surgically treated patients with hepatocellular carcinoma ( hcc ) , which represent a highly selected group , have higher survival rates compared to those of medically treated patients at a comparable stage .
however , long - term prognosis remains unsatisfactory because of the high incidence of tumor recurrence and metastasis after hepatectomy .
thus , identification of markers of poor prognosis is important in order to provide the opportunity for timely intervention .
high proliferation rate , a classic hallmark of cancer , is due to the self - sufficiency of growth signals , insensitivity to anti - growth signals , and limitless replicative potential . a variety of methods , including analysis of proliferating cell nuclear antigen , bromodeoxyuridine , argyrophilic nuclear organized regions , ki-67 nuclear antigen , and phosphorylated histone h3 , are used in evaluation of proliferative activity [ 5 - 7 ] . however , many of these methods can not be applied in daily clinical practice . in contrast
, the mitotic index , which is a useful and simple method for analysis of cell proliferation , can be easily applied to routine clinical practice .
the prognostic role of mitotic index in patient survival has been confirmed in several cancers .
in addition , mitotic index has been incorporated in the american joint committee on cancer ( ajcc ) seventh tumor staging system for malignant melanoma , gastrointestinal tumor , and neuroendocrine tumors of the gastrointestinal tract . in hccs
, previous studies indicated a potential role of high mitotic index as an adverse prognostic indicator in cohorts of fewer than 200 patients .
however , the practical utility of mitotic index as a predictor of prognosis in patients with hcc has not been determined . in this study
, we evaluated mitotic index as a possible prognostic marker in a large cohort of 282 patients with primary hcc who received long - term follow - up for 120 months .
we also attempted to determine the cutoff value for mitotic index that showed the most significant prognostic role in hcc patients .
a total of 290 patients who were pathologically confirmed to have primary hcc and underwent curative resection at samsung medical center , seoul , korea between july 2000 and may 2006 were enrolled in this study .
eight patients who received preoperative treatments , including transcatheter arterial chemoembolization , radiofrequency ablation , and radiation therapy , were excluded ; therefore , 282 patients were included in this study .
curative resection was defined as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors on computed tomography scans performed 1 month after surgery .
clinical parameters , including age , gender , date of surgery , serum -fetoprotein ( afp ) , and serum albumin , were obtained by reviewing the medical records .
when the tumor was less than 3 cm in size , all tumors were sectioned and embedded .
when the tumor was larger than 3 cm in size , at least four sections were taken for the pathologic examinations and the mean number of blocks was one for 1 cm of tumor diameter .
histopathologic features of hccs , including histologic differentiation , microvascular invasion , major portal vein invasion , intrahepatic metastasis , multicentric occurrence , and non - tumor liver pathology , were reviewed by two pathologists ( s.y.h . and c .- k.p . ) .
intrahepatic metastasis and multicentric occurrence were determined according to the criteria of the liver cancer study group of japan .
hcc recurrence within the first two years following surgery is mainly due to intrahepatic metastasis , whereas late recurrence usually results from multicentric occurrence . using 2 years as a cutoff ,
all patients were staged according to the ajcc staging system   and barcelona clinic liver cancer ( bclc ) staging classification . during follow - up ,
serum afp levels were monitored and three phase dynamic computed tomography scan or magnetic resonance imaging was performed every 3 months after surgery . the median follow - up period was 120 months ( range , 14 to 151 months ) for survivors .
recurrence - free survival ( rfs ) was measured from the date of surgery until detection of tumor recurrence .
disease - specific survival ( dss ) was defined as the interval between the date of surgery and the date of hcc - related death , which was defined as : ( 1 ) the 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 , and ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation . two liver pathologists ( s.y.h . and c .- k.p . )
counted the number of mitotic cells in 10 high - power fields ( hpfs ) of hematoxylin and eosin - stained slides , and found areas containing the most mitotic figures , the so - called hot spot . after counting the mitoses in the hot spot ,
if no hot spot could be found and mitoses were sparse and randomly scattered throughout the lesion , a representative mitosis was chosen and the count began with that field and was extended to adjacent fields . according to the criteria of mitotic figures defined by baak , mitotic cells were counted only if there was complete concordance between the two pathologists using a multi - head microscope .
the x - tile statistics package ( yale university , new haven , ct ) was used to determine the optimal cutoff value with the highest level of statistical significance related to patient survival ; cases were dichotomized into low and high mitotic index groups based on the established cutoff value .
analysis of the association between mitotic index and clinicopathologic parameters was performed using the chi - square test , fisher exact test , or cochran armitage test .
differences in survival rates were assessed using the log - rank test or breslow test .
the cox proportional hazard regression model was used to assess the association between clinicopathologic factors and survival time .
we examined the proportional hazard assumption graphically to determine whether variables in the cox proportional hazard model were constants that do not depend on time .
a total of 290 patients who were pathologically confirmed to have primary hcc and underwent curative resection at samsung medical center , seoul , korea between july 2000 and may 2006 were enrolled in this study .
eight patients who received preoperative treatments , including transcatheter arterial chemoembolization , radiofrequency ablation , and radiation therapy , were excluded ; therefore , 282 patients were included in this study .
curative resection was defined as complete resection of all tumor nodules with clear microscopic resection margins and no residual tumors on computed tomography scans performed 1 month after surgery .
clinical parameters , including age , gender , date of surgery , serum -fetoprotein ( afp ) , and serum albumin , were obtained by reviewing the medical records .
when the tumor was less than 3 cm in size , all tumors were sectioned and embedded .
when the tumor was larger than 3 cm in size , at least four sections were taken for the pathologic examinations and the mean number of blocks was one for 1 cm of tumor diameter .
histopathologic features of hccs , including histologic differentiation , microvascular invasion , major portal vein invasion , intrahepatic metastasis , multicentric occurrence , and non - tumor liver pathology , were reviewed by two pathologists ( s.y.h . and c .- k.p . ) .
intrahepatic metastasis and multicentric occurrence were determined according to the criteria of the liver cancer study group of japan .
hcc recurrence within the first two years following surgery is mainly due to intrahepatic metastasis , whereas late recurrence usually results from multicentric occurrence . using 2 years as a cutoff ,
all patients were staged according to the ajcc staging system   and barcelona clinic liver cancer ( bclc ) staging classification . during follow - up ,
serum afp levels were monitored and three phase dynamic computed tomography scan or magnetic resonance imaging was performed every 3 months after surgery . the median follow - up period was 120 months ( range , 14 to 151 months ) for survivors .
recurrence - free survival ( rfs ) was measured from the date of surgery until detection of tumor recurrence .
disease - specific survival ( dss ) was defined as the interval between the date of surgery and the date of hcc - related death , which was defined as : ( 1 ) the 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 , and ( 5 ) variceal hemorrhage with portal venous tumor thrombus proximal to the first bifurcation .
two liver pathologists ( s.y.h . and c .- k.p . ) counted the number of mitotic cells in 10 high - power fields ( hpfs ) of hematoxylin and eosin - stained slides , and found areas containing the most mitotic figures , the so - called hot spot . after counting the mitoses in the hot spot ,
if no hot spot could be found and mitoses were sparse and randomly scattered throughout the lesion , a representative mitosis was chosen and the count began with that field and was extended to adjacent fields . according to the criteria of mitotic figures defined by baak , mitotic cells were counted only if there was complete concordance between the two pathologists using a multi - head microscope .
the x - tile statistics package ( yale university , new haven , ct ) was used to determine the optimal cutoff value with the highest level of statistical significance related to patient survival ; cases were dichotomized into low and high mitotic index groups based on the established cutoff value .
analysis of the association between mitotic index and clinicopathologic parameters was performed using the chi - square test , fisher exact test , or cochran armitage test .
differences in survival rates were assessed using the log - rank test or breslow test .
the cox proportional hazard regression model was used to assess the association between clinicopathologic factors and survival time .
we examined the proportional hazard assumption graphically to determine whether variables in the cox proportional hazard model were constants that do not depend on time .
the median patient age was 53 years ( range , 17 to 76 years ) ; 234 patients were males , and 48 were females .
two hundred and eighteen patients ( 77.3% ) were infected with hepatitis b virus , and 26 ( 9.2% ) with hepatitis c virus .
two hundred and three patients ( 72.0% ) suffered from tumor recurrence ; 153 patients ( 54.3% ) from early recurrence , and 50 patients ( 17.7% ) from late recurrence .
twenty - nine of the 127 deaths were due to non - hcc  related causes .
seventeen of the 29 deaths were due to hepatic failure ; eight were due to non - hepatic causes , and four were due to unknown causes .
the mean mitotic index was 7.75 ( 95% confidence interval , 6.47 to 9.03 ) and the median value was 3.00 ( range , 0 to 60 ) . using the x - tile package , mitotic index was graded as low ( 4 or less mitoses per 10 hpfs ) or high ( 5 or more mitoses per 10 hpfs ) ( fig .
high mitotic index showed significant association with younger age ( p < 0.001 ) , larger tumor size ( p=0.022 ) , higher edmondson grade ( p < 0.001 ) , microvascular invasion ( p < 0.001 ) , major portal vein invasion ( p=0.026 ) , intrahepatic metastasis ( p < 0.001 ) , higher afp level ( p < 0.001 ) , hepatitis b virus etiology ( p=0.012 ) , and liver cirrhosis ( p=0.016 ) . as the ajcc t - stage or bclc stage increased , the frequency of high mitotic index also showed a significant increase ( p < 0.001 and p < 0.001 , respectively ) .
the 3- , 5- , 7- , and 9-year rfs rates for 282 hcc patients were 43.5% , 37.2% , 31.1% , and 30.0% , respectively .
the 3- , 5- , 7- , and 9-year dss rates were 75.1% , 67.1% , 59.6% , and 53.4% , respectively .
patients with high mitotic index had shorter dss ( p < 0.001 ) and tended to have shorter rfs ( p=0.112 ) ( fig .
2 ) . by applying the breslow test , which attributes greater weight to earlier events , patients with high mitotic index had shorter rfs ( p=0.003 ) .
in subgroup analysis among patients with a larger tumor size ( > 5 cm ) , microvascular invasion , intrahepatic metastasis , higher ajcc t - stage , and higher blcl stage , high mitotic index showed unfavorable influences on dss ( p=0.001 , p=0.008 , p=0.003 , p=0.012 , and p < 0.001 , respectively ) ( fig .
larger tumor size , edmondson grade iii , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t - stage , higher bclc stage , lower albumin level , and higher afp level showed unfavorable influences on both rfs and dss .
in addition , we found that high mitotic index showed an unfavorable influence on dss ( p < 0.001 ) ( table 2 ) . because ajcc t - stage and bclc stage were associated with vascular invasion , to avoid potential bias we did not perform multiple analyses using these indices . in multivariate analysis , intrahepatic metastasis and lower albumin level
larger tumor size and high mitotic index ( p=0.004 ) were found to be independent predictors of shorter dss .
patients with high mitotic index were more likely to suffer from disease - specific death compared to those with low mitotic index ( hazard ratio , 1.818 ) ( table 3 ) .
multivariate analysis of rfs with mitotic index could not be performed , because the survival curves according to mitotic index were crossed and the proportional hazard assumption was violated .
the median patient age was 53 years ( range , 17 to 76 years ) ; 234 patients were males , and 48 were females .
two hundred and eighteen patients ( 77.3% ) were infected with hepatitis b virus , and 26 ( 9.2% ) with hepatitis c virus .
two hundred and three patients ( 72.0% ) suffered from tumor recurrence ; 153 patients ( 54.3% ) from early recurrence , and 50 patients ( 17.7% ) from late recurrence .
twenty - nine of the 127 deaths were due to non - hcc  related causes .
seventeen of the 29 deaths were due to hepatic failure ; eight were due to non - hepatic causes , and four were due to unknown causes .
the mean mitotic index was 7.75 ( 95% confidence interval , 6.47 to 9.03 ) and the median value was 3.00 ( range , 0 to 60 ) . using the x - tile package , mitotic index was graded as low ( 4 or less mitoses per 10 hpfs ) or high ( 5 or more mitoses per 10 hpfs ) ( fig .
high mitotic index showed significant association with younger age ( p < 0.001 ) , larger tumor size ( p=0.022 ) , higher edmondson grade ( p < 0.001 ) , microvascular invasion ( p < 0.001 ) , major portal vein invasion ( p=0.026 ) , intrahepatic metastasis ( p < 0.001 ) , higher afp level ( p < 0.001 ) , hepatitis b virus etiology ( p=0.012 ) , and liver cirrhosis ( p=0.016 ) . as the ajcc t - stage or bclc stage increased , the frequency of high mitotic index also showed a significant increase ( p < 0.001 and p < 0.001 , respectively ) .
the 3- , 5- , 7- , and 9-year rfs rates for 282 hcc patients were 43.5% , 37.2% , 31.1% , and 30.0% , respectively .
the 3- , 5- , 7- , and 9-year dss rates were 75.1% , 67.1% , 59.6% , and 53.4% , respectively .
patients with high mitotic index had shorter dss ( p < 0.001 ) and tended to have shorter rfs ( p=0.112 ) ( fig .
2 ) . by applying the breslow test , which attributes greater weight to earlier events , patients with high mitotic index had shorter rfs ( p=0.003 ) .
in subgroup analysis among patients with a larger tumor size ( > 5 cm ) , microvascular invasion , intrahepatic metastasis , higher ajcc t - stage , and higher blcl stage , high mitotic index showed unfavorable influences on dss ( p=0.001 , p=0.008 , p=0.003 , p=0.012 , and p < 0.001 , respectively ) ( fig .
larger tumor size , edmondson grade iii , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher ajcc t - stage , higher bclc stage , lower albumin level , and higher afp level showed unfavorable influences on both rfs and dss .
in addition , we found that high mitotic index showed an unfavorable influence on dss ( p < 0.001 ) ( table 2 ) . because ajcc t - stage and bclc stage were associated with vascular invasion , to avoid potential bias we did not perform multiple analyses using these indices . in multivariate analysis , intrahepatic metastasis and lower albumin level
larger tumor size and high mitotic index ( p=0.004 ) were found to be independent predictors of shorter dss .
patients with high mitotic index were more likely to suffer from disease - specific death compared to those with low mitotic index ( hazard ratio , 1.818 ) ( table 3 ) .
multivariate analysis of rfs with mitotic index could not be performed , because the survival curves according to mitotic index were crossed and the proportional hazard assumption was violated .
in the current study , we elucidated the prognostic significance of mitotic index in a large cohort of patients with primary hcc who received long - term follow - up .
we attempted to establish a cutoff value for mitotic index that showed the most significant prognostic role in hcc patients .
low mitotic index was defined as four or fewer mitoses per 10 hpfs , and high mitotic index as five or more .
high mitotic index showed correlation with larger tumor size , higher edmondson grade , microvascular invasion , major portal invasion , intrahepatic metastasis , higher ajcc t - stage , higher bclc stage , higher afp level , hepatitis b virus etiology , and liver cirrhosis .
in addition , patients with high mitotic index had shorter dss and tended to have shorter rfs .
the mean time gap between recurrence and death in the high mitotic index group was significantly lower than in the low mitotic index group ( 30.5 months vs. 43.8 months , p=0.004 ) .
treatment modality after recurrence did not differ statistically between low versus high mitotic index group ( p=0.38 ) ( supplementary table 1 ) .
it is assumed that the high mitotic index group had poorer response to the salvage treatment after recurrence than the low mitotic index group . only a few studies on the prognostic role of mitotic index in hcc
haratake et al . , who divided mitotic index into three groups ( 0 - 4/10 hpfs , 5 - 9/10 hpfs , 10 and more/10 hpfs ) , observed a better prognosis for patients with hcc who exhibited low mitotic activity .
they reported that longer survival periods were observed in some patients with larger tumors if the mitotic indices were low .
ouchi et al .   showed that mitotic index was an independent significant variable , influencing the overall survival of 40 patients with hcc following hepatic resection .
in addition , they found that hcc with a high mitotic index (  5/10 hpfs ) was associated with multiple tumors and advanced tumor stage .
they also reported a highly significant correlation between mitotic index and ki-67 labelling index ( r=0.870 ) .
nanashima et al .   also reported correlation of high mitotic index (  5/10 hpfs ) with overall survival in 81 patients with hcc by univariate analysis .
they also found a significant association of high mitotic index with vascular invasion , poor histologic differentiation , and recurrence rate .
interestingly , the criteria of mitotic index used in previous studies are the same as those applied in our study , which were determined statistically for the strongest prognostic effect . to the best of our knowledge ,
this is the first report to show that high mitotic index is an independent predictor of shorter dss .
hcc recurrence within the first 2 years following surgery is mainly due to intrahepatic metastasis , whereas recurrence after 2 years following surgery usually results from multicentric occurrence of hcc .
it is assumed that the high mitotic index group did not show shorter rfs after 48 months following surgery , because recurrence after 48 months following surgery was mainly due to newly developed hcc .
the most outstanding finding of this study is that subgroup analysis among patients with larger tumor size , microvascular invasion , intrahepatic metastasis , higher ajcc t - stage , and higher blcl stage showed unfavorable influences of high mitotic index on dss . counting mitotic cells is a simple , rapid , and inexpensive method which can be routinely performed in daily practice .
our data show , for the first time , that high mitotic index is an independent predictor of shorter dss in surgically resected hccs .
prospective studies are needed to further investigate the role of mitotic index as a prognostic factor in hcc .
 purposehigh proliferation rate is a hallmark of cancer . 
 the mitotic index is a useful and simple method for analysis of cell proliferation . 
 however , the practical utility of mitotic index as a predictor of prognosis in patients with hepatocellular carcinoma ( hcc ) has not been determined . 
 therefore , we examined mitotic index as a prognostic marker in hcc patients.materials and methodswe counted the number of mitotic cells in 10 high - power fields of the tumor area on hematoxylin and eosin  stained slides representing 282 surgically resected hccs . 
 the highest number of mitotic cells was defined as the mitotic index.resultshigh mitotic index was observed in 127 of 282 hccs . 
 high mitotic index showed significant association with younger age , larger tumor size , higher edmondson grade , microvascular invasion , major portal vein invasion , intrahepatic metastasis , higher american joint committee on cancer ( ajcc ) t - stage , higher barcelona clinic liver cancer ( bclc ) stage , higher alpha - fetoprotein level , hepatitis b virus etiology , and liver cirrhosis . 
 patients with high mitotic index had shorter disease - specific survival ( dss ) ( p < 0.001 ) and tended to have shorter recurrence - free survival ( p=0.112 ) . in subgroup analysis among patients with a larger tumor size , microvascular invasion , intrahepatic metastasis , 
 higher ajcc t - stage , and higher blcl stage , high mitotic index showed unfavorable influences on dss ( p=0.001 , p=0.008 , p=0.003 , p=0.012 , and p < 0.001 , respectively ) . 
 in addition , high mitotic index was an independent predictor of shorter dss ( p=0.004).conclusionhigh mitotic index may be a novel predictor of dss in patients with hcc and may have utility as an auxiliary prognostic factor in hcc . 