomas, small cell lung carcinomas and hepatocellular carcinomas predominantly expressed sst2. The prostate carcinomas and sarcomas preferentially expressed sstl, while a majority of inactive pituitary adenomas displayed sst3 and, to a lesser extent, sst2. Growth hormone-secreting pituitary adenomas preferentially expressed sst2 and sst5; gastroenteropancreatic tumours and phaeochromocytomas frequently displayed sst2 and/or sstl. Non-neoplastic human tissues such as vessels, nerve plexus, pancreatic islets, prostatic stroma, adrenal medulla, spleen and germinal centres of the lymphoid tissues preferentially expressed sst2. However, the human gastric mucosa predominantly expressed sst1 while colonic mucosa displayed sst2. Interestingly, a minority of tumours showed a strong 125I-[Leu8, D-Trp22, Tyr25]-somatostatin-28 binding, of which less than 50% could be displaced by the sum of the five subtype-selective analogues. This observation suggests the existence of an as yet unknown subtype in selected tumours. This study is the first report to analyse the somatostatin receptor subtype expression in tumours with binding methods. We conclude that sst2, with high affinity for current radiopharmaceuticals such as Octreoscan, is predominantly expressed in a majority of tumours. Fewer tumour types (sarcomas, prostate cancers, inactive pituitary adenomas) preferentially express another subtype. This information is of importance with regard to the clinical applications and development of somatostatin analogues with distinct receptor subtype selectivities.     

Biokinetics and imaging with the somatostatin receptor PET radioligand (68)Ga-DOTATOC: preliminary data.

Somatostatin (SMS) scintigraphy is widely used for the detection and staging of neuroendocrine tumours. Because of its superior imaging properties, there is growing interest in the use of positron emission tomography (PET) technology for SMS scintigraphy. This study addressed the production of gallium-68 DOTATOC, its biokinetics and its clinical performance in detecting SMS-positive tumours and metastases. A preparation protocol was developed, yielding 40% overall incorporation of (68)Ga into the peptide (DOTATOC). After column filtration, the radiochemical purity exceeded 98%. Eight patients with histologically verified carcinoid tumours were injected with 80-250 MBq of this tracer. PET acquisition was initiated immediately after administration and carried out until 3 h post injection. Images were quantitated using standardised uptake values and target to non-target ratios. Prior to (68)Ga-DOTATOC PET, all patients underwent indium-111 octreotide planar and single-photon emission tomographic (SPET) imaging. Arterial activity elimination was bi-exponential, with half-lives of 2.0 (+/-0.3) min and 48 (+/-7) min. No radioactive metabolites were detected within 4 h in serum. Maximal tumour activity accumulation was reached 70+/-20 min post injection. Kidney uptake averaged <50% compared with spleen uptake. Of 40 lesions predefined by computed tomography and/or magnetic resonance imaging, (68)Ga-DOTATOC PET identified 100%, whereas (111)In-octreotide planar and SPET imaging identified only 85%. Tumour to non-tumour ratios ranged from >3:1 for liver ((111)In-octreotide: 1.5:1) to 100:1 for CNS ((111)In-octreotide: 10:1). With (68)Ga-DOTATOC >30% additional lesions were detected. It is concluded that PET using (68)Ga-DOTATOC results in high tumour to non-tumour contrast and low kidney accumulation and yields higher detection rates as compared with (111)In-octreotide scintigraphy.     

Correlation of immunohistopathological expression of somatostatin receptor 2 with standardised uptake values in <Superscript>68</Superscript>Ga-DOTATOC PET/CT

Purpose  In clinical routine somatostatin analogue positron emission tomography/computed tomography (PET/CT) such as ⁶⁸Ga-DOTA-Tyr-octreotide (DOTATOC)-PET/CT could substitute conventional ¹¹¹In-Octreotide scintigraphy. Immunohistochemistry (IHC) for somatostatin receptor 2 (SSTR2) might be a tool to predict positivity of ⁶⁸Ga-DOTATOC in patients where initial staging was not performed, e.g., in incidental findings. We therefore compared a score of SSTR2-IHC with the in vivo standard uptake value (SUV) of preoperative or prebiopsy ⁶⁸Ga-DOTATOC PET/CT. Materials and methods  In 18 patients, ⁶⁸Ga-DOTATOC PET/CT scans were quantified with SUV calculations and correlated to a cell membrane-based SSTR2-IHC score (ranging from 0 to 3). Results  Negative IHC scores were consistent with SUV values below 10. Furthermore, all score 2 and 3 specimens corresponded with high SUV values (above 15). Conclusion  SSTR2-IHC scores correlated well with SUV values and we propose to use SSTR2 immunohistochemistry in patients missing a preoperative PET scan to indicate ⁶⁸Ga-DOTATOC-PET/CT as method for restaging and follow-up in individual patients.     

Somatostatin receptor sst1–sst5 expression in normal and neoplastic human tissues using receptor autoradiography with subtype-selective ligands

Somatostatin receptors are known to be expressed in a large number of human tumours and represent the basis for in vivo tumour targeting. Stable somatostatin derivatives such as octreotide or lanreotide are the most frequently used radiopharmaceuticals acting through specific binding to somatostatin receptors; however, they do not bind with high affinity to all five receptor subtypes. Whereas the mRNAs for most receptor subtypes have been detected in tumours, it is in most cases unclear which of the receptor subtype proteins are expressed. Since in vitro receptor binding methods are close correlates and predictors of in vivo peptide receptor targeting, we took advantage of the recently developed subtype-selective analogues and evaluated approximately 200 tumours for their receptor subtype protein expression in specific binding assays using autoradiography with ¹²⁵I-[Leu⁸, D-Trp²², Tyr²⁵]-somatostatin-28 and displacement by subtype-selective analogues. The majority of the tested neuroblastomas, meningiomas, medulloblastomas, breast carcinomas, lymphomas, renal cell carcinomas, paragangliomas, small cell lung carcinomas and hepatocellular carcinomas predominantly expressed sst2. The prostate carcinomas and sarcomas preferentially expressed sst1, while a majority of inactive pituitary adenomas displayed sst3 and, to a lesser extent, sst2. Growth hormone-secreting pituitary adenomas preferentially expressed sst2 and sst5; gastroenteropancreatic tumours and phaeochromocytomas frequently displayed sst2 and/or sst1. Non-neoplastic human tissues such as vessels, nerve plexus, pancreatic islets, prostatic stroma, adrenal medulla, spleen and germinal centres of the lymphoid tissues preferentially expressed sst2. However, the human gastric mucosa predominantly expressed sst1 while colonic mucosa displayed sst2. Interestingly, a minority of tumours showed a strong ¹²⁵I-[Leu⁸, D-Trp²², Tyr²⁵]-somatostatin-28 binding, of which less than 50% could be displaced by the sum of the five subtype-selective analogues. This observation suggests the existence of an as yet unknown subtype in selected tumours. This study is the first report to analyse the somatostatin receptor subtype expression in tumours with binding methods. We conclude that sst2, with high affinity for current radiopharmaceuticals such as Octreoscan, is predominantly expressed in a majority of tumours. Fewer tumour types (sarcomas, prostate cancers, inactive pituitary adenomas) preferentially express another subtype. This information is of importance with regard to the clinical applications and development of somatostatin analogues with distinct receptor subtype selectivities.     

Detection of cranial meningiomas: comparison of 68Ga-DOTATOC PET/CT and contrast-enhanced MRI

Purpose

PET imaging with somatostatin receptor ligands, such as ⁶⁸Ga-DOTATOC, is a well-established method for detection and target volume definition of meningiomas prior to radiotherapy. Since DOTATOC PET delivers a higher contrast between meningiomas and surrounding tissues than MRI, we conducted a retrospective analysis to compare the diagnostic accuracy of contrast-enhanced MRI (CE-MRI) with ⁶⁸Ga-DOTATOC PET/CT in patients with cranial meningiomas prior to radiotherapy.

Methods

Over a period of 6?years, 134 patients (20–82?years of age, 107 women and 27 men) underwent cranial CE-MRI and ⁶⁸Ga-DOTATOC PET/CT. To compare the two methods, the lesions considered typical of meningiomas visually were counted and analysed with respect to their location and SUVmax.

Results

In the 134 patients investigated by both modalities, 190 meningiomas were detected by ⁶⁸Ga-DOTATOC PET/CT and 171 by CE-MRI. With knowledge of the PET/CT data, the MRI scans were reinvestigated, which led to the detection of 4 of the 19 incidental meningiomas, resulting in an overall detection rate of 92?% of the meningioma lesions that were found by PET/CT.

Conclusion

Ga-DOTATOC PET/CT demonstrated an improved sensitivity in meningioma detection when compared to CE-MRI. Tumours adjacent to the falx cerebri, located at the skull base or obscured by imaging artefacts or calcification are particularly difficult to detect by MRI. Therefore ⁶⁸Ga-DOTATOC PET/CT may provide additional information in patients with uncertain or equivocal results on MRI or could help to confirm a diagnosis of meningioma based on MRI or could help to confirm MRI-based diagnosis of meningiomas in cases of biopsy limitations. It is possible that not only radiotherapy and surgical planning, but also follow-up strategies would benefit from this imaging modality.     

68Ga-DOTA-NOC PET/CT detects somatostatin receptors expression in von hippel-lindau cerebellar disease

A case of Von-Hippel Lindau (VHL) disease has been studied using 68Ga-DOTA-NOC PET/CT. PET/CT demonstrated the presence of somatostatin receptors within 2 focal areas in the cerebellum corresponding to the lesions detected by MRI. Considering the heterogeneous lesions localizations in VHL disease, PET/CT may be a useful imaging modality for diagnosing lesions of central nervous system and neuroendocrine lesions and for direct demonstration of somatostatin receptors for targeted treatment.     

Role of 68Ga-DOTATOC PET/CT in initial evaluation of patients with suspected bronchopulmonary carcinoid

Purpose

The objective of this study was to evaluate the role of ⁶⁸Ga-DOTATOC positron emission tomography (PET)/CT scan in patients with suspected pulmonary carcinoid tumour and to compare its results with ¹⁸F-fluorodeoxyglucose (FDG) PET/CT scan.

Methods

In this prospective study, 32 patients (age 34.22?±?12.03 years; 53.1 % female) with clinical suspicion of bronchopulmonary carcinoid were evaluated with ⁶⁸Ga-DOTATOC PET/CT and ¹⁸F-FDG PET/CT. The two imaging modalities were compared, considering the tissue diagnosis as the reference standard.

Results

Based on the reference standard 26 cases were carcinoid tumours [21 typical carcinoids (TC) and 5 atypical carcinoids (AC)] and 6 cases were non-carcinoid tumours. The sensitivity, specificity and accuracy of ⁶⁸Ga-DOTATOC PET/CT in the diagnosis of pulmonary carcinoid tumour were 96.15, 100 and 96.87 % respectively, whereas those of ¹⁸F-FDG PET/CT were 78.26, 11.1 and 59.37 % respectively. The maximum standardised uptake value (SUV_max) of TC on ⁶⁸Ga-DOTATOC PET/CT scan ranged from 3.58 to 55, while that of AC ranged from 1.1 to 32.5. ¹⁸F-FDG PET/CT was true-positive in all cases of AC and false-negative in eight cases of TC (sensitivity for TC 61.9 % and for AC 100 %).

Conclusion

⁶⁸Ga-DOTATOC PET/CT is a useful imaging investigation for the evaluation of pulmonary carcinoids. ¹⁸F-FDG PET/CT scan suffers from low sensitivity and specificity in differentiating the pulmonary carcinoids from other tumours.     

Cost comparison of 111In-DTPA-octreotide scintigraphy and 68Ga-DOTATOC PET/CT for staging enteropancreatic neuroendocrine tumours

Purpose  

Although somatostatin receptor positron emission tomography (PET)/CT is gaining increasing popularity and has shown its diagnostic superiority in several studies, ¹¹¹In-diethylenetriaminepentaacetic acid (DTPA)-octreotide is still the current standard for diagnosis of neuroendocrine tumours (NET). The aim of this study was to compare the costs for the two diagnostic tests and the respective consequential costs.     

Comparison of Diagnostic Sensitivity and Quantitative Indices Between 68Ga-DOTATOC PET/CT and 111In-Pentetreotide SPECT/CT in Neuroendocrine Tumors: a Preliminary Report

Purpose

In-pentetreotide has been used for neuroendocrine tumors expressing somatostatin receptors. Recently, ⁶⁸Ga-DOTATOC PET has been used with the advantage of high image quality. In this study, we compared quantitative indices between ¹¹¹In-pentetreotide SPECT/CT and ⁶⁸Ga-DOTATOC PET/CT.

Methods

Thirteen patients diagnosed with neuroendocrine tumors were prospectively recruited. Patients underwent ¹¹¹In-pentetreotide scans with SPECT/CT and ⁶⁸Ga-DOTATOC PET/CT before treatment. The number and location of lesions were analyzed on both imaging techniques to compare lesion detectability. Additionally, the maximal uptake count of each lesion and mean uptake count of the lungs were measured on both imagings, and target-to-normal lung ratios (TNR) were calculated as quantitative indices.

Results

Among 13 patients, 10 exhibited lesions with increased uptake on ¹¹¹In-pentetreotide SPECT/CT and/or ⁶⁸Ga-DOTATOC PET/CT. Scans with SPECT/CT detected 19 lesions, all of which were also detected on PET/CT. Moreover, 16 additional lesions were detected on PET/CT (6 in the liver, 9 in the pancreas and 1 in the spleen). PET/CT exhibited a significantly higher sensitivity than SPECT/CT (100 % vs. 54 %, P < 0.001). TNR was significantly higher on PET/CT than on SPECT/CT (99.9 ± 84.3 vs. 71.1 ± 114.9, P < 0.001) in spite of a significant correlation (r = 0.692, P = 0.01).

Conclusion

Ga-DOTATOC PET/CT has a higher diagnostic sensitivity than ¹¹¹In-pentetreotide scans with SPECT/CT. The TNR on PET/CT is higher than that of SPECT/CT, which also suggests the higher sensitivity of PET/CT. ¹¹¹In-pentetreotide SPECT/CT should be used carefully if it is used instead of ⁶⁸Ga-DOTATOC PET/CT.     

Evaluation of <Superscript>68</Superscript>Ga-DOTATOC PET/MRI for whole-body staging of neuroendocrine tumours in comparison with <Superscript>68</Superscript>Ga-DOTATOC PET/CT

Objectives

To compare the diagnostic performance of ⁶⁸Ga-DOTATOC PET/MRI and ⁶⁸Ga-DOTATOC PET/CT in the whole-body staging of patients with neuroendocrine tumours (NET).

Methods

Thirty patients with histopathologically confirmed NET underwent PET/CT and PET/MRI in a single-injection protocol. PET/CT and PET/MRI scans were prospectively evaluated with regard to lesion count, localization, nature (NET/non-NET), and conspicuity (four-point scale). Histopathology and follow-up imaging served as the reference standards. The proportions of NET and non-NET lesions rated correctly were compared using McNemar’s chi-squared test. The Wilcoxon test was used to assess differences in SUVmax and lesion conspicuity. The correlation between the SUVmax for the same lesions from each modality was analysed using Pearson’s correlation coefficient (r).

Results

According to the reference standard, there were 197 lesions (142 NET, 55 non-NET). Lesion-based analysis showed a higher proportion of correctly rated NET lesions on PET/MRI than on PET/CT (90.8% vs. 86.7%, p?=?0.031), whereas on PET/CT there was a higher proportion of correctly rated non-NET lesions (94.5% vs. 83.6%, p?=?0.031). SUVmax was strongly correlated (r?=?0.86; p?<?0.001) and did not differ significantly (p?=?0.35) between the modalities. Overall conspicuity and NET lesion conspicuity were higher on PET/MRI (both p?<?0.01).

Conclusions

Ga-DOTATOC PET/MRI yielded a higher proportion of correctly rated NET lesions and should be regarded as a valuable alternative to ⁶⁸Ga-DOTATOC PET/CT in whole-body staging of NET patients.

Key Points

? ⁶⁸ Ga-DOTATOC PET/MRI correctly identified more NET lesions than ⁶⁸ Ga-DOTATOC PET/CT. ? ⁶⁸ Ga-DOTATOC PET/MRI provides better NET lesion conspicuity than ⁶⁸ Ga-DOTATOC PET/CT. ? SUVmax values from the two modalities are strongly correlated and do not differ significantly.

     

Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists.

The uptake of radiolabeled somatostatin analogs by tumor cells through receptor-mediated internalization is a critical process for the in vivo targeting of tumoral somatostatin receptors. In the present study, the somatostatin receptor internalization induced by a variety of somatostatin analogs was measured with new immunocytochemical methods that allow characterization of trafficking of the somatostatin receptor subtype 2 (sst2), somatostatin receptor subtype 3 (sst3), and somatostatin receptor subtype 5 (sst5) in vitro at the protein level. METHODS: Human embryonic kidney 293 (HEK293) cells expressing the sst2, sst3, or the sst5 were used in a morphologic immunocytochemical internalization assay using specific sst2, sst3 and sst5 antibodies to qualitatively and quantitatively determine the capability of somatostatin agonists or antagonists to induce somatostatin receptor internalization. In addition, the internalization properties of a selection of these agonists have been compared and quantified in sst2-expressing CHO-K1 cells using an ELISA. RESULTS: Agonists with a high sst2-binding affinity were able to induce sst2 internalization in the HEK293 and CHO-K1 cell lines. New sst2 agonists, such as Y-DOTA-TATE, Y-DOTA-NOC, Lu-DOTA-BOC-ATE (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; TATE is [Tyr3, Thr8]-octreotide; NOC is [1-NaI3]-octreotide; and BOC-ATE is [BzThi3, Thr8]-octreotide), iodinated sugar-containing octreotide analogs, or BIM-23244 were considerably more potent in internalizing sst2 than was DTPA-octreotide (where DTPA is diethylenetriaminepentaacetic acid). Similarly, compounds with high sst3 affinity such as KE108 were able to induce sst3 internalization. In sst2- or sst3-expressing cell lines, agonist-induced receptor internalization was efficiently abolished by sst2- or sst3-selective antagonists, respectively. Antagonists alone had no effect on sst2 or sst3 internalization. We also showed that somatostatin-28 and somatostatin-14 can induce sst5 internalization. Unexpectedly, however, potent sst5 agonists such as KE108, BIM-23244, and L-817,818 were not able to induce sst5 internalization under the same conditions. CONCLUSION: Using sensitive and reproducible immunocytochemical methods, the ability of various somatostatin analogs to induce sst2, sst3, and sst5 internalization has been qualitatively and quantitatively determined. Whereas all agonists triggered sst2 and sst3 internalization, sst5 internalization was induced by natural somatostatin peptides but not by synthetic high-affinity sst5 agonists. Such assays will be of considerable help for the future characterization of ligands foreseen for nuclear medicine applications.     

Detection of unknown primary neuroendocrine tumours (CUP-NET) using 68Ga-DOTA-NOC receptor PET/CT

Purpose

This bi-centric study aimed to determine the role of receptor PET/CT using ⁶⁸Ga-DOTA-NOC in the detection of undiagnosed primary sites of neuroendocrine tumours (NETs) and to understand the molecular behaviour of the primarily undiagnosed tumours.

Methods

Overall 59 patients (33 men and 26 women, age: 65?±?9 years) with documented NET and unknown primary were enrolled. PET/CT was performed after injection of approximately 100 MBq (46–260 MBq) of ⁶⁸Ga-DOTA-NOC. The maximum standardised uptake values (SUV_max) were calculated and compared with SUV_max in known pancreatic NET (pNET) and ileum/jejunum/duodenum (SI-NET). The results of PET/CT were also correlated with CT alone.

Results

In 35 of 59 patients (59%), ⁶⁸Ga-DOTA-NOC PET/CT localised the site of the primary: ileum/jejunum (14), pancreas (16), rectum/colon (2), lungs (2) and paraganglioma (1). CT alone (on retrospective analyses) confirmed the findings in 12 of 59 patients (20%). The mean SUV_max of identified previously unknown pNET and SI-NET were 18.6?±?9.8 (range: 7.8–34.8) and 9.1?±?6.0 (range: 4.2–27.8), respectively. SUV_max in patients with previously known pNET and SI-NET were 26.1?±?14.5 (range: 8.7–42.4) and 11.3?±?3.7 (range: 5.6–17.9). The SUV_max of the unknown pNET and SI-NET were significantly lower (p?<?0.05) as compared to the ones with known primary tumour sites; 19% of the patients had high-grade and 81% low-grade NET. Based on ⁶⁸Ga-DOTA-NOC receptor PET/CT, 6 of 59 patients were operated and the primary was removed (4 pancreatic, 1 ileal and 1 rectal tumour) resulting in a management change in approximately 10% of the patients. In the remaining 29 patients, because of the far advanced stage of the disease (due to distant metastases), the primary tumours were not operated. Additional histopathological sampling was available from one patient with bronchial carcinoid (through bronchoscopy).

Conclusion

Our data indicate that ⁶⁸Ga-DOTA-NOC PET/CT is highly superior to ¹¹¹In-OctreoScan (39% detection rate for CUP according to the literature) and can play a major role in the management of patients with CUP-NET.