Correlation of CHEK2 protein expression and c.1100delC mutation status with tumor characteristics among unselected breast cancer patients

The CHEK2 kinase is a tumor suppressor whose activation in response to DNA double-strand breaks contributes to cell cycle arrest or apoptosis. The c.1100delC mutation is associated with familial breast cancer, and tumors from mutation carriers show reduced or absent CHEK2 protein expression. We have here studied CHEK2 protein expression by immunohistochemistry on a tissue microarray of 611 unselected breast tumors and also evaluated the tumor characteristics among 1,297 unselected breast cancer patients defined for the c.1100delC germ line mutation status (2.5% carrier frequency). CHEK2 protein expression was reduced in 21.1% of the unselected breast cancers studied. Tumors with reduced CHEK2 expression had more often larger primary tumor size (pT3-4; nominal significance p = 0.002) compared to tumors with normal staining. A similar trend for larger tumor size was seen among the 37 breast tumors from c.1100delC germ line mutation carriers. Tumors from c.1100delC mutation carriers were of higher grade than those of noncarriers (nominal significance p = 0.02). The c.1100delC germ line mutation also associated strongly with bilateral breast cancer. No significant correlation was seen between CHEK2 status and hormone receptor status, histology, lymph node status, or overall survival.     

Genetic and functional analysis of CHEK2 (CHK2) variants in multiethnic cohorts

The CHEK2-1100delC mutation is recurrent in the population and is a moderate risk factor for breast cancer. To identify additional CHEK2 mutations potentially contributing to breast cancer susceptibility, we sequenced 248 cases with early-onset disease; functionally characterized new variants and conducted a population-based case-control analysis to evaluate their contribution to breast cancer risk. We identified 1 additional null mutation and 5 missense variants in the germline of cancer patients. In vitro, the CHEK2-H143Y variant resulted in gross protein destabilization, while others had variable suppression of in vitro kinase activity using BRCA1 as a substrate. The germline CHEK2-1100delC mutation was present among 8/1,646 (0.5%) sporadic, 2/400 (0.5%) early-onset and 3/302 (1%) familial breast cancer cases, but undetectable amongst 2,105 multiethnic controls, including 633 from the US. CHEK2-positive breast cancer families also carried a deleterious BRCA1 mutation. 1100delC appears to be the only recurrent CHEK2 mutation associated with a potentially significant contribution to breast cancer risk in the general population. Another recurrent mutation with attenuated in vitro function, CHEK2-P85L, is not associated with increased breast cancer susceptibility, but exhibits a striking difference in frequency across populations with different ancestral histories. These observations illustrate the importance of genotyping ethnically diverse groups when assessing the impact of low-penetrance susceptibility alleles on population risk. Our findings highlight the notion that clinical testing for rare missense mutations within CHEK2 may have limited value in predicting breast cancer risk, but that testing for the 1100delC variant may be valuable in phenotypically- and geographically-selected populations.     

CHEK2 1100delC is a susceptibility allele for HNPCC-related colorectal cancer

PURPOSE: The pathogenic CHEK2 1100delC variant is firmly established as a breast cancer susceptibility allele. Dutch CHEK2 1100delC breast cancer families frequently also include colorectal cancer cases, and the variant is particularly prevalent among breast cancer families with hereditary breast and colorectal cancer. Yet, it is still unclear whether CHEK2 1100delC also confers a colorectal cancer risk independent of its breast cancer risk. EXPERIMENTAL DESIGN: CHEK2 1100delC was genotyped in the index cases of 369 Dutch colorectal cancer families that had been excluded for familial breast cancer. The cohort included 132 cases with familial adenomatous polyposis (FAP) and FAP-related disease, and 237 cases with hereditary nonpolyposis colorectal cancer (HNPCC) and HNPCC-related disease. RESULTS: None of the FAP/FAP-related cases carried the CHEK2 1100delC variant. In contrast, CHEK2 1100delC was present in 10 of 237 (4.2%) HNPCC/HNPCC-related cases that was significantly more prevalent than the 1.0% Dutch population frequency (odds ratio, 4.3; 95% confidence interval, 1.7-10.7; P = 0.002). Nine of the 10 CHEK2 1100delC colorectal cancer cases met the revised Amsterdam and/or Bethesda criteria. The 10 CHEK2 1100delC colorectal cancer families had a high-risk cancer inheritance pattern, including 35 colorectal cancer cases, 9 cases with polyps, and 21 cases with other tumor types. CONCLUSION: Our analysis provides strong evidence that the 1100delC variant of CHEK2 confers a colorectal cancer risk in HNPCC/HNPCC-related families, supporting the hypothesis that CHEK2 is a multiorgan cancer susceptibility gene.     

CHEK2*1100delC does not contribute to risk to breast cancer among Malay, Chinese and Indians in Malaysia

A truncating mutation (1100delC) in the cell cycle checkpoint kinase-2 gene, CHEK2, has been identified as a risk factor for familial and sporadic breast cancer in some Northern and Western European populations. However, the prevalence of CHEK2*1100delC in breast cancer appears to be population dependent. We analysed the prevalence of CHEK2*1100delC in 668 breast cancer cases, of which 542 were invasive breast cancers, from a hospital-based cohort of breast cancer patients from Kuala Lumpur, Malaysia. The variant was not found in any patients in this cohort, suggesting that CHEK2*1100delC is rare in our population, and unlikely to contribute significantly to risk to breast cancer among the Malay, Chinese and Indian ethnic groups in Malaysia. This suggests that screening for this allele should not be routinely conducted in Malaysia.     

A multicenter study of cancer incidence in CHEK2 1100delC mutation carriers.

The CHEK2 1100delC protein-truncating mutation has a carrier frequency of approximately 0.7% in Northern and Western European populations and confers an approximately 2-fold increased risk of breast cancer. It has also been suggested to increase risks of colorectal and prostate cancer, but its involvement with these or other types of cancer has not been confirmed. The incidence of cancer other than breast cancer in 11,116 individuals from 734 non-BRCA1/2 breast cancer families from the United Kingdom, Germany, Netherlands, and the United States was compared with that predicted by population rates. Relative risks (RR) to carriers and noncarriers were estimated by maximum likelihood, via the expectation-maximization algorithm to allow for unknown genotypes. Sixty-seven families contained at least one tested CHEK2 1100delC mutation carrier. There was evidence of underreporting of cancers in male relatives (422 cancers observed, 860 expected) but not in females (322 observed, 335 expected); hence, we focused on cancer risks in female carriers. The risk of cancers other than breast cancer in female carriers was not significantly elevated, although a modest increase in risk could not be excluded (RR, 1.18; 95% confidence interval, 0.64-2.17). The carrier risk was not significantly raised for any individual cancer site, including colorectal cancer (RR, 1.60; 95% confidence interval, 0.54-4.71). However, between ages 20 to 50 years, the risks of colorectal and lung cancer were both higher in female carriers than noncarriers (P = 0.041 and 0.0001, respectively). There was no evidence of a higher prostate cancer risk in carriers than noncarriers (P = 0.26), although underreporting of male cancers limited our power to detect such a difference. Our results suggest that the risk of cancer associated with CHEK2 1100delC mutations is restricted to breast cancer, although we cannot rule out a small increase in overall cancer risk.     

<Emphasis Type="Italic">CHEK2</Emphasis> 1100delC and male breast cancer in the Netherlands

Mutations in the breast cancer susceptibility genes BRCA1, BRCA2, and CHEK2 are known risk factors for female breast cancer. Mutations in BRCA1 and BRCA2 also are associated with male breast cancer (MBC). Similarly, it had been suggested in the original CHEK2 identification report that the CHEK2 1100delC mutation confers an increased risk for MBC. Here, we have evaluated the risk of CHEK2 1100delC for MBC by genotyping CHEK2 1100delC in 23 familial and 71 unselected Dutch MBC cases. None of the 23 familial MBC cases carried the CHEK2 1100delC mutation. In contrast, CHEK2 1100delC was present in 3 of the 71 (4.2%) unselected MBC cases, which was significantly more prevalent than the 1.1% Dutch population frequency assessed in 1,692 individuals (P = 0.05, OR = 4.1, 95% CI 1.2–14.3). Our data suggest that, in the Netherlands, CHEK2 1100delC is associated with an increased risk for MBC.     

CHEK2 1100delC is not a risk factor for male breast cancer population

Genetic risk factors for male breast cancer (MBC) are poorly understood. High penetrance genes such as BRCA1 or BRCA2 account for only a small proportion of the disease. A 1100delC mutation in CHEK2 (previously known as CHK2), a cell-cycle checkpoint kinase, has been implicated in predisposition of Li-Fraumeni syndrome (LFS) and breast cancer in families suggestive of LFS. This 1100delC mutation has also been shown to confer a 2-fold increase of breast cancer risk in women and a 10-fold increase of risk in men. It was estimated to account for 1% of breast cancers in women and as much as 9% of breast cancers in men at the population level based on analysis of breast cancer families without BRCA1 or BRCA2 mutations. We wanted to evaluate the significance of CHEK2 1100delC in predisposition to MBC by assessing its frequency in a population-based material of 114 Finnish MBC patients. Two patients (1.8%) carried the 1100delC mutation. The mutation frequency among MBC cases was similar to that seen in population controls (26/1885, 1.4%). Our results indicate that CHEK2 1100delC variant does not substantially increase the risk of male breast cancer at the population level. We cannot exclude the fact that a small fraction of hereditary, family-positive male breast cancers could be attributable to CHEK2 mutations.     

Frequency of CHEK2 mutations in a population based, case-control study of breast cancer in young women

Introduction  

The cell-cycle checkpoint kinase (CHEK)2 protein truncating mutation 1100delC has been associated with increased risk for breast or prostate cancer. Multiple studies have found an elevated frequency of the 1100delC variant in specific stratifications of breast cancer patients with a family history of the disease, including BRCA1/BRCA2 negative families and families with a history of bilateral disease or male breast cancer. However, the 1100delC mutation has only been investigated in a few population-based studies and none from North America.     

Risk for contralateral breast cancer among carriers of the CHEK2*1100delC mutation in the WECARE Study

The protein encoded by the CHEK2 gene is involved in cellular repair of DNA damage. The truncating mutation, CHEK2*1100delC, seems to increase the risk for breast cancer. We investigated whether the CHEK2*1100delC mutation carrier status increases the risk for asynchronous contralateral breast cancer (CBC) and whether it interacts with radiation therapy (RT) or chemotherapy in regard to CBC risk. The germline mutation frequency was assessed in 708 women with CBC and 1395 women with unilateral breast cancer (UBC) in the Women's Environment, Cancer and Radiation Epidemiology (WECARE) Study whose first primary breast cancer was diagnosed before age 55 years and during 1985--1999. Seven women with CBC (1.0%) and 10 women with UBC (0.7%) were CHEK2*1100delC variant carriers (rate ratio (RR)=1.8, 95% confidence interval (CI)=0.6-5.4 for CBC vs UBC). Carriers who received RT for their first breast cancer, compared with non-carriers not treated with RT, had an RR of developing CBC of 2.6 (95% CI=0.8-8.7). We found no significant associations between the CHEK2*1100delC mutation and CBC overall or among those treated with RT. However, the sampling variability was such that modest increases in risk could not be excluded. Nonetheless, because this is a rare mutation, it is unlikely to explain a major fraction of CBC in the population.     

German populations with infrequent CHEK2*1100delC and minor associations with early-onset and familial breast cancer

CHEK2*1100delC is associated with a twofold increased breast cancer risk. This was shown in a collaborative analysis of European populations, but not in other populations from Europe and the US. Accordingly, there is a need to clarify the role of CHEK2*1100delC in breast cancer.We established its prevalence in two German populations GENICA (Northrhine-Westphalia, n = 724) and KORA (Bavaria, n = 600) and in women with breast cancer. The latter included cases (n = 688) from the GENICA breast cancer case-control study, patients with early-onset breast cancer (n = 86) and patients with familial breast cancer (n = 71). The latter patient groups were previously investigated for BRCA1/2-mutations and tested negative. Mutation analysis was performed by combined PCR/DHPLC methodology.CHEK2*1100delC was found in 0.9% of GENICA controls and was absent in the KORA controls indicating a significant difference between the two populations (P = 0.03). The frequency of CHEK2*1100delC in age-matched cases of the GENICA collection was 0.8% and thus not different from controls (OR 0.88, 95% CI 0.21–3.50). In patients with early-onset disease CHEK2*1100delC was found at a frequency of 2.3% referring to an increased breast cancer risk of 2.56 (95% CI 0.25–14.58). In patients with familial disease the frequency was 1.4% referring to an increased risk of 1.53 (95% CI 0.03–12.93).Our data showed variations in CHEK2*1100delC prevalence within German populations suggesting possible inaccuracies in breast cancer risk assessments from non population-based studies. In patients with a high-risk profile however, CHEK2*1100delC was indicative for this risk and highest for early-onset breast cancer.     

Genomic large rearrangement screening of BRCA1 and BRCA2 genes in high-risk Turkish breast/ovarian cancer patients by using multiplex ligation-dependent probe amplification assay

In this study, MLPA assay was performed for detection of large rearrangements of BRCA1 and BRCA2 genes in 16 familial, 29 early onset, 3 male breast cancer, and 2 bilateral breast/ovarian cancer high risk Turkish index cases. MLPA assay for all exons of both genes and for 1100delC variant of CHEK2 gene were performed. Analyses, revealed no large genomic rearrangements in both genes, and, no 1100del variant in CHEK2 gene. Our data which represents the first results for Turkish patients, suggest that, the frequency of BRCA1 and BRCA2 genes' large rearrangements is very low.     

Gene expression profiling assigns CHEK2 1100delC breast cancers to the luminal intrinsic subtypes

CHEK2 1100delC is a moderate-risk cancer susceptibility allele that confers a high breast cancer risk in a polygenic setting. Gene expression profiling of CHEK2 1100delC breast cancers may reveal clues to the nature of the polygenic CHEK2 model and its genes involved. Here, we report global gene expression profiles of a cohort of 155 familial breast cancers, including 26 CHEK2 1100delC mutant tumors. In line with previous work, all CHEK2 1100delC mutant tumors clustered among the hormone receptor-positive breast cancers. In the hormone receptor-positive subset, a 40-gene CHEK2 signature was subsequently defined that significantly associated with CHEK2 1100delC breast cancers. The identification of a CHEK2 gene signature implies an unexpected biological homogeneity among the CHEK2 1100delC breast cancers. In addition, all 26 CHEK2 1100delC tumors classified as luminal intrinsic subtype breast cancers, with 8 luminal A and 18 luminal B tumors. This biological make-up of CHEK2 1100delC breast cancers suggests that a relatively limited number of additional susceptibility alleles are involved in the polygenic CHEK2 model. Identification of these as-yet-unknown susceptibility alleles should be aided by clues from the 40-gene CHEK2 signature.     

Identification of a Novel BRCA2 and CHEK2 A-C-G-C Haplotype in Turkish Patients Affected with Breast Cancer

Background: Many breast cancers are caused by certain rare and familial mutations in the high or moderatepenetrance genes BRCA1, BRCA2 and CHEK2. The aim of this study was to examine the allele and genotypefrequencies of seven mutations in BRCA1, BRCA2 and CHEK2 genes in breast cancer patients and to investigatetheir isolated and combined associations with breast cancer risk. Methods: We genotyped seven mutations inBRCA1, BRCA2 and CHEK2 genes and then analyzed single variations and haplotype associations in 106 breastcancer patients and 80 healthy controls. Results: We found significant associations in the analyses of CHEK2-1100delC (p=0.001) and BRCA1-5382insC (p=0.021) mutations in breast cancer patients compared to controls.The highest risk was observed among breast cancer patients carrying both CHEK2-1100delC and BRCA2-Met784Val mutations (OR=0.093; 95%CI 0.021-0.423; p=0.001). We identified one previously undescribedBRCA2 and a CHEK2 four-marker haplotype of A-C-G-C which was overrepresented (χ2=7.655; p=0.0057)in the patient group compared to controls. Conclusion: In this study, we identified a previously undescribedBRCA2 and CHEK2 A-C-G-C haplotype in association with the breast cancer in our population. Our resultsfurther suggest that the CHEK2-1100delC mutation in combination with BRCA2-Met784Val may lead to anunexpected high risk which needs to be confirmed in larger cohorts in order to better understand their role inthe development and prognosis of breast cancer.     

Excess Risk for Contralateral Breast Cancer in CHEK2*1100delC Germline Mutation Carriers

We detected a significant excess risk for CHEK2*1100delC mutation carriers to develop a contralateral breast tumor, OR = 6.5 (95% CI 1.5-28.8, p = 0.005). The highest percentage of mutation carriers was detected among those bilateral breast cancer patients who had received radiation treatment for their first breast tumor. These results warrant prolonged medical surveillance and may indicate a clinically important interaction between CHEK2 heterozygosity and radiation in the development of contralateral breast cancer.     

CHEK2 variant I157T may be associated with increased breast cancer risk

Cell cycle checkpoint kinase 2 (CHEK2) is a transducer of cellular responses to DNA damage. The CHEK2 1100delC has previously been shown to be a low-penetrance breast cancer susceptibility allele. We have evaluated the role of another CHEK2 variant, I157T in the FHA domain of the gene, for association with breast cancer. I157T was found at a significantly higher frequency in the population-based series of breast cancer patients (77/1035, 7.4%, odds ratio [OR] = 1.43, 95% confidence interval [CI] = 1.06-1.95, p = 0.021) than among population controls (100/1885, 5.3%). The frequency in the familial breast cancer patients was not elevated (28/507, 5.5%, OR = 1.04, 95% CI = 0.68-1.61). The I157T protein, that undermines cellular responses to ionizing radiation and shows deficiency in substrate recognition in vivo, was expressed at normal level in tumor tissues as well as in cultured cells. The I157T protein was stable and it dimerized with the wild-type CHEK2 co-expressed in human cells. These functional properties of the I157T protein suggest that this variant may have negative effect on the pool of normal CHEK2 protein in heterozygous carrier cells by formation of heterodimers with wild-type CHEK2. The I157T variant may be associated with breast cancer risk, but the risk is lower than for 1100delC.     

The breast cancer low-penetrance allele 1100delC in the CHEK2 gene is not present in Spanish familial breast cancer population

Searching for low-penetrance genes involved in breast cancer susceptibility has been a field of interest in the last few years. Recently, the CHEK 2 gene, involved in DNA damage and replication checkpoints, has been pointed out as a good candidate; moreover, a specific variant in this gene,1100delC, has been found to increase breast cancer susceptibility among familial breast cancer cases not attributable to mutations in BRCA1 or BRCA2 genes. In our present study, we evaluated the role of the 1100delC variant as a susceptibility allele in breast cancer in the Spanish population. However, our results suggest that this variant is absent or very infrequent in our population, making its screening irrelevant from the practical point of view.     

Evaluation of variants in the CHEK2, BRIP1 and PALB2 genes in an Irish breast cancer cohort

It has been proposed that rare variants within the double strand break repair genes CHEK2, BRIP1 and PALB2 predispose to breast cancer. The aim of this study was to evaluate the prevalence of these variants in an Irish breast cancer cohort and determine their contribution to the development of breast cancer in the west of Ireland. We evaluated the presence of CHEK2_1100delC variant in 903 breast cancer cases and 1,016 controls. Six previously described variants within BRIP1 and five within PALB2 were screened in 192 patients with early-onset or familial breast cancer. Where a variant was evident, it was then examined in the remainder of our 711 unselected breast cancer cases. CHEK2_1100delC was found in 5/903 (0.5%) breast cancer cases compared to 1/1016 (0.1%) controls. One mutation at BRIP1 (2392 C>T) was identified in the early-onset/familial cohort. Examination of this variant in the remainder of our cohort (711 cases) failed to identify any additional cases. None of the previously described PALB2 variants were demonstrated in the early-onset/familial cohort. We show evidence of CHEK2_1100delC and BRIP1 2392 C>T within the Irish population. CHEK2_1100delC and BRIP1 mutations incidence in Ireland is similar to that found in other unselected breast cancer cohorts from northern European countries. We found no evidence to suggest that PALB2 mutation is an important breast cancer predisposition gene in this population.     

Limited relevance of the CHEK2 gene in hereditary breast cancer

To establish the importance of CHEK2 mutations for familial breast cancer incidence in the German population, we have screened all 14 of the coding exons in 516 families negative for mutations in both the BRCA1 and BRCA2 genes. We found 12 distinct variants in 30 unrelated patients (5.81%), including 5 that are novel and an additional 4 found for the first time in breast cancer. These aberrations were evaluated in 500 healthy women aged over 50 years and in the case of the 2 exon 10 mutations, 1100delC and 1214del4bp, in 1315 randomized healthy controls. According to our results, a statistically significant association for the exon 10 mutations was observed (p = 0.006). The prevalence of the 1100delC mutation in the German population, however, is significantly lower than those reported for other Caucasian populations both in familial breast cancer patients (1.6%) and controls (0.5%), and shows independent segregation with breast cancer in 2 of 4 families analyzed. The remaining 10 variants were more abundant in patients (21) compared to the controls (12) although the difference was not statistically significant. Interestingly, we found no increased breast cancer risk associated with the splice site mutation IVS2+1G-->A or the most common missense mutation I157T, which account for more than half (12/21) of the variants observed in patients. The low prevalence and penetrance of the exon 10 deletion mutations together with no, or an uncertain elevation in risk for other CHEK2 mutations suggests a limited relevance for CHEK2 mutations in familial breast cancer. Further evaluation of the unique variants observed in breast cancer is required to determine if they may play a role in a polygenic model of familial breast cancer. Nevertheless, it seems premature to include CHEK2 screening in genetic testing.     

BRCA1, BRCA2 and CHEK2 (1100 del C) Germline Mutations in Hereditary Breast and Ovarian Cancer Families in South India

Cancer of the breast is the second most common cancer seen among Indian women. This study describes the use ‍of DHPLC for mutation analysis for BRCA1, BRCA2 and CHEK2 (1100delC) in 22 patients with a family history of ‍breast and/or ovarian cancer and early onset breast cancer (<35 years of age). Three of the 22 patients were found to ‍have a non-sense mutation or a deletion, resulting in a premature stop codon, potentially leading to a truncated ‍protein. Two of these were in BRCA1 (one was a novel 5 base deletion) and one in the BRCA2 gene. No patient was ‍found in our series to have the CHEK2 (1100delC) mutation. DNA from a healthy blood donor and all but one of the ‍22 patients, demonstrated polymorphisms in BRCA1 and/or BRCA2 genes. This is the first study from South India, ‍on BRCA1, BRCA2 & CHEK2 (1100 del C) mutations in patients with a family history of breast and/or ovarian ‍cancer and early onset breast/ovarian cancer, using the sensitive DHPLC approach.