Prevalence and significance of bacterial contamination of autologous stem cell products.

There is limited and conflicting information on the prevalence of contamination of haematopoietic stem and progenitor cell products (HPCPs), and their optimal management remains unclear. The authors reviewed the microbial surveillance data of HPCPs collected between January 2002 and December 2019 for autologous transplantation at the study institution to determine the prevalence of microbial contamination and the potential infectious complications among recipients. Among 3935 HPCPs, 25 (0.6%) were contaminated. Ultimately, 22 patients received contaminated grafts, with pre-emptive antimicrobial therapy initiated in six of these patients. No patients developed subsequent infectious complications. These data suggest that microbial contamination of autologous HPCPs and associated adverse outcomes are rare.

LIGHT/LTßR signaling regulates self-renewal and differentiation of hematopoietic and leukemia stem cells.

The production of blood cells during steady-state and increased demand depends on the regulation of hematopoietic stem cell (HSC) self-renewal and differentiation. Similarly, the balance between self-renewal and differentiation of leukemia stem cells (LSCs) is crucial in the pathogenesis of leukemia. Here, we document that the TNF receptor superfamily member lymphotoxin-ß receptor (LTßR) and its ligand LIGHT regulate quiescence and self-renewal of murine and human HSCs and LSCs. Cell-autonomous LIGHT/LTßR signaling on HSCs reduces cell cycling, promotes symmetric cell division and prevents primitive HSCs from exhaustion in serial re-transplantation experiments and genotoxic stress. LTßR deficiency reduces the numbers of LSCs and prolongs survival in a murine chronic myeloid leukemia (CML) model. Similarly, LIGHT/LTßR signaling in human G-CSF mobilized HSCs and human LSCs results in increased colony forming capacity in vitro. Thus, our results define LIGHT/LTßR signaling as an important pathway in the regulation of the self-renewal of HSCs and LSCs.

NSAID treatment with meloxicam enhances peripheral stem cell mobilization in myeloma.

Chemotherapy with G-CSF is used to mobilize peripheral stem cells in multiple myeloma (MM) patients, with plerixafor as a rescue strategy for poorly mobilizing patients. Preclinical studies suggested that the nonsteroidal anti-inflammatory drug meloxicam enhances the mobilization of CD34+ cells. In this single-center study, we evaluated whether adding meloxicam to chemotherapy/G-CSF mobilization increases peripheral hematopoietic CD34+ cell levels and reduces the need of using plerixafor. We prospectively compared two consecutive cohorts of MM patients in first remission mobilized with G-CSF and non-myelosuppressive chemotherapy with vinorelbine or gemcitabine. The second cohort additionally received oral meloxicam. The cohorts comprised 84 patients without meloxicam (-M) and 66 patients with meloxicam (+M). Meloxicam was well tolerated and associated with similar hematologic engraftment after transplantation and equal survival rates. However, the meloxicam group had higher CD34+ cell levels on day 8 of the mobilization procedure (53 200 versus 35 600 CD34+ cells/mL; P=0.007), and fewer patients needed >1 collection day (+M: 6 (9%) patients versus -M: 16 (19%) patients; P=0.04). This resulted in reduced plerixafor administrations (+M: 7 (11%) patients versus -M: 18 (21%) patients; P=0.03) and less costs. Our data suggest that meloxicam enhances the mobilization of hematopoietic CD34+ blood cells in MM patients.

Assessment of imatinib as first-line treatment of chronic myeloid leukemia: 10-year survival results of the randomized CML study IV and impact of non-CML determinants.

Chronic myeloid leukemia (CML)-study IV was designed to explore whether treatment with imatinib (IM) at 400 mg/day (n=400) could be optimized by doubling the dose (n=420), adding interferon (IFN) (n=430) or cytarabine (n=158) or using IM after IFN-failure (n=128). From July 2002 to March 2012, 1551 newly diagnosed patients in chronic phase were randomized into a 5-arm study. The study was powered to detect a survival difference of 5% at 5 years. After a median observation time of 9.5 years, 10-year overall survival was 82%, 10-year progression-free survival was 80% and 10-year relative survival was 92%. Survival between IM400 mg and any experimental arm was not different. In a multivariate analysis, risk group, major-route chromosomal aberrations, comorbidities, smoking and treatment center (academic vs other) influenced survival significantly, but not any form of treatment optimization. Patients reaching the molecular response milestones at 3, 6 and 12 months had a significant survival advantage. For responders, monotherapy with IM400 mg provides a close to normal life expectancy independent of the time to response. Survival is more determined by patients' and disease factors than by initial treatment selection. Although improvements are also needed for refractory disease, more life-time can currently be gained by carefully addressing non-CML determinants of survival.

Secondary malignancies in chronic myeloid leukemia patients after imatinib-based treatment: long-term observation in CML Study IV.

Treatment of chronic myeloid leukemia (CML) has been profoundly improved by the introduction of tyrosine kinase inhibitors (TKIs). Long-term survival with imatinib is excellent with a 8-year survival rate of ∼88%. Long-term toxicity of TKI treatment, especially carcinogenicity, has become a concern. We analyzed data of the CML study IV for the development of secondary malignancies. In total, 67 secondary malignancies were found in 64 of 1525 CML patients in chronic phase treated with TKI (n=61) and interferon-α only (n=3). The most common malignancies (n⩾4) were prostate, colorectal and lung cancer, non-Hodgkin's lymphoma (NHL), malignant melanoma, non-melanoma skin tumors and breast cancer. The standardized incidence ratio (SIR) for all malignancies excluding non-melanoma skin tumors was 0.88 (95% confidence interval (0.63-1.20)) for men and 1.06 (95% CI 0.69-1.55) for women. SIRs were between 0.49 (95% CI 0.13-1.34) for colorectal cancer in men and 4.29 (95% CI 1.09-11.66) for NHL in women. The SIR for NHL was significantly increased for men and women. An increase in the incidence of secondary malignancies could not be ascertained. The increased SIR for NHL has to be considered and long-term follow-up of CML patients is warranted, as the rate of secondary malignancies may increase over time.

Long-term outcome of patients with newly diagnosed chronic myeloid leukemia: a randomized comparison of stem cell transplantation with drug treatment.

Tyrosine kinase inhibitors represent today's treatment of choice in chronic myeloid leukemia (CML). Allogeneic hematopoietic stem cell transplantation (HSCT) is regarded as salvage therapy. This prospective randomized CML-study IIIA recruited 669 patients with newly diagnosed CML between July 1997 and January 2004 from 143 centers. Of these, 427 patients were considered eligible for HSCT and were randomized by availability of a matched family donor between primary HSCT (group A; N=166 patients) and best available drug treatment (group B; N=261). Primary end point was long-term survival. Survival probabilities were not different between groups A and B (10-year survival: 0.76 (95% confidence interval (CI): 0.69-0.82) vs 0.69 (95% CI: 0.61-0.76)), but influenced by disease and transplant risk. Patients with a low transplant risk showed superior survival compared with patients with high- (P<0.001) and non-high-risk disease (P=0.047) in group B; after entering blast crisis, survival was not different with or without HSCT. Significantly more patients in group A were in molecular remission (56% vs 39%; P=0.005) and free of drug treatment (56% vs 6%; P<0.001). Differences in symptoms and Karnofsky score were not significant. In the era of tyrosine kinase inhibitors, HSCT remains a valid option when both disease and transplant risk are considered.

Clinical usefulness of therapeutic concentration monitoring for imatinib dosage individualization: results from a randomized controlled trial.

PURPOSE: This study assessed whether a cycle of "routine" therapeutic drug monitoring (TDM) for imatinib dosage individualization, targeting an imatinib trough plasma concentration (C min) of 1,000 ng/ml (tolerance: 750-1,500 ng/ml), could improve clinical outcomes in chronic myelogenous leukemia (CML) patients, compared with TDM use only in case of problems ("rescue" TDM). METHODS: Imatinib concentration monitoring evaluation was a multicenter randomized controlled trial including adult patients in chronic or accelerated phase CML receiving imatinib since less than 5 years. Patients were allocated 1:1 to "routine TDM" or "rescue TDM." The primary endpoint was a combined outcome (failure- and toxicity-free survival with continuation on imatinib) over 1-year follow-up, analyzed in intention-to-treat (ISRCTN31181395). RESULTS: Among 56 patients (55 evaluable), 14/27 (52 %) receiving "routine TDM" remained event-free versus 16/28 (57 %) "rescue TDM" controls (P = 0.69). In the "routine TDM" arm, dosage recommendations were correctly adopted in 14 patients (median C min: 895 ng/ml), who had fewer unfavorable events (28 %) than the 13 not receiving the advised dosage (77 %; P = 0.03; median C min: 648 ng/ml). CONCLUSIONS: This first target concentration intervention trial could not formally demonstrate a benefit of "routine TDM" because of small patient number and surprisingly limited prescriber's adherence to dosage recommendations. Favorable outcomes were, however, found in patients actually elected for target dosing. This study thus shows first prospective indication for TDM being a useful tool to guide drug dosage and shift decisions. The study design and analysis provide an interesting paradigm for future randomized TDM trials on targeted anticancer agents.

Velocity of early BCR-ABL transcript elimination as an optimized predictor of outcome in chronic myeloid leukemia (CML) patients in chronic phase on treatment with imatinib.

UNLABELLED: Early assessment of response at 3 months of tyrosine kinase inhibitor treatment has become an important tool to predict favorable outcome. We sought to investigate the impact of relative changes of BCR-ABL transcript levels within the initial 3 months of therapy. In order to achieve accurate data for high BCR-ABL levels at diagnosis, beta glucuronidase (GUS) was used as a reference gene. Within the German CML-Study IV, samples of 408 imatinib-treated patients were available in a single laboratory for both times, diagnosis and 3 months on treatment. In total, 301 of these were treatment-naïve at sample collection. RESULTS: (i) with regard to absolute transcript levels at diagnosis, no predictive cutoff could be identified; (ii) at 3 months, an individual reduction of BCR-ABL transcripts to the 0.35-fold of baseline level (0.46-log reduction, that is, roughly half-log) separated best (high risk: 16% of patients, 5-year overall survival (OS) 83% vs 98%, hazard ratio (HR) 6.3, P=0.001); (iii) at 3 months, a 6% BCR-ABL(IS) cutoff derived from BCR-ABL/GUS yielded a good and sensitive discrimination (high risk: 22% of patients, 5-year OS 85% vs 98%, HR 6.1, P=0.002). Patients at risk of disease progression can be identified precisely by the lack of a half-log reduction of BCR-ABL transcripts at 3 months.

Early molecular and cytogenetic response is predictive for long-term progression-free and overall survival in chronic myeloid leukemia (CML).

In the face of competing first-line treatment options for CML, early prediction of prognosis on imatinib is desirable to assure favorable survival or otherwise consider the use of a second-generation tyrosine kinase inhibitor (TKI). A total of 1303 newly diagnosed imatinib-treated patients (pts) were investigated to correlate molecular and cytogenetic response at 3 and 6 months with progression-free and overall survival (PFS, OS). The persistence of BCR-ABL transcript levels >10% according to the international scale (BCR-ABL(IS)) at 3 months separated a high-risk group (28% of pts; 5-year OS: 87%) from a group with >1-10% BCR-ABL(IS) (41% of pts; 5-year OS: 94%; P=0.012) and from a group with ≤1% BCR-ABL(IS) (31% of pts; 5-year OS: 97%; P=0.004). Cytogenetics identified high-risk pts by >35% Philadelphia chromosome-positive metaphases (Ph+, 27% of pts; 5-year OS: 87%) compared with ≤35% Ph+ (73% of pts; 5-year OS: 95%; P=0.036). At 6 months, >1% BCR-ABL(IS) (37% of pts; 5-year OS: 89%) was associated with inferior survival compared with ≤1% (63% of pts; 5-year OS: 97%; P<0.001) and correspondingly >0% Ph+ (34% of pts; 5-year OS: 91%) compared with 0% Ph+ (66% of pts; 5-year OS: 97%; P=0.015). Treatment optimization is recommended for pts missing these landmarks.

Short telomeres and high telomerase activity in T-cell prolymphocytic leukemia.

To test the role of telomere biology in T-cell prolymphocytic leukemia (T-PLL), a rare aggressive disease characterized by the expansion of a T-cell clone derived from immuno-competent post-thymic T-lymphocytes, we analyzed telomere length and telomerase activity in subsets of peripheral blood leukocytes from 11 newly diagnosed or relapsed patients with sporadic T-PLL. Telomere length values of the leukemic T cells (mean+/-s.d.: 1.53+/-0.65 kb) were all below the 1st percentile of telomere length values observed in T cells from healthy age-matched controls whereas telomere length of normal T- and B cells fell between the 1st and 99th percentile of the normal distribution. Leukemic T cells exhibited high levels of telomerase and were sensitive to the telomerase inhibitor BIBR1532 at doses that showed no effect on normal, unstimulated T cells. Targeting the short telomeres and telomerase activity in T-PLL seems an attractive strategy for the future treatment of this devastating disease.

Impact of culture conditions on the proliferative lifespan of human T cells in vitro.

BACKGROUND: In human T cells, telomerase is transiently expressed upon activation and stimulation and, as shown previously, telomerase levels are able to control the lifespan of T cells. To improve T-cell expansion it is of critical importance to understand the effects of culture parameters on telomerase activity and lifespan. METHODS: We investigated the influence of culture condition (FCS, human AB serum and autologous serum) and stimulation (PHA/feeder cells, anti-CD3/CD28 beads) on the lifespan, clonogenicity (number of positive wells), cell cycle, telomerase activity and telomere length of T cells in vitro. RESULTS: The proliferative lifespan of T cells expanded with PHA/feeder cells and autologous serum from different donors was doubled compared with stimulation with PHA/feeder cells and AB serum. No or only a small difference was found for T cells expanded with anti-CD3/CD28 beads and autologous or AB serum. The use of autologous serum also increased the clonogenicity to about three-fold compared with the use of AB serum or FCS, without any signs of differences in the fractions of cycling cells. Interestingly, T cells cultured with autologous serum exhibited a significantly higher telomerase activity at day 6 after stimulation and a reduced decline of telomerase activity compared with cultures with AB serum. DISCUSSION: The use of autologous serum combined with PHA stimulation and feeder cells remarkably extends the proliferative lifespan and clonogenicity and increases the telomerase activity of human T cells in vitro. This might be useful for applications where large numbers of specific T cells are required.

Limited telomere shortening in hematopoietic stem cells after transplantation.

The number of cell divisions in hematopoietic stem cells (HSCs) following transplantation of bone marrow or mobilized peripheral blood into myelo-ablated recipients is unknown. This number is expected to depend primarily on the number of transplanted stem cells, assuming that stem cells do not differ in engraftment potential and other functional properties. In a previous study, we found that the telomere length in circulating granulocytes in normal individuals shows a biphasic decline with age, most likely reflecting age-related changes in the turnover of HSCs. In order to study HSCs' proliferation kinetics following stem cells transplantation, we analyzed the telomere length in donor-derived nucleated blood cells in four HLA-matched bone marrow transplant recipients relative to comparable cells from the sibling donors. In each case, the telomeres in granulocytes were shorter in the recipient than in the donor. This difference was established in the first year post transplantation and did not change after that. The telomere length in naïve and memory T cells showed marked differences after transplantation, complicating the interpretation of telomere length data using unseparated nucleated blood cells. Interestingly, the telomere length in naïve T cells that were first observed six months post transplantation was very similar in donor and recipient pairs. Our observations are compatible with a limited number of additional cell divisions in stem cell populations after bone marrow transplantations and support the idea that different populations of stem cells contribute to short-term myeloid and long-term lympho myeloid hematopoiesis.

Gel-filtration of sickle erythrocytes: separation based on cell deformability.

UNLABELLED: Filtration of red blood cells (RBC) through columns of pre-swollen agarose-based beads has been evaluated using cells from subjects with sickle cell disease. Elution profiles from these gels showed elution times close to normal controls for a large fraction of sickle erythrocytes and a prolonged elution time for a sub-population of these cells. Analysis of red blood cell deformability using a computerized micropore filtration system (CTA) indicated that the deformability of sickle red blood cells in the first fraction was similar to controls but that the last fraction contained a sub-population of rigid RBC. We thus conclude that sickle red blood cell separation in columns of agarose-based beads is based upon cell deformability. Gel filtration therefore appears to be an interesting tool for the study of red blood cells in a variety of disorders with sub-populations of rigid, abnormal cells, and seems especially suited for studies in various sickle cell diseases. KEYWORDS: Deformability, gel filtration, sickle cell disease, erythrocyte

Extension of cell life-span and telomere length in animals cloned from senescent somatic cells.

The potential of cloning depends in part on whether the procedure can reverse cellular aging and restore somatic cells to a phenotypically youthful state. Here, we report the birth of six healthy cloned calves derived from populations of senescent donor somatic cells. Nuclear transfer extended the replicative life-span of senescent cells (zero to four population doublings remaining) to greater than 90 population doublings. Early population doubling level complementary DNA-1 (EPC-1, an age-dependent gene) expression in cells from the cloned animals was 3.5- to 5-fold higher than that in cells from age-matched (5 to 10 months old) controls. Southern blot and flow cytometric analyses indicated that the telomeres were also extended beyond those of newborn (<2 weeks old) and age-matched control animals. The ability to regenerate animals and cells may have important implications for medicine and the study of mammalian aging.

[Patient with recurrent gastrointestinal hemorrhage in acquired von Willebrand disease]. / Patientin mit rezidivierenden gastrointestinalen Blutungen bei akquiriertem Morbus von Willebrand.

A 61-year-old female without prior history of bleeding experienced several symptomatic episodes of gastrointestinal bleeding in the last few years. A source of gastrointestinal bleeding could not be found although several tests of occult blood in the stool were positive. Diagnostically, a significant deficiency of von Willebrand factor could be found due to a shortened half-life of the von Willebrand factor. This acquired von Willebrand factor deficiency most likely can be explained by an immunological mechanism (e.g. antibodies against von Willebrand factor). Neither an immunosuppressive therapy with steroids nor cyclophosphamide could normalize the von Willebrand factor and the bleeding continued. Only tranexamic acid (inhibiting fibrinolysis) could at last stop the symptomatic episodes of gastrointestinal bleeding despite the fact of decreasing von Willebrand factor.

Ifosfamide-induced stomatocytosis and mesna-induced echinocytosis: influence on biorheological properties of blood.

Ifosfamide is an alkylating agent which has poorly understood toxic side effects such as encephalopathy. We hypothesized that ifosfamide and concomitantly applied mesna could have an influence on the flow properties of blood, and thus carried out an in vitro study. Whole blood was incubated in vitro with increasing concentrations of ifosfamide (0-50 mg/ml), mesna (0-20 mg/ml) and combinations thereof. Chloroacetaldehyde, a major metabolite of ifosfamide, was also studied (0-5 mmol/l). Ifosfamide led to a dose-dependent stomatocytic shape transformation and mesna to an echinocytic shape transformation of erythrocytes. These shape changes were reversible upon removal of the causing agent. Both shape changes increased whole blood viscosity. Erythrocyte aggregation was decreased by both drugs at high concentration. Erythrocyte deformability, as measured with the transit time through 5-microm pores, was decreased by mesna and remained unaffected by ifosfamide. These effects were seen at concentrations which may be reached in vivo at the infusion site of the drugs into a vein and in the urinary tract. We conclude that ifosfamide and mesna interact with the lipid bilayer of the cell membrane, which may contribute to the toxicity of the compounds.

The anti-neoplastic drug 5-fluorouracil produces echinocytosis and affects blood rheology.

The anti-neoplastic agent 5-fluorouracil (5-FU) in high therapeutic doses can induce angina pectoris and myocardial infarction. The pathophysiological mechanism of this side-effect has not yet been elucidated. We analysed the influence of 5-FU on blood rheology in vitro. Whole blood, blood cell suspensions and plasma were incubated with increasing concentrations of 5-FU (final concentrations 0, 0.08, 0.4, 2, 10 and 25 mg/ml 5-FU) at 37 degrees C. Erythrocyte morphology was analysed after fixation with glutaraldehyde. Viscosity was measured at high and low shear rates (94 and 0.1 s[-1]). Erythrocyte aggregation and the cell transit times of erythrocytes through 5 microm pores and polymorphonuclear leucocytes through 8 microm pores were determined. 5-FU induced a dose-dependent formation of echinocytes within minutes and was reversible upon removal of 5-FU, which reflected a preferential intercalation of the drug in the outer hemileaflet of the cell membrane. High shear blood viscosity was increased at the highest 5-FU concentration (148 +/- 12%), and at low shear rate a dose-dependent decrease was found (0 mg/ml: 100%, 0.08 mg/ml: 87 +/- 10%, 0.4 mg/ml: 80 +/- 19%, 2 mg/ml: 70 +/- 15%, 10 mg/ml: 40 +/- 19%, 25 mg/ml: 33 +/- 5%). Erythrocyte aggregation was decreased by the 5-FU-induced echinocytosis. The transit time of erythrocytes through narrow pores was increased in a dose-dependent manner by 5-FU, whereas the transit time of polymorphonuclear leucocytes was initially decreased at 10 mg/ml and returned to control after 60 min incubation. We conclude that 5-FU interacts with the cell membrane, induces echinocytosis and vesiculation and affects blood rheology in several ways which may contribute to cardiovascular complications.

No influence of echinocytic shape transformation of erythrocytes on phagocytosis by autologous monocytes in vitro.

The influence of echinocytosis induced by ATP depletion on erythrophagocytosis was analysed. Monocytes were isolated from peripheral blood of healthy, rhesus-positive volunteers and cultured for a total time of 6 h at 37 degrees C with 5% CO2. Erythrocytes from the same donor, either fresh (discocytes) or incubated at 37 degrees C for 48 h (spheroechinocytes) were added. Phagocytosis was quantified by measuring the chemiluminescence of the ingested heme. The rate of phagocytosis was 0.03 +/- 0.15 discocytes per phagocytic cell and 0.01 +/- 0.08 spheroechinocytes per phagocytic cell. Autologous serum did not affect this rate. The addition of anti-RhD immunoglobulins, however, increased phagocytosis drastically for discocytes and spheroechinocytes (4.22 +/- 2.09 and 5.24 +/- 2.58 erythrocytes per phagocytic cell, respectively). It is concluded that spheroechinocytosis induced by metabolic depletion, certainly one of the most severe forms of erythrocyte shape abnormalities, does not stimulate monocytes to remove these cells.