다중 면역형광 패널을 이용한 하인두암의 종양 내 침윤 림프구의 분포와 같이 발현되는 면역학적 지표

Abstract

Background and Objectives: Immunological markers predicting treatment response to immunotherapeutic agents have not been revealed. Tumor infiltrating lymphocytes (TILs) are essential to activate innate or adaptive immunity against tumor. Rather than one type of TILs, the distribution of regulatory and cytotoxic T-cell markers may help to delineate the immune-profiling and to predict the response to immunotherapy. This preliminary study was aimed to evaluate the overall immunological tumor microenvironment by utilizing multiplex immunofluorescence (mIF) panels. Materials and Methods: Formalin fixed paraffin-embedded tissue (FFPE) specimens of 10 cases of hypopharyngeal squamous cell carcinoma were prepared for conventional immunohistochemistry (IHC), uniplex and multiplex IF validation. The Opal™ Multiplex IHC Kit (PerkinElmer) was used for multiple IF validation and these findings were reviewed by a pathologist. The panel including pancytokeratin, PD-L1, CD4, CD8, CD68, and DAPI was developed and simultaneous marker expressions were quantified to evaluate the expression of tumor infiltrating lymphocytes (TILs) and programmed cell death ligand 1 (PD-L1). Results: The mIF panel demonstrated the distribution of TILs and PD-L1 in the FFPE tissue. Compared to each conventional IHC, we could evaluate the distribution of PD-L1, CD4+, CD8+, CD68+ cells in one slide. Quantitative analysis demonstrated that the types of TILs were distinct from each samples as well as the amount of aggregated TILs. Conclusions: The mIF may give us an additional information presuming tumor tissue immune-profiling, elucidating main contributing TILs. This finding may help to evaluate immunological status and to choose adequate immunotherapeutic agents according to the crucial immune cells.