Impact of CD138 Magnetic Bead-based Positive Selection on Bone Marrow Plasma Cell Surface Markers.

BACKGROUND: Isolation of malignant plasma cells from bone marrow of patients with monoclonal gammopathies is critical for studies into the disease biology. The plasma cells are typically isolated by positive selection using plasma cell markers such as CD138. Here we have examined the effect of CD138 magnetic bead selection on the expression of other surface phenotypic markers on plasma cells. MATERIALS AND METHODS: Bone marrow aspirates from 16 patients were split and prepared using 2 methods before staining for flow cytometric evaluation. The first method (whole bone marrow) used an ammonium-chloride-potassium lyse of whole bone marrow followed by 2 phosphate buffered saline washes. The second method used CD138-positive magnetic sorting technology (Stem Cell Technology). The cells were run on the FACSCanto flow cytometer after staining for CD38, CD45, CD56, activation markers CD71, CD69, CD154, adhesion markers CD49d, CD49e, CD11a, CD11b, and CD66, B cell markers CD19 and CD20, and for clonality. RESULTS: There was a substantial loss in the expression of CD71, CD11b, CD11a, CD69, and CD49e on plasma cells following CD138-based sorting. Moreover, in 8 of the 16 cases, there was a nearly complete loss of the CD45-positive subset with a loss of discrimination between CD45-negative and CD45-positive plasma cell subsets in the remaining CD138-sorted preparations. CONCLUSIONS: The change in immunophenotype of the plasma cells on magnetic sorting should be kept in mind when isolating plasma cells using CD138-positive selection for analysis of plasma cells. The technique for characterizing plasma cells should be selected based on the study design to prevent loss of crucial and valuable information.

Histone deacetylase inhibition in combination with MEK or BCL-2 inhibition in multiple myeloma.

Despite recent advances in the treatment of multiple myeloma, patients with this disease still inevitably relapse and become refractory to existing therapies. Mutations in K-RAS, N-RAS and B-RAF are common in multiple myeloma, affecting 50% of patients at diagnosis and >70% at relapse. However, targeting mutated RAS/RAF via MEK inhibition is merely cytostatic in myeloma and largely ineffective in the clinic. We examined mechanisms mediating this resistance and identified histone deacetylase inhibitors as potent synergistic partners. Combining the MEK inhibitor AZD6244 (selumetinib) with the pan-histone deacetylase inhibitor LBH589 (panobinostat) induced synergistic apoptosis in RAS/RAF mutated multiple myeloma cell lines. Interestingly, this synergy was dependent on the pro-apoptotic protein BIM. We determined that while single-agent MEK inhibition increased BIM levels, the protein remained sequestered by antiapoptotic BCL-2 family members. LBH589 dissociated BIM from MCL-1 and BCL-XL, which allowed it to bind BAX/BAK and thereby initiate apoptosis. The AZD6244/LBH589 combination was specifically active in cell lines with more BIM:MCL-1 complexes at baseline; resistant cell lines had more BIM:BCL-2 complexes. Those resistant cell lines were synergistically killed by combining the BH3 mimetic ABT-199 (venetoclax) with LBH589. Using more specific histone deacetylase inhibitors, i.e. MS275 (entinostat) and FK228 (romidepsin), and genetic methods, we determined that concomitant inhibition of histone deacetylases 1 and 2 was sufficient to synergize with either MEK or BCL-2 inhibition. Furthermore, these drug combinations effectively killed plasma cells from myeloma patients ex vivo Given the preponderance of RAS/RAF mutations, and the fact that ABT-199 has demonstrated clinical efficacy in relapsed/refractory multiple myeloma, these drug combinations hold prom ise as biomarker-driven therapies.

Aurora kinase and FGFR3 inhibition results in significant apoptosis in molecular subgroups of multiple myeloma.

Aberrant expression of proteins involved in cell division is a constant feature in multiple myeloma (MM), especially in high-risk disease. Increasingly, therapy of myeloma is moving towards individualization based on underlying genetic abnormalities. Aurora kinases are important mediators of cell cycle and are up regulated in MM. Functional loss of Aurora kinases results in genetic instability and dysregulated division leading to cellular aneuploidy and growth arrest. We investigated the role of Aurora kinase inhibition in MM, using a small molecule inhibitor A1014907. Low nanomolar A1014907 concentrations induced aneuploidy in MM cell lines independent of underlying cytogenetic abnormalities by inhibiting Aurora Kinases. However, A1014907 induced more pronounced and dose dependent apoptosis in cell lines with t(4;14) translocation. Translocation t(4;14) is observed in about 15% of patients with MM leading to constitutive activation of FGFR3 in two-thirds of these patients. Further investigation of the mechanism of action of A1014907 revealed potent FGFR3 pathway inhibition only in the sensitive cell lines. Thus, our results show that aurora kinase inhibition causes cell cycle arrest and aneuploidy with minimal apoptosis whereas inhibiting both aurora kinase and FGFR3 activity induced potent apoptosis in MM cells. These results support clinical evaluation of A1014907 in MM patients with t(4;14) translocation and/or FGFR3 expression.

Smac mimetic LCL161 overcomes protective ER stress induced by obatoclax, synergistically causing cell death in multiple myeloma.

Bcl2 and IAP families are anti-apoptotic proteins deregulated in multiple myeloma (MM) cells. Pharmacological inhibition of each of these families has shown significant activity only in subgroups of MM patients. Here, we have examined a broad-spectrum Bcl2 family inhibitor Obatoclax (OBX) in combination with a Smac mimetic LCL161 in MM cell lines and patient cells. LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines. The cytotoxicity was mediated through inhibition of the IAPs, activation of caspases and up regulation of the pro-apoptotic proteins Bid, Bim, Puma and Noxa by the drug combination. In addition, we observed that OBX caused ER stress and activated the Unfolded Protein Response (UPR) leading to drug resistance. LCL161, however inhibited spliced Xbp-1, a pro-survival factor. In addition, we observed that OBX increased GRP78 localization to the cell surface, which then induced PI3K dependent Akt activation and resistance to cell death. LCL161 was able to block OBX induced Akt activation contributing to synergistic cell death. Our results support clinical evaluation of this combination strategy in relapsed refractory MM patients.

Multiple mechanisms contribute to the synergistic anti-myeloma activity of the pan-histone deacetylase inhibitor LBH589 and the rapalog RAD001.

We examined the pre-clinical activity of pan-histone deacetylase inhibitor LBH589 in combination with mTORC1 inhibitor RAD001 and observed that the drug combination strongly synergized in inducing cytotoxicity in multiple myeloma (MM) cells. LBH589 caused an increase in acetylated histones and RAD001 inhibited mTORC1 activity. RAD001 caused potent G0/G1 arrest while LBH589 induced pronounced apoptosis, both of which were enhanced when the drugs were used in combination. LBH589/RAD001 combination led to down regulation of pStat3, cyclins, CDKs and XIAP and up regulation of pro-apoptotic Bcl-2 family proteins. A clinical trial is underway using LBH589/RAD001 combination in relapsed MM patients.

Phase II study of bevacizumab in combination with sorafenib in recurrent glioblastoma (N0776): a north central cancer treatment group trial.

PURPOSE: We hypothesized that vertical blockade of VEGF signaling by combining bevacizumab with sorafenib in patients with recurrent glioblastoma would result in a synergistic therapeutic effect. We also investigated whether VEGF, VEGFR2 and hypoxia-inducible factor-1α single-nucleotide polymorphisms (SNP), circulating biomarkers of angiogenesis, and MRI markers such as apparent diffusion coefficient (ADC) are correlated with treatment efficacy and/or toxicity. EXPERIMENTAL DESIGN: Patients received bevacizumab (5 mg/kg every 2 weeks) with sorafenib (200 mg twice a day, weekly, days 1-5; group A). Due to toxicity, the starting sorafenib dose was subsequently modified to 200 mg every day (group B). RESULTS: Fifty-four patients were enrolled: 19 patients in group A and 35 in group B. Objective response rate was 18.5% with median duration of 6.7 months (range 0.5-24.1 months). Six-month progression-free survival (PFS6) was 20.4% (11/54), and median overall survival (OS) was 5.6 months [95% confidence interval (CI), 4.7-8.2]; outcome was similar between the two dose groups. We identified SNPs in the VEGF and VEGFR2 promoter regions, which were associated with PFS6 (P<0.022). Among molecular markers of angiogenesis, a higher log2 baseline level of stromal cell-derived factor-1 was associated with PFS6 success (P=0.04). Circulating endothelial cells decreased during treatment with subsequent increase at disease progression (P=0.022). Imaging analysis showed a trend associating ADC-L with poor outcome. CONCLUSIONS: The bevacizumab/sorafenib combination did not improve outcome of patients with recurrent glioblastoma versus historic bevacizumab-treated controls. Biologic markers of response and resistance to bevacizumab in gliomas were identified which merit prospective validation.

Anti-myeloma activity of Akt inhibition is linked to the activation status of PI3K/Akt and MEK/ERK pathway.

The PI3K/Akt/mTOR signal transduction pathway plays a central role in multiple myeloma (MM) disease progression and development of therapeutic resistance. mTORC1 inhibitors have shown limited efficacy in the clinic, largely attributed to the reactivation of Akt due to rapamycin induced mTORC2 activity. Here, we present promising anti-myeloma activity of MK-2206, a novel allosteric pan-Akt inhibitor, in MM cell lines and patient cells. MK-2206 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested, albeit with significant heterogeneity that was highly dependent on basal pAkt levels. MK-2206 was able to inhibit proliferation of MM cells even when cultured with marrow stromal cells or tumor promoting cytokines. The induction of cytotoxicity was due to apoptosis, which at least partially was mediated by caspases. MK-2206 inhibited pAkt and its down-stream targets and up-regulated pErk in MM cells. Using MK-2206 in combination with rapamycin (mTORC1 inhibitor), LY294002 (PI3K inhibitor), or U0126 (MEK1/2 inhibitor), we show that Erk- mediated downstream activation of PI3K/Akt pathway results in resistance to Akt inhibition. These provide the basis for clinical evaluation of MK-2206 alone or in combination in MM and potential use of baseline pAkt and pErk as biomarkers for patient selection.

Sorafenib, a multikinase inhibitor, is effective in vitro against non-Hodgkin lymphoma and synergizes with the mTOR inhibitor rapamycin.

Non-Hodgkin lymphoma (NHL) represents a heterogenous group of neoplasias originating from lymphoid cells. Increased angiogenesis and expression of Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFR) have been found to be associated with NHL disease progression. Increase in VEGF and other cytokines stimulate signaling cascades, including the Ras/Raf/Mek/Erk pathway, resulting in increased proliferation and decreased apoptosis. Here, we report the in vitro antilymphoma activity of sorafenib, an inhibitor of VEGFR and Raf kinase. Sorafenib induced potent cytotoxicity in NHL cell lines and patient samples. This induction of cytotoxicity was associated with a corresponding increase in apoptotic cell death. Mechanism of action of sorafenib was investigated in follicular (DoHH2) and Burkitt lymphoma (Raji) cell lines. pStat3, pAkt, Mcl1, and Xiap were downregulated in both cell lines, whereas pErk decreased in Raji but not in DoHH2 cells following sorafenib treatment. IL6 was unable to prevent sorafenib induced repression of pStat3, pAkt, Mcl1, and Bcl-Xl. Sorafenib in combination with an mTORC1 inhibitor rapamycin demonstrated synergy in inducing cytotoxicity in NHL cells. Sorafenib/rapamycin combination resulted in downregulation of pAkt, pmTOR, p-p70S6K, p4EBP1, pGSK3ß, Mcl1, and Bcl-Xl. On the basis of our results, a clinical trial is underway using sorafenib with everolimus in NHL patients.

Immunophenotyping in multiple myeloma and related plasma cell disorders.

Plasma cell disorders form a spectrum ranging from the asymptomatic presence of small monoclonal populations of plasma cells to conditions like plasma cell leukemia and multiple myeloma, in which the bone marrow can be replaced by the accumulation of neoplastic plasma cells. Immunophenotyping has become an invaluable tool in the management of hematological malignancies and is increasingly finding a role in the diagnosis and monitoring of plasma cell disorders. Multiparameter flow cytometry has evolved considerably during the past decade with an increasing ability to screen large numbers of events and to detect multiple antigens at the same time. This, along with a better understanding of the phenotypic heterogeneity of the clonal plasma cells in different disorders, has made immunophenotyping an indispensible tool in the diagnosis, prognostic classification and management of plasma cell disorders. This book chapter addresses the approaches taken to evaluate monoclonal plasma cell disorders, and the different markers and techniques that are important for the study of these diseases.

TG101209, a novel JAK2 inhibitor, has significant in vitro activity in multiple myeloma and displays preferential cytotoxicity for CD45+ myeloma cells.

Interaction of myeloma cells with the bone marrow microenvironment is mediated in large part through different cytokines, especially VEGF and IL6. These cytokines, especially IL6, leads to upregulation of the JAK/STAT pathway in myeloma cell, contributing to increased proliferation, decreased apoptosis, and acquired drug resistance. Here, we examined the preclinical activity of a novel JAK2 inhibitor TG101209. TG101209 induced dose- and time-dependent cytotoxicity in a variety of multiple myeloma (MM) cell lines. The induction of cytotoxicity was associated with inhibition of cell cycle progression and induction of apoptosis in myeloma cell lines and patient-derived plasma cells. Evaluation of U266 cell lines and patient cells, which have a mix of CD45 positive and negative cells, demonstrated more profound cytotoxicity and antiproliferative activity of the drug on the CD45+ population relative to the CD45- cells. Exploring the mechanism of action of TG101209 indicated downregulation of pJak2, pStat3, and Bcl-xl levels with upregulation of pErk and pAkt levels indicating cross talk between signaling pathways. TG101209, when used in combination with the PI3K inhibitor LY294002, demonstrated synergistic cytotoxicity against myeloma cells. Our results provide the rationale for clinical evaluation of TG101209 alone or in combination with PI3K/Akt inhibitors in MM.

R-(-)-gossypol (AT-101) activates programmed cell death in multiple myeloma cells.

OBJECTIVE: Bcl-2 family proteins play a critical role in malignancies by regulating the balance between cell survival and apoptosis. R-(-)-gossypol (AT-101) is a small molecule that mimics the BH3 domain of cellular Bcl-2 inhibitors and interferes with the function of prosurvival Bcl-2 proteins. We examined the cytotoxicity of AT-101 in the context of multiple myeloma, a fatal hematological malignancy. MATERIALS AND METHODS: Multiple myeloma cell lines and primary cells obtained from multiple myeloma patients were used to investigate the effects of AT-101. Cell viability, apoptosis, and apoptosis pathways were examined using conventional viability assays, flow cytometry, and immunoblots. RESULTS: AT-101 was not only cytotoxic to conventional multiple myeloma cell lines, but was also effective against drug-resistant cell lines and primary multiple myeloma patient cells. Furthermore, AT-101 decreased proliferation of multiple myeloma cell lines in the presence of marrow stromal cells, indicating that this drug may overcome the protective effect of the microenvironment that is important for multiple myeloma cell proliferation and survival. Apoptosis was activated via the mitochondrial pathway in multiple myeloma cell lines treated with AT-101 as demonstrated by an increased Bax to Bcl-2 ratio, mitochondrial membrane depolarization, and caspase activation. Finally, our studies demonstrated that AT-101 exhibits potent synergy with dexamethasone, a valuable therapeutic for multiple myeloma. CONCLUSION: These data suggest that the activity of AT-101 may be highly relevant to multiple myeloma disease biology and may represent an option for treatment of patients with this disease.

Mechanisms of regulation of CXCR4/SDF-1 (CXCL12)-dependent migration and homing in multiple myeloma.

The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1-dependent migration was regulated by the P13K and ERK/ MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.

Differential expression of vascular endothelial growth factors and their receptors in multiple myeloma.

BACKGROUND AND OBJECTIVES: Bone marrow angiogenesis is increased in patients with multiple myeloma (MM) and correlates with disease stage. DESIGN AND METHODS: Previous studies of quantifying vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFR) in plasma cells from patients at different stages of MM found no significant difference in expression between overt MM and earlier pre-malignant stages of the disease namely, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM). RESULTS: In this report we used quantitative flow cytometry to study cytoplasmic VEGF (cyVEGF) expression (measured as antibody binding capacity) in plasma cells from patients with MM (n = 22), MGUS/SMM (n = 12), and AL-amyloidosis (AL) (n = 9). CyVEGF expression was higher in MM (169,591) than in MGUS/SMM (144,858), or AL (106,011) although these differences were not statistically significant. Using an indirect VEGFR assay that measures VEGF binding, we found VEGF receptors on plasma cells from all groups of patients, with the lowest expression on plasma cells from normal individuals. We detected VEGF R1, VEGF R2, and VEGF R3 on plasma cells from all groups of patients and found receptor expression predominantly in the subset of CD45-positive plasma cells. INTERPRETATION AND CONCLUSIONS: This study supports the concept that VEGF is involved in the pathogenesis of MM, and suggests that VEGF may differentially affect a subset of plasma cells.

A phase II trial of imatinib in patients with refractory/relapsed myeloma.

Although imatinib was designed to specifically inhibit the bcr-abl gene product, it inhibits other receptor tyrosine kinases including c-kit. As pre-clinical data, 126 patients with plasma cell disorders and 19 controls were evaluated for c-kit expression. Patients were eligible for the treatment trial if they had relapsed/refractory myeloma. The primary end-point of the study was response. Of the 145 studied before the trial, c-kit expression was present on the bone marrow plasma cells of control (11%), AL amyloid (53%), MGUS (47%), SMM (67%) and MM (42%) patients. Twenty-three MM patients were enrolled on the therapeutic trial (imatinib 400 mg daily) and 52% had positive c-kit staining. There were no responses. The median duration of treatment was 48 days (range: 12-349). Patients ended treatment due to progressive disease (18 patients), death (3) and other (2). The data suggest that imatinib is not an active agent in patients with relapsed or refractory multiple myeloma.

FISH is superior to PCR in detecting t(14;18)(q32;q21)-IgH/bcl-2 in follicular lymphoma using paraffin-embedded tissue samples.

Detection of t(14;18)(q32;q21)-IgH/bcl-2, which is present in 70% to 95% of follicular lymphomas (FLs), might aid in diagnosing FL. The efficacy of routine polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) techniques in detecting t(14;18) in paraffin-embedded tissue samples was compared on 5 normal tonsils and 28 FLs demonstrated to be t(14;18)+ by previous karyotyping. There was technical failure in 14 (50%) of the FLs by PCR, likely due to B-5 fixation, and 4 (14%) of FLs by FISH, likely due to advanced specimen age. In the remaining successful cases, 5 (36%) of 14 were positive by PCR and 24 (100%) of 24 were positive by FISH. All 5 normal tonsils were negative by both methods. FISH is superior to PCR for detecting t(14;18) from paraffin-embedded tissue samples because it is more sensitive and equally specific.

Bone marrow angiogenic ability and expression of angiogenic cytokines in myeloma: evidence favoring loss of marrow angiogenesis inhibitory activity with disease progression.

We compared the angiogenic potential of bone marrow plasma and the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and their receptors on plasma cells from patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma (NMM). Cytokine and cytokine-receptor expression was studied by bone marrow immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (RT-PCR) on sorted plasma cells, and quantitative Western blot analysis. Bone marrow angiogenic potential was studied using a human in vitro angiogenesis assay. The expression levels of VEGF, bFGF, and their receptors were similar among MGUS, SMM, and NMM. Sixty-one percent of NMM samples stimulated angiogenesis in the in vitro angiogenesis assay compared with SMM (0%) and MGUS (7%) (P <.001). Importantly, 63% of MGUS samples inhibited angiogenesis compared with SMM (43%) and NMM (4%) (P <.001). The inhibitory activity was heat stable, not overcome by the addition of VEGF, and corresponded to a molecular weight below 10 kd by size-exclusion chromatography. Our results suggest that increasing angiogenesis from MGUS to NMM is, at least in part, explained by increasing tumor burden rather than increased expression of VEGF/bFGF by individual plasma cells. The active inhibition of angiogenesis in MGUS is lost with progression, and the angiogenic switch from MGUS to NMM may involve a loss of inhibitory activity.

Flow cytometric assessment of TCR-Vbeta expression in the evaluation of peripheral blood involvement by T-cell lymphoproliferative disorders: a comparison with conventional T-cell immunophenotyping and molecular genetic techniques.

Molecular genetic T-cell receptor (TCR) and flow cytometric analysis using antibodies to conventional T-cell antigens and TCR beta-chain variable region families (TCR-Vbeta) were performed in 65 peripheral blood specimens evaluated for potential involvement by a T-cell lymphoproliferative disorder (TCLPD). A normal or reactive conventional T-cell immunophenotype was present in 36 cases; TCR-Vbeta flow cytometric and molecular TCR analyses were negative for clonality in 32 and 27 of these cases, respectively. In the remaining normal and reactive cases, one or both methods seemed to detect dominant cell populations in settings with limited T-cell diversity. We identified 29 TCLPDs; all studied cases had clonal molecular TCR results; 23 TCLPDs had clonal TCR-Vbeta flow cytometric results; the remaining were suggestive of (n = 3) or negative (n = 3) for clonality. TCR-Vbeta flow cytometric analysis is a powerful clinical laboratory tool that can be used to aid in the rapid diagnosis of peripheral blood involvement by T-cell malignant neoplasms.

Flt-3 and c-kit mutation studies in a spectrum of chronic myeloid disorders including systemic mast cell disease.

We screened 115 patients with chronic myeloid disorders (CMD) for known flt-3 and c-kit mutations in both the juxtamembrane (JM) and the activation loop (AL) domains. None of the patients displayed flt-3 (JM or AL) or c-kit JM mutations. However, the c-kit AL (D816V) mutation was detected in 5 of 16 patients with systemic mast cell disease (SMCD) but in none of the remaining 99 patients with other CMD. In SMCD, the presence of D816V mutation was significantly associated with advanced age, an aggressive clinical course, increased bone marrow mast cell content, and chronic myelomonocytic leukemia.

Expression of CD52 on plasma cells in plasma cell proliferative disorders.

Multiple myeloma (MM) and primary systemic amyloidosis (AL) remain incurable disorders, and new treatments targeted to the malignant plasma cells are needed. Alemtuzumab is a humanized monoclonal antibody to CD52 and has activity in chronic lymphocytic leukemia. We examined the CD52 expression on CD45+ and CD45- plasma cell populations to evaluate the potential for using alemtuzumab for these disorders. Bone marrows from 61 patients (29 AL, 23 MM, and 9 MGUS [monoclonal gammopathies of undetermined significance]) were studied using 3-color (CD38/45/52) flow cytometry. Among those with MGUS, MM, and AL, 67%, 52%, and 35%, respectively, were positive for CD52 expression. The CD52 expression was predominantly confined to the clonal CD38+/CD45+ plasma cell fraction with median expression of 68%, 88%, and 82% in MGUS, MM, and AL, respectively, compared with 18%, 6%, and 9% among the CD45- plasma cell population. Clinical trials are warranted in these diseases to learn the therapeutic benefit of anti-CD52 immunotherapy.