Cardiomyocyte maturation alters molecular stress response capacities and determines cell survival upon mitochondrial dysfunction.

Cardiomyocyte maturation during pre- and postnatal development requires multiple intertwined processes, including a switch in energy generation from glucose utilization in the embryonic heart towards fatty acid oxidation after birth. This is accompanied by a boost in mitochondrial mass to increase capacities for oxidative phosphorylation and ATP generation required for efficient contraction. Whether cardiomyocyte differentiation is paralleled by augmented capacities to deal with reactive oxygen species (ROS), physiological byproducts of the mitochondrial electron transport chain (ETC), is less clear. Here we show that expression of genes and proteins involved in redox homeostasis and protein quality control within mitochondria increases after birth in the mouse and human heart. Using primary embryonic, neonatal and adult mouse cardiomyocytes in vitro we investigated how excessive ROS production induced by mitochondrial dysfunction affects cell survival and stress response at different stages of maturation. Embryonic and neonatal cardiomyocytes largely tolerate inhibition of ETC complex III by antimycin A (AMA) as well as ATP synthase (complex V) by oligomycin but are susceptible to complex I inhibition by rotenone. All three inhibitors alter the intracellular distribution and ultrastructure of mitochondria in neonatal cardiomyocytes. In contrast, adult cardiomyocytes treated with AMA undergo rapid morphological changes and cellular disintegration. At the molecular level embryonic cardiomyocytes activate antioxidative defense mechanisms, the integrated stress response (ISR) and ER stress but not the mitochondrial unfolded protein response upon complex III inhibition. In contrast, adult cardiomyocytes fail to activate the ISR and antioxidative proteins following AMA treatment. In conclusion, our results identified fundamental differences in cell survival and stress response in differentiated compared to immature cardiomyocytes subjected to mitochondrial dysfunction. The high stress tolerance of immature cardiomyocytes might allow outlasting unfavorable intrauterine conditions thereby preventing fetal or perinatal heart disease and may contribute to the regenerative capacity of the embryonic and neonatal mammalian heart.

USP8 Mutations in Pituitary Cushing Adenomas-Targeted Analysis by Next-Generation Sequencing.

Gain-of-function somatic mutations in the ubiquitin specific protease 8 (USP8) gene have recently been reported as a cause of pituitary adenomas in Cushing disease. Molecular diagnostic testing of tumor tissue may aid in the diagnosis of specimens obtained through therapeutic transsphenoidal surgery; however, for small tumors, availability of fresh tissue is limited, and contamination with normal tissue is frequent. We performed molecular testing of DNA isolated from single formalin-fixed and paraffin-embedded (FFPE) tissue sections of 42 pituitary adenomas from patients with Cushing disease (27 female patients and 15 male patients; mean age at surgery, 42.5 years; mean tumor size, 12.2 mm). By Sanger sequencing, we identified previously reported USP8 missense mutations in six tumors. Targeted next-generation sequencing (NGS) revealed known or previously undescribed missense mutations in three additional tumors (two with two different mutations each), with mutant allele frequencies as low as 3%. Of the nine tumors with USP8 mutations (mutation frequency, 21.4%), seven were from female patients (mutation frequency, 25.9%), and two were from male patients (mutation frequency, 13.3%). Mutant tumors were on average 11.4 mm in size, and patients with mutations were on average 43.9 years of age. The overall USP8 mutation frequency in our cohort was lower than in previously described cohorts, and we did not observe USP8 deletions that were frequent in other cohorts. We demonstrate that testing for USP8 variants can be performed from small amounts of FFPE tissue. NGS showed higher sensitivity for USP8 mutation detection than did Sanger sequencing. Assessment for USP8 mutations may complement histopathological diagnosis.

Targeting CXCR4 (CXC Chemokine Receptor Type 4) for Molecular Imaging of Aldosterone-Producing Adenoma.

Primary aldosteronism is the most frequent cause of secondary hypertension and is associated with increased morbidity and mortality compared with hypertensive controls. The central diagnostic challenge is the differentiation between bilateral and unilateral disease, which determines treatment options. Bilateral adrenal venous sampling, currently recommended for differential diagnosis, is an invasive procedure with several drawbacks, making it desirable to develop novel noninvasive diagnostic tools. When investigating the expression pattern of chemokine receptors by quantitative real-time polymerase chain reaction and immunohistochemistry, we observed high expression of CXCR4 (CXC chemokine receptor type 4) in aldosterone-producing tissue in normal adrenals, adjacent adrenal cortex from adrenocortical adenomas, and in aldosterone-producing adenomas (APA), correlating strongly with the expression of CYP11B2 (aldosterone synthase). In contrast, CXCR4 was not detected in the majority of nonfunctioning adenomas that are frequently found coincidently. The specific CXCR4 ligand 68Ga-pentixafor has recently been established as radiotracer for molecular imaging of CXCR4 expression and showed strong and specific binding to cryosections of APAs in our study. We further investigated 9 patients with primary aldosteronism because of APA by 68Ga-pentixafor-positron emission tomography. The tracer uptake was significantly higher on the side of increased adrenocortical aldosterone secretion in patients with APAs compared with patients investigated by 68Ga-pentixafor-positron emission tomography for other causes. Molecular imaging of aldosterone-producing tissue by a CXCR4-specific ligand may, therefore, be a highly promising tool for noninvasive characterization of patients with APAs.

Epitope-Specific Antitumor Immunity Suppresses Tumor Spread in Papillary Thyroid Cancer.

Context: Papillary thyroid cancer (PTC) is characterized by a lymphocytic infiltration. PTC patients with lymphocytic infiltration may have a better clinical outcome. Objective: Characterization of tumor epitope-specific immunity and correlation analyses with the clinical outcome. Patients: 150 PTC patients; 40 Hashimoto thyroiditis (HT) patients; 21 healthy controls; 27,239 healthy whites (for HLA typing). Main Outcome Measures: HLA class I restricted thyroperoxidase (TPO) and thyroglobulin (Tg) epitope-specific T cells (tetramer analyses), correlation analyses between HLA class II phenotypes, T cell immunity, and the clinical course. Results: The frequency of TPO- and Tg-specific CD8+ T cells in PTC patients was largely increased compared with healthy controls (TPO and Tg, P < 0.005 and P < 0.005) and was similar to those in HT patients. HLA-DQB1*03-positive PTC patients had a significantly lower risk [risk ratio (RR), 0.170; 95% confidence interval (CI), 0.037 to 0.755; P < 0.05] and HLA-DRB1*03-positive and HLA-DQB1*02-positive PTC patients a significantly higher risk (HLA-DRB1*03: RR, 4.400; 95% CI, 1.378 to 14.05; P < 0.05; HLA-DQB1*02: RR, 3.692; 95% CI, 1.102 to 12.38; P < 0.05) for distant metastases, compared with patients with other haplotypes. HLA-DQB1*03-positive PTC patients revealed an increased responsiveness of tumor epitopes in vitro. These tumor epitope-specific CD8+ T cells were also found in lymph node metastases of HLA-DQB1*03-positive PTC patients. Conclusion: We demonstrate a tumor epitope-specific immunity in PTC patients and the protective role of HLA-DQB1*03 against metastatic spread. These results have direct implications for new treatment options with immune checkpoint inhibitors.

Inhibitor of Apoptosis Proteins (IAPs) are commonly dysregulated in GIST and can be pharmacologically targeted to enhance the pro-apoptotic activity of imatinib.

Gastrointestinal stromal tumors (GIST) exhibit a strong oncogenic dependency on KIT and KIT inhibitors confer long lasting disease stabilization in the majority of patients. Nonetheless, KIT inhibition alone does not cure GIST as a subset of GIST cells evade apoptosis and eventually develop resistance. Inhibitors of Apoptosis Proteins (IAPs) may confer resistance to drug-induced apoptosis. We observed that the mRNA and protein of IAPs XIAP (BIRC4) and survivin (BIRC5) were highly expressed in primary GIST tumors and cell line models. Amplification of the respective gene loci (BIRC2, BIRC3, BIRC4, BIRC5) was detected in 47% of GIST studied by SNP arrays. Whole exome analyses revealed a mutation of SMAC(DIABLO) in a heavily pretreated patient. Both, survivin (rank 62-92/11.194 tested proteins) and XIAP (rank 106-557/11.194) were found to be essential proteins for survival in a synthetic lethality screen. Expression of XIAP and survivin decreased upon KIT inhibition and may play a role in KIT-regulated pro-survival signaling. SMAC-mimetic treatment with LCL161 and TL32711 reduced cIAP1 and XIAP expression. Survivin inhibitor YM155 lead to transcriptional repression of BIRC5/survivin (YM155) and induced apoptosis. Combinational treatment with KIT inhibitors (imatinib, regorafenib) enhanced the proapoptotic effect. These findings support the combination of KIT inhibition with IAP antagonists in GIST.

Metastatic Malignant Melanoma With Complete Loss of Differentiation Markers (Undifferentiated/Dedifferentiated Melanoma): Analysis of 14 Patients Emphasizing Phenotypic Plasticity and the Value of Molecular Testing as Surrogate Diagnostic Marker.

Metastatic malignant melanoma is notorious for its phenotypic diversity and loss of differentiation markers. We herein summarized our experience with 14 metastatic melanomas showing complete loss of immunohistochemical melanocytic markers (with or without heterologous differentiation). Patients included 11 men and 3 women aged 24 to 78 years (median, 67 y). Thirteen patients had histologically confirmed primary skin melanoma, and 1 had metastatic melanoma of unknown primary. Undifferentiated metastasis was diagnosed synchronous to primary tumor (n=1), following skin melanoma by 3 months to 9 years (n=11) and preceding it by 1 year (n=1). Sites of undifferentiated metastases were axillary (3), inguinal (1), or submandibular (1) lymph nodes, digestive tract (2), bone/soft tissue (2), lung/pleura (2), and disseminated (n=3). Histology of metastases mimicked undifferentiated pleomorphic or spindle cell sarcoma with variable myxoid and giant cell areas (n=10) and cytokeratin-positive undifferentiated small cell sarcoma (n=1). Three cases showed heterologous dedifferentiation: pleomorphic rhabdomyosarcoma (n=1), teratocarcinosarcoma-like with prominent rhabdomyoblasts (n=1), and adenocarcinoma-like with metaplastic bone (n=1). All cases were negative for S100, melanoma cocktail, HMB45, Melan A, and SOX10. Other markers showed following results: smooth muscle actin (1/14), p16 (1/14), TP53 (2/12), pancytokeratin (4/14), desmin (5/14), h-caldesmon (0/9), and MDM2/CDK4 (0/5). SMARCB1 was intact in 8/8 cases. Genotyping showed BRAF(V600E) mutation (5/14), NRAS mutation (5/14), and BRAF/NRAS wild-type (4/14). In conclusion, undifferentiated/dedifferentiated metastatic melanoma is likely underrecognized and frequently mistaken for undifferentiated sarcoma or other neoplasms. Diagnosis of undifferentiated sarcoma at sites where melanoma metastasis are frequent (eg, inguinal and axillary region) should be made with great caution and warrants exploration of the remote history. Genotyping is a helpful surrogate marker in classifying such difficult cases. In the light of available targeted therapies, recognition of undifferentiated/dedifferentiated metastatic melanoma is mandatory for appropriate treatment.

Urachal Carcinoma of the Bladder: Impact of Clinical and Immunohistochemical Parameters on Prognosis.

PURPOSE: Urachal carcinoma of the bladder is a rare malignancy. Its histological phenotype is similar to that of primary bladder and colorectal adenocarcinoma. The aim of this study was to explore the expression and prognostic relevance of 6 select protein markers of urachal carcinoma of the bladder, including p53, Ki67, RHAMM, BGN, IMP3 and MMP-7, which were formerly shown to be prognostic in urothelial carcinoma and colorectal adenocarcinoma. MATERIALS AND METHODS: Clinical and followup data were obtained on a total of 26 patients with urachal carcinoma of the bladder treated at 2 university hospitals. Immunohistochemical analysis of p53, Ki67, RHAMM, BGN, IMP3 and MMP-7 expression was performed in samples from 15 patients. Clinicopathological parameters and immunohistochemical results were tested for prognostic value on univariable and multivariable analyses. RESULTS: Followup was 50 months. Five-year overall and progression-free survival was 46% and 32%, respectively. On multivariable analysis a positive resection margin was an independent predictor of poor overall survival (p = 0.025). RHAMM (p = 0.0431), IMP3 (p = 0.0052), Ki67 (p = 0.0006) and p53 (p = 0.0024) expression rates were significantly increased in urachal carcinoma of the bladder cells compared to normal urothelium. IMP3 was elevated in Sheldon tumor stage IIIA compared to IIIB or greater (p = 0.0048). None of the analyzed protein markers was associated with survival. CONCLUSIONS: The independent prognostic value of a positive resection margin underlines the importance of complete surgical removal of urachal carcinoma of the bladder combined with en bloc resection of the median umbilical ligament and umbilicus. Our results in a limited number of samples show that Ki67, p53, RHAMM and IMP3 expression is enhanced but has no prognostic significance in urachal carcinoma of the bladder.

Differential proteomic and tissue expression analyses identify valuable diagnostic biomarkers of hepatocellular differentiation and hepatoid adenocarcinomas.

The exact discrimination of lesions with true hepatocellular differentiation from secondary tumours and neoplasms with hepatocellular histomorphology like hepatoid adenocarcinomas (HAC) is crucial. Therefore, we aimed to identify ancillary protein biomarkers by using complementary proteomic techniques (2D-DIGE, label-free MS). The identified candidates were immunohistochemically validated in 14 paired samples of hepatocellular carcinoma (HCC) and non-tumourous liver tissue (NT). The candidates and HepPar1/Arginase1 were afterwards tested for consistency in a large cohort of hepatocellular lesions and NT (n = 290), non-hepatocellular malignancies (n = 383) and HAC (n = 13). Eight non-redundant, differentially expressed proteins were suitable for further immunohistochemical validation and four (ABAT, BHMT, FABP1, HAOX1) for further evaluation. Sensitivity and specificity rates for HCC/HAC were as follows: HepPar1 80.2%, 94.3% / 80.2%, 46.2%; Arginase1 82%, 99.4% / 82%, 69.2%; BHMT 61.4%, 93.8% / 61.4%, 100%; ABAT 84.4%, 33.7% / 84.4%, 30.8%; FABP1 87.2%, 95% / 87.2%, 69.2%; HAOX1 95.5%, 36.3% / 95.5%, 46.2%. The best 2×/3× biomarker panels for the diagnosis of HCC consisted of Arginase1/HAOX1 and BHMT/Arginase1/HAOX1 and for HAC consisted of Arginase1/FABP1 and BHMT/Arginase1/FABP1. In summary, we successfully identified, validated and benchmarked protein biomarker candidates of hepatocellular differentiation. BHMT in particular exhibited superior diagnostic characteristics in hepatocellular lesions and specifically in HAC. BHMT is therefore a promising (panel based) biomarker candidate in the differential diagnostic process of lesions with hepatocellular aspect.

Novel somatic mutations in primary hyperaldosteronism are related to the clinical, radiological and pathological phenotype.

UNLABELLED: Aldosterone-producing adenomas (APAs) and bilateral adrenal hyperplasia are important causes of secondary hypertension. Somatic mutations in KCNJ5, CACNA1D, ATP1A1, ATP2B3 and CTNNB1 have been described in APAs. OBJECTIVE: To characterize clinical-pathological features in APAs and unilateral adrenal hyperplasia, and correlate them with genotypes. DESIGN: Retrospective study. SUBJECTS AND MEASUREMENTS: Clinical and pathological characteristics of 90 APAs and seven diffusely or focally hyperplastic adrenal glands were reviewed, and samples were examined for mutations in known disease genes by Sanger or exome sequencing. RESULTS: Mutation frequencies were as follows: KCNJ5, 37·1%; CACNA1D, 10·3%; ATP1A1, 8·2%; ATP2B3, 3·1%; and CTNNB1, 2·1%. Previously unidentified mutations included I157K, F154C and two insertions (I150_G151insM and I144_E145insAI) in KCNJ5, all close to the selectivity filter, V426G_V427Q_A428_L433del in ATP2B3 and A39Efs*3 in CTNNB1. Mutations in KCNJ5 were associated with female and other mutations with male gender (P = 0·007). On computed tomography, KCNJ5-mutant tumours displayed significantly greater diameter (P = 0·023), calculated area (P = 0·002) and lower precontrast Hounsfield units (P = 0·0002) vs tumours with mutations in other genes. Accordingly, KCNJ5-mutant tumours were predominantly comprised of lipid-rich fasciculata-like clear cells, whereas other tumours were heterogeneous (P = 5 × 10(-6) vs non-KCNJ5 mutant and P = 0·0003 vs wild-type tumours, respectively). CACNA1D mutations were present in two samples with hyperplasia without adenoma. CONCLUSIONS: KCNJ5-mutant tumours appear to be associated with fasciculata-like clear cell predominant histology and tend to be larger with a characteristic imaging phenotype. Novel somatic KCNJ5 variants likely cause adenomas by loss of potassium selectivity, similar to previously described mutations.

NOX4 in Mitochondria: Yeast Two-Hybrid-Based Interaction with Complex I Without Relevance for Basal Reactive Oxygen Species?

NADPH oxidases (NOXs) represent the only known dedicated source of reactive oxygen species (ROS) and thus a prime therapeutic target. Type 4 NOX is unique as it produces H2O2, is constitutively active, and has been suggested to localize to cardiac mitochondria, thus possibly linking mitochondrial and NOX-derived ROS formation. The aim of this study was to identify NOX4-binding proteins and examine the possible physiological localization of NOX4 to mitochondria and its impact on mitochondrial ROS formation. We here provide evidence that NOX4 can, in principle, enter protein-protein interactions with mitochondrial complex I NADH dehydrogenase subunits, 1 and 4L. However, under physiological conditions, NOX4 protein was neither detectable in the kidney nor in cardiomyocyte mitochondria. The NOX inhibitor, GKT136901, slightly reduced ROS formation in cardiomyocyte mitochondria, but this effect was observed in both wild-type and Nox4(-/-) mice. NOX4 may thus associate with mitochondrial complex I proteins, but in cardiac and renal mitochondria under basal conditions, expression is beyond our detection limits and does not contribute to ROS formation.

PRKACA mutations in cortisol-producing adenomas and adrenal hyperplasia: a single-center study of 60 cases.

OBJECTIVE: Cortisol excess due to adrenal adenomas or hyperplasia causes Cushing's syndrome. Recent genetic studies have identified a somatic PRKACA(L206R) mutation as a cause of cortisol-producing adenomas. We aimed to compare the clinical features of PRKACA-mutant lesions with those of CTNNB1 mutations, and to search for similar mutations in unilateral hyperplasia or tumors co-secreting aldosterone. DESIGN, PATIENTS, AND METHODS: In this study, 60 patients with cortisol excess who had adrenalectomies at our institution between 1992 and 2013 were assessed, and somatic mutations were determined by Sanger sequencing. A total of 36 patients had overt Cushing's syndrome, the remainder were subclinical: 59 cases were adenomas (three bilateral) and one was classified as hyperplasia. Four tumors had proven co-secretion of aldosterone. RESULTS: Among cortisol-secreting unilateral lesions without evidence of co-secretion (n=52), we identified somatic mutations in PRKACA (L206R) in 23.1%, CTNNB1 (S45P, S45F) in 23.1%, GNAS (R201C) in 5.8%, and CTNNB1+GNAS (S45P, R201H) in 1.9%. PRKACA and GNAS mutations were mutually exclusive. Of the co-secreting tumors, two (50%) had mutations in KCNJ5 (G151R and L168R). The hyperplastic gland showed a PRKACA(L206R) mutation, while patients with bilateral adenomas did not have known somatic mutations. PRKACA-mutant lesions were associated with younger age, overt Cushing's syndrome, and higher cortisol levels vs non-PRKACA-mutant or CTNNB1-mutant lesions. CTNNB1 mutations were more significantly associated with right than left lesions. CONCLUSIONS: PRKACA(L206R) is present not only in adenomas, but also in unilateral hyperplasia and is associated with more severe autonomous cortisol secretion. Bilateral adenomas may be caused by yet-unknown germline mutations.

Perfil epidemiológico dos pacientes atendidos no Centro Auditivo de Petrolina-PE / Epidemiological profile of patients treated in Petrolina-PE Auditory Center

OBJETIVO: caracterizar o perfil epidemiológico de pacientes atendidos no Centro Auditivo de Petrolina-PE segundo sexo, idade, cidade em que residem e tipo de perda auditiva no ano de 2009. MÉTODO: participaram desta pesquisa todos os pacientes atendidos no Centro Auditivo de Petrolina-PE no ano de 2009, totalizando 553 indivíduos. Foram considerados apenas os prontuários com dados completos e exames audiológicos realizados, sendo este o critério de inclusão no estudo. Foram excluídos 73 indivíduos que tinham os prontuários incompletos. RESULTADOS: amostra foi composta por 480 prontuários, destes, 55,2 por cento (265) eram do gênero feminino e 44,8 por cento (215) do gênero masculino. Em relação a faixa etária, observou-se que a média de idade foi de 49,1 anos, variando entre 4 e 93 anos. A distribuição da população segundo o tipo de perda auditiva foi de 220 (22,9 por cento) orelhas com limiares auditivos normais, 572 (59,5 por cento) com perda auditiva sensorioneural, 134 (13,9 por cento) mistas e 34 (3,5 por cento) condutivas. Em relação a cidade de origem dos participantes observou-se que 392 (81,6 por cento) indivíduos residem em Petrolina e 82 (18,4 por cento) em outras cidades de Pernambuco. CONCLUSÃO: o presente estudo demonstrou que a deficiência auditiva predominantemente foi do tipo sensorioneural. Em relação ao gênero, houve predomínio do feminino. Apresentou-se considerável número de pessoas com deficiência auditiva acima dos 60 anos, mostrando assim a necessidade de programa de reabilitação de idosos.

PURPOSE: to characterize the epidemiological profile of patients attending the Hearing Center of Petrolina-PE by gender, age, city where they reside and type of hearing loss, in 2009. METHOD: all patients treated at the Hearing Center of Petrolina-PE in 2009 took part in this study, totaling 553 subjects. We considered only the records with complete data and audiological findings, with this being the criteria for inclusion in the study. We excluded 73 subjects whose records were incomplete. RESULTS: the sample consisted of 480 medical records, namely: 55.2 percent (265) were female and 44.8 percent (215) male. Regarding age, we noted that the mean age was 49.1 years, ranging between 4 and 93 years. The population distribution according to type of hearing loss was 220 (22.9 percent) ears with normal hearing thresholds, 572 (59.5 percent) with sensorineural hearing loss, 134 (13.9 percent) and 34 mixed (3.5 percent) conductive. Regarding the city of origin of the participants, the study showed 392 (81.6 percent) subjects residing in Petrolina and 82 (18.4 percent) in other Pernambuco cities. CONCLUSION: this study showed that hearing loss was predominantly sensorineural. Regarding gender, there was a female predominance. there was a considerable number of people with hearing loss over 60 year old, and such being the case, showing the need for a rehabilitation program for the elder.