Recent genomic studies have identified chromosomal rearrangements defining new subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL), however many cases lack a known initiating genetic alteration. Using integrated genomic analysis of 1,988 childhood and adult cases, we describe a revised taxonomy of B-ALL, incorporating 23 subtypes defined by chromosomal rearrangements, sequence mutations, or heterogeneous genomic alterations, many of which show marked variation in prevalence according to age. Two subtypes have frequent alterations of the B lymphoid transcription factor gene PAX5. One, PAX5alt (7.4%), has diverse PAX5 alterations (rearrangements, intragenic amplifications or mutations), and a second subtype is defined by PAX5 p.Pro80Arg and biallelic PAX5 alterations. We show that p.Pro80Arg impairs B lymphoid development and promotes the development of B-ALL with biallelic Pax5 alteration in vivo. These results demonstrate the utility of transcriptome sequencing to classify B-ALL and reinforce the central role of PAX5 as a checkpoint in B lymphoid maturation and leukemogenesis.

Philadelphia chromosome-like (Ph-like) acute lymphoblastic leukemia (ALL) is a high-risk subtype characterized by genomic alterations that activate cytokine receptor and kinase signaling. We examined the frequency and spectrum of targetable genetic lesions in a retrospective cohort of 1389 consecutively diagnosed patients with childhood B-lineage ALL with high-risk clinical features and/or elevated minimal residual disease at the end of remission induction therapy. The Ph-like gene expression profile was identified in 341 of 1389 patients, 57 of whom were excluded from additional analyses because of the presence of - (n = 46) or - (n = 11). Among the remaining 284 patients (20.4%), overexpression and rearrangement of (- or -) were identified in 124 (43.7%), with concomitant genomic alterations activating the JAK-STAT pathway (, ,) identified in 63 patients (50.8% of those with rearrangement). Among the remaining patients, using reverse transcriptase polymerase chain reaction or transcriptome sequencing, we identified targetable ABL-class fusions (, ,, and ) in 14.1%, rearrangements or fusions in 8.8%, alterations activating other JAK-STAT signaling genes (, ,) in 6.3% or other kinases (, ,) in 4.6%, and mutations involving the Ras pathway (, ,, ) in 6% of those with Ph-like ALL. We identified 8 new rearrangement partners for 4 kinase genes previously reported to be rearranged in Ph-like ALL. The current findings provide support for the precision-medicine testing and treatment approach for Ph-like ALL implemented in Children's Oncology Group ALL trials.

Chromosomal rearrangements are initiating events in acute lymphoblastic leukaemia (ALL). Here using RNA sequencing of 560 ALL cases, we identify rearrangements between MEF2D (myocyte enhancer factor 2D) and five genes (BCL9, CSF1R, DAZAP1, HNRNPUL1 and SS18) in 22 B progenitor ALL (B-ALL) cases with a distinct gene expression profile, the most common of which is MEF2D-BCL9. Examination of an extended cohort of 1,164 B-ALL cases identified 30 cases with MEF2D rearrangements, which include an additional fusion partner, FOXJ2; thus, MEF2D-rearranged cases comprise 5.3% of cases lacking recurring alterations. MEF2D-rearranged ALL is characterized by a distinct immunophenotype, DNA copy number alterations at the rearrangement sites, older diagnosis age and poor outcome. The rearrangements result in enhanced MEF2D transcriptional activity, lymphoid transformation, activation of HDAC9 expression and sensitive to histone deacetylase inhibitor treatment. Thus, MEF2D-rearranged ALL represents a distinct form of high-risk leukaemia, for which new therapeutic approaches should be considered.

• MRD measured by flow cytometry is prognostic in childhood B-ALL even with more effective high-dose methotrexate therapy.• Intensive therapy in MRDpositive patients altered the timing of relapse but did not overcome the poor prognostic significance of MRD.Minimal residual disease (MRD) is highly prognostic in pediatric B-precursor acute lymphoblastic leukemia (B-ALL). In Children's Oncology Group high-risk B-ALL study AALL0232, we investigated MRD in subjects randomized in a 2 3 2 factorial design to receive either highdose methotrexate (HD-MTX) or Capizzi methotrexate (C-MTX) during interim maintenance (IM) or prednisone or dexamethasone during induction. Subjects with end-induction MRD ‡0.1% or those with morphologic slow early response were nonrandomly assigned to receive a second IM and delayed intensification phase. MRD was measured by 6-color flow cytometry in 1 of 2 reference labs, with excellent agreement between the two. Subjects with end-induction MRD <0.01% had a 5-year event-free survival (EFS) of 87% 6 1% vs 74% 6 4% for those with MRD 0.01% to 0.1%; increasing MRD amounts was associated with progressively worse outcome. Subjects converting from MRD positive to negative by end consolidation had a relatively favorable 79% 6 5% 5-year disease-free survival vs 39% 6 7% for those with MRD ‡0.01%. Although HD-MTX was superior to C-MTX, MRD retained prognostic significance in both groups (86% 6 2% vs 58% 6 4% for MRD-negative vs positive C-MTX subjects; 88% 6 2% vs 68% 6 4% for HD-MTX subjects). Intensified therapy given to subjects with MRD >0.1% did not improve either 5-year EFS or overall survival (OS). However, these subjects showed an early relapse rate similar to that seen in MRD-negative ones, with EFS/OS curves for patients with 0.1% to 1% MRD crossing those with 0.01% to 0.1% MRD at 3 and 4 years, thus suggesting that the intensified therapy altered the disease course of MRD-positive subjects. Additional interventions targeted at the MRD-positive group may further improve outcome. This trial was registered at www.clinicaltrials.gov as #NCT00075725. (Blood. 2015;126(8):964-971) IntroductionMinimal residual disease (MRD) is highly predictive of relapse in children, adolescents, and young adults treated for acute lymphoblastic leukemia (ALL). [1][2][3][4][5][6] MRD is typically measured either by assessment of clone-specific markers of immunoglobulin and/or T-cell receptor gene rearrangements using polymerase chain reaction (PCR) or by flow cytometry, taking advantage of the fact that leukemic cells have phenotypes that allow them to be distinguished from normal cells. The Children's Oncology Group (COG) has been assessing MRD by flow cytometry at the end of 4 weeks of induction therapy in subjects with newly diagnosed ALL since 1999, and has previously demonstrated that this is the most powerful predictor of outcome in children, adolescents, and young adults with B-precursor ALL (B-ALL). Because MRD is known to be such a strong prognostic factor, most studies of childhood ALL use this to deter...

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