key: cord-1012601-0prl0rnb authors: Rüchel, Nadine; Jepsen, Vera H.; Hein, Daniel; Fischer, Ute; Borkhardt, Arndt; Gössling, Katharina L. title: In Utero Development and Immunosurveillance of B Cell Acute Lymphoblastic Leukemia date: 2022-03-16 journal: Curr Treat Options Oncol DOI: 10.1007/s11864-022-00963-3 sha: 0d1cadd62721f4d26fc79a713388e68ddbab3094 doc_id: 1012601 cord_uid: 0prl0rnb Acute lymphoblastic leukemia (ALL) is the most frequent type of pediatric cancer with a peak incidence at 2–5 years of age. ALL frequently begins in utero with the emergence of clinically silent, preleukemic cells. Underlying leukemia-predisposing germline and acquired somatic mutations define distinct ALL subtypes that vary dramatically in treatment outcomes. In addition to genetic predisposition, a second hit, which usually occurs postnatally, is required for development of overt leukemia in most ALL subtypes. An untrained, dysregulated immune response, possibly due to an abnormal response to infection, may be an important co-factor triggering the onset of leukemia. Furthermore, the involvement of natural killer (NK) cells and T helper (Th) cells in controlling the preleukemic cells has been discussed. Identifying the cell of origin of the preleukemia-initiating event might give additional insights into potential options for prevention. Modulation of the immune system to achieve prolonged immunosurveillance of the preleukemic clone that eventually dies out in later years might present a future directive. Herein, we review the concepts of prenatal origin as well as potential preventive approaches to pediatric B cell precursor (BCP) ALL. Leukemia is a life-threatening disease caused by uncontrolled proliferation of blood and blood precursor cells. Depending on the cell type of clonal expansion, it can be segregated into different subtypes that have quite distinct incidences, pathogenesis, treatment options, and outcomes [1] . Approximately one-third of all cancers diagnosed below the age of 18 are leukemia, with about 74% of these being acute lymphoblastic leukemia (ALL, 4.3/100,000 children G15 years) in Germany [2] . ALL peaks between the age of 2 and 5 years and has a good outcome in most cases. However, about 10% of children present with poor prognosis, based on subtype and risk factors like advanced age [3, 4] . Herein, we review and discuss recent studies and concepts of prenatal pathogenesis of leukemia, with a special focus on infections or m i c r o b i o t a i n f l u e n c i n g a n t i -l e u k e m i c immunosurveillance. Childhood B-ALL arises through a complex interplay between inherited genetic background and acquired somatic alterations [4] . The genetic background of patients includes alterations in cancer-predisposing genes, single nucleotide polymorphisms (SNPs), and cancer predisposition syndromes that confer susceptibility to leukemia [5] . In addition to the underlying inherited genetics, prenatal chromosomal aberrations, such as aneuploidy and interchromosomal translocations [6] , give rise to preleukemic cells. Further oncogenic events in these clinically silent cell clones, most likely triggered by environmental factors in early childhood, are required to ultimately lead to overt leukemia [4]. Several germline mutations which confer susceptibility to leukemia development have been described [7••]. Most of the affected genes are also targets of somatic alterations in ALL. function of ETV6 [5, 11] . A cluster of mutations occurs in the DNA-binding E26 transformation-specific (ETS) domain of ETV6, leading to dominant negative effects and transcriptional repression [5, 7, 12] . PAX5, located at 9p13, encodes for the B cell lineage transcription factor PAX5 which is important for B-lymphoid lineage maturation [13] . So far, only few families with germline PAX5 mutations have been described, presenting with incomplete penetrance [14, 15] . Reported missense mutations of PAX5 occur at amino acid positions G183 (c547G9A, p.Gly183Ser) or R38 (c113G9A, p.Arg38His), both resulting in decreased PAX5-mediated transcriptional repression [14] [15] [16] . Carriers of germline PAX5 mutations are susceptible to acquiring ALL, but the presence of the mutation does not seem to be sufficient for development of overt leukemia. A second mutational hit is required, e.g., inactivation of the wild-type PAX5 allele by deletion of 9p, formation of a 9q isochromosome, or dicentric 9q chromosome [14] [15] [16] . IKZF1 encodes for the hematopoietic zinc-finger (ZF) transcription factor IKAROS. Germline IKZF1 mutations have been described in families with common variable immunodeficiency (CVID) [17] and in cases of familial and sporadic ALL [18] . Mutations include missense, nonsense, and frameshift variants and are located mostly outside the ZF motifs [5] . IKZF1 mutations within its DNA-binding domain affect transcriptional activation of its target genes, whereas truncating mutations may have an impact on dimerization [18] . The majority of identified IKZF1 germline variants are not restricted to specific functional domains and were shown to impact subcellular localization, adhesion, and anti-leukemic drug efficacy [18] . Li-Fraumeni syndrome is an autosomal dominant disorder [19] , usually caused by TP53 germline mutations, that presents with high susceptibility to cancers like breast cancer, brain tumors, and ALL, predominantly low hypodiploid ALL [7, 20, 21] . Low hypodiploidy is characterized by 32-39 chromosomes and is present in approximately 1% of childhood ALL cases [7, 22] . Occurrence of germline TP53 mutations is associated with older age at diagnosis and poor outcome [23] . TP53 encodes the tumor suppressor protein p53 and is one of the most frequently mutated genes in cancer. The majority of TP53 mutations occur in its DNAbinding or nuclear export domains [7, 20] . Children with Down syndrome or Noonan syndrome are also at higher risk of developing acute leukemia, primarily acute myeloid leukemia (AML) [24, 25] . Down syndrome is characterized by trisomy of chromosome 21, which may affect leukemia development [24] . About 1% of children with Down syndrome will develop ALL or AML [24] . Noonan syndrome is an autosomal dominant disorder that belongs to the family of RASopathies and presents with symptoms including facial dysmorphologies, growth retardation, heart defects, and skin manifestations [25] . Rarely, germline mutations in PTPN11, encoding the phosphatase SHP2, and in SOS1, encoding the guanine nucleotide exchange factor SOS1, have been observed in patients with Noonan syndrome, who subsequently developed ALL [25] . In addition to the rare but highly penetrant germline mutations and cancer predisposition syndromes described here, genome-wide association studies (GWASs) have identified further germline variations that are frequent but show low penetrance. These are mostly SNPs, which, cumulatively, may confer a higher risk for ALL development. Although these risk alleles individually produce a modest effect and may be of limited clinical significance, in aggregate they can give rise to as much as a ninefold increase in leukemia risk for subjects with risk alleles in multiple genes compared to subjects with no risk alleles [26] . Genes involved include IKZF1, CDKN2A, PIP4K2A, LHPP, ELK3, GATA3, ARID5B, CEBPE, MYC, ERG, and TP63 [7, [27] [28] [29] [30] , with the SNPs being located in the vicinity of these genes and influencing gene expression. Some of these SNPs are associated with distinct ALL subtypes or genetic ancestry. Examples are an intronic SNP in GATA3 (dbSNP: rs3824662) that is associated with Philadelphia chromosome (Ph)-like ALL and poor outcome [31] and a risk locus in TP63 (dbSNP: rs17505102) that is associated with ETV6-RUNX1 + ALL [28] . Fusion genes generated by interchromosomal translocations are recurrent genetic alterations in pediatric BCP-ALL [32] . Several studies indicate that these translocations frequently arise in utero, giving rise to preleukemic cells. The first indications that ALL has prenatal origins were reports of concordant BCP-ALL in monozygotic twins [33] [34] [35] [36] [37] . In these cases, preleukemic cell clones arising in one twin spread to the other twin via the monochorionic placenta, as confirmed via the identification of shared genetic lesions, immunoglobulin (Ig), or T cell receptor (TCR) rearrangements in the leukemic cells of both twins [38] . Identification of genomic breakpoints in neonatal blood spots (Guthrie cards) or cord blood further corroborates the prenatal origin of preleukemic lesions [39] [40] [41] [42] [43] [44] [45] . Altogether, in utero development has been shown for several BCP-ALL subtypes, including high hyperdiploid ALL, ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and KMT2A rearrangements (as reviewed in [3•]). With up to 30% of cases, high hyperdiploidy is the most common genetic subtype in childhood BCP-ALL, characterized by the gain of chromosomes (950 chromosomes) [22, 46] . While other tri-or tetrasomies have been reported, chromosomal gains typically include chromosomes X, 4, 6, 10, 14, 17, 18, and 21 [47] . The hyperdiploid genotype is likely generated by a single abnormal mitosis leading to simultaneous gain of chromosomes [48] . Leukemia susceptibility in high hyperdiploid ALL is driven by gene dosage effects [47, 49, 50] that impact chromatin architecture, e.g., by weakening topologically associating domain (TAD) boundaries [51• ]. The most common chromosomal translocation of pediatric ALL, accounting for about 20% of cases, is t(12;21)(p13;q22) [52] . This translocation leads to the fusion of two transcription factors involved in normal hematopoiesis, ETV6 and RUNX1. Although the ETV6-RUNX1 translocation has been detected in a large number of healthy neonates (1-5%), leukemia incidence among carriers is much lower (0.2-1%) [3, 43] . The fusion gene has weak oncogenic potential that manifests itself in a low concordance rate of about 10% in monozygotic twins [38] . ETV6-RUNX1 acts as an oncogenic transcription factor and leads to a specific preleukemic phenotype characterized by a partial block of B cell differentiation and aberrant co-expression of myeloid markers [53] . Recurrent postnatal, leukemia-inducing mutations include ETV6 deletions (≈70% of cases), RUNX1 gain (23%), and extra der(21)t(12;21) (10%) [54] . BCP-ALL with t(9;22)(q34;q11), also referred to as Ph + ALL, is present in ≈2% of pediatric ALL, but is significantly more common in adults [22, 55] . The majority of pediatric patients with BCR-ABL1 fusion genes harbor the p190 BCR-ABL1 subtype [56] . This chromosomal translocation leads to the formation of the BCR-ABL1 oncogene, encoding for a tyrosine kinase. While high hyperdiploidy and ETV6-RUNX1 are associated with a favorable treatment outcome [57] , BCR-ABL1 confers a poorer outcome [58] . A common cooperating oncogenic lesion in BCR-ABL1 + ALL is the deletion of the Blineage transcription factor IKZF1 (in 980% of cases) [59] . The t(1;19)(q23;p13) translocation encoding the TCF3-PBX1 fusion gene is present in ≈4% of childhood ALL cases [55, 60] . TCF3-PBX1 + ALL is associated with a good prognosis but frequent central nervous system (CNS) relapse [61] . Like ETV6-RUNX1, the TCF3-PBX1 fusion protein has low oncogenic potential and requires secondary, cooperating mutations for overt leukemia to develop [62] . KMT2A (or MLL: mixed-lineage leukemia) rearrangements of 11q23 with other chromosomes are typically found in infant BCP-ALL (children G1 year) [34, 63] . KMT2A-rearranged leukemia often present with CNS involvement and are associated with poor treatment outcome [63] . Fusion genes involving KMT2A are likely sufficient for leukemia development, as suggested by a high concordance rate in monozygotic twins [38] and rare detection of secondary, cooperative mutations [64] . Investigation of early BCP-ALL development is invaluable in identifying new targeted treatment options and approaches to preventing leukemic transformation. BCP-ALL originates in a single cell, with subsequent clonal expansion of premalignant cells that may acquire more malignant traits. Due to the covert early etiology of the disease and the complexity of prenatal leukemic development, identifying and characterizing the BCP-ALL cell of origin remains challenging. Several studies have tried to narrow down the cell in which the first preleukemia-initiating event preferentially occurs (Table 1) . Although B cell blasts of different BCP-In Utero Development and Immunosurveillance of B Cell Acute Lymphoblastic Leukemia Rüchel et al. [65, 66] . An increasing number of studies provide evidence for the in utero origin of common BCP-ALL chromosome aberrations (as reviewed in [3•]). This suggests that preleukemic cells may arise in an early progenitor cell during fetal development, e.g., in the bone marrow or fetal liver. Ig and TCR gene rearrangements in BCP-ALL blast cells have been used as markers to investigate the clonal origin of leukemic cells. These markers have been identified in a large number of BCP-ALL patients (990%) [79, 80] . However, given that recombination activating gene (RAG)-driven rearrangements take place continually during clonal evolution of BCP-ALL [81] , Ig/TCR gene status may not reflect the preleukemia-initiating cell. Shared clonal Ig and TCR gene rearrangements in twins with concordant BCP-ALL might give better insight, as shown in studies of twins with concordant ETV6-RUNX1 + ALL that identified pro B cells or RAG1/2 − stem cells as potential cells of origin [72, 82] . Lineage switching upon relapse has been described in BCP-ALL, mostly for KMT2A-rearranged or BCR-ABL1 + ALLs [83, 84] . In the latter case, a subgroup of patients carrying the fusion gene presented with chronic myeloid leukemia (CML)-like disease, pointing to a multipotent progenitor cell [75] . Likewise, ambiguous expression of lymphoid and myeloid lineage markers, as observed in many BCP-ALL patients [85] , might point to an early progenitor cell with lympho-myeloid potential. Recently, lympho-myeloid precursor origin has been suggested for ETV6-RUNX1 + ALL, due to aberrant co-expression of myeloid markers observed in an ETV6-RUNX1 + human-induced pluripotent stem cell (hiPSC) model [53] . Interleukin-7 receptor α (IL-7Rα) mutations in BCP-ALL development IL-7Rα (encoded by the IL7R gene) is an important factor for lymphoid development. Together with the interleukin-2 receptor gamma (IL-2Rγ), it forms the IL-7 receptor (IL-7R) [86] . Recently, several groups have described activating mutations in IL7R as being involved in the initiation and development of BCP-ALL [87] [88] [89] . Inactivating mutations of IL7R are associated with severe combined immunodeficiency (SCID). SCID patients lack T cells. In mice, SCID manifests in both B and T cell absence [90] . In contrast, activating IL7R mutations have been observed in ALL, especially in Ph-like and PAX5 P80R subtypes. Using a conditional knock-in mouse model, Almeida et al. showed that physiological levels of mutant IL-7Rα were sufficient to generate preleukemic B cell precursors and to initiate leukemia resembling the human Ph-like and PAX5 P80R ALL subtypes [87] . Thomas et al. generated a genetically engineered mouse model with B cell-intrinsic expression of mutant IL7R that presented with development of BCP-ALL [88] . In an elegant study, Geron et al. transduced human CD34 + hematopoietic cells with mutant IL-7Rα. After transplantation into NOD/LtSz-scid IL-2Rγ null mice, a preleukemic state with retained self-renewal capacity developed [89••] . In all three studies, additional mutations acquired during leukemia development were observed. These led to upregulation of IL-7R signaling (via the JAK/STAT5 or the PI3K/mTOR pathway), upregulation of oncogenes (e.g., MYC, BCL2), and downregulation of tumor suppressors (including IKZF1) [87] [88] [89] . Additionally, CDKN2A was silenced [89••] , and recurrent somatic KRAS mutations which cooperate with mutant IL7R were observed [87, 88] . Taking all this together, a clear leukemia-initiating effect of constitutively active IL-7Rα could be observed in different mouse models as well as in human hematopoietic progenitors, with similarities to Ph-like and/or PAX5 P80R BCP-ALL subtypes. However, further studies are needed to fully understand how the interplay with other mutations leads to the development of overt leukemia. For the development of overt leukemia, a multifactorial etiology is proposed where a combination of genetic susceptibility and external factors induces leukemic transformation. External factors such as radiation, smoking, and infections, amongst others, can play a role in utero or postnatally. Radiation and smoking have already been reviewed elsewhere [91, 92] , associating high doses of ionizing radiation with ALL development and paternal smoking preconception and during pregnancy with an elevated risk for ALL. Infection has been suggested to be a likely trigger for ALL development. As postulated in the two-hit or delayed infection hypothesis by Mel Greaves [4], overt BCP-ALL requires an initiating mutation in utero (first hit) as well as a second postnatal mutation (second hit) [4] . In this model, the second hit is triggered by a dysregulated immune response towards common infections. Depending on the timing, infections were suggested to either have a protective (early) or detrimental (late) effect [4]. Pre-and postnatal infections have therefore been investigated as potential risk factors for triggering ALL. In utero cytomegalovirus (CMV) infection was found to be more prevalent in children who later developed leukemia compared to healthy controls [93] . CMV is a member of the herpesvirus family and is known to cause hearing loss and/or growth retardation in the developing child [94, 95] . CMV can cross the placenta and thus infect the child in utero. Maternal reactivation or reinfection can also play a role, probably due to influences on immune crosstalk between mother and fetus [95] . Interestingly, CMV degrades the neonatal Fc receptor (FcRn) which is responsible for the transfer of IgG through the placenta. Thereby, CMV interferes with the immunity that is conferred from mother to child [96] . Other herpesviruses, like Epstein-Barr virus (EBV) and varicella zoster virus (VZV), may also play a role in the development of childhood BCP-ALL. A significantly increased risk for ALL development could be detected for maternal EBV infection [97] ; however, significant correlation of EBV infection and ALL development could not be shown in a follow-up study [98] . A higher childhood leukemia risk was also observed when the mothers were infected with varicella or rubella during pregnancy [99•] . A link between maternal influenza infection and an increased risk of leukemia development was found in several studies as early as the 1970s [100, 101] . In a current meta-analysis by He et al., maternal influenza infection was significantly associated with higher risk of developing ALL [99•] . In terms of postnatal infections, a possible connection to influenza was observed in two space-time clusters [102, 103] . In the UK, increases in ALL incidence were observed in the years 1976 and 1990, following winter influenza epidemics [102] . In Milan, Italy, seven newly diagnosed ALL cases occurred within 4 weeks. All of these children were seropositive for the AH1N1 swine flu virus, whose outbreak occurred 3 to 6 months prior to leukemia diagnosis [103] . A possible explanation could be that influenza infection led to a strong dysregulated inflammatory response in the predisposed children, resulting in leukemic transformation of preleukemic cells. However, it is unlikely that influenza plays a unique role in the development of childhood ALL. It seems to be more important that predisposed children show an abnormal immune response to common infections. Other space-time clusters with a high incidence of childhood leukemia cases, e.g., the one in Fallon, USA (1997 USA ( -2003 , were not linked to influenza epidemics [104] . In light of the current severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic, it will be interesting to see the influences of the virus and the infection-prevention measures (e.g., lockdown, increased hygiene) on the development of ALL cases in the future. The critical second hit for the development of overt leukemia could be infection with SARS-CoV-2, leading to an aberrant immune reaction [105] . However, it is also possible that the measures taken to prevent the spread of the disease, like closing nurseries and schools, may provide a means for reducing ALL cases. A similar scenario occurred during the SARS-CoV-1 outbreak in Hong Kong in 2003 [106] . Here, a marked decline in common infectious diseases, like chickenpox, as well as a decline in ALL incidence were observed in the same year [106] . However, the same measures could also lead to an increase of ALL cases in the next years, as children born during the current pandemic have fewer social contacts and are less exposed to common infections during the critical time period where the immune system has to be trained in order to avoid ALL development, according to the delayed infection hypothesis [4] . Thus, the next years will show the influence of the pandemic and of the lockdown measures on the development of ALL cases and may give initial insights into how to prevent the development of leukemia in the future. Taken together, infections may promote leukemia at two different stages: (1) in utero due to the oncogenic potential of a virus or due to immune responses of a not yet fully developed fetal immune system or (2) after birth due to a dysregulated immune response. The in utero and early-in-life development of the immune system has long-term consequences for efficient control of the preleukemic clone The double-hit scenario of secondary events, such as infections, triggering leukemic progression is supported by epidemiological data [4] . Additionally, animal studies showed that genetically predisposed mice developed leukemia only in a pathogen-containing environment [4, 107] . The exact mechanism remains unclear, but the lack of efficient immune cell training by microbial colonization and pathogens in utero and early in life has been suggested to be crucial for the development of ALL [5, 108] . Infections shape the immune system and thereby indirectly affect the preleukemic clone. In this context, among other innate immune cells, natural killer (NK) cells have been shown to be modulated by trained immunity. Infectious stimuli induced epigenetic reprogramming towards enhanced killing capacity of NK cells [109] . Furthermore, NK cells combine an antiviral and anti-tumor killing capacity and are thus promising candidates for modulation of preleukemic cells. NK cells were shown to gain memory functions after viral infections or after stimulation with pro-inflammatory cytokines [110, 111] . Interestingly, NK cell cytotoxicity against a leukemic cell line was also significantly enhanced after CMV infection, mediated by the NKG2C (+) NK cell subpopulation [112] . In contrast, single cell RNA sequencing of ETV6-RUNX1 + ALL cases revealed significant inhibition of NK cell activity in the tumor microenvironment [113••] . This suggests that the dual role of NK cells can be explained by taking the different NK cell subtypes into account. Recent genetic studies have provided proof that a certain genetic constitution of NK cells controls BCP-ALL [114] . Killer immunoglobulin receptors (KIR) on NK cells interact with human lymphocyte antigen (HLA) class I molecules. The inhibitory NK cell receptor KIR2DL1-a high-affinity ligand for HLA-C2-is significantly increased in BCP-ALL patients. In another study, five NK cell-related factors (KIR2DL5A, NKp46, FasL, granzyme B and PI-9) were positively associated with detection of minimal residual disease at the end of induction therapy [115] . How the inhibitory NK cell receptors' control of the preleukemic clone is determined by genetic factors or modulated by infections should be part of future studies. The importance of early and even prenatal immune training with microbial antigens is underlined by epidemiological data that refer to the hygiene hypothesis [116•] . Interestingly, the same epidemiological factors leading to a clean and hygienic environment, such as late introduction into day care, order and number of siblings, and early antibiotic treatment [117] , have been associated with a higher incidence of autoimmune diseases and allergies as well as with a higher incidence of BCP-ALL [4, 118] . These are all diseases that are predominantly mediated by T helper (Th) cells, suggesting a certain role of Th responses in the control of the preleukemic clone. Atopic disease and childhood ALL are negatively correlated. A Th2 phenotype might be protective against ALL development [119] , while pro-inflammatory Th1 cells with high interferon gamma (IFNγ) levels have been shown to migrate towards BCP-ALL cells and favor their proliferation via upregulation of CD38 and IFNγ-induced protein 10 (IP-10) production [120••] mediated by activation-induced cytidine deaminase (AID) upregulation [121] . But, what driving force skews the immune response towards one or the other direction, given the fact that early immune cell priming is lacking in both scenarios? Miedema and colleagues attributed this to a particular genetic predisposition, since they found two SNPs in the TLR6 gene associated with BCP-ALL, leading to an altered Th1/Th2 balance upon microbial exposure [122] . The immunosurveillance mechanisms are summarized in Figure 1 . Diagnosis of a severe underlying germline ALL predisposition with a high penetrance, such as TP53 mutation/Li-Fraumeni syndrome, offers the opportunity to monitor the patient closely for early cancer occurrence and clearly improves overall survival [123] . By contrast, diagnosis of a more common predisposition, like an in utero occurring somatic ETV6-RUNX1 mutation, does not provide such a benefit, as the mutation confers only a minor risk of a child developing ALL, a disease for which current chemotherapy treatment protocols achieve 80-90% overall survival without early detection being critical for its outcome. However, successful treatment comes at the price of significant acute and late toxicities, which account for a large proportion of deaths. Acute adverse effects during chemotherapy for childhood cancer can affect all organs, and two-thirds of childhood cancer survivors live with long-term effects of the toxic treatment, which can be severe (e.g., cognitive impairment, osteonecrosis, secondary cancers, infertility, depression) [124] . Therefore, there is an urgent need to employ strategies aimed at preventing children from getting cancer in the first place. In the absence of means to directly target and eliminate the preleukemic cells, general training of the immune system early in life (e.g., in child day-care, through contact with pets) is recommended and promising. More targeted approaches currently include (1) training of the innate immune response via specific vaccination or (2) modulation of the microbiome (by, e.g., probiotics) to achieve a healthier, more complex state [5] . Targeting Th1/Th2 lineage determination to prevent the clonal expansion of the preleukemic clone may be a promising alternative treatment approach to follow up on. However, differentiation programs are complex and intricately cross-linked. Side effects of pharmacologic modulation in genetically predisposed children can be severe and may outweigh potential benefits. We believe that further studies employing larger cohorts of predisposed children are clearly necessary to understand the complex interplay of genetic predisposition and environmental factors and to finally enable us to develop targeted preventive approaches. 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The Lancet The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias Sitespecific translocation and evidence of postnatal origin of the t(1;19) E2A-PBX1 fusion in childhood acute lymphoblastic leukemia Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options Cytogenetically aberrant cells are present in the CD34+CD33-38-19-marrow compartment in children with acute lymphoblastic leukemia Investigation of clonal involvement of myeloid cells in Philadelphia-positive and high hyperdiploid acute lymphoblastic leukemia Immature CD34+CD19− progenitor/stem cells in TEL/AML1-positive acute lymphoblastic leukemia are genetically and functionally normal Characterization of acute lymphoblastic leukemia progenitor cells Initiating and cancerpropagating cells in TEL-AML1-associated childhood leukemia Clonal origins of ETV6-RUNX1+ acute lymphoblastic leukemia: studies in monozygotic twins Leukemic stem cells in childhood high-risk ALL/ t(9;22) and t(4;11) are present in primitive lymphoidrestricted CD34+CD19-Cells Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia Monitoring of childhood ALL using BCR-ABL1 genomic breakpoints identifies a subgroup with CML-like biology Human chromosomal translocations at CpG sites and a theoretical basis for their lineage and stage specificity Mll-AF4 confers enhanced self-renewal and lymphoid potential during a restricted window in development Discovery of a CD10-negative B-progenitor in human fetal life identifies unique ontogenyrelated developmental programs This reference is of importance because it investigates the origins of infant leukemia, identifying fetal-restricted CD10 -prepro B progenitor cells as potential leukemia-initiating cells 79 High incidence and unique features of antigen receptor gene rearrangements in TEL-AML1-positive leukemias Crosslineage T cell receptor gene rearrangements occur in more than ninety percent of childhood precursor-B acute lymphoblastic leukemias: alternative PCR targets for detection of minimal residual disease Massive evolution of the immunoglobulin heavy chain locus in children with B precursor acute lymphoblastic leukemia Genomic analysis of different clonal evolution in a twin pair with t(12;21) positive acute lymphoblastic leukemia sharing the same prenatal clone Mutational and transcriptomic profiling of acute leukemia of ambiguous lineage reveals obscure but clinically important lineage bias Lineage switch at relapse of childhood acute leukemia: a report of four cases Frequent but nonrandom expression of myeloid markers on de novo childhood acute lymphoblastic leukemia Flip the coin: IL-7 and IL-7R in health and disease Interleukin-7 receptor alpha mutational activation can initiate precursor B-cell acute lymphoblastic leukemia Activated interleukin-7 receptor signaling drives B-cell acute lymphoblastic leukemia in mice An instructive role for Interleukin-7 receptor α in the development of human B-cell precursor leukemia This reference is of outstanding importance because it investigates the role of activating IL-7Rα mutations for the development of a preleukemic state in BCP-ALL. This study elegantly shows that activation of IL-7Rα in human hematopoietic stem and progenitor cells is sufficient for the development of preleukemic cells and leads to overt leukemia in combination with CDKN2A deletions 90 Risk factors for childhood leukemia: radiation and beyond. Front Public Health Paternal smoking before conception and during pregnancy is associated with an increased risk of childhood acute lymphoblastic leukemia: a systematic review and meta-analysis of 17 case-control studies In utero cytomegalovirus infection and development of childhood acute lymphoblastic leukemia Neuropathogenesis of congenital cytomegalovirus infection: disease mechanisms and prospects for intervention Cytomegalovirus as an immunomodulator across the lifespan Human cytomegalovirus evades antibody-mediated immunity through endoplasmic reticulum-associated degradation of the FcRn receptor Maternal herpesvirus infections and risk of acute lymphoblastic leukemia in the offspring Activation of maternal Epstein-Barr virus infection and risk of acute leukemia in the offspring This reference is of importance because it thoroughly analyzes 20 studies regarding the association between maternal infection during pregnancy and childhood leukemia. The authors conclude that specific infections during pregnancy are associated with a higher risk to develop leukemia 100 Association between malignant disease in children and maternal virus infections Childhood leukemia incidence in Britain, 1974-2000: time trends and possible relation to influenza epidemics Possible role of pandemic AH1N1 swine flu virus in a childhood leukemia cluster Unusual space-time patterning of the Fallon, Nevada leukemia cluster: evidence of an infectious etiology Leukaemia and lockdown: The delayed infection model of childhood acute lymphoblastic leukaemia and the COVID-19 pandemic Impact of SARS on development of childhood acute lymphoblastic leukaemia Infection exposure promotes ETV6-RUNX1 precursor B-cell leukemia via impaired H3K4 demethylases Stereotypic immune system development in newborn children EZH2 identifies the precursors of human natural killer cells with trained immunity About training and memory: NK-cell adaptation to viral infections Proinflammatory cytokine signaling required for the generation of natural killer cell memory Latent cytomegalovirus infection enhances anti-tumour cytotoxicity through accumulation of NKG2C+ NK cells in healthy humans Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities This reference is of outstanding importance because it unravels the regulatory cell network in the bone marrow during BCP-ALL using single cell RNA sequencing NK cell genotype and phenotype at diagnosis of acute lymphoblastic leukemia correlate with postinduction residual disease This review gives an overview of research on the hygiene hypothesis that may play a role in the development of autoimmune diseases, allergies and BCP-ALL 117 Infectious diseases in the first year of life, perinatal characteristics and childhood acute leukaemia Atopic disease and childhood acute lymphoblastic leukemia Bone marrow T helper cells with a Th1 phenotype induce activation and proliferation of leukemic cells in precursor B acute lymphoblastic leukemia patients Support of BCP-ALLcells by autologous bone marrow Th-cells involves induction of AID expression but not widespread AID off-target mutagenesis Polymorphisms in the TLR6 gene associated with the inverse association between childhood acute lymphoblastic leukemia and atopic disease Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: 11 year follow-up of a prospective observational study Late effects of childhood leukemia therapy The authors thank Stewart Boden for critical reading of the manuscript.