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

Summary

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.

Abstract

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 (≥1%, ≥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.

Introduction

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 NSCLC^1,2,3,4.

Currently, pembrolizumab is approved for treatment of PD-L1-expressing NSCLC in both treatment-naive patients with a PD-L1 expression TPS of ≥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 ≥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 institutions^6,7. 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 samples^10,11. We found a high concordance with the LDT versus the "gold standard" PD-L1 IHC 22C3 assay^10,11. This clinically validated protocol will support reliable, high-quality PD-L1 testing across regions globally.

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Protocol

All procedures have been approved by the local ethics committee (Human Research Ethics Committee, Centre Hospitalier Universitaire de Nice/Tumorothèque 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

1. 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.
2. Embed the cassette in paraffin.
   NOTE: Fixation can be achieved by perfusion or immersion immediately following dissection, and typically requires 8–10 h. The fixative volume should be 15–20x 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.
3. 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%, 85%, and 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 (44–60 °C).
4. Embed the tissue inside a mold filled with molten paraffin (see Table of Materials) and wait for solidification.
5. Section the paraffin-embedded tissue at a thickness of 3 μm on a microtome.
6. Transfer the paraffin ribbon to a positively charged microscope glass slide (see Table of Materials).
7. Dry the slide for 1 h at 37 °C.
   Note: Store the tissue sections at 2–8 °C (preferred) or at room temperature up to 25 °C to preserve antigenicity, and stain within 15 d of sectioning.

2. Preparation of Cytology Samples

1. Collect bronchial washings in a preservative solution (see Table of Materials).
   NOTE: For this, standard bronchoscopy technique is used.
   1. Lavage the distribution of the bronchus to be sampled. Collect the wash in a clean container. Label the container with the patient’s first and last name, date of birth, and specimen source.
2. 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.
3. Remove the supernatant and add 10 mL of a mucolytic solution (see Table of Material).
4. Shake the sample for 20 min at medium speed (level 9) and centrifuge it at 250 x g for 5 min at room temperature.
5. Remove the supernatant and deposit the cell pellet in collection tubes containing 10% NBF.
6. Add 4 drops of a cell block preparation reagent (see Table of Materials) to the cell pellet.
7. Transfer the cell pellet to a cassette, fix it in 10% NBF, and paraffin-embed it for cell block preparation and sectioning (see steps 1.1–1.7).

3. PD-L1 Staining Assay

1. Switch on the autostainer and the computer.
2. Double-click on the autostainer icon and choose the user.
3. Click on 'Create label' and then on 'Protocol'.
4. Double-click on the 22C3 protocol.
   NOTE: The protocol is first installed on the autostainer’s computer by clicking on 'Create Protocol'. The full protocol is provided as Supplementary File 1.
5. Write the patient’s ID on the label and click on 'Print'.
6. Stick the label on the slide.
7. Open the slide drawer by pressing on the button of the drawer that has been chosen.
8. Place the labeled slide on the thermal pad with the label facing up- and inward.
9. Close the slide drawer.
10. Remove the caps from the dispensers and load the reagents on the reagents’ racks.
11. Open the hood of the stainer and place the racks on the reagents’ carrousel making sure that they fit and hold in position.
12. Close the hood.
13. On the software, click on the instrument that will be used and click on 'Running'.
14. At the end of the IHC procedure, rinse the slide for several seconds with tap water and one drop of a cleaning solution.
15. Rehydrate the slide with one bath of ethanol 100% and a second bath of ethanol 95% for several seconds.
16. Place the slide into the coverslipping machine for an automated loading of the coverslip.

4. Interpretation of the PD-L1 Staining

NOTE: A qualified pathologist should perform the interpretation of the PD-L1 IHC test.

1. Assess the quality of the PD-L1 staining by analyzing the positive and negative controls before the examination of the patient’s specimen.
   NOTE: The results obtained on the patient’s specimen are considered invalid if the staining of the controls is not acceptable.
2. Confirm the presence of a minimum of 100 viable tumor cells under a microscope.
   NOTE: Report when the patient’s specimen has less than 100 tumor cells.
3. 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.
4. Score partial or complete cell membrane staining.
   NOTE: The cytoplasmic staining has to be excluded from interpretation.
5. 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.

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Results

Using the procedure presented here, and as described in detail in this group's recent publications^10,11, 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...

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Discussion

We have validated an optimized LDT using the 22C3 PD-L1 antibody concentrate, by comparing it with the corresponding clinically validated commercial test^10,11. 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 ≥1% TPS and the ≥50% TPS cut points. As recently recommended by the Internatio...

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Materials

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