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.

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.

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.

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.

A new murine monoclonal antibody for the diagnosis of erythroleukemia.

The authors have developed a murine monoclonal antibody, RC-82.4, against an antigen expressed by a human erythroleukemia cell line OCI-MIR. The antibody reacts with an antigen expressed by proerythroblasts, normoblasts, and some reticulocytes but not expressed in erythrocytes, granulocytes, monocytes, megakaryocytes, plasma cells, or lymphocytes. The authors have established an immunocytochemical method for studying bone marrow smears with RC-82.4. By studying bone marrow smears from 11 patients with M-6 erythroleukemia and 104 patients with various other hematologic and nonhematologic malignancies, the authors have found that RC-82.4 has great sensitivity and specificity in recognizing erythroid differentiation in blasts. The authors have used RC-82.4 and antihemoglobin antibodies to identify erythroblasts in acute and secondary acquired cases of erythroleukemia that would have been unclassifiable by morphologic and all other conventional cytochemical and immunocytochemical criteria.

Effect of sonication on paraffin-embedded tissue preparation for DNA flow cytometry.

Since the publication of paraffin block extraction procedures, flow cytometric analysis of DNA ploidy and S-phase of tumor specimens has been widely applied. DNA aneuploidy, DNA tetraploid (elevated G2/M), and elevated S-phase are clinically significant in some tumor systems. True DNA tetraploid cell lines will contain a large 4c population and perhaps an 8c population; samples with cell aggregates will also contain a 6c population. Microscopic examination of samples having a 6c peak revealed nuclei with adhering debris and doublets, triplets, and larger nuclear aggregates. After sonication, a uniform suspension of single nuclei without adherent debris was seen. In addition to reducing the percent of G2/M cells, sonication also reduced S-phase percent such that it was closer to the bromodeoxyuridine labeling index. The DNA ploidy classification of specimens was also compared pre- and post-sonication. Four of 96 breast cancer samples changed classification; all were specimens in which the histogram became cleaner and a small DNA aneuploid peak became apparent after sonication.

Simultaneous measurement of total and IgA-conjugated alpha 1-microglobulin by a combined immunoenzyme/immunoradiometric assay technique.

alpha 1-Microglobulin (alpha 1m), a glycoprotein (Mr = 30,000) found in serum and urine, is also present in serum conjugated to monomeric IgA (alpha 1-m-IgA). We have developed a simultaneous enzyme-linked immunoenzyme/immunoradiometric assay that involves three different monoclonal antibodies. Assay of serial dilutions of serum and urine demonstrated parallelism. Normal mean concentrations in serum (n = 75) were: total alpha 1m, 2.33 mumol/L; alpha 1m-IgA, 1.24 mumol/L; unconjugated (free) alpha 1m, 1.09 mumol/L; molar ratio (alpha 1m-IgA/total alpha 1m), 0.53. The mean concentration of alpha 1m in eight urine specimens from normal individuals was 0.19 mumol/L, with no detectable alpha 1m-IgA. A low urinary pH does not significantly affect assay results, unlike assays of beta 2-microglobulin. In patients with myeloma-related renal disease, total and free alpha 1m values for serum correlated well with values for creatinine and beta 2-microglobulin in serum.

Isolation and preliminary characterization of a monoclonal antibody that interacts preferentially with the liver isoenzyme of human alkaline phosphatase.

We have prepared murine monoclonal antibodies against isolated human bone alkaline phosphatase (ALP, EC 3.1.3.1). Hybridoma supernates were separately screened for reactivity against both human liver and bone ALP. Although most antibody-positive hybrids showed similar reactivity against both isoenzymes, one hybridoma produced an antibody that interacted preferentially with liver ALP. This antibody was purified and used to establish an immunoassay to differentiate liver ALP from bone ALP. When equal activities of the two isoenzymes (as determined by a conventional enzymic assay) were measured by the immunoassay, a fivefold greater response was obtained with liver than with bone ALP. The immunoassay can be used to measure the proportions of the bone and liver isoenzymes in mixtures of them. Cross reactivity with human placental and intestinal ALP is less than 3% relative to liver ALP. These findings support the feasibility of developing immunological methods to differentiate these isoenzymes in the clinical laboratory.

Development of monoclonal antibodies to proteins separated by two-dimensional gel electrophoresis.

We have developed techniques for the production of monoclonal antibodies using Coomassie blue-stained protein spots cut from high resolution 2-dimensional polyacrylamide gels. The gel spots were homogenized with Freund's adjuvant and injected sub-cutaneously into a mouse, at several places along the flank. After boosting twice the spleen cells were hybridized by standard methods. Hybrids, clones and ascitic fluids were also screened with antigen prepared from 2-dimensional gel spots. The spots were cut from gels, homogenized in the presence of guanidinium chloride, and extracted by shaking overnight. The acrylamide was removed, the sample dialyzed to remove denaturant and the protein labeled with 125I. An alternative method for the production of screening antigen using column chromatography is described. These techniques allow the production of monoclonal antibodies to specific protein components of complex mixtures, even in the presence of other immunodominant proteins.