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Introduction: Infiltration of the maxillofacial region by multiple myeloma is common and typically appears as punched out osteolytic lesions of the jaws. Although swelling, bone pain, and tooth mobility are characteristic clinical symptoms, root resorption is rare in conjunction with myeloma nests., Methods: A case of a 67-year-old man with multiple myeloma is presented. Root resorption of the second mandibular premolar and the first and second molars on the right side, with consecutive tooth mobility, was the initial manifestation of the disease and was primarily detected on a periapical radiograph. The treating dentist referred the patient to the department for further examination, which revealed multiple myeloma. The patient received tandem high-dose chemotherapy and autologous stem cell transplantation as standard myeloma treatment. Intravenous bisphosphonates were administered to curb the osteolytic lesions., Results: No signs of bisphosphonate-related osteonecrosis of the jaw were observed until the end of the follow-up period., Conclusions: Only 5 reports of myeloma-associated root resorption have been reported in the literature. In all cases, mandibular premolars or molars were involved, and the patients exhibited extensive involvement of the jaw by myeloma. This report highlights the importance of correct interpretation of clinical signs and radiographs by dental specialists in the diagnostic algorithm of systemic diseases. Furthermore, this is an example for the inevitable part of bisphosphonates in the treatment of osteolytic processes., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Background: B cell malignancies are characterized by clonal expansion of B cells expressing tumor-specific idiotypes on their surface. These idiotypes are ideal target antigens for an individualized immunotherapy. However, previous idiotype vaccines mostly lacked efficiency due to a low immunogenicity of the idiotype. The objective of the present study was the determination of the feasibility, safety and immunogenicity of a novel chemically linked phage idiotype vaccine., Methods: In the murine B cell lymphoma 1 model, tumor idiotypes were chemically linked to phage particles used as immunological carriers. For comparison, the idiotype was genetically expressed on the major phage coat protein g8 or linked to keyhole limpet hemocynanin. After intradermal immunizations with idiotype vaccines, tolerability and humoral immune responses were assessed., Results: Feasibility and tolerability of the chemically linked phage idiotype vaccine was demonstrated. Vaccination with B cell lymphoma 1 idiotype expressing phage resulted in a significant survival benefit in the murine B cell lymphoma 1 protection model (60.2±23.8 days vs. 41.8±1.6 days and 39.8±3.8 days after vaccination with wild type phage or phosphate buffered saline, respectively). Superior immunogenicity of the chemically linked phage idiotype vaccine compared to the genetically engineered phage idiotype and keyhole limpet hemocynanin-coupled idiotype vaccine was demonstrated by significantly higher B cell lymphoma 1 idiotype-specific IgG levels after vaccination with chemically linked phage idiotype., Conclusion: We present a novel, simple, time- and cost-efficient phage idiotype vaccination strategy, which represents a safe and feasible therapy and may produce a superior immune response compared to previously employed idiotype vaccination strategies.

In view of the fact that histone deacetylases (HDACs) are promising targets for myeloma therapy, we investigated the effects of the HDAC inhibitor CR2408 on multiple myeloma (MM) cells in vitro. CR2408 is a direct pan-HDAC inhibitor and inhibits all known 11 HDACs with a 50% inhibitory concentration (IC(50) ) of 12 nmol/l (HDAC 6) to 520 nmol/l (HDAC 8). Correspondingly, CR2408 induces hyperacetylation of histone H4, inhibits cell growth and strongly induces apoptosis (IC(50) =0.1-0.5 μmol/l) in MM cell lines and primary MM cells. CR2408 leads to fragmentation of cells and induces an accumulation in the subG1 phase accompanied with moderately decreased levels of cyclin D1 and cdk4 and strongly decreased levels of cdc25a, pRb and p53. Interruption of the cell cycle is reflected by inhibition of cell proliferation and is accompanied by decreased phosphorylation of 4E-BP1 and p70S6k. Treatment with CR2408 results in increased protein levels of Bim and pJNK and downregulation of Bad and Bcl-xL and activation of Caspases 3, 8 and 9. Furthermore, as HDAC inhibitors have shown synergism with other drugs, these effects were investigated and synergism was observed for combinations of CR2408 with doxorubicin and bortezomib. In conclusion, we have identified potent anti-myeloma activity for this novel HDAC inhibitor that gives further insights into the biological sequelae of HDAC inhibition in MM., (© 2011 Blackwell Publishing Ltd.)

Multiple myeloma is still uncurable. Myeloma cells become resistant to common drugs and patients eventually die of tumour progression. Therefore, new targets and drugs are urgently needed. NVP-BGT226 is a novel, orally bioavailable small-molecule inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin. Here, we show that NVP-BGT226 inhibits growth in common myeloma cell lines and primary myeloma cells at nanomolar concentrations in a time-dependent and dose-dependent manner. Western blots for the detection of caspase 3 cleavage and annexin-V-fluorescein isothiocyanate/propidium iodide assays revealed induction of apoptosis in common myeloma cells lines. Induction of apoptosis was accompanied by upregulation of proapoptotic Bim and a moderate upregulation of Mcl-1 and Bad and a downregulation of Bcl-2, Bax and Bcl-Xl. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the 5-bromo-2'-deoxyuridine assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, cyclin D2, pRb and cdc25a. NVP-BGT226 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1 in a time-dependent and dose-dependent manner. Furthermore, we show that the stimulatory effect of insulin-like growth factor 1, interleukin-6 and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BGT226. Overall, inhibition of phosphoinositol-3-kinase/mammalian target of rapamycin by NVP-BGT226 is highly effective, and NVP-BGT226 represents a potential new candidate for targeted therapy in multiple myeloma.

Background Aims: Neutropenia following high-dose chemotherapy is associated with a substantial risk of infectious complications. The aim of this study was to identify variables in residual leukocyte subsets during neutropenia that are predictive for neutropenic fever., Methods: Residual leukocytes in the peripheral blood on day 5 after autologous blood stem cell transplantation were analyzed by three-color flow cytometry in 55 consecutive patients with multiple myeloma. Furthermore, the number of T cells transfused with the autografts was determined., Results: Neutrophil counts at day 5 and neutrophil engraftment were similar in patients with and without neutropenic fever. Low absolute lymphocyte, CD4(+) CD28(+) and CD45RO(+) CD28(+) counts at day 5 were associated with neutropenic fever. T-cell counts at day 5 correlated with the CD3(+) cell number in the graft., Conclusions: Our data show that the absolute lymphocyte, CD4(+) CD28(+) and CD45RO(+) CD28(+) counts play a role in host defense during severe neutropenia. The T-cell number in the graft may help to identify patients at high risk of neutropenic infections.

Background and Aims: Dendritic cell (DC)-based vaccination can induce antitumor T cell responses in vivo. This clinical pilot study examined feasibility and outcome of DC-based tumor vaccination for patients with advanced pancreatic adenocarcinoma., Methods: Tumor lysate of patients with pancreatic carcinoma was generated by repeated freeze-thaw cycles of surgically obtained tissue specimens. Patients were eligible for DC vaccination after recurrence of pancreatic carcinoma or in a primarily palliative situation. DC were generated from peripheral blood mononuclear cells (PBMC), loaded with autologous tumor lysate, stimulated with TNF-α and PGE(2) and injected intradermally. All patients received concomitant chemotherapy with gemcitabine. Disease response was the primary endpoint. Individual immunological responses to DC vaccination were analyzed by T cell-based immunoassays using pre- and post-vaccination samples of non-adherent PBMC., Results: Twelve patients received DC vaccination and concomitant chemotherapy. One patient developed a partial remission, and two patients remained in stable disease. Median survival was 10.5 months. No severe side effects were observed. Tumor-reactive T cells could be detected prior to vaccination. DC vaccination increased the frequency of tumor-reactive cells in all patients tested; however, the degree of this increase varied. To quantify the presence of tumor-reactive T cells, stimulatory indices (SI) were calculated as the ratio of proliferation-inducing capacity of lysate-loaded versus -unloaded DC. The patient with longest overall survival of 56 months had a high SI of 6.49, indicating that the presence of a pre-vaccination antitumor T cell response might be associated with prolonged survival. Five patients survived 1 year or more., Conclusion: DC-based vaccination can stimulate an antitumoral T cell response in patients with advanced or recurrent pancreatic carcinoma receiving concomitant gemcitabine treatment.

Background: Neuroblastomas account for 97% of all neuroblastic tumors and for approximately 15% of all pediatric cancer fatalities. However, in adults neuroblastoma is a very rare finding., Case Report: Here, we present the case of a 55-year-old patient who was diagnosed with neuroblastoma stage IV one year after the false diagnosis of a non-secretory multiple myeloma., Results: The patient received six cycles of a chemotherapy protocol with cisplatin, etoposide and vindesine alternating with vincristine, dacarbazine, ifosfamide and doxorubicin, but the response to treatment was insufficient (stable disease)., Conclusion: The standard chemotherapy protocols used for children are not sufficient for adult patients. Different treatment approaches are needed to improve the prognosis of adult patients with neuroblastoma.

Two trivalent constructs consisting of single-chain Fv antibody fragments (scFvs) specific for the interleukin-3 receptor alpha chain (CD123), CD33 and the Fcgamma-receptor III (CD16) were designed and characterized for the elimination of acute myeloid leukaemia (AML) cells. The dual targeting single-chain Fv triplebody (sctb) [123 x ds16 x 33] and the mono targeting sctb [123 x ds16 x 123] both specifically bound their respective target antigens and were stable in human serum at 37 degrees C for at least 5 d. Both constructs induced potent antibody-dependent cellular cytotoxicity (ADCC) of two different AML-derived CD33- and CD123 double-positive cell lines in the low picomolar range using isolated mononuclear cells (MNCs) as effector cells. In these experiments the dual targeting molecule produced significantly stronger lysis than the mono targeting agent. In addition, the sctbs showed a high potency in mediating ADCC of primary leukaemia cells isolated from peripheral blood or bone marrow of seven AML patients. Hence, these novel molecules displayed potent anti-leukaemic effects against AML cells in vitro and represent attractive candidates for further preclinical development.

Single-chain Fv triplebodies (sctb), consisting of a single polypeptide chain with 3 single-chain antibody variable fragments connected in tandem, were generated as antileukemic agents. A CD19-specific sctb of this format has previously been shown to be superior to a bispecific single-chain Fv antibody fragment (bsscFv) for the elimination of leukemic B-lineage cells, but corresponding targeted agents for the treatment of acute myeloid leukemia are still lacking. For this purpose, both a bsscFv and a sctb specific for CD33 and the trigger molecule CD16 (FcgammaRIII) were produced. The sctb displayed 3.5-fold greater avidity for CD33 than the bsscFv 33xds16, whereas both had close to equal affinity for CD16. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, both the bsscFv 33xds16 and the sctb induced lysis of tumor cells with half maximum effective concentrations (EC50) in the low picomolar range. It is interesting to note that the sctb promoted equal lysis of human leukemia-derived cell lines at 10 to 200-fold lower concentrations than the bsscFv. Both molecules mediated ADCC of primary patient cells. In conclusion, both the bsscFv 33xds16 and the sctb 33xds16x33 eliminated acute myeloid leukemia cells in ADCC reactions, but the novel sctb format showed significantly greater specific activity.

Inhibition of histone deacetylase (HDAC) is a promising mechanism for novel, anti-myeloma agents. We investigated the effects of the novel HDAC inhibitor resminostat on multiple myeloma (MM) cells in vitro. Resminostat is a potent inhibitor of HDACs 1, 3 and 6 [50% inhibitory concentration (IC50)=43-72 nmol/l] representing HDAC classes I and II and induces hyperacetylation of histone H4 in MM cells. Low micromolar concentrations of resminostat abrogated cell growth and strongly induced apoptosis (IC50=2.5-3 micromol/l in 3 out of 4 MM cell lines) in MM cell lines as well as primary MM cells. At 1 micromol/l, resminostat inhibited proliferation and induced G0/G1 cell cycle arrest in 3 out of 4 MM cell lines accompanied with decreased levels of cyclin D1, cdc25a, Cdk4 and pRb as well as upregulation of p21. Resminostat decreased phosphorylation of 4E-BP1 and p70S6k indicating an interference with Akt pathway signalling. Treatment with resminostat resulted in increased protein levels of Bim and Bax and decreased levels of Bcl-xL. Caspases 3, 8 and 9 were activated by resminostat. Furthermore, synergistic effects were observed for combinations of resminostat with melphalan and the proteasome inhibitors bortezomib and S-2209. In conclusion, we have identified potent anti-myeloma activity for this novel HDAC inhibitor.

Bortezomib is the first approved member of a new class of anti-myeloma agents, the proteasome inhibitors. Further proteasome inhibitors are needed to optimise this promising treatment option. S-2209 [1-[1-{1-[(2,4-Dioxo-imidazolidin-1-ylimino)-methyl]-2-phenyl-ethylcarbamoyl}-2-(1H-indol-3-yl)-ethylcarbamoyl]-2-(1H-indol)] inhibits the chymotryptic activity of the human 20S proteasome (half maximal effective concentration, IC(50) approximately 220 nmol/l) which was determined by a proteasome inhibition assay. A nuclear factor kappaB inhibition assay revealed a half maximal effective concentration (EC(50)) of 0.9 micromol/l. The WST-1 growth assay showed inhibition of cell growth of all tested multiple myeloma (MM) cell lines with an IC(50) between 100 nmol/l and 600 nmol/l. Strong induction of apoptosis was seen in MM cells at nanomolar concentrations (IC(50) approximately 300 nm) as well as in primary myeloma cells. No induction of apoptosis was detected in peripheral blood mononuclear cells from healthy humans. Upregulation of p53, activation of JNK protein, and downregulation of Mcl-1 was revealed. Despite the administration of 15 mg S-2209/kg/d in wistar rats, no toxicity with respect to body weight, hepatic enzymes, creatinine or haemoglobin was seen. Proteasome inhibition in white blood cells isolated from the treated rats was higher in the S-2209 treated animals in comparison with the control animals treated with 0.1 mg/kg/d bortezomib. S-2209 is active in myeloma cells and shows a favourable toxicity profile in first in-vivo studies. S-2209 is a promising agent for further clinical development.

NVP-BEZ235 is a new inhibitor of phosphoinositol-3-kinase (PI3 kinase) and mammalian target of rapamycin (mTOR) whose efficacy in advanced solid tumours is currently being evaluated in a phase I/II clinical trial. Here we show that NVP-BEZ235 inhibits growth in common myeloma cell lines as well as primary myeloma cells at nanomolar concentrations in a time and dose dependent fashion. Further experiments revealed induction of apoptosis in three of four cell lines. Inhibition of cell growth was mainly due to inhibition of myeloma cell proliferation, as shown by the BrdU assay. Cell cycle analysis revealed induction of cell cycle arrest in the G1 phase, which was due to downregulation of cyclin D1, pRb and cdc25a. NVP-BEZ235 inhibited phosphorylation of protein kinase B (Akt), P70S6k and 4E-BP-1. Furthermore we show that the stimulatory effect of CD40-ligand (CD40L), insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6) and conditioned medium of HS-5 stromal cells on myeloma cell growth is completely abrogated by NVP-BEZ235. In addition, synergism studies revealed synergistic and additive activity of NVP-BEZ235 together with melphalan, doxorubicin and bortezomib. Taken together, inhibition of PI3 kinase/mTOR by NVP-BEZ235 is highly effective and NVP-BEZ235 represents a potential new candidate for targeted therapy in multiple myeloma.

Multiple myeloma is still an incurable disease; therefore, new therapeutics are urgently needed. A771726 is the active metabolite of the immunosuppressive drug leflunomide, which is currently applied in the treatment of rheumatoid arthritis, BK virus nephropathy, and cytomegaly viremia. Here, we show that dihydroorotate dehydrogenase (DHODH) is commonly expressed in multiple myeloma cell lines and primary multiple myeloma cells. The DHODH inhibitor A771726 inhibits cell growth in common myeloma cell lines at clinically achievable concentrations in a time- and dose-dependent manner. Annexin V-FITC/propidium iodide staining revealed induction of apoptosis of multiple myeloma cell lines and primary multiple myeloma cells. The 5-bromo-2'-deoxyuridine cell proliferation assay showed that inhibition of cell growth was partly due to inhibition of multiple myeloma cell proliferation. A771726 induced G(1) cell cycle arrest via modulation of cyclin D2 and pRb expression. A771726 decreased phosphorylation of protein kinase B (Akt), p70S6K, and eukaryotic translation initiation factor 4E-binding protein-1 as shown by Western blotting experiments. Furthermore, we show that the stimulatory effect of conditioned medium of HS-5 bone marrow stromal cells on multiple myeloma cell growth is completely abrogated by A771726. In addition, synergism studies revealed synergistic and additive activity of A771726 together with the genotoxic agents melphalan, treosulfan, and doxorubicin as well as with dexamethasone and bortezomib. Taken together, we show that inhibition of DHODH by A771726/leflunomide is effective in multiple myeloma. Considering the favorable toxicity profile and the great clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in multiple myeloma.

A novel bispecific antibody-derived recombinant protein targeting leukemias and lymphomas was designed, a single-chain Fv triple body (sctb) consisting of 1 polypeptide chain with 3 scFvs connected in tandem. The distal scFvs were specific for the tumor antigen CD19, and the central scFv for the trigger molecule CD16 (FcgammaRIII) on natural killer (NK) cells and macrophages. We had previously built a disulphide stabilized (ds) bsscFv [19 x 16] with monovalent binding for CD19 from ds components. The sctb ds[19 x 16 x 19] also used ds components and displayed 3-fold greater avidity for CD19 than the bsscFv (KD = 13 vs. 42 nM), whereas both had equal affinity for CD16 (KD = 58 nM). Plasma half-lives in mice were 4 and 2 hours for the sctb and the bsscFv, respectively. In antibody-dependent cellular cytotoxicity reactions with human mononuclear cells as effectors, the sctb promoted equal lysis of leukemic cell lines and primary cells from leukemia and lymphoma patients at 10-fold to 40-fold lower concentrations than the bsscFv. This new format may also be applicable to a variety of other tumor antigens and effector molecules. With half-maximum effective concentrations (EC50) in the low picomolar range, the sctb ds[19 x 16 x 19] is an attractive candidate for further preclinical evaluation.

Multiple myeloma is still not curable and drug combination strategies are currently being evaluated in order to achieve high remission rates with tolerable toxicity. Bortezomib has been shown to exert inhibitory effects on NFkappaB activity. NFkappaB in turn is known to be activated by cytokines, growth factors and by cellular adhesion to bone marrow stromal cells and represents an important mediator of primary and secondary drug resistance in multiple myeloma that confers to proliferation and survival. In this study we confirm that bortezomib sensitized MM cells to the DNA-damaging drugs melphalan and doxorubicin. Further, we demonstrate that the sole incubation of MM cells with melphalan or doxorubicin leads to a vast activation of NFkappaB activity. Additionally, we show that the co-incubation of bortezomib with melphalan or doxorubicin reduces activation of NFkappaB. These data suggest that the drug-sensitizing effect of bortezomib on MM cells is due to inhibition of melphalan- and doxorubicin-induced activation of NFkappaB activity. This study, therefore, supports the idea of combining a NFkappaB inhibitor with alkylating drugs in the therapy of multiple myeloma.

Although the treatment outcome of lymphoid malignancies has improved in recent years by the introduction of transplantation and antibody-based therapeutics, relapse remains a major problem. Therefore, new therapeutic options are urgently needed. One promising approach is the selective activation of apoptosis in tumor cells by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study investigated the pro-apoptotic potential of a novel TRAIL fusion protein designated scFvCD19:sTRAIL, consisting of a CD19-specific single-chain Fv antibody fragment (scFv) fused to the soluble extracellular domain of TRAIL (sTRAIL). Potent apoptosis was induced by scFvCD19:sTRAIL in several CD19-positive tumor cell lines, whereas normal blood cells remained unaffected. In mixed culture experiments, selective binding of scFvCD19:sTRAIL to CD19-positive cells resulted in strong induction of apoptosis in CD19-negative bystander tumor cells. Simultaneous treatment of CD19-positive cell lines with scFvCD19:sTRAIL and valproic acid (VPA) or Cyclosporin A induced strongly synergistic apoptosis. Treatment of patient-derived acute B-lymphoblastic leukemia (B-ALL) and chronic B-lymphocytic leukemia (B-CLL) cells resulted in strong tumoricidal activity that was further enhanced by combination with VPA. In addition, scFvCD19:sTRAIL prevented engraftment of human Nalm-6 cells in xenotransplanted NOD/Scid mice. The pre-clinical data presented here warrant further investigation of scFvCD19:sTRAIL as a potential new therapeutic agent for CD19-positive B-lineage malignancies.

Objectives: Despite high dose treatment regimes multiple myeloma (MM) disease is still not curable. Patients become resistant to cytotoxic drugs and die of disease progression. Therefore, besides new cytotoxic compounds drug sensitisers are urgently needed., Methods: The MM cell lines U266, OPM-2, RPMI-8226 and NCI-H929 were incubated with the common anti-myeloma drugs like melphalan together with protein kinase C (PKC) inhibitors. Growth inhibition was measured using the water-soluble tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1 assay), and apoptosis was determined by flow cytometry after staining with fluorescein isothiocyanate-labeled annexin V (annexin-V-FITC) and propidium iodide. Intracellular signalling was shown by western blotting., Results: In this study we show that the combination of melphalan or doxorubicin with a PKC inhibitor, Gö6976 or enzastaurin, strongly increases cell toxicity. Increase of cytotoxicity is shown to be due to increased induction of apoptosis. Furthermore, we show that the protective effect of human bone marrow stromal cells (hBMSC) is abrogated by the PKC inhibitors. Finally, western blotting experiments revealed that incubation of myeloma cells with cytotoxic drugs like melphalan or doxorubicin leads to increased phosphorylation and therefore degradation of inhibitor of nuclear factor kappa B (IkappaB) and release of nuclear factor kappa B (NFkappaB). In contrast, enzastaurin inhibits phosphorylation of IkappaB., Conclusions: We conclude that the combination of conventional drugs and PKC inhibitors might be very effective and represents a new strategy in the treatment of MM.