Figure 9 from OPG-Producing B Cells and RANKL-Expressing T Cells Define Immune Signatures Predictive of Bone Metastases in Breast Cancer

Opposing infiltration patterns of RANKL+ and OPG+ lymphocytes in primary tumors of patients with TNBC with or without bone metastases. A, Quantification of tumor-infiltrating CD3+ T cells in primary tumor samples from patients with TNBC who later developed BoneMet (n = 5) vs. those who remained No BoneMet (n = 5). Data are shown as box and whisker plots with individual values, and statistical comparison was performed by unpaired two-tailed Student t test (P = 0.0247); variance homogeneity was verified by F test (P = 0.0984). B, Representative IHC images of CD3+ T cells in primary tumors from each patient group. C, Quantification of RANKL+ lymphocytes in the same cohort, also expressed as box and whisker plots with overlaid points. Statistical analysis: Mann–Whitney U test (P = 0.0278) and confirmatory unpaired two-tailed t test (P = 0.0173), with F test confirming homogeneity of variance (P = 0.2850). D, Representative images of RANKL staining in tumor sections from BoneMet and No BoneMet patients. E, Quantification of CD20+ B cells in the same tumor samples. Although not statistically significant (P = 0.1323) by unpaired two-tailed t test, a higher median infiltration was observed in the No BoneMet group, and F test for variance (P = 0.1287). F, Representative IHC images of CD20 staining showing differences in localization and density between groups. G, Quantification of OPG+ lymphocytes in No BoneMet tumors compared with BoneMet. Median values were 50.95% (No BoneMet) and 17.31% (BoneMet). Statistical analysis: unpaired two-tailed t test (P = 0.0341) with variance verified by F test (P = 0.6365); the Mann–Whitney U test showed a consistent trend (P = 0.0556). H, Representative images of OPG+ cells from each group. Statistical assumptions were verified using the F test for variance homogeneity. Due to the limited sample size, nonparametric tests were prioritized, and parametric results were used as confirmatory evidence.