Ivana Zavrski

Bone marrow microvessel density is a prognostic factor for survival in patients with multiple myeloma.

Abstractu2002The importance of neoangiogenesis for the progressive growth and viability of solid tumors is well established. Recently, there has been growing evidence that angiogenesis might also be important in hematological malignancies, but only few data are available. In this report, we have studied the impact of bone marrow microvessel density and survival in patients with multiple myeloma (MM). Immunohistochemical CD34 stained paraffin-embedded bone marrow biopsies of 44 patients with newly diagnosed MM were studied. Microvessels were counted in 400× magnification and the mean number of vessels per area in each sample was noted as the microvessel density (MVD). The median MVD was 48 vessels/mm2, the range was 0–125 vessels/mm2. Using a cut-off value of the median MVD in the Kaplan-Meier analysis, the median survival was 22.2 months in the group with the higher MVD and was not reached in the group with the lower MVD (P<0.01). In a multivariate Cox regression analysis, using previously identified prognostic factors β2-microglobulin, C-reactive protein (CRP), and age, MVD remained significant as a prognostic factor (P<0.03).

Decrease of bone marrow angiogenesis in myeloma patients achieving a remission after chemotherapy.

Abstract: The impact of angiogenesis is well known for the growth and viability of solid tumors. Fewer studies have been published relating angiogenesis to clinical or pathological parameters in hematological malignancies. In this report, we have estimated the bone marrow microvessel density (MVD) before and after conventional‐dose or high‐dose chemotherapy with autologous stem cell transplantation. Immunohistochemical CD34‐stained paraffin‐embedded bone marrow biopsies of 21 patients with stage III multiple myeloma were studied. Microvessels were counted at 400× magnification, and the mean number of vessels per area in each sample was noted as the MVD. The median MVD of all patients was 53.1 vessels/mm2 (range 15.5–174.7 vessels/mm2) before treatment and 29.3 vessels/mm2 (range 0–221.1 vessels/mm2) after chemotherapy. The post‐treatment MVD in the two groups of patients with and without remission was significantly different (p=0.001), whereas the pretreatment MVD was not. Responders but not nonresponders showed a significant decrease of MVD after therapy in comparison to their pretreatment levels. The progression‐free survival in patients who achieved a reduction in MVD after chemotherapy was significantly longer than in patients without a decrease in MVD (P=0.006). Furthermore, we compared the MVD of patients after achievement of a remission to MVD of 15 untreated stage I myeloma patients. The MVD of patients in remission was not statistically different from the MVD in stage I myeloma. These results underscore the impact of angiogenesis in myeloma and give the first report that effective chemotherapy is accompanied by a significant decrease in bone marrow angiogenesis in this disease.

Proteasome inhibitors induce growth inhibition and apoptosis in myeloma cell lines and in human bone marrow myeloma cells irrespective of chromosome 13 deletion

PurposeIn this study, we investigated the effects of cell-permeable proteasome inhibitors MG-132, MG-262, PSI, and lactacystin on multiple myeloma cell lines OPM-2, U266, RPMI 8226-S, freshly isolated plasma cells with or without deletion of chromosome 13 from patients with multiple myeloma and plasma cell leukemia, and CD34+ human hematopoietic stem cells. The effects of proteasome inhibitors on cell cycle progression, cell growth, and apoptosis were determined.MethodsMTT-assay was used to examine the cytotoxicity, and annexin-V staining to quantify apoptosis. Cell cycle analyses were performed using 7-ADD and Ki-67 staining by flow cytometry.ResultsPSI was the most potent proteasome inhibitor among those tested with a half maximal cytotoxicity (IC50) of 5.7 nM, followed by MG-262, MG-132, and lactacystin. Growth inhibition occurred irrespective of chromosome 13 status. Cell cycle arrest occurred in a dose- and time-dependent manner. Low, subapoptotic dosages led to a partial loss of Ki-67 antigen, whereas apoptotic dosages led to reduced Ki-67 levels. Apoptosis was partially dependent on activation of caspase-3, since Ac-DEVD-cho, a caspase-3 inhibitor, could reduce apoptosis significantly. The cytotoxicity of the four proteasome inhibitors tested was significantly lower in human hematopoietic stem cells than in myeloma cells.ConclusionsOur results show that proteasome inhibitors induce time- and dose-dependent cell cycle alterations, growth inhibition, and apoptosis in human myeloma cells irrespective of chromosome 13 deletion.

Relationship between bone marrow angiogenesis and plasma cell infiltration and serum β2-microglobulin levels in patients with multiple myeloma

Abstract. There is growing evidence that angiogenesis is important not only in solid tumors but also in hematological malignancies. Recently, we found that bone marrow angiogenesis is a prognostic factor for disease-related survival in patients with multiple myeloma. In this report, we addressed the question of whether the microvessel density in bone marrow biopsies is correlated to other myeloma parameters, e.g., serum β2-microglobulin (β2-MG) and plasma cell infiltration in the bone marrow. In 22 multiple myeloma patients, immunohistochemical, CD34-stained, paraffin-embedded bone marrow biopsies before and after chemotherapy were studied. Microvessels were counted in 400× magnification, and the mean number of vessels per area in each sample was noted as the microvessel density (MVD). Pretreatment bone marrow MVD (median: 44, range: 11–175 vessels/mm2) correlated significantly with the bone marrow plasma cell infiltration (median: 30%, range: 5–90%, r=0.642, P=0.001) and β2-MG (median: 2.74, range: 1.4–26.1xa0mg/l, r=0.749, P<0.0005). In contrast, there was no correlation between posttreatment MVD and plasma cell infiltration or β2-MG (median: MVD 31, range: 0–221 vessels/mm2, median plasma cell infiltration: 15%, range: 5–80%, r=0.229, P=0.306 and median β2-MG: 2.65, range: 1–27.6xa0mg/l, r=-0.042, P=0.853). These findings show that the strong correlations between bone marrow MVD and plasma cell infiltration as well as serum β2-MG levels disappear after chemotherapy. The underlying mechanisms need further investigations.

Bone marrow angiogenesis and its correlation with other disease characteristics in multiple myeloma in stage I versus stage II-III

PurposeWe studied bone marrow angiogenesis in different stages of multiple myeloma according to the Durie and Salmon classification and its correlations with other disease characteristics.MethodsSixty-five immunohistochemical CD34-stained, paraffin-embedded bone marrow biopsies of multiple myeloma patients and 12 controls were studied. The mean number of microvessels per area in each sample was determined as the microvessel density (MVD). In addition, plasma cell infiltration of the bone marrow, serum β2-microglobulin, immunoglobulin levels, C-reactive protein, and serum calcium concentration were measured in 22 patients with stage I multiple myeloma and in 43 patients in stage II-III.ResultsIn myeloma patients, the bone marrow MVD was significantly higher than in controls (P<0.001). In 43 patients with stage II-III multiple myeloma, MVD was significantly higher than in 22 patients with stage I (median MVD 46 and 21 vessels/mm2, respectively, P=0.005). Additionally, in stage II-III the bone marrow MVD correlated positively with the bone marrow plasma cell infiltration (r=0.55, P<0.001) and the serum β2-microglobulin level (r=0.53, P<0.001), while in stage I patients no correlation could be found.ConclusionsAngiogenesis is significantly increased in stage II-III myeloma in comparison to stage I. In stages II-III, bone marrow angiogenesis is correlated with plasma cell infiltration and serum β2-microglobulin levels.

Expression of receptor activator of NF-κB ligand (RANKL) mRNA in human multiple myeloma cells

Purpose Increased bone resorption is a hallmark of multiple myeloma and a result of excessive osteoclast activation. Recently, the receptor activator of NF-κB ligand (RANKL) was found to be the critical factor for osteoclastogenesis. Studies showed that myeloma cells induce RANKL expression in bone marrow stromal cells, but it remained a controversy whether myeloma cells directly express RANKL.Methods Therefore, we analyzed the expression of RANKL mRNA in freshly isolated CD138 positive plasma cells from patients with multiple myeloma and osteolytic bone lesions, using three different primer pairs against human RANKL.Results RANKL mRNA could be detected in bone marrow plasma cells from myeloma patients with osteolytic myeloma bone disease.Conclusions These findings show that myeloma cells directly express RANKL and indicate that specific blockade of RANKL may be an effective treatment for myeloma bone disease.

Treatment of refractory chronic lymphocytic leukemia with Campath‐1H in combination with lamivudine in chronic hepatitis B infection

Campath‐1H, a monoclonal antibody against human CD52, is used for the therapy of refractory or relapsed chronic lymphocytic leukemia (CLL). Treatment with campath is associated with an increased incidence of infections and also fatal reactivation of viral infections. Reactivation of hepatitis B virus (HBV) in HBsAg‐positive patients is a well‐documented complication of cytotoxic or immunosuppressive therapy and has also been observed after treatment with rituximab. To date, there are no reports on campath treatment in HBsAg carriers. Here, we present the case of a patient with heavily pretreated CLL who was HBsAg‐positive with a high virus load (>2u2003billionu2003copies/mL). He required treatment because of progressive CLL with massive bone marrow infiltration, severe anemia and thrombocytopenia. Campath was initiated and lamivudine, an inhibitor of reverse transcriptase, was simultaneously given to prevent HBV proliferation. During the treatment, no deterioration of liver parameters was observed, and the virus load decreased. After therapy with campath, hemoglobin and platelet counts increased markedly. This report shows that lamivudine is highly effective in inhibiting HBV proliferation and can be used to prevent HBV flare‐up during campath treatment in patients tested positive for HBs antigen.

Bisphosphonates in early multiple myeloma

To the Editor: We read the recent publication by Jantunen on Bisphosphonate therapy in multiple myeloma: past, present, future with great interest (1). We would like to add some novel findings concerning the pathophysiology of the myeloma bone disease and recent developments in its treatment. Human myeloma cells express a potent osteoclastogenic factor, the receptor activator of NF-jB (RANK) ligand (2, 3). RANK ligand activates its specific receptor RANK, which is located on osteoclasts. Recent studies showed that RANK ligand is the key mediator of osteoclastogenesis and plays a crucial role in bone destruction in malignant bone disease (4). The level of RANK ligand expression on the surface of bone marrow plasma cells correlates with the bone status of the patients. Myeloma patients with osteolytic bone lesions showed a significantly higher expression of RANK ligand compared with patients without osteolysis (P < 0.0005) (5, 6). Furthermore, angiogenesis may be related to bone resorption in multiple myeloma (7, 8). As mentioned by Jantunen (1), magnetic resonance imaging (MRI) has been established as a more sensitive technique than conventional radiography to depict bone abnormalities in multiple myeloma. Although MRI is a useful and sensitive tool to discover myeloma-induced bone disease, this technique remains expensive and is not always available. We previously published that suitable markers of osteoclastic bone destruction, e.g. serum carboxy-terminal telopeptide of type-I collagen (ICTP) levels increase parallel to advanced disease stages and differ significantly between individuals with monoclonal gammopathy of undetermined significance (MGUS) and patients with myeloma in stage I (9). Furthermore, we analyzed serum ICTP levels and MRI in patients with multiple myeloma, who had no skeletal abnormalities in conventional radiographs. The positive and negative predictive value of ICTP for depiction of MRI abnormalities as a result of myeloma bone disease was about 85% (10, 11). Therefore ICTP can be used as an inexpensive parameter to identify myeloma patients with normal skeletal radiographs who have a high probability of skeletal involvement and deserve more accurate diagnostic evaluation using MRI. Recent data show that bisphosphonates may exert a direct antimyeloma effect by induction of apoptosis and cell-cycle arrest and indirect effects through their stimulation of cdT cells. Thus the effects of bisphosphonates on myeloma may not be limited to the inhibition of bone resorption. Zoledronate-induced cytotoxicity was significantly greater than that of pamidronate (12). Furthermore, enhanced osteoclastic bone resorption releases various cytokines and growth factors from the bone matrix that further stimulate myeloma cell proliferation, thus initiating and maintaining a vicious circle between osteoclasts and myeloma cells. Pamidronate and zoledronate induce a significant inhibition of the constitutive production of interleukin-6 by bone marrow stromal cells (13). In addition, recent data show that bisphosphonates may induce modifications of circulating angiogenic factor levels (14), and zoledronic acid exhibits marked antiangiogenic properties (15). Zoledronic acid treatment in mice models was shown to inhibit the development of myeloma bone disease, decrease osteolysis, tumor burden and angiogenesis, and increase survival (16, 17). Given these data and the new insights in the myeloma biology, it is time to clarify the role of bisphosphonates in stage I myeloma according to Salmon and Durie, as suggested in the review by Jantunen (1). Therefore, in 2000, the German Myeloma Study Group has initiated a prospective randomized phase III trial to answer the important question whether the treatment with a potent bisphosphonate (zoledronate) is capable of prolonging the progression-free survival in patients with stage I multiple myeloma (Principal investigators: M. Freund and O. Sezer).

Circulating hepatocyte growth factor levels in multiple myeloma

We read the study by Seidel et al (2002) with interest. The authors stated that hepatocyte growth factor (HGF) levels had not been previously examined in serum from younger myeloma patients nor in patients treated with high-dose chemotherapy. In 2001, we published an analysis of serum HGF levels in 67 patients with multiple myeloma or monoclonal gammopathy of undetermined significance (MGUS) and in 20 control subjects (Sezer et al, 2001). Furthermore, we simultaneously measured circulating serum levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), two additional angiogenic cytokines produced by myeloma cells. The age range of our myeloma patients was 39–86 years. No correlation of the HGF serum levels with age was found in either the myeloma patients or control subjects. In 32 myeloma patients treated with conventional or high-dose chemotherapy, preand post-treatment samples were analysed. Within the subgroup of patients who achieved a complete or partial response, there was a significant decrease in HGF, VEGF and bFGF values after therapy in comparison with their pretreatment levels (Sezer et al, 2001). The decrease occurred both in patients treated with conventional and high-dose chemotherapy. In contrast to this group, the patients who did not achieve remission showed no significant change in serum HGF, VEGF and bFGF levels (Sezer et al, 2000). In conclusion, these results indicated that the reduction of HGF levels is related to the chemotherapy-induced remission in multiple myeloma, irrespective of the dose level of the chemotherapy used. Christian Jakob Claudia Fleissner Ivana Zavrski Ulrike Heider Kurt Possinger Orhan Sezer Department of Haematology and Oncology, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Berlin, Germany. E-mail: sezer@charite.de