Approximately 50% of patients with Ph-like ALL harbor rearrangements of the cytokine receptor- like factor 2 (CRLF2) gene, located on the pseudoautosomal region 1 (PAR1) of chromosomes Xp22 and Yp11 [4,12,15,24]. In normal conditions CRLF2 dimerizes with the α- subunit of interleukin- 7 receptor (IL7RA) to form a heterodimeric thymic stromal lymphopoietin receptor (TSLPR) which actives downstream JAK2/STAT5 and thePI3K/AKT/mTOR pathways [26,27,28] and is implicated in early B-cell development [29]. CRLF2 deregulation results from three main mechanisms: (1) a cryptic rearrangement that juxtaposes CRLF2 to the immunoglobulin heavy chain locus (IGH); (2) a focal deletion in the pseudoautosomal region of the sex chromosomes resulting in P2Y receptor family member 8 (P2RY8)-CRLF2 fusion that positions CRLF2 under the control of the P2RY8 promoter; (3) and less frequently by an activating CRLF2 point mutation, F232C [13,17,22,23,25,30,31,32,33]. Rearrangements of CRLF2 account for 24% of pediatric patients with NCI SR Ph-like ALL [9], 55% of children with HR disease [10] and 50% to 60% of adolescent and adult patients with Ph-like ALL cases [4,11,12,13,17,18]. P2RY8-CRLF2 fusions occur more commonly in younger children and in patients with Down syndrome (DS) ALL [22,25], while IGH-CRLF2 fusions are detected more frequently in older patients and patients of Hispanic ethnicity [34]. In a genome-wide association study of CRLF2-rearranged ALL, the inherited GATA3 variant rs3824662 was associated with CRLF2 rearrangement, JAK mutation, IKZF1 deletion, variation in GATA3 expression and increased risk of relapse [35]. This variant is markedly more common in patients of Hispanic ethnicity (~40%) or Native American (~50%) genetic ancestry, while is it detected in only 14% of Europeans [35,36]. The point mutation changing phenylalanine 232 to cysteine in CRLF2 has been identified in 9% of DS-ALL patients [25] and 21% of adult B-ALL patients [23]. In in vitro assays, the expression of CRLF2 F232C in the absence of co-expression of mutant JAK2 promotes JAK2 signaling activation and cell transformation [23,25,37]. CRLF2 rearrangement and overexpression is associated with worse outcome compared to cases with lack of CRLF2 alterations [15,34,38,39]. However, the poor prognostic impact of CRLF2 overexpression is overcome by BCR-ABL1–like signature and IKZF1 deletion in the Dutch Childhood Oncology Group trials and German Cooperative ALL trials [37]. In about half of CRLF2-rearranged pediatric Ph-like ALL cases, concomitant JAK1 and JAK2 (most commonly in the pseudokinase domain at R683) mutations occur. In adults, the frequency of JAK mutations in patients with CRLF2 rearrangement is lower, with a ratio of 1:4 with JAK wild type [4,12,15,23]. In JAK1 the most common mutation is represented by V658F which is the homolog of JAK2 V617F, hotspot in myeloproliferative neoplasms. Other alterations leading to JAK/STAT activation target IL7RA, SH2B3, IL2RB, and TYK2 genes. Collectively these alterations are approximately two-fold higher in children (14%) compared to adolescents (5.0%), and adults (7.3%) [4,12,15]. IL7RA mutations occur in exon 6 and are mainly in-frame insertion/deletions in the juxtamembrane-transmembrane domain or, rarely, a serine-to-cysteine substitution at amino acid 185 in the extracellular domain [38]. Independent of CRLF2 rearrangements, JAK-STAT signaling activation can result from JAK2 (~7%) or erythropoietin receptor (EPOR, 5%) -rearrangements.

Over 20 different JAK2 gene fusion partners have been reported (most commonly EBF1, ETV6, PAX5, and BCR), making JAK2 the most promiscuous gene in Ph-like ALL. All fusions preserve the JAK2 kinase domain and result in STAT5 activation and growth factor independence, making cells expressing these fusions amenable to JAK2 inhibitors.

Common EPOR rearrangements involve juxtaposition or less frequently translocation of the EPOR gene in proximity of a strong enhancer, such as that of the immunoglobulin heavy (IGH) or kappa (IGK) loci, that drives its expression. Less frequent rearrangements involve insertion of EPOR into the upstream region of LAIR1 or the THADA loci [4,39]. All these rearrangements clip off the C-terminal cytoplasmic tail, thus preserving the proximal tyrosine requested for activation and removing almost all tyrosine sites required for shutting off the receptor signaling and down-regulate and internalize the receptor. This leads to transformation in in vivo models and sensitivity to a variety of different JAK2 inhibitors in in vitro and in vivo models. While IGH-EPOR fusion due the translocation t(14;19)(q32;p13) can be detected by fluorescence in situ hybridization (FISH) [40], the other EPOR rearrangements are cryptic and challenging to detect without using next-generation sequencing (NGS) technologies. The prevalence of EPOR rearrangements has a peak in young adults (9%) compared to children and adolescents (5% and 3%, respectively). They are rarely detected in adults (1%) [13,39]. JAK2 and EPOR rearrangements are associated with the poorest outcome compared with the other molecular Ph-like subtypes [12,13].

The ABL-class gene fusions include rearrangements of ABL proto-oncogene 1 (ABL1; e.g., to RCSD1, NUP214, LSM14A, ETV6, RANBP2, CENPC, FOXP1, SFPQ, SNX1, SNX2, SPTNA1, ZMIZ1, NUP153), ABL proto-oncogene 2 (ABL2; e.g., to RCSD2, PAG1, ZC3HAV1), colony-stimulating factor 1 receptor (CSF1R; e.g., to SSBP2, MEF2D, TBL1XR1), platelet-derived growth factor receptor beta (PDGFRB; e.g., to EBF1, ETV6, ATF7IP, SNX29, SSBP2, TNIP1, ZEB2, ZMYND8, NUMA1) and LYN (GATAD2A-LYN, NCOR1-LYN [41]), with multiple partner genes, with ABL1 and PDGFRB being the most common. The prevalence of these rearrangements is 17% in children, 9% in adolescents, 10% young adults and 9% older adults [4,12,14,15]. Patients with ABL-class fusions respond poorly to chemotherapy regimens, and the EBF1-PDGFRB fusion in particular is associated with induction failure [42,43,44]. All fusions preserve the tyrosine kinase of the ABL-class gene and promote constitutive kinase signaling that confers the ability to survive and grow independently of cytokine in vitro [45]. Imatinib, the dual ABL1/SRC inhibitor dasatinib or other TKIs inhibit the downstream signaling induced by each of these chimeric fusion proteins [4,46,47] and are currently used in clinical trials. The best and first example is provided by the inhibition of EBF1-PDGFRB fusion by imatinib [44,46,47,48,49]. The emergence of kinase domain point mutations may represent a potential mechanism of relapse in EBF1-PDGFRB or other kinase driven-subtypes in Ph-like ALL. Recently, the T681I gatekeeper mutation has been demonstrated to be the most common resistant mutation in EBF1-PDGFRB Ph-like ALL to both imatinib and dasatinib in in vitro screens and it was associated with a trend towards increased risk of relapse in patients harboring T681I subclones at diagnosis compared to T681I-negative patients [50].

Around 5% of Ph-like ALL cases harbor gene fusions or mutations involving NTRK3, BLNK, DGKH, PTK2B, FLT3, FGFR1, TYK2 and SH2B3. Among those, one percent of cases harbor the fusion between ETV6 and NTRK3 encoding a member of the tropomyosin receptor tyrosine kinase (TRK) family [51]. This fusion is not unique of Ph-like ALL since it has been identified in a range of hematological malignancies, such as acute myeloid leukemia [52], infantile sarcoma [53,54] and solid tumors [55,56,57,58]. In preclinical models, ETV6-NTRK3 has been shown to promote the development of an aggressive B-ALL and to be exquisitely sensitive to the TRK inhibitors larotrectinib (LOXO-101) or PLX7486 (Plexxikon) in both patient derived xenograft models and in B-ALL patients with ETV6-NTRK3 [55,59,60]. Recently, a clinical response to larotrectinib has been reported in an adult Ph-like ALL with cryptic ETV6-NTRK3 rearrangement and NRASGly12Asp mutation. The patient failed to respond to multiagent chemotherapy and relapse after investigational CD19-directed chimeric antigen receptor T-cell therapy with a clone positive for ETV6-NTRK3 but not anymore for the NRASGly12Asp mutation. The relapsed leukemia progressed with further chemo- and immunotherapy but showed substantial leukemic cytoreduction using the TRK inhibitor larotrectinib [61]. Fusions of the B Cell Linker Protein (BLNK) or SLP65 gene to DNTT (also known as TDT) have been also described [13,62]. BLNK encodes a cytoplasmic adapter protein important for B-cell development and function by activating BCR downstream signaling [63], while DNTT encodes a encodes a template-independent DNA polymerase that catalyzes the addition of deoxynucleotides and that is highly expressed in normal and malignant pre-B and pre-T lymphocytes during early differentiation [64].

In addition to gene fusions, RAS pathway activating mutations or deletions (KRAS, NRAS, NF1, PTPN11) and copy number aberrations in genes involved in B-cell development (IKZF1, PAX5, EBF1, and ETV6) and cell cycle regulators (CDKN2A/B, TP53, BTG1, and RB1) are recurrent. Deletions in IKZF1 occur in around 27% of pediatric cases and in approximately 70% of high-risk pediatric patients with ALL [4]. As in BCR-ABL positive ALL [6,59], IKZF1 deletions confer a poor prognostic outcome [20]. IKZF1 deletions are significantly more common in patients carrying kinase or cytokine receptor rearrangement (IGH-CRLF2) than a sequence mutation [4,13], especially in Hispanic/Latino (H/L) children with B-ALL (29% in H/L compared to 15% of non-Hispanic Whites) where both IGH-CRLF2 translocation and IKZF1 deletion provide a strong biological rationale for the higher death-rate H/L experience due to B-ALL [60].