we obtained formalin - fixed , paraffin - embedded tissue sections from 30 leiomyosarcoma patients at the university of texas md anderson cancer center ( mdacc ; houston , texas , usa ) . these included samples from the retroperitoneum ( n = 12 ) , gastrointestinal tract ( n = 9 ) , uterus ( n = 6 ) , and skin ( n = 3 ) . to validate the results obtained with the mdacc samples , another cohort of 45 formalin - fixed , paraffin - embedded tissues from patients with leiomyosarcoma were retrieved from tianjin medical university cancer institute & hospital ( tmucih ) for further study . the patients ' clinicopathologic characteristics , including sex , age , clinical stage , degree of cell differentiation , tumor location , tumor size , the presence or absence of hemorrhage / cyst , and histopathologic subtype , were also collected . the tissues had been acquired from 18 male and 27 female patients with a mean age of 55.8 years . histopathologic subtypes were assigned based on the 2002 world health organization classification of tumors of soft tissue and bone , . overall survival time was defined from the date of diagnosis or the first treatment to the date of death or last follow - up . overall survival ranged from 1 to 74 months , and the median survival was 19 months . the study protocol was approved by the institutional review boards of both hospitals , and consent was obtained from each patient . briefly , tissue sections ( 4 m ) were dewaxed with xylene , rehydrated with ethanol , soaked in citrate buffer solution ( 10 mmol / l , ph=6 ) , and boiled in a pressure cooker for 2.5 min to repair the antigen . the sections were then blocked for 20 min with normal serum ( vector laboratories , burlingame , ca ) and incubated overnight at 4c with rabbit polyclonal anti - prune2 antibody ( abcam company , abcam , cambridge , uk ; diluted 1:100 ) . secondary antibody was applied for 60 min ( one drop of biotinylated anti - rabbit with three drops of normal rabbit serum ) . dab ( dako corporation , carpinteria , ca ) staining was applied for 510 min and monitored by microscopy . two pathologists blinded to clinical information evaluated and scored prune2 staining based on staining extent and intensity . ten random high - power fields , each containing approximately 100 cells , were observed under the microscope ( 40 ) . first , staining extent was scored according to the proportion of positive tumor cells : 0% ( score 0 ) , 10% ( score 1 ) , 11% to 25% ( score 2 ) , 26% to 50% ( score 3 ) , 51% to 75% ( score 4 ) , and > 75% ( score 5 ) . second , staining intensity was scored based on the color observed : no color ( score 0 ) , yellow ( score 1 ) , tan ( score 2 ) , and brown ( score 3 ) . final scores were calculated by adding the extent and intensity scores , and the results were used to divide patients into two groups : low expression group , for final scores of 04 , and high expression group , for final scores > 4 . kaplan - meier survival analysis ( log - rank test ) and multivariate cox regression analysis were used to examine the relationship between overall survival and prune2 protein expression . we obtained formalin - fixed , paraffin - embedded tissue sections from 30 leiomyosarcoma patients at the university of texas md anderson cancer center ( mdacc ; houston , texas , usa ) . these included samples from the retroperitoneum ( n = 12 ) , gastrointestinal tract ( n = 9 ) , uterus ( n = 6 ) , and skin ( n = 3 ) . to validate the results obtained with the mdacc samples , another cohort of 45 formalin - fixed , paraffin - embedded tissues from patients with leiomyosarcoma were retrieved from tianjin medical university cancer institute & hospital ( tmucih ) for further study . the patients ' clinicopathologic characteristics , including sex , age , clinical stage , degree of cell differentiation , tumor location , tumor size , the presence or absence of hemorrhage / cyst , and histopathologic subtype , were also collected . the tissues had been acquired from 18 male and 27 female patients with a mean age of 55.8 years . histopathologic subtypes were assigned based on the 2002 world health organization classification of tumors of soft tissue and bone , . overall survival time was defined from the date of diagnosis or the first treatment to the date of death or last follow - up . overall survival ranged from 1 to 74 months , and the median survival was 19 months . the study protocol was approved by the institutional review boards of both hospitals , and consent was obtained from each patient . , tissue sections ( 4 m ) were dewaxed with xylene , rehydrated with ethanol , soaked in citrate buffer solution ( 10 mmol / l , ph=6 ) , and boiled in a pressure cooker for 2.5 min to repair the antigen . the sections were then blocked for 20 min with normal serum ( vector laboratories , burlingame , ca ) and incubated overnight at 4c with rabbit polyclonal anti - prune2 antibody ( abcam company , abcam , cambridge , uk ; diluted 1:100 ) . secondary antibody was applied for 60 min ( one drop of biotinylated anti - rabbit with three drops of normal rabbit serum ) . dab ( dako corporation , carpinteria , ca ) staining was applied for 510 min and monitored by microscopy . two pathologists blinded to clinical information evaluated and scored prune2 staining based on staining extent and intensity . ten random high - power fields , each containing approximately 100 cells , were observed under the microscope ( 40 ) . first , staining extent was scored according to the proportion of positive tumor cells : 0% ( score 0 ) , 10% ( score 1 ) , 11% to 25% ( score 2 ) , 26% to 50% ( score 3 ) , 51% to 75% ( score 4 ) , and > second , staining intensity was scored based on the color observed : no color ( score 0 ) , yellow ( score 1 ) , tan ( score 2 ) , and brown ( score 3 ) . final scores were calculated by adding the extent and intensity scores , and the results were used to divide patients into two groups : low expression group , for final scores of 04 , and high expression group , for final scores > 4 . kaplan - meier survival analysis ( log - rank test ) and multivariate cox regression analysis were used to examine the relationship between overall survival and prune2 protein expression . in the 30 leiomyosarcoma tissues from mdacc , prune2 protein staining was predominantly in the cytoplasm of cells ( figure 1 ) . low expression was discovered in 63.3% ( 19/30 ) of the samples ( figure 1a ) , while high expression was observed in 36.7% ( 11/30 ) ( figure 1b ) . to investigate the prognostic role of prune2 , we analyzed the association between prune2 protein expression and overall survival . univariate analysis showed that prune2 protein expression had no significant association with overall survival (  = 0.246 , p = 0.62 ) ( figure 2a ) . a , low expression of prune2 protein ; b , high expression of prune2 protein . to validate these results and further evaluate the prognostic role of prune2 in leiomyosarcoma , we used a larger cohort of leiomyosarcoma tissue samples from tmucih . in this set of 45 samples , prune2 was also predominantly detected in the cytoplasm ; and 62.2% ( 28/45 ) had low expression , while 37.8% ( 17/45 ) had high expression . we also analyzed the association between prune2 protein expression and clinicopathologic factors , and found that prune2 protein expression was significantly associated with tumor size ( p = 0.03 ) and the absence of hemorrhage / cyst ( p = 0.014 ) ( table 1 ) . in addition , univariate analysis showed that there was a significant association between prune2 protein expression and overall survival (  = 5.399 , p = 0.02 ) ( figure 2b ) , with longer survival noted among patients with high levels of the protein . furthermore , multivariate analysis revealed that high prune2 protein expression was an independent , favorable prognostic factor for overall survival ( hr = 0.373 , p = 0.025 ) . a , prune2 protein expression is not significantly associated with overall survival in patients from md anderson cancer center ; b , prune2 protein expression is significantly associated with overall survival in patients from tianjin medical university cancer institute & hospital . in the 30 leiomyosarcoma tissues from mdacc , prune2 protein staining was predominantly in the cytoplasm of cells ( figure 1 ) . low expression was discovered in 63.3% ( 19/30 ) of the samples ( figure 1a ) , while high expression was observed in 36.7% ( 11/30 ) ( figure 1b ) . to investigate the prognostic role of prune2 , we analyzed the association between prune2 protein expression and overall survival . univariate analysis showed that prune2 protein expression had no significant association with overall survival (  = 0.246 , p = 0.62 ) ( figure 2a ) . a , low expression of prune2 protein ; b , high expression of prune2 protein . to validate these results and further evaluate the prognostic role of prune2 in leiomyosarcoma , we used a larger cohort of leiomyosarcoma tissue samples from tmucih . in this set of 45 samples , prune2 was also predominantly detected in the cytoplasm ; and 62.2% ( 28/45 ) had low expression , while 37.8% ( 17/45 ) had high expression . we also analyzed the association between prune2 protein expression and clinicopathologic factors , and found that prune2 protein expression was significantly associated with tumor size ( p = 0.03 ) and the absence of hemorrhage / cyst ( p = 0.014 ) ( table 1 ) . in addition , univariate analysis showed that there was a significant association between prune2 protein expression and overall survival (  = 5.399 , p = 0.02 ) ( figure 2b ) , with longer survival noted among patients with high levels of the protein . furthermore , multivariate analysis revealed that high prune2 protein expression was an independent , favorable prognostic factor for overall survival ( hr = 0.373 , p = 0.025 ) . a , prune2 protein expression is not significantly associated with overall survival in patients from md anderson cancer center ; b , prune2 protein expression is significantly associated with overall survival in patients from tianjin medical university cancer institute & hospital . the most important contribution of this study is that we found a significant association between prune2 protein expression and overall survival , and that prune2 protein expression was an independent prognostic factor for leiomyosarcoma . more specifically , leiomyosarcoma patients with higher prune2 protein expression exhibited a better survival trend , suggesting that increased levels of the protein might be a favorable prognostic factor for leiomyosarcoma . similarly , in neuroblastoma and prostate cancer , prune2 protein is highly expressed and also plays a prognostic role , . on the other hand , we found no statistically significant association between prune2 mrna expression and survival time in tissue samples from patients at mdacc in our previous study . here , kaplan - meier survival analysis revealed no significant relationship between overall survival time and prune2 protein expression in samples from mdacc , which is consistent with our previous result . nevertheless , in the larger cohort of samples from tmucih , prune2 protein expression showed a significant association with overall survival and was an independent prognostic factor . possible causes are the differences in race among the patients from each center and in the number of cases . we also found that prune2 protein expression was significantly associated with tumor size and hemorrhage / cyst . specifically , prune2 protein expression was higher in the smaller tumor size group ( < 10 cm ) . because prune2 plays an important role in regulating cell differentiation and apoptosis , this result suggests that patients with larger tumors may have worse survival . leiomyosarcoma is a rare soft tissue tumor that can occur anywhere in the human body . several factors have been reported to predict poor outcomes in patients with leiomyosarcoma , including advanced age , vascular invasion , dna aneuploidy , and c - myc expression , , . in particular , c - myc expression was reported to be a marker for poor prognosis in leiomyosarcoma . in this study , we have identified that prune2 protein expression level can serve as a prognostic factor for leiomyosarcoma . in conclusion , we report the association between prune2 protein expression and prognosis in human leiomyosarcoma . survival time was longer in tmucih leiomyosarcoma patients with higher expression of prune2 protein . this suggests that prune2 may be involved in the process of leiomyosarcoma development and can be regarded as a biomarker for favorable prognosis . further study is necessary to better understand the role that racial factors play for the relationship between prune2 protein expression and prognosis in patients with leiomyosarcoma , and discover new therapeutic strategies against aggressive leiomyosarcoma .