Monika Engelhardt

Side-population cells from different precursor compartments.

The rapid efflux of the fluorescent DNA-binding dye Hoechst 33342 identifies a rare, so-called side population (SP), which rapidly expels the dye, can reconstitute the bone marrow (BM) of lethally irradiated mice, and has proven negative for most lineage markers including CD34. Because SP cells from human cell sources, such as mobilized peripheral blood [apheresis products (AP)], cord blood (CB), or BM have not been extensively characterized to date, we sought to analyze SP cells from various cell sources. We detected murine SP cells with a median frequency of 0.04% (n = 23) and a 52-fold colony-forming units (CFU) increase compared to unsorted cells (p = 0.028). The median frequency of human SP cells was 0.02% (n = 90), with highest numbers in donor AP, and lower in CB and BM. Human SP cells were mostly CD34(-) and lineage marker-negative. These showed no enrichment in CFU before expansion; however, they displayed a CFU increase after 5-7 days of cytokine-supported suspension culture (10.7-fold at day 5, 7.2-fold at day 7; n = 17) that was significant compared to both input (day 0) SP and to non-SP cells before and after expansion (p < 0.05). SP cells demonstrated a significant long-term culture-initiating cell (LTC-IC) increase of 167-fold (n = 17) as compared to non-SP cells (p = 0.002), with the highest numbers from AP specimens. We conclude that human primitive hematopoietic cells can be isolated via Hoechst staining and that SP cells of various human sources show substantial differences and represent a rare CD34(-) population with stem cell potential.

Potent in vitro and in vivo activity of sorafenib in multiple myeloma: induction of cell death, CD138-downregulation and inhibition of migration through actin depolymerization.

Despite considerable advances, multiple myeloma (MM) remains incurable and the development of novel therapies targeting the interplay between plasma cells (PCs) and their bone marrow (BM) microenvironment remains essential. We investigated the effect of various agents in vitro on the proliferation, phenotype, morphology, actin polymerization and migration of MM cells and, in vivo, the tumour growth of L363‐bearing non‐obese diabetic severe combined immunodeficient mice with a deficient interleukin‐2 receptor gamma chain (NSG). In vitro, we observed a dose‐dependent cytotoxicity with bortezomib and sorafenib. Using RPMI8226 cells co‐expressing histone 2B‐mCherry and cytochrome c‐GFP, bortezomib‐ and sorafenib‐induced apoptosis was confirmed, and both agents combined showed synergism. Sorafenib induced CD138‐downregulation and abolished CXCL12‐induced actin polymerization. L363 cells expressed CCR4 and CCR5 and migrated to their common ligand CCL5. Chemotaxis to BM stroma cells was notable and significantly reduced by sorafenib. Downregulation of phospho‐ERK appeared relevant for the inhibition of actin polymerization and chemotaxis. Sorafenib alone, and combined with bortezomib, showed substantial antitumour activity in L363‐bearing NSG. Correspondingly, sorafenib induced clinical responses in MM‐/AL‐amyloidosis patients. We conclude that, in addition to the cytotoxic and anti‐angiogenic effects of sorafenib, blocking of MM cell migration and homing represent promising mechanisms to interrupt the interplay between PCs and their supportive microenvironment.

Preclinical models of multiple myeloma: a critical appraisal

Introduction: Preclinical models, by definition, are an approximation of reality, and their use in developing anticancer drugs is eagerly explored. Positive clinical correlations have been identified with different test systems, in addition to limitations and a need to improve preclinical model systems. Predicting whether a potential new anticancer agent will have a positive therapeutic index in patients remains a challenge. Areas covered: We here review the fundamental requirements and remarkable progress of preclinical in vitro and in vivo assays used to assess the therapeutic potential of experimental anticancer drugs in multiple myeloma (MM). In MM, the interaction with the bone marrow microenvironment (BMM) plays a crucial role in disease progression, including resistance to antimyeloma agents. In vitro and in vivo approaches are, therefore, discussed with respect to their ability to mimic the important characteristics of MM and its BMM. In general, MM models should parallel the biological, genetic, etiological, immunological and therapeutic properties of the human disease. Expert opinion: All models discussed here have their defined strengths, but also limitations with respect to their predictive features. Understanding the preclinical models in a more profound way should lead to optimized clinical trials, thereby expanding the therapeutic arsenal and improving patient outcome further.

Monitoring APC/C activity in the presence of chromosomal misalignment in unperturbed cell populations

Chromosome segregation is under strict control of the spindle assembly checkpoint (SAC). The SAC regulates anaphase-promoting complex/cyclosome (APC/C)-dependent proteolysis of securin and cyclin B. Unattached or misaligned chromosomes trigger SAC-mediated mitotic delay by stabilizing securin and cyclin B due to inhibition of APC/C until the problem is solved. Here we present a hitherto unavailable model facilitating the simultaneous depiction of chromosome movements and pulse-chased cyclin B proteolysis in every single cell within a cell population. During chromosome misalignment, we observed slow cyclin B degradation, which changed to fast degradation once the SAC was satisfied, initiating chromosome separation and mitotic exit. Slow degradation during a SAC-mediated mitotic delay is part of a tightly regulated balance between cyclin B synthesis and degradation. Since chromosomal misalignment is a rare event, the ability to study entire cell populations enabled us to monitor for the first time SAC surveillance in living cells without the need of highly artificial perturbation by spindle poisons.

The Prevention of Medication-related Osteonecrosis of the Jaw.

BACKGROUNDnMedication-related osteonecrosis of the jaw (MRONJ) is a preventable complication of antiresorptive treatment. It arises in 1-20% of patients with bone metastases of solid tumors and hematologic malignancies and in 0.1-2% of patients being treated for osteoporosis with bisphosphonates. Depending on the underlying disease and medication dosage, the risk of MRONJ can be elevated even in the first year of antiresorptive treatment. The treatment of MRONJ is difficult and often involves surgery of the jaw.nnnMETHODSnWe systematically reviewed publications retrieved by a selective search for literature on the prevention of MRONJ in the PubMed and Cochrane Library databases and with the aid of the Google Scholar search engine.nnnRESULTSn15 of 559 retrieved publications were included in the analysis. The quality of the evidence in the studies was generally moderate to low, with most of them being case series. In one case series of over 1200 patients with multiple myeloma, the incidence of MRONJ was lowered from 4.6% to 0.8% through regular dental checkups and improved oral hygiene. Tooth extraction, in particular, is associated with a high risk of MRONJ. In a retrospective study, 57% of patients who underwent tooth extraction without antibiotic prophylaxis developed MRONJ, compared to 0% with antibiotic prophylaxis.nnnCONCLUSIONnBefore antiresorptive medication is begun, oral hygiene should be improved. Moreover, it seems that perioperative antibiotic prophylaxis and adequate plastic wound closure can often prevent MRONJ. In view of the fact that bisphosphonates can persist in bone for more than 15 years, patients should be thoroughly informed of the risk that antiresorptive treatment can cause MRONJ, and the measures discussed should be initiated.

Successful treatment of a patient with myelodysplastic syndrome (RAEB) with Darbepoetin-alfa in combination with Pegfilgrastim.

Dear Editor, In December 1999, a 67-year-old man presented with a mild leukopenia and thrombocytopenia. Bone marrow (BM) biopsy and aspirate revealed an abnormal proliferation and differentiation with an excess of blasts (5–10%). A refractory anaemia with excess of blasts (RAEB) according to FAB, and RAEB I according to the WHO classification system, was diagnosed. The patient received vitamin substitution with folic acid and vitamin B12, and until December 2001, no further treatment was required (Fig. 1). In January 2002, the myelodysplastic syndrome (MDS) progressed and we started with cyclosporine A. After an initial rise of peripheral blood cell (PB) counts, the MDS further progressed. Therefore, thalidomide was begun but also proved to be ineffective. The clinical course was complicated by a pneumonia and severe sepsis, whereby the patient needed mechanical ventilator support and was treated with broad spectrum antibiotics and Filgrastim (Neupogen) 300 μg/day for 5 days. Due to this treatment, leukocyte counts increased, and initially, platelet counts also improved. He recovered, but thereafter, became transfusion dependent. The serum erythropoetin (EPO) level was elevated to 453 IU/l. In order to best treat his anaemia and his MDS, he received 150 μg of Darbepoetin-alfa (DP-α; Aranesp) once weekly subcutaneously. This resulted in initially prolonged transfusion intervals; however, his MDS progressed again after a few months. In July 2004, we combined DP-α with 6 mg of Pegfilgrastim (pegFG; Neulasta) given every 4 weeks. Before this combined treatment, the PB count was as follows: leukocytes 1.2/nl [absolute neutrophil count (ANC) 0.27/nl], Hb 7.4 g/dl, platelets 23/nl. DP-α and peg-FG were well tolerated, and after 4 weeks, all three cell lines responded significantly: leukocytes were rising to 3.1/nl (ANC 1.5/nl), the Hb to 11.8 g/dl and platelets to 75/nl (Fig. 1), thereby inducing a major neutrophil, erythroid and platelet response according to the international working group response criteria [8]. The patient is now transfusion independent and no infectious complications have occurred. The quality of life has improved significantly and he is now working as a guide at a travel agency. At last follow-up, PB count was as follows: leukocytes 1.8/nl, Hb 10.4 g/dl, platelets 40/nl, and this PB improvement—due to both novel cytokines DP-α and pegFG—is now ongoing for 7 months. Reports on the treatment of MDS with recombinant erythropoetin (rhEPO) in combination with granulocyte or granulocyte/macrophage colony-stimulating factor (Gor GM-CSF) have suggested that hyperglycemic–glycogenolytic factor (HGF) combinations are more effective than rhEPO alone [3]. Response rates have been reported to occur in up to 40% with combined HGF, as compared to 15–20% with EPO alone [3]. In a recently published randomised trial, 60 patients with low-risk MDS were divided in two groups: rhEPO and G-CSF vs supportive care. The combined treatment led to responses in 41.7% as compared to no responses in the supportive care arm [1]. HellstromLindberg et al. [2] have published a validated decision model for treating the anaemia of MDS with EPO and GCSF: a serum EPO level less than 500 U/L and a transfusion need of less than 2 units/month predicted a high probability of response to treatment (61%), whereas the response rate of patients with higher EPO levels and higher transfusion needs was 14%. Most recently, in patients with low/intermediate risk MDSs, improved response rates have been reported with the long-acting erythropoiesis-stimulating molecule DP-α (40.5% [9]) and with high-dose epoetin alfa induction treatment (40,000 IU biweekly, erythroid response of 68% [10]). A. Jakob . F. W. Hirsch Department of Hematology and Oncology, Medical Center Offenburg, Ebertplatz 12, 77654 Offenburg, Germany

Proteasome inhibition enhances the efficacy of volasertib-induced mitotic arrest in AML in vitro and prolongs survival in vivo

Elderly and frail patients, diagnosed with acute myeloid leukemia (AML) and ineligible to undergo intensive treatment, have a dismal prognosis. The small molecule inhibitor volasertib induces a mitotic block via inhibition of polo-like kinase 1 and has shown remarkable anti-leukemic activity when combined with low-dose cytarabine. We have demonstrated that AML cells are highly vulnerable to cell death in mitosis yet manage to escape a mitotic block through mitotic slippage by sustained proteasome-dependent slow degradation of cyclin B. Therefore, we tested whether interfering with mitotic slippage through proteasome inhibition arrests and kills AML cells more efficiently during mitosis. We show that therapeutic doses of bortezomib block the slow degradation of cyclin B during a volasertib-induced mitotic arrest in AML cell lines and patient-derived primary AML cells. In a xenotransplant mouse model of human AML, mice receiving volasertib in combination with bortezomib showed superior disease control compared to mice receiving volasertib alone, highlighting the potential therapeutic impact of this drug combination.

Up-regulation of RUNX2 in acute myeloid leukemia in a patient with an inherent RUNX2 haploinsufficiency and cleidocranial dysplasia.

* Present address: Biozentrum, University of Basel, Switzerland † Present address: Department of Internal Medicine, Hospital Mittersill, Austria Correspondence: Ralph W ä sch, MD, Associate Professor, Department of Hematology, Oncology, and Stem Cell Transplantation, University Medical Center, Hugstetter Strasse 55, 79106 Freiburg, Germany. Tel: x02 49-761-27072890. Fax: x02 49-761-27033180. E-mail: ralph.waesch@uniklinik-freiburg.de

Frequency, severity and risk factors for oral mucositis after BEAM conditioning and autologous peripheral blood stem cell transplantation: a single center analysis and review of the literature.

High-dose chemotherapy (hd-CTx) may be complicated by various toxicities, including oral mucositis (OM) [1-4]. In addition to being painful, leading to opioid use, requirement for parenteral nutrit...

Does colorectal cancer in ulcerative colitis patients constitute a risk for chemotherapy refractoriness?:a systemic approach by detailed analysis via the electronic tumor base documentation system.

Background: Ulcerative colitis (UC) patients may develop colorectal cancer (CRC), especially with pancolitis and longer UC duration. The question whether CRC with underlying UC has a dismal prognosis remains unsettled. Patients and Methods: We performed an electronic tumor base documentation (eTBD) search of CRC and UC patients at our department to address whether (1) CRC prognosis is impaired and (2) defined risks can be determined. Results: With the inclusion of an index patient with UC and unresponsive CRC, 20 additional patients were identified via eTBD. Chemotherapy response was less substantial, with complete response or stable disease in 3 patients each, but rapidly progressing or refractory disease in 15/21 patients. 12 out of the 21 patients died. Our hazard ratio analysis revealed International Union against Cancer (UICC) stage IV and III disease, grade 3 tumors, longer latency from UC to CRC and age >60 years as potential risks. Median progression-free survival and overall survival were 48 and 82 months, respectively. Time from tumor dissemination to death was 10 months. Conclusions: The prognosis of CRC in UC patients is not necessarily impaired, albeit chemotherapy response with disseminated disease may be unfavorable. Our data should be enlarged by subsequent analyses to better elucidate whether response in UC and CRC is more challenging and defined risks can be confirmed.