crc cases were selected from patients who underwent surgical treatment at eulji university hospital from january 2000 to june 2005 .
all cases were histologically confirmed to be primary colorectal adenocarcinoma and hematoxylin and eosin slides were re - evaluated by two independent pathologists .
the tumor grade of the adenocarcinoma was classified into low grade ( 50% of tumor glands ) and high grade ( < 50% of tumor glands ) . for signet ring cell carcinoma and mucinous adenocarcinoma ,
tumor budding was defined as a single or group of less than five detached tumor cells and classified into two grades .
tumor recurrence was designated as tumor occurring at the anastomosing site , in the regional lymph nodes , and the pelvic cavity diagnosed by radiology , colonoscopy , exploratory surgical , and/or histological examination .
in addition , metastasis was defined as the presence of tumor cells outside the area of resection , including the liver , pancreas , lung , and other organs .
all cases of crc tissue with accompanying normal mucosal tissue were fixed in 10% buffered formalin for 24 hours and embedded in paraffin .
tissue sections of 34 m thickness were cut and mounted on probeon slides ( fisher scientific , pittsburgh , pa , usa ) .
sections that contained both tumor and adjacent uninvolved colonic mucosa were selected for immunohistochemistry ( ihc ) in most cases . in a few cases ,
sections were trimmed in order to decrease the surface area for an even distribution of antibodies , so that only the tumor portion was included in the ihc evaluation .
paraffin embedded tissue sections were deparaffinized and rehydrated through a series of xylene and alcohol .
slides were then treated with 10 mm / l sodium citrate buffer ( ph 6.1 ) for 15 minutes and autoclaved at 120c for antigen retrieval .
all slide sections for ihc were incubated in 3% h2o2 for 10 minutes to inactivate endogenous peroxidase , washed with 10 mm / l phosphate buffered saline buffer ( ph 7.4 ) , and then incubated with normal bovine serum to reduce false - positive staining .
saco , me , usa ) and hif-1 ( 1:50 , novus biologicals , littleton , co , usa ) were used as primary antibodies .
slide sections were incubated with primary antibodies overnight at 4c in a wet chamber and stained with diaminobenzidine as the substrate using an envision - hrp kit ( dako , glostrup , denmark ) .
sections were counterstained with mayer s hematoxylin solution and then mounted . to evaluate the expression of thymosin 4 and hif-1 in association with various clinico - pathological factors ,
the immunoreactivity of both thymosin 4 and hif-1 were analyzed in a semi - quantitative manner by two independent pathologists who were blinded to outcomes .
immunoreactivity for thymosin 4 and hif-1 was observed primarily in the cytoplasm and nuclei of normal mucosal epithelium and tumor cells , respectively .
the intensity of immunohistochemical staining was scored as 0 to 2 ( 0 , weaker staining than the normal mucosal epithelium ; 1 , staining similar to the normal mucosal epithelium ; and 2 , stronger staining than the normal mucosal epithelium ) .
the percentage of positive cells was scored as 1 ( < 25% of tumor cells ) , 2 ( 25%49% of tumor cells ) , 3 ( 50%74% of tumor cells ) , and 4 (  75% of tumor cells ) . to evaluate the statistical significance between thymosin 4 and hif-1 expression and clinico - pathological factors
, the median value ( 25% of tumor cells showing a strong positive reaction than normal epithelium ) of the series was used as the cutoff value to distinguish between tumor cells with low expression ( < 25% tumor cells ) and high expression (  25% of tumor cells ) .
the correlation between thymosin 4 and the various clinico - pathological parameters were analyzed with the pearson s chi - square test or fisher exact test . to evaluate statistical analysis ,
recurrence - free survival was defined as the duration from the date of surgery to the first date of recurrence or the date of last follow - up .
similarly , overall survival was defined as the duration from the date of surgical therapy to the date of death or date of last follow - up .
the mean follow - up duration for all patients was 53.3 months , ranging from 0.6 to 121.9 months .
using the kaplan - meier method , the recurrence - free survival curve and the overall survival curve were formulated . to examine the statistical significance of the differences in survival distribution , log - rank test was utilized .
multivariate analysis for overall survival and recurrence - free survival was performed using cox proportional hazard regression analysis . in all statistical analyses , p - values less than .05 were considered statistically significant .
the institutional review board of eulji university hospital approved the study protocol and provided all necessary ethical permission .
crc cases were selected from patients who underwent surgical treatment at eulji university hospital from january 2000 to june 2005 .
all cases were histologically confirmed to be primary colorectal adenocarcinoma and hematoxylin and eosin slides were re - evaluated by two independent pathologists .
the tumor grade of the adenocarcinoma was classified into low grade ( 50% of tumor glands ) and high grade ( < 50% of tumor glands ) . for signet ring cell carcinoma and mucinous adenocarcinoma ,
tumor budding was defined as a single or group of less than five detached tumor cells and classified into two grades .
tumor recurrence was designated as tumor occurring at the anastomosing site , in the regional lymph nodes , and the pelvic cavity diagnosed by radiology , colonoscopy , exploratory surgical , and/or histological examination .
in addition , metastasis was defined as the presence of tumor cells outside the area of resection , including the liver , pancreas , lung , and other organs .
all cases of crc tissue with accompanying normal mucosal tissue were fixed in 10% buffered formalin for 24 hours and embedded in paraffin .
tissue sections of 34 m thickness were cut and mounted on probeon slides ( fisher scientific , pittsburgh , pa , usa ) .
sections that contained both tumor and adjacent uninvolved colonic mucosa were selected for immunohistochemistry ( ihc ) in most cases . in a few cases ,
sections were trimmed in order to decrease the surface area for an even distribution of antibodies , so that only the tumor portion was included in the ihc evaluation .
paraffin embedded tissue sections were deparaffinized and rehydrated through a series of xylene and alcohol .
slides were then treated with 10 mm / l sodium citrate buffer ( ph 6.1 ) for 15 minutes and autoclaved at 120c for antigen retrieval .
all slide sections for ihc were incubated in 3% h2o2 for 10 minutes to inactivate endogenous peroxidase , washed with 10 mm / l phosphate buffered saline buffer ( ph 7.4 ) , and then incubated with normal bovine serum to reduce false - positive staining .
saco , me , usa ) and hif-1 ( 1:50 , novus biologicals , littleton , co , usa ) were used as primary antibodies .
slide sections were incubated with primary antibodies overnight at 4c in a wet chamber and stained with diaminobenzidine as the substrate using an envision - hrp kit ( dako , glostrup , denmark ) .
to evaluate the expression of thymosin 4 and hif-1 in association with various clinico - pathological factors , the immunoreactivity of both thymosin 4 and hif-1 were analyzed in a semi - quantitative manner by two independent pathologists who were blinded to outcomes .
immunoreactivity for thymosin 4 and hif-1 was observed primarily in the cytoplasm and nuclei of normal mucosal epithelium and tumor cells , respectively .
the intensity of immunohistochemical staining was scored as 0 to 2 ( 0 , weaker staining than the normal mucosal epithelium ; 1 , staining similar to the normal mucosal epithelium ; and 2 , stronger staining than the normal mucosal epithelium ) .
the percentage of positive cells was scored as 1 ( < 25% of tumor cells ) , 2 ( 25%49% of tumor cells ) , 3 ( 50%74% of tumor cells ) , and 4 (  75% of tumor cells ) . to evaluate the statistical significance between thymosin 4 and hif-1 expression and clinico - pathological factors , the median value ( 25% of tumor cells showing a strong positive reaction than normal epithelium ) of the series was used as the cutoff value to distinguish between tumor cells with low expression ( < 25% tumor cells ) and high expression (  25% of tumor cells ) .
the correlation between thymosin 4 and the various clinico - pathological parameters were analyzed with the pearson s chi - square test or fisher exact test . to evaluate statistical analysis ,
recurrence - free survival was defined as the duration from the date of surgery to the first date of recurrence or the date of last follow - up .
similarly , overall survival was defined as the duration from the date of surgical therapy to the date of death or date of last follow - up .
the mean follow - up duration for all patients was 53.3 months , ranging from 0.6 to 121.9 months .
using the kaplan - meier method , the recurrence - free survival curve and the overall survival curve were formulated . to examine the statistical significance of the differences in survival distribution , log - rank test was utilized .
multivariate analysis for overall survival and recurrence - free survival was performed using cox proportional hazard regression analysis . in all statistical analyses , p - values less than .05 were considered statistically significant .
the institutional review board of eulji university hospital approved the study protocol and provided all necessary ethical permission .
the median age of the 143 crc patients ( 75 men and 68 women ) at surgery was 62.2 years ( range , 28 to 86 years ) and the median tumor size was 5.2 cm ( range , 0.8 to 12.0 cm ) in maximum diameter .
the majority of crcs were moderately differentiated adenocarcinoma and 111 cases ( 77.6% ) were classified as low grade and 32 cases ( 22.4% ) as high grade ( poorly differentiated 19 , signet ring cell carcinoma 3 , and mucinous carcinoma 10 ) .
one hundred and four cases ( 72.7% ) showed lymphovascular tumor invasion and 106 cases ( 74.1% ) exhibited high - grade tumor budding . according to the seventh edition of the ajcc tnm system , 26 patients ( 18.2% ) were diagnosed with early - stage tumor invasion ( five cases of pt1 and 21 cases of pt2 ) and 117 patients ( 81.8% ) were diagnosed with advanced - stage tumor invasion ( 105 cases of pt3 and 12 cases of pt4 ) .
seventy - six patients ( 53.1% ) presented with regional lymph node metastasis and 22 patients ( 15.4% ) with distant metastasis .
twenty - one patients ( 14.7% ) were at ptnm stage i , 45 patients ( 31.5% ) were at stage ii , 55 patients ( 38.5% ) were at stage iii , and 22 patients ( 15.4% ) were at stage iv .
twenty - seven patients ( 18.9% ) were treated with chemoradiation after the first surgery ( data not shown ) .
clinico - pathological characteristics of the 143 crc patients are summarized in table 1 . in the normal colonic epithelium , immunoreactivity for thymosin 4 and hif-1
while thymosin 4 expression was found primarily in the cytoplasm of cancer cells , hif-1 was stained predominantly in the nuclei of tumor cells ( fig .
a high level of thymosin 4 and hif-1 expression was observed in 66 of the 143 patients ( 46.2% ) and in 67 of the 143 patients ( 46.9% ) , respectively .
we found that predictive factors for prognosis , such as lymphovascular invasion , invasion depth ( pt ) , regional lymph node metastasis ( pn ) , distant metastasis , and tnm stage showed statistically significant correlations with thymosin 4 immunoreactivity ( table 1 ) .
patients with high thymosin 4 expression levels showed a significantly greater presence of lymphovascular invasion , more frequent regional lymph node metastasis , deeper invasion depth , and more advanced tumor stage than in those with low thymosin 4 expression levels ( p<.001 ) .
we found a statistically significant correlation between high hif-1 immunohistochemical expression and clinico - pathological factors such as lymphovascular invasion ( p=.006 ) , invasion depth ( p=.002 ) , regional lymph node metastasis ( p=.007 ) , distant metastasis ( p=.029 ) , and tnm stage ( p=.002 ) ( data not shown ) .
we performed multivariate analysis to examine the correlation between thymosin 4 expression levels with recurrence - free survival and overall survival .
forty patients ( 28.0% ) presented with cancer recurrence during follow - up and 55 patients ( 38.5% ) died of crc with or without metastasis .
seven patients ( 4.9% ) died of unknown causes , 17 patients ( 11.9% ) were alive with local recurrence and/or distant metastasis , and 64 patients ( 44.8% ) remained alive and recurrence - free .
kaplan - meier analysis showed that high thymosin 4 expression was significantly correlated with decreased recurrence - free survival ( p<.001 ) ( fig .
recurrence - free survival was shorter in patients with high expression levels of thymosin 4 , with a mean duration of 37.3 months ( 95% confidence interval [ ci ] , 27.833 to 46.684 ) , and was longer in patients with low levels of thymosin 4 expression , with a mean duration of 84.6 months ( 95% ci , 73.538 to 95.571 ) .
we also found that high thymosin 4 expression significantly correlated with worse overall survival ( p=.001 ) .
thymosin 4 expression status also significantly split the cumulative overall survival curves ( fig . 2b ) . while the overall survival of crc patients with high thymosin 4 expression was mean duration of 51.7 months ( 95% ci , 41.381 to 61.939 ) , the overall survival of crc patients with low thymosin 4 expression was longer with mean duration of 96.8 months ( 95% ci , 86.952 to 106.732 ) .
multivariate analysis was also performed to assess the prognostic value of thymosin 4 expression for recurrence - free survival and overall survival using various clinico - pathological parameters .
statistically significant clinico - pathological factors that were correlated with overall survival were high thymosin 4 expression ( p=.005 ) , high tumor grade ( p=.013 ) , high tumor budding ( p=.047 ) , and presence of distant metastasis ( p=.001 ) .
the relative risk ( rr ) of death in patients with a high expression level of thymosin 4 was more than two times greater ( rr , 2.457 ; 95% ci , 1.315 to 4.592 ) than those with low thymosin 4 expression levels .
high thymosin 4 expression was also an independent and relevant factor of decreased recurrence - free survival ( p=.001 ) .
the rr of recurrence for patients with high thymosin 4 expression level was 2.540 ( 95% ci , 1.479 to 4.362 ) .
a statistically significant correlation between high hif-1 expression and high thymosin 4 expression was also found ( p<.001 ) .
specifically , of the 66 cases exhibiting high thymosin 4 expression , 49 cases ( 74.2% ) also showed high nuclear immunoreactivity of hif-1. of the 77 cases expressing low thymosin 4 expression , 59 cases ( 76.6% ) revealed corresponding low hif-1 expression ( table 3 ) .
the median age of the 143 crc patients ( 75 men and 68 women ) at surgery was 62.2 years ( range , 28 to 86 years ) and the median tumor size was 5.2 cm ( range , 0.8 to 12.0 cm ) in maximum diameter .
the majority of crcs were moderately differentiated adenocarcinoma and 111 cases ( 77.6% ) were classified as low grade and 32 cases ( 22.4% ) as high grade ( poorly differentiated 19 , signet ring cell carcinoma 3 , and mucinous carcinoma 10 ) .
one hundred and four cases ( 72.7% ) showed lymphovascular tumor invasion and 106 cases ( 74.1% ) exhibited high - grade tumor budding . according to the seventh edition of the ajcc tnm system , 26 patients ( 18.2% ) were diagnosed with early - stage tumor invasion ( five cases of pt1 and 21 cases of pt2 ) and 117 patients ( 81.8% ) were diagnosed with advanced - stage tumor invasion ( 105 cases of pt3 and 12 cases of pt4 ) .
seventy - six patients ( 53.1% ) presented with regional lymph node metastasis and 22 patients ( 15.4% ) with distant metastasis .
twenty - one patients ( 14.7% ) were at ptnm stage i , 45 patients ( 31.5% ) were at stage ii , 55 patients ( 38.5% ) were at stage iii , and 22 patients ( 15.4% ) were at stage iv .
twenty - seven patients ( 18.9% ) were treated with chemoradiation after the first surgery ( data not shown ) .
clinico - pathological characteristics of the 143 crc patients are summarized in table 1 . in the normal colonic epithelium , immunoreactivity for thymosin 4 and hif-1
while thymosin 4 expression was found primarily in the cytoplasm of cancer cells , hif-1 was stained predominantly in the nuclei of tumor cells ( fig .
a high level of thymosin 4 and hif-1 expression was observed in 66 of the 143 patients ( 46.2% ) and in 67 of the 143 patients ( 46.9% ) , respectively .
we found that predictive factors for prognosis , such as lymphovascular invasion , invasion depth ( pt ) , regional lymph node metastasis ( pn ) , distant metastasis , and tnm stage showed statistically significant correlations with thymosin 4 immunoreactivity ( table 1 ) .
patients with high thymosin 4 expression levels showed a significantly greater presence of lymphovascular invasion , more frequent regional lymph node metastasis , deeper invasion depth , and more advanced tumor stage than in those with low thymosin 4 expression levels ( p<.001 ) .
we found a statistically significant correlation between high hif-1 immunohistochemical expression and clinico - pathological factors such as lymphovascular invasion ( p=.006 ) , invasion depth ( p=.002 ) , regional lymph node metastasis ( p=.007 ) , distant metastasis ( p=.029 ) , and tnm stage ( p=.002 ) ( data not shown ) .
we performed multivariate analysis to examine the correlation between thymosin 4 expression levels with recurrence - free survival and overall survival .
forty patients ( 28.0% ) presented with cancer recurrence during follow - up and 55 patients ( 38.5% ) died of crc with or without metastasis .
seven patients ( 4.9% ) died of unknown causes , 17 patients ( 11.9% ) were alive with local recurrence and/or distant metastasis , and 64 patients ( 44.8% ) remained alive and recurrence - free .
kaplan - meier analysis showed that high thymosin 4 expression was significantly correlated with decreased recurrence - free survival ( p<.001 ) ( fig .
recurrence - free survival was shorter in patients with high expression levels of thymosin 4 , with a mean duration of 37.3 months ( 95% confidence interval [ ci ] , 27.833 to 46.684 ) , and was longer in patients with low levels of thymosin 4 expression , with a mean duration of 84.6 months ( 95% ci , 73.538 to 95.571 ) .
we also found that high thymosin 4 expression significantly correlated with worse overall survival ( p=.001 ) .
while the overall survival of crc patients with high thymosin 4 expression was mean duration of 51.7 months ( 95% ci , 41.381 to 61.939 ) , the overall survival of crc patients with low thymosin 4 expression was longer with mean duration of 96.8 months ( 95% ci , 86.952 to 106.732 ) .
multivariate analysis was also performed to assess the prognostic value of thymosin 4 expression for recurrence - free survival and overall survival using various clinico - pathological parameters .
statistically significant clinico - pathological factors that were correlated with overall survival were high thymosin 4 expression ( p=.005 ) , high tumor grade ( p=.013 ) , high tumor budding ( p=.047 ) , and presence of distant metastasis ( p=.001 ) .
the relative risk ( rr ) of death in patients with a high expression level of thymosin 4 was more than two times greater ( rr , 2.457 ; 95% ci , 1.315 to 4.592 ) than those with low thymosin 4 expression levels .
high thymosin 4 expression was also an independent and relevant factor of decreased recurrence - free survival ( p=.001 ) .
the rr of recurrence for patients with high thymosin 4 expression level was 2.540 ( 95% ci , 1.479 to 4.362 ) .
a statistically significant correlation between high hif-1 expression and high thymosin 4 expression was also found ( p<.001 ) .
specifically , of the 66 cases exhibiting high thymosin 4 expression , 49 cases ( 74.2% ) also showed high nuclear immunoreactivity of hif-1. of the 77 cases expressing low thymosin 4 expression , 59 cases ( 76.6% ) revealed corresponding low hif-1 expression ( table 3 ) .
growing tumors require oxygen and nutrient delivery through neovasculature . however , intratumoral hypoxia induced by imbalance between tumor growth and insufficient angiogenesis can lead to expression of hif-1 , which is a transcriptional factor that activates tumor survival in an unstable hypoxic tumor microenvironment [ 29 - 32 ] .
recent reports have shown that thymosin 4 stabilizes hif-1 in human cancer cells   and that thymosin 4 also induces migration and metastasis of colon cancer cells via the ilk / iqgap1/rac1 signal transduction pathway .
few studies have evaluated whether overexpression of thymosin 4 influences clinical prognosis and whether thymosin 4 is related to hif-1 in crcs . to better understand the relationship
, we analyzed the clinical significance and expression status of thymosin 4 and hif-1 in crc patients .
our study demonstrates that high thymosin 4 expression has a significant association with lymphovascular invasion , nodal status , distant metastasis , and tumor progression in crc patients ( p<.001 ) .
this finding is consistent with our previous study showing hypoxia - induced high expression of thymosin 4 , which also significantly correlated with regional lymph node metastasis in breast cancer . in a previous study
, we found thymosin 4 to be up - regulated under hypoxic conditions ( 5% o2 ) using an in vitro hypoxia - induced model to generate transcription profiles in human crc .
based on these findings , for this study we examined the association between thymosin 4 expression and hif-1 expression in crc specimens .
we then discovered that the overexpression of thymosin 4 in crc is closely related to the restricted overexpression of hif-1 in the crc cells ( p<.001 ) .
recently , there have been reports regarding the association between thymosin 4 expression with tumor development and epithelial mesenchymal transition ( emt ) .
in particular , nemolato et al .   reported high expression of thymosin 4 at the invasive front in colon cancer and discussed its associated with emt as well as invasion and metastasis of tumor cells .
however , from our study , since we were unable to find an association between thymosin 4 expression and tumor budding ( p=.118 ) and tumor border ( p=.560 ) , we found the direct association of thymosin 4 expression with emt to be weak .
the hif complex , which involves various hypoxia - regulated genes , is a group of critical gene products in the tumor microenvironment of hypoxic adaptation and in angiogenesis .
the hif complex is also an essential mediator in coordinating transcription of various factors in the tumor cells to survive in the hypoxic environment and its overexpression has been associated with increased mortality in various cancer types [ 31,35 - 37 ] . among hif
whether hif-2 , hif-3 , and hif-1 also play critical roles in the hif pathway and regulate hif target genes is not yet clearly known [ 38 - 41 ] .
hif-1 is frequently upregulated in various cancer cells and the overexpression of hif-1 correlates with advanced cancer progression or aggressiveness .
however , the clinical significance of hif-1 in crc has not been extensively studied . in this study
, we observed a significant association between thymosin 4 expression and hif-1 expression ( p<.001 ) .
this result coincides with previous studies that found overexpression of hif-1 to be associated with poor prognosis .
thymosin 4 has various functional roles in normal cell biology and its mechanism of action has recently been studied in various tumors . in this study
, we found that high cytoplasmic expression of thymosin 4 is clinically important and an independent prognostic factor for crc patients .
as our results demonstrate that high thymosin 4 expression significantly correlates with tumor recurrence and worse overall survival , we suggest that high thymosin 4 expression may be a useful prognostic factor in crc .
our results demonstrate that hif-1 is correlated with overexpression of thymosin 4 in human crc .
although further studies are necessary to further validate our findings , we suggest that thymosin 4 , has potential as a prognostic biomarker and has potential as a hif pathway target in human crc .
 backgroundthymosin 4 is a multi - functional hormone - like polypeptide , being involved in cell migration , angiogenesis , and tumor metastasis . 
 this study was undertaken to clarify the clinicopathologic implications of thymosin 4 expression in human colorectal cancers ( crcs).methodswe investigated tissue sections from 143 patients with crc by immunohistochemistry . in addition , we evaluated the expression patterns and the clinico - pathological significance of thymosin 4 expression in association with hypoxia inducible factor-1 ( hif-1 ) expression in the crc series.resultshigh expression of thymosin 4 was significantly correlated with lymphovascular invasion , invasion depth , regional lymph node metastasis , distant metastasis , and tnm stage . 
 patients with high expression of thymosin 4 showed poor recurrence - free survival ( p = .001 ) and poor overall survival ( p = .005 ) on multivariate analysis . 
 we also found that thymosin 4 and hif-1 were overexpressed and that thymosin 4 expression increased in parallel with hif-1 expression in crc.conclusionsa high expression level of thymosin 4 indicates poor clinical outcomes and may be a useful prognostic factor in crc . 
 thymosin 4 is functionally related with hif-1 and may be a potentially valuable biomarker and possible therapeutic target for crc . 
MATERIALS AND METHODS
Case selection and immunohistochemistry
Assessment of IHC staining
Statistical analysis of prognostic parameters
Ethical permission
RESULTS
Association of clinico-pathological characteristics with thymosin 
High thymosin 
DISCUSSION