Meng-yi Wang， Zhe-yu Niu， Xiang-Gao， Li Zhou，Quan Liao*， and Yu-pei Zhao*

Department of General Surgery， Peking Union Medical College Hospital，Chinese Academy of Medical Sciences & Peking Union Medical College， Beijing 100730， China

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Prognostic Impact of Cell Division Cycle Associated 2 Expression on Pancreatic Ductal Adenocarcinoma△

Meng-yi Wang， Zhe-yu Niu， Xiang-Gao， Li Zhou，Quan Liao*， and Yu-pei Zhao*

Department of General Surgery， Peking Union Medical College Hospital，Chinese Academy of Medical Sciences & Peking Union Medical College， Beijing 100730， China

cell division cycle associated 2； immunohistochemistry； prognosis；pancreatic ductal adenocarcinoma

Objective To examine the expression of cell division cycle associated 2 （CDCA 2） in pancreatic ductal adenocarcinoma （PDAC） and investigate its role in prognosis of PDAC patients.

Methods This retrospective study included 155 PDAC patients who underwent surgical treatment and complete post-operative follow-up. Clinicopathologic data were collected through clinical database. Tissue microarray was constructed and immunohistochemistry was performed to detect CDCA2 expression in the PDAC tumor tissues and adjacent non-tumor tissues. Clinicopathological characteristics between high and low CDCA2 expression were compared. Correlation of CDCA2 expressions with patients’ survival was analyzed using Kaplan-Meier method and Cox regression analysis.

Results Expression of CDCA2 in PDAC cells was significantly higher than that in adjacent non-tumor tissues （U=4056.5， P＜0.001）. Univariate analysis showed that CDCA2 expression ［hazard ratio （HR）=1.574，95% confidence interval （CI）=1.014-2.443， P=0.043］ and node metastasis （HR=1.704， 95%CI=1.183-2.454，P=0.004） were significantly associated with prognosis. Cox regression analysis showed CDCA2 expression was not an independent prognostic risk factor （HR=1.418， 95%CI=0.897-2.242， P=0.135） for PDCA patients. Stratification survival analysis demonstrated CDCA2 expression as an independent prognostic risk factor in male patients （HR=2.554， 95%CI=1.446-4.511， P=0.003） or in non-perineural invasion patients（HR=2.290， 95%CI=1.146-4.577， P=0.012）.

Conclusions CDCA2 is highly expressed in PDAC tumor tissue. Although CDCA2 is not an independent prognostic risk factor for PDAC patients， it might be used to help predict prognosis of male or non-perineural invasion patients of PDAC.

Chin Med Sci J 2016； 31（3）：149-154

P ANCREATIC ductal adenocarcinoma （PDAC） is a common malignancy that shows rapid progression and a poor prognosis. The incidence of PDAC is rising， and the 5-year survival rate is only approximately 7%.1Over half of patients cannot undergo tumor resection when diagnosed， and most patients develop chemoresistance to the chemotherapeutic agents. Therefore， identification of a new target for treatment of PDAC is urgent.2，3

For the majority of the PDAC tumor tissue is stroma，currently direct DNA sequencing approaches using tumor tissues are very difficult to perform. In our previous study，we used laser capture microdissection to purify cancer cells for whole exon genome sequencing. The results showed that in addition to p53 or kras mutations， mutation in cell division cycle associated 2 gene （CDCA2） was commonly observed in multiple samples.

CDCA2， also known as Repo-Man （recruits PP1 onto mitotic chromatin at anaphase）， is the target subunit for protein phosphatase 1（PP1）. CDCA2 participates in the dephosphorylation of histone H3 at the end of mitosis. Its main function is to help PP1 combine with chromatin in anaphase. Studies have confirmed that CDCA2 is associated with cell proliferation and DNA damage repair （DDR）， and is related to tumor progression in vitro.4CDCA2 expression has been reported in malignancies such as breast cancer，oral squamous cell carcinoma （OSCC）， melanoma， and aggressive neuroblastoma.5-9The role of CDCA2 in malignancy was considered to be its ability to promote cell cycle progression and inhibit apoptosis. To the best of our knowledge， the role of CDCA2 in PDAC has not been fully demonstrated.

In this study， we examined CDCA2 expression in PDAC tumorous and adjacent non-tumor tissues， and investigate the relationship between CDCA2 expression and prognosis of patients with PDAC.

PATIENTS AND METHODS

Patients’ information and follow-up

The retrospective cohort consisted of 155 patients with PDAC， including 98 males and 57 females. Ages ranged from 34 to 85 （median： 60） years. Patient's selection criteria： （1） histologically proven adenocarcinoma； （2）surgically resected specimens available； （3） clinicopathological data available. The diagnosis and staging were based on the Staging Manual of the American Joint Committee on Cancer （AJCC）， 7th edition.10Histological grade， peri-neural invasion （PNI）， T and N stages were given by post-operational routine pathologic examinations. Overall survival was defined as the survival time after surgery （approximately 0-87 months； median： 12.55 months）. Written informed consents for storage of tissue samples and publication of this study were obtained before surgery after a fully verbal explanation.

Tissue microarray （TMA） construction and immunohistochemistry （IHC）

TMA was constructed with a manual tissue microarray（Beecher Instruments， Sun Prairie， WI， USA） using formalin-fixed paraffin-embedded blocks. Two cores of tumor tissue and adjacent non-tumor tissue per case were sampled from representative areas using a 1.5-mm punch.

IHC was performed to detect CDCA2 expression. A rabbit anti-human CDCA2 polyclonal antibody （PAB21795，Abnova Corporation， Taiwan） and an EnVision+ two-step staining kit （Dako， Glostrup， Denmark） were used for staining. Briefly， 4-μm-thick tumor sections were fixed in formalin， embedded in paraffin， mounted and deparaffinized in xylene， and rehydrated in graded alcohol. The slides were washed with phosphate-buffered saline （PBS），immersed in 0.01 mol/L citrate buffer solution （pH 6.0），and placed in a microwave for 10 minutes for antigen retrieval. Peroxidase was quenched using 3% hydrogen peroxide for 10 minutes. After washing again with PBS， the slides were incubated at 4°C overnight with the primary antibody （1：400）. After washing with PBS four times，horseradish peroxidase-labeled secondary antibody was added to incubate for 30 minutes， using diaminobenzidine as a chromogen. Finally， the slides were counterstained with hematoxylin （Sigma-Aldrich Co.， LLC， Munich， Germany）to visualize the nuclei. Preimmune rabbit serum at the same dilution was used as the negative control.

Scoring of CDCA2 expression was conducted according to the ratio of positive cells and staining intensity. Positive cell scores were determined based on the frequency of positive cells in each sample： 0-24%， score 1； 25%-49%，score 2； 50%-74%， score 3； and over 75%， score 4. Staining intensity was scored as follows： no staining， score 0； weak staining， score 1； moderate staining， score 2； and strong staining， score 3.11Final score was determined by multiplying the positive cell score and staining intensity score. Low expression was defined as a final score≤ 3， and high expression as a final score ≥ 4. The scores were assessed independently by two senior pathologists in a blinded manner.

Statistical analysis

The Mann-Whitney U test was used to compare CDCA2 expression scores between the tumor tissues and theadjacent non-tumor tissues. The Chi-squared test was used to detect correlations of categorical variables in clinicopathological features and CDCA2 expression. The Kaplan-Meier method and log-rank test were used in the survival analysis and the COX regression model was used to analyze prognostic factors. Statistical analyses were performed using SPSS （version 17.0； SPSS Inc.， Chicago，IL， USA）. A P value of less than 0.05 was considered statistically significant.

RESULTS

CDCA2 expression in PDAC tumor tissue and adjacent non-tumor tissues

Positive staining of CDCA2 was observed in the nuclei of both PDAC tumorous and adjacent non-tumor tissues（Fig. 1）. The final scores of CDCA2 expression in PDAC cells（4.44±3.906） was significantly higher than that of the normal pancreatic duct cells in adjacent non-tumor tissue（1.50±0.501， U=4056.5， P＜0.001）.

CDCA2 expression and its clinicopathological relevance in PDAC

According to the final scores， CDCA2 in PDCAs were highly expressed in 117 （117/155， 75.5%） patients and relatively low expressed in 38 （38/155， 24.5%） patients. As shown in Table 1， there was no significant difference between the high expression group and low expression group in function of age， gender， tumor size， tumor location， T stages， node metastasis， AJCC stages， histopathological type， as well as perineural invasion status.

Figure 1. Cell division cycle associated 2 （CDCA2） expression in pancreatic ductal adenocarcinoma （PDAC） and adjacent non-tumor tissues. EnVision ×200 Images of CDCA2 staining in PDAC tumorous （the 1st and 3rd row） and adjacent non-tumor tissues （the 2nd and 4th row）. Positive cells stain in yellow.

Effect of CDCA2 expression on prognosis

Univariate analysis showed that CDCA2 expression ［low vs. high， hazard ratio （HR） =1.574， 95%confidence Interval（CI）=1.014-2.443， P=0.043］ （Fig. 2） and node metastasis（negative vs. positive， HR=1.704， 95%CI=1.183-2.454，P=0.004） were significantly associated with prognosis. The other clinicopathological characteristics we observed， such as age， gender， tumor location， tumor size， histopathologicalgrade， T stages， and perineural invasion， were not related to prognosis （Table 2）. Cox regression analysis showed that only node metastasis was an independent prognostic risk factor （negative vs. positive， HR=1.667，95%CI=1.156-2.404， P=0.006）， while CDCA2 expression was not an independent risk factor （low vs. high，HR=1.418， 95%CI=0.897-2.242， P=0.135）.

Table 1. Correlation of CDCA2 expression and clinicopathological features in PDAC patients （n=155）

Patients were grouped by the clinical features above for stratification survival analysis. We found that male patients or patients who did not have perineural invasion had a significant poor survival if CDCA2 was highly expressed， compared to those CDCA2 was low expressed. CDCA2 expression was an independent prognostic risk factor in male patients （low vs. high， HR=2.554， 95%CI= 1.446-4.511， P=0.003） and in non-perineural invasion patients （low vs. high， HR=2.290， 95%CI=1.146-4.577，P=0.012） （Fig. 3）. There was no statistical significance in other subgroups.

Figure 2. Kaplan-Meier survival curves of PDAC stratified by CDCA2 expression levels. Univariate analysis shows that patients with high CDCA2 expression was significantly associated with a poor prognosis （P=0.043）.

Table 2. Cox regression analyses of CDCA2 expression in PDAC tumor tissue and clinicopathological features （n=155）

Figure 3. Stratifid survival analysis of CDCA2 expression in male patients （A） and in patients of non-perineural invasion （B）. CDCA2 expression was demonstrated as an independent prognostic risk factor in male patients （P=0.003） or in non-perineural invasion patients （P=0.012）.

DISCUSSION

CDCA2 is localized in the nucleus and plays important roles in mitosis as well as in DDR. During anaphase， as a regulatory subunit of PP1γ， CDCA2 recruits PP1γ to histone H3 to promote dissociation of chromatin. Phosphorylation of CDCA2 by cyclin B-CDK1 prevents CDCA1 from binding to PP1γ and inhibits its subsequent binding to chromatin. CDCA2 is also controlled by Aurora B， which prevents CDCA2 from binding to histone before replication is ready.12CDCA2 influences nuclear reconstruction in mitosis by regulating nuclear envelope remodeling.13In tumor cells with reduced CDCA2 expression by short interfering RNA， both abnormal chromatin replication and irregular nuclear morphology were observed.14

Ataxia telangietisa mutant （ATM） functions in DDR progression. When DNA is damaged， ATM is phosphorylated and subsequently activates p53 and p21. Activation of p21 inhibits cyclin-CDK complexes to stop mitosis and allow time for DNA recovery. If repair fails， cell eventually undergo apoptosis. When CDCA2 recruits PP1γ to chromatin，the compound would resist ATM and other downstream proteins. So overexpression of CDCA2 in cells would decrease DDR sensitivity in cancer progression.5

A previous study of OSCC showed that CDCA2 was expressed at higher levels in cancer cells than that in adjacent non-tumor tissues， and was significantly related with tumor staging in OSCC.6In our study， we found that CDCA2 expression was significantly higher in PDAC tumor tissue than that in adjacent non-tumor tissues. These findings are consistent with CDCA2 biological function. As the facts that many patients with pancreatic mass do not come with elevated CA19-9 in serum， sometimes the mass is too small to diagnosis on imaging exams， and biopsy by endoscopic ultrasonography may not obtain enough tissue for morphological pathology examination， our results indicate that CDCA2 could be used as an indicator for diagnosis of PDAC.

However， we did not find a relationship between CDCA2 expression and T staging. This might be due to the bias in patients' selection in our study. Since in most PDACpatient， tumors are unresectable when diagnosed， while the specimens in our study came from resected tissues，thus most of the patients in our study were in relatively earlier stage compared to the general PDAC population. This bias may be inevitable given the setting of pancreatic cancer research.

Univariate analyses showed that CDCA2 expression and N staging were related with prognosis. Patients with high expression of CDCA2 showed poor prognosis. Although multivariate analysis didn't demonstrate CDCA2 as an independent risk factor， the stratification analysis showed that in male patients and non-perineural invasion patients， CDCA2 was an independent risk factor to predict prognosis. Thus， for particular subgroups of pancreatic cancer patients， performing IHC for CDCA2 after resection may be helpful for physicians to predict prognosis and choose better treatments.

In conclusion， our results showed that CDCA2 was highly expressed in PDAC tumor tissues. During tumor progression， CDCA2 overexpression likely results in accelerating proliferation of tumor cells and reducing their apoptosis. CDCA2 might be a good marker for diagnosis of PDAC and predicting prognosis.

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for publication March 18， 2016.

*Corresponding authors Quan Liao Tel： 86-10-69156007， E-mail： lqpumc@126.com， Yu-pei Zhao Tel： 86-10-69156007， E-mail： zhao8028@263.net

△Supported by the National High Technology Research and Development Program of China （863 Program） （2012AA02A212）.