Name: building up a high-throughput screening platform to assess the heterogeneity of her2 gene amplification in breast cancers
Uploaded: 2017-12-05T15:00:00
Description: Watch this Scientific Journal Video about Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers at JoVE.com

Este estudo foi aprovado pelas placas de revisão institucional da IRCCS Ca' Granda Foundation, Hospital Policlínico, Milão, Itália.
1. seleção de pacientes e espécimes de tecido
<ol><li>Recuperar os slides arquivamento de todos os casos a serem analisados, incluindo todos os disponível hematoxilina e eosina (H & E) e slides de IHC do diagnóstico original e, se estiver presente, um H & E de correspondem tecido mamário não-neoplásicos (por exemplo, margem cirúrgica) .</li...

<ol>
	<li>Allison, K. H., Dintzis, S. M., Schmidt, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+HER2+heterogeneity+by+fluorescence+in+situ+hybridization+according+to+CAP+expert+panel+recommendations:+time+for+a+new+look+at+how+to+report+heterogeneity.">Frequency of HER2 heterogeneity by fluorescence in situ hybridization according to CAP expert panel recommendations: time for a new look at how to report heterogeneity.</a> Am J Clin Pathol. 136 (6), 864-871 (2011).</li><li>Montemurro, F., Scaltriti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+drugs+targeting+HER-family+signalling+in+cancer.">Biomarkers of drugs targeting HER-family signalling in cancer.</a> J Pathol. 232 (2), 219-229 (2014).</li><li>Wolff, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+human+epidermal+growth+factor+receptor+2+testing+in+breast+cancer:+American+Society+of+Clinical+Oncology/College+of+American+Pathologists+clinical+practice+guideline+update.">Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update.</a> J Clin Oncol. 31 (31), 3997-4013 (2013).</li><li>Ng, C. K., Pemberton, H. N., Reis-Filho, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+intratumor+genetic+heterogeneity:+causes+and+implications.">Breast cancer intratumor genetic heterogeneity: causes and implications.</a> Expert Rev Anticancer Ther. 12 (8), 1021-1032 (2012).</li><li>Vance, G. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+heterogeneity+in+HER2+testing+in+breast+cancer:+panel+summary+and+guidelines.">Genetic heterogeneity in HER2 testing in breast cancer: panel summary and guidelines.</a> Arch Pathol Lab Med. 133 (4), 611-612 (2009).</li><li>Murthy, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+HER2/Neu+status+by+fluorescence+in+situ+hybridization+in+immunohistochemistry-equivocal+cases+of+invasive+ductal+carcinoma+and+aberrant+signal+patterns:+a+study+at+a+tertiary+cancer+center.">Assessment of HER2/Neu status by fluorescence in situ hybridization in immunohistochemistry-equivocal cases of invasive ductal carcinoma and aberrant signal patterns: a study at a tertiary cancer center.</a> Indian J Pathol Microbiol. 54 (3), 532-538 (2011).</li><li>Ohlschlegel, C., Zahel, K., Kradolfer, D., Hell, M., Jochum, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+genetic+heterogeneity+in+breast+carcinoma.">HER2 genetic heterogeneity in breast carcinoma.</a> J Clin Pathol. 64 (12), 1112-1116 (2011).</li><li>Chang, M. C., Malowany, J. I., Mazurkiewicz, J., Wood, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Genetic+heterogeneity'+in+HER2/neu+testing+by+fluorescence+in+situ+hybridization:+a+study+of+2,522+cases.">'Genetic heterogeneity' in HER2/neu testing by fluorescence in situ hybridization: a study of 2,522 cases.</a> Mod Pathol. 25 (5), 683-688 (2012).</li><li>Bartlett, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneous+HER2+gene+amplification:+impact+on+patient+outcome+and+a+clinically+relevant+definition.">Heterogeneous HER2 gene amplification: impact on patient outcome and a clinically relevant definition.</a> Am J Clin Pathol. 136 (2), 266-274 (2011).</li><li>Seol, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+heterogeneity+of+HER2+gene+amplification+in+breast+cancer:+its+clinicopathological+significance.">Intratumoral heterogeneity of HER2 gene amplification in breast cancer: its clinicopathological significance.</a> Mod Pathol. 25 (7), 938-948 (2012).</li><li>Hanna, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+in+situ+hybridization+in+breast+cancer:+clinical+implications+of+polysomy+17+and+genetic+heterogeneity.">HER2 in situ hybridization in breast cancer: clinical implications of polysomy 17 and genetic heterogeneity.</a> Mod Pathol. 27 (1), 4-18 (2014).</li><li>Sanguedolce, F., Bufo, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+assessment+by+silver+in+situ+hybridization:+where+are+we+now?.">HER2 assessment by silver in situ hybridization: where are we now?.</a> Expert Rev Mol Diagn. 15 (3), 385-398 (2015).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Contrasting+Role+of+p16Ink4A+Patterns+of+Expression+in+Neuroendocrine+and+Non-Neuroendocrine+Lung+Tumors:+A+Comprehensive+Analysis+with+Clinicopathologic+and+Molecular+Correlations.">The Contrasting Role of p16Ink4A Patterns of Expression in Neuroendocrine and Non-Neuroendocrine Lung Tumors: A Comprehensive Analysis with Clinicopathologic and Molecular Correlations.</a> PLoS One. 10 (12), e0144923 (2015).</li><li>Albanghali, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+tissue+microarrays+from+core+needle+biopsies+-+a+systematic+literature+review.">Construction of tissue microarrays from core needle biopsies - a systematic literature review.</a> Histopathology. 68 (3), 323-332 (2016).</li><li>Navani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+evaluation+of+tissue+microarrays+in+a+high-throughput+research+project:+The+contribution+of+Indian+surgical+pathology+to+the+Human+Protein+Atlas+(HPA)+project.">Manual evaluation of tissue microarrays in a high-throughput research project: The contribution of Indian surgical pathology to the Human Protein Atlas (HPA) project.</a> Proteomics. 16 (8), 1266-1270 (2016).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+in+gastric+cancer:+a+digital+image+analysis+in+pre-neoplastic,+primary+and+metastatic+lesions.">HER2 in gastric cancer: a digital image analysis in pre-neoplastic, primary and metastatic lesions.</a> Mod Pathol. 26 (6), 816-824 (2013).</li><li>Amin, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AJCC Cancer Staging Manual. , (2017).</li><li>Lakhani, S. R., Ellis, I. O., Schnitt, S. J., Tan, P. H., van de Vijver, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> WHO Classification of Tumours of the Breast. , (2012).</li><li>Kononen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+microarrays+for+high-throughput+molecular+profiling+of+tumor+specimens.">Tissue microarrays for high-throughput molecular profiling of tumor specimens.</a> Nat Med. 4 (7), 844-847 (1998).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resolving+quandaries:+basaloid+adenoid+cystic+carcinoma+or+breast+cylindroma?+The+role+of+massively+parallel+sequencing.">Resolving quandaries: basaloid adenoid cystic carcinoma or breast cylindroma? The role of massively parallel sequencing.</a> Histopathology. 68 (2), 262-271 (2016).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+events+in+the+progression+of+adenoid+cystic+carcinoma+of+the+breast+to+high-grade+triple-negative+breast+cancer.">Genetic events in the progression of adenoid cystic carcinoma of the breast to high-grade triple-negative breast cancer.</a> Mod Pathol. 29 (11), 1292-1305 (2016).</li><li>Fusco, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+NAB2-STAT6+gene+fusions+and+oestrogen+receptor-&#945;+expression+in+pulmonary+adenofibromas.">Recurrent NAB2-STAT6 gene fusions and oestrogen receptor-&#945; expression in pulmonary adenofibromas.</a> Histopathology. 70 (6), 906-917 (2017).</li><li>Hammond, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Society+of+Clinical+Oncology/College+Of+American+Pathologists+guideline+recommendations+for+immunohistochemical+testing+of+estrogen+and+progesterone+receptors+in+breast+cancer.">American Society of Clinical Oncology/College Of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer.</a> J Clin Oncol. 28 (16), 2784-2795 (2010).</li><li>Fusco, N., Bosari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+aberrations+and+heterogeneity+in+cancers+of+the+digestive+system:+Implications+for+pathologists+and+gastroenterologists.">HER2 aberrations and heterogeneity in cancers of the digestive system: Implications for pathologists and gastroenterologists.</a> World J Gastroenterol. 22 (35), 7926-7937 (2016).</li><li>Dowsett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Ki67+in+breast+cancer:+recommendations+from+the+International+Ki67+in+Breast+Cancer+working+group.">Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group.</a> J Natl Cancer Inst. 103 (22), 1656-1664 (2011).</li><li>Dekker, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+sensitivity+and+specificity+of+HER2+testing+in+breast+cancer+using+a+tissue+micro-array+approach.">Determining sensitivity and specificity of HER2 testing in breast cancer using a tissue micro-array approach.</a> Breast Cancer Res. 14 (3), R93 (2012).</li><li>De Sio, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+MALDI-Mass+Spectrometry+Imaging+method+applicable+to+different+formalin-fixed+paraffin-embedded+human+tissues.">A MALDI-Mass Spectrometry Imaging method applicable to different formalin-fixed paraffin-embedded human tissues.</a> Mol Biosyst. 11 (6), 1507-1514 (2015).</li></ol>

Aqui, temos as estratégias de laboratório para realizar análises SISH do gene HER2 e sua correspondente Centrómero em alto rendimento TMAs dos cancros da mama de forma heterogénea transformados detalhados. Este método é econômico e pode ser realizado na maioria dos laboratórios para o estudo da heterogeneidade de amplificação do gene HER2 em grandes coortes de mama câncer obtida forma patologia archives.
Devido a importância clínica do HER2 testes em câncer de ...

HER2 é um proto-oncogene que é overexpressed e amplificado em 15-30% de mama invasivo todos os cancros1,2. Superexpressão de HER2 é inferido pela presença de > 10% células com membrana forte imuno-histoquímica (IHC) coloração (3 +), enquanto a amplificação do gene pode ser avaliada quando a proporção de HER2/Centrómero é ≥2 ou o número de cópia do gene é ≥ 6, na contagem de pelo menos 20 células por em situ da hibridação (ISH)3.
Heterogeneidade genética intra-tumor tem sido amplamente descrita em cânceres de mama, sendo um contribuinte potencialmente adverso para avaliação de biomarcadores e tratamentos resposta4. De acordo com a faculdade de americana patologistas (CAP), HER2 heterogeneidade existe se HER2 é amplificado em > 5% e < 50% do tumor infiltrando-se células5. Lamentavelmente, a incidência real da heterogeneidade espacial HER2 no câncer de mama continua a ser um assunto de controvérsia entre os patologistas, com alguns autores de manutenção que é um evento extremamente raro e outros, sugerindo que até 40% dos casos são HER2-heterogêneos1,5,6,7,8,9,10. Apesar dos mecanismos biológicos subjacentes a esta condição não são ainda totalmente esclarecido, os impactos clínicos e prognósticos da heterogeneidade de HER2 intra-tumor são cruciais para a de pacientes de câncer de mama11.
Recentemente, técnicas moleculares brilhante-campo, tais como o cromogênico ISH (CISH) e prata ISH (SISH), têm emergido como métodos confiáveis para detectar heterogeneidade HER2 em tecidos FFPE, com algumas vantagens em comparação com fluorescente ISH (FISH)12. Lamentavelmente, a análise em massa dos casos simples continua a ser impraticável em estudos de coorte-grande investigação. Vários grupos têm sugerido que a combinação da histoquímica, IHC e ISH com tecnologias TMA poderia representar uma estratégia valiosa no estudo de câncer biologia13,14,15,16. Com este método amplamente adotado, amostras de tecido de diferentes pacientes analisáveis simultaneamente, minimizando o tecido e os reagentes empregados e, assim, fomentar a análise uniforme de uma grande série de casos14. No entanto, não há protocolos estão disponíveis para a caracterização molecular de elevado-produção simultânea de múltiplas amostras de tecido que foram submetidos a diferentes de processamento em termos de reagentes, tempos de fixação e métodos de conservação empregados, tais como arquivamento blocos.
Tendo em conta as implicações clínicas e prognósticos da heterogeneidade espacial HER2 no câncer de mama, desenvolvemos uma plataforma integrada de molecular para avaliá-la em grande série de casos de forma heterogénea transformados. Aqui, podemos retratar as estratégias de laboratório para gerar e analisar a heterogeneidade intra-tumor de amplificação do HER2 em alto rendimento TMAs do câncer de mama por meio de SISH. O protocolo a seguir foi desenvolvido para medir os tumores > 5 mm (> pT1b de acordo com o TNM 2017)17. Para lesões menores, recomendamos realizar a análise em seções seriais Full-face. Nosso processo permite a análise simultânea de IHC e SISH até 30 dos cancro da mama, englobando uma média de 6 áreas distintas (intervalo de 4-8) para cada caso. No total, 180 núcleos de tecido de 1 mm de diâmetro, com 500 µm entre os núcleos e 2 mm entre a grade e bordas serão gerados para cada bloco TMA.

<table border="0" cellpadding="0" cellspacing="0" width="1127">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Surgipath Paraplast</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">39601006</td>
			<td style="width:383px;">Tissue embedding medium, 56 &#176;C melting point</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Eosin Y&#160; 1% water solution</td>
			<td style="width:328px;">Bio Optica, Milan, Italy, EU</td>
			<td style="width:120px;">510002</td>
			<td style="width:383px;">Eosin yellowish, water-soluble</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Carazzi&#8217;s hematoxylin</td>
			<td style="width:328px;">Bio Optica, Milan, Italy, EU</td>
			<td style="width:120px;">506012</td>
			<td style="width:383px;">Alum hematoxylin ripened using potassium iodate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Diamond quality</td>
			<td style="width:328px;">Laboindustria, Arzergrande, Italy, EU</td>
			<td style="width:120px;">33533</td>
			<td style="width:383px;">26x76 mm microscope slides</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Leica CV Mount</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">14046430011</td>
			<td style="width:383px;">Mounting medium, with no monomers, based on polymers of butylmethacrylate in xylene</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">FLEX IHC microscope slides</td>
			<td style="width:328px;">Agilent Technologies (Dako),&#160; Santa Clara, CA, USA</td>
			<td style="width:120px;">K8020</td>
			<td style="width:383px;">Coated microscope slides for adhesion of FFPE for use in IHC</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:297px;">BenchMark ULTRA</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">N750-BMKU-FS</td>
			<td style="width:383px;">Slide staining system</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">CONFIRM anti-Estrogen Receptor (ER) (SP1) Rabbit Monoclonal Primary Antibody</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-4324</td>
			<td style="width:383px;">Primary antibody, ready-to-use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">CONFIRM anti-Progesterone Receptor (PR) (1E2) Rabbit Monoclonal Primary Antibody</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-2223</td>
			<td style="width:383px;">Primary antibody, ready-to-use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-4286</td>
			<td style="width:383px;">Primary antibody, ready-to-use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">PATHWAY HER2 (4B5) Rabbit Monoclonal Primary Antibody</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-4493</td>
			<td style="width:383px;">Primary antibody, ready-to-use</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">ultraView Universal DAB Detection Kit</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">760-500</td>
			<td style="width:383px;">Indirect, biotin-free system for detecting mouse IgG, mouse IgM and rabbit primary antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">INFORM HER2 Dual ISH DNA Probe Cocktail</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">780-4422</td>
			<td rowspan="3" style="width:383px;">INFORM HER2 Dual ISH assay - Dual color in situ hybridization FDA approved automated assay for determining HER2 gene status in breast cancer patients&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ultraView Silver ISH DNP Detection Kit</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">800-098</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ultraView Red ISH DIG Detection Kit</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">800-505</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">ISH Protease 3</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">780-4149</td>
			<td style="width:383px;">Used in the ISH process to remove protein that surrounds the target DNA sequences of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Hematoxylin</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">760-2021</td>
			<td style="width:383px;">Modified Gill&#39;s hematoxylin counterstain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Hematoxylin II Counterstaining</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">790-2208</td>
			<td style="width:383px;">Modified Meyer&#39;s hematoxylin counterstain reagent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Bluing reagent</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">760-2037</td>
			<td style="width:383px;">Aqueous solution of buffered lithium carbonate for bluing hematoxylin stained sections on glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">HybReady</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">780-4409</td>
			<td style="width:383px;">Formamide-based buffer for ISH assays</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">EZ Prep (10x)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-102</td>
			<td style="width:383px;">Concentrate solution for paraffin removal from tissue samples during IHC and ISH reactions, and to dilute 1:10.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:297px;">SSC Buffer (10X)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-110</td>
			<td style="width:383px;">Sodium Chloride Sodium Citrate buffer solution is used for stringency washes and to rinse slides between staining steps and provide a stable aqueous environment for the in situ hybridization reactions. Dilute 1:5.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:297px;">ULTRA LCS</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">650-210</td>
			<td style="width:383px;">Prediluted (ready-to-use) coverslip solution used as a barrier between the aqueous reagents and the air to prevent evaporation in the IHC and ISH reactions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Reaction Buffer (10x)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-300</td>
			<td style="width:383px;">Tris based buffer solution (pH 7.6 &#177; 0.2) to rinse slides between staining steps during IHC and ISH. Dilute 1:10.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ULTRA Cell Conditioning (ULTRA CC2)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-223</td>
			<td rowspan="2" style="width:383px;">Pretreatment steps in the processing of tissue samples during IHC and ISH. Ready to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">ULTRA Cell Conditioning (ULTRA CC1)</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">950-224</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">ultraView Silver Wash II</td>
			<td style="width:328px;">Ventana medical system, Tucson, AZ, USA</td>
			<td style="width:120px;">780-003</td>
			<td style="width:383px;">Ready-to-use solution to rinse slides between IHC and ISH staining steps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Microtome</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">RM 2255</td>
			<td style="width:383px;">Automated rotary microtome</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Multistainer</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">ST 5020</td>
			<td style="width:383px;">Workstation for automated staining and coverslipping</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Minicore 1</td>
			<td style="width:328px;">Alphelys, Plaisir, France, EU</td>
			<td style="width:120px;">00-MICO-1</td>
			<td style="width:383px;">Semi-automatic arrayer for TMA contruction with TMA Designer 2 embedded software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Aperio ScanScope CS2</td>
			<td style="width:328px;">Leica Biosystems, Wetzlar, Germany, EU</td>
			<td style="width:120px;">K080254</td>
			<td style="width:383px;">Image capture device - digital pathology scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue-Tek III Uni-Cassette</td>
			<td>Sakura Finetek Europe B.V</td>
			<td style="width:120px;">4135</td>
			<td>Cassette</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Tissue-Tek Paraform Standard Base Mold</td>
			<td>Sakura Finetek Europe B.V</td>
			<td style="width:120px;">7055</td>
			<td>Stainless-Steel base metal mold</td>
		</tr>
	</tbody>
</table>

building up a high-throughput screening platform to assess the heterogeneity of her2 gene amplification in breast cancers

Terapias alvo contra o receptor do fator de crescimento epidérmico humano (HER2) de 2 mudaram radicalmente os resultados dos pacientes com câncer de Mama HER2-positivo. No entanto, uma minoria de casos exibe uma distribuição heterogênea de células HER2-positivo, que gera grandes desafios clínicos. Até à data, não há protocolos confiáveis e padronizados para a caracterização e quantificação de amplificação de gene HER2 heterogênea em grandes coortes têm sido propostos. Aqui, apresentamos uma metodologia de alta produtividade para avaliar simultaneamente o status HER2 em diferentes áreas topográficas de vários cancros da mama. Em particular, ilustramos o procedimento laboratorial para construir aprimorado os microarrays do tecido (TMAs) incorporando um mapeamento alvo dos tumores. Todos os parâmetros TMA foram otimizados especificamente para a prata em situ da hibridação (SISH) de formalina-parafina (FFPE) mama tecidos fixados. Análise imunohistoquímica de biomarcadores os prognósticos e preditivos (isto é, ER, PR, Ki67 e HER2) deve ser realizada utilizando procedimentos automatizados. Um protocolo personalizado de SISH foi implementado para permitir uma análise molecular de alta qualidade através de vários tecidos que passou por diferentes procedimentos de fixação, processamento e armazenamento. Neste estudo, nós fornecemos um prova de princípio que sequências específicas de DNA pode ser câncer de mama de forma heterogénea transformados e simultaneamente localizadas em áreas topográficas distintas de múltiplos usando um método eficiente e econômico.

Em geral, 444 mama invasivo cânceres foram incorporados em 15 TMAs especificamente otimizados para análises ISH. Entre os 2.664 pontos amostrados, 2.651 (99,5%) eram representativas das áreas previamente selecionadas e, portanto, considerado favorável para análises subsequentes. Heterogeneidade de intra-tumor foi determinada por meio de IHC e SIH, com particular ênfase para as distribuições heterogêneas de clones de HER2-positivo nas áreas topográficas distintas dos tumores. <s...

Watch this Scientific Journal Video about Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers at JoVE.com

Building Up a High-throughput Screening Platform to Assess the Heterogeneity of HER2 Gene Amplification in Breast Cancers

Distribuição heterogênea de células HER2-positivo pode ser observada em um subconjunto dos cancros da mama e gera dilemas clínicas. Aqui, apresentamos um protocolo confiável e econômico para definir, quantificar e comparar a heterogeneidade genética de HER2 intra-tumor em uma grande série de cancros da mama de forma heterogénea transformados.

Construindo uma plataforma High-throughput Screening para avaliar a heterogeneidade da amplificação do Gene HER2 no câncer de mama

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast ...

Research

JoVE Journal

Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Um<em&gt; In vitro</em&gt; Modelo para estudar a heterogeneidade de diferenciação de macrófagos humanos e Polarização

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Imunologia e Infecção

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Um ensaio fenotípico microscópico para quantificação de micobactérias intracelulares adaptadas para triagem de alto rendimento/alto conteúdo

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Triagem de alta capacidade para amplo espectro químico Inibidores de Vírus RNA

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

High-throughput Gene Tagging in Trypanosoma brucei

High-throughput Gene Tagging in Trypanosoma brucei

De alto rendimento Gene Tagging no<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

A Fluorescência baseada em Linfócitos ensaio adequado para high-throughput screening de pequenas moléculas

High-throughput screening (HTS) is currently the mainstay for the identification of chemical entities capable of modulating biochemical reactions or ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Uma plataforma de alto rendimento para o rastreio de Salmonella spp. /Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

Medição de alto rendimento da membrana plasmática, efectuou a eficiência em células de mamíferos

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

A High-throughput Assay to Assess and Quantify Neutrophil Extracellular Trap Formation

Um ensaio de alta produtividade para avaliar e quantificar a formação de neutrófilos extracelular armadilha

Neutrophil extracellular traps (NETs) are immunogenic extracellular DNA structures that can be released by neutrophils upon a wide variety of triggers. ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Triagem de alto rendimento de compostos químicos para elucidar seus efeitos na persistência bacteriana

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...

High-Throughput Transcriptome Analysis for Investigating Host-Pathogen Interactions

Análise de transcriptome de alto rendimento para investigar interações host-pathogen

Pathogens can cause a wide variety of infectious diseases. The biological processes induced by the host in response to infection determine the severity of ...