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Environmental science and pollution research international
2018

Toxicological evaluation of nail polish waste discarded in the environment.

Felzenszwalb, Israel, Fernandes, Andreia da Silva, Brito, Lara Barroso, Oliveira, Gisele Augusto Rodrigues, Silva, Paula Aquino Soeiro, Arcanjo, Maria Elena, Marques, Monica Regina da Costa, Vicari, Taynah, Leme, Daniela Morais, Cestari, Marta Margarete, Ferraz, Elisa Raquel Anastacio

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Digital object identifier (DOI): 10.1007/s11356-018-1880-y

Journal of phycology
2018

Quantitative comparison of taxa and taxon concepts in the diatom genus Fragilariopsis: a case study on using slide scanning, multi-expert image annotation and image analysis in taxonomy.

Beszteri, Bánk, Allen, Claire, Almandoz, Gastón O, Armand, Leanne, Barcena, María Ángeles, Cantzler, Hannelore, Crosta, Xavier, Esper, Oliver, Jordan, Richard W, Kauer, Gerhard, Klaas, Christine, Kloster, Michael, Leventer, Amy, Pike, Jennifer, Rigual Hernández, Andrés S

Semi-automated methods for microscopic image acquisition, image analysis and taxonomic identification have repeatedly received attention in diatom analysis. Less well studied is the question whether and how such methods might prove useful for clarifying the delimitation of species that are difficult to separate for human taxonomists. To try to answer this question, three very similar Fragilariopsis species endemic to the Southern Ocean were targeted in this study: F. obliquecostata, F. ritscheri, and F. sublinearis. A set of 501 extended focus depth specimen images were obtained using a standardized, semi-automated microscopic procedure. Twelve diatomists independently identified these specimen images in order to reconcile taxonomic opinions and agree upon a taxonomic gold standard. Using image analyses, we then extracted morphometric features representing taxonomic characters of the target taxa. The discriminating ability of individual morphometric features was tested visually and statistically, and multivariate classification experiments were performed to test the agreement of the quantitatively-defined taxa assignments with expert consensus opinion. Beyond an updated differential diagnosis of the studied taxa, our study also shows that automated imaging and image analysis procedures for diatoms are coming close to reaching a broad applicability for routine use. This article is protected by copyright. All rights reserved.

Digital object identifier (DOI): 10.1111/jpy.12767

PloS one, 12, e0178877
2017

Depletion of ATP and glucose in advanced human atherosclerotic plaques.

Ekstrand, Matias, Widell, Emma, Hammar, Anna, Akyürek, Levent M, Johansson, Martin, Fagerberg, Björn, Bergström, Göran, Levin, Malin C, Fogelstrand, Per, Borén, Jan, Levin, Max

Severe hypoxia develops close to the necrotic core of advanced human atherosclerotic plaques, but the energy metabolic consequences of this hypoxia are not known. In animal models, plaque hypoxia is also associated with depletion of glucose and ATP. ATP depletion may impair healing of plaques and promote necrotic core expansion. To investigate if ATP depletion is present in human plaques, we analyzed the distribution of energy metabolites (ATP, glucose, glycogen and lactate) in intermediate and advanced human plaques. Snap frozen carotid endarterectomies from 6 symptomatic patients were analyzed. Each endarterectomy included a large plaque ranging from the common carotid artery (CCA) to the internal carotid artery (ICA). ATP, glucose, and glycogen concentrations were lower in advanced (ICA) compared to intermediate plaques (CCA), whereas lactate concentrations were higher. The lowest concentrations of ATP, glucose and glycogen were detected in the perinecrotic zone of advanced plaques. Our study demonstrates severe ATP depletion and glucose deficiency in the perinecrotic zone of human advanced atherosclerotic plaques. ATP depletion may impair healing of plaques and promote disease progression.

Digital object identifier (DOI): 10.1371/journal.pone.0178877

Appl. Sci., 7, 330
2017

Large-Scale Permanent Slide Imaging and Image Analysis for Diatom Morphometrics

Michael Kloster, Oliver Esper, Gerhard Kauer, Bánk Beszteri

Light microscopy analysis of diatom frustules is widely used in basic and applied research, notably taxonomy, morphometrics, water quality monitoring and paleo-environmental studies. Although there is a need for automation in these applications, various developments in image processing and analysis methodology supporting these tasks have not become widespread in diatom-based analyses. We have addressed this issue by combining our automated diatom image analysis software SHERPA with a commercial slide-scanning microscope. The resulting workflow enables mass-analyses of a broad range of morphometric features from individual frustules mounted on permanent slides. Extensive automation and internal quality control of the results helps to minimize user intervention, but care was taken to allow the user to stay in control of the most critical steps (exact segmentation of valve outlines and selection of objects of interest) using interactive functions for reviewing and revising results. In this contribution, we describe our workflow and give an overview of factors critical for success, ranging from preparation and mounting through slide scanning and autofocus finding to final morphometric data extraction. To demonstrate the usability of our methods we finally provide an example application by analysing Fragilariopsis kerguelensis valves originating from a sediment core, which substantially extends the size range reported in the literature.

Digital object identifier (DOI): 10.3390

Sci Rep, 6, 25658
2016

Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.

Villasenor, Roberto, Ozmen, Laurence, Messaddeq, Nadia, Grüninger, Fiona, Loetscher, Hansruedi, Keller, Annika, Betsholtz, Christer, Freskg\aard, Per-Ola, Collin, Ludovic

The Blood-Brain Barrier (BBB) restricts access of large molecules to the brain. The low endocytic activity of brain endothelial cells (BECs) is believed to limit delivery of immunoglobulins (IgG) to the brain parenchyma. Here, we report that endogenous mouse IgG are localized within intracellular vesicles at steady state in BECs in vivo. Using high-resolution quantitative microscopy, we found a fraction of endocytosed IgG in lysosomes. We observed that loss of pericytes (key components of the BBB) in pdgf-b(ret/ret) mice affects the intracellular distribution of endogenous mouse IgG in BECs. In these mice, endogenous IgG was not detected within lysosomes but instead accumulate at the basement membrane and brain parenchyma. Such IgG accumulation could be due to reduced lysosomal clearance and increased sorting to the abluminal membrane of BECs. Our results suggest that, in addition to low uptake from circulation, IgG lysosomal degradation may be a downstream mechanism by which BECs further restrict IgG access to the brain.

Digital object identifier (DOI): 10.1038/srep25658

Front Neurol, 7, 23
2016

Lesion Size Is Exacerbated in Hypoxic Rats Whereas Hypoxia-Inducible Factor-1 Alpha and Vascular Endothelial Growth Factor Increase in Injured Normoxic Rats: A Prospective Cohort Study of Secondary Hypoxia in Focal Traumatic Brain Injury.

Thelin, Eric Peter, Frostell, Arvid, Mulder, Jan, Mitsios, Nicholas, Damberg, Peter, Aski, Sahar Nikkhou, Risling, M\aarten, Svensson, Mikael, Morganti-Kossmann, Maria Cristina, Bellander, Bo-Michael

Hypoxia following traumatic brain injury (TBI) is a severe insult shown to exacerbate the pathophysiology, resulting in worse outcome. The aim of this study was to investigate the effects of a hypoxic insult in a focal TBI model by monitoring brain edema, lesion volume, serum biomarker levels, immune cell infiltration, as well as the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF).Female Sprague-Dawley rats (n = 73, including sham and naive) were used. The rats were intubated and mechanically ventilated. A controlled cortical impact device created a 3-mm deep lesion in the right parietal hemisphere. Post-injury, rats inhaled either normoxic (22\% O2) or hypoxic (11\% O2) mixtures for 30 min. The rats were sacrificed at 1, 3, 7, 14, and 28 days post-injury. Serum was collected for S100B measurements using ELISA. Ex vivo magnetic resonance imaging (MRI) was performed to determine lesion size and edema volume. Immunofluorescence was employed to analyze neuronal death, changes in cerebral macrophage- and neutrophil infiltration, microglia proliferation, apoptosis, complement activation (C5b9), IgG extravasation, HIF-1α, and VEGF.The hypoxic group had significantly increased blood levels of lactate and decreased pO2 (p < 0.0001). On MRI post-traumatic hypoxia resulted in larger lesion areas (p = 0.0173), and NeuN staining revealed greater neuronal loss (p = 0.0253). HIF-1α and VEGF expression was significantly increased in normoxic but not in hypoxic animals (p < 0.05). A trend was seen for serum levels of S100B to be higher in the hypoxic group at 1 day after trauma (p = 0.0868). No differences were observed between the groups in cytotoxic and vascular edema, IgG extravasation, neutrophils and macrophage aggregation, microglia proliferation, or C5b-9 expression.Hypoxia following focal TBI exacerbated the lesion size and neuronal loss. Moreover, there was a tendency to higher levels of S100B in the hypoxic group early after injury, indicating a potential validity as a biomarker of injury severity. In the normoxic group, the expression of HIF-1α and VEGF was found elevated, possibly indicative of neuro-protective responses occurring in this less severely injured group. Further studies are warranted to better define the pathophysiology of post-TBI hypoxia.

Digital object identifier (DOI): 10.3389/fneur.2016.00023

J Neurosci Methods, 247, 41–49
May, 2015

Assessing fibrinogen extravasation into Alzheimer's disease brain using high-content screening of brain tissue microarrays.

Narayan, Pritika J., Kim, Sue-Ling, Lill, Claire, Feng, Sheryl, Faull, Richard L M., Curtis, Maurice A., Dragunow, Michael

Tissue microarrays are commonly used to evaluate disease pathology however methods to automate and quantify pathological changes are limited.This article demonstrates the utility of the VSlide scanner (MetaSystems) for automated image acquisition from immunolabelled tissue microarray slides, and subsequent automated image analysis with MetaXpress (Molecular Devices) software to obtain objective, efficient and reproducible data from immunolabelled tissue microarray sections.Significant increases in fibrinogen immunolabelling were observed in 29 Alzheimer's disease cases compared to 28 control cases analysed from a single tissue microarray slide. Western blot analysis also demonstrated significant increases in fibrinogen immunolabelling in 6 Alzheimer's cases compared to 6 control cases. The observed changes were also validated with gold standard blinded manual H-scoring.VSlide Metafer software offers a 'tissue microarray acquisition' plugin for easy mapping of tissue cores with their original position on the tissue microarray map. High resolution VSlide images are compatible with MetaXpress image analysis software. This article details the coupling of these two technologies to accurately and reproducibly analyse immunolabelled tissue microarrays within minutes, compared to the gold standard method of manual counting using H-scores which is significantly slower and prone to inter-observer variation.Here, we couple brain tissue microarray technology with high-content screening and automated image analysis as a powerful way to address bottle necks in data generation and improve throughput, as well as sensitivity to study biological/pathological changes in brain disease.

Digital object identifier (DOI): 10.1016/j.jneumeth.2015.03.017

Stroke, 46(3), 835–842
March, 2015

Imaging of a clinically relevant stroke model: glucose hypermetabolismrevisited.

Fabian Arnberg, Jonas Grafstroem, Johan Lundberg, Sahar Nikkhou-Aski, Philip Little, Peter Damberg, Nicholas Mitsios, Jan Mulder, Li Lu, Michael Soederman, Sharon Stone-Elander, Staffan Holmin

Ischemic stroke has been shown to cause hypermetabolism of glucose in the ischemic penumbra. Experimental and clinical data indicate that infarct-related systemic hyperglycemia is a potential therapeutic target in acute stroke. However, clinical studies aiming for glucose control in acute stroke have neither improved functional outcome nor reduced mortality. Thus, further studies on glucose metabolism in the ischemic brain are warranted.We used a rat model of stroke that preserves collateral flow. The animals were analyzed by [2-(18)F]-2-fluoro-2-deoxy-d-glucose positron emission tomography or magnetic resonance imaging during 90-minute occlusion of the middle cerebral artery and during 60 minutes after reperfusion. Results were correlated to magnetic resonance imaging of cerebral blood flow, diffusion of water, lactate formation, and histological data on cell death and blood-brain barrier breakdown.We detected an increased [2-(18)F]-2-fluoro-2-deoxy-d-glucose uptake within ischemic regions succumbing to infarction and in the peri-infarct region. Magnetic resonance imaging revealed impairment of blood flow to ischemic levels in the infarct and a reduction of cerebral blood flow in the peri-infarct region. Magnetic resonance spectroscopy revealed lactate in the ischemic region and absence of lactate in the peri-infarct region. Immunohistochemical analyses revealed apoptosis and blood-brain barrier breakdown within the infarct.The increased uptake of [2-(18)F]-2-fluoro-2-deoxy-d-glucose in cerebral ischemia most likely reflects hypermetabolism of glucose meeting increased energy needs of ischemic and hypoperfused brain tissue, and it occurs under both anaerobic and aerobic conditions measured by local lactate production. Infarct-related systemic hyperglycemia could serve to facilitate glucose supply to the ischemic brain. Glycemic control by insulin treatment could negatively influence this mechanism.

Acta Neuropathol
February, 2015

Critical role of somatostatin receptor 2 in the vulnerability ofthe central noradrenergic system: new aspects on Alzheimer's disease.

Csaba Adori, Laura Glueck, Swapnali Barde, Takashi Yoshitake, Gabor G. Kovacs, Jan Mulder, Zsofia Magloczky, Laszlo Havas, Kata Boelcskei, Nicholas Mitsios, Mathias Uhlen, Janos Szolcsanyi, Jan Kehr, Annica Roennbaeck, Thue Schwartz, Jens F. Rehfeld, Tibor Harkany, Miklos Palkovits, Stefan Schulz, Tomas Hoekfelt

Alzheimer's disease and other age-related neurodegenerative disorders are associated with deterioration of the noradrenergic locus coeruleus (LC), a probable trigger for mood and memory dysfunction. LC noradrenergic neurons exhibit particularly high levels of somatostatin binding sites. This is noteworthy since cortical and hypothalamic somatostatin content is reduced in neurodegenerative pathologies. Yet a possible role of a somatostatin signal deficit in the maintenance of noradrenergic projections remains unknown. Here, we deployed tissue microarrays, immunohistochemistry, quantitative morphometry and mRNA profiling in a cohort of Alzheimer's and age-matched control brains in combination with genetic models of somatostatin receptor deficiency to establish causality between defunct somatostatin signalling and noradrenergic neurodegeneration. In Alzheimer's disease, we found significantly reduced somatostatin protein expression in the temporal cortex, with aberrant clustering and bulging of tyrosine hydroxylase-immunoreactive afferents. As such, somatostatin receptor 2 (SSTR2) mRNA was highly expressed in the human LC, with its levels significantly decreasing from Braak stages III/IV and onwards, i.e., a process preceding advanced Alzheimer's pathology. The loss of SSTR2 transcripts in the LC neurons appeared selective, since tyrosine hydroxylase, dopamine â-hydroxylase, galanin or galanin receptor 3 mRNAs remained unchanged. We modeled these pathogenic changes in Sstr2 (-/-) mice and, unlike in Sstr1 (-/-) or Sstr4 (-/-) genotypes, they showed selective, global and progressive degeneration of their central noradrenergic projections. However, neuronal perikarya in the LC were found intact until late adulthood (

Brain Struct Funct
January, 2015

Acute neuroinflammation in a clinically relevant focal cortical ischemicstroke model in rat: longitudinal positron emission tomography andimmunofluorescent tracking.

Miklos Toth, Philip Little, Fabian Arnberg, Jenny Haeggkvist, Jan Mulder, Christer Halldin, Balazs Gulyas, Staffan Holmin

<p>Adequate estimation of neuroinflammatory processes following ischemic stroke is essential for better understanding of disease mechanisms, and for the development of treatment strategies. With the TSPO (18 kDa translocator protein) positron emission tomography (PET) radioligand [<sup>11</sup>C]PBR28, we monitored longitudinally the inflammatory response post-transient cerebral ischemia in rats, using a recently developed rat stroke model that produces isolated focal cortical infarcts with clinical relevance in size and pathophysiology. Six Sprague-Dawley rats were subjected to 90 min transient endovascular occlusion of the M2 segment of the middle cerebral artery (M2CAO). Animals were imaged with a nanoScan® PET/MRI system at 1, 4, 7 and 14 days after M2CAO with a bolus injection of [<sup>11</sup>C]PBR28. In the infarct region, we found a significantly increased uptake of [<sup>11</sup>C]PBR28 on day 4, 7 and 14 compared to day 1 as well as compared to the contralateral cortex. No significant increase was detected in the contralateral cortex during the 14 days of imaging. The activation in the infarct region gradually decreased between day 4 and day 14. In an additional group of animals (<em>n</em> = 26), immunofluorescence studies were performed with antibodies for activated microglia/monocytes (Cd11b), phagocytes (Cd68), astrocytes (glial fibrillary acidic protein) and TSPO. The TSPO immunofluorescence signal indicated reactive microgliosis post injury, corresponding to PET findings. The present clinically relevant animal model and TSPO PET ligand appear to be well suited for studies on neuroinflammation after ischemic stroke.</p>

EJNMMI Res, 4(1), 17
2014

Visualization of angiogenesis during cancer development in the polyoma middle T breast cancer model: molecular imaging with (R)-[11C]PAQ.

Samén, Erik, Lu, Li, Mulder, Jan, Thorell, Jan-Olov, Damberg, Peter, Tegnebratt, Tetyana, Holmgren, Lars, Rundqvist, Helene, Stone-Elander, Sharon

Vascular endothelial growth factor receptor 2 (VEGFR2) is a crucial mediator of tumour angiogenesis. High expression levels of the receptor have been correlated to poor prognosis in cancer patients. Reliable imaging biomarkers for stratifying patients for anti-angiogenic therapy could therefore be valuable for increasing treatment success rates. The aim of this study was to investigate the pharmacokinetics and angiogenesis imaging abilities of the VEGFR2-targeting positron emission tomography (PET) tracer (R)-[11C]PAQ.(R)-[11C]PAQ was evaluated in the mouse mammary tumour virus-polyoma middle T (MMTV-PyMT) model of metastatic breast cancer. Mice at different stages of disease progression were imaged with (R)-[11C]PAQ PET, and results were compared to those obtained with [18 F]FDG PET and magnetic resonance imaging. (R)-[11C]PAQ uptake levels were also compared to ex vivo immunofluorescence analysis of tumour- and angiogenesis-specific biomarkers. Additional pharmacokinetic studies were performed in rat and mouse.A heterogeneous uptake of (R)-[11C]PAQ was observed in the tumorous mammary glands. Ex vivo analysis confirmed the co-localization of areas with high radioactivity uptake and areas with elevated levels of VEGFR2. In some animals, a high focal uptake was observed in the lungs. The lung uptake correlated to metastatic and angiogenic activity, but not to uptake of [18 F]FDG PET. The pharmacokinetic studies revealed a limited metabolism and excretion during the 1-h scan and a distribution of radioactivity mainly to the liver, kidneys and lungs. In rat, a high uptake was additionally observed in adrenal and parathyroid glands.The results indicate that (R)-[11C]PAQ is a promising imaging biomarker for visualization of angiogenesis, based on VEGFR2 expression, in primary tumours and during metastasis development.

Digital object identifier (DOI): 10.1186/2191-219X-4-17

Revista brasileira de ortopedia, 49, 62–68
2014

Effect of hyaluronic acids as chondroprotective in experimental model of osteoarthrosis.

Oliveira, Marcello Zaia, Albano, Mauro Batista, Namba, Mario Massatomo, da Cunha, Luiz Antônio Munhoz, de Lima Gonçalves, Renan Rodrigues, Trindade, Edvaldo Silva, Andrade, Lucas Ferrari, Vidigal, Leandro

<p>To analyze the effects of hyaluronic acid of different molecular weights in an experimental model of osteoarthritis in rabbits. forty-four male California rabbits were divided randomly into three groups and underwent resection of the anterior cruciate ligament in his right knee. After three weeks of the surgical procedure began three weekly intra-articular injections of hyaluronic acid native (Polireumin®)-PR, hyaluronic acid branched chain (Synvisc®)-S and 0.9% saline-P. All animals were sacrificed after twelve weeks of surgery and tibial plateau infiltrated the knees were dissected. Histological cartilage of the support areas of the tibial plateaus were stained with Alcian Blue pH 1.0, Alcian Blue pH = 2.5 and toluidine blue for research on the amount of proteoglycans. The intensity of staining was quantified on a Zeiss microscope apparatus Imager Z2 MetaSystems and analyzed by software MetaferMsearch. the effect of chondroprotetor hyaluronic acids used in the study was confirmed when compared to the control group, but the comparison made between them, there was no statistically significant difference regarding chondroprotetion. The hyaluronic acids tested had chondroprotective effect, with no statistical difference with regard to the different molecular weights.</p>

Digital object identifier (DOI): 10.1016/j.rboe.2014.01.007

Anal Cell Pathol (Amst), 33(2), 105–112
2010

Automated analysis of protein expression and gene amplification withinthe same cells of paraffin-embedded tumour tissue.

Timo Gaiser, Lissa Berroa-Garcia, Ralf Kemmerling, Aparajita Dutta, Thomas Ried, Kerstin Heselmeyer-Haddad

<p>The simultaneous detection of protein expression and gene copy number changes in patient samples, like paraffin-embedded tissue sections, is challenging since the procedures of immunohistochemistry (IHC) and Fluorescence in situ Hybridization (FISH) negatively influence each other which often results in suboptimal staining. Therefore, we developed a novel automated algorithm based on relocation which allows subsequent detection of protein content and gene copy number changes within the same cell. Paraffin-embedded tissue sections of colorectal cancers were stained for CD133 expression. IHC images were acquired and image coordinates recorded. Slides were subsequently hybridized with fluorescently labeled DNA probes. FISH images were taken at the previously recorded positions allowing for direct comparison of protein expression and gene copy number signals within the same cells/tissue areas. Relocation, acquisition of the IHC and FISH images, and enumeration of FISH signals in the immunophenotyped tumour areas were done in an automated fashion. Automated FISH analysis was performed on 13 different colon cancer samples that had been stained for CD133; each sample was scored for MYC, ZNF217 and Chromosome 6 in CD133 positive and negative glands. From the 13 cases four (31%) showed amplification for the MYC oncogene and seven of 13 (54%) cases were amplified for ZNF217. There was no significant difference between CD133 positive tumour and CD133 negative tumour cells. The technique and algorithm presented here enables an easy and reproducible combination of IHC and FISH based on a novel automated algorithm using relocation and automated spot counting.</p>

J Natl Cancer Inst, 101(10), 736–750
May, 2009

Ki67 index, HER2 status, and prognosis of patients with luminal Bbreast cancer.

Maggie C U. Cheang, Stephen K. Chia, David Voduc, Dongxia Gao, Samuel Leung, Jacqueline Snider, Mark Watson, Sherri Davies, Philip S. Bernard, Joel S. Parker, Charles M. Perou, Matthew J. Ellis, Torsten O. Nielsen

<p>Gene expression profiling of breast cancer has identified two biologically distinct estrogen receptor (ER)-positive subtypes of breast cancer: luminal A and luminal B. Luminal B tumors have higher proliferation and poorer prognosis than luminal A tumors. In this study, we developed a clinically practical immunohistochemistry assay to distinguish luminal B from luminal A tumors and investigated its ability to separate tumors according to breast cancer recurrence-free and disease-specific survival.Tumors from a cohort of 357 patients with invasive breast carcinomas were subtyped by gene expression profile. Hormone receptor status, HER2 status, and the Ki67 index (percentage of Ki67-positive cancer nuclei) were determined immunohistochemically. Receiver operating characteristic curves were used to determine the Ki67 cut point to distinguish luminal B from luminal A tumors. The prognostic value of the immunohistochemical assignment for breast cancer recurrence-free and disease-specific survival was investigated with an independent tissue microarray series of 4046 breast cancers by use of Kaplan-Meier curves and multivariable Cox regression.Gene expression profiling classified 101 (28%) of the 357 tumors as luminal A and 69 (19%) as luminal B. The best Ki67 index cut point to distinguish luminal B from luminal A tumors was 13.25%. In an independent cohort of 4046 patients with breast cancer, 2847 had hormone receptor-positive tumors. When HER2 immunohistochemistry and the Ki67 index were used to subtype these 2847 tumors, we classified 1530 (59%, 95% confidence interval [CI] = 57% to 61%) as luminal A, 846 (33%, 95% CI = 31% to 34%) as luminal B, and 222 (9%, 95% CI = 7% to 10%) as luminal-HER2 positive. Luminal B and luminal-HER2-positive breast cancers were statistically significantly associated with poor breast cancer recurrence-free and disease-specific survival in all adjuvant systemic treatment categories. Of particular relevance are women who received tamoxifen as their sole adjuvant systemic therapy, among whom the 10-year breast cancer-specific survival was 79% (95% CI = 76% to 83%) for luminal A, 64% (95% CI = 59% to 70%) for luminal B, and 57% (95% CI = 47% to 69%) for luminal-HER2 subtypes.Expression of ER, progesterone receptor, and HER2 proteins and the Ki67 index appear to distinguish luminal A from luminal B breast cancer subtypes.</p>

Nat Genet, 41(5), 619-24
2009

Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate

BS Carver, J Tran, A Gopalan, Z Chen, S Shaikh, A Carracedo, A Alimonti, C Nardella, S Varmeh, PT Scardino, C Cordon-Cardo, W Gerald, PP Pandolfi

Chromosomal translocations involving the ERG locus are frequent events in human prostate cancer pathogenesis; however, the biological role of aberrant ERG expression is controversial. Here we show that aberrant expression of ERG is a progression event in prostate tumorigenesis. We find that prostate cancer specimens containing the TMPRSS2-ERG rearrangement are significantly enriched for loss of the tumor suppressor PTEN. In concordance with these findings, transgenic overexpression of ERG in mouse prostate tissue promotes marked acceleration and progression of high-grade prostatic intraepithelial neoplasia (HGPIN) to prostatic adenocarcinoma in a Pten heterozygous background. In vitro overexpression of ERG promotes cell migration, a property necessary for tumorigenesis, without affecting proliferation. ADAMTS1 and CXCR4, two candidate genes strongly associated with cell migration, were upregulated in the presence of ERG overexpression. Thus, ERG has a distinct role in prostate cancer progression and cooperates with PTEN haploinsufficiency to promote progression of HGPIN to invasive adenocarcinoma.

Nat Genet, 40(7), 806–7; author reply
July, 2008

ESR1 gene amplification in breast cancer: a common phenomenon?

Lindsay A. Brown, Jeremy Hoog, Suet-Feung Chin, Yu Tao, Abd Alnaser Zayed, Koei Chin, Andrew E. Teschendorff, John F. Quackenbush, John C. Marioni, Samuel Leung, Charles M. Perou, Torsten O. Neilsen, Matthew Ellis, Joe W. Gray, Philip S. Bernard, David G. Huntsman, Carlos Caldas

Mod Pathol, 21(10), 1271-7
2008

High concordance between immunohistochemistry and fluorescence in situ hybridization testing for HER2 status in breast cancer requires a normalized IHC scoring system

AM Gown, LC Goldstein, TS Barry, SJ Kussick, PL Kandalaft, PM Kim, CC Tse

The American Society of Clinical Oncologists and College of American Pathologists have recently released new guidelines for laboratory testing of HER2 status in breast cancer, which require high levels (95%) of concordance between immunohistochemistry positive (3+) and fluorescence in situ hybridization-amplified cases, and between immunohistochemistry negative (0/1+) and fluorescence in situ hybridization-nonamplified cases; these required levels of concordance are significantly higher than those found in most published studies. We tested the hypothesis that a modification of the HER2 immunohistochemistry scoring system could significantly improve immunohistochemistry and fluorescence in situ hybridization concordance. A total of 6604 breast cancer specimens were evaluated for HER2 status by both immunohistochemistry and fluorescence in situ hybridization using standard methodologies. Results were compared when the standard immunohistochemistry scoring system was replaced by a normalized scoring system in which the HER2 score was derived by subtracting the score on the non-neoplastic breast epithelium from that on the tumor cells. Among the 6604 tumors, using a non-normalized immunohistochemistry scoring system, 267/872 (30.6%) of the immunohistochemistry 3+ cases proved to be fluorescence in situ hybridization nonamplified, whereas using the normalized scoring system only 30/562 (5.3%) of immunohistochemistry 3+ cases proved to be 'false positive'. The concordance rate between immunohistochemistry 3+ and fluorescence in situ hybridization-amplified cases using the normalized scoring method was 94.7%, whereas the concordance using the non-normalized method was only 69.4%. Extremely high concordance between immunohistochemistry and fluorescence in situ hybridization assessment of HER2 status in breast cancer is achievable, but to attain this high level of concordance, modification of the FDA-approved immunohistochemistry scoring system is required.

Breast Cancer Res Treat, 112(3), 453-9
2008

New cutpoints to identify increased HER2 copy number: analysis of a large, population-based cohort with long-term follow-up

KC Jensen, DA Turbin, S Leung, MA Miller, K Johnson, B Norris, T Hastie, S McKinney, TO Nielsen, DG Huntsman, CB Gilks, RB West

BACKGROUND: HER2 gene amplification and/or protein overexpression in breast cancer is associated with a poor prognosis and predicts response to anti-HER2 therapy. We examine the natural history of breast cancers in relationship to increased HER2 copy numbers in a large population-based study. PATIENTS AND METHODS: HER2 status was measured by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in approximately 1,400 breast cancer cases with greater than 15 years of follow-up. Protein expression was evaluated with two different commercially-available antibodies. RESULTS: We looked for subgroups of breast cancer with different clinical outcomes, based on HER2 FISH amplification ratio. The current HER2 ratio cut point for classifying HER2 positive and negative cases is 2.2. However, we found an increased risk of disease-specific death associated with FISH ratios of >1.5. An 'intermediate' group of cases with HER2 ratios between 1.5 and 2.2 was found to have a significantly better outcome than the conventional 'amplified' group (HER2 ratio >2.2) but a significantly worse outcome than groups with FISH ratios less than 1.5. CONCLUSION: Breast cancers with increased HER2 copy numbers (low level HER2 amplification), below the currently accepted positive threshold ratio of 2.2, showed a distinct, intermediate outcome when compared to HER2 unamplified tumors and tumors with HER2 ratios greater than 2.2. These findings suggest that a new cut point to determine HER2 positivity, at a ratio of 1.5 (well below the current recommended cut point of 2.2), should be evaluated.

Cytometry, 73, 651- 657
2008

Automated FISH analysis using dual-fusion and break-apart probes on paraffin-embedded tissue sections.

D. Alpár, J. Hermesz, L. Pótó, R. László, L. Kereskai, P. Jáksó, G. Pajor, L. Pajor, B. Kajtár

Detecting balanced translocations using tissue sections plays an important diagnostic role in cases of hematological malignancies. Manual scoring is often problematic due to truncation and overlapping of nuclei. Reports have described automated analysis using primarily tile sampling. The aim of this study was to investigate an automated fluorescent in situ hybridization analysis method using grid sampling on tissue sections, and compare the performance of dual-fusion (DF) and break-apart (BA) probes in this setting. Ten follicular, 10 mantle cell lymphoma, and 10 translocation-negative samples were used to set the threshold of false positivity using IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes. The cut-off distances of red and green signals to define fusion signals were 0.5, 1.0, and 1.2 mum for the IGH/CCND1, IGH/BCL-2 DF, and IGH BA probes, respectively. The mean false positivity of grid units was 5.3, 11.4, and 28.1%, respectively. Ten to 14 additional samples analyzed blindly and were correctly classified using each probe. Discriminating positive and negative samples using automated analysis and grid sampling was possible with each probe, although different definitions of fusion signals were required due to the different physical distances between the DNA probes. Using the DF probes resulted in lower false positivity, which was less affected by signal numbers per grid units.

Journal of Molecular Diagnostics, 9, 144- 150
2007

Analysis of HER2 gene amplification using an automated fluorescence in situ hybridization signal enumeration system.

R. Stevens, I. Almanaseer, M. Gonzalez, D. Caglar, R.A. Knudson, R.P. Ketterling, D.S. Schrock, T.A. Seemayer, J.A. Bridge

The HER2 gene, amplified in 10 to 35% of invasive human breast carcinomas, has prognostic and therapeutic implications. Fluorescent in situ hybridization is one method currently used for assessing HER2 status, but fluorescent in situ hybridization involves the time-consuming step of manual signal enumeration. To address this issue, Vysis has developed an automated signal enumeration system, Vysis AutoVysion. A multicenter, blinded study was conducted on 39 formalin-fixed, paraffin-embedded invasive breast carcinoma specimens, including 20 HER2 nonamplified and 19 HER2 amplified (weakly to highly amplified), provided in duplicate to each study site for analysis. Calculation of the HER2/CEP17 ratio and the hands-on time of both manual and automated enumeration approaches were compared. Overall agreement of HER2 classification results (positive and negative) was 92.5% (196 of 212). The Vysis AutoVysion System requires manual enumeration for cases with scanner results within the ratio range of 1.5 to 3.0. When the data in this range are excluded, the agreement between manual and scanner results is 98.8% (169 of 171). The average Vysis AutoVysion System hands-on time per slide was 4.59 versus 7.47 minutes for manual signal enumeration (savings of 2.88 minutes/slide). These data suggest that the Vysis AutoVysion System can correctly classify specimens and may increase the overall efficiency of HER2 testing.