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Niels WCJ van de Donk,1,2 Güllü Görgün,1 Richard WJ Groen,1,3 Jana Jakubikova,1 Constantine S Mitsiades,1 Teru Hideshima,1 Jacob Laubach,1 Inger S Nijhof,2 Reinier A Raymakers,2 Henk M Lokhorst,2 Paul G Richardson,1 Kenneth C Anderson11Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 2Department of Hematology, 3Department of Cell Biology, University Medical Center Utrecht, Utrecht, The NetherlandsAbstract: Lenalidomide is an amino-substituted derivative of thalidomide with direct antiproliferative and cytotoxic effects on the myeloma tumor cell, as well as antiangiogenic activity and immunomodulatory effects. Together with the introduction of bortezomib and thalidomide, lenalidomide has significantly improved the survival of patients with relapsed and refractory myeloma. The most common adverse events associated with lenalidomide include fatigue, skin rash, thrombocytopenia, and neutropenia. In addition, when lenalidomide is combined with dexamethasone or other conventional cytotoxic agents, there is an increase in the incidence of venous thromboembolic events. There is now evidence that continued treatment with lenalidomide has a significant impact on survival by improving the depth and duration of response. This highlights the value of adverse event management and appropriate dose adjustments to prevent toxicity, and of allowing continued treatment until disease progression. In this review, we will discuss the different lenalidomide-based treatment regimens for patients with relapsed/refractory myeloma. This is accompanied by recommendations of how to manage and prevent adverse events associated with lenalidomide-based therapy.Keywords: lenalidomide, multiple myeloma, immunomodulatory drugs, relapse treatment, refractory disease

Multiple myeloma (MM) is a hematological malignancy that is still considered incurable due to the development of therapy resistance and subsequent relapse of disease. MM plasma cells (PC) use NFκB signaling to stimulate cell growth and disease progression, and for protection against therapy-induced apoptosis. Amongst its diverse array of target genes, NFκB regulates the expression of pro-survival BCL-2 proteins BCL-XL, BFL-1, and BCL-2. A possible role for BFL-1 in MM is controversial, since BFL-1, encoded by BCL2A1 , is downregulated when mature B cells differentiate into antibody-secreting PC. NFκB signaling can be activated by many factors in the bone marrow microenvironment and/or induced by genetic lesions in MM PC. We used the novel signal transduction pathway activity (STA) computational model to quantify the functional NFκB pathway output in primary MM PC from diverse patient subsets at multiple stages of disease. We found that NFκB pathway activity is not altered during disease development, is irrespective of patient prognosis, and does not predict therapy outcome. However, disease relapse after treatment resulted in increased NFκB pathway activity in surviving MM PC, which correlated with increased BCL2A1 expression in a subset of patients. This suggests that BFL-1 upregulation, in addition to BCL-XL and BCL-2, may render MM PC resistant to therapy-induced apoptosis, and that BFL-1 targeting could provide a new approach to reduce therapy resistance in a subset of relapsed/refractory MM patients.

Novel combination therapies have markedly improved the lifespan of patients with multiple myeloma (MM), but drug resistance and disease relapse remain major clinical problems. Dexamethasone and other glucocorticoids are a cornerstone of conventional and new combination therapies for MM, although their use is accompanied by serious side effects. We aimed to uncover drug combinations that act in synergy and, as such, allow reduced dosing while remaining effective. Dexamethasone and the myeloid cell leukemia 1 (MCL-1) inhibitor S63845 (MCL-1i) proved the most potent combination in our lethality screen and induced apoptosis of human myeloma cell lines (HMCLs) that was 50% higher compared with an additive drug effect. Kinome analysis of dexamethasone-treated HMCLs revealed a reduction in serine/threonine peptide phosphorylation, which was predicted to result from reduced Akt activity. Biochemical techniques showed no dexamethasone-induced effects on FOXO protein or GSK3 but did show a 50% reduction in P70S6K phosphorylation, downstream of the Akt-mTORC1 axis. Replacing dexamethasone by the P70S6K1 isoform-specific inhibitor PF-4708671 (S6K1i) revealed similar and statistically significant synergistic apoptosis of HMCLs in combination with MCL-1i. Interestingly, apoptosis induced by the P70S6K1i and MCL-1i combination was more-than-additive in all 9 primary MM samples tested; this effect was observed for 6 of 9 samples with the dexamethasone and MCL-1i combination. Toxicity on stem and progenitor cell subsets remained minimal. Combined, our results show a strong rationale for combination treatments using the P70S6K inhibitor in MM. Direct and specific inhibition of P70S6K may also provide a solution for patients ineligible or insensitive to dexamethasone or other glucocorticoids., (© 2021 by The American Society of Hematology.)

Multiple myeloma is the second most frequent hematological malignancy in the western world and remains incurable, predominantly due to acquired drug resistance and disease relapse. The highly conserved Wnt signal transduction pathway, which plays a key role in regulating cellular processes of proliferation, differentiation, migration, and stem cell self-renewal, is associated with multiple aspects of disease. Bone homeostasis is severely disturbed by Wnt antagonists that are secreted by the malignant plasma cells in the bone marrow. In the vast majority of patients, this results in osteolytic bone disease, which is associated with bone pain and pathological fractures and was reported to facilitate disease progression. More recently, cumulative evidence also indicates the importance of intrinsic Wnt signaling in the survival of multiple myeloma cells. However, Wnt pathway-activating gene mutations could not be identified. The search for factors or processes responsible for Wnt pathway activation currently focuses on aberrant ligand levels in the bone marrow microenvironment, increased expression of Wnt transcriptional co-factors and associated micro-RNAs, and disturbed epigenetics and post-translational modification processes. Furthermore, Wnt pathway activation is associated with acquired cell adhesion-mediated resistance of multiple myeloma cells to conventional drug therapies, including doxorubicin and lenalidomide. In this review, we present an overview of the relevance of Wnt signaling in multiple myeloma and highlight the Wnt pathway as a potential therapeutic target for this disease.

Allogeneic stem cell transplantation (allo-SCT) has the potential to induce long-term remission in multiple myeloma (MM), but the role of allo-SCT in MM is controversial due to the high rate of treatment-related mortality (TRM). However, although proteasome inhibitors and immunomodulatory drugs have improved the outcome of patients with MM, high-risk patients still have a very poor prognosis. This indicates the need for new treatment strategies and identification of patients who might benefit from allo-SCT. We therefore analyzed the outcome of one hundred and forty-seven patients with MM who received an allo-SCT at our institution (58 in first line, 89 in relapsed/refractory setting) after a median follow-up of 88.8 months. For the first-line setting, median progression-free survival (PFS) and overall survival (OS) were remarkably good, with a CR rate of 48.3%, median PFS of 30.2 months, and 10-yr OS of 51%. We found no difference in outcome for patients with high-risk metaphase cytogenetics or FISH del(13q14), but efficacy in current standard high-risk patients could not be determined. The outcome in the relapsed/refractory setting was poor, especially in the subgroup of patients relapsing within 18 months after auto-SCT. Therefore, if applied at all in these patients, improvement of allo-SCT is needed, focusing on reduction of TRM and more effective immunotherapy., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

In a prospective multicenter phase II study, we evaluated the effect of three courses of vincristine, doxorubicin and dexamethasone followed by high-dose melphalan and autologous stem cell transplantation on an intention-to-treat basis. Sixty-nine newly diagnosed patients with amyloid light chain amyloidosis were included between November 2000 and January 2006: 37 men and 32 women with a median age of 56 years, including 46% of patients with cardiac and 22% of patients with involvement of 3 or 4 organs. Initial results presented in 2008 showed a 4-year overall survival rate of 62% among all the patients, while the 4-year survival rate after transplantation was 78%. Here we report the long-term follow-up data after a median follow up of 115 months of the patients still alive. Median survival of all patients was 96 months from registration and for the transplanted patients ten years from the date of transplantation. Twelve (12%) patients died during induction therapy with vincristine, doxorubicin and dexamethasone, including 8 patients (12%) due to treatment-related mortality. Two patients died within one month following high-dose melphalan. We conclude that vincristine, doxorubicin and dexamethasone should not be applied as induction therapy for intensification in amyloid light chain amyloidosis. However, a 2-step approach consisting of a non-intensive less toxic induction therapy followed by high-dose melphalan and autologous stem cell transplantation may result in extended survival in newly diagnosed patients with amyloid light chain amyloidosis (clinicaltrials.gov identifier: 01207094)., (Copyright© Ferrata Storti Foundation.)

During recent years, our understanding of the pathogenesis of inherited microcytic anemias has gained from the identification of several genes and proteins involved in systemic and cellular iron metabolism and heme syntheses. Numerous case reports illustrate that the implementation of these novel molecular discoveries in clinical practice has increased our understanding of the presentation, diagnosis, and management of these diseases. Integration of these insights into daily clinical practice will reduce delays in establishing a proper diagnosis, invasive and/or costly diagnostic tests, and unnecessary or even detrimental treatments. To assist the clinician, we developed evidence-based multidisciplinary guidelines on the management of rare microcytic anemias due to genetic disorders of iron metabolism and heme synthesis. These genetic disorders may present at all ages, and therefore these guidelines are relevant for pediatricians as well as clinicians who treat adults. This article summarizes these clinical practice guidelines and includes background on pathogenesis, conclusions, and recommendations and a diagnostic flowchart to facilitate using these guidelines in the clinical setting., (© 2014 by The American Society of Hematology.)

Background: X-linked sideroblastic anaemia (XLSA; OMIM#300751) is the most common inherited form of sideroblastic anaemia and is associated with several mutations in the erythroid specific 5-aminolevulinate synthase gene (ALAS₂). This gene encodes for aminolevulinic acid synthase 2 (ALAS₂), the catalytic enzyme involved in the first en rate-limiting step of haem biosynthesis.1-3 The disorder is characterised by mostly mild hypochromic microcytic anaemia with bone marrow ring sideroblasts. Even untransfused patients with mild or no anaemia are at risk for severe systemic iron overload due to ineffective erythropoiesis. To date, 61 different ALAS₂ mutations have been reported in 120 families with XLSA. Descriptions of molecularly confirmed case series from the Netherlands, however, are lacking., Methods: We reviewed age of presentation, clinical and biochemical features, ALAS₋₂ defects and treatment characteristics of 15 Dutch patients from 11 unrelated families diagnosed with XLSA., Results and Conclusions: In one family a novel pathogenic c.1412G>A (p.Cys471Tyr) mutation was found. All other families shared the previously described c.1355G>A (p.Arg452His) mutation. Haplotype analysis in seven probands with the p.Arg452His mutation strongly suggests that six of them were ancestrally related. Nevertheless, their phenotype was very different. Our patients illustrate the phenotypical heterogeneity in the presentation of XLSA patients, the effectiveness of treatment regimens and the various pitfalls associated with the diagnosis, follow-up and treatment of the disease. A timely diagnosis avoids unnecessary investigations and allows adequate treatment that can prevent systemic iron load with subsequent severe life-threatening complications. Therefore, we suggest considering XLSA in both male and female patients with unexplained iron overload and÷or (mild) microcytic anaemia, also at older age.

X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia. In affected males, it is uniformly associated with partial loss-of-function missense mutations in the erythroid-specific heme biosynthesis protein 5-aminolevulinate synthase 2 (ALAS2). Here, we report five families with XLSA owing to mutations in a GATA transcription factor binding site located in a transcriptional enhancer element in intron 1 of the ALAS2 gene. As such, this study defines a new class of mutations that should be evaluated in patients undergoing genetic testing for a suspected diagnosis of XLSA., (Copyright © 2013 Wiley Periodicals, Inc.)

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies.

Background: ATP-binding cassette (ABC) transporters protect cells against unrelated (toxic) substances by pumping them across cell membranes. Earlier we showed that many ABC transporters are highly expressed in hematopoietic stem cells (HSCs) compared to more committed progenitor cells. The ABC transporter expression signature may guarantee lifelong protection of HSCs but may also preserve stem cell integrity by extrusion of agents that trigger their differentiation. Here we have studied whether non-hematopoietic stem cells (non-HSCs) exhibit a similar ABC transporter expression signature as HSCs., Results: ABC transporter expression profiles were determined in non-hematopoietic stem cells (non-HSCs) from embryonic, neonatal and adult origin as well as in various mature blood cell types. Over 11,000 individual ABC transporter expression values were generated by Taqman Low Density Arrays (TLDA) to obtain a sensitivity comparable with quantitative real-time polymerase chain reactions. We found that the vast majority of transporters are significantly higher expressed in HSCs compared to non-HSCs. Furthermore, regardless their origin, non-HSCs exhibited strikingly similar ABC transporter expression profiles that were distinct from those in HSCs. Yet, sets of transporters characteristic for different stem cell types could be identified, suggesting restricted functions in stem cell physiology. Remarkably, in HSCs we could not pinpoint any single transporter expressed at an evidently elevated level when compared to all the mature blood cell types studied., Conclusions: These findings challenge the concept that individual ABC transporters are implicated in maintaining stem cell integrity. Instead, a distinct ABC transporter expression signature may be essential for stem cell function. The high expression of specific transporters in non-HSCs and mature blood cells suggests a specialized, cell type dependent function and warrants further functional experiments to determine their exact roles in cellular (patho)physiology.

Unrestricted somatic stem cells (USSCs) have been recently identified in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR-135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confirmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (>or=2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This finding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.

Purpose: Cytokines play a pivotal role in Hodgkin's lymphoma (HL). Because interleukin-6 (IL-6) induces expression of hepcidin, one of the principal regulators of iron metabolism, we studied the contribution of hepcidin in anemia in HL at diagnosis., Patients and Methods: Plasma samples from 65 patients with HL were analyzed for hepcidin levels using a combination of weak cation exchange chromatography and time-of-flight mass spectrometry; cytokine levels were analyzed using enzyme-linked immunosorbent assays and parameters of iron metabolism and acute-phase reaction., Results: Hepcidin plasma levels were significantly higher in HL patients when compared with controls, independent of the presence of anemia (P = .001). In the subset of patients with anemia, hepcidin levels inversely correlated with hemoglobin levels (P = .01). Analyzing parameters of iron metabolism, hepcidin levels showed a positive correlation with ferritin (P < .001) and an inverse correlation to iron and iron-binding capacity. Hepcidin strongly correlated to IL-6 levels (P < .001) but not to IL-10 or thymus and activation-regulated cytokine (TARC)/chemokine (C-C motif) ligand 17 (CCL17) levels. In a multivariate regression analysis, IL-6 and fibrinogen levels were independently associated with hepcidin. Higher hepcidin levels were observed in patients with more aggressive disease characteristics: stage IV disease (P = .01), presence of B symptoms (P = .03), and International Prognostic Score > 2 (P = .005)., Conclusion: Our findings suggest that in HL, hepcidin is upregulated by IL-6. Elevated hepcidin levels result in iron restriction and signs of anemia of chronic inflammation, although hepcidin-independent mechanisms contribute to development of anemia in HL.

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal haematopoietic stem cell malignancies. A subgroup, the so-called sideroblastic MDS, shows ring sideroblasts in the bone marrow aspirate that represent mitochondrial iron accumulation. Patients with sideroblastic MDS also develop systemic iron overload and generally have a low-risk MDS. Therefore it is important to understand the mechanisms responsible for iron accumulation and the associated toxicity in these patients. Recently, low levels of the iron-regulatory peptide hepcidin were found to contribute to body iron overload in beta-thalassaemia patients. A similar mechanism may account for systemic iron accumulation in sideroblastic MDS. Mitochondrial iron accumulation is observed in several subtypes of MDS, and predominantly in refractory anaemia with ring sideroblasts. The presence of ring sideroblasts is also the diagnostic hallmark in patients with inherited forms of sideroblastic anaemia. The ever-increasing insights into the affected pathways in inherited sideroblastic anaemia may lead to a better comprehension of the pathogenesis of mitochondrial iron accumulation in MDS patients. Overall, an improved understanding of the mechanisms responsible for iron overload in MDS will lead to novel treatment strategies to reduce both systemic and mitochondrial iron overload, resulting in less tissue damage and more effective erythropoiesis.

Stem cells are widely studied to enable their use in tissue repair. However, differences in function and differentiation potential exist between distinct stem cell populations. Whether those differences are due to donor variation, cell culture, or intrinsic properties remains elusive. Therefore, we compared 3 cell lines isolated from 3 different niches using the Affymetrix Exon Array platform: the cord blood-derived neonatal unrestricted somatic stem cell (USSC), adult bone marrow-derived mesenchymal stem cells (BM-MSC), and adult adipose tissue-derived stem cells (AdAS). While donor variation was minimal, large differences between stem cells of different origin were detected. BM-MSC and AdAS, outwardly similar, are more closely related to each other than to USSC. Interestingly, USSC expressed genes involved in the cell cycle and in neurogenesis, consistent with their reported neuronal differentiation capacity. The BM-MSC signature indicates that they are primed toward developmental processes of tissues and organs derived from the mesoderm and endoderm. Remarkably, AdAS appear to be highly enriched in immune-related genes. Together, the data suggest that the different mesenchymal stem cell types have distinct gene expression profiles, reflecting their origin and differentiation potential. Furthermore, these differences indicate a demand for effective differentiation protocols tailored to each stem cell type.

Background and Methods: To obtain efficacy and safety data on lenalidomide treatment outside of clinical trials, we analyzed the clinical data of 114 patients with refractory or relapsed multiple myeloma treated with lenalidomide on a compassionate use basis. The recommended treatment consisted of lenalidomide 25 mg given on days 1-21 of a 28-day cycle, in combination with dexamethasone. A median of 3 previous lines of therapy were given, including thalidomide in 91%. Most patients were treated until progression or intolerable toxicity., Results: The median number of cycles was 7 (range, 1-21+ cycles) with a maximum response after a median of 3 cycles (range, 1-10 cycles). The overall response rate was 69%, including complete response in 6%, very good partial response in 19%, and partial response in 44%. The response rate was not influenced by previous thalidomide and/or bortezomib treatment. The median time to progression (TTP) was 9 months and the median overall survival (OS) was 22 months. A significantly longer TTP was observed in patients who previously underwent allogeneic stem cell transplantation (12.5 months vs. 8 months; P = .036). Overall survival was significantly affected by performance status (P < .0001). Lenalidomide toxicity was predominantly hematologic (37%; Common Toxicity Criteria > or = 3) and the incidence of venous thrombotic events was low (5%) using the recommended prophylaxis., Conclusion: This analysis confirms that, outside clinical prospective trials, treatment with lenalidomide is highly effective and feasible in heavily pretreated patients with multiple myeloma.

Chronic infections are caused by microorganisms that display effective immune evasion mechanisms. Dendritic cell (DC)-dependent T cell-mediated adaptive immunity is one of the mechanisms that have evolved to prevent the occurrence of chronic bacterial infections. In turn, bacterial pathogens have developed strategies to evade immune recognition. In this study, we show that gram-negative and gram-positive bacteria differ in their ability to activate DCs and that gram-negative bacteria are far more effective inducers of DC maturation. Moreover, we observed that only gram-negative bacteria can induce loss of adhesive podosome structures in DCs, a response necessary for the induction of effective DC migration. We demonstrate that the ability of gram-negative bacteria to trigger podosome turnover and induce DC migration reflects their capacity to selectively activate TLR4. Examining mice defective in TLR4 signaling, we show that this DC maturation and migration are mainly Toll/IL-1 receptor domain-containing adaptor-inducing IFNbeta-dependent. Furthermore, we show that these processes depend on the production of PGs by these DCs, suggesting a direct link between TLR4-mediated signaling and arachidonic metabolism. These findings demonstrate that gram-positive and gram-negative bacteria profoundly differ in their capacity to activate DCs. We propose that this inability of gram-positive bacteria to induce DC maturation and migration is part of the armamentarium necessary for avoiding the induction of an effective cellular immune response and may explain the frequent involvement of these pathogens in chronic infections.

Recently various new gene defects have been identified which explain some previously unknown causes of inherited microcytic anaemias. These defects are located in genes that encode for the cellular iron importing protein Divalent Metal Transporter 1 (DMT1), the iron exporting protein ferroportin, the mitochondrial enzyme glutaredoxin-5 and the hepatocyte membrane protein matriptase-2.

Multiple myeloma is a malignant B-cell neoplasm that involves the skeleton in approximately 80% of the patients. With an average age of 60 years and a 5-years survival of nearly 45% Brenner et al. (Blood 111:2516-2520,35) the onset is to be classified as occurring still early in life while the disease can be very aggressive and debilitating. In the last decades, several new imaging techniques were introduced.The aim of this review is to compare the different techniques such as radiographic survey, multidetector computed tomography (MDCT), whole-body magnetic resonance imaging (WB-MRI), fluorodeoxyglucose positron emission tomography-(FDG-PET) with or without computed tomography(CT), and 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) scintigraphy. We conclude that both FDG-PET in combination with low-dose CT and whole-body MRI are more sensitive than skeleton X-ray in screening and diagnosing multiple myeloma. WB-MRI allows assessment of bone marrow involvement but cannot detect bone destruction, which might result in overstaging. Moreover,WB-MRI is less suitable in assessing response to the rapythan FDG-PET. The combination of PET with low-dose CT can replace the golden standard, conventional skeletal survey. In the clinical practise, this will result in upstaging,due to the higher sensitivity.

Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC [unrestricted somatic stem cells (USSCs)]. The maturation to mature DCs (mDCs) was enhanced when DCs were co-cultured with USSC, as evidenced by the up-regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin-12 production. So, USSCs mature iDCs, thereby redirecting the antigen-uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.

Recently, the antagonizing effect on the differentiation of mesenchymal stem cells (MSCs) by toll-like receptor (TLR) ligands, was described. Our study shows that on more primitive cord blood derived MSCs, the expression of TLRs and ligand-induced triggering differs from that of bone marrow derived MSCs. At the RNA level, cord blood MSCs (unrestricted somatic stem cells; USSCs) express low levels of TLR1,3,5,9 and high levels of TLR4 and TLR6. At the protein level expression of TLR5 and very low expression of TLR4 was observed. NF-kappaB translocation studies revealed that both TLR4 and TLR5 are functional, although signalling kinetics induced by the individual ligands differed. Stimulation of USSCs with either lipopolysaccharide (LPS) or flagellin resulted in a marked increase of interleukin (IL)-6 and/or IL-8 production although levels differed significantly between both stimuli. Interestingly, tumour necrosis factor (TNF)-alpha was undetectable after TLR stimulation, which appeared to be due to an inactivated TNF-alpha promoter in USSCs. Moreover, osteoblastic differentiation was enhanced after triggering USSCs with LPS and flagellin. In summary, TLR4 and 5 signalling in USSCs is slow and results in the up-regulation of a restricted number of pro-inflammatory cytokines and enhanced osteoblastic differentiation. Apparently, the outcome of TLR signalling depends on the cell type that expresses them.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of neoplastic hematopoietic disorders. Several recurrent chromosomal aberrations have been associated with MDS, but the genes affected have remained largely unknown. To identify relevant genetic lesions involved in the pathogenesis of MDS, we conducted SNP array-based genomic profiling and genomic sequencing in 102 individuals with MDS and identified acquired deletions and missense and nonsense mutations in the TET2 gene in 26% of these individuals. Using allele-specific assays, we detected TET2 mutations in most of the bone marrow cells (median 96%). In addition, the mutations were encountered in various lineages of differentiation including CD34(+) progenitor cells, suggesting that TET2 mutations occur early during disease evolution. In healthy tissues, TET2 expression was shown to be elevated in hematopoietic cells with highest expression in granulocytes, in line with a function in myelopoiesis. We conclude that TET2 is the most frequently mutated gene in MDS known so far.

Immunoglobulin A (IgA) secretion by plasma cells in the immune system is critical for protecting the host from environmental and microbial infections. However, the molecular mechanisms underlying the generation of IgA(+) plasma cells remain poorly understood. Here, we report that the B cell-expressed tetraspanin CD37 inhibits IgA immune responses in vivo. CD37-deficient (CD37-/-) mice exhibit a 15-fold increased level of IgA in serum and significantly elevated numbers of IgA(+) plasma cells in spleen, mucosal-associated lymphoid tissue, as well as bone marrow. Analyses of bone marrow chimeric mice revealed that CD37-deficiency on B cells was directly responsible for the increased IgA production. We identified high local interleukin-6 (IL-6) production in germinal centers of CD37-/- mice after immunization. Notably, neutralizing IL-6 in vivo reversed the increased IgA response in CD37-/- mice. To demonstrate the importance of CD37-which can associate with the pattern-recognition receptor dectin-1-in immunity to infection, CD37-/- mice were exposed to Candida albicans. We report that CD37-/- mice are evidently better protected from infection than wild-type (WT) mice, which was accompanied by increased IL-6 levels and C. albicans-specific IgA antibodies. Importantly, adoptive transfer of CD37-/- serum mediated protection in WT mice and the underlying mechanism involved direct neutralization of fungal cells by IgA. Taken together, tetraspanin protein CD37 inhibits IgA responses and regulates the anti-fungal immune response.

The C-type lectins DC-SIGN (CD209) and L-SIGN (CD299) recognize defined carbohydrates expressed on pathogens and cells. Those lectins are expressed on dendritic cells (DC) and/or on liver-sinusoidal endothelial cells. Both cell types modulate immune responses. In acute lymphoblastic leukemia (ALL), aberrant glycosylation of blast cells can alter their interaction with the C-type lectins DC-SIGN and L-SIGN, thereby affecting their immunological elimination. We investigated whether recombinant DC-SIGN and L-SIGN bind to blood or bone marrow cells from B- and T-ALL patients and compared that with binding of peripheral blood lymphocytes from healthy donors. It was found that increased binding of ALL cells to DC-SIGN and L-SIGN was observed compared to cells from healthy donors. Furthermore, L-SIGN bound a higher percentage of leukemic and normal cells than DC-SIGN. B-ALL bone marrow cells showed the highest binding to L-SIGN. DC-SIGN bound equally well to B-ALL and T-ALL cells. Within ALL subtypes, DC-SIGN binding was higher with mature T-ALL. Interestingly, our data demonstrate that increased binding of DC-SIGN and L-SIGN to peripheral leukemic cells from B-ALL patients is associated with poor survival. These data demonstrate that high binding of B-ALL peripheral blood cells to DC-SIGN and L-SIGN correlates with poor prognosis. Apparently, when B-ALL cells enter the blood circulation and are able to interact with DC-SIGN and L-SIGN the immune response is shifted toward tolerance. Additional studies are necessary to ascertain the possible role of these results in terms of disease pathogenesis and their potential as target to eradicate leukemic cells.

Minor histocompatibility antigens (MiHA) selectively expressed by haematopoietic cells are attractive targets for specific immunotherapy after allogeneic stem cell transplantation (SCT). Previously, we described LRH-1 as a haematopoietic-lineage restricted MiHA that is capable of eliciting an allogeneic cytotoxic T-lymphocyte (CTL) response after SCT and donor lymphocyte infusion. Importantly, the gene encoding LRH-1, P2X5, is not expressed in prominent graft-versus-host-disease target tissues such as skin, liver and gut. Here, we investigate whether LRH-1-specific immunotherapy may be exploited for the treatment of lymphoid malignancies. We examined P2X5 mRNA expression in a large panel of patient samples and cell lines from different types of lymphoid malignancies by real-time quantitative reverse transcription polymerase chain reaction. P2X5 mRNA was highly expressed in malignant cells from all stages of lymphoid development. Furthermore, all LRH-1-positive lymphoid tumour cell lines were susceptible to LRH-1 CTL-mediated lysis in flow cytometry-based cytotoxicity assays. However, interferon-gamma production was low or absent after stimulation with some cell lines, possibly due to differences in activation thresholds for CTL effector functions. Importantly, primary cells from patients with lymphoid malignancies were effectively lysed by LRH-1-specific CTL. These findings indicate that MiHA LRH-1 is a potential therapeutic target for cellular immunotherapy of lymphoid malignancies.

Enhanced proliferation of MDS progenitors is abrogated by increased apoptosis of their progeny in vivo. We investigated whether bone marrow mononuclear cells (BMMNC) of MDS patients also showed enhanced proliferation and apoptosis in vitro in comparison with acute myeloid leukemia (AML) and normal BM (NBM). NBM showed a decrease in the number of clusters in time due to apoptosis of clusters and due to development of clusters into colonies with low apoptotic level. In MDS patients, about two-fold more clusters have developed at day 4, and in contrast with NBM, the total number of clusters at day 7 remained high in spite of an increasing percentage of apoptotic clusters (from 52 to 76%) in combination with more colony formation. The number of clusters and colonies showed a sharp decrease at day 10 because of persistently high apoptosis at cluster level and increasing apoptosis in colonies. BMMNC of AML patients showed a decreased proliferation with enhanced apoptosis at cluster level in contrast to a relatively low apoptotic levels in the colony-forming cells. This data show that increased proliferation is abrogated by enhanced apoptosis in MDS, whereas AML showed decreased proliferation with a low level of apoptosis in colony-forming cells. These growth profiles of BMMNC are independent of stromal influences and may represent intrinsic features of the MDS progenitors and accessory cell interactions.

Hereditary haemochromatosis (HH) is a disease related to mutations in the HFE gene and can lead to progressive iron accumulation, especially in the liver, eventually resulting in organ damage. We have developed guidelines for the diagnosis and treatment of this disease according to CBO methodology (dutch institute for Healthcare Quality). The prevalence of clinical symptoms such as fatigue, arthropathies, impotence and diabetes mellitus among homozygotes was similar to that in a control population. Nevertheless, we recommend the assessment of serum iron indices when these symptoms remain unexplained. When transferrin saturation is >45% and ferritin exceeds local reference ranges, HFE mutations should be investigated. Homozygosity for the C282Y mutation or combined C282Y/H63d mutation confirms the diagnosis of HFE-related HH. Liver biopsy is recommended when ferritin exceeds 1000 microg/l to establish the presence or absence of cirrhosis, which will affect prognosis and management. iron accumulation confirmed by magnetic resonance imaging (MRI) in the absence of the homozygous C282Y mutation or the combined C282Y/H63d genotype may justify a search for rare hereditary forms of non-HFE HH in a specialised centre. The literature supports the benefits of adequate phlebotomy and the screening of first-degree relatives of index patients with clinically overt HH. overall, the guidelines presented here are to a great extent based on the expert opinion of the working party, as the quantity of evidence that met predefined criteria posed by the evidence-based approach was small. We therefore recommend world-wide efforts to collaboratively address these remaining issues.

The purpose of this study was to determine the role of pre-emptive donor lymphocyte infusion (pDLI) after partial T-cell-depleted allogeneic SCT in patients with multiple myeloma (MM). A cohort of 24 MM patients was treated with partial T-cell-depleted myeloablative SCT between December 1997 and April 2002. These patients were intended to receive pDLI after SCT. The overall response rate after SCT was 83% (20 of 24 patients) with 10 patients (42%) in complete remission (CR). Transplant-related mortality within 1 year after SCT was 29%. Thirteen patients (54%) received pDLI and four patients in partial remission reached CR. GVHD>grade I after pDLI developed in 4 out of 13 patients (30%). Four patients received therapeutic DLI, without preceding pDLI. Eleven patients (46%) are alive, with a median follow-up of 67 months (range, 48-100 months). Seven of these patients (29%) are in continuous CR (CCR), which was confirmed by a negative patient-specific IgH PCR in four patients. All seven patients in CCR received pDLI. Although myeloablative SCT in MM induces high toxicity, we show that the concept of T-cell depletion followed by pDLI is promising and needs to be investigated in a reduced-intensity conditioning setting.

Dendritic cells (DCs) are professional APCs of the immune system that play a key role in regulating T cell-based immunity. The capacity of DCs to activate T cells depends on their maturation state as well as their ability to migrate to the T cell areas of draining lymph nodes. In this study, we investigated the effects of DC maturation stimuli on the actin cytoskeleton and beta(1) integrin-dependent adhesion and migration. Podosomes, specialized adhesion structures found in immature monocyte-derived DCs as well as myeloid DCs, rapidly dissolve in response to maturation stimuli such as TNF-alpha and PGE(2), whereas the TLR agonist LPS induces podosome dissolution only after a long lag time. We demonstrate that LPS-mediated podosome disassembly as well as the onset of high-speed DC migration are dependent on the production of PGs by the DCs. Moreover, both of these processes are inhibited by Ab-induced activation of beta(1) integrins. Together, these results show that maturation-induced podosome dissolution and loss of alpha(5)beta(1) integrin activity allow human DCs to undergo the transition from an adhesive to a highly migratory phenotype.

In this retrospective study, we evaluated donor lymphocyte infusions given for relapsed (n=48) or persistent (n=15) myeloma following non-myeloablative allogeneic stem cell transplantation (Allo-SCT). Twenty-four of 63 patients (38.1%) responded: 12 patients (19.0%) with a partial response (PR) and 12 patients (19.0%) with a complete response (CR). Overall survival after donor lymphocyte infusions (DLI) was 23.6 months (1.0-50.7+). Median overall survival for non-responding patients was 23.6 months and has not been reached for the patients responding to DLI. In responders, progression-free survival after DLI was 27.8 months (1.2-46.2+). Patients with a PR had a median progression-free survival of 7.0 months, whereas patients with a CR to DLI had a median progression-free survival of 27.8 months. Major toxicities were acute graft-versus-host disease (GVHD) (38.1%) and chronic GVHD (42.9%). Seven patients (11.1%) died from treatment-related mortality. The only significant prognostic factors for response to DLI were the occurrence of acute and chronic GVHD. There was a trend towards significance for time between transplantation and DLI, and response. Donor lymphocyte infusion following non-myeloablative Allo-SCT is a valuable strategy for relapsed or persistent disease.

Purpose: Acute myelogenous leukemia (AML) is a disease originating from normal hematopoietic CD34+ CD38- progenitor cells. Modulation of the multidrug ATP-binding cassette transporter ABCB1 has not resulted in improved outcome in AML, raising the question whether leukemic CD34+ CD38- cells are targeted by this strategy., Experimental Design: ABCB1-mediated transport in leukemic CD34+ CD38- cells compared with their normal counterparts was assessed by quantitating the effect of specific ABCB1 modulators (verapamil and PSC-833) on mitoxantrone retention [defined as efflux index (EI), intracellular mitoxantrone fluorescence intensity in the presence/absence of inhibitor]., Results: ABCB1 was the major drug transporter in CD34+ CD38- cells in normal bone marrow (n = 16), as shown by the abrogation of mitoxantrone extrusion by ABCB1 modulators (EI, 1.99 +/- 0.08). Surprisingly, ABCB1-mediated drug extrusion was invariably reduced in CD34+ CD38- cells in AML (n = 15; EI, 1.21 +/- 0.05; P < 0.001), which resulted in increased intracellular mitoxantrone retention in these cells (mitoxantrone fluorescence intensity, 4.54 +/- 0.46 versus 3.08 +/- 0.23; P = 0.004). Active drug extrusion from these cells occurred in the presence of ABCB1 modulators in the majority of samples, pointing in the direction of redundant drug extrusion mechanisms. Residual normal CD34+ CD38- cells could be identified by their conserved ABCB1-mediated extrusion capacity., Conclusion: ABCB1-mediated drug extrusion is reduced in leukemic CD34+ CD38- progenitor cells compared with their residual normal counterparts. Redundant drug transport mechanisms confer mitoxantrone transport from leukemic progenitors. These data argue that ABCB1 modulation is not an effective strategy to circumvent drug extrusion from primitive leukemic progenitor cells and may preferentially target residual normal progenitors in AML.

The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding-cassette transporter involved in the transport of drugs used in the treatment of multiple myeloma (MM). Its expression, function and clinical significance in MM, however, are unknown. We report that BCRP is preferentially expressed and functionally active in normal plasma cells but that its function is significantly impaired in plasma cells in newly diagnosed MM. The data presented argue against a role for BCRP in primary drug resistance in MM and the utilisation as a molecular target as such but warrant research into its (patho)physiological role in normal and malignant plasma cells.

Objective: Bone marrows (BM) of myelodysplastic syndrome (MDS) patients show increased proliferation and premature programmed cell death (PCD) in vivo as well as in vitro. We explored the proliferative capacity and apoptotic propensity of CD34+ progenitor cells of MDS patients excluding accessory cell interference., Materials and Methods: CD34+/CD3-/CD19- cells of 5 MDS patients and 5 normal BM were sorted as single cells into single wells and were cultured in liquid medium. Wells were evaluated on days 4, 7, 10, and 14. PCD was determined by staining with annexin V-FITC. Growth rate and cell doubling time (Td) were calculated for each colony-forming cell., Results: Normal BM CD34+ cells formed clusters and colonies and both showed increasing PCD in time, although within colonies the degree of apoptosis was twice as high (about 25%) as compared with clusters at all time points. In MDS increased cluster formation was observed at all evaluation points when compared to normal BM, whereas the number of colonies was markedly reduced (1/7 of normal). These colonies were also smaller, usually smaller than 100 cells. Significantly enhanced levels of PCD of clusters (53-79%) in combination with longer cell doubling times explain this slower formation of smaller colonies. Surprisingly, these colonies showed considerably lower levels of PCD (7-32%) as compared to normal (1-48%, median values)., Conclusions: In the absence of stromal influences and accessory cells, this study in MDS patients showed intrinsically enhanced proliferation and apoptosis of cluster-forming cells, as the opposite was true for colony-forming cells.

Purpose: Acute myeloid leukemia (AML) is considered a stem cell disease. Incomplete chemotherapeutic eradication of leukemic CD34+38- stem cells is likely to result in disease relapse. The purpose of this study was to investigate the role of the breast cancer resistance protein (BCRP/ATP-binding cassette, subfamily G, member 2) in drug resistance of leukemic stem cells and the effect of its modulation on stem cell eradication in AML., Experimental Design: BCRP expression (measured flow-cytometrically using the BXP21 monoclonal antibody) and the effect of its modulation (using the novel fumitremorgin C analogue KO143) on intracellular mitoxantrone accumulation and in vitro chemosensitivity were assessed in leukemic CD34+38- cells., Results: BCRP was preferentially expressed in leukemic CD34+38- cells and blockage of BCRP-mediated drug extrusion by the novel fumitremorgin C analogue KO143 resulted in increased intracellular mitoxantrone accumulation in these cells in the majority of patients. This increase, however, was much lower than in the mitoxantrone-resistant breast cancer cell line MCF7-MR and significant drug extrusion occurred in the presence of BCRP blockage due to the presence of additional drug transport mechanisms, among which ABCB1 and multiple drug resistance protein. In line with these findings, selective blockage of BCRP by KO143 did not enhance in vitro chemosensitivity of leukemic CD34+38- cells., Conclusions: These results show that drug extrusion from leukemic stem cells is mediated by the promiscuous action of BCRP and additional transporters. Broad-spectrum inhibition, rather than modulation of single mechanisms, is therefore likely to be required to circumvent drug resistance and eradicate leukemic stem cells in AML.

Introduction: Epstein-Barr virus (EBV) establishes lifelong latent infection. In some patients the host-virus balance is disturbed, resulting in a chronic active EBV infection. The following case illustrates the difficulty in diagnosing and treating chronic EBV infection., Case: A 30-year-old woman was referred because of recurrent swellings of lymphatic tissue of both eyelids, orbit and lymph nodes and general malaise since the age of 19. In the past, repeated biopsies showed MALT lymphoma and nonspecific lymphoid infiltrations. Now, a biopsy of an axillary lymph node showed paracortical hyperplasia with a polymorphous polyclonal lymphoid proliferation, and large numbers of EBV-encoded small RNA (EBER) positive cells, consistent with EBV infection. Laboratory investigation showed a high EBV viral load. No evidence of immunodeficiency was found. Chronic active EBV infection (CAEBV) was diagnosed. Treatment with high-dose acyclovir did not significantly reduce the viral load. Rituximab was given in an attempt to reduce the amount of EBV-infected B lymphocytes. However, soon after the second dose the patient died of a sub-arachnoidal haemorrhage., Conclusion: This case report illustrates CAEBV as a rare manifestation of EBV-induced disease, which will be detected more frequently with the use of EBV-EBER hybridisation of lymph nodes and polymerase chain reaction (PCR) for EBV DNA. The prognosis is poor with no established therapeutic strategies.

Design of automated image processing systems to determine migration characteristics of individual cells is not trivial. Every test sample requires separate recording and the analysis of individual cell tracks in two- or three-dimensional migration systems by time-lapse microscopy is extremely laborious. Here, we describe a new Automated Cell Track System (ACTS). In addition to contrast differences, which are used by existing analysis systems, the ACTS algorithms recognize cells on the basis of morphological similarities in successive images and adapt to the continuous shape changes of individual cells during migration. The system facilitates simultaneous analysis of multiple cells and the measurement of multiple wells in one single experiment. We validated the system studying HSB-2 T cell migration in standard 96-well microtiter plates coated with ICAM-1-Fc protein or control CD14-Fc protein. Migration of HSB-2 T cells on ICAM-1-Fc is Leukocyte Function-associated Antigen-1 (LFA-1)-mediated and both the number and the speed of migrating cells depend on the ICAM-1-Fc concentration. We show that automated analysis of the migration data yields similar results as manual analysis, but in a fraction of the time. We conclude that this system is extremely well suited to precisely monitor the migratory behavior of individual cells. The analysis of multiple wells in parallel makes this set-up appropriate in high throughput screening in which multiple components are simultaneously tested for their effect on cell migration.

Background: Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux., Methods: We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively)., Results: The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively)., Conclusion: The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells., (Copyright 2002 Wiley-Liss, Inc.)

In normal bone marrow, WT1 expression is restricted to CD34+ cells. We assessed WT1 mRNA expression levels with quantitative, real-time reverse transcription polymerase chain reaction in normal, myelodysplastic (MDS) and secondary acute myeloid leukaemia (sAML) bone marrow subfractions, based on differentiation status. The highest WT1 expression was observed in the primitive CD34+ rhodamine-123 (rho) dull cells, both in healthy donors and MDS or sAML patients. In contrast to normal CD34-negative bone marrow cells, WT1 was present in CD34-negative bone marrow cells in 12 out of 13 MDS patients and two sAML samples. Further analysis of this aberrant WT1 expression was performed in the CD34-negative subfractions of three MDS patients. In one of these, WT1 expression was found exclusively in the erythroid cells. This patient was completely transfusion dependent and showed morphological dyserythropoiesis. In another MDS patient, WT1 expression was found in a non-erythroid compartment. We conclude that abnormal WT1 expression may contribute to the disturbed differentiation of haematopoietic cells in MDS patients.