Background
The
CHEK2 c.1100delC mutation, leading to premature translation termination, was discovered to be the first moderate-risk breast cancer (BC) susceptibility allele in 2002 [
1,
2]. Women who carry this germline mutation have a 2.3-fold increased risk to develop BC during their life time compared with the general population [
3,
4]. BCs from
CHEK2 mutation carriers are mostly of the luminal/ER+ subtype and are diagnosed at a younger age than sporadic BCs (median age of 50 vs 60 years) [
5‐
8]. Furthermore, BC patients carrying the c.1100delC mutation have increased risk of developing a contralateral BC and a worse survival compared to sporadic BC patients although resistance to either endocrine or chemotherapy does not appear to play a role herein [
5,
8‐
12]. To provide tailored prevention and treatment strategies for
CHEK2 mutation carriers, it is important to unravel the biological mechanism that
CHEK2 c.1100delC exploits to drive tumorigenesis.
Similar to BRCA1 and BRCA2, CHEK2 operates in the DNA damage response (DDR) pathway. Once activated, CHEK2 is able to phosphorylate more than 20 different effector proteins involved in DNA repair, cell cycle regulation, TP53 signaling and apoptosis (e.g., BRCA1, CDC25A, TP53, and PML) [
13]. Considering the central role of CHEK2 in these pathways and the merely moderate BC risk the c.1100delC mutation confers, many of its functions must be redundant in mammary epithelial cells in which CHEK2-associated BCs arise.
Functional studies and mouse models have produced conflicting results [
14‐
18]. One important reason for this is likely the use of either non-human model systems or non-mammary epithelial cell types. Considering the latter, hormonal factors seem to play an important role in the development of BC in women and mice carrying the c.1100delC mutation, since the vast majority of BCSs in women is of the luminal/ER+ subtype [
6,
7] and
Chk2 c.1100delC knock-in mice developed tumors preferentially in females [
18].
In addition, results from gene expression and copy number (CN) profiling studies on CHEK2-associated BCs have also not provided significant clues regarding CHEK2-driven tumorigenesis [
7,
19,
20]. In this respect, next-generation sequencing technology has provided much insight into the mutational processes that operate during tumorigenesis in recent years. For example, mutational profiling has identified the APOBEC-catalyzed cytidine deamination to be a major source of mutation in cancer [
21]. Moreover, homologous recombination repair deficiency (HRD), caused by loss of BRCA1 or BRCA2 function, leaves a specific genomic imprint on the DNA characterized by two single base substitution (SBS) signatures (SBS3 and SBS8), one small insertion/deletion (ID) signature (ID6) and two specific structural variant (SV) signatures (SV3 for BRCA1-type cancers and SV5 for BRCA2-type cancers) [
22‐
24]. Interestingly, BCs from women carrying truncating variants in
CHEK2 did not display a dominant HRD-related mutational signature, in contrast to BCs from
BRCA1,
BRCA2 and
PALB2 mutation carriers, but similar to BCs from
ATM mutation carriers [
25‐
27]. Both studies on CHEK2- and ATM-associated BCs used exome and targeted sequencing data, limiting resolution and precluding the analyses of larger SVs.
In the current study, we have sequenced the primary tumor and normal genomes of 20 CHEK2 c.1100delC mutation carriers as well as their tumor transcriptomes. Including pre-existing genomic data, we exhaustively compared CHEK2 primary BC (pBC) genomes to pBC genomes from BRCA1/2 mutation carriers, pBCs that displayed HRD and ER− and ER+ pBCs, totaling to 574 pBC genomes. Findings were validated in 517 metastatic BC (mBC) genomes subdivided into the same subgroups.
Discussion
Our interrogation of the somatic landscape of CHEK2 BCs revealed novel genomic features specific to CHEK2-driven BC. First, and in agreement with Mandelker et al
., we did not observe an HRD phenotype among CHEK2 BCs [
26]. Instead, CHEK2 BCs were most similar to ER+ BCs. Second, CHEK2 BC genomes that lost the wild-type
CHEK2 allele did not harbor any somatic
TP53 mutations (
i.e., 0/43 in all three cohorts combined). Third, CHEK2* BCs displayed a unique size distribution of SVs that is not simply caused by the increased chromothripsis frequency among these genomes.
There are two reasons why the latter two observations were not reported by Mandelker et al., which also represent strengths of our study. First of all, inherent to the nature of their data (from whole exome sequencing and targeted sequencing using the MSK-IMPACT panel) structural variation and related events such as chromothripsis could not be evaluated. Second, although Mandelker et al. evaluated allelic loss at the CHEK2 locus, they instead opted to stratify samples according to low and high-risk CHEK2 variants. Since our cohort consisted only of BCs from c.1100delC carriers, we did not have to prioritize classification in this respect. Another strength of our study was the availability of a second cohort for validation purposes. A disadvantage of having an mBC cohort for validation, however, is that due to disease progression and/or treatment-induced selection meaningful pBC-specific associations could have been obfuscated.
Our observation that CHEK2* pBCs do not harbor any somatic
TP53 mutations and have at least part of their biology in common with
TP53 mutant pBCs may not be completely surprising. Several studies in the past have found links between inactivation of CHEK2 and TP53 pathway signaling during tumorigenesis. However, results have often been conflicting, thus placing doubts on their validity. For example, in thymocytes from two different
Chk2−/− mouse models Chk2 seemed to regulate p53-dependent apoptosis [
14‐
16], but this was not confirmed in a knock-in
Chk2 c.1100delC mouse model [
17]. Moreover, before
CHEK2 c.1100delC was identified to be a moderate-risk BC susceptibility gene, it was actually a candidate gene for Li-Fraumeni syndrome [
1,
2,
52,
53], which is caused by germline mutations in
TP53 [
54,
55]. More recently, Boonen et al
. identified CHEK2-dependent phosphorylation of KAP1 p.S473 to be an excellent functional read-out for pathogenicity of germline
CHEK2 variants [
56]. Interestingly, KAP1 is a nuclear co-repressor that inactivates TP53 [
57]. Unfortunately, despite many links observed between CHEK2 and TP53, how precisely
CHEK2 c.1100delC could promote tumorigenesis through the TP53 pathway is still unclear. For this, functional studies in proper model systems (
i.e., ER+ human breast cells) are required.
Further supporting the shared biology between CHEK2* and
TP53 mutant BCs is the observation that CHEK2* pBCs had the highest WGD frequency among the subgroups, a feature enriched among
TP53 mutant cancers [
50]. In fact, WGD frequency of CHEK2* genomes was intermediate to
TP53 wild-type and mutant BCs, an observation fitting the incomplete penetrance of
CHEK2 c.1100delC. Considering the many roles of TP53 as well as CHEK2, and only a subset overlapping, not all roles these proteins fulfil will be relevant for tumorigenesis. Consistent though, with the high WGD frequency among CHEK2* pBCs, embryonic fibroblasts from knock-in
Chk2 c.1100delC mice showed an altered cell cycle distribution and a population of cells that are multinuclear, indicative of a cytokinesis defect [
17]. It may thus be interesting to subclassify WGD-positive cancers in those being multinucleated versus polyploid, since underlying causal mechanisms and thus players involved may be different.
Lack of somatic
TP53 mutation among CHEK2* BC genomes may also be interpreted as a lack of severity of
CHEK2 c.1100delC-driven BC instead of signaling through the TP53 pathway. However, consistent with literature [
5,
8,
10,
11,
46‐
49], we observed that BC patients with germline
CHEK2 c.1100delC or a somatic
TP53 mutation have an unfavorable clinical outcome compared to wild-type patients. In fact, we here show that
CHEK2 c.1100delC is an independent prognostic factor, whereas
TP53 mutation is an independent predictor of response to tamoxifen. This is in agreement with two previous studies showing the efficacy of chemotherapy or endocrine therapy is unlikely to account for the unfavorable survival of
CHEK2 mutation carriers [
11,
12]. However, considering the small group of
CHEK2 mutation carriers in the predictive cohort (
n = 13) and the similar overall response rates in
CHEK2 mutation carriers and patients with
TP53 mutations, power could have been an issue in this analysis. If proven irreproducible,
IGF1R could be an endocrine resistance gene to investigate further since
IGF1R overexpression has been associated with poor outcome and resistance to conventional BC therapies [
58].
Another key finding from our analyses was that CHEK2 BCs display a unique size distribution of SVs, most similar to, but significantly different from ER+ BCs. Considering previous reports associating genes with a specific SV size distribution, size distribution profiles can also be considered biological scars arising from specific mutational events. For example, combined inactivation of
TP53 and
BRCA1 produced TDs with an average length of 11 kb, while CCNE1 pathway activation and
CDK12 mutations generated TDs with an average length of 231 kb and 1.7 Mb, respectively [
59]. In addition, deletions in metastatic colorectal cancers were predominantly 10 kb to 1 Mb in size and frequently located in common fragile sites. Further analyses of breakpoints and localization of these deletions suggested transcription-dependent double-fork failure as an origin [
60]. Therefore, unravelling the underlying mechanism that generates the CHEK2-specific SV size distribution profile would be an important aspect of understanding how
CHEK2 c.1100delC promotes breast tumorigenesis. Despite the high chromothripsis frequency among CHEK2* BCs, chromothripsis did not appear to be the (sole) driver of the CHEK2-specififc SV size distribution profile. Also, a mechanistic overlap with previously published size distribution patterns is not evident [
59,
60].
CHEK2* BCs were most similar to ER+ BCs, even indistinguishable in some aspects, suggesting overlapping tumor evolution. Still,
CHEK2 c.1100delC carriers have a shorter survival and intrinsic tumor aggressiveness plays a role. To provide efficacious anti-cancer treatment and chemoprevention for these women, we need to identify the Achilles’ heel for CHEK2-driven tumorigenesis. We and others have by now firmly established that CHEK2 BCs do not display HRD and thus
CHEK2 mutation carriers will not benefit from PARP inhibitor therapy [
26,
61‐
63]. Moreover, because of the relatively low TMB we observed among CHEK2 BCs, these women are also not likely to benefit from immune checkpoint inhibitor therapy, but clinical trials investigating this are needed. The CHEK2-specific genomic features we identified here should therefore be further interrogated
in silico as well as propel further functional experiments to finally unravel the mechanism of CHEK2-driven tumorigenesis, thereby paving the way for personalized medicine for
CHEK2 mutation carriers.
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