ALBERT

Beschreibung: Contemporary treatment of pediatric acute lymphoblastic leukemia (ALL) requires the assignment of patients to specific risk groups. We have recently demonstrated that expression profiling of leukemic blasts can accurately identify the known prognostic subtypes of ALL, including T-cell lineage ALL (T-ALL), E2A-PBX1, TEL-AML1, MLL rearrangements, BCR-ABL, and hyperdiploid karyotypes with more than 50 chromosomes. As the next step toward developing this methodology into a frontline diagnostic tool, we have now analyzed leukemic blasts from 132 diagnostic samples using higher density oligonucleotide arrays that allow the interrogation of most of the identified genes in the human genome. Nearly 60% of the newly identified subtype discriminating genes are novel markers not identified in our previous study, and thus should provide new insights into the altered biology underlying these leukemias. Moreover, a proportion of the newly selected genes are highly ranked as class discriminators, and when incorporated into class-predicting algorithms resulted in an overall diagnostic accuracy of 97%. The performance of an array containing the identified discriminating genes should now be assessed in frontline clinical trials in order to determine the accuracy, practicality, and cost effectiveness of this methodology in the clinical setting.

Beschreibung: Transcriptional repression by chimeric transcription factors is emerging as a common theme in leukemogenesis and as a therapeutic target for chromatin remodeling agents. We hypothesized that rearrangements involving the MLL gene result in the inappropriate silencing of growth and survival control genes subordinate to this positive epigenetic transcriptional regulator. To identify some of these genes, we used Significance Analysis of Microarrays (SAM), a supervised learning algorithm. We found significant gene expression differences between 13 patients with MLL translocations and 12 core binding factor (CBF) rearranged patients, 2 t(8;21), 10 INV16, from a Pediatric Oncology Group AML study (POG 9421). We also analyzed gene expression data from a published study of adult AML including 8 MLL rearranged patients, 11 with t(8;21) and 15 with INV16. SAM identified 10 genes, common to both datasets, that were significantly under-expressed in the MLL rearranged patients. One of the most significant genes was osteonectin, also known as secreted protein, acidic, rich in cysteine (SPARC). This gene encodes a matricellular glycoprotein with diverse functions in cell-matrix interactions. SPARC has been identified as a target of epigenetic silencing in pancreatic cancer and addition of exogenous SPARC to cancer cell lines induces growth arrest and apoptosis. To determine if leukemia cell lines could be used as a model to study the basis for SPARC silencing and its role in cell growth and survival we measured SPARC expression in AML cell lines with rearranged and germline MLL genes. By real-time quantitative reverse transcriptase PCR (Q-RT-PCR), the cell lines THP-1 (MLL-AF-9) and ML-2 (MLL-AF6) expressed SPARC mRNA levels 40 to 1000 fold lower than Kasumi-1 (t(8;21)) and KG1a. By Western blot, SPARC was easily detectable in Kasumi-1 and KG1a but undetectable in the MLL rearranged lines. Bisulfite sequencing revealed extensive methylation of CpG dinucleotides in the promoter region and first exon of SPARC in THP-1 and ML-2 but a complete lack of methylation in KG1a. Treatment with the DNA methyltransferase inhibitor 5-aza-2′deoxycytidine (DAC) restored SPARC expression to nearly normal levels in THP-1 cells by Q-RT-PCR and Western blot, suggesting that promoter methylation is critical to the silencing of this gene. To determine if SPARC was contributing to the growth inhibitory effect of DAC, cells were cultured in varying concentrations of exogenous purified SPARC. Concentrations of SPARC that reduced the growth of ML-2 and THP-1 by 30 to 40% had no effect on the growth of KG1a cells. We conclude that SPARC, a putative tumor suppressor that is epigenetically silenced in pancreatic cancer, is also silenced by promoter methylation in AML with MLL rearrangements. These studies suggest that SPARC expression may constitute a reliable pharmacodynamic endpoint for clinical studies of chromatin remodeling agents in patients with MLL rearranged AML. Whether SPARC is a direct target of MLL and how MLL rearrangements are related to SPARC silencing are the subject of future studies.

Beschreibung: Contemporary treatment of pediatric acute myeloid leukemia (AML) requires the assignment of patients to specific risk groups. To explore whether expression profiling of leukemic blasts could accurately distinguish between the known risk groups of AML, we analyzed 130 pediatric and 20 adult AML diagnostic bone marrow or peripheral blood samples using the Affymetrix U133A microarray. Class discriminating genes were identified for each of the major prognostic subtypes of pediatric AML, including t(15;17)[PML-RARα], t(8;21)[AML1-ETO], inv16 [CBFβ-MYH11], MLL chimeric fusion genes, and cases classified as FAB-M7. When subsets of these genes were used in supervised learning algorithms, an overall classification accuracy of more than 93% was achieved. Moreover, we were able to use the expression signatures generated from the pediatric samples to accurately classify adult de novo AMLs with the same genetic lesions. The class discriminating genes also provided novel insights into the molecular pathobiology of these leukemias. Finally, using a combined pediatric data set of 130 AMLs and 137 acute lymphoblastic leukemias, we identified an expression signature for cases with MLL chimeric fusion genes irrespective of lineage. Surprisingly, AMLs containing partial tandem duplications of MLL failed to cluster with MLL chimeric fusion gene cases, suggesting a significant difference in their underlying mechanism of transformation.

Beschreibung: By using rapid flow cytometric techniques capable of detecting one leukemic cell in 104 normal cells, we prospectively studied minimal residual disease (MRD) in 195 children with newly diagnosed acute lymphoblastic leukemia (ALL) in clinical remission. Bone marrow aspirates (n = 629) were collected at the end of remission induction therapy and at 3 intervals thereafter. Detectable MRD (ie, ≥0.01% leukemic mononuclear cells) at each time point was associated with a higher relapse rate (P 

Beschreibung: To describe the clinical and biologic features of pediatric acute megakaryoblastic leukemia (AMKL) and to identify prognostic factors, experience at St Jude Children's Research Hospital was reviewed. Of 281 patients with acute myeloid leukemia treated over a 14-year period, 41 (14.6%) had a diagnosis of AMKL. Six patients had Down syndrome and AMKL, 6 had secondary AMKL, and 29 had de novo AMKL. The median age of the 22 boys and 19 girls was 23.9 months (range, 6.7-208.9 months). The rate of remission induction was 60.5%, with a 48% rate of subsequent relapse. Patients with Down syndrome had a significantly higher 2-year event-free survival (EFS) estimate (83%) than did other patients with de novo AMKL (14%) or with secondary AMKL (20%;P ≤ .038). Among patients who had de novo AMKL without Down syndrome, 2-year EFS was significantly higher after allogeneic bone marrow transplantation (26%) than after chemotherapy alone (0%;P = .019) and significantly higher when performed during remission (46%) than when performed during persistent disease (0%;P = .019). The 5-year survival estimates were significantly lower for de novo AMKL (10%) than for other forms of de novo AML (42%; P 

Beschreibung: CD38 is a transmembrane molecule whose expression varies during hematopoietic cell differentiation. We used stroma-supported cultures of human myeloid cells to assess the effects of CD38 ligation on myeloid differentiation. In 8 experiments with CD34+cells purified from normal bone marrow or cord blood, flow cytometry used with antibodies to CD34 and myeloperoxidase (MPO) identified 4 cell populations after 7 days of culture. Addition of anti-CD38 (T16) to the cultures induced a profound reduction of the most mature (CD34−MPO++) cell population, which includes promyelocytes, myelocytes and metamyelocytes; mean (± SD) cell recovery was 12.8% ± 9.8% of that in parallel cultures with an isotype-matched control antibody. The suppressive effect of CD38 ligation on phenotypically more immature normal cells was inconsistent but generally less pronounced. Recovery of CD34++MPO− cells was 63.3% ± 24.4%, recovery of CD34[+/−]MPO− cells was 95.3% ± 35.1%, and recovery of CD34−MPO+cells was 42.0% ± 18.7% of that in control cultures. However, anti-CD38 suppressed recovery of cells obtained from 6 patients with CD38+ acute myeloid leukemia; after 7-day cultures, cell recovery was 25.2% ± 21.7% of that in control cultures. Cell recovery was also reduced by F(ab′)2 or Fab fragments of anti-CD38. CD38 ligation dramatically suppressed recovery of murine 32D myeloid cells transfected with human CD38 and cocultured with stroma (3.8% ± 7.3%; n = 7). CD38 ligation of CD38 + 32D cells also induced cell aggregation, tyrosine kinase activity, and Ca++ influx. We conclude that CD38 mediates signals that culminate in suppression of myeloid cell growth and survival.

Beschreibung: By using rapid flow cytometric techniques capable of detecting one leukemic cell in 104 normal cells, we prospectively studied minimal residual disease (MRD) in 195 children with newly diagnosed acute lymphoblastic leukemia (ALL) in clinical remission. Bone marrow aspirates (n = 629) were collected at the end of remission induction therapy and at 3 intervals thereafter. Detectable MRD (ie, ≥0.01% leukemic mononuclear cells) at each time point was associated with a higher relapse rate (P 

Beschreibung: St Jude Total Therapy Study XIIIB for childhood acute lymphoblastic leukemia (ALL) incorporated more stringent risk classification, early intensification of intrathecal chemotherapy, reinduction treatment, and the addition of dexamethasone to postremission therapy to increase the proportion of event-free survivors without jeopardizing their quality of life. Cranial irradiation was reserved for the 12% of patients who had T-cell ALL and a presenting leukocyte count of 100 × 109/L or more, or CNS-3 (5 or more leukocytes/μL with identifiable blast cells in an atraumatic sample or the presence of cranial nerve palsy) status. Among the 247 consecutive patients enrolled in the study, 117 were classified as having lower-risk leukemia and received mainly antimetabolite-based continuation therapy; the 130 cases with higher-risk leukemia received more intensive continuation chemotherapy with multiple drug pairs administered in weekly rotation. The 5-year event-free survival estimate was 80.8% ± 2.6% (SE); the 8-year rate was 78.6% ± 5.8%. The 5-year cumulative risk of an isolated central nervous system (CNS) relapse was 1.7% ± 0.8%, and that of isolated plus combined CNS relapse was 3.0% ± 1.1%. The 5-year cumulative risks of etoposide-related myeloid malignancies were 1.8% ± 1.3% in the lower-risk patients who received a cumulative dose of 1.2 g/m2 and 5.0% ± 2.0% in the higher-risk patients who received a cumulative dose of up to 14.4 g/m2 (P = .18). Independent adverse prognostic features included the presence of MLL-AF4 or BCR-ABL fusion gene and minimal residual leukemia of 0.01% or more at the end of the 6-week remission induction phase. Our results suggest the efficacy of early intensification of intrathecal chemotherapy and provide the basis for studies omitting cranial irradiation altogether. (Blood. 2004;104:2690-2696)

Beschreibung: The karyotype of the leukemia cell at diagnosis is of prognostic importance. The presence of t(8;21), inv(16) and t(15;17) are currently used to make therapeutic decisions. Additionally, specific mutations such as those involving the FLT3 gene are of prognostic importance as they indicate patients likely to relapse early or fail initial induction therapy. Approximately 20% of children with AML have normal karyotypes at diagnosis and no identifiable chromosomal abnormality using standard methods of analysis. In this subgroup there is an increased incidence of FLT3 mutations (internal tandem duplications or point mutations). We observed that patients with normal karyotypes who were enrolled in the Pediatric Oncology Group (POG) study #9421 had two significantly different clinical outcomes that were associated with the expression of FLT3 mutations. We hypothesized that gene expression profiles would identify genes that cooperate with FLT3 mutations in conferring poor clinical outcome. Bone marrow and/or blood samples from an initial 103 of 622 eligible patients registered on the POG study #9421 were studied. Cytogenetic testing was carried out by clinical laboratories at institutions where AML was diagnosed; all cytogenetic reports underwent centralized review. Samples were analyzed on a 43,760-element spotted array (containing 41,751 unique genes and ESTs) from the Stanford University Microarray Core Facility. Of the 103 diagnostic samples, 20 had normal karyotypes. The FLT3-mutation status was determined by RT-PCR analysis on RNA samples (exon 14 for ITD’s and exon 20 for point mutations). Twelve patients expressed FLT3-mutations and 8 expressed wild-type FLT3 (FLT3-WT). Both groups had a mean WBC of 103x109/L at diagnosis. Probability of event free survival (EFS) was 75% for the FLT3-WT subgroup and 9% for the FLT3-mutant subgroup (P=0.0006). We used Prediction Analysis for Microarrays (PAM) to find the minimum number of genes that could identify samples with FLT3 mutations. PAM identified a 7-gene cluster that differentiated normal cytogenetic cases by clinical outcome. A cluster of 5 overexpressed genes (i.e., HIST1H2AC, HIST1H2AL, HIST1H2BD, HIST1H2BG, and HIST2H2BE) and 2 underexpressed genes (GASP and IVNS1ABP) were associated with poor outcome. In summary, children with AML, normal karyotype, and absence of FLT3 mutations have a good outcome. In contrast, when these same patients express a FLT3 mutation their outcome is poor despite intensive therapy. This poor outcome is associated with increased expression of several histone genes, which may be downstream targets of the FLT3 signal transduction pathway. We will corroborate these findings in a larger set of POG 9421 diagnostic samples with normal karyotypes.

Beschreibung: The role of allogeneic bone marrow transplant (BMT) in pediatric patients (pts) with de novo AML in first complete remission remains controversial. One of the objectives of POG 9421, a phase 3 trial for children with newly diagnosed AML, was to compare the disease free survival between pts genetically randomized between allogeneic BMT and consolidation chemotherapy. All pts enrolled on POG 9421 were randomized to standard dose (SD) ara-C or high dose (HD) ara-C during course 1 of induction prior to knowing if there was an HLA matched (5/6 or 6/6) sibling that might be a potential marrow donor. The intent of the study was for pts who achieved a CR and had an HLA matched sibling and did not have Down syndrome (DS) to receive a BMT following the second course of induction. The protocol preparative regimen included TBI (1200 cGy at 150 cGy bid for 4 days) and high dose etoposide (60 mg/kg iv over 4 hours on day -3). GVHD prophylaxis utilized methotrexate and cyclosporine. Six hundred and fifty-four patients were registered on POG 9421, and 32 were ineligible due to wrong diagnosis or major protocol violation and 57 had DS. Five hundred and one of 560 evaluable pts achieved a CR and were eligible for transplant if a matched sibling donor was identified. 83/501 or 16.5% of the patients had a donor and proceeded to transplant and were eligible for evaluation. Of the 83 pts, 39 received SD ara-c for induction and 44 received HD ara-c for induction. The median times from registration to CR were 39.0 days and 33.0 days, and the median times from CR to transplant were 39.0 days and 46.5 days for SD and HD ara-c, respectively. Twenty-nine of 83 pts had post BMT events including 10 deaths from toxicity, 18 relapses, and 1 second malignancy. Of the 29 events, 6 deaths and 6 relapses occurred in the SD arm and 4 deaths, 12 relapses, and 1 secondary malignancy in the HD arm. The 3yr DFS of patients who received SD ara-c induction was 71.8 +/− 7.6%, and was not significantly different from that of HD ara-c induction (3-year DFS 63.3 +/− 8.2%, p=0.49). Seven deaths occurred within 100 days of transplant. The rates of patients off protocol therapy due to event in cytogenetic subgroups are as follows: 7/25 for patients with normal cytogenetics; 3/10 for patients with 11q23 abnormalities; 6/13 for patients with t(8;21), and 7/18 for patients with miscellaneous abnormalities; there were no events during the protocol therapy among the 6 patients with inv16. The 3-year DFS and 3-year overall survival for the 83 transplanted patients were 67.3 +/− 5.6% and 69.7 +/− 5.4%, respectively. The DFS for the transplanted patients was superior to consolidation chemotherapy (N=418, 3-year DFS=37.5+/− 2.7%, p