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To acquire a better understanding of clonal evolution of acute myeloid leukemia (AML) and to identify the clone(s) responsible for disease recurrence, we have comparatively studied leukemia-specific mutations by whole-exome-sequencing (WES) of both the leukemia and the nonleukemia compartments derived from the bone marrow of AML patients. The T-lymphocytes, B-lymphocytes and the functionally normal hematopoietic stem cells (HSC), that is, CD34+ /CD38- /ALDH+ cells for AML with rare-ALDH+ blasts (

In acute myeloid leukaemia (AML) t(8;16)(p11;p13)/MYST3-CREBBP is a very rare abnormality. Previous small series suggested poor outcome. We report on 59 patients with t(8;16) within an international, collaborative study. Median age was 52 (range: 16-75) years. AML was de novo in 58%, therapy-related (t-AML) in 37% and secondary after myelodysplastic syndrome (s-AML) in 5%. Cytogenetics revealed a complex karyotype in 43%. Besides MYST3-CREBBP, whole-genome sequencing on a subset of 10 patients revealed recurrent mutations in ASXL1, BRD3, FLT3, MLH1, POLG, TP53, SAMD4B (n = 3, each), EYS, KRTAP9-1 SPTBN5 (n = 4, each), RUNX1 and TET2 (n = 2, each). Complete remission after intensive chemotherapy was achieved in 84%. Median follow-up was 5·48 years; five-year survival rate was 17%. Patients with s-/t-AML (P = 0·01) and those with complex karyotype (P = 0·04) had an inferior prognosis. Allogeneic haematopoietic cell transplantation (allo-HCT) was performed in 21 (36%) patients, including 15 in first complete remission (CR1). Allo-HCT in CR1 significantly improved survival (P = 0·04); multivariable analysis revealed that allo-HCT in CR1 was effective in de novo AML but not in patients with s-AML/t-AML and less in patients exhibiting a complex karyotype. In summary, outcomes of patients with t(8;16) are dismal with chemotherapy, and may be substantially improved with allo-HCT performed in CR1.

Premature aging of both CD4+ regulatory T (Treg) and CD4+ responder‐T (Tresp) cells in patients with end‐stage renal disease (ESRD) is expected to affect the success of later kidney transplantation. Both T‐cell populations are released from the thymus as inducible T‐cell co‐stimulator‐positive (ICOS +) and ICOS − recent thymic emigrant (RTE) Treg/Tresp cells, which differ primarily in their proliferative capacities. In this study, we analysed the effect of ESRD and subsequent renal replacement therapies on the differentiation of ICOS + and ICOS − RTE Treg/Tresp cells into ICOS + CD31− or ICOS − CD31− memory Treg/Tresp cells and examined whether diverging pathways affected the suppressive activity of ICOS + and ICOS − Treg cells in co‐culture with autologous Tresp cells. Compared with healthy controls, we found an increased differentiation of ICOS + RTE Treg/Tresp cells and ICOS − RTE Treg cells through CD31+ memory Treg/Tresp cells into CD31− memory Treg/Tresp cells in ESRD and dialysis patients. In contrast, ICOS − RTE Tresp cells showed an increased differentiation via ICOS − mature naive (MN) Tresp cells into CD31− memory Tresp cells. Thereby, the ratio of ICOS + Treg/ICOS + Tresp cells was not changed, whereas that of ICOS − Treg/ICOS − Tresp cells was significantly increased. This differentiation preserved the suppressive activity of both Treg populations in ESRD and partly in dialysis patients. After transplantation, the increased differentiation of ICOS + and ICOS − RTE Tresp cells proceeded, whereas that of ICOS + RTE Treg cells ceased and that of ICOS − RTE Treg cells switched to an increased differentiation via ICOS − MN Treg cells. Consequently, the ratios of ICOS + Treg/ICOS + Tresp cells and of ICOS − Treg/ICOS − Tresp cells decreased significantly, reducing the suppressive activity of Treg cells markedly. Our data reveal that an increased tolerance‐inducing differentiation of ICOS + and ICOS − Treg cells preserves the functional activity of Treg cells in ESRD patients, but this cannot be maintained during long‐term renal replacement therapy.

BACKGROUND Recent approaches in multiple myeloma (MM) treatment have targeted CD38. As antigen expression levels on plasma cells (PCs) were demonstrated to affect response to monoclonal antibody (mAb) treatment, a precise characterization of PC phenotype is warranted. METHODS Anti-CD38 mAb (isatuximab) was tested for antibody-dependent cellular cytotoxicity (ADCC) in MM cell lines. Quantification of the number of sites (NOS) of CD38 on bone marrow PCs and other immune cells obtained from light chain (AL) amyloidosis (n = 46) and smoldering multiple myeloma (SMM) patients (n = 19) was performed with two different quantitative flow cytometry (QFCM) applications. RESULTS ADCC activity of isatuximab was observed in cell lines with >100 × 103 CD38-NOS only. The average PC CD38-NOS was 153 ± 53 × 103 in AL amyloidosis and 138.7 ± 53 × 103 in SMM patients. Eight (17%) AL amyloidosis and 4 (21%) SMM patients showed a PC CD38-NOS level

BACKGROUND Granulocyte–colony-stimulating factor (G-CSF) originators such as filgrastim (Neupogen) and lenograstim (Granocyte) are widely used for peripheral blood stem cell (PBSC) mobilization. In recent years, biosimilar agents have been approved for the same indications. The aim of this retrospective study was to compare the mobilization efficiency of the three G-CSF variants originator filgrastim, lenograstim, and the biosimilar Filgrastim Hexal in a homogeneous group of multiple myeloma (MM) patients in first-line therapy. STUDY DESIGN AND METHODS Overall mobilization data of 250 patients with MM were included. Of these patients, 74 (30%), 131 (52%), and 45 (18%) were mobilized with originator filgrastim, biosimilar Filgrastim Hexal, or lenograstim, respectively, at a dose of 5 to 10 µg/kg body weight subcutaneously starting from Day 5 after chemomobilization with CAD (cyclophosphamide, doxorubicin, dexamethasone) until completion of PBSC collection. RESULTS All but one patient reached the collection goal of a minimum of at least 2 × 106 CD34+ cells/kg body weight during a median of one (range, one to three) leukapheresis session. No significant differences in CD34+ mobilization and collection yields between the filgrastim-mobilized (median, 10.5; range, 2.7-40.4), Filgrastim Hexal–mobilized (median, 9.9; range, 0.2-26.0), and lenograstim-mobilized (median, 10.7; range, 3.1-27.9 CD34+ cells × 106/kg body weight) patients were observed. CONCLUSION Concerning the clinically relevant efficiencies of PBSC mobilization and in terms of reaching the individual collection target, this retrospective study did not detect any significant differences between the three G-CSF variants in the analyzed patient cohort.

Objective Analysis of the efficiency and toxicity of cyclophosphamide-based stem cell mobilization in patients with relapsed multiple myeloma (RMM). Methods Peripheral blood stem cells (PBSCs) were mobilized with high dose cyclophosphamide (2 g/m2 daily on days 1 and 2) and G-CSF plus pre-emptive/rescue plerixafor in RMM patients (first to third relapse) treated within the ReLApsE trial of the German-Speaking Myeloma Multicenter Group (GMMG). Results Mobilization was initiated with high-dose cyclophosphamide (HD-CY) and G-CSF in 30 patients. Fifteen patients received additional pre-emptive/rescue administration of plerixafor. Stem cell collection was successful (≥2×106 CD34+ cells per kg bw) in 77% (23/30 patients). Patients with prior high-dose melphalan collected a significantly lower median total number of PBSCs than patients without prior high-dose melphalan (3.3×106 vs 17×106 CD34+ cells/kg bw). Toxicity of HD-CY was frequent with 12 serious adverse events (SAE) in 37% of patients (11/30 patients). Infections accounted for the majority of SAE reports. In two patients, SAEs were lethal (septic shock). Conclusions These data proof feasibility of PBSC collection at relapse but emphasize the importance of collection and storage of additional PBSC transplants during first-line treatment when mobilization is more efficient and less toxic.

We tested the hypothesis that proliferative activity of hematopoietic stem cells has impact on survival in newly diagnosed patients with myelodysplastic syndrome (MDS) and secondary acute myeloid leukemia (AML). RNA expression profiles of CD34(+) cells were analyzed in 125 MDS patients and compared to healthy controls. Prognostic impact on overall survival (OS) of mRNA proliferation signatures established for solid tumor cells was analyzed retrospectively. For validation on the protein level, immunofluorescence and immunohistochemistry analyses in bone marrow (BM) biopsies were performed, and an independent cohort of 223 MDS and secondary AML patients was investigated. Lower proliferative activity correlated with the expression of cyclin-dependent kinase inhibitor 1C (CDKN1C) and with shorter OS (p

Terumo BCT recently introduced a new system for mononuclear cell (MNC) collection that uses a Spectra Optia apheresis machine equipped with a redesigned disposable kit and software program (version 11.2). It allows for the continuous collection of MNCs, unlike the original Spectra Optia system (version 7.2), which included a chamber for two-step cell separation. The aim of this study was to compare the two apheresis systems in regard to specific performance parameters. A retrospective data analysis of 150 patients who had undergone peripheral blood stem cell collection between March of 2014 and May of 2015 at our institution was performed. For the matched comparison, patients were divided into two groups by diagnosis and by previous forms of therapy received: a homogeneous group of patients with multiple myeloma (MM) that had received first line therapy ("MM" group, n = 88) and a heterogeneous group that included all of the other patients ("other" group, n = 62). No significant differences in CD34+ collection yields between both collection regimens were found (pMM = 0.19, pother = 0.74) in either group. Moreover, similar performance ratios (collected/predicted CD34+ cell number in %) were observed (pMM = 0.89, pother = 0.1). No relevant variations in platelet or hemoglobin loss were found between the two systems. We conclude that the new continuous Spectra Optia MNC system is equally efficient in collecting CD34+ cells and can be used without sacrificing collection efficiency levels when treating a broad variety of autologous patients. J. Clin. Apheresis 32:27-34, 2017. © 2016 Wiley Periodicals, Inc.

The prospective, randomized phase III trial GMMG-HD2 aimed at demonstrating non-inferiority of single (Arm A) versus tandem (Arm B) high-dose melphalan followed by autologous transplantation (HDM/ASCT) with regard to 2-year event-free survival (EFS) in newly-diagnosed multiple myeloma (MM) and included 358 evaluable patients [Intention-to-treat population, (ITT), single/tandem HDM/ASCT: n = 177/181]. After a median follow-up of more than 11 years, non-inferiority of single versus tandem HDM/ASCT was demonstrated using the planned non-inferiority threshold of 15% of the 2-year EFS rate. Neither EFS (P = 0·53) nor overall survival (OS) (P = 0·33) differences were observed in the ITT population. In the tandem arm, 26% (n = 47/181) of patients refused a second HDM/ASCT due to non-medical reasons. A per-protocol (PP) analysis, including patients who received the intervention (single/tandem HDM/ASCT: n = 156/93) and patients who did not receive a second HDM/ASCT due to medical reasons (12%, n = 22/181), did not yield differences in EFS (P = 0·61) or OS (P = 0·16). In the ITT and PP set of the tandem arm, the rates of complete responses increased from first to second HDM/ASCT (both P = 0·04). Ten-year OS for the entire ITT was 34% (95% confidence interval: 29-40%). OS after first relapse was significantly shortened in the tandem arm (P = 0·04). In this study single HDM/ASCT was non-inferior to tandem HDM/ASCT in MM.

The discovery of new targets for tailored therapy is a major improvement in oncology, and tools for the rapid and reliable detection of these targets are essential. Clinical trials demonstrated the benefit of recently developed antibodies against antigens on malignant B-cells. The aim of this study was to assess patients with plasma cell (PC) disorders for expression of antigens on malignant PCs that have exhibited promise in targeted cancer therapy. We retrospectively analyzed the expression of CD20, CD22, CD27, CD30, CD38, CD52, CD81, CD138, and SLAMF7 on PCs by flow cytometry in 103 patients with PC disorders. Furthermore, we studied cytogenetic data to correlate immunophenotyping and genetic parameters. The expression frequency of CD22, CD30, and CD52 was similar to other studies (12–35%, 0–19%, and 0–8%, respectively). Unexpectedly, we observed a high CD20 expression frequency in 37% of all AL-amyloidosis cases. The presence of t(11;14) correlated positively with CD20 expression on PCs in AL-amyloidosis (p = 0.018). Furthermore, the expression level of SLAMF7 was decreased in advanced PC disorders (p = 0.025) and a diminished expression of SLAMF7 is associated with low expression of CD27 and CD81 on malignant PCs in newly diagnosed multiple myeloma. This study provides a contribution to targeted therapy options in PC disorders. Particularly, the results put an emphasis on CD20 as therapeutic target in AL-amyloidosis. Regarding the therapeutic options of the SLAMF7 antibody elotuzumab, these data advise that analysis of SLAMF7 expression before application of elotuzumab might help to estimate the efficacy of elotuzumab in clinical trials. © 2016 International Clinical Cytometry Society

Wilms' tumor gene 1 (WT1) is overexpressed in leukemia and WT1-derived CD8(+) T-cell epitopes for immunotherapies targeting WT1 have been defined. Here, we analyzed expression of WT1 in 226 peripheral blood and bone marrow samples from patients with acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) before and after allogeneic stem cell transplantation (SCT). Transcripts were assessed by quantitative polymerase chain reaction, and WT1-specific CD8+ cytotoxic T cells (CTL) were monitored by tetramer staining and enzyme-linked immunospot (ELISPOT) assays. Reduction of WT1 levels correlated with a longer survival (p

For patients with severe and refractory autoimmune diseases, high-dose chemotherapy and autologous hematopoietic stem cell transplantation has been established as a considerable therapeutic option in recent years. In this retrospective single-center analysis, we assessed the feasibility and efficacy of peripheral blood stem cells (PBSC) mobilization and collection in 35 patients with refractory autoimmune disease (AID). The mobilization data of 15 patients with systemic sclerosis (SSc), 11 patients with multiple sclerosis (MS), and 9 patients with other AID were analyzed. Stem cell mobilization with cyclophosphamide chemotherapy 2 × 2 g/m(2) (n = 16) or 1 × 2 g/m(2) (n = 17) and G-CSF followed by PBSC collection was performed between 1999 and 2015. Leukapheresis was performed in 16 inpatients and 19 outpatients. All patients reached their collection goal and no collection failures were observed. The median PBSC collection result was 12.2 (SSc), 8.0 (MS), and 8.2 (other AID) × 10(6) CD34+ cells/kg, respectively. Twenty-five of 35 (71%) patients achieved a sufficient collection with one leukapheresis session, while 6 patients (17%) required two and 4 patients (11%) required three or more leukapheresis sessions. No correlation of the collected PBSC number was observed regarding age, body weight, diagnosis, disease duration, skin sclerosis, or previous cyclophosphamide. Mobilization chemotherapy with cyclophosphamide 2 × 2 g/m(2) and 1 × 2 g/m(2) delivered comparable mobilization results with leukapheresis on day 13 or 14. In summary, we demonstrate that PBSC collection is safe and feasible in patients with AID. Mobilization chemotherapy with cyclophosphamide 1 × 2 g/m(2) and 2 × 2 g/m(2) is equally effective in those patients.

In the multicentre prospective randomized HD2002 trial, rituximab maintenance therapy (375 mg/m(2) every 3 months for 2 years) versus observation was evaluated for CD20(+) B-cell lymphoma. Out of 321 patients [161 randomized to the treatment group (TG), 160 to the observation group (OG)], 295 data sets were evaluable for statistical analysis. Estimated 5-year relapse-free survival (RFS) was 81% in the TG and 70% in the OG (logrank test, P = 0·047). In the diffuse large B-cell lymphoma (DLBCL) subgroup (n = 152), 5-year RFS was excellent, at 87% in the TG and 84% in the OG (logrank test, P = 0·35). Of note, only in male patients of the DLBCL subgroup was RFS significantly superior in the TG in comparison to the OG (5-year RFS: 88% vs. 74%; logrank test, P = 0·05). Cox regression analysis showed a significant interaction between treatment and gender regarding overall survival (OS) (P = 0·006) and RFS (P = 0·02), with a lower hazard in females than males in the OG [OS: hazard ratio (HR) (female:male) = 0·11; 95% confidence interval (CI) = 0·00-1·03; RFS: HR (female:male) = 0·27; 95% CI = 0·05-0·97], and no significant differences between males and females in the TG. We conclude that Rituximab maintenance therapy improves survival in male patients with DLBCL.

To understand the precise disease driving mechanisms in acute myeloid leukemia (AML), comparison of patient matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. In this analysis, we have examined the value of aldehyde dehydrogenase (ALDH) activity in combination with CD34 expression for the separation of HSC from LSC in 104 patients with de novo AML. The majority of AML patients (80 out of 104) had low percentages of cells with high ALDH activity (ALDH+ cells, What's new? To understand the precise disease‐driving mechanisms in acute myeloid leukemia (AML), comparison of patient‐matched hematopoietic stem cells (HSC) and leukemia stem cells (LSC) is essential. This study demonstrates the relevance of aldehyde dehydrogenase (ALDH) for the prospective identification of AML cases in which separation of functionally normal HSC from LSC is possible. Increased activity of this biomarker also characterizes a subgroup of patients with adverse outcome, which might be helpful in risk stratification prior to therapy. Overall, this study demonstrates functional heterogeneity of leukemia cells and suggests divergent roles for ALDH activity in normal HSC versus leukemia‐initiating cells.

Despite high-dose chemotherapy followed by autologs stem-cell transplantation as well as novel therapeutic agents, multiple myeloma (MM) remains incurable. Following the general trend towards personalized therapy, targeted immunotherapy as a new approach in the therapy of MM has emerged. Better progression-free survival and overall survival after tandem autologs/ allogeneic stem cell transplantation suggest a graft versus myeloma effect strongly supporting the usefulness of immunological therapies for MM patients. How to induce a powerful antimyeloma effect is the key issue in this field. Pivotal is the definition of appropriate tumor antigen targets and effective methods for expansion of T cells with clinical activity. Besides a comprehensive list of tumor antigens for T cell-based approaches, eight promising antigens, CS1, Dickkopf-1, HM1.24, Human telomerase reverse transcriptase, MAGE-A3, New York Esophageal-1, Receptor of hyaluronic acid mediated motility and Wilms’ tumor gene 1, are described in detail to provide a background for potential clinical use. Results from both closed and ongoing clinical trials are summarized in this review. On the basis of the preclinical and clinical data, we elaborate on three encouraging therapeutic options, vaccine-enhanced donor lymphocyte infusion, chimeric antigen receptors–transfected T cells as well as vaccines with multiple antigen peptides, to pave the way towards clinically significant immune responses against MM.

Background Harvest of more than one CD34+ stem cell transplant has become the standard, to ensure the option for a second autologous transplantation in patients with relapsed or progressive multiple myeloma (MM). Additional administration of the CXCR-4 inhibitor plerixafor has been shown to increase the efficiency of CD34+ stem cell harvest. However, the algorithm when to apply plerixafor is still under debate. Study Design and Methods In this retrospective study, 46 MM patients were categorized into four groups according to their CD34+ stem cell count in peripheral blood (PB) and mobilization with or without plerixafor: Group A comprised poor mobilizers with CD34+ cell counts of fewer than 20 × 106/L in PB. Group B included inadequate mobilizers with CD34+ cell counts of 20 × 106/L or more in PB and a low CD34+ stem cell yield in the first leukapheresis session. Patients receiving plerixafor preemptively (Group A1) and as a rescue strategy (Group B1) were compared to patients continuing stem cell collection with granulocyte–colony-stimulating factor alone (Groups A2 and B2). Results In both, the preemptive and the rescue settings, plerixafor enhanced the CD34+ stem cell yield significantly. Poor mobilization and administration of plerixafor was not associated with delayed engraftment. Conclusion Our data demonstrate that administration of plerixafor is safe and effective and facilitates a significantly higher CD34+ stem cell harvest. Based on the presented data, we propose an algorithm for the use of plerixafor for CD34+ stem cell mobilization and harvesting in poor mobilizing myeloma patients.

While extensive data demonstrated that plerixafor improves stem cell harvest in difficult-to-mobilize patients, economic concerns limit a broader application. We retrospectively assessed the effect of an early plerixafor rescue regimen for mobilization in patients with multiple myeloma. Patients were intended for high-dose chemotherapy followed by autologous peripheral blood stem cell transplantation (ABSCT) and therefore received cyclophosphamide-based mobilization chemotherapy and consecutive stimulation with granulocyte colony-stimulating factor (G-CSF). Fifteen patients with poor stem cell harvest in the first leukapheresis session received plerixafor. Data were compared with a matched historic control group of 45 patients who also had a poor stem cell yield in the first apheresis session, but continued mobilization with G-CSF alone. Patients in the plerixafor group collected significantly more CD34+ cells in total (median 4.9 vs. 3.7 [range 1.6–14.1 vs. 1.1–8.0] × 106 CD34+ cells /kg bw; P

We retrospectively analyzed and compared the efficacy and toxicity of azacitidine (AZA) and low-dose cytarabine (LD-Ara-C) in 65 palliative patients with acute myeloid leukemia (AML) showing high bone marrow blast counts (≥30%) before start of treatment. Twenty-seven and 38 patients received AZA and LD-Ara-C, respectively. The median patient age was 71 yr. Patient and disease characteristics did not differ between the treatment groups, except for BM blast counts, and peripheral leukocyte and blast counts which were significantly higher in the LD-Ara-C group. AZA and LD-Ara-C were first-line treatment in 12 (44%) and 17 patients (45%), respectively. Response and hematologic improvement rates were low and similar in both treatment groups. In both treatment groups, most common non-hematologic toxicities included febrile neutropenia, pneumonia, and bleedings without significant differences regarding frequencies. Estimated 1-yr survival rates were 15% (95% CI 8-22) and 13% (95% CI 7-19) in the AZA and LD-Ara-C groups, respectively, without statistically significant difference. In multivariate analysis (n = 65), previous treatment (HR 2.27, 95% CI 1.00-5.22, P = 0.05) and adverse cytogenetics (HR 2.50, 95% CI 1.20-5.22, P = 0.02) were independent predictors of poor survival. In our center and within the limitations of a retrospective study, both treatment regimens showed similar but limited efficacy in palliative patients with AML and high BM blast counts.

BACKGROUND Therapeutic options for patients with recurrent multiple myeloma after autologous stem cell transplantation (ASCT) include novel agents, conventional chemotherapy, or salvage ASCT with no standard of care. METHODS A total of 200 patients with multiple myeloma who developed disease recurrence after treatment with upfront ASCT and received an autologous retransplantation as salvage therapy at the study center over a period of 15 years were retrospectively reviewed. The objective of the current study was to evaluate the role of salvage ASCT in terms of efficacy, particularly taking into account the impact of novel agents. RESULTS The median progression-free survival (PFS) and overall survival after salvage ASCT were 15.2 months and 42.3 months, respectively. The overall response rate (a partial response or greater) was 80.4% at day 100, excluding 6 patients who died before assessment. Factors associated with improved PFS and overall survival after salvage ASCT included an initial PFS of > 18 months after upfront ASCT, bortezomib-containing or lenalidomide-containing therapies for reinduction, response to reinduction, and an International Staging System stage of I before salvage ASCT. CONCLUSIONS Salvage ASCT is capable of achieving sustained disease control in patients with multiple myeloma. The use of lenalidomide and bortezomib for reinduction has improved the results after salvage ASCT, suggesting that novel agents and salvage ASCT are complementary rather than alternative treatment approaches. Cancer 2013;119:2438-2446. © 2013 American Cancer Society.

Serological analyses within epidemiological cohort and case-control studies indicate to an association between HBV infection and risk of multiple myeloma (MM). To verify the relationship with an independent approach, we investigated the correlation between HBV positivity and chromosomal aberrations within 680 patients of the National Center for Tumor Diseases Heidelberg for which the serological HBV status (HBsAg and anti-HBc) and FISH data for five gains (1q21, 9q34, 11q23, 15q22, 19q13), five losses (6q21, 8p21, 13q14, 17p13, 22q11), and three IgH translocations [t(4,14), t(11,14), t(14,16)] were available. Deletion of 8p21 and 13q14 were shown associated with HBV positivity within hepatocellular carcinoma in other investigations. In the present evaluation, the odds ratio for loss of 8p21 was significantly elevated (OR = 2.74, 95% CL = 1.36-5.50, P = 0.0048) and for loss of 13q14 non-significantly increased (OR = 1.40, 95% CL = 0.74-2.65) in anti-HBc positive patients. The results provide further support for a role of HBV infection in the pathogenesis of MM.

Acute myeloid leukemia (AML) is considered to be a disease of stem cells. A rare defective stem cell population is purported to drive tumor growth. Similarly to their normal counterparts, leukemic stem cells (LSC) divide extreme slowly. This may explain the ineffectiveness of conventional chemotherapy in combatting this disease. Novel treatment strategies aimed at disrupting the binding of LSC to stem cell niches within the bone marrow might render the LSC vulnerable to chemotherapy and thus improving treatment outcome. This review focuses on the detection of LSC, our current knowledge about their cellular and molecular biology, and LSC interaction with the niche. Finally, we discuss the clinical relevance of LSC and prospective targeted treatment strategies for patients with AML.

Leukemia stem cells (LSCs) might originate from malignant transformation of normal hematopoietic stem cells (HSCs), or alternatively, of progenitors in which the acquired mutations have re-installed a dysregulated self-renewal program. LSCs are on top of a hierarchy and generate leukemia cells with more differentiated characteristics. While most leukemia cells are initially sensitive to chemo- and radiotherapy, LSCs are resistant and are considered to be the basis for disease relapse after initial response. Albeit important knowledge on LSC biology has been gained from xenogeneic transplantation models introducing human leukemia cells into immune deficient mouse models, the prospective identification and isolation of human LSC candidates has remained elusive and their prognostic and therapeutic significance controversial. This review focuses on the identification, enrichment and characterization of human LSC derived from patients with acute myeloid leukemia (AML). Experimental data demonstrating the clinical significance of estimating LSC burden and strategies to eliminate LSC will be summarized. For long-term cure of AML, it is of importance to define LSC candidates and to understand their tumor biology compared to normal HSCs. Such comparative studies might provide novel markers for the identification of LSC and for the development of treatment strategies that might be able to eradicate them.

Bone marrow plasma cell infiltration is a crucial parameter of disease activity in monoclonal plasma cell disorders. Until now, the only way to quantify such infiltration was bone marrow biopsy or aspiration. Diffusion-weighted imaging (DWI) is a magnetic resonance imaging-technique that may mirror tissue cellularity by measuring random movements of water molecules. To investigate if DWI is capable of assessing bone marrow cellularity in monoclonal plasma cell disease, we investigated 56 patients with multiple myeloma or monoclonal gammopathy of undetermined significance, and 30 healthy controls using DWI of the pelvis and/or the lumbar spine. In 25 of 30 patients who underwent biopsy, bone marrow trephine and DWI could be compared. Of the patients with symptomatic disease 15 could be evaluated after systemic treatment. There was a positive correlation between the DWI-parameter apparent diffusion coefficient (ADC) and bone marrow cellularity as well as micro-vessel density (P

Within 2–3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo. This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications.

Chromosomal instability (CIN), defined by an elevated frequency of the occurrence of novel chromosomal aberrations, is strongly implicated in the generation of aneuploidy, one of the hallmarks of human cancers. As for aneuploidy itself, the role of CIN in the evolution and progression of malignancy is a matter still open to debate. We investigated numerical as well as structural CIN in primary CD34-positive cells by determining the cell-to-cell variability of the chromosome content using fluorescence-in situ-hybridization (FISH). Thereby, CIN was measured in 65 patients with myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML) and control subjects. Among MDS patients, a subgroup with elevated levels of CIN was identified. At a median follow-up of 17.2 months, all patients within this ‘high CIN’ subgroup had died or progressed to AML, while 80% of MDS patients with normal CIN levels had stable disease (P < 0.001). Notably, there was no statistically significant difference between ‘normal CIN’ and ‘high CIN’ MDS patients regarding established risk factors. Hence, elevated CIN levels were associated with poor outcome, and our method provided additional prognostic information beyond conventional cytogenetics. Furthermore, in all three MDS patients for whom serial measurements were available, development of AML was preceded by increasing CIN levels. In conclusion, elevated CIN levels may be valuable as an early indicator of poor prognosis in MDS, hence corroborating the concept of CIN as a driving force in tumour progression.

To identify genetic mechanisms controlling bone marrow microcirculation and angiogenesis in patients with plasma cell disease we simultaneously performed bone marrow dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and cytogenetics (iFISH) on CD138 purified plasma cells of MGUS (n=31) and untreated multiple myeloma (MM) (n = 87) patients. The adverse cytogenetic abnormalities gain of 1q21, deletion 17p13 and deletion 13q14 significantly correlated with at least one DCE-MRI finding (aberrant “focal” microcirculation pattern, increase in median microcirculation parameter Amplitude A or exchange rate constant kep). We conclude that gain of 1q21, deletion 13q14 and deletion 17p13 trigger the angiogenic cascade in MM. Our findings will have important implications for the design, analysis and stratification for clinical studies of patients with MM in particular if compounds with antiangiogenic activity are used. © 2008 Wiley-Liss, Inc.

Introduction Atypical or nontuberculous mycobacteria are normal commensals of water, soil, dust, and cattle milk. Human infections are relatively uncommon, but an increasing number of cases have been reported in the context of human immunodeficiency virus (HIV) or therapeutic immunosuppression (1). Mycobacterium avium complex and Mycobacterium kansasii account for most episodes of nontuberculous systemic diseases. Besides many reports of patients with HIV, there are only rare published examples of patients with chronic inflammatory arthritides under immunosuppression therapy. Most of these reports describe Mycobacterium marinum infections (2–4). In the last 7 years, there have been only a few reports of M kansasii infections in patients with chronic inflammatory rheumatic diseases (5–11). Nakamura et al (8) collected references on 11 patients from the literature since 1963. Bernard et al (9) described a French retrospective study of 26 patients from their own clinic and the literature who had M kansasii septic arthritis and no underlying disease. This indicates that, overall, this infectious complication is very rare. However, the incidence might increase as we tend to use more aggressive immunosuppressants (especially tumor necrosis factor inhibitors) with a more relevant impact on the immune system. Moreover, with better diagnostic possibilities and better awareness of the necessity of an aggressive treatment, immunosuppression therapy will start much earlier. We report a patient with long-lasting seronegative rheumatoid arthritis and a more recent local tenosynovitis. We identified a local M kansasii infection as the cause for the tenosynovitis on the basis of a positive bacterial culture result. AlthoughM kansasii is second only toM avium complex as the causative agent of nontuberculous lung disease, which often involves the upper lobe, it is rarely identified as a cause of extrapulmonary infection, especially arthritis or tenosynovitis. The infection primarily infects the lungs with rare precipitation in joints, tendons, or bone (9,12). When affecting the joints, monarthritis is usually erosive (11,13). Infections with M kansasii are environmentally acquired. It has been isolated from tap water in the warm southern US states (Texas), where it can survive for up to 12 months (14,15).

BACKGROUND: Granulocyte–colony-stimulating factor (G-CSF) is known to affect functional activity and antigen expression of neutrophil granulocytes. Beside nonglycosylated filgrastim and glycosylated lenograstim, pegylated filgrastim (pegfilgrastim) has recently been introduced for single administration into clinical use. STUDY DESIGN AND METHODS: Here, granulocytes from 27 patients with nonmyeloid malignancies were compared functionally (migration, reactive oxygen species production, and G-CSF serum levels) and phenotypically (cell surface antigen expression) before and after G-CSF administration. RESULTS: After exposure to G-CSF, chemotaxis was reduced significantly in the filgrastim group. Immunophenotypically, in vivo G-CSF–primed granulocytes were more mature in the lenograstim than in the filgrastim and to lesser extent in the pegfilgrastim groups as shown by the expression profile for CD11b, CD14, and CD16. Of note, G-CSF serum levels were similar among the groups. CONCLUSION: Our data suggest that granulocytes exposed to glycosylated G-CSF in vivo seem to resemble more closely their steady-state phenotype than after treatment with nonglycosylated and to lesser extent pegylated G-CSF.

Background: We analyzed the hematopoietic reconstitution and outcome of 508 patients with multiple myeloma (MM) with respect to the number of CD34+ cells reinfused at our center. Patients and methods: Each cohort of 390 patients (unselected CD34+ cell transplant) and 118 patients (CD34+ selected transplant) was divided into four subgroups. Among the 390 transplantations, 86 patients received a high dose (HD)) of ‡6.50 · 106 unselected CD34+ cells/kg, 116 patients a low dose (LD)) of 20 · 109/L (10 d vs. 11 d) (P = 0.058). CD34+ cell-dose was significant for long-term platelet recovery at day 360 (unselected transplant P = 0.015, selected transplant P = 0.023). Number of transplanted CD34+ cells had no significant impact on transplant related mortality, overall survival or CR/PR rates within 100 d. In terms of supportive care the differences of high-/low-dose grafts were minimal. Conclusions: These results confirm that high doses of CD34+ PBSC shorten hematopoietic reconstitution and reduce hospitalization. Nevertheless secure engraftment results from transplantation of 2.00– 3.00 · 106 CD34+ cells/kg. As 60% of our pretreated patients are able to collect ‡5.00 · 106 CD34+ cells/ kg within a single leukapheresis, division into two or more freezing bags allows safe tandem transplantation in the majority of MM patients.

B-cell chronic lymphocytic leukemia (CLL) is characterized by a high rate of clonal genomic alterations and a low proliferative activity with cell cycle arrest in G0/G1 phase. Recently, centrosome aberrations have been described as a possible cause of chromosomal instability and aneuploidy in many human malignancies. To investigate whether centrosome aberrations do occur in CLL and whether they correlate with common prognostic factors and disease activity, we examined peripheral blood mononuclear cells (PBMC) from 70 patients with previously untreated CLL using an antibody to γ-tubulin. All 70 CLL samples displayed significantly more cells with centrosome aberrations (median: 26.0%, range 11.0–41.5%) as compared to peripheral blood B lymphocytes from 20 age-matched, healthy individuals (median: 2.0%, range 0–6%; p < 0.001). The extent of centrosome aberrations correlated with the proliferative activity of the CLL cases as measured by lymphocyte doubling time (p = 0.02) as well as with time to first treatment (p = 0.05). Accordingly, more centrosome aberrations were found in PHA-stimulated T lymphocytes from healthy individuals as well as in B cells from surgically removed tonsil tissue of patients with acute tonsillitis as compared to the peripheral blood B lymphocytes from the control group. In contrast, no correlation was observed between centrosome aberrations and immunoglobulin VH gene mutation status or cytogenetically defined risk groups. These findings suggest that, despite the common observation of most CLL cells remaining in G0/G1 phase, their centrosome replication process is deregulated and correlates to the proliferative activity of CLL cells. © 2007 Wiley-Liss, Inc.

Amyloid light chain (AL) amyloidosis is the result of a clonal plasma cell expansion, in which monoclonal light chains transform to amyloid deposit in various tissues and can lead to organ dysfunction and organ failure. The median survival of patients with AL amyloidosis without therapy is 10-14 months. With high-dose melphalan (HDM) and autologous stem cell transplantation (ASCT), haematological and clinical remission rates of up to 50% of treated patients have been reported from phase II studies. HDM followed by ASCT appears to prolong survival in patients, if haematological remission can be reached. In this phase II study, we evaluated vincristine, adriamycin and dexamethasone (VAD) as induction chemotherapy prior to stem cell mobilization and HDM with ASCT. The regimen was, in general, feasible in patients with AL amyloidosis, but VAD chemotherapy had a considerable World Health Organization (WHO) grade III-IV toxicity (25%) and mortality (7%) rate. VAD pretreatment did not interfere with stem cell mobilization and HDM with ASCT was possible in 86% of patients. The overall treatment efficacy was comparable with reported results of HDM and ASCT without preceding chemotherapy. We could not show an additional benefit of VAD induction in terms of increasing haematological response rate; however the 13% mortality rate after HDM and ASCT in our series was lower than the previous report.

Fludarabine in combination with cyclophosphamide is an effective treatment for newly diagnosed as well as relapsed follicular lymphoma. The anti-CD20 antibody rituximab has been employed successfully for the same indications. No such data were available on a combined use of these agents. Therefore, we conducted a phase II study to evaluate the safety and efficacy of a combination of rituximab (375 mg/m2), fludarabine (4 x 25 mg/m2) and cyclophosphamide (1 x 750 mg/m2), for the treatment of relapsed follicular lymphoma. An unexpected, severe hematologic toxicity with significant, prolonged thrombocytopenias WHO grade III/IV in 6 (35%) of 17 patients treated in total occurred, leading to early termination of the trial. Cytologic and serologic analyses point toward a direct toxic effect. Older patients (mean age 64.7 vs. 56.5 yr) were significantly (P = 0.02) more likely to suffer from this toxicity, whereas no other clinical or hematologic parameter differed statistically between the patients suffering from thrombocytopenia and those who did not. The addition of rituximab to fludarabine/cyclophosphamide employed at doses given above in relapsed follicular lymphoma may have led to this increase in thrombocytopenias. Therefore, caution should be exercised when combining these drugs for the treatment of patients with relapsed follicular lymphoma, especially when treating older patients.

To develop an effective antitumor immunotherapy for B-lineage non-Hodgkin's lymphoma, we constructed a tetravalent tandem diabody (tanDb) specific for both human CD19 (B-cell marker) and CD3 (T-cell antigen). Here, we report the effective killing of malignant primary B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) by autologous T cells induced by tanDb at very low E:T ratios. Mononuclear cells from patients with B-CLL were cultured with bispecific antibody fragments in either the presence or absence of monospecific anti-CD28 antibody. Use of tetravalent tanDbs caused almost quantitative elimination of malignant B cells from the blood samples of 19 patients and some cytotoxic activity in 3 of 23 analyzed cases. In contrast, the structurally similar but bivalent diabody and single-chain diabody demonstrated nearly no antitumor activity in an autologous system. tanDb-induced activation and proliferation of T cells occurred only in the presence of CD19+ target cells. Expression of the B7-1 (CD80) and B7-2 (CD86) molecules on the surface of leukemia cells made unnecessary the additional CD28-costimulation of T cells. When only a few tanDb molecules were present, the effect of CD28 costimulation on T-cell activation was more pronounced. Depending on the patient sample, we observed a 10- to 1,000-fold decrease of the half-maximal concentrations of tanDb for cell lysis. Upon CD28 crosslinking by agonistic MAb, specific tumor cell lysis was found at tanDb concentrations as low as 0.5 pM. These data demonstrate that the tetravalent CD19×CD3 tanDb might be a promising tool for the immunotherapy of human B-cell leukemias and lymphomas. © 2004 Wiley-Liss, Inc.

Patients with chronic lymphocytic leukemia (CLL) are at a significantly increased risk of developing a second malignant neoplasm in the course of their disease. The occurrence of large cell lymphoma [Richter's syndrome (RS)] has been described in approximately 3-5% of CLL patients. Other types of secondary lymphoid malignancies are extremely rare. Here we describe a patient, heavily pretreated, with long history of CLL who developed a secondary follicular lymphoma, transformed into a diffuse large cell lymphoma (LCL), with isolated manifestation in the bone, a very rare manifestation of RS. CLL and LCL cells were of distinct clonal origin, as documented by DNA sequencing of the CDR3 regions. Twelve months after the completion of chemo- and local radiotherapy, the patient is still in remission.

Summary. This study evaluates the immunological effects in 21 patients with multiple myeloma (MM) following allogeneic stem cell transplantation with a non-myeloablative conditioning regimen. Patients were heavily pretreated, most of them having progressed despite one to three courses of high dose chemotherapy followed by autologous stem cell rescue. For patients conditioned with low dose total body irradiation and Fludarabine, development of full (100%) donor chimerism (FDC) within the first 3–4 months strongly depended on the number of T cells transplanted. This putative graft-versus-haematopoiesis effect, however, did not coincide with clinical response. Rather, 10 of 17 patients progressed although FDC in peripheral blood was achieved. In contrast, clinical graft-versus-host disease (GvHD) of grade III–IV was associated with disease remission. Four of six patients achieved complete remission (CR) and one patient achieved a partial remission (PR) in close temporal relation with their acute GvHD. Two of four patients in CR succumbed to the consequences of this side effect. One patient in PR progressed soon after successful treatment of the GvHD, one of two patients with a long-lasting CR developed chronic GvHD. Our observations suggest the existence of two distinct immune effects in MM following allogeneic transplantations with reduced conditioning regimen. First, the putative graft-versus-haematopoiesis effect that induced FDC was observed in all patients receiving unmanipulated grafts. Secondly, the graft-versus-myeloma effect that induced CR did not correlate with FDC but was associated with grade III–IV GvHD in our group of heavily pretreated patients.

Our purpose was to assess the presence of centrosomal aberrations as measured by immunohistochemistry in primary invasive breast cancer and their association with established and proposed prognostic factors. Tissue sections of 103 primary invasive breast cancers were examined using centrosome-specific antibodies to pericentrin and gamma-tubulin. At least 3 different tumor regions per case were examined to determine maximum centrosomal aberration levels, which represent the proportion of cells with abnormal centrosomes in the region with the highest percentage of cells with centrosomal aberrations. The chi(2) test was performed to evaluate the association of maximum centrosomal aberration levels with patient age; tumor size; nodal status; nuclear grade; hormone receptor and Her2/neu expression; proportion of Ki67-, p53- and Bcl-2-positive tumor cells; DNA index; S-phase fraction; and proliferation index. With pericentrin immunohistochemistry, maximum centrosomal aberration levels >35% were detectable in 92 of the 103 breast carcinomas (89%). We found a highly significant correlation of maximum centrosomal aberration levels above 35% with axillary nodal tumor involvement (p 35% were also associated with an increased DNA index (p = 0.006). In a subset of patients, additional staining of centrosomes with a monoclonal anti-gamma-tubulin antibody essentially confirmed these results. In primary invasive breast cancer, centrosomal aberrations are associated with those factors predicting a more aggressive course of disease. This might indicate a fundamental role of centrosomal dysfunction in disease evolution, possibly as a result of chromosome missegregation during mitosis.

To identify patients at high risk of life-threatening infections, we retrospectively analysed the prevalence of infectious complications in 187 chronic lymphocytic leukaemia patients treated in our institution since 1999 and correlated them with clinical features. A questionnaire with detailed questions regarding infectious complications was mailed to patients and their general practitioners. Major infections (requiring intravenous antibiotics or inpatient treatment) were reported in 37 patients (19.8%) and minor infections (requiring oral antibiotics and outpatient treatment) in 113 patients (60.4%). Univariate analysis identified advanced disease (P = 0.02), gender (P = 0.01), duration of disease (P = 0.007), number of previous chemotherapy regimens (P < 0.001), previous therapy with purine analogues and monoclonal antibodies (P < 0.001; P = 0.019), massive splenomegaly (P = 0.03), low granulocyte count (P < 0.001), low serum immunoglobulin concentration (P = 0.005), low haemoglobin concentration (P < 0.001) and high serum lactate dehydrogenase (LDH) concentration (P < 0.001) as risk factors for major infections. In multivariable logistic regression analysis, only the number of previous chemotherapy regimens (risk ratio [RR] = 1.8; 95% confidence interval [CI] 1.2-8.0) and haemoglobin concentration (RR = 0.6; CI 0.5-0.8) remained significant for major infections. The number of previous chemotherapy regimens was the only independent risk factor for minor (RR = 7.6; CI 2.2-25.7) and varicella-zoster virus infections (RR = 2.1; CI 1.3-3.4). In untreated patients, the only risk factor for major infections was LDH concentration. Patients treated with purine analogues or autologous stem cell transplantation had a higher risk of developing viral infections. In conclusion, disease activity and pretreatment extent have a stronger impact on the risk of severe infectious complications than hypogammaglobulinaemia. Preferably, prophylactic strategies should be evaluated in patients defined by these parameters.

Our purpose was to determine the predictive value of tumor biologic parameters in patients with HRPBC who received HDCT with ASCT as first-line treatment. From September 1992 to May 2000, 149 stage II or III HRPBC patients were enrolled in a single-arm trial using a tandem HDCT regimen followed by ASCT. Her2/neu, p53, Ki67 and bcl-2 protein expression was studied using immunohistochemic staining on formalin-fixed, paraffin-embedded primary tumor sections. DNA content of tumor cells (DNA index) and tumor cell proliferation (SPF) were measured by DNA flow cytometry. The relationship between these tumor biologic parameters, on the one hand, and DFS, DDFS and OS, on the other, was analyzed. With a median follow-up of 43 months (range 7-106), p53 protein accumulation (p = 0.000004), negative combined hormone receptor status (p = 0.003) and Her2/neu overexpression (p = 0.02) were significant negative predictors of OS in univariate analysis. A poorer DFS was associated with p53 positivity (p = 0.04) and nodal ratio > or = 0.8 (p = 0.008). Poorer DDFS was associated with p53 positivity (p = 0.03). In multivariate analysis, Her2/neu overexpression (RR = 3.86, 95% CI 1.48-10.1, p = 0.006) and p53 overexpression (RR = 6.06, 95% CI 2.22-16.52, p < 0.001) proved to be independent predictors of adverse OS. p53 overexpression was the only independent predictor of DFS (RR = 2.21, 95% CI 1.07-4.57, p = 0.03). p53 overexpression and Her2/neu overexpression are independent negative predictors of survival in HRPBC treated with HDCT. The adverse impact of these biologic features was probably not altered by HDCT. For HRPBC patients with tumors not overexpressing Her2/neu or p53, HDCT may be an appropriate approach to achieve long-term survival and tumor control.

We report our long-term experience with autologous bone marrow transplantation (ABMT) for 32 adult patients with acute lymphocytic leukaemia (ALL) in second or later remission (CR), or in first CR but with high-risk. Bone marrow was purged with mafosfamide (n = 25) or with immunomagnetic beads and monoclonal antibodies (n = 7). Retrospective analysis showed that 12 out of 32 patients were in continuous complete remission (CCR) at a median of 143 months (range 66-181 months). A plateau was reached at 50 months and the disease-free and overall survival rates were both 37.5%. It was notable that durable CCR could be achieved for patients in second (three out of nine) or third (one out of six) CR. ABMT could produce durable CCR and the long-term outcome compared favourably with those reported for allogeneic transplantation.

The aim of our study was to investigate the quantitative microcirculation parameters amplitude A (hypothetical intravascular volume) and exchange rate constant k21 (hypothetical vascular permeability) by contrast-enhanced dynamic magnetic resonance imaging (dMRI) as markers of angiogenesis in multiple myeloma (MM). Therefore lumbar spine and spina iliaca superior posterior of 16 normal controls and 41 patients with active MM were assessed using a dMRI protocol with a pump controlled bolus infusion of Gadolinium-DTPA. Pharmacokinetic parameters, amplitude A and exchange rate constant k21 were calculated according to a 2-compartment model. Color-coded parameter images were generated from pharmacokinetic data analysis and superimposed onto the conventional MR images. Amplitude A and k21 parameters were significantly increased in patients with MM compared with controls (p = 0.001; median Actr, 0.2 [range, 0.09–0.4]; median AMM, 0.93 [range, 0.2–2.2]; median k21ctr, 0.09 min−1 [range, 0.03–0.9]; median k21MM, 4.58 [range, 0.22–23.8]). Within the group of MM patients the pattern of color-coded parameter images were found to be either of “diffuse” (n = 13, 31%) or “focal” (n = 28, 69%) type of distribution of microcirculation. Comparison of amplitude A in patients with “focal” vs. “diffuse” pattern of the pharmacokinetic maps revealed a significant increase in the median of amplitude A in the “focal” group. Amplitude A values allowed a classification of patients according to severe osteolytic bone involvement (p = 0.023) with the best cutoff value of 0.7 for amplitude A. Downmodulation of amplitude A was observed in a MM patient treated with standard VAD chemotherapy. Our data demonstrate that dMRI is a novel imaging technique for the detection and monitoring of MM bone lesions. It provides independent evidence for angiogenesis in MM. © 2001 Wiley-Liss, Inc.

We performed a pilot study including rituximab (Mabthera; IDEC-C2B8, Hoffmann-La Roche) with a sequential high-dose therapy protocol in 15 patients with follicular and three patients with mantle cell lymphoma and studied the potential of the chemoimmunotherapy to induce depletion of malignant B cells in vivo. Our treatment protocol included induction with three cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy, followed by peripheral blood stem cell (PBSC) mobilization using high-dose cytosine arabinoside (2 g/m2 every 12 h, days 1 and 2) and mitoxantrone (10 mg/m2, days 2 and 3) (HAM), preceeded by rituximab (375 mg/m2). The proportion of CD19+ B cells in blood and bone marrow decreased from 1·2 ± 0·4% to 0·13 ± 0·1% (P = 0·01) and from 2·7 ± 0·8% to 0·8 ± 0·5% (P = 0·03) respectively. The number of t(14;18)-positive cells in blood and bone marrow progressively decreased with treatment, as assessed by the quantitative real-time PCR assay in four patients. Conversion to PCR-negativity was achieved in the peripheral blood (PB) of seven informative patients. Leucaphereses were performed during the granulocyte colony-stimulating factor (G-CSF)-supported leucocyte recovery phase. In 17 of 18 patients, a median of 15·1 × 106 CD34+ cells/kg body weight (BW) could be harvested by a single procedure for enrichment by an immunomagnetic method. Leucapheresis products contained 51·3 ± 28·8 × 104 CD19+ B cells/kg BW (mean) and were t(14;18) PCR negative in all seven informative patients. These data compare favourably with results obtained in patients treated with the same regimen without rituximab. The high-dose therapy (n = 12 patients), including total body irradiation (14·4 Gy) and cyclophosphamide (200 mg/kg BW), was also preceeded by rituximab. Recovery of neutrophils to > 0·5 × 109/l and of platelets to > 20 × 109/l required a median of 13·5 and 11·5 d (range 11–24 and 9–24 d) respectively. In conclusion, the addition of the CD20 antibody to chemotherapy ensured tumour depletion in vivo and allowed the collection of PBSCs devoid of tumour cells and with conserved engraftment capability.

The number of circulating clonotypic B cells in patients with multiple myeloma (MM) after high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) was investigated. Peripheral CD19+ B cells have been reported to persist throughout conventional and HDT and might resemble a source of relapse in patients with MM. We assessed the proportion of malignant cells in CD20+ and CD19+ cell fractions of 14 peripheral blood (PB) samples from 12 patients after HDT and PBSCT. Nine samples were obtained from patients in continuous remission, and five patients were in progressive disease or beginning relapse. The CD20+ fractions obtained had a mean purity of 96.8%. The percentages of tumour cells were determined using a quantitative allele-specific oligonucleotide PCR assay based on the method of limiting dilutions. In the group of patients in continuous remission the median number of tumour cells in the CD20+ cell fractions was 1.9/ml (range 0-7.2 tumour cells/ml PB) higher than in the CD20- fractions (median 0; range 0-29 tumour cells/ml PB). Higher tumour cell numbers in both fractions, particularly pronounced in the negative ones, were found in patients with progressive disease or beginning relapse (CD20+: range 3.8-585; median 32 tumour cells/ml PB; CD20-: range 25-25527; median 334 tumour cells/ml PB). Enrichment with the anti-CD19 antibody as a second pan B-cell marker revealed comparable tumour cell numbers. In conclusion, an anti-CD20 antibody treatment could be a promising approach for the eradication of malignant cells in the PB of patients in continuous remission after HDT and PBSCT with low amounts of tumour cells in the B-cell compartment and an almost complete absence of tumour cells in the CD20- fractions.

BACKGROUND: It was previously reported that the combination of granulocyte-macrophage-colony-stimulating factor (GM–CSF) and granulocyte-CSF (G–CSF) for 4 days mobilized more primitive CD34+ subsets than did either G–CSF or GM–CSF alone. STUDY DESIGN AND METHODS: The studies determine the optimal number of days of growth factor dosing for mobilization and collection of peripheral blood progenitor cells, by increasing the days of administration of GM–CSF and/or G–CSF or employing the sequential administration of GM–CSF followed by G–CSF. Sixty normal subjects were given injections of G–CSF or GM–CSF alone; GM–CSF and G–CSF concurrently for 4, 5, or 6 days; or a sequential regimen of GM–CSF for 3 or 4 days followed by G–CSF for 2 or 3 days. A 10-L apheresis was performed 24 hours after the last dose. RESULTS: The three most efficacious mobilization regimens consisted of sequential GM–CSF for 3 days followed by G–CSF for either 2 or 3 days and G–CSF alone for 5 days. Each of these regimens resulted in the collection of significantly greater numbers of CD34+ cells by apheresis than any of the 4-day dosing regimens with G–CSF and/or GM–CSF (sequential GM–CSF/G–CSF: 3 days/2 days = 3.58 ± 0.53 × 106 CD34+ cells/kg; GM–CSF/G–CSF: 3 days/3 days = 4.45 ± 1.08 × 106 CD34+ cells/kg; G–CSF: 5 days = 3.58 ± 0.97 × 106 CD34+ cells/kg; all p < 0.05 vs. G–CSF and/or GM–CSF for 4 days). Clonogenic assays generally paralleled the level of CD34+ cells. Regimens containing GM–CSF resulted in a higher percentage of the cells from primitive CD34+/CD38–/HLA-DR+ subset than G–CSF alone. CONCLUSION: Compared with 4-day dosing regimens with G–CSF and/or GM–CSF, mobilization of CD34+ cells in normal subjects using sequential GM–CSF for 3 days followed by G–CSF for 2 or 3 days or using G–CSF alone for 5 days increased the number CD34+ cells that can be collected by a single 10-L apheresis 24 hours after the last dose of cytokine.

Summary. Peripheral blood lymphocyte (PBL) subsets and bone marrow biopsies were analysed in six patients with hairy cell leukaemia (HCL) treated with 2′-deoxycoformycin (pentostatin, DCF) according to a phase II trial of the EORTC Leukemia Cooperative Group. All patients responded to DCF with four complete and two partial remissions according to conventional criteria. Within the PBL subsets, major changes concerned the CD4± T cells, which during DCF therapy were distinctly suppressed to nadir values of 0–038–0–18 (median 0–126) × 109/-1. In five patients these cells returned to normal 3-0–49–5 (median 14·5) months after the last DCF injection. CD8± cells were decreased to a lesser extent, and NK cell numbers improved during treatment. Bone marrow immunohistology applying the MoAb B-ly7 demonstrated residual hairy cells (HCs) in all of the six patients following DCF treatment with nadir HC numbers of 0·2–3·0% of bone marrow cells. Immunoglobulin gene rearrangement analysis of DNA obtained from these biopsies showed only germline bands, whereas rearranged bands had been present on the pretreatment specimens. Within the observation period of 15·5–54·0 (median 47·0) months after discontinuation of DCF therapy, immunohistology demonstrated a continuous increase in HC numbers in five of the six patients with clonal rearrangement detectable in bone marrow specimens from three of these patients at last follow-up date. Although established on the basis of a small number of patients, these data suggest that DCF treatment as currently employed in HCL is unable to eradicate the malignant B cell clone.

Etoposide as a single agent is active in relapsed and refractory acute myelogenous leukemia (AML), with complete responses (CR) rates of 10% to 25%. The drug has been safely combined with cytarabine, azacytidine, vinca alkaloids, and anthracyclines, inducing remission rates of 20% to 60% in patients with previously treated AML. The experience with etoposide in acute lymphoblastic leukemia is less extensive, but the drug seems to be active in combination with cytarabine or aclacinomycin. In addition, etoposide is combined with cytarabine and anthracyclines for the primary treatment of AML. The response rates thus achieved are comparable with those obtained with standard regimens. A Phase I/II trial was initiated to study the efficacy of the NOVE combination (mitoxantrone [10 mg/m2/d, days 1 to 5] plus etoposide [100 mg/m2/d for 3, 4, or 5 days] in patients with refractory AML. The results showed that extended duration of etoposide administration is associated with higher CR rates. Overall, a CR rate of 43% was achieved in 61 patients. A sequential regimen with IDAC (idarubicin/cytarabine) and NOVE was designed for primary treatment of adult patients with AML. Cycles of IDAC or NOVE are applied depending on response. The results of the pilot study with this strategy were encouraging with 18 of 20 patients achieving CR. Further studies are under way to verify the efficacy of this strategy.

Previous study has shown that the combination of mitoxantrone (Novantrone, NO) and Ara-C (AC) (NOAC) was active in refractory non-Hodgkin's lymphoma (NHL) but myelosuppression was dose-limiting. In a pilot study, we investigated the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) after NOAC chemotherapy in patients with refractory NHL. NO was applied at a dosage of 10 mg/m2/day on days 2 and 3 and AC at 3 g/m2/12h on days 1 and 2. RhGM-CSF was administered at 250 ug/m2/day as a continuous i.v. infusion from day 6 until the neutrophils were greater than 3.0/nl for 3 consecutive days. Twenty-three patients from five of the nine participating centers were treated with NOAC chemotherapy plus rhGM-CSF, whereas 14 patients from the other four centers received chemotherapy alone. With rhGM-CSF, the median duration of severe neutropenia (less than 0.5/nl) after NOAC was 8 days versus a median of 13 days without rhGM-CSF (P = 0.0058), and that of thrombocytopenia (less than 20.0/nl), 3 days versus 7 days (P greater than 0.4, NS). The rates of infections and stomatitis were 25% and 17%, respectively, for patients treated with rhGM-CSF as compared to 53% (P = 0.0547, NS) and 60% (P = 0.0078), respectively, without rhGM-CSF. The following side effects were associated with the administration of rhGM-CSF: pleural and/or pericardial effusions in five patients, thrombosis in two patients, bone pain in two patients, and respiratory distress syndrome in one patient. A complete remission was achieved in nine of the 23 patients treated with NOAC plus rhGM-CSF, and in two of the 14 patients treated with chemotherapy alone. The median survival of patients treated with rhGM-CSF was not reached at 400 days and seemed to be longer than that of patients treated with chemotherapy alone (median, 109 days; P = 0.036). RhGM-CSF after chemotherapy can be applied safely to patients with NHL, shorten the period of severe cytopenia, reduce the rates of stomatitis, and did not seem to cause adverse effects on response.

Preface. List of Contributors. Part I Stem Cell Biology. 1 Clinical Potentials of Stem Cells: Hype or Hope? (Anthony D. Ho and Wolfgang Wagner). 1.1 Introduction. 1.2 What are Stem Cells? 1.3 Stem Cells and Regeneration. 1.4 Adult and Embryonic Stem Cells. 1.5 In the Beginning was the Hematopoietic Stem Cell. 1.6 Trans-Differentiation of ASCs. 1.7 The Plasticity of ASCs: All Hype and no Hope? 1.8 The Battle of Two Cultures: ESCs versus ASCs. 1.9 The Challenges for Stem Cell Technology. 1.10 Regulation of Self-Renewal versus Differentiation, Asymmetric Divisions. 1.11 Genotype and Expression Profiles of Primitive HSCs. 1.12 Maintaining Stemness: Interactions between HSCs and the Cellular Microenvironment. 1.13 Mesenchymal Stem Cells. 1.14 Preliminary Clinical Studies. 1.15 Concluding Remarks and Future Perspectives. 2 Alteration of Hematopoietic Stem Cell Fates by Chromatin-Modifying Agents (Nadim Mahmud, Mohammed Milhem, Hiroto Araki, and Ronald Hoffman). 2.1 Introduction. 2.2 Cytotoxicity/Antitumor Activity versus Hypomethylating Effects of 5-Azacytidine and its Analogues. 2.3 Treating HSC with 5azaD/TSA can Alter their Fate. 2.4 Are the Effects of 5azaD/TSA Due to Cytotoxicity? 2.5 Treating HSC with Valproic Acid. 2.6 Ex-vivo Expansion of HSC Using Chromatin-Modifying Agents. 2.7 Reactivation of Gene Expression by Treating Cells with Chromatin-Modifying Agents. 2.8 Alteration of Nonhematopoietic Fate by Chromatin-Modifying Agents. 2.9 Safety/Toxicity of Treating Cells with Chromatin-Modifying Agents. 2.10 Conclusion. 3 Increasing Impact of Micro RNAs in Stem Cell Biology and Medicine (Peter Wernet). 3.1 Introduction. 3.2 Biogenesis of miRNAs. 3.3 Action Modes of miRNAs. 3.4 Potential Function Modes of miRNAs. 3.5 Conclusions. Part II Standardization and Quality Assurance of Stem Cell Preparations. 4 Novel Strategies for the Mobilization of Hematopoietic Stem Cells (Stefan Fruehauf, Timon Seeger, and Julian Topaly). 4.1 Physiology of Blood Stem Cell Mobilization. 4.2 Innovative Agents for PBPC Mobilization. 5 Pluripotent Stem Cells from Umbilical Cord Blood (Gesine Kogler and Peter Wernet). 5.1 Biological Advantages of Cord Blood as a Stem Cell Resource. 5.2 The Generation and Expansion of Pluripotent Cells (USSC) from Cord Blood. 5.3 Other Multipotent Nonhematopoietic Stem Cells: Mesenchymal Cells in CB and CB Tissue. 5.4 Conclusion: Future Efforts Towards the Regenerative Capacity of CB Nonhematopoietic Cells. 6 Good Manufacturing Practices: Clinical-Scale Production of Mesenchymal Stem Cells (Luc Sensebe, Philippe Bourin, and Luc Douay). 6.1 Introduction. 6.2 Prerequisites for the Clinical-Scale Production of MSCs. 6.3 Clinical-Scale Production: the French Experience. 6.4 QA and QC. 6.5 Future Prospects. 7 The Clonal Activity of Marked Hematopoietic Stem Cells (Jingqiong Hu, Manfred Schmidt, Annette Deichmann, Hanno Glimm, and Christof von Kalle). 7.1 Introduction. 7.2 Characterization of In-vivo Clonal Activity of HSCs by Genetic Marking. 7.3 Retroviral Integration Site Analysis. 7.4 Clonality Analysis in Animal Model and Human Gene Therapy Trials. 7.5 Interaction of Retroviral Integration Site and Transgene Expression with Clonal Activity of the Respective HSC. 7.6 Clinical Interventions Affect the Clonal Activity of Marked HSCs. 7.7 Perspectives. Part III On the Threshold to Clinical Applications. 8 ALarge Animal Non-Injury Model for Study of Human Stem Cell Plasticity (Graca Almeida-Porada, Christopher D. Porada, and Esmail D. Zanjani). 8.1 Introduction. 8.2 The Uniqueness of the Fetal Sheep Model. 8.3 Differentiative Potential of Human Cells in the Fetal Sheep Model. 9 Developmental Potential of Somatic Stem Cells Following Injection into Murine Blastocysts (Michael Durr, Friedrich Harder, and Albrecht M. Muller). 9.1 Introduction. 9.2 Neurosphere Cells Generate Erythroid-Like Cells Following Injection into Early Embryos. 9.3 Hematopoietic Chimerism by Human Cord Blood-Derived HSCs. 9.4 Injection of Leukemic Cells into Blastocysts. 9.5 Discussion. 10 Testing the Limits: The Potential of MAPC in Animal Models (Felipe Prosper and Catherine M. Verfaillie). 10.1 Introduction. 10.2 Characterization of MAPCs. 10.3 In-Vitro Differentiation Potential of MAPCs. 10.4 In-Vivo Differentiation Potential of MAPCs. 10.5 Mechanisms Underlying the Phenomenon of MAPCs. 10.6 Conclusion. 11 Mesenchymal Stem Cells as Vehicles for Genetic Targeting of Tumors (Frank Marini, Brett Hall, Jennifer Dembinski, Matus Studeny, A. Kate Sasser, and Michael Andreeff). 11.1 Introduction. 11.2 The Tumor Stroma and its Components. 11.3 The Role of Tumor-Stroma Interactions in Tumor Progression. 11.4 The Similarity of MSC Tumor Tropism to Wound Healing. 11.5 The Rationale for using MSCs as Cellular Delivery Vehicles. 11.6 The Challenges in Developing MSC-Based Delivery Strategies. 11.7 Conclusions. Part IV Clinical Trials. 12 Endothelial Progenitor Cells for Cardiac Regeneration (Ulrich Fischer-Rasokat and Stefanie Dimmeler). 12.1 Characterization of Endothelial Progenitor Cells. 12.2 Functions of EPCs to Improve Cardiac Function. 12.3 Mechanisms of Homing. 12.4 Results from Clinical Studies.6 13 Stem Cells and Bypass Grafting for Myocardial and Vascular Regeneration (Christof Stamm, Dirk Strunk, and Gustav Steinhoff). 13.1 Introduction. 13.2 Coronary Artery Disease. 13.3 Indications for CABG Surgery. 13.4 The Rationale for Cell Therapy in CABG Patients. 13.5 The Role of Bone Marrow Cells. 13.6 Combination of (Stem) Cell Treatment with CABG Surgery. 13.7 Outlook. 14 Adoptive Immunotherapy: Guidelines and Clinical Practice (Hans-Jochem Kolb, Christoph Schmid, Iris Bigalke, Raymund Buhmann, Belinda Simoes, Ting Yang, Johanna Tischer, Michael Stanglmaier, Horst Lindhofer, Christine Falk, and Georg Ledderose). 14.1 Introduction. 14.2 Animal Experiments. 14.3 The First Clinical Results in CML. 14.4 The EBMT Study. 14.5 The Graft-versus-Leukemia Effect. 14.6 Cytokines. 14.7 Bispecific Antibodies. 14.8 NK and NK-T Cells and HLA-Haploidentical Transplantation. 14.9 Outlook of Adoptive Immunotherapy in Chimerism. 15 Immune Escape and Suppression by Human Mesenchymal Stem Cells (Katarina Le Blanc and Olle Ringden). 15.1 Introduction. 15.2 MSCs Escape the Immune System. 15.3 Immunosuppression by MSCs. 15.4 MSC in the Clinic. 16 Stem Cell Transplantation: The Basis for Successful Cellular Immunotherapy (Peter Dreger, Matthias Ritgen, and Anthony D. Ho). 16.1 Introduction. 16.2 Allogeneic Stem Cell Transplantation in CLL. 16.3 Graft-versus-Leukemia Effect in CLL. 16.4 Allo-SCT with Reduced-Intensity Conditioning in CLL. 16.5 RICT from Unrelated Donors. 16.6 T-Cell Depletion. 16.7 Allo-SCT in Follicular Lymphoma. 16.8 Allo-SCT in Waldenstrom's Disease. 16.9 Conclusions and Perspectives. Index.