Treatment and prognosis of childhood acute lymphoblastic leukemia on protocols ALL-BFM 90, 95 and ALL IC-BFM 2002: a retrospective single-center study from Olomouc, Czech Republic.

Great progress has been made in the diagnostics and treatment of childhood acute lymphoblastic leukemia (ALL) over the past decades. The vast majority of children are cured, however, there is need for further improvement, especially in specific patient subgroups. Our aim was to retrospectively evaluate disease characteristics and treatment outcomes of children with ALL enrolled in a single center into consecutive treatment protocols (ALL-BFM 90, ALL-BFM 95 and ALL IC-BFM 2002) between years 1990 and 2007 and comprehensively summarize diagnostic and therapeutic advances between protocols. In total, 97 patients aged 0 to 18 years were treated for ALL at University Hospital Olomouc in the Czech Republic and steadily high relapse-free survival (RFS), event-free survival (EFS) and overall survival (OS) were observed during the evaluated time period without significant difference between the protocols (RFS 80-86%, EFS 75-83% and OS 84-92%). In conclusion, our center has demonstrated survival rates comparable to leading international study groups for childhood ALL over a substantial period of time. This has been achieved namely due to advances in diagnostics, excellent collaboration on regional, national and international level, quality assurance and high overall standard of care. The acquired experience has been crucial for current participation in the best performing Berlin-Frankfurt-Münster (BFM)-based international trials for childhood ALL.

[The effectiveness of anagrelide treatment in patients with Ph negative myeloproliferative diseases: influence on the incidence of thrombosis in the data from the Registry of patients with essential thrombocythemia and thrombocythemia associated with other myeloproliferative diseases treated with Thromboreductin® to the end of 2012]. / Úcinnost lécby anagrelidem u nemocných s Ph negativními myeloproliferativními chorobami: ovlivnení výskytu trombózy ve výstupech Registru pacientu s dia-gnózou esenciální trombocytemie a trombocytemie provázející jiné myeloproliferativní onemocnení lécených Thromboreductinem® ke konci roku 2012.

In the Czech Republic, anagrelide (Thromboreductin®) [29] is used according to the recommendations of the Czech Working Group on Myeloproliferative Disorders (CZEMP) for treatment of thrombocythemia associated with Ph negative myeloproliferative disorders (MPDs). The patient data are collected in the Registry of patients with essential thrombocythemia (ET) and thrombocythemia associated with other MPDs treated with Thromboreductin®. At the end of 2012, the Registry contained data on 1,161 patients. Out of these, 1,159 patients with the dia-gnosis of a Ph negative MPD were evaluated. In 844 patients, precise WHO based dia-gnosis was known at start of therapy: 442 (52.4%) had ET, 108 (12.8%) had polycythaemia vera (PV) and 243 had primary myelofibrosis (PMF). The median age was 51 years at the time of diagnosis. At the time of the evaluation of the population, the median was 59 years. Every year, the proportion of patients newly treated with anagrelide as a firstline treatment in accordance with the CZEMP guidelines has been increasing. A growing proportion of patients has been treated with an additional cytoreducing drug, such as hydroxyurea and interferon. The majority of the patients received also an antiaggregant (or anticoagulant). More than a half of patients harbors the JAK2 mutation. A prompt decrease of platelet counts (as the response to Thromboreductin® treatment) was documented in most of the patients. After one year, 86.9% of patients had a full or partial response. In poorer responders, combination cytoreductive treatment was administered rather then the escalation of the Thromboreductin® dosage. There were 461 thrombotic manifestations in 363 patients and 61 haemorrhagic events in 57 patients recorded in the patients history. In the course of treatment (followup; F U), thrombosis was diagnosed only 179-times in 136 patients. There were more haemorrhagic events during F U: 109 events in 83 patients. Upon comparison of the number of events during F U to their numbers in history, we found a twofold decrease in arterial thrombosis, an almost twofold decrease in microvascular thrombosis and even a 6.6- fold decrease in venous thromboembolism events. Bleeding episodes increased 1.8-fold during F U. However, the vast majority of these hemorrhagic events were clinically insignificant. In conclusion, the treatment strategy according to the CZEMP guidelines incorporating anagrelide is highly effective in reducing the platelet counts, strongly prevents venous events, reduces arterial events, and leads to an increase of minor hemorrhages.

[Diagnosis and treatment of BCR/ABL-negative myeloproliferative diseases--principles and rationale of CZEMP recommendations]. / Diagnostika a lécba BCR/ABL-negativních myeloproliferativních onemocnení--principy a výchdiska doporuceni CZEMP.

In 2009, the recommendations of the Czech Collaborative Group for Ph- Myeloproliferative Diseases (CZEMP) for diagnosis and treatment of BCR/ABL-negative myeloproliferative diseases (MPD), i.e., essential thrombocythemia (ET), polycythaemia vera (PV) and primary myelofibrosis (PMF) were updated and extended. The present article gives the rationale of the recommendations in full detail. The CZEMP diagnostic criteria for ET and PMF are based on histopathological (HP) findings, which must unconditionally be in line with the given clinical and laboratory characteristics of ET or of a certain stage of PMF, respectively. The platelet count is not decisive for diagnosis. In cases lacking an adequately taken and read HP finding, the Polycythemia Vera Study Group (PVSG) criteria are recommended. The diagnosis of typical PV is based on demonstration of the V617F mutation of the JAK2 gene along with a significant increase of red cell parameters. If these are close to borderline, the demonstration of increased total red cell mass (RCM) is required. In atypical cases lacking polyglobulia or elevated RCM, the HP picture of PV (in accordance with WHO description) plus JAK2 V617F mutation is satisfactory for diagnosis, or, in cases lacking JAK2 V617F mutation, the HP picture of PV along with polyglobulia (or increased RCM) is sufficient. The treatment principles of ET and other MPDs with thrombocythemia (MPD-T; i.e., the early stages of PMF and PV) are identical. The patients are stratified by their thrombotic risk (preceding thrombosis, another thrombophilic state, jAK2 mutation), presence of disease symptoms (mainly microcirculatory), platelet count and age. Only patients up to 65 years lacking the above mentioned risks with a platelet count < 1000 x 10(9)/l are considered as low-risk and do not demand cytoreducing therapy. The others are high-risk ones and have an indication for thromboreduction. In patients older than 65 years, the potentially leukemogenic drug hydroxyurea (HU) may be used. In the younger ones, the choice is between anagrelide (ANG) or interferon-alpha (IFN). In high-risk patients, the treatment goal is to maintain platelet counts below 400, and in low-risk ones, below 600 x 10(9)/l. In PV, polycythemia itself is another thrombotic risk factor. The condition is treated by bloodletting or erythrocytaphereses. If hematocrit levels < or =45 are not achieved, cytoreductive therapy using HU in patients over 65 years, or IFN in younger individuals is required. All patients with thrombocythemia in PV are high-risk and have an indication for cytoreduction. Acetylsalicylic acid is given to all patients with MPD-T with platelets < 1000 x 10(9)/l (at higher counts, hemorrhage is imminent), and to all individuals with PV, unless contraindication is present. In case of platelet count normalization, it may be withdrawn in cases of low-risk ET or PMF, not in JAK2+ PV. The treatment of advanced stages of PMF is symptomatic, with substitution of blood derivatives being the basis. The only curative treatment is allogeneic stem cell transplantation, which should not be indicated too early seeing to its risks, but also not too late--we must not allow transition into acute leukemia, which is heralded by blasts in the blood picture. The indication is the presence of any of the following criteria: values of hemoglobin < 10 g/dl, WBC < 4 x 10(9)/l and platelets < 100 x 10(9)/l, any percentage of blasts or > or = 10% immature granulocytes in the differential picture, >1 erythroblast per 100 cells--all at repeated examinations within at least a 2-month interval, and in addition, rapid progression of hepato-/splenomegaly, presence of general symptoms of the disease, portal hypertension and extensive swellings.

[Essential thrombocythaemia and other myeloproliferative disorders with thrombocythaemia treated with Thromboreductin. A report from the database of register for the 1st quarter of 2010]. / Esenciální trombocytemie a jiné myeloproliferate s trombocytemií lécené Thromboreductinem. Výstupy z databáze Registru k 1. ctvrtletí roku 2010.

In the Czech Republic, anagrelid is used according to the recommendations of the Czech Working Group on Myeloproliferative Disorders for treatment ofthrombocythaemias associated with chronic myeloproliferative disorders--mainly essential thrombocythaemia and, regularly, reports are being presented from the Register of Patients Treated with Thromboreductin, most recently last year (Vnitr Lék 2009; 55: I-XII). The Register commenced in 2005 and from then it aims to determine detailed clinical and laboratory profiles of the patients. The structure of the Register has changed significantly in the course of its existence, reflecting the reports from each of the analyses conducted so far. Also, the data entry in the database improves every year and it reaches 97% on some of the items. The longest evaluation period in some of the patients is 108 months. By April 2010, the Register database contained data on 717 patients. Of these, 672 patients with the diagnosis of a Ph-negative chronic myeloproliferative disorder were evaluated. This year's analysis included the patients with essential thrombocythaemia, polycythaemia vera and primary myelofibrosis only. The analysis included 418 women and 254 men with median age of50 years. Unlike the first years, 2/3 of the current sample are non pretreated patients, meaning that the patients reach the specialized centres early in their treatment. Also, patients, and the older patients in particular, are more frequently treated with combined regimens including Thromboreductin. We increasingly observe hypertension as one of the monitored risk factors preceding the disease and laboratory parameters showJAK2 mutation in more than a half of patients while some form ofthrombotic diathesis is found in the anamnesis of 7-10% of patients. Some bleeding is observed in 1-5% of the registered patients. In comparison to the previous years, this is a decrease in the prevalence of clinical symptoms prior to the disease onset; this is very likely associated with an earlier patient diagnosis within the asymptomatic phase of the disease. Therapeutically, we achieve a fast treatment response but there still are 16.3% of sufficient afterone year of treatment. Thromboreductin dose is increasing but even in this group it does not exceeds the mean of 2.38 mg per 24 hours. Complications are observed in 6.2% of patients in the first year of therapy, and ofthese, thrombotic events in about 2.5% and (small) bleeding complications in 4% of patients. The data suggest that we still do not reach treatment response in a certain proportion of patients after a year of their therapy. Even though the care results from the analysed data improve every year, the Register helps to uncover some issues that still remain, such as treatment intensification and other treatment modifications.

[The results of patients with essentials thrombocythemia and other myeloproliferation-related thrombocythemia--a report of patients treated with Thromboreductin]. / Výsledky lécby nemocných s esentiální trombocytemií a dalsími myeloproliferacemi provázenými trombocytemií--zpráva z registru pacientu lécených Thromboreductinem.

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.

Natural history of recessive inheritance of DMT1 mutations.

DMT1 deficiency causes microcytic hypochromic anemia due to decreased erythroid iron utilization. Anemia is present from birth. Transferrin saturation is high and serum ferritin is mildly elevated, despite liver iron overload. DMT1 deficiency must be considered in the differential diagnosis of microcytic hypochromic anemia observed in the newborn period.

[Essential thrombocytemia and other myeloproliferations with thrombocytemia in the data of the register of patients treated with Thromboreductin till the end of 2006]. / Esenciálni trombocytemie a dalsí myeloproliferace s trombocytemií v údajích registru pacientu lécených Thromboreductinem do konce roku 2006.

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.

[Prenatal parvovirus B19 infection in fetus]. / Prenatální infekce plodu parvovirem B19.

OBJECTIVE: To analyse 3 cases of parvovirus B19 infection in a pregnant woman followed by transplacental transmission to the fetus. DESIGN: A reprospective study. SETTING: Department of Obstetrics and Gynecology, University Hospital, Olomouc. METHOD: Parvovirus B19 vertical transmission from a pregnant woman to the fetus was diagnosed in 3 cases. Serologic testing of IgG and IgM antibodies against parvovirus B19, cytological bone marrow examination and parvovirus B19 DNA analysis by PCR methods were performed. The fetal anemia was predicted by measurements of peak systolic velocities in the middle cerebral artery (MCA-PSV). RESULTS: There were three pregnancies in all (1st - single, 2nd and 3rd - dizygotic twins). In the 1st and 2nd pregnancy the diagnosis of parvovirus B19 infection was set on the basis of erythroblastopenia diagnosed in the neonatal period or early infancy. In the 2nd pregnancy (dizygotic twins) intrauterine death of one twin occured. In the 3rd case (dizygotic twins) the diagnosis was already set in the 20th week of pregnancy. Subsequently the fetal anemia was predicted by doppler examination. The fetuses did not require invasive intrauterine intervention and mild anemia was diagnosed after delivery. Progressive intrauterine growth retardation of one twin was observed. Neither cardiomegaly nor fetal hydrops did not occur. All mothers were asymptomatic during the whole pregnancy. CONCLUSION: Parvovirus B19 infection should be excluded in all cases of nonimmune fetal hydrops, severe fetal anemia, cardiomegaly, intrauterine growth retardation (IUGR) and chronic erythroblastopenia diagnosed in the neonatal period or early infancy. The presence of IgM and IgG antibodies against parvovirus B19 is highly specific but their negativity is insufficient for the exclusion of a parvovirus B19 infection. Viral DNA testing by PCR method is the only reliable method available.

Importance of using comparative genomic hybridization to improve detection of chromosomal changes in childhood acute lymphoblastic leukemia.

We used comparative genomic hybridization (CGH) and conventional cytogenetics (CC) to define chromosomal changes and to evaluate the usefulness of CGH in 65 patients having childhood acute lymphoblastic leukemia (ALL). Subsequently, fluorescence in situ hybridization (FISH) was used to evaluate the CGH and cytogenetic results. Comparative genomic hybridization revealed DNA copy number changes in 49 (75%) patients (including 7 patients with unsuccessful cytogenetics and 2 patients with normal karyotype). A total of 85 losses and 195 gains were detected. The most commonly gained chromosomes were 21 (35%), X (31%), 18 (27%), 10 (26%), 6 (25%), 17 (25%), 4 (23%), and 14 (22%). Losses were most frequently observed on chromosomes 9p (18%) and 12p (11%). Other losses were detected on chromosomes 13q (9%), 6q (9%), 7p (8%), and chromosome X (6%). Conventional cytogenetics revealed chromosomal changes in 53 (82%) patients. The employment of CGH and FISH together with CC analysis revealed chromosomal changes in 62 (95%) of the childhood ALL patients investigated. The CGH completed CC results in 36 patients; in 9 patients, the changes escaped detection without using CGH. The results of our study were compared to 6 other CGH studies previously reported. Our observations underline the benefits of supplementing routine cytogenetic investigation in childhood ALL by FISH and CGH, because small unbalanced changes may escape detection when conventional cytogenetics is the only diagnostic method used.

Complex karyotypes in childhood acute lymphoblastic leukemia: cytogenetic and molecular cytogenetic study of 21 cases.

Cytogenetic and molecular cytogenetic analysis of 79 childhood acute lymphoblastic leukemias (ALL) revealed chromosomal abnormalities in 76 (96%). Complex karyotypes (a finding of three and more chromosomal aberrations in a karyotype) were identified in 21 (26.6%) out of 79 patients. In 11 patients, complex karyotypes have included common recurrent chromosomal abnormalities, such as translocation t(12;21) in seven cases, t(9;22) in two cases, one case with t(2;1;19) and another one with translocation involving 11q23. In 10 patients, miscellaneous abnormalities were detected. Five patients displayed hyperdiploidy (47 approximately 57 chromosomes), three patients complex karyotypes with deletions of 9p, one patient with two new complex translocations t(2;4;12;13) and t(7;11;20), and the last patient with dic(12;21). The evaluation of the frequency of the chromosomal breaks (>5 per chromosome) showed that chromosomes 2, 4, 5, 7, 9, 12, 13, and 21 were most frequently affected. Survival analysis revealed statistically significant unfavorable event-free survival (EFS) (P=0.013) and decreased overall survival in the group with complex karyotypes (n=21) compared with the other cases (n=58). The evaluation of overexpression profile revealed increased occurrence of double CD13/CD33 positivity in patients with common recurrent chromosomal abnormalities (in 70% of cases); no such cases were registered in the other group (P<0.01).

Mediastinal teratoma and acute megakaryoblastic leukemia.

The association between mediastinal teratoma and acute megakaryoblastic leukemia (AMKL) in a 15-year-old boy is described. The clinical course is compared with 20 previously reported cases of AMKL in children. Chromosome studies at diagnosis of the leukemia showed multiple leukemic stem lines with numerical and structural abnormalities.

Differential antileukemic activity of prednisolone and dexamethasone in freshly isolated leukemic cells.

This study was designed to compare the antileukemic activity of prednisolone and dexamethasone in childhood acute lymphoblastic leukemia (ALL) under in vitro conditions. The chemoresistance of leukemic cells was ascertained by means of a MTT assay in 69 ALL children at diagnosis and the concentration killing 50% of leukemic cells (LCS50) was determined. The children were treated using the protocol ALL-BFM 90/95. Statistical correlations were made among prednisolone (PRED) and/or dexamethasone (DEX) LCS50 and absolute number of blast cells (ANB) on day 0/8 and a new parameter named blast cells clearance (BCC, BCC8 [%] = ANB8: ANB0 x 100) on day 8. Despite the previously published results of Ito et al. (J. Clin. Oncol. 14: 2370-2376, 1996) and Kaspers et al. (MPO 27: 114-121, 1996) on a positive correlation of DEX versus PRED LCS50 (p < 0.002), in our study, we identified 30% of children (21/69) with differential in vitro responsiveness to PRED and DEX. 16% of patients (11/69) were highly sensitive to DEX and resistant to PRED, while 14% of them (10/69) were resistant to DEX and highly sensitive to PRED. The major difference found in our and the other studies was in the processing of leukemic cells. These results were confirmed in a model experiment using the CCRF-CEM line, where we showed that sensitivity to PRED and DEX, but not to other anti-cancer drugs critically depends on manipulation with tumor cells (cryopreservation). Correlation of PRED/DEX in vitro sensitivity values with parameters of in vivo patient's response to PRED monotherapy identified significant association of PRED LCS50 with BCC8 (p < 0.02). It indicates strong linkage of in vitro sensitivity to PRED with percentage of blast cells eliminated from patient blood within the first 8 days of PRED monotherapy.

[Dendritic cells and their use in the therapy of neoplastic diseases]. / Dendritické bunky a jejich vyuzití v terapii nádorových chorob.

Dendritic cells (DC) constitute a heterogeneous leukocyte population. Their main function is to capture and process antigens (Ag) and present them to immunocompetent cells. Heterogeneity of DC is reflected at several levels. Myeloid and lymphoid lineage of the DC can be distinguished according to the precursor cell they originate from. The functional differentiation is of the great importance. DC can induce either specific immune reaction or tolerance to certain Ag. It depends upon the microenvironment where the processing of Ag takes place. Phenotypic and functional differences between the subtypes of DC are being extensively investigated for the purpose of their use in the immunotherapy of various diseases, tumors in particular. It appears that one of the causes of the specific anti-tumor immunity failure is the insufficient function of DC in vivo in patients with malignant diseases. Recent technology advances has enabled to generate and cultivate DC in sufficient amounts in vitro from their precursors. Coculturing of DC with the tumor Ag in the presence of cytokine mixture leads to the efficient Ag presentation and to the generation of specific cytotoxic lymphocytes capable of killing tumor cells. Subsequent application of these tumor Ag pulsed DC to the laboratory animals, and to the patients in first clinical studies, can induce regression of malignant disease. On the other side the ability of DC to induce tolerance to certain Ag is the subject of investigation in the field of immunotherapy of hypersensitivity states induced either by outer Ag (allergy) or inner Ag (autoimmune diseases). In this review we summarize source and ontogeny of DC, their morphology, phenotype, function and different ways of their generation in vitro. We emphasize the use of DC in the clinical practice aimed at the immunotherapy of tumor diseases.

[Improved results in children with acute lymphoblastic leukemia treated with the ALL-BFM 90 protocol in the Czech Republic]. / Zlepsení výsledku lécby detí s akutní lymfoblastickou leukémií podle protokolu ALL-BFM 90 v Ceské republice.

BACKGROUND: Prognosis of children with acute lymphoblastic leukaemia (ALL)--the most common cancer in childhood, has improved remarkably over the last 40 years. The authors report the treatment outcome in children with ALL cured according to ALL-BFM 90 Study protocol in the Czech Republic during the first half of nineties. METHODS AND RESULTS: Children aged 0-18 years were included into the study in 10 centers between 1990 to 1996. Patients were classified into standard-risk (SR), medium-risk (MR) and high-risk (HR) group according to initial leukaemic burden, early treatment response, and genotype of leukaemia. Duration of the chemotherapy was two years. Treatment results were evaluated in 352 children. With a median follow-up of 7.3 years, event-free-survival (EFS) was 71.3% and overall survival 76.4%. EFS was 80.3%, 74% and 28.2% in SR, MR and HR group, respectively. Relapse was diagnosed in 17.8% of the patients. CONCLUSIONS: The treatment outcome of children with ALL improved significantly (p = 0.0045) compared to the previous study ALL-BFM 83 (EFS 62%). These results are comparable to those achieved by leading leukaemia study groups in the world.