It is important that tumour samples collected for programmed cell death ligand-1 (PD-L1) testing are high quality, in order to more accurately reflect patient PD-L1 expression and ensure confidence in clinical decision-making.
For more information on obtaining tissue for PD-L1 testing in lung cancer, please visit the iD Lung website.
To ensure adequate accuracy of PD-L1 test results, characteristics of tumour samples need to be considered before analysis takes place. Important characteristics associated with PD-L1 testing include sample:
- Type: Tumour biopsy or cytology samples
- Age: Fresh or archival
- Location: Primary or metastatic
Routinely processed, formalin-fixed paraffin-embedded (FFPE) tumour biopsy samples containing an adequate number of viable tumour cells (≥100) are suitable for use in non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC).1, 2
The VENTANA SP263 assay is also validated for use with NSCLC FFPE fine-needle aspiration cell blocks for the assessment of tumour cell (TC) PD-L1 expression.3
The use of cytology samples for PD-L1 testing
In this short interview, Professor Keith Kerr (University of Aberdeen, Aberdeen, UK) discusses the potential use of cytology samples for PD-L1 testing.
Evidence suggests that PD-L1 staining is concordant between fresh (<3 months old) and archival (<3 years old) tumour biopsies from the same patient.4 However, samples that are older than 3 years may lead to an underestimation of PD-L1 status.5 Therefore, both fresh and archival (<3 years old) tumour samples are suitable for PD-L1 testing.
PD-L1 testing on paired primary and metastatic lesions in NSCLC and UC have shown high concordance, suggesting that tumour location does not influence PD-L1 expression.4, 6, 7
How do I test?
An overview of the different steps involved in PD-L1 testing can be seen below. Briefly, samples are initially processed and mounted onto microscope slides. Sample sections are then stained for PD-L1 by using a commercial immunohistochemistry (IHC) assay with an automated staining platform. Stained samples are then quality control (QC) checked by a trained pathologist.
Overview of the steps performed during PD-L1 testing is shown below
1. Sections cut
2. Sections mounted
3. Slides dried
4. Slides labelled
5. Automated IHC stainer
6. Staining quality checked
Before PD-L1 expression can be assessed, samples must be appropriately processed in line with the recommended PD-L1 IHC assay recommendations.
Standard tissue sample preparation and processing procedure for PD-L1 testing*3, 8-10
*The preparation instructions differ for each PD-L1 assay. For further details, refer to specific package insert for individual assays
Currently, there are five IHC assays, either commercially available or in development, which can be used to determine PD-L1 expression. Each PD-L1 assay is used with an automated platform to ensure consistent sample processing.11
The choice of PD-L1 IHC assay and the staining procedure is used in line with specific immunotherapies according to their respective diagnostic labels.
*Based on the FDA and CE-IVD diagnostic regulatory approval
BMS, Bristol-Myers Squibb; CE-IVD, Conformité Européenne-in vitro diagnostic; Dx, diagnostic; FDA, Food and Drug Administration; IHC, immunohistochemistry; MSD, Merck Sharp & Dohme; NA, not applicable; PD-L1, programmed cell death ligand-1
Quality control procedures
During PD-L1 testing, each patient sample is sectioned twice: testing section and negative control section.3, 8-10
- Testing section: Slide stained with PD-L1 antibody
- Negative reagent control section: Slide stained with a monoclonal negative control antibody
Apart from the different primary antibodies, the staining procedure used for the two sections is identical. The purpose of the negative control section is to evaluate non-specific staining.3, 8-10
A slide containing a control section is also included within each staining run in order to confirm adequate assay functionality. Examples of control tissues commonly used in PD-L1 testing include benign human tonsil tissue and placental stromal tissue and vasculature, since they contain both PD-L1 positive and negative regions.3, 8-10
PD-L1 testing interpretation
Stained slides should be scored and interpreted by a qualified pathologist using a light microscope.3,10 Before interpreting patient samples, the control and negative reagent sections should be examined in order to verify the performance of the IHC assay.
Positive controls should show moderate-to-strong PD-L1 staining; weak or lack of PD-L1 staining in positive controls is not acceptable.3
If control tissues fail to demonstrate anticipated staining, then patient samples within the same staining run are considered invalid.3
Negative reagent section
The negative reagent control section should show limited, non-specific staining within intact cells.
If background PD-L1 staining is excessive, then patient samples within the same staining run are considered invalid.3
Different compartments of the tumour sections can be
seen following PD-L1 IHC:
- TC area: The proportion of TCs with PD-L1 expression
- Immune cell (IC) area: The proportion of ICs with PD-L1 expression
- Tumour area: The whole tumour area, regardless of PD-L1 staining
Schematic representation of PD-L1 staining in different tumour compartments
IC, immune cell; PD-L1, programmed cell death ligand-1; TC, tumour cell
PD-L1 expression is determined by calculating the relative proportion of staining of the different tumour compartments using an algorithm. An algorithm-specific cut-off value is then applied to define PD-L1 expression as high or low.
PD-L1 algorithms and cut-offs
Different pharmaceutical companies have adopted their own algorithm and cut-offs to determine PD-L1 expression in different cancers, such as NSCLC and bladder cancer.
Non-small cell lung cancer
The majority of algorithms in NSCLC assess the percentage of TCs with PD-L1 expression. However, an algorithm adopted by Roche also considers PD-L1 expression on tumour infiltrating ICs.
Different PD-L1 algorithms to determine PD-L1 expression in NSCLC22-28
A range of cut-offs of PD-L1 expression have been used in NSCLC clinical trials to categorise patients into PD-L1 high and low status.
BMS, Bristol-Myers Squibb; IC, immune cell; MSD, Merck Sharp and Dohme; PD-L1, programmed cell death ligand-1; TC, tumour cell
In bladder cancer, most pharmaceutical companies have utilised their own algorithm to determine PD-L1 expression.
Different PD-L1 algorithms to determine PD-L1 expression in bladder cancer30
Each algorithm is associated with a specific cut-off value to define patients with high PD-L1 expression.
*When ICs ≤1% of the tumour area, the tumour is PD-L1 high if all ICs are stained or PD-L1 low if some/no ICs are stained3
BMS, Bristol-Myers Squibb; CPS, combined positive score; IC, immune cell; MSD, Merck Sharp and Dohme; PD-L1, programmed cell death ligand-1; TC, tumour cell
- Rebelatto MC, Midha A, Mistry A, et al. Development of a programmed cell death ligand-1 immunohistochemical assay validated for analysis of non-small cell lung cancer and head and neck squamous cell carcinoma. Diagn Pathol 2016;11:95
- Eckstein M, Cimadamore A, Hartmann A, et al. PD-L1 assessment in urothelial carcinoma: a practical approach. Ann Transl Med 2019;7:690
- Roche. VENTANA PD-L1 (SP263) Assay package insert. 2020. Available from: https://pim-eservices.roche.com/eLD/api/downloads/4b0ce998-2fb6-ea11-fc90-005056a71a5d?countryIsoCode=gb. Accessed: 5 October 2020
- Midha A, Sharpe A, Scott M, et al. PD-L1 expression in advanced NSCLC: primary lesions versus metastatic sites and impact of sample age. J Clin Oncol 2016;34:3025
- Gagné A, Wang E, Bastien N, et al. Impact of specimen characteristics on PD-L1 testing in non-small cell lung cancer: validation of the IASLC PD-L1 testing recommendations. J Thorac Oncol 2019;14:2062–2070
- Tretiakova M, Fulton R, Kocherginsky M, et al. Concordance study of PD-L1 expression in primary and metastatic bladder carcinomas: comparison of four commonly used antibodies and RNA expression. Mod Pathol 2018;31:623–632
- Munari E, Zamboni G, Lunardi G, et al. PD-L1 expression comparison between primary and relapsed non-small cell lung carcinoma using whole sections and clone SP263. Oncotarget 2018;9:30465–30471
- Roche. VENTANA PD-L1 (SP142) Assay package insert (FDA). 2016. Available from: https://www.accessdata.fda.gov/cdrh_docs/pdf16/p160006c.pdf. Accessed: 15 May 2020
- Dako. PD-L1 IHC 22C3 pharmDx package insert (FDA). 2018. Available from: https://www.accessdata.fda.gov/cdrh_docs/pdf15/P150013S011C.pdf. Accessed: 15 May 2020
- Dako. PD-L1 IHC 28-8 pharmDx package insert (FDA). 2017. Available from: https://www.accessdata.fda.gov/cdrh_docs/pdf15/P150025S003C.pdf. Accessed: 15 May 2020
- Cree IA, Booton R, Cane P, et al. PD-L1 testing for lung cancer in the UK: recognizing the challenges for implementation. Histopathology 2016;69:177–186
- Roche. VENTANA PD-L1 (SP263) Assay (CE IVD). 2020. Available from: https://diagnostics.roche.com/global/en/products/tests/ventana-pd-l1-_sp263-assay2.html. Accessed: 15 May 2020
- Roche. VENTANA PD-L1 (SP263) Assay (US FDA Approved). 2020. Available from: https://diagnostics.roche.com/global/en/products/tests/ventana-pd-l1-_sp263-assay1.html. Accessed: 15 May 2020
- Roche. VENTANA PD-L1 (SP142) Assay (CE IVD). 2020. Available from: https://diagnostics.roche.com/global/en/products/tests/ventana-pd-l1-_sp142-assay2.html. Accessed: 15 May 2020
- Roche. VENTANA PD-L1 (SP142) Assay (US FDA Approved). 2020. Available from: https://diagnostics.roche.com/global/en/products/tests/ventana-pd-l1-_sp142-assay1.html. Accessed: 15 May 2020
- Agilent. PD-L1 IHC 22C3 pharmDx Overview (EU). 2020. Available from: https://www.agilent.com/en/product/pharmdx/pd-l1-ihc-22c3-pharmdx-overview. Accessed: 15 May 2020
- Agilent. PD-L1 IHC 22C3 pharmDx Overview (US). 2020. Available from: https://www.agilent.com/en-us/product/pharmdx/pd-l1-ihc-22c3-pharmdx-overview. Accessed: 15 May 2020
- Agilent. PD-L1 IHC 28-8 pharmDx Overview (US). 2020. Available from: https://www.agilent.com/en-us/pd-l1-ihc-28-8-overview. Accessed: 15 May 2020
- Agilent. PD-L1 IHC 28-8 pharmDx Overview (EU). 2020. Available from: https://www.agilent.com/en/product/pharmdx/pd-l1-ihc-28-8-overview. Accessed: 15 May 2020
- OncLive. FDA approves PD-L1 IHC 28-8 pharmDx as companion diagnostic for nivolumab plus ipilimumab in NSCLC. 2020. Available from: https://www.onclive.com/web-exclusives/fda-approves-pdl1-ihc-288-pharmdx-as-companion-diagnostic-for-nivolumabipilimumab-in-nsclc. Accessed: 19 May 2020
- Patel MR, Ellerton J, Infante JR, et al. Avelumab in metastatic urothelial carcinoma after platinum failure (JAVELIN Solid Tumor): pooled results from two expansion cohorts of an open-label, phase 1 trial. Lancet Oncol 2018;19:51–64
- Kim H, Chung J-H. PD-L1 testing in non-small cell lung cancer: past, present, and future. J Pathol Transl Med 2019;53:199–206
- Barlesi F, Vansteenkiste J, Spigel D, et al. Avelumab versus docetaxel in patients with platinum-treated advanced non-small-cell lung cancer (JAVELIN Lung 200): an open-label, randomised, phase 3 study. Lancet Oncol 2018;19:1468–1479
- Antonia SJ, Villegas A, Daniel D, et al. Overall survival with durvalumab after chemoradiotherapy in stage III NSCLC. N Engl J Med 2018;379:2342–2350
- Reck M, Rodríguez-Abreu D, Robinson AG, et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med 2016;375:1823–1833
- Borghaei H, Paz-Ares L, Horn L, et al. Nivolumab versus docetaxel in advanced nonsquamous non-small-cell lung cancer. N Engl J Med 2015;373:1627–1639
- Peters S, Gettinger S, Johnson ML, et al. Phase II trial of atezolizumab as first-line or subsequent therapy for patients with programmed death-ligand 1-selected advanced non-small-cell lung cancer (BIRCH). J Clin Oncol 2017;35:2781–2789
- Herbst RS, Baas P, Kim D-W, et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet 2016;387:1540–1550
- Mok TSK, Wu YL, Kudaba I, et al. Pembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial. Lancet 2019;393:1819-1830
- Powles T, Walker J, Andrew Williams J, et al. The evolving role of PD-L1 testing in patients with metastatic urothelial carcinoma. Cancer Treat Rev 2020;82:101925
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