Spatial analyses of the heterogeneity of distribution of PBL signature positive cells. (A) Representative immunofluorescence image of GeoMx DSP shows the selected region of interests in PCNSL_09. Blue, nuclei stain (SYTO13); green, anti-CD45; yellow, anti-CD20; red, anti-CD68. Scale bar shows 500 μm. (B) Heat map of the estimated cell proportion in each area of interest (AOI) shows higher variation across samples than within samples. Each column represents an AOI. (C) Representative images of CD20 and CD138 IHC in 2 samples with PBL signature cells. In PCNSL_09, the distribution of CD138⁺ cells varies within the tumor, whereas in patient 10, CD138⁺ cells are uniformly distributed throughout the tumor. Small panel of CD20 staining shows an overall image of the tumor. Scale bar shows 200 μm (CD20) or 20 μm (CD138). (D) Representative density map of CD138 IHC in PCNSL_09 and PCNSL_10. Yellow lines represent CD138⁺ cells. Scale bar shows 200 μm. (E) Spatial feature plot shows the PBL signature score and B cells in publicly available data (Heming et al⁸). Patient 1 harbors PBL signature lymphoma cells and their distribution varies within the tumor, whereas other patients hardly harbor high PBL score areas. (F) Dot plot shows the logarithmic number of CD138⁺ cells per square millimeter in each sample. The dotted line denotes the determined cutoff value, derived as the geometric mean of CD138⁺ cell counts per square millimeter. The term "Diffuse" designates a uniform distribution of CD138⁺ cells throughout the tumor (the presence of CD138⁺ cells in more than two-thirds of the CD20⁺ area). "Localized" indicates a variable distribution of CD138⁺ cells within the tumor (the presence of CD138⁺ cells in less than two-thirds of the regions). “Negative” denotes instances where the count of CD138⁺ cells falls below the established cutoff value. “No overlap” indicates CD138⁺ cells are present outside the CD20⁺ regions. PBL, plasmablast; DC, dendric cell; NK, natural killer cell; Treg, regulatory T cell.