Cost-effectiveness of lenalidomide plus dexamethasone vs. bortezomib plus melphalan and prednisone in transplant-ineligible U.S. patients with newly-diagnosed multiple myeloma.

OBJECTIVE: To conduct a cost-effectiveness assessment of lenalidomide plus dexamethasone (Rd) vs bortezomib plus melphalan and prednisone (VMP) as initial treatment for transplant-ineligible patients with newly-diagnosed multiple myeloma (MM), from a U.S. payer perspective. METHODS: A partitioned survival model was developed to estimate expected life-years (LYs), quality-adjusted LYs (QALYs), direct costs and incremental costs per QALY and LY gained associated with use of Rd vs VMP over a patient's lifetime. Information on the efficacy and safety of Rd and VMP was based on data from multinational phase III clinical trials and a network meta-analysis. Pre-progression direct costs included the costs of Rd and VMP, treatment of adverse events (including prophylaxis) and routine care and monitoring associated with MM. Post-progression direct costs included costs of subsequent treatment(s) and routine care and monitoring for progressive disease, all obtained from published literature and estimated from a U.S. payer perspective. Utilities were obtained from the aforementioned trials. Costs and outcomes were discounted at 3% annually. RESULTS: Relative to VMP, use of Rd was expected to result in an additional 2.22 LYs and 1.47 QALYs (discounted). Patients initiated with Rd were expected to incur an additional $78,977 in mean lifetime direct costs (discounted) vs those initiated with VMP. The incremental costs per QALY and per LY gained with Rd vs VMP were $53,826 and $35,552, respectively. In sensitivity analyses, results were found to be most sensitive to differences in survival associated with Rd vs VMP, the cost of lenalidomide and the discount rate applied to effectiveness outcomes. CONCLUSIONS: Rd was expected to result in greater LYs and QALYs compared with VMP, with similar overall costs per LY for each regimen. Results of this analysis indicated that Rd may be a cost-effective alternative to VMP as initial treatment for transplant-ineligible patients with MM, with an incremental cost-effectiveness ratio well within the levels for recent advancements in oncology.

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.

Expression of IL-6 and IL-6 receptors by circulating clonotypic B cells in multiple myeloma: potential for autocrine and paracrine networks.

OBJECTIVE: To investigate the participation of clonotypic MM B cells in the IL-6 network in patients with multiple myeloma. METHODS: CD19(+) B cells from 45 patients with multiple myeloma and from 18 healthy donors were sorted and their expression of IL-6, IL-6 receptor (CD126) characterized by flow cytometry, in situ RT-PCR, and ELISA measurement of IL-6 and soluble IL-6R. Expression of CD31 was detected by flow cytometry. RESULTS: Interleukin-6 (IL-6) is a pleiotropic cytokine often overexpressed in multiple myeloma (MM). IL-6 induces growth and inhibits apoptosis of MM plasma cells, and upregulates the activity of osteoclasts. MM plasma cells, the most mature component of the MM clone, secrete IL-6 and induce IL-6 production from other cell types. However, the MM clone also includes circulating clonotypic B lymphocytes. Using ELISA and in situ RT-PCR we demonstrate here that, unlike the healthy control B cells, MM B cells express IL-6 mRNA and secrete IL-6 protein. In vitro, MM B cells were the major producers of IL-6 in peripheral blood mononuclear cells. On average, 50% of MM B cells express the IL-6 receptor (IL-6R, CD126), suggestive of autocrine stimulation. They also express CD31, potentially facilitating their paracrine interactions with osteoclast precursors. CONCLUSION: Secretion of IL-6 by circulating clonotypic B cells in MM may contribute to the autocrine and paracrine cytokine networks that maintain the malignant clone and are responsible for disruption of normal bone metabolism in this incurable disease.

Elevated soluble MUC1 levels and decreased anti-MUC1 antibody levels in patients with multiple myeloma.

Soluble MUC1 (sMUC1) levels are elevated in many MUC1(+) cancers. We and others have shown that MUC1 is expressed on multiple myeloma (MM) plasma cells and B cells. In this study, we measured sMUC1 levels in bone marrow (BM) plasma from 71 MM patients and 21 healthy donors (HDs), and in peripheral blood (PB) plasma from 42 MM patients and 13 HDs using an immunoassay that detects the CA27.29 epitope of MUC1. sMUC1 levels were found to be significantly greater (mean 31.76 U/mL, range 5.69 to 142.48 U/mL) in MM patient BM plasma versus HD BM plasma (mean 9.68 U/mL, range 0.65 to 39.83 U/mL) (P <. 001). Importantly, BM plasma sMUC1 levels were related to tumor burden because sMUC1 levels were significantly higher for MM patients with active disease (34.62 U/mL, range 5.69 to 142.48 U/mL) versus MM patients with minimal residual disease (16.16 U/mL, range 5.7 to 56.68 U/mL) (P =.0026). sMUC1 levels were also elevated in the PB plasma of MM patients (32.79 U/mL, range 4.15 to 148.84 U/mL) versus HDs (18.47 U/mL, range 8.84 to 42.49) (P =.0052). Lastly, circulating immunglobulin M (IgM) and IgG antibodies to MUC1 were measured in 114 MM patients and 31 HDs, because natural antibodies to MUC1 have been detected in patients with other MUC1-bearing malignancies. These studies demonstrated lower levels of circulating IgM (P <.001) and IgG (P =.078) antibodies to MUC1 in MM patients compared with HDs. Our data therefore show that in MM patients, sMUC1 levels are elevated and correlate with disease burden, whereas anti-MUC1 antibody levels are decreased.

Selective targeting of immunoliposomal doxorubicin against human multiple myeloma in vitro and ex vivo.

Circulating malignant CD19(+) B cells have been implicated in the pathogenesis and relapse of multiple myeloma (MM). This study investigated the therapeutic applicability of using long-circulating liposome-encapsulated doxorubicin (DXR) targeted against the internalizing CD19 antigens present on human MM cells. In vitro binding studies using the CD19(+) MM cell line ARH77 demonstrated that CD19-directed immunoliposomes (SIL[anti-CD19]) specifically attached to these cells. Formulations of immunoliposomal doxorubicin (DXR-SIL[anti-CD19]) showed a higher association with, and higher cytotoxicity against, ARH77 cells than did non-targeted liposomal doxorubicin (DXR-SL) or isotype-matched controls (DXR-NSIL[IgG2a]). By using the pH-sensitive fluorophore, 1-hydroxypyrene-3,6, 8-trisulfonic acid, binding of SIL[anti-CD19] to CD19 antigens was shown to trigger receptor-mediated internalization of the antibody-antigen complexes into endosomes. Targeting of SIL[anti-CD19] to CD19(+) B cells was also demonstrated in a heterogeneous mixture of peripheral blood mononuclear cells (PBMC) from MM patients. A decrease in cellular DNA (which is an indicator of apoptosis) caused by the cytotoxicity of DXR-SIL[anti-CD19] to myeloma PBMC was determined by using flow cytometry. While PBMC treatment with free DXR resulted in non-specific cytotoxicity to both B and T cells, DXR-SL were only minimally cytotoxic to either. In contrast, DXR-SIL[anti-CD19] were selectively cytotoxic for B cells in PBMC, indicating that this treatment may be effective in eliminating circulating malignant B cells in MM patients.

Treatment of multiple myeloma by antibody mediated immunotherapy and induction of myeloma selective antigens.

BACKGROUND: In view of the successful use of serotherapy in many B-cell malignancies, we and others have sought to identify tumor selective antigens for the serotherapy of plasma cell dyscrasias (PCD) including multiple myeloma (MM), and Waldenstrom's macroglobulinemia (WM). We recently identified Muc-1 core protein as a MM selective antigen. Though Muc-1 core protein is abundantly expressed on most MM plasma cells, expression of this antigen can be absent, or weak on some plasma cells which could potentially result in the selection of Muc-1 core protein negative clones following serotherapy of PCD. In addition to Muc-1 core protein, we have also been examining the use of CD20 directed serotherapy for PCD. DESIGN: As part of these efforts, we recently initiated a phase II clinical trial examining the use of Rituximab (Rituxan, MabThera) as a single agent in MM patients; as well several WM patients have been treated with Rituximab at our Institutions. RESULTS: In previous studies, we have shown that CD20 is abundantly expressed on the plasma cells of most WM patients; in contrast, CD20 is expressed on plasma cells from a minority of MM patients, and in these patients expression of CD20 can be weak or heterogeneous with both CD20+ and CD20- plasma cells present. As such, we have sought out clinically useful inducers of Muc-1 core protein, and of CD20 on malignant plasma cells. CONCLUSIONS: These efforts resulted in the identification of dexamethasone (Dex) as a potent inducer of Muc-1 core protein on MM plasma cells, and interferon-gamma (IFN-gamma) as a potent inducer of CD20 on MM plasma cells and B-cells. Importantly, these agents induced their respective antigens at pharmacologically achievable doses.

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.

Myeloma progenitors in the blood of patients with aggressive or minimal disease: engraftment and self-renewal of primary human myeloma in the bone marrow of NOD SCID mice.

The myelomagenic capacity of clonotypic myeloma cells in G-CSF mobilized blood was tested by xenotransplant. Intracardiac (IC) injection of NOD SCID mice with peripheral cells from 5 patients who had aggressive myeloma led to lytic bone lesions, human Ig in the serum, human plasma cells, and a high frequency of clonotypic cells in the murine bone marrow (BM). Human B and plasma cells were detected in BM, spleen, and blood. Injection of ex vivo multiple myeloma cells directly into the murine sternal BM (intraosseus injection [IO]) leads to lytic bone lesions, BM plasma cells, and a high frequency of clonotypic cells in the femoral BM. This shows that myeloma has spread from the primary injection site to distant BM locations. By using a cellular limiting dilution PCR assay to quantify clonotypic B lineage cells, we confirmed that peripheral myeloma cells homed to the murine BM after IC and IO injection. The myeloma progenitor undergoes self-renewal in murine BM, as demonstrated by the transfer of human myeloma to a secondary recipient mouse. For 6 of 7 patients, G-CSF mobilized cells from patients who have minimal disease, taken at the time of mobilization or after cryopreservation, included myeloma progenitors as identified by engraftment of clonotypic cells and/or lytic bone disease in mice. This indicates that myeloma progenitors are mobilized into the blood by cyclophosphamide/G-CSF. Their ability to generate myeloma in a xenotransplant model implies that such progenitors are also myelomagenic when reinfused into patients, and suggests the need for an effective strategy to purge them before transplant.

Treatment of plasma cell dyscrasias by antibody-mediated immunotherapy.

The use of serotherapy to treat patients with plasma cell dyscrasias (PCDs) has been sought by us and others. Candidate antigens that have been targeted or proposed for targeting in PCDs include the immunoglobulin idiotype, CD19, CD38, CD54, CD126, HM1.24, and Muc-1 core protein. Unfortunately, many of these antigens are not ideal for use in serotherapy since they are not selectively expressed, are either shed or secreted, or have not been fully characterized. Serotherapy with an anti-CD19 monoclonal antibody (B4) conjugated to a blocked ricin toxin had no significant activity in patients with multiple myeloma (MM). Circulating CD20+ clonotypic B cells have been detected in the circulation of most MM and Waldenstrom's macroglobulinemia (WM) patients. Plasma cells from most WM patients express CD20, but most MM patient plasma cells either lack CD20 or express it weakly. In view of recent successes with anti-CD20-directed serotherapy in other B-cell malignancies, we initiated a phase II trial to study the anti-CD20 monoclonal antibody rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc, San Francisco, CA) in patients with MM. We describe two PCD patients (one with WM and one with MM) who responded to therapy. By flow cytometric analysis, CD20+ plasma cells and B cells present in the bone marrow and peripheral blood of a patient with MM disappeared with response to rituximab therapy. However, residual CD20- tumor cells remained in the bone marrow following rituximab therapy, and after 6 months this patient progressed with CD20- myeloma cells. As a potential strategy to overcome this limitation, we demonstrated that interferon-gamma at pharmacologically achievable levels induced CD20 expression on these CD20- plasma cells, consistent with our recent findings that interferon-gamma is a potent inducer of CD20 expression on MM patient plasma cells and B cells. We also characterize a response to rituximab with a decrease in paraprotein and resolution of anemia in a patient with WM whose response to rituximab is ongoing after 19+ months. This preliminary experience supports the potential use of serotherapy targeting CD20 in PCDs. Our studies further suggest that interferon-gamma may enhance CD20 expression on MM plasma cells, thereby increasing their susceptibility to anti-CD20 monoclonal antibody therapies.

Shiga-like toxin-1 receptor on human breast cancer, lymphoma, and myeloma and absence from CD34(+) hematopoietic stem cells: implications for ex vivo tumor purging and autologous stem cell transplantation.

The ribosome-inactivating protein, Shiga-like toxin-1 (SLT-1), targets cells that express the glycolipid globotriaosylceramide (CD77) on their surface. CD77 and/or SLT-1 binding was detected by flow cytometry and immunocytochemistry on lymphoma and breast cancer cells recovered from biopsies of primary human cancers as well as on B cells or plasma cells present in blood/bone marrow samples of multiple myeloma patients. Breast cancer cell lines also expressed receptors for the toxin and were sensitive to SLT-1. Treatment of primary B lymphoma, B-cell chronic lymphocytic leukemia, and myeloma B or plasma cells with SLT-1-depleted malignant B cells by 3- to 28-fold, as measured by flow cytometry. Depletion of myeloma plasma cells was confirmed using a cellular limiting dilution assay followed by reverse transcriptase-polymerase chain reaction analysis of clonotypic IgH transcripts, which showed a greater than 3 log reduction in clonotypic myeloma cells after SLT-1 treatment. Receptors for the toxin were not detected on human CD34(+) hematopoietic progenitor cells (HPC). HPC were pretreated with a concentration of SLT-1 known to purge primary malignant B cells and cultured for 6 days. The number of HPC was comparable in toxin-treated and untreated cultures. HPC were functionally intact as well. Colony-forming units (CFU) were present at an identical frequency in untreated and SLT-1 pretreated cultures, confirming that CFU escape SLT-1 toxicity. The results suggest the ex vivo use of SLT-1 in purging SLT-1 receptor-expressing malignant cells from autologous stem cell grafts of breast cancer, lymphoma, and myeloma 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.

Muc-1 core protein is expressed on multiple myeloma cells and is induced by dexamethasone.

Monoclonal antibodies (MoAbs) that selectively identify Muc-1 core protein (MoAbs DF3-P, VU-4H5) determinants were used to identify the Muc-1 glycoform present on 7 multiple myeloma (MM) cell lines, 5 MM patient plasma cells, 12 MM patient B cells, as well as 32 non-MM cell lines and normal hematopoietic cells. Flow cytometry studies demonstrated that all MM cell lines, MM patient plasma cells, and MM patient B cells expressed Muc-1 core protein epitopes. Circulating B cells from 4 normal donors also expressed Muc-1 core protein. In contrast, Muc-1 core protein was absent on 28 of 32 non-MM neoplastic cell lines, 17 of which expressed Muc-1. Splenic and tonsillar B cells, CD34(+) stem cells, resting T cells, and bone marrow plasma cells obtained from normal donors both lacked Muc-1 glycoforms. We next studied the effects of estrogen, progesterone, and glucocorticoid receptor agonists and antagonists on Muc-1 expression, because consensus sequences for the response elements of these steroids are present on the Muc-1 gene promoter. These studies showed that dexamethasone (Dex) induced Muc-1 expression on MM cell lines, as determined by both flow cytometry and Western blot analyses. Dex also induced upregulation of Muc-1 on prostate and ovarian cancer cell lines. Time and dose-response studies demonstrated that Dex induced maximal cell surface Muc-1 expression by 24 hours at concentrations of 10(-8) mol/L. Dex induced Muc-1 upregulation could be blocked with a 10-fold excess of the glucocorticoid receptor antagonist RU486, confirming that Dex was acting via the glucocorticoid receptor. No changes in Muc-1 expression were observed on MM cells treated with estrogen and progesterone receptor agonists and antagonists or with RU486. These studies provide the framework for targeting Muc-1 core protein in vaccination and serotherapy trials in MM. In addition, the finding that Muc-1 expression on MM cells can be augmented by Dex at pharmacologically achievable levels suggests their potential utility in enhancing treatments targeting Muc-1 in MM.

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.

Drug resistance in multiple myeloma: cyclosporin A analogues and their metabolites as potential chemosensitizers.

The malignant clone in myeloma is not eradicated by chemotherapy. Cyclosporins inhibit drug transport mechanisms, particularly the multidrug transporter p-glycoprotein 170, leading to their use as chemosensitizers. In myeloma, clonotypic blood B cells represent the major drug-resistant subset. This study compares the ability of cyclosporin A analogues and metabolites to inhibit cellular transporter(s) in myeloma and normal B cells in vitro, and evaluates their potential role in vivo. Cyclosporin A (CsA), CsG, PSC 833 or SDZ 280-446, and primary CsA and CsG metabolites, were tested for their ability to inhibit drug transport mechanisms of ex vivo malignant B cells from 81 patients with multiple myeloma as compared to B cells from normal donors, as measured by the export of the dye rhodamine 123 (Rh123) using multiparameter flow cytometry. The majority of myeloma B and normal B cells had efficient transporter function as measured by their CsA-sensitive export of Rh123. CsA and CsA analogues mediated efficient inhibition of this transport. Inhibition of dye transport by normal B cells required an approximately six-fold greater concentration of the synthetic peptolide SDZ 280-446 than was needed to optimally inhibit transport by myeloma B cells. PSC 833 and CsG were inhibitory at concentrations approximately five-fold lower than were required for CsA. Assessment of inhibitory potency in vivo indicated that the in vivo chemosensitizer levels of CsA and PSC 833 exceeded the transporter inhibitory concentration by four- and 20-fold respectively. In vivo, cyclosporins are rapidly and almost completely converted to metabolites. AM1 and AM4N, primary metabolites of CsA, mediated inhibition of transport, as did CsG metabolites GM1, GM4N and GM9. AM1 and GM9 are known to reach steady-state in vivo levels that exceed the inhibitory concentration identified here by 1.1- to 1.9-fold. Thus, cyclosporin metabolites, which accumulate in the blood during infusion of CsA and other cyclosporins, are shown here to be effective chemosensitizers for normally drug-resistant myeloma cells in vitro. Cyclosporin metabolites are considered to be less toxic than the parent drugs, suggesting that novel chemosensitization strategies designed to minimize concentrations of parent drug and maximize accumulation of primary metabolites in vivo may optimize cytotoxicity to the malignant clone in myeloma.

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.

Deficient drug transporter function of bone marrow-localized and leukemic plasma cells in multiple myeloma.

Although chemotherapy effectively reduces the plasma cell burden in multiple myeloma (MM), the disease recurs. MM includes circulating and bone marrow (BM) localized components. A large majority of circulating CD11b+ MM B cells (81%) express an IgH VDJ rearrangement identical to that of autologous BM plasma cells. Unlike plasma cells, these monoclonal circulating B cells exhibit dye and drug transport activity before and throughout chemotherapy. Drug resistance was measured as the ability to export the fluorescent dye Rhodamine123 (Rh123) or the drug adriamycin, using flow cytometry. The role of P-glycoprotein 170 (P-gp), the multidrug transporter, was defined by cyclosporin A (CsA)-sensitive dye export. Only 8% to 11% of BM-localized plasma cells exported dye with the majority retaining dye, identified as bright staining. Circulating leukemic plasma cells were also unable to export dye and remained Rh123(bright). However, 53% of circulating clonotypic MM B cells exhibited CsA-sensitive dye export. BM plasma cells taken before or after initiation of first line chemotherapy were equally unable to export dye. Thus in myeloma, differentiation to the plasma cell stage is accompanied by a loss of P-gp function, although P-gp phenotypic expression is retained. In contrast, for monoclonal gammopathy of undetermined significance (MGUS), 54% of BM-localized plasma cells exported dye, comparable to the 53% of circulating MGUS B cells that also exported dye, suggesting that the apparent defect in P-gp function is unique to myeloma plasma cells. Virtually all BM plasma cells in MM retained the drug adriamycin, consistent with their initial drug sensitivity in vivo, in contrast to circulating MM B cells, or to T cells in BM or blood. Thus, circulating B cells appear to be the predominant drug resistant component of the MM B-lineage hierarchy. This report suggests that successful therapeutic strategies will be those that target circulating B cells. Chemosensitization methods involving inhibition of P-gp are likely to improve depletion of these cells by compromising their ability to exclude drug. This work suggests that circulating clonotypic B cells should be monitored in clinical trials to confirm their depletion and the overall efficacy of novel treatment strategies.

Sensitivity of acute leukemia cells to cytarabine is a correlate of cellular es nucleoside transporter site content measured by flow cytometry with SAENTA-fluorescein.

Cytarabine (araC) is converted to araC 5'-triphosphate after entering leukemia cells as a substrate for nucleoside transport processes. This study tested the relationship between araC cytotoxicity, measured in an in vitro tetrazolium dye reduction assay of cell viability, and the cellular abundance of es nucleoside transport elements, assayed by a flow cytometric method that used the es-specific stain, 5-(SAENTA-x8)-fluorescein (5-(Sx8)-F), in cultured leukemia cells and in myeloblasts and lymphoblasts (blasts) from leukemia patients. Cellular es site abundance (B(max) value for 5-(Sx8)-F binding) varied sixfold among nine leukemic myeloblast samples from patients. In cultured OCI/AML-2 myeloblasts and CCRF-CEM T-lymphoblasts, and in fresh leukemic blasts, es sites were fractionally blocked by treatment with graded concentrations of nitrobenzylthioinosine (NBMPR), an inhibitory es site ligand, to simulate the variation in es expression found in leukemic blasts from patients with acute myeloid leukemia. When the cytotoxicity of a single concentration of araC was determined in NBMPR-treated leukemia cells, cell kill correlated closely with the intensity of 5-(Sx8)-F fluorescence (r = .92 to .99), a measure of the cell surface abundance of functional es nucleoside transporter sites. Concentrations of NBMPR that achieved half-maximal reduction (4.3 to 12 nmol/L) of cellular 5-(Sx8)-F fluorescence (measured by flow cytometry) approximated IC50 values (1 to 10 nmol/L) previously found for inhibition by NBMPR of es-mediated nucleoside fluxes in several cell types, supporting the view that 5-(Sx8)-F interacted with the estransporter. The correlation of araC cytotoxicity and the B(max) for 5-(Sx8)-F binding to es sites in cultured leukemia cells and in leukemic blasts from acute leukemia patients (r = .95) suggests that the flow cytometry assay of es capacity may be useful in predicting clinical response to araC.