The Short Cosyntropin Test Revisited: New Normal Reference Range Using LC-MS/MS.

Background: The cosyntropin test is used to diagnose adrenal insufficiency (AI) and nonclassical congenital adrenal hyperplasia (NCCAH). Current cutoffs for cortisol and 17-hydroxyprogesterone (17-OHP) are derived from nonstandardized immunoassays. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers direct measurement of steroids, prompting the need to re-establish normal ranges. Objective: The goal of this study was to define cutoff values for cortisol and 17-OHP in serum by LC-MS/MS 30 and 60 minutes after intravenous administration of 250 µg tetracosactide acetate to healthy volunteers and to compare the results with LC-MS/MS with routine immunoassays. Methods: Cosyntropin testing was performed in healthy subjects (n = 138) and in patients referred for evaluation of adrenocortical function (n = 94). Steroids were assayed by LC-MS/MS and compared with two immunoassays used in routine diagnostics (Immulite and Roche platforms). The cutoff level for cortisol was defined as the 2.5% percentile in healthy subjects not using oral estrogens (n = 121) and for 17-OHP as the 97.5% percentile. Results: Cortisol cutoff levels for LC-MS/MS were 412 and 485 nmol/L at 30 and 60 minutes, respectively. Applying the new cutoffs, 13 of 60 (22%) subjects who had AI according to conventional criteria now had a normal test result. For 17-OHP, the cutoff levels were 8.9 and 9.0 nmol/L at 30 and 60 minutes, respectively. Conclusions: LC-MS/MS provides cutoff levels for cortisol and 17-OHP after cosyntropin stimulation that are lower than those based on immunoassays, possibly because cross-reactivity between steroid intermediates and cortisol is eliminated. This reduces the number of false-positive tests for AI and false-negative tests for NCCAH.

ATAD2 overexpression links to enrichment of B-MYB-translational signatures and development of aggressive endometrial carcinoma.

We have explored the potential for clinical implementation of ATAD2 as a biomarker for aggressive endometrial cancer by investigating to what extent immunohistochemical (IHC) staining for ATAD2 is feasible, reflects clinical phenotype and molecular subgroups of endometrial carcinomas. Increased expression of the ATAD2 gene has been implicated in cancer development and progression in a number of tissues, but few studies have investigated ATAD2 expression using IHC. Here we show that high ATAD2 protein expression is significantly associated with established clinical-pathological variables for aggressive endometrial cancer, also in the subset of estrogen receptor α (ERα) positive tumors. Protein and mRNA expression of ATAD2 were highly correlated (P < 0.001), suggesting that IHC staining may represent a more clinically applicable measure of ATAD2 level in routinely collected formalin fixed paraffin embedded specimens. Gene expression alterations in samples with high ATAD2 expression revealed upregulation of several cancer-related genes (B-MYB, CDCs, E2Fs) and gene sets that previously have been linked to aggressive disease and potential for new targeting therapies. Our results support that IHC staining for ATAD2 may be a clinically applicable biomarker reflecting clinical phenotype and targetable alterations in endometrial carcinomas to be further explored in controlled clinical trials.

Photopic full-field electroretinography and optical coherence tomography in type 1 diabetic retinopathy.

PURPOSE: The purpose of this study was to evaluate the role of photopic full-field electroretinography (ERG) and retinal thickness measurements by spectral-domain optical coherence tomography (SD-OCT) in the assessment of disease severity in type 1 diabetic retinopathy. METHODS: A population-based cohort of 151 patients with type 1 diabetes underwent a complete ophthalmic examination, including photopic full-field ERG and SD-OCT for retinal thickness measurements. Stereoscopic fundus photographs were taken according to the Early Treatment Diabetic Retinopathy Study protocol, and the classification of diabetic retinopathy was based on the International Clinical Diabetic Retinopathy Disease Severity Scale. Associations between photographically determined retinopathy level, b-wave amplitude and peak time of the photopic single-flash and 30-Hz flicker ERG, and central retinal thickness parameters were evaluated. RESULTS: For all ERG measurements, the amplitude decreased and peak time increased with progression of the disease, but these associations lost statistical significance after adjusting for age and excluding laser-treated patients. Mean retinal thickness was significantly associated with the b-wave amplitude of photopic single-flash and 30-Hz flicker responses (r(2) = 0.08, p = 0.006; and r(2) = 0.05, p = 0.025, respectively), but revealed no association with retinopathy level. CONCLUSIONS: Photopic full-field ERG and SD-OCT-derived retinal thickness parameters have limited clinical value in the staging of diabetic retinopathy. However, thinning of the central retina leads to significant functional impairment and may reflect an ongoing neurodegenerative process in the retinal tissue.

Increased angiogenesis is associated with a 32-gene expression signature and 6p21 amplification in aggressive endometrial cancer.

BACKGROUND: Angiogenesis is a hallmark of cancer. The aim of this study was to explore whether microvessel proliferation is associated with gene expression profiles or copy number alterations in endometrial cancer. METHODS: A prospective series of endometrial carcinomas was studied for angiogenesis markers, gene expression profiles, and gene copy number data. For validation, an independent series of endometrial carcinomas as well as an external cohort of endometrial cancer patients were examined by gene expression microarrays. RESULTS: Increased microvessel proliferation (MVP) was associated with aggressive tumor features and reduced survival, and a 32-gene expression signature was found to separate tumors with high versus low MVP. An increased 32-gene signature score was confirmed to associate with high-grade tumor features and reduced survival by independent cohorts. Copy number studies revealed that amplification of the 6p21 region was significantly associated with MVP, a high 32-gene score, as well as reduced survival. CONCLUSION: Increased MVP was significantly associated with aggressive endometrial cancer and reduced survival. Integrated analyses demonstrated significant associations between increased vascular proliferation, amplification of the 6p21 region, VEGF-A mRNA expression, and the 32-gene angiogenesis signature. Our findings indicate amplification of 6p21 as a possible driver of tumor vascular proliferation in endometrial cancer.

Carboxyl-ester lipase maturity-onset diabetes of the young is associated with development of pancreatic cysts and upregulated MAPK signaling in secretin-stimulated duodenal fluid.

Carboxyl-ester lipase (CEL) maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes and pancreatic exocrine dysfunction due to mutations in the CEL gene encoding CEL. The pathogenic mechanism for diabetes development is unknown. Since CEL is expressed mainly in pancreatic acinar cells, we asked whether we could find structural pancreatic changes in CEL-MODY subjects during the course of diabetes development. Furthermore, we hypothesized that the diseased pancreas releases proteins that are detectable in pancreatic fluid and potentially reflect activation or inactivation of disease-specific pathways. We therefore investigated nondiabetic and diabetic CEL-mutation carriers by pancreatic imaging studies and secretin-stimulated duodenal juice sampling. The secretin-stimulated duodenal juice was studied using cytokine assays, mass spectrometry (MS) proteomics, and multiplexed MS-based measurement of kinase activities. We identified multiple pancreatic cysts in all eight diabetic mutation carriers but not in any of the four nondiabetic mutation carriers or the six healthy controls. Furthermore, we identified upregulated mitogen-activated protein kinase (MAPK) target proteins and MAPK-driven cytokines and increased MAPK activity in the secretin-stimulated duodenal juice. These findings show that subjects with CEL-MODY develop multiple pancreatic cysts by the time they develop diabetes and that upregulated MAPK signaling in the pancreatic secretome may reflect the pathophysiological development of pancreatic cysts and diabetes.

Integrated genomic analysis of the 8q24 amplification in endometrial cancers identifies ATAD2 as essential to MYC-dependent cancers.

Chromosome 8q24 is the most commonly amplified region across multiple cancer types, and the typical length of the amplification suggests that it may target additional genes to MYC. To explore the roles of the genes most frequently included in 8q24 amplifications, we analyzed the relation between copy number alterations and gene expression in three sets of endometrial cancers (N = 252); and in glioblastoma, ovarian, and breast cancers profiled by TCGA. Among the genes neighbouring MYC, expression of the bromodomain-containing gene ATAD2 was the most associated with amplification. Bromodomain-containing genes have been implicated as mediators of MYC transcriptional function, and indeed ATAD2 expression was more closely associated with expression of genes known to be upregulated by MYC than was MYC itself. Amplifications of 8q24, expression of genes downstream from MYC, and overexpression of ATAD2 predicted poor outcome and increased from primary to metastatic lesions. Knockdown of ATAD2 and MYC in seven endometrial and 21 breast cancer cell lines demonstrated that cell lines that were dependent on MYC also depended upon ATAD2. These same cell lines were also the most sensitive to the histone deacetylase (HDAC) inhibitor Trichostatin-A, consistent with prior studies identifying bromodomain-containing proteins as targets of inhibition by HDAC inhibitors. Our data indicate high ATAD2 expression is a marker of aggressive endometrial cancers, and suggest specific inhibitors of ATAD2 may have therapeutic utility in these and other MYC-dependent cancers.

Lack of estrogen receptor-α is associated with epithelial-mesenchymal transition and PI3K alterations in endometrial carcinoma.

PURPOSE: We hypothesized that estrogen receptor-α (ER-α) status in endometrial carcinomas, associated with poor prognosis, is reflected in transcriptional signatures suggesting targets for new therapy. EXPERIMENTAL DESIGN: Endometrial carcinoma samples in a primary investigation cohort (n = 76) and three independent validation cohorts (n = 155/286/111) were analyzed through integrated molecular profiling. Biomarkers were assessed by immunohistochemistry (IHC), DNA oligonucleotide microarray, quantitative PCR (qPCR), single-nucleotide polymorphism (SNP) array, and Sanger sequencing in the cohorts, annotated for comprehensive histopathologic and clinical data, including follow-up. RESULTS: ER-α immunohistochemical staining was strongly associated with mRNA expression of the receptor gene (ESR1) and patient survival (both P < 0.001). ER-α negativity associated with activation of genes involved in Wnt-, Sonic Hedgehog-, and TGF-ß signaling in the investigation cohort, indicating epithelial-mesenchymal transition (EMT). The association between low ER-α and EMT was validated in three independent datasets. Furthermore, phosphoinositide 3-kinase (PI3K) and mTOR inhibitors were among the top-ranked drug signatures negatively correlated with the ER-α-negative tumors. Low ER-α was significantly associated with PIK3CA amplifications but not mutations. Also, low ER-α was correlated to high expression of Stathmin, a marker associated with PTEN loss, and a high PI3K activation signature. CONCLUSION: Lack of ER-α in endometrial cancer is associated with EMT and reduced survival. We present a rationale for investigating ER-α's potential to predict response to PI3K/mTOR inhibitors in clinical trials and also suggest EMT inhibitors to ER-α-negative endometrial carcinomas.

Lipocalin 2 expression is associated with aggressive features of endometrial cancer.

BACKGROUND: Increased expression of lipocalin 2 (LCN2) has been observed in several cancers. The aim of the present study was to investigate LCN2 in endometrial cancer in relation to clinico-pathologic phenotype, angiogenesis, markers of epithelial-mesenchymal transition (EMT), and patient survival. METHODS: Immunohistochemical staining was performed using a human LCN2 antibody on a population-based series of endometrial cancer patients collected in Hordaland County (Norway) during 1981-1990 (n = 256). Patients were followed from the time of primary surgery until death or last follow-up in 2007. The median follow-up time for survivors was 17 years. Gene expression data from a prospectively collected endometrial cancer series (n = 76) and a publicly available endometrial cancer series (n = 111) was used for gene correlation studies. RESULTS: Expression of LCN2 protein, found in 49% of the cases, was associated with non-endometrioid histologic type (p = 0.001), nuclear grade 3 (p = 0.001), >50% solid tumor growth (p = 0.001), ER and PR negativity (p = 0.028 and 0.006), and positive EZH2 expression (p < 0.001). LCN2 expression was significantly associated with expression of VEGF-A (p = 0.021), although not with other angiogenesis markers examined (vascular proliferation index, glomeruloid microvascular proliferation, VEGF-C, VEGF-D or bFGF2 expression). Further, LCN2 was not associated with several EMT-related markers (E-cadherin, N-cadherin, P-cadherin, ß-catenin), nor with vascular invasion (tumor cells invading lymphatic or blood vessels). Notably, LCN2 was significantly associated with distant tumor recurrences, as well as with the S100A family of metastasis related genes. Patients with tumors showing no LCN2 expression had the best outcome with 81% 5-year survival, compared to 73% for intermediate and 38% for the small subgroup with strong LCN2 staining (p = 0.007). In multivariate analysis, LCN2 expression was an independent prognostic factor in addition to histologic grade and FIGO stage. CONCLUSION: Increased LCN2 expression is associated with aggressive features and poor prognosis in endometrial cancer.

Oncogenic PIK3CA-driven mammary tumors frequently recur via PI3K pathway-dependent and PI3K pathway-independent mechanisms.

PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy, making phosphatidylinositol 3-kinase (PI3K) a target for cancer therapy. Despite the promise of targeted therapy, resistance often develops, leading to treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CA(H1047R). Notably, most PIK3CA(H1047R)-driven mammary tumors recurred after PIK3CA(H1047R) inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of Met or Myc (also known as c-Met and c-Myc, respectively). Whereas Met amplification led to tumor survival dependent on activation of endogenous PI3K, tumors with Myc amplification became independent of the PI3K pathway. Functional analyses showed that Myc contributed to oncogene independence and resistance to PI3K inhibition. Notably, PIK3CA mutations and c-MYC elevation co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.

Integrated genome-wide DNA copy number and expression analysis identifies distinct mechanisms of primary chemoresistance in ovarian carcinomas.

PURPOSE: A significant number of women with serous ovarian cancer are intrinsically refractory to platinum-based treatment. We analyzed somatic DNA copy number variation and gene expression data to identify key mechanisms associated with primary resistance in advanced-stage serous cancers. EXPERIMENTAL DESIGN: Genome-wide copy number variation was measured in 118 ovarian tumors using high-resolution oligonucleotide microarrays. A well-defined subset of 85 advanced-stage serous tumors was then used to relate copy number variation to primary resistance to treatment. The discovery-based approach was complemented by quantitative-PCR copy number analysis of 12 candidate genes as independent validation of previously reported associations with clinical outcome. Likely copy number variation targets and tumor molecular subtypes were further characterized by gene expression profiling. RESULTS: Amplification of 19q12, containing cyclin E (CCNE1), and 20q11.22-q13.12, mapping immediately adjacent to the steroid receptor coactivator NCOA3, was significantly associated with poor response to primary treatment. Other genes previously associated with copy number variation and clinical outcome in ovarian cancer were not associated with primary treatment resistance. Chemoresistant tumors with high CCNE1 copy number and protein expression were associated with increased cellular proliferation but so too was a subset of treatment-responsive patients, suggesting a cell-cycle independent role for CCNE1 in modulating chemoresponse. Patients with a poor clinical outcome without CCNE1 amplification overexpressed genes involved in extracellular matrix deposition. CONCLUSIONS: We have identified two distinct mechanisms of primary treatment failure in serous ovarian cancer, involving CCNE1 amplification and enhanced extracellular matrix deposition. CCNE1 copy number is validated as a dominant marker of patient outcome in ovarian cancer.

Visual attention in patients with intracranial arachnoid cysts.

OBJECTIVE: To investigate whether intracranial arachnoid cysts (AC) compromise visual attention and if so, whether surgical cyst decompression leads to improvement in visual attention performance. METHODS: The experiments were carried out on 27 patients with temporal (n=21) or frontal (n=6) AC, and 27 healthy control subjects. All subjects were tested with two different visual attention paradigms. Patients were tested one day before and a minimum of 3 months after the surgery, with the same test-retest interval for the controls. RESULTS: AC impair both automatic and effortful attention. These attention impairments were significantly improved after surgery, also when controlling for learning and practice effects from pre- to post-surgery testing. Closer analysis showed that these effects were carried by patients with right hemisphere cysts for impairment in shift of attention, and by patients with a left hemisphere cyst for visual search. CONCLUSIONS: AC may impair visual attention. Cyst location may be of importance for the development of these attention deficits, as there were significant differences between patients with right hemisphere cysts and those with left hemisphere cysts. This dyscognition appears to be reversible following surgical decompression. Surgical decompression of AC may thus relieve not only clinical symptoms and complaints, but cognitive impairments as well.

Associations between cod liver oil use and symptoms of depression: the Hordaland Health Study.

BACKGROUND: Clinical trials suggest that omega-3 fatty acids improve the outcome of depression. This study aimed to evaluate the association between intake of cod liver oil, rich in omega-3 fatty acids, and high levels of symptoms of depression and anxiety in the general population. METHODS: We used data from the "The Hordaland Health Study '97-'99" (HUSK), a population based cross-sectional health survey from Norway including 21,835 subjects aged 40-49 and 70-74 years. Symptoms of depression and anxiety were measured by The Hospital Anxiety and Depression Scale (HADS). We used logistic regression to study associations. RESULTS: Among the participants, 8.9% used cod liver oil daily. A total of 3.6% had high levels of depressive symptoms. The prevalence of such depressive symptoms among the subjects who used cod liver oil daily was 2.5%, as compared to 3.8% in the rest of the population. The users of cod liver oil were significantly less likely to have depressive symptoms than non-users after adjusting for multiple possible confounding factors (odds ratio=0.71, 95% confidence interval 0.52 to 0.97). These factors included age, gender, smoking habits, coffee consumption, alcohol consumption, physical activity, and education. In addition, we found that the prevalence of high levels of depressive symptoms decreased with increasing duration (0-12 months) of cod liver oil use (multivariate adjusted test for trend, P=0.04). We were only able to study this latter association in a subset of the population aged 40-46 years. LIMITATIONS: Data are cross sectional. CONCLUSIONS: The findings indicate that regular use of cod liver oil is negatively associated with high levels of depressive symptoms in the general population.

Obesity, dyslipidemia, and diabetes with selective serotonin reuptake inhibitors: the Hordaland Health Study.

OBJECTIVE: This study aimed to examine whether subjects taking selective serotonin reuptake inhibitors (SSRIs) are more likely to have elements of the metabolic syndrome compared with those taking no psychotropic drugs. For comparison, we also studied subjects taking antipsychotic drugs. METHOD: We used data from The Hordaland Health Study '97-'99, a general community cross-sectional health survey including 25,315 subjects aged 40 to 49 and 70 to 74 years. For the groups studied, we estimated prevalence and odds ratios (ORs) for obesity, hypercholesterolemia, low high-density lipoprotein cholesterol, hyper-triglyceridemia, and diabetes. RESULTS: We observed an association between use of SSRIs as a group (N = 461) and abdominal obesity (OR = 1.40, 95% CI = 1.08 to 1.81) and hypercholesterolemia (OR = 1.36, 95% CI = 1.07 to 1.73) after adjusting for multiple possible confounders. There was also a trend toward an association between SSRI use and diabetes. In a subgroup analysis of subjects taking SSRIs, the use of paroxetine (N = 187) was markedly associated with both general and abdominal obesity but not with hypercholesterolemia. In contrast, the use of citalopram (N = 142) was not associated with any of the metabolic outcome variables, while the use of any other SSRI (sertraline, fluoxetine, or fluvoxamine) (N = 131) as a mixed subgroup was associated with both abdominal obesity and hypercholesterolemia. We also replicated the previously reported associations between use of antipsychotics and obesity and metabolic disturbances. CONCLUSION: We have shown that use of at least some SSRIs is associated with clinical and biochemical elements of the metabolic syndrome. Our data indicate differences in the metabolic side effect profile among various SSRI drugs, although treatment bias might have influenced these results. We suggest that patients taking SSRIs be carefully monitored for obesity and dyslipidemia.

SREBP activation by antipsychotic- and antidepressant-drugs in cultured human liver cells: relevance for metabolic side-effects?

Drug-induced weight gain is a major problem in the treatment of psychiatric disorders, especially with some antipsychotic- and antidepressant drugs. We have recently demonstrated that antipsychotic- and antidepressant drugs activate the SREBP (sterol regulatory element-binding proteins) transcription factors in human- and rat glial cells, with subsequent up-regulation of downstream genes involved in cholesterol- and fatty acid biosynthesis. Since stimulation of cellular lipogenesis in the liver could be of relevance for the metabolic side effects of these drugs, we have now investigated the effects of antidepressants, antipsychotic- and mood-stabilizing drugs on cell cultures of human liver cells. For several of the drugs being strongly associated with weight gain (clozapine, imipramine, and amitriptyline), we observed a very pronounced activation of SREBP. Ziprasidone and buproprion, however, which are not associated with weight gain, did hardly stimulate the SREBP system. For haloperidol, olanzapine and mirtazapine, the correspondence between metabolic side effects and SREBP stimulation in liver cells was less obvious. The mood-stabilizers did not increase SREBP activation. The results indicate a relationship between drug-induced activation of SREBP in cultured human liver cells and weight gain side-effects of antidepressant and antipsychotic drugs.

Antidepressant drugs activate SREBP and up-regulate cholesterol and fatty acid biosynthesis in human glial cells.

Dysfunction of glial lipid metabolism and abnormal myelination has recently been reported in both schizophrenia and bipolar disorder. Cholesterol is a major component of myelin, and glia-produced cholesterol serves as a glial growth factor in synaptogenesis. We have recently demonstrated that antipsychotic drugs activate the sterol regulatory element-binding protein (SREBP) transcription factors in human and rat glial cells, with subsequent up-regulation of numerous downstream genes involved in cholesterol and fatty acid biosynthesis. Since this stimulation of cellular lipogenesis could represent a new mechanism of action of psychotropic drugs, we investigated whether antidepressants and mood-stabilizers were able to induce a similar activation of SREBP-controlled lipid biosynthesis. Cultured human glioma cells (GaMg) were exposed to the antidepressant drugs imipramine, amitriptyline, clomipramine, citalopram, fluoxetine, mirtazapine and bupropion and the mood-stabilizers/antiepileptics lithium, valproate and carbamazepine. All antidepressant drugs activated the SREBP system with subsequent up-regulation of the downstream lipogenesis-related genes, although to a markedly different extent. The mood-stabilizers did not affect the SREBPs or the downstream genes. These results link antidepressant drugs, but not mood-stabilizers, to SREBP-mediated activation of cellular lipogenesis, and demonstrate a functional similarity between antipsychotic and antidepressant molecular drug action.

Arachnoid cysts cause cognitive deficits that improve after surgery.

The authors investigated cognition in patients with arachnoid cysts by assessing 55 patients and 32 control subjects on four different cognitive tests preoperatively and 3 months postoperatively. The patients improved their performance from preoperative to postoperative testing, whereas the control subjects did not show a similar improvement.