epithelial ovarian cancer ( eoc ) represents one of the most common gynecologic malignancies worldwide .
it has the highest mortality rate among malignant tumors in female reproductive system [ 1 , 2 ] . because of the lack of specific early symptoms or effective tumor biomarkers , most patients with eoc are diagnosed at the advanced stages , and the prognosis of these patients is still poor , even though there has been great improvement on traditional treatments , such as surgery , supplemented with radiotherapy and chemotherapy .
similar with other human malignancies , tumorigenesis and tumor progression of eoc are caused by numerous reproductive , environmental , and genetic risk factors .
therefore , it is of great importance to discover and analyze the genetic changes and molecular events involving the initiation , progression , and metastasis of eoc .  
mammalian runt - related transcription factor ( runx ) family contains three members ( runx1~3 ) which form the core - binding factor ( cbf ) complex and bind dna to either activate or repress gene transcription . as part of the cbf complex ,
the runx proteins are involved in the regulation of differentiation , survival , and growth in a variety of tissues .
runx1 and runx2 are specifically essential for multiple haematopoietic lineages and osteogenesis , respectively , and runx3 is closely related with neurogenesis and gut development .
accumulating studies have reported the oncogenic and tumor suppressive functions of the runx members . in the present study
, we have chosen to focus on the role of runx2 in neoplastic disease . because runx2 plays a pivotal role in the process of bone formation or osteogenesis , it is no doubt that its deregulation is strongly associated with the development of osteosarcoma .
in addition , the overexpression of runx2 has also been identified in several human malignancies , including prostate cancer , breast cancer , pancreatic cancer , colon carcinoma , and thyroid cancer .
for example , tandon et al .   indicated that runx2 is a potential therapeutic target to block tumor suppressor gene silencing in lung cancer cells .
pratap et al .   found that the expression of runx2 in breast cancer cells may be important for tumor cell invasion through matrigel chambers .
the results of chua group revealed that runx2 nuclear staining was correlated with increased prostate - specific antigen ( psa ) levels , higher gleason scores and tumours with metastatic capability .
identified runx2 as a potent prognostic factor in colon carcinoma patients through the promotion of cell proliferation and invasion properties .
however , the expression patterns and involvement of runx2 in eoc are still unclear . therefore , the aim of this study was to investigate the clinical significance of runx2 expression in eoc .
the study was approved by the research ethics committee of general hospital of pla , china .
we collected formalin - fixed , paraffin - embedded tissues from the primary ovaries obtained during surgery from women with eoc ( n = 116 ) . as controls
, we also obtained normal ovarian tissues ( n = 5 ) from women who underwent hysterectomies for benign disease .
all operations were performed in the department of obstetrics and gynecology at general hospital of pla from january 2005 to december 2006 .
all patients with only gynecology tumor were treated without preoperative radiotherapy , chemotherapy , or hormonal therapy .
surgical staging was established according to the international federation of gynecology and obstetrics ( figo ) system .
debulking status was defined according to the size of the nodules left in the peritoneal cavity after surgery .
the paraffin - embedded tissues were cut at 3  m and then deparaffinized with xylene and rehydrated for further hematoxylin eosin ( h&e ) or 3,3-diaminobenzidine ( dab ) immunohistochemistry staining employing dako envision system ( dako diagnostics , zug , switzerland ) .
briefly , following treatment with 10% normal goat serum for 45  min to block nonspecific binding sites , the consecutive tissue sections were incubated for 180  min at 25c with mouse monoclonal antibody for human runx2 ( dilution 1  :  1000 , abnova corporation , taipei , taiwan ) .
the specificity of the primary antibody has been validated by the previous studies [ 15 , 16 ] .
after washing , peroxidase - labeled polymer and substrate - chromogen were then employed in order to visualize the staining of the interested proteins .   in tumor cells of eoc tissues ,
runx2 immunoreactivity was detected in the nucleus , and the immunoreactivity was evaluated as a labeling index ( li ) according to the methods of previous studies [ 15 , 16 ] .
briefly , runx2 immunoreactivity was evaluated in the nuclei of more than 1000 tumor cells for each case , and li was calculated as the percentage of runx2 positive cells per 1000 tumor cells counted at random in each section .
this counting was performed under a 400 magnification . the software of spss version 12.0 for windows ( spss inc . ,
il , usa ) and sas 9.1 ( sas institute , cary , nc , usa ) were used for statistical analysis .
the pearson 's chi - square and fisher 's exact tests were used to assess the statistical significance of the association between runx2 expression and clinicopathologic parameters .
kaplan - meier curves were plotted to assess the effects of runx2 expression on overall and progression - free survival .
cox proportional hazard models were used to assess the prognostic significance of runx2 expression and several clinicopathologic parameters .
the expression patterns and cellular localization of runx2 in 116 eoc and 5 normal ovarian tissues were assessed by immunohistochemical analysis .
as shown in figure 1 , runx2 immunoreactivity was predominantly localized in the nuclei of eoc cells ( figure 1(a ) ) , while almost negligible in normal ovarian tissues ( figure 1(b ) ) .
the mean value of the runx2 li in 116 eoc tissues detected was 56.3% ( range , 099% ) , which was significantly higher than that in normal ovarian tissues ( 11.7% ; range , 035.2% ; p < 0.001 ) .
all the eoc tissues ( n = 116 ) were divided into two groups : high runx2 expression group ( runx2 li  55.1% , n = 78 ) and low runx2 expression group ( runx2 li < 55.1% , n = 38 ) . 

table 1 summarized the association of runx2 expression with various clinicopathological features of eoc tissues .
the nuclear li of runx2 in tumor cells was significantly associated with the clinical stage of eoc tissues ( p = 0.001 ) .
the eoc tissues with advanced clinical stage ( iii~iv ) more frequently showed high runx2 expression than those with low clinical stage ( i~ii ) .
however , runx2 expression was not correlated with age , grade , histological type , and residual tumor after surgery ( all p > 0.05 ) . in order to investigate the prognostic implications of runx2 expression in overall survival and progression - free survival of eoc , the detailed clinical information of all 116 eoc patients in high runx2 expression and low runx2 expression groups was reviewed .
median follow - up time was 66.8 months ( range , 2.2118.9 months ; mean , 66.1 months ) . at last followup , 73 ( 62.9% ) relapsed with a median time of 22.1 months ( range , 2.885.2 months ) . as determined by the log - rank test ,
eoc patients with high runx2 li had significantly shorter overall ( p < 0.001 , figure 2(a ) ) and progression - free ( p = 0.002 , figure 2(b ) ) survival than those with low runx2 li did . moreover , the univariate analysis revealed that both the advanced stage ( p < 0.001 and p = 0.008 , resp . ) and the high runx2 expression ( p < 0.001 and p = 0.002 , resp . ) predicted poorer overall and progression - free survival of eoc patients ( table 2 ) .
furthermore , the multivariate analyses identified the clinical stage ( p = 0.01 and p = 0.03 , resp . ) and the runx2 li ( both p = 0.01 ) in eoc cells as independent prognostic factors for overall and progression - free survival ( table 3 ) .
interestingly , subgroup analyses according to clinical stage revealed that eoc patients with high clinical stages ( iii~iv ) in high runx2 expression group demonstrated a significantly worse clinical outcome than those in low runx2 expression group ( figures 3(a ) and 3(b ) ) , but patients with low clinical stages ( i~ii ) had no significantly different prognosis between high and low runx2 expression groups ( figures 3(c ) and 3(d ) ) .
as the first leading cause of cancer death in female reproductive system malignant tumors , eoc has no characteristic early symptoms or tumor markers , leading to disappointing clinical outcome .
the clinical course of remission and relapse is commonly seen in patients undergoing therapy for eoc .
discovery and analysis of the genetic changes and molecular events have contributed largely to a better understanding of the molecular mechanisms of cancer ontogenesis .
accordingly , it is of great significance to identify novel specific diagnostic or prognostic markers that contribute to progression and metastasis of eoc . in the present study , we detected the overexpression of runx2 in eoc tissues compared with normal ovarian tissues , and its upregulation was closely related with the clinical stage and poor prognosis of eoc patients , which led us to believe that runx2 might be used as a candidate biomarker for aggressive disease behavior . to the best of our knowledge ,
runx proteins have been demonstrated to play positive and negative roles in carcinogenesis according to different cancer types . as a member of runxs , runx2 , also known as core - binding factor , runt domain , a - subunit 1 , cbfa1 , aml3 , or osf2 , is a lineage - specific transcription factor and the human homologue of mouse pebp2a . during embryonic development
after birth , runx2 controls bone matrix deposition , especially collagen i , by differentiated osteoblasts . targeted disruption of runx2 in mice results in failure of osteoblast differentiation and bone formation , and runx2 haploinsufficiency in humans leads to the skeletal disorder cleidocranial dysplasia , with a similar phenotype observed in runx2 haploinsufficient mice .
. reported that runx2 transcription factor may be involved in various aspects of luteal function by directly regulating the expression of diverse luteal genes in luteinizing granulosa cells of rat ovaries . in carcinogenesis , runx2 acts as a master regulator of tumor invasion and metastasis .
nuclear runx2 is increased in malignant versus benign prostate tissue and is associated with tumor aggression in general and metastasis in particular .
runx2 has the strongest basal expression among the runxs , and a significant increase in runx2 in carcinoma samples contributes to tumorigenesis in esophageal squamous cell carcinoma .
the differential expression of runx2 has been found between invasive , and noninvasive breast cancer cells , and its upregulation is linked to the invasive phenotype of cancer cells .
runx2 also plays an important role in angiogenesis by enhancing the endothelial cell proliferation , invasion and tube formation as well as indirectly by activating the promoter region of the vascular endothelial growth factor gene .
conducted genome - wide expression studies in combination with gene - pathway analyses and cross - correlation to estrogen receptor- ( er- ) chromatin - binding sites , and demonstrated that runx2 expression was induced by er in human colorectal carcinoma cells . in the present study , our data found that runx2 significantly overexpressed in eoc tissues in contrast to normal ovarian tissues .
we also showed that the eoc tissues with advanced clinical stage more frequently tend to express high runx2 .
these findings provide evidence that the up - regulation of runx2 expression might be important in the carcinogenesis of eoc . because of the poor prognosis in eoc patients , our study also focused on the prognostic implications of runx2 for this cancer .
our survival analysis demonstrated that eoc patients whose tumors overexpressed runx2 had shorter overall and progression - free survival times than those with low runx2 expression tumors , especially those at advanced clinical stages .
more importantly , further analysis using the cox regression model confirmed that the runx2 expression was an independent factor in predicting overall and progression - free survival times for eoc patients .
these findings provide evidence that runx2 could be regarded as a biomarker for predicting the outcome of eoc patients .
the function of runx2 is related with several molecules and multiple pathways , such as angiogenic factor , matrix metalloproteinases ( mmps ) , transcription factors , the mapk pathway , the stat pathway and the pi3  k pathway , which are involved in ovarian carcinogenesis .
for example , duan et al .   indicated that the abnormal activation on pi3  k / pkb signaling pathway may be correlated with the occurrence and development of eoc ; chakrabarty and kondratick   also suggested that activation on multiple cascades of the mapk pathway may promote the invasion and metastasis of eoc . in this study , although our results demonstrate the aberrant expression and important clinical significance of runx2 in eoc patients , the exact mechanism of runx2 upregulation in eoc is still not clearly understood . in this context
, further studies are needed to determine the molecular mechanism of runx2 dysfunction in human ovarian carcinogenesis . in conclusion ,
our data suggest for the first time that runx2 overexpression is associated with advanced tumor progression and poor clinical outcome of eoc patients .
 
 aim . to investigate clinical significance of runt - related transcription factor ( runx)-2 in epithelial ovarian cancer ( eoc ) 
 . 
 methods . 
 runx2 protein expression and its subcellular localization were detected by immunohistochemistry in 116 patients with eoc . 
 
 results . 
 runx2 protein was predominantly expressed in cell nucleus of eoc tissues . 
 the expression level of runx2 in eoc tissues was significantly higher than that in normal ovarian tissues ( p < 0.001 ) . 
 in addition , the nuclear labeling index ( li ) of runx2 in tumor cells was significantly associated with the advanced clinical stage of eoc tissues ( p = 0.001 ) . moreover , 
 eoc patients with high runx2 li had significantly shorter overall ( p < 0.001 ) and progression - free ( p = 0.002 ) survival than those with low runx2 li . 
 especially , subgroup analysis revealed that eoc patients with high clinical stages ( iii~iv ) in high runx2 expression group demonstrated a significantly worse clinical outcome than those in low runx2 expression group , but patients with low clinical stages ( i~ii ) had no significantly different prognosis between high and low runx2 expression groups . 
 
 conclusions . 
 our data suggest for the first time that runx2 overexpression is associated with advanced tumor progression and poor clinical outcome of eoc patients . 
 runx2 might be a novel prognostic marker of eoc . 
1. Introduction
2. Materials and Methods 
3. Results
4. Discussion