BACKGROUND:Complete 17α-hydroxylase/17,20-lyase deficiency (17-OHD) is a rare autosomal recessive form of congenital adrenal hyperplasia caused by CYP17A1 variants. Large-scale studies integrating clinical, genetic, and functional data remain limited. METHODS:We recruited 113 genetically confirmed 17-OHD patients from 107 unrelated families. Comprehensive clinical manifestations, hormonal, and imaging examination data were collected. CYP17A1 variants were identified by Sanger sequencing, whole-exome sequencing or long-read sequencing. In vitro functional studies including enzyme activity assays and minigene splicing assays were performed to verify the pathogenicity of the variants. RESULTS:All patients presented as phenotypic females, with a median diagnostic age of 17 years. Hypertension (93.58%) and hypokalemia (74.31%) were the dominant clinical features, and 91.26% exhibited Tanner stage I breast development. We identified 50 pathogenic CYP17A1 variants, including 6 novel ones (c.286C>G, c.436+1G>T, c.1241C>T, c.1300C>T, c.1348C>T, c.1433G>T). The mutation spectrum was dominated by a founder hotspot, c.985_987delinsAA, accounting for 59.29% of alleles, with mutations clustering in exons 6 and 8. In vitro enzyme activity assays confirmed near-complete loss of both 17α-hydroxylase and 17,20-lyase activities in all variants except p.Val236Gly, which retained minimal residual activity. Notably, 5 variants (including 2 missense changes, c.1084C>T and c.1085G>A) were shown to disrupt normal pre-mRNA splicing in minigene assays, revealing a dual molecular mechanism of pathogenicity involving both protein dysfunction and aberrant RNA processing. CONCLUSION:This study defines the most comprehensive CYP17A1 variant spectrum for complete 17-OHD in China, identifies 6 novel pathogenic variants, and uncovers splicing disruption as an under-recognized mechanism in missense mutations. These findings expand the molecular and clinical understanding of 17-OHD, highlight the founder effect of c.985_987delinsAA in the Chinese population, and provide critical insights for genetic diagnosis and counseling.

This study investigates the role of m6A regulators in invasive pituitary neuroendocrine tumors (PitNETs) by defining m6A-related molecular subtypes, constructing a nomogram, and elucidating the mechanistic role of METTL14 in PitNET progression. GEO datasets were analyzed for m6A regulatory gene expression. Machine learning approaches were used to develop a nomogram. m6A molecular and gene subtypes were identified, and scRNA-seq from tumors characterized intratumoral subpopulations. Functional validation was performed using METTL14 overexpression and knockdown in PitNET cells, followed by proliferation, invasion, RT-qPCR, Western blotting, m6A-RIP, RIP, RNA stability, and luciferase reporter assays. Seven key m6A regulators were selected for the nomogram. Two m6A subtypes were identified, with Cluster B showing lower immune infiltration and higher m6A scores. Further classification revealed two gene-based subtypes with consistent patterns. scRNA-seq defined four PitNET clusters, including a proliferative TPC population with the highest invasion scores, strongly correlated with METTL14. Functional experiments confirmed that METTL14 promotes proliferation, invasion, and autophagy via PI3K/AKT activation. Mechanistically, IGF2 was identified as a novel downstream effector of METTL14, as METTL14 enhanced IGF2 expression through m6A modification, thereby activating PI3K/AKT signaling. This study provides a comprehensive characterization of m6A methylation in invasive PitNET, integrating bulk and single-cell transcriptomics. The nomogram offers clinical potential, while the discovery of TPCs and the identification of the METTL14–IGF2–PI3K/AKT regulatory axis highlight novel mechanistic insights and therapeutic targets for personalized invasive PitNET management.

OBJECTIVE:To prospectively evaluate the diagnostic performance of traditional versus optimized adrenocorticotropic hormone (ACTH) ratio cutoffs for desmopressin (DDAVP)-stimulated bilateral inferior petrosal sinus sampling (BIPSS) in differentiating Cushing disease (CD) from ectopic ACTH syndrome (EAS) in ACTH-dependent Cushing syndrome. METHODS:We conducted a prospective single-center observational cohort study of 181 consecutive patients undergoing DDAVP-stimulated BIPSS in a high-volume tertiary pituitary center (Peking Union Medical College Hospital) in China between October 2023 and December 2024. Final diagnosis was confirmed by histopathology and/or biochemical remission. Diagnostic accuracy of traditional cutoffs (basal IPS:P >2.0 or stimulated >3.0) versus optimized cutoffs (determined by receiver operating characteristic curve analysis) was compared. RESULTS:Among 167 patients with definitive diagnosis (153 CD, 14 EAS), BIPSS showed excellent overall discrimination (area under the curve, 0.994). Traditional cutoffs yielded a sensitivity of 91.5% and specificity of 100.0%. Lower cutoffs (basal IPS:P >1.49 or peak stimulated IPS:P >2.26) improved sensitivity to 97.4% while maintaining 100.0% specificity. Most peak IPS: P values occurred at 3 to 5 min post-DDAVP. False-negative results were associated with absent intersinus lateralization and low IPS ACTH levels, suggesting technical or anatomic sampling limitations rather than impaired DDAVP responsiveness. CONCLUSION:DDAVP-stimulated BIPSS showed excellent diagnostic performance for differentiating CD from EAS and provides a practical alternative stimulation strategy when corticotropin-releasing hormone is unavailable. Lower DDAVP-specific ACTH ratio cutoffs improved sensitivity without compromising specificity and may reduce false-negative diagnoses of CD in patients with ACTH-dependent Cushing syndrome.

Glucocorticoid resistance syndrome (GRS) is a rare hereditary disorder caused by pathogenic variants in NR3C1, characterized by marked phenotypic heterogeneity and frequent misdiagnosis as primary aldosteronism or subclinical Cushing's syndrome. We report a family harboring a novel NR3C1 frameshift variant and provide an integrated characterization of its clinical and biochemical features. By combining structural prediction, tissue- and single-cell-level expression analyses, in silico virtual gene perturbation, and systematic literature review, we evaluated the potential functional impact and clinical relevance of this variant. Comprehensive clinical phenotyping, steroid metabolomic profiling, adrenal CT imaging, and segregation analysis were performed in the proband and his father. AlphaFold3 was used to predict the structures of wild-type and mutant GRα proteins. Variant pathogenicity was assessed following ACMG/AMP criteria. Cross-tissue and single-cell expression patterns of NR3C1 were analyzed, and in silico virtual knockout analysis was performed to generate exploratory hypotheses regarding potentially affected biological pathways. A systematic review was conducted to summarize previously reported GRS cases, their variants, and associated phenotypes. Two affected males across successive generations presented with refractory hypertension without Cushingoid features. Steroid profiling demonstrated mild hypercortisolemia with elevated ACTH, marked increases in 11-deoxycorticosterone and 11-deoxycortisol, suppressed renin, and low-to-normal aldosterone, consistent with ACTH-driven accumulation of mineralocorticoid precursors. Sequencing identified a novel NR3C1 frameshift variant, c.923dupG (p.Ala309Serfs*6), absent from population databases and co-segregating with the familial phenotype. Structural prediction indicated premature termination within the N-terminal transactivation domain, supporting loss of the downstream DNA-binding and ligand-binding domains. According to ACMG/AMP criteria, the variant was classified as pathogenic. In silico virtual perturbation analysis suggested that NR3C1 dysfunction may extend beyond a linear defect in glucocorticoid signaling, potentially influencing neuroendocrine communication, extracellular matrix regulation, protein translation, and developmental pathways. Literature review identified 66 reported GRS cases encompassing substantial phenotypic variability, with cardiovascular events documented in some carriers of N-terminal or truncating variants. We describe a novel exon 2 NR3C1 frameshift variant that co-segregates with a characteristic GRS hormonal profile and is supported by clinical, genetic, and structural evidence. This case expands the genotype-phenotype spectrum of NR3C1-related GRS. The potential relationship between N-terminal truncating variants and cardiovascular complications remains hypothesis-generating and requires validation in larger cohorts and functional studies. Our findings underscore the importance of considering NR3C1-related GRS in patients presenting with resistant hypertension accompanied by a distinctive steroid profile.

To characterize clinical, radiological, and pathological features of double pituitary adenomas (DPAs) involving a corticotroph adenoma and evaluate challenges associated with preoperative localization and surgical management of Cushing’s disease (CD). We retrospectively reviewed four institutional cases and searched PubMed and Web of Science (1990–2025), identifying 30 publications comprising 68 additional cases. Demographic, clinical, radiological, immunohistochemical, surgical, and outcome data from the combined cohort were analyzed. In all four institutional cases, the lesion removed during initial transsphenoidal surgery (TSS) was a noncorticotroph adenoma, and hypercortisolism persisted. Follow-up pituitary magnetic resonance imaging (MRI) demonstrated a second suspected lesion in all four patients. In Cases 3 and 4, MRI abnormalities were subtle or equivocal, and 68Ga-pentixafor positron emission tomography/computed tomography (PET/CT) revealed concordant focal uptake at the suspected sites. Revision TSS confirmed an adrenocorticotropic hormone (ACTH)- and T-PIT-positive corticotroph adenoma in all four patients, and all achieved early postoperative biochemical remission. In the combined cohort (72 patients), preoperative MRI identified both lesions in 12 patients (16.7

Around 10% of pituitary tumors are estimated to be aggressive and associated with poor prognosis. Loss of heterozygosity (LOH) in chromosome 11q13 has been observed in aggressive pituitary tumors (APTs). Both MEN1 and AIP, located on 11q13 with an interval of 2.7 Mb, have been discovered as genetic markers of APTs. We aimed to investigate whether 11q13 LOH covering MEN1 and AIP in pituitary tumors associate with aggressiveness and the possible interactive mechanism of the two genes. A systematic case review was conducted to search for pituitary tumors with 11q13 LOH covering MEN1 and AIP. Individual clinical data were extracted from articles meeting the selection criteria, including demographic information, genetic variations, tumor size, tumor type, aggressive features, therapy and prognoses. Bioinformatics analysis was applied to explore underlying pathogenic mechanisms. 11q13 LOH covering MEN1 and AIP was reported in 16 pituitary tumor cases. Patients were aged 30 ± 10 years old with 10 males and 6 females. 75.00% had macroadenomas. There were 56.25% GH-, 12.50% ACTH-, and 31.25% GH-PRL-secreting tumors. 68.75% had features suggesting aggressiveness, including clinical invasiveness, post-operative persistence, recurrence, and metastasis. Concurrent dysfunction of MEN1 and AIP possibly induced the tumorigenesis by strengthening the same pathogenesis pathway. 11q13 LOH covering MEN1 and AIP in pituitary tumors relates to features suggestive of aggressiveness and poor outcome. Dual dysfunction of these two tumorigenesis genes could interact to exert pathogenic mechanisms. We were the first to propose that this concomitant mutation in pituitary tumors might be a unique genetic subtype.

Clinical severity of 21-hydroxylase deficiency (21-OHD) is primarily determined by the CYP21A2 genotype and residual enzymatic activity of mutant CYP21A2. However, CYP21A2 variants often lack functional characterization, and the pathogenic contribution of multiple variants arranged in cis remains poorly understood. We aimed to determine the residual 21-hydroxylase activity of 11 rare CYP21A2 variants, assess the combined functional impact of cis-configured double variants, and refine genotype-phenotype correlations. Among 713 patients with 21-OHD, 14 carried rare CYP21A2 variants. Targeted long-read sequencing was used to resolve allele structures and phase variants. Pathogenicity was evaluated using conservation analysis, in silico prediction, enzyme activity assays, and minigene-based splicing analysis. Clinical data were reviewed to examine genotype-phenotype correlations. We identified 11 CYP21A2 variants in 14 patients, including 10 missense variants and one deep intronic variant (c.738 + 75 C > T); four were novel (p.Ile173Phe, p.Gly179Glu, p.Asp259Val, p.Pro361Leu). Two cis-configured double-variant alleles were confirmed: p.[His63Leu; Val70Leu] and p.[Thr124Ile; Ile173Asn]. Functional assays showed that all 10 missense variants exhibited reduced activity across both substrate conversions. Both cis-configured double-variant alleles showed greater functional impairment than their corresponding single variants, a pattern consistent with a cumulative functional effect. The intronic variant c.738 + 75C > T caused partial intron 6 retention (73-bp insertion), with a predicted frameshift and likely nonsense-mediated decay. Overall concordance between genotype and phenotype was 71.4

Ectopic adrenocorticotropin syndrome (EAS) is usually associated with severe multiple complications and high mortality. Several adrenal steroidogenesis inhibitors can be used to control hypercortisolism when curative surgery is not feasible, but with different availability worldwide. It was long considered that mitotane (MTT) was not suitable for severe Cushing syndrome (CS) due to its delayed onset of action. We present a case of a 17-year-old girl with rapid-onset CS and an extremely high 24-hour urinary free cortisol (UFC) level (>300 times the upper limit of normal). An anterior mediastinal nodule with contrast enhancement was identified in computed tomography, with positive 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography with computed tomography (PET/CT) uptake (maximum standardized uptake value = 10.1), suggestive of a thymic neuroendocrine tumor as the most likely cause of EAS. Preoperative MTT monotherapy titrated to 2 g/day reduced UFC by 85% within 13 days without adverse effects, stabilized severe neuropsychiatric disturbances and opportunistic infections, thus enabling successful thymectomy. The tumor turned out to be an adrenocorticotropin-secreting thymic typical carcinoid. Other EAS cases treated with MTT reported in the literature were reviewed, and the time needed to control hypercortisolism using MTT was shorter than previously reported. Instead of an "add-on drug," we should reconsider the role of MTT in the treatment of severe hypercortisolism in EAS.

OBJECTIVE:ACTH-dependent Cushing's syndrome (CS) causes profound immune dysfunction and severe infections. This study aimed to characterize immune cell phenotypes, develop predictive models for differentiating Cushing's disease (CD) from ectopic ACTH syndrome (EAS) and predict infection risk, and evaluate immune recovery after surgical remission. DESIGN:Retrospective single-center study. METHODS:We studied 211 patients with ACTH-dependent CS (173 CD, 38 EAS) aged 11-75 years. Twelve patients were evaluated longitudinally after remission. Lymphocyte subsets were analysed by flow cytometry, severe infection incidence was documented, and machine learning models were developed for disease differentiation. RESULTS:EAS patients showed more severe immune suppression than CD, with reduced CD4+ T cells (224 [158.0-390.0] vs. 442 [313.0-611.0] cells/μL, P < .001) and CD3+ T cells (505 [342.0-815.0] vs. 930 [713.0-1246.0] cells/μL, P < .001), while elevated percentage of CD19+ B cells (18.2% [13.3%-25.5%] vs. 9.6% [6.1%-14.4%], P < .001). Machine learning-based model integrating immune and non-immune parameters achieved improved balanced accuracy of 75.4% (AUC = .840, 95% CI: 0.770-0.910) for CD/EAS differentiation. Severe infections occurred in 13.7% of patients. The multivariate model incorporating 24 h UFC, serum potassium nadir, CD19+ B cell percentage, and CD4+ T cell count predicted severe infection risk (AUC = .851, 95% CI: 0.774-0.927; sensitivity 86.2%, specificity 68.7%). Following surgical remission, profound T cell reconstitution occurred after 8 weeks, with CD4+ T cells increasing 3-fold (P < .001). CONCLUSIONS:Immune profiling distinguishes CD/EAS and predicts infection risk in ACTH-dependent CS. Surgical remission facilitates immune reconstitution.

Pituitary adenomas (PAs) are common brain tumors, accounting for about 15% of all brain neoplasms. Although generally benign, they can lead to serious complications through mass effects and hormone dysregulation. Emerging evidence suggests that collagen, a major component of the extracellular matrix (ECM), plays a pivotal role in PA pathophysiology. Collagen provides both structural integrity and biochemical cues within the tumor microenvironment (TME), influencing cellular behaviors and intercellular interactions. Recent studies indicate that collagen remodeling in PAs is dynamic, with alterations in collagen composition and organization affecting tumor growth, invasion, and hormone secretion. Collagen degradation products and collagenase activity may also facilitate tumor invasion into adjacent tissues. Additionally, collagen has been implicated in immune modulation, acting as a physical barrier that restricts immune cell infiltration and promotes immune evasion through receptor-mediated signaling. Metabolically, collagen may serve as an energy source or modulate metabolic pathways to sustain tumor proliferation. Clinically, collagen content in PAs correlates with tumor consistency, which has implications for surgical resection strategies. Moreover, serum collagen is emerging as a potential non-invasive biomarker for PA diagnosis and prognosis. Targeting collagen synthesis, degradation, or its mechanotransductive signaling pathways represents a promising therapeutic avenue.

OBJECTIVES:Cushing syndrome (CS) can significantly impair quality of life. It is often diagnosed late due to misdiagnosis as simple obesity under current strategies. Given that CS is characterized by distinctive facial features, for example, the "moon face," employing facial identification algorithms to analyze multiview facial images may aid in CS diagnosis. METHODS:A total of 42 pairs of patients with CS and age-, sex-, and body mass index-matched controls were enrolled in the training cohort, with 13 matched pairs separately included in the external validation cohort. Multiview facial images were collected and integrated. Four deep learning models-DenseNet, ResNet, Swin, and ViT-were trained using fivefold cross-validation. These models were evaluated on the external validation cohort. Diagnostic performance metrics were calculated and compared with the diagnostic assessments of 18 endocrinologists. RESULTS:Four facial recognition models were established. Within the training cohort, all 4 models demonstrated stable diagnostic performance across the 5 cross-validation folds. When applied to the external validation cohort, the models achieved an accuracy of 92.3% to 100%, sensitivity of 84.6% to 100%, specificity of 100%, and area under the curve of 94.4% to 100%. The deep learning models outperformed endocrinologists in accuracy and demonstrated sensitivity and specificity that equaled or exceeded those of the endocrinologists. CONCLUSIONS:These findings indicate that multiview facial identification using deep learning could provide an accurate and accessible diagnostic method for CS. Given that the Artificial intelligence models outperformed endocrinologists, this approach holds potential as a novel diagnostic tool to assist clinicians in identifying various types of CS.

OBJECTIVE:Cushing syndrome (CS) is characterized by excess cortisol and distinct facial features, including moon face and facial fat accumulation. However, facial fat distribution differences between CS and common obesity remain unclear. This study used 3-dimensional photogrammetry to quantify facial characteristics influenced by cortisol. METHODS:Patients with CS and controls were recruited at Peking Union Medical College Hospital from 2022 to 2024. Enrolled patients underwent standardized protocols for 3-dimensional facial imaging and clinical data collection. The images were subsequently processed, annotated, and subjected to measurement analysis. Distances, angles, contours, and area parameters were measured and calculated. Differences in facial features between patients with CS and obesity controls were compared. Clinical features related to CS facial features were analyzed. A support vector machine-based diagnostic model was developed using distinct facial features. RESULTS:A total of 42 patients with CS and 42 controls were enrolled (age, sex, and body mass index matched). Measurement values of CS facial parameters revealed significant cheek and lower face fat accumulation compared with common obesity, with more pronounced lower face accumulation. Fat accumulation in CS occurred in both horizontal and depth dimensions. Associated factors included age, sex, body mass index, waist circumference, disease duration, cortisol and adrenocorticotropic hormone levels, and etiologic subtype. The support vector machine diagnostic model showed accuracy of 91.7% and area under the curve of 0.96. CONCLUSION:Compared with common obesity, hypercortisolism induces a redistribution of facial fat toward the mid and lower face regions, resulting in the characteristic appearance. This study of excessive endogenous cortisol secretion offers new clinical and facial morphologic perspectives on CS.

e16604 Background: Ectopic ACTH and/or CRH-secreting pheochromocytomas are rare catecholamine-secreting tumors originating from adrenal medulla, which could also secrete ectopic adrenocorticotropic hormone (ACTH) and corticotrophin-releasing hormone (CRH), mimicking Cushing syndrome and complicating the diagnosis clinically. The extremely low incidence limits the comprehensive understanding and management for specific individuals accompanied with this kind of tumor. Furthermore, mechanisms of tumorigenesis and ectopic ACTH secretion in pheochromocytomas are unknown. Studying this rare type of tumor will help tailor therapies to the specifically molecular and metabolic characteristics of pheochromocytomas. Methods: Fresh tumor and peritumoral specimens were collected from three ACTH+&CRH+ pheochromocytoma patients and three ACTH-&CRH- pheochromocytoma patients, and then were digested and sent to construct single-cell library as well as sequencing. Subsequent analysis and immunohistochemical staining were conducted to explore the difference of ACTH+&CRH+ pheochromocytomas from the ACTH-&CRH- ones and normal tissues. Results: We constructed the linear trajectory of 2 types of pheochromocytomas. The cluster of intermediate cells that differentiated into mature ACTH+&CRH+ pheochromocytomas showing high expression of HES1, and the immunohistochemical staining revealed that HES1 was higher in ACTH+&CRH+ ones. Besides, Higher expression of POMC and PNMT was observed in ACTH+&CRH+ pheochromocytomas compared with ACTH-&CRH- ones, being in accordance with the clinical information that three ACTH+&CRH+ pheochromocytoma patients had relatively higher catecholamine levels. T cells in ACTH+&CRH+ pheochromocytomas were activated by signals, like HLA-B, promoting their cytotoxic functions, while T cells in ACTH-&CRH- ones were subject to inhibitory signals, especially from M2 macrophages. What's more, a population of endothelial cells marked by SELE was identified in ACTH+&CRH+ pheochromocytomas, showing strong interaction with CD8+ T cells and facilitating lymphocyte infiltration, particularly the GZMK⁺IL7R⁺ T cells. While ACTH-&CRH- pheochromocytomas were enriched in glycolysis and HIF signaling pathways, and characterized by lacking T cell infiltration, with macrophages being the dominant immune cells. Conclusions: HES1 plays a significant role in the fate transition of pheochromocytomas and may drive ACTH secretion. In addition, ACTH+&CRH+ pheochromocytomas are considerated as relatively immunologically“hot”tumors but poor in angiogenesis. In contrast, ACTH-&CRH- pheochromocytomas lack immune infiltration while exhibiting extensive angiogenesis. Immunotherapies may be effective for the former, and the latter may benefit from anti-angiogenic therapies.

To characterize the clinicopathological features and metabolic profiles of silent growth hormone pituitary adenomas (SGH-PAs). The single-center retrospective study identified SGH-PAs from 1387 surgically treated non-functioning PAs at Peking Union Medical College Hospital (2019–2023). Controls included 407 consecutive cases with non-functioning gonadotropin pituitary adenomas (NFG-PAs) and 90 age and gender-matched acromegaly patients. Baseline and postoperative hormonal assessments, pituitary imaging features, pathological classification, metabolic comorbidity, and follow-up outcome were collected and analyzed. 45 cases of SGH-PA (3.2

Growth hormone-secreting pituitary neuroendocrine tumors (GH-PitNETs) arise from the anterior pituitary gland and constitute 20-30% of all PitNETs, representing a significant subset of functional pituitary tumors. Despite their prevalence, the precise mechanisms underlying the development of these tumors remain elusive due to the complex pathophysiology of pituitary neoplasia. To investigate the potential role of the gut microbiome in GH-PitNETs, we conducted a comprehensive study involving 16S rRNA gene sequencing and metabolomics analysis of fecal and serum samples from 20 GH-PitNET patients and 30 healthy controls at Peking Union Medical College Hospital. Our findings revealed a distinct gut microbiota profile in GH-PitNET patients compared to healthy individuals, characterized by dysbiosis with increased abundance of Bacteroides and decreased abundance of Blautia and Bifidobacterium. Notably, alterations in specific bacterial taxa, including Intestinibacter bartlettii, Fusicatenibacter faecihominis, and Massilioclostridium, were observed in GH-PitNET patients. Concomitantly, serum metabolomics analysis identified 154 differentially abundant metabolites in GH-PitNET patients, with significant enrichment in pathways related to tryptophan metabolism. Among these metabolites, 3-indoleacetic acid (IAA) exhibited a obvious change, suggesting its potential research value for disease processing of GH-PitNETs. To further elucidate the mechanistic link between the gut microbiome and GH-PitNETs, we conducted in vitro and in vivo experiments, our results demonstrated that IAA could promote the proliferation of GH3 cells and significantly enhance growth hormone secretion by activating the cAMP pathway. These findings collectively suggest that gut microbiota dysbiosis may contribute to in the development and progression of GH-PitNETs by contributing to metabolic disturbances.

Bilateral inferior petrosal sinus sampling (BIPSS) is important in the differential diagnosis of adrenocorticotropic hormone (ACTH)-dependent Cushing’s syndrome, but BIPSS is invasive and is not reliable on tumor lateralization. Thus, we evaluated the noninvasive diagnostic evaluations, high-dose dexamethasone suppression test (HDDST) combined with different pituitary MRI scans (conventional contrast-enhanced MRI [cMRI], dynamic contrast-enhanced MRI [dMRI], and high-resolution contrast-enhanced MRI [hrMRI]), by comparison with BIPSS. We retrospectively analyzed 95 patients with ACTH-dependent Cushing’s syndrome who underwent HDDST, preoperative MRI scans (cMRI, dMRI and hrMRI) and BIPSS in our hospital between January 2016 and December 2021. The diagnostic performance of HDDST combined with cMRI (HDDST + cMRI), HDDST + dMRI and HDDST + hrMRI, and BIPSS was evaluated, including the sensitivity of identifying pituitary adenomas and the tumor lateralization accuracy. Compared with BIPSS (AUC, 0.98; 95

Pituitary surgical intervention remains the preferred treatment for Cushing’s disease (CD) while postoperative venous thromboembolism (VTE) is a significant risk. Whether to prescribe pharmacological thromboprophylaxis presents a clinical dilemma, balancing the benefit of reducing VTE risk with the potential for increasing hemorrhagic events in these patients. Currently, strong evidence and established protocols for routine pharmacological thromboprophylaxis in this population are lacking. Therefore, a randomized, controlled trial is warranted to determine the efficacy and safety of combined pharmacological and mechanical thromboprophylaxis in reducing postoperative VTE risk in patients with CD. This investigator-initiated, multi-center, prospective, randomized, open-label trial with blinded outcome assessment aims to evaluate the efficacy and safety of combined pharmacological and mechanical thromboprophylaxis compared to mechanical thromboprophylaxis alone in postoperative patients with CD. A total of 206 patients diagnosed with CD who will be undergoing transsphenoidal surgery will be randomized in a 1:1 ratio to receive either combined pharmacological and mechanical thromboprophylaxis (intervention) or mechanical thromboprophylaxis only (control). The primary outcome is the risk of VTE within 12 weeks following surgery. This trial represents a significant milestone in evaluating the efficacy of combined pharmacological and mechanical prophylaxis in reducing VTE events in postoperative CD patients. ClinicalTrials.gov Identifier: NCT04486859, first registered on 22 July 2020.

Bilateral inferior petrosal sinus sampling (BIPSS) is widely used to differentially diagnose ectopic ACTH syndrome (EAS) and Cushing’s disease (CD) with pituitary lesions less than 6 mm detected on MRI. This study aimed to investigate the diagnostic accuracy of BIPSS and prognosis of low-lateralization [lateralization ratio of bilateral inferior petrosal sampling (IPS) ≤ 1.4] CD with microadenoma (≤ 6 mm). This single-centre retrospective study (2011–2019) included 25 EAS and 179 CD (microadenoma ≤ 6 mm) patients whose diagnoses were confirmed by pathology. All CD patients received two to ten years of follow-up. According to the multivariable regression results, a false-negative diagnosis at baseline might be associated with a lateralization ratio ≤ 1.4 (adjusted OR = 12.69, 95

Disclosure: S.R. Chacko: None. C.A. Villavicencio Torres: None. K. Yu: None. S.G. Waguespack: None. L. Lu: None. Z. Belaya: None. T. Bandgar: None. A. Sada: None. O. Ragnarsson: None. B. Altieri: None. A. Newman: None. P. Vibhatavata: None. M. Tóth: None. E.V. Varlamov: None. C. Badiu: None. A. Paissan: None. M. St-Jean: None. H. Falhammar: None. N. Imamudeen: None. L. Haberbosch: None. M. Bobrowicz: None. A. Tabarin: None. R. Shah: None. M.R. Gadelha: None. A. Peersen: None. W.F. Young: None. J. Kaplan: None. J. Varghese: None. M.A. Habra: None. O. Golounina: None. G.A. Melnichenko: None. A.R. Lila: None. C. Yamichannaiah: None. I. Bancos: None. Context: Cushing syndrome (CS) due to ectopic ACTH secretion (EAS) is a rare condition, associated with severe hypercortisolism and consequent morbidity and mortality. Its varied and often emergent presentation make diagnosis and treatment challenging. Objectives: 1.Characterize clinical and biochemical presentation of EAS. 2.Describe the scope of hypercortisolism-related complications, overall and CS-specific mortality. 3.Identify factors associated with CS-specific mortality. Methods: We conducted a multicenter retrospective cohort study in patients with EAS at 37 centers. Outcomes were clinical/biochemical severity scores, complications, overall and CS-specific mortality. Results: In 1053 patients [median age at diagnosis 52 years (IQR 37-64), 57% women], metastatic disease was present at diagnosis in 585 (56%). The most common sources of EAS were bronchial neuroendocrine neoplasm (NEN) (304, 29%), pancreatic NEN (114, 11%) and small cell lung carcinoma (114, 11%), with the source of EAS being occult in 192 (18%). Clinical and biochemical severity scores were severe in 475 (45%) and 873 patients (92%) respectively. Hypokalemia was present in 819 patients (78%) in the year prior to diagnosis. Of 342 patients (32%) who had IPSS, 301 (88%) suggested an ectopic source, 7 (2%) a pituitary source, and 23 (7%) were non-diagnostic. Of 595 patients who had both functional and cross-sectional imaging, discordant findings were seen in 122 (21%), concordant lesion was seen on both in 326 (55%), and no lesion in 147 (25%). Of those with discordant results, cross-sectional imaging was accurate in 66 (54%) and functional imaging in 56 (46%).Management included medical therapy alone in 237 (23%), surgical excision of the source of EAS in 158 (15%), bilateral adrenalectomy in 77 (7%) and multimodal therapies in 498 patients (47%). Complications within 5 years after diagnosis included hospitalization related to CS or its therapy in 351 (33%), infection in 337 (32%), thromboembolic event in 102 (10%), cardiovascular event in 83 (8%) and cerebrovascular event in 27 (3%). During a median follow-up of 35 months (IQR 20-122) from CS diagnosis, death occurred in 462 patients (44%), with the cause of death related to CS in 149 (14%). CS-specific mortality was higher in metastatic compared with non-metastatic disease (19% vs. 7%, P<0.001). On multivariable analysis of age, sex, clinical and biochemical severity, older age (HR 1.03, 95% CI: 1.02-1.04) and lower clinical severity score (HR 0.97, 95% CI: 0.94-0.99), were associated with higher CS-specific mortality. Conclusion: Our interim analysis, the largest worldwide, found that most patients with EAS had severe hypercortisolism and hypokalemia. Metastatic disease was present in 56%, and the source of EAS was occult in 18%. CS-related death occurred in 14%, emphasizing the need for urgent management. Presentation: Monday, July 14, 2025

Pituitary neuroendocrine tumors (PitNETs) can be invasive or aggressive, yet the mechanisms behind these behaviors remain poorly understood, impeding treatment advancements. Here, we integrat single-cell RNA sequencing and spatial transcriptomics, analyzing over 177,000 cells and 35,000 spots across 57 tissue samples. This comprehensive approach facilitates the identification of PitNETs tumor populations and characterizes the reconfiguration of the tumor microenvironment (TME) as PitNETs progress and invade. We trace the trajectory of TPIT-lineage PitNETs and identify an aggressive tumor cluster marked by elevated p53-mediated proliferation and a higher Trouillas classification, both associated with tumor progression. Additionally, we document the heterogeneity of immune stromal cells within PitNETs, particularly noting the enrichment of SPP1+ tumor associated macrophages (TAMs) in invasive tumors. These TAMs facilitate tumor invasion through the SPP1-ITGAV/ITGB1 signaling pathway. Our in-depth single-cell and spatial analysis of PitNETs uncovers the molecular dynamics within the TME, suggesting potential targets for therapeutic intervention.