Implementation of flow cytometry in the diagnostic work-up of myelodysplastic syndromes in a multicenter approach: report from the Dutch Working Party on Flow Cytometry in MDS.

Flow cytometry (FC) is recognized as an important tool in the diagnosis of myelodysplastic syndromes (MDS) especially when standard criteria fail. A working group within the Dutch Society of Cytometry aimed to implement FC in the diagnostic work-up of MDS. Hereto, guidelines for data acquisition, analysis and interpretation were formulated. Based on discussions on analyses of list mode data files and fresh MDS bone marrow samples and recent literature, the guidelines were modified. Over the years (2005-2011), the concordance between the participating centers increased indicating that the proposed guidelines contributed to a more objective, standardized FC analysis, thereby ratifying the implementation of FC in MDS.

Evaluation of gene expression signatures predictive of cytogenetic and molecular subtypes of pediatric acute myeloid leukemia.

BACKGROUND: Pediatric acute myeloid leukemia is a heterogeneous disease characterized by non-random genetic aberrations related to outcome. The genetic subtype is currently detected by different diagnostic procedures which differ in success rate and/or specificity. DESIGN AND METHODS: We examined the potential of gene expression profiles to classify pediatric acute myeloid leukemia. Gene expression microarray data of 237 children with acute myeloid leukemia were collected and a double-loop cross validation approach was used to generate a subtype-predictive gene expression profile in the discovery cohort (n=157) which was then tested for its true predictive value in the independent validation cohort (n=80). The classifier consisted of 75 probe sets, representing the top 15 discriminating probe sets for MLL-rearranged, t(8;21)(q22;q22), inv(16)(p13q22), t(15;17)(q21;q22) and t(7;12)(q36;p13)-positive acute myeloid leukemia. RESULTS: These cytogenetic subtypes represent approximately 40% of cases of pediatric acute myeloid leukemia and were predicted with 92% and 99% accuracy in the discovery and independent validation cohort, respectively. However, for NPM1, CEBPA, MLL(-PTD), FLT3(-ITD), KIT, PTPN11 and N/K-RAS gene expression signatures had limited predictive value. This may be caused by a limited frequency of these mutations and by underlying cytogenetics. This latter is exemplified by the fact that different gene expression signatures were discovered for FLT3-ITD in patients with normal cytogenetics and in those with t(15;17)(q21;q22)-positive acute myeloid leukemia, which pointed to HOXB-upregulation being specific for FLT3-ITD(+) cytogenetically normal acute myeloid leukemia. CONCLUSIONS: In conclusion, gene expression profiling correctly predicted the most prevalent cytogenetic subtypes of pediatric acute myeloid leukemia with high accuracy. In clinical practice, this gene expression signature may replace multiple diagnostic tests for approximately 40% of pediatric acute myeloid leukemia cases whereas only for the remaining cases (predicted as 'acute myeloid leukemia-other') are additional tests indicated. Moreover, the discriminative genes reveal new insights into the biology of acute myeloid leukemia subtypes that warrants follow-up as potential targets for new therapies.

Complex karyotype newly defined: the strongest prognostic factor in advanced childhood myelodysplastic syndrome.

To identify cytogenetic risk factors predicting outcome in children with advanced myelodysplastic syndrome, overall survival of 192 children prospectively enrolled in European Working Group of Myelodysplastic Syndrome in Childhood studies was evaluated with regard to karyotypic complexity. Structurally complex constitutes a new definition of complex karyotype characterized by more than or equal to 3 chromosomal aberrations, including at least one structural aberration. Five-year overall survival in patients with more than or equal to 3 clonal aberrations, which were not structurally complex, did not differ from that observed in patients with normal karyotype. Cox regression analysis revealed the presence of a monosomal and structurally complex karyotype to be strongly associated with poor prognosis (hazard ratio = 4.6, P < .01). Notably, a structurally complex karyotype without a monosomy was associated with a very short 2-year overall survival probability of only 14% (hazard ratio = 14.5; P < .01). The presence of a structurally complex karyotype was the strongest independent prognostic marker predicting poor outcome in children with advanced myelodysplastic syndrome.

Low frequency of MLL-partial tandem duplications in paediatric acute myeloid leukaemia using MLPA as a novel DNA screenings technique.

Mixed-lineage leukaemia (MLL)-partial tandem duplications (PTDs) are found in 3-5% of adult acute myeloid leukaemia (AML), and are associated with poor prognosis. In adult AML, MLL-PTD is only detected in patients with trisomy 11 or internal tandem duplications of FLT3 (FLT3-ITD). To date, studies in paediatric AML are scarce, and reported large differences in the frequency of MLL-PTD, frequently utilising mRNA RT-PCR only to detect MLL-PTDs. We studied the frequency of MLL-PTD in a large cohort of paediatric AML (n=276) and the results from two different methods, i.e. mRNA RT-PCR, and multiplex ligation-dependent probe amplification (MLPA), a method designed to detect copy number differences of specific DNA sequences. In some patients with an MLL-rearrangement, MLL-PTD transcripts were detected, but were not confirmed by DNA-MLPA, indicating that DNA-MLPA can more accurately detect MLL-PTD compared to mRNA RT-PCR. In paediatric AML, MLL-PTD was detected in 7/276 patients (2.5%). One case had a trisomy 11, while the others had normal cytogenetics. Furthermore 4 of the 7 patients revealed a FLT3-ITD, which was significantly higher compared with the other AML cases (p=0.016). In conclusion, using DNA-MLPA as a novel screenings technique in combination with mRNA RT-PCR a low frequency of MLL-PTD in paediatric AML was found. Larger prospective studies are needed to further define the prognostic relevance of MLL-PTD in paediatric AML.

Acquisition of genome-wide copy number alterations in monozygotic twins with acute lymphoblastic leukemia.

Chimeric fusion genes are highly prevalent in childhood acute lymphoblastic leukemia (ALL) and are mostly prenatal, early genetic events in the evolutionary trajectory of this cancer. ETV6-RUNX1-positive ALL also has multiple ( approximately 6 per case) copy number alterations (CNAs) as revealed by genome-wide single-nucleotide polymorphism arrays. Recurrent CNAs are probably "driver" events contributing critically to clonal diversification and selection, but at diagnosis, their developmental timing is "buried" in the leukemia's covert natural history. This conundrum can be resolved with twin pairs. We identified and compared CNAs in 5 pairs of monozygotic twins with concordant ETV6-RUNX1-positive ALL and 1 pair discordant for ETV6-RUNX1 positive ALL. We compared, within each pair, CNAs classified as potential "driver" or "passenger" mutations based upon recurrency and, where known, gene function. An average of 5.1 (range 3-11) CNAs (excluding immunoglobulin/T-cell receptor alterations) were identified per case. All "driver" CNAs (total of 32) were distinct within each of the 5 twin pairs with concordant ALL. "Driver" CNAs in another twin with ALL were all absent in the shared ETV6-RUNX1-positive preleukemic clone of her healthy co-twin. These data place all "driver" CNAs secondary to the prenatal gene fusion event and most probably postnatal in the sequential, molecular pathogenesis of ALL.

Dexamethasone-based therapy for childhood acute lymphoblastic leukaemia: results of the prospective Dutch Childhood Oncology Group (DCOG) protocol ALL-9 (1997-2004).

BACKGROUND: A population-based cohort of children aged 1-18 years with acute lymphoblastic leukaemia (ALL) was treated with a dexamethasone-based protocol (Dutch Childhood Oncology Group [DCOG] ALL-9). We aimed to confirm the results of the most effective DCOG ALL protocol for non-high-risk (NHR) patients to date (ALL-6), compare results with ALL-7 and ALL-8, and study prognostic factors in a non-randomised setting. METHODS: From Jan 1, 1997, until Nov 1, 2004, patients with ALL were treated according to the ALL-9 protocol in eight Dutch academic centres with their affiliated peripheral hospitals. Patients were stratified into NHR and high risk (HR) groups. HR criteria were white-blood-cell count of 50,000 cells per microL or more, T-cell phenotype, mediastinal mass, CNS or testicular involvement, and Philadelphia chromosome or MLL rearrangement; patients who did not fulfil these criteria were deemed to be NHR. The NHR group was treated with a three-drug induction (dexamethasone, vincristine, and asparaginase) for 6 weeks, medium-dose methotrexate for 3 weeks, then maintenance therapy. HR patients received a four-drug induction (as for the NHR patients plus daunorubicin) for 6 weeks, high-dose methotrexate for 8 weeks, and two intensification courses before receiving maintenance therapy. Triple intrathecal medication was given 13 times in NHR patients, 15 times in HR patients (17 times for patients with initial CNS involvement). No patient received cranial irradiation. Maintenance therapy was given until 109 weeks for all patients and consisted of mercaptopurine and methotrexate for 5 weeks, alternated with dexamethasone and vincristine for 2 weeks. Kaplan-Meier analysis was done on an intention-to-treat basis with event-free survival as the primary endpoint. This trial is registered at trialregister.nl, number NTR460/SNWLK-ALL-9. FINDINGS: 859 patients were recruited to the study. Complete remission was achieved in 592 (98.5%) of the 601 patients in the NHR group and 250 (96.9%) of the 258 in the HR group. Five patients in the NHR group and four in the HR group died during induction. Median follow-up for patients alive was 72.2 (range 4.8-132.7) months as of August, 2008. 5-year event-free survival was 81% (SE 1%) in all patients: 84% (2%) in NHR patients, and 72% (3%) in HR patients. Isolated CNS relapses occurred in 22 (2.6%) of 842 patients. In a multivariate analysis, DNA index was the strongest predictor of outcome (<1.16 vs >or=1.16; relative risk 0.42, 95% CI 0.22-0.78), followed by age (1-9 vs >or=10 years; 2.23, 1.60-3.11) and white-blood-cell count (<50,000 vs >or=50,000 cells per microL; 1.60, 1.13-2.26). INTERPRETATION: The overall results of the dexamethasone-based DCOG ALL-9 protocol are better than those of our previous Berlin-Frankfurt-Münster-based protocols ALL-7 and ALL-8. The results for NHR patients were achieved with high cumulative doses of dexamethasone and vincristine, but without the use of anthracyclines, etoposide, cyclophosphamide, or cranial irradiation, therefore minimising the risk of side-effects. FUNDING: Dutch Health Insurers.

CD34 expression is associated with poor survival in pediatric T-cell acute lymphoblastic leukemia.

BACKGROUND: Children with T-lineage acute lymphoblastic leukemia (T-ALL) have an inferior outcome with combination chemotherapy compared to B-lineage ALL, and still about 30% of the patients relapse within the first 2 years following diagnosis. As CD34 has been related with poor outcome in ALL in general, we investigated the prognostic significance of the stem cell marker CD34, as well as the association of CD34 positivity with the expression of several multidrug resistance (MDR) genes. PROCEDURE: In this retrospective study, we investigated the prognostic significance of the expression of the early T-cell differentiation marker CD34 and the expression of MDR genes in relation to outcome in a cohort of 72 newly diagnosed pediatric T-ALL patients. RESULTS: CD34 expression was related to a poor 5-year disease-free-survival and a poor 5-year overall survival. Using the Cox proportional hazard model, CD34 expression predicted for increased risk for relapse and death. Expression of CD34 was associated with elevated MDR1 and MRP1 mRNA expression levels. For the entire T-ALL cohort, these expression levels of MDR1 or MRP1 did not independently predict for poor outcome. CONCLUSIONS: We conclude that CD34-positive T-ALL has a relatively poor survival that is not explained by the mRNA expression levels of MDR1, LRP, or MRP1.

The recurrent SET-NUP214 fusion as a new HOXA activation mechanism in pediatric T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is mostly characterized by specific chromosomal abnormalities, some occurring in a mutually exclusive manner that possibly delineate specific T-ALL subgroups. One subgroup, including MLL-rearranged, CALM-AF10 or inv (7)(p15q34) patients, is characterized by elevated expression of HOXA genes. Using a gene expression-based clustering analysis of 67 T-ALL cases with recurrent molecular genetic abnormalities and 25 samples lacking apparent aberrations, we identified 5 new patients with elevated HOXA levels. Using microarray-based comparative genomic hybridization (array-CGH), a cryptic and recurrent deletion, del (9)(q34.11q34.13), was exclusively identified in 3 of these 5 patients. This deletion results in a conserved SET-NUP214 fusion product, which was also identified in the T-ALL cell line LOUCY. SET-NUP214 binds in the promoter regions of specific HOXA genes, where it interacts with CRM1 and DOT1L, which may transcriptionally activate specific members of the HOXA cluster. Targeted inhibition of SET-NUP214 by siRNA abolished expression of HOXA genes, inhibited proliferation, and induced differentiation in LOUCY but not in other T-ALL lines. We conclude that SET-NUP214 may contribute to the pathogenesis of T-ALL by enforcing T-cell differentiation arrest.

Leukemia-associated NF1 inactivation in patients with pediatric T-ALL and AML lacking evidence for neurofibromatosis.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder caused by mutations in the NF1 gene. Patients with NF1 have a higher risk to develop juvenile myelomonocytic leukemia (JMML) with a possible progression toward acute myeloid leukemia (AML). In an oligo array comparative genomic hybridization-based screening of 103 patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL) and 71 patients with MLL-rearranged AML, a recurrent cryptic deletion, del(17)(q11.2), was identified in 3 patients with T-ALL and 2 patients with MLL-rearranged AML. This deletion has previously been described as a microdeletion of the NF1 region in patients with NF1. However, our patients lacked clinical NF1 symptoms. Mutation analysis in 4 of these del(17)(q11.2)-positive patients revealed that mutations in the remaining NF1 allele were present in 3 patients, confirming its role as a tumor-suppressor gene in cancer. In addition, NF1 inactivation was confirmed at the RNA expression level in 3 patients tested. Since the NF1 protein is a negative regulator of the RAS pathway (RAS-GTPase activating protein), homozygous NF1 inactivation represent a novel type I mutation in pediatric MLL-rearranged AML and T-ALL with a predicted frequency that is less than 10%. NF1 inactivation may provide an additional proliferative signal toward the development of leukemia.

Role of mutation independent constitutive activation of FLT3 in juvenile myelomonocytic leukemia.

FLT3 gene mutations have been identified as prognostic factors in myeloid malignancies. Furthermore, FLT3 can be activated by wild type overexpression or ligand-dependent in leukemic cells co-expressing FLT3 ligand (FLT3L). So far no data are available on FLT3/FLT3L expression and activation in JMML. In 51 clinical JMML samples, activating mutations were screened, FLT3 and FLT3L mRNA levels were assessed and the sensitivity of JMML cells to the FLT3 inhibitor PKC412 was tested by MTT assays. No evidence for constitutively activation of FLT3/FLT3L was found in JMML, indicating that FLT3 inhibitors are unlikely to be effective in JMML.

The outcome of molecular-cytogenetic subgroups in pediatric T-cell acute lymphoblastic leukemia: a retrospective study of patients treated according to DCOG or COALL protocols.

BACKGROUND AND OBJECTIVES: Subgroups of T-cell acute lymphoblastic leukemia (T-ALL), defined according to recurrent cytogenetic aberrations, may have different prognoses. The aim of this study was to determine the prognostic relevance of molecular-cytogenetic abnormalities in pediatric patients using quantitative real-time polymerase chain reaction and fluorescence in situ hybridization. DESIGN AND METHODS: The patients were assigned to TAL1, HOX11/TLX1, HOX11L2/TLX3, or CALM-AF10 subgroups. The cytogenetic subgroups were characterized in relation to immunophenotype and the expression of aberrantly expressed transcription factors. RESULTS: In our cohort study, CALM-AF10 was associated with an immature immunophenotype and poor outcome (p=0.005). HOX11L2 was associated with both immunophenotypically immature cases as well as cases committed to the gammadelta-lineage. HOX11L2 was significantly associated with poor outcome (p=0.01), independently of the expression of CD1 or the presence of NOTCH1 mutations. TAL1 abnormalities were associated with alphabeta-lineage commitment, and tended to be associated with a good outcome. Cells in HOX11 cases resembled early CD1-positive cortical thymocytes without expression of Cytbeta and TCR molecules. In relation to the expression of early T-cell transcription factors, high TAL1 levels were found in immunophenotypically-advanced cases, whereas high LYL1 levels were found in immature subgroups. INTERPRETATION AND CONCLUSIONS: The reported outcomes for HOX11L2-rearranged T-ALL cases are conflicting; the prognostic impact may depend on the therapy given. In our cohort, this cytogenetic aberration was associated with a poor outcome. Our data on CALM-AF10 rearranged T-ALL, albeit based on only three patients, suggest that this type of leukemia is associated with a poor outcome.

The cryptic chromosomal deletion del(11)(p12p13) as a new activation mechanism of LMO2 in pediatric T-cell acute lymphoblastic leukemia.

To identify new cytogenetic abnormalities associated with leukemogenesis or disease outcome, T-cell acute lymphoblastic leukemia (T-ALL) patient samples were analyzed by means of the array-comparative genome hybridization technique (array-CGH). Here, we report the identification of a new recurrent and cryptic deletion on chromosome 11 (del(11)(p12p13)) in about 4% (6/138) of pediatric T-ALL patients. Detailed molecular-cytogenetic analysis revealed that this deletion activates the LMO2 oncogene in 4 of 6 del(11)(p12p13)-positive T-ALL patients, in the same manner as in patients with an LMO2 translocation (9/138). The LMO2 activation mechanism of this deletion is loss of a negative regulatory region upstream of LMO2, causing activation of the proximal LMO2 promoter. LMO2 rearrangements, including this del(11)(p12p13) and t(11;14) (p13;q11) or t(7;11)(q35;p13), were found in the absence of other recurrent cytogenetic abnormalities involving HOX11L2, HOX11, CALM-AF10, TAL1, MLL, or MYC. LMO2 abnormalities represent about 9% (13/138) of pediatric T-ALL cases and are more frequent in pediatric T-ALL than appreciated until now.

Prognostic significance of blasts in the cerebrospinal fluid without pleiocytosis or a traumatic lumbar puncture in children with acute lymphoblastic leukemia: experience of the Dutch Childhood Oncology Group.

PURPOSE: To determine the significance of blasts in the CSF without pleiocytosis and a traumatic lumbar puncture in children with acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: We retrospectively studied a cohort of 526 patients treated in accordance with the virtually identical Dutch protocols ALL-7 and ALL-8. Patients were classified into five groups: CNS1, no blasts in the CSF cytospin; CNS2, blasts present in the cytospin, but leukocytes less than 5/microL; CNS3, blasts present and leukocytes more than 5/microL. Patients with a traumatic lumbar puncture (TLP; > 10 erythrocytes/mL) were classified as TLP+ (blasts present in the cytospin) or TLP- (no blasts). RESULTS: Median duration of follow-up was 13.2 years (range, 6.9 to 15.5 years). Event-free survival (EFS) was 72.6% (SE, 2.5%) for CNS1 patients (n = 304), 70.3% (SE, 4.7%) for CNS2 patients (n = 111), and 66.7% (SE, 19%) for CNS3 patients (n = 10; no significant difference in EFS between the groups). EFS was 58% (SE, 7.6%) for TLP+ patients (n = 62) and 82% (SE, 5.2%) for TLP- patients (n = 39; P < .01). Cox regression analysis identified TLP+ status as an independent prognostic factor (risk ratio, 3.5; 95% CI, 1.4 to 8.8; P = .007). Cumulative incidence of CNS relapses was 0.05 and 0.07 in CNS1 and CNS2 patients, respectively (not statistically significant). CONCLUSION: In our experience, the presence of a low number of blasts in the CSF without pleiocytosis has no prognostic significance. In contrast, a traumatic lumbar puncture with blasts in the CSF specimen is associated with an inferior outcome.

Immunoglobulin light chain gene rearrangements in precursor-B-acute lymphoblastic leukemia: characteristics and applicability for the detection of minimal residual disease.

We analyzed the frequency and characteristics of Vk-Jk and Vlambda-Jlambda rearrangements inpatients with precursor-B-acute lymphoblastic leukemia (ALL) and evaluated the applicability of these rearrangements as targets for minimal residual disease (MRD) detection. Using the BIOMED-2 primer sets, Vk-Jk and Vlambda-Jlambda rearrangements were detected in 30% and 17% of patients, respectively. Vk-Jk rearrangements were particularly frequent in common-ALL, children between 5-10 years, and TEL-AML1-positive patients. Vk-Jk and Vlambda-Jlambda rearrangements showed a good stability between diagnosis and relapse and reached good sensitivities in real-time quantitative polymerase chain reaction analysis. Our data show that Vk-Jk and Vlambda-Jlambda rearrangements can be successfully applied for MRD detection in a subset of patients with precursor-B-ALL.

High incidence of t(7;12)(q36;p13) in infant AML but not in infant ALL, with a dismal outcome and ectopic expression of HLXB9.

The t(7;12)(q36;p13) is a recurrent translocation involving the ETV6/TEL gene (12p13) and a heterogeneous breakpoint at 7q36. A fusion transcript between HLXB9 and ETV6 in AML with t(7;12) is occasionally found. To study the incidence of t(7;12) in infant and childhood acute leukemia, we screened 320 cases <36 months using FISH. Additionally, 28 pediatric cases >36 months with cytogenetic breakpoints at 12p and 7q were investigated. We studied the presence of an HXLB9-ETV6 fusion transcript and quantified the expression of various genes located in the 7q36 breakpoint region. In total, six AML patients carried the t(7;12) of which five were infants and one child of 18 months. Only one out of 99 infant ALL patients harbored the t(7;12). No t(7;12) was found in older children with AML or ALL. AML patients carrying a t(7;12) had a poor outcome with a 3-year EFS of 0%. A fusion of HLXB9 to ETV6 was found in four AML cases with t(7;12). The 7q36 genes NOM1, LMBR1, RNF32, and SHH were equally expressed among t(7;12)-positive AML versus t(7;12)-negative AML, t(7;12)-negative ALL, or normal bone marrow. However, the HLXB9 expression was highly increased in t(7;12)-positive cases, including those with an HLXB9-ETV6 fusion. We conclude that the t(7;12) is almost exclusively present in infant AML and covers 30% of infant AML, while it is extremely rare in infant ALL and older children. The t(7;12) is associated with a poor outcome and an ectopic expression of HLXB9 is commonly involved in this genetic subtype of leukemia.

Monitoring of inosine monophosphate dehydrogenase activity in mononuclear cells of children with acute lymphoblastic leukemia: enzymological and clinical aspects.

BACKGROUND: Inosine 5'-monophosphate dehydrogenase (IMPDH; EC1.1.1.205) catalyzes the rate-limiting step in guanine nucleotide biosynthesis, and may play an important role in treatment of patients with antipurines. METHODS: We used an HPLC method to measure the IMPDH activity in peripheral blood and bone marrow mononuclear cells (MNC). IMPDH activities were determined in children who were diagnosed with and treated for acute lymphoblastic leukemia (ALL), and in a group of control children. RESULTS: The median IMPDH activity for control children was 350 pmol/10(6) pMNC/hr (range 97-896; n = 47). No gender or age differences were observed. IMPDH activity at diagnosis of ALL was correlated with the percentage of peripheral blood lymphoblasts (r = 0.474; P < 0.001; n = 71). The median IMPDH activity at diagnosis was 410 pmol/10(6) pMNC/hr (range 40-2009; n = 76), significantly higher than for controls (P = 0.012). IMPDH activity significantly decreased after induction treatment, and during treatment with methotrexate (MTX) infusions (median 174 pmol/10(6) pMNC/hr; range 52-516; n = 21). The activity remained low during maintenance treatment with 6-mercaptopurine (6MP) and MTX, at a significantly lower level than for controls (P < 0.004). One year after cessation of treatment IMPDH activity returned to normal values. CONCLUSION: The decrease of IMPDH activity at remission of ALL seems to be at least partly due to the eradication of lymphoblasts with the type 2 isoform of the enzyme.

Effect of the histone deacetylase inhibitor depsipeptide on B-cell differentiation in both TEL-AML1-positive and negative childhood acute lymphoblastic leukemia.

The fusion protein TEL-AML1 in t(12;21)+ acute lymphoblastic leukemia (ALL) recruits co-repressors and histone deacetylases (HDAC), which transrepress AML1 target genes. Normal bone marrow cells were more resistant to HDAC inhibitor FK228 induced cell killing than were cells from ALL patients with or without t(12;21). FK228 induced differentiation in ALL, irrespective of the presence of t(12;21).

Long-term results of a randomized trial on extended use of high dose L-asparaginase for standard risk childhood acute lymphoblastic leukemia.

PURPOSE: Between September 1991 and May 1997, within the International Berlin-Frankfurt-Muenster Study Group (I-BFM-SG), a randomized study was performed aimed at assessing the efficacy of prolonged use of high-dose l-asparaginase (HD-l-ASP) during continuation therapy in children with standard risk (SR) acute lymphoblastic leukemia (ALL), treated with a reduced BFM-type chemotherapy. PATIENTS AND METHODS: The Italian, Dutch, and Hungarian groups participated in this study denominated IDH-ALL-91, and 494 children were enrolled. Treatment consisted of a BFM-type modified backbone with omission of the IB part in induction and elimination of two doses of anthracyclines during reinduction in both arms at the beginning of continuation therapy. Patients were randomly assigned to receive (YES-ASP) or not (NO-ASP) 20 weekly HD-l-ASP (25,000 IU/m2). RESULTS: The event-free-survival and overall survival probabilities at 10 years for the entire group were 82.5% (1.8) and 90.3% (1.3), respectively. Of the 490 patients eligible for random assignment, 355 (72.4%) were randomly assigned (178 YES-ASP and 177 NO-ASP). After a median follow-up of 9 years, the probability of disease-free survival at 10 years was 87.5% (SE, 2.5) for YES-ASP arm versus 78.7% (SE, 3.3) for NO-ASP arm (P = .03). In multivariate analysis, NO-ASP arm (P = .03), male sex (P = .004), and age older than 10 years (P = .0003) had a significantly adverse impact on outcome. CONCLUSION: In this subset of patients, selected with criteria not including monitoring of minimal residual disease, application of extended HD-l-ASP may improve prognosis, compensating reduced leukemia control that results from adoption of a reduced-intensity BFM-backbone for treatment of children with SR ALL.

Genetic variations in the glucocorticoid receptor gene are not related to glucocorticoid resistance in childhood acute lymphoblastic leukemia.

UNLABELLED: Glucocorticoid sensitivity is an important prognostic factor in pediatric acute lymphoblastic leukemia (ALL). For its antileukemic effect, glucocorticoid binds the intracellular glucocorticoid receptor (GR) subsequently regulating transcription of downstream genes. We analyzed whether genetic variations within the GR gene are related to differences in the cellular response to glucocorticoids. METHODS: In leukemic samples of 57 children, the GR gene was screened for nucleotide variations using a PCR/single-strand conformational polymorphism sequencing strategy. Data were linked to in vivo and in vitro glucocorticoid resistance. RESULTS: No somatic mutations were detected in the GR gene coding region, but six polymorphisms (i.e., ER22/23EK, N363S, BclI, intron mutation 16 bp upstream of exon 5, H588H, and N766N) were identified. In 67% of ALL cases, at least one minor allele of these polymorphisms was detected. Although only borderline significant, the incidence for the N363S polymorphism minor allele was higher (12% versus 6%, P = 0.06) and for the ER22/23EK minor allele lower (4% versus 7.6%, P = 0.1) than in a healthy, comparable population. The different genotypes of the polymorphisms were not related to prednisone resistance. In conclusion, polymorphisms but not somatic mutations in the GR gene coding region occur in leukemic blasts of children with ALL. Our data suggest that these genetic variations are not a major contributor for differences in cellular response to glucocorticoids in childhood ALL. The higher incidence of the N363S minor allele and the lower incidence of the ER22/23EK minor allele in our ALL population as compared with a normal population warrants further research.