hepatocellular carcinoma ( hcc ) is one of the most lethal malignancies , and surgical resection improves the survival rates for patients . however , the prognosis after surgical resection of hcc remains poor because of high recurrence rates . using biomarkers to identify patients presenting with a higher risk of poor prognosis may reduce mortality after curative hepatectomy . although there are many reports on histologic parameters for predicting hcc prognosis , molecular markers for hcc recurrence and prognosis could provide additional information . cancer cells frequently express genes that are specifically or preferentially expressed in male germ cells under normal conditions . atpase family aaa domain - containing 2 ( atad2 ) is one such , and works as an important cofactor for myc - dependent transcription . through myc and e2f transcription factors , atad2 increases the expression of proliferation - related and anti - apoptotic genes in many different types of cancer , including breast carcinoma , non - small cell lung carcinoma , and prostate carcinoma [ 5 - 8 ] . , atad2 has been identified as a candidate driver gene located within the amplified 8q24 locus . huang et al .   reported that a novel highly up - regulated exon - exon junction was detected in atad2 gene by rna - seq and the gene was highly expressed in hcc tissues . a recent study found that the high expression of atad2 in hcc was an independent predictor of shortened overall survival . however , the prognostic significance of atad2 protein expression in hcc is unclear . in the present study , we evaluated atad2 protein expression by immunohistochemistry to elucidate the prognostic role of atad2 in 182 hcc patients with long - term follow - up . hcc tissue samples were collected from 182 patients who were treated with surgical resection between july 2000 and may 2006 at samsung medical center ( seoul , korea ) . inclusion criteria were histologically confirmed hcc and curative resection of tumor without preoperative or postoperative adjuvant therapy . we defined curative resection as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors as indicated by a computed tomography scan one month after surgery . microvascular invasion was considered present when at least one or more endothelial cells or the tunica media of the vessel surrounded a neoplastic cell group . tumor stage was determined by both the american joint committee on cancer ( ajcc ) staging system   and barcelona clinic liver cancer ( bclc ) staging classification . using 2 years as the cut off , all hcc patients were followed by monitoring serum -fetoprotein levels and three phase dynamic computed tomography scans or magnetic resonance imaging every three months after surgery . the follow - up period for recurrence was at least 18 months , and the median follow - up period was 120.0 months ( range , 14.0 to 151.4 months ) for survivors . recurrence - free survival ( rfs ) was defined as from the date of resection until the detection of tumor recurrence . we chose disease - specific death ( hcc - related death ) as the clinical endpoint for survival analysis , defined as : ( 1 ) tumor occupying more than 80% of the liver , ( 2 ) portal venous tumor thrombus proximal to the second bifurcation , ( 3 ) obstructive jaundice due to the tumor , ( 4 ) distant metastases , or ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . disease - specific survival ( dss ) was defined from the date of resection to the date of hcc - related death . histologic sections were examined by two pathologists and representative tumor regions were marked in the formalin - fixed paraffin - embedded blocks . two tissue cores measuring 2.0 mm in diameter were punched from the marked areas of each block and arranged into new paraffin blocks . as controls , two cores of normal liver tissue from 12 patients with metastatic colon carcinoma of the liver were included in each array block . antigen retrieval was performed with 0.01 mol / l citrate buffer at ph 6.0 for 30 minutes in a pressure cooker . sections were incubated with a rabbit polyclonal antibody to atad2 ( 1:100 , nbp1 - 84122 , novus biologicals , littleton , co ) for 60 minutes at room temperature . the positive control ( human normal testis ) showed intense nuclear atad2 expression in spermatogenic cells of seminiferous tubules . no immunoreactivity was observed in tissue sections used as negative control where the primary antibody was replaced by preimmune rabbit serum . in order to validate the concordance between tissue microarrays and whole tumor sections , we observed atad2 expression for 40 corresponding whole tumor sections randomly chosen from the 182 cases . and h.w.h . ) who were blinded to the clinical details , and any discrepancies were resolved by consensus . a nearly homogeneous nuclear immunostaining with moderate staining intensity was observed . for determining atad2 expression , the proportion of stained tumor cells was determined semi - quantitatively and each sample was scored on a scale of 0 - 4 ( 0 , < 5% ; 1 , 5%-25% ; 2 , 26%-50% ; 3 , 51%-75% ; 4 , > 75% ) . duplicate tissue cores for each tumor showed high levels of homogeneity for proportion of stained cells . the association of atad2 expression with clinicopathologic features was examined by the chi - square test or fisher exact test . univariate and multivariate analyses of risks for tumor recurrence were performed using the logistic regression model . cumulative survival rates were calculated by the kaplan - meier method and analyzed by the log - rank test . the cox proportional hazards regression model was used to assess the association of survival time regressed upon multiple clinicopathologic variables . variables that were statistically significant in the univariate analysis were included in the multivariate analysis . hcc tissue samples were collected from 182 patients who were treated with surgical resection between july 2000 and may 2006 at samsung medical center ( seoul , korea ) . inclusion criteria were histologically confirmed hcc and curative resection of tumor without preoperative or postoperative adjuvant therapy . we defined curative resection as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors as indicated by a computed tomography scan one month after surgery . microvascular invasion was considered present when at least one or more endothelial cells or the tunica media of the vessel surrounded a neoplastic cell group . tumor stage was determined by both the american joint committee on cancer ( ajcc ) staging system   and barcelona clinic liver cancer ( bclc ) staging classification . using 2 years as the cut off , all hcc patients were followed by monitoring serum -fetoprotein levels and three phase dynamic computed tomography scans or magnetic resonance imaging every three months after surgery . the follow - up period for recurrence was at least 18 months , and the median follow - up period was 120.0 months ( range , 14.0 to 151.4 months ) for survivors . recurrence - free survival ( rfs ) was defined as from the date of resection until the detection of tumor recurrence . we chose disease - specific death ( hcc - related death ) as the clinical endpoint for survival analysis , defined as : ( 1 ) tumor occupying more than 80% of the liver , ( 2 ) portal venous tumor thrombus proximal to the second bifurcation , ( 3 ) obstructive jaundice due to the tumor , ( 4 ) distant metastases , or ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . disease - specific survival ( dss ) was defined from the date of resection to the date of hcc - related death . histologic sections were examined by two pathologists and representative tumor regions were marked in the formalin - fixed paraffin - embedded blocks . two tissue cores measuring 2.0 mm in diameter were punched from the marked areas of each block and arranged into new paraffin blocks . as controls , two cores of normal liver tissue from 12 patients with metastatic colon carcinoma of the liver were included in each array block . antigen retrieval was performed with 0.01 mol / l citrate buffer at ph 6.0 for 30 minutes in a pressure cooker . sections were incubated with a rabbit polyclonal antibody to atad2 ( 1:100 , nbp1 - 84122 , novus biologicals , littleton , co ) for 60 minutes at room temperature . the positive control ( human normal testis ) showed intense nuclear atad2 expression in spermatogenic cells of seminiferous tubules . no immunoreactivity was observed in tissue sections used as negative control where the primary antibody was replaced by preimmune rabbit serum . in order to validate the concordance between tissue microarrays and whole tumor sections , we observed atad2 expression for 40 corresponding whole tumor sections randomly chosen from the 182 cases . all sections were scored by two independent pathologists ( c .- k.p . and h.w.h . ) who were blinded to the clinical details , and any discrepancies were resolved by consensus . a nearly homogeneous nuclear immunostaining with moderate staining intensity was observed . for determining atad2 expression , the proportion of stained tumor cells was determined semi - quantitatively and each sample was scored on a scale of 0 - 4 ( 0 , < 5% ; 1 , 5%-25% ; 2 , 26%-50% ; 3 , 51%-75% ; 4 , > 75% ) . duplicate tissue cores for each tumor showed high levels of homogeneity for proportion of stained cells . when there were differences between duplicate tissue cores the association of atad2 expression with clinicopathologic features was examined by the chi - square test or fisher exact test . univariate and multivariate analyses of risks for tumor recurrence were performed using the logistic regression model . cumulative survival rates were calculated by the kaplan - meier method and analyzed by the log - rank test . the cox proportional hazards regression model was used to assess the association of survival time regressed upon multiple clinicopathologic variables . variables that were statistically significant in the univariate analysis were included in the multivariate analysis . the mean age of the 182 hcc patents was 52.4 years ( range , 17 to 76 years ) , and 82.4% of the patients were male . chronic hepatitis b virus infection was detected in 141 patients ( 77.5% ) and chronic hepatitis c virus infection in 16 patients ( 8.8% ) . the mean tumor size was 5.3 cm , and 109 of the 182 tumors ( 59.9% ) were  5 cm in size . tumor recurrence was detected in 132 patients ( 72.5% ) , early recurrence in 108 patients ( 59.3% ) , and late recurrence in 24 patients ( 13.2% ) . fifteen of the 91 deaths in this study were due to non - hcc causes . nine of the 15 deaths were due to hepatic failure , five due to non - hepatic causes , and one due to unknown cause . immunoreactivity for atad2 was observed only in the nuclei of tumor cells with moderate staining intensity . we regarded the atad2 as positive when  5% of tumor cells showed nuclear immunoreactivity ( fig . atad2 expression was significantly associated with larger tumor size ( p=0.001 ) , higher edmondson grade ( p=0.015 ) , microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p atad2 expression was associated with the early recurrence ( p < 0.001 ) , but not with the late recurrence ( p=0.619 ) ( table 1 ) . in univariate analyses , early recurrence was significantly associated with larger tumor size ( p=0.019 ) , microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher ajcc t - stage ( p < 0.001 ) , lower albumin level ( p=0.041 ) , viral etiology ( p=0.004 ) , and atad2 expression ( p < 0.001 ) . as ajcc t - stage and bclc stage were associated with vascular invasion , we did not make multiple analyses with these variables to avoid potential bias . an evaluation of the association of serum -fetoprotein level with early recurrence was not performed due to missing data ( n=175 ) . in multivariate analyses , microvascular invasion ( p=0.003 ) , intrahepatic metastasis ( p=0.003 ) , and viral etiology ( p=0.029 ) were independent predictors of early recurrence . atad2 expression tended to be independent predictor of early recurrence ( p=0.059 ) ( table 2 ) . the rfs and dss rates for 182 hcc patients were 36.1% and 75.5% at 3 years , 32.1% and 66.7% at 5 years , 27.5% and 61.1% at 7 years , and 26.6% and 55.2% at 9 years , respectively . on univariate analyses , larger tumor size , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t - stage , higher bclc stage , and lower albumin level showed unfavorable influences on both rfs and dss . the 5-year rfs rate of the atad2-positive group was significantly lower than that of the atad2-negative group ( 23.1% vs. 48.6% ) ( fig . the mean rfs of atad2-positive group was 37.6 months and the atad2-negative group was 74.0 months . subgroup analysis among patients with tumor size  5.0 cm ( n=109 ) , patients at bclc stage 0 or a ( n=92 ) , and patients with -fetoprotein  20 ng / ml ( n=61 ) , the atad2-positive groups ( n=61 , n=50 , and n=30 , respectively ) also unfavorably influenced rfs ( p=0.008 , p=0.009 , and p=0.013 , respectively ) ( fig . the 5-year dss rate of the atad2-positive group was significantly lower than that of the atad2-negative group ( 57.8% vs. 83.4% ) ( fig . the mean dss of atad2-positive group was 88.1 months and of atad2-negative group , 123.3 months . multivariate analyses indicated that intrahepatic metastasis and lower albumin level were found to be independent predictors of both shorter rfs and shorter dss . atad2 expression ( p=0.002 ) was an independent predictor of shorter rfs , but not of dss ( p=0.109 ) . atad2-positive patients were more likely to suffer from recurrence than atad2-negative patients ( hazard ratio , 1.857 ) ( table 4 ) . the mean age of the 182 hcc patents was 52.4 years ( range , 17 to 76 years ) , and 82.4% of the patients were male . chronic hepatitis b virus infection was detected in 141 patients ( 77.5% ) and chronic hepatitis c virus infection in 16 patients ( 8.8% ) . the mean tumor size was 5.3 cm , and 109 of the 182 tumors ( 59.9% ) were  5 cm in size . tumor recurrence was detected in 132 patients ( 72.5% ) , early recurrence in 108 patients ( 59.3% ) , and late recurrence in 24 patients ( 13.2% ) . fifteen of the 91 deaths in this study were due to non - hcc causes . nine of the 15 deaths were due to hepatic failure , five due to non - hepatic causes , and one due to unknown cause . immunoreactivity for atad2 was observed only in the nuclei of tumor cells with moderate staining intensity . we regarded the atad2 as positive when  5% of tumor cells showed nuclear immunoreactivity ( fig . atad2 expression was significantly associated with larger tumor size ( p=0.001 ) , higher edmondson grade ( p=0.015 ) , microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher ajcc t - stage ( p atad2 expression was associated with the early recurrence ( p < 0.001 ) , but not with the late recurrence ( p=0.619 ) ( table 1 ) . in univariate analyses , early recurrence was significantly associated with larger tumor size ( p=0.019 ) , microvascular invasion ( p < 0.001 ) , intrahepatic metastasis ( p < 0.001 ) , higher ajcc t - stage ( p < 0.001 ) , lower albumin level ( p=0.041 ) , viral etiology ( p=0.004 ) , and atad2 expression ( p < 0.001 ) . as ajcc t - stage and bclc stage were associated with vascular invasion , we did not make multiple analyses with these variables to avoid potential bias . an evaluation of the association of serum -fetoprotein level with early recurrence was not performed due to missing data ( n=175 ) . in multivariate analyses , microvascular invasion ( p=0.003 ) , intrahepatic metastasis ( p=0.003 ) , and viral etiology ( p=0.029 ) were independent predictors of early recurrence . atad2 expression tended to be independent predictor of early recurrence ( p=0.059 ) ( table 2 ) . the rfs and dss rates for 182 hcc patients were 36.1% and 75.5% at 3 years , 32.1% and 66.7% at 5 years , 27.5% and 61.1% at 7 years , and 26.6% and 55.2% at 9 years , respectively . on univariate analyses , larger tumor size , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t - stage , higher bclc stage , and lower albumin level showed unfavorable influences on both rfs and dss . the 5-year rfs rate of the atad2-positive group was significantly lower than that of the atad2-negative group ( 23.1% vs. 48.6% ) ( fig . the mean rfs of atad2-positive group was 37.6 months and the atad2-negative group was 74.0 months . subgroup analysis among patients with tumor size  5.0 cm ( n=109 ) , patients at bclc stage 0 or a ( n=92 ) , and patients with -fetoprotein  20 ng / ml ( n=61 ) , the atad2-positive groups ( n=61 , n=50 , and n=30 , respectively ) also unfavorably influenced rfs ( p=0.008 , p=0.009 , and p=0.013 , respectively ) ( fig . the 5-year dss rate of the atad2-positive group was significantly lower than that of the atad2-negative group ( 57.8% vs. 83.4% ) ( fig . the mean dss of atad2-positive group was 88.1 months and of atad2-negative group , 123.3 months . multivariate analyses indicated that intrahepatic metastasis and lower albumin level were found to be independent predictors of both shorter rfs and shorter dss . atad2 expression ( p=0.002 ) was an independent predictor of shorter rfs , but not of dss ( p=0.109 ) . atad2-positive patients were more likely to suffer from recurrence than atad2-negative patients ( hazard ratio , 1.857 ) ( table 4 ) . atad2 contributes to the development of aggressive cancer through the enhancement of myc - dependent transcription . atad2 expression is high in several human cancers , including breast carcinoma , colon carcinoma , lung carcinoma , stomach carcinoma , and uterine carcinoma . high atad2 expression is a strong predictor of rapid mortality in lung and breast cancers . atad2 depletion by rna interference knockdown reduced huh7 and hcclm3 cell proliferation and led to a g1 phase cell cycle arrest . in hcc cells , atad2 regulates cell migration / invasion via the regulation of apc and/or ctnna1 expression . in this study , we evaluated the prognostic significance of atad2 protein expression in hcc and demonstrated that atad2 expression is significantly associated with larger tumor size , higher edmondson grade , microvascular invasion , intrahepatic metastasis , higher ajcc t - stage , and higher bclc stage . moreover , atad2 expression is an independent predictor of shorter rfs and tended to predict early recurrence . in the present study , early stage hcc patients with tumor size  5.0 cm or patients at bclc stage 0 or a have favorable outcomes in both rfs and dss ( table 3 ) , but 75 of 109 ( 68.8% ) with tumor size  5.0 cm and 59 of 92 ( 64.1% ) bclc stage 0 or a developed tumor recurrence . we found within these populations , atad2 positivity unfavorably influenced rfs ( p=0.008 and p=0.009 , respectively ) . identification of patients with poor prognosis in early stage hcc is critical to the optimization of personalized treatment . thus , our findings support the possible importance of atad2 expression in hcc in detecting a phenotype that can predict recurrence risk in early stage hcc . serum -fetoprotein is widely used to screen , mainly for hcc , and is an important predictor of patient survival following tumor resection . preoperative high serum -fetoprotein level ( > 60 ng / ml ) could be a risk factor for recurrence after resection in patients with liver cirrhosis . however , there is no effective marker for monitoring recurrence for patients with normal serum -fetoprotein level (  20 ng / ml ) after hepatectomy . in this study , tumor recurrence was detected in 40 of 61 patients ( 65.6% ) with normal serum -fetoprotein level . we found that within patients with normal serum -fetoprotein level , the atad2-positive group still had significantly poorer rfs than the atad2-negative group ( p=0.013 ) . thus , our study revealed the potential use of atad2 in predicting recurrence risk in the patient group in which serum -fetoprotein levels were not prognostically predictive . our findings indicate that atad2 protein may be a novel potential predictor of rfs in hcc patients after curative resection , and atad2 may have prognostic value in hcc patients with early stage hcc or normal serum -fetoprotein level . further study is needed to examine the roles of atad2 protein expression in the development and progression of hcc .