Interestingly, in murine models immune restricted cells with lymphoid potential are observed in the yolk sac (YS), preceding the first HSCs found in FL; these have potential to produce lymphocytes and granulocyte macrophage progenitors (52) and express Il7 receptor (Il7-r/CD127). Transcriptomic data suggests that such lymphoid progenitors may also be present in human YS (9) but these have not been systematically characterized yet. In humans a potentially analogous cell has been identified in the FL, from 6 pcw; (CD34⁺CD19⁻IL7R⁺) (1, 50, 53, 54). Similar IL7R⁺ progenitors have been described in human FBM (1). FL and FBM CD34⁺CD127⁺CD19⁻CD10⁻ ELP have been characterized by functional and transcriptomic assays, and shown to generate B, T and NK cells while retaining some residual myeloid output. These fetal-specific ELP are very rare in postnatal life (1, 54). There has therefore, been considerable interest in these cells as potential target cells for childhood-ALL.

From 7 pcw the presence of two committed CD19⁺ B-progenitors downstream of ELP has been confirmed in human FL samples; PreProB (CD34⁺CD19⁺CD10⁻) and ProB (CD34⁺CD19⁺CD10⁺) progenitors; differing in their CD10 expression (1, 50, 54). Similar progenitors have been described in cord blood (55, 56). PreProB-progenitors account for ~2.5% and ProB-progenitors ~8% of FL CD34⁺ cells, and these frequencies remain fairly stable in FL between 7 and 20 pcw. These cells have also been identified by single cell transcriptomic approaches in the human FL (9).

PreProB and ProB-progenitors are also present and markedly expanded in human FBM (1). Both B-progenitor compartments undergo marked expansion in the early stages of colonization of FBM, to account for up to around 20% and 11% of FBM CD34⁺ cells, respectively, at 11 pcw. Later in the second trimester PreProB-progenitors plateau while ProB-progenitors expand further to >30% of CD34⁺ cells in FBM. By contrast, ABM CD34⁺ compartment was found to have only 0.5% PreProB-progenitors and 14% ProB-progenitors (1).

Both PreProB and ProB-progenitors lie downstream of ELP and generate exclusively B-lymphoid progeny in vitro and in vivo. Functional and molecular studies have established that FBM PreProB-progenitors lie upstream of ProB-progenitors, and are therefore the earliest B-lymphoid restricted progenitors in the fetal B-cell developmental hierarchy (1).

B cell maturation, defined by B cell receptor diversification, commences in B-lymphoid progenitors in fetal life. Fetal ELP and PreProB-progenitors show partial (D_H-J_H) IgH rearrangement (1, 54), whereas the more mature ProB-progenitors demonstrate complete V_H-D_H-J_H rearrangement (1).

CD19⁺ B-cells have been reported in FL and FBM by many groups (30, 48, 49, 57–59), and recently been characterized in greater detail (1, 9, 60, 61). Evidence of B cell maturation is demonstrable in human fetal life, with polyclonal CD19⁺IgM⁺ B-cells (60–63). Although FL and FBM immunoglobulin heavy chain repertoires are equally diversified, FL appears to be the main source of IgM natural immunity during the 2nd trimester, and this correlates with the majority of B-cells in 2nd trimester FBM being CD34⁻CD19⁺CD10⁺IgM/D⁻ PreB-cells with a relative lack of more downstream immature and transitional B-cells (60).

B cells can be further divided into B1 B-cells of the innate immune system and “conventional” B2 B-cells of the adaptive immune system. This division is well-established in mice, where sIgM⁺CD11b⁺CD5⁺ B1a B-cells were first identified (64, 65) through the search for, the still elusive, cell of origin of adult human CLL (42, 66). B1b B-cells (sIgM⁺CD11b⁺CD5⁻) were subsequently described (67); both these subtypes are seen predominantly in serous cavities. Further characterization of splenic B1 cells have identified them to be CD5^+/−CD19^hiCD1d^midCD23⁻CD43⁺IgM^hiIgD^lo (68). Murine B1 B-cell progenitors are found in the yolk sac (69) prior to the emergence of the first definitive HSCs in the FL, which have both B1 and B2 B-cell output (70). The B-cell output skews toward B2 B-cells over ontogeny, with B1 B-cell output being exceedingly rare in ABM (65, 71).

Human B1 B-cells and their upstream progenitors have been proposed as the in utero cell of origin for infant and childhood-ALL (72) and as having a role in auto-immune disease (73, 74). In humans, B1 B-cells were described in umbilical cord blood and adult peripheral blood. These cells were CD20⁺CD27⁺CD43⁺CD38^lo/int and functioned in line with murine counterparts, including spontaneous IgM secretion, constitutional BCR receptor activity and ability to induce allogeneic T cell proliferation (75). Putative B1 B-cells have also been described in human fetal hematopoiesis, with greatest frequencies in 10 pcw FL, decreasing as FBM is colonized (59). After birth, estimates of B1 B-cell populations range from 1 to 10% circulating B-cells, this frequency falls as age increases (76–78).

The progenitors of B1 B-cells in humans remain elusive and contentious. Two theories posit either a lineage (or layered) model where different subtypes arise from different progenitors or a selection model whereby there is interconversion between B1 and B2 B-cells. In humans, CD27 (one of the cell surface markers of B1 B-cells) expression in ABM ProB-cells coincides with LIN28B expression levels similar to that seen in FL. These cells mature preferentially to B1-like B-cells compared to their CD27⁻ counterparts. It is not clear whether this relates to a separate lineage or alternative differentiation potential (79).

In summary, human fetal B-lymphopoiesis starts around 6 pcw in FL, with B-cell production happening simultaneously in FL and FBM from 2nd trimester. Hematopoiesis in the FBM is skewed toward B-lymphopoiesis in 2nd trimester. In addition there are fetal-specific B-lymphoid progenitors (ELP and PreProB-progenitors), B-cells (B1 B-cells) and developmental pathways that are different from human adult life (Figure 1).

The clinical course and molecular features of iALL are distinct from childhood-ALL. iALL remains a disease with dismal event-free survival (EFS) (18, 92–94), although recent risk-stratified treatment protocols suggest that outcomes could be improved (95). In iALL, blasts are predominantly CD19⁺CD10⁻, often with aberrant myeloid cell surface markers suggestive of an immature B-progenitor, as opposed to a CD19⁺CD10⁺ Pre-B phenotype in childhood-ALL (18, 96). KMT2A gene rearrangements (KMT2A-r) is the main genetic driver for 70–80% iALL cases, as opposed to only 2–5% of childhood-ALL cases (97, 98).

Current evidence suggests that iALL (particularly KMT2A-r ALL) originates in utero and has been traced back to its fetal origin through retrospective detection of the fusion gene in neonatal blood spots (99), as well as studies in monozygotic twins with ALL (100, 101). A characteristic feature of iALL is the fact that a single hit (KMT2A-r) before birth seems to be sufficient to induce a rapidly-proliferating, therapy-resistant leukemia without the need for additional mutations (102).

Unlike iALL, many cases of childhood-ALL also originate in utero but only develop into full-blown leukemia after a second post-natal hit (15, 16). Several subtypes of childhood B-ALL have been shown to arise in utero including those characterized by KMT2A-r (103, 104), ETV6-RUNX1 (105–107), BCR-ABL (108), TCF3-PBX1 (109), TCF3-ZNF384 (110) gene fusions and high hyperdiploid ALL (111, 112) (Figure 2).

There are several properties of fetal hematopoietic cells that may underlie the pathogenesis of iALL and childhood-ALL. Firstly, fetal HSPC are more proliferative (6, 7) and have better long term repopulating ability in xenograft models (8, 113–115). Fetal-specific gene expression programs such as the LIN28B-LET-7-HMGA2 axis (79, 80, 116, 117) have been shown to drive self-renewal (118) and oncogenesis (10–12, 119). Activation of LIN28B, in particular, has been demonstrated in several cancers and results in suppression of LET-7 micro-RNAs and subsequent de-repression of an array of oncogenes including MYC, RAS, BLIMP1, ARID3A and HMGA2 (10, 120). ARID3A is necessary for fetal B lymphopoiesis and B1 cell division (121, 122), and has also been shown to promote cancers by driving higher MYC expression (123, 124). HMGA2 is a fetal-specific transcription factor that is re-expressed in many cancers. It promotes cell proliferation, and the Lin28-Let-7-HMGA2 axis maintains cancers in an undifferentiated state (125). The expression of oncogenes such as LIN28B in fetal HSPC, may therefore play a role in leukemia initiation and transformation of fetal target cells, and in particular the development of aggressive leukemias in infancy and early childhood.

Secondly, there is a higher proportion of B-progenitors in fetal life compared to adults (1, 2). B-lymphopoiesis itself changes through the human lifetime with a switch in the ratio of B-progenitors to more mature B-cells (30, 49). Regardless of the mechanism, hematopoiesis in the human FBM is skewed toward the B-lymphoid lineage with the presence of a very high frequency of B-progenitors (1, 126) thus expanding the pool of target cells for malignant transformation.

It is also possible that the fetal cell of origin for iALL and childhood-ALL are different (Figure 2). We suggest that an attractive hypothesis is that iALL arises in a unique B-progenitor found only in fetal life. Of particular interest are fetal-specific IL7R⁺ ELP (1, 50, 53, 54) and PreProB-progenitors (1, 50, 55, 56) that share immunophenotypic, transcriptomic and IgH rearrangement patterns with iALL blasts (1, 96). Compared to ABM counterparts, fetal PreProB-progenitors uniquely express known oncofetal genes, such as LIN28B, as well as genes implicated in KMT2A-r iALL, such as KLRK1 and PPP1R14A (127, 128) that have not previously been recognized as being fetal-specific (1). Fetal ELP/PreProB-progenitors also demonstrate features that could account for lineage plasticity such as an accessible chromatin pattern, together with residual expression, of myeloid and stem cell genes (1). In addition, iALL can switch to a myeloid lineage at relapse, especially after B-lymphoid directed treatment (129–132). This could either be a feature of residual preleukemic primitive progenitors that are capable of giving rise to both myeloid and lymphoid leukemia, or because of plasticity and/or reprogramming of leukemic early B-lymphoid progenitors (130, 133). For example, KMT2A-r, the most frequent genetic driver of iALL, may drive leukemogenesis by binding to accessible genes in permissive fetal progenitors; or indeed alter the chromatin accessibility and gene expression patterns of target genes. KMT2A is a lysine methyltransferase, and KMT2A-r is thought to promote leukemogenesis by activating key target genes such as HOXA9 and MEIS1 (134, 135). Although there is some heterogeneity in KMT2A-r ALL based on the specific fusion partner gene, most KMT2A-fusion proteins drive and maintain leukemia via a protein complex involving AF4/ENL/AF9/PTEF-B. KMT2A-fusion proteins bind directly to gene targets where they aberrantly upregulate gene expression, partly by increasing histone-3-lysine-79 dimethylation through DOT1L (135).

These mechanisms of KMT2A-r mediated transformation are difficult to validate without a bona fide model of iALL, which has been very difficult to generate. However, we have recently developed a novel iALL model derived by CRISPR-Cas9 mediated KMT2A-r in primary human FL HSPC (136). This demonstrates that a human fetal cell context is permissive, and indeed probably required; to give rise to an ALL that recapitulates key features of iALL. In this model, recruitment of fetal-specific genes by KMT2A-AF4 is demonstrated by KMT2A-N and AF4-C binding and H3K79me2 at these genes by ChIP-seq (136). Furthermore, maintenance of fetal-specific gene expression programs accounts for the unique molecular profile of iALL, suggesting that it is the specific fetal target cell(s) in which it arises that provide the permissive cellular context (136).

It is possible that childhood-ALL on the other hand is likely to arise from a more mature CD19⁺CD10⁺ fetal B-progenitor such as ProB-progenitors or PreB-cells. These cell populations are found in abundance in FBM and expand rapidly throughout the second trimester. As in iALL, several genes that have been implicated in the pathogenesis of childhood-ALL are also important in fetal B lymphoid development. Some of these, such as PAX5, EBF1, TCF3, and IL7R (137, 138), are expressed at higher levels in fetal B-progenitors compared to postnatal counterparts (1). This is also true for the B-cell specific gene RAG1 that may play a role in driving childhood-ALL-associated chromosomal translocations such as ETV6-RUNX1 (139). In addition, childhood-ALL is characterized by multiple lesions affecting cell cycle and B-cell differentiation genes (138). It is hypothesized that the proliferative capacity and complementary epigenetic profile (such as greater chromatin accessibility of highly expressed genes) of the cell of origin provide the right substrate for leukemic transformation (35, 140). This permissive cell-state is likely to be present in FBM ProB-progenitors where their rapid proliferation at the expense of differentiation during a particular developmental time window may make them more susceptible to oncogenic hits. Others have hypothesized that it is the fetal/neonatal BM niche that drives the lymphoid-biased phenotype of KMT2A-r infant/childhood leukemia (141).