- 0194 ) , 23 patients from the department of pathology , gangnam severance hospital , were enrolled in this study .
the patients ranged in age from 35 to 59 years old , with a mean age of 47 .
fresh surgical samples of ptc with ( 13 cases ) or without ( 10 cases ) lt were collected from the records in the department of pathology with the patients agreement and the formal permission of the irb .
the prognostic parameters ( tumor size , extra - thyroidal extension of ptc , and lymph node metastasis ) were analyzed . among the subtypes of ptc samples in this group ,
the nontumor portions were used for measuring the mrna quantities of the cytokines , and the ptc portions were subject to immunohistochemical staining of cip1 ( p21)/kip1 ( p27 ) for protein expression analysis , as described in more detail in later section .
the nontumor portions of the patient samples were used to measure the mrna levels of the following cytokines : tumor necrosis factor  ( tnf- ) , interferon  ( ifn- ) , il-2 , il-4 , il-10 , and il-1. the 13 cases with concomitant ptc and lt were referred to as the objective group , and the 10 cases of ptc without lt were used as a reference or control group .
quantitative real - time polymerase chain reaction ( pcr ) was performed according to the manual of lightcycler 480 real - time pcr system ( roche applied science , manheim , germany ) .
the reference gene value ( actin and 18s rna ) and il-1 value were measured in every sample .
although it is known to be meaningless to compare the levels of cytokines from the same specimen , the expressed cytokine levels could be ranked and compared in this research via a quantitative real - time pcr method . using the concept of relative quantification , the cytokine values were corrected for differences in quality and quantity by dividing the concentration of a target rna by the concentration of a reference rna in the same sample ( relative ratio = concentration of target / concentration of reference ) .
the most common way to compare expression levels of different samples is to designate one of the samples as calibrator , where all other samples are compared to this calibrator . for normalization of the final results ,
the target / reference ratio of each sample is divided by the target / reference ratio of the calibrator sample : calibrator normalized ratio=(sample ; concentration of target / concentration of reference)/(calibrator ; concentration of target / concentration of reference ) . in the present work
 this means that the reference gene was calculated , but the target gene could not be detected despite many pcr amplification processes . in these cases , we adjusted these  not detected  values to zero ( 0 ) .
the immunohistochemistry ( ihc ) of the p27 protein expression in the ptc tumor tissue from all 23 cases was analyzed using paraffin - embedded tissue .
formalin - fixed paraffin - embedded sections ( 3 m thick ) were dewaxed in xylene and rehydrated through graded alcohols to water .
antigen retrieval was performed in citrate buffer ( ph 6.0 ) within a microwave pressure cooker , and endogenous biotin detection was blocked with the avidin - biotin blocking kit ( vector laboratories inc . , burlingame , ca , usa ) .
optimum primary antibody dilutions were predetermined , and appropriate positive control samples ( tissues known to be positive for the immunohistochemical marker ) and negative control samples ( test tissue sections without the addition of primary antibody ) were used for p27 ( novocastra , newcastle upon tyne , uk ) .
after incubation , the slide was washed with phosphate buffered saline , and a secondary incubation was carried out with biotin anti - mouse / anti - rabbit igg followed by streptavidin - hrp ( signet pathology system , dedham , ma , usa ) for 30 minutes .
when p27 immunoreaction was interpreted , only the nuclear staining on ptc cells was regarded as positive .
its measurement was evaluated by the following two - tiered grading system : low grade ( negative staining or faint staining in less than 30% of the tumor cells ) and high grade ( positive staining in more than 30% of the tumor cells ) ( fig .
nuclear p27 immunoreactivity was considered altered when the expression was less than 30% of previously published , clinically relevant levels . in 21 cases ,
the results of ihc were summarized in two groups : the cases with lt ( n=13 ) and the cases without lt ( n=8 ) .
two cases were not performed , for the ihc sections for p27 were devoid of tumor tissue .
the pearson chi - square test was used to examine the relationships of cytokines ( tnf- , ifn- , il-2 , il-4 , il-10 , and il-1 ) and p27 expression with the clinicopathological characteristics .
all reported p - values were 2-sided , and the significance was set at .05 .
all statistical tests were performed with spss ver . 17.0 ( spss inc . , chicago , il , usa ) .
- 0194 ) , 23 patients from the department of pathology , gangnam severance hospital , were enrolled in this study .
the patients ranged in age from 35 to 59 years old , with a mean age of 47 .
fresh surgical samples of ptc with ( 13 cases ) or without ( 10 cases ) lt were collected from the records in the department of pathology with the patients agreement and the formal permission of the irb .
the prognostic parameters ( tumor size , extra - thyroidal extension of ptc , and lymph node metastasis ) were analyzed . among the subtypes of ptc samples in this group ,
the nontumor portions were used for measuring the mrna quantities of the cytokines , and the ptc portions were subject to immunohistochemical staining of cip1 ( p21)/kip1 ( p27 ) for protein expression analysis , as described in more detail in later section .
the nontumor portions of the patient samples were used to measure the mrna levels of the following cytokines : tumor necrosis factor  ( tnf- ) , interferon  ( ifn- ) , il-2 , il-4 , il-10 , and il-1. the 13 cases with concomitant ptc and lt were referred to as the objective group , and the 10 cases of ptc without lt were used as a reference or control group .
quantitative real - time polymerase chain reaction ( pcr ) was performed according to the manual of lightcycler 480 real - time pcr system ( roche applied science , manheim , germany ) .
the reference gene value ( actin and 18s rna ) and il-1 value were measured in every sample .
although it is known to be meaningless to compare the levels of cytokines from the same specimen , the expressed cytokine levels could be ranked and compared in this research via a quantitative real - time pcr method . using the concept of relative quantification
, the cytokine values were corrected for differences in quality and quantity by dividing the concentration of a target rna by the concentration of a reference rna in the same sample ( relative ratio = concentration of target / concentration of reference ) .
the most common way to compare expression levels of different samples is to designate one of the samples as calibrator , where all other samples are compared to this calibrator . for normalization of the final results ,
the target / reference ratio of each sample is divided by the target / reference ratio of the calibrator sample : calibrator normalized ratio=(sample ; concentration of target / concentration of reference)/(calibrator ; concentration of target / concentration of reference ) . in the present work
 this means that the reference gene was calculated , but the target gene could not be detected despite many pcr amplification processes . in these cases
the immunohistochemistry ( ihc ) of the p27 protein expression in the ptc tumor tissue from all 23 cases was analyzed using paraffin - embedded tissue .
formalin - fixed paraffin - embedded sections ( 3 m thick ) were dewaxed in xylene and rehydrated through graded alcohols to water .
antigen retrieval was performed in citrate buffer ( ph 6.0 ) within a microwave pressure cooker , and endogenous biotin detection was blocked with the avidin - biotin blocking kit ( vector laboratories inc . , burlingame , ca , usa ) .
optimum primary antibody dilutions were predetermined , and appropriate positive control samples ( tissues known to be positive for the immunohistochemical marker ) and negative control samples ( test tissue sections without the addition of primary antibody ) were used for p27 ( novocastra , newcastle upon tyne , uk ) .
after incubation , the slide was washed with phosphate buffered saline , and a secondary incubation was carried out with biotin anti - mouse / anti - rabbit igg followed by streptavidin - hrp ( signet pathology system , dedham , ma , usa ) for 30 minutes .
when p27 immunoreaction was interpreted , only the nuclear staining on ptc cells was regarded as positive .
its measurement was evaluated by the following two - tiered grading system : low grade ( negative staining or faint staining in less than 30% of the tumor cells ) and high grade ( positive staining in more than 30% of the tumor cells ) ( fig .
nuclear p27 immunoreactivity was considered altered when the expression was less than 30% of previously published , clinically relevant levels . in 21 cases ,
the results of ihc were summarized in two groups : the cases with lt ( n=13 ) and the cases without lt ( n=8 ) .
two cases were not performed , for the ihc sections for p27 were devoid of tumor tissue .
the pearson chi - square test was used to examine the relationships of cytokines ( tnf- , ifn- , il-2 , il-4 , il-10 , and il-1 ) and p27 expression with the clinicopathological characteristics .
all reported p - values were 2-sided , and the significance was set at .05 .
all statistical tests were performed with spss ver . 17.0 ( spss inc . , chicago , il , usa ) .
the subtypes of the ptcs with lt were determined to be the most conventional ( 11/13 , 84.61% ) , and those of only two cases were follicular variants ( 2/13 , 15.38% ) .
the tumor sizes ranged from 0.2 to 1.1 cm , with an average of 0.55 cm .
of the 13 cases of this type , six cases showed extrathyroidal tumor extension ( et ) ( 6/13 , 46.15% ) , and two cases presented with lymph node metastasis ( 2/13 , 15.38 % ) .
the subtypes of the ptcs with lt were all conventional ( 10/10 , 100% ) .
tumor sizes ranged from 0.1 to 1.5 cm , with an average of 0.61 cm .
there were three cases with lymph node metastasis ( 3/10 , 30% ) . in all 23 cases ,
the cytokine immune profiles were summarized into two groups : the cases with lt ( n=13 ) and the cases without lt ( n=10 ) .
the cytokines could have originated from inflammatory cells , thyroid tissue , and endothelial cells . in this research ,
after manipulating the data using the concept of a calibrator with il-1 , each sample of ptc with lt was compared ( table 1 ) . among the six cytokines analyzed ,
based on these factors , the cytokine expression in the ptc samples with lt represented a mixed th1 ( tnf- and ifn- ) and th2 ( il-4 and il-10 ) immunity . after manipulating data using the concept of a calibrator with il-1 , each sample of ptc without lt
in contrast to the cases of ptc with lt , the cases without lt frequently exhibited nondetectable cytokine levels . among the six cytokines analyzed , the expression levels of ifn- , il-10 , and il-2 levels were too low to be measured . based on these data ,
the cytokines expressed in ptc without lt represented a mixed th1 ( tnf- ) and th2 ( il-4 and il-1 ) immunity . the prognostic parameters ( tumor size , et , and lymph node metastasis ) were not statistically related to lt , but the cases without lt had a tendency for lymph node metastasis ( table 3 ) .
the cases with lt had enhanced expression of the cytokines ( tnf- , ifn- , il-4 , il-10 , and il-2 ) compared to the samples of ptc without lt . among the five cytokines ,
the expression levels of tnf- , ifn- , and il-4 were significantly higher in the cases with lt than in those without lt ( p<.05 ) . in terms of p27 as a surrogate marker for lymph node metastasis , the degree of p27 expression was not correlated with lymph node metastasis in this study ; high - grade expression of p27 had a tendency to occur in ptc cases with lt .
in contrast , lymph node metastasis occurred slightly more frequently in ptc cases without lt . conclusively
, the degree of p27 expression did not show any correlation with lymph node metastasis in this study ( p=.15 ) ( table 4 ) . due to this unexpected
finding , which revealed in the insufficiency of the ptc cases without lt to serve as a reference group , this study adopted a new goal to use the ptc cases with lt to analyze the relationship between cytokine immune profiles and prognostic parameters .
in addition , the ptc cases in this study were mostly microcarcinomas , which might represent the incipient phase of the tumors .
the incipient phase of tumors must always have a limitation in representing proper tumor staging , such as tumor size , nodal metastasis , and tumor extension .
hence , in this study , p27 was used as a surrogate marker of lymph node metastasis in the cases of microcarcinoma ( as an incipient carcinoma ) .
therefore , the statistical analysis of this profiles solely made use of the ptc cases with lt .
except for the il-4 cytokine , the expression levels of the cytokines tnf- , ifn- , and il-10 were lower in the cases with low - grade expression of p27 than those with high - grade expression of p27 , but there was no statistical significance ( table 5 ) .
the cases with et had a tendency to demonstrate higher levels of cytokine expression than those with intrathyroidal tumor confinement ( table 6 ) .
again , there was no statistical significance . in summary , although the data were not statistically significant , the trends follow a pattern where higher cytokine levels were present in the cases with high grade expression of p27 ( except for il-4 ) and et .
the subtypes of the ptcs with lt were determined to be the most conventional ( 11/13 , 84.61% ) , and those of only two cases were follicular variants ( 2/13 , 15.38% ) .
the tumor sizes ranged from 0.2 to 1.1 cm , with an average of 0.55 cm .
of the 13 cases of this type , six cases showed extrathyroidal tumor extension ( et ) ( 6/13 , 46.15% ) , and two cases presented with lymph node metastasis ( 2/13 , 15.38 % ) .
the subtypes of the ptcs with lt were all conventional ( 10/10 , 100% ) .
tumor sizes ranged from 0.1 to 1.5 cm , with an average of 0.61 cm .
there were three cases with lymph node metastasis ( 3/10 , 30% ) . in all 23 cases ,
the cytokine immune profiles were summarized into two groups : the cases with lt ( n=13 ) and the cases without lt ( n=10 ) .
the cytokines could have originated from inflammatory cells , thyroid tissue , and endothelial cells . in this research ,
after manipulating the data using the concept of a calibrator with il-1 , each sample of ptc with lt was compared ( table 1 ) . among the six cytokines analyzed ,
based on these factors , the cytokine expression in the ptc samples with lt represented a mixed th1 ( tnf- and ifn- ) and th2 ( il-4 and il-10 ) immunity . after manipulating data using the concept of a calibrator with il-1 , each sample of ptc without lt
in contrast to the cases of ptc with lt , the cases without lt frequently exhibited nondetectable cytokine levels . among the six cytokines analyzed , the expression levels of ifn- , il-10 , and il-2 levels were too low to be measured . based on these data ,
the cytokines expressed in ptc without lt represented a mixed th1 ( tnf- ) and th2 ( il-4 and il-1 ) immunity .
the subtypes of the ptcs with lt were determined to be the most conventional ( 11/13 , 84.61% ) , and those of only two cases were follicular variants ( 2/13 , 15.38% ) .
the tumor sizes ranged from 0.2 to 1.1 cm , with an average of 0.55 cm .
of the 13 cases of this type , six cases showed extrathyroidal tumor extension ( et ) ( 6/13 , 46.15% ) , and two cases presented with lymph node metastasis ( 2/13 , 15.38 % ) .
the subtypes of the ptcs with lt were all conventional ( 10/10 , 100% ) . three cases with et were present ( 3/10 , 30% ) .
tumor sizes ranged from 0.1 to 1.5 cm , with an average of 0.61 cm .
in all 23 cases , the cytokine immune profiles were summarized into two groups : the cases with lt ( n=13 ) and the cases without lt ( n=10 ) .
the cytokines could have originated from inflammatory cells , thyroid tissue , and endothelial cells . in this research ,
after manipulating the data using the concept of a calibrator with il-1 , each sample of ptc with lt was compared ( table 1 ) . among the six cytokines analyzed ,
tnf- , il-4 , and ifn- were relatively well expressed . based on these factors , the cytokine expression in the ptc samples with lt represented a mixed th1 ( tnf- and ifn- ) and th2 ( il-4 and il-10 ) immunity .
after manipulating data using the concept of a calibrator with il-1 , each sample of ptc without lt was compared ( table 2 ) .
in contrast to the cases of ptc with lt , the cases without lt frequently exhibited nondetectable cytokine levels . among the six cytokines analyzed , the expression levels of ifn- , il-10 , and il-2 levels were too low to be measured . based on these data ,
the cytokines expressed in ptc without lt represented a mixed th1 ( tnf- ) and th2 ( il-4 and il-1 ) immunity .
the prognostic parameters ( tumor size , et , and lymph node metastasis ) were not statistically related to lt , but the cases without lt had a tendency for lymph node metastasis ( table 3 ) .
the cases with lt had enhanced expression of the cytokines ( tnf- , ifn- , il-4 , il-10 , and il-2 ) compared to the samples of ptc without lt . among the five cytokines ,
the expression levels of tnf- , ifn- , and il-4 were significantly higher in the cases with lt than in those without lt ( p<.05 ) . in terms of p27 as a surrogate marker for lymph node metastasis , the degree of p27 expression was not correlated with lymph node metastasis in this study ; high - grade expression of p27 had a tendency to occur in ptc cases with lt .
in contrast , lymph node metastasis occurred slightly more frequently in ptc cases without lt . conclusively
, the degree of p27 expression did not show any correlation with lymph node metastasis in this study ( p=.15 ) ( table 4 ) .
due to this unexpected finding , which revealed in the insufficiency of the ptc cases without lt to serve as a reference group , this study adopted a new goal to use the ptc cases with lt to analyze the relationship between cytokine immune profiles and prognostic parameters .
in addition , the ptc cases in this study were mostly microcarcinomas , which might represent the incipient phase of the tumors .
the incipient phase of tumors must always have a limitation in representing proper tumor staging , such as tumor size , nodal metastasis , and tumor extension .
hence , in this study , p27 was used as a surrogate marker of lymph node metastasis in the cases of microcarcinoma ( as an incipient carcinoma ) .
therefore , the statistical analysis of this profiles solely made use of the ptc cases with lt .
except for the il-4 cytokine , the expression levels of the cytokines tnf- , ifn- , and il-10 were lower in the cases with low - grade expression of p27 than those with high - grade expression of p27 , but there was no statistical significance ( table 5 ) .
the cases with et had a tendency to demonstrate higher levels of cytokine expression than those with intrathyroidal tumor confinement ( table 6 ) .
again , there was no statistical significance . in summary , although the data were not statistically significant , the trends follow a pattern where higher cytokine levels were present in the cases with high grade expression of p27 ( except for il-4 ) and et .
this study revealed the presence of mixed th1 ( ifn- , tnf- , and il-2 ) and th2 ( il-4 and il-10 ) immunity in lymphocytes in cases of ptc with lt .
although there is no statistical significance with a limited case number , this study could infer some meaningful results . in the cases with lt ,
the higher expression of cytokine levels had a tendency to be associated with high p27 immuno - reactivity ( table 5 ) .
furthermore , except for il-4 , all cytokines were decreased in the cases with lt and p27 underexpression , which represents the increased possibility of lymph node metastasis . because il-4 is associated with aggressive ptc , the fact that all cytokine except il-4 were highly expressed in the cases with lt and increased p27 expression supports the hypothesis that high expression of cytokines except il-4 may contribute to anticancer effect on the ptc in terms of lymph node metastasis .
in contrast , the cases with et revealed the higher expression of cytokines than those without et . based on these results ,
we propose that the higher expression of cytokines tnf- , ifn- , il-2 , and il-1 might inhibit nodal metastasis but not inhibit ( or possibly enhance ) the extrathyroidal extension . in view of helper t cell immunity , mixed th1 and th2 immunity seems to play a role in anticancer activity by inhibiting lymph node metastasis .
there have been several studies on the immune profiles of thyroiditis . among them , phenekos et al .  
reported that ht and graves disease have two different helper t cell immunities . in their report , with the preferential expression of il-2 , ifn- , il-12 , and il-18 , a th1 pattern of immune response which is characteristic of cellular immunity , is dominant in ht .
in contrast , ajjan et al .   reported a mixed th1 and th2 immune response in ht cases .
they reported that reverse transcription polymerase chain reaction results showed both th1 and th2 immunity .
our findings demonstrated that il-4 expression , together with other cytokines , has a tendency to be higher in the cases with et .
in contrast , in the cases with underexpression of p27 , which may represent lymph node metastasis , il-4 was higher than other cytokines .
this may imply that il-4 contributes to the lymphatic spread of the tumor , a concept that is supported by vella et al .   who demonstrated that il-4 levels were augmented in aggressive ptcs .
they suggested that ptc cells receive protection from apoptosis by il-4 production in the activated t lymphocytes of thyroid glands . to determine which of these conflicting results in terms of both nodal metastasis and capsular tumor extension are correct
, further study may be necessary to disclose the role of il-4 in ptc biological behavior . a previous work by yip et al
.   demonstrated with in vitro tests that il-1 was an anticancer factor , which suppressed the proliferation and reduced the invasive potential of human ptc cells . in this study , il-1 was found to be constitutively expressed in all cases and was used as a calibrator to compare cytokine levels among the cases .
unfortunately , due to its use as a calibrator in this study , il-1 expression levels in each case could not be compared .
it is generally accepted that there is a beneficial relationship between chronic lt and the biological behavior of ptc , which results in improved prognoses .
paulson et al .   suggested that chronic lt might have a protective role in tumor spread . supporting this evidence , mitsiades et al .   reported that th1 cytokines , such as ifn- and tnf- , increase the sensitivity of both normal and neoplastic thyrocytes to fasl and trail , which lead to apoptosis .
established that ht was associated with ptc , as was chronic inflammation with cancer in other locations .
they also mentioned that the coexistence of ht in ptc cases introduced favorable clinical outcomes compared with those of ptc without ht .
determined that patients with ptc and chronic lt had smaller tumor sizes , a lower incidence of capsular invasion , and a significantly lower incidence of lymph node metastases compared to patients without chronic lt .
several studies have also indicated that antithyroid antibodies are able to recognize these malignant cells and destroy them in the same way that they destroy normal follicular cells , contributing to the low rate of clinical progression of these lesions .
the p27 protein was first identified as an inhibitor of cyclin e / cdk2 complexes during transforming growth factor induced g1 arrest .
the inhibitory roles of p27 towards cyclin / cdk complexes are weakened by phosphorylation directed by certain signal transduction pathways .
the current model delineates that p27 suppresses tumorigenesis by inhibiting cyclin / cdk activity in the nucleus , but it exerts other functions in the cytoplasm that are potentially oncogenic .
there have been several clinical studies describing the relationship between the expression of p27 and lymph node metastasis .
reported that p27 expression in nonmetastasizing ptc was lower than that in normal thyroid tissue and higher than that in metastasizing ptc .
our ihc analysis showed that p27 expression did not demonstrate any relationship to lymph node metastasis in ptc with lt , but this may be attributed to the early stage of the tumors , i.e. , our study mainly included patients with microcarcinomas .
there may be a chance that even ptc cases with low expression of p27 were too incipient to reveal lymph node metastasis . with this limitation in the evaluation of the relationship between cytokine levels and nodal metastasis ,
the common p27 marker was used as a surrogate marker to represent the possibility of nodal metastasis in early phase ptcs .
as aforementioned , after focusing on the cases with lt , the cases with low - grade expression of p27 tended to be associated with lower levels of cytokines than those with high - grade expression of p27 .
this may implicate that the cases with lower levels of mixed th1 and th2 cytokines have a higher probability of having lymph node metastasis . considering the low cytokine expression in the cases with underexpression of p27 ( except for il-4 ) , our results indicate that mixed th1 and th2 immune cytokines have a tendency toward anticancer effects in terms of lymph node metastasis . to predict tumor prognosis from cytokine levels
 backgroundpapillary thyroid carcinoma ( ptc ) is frequently accompanied by lymphocytic thyroiditis ( lt ) . 
 some reports claim that hashimoto s thyroiditis ( the clinical form of lt ) enhances the likelihood of ptc ; however , others suggest that lt has antitumor activity . 
 this study was aimed to find out the relationship between the patterns of helper t cell ( th ) cytokines in thyroid tissue of ptc with or without lt and the clinicopathological manifestation of ptc . 
 methodsfresh surgical samples of ptc with ( 13 cases ) or without ( 10 cases ) lt were used . 
 the prognostic parameters ( tumor size , extra - thyroidal extension of ptc , and lymph node metastasis ) were analyzed . 
 the mrna levels of two subtypes of th cytokines , th1 ( tumor necrosis factor  [ tnf- ] , interferon  [ ifn- ] , and interleukin [ il ] 2 ) and th2 ( il-4 and il-10 ) , were analyzed . because most ptc cases were microcarcinomas and recent cases without clinical follow - up , negative or faint p27 immunoreactivity was used as a surrogate marker for lymph node metastasis . 
 resultsptc with lt cases showed significantly higher expression of tnf- ( p = .043 ) , ifn- ( p < .010 ) , il-4 ( p = .015 ) than those without lt cases . 
 although the data were not statistically significant , all analyzed cytokines ( except for il-4 ) were highly expressed in the cases with higher expression of p27 surrogate marker . 
 conclusionsthese results indicate that mixed th1 ( tnf- , ifn- , and il-2 ) and th2 ( il-10 ) immunity might play a role in the antitumor effect in terms of lymph node metastasis . 