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PURPOSE Infant acute lymphoblastic leukemia (ALL) is characterized by a high incidence of KMT2A gene rearrangements and poor outcome. We evaluated the value of minimal residual disease (MRD) in infants with KMT2A-rearranged ALL treated within the Interfant-06 protocol, which compared lymphoid-style consolidation (protocol IB) versus myeloid-style consolidation (araC, daunorubicin, etoposide/mitoxantrone, araC, etoposide). MATERIALS AND METHODS MRD was measured in 249 infants by DNA-based polymerase chain reaction of rearranged KMT2A, immunoglobulin, and/or T-cell receptor genes, at the end of induction (EOI) and end of consolidation (EOC). MRD results were classified as negative, intermediate (< 5 × 10−4), and high (≥ 5 × 10−4). RESULTS EOI MRD levels predicted outcome with 6-year disease-free survival (DFS) of 60.2% (95% CI, 43.2 to 73.6), 45.0% (95% CI, 28.3 to 53.1), and 33.8% (95% CI, 23.8 to 44.1) for infants with negative, intermediate, and high EOI MRD levels, respectively ( P = .0039). EOC MRD levels were also predictive of outcome, with 6-year DFS of 68.2% (95% CI, 55.2 to 78.1), 40.1% (95% CI, 28.1 to 51.9), and 11.9% (95% CI, 2.6 to 29.1) for infants with negative, intermediate, and high EOC MRD levels, respectively ( P < .0001). Analysis of EOI MRD according to the type of consolidation treatment showed that infants treated with lymphoid-style consolidation had 6-year DFS of 78.2% (95% CI, 51.4 to 91.3), 47.2% (95% CI, 33.0 to 60.1), and 23.2% (95% CI, 12.1 to 36.4) for negative, intermediate, and high MRD levels, respectively ( P < .0001), while for myeloid-style–treated patients the corresponding figures were 45.0% (95% CI, 23.9 to 64.1), 41.3% (95% CI, 23.2 to 58.5), and 45.9% (95% CI, 29.4 to 60.9). CONCLUSION This study provides support for the idea that induction therapy selects patients for subsequent therapy; infants with high EOI MRD may benefit from AML-like consolidation (DFS 45.9% v 23.2%), whereas patients with low EOI MRD may benefit from ALL-like consolidation (DFS 78.2% v 45.0%). Patients with positive EOC MRD had dismal outcomes. These findings will be used for treatment interventions in the next Interfant protocol.

Infants with KMT2A‐rearranged acute lymphoblastic leukemia (KMT2A-r ALL) have a poor prognosis. KMT2A-r ALL overexpresses FLT3, and the FLT3 inhibitor (FLT3i) lestaurtinib potentiates chemotherapy‐induced cytotoxicity in preclinical models. Children’s Oncology Group (COG) AALL0631 tested whether adding lestaurtinib to post‐induction chemotherapy improved event-free survival (EFS). After chemotherapy induction, KMT2A-r infants received either chemotherapy only or chemotherapy plus lestaurtinib. Correlative assays included FLT3i plasma pharmacodynamics (PD), which categorized patients as inhibited or uninhibited, and FLT3i ex vivo sensitivity (EVS), which categorized leukemic blasts as sensitive or resistant. There was no difference in 3-year EFS between patients treated with chemotherapy plus lestaurtinib (n = 67, 36 ± 6%) vs. chemotherapy only (n = 54, 39 ± 7%, p = 0.67). However, for the lestaurtinib-treated patients, FLT3i PD and FLT3i EVS significantly correlated with EFS. For FLT3i PD, EFS for inhibited/uninhibited was 59 ± 10%/28 ± 7% (p = 0.009) and for FLTi EVS, EFS for sensitive/resistant was 52 ± 8%/5 ± 5% (p

Simple Summary Infant acute myeloid leukemia (AML) is a rare subgroup of AML of pediatric patients under two years of age. For a long time, they have been included in pediatric forms of AML. However, infant AML demonstrates unusual clinical and biological characteristics, and its prognosis differs from AML in older children. Most treatment protocols do not distinguish age subgroups in pediatric AML, when in reality these unusual forms of infancy might benefit from specific targeted therapeutics. Herein we summarize the various specificities of infant AML. Abstract Infant acute myeloid leukemia (AML) is a rare subgroup of AML of children

Infants with MLL-rearranged infant acute lymphoblastic leukemia (MLL-r iALL) undergo intense therapy to counter a highly aggressive malignancy with survival rates of only 30–40%. The majority of patients initially show therapy response, but in two-thirds of cases the leukemia returns, typically during treatment. The glucocorticoid drug prednisone is established as a major player in the treatment of leukemia and the in vivo response to prednisone monotreatment is currently the best indicator of risk for MLL-r iALL. We used two different single-cell RNA sequencing technologies to analyze the expression of a prednisone-dependent signature, derived from an independent study, in diagnostic bone marrow and peripheral blood biopsies. This allowed us to classify individual leukemic cells as either resistant or sensitive to treatment and show that quantification of these two groups can be used to better predict the occurrence of future relapse in individual patients. This work also sheds light on the nature of the therapy-resistant subpopulation of relapse-initiating cells. Leukemic cells associated with high relapse risk are characterized by basal activation of glucocorticoid response, smaller size, and a quiescent gene expression program with cell stemness properties. These results improve current risk stratification and elucidate leukemic therapy-resistant subpopulations at diagnosis.

The treatment of children diagnosed with acute lymphoblastic leukemia (ALL) has improved significantly over recent decades, with 5-year event-free survival (EFS) approaching 85% [1]. However, 5-year EFS for infants diagnosed at less than 1 year of age with MLL-rearranged ALL remains less than 40% [2]. MLL-rearranged infant ALL is both clinically and biologically distinct from other childhood ALL subtypes, which is reflected by a unique gene expression profile [3], a remarkably silent mutational landscape [4], and pronounced resistance to currently applied chemotherapeutics [5].

Chimeric antigen receptor (CAR) T-cells, engineered autologous T-cells that target antigens found in leukemia, have shown durable remissions in relapsed acute lymphoblastic leukemia (ALL). Infant ALL with KMT2A rearrangements (KMT2Ar) is a rare, aggressive form of leukemia associated with extramedullary disease both at diagnosis and at relapse, and overall outcomes for these patients are dismal. Here we report the successful use of tisagenlecleucel, a CAR T-cell product approved for relapsed/refractory ALL, in a patient with KMT2Ar infant ALL who was treated for combined marrow and extramedullary (renal) relapse.

Infant acute lymphoblastic leukemia (ALL) comprises 2.5%-5% of pediatric ALL with inferior survival compared to older children. A majority of infants (80%) with ALL harbor KMT2A gene rearrangement, which portends a poor prognosis. Approximately 94 different partner genes have been identified to date. The common rearrangements include t(4;11)(q21;q23)KMT2A-AFF1,t(11;19) (q23;p13.3)KMT2A-MLLT1 and t(9;11)(p22;q23)KMT2A-MLLT3. We report a novel translocation t(5;11)(q35;q23)KMT2A-MAML1 in newly diagnosed infant precursor B-ALL. Long-term follow-up and a larger number of patients are needed to better understand its prognostic significance.

Purpose Despite advances in childhood cancer care, some patients die soon after diagnosis. This population is not well described and may be under-reported. Better understanding of risk factors for early death and scope of the problem could lead to prevention of these occurrences and thus better survival rates in childhood cancer. Methods We retrieved data from SEER 13 registries on 36,337 patients age 0 to 19 years diagnosed with cancer between 1992 and 2011. Early death was defined as death within 1 month of diagnosis. Socioeconomic status data for each individual’s county of residence were derived from Census 2000. Crude and adjusted odds ratios and corresponding 95% CIs were estimated for the association between early death and demographic, clinical, and socioeconomic factors. Results Percentage of early death in the period was 1.5% (n = 555). Children with acute myeloid leukemia, infant acute lymphoblastic leukemia, hepatoblastoma, and malignant brain tumors had the highest risk of early death. On multivariable analysis, an age younger than 1 year was a strong predictor of early death in all disease groups examined. Black race and Hispanic ethnicity were both risk factors for early death in multiple disease groups. Residence in counties with lower than median average income was associated with a higher risk of early death in hematologic malignancies. Percentages of early death decreased significantly over time, especially in hematologic malignancies. Conclusion Risk factors for early death in childhood cancer include an age younger than 1 year, specific diagnoses, minority race and ethnicity, and disadvantaged socioeconomic status. The population-based disease-specific percentages of early death were uniformly higher than those reported in cooperative clinical trials, suggesting that early death is under-reported in the medical literature. Initiatives to identify those at risk and develop preventive interventions should be prioritized.

// Mark Kerstjens 1, * , Emma M.C. Driessen 1, * , Merel Willekes 1 , Sandra S. Pinhancos 1 , Pauline Schneider 1 , Rob Pieters 1, 2 , Ronald W. Stam 1 1 Department of Pediatric Oncology/Hematology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands 2 Princess Maxima Center for Pediatric Oncology, Utrecht, The Netherlands * These authors have contributed equally to this work Correspondence to: Ronald W. Stam, email: r.stam@erasmusmc.nl Keywords: MLL-rearrangements, RAS-pathway, leukemia, MEK inhibitors Received: May 31, 2016 Accepted: August 13, 2016 Published: August 31, 2016 ABSTRACT Acute lymphoblastic leukemia (ALL) in infants is an aggressive malignancy with a poor clinical outcome, and is characterized by translocations of the Mixed Lineage Leukemia (MLL) gene. Previously, we identified RAS mutations in 14-24% of infant ALL patients, and showed that the presence of a RAS mutation decreased the survival chances even further. We hypothesized that targeting the RAS signaling pathway could be a therapeutic strategy for RAS -mutant infant ALL patients. Here we show that the MEK inhibitors Trametinib, Selumetinib and MEK162 severely impair primary RAS -mutant MLL -rearranged infant ALL cells in vitro . While all RAS -mutant samples were sensitive to MEK inhibitors, we found both sensitive and resistant samples among RAS -wildtype cases. We confirmed enhanced RAS pathway signaling in RAS -mutant samples, but found no apparent downstream over-activation in the wildtype samples. However, we did confirm that MEK inhibitors reduced p-ERK levels, and induced apoptosis in the RAS -mutant MLL -rearranged ALL cells. Finally, we show that MEK inhibition synergistically enhances prednisolone sensitivity, both in RAS -mutant and RAS -wildtype cells. In conclusion, MEK inhibition represents a promising therapeutic strategy for MLL -rearranged ALL patients harboring RAS mutations, while patients without RAS mutations may benefit through prednisolone sensitization.

Background Infant acute lymphoblastic leukemia (ALL) is an infrequent disease characterized by clinical and biological features related to poor prognosis. Adapted therapies were designed without a clear consensus regarding the best treatment options. We aimed to compare the outcome between infant ALL cases receiving Interfant versus BFM-based protocols. Procedure This is a retrospective observational study. From April 1990 to June 2018, infant ALL cases were enrolled in one of the five consecutive treatment protocols. Clinical, demographic, and biological features and outcome were evaluated. A comparative analysis was performed between Interfant protocols and BFM-based protocols. Results During the studied period, 1913 ALL patients were admitted and 116 (6%) were infants. Treatment administered was: ALL-BFM'90 (n = 16), 1-ALL96-BFM/HPG (n = 7), Interfant-99 (n = 39), Interfant-06 (n = 35), and ALLIC-BFM'2009 (n = 19). The 5-year event-free survival probability (EFSp) was 31.9(standard error [SE] 4.6)% for the entire population, with a significant difference among risk groups according to Interfant-06 criteria (P = .0029). KMT2A-rearrangement status was the strongest prognostic factor (P = .048), independently of the protocol strategy. The median time for relapse was 24.1 months for patients with minimal residual disease (MRD)-negative versus 11.5 months for those with MRD-positive (P = .0386). EFSp and cumulative relapse risk probability (CRRp) were similar. Interfant protocols showed comparable induction (8.1% vs 7.1%, P = .852) and complete remission mortality (21.6% vs 28.6%, P = .438), failing to reduce the relapse rate (48.5% vs 30.7%, P = .149). Conclusions Interfant protocols and BFM-based protocols presented comparable results. The risk group stratification proposed by Interfant-06 was validated by our results, and MRD seems useful to identify patients with an increased risk of early relapse.

10030 Background: Infant acute lymphoblastic leukemia (ALL) is the only subtype of childhood ALL whose outcome has not improved over the past two decades. The most important prognosticator is the presence of rearrangements in the Mixed Lineage Leukemia gene (MLL-r), however, many patients present with high-risk clinical features but without MLL-r. We recently identified two cases of infant ALL with high-risk clinical features resembling MLL-r, but were negative for MLL-r by conventional diagnostics. RNA sequencing revealed a partial tandem duplication in MLL (MLL-PTD). We thus aimed to determine if MLL-PTD, other MLL abnormalities, or other genetic or transcriptomic features were driving this subset of high-risk infant ALL without MLL-r. Methods: We obtained 19 banked patient samples from the Children’s Oncology Group (COG) infant ALL trial (AALL0631) from MLL wildtype patients as determined by FISH and cytogenetics. Utilizing deep RNA-sequencing, we manually inspected the MLL gene for MLL-PTD, while also performing automated fusion detection and gene expression profiling in search of defining features of these tumors. Results: 3 additional MLL-PTDs were identified, all in patients with infant T-cell ALL, whereas both index cases were in patients with infant B-cell ALL. Gene expression profiling analysis revealed that all five MLL-PTD infants clustered together. Eight infants (7 with B-cell ALL) were found to have Ph-like expression. Five of these 8 infants were also found to have an IKZF1/JAK2 expression profile; one of these five had a PAX5-JAK2 fusion detected. Two infants (including the one noted above) had novel PAX5 fusions, known drivers of B-cell leukemia. Additional detected fusions included TCF3-PBX1 and TCF4-ZNF384. Conclusions: MLL-PTDs were found in both B- and T-cell infant ALL. Though Ph-like ALL has been described in adolescents and young adults, we found a substantial frequency of Ph-like expression among MLL-WT infants. Further characterization of these infants is ongoing. If replicated in other infant cohorts, these two findings may help explain the poor prognosis of MLL-WT ALL when compared to children with standard risk ALL, and offer the possibility of targeted therapy for select infants.

Despite vast improvements that have been made in the treatment of children with acute lymphoblastic leukemia (ALL), the majority of infant ALL patients (~80 %, < 1 year of age) that carry a chromosomal translocation involving the mixed lineage leukemia (MLL) gene shows a poor response to chemotherapeutic drugs, especially glucocorticoids (GCs), which are essential components of all current treatment regimens. Although addressed in several studies, the mechanism(s) underlying this phenomenon have remained largely unknown. A major drawback of most previous studies is their primary focus on individual genes, thereby neglecting the putative significance of inter-gene correlations. Here, we aimed at studying GC resistance in MLL-rearranged infant ALL patients by inferring an associated module of genes using co-expression network analysis. The implications of newly identified candidate genes with associations to other well-known relevant genes from the same module, or with associations to known transcription factor or microRNA interactions, were substantiated using literature data. A weighted gene co-expression network was constructed to identify gene modules associated with GC resistance in MLL-rearranged infant ALL patients. Significant gene ontology (GO) terms and signaling pathways enriched in relevant modules were used to provide guidance towards which module(s) consisted of promising candidates suitable for further analysis. Through gene co-expression network analysis a novel set of genes (module) related to GC-resistance was identified. The presence in this module of the S100 and ANXA genes, both well-known biomarkers for GC resistance in MLL-rearranged infant ALL, supports its validity. Subsequent gene set net correlation analyses of the novel module provided further support for its validity by showing that the S100 and ANXA genes act as ‘hub’ genes with potentially major regulatory roles in GC sensitivity, but having lost this role in the GC resistant phenotype. The detected module implicates new genes as being candidates for further analysis through associations with known GC resistance-related genes. From our data we conclude that available systems biology approaches can be employed to detect new candidate genes that may provide further insights into drug resistance of MLL-rearranged infant ALL cases. Such approaches complement conventional gene-wise approaches by taking putative functional interactions between genes into account.

To address the poor prognosis of mixed lineage leukemia (MLL)-rearranged infant acute lymphoblastic leukemia (iALL), we generated a panel of cell lines from primary patient samples and investigated cytotoxic responses to contemporary and novel Food and Drug Administration-approved chemotherapeutics. To characterize representation of primary disease within cell lines, molecular features were compared using RNA-sequencing and cytogenetics. High-throughput screening revealed variable efficacy of currently used drugs, however identified consistent efficacy of three novel drug classes: proteasome inhibitors, histone deacetylase inhibitors and cyclin-dependent kinase inhibitors. Gene expression of drug targets was highly reproducible comparing iALL cell lines to matched primary specimens. Histone deacetylase inhibitors, including romidepsin (ROM), enhanced the activity of a key component of iALL therapy, cytarabine (ARAC) in vitro and combined administration of ROM and ARAC to xenografted mice further reduced leukemia burden. Molecular studies showed that ROM reduces expression of cytidine deaminase, an enzyme involved in ARAC deactivation, and enhances the DNA damage-response to ARAC. In conclusion, we present a valuable resource for drug discovery, including the first systematic analysis of transcriptome reproducibility in vitro, and have identified ROM as a promising therapeutic for MLL-rearranged iALL.

Background Infant acute lymphoblastic leukemia (ALL) has never occurred in families except for the ∼100% concordant cases in monozygous twins attributed to twin-to-twin metastases. We report the first kindred with infant ALL in non-twin siblings. The siblings were diagnosed with MLL-rearranged (MLL-R) ALL 26 months apart. The second affected sibling had an unaffected dichorionic monozygous co-twin. Both had fatal outcomes. Procedures Translocations were characterized by karyotype, FISH, multiplex FISH, and MLL breakpoint cluster region (bcr) Southern blot analysis. Breakpoint junctions and fusion transcripts were cloned by PCR. TP53 mutation and NADPH quinone oxidorecuctase 1 (NQO1) C609T analyses were performed, and pedigree history and parental occupations were ascertained. The likelihood of chance occurrence of infant ALL in non-twin siblings was computed based on a binomial distribution. Zygosity was determined by single nucleotide polymorphism (SNP) array. Results The translocations were not related or vertically transmitted. The complex karyotype of the proband's ALL had chromosome 2, 3, 4, and 11 abnormalities causing a 5′-MLL-AFF1-3′ fusion and a non-productive rearrangement of 3′MLL with a chromosome 3q intergenic region. The affected twin's ALL exhibited a simple t(4;11). The complex karyotype of the proband's ALL suggested a genotoxic insult, but no exposure was identified. There was no germline TP53 mutation. The NQO1 C609T risk allele was absent. The likelihood of infant ALL occurring in non-twin siblings by chance alone is one in 1.198 × 109 families. Conclusions Whether because of a deleterious transplacental exposure, novel predisposition syndrome, or exceedingly rare chance occurrence, MLL-R infant ALL can occur in non-twin siblings. The discordant occurrence of infant ALL in the monozygous twins was likely because they were dichorionic.

Introduction: Infant acute lymphoblastic leukemia (ALL), is a particularly aggressive subtype of leukemia with an early onset and unfavorable clinical outcome. Most (~70%) cases of infant ALL involve chromosomal rearrangement of KMT2A (KMT2A-r) on chromosome 11q23, the strongest independent predictor of a poor prognosis. To date, genomics studies have consistently demonstrated KMT2A-r infant ALL to have a strikingly silent landscape of DNA mutations, aside from the KMT2A-r itself. Germline mutations in cancer predisposition genes are found in 8.6% of pediatric malignancies and 4.4% of pediatric leukemias, compared to 1.1% in persons in the 1000 Genomes Project (Zhang J et al., N Engl J Med 2015). We hypothesized that germline variants may contribute to the development of KMT2A-r ALL in infants. We examined the germline variants in remission blood samples from a large cohort of infants with KMT2A-r ALL who were enrolled in Children's Oncology Group (COG) trial AALL15P1. Methods: We performed whole genome sequencing (WGS) and whole exome sequencing (WES) on DNA isolated from peripheral blood from 36 KMT2A-r cases at time of remission. Sequencing was performed using an Illumina Hiseq 4000 or 2500 to a minimum depth of 90Gb (WGS) and 15Gb (WES). Alignment and variant calling were performed using the Dragon Bio-IT platform (v 3.2.8, Illumina). Blueprint Genetics clinical panels and the medical literature (Xa M et al., Nature 2018) were used to comprise a list of 346 genes associated with cancer predisposition and bone marrow failure syndromes. From this gene pool, variants were selected for analysis based on a variant allele frequency of ~50% and minor allele frequency Results: Of 351 variants initially identified, we found 3 likely pathogenic (LP) and 6 pathogenic(P) non-synonymous germline variants (for a total of 9 LP/P variants) and 144 variants of unknown significance (VUS). In total, 19.4% (n=7) of patient samples displayed at least one LP/P variant. Two patient samples contained 2 variants each. Variants classified as VUS, LP, or P were further characterized by possible causative pathway: 37.9% (n=58) of variants were in genes associated with bone marrow failure (BMF), 17.6% (n=27) in driver genes, 13.1% (n=20) in genes associated with inherited leukemias, 11.1% (n=17) in tumor suppressor genes, 7.8% (n=12) in tyrosine kinase genes, and 29.4% (n=45) in other predisposition genes . Many variants were present in more than one pathway and are represented as such. Table 1 demonstrates the genetic characteristics of the 9 P/LP variants found in our cohort. ERCC2 was the only gene with multiple LP/P variants across samples, accounting for 2 (1 LP, 1 P) of the 9 deleterious variants identified (22%). Conclusion: We identified germline variants in cancer predisposition genes in 19.4% of this cohort of infant ALL patients, a higher mutation rate than has previously been reported. Among pathways evaluated, variants in genes associated with bone marrow failure predisposition were the most frequent. Interestingly, variants in ERCC2, which encodes a protein involved with repair of damaged DNA, were recurrent among infants with KMT2A-r ALL. Future directions include comparison to germline variants in cancer predisposition genes in other infant and non-infant ALL cohorts. Disclosures Brown: Novartis: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Guest:Syndax Pharmaceuticals: Consultancy.

Treatment of infants with acute lymphoblastic leukemia (ALL), especially those with mixed lineage leukemia (MLL) rearrangement (MLL-r), which account for approximately 80% of cases, is still a major challenge for pediatric hematologists and oncologists worldwide. Continuing efforts by collaborative clinical study groups in Europe, North America, and Japan have rescued approximately half of the MLL-r ALL patients with intensive chemotherapy with or without allogeneic hematopoietic stem cell transplantation. Recent progress has clarified the unique mechanism of MLL-r ALL: the aberrant methylation and histone modifications via DOT1L and other related molecules by MLL fusion proteins lead to leukemogenetic gene expression, thus to overt leukemia. In order to overcome this dismal subtype of ALL, novel targeted therapy based on leukemia biology is urgently needed. Due to the extreme rarity of the disease, collaboration between the study groups in Europe (Interfant), North America (Children's Oncology Group), and Japan (Japanese Pediatric Leukemia/Lymphoma Study Group) is under way.

Correction to: Leukemia (2016); 30(5), 1184–1187; doi:10.1038/leu.2015.246 Following the publication of this article the authors noted that the labels in Figures 1b and d have been switched. The correct labels in Figure 1b are; mixed lineage leukemia rearranged (MLL rearranged; dotted line) and unknown (thin solid line).

Background Infants with acute lymphoblastic leukemia (ALL) have a poor prognosis. Intensification of therapy has resulted in fewer relapses but increased early deaths, resulting in failure to improve survival.

Distinct from other forms of acute lymphoblastic leukemia (ALL), infant ALL with mixed lineage leukemia (MLL) gene rearrangement, the most common leukemia occurring within the first year of life, might arise without the need for cooperating genetic lesions. Through Ig/TCR rearrangement analysis of MLL-AF4+ infant ALL at diagnosis and xenograft leukemias from mice transplanted with the same diagnostic samples, we established that MLL-AF4+ infant ALL is composed of a branching subclonal architecture already at diagnosis, frequently driven by an Ig/TCR-rearranged founder clone. Some MLL-AF4+ clones appear to be largely quiescent at diagnosis but can reactivate and dominate when serially transplanted into immunodeficient mice, whereas other dominant clones at diagnosis can become more quiescent, suggesting a dynamic competition between actively proliferating and quiescent subclones. Investigation of paired diagnostic and relapse samples suggested that relapses often occur from subclones already present but more quiescent at diagnosis. Copy-number alterations identified at relapse might contribute to the activation and expansion of previously quiescent subclones. Finally, each of the identified subclones is able to contribute to the diverse phenotypic pool of MLL-AF4+ leukemia-propagating cells. Unraveling of the subclonal architecture and dynamics in MLL+ infant ALL may provide possible explanations for the therapy resistance and frequent relapses observed in this group of poor prognosis ALL.

Background/Objectives Infants with ALL have poor outcomes, especially the 70-80% with KMT2A rearrangements (KMT2A-r). Intensification of chemotherapy has reached the limit of tolerability, and stem cell transplantation (SCT) has not improved outcomes. The FLT3 kinase is overexpressed and constitutively activated in KMT2A-r ALL. FLT3 inhibitors (FLT3i) selectively kill KMT2A-r ALL cells in vitro and in vivo, and synergize with chemotherapy. COG AALL0631 was a randomized trial that tested the hypothesis that the addition of the FLT3i lestaurtinib to post-induction chemotherapy would improve outcomes for KMT2A-r ALL.As previously reported, the addition of lestaurtinib did not improve outcomes [3-year disease-free/overall survival (DFS/OS) 39%/46% without lestaurtinib vs. 37%/45% with lestaurtinib, log-rank p=0.78/0.92]. We hypothesized that correlative laboratory measures of FLT3i pharmacodynamics and ex vivo FLT3i sensitivity would enhance our understanding of the clinical responses for KMT2A-r infants treated with lestaurtinib. Design/Methods FLT3i pharmacodynamics (PD) were determined using plasma inhibitory activity (PIA) measured at 5 trough time points during lestaurtinib treatment, using Western blotting and densitometry (normalized to pre-treatment levels). PIA ranged from 0% (no evidence of FLT3 inhibition) to 100% (full inhibition). Mean PIA of 5 troughs was calculated for each patient, and was categorized based on pre-defined protocol criteria as "adequate", or FLT3 inhibited (≥85%) vs "inadequate", or FLT3 uninhibited ( Results Of 210 eligible patients, 146 were KMT2A-r, and 78 were assigned to receive lestaurtinib. Of these, data were available for FLT3i PD (n=73), FLT3i EVS (n=62), MRD (n=55), and DFS/OS (n=78). FLT3i PD and FLT3i EVS significantly correlated with both survival and MRD. For FLT3i PD, 27 were "FLT3 inhibited" and 46 were "FLT3 uninhibited". The DFS/OS for inhibited was 59%/67% vs. 28%/37% for uninhibited (p=0.01/0.02). The week 10 MRD-negative rate was 90% for inhibited vs. 52% for uninhibited (p=0.003). For FLT3i EVS, of the 62 lestaurtinib-treated patients with data, 42 were "sensitive" and 20 were "resistant". The DFS/OS for sensitive was 52%/62% vs. 5%/11% for resistant (p Conclusion While the addition of lestaurtinib to chemotherapy did not improve outcomes, patients achieving potent pharmacodynamic inhibition of FLT3 and those whose leukemia cells were sensitive to ex-vivo FLT3-inhibitor induced cytotoxicity did benefit from the addition of lestaurtinib. Selection of patients with FLT3-sensitive leukemia, real-time intra-patient dose escalation of patients with inadequate pharmacodynamics, and/or using a more potent FLT3 inhibitor may enhance the efficacy of FLT3 inhibition for KMT2A-r infant ALL. Disclosures Raetz: Pfizer: Research Funding. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Hunger:Bristol Myers Squibb: Consultancy; Amgen: Consultancy, Equity Ownership; Novartis: Consultancy; Jazz: Honoraria. Borowitz:Beckman Coulter: Honoraria. Small:Pharos I, B & T: Consultancy, Research Funding; InSilico Medicine: Membership on an entity's Board of Directors or advisory committees.

Background Infant acute lymphoblastic leukemia (ALL) is a malignant disorder with poor clinical outcome. It is well-known that infant ALL cases with rearrangement of the KMT2A gene (KMT2A-r) have an even poorer prognosis than non-KMT2A-r cases. Interestingly, KMT2A-r infant ALL cases have remarkably few other genetic alterations. We hypothesized that non-coding events in cancer genomes (e.g. loss of expression or methylation) may play a role in this disease. Such events can be captured using genomic analyses in haploid genomes using analytical approaches for allelic imbalance quantification. Here, we examine whether allelic imbalance is a feature of infant ALL. Methods We performed whole genome sequencing (WGS) and RNA sequencing on peripheral blood or bone marrow specimens from 29 KMT2A-r cases and 14 non-KMT2A-r cases at diagnosis (DX), remission (MD), and relapse (RL) as applicable. WGS data from MD samples was phased using a 1000 Genomes reference panel. Biallelic expression was measured on the phased genome for transcripts with at least 2 SNPs and at least 15 aligned reads. Lesser allele fraction (LAF; allele fraction for the less prevalent allele) for DX/RL versus MD samples was compared for transcripts that had LAFs available in at least 3 cases using t-test. Transcript-level p-values were aggregated at the gene level using the Sidak method. Results Allelic imbalance in expression (LAF Discussion Our study suggests that allelic imbalance quantification may help uncover novel molecular mechanisms in infant ALL, especially KMT2A-r cases. However, similar to gene expression, the patterns of allelic imbalance in KMT2A-r cases at DX do not allow efficient prediction of which patients go on to relapse. Citation Format: Byunggil Yoo, Midhat S. Farooqi, Rumen Kostadinov, Warren Cheung, Emily Farrow, Shannon Kelley, Neil Miller, Bing Ge, Margaret Gibson, Patrick Brown, Erin M. Guest, Tomi Pastinen. Allelic imbalance in KMT2A-rearranged infant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3649.

Background Infants (

MLL-rearranged infant acute lymphoblastic leukemia (ALL) is an aggressive type of leukemia characterized by a unique gene-expression profile. We uncovered that the activation of particular (proto-onco)genes is mediated by promoter hypomethylation. In search for therapeutic agents capable of targeting these potential cancer-promoting genes, we applied connectivity mapping on a gene expression signature based on the genes most significantly hypomethylated in t(4;11)-positive infant ALL as compared with healthy bone marrows. This analysis revealed histone deacetylase (HDAC) inhibitors as suitable candidates to reverse the unfavorable gene signature. We show that HDAC inhibitors effectively induce leukemic cell death in t(4;11)-positive primary infant ALL cells, accompanied by downregulation of MYC, SET, RUNX1, RAN as well as the MLL-AF4 fusion product. Furthermore, DNA methylation was restored after HDAC inhibitor exposure. Our data underlines the essential role for epigenetic de-regulation in MLL-rearranged ALL. Furthermore, we show, for the first time, that connectivity mapping can indirectly be applied on DNA methylation patterns, providing a rationale for HDAC inhibition in t(4;11)-positive leukemias. Given the presented potential of HDAC inhibitors to target important proto-oncogenes including the leukemia-specific MLL fusion in vitro, these agents should urgently be tested in in vivo models and subsequent clinical trials.

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants is characterized by a poor clinical outcome and resistance to glucocorticoids (for example, prednisone and dexamethasone). As both the response to prednisolone in vitro and prednisone in vivo are predictive for clinical outcome, understanding and overcoming glucocorticoid resistance remains an essential step towards improving prognosis. Prednisolone-induced apoptosis depends on glucocorticoid-evoked Ca(2+) fluxes from the endoplasmic reticulum towards the mitochondria. Here, we demonstrate that in MLL-rearranged infant ALL, over-expression of S100A8 and S100A9 is associated with failure to induce free-cytosolic Ca(2+) and prednisolone resistance. Furthermore, we demonstrate that enforced expression of S100A8/S100A9 in prednisolone-sensitive MLL-rearranged ALL cells, rapidly leads to prednisolone resistance as a result of S100A8/S100A9 mediated suppression of prednisolone-induced free-cytosolic Ca(2+) levels. In addition, the Src kinase inhibitor PP2 markedly sensitized MLL-rearranged ALL cells otherwise resistant to prednisolone, via downregulation of S100A8 and S100A9, which allowed prednisolone-induced Ca(2+) fluxes to reach the mitochondria and trigger apoptosis. On the basis of this novel mechanism of prednisolone resistance, we propose that developing more specific S100A8/S100A9 inhibitors may well be beneficial for prednisolone-resistant MLL-rearranged infant ALL patients.

Infant acute lymphoblastic leukemia (ALL) involving mixed-lineage leukemia (MLL) fusions has attracted a huge interest in basic and clinical research because of its prenatal origin, mixed-lineage phenotype, dismal prognosis and extremely short latency. Over 90% of infant ALLs are pro-B ALL harboring the leukemic fusion MLL-AF4. Despite the fact that major achievements have provided a better understanding about the etiology of infant MLL-AF4+ ALL over the last two decades, key questions remain unanswered. Epidemiological and genetic studies suggest that the in utero origin of MLL rearrangements in infant leukemia may be the result of prenatal exposure to genotoxic compounds. In fact, chronic exposure of human embryonic stem cells (hESCs) to etoposide induces MLL rearrangements and makes hESC more prone to acquire subsequent chromosomal abnormalities than postnatal CD34(+) cells, linking embryonic exposure to topoisomerase II inhibitors to genomic instability and MLL rearrangements. Unfortunately, very little is known about the nature of the target cell for transformation. Neuron-glial antigen 2 expression was initially claimed to be specifically associated with MLL rearrangements and was recently shown to be readily expressed in CD34+CD38+, but not CD34+CD38- cells suggesting that progenitors rather than stem cells may be the target cell for transformation. Importantly, the recent findings showing that MLL-AF4 rearrangement is present and expressed in mesenchymal stem cells from infant patients with MLLAF4+ ALL challenged our current view of the etiology and cellular origin of this leukemia. It becomes therefore crucial to determine where the leukemia relapses come from and how the tumor-stroma relationship is defined at the molecular level. Finally, MLL-AF4 leukemogenesis has been particularly difficult to model and bona fide MLL-AF4 disease models do not exist so far. It is likely that the current disease models are missing some essential ingredients of leukemogenesis in the human embryo/fetus. We thus propose modeling MLL-AF4+ infant pro-B ALL using prenatal hESCs.

MLL-rearranged acute lymphoblastic leukemia (ALL) in infants (

MLL/AF4 and AML/MTG8 represent two leukemic fusion genes, which are most frequently found in infant acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML), respectively. We examined the influence of MLL/AF4 and AML1/MTG8 fusion genes on the expression of TERT coding for the telomerase protein subunit, and subsequently telomerase activity in t(4;11)-positive ALL and t(8;21)-positive cell lines, respectively. MLL/AF4 suppression diminished telomerase activity and expression of TERT. Blocking pro-apoptotic caspase activation in conjunction with MLL/AF4 knockdown enhanced the inhibition of TERT gene expression, which suggests that MLL/AF4 depletion does not reduce TERT expression levels by inducing apoptosis. Knockdown of HOXA7, a direct transcriptional target of MLL/AF4 fusion gene, caused a reduction of telomerase and TERT to an extent similar to that observed with MLL/AF4 suppression. Chromatin immunoprecipitation of SEM cells, using ectopically expressed FLAG-tagged Hoxa7, indicates HOXA7 binding site in the TERT promoter region. Furthermore, suppression of the AML1/MTG8 fusion gene was associated with severely reduced clonogenicity, induction of replicative senescence, impaired TERT expression and accelerated telomere shortening. We thus present findings that show a mechanistic link between leukemic fusion proteins, essential for development and maintenance of leukemia, and telomerase, a key element of both normal and malignant self-renewal.

Though childhood acute lymphoblastic leukemia (ALL) is highly treatable, there remain subsets of pediatric ALL with very poor prognoses. Infant ALL, found in children under the age of 1, is difficult to treat due to the scarcity of cases impeding the ability of even the largest pediatric oncology centers from gaining experience in treating the disease, the more aggressive initial clinical presentation, as well as the inability for these young patients to tolerate toxicities associated with chemotherapeutic regimens and procedures. Despite being only 5% of total ALL cases, 80% of infant ALL cases are marked by mixed lineage leukemia (MLL/KMT2A) rearrangements. In KMT2A rearranged (KMT2Ar) ALL, FLT3 is the most differentially expressed gene that distinguishes KMT2Ar ALL from non-KMT2Ar ALL making FLT3 an attractive target for infant ALL. CD19 and CD22 targeting chimeric antigen receptor (CAR) T cell therapy has demonstrated outstanding responses in phase 1 clinical trials for relapsed/refractory B ALL, leading to tremendous interest in testing other CAR targets. Here we explore FLT3 CAR as a potential treatment for B ALL and the unexpected finding that FLT3 CAR T cells induce lineage switch of an infant ALL from a B to T cell phenotype. We generated a FLT3-targeting CAR consisting of a FLT3 binding domain derived from a well-characterized anti-human FLT3 antibody coupled to 4-1BB costimulatory and CD3-zeta activation domains. In vitro studies confirmed that human T cells expressing the FLT3 CAR produced interferon-gamma and interleukin-2 after co-culture with KMT2Ar B ALL SEM and infant B ALL KOPN8. FLT3 CAR T cells eliminated ALL in vivo in NOD-SCID-IL2Rγc-/- (NSG) mice engrafted with high FLT3 expressing SEM. KOPN8, which expresses lower levels of FLT3 when treated with FLT3 CAR T cells showed an initial clearance of leukemia in NSG mice, however relapsed with ALL that no longer expressed FLT3 or B cell marker CD19. Interestingly, this loss of FLT3 and CD19 happened concurrently with gain of T cell markers (CD3+ and either CD4+ or CD8+). The durability of this T cell phenotype was transient because when T lineage switched KOPN8 was cultured ex vivo without immune pressure, the KOPN8 cells reverted to the parental B ALL phenotype (FLT3+, CD19+, CD3 neg, CD4 neg, CD8 neg) suggesting that the ability to lineage switch is not a selection of a clone that genetically does not express B cell markers while expressing T cell markers, but rather a potential epigenetic mechanism driving the cell lineage change. Contrary to reports from CD19 CAR treated KMT2Ar B ALL that switched to a myeloid phenotype, these cells did not upregulate myeloid markers (CD33, CD11b). Taken together these data imply that lineage switch driven by CAR T cell immune pressure may cause different types of lineage switch based on the target of the CAR. Furthermore, using CAR T cell immunotherapy we may be able to interrogate the biology of leukemia. Citation Format: Christopher D. Chien, Lila Yang, Sang M. Nguyen, Christopher T. Sauter, Kazusa Ishii, Feng Shen, Sarah K. Tasian, Terry J. Fry. FLT3 chimeric antigen receptor T cell therapy induces B to T cell lineage switch in infant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1630.

Introduction: Rearrangements involving the MLL (mixed lineage leukemia, KMT2A) gene (MLLr) occur broadly in acute leukemia, in ~80% of infant acute lymphoblastic leukemia (ALL) cases, and are associated with poor outcome. Menin is a ubiquitously expressed nuclear protein, and the MLLr-menin interaction is the key impetus for transformation of MLLr-expressing cells. VTP-50469 is a potent, orally available, small molecule inhibitor of the MLL-menin interaction with pM binding affinity for menin. Therefore, it was of interest to test the in vivo efficacy of VTP-50469 against preclinical models of infant MLLr-ALL. Methods: Infant MLLr-ALL patient-derived xenografts (PDXs) grew in an orthotopic manner in NSG mice. Engraftment and response to treatment were assessed by enumeration of the % human leukemic blasts in the murine peripheral blood (%huCD45+). Treatment commenced when the median %huCD45+ exceeded 1%, and mice received VTP-50469 (120 mg/kg by oral gavage twice daily x 28) or vehicle. An event was defined as the %huCD45+ >25% or leukemia-related morbidity. The Kaplan-Meier method was used to compare event-free survival (EFS) between treated (T) and control (C) groups. Stringent objective response measures were assigned to each mouse and reported as group medians (Houghton et al, Pediatr. Blood Cancer, 2007;49:928-40). Leukemia infiltration into the femoral bone marrow was also assessed at Day 28 following treatment initiation. VTP-50469 was provided by Vitae Pharmaceuticals. Results: VTP-50469 was well tolerated, with maximum average weight losses of 1.6-6.4% across treatment groups compared to 0-2.0% in vehicle control treated groups. VTP-50469 induced significant differences in EFS distribution compared to control in 6 of 6 (100%) of the evaluable MLLr-ALL PDXs. VTP-50469 T-C values in MLLr-ALL PDXs ranged from 1.2 to 100 days (T/C 1.3-21.1), and Maintained Complete Responses (MCRs) were observed in 5 of 6 PDXs. Two of 8 mice engrafted with an MLLr-ALL harboring the MLL-AFF1 (t4;11) translocation had not reached event >230 days following treatment initiation. A significant reduction (P Conclusions: VTP-50469 exerted profound in vivo efficacy against ALL PDXs derived from infants harboring MLL-AFF1, MLL-GAS7, and MLL-ENL translocations, and significantly reduced leukemia infiltration in the bone marrow. VTP-50469 was also effective across a broad dose range, indicating that it may represent a novel treatment for MLLr leukemia. (Supported by NCI Grants CA199222 & CA199000) Citation Format: Richard B. Lock, Kathryn Evans, Tara Pritchard, Stephen W. Erickson, Yuelong Guo, David A. Claremon, Gerard M. McGeehan, Beverly A. Teicher, Malcolm A. Smith. Pediatric Preclinical Testing Consortium evaluation of the menin inhibitor, VTP-50469, against xenograft models of MLL-rearranged infant acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3187.

Bimodal distribution of genomic MLL breakpoints in infant acute lymphoblastic leukemia treatment

The pathogenesis of infant acute lymphoblastic leukemia (ALL) is still not well defined. Short latency to leukemia and very high concordance rate for ALL in Mixed-Lineage Leukemia (MLL)-positive infant twins suggest that the MLL rearrangement itself could be sufficient for overt leukemia. Attempts to generate a suitable mouse model for MLL-AF4-positive ALL did not thoroughly resolve the issue of whether cooperating mutations are required to reduce latency and to generate overt leukemia in vivo. In this study, we applied single-nucleotide polymorphism array technology to perform genomic profiling of 28 infant ALL cases carrying t(4;11) to detect MLL-cooperating aberrations hidden to conventional techniques and to gain new insights into infant ALL pathogenesis. In contrast to pediatric, adolescent and adult ALL cases, the MLL rearrangement in infant ALL is associated with an exceptionally low frequency of copy-number abnormalities, thus confirming the unique nature of this disease. By contrast, additional genetic aberrations are acquired at disease relapse. Small-segmental uniparental disomy traits were frequently detected, mostly constitutional, and widely distributed throughout the genome. It can be argued that the MLL rearrangement as a first hit, rather than inducing the acquisition of additional genetic lesions, has a major role to drive and hasten the onset of leukemia. Leukemia (2010) 24, 169-176; doi:10.1038/leu.2009.203; published online 12 November 2009

Compared with acute lymphoblastic leukemia (ALL) in older children, ALL in infants has a dismal outcome because rearrangements of the mixed-lineage leukemia (MLL) gene occur in about 80% of these patients, leading to an aggressive type of leukemia. With most recent therapies, about 50% long-term event-free survival is achieved, but early bone marrow relapse remains a major problem. Early intensification of chemotherapy and new innovative therapies are necessary to improve outcome. Bone marrow transplantation should be limited to a small subset of well-recognized ALL patients with a very poor prognosis. New genetic and epigenetic insights into the biology of MLL-rearranged ALL suggest new possibilities for therapies.

The treatment outcome for infant acute lymphoblastic leukemia (ALL) with positive MLL gene rearrangements remains poor. We analyzed whether additional chromosomal abnormalities (ACA) other than 11q23 translocation could affect the disease behavior and its prognosis. Eighteen of seventy-four patients with infant acute lymphoblastic leukemia showed ACA, including three-way translocations in four, other novel translocations in four, and complex structural chromosomal changes in four. Only age less than 6 months and positive central nervous system leukemia were significant prognostic factors by multivariate analysis. However, overall survival rates were worse in patients with ACA compared to those with non-ACA. Genetic alterations induced by additional chromosomal changes may be associated with disease progression and poorer overall survival rates in infants with MLL-rearranged ALL.

We evaluated the efficacy of a treatment strategy in which infants with acute lymphoblastic leukemia (ALL) were stratified by their MLL gene status and then assigned to different risk-based therapies. A total of 102 patients were registered on two consecutive multicenter trials, designated MLL96 and MLL98, between 1995 and 2001. Those with a rearranged MLL gene (MLL-R, n=80) were assigned to receive intensive chemotherapy followed by hematopoietic stem cell transplantation (HSCT), while those with germline MLL (MLL-G, n=22) were treated with chemotherapy alone. The 5-year event-free survival (EFS) rate for all 102 infants was 50.9% (95% confidence interval, 41.0-60.8%). The most prominent late effect was growth impairment, observed in 58.9% of all evaluable patients in the MLL-R group. This plan of risk-based therapy appears to have improved the overall prognosis for infants with ALL, compared with previously reported results. However, over half the events in patients with MLL rearrangement occurred before the instigation of HSCT, and that HSCT-related toxic events comprised 36.3% (8/22) of post-transplantation events, suggesting that further stratification within the MLL-R group and the development of more effective early-phase intensification chemotherapy will be needed before the full potential of this strategy is realized.

The most common chromosomal abnormality of infant acute lymphoblastic leukemia (ALL) is the t(4;11)(q21;q23), which gives rise to the MLL-AF4 fusion gene [1]. MLL (also known as ALL-I, HTRX or HRX)...

Based on observational studies, early age leukemia (EAL) was associated with maternal hormone exposure during pregnancy. We studied the association between genetic polymorphisms of estrogen metabolism and EAL. Using data from the Brazilian Collaborative Study Group of Infant Acute Leukemia (2000–2012), 350 cases and 404 age-matched controls and 134 mothers of cases and controls were genotyped to explore polymorphisms in genes of the estrogen metabolism pathway: CYP1B1 (c.1294C>G, rs1056836), CYP3A4 (c.-392A>G, rs2740574), CYP3A5 (c.219-237G>A, rs776746), GSTM1/GSTT1 deletions, and SULT1A1 (c.638G>A, rs9282861; and c.667A>G, rs1801030). Logistic regression was used to calculate the odds ratios (OR) with 95% confidence intervals (CIs), and unconditional logistic regression was used to estimate adjusted odds ratios (aORs) by ethnicity. Because of multiple testing, p values < 0.01 were significant after Bonferroni correction. SULT1A1 (c.638G>A) was associated to infant acute lymphoblastic leukemia and acute myeloid leukemia (AML) risk in males (additive model: aOR = 0.52; 95% CI: 0.29–0.95, p = 0.03; dominant model: aOR = 2.18; 95% CI: 1.17–4.05, p = 0.01, respectively). CYP1B1 polymorphism was associated with a decreased risk of AML either for non-white or female children (additive model: OR = 0.24; 95% CI: 0.08–0.76, p < 0.01; additive model: aOR = 0.27; 95% CI: 0.08–0.89, p = 0.03, respectively). Since polymorphisms of Cytochrome P450 genes presented gender-specific risk associations, we also investigated their expression. CYP1B1 was not expressed in 57.1% of EAL cases, and its expression varied by genotype, gender, and leukemia subtype. Maternal-fetal GSTT1 null genotype was associated with risk of EAL. This study shows that polymorphisms in genes of estrogen metabolism confer genetic susceptibility to EAL, mainly in males, and maternal susceptibility genes modify the risk for developing EAL in newborns.

The antimetabolite cytosine arabinoside (Ara-C) is a deoxycytidine analog that is used as a therapeutic agent in many leukemia treatment regimens. In combination with anthracyclines, Ara-C is the most effective agent in the treatment of acute myeloid leukemia (AML). In the treatment of acute lymphoblastic leukemia (ALL), the use of Ara-C is limited. However, leukemic cells from infants (

MLL rearranged acute lymphoblastic leukemia (MLL) is an aggressive type of acute lymphoblastic leukemia (ALL), diagnosed predominantly in infants (1 years of age). Since current chemotherapy fails in50% of patients with MLL, new therapeutic strategies are desperately needed. For this, understanding the biological features characterizing MLL is necessary. Analysis of gene expression profiles revealed that the expression of the tumor suppressor gene FHIT is reduced in children with MLL rearranged ALL as compared to ALL patients carrying germ line MLL. This finding was confirmed by quantitative real-time PCR. In 100% of the infant MLL cases tested, methylation of the FHIT 5'CpG region was observed, resulting in strongly reduced mRNA and protein expression. In contrast, FHIT methylation in infant and non-infant ALL patients carrying germ line MLL was found in only approximately 60% (Por =0.004). FHIT expression was restored upon exposing leukemic cells to the demethylating agent decitabine, which induced apoptosis. Likewise and more specifically, leukemic cell death was induced by transfecting MLL rearranged leukemic cells with expression vectors encoding wild-type FHIT, confirming tumor suppressor activity of this gene. These observations imply that suppression of FHIT may be required for the development of MLL, and provide new insights into leukemogenesis and therapeutic possibilities for MLL.

The authors describe an infant presenting at 2 weeks of age with congenital acute lymphoblastic leukemia who had previously received routine bacille Calmette-Guérin (BCG) vaccination at birth. The risk of BCG dissemination in immunocompromised infants is discussed and the use of antimycobacterial prophylaxis in such cases considered.

Acute lymphoblastic leukemia (ALL) in infants under 1 year is strongly associated with translocations involving 11q23 (MLL gene), CD10-negative B-lineage (proB) immunophenotype, and poor outcome. The present study analyses the relationship between age, MLL rearrangements, proB-lineage, and in vitro drug resistance determined using the MTT assay. Compared to 425 children aged over 1 year with common/preB (c/preB) ALL, the 44 infants were highly resistant to steroids (for prednisolone (PRED) more than 580-fold, P=0.001) and L-asparaginase (L-ASP) (12-fold, P=0.001), but more sensitive to cytarabine (AraC) (1.9-fold, P=0.001) and 2-chlorodeoxyadenosine (2-CdA) (1.7-fold, P0.001). No differences were found for vincristine, anthracyclines, thiopurines, epipodophyllotoxines, or 4-hydroperoxy (HOO)-ifosfamide. ProB ALL of all ages had a profile similar to infant ALL when compared with the group of c/preB ALL: relatively more resistant to L-ASP and PRED (and in addition thiopurines), and more sensitive to AraC and 2-CdA. Age was not related to cellular drug resistance within the proB ALL group (1 year, n=32, vs/=1 year, n=19), nor within the MLL-rearranged ALL (1 year, n=34, vs/=1 year, n=8). The translocation t(4;11)(q21;q23)-positive ALL cases were more resistant to PRED (7.4-fold, P=0.033) and 4-HOO-ifosfamide (4.4-fold, P=0.006) than those with other 11q23 abnormalities. The expression of P-glycoprotein, multidrug-resistance protein, and lung-resistance protein (LRP) was not higher in infants compared to older c/preB ALL patients, but LRP was higher in proB ALL and MLL-rearranged ALL of all ages. In conclusion, infants with ALL appear to have a distinct in vitro resistance profile with the proB immunophenotype being of importance. The role of MLL cannot be excluded, with the t(4;11) being of special significance, while age appears to play a smaller role.

An inactivating polymorphism at position 609 in the NAD(P)H:quinone oxidoreductase 1 gene (NQO1 C609T) is associated with an increased risk of adult leukemia. A small British study suggested thatNQO1 C609T was associated with an increased risk of infant leukemias with MLL translocations, especially infant acute lymphoblastic leukemia (ALL) with t(4;11). We explored NQO1 C609Tas a genetic risk factor in 39 pediatric de novo and 18 pediatric treatment-related leukemias with MLL translocations in the United States. Children with de novo B-lineage ALL withoutMLL translocations and a calculation of the expected genotype distribution in an ethnically matched population of disease-free subjects served as the comparison groups. Patients with de novo leukemias with MLL translocations were significantly more likely to be heterozygous at NQO1 C609T (odds ratio [OR] = 2.77, 95% confidence intervals [CI] 1.17-6.57;P = .02), and significantly more likely to have low/null NQO1 activity than patients with de novo B-lineage ALL withoutMLL translocations (OR = 2.47, 95% CI 1.08-5.68;P = .033). They were also significantly more likely to have low/null NQO1 activity than expected in an ethnically matched population of disease-free subjects (OR = 2.50,P = .02). Infants younger than 12 months old at diagnosis of leukemia with t(4;11) were most likely to have low/null NQO1 activity (OR > 10.0). Conversely, the distribution ofNQO1 genotypes among patients with treatment-related leukemias with MLL translocations was not statistically different than in the comparison groups. The inactivating NQO1polymorphism is associated with an increased risk of de novo leukemia with MLL translocations in infants and children.

We used karyotyping, fluorescence in situ hybridization (FISH), Southern blotting, and RT-PCR in order to analyze prospectively 77 infants (less than 1 year of age) with acute lymphoblastic leukemia for the occurrence of 11q23/MLL rearrangements and/or other cytogenetic abnormalities. Out of the 69 informative samples we found an 11q23/MLL rearrangement in 42 cases (61%). Regarding only pro-B ALL cases, the incidence of 11q23/MLL rearranged cases, however, reached more than 90% The infants were treated within the therapy studies ALL-BFM90, ALL-BFM95 and CoALL-05-92. For patients with an adequate follow-up of 4 years the event-free survival of the 11q23/MLL-positive and 11q23/MLL-negative group was 0.2 or 0.64, respectively (P = 0.024). The monoclonal antibody 7.1. (moab 7.1) does not react with normal hematopoetic precursors or mature blood cells but was shown to specifically react with leukemic cells bearing a rearrangement of chromosome 11q23 or the MLL gene, respectively. We, therefore, specifically addressed the question whether the reactivity of moab 7.1, as determined by flow cytometry, may substitute for molecular testing of an 11q23/MLL rearrangement in this cohort of infant ALLs. Reactivity of moab 7.1 indicated a 11q23/MLL rearrangement with a specificity of 100%. However, five of the 11q23/MLL-positive cases did not react with moab 7.1 indicating a sensitivity of 84% only. Three of these five moab 7.1-negative but 11q23/MLL-positive cases could be identified by their unique expression pattern of CD65s and/or CD15. Thus, 95% of all 11q23/MLL-positive ALL cases in infancy may be identified by flow cytometry based on their expression of CD15, CD65s and/or moab 7.1.

Summary Background The prognosis and optimum treatment of childhood acute lymphoblastic leukaemia (ALL) with abnormalities of chromosomal band 11q23 are controversial. We aimed to identify prognostic factors that might help in planning future therapy, and to assess the effectiveness of haemopoietic stem-cell transplantation in patients with the t(4;11) translocation, which is associated with a particularly poor outcome. Methods We reviewed data on 497 children and young adults who had ALL with various 11q23 abnormalities, including the translocations t(4;11), t(9;11), and t(11;19). All patients were treated with intensive chemotherapy, with or without haemopoietic stem-cell transplantation in first complete remission, by 11 study groups and single institutions from 1983 to 1995. Findings Age was the most important prognostic factor. In a Cox's proportional-hazard model stratified by 11q23 abnormalities, infants younger than 1 year fared significantly worse than patients 1 year of age or older (hazard ratio for event-free survival 1·84 [95% CI 1·38–2·47], p=0·0001). Among infants, any category of 11q23 abnormality conferred a dismal outcome, whereas in older patients, t(4;11) and t(9;11) were associated with a worse outcome than were other 11q23 changes. In the largest subgroup–256 patients with t(4;11)–any type of transplantation was associated with significantly worse disease-free survival (1·61 [1·10–2·35], p=0·014) and overall survival (1·76 [1·08–2·45], p=0·004) compared with chemotherapy only. Even transplantation with stem cells from HLA-matched related or HLA-matched unrelated donors tended to be associated with a worse outcome than chemotherapy alone. Interpretation The prognosis of acute lymphoblastic leukaemia with an 11q23 abnormality is particularly dismal in infants. Allogeneic transplantation with haemopoietic stem cells from an HLA-matched related donor does not seem to improve the clinical outcome in patients with t(4;11)-positive leukaemia.

Background: Infant acute lymphoblastic leukemia (ALL) is a high-risk cancer with poor survival due to treatment failure and relapse. The majority of infant ALL cases possess an MLL (KMT2A) translocation (MLL-R), where MLL joins with AF4 (AFF1), ENL (MLLT1), or another partner gene to create a novel fusion gene. A subset of infant ALL cases do not involve an MLL rearrangement (non-MLL-R). Gene expression profiling delineates MLL-R from non-MLL-R cases. Here, we describe transcriptome profiling and cluster analysis of MLL-R and non-MLL-R cases and compare between partner genes. Methods: We performed RNA sequencing (RNA-seq) on diagnostic blood or bone marrow samples from 40 infants with ALL. Cohort A included 16 patients with MLL-R ALL known to have relapsed; cohort B, 10 patients with MLL-R ALL not known to have relapsed; and cohort C, 14 patients with non-MLL-R ALL whom to date have also not relapsed. RNA-seq was performed to an average of 8.8Gb and analyzed using Bowtie2, RSEM, and limma-voom. Multiple different groups of cases (e.g. cohort A versus B) were compared for differential gene expression; the resulting genes were ranked by adjusted P value with a significance cutoff of 0.01 and further evaluated for pathway analysis with Ingenuity software. Hierarchical clustering of samples was conducted using distance measures computed from normalized gene expression. Results: Transcriptome analysis of MLL-R versus non-MLL-R cases found 2,334 differentially expressed genes. Ingenuity pathway analysis of these genes showed enrichment in specific gene sets, such as those involved in PTEN, EGF, and NF-κB signaling. Ingenuity predicted increased PTEN signaling and diminished EGF and NF-κB signaling in MLL-R cases relative to non-MLL-R cases. Transcriptome analysis of relapsed versus non-relapsed MLL-R cases found no differentially expressed genes meeting significance criteria, while analysis of MLL fusion partners identified 46 differentially expressed genes including BMI1 and MYC. Cluster analysis demonstrated a distinct grouping of non-MLL-R cases relative to MLL-R cases. Additionally, MLL-R samples sub-clustered according to their MLL fusion gene partners. However, no difference in clustering was appreciated between MLL-R cases that later went on to relapse versus those that did not. Conclusions: Our findings show that MLL-R infant ALL has a distinct gene expression signature at diagnosis that correlates with the type of MLL translocation present. This is important because it suggests treatment strategies could be tailored to the MLL partner gene. Risk of relapse in MLL-R cases could not be predicted by this signature due to a paucity of differentially expressed genes between relapsed and non-relapsed groups at diagnosis. Similar to prior profiling studies, we find that non-MLL-R cases independently cluster together and have a gene expression signature distinct from MLL-R cases.[B.Y. and M.F. contributed equally to this work.] Citation Format: Byunggil Yoo, Midhat S. Farooqi, Rumen Kostadinov, Neil Miller, Jeff Johnston, Emily Farrow, Shannon Kelley, Margaret Gibson, Patrick Brown, Erin Guest. Transcriptome and cluster analysis of infant acute lymphoblastic leukemia cases with and without MLL (KMT2A) rearrangement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2416. doi:10.1158/1538-7445.AM2017-2416

Systemic adenovirus infection in the immunocompromised host is often fatal and therapeutic options are limited. We report an infant with acute lymphoblastic leukemia who developed disseminated adenovirus infection while lymphopenic during maintenance chemotherapy 6 months following a bout of adenoviral diarrhea. His serum adenoviral load peaked at 35 million copies/mL and was associated with pancytopenia and hepatic injury. Treatment with cidofovir was effective although associated with mild renal injury. The patient recovered fully and completed chemotherapy for infant acute lymphoblastic leukemia.

Conditioning regimens for pediatric acute lymphoblastic leukemia (ALL) usually include total body irradiation (TBI), but TBI may result in serious sequelae. Busulfan and cyclophosphamide have been used as an alternative to TBI. Etoposide also has been widely used to enhance antileukemic effect. However, toxicities have been reported in some studies using busulfan, cyclophosphamide, and etoposide regimen. A reduced toxicity myeloablative regimen using busulfan and fludarabine showed promising results. Also, therapeutic drug monitoring (TDM) and administration of targeted doses of busulfan have been recommended to improve the outcome of hematopoietic stem cell transplantation (HSCT). In this study, we evaluated the outcome of HSCT using a targeted once-daily i.v. busulfan–fludarabine–etoposide (BuFluVP) regimen in pediatric and infant ALL. Busulfan (age ≥ 1 year, 120 mg/m2; age < 1 year, 80 mg/m2) was administered once daily as the first dose on day −8, and a targeted dose of busulfan was used according to the TDM results on days −7 to −5. Forty-four patients were evaluated. Donor-type neutrophil engraftment was achieved in all patients. Veno-occlusive disease occurred in 7 patients (15.9%), but all patients were successfully treated. Cumulative incidence of treatment-related mortality and relapse were 9.1% and 9.9%, respectively. One-year overall survival and event-free survival rates of all patients were 86.2% and 83.8%, respectively. Twelve patients (27.3%) were infants at diagnosis, and their 1-year overall survival rate was 83.3%. Our study demonstrated that HSCT using a targeted once-daily i.v. BuFluVP regimen showed favorable outcomes and could be an option for HSCT in pediatric and infant ALL.

Acute lymphoblastic leukemia (ALL) in infants has a significantly inferior outcome in comparison with older children. Despite initial improvements in survival of infants with ALL since establishment of the first pediatric cooperative group ALL trials, the poor outcome has plateaued in recent years. Historically, infants were treated on risk-adapted childhood ALL protocols. These studies were pivotal in identifying the need for infant-specific protocols, delineating prognostic categories and the requirement for a more unified approach between study groups to overcome limitations in accrual because of low incidence. This subsequently led to the development of collaborative infant-specific studies. Landmark outcomes have included the elimination of cranial radiotherapy following the discovery of intrathecal and high-dose systemic therapy as a superior and effective treatment strategy for central nervous system disease prophylaxis, with improved neurodevelopmental outcome. Universal prospective identification of independent adverse prognostic factors, including presence of a mixed lineage leukemia rearrangement and young age, has established the basis for risk stratification within current trials. The infant-specific trials have defined limits to which conventional chemotherapeutic agents can be intensified to optimize the balance between treatment efficacy and toxicity. Despite variations in therapeutic intensity, there has been no recent improvement in survival due to the equilibrium between relapse and toxicity. Ultimately, to improve the outcome for infants with ALL, key areas still to be addressed include identification and adaptation of novel prognostic markers and innovative therapies, establishing the role of hematopoietic stem cell transplantation in first complete remission, treatment strategies for relapsed/refractory disease and monitoring and timely intervention of late effects in survivors. This would be best achieved through a single unified international trial.