Persistent preswitch clonotypic myeloma cells correlate with decreased survival: evidence for isotype switching within the myeloma clone.

Multiple myeloma (MM) is identified by unique immunoglobulin heavy chain (IgH) variable diversity joining region gene rearrangements, termed clonotypic, and an M protein termed the "clinical" isotype. Transcripts encoding clonotypic pre and postswitch IgH isotypes were identified in MM peripheral blood mononuclear cells (PBMCs), bone marrow (BM), and mobilized blood. For 29 patients, 38 BM, 17 mobilized blood, and 334 sequential PBMC samples were analyzed at diagnosis, before and after transplantation for 2 to 107 months. The clinical clonotypic isotype was readily detectable and persisted throughout treatment. Eighty-two percent of BM and 38% of PBMC samples also expressed nonclinical clonotypic isotypes. Clonotypic immunoglobulin M (IgM) was detectable in 68% of BM and 25% of PBMC samples. Nonclinical clonotypic isotypes were detected in 41% of mobilized blood samples, but clonotypic IgM was detected in only 12%. Patients with persistent clonotypic IgM expression had adverse prognostic features at diagnosis (lower hemoglobin, higher beta(2)-microglobulin) and higher numbers of BM plasma cells compared with patients with infrequent/absent clonotypic IgM. Patients with persistent clonotypic IgM expression had significantly poorer survival than patients with infrequent IgM expression (P <.0001). In a multivariate analysis, persistent clonotypic IgM expression in the blood correlated independently with poor survival (P =.01). In nonobese diabetic severe combined immunodeficiency mice, xenografted MM cells expressed clinical and nonclinical postswitch clonotypic isotypes. MM expressing clonotypic IgM engrafted both primary and secondary mice, indicating their persistence within the murine BM. This study demonstrates that MM clonotypic cells expressing preswitch transcripts are tied to disease burden and outcomes. Because MM pathology involves postswitch plasma cells, this raises the possibility that IgH isotype switching in MM may accompany worsening disease.

Warfarin monitoring in patients with anticardiolipin antibodies, but without lupus anticoagulants.

Misleading international normalized ratios (INR) may be obtained from anticoagulated patients with lupus anticoagulants (LA). As a result, special precautions have been suggested for oral anticoagulant dosing. It is possible that plasma from subjects with an anticardiolipin antibody (ACA) without an LA could also affect the INR. We have studied nine such subjects who were anticoagulated and compared them with 11subjects who had neither antibody. The INR was performed, using local specific ISIs, with 11 different thromboplastins. No substantial difference was seen between the ACA-positive and ACA-negative patients, for either individual thromboplastins or patients. We similarly measured INRs in eight nonanticoagulated patients with ACAs. No effect on the INR results was observed. Thus, in these anticoagulated and nonanticoagulated patients we detected no evidence of any effect of an ACA on the INR.

Intraoperative heparin in addition to postoperative low-molecular-weight heparin for thromboprophylaxis in total knee replacement.

The administration of heparin during operation has been reported to enhance the efficacy of thromboprophylaxis in patients undergoing total hip replacement. We have performed a small pilot study in which intraoperative doses of heparin were given in addition to the usual postoperative thromboprophylaxis with enoxaparin in 32 patients undergoing total knee replacement. The primary endpoint was deep-vein thrombosis (DVT) as demonstrated by bilateral venography on 6 +/- 2 days after operation. Sixteen patients developed DVT; in two the thrombosis was proximal as well as distal and in one the occurrence was bilateral. There was one major haemorrhage. These results are similar to those obtained with the use of postoperative thromboprophylaxis with enoxaparin alone. They do not provide support for the initiation of a larger randomised trial of this approach to management.

In multiple myeloma, circulating hyperdiploid B cells have clonotypic immunoglobulin heavy chain rearrangements and may mediate spread of disease.

DNA aneuploidy characterizes a proportion of malignant bone marrow (BM)-localized plasma cells in multiple myeloma (MM). This analysis shows that for most MM patients, circulating clonotypic B cells in MM are also hyperdiploid. Although all normal B cells and some malignant B cells are diploid, hyperdiploidy is likely to be exclusive to those that are malignant. Hyperdiploid MM B cells express CD34 and have clonotypic IgH transcripts, confirming them as part of the malignant clone. For MM, 92% (70/76) of patients had a DNA hyperdiploid subset [5-30% of peripheral blood mononuclear cells (PBMCs)] of CD19+ B cells. All CD19+ PBMCs in MM expressed CD19 and IgH variable diversity joining (VDJ) transcripts, confirming them as B cells. DNA aneuploid cells were undetectable in T or B lymphocytes from normal blood, spleen or thymus, or in blood from patients with B chronic lymphocytic leukemia. In MM, untreated patients had the highest DNA index (1.12). DNA hyperdiploid PBMCs were most frequent among untreated patients and were significantly reduced after chemotherapy. Diploid B cells were significantly more frequent after chemotherapy than at diagnosis. Of the hyperdiploid PBMCs, 81 +/- 3% expressed CD34 and CD19. In contrast to circulating CD34+ B cells, CD34- B cells in MM are diploid. In MM, unlike hyperdiploid PBMC B cells, hyperdiploid BM plasma cells lack both CD34 and CD19, suggesting that loss of CD34 correlates with differentiation and BM anchoring. In situ reverse transcription-PCR of the CD34+ (hyperdiploid) and CD34- (diploid) PBMC B-cell subsets was performed using patient-specific primers to amplify clonotypic IgH VDJ transcripts. Confirming previous work, CD34+ hyperdiploid MM PBMCs were clonotypic (86 +/- 5%). In contrast, CD34- diploid MM PBMCs had few monoclonal cells (4.8 +/- 2%). The lack of hyperdiploidy, together with the relative absence of cells having clonotypic transcripts, suggests these polyclonal CD34- B cells are normal. After culture in colchicine to arrest mitosis, hyperdiploid B cells were reduced and MM B cells accumulated in a diploid G2-M, suggesting that hyperdiploid in MM may represent a transient S-phase arrest rather than an aneuploid G0 phase. The DNA hyperdiploidy of CD34+ clonotypic B cells suggests these cells may be clinically important constituents of the myeloma clone and that they may play a direct role in the spread of myeloma.

Splenectomy during partial remission in thrombotic thrombocytopenic purpura with prolonged plasma exchange dependency.

Some patients with thrombotic thrombocytopenic purpura (TTP) remain plasma-exchange-dependent for prolonged periods of time. This exposes patients to risk, uses substantial resources, and requires prolonged hospitalization. We have splenectomized 7 such patients following 25-42 plasma exchanges while patients were in partial remission only and were clinically stable. In 6 patients, including 1 with TTP secondary to mitomycin C, thrombocytopenia promptly resolved. Relapse has not occurred during 18 or more months of observation. The seventh patient did not respond. We conclude that splenectomy should be considered as an alternative to continued plasma-exchange therapy in such patients.

Potential role for hyaluronan and the hyaluronan receptor RHAMM in mobilization and trafficking of hematopoietic progenitor cells.

Although the mechanism(s) underlying mobilization of hematopoietic progenitor cells (HPCs) is unknown, detachment from the bone marrow (BM) microenvironment and motility are likely to play a role. This work analyzes the motile behavior of HPCs and the receptors involved. CD34(+)45(lo/med)Scatterlo/med HPCs from granulocyte colony-stimulating factor (G-CSF)-mobilized blood and mobilized BM were compared with steady-state BM for their ability to bind hyaluronan (HA), their expression of the HA receptors RHAMM and CD44, and their motogenic behavior. Although RHAMM and CD44 are expressed by mobilized blood HPCs, function blocking monoclonal antibodies (MoAbs) identified RHAMM as a major HA binding receptor, with a less consistent participation by CD44. Permeabilization of mobilized blood HPCs showed a pool of intracellular (ic) RHAMM and a smaller pool of icCD44. In contrast, steady-state BM HPCs have significantly larger pools of icRHAMM and icCD44. Also, in contrast to mobilized blood HPCs, for steady-state BM HPCs, MoAbs to RHAMM and CD44 act as agonists to upregulate HA binding. The comparison between mobilized and steady-state BM HPCs suggests that G-CSF mobilization is associated with depletion of intracellular stores of HA receptors and modulates HA receptor usage. To confirm that mobilization alters the HA receptor distribution and usage by HPCs, samples of BM were collected at the peak of G-CSF mobilization in parallel with mobilized blood samples. HA receptor distribution of mobilized BM HPCs was closely matched with mobilized blood HPCs and different from steady-state BM HPCs. Mobilized BM HPCs had lower pools of icHA receptors, similar to those of mobilized blood HPCs. Treatment of mobilized BM HPCs with anti-RHAMM MoAb decreased HA binding, in contrast to steady-state BM HPCs. Thus, G-CSF mobilization may stimulate an autocrine stimulatory loop for HPCs in which HA interacts with basal levels of RHAMM and/or CD44 to stimulate receptor recycling. Consistent with this, treatment of HPCs with azide, nystatin, or cytochalasin B increased HA binding, implicating an energy-dependent process involving lipid rafts and the cytoskeleton. Of the sorted HPCs, 66% were adherent and 27% were motile on fibronectin plus HA. HPC adherence was inhibited by MoAbs to beta1 integrin and CD44, but not to RHAMM, whereas HPC motility was inhibited by MoAb to RHAMM and beta1 integrin, but not to CD44. This finding suggests that RHAMM and CD44 play reciprocal roles in adhesion and motility by HPCs. The G-CSF-associated alterations in RHAMM distribution and the RHAMM-dependent motility of HPCs suggest a potential role for HA and RHAMM in trafficking of HPCs and the possible use of HA as a mobilizing agent in vivo.

Overexpression of the receptor for hyaluronan-mediated motility (RHAMM) characterizes the malignant clone in multiple myeloma: identification of three distinct RHAMM variants.

The receptor for hyaluronan (HA)-mediated motility (RHAMM) controls motility by malignant cells in myeloma and is abnormally expressed on the surface of most malignant B and plasma cells in blood or bone marrow (BM) of patients with multiple myeloma (MM). RHAMM cDNA was cloned and sequenced from the malignant B and plasma cells comprising the myeloma B lineage hierarchy. Three distinct RHAMM gene products, RHAMMFL, RHAMM-48, and RHAMM-147, were cloned from MM B and plasma cells. RHAMMFL was 99% homologous to the published sequence of RHAMM. RHAMM-48 and RHAMM-147 variants align with RHAMMFL, but are characterized by sequence deletions of 48 bp (16 amino acids [aa]) and 147 bp (49 aa), respectively. The relative frequency of these RHAMM transcripts in MM plasma cells was determined by cloning of reverse-transcriptase polymerase chain reaction (RT-PCR) products amplified from MM plasma cells. Of 115 randomly picked clones, 49% were RHAMMFL, 47% were RHAMM-48, and 4% were RHAMM-147. All of the detected RHAMM variants contain exon 4, which is alternatively spliced in murine RHAMM, and had only a single copy of the exon 8 repeat sequence detected in murine RHAMM. RT-PCR analysis of sorted blood or BM cells from 22 MM patients showed that overexpression of RHAMM variants is characteristic of MM B cells and BM plasma cells in all patients tested. RHAMM also appeared to be overexpressed in B lymphoma and B-chronic lymphocytic leukemia (CLL) cells. In B cells from normal donors, RHAMMFL was only weakly detectable in resting B cells from five of eight normal donors or in chronically activated B cells from three patients with Crohn's disease. RHAMM-48 was detectable in B cells from one of eight normal donors, but was undetectable in B cells of three donors with Crohn's disease. RHAMM-147 was undetectable in normal and Crohn's disease B cells. In situ RT-PCR was used to determine the number of individual cells with aggregate RHAMM transcripts. For six patients, 29% of BM plasma cells and 12% of MM B cells had detectable RHAMM transcripts, while for five normal donors, only 1. 2% of B cells expressed RHAMM transcripts. This work suggests that RHAMMFL, RHAMM-48, and RHAMM-147 splice variants are overexpressed in MM and other B lymphocyte malignancies relative to resting or in vivo-activated B cells, raising the possibility that RHAMM and its variants may contribute to the malignant process in B-cell malignancies such as lymphoma, CLL, and MM.

Drug resistance in multiple myeloma: novel therapeutic targets within the malignant clone.

Myeloma is incurable because the malignant stem cell is not eradicated by treatment. Thus, identification of the malignant hierarchy of B lineage cells in myeloma is required to identify potentially generative components and to evaluate their drug resistance properties. BM plasma cells are usually depleted by chemotherapy, but clonotypic B cells survive melphalan/prednisone as well as combination chemotherapy. In vitro, circulating and bone marrow-localized myeloma plasma cells show defective drug export, despite their phenotypic expression of P-glycoprotein, the mdr1 gene product. In contrast to plasma cells, circulating myeloma clonotypic B cells exhibit very efficient drug export. This suggests that circulating clonotypic MM B cells comprise a reservoir of drug resistant disease in myeloma although their stem cell potential remains to be confirmed. The malignant clone in each myeloma patient is defined by a unique IgH VDJ gene rearrangement. Using methods that exclude the possibility that a frequent but non-malignant clone has inadvertently been identified, and after confirming that the sequence identified is expressed by nearly all bone marrow plasma cells, we show that the drug resistant set of myeloma B cells is clonally related to the malignant plasma cells in myeloma. Clonotypic MM B cells survive chemotherapy, persist during clinically defined "minimal residual disease" and remain after autologous transplantation. Thus their malignant status is an important consideration. If malignant, they must be considered in the design of therapy. If non-malignant, they would be expected to have minimal impact on the disease process. A variety of evidence provides strong support for the view that clonotypic drug resistant B cells are malignant and may include the generative compartment of myeloma. The P-gp+ set of clonotypic B cells is extensively DNA aneuploid, an attribute of malignancy. All clonotypic B cells overexpress RHAMM, a novel oncogene involved in malignant spread. Finally, the population of clonotypic B cells lacks intraclonal heterogeneity. Since intraclonal heterogeneity is driven by the response to antigens, its absence in these cells indicates that they are no longer antigen-responsive. Since antigen-independent clonal expansion is characteristic of lymphoid malignancies, these observations provide further proof that clonotypic B cells in myeloma are malignant. Thus, the drug resistance of these cells is highly relevant to understanding why myeloma remains incurable despite the initial chemosensitivity of most bone marrow plasma cells.

A high frequency of circulating B cells share clonotypic Ig heavy-chain VDJ rearrangements with autologous bone marrow plasma cells in multiple myeloma, as measured by single-cell and in situ reverse transcriptase-polymerase chain reaction.

In multiple myeloma (MM), the VDJ rearrangement of the immunoglobulin heavy chain expressed by MM plasma cells provides a unique clonotypic marker. Although clonotypic MM cells have been found in the circulation, their number has been controversial. Our objective was to provide direct evidence, using single-cell assays, for the frequency of clonotypic cells in blood of 18 MM patients, and to confirm their identity as B cells. The clonotypic Ig heavy-chain (IgH) VDJ was determined from single plasma cells using consensus reverse transcriptase-polymerase chain reaction (RT-PCR), subcloning, and sequencing. For all patients, using patient-specific primers, clonotypic transcripts were amplified from 10 or more individual plasma cells. Using in situ RT-PCR, for all patients greater than 80% of plasma cells were found to be clonotypic. Three separate methods, RT-PCR, single-cell RT-PCR, and in situ RT-PCR, were used to analyze clonotypic cells in peripheral blood mononuclear cells (PBMC) from MM patients. Sequencing of the IgH transcripts expressed by individual cells obtained by limiting dilution of freshly isolated PBMC from a MM patient showed that all B cells expressed an identical CDR3. This intraclonal homogeneity indicates an escape from antigenic-selection, characteristic of malignant B cells. For this patient, the frequency of clonotypic PBMC, about 25%, was comparable to the number of PBMC B cells (34%). Because the PBMC included less than 1% plasma cells, virtually all clonotypic PBMC must be B cells. Using single-cell RT-PCR, clonotypic IgH transcripts were identified in individual sorted B cells from blood. To accurately quantify the number of clonotypic B cells, sorted B cells derived from 18 MM patients (36 samples) and 18 healthy donors (53 samples) were analyzed using in situ RT-PCR with patient-specific primers. Clonotypic transcripts were not detectable among normal B cells. For the 18 MM patients, a mean of 66% +/- 4% (SE) of blood B cells were clonotypic (range, 9% to 95%), with mean absolute number of 0.15 +/- .02 x 10(9)/L blood. Over time in individual patients, conventional chemotherapy transiently decreased circulating clonotypic B cells. Their numbers were increased in granulocyte colony-stimulating factor (G-CSF)- mobilized blood of one patient. However, clonotypic B cells of a one patient became undetectable after allogeneic transplant, correlating with complete remission. Although contributions to MM spread and progression is likely, their malignant status and impact has yet to be clarified. Their high frequency in the blood, and their resistence to conventional chemotherapy suggests that the number of circulating clonotypic cells should be clinically monitored, and that therapeutic targeting of these B cells may benefit myeloma patients.

Es nucleoside transporter content of acute leukemia cells: role in cell sensitivity to cytarabine (araC).

Nucleoside analogs are important components of treatment regimens for acute leukemia in adults. Plasma membrane permeation of the nucleoside analog molecules, the initial event in the cellular conversion of nucleosides to active agents, is mediated by nucleoside-specific membrane transporters. The widely-expressed es nucleoside transporter accepts as substrates diverse nucleoside analogs, including cytarabine (araC), 2-chlorodeoxyadenosine, and fludarabine. The cellular content of es transporter sites has been measured in blasts from patients with acute lymphoblastic leukemia and acute myelogenous leukemia, by a sensitive, quantitative flow cytometry assay that employs the tightly-bound es ligand, SAENTA fluorescein. Values for es transporter expression varied ten-fold among samples from patients with acute myelogenous leukemia. In this article, we review current findings that document, in confocal fluorescence microscopy images and in flow cytometry assays of SAENTA fluorescein-stained cells, the patient-to-patient variance of es transporter expression in leukemic blasts from patients. Our data show a correlation between the expression of es transporters and the in vitro sensitivity to nucleoside drugs of blasts from acute leukemia patients. These findings show that the flow cytometry assay of es expression provides a facile means of predicting resistance of leukemia cells to the cytotoxicity of araC and other nucleosides.

Platelet activation by a novel solid-phase agonist: effects of VWF immobilized on polystyrene beads.

The interaction between platelets stirred in suspension and VWF immobilized on polystyrene beads was studied. Platelets aggregated and released ATP in response to stirring with VWF beads. Closer examination of the interaction using transmission electron microscopy revealed that the platelets did not simply aggregate with one another but initially adhered to the beads and spread. Platelets in suspension then bound to the bead-adherent platelets forming layers of platelets associated with each bead. The VWF bead-induced platelet activation was completely inhibited by addition of monoclonal antibody (mAb) to GPIb or GPIIb/IIIa. In addition, the activation response was inhibited in the presence of aspirin, indomethacin or the thromboxane receptor antagonist BM13.177, demonstrating a dependence on an intact cyclo-oxygenase pathway. Platelet function studies were carried out on 30 patients with a history of mild bleeding using conventional optical aggregation and VWF bead-induced platelet activation. 12 patients were abnormal by conventional optical aggregometry, whereas 27 patients showed depressed ATP release in response to VWF beads. The results suggest that easily-bruised patients may have a platelet function defect rather than a vascular-based abnormality and that VWF bead-induced platelet activation is a more sensitive test for detecting platelet dysfunction.

Adhesion of multiple myeloma peripheral blood B cells to bone marrow fibroblasts: a requirement for CD44 and alpha4beta7.

We have earlier described the presence of phenotypically unusual monoclonal B cells within the peripheral blood of multiple myeloma (MM) patients. To determine the biological properties of these B cells as compared to B cells from normal donors, we investigated the potential of CD19+ MM blood B cells to adhere to endothelial cell and bone marrow (BM)-fibroblast monolayers. We find that 30-60% of freshly isolated CD19+ MM blood B cells adhere to endothelial cell monolayers, and 50-80% adhere to BM fibroblast monolayers. The adhesion of MM blood B cells to either monolayer was not increased by in vitro activation, suggesting that these cells were activated in vivo. In contrast, fewer than 10% of CD19+ B cells from peripheral blood of normal donors adhered. Function-blocking monoclonal antibodies (mAbs) were used to determine which adhesion receptors were involved in CD19+ MM blood B cell interaction with BM fibroblasts. mAbs against very late antigen 4, the beta7-integrin subunit, and CD44, but not mAbs against very late antigen 5 and beta1, inhibited adhesion 61, 50, and 30%, respectively. The lack of inhibition with mAbs against beta1 implicates alpha4beta7 but not alpha4beta1 in adhesion of CD19+ MM blood B cells. To determine the alpha4beta7 ligand that mediated MM blood B cell adhesion, mAbs against vascular cellular adhesion molecule 1 and fibronectin, as well as CS1 and RGD peptides, were used as inhibitors. These were unable to reduce the adhesion of CD19+ MM blood B cells to BM fibroblasts, suggesting that fibronectin and vascular cellular adhesion molecule 1 are not involved in adhesion. Also, adhesion of MM blood B cells to mucosal addressin cell adhesion molecule 1-transfected Chinese hamster ovary cells was not enhanced compared to control-transfected Chinese hamster ovary cells, suggesting that mucosal addressin cell adhesion molecule 1 was not promoting adhesion of these cells. These data implicate CD44:HA interactions, as well as alpha4beta7 and an as yet unidentified ligand in the adhesion of in vivo activated MM blood B cell adhesion to BM fibroblasts. The adhesion properties of MM CD19+ B cells distinguishes them from normal B cells. Although the malignant status of these cells is as yet undefined, their adhesion properties implicate MM blood B cells in migratory spread of the disease.

Circulating clonotypic B cells in the biology of multiple myeloma: speculations on the origin of myeloma.

The population of circulating B cells in myeloma patients includes an apparently large but variable subset with the IgH VDJ rearrangement diagnostic for the malignant clone of plasma cells in individual myeloma patients. Although the biological significance is at present unknown, it is likely that they include both malignant and non-malignant clonal relatives of the myeloma plasma cells. This article presents speculations on the significance of these cells in the origin of myeloma and the relationship between monoclonal gammopathy of undetermined significance (MGUS) and frank myeloma. MGUS appears to represent the establishment of clonal dominance probably by a chronically antigen-stimulated B cell clone. It seems likely that malignant transformation event(s) occurring in a clonal daughter cell give rise to myeloma. If correct, this implies that in a myeloma patient, non-malignant antigen-responsive B cells expressing the patient-specific IgH rearrangement coexist in the circulation and probably all lymphoid tissues, with their malignant antigen-independent relatives. However, the significance one attributes to the clonotypic B cells detected in the blood of myeloma patients depends in part on the view one takes of the progression from MGUS to myeloma. An alternative perspective is that MGUS represents a dormant state of malignancy held in check by controlled apoptosis, arrested cell cycling, and/or by immunoregulatory networks. Although lacking in experimental support, if this interpretation were correct, myeloma would occur when the regulatory mechanisms fail, allowing uncontrolled malignant cell renewal. This alternative view would imply that the majority of circulating clonotypic B cells might be malignant. Thus, an analysis of the biology of these clonotypic circulating B cells, with an emphasis on measures of malignancy, is likely to shed considerable light on the events underlying myeloma genesis, progression and spread.

Hyaluronan-dependent motility of B cells and leukemic plasma cells in blood, but not of bone marrow plasma cells, in multiple myeloma: alternate use of receptor for hyaluronan-mediated motility (RHAMM) and CD44.

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.

Bleeding risks, risk factors and management of bleeding complications after treatment with anticoagulants, specific antithrombins, thrombolytics IIb-IIIa receptor blockers.

Assessment of the risks of new antithrombotic therapies is best undertaken by evaluating risk factors for bleeding in individual patients, and risks associated with specific antithrombotic agents. This forms the basis for the development of a management strategy for major bleeding complications. Patient-related risk factors for bleeding with oral anticoagulants include: trauma, invasive procedures, history of bleeding disorder, high anticoagulant intensity, concomitant use of antiplatelet drugs, presence of underlying severe disease, advanced age, and prior history of cerebrovascular accident, or gastrointestinal bleeding. Weight-adjusted and other nomograms are more successful in achieving a balance between therapeutic effect and safety with intravenous heparin. The most important complication of thrombolytic therapy is intracranial haemorrhage, and the risks increase with age > 65 years, weight under 70 k, hypertension on admission and the use of tissue plasminogen activator: this profile is helpful in assessing risk-benefit ratio amongst individual patients. Recent experience with the experimental use of antithrombin agents such as hirudin, indicates a delicate dose-response relationship as it relates to the risk of cerebral haemorrhage, when used in conjunction with thrombolytic agents. A definitive answer regarding the role of hirudin and the balance of safety and efficacy awaits completion of ongoing trials. Novel IIb/IIIa platelet inhibitors appear to offer a significant therapeutic advance: major bleeding is variable and depends in part on the use of concomitant procedures, and heparin therapy. It is important to identify the source and severity of bleeding with the use of antithrombotic therapy and its haemodynamic consequences in constructing a management plan. Well developed treatment algorithms for patients with severe bleeding exist, and although laboratory testing may be helpful, it is on balance of marginal benefit since patients usually require urgent therapy. Future investigation promises more readily available, rapid and specific laboratory testing, and newer antithrombotic agents that are easier to administer and monitor. Molecular targeting with fusion proteins that attract to a specific antigen, thereby delivering more effective and safe therapy, offer new promise.

In multiple myeloma, clonotypic B lymphocytes are detectable among CD19+ peripheral blood cells expressing CD38, CD56, and monotypic Ig light chain.

Multiple myeloma (MM) is characterized by a plasma cell infiltrate of the bone marrow (BM). However, late-stage monotypic B cells have been detected in the blood. This work analyzes the effects of clinical treatment on late stage CD19+ B cells present in 752 blood samples from 152 MM patients. MM patients have 2 to 8 times as many circulating CD19+ cells as do normal donors. Analysis of the Ig heavy chain (IgH) gene rearrangements using polymerase chain reaction indicates that the CD19+ population includes cells sharing the same clonotypic CDR3 region as is detected in the BM plasma cells, for patients analyzed during chemotherapy or in relapse. They are also monotypic as defined by their cytoplasmic or surface expression of Ig kappa or lambda light chain. The light chain restriction is the same as that of the BM plasma cells. Individual patients observed over 1- to 2-year periods exhibit considerable variation in the number of B cells present in blood; this number does not correlate with the concentration of serum monoclonal Ig. The monoclonal blood CD19+ cells are not eliminated by any of the chemotherapy regimens analyzed and remain at high levels during transient remissions. Patients in the progressive phase of disease or in relapse have significantly higher numbers of B cells than do patients in transient remission or untreated patients. During periods when the quantity of blood B cells approaches normal, phenotypically their quality is highly abnormal, with physical and phenotypic heterogeneity. Most B cells express CD45R0, a high density of CD38, and CD56 characteristic of late-stage B or pre-plasma cells. CD38hi blood B cells had a cyclical presence. We conclude that monoclonal B cells in the blood of myeloma patient populations include drug-resistant reservoirs of clonotypic cells that may underlie relapse.