formalin - fixed , paraffin - embedded tissue samples from 61 patients with primary localized esfts were obtained in korea , brazil , and argentina .
fifty - five of the 61 tissue samples were obtained by surgical biopsy and the other six were obtained by surgical excision . at the time of tissue sampling , none of the patients had a history of chemotherapy or radiation therapy and there was no evidence of metastatic disease .
briefly , they are small round cell sarcomas showing diffuse membranous cd99 immunostaining , cytoplasmic periodic acid  schiff staining , and ewsr1 gene translocation as demonstrated with fluorescence in situ hybridization ( zytolight spec ewsr1 dual color break apart probes , zytovision , bremerhaven , germany ) .
however , lack of an ewsr1 gene translocation was not considered as grounds for exclusion if a tumor showed the typical immunophenotypes , which are inconsistent with other small round cell tumors in the differential diagnosis of diseases such as lymphoma and rhabdomyosarcoma .
data , including the follow - up period and overall survival , were available for 48 patients . during follow - up ,
the patients were grouped into dead of disease , alive with disease , and no evidence of disease ( ned ) .
the classification of patients as ned was established when the follow - up period had passed more than 24 months .
our study protocol was reviewed and approved by the kyung hee university institutional review board .
idh1/2 mutations were tested using the pentose nucleic acid ( pna ) clamp idh1/2 mutation detection kit ( panagene , daejeon , korea ) .
all reactions had a total reaction volume of 20 l and contained template dna , primer and pna probe sets , and fluorescent polymerase chain reaction ( pcr ) master mix .
real - time pcr reactions of pna - mediated clamping pcr were performed using a cfx 96 ( bio - rad , hercules , ca , usa ) .
pcr cycling conditions were as follows : 5 minutes at 94c followed by 40 cycles of 94c for 30 seconds , 70c for 20 seconds , 63c for 30 seconds , and 72c for 30 seconds . in this assay
the pna probe , which is complementary to the wild - type sequence , suppresses amplification of the wild - type target .
this suppression results in preferential amplification of mutant sequences by competitively inhibiting the binding of dna primers to wild - type dna .
calculations of the delta ct ( ct ) value were done as follows : ct1=(standard ct)(sample ct ) , ct2=(sample ct)(non - pna mix ct ) .
the gene was considered to be mutated when ct1 values were more than 2.0 . when ct1 values were between 0 and 2 ,
genomic pcr for sequencing was performed in 20-l volumes using 30 ng of template dna and 2 taq pcr smart mix ( solgent , daejeon , korea ) .
the pcr primers used for idh1/2 amplification were as follows : idh1 forward primer ( 5'-cggtcttcagagaagccatt-3 ' ) and idh1 reverse primer ( 5'-gcaaaatcacattattgccaac-3 ' ) .
idh2 forward primer ( 5'-ccaatggaactatccg-3 ' ) and idh2 reverse primer ( 5'-ctccaccctggcctacctg-3 ' ) .
pcr cycling commenced with a 10 minutes hold at 95c , followed by 40 cycles of 95c for 30 seconds , 58c for 40 seconds , and 72c for 60 seconds , terminating with 72c for 5 minutes .
each amplified product was purified using a pcr clean - up kit ( macherey - nagel , duren , germany ) and sequenced in duplicate , in both the forward and reverse directions , using a bigdye terminator kit ( applied biosystems , carlsbad , ca , usa ) on an abi prism 3100 station ( applied biosystems ) , according to the manufacturer s instructions .
the primary antibody that is specific for the idh1 r132h point mutation ( 1:200 , histonova dia - h09 , dianova , hamburg , germany ) was used for the samples bearing idh1 mutations , revealed by either direct pcr or pna clamping .
the bond polymer intense detection system ( vision biosystems , melbourne , australia ) was used according to the manufacturer 's instructions with minor modifications .
pearson s chi - square test or fisher exact test were performed to determine correlations between idh mutation status and clinicopathological parameters .
formalin - fixed , paraffin - embedded tissue samples from 61 patients with primary localized esfts were obtained in korea , brazil , and argentina .
fifty - five of the 61 tissue samples were obtained by surgical biopsy and the other six were obtained by surgical excision . at the time of tissue sampling , none of the patients had a history of chemotherapy or radiation therapy and there was no evidence of metastatic disease .
briefly , they are small round cell sarcomas showing diffuse membranous cd99 immunostaining , cytoplasmic periodic acid  schiff staining , and ewsr1 gene translocation as demonstrated with fluorescence in situ hybridization ( zytolight spec ewsr1 dual color break apart probes , zytovision , bremerhaven , germany ) .
however , lack of an ewsr1 gene translocation was not considered as grounds for exclusion if a tumor showed the typical immunophenotypes , which are inconsistent with other small round cell tumors in the differential diagnosis of diseases such as lymphoma and rhabdomyosarcoma .
data , including the follow - up period and overall survival , were available for 48 patients . during follow - up ,
the patients were grouped into dead of disease , alive with disease , and no evidence of disease ( ned ) .
the classification of patients as ned was established when the follow - up period had passed more than 24 months .
our study protocol was reviewed and approved by the kyung hee university institutional review board .
idh1/2 mutations were tested using the pentose nucleic acid ( pna ) clamp idh1/2 mutation detection kit ( panagene , daejeon , korea ) .
all reactions had a total reaction volume of 20 l and contained template dna , primer and pna probe sets , and fluorescent polymerase chain reaction ( pcr ) master mix .
real - time pcr reactions of pna - mediated clamping pcr were performed using a cfx 96 ( bio - rad , hercules , ca , usa ) .
pcr cycling conditions were as follows : 5 minutes at 94c followed by 40 cycles of 94c for 30 seconds , 70c for 20 seconds , 63c for 30 seconds , and 72c for 30 seconds . in this assay
the pna probe , which is complementary to the wild - type sequence , suppresses amplification of the wild - type target .
this suppression results in preferential amplification of mutant sequences by competitively inhibiting the binding of dna primers to wild - type dna .
calculations of the delta ct ( ct ) value were done as follows : ct1=(standard ct)(sample ct ) , ct2=(sample ct)(non - pna mix ct ) .
the gene was considered to be mutated when ct1 values were more than 2.0 . when ct1 values were between 0 and 2 , a ct2 value was then calculated .
genomic pcr for sequencing was performed in 20-l volumes using 30 ng of template dna and 2 taq pcr smart mix ( solgent , daejeon , korea ) .
the pcr primers used for idh1/2 amplification were as follows : idh1 forward primer ( 5'-cggtcttcagagaagccatt-3 ' ) and idh1 reverse primer ( 5'-gcaaaatcacattattgccaac-3 ' ) .
idh2 forward primer ( 5'-ccaatggaactatccg-3 ' ) and idh2 reverse primer ( 5'-ctccaccctggcctacctg-3 ' ) .
pcr cycling commenced with a 10 minutes hold at 95c , followed by 40 cycles of 95c for 30 seconds , 58c for 40 seconds , and 72c for 60 seconds , terminating with 72c for 5 minutes .
each amplified product was purified using a pcr clean - up kit ( macherey - nagel , duren , germany ) and sequenced in duplicate , in both the forward and reverse directions , using a bigdye terminator kit ( applied biosystems , carlsbad , ca , usa ) on an abi prism 3100 station ( applied biosystems ) , according to the manufacturer s instructions .
the primary antibody that is specific for the idh1 r132h point mutation ( 1:200 , histonova dia - h09 , dianova , hamburg , germany ) was used for the samples bearing idh1 mutations , revealed by either direct pcr or pna clamping .
the bond polymer intense detection system ( vision biosystems , melbourne , australia ) was used according to the manufacturer 's instructions with minor modifications .
pearson s chi - square test or fisher exact test were performed to determine correlations between idh mutation status and clinicopathological parameters .
using the pna clamping method , idh1/2 mutations were detected in three of the 61 patients ( 5% ) . of these three samples , two were idh1 mutants and one sample was an idh2 mutant . by direct sequencing , idh1/2 mutations were detected in two of the 61 patients ( 3% ) , of which , one sample was an idh1 mutant and one sample was an idh2 mutant . in total , four cases out of 61 ( 6% ) harbored idh1/2 mutations by at least one of the two methods employed , and the numbers of idh1 and idh2 mutants were equal ( table 1 ) .
table 2 summarizes the clinicopathologic characteristics of the four mutant cases . in one of four cases
, the idh1 mutation was found by both the pna clamping method and direct sequencing ( case no
, the idh1/2 mutation was found only by the pna clamping method ( cases nos . 2 and 3 ) .
in one of four cases , examination by the pna clamping method showed equivocal results , but direct sequencing showed an idh2 mutation ( case no .
. the overall concordance rate of both methods was over 95% ( 58 of 61 ) and the discordance rate was less than 5% ( 3 of 61 ) . in mutant cases ,
the concordance rate was 25% ( 1 of 4 ) and the discordance rate was 75% ( 3 of 4 ) , although case no . 4 showed equivocal results by the pna clamping method .
immunohistochemistry with antibody to dia - h09 in the four cases bearing idh1/2 mutations showed positive reactions only in case no .
in the four cases bearing idh1/2 mutations , three patients were korean and one patient was brazilian , and the male / female ratio was 1:1 .
three of the four patients were in their second decade and one patient was in the fifth decade .
there was no evidence of distant metastasis in all patients and only one patient died during follow - up .
statistical analyses showed that idh1/2 mutant cases had stronger associations with korean patients than with south american patients ( p=.009 ) .
there was no significant association between idh1/2 mutations and any of the other characteristics of tumors or patients ( table 3 ) .
research on idh1/2 mutations in human tumors has been active in recent years and has revealed that various tumors of different origins bear idh1/2 mutations . following increased interest in idh1/2 mutations in soft tissue tumors , a rudimentary study on sarcoma cell lines demonstrated idh mutations in fibrosarcoma .
subsequently , a study on chondrogenic tumors demonstrated idh1/2 mutations in 81 of 145 ( 56% ) cases with an idh1:idh2 mutation ratio of 10.6:1 .
this study also included the evaluation of idh1/2 mutations in 222 osteosarcomas , 79 chordomas , and 25 esfts , and no mutations were found .
furthermore , previous studies support the value of examining idh mutations for the purpose of differentiating chondrosarcoma from chondroblastic osteosarcoma   and chordoma .
however , a recent study in japan showed that three of 12 osteosarcomas ( 25% )   and 16 of 20 giant cell tumors ( 80% ) harbor idh2 mutations , suggesting the possibility of idh1/2 mutations in various soft tissue tumors in addition to chondrogenic tumors .
the pna clamping method is known to be sensitive , rapid , and simple to perform and can detect mutant alleles when present at levels 100-fold lower than those of wild - type alleles .
in contrast , the minimum percentage of mutant dna required for analysis by direct sequencing is more than 25% .
the two cases harboring idh mutations , found only by pna clamping , might have had less than 25% mutant dna .
it is worth noting that three osteosarcomas bearing idh mutations were found in japanese , and three esft bearing idh mutations were found in korean patients .
although it is still early to remark on the background responsible for these findings , it is possible that asian populations may be predisposed to idh mutations in these tumors and therefore should be further evaluated .
idh1 r132h represents the most common type in gliomas , and idh2 r140q is exclusively found in acute myeloid leukemia . whereas idh1 r132c represents the most common type in cartilaginous tumors , idh2 r172s is the dominant type in osteosarcomas   and giant cell tumors . in our study
, esfts demonstrated equal numbers of idh1 and idh2 mutations in which one case of r132h and one case of r172k were found . a previous study on cartilaginous tumors demonstrated that idh mutations are frequent in acral - based tumors without any other association with other factors .
idh mutations in osteosarcomas and giant cell tumors did not show any association with other clinical parameters .
our study also did not find a significant association between idh mutations and clinical parameters of esfts . in conclusion
it provides evidence that esfts can harbor idh mutations in previously known hot - spot regions , although its incidence is rare . to provide generalized knowledge , our study is still lacking enough data about these mutations in esfts .
further validation with a larger case - based study would establish more reliable and significant data on prevalence rate and the biological significance of idh1/2 mutation status in esfts .
 background : isocitrate dehydrogenase ( idh ) catalyzes the oxidative decarboxylation of isocitrate to yield -ketoglutarate ( -kg ) with production of reduced nicotinamide adenine dinucleotide ( nadh ) . 
 dysfunctional idh leads to reduced production of -kg and nadh and increased production of 2-hydroxyglutarate , an oncometabolite . 
 this results in increased oxidative damage and stabilization of hypoxia - inducible factor  , causing cells to be prone to tumorigenesis.methods:this study investigated idh mutations in 61 ewing sarcoma family tumors ( esfts ) , using a pentose nucleic acid clamping method and direct sequencing.results:we identified four cases of esfts harboring idh mutations . 
 the number of idh1 and idh2 mutations was equal and the subtype of idh mutations was variable . 
 clinicopathologic analysis according to idh mutation status did not reveal significant results.conclusions:this study is the first to report idh mutations in esfts . 
 the results indicate that esfts can harbor idh mutations in previously known hot - spot regions , although their incidence is rare 
 . further validation with a larger case - based study would establish more reliable and significant data on prevalence rate and the biological significance of idh mutations in esfts . 