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To analyze the drug-resistance profile at first and subsequent relapse in children with acute lymphoblastic leukemia (ALL).A total of 154 pediatric ALL samples were tested for ex vivo chemosensitivity for up to 19 drugs. Their combined drug resistance profile (PVA score) was analyzed.The median relative resistance scores between patients with multiple relapse and those with first relapse considering all drugs was 2.0. The median PVA score at subsequent relapses was 8 vs. 6 at first relapse (p=0.004). Samples from multiple-relapsed ALL were more drug resistant to: prednisolone (1.9-fold), dexamethasone (1.5-fold), vincristine (3.1-fold), L-asparaginase (5-fold), mitoxantrone (2.4-fold), cytarabine (4.3-fold), mercaptopurine (2.2-fold), thioguanine (4.8-fold), etoposide (2.6-fold) and melphalan (2.7-fold). Lymphoblasts at multiple relapse were comparably resistant to: daunorubicin, doxorubicin, cyclophosphamide, ifosfamide, busulfan, treosulfan, fludarabine, clofarabine and bortezomib.In comparison to first relapse, subsequent relapsed childhood ALL is more ex vivo-resistant to most tested drugs.

Keratinocyte growth factor (palifermin) is used for prevention of mucositis in adults following autologous and allogeneic hematopoietic stem cell transplantation (HSCT). It is known that palifermin decreases length of initial hospital stay, mean number of days of total parenteral nutrition (TPN) and the use of opioids for pain control in oral mucositis in adults. There are limited data evaluating palifermin use in children following autologous HSCT.The objective of the present study was the analysis of efficacy and safety of palifermin in children undergoing auto-HSCT.This matched-pair analysis study included 62 pediatric patients undergoing first auto-HSCT receiving palifermin on a compassionate-use basis (study group, n=31) or not (control group, n=31).Palifermin decreased the incidence of severe (grade 3-4 WHO) oral mucositis (p=0.041), length of hospitalization (p=0.047) and contributed to the shorter duration of oral mucositis (p=0.035) and lower incidence of clinically or microbiologically documented infections (p=0.038). There were no differences between groups in opioid use, neutrophil and platelet recovery, TPN use and gastrointestinal hemorrhage.Palifermin decreases the incidence and severity of oral mucositis in children undergoing autologous HSCT.

Analysis of risk factors for survival in long-term follow-up of children treated at a single pediatric center in Poland.Out of 623 children diagnosed with cancer between 1995-2005, 110 were treated for brain tumors and followed-up, with a mean survival of 11.4 years.Overall 5-year survival in the whole cohort was 60.9±4.7%, while 10-year survival was 58.2±4.7%. No relapse, progression or death occurred after six years from initial diagnosis. Survival was 48.1±9.6% for patients with medulloblastoma and primitive neuroectodermal tumors; 83.3±6.2% for low-grade astrocytoma; 56.6±16.6% for ependymoma, while 0% at 72 months for high-grade glioma. Patients with cerebellar tumors had a survival rate 69.0±7.1% at 10 years. Multivariate analysis showed that factors predicting poor outcome were: grade III-IV tumor, incomplete surgical resection, and complications after surgical resection, while diagnosis of low-grade glioma was the only factor predicting good outcome. Progression of the disease during therapy was an additional independent adverse risk factor for survival.Long-term survival was achieved by 58% of children with brain tumors. Advanced tumor stage, incomplete surgical resection, complications of surgical treatment, and progression of the disease during treatment predicted poor outcome.

The analysis of the individualized tumor response testing (ITRT) at first and subsequent relapse in children with acute myeloid leukemia (AML).A total of 76 pediatric AML samples underwent ITRT for up to 21 drugs.No significant differences between ITRT at first and subsequent relapse were found, and no drug was found, for which significantly higher resistance of myeloblasts was observed at subsequent relapse, when compared to first relapse of AML. For most tested drugs, patients with relapse had higher IRTR than those with de novo AML. The median relative resistance value between patients with relapse and those with de novo diagnosis for all 21 drugs tested was 1.6. Samples of relapsed AML samples were significantly more resistant to: Idarubicin (1.8-fold), etoposide (5.9-fold), cytarabine (1.7-fold), fludarabine (3.7-fold) and busulfan (4.3-fold).ITRT in relapsed AML is higher in comparison to that at initial diagnosis, while no differences in ITRT between first and subsequent relapse of AML were found.

The analysis of the prognostic impact of residual disease at day 15 of induction therapy, individual tumor response testing (ITRT) at diagnosis, initial factors and initial therapy response to the risk of relapse in children with precursor B-cell acute lymphoblastic leukemia (ALL).A total of 87 children were tested at diagnosis for ITRT and for persistence of blasts in bone marrow at day 15 (BML150.5%) and were followed-up in long-term analysis.The probability of disease-free survival (pDFS) was significantly better for patients with an ITRT profile showing sensitivity to prednisolone, vincristine, daunorubicin, and L-asparaginase. Patients with BML150.5% had higher ITRT for prednisolone, daunorubicin, L-asparaginase, and etoposide. Three factors had predictive impact for relapse: BML150.5%, ITRT for prednisolone and high combined ITRT profile for prednisolone, vincristine and L-asparaginase (PVA score).Persistence of blasts in bone marrow at day 15, ITRT showing resistance to prednisolone and high PVA score were the strongest and comparable prognostic factors predicting relapse in childhood ALL.

The predictive value of residual disease measured by flow cytometry at day 15 of induction therapy was analyzed in 182 children treated for acute lymphoblastic leukemia (ALL).Peripheral blood (PB) and bone marrow were assessed for leukemia cells by morphology and flow cytometry at days 0, 8, and 15.Absolute blast count (ABC)200/μl in PB by day 15 assessed by flow cytometry predicted a lower probability of disease free survival (pDFS) (p=0.056). Patients with bone marrow lymphoblast (BML)0.5% had a lower pDFS (p=0.002). Cumulative relapse incidence for patients with BML0.5% was 8.9% vs. 47.1% (OR=4.6, p=0.036). In common/pre-B-ALL patients aged10 years with BML0.5%, pDFS value was significantly lower. In the multivariate analysis, the only significant factor with adverse prognostic value for pDFS was BML0.5% (HR=5.3 p=0.030).BML0.5% analyzed by flow cytometry at day 15 is possibly the strongest prognostic factor in pediatric ALL.

The proteasome inhibitor, bortezomib, is known to be effective in the therapy of various neoplasms. The objective of this study was the analysis of the ex vivo activity of bortezomib in paediatric acute lymphoblastic leukaemia (ALL), in comparison to paediatric acute myeloid leukaemia (AML). A total of 159 patients entered the study, including 106 ALL (including 86 precursor-B-cell ALL, and 20 T-cell ALL) and 53 AML children. The ex vivo sensitivity to bortezomib and 16 other drugs was studied by MTT assay. Paediatric AML samples were more resistant than paediatric ALL samples to most of the tested drugs, except for cytarabine and thioguanine. With respect to immunophenotype, ex vivo drug resistance in T-cell ALL (T-ALL) was higher for most of the drugs. No differences in drug resistance between T-ALL and common/pre-B-cell-ALL were found for daunorubicin, mitoxantrone and 6-thioguanine. Bortezomib was the only compound which was more active in T-ALL than in common/pre-B-ALL paediatric samples. In conclusion, bortezomib had good ex vivo activity in paediatric T-ALL samples.

Clofarabine is a second-generation nucleoside analogue. The aim of the study was the analysis of ex vivo activity of clofarabine and 14 other anticancer drugs in pediatric and adult acute lymphoblastic (ALL) and myeloid (AML) leukemia.The ex vivo drug resistance profile was analyzed in 282 patients, including 201 children with ALL de novo, 24 children with relapsed ALL, 25 children with AML de novo and 32 adults with AML. Cellular ex vivo drug resistance was tested by means of the MTT assay.Clofarabine had comparable ex vivo activity against lymphoblasts and myeloblasts, both on initial diagnosis and at relapse, both in children and in adults. Its activity in acute myeloid leukemia was independent of patient age. No significant differences in drug resistance to clofarabine between pediatric age-based subgroups of ALL were detected, while it was observed for most of other drugs. An activity of clofarabine in relapsed pediatric ALL patients was as good as in newly-diagnosed ones.In comparison to childhood acute lymphoblastic leukemia, lack of differences in ex vivo activity gives rationale for use of clofarabine in refractory and relapsed pediatric and adult patients with acute myeloid leukemia.

Bortezomib is an inhibitor of proteasome and NF-kappaB, with activity in various solid tumors and hematological malignancies.The aim of the study was the analysis of in vitro drug resistance to bortezomib and other anticancer drugs in de novo and relapsed adult acute myeloid leukemia (AML).The leukemic cells of 46 adult patients with AML were tested for the in vitro drug resistance profile. The group included 20 de novo and 26 relapsed AML patients, among whom, 12 relapsed after allogeneic hematopoietic stem cell transplantation (HSCT) and 4 after autologous HSCT. The MTT assay was performed for 21 drugs. Expression of P-glycoprotein (PGP), multidrug resistance-associated protein-1 (MRP1) and lung resistance protein (LRP) proteins was measured by flow cytometry.No significant differences in drug resistance were found for all tested drugs between de novo and relapsed AML samples, while expression of PGP, MRP1 and LRP was higher in relapsed patients. Patients with refractory or relapsed disease, had higher resistance of myeloblasts to cyclophosphamide (RR = 2.4, p = 0.050), and better sensitivity to busulfan (RR = 0.4, p = 0.054) and topotecan (RR = 0.4, p = 0.031). Those who have died due to refractory/relapsed disease (n = 16) had better sensitivity to bortezomib (RR = 0.6, p = 0.046) and treosulfan (RR = 0.1, p = 0.018).In vitro drug resistance in relapsed adult AML is comparable to that in de novo disease. Activity in vitro of bortezomib might be a rationale for its use in refractory/relapsed AML adult patients.

The prognostic role of the ex vivo drug resistance profile has not yet been proved in childhood acute myeloid leukemia (AML). The aim of the study was the analysis of the impact of the ex vivo drug resistance profile in a cohort of 44 children with AML undergoing hematopoietic stem cell transplantation (HSCT).Myeloblasts for drug resistance testing were obtained from the bone marrow either on diagnosis or at relapse, before the HSCT procedure and were tested by the MTT assay.Children who relapsed after transplantation showed higher ex vivo resistance of the leukemic blasts to etoposide, mercaptopurine, thioguanine, fludarabine, mitoxantrone and treosulfan than those who stayed in remission. Despite being nondiscriminative, the combined ex vivo drug resistance profile to fludarabine, treosulfan and etoposide (FTE score) was the strongest prognostic factor by multivariate analysis.The combined drug resistance profile to fludarabine, treosulfan and etoposide may be useful for better stratification of children with AML undergoing stem cell transplantation or to indicate the necessity for additional post-transplant therapy.

Glucocorticoids are the most important group of drugs used in the treatment of childhood acute lymphoblastic leukemia (ALL), however, resistance to this group remains the main obstacle in curing the disease. One of the possibilities to circumvent glucocorticoid resistance is the use of new compounds, such as cortivazol (CVZ), which has two binding sites for the glucocorticoid receptor.Analysis of ex vivo sensitivity to cortivazol and other glucocorticoids in childhood acute lymphoblastic leukemia, as well as the relationship to anticancer therapy outcome.Leukemic samples from 60 children with ALL were tested by the M7T assay for glucocorticoid resistance. Cell cycle before and after ex vivo glucocorticoid treatment was analyzed by flow cytometry.Although all tested glucocorticoids presented significant cross-resistance, CVZ showed high antileukemic activity. The equivalent activity of CVZ was 165-fold higher than prednisolone, 7.ZS5-fold higher than dexamethasone and 2.8-fold higher than betamethasone. CVZ showed relatively better cytotoxicity than other glucocorticoids in prednisolone-poor-responders. CVZ, like other glucocorticoids, caused cell cycle arrest in the GI-phase, and increased the percentage of apoptotic cells to a greater extent than other glucocorticoids. The results of antileukemic therapy were strongly related to the ex vivo resistance to all tested glucocorticoids.Cortivazol has potent antileukemic activity in childhood ALL. Its activity is related to cell cycle arrest and induction of apoptosis.

Relapsed pediatric patients with acute myeloid leukemia (AML) have a poor clinical prognosis. The aim of this study was the analysis of the ex vivo drug resistance profile on relapse in childhood AML in comparison to newly diagnosed AML. The results of 98 pediatric AML samples tested by the MTT assay were analyzed. Eighteen samples (18%) were excluded from the further analysis due to spontaneous apoptosis of blasts in 4-days culture, low percentage of myeloblasts in the sample either in the beginning or at the end of the assay, infection, or formation of clots in the sample. Finally, ex vivo drug resistance of 20 relapsed samples were compared with that of 60 de novo AML, including 9 matched pairs. Up to 18 drugs were tested for each patient. No significant differences between drug resistance at diagnosis and at relapse in AML was found, neither for the whole groups of patients, nor for matched pairs only. Possibly, relatively good sensitivity of myeloblasts on relapse was found against melphalan, thiotepa, 4-HOO-ifosfamide, and cladribine. In summary, cellular drug resistance in childhood AML at relapse is not higher than at first diagnosis. These observations suggest that other, than cellular drug resistance, factors play a key role in therapy failure of relapsed childhood AML.

Glufosfamide is a new agent for cancer chemotherapy. The objective of the study was the comparison of the in vitro drug resistance profile of glufosfamide with other oxazaphosphorines in 106 samples of childhood acute leukemia by means of the MTT assay. The following drugs were tested: glufosfamide, 4-HOO-ifosfamide, 4-HOO-cyclophosphamide, mafosfamide cyclohexylamine salt, prednisolone, vincristine, L-asparaginase, daunorubicin and cytarabine. In the group of initial Acute Lymphoblastic Leukemia (ALL) samples, equivalent cytotoxicity values for glufosfamide, 4-HOO-ifosfamide, 4-HOO-cyclophosphamide and mafosfamide were 5.95, 9.92, 4.60 and 3.90 microg/ml, respectively. In comparison to initial ALL samples, the relative resistance for glufosfamide and 4-HOO-ifosfamide in relapsed ALL samples were 1.9 (p=0.049) and 1.3 (ns), and in initial Acute Myeloblastic Leukemia (AML) samples, respectively, 31 (p0.001) and 5 (p=0.001). All oxazaphosphorines showed highly significant cross-resistance. In conclusion, in vitro activity of glufosfamide is comparable to ifosfamide. Glufosfamide shows high activity against lymphoblasts both on diagnosis and on relapse, however it cannot circumvent resistance to other oxazaphosphorines.