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SAT-173 SAMD9 (Sterile Alpha Motif Domain-Containing 9) Expression in Adrenocortical Tumors

Abstract Introduction: SAMD9 variants are associated with colon, breast and lung tumors. SAMD9 mutations result in adrenal hypoplasia, suggesting its importance in adrenal development. We investigated the contribution of abnormal expression of SAMD9 and its homologous, SAMD9L, to the pathogenesis of pediatric adrenocortical tumors (pACT) Objective: To evaluate the involvement of SAMD9 and SAMD9L in normal human adrenal cortex development and adrenocortical tumorigenesis, as well as to evaluate their association with tumor presentation and patient outcome. Methods: pACT samples (n= 72), normal pediatric adrenal cortices (n= 11), and normal fetal and post-natal adrenals (20 weeks of gestation to 10 years of age; n= 51) were enrolled. Protein expression of SAMD9 (immunohistochemistry) and SAMD9/SAMD9L mRNA levels were evaluated (qPCR). The associations between SAMD9/SAMD9L expression in pACT with tumor presentation (P53 p.R337H genotype and metastasis occurrence) and patient outcome (Overall (OS) and Disease-Free Survival) were analyzed. In silico, publicly available data from pediatric patients with ACT available in the Gene Expression Omnibus were used to evaluate the aforementioned associations with SAMD9 (GSE76021) and SAMD9L (GSE76019) mRNA levels. In vitro, SAMD9 subcellular localization pattern was investigated in the ACT cell line NCI-H295R (immunofluorescence). Results: Nuclear and cytoplasmic SAMD9 expression was observed throughout all different phases of adrenal development evaluated. However, in pACT samples, 26% presented nuclear and 86% cytoplasmic immunostaining. In line, NCI-H295R cells presented mostly cytoplasmic SAMD9 immunostaining under basal conditions. No differential expression was observed between SAMD9 or SAMD9L mRNA levels in pACT and in normal pediatric adrenals. Moreover, no association between SAMD9 immunostaining, SAMD9 or SAMD9L mRNA levels, tumor presentation and patient outcome were observed in our cohort, nor in the in silico evaluation. Conclusion: SAMD9 is nuclear and cytoplasmic expressed during adrenal cortex adrenal development and its loss of function is associated with adrenal hypoplasia. On the other hand, SAMD9 is mostly cytoplasmic expressed in pACT and immortalized NCI-H295R cells. No differential expression of SAMD9 nor its counterpart SAMD9L mRNA were associated with tumor presentation and pediatric patient outcome.


MON-251
The patient is a 54-year-old male with type 2 diabetic treated with insulin who presented with a two-week history of fever nausea and vomiting. He subsequently developed seizure episodes with changes in mental status prompting a visit to the emergency room. Upon admission the patient was hypotensive and lethargic. After an initial improvement, he developed polyuria (14-15 liters per day) in the context of eunatremia and specific gravity of 1010. His initial evaluation revealed a serum a.m. cortisol of 1.2 mcg/dL (6.2 to 19.4), low free T4 of 0.64 Ng/ dl) 0.9-1.7) and secondary hypogonadism with a total testosterone of less than 2.5 ng/dL (181-772). Hormonal replacement was initiated. MRI of the brain revealed heterogeneous sellar lesion measuring 2.1 x 2.0 x 2.0 cm with peripheral enhancement following contrast administration and deviation of the pituitary stalk to the right. The lesion invaded the left cavernous sinus (Knosp III) and abutted the optic chiasm. He underwent an uneventful transsphenoidal tumorectomy revealing extensive coagulation necrosis with chronic lymphocytic infiltration, numerous macrophages and multinucleated giant cells with deposits of hemosiderin, cellular debris and isolated cholesterol crystals surrounded by granulomatous tissue. The pathology was compatible with xanthogranulomatous hypophysitis. We report a rare case of Xanthogranulomatous hypophysitis (XGH), the least common subtype of pituitary hypophysitis often mistaken by a neoplastic lesion. It is more common in young adults occurring in isolation, as a secondary reaction to a local process such as apoplexy part or as part of a systemic condition with autoimmune aetiology, often resulting in variable degrees of anterior and posterior pituitary deficiencies.

SAT-173
Introduction: SAMD9 variants are associated with colon, breast and lung tumors. SAMD9 mutations result in adrenal hypoplasia, suggesting its importance in adrenal development. We investigated the contribution of abnormal expression of SAMD9 and its homologous, SAMD9L, to the pathogenesis of pediatric adrenocortical tumors (pACT) Objective: To evaluate the involvement of SAMD9 and SAMD9L in normal human adrenal cortex development and adrenocortical tumorigenesis, as well as to evaluate their association with tumor presentation and patient outcome. Methods: pACT samples (n= 72), normal pediatric adrenal cortices (n= 11), and normal fetal and post-natal adrenals (20 weeks of gestation to 10 years of age; n= 51) were enrolled. Protein expression of SAMD9 (immunohistochemistry) and SAMD9/SAMD9L mRNA levels were evaluated (qPCR). The associations between SAMD9/SAMD9L expression in pACT with tumor presentation (P53 p.R337H genotype and metastasis occurrence) and patient outcome (Overall (OS) and Disease-Free Survival) were analyzed. In silico, publicly available data from pediatric patients with ACT available in the Gene Expression Omnibus were used to evaluate the aforementioned associations with SAMD9 (GSE76021) and SAMD9L (GSE76019) mRNA levels. In vitro, SAMD9 subcellular localization pattern was investigated in the ACT cell line NCI-H295R (immunofluorescence). Results: Nuclear and cytoplasmic SAMD9 expression was observed throughout all different phases of adrenal development evaluated. However, in pACT samples, 26% presented nuclear and 86% cytoplasmic immunostaining. In line, NCI-H295R cells presented mostly cytoplasmic SAMD9 immunostaining under basal conditions. No differential expression was observed between SAMD9 or SAMD9L mRNA levels in pACT and in normal pediatric adrenals. Moreover, no association between SAMD9 immunostaining, SAMD9 or SAMD9L mRNA levels, tumor presentation and patient outcome were observed in our cohort, nor in the in silico evaluation. Conclusion: SAMD9 is nuclear and cytoplasmic expressed during adrenal cortex adrenal development and its loss of function is associated with adrenal hypoplasia. On the other hand, SAMD9 is mostly cytoplasmic expressed in pACT and immortalized NCI-H295R cells. No differential expression of SAMD9 nor its counterpart SAMD9L mRNA were associated with tumor presentation and pediatric patient outcome.

In Vitro Modeling of Brain-Bone Communication: Factors Secreted from Immortalized Female Kisspeptin Neurons Modulate Gene Expression in Osteoblasts
Teagan James, BS, Marcus Weinman, BS, Patrick Everett Chappell, PHD. Oregon State Univ, Corvallis, OR, USA.

SUN-263
While the sex steroid hormone estrogen (E2) is essential for maximum reproductive fitness, circulating E2 is also involved in maintaining skeletal homeostasis in females. E2 in male and female animals has been shown to stimulate cancellous bone formation via activation of estrogen receptor alpha (ERα), and it was widely thought that this effect was mediated directly at the level of bone. A recent study, however, demonstrated a large increase in bone density in female mice in which ERα was deleted from specific neuroendocrine neurons in the arcuate nucleus of the hypothalamus, specifically those expressing kiss1, a population required for fertility and pubertal progression. Bone from transgenic Kiss1-cre X ERα floxed females showed higher osteoblast functioning, accompanied by increases in the expression of sp7 and runx2, positing the existence of a direct neural-bone regulatory axis that is altered by circulating E2 at the level of the brain (Herber et al., Nat Commun 2019). Our laboratory recently published a study demonstrating that GnRH and Kisspeptin, typically thought to act primarily within the neuroendocrine reproductive axis, are synthesized and secreted in an autocrine fashion by canine osteosarcoma cells in vitro, and that these neuropeptides can stimulate tumor cell proliferation (BMC Cancer 2019). Separately, our lab has also recently generated two immortalized Kiss1-expressing and -secreting cell lines, KTaR-1 (representative of female arcuate Kiss-1 neurons) and KTaV-3 cells (representative of female AVPV Kiss-1 neurons) (Endocrinology 2017). In the current study, we have combined these two in vitro models to explore if factors secreted by female ARC-derived KTaR-1 cells may affect multiple parameters of osteoblast function, including sp7 and runx2 expression (evaluated by qPCR), and ability to form bone matrix (evaluated by Alizarin Red assay). Preliminary results suggest that exposure canine osteosarcoma (COS) cells to conditioned media from KTaR-1 cells leads to increases in sp7 expression in an E2-dependent manner, such that 24h E2-deprivation of these neurons stimulates secretion of osteogenic factors. Additionally, media from both KTaR-1 and KTaV-3 cells stimulated Ca2+ production from cultured osteoblasts, as evaluated by Alizarin Red. We are continuing to explore these in vitro interactions using COS cells, as well as normal osteoblasts (cNOB) and immortalized cNOBs, and are evaluating media and exosomal proteomics to further characterize putative factors. While further study is required, these initial results suggest that our immortalized neuronal KP cell models may provide useful molecular tools to explore the regulation of this newly-proposed neural-bone axis.

MON-657
Abstract: We previously reported a nonrandomized case control study that showed significant improvements in BMI, HbA1c and HDL levels in 30 diabetic veterans who underwent sleeve gastrectomy (SG) through 2 yrs, compared to 23 controls receiving medical management. BMI decreased from 46 to 34 over two yrs and HbA1c from 7.3 to 5.9% in the SG group. We now report the 8 yr follow up. Since then, bariatric surgery has increasingly become an option for the management of not only obesity but also type 2 diabetes. More data is needed on long term outcomes of bariatric surgery compared to medical care. Our cohort is mainly men with more comorbidities and higher average age than typically included in other cohorts that usually include more women. Objectives: The main objective was to compare long term changes in weight, HbA1C, lipids and creatinine in SG group vs. controls. Secondary objectives analyzed were cardiovascular (CV) and mental health outcomes in SG cases and controls. Methods: Charts for diabetic veterans 18 to 80 yrs were reviewed over an 8 yr period. Data was analyzed using Friedman Test, Wilcoxon Signed Rank test with 2-tailed significance level set at p<05. Results: 23 cases and 22 controls were included in the 8-year data analysis. Mean age was 56 and 96% were male. The mean BMI and HbA1C at baseline were 45 and 7.3 in SG group with 41% on insulin and 42 and 8.4 for controls