Beata Kuryło-Rafińska

Phenotype of NK Cells Determined on the Basis of Selected Immunological Parameters in Children Treated due to Acute Lymphoblastic Leukemia.

AbstractAcute lymphoblastic leukemia (ALL) is the most frequent pediatric malignancy. The chemotherapy for ALL is associated with a profound secondary immune deficiency.We evaluated the number and phenotype of natural killer (NK) cells at diagnosis, after the intensive chemotherapy and following the completion of the entire treatment for patients with ALL. The fraction, absolute number, and percentage of NK cells expressing interferon-&ggr; were determined in full blood samples. The fraction of NK cells expressing CD158a, CD158b, perforin, A, B, and K granzymes was examined in isolated NK cells.We have shown that patients assessed at ALL diagnosis showed significantly lower values of the fraction of NK cells and percentage of NK cells with the granzyme A expression. Additionally, the absolute number of NK cells, the expression of CD158a, CD158b, perforin, and granzyme A were significantly lower in patients who completed intensive chemotherapy. Also, there was a significantly higher fraction of NK cells expressing granzyme K in patients who completed the therapy.Abnormalities of NK cells were found at all stages of the treatment; however, the most pronounced changes were found at the end of intensive chemotherapy.

Individualized tumor response testing profile has a prognostic value in childhood acute leukemias: multicenter non-interventional long-term follow-up study

Abstract A total number of 817 children with acute lymphoblastic leukemia (ALL) and 181 with acute myeloblastic leukemia (AML) were assessed for individualized tumor response testing (ITRT) profile as a prognostic factor in long-term follow-up. For each patient, ITRT, initial response to therapy and long-term outcome were assessed. In initial ALL, an impact on long-term response was shown in ITRT for 13 drugs, while in initial AML only for cytarabine. For patients with ALL, a combined five-drug ITRT profile for prednisolone, l-asparaginase, vincristine, cytarabine and daunorubicin or doxorubicin had predictive value for probability of disease-free survival (pDFS) in univariate analysis, whereas in multivariate analysis, bone marrow response by day 33 was the only prognostic factor. For patients with AML, no factor had prognostic value for pDFS in univariate analysis, while ITRT to cytarabine almost reached significance. In conclusion, ITRT can possibly be regarded as a risk factor in childhood acute leukemias.

Differential ex vivo drug resistance profile in first and subsequent relapsed childhood acute myeloid leukemia in comparison to initial diagnosis

Background. Current cure rate reach 50-60% of long-term survival in childhood acute myeloblastic leukemia (AML). In spite of continuous progress in therapy of AML, relapses still occur frequently in both children and adolescents. The aim of this study was the analysis of the ex vivo drug resistance profile first and subsequent relapse in childhood AML in comparison to newly diagnosed AML. Methods. The results of 76 pediatric AML samples tested for drug resistance by the MTT assay were analyzed. Up to 22 drugs were tested for each patient. Results. No significant differences between ex vivo drug resistance at first and subsequent relapse of childhood AML 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, relapsed patients had higher ex vivo drug resistance profile than de novo AML patients. The median RR (relative resistance between relapsed and de novo diagnosed patients) value of all 22 drugs tested was 1.6. For five drugs, RR was significantly higher at relapse: idarubicin (1.8-fold), etoposide (5.9-fold), cytarabine (1.7-fold), fludarabine (3.7-fold) and busulfan (4.3-fold). For other four drugs, a trend for higher resistance at relapse was observed: for daunorubicin, mitoxantrone, L-asparaginase and cladribine. Conclusion. Ex vivo drug resistance profile in relapsed childhood AML is higher in comparison to initial diagnosis, however we did not find differences in ex vivo drug resistance between first and subsequent relapse of AML.

Impact of CMV and EBV on Immune Recovery After Allogeneic Hematopoietic Cell Transplantation in Children

Background/Aim: Immune recovery is a key factor in the management of patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This study analyzed the factors contributing to immune reconstitution after allo-HSCT. Patients and Methods: Overall, 65 children with malignant or non-malignant diseases were included in multivariate analyses. Results: The following factors contributed to a faster immune recovery: peripheral blood as a stem cell source and reactivation of CMV infection for CD3+ and CD4+ lymphocyte subpopulations; reactivation of CMV infection for CD8+ subset; donor EBV-IgG+ and no EBV reactivation for CD19 lymphocytes; recipient age below 10 years and peripheral blood as a stem cell source for NK cells. For CD2 and CD4/CD8 ratio no factor was significant in multivariate analysis. Conclusion: Patients receiving a graft from an EBV-IgG-positive donor and not having early EBV post-transplant viremia show faster recovery of the B-cells, while patients with early CMV-DNA-emia have a better re-establishment of T-cell subsets.

Lymphocyte subpopulations in children after allogenic stem cell transplantation

B a c k g r o u n d . Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a method of treatment in a variety of diseases, which involves replacing the recipients bone marrow by healthy donor stem cells. O b j e c t i v e . The aim of the study was the analysis of immune reconstitution in children and adolescents after allo- HSCT. P a t i e n t s a n d m e t h o d s . We analyzed lymphocyte subpopulations in 67 children after allo-HSCT for hematological malignancies and non-malignant diseases. B-cells, T-cells, NK (natural killer cells) and CD4/CD8 ratio were assayed in peripheral blood by flow cytometry using a set of antibodies CD2/CD19, CD3/CD4/CD8, CD3/CD16CD56 labeled with different fluorescent dyes. The tests were performed at 1, 3, 6, 9, 12, 15, 18 and 24 months after the HSCT. R e s u l t s . Normalization of the number of lymphocytes occurred rapidly in the population of NK cells; however, significant decrease in the proportion of NK cells was observed at 6 months after HSCT (p=0.028). The recovery of T cells occurred after 3 months for CD2 and after 6 months for CD3 cells. Within the B-cell population, a significant delay of reconstitution was observed: CD19 cell percentage exceeded 5% (p<0.001) in the ninth month after HSCT, while it was above 10% afterwards. The normalization of CD4/CD8 ratio was very slow and delayed and did not achieve normal values within 24 months after allo-HSCT. C o n c l u s i o n s . Reconstitution of the immune system in children after allo-HSCT is the fastest for NK cells. Normal values of T- and B-lymphocytes were reached after 6 and 9 months, respectively. CD4/CD8 ratio is a parameter the normalization of which requires at least two years.

IN VITRO DRUG RESISTANCE IN CHILDHOOD MATURE B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

B a c k g r o u n d . Mature B-cell acute lymphoblastic leukemia (B-ALL) is a rare type of leukemia in children and counts for 1-2% of all leukemia cases. Pediatric B-ALL is treated according to protocols for non-Hodgkin lymphomas. O b j e c t i v e . The aim of the study was to analyze the in vitro drug resistance in children with B-ALL compared to ALL with other phenotypes. M a t e r i a l a n d m e t h o d s . A total number of 15 children with B-ALL (6 girls and 9 boys, median age 8 years, range 1.9-15 years) were included into the analysis. The in vitro drug resistance tests on leukemic cells were performed by means of the MTT assay. The results of B-ALL patients were compared to those obtained in patients with pre- B/common ALL (479 cases), pro-B-ALL (31cases) and T-ALL (87 cases). Results: B-ALL cells were more resistant than pre-B/common-ALL cells to dexamethasone and idarubicin. In comparison to pro-B-ALL phenotype, B-ALL blasts were more resistant to idarubicin and more sensitive to treosulfan. No significant differences were found in the in vitro drug resistance between B-ALL and T-ALL. Blasts of T-ALL were more resistant than pre-B/common-ALL cells to most of tested drugs. Conclusion: From the clinical point of view, B-ALL cells have similar in vitro drug sensitivity when compared to T-ALL, and higher drug resistance to dexamethasone than pre-B/common-ALL.

Bortezomib has little ex vivo activity in chronic myeloid leukemia: individual tumor response testing comparative study in acute and chronic myeloid leukemia.

Aim of the study Resistance to imatinib is one of the most important issues in treatment of chronic myeloid leukemia (CML) patients. The objective of the study was to analyze the ex vivo drug resistance profile to bortezomib and 22 other antileukemic drugs, including three tyrosine kinase inhibitors (TKIs), in CML in comparison to acute myeloid leukemia (AML). Material and methods A total of 82 patients entered the study, including 36 CML and 46 AML adults. Among CML patients, 19 had advanced disease, 16 were resistant to imatinib, and 6 had ABL-kinase domain mutations. The ex vivo drug resistance profile was studied by the MTT assay. Results CML cells were more resistant than AML blasts to the following drugs: prednisolone, vincristine, doxorubicin, etoposide, melphalan, cytarabine, fludarabine, thiotepa, 4-HOO-cyclophosphamide, thioguanine, bortezomib, topotecan, and clofarabine. CML cells were 2-fold more sensitive to busulfan than AML cells. CML patients with clinical imatinib resistance had higher ex vivo resistance to vincristine, daunorubicin, etoposide, and busulfan. No significant differences to all tested drugs, including TKIs, were observed between CML patients with non-advanced and advanced disease. CML patients with mutation had higher ex vivo resistance to vincristine, idarubicin, thiotepa, and busulfan. Conclusions CML cells are ex vivo more resistant to most drugs than acute myeloid leukemia blasts. Busulfan is more active in CML than AML cells. In comparison to AML cells, bortezomib has little ex vivo activity in CML cells. No differences between CML subgroups in sensitivity to 3 tested TKIs were detected.