Comparison of the immunomodulatory effect of single MSC batches versus pooled MSC products.

Severe corticosteroid-refractory graft-versus-host-disease (GVHD) is a major non-relapse cause of mortality and morbidity after an allogeneic hematopoietic stem cell transplantation (allo-HSCT). One of the most promising treatment options is using advanced therapy medicinal products based on mesenchymal stem cells (MSCs) immunomodulation ability. The protocols of MSC application differ in many parameters including a source of MSC, a dose, a number of doses or way of preparation of the medicinal product. The process is limited by the need for laborious and expensive manufacturing processes fraught with batch-to-batch variability. In our study, we compared the immunomodulatory effects of different MSC batches versus pooled MSC, specifically the influence on lymphocyte proliferation, the metabolic activity, and the expression of activation markers on T cells. Our goal was to determine whether the effect depends on donor-to-donor heterogeneity and if pooling of MSCs could increase their immunomodulatory ability. All tested batches showed an immunomodulatory effect, with no significant differences between the groups. Our study suggests that immunosuppressive potential is comparable in single batches and pooled products, and the use of products got from individual donors is suitable to treat corticosteroid-refractory GVHD.

Growth of colorectal liver metastases is not accelerated by intraportal administration of stem cells after portal vein embolization.

INTRODUCTION: Future liver remnant volume (FLRV) is a crucial factor impacting resectability of colorectal liver metastases (CLM). In case of low FLRV, augmentation can be done by performing portal vein embolization (PVE). However, there is a risk of progression of CLM between PVE and resection. Intraportal application of autologous hematopoietic stem cells (HSC) is a possibility to accelerate the growth of FLRV. The effect of thus applied SC on CLM progression still remains unclear, though. METHODS: 63 patients underwent PVE between 2003 and 2015. In 20 patients a product with HSC was applied intraportally on the first day after PVE (PVE HSC group). HSC were gained from peripheral blood (10 patients) or bone marrow (10 patients). FLRV and volume of liver metastases (VLM) were evaluated by CT volumetry. The gained data were statistically evaluated in relation to the disease free interval (DFI), overall survival (OS), achievement of CLM resectability and progression of extrahepatic metastases. We compared the PVE HSC group with the group of patient undergoing simple PVE. RESULTS: No significant difference in FLRV and VLM growth was observed between the study groups. The percentage of exploratory laparotomies was smaller in the group with PVE and HSC application. Patients with simple PVE had a significantly higher incidence of extrahepatic metastases during follow up. We did not observe any significant differences in DFI and OS between the groups. CONCLUSION: HSC application did not accelerate CLM growth in comparison with PVE alone. PVE and HSC application had a higher percentage of patients undergoing liver resection and a lower incidence of extrahepatic metastases.

Development of Resistant GvHD in a Patient Treated with Nivolumab for Hodgkins Lymphoma Relapse after Allogeneic Unrelated Transplantation.

BACKGROUND: Allogeneic hematopoietic stem cell transplantation is one of the therapeutic options for patients with relapsed or refractory classic Hodgkins lymphoma (cHL). In the case of dis-ease relapse after transplant, other treatment options are still limited (for example donor lymphocyte infusion, and chemother-apy with brentuximab, bendamustine, or other agents) with uncertain outcomes in terms of patient tolerance and long-term dis-ease remission. One way to achieve remission is administration of the PD-1 inhibitor nivolumab, a PD-1 checkpoint inhibitor. Nivolumab is also indicated for the treatment of cHL relapses after autologous hematopoietic stem cell transplantation. Since September 2018, nivolumab has been approved by the State Institute for Drug Control in the Czech Republic for treatment of cHL autologous hematopoietic stem cell transplantation relapse; however, treatment with nivolumab is accompanied by an increased risk of develop-ing fatal, acute graft-versus-host dis-ease. CASE: The article describes the development of resistant acute graft-versus-host disease in a patient who had received allogeneic-unrelated transplantation and nivolumab treatment for Hodgkins lymphoma relapse. CONCLUSION: Our case study, as well as the literature review, demonstrates the excellent efficacy of PD-1 inhibitors, but also cautions against the administration of these agents in patients follow-ing allogeneic hematopoietic stem cell transplantation. Administration of nivolumab to these patients should be done on a strictly individual basis in the context of known risks, and consideration should be given to other treatment options. Key words Hodgkins lymphoma -  PD-1 inhibitor -  nivolumab -  GvHD -  transplantation.

Precise determination of primary cytogenetic abnormalities provides added value for stratification of chronic lymphocytic leukemia patients.

Cytogenetic analysis has become a standard procedure in the management of newly diagnosed chronic lymphocytic leukemia patients. Prognostic information is reported based on the presence of certain abnormalities and karyotype complexity after conventional karyotyping and/or fluorescence in situ hybridization (FISH). The information on cytogenetic abnormalities occurring in isolation is robust; however, the performance of patients with two or more cytogenetic abnormalities is heterogeneous and information is scarce. This retrospective study analyzed whether information on the precise determination of primary cytogenetic abnormalities can have some added value in terms of risk stratification in chronic lymphocytic leukemia (CLL) patients. The study cohort was 121 patients without the need to start treatment for CLL immediately after diagnosis but had completed initial cytogenetic analysis. Results from conventional karyotyping after stimulation of CLL cells and FISH analysis were combined. Risk stratification based purely on the determination of primary cytogenetic abnormalities was effective in CLL patients, with comparable results in stratification based on the presence of certain abnormalities and karyotype complexity. It is recommended that information on suspected primary abnormalities is included in cytogenetic reports, especially in patients with two or more abnormalities, because this can provide valuable additional information.

Comparison of autologous hematopoietic cell transplantation performed in tandem and in disease relapse in multiple myeloma patients.

Multiple myeloma is a malignant hemato-oncological malignancy that affects up to 600 people in the Czech Republic every year. Treatment options are under constant improvement and the autologous hematopoietic cell transplantation (Tx) remains a part of treatment protocols. Despite modern drug administration, the autologous Tx keeps its irreplaceable position and when ensuring two autologous Tx, the studies confirm a survival time more than twice as long as in non-transplant patients. However, there are no standardized procedures specifying the period in between the transplantations in more detail. Within our group, we compared the total of 66 patients who were administered a double transplant. One group underwent both planned tandem autologous Tx within a median of six months and mostly achieved just partial remission (PR) and less after the first transplant and out of disease progression. The other group only underwent the second Tx within a median of up to 14 months during a progression period or disease relapse. Both groups were comparable as far as basic parameters are concerned (age, type of induction therapy and cytogenetic risk). A significantly better treatment free survival (TFX) and overall survival (OS) were observed in the group where tandem Tx was administered. TFS was 18 months and median OS was not reached for the group of patients who received tandem Tx, while TFS was 10 months (p=0.04) and median OS was 57 months (p=0.005) for those who received delayed second Tx. In the group of patients who received second Tx during relapse, we observed that TFS and OS were shorter in those with a higher paraprotein level, thus suggesting the potential role of paraprotein level as a prognostic marker. The TFS in the subgroup with a high initial level was 4 months vs. 11 months (p=0.0016) and OS 44 months vs. 65 months (p=0.03).

2DEP cytometry: distributed dielectrophoretic cytometry for live cell dielectric signature measurement on population level.

In this work, a novel force equilibrium method called distributed dielectrophoretic cytometry (2DEP cytometry) was developed. It uses a dielectrophoresis (DEP)-induced vertical translation of live cells in conjunction with particle image velocimetry (PIV) in order to measure probabilistic distribution of DEP forces acting on an entire cell population. The method is integrated in a microfluidic device. The bottom of the microfluidic channel is lined with an interdigitated electrode array. Cells passing through the micro-channel are acted on by sedimentation forces, while DEP forces either oppose sedimentation, support sedimentation, or neither, depending on the dielectric (DE) signatures of the cells. The heights at which cells stabilize correspond to their DE signature and are measured indirectly using PIV, which enables simultaneous and high-throughput collection of hundreds of single-cell responses in a single PIV frame. The system was validated using polystyrene micro-particles. Preliminary experimental data quantify the DE signatures of immortalized myelogenous leukemia cell lines K562 and KG1. We show DEP-induced cell translation along the parabolic velocity profile can be measured by PIV with sub-micron precision, enabling identification of individual cell DE signatures. DE signatures of the selected cell lines are distinguishable. Throughput of the method enables measurement of DE signatures at 10 different frequencies in almost real time.

Beneficial effects of mesenchymal stem cells on adult porcine cardiomyocytes in non-contact co-culture.

Mesenchymal stem cells (MSCs) have been reported to improve survival of cardiomyocytes (CMCs) and overall regeneration of cardiac tissue. Despite promising preclinical results, interactions of MSCs and CMCs, both direct and indirect, remain unclear. In this study, porcine bone marrow MSCs and freshly isolated porcine primary adult CMCs were used for non-contact co-culture experiments. Morphology, viability and functional parameters of CMCs were measured over time and compared between CMCs cultured alone and CMCs co-cultured with MSCs. In non-contact co-culture, MSCs improved survival of CMCs. CMCs co-cultured with MSCs maintained CMCs morphology and viability in significantly higher percentage than CMCs cultured alone. In viable CMCs, mitochondrial respiration was preserved in both CMCs cultured alone and in CMCs co-cultured with MSCs. Comparison of cellular contractility and calcium handling, measured in single CMCs, revealed no significant differences between viable CMCs from co-culture and CMCs cultured alone. In conclusion, non-contact co-culture of porcine MSCs and CMCs improved survival of CMCs with a sufficient preservation of functional and mitochondrial parameters.

The Quality Control of Mesenchymal Stromal Cells by in Vitro Testing of Their Immunomodulatory Effect on Allogeneic Lymphocytes.

Mesenchymal stromal cells (MSC) represent a promising treatment of graft-versus-host disease (GVHD) in patients after allogeneic haematopoietic stem cell transplantation. We performed co-cultivation experiments with non-specifically stimulated lymphocytes to characterize the immunosuppressive activity of MSC. MSC influenced expression of some activation antigens. CD25 expression was lower with MSC and reached 55.2 % vs. 84.9 % (CD4+, P = 0.0006) and 38.8 % vs. 86.6 % (CD8+, P = 0.0003) on day +4. Conversely, CD69 antigen expression remained higher with MSC (73.3 % vs. 56.8 %, P = 0.0009; 59.5 % vs. 49.7 %, ns) and its down-regulation along with the culture time was less pronounced. MSC reduced proliferation of the stimulated lymphocytes. The cell percentages detected in daughter generations were decreased (32.82 % vs. 10.68 % in generation 4, P = 0.0004 and 29.85 % vs. 10.09 % in generation 5, P = 0.0008), resulting in a lower proliferation index with MSC (1.84 vs. 3.65, P < 0.0001). The addition of MSC affected expression of some cytokines. Production of pro-inflammatory cytokines was decreased: IL-6 (19.5 vs. 16.3 MFI; P < 0.0001 in CD3+/CD4+ and 14.5 vs. 13.2 MFI; P = 0.0128 in CD3+/CD8+), IFN-γ (13.5 vs. 12.0 MFI; P = 0.0096 in CD3+/CD4+). Expression of anti-inflammatory IL-10 was only slightly increased after the addition of MSC (ns). The analysis confirmed the immunomodulatory activity of MSC. The functional tests have proved to be an important part of the quality control of the advanced therapy cellular product intended for GVHD treatment. Future research should focus on the interaction between MSC and the patient immune environment more closely.

Prognostic factors to predict outcome of reduced intensity allogeneic haematopoietic cell transplantation for chronic lymphocytic leukemia.

Despite advances in immunochemotherapy CLL remains an incurable disease.. Allogeneic haematopoietic cell transplantation (HCT) has proven curative potential with ability to overcome adverse prognostic factors, however due to its toxicity it is generally perceived as the last option. We performed retrospective study to explore the outcomes and possible determinants of survival in the unselected consecutive cohort of 68 CLL patients (median age 59 years) receiving reduced intensity HCT as a part of salvage therapy in 2 Czech centers. The median interval from diagnosis to HCT was 69 months with median 3 of prior regimens, all patients were refractory to purine analogues. 49% of patients were transplanted with advanced (i.e. refractory or progressive disease or CR/PR>3), 38% had high risk cytogenetics. With median follow-up of 35 months the 3-year Kaplan-Meier survival probability for OS and PFS were 39% and 26%, respectively. Altogether 18 patients (26%) have relapsed or progressed. During the follow-up 41 patients died, 32 (78%) of transplant related factors (NRM), the others of relapse or disease progression.Univariate analysis failed to identify any clinical and pre- or post-transplant variables having clear prognostic significance for OS or PFS. The marginal OS advantage favoring HCT performed recently was detected (3-year OS: 31% for HCT until 2006 and 47% thereafter, p=0.0923). In multivariable hazards model only the female donors were associated with shorter OS (HR 2.278, p=0.016) whereas transplanted T-cell> 2.75x108/kg predicted inferior PFS(HR 1.957, p=0.035). No prognostic impact of donor type, age of donor and recipient, HLA mismatch, disease status pre-HCT, number of previous therapy lines, interval from dg. to HCT and number of transplanted hematopoietic cells was found. Our findings support the conclusion that alloHCT is able to overcome well known negative cytogenetic prognostic factors and that preferring male to female donors could be beneficial.

[Treatment of Chronic Lymphocytic Leukemia with TP53 Aberrations]. / Lécba chronické lymfocytární leukemie s aberací TP53.

Patients with chronic lymphocytic leukemia with deletion of the short arm of chromosome 17 (17p -) or mutation of the TP53 gene have significantly worse prognosis with a higher risk of progression to symptomatic disease, worse and shorter responses to chemo immunotherapy, and more frequent occurrence of Richters syndrome. TP53 deletion/ mutation is currently the only genetic abnormality that independently predicts response to treatment and also affects the choice of therapeutic approach in chronic lymphocytic leukemia. This work summarizes treatment options available for this poor prognosis variant of chronic lymphocytic leukemia. Traditional chemo immunotherapy (e. g. FCR) does not offer longterm disease control, and patients with TP53 deletion/ mutation were usually considered to undergo allogeneic bone marrow transplantation. New molecules from the group of BCR inhibitors or BCL2 antagonists achieve excellent efficacy in chronic lymphocytic leukemia with del17p even in relapsed/ refractory (R/ R) cases, with a higher percentage of responses and prolonged survival without progression. Clinical trials are ongoing to determine optimal therapeutic approach and to induce longterm remission of the disease. The new molecules change algorithms for treatment of patients with TP53 aberration, including indication for allogeneic transplantation. Especially younger patients should be consulted in centers of intensive hematological care to consider their inclusion into clinical trials testing new molecules or to indicate allogeneic transplantation at the optimal time.

Allogeneic stem cell transplantation can improve outcome of AML patients without complete cytogenetic response after induction and consolidation treatment.

UNLABELLED: Our retrospective analysis was performed on 376 consecutive patients diagnosed with AML. A total of 256 (68%) were treated with standard "7+3" induction and high-dose cytarabine and mitoxantrone containing "4+3" consolidation/intensification regimens. Our study focused on patients with presumably very poor prognosis - patients, who did not achieve complete cytogenetic remission (CRc). Twenty-five AML patients without CRc were further analysed for clinical and laboratory parameters. Firstly, the subgroups with or without morphologic CR were compared. Similar cytogenetic abnormalities were observed in both with myelodysplasia related changes being the most common. Complex karyotype with deletion of 5q constituted approximately a third of all karyotypes in both subgroups. There were 1 patient with intermediate risk cytogenetics in the subgroup without morphologic CR and 5 patients in the subgroup with morphologic CR. Interestingly, in 4/25 patients subclones were diminished by the chemotherapy treatment, however cytogenetically less advanced clones proliferated. Secondly, transplanted or nontransplanted patients were analysed. Allogeneic stem cell transplantation (allo-SCT) was found to be the only curative treatment for patients without CRc after 7+3 and 4+3 regimens. In our cohort, 40% of the patients, who underwent allo-SCT, are alive. Importantly, 67% of the patients, who died after allo-SCT, died of causes unrelated to progression of AML. Nonrelapse mortality is therefore one of the fields where survival could be further improved. KEYWORDS: acute myeloid leukaemia, complete cytogenetic remission, cytogenetic abnormalities, stem cell transplantation, nonrelapse mortality.

Discontinuation of tyrosine kinase inhibitors in chronic myeloid leukemia patients - worldwide battlefield.

UNLABELLED: Advances in understanding the pathogenesis of chronic myeloid leukemia (CML) and implementation of the therapy with tyrosine kinase inhibitors (TKI) could be considered as a prototype of successful fight against cancer. However, for an optimally responding patient it is recommended to follow the TKI therapy indefinitely. The question about the possibility of safe TKI treatment discontinuation in certain clinical situations was raised and is currently under close investigation worldwide. Currently, imatinib discontinuation trails have shown that about 60% of eligible patients experienced molecular recurrence within 6 months of treatment discontinuation, while the remaining 40% remained in defined deep molecular response throughout the duration of mostly two years follow-up. Interestingly, retreatment with the same TKI or another TKI was successful in the vast majority of patients demonstrating molecular recurrence of the disease. These findings support the concept of safe TKI treatment discontinuation and its usefulness for a specific subset of CML patients. However, recent data are not sufficient for TKI discontinuation attempts outside clinical trials yet. Because of the high risk of potentially problematic molecular recurrences of the pathological clones, the key question is to find the right predictive marker of TKI discontinuation success, however it stays unsolved yet. This minireview brings a concise summary of this hot topic with a realistic view from clinical routine. KEYWORDS: tyrosine kinase inhibitors, imatinib, discontinuation, chronic myeloid leukemia.

[Ultra high risk chronic lymphocytic leukemia -  characteristics and treatment options]. / Vysoce riziková chronická lymfocytární leukemie -  charakteristika a lécebné moznosti.

Chronic lymphocytic leukemia (CLL) is the most common form adult leukemia in western world. The disease is typically cha-racterized by heterogeneous clinical behavior ranging from indolent course to rapidly progressive disease. Using clinical and bio-logical factors we can stratify patients with CLL and prospectively identify those who can be expected unfavorable course. There is a special group known as ultra highrisk chronic lymphocytic leukemia with an extremely poor prognosis. These are about 10- 15% of all patients with CLL. They do not respond to standard treatment and their survival is short with a median of 2- 3 years. For highrisk patients are considered: patients with a proven TP53 defect, refractory to purine analogues or with early relapse after chemoimmunotherapy based on fludarabine ( 24 months). While the standard 1st line treatment protocol in younger patients is chemoimmunotherapy FCR, in case of ultra highrisk CLL other methods like allogeneic hematopoietic stem cell transplantation or clinical trials testing the new drugs should be considered. In particular, allogeneic hematopoietic stem cell transplantation is a very promising treatment modality that offers longterm disease control and cure regardless of the unfavorable CLL subtype. Transplantation treatment should be therefore considered in all younger patients with ultra highrisk CLL, who should be without delay referred to a center for intensive hematological treatment.

Portal vein embolization and application of autologous stem cells in patients with primary unresectable liver tumours.

BACKGROUND: Only 15-20 % of patients with liver tumours can undergo radical surgery. Insufficient future liver remnant volume (FLRV) is one of the main causes of tumours unresectability. Portal vein embolization (PVE) together with administration of haematopoietic stem cells (HSC) may expand the operability of primary unresectable liver tumours. METHODS: In this pilot study, the authors reported on five patients (1 hepatocellular carcinoma, 4 colorectal cancer metastases) with FLRV <30 %, who underwent PVE on the side of the tumour with a subsequent application of HSC to the non-embolized branch of portal vein. RESULTS: PVE with HSC application was without any complications. In three patients, a sufficient increase of FLRV occurred within 2-4 weeks followed by a liver resection. All patients were between 5-12 months after the surgery in good condition; one of them was diagnosed with pulmonary metastasis after nine months that was successfully treated with laser metastasectomy. In one patient with hepatocellular carcinoma, an increase of FLRV and progression of the tumour in the liver occurred following the PVE with administration of HSC and the patient was treated only symptomatically. Despite an adequate increase of FLRV, severe intraabdominal adhesions hampered liver resection in one patient. CONCLUSIONS: Combination of PVE with HSC administration appeared to be a promising method that stimulated growth of FLRV with a subsequent possibility of an early radical liver resection. The issue is a danger of tumour progression in the liver parenchyma following the PVE with HSC. The current randomized study should answer these questions (Tab. 1, Fig. 4, Ref. 38).

[Spontaneous remission of acute myeloid leukemia - a single center case reports]. / Spontánní remise akutní myeloidní leukemie - klinické prípady jednoho centra.

BACKGROUND: Acute myeloid leukemia is a malignant disease characterized by clonal expansion of immature hematopoietic cells - myeloblasts - in the bone marrow. Intensive chemotherapy treatment in elderly patients (over 60) has disappointing results. In these patients, conservative treatment, including compensation of deficiency of red blood cells and platelets by transfusions and treatment of infectious complications is recommended. Also, relatively new treatment with hypometyl agents (azacytidine, decitabine) could be used. DESIGN: The idea of this article is to present a spontaneous remission phenomenon, which has not been published in Czech literature yet. In this article, we present 2 case studies of our patients who were diagnosed with acute myeloid leukemia, were not treated with chemotherapy and spontaneously reached remission of acute myeloid leukemia. CONCLUSION: The mechanisms of the spontaneous remission remain unclear, but we assume positive effect of a severe systemic infection or previous applications of blood transfusions. Antibodies in blood transfusions and a strong immune response to sepsis may have contributed to spontaneous remission.

CD34⁺ cell content in unrelated allogeneic peripheral blood stem cell grafts transported internationally. Does the inter-laboratory variability affect comparability of graft quality data?

BACKGROUND: Increasing numbers of unrelated hematopoietic stem cell grafts are transported internationally and evaluated concurrently in different laboratories. The graft quality assessment using the CD34(+) enumeration could be influenced by inter-laboratory variability. METHODS: We retrospectively analyzed the content of CD34(+) cells in 154 consecutive collections being performed in different transplant centers during two periods (2003-2004, 2007-2010). All samples were tested twice in our own and partner laboratories. CD34(+) percentage and absolute number were compared. RESULTS: The percentage and the total CD34(+) content correlated well in both observed periods (CD34(+)%: r=0.899 and r=0.922; CD34(+)×10(8)/kg: r=0.966 and r=0.880; p<0.0001). Median CD34(+) percentages obtained in our centre in comparison with other laboratories were 0.54% vs. 0.46% in 2003-2004 and 0.69% vs. 0.70% in 2007-2010 period. The degree of laboratory compliance was affected by the laboratory identity. CD34(+) percentage reported by one laboratory and CD34(+)×10(8)/kg reported by three from twelve laboratories lacked statistically significant correlation with our own data. CONCLUSIONS: The study documented that results of CD34(+) cell dose assessment of the same grafts reported by different transplant centers are comparable. The graft quality data and the CD34(+) enumeration possess a limited level of inter-laboratory variability.

Distribution of KIR genes in the population of unrelated individuals homozygous for ancestral haplotype AH8.1 (HLA-A1B8DR3).

Despite the independent segregation of genes encoding killer immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA), there is some evidence of some kind of co-evolution. Therefore, one could expect reduced KIR diversity within the HLA restricted population. A total of 41 unrelated individuals homozygous for ancestral HLA haplotype AH8.1 (HLA-A*0101-Cw*0701-B*0801-DRB1*0301-DQB1*0201) were genotyped for KIRs. Over all, 14 different genotypes were identified. The KIR genes and genotypes repertoire generally mirror the published frequencies in Caucasians. Except for KIR2DS4, all activating genes presented frequencies below 50%. KIR2DS5 was the least frequent among activating genes (17%), whereas KIR2DL5 (37%) among inhibitory ones. The most frequent (39%) was AA genotype. Twenty-two individuals (54%) had a copy of KIR haplotypes A and B (AB genotype), whereas three (7%) were homozygous for B (BB genotype). Nine of fourteen reported genotypes occurred only in one individual. Five genotypes were reported in less than twenty individuals worldwide and one genotype was reported so far only once. Conversely, the three most frequent genotypes account for 68% of all detected genotypes. The results show the unrestricted KIR diversity in this HLA uniform group and support the fact that the driving force for KIR evolution is not exclusively a major histocompatibility complex.

[Effect of growth factor on the phenotype of subpopulations and on the kinetics of CD34+ cells in the peripheral blood and in grafts of peripheral stem cells in patients with non-Hodgkin's lymphoma indicated for autologous peripheral blood stem cell transplantation]. / Vliv typu rustového faktoru na fenotyp subpopulací a kinetiku CD34+ bunek v periferní krvi a stepech periferních kmenových bunek pacientu s non-hodgkinskými lymfomy indikovaných k autologní transplantaci krvetvorby.

BACKGROUND: Peripheral blood stem cells are the preferred source for transplantation of hematopoiesis in patients with non-Hodgkin's lymphoma. Application of hematopoietic growth factors is a part of the mobilization chemotherapy regimen. Time overlap of the highest leukocyte and CD34+ cell count is required for optimal graft collection. Authors analyzed the effect of two growth factors (leridistim and filgrastim) on the kinetics and phenotype of CD34+ cells in patients with non-Hodgkin's lymphoma indicated for autologous peripheral blood stem cell transplantation. METHODS AND RESULTS: Authors analyzed phenotype of CD34+ cell subpopulations and their kinetics in peripheral blood and leukapheresis products by flow cytometry during mobilization and graft collection. Statistically significant differences in expression of lineage-committed antigens between growth factors were found (CD3, CD5--T-lineage, CD56 NK-lineage, CD20 for B-lineage, p < 0.05), as well as for lineage non-specific antigens (CD38, p < 0.05 and CD54, p < 0.01). The most significant divergence was observed between CD34+CD19+ subpopulations of leridistim and filgrastim stimulated blood and graft (p < 0.001). CONCLUSIONS: Expression of lineage-committed antigens on CD34+ subpopulations between two growth factors was statistically different. Kinetics of CD34+ cells during mobilization regimen with leridistim was not superior to filgrastim concerning the quality of graft.

Mobilization of peripheral blood stem cells in CLL patients after front-line fludarabine treatment.

Autologous peripheral blood stem cell transplantation is performed in an increasing number of chronic lymphocytic leukaemia (CLL) patients who are in the first remission following fludarabine treatment. There are contradictory data about the adverse impact of fludarabine on stem cell harvest. We analysed retrospectively mobilization results in 56 poor-risk CLL patients (median age: 56 years) who underwent first-line treatment with fludarabine and cyclophosphamide. The mobilization, consisting of cyclophosphamide 3 g/m(2) and granulocyte colony-stimulating factor (G-CSF) 10 microg/kg per day, was performed with a median of 77 days following the last fludarabine course. The target yield was >or=2.0x10(6) CD34+ cells/kg. The procedure was successful in 23 (41%) patients. A median of 3.3x10(6) CD34+ cells/kg was collected per patient. The successful mobilization was associated with a longer interval from the last chemotherapy (>2 months). The mobilization result was not influenced by the number of fludarabine cycles. No correlation was found in other parameters such as disease stage at diagnosis, disease status at stimulation or age. The poorly mobilized patients had significantly lower prestimulation blood counts (platelets, WBC and haemoglobin). Our data show that fludarabine does not generally prevent the stem cell mobilization; nevertheless, mechanisms related to the impact of fludarabine on stem cell harvest must be further investigated.