Unraveling the biology of multiple myeloma disease: cancer stem cells, acquired intracellular changes and interactions with the surrounding micro-environment.

Cancerogenesis is believed to be a multistep process composed of different alterations that drive the transformation of normal human cells into highly malignant derivatives. These changes include self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, the evasion of programmed cell death, a limitless replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Some evidence suggests that these cancer cells are derived from immature, proliferative precursors that feed the cancer cell compartment. Both, precursors and cancer cells, intensively interact with their surroundings, commonly described as their micro-environment. The processes above are also important in the biology of multiple myeloma, a malignant bone marrow disorder consisting of monoclonal plasma cells accumulation. In the current review we describe the biology of this disease, stressing the origin of myeloma cells, their acquired genetic changes and interactions with their bone marrow microenvironment.

Extravasation and homing mechanisms in multiple myeloma.

Multiple myeloma (MM) is a malignant B-cell disorder characterized by a monoclonal expansion of plasma cells (PC) in the bone marrow (BM). During the main course of disease evolution, MM cells depend on the BM microenvironment for their growth and survival. Reciprocal interactions between MM cells and the BM mediate not only MM cell growth, but also protect them against apoptosis and cause bone disease and angiogenesis. A striking feature of MM represents the predominant localization and retention of MM cells in the BM. Although BM PC indeed represent the main neoplastic cell type, small numbers of MM cells can also be detected in the peripheral blood circulation. It can be assumed that these circulating cells represent the tumour-spreading component of the disease. This implicates that MM cells have the capacity to (re)circulate, to extravasate and to migrate to the BM (homing). In analogy to the migration and homing of normal leucocytes, the BM homing of MM cells is mediated by a multistep process of extravasation with adhesion to the endothelium, invasion of the subendothelial basement membrane, followed by further migration within the stroma, mediated by chemotactic factors. At the end stage of disease, MM cells are thought to develop autocrine growth supporting loops that enable them to survive and proliferate in the absence of the BM microenvironment and to become stroma-independent. In this stage, the number of circulating cells increases and growth at extramedullary sites can occur, associated with alteration in adhesion molecule and chemokine receptor expression. This review summarizes the recent progress in the study of the extravasation and homing mechanisms of MM cells.

The involvement of osteopontin and its receptors in multiple myeloma cell survival, migration and invasion in the murine 5T33MM model.

Multiple myeloma (MM) is a malignancy characterised by the accumulation of monoclonal plasma cells in the bone marrow. Different reports indicate the expression of CD44 variant isoforms (CD44v) by MM cells. Osteopontin (OPN), which is expressed by MM cells, is known to be a ligand for CD44v6. In this study, we investigated the role of OPN with emphasis on a functional correlation between OPN and CD44v in the 5T33MM model. Our group reported the expression of CD44v by 5T33MM cells. Using this model, we have demonstrated the secretion of OPN by 5T33MM cells. OPN affected 5T33MM cell survival by increasing proliferation and inhibiting apoptosis. OPN also stimulated 5T33MM cell migration, transendothelial migration and matrix metalloproteinase-9 activity. We confirmed the proliferative and migratory effects of OPN on human MM cells. By applying inhibiting anti-CD44v6 antibodies, we found that OPN stimulated cell proliferation by engaging this isoform. Anti-CD44v antibodies and RGD peptides both inhibited cell migration, suggesting an involvement of both, CD44v isoforms and integrins. In conclusion, OPN may act as a mediator of MM cell survival by engaging CD44v. The protein is further involved in migration and invasion of MM cells through the activation of either alphavbeta3 integrin or CD44v isoforms.

Role of the hypoxic bone marrow microenvironment in 5T2MM murine myeloma tumor progression.

BACKGROUND AND OBJECTIVES: Unlike most other tumors, multiple myeloma (MM) cells have to survive and to grow in a bone marrow (BM) microenvironment which is already hypoxic by nature. BM hypoxia is crucial for normal hematopoiesis. However, how BM hypoxia and MM affect each other is unknown. We addressed this topic in the 5T2MM mouse model. DESIGN AND METHODS: Levels of hypoxia in the BM of control and tumor-bearing mice were analyzed by flow cytometric analysis of pimonidazole hypoxyprobe binding and hypoxia-inducible factor 1alpha (HIF-1alpha) expression. Micro-vessel density was measured by CD31 staining of BM sections for immunohistochemistry. Apoptosis sensitivity of CD45 5T2MM subsets in hypoxic conditions were analyzed by detection of active caspase-3. RESULTS: Analysis of control and 5T2MM diseased mice injected with pimonidazole hypoxyprobe indicated that both normal BM and myeloma-infiltrated BM are hypoxic. However, the hypoxia in the myelomatous BM was significantly decreased. Analysis of HIF-1alpha expression, a surrogate marker of hypoxia, also demonstrated significantly lower levels of hypoxia in myeloma-infiltrated BM. HIF-1alpha expression was inversely correlated with the micro-vessel density. In vitro culture of 5T2MM cells under hypoxic conditions indicated induction of apoptosis in the CD45- MM-fraction, but not in the CD45+ 5T2MM-cells. INTERPRETATION AND CONCLUSIONS: These data suggest that native BM hypoxia is advantageous for the tumor-initiating CD45+ 5T2MM-cells. Together with the decreased hypoxia in myeloma-infiltrated BM it also indicates that myeloma-associated angiogenesis is functional and permissive for the expansion of CD45- 5T2MM-cells. All together the data raise the possibility of an important role of BM hypoxia in myeloma tumor progression.

Catheter tip position as a risk factor for thrombosis associated with the use of subcutaneous infusion ports.

AIMS: The use of subcutaneous infusion ports has become standard practice to provide a long-term venous access in oncological patients. The aim of this retrospective study was to assess the different complications of infusion ports in our population and to identify predisposing factors. PATIENTS AND METHODS: We reviewed the medical records of 437 patients who were followed at the Oncology/Haematology Department of our hospital. Of these patients, there were 370 (84.4%) with solid tumours and 58 (13.2%) with haematological disease. The position of the catheter tip was evaluated by reviewing the available chest radiographs or phlebographies. MAIN RESULTS: Analysis of the records showed that 346 patients (79.17%) had no complications. The most common complications after implantation were thrombosis (8.46%), catheter dysfunction (4.8%) and infections (4.4%). Univariate and multivariate analysis showed that catheter tip positioning was the most important predisposing risk factor for thrombosis. Catheter tips positioned in the brachiocephalic vein or in the cranial part of the superior vena cava were associated with a high risk of thrombosis. Other significant risk factors were gender and initial diagnosis. Female patients and patients with lung cancer also had an elevated risk of developing a thrombosis. CONCLUSIONS: Compared to other reports, we noted a higher rate of thrombosis and port dysfunction. Since catheter tip position was a predisposing factor for developing a thrombosis, correct catheter position has to be ensured during placement. Prophylactic antithrombotic treatment might be beneficial in the event of failure to position the catheter correctly.

Post-transplantation tumour load in bone marrow, as assessed by quantitative ASO-PCR, is a prognostic parameter in multiple myeloma.

High-dose therapy (HDT) and autologous transplantation prolongs remission duration and survival in multiple myeloma (MM), but relapse still occurs at a median of 2 years post-HDT. In order to investigate whether the number of residual tumour cells in the bone marrow (BM) after transplantation can predict the duration of response, a quantitative allele-specific oligonucleotide polymerase chain reaction (qASO-PCR) assay was used to measure tumour load in BM at 3-6 months post-HDT in 67 patients. The method of maximally selected log-rank statistics was used to test for the existence of a cut-off value in the BM tumour load data set. A cut-off value with respect to progression-free survival (PFS) was identified (P = 0.001). The estimated threshold for placing patients into a "good" or "bad" prognostic group was 0.015% (n = 22 and 38 respectively) with a median PFS of 64 months vs. 16. Multivariate analysis showed grouping by PCR result to be an independent prognostic factor for PFS (estimated hazard ratio after shrinkage, 3.91). This study identifies for the first time a threshold of the post-HDT tumour load with prognostic significance for PFS in MM. Quantitative molecular assessment thus may help to identify those patients who are in need of further treatment early after autologous transplantation.

Angiogenic switch during 5T2MM murine myeloma tumorigenesis: role of CD45 heterogeneity.

The active role of angiogenesis during disease progression is well recognized in solid tumors. In hematologic malignancies such as multiple myeloma (MM), it is not known whether tumor neovascularization is an epiphenomenon or whether it is actively involved in disease progression. At clinical presentation, myeloma disease and the associated angiogenesis are both well established. Here the 5T2MM murine model was used to analyze angiogenesis during preclinical myeloma stages. Bone marrow (BM) of 5T2MM-inoculated mice was analyzed at weekly intervals until the end stage of the disease. Histologic analysis and assessment of microvessel density (MVD) by CD31 staining demonstrated a preangiogenic stage of small tumor aggregates followed by an angiogenic switch and subsequently an angiogenic stage of progressive tumor growth and large, confluent tumor nodules. Flow cytometric analysis that indicated an increase in percentage CD45- MM cells preceded the angiogenic switch. Real-time polymerase chain reaction (RT-PCR) of sorted CD45+ and CD45- MM cells indicated higher vascular endothelial growth factor 120 (VEGF120) and VEGF164 transcripts in CD45- MM cells. VEGF enzyme-linked immunosorbent assay (ELISA) revealed high secretion by CD45- MM cells but no protein secretion by CD45+ MM cells, indicating angiogenic heterogeneity among the MM cells. These data suggest that, like in solid tumors, angiogenic switch and angiogenic heterogeneity exist in MM.

Delayed in vivo disease progression is associated with high proportions of CD45+ myeloma cells in the 5T2MM murine model.

Both CD45(+) and CD45(-) multiple myeloma (MM) cells are observed in the bone marrow (BM) of MM patients; however, their impact on the outcome of the disease is unknown. Most (92%) of the mice injected with murine 5T2MM cells develop myeloma in 10-12 weeks and show hind leg paralysis at the end phase of the disease. In the end stage 5T2MM cells are predominantly CD45(-), in analogy to the common human situation. Herein we report that 8% of the mice have a delayed tumor progression (14-24 weeks) with a complete different feature in the end stage of the disease. These animals had typically a bowed back and never got paralyzed. The MM cells in the BM of these mice were predominantly CD45(+). These data indicate that CD45 subsets are associated with the final outcome of myeloma disease.

Multiple myeloma tumor progression in the 5T2MM murine model is a multistage and dynamic process of differentiation, proliferation, invasion, and apoptosis.

At clinical presentation, multiple myeloma (MM) is already a well-established disease. The processes involved in earlier stages are, however, unknown. Here the 5T2MM murine model was used to analyze differentiation, proliferation, invasion, and apoptosis of MM cells during disease progression. Naive mice were injected with 5T2MM cells and from the onset of the experiment 3 mice were killed each week until the end stage. Myeloma cells were isolated from the bone marrow and selected by sequential gating of 5T2MM idiotype(+) cells by flow cytometry. Microscopic analysis of these sorted 5T2MM idiotype(+) cells confirmed their identity as true myeloma cells. Based on serum paraprotein concentration and bone marrow tumor load, 3 disease stages were distinguished: a quiescent stage, an intermediate stage, and an end stage, of slow, moderate, and accelerated tumor progression, respectively. In the quiescent stage, the majority of the myeloma cells were CD45(+)CD138(-)IL-6R alpha(+), corresponding to an immature, invasive, and apoptosis-resistant phenotype. In the end stage the majority of the myeloma cells had differentiated into CD45(-)CD138(+)IL-6R alpha(-) cells, corresponding to a mature, less invasive, and apoptosis-sensitive phenotype. In the intermediate stage a gradual transition from the quiescent toward the end stage was observed. In line with these data, analysis of sorted 5T2MM cells demonstrated a significant decrease in invasive capacity and a significant increase in (dexamethasone-induced) apoptosis sensitivity and in proliferation during the disease progression. These data suggest that myeloma disease progression is a multistage and dynamic process of differentiation, proliferation, invasion, and apoptosis.