Name: automated multiplex immunofluorescence panel for immuno-oncology studies on formalin-fixed carcinoma tissue specimens
Uploaded: 2019-01-21T13:00:00
Description: Watch this Scientific Journal Video about Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens at JoVE.com

Editorial support was provided by Deborah Shuman of MedImmune.

NOTE: The protocol presented here describes how to perform immunoprofiling of an mIF panel by using TSA for six antibodies (CD68, ki67, PD-L1, PD-1, CD8, and AE1/AE3) on an automated stainer (see Table of Materials). The protocol also describes how to perform the drop controls for a quality control of a new mIF panel (see Supplemental Materials). In this protocol, staining is performed with eight unstained FFPE slides from human tonsil (positive control) and eight unstai...

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	<li>Bethmann, D., Feng, Z., Fox, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoprofiling+as+a+predictor+of+patient's+response+to+cancer+therapy-promises+and+challenges.">Immunoprofiling as a predictor of patient's response to cancer therapy-promises and challenges.</a> Current Opinion in Immunology. 45, 60-72 (2017).</li><li>Taube, J. 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=Implications+of+the+tumor+immune+microenvironment+for+staging+and+therapeutics.">Implications of the tumor immune microenvironment for staging and therapeutics.</a> Modern Pathology. 31 (2), 214-234 (2018).</li><li>Idikio, H. 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R., 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=Immunohistochemical+and+image+analysis-based+study+shows+that+several+immune+checkpoints+are+co-expressed+in+non-small+cell+lung+carcinoma+tumors.">Immunohistochemical and image analysis-based study shows that several immune checkpoints are co-expressed in non-small cell lung carcinoma tumors.</a> Journal of Thoracic Oncology. 13 (6), 779-791 (2018).</li><li>Rehman, J. A., 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=Quantitative+and+pathologist-read+comparison+of+the+heterogeneity+of+programmed+death-ligand+1+(PD-L1)+expression+in+non-small+cell+lung+cancer.">Quantitative and pathologist-read comparison of the heterogeneity of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer.</a> Modern Pathology. 30 (3), 340-349 (2017).</li><li>Dixon, A. R., 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=Recent+developments+in+multiplexing+techniques+for+immunohistochemistry.">Recent developments in multiplexing techniques for immunohistochemistry.</a> Expert Review of Molecular Diagnostics. 15 (9), 1171-1186 (2015).</li><li>Stack, E. C., Wang, C., Roman, K. A., Hoyt, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+immunohistochemistry,+imaging,+and+quantitation:+a+review,+with+an+assessment+of+Tyramide+signal+amplification,+multispectral+imaging+and+multiplex+analysis.">Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis.</a> Methods. 70 (1), 46-58 (2014).</li><li>Feng, Z., 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=Multiparametric+immune+profiling+in+HPV-+oral+squamous+cell+cancer.">Multiparametric immune profiling in HPV- oral squamous cell cancer.</a> JCI Insight. 2 (14), (2017).</li><li>Feng, Z., 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=Multispectral+imaging+of+formalin-fixed+tissue+predicts+ability+to+generate+tumor-infiltrating+lymphocytes+from+melanoma.">Multispectral imaging of formalin-fixed tissue predicts ability to generate tumor-infiltrating lymphocytes from melanoma.</a> Journal for ImmunoTherapy of Cancer. 3, 47 (2015).</li><li>Granier, 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=Multiplexed+immunofluorescence+analysis+and+quantification+of+intratumoral+PD-1++Tim-3++CD8++T+cells.">Multiplexed immunofluorescence analysis and quantification of intratumoral PD-1+ Tim-3+ CD8+ T cells.</a> Journal of Visualized Experiments. (132), e56606 (2018).</li><li>Parra, E. R., 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=Validation+of+multiplex+immunofluorescence+panels+using+multispectral+microscopy+for+immune-profiling+of+formalin-fixed+and+paraffin-embedded+human+tumor+tissues.">Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.</a> Scientific Reports. 7 (1), 13380 (2017).</li><li>Ribas, A., Wolchok, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunotherapy+using+checkpoint+blockade.">Cancer immunotherapy using checkpoint blockade.</a> Science. 359 (6382), 1350-1355 (2018).</li><li>Gorris, M. A. 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=Eight-color+multiplex+immunohistochemistry+for+simultaneous+detection+of+multiple+immune+checkpoint+molecules+within+the+tumor+microenvironment.">Eight-color multiplex immunohistochemistry for simultaneous detection of multiple immune checkpoint molecules within the tumor microenvironment.</a> Journal of Immunology. 200 (1), 347-354 (2018).</li><li>Parra, E. 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The ongoing cancer immunotherapy revolution is opening novel and promising therapeutic options for cancer patients13. Advances in the field of immuno-oncology will require increased knowledge of the inflammatory tumor microenvironment, not only to understand the biology of the immunological mechanisms involved in carcinogenesis but also to find predictive biomarkers for new immunotherapy-based treatments1,2. Due to the complex biology of c...

Immunoprofiling analysis of FFPE tumor tissue samples has become an essential component of immuno-oncology studies, particularly for the discovery and validation of novel predictive biomarkers for cancer immunotherapy in the context of clinical trials1,2. Chromogenic IHC, using chemical chromogens such as diaminobenzidine, remains the standard technique in diagnostic pathology for the immunolabeling of biopsy tissue3. Standard IHC can also be used for cancer tissue immunoprofiling, including the quantitation of subpopulations of tumor-associated lymphocytes and the assessment of expression levels of immune checkpoints such as programmed cell death ligand 1 (PD-L1)4,5. Standard IHC is limited, however, in that only one antigen can be labeled per tissue section. Because immunoprofiling studies typically require the analysis of the combined expression of several markers, the use of standard IHC would require the staining of multiple tissue sections, each stained with a single marker, and would, therefore, be substantially limited for the analysis of small tissue samples such as core needle biopsies. Standard IHC methods are also limited for the assessment of markers that are coexpressed by diverse cell populations, as is common with immune checkpoint markers such as PD-L1, which is expressed by both tumor-associated macrophages and cancer cells. This limitation has been reported in, for instance, the use of standard monoplex IHC by pathologists for the quantitative analysis of an IHC marker expressed by diverse cell types6. The development of multiplex chromogenic IHC techniques employing diverse colored chromogens on the same tissue section represents an advancement over the standard IHC monoplex method,7 although they remain limited by the immunolabeling of just a few markers and also present an important technical challenge for the proper evaluation of markers expressed in the same subcellular compartments of the same cell populations.
The aforementioned caveats regarding tissue availability from clinical samples, as well as the limitations of multiplex chromogenic IHC techniques, have given rise to the need to develop improved multiplex methods for immuno-oncology studies based on fluorescent labeling combined with imaging systems that can effectively separate the signals of multiple fluorophores from the same slide. One such technique is based on tyramide signal amplification (TSA) combined with multispectral microscopy imaging for efficient color separation8. A commercially available TSA-based kit employs fluorophores optimized for multispectral imaging8 (see Table of Materials). A critical advantage of this system is its compatibility with the same unlabeled primary antibodies that have already been validated and optimized for standard chromogenic IHC9,10,11. This allows not only faster optimization but also flexibility in the optimization and panel modifications incorporating new targets. Furthermore, the multiplex immunofluorescence (mIF) TSA method can be optimized for commercially available automated IHC stainer systems, allowing for a straightforward transfer from monoplex chromogenic IHC to mIF.
Here we present a protocol for an mIF panel for immuno-oncology studies that is based on automated mIF TSA staining and uses a multispectral scanner for imaging. This protocol can be adapted and modified by any laboratory user with access to the described instrumentation and reagents. The protocol includes a panel of six primary antibodies for the immunoprofiling of carcinomas: PD-L1, PD-1, CD68 (as a pan-macrophage marker), CD8 (T-cytotoxic cells), Ki-67, and AE1/AE3 (pan-cytokeratin, used as an epithelial marker for the identification of carcinoma cells). A recent study describes the optimization of a manual TSA mIF protocol by using chromogenic IHC as a standard reference to validate the multiplex staining12. The updated method presented here has been developed by using a commercially available, seven-color TSA kit optimized in an automated stainer, drastically shortening the staining time from 3&#8211;5 days to 14 h, while also improving the consistency of the staining. In addition to the detailed main protocol presented here, a Supplemental Materials section includes the &#34;drop-control&#34; method, an additional quality control process to evaluate a new mIF panel, as well as technical notes for the optimization, troubleshooting, and development of new multiplex panels to help the laboratory user to set up and optimize the mIF TSA method for customized mIF panels.

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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:273px;">&#34;InForm 2.4.2&#34; Software for Spectral Unmixing and Image Analysis</td>
			<td style="width:123px;">PerkinElmer</td>
			<td style="width:119px;">CLS151066</td>
			<td style="width:299px;">Called &#34;spectral unmixing software&#34; in text</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">&#34;Phenochart 1.0.9&#34; QPTIFF Software for Selection of MSI and Overall Slide Scan Viewing</td>
			<td style="width:123px;">PerkinElmer</td>
			<td style="width:119px;">CLS151067</td>
			<td>Called &#34;QPTIFF software&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">#1.5 Coverslips</td>
			<td>Sigma Aldrich</td>
			<td>2975246</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 Proof Ethanol</td>
			<td>Koptec</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20x Tris-Buffered Saline</td>
			<td>VWR</td>
			<td>J640-4L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Antibody Diluent</td>
			<td>DAKO</td>
			<td>S2203</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD68 Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M087601-2</td>
			<td>Clone PG-M1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CD8 Rabbit Monoclonal</td>
			<td>Ventana</td>
			<td>M5392</td>
			<td>Clone SP239</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-CK Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M351501-2</td>
			<td>Clone AE1/AE3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-ki67 Mouse Monoclonal</td>
			<td>DAKO</td>
			<td>M724001-2</td>
			<td>Clone MIB-1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PD-1 Rabbit Monoclonal</td>
			<td>Cell Signaling</td>
			<td>#86163</td>
			<td>Clone D4W2J</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-PD-L1 Rabbit Monoclonal</td>
			<td>Ventana</td>
			<td>790-4905</td>
			<td>Clone SP263</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Dewax Solution</td>
			<td>Leica</td>
			<td>AR9222</td>
			<td>Called &#34;dewax solution&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Epitope Retrieval Solution 1</td>
			<td>Leica</td>
			<td>AR9961</td>
			<td>Called &#34;ER1&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Epitope Retrieval Solution 2</td>
			<td>Leica</td>
			<td>AR9640</td>
			<td>Called &#34;ER2&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Open Containers, 30 mL</td>
			<td>Leica</td>
			<td>OP309700</td>
			<td>Called &#34;30 mL open containers&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Open Containers, 7 mL</td>
			<td>Leica</td>
			<td>OP79193</td>
			<td>Called &#34;7 mL open containers&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Bond Polymer Refine Detection</td>
			<td>Leica</td>
			<td>DS9800</td>
			<td>Called &#34;chromogenic detection kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Research Detection Kit</td>
			<td>Leica</td>
			<td>DS9455</td>
			<td>Called &#34;research detection kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Titration Kit</td>
			<td>Leica</td>
			<td>OPT9049</td>
			<td>Called &#34;titration kit&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Universal Covertile Novocastra</td>
			<td>Leica</td>
			<td>S21.2001</td>
			<td>Called &#34;covertiles&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bond Wash Solution 10X Concentrate</td>
			<td>Leica</td>
			<td>AR9590</td>
			<td>Called &#34;10x wash solution&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BondRX Autostainer</td>
			<td>Leica</td>
			<td style="width:119px;"></td>
			<td>Called &#34;automated stainer&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">BondRX Software Version 5.2.1.204</td>
			<td>Leica</td>
			<td style="width:119px;"></td>
			<td>Called &#34;automated stainer software&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Opal 7-Color Automation IHC Kit</td>
			<td style="width:123px;">PerkinElmer</td>
			<td>NEL801001KT</td>
			<td style="width:299px;">Called &#34;multispectral staining kit&#34; in text</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase Block</td>
			<td>Leica</td>
			<td>RE7101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond Antifade Mountant</td>
			<td>Thermo</td>
			<td>P36965</td>
			<td>Called &#34;slide mountant&#34; in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:273px;">Starfrost Slides</td>
			<td style="width:123px;">Fisher</td>
			<td style="width:119px;">15-183-51</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:273px;">Vectra Polaris Multispectral Microscope with &#34;Vectra 3.0.5&#34; Software for Multispectral Microscope Control</td>
			<td style="width:123px;">PerkinElmer</td>
			<td>CLS143455</td>
			<td>Called &#34;microscope control software&#34; in text</td>
		</tr>
	</tbody>
</table>

automated multiplex immunofluorescence panel for immuno-oncology studies on formalin-fixed carcinoma tissue specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

The protocol described here will provide results like those shown in Figure 2. Start with an evaluation of the staining in the tonsil control, beginning with the surface squamous cell epithelium. The histology of the tonsil sample can be reviewed with a pathologist, using the H&#38;E slide as a reference. If chromogenic IHC sections are performed with the same markers on the same tissue block, then these can be used to confirm the density and distribution of ...

Watch this Scientific Journal Video about Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens at JoVE.com

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

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