Retrospective studies have demonstrated that GEP can be used effectively to classify ALL into prognostically important subtypes. BCR-ABL1-like ALL is a newly described ALL subtype lacking BCR-ABL1, but with a similar expression profile to BCR-ABL1-positive leukemia. This new “BCR-ABL1-like” (“Ph-chromosome-like”) ALL includes approximately 15% of all precursor B-cell ALL in children and 30% to 40% of adults with ALL and is associated with higher relapse rate and lower EFS (see Fig. 1 and Table 1). The prevalence of Ph-like ALL increases with age ranging from 10% to 15% of children to 20% to 25% of young adults (21 to 39 years) and adults (>40 years) with ALL. The WHO (2017) endorsed BCR-ABL1-like B-ALL as a provisional entity with the gene expression pattern “very similar” to that seen in Ph-positive/BCR-ABL1 fusion-positive ALL. Both entities commonly harbor genetic alterations targeting B –lymphoid transcription factors, including IKZF1. Large-scale genomic profiling and sequencing studies of over 1700 childhood and young adult patients with ALL has revealed rearrangements of ABL-class genes, rearrangements of JAK2, EPOR, and CRLF2. ABL-class rearrangements result in the expression of fusion genes that activate ABL1, ABL2, CSFR1, and PDGFRB. In this group of patients IKZF-1 deletions occur in 40% of pediatric and in 46% of adult patients. CRLF2 (Xp22.33/p11.3) rearrangements are observed in approximately 50% of patients with BCR-ABL1-like ALL with frequent concomitant mutations of JAK2, and as mentioned above, are potentially amenable to treatment with JAK2 inhibitors. Two forms of CRLF2 rearrangements have been identified which occur either as translocations, CRLF2-IGH, or through focal deletion of the PAR1, a pseudo-autosomal region of chromosome X/Y resulting in P2RY8-CRLF2 fusion. The gene encodes for cytokine receptor-like factor 2, also known as thymic stromal–derived lymphopoietin receptor which in combination with the interleukin-7 receptor forms the receptor for the thymic stromal lymphopoietin. CRLF2-rearranged leukemic cells have activated JAK-STAT and PI3K signaling pathways. Detection of rearrangements involving ABL1, ABL2, CRLF2, EPOR, PDGFRB, and CSF1R using FISH analysis is very useful to identify BCR-ABL1-like ALL.
Fig1. ESTIMATED FREQUENCY OF SPECIFIC GENETIC SUBTYPES IN CHILDHOOD ACUTE LYMPHOBLASTIC LEUKEMIA. The pie chart depicts the estimated frequencies of each subtype of acute lymphoblastic leukemia (ALL) among patients treated in St. Jude Total Therapy Study, with modifications to account for discoveries of novel genetic abnormalities. T cell ALL (T-ALL) subtypes are depicted in blue. Among the B cell precursor ALL (B-ALL) subgroup, low-risk subtypes are in yellow, intermediate-risk subtypes are in green, and high-risk subtypes are in red; however, the prognosis of genetic subtypes depends on treatment efficacy and, hence, might differ between various clinical trials. Moreover, the prognosis of the 3% of patients with B-ALL who cannot be classified into one of these subgroups (orange segment) cannot be predicted at present. DUX4/ERG, DUX4 rearranged and ERG deregulated; iAMP21, intrachromosomal amplification of chromosome 21. (Reproduced with permission from Pui C-H, et al. Somatic and germline genomics in paediatric acute lymphoblastic leukaemia. Nat Rev Clin Oncol. 2019;16(4):227–240.)
Table1. Clinical and Biological Characteristics of Newly Identified Molecular Subtypes in Childhood Acute Lymphoblastic Leukemia
t(1;19)(q23;p13.3)/E2A-PBX1 occurs in 5% to 6% of patients with B-cell precursor childhood and adult ALL. However, among patients with a pre-B (cytoplasmic Ig-positive) t(1;19) is found in approximately 25% of cases (see Fig. 2D). Cytogenetically, two forms of t(1;19) have been identified: 25% of cases have a balanced reciprocal t(1;19), whereas 75% have a rearrangement of unbalanced der(19)t(1;19)(q23;p13.3). The unbalanced der(19)t(1;19) arises from the initial trisomy of chromosome 1 followed by the t(1;19) translocation, with subsequent loss of the derivative chromosome 1. More than 95% of t(1;19) are associated with the E2A–PBX1 chimeric gene protein product which arrests cell differentiation. The E2A gene (originally identified by the binding of E2A proteins to the kE2DNA sequence motif contained in the Ig κ light-chain enhancer) on chromosome 19, band p13.3, is fused to the PBX1 (homeobox) gene on chromosome 1, band q23. Approximately 1% of pediatric patients with B-cell-ALL have a variant t(17;19)(q21–q22;p13)/E2A HLF translocation resulting with two different genomic rearrangements. The first is the fusion between the HLF gene (breakpoint in intron 3) on chromosome 17 and the E2A gene (within intron 13) on chromosome 19, associated with disseminated intravascular coagulopathy. The second is the breakpoint in intron 12 of E2A and intron 3 of HLF, which is associated with hypercalcemia.
Fig2. PROGNOSTIC CYTOGENETIC CATEGORIES IN ACUTE LYMPHOBLASTIC LEUKEMIA. (A) Localization of TEL/ETV6 and AML1 fluorescence probes to chromosomes from a normal bone marrow metaphase cell. TEL/ETV6 is on 12p13 (green) and AML1 is on 21q (red). (B) Partial karyotype showing t(12;21)(p13;q22) (arrows). The short arrow at 12p indicates a possible TEL deletion from normal chromosome 12. (C) FISH study showing loss of TEL (green) from normal 12 homologue in interphase nucleus, a frequent subclonal evolution in patients with t(12;21). (D) Hyperdiploidy, specifically trisomies for chromosomes 4 (green), 10 (red), and 17 (aqua), is associated with low-risk cytogenetic category (see text for details) and is present in disomy in interphase cells (top left). (E) Partial G-banded karyotype showing t(1;19)(q23;p13.3), which occurs in approximately 6% of patients with B-cell precursor childhood acute lymphoblastic leukemia. (F) FISH hybridization to bone marrow nucleus showing BCR-ABL fusion (yellow). (G) As a consequence of t(9;22), occurring in 5% of children and 20% to 25% of adults with acute lymphoblastic leukemia. (H) Interphase nucleus after FISH study with tricolor probe. Dual-color/breakapart MLL shows separation of the 3′ end and the 5′ end as a result of 11q23 rearrangement. CEP11 (aqua) indicates disomy of chromosome 11, used as internal control. MLL rearrangements in acute lymphoblastic leukemia are associated with unfavorable prognosis. FISH, fluorescence in situ hybridization.
Currently, it is thought that an unbalanced der(19) in pediatric ALL is associated with significantly improved outcome as compared with patients with balanced t(1;19). In adults as the sole abnormality, t(1;19)/der(19)t(1;1.9) is associated with an intermediate prognosis; however, within the context of a hyperdiploid karyotype, it is associated with a poor prognosis. Patients with E2A-PBX1 fusion also display PAX5 (19p13.2) haploinsufficiency, detectable both by conventional and molecular cytogenetics. A variant t(17;19) rear rangement is associated with a poor prognosis. A recent study of adult patients with ALL has suggested that prognosis of patients with t(1;19) can be substantially improved by the treatment with the hyper-CVAD regimen. Both t(1;19) and t(17;19) are easily identifiable by conventional cytogenetics, FISH, and RT-PCR; the latter two methodologies are particularly useful in post-treatment specimens that are cytogenetically normal. Similar to the ETV6-RUNX1 fusion, in utero origin of E2A-PBX1 (see later) was recently documented as well as its presence in approximately 0.6% of healthy newly born and therefore they either convert to overt leukemia or, in most cases, die out later in life. Recent molecular studies underlying leukemogenesis of t(1;19) proposed the following model by which E2A-PBX1 directly interacts, through a region spanning the PBX1 homeodomain, with RUNX1 gene that specifically targets a subset of RUNX1-occupied genes and 2 enhancers in the RUNX1 locus. Gene-expression analysis confirmed that E2A-PBX1 directly controls RUNX1 activation in pre-B ALL. Therefore RUNX1 acts as a master regulator in the establishment of hematopoietic stem cells. It is the physical interaction of E2A-PBX1 with RUNX1 but not the DNA binding activity that plays the crucial event in the activation of RUNX1-related gene programs both by direct activation of RUNX1 targets and by reinforcing RUNX1 transcriptional autoregulatory loop and thereby contributing to ALL pathogenesis.
In ALL, the most frequent KMT2A translocations include t(4;11) (1% to 2% incidence in children and two-thirds of KMT2A-positive adults) (Fig. 3) leading to an MLL-AFF1 (AF4) fusion and t(11;19)(q23;p13.3) resulting in KMT2A/MLL MLLT1 (ENL) fusion. These abnormalities are present in more than 80% of patients with infant leukemia and 10% of childhood and adult KMT2A-positive leukemia. KMT2Ar is associated with a poor out come in both children and adults. One-third of patients with t(4;11) have secondary abnormalities; the most frequent are +X, i(7q), abnormalities of 9p, including i(9q), and +8. The outcomes of patients with t(11;19) is generally poor, especially in children younger than 1 year of age. The most frequent additional abnormalities in patients with t(11;19) are +X, +8, and del(6q). Other less common KMT2A/MLL translocations include t(9;11)(p22;q23)/KMT2A/MLL-MLLT3, t(10;11)(p13–15;q23)/KMT2A-MLLT10, and others. A large multi institutional study has determined that secondary aberrations do not affect the prognosis of children with ALL showing t(4;11), t(11;19) or other KMT2A translocations.
Fig3. A HYPERDIPLOID B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA WITH TWO COPIES OF t(4;11). A diagnostic karyotype, in 100% of examined bone marrow metaphase cells, had 49,XX, +der(4)t(4;11) (q21;q23),t(4;11), del(5)(q15q34),+6,+13 karyogram. These findings are consistent with a hyperdiploid karyotype including trisomies of chromosomes 6 and 13. The patient also had a balanced translocation between the long arms of chromosomes 4 and 11 followed by a replication of the abnormal derivative chromosome 4. (B) A second abnormal chromosome der(4) is shown within the metaphase cell after FISH probing using a KMT2A breakapart FISH probe. Red arrows point to a gain of 3′ KMT2A localized on two der(4) chromosomes while the green arrow indicates that the 5′ of KMT2a remained on the abnormal 11. In adult ALL, a hyperdiploid karyotype involving t(4;11) is considered a high risk category. Of note, del(5q) is typically observed in myeloid disorders but it has been reported rarely in lymphoid diseases as well. ALL, acute lymphoblastic leukemia; FISH, fluorescence in situ hybridization.
Secondary or therapy-related forms of ALL are rarely reported but the majority of them are associated with MLL rearrangements, the most frequent being t(4;11). Most of these patients have received topoisomerase II inhibitors which are known to cause double-strand DNA breaks.
t(8;14)(q24;q32) is seen in fewer than 5% of all patients with ALL (children and adults) (Fig. 4). Variant translocations t(8;22) (q24;q11) and t(2;8)(p12;q24) are seen in less than 1% of children and adults. These cells express CD10, CD19, CD20, and surface IgM immunophenotype. This form of ALL has an extremely poor prognosis. The same translocation is found in Burkitt lymphoma (BL), and both entities likely represent the same disease with different manifestations. The majority of adult patients with t(8;14) die within 1 year of diagnosis. Approximately 4% of children and 11% of adult pre–B-cell precursor ALL have recurrent IGH translocations usually identified by cytogenetics or FISH. The CRLF2 gene, which maps to the pseudo-autosomal region 1 of the sex chromosomes, is the gene that is most frequently targeted in 20% to 26% of IGH translocations in pre–B-cell precursor ALL. IGH-CRLF2 rearrangements result from the cryptic t(Y;14)(p11;q32) or t(X;14)(p22;q32). The second most frequent IGH translocation is t(14;19)(q32;q13) and IGH-CEBP chimeric fusion. The third most frequent translocation partner involves CEBPD gene (8q11) resulting in t(8;14) (q11;q32) and IGH-CEBPD fusion, which is primarily found in children and young adults and is strongly associated with DS (about 30% of cases). This subgroup frequently has a gain of X chromosome, trisomy 21 as an acquired abnormality, and the Ph chromosome/t(9;22) (q34;q11). The t(14;19)(q32;q13)/IGH-EPOR appears to be causally related to pre–B-cell precursor ALL because it always appears as the single abnormality. The inv(14) (q11q32)/ins(14;14)(q11;q32) leads to IGH-TRA/D, which represent a rearrangement mediated by an interlocus site-specific recombination event between IGH and the TRA and TRB genes. The t(14;20)(q32;q13) results in IGH-CEBPB. Collectively, patients with IGH translocations may be included in a subgroup of pre–B-cell precursor ALL.
Fig4. t(8;14)(q24;q32) IN ACUTE LYMPHOBLASTIC LEUKEMIA. Top panel shows a partial G-banded karyotype of chromosomes 8 and 14 with arrows indicating the breakpoints on each chromosome. The bottom panel is a partial metaphase (chromosomes are stained blue with DAPI) after FISH testing using three probes: IGH signal (green) is seen on normal chromosome 14, aqua signals for centromere 8 are observed on both chromosomes 8, MYC (red) is seen on both chromosomes 8 but note a more bold signal on one chromosome 8 when compared to the other. Because MYC is broken as a result of t(8;14), the third copy of MYC is on chromosome 14 where it is fused to IGH and seen as a yellow signal.
Abnormalities of the short arms of chromosome 9 (p21–22) occur at a frequency of 7% to 13%. In adults the presence of del(9p) appears to be associated with a favorable outcome, whereas in children with ALL, del(9p) is associated with poor outcome. The most frequent abnormalities are co-deletions of two genes, CDKN2A and CDKN2B, as well as the interferon α and β genes found in many cases. Among the structural rearrangements involving the short arms of chromo some 9, t/dic(9;12)(p11–12;p11–13) is a rare recurrent abnormality associated with L1 morphology (FAB classification), a pre–B-cell phenotype, and an excellent prognosis.
The IKZF1 gene at 7p12.2 codes for IKAROS, an essential transcription factor in hematopoiesis primarily involved in lymphoid differentiation and is an essential player in the regulation of both T- and B-cell lineage specification. The gene is composed of eight exons, spanning a total of 6.2 kb and coding for a 519-amino acid protein. Deletions of IKZF1 have been reported in 15% of pediatric and 30% to 50% of adult patients with ALL, and in 75% of Ph-positive B-cell ALL. Both focal and nonfocal IKZF1 deletions have been shown to be associated with an increased risk of relapse and decreased EFS in both pediatric and adult ALL. Most laboratories use SNP array technique to detect IKZF1 deletion. Recent genomic studies of patients with ALL have confirmed a higher hazard of relapse in adult ALL with focal IKZF1 deletions.
Other chromosomal abnormalities detected in nonrandom fashion in adult patients with ALL include deletions, both terminal and interstitial, of the long arm of chromosome 6, and isochromosomes of 7q, 9q, 17q, and 21q. Adult patients with ALL harboring t(9;22), t(4;11), t(8;14), -7, +8 chromosomal aberrations have a poorer prognosis and significantly lower probability of long-term CR and survival than do patients with a normal karyotype or patients with other chromosomal rearrangements.
