Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing.

BACKGROUND: The external quality assurance (EQA) process aims at establishing laboratory performance levels. Leading European groups in the fields of EQA, Pathology, and Medical and Thoracic Oncology collaborated in a pilot EQA scheme for somatic epidermal growth factor receptor (EGFR) gene mutational analysis in non-small-cell lung cancer (NSCLC). METHODS: EQA samples generated from cell lines mimicking clinical samples were provided to participating laboratories, each with a mock clinical case. Participating laboratories performed the analysis using their usual method(s). Anonymous results were assessed and made available to all participants. Two subsequent EQA rounds followed the pilot scheme. RESULTS: One hundred and seventeen labs from 30 countries registered and 91 returned results. Sanger sequencing and a commercial kit were the main methodologies used. The standard of genotyping was suboptimal, with a significant number of genotyping errors made. Only 72 out of 91 (72%) participants passed the EQA. False-negative and -positive results were the main sources of error. The quality of reports submitted was acceptable; most were clear, concise and easy to read. However, some participants reported the genotyping result in the absence of any interpretation and many obscured the interpretation required for clinical care. CONCLUSIONS: Even in clinical laboratories, the technical performance of genotyping in EGFR mutation testing for NSCLC can be improved, evident from a high level of diagnostic errors. Robust EQA can contribute to global optimisation of EGFR testing for NSCLC patients.

Cloning of PBDX, an MIC2-related gene that spans the pseudoautosomal boundary on chromosome Xp.

The pseudoautosomal boundaries are the interface between pseudoautosomal and sex chromosome-specific DNA sequences. We have isolated a gene, PBDX, from the human pseudoautosomal boundary region of Xp. The three exons at the 5' end of PBDX are situated in the pseudoautosomal region immediately downstream of MIC2, whereas the other seven exons are in the X-specific region. Hence, PBDX is inherited in two modes: its 5' end is pseudoautosomally inherited and its 3' end is X-linked. The predicted amino acid sequence of the 540 bp coding region is 48% homologous to 12E7, the product of MIC2. By virtue of its position, PBDX becomes an excellent candidate for the XG blood group gene.

Hypoxia-inducible factor pathway activation in restless legs syndrome patients.

BACKGROUND AND PURPOSE: These studies tested the hypothesis that hypoxia inducible factor-1α (HIF-1α) pathway activation occurs in substantia nigra neurons and brain microvasculature in patients with restless legs syndrome. METHODS: Immunohistochemical analyses of substantia nigra tissue from six RLS and six control subjects were analyzed for HIF-1α, neuronal nitric oxide synthase (nNOS) and nitrotyrosine immunoreactivity. Microvessel lysates were obtained from cortex tissue from four RLS and four control subjects and the lysates were quantified for HIF-2α and vascular endothelial growth factor (VEGF) expression using immunoblot analyses. HIF-1α activation of peripheral blood monocyte cells (PBMCs) (14 RLS and 9 control) was determined through immunoblot analysis of PBMC lysates for EPO. RESULTS: HIF-1α immunoreactivity in substantia nigra neurons was significantly increased in five of six RLS patients as compared with controls. In addition, nNOS and nitrotyrosine expression are up-regulated in the substantia nigra of four of six RLS patients as compared with controls. HIF-2α and VEGF expression are significantly up-regulated in the microvasculature lysates from four RLS cortical brain tissue as compared with controls. Erythropoietin levels are significantly increased in RLS PBMCs. CONCLUSIONS: These results demonstrate that the hypoxia pathway is activated in multiple cell types in individuals with RLS. Increased nNOS and nitrotyrosine suggests that nitric oxide is involved in the activation. Activation of the hypoxia pathway can result from or contribute to cellular iron deficiency. These observations suggest a novel direction to explore in RLS that is tied to the iron deficiency model but better explains the findings in postmortem studies.

Gender differences in functional connectivity during emotion regulation.

Gender differences in emotion regulation (ER) have been postulated, yet their neural basis remains poorly understood. The goal of this study was to investigate this issue from a functional connectivity (FC) perspective. Utilizing a region of interest (ROI) analysis, we investigated whether men and women (N = 48) differed in their FC pattern while viewing versus regulating negative emotion induced by highly salient pictures, and whether this pattern related to their self-reported negative affect and suppression success. Despite women reporting more negative affect, both genders had comparable suppression success. Moreover, differences emerged between men and women's FC patterns. During the regulation of negative emotion, better suppression in women was associated with stronger FC within a cingulo-opercular network, while men exhibited stronger FC within posterior regions of the ventral attentional network. We conclude that due to their propensity for higher emotional reactivity, women may employ a frontal top-down control network to downregulate negative emotion, while men may redirect attention away from the negative stimulus by using posterior regions of the ventral attention network. The findings may have significant implications for understanding women's vulnerability for developing affective disorders and developing targeted individualized treatment.

Biochemical and morphological comparison of plasma membrane and milk fat globule membrane from bovine mammary gland.

Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids. The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components, beta-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane. Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes. Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources. Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material. Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents.

Saturation in milk and meat fats.

Meat and milk products from ruminants (cows, goats, sheep, and beef animals) contribute 35 to 40 percent of the fat in the average American diet. Such fat is highly saturated, containing less than about 4 percent polyunsaturated fatty acids. The unsaturated plant lipids (fats) ordinarily consumed by the ruminant are hydrogenated (saturated) in the rumen. Transport and incorporation of this hydrogenated fat into meat and milk follows. Rumen hydrogenation does not take place until the fat is broken down to free fatty acids, thus establishing the fact that lipolysis is an essential feature of the process. Circumvention of this lipolysis may lead to more-unsaturated meat and milk fat.

Food value of red tide (Gonyaulax polyedra).

Two harvests of ocean-growing red tide, comprised mainly of Gonyaulax polyedra, were evaluated in limited trials of rat feeding. The protein of red tide (25 to 30 percent, dry basis) supported growth satisfactorily. The essential amino acid composition of the protein closely resembles that of casein, the major protein of milk. As a marine resource, plankton represents a challenge for research.

Quality of diagnostic mutation analyses for phenylketonuria.

DNA sequence analyses have become a major component in the diagnostic work-up of patients; however, limited consideration appears to be given to the possibility that reported results may in fact be wrong. Over the last four years we have carried out an External Quality Assessment scheme for mutation analysis in phenylketonuria. Each year, three DNA samples with previously characterized genotypes were mailed to participating laboratories. Indications for testing were either confirmation of diagnosis and prediction of disease severity, or carrier analysis. Each year there were several laboratories that failed to identify mutations because of methodological limitations. Of the participating laboratories that used comprehensive mutation detection methods, each year there was at least one that missed at least one mutation. Indeed, in the 2007 scheme almost 8% of reports from laboratories that used comprehensive mutation detection methods such as sequencing of all exons of the PAH gene contained incorrect genotypes. There were also serious deficiencies in the interpretation of genotype data: in the 2007 scheme, 6 out of 10 laboratories that obtained full genotyping marks for interpretation incurred a reduction of marks because information on the expected phenotype was missing or wrong. Several laboratories failed to appreciate the clinical relevance of a mutation associated with mild hyperphenylalaninaemia, which does not require treatment, and some discussed the option of prenatal diagnosis in the respective case. In conclusion, mutation analyses may be prone to errors and this demands careful interpretation of results in relation to clinical and biochemical findings.

Tetrahydrobiopterin alters superoxide and nitric oxide release in prehypertensive rats.

Constitutive nitric oxide synthase (cNOS) with insufficient cofactor (6R)-5,6,7,8-tetrahydrobiopterin (H4B) may generate damaging superoxide (O2-). This study was designed to determine whether cNOS-dependent generation of O2- occurs in spontaneously hypertensive rats (SHR) before the onset of hypertension. Aortas from 4-wk-old SHR and Wistar-Kyoto rats were used. cNOS was stimulated by calcium ionophore A23187. In situ measurements of nitric oxide and hydrogen peroxide by electrochemical sensors and O2- production by chemiluminescence method were performed. Isometric tension was continuously recorded. H4B by high performance liquid chromatography and [3H]citrulline assay were determined in homogenized tissue. The A23187-stimulated production of O2- and its superoxide dismutase product hydrogen peroxide were significantly higher, whereas nitric oxide release was reduced in SHR aortas, with opposite results in the presence of exogenous H4B. Furthermore, NG-monomethyl-L-arginine inhibited the generation of cNOS-dependent O2- by approximately 70%. Natural H4B levels were similar in both strains; however, equivalent cNOS activity required additional H4B in SHR. The endothelium-dependent relaxations to A23187 were significantly inhibited by catalase, and enhanced by superoxide dismutase, only in SHR; however, these enzymes had no effect in the presence of H4B. Thus, dysfunctional cNOS may be a source of O2- in prehypertensive SHR and contribute to the development of hypertension and its vascular complications.

Cost-effectiveness of angiotensin-converting enzyme inhibitors versus angiotensin II receptor blockers as first-line treatment in autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a rare kidney disorder impacting ∼1:2,500 individuals among the general US population. Hypertension is a significant predictor of ADPKD progression, and a risk factor for development of cardiovascular disease (CVD), the most common cause for mortality among ADPKD patients. Angiotensin-converting enzymes inhibitors (ACE-I) are widely used as first-line treatment in ADPKD for the management of hypertension. However, their cost-effectiveness relative to other hypertensive medications, such as angiotensin II receptor blockers (ARB), has never been assessed. OBJECTIVE: To determine if ARB are more cost-effective than ACE-Is as first-line treatment in ADPKD. METHODS: A Markov-state decision model was constructed for estimation of cost and outcome benefits in hypertensive ADPKD patients. Transition probabilities were extrapolated from a retrospective cohort study comparing chronic kidney disease (CKD) stage transitions in ADPKD patients. Annual pharmaceutical costs per average daily dose per CKD stage were extracted from a US healthcare claims database. Median total healthcare costs per CKD stage or transplant were extracted from the published literature. The time horizon was set to 30 years, with 1-year duration to cycle shift. A cost-effectiveness analysis was conducted to estimate the incremental cost-effectiveness ratio (ICER) of ACE-I vs ARB per additional year of prevented transplant and/or death. A one-way probabilistic sensitivity analysis was conducted, with 10% variation in probabilities and cost. RESULTS: Total annual healthcare costs accrued after 30 years among ADPKD patients taking ACE-Is was estimated to be $3,505,028.41, compared to ARB at $3,644,327.65. Life expectancy was increased by 1.39 years among patients taking ACE-I. Approximate 10-year survival in patients taking ACE-Is was 47% compared to ARB at 34%. CONCLUSIONS: ACE-I dominated ARB and displayed greater cost-effectiveness due to lower cost and increased capacity to prolong years of life without transplant or death among hypertensive ADPKD patients. This model strengthens the value of ACE-I over ARB as first-line treatment for hypertension management in ADPKD patients.

Activation of the ras-mitogen-activated protein kinase pathway and phosphorylation of ets-2 at position threonine 72 in human ovarian cancer cell lines.

The activation status of the ras pathway was studied in eight ovarian tumor cell lines. Three biochemical parameters indicative of ras activation were tested: (a) the ratio of the ras-GTP:ras-GDP complex; (b) the activity of mitogen-activated protein kinases p42/p44; and (c) ets-2 phosphorylation at position threonine 72, a mitogen-activated protein kinase phosphorylation site in vivo. Four of the ovarian tumor cell lines had an activated ras pathway by these three parameters, whereas only one of these contained a mutated ras gene. In addition, ras/ets-2 responsive genes such as the urokinase plasminogen activator (uPA) were activated in these four cell lines. Transient transfection assays indicated that the compound ets-AP1 oncogene responsive enhancer present in the uPA gene was the target of ras signaling in ovarian tumor cells and that the combination of activated ras and ets-2 could superactivate the uPA enhancer element. Coexpression of the dominant-negative ras-Asn17 cDNA gene abrogated activity of this uPA element in ovarian tumor cells. These data indicate that ets-2 is a nuclear target of ras action in ovarian tumor cell lines and that ras signaling pathways may be activated in ovarian cancer by mechanisms independent of direct genetic damage to ras genes.

Release of remnant plasma membrane from milk fat globules by Triton X-100.

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material (50 000 X g, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-releasing material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20-22 degrees C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.

Incidence and characteristics of cell pieces on human milk fat globules.

For biochemical and nutritional purposes, milk fat globules of a species are assumed to be more or less uniform. The incidence of organelle-containing cytoplasm (crescents) on human milk fat globules was determined by fluorescence microscopy employing acridine orange. The percentage of globules with crescents in 20 predominantly morning samples from 17 donors ranged from 1 to 38% with three-quarters falling between 3 and 8%. However, analysis of morning and night samples from eight donors showed a significant nocturnal rise in the proportion of globules with crescents. Values for night samples were, on average, twice those for morning samples. Crescents deteriorate in human milk. These changes begin in the breast, with marked disintegration occurring in milk during 36-h storage at 2 degrees C. Crescents were isolated from globules by churning and separated from the released membrane by selective centrifugation. SDS-polyacrylamide gel electrophoresis showed that crescents contribute bands which render the globule protein pattern more complex. The significance of crescents in globule secretion, globule membrane preparation, infant nutrition and as a source of mammary cell components is discussed.

In vivo effects of colchicine on milk fat globule membrane.

Milk secretion in lactating goats was suppressed reversibly by infusing colchicine (2.5 to 5 mg) into one half of the udder via the teat canal. Fat globules were isolated from milks before, during and after (96 h post-infusion) this suppression. Protein, phospholipid, cholesterol (free and esterified), 5'-nucleotidase activity and peptide patterns by gel electrophoresis of these globule samples were determined. Association of [14C]colchicine with milk fat globules in vivo and in vitro also was investigated. Amounts of protein, phospholipid and free cholesterol per g of globule and 5'-nucleotidase per mg of globule protein fall following colchicine infusion. The nature of these changes suggests that the supply of membrane for milk secretion is restricted as a result of the drug treatment. Patterns of globule peptides by gel electrophoresis were qualitatively similar during the experimental period. However, a major globule glycoprotein, Mr = 52 000, showed a significant (3-fold) increase relative to the other principal peptide bands during the period of reduced milk flow. Analysis of milks for radioactivity following infusion of [14C]colchicine revealed that a portion of activity returning in milk is associated with fat globules. This activity peaked at 72 h post-infusion. Evaluation of [14C]colchicine binding to milk fat globules in vitro yielded evidence that the drug binds to the cytoplasmic, but not the exterior surface of the globule membrane. Colchicine's inhibition of milk synthesis and secretion is discussed.

The epithelial mucin, MUC1, of milk, mammary gland and other tissues.

MUC1 is a mucin-type glycoprotein that is integrally disposed in the apical plasma membrane of the lactating epithelial cell and protrudes from the cell surface into the alveolar lumen where milk is stored. Envelopment of milk fat globules by this membrane accomplishes their secretion and conveys MUC1 into milk. The human form of this mucin has been detected in many other organs, tissues and body fluids. It projects from the cell surface as long filaments. In the human and a number of other species, MUC1 is polymorphic due to variable numbers of a tandemly repeated segment 20 amino acids in length. The individual codominantly expresses two alleles for the mucin so that differences in its size among individuals and between the two forms of an individual are observed. The tandem repeats are rich in serines and threonines which serve as O-glycosylation sites. Carbohydrate content of MUC1, as isolated from milk of human, bovine and guinea pig, is approximately 50%. The oligosaccharides carry substantial sialic acid at their termini and this accounts for two putative functions of this mucin, i.e., to keep ducts and lumens open by creating a strong negative charge on the surface of epithelial cells which would repel opposite sides of a vessel, and to bind certain pathogenic microorganisms. MUC1 is protease resistant (trypsin, chymotrypsin and pepsin) and large fragments of it can be found in the feces of some but not all breast-fed infants. MUC1 has a highly varied structure because of its polymorphism, qualitative and quantitative variations in its glycosylation between tissues, individuals and species, and differences due to divergence in the nucleotide sequences among species. Sequencing of the MUC1 gene for various species is showing promise of revealing unique evolutionary relationships and has already indicated conserved aspects of the molecule that may be functionally important. Among these are positions of serine, threonine and proline in the tandem repeats and a high degree of homology in the transmembrane and cytoplasmic segments of the molecule.

The supression of milk fat globule secretion by clochicine: an effect coupled to inhibition of exocytosis.

The effects of colchicine on release of milk lipids from mammary tissue were evaluated by biochemical analysis of milk and morphological study of the tissue following intramammary infusions of the alkaloid into lactating goats. Colchicine produces a reversible drop in milk yield. As the flow of milk resumes, 36--48 h after infusion, the fat content of the milk increases, phospholipid per g of total globule lipid falls, mean size of milk fat globules increases and diameters of fat droplets (presecretory milk fat globules) within lactating cells approximately double. These observations are consistent with the conclusion that colchicine suppresses milk fat globule secretion but that globules continue to grow in size wihtin cells during the suppression period. These findings indicate that secretion of milk fat globules and the skim milk phase are coupled.

Differences between individuals in high-molecular weight glycoproteins from mammary epithelia of several species.

Milk fat globules are secreted by envelopment in plasma membrane of the lactating cell. SDS-gel electrophoresis of proteins from this membrane has revealed differences between milk donors in two mucin-like glycoproteins. One of these glycoproteins resolves in 3% acrylamide stacking gel and the other in 4% running gel. The proteins vary in number of bands (one or two) and band mobilities. This polymorphism arises, at least in part, from expression of hypervariable genes. In this study, gel electrophoretic evidence of similar polymorphism in glycoproteins from cow, chimpanzee, horse and human milks is presented. In distinction to the other species, the cow expressed only one of these proteins which was detected in the running gel at Mr 180,000 to 200,000. The electrophoresis pattern for this protein from six cows was highly varied with respect to number (one or two) and position of bands. Peanut agglutinin, wheat germ agglutinin and concanavalin A all were bound specifically by bands of the bovine glycoprotein. Binding of concanavalin A distinguishes the bovine protein from the two human glycoproteins. Further studies of species differences should help shed light on the evolution of these unique glycoproteins and their possible functions in mother and young.

Antiserum to the milk fat globule membrane. Preparation and capacity to suppress milk secretion.

A procedure is described for preparing rabbit antiserum to goat milk fat globule membrane. This membrane is derived from the secretory surface of the lactating cell. Immunoelectrophoresis and enzyme-linked immunosorbent assay showed that antibody development reached maximal levels in about 6--8 weeks. Infusion of 5--10 ml of this antiserum into the lactating mammary gland of goats via the teat canal depressed milk yields temporarily on the infused side to 60--80% of normal. Ordinary serum from rabbit, goat or human did not evoke such a response and rabbit complement was not essential for the effect. Fractionation showed that the globulin fraction of the antiserum contained the milk-suppressing principle. Milk from the antiserum-infused side of the udder showed extensive and tenacious clumping of fat globules on standing 12--24 h. The inhibition of milk flow by antibodies to the secretory membrane resembles a previously observed inhibition following infusion of concanavalin A or its succinyl derivative. Binding of antibodies or lectins which recognize specific surface protein components of the lactating cell appears to be involved in the suppression mechanism. The possible relevance of our findings to autoimmune suppression of exocytosis is noted.

Suppression of milk secretion (exocytosis) by concanavalin a in vitro.

The observation that concanavalin A can inhibit milk secretion was evaluated in an in vitro system employing minced mammary gland or isolated alveoli from lactating rats. Release of milk constituents (casein, lactose and fat globules) into the medium in the presence and absence of concanavalin A was measured during 1 or 2 h incubations. The effect of concanavalin A on glucose uptake and CO2 production of the minced tissue was also studied. Concanavalin A suppressed release of milk components at a concentration as low as 80 micrograms/ml of medium. Respiration of minced mammary tissue in the presence of concanavalin A (100 micrograms/ml of medium) was essentially the same as that of the control. The data are evidence that concanavalin A acts directly on the mammary cell in suppressing milk secretion and that the effect is not due to cytotoxicity.

Secretion of prosaposin, a multifunctional protein, by breast cancer cells.

Western blotting and immunodetection with three antibodies were used to probe conditioned media of breast cancer cells (MDA231, MDA435, MCF-7) for prosaposin, a lysosomal protein that occurs in milk. It was readily detected in media from these cells, and from that of an sv40-transformed mammary epithelial cell, HBL100, but not from medium of human neural tumor cells (SK-N-MC). In cultures of MCF-7 cells, the prosaposin pattern of secretion over time closely resembled that of procathepsin D, another lysosomal protein occurring in milk. Supplementing medium with 17beta-estradiol (0. 1-100 nM) dose dependently increased secretion of both proteins after 48 h without changes in cell viability. The influence of 17beta-estradiol on secretion could play a role in the trophic activity of prosaposin in cellular differentiation and cell death protection. In concert with other lysosomal proteins in the tumor environment, such as procathepsin D, prosaposin may be a factor in eliminating barriers to tumor metastasis by facilitating hydrolysis of membrane glycolipids. The number of milk proteins known to be secreted by breast cancer cells is growing. There is evidence that at least some of these may be secreted in an endocrine manner in the normal, non-lactating breast.