this study was approved by the institutional review board of seoul national university bundang hospital ( b-1502 - 286 - 302 ) .
one hundred and ninety consecutive cases of surgically resected or explanted hccs were enrolled in this study , and were retrieved from the surgical pathology files of seoul national university bundang hospital from may 2003 to april 2010 .
the electronic medical records , surgical pathology reports , and the hematoxylin and eosin - stained slides were reviewed for each case , and the clinicopathological variables noted included tumor size , multiplicity ( including intrahepatic metastasis / satellite nodules and multicentric occurrences ) , histological differentiation according to the edmondson - steiner grade ( the highest grade in tumors with heterogeneity ) , the presence of microvascular or major vascular invasion , the patient demographics ( age and sex ) , and the presence of an underlying etiology ( e.g. , hepatitis b , c , alcohol ) .
follow - up data was also obtained from the medical records , including recurrences ( including local recurrence and distant metastasis ) and death .
the median follow - up period was 52 months ( range , 0 to 133 months ) .
overall survival was defined as thfe interval from initial surgical treatment for hcc to the date of death , and disease - free survival as the interval from initial treatment to local or distant recurrence .
tissue cores measuring 2 mm in diameter were sampled from formalin - fixed paraffin - embedded hcc tissues and arranged in recipient tissue array blocks using a trephine apparatus ( superbiochips laboratories , seoul , korea ) .
two or three cores were sampled from each hcc , depending on the amount of histological heterogeneity present in the tumor .
four m - thick sections were obtained from the tissue microarray blocks and subjected to immunohistochemical staining for sall4 ( 1:100 , mouse monoclonal antibody , clone ee-30 , santa cruz technologies inc . , santa cruz , ca , usa ) , k19 ( 1:150 , mouse monoclonal antibody , clone ba17 , dako , glostrup , denmark ) and epcam ( 1:3,000 , mouse monoclonal antibody , clone vu-1d9 , calbiochem , darmstadt , germany ) .
briefly , tissue sections were deparaffinized in xylene , rehydrated in graded alcohol , and antigen retrieval was performed using citrate buffer ( ph 6.0 ) for 15 minutes .
sections were incubated with the primary antibodies at room temperature for 30 minutes , and then incubated with secondary antibodies ( envision detection system , dako ) .
sall4 was expressed in the tumor cell nuclei , and positivity for sall4 expression was defined as nuclear staining for the protein in more than 10% of the tumor cells after calculating the sall4-labeling index ( a ratio of positive nuclei / total nuclei under the 400 field magnification ) with the help of the image j software ( downloaded from http://imagej.nih.gov/ij ) .
membranous and/or cytoplasmic staining in the tumor cells were counted as positive for epcam and k19 .
chi - square , fisher s exact and t tests were performed as deemed appropriate .
univariable analyses for overall survival and disease - free survival were performed using the kaplan - meier method and log - rank tests .
statistically significant variables from the univariable analysis were entered into the multivariable analyses using the cox proportional hazard method .
this study was approved by the institutional review board of seoul national university bundang hospital ( b-1502 - 286 - 302 ) .
one hundred and ninety consecutive cases of surgically resected or explanted hccs were enrolled in this study , and were retrieved from the surgical pathology files of seoul national university bundang hospital from may 2003 to april 2010 .
the electronic medical records , surgical pathology reports , and the hematoxylin and eosin - stained slides were reviewed for each case , and the clinicopathological variables noted included tumor size , multiplicity ( including intrahepatic metastasis / satellite nodules and multicentric occurrences ) , histological differentiation according to the edmondson - steiner grade ( the highest grade in tumors with heterogeneity ) , the presence of microvascular or major vascular invasion , the patient demographics ( age and sex ) , and the presence of an underlying etiology ( e.g. , hepatitis b , c , alcohol ) .
follow - up data was also obtained from the medical records , including recurrences ( including local recurrence and distant metastasis ) and death .
the median follow - up period was 52 months ( range , 0 to 133 months ) .
overall survival was defined as thfe interval from initial surgical treatment for hcc to the date of death , and disease - free survival as the interval from initial treatment to local or distant recurrence .
tissue cores measuring 2 mm in diameter were sampled from formalin - fixed paraffin - embedded hcc tissues and arranged in recipient tissue array blocks using a trephine apparatus ( superbiochips laboratories , seoul , korea ) .
two or three cores were sampled from each hcc , depending on the amount of histological heterogeneity present in the tumor .
four m - thick sections were obtained from the tissue microarray blocks and subjected to immunohistochemical staining for sall4 ( 1:100 , mouse monoclonal antibody , clone ee-30 , santa cruz technologies inc . , santa cruz , ca , usa ) , k19 ( 1:150 , mouse monoclonal antibody , clone ba17 , dako , glostrup , denmark ) and epcam ( 1:3,000 , mouse monoclonal antibody , clone vu-1d9 , calbiochem , darmstadt , germany ) .
briefly , tissue sections were deparaffinized in xylene , rehydrated in graded alcohol , and antigen retrieval was performed using citrate buffer ( ph 6.0 ) for 15 minutes . sections were incubated with the primary antibodies at room temperature for 30 minutes , and then incubated with secondary antibodies ( envision detection system , dako ) .
sall4 was expressed in the tumor cell nuclei , and positivity for sall4 expression was defined as nuclear staining for the protein in more than 10% of the tumor cells after calculating the sall4-labeling index ( a ratio of positive nuclei / total nuclei under the 400 field magnification ) with the help of the image j software ( downloaded from http://imagej.nih.gov/ij ) .
membranous and/or cytoplasmic staining in the tumor cells were counted as positive for epcam and k19 .
all statistical analyses were conducted using ibm spss ver . 21 ( ibm corp . , armonk , ny , usa ) .
chi - square , fisher s exact and t tests were performed as deemed appropriate .
univariable analyses for overall survival and disease - free survival were performed using the kaplan - meier method and log - rank tests .
statistically significant variables from the univariable analysis were entered into the multivariable analyses using the cox proportional hazard method .
the immunohistochemical stain results and the expression frequencies of sall4 , k19 , and epcam are summarized in figs . 1 and 2 , respectively .
the sall4 nuclear labeling index ranged from 0% to 78.9% ( meanstandard deviation , 8.919.2% ) in the 190 hccs , and 39 cases ( 20.5% ) were deemed positive for sall4 expression ( labeling index 10% ) .
although a definite nuclear staining was present in the tumor cells , the intensity of staining was generally not as strong as in the positive control tissue ( testicular seminoma ) ( fig .
sall4 positivity was generally uniformly distributed in the hccs without predilections for a particular morphological tumor cell : some sall4-positive tumor cells were small with increased nuclear / cytoplasmic ratios reminiscent of the stem / progenitor cell phenotype , while others were more typical hccs with various degrees of differentiation . in the adjacent non - neoplastic liver , the hepatocytes and bile ducts did not stain for sall4 , and interestingly , sall4 positivity was seen in occasional ductular reactions , although the staining was very faint and barely visible ( fig .
k19 and epcam expression data were available for 189 cases ( due to tissue core loss in 1 case ) .
k19 and epcam were expressed in 30 ( 15.9% ) and 92 ( 48.7% ) cases , respectively , in the typical membranous / cytoplasmic pattern , and they were also expressed in the bile ducts and ductular reactions in the non - neoplastic livers .
a significant positive correlation was seen between epcam and k19 expression : k19 positivity was seen in 27 out of 92 epcam - positive hccs ( 29.3% ) compared to 3 out of 97 epcam negative hccs ( 3.1% ) ( p<.001 ) .
sall4 expression was more frequently seen in epcam - positive hccs ( 26/92 , 28.3% ) compared to epcam - negative hccs ( 13/97 , 13.4% ) ( p=.013 ) ( fig .
however , there was no significant correlation between sall4 and k19 expression : only 3.2% of the hccs demonstrated positivity for both sall4 and k19 ( fig .
the relationships between the expression of sall4 , k19 , and epcam in hccs are summarized in a venn diagram in fig .
univariable analysis demonstrated that sall4 expression was significantly associated with reduced overall survival ( p=.014 ) ( fig .
in addition to sall4 expression , larger tumor size ( > 3 cm , p=.045 ) , multiplicity of tumor ( p=.001 ) , major vascular invasion ( p=.046 ) and higher t stage ( stage 3 or 4 , p<.001 ) were significantly associated with decreased overall survival . in comparison ,
3b ) , while larger size ( > 3 cm , p=.009 ) , multiplicity ( p=.002 ) and higher t stage ( stage 3 or 4 , p=.019 ) were significantly associated with reduced disease - free survival . hccs with k19 expression had a tendency for decreased overall survival , although this result was not statistically significant ( p=.063 ) , while disease - free survival was significantly decreased for k19 expressing hccs ( p=.001 ) ( fig .
epcam expression was associated with a tendency for decreased overall survival ( p=.053 ) ; however , it was not associated with disease - free survival .
interestingly , when we performed the survival analysis after combining epcam and sall4 expression status , hccs that expressed both sall4 and epcam were associated with significantly decreased overall survival , compared to those cases which were negative for both these markers ( p=.031 ) ( fig .
4 ) . in addition , while epcam - negative hccs were associated with a relatively favorable outcome compared to epcam - positive hccs during the earlier follow - up period ( < 5 years ) , epcam - negative hccs with sall4-positivity showed an abrupt decrease in overall survival after 5 years of follow - up .
on multivariable analysis , sall4 ( p=.004 ) , multiplicity ( p=.016 ) and higher t stage ( p=.004 ) remained as significant independent predictors of decreased overall survival ( table 2 ) . for disease - free survival , larger tumor size ( > 3 cm ,
sall4 expression was not significantly associated with other clinicopathological parameters of tumor aggressiveness , such as presence of vascular invasion , larger tumors , and multiplicity ( table 3 ) . on the other hand ,
epcam expression in hccs was more frequently associated with poor histological differentiation ( p=.019 ) , microvascular invasion ( p=.011 ) , younger age ( p=.020 ) , b - viral etiology ( p=.010 ) , and high serum -fetoprotein ( afp ) levels ( p=.023 ) , and k19 expression was more frequently associated with higher serum afp levels ( p=.032 ) , major vascular invasion ( p=.010 ) , and higher pathological t stage ( 3 or 4 , p=.011 ) . increased tumor size was also more frequent in k19-positive hccs , although not statistically significant ( p=.076 ) .
the immunohistochemical stain results and the expression frequencies of sall4 , k19 , and epcam are summarized in figs . 1 and 2 , respectively .
the sall4 nuclear labeling index ranged from 0% to 78.9% ( meanstandard deviation , 8.919.2% ) in the 190 hccs , and 39 cases ( 20.5% ) were deemed positive for sall4 expression ( labeling index 10% ) .
although a definite nuclear staining was present in the tumor cells , the intensity of staining was generally not as strong as in the positive control tissue ( testicular seminoma ) ( fig .
sall4 positivity was generally uniformly distributed in the hccs without predilections for a particular morphological tumor cell : some sall4-positive tumor cells were small with increased nuclear / cytoplasmic ratios reminiscent of the stem / progenitor cell phenotype , while others were more typical hccs with various degrees of differentiation . in the adjacent non - neoplastic liver , the hepatocytes and bile ducts did not stain for sall4 , and interestingly , sall4 positivity was seen in occasional ductular reactions , although the staining was very faint and barely visible ( fig .
k19 and epcam expression data were available for 189 cases ( due to tissue core loss in 1 case ) .
k19 and epcam were expressed in 30 ( 15.9% ) and 92 ( 48.7% ) cases , respectively , in the typical membranous / cytoplasmic pattern , and they were also expressed in the bile ducts and ductular reactions in the non - neoplastic livers .
a significant positive correlation was seen between epcam and k19 expression : k19 positivity was seen in 27 out of 92 epcam - positive hccs ( 29.3% ) compared to 3 out of 97 epcam negative hccs ( 3.1% ) ( p<.001 ) .
sall4 expression was more frequently seen in epcam - positive hccs ( 26/92 , 28.3% ) compared to epcam - negative hccs ( 13/97 , 13.4% ) ( p=.013 ) ( fig .
however , there was no significant correlation between sall4 and k19 expression : only 3.2% of the hccs demonstrated positivity for both sall4 and k19 ( fig .
the relationships between the expression of sall4 , k19 , and epcam in hccs are summarized in a venn diagram in fig .
univariable analysis demonstrated that sall4 expression was significantly associated with reduced overall survival ( p=.014 ) ( fig .
in addition to sall4 expression , larger tumor size ( > 3 cm , p=.045 ) , multiplicity of tumor ( p=.001 ) , major vascular invasion ( p=.046 ) and higher t stage ( stage 3 or 4 , p<.001 ) were significantly associated with decreased overall survival . in comparison ,
3b ) , while larger size ( > 3 cm , p=.009 ) , multiplicity ( p=.002 ) and higher t stage ( stage 3 or 4 , p=.019 ) were significantly associated with reduced disease - free survival .
hccs with k19 expression had a tendency for decreased overall survival , although this result was not statistically significant ( p=.063 ) , while disease - free survival was significantly decreased for k19 expressing hccs ( p=.001 ) ( fig .
epcam expression was associated with a tendency for decreased overall survival ( p=.053 ) ; however , it was not associated with disease - free survival . interestingly ,
when we performed the survival analysis after combining epcam and sall4 expression status , hccs that expressed both sall4 and epcam were associated with significantly decreased overall survival , compared to those cases which were negative for both these markers ( p=.031 ) ( fig .
4 ) . in addition , while epcam - negative hccs were associated with a relatively favorable outcome compared to epcam - positive hccs during the earlier follow - up period ( < 5 years ) , epcam - negative hccs with sall4-positivity showed an abrupt decrease in overall survival after 5 years of follow - up . on multivariable analysis ,
sall4 ( p=.004 ) , multiplicity ( p=.016 ) and higher t stage ( p=.004 ) remained as significant independent predictors of decreased overall survival ( table 2 ) . for disease - free survival , larger tumor size ( > 3 cm ,
sall4 expression was not significantly associated with other clinicopathological parameters of tumor aggressiveness , such as presence of vascular invasion , larger tumors , and multiplicity ( table 3 ) . on the other hand ,
epcam expression in hccs was more frequently associated with poor histological differentiation ( p=.019 ) , microvascular invasion ( p=.011 ) , younger age ( p=.020 ) , b - viral etiology ( p=.010 ) , and high serum -fetoprotein ( afp ) levels ( p=.023 ) , and k19 expression was more frequently associated with higher serum afp levels ( p=.032 ) , major vascular invasion ( p=.010 ) , and higher pathological t stage ( 3 or 4 , p=.011 ) . increased tumor size was also more frequent in k19-positive hccs , although not statistically significant ( p=.076 ) .
stemness-related marker expression  those that have the histomorphological features of conventional hccs but express markers related to stemness on immunohistochemistry  have been receiving increasing interest over the past several years , as there is accumulating evidence that they are associated with aggressive behavior and poor prognosis , compared to conventional hccs that do not express these markers .
the more frequently discussed markers of stemness in hccs include k19 , epcam , and cd133 ; however , it is still uncertain which marker best represents stemness in hccs , and there is still a growing number of putative markers of stemness in the literature , including the recently proposed sall4 , which need further validation .
we were interested to see whether sall4 expression in hccs is indeed associated with the expression of other more established markers of stemness ( k19 and epcam ) and whether it has clinicopathological implications in our cohort of resected hccs .
interestingly , we found sall4 expression in 20.5% of hccs , that sall4 expression is positively correlated with epcam expression , and also that it has prognostic implications .
sall4 is a zinc finger transcriptional activator located on chromosome 20q13.13 - 13.2 that is required for the maintenance of pluripotent embryonic stem cells , potentially through interactions with oct3/4 , sox2 , and nanog [ 12 - 14 ] .
mutation in sall4 results in the okihiro syndrome , a rare autosomal dominant disorder characterized by multiple organ defects .
sall4 has been found to be expressed in leukemias and hematopoietic stem cells , and also in solid tumors such as germ cell tumors and afp - producing gastric cancers , suggesting that sall4 may be a marker of embryonic stem cells and also cancer stem cells [ 15 - 18 ] . in the fetal murine liver
, sall4 has been shown to be expressed in hepatic stem / progenitor cells , but the expression diminishes gradually during development and is not seen in adult hepatocytes .
these findings suggest that sall4 may be a good marker of hepatic stem / progenitor cells and hcc cancer stem cells . over the past two years
, there has been increasing interest in sall4 as a potential marker for identifying hccs with features of 
demonstrated a decreased overall survival in their profiling analysis of 110 hccs ( from the microarray dataset published by lee et al . ) , and also showed that the overexpression of sall4 in liver cancer cells induced the expression of k19 and epcam , suggesting that sall4 may be a marker of stem cells .
zeng et al .   demonstrated a decreased recurrence - free survival in sall4-positive hccs , and sall4 activation in cell lines resulted in the up - regulation of hepatic stem cell markers krt19 , epcam , and cd44 .
another extensive study showed a poor overall survival in sall4-positive hccs in two independent cohorts ( singapore , n=179 ; hong kong , n=228 ) , in addition to an enrichment of progenitor - like gene signatures and overexpression of proliferative and metastatic genes on gene expression analysis .
in another recent analysis of western hccs , higher tumor grade , more frequent lymphovascular invasion and shorter recurrence - free and overall survivals were seen in sall4-positive hccs , although the sall4-positivity was only seen very rarely ( 1.3% ) , suggesting differences in sall4 expression status according to the etiology of hcc .
the expression frequency of sall4 in hcc has in fact varied widely in different reports , ranging from 1.3% to 85% [ 9 - 11,20,21 ] .
it is plausible that a higher prevalence of hepatitis b virus infection may account for the higher frequency of sall4-positivity in eastern hccs ; interestingly , sall4-positivity has been shown to be more frequently associated with hepatitis b virus infection .
in addition , the differences in the antibodies , immunohistochemistry protocols , and the interpretation methods for defining  sall4-positivity  are also likely to account for the wide range of sall4-positivity reported in the literature .
as it has been previously demonstrated that nuclear labeling for sall4 in liver tissues is seen with high ph antigen retrieval and not as clearly with the conventional citrate buffer , we also stained the same tissue microarrays for sall4 using a different antigen retrieval method ( tris - edta buffer ; ph 9.0 ) to see if there were differences in the staining pattern or frequency ; however , the results were identical ( data not shown ) . therefore , sall4 immunostaining is feasible using the citrate buffer ( ph 6.0 ) for antigen retrieval .
we found that the nuclear staining for sall4 in the testicular seminoma was very intense , diffuse and finely granular , in comparison to most sall4-positive hccs in which the nuclear staining was weaker and punctate or clumped , similar to a previous report . in practice ,
when faced with the occasional diagnostic challenge of discriminating between hcc and yolk sac tumor  which may have similar morphological features and also positivity for afp and glypican-3sall4 immunohistochemistry may play a role , as the strong and diffuse staining for sall4 is rarely seen in hccs .
however , as for the utility of sall4 in hccs as a prognostic marker and a marker of 
stemness ,  further validation in independent cohorts of hcc would be necessary , as the staining is not as intense , and there is likely to be interobserver variation in the interpretation of sall4 positivity .
although we found a significantly decreased overall survival and a higher frequency of epcam positivity in sall4-positive hccs compared to sall4-negative hccs , we performed a semi - manual count of the sall4-labeling index with the help of an image analysis software , which is not always a feasible method in routine pathology practice in comparison to the distinct cytoplasmic staining for k19 or epcam .
our finding of a positive correlation between sall4 and epcam expression in hccs , poor overall survival in sall4-positive hccs , and positive ( albeit weak and sporadic ) staining in the ductular reactions support the recent literature that sall4 may be a useful marker of  stemness  in hepatic stem / progenitor cells and hccs .
however , in contrast to k19 or epcam , the nuclear staining is not as easily appreciable by immunohistochemistry , and other than being a prognostic indicator of poor overall survival , there were no significant correlations between sall4 positivity and the clinicopathological parameters suggestive of aggressiveness .
therefore , while sall4 is an independent prognostic factor for decreased overall survival in hcc patients and may be a potential marker for identifying the aggressive subgroup of hccs with  stemness  features , further validation in larger hcc cohorts is required prior to further consideration of sall4 as a good marker of  stemness  in liver specimens .
 background : there is increasing interest in hepatocellular carcinomas ( hcc ) expressing  stemness-related markers , as they have been associated with aggressive behavior and poor prognosis . in this study , we investigated the usefulness of sal - like protein 4 ( sall4 ) , a recently proposed candidate marker of  
 stemness.methods:immunohistochemical stains were performed for sall4 , k19 , and epithelial cellular adhesion molecule ( epcam ) on tissue microarrays constructed from 190 surgically resected hccs , and the results were correlated with the clinicopathological features and patient survival data.results:nuclear sall4 expression was observed in 39/190 hccs ( 20.5% ) , while k19 and epcam were expressed in 30 ( 15.9% ) and 92 ( 48.7% ) hccs , respectively . 
 the nuclear expression was generally weak , punctate or clumped . 
 sall4 expression was significantly associated with a poor overall survival compared to sall4-negative hccs ( p = .014 ) compared to sall4-negative hccs . 
 on multivariate analysis adjusted for tumor size , multiplicity , vascular invasion , and pathological tumor stage , sall4 remained as a significant independent predictor of decreased overall survival ( p= .004 ) . 
 sall4 expression was positively correlated with epcam expression ( p = .013 ) but not with k19 expression . 
 hccs that expressed both sall4 and epcam were associated with significantly decreased overall survival , compared to those cases which were negative for both of these markers ( p = .031).conclusions : although sall4 expression was not significantly correlated with other clinicopathological parameters suggestive of tumor aggressiveness , sall4 expression was an independent predictor of poor overall survival in human hccs , and was also positively correlated with epcam expression . 
MATERIALS AND METHODS
Case selection and review
Tissue microarray construction and immunohistochemistry
Statistical analysis
RESULTS
SALL4, K19, and EpCAM expression in HCC
Correlation with clinicopathological variables and survival
DISCUSSION