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Introduction
Cellular origin and clinical prognostic markers of infant MLLr B-ALL Methods
B-cell precursor acute lymphoblastic leukemia (BCP- ALL) is the most frequent cancer in children.1 Current 5- year survival rates in pediatric BCP-ALL approach 90%. However, BCP-ALL in infants (iBCP-ALL; <1 year of age) remains clinically challenging with an aggressive early clinical presentation in uniquely vulnerable hosts.2 Approximately 80% of iBCP-ALL are diagnosed with chromosomal rearrangements involving the mixed-lin- eage leukemia (KMTA2, also called MLL) gene, located on 11q23,3–5 which confers a dismal prognosis especially in patients carrying the t(4;11)/KMT2A-AFF1+ (MLL- AF4+).6–8
MLL is a H3K4 histone methyltransferase required for normal hematopoiesis and HOX gene expression.9,10 Leukemia transformation by MLL fusions requires the recruitment of the H3K79 histone methyltransferase Dot1L to the MLL transcriptional complex.11,12 Indeed, an H3K79 methylation profile defines both mouse and human t(4;11)/MLL-AF4+ BCP-ALL.13 Importantly, MLL rearrangements (MLLr) occur prenatally during embryon- ic/fetal hematopoiesis, and the concordance rate for iBCP-ALL in identical twins with a monochorionic pla- centa is close to 100%.14–17 This, coupled to the extremely short latency, suggests that MLL fusions might be suffi- cient for leukemogenesis.4 Accordingly, genome-wide studies using both single nucleotide polymorphism arrays and whole-genome sequencing revealed that MLLr iBCP-ALL has a very low frequency of somatic mutations with the predominant clone carrying ~1.3 non-silent mutations and one copy number alteration.18– 20 Although these studies were performed at low cover- age sequencing they reinforce the concept that MLLr iBCP-ALL requires few additional mutations to induce full transformation. In contrast, MLL-AF4-induced leuke- mogenesis has proven difficult to model.4,9 With the exception of a recent work by Lin et al.21,22 who fused human MLL to murine Af4, creating an artificial leuke- mogenic human-mouse chimeric fusion, current murine and humanized models of MLL-AF4+ BCP-ALL do not faithfully recapitulate the disease pathogenesis/pheno- type, suggesting that MLL-AF4 per se is insufficient to ini- tiate leukemogenesis.23–28
The few mutations and copy number alterations pres- ent in MLLr iBCP-ALL seem subclonal and not always retained at relapse.20 Intratumor heterogeneity drives clonal evolution in response to microenvironmental cues and cytotoxic treatment and therefore recurrent muta- tions at diagnosis and relapse may be found in minor but clinically relevant subclones.29 Here we aimed to address the clinical relevance of subclonal mutations and gene expression signatures in a large cohort of iBCP-ALL. To do this, we performed deeper exome sequencing along with whole-genome DNA- and RNA-sequencing on a large cohort of 50 MLLr and non-MLL iBCP-ALL patients uniformly treated and followed up according to an Interfant treatment protocol.30 Similarly to Anderson et al.,20 we report a silent mutational landscape in iBCP-ALL irrespective of the MLL rearrangement/status. However, strikingly, our genome-wide DNA and RNA analyses revealed new, clinically relevant information about dis- ease outcome and cell-of-origin for t(4;11) and RAS mutations.
Patients
Bone marrow or peripheral blood samples from 124 infants (<12 months old) diagnosed with either pro-B or pre-B-cell ALL were used in this study. The discovery cohort of patients was composed of 42 de novo cases: 27 with the t(4;11) encoding for MLL-AF4, five with the t(9;11) encoding for KMT2A-MLLT3 (MLL-AF9) and ten without MLLr (non-MLL B-other BCP-ALL without numerical or structural chromosomal abnormalities reported at diagnosis). Additionally, for eight MLL-AF4+ iBCP-ALL patients matched diagnostic-relapse samples were available allow- ing for longitudinal studies. MLL rearrangements were confirmed by fluorescence in-situ hybridization.31,32 For validation, an addi- tional cohort of patients, comprising 43 MLL-AF4+, 11 MLL-AF9+, and 28 non-MLL iBCP-ALL cases, was used. All patients were enrolled in the Interfant99 treatment study. Bone marrow samples were collected at Erasmus MC-Sophia Children’s Hospital (Rotterdam, the Netherlands), Armand Trousseau Hospital (Paris, France), and San Gerardo Pediatric Hospital (Monza, Italy). Complete remission bone marrow samples were available for all patients. The clinical and genetic features of the patients are pre- sented in Online Supplementary Table S1. As a control for the RNA- sequencing studies, CD34+CD19+ healthy B-cell progenitors were purified by fluorescence-activated cell sorting (FACS) from 22- week old human fetal livers (FL) as previously described.32 FL hematopoietic stem and progenitor cells (HSPC) were processed and FACS-purified from second trimester human FL as previously described.33 Briefly, cells were processed and stained for flow cytometry with up to ten fluorophore-conjugated monoclonal antibodies [antibodies (clone): CD34PECy7 (8G12), CD45RA FITC (HI100), CD19APC (HIB19), CD123PE (9F5), CD90 PECy5 (5E10), CD38 Pacific blue (HIT2), lineage cocktail APC (CD2 (RPA-2.10)/CD3 (OKT3)/CD14 (61D3)/ CD16(CB16)/ CD19 (HIB19)/CD56 (TULY56)/CD235a (HIR2)]. FACS was performed using a BD FACSAria II (Becton Dickinson). Gates were set with unstained and fluorescence minus one controls, on viable cells. Data were analyzed using FlowJo software (Tree Star). Gating strategies are as described in the results section. The study was approved by the Barcelona Clinic Hospital (2013/8529) and Hammersmith and Queen Charlotte’s Hospital (04/Q0406/145) research ethics committees.
Statistical analysis
For quantitative variables, a one-tailed t-test was used to identi- fy significant differences between groups. For qualitative vari- ables, a Fisher exact test was used in order to identify significant differences between groups of patients. Software for analysis of mutations and gene expression have their own statistical models explained in detailed in the references. Where multiple tests were performed the significance is shown corrected for multiple testing. Mutation allele frequency evolution was plotted with the R pack- age distribution Fishplot. Patterns Fisher exact test was used to assess the association between clinical characteristics and pres- ence of RAS mutations or AF4-MLL expression. Event-free sur- vival was defined as time from diagnosis to first event, i.e. resist- ance, relapse, death from any cause, or second malignant neo- plasm. Observation periods were censored at the time of last con- tact when no events were reported. Event-free survival curves were estimated with the Kaplan-Meier method and standard errors (SE) were calculated according to Greenwood. Differences in event-free survival and overall survival between groups were compared with the log-rank test. Analysis of the prognostic rele- vance of AF4-MLL/HOXA expression in combination with risk
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