*2.1.4. Somatic hypermutation (SHM)*

(MMR). After the formation of DSBs in the S regions (donor and acceptor), these S regions are

During normal B-cell development, the DNA repair pathways (BER and MMR) reduce the effect of off-target AID activity. However, several external factors like cellular stress, hypoxia, and viral infections; or intrinsic factors such as alterations in repair pathways may change the

One evident deviation of the normal V(D)J recombination and CSR processes is the possibility of rearrangements between segments belonging to different genes. In fact, reciprocal chromosomal translocations are the most common recurrent genetic anomalies in lymphoid malignancies and the newly formed junctions generated in most human lymphoid transloca-

One paradigmatic example is follicular (FL), a lymphoid neoplasm characterized by the t(14;18)(q32;q21) translocation that juxtaposes the anti-apoptotic proto-oncogene BCL2 to the immunoglobulin heavy chain locus [16]. The functional result of this translocation is constitutive transcriptional upregulation of BCL2. Although this translocation is considered the founding event in FL pathogenesis, t(14;18)-positive B cells can be detected in many healthy individuals [17]. Therefore, this genetic event alone seems insufficient to cause lymphoma.

The t(11;14)(q13;q32) translocation, a hallmark of mantle cell lymphoma (MCL), results in the overexpression of cyclin D1 and also appears to be a V(D)J-mediated translocation [18]. As in FL, the sole constitutive overexpression of this cell cycle regulator is insufficient to explain

Whereas the t(14;18) or t(11;14) translocations result from a mistake during V(D)J recombination, some translocations involve the IgH class switch regions in a failed CSR event. Translocations at the IgH class switch regions seem to depend on AID activity and commonly involve c-MYC and BCL-6 [19]. BCL6 is the most commonly rearranged gene in activated B cell (ABC) diffuse large B-cell lymphoma (DLBCL) and c-MYC rearrangements can be

BCL6 is a proto-oncogene encoding a transcriptional repressor expressed during B cell differentiation in germinal centers. A block in the normal downregulation of BCL6, through its translocation with more than 20 possible partner genes, might favor differentiation arrest, continuous cell proliferation, survival, and genetic instability [20]. BCL6 also suppresses the activity of the tumor suppressor gene TP53, which allows BCL6-expressing cells to escape apoptosis [21].

The c-MYC gene at 8q24 is involved in three translocations observed in DLBCL, most commonly t(8;14) (q24;q32), and less often t(2;8) (p12;q24) and t(8;22) (q24;q11) [21]. In the t(8;14) (q24;q32) translocation, also observed in BL, the gene segments from the IgH locus are joined with various regions around and within the c-MYC proto-oncogene [22]. As a result, IgH

regulatory elements are misplaced upstream, of the c-MYC proto-oncogene [23].

*2.1.3. V(D)J recombination, class switch recombination, and neoplastic transformation*

recombined by non-homologous end joining (NHEJ) [13].

outcome of AID-induced lesions [14].

20 Hematology - Latest Research and Clinical Advances

tions have the canonical features of NHEJ [15].

observed in Burkitt lymphoma (BL) and DLBCL.

malignant transformation.

Somatic hypermutation (SHM) is the biological underlying mechanism for the generation of the secondary antibody repertoire. AID is the single enzyme that is responsible for the initiation of this process [25].

SHM is a post-rearrangement diversification process that introduces point mutations in the variable regions of the Ig loci, which can alter the antibody binding to its cognate antigen. AID acts enzymatically as a cytosine deaminase that converts cytosine to uracil. Uracil is mutagenic when paired with guanosine, this U:G mismatch triggers error-prone DNA repair in B cells. SHM results in a mutation rate of circa 1 mutation/1000 bp per cell generation. This mutation frequency is a million-fold higher than spontaneous mutation rate in somatic cells [26]. Highly selected antibodies with neutralizing activity against influenza virus can accumulate 30–40 mutations, and broadly neutralizing antibodies against HIV more than 100 mutations [27, 28].

AID acts on a single strand, thus its activity is probably generated during at transcription bubbles (**Figure 1**). Once AID produced deamination of dC to dU the error-prone processing begins. First AID-catalyzed uracils in the DNA are recognized by either the uracil-DNA glycosylase (UNG)—triggering the base excision repair (BER) pathway—or by the mismatch recognition heterodimer MutSα—initiating the mismatch repair (MMR) pathway. In BER, UNG binds to the U:G mispair and produces an abasic site, then this site is cleaved by the apurinic/apyrimidinic endonuclease (APE1), which removes the abasic site nucleotide and the DNA polymerase Polβ resynthesizes the DNA strand [29]. In the MMR pathway, the proteins MSH2 and MSH6 bind to the U:G mismatch and recruit DNA Polη, a low fidelity polymerase, that introduces error during nucleotide synthesis [30].

The processing of uracils by BER and MMR may result in different outcomes. The introduced uracils may (1) be replaced by another nucleotide, (2) expose DNA to further mutations in its vicinity like mutations at A:T pairs or (3) can be converted into DNA DSBs. The latter seems to be necessary for CSR.

Because of its mutagenic potential, SHM has multiple layers of regulation and competition between alternative pathways that define the level of SHM [31]. There is also increasing evidence that epigenetic factors, such as DNA methylation and post-translational histone modifications play major roles in regulating SHM [32]. Its implications in lymphoma development remain elusive.

When SHM affect off-target genes, it is referred to as aberrant SHM. Aberrant SHM can be mainly detected in FL, BL, DLBCL, and CLL [33–35]. This topic has been extensively reviewed elsewhere [36–39].

AID expression is detected in a tumor subset in the peripheral blood of CLL patients [43, 44]. These cases display the dissociation between CSR and SHM, an observation that resembles our findings in FL. Our data also suggest that functional AID expression in CLL correlates

The Antigen Receptor as a Driver of B-Cell Lymphoma Development and Evolution

http://dx.doi.org/10.5772/intechopen.72122

23

Analysis of whole genome and whole exome sequencing data classified by the trinucleotide context of single nucleotide variants in so-called mutation signatures can help to elucidate underlying mutagenic mechanisms in tumor samples [46]. Our data indicate that the mutational landscape of both CLL and FL seems to be strongly shaped by AID activity. In FL, AID-induced mutations are mainly restricted to canonical AID hotspots and CpG methylation-dependent mutagenesis sites. In strong contrast, both canonical and non-canonical AID

SHM may not only contribute to lymphomagenesis by acting on oncogenes and proto-oncogenes, but also may provide adaptive advantages. As suggested by our data, BCR editing

Antibody molecules, when expressed on the cell surface, constitute the binding moiety of a molecular complex known as B-cell antigen receptor (BCR). Signals from the BCR regulate the development and function of B cells. However, the ability of the BCR signaling pathway to induce cell survival and proliferation could be adopted and distorted by malignant

The BCR immunoglobulin consists of a heavy chain and a light chain, whereas its precursor, the pre-BCR, consists of a heavy chain and a surrogate light chain. The transmembrane domain of the heavy chains anchors the BCR to the cell membrane, where each BCR molecule associates with the signaling subunit. The signaling subunit is constituted by a heterodimer of Igα (CD79A) and Igβ (CD79B) [51]. Within their cytoplasmic tails, Igα and Igβ harbor 2 conserved tyrosine residues as part of a 26 amino acid-long sequence, also referred to as an immunoreceptor tyrosine-based activation motif (ITAM) [52]. Phosphorylation of ITAM through kinases, such as Lck/Yes-related novel protein tyrosine kinase (LYN), B-lymphoid kinase (BLK), or spleen tyrosine kinase (SYK), marks the first step in signal transduction from the BCR to the nucleus [53]. SYK in conjunction with PI3K recruits Burton's tyrosine kinase (BTK). Upon activation of the BCR pathway, BTK binds to PIP3 and attaches to the plasma membrane [54]. These events contribute to BCR-induced calcium release, cell proliferation,

In pre-B cells, the BCR signaling cascade is activated through autonomous signaling, a mechanism that relies on the structural conformation of the pre-BCR which is constituted by a heavy chain and a surrogate light chain [56, 57]. While pre-B cells rely on autonomous BCR signaling, immature and mature B-cells receive two types of signals from their BCRs: the antigen-dependent, and the antigen-independent "tonic" signals. The antigen-dependent signal is generated by binding of an external antigen to the BCR and results in the clustering and

with a distinctive genomic landscape and disease evolution [45].

motifs seem to contribute to the mutational landscape of CLL [47].

**2.2. Pathogenic activation of the B-cell receptor signaling cascade**

and activation of the NF-κB pathway (**Figure 2A**) [55].

cells.

through SHM may allow FL cells to escape from immunosurveillance [48–50].

**Figure 1.** Molecular mechanism of somatic hypermutation (SHM). AID requires a single strand to initiate the SHM process. Transcription by RNA polymerase II (RNA Pol II) exposes the single-stranded DNA template for AID. AID deaminates a cytosine to create an uracil, which can then be processed by different pathways. Replication over the uracil results in C to T or G to A transition mutations. Processing by uracil DNA glycosylase (UNG) generates an abasic site (Φ) that is cleaved by the apurinic/apyrimidinic endonuclease (APE1), which removes this site and then Polβ resynthesizes the DNA. Recognition of the U-G mismatch by MutSα (represented by a torus shape) followed by the action of Exo1 and Polη spreads mutations (indicated as "N") to surrounding A-T nucleotides. UNG and Msh2/Msh6 can also act in the context of high fidelity base excision repair (BER) and mismatch repair (MMR) pathways, which results in error-free repair.

We have recently described that, in IgM expressing FL, the mutation load of the Ig genes can be described as a function of the AID expression level. In contrast, in FL cases that underwent class switch recombination (i.e., IgG expressing lymphomas) AID expression and SHM of immunoglobulin genes are dissociated [40, 41]. The distinctive patterns induced by SHM may also have implications for the clinical evolution of the disease [42].

AID expression is detected in a tumor subset in the peripheral blood of CLL patients [43, 44]. These cases display the dissociation between CSR and SHM, an observation that resembles our findings in FL. Our data also suggest that functional AID expression in CLL correlates with a distinctive genomic landscape and disease evolution [45].

Analysis of whole genome and whole exome sequencing data classified by the trinucleotide context of single nucleotide variants in so-called mutation signatures can help to elucidate underlying mutagenic mechanisms in tumor samples [46]. Our data indicate that the mutational landscape of both CLL and FL seems to be strongly shaped by AID activity. In FL, AID-induced mutations are mainly restricted to canonical AID hotspots and CpG methylation-dependent mutagenesis sites. In strong contrast, both canonical and non-canonical AID motifs seem to contribute to the mutational landscape of CLL [47].

SHM may not only contribute to lymphomagenesis by acting on oncogenes and proto-oncogenes, but also may provide adaptive advantages. As suggested by our data, BCR editing through SHM may allow FL cells to escape from immunosurveillance [48–50].
