Validation of the digital PCR system in tyrosine kinase inhibitor-resistant EGFR mutant non-small-cell lung cancer.

The aim of this study was to compare the accuracy of the QuantStudio 3D Digital polymerase chain reaction (dPCR) system and a PCR-based next generation sequencing (NGS) system for detecting a secondary mutation in the epidermal growth factor receptor (EGFR) gene T790M in non-small cell lung cancer (NSCLC) patients previously diagnosed with EGFR-activating mutations. Twenty-five patients with NSCLC previously treated with EGFR-TKIs were examined. The patients were treated daily with either erlotinib or gefitinib. New biopsies, followed by DNA sequencing on an Ion Torrent systems using the Ion Torrent AmpliSeq Cancer Hotspot Panel and dPCR were performed. A comparison of NGS, sensitive PCR, and dPCR revealed that the sensitivities of NGS and dPCR were similar in this study. As well, T790M was detected in as low as about 5% of mutant allelic frequencies, which represented 5% of the total reads on site mapped reads in NGS and greater than 5% of the dPCR reads, which represented mutant and wild type copies. The strategy in which NGS sequencing is followed by revealed acquired mutation with dPCR may be a reasonable one. We demonstrated the utility of combining NGS and dPCR as a tool for monitoring T790M. NGS and dPCR with formalin-fixed paraffin-embedded (FFPE) specimens might become a standard genomic test for exploring acquired resistance to targeted molecular medicines.

Intentions and Factors Regarding Selection of Workplace by Postgraduate Dental Trainees after Clinical Training.

The purpose of the present study was to investigate the reasons governing selection of geographical location of workplace by postgraduate dental trainees. We believe that such data would aid in the development of measures aimed at reducing regional disparities in the number of dentists. A total of 10 dental colleges and faculties in Japan were invited to participate in the study. Anonymous, self-completed questionnaire surveys were given to 739 dentists at 13 clinical training facilities following training in 2012 and 2013; 713 responses were analyzed. The items surveyed included the local population in the area in which they wished to work and whether they wanted to work in rural areas or remote islands. The most common response to the question regarding population was "medium-size cities" with a population of 100,000 to 300,000 people (67.3%). The largest number of respondents stated that they hoped to work in their hometown or a neighboring area (49.9%). A large percentage wished to work in medium-size cities, which may reflect the population of their hometowns. The current system allows dentists a free choice in becoming practitioners or hospital dentists on completion of training. This may explain why so many expressed a preference to work in their hometown or a neighboring area here. These results suggest that policies relying on increasing the supply of dentists as the sole solution to rural shortages are flawed.

Next-generation sequencing of tyrosine kinase inhibitor-resistant non-small-cell lung cancers in patients harboring epidermal growth factor-activating mutations.

BACKGROUND: The aim of this study was to detect the epidermal growth factor receptor (EGFR)-activating mutations and other oncogene alterations in patients with non-small-cell lung cancers (NSCLC) who experienced a treatment failure in response to EGFR-tyrosine kinase inhibitors (TKIs) with a next generation sequencer. METHODS: Fifteen patients with advanced NSCLC previously treated with EGFR-TKIs were examined between August 2005 and October 2014. For each case, new biopsies were performed, followed by DNA sequencing on an Ion Torrent Personal Genome Machine (PGM) system using the Ion AmpliSeq Cancer Hotspot Panel version 2. RESULTS: All 15 patients were diagnosed with NSCLC harboring EGFR-activating mutations (seven cases of exon 19 deletion, seven cases of L858R in exon 21, and one case of L861Q in exon 21). Of the 15 cases, acquired T790M resistance mutations were detected in 9 (60.0%) patients. In addition, other mutations were identified outside of EGFR, including 13 cases (86.7%) exhibiting TP53 P72R mutations, 5 cases (33.3%) of KDR Q472H, and 2 cases (13.3%) of KIT M541L. CONCLUSIONS: Here, we showed that next-generation sequencing (NGS) is able to detect EGFR T790M mutations in cases not readily diagnosed by other conventional methods. Significant differences in the degree of EGFR T790M and other EGFR-activating mutations may be indicative of the heterogeneity of disease phenotype evident within these patients. The co-existence of known oncogenic mutations within each of these patients may play a role in acquired EGFR-TKIs resistance, suggesting the need for alternative treatment strategies, with PCR-based NGS playing an important role in disease diagnosis.

Ectopic ACTH syndrome caused by desmopressin-responsive thymic neuroendocrine tumor.

A 32-year-old Chinese woman with rapid weight gain and progressive edema was found to have typical Cushingoid features. Her endocrine data were consistent with a diagnosis of ACTH-dependent Cushing's syndrome. To differentiate ectopic ACTH syndrome (EAS) from Cushing's disease (CD), various dynamic endocrine and imaging tests were performed. Her ACTH response was negative to corticotropin-releasing hormone (CRH) and positive to desmopressin. Magnetic resonance imaging of the pituitary showed no mass lesion. Computed tomography scan of the chest revealed a large mass (21 × 15 mm) in the anterior mediastinum, where positron emission tomography showed accumulation of [(18)F] fluorodeoxyglucose. Selective venous sampling showed marked step-up in ACTH level in the internal thoracic vein but not in the cavernous sinus after CRH stimulation. These data are compatible with the diagnosis of EAS. The resected tumor was pathologically consistent with thymic neuroendocrine tumor (NET) positive for ACTH by immunohistochemistry and abundant V1b receptor gene expression by RT-PCR. Postoperatively, her circulating ACTH/cortisol levels became normalized, and responded to stimulation with CRH but not with desmopressin. Her Cushingoid appearance gradually disappeared, and she was free from recurrence 5 years after surgery. This is a rare case of desmopressin-responsive EAS caused by thymic NET with predominant V1b gene expression, which was successfully localized by imaging modalities combined with selective venous sampling.

Adult Oral Health Programs in Japanese Municipalities: Factors Associated with Self-Rated Effectiveness.

Health Japan 21 plan establishes specific targets for aspects of health including oral health for 2010, in an effort to increase health expectancy. Despite this, there has been insufficient improvement in oral health status in adults. The objective of this cross-sectional study was to determine the factors associated with effective oral health programs for adults in Japanese municipalities. Questionnaires were mailed to all 1,472 municipalities in Japan and responses were obtained from 862 municipalities (response rate: 58.6%). After excluding 71 municipalities with "unknown" answer, no answer, or lack of relevant information, we analyzed the data from the remaining 791 municipalities with or without oral health programs for adults self-reported as effective within three years. Multilevel Poisson regression models were used to examine the associations of effective programs with oral health personnel, contact with related agencies, the establishment of Health Japan 21 goals, financial status, the density of dentists and population density at the municipality level, and having oral health personnel at the prefecture level. Three hundred and fifty-four municipalities reported having effective programs. In the fully adjusted model, having dental hygienists in the municipal office (P < 0.05) and a high number of contacts with related agencies (P < 0.05) were significantly associated with having effective programs. These results suggest that having dental hygienists and contact with related agencies such as residents, local dental associations, companies, community general support centers, or medical, nursing or welfare facilities are promoting factors for effective adult oral health programs in Japanese municipalities.

Association between the longest job and oral health: Japan Gerontological Evaluation Study project cross-sectional study.

BACKGROUND: Inequality in oral health is a major challenge. Oral diseases and their risk factors accumulate throughout life. The objective of this cross-sectional study was to examine the association of longest job with oral health status and oral health behavior among older Japanese. METHODS: Subjects were a total of 23,191 (11,310 males and 11,881 females) community-dwelling individuals aged 65 or over, living independently and able to perform daily activities from 30 municipalities across Japan. The outcome variables were oral health status (number of teeth, use of denture or bridge and subjective oral health status) and oral health behavior (dental visit for treatment and use of interdental brush or dental floss). The longest job was used as an explanatory variable. Age, educational attainment, equivalent income, and densities of dentists and population in municipalities were used as covariates. Two-level (first level: individual, second level: municipality) multilevel Poisson regression analyses were performed for each sex. RESULTS: Multilevel Poisson regression analyses showed that all variables of oral health status and oral health behavior were significantly associated with longest job after adjusting for all covariates except denture/bridge use and dental visit for females. People whose longest jobs were sales/service, skilled/labor, agriculture/forestry/fishery or others, or who had no occupation were more likely to have poor oral health status and oral health behavior compared to those whose longest jobs were professional/technical. CONCLUSIONS: The longest job may be one of the major determinants of oral health status and oral health behavior in Japanese older people.

Social determinants of denture/bridge use: Japan gerontological evaluation study project cross-sectional study in older Japanese.

BACKGROUND: Studies suggest that using a denture/bridge may prevent disability in older people. However, not all older people with few remaining teeth use a denture/bridge. This cross-sectional study aimed to examine the social determinants which promote denture/bridge use among older Japanese. METHODS: A total of 54,388 (25,630 males and 28,758 females) community-dwelling individuals aged 65 or over, living independently, able to perform daily activities, and with 19 or fewer teeth. The dependent variable was denture/bridge use. Socio-demographics, number of teeth, present illness, social participation, social support, and social networks were used as individual-level independent variables. Data for social capital were aggregated and used as local district (n = 561 for males, n = 562 for females) -level independent variables. Number of dentists working in hospitals/clinics per population and population density were used as municipality (n = 28) -level independent variables. Three-level multilevel Poisson regression analysis was performed for each sex. RESULTS: High equivalent income, low number of teeth, present illness, and living in a municipality with high population density were significantly associated with denture/bridge use in both sexes in the fully adjusted models (p < 0.05). Denture/bridge use was significantly associated with high educational attainment in males and participating in social groups in females in the fully adjusted model (p < 0.05). No significant associations were observed between denture/bridge use and social capital. CONCLUSIONS: Denture/bridge use was significantly associated with high economic status and present illness in both sexes, high educational attainment in males, and participation in social groups in females among community-dwelling older Japanese after adjusting for possible confounders.

Dental health behavior of parents of children using non-fluoride toothpaste: a cross-sectional study.

BACKGROUND: One of the dental health goals of Health Japan 21, in which the Japanese government clarified its health policy, was to ensure the use of fluoride toothpaste in 90% or more of schoolchildren. This goal was not achieved. The aim of this cross-sectional questionnaire study was to evaluate the characteristics of parents whose children use non-fluoride toothpaste. METHODS: In December 2010, questionnaire forms were sent to 18 elementary schools or school dentists. Students (6-12 years old) were asked to take the forms home for their parents to fill in, and to bring the completed questionnaire to school. The collected questionnaires were mailed from schools to the author's institution by the end of March 2011. The relationship between fluoride in toothpaste and reasons for choice of toothpaste, the child's toothbrushing habits, and attitude toward child caries prevention was examined in the 6,069 respondents who answered all the questions for the analyses and indicated that their children use toothpaste. RESULTS: Non-fluoride toothpaste users accounted for 5.1% of all toothpaste users. Among the children using non-fluoride toothpaste, significantly greater numbers gave 'anti-gingivitis', 'halitosis prevention' or 'tartar control' as reasons for choice of toothpaste; did not give 'has fluoride', 'is cheaper' or 'tastes good' as reasons for choice of toothpaste; or used toothpaste sometimes, or were in 4th - 6th grades. There was no significant relationship between use of non-fluoride toothpaste and measures taken for caries prevention in children. Multilevel (first level: individual, second level: school) logistic regression analysis indicated that use of non-fluoride toothpaste was significantly related to: giving 'anti-gingivitis' (odds ratio: 1.44) as a reason for choice of toothpaste; not giving 'has fluoride' (0.40), 'tastes good' (0.49) or 'is cheaper' (0.50) as the reason for choice of toothpaste; to toothbrushing less often (twice a day: 1.34, once a day or less: 1.46) and to using toothpaste less often (sometimes: 1.39). CONCLUSIONS: It is necessary to teach parents that dental caries is the dental health issue with the highest priority for children, and therefore fluoride toothpaste should be used.

Association between self-reported dental health status and onset of dementia: a 4-year prospective cohort study of older Japanese adults from the Aichi Gerontological Evaluation Study (AGES) Project.

OBJECTIVES: Studies have shown that people with cognitive impairment have poor dental health. However, the direction of causality remains unknown. This prospective cohort study aimed to determine the association between four self-reported dental health variables and dementia onset in older Japanese people. METHODS: Analysis was conducted on 4425 residents 65 years or older. Four self-reported dental health variables included the number of teeth and/or use of dentures, ability to chew, presence/absence of a regular dentist, and taking care of dental health. Data were collected using self-administered questionnaires given in 2003. Records of dementia onset during 2003 to 2007 were obtained from municipalities in charge of the public long-term care insurance system. Age, income, body mass index, present illness, alcohol consumption, exercise, and forgetfulness were used as covariates. RESULTS: Dementia onset was recorded in 220 participants. Univariate Cox proportional hazards models showed significant associations between the dental health variables and dementia onset. In models fully adjusted for all covariates, hazard ratios (95% confidence intervals) of dementia onset of respondents were as follows: 1.85 (1.04-3.31) for those with few teeth and without dentures; 1.25 (0.81-1.93) for those who could not chew very well; 1.44 (1.04-2.01) for those who did not have a regular dentist; and 1.76 (0.96-3.20) for those who did not take care of their dental health. CONCLUSIONS: Few teeth without dentures and absence of a regular dentist, not poor mastication and poor attitudes toward dental health, were associated with higher risk of dementia onset in the older Japanese cohort even after adjustment for available covariates.

Evaluation of salivary cortisol measurements for the diagnosis of subclinical Cushing's syndrome.

Late-night salivary cortisol (NSC) has been recognized as a sensitive and easy-to-perform screening test for the diagnosis of overt Cushing's syndrome (CS). However, there have been few reports on the diagnostic utility of salivary cortisol (SC) measurement in the diagnosis of subclinical Cushing's syndrome (SCS). Therefore, the present study was designed to evaluate the usefulness of SC measurements at late-night and after overnight 1 mg dexamethasone suppression test (DST) for the diagnosis of SCS in 42 patients with adrenal incidentaloma. We evaluated 16 patients with SCS, 12 with nonfunctioning adenoma (NFA), 8 with primary aldosteronism (PA), and 6 with pheochromocytoma (Pheo). NSC levels in SCS patients (0.238 ± 0.106 µg/dL) were significantly (P < 0.05) higher than those in NFA patients (0.154 ± 0.104 µg/dL); the cutoff value (0.11 µg/dL) by ROC analysis gave high sensitivity (100%) with low specificity (50%). Post DST SC levels in SCS patients (0.238 ± 0.116 µg/dL) were significantly (P = 0.0081) higher than those in NFA patients (0.136 ± 0.110 µg/dL); the cutoff value (0.12 µg/dL) by ROC analysis gave high sensitivity (93.8%) with somewhat improved specificity (58.3%). Both NSC and post DST SC levels were comparable between NFA, PA, and Pheo patients. In conclusion, our study revealed that measurements of NSC and/or post DST SC among patients with adrenal incidentaloma prove to have high sensitivities, but low specificities for the diagnosis of SCS from NFA, suggesting its possible alternative option before the screening tests for SCS currently employed in Japan.

Differential expression of genes related to drug responsiveness between sparsely and densely granulated somatotroph adenomas.

There are two main subtypes of GH-producing pituitary adenoma: densely granulated (DG-type) and sparsely granulated (SG-type). Despite the difference in drug responsiveness between the two subtypes, their molecular mechanisms remain unknown. The aim of this study is to evaluate the differential expression of genes related to drug responsiveness between the two subtypes of somatotroph adenoma, and their relationship to the clinical characteristics. Eighty-two acromegaly patients (44 DG-type, 38 SG-type) were studied retrospectively. Clinical characteristics were compared between the two subtypes. Among them, 36 tumor tissue specimens (19 DG-type, 17 SG-type) were available for investigation of the expression of SSTR2, SSTR5 and D2R that are reported to be involved in drug responsiveness by realtime RT-PCR. Protein level was evaluated by immunohistochemical study. Patients with SG-type adenomas were younger in age and showed greater GH suppression by octreotide, but not by bromocriptin, and bigger in size and more invasiveness than DG-type adenomas. The mRNA expression of SSTR2 in DG-type adenomas were greater than those in SG-type adenomas and showed significantly positive correlation with GH suppression by octreotide. There was positive correlation between mRNA and protein levels of SSTR2. These data suggested that the differences of responsiveness to octreotide between DG- and SG-type adenomas are based on the expression levels of SSTR2.

Salusins: newly identified bioactive peptides with hemodynamic and mitogenic activities.

The discovery of endogenous bioactive peptides has typically required a lengthy identification process. Computer-assisted analysis of cDNA and genomic DNA sequence information can markedly shorten the process. A bioinformatic analysis of full-length, enriched human cDNA libraries searching for previously unidentified bioactive peptides resulted in the identification and characterization of two related peptides of 28 and 20 amino acids, which we designated salusin-alpha and salusin-beta. Salusins are translated from an alternatively spliced mRNA of TOR2A, a gene encoding a protein of the torsion dystonia family. Intravenous administration of salusin-alpha or salusin-beta to rats causes rapid, profound hypotension and bradycardia. Salusins increase intracellular Ca2+, upregulate a variety of genes and induce cell mitogenesis. Salusin-beta stimulates the release of arginine-vasopressin from rat pituitary. Expression of TOR2A mRNA and its splicing into preprosalusin are ubiquitous, and immunoreactive salusin-alpha and salusin-beta are detected in many human tissues, plasma and urine, suggesting that salusins are endocrine and/or paracrine factors.

Differential gene expression profiles of POMC-related enzymes, transcription factors and receptors between non-pituitary and pituitary ACTH-secreting tumors.

The differential gene expression of proopiomelanocortin (POMC)-related processing enzymes, transcription factors, and receptors responsible for ACTH secretion between non-pituitary and pituitary ACTH-secreting tumors remains obscure. This study was attempted to determine the gene expression profiles of transcription factors (Tpit, NeuroD1 and IKZF1), proprotein convertase (PC) 1/3 and PC2, and several key receptors linked to ACTH secretion, including corticotrophin releasing hormone receptor (CRHR1), vasopressin receptor 1b (V1bR), somatostatin receptor (SSTR) subtype-2, -5 and dopamine receptor type 2 (D2R) in non-pituitary and pituitary ACTH-secreting tumors. Surgical tissue specimens from carcinoid tumors causing ectopic ACTH syndrome (EAS: n=4) and pituitary tumors causing Cushing's disease (CD: n=13), were subjected to real-time RT-PCR for measurements of each mRNA levels. POMC and CRHR1 mRNA levels in CD were far greater than those in EAS, whereas IKZF1, PC2, SSTR-2 and -5 mRNA levels in EAS were significantly greater than those in CD. NeuroD1, Tpit, PC1/3, V1bR and D2R mRNA levels were comparable between EAS and CD. In conclusion, differential gene expression profiles revealed more abundant mRNA expression in EAS than in CD of 1) IKZF1 with its potential implication of cell differentiation and hormone secretion, 2) PC2 with its possible enhanced processing activity of mature ACTH, and 3) SSTR-2 and -5 with their potential therapeutic application of more selective agonists in EAS patients.

Clinicopathological features of primary aldosteronism associated with subclinical Cushing's syndrome.

Primary aldosteronism (PA), an autonomous aldosterone hypersecretion from adrenal adenoma and/or hyperplasia, and subclinical Cushing syndrome (SCS), a mild but autonomous cortisol hypersecretion from adrenal adenoma without signs or symptoms of Cuhing's syndrome, are now well-recognized clinical entities of adrenal incidentaloma. However, the clinicopathological features of PA associated with SCS (PA/SCS) remain unknown. The present study was undertaken to study the prevalence of PA/SCS among PA patients diagnosed at our institute, and characterize their clinicopathlogical features. The prevalence of PA/SCS was 8 of 38 PA patients (21%) studied. These 8 PA/SCS patients were significantly older and had larger tumor, higher serum potassium levels, lower basal plasma levels of aldosterone, ACTH and DHEA-S as well as lower response of aldosterone after ACTH stimulation than those in 12 patients with aldosterone-producing adenoma without hypercortisolism. All 8 PA/SCS patients showed unilateral uptake by adrenal scintigraphy at the ipsilateral side, whereas the laterality of aldosterone hypersecretion as determined by adrenal venous sampling varied from ipsilateral (3), contralateral (2), and bilateral side (2). 6 PA/SCS patinets who underwent adrenalectomy required hydrocortisone replacement postoperatively. Histopathological analysis of the resected adrenal tumors from 5 PA/SCS patients revealed a single adenoma in 3, and double adenomas in 2, with varying degrees of positive immunoreactivities for steroidgenic enzymes (3ß-HSD, P450(C17)) by immunohistochemical study as well as CYP11B2 mRNA expression as measured by real-time RT-PCR. In conclusion, PA/SCS consists of a variety of adrenal pathologies so that therapeutic approach differs depending on the disease subtype.

Clinicopathological features, biochemical and molecular markers in 5 patients with adrenocortical carcinoma.

Adrenocortical carcinoma (ACC) is a very rare malignant tumor with poor prognosis. To gain insight into the pathogenic significance of ACC, we studied clinicopathological features and gene expression profile in ACC. We analyzed five ACC cases (two men and three women) with the median age of 45-year-old who underwent adrenalectomy at our institute. Endocrine studies revealed that two cases had subclinical Cushing's syndrome (SCS) and one with concomitant estrogen-secreting tumor, while the rest of three cases had non-functioning tumors. Analysis of urinary steroids profile by gas chromatography/mass spectrometry showed increased metabolites of corticosteroid precursors, such as 17-OH pregnenolone, 17-OH progesterone, dehydroepiandorosterone (DHEA), and 11-deoxycortisol in all five cases. The pathological diagnosis of ACC was based on Weiss's criteria with its score ≥ 3. The mean size of the resected tumors was 87 mm and Ki67/MIB1 labeling index, a proliferative marker, was 3-27%. Immunohistochemical analysis revealed a disorganized expression of several steroidogenic enzymes, such as 3ß-hydroxysteroid dehydrogenase, 17α-hydroxylase, and DHEA-sulfotransferase. Among several genes determined by RT-PCR, insulin-like growth factor (IGF)-II mRNA was consistently and abundantly expressed in all 5 tumor tissues. Postoperatively, two cases with SCS developed local recurrence and liver metastasis. The present study suggests that the disorganized expression of steroidogenic enzymes and the overexpression of IGF-II by the tumor are hallmarks of ACC, which could be used as biochemical and molecular markers for ACC.

The cathepsin L gene is a direct target of FOXO1 in skeletal muscle.

FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominant-negative form of FOXO1 or in skeletal-muscle-specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy.

Endothelial dysfunction aassociated with hypercortisolism is reversible in Cushing's syndrome.

Cushing's syndrome (CS) as represented by chronic glucocorticoid excess is associated with increased rate of cardiovascular morbidity and mortality. Because endothelial dysfunction is an early event of atherosclerosis, we investigated whether endothelial dysfunction is associated with CS and reversible. Twenty-one CS patients due to different causes were studied for vascular endothelial function by ultrasound measurement of flow-mediated vasodilation (FMD), among whom 12 patients were re-evaluated after surgical and medical treatment; 12 age- and gender-matched subjects served as control. Percent (%) FMD in CS patients (5.8+/-1.9 %) was significantly (p =0.0014) lower than that in control subjects (8.1+/-1.7 %). In CS patients, %FMD showed significant (p < 0.01) negative correlations with morning serum cortisol levels (r =-0.58) and 24-h urinary free cortisol excretion (r =-0.58). After surgical and medical treatment in CS patients, morning cortisol levels significantly (p =0.0025) decreased from 23.4 [15.6-37.3] to 10.2 [7.7-12.9] microg/dL, whereas %FMD significantly (p =0.0024) increased from 5.2+/-1.9 to 7.8+/-2.3 %; changes of %FMD after treatment significantly (p =0.0004) and inversely correlated with those of morning cortisol levels (r =-0.85), but not with those of body mass index, blood pressure, glycemic or lipid profiles. Taken together, the present study clearly revealed that endothelial dysfunction in CS patients is related to hypercortisolemia and reversible after treatment, suggesting the possible role of cortisol excess in the development of endothelial dysfunction, thereby possibly leading to increased cardiovascular complications.

Ectopic ACTH syndrome caused by bronchial carcinoid tumor indistinguishable from Cushing's disease.

A 75-year-old woman was admitted to our hospital because of a poor glycemic control. She was found to have Cushingoid feature and dynamic endocrine tests showed elevated plasma ACTH and cortisol levels, lack of their circadian rhythm, non-suppressibility to high-dose dexamethasone, responsiveness to CRH, but not to DDAVP, and suppression to octreotide. Pituitary MRI showed an equivocal small lesion. CT scan of the chest showed two nodular lesions in the right lung (S5, S7), while a mild uptake was noted only in S5 lesion by FDG-PET, but positive uptake was only in S7 lesion by somatostatin receptor scintigraphy (SRS). Inferior petrosal sinus sampling revealed a gradient of plasma ACTH after CRH stimulation, consistent with the diagnosis of Cushing' s disease. She underwent middle and inferior lobectomy of the right lung. The resected tumor in S7 was consistent with the diagnosis of a bronchial carcinoid tumor with positive ACTH immunoreactivity, while that of S5 was cryptococcal granuloma. RT-PCR revealed abundant expressions of POMC and SSTR (-1, -2, -5), but not of CRHR and V1bR. Postoperatively, abnormal endocrine data were normalized along with improvement of hypertension and diabetes. This was a diagnostic challenging case with ectopic ACTH syndrome indistinguishable from Cushing' s disease by various endocrine and imaging tests, among which SRS successfully localized the tumor responsible for ectopic ACTH secretion.

Clinical features and management of ectopic ACTH syndrome at a single institute in Japan.

Ectopic ACTH syndrome (EAS) is a diagnostic challenge because it is often indistinguishable from Cushing's disease. We describe our series of EAS patients referred to us during 1992-2009. Among 16 cases (9 females / 7 males), with mean age of 58.4 ± 19.0yr, the ectopic source was identified in ten (proven EAS), whereas unidentified in six (occult/unknown EAS). Their salient clinical manifestations included Cushingoid feature (88%), skin pigmentation (88%), profound hypokalemia (88%), hypertension (75%), diabetes/impaired glucose tolerance (75%), hyperlipidemia (69%), and severe infection (44%). Dynamic endocrine tests revealed markedly elevated plasma ACTH levels (211 ± 116pg/mL) and cortisol levels (60.9 ± 30.1µg/dL) which showed resistance to overnight high-dose (8mg) dexamethasone suppression test in 15 (94%) and unresponsiveness to CRH stimulation in 12 (75%). No ACTH gradient during inferior petrosal sampling was noted in 13 of 15 (87%). Imaging tests by CT/MRI identified the tumors in 8 of 16 (50%), in 4 of 11 (36%) and 4 of 6 (66.7%) octreotide-responders by somatostatin receptor scintigraphy, but in only one of 9 (11.1%) by FDG-PET scan. Six cases deceased, including small cell carcinoma (2) and adenocarcinoma (1) of lung, neuroendocrine carcinoma of pancreas (1) and stomach (1), and olfactory neuroblastoma (1), whereas 4 cases survived after removal of the tumors, including bronchial carcinoid tumor (3) and thymic hyperplasia (1). Six occult/unknown EAS patients survived for 67.5 months after medical treatment with metyrapone to control hypercortisolism. Thus, various endocrine tests combined with imaging studies are required to correctly localize the tumors. Control of hypercortisolemia by metyrapone, even if tumor is unrecognized, is critical for better prognosis, and the long-term follow-up by repeated endocrine and imaging tests is mandatory.

Successful localization of ectopic ACTH-secreting bronchial carcinoid by selective pulmonary arterial sampling.

Among patient with ACTH-dependent Cushing's syndrome, about 10-20% of those with ectopic ACTH syndromes (EAS) have occult or unknown tumors. Despite the intensive search for the culprit tumors by dynamic endocrine tests and imaging tests, it is often difficult to localize and confirm the source of occult ectopic ACTH secretion. We report a patient with EAS caused by a small bronchial carcinoid tumor, which was successfully localized by a selective pulmonary arterial sampling for the first time. A 69-year-old woman presented with typical Cushingoid features and elevated plasma ACTH and cortisol levels, which showed lack of circadian rhythm, no suppression by high-dose dexamethasone, and no response to CRH stimulation. No mass lesion was detected by pituitary MRI, and inferior petrosal sinus sampling showed no central to peripheral ACTH gradient. Although CT scan of the chest revealed a very small nodule in the right lung, it could not be confirmed by either somatostatin receptor scintigraphy or fluorodeoxyglucose positron emission tomography. Selective pulmonary arterial sampling of the wedged blood from a pulmonary artery branch affecting the nodule showed a marked ACTH gradient, and the lobectomy of the right middle lung resulted in dramatic decreases in plasma ACTH and cortisol levels. The resected tumor was diagnosed as a bronchial carcinoid tumor with positive immunostaining for ACTH. Thus, selective pulmonary arterial sampling, because of its more site-selective measurement of hormonal secretion, could be one of the useful tools to localize and confirm the ectopic ACTH production by a small pulmonary tumor.