Measurable residual disease analysis in paediatric acute lymphoblastic leukaemia patients with ABL-class fusions.

BACKGROUND: ABL-class fusions including NUP214-ABL1 and EBF1-PDGFRB occur in high risk acute lymphoblastic leukaemia (ALL) with gene expression patterns similar to BCR-ABL-positive ALL. Our aim was to evaluate new DNA-based measurable residual disease (MRD) tests detecting these fusions and IKZF1-deletions in comparison with conventional immunoglobulin/T-cell receptor (Ig/TCR) markers. METHODS: Precise genomic breakpoints were defined from targeted or whole genome next generation sequencing for ABL-fusions and BCR-ABL1. Quantitative PCR assays were designed and used to re-measure MRD in remission bone marrow samples previously tested using Ig/TCR markers. All MRD testing complied with EuroMRD guidelines. RESULTS: ABL-class patients had 46% 5year event-free survival and 79% 5year overall survival. All had sensitive fusion tests giving high concordance between Ig/TCR and ABL-class fusion results (21 patients, n = 257 samples, r2 = 0.9786, P < 0.0001) and Ig/TCR and IKZF1-deletion results (9 patients, n = 143 samples, r2 = 0.9661, P < 0.0001). In contrast, in BCR-ABL1 patients, Ig/TCR and BCR-ABL1 tests were discordant in 32% (40 patients, n = 346 samples, r2 = 0.4703, P < 0.0001) and IKZF1-deletion results were closer to Ig/TCR (25 patients, n = 176, r2 = 0.8631, P < 0.0001). CONCLUSIONS: MRD monitoring based on patient-specific assays detecting gene fusions or recurrent assays for IKZF1-deletions is feasible and provides good alternatives to Ig/TCR tests to monitor MRD in ABL-class ALL.

Whole-genome sequencing facilitates patient-specific quantitative PCR-based minimal residual disease monitoring in acute lymphoblastic leukaemia, neuroblastoma and Ewing sarcoma.

BACKGROUND: Minimal residual disease (MRD) measurement is a cornerstone of contemporary acute lymphoblastic leukaemia (ALL) treatment. The presence of immunoglobulin (Ig) and T cell receptor (TCR) gene recombinations in leukaemic clones allows widespread use of patient-specific, DNA-based MRD assays. In contrast, paediatric solid tumour MRD remains experimental and has focussed on generic assays targeting tumour-specific messenger RNA, methylated DNA or microRNA. METHODS: We examined the feasibility of using whole-genome sequencing (WGS) data to design tumour-specific polymerase chain reaction (PCR)-based MRD tests (WGS-MRD) in 18 children with high-risk relapsed cancer, including ALL, high-risk neuroblastoma (HR-NB) and Ewing sarcoma (EWS) (n = 6 each). RESULTS: Sensitive WGS-MRD assays were generated for each patient and allowed quantitation of 1 tumour cell per 10-4 (0.01%)-10-5 (0.001%) mononuclear cells. In ALL, WGS-MRD and Ig/TCR-MRD were highly concordant. WGS-MRD assays also showed good concordance between quantitative PCR and droplet digital PCR formats. In serial clinical samples, WGS-MRD correlated with disease course. In solid tumours, WGS-MRD assays were more sensitive than RNA-MRD assays. CONCLUSIONS: WGS facilitated the development of patient-specific MRD tests in ALL, HR-NB and EWS with potential clinical utility in monitoring treatment response. WGS data could be used to design patient-specific MRD assays in a broad range of tumours.

Outcomes for Australian children with relapsed/refractory acute lymphoblastic leukaemia treated with blinatumomab.

We report on the Australian experience of blinatumomab for treatment of 24 children with relapsed/refractory precursor B-cell acute lymphoblastic leukaemia (B-ALL) and high-risk genetics, resulting in a minimal residual disease (MRD) response rate of 58%, 2-year progression-free survival (PFS) of 39% and 2-year overall survival of 63%. In total, 83% (n = 20/24) proceeded to haematopoietic stem cell transplant, directly after blinatumomab (n = 12) or following additional salvage therapy (n = 8). Four patients successfully received CD19-directed chimeric antigen receptor T-cell therapy despite prior blinatumomab exposure. Inferior 2-year PFS was associated with MRD positivity (20%, n = 15) and in KMT2A-rearranged infants (15%, n = 9). Our findings highlight that not all children with relapsed/refractory B-ALL respond to blinatumomab and factors such as blast genotype may affect prognosis.

CKLF and IL1B transcript levels at diagnosis are predictive of relapse in children with pre-B-cell acute lymphoblastic leukaemia.

Disease relapse is the greatest cause of treatment failure in paediatric B-cell acute lymphoblastic leukaemia (B-ALL). Current risk stratifications fail to capture all patients at risk of relapse. Herein, we used a machine-learning approach to identify B-ALL blast-secreted factors that are associated with poor survival outcomes. Using this approach, we identified a two-gene expression signature (CKLF and IL1B) that allowed identification of high-risk patients at diagnosis. This two-gene expression signature enhances the predictive value of current at diagnosis or end-of-induction risk stratification suggesting the model can be applied continuously to help guide implementation of risk-adapted therapies.

The application of RNA sequencing for the diagnosis and genomic classification of pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy, and implementation of risk-adapted therapy has been instrumental in the dramatic improvements in clinical outcomes. A key to risk-adapted therapies includes the identification of genomic features of individual tumors, including chromosome number (for hyper- and hypodiploidy) and gene fusions, notably ETV6-RUNX1, TCF3-PBX1, and BCR-ABL1 in B-cell ALL (B-ALL). RNA-sequencing (RNA-seq) of large ALL cohorts has expanded the number of recurrent gene fusions recognized as drivers in ALL, and identification of these new entities will contribute to refining ALL risk stratification. We used RNA-seq on 126 ALL patients from our clinical service to test the utility of including RNA-seq in standard-of-care diagnostic pipelines to detect gene rearrangements and IKZF1 deletions. RNA-seq identified 86% of rearrangements detected by standard-of-care diagnostics. KMT2A (MLL) rearrangements, although usually identified, were the most commonly missed by RNA-seq as a result of low expression. RNA-seq identified rearrangements that were not detected by standard-of-care testing in 9 patients. These were found in patients who were not classifiable using standard molecular assessment. We developed an approach to detect the most common IKZF1 deletion from RNA-seq data and validated this using an RQ-PCR assay. We applied an expression classifier to identify Philadelphia chromosome-like B-ALL patients. T-ALL proved a rich source of novel gene fusions, which have clinical implications or provide insights into disease biology. Our experience shows that RNA-seq can be implemented within an individual clinical service to enhance the current molecular diagnostic risk classification of ALL.

IKZF1 Deletions with COBL Breakpoints Are Not Driven by RAG-Mediated Recombination Events in Acute Lymphoblastic Leukemia.

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in both childhood and adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Previously, we revealed that COBL is a hotspot for breakpoints in leukemia and could promote IKZF1 deletions. Through an international collaboration, we provide a detailed genetic and clinical picture of B-ALL with COBL rearrangements (COBL-r). Patients with B-ALL and IKZF1 deletion (n = 133) were included. IKZF1 ∆1-8 were associated with large alterations within chromosome 7: monosomy 7 (18%), isochromosome 7q (10%), 7p loss (19%), and interstitial deletions (53%). The latter included COBL-r, which were found in 12% of the IKZF1 ∆1-8 cohort. Patients with COBL-r are mostly classified as intermediate cytogenetic risk and frequently harbor ETV6, PAX5, CDKN2A/B deletions. Overall, 56% of breakpoints were located within COBL intron 5. Cryptic recombination signal sequence motifs were broadly distributed within the sequence of COBL, and no enrichment for the breakpoint cluster region was found. In summary, a diverse spectrum of alterations characterizes ΔIKZF1 and they also include deletion breakpoints within COBL. We confirmed that COBL is a hotspot associated with ΔIKZF1, but these rearrangements are not driven by RAG-mediated recombination.

A risk score including microdeletions improves relapse prediction for standard and medium risk precursor B-cell acute lymphoblastic leukaemia in children.

To prevent relapse, high risk paediatric acute lymphoblastic leukaemia (ALL) is treated very intensively. However, most patients who eventually relapse have standard or medium risk ALL with low minimal residual disease (MRD) levels. We analysed recurrent microdeletions and other clinical prognostic factors in a cohort of 475 uniformly treated non-high risk precursor B-cell ALL patients with the aim of better predicting relapse and refining risk stratification. Lower relapse-free survival at 7 years (RFS) was associated with IKZF1 intragenic deletions (P < 0·0001); P2RY8-CRLF2 gene fusion (P < 0·0004); Day 33 MRD>5 × 10-5 (P < 0·0001) and High National Cancer Institute (NCI) risk (P < 0·0001). We created a predictive model based on a risk score (RS) for deletions, MRD and NCI risk, extending from an RS of 0 (RS0) for patients with no unfavourable factors to RS2 +  for patients with 2 or 3 high risk factors. RS0, RS1, and RS2 +  groups had RFS of 93%, 78% and 49%, respectively, and overall survival (OS) of 99%, 91% and 71%. The RS provided greater discrimination than MRD-based risk stratification into standard (89% RFS, 96% OS) and medium risk groups (79% RFS, 91% OS). We conclude that this RS may enable better early therapeutic stratification and thus improve cure rates for childhood ALL.

Targeted Next-Generation Sequencing for Detecting MLL Gene Fusions in Leukemia.

Mixed lineage leukemia (MLL) gene rearrangements characterize approximately 70% of infant and 10% of adult and therapy-related leukemia. Conventional clinical diagnostics, including cytogenetics and fluorescence in situ hybridization (FISH) fail to detect MLL translocation partner genes (TPG) in many patients. Long-distance inverse (LDI)-PCR, the "gold standard" technique that is used to characterize MLL breakpoints, is laborious and requires a large input of genomic DNA (gDNA). To overcome the limitations of current techniques, a targeted next-generation sequencing (NGS) approach that requires low RNA input was tested. Anchored multiplex PCR-based enrichment (AMP-E) was used to rapidly identify a broad range of MLL fusions in patient specimens. Libraries generated using Archer FusionPlex Heme and Myeloid panels were sequenced using the Illumina platform. Diagnostic specimens (n = 39) from pediatric leukemia patients were tested with AMP-E and validated by LDI-PCR. In concordance with LDI-PCR, the AMP-E method successfully identified TPGs without prior knowledge. AMP-E identified 10 different MLL fusions in the 39 samples. Only two specimens were discordant; AMP-E successfully identified a MLL-MLLT1 fusion where LDI-PCR had failed to determine the breakpoint, whereas a MLL-MLLT3 fusion was not detected by AMP-E due to low expression of the fusion transcript. Sensitivity assays demonstrated that AMP-E can detect MLL-AFF1 in MV4-11 cell dilutions of 10-7 and transcripts down to 0.005 copies/ng.Implications: This study demonstrates a NGS methodology with improved sensitivity compared with current diagnostic methods for MLL-rearranged leukemia. Furthermore, this assay rapidly and reliably identifies MLL partner genes and patient-specific fusion sequences that could be used for monitoring minimal residual disease. Mol Cancer Res; 16(2); 279-85. ©2017 AACR.

Differential expression of MUC4, GPR110 and IL2RA defines two groups of CRLF2-rearranged acute lymphoblastic leukemia patients with distinct secondary lesions.

CRLF2-rearrangements (CRLF2-r) occur frequently in Ph-like B-ALL, a high-risk ALL sub-type characterized by a signaling profile similar to Ph + ALL, however accumulating evidence indicates genetic heterogeneity within CRLF2-r ALL. We performed thorough genomic characterization of 35 CRLF2-r cases (P2RY8-CRLF2 n = 18; IGH-CRLF2 n = 17). Activating JAK2 mutations were present in 34% of patients, and a CRLF2-F232C mutation was identified in an additional 17%. IKZF1 deletions were detected in 63% of cases. The majority of patients (26/35) classified as Ph-like, and these were characterized by significantly higher levels of MUC4, GPR110 and IL2RA/CD25. In addition, Ph-like CRLF2-r samples were significantly enriched for IKZF1 deletions, JAK2/CRLF2 mutations and increased expression of JAK/STAT target genes (CISH, SOCS1), suggesting that mutation-driven CRLF2/JAK2 activation is more frequent in this sub-group. Less is known about the genomics of CRLF2-r cases lacking JAK2-pathway mutations, but KRAS/NRAS mutations were identified in 4/9 non-Ph-like samples. This work highlights the heterogeneity of secondary lesions which may arise and influence intracellular-pathway activation in CRLF2-r patients, and importantly presents distinct therapeutic targets within a group of patients harboring identical primary translocations, for whom efficient directed therapies are currently lacking.

Monitoring of childhood ALL using BCR-ABL1 genomic breakpoints identifies a subgroup with CML-like biology.

We used the genomic breakpoint between BCR and ABL1 genes for the DNA-based monitoring of minimal residual disease (MRD) in 48 patients with childhood acute lymphoblastic leukemia (ALL). Comparing the results with standard MRD monitoring based on immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements and with quantification of IKZF1 deletion, we observed very good correlation for the methods in a majority of patients; however, >20% of children (25% [8/32] with minor and 12.5% [1/8] with major-BCR-ABL1 variants in the consecutive cohorts) had significantly (>1 log) higher levels of BCR-ABL1 fusion than Ig/TCR rearrangements and/or IKZF1 deletion. We performed cell sorting of the diagnostic material and assessed the frequency of BCR-ABL1-positive cells in various hematopoietic subpopulations; 12% to 83% of non-ALL B lymphocytes, T cells, and/or myeloid cells harbored the BCR-ABL1 fusion in patients with discrepant MRD results. The multilineage involvement of the BCR-ABL1-positive clone demonstrates that in some patients diagnosed with BCR-ABL1-positive ALL, a multipotent hematopoietic progenitor is affected by the BCR-ABL1 fusion. These patients have BCR-ABL1-positive clonal hematopoiesis resembling a chronic myeloid leukemia (CML)-like disease manifesting in "lymphoid blast crisis." The biological heterogeneity of BCR-ABL1-positive ALL may impact the patient outcomes and optimal treatment (early stem cell transplantation vs long-term administration of tyrosine-kinase inhibitors) as well as on MRD testing. Therefore, we recommend further investigations on CML-like BCR-ABL1-positive ALL.

Heterogeneity in mechanisms of emergent resistance in pediatric T-cell acute lymphoblastic leukemia.

Relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) remains a significant clinical problem and is thought to be associated with clonal selection during treatment. In this study we used an established pre-clinical model of induction therapy to increase our understanding of the effect of engraftment and chemotherapy on clonal selection and acquisition of drug resistance in vivo. Immune-deficient mice were engrafted with patient diagnostic specimens and exposed to a repeated combination therapy consisting of vincristine, dexamethasone, L-asparaginase and daunorubicin. Any re-emergence of disease following therapy was shown to be associated with resistance to dexamethasone, no resistance was observed to the other three drugs. Immunoglobulin/T-cell receptor gene rearrangements closely matched those in respective diagnosis and relapse patient specimens, highlighting that these clonal markers do not fully reflect the biological changes associated with drug resistance. Gene expression profiling revealed the significant underlying heterogeneity of dexamethasone-resistant xenografts. Alterations were observed in a large number of biological pathways, yet no dominant signature was common to all lines. These findings indicate that the biological changes associated with T-ALL relapse and resistance are stochastic and highly individual, and underline the importance of using sophisticated molecular techniques or single cell analyses in developing personalized approaches to therapy.

COBL is a novel hotspot for IKZF1 deletions in childhood acute lymphoblastic leukemia.

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in childhood B-cell precursor acute lymphoblastic leukemia. Because of its clinical importance, we previously mapped breakpoints of intragenic deletions and developed a multiplex PCR assay to detect recurrent intragenic ΔIKZF1. Since the multiplex PCR was not able to detect complete deletions (IKZF1 Δ1-8), which account for ~30% of all ΔIKZF1, we aimed at investigating the genomic scenery of IKZF1 Δ1-8. Six samples of cases with IKZF1 Δ1-8 were analyzed by microarray assay, which identified monosomy 7, isochromosome 7q, and large interstitial deletions presenting breakpoints within COBL gene. Then, we established a multiplex ligation-probe amplification (MLPA) assay and screened copy number alterations within chromosome 7 in 43 diagnostic samples with IKZF1 Δ1-8. Our results revealed that monosomy and large interstitial deletions within chromosome 7 are the main causes of IKZF1 Δ1-8. Detailed analysis using long distance inverse PCR showed that six patients (16%) had large interstitial deletions starting within intronic regions of COBL at diagnosis, which is ~611 Kb downstream of IKZF1, suggesting that COBL is a hotspot for ΔIKZF1. We also investigated a series of 25 intragenic deletions (Δ2-8, Δ3-8 or Δ4-8) and 24 relapsed samples, and found one IKZF1-COBL tail-to-tail fusion, thus supporting that COBL is a novel hotspot for ΔIKZF1. Finally, using RIC score methodology, we show that breakpoint sequences of IKZF1 Δ1-8 are not analog to RAG-recognition sites, suggesting a different mechanism of error promotion than that suggested for intragenic ΔIKZF1.

Integration of genetic and clinical risk factors improves prognostication in relapsed childhood B-cell precursor acute lymphoblastic leukemia.

Somatic genetic abnormalities are initiators and drivers of disease and have proven clinical utility at initial diagnosis. However, the genetic landscape and its clinical utility at relapse are less well understood and have not been studied comprehensively. We analyzed cytogenetic data from 427 children with relapsed B-cell precursor ALL treated on the international trial, ALLR3. Also we screened 238 patients with a marrow relapse for selected copy number alterations (CNAs) and mutations. Cytogenetic risk groups were predictive of outcome postrelapse and survival rates at 5 years for patients with good, intermediate-, and high-risk cytogenetics were 68%, 47%, and 26%, respectively (P < .001). TP53 alterations and NR3C1/BTG1 deletions were associated with a higher risk of progression: hazard ratio 2.36 (95% confidence interval, 1.51-3.70, P < .001) and 2.15 (1.32-3.48, P = .002). NRAS mutations were associated with an increased risk of progression among standard-risk patients with high hyperdiploidy: 3.17 (1.15-8.71, P = .026). Patients classified clinically as standard and high risk had distinct genetic profiles. The outcome of clinical standard-risk patients with high-risk cytogenetics was equivalent to clinical high-risk patients. Screening patients at relapse for key genetic abnormalities will enable the integration of genetic and clinical risk factors to improve patient stratification and outcome. This study is registered at www.clinicaltrials.org as #ISCRTN45724312.

The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

Effective targeting of the P53-MDM2 axis in preclinical models of infant MLL-rearranged acute lymphoblastic leukemia.

PURPOSE: Although the overall cure rate for pediatric acute lymphoblastic leukemia (ALL) approaches 90%, infants with ALL harboring translocations in the mixed-lineage leukemia (MLL) oncogene (infant MLL-ALL) experience shorter remission duration and lower survival rates (∼50%). Mutations in the p53 tumor-suppressor gene are uncommon in infant MLL-ALL, and drugs that release p53 from inhibitory mechanisms may be beneficial. The purpose of this study was to assess the efficacy of the orally available nutlin, RG7112, against patient-derived MLL-ALL xenografts. EXPERIMENTAL DESIGN: Eight MLL-ALL patient-derived xenografts were established in immune-deficient mice, and their molecular features compared with B-lineage ALL and T-ALL xenografts. The sensitivity of MLL-ALL xenografts to RG7112 was assessed in vitro and in vivo, and the ability of RG7112 to induce p53, cell-cycle arrest, and apoptosis in vivo was evaluated. RESULTS: Gene-expression analysis revealed that MLL-ALL, B-lineage ALL, and T-ALL xenografts clustered according to subtype. Moreover, genes previously reported to be overexpressed in MLL-ALL, including MEIS1, CCNA1, and members of the HOXA family, were significantly upregulated in MLL-ALL xenografts, confirming their ability to recapitulate the clinical disease. Exposure of MLL-ALL xenografts to RG7112 in vivo caused p53 upregulation, cell-cycle arrest, and apoptosis. RG7112 as a single agent induced significant regressions in infant MLL-ALL xenografts. Therapeutic enhancement was observed when RG7112 was assessed using combination treatment with an induction-type regimen (vincristine/dexamethasone/L-asparaginase) against an MLL-ALL xenograft. CONCLUSIONS: The utility of targeting the p53-MDM2 axis in combination with established drugs for the management of infant MLL-ALL warrants further investigation.

Persistent MRD before and after allogeneic BMT predicts relapse in children with acute lymphoblastic leukaemia.

Minimal residual disease (MRD) during early chemotherapy is a powerful predictor of relapse in acute lymphoblastic leukaemia (ALL) and is used in children to determine eligibility for allogeneic haematopoietic stem cell transplantation (HSCT) in first (CR1) or later complete remission (CR2/CR3). Variables affecting HSCT outcome were analysed in 81 children from the ANZCHOG ALL8 trial. The major cause of treatment failure was relapse, with a cumulative incidence of relapse at 5 years (CIR) of 32% and treatment-related mortality of 8%. Leukaemia-free survival (LFS) and overall survival (OS) were similar for HSCT in CR1 (LFS 62%, OS 83%, n = 41) or CR2/CR3 (LFS 60%, OS 72%, n = 40). Patients achieving bone marrow MRD negativity pre-HSCT had better outcomes (LFS 83%, OS 92%) than those with persistent MRD pre-HSCT (LFS 41%, OS 64%, P < 0·0001) or post-HSCT (LFS 35%, OS 55%, P < 0·0001). Patients with B-other ALL had more relapses (CIR 50%, LFS 41%) than T-ALL and the main precursor-B subtypes including BCR-ABL1, KMT2A (MLL), ETV6-RUNX1 (TEL-AML1) and hyperdiploidy >50. A Cox multivariate regression model for LFS retained both B-other ALL subtype (hazard ratio 4·1, P = 0·0062) and MRD persistence post-HSCT (hazard ratio 3·9, P = 0·0070) as independent adverse prognostic variables. Persistent MRD could be used to direct post-HSCT therapy.

Improving the identification of high risk precursor B acute lymphoblastic leukemia patients with earlier quantification of minimal residual disease.

The stratification of patients with acute lymphoblastic leukemia (ALL) into treatment risk groups based on quantification of minimal residual disease (MRD) after induction therapy is now well accepted but the relapse rate of about 20% in intermediate risk patients remains a challenge. The purpose of this study was to further improve stratification by MRD measurement at an earlier stage. MRD was measured in stored day 15 bone marrow samples for pediatric patients enrolled on ANZCHOG ALL8 using Real-time Quantitative PCR to detect immunoglobulin and T-cell receptor gene rearrangements with the same assays used at day 33 and day 79 in the original MRD stratification. MRD levels in bone marrow at day 15 and 33 were highly predictive of outcome in 223 precursor B-ALL patients (log rank Mantel-Cox tests both P<0.001) and identified patients with poor, intermediate and very good outcomes. The combined use of MRD at day 15 (≥ 1 × 10(-2)) and day 33 (≥ 5 × 1(-5)) identified a subgroup of medium risk precursor B-ALL patients as poor MRD responders with 5 year relapse-free survival of 55% compared to 84% for other medium risk patients (log rank Mantel-Cox test, P = 0.0005). Risk stratification of precursor B-ALL but not T-ALL could be improved by using MRD measurement at day 15 and day 33 instead of day 33 and day 79 in similar BFM-based protocols for children with this disease.