Expression Of Somatostatin Receptor Subtypes Research Articles

Correlation between tumor invasion and somatostatin receptor subtypes in acromegaly.

The low expression of somatostatin receptor (SSTR) subtypes in somatotropinoma is associated with a poor response to somatostatin analogs (SSAs). However, the correlation between SSTRs and tumor invasion has not yet been clarified. Therefore, the authors aimed to investigate the relationship between SSTRs and tumor invasion, as well as the correlation between tumor invasiveness and pharmacological response to SSAs. A total of 102 patients with acromegaly who underwent surgery between December 2016 and December 2021 at the largest pituitary tumor surgery center in southern China were included in this retrospective study. Patients were divided into the noninvasive tumor group (Knosp grades 0-2 and Hardy-Wilson grade I or II) and invasive group (either Knosp grade 3 or 4 or Hardy-Wilson grade III or IV). The positive response to SSAs was defined by the following criteria after at least 3 months of SSA treatment: 1) ≥ 50% reduction or age- and sex-adjusted normal range of insulin-like growth factor-1 (IGF-1) level; 2) ≥ 80% reduction in or normal range of growth hormone (GH) level; or 3) > 20% reduction in tumor volume. The reference for the normal range of age- and sex-adjusted serum IGF-1 levels was derived from a survey of 2791 healthy adults (1339 males and 1452 females) in China. Demographics and clinical characteristics including tumor size, biochemical assessment, expression levels of SSTRs, and response to preoperative SSAs were compared between the invasive group and noninvasive group. Receiver operating characteristic (ROC) curve analysis was performed to assess the association between SSTR2 and tumor invasion. Compared with the noninvasive group, the invasive group presented with a larger tumor size (9.99 ± 10.41 cm3 vs 3.50 ± 4.02 cm3, p < 0.001), relatively lower SSTR2 expression (p < 0.001), and poorer response to SSAs (36.4% vs 91.7%, p < 0.001). In addition, there was a significant negative correlation between SSTR2 mRNA level and tumor size (r = -0.214, p = 0.031). However, there were no statistically significant differences in the expression of SSTR1, SSTR3, and SSTR5 between the groups. ROC analysis revealed that the low SSTR2 mRNA level was closely associated with tumor invasion (area under the curve 0.805, p < 0.0001). Tumor invasion is negatively correlated with SSTR2 level but is not associated with other SSTR subtypes. Patients with invasive tumors have a poorer response to SSA therapy, which may be due to the low level of SSTR2 expression. Therefore, SSTR2 could be considered as a routine investigative marker for aiding management of postoperative residual tumors.

Abstract 5165: Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies

Abstract Background: Expression of Somatostatin Receptor subtype 2 (SSTR2) is observed in multiple cancers, including breast and colorectal cancer. Its role as a prognostic or predictive marker is cancer-type dependent. Recently, there has been renewed interest in assessing this biomarker since it plays a critical role as a target for radiotherapeutics (peptide receptor radionuclide therapy - PRRT) in patients with neuroendocrine tumors (NETs). Blood-based liquid biopsies are a non-invasive tool with proven effectiveness in characterizing cancer biomarker expression in patients. In this study, we describe the development and validation of a novel assay to analyze circulating tumor cells (CTCs) for expression of SSTR2 using the RareCyte platform. Experimental Procedures: Using AccuCyte®, an unbiased density-based method for collecting nucleated cells from whole blood, we transferred nucleated cells to slides and stained for SSTR2, CD45, and CK/EpCAM to identify CTCs (CK+/EpCAM+, CD45-), and evaluate SSTR2 expression. Slides were imaged with CyteFinder®, an automated multiparameter immunofluorescent (IF) microscopy system that applies machine learning algorithms for rare cell identification. Both clinical and spike-in samples were used for assay validation. Single-cell SSTR2 mean fluorescence intensities (MFI) were analyzed to determine protein expression levels. Colorectal cancer with neuroendocrine features and Merkel cell cancer (neuroendocrine tumor of the skin) patient samples were evaluated for CTC enumeration and SSTR2 expression. Results: The assay’s performance metrics for SSTR2 expression were 90+% accuracy, 85% sensitivity, 100% specificity with repeatability and intermediate precision CVs of 14.4% and 16.4%, respectively. A small cohort of colorectal cancer (1) and Merkel cell cancer (2) patient blood samples were tested using the assay. All 3 patients had high CTC levels (300+ per 7.5 mL). The percentage of SSTR2-positive CTCs ranged from 20-60%, reflective of tumor heterogeneity. Conclusions: SSTR2 is an important target now in the advent of theranostics - PRRT. Prognostic and therapeutic implications of SSTR2 expression is an area of active clinical research. SSTR2 targeted therapeutics like 177Lu-dotatate illustrate the possible utility of SSTR2 as a predictive biomarker of treatment response. To date, most of the work has been focused on assessing SSTR2 on tissue. Herein, we show how a CTC platform can be adapted to assess a clinically relevant cell surface protein biomarker. This is now included as an important correlative in a clinical trial for Merkel Cell Cancer patients called iPRRT that is actively accruing (NCT05583708). Citation Format: Erin G. Bayer, Rachel N. Ponting, Ciara M. Kelly, Areeb Lutfi, Maaz K. Afghan, Arturo B. Ramirez, Pashtoon M. Kasi. Development and validation of an assay for the characterization of SSTR2 expression on CTCs from liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5165.

FRI265 I123-MIBG Positive, CU64-Dotatate PET Negative Pheochromocytoma In A Patient With Bilateral Adrenal Incidentalomas

Abstract Disclosure: P. Pagadala: None. V. Patel: None. Bilateral incidental adrenal nodules represent 10-23% of all incidental adrenal nodules. Most are benign nonfunctioning adenomas however these can be catecholamine-secreting tumors that arise from chromaffin cells of the adrenal medulla called pheochromocytomas. Identifying pheochromocytoma using anatomic imaging alone in cases of bilateral adrenal nodules is challenging. Functional imaging with radioisotope tracers can be helpful such as MIBG (meta-iodobenzylguanidine) and DOTATATE scans. We describe a case of pheochromocytoma, which was negative on DOTATATE but positive on MIBG scan which localized the pheochromocytoma. A 65-year-old male with a history of hypertension, chronic kidney disease and obstructive sleep apnea was found to have bilateral adrenal nodules on routine imaging. MRI abdomen showed a right adrenal nodule 1.7cm x 1.8cm x 1.7cm and a left adrenal nodule 2.3 cm in maximum dimension. He had no apparent symptoms of catecholamine excess. Biochemical testing revealed elevated total plasma metanephrines on serial assessments along with elevated 24-hour urine dopamine levels. A CT scan with adrenal protocol further characterized the right adrenal nodule with precontrast density of 43 Hounsfield Units (HU) with a rapid washout of 75% within 5 minutes, consistent with benign adrenal adenoma. The left adrenal gland had 2 nodules. A smaller superior nodule 1.3 cm AP x 1.0 cm transverse x 0.9 cm craniocaudal in size, density of -3 HU on the precontrast images, with approximately 85% washout most consistent with a benign adrenal adenoma. A larger inferior nodule was 2.3 cm AP x 2.2 cm transverse x 2.3 cm craniocaudal in size and precontrast density of 32 HU, rising to 58 HU during the portal venous phase and remaining at about 58 HU on the delayed images, indicating no significant washout and is indeterminate. A Cu64 labeled DOTATATE scan showed the 1.6 cm right adrenal nodule demonstrated activity similar to that of contralateral background adrenal activity. There was no comment regarding the two left adrenal nodules. A I123 MIBG scan showed the larger of the two left adrenal nodules had a strong uptake of the MIBG highly likely to be a pheochromocytoma. The smaller left adrenal nodule and the right adrenal nodule had no significant MIBG uptake, making these adrenal adenomas. The patient underwent left robotic adrenalectomy and pathology confirmed a 2.3 cm pheochromocytoma in addition to adrenocortical nodular disease. Three months post-surgery, plasma and 24-hour urinary measurements for metanephrines and catecholamines had normalized. Further research is needed to assess the benefit of I123MIBG when DOTATATE is negative. A potential cause of false negatives on DOTATATE is variability in somatostatin receptor subtype expression. Better imaging modalities for pheochromocytomas and guidelines for the management of bilateral adrenal nodules would assist in treatment decisions. Presentation: Friday, June 16, 2023

Somatostatin receptor subtype expression and radiomics from DWI-MRI represent SUV of [68Ga]Ga-DOTATOC PET in patients with meningioma

ObjectiveThis retrospective study aimed to analyse the correlation between somatostatin receptor subtypes (SSTR 1–5) and maximum standardized uptake value (SUVmax) in meningioma patients using Gallium-68 DOTA-D-Phe1-Tyr3-octreotide Positron Emission Tomography ([68Ga]Ga-DOTATOC PET). Secondly, we developed a radiomic model based on apparent diffusion coefficient (ADC) maps derived from diffusion weighted magnetic resonance images (DWI MRI) to reproduce SUVmax.MethodThe study included 51 patients who underwent MRI and [68Ga]Ga-DOTATOC PET before meningioma surgery. SUVmax values were quantified from PET images and tumour areas were segmented on post-contrast T1-weighted MRI and mapped to ADC maps. A total of 1940 radiomic features were extracted from the tumour area on each ADC map. A random forest regression model was trained to predict SUVmax and the model’s performance was evaluated using repeated nested cross-validation. The expression of SSTR subtypes was quantified in 18 surgical specimens and compared to SUVmax values.ResultsThe random forest regression model successfully predicted SUVmax values with a significant correlation observed in all 100 repeats (p < 0.05). The mean Pearson’s r was 0.42 ± 0.07 SD, and the root mean square error (RMSE) was 28.46 ± 0.16. SSTR subtypes 2A, 2B, and 5 showed significant correlations with SUVmax values (p < 0.001, R2 = 0.669; p = 0.001, R2 = 0.393; and p = 0.012, R2 = 0.235, respectively).ConclusionSSTR subtypes 2A, 2B, and 5 correlated significantly with SUVmax in meningioma patients. The developed radiomic model based on ADC maps effectively reproduces SUVmax using [68Ga]Ga-DOTATOC PET.

Leveraging Transcriptomics to Grade Pancreatic Neuroendocrine Neoplasms(NENs) and Assess Molecular Alterations Associated with Somatostatin Receptor(SSTR) Subtype Expression

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Molecular targets in acromegaly.

Molecular therapeutic targets in growth hormone (GH)-secreting adenomas range from well-characterized surface receptors that recognize approved drugs, to surface and intracellular markers that are potential candidates for new drug development. Currently available medical therapies for patients with acromegaly bind to somatostatin receptors, GH receptor, or dopamine receptors, and lead to attainment of disease control in most patients. The degree of control is variable: however, correlates with both disease aggressiveness and tumor factors that predict treatment response including somatostatin receptor subtype expression, granulation pattern, kinases and their receptors, and other markers of proliferation. A better understanding of the mechanisms underlying these molecular markers and their relationship to outcomes holds promise for expanding treatment options as well as a more personalized approach to treating patients with acromegaly.

Characterization of sporadic somatotropinomas with high GIP receptor expression

To analyze the expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in somatotropinomas specimens and compare clinical, biochemical, radiological, therapeutic, molecular, and pathological data among those who overexpressed (GIPR +) and those who did not overexpress (GIPR-) GIPR. Clinical, biochemical, radiological, molecular, and pathological data were collected. GNAS1 sequencing was performed with the Sanger method. Protein expression of somatostatin receptor subtypes 2 and 5 and CAM 5.2 were analyzed by immunohistochemistry. Quantitative real-time PCR was performed to analyze the mRNA expression of GIPR with the TaqMan® method. Positive expression was considered when the fold change (FC) was above 17.2 (GIPR +). A total of 74 patients (54% female) were included. Eighteen tumors (24%) were GIPR + . Gsp mutation was detected in 30 tumors (40%). GIPR + tumors were more frequently densely granulated adenomas (83% vs 47%, p = 0.028). There was no difference in clinical, biochemical, radiological, therapeutic (surgical cure or response to medical therapy), or other pathological features between GIPR + and GIPR- tumors. Twenty-eight out of 56 (50%) GIPR- tumors harbored a gsp mutation, whereas two out of 18 (11%) GIPR + tumors harbored a gsp mutation (p = 0.005). We described, for the first time, that GIPR + and gsp mutations are not mutually exclusive, but gsp mutations are less common in GIPR + tumors. GIPR + and GIPR- tumors have similar clinical, biochemical, radiological, therapeutic, and pathological features, with the exception of a high frequency of densely granulated adenomas among GIPR + tumors.

Differential expression of somatostatin receptor (SSTR) subtypes across a spectrum of neuroendocrine neoplasms (NENs).

3071 Background: Targeted therapy of NENs based on the presence of SSTRs fills a unique niche in tumor biology and clinical treatment of patients with solid tumors. SSTRs have multiple isoforms and are collectively expressed in the majority of NENs. However, subtypes are still not routinely tested and thus not assessed for clinical decision-making, especially for patients meriting consideration of targeted radionucleotide therapy. Clarifying the landscape of SSTR subtypes using molecular techniques more sensitive than immunohistochemistry (IHC)-the standard of testing, and identifying associated genomic biomarkers that differ between them, will pave the way for more sophisticated decision-making in the future. Additionally, leveraging transcriptomics to better assess mitotic markers such as Ki-67 to assess tumor grade, would increase diagnostic accuracy. Here we provide initial validation across a spectrum of NENs. Methods:1595 NENs were analyzed using Next Generation Sequencing (592 gene panel, NextSeq), Whole Exome and Transcriptome Sequencing (NovaSeq), and IHC at Caris Life Sciences (Phoenix, AZ). Significance was determined using chi-square, Fisher-Exact or Mann-Whitney U and p-adjusted for multiple comparisons (q&lt;0.05) where applicable. Results: In a subset of 492 NENs with accompanying tumor grading information, a median MKI67 (gene encoding Ki-67) TPM value of 2.27 for low-grade (LG-), and 38.7 for high-grade NENs (HG-NENs) was observed (q&lt;0.05). Using ROC curve analysis, a threshold of MKI67 expression (13.4375 TPM) differentiated LG- from HG-NENs, with a true positive rate of 86.84%, a false positive rate of 11.9% and an AUC of 95% and was subsequently applied to the entire cohort to infer HG/LG. Compared to HG-NENs (n = 862), LG-NENs(n = 733) expressed higher levels of SSTR 1(3.5-fold),2 (2.9-fold) and 5 (1.67-fold) and lower levels of SSTR4 (0.28-fold)(q&lt;0.05). Further, the expression of SSTRs 3 and 4 in HG-NEN (rs= 0.63) and SSTRs 1 and 2 in LG-NENs (rs= 0.64) were positively correlated. Overall, the prevalence of altered TP53, RB1, PIK3CA, APC, KRAS was higher and MEN1 was lower in HG-vs LG-NENs (q&lt;0.05). For each SSTR subtype, we established high and low cohorts based on median expressions. In LG-NENs, increased alterations in TP53 and RB1 were associated with increased expression of SSTRs 1 and 2 and reduced expression of SSTRs 3 and 4. In HG-NENs, increased alterations in APC were associated with increased expression of SSTR 1 and 4 and reduced expression of SSTRs 3 and 5. Additional subtype- and grade-specific alterations were also observed. Conclusions: This study provides evidence that WTS and NGS can be leveraged to predict grade of NENs and define characteristic differences in the genomic landscape across SSTR subtypes in HG and LG NENs. Incorporating the molecular profiling of NENs can thus aid in advancing the development of more tailored therapeutic strategies.

Heterogeneity of SSTR2 Expression Assessed by 68Ga-DOTATOC PET/CT Using Coefficient of Variation in Patients with Neuroendocrine Tumors.

High levels of somatostatin receptor subtype 2 (SSTR2) are a prerequisite for therapy with unlabeled or labeled somatostatin analogs. However, it is still unclear how the heterogeneity of SSTR2 expression may affect tumor response to therapy. The aim of our study was to test the ability of an imaging parameter such as coefficient of variation (CoV) derived from PET/CT with 68Ga-peptides in the evaluation and quantification of the heterogeneity of SSTR2 expression within primary and metastatic lesions of patients with neuroendocrine tumors. Methods: Thirty-eight patients with pathologically proven neuroendocrine tumors who underwent 68Ga-DOTATOC PET/CT were studied. Primary tumors were localized in the gastroenteropancreatic, bronchopulmonary, and other anatomic districts in 25, 7, and 6 patients, respectively. Malignant lesions were segmented using an automated contouring program and an SUV threshold of more than 2.5 or, in the case of liver lesions, a threshold of 30% of the SUVmax The imaging parameters SUVmean, CoV, SUVmax, receptor-expressing tumor volume, and total lesion receptor expression were obtained for each lesion. SUVmean, CoV, and SUVmax were also obtained for representative volumes of normal liver and spleen, as well as for the whole pituitary gland. Results: In total, 107 lesions were analyzed, including 35 primary tumors, 32 metastatic lymph nodes, and 40 distant metastases. Average CoVs were 0.49 ± 0.20 for primary tumors, 0.57 ± 0.26 for lymph node metastases, and 0.44 ± 0.20 for distant metastases. The CoVs of malignant lesions were up to 4-fold higher than those of normal tissues (P ≤ 0.0001). Among malignant lesions, the highest CoV was found for bone metastases (0.68 ± 0.20), and it was significantly greater than that of primary lesions (P = 0.01) and liver metastases (P < 0.0001). On the other hand, the lowest CoV was found for liver lesions (0.32 ± 0.07), probably because of the high background uptake. Conclusion: Our findings indicate that the heterogeneity of uptake, reflecting that of SSTR2, varies with the type and site of malignant lesions as assessed by CoVs obtained from 68Ga-DOTATOC PET/CT scans. These observations may be related to different biologic characteristics of tumor lesions in the same patient-differences that may affect their response to treatment with both labeled and unlabeled somatostatin analogs.

Glucocorticoid Receptor Antagonism Upregulates Somatostatin Receptor Subtype 2 Expression in ACTH-Producing Neuroendocrine Tumors: New Insight Based on the Selective Glucocorticoid Receptor Modulator Relacorilant.

Somatostatin exhibits an inhibitory effect on pituitary hormone secretion, including inhibition of growth hormone and adrenocorticotropic hormone (ACTH), and it can have antisecretory and antitumor effects on neuroendocrine tumors (NETs) that express somatostatin receptors. Although the precise mechanism remains unclear, the finding that glucocorticoids downregulate somatostatin receptor subtype 2 (SSTR2) expression has been used to explain the lack of efficacy of traditional SSTR2-targeting analogs in patients with ACTH-secreting NETs. Glucocorticoid receptor (GR) antagonism with mifepristone has been shown to reverse the glucocorticoid-induced downregulation of SSTR2; however, the effects of GR modulation on SSTR2 expression in ACTH-secreting NETs, particularly corticotroph pituitary tumors, are not well known. The current study presents new insight from in vitro data using the highly selective GR modulator relacorilant, showing that GR modulation can overcome dexamethasone-induced suppression of SSTR2 in the murine At-T20 cell line. Additional data presented from clinical case observations in patients with ACTH-secreting NETs suggest that upregulation of SSTR2 via GR modulation may re-sensitize tumors to endogenous somatostatin and/or somatostatin analogs. Clinical, laboratory, and imaging findings from 4 patients [2 ACTH-secreting bronchial tumors and 2 ACTH-secreting pituitary tumors (Cushing disease)] who were treated with relacorilant as part of two clinical studies (NCT02804750 and NCT02762981) are described. In the patients with ectopic ACTH secretion, SSTR2-based imaging (Octreoscan and 68Ga-DOTATATE positron emission tomography) performed before and after treatment with relacorilant showed increased radiotracer uptake by the tumor following treatment with relacorilant without change in tumor size at computed tomography. In the patients with Cushing disease who received relacorilant prior to scheduled pituitary surgery, magnetic resonance imaging after a 3-month course of relacorilant showed a reduction in tumor size. Based on these findings, we propose that GR modulation in patients with ACTH-secreting NETs upregulates previously suppressed SSTR2s, resulting in tumor-specific antisecretory and anti-proliferative effects. The effect of relacorilant on pituitary corticotroph tumors is being investigated in an ongoing phase 3 study (NCT03697109; EudraCT 2018-003096-35).

Differences in somatostatin receptor subtype expression in patients with acromegaly: new directions for targeted therapy?

PurposeTo analyze the expression of somatostatin receptor (SSTR)2a and 5 by immunohistochemistry (IHC) in surgically resected somatotrophic pituitary adenomas and to associate expression rates with tumor size and clinical, biochemical, and histological parameters and response to somatostatin analog (SA) therapy.MethodsForty-three microsurgically treated patients with histopathologically proven growth hormone (GH)–producing pituitary adenoma were included (WHO 2017). SSTR subtype expression was analyzed in adenoma tissues using monoclonal antibodies (Abcam, SSTR2a-UMB1, SSTR5-UMB4). Expression rates were classified as low (≤ 20% staining positivity), moderate (21–50%), and high (> 50%). Furthermore, biochemical parameters such as human growth hormone (hGH) and insulin-like growth factor-1 (IGF-1) levels were measured and clinical, biochemical, radiological, and histological data were evaluated.ResultsOf the 43 patients included in this study, 28 were female and 15 were male. The median age was 52 years (range 17–72 years). The median tumor size was 1.2 cm (range: 0.13–3.93 cm). All resected tumors showed positivity for somatotrophic hormone (STH). In all tissue samples, SSTR2a signal expression was detectable in immunohistochemistry, while only 39 samples were positive for SSTR5. Thirty-six samples had a high expression of SSTR2a, while three had a moderate and four a low SSTR2a signal. In comparison, SSTR5 signal was high in 26 out of 43 samples, while seven adenomas showed a moderate and six cases a low expression rate of SSTR5. The median IGF-1 was 714.2 µg/l and the median GH 19.6 mU/l (= 6.53 µg/l). The present study indicates that there is no significant relationship between the expression rates of receptor subtypes and the parameters we analyzed. However, our study revealed that smaller adenomas have a lower baseline GH level (p = 0.015),ConclusionIHC with monoclonal antibodies appears to be a suitable method to determine the expression rates of SSTR2a and 5 at protein levels, as it is not possible to draw conclusions regarding receptor subtypes solely on the basis of the parameters analyzed.

Abstract LB120: A novel peptide-drug conjugate induces DNA damage in SSTR2-expressing cells

Abstract Introductory statement: To overcome enzyme-mediated resistance mechanisms associated with the DNA alkylating agent, temozolomide (TMZ), we seek to develop a peptide-drug conjugate (PDC) for selective drug delivery in cells that express the somatostatin receptor subtype-2 (SSTR2). Material and methods: The clinically used somatostatin analog, 68Ga-DOTA-TOC, was used as a model for development of the SSTR2-targeted PDC. Synthesis was performed by (i) replacing DOTA with a multimodality chelator (MMC), (ii) attaching a modified TMZ analog to MMC, and (iii) conjugating the payload moiety to TOC on solid-phase. The resulting product, MMC(TMZ)-TOC, was radiolabeled with 67Ga using cation exchange chromatography methods optimized for 68Ga. Retention of SSTR2 binding was examined in cell lines with different expression level of SSTR2 with and without blocking doses of octreotide. A membrane acid wash was performed to evaluate internalization efficiency. The cytotoxicity of MMC(TMZ)-TOC was tested in IMR-32 cells that endogenously express SSTR2, and potency was compared to free-TMZ. An alkaline comet assay was then performed to assess the DNA-damaging properties of the PDC in the presence and absence of SSTR2 blocking. Unpublished results: MMC(TMZ)-TOC was efficiently produced with chemical purity &amp;gt;90% as shown by high-performance liquid chromatography (HPLC). Radiochemical purity following 67Ga labeling was &amp;gt;95% and indicates the feasibility of using the MMC to directly label the drug conjugates. Cell-based experiments showed that specific binding of the 67Ga-labeled PDC was similar to 67Ga-DOTA-TOC and correlated with SSTR2 expression. In HCT116-SSTR2 cells that overexpress SSTR2, 14.8±4.8% of 67Ga-MMC(TMZ)-TOC and 17.0±4.2% of 67Ga-DOTA-TOC were taken up by cells. Blocking the excess octreotide reduced binding to near background levels, illustrating receptor-mediated uptake of the PDC. Acid-washing demonstrated internalization of 67Ga-MMC(TMZ)-TOC after receptor-binding, indicating retention of the agonist properties of TOC after payload conjugation. Results from the cell cytotoxicity study demonstrated that the PDC inhibited cell growth in a dose-dependent manner that was similar to free-TMZ, with the IC50 values of 81.6 and 75.6 µM for free-TMZ and MMC(TMZ)-TOC, respectively. The comet assay revealed that MMC(TMZ)-TOC was effective in causing DNA damage in cells that express SSTR2, as shown by reduced cytotoxic effects when co-incubated with octreotide. Conclusions: We showed that a clinically used SSTR2-targeted radiopharmaceutical can be converted into a PDC that retains receptor-binding and internalization properties. We also showed that the cytotoxic effects of TMZ are maintained by the PDC. Based on these results, in vivo evaluation of MMC(TMZ)-TOC is warranted to assess its potential as a targeted therapy for NETs. Citation Format: Solmaz AghaAmiri, Sukhen C. Ghosh, Lea Stitzlein, Servando Hernandez Vargas, Bruno Perlatti, Daniel M. Halperin, Ali Azhdarinia. A novel peptide-drug conjugate induces DNA damage in SSTR2-expressing cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB120.

Giant silent corticotrope pituitary adenoma in a patient with complicated clinical course

Introduction Somatostatin analogues are a cornerstone in the treatment of acromegaly. Somatostatin analogues of the first generation, mainly targeting the somatostatin receptor (SSTR) subtype 2, proofed to be efficient in most patients with acromegaly. With the development of somatostatin analogues targeting the subtype 5 additionally to the subtype 2, i.e. pasireotide, an efficient drug for ACTH secreting pituitary tumors and suboptimal responding acromegaly patients became available. Methods We investigated immunohistochemically SSTR subtypes expression in three pituitary adenomas: two from operated acromegaly patients with clinical relapse and one from a patient with clinically silent ACTH-positive macroadenoma with unfavourable clinical course. Results The predictive value of SSTR subtypes immunhistochemical analysis for the therapeutic response was discussed. The one silent corticotroph adenoma case presented here was negative for subtype 2 as well as for subtype 5 and therefore we did not see an indication for somatostatin analogue therapy. In acromegaly high Ki-67 appeared to have a negative impact upon therapeutic response in the two cases. Conclusion The immunohistochemical analysis of SSTR subtypes expression in pituitary adenoma patients especially with acromegaly and complicated clinical course might be helpful for decision making or prognostic significance. Our case reports point to the need of clinical trials for further investigation.

A Prospective Trial With Ketoconazole Induction Therapy and Octreotide Maintenance Treatment of Cushing’s Disease

Abstract Context: The lack of efficacy of octreotide in the medical treatment of Cushing’s disease (CD) may result from suppressive effects of hypercortisolism on somatostatin receptor subtype 2 (SST2) expression by corticotroph adenomas. We previously demonstrated that SST2 mRNA expression levels in corticotroph tumor cells from patients preoperatively treated with cortisol-lowering therapy are significantly higher compared to those from patients with uncontrolled CD at time of surgery. It may be hypothesized that control of cortisol production induced by steroidogenesis inhibitors may reverse SST2 expression and increase efficacy of tumor-directed therapy with octreotide. Objective: To evaluate the efficacy of a sequential strategy of initiation treatment with ketoconazole (KTC) to reduce cortisol levels, followed by octreotide as maintenance therapy in patients with CD. Patients and Design: 14 adult patients with CD were prospectively enrolled. All patients started on KTC (600-800 mg/day), and once cortisol levels were normalized, octreotide 20 mg/4 weeks was initiated which could eventually be increased to 30 mg/4 weeks. After two months of combined therapy, patients were maintained on octreotide monotherapy until the end of the study period (9 months). Treatment success was assessed by the mean of 2 collections of urinary free cortisol (UFC) levels. Results: The mean age of our study population (14 patients) was 48.6 years, 64% (n=8) were female, 85% (n=12) were newly diagnosed and naïve in treatment. Ketoconazole was able to normalize UFC level in 11 (79%) patients. Subsequently, octreotide effectively sustained normal UFC levels in 3 patients (27%) (responders). Four (36%) other patients showed a partial response to octreotide. In 3 patients, normal UFC levels were sustained for one- or two-months following discontinuation of KTC and in the other partial responder, the UFC levels at follow-up decreased by at least 50% of the baseline levels. The remaining 4 (36%) patients developed hypercortisolism as soon as ketoconazole was stopped (non-responders). Responders to octreotide had lower UFC levels at baseline when compared to partial and non-responders (1.40 ± 0.06 vs. 2.05 ± 0.20 ULN, p=0.08). Two of three responders showed improvement in weight, waist circumference, and systolic and diastolic blood pressure during the treatment period. In terms of side effects, one patient discontinued KTC because of gastrointestinal intolerance and 5 patients had a transient increase in liver enzymes. Conclusions: This proof-of-concept study shows that the sequential treatment with ketoconazole to lower cortisol levels followed by octreotide to maintain normal cortisol production seems effective in a subset of patients with mild CD. Ongoing studies aim to evaluate whether this is the result from increased SST2 expression in corticotroph adenomas.

Inhibitory effects of somatostatin analogue in bleomycin-induced pulmonary fibrosis

Background and objectives Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease. An increased expression of somatostatin receptor subtype 2 in patients with IPF was identified and lung fibroblasts expressed somatostatin receptors in vitro. In addition, somatostatin analogue inhibits the expression of transforming growth factor-β, insulin-like growth factor (IGF) −1, platelet-derived growth factor, and basic fibroblast growth factor. Therefore, we examined the effects of somatostatin analogue on bleomycin-induced pulmonary fibrosis in mice. In a similar model, it has been reported that administration of high-dose somatostatin analogs suppressed acute inflammation and subsequent pulmonary fibrosis. However, it was clarified that the same effect can be obtained even at the dose used in clinical practice. Methods C57BL/6 mice received a single tracheal instillation of bleomycin. After randomly allocated, mice were treated with subcutaneous injection of either normal saline or somatostatin analogue. Results Somatostatin analogue reduced the number of neutrophils and lymphocytes in bronchoalveolar lavage (BAL) and IGF-1 level in serum and BAL fluid and attenuated weight loss. The hydroxyproline content of the lung homogenates in somatostatin analogue treatment group was significantly lower than in that of normal saline treatment group. Conclusions These results suggest that somatostatin analogue may attenuate pulmonary fibrosis after bleomycin treatment at the dose used in clinical practice.

Evaluation of Ac-Lys0(IRDye800CW)Tyr3-octreotate as a novel tracer for SSTR2-targeted molecular fluorescence guided surgery in meningioma

PurposeMeningioma recurrence rates can be reduced by optimizing surgical resection with the use of intraoperative molecular fluorescence guided surgery (MFGS). We evaluated the potential of the fluorescent tracer 800CW-TATE for MFGS using in vitro and in vivo models. It targets somatostatin receptor subtype 2 (SSTR2), which is overexpressed in all meningiomas.MethodsBinding affinity of 800CW-TATE was evaluated using [177Lu] Lu-DOTA-Tyr3-octreotate displacement assays. Tumor uptake was determined by injecting 800CW-TATE in (SSTR2-positive) NCI-H69 or (SSTR2-negative) CH-157MN xenograft bearing mice and FMT2500 imaging. SSTR2-specific binding was measured by comparing tumor uptake in NCI-H69 and CH-157MN xenografts, blocking experiments and non-targeted IRDye800CW-carboxylate binding. Tracer distribution was analyzed ex vivo, and the tumor-to-background ratio (TBR) was calculated. SSTR2 expression was determined by immunohistochemistry (IHC). Lastly, 800CW-TATE was incubated on frozen and fresh meningioma specimens and analyzed by microscopy.Results800CW-TATE binding affinity assays showed an IC50 value of 72 nM. NCI-H69 xenografted mice showed a TBR of 21.1. 800CW-TATE detection was reduced after co-administration of non-fluorescent DOTA-Tyr3-octreotate or administration of IRDye800CW. CH-157MN had no tumor specific tracer staining due to absence of SSTR2 expression, thereby serving as a negative control. The tracer bound specifically to SSTR2-positive meningioma tissues representing all WHO grades.Conclusion800CW-TATE demonstrated sufficient binding affinity, specific SSTR2-mediated tumor uptake, a favorable biodistribution, and high TBR. These features make this tracer very promising for use in MFGS and could potentially aid in safer and a more complete meningioma resection, especially in high-grade meningiomas or those at complex anatomical localizations.

The role of [68\xa0Ga]Ga-DOTATATE PET/CT in wild-type KIT/PDGFRA gastrointestinal stromal tumours (GIST)

Background[68 Ga]Ga-DOTATATE PET/CT is now recognised as the most sensitive functional imaging modality for the diagnosis of well-differentiated neuroendocrine tumours (NET) and can inform treatment with peptide receptor radionuclide therapy with [177Lu]Lu-DOTATATE. However, somatostatin receptor (SSTR) expression is not unique to NET, and therefore, [68 Ga]Ga-DOTATATE PET/CT may have oncological application in other tumours. Molecular profiling of gastrointestinal stromal tumours that lack activating somatic mutations in KIT or PDGFRA or so-called ‘wild-type’ GIST (wtGIST) has demonstrated that wtGIST and NET have overlapping molecular features and has encouraged exploration of shared therapeutic targets, due to a lack of effective therapies currently available for metastatic wtGIST.AimsTo investigate (i) the diagnostic role of [68 Ga]Ga-DOTATATE PET/CT; and, (ii) to investigate the potential of this imaging modality to guide treatment with [177Lu]Lu-DOTATATE in patients with wtGIST.Methods[68 Ga]Ga-DOTATATE PET/CT was performed on 11 patients with confirmed or metastatic wtGIST and one patient with a history of wtGIST and a mediastinal mass suspicious for metastatic wtGIST, who was subsequently diagnosed with a metachronous mediastinal paraganglioma. Tumour expression of somatostatin receptor subtype 2 (SSTR2) using immunohistochemistry was performed on 54 tumour samples including samples from 8/12 (66.6%) patients who took part in the imaging study and 46 tumour samples from individuals not included in the imaging study.Results[68 Ga]Ga-DOTATATE PET/CT imaging was negative, demonstrating that liver metastases had lower uptake than background liver for nine cases (9/12 cases, 75%) and heterogeneous uptake of somatostatin tracer was noted for two cases (16.6%) of wtGIST. However, [68 Ga]Ga-DOTATATE PET/CT demonstrated intense tracer uptake in a synchronous paraganglioma in one case and a metachronous paraganglioma in another case with wtGIST.ConclusionsOur data suggest that SSTR2 is not a diagnostic or therapeutic target in wtGIST. [68 Ga]Ga-DOTATATE PET/CT may have specific diagnostic utility in differentiating wtGIST from other primary tumours such as paraganglioma in patients with sporadic and hereditary forms of wtGIST.

Inter and intra-tumor somatostatin receptor 2 heterogeneity influences peptide receptor radionuclide therapy response.

Patients with neuroendocrine tumors (NETs) can be treated with peptide receptor radionuclide therapy (PRRT). Here, the somatostatin analogue octreotate radiolabeled with lutetium-177 is targeted to NET cells by binding to the somatostatin receptor subtype 2 (SST2). During radioactive decay, DNA damage is induced, leading to NET cell death. Although the therapy proves to be effective, mortality rates remain high. To appropriately select more optimal treatment strategies, it is essential to first better understand the radiobiological responses of tumor cells to PRRT.Methods: We analyzed PRRT induced radiobiological responses in SST2 expressing cells and xenografted mice using SPECT/MRI scanning and histological and molecular analyses. We measured [177Lu]Lu-DOTA-TATE uptake and performed analyses to visualize induction of DNA damage, cell death and other cellular characteristics.Results: The highest accumulation of radioactivity was measured in the tumor and kidneys. PRRT induced DNA damage signaling and repair in a time-dependent manner. We observed intra-tumor heterogeneity of DNA damage and apoptosis, which was not attributed to proliferation or bioavailability. We found a strong correlation between high DNA damage levels and high SST2 expression. PRRT elicited a different therapeutic response between models with different SST2 expression levels. Heterogeneous SST2 expression levels were also confirmed in patient NETs.Conclusion: Heterogeneous SST2 expression levels within NETs cause differentially induced DNA damage levels, influence recurrent tumor phenotypes and impact the therapeutic response in different models and potentially in patients. Our results contribute to a better understanding of PRRT effects, which might impact future therapeutic outcome of NET patients.

Expression of Somatostatin Receptor Subtypes (SSTR-1-SSTR-5) in Pediatric Hematological and Oncological Disorders.

Hematological and oncological disorders represent leading causes of childhood mortality. Neuropeptide somatostatin (SST) has been previously demonstrated in various pediatric tumors, but limited information exists on the expression and characteristics of SST receptors (SSTR) in hematological and oncological disorders of children. We aimed to investigate the expression of mRNA for SSTR subtypes (SSTR-1–5) in 15 pediatric hematological/oncological specimens by RT-PCR. The presence and binding characteristics of SSTRs were further studies by ligand competition assay. Our results show that the pediatric tumor samples highly expressed mRNA for the five SSTR subtypes with various patterns. The mRNA for SSTR-2 was detected in all specimens independently of their histological type. A Hodgkin lymphoma sample co-expressed mRNA for all five SSTR subtypes. SSTR-3 and SSTR-5 were detected only in malignant specimens, such as rhabdomyosarcoma, Hodgkin lymphoma, acute lymphoblastic leukemia, and a single nonmalignant condition, hereditary spherocytosis. The incidence of SSTR-1 and SSTR-4 was similar (60%) in the 15 specimens investigated. Radioligand binding studies demonstrated the presence of specific SSTRs and high affinity binding of SST analogs in pediatric solid tumors investigated. The high incidence of SSTRs in hematological and oncological disorders in children supports the merit of further investigation of SSTRs as molecular targets for diagnosis and therapy.

Multicenter, Observational Study of Lanreotide Autogel for the Treatment of Patients with Acromegaly in Routine Clinical Practice in Germany, Austria and Switzerland.

Evidence from controlled trials has shown that lanreotide autogel is effective in achieving biochemical and symptom control in patients with acromegaly. However, it is important to better understand the real-world patient population receiving lanreotide autogel treatment. In this non-interventional study the long-term treatment response to lanreotide autogel in adult patients with acromegaly from office-based centers or clinics in Germany, Austria and Switzerland was studied. Assessments included growth hormone and insulin-like growth factor-I levels, symptoms, quality of life, lanreotide plasma levels and tumor somatostatin receptor subtype expression. The primary endpoint was achievement of full biochemical control, defined as growth hormone ≤2.5 µg/L and insulin-like growth factor I normalization at month 12. 76 patients were enrolled from 21 sites. 7/51 (13.7%) patients of the efficacy population had full biochemical control at baseline, 15/33 (45.5%) at month 12 and 10/26 (38.5%) at month 24 of treatment. At 12 months of treatment higher rates of biochemical control were observed in the following subgroups: older patients (>53 years [median]), females, treatment-naïve patients, and patients with a time since diagnosis of longer than 1.4 years (median). No clinically relevant differences in acromegaly symptoms or quality of life scores were observed. Median fasting blood glucose and glycated hemoglobin levels remained unchanged throughout the study. No new safety signals were observed. Overall tolerability of treatment with lanreotide autogel was judged by 80.8% of the enrolled patients at month 12 as 'very good' or 'good'. Treatment with lanreotide autogel in a real-world setting showed long-term effectiveness and good tolerability in patients with acromegaly.