MM rBM has an abnormal tissue architecture and exhibits clonal expansion of MM cells with chromosomal abnormalities. (A) BMCs from normal donors (n = 5) and MM patients (n = 10) were grown in rBM for 21 days followed by assessment of the overall culture architecture by brightfield microscopy (top, original magnification 50×; bottom, original magnification 200×). Cells localized to “tracks” are TRAP⁺ osteoclasts (not shown). Representative images are shown with the plane of focus set to the bottom of the plate representing reconstructed endosteum. (B) BM cells (n = 16 patients) were grown in rBM for the indicated number of days, and the extent of the malignant outgrowth of the MM clone was measured by RQ-PCR using patient specific primers. Each malignant cell has only one copy of the IgH VDJ rearranged template; RQ-PCR determines the number of rearranged IgH VDJ templates compared with a β2-microglobulin standard curve and an IgH VDJ positive control curve. Percent clonal cells corresponds to the percentage clonal VDJ templates present in the sample normalized to the β2-microglobulin gene (*P = .006, **P = .001, ***P = .002, ****P = .003). Error bars represent SEM. (C) Absolute numbers of BMCs and PCs, which were originally placed into and recovered from rBM (mean values for 3 different patients). (D) FISH analysis of cells harvested from 3-D culture. Cells from 3-D cultures were screened for the chromosomal abnormalities (marked with arrows, and percentage plasma cells with each abnormality stated at the top left) detected in preculture BM PCs, using cytospins from the matching D21 rBM (n = 5 patients) stained by interphase FISH. Abnormal cells are indicated by arrows. (Left panel) PCs with a deletion of 13q34 locus (1 green signal) and a normal cell (2 green signals). (Middle panel) PCs with an amplification of 1q21 (3 red signals) and a normal cell (2 red signals). (Right panel) PCs with t(11;14) translocation with 2 derivative chromosomes (2 yellow signals), 1 normal chromosome 11 (1 red signal), and 1 normal chromosome 14 (1 green signal; original magnification 630×). For additional image acquisition information, see “Microscopy” and “Fluorescent in situ hybridization.”