[Results of immunosupressive therapy in children with severe aplastic anaemia. Report of the Polish Paediatric Haematology Group]. / Wyniki leczenia ciezkiej postaci anemii aplastycznej u dzieci. Raport Polskiej pediatrycznej grupy do spraw nienowotworowych chorób ukladu krwiotwórczego.

INTRODUCTION: Bone marrow transplantation from HLA identical family donors is the treatment of choice for children with severe aplastic anaemia (SAA). When there is no donor available, combined immunosuppressive therapy is given. AIM: evaluation of results of immunosupressive therapy in children with severe aplastic anaemia. MATERIAL AND METHODS: SAA was diagnosed in 105 children (42 girls, 73 boys), aged 2-18 years, in the eleven haematological centres in Poland, between 1993-2007. All patients received the Severe Aplastic Anaemia Working Party of the EBMT protocol which included: antilymphocyte globulin or antithymocyte globulin, cyclosporin A, prednisolone. Granulocyto- or granulocytomacrophagic-cell stimulation factor was additionally administered during deep neutropenia. Haematological response was evaluated on day 84 or 112 and 180 of the therapy. RESULTS: complete remission occurred in 53 patients (51.5%), partial remission in 27 (24.7%), no response was obtained in 25 children (23.8%) on day 180, of the therapy. Period of observation was from 12 months to 12.5 years. During this time relapse occurred in 10 patients (9.5%). We observed 22 deaths: 8 early, during the first 3 months of IS and 14 after the first 3 months of immunosuppresive therapy (IS). At present 70 children (66.6%) are in first remission with lasts from 12 months to 12.5 years. The survival at 12.5-years is 78.6%. During the 12.5 years of follow-up we had two cases with a late clonal complication (PNH and MDS). Transformation to acute nonlymphoblastic leukaemia was observed in two of our patients. CONCLUSIONS: 1. Immunosuppresive therapy (IS) in children with SAA, without bone marrow family donors, is more effective after introduction of combined IS (12.5 years survival in this study was 80% for children with very severe aplastic anaemia (v SAA). 2. In our studies among the children followed up after IS therapy, there were: 1 case of periodic nocturnal haemoglobinuria (PNH), 1 case of myelodysplastic syndrome (MDS) and 2 cases of myeloid leukaemia (probability of incidence was 3.8%).

[Does the residual mediastinal mass have prognostic significance in children with Hodgkin's disease (HD)?]. / Czy obecnosc przetrwalej zmiany w sródpiersiu ma znaczenie prognostyczne w chorobie Hodgkina (HD) u dzieci?

Prognostic significance of residual mediastinal tumor mass in children treated for HD as well as the choice of the optimal management of these cases still remains unknown. In years 1994-2001 in 10 PPLLSG participating centers 480 children (age 2-19.7 years) were treated for HD (stages I-IV). In 338 cases initial mediastinal/lung hilus involvement was present. All patients with initial mediastinal/lung hilus involvement were treated with multidrug chemotherapy combined with involved field radiotherapy. In five cases remission was not achieved. Complete remission (CR) was achieved in 226 patients and uncertain complete remission (UCR) in 107 patients, in whom after completion of planned treatment residual changes in mediastinum/lung hilus were identified in radiological examinations. Twenty four children with persistent mediastinal tumor underwent thoracoscopy or thoracotomy. In only one case histopathological examination revealed the presence of neoplastic cells in mediastinal mass tissue, in 2 another cases cystic changes in mediastinum were present, in one case thymic tissue was identified, necrotic tissue was present in 20 cases. Among 107 children with residual mediastinal tumor and 226 patients with CR achieved, relapses occurred in 6 and 18 patients respectively. Over 5-year relapse-free survival was 92.4% and 91.3% respectively. Patients with the presence of mediastinal/lung hilus tumor after the completion of the treatment do not have an increased risk of relapse, but before the completion of therapy they require careful, clear-sighted and repeated examinations including computed tomography (CT), magnetic resonance imaging (MRI) and especially positron emission tomography (PET) to evaluate the nature of persistent lesions. Only in clinically and radiologically doubtful cases tumor biopsy with subsequent histopatological examination should be performed.

[Results of immunosuppressive therapy in children with severe aplastic anaemia. Report by the Polish Paediatric Leukaemia and Lymphoma Study Group]. / Efekty leczenia immunosupresyjnego ciezkiej postaci anemii aplastycznej u dzieci. Raport polskiej pediatrycznej grupy ds. leczenia bialaczek i chloniaków.

INTRODUCTION: Bone marrow transplantation from HLA identical family donors is the treatment of choice for children with severe aplastic anaemia (SAA). When no donor is available, combined immunosuppressive therapy is given. AIM: Evaluation of results of immunosuppressive therapy in children with severe aplastic anaemia. MATERIAL AND METHODS: SAA was diagnosed in 85 children (31 girls, 54 boys) aged 2-17.5 years in the eleven centres of the Polish Paediatric Leukaemia and Lymphoma Study Group (PPLLSG) in Poland between 1993-2003 years. All patients received protocol of the Severe Aplastic Anaemia Working Party of the Europe Bone Marrow Transplant (EBMT): antilymphocyte globulin or antithymocyte globulin, cyclosporin A, prednisolone and granulocyto- or granulocyto-macrophagic-cell stimulation factor was additionally administered during deep neutropenia. Haematological response was evaluated on day 84, 112 or 180 of the therapy. RESULTS: complete remission occurred in 43 patients (50.5%), partial remission in 22 (25.4%), no response was obtained in 20 children (23.7%) in 180 day of the therapy. Period of observation was from 12 months to 10.5 years. During this time relapse occurred in 6 patients (7%). We observed 16 deaths: 7 early during the first 3 months of immunosuppressive therapy (IS) and 9 after the first 3 months of IS. CONCLUSION: the actual survival at 10-years, after immunosuppressive therapy is 81.2% in our group. Transformation to leukaemia or myelodysplastic syndrome (MDS) was not observed in any of our patients. We observed one case with paroxysmal nocturnal haemoglobinuria (PNH).

RNA and protein evidence for haplo-insufficiency in Diamond-Blackfan anaemia patients with RPS19 mutations.

The genetic basis of Diamond-Blackfan anaemia (DBA), a congenital erythroid hypoplasia that shows marked clinical heterogeneity, remains obscure. However, the fact that nearly one-quarter of patients harbour a variety of mutations in RPS19, a ribosomal protein gene, provides an opportunity to examine whether haplo-insufficiency of RPS19 protein can be demonstrated in certain cases. To that end, we identified 19 of 81 DBA index cases, both familial and sporadic, with RPS19 mutations. We found 14 distinct insertions, deletions, missense, nonsense and splice site mutations in the 19 probands, and studied mutations in 10 patients at the RNA level and in three patients at the protein level. Characterization of the mutations in 10 probands, including six with novel insertions, nonsense and splice site mutations, showed that the abnormal transcript was detectable in nine cases. The RPS19 mRNA and protein in CD34+ bone marrow cells identified haplo-insufficiency in three cases predicted to have one functional allele. Our data support the notion that, in addition to rare DBA patients with the deletion of one allele, the disease in certain other RPS19 mutant patients is because of RPS19 protein haplo-insufficiency.

Antibody response to influenza vaccination in splenectomized patients in Poland.

The aim of this study was to assess antibody response in 62 splenectomized patients and in 55 healthy subjects vaccinated with split influenza vaccine ("Fluarix," SmithKline Beecham). Response to hemagglutinin and neuraminidase was assessed before vaccination and after 1 month by hemagglutination inhibition test and neuraminidase inhibition test. After vaccination, antibody titers significantly increased in both groups. Postvaccination protection rates ranged from 62.9 to 90.3% in the splenectomized patients and from 81.8 to 94.5% in the control group. Response rates ranged from 50.0 to 75.8% and from 60.0 to 70.9%, respectively. Splenectomized patients produced high antibody levels regardless of the time elapsed from the operation to the vaccination. All requirements of the Committee for Proprietary Medicinal Products regarding humoral response to influenza vaccination in healthy people were fulfilled in both groups. The response in the splenectomized patients was comparable to that in the control group.

Incidence and clinical characteristics of second malignant neoplasms in children: a multicenter study of a polish pediatric leukemia/lymphoma group.

BACKGROUND: The development of second malignant neoplasms (SMNs) in patients receiving chemotherapy and radiation therapy for primary cancers is one of the limitations to the quality and length of survival. The present study was undertaken to examine various characteristics of children who developed SMNs following successful therapy for primary leukemia or Hodgkin's disease (HD). MATERIAL/METHODS: A total of 3252 children with various forms of leukemia and 849 children with HD treated between, 1970-1997 at 7 pediatric centers of the Polish Pediatric Hematology/Oncology Group and subsequently followed-up entered the study. A second malignancy was diagnosed in 36 of these children. RESULTS: Of the 3252 patients diagnosed as having acute leukemia during this period, 16 developed SMNs (estimated frequency 0.49%). SMNs developed in 20 of the 849 children treated for HD (2.36%). The most frequent SMNs were soft tissue sarcoma and thyroid carcinomas, mainly following Hodgkin's disease. Other tumors occurred at about the same frequencies in both groups. The interval from the end of initial treatment to diagnosis of an SMN ranged from 2 years 7 months to 17 years 6 months, with a median of 7 yrs 4 mo. for acute lymphoblastic leukemia (ALL) patients and 10 years for children with HD. The estimated accumulated risk of SMN following acute leukemia is 0.95% at 15 years and, for HD, 5.1% at 20 yrs and 7% at 25 yrs. CONCLUSIONS: Children who have been successfully treated for one cancer have a higher than expected incidence of additional tumors.

A novel mutation in the glucose-6-phosphate dehydrogenase gene in a subject with chronic nonspherocytic hemolytic anemia--characterization of enzyme using yeast expression system and molecular modeling.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common human enzymopathy. Human G6PD gene is highly polymorphic, with over 130 mutations identified, many of which cause hemolytic anemia. We studied a novel point mutation in the G6PD gene 1226 C-->G, predicting the proline 409 to arginine substitution (G6PD Suwalki). We expressed the human wild-type and mutated G6PD gene in yeast Saccharomyces cerevisiae which allowed the characterization of the Suwalki variant. We showed that human wild-type, as well as the mutated (1226 C-->G) G6PD gene, functionally complemented the phenotype displayed by the yeast strain with disruption of the ZWF1 gene (homologue of the human G6PD gene). Comparison of wild-type (wt) human G6PD purified from yeast and from blood shows no significant differences in the Km values for G6P and in the utilization rate for the substrate analogue, 2-deoxyG6P. The P409R substitution leads to drastic changes in G6PD kinetics. The specific activity as well as stability of mutated G6PD is also significantly reduced. Besides this, the effect of this mutation was analyzed using a model of the tertiary structure of the human enzyme. The localization of the P409R mutation suggests that it may influence the stability of the whole protein by changing tetramer interactions and disturbing the binding of structural NADP+.

[Preliminary results of the use of MRD test among children with acute lymphoblastic leukaemia]. / Wstepne wyniki wykorzystania testu MRD u dzieci z ostra bialaczka limfoblastyczna.

UNLABELLED: The purpose of the study was to monitor minimal residue disease (MRD) among children with ALL and to evaluate the possibility of using this test to detect and monitor the minimal residual disease (MRD test) in the stratification of risk groups on a parallel basis with other recognised prognostic factors. MATERIALS AND METHODS: 56 children, with de novo diagnosed ALL, comprised test group. Control group consisted of 10 healthy persons. DNA was isolated from peripheral blood and bone marrow. During research applied was the PCR method with the use of starters specific for conservative IgH and TCR delta gene fragments. ALL therapy was monitored by evaluation of MRD in the 15th and 33rd day of treatment and prior to supportive treatment. Stratification into risk groups, based on the MRD test results was compared with the stratification conducted in accordance with the recognised prognostic factors, such as: primary leucocytosis, steroid therapy response, drug resistance and karyotype. RESULTS: Among 52 children (92.8%) of the 56, which were tested prior to the start of treatment, obtained the sought for rearrangement, 90-120bp size stripe being an amplified regrouping of the VDJ fragment. Among all the tested healthy persons, the test result was negative. Testing the IgH and TCR delta gene regrouping allowed to demonstrate clonality of the neoplastic process during the diagnosis in 92.8% of the cases. In some of them the regrouping had a bi- or oligoclonal character. The number of patients with a positive MRD test result was dependent on the phase of treatment. Positive results were recorded among 10% of the patients (4/39) during clinical remission, among 86% (6/7) deceased and among 80% (4/5) suffering from relapse. Among 16.7% (2/13) of the patients with determined relapse or who died due to the course of illness process were also observed negative MRD test results. Steroid resistance was stated among 18/56 children, out of which 8/56 obtained a positive MRD test result in the 33rd day of treatment. Among 3 of them the result was maintained prior to supportive treatment. Leucocytosis during diagnosis above 50 thousand was recorded among 10/56 patients. Among 5 of them determined was rearrangement on the 33rd day of treatment, and for 1 prior to supportive treatment. Cytogenetic tests showed a correct karyotype for 19 out of 56 tested children. In this group steroid resistance was determined among 8 children, high preliminary leucocytosis among 5 and in 17 cases of the tested rearrangement. Stratification into therapeutic groups based on classical prognostic factors in most cases was the same with the stratification based on the MRD test results. The accuracy of the latter method was greater. CONCLUSIONS: The use of MRD test has its application in risk group qualification. MRD test is helpful in determining relapses prior to the appearance of clinical symptoms. Among the tested group of children with ALL a positive MRD test result was a bad prognostic factor. It seams that the use of a combined analysis of the risk factors and the MRD test results in a significant manner contribute to a more complex evaluation of the patient's condition.

[Implementation of the unified model of presenting cancer diagnosis in Polish pediatric onco-hematology centers]. / Wdrazanie jednolitego modelu informowania dzieci o rozpoznaniu choroby nowotworowej w polskich osrodkach onko/hematologii dzieciecej.

Presentation of full information related to diagnosis of children with cancer should be one of principles in pediatric oncology. Multidisciplinary approach to each newly diagnosed child and its parents contributes to improving this standard. The Polish Pediatric Leukemia and Lymphoma Group is engaged in these activities since 1998 and it resulted in implementation of several SIOP recommendations in most of Polish pediatric oncohematology centers. The unified model of presentation of diagnosis for a child, parents and family was of an importance and the efforts to introduce it in all cooperating centers was undertaken. Proposed model of informing consists of several steps. Procedure should be individually tailored according to natural history of the disease and characteristics of the family. The purpose of the study was to evaluate the informing procedure in 60 children with newly diagnosed neoplasmatic disease.

[Evaluation of systolic and diastolic function of the left ventricle in children with acute lymphoblastic leukaemia before treatment]. / Ocena czynnosci skurczowej i rozkurczowej lewej komory serca u dzieci z ostra bialaczka limfoblastyczna przed rozpoczeciem leczenia.

Between 1995 and 2001 echo-cardiography was performed in 244 children (128 boys, 116 girls) with acute lymphoblastic leukaemia (ALL) before the beginning of therapy with anthracyclines (medium 5.4 days after the diagnosis). The mean age at diagnosis was 5.4 years (range 9 months to 17.7 years). 189 children (97 boys and 92 girls) were included into the standard and medium risk groups and 55 (31 boys and 24 girls) into the high risk group. 29% of ALL children had disturbances in ECG. Changes in the thickness of the intraventricular septum (%IVSTh) and left ventricular posterior wall (%LVPWTh) were statistically lower, especially in children under 7 years of age. Some children showed lowering of shortening fraction (%FS - 8.6%), ejection fraction (%EF - 10.2%) and corrected velocity of fibber-shortening (Vcfc - 25.8%). Children with decreased shortening fraction (%FS) had left ventricular posterior wall thickness (%LVPWTh) impairment. Changes in diastolic function indicate impaired relaxation and compliance of the left ventricle. Decreased peak early filling velocity (E) was found. There were also longer deceleration time (EDecT) and decreased deceleration from peak E velocity (E/Dec) and longer isovolumetric relaxation time in children in standard and medium risk groups. Shorter acceleration time (EAccT) was seen in the high risk group. Evaluation of cardiac function before anthracycline chemotherapy will allow to select patients with pre-existing cardiac impairment for whom cardioprotective treatment is absolutely necessary.

[Efficacy of idarubicin in the treatment of childhood acute non-lymphoblastic leukemia: report of Polish Pediatric Leukemia/Lymphoma Study Group]. / Zastosowanie idarubicyny w leczeniu ostrej bialaczki nielimfoblastycznej u dzieci--Raport Polskiej Grupy Pediatrycznej ds Bialaczek i Chloniaków.

UNLABELLED: Results of treatment of childhood ANLL remained unsatisfactory for a long time, and introduction of a new drug seemed justified as the EFS achieved in this disease between 1993-97 was 42%. In 1998 a new protocol containing idarubicine in dose 12 mg/m2 (each dose regardless of treatment phase) was introduced. Between 1998 and 2001, 137 children with ANLL were referred to nine participating centers of PPLLSG. Thirty nine were uneligible, so 98 were evaluated. Among them 56 were qualified to standard risk group (SRG) and 35 to high risk group (HRG). In 7 children the risk group was not established due to early death. Remission rate was 79% in the whole group and 98% and 63% in SRG and HRG, respectively. The actuarial 3-year overall survival (OS), 3-year event-free survival (EFS) and 3-year event-free interval (EFI) are respectively: In the whole group 61%, 54% and 67%; in SRG 81%, 70% and 71%; in HRG 40%, 40%, and 53%. CONCLUSIONS: Due to introduction if idarubicine significant improvement of the results was achieved in SRG (better OS, EFS and EFI). Better new treatment methods are required in HRG.

[Advances in the treatment of children with high risk acute lymphoblastic leukemia (ALL) treated with modified "NEW YORK" protocols between 1987 and 2002]. / Postepy w leczeniu dzieci z ostra bialaczka limfoblastyczna (ALL) wysokiego ryzyka wg modyfikowanych programów "NOWY JORK" w latach 1987-2002.

From 1981 to 1986, in children with ALL and initial WBC > or = 50,000/mm3, over 6-year disease-free survival was significantly lower (33%) than in children with WBC < 50,000/mm3 (60%). In attempt to improve this unsatisfactory results, three modified American protocols named: "New York", "New York I", and "New York II", "New York I", and "New York II" were introduced consecutively in the centers of Polish Pediatric Leukemia Lymphoma Study Group (respectively, in 1987, 1997, and 1999). The treatment results achieved in three consecutive therapeutic groups of children with ALL and initial WBC > or = 50,000/mm3: group I--213 children (1987-1996), group II--58 children (1997-1999), and group III--52 children (1999-2001) are presented. The observation was completed in December 31, 2002. In three evaluated groups the first complete remissions (CRs) were achieved in 90.6%. 94.8%. and 94.2% of patients, respectively. Relapses occurred in 71 patients of group I (37%), in 9 patients of group II (16%), and in 6 patients of group III (12%). The complications of treatment caused death in 7 children of group I, in 1 child of group II, and in 2 children of group III. Eighty-one (38%), 11 (18.9%), and 9 (17.3%) patients, respectively, died due to progression of disease. The event-free survival (EFS) in three evaluated groups did not depend on age of children and WBC. The rates of 2-, 5-, and 10-year event-free survival (EFS) in group I were: 69.9%, 55.3%, and 53.6%, respectively and the rates of 2- and 5-year EFS in group II were: 80.7% and 72.7%, respectively. The rate of 2-year EFS in group III was 71.6%. The analysis of achieved treatment results in three evaluated groups shows the gradual improvement of the prognosis in children with ALL and initial WBC > or = 50,000/mm3 treated with the use of modified protocols "New York" and "New York I" in comparison with patients treated before 1987. Longer observation is needed for evaluation of efficacy and complications of "New York II" protocol.

[Chromosomal aberrations in differential diagnosis and prognosis in childhood acute leukaemias]. / Aberracje chromosomowe w diagnostyce róznicowej i prognozowaniu w ostrych bialaczkach u dzieci.

The purpose of our study was to determine the frequency of specific, somatic chromosomal abnormalities in children with acute leukaemia and to evaluate the usefulness of cytogenetic study and DNA analysis as diagnostic and prognostic tools in these diseases. Among 63 children with acute lymphoblastic leukaemia (ALL) and 13 with de novo acute myeloblastic leukaemia (AML), hyperdiploidy was found in 25% and hypodiploidy in 6% of patients. Normal karyotype was found in 44% whereas pseudodiploidy in 25% of children with ALL. In the group of children with AML, pseudodiploidy was found in 2 cases and normal karyotype in 11. Translocations t(12;21), t(4;11), t(6;11) and t(9;11) failed to be detected by conventional cytogenetics. They were found by molecular methods. On the other hand, the t(1;14) and t(8;14) translocations were detected exclusively by karyotype analysis. The probability of event-free survival (EFS) in the group of children with ALL and genetic abnormalities of favourable prognosis was 96% whereas in the group of children with unfavourable prognosis it was 55%. Classical cytogenetic methods together with more sensitive molecular tests allow to detect diagnostically and prognostically relevant chromosomal aberrations in childhood acute leukaemias.

Incidence of childhood cancers in Poland in 1995-1999.

BACKGROUND: The annual rate for childhood cancers in developed countries amounts to 105-130 new cases per 1 million children. The Polish population aged 0-17 years is estimated at approximately 10 million children and adolescents, thus ca. 1100-1300 new cases can be expected every year. In 1995, we started a national childhood cancer registry. MATERIAL/METHODS: Information on the new diagnoses of childhood cancers was collected in 11 regional centers and submitted to the national center in Lublin. All data were verified carefully and standardized incidence rates were calculated. RESULTS: In 1995, we registered 1028 newly diagnosed malignant neoplasms, in 1996 and 1997 - 1036 cases, in 1998 - 1007, and in 1999 - 1158 new cases. The estimated incidence rates were: 102.4; 109.5; 111.9; 111.6 and 118.3 per 1 million children, respectively. The most frequent childhood cancers include leukemia, which accounts for 28% of cancer cases, lymphoma (14.3%) and C. N. S. tumors (16.3%). CONCLUSIONS: Neoplasms of the hematopoietic system (leukemias and lymphomas) account for about 42% of all childhood cancers. Malignant lymphomas, bone tumors and germinal tumors are more frequently diagnosed in Poland, but the incidence of central nervous system tumors is lower than in other countries.

In vitro activity of oxazaphosphorines in childhood acute leukemia: preliminary report.

Glufosfamide (beta-D-glucosyl-ifosfamide mustard) is a new agent for cancer chemotherapy. Its pharmacology is similar to commonly used oxazaphosphorines, but it does not require activation by hepatic cytochrome P-450 and preclinically demonstrates lower nephrotoxicity and myelosuppression than ifosfamide. The aim of the study was a comparison of the drug resistance profiles of glufosfamide and other oxazaphosphorines in childhood acute leukemias. Leukemic cells, taken from children with ALL on diagnosis (n = 41), ALL on relapse (n = 12) and AML on diagnosis (n = 13) were analyzed by means of the MTT assay. The following drugs were tested: glufosfamide (GLU), 4-HOO-ifosfamide (IFO), 4-HOO-cyclophosphamide (CYC) and mafosfamide cyclohexylamine salt (MAF). In the group of initial ALL samples median cytotoxicity values for GLU, IFO, CYC and MAF were 15.5, 33.8, 15.7 and 7.8 microM, respectively. In comparison with initial ALL samples, the relative resistance for GLU and IFO in relapsed ALL samples was 1.9 (p = 0.049) and 1.3 (ns), and in initial AML samples 31 (p < 0.001) and 5 (p = 0.001), respectively. All oxazaphosphorines presented highly significant cross-resistance. Glufosfamide presented high activity against lymphoblasts both on diagnosis and on relapse.

Cross-resistance to five glucocorticoids in childhood acute lymphoblastic and non-lymphoblastic leukemia samples tested by the MTT assay: preliminary report.

In vitro antileukemic activity of five glucocorticoids and their cross-resistance pattern in childhood acute lymphoblastic and non-lymphoblastic leukemia were determined by means of the MTT assay in 25 leukemia cell samples of childhood acute leukemias. The equivalent antileukemic concentrations of the drugs tested were: 34 microM hydrocortisone (HC), 8 microM prednisolone (PRE), 1.5 microM methylprednisolone (MPR), 0.44 microM dexamethasone (DX) and 0.22 microM betamethasone (BET). In comparison with initial ALL cell samples, the relapsed ALL group was more resistant to PRE (38-fold, p = 0.044), DX (> 34-fold, p = 0.04), MPR (38-fold), BET (45-fold) and HC (33-fold). The AML cell samples were even more resistant to: PRE (> 85-fold, p = 0.001), DX (> 34-fold, p = 0.004), MPR (> 69-fold, p = 0.036), BET (> 69-fold, p = 0.038) and HC (54-fold, p = 0.059) when compared with ALL on initial diagnosis. A significant cross-resistance among all the glucocorticoids used was found. Only in some individual cases the cross-resistance was less pronounced.

The influence of intracellular idarubicin and daunorubicin levels on drug cytotoxicity in childhood acute leukemia.

Uptake and efflux of two anthracyclines, idarubicin (IDA) and daunorubicin (DNR), was studied in childhood acute leukemia samples. A comparison of IDA and DNR transport phenomena in relation to drug cytotoxicity and expression of P-glycoprotein (PGP) was made. Intracellular content of IDA/DNR was determined by flow cytometry using the fluorescent properties of the drugs. In vitro drug cytotoxicity was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. PGP expression was analysed by flow cytometry. The uptake and efflux rates were non-significantly higher for IDA than DNR. There were no differences between three types of leukemia with respect to drug content during accumulation and retention. After correction for the cell volume, intracellular concentration of both drugs in each moment of uptake and efflux was significantly lower in relapsed ALL and AML samples in comparison with initial ALL cells. Efflux, but not uptake, of both drugs was inversely correlated with PGP expression and IDA, but not DNR, cytotoxicity. The cytotoxicity was correlated with drug accumulation for both drugs and with drug retention for IDA. In conclusion, it seems that (1) intracellular content was related to the lipophilic properties of the drugs rather than to the type of leukemia, (2) decreased intracellular concentration of both drugs might have an impact on compromised therapy results in AML and relapsed ALL children, (3) IDA presents higher cytotoxicity, which possibly might be decreased by the presence of PGP. These results might have a practical impact on the rational design of new chemotherapy protocols.

Apoptosis and pH of blasts in acute childhood leukemia.

BACKGROUND: The purpose of our study was to determine whether spontaneous and post-initial therapy apoptosis indexes (AI) and the intracellular pH of blasts have prognostic value in the treatment of children with acute leukemia. MATERIAL/METHODS: Blasts from 38 children with acute leukemia (31 with ALL and 7 with AML) were tested for apoptotic index (Annexin V) and intracellular pH (SNARF). Of the ALL patients, 9 were low risk, 7 medium risk, and 15 high risk. Among the AML patients, 1 was low risk and 6 were high risk. Follow-up ranged from 6 to 120 weeks (median 60 weeks). RESULTS: The mean spontaneous AI was 19 +/-16%. The pH of leukemic blasts before treatment varied between 6.6 and 7.9 (mean 7.2 +/- 0.5). For patients < 10 years old, the markers for good prognosis were a WBC count below 50 x 10(3)/microml, good response to prednisone therapy at day 8, and remission at or before day 33. Univariate analysis showed that pH < 7 had favorable prognostic significance. Overall, the probability of EFS for patients with pH < 7 was 1.0, as opposed to 0.49 for those with pH > 7 (p=0.049, n=36). The probability of EFS for patients with AI below the median was not significantly different from those with AI above the median (0.4 vs 1.0, NS). CONCLUSIONS: The assessment of intracellular pH in blasts may be an important prognosticator for ALL patients. Children with low spontaneous AI or high pH of leukemic blasts appear to have an unfavorable prognosis.

Efficacy of subunit trivalent influenza vaccine in previously vaccinated children suffering from hemophilia.

OBJECTIVE: To assess humoral immune response to subunit trivalent influenza vaccine in children suffering from hemophilia who had been immunized for the first time in 1993-94 and then in 1996-97. METHODS: In autumn 1996-97, 38 previously vaccinated hemophiliac patients were subcutaneously immunized with a single 0.5-mL dose of subunit influenza vaccine containing the following three virus strains: A/Singapore/6/86 (H1N1), A/Wuhan/359/95 (H3N2) and B/Beijing/184/93 (HB). Antibody response to influenza vaccine was measured before vaccination, 3 weeks after vaccination and 6 months after vaccination, by use of hemagglutinin- and neuraminidase-inhibition tests. To present the level of seroconversion, geometric mean titers of anti-influenza antibodies, mean fold increase, protection rate and conversion rate were determined. All results were compared with the control group of 23 healthy persons who had never been vaccinated against influenza and for whom the same serologic tests were carried out as for the vaccinated group. RESULTS: Three weeks after immunization, antihemagglutinin antibody levels were 3.9-10.9 times higher than before vaccination, but the highest mean fold increase values were recorded 6 months after vaccination, ranging from 8.4 to 28.6. In the case of neuraminidase, mean fold increases of antibodies reached values of 3.6-12.3 three weeks after vaccination and 7.1-29.1 six months after vaccination. The highest proportion of subjects protected was observed 6 months after immunization and ranged from 76.3% to 97.4%, compared to 52.6-60.5% 3 weeks after vaccination. Similar values were obtained for conversion rate: 71.1-86.8% 6 months after vaccination, in comparison with 39.5-42.1% 3 weeks after immunization. CONCLUSIONS: All data obtained in the present study indicate a significant immune response to subunit trivalent influenza vaccine in patients suffering from hemophilia; this is additionally confirmed by the fact that none of the vaccinated children were infected with the influenza virus and no serious adverse reactions were observed after administration of the vaccine.