Inhibitor of apoptosis proteins as therapeutic targets in multiple myeloma.

The inhibitor of apoptosis (IAP) proteins have a critical role in the control of apoptotic machinery, and has been explored as a therapeutic target. Here, we have examined the functional importance of IAPs in multiple myeloma (MM) by using a Smac (second mitochondria-derived activator of caspases)-mimetic LCL161. We observed that LCL161 was able to potently induce apoptosis in some MM cell lines but not in others. Examining the levels of X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein 1 (cIAP1) and cellular inhibitor of apoptosis protein 2 (cIAP2) post LCL161 treatment indicated clear downregulation of both XIAP activity and cIAP1 levels in both the sensitive and less sensitive (resistant) cell lines. cIAP2, however, was not downregulated in the cell line resistant to the drug. Small interfering RNA-mediated silencing of cIAP2 significantly enhanced the effect of LCL161, indicating the importance of downregulation of all IAPs simultaneously for induction of apoptosis in MM cells. LCL161 induced marked up regulation of the Jak2/Stat3 pathway in the resistant MM cell lines. Combining LCL161 with a Jak2-specific inhibitor resulted in synergistic cell death in MM cell lines and patient cells. In addition, combining LCL161 with death-inducing ligands clearly showed that LCL161 sensitized MM cells to both Fas-ligand and TRAIL.

MRK003, a γ-secretase inhibitor exhibits promising in vitro pre-clinical activity in multiple myeloma and non-Hodgkin's lymphoma.

Notch-stimulated signaling cascade results in transcriptional regulation of genes involved in cell fate decision, apoptosis and proliferation and has been implicated in various malignancies. Here, we investigated the impact of MRK003, an inhibitor of this pathway, on myeloma and lymphoma cells. We first studied the expression patterns of notch receptors and ligands on multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL) cell lines. Next, we used a γ-secretase inhibitor, MRK003 to test the importance of notch-stimulated pathways in MM and NHL disease biology. We observed expression of notch receptors and ligands on MM and NHL cell lines. MRK003 treatment induced caspase-dependent apoptosis and inhibited proliferation of MM and NHL cell lines and patient cells. Examination of signaling events after treatment showed time-dependent decrease in levels of the notch intracellular domain, Hes1 and c-Myc. MRK003 downregulated cyclin D1, Bcl-Xl and Xiap levels in NHL cells and p21, Bcl-2 and Bcl-Xl in MM cells. In addition, MRK003 caused an upregulation of pAkt, indicating crosstalk with the PI3K/Akt pathway. We evaluated MRK003 in combination with Akt1/2 kinase inhibitor and observed synergy in killing MM and NHL cell lines examined.

Sorafenib, a dual Raf kinase/vascular endothelial growth factor receptor inhibitor has significant anti-myeloma activity and synergizes with common anti-myeloma drugs.

Multiple myeloma is characterized by increased bone marrow neovascularization driven in part by vascular endothelial growth factor (VEGF). In addition, the Ras/Raf/MEK/ERK pathway is critical for the proliferation of myeloma cells and is often upregulated. Sorafenib (Nexavar) is a novel multi-kinase inhibitor that acts predominantly through inhibition of Raf-kinase and VEGF receptor 2, offering the potential for targeting two important aspects of disease biology. In in vitro studies, sorafenib-induced cytotoxicity in MM cell lines as well as freshly isolated patient myeloma cells. It retained its activity against MM cells in co-culture with stromal cells or with interleukin-6, VEGF or IGF; conditions mimicking tumor microenvironment. Examination of cellular signaling pathways showed downregulation of Mcl1 as well as decreased phosphorylation of the STAT3 and MEK/ERK, as potential mechanisms of its anti-tumor effect. Sorafenib induces reciprocal upregulation of Akt phosphorylation; and simultaneous inhibition of downstream mTOR with rapamycin leads to synergistic effects. Sorafenib also synergizes with drugs such as proteasome inhibitors and steroids. In a human in vitro angiogenesis assay, sorafenib showed potent anti-angiogenic activity. Sorafenib, through multiple mechanisms exerts potent anti-myeloma activity and these results favor further clinical evaluation and development of novel sorafenib combinations.

ABT-737, an inhibitor of Bcl-2 family proteins, is a potent inducer of apoptosis in multiple myeloma cells.

Disruption of pathways leading to programmed cell death plays a major role in most malignancies, including multiple myeloma (MM). ABT-737 is a BH3 mimetic small-molecule inhibitor that binds with high affinity to Bcl-2 and Bcl-xL, preventing the sequestration of proapoptotic molecules and shifting the cell survival/apoptosis balance toward apoptosis induction. In this study, we show that ABT-737 is cytotoxic to MM cell lines, including those resistant to conventional therapies, and primary tumor cells. Flow cytometric analysis of intracellular levels of Bcl-2 family proteins demonstrates a clear inversion of the Bax/Bcl-2 ratio leading to induction of apoptosis. Activation of the mitochondrial apoptosis pathway was indicated by mitochondrial membrane depolarization and caspase cleavage. Additionally, several signaling pathways known to be important for MM cell survival are disrupted following treatment with ABT-737. The impact of ABT-737 on survival could not be overcome by the addition of interleukin-6, vascular endothelial growth factor or insulin-like growth factor, suggesting that ABT-737 may be effective in preventing the growth and survival signals provided by the microenvironment. These data indicate that therapies targeting apoptotic pathways may be effective in MM treatment and warrant clinical evaluation of ABT-737 and similar drugs alone or in combination with other agents in the setting of MM.

Thymoglobulin targets multiple plasma cell antigens and has in vitro and in vivo activity in multiple myeloma.

Multiple myeloma is characterized by the proliferation of clonal plasma cells that have a heterogeneous expression of various cell surface markers, precluding successful use of monoclonal antibodies for therapeutic targeting of the tumor cell. Thymoglobulin (rabbit-derived polyclonal anti-thymocyte globulin), by virtue of its method of preparation, contains antibodies against several B-cell and plasma cell antigens and offers an attractive option for immunotherapy of myeloma. Here, we demonstrate potent anti-myeloma activity of the rabbit anti-thymocyte globulin preparation Thymoglobulin in vitro and in vivo in an animal model of myeloma. Thymoglobulin was able to induce dose- and time-dependent apoptosis of several myeloma cell lines, including those resistant to conventional anti-myeloma agents. Importantly, the anti-myeloma activity was preserved even when myeloma cells were grown with different cytokines demonstrating the ability to overcome microenvironment-mediated resistance. Thymoglobulin induced apoptosis of freshly isolated primary myeloma cells from patients. Using a competitive flow cytometric analysis, we were able to identify the potential antigen targets for Thymoglobulin preparation. Finally, in a plasmacytoma mouse model of myeloma, Thymoglobulin delayed the tumor growth in a dose-dependent manner providing convincing evidence for continued evaluation of this agent in the clinic in patients with myeloma, either alone or in combination with other agents.

CD45 expression by bone marrow plasma cells in multiple myeloma: clinical and biological correlations.

Multiple myeloma (MM) is characterized by accumulation of clonal plasma cells (PCs). CD45, a key regulator of antigen-mediated signaling and activation in lymphocytes, is present in early stages of PCs development. We studied CD45 expression on MM PCs by flow cytometry, correlating it to important biological disease characteristics. Additionally, we examined the expression of various adhesion molecules on PCs. A total of 75 patients with untreated MM (29), relapsed MM (17), smoldering MM (12), and monoclonal gammopathy of undetermined significance (MGUS) (17) were studied. The proportion of PCs expressing CD45 was higher among those with early disease (MGUS or smoldering MM) compared to those with advanced disease (new or relapsed MM) (43 vs 22%; P=0.005). Among those with advanced disease, patients with bone lesions had a lower percentage of CD45-positive (CD45+) PCs; 14 vs 34% (P=0.02). Patients with high-grade angiogenesis had a lower percentage of CD45+ PCs; 13 vs 31% (P=0.03). The median overall survival for the CD45+ group (>20% PCs positive) was 39 vs 18 months for the CD45-negative (CD45-) group (P=0.07). The expression of CD138, CD56 and CD54 were higher among the CD45- PCs. This study demonstrates important biological correlates of CD45 expression on myeloma cells.

Bone marrow mast cell immunophenotyping in adults with mast cell disease: a prospective study of 33 patients.

The aberrant co-expression of CD2 and CD25 antigens is the immunophenotypic hallmark of neoplastic mast cells, and has been consistently identified on bone marrow mast cells from patients with indolent mast cell disease (MCD). We prospectively analyzed the bone marrow mast cell immunophenotype by multiparametric flow cytometry (FC) for 33 MCD cases, to examine the role of CD2 and CD25 expression in establishing diagnosis, detecting histologically occult bone marrow mast cell infiltration, and assessing treatment response. While CD25 was almost uniformly expressed, only 6 of 13 patients with indolent MCD, 1 of 8 with aggressive MCD, 2 of 7 with MCD and an associated hematological disorder, and none of the 2 patients with either mast cell leukemia or smoldering systemic mastocytosis, expressed CD2. One of three patients with cutaneous mastocytosis had an aberrant CD2+/CD25+ mast cell population suggesting histologically occult bone marrow involvement. CD25 expression was lost in one patient who achieved complete histologic remission with therapy, but not in two patients who achieved a partial remission. In conclusion, CD25, but not CD2, is a reliable marker for neoplastic mast cells, and CD25 expression indicates histologically occult bone marrow infiltration and residual disease after therapy.

Clinicopathologic, immunophenotypic, and molecular characterization of primary cutaneous follicular B-cell lymphoma.

OBJECTIVE: To determine the clinicopathologic, immunophenotypic, and molecular characteristics of primary follicular cutaneous B-cell lymphoma (CBCL) as defined by the revised European-American lymphoma classification. DESIGN: A retrospective survey of the medical records, an immunohistochemical study of archival biopsy specimens. and molecular studies of preserved DNA of all patients with follicle center lymphoma-follicular (FCL-F) primary CBCL from 1987 to 1997. SETTING: A single-center outpatient specialty clinic at an academic medical center. PATIENTS: Twenty-one patients (68% of all new primary CBCL cases), including 14 men and 7 women (age range, 33-88 years; mean, 55 years). RESULTS: The head and neck region was the most frequent primary site. Following treatment, recurrences were relatively frequent, but the overall mortality rate during 1.0 to 11.3 years (mean, 6.3 years) of follow-up was 4.8%. Immunohistochemical analysis for B- and T-cell lineages was helpful in enhancing the folliclelike structures. CD10, bcl-2, and CD43 were expressed by the neoplastic cells in 9 (47%) of 19 cases, 4 (21%) of 19 cases, and 2 (13%) of 16 cases, respectively. Immunohistochemical detection of cytoplasmic immunoglobulin light chains, using steaming in EDTA as the antigen-retrieval technique, was successful in 12 (71%) of 17 cases. The Ig heavy-chain gene rearrangements, using the Southern blot technique, detected clonality in 17 (94%) of 18 cases. The bcl-2 gene rearrangements were detected in only 2 (13%) of 15 of the primary cutaneous FCL-F cases, compared with 9 (75%) of 12 of the primary nodal FCL-F cases (P =.002). CONCLUSIONS: Primary cutaneous FCL-F is a relatively common subtype of CBCL, with a relatively indolent course. It has many features in common with primary nodal FCL-F, except for low rates of bcl-2 expression and bcl-2 gene rearrangements.

Image analysis and flow cytometric DNA studies of benign and malignant body cavity fluids: reappraisal of the role of current methods in the differential diagnosis of reactive versus malignant conditions.

Cytologic examination of body fluids is commonly performed in the clinical laboratory. Determination of the presence of malignancy may sometimes be difficult. In this study, we prospectively studied 60 body fluids with a panel of antibodies, including MOC-31, epithelial membrane antigen, carcinoembryonic antigen, B72.3, keratin, desmin, and CA-125. DNA and S-phase studies were performed both by flow cytometry and image analysis. Thirty-seven fluids were classified as benign and 23 were classified as malignant. The sensitivity of the antibodies for identification of carcinoma in descending order of percentage detection rate were MOC-31 (95%), epithelial membrane antigen (93%), B72.3 (84%), and carcinoembryonic antigen (80%). Desmin stained mesothelial cells in all cases. CA-125 gave similar results but was less specific. Flow cytometry detected 14 of 20 malignant fluids and image analysis 17 of 23 by identifying an aneuploid population. Benign reactive mesothelial cells were not aneuploid. Tetraploidy due to reactive mesothelial cells was found in 9 of 37 body fluids. Their S-phase fraction was low (average, 3.2%). Tetraploidy in malignant cells was distinguished from the reactive mesothelial cells by high S-phase (average, 25.95). S-phase had some use as a discriminating factor, because no benign reactive cases had more than 17%. However, 7 of 23 malignant cases had a value below 17%. DNA analysis by image was more sensitive and specific than flow. Either may be used when immunocytochemistry is nondiagnostic or cannot be performed.

Measurement of the cell proliferation rate of bone marrow erythroid precursors by flow cytometry: initial applications to multiple myeloma.

To investigate the pathophysiology of anemia, a two-color flow cytometric method was developed that measures the proliferative rate of the marrow erythroid cells (EPR). The method uses a monoclonal antibody, RC17.2, to identify erythroid precursors and propidium iodide to determine the %S-phase. This technique was then used to test the hypothesis that a decrease in the proliferative rate of the marrow erythroid precursors contributes to the anemia of multiple myeloma. The EPR was determined on the marrow aspirate from 56 patients and the mean EPR was 31.2% (median, 31: range, 14-55). Patients with anemia (n = 36) had a median EPR of 27% compared to 35% for those patients with a normal Hgb (p = < 0.001); however, there was no difference in the % marrow erythroid precursors (p = 0.96) or % marrow plasma cells (p = 0.08) between the two groups. These results suggest that one possible cause for the anemia of myeloma is a decrease in the EPR. This flow cytometric technique may also be useful in studying other anemias.

Syndecan-1 expression on malignant cells from the blood and marrow of patients with plasma cell proliferative disorders and B-cell chronic lymphocytic leukemia.

Syndecan-1 is a low-affinity receptor for basic fibroblast growth factor (bFGF). In this study, we used flow cytometry to examine expression of syndecan-1 on monoclonal cells from the blood (n = 37) and marrow (n = 81) of patients with plasma cell (PC) proliferative disorders (PCPD) and blood cells from patients (n = 39) with B cell chronic lymphocytic leukemia (B-CLL). The marrow CD38+CD45- and CD38+CD45+ PC were syndecan-1 positive in all patients with PCPD and there was no difference between patients with monoclonal gammopathy of undetermined significance (MGUS) vs multiple myeloma or cases with vs without bone lesions. In 38% of cases, syndecan-1 expression on the PC was heterogeneous with > or =25% of PC syndecan-1 negative. We found similar syndecan-1 expression on blood and marrow PC in the 36 cases with paired samples. CLL cells were syndecan-1 negative in 97% (38/39) of the cases. Syndecan-1 is a useful marker to detect malignant plasma cells in the blood or marrow; however, it is not helpful in distinguishing MGUS from active myeloma. In addition, syndecan-1 is present on the less mature (CD45+) PC, and there is heterogeneity of expression within and between patients. The relevance of the bFGF bound to myeloma cells via syndecan-1 remains to be elucidated.

Is multidrug resistance (P-glycoprotein) an intrinsic characteristic of plasma cells in patients with monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma and amyloidosis?

Multiple myeloma is not a curable disease, and most patients relapse after plateau phase. Drug resistance is a major problem in effective chemotherapy in this kind of disease. Current approaches are aimed at reversing or preventing drug resistance late in the disease. We studied a drug resistance marker, P-glycoprotein (P-gp), in a total of 43 patients with monoclonal gammopathy. This group included eight with monoclonal gammopathy of undetermined significance (MGUS), five with plasmacytoma (PCM), nineteen with multiple myeloma (MM; six newly diagnosed, seven plateau, five refractory, one relapse) and eleven amyloidosis (seven newly diagnosed, four after treatment). Using 3-color flow cytometry, a plasma cell gate was selected on the basis of CD38+/45-(dim) staining and the population was examined for the expression of P-gp using two monoclonal antibodies (MRK16 and UIC2). P-gp expression was positive on marrow plasma cells in 42/43 patients. The resistance index (RI) in these cases (range 2.0-7.07) is comparable to that in the positive cell line KG-1A (3.05-3.08). In 2 of 5 patients with refractory MM, the RI for P-gp (5.4, 6.36) was higher than in plateau phase. These data suggest that relative resistance due to the P-gp mechanism is likely to be an intrinsic property of plasma cells in monoclonal gammopathies and may provide a partial explanation as to why these diseases always relapse. The results of our study support strategies for MDR reversal earlier in the course.

The role of conventional cytology, immunocytochemistry, and flow cytometric DNA ploidy in the evaluation of body cavity fluids: a prospective study of 52 patients.

Fifty-two specimens of body cavity fluids from 52 patients were analyzed with conventional cytology, immunocytochemistry, and flow cytometric DNA ploidy methods to evaluate the most appropriate way of applying and interpreting immunocytochemistry and to evaluate the contribution of DNA ploidy analysis to conventional cytology in the diagnosis of body cavity fluids. The results suggest that conventional cytology still has an important role in the diagnosis of body cavity fluids. MOC 31 is the most sensitive monoclonal antibody for distinguishing benign mesothelial cells from malignant epithelial cells. Immunocytochemistry with the combination of cytokeratin, desmin, and MOC 31 with or without epithelial membrane antigen is suggested as a helpful ancillary method for the differential diagnosis of body cavity fluids. Flow cytometric DNA ploidy analysis also provides additional information in some difficult cases. Appropriate integration of clinical information and results of conventional cytology, immunocytochemistry, and flow cytometry are necessary to achieve the most accurate diagnosis in patients with effusion involving a body cavity.

Expression of the hematopoietic stem cell antigen CD34 on blood and bone marrow monoclonal plasma cells from patients with multiple myeloma.

Monoclonal plasma cells (CD38+CD45-/dim) are typically present in the blood of patients with active myeloma and can contaminate stem cell harvests. This has led to strategies that select CD34+ cells for use in autologous stem cell transplantation with the goal of decreasing tumor cell contamination. The aim of this study was to learn if the CD34 antigen is expressed on monoclonal plasma cells in the blood or marrow of patients with multiple myeloma. We used three-color flow cytometry (surface CD38;CD45 and cytoplasmic kappa or lambda) to identify monoclonal plasma cells in the blood (n = 24) and marrow (n = 37) from patients with plasma cell proliferative disorders. In each case the CD38+CD45- and CD38+CD45dim+ monoclonal populations were then analyzed for CD34 expression. In all 24 blood and 37 marrow samples, the CD38+CD45-monoclonal plasma cells were negative for CD34 expression. CD38+CD45dim+ monoclonal cells were documented in the blood of 11 patients and in the marrow of 33 patients and this cell population was also CD34-negative in all cases. These results indicate that CD34 is usually not expressed on the CD38+CD45-CD45dim+ monoclonal plasma cells in the blood or marrow of patients with plasma cell proliferative disorders. CD34 selection methods should therefore decrease the chance of tumor cell contamination of the stem cell product.

Clonal circulating cells are common in plasma cell proliferative disorders: a comparison of monoclonal gammopathy of undetermined significance, smoldering multiple myeloma, and active myeloma.

The blood of most patients with active multiple myeloma (MM) contains cells related to the bone marrow tumor. However, identifying clonal cells in the blood of patients with monoclonal gammopathy of undetermined significance (MGUS) has been difficult. In this study, we analyzed blood mononuclear cells (BMNCs) from 16 patients with MGUS, 2 with amyloidosis, 8 with smoldering MM (SMM), 2 with indolent MM (IMM), and 15 with active MM using three different methods to detect and quantitate clonal cells, ie, immunofluorescence microscopy (IM) for monoclonal plasma cells, three-color flow cytometry (FC) for CD38(+)CD45- CD45(dim) cells, and the allele-specific oligonucleotide polymerase chain reaction (ASO-PCR). Using ASO-PCR, we were able to detect clonal cells in the blood in 13 of 16 patients with MGUS, 2 of 2 with amyloid, 6 of 8 with SMM, 2 of 2 with IMM, and 13 of 15 with MM. In 9 of the 13 patients with MGUS with blood involvement, the number of clonal cells was very small ( < 0.04% of the BMNCs). The median percentage of clonal cells as determined by ASO-PCR was 0.02 for MGUS, 0.02 for SMM, and 0.24 for MM. Clonal plasma cells or CD38+CD45-CD45(dim) cells were identified by IM or FC in 6 of 16 MGUS patients, 4 of 8 with SMM, and 11 of 15 with MM. In all cases in which IM or FC detected clonal cells, the ASO-PCR was positive. This study shows that, by using ASO-PCR, clonal cells can be found at very low levels in the blood in most patients with MGUS. However, the number of clonal cells in the blood of MGUS patients is less than those with overt MM (P = .006). In contrast to MGUS, patients with active MM are more likely to have identifiable clonal circulating plasma cells (P = .05).

Detection of myeloma cells in the peripheral blood by flow cytometry.

Bone marrow plasma cells (PC) from patients with multiple myeloma (MM) express monoclonal cytoplasmic immunoglobulin (clg) light chain, strongly express CD38, and usually lack or dimly express CD45. The detection of malignant plasma cells in the peripheral blood (PB) by immunofluorescence microscopy (IM) distinguishes patients with active MM from those with stable disease. The aim of this study was to learn whether two-color (CD38 and CD45) flow cytometry (FC) on whole blood specimens (WBFC) and three-color FC (CD38, CD45, and anti-kappa or lambda clg) on mononuclear cells could identify circulating PC as well as the standard, more labor intensive IM technique. Split-samples of PB from 73 patients with plasma cell proliferative disorders were examined by both techniques. WBFC detected CD38+ CD45- cells in 94% (33/35) of patients with circulating monoclonal PC detected by IM and three-color FC detected monoclonal CD38+ CD45- cells in 77% (27/35) of these cases. The absolute number of monoclonal PC detected by IM was compared to the FC methods and the Spearman rank correlations were 0.77 with WBFC and 0.80 with three-color FC. This study indicates that WBFC, using antibodies to CD38 and CD45, offers a practical and reliable method to detect and quantify circulating malignant PC in patients with MM.

Concordance of DNA ploidy pattern as measured by flow cytometry in primary, metastatic, and persistent ovarian carcinoma.

In an attempt to evaluate the stability of DNA content in ovarian carcinoma, 66 tumor specimens from 25 patients with stage III disease were analyzed by flow cytometry. For all patients, both primary tumor and omental metastasis were available, and for 16 patients the persistent tumor found at second-look operation was also available. The concordance of the tumor DNA content in the primary tumor and in the corresponding omental metastasis was 72%; the concordance in persistent tumor at second-look operation was 63%. When diploid and aneuploid tumors with very low DNA index were considered together, the concordance of the DNA content in primary and metastatic tumors reached 84%. According to our data, if DNA ploidy is used to differentiate patients with a favorable prognosis (DNA diploid and DNA aneuploid with low DNA index) from those with an unfavorable prognosis (DNA aneuploid with high DNA index), a 16% risk of misclassification appears unacceptable in prospective studies. Hence, the assessment of two specimens (preferably the primary lesion and a metastatic site) to determine the most abnormal DNA ploidy result would reduce the risk of underclassification of tumor.

Terminal component of complement (C9) in the cerebrospinal fluid of patients with multiple sclerosis and neurologic controls.

We measured the concentration of C9 in the CSF and plasma of 93 consecutive patients referred for CSF examination in an outpatient multispecialty clinic. We noted no differences in CSF C9 or C9 index between patients with multiple sclerosis and neurologic controls.