GNAS Mutations Research Articles

Kinome profiling identifies MARK3 and STK10 as potential therapeutic targets in uveal melanoma

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the “dark kinome”. Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

A Clinical-Molecular Prognostic Scoring System for Myelodysplastic Syndrome in Asia - a Multicenter Study of the Asian Myeloid Working Group (AMWG)

Background: In Asia, treatment decisions in myelodysplastic neoplasms (MDS) are conventionally based on the Revised International Prognostic Scoring system (IPSS-R). A unique clinical-molecular prognostic model, the IPSS-molecular (IPSS-M), has been developed based on 2957 patients with de-novo MDS, therapy-related MDS, and MDS/myeloproliferative neoplasm (MPN) overlap syndrome. The IPSS-M comprises haematological parameters, karyotypic abnormalities and somatic mutations in a 31-gene panel. Validation of the IPSS-M in large multicentre studies in Asian has however not been performed. Hence, prognostic models taking into account the unique clinical-molecular characteristics of Asian MDS patients remain undefined. Aims: The objectives of this multicenter cohort study were: 1. to define the haematological, cytogenetic and molecular characteristics of Asian MDS patients; and 2. to develop a prognostic model based on these characteristics. Methods: Consecutive MDS patients in Hong Kong, Singapore and Taiwan diagnosed between 2004 to 2021 were studied. Data were censored on 17 September 2022. Patients with MDS/MPN overlap syndrome were excluded. A panel of 54 myeloid-related genes was sequenced in the diagnostic bone marrow by next-generation sequencing. Hematological, pathological, cytogenetic and molecular variables were evaluated for their association with overall survival (OS), leukemia-free survival (LFS), and time to progression to secondary acute myeloid leukemia (TTP-sAML). Variable selection was performed to determine the confounding variable and independent prognostic variables. The relative weights of the selected variables were estimated using a Cox multivariable model adjusted for confounders. According to the weights of each prognostic variable, individual patient-specific prognostic scores were calculated. Based on the scores of the entire cohort, six prognostic subgroups were defined. The statistical predictive power of this prognostic model was assessed by the concordance index (C-index). The prognostic model was validated with publicly available data from the IPSS-M cohort. Results: Seven hundred and seventy-eight men (63.5%) and 447 women (36.5%) at a median age of 68.4 (interquartile range, IQR: 57-77) years were studied. After a median follow-up of 2.85 (IQR: 0.97-7.06) years, there were 693 deaths (56.6%) and 284 transformations to sAML (23.2%). Three-hundred and ninety-four patients (32.2%) received hypomethylating agents (azaciticine, N=367; decitabine, N=27), and 158 patients (12.9%) underwent allogeneic haematopoietic stem cell transplantation. All patients showed at least one genetic alteration in the 54-gene panel (Figure 1A). Prognostic scoring systems (each with 6 prognostic subgroups) were established for OS (C-index: 0.72) (Figure 1A), LFS (C-index:0.71) (Figure 1B) and TTP-sAML (C-index: 0.74) (Figure 1C). Genomic factors significantly associated with inferior outcomes were monosomy 7, del(5q), and mutations in GNAS and TP53 for OS; trisomy 19, del(5q), monosomy 7, and mutations in GNAS, PTPN11 and TP53 for LFS, and i(17q), del(5q), and mutations in NPM1, NRAS, GNAS, IDH2, SF3B1, and RUNX1 for TTP-sAML. Using the IPSS-M publically available dataset as the validation cohort, our prognostic model gave C-indices of 0.70, 0.71 and 0.74 for OS, LFS and TTP-sAML respectively. Finally, this prognostic model had superior prognostic power as compared with the IPSS-R and IPSS-M for the current Asian cohort (Figure 1D). Conclusion: Combining genomic with hematological and cytogenetic parameters, the Asian clinical-molecular prognostic model improved the risk stratification of patients with MDS in Asia, potentially improving clinical decision-making.

Gene Rearrangement and Expression of PRKACA and PRKACB Govern Morphobiology of Pancreatobiliary Oncocytic Neoplasms

Intraductal oncocytic papillary neoplasms (IOPNs) are distinct from intraductal papillary mucinous neoplasms based on characteristic morphologic and genetic features represented by fusion genes involving PRKACA or PRKACB (PRKACA/B). However, pancreatic and biliary tumors with partial oncocytic features are often encountered clinically, and their molecular features are yet to be clarified. This study included 80 intraductal papillary neoplasms: 32 tumors with mature IOPN morphology (typical), 28 with partial or subclonal oncocytic features (atypical), and 20 without oncocytic features (control). We analyzed PRKACA/B fusion genes, including ATP1B1::PRKACA, DNAJB1::PRKACA, and ATP1B1::PRKACB, by reverse-transcription PCR; mRNA expression of fusion genes and nonrearranged PRKACA/B genes by quantitative reverse-transcription PCR; mutations in KRAS, BRAF, and GNAS by targeted sequencing or droplet digital PCR; and the expression of cyclic adenosine monophosphate (cAMP)–dependent protein kinase catalytic subunits α (PRKACA) and β (PRKACB), phosphorylated cAMP response element-binding protein, and aberrations of p16, p53, SMAD4, STK11, and β-catenin by immunohistochemistry. PRKACA/B fusion genes were detected in 100% (32/32) of typical, 46% (13/28) of atypical, and 0% (0/20) of control (P < .05). Expression of PRKACA, PRKACB, and phosphorylated cAMP response element-binding protein was upregulated in neoplasms with PRKACA/B fusion genes (P < .05). mRNA expression of the PRKACA/B fusion genes and protein expression of PRKACA or PRKACB tended to be higher in typical than in atypical cases (mRNA, P = .002; protein expression, P = .054). In some atypical neoplasms with mixed subtypes, PRKACA/B fusion genes were superimposed exclusively on oncocytic components. Typical IOPNs harbored fewer KRAS and GNAS mutations than control samples and fewer alterations in p53 and STK11 than atypical samples (P < .05). In conclusion, PRKACA/B fusion genes not only are the characteristic drivers of IOPNs but also play a crucial role in the development of subclonal oncocytic neoplasms. Moreover, oncocytic morphology is strongly associated with upregulation of PRKACA/B, which may provide clues for potential therapeutic options.

Recognition of GNAS and KRAS mutations in endoscopic ultrasound – fine needle aspiration (EUS-FNA) fluid samples from the main pancreatic duct (MPD) accurately discriminates intraductal papillary mucinous neoplasm (IPMN) from benign conditions with MPD dilatation

Recognition of GNAS and KRAS mutations in endoscopic ultrasound – fine needle aspiration (EUS-FNA) fluid samples from the main pancreatic duct (MPD) accurately discriminates intraductal papillary mucinous neoplasm (IPMN) from benign conditions with MPD dilatation

Neonatal cholestasis as the onset symptom of McCune-Albright syndrome: case reports and a literature review.

This study aimed to summarize and show the characteristics and evolutionary process of neonatal cholestasis caused by McCune-Albright syndrome (MAS), as neonatal cholestasis may be the initial manifestation of MAS before other classic clinical features appear. The clinical characteristics, treatment methods, and outcomes of three neonatal cholestasis cases caused by MAS in our center were retrospectively studied. In addition, all the reported cases of MAS combined with cholestasis were reviewed and summarized to show the cholestatic features in them. We have confirmed three MAS cases in our center, presenting onset symptoms of jaundice, pale stool, and neonatal cholestasis soon after birth. The cholestasis subsided spontaneously at around the sixth month. The literature review showed that the levels of total bilirubin, conjugated bilirubin, ALT, AST, and GGT in neonatal MAS cholestasis cases were 207 μmol/L (range 65-445 μmol/L), 162 μmol/L (range 46-412 μmol/L), 821 U/L (range 85-3,597 U/L), 532 U/L (range 127-3,633 U/L), and 244 U/L (range 79-3,800 U/L), respectively. Liver histology showed canalicular and hepatocellular cholestasis, giant hepatic cell transformation, and bile paucity. Extrahepatic manifestations such as café-au-lait pigmented skin lesions, Cushing's syndrome, hyperthyroidism, renal tubular dysfunction, and skeletal abnormalities could occur simultaneously when jaundice occurred. GNAS mutations had a high positive rate (83.3%-100%) in liver tissue with cholestasis. Neonatal cholestasis caused by MAS could be self-resolved, but hepatic lesions persist and have malignant potential. MAS can be one of the causes of neonatal cholestasis, which may be the first manifestation of the disease. Extrahepatic coexisting symptoms of MAS and liver histology can help to distinguish MAS from other etiology of cholestasis. Detecting GNAS mutations in liver tissue may shorten diagnostic time and is of particular interest in the partial and atypical forms of MAS with neonatal cholestasis. Neonatal cholestasis in children with MAS can self-resolve, but liver dysfunction and malignant lesions persist.

Prognostic fine needle aspiration biopsy of uveal melanoma: Molecular and genetic factors of metastasis risk

Introduction. Molecular genetic testing is actively used for prognostication in patients with uveal melanoma (UM). Tissue for genetic analysis may be obtained either by surgical excision or through fine-needle aspiration biopsy (FNAB). Performing genetic testing and FNAB in each institution can differ in surgical techniques and laboratory methodologies.Aim. To present our own experience of performing FNAB-based molecular genetic testing for prognostication in patients with uveal melanoma.Materials and methods. Prognostic FNAB (n = 151) were combined with brachytherapy or stereotactic surgery. Genetic testing was performed by methods based on polymerase chain reaction (GNAQ, GNA11, EIF1AX and SF3B1 mutations) and fluorescence in situ hybridization (copy numbers of PPARG and MYC genes); cytology of FNAB material was also assessed.Results. Fine-needle aspiration biopsy material was informative in 91 % of cases. At the median follow-up of 36 months, 12 cases of distant metastases were detected. Occurrence of the assessed mutations and copy numbers were related to other representative studies. PPARG deletion was shown to be a significant prognostic factor for metastasis-free survival (p &lt;0,01), which was demonstrated for the first time; EIF1AX and SF3B1 mutations, MYC amplification and cytological class were not shown to be significantly associated with survival in our study.Conclusion. FNAB-based molecular genetic testing for prognostication in patients with uveal melanoma was shown to be a reliable and highly informative approach. Some of the prognostic factors need to be evaluated further with longer follow-up.

OR20-04 Genetic Profiling Using A Gene Panel Identifies Pathogenic Variants And Copy Number Variations In Pituitary Adenomas

Abstract Disclosure: T. Silva: None. D. Mohan: None. J. Mebarak: None. D. Meredith: None. W. Bi: Speaker; Self; Stryker. R. Carroll: None. U.B. Kaiser: None. A. Marcondes Lerario: None. A. Abreu: None. Introduction: Pituitary adenomas (PA) are the second most common central nervous system neoplasm and arise from different cell lineages. PA are benign tumors, although a subset of them may present with aggressive behavior, resulting in significant morbidity. Several genetic alterations have been implicated in PA tumorigenesis, such as USP8 mutations in Cushing disease (CD) and GNAS mutations in somatotroph adenomas (SA). However, the genetic profiles of PA are not completely understood. The aim of this study was to identify genetic alterations associated with PA using a gene panel. Methods: Targeted next generation exome sequencing (OncoPanel) was performed to identify single nucleotide variants (SNV) and copy number variations (CNV) associated with PA. OncoPanel version 1 included 275 genes, version 2 had 300 genes, and the third, current version 447 genes. USP8 was included only in the most recent version. Patients undergoing surgery at our institution between 2013-2020, who agreed to participate in the OncoPanel initiative and who had samples with good quality DNA were enrolled. Classification of PA into subtypes was performed by combining clinical and histopathological data. A strict algorithm was used to assess SNV pathogenicity. Chi-square, Mann Whitney and Kruskal-Wallis tests were used for statistical analyses. Results: Our cohort included 171 patients with following PA subtypes: TPIT lineage - 18 CD, 14 silent ACTH; PIT-1 lineage - 29 SA, 21 lactotroph and 4 silent poorly differentiated PIT-1 adenomas; SF-1 lineage - 77 gonadotroph and 8 null cell adenomas (negative immunostaining for hormones and TF). GNAS mutations were found in 28% of SA and USP8 mutations in 50% of CD screened for USP8. Pathogenic variants in MECOM were associated with silent ACTH, in FGFR4 and NF2 with CD, and in NOTCH2 with lactotroph adenomas, whereas pathogenic variants in TP53, EGFR, MEN1, BRCA1, FH, ATR, BLM, and XPC were identified in multiple PA subtypes. The number of CNV varied significantly between different subtypes of adenomas and were higher in lactotroph (median, 46), poorly differentiated PIT-1 (41), SA (26) and silent ACTH (17) compared to CD (3), gonadotroph (1) and null cell (0.5) (p&amp;lt; 0.001). MIB-1 proliferation index &amp;gt;3% was associated with higher CNV burden (p=0.022). Interestingly, copy number loss of chromosome 16 and 1q combined were exclusively identified in SA without GNAS mutations. Conclusion: OncoPanel analyses identified pathogenic variants in PA, some well recognized variants such as USP8 and GNAS, and some rare events such as NF2 in CD. In addition, our findings highlight subtype-specific patterns of genomic instability. The association between high CNV burden and the MIB-1 proliferation index may help to predict adenoma behavior. Genomic profile using a gene panel has the potential to better classify PA and contribute to clinical management. Presentation: Saturday, June 17, 2023

THU235 Usefulness Of Digital Droplet Polymerase Chain Reaction (ddPCR) For Molecular Diagnosis Of McCune-Albright Syndrome In DNA From Peripheral Blood Leukocytes

Abstract Disclosure: A.G. de Faria: None. L.R. Montenegro: None. A.A. Jorge: None. R.S. Jallad: None. R.M. Martin: None. M.C. Fragoso: None. A.P. Canton: None. C.E. Seraphim: None. F.R. Tinano: None. N.C. Pinto: None. B.B. Mendonca: None. A. Latronico: None. V.N. Brito: None. Background: McCune-Albright Syndrome (MAS) is a rare congenital disorder caused by post-zygotic activating mutations in GNAS gene. Due to the mosaic pattern of this disease, mutation abundance is frequently low in several tissues, including blood cells. The emergence of digital droplet Polymerase Chain Reaction (ddPCR) is a breakthrough technology of quantitative PCR useful for a targeted detection and quantification of rare events. Objective: This study aims to identify the GNAS mutation in peripheral blood leukocytes of patients with MAS using ddPCR. Patients and Methods: Thirty-four patients (25 female and 9 male) with typical (classic triad) or atypical (one or two features) MAS were included. DNA was extracted from peripheral blood and was analyzed through ddPCR method. Two ddPCR mutation detection assays were used, each one targeting the p.R201C or p.R201H mutations. For each assay, the Taqman PCR system was used with two fluorescence probes: the first labeled with HEX, targeting de (WT allele), and the second labeled 6-FAM, targeting the (mutated allele). The mixture (reagents + DNA) was divided into 20,000 nanodrops, which was placed on a PCR plate and amplified by PCR. After amplification by PCR, the fluorescence of each droplet was read on the 200 Droplet Digital PCR system (Bio-Rad), being classified as positive (drop containing the target sequence) or negative. Results were analyzed using Quantasoft software (Bio-Rad) to determine the total amount of positive droplets in the original sample. The results were reported in fractional abundance (FA), corresponding to the percentage of mutated alleles within the total alleles. Results: Among all patients, the most common MAS feature was osteofibrous dysplasia (OFD) (73.5%), followed by peripheral precocious puberty (PPP) (64.7%), and café-au-lait skin spots (52.9%). Acromegaly or hyperthyroidism was diagnosed in 6 and 4 patients, respectively. Classical MAS triad, 2 clinical features, or 1 clinical feature (PPP or OFD) were present in 15, 9, and 10 patients, respectively. GNAS mutations were identified in 12/34 (35.3%) MAS patients: the p.R201C in 5 and the p.R201H in 7. Among them, 8 patients presented typical MAS and 4 patients with two MAS clinical features, respectively. Considering only typical MAS group, the positive molecular test rate was 53.3% (8/15). GNAS mutations were not identified in patients with only one MAS feature. There was a positive correlation between a positive molecular test and the number of MAS clinical features (p = 0.01). Median FA was 1.45%, ranged from 0.25% to 8.4%. There was no association between FA rate and the number of MAS clinical features (p=1.0). Conclusion: ddPCR representeds a more sensitive tool for identifying GNAS mutations in peripheral blood leukocytes, even with low abundance, in patients with classical and atypical MAS. Presentation: Thursday, June 15, 2023

FRI597 Impact Of Theophylline On BMI In Pseudohypoparathyroidism Type 1a - Results Of An Open Label Extension Trial

Abstract Disclosure: A.H. Shoemaker: None. J. Tamaroff: None. H. Jueppner: None. Background: Pseudohypoparathyroidism type 1a (PHP1A) is a rare genetic disorder characterized by early onset obesity, short stature and multi hormone resistance. The underlying causes are maternally inherited, heterozygous inactivating mutations in GNAS, which encodes the α-subunit of stimulatory G protein (Gsα). Due to tissue specific silencing of paternal Gsα expression, maternal GNAS mutations lead to impaired Gsα-protein coupled receptor (GPCR) function and multihormone resistance. The GPCR signaling cascade begins with increased cAMP which is rapidly degraded by phosphodiesterase (PDE). We hypothesized that paternal Gsα expression is reduced, but not absent, and therefore the nonselective PDE inhibitor theophylline may potentiate the cAMP/PKA-dependent cellular events down-stream of GPCRs. We hypothesized that theophylline would reduce BMI in PHP1A patients while being safe and well tolerated. Methods: Children and adults with PHP1A who completed the randomized, placebo controlled, double-blind clinical trials (RCT) NCT03029429 and NCT04551170 were eligible for enrollment in an open-label extension (OLE) study of theophylline treatment (NCT04240821). As the RCT is ongoing and treatment assignment is blinded, we compared baseline enrollment data (prior to any exposure to theophylline or placebo) with the last study visit in the OLE study. The RCTs are 1 year long and then patients in the OLE study are followed for up to 2 additional years. The primary outcome is change in BMI, expressed as % of the 95th %ile (BMI95) to account for differences in gender and age. Results: To date, 23 patients have enrolled in, and 16 patients have completed the RCTs. Two patients completed RCTs before the OLE was available. One patient was ineligible due to study drug noncompliance. One patient was Canadian and unable to participate due to COVID-19 travel restrictions. The remaining 12 completers enrolled in the OLE. One withdrew prior to OLE visit 2 due to difficulty with travel and is not included in the analysis. Patients were 17.2 ± 12.9 years old (range 9-55 years) and 10 of 11 were female. All participants were white, one patient was Hispanic. Patients were followed for an average of 30 months. Baseline BMI95 was 133.5 ± 19.9% and at the last OLE visit, BMI95 had decreased to 124.7 ± 18.6% (p= 0.06 by paired t-test). A decrease in BMI95 was seen in 73% of patients and 36% had a decrease in BMI95 &amp;gt;20%. Review of the 3 patients without improvement in BMI95 showed one had an increasing BMI95 during the RCT, followed by decreasing BMI95 during the OLE, and another patient had persistently sub-therapeutic theophylline levels. The most common adverse event attributed to the drug was transient nausea/vomiting. There were no OLE withdrawals due to adverse events. Conclusion: Most patients with PHP1A in a long term, OLE study of theophylline demonstrated an improvement in BMI95. Theophylline was safe and well tolerated. Presentation: Friday, June 16, 2023

SAT218 Refractory Hypocalcemia From Combined Autosomal Dominant Hypocalcemia Type 2 And Postsurgical Hypoparathyroidism

Abstract Disclosure: M. Sonmez Ince: None. F. Hannan: None. N. Esfandiari: None. M. Hussain: None. M.R. Haymart: None. R.V. Thakker: None. G.A. Clines: None. Background: Autosomal dominant hypocalcemia type 2 (ADH2), due to activating mutations in GNA11, is a rare disorder that causes hypocalcemia with low or inappropriately normal PTH. Postsurgical hypoparathyroidism (PSHP) is the most common etiology for hypoparathyroidism. The patient described was afflicted by both conditions and had persistent hypocalcemia despite aggressive management. Clinical Case: A 50-year-old female was found to have an enlarged thyroid on exam and subsequently diagnosed with papillary thyroid cancer (PTC). She underwent a total thyroidectomy without parathyroid autotransplantation since parathyroid glands were not identified during intraoperative pathologic survey. Surgical pathologic examination confirmed PTC with the identification of a single and small intrathyroidal parathyroid gland. Due to the patient’s low risk (T1N0M0), radioactive iodine ablation was not performed. Before thyroidectomy, serum corrected calcium was 6.4-8.5 mg/dL (8.6-10.3 mg/dL) and PTH was 9-21 pg/mL (10-65 pg/mL) during the previous 20 years. Following thyroidectomy, the patient had multiple emergency department visits due to hypocalcemia, hand cramping, and paresthesias. The patient was diagnosed with postsurgical hypoparathyroidism: corrected calcium 5.6-6.7 mg/dL, 24-hour urine calcium 112 mg (100-300 mg/24hour), magnesium 1.4-1.9 mg/dL (1.6-2.6 mg/dL), phosphorus 3-8.2 mg/dL (2.7-4.6 mg/dL). PTH remained &amp;lt;6 pg/mL. The patient was then referred to an academic endocrinology. TSH, AM cortisol, 25-vitamin D, iron, ceruloplasmin, copper, and 21-hydroxylase antibody concentrations were normal. Genetic testing revealed a pathogenic GNA11 variant (c.178C&amp;gt;T, p.(Arg60Cys)); no mutations were identified with CASR or AP2S1. Despite aggressive management with total daily oral calcitriol up to 12 mcg daily, calcium 8 grams, and chlorthalidone, the patient required calcium infusions thrice weekly. Despite the addition of teriparatide 20 mcg BID, the patient remained dependent on calcium infusions, with improvement after the addition of IV calcitriol. Recently, continuous teriparatide delivery was started using an Omnipod insulin pump that has significantly reduced the patient’s dependence on calcium infusions with serum calcium that are now 8-9 mg/dL. Conclusion: ADH2, due to gain-of-function mutations of GNA11, that encodes the Gα11 protein, causes life-long hypocalcemia that is often asymptomatic and undiagnosed. We report here the first documented case of combined ADH2 and PSHP. This case highlights that patients with ADH2 likely have hypoplastic parathyroid glands and that caution is required with procedures that may disrupt parathyroid gland function. Furthermore, the refractory nature of this patient’s hypocalcemia may suggest that GNA11 mutations in the kidney and skeleton reduce the effectiveness of standard hypocalcemia treatments. Presentation: Saturday, June 17, 2023

THU463 Etiology Of Hypophosphatemia In A Patient With Co-existing Mesenchymal Tumor And Fibrous Dysplasia

Abstract Disclosure: M. Patil: None. S. Wangsiricharoen: None. A. Lazar: None. T.A. Guise: None. Tumor induced osteomalacia (TIO) is a rare paraneoplastic phenomenon. Fibroblast growth factor-23 (FGF23) secretion from phosphaturic mesenchymal tumor (PMT) is implicated as the cause for hypophosphatemia and osteomalacia. Other rare conditions associated with dysregulated FGF23 production include fibrous dysplasia (FD). Coexistence of two unusual conditions which could increase FGF23 concentrations is rare. We report a case of PMT causing TIO in a patient with evidence of FD. A 79-year-old male was evaluated for worsening rib and right flank pain of more than one year duration. He also complained of generalized muscle weakness. His total alkaline phosphatase (ALP) levels were noted to be increased for many years. Computed tomography (CT) showed scattered sclerosis within ribs, and lytic lesions with cortical bone destruction in right iliac bone (4.3 x1.5 cm) and L4 vertebra. Biopsy of the right iliac lesion revealed findings compatible with PMT. Positron Emission Tomography (PET) showed fluorodeoxyglucose (FDG) avid lesion of the right anterior iliac wing, diffuse osteomalacia, insufficiency fractures of the ribs, as well as FDG avid lesions in left supra-acetabular area and in L4 vertebral body concerning for metastatic disease. Laboratory evaluation revealed ALP 306 (40-129 U/L), bone specific ALP 99 (0-20 mcg/L), phosphorus 1.5 (2.5-4.5 mg/dL), 25-(OH)-vitamin D 27 (30- 100 ng/mL), 24 hour urinary phosphorus 0.6 (0.4-1.3 g/24 hr), PTH 51.3 (15-65 pg/mL), FGF23 303 (&amp;lt;180 RU/mL), Calcium 10.3 (8.4-10.2 mg/dL), albumin 4.4 (3.5- 5.2 gm/dL), C-terminal telopeptide of type 1 collagen (CTX) 1611 (10-854 pg/mL). CT-guided cryoablation of the right iliac lesion was performed. Also, biopsy and cryoablation of the left supra-acetabular lesion was performed; pathology showed fibrous dysplasia (FD). Mutation analysis confirmed a mutation in exon 8 of GNAS gene. Post ablation, FGF23 dropped to 102 RU/mL and phosphorus increased to 3.0 mg/dL. The patient had significant improvement in bone pain and weakness. The patient with classic features of TIO improved with simultaneous cryoablation of both the PMT as well as the FD lesions. The unusual feature of the case is co-existence of PMT and FD. Malignant transformation of FD lesions is a rare complication. We confirmed that the PMT did not have GNAS mutation and is unlikely to arise from FD. And FGF23 in situ hybridization was strongly positive in the PMT and negative in the FD, ruling out FD as a contributor to patient’s hypophosphatemia. We plan to perform DOTATATE scan to differentiate PMT from FD in the L4 lesion. This is a unique case of coexistence of two separate conditions, both of which could secrete intact FGF23. Our studies suggest that PMT is the only etiology of hypophosphatemia and TIO. Presentation: Thursday, June 15, 2023

THU452 Polyostotic Fibrous Dysplasia Complicated By Aneurysmal Bone Cyst In A Patient With McCune-Albright Syndrome

Abstract Disclosure: E. Milazzo: None. K.L. Hermayer: None. Introduction: McCune-Albright Syndrome (MAS) is a rare mosaic disorder with GNAS mutations, encoding the alpha-subunit of the Gs G-coupled protein receptor, activating it, and inappropriately producing cAMP. The hyperfunctioning endocrinopathies associated with MAS are due to signaling through the TSH, LH, FSH, GHRH, and ACTH receptors. MAS clinical presentation occurs with café-au-lait skin macules, precocious puberty, and fibrous dysplasia (FD) of the bone. FD is a benign tumor that leads to increased rates of bone fractures and if it causes facial deformities, can be complicated with orbital neuropathies. An aneurysmal bone cyst (ABC) is a blood-filled cyst that can be a primary or secondary lesion due to co-existing lesions. ABC is a rare complication of FD. We report a rare case of ABC in a MAS patient who presented with acute visual disturbance and enlargement of the forehead and was successfully treated with surgery. Clinical Case: A 20-year-old female diagnosed with MAS at the age of 2 who had precocious puberty by the 3rd year of life and developed polyostotic FD in the right arm, left femur, cranial, and skull bones with multiple long bone fractures by the age of 5-6 and required numerous orthopedic surgeries. She presented with the characteristic café-au-lait skin macules on the back and right arm. In 2018, a slow decline of left eye vision required left cranial decompression of the optic nerve, with pathology showing fibrous tissue compatible with FD. In 2021, she complained of acute right forehead enlargement, compressing her right eye with blurry vision. An MRI showed FD involving the facial and skull bones with a new expansible lesion in the right frontal bone with internal layering fluid, exerting a mild mass effect on the frontal lobe and extending superiorly to the orbit with inferior displacement of the globe. These findings were consistent with an ABC. Right frontal craniectomy and cranioplasty were performed in 12/2021, with pathology showing a fibrous pseudocyst wall with associated lymphoplasmacytic inflammation and evidence of prior hemorrhage with Ki-67 in the cyst wall at 15%. Lab work showed chronic alkaline phosphatase elevation at 567IU/L (42-106IU/L), normal calcium at 9.2mg/dl (8.4-10.3mg/dl), phosphate at 3.3mg/dl (3.3-5.1mg/dl), and PTH at 26pg/ml (15-65mg/dl), with Vitamin D insufficiency at 25.1ng/ml (30-100ng/ml). Endocrinology workup was negative for thyroid, adrenal, gonadotrophic, and GH dysfunction. 5 months post-surgery, the patient demonstrated complete recovery, and MRI showed no recurrence of the ABC. Conclusion: ABC is a benign solitary tumor generally arising from the metaphysis of long bones and rarely involves the cranium. ABC associated with FD has only been reported in a few cases. The treatment is surgical excision, with radiation and cryotherapy used in nonorbital cases. Presentation: Thursday, June 15, 2023

FRI137 Histologic And Genetic Characteristics Of Primary Aldosteronism In Late Adulthood

Abstract Disclosure: A. Blinder: None. K. Nanba: None. D. Sapiro: None. J. Baker: None. C. Liu: None. I. Bancos: None. M. Lyden: None. W. Young: None. H. Wachtel: None. D. Cohen: None. J. Luther: None. T.A. Williams: None. M. Reincke: None. T. Else: None. T.J. Giordano: None. A. Udager: None. W.E. Rainey: None. Background: There is accumulating evidence that changes in adrenal histology and regulation of renin-angiotensin-aldosterone system occur with age. Primary aldosteronism (PA) is the most common cause of endocrine hypertension. Recent clinical studies suggest unique characteristics of PA in elderly patients, including high prevalence of comorbidities and low clinical cure rate after adrenalectomy. However, histologic and genetic features of PA in late adulthood have not been well elucidated. Objective: To determine histologic and genetic characteristics of PA in late adulthood. Methods: Seventy-six patients who underwent unilateral adrenalectomy at the age of 60 or older were included in this study. Formalin-fixed, paraffin-embedded (FFPE) sections of resected adrenal tissue were used for the analysis. Aldosterone synthase (CYP11B2) immunohistochemistry (IHC) was performed to identify the source of inappropriate aldosterone production. Histologic classification was made according to the HISTALDO (histopathology of primary aldosteronism) consensus. CYP11B2 IHC-guided DNA capture followed by sequencing analysis using direct Sanger sequencing or ion torrent based next-generation sequencing (NGS) was performed. Results: Median age of the studied patients was 70 (interquartile range: 64-73). Based on the CYP11B2 IHC, histologic diagnosis was made as follows; aldosterone-producing adenoma (APA, n=23), aldosterone-producing nodule (APN, n=41), and non-functioning adrenocortical adenoma (NFA, n=6). The remaining six cases had multiple CYP11B2-expressing neoplastic lesions, including APA with APN (n=2) and multiple APNs (n=4). Co-existence of NFA with APA or APN(s) was also observed in 13. Sequencing analysis revealed that somatic KCNJ5 mutation was the most frequent genetic alteration in APA (7/23, 30%), whereas somatic CACNA1D mutation was the most common genetic cause of APN (20/39 with successful sequencing, 51%). Of the 18 sequenced NFA DNA samples, two had somatic mutations in CTNNB1 (n=1) and GNAS (n=1). Conclusion: APN is the major histologic feature of PA in late adulthood. Co-existence of NFA also appears to be a frequent observation in this age population. Somatic CACNA1D and KCNJ5 mutations are common in APN and APA, respectively. Presentation: Friday, June 16, 2023

FRI144 Using Single Cell Technology To Predict The Cell Of Origin Of Aldosterone-producing Adrenal Adenomas

Abstract Disclosure: K. Laycock: None. C. Cabrera: None. E. Wozniak: None. C. Mein: None. E. Azizan: None. X. Wu: None. G. Argentesi: None. E. Goodchild: None. W. Drake: None. M.J. Brown: None. Background: Aldosterone-producing adrenal adenomas (APAs) are the commonest surgically curable cause of hypertension, but individual prediction of postoperative outcome is challenging. Emerging data suggest that outcomes are influenced by APA genotype; identifying the cell of origin of APAs could facilitate a greater understanding of why certain genotypes are more/less likely to be associated with residual/recurrent autonomous aldosterone production(1, 2). Aims: 1. To develop an adult adrenal single cell atlas. 2. To identify the likely cell of origin for APAs with different mutations. Methodology: Bulk RNA sequencing from 67 APAs discovered known or novel somatic genotypes in 55, and replicated or discovered distinctive transcriptomes for each genotype. Single nuclei RNA sequencing was performed on 13 adrenals adjacent to the genotyped APAs. After aggregation of all 13 datasets, cluster analysis was undertaken to identify cell types and some of the transitions between these. Similarities were sought between the genotype-enriched transcriptomes of APAs, and the transcriptomes of individual cell clusters in adjacent adrenal. Results: A single nuclei atlas of 56,442 nuclei was developed. Clusters included each of the 3 cortical zones, aldosterone-producing micronodules, capsular cells, adrenal medulla, macrophages, T cells, stromal cells, endothelial cells and neurones. Several zona glomerulosa selective transcripts in previous laser-capture studies(3, 4) clustered separately from CYP11B2-positive cells (e.g. LGR5, GABBR2) or in distinct non-steroidogenic clusters (e.g. KIAA1210, RSPO3, PTGDS).A sub-cluster of stromal cells, positive for KIAA1210, RSPO3, CDH13 and MFAP5 (each p&amp;lt;10-254 cf other clusters) is likely to represent the distinctive KIAA1210+ capsular/interstitial cells on adrenal immunohistochemistry (www.proteinatlas.org). Bulk RNA sequencing of a rare CLCN2-mutant APA showed 13-30-fold higher expression of these and multiple other genes localising to the single nuclei stromal-cell cluster. Conclusion: Single nuclei sequencing documents the heterogeneity of adrenal cells, and potential for multiple different cell-types to develop into APAs. Ongoing analysis is underway to replicate the CLCN2-mutant transcriptome and identify cells-of-origins for tumours with commoner mutations. 1.Wu X et al. 11C metomidate PET-CT versus AVS for diagnosing surgically curable PA: a prospective within-patient trial. Nat Med. In press 2.Zhou J et al. Somatic mutations of GNA11 and GNAQ in CTNNB1-mutant APAs presenting in puberty, pregnancy or menopause. Nat Genet. 2021 3.Nishimoto K et al. Aldosterone-stimulating somatic gene mutations are common in normal adrenal glands. Proc Natl Acad Sci USA. 2015 4.Shaikh LH et al. LGR5 Activates Noncanonical Wnt Signaling and Inhibits Aldosterone Production in the Human Adrenal. J Clin Endocrinol Metab. 2015 Presentation: Friday, June 16, 2023

THU234 An Adolescent Patient With Asymptomatic Hypocalcemia And Extensive Cerebral Calcification Due To Pseudohypoparathyroidism Type 1B: A Care Report

Abstract Disclosure: R. Alemzadeh: None. J.D. Ross: None. D. Guillen-Mendoza: None. Background: Pseudohypoparathyroidism (PHP) is a heterogeneous group of disorders due to renal resistance to parathyroid hormone (PTH) resulting in biochemical hypoparathyroidism but with skeletal sensitivity to PTH. Case Presentation: A 15-year-old African American male was incidentally found to have biochemical evidence of severe hypocalcemia during routine evaluation of his bilateral slipped capital-femoral epiphyses. The patient was at age-appropriate stage of puberty with normal height and without any features of hereditary osteodystrophy. However, he had history of mild developmental delay requiring school accommodations. On physical exam, there was some mild dyscoordination with finger and toe-tapping and a Myerson’s sign, negative tetany signs. The biochemical screening revealed markedly low serum calcium (4.9 mg/dL) with elevated phosphorus (7.8 mg/dL) and PTH (1314 pg/mL) levels. His hypocalcemia was accentuated by the presence of severe 25 hydroxy-vitamin D deficiency (7.5 ng/mL) with mild hypomagnesemia (1.4 mg/dL). He had normal renal and thyroid function tests with normal growth factors. There were minimal plasma cyclic AMP responses to a single dose of intravenous teriparatide (Ellsworth-Howard test) consistent with diagnosis of pseudohypoparathyroidism. The skeletal survey showed bowing and under-tubulation of the radius, ulna, tibia, and fibula as well as slipped capital femoral epiphysis bilaterally. His brain CT scan showed symmetrical severe dense calcifications in the basal ganglia, bilateral frontal, parietal and temporal subcortical white matter, pons and right dentate nucleus. Genetic testing did not reveal any GNAS mutations. The patient was initially treated with parenteral calcium followed by high-dose oral calcium supplements and calcitriol. His coexisting hypovitaminosis D and hypomagnesemia were corrected with oral high-dose cholecalciferol and magnesium sulfate, respectively. Conclusions: An incidental finding of severe asymptomatic hypocalcemia and elevated PTH without hereditary osteodystrophy phenotype raised the clinical suspicion of pseudohypoparathyroidism type 1B. This was associated with cerebral calcifications with increasing risk of worsening learning disabilities and minimal neurological findings. Presentation: Thursday, June 15, 2023

OR17-05 Lysine Demethylase KDM1A And Ectopic Expression Of GIP-Receptor In Somatotropinomas Of Patients With Paradoxical Response To Oral Glucose

Abstract Disclosure: F. Chasseloup: None. L. Tosca: None. D. Regazzo: None. A. Proust: None. M. Hage: None. E. Kuhn: None. C. Jublanc: None. K. Mokhtari: None. S. Salenave: None. S. Gaillard: None. F. Parker: None. A. Boch: None. G. Tachdjian: None. P. Chanson: None. J. Bouligand: None. G. Occhi: None. P. Kamenicky: None. Introduction: Paradoxical increase of GH following oral glucose load has been described in ∼30% of patients with acromegaly and has been related to the ectopic expression of the glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) in somatotropinomas. Recently, we identified germline pathogenic variants of lysine demethylase 1A (KDM1A) in patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing’s syndrome. Most patients also displayed a deletion of chromosome 1p, including the KDM1A locus in their adrenal tissues, resulting in complete loss of KDM1A expression. The ectopic expression of GIPR in both adrenal and pituitary lesions suggests a common molecular mechanism. The aim of our study was to search for genetic abnormalities of KDM1A in somatotroph pituitary adenomas. Methods: We collected somatotropinoma specimens from acromegalic patients followed in two tertiary endocrine centers in France and one in Italy. Somatic DNA was studied by targeted exome NGS and array-CGH. GIPR and KDM1A expression was quantified in the tumors using digital droplet PCR. Results: We included 186 patients: 108 patients (70.6 %) had a classic pathological GH response after oral glucose load, whereas 45 patients (29.4%) displayed a paradoxical rise of GH concentrations. Patients with a paradoxical response displayed higher IGF-1 levels (360 ± 111.8 % above ULN vs. 309.8 ± 107.3 %, p= 0.0130) and less invasive and smaller tumors (14.5 ± 5.58 mm vs. 18.5 ± 8.73 m, p= 0.0066). Amongst the 146 somatotropinoma specimens analyzed by targeted-NGS, no tumor harbored a KDM1A pathogenic variant, and 35 tumors harbored GNAS mutations (6 with and 29 without paradoxical GH responses). We identified a recurrent 1p deletion encompassing the KDM1A locus in 26 tumors, which were more frequently but not exclusively found in patients with paradoxical GH response compared to those with classic GH response. Somatic deletion of one KMD1A allele was associated with lower KDM1A expression (p=4.5e-5) and higher GIPR expression (p=0.0005). Discussion: Unlike in GIP-dependent PBMAH, we did not identify KDM1A genetic variants in a large cohort of acromegalic patients, independently of their GH response pattern to oral glucose loading. We identified recurrent 1p deletion in some tumors. Pituitary adenomas with a loss of one KDM1A copy due to chromosome 1p deletion harbored higher levels of GIPR transcripts than adenomas diploid for the KDM1A locus. If KDM1A haploinsufficiency leads to partial transcriptional derepression at the GIPR locus and a paradoxical rise of GH after glucose load warrants further investigations. Presentation: Saturday, June 17, 2023

GNAQ/GNA11 Mosaicism Is Associated with Abnormal Serum Calcium Indices and Microvascular Neurocalcification

Mosaic mutations in genes GNAQ or GNA11 lead to a spectrum of diseases including Sturge-Weber syndrome and phakomatosis pigmentovascularis with dermal melanocytosis. The pathognomonic finding of localized "tramlining" on plain skull radiography, representing medium-sized neurovascular calcification and associated with postnatal neurological deterioration, led us to study calcium metabolism in a cohort of 42 children. In this study, we find that 74% of patients had at least one abnormal measurement of calcium metabolism, the commonest being moderately low serum ionized calcium (41%) or high parathyroid hormone (17%). Lower levels of ionized calcium even within the normal range were significantly associated with seizures, and with specific antiepileptics despite normal vitamin D levels. Successive measurements documented substantial intrapersonal fluctuation in indices over time, and DEXA scans were normal in patients with hypocalcemia. Neurohistology from epilepsy surgery in five patients revealed not only intravascular, but perivascular and intraparenchymal mineral deposition and intraparenchymal microvascular disease in addition to previously reported findings. Neuroradiology review clearly demonstrated progressive calcium deposition in individuals over time. These findings and those of the adjoining paper suggest that calcium deposition in the brain of patients with GNAQ/GNA11 mosaicism may not be a nonspecific sign of damage as was previously thought, but may instead reflect the central postnatal pathological process in this disease spectrum.

Radiation Therapy for Ocular Melanoma – a Narrative Review with Insides from TRIUMF, Canada’s Only Proton Beam Therapy Center

"Ocular melanoma (OM) originates from melanocytes in the eye, predominantly in the uvea, particularly the choroid. The yearly incidence is around six cases per million. OM is not primarily driven by ultraviolet exposure like skin melanoma, but is usually caused by mutations in GNAQ or GNA11. Symptoms like blurry vision and visual field defects appear late. Diagnosis is often made via eye exams, specialized ultrasound, and rarely biopsy. This narrative review describes the radiation treatment modalities of OM and highlights the landscape of proton beam irradiation in Canada. Historically, enucleation was the standard of care for OM. However, current strategies consider tumor size, location, patient age, visual potential, and metastatic presence. Primary treatments include radiation therapy and surgery. Radiation therapy includes plaque brachytherapy (PB), proton beam irradiation (PBI), stereotactic radiosurgery (SRS), and stereotactic radiotherapy (SRT). Surgery includes endoresection, exoresection, and enucleation. Tebentafusp-tebn has been FDA-approved for metastatic cases. PB, the most common radiation therapy for OM, involves radioisotopes delivering radiation into the tumor. Comparable survival rates between PB and enucleation for medium choroidal melanoma have made PB the standard of care. PB has certain limitations, mainly surgical complications. PBI uses a particle accelerator for focused, high-energy proton radiation, yielding high tumor control and survival rates, though the availability of proton facilities is a significant limitation. Vancouver is the only center in Canada for PBI, administered not in a healthcare facility but at TRIUMF (Tri-University Meson Facility). TRIUMF, the world's largest cyclotron particle accelerator, in partnership with BC Cancer and UBC Department of Ophthalmology and Eye Care Center, has treated over 200 ocular melanoma patients between 1995 and 2017, achieving a 91% tumor control rate and 82% five-year survival rate. Emerging combination therapies like Ataxia Telangiectasia Mutated (ATM) protein kinase inhibition before PBI show potential, possibly reducing radiation dose and resistance."

Papillary Hemangioma Harbors Somatic GNA11 and GNAQ Mutations.

Papillary hemangioma (PH) is a small, primarily dermal lesion occurring predominantly in the head and neck in both children and adults. Its signature characteristics are dilated thin-walled channels containing papillary clusters of mainly capillary-sized vessels and endothelial cytoplasmic eosinophilic inclusions. Given certain histopathologic similarities to congenital hemangioma which harbor mutations in GNAQ and GNA11 , we investigated whether similar mutations are present in PH. Seven PH specimens were studied. All presented in the first 4 years of life, with one being noted at birth. With the exception of one lesion, all were in the head and neck. Lesions were bluish and ranged in size from 0.5 to 2.8cm. Four samples had GNA11 p.Q209L and 3 had GNAQ p.Q209L missense mutations. Mutations in GNA11 and GNAQ are associated with other types of somatic vascular lesions including capillary malformation, congenital hemangioma, anastomosing hemangioma, thrombotic anastomosing hemangioma, and hepatic small cell neoplasm. Shared mutations in GNA11 and GNAQ may account for some overlapping clinical and pathologic features in these entities, perhaps explicable by the timing of the mutation or influence of the germline phenotype.

DNA Methylation Pattern in Somatotroph Pituitary Neuroendocrine Tumors

Introduction: Growth hormone secretion by sporadic somatotroph neuroendocrine pituitary tumors (PitNETs) is a major cause of acromegaly. These tumors are relatively heterogenous in terms of histopathological and molecular features. Our previous transcriptomic profiling of somatotroph tumors revealed three distinct molecular subtypes. This study aimed to investigate the difference in DNA methylation patterns in subtypes of somatotroph PitNETs and its role in distinctive gene expression. Methods: Genome-wide DNA methylation was investigated in 48 somatotroph PitNETs with EPIC microarrays. Gene expression was assessed with RNAseq. Bisulfite pyrosequencing and qRT-PCR were used for verifying the results of DNA methylation and gene expression. Results: Clustering tumor samples based on methylation data reflected the transcriptome-related classification. Subtype 1 tumors are densely granulated without GNAS mutation, characterized by high expression of NR5A1 (SF-1) and GIPR. The expression of both genes is correlated with specific methylation of the gene body and promoter. This subtype has a lower methylation level of 5′ gene regions and CpG islands than the remaining tumors. Subtype 2 PitNETs are densely granulated and frequently GNAS-mutated, while those in subtype 3 are mainly sparsely granulated. Methylation/expression analysis indicates that ∼50% genes located in differentially methylated regions are those differentially expressed between tumor subtypes. Correlation analysis revealed DNA methylation-controlled genes, including CDKN1B, CCND2, EBF3, CDH4, CDH12, MGMT, STAT5A, PLXND1, PTPRE, and MMP16, and genes encoding ion channels and semaphorins. Conclusion: DNA methylation profiling confirmed the existence of three molecular subtypes of somatotroph PitNETs. High expression of NR5A1 and GIPR in subtype 1 tumors is correlated with specific methylation of both genes.