bluuebunny/crossref_metadata_2025 huggingface dataset:
Do Not Miss MEN 1 Screening When Patients Are Newly Diagnosed With Pancreatic Neuroendocrine Tumor
Authors: Bai Ravisha , Izuora Kenneth E
Published: May 2021

Background: Multiple endocrine neoplasia type 1(MEN 1) is a rare genetic disorder characterized by the combined occurrence of parathyroid, pituitary, and duodeno-pancreatic neuroendocrine tumors (NETs) along with other characteristic tumors. Here, we present a patient with history of pancreatic NET who presented 2 years after her initial diagnosis with primary hyperparathyroidism and prolactinoma, consistent with MEN 1.

Case Report: A 60-year-old African American woman was admitted to our hospital with nausea, vomiting and headache for a week. On presentation, she was found to be hypotensive and tachycardic, both responded to IV fluids. Workup for infectious etiology came back negative. Her physical exam was benign except for dry oral mucosa, dry skin and epigastric tenderness. Laboratory evaluation showed serum calcium 11.8 mg/dL (8.4 - 10.2 mg/dL), which prompted further work up. Her PTH level was 1,079 pg/ml (8.5 - 72.5 pg/ml), Phosphorus 2.1 mg/dL (2.3 - 4.7 mg/dL), and 25 Hydroxy Vitamin D 10.2 ng/ml (7.4 - 44.0 ng/ml). Additional history and review of her records revealed she was hospitalized 2 years earlier with nausea, vomiting and abdominal pain. Then she was found to have esophageal and gastric ulcers on EGD. Endoscopic ultrasound revealed an 8 mm hypoechoic lesion in the body of pancreas found to be a pancreatic NET on biopsy. She had elevated gastrin level of 470 pg/ml (0 - 115 pg/ml) and was diagnosed with a gastrinoma. She was discharged on a PPI and was lost to follow up. Patient had no significant family history. Given her past history and current presentation, we assessed her pituitary hormonal function which was unremarkable except for prolactin level of >1,000 ng/ml (post-menopausal 1.8 - 20.3 ng/ml). Pituitary MRI showed a 1.8 cm macroadenoma. The optic nerve and optic chiasm were normal. Based on her clinical presentation and radiologic findings, she was diagnosed with MEN 1. Her Calcium level trended down with IV hydration, and her presenting symptoms resolved. She was started on bromocriptine, pantoprazole and vitamin D and was discharged with out-patient appointments scheduled with gastroenterology and endocrinology clinics.

Conclusion: This case highlights the importance of having a high index of suspicion to prompt screening for MEN 1 in patients newly diagnosed with a pancreatic NET.

Early diagnosis and timely treatment of the associated disease conditions in MEN 1 can prevent significant morbidity and mortality associated with these conditions.
Hypoglycemia, Something Lost in Translation
Authors: Al-Naqeeb Ghadah , Neupane Rabin , Baral Neelam , Gao Faye , Steves Mark , Wexler Jason Adam
Published: May 2021

Introduction: Insulinoma is a rare neuroendocrine tumor that may occur sporadically or as part of multiple neuroendocrine neoplasia type 1, with an incidence of 4 per million per year. Given the nonspecific symptoms of hypoglycemia, the diagnosis of insulinoma may be delayed by months, years or even decades. Case Presentation: A 50 year-old Hispanic, Spanish speaking male with a history of hypertension presented with progressive epigastric pain, nausea and bilious vomiting for 2 days. Physical exam was notable for abdominal distention and tenderness. Initial labs showed white blood cells of 19.4 k/uL (4-10.8), glucose of 85 mg/dl (65-140). CT abdomen showed findings consistent with moderate grade small bowel obstruction and normal pancreas. The patient was managed conservatively with nasogastric tube placement and nothing per oral (NPO). Within 8 hours started experience sweating, restlessness, fingerstick glucose was 40 mg/dl. Upon questioning, he reported episodes of lightheadedness, sweating and shakiness 8 years prior to this hospitalization, and he recalled being told that he was hypoglycemic during prior hospitalizations at another institution. He stated he was told to drink juice upon experiencing such symptoms and no work up or diagnostic investigation was recommended. We initiated a hypoglycemia investigation when he was NPO, when serum glucose level of 40 mg/dl. Labs showed insulin level 34 uIU/mL (3-19), free insulin 25 uIU/mL (3-19), C-peptide 3.32 ng/mL (0.81-3.85), beta hydroxybutyrate <0.02 mmol/L (0.02-0.27) and proinsulin of 36.6 pmol/L (3-50) and hemoglobin a1C of 5% (3.8-5.6). Serum screening for hypoglycemic agents was negative. When MRI abdomen was negative for a pancreatic mass. We proceeded with advanced diagnostic imaging with endoscopic ultrasound (EUS) which showed a 26 mm x 19 mm ovoid, hypoechoic mass at the junction of the pancreatic body and tail. Tissue specimen obtained with fine needle aspiration showed grade 1 neuroendocrine neoplasm. The patient underwent distal pancreatectomy and splenectomy with suture repair of the small bowel and left wedge liver biopsy. The pathology showed a well-differentiated neuroendocrine tumor of the pancreas, positive for synaptophysin, chromogranin and CD56 and Ki-67 with proliferation index <2% and no evidence of metastasis to the liver. The patient’s hypoglycemia resolved following surgery and rose to > 200 mg/dL requiring us to begin insulin therapy during the hospital stay before ultimately discharging the patient on metformin for type 2 diabetes. Conclusion: Insulinoma is a rare but treatable neuroendocrine tumor. Early diagnosis is important to avoid adverse clinical events related to hypoglycemia. It is important to maintain a strong clinical suspicion in order to identify and pursue the cause of unexplained hypoglycemia to improve the quality of life of patients with this rare condition.
Metastatic Non-Functional Pancreatic Neuroendocrine Tumor in a Patient With a Pathogenic TSC1 Mutation
Authors: Sliwinska Aleksandra , McFadden David , Malchoff Carl D
Published: May 2021

Introduction: Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem disorder caused by mutations in two tumor suppressor genes (TSC1 and TSC2) encoding proteins critical in cell growth and proliferation and is associated with an increased risk for benign and malignant tumors. Pancreatic neuroendocrine tumors (PNET) occur in 1.5-1.8% of patients with TSC. They have been primarily reported in TSC2, with only a few cases in the context of TSC1. We describe a non-functional metastatic PNET in a patient with a known pathogenic TSC1 mutation.

Case Description: A 49-year old female with a past medical history of bipolar disease, a family history of TSC1 in a daughter, a known pathologic TSC1 mutation (c.2356C>T), and previous history of renal cell carcinoma (RCC) was evaluated for a pancreatic lesion with likely liver metastases. Two years prior to the presentation, a 10 mm pancreatic lesion was initially noted on the CT scan during a renal mass evaluation. Nephrectomy confirmed the RCC. Two years later, surveillance imaging revealed two new lesions in the liver, and the pancreatic lesion had enlarged to 1.3 cm. She denied symptoms suggestive of a functional PNET, and her physical exam was unremarkable. Gallium-68 dotatate (GA-68) PET/CT scan demonstrated avid lesions of both the pancreatic tail and left hepatic lobe. Biopsy of the 3 cm liver mass revealed a metastatic high-grade neuroendocrine carcinoma (mib-1 proliferation index of 15%) favoring a primary PNET. Immunohistochemical stains were positive for chromogranin A, synaptophysin, EMA, PAX 8, CK 20, and villin. Stains for CA 19-9 and CK 7 were negative. Biochemical testing suggested that the PNET was non-functional. Specifically, serum concentrations of gastrin, somatostatin, glucagon, glucose, C-peptide, proinsulin, pancreatic polypeptide, chromogranin A, and vascular endothelial growth factor were within normal limits. The patient underwent distal pancreatectomy and resection of the liver mass. Pathological specimens confirmed the diagnosis of a PNET. The patient remained asymptomatic and was monitored regularly with MRI and GA-68 PET/CT scans without further evidence of the disease for 1.5 years. Conclusions: We conclude that this patient harboring a c.2356C>T TSC1 mutation developed a non-functional metastatic PNET. PNET is a likely component of TSC1.

References: 1.Lodish, M. B., & Stratakis, C. A. (2010). Endocrine tumors in neurofibromatosis type 1, tuberous sclerosis, and related syndromes. Best practice & research Clinical endocrinology & metabolism, 24(3), 439-449.2.Dworakowska, D., & Grossman, A. B. (2009). Are neuroendocrine tumors a feature of tuberous sclerosis? A systematic review. Endocrine-Related Cancer, 16(1), 45.
The Diagnosis and Management of a Rare ACTH-Producing Neuroendocrine Tumor Causing Cushing’s Syndrome
Authors: Findlay Ryan , Gray Daryl , Shepherd Jessica , Joseph Mariamma , Hussain Nadeem , Laidley David , Van Uum Stan
Published: May 2021

A 57-year-old woman presented with a three-month history of worsening lethargy, weight gain, central adiposity, proximal muscle weakness, and hypokalemic hypertension. Initial investigations were consistent with ACTH-dependent Cushing’s Syndrome. 24-hour urine cortisol: 711nmol/day (normal<275), cortisol after 1 mg dexamethasone suppression test 960nmol/L (normal<50)), 2-day dexamethasone suppression cortisol: 575 and ACTH: 19 pmol/L. MRI sella was normal. She required several hospital admissions for worsening proximal muscle weakness and falls. She was started on ketoconazole and spironolactone. Basal insulin was started for dysglycemia. Petrosal vein sampling indicated ectopic ACTH production. CT scan of chest/abdomen/pelvis showed no intrathoracic or intra-abdominal abnormality. Thyroid ultrasound and whole-body octreotide scan showed no abnormalities. A Gallium 68-dotatate PET scan showed focal tracer uptake in the pancreatic tail suspicious for a well-differentiated neuroendocrine tumor (NET). MRI pancreas showed a slightly truncated pancreatic tail with no discrete lesion. We proceeded with Endoscopic Ultrasound (EUS) which revealed an irregular hypoechoic mass in the pancreatic tail. EUS–guided fine-needle biopsy (EUS-FNB) using a 22G SharkCore needle, revealed a neuroendocrine neoplasm staining positively for ACTH. She underwent distal pancreatectomy with pathology showing a 1.4cm well-differentiated NET, staining positive for ACTH with a Ki67 index of 10%. Postoperatively, her ketoconazole, spironolactone, and insulin were discontinued. Her dysglycemia, hypertension and other clinical symptoms gradually improved. Immediately post-operatively, her AM cortisol was 403 and her 24-hour urine cortisol was elevated 959nmol/day. Repeat AM cortisol and 24-hour urine cortisol one-month post-surgery were 392 nmol/L and 72 nmol/day respectively, confirming biochemical remission. Discussion: ACTH-producing pancreatic NETs are rare, accounting for less than 15% of reported cases of EAS. They are often aggressive, with 78.7% of reported cases having distant metastasis at diagnosis (1). They are associated with significant morbidity and mortality. Diagnosis and management can be difficult and require multiple investigations and multidisciplinary team involvement. In our case, we utilized endoscopic ultrasound to ensure a correct preoperative localization. We discuss the systematic approach for diagnosis and the management of an ACTH-producing pancreatic NET. References: 1. Maragliano R, Vanoli A, Albarello L, Milione M, Basturk O, et al. ACTH-secreting pancreatic neoplasms associated with Cushing syndrome: clinicopathologic study of 11 cases and review of the literature. Am J Surg Pathol. 2015 Mar;39(3):374-82. doi: 10.1097/PAS.0000000000000340. PMID: 25353285.
Chemically-Modified Tetraiodothyroacetic Acid (Tetrac) Induces Cancer Cell Apoptosis and Facilitates Clearance of Apoptotic Debris (Efferocytosis)
Authors: Godugu Kavitha , Lin Hung-yun , Mousa Shaker A , Davis Paul J
Published: May 2021

Tetraiodothyroaetic acid (tetrac) is a derivative of L-thyroxine with anticancer properties. By multiple molecular mechanisms, tetrac and chemically-modified tetrac induce apoptosis in a variety of human cancer cells in vitro and in xenografts. The anticancer activities of tetrac are initiated at the thyroid hormone analogue receptor on the extracellular domain of plasma membrane integrin αvβ3 (PJ Davis et al., Physiol Rev 101:319-352, 2021). Induction of apoptosis in glioblastoma xenograft with chemically modified tetrac (P-bi-TAT) has yielded 90% in volume of grafts that continues after discontinuation of tetrac. In the present study, we show that human glioblastoma xenograft shrinkage in response to P-bi-TAT is associated with local appearance of phagocytic monocytes and clearance of apoptotic debris (efferocytosis). Primary culture xenograft of glioblastoma cells (GBM 052814, kindly provided by the University of Pittsburgh Medical Center, Department of Neurosurgery) and U87-luc (ATCC, Manassas, VA) xenografts were generated in 5-member groups of nude mice for each tumor cell type and for controls. Five days post-implantation, injection of animals was begun with PBS (control) or P-bi-TAT (10 mg/kg body weight). Injection was continued X21 days and animals were then maintained off-treatment for an additional 21 days. Tumors were harvested, formalin-fixed and slide-mounted, then analyzed by TUNEL assay for apoptosis and by anti-CD68 staining for monocytic macrophage content. Histologic analysis (H&E staining) was also carried out. TUNEL analysis and histopathology of both xenograft models revealed more than 90% apoptotic change with 21-days of P-bi-TAT treatment (P <0.001) and persistence of 40% apoptotic change 3 weeks post-discontinuation of drug (P<0.001 vs. end of treatment change). By H&E histology and CD68 analysis, monocytes accounted for more than 90% of the viable cells after 3 weeks’ drug treatment. Sixty percent of the end-of-treatment monocyte population persisted 3 weeks after discontinuation of P-bi-TAT (P <0.001). Histology revealed negligible cell debris after 3 weeks of drug treatment and at 3 weeks post-discontinuation of P-bi-TAT. Thus, the anticancer/pro-apoptotic action of tetrac-containing P-bi-TAT is associated with efferocytosis that contributes to the frank tumor shrinkage that results from P-bi-TAT treatment of human glioblastoma xenografts. This is the first documentation of efferocytosis regulated from the thyroid hormone analogue receptor on tumor cell integrin αvβ3.
Cholecalciferol Mediates Apoptosis in Siha Cervical Cancer Line via Autocrine Mechanisms
Authors: Bhoora Sachin , Pillay Tahir S , Punchoo Rivak
Published: May 2021

Cervical cancer disproportionately affects low-resource countries and is a significant health burden in South Africa. Pre-clinical studies have demonstrated numerous anti-cancer actions of vitamin D metabolites. Here, the anti-cancer action of the vitamin D precursor, cholecalciferol, was investigated in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Apoptotic cell death was investigated by flow cytometry, which measured mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and the expression of DNA damage markers. Additionally, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis. Expression of the vitamin D metabolising system (VDMS) – consisting of cholecalciferol activating (CYP2R1 and CYP27A1), calcidiol activating (CYP27B1) and calcidiol inactivating (CYP24A1) enzymes, and the vitamin D receptor (VDR) – was assessed by qPCR and Western blots. Data were analysed using a one-way ANOVA and Bonferroni post-hoc tests and p < 0.05 was considered statistically significant. Significant decreases in cell count (p = 0.011) and cell viability (p < 0.0001) were identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, biochemical markers at 2600 nM treatment were significant for apoptosis, and included decreased ∆Ψm (p = 0.0145); increased PS externalisation (p = 0.0439); terminal caspase activation (p = 0.0025); and nuclear damage (p = 0.004). Moreover, biochemical apoptosis was corroborated by classical apoptotic features observed by brightfield microscopy and TEM. Additionally, a significant increase in CYP2R1 gene (p < 0.0001) and protein (p = 0.021) expression, and a converse significant decrease in CYP27B1 gene (p = 0.003) and protein expression (p = 0.031) were observed at 2600 nM cholecalciferol treatment. Furthermore, significant increases in VDR gene (p = 0.033) and protein (p = 0.04) expression, and CYP24A1 gene (p < 0.0001) and protein (p = 0.0274) expression were observed at 2600 nM cholecalciferol. In summary, high-dose cholecalciferol treatment of SiHa cervical cancer cells inhibits cell growth, induces apoptosis, and furthermore, upregulates CYP2R1 and VDR expression. Taken together, these findings suggest that autocrine activation of cholecalciferol to calcidiol may mediate VDR signalling of cell growth inhibition, and apoptosis in SiHa experimental cultures.
Comprehensive Analysis of Clinical Features in Index Cases With Multiple Endocrine Neoplasia Type 1 Refine the Risk Rate for Detection of Mutation Distinguishing Negative-Mutation (Phenocopies) and Positive-Mutation Cases
Authors: Urtremari Betsaida , Carvalho Rafael A , Santana Lucas S , Antonio Louise V , Ribeiro Flavio G , Lugeiro Palloma C , Quedas Elisangela P S , Lourenco Jr Delmar M
Published: May 2021

Background: Index cases clinically diagnosed with multiple endocrine neoplasia type 1 (MEN1) are declared as MEN1 phenocopies if no germline MEN1 mutation is identified. In comparison with positive-mutation cases, most phenocopies have been diagnosed in older age, mainly by association of primary hyperparathyroidism (HPT) and pituitary adenoma (PIT), with HPT predominantly diagnosed as uniglandular disease. Besides that, phenocopies rarely develop a third MEN1-related tumor and are associated with lower morbidity and longer survival. However, all these data are mainly derived of genetic studies by Sanger targeted to MEN1 gene from a strict number of MEN1 phenocopies. Objectives: to recognize strong clinical profiles capable of predicting the occurrence or absence of germline MEN1 mutation refining the clinical differentiation of phenocopies and mutation-positive cases before genetic testing disclosure. Casuistic/Methods: 143 MEN1 index cases: 87 MEN1-positive and 56 true MEN1 phenocopies (excluded mutations for MLPA assay and by a mini-painel based on long-range PCR and next generation sequencing of 6 MEN1-related genes (MEN1, p15, p18, p21, p27, AIP) covering full coding and non-coding regions. Results: High detectability rate of MEN1 mutation was associated with the presence of ≥ 4 organs affected for primary tumors (100%), association of HPT/PET (neuroendocrine pancreatic tumor)/PIT (93%), HPT/PET (81%), positive familial history (88% vs. 48%), presence of PET (84%) as malign (80%) as multifocal (95%), two different PETs (100%), multiglandular HPT (81%) and diagnosis of one MEN1-related tumor (93%) or of two/three MEN1 tumors diagnosed before 21y (100%). The combination HPT/PIT has the lowest rate of detection of mutation (33%), it is even lower if PIT was acromegaly (12%) or age at the diagnosis of HPT and PIT was, respectively, > 45y and >30y (8%) and absent if it is added uniglandular HPT (0%) or if there was association HPT/PIT (age-independent) with uniglandular HPT (0%). The prediction for detection of mutation increases if these HPT (> 45y)/PIT (> 30y) cases have multiglandular HPT (20%) and it is 100% with association HPT (< 30y)/PIT (< 21y). A p <0.05 was observed to all data above. Conclusions: By integration of phenotypic clues and full genetic analysis applied to the largest MEN1 phenocopy series, we identified strong clinical predictors capable of anticipate the potential risk rate for mutation detection revealing the estimated chance of an index case harbor a mutation or be classified as phenocopy. By their peculiarities, the management/treatment of phenocopies should potentially be different of that recommended to mutation-positive cases.
Functional Characterization of the Orphan Nuclear Receptor TLX in Triple Negative Breast Cancer
Authors: Nelson Adam , Wang Yu , Ma Liqian , He Sisi , Henn Madeline , Shahoei Sayyed Hamed , Saleh Tareq , Carpenter Valerie , Gewirtz David , Spinella Michael J , Nelson Erik Russell
Published: May 2021

Despite the development of various therapeutic strategies, breast cancer persists as the second leading cause of cancer-related death among women in the United States. While endocrine modulation and monoclonal antibody therapy have proved to be indispensable modes of intervention for hormone receptor (HR)-positive and HER-2 positive patients, the triple negative breast cancer (TNBC) patient population do not respond to these therapies. As TNBC is considered one of the most challenging subtypes of breast cancer to treat, there is a significant need for the development of targeted therapeutics. Due to their well-known amenability to small-molecule modulation, we investigated whether any nuclear receptors beyond those that are traditionally studied in breast cancer (e.g. ER, PR, and AR), may represent a novel target in the TNBC patient population. Analysis of clinical data revealed that expression of the orphan nuclear receptor TLX (NR2E1) was positively correlated with relapse-free survival, distant metastasis-free survival, and overall survival in both ER-negative and basal-like breast cancer patients. Therefore, we hypothesized that TLX could influence the pathophysiology of TNBC. To interrogate this hypothesis, we established TNBC cells with stable expression of TLX in order to identify direct regulatory targets, as well as the precise physiological mechanism(s) TLX may be regulating. To date, our work has revealed that TLX inhibits proliferation, slows migration, alters chemosensitivity, and impairs cell cycle progression in TNBC cells. In agreement with these findings, our work has also revealed that TLX is capable of modulating the expression of several genes that are known to regulate the processes of growth, migration, and cell cycle. Taken together, our early work supports our hypothesis, and provides valuable insight into the potential pro-survival function of TLX in TNBC. Ongoing work will continue to probe the mechanisms by which TLX impacts breast cancer biology, and establish whether the growth-inhibitory effects translate to in vivo models. As prior work has demonstrated that TLX’s transcriptional activity can be regulated by both synthetic and natural ligands, the results of our work would provide the foundational data necessary for the development of a TLX-based therapy for a patient population with limited therapeutic options and a poor prognostic outlook.
A Multifocal Paraganglioma in an African Male Due to an Underlying SDHB Mutation
Authors: Siddiqui Nida Mishraz , Seedat Faheem , Bulbulia Saajidah , Krause Amanda , Daya Reyna , Bayat Zaheer
Published: May 2021

Pheochromocytomas and paragangliomas (PPGL) are rare neuroendocrine tumors arising from adrenal and extra-adrenal paraganglia. Up to forty percent are due to an underlying germline mutation. Mutations in the subunit B gene of the SDH complex (SDHB) are associated with PGL syndrome four. A 34-yr-old African man from Southern Africa presented with a two-year history of sustained hypertension associated with the classic triad of sweating, headaches and palpitations. Family history was contributory towards early unexpected deaths of his father (age 42) and two younger brothers (ages 13 and 14 respectively). On examination his blood pressure was persistently elevated measuring 146/87mmHg. In view of the classic presenting symptoms and hypertension onset at a young age, a PPGL was suspected. Biochemical investigations were positive with an elevated 24-hour urine normetanephrine level of 35807 (480-2424nmol/24hours), normal metanephrine level of 689 (264-1729nmol/24hours), an elevated normetanephrine:creatinine ratio of 3270 (28-158nmol/mmol creatinine) and an elevated methoxytyramine level 4941.69nmol/24 hours (<800nmol/24 hours). Computed tomography of the abdomen and neck revealed a homogenous soft tissue mass measuring 5.9cm x 3.6cm x 6.6cm anterior to the right kidney and separate from the right adrenal gland and a carotid body tumor measuring 3.6cm x 2.9cm x 4.1cm. Both were radio-avid on a [68Gallium]-DOTATATE-Positron Emission Tomography (PET)-CT. There were no features to suggest metastatic disease. Genetic testing is not available in South Africa; therefore, testing was done at an international laboratory. This revealed a pathological SDHB mutation variant, c.724C>A p.(Arg242Ser) and hence PGL4 syndrome. The patient underwent staged surgery with successful removal of the intra-abdominal tumor. Unfortunately, due to peri-operative complications associated with the second surgery, the patient demised. Histopathological examination of both tumors was consistent with a paraganglioma. Genetic counselling and testing were offered to all living first-degree relatives. His sister tested positive for the same pathological variant. His 6-week-old son will be offered counselling and testing at a later stage. To the best of our knowledge, we are the first to describe the missense SDHB mutation (pathogenic variant c.724C>A p.[Arg242Ser]) and the occurrence of an SDHB associated PGL in a family of African ethnicity. This case highlights the importance of genetic counselling and testing in patients with a confirmed PPGL. Due to resource limitation the African population remains under represented in genetic studies which limits the utility of precision medicine in this group. As such, our case is an important addition to the body of knowledge in this growing field and highlights the need for cost effective genetic screening tools in low resourced settings.
Hormonal Regulation of Semaphorin 7a Promotes Therapeutic Resistance in Breast Cancer
Authors: Crump Lyndsey , Richer Jennifer K , Porter Weston , Lyons Traci
Published: May 2021

Background: The majority of all breast cancers (BC) are estrogen receptor positive (ER+). While ER-targeting endocrine therapies have improved patient survival, many of these tumors develop drug resistance and recur within 20 years. Therefore, novel targets are needed to predict for recurrence and to treat recurrent ER+BC. Previous reports describe a tumor-promotional role for Semaphorin 7A (SEMA7A) in ER- disease; yet, the role of SEMA7A in ER+ disease is poorly characterized. Hypothesis: SEMA7A promotes cell survival and drug resistance in ER+ BC. Methods: We overexpressed SEMA7A in ER+ BC cells, then used the ER-targeting agents tamoxifen and fulvestrant to test how SEMA7A-expressing cells respond to endocrine therapy. In vitro, we used proliferation and cell survival assays. In vivo, we implanted ER+ BC cells, then treated the animals with fulvestrant to measure how SEMA7A affects tumor growth and metastasis. We also utilized drug resistant cells, which have high endogenous SEMA7A levels, to measure markers of stemness and multi-drug resistance via flow cytometry. Results: We first found that SEMA7A expression correlates with decreased relapse free survival in patients with ER+BC who received endocrine therapy (Kmplotter; p=0.042). We also observe that SEMA7A is hormonally regulated in ER+BC, but its expression does not uniformly decrease with endocrine therapy agents. Instead, long term estrogen deprivation and ER-targeting drug treatments increase SEMA7A expression, likely through the action of other hormone receptors such as the androgen receptor, which also increases with long term estrogen deprivation. Further, in ER+ cell lines, overexpression of SEMA7A promotes in vitro growth in the face of estrogen-deprivation, tamoxifen, or fulvestrant treatments. In vivo, SEMA7A promotes fulvestrant resistance in the primary tumor and induces lung metastases. Finally, we report that pro-survival signaling is a therapeutic vulnerability of ER+SEMA7A+ tumors. Conclusion: These studies describe that SEMA7A promotes drug resistance in ER+ BC. We propose that targeting pro-survival signaling may prove efficacious for treating SEMA7A+ tumors, which are less likely to respond to endocrine therapies.
Incidence and Phenotypes of Endocrinopathies in Association With Immune Checkpoint Inhibitors: The Experience of Integrated Health Network
Authors: Mehmood Hassan , Hasan Farhad
Published: May 2021

Introduction: Since 2011 FDA has approved several immune checkpoint inhibitors (ICI) as antineoplastic agents with very promising results. The side effects profile of ICI is different from that of conventional cancer therapies and predominated by immune-related adverse effects (IRAEs). Autoimmune endocrinopathies (AEs), such as disorders of the thyroid, pituitary, and adrenal glands are increasingly recognized IRAEs of ICI. If not diagnosed and treated early, AEs may cause serious harm in an already compromised patient by advanced malignancy. Therefore, it is necessary to recognize the incidence, phenotypes, and time to onset of various AEs in association with ICI. This retrospective study of a large cancer population treated at an integrated health network aims to do so. Methods: We conducted a retrospective chart review of adult patients treated with ICI as cancer therapy at Allegheny Health Network—a major healthcare provider in Western Pennsylvania—between 1/1/2016 and 6/30/2019. Inclusion criteria were ages 18-90 years who received one or more ICI within the study period, laboratory data available for a minimum of 6 months from date of the first ICI dose, and no pre-existing outcome of interest at the time of, or prior to, receiving the first ICI dose. Exclusion criteria were pre-existing thyroid, pituitary, or adrenal diseases. Results: We screened the records of 1200 patients who were treated for 47 different cancers for AEs. Of this sample, 57% were males 92.5% were White. Follow up period ranged between 6-57 months, during which 56% of patient died at a mean age of 67.1 years. The most common cancer diagnoses were non-small cell lung cancer (45.4%), malignant melanoma (11.7%), renal cell carcinoma (6.3%), and small cell lung cancer (6.2%). These patients received one or more agents from the three classes of ICI, including PD-1 (75.86%), PDL-1 (12.57%), and CTLA-4 (11.57%). The overall incidence of AEs was 18%. The distribution of AEs was as follows: primary hypothyroidism 15.3%, secondary hypothyroidism 0.5%, primary hyperthyroidism 3.5%, primary adrenal insufficiency 0.2%, secondary adrenal insufficiency 2.7%, and type 1 diabetes mellitus 0.2%. Of the 42 patients diagnosed with hyperthyroidism, 28 (66.7%) later developed primary hypothyroidism within a mean time of 9 weeks. There was no statistically significant association between the incidence of AEs and gender (P=0.748) or age (P=0.998). Conclusion: AEs are common sequelae of ICI and primary hypothyroidism is the most prevalent AEs among our study sample. This study should help increase awareness about the importance of routine screening of the AEs in cancer patients receiving ICI.
Inhibiting Growth of Human Triple-Negative Breast Cancer Cells by Activating Mutant p53 Protein Alone or in Combination With a Phosphatidylserine-Targeting Antibody
Authors: Liang Yayun , Brekken Rolf A , Hyder Salman M
Published: May 2021

Triple-negative human breast cancers (TNBC) lack three proteins commonly targeted by chemotherapy; estrogen and progesterone receptors, and her-2-neu. Most current protocols used to treat TNBC are largely ineffective and aggressive tumors frequently re-emerge, leading to metastasis and patient death. Thus, new therapies for TNBC are needed. Recent studies show that around 80% of TNBC express mutant p53 (mtp53), a functionally defective form of the p53 tumor suppressor protein. If mtp53 is converted into the active wild-type protein (wtp53), tumor suppressor functions are recovered. Most p53 mutations occur in the DNA-binding domain, causing normal regulation of p53 target genes involved in apoptosis, cell-cycle arrest, and angiogenesis to be blocked. This promotes metastasis and renders tumors resistant to chemotherapy. APR-246 is a small-molecule drug that re-activates mtp53 by covalent modification of the DNA-binding core domain of the mutant protein through alkylation of thiol groups and has been shown to reactivate mtp53 and restore p53 function. We examined whether APR-246 could inhibit TNBC growth, both in vitro and in vivo. Cell viability assays and FACS were used to measure in vitro TNBC cell growth and apoptosis respectively, in MDA-MB-231 and MDA-MB-468 cells, with MCF-7 cells (which express wtp53) as controls. Analysis of TNBC growth in vivo was assessed in a mouse model of MDA-MB-231 derived xenografts. Nuclear extracts of APR-246-treated TNBC cells exhibited significantly increased p53 DNA binding compared with untreated cells, indicating that APR-246 converts mtp53 to wtp53 in these cells. APR-246 significantly reduced TNBC cell viability in vitro, but had no effect on normal mammary cells or wtp53-expressing MCF-7 cells. Pro-apoptotic proteins, Bax, p21 and caspase-3 were elevated in APR-246 treated cells, while the cell survival protein Bcl-2 was suppressed. In the xenograft model, animals were given an intravenous (iv) tail vein injection of APR-246 alone (100 mg/kg/day) once tumors reached 100 mm3. A second group received an intraperitoneal (ip) injection of 2aG4 antibody (100 µg/mouse/day), which targets phosphatidylserine and disrupts tumor blood vessel formation. A third group was given both APR-246 and 2aG4 using the same doses above. A control group received antibody C44 (100 µg/mouse/day, ip) and/or PBS (0.1 mL/day, iv). A total of 18 treatments were used. Administration of APR-246 alone or in combination with 2aG4, significantly reduced TNBC tumor growth, as well as two markers of angiogenesis (vascular endothelial growth factor expression and blood-vessel density). APR-246 in combination with 2aG4 completely eradicated almost 20% of the TNBC tumors. We conclude that TNBC is inhibited by APR-246 and 2aG4. Such treatment could represent an effective and innovative means of combating these particularly aggressive and deadly types of cancer.
Microtubule-Associated Protein 2 as a DHEA Binding Protein in Endometrial Cancer
Authors: Miki Yasuhiro , Iwabuchi Erina , Takagi Kiyoshi , Suzuki Takashi , Sasano Hironobu , Ito Kiyoshi
Published: May 2021

Dehydroepiandrosterone (DHEA) is an androgen secreted by the adrenal glands, but its binding affinity for the androgen receptor is very low. DHEA is transformed into androstenedione by 3β-hydroxysteroid dehydrogenase (HSD) and then into testosterone by 17β-HSD type 5, or into estrone by aromatase. DHEA is also converted into androstenediol by 17β-HSD type 1. Therefore, DHEA is considered to play an important role as a precursor hormone for sex steroid hormones. We performed a search for a protein having an amino acid sequence homology to the DHEA binding site of 17β-HSD type 1, and found that microtubule-associated protein 2 (MAP2) binds to DHEA (Laurine E et al., J Biol Chem. 2003). MAP2 expression is necessary for neurite extension and cessation of cell division. MAP2 is known to suppress migration and invasion and affect the assembly, stabilization, and bundling of microtubules in melanoma cells, but the function of MAP2 in endometrial cancer has not been clarified. In this study, we investigated the expression of MAP2 and its association with DHEA in order to clarify the direct non-receptor action of DHEA in endometrial cancer. We employed frozen and formalin-fixed paraffin-embedded (FFPE) tissues of 35 endometrial cancer tissues (G1, n=12; G2, n=10; G3, n=9; Serous, n=4). Hormone concentrations were measured by liquid chromatograph-tandem mass spectrometer from the frozen sample, and immunohistochemistry of MAP2 was performed using FFPE tissues. We also examined MAP2 immunoreactivity using 59 normal endometrial tissues (proliferative phase, n=33; secretory phase, n=26) of FFPE tissue microarray slides. MAP2 immunoreactivity was found in the cytoplasm of endometrial cancer cells, and the MAP2-positive rate was significantly higher in type 1 (G1 and G2) than in type 2 (G2 and G3). The cell proliferation marker Ki-67 index was significantly lower in the MAP2-positive group. MAP2 was also detected in the glandular epithelial cells of the normal endometrium. The MAP2-positive rate was lower in the proliferative phase than in the secretory phase. Furthermore, the concentration of DHEA in the cancer tissue was significantly higher in the MAP2-positive group than in the MAP2-negative group. MAP2 is known to act on the stability of microtubules and is thought to be involved in the suppression of proliferation and infiltration in cancer cells. It was suggested that DHEA is involved in the stabilization of MAP2 and suppresses the progression of cancer in a hormone receptor-independent manner.
Systemic Immune Response in Murine Bilateral Pheochromocytoma Model During Immunotherapy Based on a Combination of Mannan-BAM, TLR Ligands and Anti-CD40 Antibodies (MBTA Therapy)
Authors: Uher Ondrej , Huynh Thanh-Truc , Zhu Boqun , Horn Lucas A , Medina Rogelio , Wang Herui , Palena Claudia , Chmelar Jindrich , Zhuang Zhengping , Zenka Jan , Pacak Karel
Published: May 2021

Immunotherapy has become an essential component of cancer treatment, however, a majority of patients with solid metastatic cancers, such as pheochromocytoma (PHEO), do not respond to this type of therapy. Recently, we developed an intratumoral (i.t.) immunotherapy based on the unique combination of TLR ligands, anti-CD40 antibodies, and mannan, which is artificially bound to tumor cells via an anchor (MBTA therapy). This therapy resulted in the complete eradication of aggressive subcutaneous PHEO in 67% of mice and demonstrated a systemic antitumor immune response and regression of non-treated lesions in the metastatic model (1). To further evaluate this systemic effect generated during MBTA therapy, we established a murine bilateral PHEO model, where MBTA therapy was i.t. injected into one tumor, and the distant (non-treated) tumor was monitored for changes in size and immune cell infiltration. The growth of both MBTA-treated and distant tumors was reduced compared to that of the control. Interestingly, survival of the MBTA-treated mice was twice as long compared to the control mice. Moreover, we have made several unique observations during the experiments which were focused on the tumor microenvironment. Flow cytometry analysis revealed the ability of MBTA therapy to significantly increase the infiltration of innate immune cells (monocytes, DCs, macrophages, NK cells) not only in MBTA-treated tumors, but also in distal tumors, despite the fact that MBTA therapy was designed to elicit only local inflammation. An analysis of the macrophage phenotype revealed a switch from protumor M2 to antitumor M1 macrophages in both tumors during the entire MBTA therapy treatment. Analysis of splenic adaptive immune cells revealed that naïve CD4+ or CD8+ T cells differentiated into central memory cells and effector memory cells. CD4+ and CD8+ T cells were elevated in MBTA-treated and distant tumors with a significantly higher frequency of CD8+ effector memory T cells. Moreover, the adoptive transfer of CD4+ and CD8+ T cells revealed that immune memory, after tumor rechallenging, was driven by CD4+ T cells. Collectively, these results illustrate the ability of MBTA therapy to activate both parts of the immune system and render a systemic antitumor response against non-treated metastases. We believe that our results could lead to the use of MBTA therapy in patients with aggressive, metastatic lesions. Reference: Caisova et al., Cancers (Basel), 2019. 11(5).
Triple Negative Breast Cancer is Dependent on the Lysosomal Cholesterol Transporter NPC1
Authors: O’Neill Kathleen
Published: May 2021

Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer (BC) with peak rate of metastasis within the first few years post diagnosis and few targeted therapies. Normal epithelial cells and estrogen receptor alpha (ER) positive BC express the microRNA-200c (miR-200c), a potent suppressor of epithelial-to-mesenchymal transition (EMT). However, miR-200c is silenced or lost in TNBC, allowing aberrant expression of genes conferring a de-differentiated, non-epithelial phenotype that confers invasive and chemo-resistant properties. Recent literature demonstrated that EMT also promotes altered tumor cell metabolism. Hypothesis: We postulate that EMT reversal in TNBC will reveal selective advantages and identify novel therapeutic vulnerabilities. Methods: We used restoration of miR-200c as a tool to identify selective advantages conferred by EMT. In addition to driving global metabolic changes, miR-200c-repressed key cholesterol metabolism genes that support the uptake of dietary cholesterol, which is delivered via low-density-lipoproteins (LDL) and processed by the lysosomal cholesterol transporter, Niemann-Pick Type C1 (NPC1). Manipulation of NPC1 by genetic and pharmacological means was used to determine if and how TNBC are reliant on this pathway. Results: We determined that NPC1 is overexpressed in TNBC relative to ER+BC (Nature Metabric P<0.0001). Restoration of miR-200c directly targets the NPC1 3’UTR and represses NPC1 by two-fold (p=0.01). While silencing of NPC1 in ER+ BC cells led to slowed proliferation, TNBC cell lines died within 48-72 hours. NPC1 is associated with mitochondrial dysfunction and mTOR suppression. Intracellular cholesterol homeostasis is critical for cell survival and is carefully regulated, but how these homeostatic mechanisms adapt during tumor progression is poorly understood. Conclusions: This study demonstrates that while mesenchymal-like TNBC cells do not require exogenous cholesterol from the microenvironment, this cancer type is sensitive to the loss of NPC1. Overall, this work identifies NPC1 as a novel target in TNBC and sheds light on how lysosomes and mitochondria interact to sense cholesterol and drive cell survival.
Using Multimodal Functional Imaging in the Management of SDHx-Related Pheochromocytoma and Paraganglioma
Authors: White Gemma , Anandappa Samantha , Masucci Michael , Hassan Fahim-Ul , Breen Louise , Joshi Mamta , McGowan Barbara , Hubbard Jonathan , Obholzer Rupert , Touska Philip , Izatt Louise , Velusamy Anand , Carroll Paul
Published: May 2021

Background: Succinate Dehydrogenase (SDH) subunit pathogenic variants predispose to Pheochromocytoma and Paraganglioma (PPGL). Functional imaging harnesses the innate receptor expression and the aberrant cellular pathways in PPGLs to improve diagnostic accuracy & guide treatments, including nuclear medicine therapies. Currently commonly available functional imaging modalities include 18F-FDG PET, 123I-MIBG and 68Ga-DOTATATE. Aims: To analyze the use of 123I-MIBG, 18F-FDG PET and 68Ga-DOTATATE in patients harboring SDHB & SDHD pathogenic variants and determine the detection rates for both primary tumors and metastatic sites of disease. Methods: Retrospective review of patient records and imaging reports allowed tumor characteristics and imaging features of 21 patients with SDH-related PPGL to be recorded. Contrast enhanced CT/MRI were used as control to calculate the sensitivity of each functional imaging modality. Avidity of the primary lesion and metastatic deposits were used to calculate detection rates. 123I-MIBG imaging was available for 22 primary tumors (8 SDHB, 14 SDHD), 18FDG-PET for 24 (9 SDHB, 15 SDHD) and 68Ga-DOTATATE for 6 (2 SDHB, 4 SDHD) respectively. Results: 29 PPGLs (primary and metastases, 13 SDHB, 16 SDHD) were identified in 21 patients. 123I-MIBG detected 14/22 (64%) primary tumors; 5/8 (63%) SDHB and 9/14 (64%) SDHD-related PPGL. According to tumor location, 3/3 PCCs, 6/8 HNPGLs and 4/11 non-HNPGLs demonstrated 123I-MIBG avidity. Both18F-FDG PET and 68Ga-DOTATATE detected all PPGLs imaged; 24 (9 SDHB, 15 SDHD) and 6 (2 SDHB, 4 SDHD) respectively, demonstrating 100% sensitivity in the detection of the primary PPGL in all the above locations. 6 metastatic deposits (located in bone, lungs, liver and local lymph nodes) in 4 patients were imaged using all 3 modalities (3 SDHB, 1 SDHD), all of which were avid on 18F-FDG PET and 68Ga-DOTATATE whereas only 50% demonstrated avidity on 123I-MIBG imaging. Discussion: Recent guidelines promote preferential use of 68Ga-DOTATATE and 18F-FDG PET as initial functional imaging modalities in SDHx-related disease over 123I-MIBG. The results from our patient cohort indicate superior sensitivity (100%) for detection of SDHx-related disease with FDG and Dotatate compared with MIBG. In contrast to the current literature, a high proportion (75%) of HNPGLs in our series demonstrated MIBG avidity. Further prospective studies are needed to further evaluate these and various other novel tracers to inform diagnostic and therapeutic strategy in PPGLs arising from SDHx and various other germline and somatic pathogenic variants.
A Rare Case Report of Acquired Bartters With Positive Anti Scl70 Antibody
Authors: Pande Arunkumar Ramanarth , Mohanty Deepali
Published: May 2021

Introduction: Bartter syndrome autosomal recessive renal tubular disorders usualy presents in infant age group or antenatallyCase Description64 years housewife, a known case of hypothyroidism with in emergency with generalized weakness and hypotension. She had an small adrenal nodule work up which showed that it was non functional. On evaluation at time of stress (blood pressure 70/50) her serum cortisol was 300nmol/l and she was therefore started on hydrocortisone. Her records revealed hypokalemia for last 2 years. Workup of hypokalemia showed kaliuresis, metabolic alkalosis, hypercalciuria and excess chloride excretion in the urine, suggestive of bartter’s syndrome. But this needed further confirmation as bartter’s is rare in this age group and also the test was done at the time of stress. She was started on spironolactone and oral potassium supplementation. CECT head done for evaluation of postural dizziness showed chronic cortical venous thrombosis. On follow up ACTH stimulated cortisol was done which showed normal cortisol level so steroid was stopped. She was readmitted one month later with hypokalemia(K-1.8 meq/l) while being on same dosage of spironolactone and potassium. Workup of hypokalemia showed same feature of bartter’s syndrome. Considering the chain of events, a suggestion of bartter’s and response to steroid, a diagnosis of acquired autoimmune bartter’s syndrome along with hypothyroidism was made. An autoimmune work up showed anti SCL 70 positive2+(++). Patient responded to steroid, spironolactone, indomethacin and oral potassium supplementation. Discussion: Bartter’s syndrome can rarely happen in old age too. Seek for autoimmune cause once we suspect acquired bartter’s. To best of our knowledge previously only one case has been reported of acquired Bartter with systemic sclerosis. Patient may develop systemic sclerosis in future as anti SCL70 antibody is very specific for it. It is positive in less than 1 % of general population. Anti SCL70 antibody is rarely positive in Sjogrens syndrome. Our patient did not feature of sjogren or systemic sclerosis even after 4 years follow up