Development of a Companion Diagnostic PD-L1 Immunohistochemistry Assay for Pembrolizumab Therapy in Non-Small-cell Lung Cancer

Appl Immunohistochem Mol Morphol. 2016 Jul;24(6):392-7. doi: 10.1097/PAI.0000000000000408.


A companion diagnostic assay was codeveloped by Dako for pembrolizumab non-small-cell lung cancer clinical trials to detect PD-L1 expression by immunohistochemistry (IHC). This automated IHC assay has been analytically verified and validated using Dako's autostainer Link 48 and 22C3 mouse anti-PD-L1 monoclonal antibody to detect the PD-L1 expression in formalin-fixed paraffin-embedded human tumor tissue specimens. The PD-L1 22C3 IHC assay was optimized for high sensitivity and specificity. Repeatability and reproducibility studies were conducted at Dako and at 3 Clinical Laboratory Improvement Amendments certified laboratories during assay development. The studies included: intersite and intrasite, interobserver and intraobserver, interinstrument, interoperator, interday, and interlot, and intraday and intrarun. All precision studies performed at Dako and external laboratories achieved >85% point-estimate agreements for all 3 agreement types (negative, positive, and overall). A clinical cutoff (tumor proportion score ≥50%) of PD-L1 expression was determined and evaluated through a phase 1 clinical trial (KEYNOTE-001) for advanced non-small-cell lung cancer patients treated with pembrolizumab. The treatment effect of pembrolizumab in the 61 subjects who had a tumor PD-L1 of tumor proportion score ≥50% was substantial, with an overall response rate of 41% (95% confidence interval, 28.6-54.3) as compared with 20.6% (95% confidence interval, 15.5-26.5) observed in the 223 subjects irrespective of PD-L1 status. PD-L1 IHC 22C3 pharmDx is a sensitive, precise, and robust companion diagnostic assay, which will facilitate safe and effective use for pembrolizumab in cancer patients.

Publication types

  • Clinical Trial, Phase I
  • Multicenter Study
  • Validation Study

MeSH terms

  • Antibodies, Monoclonal, Humanized / therapeutic use*
  • Antineoplastic Agents / therapeutic use*
  • B7-H1 Antigen / metabolism*
  • Biomarkers, Tumor / metabolism*
  • Carcinoma, Non-Small-Cell Lung / drug therapy*
  • Humans
  • Immunohistochemistry
  • Limit of Detection
  • Lung Neoplasms / drug therapy*
  • Reproducibility of Results


  • Antibodies, Monoclonal, Humanized
  • Antineoplastic Agents
  • B7-H1 Antigen
  • Biomarkers, Tumor
  • CD274 protein, human
  • pembrolizumab