Summary The aim of this study was to evaluate the tissue infiltration and phenotypic adhesion profile of 5T2 multiple myeloma (MM) and 5T33 MM cells and to correlate it with that observed in human disease. For each line, 30 mice were intravenously inoculated with myeloma cells and at a clear-cut demonstrable serum paraprotein concentration; mice were sacrificed and a number of organs removed. The haematoxylin-eosin stainings on paraffin sections were complemented with immunohistochemistry using monoclonal antibodies developed against the specific MM idiotype. When analysed over time, 5T2 MM cells could be observed in bone marrow samples from week 9 after transfer of the cells. For the 5T33 MM, a simultaneous infiltration was observed in bone marrow, spleen and liver 2 weeks after inoculation. Osteolytic lesions consistently developed in the 5T2 MM, but this was not consistent for 5T33 MM. PCNA staining showed a higher proliferative index for the 5T33 MM cells. The expression of adhesion molecules was analysed by immunohistochemistry on cytosmears: both 5T2 MM and 5T33 MM cells were LFA-1, CD44, VLA-4 and VLA-5 positive. We conclude that both lines have a phenotypic adhesion profile analogous to that of human MM cells. As the 5T2 MM cells are less aggressive than the 5T33 MM cells, their organ distribution is more restricted to the bone marrow and osteolytic lesions are consistently present, the former cell line induces myeloma development similar to the human disease.Keywords: multiple myeloma; adhesion molecules; organ involvement; 5T2; 5T33 Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal expansion of malignant plasma cells secreting a monoclonal immunoglobulin (Ig). The disease is mainly localized in the bone marrow. In this microenvironment the myeloma plasma cells receive signals necessary for their proliferation, terminal differentiation and for the secretion of osteoclast-activating factors. The osteoclast-activating factors recruit osteoclasts, which induce in situ osteolytic bone lesions (Bataille et al, 1989;Alsina et al, 1996); this is one of the major characteristics of the disease. It has been suggested that both cytokines and adhesion molecules are involved in this complex network of signals (Van Riet and Van Camp, 1993).To elucidate the exact mechanisms described above, an in vivo MM model is necessary. Radl et al (1979) found that 0.5% of ageing C57BL/KaLwRij mice spontanously developed a disease reminiscent of MM. The MM cells isolated from the bone marrow of different mice (5T MM) did not grow in vitro but could be transplanted by intravenous injection into young recipients of the same strain. This transplantable model resembles the human disease in several aspects (Radl et al, 1988): myeloma occurred spontaneously, the frequency of development of the disease is age related, tumour load can be assessed by paraproteinaemia and the (Radl et al, 1988).In order to understand the homing mechanisms of the 5T MM cells to the bone marrow, it was essential to determine accurately the...

Hypoxia is associated with increased metastatic potential and poor prognosis in solid tumors. In this study, we demonstrated in the murine 5T33MM model that multiple myeloma (MM) cells localize in an extensively hypoxic niche compared with the naive bone marrow. Next, we investigated whether hypoxia could be used as a treatment target for MM by evaluating the effects of a new hypoxiaactivated prodrug TH-302 in vitro and in vivo. In severely hypoxic conditions, TH-302 induces G 0 /G 1 cell-cycle arrest by down-regulating cyclinD1/2/3, CDK4/6, p21 cip-1 , p27 kip-1 , and pRb expression, and triggers apoptosis in MM cells by upregulating the cleaved proapoptotic caspase-3, -8, and -9 and poly ADP-ribose polymerase while having no significant effects under normoxic conditions. In vivo treatment of 5T33MM mice induces apoptosis of the MM cells within the bone marrow microenvironment and decreases paraprotein secretion. Our data support that hypoxia-activated treatment with TH-302 provides a potential new treatment option for MM. (Blood. 2010;116(9):1524-1527) IntroductionMultiple myeloma (MM) is an incurable clonal B-cell malignancy characterized by the accumulation of neoplastic plasma cells in the bone marrow (BM). 1 Studies have shown that the intimate reciprocal relationship between tumor cells and the cellular and noncellular microenvironment plays a pivotal role in MM growth and survival. 2,3 Hypoxia, one of the important microenvironmental factors, is well known to be highly associated with increased angiogenesis and metastatic potential as well as poor prognosis in solid tumors. More recently, hypoxia has been demonstrated to be crucial for normal marrow hematopoiesis. [4][5][6] However, the role of hypoxia in the etiology, pathogenesis, and possible treatment of hematologic malignancies, such as MM, is still unknown.Given very low oxygen levels, as found in tumors, are rarely observed in normal tissues, the presence of hypoxic tumor cells is therefore regarded not only as an adverse prognostic factor but also as a potential target for tumor-specific treatment. Currently, several hypoxia-targeted therapeutics are under development. 7-12 TH-302 is a new hypoxia-activated prodrug that is being evaluated in phase 1/2 clinical trials for the treatment of solid tumors as a monotherapy and in combination with 4 chemotherapeutic agents (gemcitabine, pemetrexed, doxorubicin, and docetaxel). TH-302 is a 2-nitroimidazole prodrug of the cytotoxin bromo-isophosphoramide mustard, with a favorable physicochemical, metabolic, and pharmacokinetic profile and exhibits hypoxiaselective cytotoxicity across a broad spectrum of human cancer cell lines in vitro and in vivo efficacy in a large panel of human tumor xenografts. 13,14 The doses used in the clinical studies are in the same range as the doses demonstrating efficacy in both in vitro and in vivo preclinical models.In this study, we investigated the hypoxic nature of MM by staining the BM of naive and 5T33MM mice with the exogenous hypoxia marker pimonidazole and endogenou...

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.

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There is still much controversy about the precursor cell type in multiple myeloma (MM). Some authors claim that it is a pre-B cell, others state that it is a memory B cell or plasmablast. We have recently shown that the VDJ region of the MM immunoglobulin heavy chain gene is somatically hypermutated and antigen selected, without intraclonal variation or evolution in time. By using a patient-specific PCR approach we have now obtained evidence that the premyeloma cell can be situated in the pre-switched B-cell compartment and that heavy chain switching can occur without further somatic mutation. Based on the MM immunoglobulin sequences derived from the bone marrow, patient-specific CDR2 and CDR3 oligonucleotides were designed. B lymphocytes were separated from plasma cells based on the expression of CD19 and HLA class II or surface bound IgM using immunomagnetic beads. The expressed Ig sequences were amplified by RT-PCR using patient specific CDR2 primers and isotype specific primers (C mu, C gamma, and C alpha). Myeloma-specific Ig sequences were detected by a myeloma-specific CDR3 probe and sequenced. In one out of five cases we found in the peripheral blood clonally related IgM and IgA sequences with the same somatic mutations as the MM-IgG sequence. In another case of an IgG MM we found in the bone marrow clonally related IgA sequences with the same somatic mutations. These findings, together with the fact that myeloma-Ig genes contain somatic mutations without intraclonal variation, suggest that the clonogenic cell in multiple myeloma can originate from a pre-switched but somatically mutated B cell.