bluuebunny/crossref_metadata_2025 huggingface dataset:
8636 Characterization of the Role of Neutrophils in Promoting Progression of Lymphangioleiomyomatosis
Authors: Henson Thomas R , Minor Briaunna , Hammes Stephen R
Published: October 2024

Disclosure: T.R. Henson: None. B. Minor: None. S.R. Hammes: None.

Lymphangioleiomyomatosis (LAM) is a rare, multisystemic disease that is associated with cystic lung destruction. LAM is caused by small smooth muscle cell-like tumors throughout the lung that contain deleterious mutations in the tuberous sclerosis complex genes TSC1 or TSC2. This attenuates the activity of the mammalian target of rapamycin complex 1 (mTORC1) causing increased proliferation of the smooth muscle cells. LAM is a sexually dimorphic disease that primarily affects genetic females. Clinical observations implicate the role of estrogen in disease progression, as it is exacerbated during pregnancy and by use of exogenous estrogens in birth control. LAM tumor-derived cells lines have shown that direct estrogen stimulation of cell proliferation is modest compared to the dramatic in vivo effects of estradiol (E2) stimulation in a uterine-specific TSC2-null mouse model. Creation of a monoclonal cell line from these mice resulted in the generation of the LTM3 cell line, which also demonstrates low response to direct estrogen stimulation in vitro. This indicates that direct E2 stimulation may act upon other cell types in the tumor microenvironment. Immunophenotyping of uterine-specific TSC2-null mice has shown that the tumor microenvironment is enriched with myeloid cells specifically neutrophils. Functional analysis of neutrophil suppression in vivo has shown a reduction in myometrial growth in the uterine-specific TSC2-null mice, indicating a key role for neutrophil-mediated LAM tumor growth. Altered transcriptional regulation of neutrophils further highlights the importance of neutrophils in LAM progression. Bulk messenger RNA sequencing revealed E2 dependent increase in the neutrophil derived serine protease neutrophil elastase (NE). NE in vitro has been shown to increase migration, invasion, and proliferation of LAM cell lines, while inhibition in vivo has shown a reduction to the myometrial growth. Previously published data by the lab has shown that E2 increases tumor induced expansion of neutrophils in the bone marrow. Isolation and purification of Neutrophils from mouse bone marrow stimulated with tumor conditioned media from the LTM3 cells has shown signs of activation via upregulated gene expression. The focus of the current research has been the characterization of bone marrow derived neutrophils. Currently working with an in vitro system to study the proliferation of LTM3 and the rat leiomyoma (ELT3) cell lines when they are co-cultured with neutrophils. Understanding the mechanism by which neutrophils are activated and interact with LAM cells can give understanding to disease progression may provide insight into future treatment options.

Presentation: 6/3/2024
7277 Diabetic Ketoacidosis as the First Manifestation of Ectopic Cushing’s Syndrome
Authors: Saudade Medeiros Regina , Pereira Bernardo , Pataco Andreia , Senra Catarina , João Ponte Maria , Anselmo João , Sousa Isabel
Published: October 2024

Disclosure: R.S. Medeiros: None.

Introduction: Ectopic Cushing’s Syndrome (ECS) represents 10-20% of all ACTH-dependent CS, and the majority of cases arise from lung carcinoids. These indolent lesions usually evolve clinically over 6-24 months, whereas carcinomas are associated with faster onset of ECS. Diabetic Ketoacidosis (DKA) as a presenting manifestation of Cushing’s Syndrome (CS) is very rare, with the few cases published to date related to Cushing’s disease. Clinical Case: A 42-year-old woman was admitted in the emergency room with DKA and COVID-19. During stabilization, florid CS was clinically suspected. She reported a 6-month history of secondary amenorrhea, lack of concentration and memory, easy bruising, proximal myopathy with frequent falls evolving to daily life dependency from relatives. She also reported a 2-month diagnosis of dyslipidemia, Diabetes and hypertension, and a diagnosis of atypical pneumonia 6 months ago. Physical exam showed typical cushingoid appearance, especially for marked limb sarcopenia and proximal myopathy. An overnight dexamethasone suppression test (morning cortisol: 25.7 ug/dL), 24h urinary free cortisol (UFC; 1072.5 ug/dL; reference: <176) and serum cortisol at 23h00 (25.5 ug/dL) confirmed CS. ACTH-dependent CS was diagnosed. She had non-suppressible serum cortisol in high dose dexamethasone suppression test, and CRH stimulation test was suggestive of Cushing’s disease only by a 20% rise of cortisol at 30 minutes. Pituitary MRI was normal. Inferior Petrosal Sinus sampling was postponed for several months due to healthcare strikes. The patient started 750 mg of metyrapone in three divided doses but rapidly decreased the dose and stopped it due to clinical resolution of CS, a dramatic improvement of all glucocorticoid-related comorbidities leading to discontinuation of statin, insulin, main oral anti-hyperglycemic and antihypertensive medications, and normal midnight salivary cortisol and 24h UFC. She remained in this eucortisolemic state for 12 months. Thoracic CT scan revealed thymic hyperplasia and a 25x15 mm well-defined nodule in the lingula. A [6][8]Ga-DOTANOC PET/CT showed a single uptake in the same lung region. The patient underwent lingulectomy plus excisional biopsy of interlobar lymph nodes, and pathology revealed a low-grade neuroendocrine tumor (Ki67<2%, <2 mitosis) without involved lymph nodes. The patient is weaning off from physiologic doses of hydrocortisone. Conclusion: DKA can rarely be the presenting manifestation of severe CS, including ECS. Clinicians should suspect on this etiology of DKA based on clinical signs and cumulative morbidity not typical of a young patient. This hint would lead to the expedite implementation of targeted therapies for initial stabilization of the CS patient, and the timely institution of a proper diagnostic approach and definitive treatment of this challenging patients.

Presentation: 6/2/2024
7232 Direct m6A mRNA-Seq in Endocrine- Resistant and Sensitive Breast Cancer Cells
Authors: Petri B J , Valdes B L , Piell K M , Rouchka E C , Klinge C M
Published: October 2024

Disclosure: B.J. Petri: None. B.L. Valdes: None. K.M. Piell: None. E.C. Rouchka: None. C.M. Klinge: None.

Acquired resistance to endocrine therapies (ET) in patients with estrogen receptor α (ER+) breast tumors results in metastatic spread. RNA modifications including N6-methyladenosine (m6A) play crucial roles in the post-transcriptional regulation of gene expression and have been implicated in cancer progression. We hypothesized that m6A epitranscriptomic alterations are associated with pathways in ET resistance. Direct-RNA sequencing technology (nanopore) was used to detect and map m6A modifications at single-nucleotide resolution to comprehensively profile m6A modifications in ET-resistant LCC9 and ET -sensitive MCF-7 breast cancer cell lines with or without 4-hydroxytamoxifen (4-OHT) treatment. Additionally, MCF-7 cells were treated with the METTL3 inhibitor STM2457 to identify m6A positions and transcripts directly regulated by METTL3 activity. We developed a new statistical method by integrating m6Anet, an existing machine-learning algorithm designed to call m6A modified bases, with a generalized linear model following a binomial distribution analysis to identify significant differential m6A modification ratios (DMR). We identified 61 transcripts with DMR between vehicle-treated LCC9 compared to MCF-7 cells and 323 transcripts with DMRs between 4-OHT-treated LCC9 and MCF-7 cells. Fewer transcripts showed DMR after 4-OHT treatment in MCF-7 (41) versus LCC9 cells (399). Thus, more changes in m6A DMRs were detected in response to 4-OHT in the ET-resistant LCC9 versus ET-sensitive MCF-7 cells with “translation elongation” as the top pathway in enrichment analysis. STM2457 treatment resulted in significant changes to DMRs in MCF-7 cells with or without 4-OHT treatment and altered gene expression patterns associated with “cytosolic ribosome” in enrichment analysis. We identified many genes with multiple m6A modifications, e.g., AMIGO2 with 14 m6A sites at METTL 3 target DRACH motifs in MCF-7 cells, compared to no m6A modification in AMIGO2 in LCC9 cells. Our findings reveal distinct m6A modification patterns in ET-resistant LCC9 breast cancer cells compared to their ET-sensitive parental MCF-7 cells and in response to 4-OHT, suggesting a potential role for epitranscriptomic alterations in the development of ET resistance.

Presentation: 6/3/2024
9218 Highly Multiplexed Mapping of the Ductal Carcinoma In Situ Ecosystem to Predict Disease Outcome
Authors: Wilson Gemma M , Doan Tram B , Guild Barbara J , Pathmanathan Nirmala , Graham J Dinny
Published: October 2024

Disclosure: G.M. Wilson: None. T.B. Doan: None. B.J. Guild: None. N. Pathmanathan: None. J.D. Graham: None.

Mammographic screening programs have resulted in remarkable improvements in breast cancer survival due to early-stage detection. However, this has resulted in a dramatic increase in the detection of non-invasive ductal carcinoma in situ (DCIS), a non-obligate precursor of invasive ductal carcinoma (IDC), which is confined to the ducts and surrounded by a normal myoepithelial cell layer. Many DCIS cases will not progress to IDC within the lifetime of the patient. However, there are limited tools to accurately predict progression, thus virtually all DCIS is surgically removed, and most patients receive adjuvant radiotherapy as it halves recurrence rates. This likely represents overtreatment for a majority of patients. Emerging evidence has revealed that the adjacent microenvironment becomes progressively altered in DCIS and IDC compared to normal tissue, and this is correlated with disease outcome. The objective of this study was to investigate changes to the DCIS microenvironment during disease progression and identify disease states that predict patient outcome. We used immunohistochemistry and imaging mass cytometry with a well-annotated cohort of DCIS, with or without adjacent IDC, to measure a panel of 40 phenotypic and functional markers that allowed us to distinguish the diverse cell populations present in the tissues. We observed striking changes to almost all cell types within the DCIS tissue when comparing pure DCIS cases to DCIS adjacent to IDC. In particular, there were considerable changes to tissue-resident immune populations, with differences observed in the type, location and population size of a variety of immune cell subsets, in IDC-associated DCIS compared with DCIS-only cases. Computational mapping of cell type communities in pure and IDC-adjacent DCIS tissues revealed specific differences that could be used to predict the emergence of invasive disease, informing clinical management of this good prognosis malignancy and potentially avoiding over-treatment.

Presentation: 6/3/2024
7076 Inhibiting Oxidosqualene Cyclase, an Enzyme in the Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis of Triple-negative Breast Cancer Cells
Authors: Liang Yayun , Hyder Salman M
Published: October 2024

Disclosure: Y. Liang: None. S.M. Hyder: None.

Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 10[5] cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 10[5] MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p<0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds.

Presentation: 6/3/2024
5112 Late-Onset And Reversible Adrenal Insufficiency In Survivors Of Coronavirus Disease 2019: Results From A 24-Month Longitudinal Study
Authors: Kumar Sahoo Saroj , Menon Jayakrishnan C , Yadav Subhash B
Published: October 2024

Disclosure: S.K. Sahoo: None. J.C. Menon: None. S.B. Yadav: None.

Purpose We studied the temporal course of hypothalamic-pituitary-adrenal (HPA) dysfunction in patients with corona-virus disease (COVID19). Methods Three-hundred and two patients (median age 54 years [interquartile range 42-64], 76% males, 97 with severe/critical illness) were recruited. HPA axis was evaluated by adrenocorticotrophic hormone (ACTH)-stimulation test (APST) at admission in 243, and 12-months after discharge in 90 patients. Those with adrenal insufficiency (AI) at 12-months were further assessed by APST 6-monthly for recovery of hypoadrenalism. Results The median 0-min cortisol, peak cortisol, and ACTH during COVID19 were 295 (IQR 136-461) nmol/L, 846 (719-1088) nmol/L, and 3.9 (0.8-6.9) pmol/L respectively. AI was present in 21 (8.6%) patients. Plasma ACTH was elevated in five patients with AI, while it was low or inappropriately-normal in rest. Additionally, CIRCI was present in 22% (19/87) patients who had severe illness. At 12-months, AI was seen in 13% (12/90) patients and was hypothalamic-pituitary in origin in all. Nine (75%) of these had late-onset AI, who had normal HPA axis during COVID19. AI diagnosed at admission persisted at 12-months in three patients and resolved in six; the remaining 12 patients were lost to follow-up. The presence of AI at 12-months was independent of severity, and steroid use during COVID19. All the patients showed recovery of HPA axis in ensuing 6-12 months. A lower morning cortisol during COVID19 predicted presence of AI at one year. Conclusion AI was common during acute COVID19 and at 12-months of follow-up. AI can be late-onset, developing after recovery from COVID19. The AI was predominantly hypothalamic-pituitary in origin and resolved in the ensuing 6-12 months.

Presentation: 6/1/2024
9260 Insulin Receptor Signaling and Racial Disparities in Triple Negative Breast Cancer
Authors: Joy Engel Alexis , Samuel Krupa , Bass Ilana R , Lin Sylvia , Markella Antoniou Irini , Yagnik Radhi , LeRoith Derek , Bickell Nina A , Jane Gallagher Emily
Published: October 2024

Disclosure: A.J. Engel: None. K. Samuel: None. I.R. Bass: None. S. Lin: None. I. Antoniou: None. R. Yagnik: None. D. LeRoith: None. N.A. Bickell: None. E.J. Gallagher: Consulting Fee; Self; Novartis Pharmaceuticals.

Introduction: Obesity and type 2 diabetes are associated with poor breast cancer (BC) prognosis. These disorders, along with hyperinsulinemia are more common in Black than in White women. Black women also have higher rates of triple negative breast cancer (TNBC), and greater breast cancer mortality than White women. We hypothesized that Black women with TNBC have worse outcomes in part due to tumor promoting effects of hyperinsulinemia via the insulin receptor (IR) signaling pathway. Methods: From our IRB-approved, multi-institutional, cross-sectional, NIH-funded, study of women with newly diagnosed BC, we identified women with TNBC. We performed immunohistochemistry (IHC) analysis of formalin-fixed-paraffin-embedded tumor samples from 45 self-identified Black women and 48 self-identified White women. We quantified the BC expression of IR (n=92), IGF1R (n=92), pERK (n=91), FOXO3a (n=90) for intensity (none=0, weak=1, moderate=2, intense=3). In subsequent analysis, 0 and 1 intensity IHC staining were considered negative, and 2 and 3 were considered positive. Quantification was performed with reviewers blinded to clinical information. Clinical information (age, race, body mass index [BMI], waist circumference, fasting insulin, BC Nottingham Prognostic Index [NPI]) was evaluated after IHC scoring. Statistical proportions were compared using Fishers Exact test, and continuous variables were evaluated using parametric or non-parametric tests, as appropriate. Results: Positive IR staining was seen in 64% of samples (n=59), 74% (n=68) for IGF1R, 68% (n=62) for FOXO3a and 43.3% (n=39) for pERK. Positive IR staining was more prevalent in Black women than White women (P=0.003) but was not significantly associated with age or NPI. IGF1R, FOXO3a, pERK positive staining were not associated with race or NPI. Positive IR staining was also associated with higher BMI, waist circumference, and fasting insulin (P<0.05 for all). Positive IGF1R, FOXO3a, pERK staining were not associated with any of these factors. Conclusions: We found that 64% of TNBCs had positive expression of IR, and IR expression was more prevalent in tumors from Black women and associated with higher insulin levels, suggesting that tumors from Black women may be more sensitive to the tumor promoting effects of systemic hyperinsulinemia.

Presentation: 6/2/2024
9304 Integration of Glucocorticoid and Mineralocorticoid Receptor Signaling in Triple-negative Breast Cancer Models
Authors: Harshita Posani Sai , Lange Carol A
Published: October 2024

Disclosure: S. Posani: None. C.A. Lange: None.

Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes.

Presentation: 6/2/2024
7508 Primary Hyperparathyroidism Caused By A CDC73-related Atypical Parathyroid Tumor
Authors: Cardenas Ramiro , Tariq Sarah , Habibi Roshanak , Ebrahimi Neda , Astiazaran-Symonds Esteban
Published: October 2024

Disclosure: R. Cardenas: None. S. Tariq: None. R. Habibi: None. N. Ebrahimi: None. E. Astiazaran-Symonds: None.

Background: Primary hyperparathyroidism (PHPT) is a common endocrine disease, causes include: parathyroid adenomas (80-85%), parathyroid hyperplasia (10-15%), atypical parathyroid adenomas (APA) (1.2 %) and parathyroid carcinomas (PC) (1%). APA share many anatomic and histopathologic features with PC making the distinction between the two challenging. Although most are sporadic, some APA are caused by germline mutations in the CDC73 gene, which is associated with hyperparathyroidism-jaw tumor (HPT-JT) syndrome PC and familial isolated PHPT with APA. Clinical Case: A 42-year-old female presented to the clinic for evaluation of recurrent PHPT. She was diagnosed at age 22 when she experienced fatigue, polydipsia with hypercalcemia (12.9mg/dl). She underwent superior bilateral parathyroidectomy with pathology consistent with parathyroid adenoma. Twenty years later, symptoms recurred and tests showed hypercalcemia (13.0 mg/dl), elevated PTH (252 pg/dl), kidney function preserved (GFR: 82.5 ml/min), normal vitamin D 25 OH (31 ng/dl). Patient denied history of nephrolithiasis or fractures. Family history was positive for kidney stones in father and siblings, also history of a jaw tumor in niece. Sestamibi scan showed a left inferior parathyroid adenoma. Patient underwent resection of the adenoma; pathology revealed a left parathyroid adenoma with atypical parathyroid tumor, >95% cellularity with cytologic features concerning for malignancy, prominent macronucleoli, necrosis, elevated mitotic activity and Ki67 with 15% tumor cells without vascular invasion. Post-op Calcium and PTH normalized (9.6 mg/dl and 10.8 pg/ml, respectively). Genetic testing via multi-gene panel identified a whole gene deletion of CDC73, consistent with hyperparathyroidism-jaw tumor syndrome. Panoramic jaw x-rays did not show jaw tumors. Genetic testing for family members is underway. Conclusion: Although somatic mutations in CDC73 are frequently found in sporadic APA and PC, germline mutations are a rare cause of PHPT associated with these tumors. Histopathological examination is necessary to confirm the diagnosis of APA and to differentiate it from PC, which has a recurrence rate >50%. APA should be treated as a “tumor of uncertain malignant potential” and needs to be carefully and periodically followed up with measurement of Ca, PTH and vitamin D. Confirmation of a CDC73 mutation by genetic testing helped identify the increased risk for other associated tumors in jaw, kidneys and uterus prompting initiation of surveillance with panoramic jaw x-rays with neck shielding, renal US and pelvis US. Genetic testing of family members will allow timely detection and treatment of individuals at risk.

Presentation: 6/3/2024
7323 Prostatic Acid Phosphatase Is Negatively Regulated by Androgens and Persists in Castration-resistant Prostate Cancer
Authors: Kirschenbaum Alexander , Cheung Pamela , Yao Shen , Cheung Pamela
Published: October 2024

Disclosure: A. Kirschenbaum: None. P. Cheung: None. S. Yao: None. P. Cheung: None.

Background: Prostate cancer (PCa) is the most common cancer and second leading cause of cancer death in American men. Most patients with metastatic PCa respond initially to androgen deprivation therapy (ADT) but the majority will progress to lethal, metastatic, castrate-resistant PCa (mCRPC). The identification of markers of mCRPC that are not regulated or negatively regulated by androgens can serve as markers and therapeutic targets in mCRPC. We hypothesized that prostatic acid phosphatase (PAP), a protein phosphatase and 5’ectonucleotidase that is expressed in both the cytoplasm and transmembrane (TM-PAP) of PCa cells, is negatively regulated by androgen signaling and can serve as a therapeutic target. Methods: PAP protein expression was assessed by immunohistochemistry (IHC) in PCa cell line spheroids (VCaP, 22Rv1, LNCaP, C42B) +/- androgen addition as well as in LNCaP s.c. xenografts +/- castration. Protein expression of PAP was assessed with western blotting in PCa cell lines after treatment with androgens (DHT) and anti-androgen (enzalutamide). Results: In spheroids derived from PCa cell lines, there is some IHC expression of PAP with the strongest staining in VCaP and 22Rv1 spheroids. DHT decreased PAP IHC expression in all spheroids. LNCaP and VCaP s.c. xenografts demonstrated that castration decreased tumor growth rates but that in castrate as well as control tumors PAP expression persisted. All forms of PAP including TM-PAP are expressed in VCaP cell line and were negatively regulated by androgen addition. Conclusions: The identification of markers of mCRPC that are not regulated or are negatively regulated by androgen could help identify recalcitrant tumor cells and mount targeted delivery of therapeutic agents. In several androgen sensitive PCa cell lines, DHT addition decreased PAP expression in spheroids. In vivo, PAP expression persists after castration in spite of decreases in tumor growth rates. Finally, DHT decreases and enzalutamide restores PAP expression in vitro. These data demonstrate that PAP is a more progenitor cell marker in PCa that persists after castration and may be targeted in castration-resistant disease.

Presentation: 6/3/2024
8126 PTH-independent, Calcitriol-mediated Hypercalcemia in Renal Cell Carcinoma
Authors: Rai Gur Kiratt K , Rosairo Anne De , Madani Kulsum , Hernan Sacoto Daniel , Franco-Akel Alberto A , Belokovskaya Regina
Published: October 2024

Disclosure: G.K. Rai: None. A. De Rosairo: None. K. Madani: None. D. Sacoto: None. A.A. Franco-Akel, MD: None. R. Belokovskaya: None.

Hypercalcemia of malignancy is a known complication of advanced cancer. It occurs through various mechanisms, one of which is via 1,25-dihydroxyvitamin D (calcitriol). Calcitriol-mediated hypercalcemia accounts for approximately 1% of cases of hypercalcemia in the setting of malignancy and is usually seen in lymphomatous solid tumors. We present a gentleman with clear cell renal cell carcinoma associated with hypercalcemia with normal levels of intact parathyroid hormone (PTH), PTH-related protein (PTHrP), and 25-hydroxyvitamin D but elevated calcitriol levels. A 48 year-old-male with no significant past medical history with complaints of fatigue, hematuria, and weight loss of 38 pounds in 3 months was following urology for an elective right-sided nephrectomy. MRI of the abdomen and pelvis found a 16.1 cm renal mass with necrotic areas, causing mass effects on the liver, pancreas, and IVC. Preoperative laboratory showed hypercalcemia of 13.7 mg/dL (corrected 13.9 mg/dL) with microcytic anemia (hemoglobin 8.8 g/dL, MCV 72 fl). Workup showed low iPTH (3 pg/dL), 25-hydroxyvitamin D (16.9 ng/dL), and phosphorus level (1.7mg/dL) with elevated calcitriol (94.3 pg/dL) and a normal PTHrP. CT chest showed multiple pulmonary nodules on bilateral lobes, with the largest nodule ∼ 4.5 mm. Hypercalcemia was managed with IV fluids, calcitonin 300 units Q12 for two days, Denosumab IV infusion, prednisone 60 mg for three days, and oral ergocalciferol 50,000 units weekly. Calcium levels normalized, after which the patient underwent nephrectomy. Pathology revealed renal cell carcinoma grade G4 with mixed features of clear cell carcinoma with sarcomatoid and rhabdoid features. We report an incidental finding of hypercalcemia in a patient with renal mass. The elevated calcitriol levels can be attributed to ectopic calcitriol production through the renal mass. Post nephrectomy calcium levels remained within normal range for consecutive weeks. With this, we concur that hypercalcemia most likely originated from the renal mass that had ectopic production of calcitriol. Dysregulated calcitriol production is a well-known complication of lymphomas and granulomatous disease but is seldom reported with solid tumors. Usually, the etiology for hypercalcemia in patients with renal cell carcinoma is the overproduction of PTHrP, which was not the case with our patient. The ultimate resolution of hypercalcemia of malignancy was attained by treating the underlying malignancy.

Presentation: 6/1/2024
8743 Regulation of Tryptophan 2,3-Dioxygenase 2 and Androgen Receptor in Triple-Negative Breast Cancer
Authors: Kuo Li-Wei , Lacey Kimaya , Spoelstra Nicole , Richer Jennifer K
Published: October 2024

Disclosure: L. Kuo: None. K. Lacey: None. N. Spoelstra: None. J.K. Richer: None.

Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype with a peak recurrence rate within 3-5 years after initial diagnosis. Androgen receptor (AR) and Tryptophan 2,3-dioxygenase 2 (TDO2) are elevated in TNBC cells grown under anchorage-independent conditions and facilitate anoikis resistance. Several studies reported that the luminal AR subset of TNBC showed a positive enrichment of the tryptophan (TRP) catabolism pathway gene signature. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) inhibition decreases AR promoter transcriptional activity in prostate cancer. We have observed that TNBC with high AR have high levels of genes/proteins in the tryptophan catabolism pathway including the rate limiting enzyme TDO2. Hypothesis: TDO2 activity regulates AR expression through the downstream tryptophan catabolite kynurenine (KYN), which binds and activates AhR and AhR and AR may work together to promote TNBC progression. Methods: Human TNBC cell lines: MDA-MB-453, BT549, SUM159PT ​ were used in this study and the expression of TDO2 and AR were examined by immunohistochemistry (IHC) and western blot. TDO2 was stably knocked down (KD) via shRNA or overexpressed (OE) using lentivirus transduction. Mass spectrometry-based metabolomics were used to measure changes in TRP catabolite levels, and a cytokine array (R&D) was utilized to analyze conditioned media. Results: TDO2 and AR protein are positively correlated in human TNBC cell lines. Although overexpression of TDO2 did not alter AR protein expression in SUM159PT, in the human MDA-MB-453, which have high levels of AR, knockdown of TDO2 (shTDO2) reduced AR protein by 40% and production of kynurenine (KYN) was reduced by 50% (p<0.0001). This suggests that TDO2 activity, specifically KYN binding to AhR, may influence AR protein levels. Conditioned media (CM) from TDO2 OE and KD respectively increased and decreased CXCL5 and IGFBP2. Lastly, TCGA database analysis of TNBC tumors with high levels of both AhR and AR showed significantly shorter patient survival (HR=1.47, log rank p=0.046) compared to those with tumors with lower amounts of both genes. Conclusion: TDO2 and AR are positively correlated in human TNBC cell lines. Tumors with high levels of both AR and AhR result in significantly shorter survival time for TNBC patients. Knockdown of TDO2 significantly reduced KYN production and AR expression in luminal AR TNBC cell lines and future experiments will focus on the underlying mechanism behind this and whether it is dependent on KYN-mediated activation of AhR and if AhR stabilizes AR in TNBC.

Presentation: 6/3/2024
7036 Luteinizing Hormone/Human Chorionic Gonadotropin Receptor And Cyclooxygenase-2 Expression In Adrenocortical Carcinoma
Authors: Sheskier Rachel , Kirschenbaum Alexander , Zappi Isabella , Yao Shen , Levine Alice C
Published: October 2024

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
7822 Mild Autonomous Cortisol Secretion In High Suspicious Adrenal Incidentaloma
Authors: Zavrashvili Nino , Margvelashvili Natia , Chanturishvili Ketevan
Published: October 2024

Disclosure: N. Zavrashvili: None. N. Margvelashvili: None. K. Chanturishvili: None.

Mild Autonomous Cortisol Secretion in High Suspicious Adrenal IncidentalomaIntroduction: Incidentally discovered adrenal masses are common and mostly benign and non-functioning adenomas. However, evolving evidence suggests that a notable proportion of these adrenal adenomas may demonstrate mild autonomous cortisol secretion (MACS), which has been associated with an increased risk for hypertension, hyperglycemia, obesity, dyslipidemia, vertebral fractures, adverse cardiovascular events, and mortality. 55-year-old women with 5-year history of hypertension and hyperlipidemia. She was treated with a three-drug program: verapamil 250 mg daily, combined B-blocker, atenolol 100 mg and chlorthalidone 25 mg daily. Blood pressure control remained suboptimal. Her body mass index was 29,5 kg/m2. She had no signs or symptoms of overt Cushing syndrome.She was referred to CT coronary angiogram by her cardiologist, which revealed adrenal incidentaloma.Adrenal dedicated CT showed left-sided 3,4 cm high density node (41HU), with absolute washout <60. She was consulted by oncologist to exclude adrenal metastasis. The baseline laboratory test results excluded PA and Pheochromocytoma. The diagnosis of glucocorticoid secretory autonomy was based on positive low-dose dexamethasone suppression test (DST), low serum ACTH concentration, low-normal DHEA-S and lack of complete suppression of serum cortisol with an overnight 8-mg DST. Patient was referred to left-sided laparoscopic adrenalectomy with perioperative glucocorticoid coverage (100 mg hydrocortisone administered IV and again 8 hours later). Conclusion: Mild autonomous cortisol secretion is a term used to describe biochemical evidence of abnormal cortisol secretion in patients with adrenal incidentaloma, but without the classical external manifestations of overt CS, such as central muscle weakness, adipose tissue redistribution, and skin fragility. The management of patients with an incidentally discovered adrenal adenoma who do not have overt Cushing’s syndrome but who have MACS is more controversial as the available studies comparing outcomes of adrenalectomy vs conservative management have been generally small. It is associated with an increased risk of Type 2 diabetes mellitus, HBP, obesity, dyslipidemia, and global cardiovascular risk. Therefore, it is of great importance to correctly identify these patients. There are different tests for the screening of hypercortisolism. However, in the absence of a single test considered as the gold standard, it is necessary to combine the different tests to reach an adequate diagnostic capacity. The indication of surgery in ACS patients should be based mainly on the hormonal evaluation and the comorbidities potentially mediated by cortisol.

Presentation: 6/1/2024
7888 Mitotane Induces Different Modes Of Cell Death In Treatment Resistant And Sensitive Models Of Adrenocortical Carcinoma
Authors: Feely Sarah , Mullen Nathan , Hong Cong , Hantel Constanze , Rainey William E , Dennedy Michael C
Published: October 2024

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>0.0001), thereby demonstrating treatment-sensitivity. In HAC15 cells, a supratherapeutic dose of 200uM was needed to significantly reduce viability (p>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
9266 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing
Authors: Hye Kim Ja , Park Soyoung , Yoon Ji-Hee , Kim Dohyung , Hwang Soojin , Kim Gu-Hwan , Kim Jun , Choi Jin-Ho , Yoo Han-Wook
Published: October 2024

Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None.

Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome.

Presentation: 6/1/2024
7030 Molecular Analysis Of Congenital Adrenal Hyperplasia-X Syndrome And Development Of A Pilot Panel For Analyzing Structural Variations With Long-Read Sequencing
Authors: Kim J , Park S , Yoon J H , Kim D , Hwang S , Kim G H , Kim J , Choi J H , Yoo H W
Published: October 2024

Disclosure: J. Kim: None. S. Park: None. J. Yoon: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None.

Purpose: Genomic recombination events between the CYP21A2 and CYP21A1P genes are frequent due to their high sequence homology. Deletions in both the CYP21A2 and TNXB genes result in a chimeric TNXA/TNXB gene, leading to congenital adrenal hyperplasia (CAH)-X syndrome, characterized by a hypermobile form of Ehlers-Danlos syndrome. The aim of study was to determine the frequency of CAH-X syndrome and to develop a pilot panel for structural variation analysis using long-read sequencing technology. Methods: Among the 165 unrelated families with confirmed genetic mutations in the CYP21A2 gene, 44 probands had large deletions in one or both alleles. Long-range PCR was performed in 12 patients out of 44 probands using a forward primer for the 5’-UTR of the CYP21A2 and CYP21A1P and a reverse primer specific for exon 32 of TNXB. The amplified PCR products were then subjected to Sanger sequencing. We constructed a custom panel of 140 kb containing the RCCX modules arranged as STK19-C4A-CYP21A1P-TNXA-STK19B-C4B-CYP21A2-TNXB. In 4 patients with deletions in both the CYP21A2 and TNXB genes, long-read sequencing was performed using the custom panel on the PacBio Sequel II platform. Results: Out of 12 probands, eight harbored TNXA/TNXB chimeras. One patient was homozygous for CAH-X CH1 with a 120 bp deletion in exon 35 and had a Beighton score of 6 for joint hypermobility and multiple diverticula in the ascending colon. Two patients were heterozygous for CAH-X CH1, while three were heterozygous for the CAH-X CH2 with an intact exon 35 and the c.P4058W in exon 40. Two patients carried heterozygous mutations p.S4175N in exon 43, which is an unclassified type. Using long-read sequencing with a custom panel, missense mutations and TNXA/TNXB chimeras were detected in four patients previously identified by conventional direct sequencing and MLPA. Conclusions: This study identified 8 patients with CAH-X syndrome carrying four different TNXA/TNXB chimeras by long-range PCR. Long-read sequencing emerged as a comprehensive and efficient method for the molecular analysis of CAH, especially for the identification of structural variations. Biallelic deletion of TNXB was associated with severe clinical manifestations. Therefore, screening for CAH-X is essential for clinical management and prevention of the long-term consequences of CAH-X syndrome.

Presentation: 6/1/2024