Clinical Correlation to E-cadherin and Granulation Patterns in Corticotroph Tumors

Corticotroph adenomas, either secretory or silent, are associated with significant disease persistence and/or recurrence. Surgical resection is the first line treatment; however, recurrence rates range from 30 to 60%. Although several clinical parameters (i.e. postoperative cortisol level, tumor invasion) have been reported to predict tumor recurrence, none have high diagnostic accuracy. Granulation pattern classifies corticotroph tumors as densely granulated (DG) or sparsely granulated (SG) types, with the latter usually larger and more aggressively behaving. E-cadherin, a calcium-dependent adhesion molecule strongly expressed in normal pituitary cells, plays a role in epithelial cell behavior, tissue development, and suppresses epithelial-mesenchymal transition. Since loss of E-cadherin expression in sparsely granulated somatotroph pituitary tumors correlates with a more aggressive disease course, we sought to examine correlation between E-cadherin expression and behavior in densely versus sparsely granulated corticotroph tumors. A retrospective chart review of adult patients with corticotroph adenomas, seen at our institution between January 2012 - 2020 yielded 62 patients: 18 (29%) male and 44 female (71%), with median age at diagnosis of 49 years (range 25-82). Inclusion criteria required sufficient tissue for E- cadherin immunostaining (IHC). Microadenomas were identified in 19/62 (31%) patients, and 38/62 (52%) patients had clinical and biochemical findings consistent with excess cortisol secretion. Pre-operative imaging showed that 22/62 (35%) tumors were invasive into surrounding structures. After further classification as to densely granulated (DG) or sparsely granulated (SG) types by ACTH granulation pattern on IHC, 19/56 (34%) adenomas were SG, 37/56 (66%) were DG and 6 were not classified. E-cadherin staining was absent in 7/62 tumors (11%) and diminished in 5/62 (8%) tumors and staining did not correlate with dense versus sparse corticotroph types. Chi-squared analysis found a significant association between tumor size (greater than or less than 1cm) and secretion, with hormonally active more likely tumors to be microadenomas (p=0.004). Microadenomas were exclusively DG tumors (p<0.001). Further analysis did not find correlation between presence or absence of E-cadherin expression and tumor invasion into adjacent structures, or recurrence. In summary, the data suggests that, unlike somatotroph corticotroph adenomas for recurrence or invasion, nor does it correlate strongly with granulation status.

201 Correlation between metaphase II oocyte cytoplasmic granulation patterns and intracytoplasmic sperm injection fertilization outcome in older patients

Cytoplasmic granulation is frequently observed in human MII oocytes in fertility clinics from women requiring early egg retrieval (also called highly individualized egg retrieval, with hCG trigger at lead follicle size 16.0-18.0mm or even less) to avoid premature luteinization. Early retrieval is mostly done for older women (≥36 years old) or younger women with premature ovarian aging. However, only limited reports have focused on detailed analysis of the patterns of cytoplasmic granulation and their correlation with the quality of human oocytes. The aim of this report was to evaluate correlation between granulation patterns of human MII oocytes from early retrieval and the corresponding fertilization rates from intracytoplasmic sperm injection (ICSI). Each MII oocyte was imaged when ICSI was performed. The granulation pattern of each oocyte was reviewed, evaluated, and categorized by the same person as non-granulation (NG, homogeneous, sandy pattern, no granulation or granulation area <1/8 of total cytoplasm), central granulation (CG, ring pattern, a cluster of granules in centre with size >1/4 of oocyte diameter), uneven granulation (UG, sandy-rocky pattern, granulation area >1/8 but <3/4 of total cytoplasm), and dispersed granulation (DG, rocky pattern, granulation area >3/4 of total cytoplasm). Fertilization (pronucleus count, PN) results were then extracted from medical records and matched to the granulation categories. In total, 1759 oocytes done with ICSI in year 2018 were analysed. Fertilization (<2PN, 2PN or >2PN) rates of each group (n=381, n=1222, n=156, respectively) were calculated. Student's t-test was used for group-group comparison analysis. Compared with the NG group, the UG and CG groups had lower 2PN rates (67.2% vs. 84.6%; P<0.001; 73.0% vs. 84.6%; P<0.05; respectively) and the DG group had a much lower 2PN rate (43.3% vs. 84.6%; P<0.001). The UG and CG groups had higher <2PN rates (24.8% vs. 12.1%; P<0.001; 24.3% vs. 12.1%; P<0.001; respectively), and the DG group had a much higher <2PN rate (41.1% vs. 12.1%; P<0.001) than the NG group. For >2PN rates, both UG and DG groups were higher than the NG group (8.0% vs. 3.3%; P<0.05; 15.6% vs. 3.3%; P<0.001; respectively), whereas the DG group was higher than the UG group (15.6% vs. 8.0%). These data demonstrated that dispersed granulation was the worst pattern for fertilization. Interestingly, many of the uneven-granulation group oocytes could become homogeneous (non-granulation) after hours or overnight IVM culture, suggesting that uneven granulation might be a sign of incomplete cytoplasmic maturity. Technically, performing ICSI through the less-granulation side was easier because of less resistance to membrane breaking and to pushing sperm out. Discovering the molecular nature of the granules and related cytoskeleton structures would extend our understanding of their existence and improve treatments by adjusting stimulation and trigger accordingly.

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

Purpose Little 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. Method In 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. Results Median 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. Conclusion Pasireotide-LAR may represent a suitable therapeutic alternative in a subset of acromegalic patients requiring combination therapy involving a 1gSRL

Morphological and genetic analysis of Milnesium cf. granulatum (Tardigrada: Milnesiidae) from Northeastern North America‡

Specimens of a limnoterrestrial tardigrade collected from moss in New Hampshire, USA and Newfoundland, Canada were identified as belonging to the genus Milnesium. Morphometric data and genetic analysis of the COI gene demonstrated that animals collected from these sites represent the same species. Specimens most closely resemble Milnesium granulatum, a South American species that has been reported also from Great Smoky Mountains National Park (GSMNP), USA. New Hampshire and Newfoundland animals have the same adult claw configuration and cuticular granulation pattern as M. granulatum. Theira* (i.e., normalized to correct for allometric effects) pt values differ from GSMNP M. granulatum in having a more posterior stylet support insertion point. However, all their morphometric values overlap in range with GSMNP specimens. Unlike some other species in the genus, the claw configuration of New Hampshire Milnesium cf. granulatum shows no ontogenetic change in claw configuration. A short (269 bp on average) fragment of the internal transcribed spacer region 2 (ITS-2) was sequenced for 20 New Hampshire adults and from siblings hatched in the laboratory from 8 egg clutches. Genetic diversity among New Hampshire adults was 0.0–3.7%. Multiple haplotypes were found between siblings in a single clutch.

Turbulent diffusion in the photosphere as observational constraint on dynamo theories

We utilized line-of-sight magnetograms acquired by HMI/SDO to derive the value of turbulent magnetic diffusivity in undisturbed photosphere. Two areas, a coronal hole area (CH) and an area a super-granulation pattern, SG, were analyzed. The behavior of the turbulent diffusion coefficient on time scales of 1000-40000 s and spatial scales of 500-6000 km was explored. Small magnetic elements in both CH and SG areas disperse in the same way and they are more mobile than the large elements. The regime of super-diffusivity is found for small elements (the turbulent diffusion coefficient K growths from 100 to 300 km2 s−1). Large magnetic elements disperse differently in the CH and SG areas. Comparison of these results with the previously published shows that there is a tendency of saturation of the diffusion coefficient on large scales, i.e., the turbulent regime of super-diffusivity gradually ceases so that normal diffusion with a constant value of K ≈ 500 km2 s−1 might be observed on time scales longer than a day. The results show that the turbulent diffusivity should not be considered in modeling as a scalar, the flux- and scale-dependence is obvious.

Somatostatin receptor ligands and resistance to treatment in pituitary adenomas

Somatostatin (SST), an inhibitory polypeptide with two biologically active forms SST14 and SST28, inhibits GH, prolactin (PRL), TSH, and ACTH secretion in the anterior pituitary gland. SST also has an antiproliferative effect inducing cell cycle arrest and apoptosis. Such actions are mediated through five G-protein-coupled somatostatin receptors (SSTR): SSTR1–SSTR5. In GH-secreting adenomas, SSTR2 expression predominates, and somatostatin receptor ligands (SRLs; octreotide and lanreotide) directed to SSTR2 are presently the mainstays of medical therapy. However, about half of patients show incomplete biochemical remission, but the definition of resistanceper seremains controversial. We summarize here the determinants of SRL resistance in acromegaly patients, including clinical, imaging features as well as molecular (mutations, SSTR variants, and polymorphisms), and histopathological (granulation pattern, and proteins and receptor expression) predictors. The role of SSTR5 may explain the partial responsiveness to SRLs in patients with adequate SSTR2 density in the cell membrane. In patients with ACTH-secreting pituitary adenomas, i.e. Cushing's disease (CD), SSTR5 is the most abundant receptor expressed and tumors show low SSTR2 density due to hypercortisolism-induced SSTR2 down-regulation. Clinical studies with pasireotide, a multireceptor-targeted SRL with increased SSTR5 activity, lead to approval of pasireotide for treatment of patients with CD. Other SRL delivery modes (oral octreotide), multireceptor-targeted SRL (somatoprim) or chimeric compounds targeting dopamine D2 receptors and SSTR2 (dopastatin), are briefly discussed.

Epithelial Splicing Regulator Protein 1 and Alternative Splicing in Somatotroph Adenomas

Somatotroph adenomas secrete supraphysiological amounts of GH, causing acromegaly. We have previously hypothesized that epithelial mesenchymal transition (EMT) may play a central role in the progression of these adenomas and that epithelial splicing regulator 1 (ESRP1) may function prominently as a master regulator of the EMT process in pituitary adenomas causing acromegaly. To further elucidate the role of ESRP1 in somatotroph adenomas and in EMT progression, we used RNA sequencing (RNAseq) to sequence somatotroph adenomas characterized by high and low ESRP1 levels. Transcripts identified by RNAseq were analyzed in 65 somatotroph adenomas and in GH-producing pituitary rat cells with a specific knockdown of Esrp1. The clinical importance of the transcripts was further investigated by correlating mRNA expression levels with clinical indices of disease activity and treatment response. Many of the transcripts and isoforms identified by RNAseq and verified by quantitative PCR were involved in vesicle transport and calcium signaling and were associated with clinical outcomes. Silencing Esrp1 in GH3 cells resulted in changes of gene expression overlapping the data observed in human somatotroph adenomas and revealed a decreased granulation pattern and attenuated GH release. We observed an alternative splicing pattern for F-box and leucine-rich repeat protein 20, depending on the ESPR1 levels and on changes in circulating IGF-I levels after somatostatin analog treatment. Our study indicates that ESRP1 in somatotroph adenomas regulates transcripts that may be essential in the EMT progression and in the response to somatostatin analog treatment.