Ikki Sakuma

Cushing Syndrome Due to ACTH-Secreting Pheochromocytoma, Aggravated by Glucocorticoid-Driven Positive-Feedback Loop

CONTEXT Pheochromocytoma is a catecholamine-producing tumor that originates from adrenal chromaffin cells and is capable of secreting various hormones, including ACTH. CASE DESCRIPTION A 56-year-old female presented with Cushingoid appearance and diabetic ketoacidosis. Endocrinological examinations demonstrated ectopic ACTH production with hypercortisolemia and excess urinary cortisol accompanied by elevated plasma and urine catecholamines. Computed tomography revealed a large left adrenal tumor with bilateral adrenal enlargement. Metaiodobenzylguanidine scintigraphy revealed abnormal accumulation in the tumor, which was eventually diagnosed as pheochromocytoma with ectopic ACTH secretion with subsequent manifestation of Cushings syndrome. Ectopic ACTH secretion and catecholamine production were blocked by metyrapone treatment, whereas dexamethasone paradoxically increased ACTH secretion. Left adrenalectomy resulted in complete remission of Cushings syndrome and pheochromocytoma. IN VITRO STUDIES Immunohistological analysis revealed that the tumor contained two functionally distinct chromaffin-like cell types. The majority of tumor cells stained positive for tyrosine hydroxylase (TH), whereas a minor population of ACTH-positive tumor cells was negative for TH. Furthermore, gene expression and in vitro functional analyses using primary tumor tissue cultures demonstrated that dexamethasone facilitated ACTH as well as catecholamine secretion with parallel induction of proopiomelanocortin (POMC), TH, and phenylethanolamine N-methyltransferase mRNA, supporting a glucocorticoid-dependent positive-feedback loop of ACTH secretion in vivo. DNA methylation analysis revealed that the POMC promoter of this tumor, particularly the E2F binding site, was hypomethylated. CONCLUSION We present a case of ectopic ACTH syndrome associated with pheochromocytoma. ACTH up-regulation with paradoxical response to glucocorticoid, possibly through the hypomethylation of the POMC promoter, exacerbated the patients condition.

GERMLINE DELETION OF ARMC5 IN FAMILIAL PRIMARY MACRONODULAR ADRENAL HYPERPLASIA

OBJECTIVE Primary macronodular adrenal hyperplasia (PMAH) is considered a predominantly sporadic disease, but familial forms are well recognized. Genetic studies revealed germline mutations in the armadillo repeat containing 5 gene (ARMC5) in the majority of PMAH cases. Furthermore, somatic ARMC5 mutations, as different types of second-hit mutations and loss of heterozygosity have been reported in each adrenal nodule in PMAH. Here, we describe the involvement of ARMC5 alteration in a familial case of PMAH. METHODS In our study, we performed clinical and genetic evaluations in a mother and her son with familial PMAH. To search for mutations and deletion of ARMC5, we used Sanger sequencing and droplet digital polymerase chain reaction (ddPCR), respectively. RESULTS Both patients showed the same phenotype of subclinical Cushing syndrome, with mild excess of mineralocorticoids and vasopressin-responsive cortisol secretion. The ddPCR analysis demonstrated that both mother and son had germline deletions in exons 1 to 5 of the ARMC5 gene locus. Furthermore, Sanger sequencing of DNA from the right and left adrenal nodules as well as peripheral blood of the son revealed the presence of another germline, missense mutation in ARMC5 exon 3 (p.P347S). CONCLUSION This is the first report demonstrating germline deletion of ARMC5 in familial PMAH. In addition to investigating mutations, germline and somatic deletions of ARMC5 could be examined by ddPCR, which permits rapid and accurate evaluation of the ARMC5 allelic status.

Pulmonary arterial sampling was useful for localizing ectopic ACTH production in a patient with bronchial carcinoid causing Cushing syndrome.

OBJECTIVEWe report a 44-year old man with ectopic adrenocorticotropic hormone (ACTH) syndrome caused by bronchial carcinoid that developed cushing syndrome.METHODSWe performed several imaging studies, including chest and abdominal CT, for exploration of nodules and selective pulmonary arterial sampling for localizing a source of ectopic ACTH production.RESULTSThe patient was diagnosed as Cushing syndrome due to ectopic production of ACTH without identification of its source(s). After 2 years’ follow-up with repeated CT scans every 6–12 months and treatment with metyrapone, chest CT revealed two small nodules respectively in the segment (S) 4 and 10 of the right lung. We performed selective pulmonary arterial sampling from branches of the right pulmonary artery to obtain blood from the nodules in a reverse flow fashion: wedged sampling from the basal branch (A8, 9 and 10) revealed significant elevation of ACTH, whereas sampling from the lateral branch (A4) did not, indicating that the S10 nodule produced ACTH ectopically. The video-assisted thoracoscopic surgery removing the right inferior lobe normalized plasma ACTH, serum cortisol and 24-hour urinary free cortisol. The S10 nodule was histologically diagnosed as atypical bronchial carcinoid containing immunoreactive ACTH.CONCLUSIONSSelective pulmonary arterial sampling was useful for localizing the lesion of ectopic ACTH production and helped make the decision for its surgical removal. This procedure should be considered once lung nodules suspicious for ectopic ACTH production are identified in patients with EAS.

Multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells causing acromegaly associated with subclinical Cushing’s disease: a case report

BackgroundA functional pituitary adenoma can produce multiple anterior-pituitary hormones, such as growth hormone (GH) -producing adenomas (GHoma) with prolactin or thyrotropin stimulating hormone production in the same lineage. However, it is very rare that acromegaly shows subclinical Cushing’s disease (SCD) beyond the lineage. Here we describe the involvement of intratumoral coexistence with 2 types of hormone-producing cells associated with different lineage in acromegaly concomitant with SCD.Case presentationIn our study, we performed clinical evaluation of the patient showing acromegaly with SCD. To elucidate the mechanisms of this pathology, we analyzed immunohistochemistry and gene expression of anterior-pituitary hormones and transcriptional factors in the resected pituitary tumor. On immunohistochemical staining, most of the tumor cells were strongly stained for GH antibody, while some cells were strongly positive for adrenocorticotropic hormone (ACTH). Gene expression analysis of a transsphenoidal surgery sample of the pituitary gland revealed that ACTH-related genes, such as POMC, Tpit, and NeuroD1 mRNA, had higher expression in the tumor tissue than the nonfunctional adenoma but lower expression compared to an adenoma of typical Cushing’s disease. Further, double-labeling detection methods with a fluorescent stain for ACTH and GH demonstrated the coexistence of ACTH-positive cells (GH-negative) among the GH-positive cells in the tumor. Additionally, Pit-1 expression was reduced in the ACTH-positive cells from tumor tissue primary culture.ConclusionHere we described a case of a pituitary tumor diagnosed with acromegaly associated with SCD. We performed quantitative-expression analyses of transcriptional factors of the tumor tissue and immunohistochemistry analysis of tumor-derived primary culture cells, which suggested that the multihormonal pituitary adenoma concomitant with Pit-1 and Tpit lineage cells caused acromegaly associated with SCD.

Prednisolone-responsive Postpartum IgG4-related Hypophysitis

We herein report the case of a 25-year-old woman who presented with severe headache and visual field defects after childbirth. Magnetic resonance imaging revealed marked swelling of the pituitary gland, and an endocrinological examination revealed panhypopituitarism and diabetes insipidus. An immunohistological analysis of a transsphenoidal biopsy sample of the pituitary gland showed the significant accumulation of an immunogloblin G4 (IgG4)-positive population, leading to the diagnosis of IgG4-related hypophysitis. The patient was treated with prednisolone, which markedly reduced the swelling of the pituitary gland, in association with recovery of the pituitary function. This is a rare case of biopsy-proven IgG4-related hypophysitis with a postpartum onset.

A Unique Case of Renovascular Hypertension due to Fibromuscular Dysplasia in an Extra-renal Artery

A 33-year-old man was admitted to our hospital to undergo an evaluation to determine the cause of secondary hypertension. Computerized tomography angiography (CTA) showed bilateral multiple renal arteries with significant stenosis of the right extra-renal artery due to fibromuscular dysplasia and segmental impairment of renal perfusion. Although the plasma aldosterone concentration and plasma renin activity were within the normal ranges, percutaneous balloon dilatation of the stenotic lesion resolved his hypertension, leading to a diagnosis of renovascular hypertension caused by segmental renal ischemia due to extra-renal artery stenosis. CTA should be considered during the examination of patients with early-age hypertension, even if the plasma renin activity is not sufficiently elevated.

Multiple Sampling from the Central Veins with their Tributaries can Detect Bilateral Hyperaldosteronism with a Cortisol-Producing Adenoma in a Hypertensive Patient

A 52-year old woman was admitted to our hospital for evaluation of left adrenal incidenataloma. Endocrinological examination showed Cushing’s syndrome (CS) complicated with masked primary aldosteronism (PA). On the other hand, multiple sampling from the central veins and one or two tributaries of the adrenal veins before and after ACTH-stimulation (multiple AVS) clearly revealed bilateral hyperaldosteronism with excess cortisol secretion from the left adrenal. Thus, we diagnosed this case as CS due to left adrenal tumor with bilateral hyperaldosteronism, and left adrenalectomy was done. Immunohistochemical analysis of the removed left adrenal showed cortisol-producing adenoma and multiple aldosterone-producing cell clusters (APCCs) expressing CYP11B2 within the attached adrenal. Bilateral PA is mostly diagnosed as idiopathic hyperaldosteronism (IHA). IHA has not been examined enough pathologically. We first describe here a possible involvement of APCCs inducing hyperaldosteronism in a case of bilateral PA with a cortisol-producing-adenoma.

Glucagonoma With Necrolytic Migratory Erythema: Metabolic Profile and Detection of Biallelic Inactivation of DAXX Gene

Context Necrolytic migratory erythema (NME) occurs in approximately 70% of patients with glucagonoma syndrome. Excessive stimulation of metabolic pathways by hyperglucagonemia, which leads to hypoaminoacidemia, contributes to NME pathogenesis. However, the molecular pathogenesis of glucagonoma and relationships between metabolic abnormalities and clinical symptoms remain unclear. Patient A 53-year-old woman was referred to our hospital with a generalized rash and weight loss. NME was diagnosed by histopathological examination of skin biopsy tissue. Laboratory tests revealed diabetes, hyperglucagonemia, marked insulin resistance, severe hypoaminoacidemia, ketosis, and anemia. Enhanced computed tomography scans detected a 29-mm pancreatic hypervascular tumor, which was eventually diagnosed as glucagonoma. Preoperative treatment with octreotide long-acting release reduced the glucagon level, improved the amino acid profile, and produced NME remission. Surgical tumor excision normalized the metabolic status and led to remission of symptoms, including NME. Interventions Whole-exome sequencing (WES) and subsequent targeted capture sequencing, followed by Sanger sequencing and pyrosequencing, identified biallelic alteration of death-domain associated protein (DAXX) with a combination of loss of heterozygosity and frameshift mutations (c.553_554del:p.R185fs and c.1884dupC:p.C629fs) in the glucagonoma. Consistently, immunohistochemistry confirmed near-absence of DAXX staining in the tumor cells. Tumor expression of glucagon and somatostatin receptor subtype 2 and 3 messenger RNA was markedly upregulated. Conclusions This is a report of glucagonoma with biallelic inactivation of DAXX determined by WES. The tumor manifested as glucagonoma syndrome with generalized NME. This case showed the relationship between hypoaminoacidemia and NME status. Further investigations are required to elucidate the underlying mechanisms of NME onset and glucagonoma tumorigenesis.

p53-inducible DPYSL4 associates with mitochondrial supercomplexes and regulates energy metabolism in adipocytes and cancer cells

Significance We herein performed RNA sequencing to show that DPYSL4 is a p53-inducible regulator of energy metabolism in both cancer cells and normal cells, such as adipocytes. DPYSL4 was found to localize in both cytosol and mitochondria, particularly in associations with mitochondrial supercomplexes, providing a potential mechanism for its regulation of OXPHOS and cellular energy supply. Furthermore, DPYSL4 expression suppressed tumor growth and metastasis in vivo. Together, these results suggest a potential link between p53-inducible DPYSL4 and the pathophysiology of cancer and metabolic disorders, possibly via its energy-regulating function. The tumor suppressor p53 regulates multiple cellular functions, including energy metabolism. Metabolic deregulation is implicated in the pathogenesis of some cancers and in metabolic disorders and may result from the inactivation of p53 functions. Using RNA sequencing and ChIP sequencing of cancer cells and preadipocytes, we demonstrate that p53 modulates several metabolic processes via the transactivation of energy metabolism genes including dihydropyrimidinase-like 4 (DPYSL4). DPYSL4 is a member of the collapsin response mediator protein family, which is involved in cancer invasion and progression. Intriguingly, DPYSL4 overexpression in cancer cells and preadipocytes up-regulated ATP production and oxygen consumption, while DPYSL4 knockdown using siRNA or CRISPR/Cas9 down-regulated energy production. Furthermore, DPYSL4 was associated with mitochondrial supercomplexes, and deletion of its dihydropyrimidinase-like domain abolished its association and its ability to stimulate ATP production and suppress the cancer cell invasion. Mouse-xenograft and lung-metastasis models indicated that DPYSL4 expression compromised tumor growth and metastasis in vivo. Consistently, database analyses demonstrated that low DPYSL4 expression was significantly associated with poor survival of breast and ovarian cancers in accordance with its reduced expression in certain types of cancer tissues. Moreover, immunohistochemical analysis using the adipose tissue of obese patients revealed that DPYSL4 expression was positively correlated with INFg and body mass index in accordance with p53 activation. Together, these results suggest that DPYSL4 plays a key role in the tumor-suppressor function of p53 by regulating oxidative phosphorylation and the cellular energy supply via its association with mitochondrial supercomplexes, possibly linking to the pathophysiology of both cancer and obesity.

SEVEN FAMILIAL DYSALBUMINEMIC HYPERTHYROXINEMIA CASES IN THREE UNRELATED JAPANESE FAMILIES AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS OF THE THYROXINE BINDING PROFILE

OBJECTIVE Familial dysalbuminemic hyperthyroxinemia (FDH) is caused by abnormal human serum albumin (HSA) with an increased thyroxine (T4) affinity leading to euthyroid hyperthyroxinemia. One- and 2-step immunoassays of serum samples from FDH patients (e.g., Japanese patients) with the HSA R218P mutation can yield false-positive free thyroxine (FT4) results. Therefore, it is difficult to distinguish FDH from syndrome of inappropriate secretion of thyroid-stimulating hormone (TSH) (e.g., syndrome of resistance to thyroid hormone, TSH-producing pituitary adenoma), even when multiple assays are used. To investigate T4 to HSA binding, we examined serum samples from 7 patients from 3 Japanese families with FDH. Clinically, abnormal thyroid function tests were noted in pregnant Patient 1. Patients 2 and 3 had histories of inappropriate treatment with antithyroid drugs and surgery. METHODS All patients and affected family members were diagnosed with FDH using direct sequencing analysis. Gel filtration high-performance liquid chromatography was used for the biochemical analyses. RESULTS The genomic analysis revealed a heterozygous missense mutation in HSA (R218P). In FDH patient sera, the albumin effluent corresponded to the peaks for total T4 (TT4); approximately 60% of the T4 in the effluent was detected as FT4. The results for the albumin effluent from healthy volunteer and TSHoma patient sera showed no corresponding TT4 peak. CONCLUSION In the FDH patients, a relatively larger quantity of T4 was bound to abnormal HSA. This bound T4 was measured as FT4 during the analysis. ABBREVIATIONS F = free; FDH = familial dysalbuminemic hyperthyroxinemia; HPLC = high-performance liquid chromatography; HSA = human serum albumin; PCR = polymerase chain reaction; SITSH = syndrome of inappropriate secretion of TSH; T = total; T3 = triiodothyronine; T4 = thyroxine; TSH = thyroid-stimulating hormone; WT = wild-type.