557 Yeong J, et al. J Clin Pathol 2020;73:557–562. doi:10.1136/jclinpath-2019-206252 Multiplex immunohistochemistry/ immunofuorescence (mIHC/IF) for PD-L1 testing in triple-negative breast cancer: a translational assay compared with conventional IHC Joe Yeong , 1,2,3 Tira Tan, 4 Zi Long Chow, 1,5 Qing Cheng, 6 Bernett Lee, 3 Amanda Seet, 4 Johnathan Xiande Lim, 1 Jeffrey Chun Tatt Lim, 2 Clara Chong Hui Ong, 1,7 Aye Aye Thike, 1 Sahil Saraf, 1 Benjamin, Yong Cheng Tan, 1 Yong Cheng Poh, 8 Sidney Yee, 8 Jin Liu, 6 Elaine Lim, 4 Jabed Iqbal, 1 Rebecca Dent, 4 Puay Hoon Tan 1 Original research To cite: Yeong J, Tan T, Chow ZL, et al. J Clin Pathol 2020;73:557–562. ► Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jclinpath-2019-206252). For numbered affliations see end of article. Correspondence to Dr Joe Yeong, Pathology, Singapore General Hospital, Singapore 169856, Singapore; joe.yeong.p.s@sgh.com.sg Professor Puay Hoon Tan; tan. puay.hoon@singhealth.com.sg JY and PHT are joint senior authors. Received 6 October 2019 Revised 23 December 2019 Accepted 31 December 2019 Published Online First 22 January 2020 © Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ. ABSTRACT 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/immunofuorescence (mIHC/ IF) that allowed simultaneous quantifcation 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 coeffcient 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 signifcant advance in the management of therapeutically challenging cancers. Further analysis and assay harmonisation are essential for translation to a routine diagnostic setting. INTRODUCTION Triple-negative breast cancer (TNBC) is character- ised by a lack of cerbB2-(HER2), oestrogen and progesterone receptor expression, and has a rela- tively poor prognosis compared with other types of breast cancer. For this reason, novel and effective treatments are urgently required. The recent phase III IMpassion130 trial demonstrated progression- free survival (PFS) in patients with TNBC treated with upfront atezolizumab–nab-paclitaxel treat- ment, compared with placebo-nab-paclitaxel treatment. 1 As a result, The United States Food and Drug Administration (FDA) granted acceler- ated approval for atezolizumab in combination with nab-paclitaxel in programmed death-ligand 1 (PD-L1) + , non-resectable TNBC. 2 However, the IMpassion130 trial conducted immunohistochem- istry (IHC) with the SP142 PD-L1 antibody clone to identify PD-L1 + patients, as a companion diag- nostic kit. This particular clone was found to be an outlier in previous IHC harmonisation studies performed in lung cancer, 3–6 compared with SP263, 22C3 and other clones. Schats et al 78 reported that the SP142 clone uniquely binds to PD-L1 isoform 2, which lacks AA 19–132. Whether breast cancer cells express this particular isoform remains unclear. However, in contrast to other clones, SP142 tends to recognise PD-L1 expression on immune cells; other clones predominantly label PD-L1 on tumour cells. Furthermore, PD-L1 expression on immune cells, not tumour cells, predicts outcome in atezolizumab-treated TNBC, 1 but accurate scoring of PD-L1 expression in immune cells remains chal- lenging for pathologists. 4 6 7 9 Previous studies have shown that pathologists are typically unable to report the immune count (IC) accurately or repro- ducibly, with an interclass correlation coefficient between 0.19 and 0.28. 4 6 10 In addition, the staining protocol for the SP142 clone differs from that used for other clones in terms of both methodology and equipment. At present, three IHC-based companion diagnostic assays have been approved for PD-L1 monoclonal antibody therapies, which use separate staining platforms and scoring systems to predict patient response to anti-PD-1/PD-L1 immunotherapy. The 22C3 clone is detected using the Dako Autostainer Link 48 platform (Dako; Agilent Technologies, Santa Clara, California, USA), whereas the SP263 and SP142 clones are detected with the Ventana BenchMark ULTRA platform (Ventana Medical Systems; Roche Diagnostics, Basel, Switzerland). However, all these on November 29, 2021 by guest. Protected by copyright. http://jcp.bmj.com/ J Clin Pathol: first published as 10.1136/jclinpath-2019-206252 on 22 January 2020. Downloaded from