A comparability study of immunohistochemical assays for PD-L1 expression in hepatocellular carcinoma


Programmed death ligand 1 (PD-L1) protein expression by immunohistochemistry is a promising biomarker for PD-1/PD-L1 blockade in hepatocellular carcinoma. There are a number of commercially available PD-L1 assays. Our study aimed to compare the analytical performance of different PD-L1 assays and evaluate the reliability of pathologists in PD-L1 scoring. Consecutive sections from tumor samples from 55 patients with surgically resected primary hepatocellular carcinoma were stained with four standardized PD-L1 assays (22C3, 28–8, SP142, and SP263). We also correlated the PD-L1 protein level by immunohistochemistry with the mRNA level of those genes associated with tumor immune microenvironment by the NanoString platform. Five pathologists independently assessed PD-L1 expression on tumor cells [tumor proportion score] together with tumor-infiltrating immune cells (combined positive score). The 22C3, 28–8, and SP263 assays had comparable sensitivity in detecting PD-L1 expression, whereas the SP142 assay was the least sensitive assay. The inter-assay agreement measured by intraclass correlation coefficients for the tumor proportion score and combined positive score were 0.646 and 0.780, respectively. The inter-rater agreement was good to excellent (the overall intraclass correlation coefficient for the tumor proportion score and combined positive score was 0.946 and 0.809, respectively). Pathologists were less reliable in scoring combined positive score than tumor proportion score, particularly when using the SP142 assay. Up to 18% of samples were misclassified by individual pathologists in comparison to the consensus score at the cutoff of combined positive score ≥ 1. The combined positive score by the 22C3 assay demonstrated the strongest correlation with immune-related gene mRNA signatures, closely followed by combined positive scores by the 28–8 and SP263 assays. In conclusion, the 22C3, 28–8, and SP263 assays are highly concordant in PD-L1 scoring and suggest the interchangeability of these three assays. Further improvement of the accuracy in assessing PD-L1 expression at a low cutoff is still necessary.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. 1.

    Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372:2018–28.

    Article  Google Scholar 

  2. 2.

    Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science. 2017;357:409–13.

    CAS  Article  Google Scholar 

  3. 3.

    Powles T, Duran I, van der Heijden MS, Loriot Y, Vogelzang NJ, De Giorgi U, et al. Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2018;391:748–57.

    CAS  Article  Google Scholar 

  4. 4.

    Schachter J, Ribas A, Long GV, Arance A, Grob JJ, Mortier L, et al. Pembrolizumab versus ipilimumab for advanced melanoma: final overall survival results of a multicentre, randomised, open-label phase 3 study (KEYNOTE-006). Lancet. 2017;390:1853–62.

    CAS  Article  Google Scholar 

  5. 5.

    El-Khoueiry AB, Sangro B, Yau T, Crocenzi TS, Kudo M, Hsu C, et al. Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet. 2017;389:2492–502.

    CAS  Article  Google Scholar 

  6. 6.

    Zhu AX, Finn RS, Edeline J, Cattan S, Ogasawara S, Palmer D, et al. Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial. Lancet Oncol. 2018;19:940–52.

    Article  Google Scholar 

  7. 7.

    Hirsch FR, McElhinny A, Stanforth D, Ranger-Moore J, Jansson M, Kulangara K, et al. PD-L1 immunohistochemistry assays for lung cancer: results from phase 1 of the blueprint PD-L1 IHC assay comparison project. J Thorac Oncol. 2017;12:208–22.

    Article  Google Scholar 

  8. 8.

    European Association for the Study of the Liver. EASL clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol. 2018;69:182–236.

    Article  Google Scholar 

  9. 9.

    Heimbach JK, Kulik LM, Finn RS, Sirlin CB, Abecassis MM, Roberts LR, et al. AASLD guidelines for the treatment of hepatocellular carcinoma. Hepatology. 2018;67:358–80.

    Article  Google Scholar 

  10. 10.

    Nault JC, Galle PR, Marquardt JU. The role of molecular enrichment on future therapies in hepatocellular carcinoma. J Hepatol. 2018;69:237–47.

    CAS  Article  Google Scholar 

  11. 11.

    Brunnstrom H, Johansson A, Westbom-Fremer S, Backman M, Djureinovic D, Patthey A, et al. PD-L1 immunohistochemistry in clinical diagnostics of lung cancer: inter-pathologist variability is higher than assay variability. Mod Pathol. 2017;30:1411–21.

    Article  Google Scholar 

  12. 12.

    Adam J, Le Stang N, Rouquette I, Cazes A, Badoual C, Pinot-Roussel H, et al. Multicenter harmonization study for PD-L1 IHC testing in non-small-cell lung cancer. Ann Oncol. 2018;29:953–8.

    CAS  Article  Google Scholar 

  13. 13.

    Chan AWH, Tong JHM, Kwan JSH, Chow C, Chung LY, Chau SL, et al. Assessment of programmed cell death ligand-1 expression by 4 diagnostic assays and its clinicopathological correlation in a large cohort of surgical resected non-small cell lung carcinoma. Mod Pathol. 2018;31:1381–90.

    CAS  Article  Google Scholar 

  14. 14.

    Tsao MS, Kerr KM, Kockx M, Beasley MB, Borczuk AC, Botling J, et al. PD-L1 immunohistochemistry comparability study in real-life clinical samples: results of blueprint phase 2 project. J Thorac Oncol. 2018;13:1302–11.

    Article  Google Scholar 

  15. 15.

    Pinato DJ, Mauri FA, Spina P, Cain O, Siddique A, Goldin RD, et al. Quantitative comparison of PD-L1 immuno-histochemical assays in hepatocellular carcinoma: the blueprint-HCC study. J Clin Oncol. 2018;36:91.

    Article  Google Scholar 

  16. 16.

    Theise ND, Curado MP, Franceshi S, Hytiroglou P, Kudo M, Park YN, et al. Hepatocellular carcinoma, In: Bosman FT, Carneiro F, Hruban RH, Theise ND, (eds). WHO Classification of Tumours of the Digestive System International Agency for Research on Cancer: Lyon; 2010. pp 205–16.

  17. 17.

    Chan AW, Tong JH, Pan Y, Chan SL, Wong GL, Wong VW, et al. Lymphoepithelioma-like hepatocellular carcinoma: an uncommon variant of hepatocellular carcinoma with favorable outcome. Am J Surg Pathol. 2015;39:304–12.

    Article  Google Scholar 

  18. 18.

    Chan AW, Yu S, Yu YH, Tong JH, Wang L, Tin EK, et al. Steatotic hepatocellular carcinoma: a variant associated with metabolic factors and late tumour relapse. Histopathology. 2016;69:971–84.

    Article  Google Scholar 

  19. 19.

    Kulangara K, Zhang N, Corigliano E, Guerrero L, Waldroup S, Jaiswal D, et al. Clinical utility of the combined positive score for programmed death ligand-1 expression and the approval of pembrolizumab for treatment of gastric cancer. Arch Pathol Lab Med. 2019;143:330–7.

    Article  Google Scholar 

  20. 20.

    Cesano A. nCounter((R)) PanCancer Immune Profiling Panel (NanoString Technologies, Inc., Seattle, WA). J Immunother Cancer. 2015;3:42.

    Article  Google Scholar 

  21. 21.

    Ayers M, Lunceford J, Nebozhyn M, Murphy E, Loboda A, Kaufman DR, et al. IFN-gamma-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest. 2017;127:2930–40.

    Article  Google Scholar 

  22. 22.

    Cancer Genome Atlas Research Network. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell. 2017;169:1327–41 e23.

    Article  Google Scholar 

  23. 23.

    Bellmunt J, de Wit R, Vaughn DJ, Fradet Y, Lee JL, Fong L, et al. Pembrolizumab as second-line therapy for advanced urothelial carcinoma. N Engl J Med. 2017;376:1015–26.

    CAS  Article  Google Scholar 

  24. 24.

    Burtness B, Harrington KJ, Greil R, Soulières D, Tahara M, De Castro G, et al. LBA8_PRKEYNOTE-048: phase III study of first-line pembrolizumab (P) for recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). Ann Oncol. 2018;29:mdy424.045.

    Article  Google Scholar 

  25. 25.

    Rimm DL, Han G, Taube JM, Yi ES, Bridge JA, Flieder DB, et al. A prospective, multi-institutional, pathologist-based assessment of 4 immunohistochemistry assays for PD-L1 expression in non-small cell lung cancer. JAMA Oncol. 2017;3:1051–8.

    Article  Google Scholar 

  26. 26.

    Koo TK, Li MY. A guideline of selecting and reporting intraclass correlation coefficients for reliability research. J Chiropr Med. 2016;15:155–63.

    Article  Google Scholar 

  27. 27.

    Calderaro J, Rousseau B, Amaddeo G, Mercey M, Charpy C, Costentin C, et al. Programmed death ligand 1 expression in hepatocellular carcinoma: relationship with clinical and pathological features. Hepatology. 2016;64:2038–46.

    CAS  Article  Google Scholar 

  28. 28.

    Wang C, Hahn E, Slodkowska E, Eskander A, Enepekides D, Higgins K, et al. Reproducibility of PD-L1 immunohistochemistry interpretation across various types of genitourinary and head/neck carcinomas, antibody clones, and tissue types. Hum Pathol. 2018;82:131–9.

    CAS  Article  Google Scholar 

  29. 29.

    Taylor CR, Jadhav AP, Gholap A, Kamble G, Huang J, Gown A, et al. A multi-institutional study to evaluate automated whole slide scoring of immunohistochemistry for assessment of programmed death-ligand 1 (PD-L1) expression in non-small cell lung cancer. Appl Immunohistochem Mol Morphol. 2019;27:263–9.

    CAS  Article  Google Scholar 

  30. 30.

    Koelzer VH, Gisler A, Hanhart JC, Griss J, Wagner SN, Willi N, et al. Digital image analysis improves precision of PD-L1 scoring in cutaneous melanoma. Histopathology. 2018;73:397–406.

    Article  Google Scholar 

  31. 31.

    Lu LC, Lee YH, Chang CJ, Shun CT, Fang CY, Shao YY, et al. Increased expression of programmed death-ligand 1 in infiltrating immune cells in hepatocellular carcinoma tissues after Sorafenib treatment. Liver Cancer. 2019;8:110–20.

    CAS  Article  Google Scholar 

  32. 32.

    Yarchoan M, Xing D, Luan L, Xu H, Sharma RB, Popovic A, et al. Characterization of the immune microenvironment in hepatocellular carcinoma. Clin Cancer Res. 2017;23:7333–9.

    CAS  Article  Google Scholar 

  33. 33.

    Veldman-Jones MH, Lai Z, Wappett M, Harbron CG, Barrett JC, Harrington EA, et al. Reproducible, quantitative, and flexible molecular subtyping of clinical DLBCL samples using the NanoString nCounter system. Clin Cancer Res. 2015;21:2367–78.

    CAS  Article  Google Scholar 

  34. 34.

    Reis PP, Waldron L, Goswami RS, Xu W, Xuan Y, Perez-Ordonez B, et al. mRNA transcript quantification in archival samples using multiplexed, color-coded probes. BMC Biotechnol. 2011;11:46.

    CAS  Article  Google Scholar 

  35. 35.

    Ott PA, Bang YJ, Piha-Paul SA, Razak ARA, Bennouna J, Soria JC, et al. T-cell-inflamed gene-expression profile, programmed death ligand 1 expression, and tumor mutational burden predict efficacy in patients treated with pembrolizumab across 20 cancers: KEYNOTE-028. J Clin Oncol. 2019;37:318–27.

    Article  Google Scholar 

  36. 36.

    Lee KH, Hsu CH, Lee MS, Ryoo BY, Verret W, He AR, et al. 150OAtezolizumab+bevacizumab in hepatocellular carcinoma (HCC): safety and clinical activity results from a phase Ib study. Ann Oncol. 2018;29:mdy432.002.

    Google Scholar 

Download references


This work was supported in part by a Collaborative Research Fund from the Hong Kong Research Grants Council (Ref. No.: C4041–17).

Author information



Corresponding author

Correspondence to Anthony Wing-Hung Chan.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shi, L., Zhang, S., Chen, J. et al. A comparability study of immunohistochemical assays for PD-L1 expression in hepatocellular carcinoma. Mod Pathol 32, 1646–1656 (2019). https://doi.org/10.1038/s41379-019-0307-8

Download citation

Further reading

  • NF-κB and Its Role in Checkpoint Control

    • Annika C. Betzler
    • , Marie-Nicole Theodoraki
    • , Patrick J. Schuler
    • , Johannes Döscher
    • , Simon Laban
    • , Thomas K. Hoffmann
    •  & Cornelia Brunner

    International Journal of Molecular Sciences (2020)