The Wnt pathway protein Dvl1 targets somatostatin receptor 2 for lysosome-dependent degradation.

The Somatostatin receptor 2 (Sstr2) is a heterotrimeric G protein-coupled receptor that is highly expressed in neuroendocrine tumors and is a common pharmacological target for intervention. Unfortunately, not all neuroendocrine tumors express Sstr2, and Sstr2 expression can be downregulated with prolonged agonist use. Sstr2 is rapidly internalized following agonist stimulation and, in the short term, is quantitatively recycled back to the plasma membrane. However, mechanisms controlling steady state expression of Sstr2 in the absence of agonist are less well described. Here, we show that Sstr2 interacts with the Wnt pathway protein Dvl1 in a ligand-independent manner to target Sstr2 for lysosomal degradation. Interaction of Sstr2 with Dvl1 does not affect receptor internalization, recycling, or signaling to adenylyl cyclase but does suppress agonist-stimulated ERK1/2 activation. Importantly, Dvl1-dependent degradation of Sstr2 can be stimulated by overexpression of Wnts and treatment of cells with Wnt pathway inhibitors can boost Sstr2 expression in neuroendocrine tumor cells. Taken together, this study identifies for the first time a mechanism that targets Sstr2 for lysosomal degradation that is independent of Sstr2 agonist and can be potentiated by Wnt ligand. Intervention in this signaling mechanism has the potential to elevate Sstr2 expression in neuroendocrine tumors and enhance Sstr2-directed therapies.

Structural modifications toward improved lead-203/lead-212 peptide-based image-guided alpha-particle radiopharmaceutical therapies for neuroendocrine tumors.

PURPOSE: The lead-203 (203Pb)/lead-212 (212Pb) elementally identical radionuclide pair has gained significant interest in the field of image-guided targeted alpha-particle therapy for cancer. Emerging evidence suggests that 212Pb-labeled peptide-based radiopharmaceuticals targeting somatostatin receptor subtype 2 (SSTR2) may provide improved effectiveness compared to beta-particle-based therapies for neuroendocrine tumors (NETs). This study aims to improve the performance of SSTR2-targeted radionuclide imaging and therapy through structural modifications to Tyr3-octreotide (TOC)-based radiopharmaceuticals. METHODS: New SSTR2-targeted peptides were designed and synthesized with the goal of optimizing the incorporation of Pb isotopes through the use of a modified cyclization technique; the introduction of a Pb-specific chelator (PSC); and the insertion of polyethylene glycol (PEG) linkers. The binding affinity of the peptides and the cellular uptake of 203Pb-labeled peptides were evaluated using pancreatic AR42J (SSTR2+) tumor cells and the biodistribution and imaging of the 203Pb-labeled peptides were assessed in an AR42J tumor xenograft mouse model. A lead peptide was identified (i.e., PSC-PEG2-TOC), which was then further evaluated for efficacy in 212Pb therapy studies. RESULTS: The lead radiopeptide drug conjugate (RPDC) - [203Pb]Pb-PSC-PEG2-TOC - significantly improved the tumor-targeting properties, including receptor binding and tumor accumulation and retention as compared to [203Pb]Pb-DOTA0-Tyr3-octreotide (DOTATOC). Additionally, the modified RPDC exhibited faster renal clearance than the DOTATOC counterpart. These advantageous characteristics of [212Pb]Pb-PSC-PEG2-TOC resulted in a dose-dependent therapeutic effect with minimal signs of toxicity in the AR42J xenograft model. Fractionated administrations of 3.7 MBq [212Pb]Pb-PSC-PEG2-TOC over three doses further improved anti-tumor effectiveness, resulting in 80% survival (70% complete response) over 120 days in the mouse model. CONCLUSION: Structural modifications to chelator and linker compositions improved tumor targeting and pharmacokinetics (PK) of 203/212Pb peptide-based radiopharmaceuticals for NET theranostics. These findings suggest that PSC-PEG2-TOC is a promising candidate for Pb-based targeted radionuclide therapy for NETs and other types of cancers that express SSTR2.

Combining [177Lu]Lu-DOTA-TOC PRRT with PARP inhibitors to enhance treatment efficacy in small cell lung cancer.

PURPOSE: Small cell lung cancer (SCLC) is a highly aggressive tumor with neuroendocrine origin. Although SCLC frequently express somatostatin receptor type 2 (SSTR2), a significant clinical benefit of SSTR2-targeted radionuclide therapies of SCLC was not observed so far. We hypothesize that combination treatment with a PARP inhibitor (PARPi) could lead to radiosensitization and increase the effectiveness of SSTR2-targeted therapy in SCLC. METHODS: SSTR2-ligand uptake of the SCLC cell lines H69 and H446 was evaluated in vitro using flow cytometry, and in vivo using SPECT imaging and cut-and-count biodistribution. Single-agent (Olaparib, Rucaparib, [177Lu]Lu-DOTA-TOC) and combination treatment responses were determined in vitro via cell viability, clonogenic survival and γH2AX DNA damage assays. In vivo, we treated athymic nude mice bearing H69 or H446 xenografts with Olaparib, Rucaparib, or [177Lu]Lu-DOTA-TOC alone or with combination treatment regimens to assess the impact on tumor growth and survival of the treated mice. RESULTS: H446 and H69 cells exhibited low SSTR2 expression, i.e. 60 to 90% lower uptake of SSTR2-ligands compared to AR42J cells. In vitro, combination treatment of [177Lu]Lu-DOTA-TOC with PARPi resulted in 2.9- to 67-fold increased potency relative to [177Lu]Lu-DOTA-TOC alone. We observed decreased clonogenic survival and higher amounts of persistent DNA damage compared to single-agent treatment for both Olaparib and Rucaparib. In vivo, tumor doubling times increased to 1.6-fold (H446) and 2.2-fold (H69) under combination treatment, and 1.0 to 1.1-fold (H446) and 1.1 to 1.7-fold (H69) in monotherapies compared to untreated animals. Concurrently, median survival was higher in the combination treatment groups in both models compared to monotherapy and untreated mice. Fractionating the PRRT dose did not lead to further improvement of therapeutic outcome. CONCLUSION: The addition of PARPi can markedly improve the potency of SSTR2-targeted PRRT in SCLC models in SSTR2 low-expressing tumors. Further evaluation in humans seems justified based on the results as novel treatment options for SCLC are urgently needed.

Nanotechnology-Based Strategy for Enhancing Therapeutic Efficacy in Pancreatic Cancer: Receptor-Targeted Drug Delivery by Somatostatin Analog.

A novel nanotechnology-based drug delivery system (DDS) targeted at pancreatic cancer cells was developed, characterized, and tested. The system consisted of liposomes as carriers, an anticancer drug (paclitaxel) as a chemotherapeutic agent, and a modified synthetic somatostatin analog, 5-pentacarbonyl-octreotide, a ligand for somatostatin receptor 2 (SSTR2), as a targeting moiety for pancreatic cancer. The cellular internalization, cytotoxicity, and antitumor activity of the DDS were tested in vitro using human pancreatic ductal adenocarcinoma (PDAC) cells with different expressions of the targeted SSTR2 receptors, and in vivo on immunodeficient mice bearing human PDAC xenografts. The targeted drug delivery system containing paclitaxel exhibited significantly enhanced cytotoxicity compared to non-targeted DDS, and this efficacy was directly related to the levels of SSTR2 expression. It was found that octreotide-targeted DDS proved exceptionally effective in suppressing the growth of PDAC tumors. This study underscores the potential of octreotide-targeted liposomal delivery systems to enhance the therapeutic outcomes for PDAC compared with non-targeted liposomal DDS and Paclitaxel-Cremophor® EL, suggesting a promising avenue for future cancer therapy innovations.

Pituitary Adenoma: SSTR2 rs2236750, SSTR5 rs34037914, and AIP rs267606574 Genetic Variants, Serum Levels, and Ki-67 Labeling Index Associations.

Background and Objectives: This study explores the complex pathogenesis of pituitary adenomas (PAs), prevalent intracranial tumors in the pituitary gland. Despite their generally benign nature, PAs exhibit a diverse clinical spectrum involving hormone hypersecretion and varying invasiveness, hinting at multifaceted molecular mechanisms and abnormalities in tumorigenesis and gene regulation. Materials and Methods: The investigation focuses on the Ki-67 labeling index, SSTR2 rs2236750, SSTR5 rs34037914, and AIP rs267606574 polymorphisms, alongside serum levels of SSTR2, SSTR5, and AIP, to discern their association with PAs. The Ki-67 labeling index was assessed using immunohistochemical analysis with the monoclonal antibody clone SP6, representing the percentage of tumor cells showing positive staining. Genotyping was performed via real-time polymerase chain reaction, and serum levels were analyzed using ELISA. The study included 128 PA patients and 272 reference group subjects. Results: The results derived from binary logistic regression analysis revealed an intriguing correlation between the SSTR2 rs2236750 AG genotype and approximately a 1.6-fold increased likelihood of PA occurrence. When analyzing SSTR5 rs34037914, statistically significant differences were found between Micro-PA and the reference group (p = 0.022). Additionally, the SSTR5 rs34037914 TT genotype, compared with CC + CT, under the most robust genetic model (selected based on the lowest AIC value), was associated with a 12-fold increased odds of Micro-PA occurrence. However, it is noteworthy that after applying Bonferroni correction, these findings did not retain statistical significance. Conclusions: Consequently, while this study hinted at a potential link between SSTR2 rs2236750 and pituitary adenoma development, as well as a potential link between SSTR5 rs34037914 and Micro-PA development, it underscored the need for further analysis involving a larger cohort to robustly validate these findings.

Atretic cephalocele and encephalocele: A single-institution clinicopathological study.

BACKGROUND: Encephaloceles are neural tube defects characterized by herniation of meninges, neural tissue and cerebrospinal fluid, while atretic cephaloceles denote a rudimentary connection to the intracranial space with absence of herniated neural tissue and represent an infrequent dermatopathologic diagnosis. Limited reports of these entities confound the challenge in their histopathologic distinction. Accurate classification is important given associated anomalies and neurologic manifestations that impact prognosis. METHODS: We describe the clinicopathological and immunohistochemical [glial fibrillary acidic protein (GFAP), S100, epithelial membrane antigen (EMA), and somatostatin receptor subtype 2A (SSTR2A)] features in a retrospective series encountered at a single institution between 1994 and 2020. RESULTS: We identified 13 cases classified as atretic cephalocele (n = 11) and encephalocele (n = 2). Hamartomatous changes and multinucleated cells were unique to atretic cephaloceles while myxoid areas were unique to encephaloceles. At least focal staining for SSTRA was seen in all atretic cephaloceles with the majority (87.5%) staining for EMA; negative staining for GFAP and S100 confirmed absence of neural tissue. Encephaloceles were GFAP and S100 positive, and negative for SSTR2 and EMA. Atretic cephaloceles had a favorable prognosis compared to encephaloceles, with severe morbidity present in both encephalocele cases. CONCLUSION: Our study raises awareness of atretic cephalocele and encephalocele among dermatopathologists and reveals a mutually exclusive immunophenotype that facilitates their distinction for prognostication and management.

Somatostatin receptor imaging with [68Ga]Ga-DOTATATE positron emission tomography/computed tomography (PET/CT) in patients with nasopharyngeal carcinoma.

PURPOSE: To explore the feasibility of [68Ga]Ga-DOTATATE positron emission tomography/computed tomography (PET/CT) in patients with non-keratinizing nasopharyngeal carcinoma (NPC) and to evaluate whether [68Ga]Ga-DOTATATE PET/CT could be used for non-invasive determination of somatostatin receptor 2 (SSTR2) expression in NPC. METHODS: This prospective study included patients with NPC who underwent [68Ga]Ga-DOTATATE PET/CT between February and May 2021. The [68Ga]Ga-DOTATATE and [18F]FDG uptakes in primary and metastatic NPC lesions were calculated and compared, and the [68Ga]Ga-DOTATATE uptake between SSTR2 score groups was analysed. RESULTS: A total of 36 participants (25 patients, initial staging; 11 patients, recurrence detection) were included; 33 patients also underwent [18F]FDG PET/CT for staging/restaging as a part of their routine diagnostic workup. [68Ga]Ga-DOTATATE PET/CT showed an intense tracer uptake in primary and metastatic NPC lesions. The radiotracer uptake was higher with [68Ga]Ga-DOTATATE than with [18F]FDG PET in primary NPC lesions (SUVmax: 12.03 vs. 10.07, P = 0.048; tumour-to-brain ratio: 36.16 vs. 0.86, P < 0.001) and regional lymph node metastases (median SUVmax: 9.11 vs. 6.12, P < 0.001) and comparable in bone and visceral metastases. Importantly, most NPC lesions showed intense SSTR2 expression (85.7%), which was strongly correlated with the [68Ga]Ga-DOTATATE uptake. The SUVmax of SSTR2-negative lesions was significantly lower than that of SSTR2-positive lesions (SUVmax: 4.95 vs. 12.61, P = 0.013). CONCLUSION: [68Ga]Ga-DOTATATE PET/CT is a promising imaging modality for detecting primary and metastatic NPC, with favourable image contrast and comparable diagnostic efficacy when compared to [18F]FDG PET/CT. An intense SSTR2 expression was observed in most NPCs, and this expression was significantly correlated with the [68Ga]Ga-DOTATATE uptake.

TGF-ß1/Smad2/3 signaling pathway modulates octreotide antisecretory and antiproliferative effects in pituitary somatotroph tumor cells.

Octreotide (OCT) is used to inhibit hormone secretion and growth in somatotroph tumors, although a significant percentage of patients are resistant. It has also been tested in nonfunctioning (NF) tumors but with poor results, with these outcomes having been associated with SSTR2 levels and impaired signaling. We investigated whether OCT inhibitory effects can be improved by TGF-ß1 in functioning and nonfunctioning somatotroph tumor cells. OCT effects on hormone secretion and proliferation were analyzed in the presence of TGF-ß1 in WT and SSTR2-overexpressing secreting GH3 and silent somatotroph tumor cells. The mechanism underlying these effects was assessed by studying SSTR and TGFßR signaling pathways mediators. In addition, we analyzed the effects of OCT/TGF-ß1 treatment on tumor growth and cell proliferation in vivo. The inhibitory effects of OCT on GH- and PRL-secretion and proliferation were improved in the presence of TGF-ß1, as well as by SSTR2 overexpression. The OCT/TGF-ß1 treatment induced downregulation of pERK1/2 and pAkt, upregulation of pSmad3, and inhibition of cyclin D1. In vivo experiments showed that OCT in the presence of TGF-ß1 blocked tumor volume growth, decreased cell proliferation, and increased tumor necrosis. These results indicate that SSTR2 levels and the stimulation of TGF-ß1/TGFßR/Smad2/3 pathway are important for strengthening the antiproliferative and antisecretory effects of OCT.

Transcriptome analysis of the transdifferentiation of canine BMSCs into insulin producing cells.

BACKGROUND: Bone marrow mesenchymal stem cells are a potential resource for the clinical therapy of certain diseases. Canine, as a companion animal, living in the same space with human, is an ideal new model for human diseases research. Because of the high prevalence of diabetes, alternative transplantation islets resource (i.e. insulin producing cells) for diabetes treatment will be in urgent need, which makes our research on the transdifferentiation of Bone marrow mesenchymal stem cells into insulin producing cells become more important. RESULT: In this study, we completed the transdifferentiation process and achieved the transcriptome profiling of five samples with two biological duplicates, namely, "BMSCs", "islets", "stage 1", "stage 2" and "stage 3", and the latter three samples were achieved on the second, fifth and eighth day of induction. A total of 11,530 differentially expressed transcripts were revealed in the profiling data. The enrichment analysis of differentially expressed genes revealed several signaling pathways that are essential for regulating proliferation and transdifferentiation, including focal adhesion, ECM-receptor interaction, tight junction, protein digestion and absorption, and the Rap1 signaling pathway. Meanwhile, the obtained protein-protein interaction network and functional identification indicating involvement of three genes, SSTR2, RPS6KA6, and VIP could act as a foundation for further research. CONCLUSION: In conclusion, to the best of our knowledge, this is the first survey of the transdifferentiation of canine BMSCs into insulin-producing cells according with the timeline using next-generation sequencing technology. The three key genes we pick out may regulate decisive genes during the development of transdifferentiation of insulin producing cells.

A Cyanine-Bridged Somatostatin Hybrid Probe for Multimodal SSTR2 Imaging in Vitro and in Vivo: Synthesis and Evaluation.

Multimodal imaging probes have attracted the interest of ongoing research, for example, for the surgical removal of tumors. Modular synthesis approaches allow the construction of hybrid probes consisting of a radiotracer, a fluorophore and a targeting unit. We present the synthesis of a new asymmetric bifunctional cyanine dye that can be used as a structural and functional linker for the construction of such hybrid probes. 68 Ga-DOTATATE, a well-characterized radiopeptide targeting the overexpressed somatostatin receptor subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our cyanine dye, thus providing additional information along with the data obtained from the radiotracer. We tested the SSTR2-targeting and imaging properties of the resulting probe 68 Ga-DOTA-ICC-TATE in vitro and in a tumor xenograft mouse model. Despite the close proximity between dye and pharmacophore, we observed a high binding affinity towards SSTR2 as well as elevated uptake in SSTR2-overexpressing tumors in the positron emission tomography (PET) scan and histological examination.

Design, synthesis, and biological evaluation of novel somatostatin receptor subtype-2 agonists: Optimization for potency and risk mitigation of hERG and phospholipidosis.

Somatostatin receptors are members of G-protein coupled receptor superfamily. Receptors can be classified into five subtypes, SSTR1 to 5. The highly potent and orally active SSTR2 agonist 7, which had been identified by our group, was found out to have toxicological liabilities such as hERG inhibition and phospholipidosis (PLD). We investigated the relationship between in silico physicochemical properties and hERG and PLD, and explored well-balanced agonists to identify amide 19 and benzimidazole 30. As a result of this exploration, we found out that the value of (cLogP) [2] + (pKa) [2] needs to be less than 110 to mitigate the liabilities.

Evaluation of 68Ga-DOTATATE uptake at the pituitary region and the biochemical response to somatostatin analogs in acromegaly.

PURPOSE: Acromegaly is associated with many comorbidities and increased mortality. The first-line treatment is transsphenoidal surgery. However, many patients also need adjuvant drug treatment after surgery. Somatostatin analog (SSA), which suppresses GH secretion by somatotrophs by binding to the SSTR2 receptor, is the first choice. Nevertheless, 50% of patients are partially or totally resistant to SSA, so predictive factors of response are helpful to individualize drug treatment. 68GaDOTATATE PET/CT has emerged as the gold-standard method in the diagnosis and follow-up of gastroenteropancreatic neuroendocrine tumors, which also express SSTR. Our objective was to evaluate whether 68Ga-DOTATATE uptake (SUV max) at the pituitary region of patients on SSA therapy would be useful as a drug response predictor without the need of tumoral tissue. METHODS: Fifteen acromegalics patients on SSA treatment for at least 6 months were underwent to 68Ga-DOTATATE PET/CT at the nuclear medicine service. There was an SSA complete response group (n = 5), defined as GH < 1 µg/L and IFG-1 in the normal range for gender and age, and a group that did not meet these criteria (n = 10). RESULTS: As a result, we did not find out a significantly higher SUV max in the complete response group (p = 0.0576) to SSA. However, we found a significant inverse relationship between postoperative GH values and the SUVmax at the sella turcica (p = 0.0188), probably reflecting tumor SSTR2 expression. CONCLUSION: Thus, after this initial evaluation, 68GaDOTATATE PET/CT should be better studied to assess its usefulness in the follow-up of acromegalic patients.

A new pathological classification of intrahepatic cholangiocarcinoma according to protein expression of SSTR2 and Bcl2.

BACKGROUND: No universal classification method for intrahepatic cholangiocarcinoma (IHCC) has been reported based on the embryological origin of biliary epithelial cells. The aim of this study was to classify IHCC according to protein expression levels of somatostatin receptor 2 (SSTR2) and b-cell leukemia/lymphoma 2 (Bcl2) and to elucidate the clinicopathological features of each group. METHODS: Fifty-two IHCC patients who underwent hepatic resection were enrolled in this study. Protein expression levels of SSTR2 and Bcl2 were examined using immunohistochemistry. Clinicopathological factors were compared between the three groups and prognostic factors were investigated. RESULTS: The patients were divided into three groups: SSTR2 positive and Bcl2 negative (p-Group H, n = 21), SSTR2 negative and Bcl2 positive (p-Group P, n = 14), and the indeterminate group (p-Group U, n = 17) for cases where SSTR2 and Bcl2 were both positive or both negative. All p-Group P cases displayed curability A or B. The 5-year survival rates of p-Group H and U patients were worse than those in p-Group P. p-Group H had higher T-factor, clinical stage, and incidence of periductal infiltration than p-Group P. CONCLUSIONS: This method could be used to classify IHCC into peripheral and perihilar type by embryological expression patterns of SSTR2 and Bcl2.

Perineurioma versus meningioma. A multi-institutional immunohistochemical and ultrastructural studY.

The differential diagnosis between perineurioma (PN) and meningioma (MEN) can be difficult by histology and immunohistochemistry (IHC) because the perineurium and arachnoid have the same embryological origin. However, there are no comparative studies determining conclusive parameters for the differential diagnosis. The aim of this study is to compare IHC of PN and MEN and their ultra-structural characteristics to elucidate which are the useful data that allow differentiate both entities. Thirty-five MEN were analyzed, and 15 PN, (11 skin and soft tissues and four oral cavity). IHC for epithelial membrane antigen (EMA), Claudin-1, GLUT-1, somatostatin-2 receptor (SSTR-2), and progesterone receptor (RP) was performed. Ultrastructural studies were performed on 8 MEN and 15 PN. Only in PN Claudin-1 was positive in 9/11 (90%) cases and GLUT-1 in 7/11 (63%) cases. In MEN, the progesterone receptor was expressed in 21/35 (60%) cases and no case expressed Claudin-1 and GLUT-1; EMA was expressed in all MEN cases and 93% of PN. SSTR-2 was expressed weakly in six cases of MEN (17%), and it was not considered useful for differential diagnosis. On ultrastructure, PN showed thin and parallel processes, some caveolae, and lacked cell junctions. The cellular processes were surrounded by a collagenous stroma in 94% of the cases. MEN were characterized by curved cytoplasmic cell processes showing desmosomes in 75% of cases. Ultrastructural findings aid in the differential diagnosis between PN and MEN, especially if molecular studies are not available.

Clinicopathologic features and BRAF mutation status of tracheal glomus tumors - Characterization of 4 cases and the distinction from low-grade neuroendocrine tumors.

BACKGROUND: Glomus tumors are uncommon and mostly benign mesenchymal neoplasms of the perivascular family. To date, only a few cases of glomus tumors occurring in the trachea have been reported. Tracheal glomus tumors simulated low-grade neuroendocrine tumors on clinical and histomorphological examination, so the differential diagnosis between these two entities is very necessary. The latest studies showed that BRAF mutation may be associated with a malignant phenotype of glomus tumors. METHODS: We investigated the clinical, histopathologic, immunohistochemical, and BRAF V600E mutation status of four cases of tracheal glomus tumors. RESULTS: The cases showed a female predilection (male:female, 1:3) with a median age of 35.5. All of the cases had the typical morphological characteristics of glomus tumors, such as uniform round tumor cells with nest-like distribution surrounding thin-walled vessels; two of them met the malignant diagnostic criteria based on the 5th edition of WHO classification, including marked nuclear atypia and any level of mitotic activity. Immunohistochemistry showed diffusely positive for vimentin (4/4), α-SMA (4/4) and collagen IV (4/4), variably reactive for synaptophysin (3/4) and SSTR2 (2/2), and negative for AE1/AE3 (0/4) and chromogranin A (0/4). Three tested cases harbored no BRAF V600E mutation. Three follow-up cases were alive and free of disease with an average follow-up of 89.3 months. CONCLUSIONS: Tracheal glomus tumors are rare mesenchymal tumors that have overlapping morphologic and immunohistochemical features with neuroendocrine neoplasms. Our cases highlight the importance of careful histomorphological examination and comprehensive immunohistochemical study in reaching a correct diagnosis of glomus tumors of the trachea. Other than BRAF mutation, malignant glomus tumors may have a complex mutational profile.

Quantitative digital image analysis of somatostatin receptor 2 immunohistochemistry in pancreatic neuroendocrine tumors.

Immunohistochemical analysis of somatostatin receptor 2 (SSTR2) provides important information regarding the potential therapeutic efficacy of somatostatin analogues (SSAs) in patients with neuroendocrine tumors. HER2 scoring has been proposed to interpret SSTR2 immunoreactivity but their reproducibility was relatively low because of its intrinsic subjective nature. Digital image analysis (DIA) has recently been proposed as an objective and more precise method of evaluating immunoreactivity. Therefore, in this study, we used DIA for analyzing SSTR2 immunoreactivity in pancreatic neuroendocrine tumors (PanNETs) to obtain its H score and "(%) strong positive cells" and compared the results with those of manually obtained HER2 scores. Membranous SSTR2 immunoreactivity evaluated by DIA was calculated by two scales as: "Membrane Optical Density" and "Minimum Membrane Completeness". PanNETs with HER2 score of > 2 demonstrated the highest concordance with results of "(%) strong positive cells" obtained by DIA when "Minimum Membrane Completeness" was tentatively set at 80%. The SSTR2 immunoreactivity, evaluated based on all scoring systems, was different between grades G1 and G2 in insulinoma but not in non-functional PanNETs. DIA provided reproducible results of SSTR2 immunoreactivity in PanNETs and yielded important information as to the potential application of SSAs.

Pharmacokinetic analysis of [68Ga]Ga-DOTA-TOC PET in meningiomas for assessment of in vivo somatostatin receptor subtype 2.

PURPOSE: DOTA-D-Phe1-Tyr3-octreotide with gallium-68 ([68Ga]Ga-DOTA-TOC) is one of the PET tracers that forms the basis for peptide receptor radionuclide therapy based on somatostatin receptor subtype 2 (SSTR2) expression in meningiomas. Yet, the quantitative relationship between [68Ga]Ga-DOTA-TOC accumulation and SSTR2 is unknown. We conducted a correlative analysis of a range of [68Ga]Ga-DOTA-TOC PET metric(s) as imaging surrogate(s) of the receptor binding in meningiomas by correlating the PET results with SSTR2 expression from surgical specimens. We additionally investigated possible influences of secondary biological factors such as vascularization, inflammation and proliferation. METHODS: Fifteen patients with MRI-presumed or recurrent meningiomas underwent a 60-min dynamic [68Ga]Ga-DOTA-TOC PET/CT before surgery. The PET data comprised maximum and mean standardized uptake values (SUVmax, SUVmean) with and without normalization to reference regions, and quantitative measurements derived from kinetic modelling using a reversible two-tissue compartment model with the fractional blood volume (VB). Expressions of SSTR2 and proliferation (Ki-67, phosphohistone-H3, proliferating cell nuclear antigen) were determined by immunohistochemistry and/or quantitative polymerase chain reaction (qPCR), while biomarkers of vascularization (vascular endothelial growth factor A (VEGFA), endothelial marker CD34) and inflammation (cytokine interleukin-18, microglia/macrophage-specific marker CD68) by qPCR. RESULTS: Histopathology revealed 12 World Health Organization (WHO) grade I and three WHO grade II meningiomas showing no link to SSTR2. The majority of [68Ga]Ga-DOTA-TOC PET metrics showed significant associations with SSTR2 protein, while all PET metrics were positively correlated with SSTR2 mRNA with the best results for mean tumour-to-blood ratio (TBRmean) (r = 0.757, P = 0.001) and SUVmean (r = 0.714, P = 0.003). Significant positive correlations were also found between [68Ga]Ga-DOTA-TOC PET metrics, and VEGFA and VB. SSTR2 mRNA was moderately correlated with VEGFA (r = 0.539, P = 0.038). Neither [68Ga]Ga-DOTA-TOC PET metrics nor SSTR2 were correlated with proliferation or inflammation. CONCLUSION: [68Ga]Ga-DOTA-TOC accumulation in meningiomas is associated with SSTR2 binding and vascularization with TBRmean being the best PET metric for assessing SSTR2.

Advances in Molecular Classification and Therapeutic Opportunities in Meningiomas.

PURPOSE OF REVIEW: Our understanding of the genetic and epigenetic alterations in meningioma and the underlying tumor biology of meningioma has significantly changed over the past decade and resulted in revision of prognostically relevant meningioma subclasses within and beyond the WHO classification of CNS tumors. RECENT FINDINGS: The 2016 WHO classification of CNS tumors recognizes WHO grade I, II, and III based on histopathological features. Recent work has identified genetic alterations with prognostic implications, including mutations of the TERT promoter, loss of function of the DMD gene, and inactivation of the tumor suppressor BAP-1. Studies of DNA methylation patterns in meningiomas have resulted in a novel and prognostically relevant meningioma subclassification schema. There have been major advances in our understanding of prognostically relevant genetic and epigenetic changes in meningioma which will hopefully allow for improvement in clinical trial design and the development of more effective therapies for meningioma.

Clinicopathologic characteristics and novel biomarkers of aggressive B-cell lymphomas in the nasopharynx.

BACKGROUND: The most common nasopharyngeal lymphoma in the United States are B-cell non-Hodgkin lymphomas (B-NHL). Relatively little is known about the clinicopathologic features of these cases. In this study, we characterize a bi-institutional cohort of aggressive B-NHL primary to the nasopharyngeal area. We compare and contrast EBV-positive versus EBV-negative cases and evaluate expression of SSTR2, CD30, and PD-L1, potential markers for targeted therapeutics. METHODS AND RESULTS: We retrieved 53 cases of aggressive B-NHL from the two institutions. Staining was performed for in situ EBV (EBER), CD30, SSTR2 and PD-L1. The response to initial therapy, disease-free interval, and survival at two- and five-year following initial diagnosis were used as primary clinical outcome. Overall, 13 out of 53 cases (23%) were EBV positive. CD30 expression was more frequent in EBV-positive than in EBV-negative cases (4/6 vs 1/17). Seven of 14 (50%) cases tested demonstrated expression of PD-L1 within tumor cells; the two EBV-positive DLBCL tested showed substantial PD-L1 reactivity. Six of 15 (40%) cases tested were positive for SSTR2. The three EBV-positive patients with available outcome data died within one year of diagnosis; in contrast, the EBV-negative cases showed survival rate of 100% (8/8) and 83% (5/6) at two- and five-year follow-up, respectively. DISCUSSION: The aggressive B-NHLs of the nasopharynx show differences between EBV-positive versus EBV-negative cases. The association of EBV-positive cases with expression of CD30 and PD-L1 may be particularly informative for targeted therapies. A significant number of cases expresses SSTR2, which could render them susceptible to somatostatin analogue and peptide receptor radionuclide therapies. Finally, our limited case series suggest that EBV negativity may be associated with a better prognosis.

The role of somatostatin in GLP-1-induced inhibition of glucagon secretion in mice.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) receptor agonists are currently used for the treatment of type 2 diabetes. Their main mechanism of action is enhancement of glucose-induced insulin secretion (from increased beta cell glucose sensitivity) and inhibition of glucagon secretion. The latter has been demonstrated to account for about half of their blood glucose-lowering activity. Whereas the effect of GLP-1 on insulin secretion is clearly dependent on ambient glucose concentrations and has been described in detail, the mechanism responsible for the inhibitory effect of GLP-1 on glucagon secretion is heavily debated. Glucagon inhibition is also said to be glucose-dependent, although it is unclear what is meant by this. We hypothesise here that GLP-1 does not inhibit glucagon secretion during hypoglycaemia because the inhibition depends on somatostatin secretion, which in turn is dependent on glucose levels. METHODS: We used the perfused mouse pancreas model to investigate this hypothesis. RESULTS: We found that, in this model, GLP-1 was able to significantly inhibit glucagon secretion from pancreatic alpha cells at all glucose levels tested: 6.0, 1.5 and 0.5 mmol/l (-27.0%, -37.1%, and -23.6%, respectively), and the decrease in glucagon secretion was invariably accompanied by an increase in somatostatin secretion (+286.8%, +158.7%, and +118.8%, respectively). Specific blockade of somatostatin receptor 2 increased glucagon secretion (+118.8% at 1.5 mmol/l glucose and +162.9% at 6.0 mmol/l glucose) and completely eliminated the inhibitory effect of GLP-1. CONCLUSIONS/INTERPRETATION: We have shown here that the glucagon-lowering effect of GLP-1 is entirely mediated through the paracrine actions of somatostatin in the perfused mouse pancreas. However, in this model, the inhibitory effect of GLP-1 was preserved at hypoglycaemic levels, leaving unanswered the question of how this is avoided in vivo in individuals treated with GLP-1 receptor agonists.