Rearrangement of NUTM1 gene (NUTM1r) is one of the most frequent aberrations occurring in infants (younger than 1 year at diagnosis) with B-cell precursor Acute Lymphoblastic Leukaemia (BCP-ALL). In this study we had the unique opportunity to analyze the umbilical cord blood (UCB) sample from one infant patient with NUTM1r BCP-ALL. Herein we reported for the first time that NUTM1r infant ALL arise prenatally, as both the patient-specific CUX1::NUTM1 fusion gene, as well as two IG/TR leukaemic markers were already present and detectable in the patient's UCB at birth. Our results clearly demonstrate the prenatal origin of NUTM1r infant BCP-ALL.

KMT2A rearrangements are associated with a poor clinical outcome in infant, pediatric, and adult acute lymphoblastic and myeloid leukemia. Here, we present a protocol to reconstruct chromosomal translocations with different partner genes of KMT2A in vitro. We describe steps for patient-specific single guide RNA (sgRNA) design, optimized sgRNA in vitro transcription, detailed purification of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB), and CRISPR-Cas9 editing of the test cell line K562 as well as UCB HSPCs. The provided methodology is donor independent.

Measurable residual disease (MRD) monitoring in childhood acute myeloid leukemia (AML) is used to assess response to treatment and for early detection of imminent relapse. In childhood AML, MRD is typically evaluated using flow cytometry, or by quantitative detection of leukemia-specific aberrations at the mRNA level. Both methods, however, have significant limitations. Recently, we demonstrated the feasibility of MRD monitoring in selected subgroups of AML at the genomic DNA (gDNA) level. To evaluate the potential of gDNA-based MRD monitoring across all AML subtypes, we conducted a comprehensive analysis involving 133 consecutively diagnosed children. Integrating next-generation sequencing into the diagnostic process, we identified (presumed) primary genetic aberrations suitable as MRD targets in 97% of patients. We developed patient-specific quantification assays and monitored MRD in 122 children. The gDNA-based MRD monitoring via quantification of primary aberrations with a sensitivity of at least 10 −4 was possible in 86% of patients; via quantification with sensitivity of 5 × 10 −4 , of secondary aberrations, or at the mRNA level in an additional 8%. Importantly, gDNA-based MRD exhibited independent prognostic value at early time-points in patients stratified to intermediate-/high-risk treatment arms. Our study demonstrates the broad applicability, feasibility, and clinical significance of gDNA-based MRD monitoring in childhood AML.

Pediatric acute myeloid leukemia (AML) is a highly heterogeneous disease making standardized measurable residual disease (MRD) assessment challenging. Currently, patient-specific DNA-based assays are only rarely applied for MRD assessment in pediatric AML. We tested whether quantification of genomic breakpoint-specific sequences via quantitative polymerase chain reaction (gDNA-PCR) provides a reliable means of MRD quantification in children with non-standardrisk AML and compared its results to those obtained with state-of-the-art ten-color flow cytometry (FCM). Breakpointspecific gDNA-PCR assays were established according to Euro-MRD consortium guidelines. FCM-MRD assessment was performed according to the European Leukemia Network guidelines with adaptations for pediatric AML. Of 77 consecutively recruited non-standard-risk pediatric AML cases, 49 (64%) carried a chromosomal translocation potentially suitable for MRD quantification. Genomic breakpoint analysis returned a specific DNA sequence in 100% (41/41) of the cases submitted for investigation. MRD levels were evaluated using gDNA-PCR in 243 follow-up samples from 36 patients, achieving a quantitative range of at least 10-4 in 231/243 (95%) of samples. Comparing gDNA-PCR with FCM-MRD data for 183 bone marrow follow-up samples at various therapy timepoints showed a high concordance of 90.2%, considering a cut-off of ≥0.1%. Both methodologies outperformed morphological assessment. We conclude that MRD monitoring by gDNA-PCR is feasible in pediatric AML with traceable genetic rearrangements and correlates well with FCM-MRD in the currently applied clinically relevant range, while being more sensitive below that. The methodology should be evaluated in larger cohorts to pave the way for clinical application.

Background The BCR::ABL1 is a hallmark of chronic myeloid leukemia (CML) and is also found in acute lymphoblastic leukemia (ALL). Most genomic breaks on the BCR side occur in two regions - Major and minor - leading to p210 and p190 fusion proteins, respectively. Methods By multiplex long-distance PCR or next-generation sequencing technology we characterized the BCR::ABL1 genomic fusion in 971 patients (adults and children, with CML and ALL: pediatric ALL: n = 353; pediatric CML: n = 197; adult ALL: n = 166; adult CML: n = 255 patients) and designed "Break-App" web tool to allow visualization and various analyses of the breakpoints. Pearson's Chi-Squared test, Kolmogorov-Smirnov test and logistic regression were used for statistical analyses. Results Detailed analysis showed a non-random distribution of breaks in both BCR regions, whereas ABL1 breaks were distributed more evenly. However, we found a significant difference in the distribution of breaks between CML and ALL. We found no association of breakpoints with any type of interspersed repeats or DNA motifs. With a few exceptions, the primary structure of the fusions suggests non-homologous end joining being responsible for the BCR and ABL1 gene fusions. Analysis of reciprocal ABL1::BCR fusions in 453 patients showed mostly balanced translocations without major deletions or duplications. Conclusions Taken together, our data suggest that physical colocalization and chromatin accessibility, which change with the developmental stage of the cell (hence the difference between ALL and CML), are more critical factors influencing breakpoint localization than presence of specific DNA motifs.

KMT2A-rearranged acute lymphoblastic infant leukemia (KMT2A-r iALL) is associated with outsize risk of relapse and relapse mortality. We previously reported strong upregulation of the immediate early gene EGR3 in KMT2A::AFF1 iALL at relapse; now we provide analyses of the EGR3 regulome, which we assessed through binding and expression target analysis of an EGR3-overexpressing t(4;11) cell culture model. Our data identify EGR3 as a regulator of early B-lineage commitment. Principal component analysis of 50 KMT2A-r iALL patients at diagnosis and 18 at relapse provided strictly dichotomous separation of patients based on the expression of four B-lineage genes. Absence of B-lineage gene expression translates to more than two-fold poorer long-term event-free survival. In conclusion, our study presents four B-lineage genes with prognostic significance, suitable for gene expression-based risk stratification of KMT2A-r iALL patients.

Expression of the exon 10 fusion transcript in patient 17 and patient 32. Standard curve was established using patient 32 sample in a range from 25ng/µl to 0.1 ng/µl in 1 in 2 serial dilutions.

Infant acute lymphoblastic leukemia is a devastating disease which arises mainly due to the occurrence of KMT2A rearrangements (KMT2A-r). In this context, risk stratification is an indispensable pre-treatment step enabling assignment of patients to appropriate intervention subgroups. KMT2A-r, young age, poor Glucocorticoid response rates, and high blast counts are the primarily applied risk-determining factors. Our group recently described 100-fold increased expression of the immediate early gene EGR3 at time of relapse compared to diagnosis. Here, we present the EGR3 regulome of infant KMT2A-r ALL which was assessed through integration of ChIP-seq and MACE-seq data of an EGR3-overexpressing KMT2A::AFF1 cell culture model. Binding and expression target analysis followed by gene set enrichment analysis, motif scanning, and flow cytometry identified EGR3 as a mediator of B lineage commitment. Moreover, EGR3 was found to collaborate with a set of B lineage transcription factors including PAX5. Gene expression analysis of patient cohorts from diagnosis (n=50) and relapse (n=18) uncovered strict bimodal clustering of patients determined by four EGR3-regulated B lineage marker genes. Although the whole diagnosis cohort was assigned to the high-risk group, event free survival analysis of patient clusters demonstrated absence of B lineage gene expression translating to clearly inferior long-term event-free survival. Possible confounders including age, blast percentage, and HOXA status did not affect dichotomous separation of patients. In conclusion, our study identified EGR3 as a mediator of B lineage specification and commitment in infant KMT2A::AFF1 ALL and characterized the EGR3 regulome in detail. Furthermore, we present four B lineage markers whose gene expression can be applied for substratification of high-risk patients potentially improving clinical decision making. Infant acute lymphoblastic leukemia is a devastating disease which arises mainly due to the occurrence of KMT2A rearrangements (KMT2A-r). In this context, risk stratification is an indispensable pre-treatment step enabling assignment of patients to appropriate intervention subgroups. KMT2A-r, young age, poor Glucocorticoid response rates, and high blast counts are the primarily applied risk-determining factors. Our group recently described 100-fold increased expression of the immediate early gene EGR3 at time of relapse compared to diagnosis. Here, we present the EGR3 regulome of infant KMT2A-r ALL which was assessed through integration of ChIP-seq and MACE-seq data of an EGR3-overexpressing KMT2A::AFF1 cell culture model. Binding and expression target analysis followed by gene set enrichment analysis, motif scanning, and flow cytometry identified EGR3 as a mediator of B lineage commitment. Moreover, EGR3 was found to collaborate with a set of B lineage transcription factors including PAX5. Gene expression analysis of patient cohorts from diagnosis (n=50) and relapse (n=18) uncovered strict bimodal clustering of patients determined by four EGR3-regulated B lineage marker genes. Although the whole diagnosis cohort was assigned to the high-risk group, event free survival analysis of patient clusters demonstrated absence of B lineage gene expression translating to clearly inferior long-term event-free survival. Possible confounders including age, blast percentage, and HOXA status did not affect dichotomous separation of patients. In conclusion, our study identified EGR3 as a mediator of B lineage specification and commitment in infant KMT2A::AFF1 ALL and characterized the EGR3 regulome in detail. Furthermore, we present four B lineage markers whose gene expression can be applied for substratification of high-risk patients potentially improving clinical decision making.

Abstract IKZF1 deletions are associated with an increased risk of relapse in B-cell precursor acute lymphoblastic leukemia (B-ALL), and their accurate detection has great clinical impact. Here, we included four international cohorts of pediatric and adult patients with B-ALL, and reviewed literature to illustrate the recombination map of IKZF1 deletions, with a focus at non-recurrent deletions. We provide a substantial basis for the improvement of diagnostic methods based on MLPA and multiplex PCR for the identification of IKZF1 deletions, and also demonstrate that rare IKZF1 deletions increase the incidence of relapse in these patients. Of note, non-recurrent deletions comprised a wide range of alterations, but the majority were Δ1 and Δ1–3. They were often associated with reciprocal IKZF1 fusions. So far, a total of 23 IKZF1 gene fusions were identified in B-ALL. We also verified the occurrence of the heptamer sequence (E-value: 9.9 x 10− 9) and an enrichment of GC nucleotides (71% versus 56%; P value = 4.9 x 10− 3) exclusively within breakpoint clusters, suggesting that RAG recombination and TdT activity may promote the majority of IKZF1 deletions, although rare types of alterations may be associated with other molecular mechanism of leukemogenesis, such as microhomology-mediated end joining.

KMT2A-rearranged (KMT2A-r) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is widely recognized as a high-risk leukemia in both children and adults. However, there is a paucity of data on adults treated in recent protocols, and the optimal treatment strategy for these patients is still a matter of debate. In this study, we set out to refine the prognosis of adult KMT2A-r BCP-ALL treated with modern chemotherapy regimen and investigate the prognostic impact of comutations and minimal residual disease (MRD). Of 1091 adult patients with Philadelphia-negative BCP-ALL enrolled in 3 consecutive trials from the Group for Research on Adult Acute Lymphoblastic Leukemia (GRAALL), 141 (12.9%) had KMT2A-r, with 5-year cumulative incidence of relapse (CIR) and overall survival (OS) rates of 40.7% and 53.3%, respectively. Molecular profiling highlighted a low mutational burden in this subtype, reminiscent of infant BCP-ALL. However, the presence of TP53 and/or IKZF1 alterations defined a subset of patients with significantly poorer CIR (69.3% vs 36.2%; P = .001) and OS (28.1% vs 60.7%; P = .006) rates. Next, we analyzed the prognostic implication of MRD measured after induction and first consolidation, using both immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements and KMT2A genomic fusion as markers. In approximately one-third of patients, IG/TR rearrangements were absent or displayed clonal evolution during the disease course, compromising MRD monitoring. In contrast, KMT2A-based MRD was highly reliable and strongly associated with outcome, with early good responders having an excellent outcome (3-year CIR, 7.1%; OS, 92.9%). Altogether, our study reveals striking heterogeneity in outcomes within adults with KMT2A-r BCP-ALL and provides new biomarkers to guide risk-based therapeutic stratification.

Cellular ontogeny and MLL breakpoint site influence the capacity of MLL-edited CD34+ hematopoietic cells to initiate and recapitulate infant patients' features in pro-B-cell acute lymphoblastic leukemia (B-ALL). We provide key insights into the leukemogenic determinants of MLL-AF4+ infant B-ALL.

This file contains Supplementary Methods describing the Pediatric Preclinical Testing Program (PPTP) scoring method. Supplementary Figure S1: Engraftment rates and organ infiltration of MLL-ALL xenografts. Supplementary Figure S2: Comparison of expression of specific genes between MLL-ALL and BCP-ALL xenografts. Supplementary Figure S3: Comparison of HOXA gene expression between MLL-ALL and BCP-ALL xenografts. Supplementary Figure S4: RG7112 induces caspase-dependent cell death in RS4;11 cells. Supplementary Figure S5: In vivo efficacy of RG7112 against MLL-ALL xenografts. Supplementary Figure S6: Effects of RG7112 on mouse weight and haematological parameters. Supplementary Figure S7: Immunoblots of spleen-derived cells from replicate mice showing the effects of RG7112 on MLL-14 xenograft cells. Supplementary Figure S8: Relationships between TP53 and MDM2 gene expression and in vivo responses of ALL xenografts to RG7112. Supplementary Table S1: Rates of serial engraftment of MLL-ALL xenografts. Supplementary Table S2: The top 100 genes that distinguish MLL-rearranged ALL xenografts from BCP-ALL xenografts. Supplementary Table S3: Statistical analysis of genes identified by Armstrong et al (2002) between MLL-ALL and BCP-ALL xenografts. Supplementary Table S4: Details of individual mouse responses to in vivo treatment with RG7112. Supplementary Table S5: In vitro Combination Indices of RG7112 with established drugs used to treat ALL. Supplementary Table S6: In vivo efficacy of RG7112 in combination with a VXL induction-type regimen. Supplementary Table S7: Details of individual mouse responses to in vivo treatment with RG7112 combined with VXL against MLL-ALL xenografts.

The BCR::ABL1 fusion gene is a hallmark of chronic myeloid leukemia (CML) but is also found in patients with acute lymphoblastic leukemia (ALL). There are two main breakpoint clusters in BCR - “minor” (between exons 1-2, resulting in p190 fusion protein, prevalent in ALL and scarce in CML) and “Major” (between exons 13-15, resulting in p210 protein). On the ABL1 side, the breakpoints are mostly localized between exons 1-3. Due to large intronic areas where the breakpoints occur, only a few papers have been published focusing on the structure of BCR::ABL1 fusions. Moreover, with a single exception, these studies included only the Major- BCR::ABL1 patients. Here, we focused on the analysis of BCR::ABL1 breakpoints involving CML and ALL, children and adults, and Major and minor forms of fusion. To our knowledge, we present data on the largest cohort of patients with BCR::ABL1 fusion identified at the DNA level described to date. Genomic breakpoints were found in 884 patients with BCR::ABL1+ ALL (n=463) and CML (n=421) by multiplex long distance PCR/Sanger sequencing or by NGS Custom Target Enrichment. The RSS database, MEME software and RepeatMasker were used to search RSS, specific motifs known for mediation of DNA breaks (59 motifs) and interspersed and other types of repeats within particular DNA areas. The uniformity test performed on breakpoint distribution within the minor (n=356) and Major (n=528) BCR areas revealed a non-random pattern (p=2.34e-21 and p=2.56e-10, respectively) with breakpoints accumulated in the second halves of the intron 1 (minor BCR) and intron 13 (Major BCR). Within the ABL1 area, breakpoint sites were distributed more randomly (p=9.28e-03); however, the breakpoint distribution within the ABL1 differed significantly between patients with CML and ALL (p=7.43e-05; see Figure) with higher accumulation of breakpoints near the 5‘ end of the ABL1 intron 1 in CML and near the 3‘ end of the intron 1 in ALL. The difference was not driven by the type of fusion (minor vs. Major BCR) as it was still visible even if only Major BCR::ABL1+ patients were analysed (414 CML and 108 ALL; p=2.28e-03). Comparison between various groups of patients (females vs. males; adults vs. children; various age groups, minor vs. Major BCR fusion [in ALL patients]), did not reveal a significantly distinct breakpoint distribution in any BCR or ABL1 area. Analysis of genomic breakpoints showed that fusions are mostly formed in loci with short homologies (49%; 1-71bp, median=2bp), by blunt end junctions (36%) or by a junction with insertion of random nucleotides (13%; 1-42bp, median=3bp) strongly suggesting NHEJ mechanism of double-strand break repair in vast majority of cases. In 313 patients we compared BCR::ABL1 and the reciprocal ABL1::BCR fusion. Vast majority of the BCR::ABL1 and ABL1::BCR breakpoints (81% and 79% on BCR and ABL1 area, respectively) were located within ±100bp window, showing almost precise reciprocal translocation, with negligible losses/gains of DNA resulting from the fusion. Analysis of the association of breakpoints with DNA motifs and chromatin structure did not reveal any significant association with the localization of breakpoints. In conclusion, we show different breakpoint distribution in ABL1 between CML and BCR::ABL1+ ALL. The double-strand breaks are repaired by NHEJ; however, we have not found any DNA or epigenetic motif, which might be responsible for the breakpoints. Our data suggest that the differences in breakpoint distribution are due to different chromatin accessibility and/or different relative positions of the BCR and ABL1 genes throughout the cell development, reflecting the origin of CML in earlier stages of hematopoiesis compared to ALL. Supported by AZV (NU21-03-00128) and NICR No. LX22NPO5102.

Monitoring of minimal residual disease (MRD) in childhood acute myeloid leukemia (AML) can assess response to treatment and predict relapse. Current MRD monitoring methods in childhood AML - flow cytometry and quantitation of fusion transcripts - have significant shortcomings and do not cover the entire spectrum of patients. We demonstrated (Lukes et al, Hemasphere 2020) the feasibility of MRD monitoring using DNA-based primary targets in selected subgroups of AML. Now we extended this approach to the unselected consecutive population of pediatric AML. We included 133 children (0-18) out of 135 consecutively diagnosed with AML in Czechia (2012-2022), all treated on non-MRD-based protocols. For genetic characterization, we used algorithm consisting of screening for recurrent gene fusions and mutations by PCR and targeted-NGS followed by transcriptome sequencing (WTS). Targeted-NGS (or PCR) was performed to identify genomic fusion sequences. 130 patients were eligible for MRD monitoring (3 patients died early). MRD was preferentially monitored by DNA-based qPCR (alternatively by amplicon NGS or qRT-PCR). MRD levels were expressed relative to diagnosis. Primary genetic aberration was found by targeted screening in 102 children; remaining 31 were further investigated using WTS. In 27 children, WTS identified rare, novel or atypical primary genetic aberrations not covered by the targeted screening. Primary genetic aberration was found in 97% of cases (129/133). Majority of AMLs (81%) were classified into common subtypes: AML with KMT2Ar, PML::RARA, RUNX1::RUNX1T1, CBFB::MYH11, mutation (m) of GATA1 , CEBPA or NPM1. Recurrent but rare aberrations in pediatric AML were found in 16 patients: UBTFm, RUNX1m, HOXA10 translocation, KAT6A::CREBBP, KAT6A::LEUTX, DEK::NUP214, BCR::ABL1, NUP98::NSD1 and CBFA2T3::GLIS2. In 5 patients, fusion genes were found, previously described sporadically (SPFQ::ZFP36L216, XPO1::TNRC1817) or not at all in AML (ETV6::CTNNB1, FUS::FEV, ZEB2::RUNX1). Only secondary aberrations were identified in remaining 4 patients. In 82 patients (of 84 examined), genomic fusion sequence was found and qPCR designed and used. Of 41 children carrying genetic aberrations other than fusions, in 29 quantification system was successfully implemented. Altogether, quantification system for MRD monitoring was established in 122 patients, with primary aberrations as DNA targets in 116 patients, reaching sensitivity of 10 -4 in 112 and 5x10 -4 in 4 patients. Four and two children were monitored with sensitivity of 10 -4 using secondary aberrations as DNA targets or by qRT-PCR-based quantification of fusion transcripts, respectively. MRD clearance significantly differed among genetic subtypes: while GATA1m AML had the fastest response, in contrast to patients with prognostically favorable genetic subtypes - CBFB::MYH11, RUNX1::RUNX1T1, CEBPAm and NPM1m - who were treated predominantly on the standard risk (SR) arm of the AML-BFM 2012 Registry Protocol and none achieved molecular remission (mREM) at D28 (70% vs 0%, p < 0.0001). Significant proportion of patients (25-89% within individual subtypes) did not achieve mREM even after the last block of chemotherapy. The response of patients with KMT2Ar AML and AML classified into remaining subtypes (predominantly intermediate- and high-risk) was overall faster compared to prognostically favorable subtypes on SR arm (KMT2Ar D28 mREM 33% vs 0% at D28, p < 0.0001). KMT2A::MLLT10/3 had faster clearance then other KMT2Ar (mREM D28 44% vs. 0% in other KMT2Ar, p = 0.02). In a multivariate analysis including (cyto)genetic risk and treatment, D28 MRD was the only significant predictor of outcome using both the 10 -3 (p = 0.006 for EFS and 0.012 for OS) and 10 -2 levels (p = 0.004 for EFS and 0.01 for OS) for stratification. Similarly, in an alternative multivariate analysis model, D56 MRD (positive at any level versus negative) was also the only significant predictor of outcome independent of risk and treatment (p = 0.005 for EFS and 0.01 for OS) In summary, we present a strategy for MRD monitoring in pediatric AML that is technologically feasible, real-life applicable to vast majority of all patients, and has clear prognostic significance. Supported by National Institute of Cancer Research No. LX22NPO5102 funded by the European Union-Next Generation EU, and AZV grant NU20-07-00322.

Background:IKZF1 deletions are associated with an increased risk of relapse in patients with B-cell precursor acute lymphoblastic leukemia (B-ALL), and their accurate detection has great clinical impact. Aims: We illustrate the recombination map of IKZF1 deletions, with a focus on non-recurrent deletions (neither Δ1-8, Δ2-3, Δ2-7, Δ2-8, Δ4-7, nor Δ4-8). In addition, we evaluate whether rare deletions are associated with a dismal prognosis for B-ALL patients, and provide a substantial basis for the improvement of diagnostic methods based on MLPA and multiplex PCR (M-PCR) to identify IKZF1 deletions. Methods: We included pediatric and adult patients with B-ALL of four international cohorts. IKZF1 deletions were screened using MLPA, and non-recurrent deletions were verified by NGS. After mapping the breakpoints, we identified breakpoint clusters (BC) to inspect genetic signatures associated with these DNA breaks. We used MEME for the agnostic motif search and FIMO for the identification of motifs in individual samples. We also annotated the presence of filler DNA (additional nucleotides) or microhomologies at the deletion junctions. The sequence data was used to update MLPA and M-PCR for the detection of IKZF1 deletions. Last, we compared the overall survival (OS) and cumulative incidence of relapse (CIR) of (1) non-recurrent IKZF1 deletions vs. (2) IKZF1 wild-type or (3) IKZF1 Δ4-7. The OS was compared by the log-rank test, while the CIR was analyzed by the Gray test. All data analyses were performed using R studio. Results: From 1,608 B-ALL samples, 17% had IKZF1 deletions. Non-recurrent deletions comprised 7% of them, and displayed a wide range of alterations: Δ1, Δ1-2, Δ1-3, Δ1-4, Δ1-5, Δ1-7, Δ2, Δ3, Δ4-6, Δ5, Δ5-7, Δ5-8 and Δ6-8. They included a total of 23 IKZF1 fusions. Most of them were reciprocal, leading to Δ1 and Δ1-3. After mapping the breakpoints, we defined 24 BCs. Four BCs (5’BC01, 5’BC02, 3’BC01, and 3’BC02) promoted rare rearrangements. The majority of ∆1 (9 out of 13) were associated with 3’BC01. This information allowed us to determine one commonly deleted region within IKZF1 exon 1, which is ideal for designing MLPA probes for detection of such deletions. In addition, it provided fundamental information to design an updated M-PCR to detect rare (∆1, ∆1-2, ∆1-3 and ∆2-3) and the most recurrent (∆2-7, ∆2-8, ∆4-7 and ∆4-8) deletions. Of note, our novel M-PCR extends the coverage of previous assays, which frequently missed the detection of ∆2-8 and ∆4-8. We also verified the occurrence of a heptamer-like sequence of RAG1 (E-value: 9.9 x 10-9) within BCs and an enrichment of GC nucleotides filling their deletion junctions (95% vs. 47% in deletions outside any BC). The three IKZF1 status groups had similar OS rates (P value = 0.60). On the other hand, we observed a higher 10-yrs CIR in patients with non-recurrent IKZF1 deletion compared to wild-type (14.7% vs. 50.1%; P value = 0.09). Summary/Conclusion: This study provides a wide spectrum of structural alterations that affect the IKZF1 gene, and point out methodological adjustments in MLPA and M-PCR to fine-tune the detection of IKZF1 deletions. Also, we summarize several levels of evidence that support the idea that RAG and TdT often mediate these alterations. Last, our results highlight that rare and recurrent deletions impact the prognosis of B-ALL patients. Therefore, their identification may contribute to appropriate risk stratification and therapeutic benefits for them. Keywords: Gene fusion, Ikaros, B cell acute lymphoblastic leukemia

Comparison of MLL fusions detected by cytogenetics, fluorescent in situ hybridization (FISH), Long distance Inverse polymerase chain reaction (LDI-PCR) and Anchored multiplex PCR enrichment (AMP-E).