Recruiting refugees and migrants as potential hematopoietic stem cell donors to serve patients of comparable ethnicities with rare human leucocyte antigen patterns - The BluStar.NRW project in North Western Germany.

Currently, approximately 19 million people with a migration background live in Germany. The majority of those descend from regions where the population has a genetically different distribution of HLA antigens when compared to the HLA frequencies usually found in North Western Europe. In case of severe haematological disorders of these individuals, allogeneic stem cell transplantation may be the treatment of choice. However, finding appropriate histocompatible hematopoietic stem cell donors continues to be a major challenge. If no matching sibling donors are available, there are only few suitable donors with a similar genetic background available in international blood stem cell donor registries. The "BluStar.NRW" project aimed to recruit new blood and hematopoietic stem cell donors with a migration background and to noticeably increase the number of suitable donors for patients within this group. Since December 2017, a total number of 9100 blood and stem cell donors with a migration background were recruited and typed for this project. HLA typing for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 was performed by Next Generation Sequencing. We assessed the proportion of rare alleles according to HLA frequency tables, as defined by a frequency of <1:1000. The rare HLA allele frequencies according to HLA frequency tables of the BluStar.NRW cohort were compared with a matched control donor cohort: Rare HLA-A, -B, -C, -DRB1 and -DQB1 alleles occurred three times more frequent than in the control group, but rare HLA-DPB1 alleles occurred more frequently in the control cohort. This difference was highly significant for all HLA alleles (p < 0.0001 for HLA-A, -B, -C, -DRB1, -DPB1; p = 0.0002 for HLA-DQB1). In addition, the distribution of rare alleles differed between the two groups. To date, 29 work-ups were initiated, 12 PBSC, one BM and three DLI were collected so far out of the BluStar.NRW cohort. The apheresis probability is twofold higher (0.18% vs. 0.07%) compared to the control group which clearly shows a serious medical need. However, 13 work-ups were cancelled in the BluStar.NRW donor cohort which represents an almost twice as higher cancellation rate (45% vs. 25%). This single registry analysis with a large sample cohort clearly indicates that hematopoietic stem cell donors with a migration background represent an adequate donor pool to serve patients of comparable ethnicity.

Cardiovascular risk factors: The effects of ageing and smoking on the immune system, an observational clinical study.

Currently immunomodulatory compounds are under investigation for use in patients with cardiovascular disease, caused by atherosclerosis. These trials, using recurrent cardiovascular events as endpoint, require enrollment of large patient groups. We investigated the effect of key risk factors for atherosclerosis development, ageing and smoking, on the immune system, with the objective to identify biomarkers differentiating between human populations, and potentially serving as endpoints for future phase 1B trials with immunomodulatory compounds. Blood was collected from young healthy volunteers (aged 18-25 years, n=30), young smokers (18-25 years, n=20), elderly healthy volunteers (>60 years, n=20), heavy smokers (>45 years, 15 packyears, n=11) and patients with stable coronary artery disease (CAD) (>60 years, n=27). Circulating immune cell subsets were characterized by flow cytometry, and collected plasma was evaluated by proteomics (Olink). Clear ageing effects were observed, mostly illustrated by a lower level in CD8+ and naïve CD4+ and CD8+ T cells, with an increase in CD4+ and CD8+ effector memory T cells in elderly healthy volunteers compared to young healthy volunteers. Heavy smokers showed a more inflammatory cellular phenotype, especially a shift in Th1/Th2 ratio: higher Th1 and lower Th2 percentages compared to young healthy volunteers. A significant decrease in circulating atheroprotective oxLDL-specific IgM was found in patients with CAD compared to young healthy volunteers. Elevated pro-inflammatory and chemotactic proteins TREM1 and CCL11 were observed in elderly volunteers compared to young volunteers. In addition, heavy smokers had an increase in pro-inflammatory cytokine IL-6 and lysosomal protein LAMP3. These data show that ageing and smoking are associated with an inflammatory immunophenotype, and that heavy smokers or aged individuals may serve as potential populations for future clinical trials investigating immunomodulatory drugs targeted for cardiovascular disease.

Expression of nuclear progesterone receptor, progesterone receptor membrane components 1 and 2 and prostaglandin-endoperoxide synthase 2 in the endometrium and oviduct of spontaneously ovulating cats.

Although ovulations not followed by pregnancy occur regularly in cats, differences in endometrial function between cats in the luteal and non-luteal phase have not been studied so far. Progesterone exerts its effects through a nuclear progesterone receptor (PGR) and via cell-membrane bound receptors referred to as progesterone receptor membrane component (PGRMC) 1 and 2. Progesterone receptor expression is regulated by gonadal steroid hormones and therefore may change throughout the oestrous cycle. Protein expression of PGR, PGRMC-1 and 2 and prostaglandin-endoperoxide synthase 2 (PTGS2) was analysed in the endometrium and oviduct of non-pregnant female cats in the follicular (n = 8) and luteal phase (n = 9). We hypothesized that the presence of corpora lutea (CL) is associated with downregulation of progesterone receptors and PTGS2. Cells of the luminal endometrial epithelium, endometrial stroma and oviductal epithelium were assessed by immunohistochemistry. The PGR protein expression was more pronounced in the endometrial epithelium than stroma (p < 0.001) and less pronounced in cats with a CL than without CL (p < 0.001) but did not differ between groups in the oviduct. The PTGS2 was localized only in the endometrial and oviductal epithelium and its expression was reduced in cats with CL (p = 0.001). In the endometrial epithelium, PGRMC-1 expression was reduced in cats with CL (p < 0.05). Expression of PGRMC-2 was highest in the endometrial epithelium and lowest in the endometrial stroma (p = 0.01) but did not differ between cats with and without CL. In conclusion, progesterone receptor and PTGS2 downregulation in the female cat closely resembles findings in other spontaneously ovulating domestic animal species.

Can LDL cholesterol be too low? Possible risks of extremely low levels.

Following the continuous accumulation of evidence supporting the beneficial role of reducing low-density lipoprotein cholesterol (LDL-C) levels in the treatment and prevention of atherosclerotic cardiovascular disease and its complications, therapeutic possibilities now exist to lower LDL-C to very low levels, similar to or even lower than those seen in newborns and nonhuman species. In addition to the important task of evaluating potential side effects of such treatments, the question arises whether extremely low LDL-C levels per se may provoke adverse effects in humans. In this review, we summarize information from studies of human cellular and organ physiology, phenotypic characterization of rare genetic diseases of lipid metabolism, and experience from clinical trials. Specifically, we emphasize the importance of the robustness of the regulatory systems that maintain balanced fluxes and levels of cholesterol at both cellular and organismal levels. Even at extremely low LDL-C levels, critical capacities of steroid hormone and bile acid production are preserved, and the presence of a cholesterol blood-brain barrier protects cells in the central nervous system. Apparent relationships sometimes reported between less pronounced low LDL-C levels and disease states such as cancer, depression, infectious disease and others can generally be explained as secondary phenomena. Drug-related side effects including an increased propensity for development of type 2 diabetes occur during statin treatment, whilst further evaluation of more potent LDL-lowering treatments such as PCSK9 inhibitors is needed. Experience from the recently reported and ongoing large event-driven trials are of great interest, and further evaluation including careful analysis of cognitive functions will be important.

Beneficial effects of levosimendan on survival in patients undergoing extracorporeal membrane oxygenation after cardiovascular surgery.

BACKGROUND: The impact of levosimendan treatment on clinical outcome in patients undergoing extracorporeal membrane oxygenation (ECMO) support after cardiovascular surgery is unknown. We hypothesized that the beneficial effects of levosimendan might improve survival when adequate end-organ perfusion is ensured by concomitant ECMO therapy. We therefore studied the impact of levosimendan treatment on survival and failure of ECMO weaning in patients after cardiovascular surgery. METHODS: We enrolled a total of 240 patients undergoing veno-arterial ECMO therapy after cardiovascular surgery at a university-affiliated tertiary care centre into our observational single-centre registry. RESULTS: During a median follow-up period of 37 months (interquartile range 19-67 months), 65% of patients died. Seventy-five per cent of patients received levosimendan treatment within the first 24 h after initiation of ECMO therapy. Cox regression analysis showed an association between levosimendan treatment and successful ECMO weaning [adjusted hazard ratio (HR) 0.41; 95% confience interval (CI) 0.22-0.80; P=0.008], 30 day mortality (adjusted HR 0.52; 95% CI 0.30-0.89; P=0.016), and long-term mortality (adjusted HR 0.64; 95% CI 0.42-0.98; P=0.04). CONCLUSIONS: These data suggest an association between levosimendan treatment and improved short- and long-term survival in patients undergoing ECMO support after cardiovascular surgery.

An unexpected intriguing effect of Toll-like receptor regulator RP105 (CD180) on atherosclerosis formation with alterations on B-cell activation.

OBJECTIVE: In atherosclerosis, Toll-like receptors (TLRs) are traditionally linked to effects on tissue macrophages or foam cells. RP105, a structural TLR4 homolog, is an important regulator of TLR signaling. The effects of RP105 on TLR signaling vary for different leukocyte subsets known to be involved in atherosclerosis, making it unique in its role of either suppressing (in myeloid cells) or enhancing (in B cells) TLR-regulated inflammation in different cell types. We aimed to identify a role of TLR accessory molecule RP105 on circulating cells in atherosclerotic plaque formation. APPROACH AND RESULTS: Irradiated low density lipoprotein receptor deficient mice received RP105(-/-) or wild-type bone marrow. RP105(-/-) chimeras displayed a 57% reduced plaque burden. Interestingly, total and activated B-cell numbers were significantly reduced in RP105(-/-) chimeras. Activation of B1 B cells was unaltered, suggesting that RP105 deficiency only affected inflammatory B2 B cells. IgM levels were unaltered, but anti-oxidized low-density lipoprotein and anti-malondialdehyde-modified low-density lipoprotein IgG2c antibody levels were significantly lower in RP105(-/-) chimeras, confirming effects on B2 B cells rather than B1 B cells. Moreover, B-cell activating factor expression was reduced in spleens of RP105(-/-) chimeras. CONCLUSIONS: RP105 deficiency on circulating cells results in an intriguing unexpected TLR-associated mechanisms that decrease atherosclerotic lesion formation with alterations on proinflammatory B2 B cells.

CHO(E)P-14 followed by alemtuzumab consolidation in untreated peripheral T cell lymphomas: final analysis of a prospective phase II trial.

The rate of long-term remissions after treatment of peripheral T cell lymphomas (PTCL) with standard CHOP-like protocols is unsatisfactory. A prospective multicenter phase II trial was initiated in untreated patients with PTCL of all International Prognostic Index-risk groups, evaluating alemtuzumab consolidation in patients with complete or good partial remission after CHO(E)P-14 induction. Twenty-nine (70.7 %) of the 41 enrolled patients received alemtuzumab consolidation (133 mg in total). The main grades 3-4 toxicities during alemtuzumab therapy were infections and neutropenia with one potentially treatment-related death. Complete responses were seen in 58.5 %, partial responses in 2.4 % and 29.3 % had progressive disease. After a median observation time of 46 months, 19 patients have died, 16 of them due to lymphoma and/or salvage therapy complications. Event-free and overall survival at 3 years in the whole intent to treat population are 32.3 and 62.5 %, respectively, and 42.4 and 75.1 % in the patients who received alemtuzumab. In conclusion, application of a short course of alemtuzumab after CHO(E)P-14 induction is feasible although complicated by severe infections. A current phase III trial, applying alemtuzumab as part of the initial chemotherapy protocol to avoid early progression, will further clarify its significance for the therapeutic outcome.

Nuclear LEF1/TCF4 correlate with poor prognosis but not with nuclear ß-catenin in cerebral metastasis of lung adenocarcinomas.

An essential function of the transcription factors LEF1/TCF4 in cerebral metastases of lung adenocarcinomas has been described in mouse models, suggesting a WNT/ß-catenin effect as potential mechanism. Their role in humans is still unclear, thus we analyzed LEF1, TCF4, ß-catenin, and early stage prognostic markers in 25 adenocarcinoma brain metastases using immunohistochemistry (IHC). IHC revealed nuclear TCF4 in all adenocarcinoma samples, whereas only 36 % depicted nuclear LEF1 and nuclear ß-catenin signals. Samples with nuclear LEF1 as well as high TCF4 (++++) expression were associated with a shorter survival (p = 0.01, HR = 6.68), while nuclear ß-catenin had no significant impact on prognosis and did not significantly correlate with nuclear LEF1. High proliferation index Ki67 was associated with shorter survival in late-stage disease (p = 0.03, HR 3.27). Additionally, we generated a LEF1/TCF4 as well as an AXIN2 signature, the latter as representative of WNT/ß-catenin activity, following a bioinformatics approach with a gene expression dataset of cerebral metastases in lung adenocarcinoma. To analyze the prognostic relevance in primary lung adenocarcinomas, we applied both signatures to a microarray dataset of 58 primary lung adenocarcinomas. Only the LEF1/TCF4 signature was able to separate clusters with impact on survival (p = 0.01, HR = 0.32). These clusters displayed diverging enrichment patterns of the cell cycle pathway. In conclusion, our data show that LEF1/TCF4, but not ß-catenin, have prognostic relevance in primary and cerebrally metastasized human lung adenocarcinomas. In contrast to the previous in vivo findings, these results indicate that LEF1/TCF4 act independently of ß-catenin in this setting.

ß-catenin-independent WNT signaling in basal-like breast cancer and brain metastasis.

A role of WNT signaling for primary breast cancers of the basal-like subtype and as a predictor of brain metastasis has been described. However, a responsible WNT ligand has not been identified. To further clarify this question, we comparatively investigated 22 human breast cancer brain metastases as well as the highly invasive human breast cancer cell line MDA-MB-231 and the weakly motile MCF-7 as models for the basal-like and the luminal A subtype. WNT5A and B were found overexpressed in MDA-MB-231 cells as compared with MCF-7. This corresponded to reduction of MDA-MB-231 invasiveness by WNT inhibitors, whereas MCF-7 invasion was enhanced by recombinant WNT5B and abolished by WNT and Jun-N-terminal kinase antagonists. Expression and subcellular distribution of ß-catenin remained uninfluenced. Consistently, ß-catenin was not localized in the nuclei of brain metastases while there was strong nuclear c-Jun staining. Similar to MDA-MB-231, metastases showed expression of WNT5A/B and the alternative WNT receptors ROR1 and 2. These findings were validated using external gene expression datasets (Gene Expression Omnibus) of different breast cancer subtypes and brain metastases. Hierarchical cluster analysis yielded a close relation between basal-like cancers and brain metastases. Gene set enrichment analyses confirmed WNT pathway enrichment not only in basal-like primaries but also in cerebral metastases of all subtypes. In conclusion, WNT signaling seems highly relevant for basal-like and other subtypes of breast cancers metastasizing into the brain. ß-catenin-independent WNT signaling, presumably via ROR1-2, plays a major role in this context.

Natural antibodies, intravenous immunoglobulin and their role in autoimmunity, cancer and inflammation.

Natural antibodies are produced by B lymphocytes in the absence of external antigen stimulation. With their ability to recognize self, altered self and foreign antigens, they comprise an important first-line defence against invading pathogens, but are also important for tissue homeostasis. By recognizing oligosaccharides expressed on tumour cells and modified cell surface structures accompanying necrosis, natural antibodies have an important anti-tumorigenic function. IVIg contains a wide spectrum of specificities presented in normal plasma including natural antibodies and has been shown to exert inhibitory effects on tumour cells through a subfraction of anti-vascular endothelial growth factor immunoglobulin (Ig)G antibodies with anti-angiogenic properties. IgA antibodies also have potent immunomodulatory properties, being able to both induce and suppress immune responses. IgA-mediated inhibitory function is able to inhibit several inflammatory diseases including asthma and glomerulonephritis. Autoantibodies of the IgM type, on the other hand, have shown promising results in the treatment of multiple sclerosis. These autoantibodies promote remyelination rather than modulating inflammation. Oxidation-specific epitopes, as found in atherosclerotic lesions and on apoptotic cells, comprise one important target of natural antibodies. By recognizing these epitopes, natural antibodies neutralize proinflammatory responses and mediate atheroprotection.

Wnt 5a signaling is critical for macrophage-induced invasion of breast cancer cell lines.

Interactions between neoplastic and stromal cells contribute to tumor progression. Wnt genes, involved in cell migration and often deregulated in cancers, are attractive candidates to regulate these effects. We have recently shown that coculture of breast cancer cells with macrophages enhances invasiveness via matrix metalloproteases and TNF-alpha. Here we demonstrate that coculture of MCF-7 cells and macrophages leads to up-regulation of Wnt 5a in the latter. This was accompanied by activation of AP-1/c-Jun in MCF-7. Recombinant Wnt 5a mimicked the coculture effect. Wnt 5a was also detectable in tumor-associated macrophages in primary breast cancers. Experiments with agonists and antagonists of Wnt signaling revealed that a functional canonical pathway in the tumor cells was a necessary prerequisite; however, noncanonical signaling via Wnt 5a and the Jun-N-terminal kinase pathway was critical for invasiveness. It was also responsible for induction of matrix metalloprotease-7, known to release TNF-alpha. All these effects could be antagonized by dickkopf-1. Our results indicate that Wnt 5a is essential for macrophage-induced invasiveness, because it regulates tumor cell migration as well as proteolytic activity of the macrophages. The function of Wnt 5a as either a suppressor or promoter of malignant progression seems to be modulated by intercellular interactions. Wnt 5a detection in tumor-associated macrophages in breast cancer biopsies supports the assumption that similar events play a role in vivo.

Elevated concentrations of serum relaxin are associated with metastatic disease in breast cancer patients.

Relaxin (RLX) is known to induce remodeling of benign stromal tissues through upregulation of matrix metalloproteases (MMPs). Recently, we could show that RLX also induces MMPs in breast cancer cells and enhances in vitro invasiveness. To investigate its potential role for progression of breast cancer in vivo, RLX serum concentrations were determined in 160 breast cancer patients during post-surgical follow-up. RLX concentrations in cancer patients were significantly higher than in a control population of healthy blood donors and patients with various other diseases (0.47 versus 0.29 ng/ml, p < 0.0001). There was a significant difference between patients with metastases (0.62 ng/ml) and those without (0.38 ng/ml, p < 0.0001). Overall survival was shorter in RLX-positive ( > 0.4 ng/ml) than in RLX-negative patients (p = 0.016). Cox regression analysis showed that RLX was not an independent variable, in contrast to metastatic disease and primary lymph node involvement. Taken together, the detection of elevated RLX concentrations especially in patients with metastases supports the assumption that there is a role for RLX in tissue remodeling during breast cancer progression.

Procalcitonin: a useful discriminator between febrile conditions of different origin in hemato-oncological patients?

Plasma concentrations of procalcitonin (PCT) have been shown to be elevated in bacterial and fungal infections. In contrast to C-reactive protein (CRP), PCT is not elevated in inflammations of noninfectious origin. Febrile inflammatory conditions are frequent in patients with hemato-oncological diseases. A reliable marker to discriminate infectious inflammations from drug-related and tumor-associated fever is still lacking. To evaluate the impact of PCT in this setting, PCT and CRP were prospectively measured in 95 febrile hemato-oncological patients. Infections could be identified in 40 of 95 patients: 38 of 95 had fever of unknown origin (FUO), 9 patients were suspected to suffer from drug-related fever, and 8 patients from tumor-associated fever. In the noninfection group (drug-related and tumor-associated fever), PCT levels were significantly lower than in patients with infections (P<0.001) or FUO (P<0.001). Differences were still highly significant comparing patients with suspected drug-related or tumor-associated fever alone with the infection or the FUO cohort. All eight patients with tumor-associated fever as well as eight of the nine patients with drug-related fever had PCT levels within the normal range (<0.5 micro g/l). CRP values only partially allowed discrimination between the various subgroups. Differences were significant between patients with drug-related fever and the infection (P=0.001) or FUO group (P=0.004). However, as CRP levels were far above the normal range also in the patients with drug-related fever, the significance of individual values was rather limited. In conclusion, PCT may provide useful additional information to assess the clinical significance of febrile conditions. PCT may facilitate the decision on when to initiate antimicrobial or cytotoxic therapy.

Relaxin enhances in-vitro invasiveness of breast cancer cell lines by up-regulation of matrix metalloproteases.

Until recently, relaxin (RLX) has been known predominantly for its effects on the reproductive system, where it induces remodelling of the extracellular matrix and up-regulation of matrix metalloproteases (MMPs). In solid cancers, tissue remodelling and MMP activation are essential for invasion and metastasis. We therefore investigated the effect of RLX on invasiveness and MMP expression of human breast cancer cell lines. Upon incubation with porcine RLX, the invasiveness of SK-BR3 cells was significantly increased. Similar effects could be achieved in MCF-7 cells, especially when RLX was combined with epidermal growth factor. Enhanced invasiveness was accompanied by up-regulation of MMP production and could be almost completely blocked by the MMP inhibitor FN 439. Zymography revealed increased secretion of MMP-2, -7 and -9, associated with up-regulated mRNA concentrations of MMP-2, -9, -13 and -14. mRNA expression levels of MMP-1, -3, -7, -8, -10, -11, -12 and of tissue inhibitors of metalloproteases-1, -2, -3 and -4 were either very low or not detectably influenced by RLX. Taken together, RLX enhances in-vitro invasiveness of breast cancer cell lines by induction of MMP expression. It remains to be clarified whether RLX might play a similar role in vivo and promote tumour progression.

mRNA expression of matrix metalloproteases and their inhibitors differs in subtypes of renal cell carcinomas.

Altered expression of matrix metalloproteases (MMPs) and their inhibitors, the tissue inhibitors of matrix metalloproteases (TIMPs), has been demonstrated in various tumour tissues. mRNA expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, MMP-11, MMP-12, MMP-14 and TIMP-1, TIMP-2, TIMP-3 and TIMP-4 were evaluated by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in 30 renal cell carcinomas (RCC), as well as in the surrounding tissues. Expression of the MMPs was significantly stronger in the carcinomas than in non-malignant tissues. High levels were demonstrated particularly in clear cell RCCs (CC-RCC). Except for MMP-1, MMP expression in the papillary RCCs (P-RCC) was, for most MMPs, significantly lower. Expression of the TIMPs in malignant cells of both subtypes was weak, with the exception of TIMP-4 which was strongly expressed in the P-RCCs and downregulated in the CC-RCCs. The latter was correlated with chromosomal loss of 3p, harbouring the TIMP-4 gene locus. In conclusion, deregulated expression of the MMPs and TIMPs in RCCs differs according to histology, grade, size and cytogenetic characteristics, suggesting that MMP and TIMP expression patterns play an important role for the typical histomorphological features of RCC subtypes and their respective biological behaviour.

Effects of hexadecylphosphocholine on thrombocytopoiesis.

Hexadecylphosphocholine (HePC) is the first representative of the alkylphosphocholines, a novel group derived from the cytotoxic etherlysophospholipids. HePC shows a broad spectrum of antiproliferative effects in neoplastic cells in vitro and in vivo. HePC has been tested successfully in several clinical studies. One of the remarkable features of this compound has been the induction of a leucocytosis and a thrombocytosis in most of the patients receiving HePC systemically. In this paper, we have investigated the biological and molecular mechanisms by which HePC exerts this interesting effect. We found that HePC acts as an unspecific costimulator on human megakaryocytic proliferation in a soft agar assay system predominantly together with thrombopoietin (TPO). Furthermore, HePC leads to the synthesis and secretion of several haematopoietic growth factors in monocytes and bone marrow fibroblasts, determined by the direct measurement of growth factors in cellular supernatants and by the measurement of growth factor mRNA in cell extracts. Thus, HePC seems to produce the increase of blood platelets in tumour patients by two different mechanisms.

Successful transplantation and engraftment of peripheral blood stem cells after cryopreservation, positive and negative purging procedures, and a second cryopreservation cycle.

Transplantation of peripheral blood stem cells (PBSC), positively and/or negatively selected immediately after harvest, has become a widely applied therapeutic option in hematological or oncological patients. The following case of peripheral blood stem cell transplantation represents the first case of successful transplantation of PBSC, cryopreserved twice and purged after cryopreservation. PBSC were harvested in a 44-year-old female patient with a low-grade non-Hodgkin's lymphoma stage IV after mobilization with chemotherapy and G-CSF. A total number of 15.2 x 10(6) CD34+ cells/kg bodyweight was harvested with a 36.9% contamination of tumor cells coexpressing CD5 and CD20. After subsequent chemotherapy cycles and cyclophosphamide mobilization, only 0.77 x 10(6) CD34+ cells/kg bodyweight, not sufficient for transplantation, were achieved after positive selection. Therefore, 10.8 x 10(6) cryopreserved CD34+ cells/kg bodyweight were thawed and a positive selection was carried out with the BAXTER Isolex 300i machine. Before additional negative selection, the 0.77 x 10(6) positively selected CD34+ cells/kg bodyweight from the second mobilization were added. A total quantity of 4.4 x 10(6) CD34+ cells/kg bodyweight with a purity of 93.1% representing a recovery of 38% was obtained. Cells were again cryopreserved, stored and retransfused after conditioning the patient with TBI and high-dose cyclophosphamide. The patient engrafted with a WBC count > 1000/microliter on day eight and a platelet count > 20,000/microliter without transfusion support on day 12 post-transplantation. This case indicates that purging procedures can successfully be carried out with cryopreserved cell material and that purified CD34+ cells can be cryopreserved a second time before transplantation, without affecting their hematopoietic capacity.

Effects of amifostine in a patient with an advanced-stage myelodysplastic syndrome.

We report on a 63-year-old man with myelodysplastic syndrome at the stage of a refractory anemia with an excess of blasts in transformation (MDS-RAEB-T), first diagnosed in December 1996. After a period of stability, with no need for transfusions, the MDS progressed into acute myeloid leukemia (AML) in August 1998 with the emergence of a cytogenetic abnormality (11q-). Two courses of chemotherapy were given, resulting in prolonged pancytopenia; however, no clearance of bone marrow (BM) blasts was achieved. At that time, severe infections and daily epistaxis occurred. Frequent transfusions of packed red blood cells (RBC) and platelets (2-3/week) were necessary. After 2 months of persisting severe pancytopenia, we started a therapy with amifostine: 4 x 250 mg intravenously (i.v.) weekly for 1 month, followed by a maintenance therapy with 500 mg once weekly. After 2 weeks of amifostine therapy, hematopoiesis began to improve. In the subsequent 2 months, the patient became completely independent of the platelet transfusions; the transfusion frequency of RBC was permanently reduced (2 RBC transfusions/month) and a significant decrease of BM blasts was achieved. After 30 weeks of amifostine therapy, the morphology of the MDS switched to a chronic myelomonocytic leukemia (CMML)-like appearance, with continuously increasing leukocytes, so that we discontinued amifostine therapy for 1 month to exclude a possible side effect of amifostine. At that time, leukocytes further increased to 74,000/microl; thus, we decided to perform a cytoreductive chemotherapy (hydroxycarbamide) and continued weekly amifostine infusions. During 1 year of amifostine therapy, the patient had a good quality of life, with no need for hospitalization and a complete cytogenetic remission. We conclude that, in this case, amifostine had two effects: a significant improvement of impaired hematopoiesis and a slowing down of disease progression. Thus, amifostine might be a therapeutic option in older patients with advanced MDS.

Cytogenetic subgroups in acute myeloid leukemia differ in proliferative activity and response to GM-CSF.

The current study was undertaken to search for differences in the biology of cytogenetic subgroups in patients with de novo acute myeloid leukemia (AML). In addition, factors influencing the metabolism of cytosine arabinoside (araC) as the key agent of antileukemic activity were assessed. Bone marrow aspirates from 91 patients with newly diagnosed AML in whom karyotypes were successfully obtained were analyzed: (1) for spontaneous proliferative activity by 3H-thymidine (3H-TdR) incorporation; (2) proliferative response to GM-CSF by in vitro incubation of blasts for 48 h with or without GM-CSF (100 U/ml) followed by an additional 4-h exposure to 3H-TdR (0.5 microCi/ml); and (3) parameters of araC metabolism comprising 3H-araC uptake in vitro and the activities of polymerase alpha (poly alpha), deoxycytidine kinase (DCK) and deoxycytidine deaminase (DCD). According to the results of chromosome analyses four cytogenetic subgroups were discriminated: (I) normal karyotypes (n = 38); (II) favorable karyotypes [t8;21), t(15;17), inv(16)] (n = 16); (III) unfavorable karyotypes [inv (3), -5, 5q-, t(6;9), +8, t (9;11), complex abnormalities] (n = 20); (IV) karyotypes of unknown prognostic significance (n = 17). Proliferative activity of leukemic blasts was significantly higher in favorable karyotypes (group II) as compared to cases with unfavorable cytogenetics (group III) with median values and range for 3H-TdR uptake in group II of 2.48 pmol/10(5) cells (0.28-25.8) and in group III of 0.51 pmol/10(5) cells (0.04-7.6) (P = 0.0096). The respective values in group I and group IV were 0.7pmol/10(5) cells (0.0-6.7) and 0.98 pmol/10(5) cells (0.0-4.0), respectively. Inversely, response to GM-CSF, as defined by an increase in 3H-TdR incorporation >1.5- fold over control values after 48h of GM-CSF exposure, was significantly lower for patients with a favorable karyotype (group II) as compared to group I (P = 0.04) and group III (P = 0.013). No significant differences between karyotype groups I, II, III and IV were found for 3H-araC incorporation, nor for the activities of poly alpha, DCK and DCD. These data demonstrate differences in the biology of cytogenetic subgroups in AML which may partly explain the well established differences in clinical outcome.