Name: using 22c3 anti-pd-l1 antibody concentrate on biopsy and cytology samples from non-small cell lung cancer patients
Uploaded: 2018-09-25T15:00:00
Description: Watch this Scientific Journal Video about Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients at JoVE.com

This study was sponsored by Merck &#38; Co., Inc., Kenilworth, NJ, USA. The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All procedures have been approved by the local ethics committee (Human Research Ethics Committee, Centre Hospitalier Universitaire de Nice/Tumoroth&#232;que BB-0033-00025).
NOTE: This protocol is specifically adjusted for the use of the 22C3 antibody concentrate on a commercially available automated IHC stainer (referred to as autostainer here, see the Table of Materials) for tumor biopsies and cytology samples.
1. Preparation of Tumor Tissue Samples<...

<ol>
	<li>Garon, E. 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=Pembrolizumab+for+the+treatment+of+non-small-cell+lung+cancer.">Pembrolizumab for the treatment of non-small-cell lung cancer.</a> The New England Journal of Medicine. 372 (21), 2018-2028 (2015).</li><li>Herbst, R. 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=Pembrolizumab+versus+docetaxel+for+previously+treated,+PD-L1-positive,+advanced+non-small-cell+lung+cancer+(KEYNOTE-010):+a+randomised+controlled+trial.">Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial.</a> The Lancet. 387 (10027), 1540-1550 (2016).</li><li>Reck, 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=Pembrolizumab+versus+Chemotherapy+for+PD-L1-Positive+Non-Small-Cell+Lung+Cancer.">Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer.</a> The New England Journal of Medicine. 375 (19), 1823-1833 (2016).</li><li>Langer, C. 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=Carboplatin+and+pemetrexed+with+or+without+pembrolizumab+for+advanced,+non-squamous+non-small-cell+lung+cancer:+a+randomised,+phase+2+cohort+of+the+open-label+KEYNOTE-021+study.">Carboplatin and pemetrexed with or without pembrolizumab for advanced, non-squamous non-small-cell lung cancer: a randomised, phase 2 cohort of the open-label KEYNOTE-021 study.</a> The Lancet Oncology. 17 (11), 1497-1508 (2016).</li><li>Buttner, 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=Programmed+Death-Ligand+1+Immunohistochemistry+Testing:+A+Review+of+Analytical+Assays+and+Clinical+Implementation+in+Non-Small-Cell+Lung+Cancer.">Programmed Death-Ligand 1 Immunohistochemistry Testing: A Review of Analytical Assays and Clinical Implementation in Non-Small-Cell Lung Cancer.</a> Journal of Clinical Oncology. 35 (34), 3867-3876 (2017).</li><li>Folch, E., Costa, D. B., Wright, J., VanderLaan, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+cancer+diagnosis+and+staging+in+the+minimally+invasive+age+with+increasing+demands+for+tissue+analysis.">Lung cancer diagnosis and staging in the minimally invasive age with increasing demands for tissue analysis.</a> Translational Lung Cancer Research. 4 (4), 392-403 (2015).</li><li>Padmanabhan, V., 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=Improving+Adequacy+of+Small+Biopsy+and+Fine-Needle+Aspiration+Specimens+for+Molecular+Testing+by+Next-Generation+Sequencing+in+Patients+With+Lung+Cancer:+A+Quality+Improvement+Study+at+Dartmouth-Hitchcock+Medical+Center.">Improving Adequacy of Small Biopsy and Fine-Needle Aspiration Specimens for Molecular Testing by Next-Generation Sequencing in Patients With Lung Cancer: A Quality Improvement Study at Dartmouth-Hitchcock Medical Center.</a> Archives of Pathology &amp; Laboratory Medicine. 141 (3), 402-409 (2017).</li><li>Tsao, M. 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=.">.</a> IASLC ATLAS of PD-L1 Immunohistochemistry Testing in Lung Cancer. , (2017).</li><li>Thunnissen, E., de Langen, A. J., Smit, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PD-L1+IHC+in+NSCLC+with+a+global+and+methodological+perspective.">PD-L1 IHC in NSCLC with a global and methodological perspective.</a> Lung Cancer. , 102-105 (2017).</li><li>Ilie, 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=Use+of+the+22C3+anti-PD-L1+antibody+to+determine+PD-L1+expression+in+multiple+automated+immunohistochemistry+platforms.">Use of the 22C3 anti-PD-L1 antibody to determine PD-L1 expression in multiple automated immunohistochemistry platforms.</a> PLoS One. 12 (8), e0183023 (2017).</li><li>Ilie, 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=Use+of+the+22C3+anti-programmed+death+ligand+1+antibody+to+determine+programmed+death+ligand+1+expression+in+cytology+samples+obtained+from+non-small+cell+lung+cancer+patients.">Use of the 22C3 anti-programmed death ligand 1 antibody to determine programmed death ligand 1 expression in cytology samples obtained from non-small cell lung cancer patients.</a> Cancer Cytopathology. 126 (4), 264-274 (2018).</li><li>Skov, B. G., Skov, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+Comparison+of+PD-L1+Expression+on+Cytologic+and+Histologic+Specimens+From+Malignancies+in+the+Lung+Assessed+With+PD-L1+IHC+28-8pharmDx+and+PD-L1+IHC+22C3pharmDx.">Paired Comparison of PD-L1 Expression on Cytologic and Histologic Specimens From Malignancies in the Lung Assessed With PD-L1 IHC 28-8pharmDx and PD-L1 IHC 22C3pharmDx.</a> Applied Immunohistochemistry &amp; Molecular Morphology. 25 (7), 453-459 (2017).</li><li>Russell-Goldman, E., Kravets, S., Dahlberg, S. E., Sholl, L. M., Vivero, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytologic-histologic+correlation+of+programmed+death-ligand+1+immunohistochemistry+in+lung+carcinomas.">Cytologic-histologic correlation of programmed death-ligand 1 immunohistochemistry in lung carcinomas.</a> Cancer Cytopathology. 126 (4), 253-263 (2018).</li><li>Heymann, J. 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=PD-L1+expression+in+non-small+cell+lung+carcinoma:+Comparison+among+cytology,+small+biopsy,+and+surgical+resection+specimens.">PD-L1 expression in non-small cell lung carcinoma: Comparison among cytology, small biopsy, and surgical resection specimens.</a> Cancer Cytopathology. 125 (12), 896-907 (2017).</li><li>Stoy, S. P., Rosen, L., Mueller, J., Murgu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmed+death-ligand+1+testing+of+lung+cancer+cytology+specimens+obtained+with+bronchoscopy.">Programmed death-ligand 1 testing of lung cancer cytology specimens obtained with bronchoscopy.</a> Cancer Cytopathology. 126 (2), 122-128 (2018).</li><li>Ilie, M., Hofman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+PD-L1+assessment+in+non-small+cell+lung+cancer-know+your+limits+but+never+stop+trying+to+exceed+them.">Reproducibility of PD-L1 assessment in non-small cell lung cancer-know your limits but never stop trying to exceed them.</a> Translational Lung Cancer Research. 6 (Suppl 1), S51-S54 (2017).</li></ol>

We have validated an optimized LDT using the 22C3 PD-L1 antibody concentrate, by comparing it with the corresponding clinically validated commercial test10,11. The 22C3 concentrated antibody-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to the PD-L1 IHC expression determined using the PD-L1 IHC 22C3 assay at both &#8805;1% TPS and the &#8805;50% TPS cut points. As recently recommended by the Internatio...

Recent clinical trials have demonstrated the efficacy of pembrolizumab, a humanized monoclonal IgG4 kappa isotype antibody that blocks the interaction between programmed cell death 1 (PD-1) and its ligands, PD-L1 and PD-L2, in the treatment of patients with advanced NSCLC1,2,3,4.
Currently, pembrolizumab is approved for treatment of PD-L1-expressing NSCLC in both treatment-naive patients with a PD-L1 expression TPS of &#8805;50% and no epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) genomic tumor aberrations3 and for previously treated patients with a PD-L1 TPS of &#8805;1%1.
PD-L1 protein expression detected by IHC has been widely used as a predictive biomarker assay for anti-PD-1/PD-L1 therapies. In pembrolizumab clinical trials, the PD-L1 TPS obtained with formalin-fixed paraffin-embedded (FFPE) tissue samples was determined using the PD-L1 IHC 22C3 companion assay5. This assay has been approved by the US Food and Drug Administration (FDA) and has been CE-marked in Europe for the determination of the tumor PD-L1 TPS5.
Additional global options across institutions for reliable and high-quality evaluations of the PD-L1 TPS with LDTs which use the 22C3 antibody concentrate are essential to support clinical decisions made regarding patient eligibility for pembrolizumab treatment. A large number of pathology laboratories do not have access to the companion diagnostic PD-L1 IHC 22C3 assay. Therefore, the development of reliable and consistent LDTs compatible with additional, widely available IHC autostainer platforms is essential.
Moreover, there is a need to establish LDTs using cytology samples that are the only specimen type frequently available from NSCLC patients. The PD-L1 IHC 22C3 companion assay is validated for resections, core needle biopsies, and bronchoscopies only if the bronchoscopy yields 100 tumor cells. Although the above sample types are frequently obtained, cytology samples are more easily collected and are the most commonly available sample type in some institutions6,7. However, there is currently no validated diagnostic assay available for the evaluation of the PD-L1 expression in cytology samples; reliable LDTs compatible with cytology samples would further facilitate high-quality PD-L1 testing.
Furthermore, when establishing the clinical validation of an LDT, the IHC should be performed in a similar way to the corresponding clinically validated commercial test8. For instance, several critical steps should be verified to obtain the same signal in serial sections such as antibody titration, pretreatment delays, incubation time, and amplification systems9.
We recently developed an optimized LDT that uses the 22C3 antibody concentrate to evaluate the PD-L1 expression on tumor biopsies and cytology samples10,11. We found a high concordance with the LDT versus the &#34;gold standard&#34; PD-L1 IHC 22C3 assay10,11. This clinically validated protocol will support reliable, high-quality PD-L1 testing across regions globally.

<table>
	<tbody>
		<tr>
			<td>NovaPrep HQ1</td>
			<td>Novacyt</td>
			<td>NA</td>
			<td>Preservative solution for cytology specimens</td>
		</tr>
		<tr>
			<td>Novaprep&#174; HQ+ B</td>
			<td>Novacyt</td>
			<td>NA</td>
			<td>Mucolytic solution</td>
		</tr>
		<tr>
			<td>Tissue-Tek VIP 6</td>
			<td>Sakura</td>
			<td>6042 VIP 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek TEC 5 Tissue Embedding Console System</td>
			<td>Sakura</td>
			<td>5229 TEC 5</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope glass slide SuperFrost Plus</td>
			<td>Thermo Fisher Scientific</td>
			<td>4951PLUS4</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol powder</td>
			<td>Sigma-Aldrich</td>
			<td>D3801</td>
			<td></td>
		</tr>
		<tr>
			<td>Heidolph Multi Reax Vortex Shaker</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-889-410</td>
			<td></td>
		</tr>
		<tr>
			<td>Shandon Cytoblock</td>
			<td>Thermo Fisher Scientific</td>
			<td>7401150</td>
			<td></td>
		</tr>
		<tr>
			<td>22C3 anti-PD-L1 concentrate antibody</td>
			<td>Agilent Dako</td>
			<td>#M365329</td>
			<td></td>
		</tr>
		<tr>
			<td>PD-L1 IHC 22C3 pharmDx</td>
			<td>Agilent Dako</td>
			<td>SK006</td>
			<td></td>
		</tr>
		<tr>
			<td>Autostainer Link 48</td>
			<td>Agilent Dako</td>
			<td>AS480</td>
			<td></td>
		</tr>
		<tr>
			<td>BenchMark ULTRA autostainer</td>
			<td>Ventana</td>
			<td>#750-600</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiView HQ Universal Linker</td>
			<td>Ventana</td>
			<td>#760-700</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiView HRP Multimer</td>
			<td>Ventana</td>
			<td>#760-700</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiView Amplification H2O2</td>
			<td>Ventana</td>
			<td>#760-099</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiView Amplifier</td>
			<td>Ventana</td>
			<td>#760-099</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiView Amplification Multimer</td>
			<td>Ventana</td>
			<td>#760-100</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiView DAB</td>
			<td>Ventana</td>
			<td>#760-700</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiView Copper</td>
			<td>Ventana</td>
			<td>#760-700</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin II</td>
			<td>Ventana</td>
			<td>#790-2208</td>
			<td></td>
		</tr>
		<tr>
			<td>Bluing Reagent</td>
			<td>Ventana</td>
			<td>#760-2037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Conditioning 1 (CC1)</td>
			<td>Ventana</td>
			<td>#950-124</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Film Coverslipper</td>
			<td>Sakura</td>
			<td>4742</td>
			<td></td>
		</tr>
	</tbody>
</table>

using 22c3 anti-pd-l1 antibody concentrate on biopsy and cytology samples from non-small cell lung cancer patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

Using the procedure presented here, and as described in detail in this group&#39;s recent publications10,11, the optimized LDT was clinically validated with 120 archival FFPE NSCLC biopsy samples from patients who underwent surgical resection or a biopsy at the Pasteur University Hospital, Nice, between March 2007 and March 2016. Moreover, for the evaluation of PD-L1 expression of cytology samples, TPS was evaluated in 70 paired t...

Watch this Scientific Journal Video about Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients at JoVE.com

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

This method using the 22C3 antibody concentrate to determine PD-L1 expression in both tumor tissue and cytology specimen will expand the ability of laboratories to assess selection of patients with non-small cell lung cancer for immunotherapy in a reliable and reproducible manner. The main advantage of this technique is that it's highly concordant with the gold standard assay and will support reliable, high-quality PD-L1 testing across regions worldwide. In general, pathologists new to this method will struggle because of the granular staining occasionally observed.Moreover, the staining of the human cells is somewhat more intense than that observed with the gold standard. Therefore training of pathologists is necessary to obtain correct interpretation of PD-L1 staining with the 22C3 LDT. Demonstrating the procedure will be Mrs.Marame Hamila, a technician from my lab. To begin, fix the tumor tissue in 10%neutral-buffered formalin in a cassette and embed the cassette in paraffin as outlined in the text protocol. Embed the infiltrated tissue inside a mold that is filled with molten paraffin.Let the tissue stay in the mold until the paraffin has solidified. After this, use a microtome to section the paraffin-embedded tissue at a thickness of three micrometers. Transfer the paraffin ribbon to a positively-charged glass microscope slide.Then, dry the slide for one hour at 37 degrees Celsius. First, collect the bronchial washings in a preservative solution. Transfer the washings to a 50-milliliter conical tube.Add two grams of DL-Dithiothreitol and vortex the tube for 30 minutes, then centrifuge at 250 times g for five minutes at room temperature. Remove the supernatant and add 10 milliliters of a mucolytic solution. Shake the sample at medium speed for 20 minutes and centrifuge at 250 times g for five minutes at room temperature.After this, remove the supernatant and deposit the cell pellet into a collection tube containing 10%neutral-buffered formalin. Add four drops of a cell block preparation agent. Transfer the cell pellet to a cassette.Fix the cell pellet for paraffin embedding and sectioning using the process previously described for preparing tumor tissue samples. To begin, power on the autostainer and the computer. Double click on the autostainer icon and choose the user.Click on Create Label and then on Protocol. Double click on the 22C3 protocol. Fill in the patient's ID and click Print.After this, stick the label onto the slide. Press the button on the chosen slide drawer to open it and place the labeled slide on the thermal pad with the label facing upwards and inwards. Close the slide drawer.Next, remove the caps from the dispensers. Load the reagents on the reagents rack. Open the hood of the stainer and place the reagent racks on the reagents carousel, making sure that they fit properly and hold in position, then close the hood.In the software, click on the instrument that will be used and then click Running. At the end of the IHC procedure, use tap water with a single drop of cleaning solution to rinse the slide for several seconds. Dehydrate the slide by immersing it sequentially in two ethanol baths for several seconds each, then place the slide into a coverslipping machine to automatically load the cover slip.To begin assessing the quality of the PD-L1 staining, analyze the positive and negative controls before examining the patient specimen. Using a microscope, confirm the presence of at least 100 viable tumor cells. At a low magnification of 4 times, evaluate all well-preserved positive and negative tumor areas.After this, score partial or complete cell membrane staining and calculate the tumor proportion score by assessing the proportion of PD-L1-positive tumor cells relative to all tumor cells present in the well-preserved tumor areas. In this study, the tumor proportion score or TPS is evaluated for paired-tissue biopsy samples in cells blocks prepared from bronchial washes or pleural effusions. Representative staining patterns with biopsy specimens are used to compare the 22C3-antibody concentrate against the PD-L1 IHC 22C3 companion assay.The intraclass correlation coefficient, which is used to measure the correlation of the TPS score as a continuous variable, is 99%between the LDT and the gold standard. Representative staining patterns with cytology specimens are also used to compare the 22C3-antibody concentrate against the PD-L1 IHC 22C3 companion assay. The concordance rate of biopsy and cytology samples with the LDT is greater than 95%when using either of the TPS cut points.The intraclass correlation coefficient while using TPS as a continuous variable is between 0.88 and 0.90. Once mastered, this technique gives a high concordance rate with the gold standard in evaluate the cutoff for positivity or tumor histology for both tissue and cytology specimens. While attempting this procedure, it's important to remember that the samples must be evaluated with reference to the HE slide and in some difficult cases complementary stains to assess immune cells may be performed to exclude misinterpretation of the PD-L1 expression in tumor cells only.Following this procedure, other specimens like fine-needle biopsies, EUS-FNAs or bronchial brushes can be evaluated. After its setting in the clinics, the assay robustness needs to be maintained over time by seeking for accreditation, by following standard operating procedures, and by regularly participating in external quality assessment schemes. After watching this video, you should have a good understanding of how to implement the optimized protocol using the 22C3 anti-PD-L1 antibody concentrate on a widely available IHC autostainer both biopsy and cytology samples from patients with non-small cell lung cancer.

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