Integrated analysis of DNA sequencing and microarray data enabled us to discover an association between mutations of a specific gene, i.e. TP53
, with chromothripsis. The unexpected germline nature of mutant TP53
in several LFS-associated SHH-MBs means that the TP53
mutation must precede the massive shattering and chromosome rearrangements in these. This led us to propose a novel role for p53 in the initiation of, or cellular reaction to, chromothripsis – with TP53
mutations predisposing cells for chromothripsis, or facilitating cell survival following catastrophic DNA rearrangements (). The high frequency of TP53
germline mutations specifically in SHH tumors further suggests that LFS patients may be predisposed to SHH-MB, rather than to medulloblastoma per se
. This finding has clinical implications, and we recommend assessing the merit of testing SHH-MB patients with chromothripsis for germline TP53
mutations, since regular screening in LFS families was recently shown to lead to a survival benefit (Villani et al., 2011
). Additionally, particular care may be required with respect to current treatment regimens, which in most cases include DNA damaging agents and radiotherapy. These may induce therapy resistance in the primary tumor or trigger secondary malignancies in the context of constitutional TP53
mutations and deficient DNA repair.
The frequent occurrence of chromothripsis in the context of germline alterations of TP53 points to a possible requirement of TP53 mutations in the tumor cell-of-origin, or of TP53 mutations acquired early in tumor development. The identification of chromothripsis in the context of somatic TP53 mutations in an SHH-MB patient and in several AMLs additionally implicates acquired, tumor-specific TP53 mutations in chromothripsis.
Possible explanations for the absence of chromothripsis in several malignancies with TP53 mutations, including several AMLs and WNT medulloblastomas, are: their comparably late acquisition of such mutations; the occurrence of TP53 mutations in tumor subpopulations; and cell context-specific factors, including distinct gene expression programs. Further to this, selective growth advantages are likely to determine tumor development following chromothripsis. The higher rate of chromothripsis in TP53-mutated SHH-MBs (all cases we analyzed) compared to TP53-mutated AMLs (approximately half of the cases we analyzed) is striking. It is tempting to speculate that this difference is connected with the abundance of high-level oncogene amplifications, typically involving double minute chromosomes, in SHH-MBs with chromothripsis. The selective advantage of such oncogene amplification is likely higher than the selective advantage conferred by hemizygous tumor suppressor gene loss observed in AML. Furthermore, rare cases of chromothripsis in AMLs harboring wild-type TP53, and in medulloblastomas with hemizygous TP53 deletion, show that chromothripsis can also occur in the absence of TP53 point mutations – perhaps in association with low TP53 gene dosage, or other genetic or epigenetic mechanisms causing p53 pathway dysregulation.
Our analyses of rearrangement breakpoints are in support of a model of massive DNA double strand breaks (Stephens et al., 2011
), followed by NHEJ-mediated repair (). The replication-associated mechanism MMBIR can also generate alterations with multiple breakpoints (Hastings et al., 2009a
). The lack of templated insertions at the breakpoint junctions, however, which are thought to result from abortive attempts to use another template during replication (Hastings et al., 2009b
; Howarth et al., 2011
), does not substantiate the involvement of a replication-associated rearrangement mechanism in the complex alterations we observed.
Stephens et al. (2011)
have discussed two possible scenarios that may underlie the chromosome shattering and rearrangement seen in chromothripsis. Both scenarios involve damage occurring in mitotic chromosomes, since the compaction of chromosomes during mitosis could explain the highly localized nature of the DNA breaks, which are often focused on a single chromosome. We note, however, that the known spatial organization of chromosomes maintained during interphase (Cremer and Cremer, 2001
; Lichter et al., 1988
) might similarly represent a structural basis for the local occurrence of DNA shattering. In one scenario, it was proposed by Stephens et al. that ionizing radiation may lead to a catastrophic series of DNA double strand breaks. Based on our observations, these breaks might be preferentially repaired by low-fidelity mechanisms, such as error-prone NHEJ, as these are known to play a greater role when levels of p53 activity are reduced (Dahm-Daphi et al., 2005
). An additional result of impaired p53 activity could be an increased rate at which cells are able to survive, and divide, after acquiring catastrophic chromosome alterations.
In a second possible scenario, critical telomere shortening followed by chromosome end-to-end fusions (which are prone to occur in association with uncapped telomeres (Tusell et al., 2010
)) and subsequent breakage could lead to chromothripsis. Furthermore, dividing cells with uncapped telomeres are delayed at the G2/M transition in a p53-dependent manner, and unprotected telomeres in p53-deficient cells undergoing mitosis were found to be shorter than average, and prone to form end-to-end fusions (Thanasoula et al., 2010
). Intriguingly, LFS patients harbor shortened telomeres compared to unaffected individuals of the same age group, with average telomere length decreasing from generation to generation in affected families, and the age of onset of cancer showing an association with average telomere length (Tabori et al., 2007
; Trkova et al., 2007
). As two different chromosomes may participate in an end-to-end fusion, such events could explain our observations of chromothripsis leading to complex inter-chromosomal rearrangements (with derived double minute chromosomes), which involved no more than two chromosomes. Thus, critical telomere shortening followed by end-to-end fusions and subsequent tearing apart during mitosis provides a plausible explanation for the link between TP53
mutations and chromothripsis reported in this study. The finding that chromothripsis frequently occurred in individuals with advanced age at diagnosis in TP53
-mutant AML is also of note, given the progressive shortening of telomeres with age.
Additional contributions of p53 in controlling the G2/M transition checkpoint (Kastan and Bartek, 2004
) suggest that the involvement of a different mechanism – premature chromosome compaction – may also be a possibility. In this process chromosomes from an S-phase nucleus condense prematurely and, as a result, may become shattered (Meyerson and Pellman, 2011
). Given the multiple and varied roles that have been assigned to p53, it is possible that the link between TP53
and chromothripsis is associated with more than one of the aforementioned functions acting in concert ().
In contrast to earlier reports on the rarity of chromothripsis (Kloosterman et al., 2011
; Stephens et al., 2011
), we found that in specific biological contexts this phenomenon occurs at high frequency. In conclusion, results from our study – which reported the first whole-genome sequence data for tumors from LFS patients and for pediatric brain tumors – indicate a new role for p53, a protein also known as the ‘guardian of the genome’. Results from upcoming large-scale cancer genome sequencing studies (The International Cancer Genome Consortium, 2010
) may shed light on additional factors associated with this catastrophic genomic phenotype.