Malgorzata Kubicka

Unusual profiles of pediatric acute lymphoblastic leukemia with MLL gene rearrangement

The most common chromosomal abnormality of infant acute lymphoblastic leukemia (ALL) is the t(4;11)(q21;q23), which gives rise to the MLL-AF4 fusion gene [1]. MLL (also known as ALL-I, HTRX or HRX) gene translocations are among the most common chromosomal abnormalities recognized in both B-lineage acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Translocations interrupting the mixed lineage leukemia gene (MLL) occur in 7 – 10% of acute lymphoblastic leukemia and 5 – 6% of acute myeloid leukemia cases [2]. The MLL gene, which is located at the 11q23, has been cloned and has homology to the Drosophila trithorax gene [3]. MLL is required for the proper maintenance of HOX gene expression during development and hematopoiesis. About 50 different MLL fusion partners have been isolated to date, and while similarities exist between groups of partners, there exists no unifying property shared by all the partners. MLL gene rearrangements are found in leukemias with both lymphoid and myeloid phenotypes, and are often associated with infant and secondary leukemias. The immature phenotype of the leukemic blasts suggests an important role, including therapyrelated leukemias, for MLL in the early stages of hematopoietic development [4 – 6]. Rearrangements of chromosome band 11q23 are common in infant leukemias, comprising more than 70% of the observed chromosome abnormalities in children less than 1 year of age [3]. Mixed lineage leukemia (MLL) abnormalities occur in *50% of childhood pre-pre-B acute lymphoblastic leukemia [7]. In spite of the fact that it is one of the most common nonrandom chromosomal abnormalities, there are scant data showing occurrence of MLL rearrangement in childhood ALL with different phenotype, such as common-ALL or T-ALL. We analyzed 121 consecutive pediatric ALL cases diagnosed and treated in one center. All children were screened at diagnosis for immunophenotype, DNA index, karyotype by G-banding method, MLL, and BCR-ABL gene rearrangements by FISH (fluorescence in situ hybridization) and RT-PCR. Both conventional cytogenetics and FISH were carried out. FISH analysis was performed utilizing commercially available DNA probes, including whole chromosome painting probes, locus specific probes, and specific dual color translocation fusion probes. As far as MLL rearrangements are concerned, FISH can not only detect reciprocal translocation but also a deletion of at least 190 kb from the 30 region of the MLL gene [8]. Common/ pre-B-ALL immunophenotype was diagnosed as CD19þ, HLA-DRþ, CD10þ, and surface immunoglobulin7 [sIg7]; pre-pre-B-ALL (pro-B-ALL) immunophenotype as (CD19þ, HLA-DRþ, CD107, cytoplasmic m chain7 and surface immunoglobulin7 [sIg7]). Mature B-ALL cells characterized

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.

Hiperdiploidia limfoblastów i jej związek z immunofenotypem w ostrej białaczce limfoblastycznej

Wstep Do najwazniejszych badan diagnostycznych w ostrej bialaczce limfoblastycznej (ALL) zalicza sie: badania morfologiczne, badania cytochemiczne, immunofenotypowanie, badania indeksu DNA i analize cyklu komorkowego oraz badania cytogenetyczne i molekularne aberracji chromosomowych. Cel pracy Analiza związku wynikow badania immunologicznego i analizy faz cyklu komorkowego określanych metodą cytometrii przeplywowej z obrazem cytogenetycznym przy rozpoznaniu ostrej bialaczki limfoblastycznej u dzieci. Pacjenci i metodyka Analize profilu diagnostycznego pacjentow z nowo rozpoznaną ALL przeprowadzono u 151 dzieci. Za kryterium rozpoznania ostrej bialaczki limfoblastycznej przyjeto stwierdzenie w szpiku kostnym co najmniej 20% blastow bialaczkowych. Immunofenotyp blastow i indeks DNA oznaczano metodą cytometrii przeplywowej. Badania cytogenetyczne wykonano metodą prązkową, a badania molekularne metodami FISH i PCR. Wyniki i wnioski Pacjenci z ostrą bialaczką limfoblastyczną z wartością indeksu DNA limfoblastow powyzej 1,16 mają immunofenotyp charakterystyczny dla prekursorow limfocyta B, w przewazającej wiekszości przypadkow z obecnością antygenu common CD10. Aberracje cytogenetyczne limfoblastow o charakterze hiperdiploidii lub rearanzacji genow TEL-AML1 stwierdzono wylącznie wśrod pacjentow z immunofenotypem common-ALL. Nie stwierdzono znaczenia prognostycznego wartości indeksu DNA limfoblastow. Korzystny profil cytogenetyczny wyrazający sie hiperdiploidią limfoblastow lub obecnością translokacji t(12;21) lub rearanzacji TEL-AML1 ma bardzo korzystne znaczenie rokownicze w ostrej bialaczce limfoblastycznej.

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.

Profil diagnostyczny wznowy ostrej białaczki limfoblastycznej

Ostra bialaczka limfoblastyczna (ALL) jest najczestszym nowotworem u dzieci. Pomimo stalego postepu w terapii ALL, u okolo 20% pacjentow z tą chorobą dochodzi do wznowy. Szacuje sie, ze wznowa ostrej bialaczki limfoblastycznej moze byc traktowana jako czwarty co do czestości nowotwor wieku dzieciecego. Cel pracy Analiza profilu diagnostycznego ALL w okresie rozpoznania i we wznowie szpikowej. Pacjenci i metodyka Badaniom poddano 24 pacjentow ze wznową szpikową ALL, u ktorych przeprowadzono analize profilu diagnostycznego choroby w momencie rozpoznania i jej wznowy (morfologia limfoblastow wg klasyfikacji FAB, badania cytochemiczne, analiza immunofenotypu metodą cytometrii przeplywowej, ocena indeksu DNA i analiza cyklu komorkowego, analiza wynikow badan cytogenetycznych i molekularnych). Wyniki We wznowie stwierdzono zmiany w morfologii blastow (u 3 pacjentow z L1 na L2), zmiany reakcji PAS lącznie u 4 dzieci, zmiany w immunofenotypie u 7 pacjentow, w tym u 4 z ALL z linii B-komorkowej oraz u 3 z rozpoznaniem ALL linii T-komorkowej. Zmiana immunofenotypu byla związana ze zmianą wartości indeksu DNA w 3 przypadkach. Stwierdzono 2,4-krotnie wieksze ryzyko wznowy u pacjentow, u ktorych na limfoblastach nie wystepowal antygen CD10 (tj. pre-pre-B-ALL lub T-ALL) niz u pacjentow z obecnością CD10 (tj. common-pre-B-ALL). We wznowie stwierdzono nieprawidlowy kariotyp limfoblastow u wiekszości pacjentow. Wniosek We wznowie ALL u dzieci stwierdza sie zmiany w morfologii i immunofenotypie limfoblastow, czestsze wystepowanie bardziej niedojrzalego immunofenotypu, zlozonych zaburzen cytogenetycznych oraz rzadkie wystepowanie korzystnych zmian cytogenetycznych.

Komórki hematopoetyczne w szpiku i we krwi obwodowej u chorych na ostrą białaczkę limfoblastyczną, guzy lite oraz u dzieci zdrowych ☆ ☆☆ ◊

Wstep Na powierzchni komorek hematopoetycznych szpiku znajdują sie antygeny powierzchniowe. Najbardziej charakterystyczne markery komorek hematopoetycznych to CD34 i CD133. Cel pracy Analiza ekspresji CD34 i CD133 w roznych populacjach komorek szpiku kostnego i krwi obwodowej. Metody Ekspresje antygenow komorek hematopoetycznych CD34 i CD133 oceniono w 281 probkach szpiku kostnego i krwi obwodowej, pobranych od 101 pacjentow: z ostrą bialaczką limfoblastyczną (acute lymphoblastic leukemia – ALL), guzami litymi oraz w grupie kontrolnej dzieci z prawidlowym obrazem szpiku kostnego i krwi obwodowej. Wyniki Stwierdzono wiekszy odsetek komorek CD34 i CD133 w szpiku niz we krwi obwodowej: u dzieci zdrowych, w trakcie chemioterapii oraz po stymulacji czynnikami hematopoetycznymi. Po miesiącu od przeszczepienia autologicznych komorek hematopoetycznych, odsetek komorek CD34 byl nieznacznie wyzszy niz w szpiku w grupie kontrolnej. U dzieci z ALL odsetek komorek CD133 w szpiku byl wyzszy niz u dzieci zdrowych, natomiast u pacjentow z ALL w remisji choroby byl podobny jak u dzieci zdrowych. We wszystkich badanych grupach mediana odsetka komorek CD133 byla wyzsza niz mediana komorek CD34. W szpiku kostnym pacjentow poddawanych stymulacji G-SCF, jak rowniez po przeszczepieniu komorek hematopoetycznych, w wielu przypadkach zaobserwowano znaczną rozbieznośc w nasileniu ekspresji antygenow CD34 i CD133. Stwierdzono korelacje odsetka komorek z ekspresją CD34 i z ekspresją CD133. Wnioski Ekspresja antygenow CD34 i CD133 jest skorelowana, przy czym zazwyczaj stwierdza sie wyzszy odsetek komorek z ekspresją antygenu CD133. Odsetek komorek CD34 i CD133 w szpiku kostnym i krwi obwodowej wzrasta wielokrotnie po stymulacji czynnikami krwiotworczymi. Regeneracja hematopoezy po przeszczepieniu autologicznych komorek hematopoetycznych jest związana ze wzrostem odsetka komorek CD133-dodatnich.