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Assay selection and delivery

Assay selection

Multiple assays to assess tumour PD-L1 expression have been, or are currently being, developed. These include: VENTANA PD-L1 (SP263) Assay, Dako PD-L1 IHC 22C3 pharmDx, Dako PD-L1 IHC 28-8 pharmDx, VENTANA PD-L1 (SP142) Assay and laboratory developed tests (LDTs).1-7 These assays utilise a number of antibody clones, including: SP263, 22C3, 28-8, SP142, E1L3N and 73-10.1-7

The availability of multiple assays is associated with benefits and challenges

All of the commercially available assays utilise automated platform-specific systems, which facilitate greater standardisation of PD-L1 IHC staining compared with LDTs.6

Image: example automated IHC slide staining system


PD-L1 immunohistochemistry staining procedure

Key steps:1–4

  1. De-paraffinisation
  2. Antigen retrieval
  3. Application of primary antibody
  4. Application of secondary antibody
  5. Application of enzyme-bound tertiary antibody
  6. Counterstain/post-counterstain and mounting


These steps are specific to the assay used.

PD-L1 assay design

The use of controls is important to monitor the assay and confirm whether tissues have been correctly prepared and processed.1

PD-L1 IHC assay requirements1–4

Assay requirement

Negative reagent control

A negative reagent control needs to be performed on each patient sample tested in order to confirm acceptable background staining.1

Positive control tissue

A tissue control should be included with each staining procedure. Positive control tissue can be stained as a separate slide in each run (run control) or as an on slide control next to the patient sample being tested (slide control) depending on manufacturer requirements. Control tissue should be fixed in a timely manner and processed alongside patient tumour samples.1–4

Control tissues

  • Tonsil tissue contains positive and negative staining elements for the PD-L1 protein and is therefore suitable for use as a tissue control3
  • Placental stromal tissue and vasculature can be used for the assessment of background staining1
  • Commercial reference samples are available to purchase (e.g. www.horizondiscovery.com/reference-standards/ihc/pd-l1-reference-standards)
  • In-house archival tumour cases with well characterised PD-L1 expression may be suitable8
  • Note: controls with low PD-L1 expression are more likely to highlight protocol failures than controls with high expression9,10

Assay verification

Control tissue should also be used to verify antibody specificity prior to antibody use in any diagnostic procedure. This process should be repeated for each new antibody batch, or whenever there is a change in assay parameters.6 Negative reagent controls should be evaluated in each to confirm acceptable background staining.2

Assay Validation

Laboratory developed tests require additional validation steps and extensive testing to determine their accuracy before any implementation in clinical routine.9,10

The tiers of technical validation and their respective tissue tools are summarized in the figure below.9

Assay Validation

Reproduced with permission from Cheung et al. Evolution of Quality Assurance for Clinical Immunohistochemistry in the Era of Precision Medicine: Part 4: Tissue Tools for Quality Assurance in Immunohistochemistry. Appl Immunohistochem Mol Morphol 2017;25(4):228–230. http://journals.lww.com/appliedimmunohist/pages/default.aspx

Tissue tools in relation to tiers of technical validation of the immunohistochemistry protocol. iCAPCs indicates Immunohistochemistry Critical Assay Performance Controls; LOD, limit of detection; TMA, tissue microarray.

Immunohistochemistry Critical Assay Performance Controls (iCAPCs) are informative for both validation and daily QC monitoring of IHC protocols.9 Appropriately selected iCAPCs can be used to characterise IHC protocols, including technical sensitivity and specificity and have been described as ‘Gold standard controls’, based on expert agreement.10

For further reading, please see: Principles of Analytic Validation of Immunohistochemistry Assays by the College of American Pathologists, available here.

The challenges associated with multiple assays

The approval of numerous PD-L1 IHC assays in oncology indications is associated with a significant challenge to patients and care providers with respect to clinical application of PD-L1 testing and treatment decision-making.11

Running a different test for each drug is impractical due to limited tumour tissue and the requirement for a rapid turnaround time.6,10

Equally, using one test for every drug is impractical:1–7

  • Different assays use different platforms
  • Performance characteristics are unique to the selected assay
  • Scoring and interpretation guidelines vary
  • Clinical responses differ between drugs

Such challenges have led to the fruition of a number of comparability initiatives.7,11–15

Comparability initiatives

A number of cross-industry comparability initiatives are underway to help the clinical and testing community understand the comparative analytical performance of each PD-L1 assay.7,11–15

review table

The currently available comparative data are promising, providing evidence that physicians may, in the future, be able to use the tests interchangeably.7,11–15 Further research is needed to establish this in practice.

Offering a choice of validated PD-L1 tests may facilitate reliable, quality testing and informed treatment decisions.

Professor Keith Kerr discusses the potential interchangeability of validated PD-L1 assays

This short animation explores the importance of concordance in NSCLC diagnostics testing with PD-L1


  1. VENTANA PD-L1 (SP263) Assay package insert.
    https://www.accessdata.fda.gov/cdrh_docs/pdf16/P160046C.pdf (accessed July 2018).
  2. Dako PD-L1 IHC 28-8 pharmDx package insert.
    http://www.accessdata.fda.gov/cdrh_docs/pdf15/P150025c.pdf (accessed July 2018).
  3. Dako PD-L1 IHC 22C3 pharmDx package insert.
    http://www.accessdata.fda.gov/cdrh_docs/pdf15/P150013c.pdf (accessed July 2018).
  4. VENTANA PD-L1 (SP142) Assay package insert.
    http://www.accessdata.fda.gov/cdrh_docs/pdf16/P160002c.pdf (accessed July 2018).
  5. Patel MR et al. Lancet Oncol 2018;19:51–64.
  6. Cree IA, et al. Histopathology 2016;69:177–86.
  7. Adam J, et al. Ann Oncol 2018;29:953–58.
  8. Dolled-Filhart M, et al. Arch Pathol Lab Med 2016;140:1259–66.
  9. Cheung CC, et al. Appl Immunohistochem Mol Morphol 2017;25:227–30.
  10. Torlakovic EE, et al. Appl Immunohistochem Mol Morphol 2015;23:1–18.
  11. Hirsch FR, et al. J Thorac Oncol 2016;12:208–22.
  12. Tsao MS, et al. J Thorac Oncol 2018; doi: 10.1016/j.jtho.2018.05.013 [Epub ahead of print].
  13. Ratcliffe MJ, et al. Clin Cancer Res 2017;23:3585-91.
  14. Ratcliffe MJ, et al. Poster presentation at ESMO 2016 (Abstract 955).
  15. Rimm DL, et al. JAMA Oncol 2017;3:1051–58.
  16. CLOSE