Elisabeth R. van Wering

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.

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.

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.

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.

Regenerating normal B-cell precursors during and after treatment of acute lymphoblastic leukaemia: Implications for monitoring of minimal residual disease

We studied 57 childhood acute lymphoblastic leukaemia (ALL) patients who remained in continuous complete remission after treatment according to the Dutch Childhood Leukaemia Study Group ALL‐8 protocols. The patients were monitored at 18 time points during and after treatment [640 bone marrow (BM) and 600 blood samples] by use of cytomorphology and immunophenotyping for the expression of TdT, CD34, CD10 and CD19. Additionally, 60 BM follow‐up samples from six patients were subjected to clonality assessment via heteroduplex polymerase chain reaction (PCR) analysis of immunoglobulin V h‐J h gene rearrangements. We observed substantial expansions of normal precursor B cells in regenerating BM not only after maintenance therapy but also during treatment. At the end of the 2‐week intervals after consolidation and reinduction treatment, B‐cell‐lineage regeneration was observed in BM with a large fraction of immature CD34+/TdT+ B cells. In contrast, in regenerating BM after cessation of maintenance treatment, the more mature CD19+/CD10+ B cells were significantly increased, but the fraction of immature CD34+/TdT+ B cells was essentially smaller. Blood samples showed a profound B‐cell lymphopenia during treatment followed by a rapid normalization of blood B cells after treatment, with a substantial CD10+ fraction (10–30%). Heteroduplex PCR analysis confirmed the polyclonal origin of the expanded precursor B cells in regenerating BM. This information regarding the regeneration of BM is essential for the correct interpretation of minimal residual disease studies.

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.

Genetic Variations in the Glucocorticoid Receptor Gene Are Not Related to Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia

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.

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.

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.

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.