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. 
	NOTE: For this task, at least two pathologists with experience in breast pathology should discuss the diagnostic slides and resolve disagreements collegially. Use either a conventional microscope or telepathology tools (favor the latter in multicentric studies). If any, exclude all discordant cases.
	<ol>
		<li>Perform the histological re-classification of all cases according to the latest edition of the World Health Organization (WHO) classification of tumors of the breast18.</li>
		<li>Ensure that the normal tissue for each case, if present in the archival clinical samples, comprises at least one non-neoplastic terminal ductal-lobular unit.</li>
		<li>Identify all neoplastic areas that show heterogeneous cytological, architectural, or IHC features using a bright-field microscope (to be jointly performed by a pathologist and the technician(s) who will construct the TMAs).</li>
	</ol>
	</li>
	<li>Highlight the discrete topographic areas with a marker on the glass slide or with the proper tool on a digital slide software (Figure 1A).</li>
	<li>Based on the selected slides, retrieve the corresponding FFPE blocks from the archives. 
	NOTE: To optimize the TMA construction, to avoid tissue depletion, and to preserve the TMA punch structural integrity, cases with &#60;1 mm-thick residual tissue should be excluded.</li>
</ol>
2. Design and Construct TMAs Based on the Kononen Technique
<ol>
	<li>Create and open a new spreadsheet file incorporating the records of the distinct areas of each case. Include the following data on separate columns, as exemplified in Table 1: Case ID, Block ID, Area ID, tissue type (e.g., normal tissue, invasive component histotype, in situ component histotype), topography (e.g., tumor core, invasive front), morphology (e.g., nuclear grade, architecture, mitoses, cytological features), biomarkers status (i.e., estrogen receptor (ER), progesterone receptor (PR), Ki67, and HER2), and all other IHC features available.
	<ol>
		<li>Save the document.</li>
	</ol>
	</li>
	<li>Create a TMA project.
	<ol>
		<li>Install and open the TMA designer software.</li>
		<li>Define the TMA recipient block(s): access through the menu File &#62; New &#62; Block or by using CTRL+ B.</li>
		<li>Click in the first field to fill or press the Tab key, and type in dimensions of the paraffin block: width 35 (mm), height 20 (mm). Type &#34;2&#34; as the &#34;margin&#34; value (i.e., distance from paraffin edge in mm) (Figure 1B). Save the data by clicking the proper button. The new template of recipient block is now saved and ready to be used in new tissue array templates.</li>
		<li>Create a tissue array project: access to the menu File &#62; New &#62; Tissue Array or by using CTRL+ T.</li>
		<li>Fill in the name of the Tissue array, comments, and author, then click &#34;next.&#34;&#160;Click the &#34;import&#34; icon, choose the template file of recipient block previously created, and click &#34;next.&#34;</li>
		<li>Choose 1 mm as the spot size by clicking the round button. Choose 500 &#181;m as spot spacing which is the space between two centers of neighboring spots. Click on the calculator icon to calculate the total number of spots created, which should be 231. Click &#34;next.&#34;</li>
		<li>Define the spot numbering mode by clicking once on it. Click on the button &#34;define&#34; to create grids and sub-grids. Delete the central line (line 6) and the columns 8 and 15. Maintain 3 spots of line 1 for orientation. The final map should encompass a total number of 183 spots, as represented in Figure 2.</li>
		<li>Define the list of tissue types: access through the menu File &#62; New &#62; List of tissue types or by using CTRL+ L.</li>
		<li>Insert the tissue descriptions previously defined. Press down arrow or click on the + button to add a line.</li>
		<li>Define the project: access through the menu File &#62; New &#62; Booster project or by using CTRL+ P.</li>
		<li>Fill in the name of the tissue array, comments, and author, then click &#34;next.&#34;&#160;Import the spreadsheet file, list of tissue types, and the tissue array model previously created by clicking the proper icons.</li>
		<li>Click &#34;select all&#34; and define the number of spots for each tissue type; click on the &#34;match&#34; button to apply settings. A table listing the total number of cores that will be sampled and transferred to recipient blocks in this project will appear. Save the project and close the program.</li>
	</ol>
	</li>
	<li>Prepare the acceptor block(s) (Figure 1B).
	<ol>
		<li>Fill cleansed metal molds with elasticized paraffin at 65 &#176;C.</li>
		<li>Leave the paraffin blocks to cool at room temperature overnight inside of the metal molds.</li>
		<li>Extract the paraffin blocks from the metal molds. If cracks occur, discard the block and repeat steps 2.3.1-2.3.3.</li>
	</ol>
	</li>
	<li>Construct the TMA using an automated or semi-automated arrayer19.
	<ol>
		<li>Retrieve all selected FFPE blocks and the corresponding H&#38;E sections with annotations.</li>
		<li>Turn on the arrayer and insert the acceptor block(s) on the arrayer carousel.</li>
		<li>Open the arrayer control station software, click on &#34;New Tissue Array&#34;, and load the project previously created (Figure 1C).</li>
		<li>Click &#34;Continue&#34; and select the position of the donor block(s) from the menu.</li>
		<li>Define the starting position onto each recipient block: use the arrow keys to move the laser light and align it with the top-left edge on the paraffin of the recipient block.</li>
		<li>Click &#34;next&#34; and repeat for the vertical alignment.</li>
		<li>Click &#34;next&#34; to automatically create a hole in the previously defined coordinate of the recipient (acceptor) block.</li>
		<li>Empty the punch.</li>
		<li>Position the donor block for sampling and remove a tissue core from the previously annotated area of interest.</li>
		<li>Insert the donor tissue core into the hole in the recipient (acceptor) block.</li>
		<li>Remove the donor block from the arrayer.</li>
		<li>Repeat steps 2.4.5-2.4.11 until the TMA is completed.</li>
	</ol>
	</li>
	<li>Put the tissue side of the newly generated TMA block on a sterile blank slide and place them in the lab oven at 45 &#176;C for 5 min.</li>
	<li>Gently press the block on the slide for 5 s to flatten the tissue cores. Repeat once (for a total of 2 times).</li>
	<li>Remove the TMA block together with the slide from the oven, flip them, and gently detach the block from the slide.</li>
	<li>Cover the TMA tissue surface with a thin layer of elasticized paraffin at 65 &#176;C.</li>
	<li>Leave the TMA block at room temperature for 2 h.</li>
	<li>Transfer the TMA block at 4 &#176;C for 24 h. 
	NOTE: The TMA block can be stored at 4 &#176;C until exhaustion.</li>
</ol>
3. Histochemical and Immunohistochemical Analyses
<ol>
	<li>Cut TMA sections.
	<ol>
		<li>Place the TMA acceptor block(s) with the paraffin-side down on a -10 &#176;C surface for 10 min to chill the paraffin.</li>
		<li>Fill a floatation bath with ultrapure water and set heat to 38 &#176;C.</li>
		<li>Place a new blade on a rotary microtome and insert the block into the microtome chuck so the wax block faces the blade and is aligned in the vertical plane.</li>
		<li>Approach the block carefully with the blade and cut a few thin sections to ensure the positioning is correct; adjust if necessary.</li>
		<li>Cut 3 &#181;m-thick sections.</li>
		<li>Pick up the sections using tweezers and float them on the water bath surface.</li>
		<li>Pick the sections out the water bath on regular and IHC/ISH glass slides.</li>
		<li>Place the slides on a rack and store them in the oven at 45 &#176;C overnight. 
		NOTE: Once prepared, the TMA sections should be stained within 24 h.</li>
	</ol>
	</li>
	<li>Perform histochemical staining (H&#38;E) using an autostainer.
	<ol>
		<li>Insert the slides in the autostainer rack and place them at 60 &#176;C for 10 min.</li>
		<li>Open the load drawer of the autostainer and insert the rack with the TMA slides in one of the load stations with a standard H&#38;E clip facing outwards.</li>
		<li>Once the drawer is closed, the clip will be automatically recognized by the system and the following protocol will start: oven station at 37 &#176;C (6 min), xylene (2 min), xylene (2 min), ethanol 100% (2 min), ethanol 100% (2 min), ethanol 95% (2 min), ethanol 95% (2 min), distilled water (4 min), Carazzi&#39;s hematoxylin (9 min), low-pressure running water (2 min), eosin 1% (1 min), low-pressure running water (2 min), ethanol 95% (20 s), ethanol 95% (20 s), ethanol 95% (20 s), ethanol 95% (20 s), ethanol 100% (15 s), ethanol 100% (15 s), xylene (30 s), xylene (30 s).</li>
		<li>Unload the racks from the drawer and mount the slide with a cover slip.
		<ol>
			<li>Collect a drop of mount with a pipette and put it on an outer edge of the cover slide.</li>
			<li>Place the wet side of the cover slip on the slide and let the mount dry for 30 min.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Perform IHC staining for ER, PR, Ki67, and HER2 using an automated immunostainer. 
	NOTE: Before beginning a staining run, start up the system, and check the consumables reagent bottles (Table of Materials) and waste containers.
	<ol>
		<li>Place the TMA slides to analyze at 60 &#176;C for 10 min.</li>
		<li>Start the immunostainer instrument and software.</li>
		<li>At the Home View, click the Protocols button, and then click Create/Edit Protocols.</li>
		<li>Select the standard DAB (3, 3&#39;-diaminobenzidine) IHC procedure to modify the basic template.</li>
		<li>Flag &#34;Deparaffinization&#34; in the box list and set the Medium Temperature at 72 &#176;C.</li>
		<li>Flag the cc1 unmasking solution, set the Medium Temperature at 95 &#176;C, and the incubation time at 36 min.</li>
		<li>Flag the Primary Antibody box, insert the ID of the ER inline dispenser (the instrument software will recognize the dispenser on the reagent carousel), and set the incubation time at 16 min.</li>
		<li>Flag the Counterstaining box, select Hematoxylin I, and set the incubation time at 12 min.</li>
		<li>Click the &#34;Save As&#34; button and name the protocol.</li>
		<li>Repeat the steps 3.3.3-3.3.9 for the remaining antibodies using the following primary antibodies incubation times: PR 16 min, Ki67 16 min, and HER2 12 min. Use pre-diluted ready-to-use (RTU) antibodies.</li>
		<li>At the Home View, click the Create Label button.</li>
		<li>Click the Protocols button and add the ER, PR, Ki67, and HER2 protocols for each of the TMA to analyze.</li>
		<li>Click the Close /Print button.</li>
		<li>As the label template appears, enter the TMA IDs for the label.</li>
		<li>Click the Print button to print the labels and then apply them on the TMA slides.</li>
		<li>Click the instrument icon and set the instrument startup mode as &#34;Ready&#34;.</li>
		<li>Open the hood that covers the reagents carousel, place the detection system, and primary antibodies, then close the hood.</li>
		<li>Click the front button on a slide drawer to open it, load the TMA slides, and close the drawer by clicking the same button.</li>
		<li>Set the instrument startup mode as &#34;Running&#34; and follow the instructions.</li>
		<li>At the end of the run, unload the TMA slides by pressing the Open All Drawers with Completed Slides button on the instrument Slide Control and rinse them in warm soapy water to remove any remaining reagents.</li>
		<li>Wash the slides under low-pressure cold running water, dehydrate the TMA slides, and apply a glass coverslip to each slide.</li>
	</ol>
	</li>
	<li>Perform IHC analysis of ER, PR, and HER2 following the American Society of Clinical Oncology (ASCO)/CAP guidelines, and of Ki67 according to the recommendations of the International Ki67 in Breast Cancer working group (Table 2). 
	NOTE: This analysis should be performed separately in the discrete tissue spots by a pathologist with an experience in breast pathology.
	<ol>
		<li>Assess the ER expression as positive if &#8805; 1% of tumor cell nuclei are immunoreactive20,21,22,23.</li>
		<li>Assess the PR expression as positive if &#8805; 1% of tumor cell nuclei are immunoreactive20,21,22,23.</li>
		<li>Assess the HER2 expression as: a) score 3+ if circumferential membrane staining that is complete and intense, b) score 2+ if circumferential membrane staining is incomplete and/or weak/moderate and within &#62; 10% of the invasive tumor cells or complete and circumferential membrane staining is intense and within &#8804; 10% of the invasive tumor cells, c) score 1+ if incomplete membrane staining is faint/barely perceptible and within &#62; 10% of the invasive tumor cells, d) negative if no staining is present or membrane staining is incomplete and faint/barely perceptible and within &#8804; 10% of the invasive tumor cells3,24.</li>
		<li>Assess the Ki67 index as the percentage of cells with nuclear staining in at least 1,000 neoplastic cells randomly selected over 10 high-power fields (magnification, 400X)25.</li>
	</ol>
	</li>
</ol>
4. SISH Analysis of HER2
<ol>
	<li>Cut TMA sections (repeat step 3.1).</li>
	<li>Perform SISH for HER2 using an automated immunostainer.
	<ol>
		<li>Place the TMA slides to analyze at 60 &#176;C for 10 min.</li>
		<li>Start the immunostainer instrument and software.</li>
		<li>At the Home View, click the Protocols button, and then click Create/Edit Protocols.</li>
		<li>Select the &#34;U Dual ISH CKT&#34; procedure to modify the basic template.</li>
		<li>Flag &#34;Drying&#34; in the box list and set the temperature at 63 &#176;C for 20 min.</li>
		<li>Flag &#34;Cell Conditioning&#34; in the box list, then &#34;Cell Conditioning CC2&#34;, and set the temperature at 86 &#176;C and incubation time at 4 min.</li>
		<li>Flag CC2 Mild (Cycle 1), Standard (Cycle 2), and Extended (Cycle 3) for 8 min, 12 min, and 8 min, respectively.</li>
		<li>Flag &#34;ISH-Protease 3&#34; in the box list and set the incubation time at 16 min.</li>
		<li>Select the probes HER2DNP and CHR17DIG and set the incubation time at 6 h.</li>
		<li>Set the washing at 76 &#176;C for 8 min.</li>
		<li>Set the SISH multimer (SIL ISH DNP HRP) incubation time at 32 min.</li>
		<li>Set the silver chromogen (SIL ISH DNP CHRC) incubation time at 4 min.</li>
		<li>Set the red multimer (RED ISH DIG AP) incubation time at 24 min.</li>
		<li>Set the red chromogen (RED ISH DIG FR) incubation time at 8 min.</li>
		<li>Flag &#34;Counterstaining&#34; in the box list, select &#34;HEMATOXYLIN II&#34;, and set the incubation time at 8 min.</li>
		<li>Flag &#34;Post-Counterstaining&#34; in the box list, select &#34;BLUING REAGENT&#34;, and set the incubation time at 4 min.</li>
		<li>Repeat steps from 3.3.11-3.3.15 (select modified U Dual ISH CKT protocol).</li>
		<li>Open the hood that covers the reagents carousel, place the washing and the ISH detection systems, then close the hood.</li>
		<li>Repeat steps from 3.3.18-3.3.21.</li>
	</ol>
	</li>
	<li>Perform SISH analysis for HER2: assess the HER2 gene amplification as positive if the HER2/CEP17 ratio is &#8805;2.0 with an average HER2 copy number &#8805;4.0 signals per cell, and negative if the HER2/CEP17 ratio is &#60;2.0 with an average HER2 copy number &#60;4.0 signals/cell3,24. 
	NOTE: Similar to IHC, this analysis should be performed separately in the discrete tissue spots by a pathologist with experience in breast pathology.</li>
</ol>

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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><tbody><tr><td>Surgipath Paraplast</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>39601006</td><td>Tissue embedding medium, 56 &amp;deg;C melting point</td></tr><tr><td>Eosin Y&amp;nbsp; 1% water solution</td><td>Bio Optica, Milan, Italy, EU</td><td>510002</td><td>Eosin yellowish, water-soluble</td></tr><tr><td>Carazzi&amp;rsquo;s hematoxylin</td><td>Bio Optica, Milan, Italy, EU</td><td>506012</td><td>Alum hematoxylin ripened using potassium iodate</td></tr><tr><td>Diamond quality</td><td>Laboindustria, Arzergrande, Italy, EU</td><td>33533</td><td>26x76 mm microscope slides</td></tr><tr><td>Leica CV Mount</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>14046430011</td><td>Mounting medium, with no monomers, based on polymers of butylmethacrylate in xylene</td></tr><tr><td>FLEX IHC microscope slides</td><td>Agilent Technologies (Dako),&amp;nbsp; Santa Clara, CA, USA</td><td>K8020</td><td>Coated microscope slides for adhesion of FFPE for use in IHC</td></tr><tr><td>BenchMark ULTRA</td><td>Ventana medical system, Tucson, AZ, USA</td><td>N750-BMKU-FS</td><td>Slide staining system</td></tr><tr><td>CONFIRM anti-Estrogen Receptor (ER) (SP1) Rabbit Monoclonal Primary Antibody</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-4324</td><td>Primary antibody, ready-to-use</td></tr><tr><td>CONFIRM anti-Progesterone Receptor (PR) (1E2) Rabbit Monoclonal Primary Antibody</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-2223</td><td>Primary antibody, ready-to-use</td></tr><tr><td>CONFIRM anti-Ki-67 (30-9) Rabbit Monoclonal Primary Antibody</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-4286</td><td>Primary antibody, ready-to-use</td></tr><tr><td>PATHWAY HER2 (4B5) Rabbit Monoclonal Primary Antibody</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-4493</td><td>Primary antibody, ready-to-use</td></tr><tr><td>ultraView Universal DAB Detection Kit</td><td>Ventana medical system, Tucson, AZ, USA</td><td>760-500</td><td>Indirect, biotin-free system for detecting mouse IgG, mouse IgM and rabbit primary antibodies</td></tr><tr><td>INFORM HER2 Dual ISH DNA Probe Cocktail</td><td>Ventana medical system, Tucson, AZ, USA</td><td>780-4422</td><td>INFORM HER2 Dual ISH assay - Dual color in situ hybridization FDA approved automated assay for determining HER2 gene status in breast cancer patients&amp;nbsp;</td></tr><tr><td>ultraView Silver ISH DNP Detection Kit</td><td>Ventana medical system, Tucson, AZ, USA</td><td>800-098</td><td></td></tr><tr><td>ultraView Red ISH DIG Detection Kit</td><td>Ventana medical system, Tucson, AZ, USA</td><td>800-505</td><td></td></tr><tr><td>ISH Protease 3</td><td>Ventana medical system, Tucson, AZ, USA</td><td>780-4149</td><td>Used in the ISH process to remove protein that surrounds the target DNA sequences of interest</td></tr><tr><td>Hematoxylin</td><td>Ventana medical system, Tucson, AZ, USA</td><td>760-2021</td><td>Modified Gill&#039;s hematoxylin counterstain reagent</td></tr><tr><td>Hematoxylin II Counterstaining</td><td>Ventana medical system, Tucson, AZ, USA</td><td>790-2208</td><td>Modified Meyer&#039;s hematoxylin counterstain reagent</td></tr><tr><td>Bluing reagent</td><td>Ventana medical system, Tucson, AZ, USA</td><td>760-2037</td><td>Aqueous solution of buffered lithium carbonate for bluing hematoxylin stained sections on glass slides</td></tr><tr><td>HybReady</td><td>Ventana medical system, Tucson, AZ, USA</td><td>780-4409</td><td>Formamide-based buffer for ISH assays</td></tr><tr><td>EZ Prep (10x)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-102</td><td>Concentrate solution for paraffin removal from tissue samples during IHC and ISH reactions, and to dilute 1:10.</td></tr><tr><td>SSC Buffer (10X)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-110</td><td>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><td>ULTRA LCS</td><td>Ventana medical system, Tucson, AZ, USA</td><td>650-210</td><td>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><td>Reaction Buffer (10x)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-300</td><td>Tris based buffer solution (pH 7.6 &amp;plusmn; 0.2) to rinse slides between staining steps during IHC and ISH. Dilute 1:10.</td></tr><tr><td>ULTRA Cell Conditioning (ULTRA CC2)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-223</td><td>Pretreatment steps in the processing of tissue samples during IHC and ISH. Ready to use.</td></tr><tr><td>ULTRA Cell Conditioning (ULTRA CC1)</td><td>Ventana medical system, Tucson, AZ, USA</td><td>950-224</td><td></td></tr><tr><td>ultraView Silver Wash II</td><td>Ventana medical system, Tucson, AZ, USA</td><td>780-003</td><td>Ready-to-use solution to rinse slides between IHC and ISH staining steps</td></tr><tr><td>Microtome</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>RM 2255</td><td>Automated rotary microtome</td></tr><tr><td>Multistainer</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>ST 5020</td><td>Workstation for automated staining and coverslipping</td></tr><tr><td>Minicore 1</td><td>Alphelys, Plaisir, France, EU</td><td>00-MICO-1</td><td>Semi-automatic arrayer for TMA contruction with TMA Designer 2 embedded software</td></tr><tr><td>Aperio ScanScope CS2</td><td>Leica Biosystems, Wetzlar, Germany, EU</td><td>K080254</td><td>Image capture device - digital pathology scanner</td></tr><tr><td>Tissue-Tek III Uni-Cassette</td><td>Sakura Finetek Europe B.V</td><td>4135</td><td>Cassette</td></tr><tr><td>Tissue-Tek Paraform Standard Base Mold</td><td>Sakura Finetek Europe B.V</td><td>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 ...

building-up-high-throughput-screening-platform-to-assess

Research

JoVE Journal

Immunology and Infection

12.6K Views.  Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico. Heterogene verdeling van de HER2-positieve cellen kan worden waargenomen in een subset van kanker van de borst en genereert klinische dilemma's. Hier introduceren we een betrouwbare en voordelige protocol definiëren, meten en vergelijken van HER2 intra-tumor genetische heterogeniteit in een groot aantal ongelijkmatig verwerkte borstkankers.

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

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Bioengineering

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Medicine

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Cancer Research

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells ...

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

Validated Immunochemical Assay for Comprehensive Determination of the Human Epidermal Growth Factor Receptor 2 Released from and Bound to Cells

Human epidermal growth factor receptor 2 (HER2) is a well-established cancer marker. It became a very successful diagnostic and therapeutic target, especially in breast cancer and other HER2-expressing cancer types. In the clinic, the gold-standard immunohistochemical diagnostic methods employing the specific anti-HER2 antibodies are used to measure the expression level of the membrane-bound receptor. The soluble extracellular domain (ECD) of HER2 that is released from the overexpressing cells circulates in the blood and can reflect the tissue expression of the receptor. There is a need for accurate and validated assays to correlate the concentration of the circulating HER2 protein with disease clinical manifestations.
Our team has developed and validated the novel sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of the membrane-bound and the released from cells ECD domain of HER2. The assay uses two unique monoclonal antibodies specific to HER2 developed previously. The quantitation range includes HER2 concentration from 1.56-100 ng/mL, which is expected for cancer cells cultured in vitro and shows sensitivity at the level of 0.5 ng/mL. The satisfactory intra- and inter-assay precision and accuracy of the method make it applicable for HER2 quantification in various types of biological samples, including cell culture medium, serum, and solid tumor tissue. Here, we focus on the comprehensive determination of the receptor-associated and secreted by the in vitro cultured cancer cells. The paper presents a step-by-step protocol for the quantification of HER2 protein that can be employed for testing a variety of cell lines, blood, and tissues.

We present a validated sandwich ELISA assay using novel anti-HER2 monoclonal antibodies. This assay enables precise quantification of cell-bound and released HER2 protein from in vitro cultured cells and other samples, including blood and tissues.

Human epidermal growth factor receptor 2 (HER2) is a well-established cancer marker. It became a very successful diagnostic and therapeutic target, ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

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

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