Drug-resistance and quiescence of Pre-PCs in vivo. (A) Frequencies of BM clonotypic cells at diagnosis (D), remission (Rem), and relapse (Rel). (B) Frequency (top panels) and fold change (bottom panels) of clonotypic fractions in 8 patients achieving clinical remission after treatment. Top panels: Absolute frequency changes in the clonotypic fractions. Bottom panel: Relative changes in the frequency of the clonotypic cells. (C) The ratio of PC/Pre-PC fold reduction (median, 10.3; range, 4.4-332; P = .008) as estimated in each patient from panel C. (D) Cell cycle analysis after DAPI staining and multiparameter flow cytometry of BM cells shows that a significantly lower fraction of Pre-PCs than PCs are in S phase. An example and cumulative data from 7 patients are shown. (E) Left: Flow cytometry histograms showing rhodamine 123 dye exclusion by myeloma Pre-PCs and PCs compared with PBLs, naive, and memory B cells. Unstained PCs are shown as a negative control. PCs and Pre-PCs retain comparable levels of rhodamine 123 as assessed by MFI. Right: P-glycoprotein (ABCC1) and ABCG2 are not expressed in Pre-PCs or PCs as assessed by flow cytometry in BM samples. Representative of 10 patient samples. (F) A model of clonotypic hierarchy and myeloma-propagating activity. Memory B cells are at the apex of the clonotypic hierarchy in MM. However, myeloma-propagating activity is detected in the terminally differentiated B lineage cell that through an epigenetic, bidirectional transition can assume the morphologically and immunophenotypically distinct states of Pre-PCs and PCs. In most patients, the equilibrium of Pre-PC to PC transition favors PCs. Although both are enriched in myeloma-propagating activity, Pre-PCs are relatively more quiescent and treatment-resistant than PCs and in vivo are preferentially present in spleen and liver, whereas PCs are the dominant population in BM.