endometrial cancer is the fourth most common cancer among women in the united states . according to the latest data from the national cancer institute , approximately 40,100 new cases and 7,470 deaths from uterine cancer are expected in 2008 . in shanghai , china , the age - adjusted incidence of endometrial cancer increased by 4.41% per year between 1973 to 1975 and 1997 to 1999 [ 24 ] . endometrial cancer has been divided into two subtypes ( type i and ii ) based on different morphological appearance and   clinicalpathological characteristics [ 57 ] . type i , which is endometrioid histology with relatively low - grade features and good prognoses , occurs most often in postmenopausal women and is associated with unopposed estrogen exposure . in contrast , type ii endometrial cancer comprises of nonendometrial histology with an aggressive clinical course and poor prognoses . uterine papillary serous carcinoma ( upsc ) is the most common subtype of type ii endometrial cancer . it was first described by hendrickson et al .   in 1982 , and histologically similar to high - grade ovarian cancer . the microscopic criteria for diagnosis were described as neoplastic epithelium , characterized by serous differentiation with psammoma bodies present and predominantly papillary architecture . although upsc represents only about 10% of endometrial cancers , they account for over 50% of recurrences and deaths caused by endometrial cancer . the 5-year overall survival ( os ) for women with upsc is only 46% for all stages . comprehensive surgical staging is recommended for all patients with upsc , regardless of the depth of myometrial invasion of the tumor [ 9 , 10 ] . chemotherapy is recommended after the surgery , but unlike ovarian papillary serous carcinomas , upsc is a chemoresistant disease from onset , with low response rates and a short duration of response [ 11 , 12 ] . given the overall poor prognosis of these patients , new treatment modalities are urgently needed for upsc . her-2/neu , also known as c - erbb2 , is a member of the epidermal growth factor receptor transmembrane receptor tyrosine kinase family . 25% to 30% of breast cancers were found to have her-2/neu overexpression , which is considered as a negative prognostic factor [ 14 , 15 ] as well as a predictive marker of resistance to tamoxifen therapy , benefit from doxorubicin - based adjuvant chemotherapy , and trastuzumab ( anti - her-2 monoclonal antibody ) [ 1618 ] . recently , a striking overexpression of the her-2/neu in upsc has been identified by santin et al , with the rates ranging from 18% to 80% in different studies [ 1921 ] . in this study , we conducted a single institutional review of 10 years summary of this rare disease , which , to our knowledge , is the largest one so far analysing the clinicopathological features , prognostic factors , and her-2/neu status of chinese patients with upsc . the purpose of this study is to better understand clinicopathological features of upsc and the relationship with her-2/neu abnormality . all the slides of the patients with endometrial carcinoma who underwent surgery at cancer hospital , fudan university , from january 1996 to january 2006 , were reviewed by two pathologists separately . if there was dispute in diagnosis between them , slides would be reviewed by another pathologist who made the final diagnosis . altogether 457 patients with endometrial cancer were treated in our hospital during the last 10 years and 36 cases of upsc were rediagnosed , which accounted for about 8% . the information on survival was obtained in the medical record and death certificate . if this information was not available , the patient was censored after her last contact . formalin - fixed , paraffin - embedded specimens were collected from tissue bank of cancer hospital , fudan university . for all cases , a routine hematoxylin and eosin slide was evaluated to ensure that the specimen evaluated had serous carcinoma . ihc staining was performed on 4-um - thick paraffin - embedded sections according to the manufacturer 's instructions . briefly , after pretreatment with 10  mm citrate buffer at ph 6.0 using a steamer , they were incubated with c - erbb-2/her-2/neu ab at 37c for 2 hours , then incubated with immunoperoxidase anti - rabbit ab ( k4003 , dako corp . , denmark ) for half an hour and followed by substrate - chromogen solution ( dab ) . the intensity of immunostaining was graded as follows : negative ( 0 ) ; incomplete membranous staining or complete membranous staining in less than 10% of the tumor cells ( 1 + ) ; moderate intensity , complete membranous staining in greater than 10% of the tumor cells ( 2 + ) ; or strong intensity , complete membranous staining in greater than 10% of the tumor cells tumors with 2 + or 3 + staining were considered her-2/neu overexpression [ 23 , 24 ] . the sections were examined by light microscopy by two different pathologists without knowledge of the clinical outcome . cish was done using zymed spot - light her-2 cish kit ( 84 - 0146 ) according to instructions of manufacture . sections of 3  m were cut from paraffin - embedded archival tissue from tissue bank . the sections were deparaffinized and pretreated in a microwave oven at 100c for 15 minutes . after a brief rinsing with tris - buffered saline ( 0.05  mol / l tris / hcl saline , ph 7.47.6 ) , the slides were digested with enzyme for 5 to 10 minutes ( the time for each slide was different ) , followed by rinsing with distilled water for 3 times , and dehydrating with graded ethanols . the digoxigenin - labelled her-2/neu probe ( zymed ) was applied onto the slides , covered with coverslips and denatured at 95c for 5 minutes . the slides were then washed with standard saline citrate ( ssc ) in room temperature briefly then with ssc at 75c for 5 minutes . immunodetection was performed by the labelled avidin - biotin complex peroxidase system according to the manufacturer 's instructions . her-2/neu gene signals were scored in histological sections , in at least 150 neoplastic cells , using a conventional microscope ( olympus ) . tumours were classified , depending on the number of gene copies in the nuclei , as normal ( 15 copies ) and amplified ( > 6 copies or when large gene copy clusters were seen in at least 50% ) . in addition , amplified tumours were classified as low - level ( 610 copies ) or high level ( > 10 copies ) . cox 's proportional hazards regression was used to model survival with her-2/neu ihc overexpression and clinicopathological variables typically associated with prognosis . spearman correlation was used to analyze the relation between the results of ihc and cish . the median age of the patients was 63 years ( mean 64 years , range 45~81 years ) . the median bwi ( body weight index ) was 23.8  kg / m ( mean 23.73 , range 17.6~33.8 ) . the main presenting symptoms were abnormal vaginal bleeding ( 86% , 31/36 ) , while other 4 patients first presented with abdominal symptoms such as abdominal mass and ascites , and 1 with vaginal discharge . 16 patients ( 45.7% ) were complicated with hypertension and 4 patients ( 11.4% ) with diabetes . 8 cases or relatives had history of other cancers , including 1 with ovarian mucous adenocarcinoma , 2 with breast cancer , 1 with endometrial cancer , 3 with gastrointestinal cancer , and 1 with thyroid neoplasms . of these 36 patients , 11 ( 31% ) were stage i ( 1 ia , 6 ib , 4 ic ) , 3 ( 8% ) were stage ii ( 1 iia , 2 iib ) , 9 ( 25% ) were stage iii ( 4 iiia , 1 iiib , 4 iiic ) , and 13 ( 36% ) were stage iv ( 13 ivb ) . altogether 39% were early stages ( stage i / ii ) and 61% were late stages ( stage iii / iv ) . 21 tumors invaded more than half of myometrium , in which 11 invaded to serosa of the uterus . twenty - seven tumors ( 75% ) had overexpression of p53 , while positive er expression was only detected in 10 cases ( 27.8% ) , and pr expression in 7 cases ( 19.4% ) . all patients underwent exploratory surgery , hysterectomy , bilateral salpingo - oophorectomy , omentectomy , pelvic lymphadenectomy , and/or para - aortic lymphadenectomy . after the surgery , 14 patients received platinum - based chemotherapy including cisplatin , cyclophosphamide and anthracycline , or carboplatin and paclitaxel , and so forth . 4 patients received whole pelvic radiotherapy with 2 extended to the para - aortic field . 6 patients had oral tamoxifen or progestin , and 8 patients were observed after the surgery . of the 36 cases , 13 ( 36.1% ) patients had her-2/neu protein overexpression by ihc . in this 13 patients , 10 ( 27.8% ) showed 2 + and 3 ( 8.3% ) showed 3 + . overexpression of her-2/neu was seen in 50.0% ( 11/22 ) of upsc patients with advanced disease ( stage iii~iv ) , compared to 14.3% ( 2/14 ) of patients with early disease ( stage i~ii ) . patients with overexpression of her-2/neu were significantly more likely to have advanced - stage disease when compared to her-2/neu negative patients ( or = 6.0 , 95%ci : 1.0833.32 , p = .03 ) . four out of the 36 ( 11.1% ) cases were found with her-2/neu gene amplification , in which 3 amplified in high level and 1 in low level ( figure 1 ) . all ihc 3 + ( 100% , 3/3 ) cases had her-2/neu gene amplification . and all ihc negative cases had normal her-2/neu gene copies ( 15 copies ) . therefore , complete concordance between ihc and cish was observed in ihc 3 + and negative cases ( p = .001 , spearman correlation ) . only one out of 10 cases scored as 2 + by ihc ( 10% , 1/10 ) was found with gene amplification by cish . the median follow - up time for all of the patients was 31 months ( range , < 1 to 115 months ) . during the follow - up time , 17 patients died ( 47.2% ) , and 15 of these deaths were related to this disease . nine patients were observed to have disease progression during followup , with 4 in pelvis , 2 in liver , 1 in brain , 1 in both liver and pelvis , and 1 in both liver and brain . the 1-year , 3-year , and 5-year overall survival for all patients was 73.1% , 51.9% , and 43.9% , respectively ( figure 2 , ) . the results of univariate survival analysis of her-2/neu protein expression and other clinicopathological factors are shown in table 3 . the 5-year overall survival rate for her-2/neu ihc positive and negative cases was 12.9% and 68.6% , respectively . poorer outcome ( kaplan  meier ) was observed for patients with her-2/neu overexpression positive than negative patients ( p = .0023 ) ( figure 3 ) . the 5-year overall survival rate for early and advanced stage cases was 60.4% and 16% , respectively . advanced stages ( p = .0006 ) and deep myometrial invasion ( p = .0138 ) were also significantly associtated with a shorter os ( figures 4 and 5 ) . upsc is a rare type of endometrial cancer , whose clinical features are different from endometrioid endometrial adenocarcinoma . the median age of upsc was 64 years in this study , similar to previous report , but older than the median age of women with endometrioid endometrial cancers in china . unlike endometrioid endometrial cancers the median bwi was 23.8  kg / m in this study . and most important of all , compared to patients with endometrioid endometrial adenocarcinomas , patients with upsc tend to have deep myometrial invasion , high - stage diseases , and worse overall survival , as we had discussed previously in . in our study , the 1-year , 3-year , and 5-year overall survival was 73.1% , 51.9% , and 43.9% , respectively . although racial differences in the uterine cancer have been previously published , most of these studies have focused on disparities between african americans and whites . noted that black patients with upsc tended to be younger , had higher her-2/neu expression and short survival than white . slomovitz   summarized the clinical features of 129 cases with upsc . in his study , 93% patients were caucasian , and only 2% were asian . zhang et al .   compared 2,144 asians and 32,999 whites with corpus cancer in the united states , and found that asians presented at a younger age , with more advanced stage disease and higher 5-year survival rates than whites . in our study , similar to whites , main presenting symptoms were abnormal vaginal bleeding and with median bwi of 23.8  kg / m , patients with upsc ware not associated with obesity . on the other hand , asian patients with upsc had younger age ( 64 years ) and more late stage diseases ( 61% ) than whites compared to the previous report , in which median age was 68 years for whites and 56% patients were in late stages . the 3-year and 5-year overall survival in our study was 51.9% and 43.9% , respectively , which was a little worse than whites , whose 3-year and 5-year overall survival was 62.6% and 45.9% , respectively . .   found that upsc had more her-2/neu overexpression than all other types of endometrial cancers ( 23 of 38 , 61% versus 81 of 196 , 41% , resp . [ 20 , 21 ] reported that 62%~80% of upsc overexpressed her-2/neu protein , and others reported overexpression rates varyng from 40% to 48% [ 2931 ] . in contrast , slomovitz et al .   reported overexpression of her-2/neu in only 18% ( 12 of 68 ) of upsc patients . table 4 summmariued the results and methods of the studies on her-2/neu status in upsc . in our study , we demonstrated that 36% of patients with upsc overexpressed her-2/neu protein . the differences of the rate of her-2/neu overexpression , might be due to different antibody , inherent intraobserver variability , and most important of all , racial disparity . in our study , patients with her-2/neu overexpression were significantly more likely to have advanced stage disease when compared to her-2/neu negative patients ( p = .03 ) . overexpression of her-2/neu was seen in 50.0% ( 11/22 ) of upsc patients with advanced disease , compared to 14.3% ( 2/14 ) of patients with early disease . similar findings were reported by slomovitz , santin , and daz - montes et al . , in which overexpression of her-2/neu in patients with advanced disease were 24%~81.8% , compared to 8%~28.6% of patients with early disease [ 19 , 20 , 30 ] . daz - montes et al . also demonstrated that overexpression of her-2/neu was associated with higher ki-67 index , larger tumor sizes , and worse survival outcome . in survival analysis , we found that the overall survival was worse for patients with her-2/neu protein positive tumors than her-2/neu negative ones ( p = .0023 ) . the 5-year overall survival for her-2/neu ihc positive and negative cases was 12.9% and 68.6% , respectively . .   also revealed that the 5-year overall survival in upsc patients was 0% in her-2/neu ihc positive cases versus 45% in her-2/neu negative ones ( p = .008 ) . and santin et al .   demonstrated that short survival was associated significantly with her-2/neu overexpression compared with ihc expression 0~1+(p = .002 ) . all of these findings suggested that her-2/neu overexpression was a useful prognostic factor for this aggressive subtype of endometrial cancer . besides her-2/neu ihc overexpression , we also found that deep myometrial invasion ( p = .0138 ) and late stages ( p = .003 ) were associtated with a shorter os . we used cish to detect the her-2/neu gene amplification in our study and found her-2/neu gene amplification in four cases ( 11.1% ) . .   revealed that her-2/neu gene amplification was correlated with a poor survival outcome in patients with upsc ( p = .0084 ) . because of the small size of her-2/neu amplification in our study , we did not find the effect of her-2/neu amplification on overall survival . gene amplification was found in all ihc 3 + cases ( 100% , 3/3 ) . but in ihc 2 + cases , only one out of ten cases had her-2/neu gene amplification . therefore , like breast cancer , the entry criteria of trastuzumab in upsc should be confined to ihc 3 + cases , excluding the 2 + cases unless it is proved to have her-2/neu amplification by cish or fish . the gynecologic oncology group conducted a phase ii study using trastuzumab as a single agent in patients with advanced stage or recurrent endometrial cancer with her-2/neu ihc 2 + or 3 + staining , but found no activity of trastuzumab in heavily pretreated patients . entry criteria were then revised to include only those patients whose tumors were fish positive , but the study was finally closed due to slow enrollment . santin et al .   studied the effects of trastuzumab in upsc cell lines and found that upsc cell lines were highly sensitive to herceptin mediated antibody - dependent cellular cytotoxicity ( adcc ) and cell proliferation was inhibited . .   tried to use trastuzumab therapy ( 4  mg / kg intravenously over 90  min , then maintenance dose of 2  mg / kg intravenously over 30  min weekly until progression of disease ) in patients with upsc who expressed her-2/neu 3 + by ihc . one patient with ivb disease had complete response and the other with iiic disease had stable disease for 3 months . targeting her-2/neu might be beneficial for a select group of patients with upsc . in conclusion , our data revealed that chinese patients with upsc had characteristics of deep myometrial invasion , advanced stages and poor overall survival . overexpression , rather than her / neu gene amplification , is associated with advanced stage and poor survival outcome . her-2/neu might be a therapeutic target in refractory upsc patients , but it should be further evaluated by randomized clinical trials .
objective . to analyze clinico - pathological features of chinese patients with upsc , and investigate roles of her-2/neu protein expression and gene amplification in upsc prognosis 
 . methods . 
 thirty - six patients with upsc treated in cancer hospital of fudan university from 1996 to 2006 were analysed retrospectively . 
 chromogenic in situ hybridization ( cish ) and immunohistochemistry ( ihc ) were performed to evaluate her-2/neu gene amplification and protein expression respectively 
 . 
 results . 
 the median age was 63 years , and 61% ( 22/36 ) were late stages ( stage iii / iv ) . 
 the 1-year , 3-year , and 5-year overall survival ( os ) was 73.1% , 51.9% and 43.9% , respectively . 
 advanced stages ( p = .0006 ) and deep myometrial invasion ( p = .0138 ) were significantly associtated with a shorter os . in 36 cases , 27.8% ( 10/36 ) showed 2 + staining and 8.3% ( 3/36 ) showed 3 + by ihc . 
 amplification of the her-2/neu gene was observed in 11.1% ( 4/36 ) cases . 
 the 5-year overall survival rate in her-2/neu ihc 2 + 3 + and 0 ~ 1 + cases was 12.9% and 68.6% respectively . 
 her-2/neu 
 protein expression 2 + 3 + was significantly associated with advanced surgical stage and worse overall survival ( p = .03 and p = .0023 , resp . ) . 
 conclusion . 
 chinese patients with upsc showed characteristics of deep myometrial invasion , advanced stages and poor overall survival . 
 her-2/neu 
 protein overexpression is associated with advanced stage and poor survival outcome .