archival tissues of formalin - fixed , paraffin - embedded ( ffpe ) tumor blocks were retrieved from consecutively archived specimens .
a set of 177 t / nk - cell lymphomas diagnosed in severance hospital from 1999 to 2013 were included .
the subtypes of t / nk - cell lymphomas were as follows : extranodal nk / t - cell lymphoma , nasal type ( nktl ; n=60 ) , peripheral t - cell lymphoma , not otherwise specified ( ptcl - nos ; n=44 ) , angioimmunoblastic t - cell lymphoma ( aitl ; n=16 ) , anaplastic large cell lymphoma ( alcl ; anaplastic lymphoma kinase [ alk]-positive , n=16 ; alk - negative , n=17 ) , t lymphoblastic leukemia / lymphoma ( t - lbl ; n=20 ) and other types ( n=4 ; hepatosplenic t - cell lymphoma , n=2 ; subcutaneous panniculitis - like t - cell lymphoma , n=1 ; cutaneous gamma - delta t - cell lymphoma , n=1 ) .
a set of 151 diffuse large b cell lymphoma ( dlbcl ) and other types of 31 b - cell lymphomas , which were diagnosed in severance hospital from 2005 to 2011 , were included . other types of b - cell lymphomas were as follows : follicular lymphoma ( n=14 ) , mantle cell lymphoma ( n=5 ) , marginal zone b - cell lymphoma ( n=7 ) , and burkitt lymphoma ( n=5 ) .
hyperplastic palatine tonsil tissues of cancer - free individuals ( n=20 ) were randomly retrieved .
all tumor samples were reviewed by hematopathologists based on the current world health organization ( who ) criteria .
sections of ffpe tissues were prepared and stained with hematoxylin and eosin . under the microscope , the representative area was confirmed and selected for a 3-mm - core sized tissue microarray ( tma ) .
one or two different representative areas per case were selected and 29 cores were embedded in a tma block .
immunohistochemistry of nova1 ( 1:500 , abcam , cambridge , uk ) was performed on 4-m tissue sections using the ventana benchmark xt autostainer ( ventana medical systems , tucson , az , usa ) .
immunohistochemistry with other primary antibodies was performed for diagnostic purposes and the tested antibodies are listed in appendix 2 .
nova1 expression was semiquantitatively determined according to the h - score method with a total score range of 0300 .
the dominant intensity score of nuclear staining ( 0 , absent ; 1 , weak or barely detectable ; 2 , distinct brown ; and 3 , strong dark brown ) was multiplied by the percentage of positive tumor cell nuclei .
the nova1 protein expression score ranged from 0 to 300 , as high ( 200<h - score300 ) , intermediate ( 100<h - score 200 ) , or low ( 0h - score100 ) expression .
jurkat , a t - lbl cell line , was maintained in rpmi1640 medium ( 22400 - 089 , gibco , life technologies , carlsbad , ca , usa ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs ; 6000 - 044 , gibco , life technologies ) .
hh , a cell line derived from cd30-positive cutaneous t - cell lymphoma , and mac1 , a cell line derived from cd30-positive alk - negative alcl , were maintained in rpmi1640 medium supplemented with 20% heat - inactivated fbs .
snk6 , derived from epstein - barr virus ( ebv)-positive nktl , was cultured in rpmi1640 medium supplemented with 10% heat - inactivated human plasma and 700 u / ml of recombinant interleukin 2 .
yt , an ebv - positive human nk - like leukemic cell line , was cultured in iscove s modified dulbecco s medium ( 12440 - 046 , gibco ) supplemented with 20% heat - inactivated fbs .
all the cell lines derived from t- or nk - cell lymphomas were kindly provided by prof .
toledo , a dlbcl cell line , was purchased from the american type culture collection ( atcc , manassas , va , usa ) and maintained in rpmi1640 supplemented with 10% heat - inactivated fbs .
rna isolation was performed using an rneasy plus mini kit ( 74134 , qiagen , hilden , germany ) , and cdna was constructed using a cdna synthesis kit ( 11754 - 050 , invitrogen , carlsbad , ca , usa ) .
the taqman probes used were as follows : gapdh ( hs99999905-m1 , ab , life technologies ) and nova1 ( hs00359592-m1 , ab , life technologies ) . quantitative polymerase chain reaction was performed using abi steponeplus under the following conditions : 50c for 2 minutes , 95c for 5 minutes , 40 cycles of 95c for 15 seconds , and 60c for minutes .
relative mrna expression levels of nova1 were determined by the comparative method ( 2 ) against the reference gapdh .
the kruskal - wallis ( k - w ) , mann - whitney u ( m - w ) , t and  tests were used to analyze differences between the variables examined .
survival rates were analyzed with the kaplan - meier method , and differences were compared using the log - rank test .
archival tissues of formalin - fixed , paraffin - embedded ( ffpe ) tumor blocks were retrieved from consecutively archived specimens .
a set of 177 t / nk - cell lymphomas diagnosed in severance hospital from 1999 to 2013 were included .
the subtypes of t / nk - cell lymphomas were as follows : extranodal nk / t - cell lymphoma , nasal type ( nktl ; n=60 ) , peripheral t - cell lymphoma , not otherwise specified ( ptcl - nos ; n=44 ) , angioimmunoblastic t - cell lymphoma ( aitl ; n=16 ) , anaplastic large cell lymphoma ( alcl ; anaplastic lymphoma kinase [ alk]-positive , n=16 ; alk - negative , n=17 ) , t lymphoblastic leukemia / lymphoma ( t - lbl ; n=20 ) and other types ( n=4 ; hepatosplenic t - cell lymphoma , n=2 ; subcutaneous panniculitis - like t - cell lymphoma , n=1 ; cutaneous gamma - delta t - cell lymphoma , n=1 ) .
a set of 151 diffuse large b cell lymphoma ( dlbcl ) and other types of 31 b - cell lymphomas , which were diagnosed in severance hospital from 2005 to 2011 , were included . other types of b - cell lymphomas were as follows : follicular lymphoma ( n=14 ) , mantle cell lymphoma ( n=5 ) , marginal zone b - cell lymphoma ( n=7 ) , and burkitt lymphoma ( n=5 ) .
hyperplastic palatine tonsil tissues of cancer - free individuals ( n=20 ) were randomly retrieved .
all tumor samples were reviewed by hematopathologists based on the current world health organization ( who ) criteria .
sections of ffpe tissues were prepared and stained with hematoxylin and eosin . under the microscope , the representative area was confirmed and selected for a 3-mm - core sized tissue microarray ( tma ) .
one or two different representative areas per case were selected and 29 cores were embedded in a tma block .
immunohistochemistry of nova1 ( 1:500 , abcam , cambridge , uk ) was performed on 4-m tissue sections using the ventana benchmark xt autostainer ( ventana medical systems , tucson , az , usa ) .
immunohistochemistry with other primary antibodies was performed for diagnostic purposes and the tested antibodies are listed in appendix 2 .
nova1 expression was semiquantitatively determined according to the h - score method with a total score range of 0300 .
the dominant intensity score of nuclear staining ( 0 , absent ; 1 , weak or barely detectable ; 2 , distinct brown ; and 3 , strong dark brown ) was multiplied by the percentage of positive tumor cell nuclei .
the nova1 protein expression score ranged from 0 to 300 , as high ( 200<h - score300 ) , intermediate ( 100<h - score 200 ) , or low ( 0h - score100 ) expression .
jurkat , a t - lbl cell line , was maintained in rpmi1640 medium ( 22400 - 089 , gibco , life technologies , carlsbad , ca , usa ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs ; 6000 - 044 , gibco , life technologies ) .
hh , a cell line derived from cd30-positive cutaneous t - cell lymphoma , and mac1 , a cell line derived from cd30-positive alk - negative alcl , were maintained in rpmi1640 medium supplemented with 20% heat - inactivated fbs .
snk6 , derived from epstein - barr virus ( ebv)-positive nktl , was cultured in rpmi1640 medium supplemented with 10% heat - inactivated human plasma and 700
yt , an ebv - positive human nk - like leukemic cell line , was cultured in iscove s modified dulbecco s medium ( 12440 - 046 , gibco ) supplemented with 20% heat - inactivated fbs .
all the cell lines derived from t- or nk - cell lymphomas were kindly provided by prof .
toledo , a dlbcl cell line , was purchased from the american type culture collection ( atcc , manassas , va , usa ) and maintained in rpmi1640 supplemented with 10% heat - inactivated fbs .
rna isolation was performed using an rneasy plus mini kit ( 74134 , qiagen , hilden , germany ) , and cdna was constructed using a cdna synthesis kit ( 11754 - 050 , invitrogen , carlsbad , ca , usa ) .
the taqman probes used were as follows : gapdh ( hs99999905-m1 , ab , life technologies ) and nova1 ( hs00359592-m1 , ab , life technologies ) . quantitative polymerase chain reaction was performed using abi steponeplus under the following conditions : 50c for 2 minutes , 95c for 5 minutes , 40 cycles of 95c for 15 seconds , and 60c for minutes .
relative mrna expression levels of nova1 were determined by the comparative method ( 2 ) against the reference gapdh .
the kruskal - wallis ( k - w ) , mann - whitney u ( m - w ) , t and  tests were used to analyze differences between the variables examined .
survival rates were analyzed with the kaplan - meier method , and differences were compared using the log - rank test .
the nova1 expression patterns in the cell nuclei were similar to those reported in a previous study .
almost all b cells of the germinal centers or outside the germinal centers were negative for nova1 . as for t cells , occasional expression of nova1 ( intensity grade 23 with 5% or less percentage of t cells ) was noted in the paracortical area ( table 1 ) .
t / nk - cell lymphomas showed low to high nova1 expression and 99 ( 56.5% ) showed high nova1 expression ( table 1 , fig .
the expression level ( score ) of nova1 was higher in tumor cells of t / nk - cell lymphomas when compared to normal t cells of tonsil tissues ( mean score , 209 vs. 7 ; p<.001 ; non - parametric m - w ) ( fig .
. within t / nk - cell lymphomas , precursor t - lbl showed a relatively higher level of nova1 expression than did nktl or mature ( peripheral ) t - cell lymphomas , although a statistically significant difference was not noted ( p=.226 ; k - w ) ( fig .
according to the specific subtypes of t / nk - cell lymphomas , the expression level of nova1 in tumor cells varied ( p=.011 ; k - w ) .
aitl , alk - negative alcl , and t - lbl showed relatively higher nova1 expression levels when compared to other subtypes ( table 1 , fig .
2c ) . specifically , alk - positive alcl revealed a lower level of nova1 expression when compared to alk - negative alcl ( mean score , 156 vs. 259 ; p=.027 ; m - w ) and alk - positive alcl showed the lowest expression level among subtypes of t / nk - cell lymphomas ( table 1 , fig . 2c ) .
the rate of high , intermediate , or low expression of nova1 was also different according to subtype ( p < .001 ) ; high expression was relatively more frequent in aitl , alk - negative alcl , and t - lbl cells , while low expression was relatively more frequent in nktl and alk - positive alcl ( table 1 , fig .
the tested 151 dlbcl cases revealed an expression pattern with a score less than 10 .
furthermore , in the various types of other b - cell lymphomas , high nova1 expression was not noted ( table 1 , fig .
nova1 mrna was expressed only in the jurkat cell line , which corresponds to t - lbl .
nova1 mrna was not detected in other tested cell lines of hh ( correspondent to peripheral , mature t cell lymphoma ) , mac1 ( peripheral , mature t - cell lymphoma ) , snk6 ( nktl ) , yt ( nk cell leukemia ) , or toledo ( dlbcl ) ( fig .
the nova1 expression level in tumor cells was not different according to the organ / sites where lymphomas were growing ( appendix 3 ) .
the known clinicopathological prognostic factors such as age , lactate dehydrogenase level , ann - arbor stage , or international prognostic index ( ipi ) risk scores showed no correlation with nova1 expression . in cases of mature ( peripheral ) t - cell lymphomas , high expression of nova1 tended to be related to shorter overall survival rate in comparison to low or intermediate nova1 expression , although statistical significance was not observed ( fig .
5a , b ) . in other subtypes , such as nktl and t - lbl
the nova1 expression patterns in the cell nuclei were similar to those reported in a previous study .
almost all b cells of the germinal centers or outside the germinal centers were negative for nova1 . as for t cells , occasional expression of nova1 ( intensity grade 23 with 5% or less percentage of t cells ) was noted in the paracortical area ( table 1 ) .
t / nk - cell lymphomas showed low to high nova1 expression and 99 ( 56.5% ) showed high nova1 expression ( table 1 , fig .
the expression level ( score ) of nova1 was higher in tumor cells of t / nk - cell lymphomas when compared to normal t cells of tonsil tissues ( mean score , 209 vs. 7 ; p<.001 ; non - parametric m - w ) ( fig .
within t / nk - cell lymphomas , precursor t - lbl showed a relatively higher level of nova1 expression than did nktl or mature ( peripheral ) t - cell lymphomas , although a statistically significant difference was not noted ( p=.226 ; k - w ) ( fig .
according to the specific subtypes of t / nk - cell lymphomas , the expression level of nova1 in tumor cells varied ( p=.011 ; k - w ) .
aitl , alk - negative alcl , and t - lbl showed relatively higher nova1 expression levels when compared to other subtypes ( table 1 , fig . 2c ) .
specifically , alk - positive alcl revealed a lower level of nova1 expression when compared to alk - negative alcl ( mean score , 156 vs. 259 ; p=.027 ; m - w ) and alk - positive alcl showed the lowest expression level among subtypes of t / nk - cell lymphomas ( table 1 , fig . 2c )
the rate of high , intermediate , or low expression of nova1 was also different according to subtype ( p < .001 ) ; high expression was relatively more frequent in aitl , alk - negative alcl , and t - lbl cells , while low expression was relatively more frequent in nktl and alk - positive alcl ( table 1 , fig .
the tested 151 dlbcl cases revealed an expression pattern with a score less than 10 .
furthermore , in the various types of other b - cell lymphomas , high nova1 expression was not noted ( table 1 , fig .
nova1 mrna was expressed only in the jurkat cell line , which corresponds to t - lbl .
nova1 mrna was not detected in other tested cell lines of hh ( correspondent to peripheral , mature t cell lymphoma ) , mac1 ( peripheral , mature t - cell lymphoma ) , snk6 ( nktl ) , yt ( nk cell leukemia ) , or toledo ( dlbcl ) ( fig .
the nova1 expression level in tumor cells was not different according to the organ / sites where lymphomas were growing ( appendix 3 ) .
the known clinicopathological prognostic factors such as age , lactate dehydrogenase level , ann - arbor stage , or international prognostic index ( ipi ) risk scores showed no correlation with nova1 expression . in cases of mature ( peripheral ) t - cell lymphomas , high expression of nova1 tended to be related to shorter overall survival rate in comparison to low or intermediate nova1 expression , although statistical significance was not observed ( fig .
in other subtypes , such as nktl and t - lbl , no correlation was noted between nova1 expression and overall survival rate .
in this study , we found that nova1 was frequently and highly expressed in various subtypes of t / nk - cell lymphomas , with about 60% of t / nk - cell lymphoma cases showing high nova1 expression , whereas only a small number of t cells expressed nova1 in the hyperplastic palatine tonsils .
as for b - cell lymphomas , nova1 was negative in various types of b - cell lymphomas other than dlbcl , or normal b cells . in dlbcl , nova1 was expressed in a few cases ; however , high expression was not noted in any cases .
our previous study   revealed high expression of nova1 in activated t cells and the present study also confirmed that finding .
moreover , nova1 was upregulated in lymphomas derived from immature t cells such as precursor t - lbl and nktl .
however , in contrast to t / nk cells , nova1 might not be involved in b cell lesions , either malignant lymphomas or non - neoplastic b cells , as shown in our studies . among subtypes of t
regarding the different biological characteristics and pathogenesis of each subtype of t / nk - cell lymphoma , nova1 expression may be affected by various intrinsic biological factors related to each subtype .
the aitl , alk - negative alcl , and t - lbl subtypes , which showed distinctively high nova1 expression , are biologically characteristic entities .
interestingly , the nova1 expression pattern was different even within alcl specimens , according to alk expression status .
alk - negative alcl showed distinctively high nova1 expression while alk - positive alcl exhibited low nova1 expression .
both types of alcl share common morphologic features of cd30 positivity , large anaplastic  hallmark  cells , frequent loss of pan - t - cell antigens , and expression of cytotoxic molecules , and are classified as the same disease .
however , alk - positive alcl and alk - negative alcl are thought to be different entities ; the clinical features , prognosis , and gene expression signatures have been noted to be different   therefore , the differences in nova1 expression may be related to the intrinsic biologic characteristics of these two types .
there are no known studies that can explain the distinctively low nova1 expression in alk - positive alcl .
we thought that nucleophosmin ( npm ) , the fusion partner of alk - npm translocation / t(2;5 ) in alk - positive alcls , may be associated .
mutated npm molecules are abnormally localized in the cytoplasm because of impaired nuclear localization , and this abnormal localization of npm is thought to be associated with abnormal ribosome biogenesis .
the aberrant function of mutated npm may be related to inhibiting nova1 function in the splicing process of pre - rna molecules via abnormal ribosome biogenesis .
the proportion of cases showing an intermediate level of nova1 expression was relatively higher than that in other subtypes .
this difference may be because ptcl - nos is a disease group of heterogeneous entities of mature ( peripheral ) t - cell lymphomas .
these findings indicate that nova1 expression may be primarily dependent on the intrinsic characteristics of the tumor cell itself rather than extrinsic factors such as the organ - specific microenvironment , which is pre - formed before tumor growth . in association with patient prognosis ,
however , in mature ( peripheral ) t - cell lymphomas , high nova1 expression tended to be related to an inferior overall survival rate . in our previous study
, high nova1 expression levels in reactive , non - neoplastic ( mature ) t cells , which accumulate within the tertiary lymphoid structures of gastric carcinoma microenvironment , were associated with favorable patient prognosis .
suppression of nova1 in such reactive t cells was related with poor patient prognosis , and this effect is thought to be related to the suppression of regulatory t cell functions . from these findings ,
the role of nova1 in tumor t cells of malignant lymphomas and non - tumor t cells of tissues seems to be different . in normal physiologic conditions , nova1 upregulation seems to be involved in normal immune regulation . in neoplastic t cells
alternative splicing of pre - mrna is the main mechanism of transcriptome and proteome diversity , and the splicing program relies on the action of splicing factors   like nova1 .
the action of splicing factors is cell type - specific and independent of organ origin .
previous studies have suggested or shown that normal and tumorous hematopoietic cells are regulated by specific alternative splicing programs and alternative transcript variants such as c - myb , a transcription factor of hematopoietic cells ; cd45 , a transmembrane protein tyrosine phosphatase of t cells , b cells , and other hematopoietic cells ; and cd20 , a surface marker of mature b cells [ 15 - 17 ] .
likewise , nova1 may work as an alternative splicing factor in the lymphoreticular system and in lymphomagenesis of t / nk - cell lineage . among the tested cell lines , only jurkat cells , which corresponded to immature t cells
the functional role of nova1 in lymphoid cells and target genes regulated by nova1 in lymphomagenesis should be identified through further studies . in summary
, nova1 was frequently upregulated in various subtypes of t / nk - cell lymphomas .
nova1 expression may be a new biomarker for t / nk - cell lymphomas and should be studied as a possible therapeutic target for this type of lymphoma .
 background : recent studies have revealed that the splicing factor neuro - oncological ventral antigen 1 ( nova1 ) is enriched in fibroblasts and accumulated t cells of tertiary lymphoid structures . in the present study , 
 we investigated nova1 expression in various subtypes of mature and immature t- and natural killer ( nk)-cell lymphomas as well as in various b - cell lymphoma subtypes . 
 methods : nova1 immunoexpression was evaluated in hyperplastic palatine tonsils ( n = 20 ) , t- and nk - cell lymphomas ( n = 177 ) , diffuse large b - cell lymphomas ( n = 151 ) , and other types of b cell lymphomas ( n = 31 ) . 
 nuclear staining intensity and percentage of positive tumor cells were graded . 
 nova1 mrna expression was analyzed in various lymphoma cell lines.results:tumor cells of t- and nk - cell lymphomas showed higher expression levels of nova1 than did normal paracortical t cells , and 56.5% of t- and nk - cell lymphoma cases showed diffuse and strong expression . 
 the nova1 expression level varied according to the subtype ; it was higher in angioimmunoblastic t - cell lymphoma , anaplastic lymphoma kinase ( alk)-negative anaplastic large cell lymphoma ( alcl ) , and t lymphoblastic leukemia / lymphoma ( t - lbl ) , but it was lower in alk - positive alcl . in almost all b - cell lymphomas , nova1 expression was very low or negative . 
 nova1 mrna was also expressed in jurkat , a t - lbl cell line.conclusions:the present findings suggest that nova1 upregulation may be involved in certain subtypes of t- and nk - cell lymphomas , but not in b - cell lymphomas . upregulated nova1 expression seems to be a specific biological feature of activated t cells such as t- and nk - cell lymphomas . 