Amplifications and target genes in diffuse large B-cell lymphoma: real targets or consequences of structural features of the genome?

The most frequent primary genetic alteration of the B-cell neoplasms are chromosomal translocations that activate oncogenes and are essential for tumor initiation, whereas gene amplification represents another mechanism of oncogene overexpression and is found as a secondary genetic alteration. B-cell lymphomas harboring amplifications show complex and numerous alterations and are associated with an aggressive clinical behavior [1]. Metaphase- and array-comparative genomic hybridization (CGH) techniques have been very useful as a genomic tool for the screening of high-level DNA amplifications. Nevertheless, most of the target genes still remain to be elucidated. In that sense, the use of CGH/array-CGH in diffuse large B-cell lymphoma (DLBCL) has allowed the identification of recurrent amplifications and target genes, namely BCL2 and MALT1 (18q21); REL and BCL11A (2p16); CDK4, MDM2, GLI, and SAS (12q13); MIRHG1 (microRNA-17-92 cluster) (13q31.3), JAK2 (9p24); MYC (8q24); RFC4 and BCL6 (3q27); and CCND3 and BYSL (6p21). However, there are still a large number of additional amplified regions in DLBCL with elusive target genes (1q21, 1q25–q31, 2q22–q24, 4p15, 5p15, 5q34, 6p25, 9q33–q34, 10p13–p15, 10q11, 11q22–q24, 16p12, 16q24, 17p11, 22q12, Xp22, and Xq28).

In this issue of Leukemia and Lymphoma, Nagel et al. [2] identify the kinase SIK2 as a target of 11q23 amplification in a DLBCL cell line, Karpas-422. Furthermore, the authors perform functional analyses which indicate that SIK2 regulates survival and glucose metabolism via the pro-apoptotic gene BIM, and discuss that SIK2 and its downstream effectors could represent potential novel therapeutic targets. Nevertheless, although the gain and occasional amplification of the 11q22–q24 region is found in ∼8–15% of DLBCL, SIK2 amplification and overexpression could only be demonstrated in this cell line. In addition, SIK2 overexpression could not be detected in any DLBCL primary tumors. Interestingly, a similar situation has been reported for homozygous deletions, where validated target genes have been identified only in cancer cell lines but not in human tumors of the same entity [3,4].

Conventional cytogenetics [5] and FISH analysis [6] of Karpas-422 have reported a complex derivative 11 chromosome formed through terminal deletion and four inverted tandem repeats. These results are fully concordant with the data generated by SNP-array 6.0 (www.sanger.ac.uk) and also by the polymerase chain reaction (PCR) results of Nagel et al.: four copies of 11q23, a peak of eight-fold amplification of SIK2 gene, and a terminal 11q24–q25 deletion. Of note, Karpas-422 has a functionally inactivated TP53 gene (point mutation plus deletion), and most of the chromosomes showed multiple structural alterations, including another focal amplification in 16p.

The loss of TP53 function is strongly associated with genomic instability in human tumors, and one of the manifestations of genomic instability is DNA amplification. One of the mechanisms proposed for gene amplification is the breakage–fusion–bridge (BFB) cycle, which requires two or more breaks: the first leads to a telomere loss and is responsible for the initiation of several rounds of amplification, whereas the second break takes place during cell division when a chromatin bridge is formed and subsequently broken [7]. To exit a BFB cycle, the cell needs to reactivate the telomerase or to capture an intact telomere from another chromosome, thus avoiding DNA repair checkpoints and perpetuating the genomic instability.

In Karpas-422, the inverted nature of the 11q repeats and the telomeric loss strongly suggest that 11q amplification is likely to be originated thought BFB cycles. However, this gene amplification can be either due to gene (SIK2) selection or as a consequence of a specific chromosome structure that results in increased susceptibility to DNA rearrangements. Noteworthy, this latter possibility was also postulated for homozygous deletions in a survey of 636 human cancer cell lines [8], which found that 63% of the 281 homozygous deletions were located in genomic regions with no tumor suppressor genes, fragile sites, or copy-number polymorphisms (CNV). Remarkably, these homozygous deletions were not random, and clustered in regions of the genome with certain architectural features, including low gene density, low repeat frequency, and high DNA flexibility.

In that sense, SIK2 gene location at 11q23 is not flanked by CNV or segmental duplications, but is near the common fragile site FRA11G and the rare fragile site FRA11B, both located at 11q23 [9]. Moreover, the 11q23 cytoband is a hotspot of oncogenic recombination implicated in several recurrent alterations of different cancer types.

Thus, in the biology of some tumors, the presence of multiple alterations (homozygous deletions or amplifications) may reflect the complexity of break-prone DNA regions related to certain structural/architectural features rather than the positive gene selection, thus pointing to an underlying inherent genomic instability that in most cells may be allowed by the loss of functional DNA repair checkpoints.

Acknowledgements

This work is supported by the ‘Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias’ FIS06/0150 and PI08/0077.