PD-1 expression on HCC-infiltrating B cells and its clinical significance. PD-1 expression on HCC-infiltrating B cells and its clinical significance. A–H, FACS analysis of PD-1 expression on B cells from the samples as follows: normal blood from 20 healthy individuals; paired blood and normal liver tissue from 6 hepatic hemangioma (Hem) patients; paired blood, peritumoral liver tissue, and tumor tissue from 43 patients with HBV-related HCC, 6 with HCV-related HCC, and 4 with HCC but no HBV or HCV infection. Representative FACS plots of B-cell PD-1 expression are shown in A, and the values within dashed boxes indicate the proportions of PD-1hi B cells to total B cells. Frequencies of PD-1+ and PD-1hi B cells relative to total B-cell frequencies and the ratio of PD-1hi B cells to PD-1+ B cells are shown in B–D. Associations of tumor PD-1+ and PD-1hi B cells with patients' TNM stage and recurrence are shown in E–H. In G and H, patients were divided into two groups (Less/More) according to the median values of tumor PD-1+ or PD-1hi B-cell percentages shown in B and C, respectively; statistical comparisons were done by the log-rank test. I, confocal microscopy analysis of PD-1+ cells (green) and CD79a+ B cells (red) in HCC tissue. Results represent four independent experiments (N = 8). Scale bar, 100 μm. J–L, immunohistochemical staining of CD79a+ B cells in paraffin-embedded HCC (N = 53). Distribution of B cells is shown in J and K, and micrographs at higher magnification show stained peritumoral liver (1), the peritumoral stromal region (2), and cancer nest (3). Scale bar, 100 μm. Association of peritumoral stromal B cells with patients' TNM stage is shown in L. Horizontal bars indicate median values (E, F, K, and L). *, P < 0.05; **, P < 0.01; and ***, P < 0.001 (Student t test). Xiao Xiao et al. Cancer Discov 2016;6:546-559 ©2016 by American Association for Cancer Research