Background
CHEK2 (Cell-cycle-checkpoint kinase 2, also known as
CHK2) encodes a multifunctional kinase that is activated mainly by the ataxia-telangiectasia mutated (ATM) protein in response to DNA double-strand breaks [
1-
4]. Activated
CHEK2 in turn phosphorylates several critical cell-cycle proteins, including p53, Cdc25 and BRCA1, which trigger cell-cycle arrest, apoptosis, and the activation of DNA repair [
5-
7].
Numerous studies have demonstrated that
CHEK2 is a moderate breast cancer susceptibility gene [
8-
12].
CHEK2 1100delC, a truncating mutation that abrogates the kinase activity of the protein, confers an approximately 2-fold increase in breast cancer risk [
8,
13-
15]. However, the prevalence of
CHEK2 1100delC mutation varies widely among ethnic groups [
8,
11,
16-
19]. The mutation is mostly found in the Dutch population [
8,
20], and it is absent or very rare in other populations [
16-
19]. We previously screened 2255 Chinese women (1027 breast cancer cases and 1228 healthy controls) for
CHEK2 1100delC and failed to find this mutation in this population. However, a novel recurrent
CHEK2 mutation near the
CHEK2 1100delC mutation,
CHEK2 1111C > T (H371Y), was found in Chinese women [
21].
CHEK2 H371Y is within the activation loop of the
CHEK2 protein kinase domain, which is essential for the activation of
CHEK2 in response to DNA damage. Functional analysis reveals that the
CHEK2 H371Y mutation produces a dramatic decline in
CHEK2 activity and is a pathogenic mutation [
21].
CHEK2 H371Y confers a 2.43-fold increase in breast cancer risk in Chinese women.
The disruption of
CHEK2 kinase activity may not only contribute to breast cancer development but also influence breast cancer survival or response to the adjuvant therapy. Two studies have suggested that the
CHEK2 1100delC mutation is associated with poor recurrence-free survival in breast cancer [
22,
23], indicating that patients with
CHEK2 1100delC mutation have an aggressive phenotype.
No previous studies have investigated the association between CHEK2 germline mutation and response to neoadjuvant chemotherapy in breast cancer. Therefore, in the current study, we investigated whether CHEK2 H371Y mutation carriers are more likely to respond to neoadjuvant chemotherapy in terms of pathologic complete response (pCR) in a large cohort of 2334 breast cancer patients who received neoadjuvant chemotherapy and further explored the association between CHEK2 H371Y mutation status and distant recurrence-free survival (DRFS).
Methods
Study population
A total of 2382 operable primary breast cancer patients with stage I-III were treated with neoadjuvant chemotherapy at the Breast Center of Peking University Cancer Hospital from October 2003 to December 2010. The mean age of the subjects was 47.6 years (range, 22–75 years). Tumor stage was classified according to the tumor-node-metastasis classification of the Union Internationale Contre le Cancer. Tumor size was defined as the maximum tumor diameter measured on the mammogram and/or ultrasonogram at the time of diagnosis. The tumors were graded according to the modified Bloom-Richardson system. Written consent was obtained from all subjects. This study was approved by the Research and Ethical Committee of Peking University Cancer Hospital.
CHEK2 H371Y germline mutations
Peripheral blood samples were collected from all patients. Genomic DNA was extracted from the leukocyte pellet by proteinase K digestion followed by phenol-chloroform extraction. The
CHEK2 H371Y mutation was detected by polymerase chain reaction (PCR) followed by denaturing high-performance liquid chromatography (DHPLC) and sequencing or directed sequencing as described previously [
21]. We screened all 2382 patients for the germline
CHEK2 H371Y mutation.
CHEK2 H371Y status was not readable for 46 patients, and 41 of the 2336 patients were found to carry the mutation. We then screened these 41
CHEK2 H371Y mutation carriers for germline mutations in
BRCA1/2; two patients also carried a
BRCA2 germline mutation and were excluded from this study. Therefore, 2334 patients, 39 of whom were
CHEK2 H371Y carriers, were included in the final analysis in the current study.
Estrogen receptor (ER), progesterone receptor (PR), and HER2 status
ER, PR, and HER2 status were determined in the core-needle biopsy breast cancer tissue obtained before the initiation of neoadjuvant chemotherapy as described previously [
24].
Neoadjuvant chemotherapy regimens
Among the 2334 patients who received neoadjuvant chemotherapy, 94% received 4–8 cycles. Treatments were categorized in three subgroups as follows:
(1)
859 patients received an anthracycline-based regimen. The detail of the regimens are described previously [
24]. Of these, 537 patients received a CTF regimen; 247 patients received an FEC regimen; 59 patients received a CAF regimen; the remaining 16 patients received other types of anthracycline regimens.
(2)
882 patients received an anthracycline-taxane containing regimen. Of these, 682 patients received two cycles of anthracycline followed by 4 cycles of paclitaxel alone (80 mg/m2 IV once per week for 12 weeks) or paclitaxel plus carboplatin (paclitaxel 175 mg/m2 IV on day 1 or paclitaxel 60 mg/m2 IV on day 1, day 8, and day 15, and carboplatin AUC 6 IV on day 1 every three weeks); 181 patients received 4 cycles of paclitaxel alone or docetaxel plus cyclophosphamide (docetaxel 75 mg/m2 IV on day 1 and cyclophosphamide 600 mg/m2 IV on day 1 every three weeks), followed by 2 to 4 cycles of anthracyclines. The remaining 19 patients received other types of anthracycline/taxane containing regimens, i.e., a TE regimen (docetaxel plus epirubicin) or TAC regimen (docetaxel, doxorubicin, and cyclophosphamide).
(3)
593 patients received a taxane-based regimen without anthracyclines. Of these, 494 patients received 4 cycles of paclitaxel (80 mg/m2 IV once a week for 12 weeks); 76 patients received paclitaxel plus carboplatin (paclitaxel 60 mg/m2 IV on day 1, day 8, and day 15, and carboplatin AUC 6 IV on day 1 every three weeks). The remaining 23 patients received docetaxel plus cyclophosphamide (docetaxel 75 mg/m2 IV on day 1 and cyclophosphamide 600 mg/m2 IV on day 1 every three weeks).
In this cohort of 2334 patients, 108 patients received intravenous trastuzumab in combination with neoadjuvant chemotherapy.
After completion of neoadjuvant chemotherapy, patients were treated with mastectomy (n = 1351) or breast-conserving surgery (n = 983) depending on the tumor size, presence of multiple lesions or patient preference. pCR was defined as the absence of invasive breast cancer cells in the breast after the completion of neoadjuvant chemotherapy [
25,
26].
Sixty-two percent of patients received adjuvant chemotherapy with the same or alternative regimens after operation; patients with axially positive lymph nodes and/or breast-conserving therapy received radiotherapy; patients with ER and/or PR-positive disease received endocrine therapy (20 mg/d tamoxifen for 5 years or 1 mg/d anastrozole for 5 years).
Statistical analysis
The differences in clinicopathological characteristics between CHEK2 H371Y carriers and non-carriers were determined by Pearson’s chi-squared test. The associations between CHEK2 H371Y mutation status, clinicopathologic characteristics, and pathological response to neoadjuvant chemotherapy were determined by Pearson’s chi-squared test or Fisher’s exact test when the number of patients was small. A logistic regression model was applied to determine whether a factor was an independent predictor of pCR in a multivariate analysis. Distant recurrence-free survival (DRFS) was defined as the time from the date of diagnosis to first distant recurrence (not including second primary malignancies) or death from breast cancer without a recorded relapse. Survival curves were derived from Kaplan–Meier estimates and compared using log-rank tests. All statistical tests were two-sided, and P values <0.05 were considered statistically significant. The statistical analyses were performed using SPSS 16.0 software (Chicago, IL, USA).
Discussion
In our previous study, we identified a novel recurrent
CHEK2 H371Y mutation in Chinese women, and this mutation decreases
CHEK2 activity and confers an approximately 2.4-fold increase in breast cancer risk [
21]. In the present study, we investigated the association between
CHEK2 H371Y and pathologic response in 2334 women who received neoadjuvant chemotherapy. To the best of our knowledge, this is the first study to report that
CHEK2 H371Y mutation carriers are more likely to respond to neoadjuvant chemotherapy than are non-carriers and that the H371Y mutation status was an independent favorable predictor of pCR in a multivariate analysis.
In the subgroup analyses, CHEK2 H371Y mutation carriers had a higher pCR rate than did non-carriers in both neoadjuvant anthracycline-based regimens and taxane-based regimens, although the difference did not reach statistical significance. However, the CHEK2 mutation carriers had a significantly higher pCR than did non-carriers among the subgroup of women who received neoadjuvant anthracycline/taxane-containing regimen.
CHEK2 is involved in various DNA damage responses, including cell-cycle checkpoints, genome maintenance, DNA repair and apoptosis [
27]. Anthracyclines induce double-strand DNA breaks [
28,
29], the repair of which is impaired in the deficiency of
CHEK2 protein [
21,
30,
31]. Tumor cells that express mutated
CHEK2 347 exhibited a 2-to 4-fold increase in apoptosis upon treatment with adrimycin [
6].
CHEK2 kinase activity is also required for proper mitotic spindle assembly and chromosome stability [
32], and
CHEK2 deficient lymphoma cells are more sensitive to taxol [
33]. In line with these findings, our results suggested that
CHEK2 H371Y was sensitive to both anthracycline and taxane.
Previous studies showed that breast cancer patients with the
CHEK2 1100delC mutation had a worse disease-free survival than did patients without this mutation [
22,
23]. One recent study suggested that breast cancer patients with
CHEK2 1100delC mutation had a worse survival beyond 6 years after diagnosis than did non-carriers [
34]. In the current study,
CHEK2 H371Y carriers showed a slightly poorer DRFS than non-carriers in the entire study cohort with 5-years, it is worth to investigate the survival impact of the
CHEK2 H371Y mutation in a long-term follow-up. However,
CHEK2 carriers or non-carriers who achieved a pCR had a significant better DRFS than those who did not, whereas
CHEK2 H371Y carriers who did not reach a pCR had the worst DRFS in the four subgroups. Although
CHEK2 H371Y carriers were more likely to respond to neoadjuvant chemotherapy, only small subset of mutation carriers achieved a pCR, the majority of
CHEK2 H371Y carriers who did not reach a pCR might have a particularly aggressive phenotype.
Germline
BRCA1 mutation carriers are sensitive to anthracycline or cisplatin neoadjuvant chemotherapy [
35-
37]; Byrski et al. recently reported that
BRCA1 mutation carriers are extremely sensitive to cisplatin-based neoadjuvant chemotherapy (pCR rate 61%, 65 out of 107 patients) [
38].
CHEK2 H371Y mutation may share some similarity to
BRCA1 mutation, therefore, an interest issue is to see whether
CHEK2 H371Y mutation carriers are responsive to cisplatin. Recent clinical trials showed that
BRCA1 mutation carriers are sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors [
39,
40]. In vitro studies suggested that tumor cells with silenced
CHEK2 expression showed an increased sensitivity to the PARP 1 inhibitor [
33,
41]. Therefore,
CHEK2 H371Y mutation carriers may be potential candidates for treatment with PARP1 inhibitors.
Although the entire study population is large, the number of individuals with CHEK2 H371Y is relatively small; particularly when the mutation carriers were stratified in several treatment groups, therefore, the results are premature and should be interpreted cautiously.
Nevertheless, our study suggests that patients with a deficiency of CHEK2 activity due to germline mutation, like H371Y, are sensitive to neoadjuvant chemotherapy. It would be of great interest to explore whether other CHEK2 germline mutations (i.e., CHEK2 1100delC) are similarly responsive to neoadjuvant chemotherapy.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conceived and designed the experiments: YX. Performed the experiments: YL YX. Analyzed the data: YL YX. Contributed reagents/materials/analysis tools: TO JL TW ZF TF BL. Wrote the paper: YX YL. All authors read and approved the final manuscript.