The peripheral blood lymphocytes from 42 patients with multiple myeloma (MM) and 13 patients with monoclonal gammopathy of undetermined significance (MGUS) were studied by three-color immunofluorescence (IF) using antibodies directed to a broad range of B-cell markers (CD19, CD20, CD21, CD24), CALLA (CD10), PCA-1 (a plasma cell marker), and to the high and low molecular weight isoforms of the leukocyte common antigen, CD45RA (p205/220) and CD45RO (p 180). CD45RA is expressed on pre-B and B cells, and a transition from CD45RA to CD45RO defines differentiation towards plasma cells. Peripheral blood mononuclear cells (PBMC) from patients with myeloma included a large subset of B- lineage cells (mean of 39% to 45%) that were CALLA+ and PCA-1+ in all patients studied, including newly diagnosed patients and patients undergoing chemotherapy. Southern blot analysis indicated the presence of monoclonal Ig rearrangements in PBMC and a substantial reduction in the germ-line bands consistent with the presence of a large monoclonal B-cell subset. Avoidance of purification methods involving depletion of adherent cells was essential for detection of the abnormal B cells. Phenotypically, this abnormal B-cell population corresponded to late B or early pre-plasma cells (20% to 80% of PBMC), as defined by the concomitant expression of low densities of CD19 and CD20, moderate densities of CALLA and PCA-1, and strong expression of CD45RO on all B cells, with weakly coexpressed CD45RA on a small proportion. Heterogeneity in the expression of CD45RA and CD45RO within the abnormal B-cell population from any given patient suggested multiple differentiation stages. Abnormal B cells similar to those in MM were also detected in MGUS, although as a lower proportion of PBMC (26%). Abnormal B cells from patients with MGUS expressed predominantly the CD45RO isoform, but had a lower proportion of CALLA+ and PCA-1+ cells than were found on B cells from MM. This work indicates that the large subset of circulating monoclonal B lymphocytes from myeloma patients are at a late stage in B-cell differentiation, continuously progressing towards the plasma cell stage.

Migration through extracellular matrix is fundamental to malignant invasion. A receptor for hyaluronan-mediated motility (RHAMM) has previously been shown to play a fundamental role in locomotion of ras- transformed cells as well as functioning in signal transduction. Expression of RHAMM was characterized on B lymphocytes from normal and malignant lymphoid tissues using multiparameter phenotypic immunofluorescence analysis as well as functional analysis of its role in locomotion of malignant hairy cell leukemia B cells. RHAMM is not detectable on most normal B cells located in blood, spleen, or lymph node, but it is detectable on bone marrow and thymic B cells. Among B- cell malignancies, it is expressed on most terminally differentiated B cells from multiple myeloma bone marrows, is present on a subset of non- Hodgkin's lymphomas, and is absent on B chronic lymphocytic leukemia. Activation of peripheral blood B cells by Staphylococcus A cowan (SAC), but not by pokeweed mitogen, induced transient expression of RHAMM at day 3 of culture, suggesting RHAMM may be used by antigen-activated normal B cells. For malignant cells, expression of RHAMM increased on long-term culture of bone marrow plasma cells from multiple myeloma patients, indicating prolonged expression in contrast to the transient expression on SAC-activated normal B cells. Intriguingly, RHAMM was expressed on hairy leukemia cells located in spleen but absent from those in peripheral blood of the same patient. RHAMM, as expressed on splenic hairy cells, was a 58-Kd molecule that binds hyaluronan, is encoded by a 5.2-kb messenger RNA, and participates in locomotion by these cells. Hairy cells locomoted in response to hyaluronan at 4 mu per minute. Monoclonal antibody to RHAMM inhibited this locomotion almost completely as detected using video time-lapse cinemicrography. These observations are consistent with a role for RHAMM in malignant invasion and metastatic growth.

We investigated the ability of blood B cells, bone marrow (BM) plasma cells, and terminal leukemic plasma cells (T-PCL) from patients with multiple myeloma (MM) to migrate on extracellular matrix proteins. Hyaluronan (HA), but not collagen type I, collagen type IV, or laminin, promoted migration of MM blood B cells, as determined by time-lapse video microscopy. Between 13% and 20% of MM blood B cells migrated on HA with an average velocity of 19 micron/min, and greater than 75% of MM blood B cells exhibited vigorous cell movement and plasma membrane deformation, as did circulating T-PCL and extraskeletal plasma cells from patients with MM. In contrast, plasma cells obtained from BM of patients with MM lacked motility on all substrates tested and did not exhibit cell membrane protrusions or cellular deformation. MM blood B cells and MM plasma cells from all sources examined expressed the HA- binding receptors receptor for HA-mediated motility (RHAMM) and CD44. On circulating MM B cells, both RHAMM and CD44 participated in HA- binding, indicating their expression ex vivo in an activated conformation. In contrast, for the majority of BM plasma cells in the majority of patients with MM, expression of RHAMM or CD44 was not accompanied by HA binding. A minority of patients did have HA-binding BM plasma cells, involving both RHAMM and CD44, as evidenced by partial blocking with monoclonal antibodies (MoAbs) to RHAMM or to CD44. Despite HA binding by both RHAMM and CD44, migration of MM blood B cells on HA was inhibited by anti-RHAMM but not by anti-CD44 MoAbs, indicating that RHAMM but not CD44 mediates motility on HA. Thus, circulating B and plasma cells in MM exhibit RHAMM- and HA-dependent motile behavior indicative of migratory potential, while BM plasma cells are sessile. We speculate that a subset(s) of circulating B or plasma cells mediates malignant spread in myeloma.

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Multiple myeloma is basically an incurable cancer. Most patients respond initially to chemotherapy with reduction in bone marrow (BM) plasma cells and monoclonal Ig levels, but the disease nearly always recurs and becomes refractory to therapy. The objective of this study was to characterize the expression of the multidrug transport pump, P- glycoprotein 170 (P-gp), in myeloma. The great majority of B cells from peripheral blood mononuclear cells (PBMCs) in myeloma express P-gp, detected by the monoclonal antibody MRK-16. P-gp+ blood B cells exhibit extensive DNA hyperdiploidy, suggesting replicative abnormality characteristic of malignant growth. We speculate these represent a stem cell population in myeloma. The proportion of B cells expressing P-gp was comparable among untreated myeloma patients and those treated with chemotherapy, biologic response modifiers, or off treatment. Among BM cells, P-gp was absent or low in untreated myeloma patients but was expressed at high levels on BM cells from patients previously treated with chemotherapy. For untreated patients the majority of B/plasma cells expressing P-gp are located in PBMCs, not the BM cells. Flow cytometric analysis of rhodamine 123 dye efflux indicated a functional P-gp that was efficiently blocked by verapamil or cyclosporin A (CsA). Both the CD11bhi CD19+ B cells and the T cells in myeloma PBMCs had active CsA-inhibited dye efflux, but monocytes lacked the ability to efflux dye. Nearly all CD38hi plasma cells from myeloma BM cells retained dye, indicating their lack of a functional transport pump. Thus, PBMC B cells may be the predominant set of drug-resistant tumor cells. Myeloma PBMC B cells were cultured with Adriamycin with or without CsA and drug toxicity evaluated by the induction of apoptosis, using flow cytometry to quantitate DNA disruption. No apoptosis was detectable at 0.01 microgram/mL adriamycin, the in vivo steady-state level, with or without CsA. With 0.1 microgram adriamycin, no apoptosis was detectable in the absence of CsA, but with CsA, 66% of B cells initiated DNA disruption, whereas most T cells were spared. This work suggests that currently used drug dosages are too low to effect P-gp+ B- cell death, even in the presence of CsA. We suggest that blood B cells comprise a highly drug-resistant subset of the myeloma B lineage that escapes conventional chemotherapy and may underlie the almost uniform fatal relapse in myeloma patients.