Fig. 2

Analysis of biomarkers expression and spatial distribution comparing bone marrow (BM) and extramedullary disease (EMD). A-B: Visualization of biomarker expressions specific to disease biology (BCL-2, CD56, CD200, EZH2, Ki-67 and CD38) and plasma cells (PCs) expression levels as strip-violin plot after segmentation and annotation in the selected region of interest (ROI) of BM#5 sample. Expression levels of biomarkers between BM PCs and EMD PCs were statistically different (p-value < 0.0001, Mann-Whitney test). C-D: Visualization of biomarker expressions specific to disease biology (BCL-2, CD56, CD200, EZH2, Ki-67 and CD38) and expression levels as strip-violin plot after segmentation and annotation in the selected region of interest (ROI) of EMD#5 sample. Expression levels of biomarkers between BM PCs and EMD PCs were statistically different (p-value < 0.0001, Mann-Whitney test). E: Table summarizing results of statistical analysis regarding data on CD38 expression in a cohort of 5 patients after cell segmentation and PCs annotation: median value of single PCs fluorescence intensity is reported for paired BM and EMD sample. Multiple t-tests were performed and revealed statistically significant differences between samples (p-value < 0.0001). F-G: Visualization by MICS technology of CD38 (light blue), BCL-2 (red) and Ki 67 (yellow) markers in MM#5 paired samples. H-O: Immunohistochemistry validation of CD38, BCL-2 and Ki 67 expression both in BM#5 (H-L-N) and EMD#5 (I-M-O). P-Q: Cell type assortativity in neighbourhood graphs for MM#5 paired samples. Heatmaps show the fraction of links connecting all possible cell type pairs, indicating cell type mixing. R-S: Butterfly graph comparing PCs distance from CD3⁺ T-cells (R) and M2-like cells (S) in representative MM#5 paired samples. The percentage of PCs in the BM (left/blue) and EMD (right/purple) are simultaneously shown at selected distance ranges. Spatial analysis to extract distance values was performed after segmentation and annotation of PCs, T-cells and M2-like cells in each sample. Difference was confirmed as statistically significant (p < 0.0001, Mann-Whitney test)