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Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal expansion of mature B-lymphocytes. Mutational status of the immunoglobulin variable heavy chain region (IGHV) gene stratifies CLL patients into two prognostic groups. We performed microarray analysis of CLL cells using the Agilent platform to detect the most important gene expression differences regarding IGHV status in CLL cells. We analyzed a cohort of 118 CLL patients with different IGHV mutational status and completely characterized all described prognostic markers using expression microarrays and quantitative real-time RT-PCR (reverse transcription PCR). We detected lymphocyte-activation gene 3 (LAG3) as a novel prognostic marker: LAG3 high expression in CLL cells correlates with unmutated IGHV (P < 0.0001) and reduced treatment-free survival (P = 0.0087). Furthermore, quantitative real-time RT-PCR analysis identified a gene-set (LAG3, LPL, ZAP70) whose overexpression is assigned to unmutated IGHV with 90% specificity (P < 0.0001). Moreover, high expression of tested gene-set and unmutated IGHV equally correlated with reduced treatment-free survival (P = 7.7 * 10(-11) vs. P = 1.8 * 10(-11)). Our results suggest that IGHV status can be precisely assessed using the expression analysis of LAG3, LPL, and ZAP70 genes. Expression data of tested markers provides a similar statistical concordance with treatment-free survival as that of the IGHV status itself. Our findings contribute to the elucidation of CLL pathogenesis and provide novel prognostic markers for possible application in routine diagnostics.

Acute promyelocytic leukemia (APL) with atypical breakpoints in the promyelocytic leukemia (PML) and retinoic acid receptor-alpha (RARA) genes represents a rare leukemic event, which occurs preferentially in patients with variant types of the PML/RARA fusion gene. Here we report on a patient with APL with a unique PML/RARA fusion transcript that harbors a short type of this fusion gene, exhibiting unexpected results of standard PCR diagnostics. The detected transcript originates from fusion of PML exon 4 and a truncated form of transcription variant 2 of the RARA gene, with an additional 9 bp insertion. According to our knowledge, this differs from all previously described fusion transcripts.

Introduction: We present two years' experience in the treatment of adult acute lymphoblastic leukemia (ALL) according to the German GMALL 07/2003 study protocol at CELL (Czech leukemia study group--for life) hematological centers in the Czech Republic., Methods: A total number of 37 patients were included in this analysis. We evaluated complete remission and molecular remission rate, incidence of relapse, patients' status at the end of the follow-up period, incidence of chemotherapy-related adverse events and causes of death. A statistical analysis of risk factors affecting survival was carried out., Results: Complete remission was achieved in 36 (97%) patients and molecular remission in 16 (62%) of 26 evaluable patients. Disease relapse occurred in 5 (14%) patients. At the end of the follow-up period with a median of 261 days, 28 (76%) patients were alive in complete remission, one (3%) with relapsed disease and 8 (22%) dead. Treatment toxicity resulted in death in 5 cases, relapse or progression of ALL in 3 patients. Adverse events most often followed consolidation I, induction phase I, consolidation II and induction phase II. Infectious complications in the context of febrile neutropenia, GIT mucositis and side effects of PEG-asparaginase were the most common adverse events observed. The toxicity of allogeneic transplantation was not unexpected, four (25%) patients died after transplantation. Two-year progression-free and overall survival were 66% and 70%, respectively. High risk ALL, age over 35 years, CNS infiltration, disease relapse and permanent minimal residual disease were identified as the major adverse prognostic risk factors. Practical experiences and possible pitfalls of the protocol are described in the discussion., Conclusion: Our initial impression is promising. The treatment is feasible, the results very good and the toxicity acceptable. Patients at high risk should be headed to allogeneic transplantation, since the results ofconsolidation chemotherapy alone are very poor in this group. We believe that this study protocol could become a standard adult acute lymphoblastic leukemia treatment in the Czech Republic.

Background: Aplastic anemia (AA) is rarely described after a diagnosis of autoimmune disease (aID)., Aims: To assess the prevalence of prior aID in patients with AA recorded in the registry of the European Group for Blood and Marrow Transplantation (EBMT) and to evaluate treatment and outcome., Methods: 1,251 AA patients from 18 EBMT centers were assessed., Results: Fifty patients (4%) were eligible: 22 males and 28 females with a median age of 46 years at the diagnosis of aID and of 51 years at the diagnosis of AA. Information on the treatment of AA was available in 49 patients: 38 received only immunosuppressive therapy (IST), 8 patients underwent hematopoietic stem cell transplantation (HSCT) - 6 as first-line therapy and 2 after failure of IST - whilst 3 patients had a spontaneous recovery. After a median follow-up of 3.19 years, 32 patients were alive, including 7 of the 8 patients who underwent HSCT. Only 6 of 32 patients who were alive at the last follow-up were receiving IST for AA., Conclusions: Most cases of AA following aID benefitted from IST or HSCT if a matched donor was available. Further prospective investigation is needed to assess the effects of IST on the outcome of underlying aID., (Copyright 2010 S. Karger AG, Basel.)

Deletion of TP53 gene, under routine assessment by fluorescence in situ hybridization analysis, connects with the worst prognosis in chronic lymphocytic leukemia (CLL). The presence of isolated TP53 mutation (without deletion) is associated with reduced survival in CLL patients. It is unclear how these abnormalities are selected and what their mutual proportion is. We used methodologies with similar sensitivity for the detection of deletions (interphase fluorescence in situ hybridization) and mutations (yeast functional analysis) and analyzed a large consecutive series of 400 CLL patients; a subset of p53-wild-type cases (n = 132) was screened repeatedly during disease course. The most common type of TP53 inactivation, ie, mutation accompanied by deletion of the remaining allele, occurred in 42 patients (10.5%). Among additional defects, the frequency of the isolated TP53 mutation (n = 20; 5%) and the combination of 2 or more mutations on separate alleles (n = 5; 1.3%) greatly exceeded the sole deletion (n = 3; 0.8%). Twelve patients manifested defects during repeated investigation; in all circumstances the defects involved mutation and occurred after therapy. Monoallelic defects had a negative impact on survival and impaired in vitro response to fludarabine. Mutation analysis of the TP53 should be performed before each treatment initiation because novel defects may be selected by previous therapies.

The registry of patients treated with Thromboreductin (anagrelide) in the Czech Republic contains data concerning patients that have been treated using this drug since 2004. As of June 2009, the total number of patients was 549. The current analysis focused mainly on evaluation of anagrelide dosage needed to achieve a complete response in high-risk patients: reduction in platelet count to below 400 x 10(9)/l, which was also considered as reaching the therapeutic goal. The outcomes of the registry confirm that although anagrelide (Thromboreductin) is a very effective platelet-reducing agent, the administration of which is related to a low incidence of adverse effects and complications, the therapeutic goal is not achieved in all cases and or despite a quick treatment response, the therapeutic goal is achieved more slowly.

The role of autologous hematopoietic stem cell transplantation (autoHSCT) in adult acute lymphoblastic leukemia (ALL) is still unclear. We retrospectively analyzed the results of the autoHSCT and maintenance therapy, with oral 6-mercaptopurine and methotrexate, in comparison to conventional-dose chemotherapy in the consolidation treatment of adult ALL and lymphoblastic lymphoma (LBL). The patients, with HLA identical sibling donor, underwent allogeneic transplantation, while the others were treated with autoHSCT and maintenance therapy with oral 6-mercaptopurine and methotrexate, or by conventional-dose chemotherapy (patient's decision, no autologous hematopoietic stem cells harvest). Sixty consecutive adult patients (median age 35.2 years; range 17.3 to 70.7) with ALL (n = 52), LBL (n = 7), and acute biphenotypic leukemia (n = 1) were treated in our center from 1997 to 2007. Patients treated with chemotherapy alone (n = 35) had a shorter median progression-free survival (PFS) compared to patients who underwent autoHSCT plus maintenance therapy (n = 18), 8.4 and 46.8 months, respectively (p = 0.017). Patients treated with chemotherapy alone had also a shorter median overall survival (OS) compared to patients treated with autoHSCT: 13.0 vs. 46.8 months (p = 0.046). The differences remained statistically significant even after excluding patients with Ph positivity. We can conclude that, in our case, autoHSCT followed by maintenance chemotherapy is a good option for adult patients with ALL and, in standard-risk and high-risk patients, provides more favorable OS and PFS rates compared to patients treated by chemotherapy alone. However, we are aware of the fact that our analysis may have been distorted by the fact that the analysis is retrospective, that treatment with autoHSCT was based on patient's decision, and that chemotherapy may have been administered to negatively selected patients.

Unlabelled: Alemtuzumab in chronic lymphocytic leukemia treatment: retrospective analysis of outcome according to cytogenetics, Summary: Alemtuzumab is effective in B-cell chronic lymphocytic leukemia (CLL) with 17p deletion, which responds poorly to chemotherapeutic agents. Our retrospective study evaluated the benefit of alemtuzumab monotherapy in unselected patients with advanced CLL, categorized by cytogenetic profile. Data were collected from 74 consecutive who had received alemtuzumab. Median of previous therapies was 2. The incidence of cytogenetic abnormalities was: trisomy 12, 10%; 13q deletion, 13%; 11q deletion 25%; 17p deletion, 26%; none of these, 26%. The overall response rate was 65% (11% complete remission, 54% partial remission) in the whole cohort. From start of alemtuzumab therapy, median progression-free survival was 217 days, median time to alternative treatment was 287 days, and median overall survival was 999 days in the total cohort, respectively. Alemtuzumab was effective across all cytogenetic categories evaluated. There were no statistically significant differences between subgroups in the level of efficacy.

Objective: Our objective was to determine the value of frequent minimal residual disease (MRD) monitoring in acute myeloid leukemia (AML) as a robust marker of impending relapse, and whether treatment benefits patients during the MRD-positive phase of their disease., Materials and Methods: Frequent MRD monitoring was performed in all AML treatment phases using real-time quantitative polymerase chain reaction for fusion transcripts (CBFB/MYH11; RUNX1/RUNX1T1 fusion transcripts of MLL gene) and for the Wilms' tumor (WT1) gene. A total of 2,664 samples, taken from 79 AML patients and 6 healthy volunteers, were examined. Presence of fusion gene was detected in 25 of 79 examined patients., Results: Vast correlation was discovered for fusion transcripts as well as for the WT1 gene between levels in bone marrow (BM), peripheral blood, CD34(+) BM cells, and CD34(-) BM cells. WT1 expression, however, was usually positive for cases showing fusion transcripts negativity and in healthy volunteers. Moreover, no universal value of the WT1 expression could unequivocally discriminate between remission and relapse. Therefore, detection of molecular relapses relied on fusion transcripts only and was characterized by strong expression in CD34(+) cells. Considering relapsed patients, duration from molecular to hematological relapse was 8 to 79 days (median: 25.5 days). Twelve patients were treated (chemotherapy, gemtuzumab ozogamicin, or immunomodulation after allogeneic transplantation) for 21 molecular relapses and 14 responses to treatment were observed., Conclusions: Frequent quantitative monitoring of fusion transcripts is useful for reliably predicting hematological relapse in AML patients. Treatment for molecular relapse of AML can be successful.

Objective: Corticosteroid-resistant graft-vs-host disease (GVHD) is difficult to manage and is associated with high morbidity and mortality. No standard treatment exists. We have previously seen good results with pulse cyclophosphamide (Cy) in the treatment of liver GVHD in contrast to gastrointestinal GVHD, and here we report results of pulse Cy protocol in the treatment of steroid-refractory hepatitic variant of liver GVHD, with no association to the gut., Materials and Methods: Cy was infused at a dose of 1,000 mg/m(2). Twenty-nine cyclophosphamide administrations were given to 21 patients. Median time of GVHD onset and Cy administration after transplantation, or donor lymphocyte infusion, were 58 and 69 days, respectively., Results: Eleven patients (52%) achieved complete remission and 6 patients (29%) achieved partial remission. Four patients (19%) did not respond, however, their condition stabilized and, upon additional therapy, three achieved partial remission and one complete remission. Overall survival of all 21 patients is 86%, with median and maximal follow-up of 33 and 81 months, respectively. Toxicity was mild and easily manageable without influencing chimerism or disease status., Conclusions: Pulse Cy seems to be an effective treatment for steroid-refractory hepatitic variant of liver GVHD with a good toxicity profile, which may favor its use instead of drugs with more pronounced immunosuppressive effects.

Objective: To summarize available data concerning hematologic malignancies in pregnancy., Design: Review article., Setting: Department of Obstetrics and Gynekology, Fakulty of Medicine, Masaryk University and University Hospital Brno., Methods: Compilation of published data from scientific literature., Conclusions: Cancer complicating pregnancy is a rare coexistence. The incidence is approximately 1 in 1,000 pregnancies. The most frequent hematologic malignant tumor is Hodgkin's lymphoma, leukemia is less frequent and myeloproliferative diseases complicating pregnancy are sporadic coexistence. Symptoms of these deseases are often nonspecific and disguised in pregnancy, then the diagnosis can be late. It is imperative that a multidisciplinary team involving hematooncologist and obstetrician (pediatric specialist) care for patient with hematologic malignancies. Cleary, every patient have to know whole prognosis and all risk factors of treatment. Optimum timing of delivery is after 36th week of pregnancy (when chemotherapy is ended more than two weeks ago). We prefer vaginal delivery to caesarean section.

Cell therapy of myocardial infarction (MI) is under clinical investigation, yet little is known about its underlying mechanism of function. Our aims were to induce a sub-lethal myocardial infarction in a rabbit, to evaluate the abilities of labeled bone marrow mononuclear cells to migrate from the vessel bed into extracellular space of the myocardium, and to evaluate the short-term distribution of cells in the damaged left ventricle. Sub-lethal myocardial infarction was induced in rabbits by ligation of the left coronary vessel branch (in vivo). The Langendorff heart perfusion model (ex vivo) was used in the next phase. The hearts subjected to MI induction were divided into 3 groups (P1-P3), and hearts without MI formed a control group (C). Nanoparticles-labeled bone marrow mononuclear cells were injected into coronary arteries via the aorta. Perfusion after application lasted 2 minutes in the P1 group, 10 minutes in the P2 and C groups, and 25 minutes in the P3 group. The myocardium of the left ventricle was examined histologically, and the numbers of labeled cells in vessels, myocardium, and combined were determined. The numbers of detected cells in the P1 and C groups were significantly lower than in the P2 and P3 groups. In the P2 and P3 groups, the numbers of cells found distally from the ligation were significantly higher than proximally from the ligation site. Bone marrow mononuclear cells labeled with iron oxide nanoparticles proved the ability to migrate in the myocardium interstitium with significantly higher affinity for the tissue damaged by infarction.

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease with a highly variable clinical course. A proportion of patients eventually progress to a higher stage of malignancy. A recent association has been observed between the presence of aberrant somatic hypermutations in leukemic cells (hypermutations occurring outside of the immunoglobulin locus) and the transformation to a diffuse large B-cell lymphoma or prolymphocytic leukemia. In this study, we report on the rarely observed blastic transformation in a CLL patient who had previously been shown to harbor aberrant somatic hypermutations in the TP53 tumor-suppressor gene (Mol Immunol 2008;45:1525-29). The enzyme responsible, the activation-induced cytidine deaminase, was still active within the transformation, as evidenced by the ongoing class-switch recombination of cytoplasmic immunoglobulins. The transformation was accompanied by a complete p53 inactivation, as well as complex karyotype changes including prominent amplification of MYCN oncogene. Our case-study supports the view that the aberrant somatic hypermutation is associated with transformation of CLL to a more aggressive malignancy.

Objectives: Abnormalities of the TP53 or ATM, cooperating tumor-suppressor genes, significantly worsen the treatment options for chronic lymphocytic leukemia (CLL) patients. Although the aberrations seem to be mutually exclusive in this leukemia, inactivation of the former gene leads to worse prognosis. We tested the in vitro sensitivity of the CLL samples with heterozygous ATM deletion to fludarabine and combination of fludarabine and rituximab; the responses were compared with the TP53-abnormal and wild-type (wt) cells to delimitate relative significance of ATM deletion., Methods: In vitro analysis was performed on fifty-nine characterized CLL samples using viability assay WST-1. Western blot and real-time RT-PCR were used to monitor the activation of the ATM/p53 pathway., Results and Conclusions: At the clinically relevant concentration of fludarabine, TP53-abnormal samples exhibited markedly higher resistance to fludarabine than the remaining CLL samples (P = 0.012); cohort with ATM deletion was not more resistant than wt cells. A similar induction of the p53 protein and its downstream target genes PUMA and BAX in ATM-deleted and wt cells confirmed that the former subgroup has preserved a critical pro-apoptotic response. Proportions of the samples, which had been sensitized to fludarabine by rituximab pretreatment, were insignificantly lower (P = 0.22) in the TP53-abnormal and ATM-deleted subgroups compared to the wt cases (30%; 29%; 50%, respectively). The presence of ATM (11q22-23) deletion in the CLL cells should not be considered an indication of resistance to fludarabine or its combination with rituximab.

cDNA microarray technology is widely used in various biological and medical disciplines to determine gene expression profiles. Unfortunately, this technology requires a large quantity of input RNA. Since there is an increasing need for more precise analyses of defined cell subpopulations with low cell counts, working protocols using a minimal number of input cells are required. Optimal RNA isolation and its accurate amplification are crucial to the success of these protocols. The HL-60 cell line was used in the search for a suitable protocol that can be used for clinical samples of CD34+ haematopoietic cells obtained from bone marrow. The goal was to discover the best method for isolating and amplifying RNA from a small number of cells. Our evaluation of various methods and kits available in the market revealed that the combination of RNAqueous Kit for RNA isolation and the SenseAmp Plus Kit for one-round RNA amplification produced the best results. This article presents a verified protocol describing a reliable and reproducible method for obtaining enough input RNA for microarray experiments when the number of cells is limited.

Objectives: Intracoronary cell transplantation during catheter balloon inflations may be associated with adverse events. We studied the effectiveness of an alternative transplantation technique--intracoronary cell infusion., Methods: Fourteen pigs, which had survived acute myocardial infarction, were randomized into 2 treatment groups and 2 controls. Three days after infarction, 12 pigs underwent allogeneic intracoronary mononuclear bone marrow cell transplantation using either the standard technique (short-term cell injections during repeat balloon inflations, technique A, n = 6) or continuous intracoronary cell infusion without balloon inflations (technique B, n = 6). Implanted cells were stained with fluorescent dye. After transplantation, the pigs were euthanized and myocardial samples were analyzed by fluorescent microscopy., Results: The mean numbers of fluorescently labeled bone marrow cells in the infarction border zone, in the infarction mid-area and in the center of myocardial infarction were 84, 72 and 55 using technique A, and 29, 57 and 46 using technique B, respectively. The mean cell retention in the infarction border zone of 84 cells for technique A and 29 cells for technique B differed significantly (p = 0.034, two-tailed t test)., Conclusion: The continuous intracoronary cell infusion technique is a less efficient cell delivery technique as compared with the standard technique using repeat intracoronary balloon inflations., ((c) 2008 S. Karger AG, Basel.)

The registry of patients treated with Thromboreductine (anagrelid) in the contributing centres in the Czech Republic has been updated with data on the patients receiving this medication since 2004. The original purpose of the registry was to record responses to Thromboreductine therapy and adverse drug reactions in patients with essential thrombocytopenia. However, data on additional Ph negative myeloproliferations, as well as data on cytoreductive therapies other than exclusively that using Thromboreductine has also been recorded in the course of its compilation, including data on combined regimes. At present, the database contains data on 421 patients, and valid conclusions can be drawn if the level of data filling is enhanced. Evaluation has been currently focused on the analysis of the risk of development of clinical symptoms of thrombosis and on the standards of treatment from the viewpoint of the achieved treatment response. Analyses of data from the registry corroborate the special importance of the proof of JAK2 mutation, and of the test for factor V Leiden mutation, and of protein of S for the assessment of the risk of thromboembolic complications. The output of the analysis confirms that anagrelid is a very efficient thromboreductive agent the administration of which is associated with a low incidence of non-serious adverse effects (10.9%). However, in spite of a fast response to therapy, the therapeutic goal consisting in the reduction of the platelet count below 400 (or below 600) x 10(9)/l, i.e. the complete (or partial) treatment response, is relatively slow to achieve. This is likely to be due to lack of radical corrections in the dosage of the drug for different reasons.

MYH9-related disease (MYH9-RD) is a rare autosomal-dominant disorder caused by mutations in MYH9, the gene for the heavy chain of nonmuscle myosin IIA (NMMHC-IIA). All patients present from birth with macrothrombocytopenia, but in infancy or adult life, some of them develop sensorineural deafness, presenile cataracts, and/or progressive nephritis leading to end-stage renal failure. No consistent correlations have been identified between the 27 different MYH9 mutations identified so far and the variable clinical evolution of the disease. We have evaluated 108 consecutive MYH9-RD patients belonging to 50 unrelated pedigrees. The risk of noncongenital manifestations associated with different genotypes was estimated over time by event-free survival analysis. We demonstrated that all subjects with mutations in the motor domain of NMMHC-IIA present with severe thrombocytopenia and develop nephritis and deafness before the age of 40 years, while those with mutations in the tail domain have a much lower risk of noncongenital complications and significantly higher platelet counts. We also evaluated the clinical course of patients with mutations in the four most frequently affected residues of NMMHC-IIA (responsible for 70% of MYH9-RD cases). We concluded that mutations at residue 1933 do not induce kidney damage or cataracts and cause deafness only in the elderly, those in position 702 result in severe thrombocytopenia and produce nephritis and deafness at a juvenile age, while alterations at residue 1424 or 1841 result in intermediate clinical pictures. These findings are relevant not only to patients' clinical management but also to the elucidation of the pathogenesis of the disease.

Abnormalities of the TP53 gene are associated with a particularly severe prognosis in patients with B-cell chronic lymphocytic leukemia (B-CLL). This tumor-suppressor is mostly inactivated by the deletion of one and point mutation of the other allele and has not been previously shown to be hypermutated in B-CLL. We identified two patients whose lymphocytes showed repeatedly an extensive proportion of TP53 mutated cells by FASAY analysis (the yeast functional assay) and harbored various TP53 mutations, mostly single-base substitutions, in individual cells. The mutation targeting exhibited characteristic traits of the somatic hypermutation process. In the first patient (harboring the unmutated IgVH locus) a significant bias to point mutations at CG pairs (21/25; P=0.009), their remarkable preference for the RGYW/WRCY motives (28%) and the highest expression of the activation-induced cytidine deaminase (AID) mRNA among the 34 tested B-CLL samples. In the second patient no CG bias was observed but the targeting of point mutations into the RGYW/WRCY motives was even more prominent here (7/16; 44%). Moreover, six out of eight point mutations affecting AT pairs were localized in the WA/TW motives, which are also characteristic for the somatic hypermutations. This patient, who was IgVH-mutated, already did not express any significant amount of the AID transcript. Our findings add a new aspect to the mosaic of the p53 mutability in B-CLL.

Chronic myeloid leukemia has got an outstanding position among the other oncology diseases, particularly due to specific chromosomal abnormality called Philadelphia chromosome, its molecular consequence BCR/ABL fusion gene and its biologic product BCR/ABL tyrosinekinase, which is responsible for the leukemic transformation of the hematopoetic cells. We can see a real revolution in the treatment of the chronic myeloid leukemia in the last few years. The authors pay attention to the developement of the new drugs in the large historical consequences from the beginning in the 19th century (describing leukemia first time) to the introduction of the new small specific molecules into the clinical practice.

CD4+56+ hematodermic neoplasm or leukemia from early plasmocytoid dendritic cells type DC2 was recognized by WHO-EORTC classification of cutaneous lymphomas as a separate entity related to the plasmacytoid precursor dendritic cell (pDC). This diagnosis is based on expression of CD4 and CD56 antigens and absence of B, T or myeloid lineage markers. Immunohistochemistry and flow cytometry are the only methods, which allow identification of this disease, either in isolated skin lesions or in a leukemic form. Although the co-expression of CD4 and CD56 is rare and the number of described cases is low, this group bears similar characteristics in a clinical course of disease. It is a very aggressive leukemia/lymphoma, usually with primary skin involvement, in half of the cases infiltrating bone marrow or lymph nodes. Despite high rate of initial response to treatment, early and widespread relapses occur and patients die of disease progression. Although the physiological counterpart of tumour cells was identified, the origin of the disease is still discussed because of aberrant expression of cell markers. Optimal treatment is not known. However, this aggressive disease requires radical approach with intensive chemotherapy regimens, prophylaxis of CNS involvement and early indication of allogeneic bone marrow transplantation. Two case reports are described.

Since 2005, registers of patients treated with Thromboreductin (anagrelid) kept by some centres in the Czech Republic have been supplied with data concerning patients whose treatment with this preparation started in 2004. The purpose of the register is to record responses to therapy by Thromboreductin and adverse events in patients with essential thrombocytemia and other myeloproliferations, and to subsequently analyse the data. Another objective is to detect predisposition to clinical symptomatology and disease complications. Apart from thrombocyte count, additional risk factors are monitored. The database currently contains data for 336 patients. Initial analyses of data from the register point to the fact that anagrelid is a highly effective thromboreductive agent the administration of which is associated with relatively low incidence of adverse events (11.8 %) of mild and usually transitory nature. The therapeutic objective is attained at a relatively slow rate (according to overall stratification under 400 or under 600 x 10(9)/l thrombocytes), which is probably due to insufficient dose adjustment.

To assess the prognostic relevance of activating mutations of FLT3 gene on outcome of allogeneic transplantations in AML patients, we performed an analysis of all patients with FLT3 mutations registered in the Czech Acute Leukemia Clinical Register (ALERT) from 2003 till the end of 2005. Within the mentioned period 170 patients with AML of median age 56 years (23-77) were investigated for FLT3 mutation, within them 36 cases (21%) with FLT3 mutations (32 FLT3 ITD and 4 FLT3 D835) were found. Out of FLT3 ITD positive patients 13 had allogeneic transplantation, 20 patients with mutations of FLT3 were treated with chemotherapy without transplantation. Results of the treatment of these patients were compared with the results of the group of patients without FLT3 mutation, which was according to other characteristics identical with the group of patients with FLT3 mutations (n=134). Median overall survival (OS) was significantly shorter for patients with FLT3 ITD (34.8 weeks) than for those without FLT3 mutations (67.7 weeks; P=0.028). Median OS of patients with FLT3 ITD who had allogeneic transplantation was 42.5 weeks; median OS of patients with FLT3 ITD treated only with chemotherapy was 29.6 weeks (P=0.362). After allogeneic transplantation, median OS of FLT3 mutations negative patients was similar to FLT3 ITD positive patients (46.7 versus 42.5 weeks; P=0.443). Our results suggest that at present there is no strong evidence that FLT3 status alone should influence the decision to proceed to allogeneic transplantation in AML patients. Decision to proceed to alogeneic transplantation should not be based on the FLT3 status only, but it should also consider other prognostic factors.

A reduced-intensity conditioning allogeneic stem cell transplantation was given to 19 patients (aged 15-59 years) in the first chronic phase and one patient in the accelerated phase with chronic myeloid leukemia (CML) after a regimen consisting of fludarabine (Flu), busulfan (Bu) and ATG Fresenius. The median follow-up was 27 months. Until day +100, no transplant-related mortality was recorded. The incidence of acute and chronic graft-versus-host disease (GvHD) was 55 and 75%, respectively. Two patients (10%) died from GvHD. Fourteen (70%) patients achieved molecular remission. Additional post-transplant intervention (donor lymphocyte infusion, imatinib) was necessary, however, in 10 patients (50% of the patients; non-achievement of stable molecular remission or later relapses). The total direct cost of the transplantation treatment for all of the patients came to 1,572,880 euro. If the patients had been treated with imatinib and followed-up with the same time period as they were following a transplantation, the direct cost of the imatinib treatment would have been 2,005,117 euro. The transplantation treatment appears to be less expensive after approximately 2 years of follow-up. Flu+Bu+ATG is a low-toxicity regimen for patients with CML. However, a close follow-up is necessary and about 50% of the patients require further therapeutic intervention.

Anagrelide hydrochloride is an effective drug used in patients with ET and other myeloproliferative disorders with thrombocythemia to selectively decrease the number of thrombocytes. Indications for use of anagrelide were described in detail in Czech medical literature. Since 2005 data concerning treatment with anagrelide in some medical clinics have been collected in patient register showing course of treatment from 2004, when the medicament obtained marketing authorization from State Institute for Drug Control to be used in the treatment of thrombocythemia in myeloproliferative disorders. Aim of patient register is to monitor medical effect of anagrelide therapy and incidence of adverse effects in patients with ET and other myeloproliferative disorders and subsequent analysis of collected data. At the moment patient register contains data from 154 patients.

The rationale of the Czech Hematological Society guidelines for diagnosis and treatment of Philadelphia chromosome-negative myeloproliferative disorders with thrombocythemia (MPD-T) is reviewed. For diagnosis of MPD-T, the classification according to the World Health Organization or to the Rotterdam criteria is preferred because they distinguish true essential thrombocythemia from prefibrotic or early fibrotic idiopathic myelofibrosis and prepolycythemic polycythemia vera. The histopathology-based nosological distinction provided by these classifications yields valuable information on prognosis (including the risks of transition into secondary acute myeloid leukemia and myelofibrosis). Another serious complication in MPD-T is thrombosis (arterial or venous), the main risk factors of which are age, previous thrombosis, platelet counts 350 to 2,200 x 10 (9)/L (peak at approximately 900 x 10 (9)/L) and the presence of additional thrombophilic risk factors (hereditary thrombophilia, any hypercoagulable state, cardiovascular disease). The hemorrhagic risk starts increasing progressively at platelet counts > 1,000 x 10 (9)/L. Treatment should be stratified with respect to the thrombotic and hemorrhagic risks. In high-risk patients, thromboreductive therapy is warranted. All of the cytostatic drugs, including hydroxyurea, may be leukemogenic and should be given only to patients > 60 years old, whereas anagrelide or interferon alpha are preferred in younger individuals. In low-risk patients, antiaggregation therapy is sufficient, unless the platelet count exceeds 1,000 x 10 (9)/L, which is another indication for thromboreduction. Thrombopheresis is indicated in thrombocythemia > 2,000 x 10 (9)/L.

Background: Chronic lymphocytic leukemia is a heterogeneous disease manifesting with a variable clinical course. It is evident from many studies, that the division into two main prognostic categories is possible on the basis of mutation status of the immunoglobulin heavy-chain gene. The objective of our work was to identify a presence or absence of IgVH gene mutations in B-CLL patients which are monitored or treated on hematological clinics and to determine the presence of individual D and J, subgenes in malignant population of B-cells., Methods and Results: A nucleotide sequence of IgVH gene of neoplastic cells was analyzed by appropriate molecular-genetic methods. RNA/cDNA was collected from 358 patients and a spectrum of individual subgenes translocations was identified. Our results show that 56.3% of patients manifested an unmutated variable (VH) segment. It is expected from the published data that this group of patients will suffer from aggressive course of the disease and will exhibit a substantially shorter survival in comparison to patients possessing somatic hypermutations. An expanded population of leukemic B-cells showed increased occurrence of clones whose variable segments belong to three different families. VH3 alleles are the ones most frequently used. A frequency of unmutated alleles is prominently shifted into families with V I homology. The preferred "diversity and joining" segments are D3, D2 and JH 4 and JH 6., Conclusions: The analysis of heavy chain immunoglobulin gene after recombinant VH-D-J11 segments translocation belongs to a standard hematooncological investigation. The results are an important prognostic criterion for prediction of expected disease aggressivity and for a minimal residual disease monitoring.

Background: Rituximab is being used successfully in the treatment of patients with chronic B-cell lymphoproliferative diseases. The success of treatment by rituximab is influenced, among other factors, by the antigen density on tumor cells. Therefore, the authors analyzed and compared the densities of the CD20 antigen in patients with chronic lymphoproliferative diseases., Methods and Results: Previously untreated patients with B-chronic lymphocytic leukemia (B-CLL), mantle-cell lymphoma (MCL), and small-cell lymphocytic lymphoma (SCLL) were evaluated by flow cytometry. The control group consisted of blood donors. The CD20 density was measured on tumor cell populations in patients and on the B-lymphocytes of the control group. The density was expressed in MESE In the patients with B-CLL and SCLL, the CD20 density was low (25,300 vs. 36,100 MESF) and it was significantly lower than in donors (172,800 MESF; p<0.001). The difference between B-CLL and SCLL patients was not statistically significant. The density in MCL patients (196,300 MESF) was comparable to that of donors., Conclusions: We did not prove statistical different density of CD20 antigen in patients with SCLL when compared with B-CLL patients. High density in MCL patients may be helpful in differential diagnosis against B-CLL and

Corticosteroid-resistant graft-versus-host disease (GvHD) is difficult to manage and is associated with high morbidity and mortality. The purpose of our study was to evaluate the safety and efficacy of mycophenolate mofetil (MMF) as the salvage therapy for steroid-refractory GvHD. Twenty one patients (10 with acute GvHD and 11 with chronic GvHD) were studied retrospectively. Steroid-resistant GvHD was defined as acute or chronic GvHD not responding to a first-line regimen of cyclosporine A and corticosteroids in a dose equivalent to 2 mg/kg methylprednisolone for at least 7 days. MMF was added at a dose of 2 g daily, and corticosteroids were tapered. Thirteen (62%) of 21 patients responded to the treatment with MMF, including 6 (60%) of 10 patients with acute refractory GvHD and 7 (64%) of 11 patients with chronic refractory GvHD. The most common adverse effects were infectious complications (67%, 14 of 21 patients) and hematological toxicity (29%, 6 of 21 patients). Median duration of MMF administration was 6 months (range 1-27 months). Sixteen of 21 patients were alive after the median follow-up of 27 months (range 1-72 months) after the initiation of MMF therapy. All 16 surviving patients were in good clinical condition and in remission of their hematological malignancy. Five patients died--two of relapses of leukemia and three of refractory intestinal GvHD. These results suggest that MMF can be an effective treatment for some cases of steroid-refractory GvHD.

Corticosteroid-resistant GVHD is difficult to manage and is associated with high morbidity and mortality. Cyclophosphamide (Cy) is an established immunosuppressive and cytotoxic drug widely used as part of pretransplant conditioning regimens. In a retrospective study of 15 patients who had not responded to corticosteroids (nine with acute GVHD, three with GVHD after donor leukocyte infusion, and three progressive chronic GVHD), pulse Cy at a median dose of 1 g/m(2) was very effective in the treatment of skin (100% response), liver (70% response), and the oral cavity (100% response). Severe intestinal GVHD responded poorly. The toxicity profile was acceptable, with manageable, short-term myelosuppression in some patients. The risk of opportunistic infections, mixed chimerism, relapses, or post-transplant lymphoproliferative disease was not increased. Overall survival was 57%, with median and maximum follow-up of 9 and 37 months, respectively. The cost of the drug was negligible, especially when compared to monoclonal antibodies. Pulse Cy requires further investigation in corticosteroid-resistant GVHD.

Background and Aim: Amphotericin B (AmB) desoxycholate remains as one of the most efficacious agents currently available for the treatment of systemic fungal infections; however, amphotericin B colloidal dispersion (ABCD) has been developed because of AmB desoxycholate nephrotoxicity. The goal of our study was to compare the effect of administration of AmB desoxycholate and ABCD on renal functions and renal morphology in rats., Results: Amophotericin B desoxycholate as well as ABCD causes damage to renal tubuli and polyuria. Amophotericin desoxycholate causes considerably more severe damage to tubuli than ABCD, but the morphological damage to renal glomeruli is minimal in both formulas. In tubular cells, AmB desoxycholate causes severe damage to mitochondria, vacuolation of cytoplasm, and increased values of volume density of peroxisomes., Conclusion: None of these formulas causes a decrease in glomerular filtration in rats when animals are properly hydrated.

State-of-art of aggressive treatment of acute myeloid leukemia (AML) in patients older than 60 years is one of the least satisfactory topics of present-day hematology. This fact led us to ask the following questions: Does it make sense to administer aggressive treatment to older patients with AML? Could it be that we only complicate the rest of the life of these older patients with AML, by using aggressive treatment? Would they not benefit more from palliative or symptomatic therapy? What is the quality of life of older patients with AML like? Therefore, to try to answer these questions, we performed the next analysis. A retrospective analysis was performed including (without any selection) all consecutive patients over 60 years of age who were treated with AML in our centre from 1998 till 2003. We have analyzed data from 137 elderly patients who were diagnosed with AML (excluding acute promyelocytic leukemia). Median survival from diagnosis in the aggressive (curative) therapy group was 4 months, in palliative therapy group 2 months and in symptomatic therapy group 0.8 months. Patients receiving curative therapy spent in a hospital (in-patient stay) 70% (median) of their life after diagnosis of AML, patients receiving palliative treatment 64% (median) of their life after diagnosis, and patients receiving symptomatic treatment 100% (median), respectively. Only marginal advantage in the median overall survival is observed in the group of aggressively treated patients.

Amphotericin B deoxycholate (AmB) or Amphotericin B colloidal dispersion (ABCD) are used in clinics for the treatment of systemic fungal infections. The goal of our study was to compare the nephrotoxicity of these drugs in rat kidney. The effects of AmB and ABCD on the ultrastructure of the epithelium of renal tubules were studied and evaluated using morphometric and statistical methods. Two groups of 3 animals were established: group 1 was treated with AmB desoxycholate and group 2, to which ABCD was applied. AmB caused more than ABCD ultrastructural changes in the cytoplasm of the epithelial cells: damage to mitochondria, vacuolation of cytoplasm, and increased values of volume density of peroxisomes. However, we failed to observe significant differences in morphology and density of the other cell organelles. The proximal tubules seemed to be more sensitive to the nephrotoxic influence of both formulas than the distal tubules of rat kidney. Although, AmB causes more severe damage than ABCD, both drugs cause damage to renal tubuli.

The nuclear and nucleolar ultrastructure was studied by means of conventional transmission electron microscopy to provide more and complementary information on nucleolar changes accompanying the apoptotic process in leukaemic granulocytic precursors (HL-60 cells) produced by PDT without previous terminal differentiation. PDT induced the apoptotic process using BL irradiation and ALA as a precursor of the photosensitizer protoporphyrin IX. PDT produced marked changes of the nucleolar ultrastructure in apoptotic cells, such as reduction of the number and loss of fibrillar centres surrounding dense fibrillar components. Such nucleolar changes are known to reflect an alteration of nucleolar biosynthetic activities, which are believed to be located at the periphery of fibrillar centres. Some electron micrographs also indicated that fibrillar centres apparently migrated out from nucleolar bodies.

Previously, we showed that expression of myeloma-associated (proto)oncogene fibroblast growth factor receptor 3 (FGFR-3) is increased in white blood cells from patients with chronic myeloid leukemia (CML). The abnormal expression was returned back to the normal levels as soon as these patients reconstituted their hematopoiesis following transplantation of allogeneic peripheral blood stem cells. The aims of this study were: (1) to define population(s) of cells overexpressing FGFR-3, and (2) to determine the expression of FGFR-3 during the clinical course of the disease. We show that the vast majority of FGFR-3 transcripts as well as FGFR-3 protein arise from CD34+ BCR-ABL+ cells. Although increased levels of FGFR-3 were found in majority of late chronic phase patients treated with interferon alpha or hydroxyurea, the expression of FGFR-3 was always lowered following treatment with BCR-ABL tyrosine kinase inhibitor STI571. Compared to unstimulated cells, high levels of FGFR-3 were also identified in CD34+ cells from granulocyte colony-stimulating factor-mobilized blood stem cell harvests from healthy donors, suggesting a potential growth factor-dependent basis for elevated expression of FGFR-3 in CML. These findings have implications for the involvement of FGFR-3 in malignant hematopoiesis and depict FGFR-3 tyrosine kinase in CD34+ leukemic cells as a possible target for tyrosine kinase inhibitors.

Dendritic cells (DCs) are antigen-presenting cells that play a critical role in the induction of cytotoxic T-lymphocytes. An optimal method for the generation of DC for clinical use remains to be established. The aim of our study was to find an optimal cytokine combination for DC generation from peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) in serum-free conditions. Serial immunophenotyping enabled us to observe changes in DC content during the culture as well as the development of maturation and activation markers. As a source for DC culture, we used PBSC from patients with multiple myeloma after stem cell mobilization using cyclophosphamide and G-CSF, or PBMC from healthy donors without mobilization. The cells were cultured in a serum-free medium with different cytokine combinations including GM-CSF, TNF-alpha, Flt-3, CD40L, IFN-gamma, IL-1alpha, IL-6, PGE1, and IL-4. The cell cultures were evaluated by immunophenotyping. For PBMC, interleukin-12 assay was performed. For PBSC, the yield of DC as determined by CD83+ cell count ranged from 0. 6 x 10(5) to 30.1 x 10(4) (mean: 9.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated nucleated cells from apheresis. This yield corresponded to (0.3-19.1) x 10(5) (mean: 4.3 x 10(5)) per 1 x 10(6) of CD34+ cells in the apheresis products. For PBMC, the yield was (0.4-24.8) x 10(4) (mean: 2.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated mononuclear cells from venous blood. The cultured cells expressed the mature immunophenotype. No significant differences in cell yield or immunophenotype were detected when comparing different cytokine combinations.

Interleukin-2 (IL-2) is able to generate nonspecific cytotoxic effectors from hematopoietic precursors. We evaluated the feasibility and efficacy of chronic myeloid leukemia (CML) treatment with autologous hematopoietic stem cell transplantation (HSCT) using grafts cultured in IL-2 followed by immunotherapy with IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon (IFN)-alpha. Eight patients with CML were enrolled: five in an accelerated phase and three in a chronic phase. They received peripheral blood stem cells (PBSC) or bone marrow (BM) cultured in a medium containing IL-2 for 24 h. A median of 1.29 x 10(6) CD34+ cells/kg were infused after conditioning with busulfan (12 16 mg/kg) in PBSC recipients. BM was infused without prior myeloablative therapy. The engraftment occurred with a median of 15 d. Engraftment failure developed in one patient. The transplantation was followed by a 1-mo regimen of IL-2 (0.5 x 10(6) IU/m(2) daily) and GM-CSF, and 6 mo of IFN-alpha. One complete and one transient minor cytogenetic remission were observed. At 24 mo after transplantation, two patients had died of progressive disease and one of infection. Five patients had stable disease in the chronic phase. Autologous transplantation using IL-2-activated graft is feasible and the subsequent IL-2, GM-CSF, and IFN-alpha administration has acceptable toxicity. However, no benefits in comparison with conventional autologous transplantation for CML were identified in our study.

Hereditary thrombocytopenias are a heterogeneous group of extremely rare diseases characterized by a reduced number of blood platelets and by bleeding tendency of variable severity. Some of these diseases are exclusive to platelets, while in others the pathology extends to other cell types. Although rare, hereditary thrombocytopenias should be considered in the diagnosis. Hereditary thrombocytopenias have been classified into three groups depending on platelet volume. Hereditary thrombocytopenias with giant platelets form one of these groups. About fourteen clinical entities of inherited giant platelet disorders have been described. Bernard-Soulier syndrome, grey platelet syndrome and May-Hegglin anomaly are the most common giant platelet thrombocytopenias. May-Hegglin anomaly is condition characterized by the triad of thrombocytopenia, giant platelets, and pale-blue inclusions in leukocytes. May-Hegglin anomaly has an autosomal-dominant mode of inheritance. We described clinical and laboratory features of three adult women from one family with recently diagnosed May-Hegglin anomaly. Described cases are probably the first observed cases of May-Hegglin anomaly in the Czech lands.

Amphotericin B (AmB)-treated rats develop severe polyuria, polydypsia, impairment of renal concentrating ability, and morphologic signs of tubular damage. However, renal insufficiency develops quickly only in animals in which water intake is restricted to the median volume drunk by rats of the control group. Therefore, vigorous hydration seems crucial for prevention of AmB-induced nephrotoxicity. In a clinical study, 61 patients with hematologic malignancies receiving AmB therapy were massively hydrated to ensure urine output of > or =4000 mL/day. Urine sodium, potassium, and magnesium were also measured, and all losses were supplemented (potassium as a 7.45% solution via central venous catheter). AmB-treated patients developed signs of renal tubular damage (increased fractional excretion of sodium and potassium) and required large amounts of ion supplementation. The serum ion concentration and creatinine clearance remained stable. No clinically significant renal damage developed to force premature cessation of AmB treatment.

Background: During the last few years, improvement in prognosis of the adult acute lymphoblastic leukaemia (ALL) has been modest. The probability of leukemia-free survival is 20-40%. Philadelphia-chromosome positive (BCR-ABL positive) ALL has the worse prognosis. A single centre experience with treatment of ALL in adults is reported., Methods and Results: Between April 1997 and July 2000, 15 consecutive patients with de novo adult ALL (7 T-lineage ALL, 7 B-lineage ALL, 1 null ALL) begin their treatment with the seven-drug induction regimen (in phase I, daunorubicin, vincristine, L-asparaginase, i.v., and prednisone, p.o.; in phase II, 6-mercaptopurine, p.o., cytosine arabinoside and cyclophosphamide, i.v.) and central nervous system (CNS) prophylaxis (methotrexate and CNS irradiation in patients without total body irradiation in conditioning regimen), with intensive consolidation (three times high-dose methotrexate and high-dose-cytarabine, i.v.), and with/out autologous peripheral blood stem cell transplantation (PBSCT) followed by maintenance chemotherapy (6-mercaptopurine and methotrexate, p.o.). Seven patients received autologous PBSCT. Median patient age was 30 years. Three patients were BCR-ABL positive at diagnosis. With median follow-up 14 month (range 0.1-46 month), seven (4 T-lineage ALL, 2 B-lineage ALL, 1 null ALL) out of 15 patients are alive in remission (four of them receiving autologous PBSCT). Causes of death were relapse (n = 3), chemotherapy related toxicity (n = 2), infection (n = 1), and acute myeloid leukaemia developed 10 months after autologous PBSCT (n = 1). All BCR-ABL positive patients died., Conclusions: Chemotherapy alone and autologous PBSCT with maintenance therapy may be curative for adult patients with ALL. We can recommend these treatment options for patients without risk factors in particular.

In cancer patients with persistent fever and neutropenia, amphotericin B is administered for the early treatment and prevention of the clinically occult invasive fungal infections. The major drawback of the conventional amphotericin B is its nephrotoxicity. We have previously showed that massive potassium, sodium, and magnesium supplementation, which corresponds to the amount lost in kidney during amphotericin B administration, as well as vigorous hydration, can markedly reduce nephrotoxicity of conventional amphotericin B. Clinical courses of four oncological patients treated with conventional amphotericin B suspected or proven fungal infection for a period of 24 days (two patients), 39 days, and 47 days are described. During the long-term amphotericin B administration with nephroprotective measures, no severe renal function decrease was observed in any of our patients. Massive ion supplementation corresponding to the amounts lost in kidney, as well as the vigorous hydration are effective in the prevention of the renal function decrease induced by the long-term conventional amphotericin B therapy.

Profound and prolonged neutropenia following chemotherapy is a major risk factor for systemic fungal infections. It is still unclear how effective the various measures that are currently being used are in preventing serious fungal infection. Although a number of studies have been published on antifungal prophylaxis in neutropenic patients, there are a lot of conflicting data. At the present time, it seems that antifungal prophylaxis is more a matter of faith rather than science. The prophylactic use of fluconazole or itraconazole may reduce the incidence of systemic fungal infections in high-risk patient groups, mainly in patients with haematological malignancies. The antifungals currently used for prophylaxis in immunocompromised patients, and the toxicity of the antifungals are reviewed.