B-ALL with DUX4 rearrangement is one of the newer described subtypes of B-ALL. DUX4-rearranged B-ALL comprises 16% of the B-other cases or 4% of all pediatric B-ALL [28]. In B-ALL in AYA in Japan, DUX4, ZNF384, and MEF2D fusion genes account for about 40% of Ph-negative cases [29]. In B-ALL in Malaysia and Singapore, DUX4-rearranged B-ALL is the third-most-common subtype [130]. This leukemia has a favorable prognosis, similar to B-ALL, with high hyperdiploidy and ETV6::RUNX1 fusion, despite the presence of high MRD levels [130,131].

The DUX4 gene encodes a homeobox-containing protein and is located within a subtelomeric D4Z4 repeat region on 4q and 10q. The gene is present in 11–100 copies on each allele and is epigenetically silent in somatic tissues. The DUX4 rearrangement occurs most frequently with IGH and less frequently with the ERG gene. In the IGH::DUX4 fusion, a segment of the DUX4 gene is relocated to IGH, leading to the overexpression of DUX4. This rearranged form of DUX4 binds with a genetic region in the ETS-family transcription factor ERG (ETS-related gene), which leads to the expression of an ERG protein fragment that inhibits normal ERG function and causes leukemic transformation. ERG deletions are frequent secondary alterations in DUX4-rearranged B-ALL [28,29,132]. Also, IKZF1 deletions co-occur with ERG deletions in DUX4-rearranged B-ALL. As the prognosis of IKZF1 deletions depends on the co-occurring mutations in B-ALL, the usually adverse prognosis of IKZF1 deletions can be overcome in these patients by chemotherapy based on MRD evaluation [133].

DUX4 rearrangements in B-ALL are complex and different from those in CIC::DUX4 fusion-positive (non-Ewing) round-cell sarcoma (sarcoma described in [134]) [28]. The complexity of the genetic rearrangement is likely to be the reason why these abnormalities were not detected in the pre-genomics era. The gene expression profile for DUX4-rearranged B-ALL is distinctive [28]. Flow cytometry showed strong (aberrant) surface expression of CD371 on the leukemic cells in DUX4-rearranged B-ALL, which, when combined with the expression of CD2, diagnosed all cases of this type of B-ALL [135]. CD371 is predominantly expressed on myeloid cells [135] and is not expressed on mature lymphocytes (see image in [36]). DUX4-rearranged leukemic cells may also express CD66c, and the co-expression of CD66c and CD2 was almost exclusively found in DUX4 fusion-positive B-ALL [136]. An immunohistochemical stain for detecting DUX4 fusions showed immunohistochemical positivity in five of six molecularly-positive cases and negativity in three of three molecularly-negative cases [137]. DUX4-rearranged B-ALL leukemic cells may switch to monocyte-like cells, which is a feature of CD371 expression [135,137], and this switch does not lead to a worse outcome [138].

The WHO-HAEM5 diagnostic criteria require RNA or DNA sequencing by NGS to diagnose this type of B-ALL. The desirable criteria include confirming the DUX4 gene rearrangement, the presence of CD371 expression on leukemic cells by FCI, or both [36]. It is noteworthy that while RNA sequencing can diagnose DUX4 fusions, the most extensive study of DUX4-rearranged B-ALL patients examined by whole-genome sequencing (WGS) in a single clinical trial in the U.K. showed that whole-transcriptome sequencing alone could not be relied upon to identify all DUX4-rearranged B-ALL cases in the absence of WGS. These investigators established an automated bioinformatics pipeline that improved the detection of DUX4 fusions by WGS [139].

The zinc finger protein 384, ZNF384, gene is located on the chromosomal locus 12p13.31. The gene encodes for a zinc finger transcription factor that is ubiquitously expressed in the bone marrow and other tissues. The transcription factor appears to bind and regulate the promoters of the extracellular matrix genes [140]. ZNF384 rearrangements may occur with at least ten different gene partners in about 5% of childhood B-ALLs, 10% of adult B-ALLs, and 48% of mixed-phenotype acute leukemia, B/myeloid-type [141,142].

In Japan, ZNF384-related fusion genes were identified in 4.1% of 291 B-ALL or about 9% of B-other ALL patients. All ZNF384-related gene fusions, including TCF3::ZNF384 and EP300::ZNF384, showed weak or negative CD10 expression with aberrant CD13 and CD33 expression. But the clinical features differed depending on the specific fusion gene. Higher cell counts, younger age (median age five years), and more frequent relapses were present in TCF3::ZNF384-positive than in EP300::ZNF384-positive B-ALL patients. The latter group of B-ALL patients had a median age of 11 years [30,142]. FISH with break-apart probes or genomic sequencing (RNA or DNA) is required to diagnose the cryptic ZNF384 rearrangement [36].

Myocyte-enhancer factor 2 (Mef2) transcription factors are necessary for early B-cell development [143]. MEF2D, located on 1q22, encodes one of these transcription factors. MEF2D was found to be rearranged in about 5% of pediatric B-ALL without recurring genetic abnormalities. MEF2D can rearrange with multiple genes (BCL9, CSF1R, DAZAP1, HNRNPUL1, and SS18), with BCL9, located on 1q21, being the most frequent.

MEF2D::BCL9-rearranged B-ALL presents at a median age of 14 years. Morphologically, the leukemic cells appear to be mature B-cell leukemia-like cells with high expression of HDAC [144]. They have a characteristic immunophenotype with weak or absent CD10, CD38 positivity, and cytoplasmic IgM positivity. The cytogenetic rearrangement is cryptic by karyotyping, and diagnosis requires FISH, gene expression profiling, or genomic sequencing. There is resistance to chemotherapy, with very early relapse in this high-risk leukemia [32,35,144].

The PAX5 gene encodes for a transcription factor that regulates numerous genes essential for normal B cell development. B-ALL with PAX5alt and B-ALL with PAX5 p.P80R refer to two distinct types of B-ALL. Both of these types of B-ALL harbor molecular genetic abnormalities in PAX5, which lead to a loss of the normal PAX5 protein, initiating a precursor B lymphoblastic leukemia.

B-ALL with PAX5 p.P80R is unique because this subtype of B-ALL is characterized by a single point mutation in PAX5 instead of the other types of abnormalities that are common in B-ALL, such as deletions and translocations. This point mutation, c.239C>G, p.P80R, causes a substitution of proline to arginine in the DNA-binding domain of PAX5. In a cohort of 170 adult B-ALL cases that were negative for the known genetic abnormalities in B-ALL, gene expression data profiling showed four clusters corresponding to B-ALL with rearranged ZNF384, DUX4, KMT2A, and BCR::ABL1-like features [145]. A fifth cluster in this study comprised 14 patients with PAX5 p.P80R and lacked any fusion gene. Sanger sequencing identified 16 additional cases with PAX5 p.P80R in another cohort [145]. Cytogenetics showed structural rearrangements of 9p or 7p, including dic(9;20) and der(7;9). The second allele was deleted or inactivated, leading to biallelic loss of PAX5 [34,145]. Mutations of genes in the RAS pathway were also present [34,145].

B-ALL with PAX5alt includes leukemia-causing genetic abnormalities other than PAX5 p.P80R. This type of B-ALL has a gene expression profile distinct from that of B-ALL with PAX5 p.P80R [34]. It comprises about 3–5% of childhood ALLs and 9.6% of adult B-ALLs.

In contrast, B-ALL with PAX5 p.P80R comprises about 1% of childhood B-ALLs and up to 5% of adult B-ALLs. By FCI, B-ALL with PAX5 p.P80R shows a pro-B immunophenotype, with low CD20 and high CD45 expression on the leukemic cells. The leukemic cells are CD13-negative, CD33-positive, and CD2-positive and show stronger intensity CD10 expression than in KMT2A-rearranged B-ALL. The prognosis of B-ALL with PAX5 p.P80R is better than that of B-ALL with PAX5alt abnormalities [33,34,131]. The diagnosis of these subtypes requires genomic sequencing.

This rare leukemia occurs in <1% of children, 1–2% of AYA, and 2–3% of adult B-ALLs [36]. These cases have a precursor B-ALL immunophenotype, including no expression of surface immunoglobulins, but they harbor MYC rearrangement. According to gene expression profiling, these leukemias cluster with precursor B cells and other B-ALLs, but not with Burkitt leukemia [146]. In adults, these leukemias are considered high-risk B-ALLs with poor prognoses [131]. Children with MYC-rearranged B-ALLs are usually treated with Burkitt lymphoma therapy, with a better outcome than adults with MYC-rearranged B-ALL [36].

NUTM1 rearrangement is more frequent in infants (about 3–5%) than in children (0.4–0.9%) with B-ALL, and this rearrangement has not yet been detected in adults with B-ALL [147].

The nuclear protein in the testes (NUT) is normally located in post-meiotic spermatogenic cells, wherein a global increase in hyperacetylation occurs for spermatogenesis. The NUT Midline carcinoma family member 1 (NUTM1) gene (also known as NUT), located on 15q14, was first discovered as a part of the fusion gene in a rare and aggressive carcinoma called NUT carcinoma [148,149]. NUT carcinoma harbors a reciprocal t(15;19)(q14;p13.1) translocation between NUTM1 on chromosome 15q14 and the BET family gene BRD4 on chromosome 19p13.1, leading to an in-frame BRD4::NUT fusion oncogene driven by the BRD4 promoter [149]. Subsequently, with the increased evaluation of tumors by genomic sequencing approaches, the NUTM1 gene was found to also be present with other fusion partner genes in different types of cancers, including sarcomas and B-ALL [150]. These fusions lead to aberrant NUTM1 overexpression, and the altered global chromatin acetylation might confer sensitivity to histone deacetylase inhibitors and possibly to bromodomain inhibitors for NUTM1::BRD9 fusion cases [36].

Intriguingly, while NUT carcinoma is a highly aggressive cancer, NUTM1-rearranged B-ALL has a favorable prognosis. This type of B-ALL occurs in infants and comprises 21.7% to 30% of non-KMT2A-rearranged (or KMT2A germline) B-ALL in infants [147,151]. Among nine NUTM1-rearranged B-ALL patients with a median age of 8.8 months, ACIN1 (n = 5), CUX1 (n = 2), BRD9 (n = 1), and ZNF618 (n = 1) were identified as fusion partners [151]. Interestingly, this same study also identified other KMT2A-germline infant B-ALL patients with a median age of about 11 months who harbored PAX5 fusion; those patients had a poor prognosis [151].

By immunophenotype, the leukemic cells in NUTM1-rearranged B-ALL may be CD10-positive or CD10-negative, in contrast with CD10-negative leukemic cells in KMT2A-rearranged B-ALL [147,151]. However, KMT2A-rearranged B-ALL may also be positive for CD10 [136], indicating that CD10 expression alone cannot be used to distinguish these two subtypes of ALL. The diagnosis can be made by FISH using a break-apart NUTM1 probe or RNA or DNA sequencing [36,151].

This leukemia subtype comprises about 1–3% of childhood ALL [28] and about 2% of adult B-ALL [131]. Similar to Ph-like B-ALL, B-ALL with ETV::RUNX1-like features lacks the ETV6::RUNX1 fusion, but the gene expression profile is similar to that of B-ALL with ETV::RUNX1 fusion (see figure in [36]).

FCI shows the leukemia cells are CD24-positive and CD44-negative or low. However, note that this immunophenotype is not specific to this subtype of B-ALL and was also identified in B-ALLs with other genetic subtypes diagnosed by gene expression profiling [31]. Molecular analysis reveals combined ETV6 and IKZF1 alterations (rearrangements and deletions) in this type of leukemia [28]. Further, recent genomic studies showed biallelic ETV6 inactivation [139] and the APOBEC mutational signature in ETV6::RUNX1-like childhood B-ALL patients [139,152]. Of note, B-ALL with ETV6::RUNX1-like features may also arise in patients with germline ETV6 alterations [152].