This study was approved by the Institutional Review Boards from IRCCS Ca&#39; Granda Foundation, Policlinico Hospital, Milan, Italy.
1. Selection of Patients and Tissue Specimens
<ol>
	<li>Retrieve the archival slides of all cases to be analyzed, including all the available hematoxylin and eosin (H&#38;E) and IHC slides from the original diagnosis and, if present, one H&#38;E of matched non-neoplastic breast tissue (e.g., surgical margin).</li>
	<li>Perform case reviews.</st...

<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>

The authors have no conflicts of interests.

Here, we have detailed the laboratory strategies to perform SISH analyses of the HER2 gene and its corresponding centromere in high-yield TMAs of heterogeneously processed breast cancers. This method is cost-effective and can be carried out in most laboratories for the study of HER2 gene amplification heterogeneity in large cohorts of breast cancers retrieved form pathology archives.
Due to the clinical importance of HER2 testing in breast cancer and the challenges generated ...

HER2 is a proto-oncogene that is overexpressed and amplified in 15 - 30% of all invasive breast cancers1,2. HER2 overexpression is inferred by the presence of &#62;10% cells with strong membrane immunohistochemical (IHC) staining (3+), while the gene amplification can be assessed when either the HER2/centromere ratio is &#8805;2 or the gene copy number is &#8805;6, on counting at least 20 cells by in situ hybridization (ISH)3.
Intra-tumor genetic heterogeneity has been widely described in breast cancers, being a potentially adverse contributor to biomarkers evaluation and treatments response4. According to the College of American Pathologists (CAP), HER2 heterogeneity exists if HER2 is amplified in &#62;5% and &#60;50% of infiltrating tumor cells5. Regrettably, the actual incidence of HER2 spatial heterogeneity in breast cancers remains a subject of controversy among pathologists, with some authors maintaining that it is an exceedingly rare event, and others suggesting that up to 40% of cases are HER2-heterogeneous1,5,6,7,8,9,10. Despite the biological mechanisms that underpin this condition are not yet fully clarified, the prognostic and clinical impacts of intra-tumor HER2 heterogeneity are crucial for breast cancer patients11.
Recently, bright-field molecular techniques, such as chromogenic ISH (CISH) and silver ISH (SISH), have emerged as reliable methods to detect HER2 heterogeneity in FFPE tissues, with some advantages compared to fluorescent ISH (FISH)12. Regrettably, the bulk analysis of single cases remains impractical in large-cohort research studies. Several groups have suggested that the combination of histochemistry, IHC, and ISH with TMA technologies could represent a valuable strategy in the study of cancer biology13,14,15,16. With this widely adopted method, tissue samples from different patients can be analyzed concurrently, minimizing the tissue and reagents employed and thereby fostering the uniform analysis of a large series of cases14. However, no protocols are available for the simultaneous high-throughput molecular characterization of multiple tissue samples that underwent different processing in terms of reagents, fixation times, and conservation methods employed, such as archival blocks.
Given the prognostic and clinical implications of HER2 spatial heterogeneity in breast cancers, we developed an integrated molecular platform to assess it in large series of heterogeneously processed cases. Here, we portray the laboratory strategies to generate and analyze the intra-tumor heterogeneity of HER2 amplification in high-yield TMAs of breast cancer by means of SISH. The following protocol has been developed for tumors measuring &#62;5 mm (&#62;pT1b according to the TNM 2017)17. For smaller lesions, we recommend performing the analysis on full-face serial sections. Our procedure allows for the simultaneous IHC and SISH analysis of up to 30 breast cancers, encompassing a mean of 6 distinct areas (range 4 - 8) for each case. Altogether, 180 tissue cores of 1 mm in diameter, with 500 &#181;m between the cores, and 2 mm between the grid and edges will be generated for each TMA block.

<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

Targeted therapies against the human epidermal growth factor receptor 2 (HER2) have radically changed the outcome of patients with HER2-positive breast cancers. However, a minority of cases displays a heterogeneous distribution of HER2-positive cells, which generates major clinical challenges. To date, no reliable and standardized protocols for the characterization and quantification of HER2 heterogeneous gene amplification in large cohorts have been proposed. Here, we present a high-throughput methodology to simultaneously assess the HER2 status across different topographic areas of multiple breast cancers. In particular, we illustrate the laboratory procedure to construct enhanced tissue microarrays (TMAs) incorporating a targeted mapping of the tumors. All TMA parameters have been specifically optimized for the silver in situ hybridization (SISH) of formalin-fixed paraffin-embedded (FFPE) breast tissues. Immunohistochemical analysis of the prognostic and predictive biomarkers (i.e., ER, PR, Ki67, and HER2) should be performed using automated procedures. A customized SISH protocol has been implemented to allow a high-quality molecular analysis across multiple tissues that underwent different fixation, processing, and storage procedures. In this study, we provide a proof-of-principle that specific DNA sequences could be localized simultaneously in distinct topographic areas of multiple and heterogeneously processed breast cancers using an efficient and cost-effective method.

Overall, 444 invasive breast cancers were incorporated in 15 TMAs specifically optimized for ISH analyses. Among the 2,664 spots sampled, 2,651 (99.5%) were representative of the previously selected areas and therefore considered amenable for subsequent analyses. Intra-tumor heterogeneity was determined by means of IHC and SIH, with a particular focus on the heterogeneous distributions of HER2-positive clones in the distinct topographic areas of the tumors. Table 3 depict...

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

Heterogeneous distribution of HER2-positive cells can be observed in a subset of breast cancers and generates clinical dilemmas. Here, we introduce a reliable and cost-effective protocol to define, quantify, and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers.

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

The overall goal of this laboratory procedure is to simultaneously define, quantify and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers by means of high-throughput silver in situ hybridization. This method can help address key choosing breast cancer translation and research such as the study of HER2 gene amplification heterogeneity, a large cohort of cases retrieved from pathology archives. The main advantage of this technique is that it allows for high-throughput HER2 schizoanalysis across different topographic areas of multiple and heterogeneously processed breast cancers.Generally, individuals new to in situ hybridization will struggle because retrospective studies typically require the analysis of archival samples that underwent different fixation, processing and storage procedure. We first had the idea for this method when we decided to reclassify all breast cancers from our institutional database since 2003. Demonstrating this procedure will be Giulia Ercoli, a technician from our laboratory.Begin this protocol with selection of patients and tissue specimens as described in the text protocol. Create and open a new spreadsheet file, incorporating the records of the distinct areas of each case. Include separate columns for case ID, block ID, area ID, tissue type, topography, morphology, biomarker status and all other immunohistochemical features available.Create a TMA project. Define the PMA recipient blocks by navigating to file, new, block. Click on the first field to fill and type in the dimensions of the paraffin block.Next create a tissue array project by navigating to file, new, tissue array. Click the import icon, then choose the template file of the recipient block previously created. Click next.Then choose one millimeter as the spot size by clicking the round button. Choose 500 microns as the spot spacing, which is the space between two centers of neighboring spots. Then click on the calculator icon to calculate the total number of spots created, which should be 231.Then click on the button define to create grids and subgrids. Delete columns eight and 15 and the central line. Maintain three spots of line one for orientation.The final map should encompass a total number of 183 spots. Define the list of tissue types by navigating to file, new, list of tissue types. Then define the project by navigating to file, new, booster project.Next prepare the acceptor blocks. Fill cleansed metal molds with elasticized paraffin at 65 degrees Celsius. Leave the paraffin blocks to cool at film temperature overnight inside of the metal molds.The next day, extract the paraffin blocks from the metal molds. Construct the TMA using an automated or semi-automated arrayer by first turning on the arrayer and then inserting the accepter blocks on the arrayer carousel. Define the starting position onto each recipient block by using the arrow keys to move the laser light and align it with the top left edge on the paraffin of the recipient block.Click next and repeat for the vertical alignment. Click next again to automatically create a hole in the previously defined coordinate of the recipient block. Empty the punch.Position the donor block for sampling and remove a tissue core from the previously annotated area of interest. Insert the donor tissue core into the hole in the recipient block. Then remove the donor block from the arrayer.Repeat these steps until the TMA is completed. Put the tissue side of the newly generated TMA block on a sterile blank slide and place them in the lab oven at 45 degrees Celsius for five minutes. Gently press the block on the slide for five seconds to flatten the tissue cores.Repeat once for a total of two times. Next cover the TMA tissue surface with a thin layer of elasticized paraffin at 65 degrees Celsius. Leave the TMA block at room temperature for two hours.Finally, transfer the TMA block at four degrees Celsius for 24 hours before performing histochemical and immunohistochemical analyses as described in the text protocol. Place the TMA slides to analyze at 60 degrees Celsius for 10 minutes. Then start the immunostainer instrument and software.At the home view, click the protocols button and then click create/edit protocols. Select the dual ISH cocktail procedure to modify the basic template. Flag baking in the box list and set the temperature at 63 degrees Celsius for 20 minutes.Then flag deparaffinization. Next flag cell conditioning in the box list. Then flag cell conditioning CC2 and set the temperature at 86 degrees Celsius and the incubation time at four minutes.Flag mild CC2 for eight minutes, standard for 12 minutes and extended for eight minutes. Flag ISH-Protease three in the box list and set the incubation time at 16 minutes. Select the probe's HER2 dinitrophenyl and chromosome 17 digoxigenin.Incubate for 16 minutes and then set the denaturation for 20 minutes at 80 degrees Celsius. Set the probe's incubation time at six hours. Then set the SISH multimer incubation time at 32 minutes.Set the silver chromate and incubation time at four minutes. Next set the red multimer incubation time at 24 minutes. Set the red chromate and incubation time at eight minutes.Then flag counterstaining in the box list, select hematoxylin two and set the incubation time at eight minutes. Flag post counterstaining in the box list, select bluing reagent and set the incubation time at four minutes. Finally, perform schizoanalysis for HER2.Assess the HER2 gene amplification as positive if the HER2 chromosome enumeration probe 17 ratio is greater than or equal to 2.0 with an average HER2 copy number greater than or equal to 4.0 signals per cell. Assess as negative if the HER2 chromosome enumeration probe 17 ratio is less than 2.0 with an average HER2 copy number less than 4.0 signals per cell. Shown here are representative micrographs showing the silver in situ hybridization analysis of a HER2 heterogeneous breast cancer.In this paradigmatic example, two distinct areas of high-grade invasive breast cancer of no special type show different results in terms of HER2 gene amplification, visualized as black dots, compared to the centromeric enumeration probe 17, visualized as red dots. In particular, one area belonging to the tumor core and showing cytokeratin seven irregular staining pattern was HER2 amplified, while a single spot front the invasive front displayed a wild-type HER2 pattern. After watching this video, you should have a good understanding of how to perform readable schizoanalysis of tissue marked arrays incorporating multiple and heterogeneously processed breast cancers.Once mastered, this technique can be done in two working days from the case selection. Though this method is optimized for breast cancer, it can be translated to other types of cancer in which HER2 heterogeneity is critically relevant such as gastric cancer. The implications of this technique extend toward the simple assessment of HER2 because it is designed for the comprehensive mapping of multiple topographic areas in larger series of breast cancers.While attending this procedure, it's important to avoid any crack on the TMA block. Even a minimal crack in the paraffin infers the quality, reproducibility and the overall clarity of the in situ hybridization. Following this procedure, MALDI mass spectrometry imaging can be performed in order to answer additional questions on the proteomics of breast cancer, particularly in terms of intra and inter-tumor heterogeneity.Don't forget that working with TMA punches and hot paraffin wax can be hazardous and precautions such as wearing disposable nitrile gloves should always be taken while performing this procedure.

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Research

JoVE Journal

Immunology and Infection

12.3K Views.  Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico. The overall goal of this laboratory procedure is to simultaneously define, quantify and compare HER2 intra-tumor genetic heterogeneity in a large series of heterogeneously processed breast cancers by means of high-throughput silver in situ hybridization. This method can help address key choosing breast cancer translation and research such as the study of HER2 gene amplification heterogeneity, a large cohort of cases retrieved from pathology archives. The main advantage of this technique is th...