Antitumour and antiangiogenic effects of Aplidin in the 5TMM syngeneic models of multiple myeloma.

Aplidin is an antitumour drug, currently undergoing phase II evaluation in different haematological and solid tumours. In this study, we analysed the antimyeloma effects of Aplidin in the syngeneic 5T33MM model, which is representable for the human disease. In vitro, Aplidin inhibited 5T33MMvv DNA synthesis with an IC(50) of 3.87 nM. On cell-cycle progression, the drug induced an arrest in transition from G0/G1 to S phase, while Western blot showed a decreased cyclin D1 and CDK4 expression. Furthermore, Aplidin induced apoptosis by lowering the mitochondrial membrane potential, by inducing cytochrome c release and by activating caspase-9 and caspase-3. For the in vivo experiment, 5T33MM-injected C57Bl/KaLwRij mice were intraperitoneally treated with vehicle or Aplidin (90 microg kg(-1) daily). Chronic treatment with Aplidin was well tolerated and reduced serum paraprotein concentration by 42% (P<0.001), while BM invasion with myeloma cells was decreased by 35% (P<0.001). Aplidin also reduced the myeloma-associated angiogenesis to basal values. This antiangiogenic effect was confirmed in vitro and explained by inhibition of endothelial cell proliferation and vessel formation. These data indicate that Aplidin is well tolerated in vivo and its antitumour and antiangiogenic effects support the use of the drug in multiple myeloma.

Novel Tropheryma species in a lung biopsy sample from a kidney transplant recipient.

OBJECTIVES: A kidney transplant recipient with recurrent pleuritis underwent an open lung biopsy, the results of which revealed multiple nodular infiltrates. Grocott and periodic acid-Schiff staining were positive. Fungal and Tropheryma whipplei PCR were, however, negative. Further identification was needed. METHODS: Formalin-fixed, paraffin-embedded (FFPE) extraction was performed using an FFPE extraction kit. T. whipplei was searched for using a real-time PCR targeting the noncoding repeat specific for T. whipplei. Identification of the bacteria in the extract was done using 16S rDNA and 23S rDNA sequencing and BLAST analysis. Internal transcribed spacer PCR was used for fungal DNA identification. RESULTS: The FFPE extract was negative for fungi and T. whipplei. 16S rDNA sequence analysis of a 1375 bp fragment gave T. whipplei as the best match with 26 mismatches, resulting in only 98% agreement. Sequence analysis of the 23S rDNA gene again gave T. whipplei as the best match, but with only 91% agreement. A pan-Tropheryma 16S rDNA real-time PCR was developed, and both the biopsy sample and a respiratory sample of the patient were strongly positive. The patient received antimicrobial treatment targeting T. whipplei with good clinical outcome. CONCLUSIONS: 16S and 23S rDNA sequencing gave T. whipplei as the best hit, although with limited agreement. These findings suggest that a novel Tropheryma species that lacks the noncoding repeat, most frequently used for molecular detection of Whipple disease, might be the cause of the pulmonary disease. Adaptation of current PCR protocols is warranted in order to detect all Tropheryma species.

Endothelial cell-driven regulation of CD9 or motility-related protein-1 expression in multiple myeloma cells within the murine 5T33MM model and myeloma patients.

The cell surface expression of CD9, a glycoprotein of the tetraspanin family influencing several processes including cell motility and metastasis, inversely correlates with progression in several solid tumors. In the present work, we studied the expression and role of CD9 in multiple myeloma (MM) biology using the 5T33MM mouse model. The 5T33MMvitro cells were found to be CD9 negative. Injection of these cells in mice caused upregulation of CD9 expression, while reculturing them resulted in downregulation of CD9. Coculturing of CD9-negative 5T33MMvitro cells with BM endothelial cells (BMECs) resulted in a partial retrieval of CD9. Laser microdissection followed by real-time polymerase chain reaction and immunohistochemistry performed on bone sections of 5T33MMvivo diseased mice demonstrated strong local expression of CD9 on MM cells in contact with BMEC compared to MM cells further away. These findings were also confirmed by immunohistochemistry in MM patients. Neutralizing anti-CD9 antibodies inhibited transendothelial invasion of CD9-expressing human MM5.1 and murine 5T33MMvivo cells. In conclusion, we provide evidence that CD9 expression by the MM cells is upregulated in vivo by close interaction of the cells with BMEC and that CD9 is involved in transendothelial invasion, thus possibly mediating homing and/or spreading of the MM cells.

Asymptomatic pheochromocytoma in the posterior mediastinum.

A 66-year-old female patient was treated for a posterior mediastinal tumour with unknown histology. During thoracotomy, repetitive hypertensive crises had to be treated. The tumour was completely resected. Pathology revealed an extra-adrenal pheochromocytoma. Diagnosis of pheochromocytoma is usually made on the basis of clinical presentation and elevated catecholamine levels in serum or urine. Imaging is used primarily for localizing tumours prior to surgery. Complete surgical excision is the primary treatment. The only absolute indicator of malignancy is the identification of distant metastases to bone, liver, lung or lymph nodes.

A unique pathway in the homing of murine multiple myeloma cells: CD44v10 mediates binding to bone marrow endothelium.

Our group recently reported that multiple myeloma (MM) cells preferentially adhere to bone marrow (BM) endothelial cells and selectively home to the BM, suggesting the involvement of specific adhesive interactions in this process. The highly regulated expression of CD44 variant isoforms (CD44v) on the MM cells makes them good candidate adhesion molecules involved in this homing. We addressed this in the 5T experimental mouse model of myeloma. Fluorescence-activated cell sorting analysis demonstrated expression of CD44v6, CD44v7, and CD44v10 on the in vivo growing 5T2MM and 5T33MM myeloma lines. Antibody blocking experiments revealed the involvement of CD44v10 in the adhesion of 5T2MM and 5T33MM cells to BM endothelial cells. Coinjection of anti-CD44v10 antibodies with the myeloma cells into syngeneic mice demonstrated a selective blocking of their BM homing which resulted in a decreased BM tumor load and serum paraprotein at the end stage of the disease. The highly restricted expression of CD44v10 on MM cells, the blocking of MM adhesion to BM endothelial cells and of homing to BM by anti-CD44v10, and the decreased BM tumor load suggest that myeloma cells home to the BM via interactions mediated by this specific region of the adhesion molecule CD44.

In vivo induction of insulin-like growth factor-I receptor and CD44v6 confers homing and adhesion to murine multiple myeloma cells.

One of the main characteristics of multiple myeloma (MM) cells is their specific homing and growth in the bone marrow (BM). Differences between stroma-dependent and -independent MM cell lines may reveal key molecules that play important roles in their homing to the BM. We addressed this topic with a murine MM model, including the in vivo 5T33MM (5T33MMvv) stroma-dependent cell line and its in vitro stroma-independent variant (5T33MMvt). Fluorescence-activated cell-sorting analysis showed expression of insulin-like growth factor (IGF)-I receptor and CD44v6 on all 5T33MMvv cells but not on 5T33MMvt cells. Checkerboard analysis and adhesion assays revealed IGF-I-dependent chemotaxis toward BM-conditioned medium and involvement of CD44v6 in the adhesion to BM stroma of only 5T33MMvv cells. However, when 5T33MMvt cells were injected in vivo (5T33MMvt-vv), after 18 h the MM cells harvested from BM were IGF-I receptor and CD44v6 positive. This up-regulation was confirmed in 5T33MMvt-vv cells isolated from terminally diseased animals. These ST33MMvt-vv cells exhibited IGF-I-dependent chemotaxis and CD44v6-dependent adhesion to BM stroma. In vitro culture of the 5T33MMvt-vv cells could completely down-regulate IGF-I receptor and CD44v6. In fact, we could show that direct contact of 5T33MMvt cells with BM endothelial cells is a prerequisite for IGF-I receptor and CD44v6 up-regulation. These data indicate that the BM microenvironment is capable of up-regulating molecules such as IGF-I receptor and CD44v6, which facilitate homing of MM cells to the BM and support their adhesion to BM stroma.

Bortezomib-induced Sweet's syndrome.

Sweet's syndrome is an uncommon acute skin disease, associated with a variety of medical problems. The drug-induced variant is even rarer. We describe two cases of this syndrome associated with the administration of the proteasome inhibitor bortezomib. The diagnostic criteria for drug-induced Sweet's syndrome as proposed by Walker and Cohen were fulfilled. Vasculitis and neutrophilic eccrine hidradenitis were excluded.

The role of the bone marrow microenvironment in multiple myeloma.

Multiple myeloma (MM) is a malignant disease that results from an excess of monotypic plasma cells in the bone marrow (BM). This malignancy is characterised by complex karyotypic aberrancies. In 60% of all MM there are recurrent primary translocations involving the heavy chain gene locus. The MM cells strongly interact with the BM microenvironment, which is composed of endothelial cells, stromal cells, osteoclasts, osteoblasts, immune cells, fat cells and the extracellular matrix. This interaction is responsible for the specific homing in the BM, the proliferation and survival of the MM cells, the resistance of MM cells to chemotherapy, the development of osteolysis, immunodeficiency and anaemia. New therapeutic agents target both the MM, as well as the interaction MM cell - BM microenviroment.

Myeloma isotype-switch variants in the murine 5T myeloma model: evidence that myeloma IgM and IgA expressing subclones can originate from the IgG expressing tumour.

Isotype-switch variants can easily be detected in a significant proportion of multiple myeloma (MM) patients. The biological significance of these isotype-switch variants remains obscure. Therefore, we studied the appearance of these isotype-switch variants in two murine MM models, 5T2MM and 5T33MM, both of IgG isotype. With a MM-specific PCR assay we could detect isotype-switch variants in the bone marrow of both the 5T2MM and the 5T33MM bearing mice, reflecting again the close resemblance of this mouse model to the human MM. These isotype-switch variants were not found in an in vitro stroma-independent variant of the 5T33MM line. However, when this 5T33MMvitro line was injected into young syngeneic mice, isotype-switch variants appeared thereafter in the isolated tumour cells. These isotype-switch variants could only originate from the MM-IgG expressing cell since IgG subclones from the 5T33MMvitro line again gave rise to isotype-switch variants. The appearance of IgA cells can be explained by down-stream switching of IgG to IgA, while the emergence of IgM cells have to occur via trans-switching to the sister chromatid as the Cmu region is deleted from the CIS-chromosome. This study demonstrates that isotype-switch variants originate from the major tumour clone suggesting no role for the MM-IgM expressing cell as a pre-switch precursor MM cell. The appearance of isotype-switch variants should be considered as a rare but normal event now becoming visible due to the high number of clonal cells present in MM.

In vivo homing and differentiation characteristics of mature (CD45-) and immature (CD45+) 5T multiple myeloma cells.

Multiple myeloma, a plasma cell malignancy, is predominantly localized in the bone marrow. These tumoral cells display a heterogeneous expression of CD45. It is, however, unclear which subpopulation is responsible for the homing and outgrowth of the myeloma cells. In this work, we investigated the in vivo homing, proliferation, and differentiation of both CD45+ and CD45- cells in two murine myeloma models.5T2MM and 5T33MM in vivo lines of murine multiple myeloma were used. CD45 and IGF-I receptor expression was analyzed by FACS. Proliferative capacity was assessed by in vivo bromodeoxyuridine incorporation. 5TMM cells were separated into CD45+ and CD45- fractions by MACS. Initial homing was investigated in vivo by tracing of radioactively labeled cells. Myeloma cells were detected by FACS and histology. Osteolytic lesions were analyzed by radiography. Both CD45+ and CD45- 5TMM cells were able to home to the bone marrow, although the migration of the latter subset was lower, which was related to a low IGF-I receptor expression. Recipients of both fractions developed myeloma as evidenced by the presence of serum paraprotein, osteolytic lesions, and bone marrow infiltration by myeloma cells. The tumor load in the recipients of CD45- cells was higher than the CD45+ cells, which could be explained by a lower proliferation rate of the latter population. While the separated cells before injection had a homogenous expression of CD45, cells isolated from the bone marrow of these terminally diseased mice had a heterogeneous expression pattern, indicating an in vivo differentiation pattern of CD45- to CD45+ cells and vice versa. We conclude that both CD45+ and CD45- 5TMM subpopulations contain clonogenic myeloma cells with bone marrow homing and proliferative capacity.

Angiogenesis and the role of bone marrow endothelial cells in haematological malignancies.

Increased microvessel density (MVD) has been observed in the bone marrow (BM) of patients with multiple myeloma (MM), acute lymphoblastic leukaemia, acute myeloid leukaemia, and myelodysplastic and myeloproliferative syndrome. The MVD is the net result of cumulative phases of angiogenesis and angio-regression and is as such not an indicator of the ongoing angiogenesis at the time of biopsy. There is, therefore, a need for additional methods that allow the estimation of ongoing angiogenesis. Double immunostainings for CD34 and Ki-67 can be used on paraffin-embedded tissue to determine the endothelial proliferation fraction. The BM endothelial cells, as a component of the BM stroma, have a close interaction with the malignant cells. In MM, for example, they are involved in the specific homing and are a source of paracrine growth factors. Targeting the BM microvessels will not only influence the nutrient and oxygen supply, but will in addition reduce the growth stimuli provided by the EC.

Microvessel density, endothelial-cell proliferation and carbonic anhydrase IX expression in haematological malignancies, bone-marrow metastases and monoclonal gammopathy of undetermined significance.

The aim of the study was to compare the angiogenic status, potential qualitative differences in microvessels and carbonic anhydrase IX expression in bone-marrow (BM) metastases and different haematological tumours at time of diagnosis. The microvessel density (MVD), endothelial-cell proliferation (ECP) and carbonic anhydrase IX (CA IX) immunoreactivity were determined on 210 trephine biopsies from 57 patients with multiple myeloma (MM), 13 with acute myeloid leukaemia (AML), 48 with chronic myeloproliferative syndrome (CMPS), 26 with chronic lymphocytic leukaemia (CLL), 25 with epithelial BM metastases, 18 with monoclonal gammopathy of undetermined significance (MGUS) and from a control group composed of 23 patients without haematological neoplasm. There was an increased MVD and ECP in epithelial BM metastases, MM, AML, CMPS and in a part of CLL. While an ECP greater than 0 was detected in 72% of MM, 75% of CMPS and 92% of AML, it was invariably observed (100%) in the BM metastases. The absence of ECP together with a MVD comparable with the control group in our MGUS cases supports the view that MGUS is a pre-angiogenic condition. Qualitative differences in microvessels were associated with growth patterns in MM and CLL and were observed between the different entities of CMPS. In one-third of the epithelial BM metastases, there was a focal CA IX immunoreactivity, which was never observed in the haematological diseases.

Assessment of the ear swelling test and the local lymph node assay in hamsters.

In a hamster model, we compared contact sensitivity to the metal salt, potassium dichromate, to that of oxazolone, a well-known strong sensitizing agent. Using the ear swelling test, originally developed in mice, no significant differences could be observed between animals treated with potassium dichromate and controls, although oxazolone-treated animals showed a significant increase in ear thickness compared to controls. These observations were confirmed using the local lymph node assay (LLNA) where oxazolone proved to be a strong sensitizing agent, and potassium dichromate only resulted in a weak response. When the draining auricular lymph nodes were compared with the inguinal lymph nodes in the LLNA, more pronounced effects were obtained with the auricular lymph nodes. This study indicates that, also in hamsters, the LLNA is a feasible sensitization test system.

Cystosarcoma phyllodes of the prostate with rhabdomyoblastic differentiation.

A 36-year-old man presented with recurrent urinary obstruction and an enlarged, partially cystic prostate tumour on ultrasonography. Microscopically, the tumour was composed of cystically dilated ducts with leaf-like stromal projections in the lumen. A portion of the stroma was very cellular with atypia and a high mitotic rate. In addition, there was a small focus with well differentiated rhabdomyoblasts. This case is the second description of a cystosarcoma phyllodes of the prostate with rhabdomyoblasts differentiation. With radical surgery and chemotherapy only partial remission could be obtained.

Malignant large cell calcifying Sertoli cell tumor of the testis.

A 45-year old man presented with a slow-growing, unilateral beige testicular mass, with a diameter of 4 cm. The testosterone, FSH, LH, estradiol and betahCG serum levels were within normal limits, and there were no associated hormonal syndromes. The patient was treated with inguinal orchidectomy. Microscopically, the tumor was composed of nests of cells with large eosinophilic, slightly granular cytoplasm. There was only a mild degree of atypia and no mitotic activity. The tumor extended into the rete testis. There were intratumoral calcifications, and in the vicinity of the tumor, there was intratubular growth. Although this case is histologically similar to the three previously reported cases of clinically benign large cell calcifying Sertoli cell tumor of the testis with rete testis involvement, the current patient developed right sided para-aortic lymph node metastases 18 months after the initial diagnosis.

Primary ovarian small cell carcinoma of the pulmonary type: a case report and review of the literature.

Small cell carcinoma of the ovary is a rare type of ovarian carcinoma with a poor prognosis. Two types should be distinguished: the hypercalcemic type and the pulmonary type. We report the case history of a 54-year-old woman with both a Stage IIIC small cell carcinoma, pulmonary type and a well-differentiated endometrioid adenocarcinoma of the left ovary in combination with a Brenner tumor in the right ovary. A review of the literature on small cell carcinoma of the ovary is given and the findings of our patient are brought into perspective in terms of both histopathogenesis and treatment outcome.

Venous aneurysm four years after greater saphenous vein stripping.

This report describes the diagnosis and surgical treatment of a true femoral venous aneurysm. Its aetiology remains unknown. These lesions may be the source of pulmonary emboli and therefore prophylactic surgery is strongly recommended for patients with lower extremity deep venous aneurysms.

The effects of JNJ-26481585, a novel hydroxamate-based histone deacetylase inhibitor, on the development of multiple myeloma in the 5T2MM and 5T33MM murine models.

Multiple myeloma (MM) is a B-cell malignancy, which often remains incurable because of the development of drug resistance governed by the bone marrow (BM) microenvironment. Novel treatment strategies are therefore urgently needed. In this study, we evaluated the anti-MM activity of JNJ-26481585, a novel 'second-generation' pyrimidyl-hydroxamic acid-based histone deacetylase inhibitor, using the syngeneic murine 5TMM model of MM. In vitro, JNJ-26481585 induced caspase cascade activation and upregulation of p21, resulting in apoptosis and cell cycle arrest in the myeloma cells at low nanomolar concentrations. Similar results could be observed in BM endothelial cells using higher concentrations, indicating the selectivity of JNJ-26481585 toward cancer cells. In a prophylactic and therapeutic setting, treatment with JNJ-26481585 resulted in an almost complete reduction of the tumor load and a significant decrease in angiogenesis. 5T2MM-bearing mice also developed a MM-related bone disease, characterized by increased osteoclast number, development of osteolytic lesions and a reduction in cancellous bone. Treatment of these mice with JNJ-264815 significantly reduced the development of bone disease. These data suggest that JNJ-26481585 has a potent anti-MM activity that can overcome the stimulatory effect of the BM microenvironment in vivo making this drug a promising new anti-MM agent.

Current diagnostic criteria for the chronic myeloproliferative disorders (MPD) essential thrombocythemia (ET), polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF).

The clinical criteria for the diagnosis of essential thrombocythemia (ET) according to the polycythemia vera study group (PVSG) do not distinguish between ET and thrombocythemia associated with early stage PV and prefibrotic chronic idiopathic myelofibrosis (CIMF). The clinical criteria of the PVSG for the diagnosis of polycythemia vera (PV) only detects advanced stage of PV with increased red cell mass. The bone marrow criteria of the World Health Organization (WHO) are defined by pathologists to explicitly define the pathological criteria for the diagnostic differentiation of ET, PV, and prefibrotic and fibrotic CIMF. As the clinical PVSG and the pathological WHO criteria show significant shortcomings, an updated set of European Clinical and Pathological (ECP) criteria combined with currently available biological and molecular markers are proposed to much better distinct true ET from early PV mimicking ET, to distinguish ET from thrombocythemia associated with prefibrotic CIMF, and to define the various clinical and pathological stages of PV and CIMF that has important therapeutic and prognostic implications. Comparing the finding of clustered giant abnormal megakaryocytes in a representative bone marrow as a diagnostic clue to MPD, the sensitivity for the diagnosis of MPD associated with splanchnic vein thrombosis was 63% for increased red cell mass, 52% for low serum EPO level, 72% for EEC, and 74% for splenomegaly indicating the superiority of bone marrow histopathology to detect masked early and overt MPD in this setting. The majority of PV and about half of the ET patients have spontaneous EEC, low serum EPO levels and PRV-1 over-expression and are JAK2 V617F positive. The positive predictive value for the diagnosis of PV of spontaneous growth of endogenous erythroid colonies (EEC) of peripheral blood (PB) and bone marrow (BM) cells is about 80-85% when either PB or BM EEC assays, and up to 94% when BM and PB EEC assays were performed. The diagnostic impact of low serum EPO levels (ELISA assay) in a large study of 186 patients below the normal range (<3.3 IU/l) had a sensitivity specificity and positive predictive value of 87%, 97% and 97.8%, respectively, for the diagnosis of PV. There is a significant overlap of serum EPO levels in PV versus control and controls versus SE. The specificity of a JAK2 V617F PCR test for the diagnosis of MPD is high (near 100%), but only half of ET and MF (50%) and the majority of PV (up to 97%) are JAK2 V617F positive. The use of biological markers including JAK2 V617 PCR test, serum EPO, PRV-1, EEC, leukocyte alkaline phosphatase score and peripheral blood parameters combined with bone marrow histopathology has a high sensitivity and specificity (almost 100%) to diagnose the early and overt stages of ET, PV and CIMF in JAK2 V617F positive and negative MPDs.