Figures
Figure 1. Relationship between tumor BTK expression and breast cancer aggressiveness and survival. BTK mRNA expression in three cohorts was examined. (a) Tukey boxplots of BTK expression among tumors categorized by overall AJCC stage and status for tumor size (T), lymph node (N), and metastasis (M). P values from Kruskal-Wallis test are shown. (b) Estimated survival (Kaplan-Meier) with log-rank test P value and hazard ratio (HR) of disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS) for groups of patients with low and high BTK expression within the cohorts, using median value as cut-off.
Figure 2. Associations of tumor BTK expression with Nottingham histological grade, MKI67 gene expression, and cell proliferation pathways in breast cancer. BTK mRNA expression in three cohorts was examined. (a) Tukey boxplots of BTK expression in different histological grades of breast cancer. (b) Tukey boxplots of tumor MKI67 expression in tumor groups with low and high BTK expression, using median value as cut-off. (c) Gene set enrichment analysis (GSEA) of cell proliferation-related Hallmark gene sets (E2F targets, G2M checkpoint, Myc targets v1 and v2, mitotic spindle), comparing tumor groups with low and high BTK expression, using median value as cut-off. (d) Tukey boxplots of BTK expression in different cancer subtypes. (e) GSEA analyses for different cancer subtypes as in (c). GSEA NES and FDR values were calculated using GSEA software. P values shown with boxplots are from Kruskal Wallis or Mann-Whitney U tests. NES: normalized enrichment score; FDR: false discovery rate.
Figure 3. Associations of tumor BTK expression with mutation burden and immune cell infiltration in breast cancer. Tumor groups with low and high BTK expression, using median value as cut-off, were compared. (a) Tukey boxplots of mutation- and immune infiltration-related features in the TCGA cohort. BCR: B-cell receptor; LYM: lymphocyte; Mut: mutation; SNV: single nucleotide variant; TCR: T-cell receptor; TIL: tumor-infiltrating lymphocyte. (b) Gene set enrichment analysis (GSEA) comparing high- and low-BTK groups for three cohorts across whole cohorts or by subtypes. Immune-related Hallmark gene sets are examined with GSEA software. NES: normalized enrichment score; FDR: false discovery rate.
Figure 4. Single cell sequencing and spatial transcriptomic analyses of BTK expressing cells in breast cancer. (a) Heatmaps and dot plots represent BTK expression patterns across various cell types in SCP1039 and SCP1106 single cell sequencing cohorts. PVL: perivascular-like cells; CAF: cancer-associated fibroblast. The dot size corresponds to the percentage of cells expressing BTK, while the color intensity represents the average expression level of BTK within those cell populations. (b) Heatmaps and dot plots displaying the expression of BTK gene in various cell populations, including tumor cells, T cells, plasma cells, myeloid, fibroblast, endothelial and B cells. The dot size corresponds to the percentage of cells expressing BTK, while the color intensity represents the average expression level of BTK within those cell populations. (c) Spatial mapping of BTK gene expression and major cell types in human breast cancer tissue. The image displays data from Visium HD gene expression library generated from a human breast tissue sample (fixed frozen) with invasive ductal carcinoma, sourced from a 10x Genomics webpage. The left panel shows the expression levels of the BTK gene, color-coded from low (purple/blue) to high (yellow/red). The right panel shows the spatial distribution of annotated major cell types, including B cells, endothelial cells, fibroblasts, myeloid cells, T cells, and tumor cells. Data were binned to an 8 × 8 µm resolution for visualization and analysis.
Figure 5. Association of tumor BTK expression with immune cell composition and with MDSC gene expression signatures. Immune cell composition was estimated from tumor transcriptomes with xCell algorithm and compared between tumor groups with low and high BTK expression, using median value cut-off, for three cohorts. Tukey boxplots are shown for various immune cell-types and MDSC gene expression signatures, including Wang, Alshetaiwi, Cristecu granulocytic MDSC (gMDSC), Cristecu monocytic MDSC (mMDSC), and Kobayashi. Mann-Whitney U test was used to determine P values. MDSCs: myeloid derived suppressor cells; Cyt: cytolytic activity score; DC: dendritic cell; NK: natural killer cell; Th: T helper cell.
Figure 6. Association of pre-treatment tumor BTK expression with response to neoadjuvant chemotherapy. Tukey boxplots of BTK expression are shown for patient groups with either complete response (CR) or residual disease (RD) after neoadjuvant chemotherapy for different cancer subtypes. Mann-Whitney U test: *P < 0.05, **P < 0.005, ns > 0.05.
Figure 7. Association of pre-treatment tumor BTK expression with immune checkpoint molecule gene expressions, with response to immunotherapy or chemotherapy, with immune cell infiltrations and with immune-related gene signatures in the I-SPY2 cohort. (a) PD-1 or PD-L1 expressions by low and high BTK expressions, using median value as cut-off, for TCGA, METABRIC, SCAN-B, and I-SPY2 cohorts by subtypes. (b) Tukey boxplots of BTK expression by pathological complete response (pCR) or residual disease (RD) after treatment (durvalumab/olaparib and chemotherapy, n = 71, left panel) or control (chemotherapy alone, n = 34, right panel) arms by subtypes in the I-SPY2 trial. The “ns” (not significant) Mann-Whitney U test P value > 0.05. (c) Tukey boxplots display the relative expression of various immune cell populations in pretreatment tumor biopsies from patients in the I-SPY2 trial by subtypes (ER+/HER2- and TNBC). (d) Tukey boxplots display the relative expression levels of 12 immune-related gene signatures in tumors from I-SPY2 trial. The “ns” (not significant) and asterisks above the plots indicate the statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001.