Cytogenetic and Chromosomal Rearrangements

Cytogenetic abnormalities, detectable in approximately 50–60% of newly diagnosed AML patients, are categorized as non-random chromosomal rearrangements. These aberrations are further classified into three categories; AML with balanced translocations/inversions (Table 2), AML with various cytogenetic abnormalities (un-balanced translocations, eg deletions, monosomies, and trisomies) and AML with complex karyotype (representing at least 3 acquired chromosomal aberrations) (Table 3).

Cytogenetically, AML has three prognostic categories: favorable, intermediate and poor-risk group. The first group includes balanced translocations with a favorable outcome. The poor risk group has a complex aberrant karyotype that confers a poor clinical outcome. The intermediate prognosis group includes normal karyotype and other karyotypic abnormalities (Table 1).6,7

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Balanced Chromosomal Rearrangements (Translocations/Inversions)

Balanced translocations and inversions include inv(16)(p13.1q22)/t(16;16) (p13.1;q22), t(8;21)(q22;q22), inv(3)(q21q26.2)/t(3;3)(q21;q26.2), t(6;9)(p23;q34), t/inv (11q23) and t(15;17)(q24;q21). These lesions on their own are not sufficient to induce leukemia, and there is a requirement for additional secondary genetic lesions to accompany the balanced translocation/inversion. For example, trisomy 22 in AML with inv(16)/t(16;16), deletion 9q in AML with t(8;21), or monosomy 7 in AML with inv(3)/t(3;3) (Table 2).2,15

The results of t(8;21) and inv(16)/t(16;16) are, respectively, fusion proteins RUNX1-RUNX1T1 (AML1-ETO) and CBFβ-MYH11 which bock the transactivation of tumor-suppressor genes, and associate with favorable outcome in AML. Accordingly, the fusion proteins RUNX1/AML1 and CBFB-MYH11 impair myeloid differentiation and induce self-renewal properties of HPCs but on their own do not cause an overt leukemic phenotype. Rather, there should be further rare mutations in the germline of affected patients to predispose these persons for developing AML. More than one genetic change should occur in HPCs to show overt leukemia phenotype (Figures 2 and 3).15–9

Figure 2

Figure 3

The reciprocal t(8;21)(q22;q22) between the chromosomes 8 and 21 results in the fusion gene AML1-ETO (RUNX1-RUNX1T1) whose product is the fusion protein AML1-ETO. The RUNX protein family or core-binding factors (CBFs) constitutes of a group of heterodimeric transcription factors that are composed of an α-subunit (CBFα; encoded by 3 distinct genes: Runx1/Runx2/Runx3) and a β-subunit (CBFβ; encoded by CBFβ). This family is also known as the polyomavirus enhancer-binding protein-2 (PEB-2).10 At the molecular level, RUNX1 (AML1) is a DNA-binding factor, and a master regulator of HPCs, while RUNX1T1 (ETO) is a transcription factor with the repressor activity (an oncogene). The N-terminal domain of AML1 fuses to the 577 residues from the ETO C-terminal domain. The fusion protein AML1-ETO recognizes AML1 consensus binding sites and heterodimerizes with CBFβ, wherein harboring transcriptional repressor activities.1,2 The fusion protein acts as a transcriptional repressor to block AML1-dependent transactivation and transcription of tumor suppressors. In fact, the translocation interrupts AML1 protein between the N-terminal and transactivation domains, while leaving the transactivation domain of ETO protein intact.5,6 This type of translocation has the highest incidence in childhood AML (~12% of AML cases in children) (Figure 4), and profoundly associates with M2 FAB subtype (AML with maturation), but rarely seen with AML M1 or M4 subtypes. This kind of cytogenetic aberration is highly significant for diagnosis and therapy management, since on its own associates with favorable outcomes, high remission rates and long median survival, whereas c-Kit mutations occurring concurrently with t(8;21)(q22; q22), compose an independent adverse prognostic biomarker (Table 2, Figure 2). In addition, in pediatric AML M2 subtype with t(8;21), the X-chromosome loss may occur as the secondary cytogenetic events with no clinical significance, whereas the loss of the Y chromosome, as the secondary genetic event, forms a critical mutational event. Generally, conventional cytogenetic analysis FISH, or RT-PCR can readily predict AML1-ETO outcomes (Table 1).4–6 Patients with t(8;21) have a favorable prognosis with good response to conventional chemotherapy (the combination of an anthracycline and cytarabine, the “3 + 7” regimen) and show complete remissions (Tables 1 and 2). At least three or four cycles of intensive post-remission therapy with high doses of cytarabine (HDAC) are recommended for adults, improving substantially the outcome and maximizing the gain of chemotherapy (Table 2).6,15

However, in de novo AML cases, loss of function mutations (missense, nonsense or frameshift mutations) in RUNX1 gene are classified as intermediate-risk AML, rather than as the favorable risk group, and highly associate with AML M0. The RUNX1 mutations associate with higher chemotherapy resistance (where refractory rates are about 30%), and lower event-free, relapse-free, and overall survival rates.3

Inv(16)(p13;q22) leads to the formation of oncogene CBFβ-MYH11 whose product is the oncoprotein CBFβ-MYH11. The fusion protein acts as a transcriptional repressor cooperating with AML1 to repress transcription of tumor-suppressor genes PTEN, Bcl-2, CEBPA, ARF and PSGL-1. In the rearrangement, the C-terminal coiled-coil region of the smooth muscle myosin heavy chain 11 (MYH11) fuses with the first 165 residues of CBFβ.1,15 CBFβ-MYH11 fusion gene forms at least eight different transcripts in progenitor cells wherein fusion protein heterodimerizes with RUNX1 (CBFα).

In progenitor cells, RUNX1 (CBFα) and CBFβ form a complex regulating gene expression, at the Runx-binding sites on DNA. In a dominant negative manner by competing for heterodimerization and/or for DNA binding at Runx-binding sites, CBFβ-MYH11 and RUNX1-ETO fusion proteins inhibit the function of normal RUNX1 (CBFα)/CBFβ complex. CBFs are required in the hematopoietic ontogeny as the key regulators of different steps of hematopoiesis; therefore, the abnormal fused products disrupt their function and inhibit hematopoietic differentiation.3,4 The balanced rearrangements t(8;21)(q22;q22) and inv(16)(p13.1q22)/t(16;16)(p13.1;q22) are assigned as core-binding transcription factor leukemia (CBF-AML), and are the most common events found in AML, representing approximately 15–25% of all AML cases.3,6

Now, RQ-PCR and nested PCR techniques are sensitive and efficient approaches for the detection of these rearrangements (sensitivities ranging from 10 −4–10 −6).5,10 CBFβ-MYH11 has been linked with the AML M4Eo and a higher incidence in adults (Figure 4).3,4

Figure 4

Patients with inv(16)/t(16;16) have a favorable prognosis, good response to conventional chemotherapy (the combination of an anthracycline and cytarabine, the “3 + 7” regimen) and complete remissions (Tables 1 and 2). Likewise, t(8;21), receiving 3–4 cycles of HDAC is efficient and recommended for these patients (Table 2). The improved outcome with repeated therapy with cytarabine is addressed to the incorporation of cytarabine into the genomic DNA where increased sensitivity of the leukemic cells to cytarabine and inducing apoptosis.6,15

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