collected at 4 european centers , a total of 113 specimens from 105 patients were included in the present study .
the material comprises 91 mpnsts , 21 neurofibromas , and 1 tumor - free sciatic nerve from a chondrosarcoma patient serving as a nonneoplastic nerve sheath control ( supplementary table s1 ) .
all specimens have been examined by at least one pathologist , and in case of uncertainty of the diagnosis , the samples were excluded from the study .
the mean age at diagnosis for patients with malignant disease was 48 years ( range : 1179 ) for those with sporadic tumors ( n = 47 ) and 33 years ( range : 1470 ) for patients with nf1 ( n = 44 ) .
the median age for patients with neurofibromas ( n = 21 ) was 26 years ( range : 1075 ) .
the patients were diagnosed during 19732008 , and samples were collected from oslo university hospital , norway ; skne university hospital , lund , sweden ; university medical center groningen , netherlands ; and the istituto ortopedico rizzoli , bologna , italy . the norwegian biobank is registered at the institute for public health , biobank registry no .
the study was approved by the regional committee for medical and health research ethics south east , and patients were included following informed consent .
sampling , storage , and analysis of the swedish material were approved by the regional ethics committee of lund university , following informed consent from the patients .
the dutch material was approved according to the university medical center groningen , whereas the rizzoli institute ethics committee approved the italian material , and according to institutional guidelines , adult patients or guardians for minors signed the informed consent form .
dna was extracted either by phenol / chloroform followed by ethanol precipitation as described by the manufacturer of the nucleic acid extractor ( applied biosystems ) or by the qiagen allprep dna / rna mini kit according to the manufacturer 's protocol .
further , the dna was treated with sodium bisulfite using a previously described protocol and/or the epitect bisufite kit ( qiagen ) , followed by qualitative and/or quantitative methylation - specific polymerase chain reaction ( msp and/or qmsp , respectively ) .
the msp reactions were carried out in a total volume of 25 l , containing 20 ng of bilsufite - treated template , 1  pcr buffer ( qiagen ) , 0.2 mm deoxyribonucleotide triphosphate ( roche diagnostics ) , 0.8 mm of each primer , and 0.651 u hotstartaq dna polymerase ( qiagen ) .
the sequences are listed in supplementary table s2 and a schematic drawing of the promoter region with primer locations is depicted in supplementary fig . s1 .
human placental dna ( sigma - aldrich ) treated with sssi methyltransferase ( new england biolabs ) served as a positive control for the methylated reaction , whereas bisulfite - modified dna from normal lymphocytes was used as positive control for the unmethylated reaction .
the pcr products were separated using 2% agarose gels ( biorad ) stained with ethidium bromide ( sigma - aldrich ) and visualized by uv light using a genegenius gel documentation system ( syngene ) .
the results were independently scored by visual inspection by 2 of the authors , and all methylated samples were verified by a second , independent round of msp .
the rassf1a gene promoter was further subjected to qmsp analysis using a protocol previously described .
primers and probe sequences are listed in supplementary table s2 and illustrated in supplementary fig .
s1 . to normalize for potential variations in the amount of input dna ( 30 ng of bisulfite - treated template ) ,
dna methylation was calculated as percent of methylated reference ( pmr ) using the following equation:[(rassf1a / aluc4)sample(rassf1a / aluc4)positivecontrol]100
. 
  samples with pmr values exceeding the highest values among the benign neurofibromas and the nonneoplastic nerve sheath control ( ie , pmr > 0 ) were scored as methylation positive .
total rna was isolated from 5 neurofibromas and 27 mpnsts using trizol ( invitrogen ) and converted to cdna using the high capacity rna - to - dna kit ( applied biosystems ) according to the manufacturer 's protocol .
twenty nanograms of cdna was amplified in a 20-l reaction volume containing 1 taqman universal pcr mastermix ( applied biosystems ) and predesigned 1 taqman gene expression assays ( applied biosystems ; rassf1 , hs 00200394_m1 ; rassf1 isoform a , hs 00945257_m1 ) using the following pcr program : 50c for 2 min , 95c for 10 min , then 45 cycles of 95c for 15 s followed by 60c for 1 min . the quantitative gene expression of rassf1/rassf1a was measured in real time using the 7900 ht sequence detection system ( taqman , applied biosystems ) .
all samples were analyzed in triplicate , and the median value was used for further data analyses .
universal human reference rna ( a mixture of total rna from 10 different cell lines ; agilent ) was used to generate a standard curve , and the quantitative expression levels of rassf1 and rassf1a were normalized against the mean value of the endogenous controls actb ( hs99999903_m1 ) and gusb ( hs99999908_m1 ) .
fisher 's exact test was used for assessment of associations between categorical variables , while the mann - whitney - wilcoxon test was used to examine association between the methylation status of rassf1a and continuous variables .
associations to patient outcome was evaluated only for patients with available tissue from the primary tumor , and disease - specific survival was used as the endpoint where deaths of mpnst were considered events and patients who died of other causes were censored at the time of death .
all mpnst cases included in the present study were monitored closely at the same institution where they were treated , thus the cause of death could be identified with great certainty .
the survival curves were generated and compared using the kaplan  meier method with the breslow test , and cox regression with the wald test was used to generate and test uni- and multivariate proportional hazards .
collected at 4 european centers , a total of 113 specimens from 105 patients were included in the present study .
the material comprises 91 mpnsts , 21 neurofibromas , and 1 tumor - free sciatic nerve from a chondrosarcoma patient serving as a nonneoplastic nerve sheath control ( supplementary table s1 ) .
all specimens have been examined by at least one pathologist , and in case of uncertainty of the diagnosis , the samples were excluded from the study .
the mean age at diagnosis for patients with malignant disease was 48 years ( range : 1179 ) for those with sporadic tumors ( n = 47 ) and 33 years ( range : 1470 ) for patients with nf1 ( n = 44 ) .
the median age for patients with neurofibromas ( n = 21 ) was 26 years ( range : 1075 ) .
the patients were diagnosed during 19732008 , and samples were collected from oslo university hospital , norway ; skne university hospital , lund , sweden ; university medical center groningen , netherlands ; and the istituto ortopedico rizzoli , bologna , italy . the norwegian biobank is registered at the institute for public health , biobank registry no .
the study was approved by the regional committee for medical and health research ethics south east , and patients were included following informed consent .
sampling , storage , and analysis of the swedish material were approved by the regional ethics committee of lund university , following informed consent from the patients .
the dutch material was approved according to the university medical center groningen , whereas the rizzoli institute ethics committee approved the italian material , and according to institutional guidelines , adult patients or guardians for minors signed the informed consent form .
dna was extracted either by phenol / chloroform followed by ethanol precipitation as described by the manufacturer of the nucleic acid extractor ( applied biosystems ) or by the qiagen allprep dna / rna mini kit according to the manufacturer 's protocol .
further , the dna was treated with sodium bisulfite using a previously described protocol and/or the epitect bisufite kit ( qiagen ) , followed by qualitative and/or quantitative methylation - specific polymerase chain reaction ( msp and/or qmsp , respectively ) .
the msp reactions were carried out in a total volume of 25 l , containing 20 ng of bilsufite - treated template , 1  pcr buffer ( qiagen ) , 0.2 mm deoxyribonucleotide triphosphate ( roche diagnostics ) , 0.8 mm of each primer , and 0.651 u hotstartaq dna polymerase ( qiagen ) .
the sequences are listed in supplementary table s2 and a schematic drawing of the promoter region with primer locations is depicted in supplementary fig . s1 .
human placental dna ( sigma - aldrich ) treated with sssi methyltransferase ( new england biolabs ) served as a positive control for the methylated reaction , whereas bisulfite - modified dna from normal lymphocytes was used as positive control for the unmethylated reaction .
the pcr products were separated using 2% agarose gels ( biorad ) stained with ethidium bromide ( sigma - aldrich ) and visualized by uv light using a genegenius gel documentation system ( syngene ) .
the results were independently scored by visual inspection by 2 of the authors , and all methylated samples were verified by a second , independent round of msp .
the rassf1a gene promoter was further subjected to qmsp analysis using a protocol previously described .
primers and probe sequences are listed in supplementary table s2 and illustrated in supplementary fig .
s1 . to normalize for potential variations in the amount of input dna ( 30 ng of bisulfite - treated template )
dna methylation was calculated as percent of methylated reference ( pmr ) using the following equation:[(rassf1a / aluc4)sample(rassf1a / aluc4)positivecontrol]100
. 
  samples with pmr values exceeding the highest values among the benign neurofibromas and the nonneoplastic nerve sheath control ( ie , pmr > 0 ) were scored as methylation positive .
total rna was isolated from 5 neurofibromas and 27 mpnsts using trizol ( invitrogen ) and converted to cdna using the high capacity rna - to - dna kit ( applied biosystems ) according to the manufacturer 's protocol .
twenty nanograms of cdna was amplified in a 20-l reaction volume containing 1 taqman universal pcr mastermix ( applied biosystems ) and predesigned 1 taqman gene expression assays ( applied biosystems ; rassf1 , hs 00200394_m1 ; rassf1 isoform a , hs 00945257_m1 ) using the following pcr program : 50c for 2 min , 95c for 10 min , then 45 cycles of 95c for 15 s followed by 60c for 1 min . the quantitative gene expression of rassf1/rassf1a was measured in real time using the 7900 ht sequence detection system ( taqman , applied biosystems ) .
all samples were analyzed in triplicate , and the median value was used for further data analyses .
universal human reference rna ( a mixture of total rna from 10 different cell lines ; agilent ) was used to generate a standard curve , and the quantitative expression levels of rassf1 and rassf1a were normalized against the mean value of the endogenous controls actb ( hs99999903_m1 ) and gusb ( hs99999908_m1 ) .
fisher 's exact test was used for assessment of associations between categorical variables , while the mann - whitney - wilcoxon test was used to examine association between the methylation status of rassf1a and continuous variables .
associations to patient outcome was evaluated only for patients with available tissue from the primary tumor , and disease - specific survival was used as the endpoint where deaths of mpnst were considered events and patients who died of other causes were censored at the time of death .
all mpnst cases included in the present study were monitored closely at the same institution where they were treated , thus the cause of death could be identified with great certainty .
the survival curves were generated and compared using the kaplan  meier method with the breslow test , and cox regression with the wald test was used to generate and test uni- and multivariate proportional hazards .
in an initial ( qualitative ) msp screening study of several candidate genes ( not shown ) , the rassf1a promoter was found methylated in 23 of 44 mpnst samples ( 52% ) , in contrast to complete absence of methylation in 9 neurofibromas and a normal sciatic nerve ( supplementary table s1 ) .
no associations were found between rassf1a methylation and clinicopathological variables , including nf1 status , grade , localization , or size of the tumors , or patients ' age , sex , or nationality . due to interesting results from survival analyses ,
we subsequently reanalyzed the rassf1a gene promoter by qmsp and expanded the sample series to a total of 91 mpnsts , 21 neurofibromas , and the sciatic nerve . sixty percent of the mpnsts were methylated , whereas all neurofibromas and the sciatic nerve remained unmethylated ( supplementary table s1 ) .
the methylation frequency was slightly , but not significantly , higher among the nf1 patients compared with the non - nf1 patients ( 66% and 54% , respectively ; p = .3 ) .
in concordance with the results from the qualitative analysis , no associations were found between the presence of quantitatively measured methylation of the rassf1a promoter and clinicopathological variables ( supplementary table s3 ) . for samples where rna was obtainable ( n = 27 ) , quantitative gene expression measurements of 2 assays amplifying different transcripts of the rassf1 gene were performed . for mpnsts
, there was a strong association between promoter hypermethylation and reduced gene expression , p = .001 for the most specific assay , amplifying only rassf1a ( fig .  1 ) .
this effect was significant both for nf1 ( n = 15 , p = .03 ) and for non - nf1 patients
( n = 12 , p = .003 ) . both mpnsts with methylated and unmethylated rassf1a promoter had lower expression than the benign neurofibromas ( p = .0001 and p = .05 , respectively ) , suggesting that additional mechanisms are involved in silencing the rassf1a gene in mpnst
1.the gene expression of rassf1a ( assay hs 00945257_m1 detecting nm_007182.4 , rassf1a ) is significantly downregulated in mpnsts with promoter hypermethylation compared with tumors without rassf1a methylation , and also in mpnsts compared with neurofibromas independently of methylation .
the gene expression of rassf1a ( assay hs 00945257_m1 detecting nm_007182.4 , rassf1a ) is significantly downregulated in mpnsts with promoter hypermethylation compared with tumors without rassf1a methylation , and also in mpnsts compared with neurofibromas independently of methylation . for the mpnsts ,
a borderline significance was seen with the second assay , between promoter hypermethylation and reduced gene expression ( p = .06 ; data not shown ) .
in contrast to the first assay , the second assay amplified several transcript variants of the rassf1 gene . in the initial msp ( qualitative ) analyses ,
follow - up data for 36 patients with primary tumors were available , and stratification according to nf1 status suggested that rassf1a methylation could distinguish a subgroup of nf1 patients with worse disease - specific 5-year survival ( n = 20 , p = .011 ) .
this could be seen neither among patients with sporadic disease ( n = 16 , p = .68 ) nor in the unstratified mpnst patient cohort ( n = 36 , p = .11 ) ( data not shown ) .
based on these initial findings , we subsequently expanded the sample series and used a quantitative and more standardized analysis , qmsp .
for this analysis , long - term follow - up data were available for 60 specimens with primary tumor , out of the 90 mpnst patients included , and we confirmed that nf1 patients with rassf1a methylated tumors ( n = 20 ) had a significantly worse prognosis than did patients with unmethylated tumors ( n = 12 , p = .014 ; fig . 
the mean disease - specific survival for nf1 patients with methylation was 27.3 months ( 95% ci : 17.237.4 ) compared with 47.4 months ( 95% ci : 37.557.2 ) for nf1 patients without methylated tumors .
no prognostic value of rassf1a could be found for patients with sporadic disease ( n = 28 , p = .85 ; fig . 
2.disease-specific survival based on rassf1a promoter methylation status for ( a ) patients with nf1-associated mpnsts and ( b ) patients with sporadic disease .
disease - specific survival based on rassf1a promoter methylation status for ( a ) patients with nf1-associated mpnsts and ( b ) patients with sporadic disease .
a multivariate cox regression analysis revealed that methylation status of rassf1a was the strongest predictor of disease - specific survival ( hazard ratio : 5.2 ; 95% ci : 1.419.4 ; p = .013 ) compared with tumor size , metastasis at time of diagnosis , and tumor location ( p = .052 , p = .054 , and p = .076 , respectively ; table  2 ) . 

table 2.prognostic factors for 5-year disease - specific survival of nf1 patients with mpnstunivariatemultivariateparameterno . of patientshr95% ciphr95%
ciprassf1a2.8991.0328.143.04.013unmethylated12refmethylated205.2351.41319.397tumor size , cm301.0530.9921.118.091.0890.9991.187.05tumor location0.9360.3802.309.87.08nonextremities15refextremities170.3360.1011.121metastasis at time of diagnosis4.2991.45812.677.008.05no27refyes53.9760.97416.233abbreviations : hr , hazard ratio ; ref , reference category.continuous variable . prognostic factors for 5-year disease - specific survival of nf1 patients with mpnst abbreviations : hr , hazard ratio ; ref , reference category .
in an initial ( qualitative ) msp screening study of several candidate genes ( not shown ) , the rassf1a promoter was found methylated in 23 of 44 mpnst samples ( 52% ) , in contrast to complete absence of methylation in 9 neurofibromas and a normal sciatic nerve ( supplementary table s1 ) .
no associations were found between rassf1a methylation and clinicopathological variables , including nf1 status , grade , localization , or size of the tumors , or patients ' age , sex , or nationality . due to interesting results from survival analyses ,
we subsequently reanalyzed the rassf1a gene promoter by qmsp and expanded the sample series to a total of 91 mpnsts , 21 neurofibromas , and the sciatic nerve . sixty percent of the mpnsts were methylated , whereas all neurofibromas and the sciatic nerve remained unmethylated ( supplementary table s1 ) .
the methylation frequency was slightly , but not significantly , higher among the nf1 patients compared with the non - nf1 patients ( 66% and 54% , respectively ; p = .3 ) .
in concordance with the results from the qualitative analysis , no associations were found between the presence of quantitatively measured methylation of the rassf1a promoter and clinicopathological variables ( supplementary table s3 ) .
for samples where rna was obtainable ( n = 27 ) , quantitative gene expression measurements of 2 assays amplifying different transcripts of the rassf1 gene were performed . for mpnsts
, there was a strong association between promoter hypermethylation and reduced gene expression , p = .001 for the most specific assay , amplifying only rassf1a ( fig .  1 ) .
this effect was significant both for nf1 ( n = 15 , p = .03 ) and for non - nf1 patients
( n = 12 , p = .003 ) . both mpnsts with methylated and unmethylated rassf1a promoter had lower expression than the benign neurofibromas ( p = .0001 and p = .05 , respectively ) , suggesting that additional mechanisms are involved in silencing the rassf1a gene in mpnst . 

fig .  1.the gene expression of rassf1a ( assay hs 00945257_m1 detecting nm_007182.4 , rassf1a ) is significantly downregulated in mpnsts with promoter hypermethylation compared with tumors without rassf1a methylation , and also in mpnsts compared with neurofibromas independently of methylation .
the gene expression of rassf1a ( assay hs 00945257_m1 detecting nm_007182.4 , rassf1a ) is significantly downregulated in mpnsts with promoter hypermethylation compared with tumors without rassf1a methylation , and also in mpnsts compared with neurofibromas independently of methylation . for the mpnsts ,
a borderline significance was seen with the second assay , between promoter hypermethylation and reduced gene expression ( p = .06 ; data not shown ) .
in contrast to the first assay , the second assay amplified several transcript variants of the rassf1 gene .
in the initial msp ( qualitative ) analyses , follow - up data for 36 patients with primary tumors were available , and stratification according to nf1 status suggested that rassf1a methylation could distinguish a subgroup of nf1 patients with worse disease - specific 5-year survival ( n = 20 , p = .011 ) .
this could be seen neither among patients with sporadic disease ( n = 16 , p = .68 ) nor in the unstratified mpnst patient cohort ( n = 36 , p = .11 ) ( data not shown ) .
based on these initial findings , we subsequently expanded the sample series and used a quantitative and more standardized analysis , qmsp .
for this analysis , long - term follow - up data were available for 60 specimens with primary tumor , out of the 90 mpnst patients included , and we confirmed that nf1 patients with rassf1a methylated tumors ( n = 20 ) had a significantly worse prognosis than did patients with unmethylated tumors ( n = 12 , p = .014 ; fig . 
the mean disease - specific survival for nf1 patients with methylation was 27.3 months ( 95% ci : 17.237.4 ) compared with 47.4 months ( 95% ci : 37.557.2 ) for nf1 patients without methylated tumors .
no prognostic value of rassf1a could be found for patients with sporadic disease ( n = 28 , p = .85 ; fig . 
2.disease-specific survival based on rassf1a promoter methylation status for ( a ) patients with nf1-associated mpnsts and ( b ) patients with sporadic disease .
disease - specific survival based on rassf1a promoter methylation status for ( a ) patients with nf1-associated mpnsts and ( b ) patients with sporadic disease .
a multivariate cox regression analysis revealed that methylation status of rassf1a was the strongest predictor of disease - specific survival ( hazard ratio : 5.2 ; 95% ci : 1.419.4 ; p = .013 ) compared with tumor size , metastasis at time of diagnosis , and tumor location ( p = .052 , p = .054 , and p = .076 , respectively ; table  2 ) . 

table 2.prognostic factors for 5-year disease - specific survival of nf1 patients with mpnstunivariatemultivariateparameterno . of patientshr95% ciphr95%
ciprassf1a2.8991.0328.143.04.013unmethylated12refmethylated205.2351.41319.397tumor size , cm301.0530.9921.118.091.0890.9991.187.05tumor location0.9360.3802.309.87.08nonextremities15refextremities170.3360.1011.121metastasis at time of diagnosis4.2991.45812.677.008.05no27refyes53.9760.97416.233abbreviations : hr , hazard ratio ; ref , reference category.continuous variable .
prognostic factors for 5-year disease - specific survival of nf1 patients with mpnst abbreviations : hr , hazard ratio ; ref , reference category .
. studies on a handful of genes have been reported ( table  1 ) in addition to a recent paper where the methylomes of 10 pooled benign and 10 pooled malignant tumors were compared . here
, we add a detailed study analyzing the promoter methylation of rassf1a . in a large european series of mpnsts we have demonstrated rassf1a to be commonly methylated in this malignancy ( 60% ) , and at a frequency considerably higher than
all normal and benign samples were unmethylated , underscoring that rassf1a promoter methylation is cancer specific . furthermore ,
expression of the rassf1a gene was significantly reduced in mpnst samples with promoter hypermethylation compared with unmethylated samples .
this indicates that methylation causes reduced gene activity , or gene silencing , which is in agreement with reports on other cancer types .
notably , however , also the mpnsts with unmethylated rassf1a promoter had significantly lower gene expression than in benign tumors , indicating that additional mechanisms of gene regulation are involved .
regulation of microtubules has been suggested to be one of the essential tumor suppressor activities of rassf1a , thereby ensuring and maintaining chromosomal stability .
the impaired function of rassf1a in mpnst might contribute to the increased dna copy number variation and complex karyotypes that are key characteristics of mpnsts .
rassf1a methylation has been suggested to be preferentially present in the most aggressive tumors of various kinds , suggesting that it might serve as a marker for tumor progression and metastasis . in the current series of mpnsts ,
however , presence of rassf1a methylation was equally distributed among primary , relapsed , and metastatic lesions and between individuals with and without nf1 .
it was also independent of tumor size and site ( extremities or trunk ) , as well as the patient 's country of origin , age , and gender .
interestingly , survival analyses showed that rassf1a methylation in mpnsts was a surrogate marker for poor prognosis among nf1 patients only and independently of clinical risk factors such as tumor size and metastasis . to the best of our knowledge , no molecular marker in mpnsts has previously been shown to discriminate consistently between inferior and good prognosis selectively for nf1 patients .
clinical factors such as large tumor size , incomplete resection , and distant metastasis are shown to be associated with inferior prognosis , which was also true for the present study .
however , the multivariate survival analyses identified rassf1a methylation as the strongest predictor of outcome among these factors , emphasizing the clinical potential of our finding . biomarkers that are able to predict prognosis could be valuable in decision making when considering treatment alternatives . in the case of mpnsts , one could argue that nf1 patients with methylated rassf1a , predicting inferior prognosis , would benefit from an extended follow - up protocol and adjuvant treatment .
although our study includes a large number of specimens , taking into account the rareness of the disease and the fact that the findings seem valid only for nf1 patients with mpnst , it results in quite small statistical categories .
thus , the prognostic value of rassf1a promoter methylation needs to be validated in an independent sample series .
nevertheless , since methylation of the same gene has been suggested as a prognostic marker for various tumor types , including different sarcomas , the methylation status of rassf1a holds great potential to differentiate patients with both nf and mpnst into groups of good and poor prognosis .
this work was supported by grants from the norwegian cancer society ( pr-2006 - 0442 to r.a.l . , financing s.a.d . as postdoctoral fellow , pr-2008 - 0163 to g.e.l .
, pr-51260 - 2012 to r.a.l . , financing m.k . as a staff scientist ) and the faculty of medicine , university of oslo ( to r.a.l . , financing m.h . as a ph.d student ) .
 backgroundmalignant peripheral nerve sheath tumor ( mpnst ) is a rare and highly aggressive disease with no evidence of effect from adjuvant therapy . 
 it is further associated with the hereditary syndrome neurofibromatosis type 1 ( nf1 ) . 
 silencing of the tumor suppressor gene rassf1a through dna promoter hypermethylation is known to be involved in cancer development , but its impact in mpnsts remains unsettled.methodsthe rassf1a promoter was analyzed by methylation - specific pcr in 113 specimens , including 44 nf1-associated mpnsts , 47 sporadic mpnsts , 21 benign neurofibromas , and 1 nonneoplastic nerve sheath control.resultsrassf1a methylation was found only in the malignant samples ( 60% ) and identified a subgroup among patients with nf1-associated mpnst with a poor prognosis . 
 these patients had a mean 5-year disease - specific survival of 27.3 months ( 95% ci : 17.237.4 ) versus 47.4 months ( 95% ci : 37.557.2 ) for nf1 patients with unmethylated promoters , p = 0.014 . 
 in multivariate cox regression analysis , methylated rassf1a remained an adverse prognostic factor independent of clinical risk factors , p = .013 ( hazard ratio : 5.2 ; 95% ci : 1.419.4).conclusiona considerable number of mpnst samples display hypermethylation of the rassf1a gene promoter , and for these tumors , this is the first molecular marker that if validated can characterize a subgroup of patients with inferior prognosis , restricted to individuals with nf1 . 