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>Fix tumor tissue in 10% neutral buffered formalin (NBF) in a cassette. 
	NOTE: Tumor tissue from proximal lung tumors is generally obtained by bronchoscopy, performed under moderate sedation by a pulmonologist, or lung specialist. For peripheral lesions and diffuse lung disease, a transbronchial or needle biopsy is indicated. These procedures are usually done in a surgery room or intensive care unit.</li>
	<li>Embed the cassette in paraffin. 
	NOTE: Fixation can be achieved by perfusion or immersion immediately following dissection, and typically requires 8&#8211;10 h. The fixative volume should be 15&#8211;20x higher than the specimen volume. It is not recommended to fix biopsy tissue for more than 10 h, because overfixation can cause masking of the antigen. The fixation speed is about 1 mm/h at room temperature.</li>
	<li>Infiltrate the fixed tissue sample with wax in the tissue processor (see Table of Materials). 
	NOTE: Dehydration is performed in three alcohol baths with increasing concentrations 70%,&#160;85%, and&#160;90%. Water is finally removed by three final absolute alcohol baths. The clearing is performed in three toluene baths, and the wax infiltration in hot wax baths (44&#8211;60 &#176;C).</li>
	<li>Embed the tissue inside a mold filled with molten paraffin (see Table of Materials) and wait for solidification.</li>
	<li>Section the paraffin-embedded tissue at a thickness of 3 &#956;m on a microtome.</li>
	<li>Transfer the paraffin ribbon to a positively charged microscope glass slide (see Table of Materials).</li>
	<li>Dry the slide for 1 h at 37 &#176;C. 
	Note: Store the tissue sections at 2&#8211;8 &#176;C (preferred) or at room temperature up to 25 &#176;C to preserve antigenicity, and stain within 15 d of sectioning.</li>
</ol>
2. Preparation of Cytology Samples
<ol>
	<li>Collect bronchial washings in a preservative solution (see Table of Materials). 
	NOTE: For this, standard bronchoscopy technique is used.
	<ol>
		<li>Lavage the distribution of the bronchus to be sampled. Collect the wash in a clean container. Label the container with the patient&#8217;s first and last name, date of birth, and specimen source.</li>
	</ol>
	</li>
	<li>Transfer the bronchial washings to a 50 mL conical tube and add 2 g of DL-Dithiothreitol powder (see Table of Materials), vortex the tube for 30 min, and then centrifuge it at 250 x g for 5 min at room temperature.</li>
	<li>Remove the supernatant and add 10 mL of a mucolytic solution (see Table of Material).</li>
	<li>Shake the sample for 20 min at medium speed (level 9) and centrifuge it at 250 x g for 5 min at room temperature.</li>
	<li>Remove the supernatant and deposit the cell pellet in collection tubes containing 10% NBF.</li>
	<li>Add 4 drops of a cell block preparation reagent (see Table of Materials) to the cell pellet.</li>
	<li>Transfer the cell pellet to a cassette, fix it in 10% NBF, and paraffin-embed it for cell block preparation and sectioning (see steps 1.1&#8211;1.7).</li>
</ol>
3. PD-L1 Staining Assay
<ol>
	<li>Switch on the autostainer and the computer.</li>
	<li>Double-click on the autostainer icon and choose the user.</li>
	<li>Click on &#39;Create label&#39; and then on &#39;Protocol&#39;.</li>
	<li>Double-click on the 22C3 protocol. 
	NOTE: The protocol is first installed on the autostainer&#8217;s computer by clicking on &#39;Create Protocol&#39;. The full protocol is provided as&#160;Supplementary File 1.</li>
	<li>Write the patient&#8217;s ID on the label and click on &#39;Print&#39;.</li>
	<li>Stick the label on the slide.</li>
	<li>Open the slide drawer by pressing on the button of the drawer that has been chosen.</li>
	<li>Place the labeled slide on the thermal pad with the label facing up- and inward.</li>
	<li>Close the slide drawer.</li>
	<li>Remove the caps from the dispensers and load the reagents on the reagents&#8217; racks.</li>
	<li>Open the hood of the stainer and place the racks on the reagents&#8217; carrousel making sure that they fit and hold in position.</li>
	<li>Close the hood.</li>
	<li>On the software, click on the instrument that will be used and click on &#39;Running&#39;.</li>
	<li>At the end of the IHC procedure, rinse the slide for several seconds with tap water and one drop of a cleaning solution.</li>
	<li>Rehydrate the slide with one bath of ethanol 100% and a second bath of ethanol 95% for several seconds.</li>
	<li>Place the slide into the coverslipping machine for an automated loading of the coverslip.</li>
</ol>
4. Interpretation of the PD-L1 Staining
NOTE: A qualified pathologist should perform the interpretation of the PD-L1 IHC test.
<ol>
	<li>Assess the quality of the PD-L1 staining by analyzing the positive and negative controls before the examination of the patient&#8217;s specimen. 
	NOTE: The results obtained on the patient&#8217;s specimen are considered invalid if the staining of the controls is not acceptable.</li>
	<li>Confirm the presence of a minimum of 100 viable tumor cells under a microscope. 
	NOTE: Report when the patient&#8217;s specimen has less than 100 tumor cells.</li>
	<li>At a low magnification of 4X, evaluate all well-preserved positive and negative tumor areas. 
	NOTE: At a low magnification, partial membrane staining or membrane staining of weak intensity (1+) may be difficult to recognize.</li>
	<li>Score partial or complete cell membrane staining. 
	NOTE: The cytoplasmic staining has to be excluded from interpretation.</li>
	<li>Calculate the TPS by assessing the proportion of PD-L1 positive tumor cells relative to all tumor cells present in the well-preserved tumor areas. 
	NOTE: Only viable tumor cells should be examined. All other cellular elements, such as immune cells, necrotic cells, normal cells, and artifacts, have to be excluded from the examination.</li>
</ol>

<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>

The authors have nothing to disclose.

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&amp;reg; 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 H&lt;sub&gt;2O2&lt;/sub&gt;</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 ...

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Cancer Research

10.5K Views.  Université Côte d’Azur. Deze methode met behulp van de 22C3 antilichaam concentraat om PD-L1 expressie te bepalen in zowel tumorweefsel en cytologie monster zal het vermogen van laboratoria om de selectie van patiënten met niet-kleincellige longkanker voor immunotherapie te beoordelen op een betrouwbare en reproduceerbare manier uit te breiden. Het belangrijkste voordeel van deze techniek is dat het zeer in overeenstemming is met de gouden standaardtest en betrouwbare PD-L1-tests van hoge kwaliteit in verschillende...

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

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it is now well established that the immune infiltrate in human tumors can correlate with prognosis and response to therapy. The analysis of the immune infiltrate in the tumor microenvironment has become a major challenge for the classification of patients and the response to treatment.
The co-expression of inhibitory receptors such as Program Cell Death Protein 1 (PD1; also known as CD279), Cytotoxic T Lymphocyte Associated Protein 4 (CTLA-4), T-Cell Immunoglobulin and Mucin Containing Protein-3 (Tim-3; also known as CD366), and Lymphocyte Activation Gene 3 (Lag-3; also known as CD223), is a hallmark of T cell exhaustion. We developed a multiparametric in situ immunofluorescence staining to identify and quantify at the cellular level the co-expression of these inhibitory receptors. On a retrospective series of frozen tissue of renal cell carcinomas (RCC), using a fluorescence multispectral imaging technology coupled with an image analysis software, it was found that co-expression of PD-1 and Tim-3 on tumor infiltrating CD8+ T cells is correlated with a poor prognosis in RCC. To our knowledge, this represents the first study demonstrating that this automated multiplex in situ technology may have some clinical relevance.

Multiplexed Immunofluorescence Analysis and Quantification of Intratumoral PD-1+ Tim-3+ CD8+ T Cells

Studying tumor microenvironment may identify prognostic or predictive biomarkers of clinical response to immunotherapy. Presented here, is an innovative method based on in situ fluorescence multispectral imaging to analyze and count automatically various subpopulations of CD8+ T cells. This reproducible and reliable technique is suitable for large cohort analyses.

Immune cells are important components of the tumor microenvironment and influence tumor growth and evolution at all stages of carcinogenesis. Notably, it ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Murine Model for Studying Leptomeningeal Disease Using Circulating Tumor Cells

Ex Vivo Culture of Circulating Tumor Cells in the Cerebral Spinal Fluid from Melanoma Patients to Study Melanoma&#45;Associated Leptomeningeal Disease

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the meninges, the membrane layers that protect the brain and the spinal cord. Once established, the prognosis for M-LMD patients is dismal, with overall survival ranging from weeks to months. This is primarily due to a paucity in our understanding of the disease and, as a consequence, the availability of effective treatment options. Defining the underlying biology of M-LMD will significantly improve the ability to adapt available therapies for M-LMD treatment or design novel inhibitors for this universally fatal disease. A major barrier, however, lies in obtaining sufficient quantities of CTCs from the patient-derived CSF (CSF-CTCs) to conduct preclinical experiments, such as molecular characterization, functional analysis, and in vivo efficacy studies. Culturing CSF-CTCs ex vivo has also proven to be challenging. To address this, a novel protocol for the culture of patient-derived M-LMD CSF-CTCs ex vivo and in vivo is developed. The incorporation of conditioned media produced by human meningeal cells (HMCs) is found to be critical to the procedure. Cytokine array analysis reveals that factors produced by HMCs, such as insulin-like growth factor-binding proteins (IGFBPs) and vascular endothelial growth factor-A (VEGF-A), are important in supporting CSF-CTC survival ex vivo. Here, the usefulness of the isolated patient-derived CSF-CTC lines is demonstrated in determining the efficacy of inhibitors that target the insulin-like growth factor (IGF) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, the ability to intrathecally inoculate these cells in vivo to establish murine models of M-LMD that can be employed for preclinical testing of approved or novel therapies is shown. These tools can help unravel the underlying biology driving CSF-CTC establishment in the meninges and identify novel therapies to reduce the morbidity and mortality associated with M-LMD.

Author Spotlight: Assessing the Potential of Circulating Tumor Cells in Leptomeningeal Disease Research

This article describes a protocol for the propagation of cerebral spinal fluid-circulating tumor cells (CSF-CTCs) collected from patients with melanoma-associated leptomeningeal disease (M-LMD) to develop preclinical models to study M-LMD.

Melanoma-associated leptomeningeal disease (M-LMD) occurs when circulating tumor cells (CTCs) enter into the cerebral spinal fluid (CSF) and colonize the ...

Monitoring PD-1-Blocking Antibodies Bound to T Cells Derived from a Drop of Peripheral Blood

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The pharmacological efficacy of these immunotherapies is long lasting, extending even beyond the discontinuation of their injections, due to persistent blood concentrations. Here we developed a simple flow cytometry assay to evaluate the T cell binding status of the PD-1&#8211;blocking antibodies nivolumab and pembrolizumab. Like a glucose test, this assay requires just a single drop of peripheral blood. Visualizing antibody binding on T cells is more reliable than measuring antibody blood concentrations. In addition, if necessary, we can potentially analyze many distinctive immune-related markers on T cells bound to PD-1&#8211;blocking antibodies. Thus, this is a simple and minimally invasive strategy to analyze the pharmacological effect of PD-1&#8211;blocking antibodies in cancer patients.

We developed a simple flow cytometry assay for evaluating the binding of PD-1&#8211;blocking antibodies to T cells, requiring only a drop of peripheral blood from cancer patients.

Immune checkpoint inhibitors, including PD-1&#8211;blocking antibodies, have significantly improved treatment outcomes in various types of cancer. The ...

Medicine

Advancements in Pulmonary Lesion Biopsy Through Robotic Bronchoscopy

Robotic-assisted Bronchoscopy Combined with Multimodal Imaging for Targeted Lung Cryobiopsies

Robotic-assisted bronchoscopy (RAB) allows for targeted bronchoscopic biopsy in the lung. A robotic-assisted bronchoscope is navigated through the airways under direct vision after establishing a pathway to a target lesion based on mapping performed on a 3-dimensional (3D) lung and airway reconstruction obtained from a pre-procedure thin-slice computed tomography chest. RAB has maneuverability to distal airways throughout the lung, precise catheter tip articulation, and stability with the robotic arm. Adjunct imaging tools such as fluoroscopy, radial endobronchial ultrasound (r-EBUS), and cone beam computed tomography (CBCT) can be used with RAB. Studies using shape-sensing robotic-assisted bronchoscopy (ssRAB) have shown favorable diagnostic outcomes and safety profiles in both malignant and non-malignant processes for the biopsy of peripheral pulmonary lesions (PPLs). A 1.1 mm cryoprobe combined with ssRAB has been shown to be safe and effective for the diagnosis of PPLs compared to a traditional bronchoscopy with forceps biopsy. This technique can also be used for targeted lung sampling in benign processes. The aim of this article is to describe a stepwise approach to performing RAB combined with fluoroscopy, r-EBUS, and CBCT to obtain targeted transbronchial lung cryobiopsies (TBLC).

Author Spotlight: Expanding Interventional Pulmonology Research with Robotic-Assisted Bronchoscopy

This article aims to describe a stepwise approach to performing robotic-assisted bronchoscopy combined with fluoroscopy, radial endobronchial ultrasound, and cone beam computed tomography to obtain targeted transbronchial lung cryobiopsies.&#160;

Robotic-assisted bronchoscopy (RAB) allows for targeted bronchoscopic biopsy in the lung. A robotic-assisted bronchoscope is navigated through the airways ...

CTC Immunofluorescence Assay : A Technique to  Characterize Circulating Tumor Cells

Source: Garcia, J., et al. <a href="https://www.jove.com/v/59873/semi-automatic-pd-l1-characterization-enumeration-circulating-tumor">Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence.</a>&#160;J. Vis. Exp.&#160;(2019)
This video describes the Circulating Tumor Cells (CTC) Immunofluorescence assay, a technique to characterize the biomarkers on circulating tumor cells in non-small cell lung cancer patient samples. This assay could be used to monitor dynamic changes of the tumor and immune response, which may reflect the response to chemotherapy, radiation, and possible immunotherapy treatments.

Source: Garcia, J., et al. Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by ...

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

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