Stimulated emission depletion (STED) microscopy is one of the optical superresolution microscopy (SRM) techniques, more recently also referred to as nanoscopy, that have risen to popularity among biologists during the past decade. These techniques keep pushing the physical boundaries of optical resolution toward the molecular scale. Thereby, they enable biologists to image cellular and tissue structures at a level of almost molecular detail that was previously only achievable using electron microscopy. All the while, they retain the advantages of light microscopy, in particular with regards to sample preparation and flexibility of imaging. Commercially available SRM setups have become more and more available and also increasingly sophisticated, both in terms of optical performance and, importantly, ease of use. Institutional microscopy core facilities now offer widespread access to this type of systems. However, the field has grown so rapidly, and keeps growing, that biologists can be easily overwhelmed by the multitude of available techniques and approaches. From this vast array of SRM modalities, STED stands out in one respect: it is essentially an extension to an advanced confocal microscope. Most experienced users of confocal microscopy will find the transition to STED microscopy relatively easy as compared with some other SRM techniques. This also applies to STED sample preparation. Nonetheless, because resolution in STED microscopy does not only depend on the wavelength of the incident light and the numerical aperture of the objective, but crucially also on the square root of the intensity of the depletion laser and, in general, on the photochemical interaction of the fluorophore with the depletion laser, some additional considerations are necessary in STED sample preparation. Here we describe the single color staining of the somatostatin receptor subtype 2A (SSTR2A) and dual color staining of the trans-Golgi-network protein TGN 38 and the t-SNARE syntaxin-6 for STED in the endocrine cell line AtT20 and STED imaging of the samples, providing the protocols in as general a form as possible. The protocols in this chapter are used in this way in an institutional microscopy core facility.

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic disorder caused by excessive fibroblast growth factor 23 (FGF23) secretion. FGF23 immunohistochemistry (IHC) is proposed as a useful adjunctive marker to confirm TIO diagnosis. However, it often stains focally, limiting its diagnostic utility.
This work aimed to compare the diagnostic performance between somatostatin receptor 2A (SSTR2A) and FGF23 IHC for TIO.
We retrospectively reviewed TIO-diagnosed patients in Severance Hospital between July 2006 and May 2020. Histologic evaluation was performed using histoscore (H score) (expression area proportion score [0-2] × intensity score [1-3], [total, 0-6]). FGF23 and SSTR2A IHC were performed using unstained slides from 18 localized TIO patients and 9 and 15 non-TIO controls with bone and soft-tissue tumors, respectively. SSTR2A positivity was defined as cytoplasmic, membranous, or Golgi staining in more than 1% of tumor cells, and negativity as nonspecific nuclear staining. FGF23 positivity was defined as cytoplasmic expression in more than 1% of the tumor area and negativity as nonspecific nuclear staining.
Suspicious lesions were successfully detected in 14 of 15 patients who underwent 68Ga-DOTATOC scans. Diffuse cytoplasmic SSTR2A expression was identified in all TIO patients and focal weak nuclear staining in 12 of 15 controls. FGF23 cytoplasmic expression was identified in 11 of 18 TIO patients and diffuse nuclear staining in 9 of 9 controls. The H score was higher in SSTR2A than in FGF23 IHC (median [interquartile range]: 6 [6-6] vs 1 [0-2], P < .001).
SSTR2A IHC with H-score quantification might be a more sensitive, adjunctive diagnostic tool than FGF23 IHC for TIO diagnosis.

Phosphaturic mesenchymal tumour (PMT) is a rare tumour that occurs in bone or soft tissue and is associated with production of fibroblast growth factor 23 (FGF23) leading to tumor-induced osteomalacia. We report three cases of PMT involving the head and neck that highlight the broad spectrum of clinical and histologic features of PMT. One of these lesions from the hard palate demonstrated an admixture of epithelial and mesenchymal elements, a feature that can pose a diagnostic challenge. The diagnostic utility of immunohistochemistry including FGF23, somatostatin receptor 2A, SATB2, ERG and CD56 is discussed. The biochemical pathway in the development of PMT associated tumor induced osteomalacia and its role in investigations and management of PMT is also described.

Although canine pancreatic neuroendocrine neoplasms (PanNENs) have been proposed as a model for the counterpart human neoplasms, the type or grade of human PanNEN that they resemble is unclear. PanNENs in animals are classified as adenoma or carcinoma, whereas in humans they are classified as pancreatic neuroendocrine tumour (PanNET) if well-differentiated, or as pancreatic neuroendocrine carcinoma (PanNEC) if poorly differentiated. We evaluated 16 canine primary PanNENs and two metastases histologically and immunohistochemically, and graded them using the animal and human grading systems. All neoplasms had local or vascular invasion and were classified as pancreatic islet cell carcinomas according to the current WHO classification. The Ki-67 index was low in all cases (0.01-1.50%). All had cytoplasmic expression of synaptophysin and insulin but were immunonegative for glucagon, confirming a functional diagnosis of canine insulinoma. Membranous expression of SSTR2A and nuclear expression of ATRX, but no p53 expression, was found in all neoplasms. One primary tumour was diagnosed as a mixed neuroendocrine-non-neuroendocrine neoplasm, which is the first report of this neoplasm in dogs. The other 15 primary tumours and both metastatic tumours were graded as PanNET G1, according to the human WHO classification. We conclude that canine PanNENs share well-differentiated histomorphology, SSTR2A expression and absence of nuclear p53 immunolabelling with human PanNETs G1. However, they differ in ATRX gene expression and functionality, and seem to have a worse prognosis than human PanNETs G1, although their generally low Ki-67 index precludes more precise assessment of prognosis. Membranous SSTR2A expression renders canine PanNENs potentially amenable to treatment with somatostatin analogues or SSTR targeted in-vivo imaging methods.

Somatostatin receptor 2a (SSTR2a) is an important diagnostic and scintigraphic marker in several tumors, as well as a potential therapeutic target. However, the expression and clinicopathologic significance of SSTR2a in nasopharyngeal carcinoma (NPC) remain unknown. The expression of SSTR2a was retrospectively analyzed in a large series of NPC tissue samples (106 primary NPC samples, comprising 99 primary non-keratinizing NPC (NK-NPC) and 7 keratinizing NPC (K-NPC) samples, and 41 metastatic NPC samples) by immunohistochemistry, with 24 cases of normal nasopharyngeal mucosa tissues used as a control group. Normal epithelia in nasopharyngeal mucosa were negative for SSTR2a in all 24 cases. The expression of SSTR2a in primary NPC was correlated to the histological subtype. Most cases of primary NK-NPC showed expression of SSTR2a (93.9%, 93/99 cases). The percentage of SSTR2a-positive tumor cells ranged from 10 to 100%, while the intensity ranged from 2+ to 4+. None of the primary K-NPC samples showed SSTR2a expression (0/7, 100%). All cases of NPC showed negative expression of other neuroendocrine markers, including synaptophysin, chromogranin A, and CD56. Of all 41 cases of metastatic NK-NPC lesions, SSTR2a expression is concordant with that of the primary lesions, which shows statistical significance (p < 0.001). Our observations expand the spectrum of recognized SSTR2a-positive tumors and demonstrate for the first time that SSTR2a is frequently expressed in primary and metastatic NK-NPC, highlighting its potential as a scintigraphic and therapeutic target in this disease.

OBJECTIVE: Although ultrastructural studies showed that minute pulmonary meningothelial-like nodules (MPMNs) cells closely resembled meningothelial cells, their immunophenotype has not been well characterised, partly due to their rarity.
METHODS: Somatostatin receptor 2a (SSTR2a) and other markers of meningioma, including epithelial membrane antigen (EMA), progesterone receptor (PR) and S100, were analysed retrospectively in 19 MPMN cases from two institutions in China.
RESULTS: The median age of patients with MPMNs was 62.5 years (32-73 years), with a male-to-female ratio of 1:8.5. Most (15/19) patients with MPMNs had coexisting diseases, including adenocarcinomas (12 cases), bronchiectasis (1 case) and tuberculosis (2 cases). Just over half of the cases (10/19) were multifocal lesions (2-5 lesions). An additional 53 cases with 123 lesions from the literature were reviewed with reported immunophenotype information. In total, 162 lesions were included in the analysis. The size of nodules was 1-4 mm. All MPMN lesions (39/39) in the 19 cases showed strong and diffuse cytoplasmic expression of SSTR2a. The expression rate of SSTR2a was higher than that of conventional markers of meningioma, including EMA (86/138), PR (32/68) and S100 (1/125).
CONCLUSIONS: Our observations expand the spectrum of recognised SSTR2a-positive lesions and once again demonstrated that MPMNs show immunohistochemical characteristics similar to meningothelial cells.

Higher-grade meningiomas (WHO grade II and III) represent a diagnostic and prognostic challenge. We assessed the pathological and molecular characteristics of 94 higher-grade meningiomas (85 grade II, 9 grade III) to identify novel prognostic parameters. Higher mitotic count (p = 0.018), diffuse (≥50%) prominent nucleoli (p < 0.001), and sheeting (p < 0.001) were associated with recurrence. Lower SSTR2a-positive cells median rate (p = 0.048) and TERT promoter mutations (p = 0.014) were associated with recurrence and patient death, respectively; further analyses did not identify other outcome associations. Presence of Ki67 hot spots was associated with a shorter progression-free survival (PFS), independently of WHO grade at multivariate analysis (HR = 3.35, p = 0.008). Necrosis was related to a poorer overall survival (OS) at univariate (focal: HR = 4.55, p = 0.041 and diffuse: HR = 7.38, p = 0.020) and Kaplan-Meier analyses. A prognostic score was designed based on previous results: Presence of diffuse (≥50%) prominent nucleoli (0/1 point), diffuse (≥50%) sheeting (0/1 point), focal (<50%) or diffuse (≥50%) necrosis (0/1/2 points), and Ki67 hot spots (0/1 point). A total score ≥4 predicted poorer PFS and OS by Kaplan-Meier (PFS: 1.7 vs 6.4 years, p < 0.001 and OS: 5.2 vs 10.8 years, p = 0.001) and multivariate (PFS: HR = 5.98, p < 0.001 and OS: HR = 2.99, p = 0.048) analyses. These results were confirmed in an independent series of 58 grade II meningiomas (PFS: HR = 7.22, p = 0.002 and OS: HR = 9.69, p = 0.003). These associations and the integrated score could complement WHO grading.

Acromegaly is a neuroendocrine disorder caused by excess secretion of GH by somatotroph tumor cells. It is often treated with somatostatin receptor (SSTR) 2 agonists, which suppress GH secretion. SOM230 is a somatostatin analogue that targets multiple SSTRs and was recently approved for patients with treatment-resistant acromegaly. Previous reports indicate that SOM230 may function as a biased agonist, suggesting that its ability to selectively activate SSTR-dependent signaling events may contribute to its therapeutic efficacy. To better understand how SOM230 modulates Sstr2A function, which is the most commonly expressed SSTR in somatotrophs, we used real-time assays to study SOM230-dependent signaling in rat pituitary tumor cells. We observed that SOM230 suppressed cAMP production in a Gαi-dependent manner, similar to conventional Sstr2A agonists. However, it did not cause receptor internalization as would be expected for an Sstr2A agonist. Surprisingly, SOM230 did not cause membrane hyperpolarization, which is an important mechanism by which Sstr2a activation suppresses intracellular calcium (Ca2+) accumulation and GH secretion. In fact, SOM230 inhibited the ability of conventional somatostatin analogues to control membrane potential. However, SOM230 still inhibited intracellular Ca2+ accumulation in a novel, Gβγ-dependent manner. These studies show that SOM230 exhibits strong agonist bias in regulating signaling pathways downstream of Sstr2A that control GH secretion.

Olfactory neuroblastoma (ONB) is a malignant neuroendocrine neoplasm with a usually slow course, but with considerable recurrence rate. Many neuroendocrine tumors have shown good response to the treatment with somatostatin analogs and somatostatin radioreceptor therapy. In ONBs, there are scarce data on somatostatin-based treatment and the cellular expression of somatostatin receptors (SSTR), the prerequisite for binding and effect of somatostatin on normal and tumor cells. The aim of our study was to investigate the immunohistochemical expression of SSTR2A and SSTR5 in a cohort of 40 ONBs. In addition, tissue microarrays containing 40 high-grade sinonasal carcinomas as well as 6 sinonasal lymphomas, 3 rhabdomyosarcomas, and 3 Ewing sarcomas were evaluated. Volante system was applied for staining evaluation. Thirty cases (75%) were immunopositive for SSTR2A and 3 (7.5%) for SSTR5. Among the 30 SSTR2A-positive ONBs, 19 tumors (63.3%) scored 2+ and 11 (36.7%) scored 3+. All SSTR5-positive ONBs scored 2+. Neither sinonasal carcinomas nor sinonasal small round blue cell neoplasms expressed SSTR2A or SSTR5. The frequent expression of SSTR2A provides a rationale for radioreceptor diagnosis and therapy with SST analogs in ONBs. SSTR2A expression in ONBs is a helpful adjunct in the differential diagnosis of ONBs.

BACKGROUND: The distinction between meningioma, schwannoma and solitary fibrous tumour/ hemangiopericytoma can be challenging in some cases. This study evaluates the expression of Somatostatin receptor 2A (SSTR2A) and Claudin-1 in these different tumours.
METHODS: Thirty-five cases of meningioma, 10 cases of intracranial schwannoma and 10 cases of hemangiopericytoma were assessed. The immunohistochemical expression of SSTR2A and Claudin-1 was evaluated and scored according to the percentage of immunostained tumour cells (0: 1+, 2+ and 3). The intensity of staining was classified as weak, moderate and strong.
RESULTS: Positivity for SSTR2A and Claudin-1 was encountered in 89% and 49% of meningiomas respectively. None of the schwannomas or hemangiopericytomas was positive for any of both markers. All grade I and II meningiomas were positive for SSTR2A, and only 20% of grade III showed positive staining (p < 0.05). Claudin-1 positivity was detected in 50%, 43% and 60% of grade I, II and III meningioma respectively, with significantly higher intensity in grade III (p < 0.05).
CONCLUSIONS: SSTR2A is highly sensitive and specific for meningioma. Claudin-1 is highly specific for meningioma, with low sensitivity. The adjunctive use of both markers can be very helpful in the diagnosis of meningioma and its distinction from schwannoma and hemangiopericytoma.