Jan Eucker

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).

Anti-tumor effect of honokiol alone and in combination with other anti-cancer agents in breast cancer

Honokiol, an active component isolated and purified from Chinese traditional herb magnolia, was demonstrated to inhibit growth and induce apoptosis of different cancer cell lines such as human leukaemia, colon, and lung cancer cell lines; to attenuate the angiogenic activities of human endothelial cells in vitro; and to efficiently suppress the growth of angiosarcoma in nude mice. In this study, we have demonstrated that treatment of different human breast cancer cell lines with honokiol resulted in a time- and concentration-dependent growth inhibition in both estrogen receptor-positive and -negative breast cancer cell lines, as well as in drug-resistant breast cancer cell lines such as adriamycin-resistant and tamoxifen-resistant cell lines. The inhibition of growth was associated with a G1-phase cell cycle arrest and induction of caspase-dependent apoptosis. The effects of honokiol might be reversely related to the expression level of human epidermal growth receptor 2, (HER-2, also known as erbB2, c-erbB2) since knockdown of her-2 expression by siRNA significantly enhanced the sensitivity of the her-2 over-expressed BT-474 cells to the honokiol-induced apoptosis. Furthermore, inhibition of HER-2 signalling by specific human epidermal growth receptor 1/HER-2 (EGFR/HER-2) kinase inhibitor lapatinib synergistically enhanced the anti-cancer effects of honokiol in her-2 over-expressed breast cancer cells. Finally, we showed that honokiol was able to attenuate the PI3K/Akt/mTOR (Phosphoinositide 3-kinases/Akt/mammalian target of rapamycin) signalling by down-regulation of Akt phosphorylation and upregulation of PTEN (Phosphatase and Tensin homolog deleted on chromosome Ten) expression. Combination of honokiol with the mTOR inhibitor rapamycin presented synergistic effects on induction of apoptosis of breast cancer cells. In conclusion, honokiol, either alone or in combination with other therapeutics, could serve as a new, promising approach for breast cancer treatment.

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.

Coincidence of Gaucher's disease due to a 1226G/1448C mutation and of an immunoglobulin G lambda multiple myeloma with Bence-Jones proteinuria

Abstractu2002We report about a 58-year-old female with coexisting type-I Gauchers disease (GD) and multiple myeloma (MM). The diagnosis of GD was made in early childhood by means of bone marrow biopsy and was recently confirmed by analysis of the patients genomic DNA for the underlying glucocerebrosidase mutations and the identification of the 1226G/1448C genotype. At the age of 24u2009years, the patient developed massive splenomegaly. Therefore, a splenectomy was performed. No further therapy was necessary for the next 34u2009years until 1999 when progressive anemia and thrombocytopenia occurred. Additional laboratory analysis revealed high serum protein and immunoglobulin (Ig) G levels and evidence of monoclonal gammopathy and lambda light-chain proteinuria, indicating plasma cell dyscrasia. This diagnosis was confirmed by the detection of osteolytic lesions in skeletal X-rays and a bone marrow biopsy showing an extensive infiltration with Gaucher cells and an increase of plasma cells, which expressed lambda light chains. When examined by means of electron microscopy, typical Gaucher cells, i.e., histiocytes containing tubular-structured cytoplasmatic material and spots of plasma cells with an increase of the endoplasmic reticulum, were found. GD associated with acquired MM has been described 13 times in the literature from 1968 to 1997. Only three of the patients were suffering from IgG myeloma. This distribution of the monoclonal component is in contrast to that of patients suffering from MM alone.

Achievement of Complete Remission in Refractory Hodgkin's Disease with Prolonged Infusion of Gemcitabine

Although, in recent decades effectivechemotherapy regimens have been developed forthe treatment of Hodgkins disease, theprognosis of patients who experiencedisease progression is still very poor. Newtreatment approaches are urgently required tosalvage such patients. In a patient withHodgkins disease who failed to achievecomplete remission with the escalatedBEACOPP protocol, progression with bonemarrow infiltration and B symptomsdeveloped despite further treatment.Subsequently, gemcitabine was administeredin a novel schedule as a four-hour infusion of250 mg/m2 on days 1, 8, and 15, every four weeks.After the first cycle, the dose was reducedto 200 mg/m2 because of grade 3neutropenia. The condition of the patientimproved after the second cycle and notoxicity was observed during cycles 3–6.Complete remission was achieved. Twoyears after the end of gemcitabinetherapy, the patient is in good clinicalcondition and in continuous completeremission without further treatment. Thisis the first report of the prolonged infusionof gemcitabine as a salvage therapy inHodgkins 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.

Delayed release of chemokine CCL25 with bioresorbable microparticles for mobilization of human mesenchymal stem cells

Chemokines are guiding cues for directional trafficking of mesenchymal stem cells (MSC) upon injury and local chemokine delivery at injury sites is an up-to-date strategy to potentiate and prolong recruitment of MSC. In this study we present the chemokine CCL25, also referred to as thymus-expressed chemokine, to mobilize human MSC along positive but not along negative gradients. We hence proceeded to design a biodegradable and injectable release device for CCL25 on the basis of poly(lactic-co-glycolic acid) (PLGA). The conducted studies had the objective to optimize PLGA microparticle fabrication by varying selected formulation parameters, such as polymer type, microparticle size and interior phase composition. We found that microparticles of DV,50∼75u202fµm and fabricated using end-capped polymers, BSA as carrier protein and vortex mixing to produce the primary emulsion yielded high chemokine loading and delayed CCL25 release. To determine bioactivity, we investigated CCL25 released during the microparticle erosion phase and showed that deacidification of the release medium was required to induce significant MSC mobilization. The designed PLGA microparticles represent an effective and convenient off-the-shelf delivery tool for the delayed release of CCL25. However, continuative in vivo proof-of-concept studies are required to demonstrate enhanced recruitment of MSC and/or therapeutical effects in response to CCL25 release microparticles.nnnSTATEMENT OF SIGNIFICANCEnWith the discovery of chemokines, particularly CXCL12, as stimulators of stem cell migration, the development of devices that release CXCL12 has proceeded quickly in the last few years. In this manuscript we introduce CCL25 as chemokine to induce mobilization of human MSC. This study proceeds to demonstrate how selection of key formulation parameters of CCL25 loading into PLGA microparticles exerts considerable influence on CCL25 release. This is important for a broad range of efforts in in situ tissue engineering where the candidate chemokine and the delivery device need to be selected carefully. The use of such a cell-free CCL25 release device may provide a new therapeutic option in regenerative medicine.