Analysis Plan for Secondary Endpoints
An Olink multiplex proximity extension assay platform with an immuno-oncology panel (Olink Bioscience) was used according to the manufacturer's instructions. The panel includes 92 proteins associated with immune responses, inflammatory cytokines, chemokines, and soluble immune checkpoint molecules. Peripheral blood mononuclear cells were analyzed by cytometry using time-of-flight (CyTOF) with reference sample spike-in and palladium-based mass tag cell barcoding of individual samples. The Astrolabe platform was used to annotate and analyze specific cell populations. Plasma cytokines, chemokines, and circulating immune cells were assessed at three discrete timepoints (pretreatment baseline and two timepoints after avelumab treatment). Potential biomarkers associated with the activity of avelumab alone and LITT followed by avelumab with or without bevacizumab were investigated separately and correlated with survival. Normalized protein expression (NPX) and phenotypic frequency were used to identify changes in individual proteins across the sample set.Formalin-fixed paraffin-embedded 3-μm sections from archival tissue at initial diagnosis and when available from LITT procedure or a subsequent re-resection were used for single, dual, or multi-chromogen sequential IHC. IHC was performed using the Ventana Discovery Ultra (Roche Diagnostics). This system allows automated baking, deparaffinization, and cell conditioning. Single staining was performed using the RUO Discovery Multimer V2 (v0. 00.0083) and dual and multiplex chromogens using RUO Discovery Universal (v21.00.0019). Prediluted primary antibodies were obtained from Roche Diagnostics (The HALO image analysis platform was used for quantitative tissue analysis (Indica Labs, Inc.), using the Multiplex IHC module and color deconvolution to separate chromogenic stains to prepare for quantitative analysis.