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Multiplex immunohistochemistry/immunofluorescence (mIHC/IF) for PD-L1 testing in triple-negative breast cancer: a translational assay compared with conventional IHC
  1. Joe Yeong1,2,3,
  2. Tira Tan4,
  3. Zi Long Chow1,5,
  4. Qing Cheng6,
  5. Bernett Lee3,
  6. Amanda Seet4,
  7. Johnathan Xiande Lim1,
  8. Jeffrey Chun Tatt Lim2,
  9. Clara Chong Hui Ong1,7,
  10. Aye Aye Thike1,
  11. Sahil Saraf1,
  12. Benjamin, Yong Cheng Tan1,
  13. Yong Cheng Poh8,
  14. Sidney Yee8,
  15. Jin Liu6,
  16. Elaine Lim4,
  17. Jabed Iqbal1,
  18. Rebecca Dent4,
  19. Puay Hoon Tan1
  1. 1 Division of Pathology, Singapore General Hospital, Singapore
  2. 2 Integrative Biology for Theranostics, Institute of Molecular Cell Biology, Agency of Science, Technology and Research (A*STAR), Singapore
  3. 3 Singapore Immunology Network (SIgN), Agency of Science, Technology and Research (A*STAR), Singapore
  4. 4 Division of Medical Oncology, National Cancer Centre Singapore, Singapore
  5. 5 University of Tasmania, Hobart, Tasmania, Australia
  6. 6 Duke-NUS Medical School, Duke-NUS Medical School, Singapore
  7. 7 Department of Anatomical Pathology, Singapore General Hospital, Singapore
  8. 8 Diagnostics Development (DxD) Hub, Agency of Science, Technology and Research (A*STAR), Singapore
  1. Correspondence to Dr Joe Yeong, Pathology, Singapore General Hospital, Singapore 169856, Singapore; joe.yeong.p.s{at}; Professor Puay Hoon Tan; tan.puay.hoon{at}


Background Programmed death-ligand 1 (PD-L1) monoclonal antibody therapy has recently gained approval for treating metastatic triple-negative breast cancer (TNBC) -, in particular in the PD-L1+ patient subgroup of the recent IMpassion130 trial. The SP142 PD-L1 antibody clone was used as a predictive assay in this trial, but this clone was found to be an outlier in previous harmonisation studies in lung cancer.

Aims To address the comparability of PD-L1 clones in TNBC, we evaluated the concordance between conventional immunohistochemistry (IHC) and multiplex immunohistochemistry/immunofluorescence (mIHC/IF) that allowed simultaneous quantification of three different PD-L1 antibodies (22C3, SP142 and SP263).

Methods Our cohort comprised 25 TNBC cases, 12 non-small-cell lung carcinomas and 8 other cancers. EpCAM labelling was used to distinguish tumour cells from immune cells.

Results Moderate-to-strong correlations in PD-L1 positivity were found between results obtained through mIHC/IF and IHC. Individual concordance rates in the study ranged from 67% to 100%, with Spearman’s rank correlation coefficient values up to 0.88.

Conclusions mIHC/IF represents a promising tool in the era of cancer immunotherapy, as it can simultaneously detect and quantify PD-L1 labelling with multiple antibody clones, and allow accurate evaluation of tumour and immune cells. Clinicians and pathologists require this information to predict patient response to anti-PD-1/PD-L1 therapy. The adoption of this assay may represent a significant advance in the management of therapeutically challenging cancers. Further analysis and assay harmonisation are essential for translation to a routine diagnostic setting.

  • breast pathology
  • immunohistochemistry
  • immunopathology

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  • JY and PHT are joint senior authors.

  • Handling editor Cheok Soon Lee.

  • Contributors PHT and JY: designed and directed the study; drafted the manuscript, which was commented on and revised by all authors. JY, TT and ZLC: coordinated the study. JCTL, JL, AAT and CO: acquired the data. JY, SS, YCT and PHT: did conventional IHC and manual scoring. CQ, BL and JL: did analysis. JI, YCP and SY: provided advice from technical perspectives. TT, AS, EL and RD: provided advice from clinical perspectives.

  • Funding This study was funded by the A*STAR Biomedical Research Council, National Medical Research Council Stratified Medicine Programme Office (grant no. SMPO201302), awarded to Dr PHTn. Dr JI is a recipient of the Transition Award from the Singapore National Medical Research Council (grant no. NMRC/TA/0041/2015).

  • Competing interests ZLC is a medical student from the University of Tasmania on a research elective posting with the Department of Anatomical Pathology Singapore General Hospital, supported by the Royal College of Pathologists of Australasia (RCPA) medical students’ scholarship. BL is part of the SIgN Immunomonitoring platform (supported by a BMRC IAF 311006 grant and BMRC transition funds #H16/99/b0/011).

  • Patient consent for publication Not required.

  • Ethics approval The Centralised Institutional Review Board of Singhealth provided ethical approval for the use of patient materials in the present study (CIRB ref: 2013/664/F, 2015/2199 and 2011/441/B).

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available upon reasonable request. Data will be made available upon reasonable request to correspondent authors, JY and PHT.