**2.3 Polymerase chain reaction (PCR)**

Polymerase chain reaction (PCR) is a fast and cheap technique used to "amplify" small segments of DNA by copying them. Because significant amounts of a sample of DNA are needed for molecular and genetic analysis, without PCR amplification, studies of isolated pieces of DNA are almost impossible. The PCR method is based on a cell's natural processes for replicating a new DNA strand. For PCR, only a few biological ingredients are needed. The template DNA—that is, the DNA that contains the region to be copied, such as a gene—is an essential component. A prototype can be as small as one DNA molecule. The sequence of two short regions of nucleotides (DNA subunits) at either end of the region of interest is all that is needed for this fragment to be replicated. The primers bind to the template at their complementary sites, or anneal, and serve as the starting point for copying. The replication of the desired intervening sequence is achieved when DNA synthesis at one primer is guided toward the other. Free nucleotides and a DNA polymerase, an enzyme that builds new DNA strands by sequentially adding on free nucleotides according to the template's instructions, are also needed.

## **2.4 Next-generation sequencing (NGS)**

NGS requires sequencing of millions of DNA molecules concurrently to produce sequence reads. In order to detect small insertions/deletions (indels) and structural

**105**

*Genetic Abnormalities in ALL*

*DOI: http://dx.doi.org/10.5772/intechopen.97429*

variants (SVs) of 450 bp, sequence reads are aligned with the reference genome and base variants. Overall, NGS has the potential to generate up to one billion short

**3. Chromosomal and molecular abnormalities associated with ALL-B**

60–80% of patients with ALL have abnormalities in chromosome number or structural rearrangements (translocations), whereas the remaining 20–40% have normal karyotype [9, 10]. Besides those with a normal karyotype, t(9;22)(q34,q11); BCR/ABL (BCR-ABL1), t(12;21)(p13;q22);TEL/AML1 (ETV6-RUNX1), t(4;11) (q21;q23); MLL/AF4 (KMT2A/AFF1), t(1;19)(q23;p13); E2A/PBX1 (TCF3-PBX1),

The BCR/ABL1 or Philadelphia (Ph) chromosome is a t(9;22) product that fuses the chromosome 9 Abelson kinase gene (ABL1) with the chromosome 22 breakpoint cluster region (BCR) that expresses the fusion protein BCR-ABL1: a constitutively active tyrosine kinase. The breakpoint occurs between exons 1 and 2 (e1 and e2) of the BCR gene in the minor breakpoint cluster region, m-BCR, in the majority of Ph positive ALL patients, and between exons 1 and 2 of the ABL gene in the majority of Ph positive ALL patients (e1a2). This results in the development of a 7-kb mRNA and the expression of the p190 protein [13]. This transcribes an 8.5 kb mRNA that codes for a chimeric p210 protein [14]. The Philadelphia chromosome is the most important cytogenetic abnormality. It is seen in 3% of pediatric patients, and almost 25% of adults, and rises with age, reflecting about half of the cases of patients older than 60 years of age, and although historically associated with poor prognosis, results have been markedly improved with the use of tyrosine kinase inhibitors (TKIs) [15].

The most prevalent translocation in childhood acute lymphoblastic leukemia is TEL-AML1 gene fusion, induced by t(12;21) (p12;q22). However, this anomaly is rare among adults. The translocation of t(12;21) is cryptic by normal G-banding and includes FISH examination for cytogenetic detection [16]. ETV6-RUNX1 patients were thought to have a good prognosis at first, and they were associated with favorable risk factors including female gender, young age, low white cell count, and CD10+ immunophenotype [17]. However, some studies found no gain for ETV6-RUNX1 patients [18], while others found a high incidence of gene fusion in relapse patients and a predilection for late relapse [19, 20]. However, it is now clear that the initial optimism was justified. Almost every major clinical trial group in the world has confirmed that children with the ETV6-RUNX1 fusion have excellent overall survival and very low relapse rates [21, 22], and the presence of added cytogenetic or molecular abnormalities does not modify this good prognosis.

A transcriptional coactivator with methyltransferase activity encodes the gene KMT2A. The rearrangements result in the fusion of the 5′ portion of KMT2A, including the methyltransferase domain, to the 3′ region of the partner genes.

reads per instrument cycle, an immense amount of data cheaply.

are the most common cytogenetic subtypes in ALL [10–12].

**3.1 Structural chromosomal abnormalities**

*3.1.1 The t(9;22)(q34;q11.2)(BCR-ABL1)*

*3.1.2 The t(12;21)(p13;q22)(ETV6-RUNX1)*

*3.1.3 The KMT2A (MLL) Gene Rearrangements (11q23)*

variants (SVs) of 450 bp, sequence reads are aligned with the reference genome and base variants. Overall, NGS has the potential to generate up to one billion short reads per instrument cycle, an immense amount of data cheaply.
