Keywords

Introduction

Recurrent cytogenetic abnormalities have been used to subtype AML for many years, providing important prognostic information, as well as identifying potential molecular targets to guide therapy. The advent of new technology has generated abundant data, revealing a large number of genetic abnormalities existing in different AML cases. These findings provide ever-increasing evidence for subtyping in AML and include, but are not limited to, t(15;17)( q24.1;q21.2);PML-RARA, t(8;21)(q22;q22);RUNX1-RUNXT1, inv(16)(p13q22);CBFB-MYH11, t(9;11)(p22;q23);KMT2A-MLLT3, t(6;9)(p23;q34);DEK-NUP214, inv(3)(q21;q26.2);RPN1-MECOM, and t(1;22)(p13;q13);RBM15-MKL1 [1,2,3] (please see Table 1.1 for a summary) . Another example is the BCR-ABL1 translocation , the disease-defining genetic alteration in chronic myeloid leukemia (CML) , which has also been described in acute lymphocytic leukemia (ALL) and was later reported in AML. Now, the evidence indicates that de novo AML with a BCR-ABL1 translocation should be considered as a provisional category of AML [2]. Many other translocations and inversions have been found in AML, which are sometimes recurring. These recurring cytogenetic abnormalities are less frequent, more often seen in pediatric patients with uncertain prognostic or therapeutic significance. AML with such cytogenetic abnormalities are not included in this category at this time [2]. Some translocations and inversions are seen in therapy-related myeloid neoplasms and are also excluded from this category [1]. Typically, a recurrent cytogenetic abnormality will create a fusion gene encoding a chimeric protein, with one exception that has been recognized: AML with inv(3)(q21.3;q26.2) [2, 4, 5]. This chapter will describe the recurrent genetic abnormalities in AML.

Table 1.1 Recurrent cytogenetic abnormalities in AML
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Acute Myeloid Leukemia with t(15;17)(q24.1;q21.2) ; PML-RARA

AML with PML-RARA is also called acute promyelocytic leukemia (APL) and comprises 5–8% of all AML cases. The key cytogenetic abnormality is formation of the PML-RARA fusion gene, most commonly caused by a t(15;17)(q24.1;q21.2), although cryptic and variant translocations also account for a minority of cases [2].

APL presents with myeloblasts and abnormal promyelocytes (which are considered blast equivalents) in the bone marrow and peripheral blood. On occasion, the blast count may be less than 20%, but the presence of a PML-RARA is sufficient for the diagnosis of APL. There are two morphologic variants of APL: the hypergranular (classic) and the microgranular variant. The abnormal promyelocytes in the hypergranular variant of APL typically show indented or bilobated “butterfly-shaped” nuclei and intense azurophilic granulation. Single or bundles of Auer rods may also be appreciated (Fig. 1.1). This contrasts with the microgranular variant of APL, in which the abnormal promyelocytes have very small and indistinct azurophilic granules and show predominantly bilobed nuclei. The blast/blast equivalent count in the microgranular variant of APL also tends to be higher than in the hypergranular variant of APL. The azurophilic granules in both types are positive for myeloperoxidase (MPO) [1].

Fig. 1.1
figure 1

Acute promyelocytic leukemia, hypergranular variant showing prominent azurophilic granules bilobed nuclei and bundles of Auer rods

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The flow immunophenotype of APL is also characteristic and often aids in the diagnosis, especially in cases in which the morphologic features are less developed. The hypergranular variant of APL is classically CD34 and HLA-DR negative, shows high side scatter (where granulocytes typically are seen) and bright MPO, and will often show dim CD64 expression, without CD16 expression. The microgranular variant of APL, on the other hand, frequently expresses CD34 and occasionally HLA-DR (usually subset of neoplastic cells and week), shows a lower side scatter than the hypergranular variant of APL (residing in the normal blast area on the CD45/SSC plots), and frequently shows dim CD2 expression.

Cytogenetic Abnormality

At the genome level, APL with PML-RARA is characterized by a t(15;17)(q24.1;q21.2) translocation (Fig. 1.2) [1]. The breakpoint in the RARA gene is within intron 2. The breakpoints in the PML gene can occur at three different locations resulting in different sized PML-RARA transcripts . Breakpoint 1 (BCR1) at intron 6 will result in a long transcript; breakpoint 2 (BCR2) at exon 6 will result in variable transcripts, which can be of different sizes in different patients; and breakpoint 3 (BCR3) at intron 3 will result in short transcript [6, 7]. As a result, a nuclear regulatory gene (promyelocytic leukemia or PML gene) on 15q24.1 is translocated to the vicinity of the retinoic acid receptor alpha gene on 17q21.2, forming a PML-RARA fusion gene.

Fig. 1.2
figure 2

APL with PML-RARA is characterized by a t(15;17)(q24.1;q21.2) translocation

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The translocation can be detected by karyotyping and FISH assay . Different FISH assay designs have been used. One assay is called dual-color dual-fusion fluorescence FISH assay [8]. In this assay, two probes are designed to hybridize to PML at 15q24 and RARA at 17q21, respectively, overlapping the breakpoints on each chromosome (Fig. 1.3). The two probes can be labeled with different fluorescent dyes. For example, the probe specific for PML at 15q24 can be labeled with red fluorescent dye and the probe for RARA at 17q21 green. A normal cell will show two red and two green dots (Fig. 1.3). A cell with t(15;17)(q24.1;q21.2) will show one red, one green, and two yellow dots (Fig. 1.3). This assay works very well in detecting t(15;17)(q24.1;q21.2) PML-RARA translocation. This translocation is the most common change found in APL with PML-RARA . However, the RARA gene has been found to have other translocation partner genes in some cases. Such translocations are called variant RARA translocations . These variants include t(5;17)(q35;q21) NPM1-RARA [9], t(11;17)(q23;q21) PLZF (also known as ZBTB16)-RARA [10, 11], t(11;17)(q13;q21) NUMA-RARA [12], t(4;17)(q12;q21) FIP1L1-RARA [13, 14], t(2;17)(q32;q21) OBFC2A-RARA [15], t(7;17)(q11;q21) GTF2I-RARA [16], t(1;17)(q42;q21) IRF2BP2-RARA [17, 18], der(17) with duplication of 17q21.3-q23 STAT5b-RARA [19], and der(17) PRKAR1A-RARA with del(17)(q21) [20]. Generally speaking, the dual-color dual-fusion FISH assay does not work well for these variants. Therefore, a RARA dual-color break-apart FISH assay has been designed to address this issue. This assay uses dual-color probe, red and green. The red part of the probe hybridizes to RARA centromeric to the breaking point and the green part of the probe telemetric to the breaking point (Fig. 1.4). A normal interphase cell will show two yellow dots (Fig. 1.4). An interphase cell with RARA translocation, regardless of the partner genes, will show one yellow, one red, and one green dot (Fig. 1.4). This assay works better than dual-color dual-fusion assay for different RARA translocation variants. In rare cases, cryptic PML-RARA translocations cannot be detected by karyotyping or FISH assay. For such cases, RT-PCR assay can be used to detect translocations [21, 22]. RT-PCR assay can also be used to detect minimal residual disease (MRD) with PML-RARA [23]. Different RT-PCR designs have been used to improve the performance [7, 24]. In general, RNA is extracted from patients’ blood or bone marrow samples. Random hexamer primers are used to make cDNA. The target in cDNA is amplified using different primer pairs flanking fusion site of PML-RARA [23].

Fig. 1.3
figure 3

Two probes are designed to hybridize to PML at 15q24 and RARA at 17q21, respectively, overlapping the breakpoints on each chromosome. The two probes can be labeled with different fluorescent dyes. For example, the probe specific for PML at 15q24 can be labeled with red fluorescent dye and the probe for RARA at 17q21 green. A normal cell will show two red and two green dots. A cell with t(15;17)(q24.1;q21.2) will show one red, one green, and two yellow dots

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Fig. 1.4
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A normal interphase cell will show two yellow dots. A cell with translocation will show one green, one red and one yellow dots

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Somatic Gene Mutations

Somatic gene mutations in FLT3 are relatively common (50% of cases) with the FLT3-ITD being most common (30–40%) and the FLT3-D835 mutation occurring in approximately 10% of cases. However, the prognostic significance of FLT3 mutation remains unclear [25, 26].

Other gene mutations including alterations in KIT and RAS genes have been described relatively rare (generally reported frequency of 5% or less) [27].

Clinical Significance

Clinically, patients with APL often develop disseminated intravascular coagulation (DIC) , which may result in rapidly unfavorable consequences (e.g., intracranial hemorrhage). However, APL is generally sensitive to the treatment using all-trans-retinoic acid (ATRA) . Therefore, early diagnosis of APL is critical for improving patient outcome. Morphologic and immunophenotypic evaluation, in combination with FISH and PCR assays, plays an important role in patient care. Resistance to ATRA can occur due to genetic mutations in the retinoic acid receptor alpha (RARA) ligand-binding domain or the PML-B2 domain of PML-RARA. Additionally, cases of APL with variant RARA translocations may exhibit ATRA resistance [28].

Acute Myeloid Leukemia with t(8;21)(q22;q22);RUNX1-RUNX1T1

AML with t(8;21)(q22;q22);RUNX1-RUNX1T1 is one of the AMLs with recurrent genetic abnormalities, first described in 1973 [29, 30], and comprises approximately 5% of all AML cases. The key cytogenetic abnormality is a RUNX1-RUNX1T1 fusion gene [1]. RUNX1 (runt-related transcription factor 1) is also known as acute myeloid leukemia 1 gene (AML1) , polyomavirus enhancer binding protein 2 subunit a (PEBP2a) , and core-binding factor subunit A2 (CBFA2) [29]. RUNX1T1 (runt-related transcription factor 1 translocated to 1) is also known as ETO (eight twenty one).

Morphologically , AML with t(8;21)(q22;q22) typically shows large myeloblasts with abundant basophilic cytoplasm, perinuclear hofs, and azurophilic granules, some of which coalesce into larger salmon-colored granules (pseudo-Chédiak-Higashi granules ) (Fig. 1.5). In rare cases , the blasts may be less than 20%, which should not invalidate the diagnosis of AML with t(8;21)(q22;q22). Apart from myeloid blasts, the granulocytic lineage may show features of dysplasia, including hypogranulation or irregular lobation. Dysplasia in other lineages is not typically seen. Eosinophilic precursors are often increased, and occasionally basophils and mast cells are present in excess. [1, 30, 31].

Fig. 1.5
figure 5

Acute myeloid leukemia with t(8;21) showing basophilic cytoplasm and paranuclear hofs

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By immunophenotypic evaluation , AML with t(8;21)(q22;q22)is positive for CD34, HLA-DR, MPO, and CD13, with dim CD33. Of note, these leukemias can often express lymphoid markers, including CD19, CD79a, PAX5, and on occasion dim Tdt. A diagnosis of biphenotypic leukemia should not be made in this setting.

Cytogenetic Abnormality

At the genome level, AML with t(8;21)(q22;q22) is characterized by a chromosome 8 and 21 translocation (Fig. 1.6) resulting in a RUNX1-RUNX1T1 fusion gene [1]. The RUNX1 gene is on chromosome 21 and RUNX1T1 gene on chromosome 8. The breakpoint in the RUNX1T1 gene is within an intron at the 5′ end of RUNX1T1. The breakpoint in the RUNX1 gene occurs between the exon 5 and 6. Therefore, the fusion gene contains the N-terminal 177 amino acids of RUNX1, which is fused in frame to nearly full-length RUNX1T1 protein [6, 32]. The fusion gene resides on derivative chromosome 8 after the translocation [6] (Fig. 1.6).

Fig. 1.6
figure 6

At the genome level, AML with t(8;21)(q22;q22) is characterized by a chromosome 8 and 21 translocation

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RUNX1-RUNX1T1 translocation can be detected using karyotyping and FISH. One of the FISH assay designs is a dual-color dual-fusion FISH assay. In this assay, two probes are designed to hybridize to RUNX1T1 at 8q22 and RUNX1 at 21q22, respectively, overlapping the breakpoints on each chromosome. The two probes can be labeled with different fluorescent dyes. For example, the probe specific for RUNX1T1 can be labeled with red fluorescent dye and the probe for RUNX1 green. A normal cell will show two red and two green dots (Fig. 1.7). A cell with t(8;21)(q22;q22) will show one red, one green, and two yellow dots (Fig. 1.7). This assay is typically more sensitive than karyotyping.

Fig. 1.7
figure 7

The probe specific for RUNX1T1 can be labeled with red fluorescent dye and the probe for RUNX1 green. A normal cell will show two red and two green dots. A cell with t(8;21)(q22;q22) will show one red, one green, and two yellow dots

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In rare cases, cryptic RUNX1-RUNX1T1 translocations may not be detected by karyotyping or FISH assays. RT-PCR assays in these instances can be used to detect such cryptic translocations [30]. RT-PCR assays may also be used to detect minimal residual disease (MRD) of RUNX1-RUNX1T1 with a sensitivity of 10−3 to 10−4. A nested PCR design can reach a sensitivity of 10−4 to 10−5 [30].

Somatic Mutations

Several genes additionally deserve discussion here with regard to AML with t(8;21)(q22;q22): KIT, FLT3, and the RAS genes, NRAS and KRAS. KIT mutations are the most frequent and vary in incidence in these AMLs (5–30%); most mutations are within the tyrosine kinase domain [33,34,35,36,37,38,39,40,41,42,43,44].

These mutations in KIT are activating mutations which increase cell proliferation and survival.

Three other genes infrequently mutated in AML with t(8;21)(q22;q22) are FLT3 and the RAS genes KRAS and NRAS. As in many other AML subtypes, FLT3 internal tandem duplication (ITD) mutations may be seen, though at a low frequency (~5%) in AML with t(8;21)(q22;q22) [36].

In vivo mouse models have shown a cooperative effect in FLT3-ITD mutations in leukemogenesis [45].

FLT3 D835 mutations are additionally rare though the significance of these mutations in AML with t(8;21)(q22;q22) is even less certain. Similarly, NRAS and KRAS mutations are relatively rare and like FLT3-ITD mutations may have a role in leukemogenesis without impacting chemosensitivity [36].

Clinical Significance

AML with the RUNX1-RUNX1T1 translocation is considered to have a favorable prognosis when compared with other AML subtypes [46, 47]. Somatic gene mutations in KIT and FLT3-ITD mutations may modify the prognostic outcomes of patients. Several studies have shown that patients with AML with the RUNX1-RUNX1T1 translocation and KIT mutations may have an adverse outcome though some studies have shown no prognostic impact [33,34,35,36,37, 39, 41, 42].

FLT3-ITD mutations are rare but are associated with poor outcomes in patients with t(8;21)(q22;q22) [38].

Acute Myeloid Leukemia with inv(16)(p13.1q22);CBFB-MYH11 or t(16;16)(p13.1;q22);CBFB-MYH11

AML with inv(16)(p13.1q22);CBFB-MYH11 comprises 5–8% of AML cases and is predominantly seen in younger patients [1]. Morphologically , these leukemias demonstrate myelomonocytic differentiation, but more uniquely, the marrow shows increased eosinophils, which can show dysplastic features, including prominent granulation with aberrant purple-blue coloration and, on occasion, nuclear hyposegmentation (Fig. 1.8). While most cases show eosinophilia , occasionally, eosinophils are not increased, and myelomonocytic differentiation may be the only feature present. Like APL with PML-RARA and AML with t(8;21)(q22;q22), the translocation is diagnostic, and a blast count of greater than or equal to 20% is not required.

Fig. 1.8
figure 8

Acute myeloid leukemia with inv(16) showing abnormal eosinophils with violet-blue granules

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The immunophenotype of these leukemias may be heterogeneous, reflecting its myelomonocytic differentiation. It can include an immature blast population expression CD34 and CD117, as maturing granulocytes expressing CD13, CD33, CD65, and MPO and a monocyte population expressing CD4, CD14, and CD64.

Cytogenetic Abnormality

At the genome level, AML with inv(16)(p13.1q22) is characterized by an inversion of a segment of chromosome 16 resulting in CBFB-MYH11 fusion gene. CBFB is the core binding factor beta subunit located at 16q22. The MYH11 gene encodes a smooth muscle myosin heavy chain, which is normally located at 16p13.1 [1, 48]. The breakpoints at CBFB are near the 3-prime end of the coding region of CBFB and are found to be the same in different cases, though the breakpoints at MYH11 are more variable. The N-terminus and most of CBFB gene are fused to the C-terminus of MYH11 gene with its multimerization domain. All rearrangements maintain the reading frame of the fusion transcript [49, 50]. This chromosome 16 inversion disrupts CBFB resulting in impaired differentiation [51]. A similar fusion gene can also result from t(16;16)(p13.1;q22) and bears a similar consequence. The common chromosome 16 inversion is depicted in Fig. 1.9. Such an inversion comprises about 95% of cases generating a CBFB-MYH11 fusion gene. Karyotyping is a common method used and is capable of detecting most cases. A FISH assay has been used in detecting AML with inv(16)(p13.1q22) with higher sensitivity when compared to karyotyping. Generally speaking, RT-PCR assay has the highest sensitivity since it can detect subtle CBFB-MYH11 fusion transcripts [52,53,54,55].

Fig. 1.9
figure 9

The common chromosome 16 inversion

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Somatic Gene Mutations

Similar to AML with t(8;21)(q22;q22), AML with inv(16)(p13.1q22) also has frequent mutations in the KIT gene (10–40%); additionally mutations in FLT3 are seen with lesser frequency (10%) [34, 36, 42, 43, 56, 57].

RAS mutations are frequently seen in AML with inv(16)(p13.1q22) (35–50%); mutations in the RAS family of genes may function cooperatively with RUNX1-RUNX1T1 to promote leukemogenesis [36, 56, 58].

Clinical Significance

Like AML with RUNX1-RUNX1T1, AML with CBFB-MYH11 has a favorable prognosis when compared with other AML subtypes [59]. The significance of KIT and RAS mutations in these AMLs is uncertain; however, FLT3-ITD mutations have been shown in some studies to be associated with poorer prognosis within this category [34, 36, 42, 43, 56, 57].

Acute Myeloid Leukemia with t(9;11)(p22;q23);KMT2A-MLLT3

AML with t(9;11)(p22;q23) results in the fusion of KMT2A, also known as MLL, to MLLT3 and is more often seen in the pediatric setting, comprising 9–12% of pediatric AMLs and 2% of adult AMLs [1]. Morphologically , AML with t(9;11)(p22;q23) is characterized primarily by monocytic differentiation. AML with KMT2A-MLLT3 is often associated with acute monocytic and myelomonocytic leukemias [1]. Monoblasts, as well as promonocytes (considered blast equivalents), predominate. Monoblasts demonstrate round/oval nuclei, delicate chromatin with prominent nucleoli, basophilic cytoplasm, and rare azurophilic granules. Promonocytes , on the hand, have more convoluted or indented nuclei and less basophilic cytoplasm but can also demonstrate delicate chromatin and visible nucleoli.

In terms of the immunophenotype , the most commonly expressed markers in AML with t(9;11)(p22;q23) include CD13, CD33, CD15, CD38, CD64, CD4, and HLA-DR, whereas CD14, CD117, and MPO are less frequent. Interestingly, pediatric patients will often lack CD14 and CD34 expression, while adult patients will more frequently show CD14 expression and variable CD34.

Cytogenetic Abnormality

At the genome level, AML with t(9;11)(p22;q23) involves MLLT3 (mixed-lineage leukemia translocated to 3) and KMT2A genes. MLLT3 is located at 9p22 and KMT2A gene at 11q23. The breakpoints of KMT2A gene are clustered in a region that spans the 3′-portion of exon 8, exons 9–13 and the 5′-portion of exon 14 [60, 61]. The breakpoints of MLLT3 are clustered in introns 4, 7, and 8 [60, 62]. The translocation results in a MLLT-KMT2A fusion gene, which consists of the 5′ portion of KMT2A gene and the 3′ portion of MLLT3 gene [60]. Although MLLT3 is a major partner gene in the translocation associated with KMT2A, it is well known that KMT2A has many other translocation partner genes. About 80 of all partner genes have been identified [63,64,65]. In general, karyotyping can detect the majority of translocations. However, since KMT2A may have different partner genes and may have cryptic translocations, a significant portion of the translocations may be missed by karyotyping. FISH assays are a good compliment in detecting those cryptic or variant translocations. Considering the fact that KMT2A has many possible partner genes, FISH assays usually use dual-color, break-apart designs. The probes span over the KMT2A gene and cover the region on either side of KMT2A breakpoints. The probe on one side of the breakpoints will be labeled with red florescent dye, and the probe on the other side of the breakpoints will be labeled with green florescent dye (Fig. 1.10). Normal interphase cells will show two yellow dots. The cells with KMT2A translocation will show one yellow dot, one green dot, and one red dot. Next-generation sequencing (NGS) offers a new opportunity for KMT2A translocation testing as well .

Fig. 1.10
figure 10

FISH assays usually use dual-color, break-apart designs . The probes span over the KMT2A gene and cover the region on either side of KMT2A breakpoints. The probe on one side of the breakpoints will be labeled with red florescent dye, and the probe on the other side of the breakpoints will be labeled with green florescent dye

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Clinical Significance

AML with KMT2A translocations are often associated with an unfavorable prognosis when compared to other AML subtypes; however, AML with 11q23 translocations can also be seen in therapy-related leukemias after topoisomerase II inhibitor therapy [66, 67]; but such cases should be diagnosed as therapy-related leukemias [1].

Acute Myeloid Leukemia with t(6;9)(p23;q34);DEK-NUP214

AML with t(6;9)(p23;q34) can be seen in both children and adults , consisting of approximately 1% of total AML [1]. AML with t(6;9)(p23;q34) can be seen in any FAB subtype of AML. Patients usually present with anemia, pancytopenia, and thrombocytopenia. In addition to general AML morphology, this subtype is often associated with basophilia (often >2%) and multilineage dysplasia [1].

Morphologically , AML with t(6;9)(p23;q34) can be seen in any FAB subtype of AML, except APL and acute megakaryoblastic leukemia. In addition, this subtype is often associated with marrow and peripheral blood basophilia (defined as >2%). Multilineage dysplasia is often seen, with granulocytic and erythroid dysplasia being more common than megakaryocytic dysplasia. Ringed sideroblasts are present in a subset of cases [1].

The immunophenotype of AML with t(6;9)(p23;q34) is fairly nonspecific, showing typical myeloblast markers including CD34, CD117, MPO, CD13, CD33, and CD15. A subset of cases also express CD64, and Tdt expression is not uncommon.

Cytogenetic Abnormality

At the genome level, AML with t(6;9)(p23;q34) translocation results in DEK-NUP214 fusion gene on derivative chromosome 6 [6, 68, 69]. The DEK gene is located at 6p23 and the NUP214 (nuclear pore complex protein 214 kDa) gene at 9q34. The NUP214 gene is also known as the CAN gene. The breakpoints in the DEK and NUP214 genes occur in the intron of each gene. The DEK-NUP214 fusion gene consists of the 3′ portion of the NUP214 gene from chromosome 9 and the 5′ portion of the DEK gene from chromosome 6. The fusion gene contains the open reading frames of both genes [69,70,71]. Since most translocation breakpoints occur in a single intron of the NUP214 gene and in a single intron of the DEK gene, most translocations can be detected using FISH and PCR assays. PCR assays usually have higher sensitivity and real-time PCR assays can be used to follow patients for minimal residual disease [72,73,74,75].

Somatic Gene Mutations

AML with t(6;9)(p23;q34) is frequently associated with a FLT3 mutation, often FLT3-ITD (as high as 70% of cases); other mutations in myeloid mutation-associated genes are much rarer in this AML [76].

Clinical Significance

Overall, AMLs with t(6;9)(p23;q34) usually have an unfavorable prognosis, and FLT3-ITD mutations confer even faster relapse than those patients without FLT3-ITD mutation [76].

Acute Myeloid Leukemia with inv(3)(q21q26.2) or t(3;3)(q21;q26.2)

AMLs with inv(3)(q21q26.2) are rare (2% of AMLs) but usually seen adults [1]. Patients usually present with anemia, thrombocytopenia, or pancytopenia. Morphologically , AML with t(6;9)(p23;q34) can show variable features, but promyelocytic and megakaryoblastic differentiation are not typically seen. In addition, this subtype is often associated with marrow and peripheral blood basophilia (defined as >2%). Multilineage dysplasia is frequently present, with granulocytic and erythroid dysplasia being more common than megakaryocytic dysplasia. Ring sideroblasts can also be demonstrated in a subset of cases [1]. The immunophenotype of AML with t(6;9)(p23;q34) is fairly nonspecific, showing typical myeloblast markers including CD34, CD117, MPO, CD13, CD33, and CD15. A subset of cases also express CD64, and Tdt expression is not uncommon.

Cytogenetic Abnormality

At the genome level, the result of inv(3)(q21q26.2) brings a GATA2 distal hematopoietic enhancer (G2DHE) to the vicinity of the EVI1 gene, causing aberrant EVI1 expression and GATA2 haploinsufficiency [2, 4, 5]. This is unlike other AML translocations that usually result in fusion genes. The breakpoints in 3q26 are distributed over several kilobases [77,78,79,80,81]. The breakpoints in 3q21 are distributed over 100 kb [81,82,83]. The inv(3)(q21q26.2) and t(3;3)(q21;q26.2) can be detected using karyotyping; however, karyotyping may miss some cryptic changes. A dual-color, double-fusion FISH assay has better sensitivity than that of karyotyping [84]. As in other translocations, RT-PCR assays can be used to detect the inversion and translocation [83, 85, 86]. Due to the wide spread of breakpoints in the inversion and translocation, designing a multiplex RT-PCR to reach a good sensitivity is difficult. However, RNA NGS assays may overcome such challenges and offer practical solutions with good sensitivity and specificity .

Somatic Gene Mutations

The somatic gene mutational landscape of AML with inv(3)(q21q26.2) has been preliminarily mapped, and mutations in RAS genes (NRAS, KRAS) and RUNX1 are most common, with 40–50% and 20% of cases, respectively. NF1 deletions, FLT3, and CBL and IDH1 mutations are less frequent, with 10%, 10%, 10%, and 5% of cases, respectively [87, 88].

Clinical Significance

The AML patients with inv(3)(q21q26.2) and t(3;3)(q21;q26.2) tend to have unfavorable prognosis with short overall survival and poor response to conventional chemotherapy [77, 78, 87]. The role of other somatic gene mutations in AML patients with inv(3)(q21q26.2) and t(3;3)(q21;q26.2) such as those in NRAS, KRAS, RUNX1, NF1, FLT3, CBL, and IDH1 is less certain .

Acute Myeloid Leukemia with t(1;22)(p13;q13);RBM15-MKL1

AML with t(1;22)(p13;q13) is usually seen in infants without Down syndrome , more often in female infants. It consists of less than 1% of all AML cases [1] and about 10% of pediatric AMLs [89,90,91,92]. Morphologically, the blasts in AML with t(1;22)(p13;q13) present as small or large megakaryoblasts resembling those of acute megakaryoblastic leukemia [1].

Cytogenetic Abnormality

At the genome level, t(1;22)(p13;q13) results in a fusion gene, RBM15-MKL1. RBM15 (RNA-binding motif protein 15) is located at 1p13 and is also known as OTT (one twenty-two). MKL1 (megakaryocyte leukemia 1) is located at 22q13. The breakpoints on chromosome 1p13 are located at a 6 kb genomic region. The breakpoints on chromosome 22q13 are located in a 28 kb intron [93]. Although both reciprocal fusion genes are expressed, RBM15-MKL1 fusion gene has all the functional motifs encoded by each partner gene [93]. The translocation can be detected by karyotyping, FISH, and PCR [93], with PCR assays being used to follow minimal residual disease after treatment [94].

Clinical Significance

The early studies suggested that AMLs with t(1;22)(p13;q13) carry an unfavorable prognosis [1, 95, 96]; however, more intensive therapy has improved the long-term survival of such patients [97, 98].

De Novo Acute Myeloid Leukemia with t(9;22)(q34;q11.2)

AML with t(9;22)(q34;q11.2) is seen in 0.5–3.0% of all AML cases . In the cases of de novo AML , the patients should not have a history of BCR-ABL1 positive chronic myeloid leukemia (CML), as such splenomegaly and peripheral basophilia are less common than in CML in blast crisis. The translocation t(9;22)(q34;q11.2) results in a BCR-ABL1 fusion gene and is often the specific Philadelphia chromosome [99,100,101,102,103,104,105]. It is now considered a provisional category of AML with recurrent genetic abnormalities [2].

As it is a newly defined provisional entity, our experience with AML with t(9;22)(q34;q11.2) is somewhat limited. To date, no specific histologic or immunophenotypic features which distinguish this entity from typical AML have been identified [99,100,101,102,103,104,105].

Cytogenetic Abnormality

At the genome level, the t(9;22)(q34;q11.2) in this AML is the same as that seen in CML. Karyotyping, FISH, and PCR assays can be used to detect the translocation. As one may expect, FISH has higher sensitivity than karyotyping. Quantitative PCR assay is the most sensitive among the three assays and can be used to monitor minimal residual disease. Differentiating between the blast phase of CML and AML with t(9;22)(q34;q11.2) has been difficult and heavily depends on the patient’s history. However, genomic analysis of de novo cases in comparison to the blast phase of CML has demonstrated specific deletions in IKZF1 and CDNK1A/B as well as loss of IGH/VDJ and TARP regions [104].

Clinical Significance

These cases are so rare that tailored therapies are still in their infancy. It is thought that, like CML in blast phase crisis, AML with t(9;22)(q34;q11.2) may benefit from TKI (tyrosine kinase inhibitor) therapy [103,104,105,106].

Conclusion

Understanding cytogenetic changes in acute myeloid leukemia is critical not only for diagnostic subtyping but also for understanding the pathogenesis and potential clinical progression of patients. While the majority of cytogenetically defined categories of AML have been elucidated, understanding how these genetic changes result in leukemia is still far from complete. Additionally, the significance and prevalence of other somatic gene mutations in these leukemias require further study.