Adrenocortical Carcinoma Research Articles

Identification of DLK1, a Notch ligand, as an immunotherapeutic target and regulator of tumor cell plasticity and chemoresistance in adrenocortical carcinoma.

Immunotherapeutic targeting of cell surface proteins is an increasingly effective cancer therapy. However, given the limited number of current targets, the identification of new surface proteins, particularly those with biological importance, is critical. Here, we uncover delta-like non-canonical Notch ligand 1 (DLK1) as a cell surface protein with limited normal tissue expression and high expression in multiple refractory adult metastatic cancers including small cell lung cancer (SCLC) and adrenocortical carcinoma (ACC), a rare cancer with few effective therapies. In ACC, ADCT-701, a DLK1 targeting antibody-drug conjugate (ADC), shows potent in vitro activity among established cell lines and a new cohort of patient-derived organoids as well as robust in vivo anti-tumor responses in cell line-derived and patient-derived xenografts. However, ADCT-701 efficacy is overall limited in ACC due to high expression and activity of the drug efflux protein ABCB1 (MDR1, P-glycoprotein). In contrast, ADCT-701 is extremely potent and induces complete responses in DLK1 + ACC and SCLC in vivo models with low or no ABCB1 expression. Genetic deletion of DLK1 in ACC dramatically downregulates ABCB1 and increases ADC payload and chemotherapy sensitivity through NOTCH1-mediated adrenocortical de-differentiation. Single cell RNA-seq of ACC metastatic tumors reveals significantly decreased adrenocortical differentiation in DLK low or negative cells compared to DLK1 positive cells. This works identifies DLK1 as a novel immunotherapeutic target that regulates tumor cell plasticity and chemoresistance in ACC. Our data support targeting DLK1 with an ADC in ACC and neuroendocrine neoplasms in an active first-in-human phase I clinical trial ( NCT06041516 ).

EBNA1BP2 identified as potential prognostic biomarker for multiple tumor types in pan-cancer analysis

BackgroundEpstein-Barr virus (EBV) infection has been closely linked to the development of various types of cancer. EB nuclear antigen 1 binding protein 2 (EBNA1BP2) is a crucial molecule for stable isolation of EBV in latent infection. However, the role of EBNA1BP2 in multiple tumor types is remains unclear. In this study, we comprehensively analyzed the functional characteristics of EBNA1BP2 and investigate its potential as a prognostic biomarker in pan-cancer.MethodsWe utilized data from TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) databases and employed various bioinformatics analysis tools, including TIMER2.0, HPA, GEPIA2.0, PrognoScan, cBioPortal, CancerSEA, and BioGRID to explore the expression pattern, prognostic value, immune infiltration, and methylation level of EBNA1BP2 in pan-cancer. Additionally, we conducted enrichment analysis of genes associated with EBNA1BP2 to identify potential biological functions and pathways.ResultsOur analysis revealed that EBNA1BP2 expression was significantly higher in tumor tissues compared to tumor-adjacent tissues. We observed that lower expression of EBNA1BP2 in adrenocortical carcinoma (ACC), brain lower grade glioma (LGG), sarcoma (SARC), and uterine carcinosarcoma (UCS) was significantly associated with improved overall survival (OS) and disease-free survival (DFS). Furthermore, the promoter methylation level of EBNA1BP2 was downregulated in the majority of cancer types. At the single-cell level, EBNA1BP2 was found to be positively correlated with cell cycle and DNA repair processes, while negatively correlated with hypoxia. Additionally, EBNA1BP2 was associated with the infiltration of immune cells such as B cells, cancer-associated fibroblast cells, and CD8+ T cells. Gene enrichment analysis indicated that EBNA1BP2 was mainly involved in nucleoplasm and RNA binding pathways.ConclusionOur findings suggest that EBNA1BP2 may serve as a potential prognostic biomarker for survival in pan-cancer. Further experimental studies are needed to validate these findings and explore the underlying mechanisms by which EBNA1BP2 contributes to tumorigenesis.

Diagnosis of sarcomatoid adrenal cortical carcinoma in the era of personalized medicine

Diagnosis of sarcomatoid adrenal cortical carcinoma in the era of personalized medicine

The molecular genetics of adrenal cushing.

Adrenal Cushing represents 20% of cases of endogenous hypercorticism. Unilateral cortisol-producing adenoma (CPA), a benign tumor, and adrenocortical carcinoma (ACC), a malignant tumor, are more frequent than bilateral adrenal nodular diseases (primary bilateral macronodular adrenal hyperplasia (PBMAH) and primary pigmented nodular adrenal disease (PPNAD)).In cortisol-producing adrenal tumors, the signaling pathways mainly altered are the protein kinase A and Wnt/β-catenin pathways. Studying components of these pathways and exploring syndromic and familial cases of these tumors has historically enabled identification of many of the predisposing genes. More recently, pangenomic sequencing revealed alterations in sporadic tumors.In ACC, mainly due to TP53 alterations causing Li-Fraumeni syndrome, germline predisposition is frequent in children, while it is rare in adults. Pathogenic variants in the DNA mismatch repair genes MLH1, MSH2, MSH6, and PMS2, which cause Lynch syndrome or alterations of IGF2 and CDKN1C (11p15 locus) in Beckwith-Wiedemann syndrome, can also cause ACC. Rarely, ACC is described in other hereditary tumor syndromes due to germline pathogenic variants in MEN1 or APC and, in very rare cases, NF1, SDH, PRKAR1A, or BRCA2. Concerning ACC somatic alterations, TP53 and genetic or epigenetic alterations at the 11p15 locus are also frequently described, as well as CTNNB1 and ZNRF3 pathogenic variants.CPAs mainly harbor somatic pathogenic variants in PRKACA and CTNNB1 and, less frequently, PRKAR1A, PRKACB, or GNAS1 pathogenic variants. Isolated PBMAH is due to ARMC5 inactivating pathogenic variants in 20 to 25% of cases and to KDM1A pathogenic variants in food-dependent Cushing. Syndromic PBMAH may be due to germline pathogenic variants in MEN1, APC, or FH, causing type 1 multiple endocrine neoplasia, familial adenomatous polyposis, or hereditary leiomyomatosis-kidney cancer syndrome, respectively. PRKAR1A germline pathogenic variants are the main alteration causing PPNAD (isolated or part of Carney complex).

Texture analysis can predict response to etoposide-doxorubicin-cisplatin in patients with adrenocortical carcinoma.

The adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy originating from the adrenal cortex. These patients usually undergo chemotherapy with etoposide, doxorubicin, cisplatin and mitotane (EDP-M) in case of locally advanced or metastatic ACC. Computed tomography (CT) radiomics showed to be useful in adrenal pathologies. The study aimed to analyze the association between response to EDP-M treatment and CT textural features at diagnosis in patients with locally advanced or metastatic ACCs. We enrolled 17 patients with advanced or metastatic ACC who underwent CT before and after EDP-M therapy. The response to treatment was evaluated according to RECIST 1.1, Choi, and volumetric criteria. Based on the aforementioned criteria, the patients were classified as responders and not responders. Textural features were extracted from the biggest lesion in contrast-enhanced CT images with LifeX software. ROC curves were drawn for the variables that were significantly different (p < 0.05) between the two groups. Long-run high grey level emphasis (LRHGLE_GLRLM) and histogram kurtosis were significantly different between responder and not responder groups (p = 0.04) and the multivariate ROC curve combining the two features showed a very good AUC (0.900; 95%IC: 0.724-1.000) in discriminating responders from not responders. More heterogeneous tissue texture of initial staging CT in locally advanced or metastatic ACC could predict the positive response to EDP-M treatment. Adrenal texture is able to predict the response to EDP-M therapy in patients with advanced ACC.

A Decade of Insights into Bilateral Adrenal Masses: Epidemiology, Clinical Profiles, and Therapeutic Approaches

Bilateral adrenal masses (BAMs) are rare, accounting for 10-15% of adrenal masses. Their diagnostic and therapeutic approaches differ from those of unilateral masses. This study aimed to analyze the epidemiological, clinical, paraclinical, and therapeutic profiles of BAMs. It is based on a retrospective analysis of 30 patient records from the Endocrinology, Diabetology, and Nutrition Department at CHU Hassan II in Fez, over a 12-year period. The average age of the patients was 53 years, with a male predominance (53.3%). BAMs were symptomatically revealed in 46.7% of cases, incidentally discovered in 23.3%, identified during tumor staging in 20%, and associated with a genetic syndrome in 10% of cases. Clinical examination revealed hypertension in 23.3% of patients, an abdominal mass in 10%, signs of adrenal insufficiency in 10%, hypercortisolism in 6.67%, and hyperandrogenism in 16% of cases. Biological assessment showed elevated urinary or plasma metanephrine derivatives in 9 patients, hypercortisolism in 3 patients, adrenal insufficiency in 3 patients, 11-β-hydroxylase enzyme block in 3 patients, and primary hyperaldosteronism in 1 patient. Adrenal computed tomography (CT) was performed in 29 patients. A staging workup, including PET scan, was requested in 20% of cases. MIBG scintigraphy was performed in 23.3% of patients. The investigations revealed 53% of BAMs were secreting and 47% non-secreting. There were 9 cases of pheochromocytomas, including 5 associated with a syndrome (3 MEN2a, 1 MEN2b, 1 VHL), 8 cases of non-secreting adenomas, 3 cases of congenital adrenal hyperplasia due to 11-β-hydroxylase enzyme block, 2 cases of adrenal metastases, 2 cases of adrenocortical carcinoma, 2 cases of adrenal cysts, 1 case of Conn’s adenoma, 1 case of cortisol-secreting adenoma, 1 case of primary adrenal lymphoma, and 1 case of adrenal hematoma. Treatment included the management of emergencies and preoperative preparation for patients eligible for surgery. Bilateral adrenalectomy was performed in 33.3% of patients, and unilateral adenectomy in 1 case of cortisol-secreting adenoma. Medical treatment was initiated in 16.6% of cases, and palliative treatment in 13.33% of cases. Surveillance alone was implemented in the remaining 33.3%. Prognosis depends on the type of adrenal mass, with outcomes marked by the death of one case of malignant adrenocortical carcinoma and one case of primary adrenal lymphoma. Our study, involving 30 cases of BAMs, highlights their clinical, biological, radiological, therapeutic, and prognostic characteristics. Data in the literature are limited compared to unilateral adrenal masses. Future larger studies are desirable for optimal management.

Hipertensão arterial resistente: um desafio diagnóstico

Introduction: Primary hyperaldosteronism is a leading cause of secondary arterial hypertension, affecting approximately 10% to 20% of the hypertensive population worldwide and in Brazil. This condition involves excessive aldosterone production due to factors such as adrenal adenoma, hyperplasia, or, less commonly, adrenal carcinoma and familial hyperaldosteronism. This study reports a case of resistant hypertension secondary to aldosterone-producing adenoma. Clinical Case: A 70-year-old male patient presented with episodes of dizziness, syncope, cramps, and asthenia. His medical history included resistant hypertension for over 30 years, despite treatment with five classes of antihypertensive medications. Additionally, he had a history of dyslipidemia, type 2 diabetes mellitus, non-dialysis chronic renal failure, and obstructive coronary arterydisease. Physical examination showed a BMI of 29 kg/m² (overweight), mean blood pressure of 140/90 mmHg (right arm) and 150/100 mmHg (left arm), and palpable nodules in both thyroid lobes, classified as Bethesda V based on aspiration cytopathology. Subsequently, he underwent total thyroidectomy and lymph node dissection. Laboratory tests revealed high plasma aldosterone levels with suppressed renin and an elevated aldosterone-renin ratio, raising suspicion of primary hyperaldosteronism. This diagnosis was confirmed by a computed tomography scan, which identified a 1.2 cm nodule in the right adrenal gland consistent with an adenoma. The patient underwent unilateral laparoscopic adrenalectomy. Two years post-surgery, the patient’s blood pressure was well-controlled with three antihypertensive medications, and his blood sugar, aldosterone, and renin levels were normal. Discussion and Conclusion: Most patients undergoing unilateral adrenalectomy for aldosterone-producing adenomas experience significant clinical improvement. Early screening and diagnosis of primary hyperaldosteronism are crucial for effective management, reducing the risk of complications in the cardiovascular and renal systems.

7036 Luteinizing Hormone/Human Chorionic Gonadotropin Receptor And Cyclooxygenase-2 Expression In Adrenocortical Carcinoma

Abstract Disclosure: R. Sheskier: None. A. Kirschenbaum: None. I. Zappi: None. S. Yao: None. A.C. Levine: None. Adrenocortical carcinoma (ACC) is a rare malignancy that has a poor prognosis. The current mainstays of therapy are surgical resection of primary and oligometastases, and systemic therapy with mitotane with or without combination of etoposide, doxorubicin, and cisplatin for more advanced disease. New therapeutic targets are needed to increase options for medical management of mitotane-resistant disease. Expression and function of the luteinizing hormone/human chorionic gonadotropin receptor (LH/hCG-R) have been demonstrated in non-gonadal tissues and malignancies (1). In the adrenal gland, LH/hCG-R expression has been noted in some secretory benign adenomas but its expression in ACC is underexplored (1, 2). Cyclooxygenase-2 (COX-2) is expressed in a variety of cancers, but has not been previously evaluated in ACC (3). We examined the immunohistochemical expression of LH/hCG-R and COX-2 in 15 tissue samples from 7 patients with ACC of varying ENSAT stages with 5 samples from primary ACC tumors and 10 metastatic samples from varying sites. LH/hCG-R was expressed in 13/15 ACC tissue samples (6/7 patients; 4/5 primary ACC; 9/10 metastases). COX-2 expression was demonstrated in 12/15 ACC tissue samples (6/7 patients; 4/5 primary ACC; 8/10 metastases with one metastasis post-mitotane negative for COX-2). Specimens from one patient were also examined for LH/hCG-R and COX-2 expression prior to and following treatment with mitotane. Compared to the primary ACC tumor pre-treatment with mitotane, the post-mitotane ACC metastasis showed an increase in LH/hCG-R expression and a decrease in COX-2 expression. In comparison, a liver ACC metastasis from a separate patient with mitotane-resistant disease demonstrated persistent LH/hCG-R and COX-2 expression. Our findings suggest that LH/hCG-R and COX-2 are expressed in the majority of ACC primary and metastatic lesions. Our results further imply that LH/hCG-R expression persists in mitotane-resistant disease and COX-2 expression initially decreases with mitotane therapy, but is re-expressed in mitotane-resistant disease. Both LH/hCG-R and COX-2 may therefore serve as potential therapeutic targets in the management of advanced ACC. (1) Carlson HE. Human adrenal cortex hyperfunction due to LH/hCG. Mol Cell Endocrinol. 2007 Apr 15;269(1-2):46-50. (2) Doroszko DM, Chrusciel M, Stelmaszewska J, Slezak T, Anisimowicz S, Plöckinger U, Quinkler M, Bonomi M, Wolczynski S, Huhtaniemi I, Toppari J, Rahman NA. GnRH antagonist treatment of malignant adrenocortical tumors. Endocr Relat Cancer. 2019 Jan 1;26(1):103-117. (3) Hu Z, Yang Y, Zhao Y, Huang Y. The prognostic value of cyclooxygenase-2 expression in patients with esophageal cancer: evidence from a meta-analysis. Onco Targets Ther. 2017 Jun 9;10:2893-2901. Presentation: 6/1/2024

7427 Cellular Senescence As A Pro-Tumorigenic Process And Therapeutic Target In Adrenocortical Carcinoma

Abstract Disclosure: L. Smith: None. K. Warde: None. S. Haston: None. J. Martinez-Barbera: None. K. Basham: None. Adrenocortical carcinoma (ACC) is a rare, yet highly aggressive malignancy that arises in the adrenal cortex. Advanced age is a major risk factor for ACC, with the peak age incidence between 50 and 60 years old. Older individuals above age 50 also have significantly worse survival outcomes. A better understanding of how aging contributes to the development and progression of ACC will uncover new treatment strategies. A major hallmark of aging is the systemic accumulation of senescent cells. While senescent cells have been implicated in various age-related diseases, including cancer, their role in ACC remains poorly understood. ACC tumors from older patients have an enrichment of senescence-associated gene signatures. Moreover, advanced ACC patients are treated with etoposide, doxorubicin, and cisplatin (EDP), which are chemotherapies that induce senescence. While EDP offers short-term benefit in some patients, more than half will progress within 6-months. We hypothesize that the accumulation of senescent cells that arise after EDP treatment are ultimately tumor-supportive long-term. As a result, we postulate that the use of senolytic compounds that clear away senescent cells after EDP treatment will lead to improved survival outcomes for ACC patients. We developed a new ACC mouse model that provides a window to study the role of senescence in ACC tumor progression and treatment. Our model is based on adrenal specific loss of ZNRF3, which is a Wnt inhibitor that is frequently inactivated in human ACC tumors. Despite early adrenal hyperplasia, we showed that Znrf3 conditional knockout (cKO) mice do not develop adrenal tumors until 18-months of age, which corresponds to the peak age incidence in humans. Additionally, the adrenals of Znrf3 cKO mice show a robust senescence activation phenotype that precedes adrenal tumorigenesis. We hypothesize that the accumulation of senescent cells is pro-tumorigenic long-term. To test our hypothesis, we combined Znrf3 cKO mice with a newly established mouse model that enables selective ablation of senescent cells (ZKO-FDR mice). Ongoing aging studies will determine whether the removal of early and late senescent cells in ZKO-FDR mice reduces adrenal tumorigenesis. Further, we established new primary tumor cell lines from aged ZKO-FDR mice. These cell lines will facilitate testing of the pro-tumor mechanisms of senescence. Additionally, they will permit the removal of senescent cells, both genetically and pharmacologically with senolytics, as a new treatment strategy in ACC. Overall, this project aims to investigate the pro-tumor biology of age- and therapy-induced senescence in ACC. Results from these studies will advance our understanding of senescence in cancer and aid in the development of new therapeutic strategies for ACC, including senolytics. Presentation: 6/2/2024

8468 Development And Characterisation Of Three-Dimensional Cell Culture Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. P. Donlon: None. C. Hantel: None. M.C. dennedy: None. Adrenocortical carcinoma (ACC) is a rare malignancy carrying a poor prognosis and limited treatment options. Surgical resection is the only option for cure; but unsuitable for advanced disease. Mitotane is the approved medical therapy, but associated with treatment resistance and adverse effects. Newer therapies, such as immune checkpoint inhibitors, have limited success. Development of improved therapies is limited by current pre-clinical disease models. No available model reflects the tumour micro-environment. Three-dimensional (3D) cell culture models are increasingly used in cancer research (6), with a paucity of developed models in ACC. In the current study, we developed novel 3D models of ACC using a type-1 collagen matrix. We then characterised these as representative ACC disease models . 3D cell culture models of ACC were optimised by embedding ACC cell lines, MUC-1, HAC15 and H295R within a type-1 Collagen matrix at differing cell numbers. Expression of type-1 collagen in ACC primary tumours was determined using immunohistochemistry (n=4). Cell viability for each model was assessed by evaluating sytox blue staining by Flow Cytometry. Metabolic activity of ACC cells within the matrix was determined using AlamarBlue staining. Prolferative activity was assessed using Ki67. Steroidogenesis was evaluated by measuring cortisol, aldosterone and androstenedione using LC-MS/MS.. Expression of CYP11B1, CYP11B2 CYP17, and StAR was measured using RT-qPCR. Type 1 collagen is strongly expressed in ACC tumour microenvironment. MUC-1, H295R and HAC15 cells were successfully cultured in a type 1 collagen matrix. MUC-1 cells cultured in the collagen matrix remain viable throughout the period of experimentation with optimum viability between 14 and 21 days. H295R cells also remain viable with optimum viability at an earlier stage of 7-14 days. HAC15 cells demonstrate high resilience in 3D cell culture with maximum viability throughout the entire period of experimentation up to 21 days. All three models increase their metabolic and proliferative activity over time. All three steroids are expressed by all three models with an increase in aldosterone secretion observed in 3D HAC15 and H295R models compared to monolayer (p&amp;gt;0.001) at days 7 and 14. All models express key steroidogenic enzymes. 3D ACC cell culture models were developed successfully using collagen type 1 abundant in ACC. Typical characteristics of the ACC tumour environment included (i) the presence of live and necrotic cells (ii) high metabolic activity and proliferation, reflective of cell turnover.. Capacity of cells to spread and proliferate within the 3D cell culture models was limited by available collagen substrate. Steroidogenic capacity was demonstrated in 3D cell culture. This model therefore retained key characteristics of ACC and shows promise as an animal-sparing, pre-clinical model. Presentation: 6/1/2024

8180 Rare Variants Of Adrenocortical Carcinoma: A Retrospective Cohort Study

Abstract Disclosure: C.A. Villavicencio Torres: None. I. Bancos: None. Background: Adrenocortical carcinoma (ACC) is a rare malignancy with an estimated annual incidence of 0.7-2 cases per million per year. Scarce data are available on rare histological variants of ACC. Objective: To describe the clinical presentation and outcomes of patients diagnosed with oncocytic, sarcomatoid and myxoid ACC variants. Methods: Retrospective single center cohort study, 1992-2021 of patients with rare histological ACC variant. Results: A total of 20 patients were diagnosed with oncocytic (17, 85%), sarcomatoid (2, 10%), or myxoid (1, 5%) ACC. Median age of diagnosis was 47 years (range 17-80), and 14 (70%) were women. All patients had unilateral ACC (12, 60% left) with a median tumor size of 11 cm (range 4-29). Adrenal hormone excess was documented in 10 (50%) patients, mostly with either glucocorticoid or androgen excess. ENSAT stage I, II, III, and IV was documented in 2 (10%), 10 (50%), 6 (30%), and 2 (10%) patients, respectively. All patients were treated with adrenalectomy (laparoscopic in 5 and open in 15). In addition, patients were treated with mitotane (10, 50%), radiation (7, 35%), and chemotherapy (5,25%). Mitotane was administered for a median of 12 months (range 4-31) and was discontinued mainly due to side effects in 9 (90%) patients. Patients were followed for a median follow up of 6.5 years (range 0.4-12). In addition to 2 patients who presented with ENSAT stage IV metastatic disease, metastatic disease developed in additional 6 (30%) patients who initially presented with localized ACC (3 stage 2, 3 stage 3, 5 oncocytic, 1 sarcomatoid). Metastatic sites included lung (n=2), omentum (n=1), duodenum (n=1), liver (n=4), and bones (n=1). At the end of follow up, 11 (55%) patients remained disease free (53% oncocytic, 50% sarcomatoid, 100% myxoid), 5 (25%) patients were alive with metastatic ACC (23% oncocytic, 50% sarcomatoid), and 4 patients died of ACC (median time to death 21 months (range 10-50) post-adrenalectomy). Conclusions: Patients with oncocytic, sarcomatoid, and myxoid ACC may have a better prognosis than in reported literature. Larger multicenter studies are needed to identify patient and tumor-specific factors that guide management to assure improved outcomes in these very rare patients. Presentation: 6/2/2024

8123 Identification Of Glutamine Metabolism As A Druggable Therapeutic Vulnerability For Adrenocortical Carcinoma

Abstract Disclosure: V. Chortis: None. C. Yao: None. C. Ribeiro: None. L. Kelley: None. L. Nagano: None. M. Berber: None. S. Raveenthiraraj: None. P. Vendramini: None. K. Kiseljak-Vassiliades: None. D.L. Carlone: None. M.C. Haigis: None. K.S. Borges: None. D.T. Breault: None. Dysregulation of cellular metabolism is a hallmark feature of cancer that can be targeted therapeutically but remains underexplored in adrenocortical carcinoma (ACC), despite the urgent need for improved treatments. We employed a recently developed transgenic mouse model of ACC (BPCre), driven by the two most common mutations in human ACC (β-catenin gain-of-function and p53 loss-of-function), aiming to characterize in vivo ACC metabolism at a tissue level and to identify metabolic vulnerabilities. We employed metabolomics (liquid chromatography-mass spectrometry) and transcriptomics (bulk RNA-seq) to compare tissue metabolism in spontaneous BPCre tumors with control adrenals. Polar metabolites were analyzed using a library that provides a representative cross-section of major carbon and nitrogen-handling pathways. We identified widespread dysregulation of tumor metabolism (64/175 metabolites significantly dysregulated, FDR&amp;lt;0.05), with the most significantly dysregulated pathways including purine and pyrimidine metabolism, proline and glutamate metabolism, glycolysis, and the pentose phosphate pathway (FDR&amp;lt;0.05). Orthogonal analysis by RNA-seq revealed congruent metabolic pathway alterations. Comparison of the most significantly dysregulated pathways between BPCre mouse tumors and human ACC using publicly accessible human transcriptomic datasets (TCGA and GTEx) revealed extensive overlap. Given the prominent dysregulation of several glutamine (Gln)-dependent metabolic pathways in mouse and human ACC, we hypothesized that Gln catabolism is likely to represent a targetable metabolic vulnerability in ACC. Pharmacological targeting of Gln metabolism using the Gln antagonist 6-Diazo-5-Oxo-L-Norleucine (L-DON) induced marked cytotoxicity in two cell lines derived from BPCre tumors and three human ACC cell lines (NCI-H295R, CU-ACC1-2), as assessed by cell viability and clonogenic assays. Remarkably, this effect was rescued only by nucleoside supplementation, suggesting that Gln’s contribution to de novo purine and pyrimidine biosynthesis is the critical metabolic dependency in ACC cells. Treatment with the L-DON pro-drug JHU-083, in vivo, led to marked growth inhibition of subcutaneous mouse ACC tumor implants in both immunocompetent and immunodeficient mice. As expected, tissue metabolomics of JHU-083-treated tumors confirmed extensive depletion of purine metabolites. Tumor immunophenotyping by flow cytometry revealed significantly increased infiltration with Natural Killer cells, likely potentiating the anti-tumor effect of glutamine antagonism in immunocompetent mice. JHU-083 was also effective against human NCI-H295R xenografts, in vivo. This work reveals important new insights into ACC metabolism and provides pre-clinical evidence supporting Gln antagonism as a new metabolic treatment approach for ACC. Presentation: 6/2/2024

12673 Epigenetic Subtyping Of Adrenocortical Carcinoma In Archival Material And Implications For Disease Evolution

Abstract Disclosure: D. Mohan: None. A.M. Lerario: None. M.Q. Almeida: None. B.B. Mendonca: None. A. Latronico: None. M.C. Fragoso: None. G.D. Hammer: None. Adrenocortical carcinoma (ACC) is a rare cancer of the adrenal cortex, to date cured only by surgery. Pangenomic studies demonstrate that ACC is comprised of three molecular subtypes with distinct clinical outcomes. CIMP-high ACC, the most aggressive molecular subtype, is defined by abnormal epigenetic patterning including inappropriate DNA methylation. CIMP-high status of the primary tumor accounts for 30% of all ACC, but 70% of metastatic relapses. It remains unclear if ACC transitions molecular subtypes, e.g. acquires CIMP-high status, during metastatic dissemination. We and others have demonstrated that G0S2 methylation is a sensitive and specific single-locus biomarker capturing CIMP-high ACC. However, our prior studies utilized frozen tissues, often only available at specialized centers. Here, we measured G0S2 methylation in an expanded cohort of n=73 formalin-fixed paraffin-embedded (FFPE) primary, recurrent, and metastatic ACC samples. We show that G0S2 methylation status is stable in archival FFPE material, concordant with measurement in frozen specimen (Pearson r=0.94, n=28). We identify that, within individual patients, the G0S2 methylation status of the primary is often preserved in the metastasis or recurrence. These data suggest that ACC subtype switching is not required for metastatic evolution. In cases of advanced ACC with accessible lesions amenable to biopsy, epigenetic subtypes can be diagnosed prior to oncologic resection, enabling subtype-specific therapeutic interventions in the neoadjuvant setting. Presentation: 6/1/2024

12540 Epigenetic Subtyping Of Adrenocortical Carcinoma In Archival Material And Implications For Disease Evolution

Abstract Disclosure: D. Mohan: None. A.M. Lerario: None. M.Q. Almeida: None. B.B. Mendonca: None. A. Latronico: None. M.C. Fragoso: None. G.D. Hammer: None. Adrenocortical carcinoma (ACC) is a rare cancer of the adrenal cortex, to date cured only by surgery. Pangenomic studies demonstrate that ACC is comprised of three molecular subtypes with distinct clinical outcomes. CIMP-high ACC, the most aggressive molecular subtype, is defined by abnormal epigenetic patterning including inappropriate DNA methylation. CIMP-high status of the primary tumor accounts for 30% of all ACC, but 70% of metastatic relapses. It remains unclear if ACC transitions molecular subtypes, e.g. acquires CIMP-high status, during metastatic dissemination. We and others have demonstrated that G0S2 methylation is a sensitive and specific single-locus biomarker capturing CIMP-high ACC. However, our prior studies utilized frozen tissues, often only available at specialized centers. Here, we measured G0S2 methylation in an expanded cohort of n=73 formalin-fixed paraffin-embedded (FFPE) primary, recurrent, and metastatic ACC samples. We show that G0S2 methylation status is stable in archival FFPE material, concordant with measurement in frozen specimen (Pearson r=0.94, n=28). We identify that, within individual patients, the G0S2 methylation status of the primary is often preserved in the metastasis or recurrence. These data suggest that ACC subtype switching is not required for metastatic evolution. In cases of advanced ACC with accessible lesions amenable to biopsy, epigenetic subtypes can be diagnosed prior to oncologic resection, enabling subtype-specific therapeutic interventions in the neoadjuvant setting. Presentation: 6/1/2024

Which lymphadenectomy for adrenocortical carcinoma?

BackgroundLymph node dissection improves adrenocortical carcinoma staging, but remains anatomically poorly defined. This ambiguity stems from limited knowledge of the adrenals lymphatic network. This work aims to define lymph node dissection for adrenocortical carcinoma through a systematic review and anatomical study. MethodFirst, an anatomical study was conducted on fresh cadavers by injecting blue dye into each adrenal gland before dissection. Concurrently, a systematic review of anatomical and clinical studies was performed, focusing on adrenals lymphatic network, lymph node dissection, and location of invaded lymph nodes in surgical series. ResultsTwelve adrenals from 6 cadavers were resected en bloc with a median of 3 lymph nodes (1.5–6) removed. Screening of 6,506 studies revealed (1) 18 anatomical studies on cadavers detailing a 3-stage compartmentalized adrenals lymphatic network with distinct right/left lymph nodes relays; (2) 4 clinical studies highlighting discrepancies in lymph node involvement in adrenocortical carcinoma patients compared with anatomical description of adrenals lymphatic network, notably: lower implication of celiac lymph node, preponderance of ipsilateral renal hilum lymph nodes, potential contralateral involvement; (3) 21 series of adrenocortical carcinoma surgery demonstrating the heterogeneity of lymph node dissection practice (22% ± 4% lymph node dissection rate), with an average of 2.7 ± 0.6 lymph nodes removed, already fewer than in our cadaveric study. ConclusionSynthesis of anatomical and clinical studies suggest the following lymph node dissection protocol during adrenocortical carcinoma resection: capsular, renal hilum, para-cava, and inter-aortic-cava lymph nodes (right adrenocortical carcinoma); and capsular, renal hilum, para-aortic, and inter-aortic-cava lymph nodes (left adrenocortical carcinoma).

12490 Management Of Autoimmune Hepatitis Under Mitotane Therapy: Reintroduction Of Mitotane Under High Dose Corticosteroids For Targeted Mitotane Therapeutic Levels In A Patient With Recurrent Myxoid Adrenocortical Carcinoma

Abstract Disclosure: T. Hristova: None. D. Fenyves: None. C. Cloutier: None. M. Latour: None. Z. El-haffaf: None. P. Karakiewicz: None. H.J. Olney: None. I. Bourdeau: None. Histologically rare variants of adrenocortical carcinoma (ACC) include myxoid, oncocytic and sarcomatoid subtypes. There are about 50 reported cases of myxoid variants up to now that are described as more aggressive than classical ACC with poorer overall survival. Surgical resection offers the best chance of survival, with mitotane being the first line of treatment after surgery. Mitotane is currently the only approved adrenolytic drug for ACC. Among mitotane adverse effects, auto-immune hepatitis (AIH) is reported in monography, yet barely anything is reported in the literature about it. The precise mechanism of AIH has not been reported yet, but the mainstem therapy of AIH is corticosteroids alone or in combination with azathioprine. We report the case of a 43-year-old male who was diagnosed with non-secreting myxoid ACC subtype stage 2 (7x6x5,5 cm, ki67 18%, Weiss score 6/9). Multigene panel testing revealed a germline pathogenic variant in the ATM gene. Post adrenalectomy, mitotane was initiated within 2 months and increased to 2,5g daily within 6 weeks. 11 weeks after starting mitotane, liver enzymes were as follows: ALT 127 U/L (N: 10-39), AST 54 U/L (N: 13-39), ALP 165 U/L (N: 36-110), GGT 489 U/L (N: 9-47), while pre-mitotane levels were normal. Although mitotane was stopped, ALT increased to 255 U/L and remained high for 10 weeks after its cessation. Liver biopsy showed chronic hepatitis with moderate inflammation which was compatible with toxic hepatitis vs AIH. As kinetic liver enzyme tests increased after mitotane cessation, AIH was the retained diagnosis. Treatment with 40mg prednisone daily was then initiated. Pre-prednisone liver enzymes were ALT 202 U/L, AST 64 U/L, ALP 154 U/L, GGT 216 U/L, whereas 8 weeks later values had decreased to ALT 74 U/L, AST 20 U/L, ALP 108 U/L, GGT 84 U/L, and prednisone was lowered to 15mg daily. ACC recurred 3 months later, about one year after the initial adrenalectomy. After surgical resection, mitotane was restarted at 500mg daily with prednisone 40mg daily. As liver enzymes were within normal ranges, mitotane was slowly increased to 2.5g daily, with mitotane plasma levels of 11.3mg/L, whereas prednisone was decreased gradually to 12.5mg daily. Second ACC recurrence occurred one year later and mitotane was further increased to 3g daily under prednisone 12.5mg reaching mitotane levels up to 21.2 mg/L with surgical debulking. Further therapeutic avenues are being considered for a rapid recent ACC recurrence, such as PARP inhibitors for targeted therapy in the context of ATM gene mutation, but further considerations are required due to previous AIH. To the best of our knowledge this is the first reported case of AIH due to mitotane in a rare ACC subtype in a patient carrying a germline ATM gene mutation. We also report restart of mitotane therapy with concomitant high dose prednisone in the context of ACC recurrence that allowed to reach targeted therapeutic levels. Presentation: 6/1/2024

7652 Functional Adrenocortical Carcinoma Presenting As Uncontrolled Hypertension

Abstract Disclosure: B.R. Bedell: None. M. Fariduddin: None. J. Sanchez Perez: None. S.C. Kukreja: None. R.M. Sargis: None. Adrenocortical carcinoma is a rare malignancy, with approximately 2,000 cases reported in the United States since 1970.[1] Many secrete excess adrenocortical hormones, adding to morbidity and mortality. Surgery can be curative or prolong survival if diagnosis is made early, but prognosis remains poor due to delayed detection.[2] We present a case of metastatic functional adrenocortical carcinoma that presented with consequences of long-standing uncontrolled hypertension. A 59-year-old male with history of hypertension and CAD presented to the hospital with worsening bilateral pitting edema, dyspnea on exertion, palpitations, headaches, and proximal muscle weakness over for multiple months. His hypertension (HTN) was noted to be uncontrolled despite use of multiple antihypertensives (daily Lasix 20 mg, Lisinopril 40 mg, Metoprolol Succinate 100 mg and Amlodipine 10 mg). Hypokalemia of 3.0 mmol/l (3.5 - 5.2 mmol/l) was noted. Echocardiogram revealed new heart failure with preserved ejection fraction. A subsequent nuclear stress test incidentally revealed a right-sided 15 cm retroperitoneal mass, invading the IVC and the right hepatic lobe. An AM dexamethasone suppression test was notable for a cortisol of 39 ug/dl (&amp;lt;1.8 ug/dl) and an undetectable ACTH level of &amp;lt;1.5 pg/ml (7.2 - 63.3 pg/mL), suggestive of primary hypercortisolism. 24-hour urine cortisol was 930.6 ug/d, (&amp;lt;=60.0 ug/d), DHEA-s 1105 ug/dL (52 - 295 ug/dL), Androstenedione 2.641ng/mL (0.230 - 0.890 ng/mL), and 17-Hydroxyprogesterone 299.87 ng/dL (&amp;lt;=138.00 ng/dL). Plasma metanephrines were normal and aldosterone levels were undetectable, confirming cortisol and its precursor excess as the cause of hypertension. Biopsy of the mass was consistent with adrenocortical carcinoma. Due to disease extent, the patient was not a candidate for surgical debulking, and radiation therapy was commenced. Ketoconazole 200 mg and Spironolactone 150 mg twice daily led to improvement in HTN and hypokalemia. His course was further complicated by bacteremia and acute pulmonary embolism. The patient eventually elected to pursue hospice care. Adrenocortical carcinoma remains challenging to detect at early stages given a paucity of symptoms. This case highlights the need to have a high index of suspicion for hypercortisolism in patients with uncontrolled HTN and hypokalemia, as this may be indicative of a functional adrenal cancer. This could be a potential strategy for earlier diagnosis, thereby increasing survival rates by improving disease amenability to surgical intervention. (1.) Sharma E, Dahal S, Sharma P, et al. The Characteristics and Trends in Adrenocortical Carcinoma: A United States Population Based Study. J Clin Med Res. 2018;10(8):636-640. doi:10.14740/jocmr3503w (2.) Vaidya A, Nehs M, Kilbridge K. Treatment of Adrenocortical Carcinoma. Surg Pathol Clin. 2019;12(4):997-1006. doi:10.1016/j.path.2019.08.010 Presentation: 6/2/2024

7888 Mitotane Induces Different Modes Of Cell Death In Treatment Resistant And Sensitive Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. C. Hong: None. C. Hantel: None. W.E. Rainey: None. M.C. Dennedy: None. Adrenocortical carcinoma (ACC) is a rare aggressive cancer with poor survival. Adjuvant mitotane is the only approved drug for treatment of ACC. It improves survival but its use is limited by poor tolerability and drug resistance. For metastatic ACC, combination chemotherapy, using mitotane and etoposide, doxorubicin, and cisplatin improves survival, but efficacy is limited. Better understating the cytotoxic mechanisms of mitotane offers potential to develop improved therapeutic options for ACC. To investigate the underlying mechanisms in vitro, human ACC cells (HAC15 and H295R), in monolayer cell culture were treated with increasing concentrations of mitotane. Cell death was evaluated at 6 and 24 hours using flow cytometry analysis of Annexin V and Sytox Blue. This was complemented by Incucyte ® Live-Cell Analysis System imaging of annexin V and Sytox Blue staining. Expression of the markers of apoptosis, cleaved caspase 3, and necroptosis, phosphorylated MLKL and RIPK1 was measured at 6 and 24 hours. Mitotane significantly reduced the viability of H295R cells at 6 and 24 hours at the therapeutic concentration of 50uM (p&amp;gt;0.0001), thereby demonstrating treatment-sensitivity. In HAC15 cells, a supratherapeutic dose of 200uM was needed to significantly reduce viability (p&amp;gt;0.05) at 24 hours, indicating resistance to mitotane treatment at therapeutic concentrations. There was increasing Annexin V fluorescence with increasing mitotane concentration in H295R and HAC15 cells ar 6 and 24 hours without higher coinciding staining with Sytox Blue. Video analysis demonstrated these findings. Cleaved caspase 3 expression was associated with mitotane-induced cell death in H295R cells at 6 and 24 hours, indicating apoptosis. HAC15 cells demonstrated cleaved caspase 3 expression only at the highest concentration of mitotane exposure (600uM) at 6 hours, without appreciable expression at 24 hours. Phosphorylated-MLKL and RIPK1 expression was seen in controls and all mitotane doses in both cell lines at both timepoints, indicating necroptosis. In this study, H295R cells, representing a treatment-sensitive model of Mitotane undergo both apoptotic and necroptotic cell death at 6 and 24 hours of mitotane exposure. In contrast, HAC15 cells, demonstrated relative treatment resistance to mitotane, and undergo necroptotic cell death only. These results highlight the importance of understanding and targeting non-apoptotic and necroptotic cell death pathways, particularly when evaluating mechanisms of treatment resistance in ACC. Given the difference in the mechanism of cell death in treatment-sensitive and treatment-resistant cells, further exploration of necroptosis is necessary to better understand how ACC cells may manifest treatment resistance. Presentation: 6/1/2024

7703 Adrenal Cortical Carcinoma With Vascular Involvement - Complex Case Requiring Life-Saving Surgery With Bovine Pericardium- IVC Reconstruction

Abstract Disclosure: V. mehta: None. M. Renzu: None. K.N. Pereira: None. S. Sampath: None. S. Sruthi: None. E.B. Ruby: None. Adrenocortical carcinomas (ACCs) are rare endocrine tumors that exhibit unusual biological characteristics. Roughly 66% of cases are functional and have excessive hormone release causing classic symptoms of hypercortisolism, including plethora, diabetes, muscle weakness/atrophy, virilization, and osteoporosis. Few present with vague signs due to mass effects, including but not limited to abdominal/flank pain, fullness, or early satiety; the rest are incidental findings. We present a case of a 55-year-old female with no medical history presented following weeks of progressive abdominal pain with abdominal wall erythema, leg swelling, and dyspnea along with hirsutism, leukocytosis, elevated BNP, hematuria concerning for abdominal wall cellulitis and new-onset CHF. Initial CT chest revealed incompletely visualized incidental adrenal mass. Further investigations with CT abdomen and lab work revealed a left adrenal mass suspicious for ACC, extending into the left renal vein and IVC, elevated urine metanephrines and normetanephrine, and elevated cortisol levels with low ACTH. The dexamethasone suppression test confirmed inadequate cortisol suppression. Patient underwent aggressive surgery with exploratory laparotomy revealing tumor thrombus in left renal vein extending into Inferior vena cava (IVC). A left adrenalectomy, nephrectomy, and IVC reconstruction using a Bovine Pericardial Patch (BPP), which is majorly used for arterial reconstruction, was performed. Pathology results indicated ACC, measuring 13.5 cm, with extension into renal vein and vascular invasion, staged as pT4pN0 cM0 without evidence of deficient mismatch repair protein expression. ACCs are rare and profoundly aggressive malignancies with an incidence rate of 0.7–2.0 per million per year with bimodal age distribution. Disease peaks before the age of five and in the fourth to fifth decade of life, with increased incidence in females. Diagnosis cannot be conclusively established by a single histological technique, imaging, hormonal work-up, or immunohistochemical labeling, but surgical excision is the gold standard. Complete surgical resection is the only possible approach for curing or improving survival chances in patients with adrenal mass/metastasis. A retrospective case study of 36 venous reconstructions using BPP showed few complications like thrombosis in 14% (versus 11% in autologous venous repair), 11% reoperated, 3-4% developed partial thrombus and hematoma. Our case exemplifies the successful management of challenging ACC with vascular involvement using BPP, which is mostly used for arterial repairs and is now expanding to venous repairs. Outcomes stress the ongoing need for research and collaborative efforts to refine and standardize the ideal approach to ACC patients with IVC thrombosis and the use of BPP in venous repair. Presentation: 6/1/2024

7959 Combination Therapy Of Multi-Targeted Tyrosine Kinase Inhibitors And Immune Checkpoint Inhibitors In Advanced Adrenocortical Carcinoma

Abstract Disclosure: F. Yaylaci Mert: None. V. Balderrama Brondani: None. L.P. Marcal: None. M. Campbell: None. C. Jimenez: None. J. Varghese: None. A. Shah: None. M.A. Habra: None. Background: Adrenocortical carcinoma (ACC) is a rare cancer with limited response to systemic chemotherapies. Monotherapy with multi-targeted tyrosine kinase inhibitors (TKIs) or immune checkpoint inhibitors (ICIs) in ACC patients has limited efficacy. Combining TKIs with ICIs increased response rates in other malignancies but the efficacy of this approach in ACC is still unknown. This retrospective study aims to evaluate the efficacy and safety of the combined use of TKIs and ICIs in patients with advanced ACC. Methods: A single-center, retrospective cohort study in patients with advanced ACC treated with the combination of TKIs and ICIs. Response rates, progression-free survival (PFS), overall survival (OS) calculated from the time of combination, disease control rate (DCR), and safety profile were the main study objectives. Results: Seventeen patients were treated with combination therapy of TKI (lenvatinib or cabozantinib) and ICI (pembrolizumab) between January 2017 and December 2023. Eleven patients were women, the median age was 43.4 years (range 21.9-66.6), and 8 (47.1%) patients presented cortisol-producing tumors. All patients had prior systemic therapy, median of 3 (range 1-9) lines of therapy. The median duration of TKI+ICI therapy was 7.1 months (range 0.7-69.7). The treatment was discontinued in one patient due to a grade 3 adverse event (gastrointestinal fistula) after 1.6 months of treatment. Best overall responses were as follows: partial response 2 (13%), stable disease 8 (53%), and progressive disease 5 (34%). DCR was 58.8%. Median PFS was 7.1 months (range 0.7-66.4). One death occurred during the study due to disease progression. The median OS was 21.3 months (range 3.3-70.3). At the time of this report, 7 patients (41%) were still alive. Conclusion: The combined use of TKIs and ICIs in heavily treated ACC patients is associated with occasional durable responses and has a predictable and manageable side effect profile. This combination is worthy of a prospective clinical trial to validate the findings and establish new lines of therapy for this rare disease. Presentation: 6/2/2024