Determinants of variability of five programmed death ligand-1 immunohistochemistry assays in non-small cell lung cancer samples

NSCLC is the collective name for a group of epithelial cancers of the lung including adenocarcinoma, squamous cell carcinoma and large cell carcinoma. Lung adenocarcinomas make up 85% of cases for a disease that accounts for approx. 15% of cancers. Five year survival rates range from 47-1% depending upon the stage and as NSCLC is generally quite resistant to chemotherapy treatment is mostly surgical resection.

PDL-L1 is a transmembrane protein that plays a major role in immune-modulation. It binds to its receptor PD-1, present on B-cells, T-cells and myeloid cells to regulate immune signalling. Binding of PD-1 and PD-L1 results in reduced proliferation of antigen-specific immune cells such as Th-cells and also leads to an increase in anti-inflammatory T-reg cells.

Upregulation of PD-L1 is associated with increased risk and tumourigenicity of cancers, helping many escape immune surveillance. A number of immunotherapies have now been developed exploiting this mechanism including a number for NSCLC. In 2015, Nivolumab, an anti-PD-1 therapy was approved by the FDA for advanced or metastatic squamous cell carcinoma and Pembrolizumab for NSCLC - tumours expressing PD-L1 was also approved. Pembrolizumab was the first immunotherapy to be used as a first line treatment for NSCLC tumours overexpressing PD-L1 and must be conducted with a validated companion diagnostic.

PD-1/PD-L1 expression has been suggested as a possible biomarker for identifying patients that may be sensitive to PD1/PD-L1 immunotherapies. Immunohistochemical stains have been identified to accomplish this, however a number of different methods have been used such as the 22C3 antibody (Ab) clone or the 28-8 clone with the Dako Link 48 autostainer. Others have used the SP263 or SP142 antibody clone with the Ventana BenchMark Ultra autostainer. Other in-house or laboratory tests have also been developed for clinical assessments of PD-L1 expression.

This study aimed to simplify the process by assessing the different methods on clinically-relevant NSCLC samples, to identify factors relevant for PD-L1 detection and make assays more robust and reproducible.

A summary of their findings:-

In this study they assessed five different markers/protocols for PD-L1, SP263/Benchmark, 22C3/Link48, 28-8/BOND-MAX, E1L3N/BOND-MAX and SP142/BenchMark.

All five protocols successfully identified epithelial cells in placental tissue. In NSCLC samples all markers identified tumour cells, with predominantly membraneous localisation.

The SP263/Benchmark had the most intense staining, 22C3/Link48, 28-8/BOND-MAX and E1L3N/BOND-MAX showed moderate staining and weak staining was observed with SP142/BenchMark.

Staining of tumour infiltrating immune cells was possible in resected and small biopsy samples using all 5 protocols, however the small sample number prevented accurate assessment.

NSCLC samples were analysed using each method and Tumour cell frequency was estimated at 72%, 33%, 61%, 56%, and 33% for 22C3/Link48, 28-8/BOND-MAX, E1L3N/BOND-MAX, SP142/BenchMark, and SP263/BenchMark, respectively. Comparison of the different methods determined that 22C3/Link48 and SP142/BenchMark showed the best pairwise agreement with a 94% correlation on one set of samples.

A pairwise correlation between all samples and methods showed significant variation in tumour cell labelling, the best correlation being 22C3/Link48 and E1L3N/BOND-MAX (R2= 0.71).

As an independent reference for PD-L1 expression samples were analysed for RNA using RNA Scope. This identified only 33% of the samples as having PD-L1 RNA expression and only the E1L3N/BOND-MAX IHC method correlated with this expression.

The authors optimised a 7-plex multi-marker assay using the 5 PD-L1 antibodies, a CD-3 antibody and DAPI on a single section. This assay demonstrated a high degree of co-localisation across all 5 antibodies and identified tumour cells in 33% of NSCLC samples.

The development of PD1 and PD-L1 as immunotherapy targets has been supported by their expression being investigated as a predictor of response. This study with support from many others demonstrates that current PD1/PD-L1 testing procedures may not be sufficient to distinguish responders. The number and type of assay varies between labs and, as this study demonstrated, the immunostaining protocol could lead to significant variability regardless of the biology. The authors call for standardisation of PD1/PD-L1 protocols and antibodies to reduce confusion and better evaluate treatment response.

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Epistem's Expertise

At Epistem we can offer a validated orthotopic imaging model of leukaemic and solid tumours for drug development studies including a validated model of lung carcinoma. We have extensive experience in in vivo imaging to monitor treatment effects in the same animal over time and to detect treatment effects/potentiation and disease regression using single drug or a combination of additional therapeutics, including CAR‑T cell therapies.

We are currently expanding our expression services with our new NextSeq 550 NGS machine and development of RNA Scope methods. We also have GCLP-accredited labs for IHC and gene expression (Microarray and qRT-PCR). We also offer RNA-friendly laser capture microdissection ideal for isolating RNA from tumour infiltrating immune cells for expression analysis.

Soo et al., Determinants of variability of five programmed death ligand-1 immunohistochemistry assays in non-small cell lung cancer samples. Oncotarget, 2018, Vol. 9, (No. 6), pp: 6841-6851

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