Representative ROI selection and segmentation process. Using fluorescently labeled morphology probes, ROIs were selected to capture target cells while avoiding areas of histologic and staining artifacts. Segments containing specific cell types were hierarchically defined and are shown in the last column as: orange (CD138⁺ plasma cells), teal (CD3⁺ T cells), pink (CD45⁺ cells other than CD138⁺ plasma cells and CD3⁺ T cells), and green (all other cells not previously selected) zones. The top 2 rows show representative ROIs from pretreatment and posttreatment biopsy samples of a patient from arm 5 (SDd). The pretreatment sample shows plasma cell aggregates, whereas the posttreatment sample, taken during response, did not have any evidence of residual myeloma. The bottom 2 rows show representative ROIs from pretreatment and posttreatment biopsy samples of a patient from arm 6 (SKd). Unlike most patients who showed rare to no plasma cell involvement at the end of treatment, this patient showed persistently high disease burden with sheets of plasma cells at both time points. Scale bar, 100 μm.