Fig. 5: The immunomodulatory role of anti-RANKL in BC.
a Change from baseline in serum levels of free-sRANKL (n = 23, p = 2.384e-07) and CTX (n = 17, p = 1.526e-05) (significance assessed by the two-tailed sign test), the percentage of Ki-67-positive cells (p = 0.485) and the staining of activated caspase-3 (p = 0.391) (H-score) (n = 24) (significance assessed by two-tailed paired t-tests). Boxplots display median line, IQR boxes, 1.5 × IQR whiskers, and data points. b Each bar plot shows the change from baseline (Δ; post- minus pretreatment values) of the immune parameters assessed using HE (TILs) and IHC (CD3, CD20, CD8, FOXP3, proliferative TILs (TILsKi67+), CD68, and CD163). Each bar represents one patient, which are ranked by their increase in stromal TIL levels. Geometric mean changes, 95% CIs, and p-values are shown below each bar plot. For each measured parameter, the corresponding boxplot is displayed on the right-hand side. Boxplots display median line, IQR boxes, 1.5 × IQR whiskers, and data points. Tumor characteristics and tumor RANK metagene expression at baseline are shown above. p; p-values derived from two-tailed paired t-tests (*p < 0.05)#. c Representative micrographs of multiplex IHC of pre- and posttreatment tumor sections from the four patients with the highest immunomodulatory response. White scale bar, 100 μm. d Top 20 significantly enriched pathways after denosumab treatment, identified by GAGE. e Comparison of baseline serum levels of sRANKL between non-responders (NR; n = 13) vs. responders (R; n = 11) and comparison of baseline percentage of regulatory T cells (Tregs) as inferred from CIBERSORT. Boxplots display median line, IQR boxes, 1.5 × IQR whiskers, and data points. Significance determined by the two tailed Mann–Whitney U-test. f Comparison of baseline mRNA expression levels of indicated genes (normalized counts) between non-responder (NR; n = 11) and responder (R; n = 11) groups. Boxplots display median line, IQR boxes, 1.5 × IQR whiskers, and data points. Significance determined by the two-tailed Mann–Whitney U-test p-values: FOXP3 (p = 1.61e − 05), IL7R (p = 1.53e − 07), MS4A1 (p = 1.00E − 06), CD28 (p = 5.63e − 06), IFNG (p = 4.15e − 05). g Comparison of baseline RANK metagene and RANKL-treated PyMT tumor acini-derived gene signature between non-responder (NR; n = 11) and responder (R; n = 11) patients. Significance determined by the two tailed Mann–Whitney U-test. For a, b: each colored line represents one patient and indicates increase (red), decrease (blue), or no change (black) relative to baseline. Note that all variables were analyzed for all patients, but values for some lines overlap or the indicated population was not detected. Boxplots display median line, IQR boxes, 1.5 × IQR whiskers, and data points. #Responder patients are those with ≥10% increase in TIL infiltration after denosumab treatment. Significance determined by the two-tailed Mann–Whitney U-test.
