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) gen....

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 ....

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.......

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.......

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