SSTR2 Expression Research Articles

Effects of Somatostatin Analogs on Catecholamine Biosynthesis Regulated by Corticosteroids and Bone Morphogenetic Proteins in Rat Pheochromocytoma Cells

Regulatory roles of SSAs in catecholamine synthesis have not been elucidated. To clarify the actions of SSAs on catecholamine biosynthesis, we investigated the mutual interactions among SSAs including octreotide and pasireotide, steroids and BMPs using rat pheochromocytoma PC12 cells. Treatment with octreotide and pasireotide (10 nM to 10 μM) had no significant effect on mRNA levels of Th, DOPA decarboxylase and dopamine-β-hydroxylase in PC12 cells. Regarding the interaction with steroids, treatments with SSAs also had no effect on dexamethasone- or aldosterone-induced Th mRNA expression, while pasireotide reduced mRNA expression of the GR. As for the interaction with BMP-4, which can suppress Th mRNA expression by PC12 cells, SSAs did not affect Th expression reduced by BMP-4 and Id1 or Smad1/5/9 activation induced by BMP-4. However, BMP-4 treatment up-regulated MR expression, while treatment with noggin, which neutralizes endogenous BMPs, downregulated MR expression, and the presence of noggin also attenuated aldosterone-induced Th expression, suggesting that endogenous BMPs act to enhance MR activity. Moreover, BMP-4 treatment suppressed the expression of somatostatin receptors including Sstr2 and Sstr5 in PC12 cells, while treatment with noggin up-regulated the expression of Sstr2 and Sstr5, suggesting that BMPs play a desensitizing role in SSA actions. Collectively, the results revealed that SSAs have no direct effect on catecholamine synthesis; however, adrenomedullar BMPs could be modulators for the responsiveness to MR and SSTRs.

Enhancing the anti-tumour activity of 177Lu-DOTA-octreotate radionuclide therapy in somatostatin receptor-2 expressing tumour models by targeting PARP

Peptide receptor radionuclide therapy (PRRT) is an important treatment option for patients with somatostatin receptor-2 (SSTR2)-expressing neuroendocrine tumour (NET) though tumour regression occurs in only a minority of patients. Therefore, novel PRRT regimens with improved therapeutic activity are needed. Radiation induced DNA damage repair is an attractive therapeutic target to increase PRRT efficacy and consequently, we have characterised a panel of preclinical models for their SSTR2 expression, in vivo growth properties and response to 177Lu-DOTA-octreotate (LuTate) PRRT to identify models with features suitable for evaluating novel therapeutic combinations. In vitro studies using the SSTR2 expressing AR42J model demonstrate that the combination of LuTate and the small molecule Poly(ADP-ribose) polymerase-1 (PARP) inhibitor, talazoparib led to increased DNA double strand breaks, as assessed by γ-H2AX foci formation, as compared to LuTate alone. Furthermore, using the AR42J tumour model in vivo we demonstrate that the combination of LuTate and talazoparib significantly improved the anti-tumour efficacy of LuTate alone. These findings support the clinical evaluation of the combination of LuTate and PARP inhibition in SSTR2-expressing NET.

Investigation of somatostatin receptor profile of neuroendocrine carcinomas of the breast

Carcinomas of the breast with neuroendocrine features are rare primary neoplasms positive for neuroendocrine markers. According to the WHO classification of tumours of the breast they are divided into three morphologically distinct categories. They comprise neuroendocrine tumour (NET), neuroendocrine carcinoma (NEC) and carcinoma with neuroendocrine differentiation (NED). The purpose of this study was to investigate for the first time the full spectrum of sstr expression status in breast carcinomas with neuroendocrine features.Fifteen primary breast carcinomas with histological and immunohistochemical neuroendocrine features were studied. Four of them were classified as NETs and two as NECs, and the remaining 9 as carcinomas with NED. All six types of somatostatin receptor (sstr) types (sstr1, sstr 2A, sstr2B, sstr3, sstr4 and sstr5) were investigated by immunohistochemistry. To assess the distribution and intensity of membranous receptor immunoreactivity, a four-scale scoring system was used. Overall predominant receptors were sstr2A, sstr2B, sstr3 and sstr5 showing the highest membranous staining scores 3+ and 2 + . The sstr1 was not detected.Given that carcinomas with neuroendocrine features represent distinct entities, patients with such tumours may benefit from sstr targeting therapies. Immunohistochemistry for sstrs can predict the effectiveness of administration of SST analogues to those patients, thus contributing to achieve the maximum therapeutic outcome, particularly in NETs and NECs with scores 2+ and 3 + .

T2-signal intensity, SSTR expression, and somatostatin analogs efficacy predict response to pasireotide in acromegaly.

T2-signal intensity and somatostatin (SST) receptor expression are recognized predictors of therapy response in acromegaly. We investigated the relationship between these predictors and the hormonal and tumoral responses to long-acting pasireotide (PAS-LAR) therapy, which were also compared with responsiveness to first-generation somatostatin receptor ligands (SRLs). The PAPE study is a cohort study. We included 45 acromegaly patients initially receiving SRLs, followed by combination therapy with pegvisomant, and finally PAS-LAR. We assessed tumor volume reduction (≥25% from baseline), IGF-1 levels (expressed as the upper limit of normal), and T2-weighted MRI signal and SST receptor expression of the adenoma. Patients with significant tumor shrinkage during PAS-LAR showed higher IGF-1 levels during PAS-LAR (mean (S.D.): 1.36 (0.53) vs 0.93 (0.43), P = 0.020), less IGF-1 reduction after first-generation SRLs (mean (S.D.): 0.55 (0.71) vs 1.25 (1.07), P = 0.028), and lower SST2 receptor expression (median (IQR): 2.0 (1.0-6.0) vs 12.0 (7.5-12.0), P = 0.040). Overall, T2-signal intensity ratio was increased compared with baseline (mean (S.D.): 1.39 (0.56) vs 1.25 (0.52), P = 0.017) and a higher T2-signal was associated with lower IGF-1 levels during PAS-LAR (β: -0.29, 95% CI: -0.56 to -0.01, P = 0.045). A subset of PAS-LAR treated patients with increased T2-signal intensity achieved greater reduction of IGF-1 (mean (S.D.): 0.80 (0.60) vs 0.45 (0.39), P = 0.016). Patients unresponsive to SRLs with a lower SST2 receptor expression are more prone to achieve tumor shrinkage during PAS-LAR. Surprisingly, tumor shrinkage is not accompanied by a biochemical response, which is accompanied with a higher T2-signal intensity.

Molecular profiling for acromegaly treatment: a validation study.

Pharmacologic treatment of acromegaly is currently based upon assay-error strategy, the first-generation somatostatin receptor ligands (SRL) being the first-line treatment. However, about 50% of patients do not respond adequately to SRL. Our objective was to evaluate the potential usefulness of different molecular markers as predictors of response to SRL. We used somatotropinoma tissue obtained after surgery from a national cohort of 100 acromegalic patients. Seventy-one patients were treated with SRL during at least 6 months under maximal therapeutic doses according to IGF1 values. We analyzed the expression of SSTR2, SSTR5, AIP, CDH1 (E-cadherin), MKI67 (Ki-67), KLK10, DRD2, ARRB1, GHRL, In1-Ghrelin, PLAGL1 and PEBP1 (RKIP) by RT-qPCR and mutations in GNAS gene by Sanger sequencing. The response to SRL was categorized as complete response (CR), partial (PR) or non-response (NR) if IGF1 was normal, between >2<3 SDS or >3 SDS IGF1 at 6 months of follow-up, respectively. From the 71 patients treated, there were 27 CR (38%), 18 PR (25%) and 26 NR (37%). SSTR2, Ki-67 and E-cadherin were associated with SRL response (P < 0.03, P < 0.01 and P < 0.003, respectively). E-cadherin was the best discriminator for response prediction (AUC = 0.74, P < 0.02, PPV of 83.7%, NPV of 72.6%), which was validated at protein level. SSTR5 expression was higher in patients pre-treated with SRL before surgery. We conclude that somatotropinomas showed heterogeneity in the expression of genes associated with SRL response. E-cadherin was the best molecular predictor of response to SRL. Thus, the inclusion of E-cadherin in subsequent treatment-decision after surgical failure may be useful in acromegaly.

SAT-LB24 Association of Expression of Somatostatin Receptor 2 Subtype in Non-Pituitary Neuroendocrine Tumors With Response to Treatment With Somatostatin Analogues

Background: The WHO classification of non-pituitary neuroendocrine tumors (NETs) includes grading (Ki67 proliferation index and mitosis index) and staging, and has been shown to be associated with outcome in this patient population. Neuroendocrine tumors show different somatostatin receptor (SSTR) subtypes expression, but their role has not been considered in tumor progression and prognosis. We hypothesize patients with non-pituitary NET, whose tumor tissues have higher specific SSTR2 expression patterns will experience better outcomes when treated with Somatostatin Analogues (SSA) compared to those with lower or absent SSTR2 expression. Methods and patients: City of Hope patients with non-pituitary NET who received SSA (Octreotide and Lanreotide) for over 1 year as treatment. The progression free survival was based on imaging studies (MRI, CAT scan of index tumor) on RECIST criteria, and expression of SSTR2 on the tumor obtained by surgery at the time of diagnosis. Immunohistochemical staining with a monoclonal antibody to SSTR2 performed at core pathology lab at City of Hope. H-scoring (intensity of immunostaining and percent of SSTR2 expression) was performed by one pathologist, and divided into four categories of 0-4. Pearson Chi-square test was used to assess the correlation between the SSTR2 score and 1-year progression. Outcome Measures: The effect of SSA therapy on 1 year progression-free survival (PFS), among patients with high intensity and percent of SSTR2 expression on the resected tumor tissue was compared to those with low or absent SSTR2 expression. Patient population: Of the 297 patients identified with non-pituitary neuroendocrine tumor, 70 patients satisfied the inclusion criteria and a total of 52 patients had data that was completely available for analysis. Results: Mean age of study subjects was 61+1.8 years. 26 were female and 26 were male. The primary sites for NET were: pancreas 17, jejunum 16, ileum 9; paraganglioma and pheochromocytomas 3, lung and thymus 3, gastric 2, and medullary thyroid carcinoma 1. The one-year progression was not significantly different among those with low and absent SSTR2 compared to high expression of SSTR2. Step wise analysis based on the scoring of the SSRT2 did not show significant 1 year progression. Age was not associated with PFS. However, the need for additional therapy (liver embolization, surgical resection) was signigicantly reduced amongst patients with presence of SSTR2. Conclusion: In this preliminary study there was no significant association between SSTR2 expression and Progression free survival, however the need for additional therapy in patients on somatostatin analogues were reduced among subjects with presence of SSTR2 expression

SAT-723 Effects of Organohalogenated Endocrine Disrupting Chemicals on Cell Proliferation and Gene Expression in GH3 Somatolactotropes

Endocrine Disrupting Chemicals (EDCs) are substances that have been increasingly implicated in many serious pathologies, such as tumor formation, metabolic, growth and reproductive disorders. The economic and health burden of exposure to these compounds has an annual predicted cost in excess of €150 billion, across the EU regions alone. Of the growing list of compounds that act as EDCs, the organohalogenated compounds (OHCs), polybrominated diphenyl ethers (PBDEs) and polychlorinated biphenyls (PCBs) have been associated with an increased risk of pituitary disease. We have previously reported that feline patients with hypersomatotropism (acromegaly) are exposed to elevated levels of PBDEs and PCBs in their environment. However, the mechanisms by which these compounds might directly influence somatotroph function have yet to be established. In this study, we use the GH3 rat somatolactotrope cell line to investigate how two PCB congeners - 138 and 153 - influence cell proliferation (using a Crystal Violet assay) and somatotrope gene expression (using a multiplex RT-qPCR approach to examine expression of Esr1, Esr2, Sstr1, Sstr2, Sstr3, Sstr4, Sstr5, Insr, Tshr, Pou1f1, Ghrhr2, Gh). GH3 cells were treated with Phenol Red-free media in the absence or presence of either PCB138 or 153 (-10 to -6 M), or in combination (-10 to -6M) for up to 72h. Treatment with either PCB alone, or in combination, caused significant concentration-dependent, biphasic changes in cell proliferation at each time point, but with a different profile of response on each day (significantly increased at high pM/low nM concentrations); there was no evidence of toxicity at maximum concentrations (-6M). Gene expression changes were determined in GH3 cells treated in the absence or presence of either -8M or -6M PCB138 or 153 for 24h. Differential effects of these compounds were seen on the expression of Sstr3, Sstr4, Sstr5 and Insr; all other gene transcripts were unaffected. These findings reveal that GH3 cells exposed to physiologically relevant concentrations of PCB138 and 153, alone or in combination, show concentration-dependent increases in cell proliferation; furthermore, the expression of genes associated with therapeutic targets for the treatment of acromegaly (i.e. SSTRs) are differentially affected by exposure to PCB138 and 153. Our data indicate a potential mechanism for EDCs in the onset of acromegaly, that require further, in vivo, investigations.

SAT-LB61 METTL3 Promotes Sparsely Granulated GH-Secreting Pituitary Adenomas to Behavior as Densely Granulated Adenomas

Background: Growth hormone (GH)-secreting pituitary adenomas can be divided into densely and sparsely granulated subtypes, based on electron microscopic studies. The latter are frequently associated with more invasive behavior, and respond worse to somatostatin analogues. The underlining mechanisms are largely unknown. Increasing evidence showed that N6-methyladenosine (m6A) of messenger RNAs (mRNAs) participated in the development of various tumors. We aimed to investigate the role of RNA m6A modification in the classification of GH-secreting pituitary adenomas. Methods: The main components of m6A methyltransferase complex, demethylase, and RNA m6A levels were compared between sparsely and densely GH-secreting tumors. The role of METTL3 was functionally studied. Results: The level of m6A methyltransferases (METTL3, WTAP and METTL14) and demethylase (FTO and ALKBH5) were significantly downregulated in GH adenomas, comparing to the normal pituitary tissues. However, only METTL3 and METTL14 were shown to significantly higher in densely granulated tumors than those in sparsely ones. Consistently, the level of RNA m6A was markedly increased in densely granulated GH adenomas. In addition, the expression of METTL3 was positively correlated with the level of RNA m6A among tumor samples, and METTL3 silencing decreased RNA m6A of GH3 cells. METTL3 was demonstrated to function as a tumor suppressor based on in vivo and in vitro evidence, using patient-derived and GH3 cells. Moreover, the sensitivity of GH3 cells to pasireotide was increased with METTL3 overexpression, but decreased when METTL3 was silenced. Consistently, METTL3 silencing inhibited GH secretory, and decreased the expression of SSTR2 and SSTR5. Conclusions: METTL3 functions as a tumor suppressor in GH secreting adenomas, and enhance tumor cells sensitivity to medical treatment. Our work uncovers the critical roles of METTL3 in the pathogenesis of GH adenomas, since it potentially promotes the transition from sparsely to densely granulated subtypes.

SUN-118 Somatostatin Agonist Conjugated to the Evans Blue Moiety Is a Superior Analog in the Diagnosis and Treatment of Tumors Characterized by High Somatostatin Receptor Expression

Background: Radiolabeled somatostatin (SST) analogs have been proven to be effective in the diagnosis and treatment of neuroendocrine tumors (NETs), which are characterized by high somatostatin receptor (SSTR2) expression. At present, there are several SST analogs available that differ from each other in the affinity for SSTR2 and tumor retention time. To date, only a single SST agonist -DOTA-TATE- has been approved by the FDA for imaging and treatment of NETs. Recent studies have shown that addition of Evans blue (EB) moiety to an SST agonist results in superior uptake and increased retention time within the tumors. The goal of our study was to compare the diagnostic and therapeutic efficacy of three different radiolabeled SST analogs in a tumor mice model: EB-TATE - a novel modified agonist; DOTA-TATE - an agonist and JR11 - an antagonist. Methods: A rat pancreatic cell line (AR42J), characterized by high SSTR2 expression, was used to create a subcutaneous xenograft mice model. The AR42J cells formed sizable tumors within two weeks post-injection. The 86Y-EB-TATE, 68Ga-DOTA-TATE, and 68Ga-DOTA-JR11 were used to determine standard uptake values by positron emission tomography (PET) imaging. For treatment purposes, the SST analogs were labeled with 177Lu to generate 177Lu-EB-TATE, 177Lu-DOTA-TATE and 177Lu-DOTA-JR11. The mice were assigned to treatment groups based on comparable tumor volume at baseline and received two doses (0.5mCi) of the 177Lu-labeled analogs one week apart. Tumor measurements were performed twice per week and the mice were euthanized if their tumor burden exceeded 2 cm at any point in the study or after 6 weeks - landmark of the end of the study. Results: Among the three analogs tested, the novel SST analog 86Y-EB-TATE was characterized by 4.3- and 3.7- fold higher tumor uptake in comparison to 68Ga-DOTA-TATE (p<0.001) and 68Ga-DOTA-JR11 (p<0.001), respectively. There was no significant difference between the uptake of 68Ga-DOTA-TATE and 68Ga-DOTA-JR11 (p=0.9). Consistently with higher tumor uptake on imaging, 177Lu-EB-TATE-treated mice responded to the treatment with an overall 86.5±13.2% reduction in the tumor volume after two weeks post-therapy. On the contrary, despite therapy with 177Lu-DOTA-TATE and 177Lu-DOTA-JR11, the mice treated with these agents presented with tumor progression exceeding 2 cm and were euthanized. Consequently, the progression-free survival (PFS) was significantly longer in 177Lu-EB-TATE group (24±0 days) compared with 177Lu-DOTA-TATE (7.7±2.6 days, p<0.001) and 177Lu-DOTA-JR11 (6.3±3 days, p<0.001). There was no difference in PFS between 177Lu-DOTA-TATE and 177Lu-DOTA-JR11-treated mice (p=0.3). Conclusion: EB-TATE is characterized by superior diagnostic and therapeutic efficacy in comparison to DOTA-TATE and DOTA-JR11. EB-TATE might be used as imaging and therapeutic agent in tumors characterized by high SSTR2 expression.

Differentiating dural metastases from meningioma: role of 68Ga DOTA-NOC PET/CT.

To assess the ability of Ga DOTA-NOC PET/computed tomography (CT) to differentiate dural metastases from meningioma. Patients who underwent a Ga DOTA-NOC PET/CT for differentiating meningiomas from dural metastases were included in the study. A visual score was assigned to the dural lesions (visual score - 1 to 3) in relation to the uptake in liver and spleen and variation in the visual score was evaluated. SUVmax of the dural lesions was also noted and difference in the values of the two pathologies were compared for statistical significance using nonparametric statistical tests. Final diagnosis was decided by histopathological confirmation whenever available. Imaging, histopathology or follow-up data of 42 patients was available for analysis. Meningioma was the final diagnosis in 31 (73.8%) patients, whereas dural metastases were diagnosed in 9 (21.4%) patients. In two patients, histopathology revealed inflammatory pseudotumor and hemangioblastoma. Meningiomas showed intense tracer uptake in 30/31 patients (visual score 3). All metastatic lesions showed some degree of tracer uptake though the intensity was lesser compared to meningioma (visual score 1, 2). Meningiomas showed a significantly higher median SUV max value compared to metastases (12.7 vs. 6.0, P = 0.001). Meningiomas can be differentiated from dural metastases by virtue of their higher uptake of Ga-labeled DOTA peptides reflecting higher SSTR expression. An asymptomatic meningeal based lesion with a high visual score (Visual score 3) has a very high probability to be a meningioma rather than dural metastasis.

Immunohistochemical Profile and 47-Gene Next-Generation Sequencing (NGS) Solid Tumor Panel Analysis of a Series of 13 Strumal Carcinoids

Strumal carcinoid is an extraordinary rare tumor of the ovary consisting of thyroid tissue intermixed with neuroendocrine tumor component. The cellular origin of strumal carcinoids has been an area of debate. There is also little data on detailed immunohistochemical and molecular characteristics of these neoplasms. For this reason, this series investigated the characteristics of a series of 13 strumal carcinoids using immunohistochemical markers and a 47-gene next-generation sequencing (NGS) solid tumor panel analysis. Both cellular components showed thyroglobulin expression in all tumors. TTF-1 expression was noted in both cellular components of 11 cases. Chromogranin A was positive in both components of most tumors (n = 12, 92.3% in the neuroendocrine component and n = 10, 76.9% in the thyroid follicular component). Synaptophysin stained the neuroendocrine component of all cases, and it was also identified in the follicular thyroid component of a single case. All tumors were negative for CDX2 and calcitonin. ISLET1 was positive in the neuroendocrine component of 8 cases (6.5%). With the exception of one case, all tumors were positive for SSTR2a. The tumors were associated with a low Ki67 labeling index. All cases were microsatellite stable and no pathogenic mutations were identified using a 47-gene NGS solid tumor analysis. This series underscored that strumal carcinoids are distinct neuroendocrine tumors. The synchronous expression for thyroid follicular epithelial and neuroendocrine differentiation biomarkers may suggest a precursor cell origin displaying mixed-amphicrine differentiation. While strumal carcinoids can be diagnosed by their typical morphology and immunohistochemical profile, frequent SSTR expression may serve as a potential theranostic biomarker in the management of affected patients. In addition, the absence of common driver mutations in the NGS solid tumor panel may suggest that these neoplasms seem to be genetically unrelated to follicular epithelial–derived thyroid tumors and potentially different than other commonly identified well-differentiated neuroendocrine neoplasms. Therefore, further studies focusing on molecular characteristics of this entity are still needed.

1386PD - Longitudinal increase in Ki67 and high-grade transformation in pancreatic neuroendocrine tumours (PNETs)

BackgroundTumor proliferation and grade are important prognostic factors at diagnosis of PNETs. Little is known about how these factors change over time and if longitudinal increase in Ki67-index is associated with prognosis. The purpose of this work was to describe longitudinal changes in Ki67-index and tumor grade, as well as its impact on overall survival, in PNETs. MethodsPlatelet free plasma from 68 GEP-NET patients was collected. Whole miRNOME NGS analysis was performed on exome enriched small-RNAs fraction of 24 patients: 12 18PET/FDG+ and 12 18PET/FDG-. MiRNAs significantly associated with 18PET/FDG outcome has been identified and validated by RT/qPCR on overall case series. Target miRNAs fold enrichment were then combined to create predictors (pAB, pAC, pBC and pABC). Man-Whitney test was applied and validated target miRNAs had been correlated with clinical outcome and clinical parameters (ki-67, grading, tumor burden and 68Gallium-PET SUVmax). ResultsNGS whole miRNOME analysis revealed 10 target miRNAs able to distinguish 18FDG/PET positive from negative Pancreatic-NETs (PNETs). Subsequently, mir-A, mir-B, mir-C (patent pending) have been validated on overall case series by multiplex RT/qPCR (p<0,0047 and p<0,0001, respectively), on PNETs case series and pBC and pABC resulted to be the best predictors (p<0,0001). All validated miRNAs and derived predictors, especially mir-B, result significantly increased in small intestine (SINETs) and in PNETs patients when compared to healthy controls. Correlation analysis between target miRNAs and clinical parameters also revealed that mir-B negatively correlates with 68Ga-PET SUVmax (p<0,04), suggesting interference with SSTR expression. ConclusionsWe defined a 3 miRNAs signature able to correlate with 18FDG/PET status. Over expression of mir-A, mir-B, mir-C or combined predictors in PNETs can provide prognostic information on tumor aggressiveness and might help to identify PRRT non-responders. In addition mir-B negatively correlates with clinical outcome and 68Ga-PET SUVmax. We are investigating if mirB interfere with SSTR expression, affecting PRRT efficacy. Legal entity responsible for the studyUppsala University. FundingEuropean Neuroendocrine Tumor Society, Uppsala University. DisclosureJ. Botling: Honoraria (self): Novartis. A. Lamarca: Honoraria (self): Merck; Honoraria (self): Pfizer; Honoraria (self): Ipsen; Advisory / Consultancy: EISAI; Advisory / Consultancy: Nutricia; Travel / Accommodation / Expenses: Ipsen; Travel / Accommodation / Expenses: Pfizer; Travel / Accommodation / Expenses: Bayer; Travel / Accommodation / Expenses: AAA; Travel / Accommodation / Expenses: SirtEx; Travel / Accommodation / Expenses: Novartis; Travel / Accommodation / Expenses: Mylan; Travel / Accommodation / Expenses: Delcath. G. Rindi: Honoraria (self): Novartis; Honoraria (self): Ipsen. J. Crona: Honoraria (self): Novartis; Honoraria (self): Ipsen. All other authors have declared no conflicts of interest.

A growth model of neuroendocrine tumor surrogates and the efficacy of a novel somatostatin-receptor–guided antibody-drug conjugate: Perspectives on clinical response?

BackgroundAs patient-derived xenografts and other preclinical models of neuroendocrine tumors for testing personalized therapeutics are lacking, we have developed a perfused, 3D bioreactor model to culture tumor surrogates from patient-derived neuroendocrine tumors. This work evaluates the duration of surrogate culture and surrogate response to a novel antibody-drug conjugate. MethodsTwenty-seven patient-derived neuroendocrine tumors were cultured. Histologic sections of a pancreatic neuroendocrine tumor xenograft (BON-1) tumor were assessed for SSTR2 expression before tumor implantation into 2 bioreactors. One surrogate was treated with an antibody-drug conjugate composed of an anti-mitotic Monomethyl auristatin-E linked to a somatostatin receptor 2 antibody. Viability and therapeutic response were assessed by pre-imaging incubation with IR-783 and the RealTime-Glo AnnexinV Apoptosis and Necrosis Assay (Promega Corporation, Madison, WI) over 6 days. A primary human pancreatic neuroendocrine tumor was evaluated similarly. ResultsMean surrogate growth duration was 34.8 days. Treated BON-1 surrogates exhibited less proliferation (1.2 vs 1.9-fold) and greater apoptosis (1.5 vs 1.1-fold) than controls, whereas treated patient-derived neuroendocrine tumor bioreactors exhibited greater degrees of apoptosis (13- vs 9-fold) and necrosis (2.5- vs 1.6-fold). ConclusionPatient-derived neuroendocrine tumor surrogates can be cultured reliably within the bioreactor. This model can be used to evaluate the efficacy of antibody-guided chemotherapy ex vivo and may be useful for predicting clinical responses.

68Ga-DOTATATE PET/CT parameters predict response to peptide receptor radionuclide therapy in neuroendocrine tumours

Purpose[177Lu]DOTATATE prolongs progression free survival (PFS) in metastatic neuroendocrine tumours (NETs). However, objective response rate is low. This, coupled with long duration of therapy and expense suggest need for better selection. We aim to assess whether baseline [68Ga]DOTATATE-PET/CT parameters, and whether response assessment by PET accurately predicts clinical outcome to [177Lu]DOTATATE. Experimental designRetrospective study of patients receiving [177Lu]DOTATATE was conducted. Patients were followed 3-monthly until disease progression. Four [68Ga]DOTATATE-PET parameters (single lesion SUVmax, tumour to spleen and liver SUV ratios, and SUVmax-av using up to five target lesions in multiple organ sites) were determined at baseline and follow-up. The association between these PET parameters either at baseline, or any changes following treatment, and PET response criteria (PERCIST and modified PERCIST) to predict PFS were determined. Patients were followed 3-monthly until disease progression. Response was determined using RECIST 1.1. Baseline SSTR2 expression was assessed and compared with PET parameters. Results55 patients with metastatic NETs were identified predominantly small bowel (N = 18) and pancreatic (N = 8) in origin. 16 were low grade, 15 intermediate and 3 high grade. Response to PRRT (N = 47): partial response (PR) 28%, stable disease (SD) 60% progressive disease (PD) 13%. Response to PRRT predicted PFS: PR 71.8 months (95%CI: not achieved), SD 29.1 months (95%CI: 15.2–43.1), and PD 9.7 months (95%CI: 0–21.02). Baseline, single lesion SUVmax predicted both response and PFS with SUV cut-off of 13.0 giving high sensitivity and specificity. Tumoural SUVmax correlated with SSTR2 expression, Spearman’s rho – 0.69, p < 0.01. ConclusionsBaseline single lesion SUVmax and SUVmax-av predicts response to [177Lu]DOTATATE. Objective response following PRRT defines a subset of patients with markedly improved PFSBaseline SUVmax 13.0 defines a threshold below which patients have poor response to PRRT and worse PFS. SUV threshold analysis should be taken forward into prospective studies.

Pasireotide-LAR in acromegaly patients treated with a combination therapy: a real-life study.

PurposeLittle data are available regarding the safety and efficacy of switching to Pasireotide-LAR monotherapy in acromegaly patients with partial resistance to first-generation somatostatin agonists (1gSRL) who require combination treatment with cabergoline or pegvisomant.MethodIn this monocentric prospective study within a tertiary university hospital, 15 consecutive acromegalic adults partially resistant to 1gSRL treated with octreotide LAR or lanreotide SR, and cabergoline (n = 4, 3.5 mg/week) or pegvisomant (n = 11, median dose 100 mg/week), were switched to Pasireotide-LAR (8 with 40 mg/month; 7 with 60 mg/month). Immunohistochemical expression level of SSTR5 and the granulation pattern of nine somatotroph adenomas were retrospectively determined to test for a correlation with the therapeutic efficacy of Pasireotide-LAR.ResultsMedian IGF-1 concentration at the first evaluation (median 3 months) was similar to baseline (1.0 vs 1.1 ULN). 11/15 patients had IGF-1 levels ≤1.3 ULN before and after the switch but individual changes were variable. Hyperglycemia was frequent and greater in diabetic patients. 7/15 patients stopped Pasireotide-LAR due to lack of control of IGF-1 or intolerance. 8/15 patients received Pasireotide-LAR for a median of 29 months with IGF-1 levels ≤1.3 ULN and acceptable glucose tolerance (median HbA1c 6.1%). Two patients required initiation of oral antidiabetic treatment. The intensity of SSTR5 expression and the granulation pattern of adenomas were of limited value for the prediction of Pasireotide-LAR effectiveness.ConclusionPasireotide-LAR may represent a suitable therapeutic alternative in a subset of acromegalic patients requiring combination therapy involving a 1gSRL

Immunohistochemical Analysis of Somatostatin Receptors in Endometriosis Tissue Samples: A Retrospective Study.

Three types of endometriosis are described: superficial peritoneal endometriosis (SPE), ovarian endometrioma (OMA), and deep infiltrating endometriosis (DIE). The expression of somatostatin receptors (SSTR1, 2, and 5) in human endometrial tissue and its ectopic form has been previously studied and may be different in each type of endometriosis. The aim of this study was to assess the immunohistochemical expression of SSTR1, 2, and 5 in tissue samples of SPE, OMA, and DIE. We performed a retrospective analysis in the pathology department database. Patients aged <50 yr and diagnosed with endometriosis have been identified and sorted into 3 groups according to their endometriosis type: SPE, OMA, and DIE. For each selected patient, formalin-fixed paraffin-embedded blocks were retrieved in order to make new sections to be incubated with polyclonal rabbit antibodies anti-SSTR1, 2, and 5. Receptor status was considered as positive on the sections when >50% of the cells showed immunostaining. Seventy-six patients were included in the analysis. SSTR1 and 5 were expressed in 95.4% and 77.2% of SPE, respectively, in 95.8% and 83.3% of OMA, respectively, and in 100% and 80% of DIE, respectively. There was no significant difference between SPE, OMA, and DIE with regard to the SSTR1 (P=0.5) and SSTR5 (P=0.9) expression. We observed a significant difference between SPE (9.0%), OMA (16.6%), and DIE (63.3%) with regard to SSTR2 expression (P<0.05). The present study identifies 2 different immunohistochemical patterns of endometriosis lesions with regard to their SSTR expression: SSTR1+/SSTR2-/SSTR5+ for SPE and OMA, and SSTR1+/SSTR2+/SSTR5+ for DIE.

Interaction between somatostatin analogues and targeted therapies in neuroendocrine tumor cells.

Somatostatin analogues (SSA) represent the standard of care for symptom control in patients with functional gastro-entero-pancreatic neuroendocrine tumors (GEP-NET). In addition, SSA exert significant anti-proliferative effects in mid-gut and pancreatic NET (PanNET). In parallel, molecularly targeted therapies (MTT) have been shown to improve progression free survival (PFS) in patients with PanNET. However, due to either primary or acquired resistance to MTT, their impact on overall survival (OS) remains unclear. To date, various hypotheses exist to explain differences in patient responsiveness to SSA and MTT. However, data addressing one of the most pivotal questions, whether combining SSA with novel MTT will result in synergistic or additive efficacy compared to monotherapy, are lacking. The aim of this study is to characterize the interaction, optimal sequence and dosing of SSA-based and molecularly targeted therapies in PanNET. Somatostatin receptor subtypes 1–5 (SSTR) were evaluated in the neuroendocrine cell lines Bon1, QGP1 and Ins-1 via immunoblot and qRT-PCR. The impact of the SSA-analogue lanreotide alone or in combination with the MTT sunitinib, everolimus and regorafenib on intracellular signalling, hormone secretion and cell proliferation was determined in cell lysates and supernatants. In addition, synergistic effects of SSA and MTT in various sequential therapeutic approaches were investigated. SSTR were differently expressed in the examined neuroendocrine tumor cell lines. SSTR modulation via lanreotide moderately influenced proliferation, mainly via modulating AKT and ERK signalling, which was paralleled by decreased chromogranin A (CgA) expression and secretion. Interestingly, MTT treatment with regorafenib upregulated the expression of SSTR-2 and -5, while sunitinib and everolimus did not significantly alter SSTR expression. Cell viability was significantly reduced by all MTT, with regorafenib exerting the most significant effects. However, compared to the marked effects of MTT alone, synergistic effects of combined MTT and lanreotide treatment were only modest and time- and dose-dependent. SSTR are differentially expressed in various NEN cell lines. Their expression is influenced by MTT treatment. Various sequential or simultaneous combinations of lanreotide and MTT did not lead to significant synergistic effects.

Pasireotide and Pegvisomant Combination Treatment in Acromegaly Resistant to Second-Line Therapies: A Longitudinal Study.

The treatment of acromegaly resistant to first- and second-line therapies can be extremely challenging. We have described six patients who were successfully treated with a combination therapy of pasireotide and pegvisomant and compared them with a control group of patients resistant to conventional somatostatin analogs (SSAs), whose disease was controlled with other treatment, such as pasireotide (as monotherapy) or pegvisomant (as monotherapy or combined with conventional SSAs). In these six patients, acromegaly was controlled with combined pasireotide and pegvisomant treatment after failure of all other treatments. Compared with the 49 patients in the control group, these six patients had giant and invasive pituitary adenomas (at both the cavernous sinus and other structures). Although not statistically significant, higher growth hormone levels, more elevated Ki-67 expression, greater somatostatin receptor (SSTR) subtype 5 expression, and lower SSTR subtype 2 expression at the diagnosis of acromegaly were detected in patients receiving combination treatment with pasireotide and pegvisomant compared with the control group. Our data have reinforced the importance of personalized treatment of patients with acromegaly according to the clinical, biochemical, molecular, and morphological disease markers and suggest that combined treatment with pasireotide and pegvisomant can induce disease control in tumors with low SSTR2 expression, resistant to conventional SSAs (alone or combined with pegvisomant) and to new-generation SSAs alone (pasireotide).

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

BackgroundThe 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 resultsWe 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. DiscussionThe 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.

MON-467 Expression of Somatostatin Receptors in Aggressive Non-Functioning Pituitary Adenomas

INTRODUCTION Non-functioning pituitary adenomas (NFPAs) are usually benign. However, a substantial proportion of pituitary adenomas show an aggressive behavior with local invasion and increased risk of regrowth or recurrence after surgery. The treatment of choice for these adenomas is transsphenoidal resection but it is not always successful. NFPAs express somastostatin receptors (SSTRs) thus providing a theoretical rationale for the pharmacological treatment with somatostatin analogues. However, a consistent method for evaluating SSTR expression needs to be implemented. AIM The aim of the study was to systematically evaluate the expression of SSTRs in NFPAs by quantitative PCR and by immunohistochemistry with reliable commercial antibodies and analyze their potential association with aggressive features. METHODS. In this retrospective descriptive study, 114 patients that underwent transsphenoidal surgery were evaluated. Clinical variables, histological subtype (including classification of agressive histological subtypes) and need for radiotherapy were collected to analyze potential associations between these variables and SSTR expression. Tumor size and cavernous sinus invasion data were obtained from magnetic resonance images. Cavernous sinus invasion was evaluated using the Knosp classification. Knosp grade 3 and 4 were defined as invasive. The expression of SSTRs (SSTR2. SSTR3 and SSTR5) was evaluated using a score system previously described [Venegas-Moreno et al. J Cell Mol Med. 2018;22(3):1640-1649]. RESULTS Quantitative PCR analysis revealed that SSTR3 was the predominant SSTR subtype detected, followed by SSTR2, SSTR5, and SSTR1. Immunohistochemistry data showed a similar expression pattern. However, The expression of the different SSTRs displayed a notable variability among NFPAs. Thus, 28,9% of NFPAs showed substantial SSTR2 expression, as indicated by high immunohistochemistry scores. Also, 12,3% of NFPAs displayed high SSTR5 expression while 86,8% of NFPAs displayed high SSTR3 levels. No significant associations between SSTR expression and sex, tumor size, invasion and surgical cure. Patients with elevated SSTR2 expression were younger but this association was not observed with SSTR3 and SSTR5. CONCLUSIONS No major association between SSTR expression and aggressive features was observed in NFPAs. A proportion of NFPAs displayed marked expression of SSTR2 and SSTR5 and thus, they might be considered potential candidates for therapy with somatostatin analogues. Sources of Research Support: Spanish Ministry of Health, ISCIII co-funded with Fondos FEDER (PI16/00175) and Novartis Oncology Spain.