Introduction: We analyzed the expression of PD-L1 in human lymphomas using hyperspectral imaging (HSI) compared to visual assessment (VA) and conventional digital image analysis (DIA) to strengthen further the value of HSI as a tool for the evaluation of brightfield-based immunohistochemistry (IHC). In addition, fluorescent multiplex immunohistochemistry (mIHC) was used as a second detection method to analyze the impact of a different detection method.
Material and methods: 18 cases (6 follicular lymphomas and 12 diffuse large B-cell lymphomas) were stained for PD-L1 by IHC and for PD-L1, CD3, and CD8 by fluorescent mIHC. The percentage of positively stained cells was evaluated with VA, HSI, and DIA for IHC and VA and DIA for mIHC. Results were compared between the different methods of detection and analysis.
Results: An overall high concordance was found between VA, HSI, and DIA in IHC (Cohens Kappa = 0.810VA/HSI, 0.710 VA/DIA, and 0.516 HSI/DIA) and for VAmIHCversus DIAmIHC (Cohens Kappa = 0.894). Comparing IHC and mIHC general agreement differed depending on the methods compared but reached at most a moderate agreement (Coheńs Kappa between 0.250 and 0.483). This is reflected by the significantly higher percentage of PD-L1+ cells found with mIHC (pFriedman = 0.014).
Conclusion: Our study shows a good concordance for the different analysis methods. Compared to VA and DIA, HSI proved to be a reliable tool for assessing IHC. Understanding the regulation of PD-L1 expression will further enlighten the role of PD-L1 as a biomarker. Therefore it is necessary to develop an instrument, such as HSI, which can offer a reliable and objective evaluation of PD-L1 expression.
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