United States

Introduction: Paragangliomas (PGL) are rare neuroendocrine tumours and account for approximately 10% of all chromaffin tumours. They may occur in the retroperitoneum, mediastinum, skull and the urinary bladder. Paragangliomas of the urinary bladder are rare entities accounting for 10% of all paragangliomas and 0.06% of all bladder tumours. Approximately 10% of urinary bladder paragangliomas are malignant. Bladder paragangliomas can be either functional or non-functional. Main symptoms of functional PGLs are due to catecholamine excess with palpitations, paroxysmal hypertension during micturition, sweating and headaches. Functional bladder PGL are diagnosed on biochemical analysis. Non-functional bladder PGLs present with haematuria and are often misdiagnosed as bladder cancer as they may cystoscopically and histologically resemble a urothelial neoplasm. Clinical cases: We present a case series of 4 surgically treated patients with urinary bladder paragangliomas who presented to Cambridge University Hospital over the last 3 years. All patients were female with an age range between 21 and 44 years. All four patients had SDHB deficient tumours, and all four patients were diagnosed with a pathogenic variant in the SDHB gene. Three patients had no family history of SDHx associated neoplasia whereas one had a paternal aunt who died from a metastatic pheochromocytoma. Three patients had presented with palpitations and hypertensive episodes, one of whom experienced symptoms while micturating. Biochemical analysis revealed elevated plasma normetadrenaline levels in all patients. On presentations, one patient had a synchronous paratracheal paraganglioma while two patients had lymph node metastasis. All patients were surgically treated with two patients undergoing fertility preserving cystectomy and bladder substitution, one undergoing partial cystectomy, radical hysterectomy and ureteric implantation and one managed with robot-assisted partial cystectomy. None of the patients have evidence of biochemical or radiological tumour recurrence on follow-up with a mean follow-up time of 23 months (range 12 to 40 months). Conclusion and points for discussion: The diagnosis of urinary bladder PGL is challenging. Biochemical assessment to assess catecholamine production and imaging studies like CT, MRI and NM Ga68 DOTATATE PET CT are helpful modalities in diagnosis. Patients presenting with extra adrenal paragangliomas including bladder paragangliomas require genetic testing because of the strong hereditary predisposition, with mutations in the SDHx genes being most common with this clinical phenotype. The surgical approach should be tailored to the individual patient, agreed by a multi-disciplinary team and the risk of malignancy should be weighed against the potential morbidity associated with a curative oncological surgery. As urinary bladder PGLs have malignant potential and are frequently hereditary (as demonstrated in this series), lifelong follow-up is required to screen for synchronous tumours and tumour recurrence.

ENDO 2022

Urinary bladder Paragangliomas – A Case series

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ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

technical paper

Multiple studies have shown that approximately 50-70% of patients with MEN1 die of causes directly related to MEN1 particularly gastroenteropancreatic (GEP) neuroendocrine tumors (NETs). While non-functional GEP-NETs are the most common in the general population, gastrinomas (40%) are the most common functional GEP-NETs in patients with MEN1. c-MET is a proto-oncogene that encodes for c-MET, a tyrosine kinase receptor which promotes tumor cell motility, proliferation, survival, invasion, and metastasis. Studies in patients with sporadic gastrinomas and pancreatic NETs (PNETs) have shown that c-MET expression correlates with decreased survival.
While c-MET inhibitors are currently in various stages of investigation for treatment of carcinoids and sporadic PNETs, data regarding their efficacy in patients with MEN1-related GEP NETs is lacking. The majority of trials in patients with GEP-NETs exclude or do not report the number of patients with MEN1. Importantly, somatic MEN1 mutations are observed in 20-40% of sporadic NETs (gastrinomas, PNETs, lung NETs, etc. ) but correlation of cMET expression with the presence of somatic or germline MEN1 mutations has not been reported. We sought to investigate the expression of c-MET in tumor tissue from germline MEN1 patients with metastatic GEP-NETs.
Methods
We identified subjects with a germline positive MEN1 mutation and pathologically confirmed distant metastasis who had a follow-up visit between 2018-2020. Of these, we selected subjects with available tissue specimens (including either multiple organ sources or different tumor types). Where available, we identified specimens from multiple source or tumor types. Immunohistochemistry (IHC) to detect c-MET was performed with anti-MET (Cell Signaling) using the DAKO IHC kit (Agilent). IHC slides were imaged and observed to score the level of c-MET staining (-, 1+ to 5+). A score of 3+ or higher was considered consistent with overexpression. We investigated if age at initial GEP-NET presentation, tumor type, tissue source, tumor grade, total number of surgeries for GEP-NET, number of sites of distant metastasis and disease status from overall GEP-NET burden over the preceding 12 months (stable/progressive) predicted c-MET expression.
Results
Eight subjects with available tissue specimens were identified, of which six had tissue from multiple organs while five had tissue from multiple tumor types. Six subjects (75%) showed increased expression of c-MET in one or more tumor specimen(s). The frequency of c-MET overexpression varied with tumor types – carcinoids (n=2/2; 100%), gastrinomas (n=3/5; 60%) and non-functional tumors (n=3/6; 50%). c-MET expression also varied among different tumors in the same patient. Tumor tissue from liver (n=2/2), duodenum (n=3/4), stomach (n=1/1), ovary (n=1/1), pancreas (n= 1/5), and lymph nodes (n=1/3), all showed over-expression of c-MET. No clear predictors of c-MET overexpression emerged.
Conclusion
Our finding suggests a role for c-MET expression in personalizing therapy for patients with MEN1-related NETs with distant metastases.

c-MET expression in MEN1-associated neuroendocrine tumors

Adrenocortical carcinoma (ACC) can occur as part of hereditary cancer syndromes, particularly Lynch syndrome and Li-Fraumeni syndrome. Here we describe 5 patients (3 women) with adrenal tumors and Birt-Hogg-Dubé syndrome (BHDS). BHDS is a hereditary tumor syndrome characterized by fibrofolliculomas/trichodiscomas, renal tumors, lung cysts, and spontaneous pneumothoraces.
All five patients had a confirmed germline FLCN pathogenic variant. One case was previously reported by our institution. Age of diagnosis (AD) ranged from 36 to 62 years with a mean AD of 46 years. All patients identified as Caucasian. Three patients had non-functioning adrenal tumors, while the remaining two patients did not undergo biochemical assessment preoperatively but did not have symptoms or history of adrenal hormone excess.
Histologically, four patients had confirmed ACC while one had an adrenocortical tumor of uncertain malignant potential, most consistent with an oncocytoma. All patients were initially treated with mitotane. Three patients developed metastatic disease requiring additional systemic chemotherapy, immunotherapy, and/or radiation.
A total of 8 confirmed ACCs in patients with BHDS have been reported to date, raising suspicions that these tumors might be a part of the BHDS tumor spectrum. When BHDS patients are screened for renal malignancies, special attention should be paid to the adrenal glands. Adrenal nodules should undergo prompt assessment as >50% of reported cases developed metastatic disease. Given possible association with BHDS, testing for FLCN pathogenic variants should be considered during the genetic evaluation of patients with ACC.

Adrenocortical Carcinoma as a Rare Manifestation of Birt-Hogg-Dubé Syndrome

Introduction:Insulinoma is a rare pancreatic neuroendocrine tumor (PNET) accounting for 1-2% of pancreatic neoplasms. Usually benign, even small tumors can cause significant morbidity due to hypoglycemia. Surgical resection is preferable choice of treatment, but diagnosis is usually delayed and localization can be evasive. We present a case of insulinoma in a patient who battled his symptoms with high carbohydrate intake leading to massive weight gain associated with severe insulin resistance.Case:A 70 year old male presented to the ED after being found unconscious in his home. Blood glucose was 34 mg/dl after missing a meal. He had a remote history of diabetes mellitus, which gradually resolved. Five years ago, he began experiencing sweats, tremors, headaches and confusion early in the morning or when fasting that improved with eating. He was hospitalized four years ago due to hypoglycemia where he was suspected to have endogenous hyperinsulinemia. CT abdomen at that time was unremarkable. Possibility of insulinoma was raised, but patient did not pursue further investigations. In the last 4 years, he avoided severe hypoglycemic events with frequents meals, but had gained more than 50 lbs with resultant BMI of 51 kg/m2. While admitted, he underwent a 72h fasting test showed glucose 42 mg/dl, insulin level was 84.8 IU/L (range <3), proinsulin was 105.7 (pmol/l) and C-peptide was 4.83 ng/ml (ref <0.2). EUS revealed 7 mm x 4 mm mass in pancreatic body. FNAB confirmed well differentiated PNET, grade 1. CT abdomen remained unrevealing. He was started on weight based diazoxide therapy with resolution of hypoglycemic events and persistent hyperglycemia, which required high dose insulin therapy and improved with significant decrease in diazoxide dose. Hospitalization was complicated by hypoxic hypercarbic respiratory failure due to obesity hypoventilation syndrome, COVID19 infection and new onset rapid atrial fibrillation. Patient was deemed not to be a surgical candidate due to multiple comorbidities and his medical management was switched to octreotide with low dose insulin therapy, which patient tolerated well.Conclusion:Diagnosis of insulinoma can be significantly delayed in cases with mild or slowly progressive course of the disease. We suggest that progressive development of severe insulin resistance in patients with baseline obesity and insulin resistance-driven DM2 can mask endogenous hyperinsulinemia for extended period of time. This gives a false sense of slower progression or even resolution of symptomatology, as well as diversion to alternative diagnoses, like postprandial hypoglycemia. We also argue that "resolution" of diabetes with significant weight gain, especially when associated with increased calorie intake through frequent meals and symptomatology suggestive of hypoglycemia should be a red flag for workup towards possible insulinoma, akin to suspecting adrenal insufficiency in diabetic patients with an unexplained decrease in insulin requirements.

Surviving Insulinoma: Severe Insulin Resistance in the Face of Endogenous Hyperinsulinemia

Introduction: There is a clinical need to develop novel and better biomarkers to monitor patients with neuroendocrine neoplasms (NENs). Circulating cell free tumor derived DNA (ctDNA), a form of liquid biopsy, is finding clinical utility in an ever increasing number of malignancies however has not been widely tested in patients with NENs.Aims: Our aim was to identify and track plasma ctDNA in a cohort of patients with NENs using a personalized, patient specific approach.Materials and methods: A total of 35 serial plasma samples were collected from 9 patients with metastatic, well differentiated NENs (6 small intestinal and 1 lung, 1 ovarian and 1 pelvic; range 2-5 plasma samples per patient) over the space of 2-25 months. For each patient, NEN specific somatic mutations (single nucleotide variants and insertions/deletions) were identified through whole exome sequencing of paired tumor-leucocyte DNA and were used to design a bespoke multi-variant Ampliseq™ HD ctDNA panel (5-20 variants per patient) for targeted next generation sequencing. Imaging and treatment were provided as per usual clinical care.Results: ctDNA was detectable in 6/9 patients and in 19/35 plasma samples. A rise in the number of ctDNA target variants and/or variant allele frequency was seen in 4/6 patients who experienced disease progression. Two of these patients received peptide receptor radionuclide therapy after which ctDNA disappeared in one patient and substantially reduced in the other, which correlated with treatment response. The 3 patients who did not have detectable ctDNA at any time point all had grade 1 small intestinal NENs with stable disease during the observation period.Discussion: Our data provide exciting evidence for the feasibility of using ctDNA as a biomarker in NENs. By targeting multiple individualized variants using next generation sequencing, we have demonstrated that ctDNA can track changes in disease burden and can monitor response to treatment in patients. Of equal importance, ctDNA was not detectable in patients with quiescent disease. This could help identify patients who do not need intensive monitoring. Targeting bespoke, multiple variants per patient is a novel and powerful approach for NENs, especially given their heterogenous genetic landscape. This study provides important early evidence that ctDNA may be a clinically useful biomarker for detection and surveillance of NENs.

Liquid Biopsies - Detecting and Tracking Circulating Cell Free Tumor Derived DNA in Patients with Neuroendocrine Neoplasms

Background: The 5-year survival for metastatic pancreatic neuroendocrine tumours (PNETs) is <50%, and current therapies are not effective. Thus, there is an unmet clinical need for new therapies for patients with PNETs. PNETs frequently have mutations in chromatin remodelling genes, including in Multiple Endocrine Neoplasia type 1 (MEN1), encoding menin, which is mutated in up to 40% of sporadic PNETs, and is part of multiple histone modifying complexes including the SET1/MLL methyltransferases.Aim: To examine multiple classes of epigenetic inhibitors and determine which may be effective in treating PNETs.Methods: BON1 and QGP1 PNET cell lines, the bronchial NET cell line H727, and the non-NET cell line HEK293T were utilised to assess >150 small molecule inhibitors targeting: histone deacetylases (HDACs); histone acetyltransferases; histone demethylases (KDMs); histone methyltransferases; bromodomain-containing proteins; menin; and DNA methyltransferases (DNMTs). MTOR inhibitors, already used clinically for PNET patients, as well as drugs targeting other tumorigenic pathways e.g. the proto-oncogene SRC, were used as comparators. Efficacy of compounds was evaluated using the Cell Titer Blue cell viability assay, and the Caspase Glo 3/7 apoptosis assay.Results: At a dose of 1mM for 7 days, 26 compounds (including HDAC, KDM, bromodomain and mTOR inhibitors) significantly decreased the viability of both the BON1 and QGP1 cell lines by >50%, compared to DMSO control treated cells. Upon increasing the dose to 10mM for 7 days, 34 additional compounds, making a total of 60 compounds (including inhibitors of menin and DNMTs), decreased viability of both BON1 and QGP1 cells by >50%; and 56 and 46 compounds also reduced viability of H727 and HEK293 cells by >50%, respectively. Apoptosis assays utilising the 60 above compounds, at 10mM for 7 days, revealed that 25, 18, 14, and 9 of them could increase apoptosis by >4-fold of BON1, QGP1, H727 and HEK293 cells, respectively. Ten compounds (2 KDM, 5 HDAC and 3 bromodomain inhibitors) that were effective in reducing viability by >50% as well as increasing apoptosis by >4-fold of NET cells, were selected for further dosing (0.1-10mM) and time course investigations (3-7 days). All 10 compounds significantly reduced viability of BON1 and QGP1 cells (p<0.01) after 3 days of treatment. The KDM and HDAC inhibitors showed the greatest efficacy, by significantly (p<0.05) reducing BON1 and QGP1 cell viability at as low as 25nM treatment for 3 days. The efficacy of bromodomain inhibitors was not attained until 1000nM treatment, however these inhibitors had very little effect on the non-NET HEK293 cells.Conclusions: Compounds targeting HDACs, KDMs or bromodomain-containing proteins are effective in reducing viability by >50% and increasing apoptosis by >4-fold of NET cells , and thus provide novel agents for studying anti-cancer mechanisms and potential therapeutic options for PNETs.

Histone eraser and reader targeting epigenetic inhibitors are effective in pancreatic neuroendocrine tumours

Adrenal homeostasis relies on the centripetal differentiation and migration of peripheral adrenocortical progenitors to replenish the steroidogenic cells in the cortex. This homeostatic process is essential for steroidogenic functions and its dysregulation has been implicated in many adrenal diseases. Steroidogenic Factor 1(SF1) is a nuclear receptor which functions as a transcriptional factor to regulate global steroidogenic gene expression and adrenal development. However, the regulation of SF1 in the processes of differentiation (steroidogenesis) versus self-renewal of progenitors necessary for adult adrenal homeostasis remains largely unknown. Transcriptional activity of SF1 is regulated by post-translational modifications including phosphorylation (S203) and SUMOylation (K119 and K194). SUMOylation inhibits CDK7-mediated SF1 phosphorylation and SUMO-defective SF1 preferentially activates "SUMO-sensitive" target genes including Shh, rather than many well-characterized "SUMO-insensitive" target genes mediating differentiation. Based on these findings, together with data that CDK7 uniquely links cell cycle to transcriptional programs engaged in proliferating cells and the marked stem cell failure reported in multiple tissues of CDK7-KO mice, we hypothesized that CDK7-mediated SF1 phosphorylation in Shh-expressing adrenocortical progenitors regulates a unique transcriptional program to maintain their undifferentiated proliferative state during adrenal homeostasis. We engineered SF1-S203A/D mouse models which contain biallelic non-phosphorylatable/phosphomimetic germline mutations of SF1 at Serine 203. We crossed S203D mice with a) Shh-LacZ reporter strain to visualize Shh-expressing progenitors locked in an SF1 phosphomimic state and b) Shh-CreERT2;mTmG strain to GFP label Shh progenitors for lineage tracing. We observed increased beta-galactosidase staining in the zG of SF1-S203D reporter mice compared to controls, indicating an expanded Shh progenitor pool when SF1 is phosphorylated at S203. After 14 days of lineage tracing, GFP was co-stained with Cyp11B2, a marker for zG steroidogenic lineage, to quantify the percentage of Shh-expressing progenitors that differentiated into Cyp11B2-expressing zG cells. SF1-S203D mice exhibited a markedly lower differentiation rate of Shh-expressing progenitors compared to wild type, suggesting that SF1 phosphorylation preserved their undifferentiated state. Moreover, SF1-S203A mutation leads to a decreased adrenal/body weight ratio as well as downregulation of Shh. For insights into the role of CDK7 in SF1 phosphorylation during adrenocortical homeostasis, we generated conditional CDK7-KO mice. Adrenocortical-restricted CDK7-KO mice had decreased adrenal size, proliferation rate and Shh expression. Lineage tracing revealed increased differentiation of Shh-expressing progenitors when CDK7 was deleted specifically and temporally in Shh-expressing progenitors. Finally, we compared the differentiation rate in WT, CDK7-KO and CDK7-KO on an S203D background. The rapid differentiation induced by CDK7 loss was partially rescued by SF1-S203D, confirming the contribution of SF1 phosphorylation in mediating the functions of CDK7 in adrenocortical progenitors. Based on these findings, we propose a novel model in which CDK7-mediated SF1 phosphorylation regulates a unique transcriptional program that contributes to maintaining the undifferentiated proliferative state of adrenocortical progenitors.


Unravelling the Role of CDK7-mediated SF1 Serine 203 Phosphorylation in Adrenal Homeostasis

Introduction
Twenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under adrenal-directed medical treatment. In CS patients without medical treatment, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of interference by cross-reacting steroid metabolites. However, it is unknown how induced changes by Ketoconazole (KTZ) or Metyrapone (MTP) on the excretion of urinary steroid metabolites alter the IA cross-reactivity interference of 24h-UFC determinations and what are its clinical implications.
Methods
24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ and 39 during MTP) from 34 CS patients. In addition, 35 urinary steroids were analyzed by GC-MS on each patient before and during treatment.
Results:
Before treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75: 1 (95% CI 1.60 - 1.94) compared to those by GC-MS. During treatment with KTZ, the cross-reactivity interference of the 24h-UFC results by IA decreased in a dose-dependent manner (p = 0.000). Thus, in patients taking KTZ >400mg/day, 24h-UFC*IA determinations were no longer overestimated, giving similar results to those by GC-MS (factor of 0.98: 1, 95% CI, 0.83 – 1.20). Baseline 24h-UFC and upper limit normal (ULN) reductions were magnified (>25%) when assessed by IA vs GC-MS leading to higher falsely KTZ efficacy and misleading biochemical classification of 15% of patients. Despite dosage, in patients taking MTP the IA cross-reactivity only decreased 0.55 which resulted in persistence of overestimated 24h-UFC results (factor of 1.33: 1, 95% CI, 1.09 -1.76). When comparing MTP efficacy among IA vs GC-MS results, ULN baseline reductions were >50% different in some patients, while in others, similar reductions were found. Acceptable 24h-UFC method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. 6β-Hydroxy-cortisol (6β-OH-cortisol) was the metabolite determining most of the 24h-UFC cross-reactivity variability (R2= 48.3%, p = 0.000) followed by 20α-dihydrocortisol (20α-DHF) (R2= 24.4%, p = 0.000). Each treatment induced a unique urinary steroid signature (p <0.001) and larger excretion reductions of 6β-OH-cortisol, 20α-DHF and 18-Hydroxy-cortisol in patients with KTZ elucidated the higher decrement of interference in the 24h-UFC by IA, compared to MTP treated patients (p = 0.001).
Conclusion:
KTZ and MTP alter distinctly the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment in a unique manner. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of ULN.

Urinary adrenal steroidome modifications by Ketoconazole and Metyrapone alter the urinary free cortisol immunoassay reliability in Cushing Syndrome

Glucocorticoids (GC) are an integral part of anti-inflammatory therapy. However, their therapeutic effectiveness is limited by their deleterious adverse effects such as the development of obesity, diabetes, and cardiometabolic disease. GC-induced obesity arises from excessive fat accumulation in white adipose tissue (WAT) as well as suppressed thermogenic function of brown adipose tissue (BAT). The sympathetic nervous system (SNS) plays a key role in the regulation of both WAT as well as BAT biology. As part of this project, we investigate the interaction between SNS and GC signaling in the adipose organ.
To that end, we modulated the SNS activity in adipose tissues of mice by altering the housing temperature of corticosterone-treated mice and respective controls to either 29°C, 22°C, or 13°C. Following the four-week treatment course, GC-treated mice housed at 29°C and 22°C gained more weight and accumulated more visceral and subcutaneous white fat than their temperature-matched controls. Interestingly, maintaining mice in the 13°C environment protected animals from GC-induced weight gain and obesity. Additionally, cold adaptation effectively prevented the development of GC-driven hyperglycemia, hyperinsulinemia and hyperleptinemia, all of which were readily observed at 29°C and 22°C. Furthermore, the sympathetic innervation as well as the thermogenic capacity of BAT were preserved in cold-acclimated mice in spite of corticosterone treatment, whereas mice housed at higher temperatures showed marked suppression of the thermogenic function in BAT following GC exposure. To further test whether sympathetic activation is responsible for the prevention of GC-induced dysmetabolism by prolonged adaption to cold, we investigated the effect of a pharmacological β3-AR agonist, CL316,243 during GC excess. Similar to cold exposure, selective activation of the β3-AR not only averted the development of GC-induced fat accumulation and hyperinsulinemia, but also preserved the thermogenic capacity in BAT despite exposure to supra-physiological levels of corticosterone.
Taken together, our data indicate that both cold-acclimation as well as β3-AR activation counteract GC-induced obesity, adipose tissues dysfunction, and related metabolic comorbidities. Thus, targeting the β3-AR may represent a potential therapeutic approach to alleviate GC-induced metabolic abnormalities.

The beta3-Adrenergic Receptor as a Target for the Prevention of Glucocorticoid-induced Adipose Tissue Dysfunction in Rodents

Context:  About 5-10% of  adrenocortical cancer (ACC) arise in patients with genetic predisposition syndromes, including Li-Fraumeni, Lynch, MEN1, or Familial Adenomatous Polyposis (APC). Recent European (2018) and American guidelines (2020) recommend that all adults with ACC should be offered clinical genetic counseling. There is a paucity of data regarding systematic germline testing in adults with ACC and the extent of the genetic evaluation is unclear.

  Objective: To describe the germline genetic characteristics in a cohort of adult patients with ACC  evaluated in  a tertiary clinical care center.

  Methods: Data including demographics, personal and family history of neoplasia, pathology reports, clinical features and genetic testing were retrospectively collected from charts of patients treated at Centre hospitalier de l'Université de Montréal (CHUM). After genetic counseling, genetic testing was proposed to patients with a pathologic diagnosis of ACC. From 2005 to 2015, TP53 gene analysis was performed using direct sequencing and MLPA. Since 2016, multigene testing was performed for a progressively increasing number of oncogenic genes using a custom next-generation panel for germline leucocyte DNA including  at least the  TP53, MSH2, MSH6, MLH1,PMS2, EPCAM, MEN1, BRCA1 and BRCA2 genes (Invitae, CA). An extended panel, based on past medical and familial history, was performed at the discretion of the geneticist. Patient data was retrospectively investigated.

  Results: We analyzed data from 44 patients with available germline genetic results. Median age of the patients was 46 years (ranging from 22 to 79 years), including 11 males (25.0%) and 33 females (75.0%). Ten patients (22.7%)  underwent only TP53 gene analysis and 32 patients (72.7%) were studied using the larger oncogenic genetic panel.
Germline pathogenic or likely pathogenic variants were identified in 5 of the 44 patients (11.4%) while genetic variants of unknown significance (VUS) were found in 7 of the 44 patients (15.9%). Among patients with pathogenic variants, one had a family history and known germline mutation in the BRCA2 gene (8765delAG, p.Glu2846GlyfsX23). One patient had a personal and familial medical history suggesting a MEN1 syndrome that was confirmed with the finding of a germline MEN1 mutation (c.1556delC, p.Pro519Leu fs40). Unsuspected germline pathogenic variants were found in three patients: 1) TP53 (c.425delC, p.Pro142fs), 2) MUTYH (c.536A>G, p.Tyr179Cys) and 3) MSH6 (c.3649-3655, p.Arg1217Leu fs9) genes. Variants of unknown significance (VUS) were found in the following genes: POT1, MSH3, PALB2, RAD51d, APC, ATM and BRCA2.
  Conclusions: Germline pathogenic variants were found in 11.4% of our cohort of patients with ACC. VUS were found in 15.9% of patients but their significance remains to be determined. Genetic counseling and germline genetic testing should be offered to all patients with ACC; however the optimal use and extent of oncogenic gene panels need to be better defined.

Germline Genetic Testing in a Cohort of Adults with Adrenocortical Carcinoma : Insights From a Clinical Care Setting

Congenital adrenal hyperplasia (CAH) is a set of defects in cortisol synthesis due to mutations in genes encoding steroidogenic enzymes. Consequently, the loss of glucocorticoid-mediated negative feedback on the HPA axis leads to chronically elevated ACTH (Adrenocorticotropin Hormone) secretion. The sustained ACTH elevation, in turn, stimulates proliferation and hypertrophy of the adrenal cortex and leads to overproduction of androgens.
To study the complexity of the ACTH-responsive cell population in the adrenal cortex, we performed single-cell RNA sequencing of the steroidogenic lineage in the adult mouse adrenal. We identified the transcription factor HHEX as a gene with restricted expression in the ACTH-responsive zona fasciculata. Although the role of HHEX in adrenal biology is completely unknown, a meta-analysis identified a germ-line variant of uncertain significance near the gene HHEX associated with increased DHEAS (adrenal-specific androgen) in human serum samples, which led us to hypothesize that HHEX contributes to the unique function of the ACTH-responsive inner cortex.
To investigate the mechanisms by which HHEX controls adrenal differentiation and homeostasis, we generated genetically modified mice harboring a deletion of the Hhex gene in the adrenal cortex. Hhex KO mice exhibited progressive adrenomegaly by 15 weeks of age, accompanied by hypertrophy prominent in the inner cortex and disruption of the endothelial organization. Expression of the transcription factor Nr5a1/Sf‑1 and steroidogenic enzymes involved in corticosterone production were significantly upregulated at 6 weeks old, prior to adenomegaly, suggesting a precocious increase in cell production of glucocorticoids. For insights into the signaling pathways controlled by HHEX, we analyzed global transcriptional changes during loss of Hhex and found dramatic downregulation of members of the membranous progesterone receptor family (PAQR). PAQR signaling has been implicated in the downregulation of cAMP, a primary mediator of ACTH signaling, and thereby could, in part, drive the increase in steroidogenesis in Hhex KO mice.
These findings suggest that the inner cortex contains a progesterone-responsive cell population in which HHEX provides additional fine-tuning of ACTH-driven cAMP signaling by progesterone and derivatives. Since we observed an increase in Hhex expression in a mouse model of CAH, experiments are now ongoing to assess the implication of HHEX and autocrine progesterone signaling in contributing to the hypertrophy and steroidogenic phenotype observed in CAH.

Urinary bladder Paragangliomas – A Case series

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