Breast cancer morbidity has increased steadily in China in the last decade. Although the same treatment regimens are used for Chinese patients as in western countries, little is known that whether the specific biomarkers used to guide treatment methods in western countries are also useful to guide treatment in China. Currently, gene expression profiles and biomarkers are used to predict prognosis and recurrence risk1-5 However, these biomarkers cannot identify high or low recurrence risks in all patients. For example, HER2 is one of the most important prediction factors, but only 25%-30% of breast cancer with human epidermal growth factor 2 (HER2) positive6. It is unknown whether there are other molecular markers that could be used to predict prognosis and recurrence in HER2 negative patients.
Recent studies have shown that RAS pathway-related proteins such as epidermal growth factor receptor 1 (EGFR1),2 HER2 and protein kinase AKT3, and cell cycle regulation, senescence proteins such as cyclin A, D1 and E1, CDK2, CDK4 and CDK6, are all associated with tumor growth7-9 We recently found increased cyclin A2 in breast cancer and normal breast tissue at both mRNA and protein levels10. However, the role of cyclin A2 in breast cancer is still unclear.11,12 It has also been shown that high cyclin A2 expression is independently associated with improved survival in colon cancer.13 Therefore, in this study we investigated whether cyclin A2 can serve as a prognostic factor for breast cancer. Identification of a recurrence biomarker in HER2 negative patients could improve selection of treatment regimens, and might also be useful as a target for the development of new treatments.
Records of 281 Chinese patients diagnosed with primary invasive breast cancer between 1999 and 2007 were retrieved from the archived database at the Peking University Third Hospital, Beijing, China. The demogra-phics and tumor characteristics are shown in Table 1. Patients follow-up consisted of clinical examinations with laboratory tests and imaging studies every 3 months for the first 2 years, every 6 months for the following 3 years, and then once every year. There were 51 patients with recurrences and 230 patients without recurrences. Benign breast lumps from 60 patients were used as normal breast tissue controls. Each patient signed informed consent to participate in the study, which was approved by the Ethics and Scientific Committees of Peking University.
Tissues for gene analysis and RT-PCR
Samples for gene analysis and RT-PCR were obtained from the tissue bank in Peking University Third Hospital. The tissues were from 6 patients with recurrences, 6 without recurrences for 3 years post operative, and 5 patients who had benign disease but underwent surgery (as the normal controls). Tumor tissues were treated with hematoxylin and eosin staining, and analyzed by an experienced breast pathologist. Eligible samples had to include at least 90% of the tumor cells, and were quickly frozen in liquid nitrogen when the tissue was collected.
Immunohistochemical staining for cyclin A2, HER2, estrogen receptors and progesterone receptors
The detailed procedures of the immunohistochemical staining with the SABC method were described as previously reported.12 The detailed information of the patients was shown in Table 1. The expression of cyclin A2, HER2, estrogen receptors (ER) and progesterone receptors (PR) in the primary tumors were detected using monoclonal antibodies for cyclin A2 (Neomarkers, 1:20 dilution, clone 6E6), HER2 (DAKO, 1:400 dilution, clone A0458), ER (ZETA, 1:400, SP-1), and PR (ZETA, 1:400, SP-2), respectively. Cyclin A2 expression in 60 normal breast tissues was also examined. The final value of protein expression was evaluated independently by two pathologists.
Evaluation of immunohistochemistry results
Cyclin A2, ER and PR immunoreactivities were evaluated semiquantitatively according to the percentage of positive cells showing distinct nuclear immunohistochemical reactions. Nuclear immunoreactivities were assessed in at least 5 high-power fields by counting 200 cells. Protein expression was divided into 4 levels with the following categories: 0-5% cell are stained (0), 6%-25% (1+), 26%-50% (2+), >50% (3+). As tumors showed heterogeneous staining, the dominant pattern was used for scoring. A cut-off value were defined if more than 5% of the cells stained was positive, while less than 5% was considered negative.11-13
The intensity of HER2 immunoreactivity on tumor cell membrane was scored using a widely accepted assessment system. The criteria are as follows: score 0, any detectable membrane signal in <10% of the tumor cells; score 1, weak incomplete membrane staining in >10% of the tumor cells; scores 2 and 3, moderate to strong membrane positivity in >10% of the tumor cells. Tumors with scores 0 or 1 were defined as negative, and those with a score of 2 or 3 were defined as positive14.
Gene expression analysis
RNA was isolated from tumor biopsy samples with Trizol (Invitrogen, Carlsbad, CA, USA) followed by further purification using the RNA Clean-up Kit. An equal amount of RNA isolated from normal breast tissue was used as the control. For gene expression profiling, the human genome 70-mer oligonucleotide microarray (CapitalBio, Beijing, China) was used following the protocol described previously.15 To identify differentially expressed genes, the Significance Analysis of Microarrays (SAM, version 3.0, CapitalBio, Beijing, China) analysis were performed using the two-class unpaired comparison method.
Quantitative real-time RT-PCR analysis
To validate the microarray results, quantitative reverse transcription-PCR (qRT-PCR) was performed using the LightCycler II Real-Time PCR System (Roche) and LightCycler-FastStart DNA master Plus SYBR Green I PCR kit, SYBR Green I PCR kit (Roche) in a Roche LightCycler II Real-Time PCR System according to the operator's manual. Cyclin A2 was measured in each specimen. The primer sequences used in this study were designed with the Premier 5.0 software (Premier, Canada) and synthesized at Invitrogen16. The sequence of the reverse primer was 5′-CAGGCTGCTGATGCAG-AAAG-3′ and the forward primer had the sequence of 5′-GAACAGACCAACCTGGCATTA-3′. The expression level of each gene was normalized with the expression level of the housekeeping gene GAPDH.
The correlations between protein expression levels and survival were analyzed. The time from therapy to the day of the first recurrence, either regionally or distantly was defined as a disease-free survival (DFS), and the time from thrapy to death was defined as an overall survival (OS); DFS and OS curves for the subgroups of patients were constructed using the Kaplan-Meier product limit estimation method and were compared by the Log-rank test to provide a univariate assessment of the prognostic value of selected clinical risk factors, which were measured at the time of entry into treatment. A value of P <0.05 was considered statistically significant. Cox proportional hazard ratios were assigned with confidence limits from parameter estimates. The consistency of cyclin A2 and HER2 was analyzed and the value of consensus was defined at 0.7-1.0. The SPSS 15.0 (SPSS Inc, Chicago, USA) software was used for the analysis.
Immunohistochemical staining of cyclin A2 and HER2
Immunohistochemical staining of cyclin A2 were negative in 91.6% and weakly positive (level 1+) in 8.3% of the 60 patients with benign breast lumps. We also observed that cyclin A2 staining was negative in the surrounding noncancerous tumor cells of invasive breast cancer samples. In the breast cancer specimens, the percentage of cells with positive cyclin A2 expression ranged from 0 to more than 80%, and strong positive staining was present in both nucleus and cytoplasm. The cyclin A2+ cells frequently appeared in clusters with heterogeneous staining. Of the tumor samples, 76.5% showed strong immunoreactivity to cyclin A2 (Figures 1 and 2). For the expression of HER2, there were 39 patients at level 0, 78 patients at level 1+, 75 at level 2+, and 88 at level 3+. Therefore, HER2 was highly overexpressed in 31.3% of the patients (88/281). Detailed staining results were shown in Table 1. One sample failed to show either cyclin A2 or HER2 staining.
Gene expression analysis and RT-PCR amplification of cyclin A2
We identified 82 genes that were upregulated and 92 genes downregulated. Cyclin A2 (CCNA2) was upregulated in all 11 examined invasive breast cancer tissues (one sample failure for detection) compared with the normal breast tissues; the average increase was 3.1-fold. Moreover, the increase of cyclin A2 expression in patients with and without recurrence were 3.7- and 2.7-fold, respectively (Figure 3 A-C). RT-PCR confirmed that the gene expression profile of cyclin A2 was increased by 2.1-fold in the tumors compared with the normal tissue, and 2.5-fold in the tumors with recurrence compared with those without recurrence.
Prognostic value of cyclin A2 and HER2 for breast cancer patients
The median follow-up time was 33 months (ranging from 8 to 103 months). Fifty-one of the 281 patients (18.1%) relapsed and 25 patients (8.8%) died due to disease progression. All of the surviving patients were followed up more than 12 months, and 50.8% over 3 years. We divided patients into four groups according to cyclin A2 expression level; the percentage of each group were listed in Table 1. We observed a statistically significant correlation between DFS and cyclin A2 expression by Log rank test (P=0.047). However, no significant correlation was observed between cyclin A2 expression and OS (P=0.6, Figure 4A). Moreover, we also observed that the HER2 expression was strongly associated with DFS (P=0.05, Figure 4B). The patients in this study did not receive trastuzumab treatment. We observed that relapse and death were more frequent in patients with intermediate cyclin A2 expression levels. Recurrence rates were 7.6%, 21.8%, 25.3% and 10% in cyclin A2-, levels 1+, 2+ and 3+, respectively (Table 2). A similar phenomenon was observed with HER2 as well. We did not perform subgroup analysis because of the small number of samples.
ER and PR expression and clinical outcome
The ER and PR levels were obtained from regular pathology diagnosis reports (Table 1). Expression levels of immunohistochemical evaluations of ER and PR in 281 patients were presented in Table 1. ER and PR expression levels were also significantly correlated with DFS (P=0.024 and 0.009, respectively), but neither was correlated with OS (Figure 4C and D).
Multivariate analysis and Kappa test
In unvaried analysis, biomarkers cyclin A2, HER2, ER and PR were significantly associated with DFS in the entire patient cohort. In multivariate analysis including cyclin A2, HER2, ER, PR, and lymph node status, no significant correlation between any of the factors and DFS except lymph node status (P=0.026,Table 3). Cyclin A2 and HER2 were not consistent when we performed Kappa testing with the value of 0.37, suggesting that cyclin A2 and HER2 are independently associated with DFS.
Cyclin A2 subgroups analysis based on HER2 status
For the 117 patients with negative HER2 expression (level 0 or 1+ detected by immunohistochemical staining), the cyclin A2 expression level was not significantly correlated with DFS (P=0.34), but patients with different levels of cyclin A2 did show a difference in survival.
By analyzing the correlation between biomarkers and the clinical outcome and long-term follow-up data of well-controlled patients with and without relapse, we found that cyclin A2 expression was increased in early recurrent breast cancer patients from a small tissue sample. We further found that there was a significant difference in the cyclin A2 levels of tumors with different grades of malignancy in a large number of patients. The recurrence rate occurred more frequently in cyclin A2+ patients (21.4%) compared with cyclin A2- patients (7.6%). Furthermore, we observed that DFS was longer in patients with the highest expression of cyclin A2, compared with patients with low and median levels of cyclin A2 expression. Recurrence rates were 21.8%, 25.3%, and 10% in patients with cyclin A2 expression at levels 1+, 2+, and 3+, respectively. No subgroup analysis was done due to the limited number of patients, but there was an obvious trend of lower recurrence rate in patients with the highest level of cyclin A2. When patients who express very high cyclin A2 were not removed in statistic analysis, the prognostic role of cyclin A2 would be obscured. The different cutoff values used could be one of the reasons for the conflicting results that have been obtained in several studies.11-13 Patients expressing cyclin A2 were reported had longer median overall survival periods and times to progression, and better responses to chemotherapy.17,18
Previously, cyclin A2 was considered to be a typical co-expression protein with proliferation markers such as proliferative cell nuclear antigen and ki67.19 Our results suggest that cyclin A2 is not only a cell proliferation marker, but also could be a prognostic factor.
In previous studies, 1:300 or 1:100 dilution of the cyclin A2 antibody was used and the obtained average expression rate was 14.5% (range from 1.2% to 45%). In our study, we used 1:20 dilution and the positive expression range was from 5% to 80%. Under this higher concentration of cyclin A2 antibody, the detected cyclin A2 expression in normal breast tissues and tissues surrounding the tumor cells were negative, indicating that the increased cyclin A2 antibody concentration did not lead to false positive detection of cyclin A2. Another aspect of this study was that we used the entire paraffin-embedded tumor slides instead of tissue microarrays for immunostaining to minimize the effect of heterogeneity of tumor when only a small number of tumor tissues were studied. Therefore, it was possible for us to observe cyclin A2 expression in different regions of the tissue.20,21
Our results confirmed that HER2 is a strong prognostic factor. We observed no significant correlation between cyclin A2 and HER2 when Kappa analysis was performed; the Kappa value was 0.37. This result suggested that cyclin A2 could be used as a biomarker to complement the additional function of HER2, especially when HER2 expression is negative. For patients with HER2 negative, 26% of them were at cyclin A2 expression levels 1+ and 2+; their recurrence rates were 21.8% and 25.3%, respectively. For patients with HER2 expression at level 1+, 22% of them were at cyclin A2 level 2+; their recurrence rate was 25.3%. In the clinical practice, patients with levels negative and 1+ of HER2 were treated the same way. Our results suggested that patients with cyclin A2 expression at levels of 1+ or 2+ should be treated more aggressively, as the same as the patients with the highest HER2 expression, even though their HER2 expression is negative.
We also observed that patients with HER2 expression at level 3+ showed lower recurrence rates than patients with HER2 expression at level 2+, similar to cyclin A2. A majority of reports have shown that HER2 overexpression is associated with a higher risk of relapse. The 10-year relapse-free survival (RFS) rates for HER2 overexpressing tumors and non-overexpressing tumors were 65.9% and 75.1%, respectively (P = 0.01). A worse outcome is observed in patients with HER2+ tumors compared with HER2- tumors for 10-year RFS (65.9% vs. 75.5%, P< 0.01) and distant RFS (71.2% vs. 81.8%, P < 0.004). In our study, the majority of patients (75.4%) received adjuvant therapy including taxanes and anthracyclines. But when the type of chemotherapy was taken into consideration, regimens that included different agents reversed the visual trend of relapse rate, with fewer relapses in patients of HER2 2+ (26.6%) and 3+ (20.5%) than in the HER2 1+ (27.7%) and 0 (28.5%). Treatment with anthracyclines alone decreased the relapse rate in the HER2 3+ patients (23.9%) compared with HER2 2+ patients (30.2%). In our study, a majority of patients (75.4%) received adjuvant therapy including taxanes and anthracyclines. Such high sensitivity to doxorubicin is not surprising since HER2 is frequently coexpressed with topoisomerase II, a known target of anthracyclines. This could be a possible explanation for the lower relapse rate of patients with the highest levels of HER2.22-25
Cyclin A2 and HER2 might be independent prognostic factors. We performed Kaplan-Meier analysis on ER and PR as a control, a positive prognostic correlation was obtained, suggesting the patients in this study were valid for correlation analysis. All these markers correlated with DFS independently. However, the correlations of cyclin A2, HER2, ER and PR with DFS no longer significant, except for lymph node status, when multivariate analysis was performed, emphasizing the need to include multiple factors.
In summary, we showed that both cyclin A2 and HER2 can predict the high risk of recurrence, and that the prediction is more effective when they are combined together. The expression of cyclin A2 can be conveniently assessed by nuclear staining and it might be a useful biomarker to predict treatment for HER2 negative Chinese patients.
Acknowledgements: We thank DU Cai and WANG Li-na for their laboratory and computer support, LÜ Jing-qiao and ZHAO Jian-qing for data analysis, and Professors HUNG Mien-chie and Stephanie Ann Miller for paper comments.
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