the presentation of nb can range from localized tumors to disseminated forms , which have different prognoses and require different treatment . therefore , determining the disease extent and stage at the time of diagnosis is important . a number of genetic and biological markers have been investigated for detection of minimal levels of nb cells . tyrosine hydroxylase ( th ) , which is mainly expressed in sympathetic nervous tissues , has been suggested as a candidate marker for detecting minimal levels of neuroblastoma cells in bone marrow ( bm ) and peripheral blood ( pb ) . molecular detection of th mrna using reverse transcription quantitative real - time polymerase chain reaction ( rt - qpcr ) has been proposed as a specific and sensitive method for detection of minimal disease in nb [ 5 - 8 ] . according to our preliminary reports however , the number of patients was small and the follow - up duration was short . in the current study , we evaluated the significance of th expression in a larger cohort of patients with a longer follow - up period as well as the clinical implications of the level of th expression among th - positive patients . written informed consent for th analysis was obtained at diagnosis from all patients or guardians . this retrospective study was approved by the institutional review board ( irb ) of samsung medical center and the requirement for informed consent was waived ( irb 2014 - 12 - 090 ) . medical records of all patients who were newly diagnosed with nb over a 10-year period from july 2005 to june 2015 were reviewed retrospectively . patients were staged according to the international neuroblastoma staging system   and disease extent was evaluated using computerized tomography ( ct ) , magnetic resonance imaging ( mri ) , technetium-99 bone scans , bilateral bm aspirates and biopsy specimens , and i- or i - metaiodobenzylguanidine ( mibg ) scanning . mycn amplification was determined using competitive polymerase chain reaction ( pcr ) , rt - qpcr , or fluorescence in situ hybridization . tumors were classified as histologically favorable or unfavorable according to the international neuroblastoma pathology classification . measurement of serum lactic dehydrogenase ( ldh ) , ferritin , neuron - specific enolase ( nse ) , and urine vanillylmandelic acid ( vma ) was also included in routine evaluation at diagnosis . patients were stratified into low - risk , intermediate - risk , and high - risk groups based on age , stage , and mycn status . stage 1 , 2 , and 4s tumors without mycn amplification were stratified as low - risk tumors whereas stage 4 tumors in patients older than 12 months or any tumors with amplified mycn were stratified as high - risk tumors . otherwise , incisional or percutaneous needle biopsy was performed with definitive surgery after six chemotherapy cycles . alternating cedc ( cisplatin+etoposide+doxorubicin+cyclophosphamide ) and ice ( ifosfamide+carboplatin+etoposide ) regimens were used for chemotherapy ( table 1 ) . patients with low - risk localized tumors underwent surgery with ( stage 2 ) or without ( stage 1 ) six cycles of preoperative or postoperative chemotherapy . patients with low - risk stage 4s tumors were treated only when there were life - threatening symptoms or the tumor progressed during observation . patients with intermediaterisk tumors received six cycles of preoperative chemotherapy , surgery , three cycles of postoperative chemotherapy , and 12 cycles of 13-cis - retinoic acid ( 125 mg / m / day for 14 days every 4 weeks ) . local radiotherapy to the primary site was administered if gross residual tumor remained after surgery . patients with high - risk tumors received tandem high - dose chemotherapy and autologous stem cell transplantation ( hdct / auto - sct ) after induction treatment ( six cycles of preoperative chemotherapy , surgery , and three cycles of postoperative chemotherapy ) . the second hdct regimen was tm - tbi ( thiotepa+melphalan+total body irradiation ) for patients diagnosed up to december 2008 . for patients diagnosed from january 2009 onwards , tbi was replaced with high - dose i - mibg ( 12 or 18 mci / kg ) to reduce late adverse effects . local radiotherapy was applied to the primary site in all high - risk patients approximately 6 weeks after the second hdct / auto - sct . differentiation therapy involving 13-cis - retinoic acid with immunotherapy using interleukin 2 ( 210 u / m / day for 5 days every 4 weeks ) was administered until 1 year after hdct / auto - sct to reduce relapse from minimal residual tumor cells . pb samples were collected at diagnosis in ethylenediaminetetraacetic acid tubes and mononuclear cells were separated and lysed in trizol reagent ( invitrogen corporation , carlsbad , ca ) . rna was extracted according to the manufacturer s instructions and 1 g of rna was used to generate 20 l of complementary dna using an rna pcr kit ( applied biosystems , foster city , ca ) . rt - qpcr was performed using real - q th quantification kits ( biosewoom , seoul , korea ) on a lightcycler ( roche diagnostics , mannheim , germany ) . rt - qpcr was performed under the following cycling conditions : denaturation at 95c for 10 minutes , 45 cycles at 95c for 10 seconds , 60c for 10 seconds , and 72c for 30 seconds , followed by a cooling step at 40c for 30 seconds . reactions were performed in a 20-l volume containing pcr reaction mixture , taqman probe ( 5'-fam agcgcaggaagctgattgctgagtamra-3 ' ) , primer mixture ( forward , 5'-tcatcacctggtcaccaagtt-3 ' ; reverse , 5'-ggtcgccgtgcctgtact-3 ' ) , 2 l cdna , and sterile water . if the expression value of abl in the rt - qpcr amplification was less than a copy number of 104 , the sample was considered inadequate for rt - qpcr analysis and the rna extraction was repeated . the quantity of th transcripts was normalized to the abl transcript level , and the result was expressed as the ratio of the th to abl transcript copy number . the mann - whitney u test was used for comparison of continuous variables between groups . progression - free survival ( pfs ) and overall survival ( os ) rates with standard errors were estimated using the kaplan - meier method . multivariate analyses of prognostic factors for pfs and os were performed using cox regression analysis . medical records of all patients who were newly diagnosed with nb over a 10-year period from july 2005 to june 2015 were reviewed retrospectively . patients were staged according to the international neuroblastoma staging system   and disease extent was evaluated using computerized tomography ( ct ) , magnetic resonance imaging ( mri ) , technetium-99 bone scans , bilateral bm aspirates and biopsy specimens , and i- or i - metaiodobenzylguanidine ( mibg ) scanning . mycn amplification was determined using competitive polymerase chain reaction ( pcr ) , rt - qpcr , or fluorescence in situ hybridization . tumors were classified as histologically favorable or unfavorable according to the international neuroblastoma pathology classification . measurement of serum lactic dehydrogenase ( ldh ) , ferritin , neuron - specific enolase ( nse ) , and urine vanillylmandelic acid ( vma ) was also included in routine evaluation at diagnosis . patients were stratified into low - risk , intermediate - risk , and high - risk groups based on age , stage , and mycn status . stage 1 , 2 , and 4s tumors without mycn amplification were stratified as low - risk tumors whereas stage 4 tumors in patients older than 12 months or any tumors with amplified mycn were stratified as high - risk tumors . otherwise , incisional or percutaneous needle biopsy was performed with definitive surgery after six chemotherapy cycles . alternating cedc ( cisplatin+etoposide+doxorubicin+cyclophosphamide ) and ice ( ifosfamide+carboplatin+etoposide ) regimens were used for chemotherapy ( table 1 ) . patients with low - risk localized tumors underwent surgery with ( stage 2 ) or without ( stage 1 ) six cycles of preoperative or postoperative chemotherapy . patients with low - risk stage 4s tumors were treated only when there were life - threatening symptoms or the tumor progressed during observation . patients with intermediaterisk tumors received six cycles of preoperative chemotherapy , surgery , three cycles of postoperative chemotherapy , and 12 cycles of 13-cis - retinoic acid ( 125 mg / m / day for 14 days every 4 weeks ) . local radiotherapy to the primary site was administered if gross residual tumor remained after surgery . patients with high - risk tumors received tandem high - dose chemotherapy and autologous stem cell transplantation ( hdct / auto - sct ) after induction treatment ( six cycles of preoperative chemotherapy , surgery , and three cycles of postoperative chemotherapy ) . the second hdct regimen was tm - tbi ( thiotepa+melphalan+total body irradiation ) for patients diagnosed up to december 2008 . for patients diagnosed from january 2009 onwards , tbi was replaced with high - dose i - mibg ( 12 or 18 mci / kg ) to reduce late adverse effects . local radiotherapy was applied to the primary site in all high - risk patients approximately 6 weeks after the second hdct / auto - sct . differentiation therapy involving 13-cis - retinoic acid with immunotherapy using interleukin 2 ( 210 u / m / day for 5 days every 4 weeks ) was administered until 1 year after hdct / auto - sct to reduce relapse from minimal residual tumor cells . pb samples were collected at diagnosis in ethylenediaminetetraacetic acid tubes and mononuclear cells were separated and lysed in trizol reagent ( invitrogen corporation , carlsbad , ca ) . rna was extracted according to the manufacturer s instructions and 1 g of rna was used to generate 20 l of complementary dna using an rna pcr kit ( applied biosystems , foster city , ca ) . rt - qpcr was performed using real - q th quantification kits ( biosewoom , seoul , korea ) on a lightcycler ( roche diagnostics , mannheim , germany ) . rt - qpcr was performed under the following cycling conditions : denaturation at 95c for 10 minutes , 45 cycles at 95c for 10 seconds , 60c for 10 seconds , and 72c for 30 seconds , followed by a cooling step at 40c for 30 seconds . reactions were performed in a 20-l volume containing pcr reaction mixture , taqman probe ( 5'-fam agcgcaggaagctgattgctgagtamra-3 ' ) , primer mixture ( forward , 5'-tcatcacctggtcaccaagtt-3 ' ; reverse , 5'-ggtcgccgtgcctgtact-3 ' ) , 2 l cdna , and sterile water . if the expression value of abl in the rt - qpcr amplification was less than a copy number of 104 , the sample was considered inadequate for rt - qpcr analysis and the rna extraction was repeated . the quantity of th transcripts was normalized to the abl transcript level , and the result was expressed as the ratio of the th to abl transcript copy number . the mann - whitney u test was used for comparison of continuous variables between groups . progression - free survival ( pfs ) and overall survival ( os ) rates with standard errors were estimated using the kaplan - meier method . multivariate analyses of prognostic factors for pfs and os were performed using cox regression analysis . a total of 210 patients ( 118 boys and 92 girls ) were evaluated for the presence of th mrna in pb at diagnosis . the median age at diagnosis was 22 months ( range , 0 to 232 months ) and median follow - up duration was 51 months ( range , 1 to 122 months ) . at diagnosis , 134 patients were older than 12 months , 103 patients had metastatic tumors including four stage 4s tumors , 90 had histologically unfavorable tumors , and 34 had mycn amplified tumors . among 89 patients with high - risk tumors , 30 patients used tm - tbi regimen in the second hdct / auto - sct and the remaining 59 patients used i - mibg - tm regimen . th expression was positive in pb at diagnosis in 60 of 210 patients ( 28.6% ) . the median expression level ( th / abl ratio ) of th mrna transcript detected in pb was 1,58010 ( range , 210 to 3,711,86410 ) . positive th expression was associated with high - risk features ( i.e. , advanced stage , older age , unfavorable pathology , and mycn amplification ) . the levels of serum ldh , ferritin , nse , and urine vma were also higher in th - positive patients ( table 2 ) . among 60 th - positive patients , 50 had metastatic tumors and the remaining 10 had localized tumors ( stage 1 in three , stage 2 in two , and stage 3 in five patients ) . among the th - positive patients , a higher th expression level was associated with advanced stage and high - risk tumor , but not with older age (  12 months ) or mycn amplification ( fig . 1a - d ) . relapse or progression occurred in 19 of 60 th - positive patients ( 31.7% ) , compared with only six of 150 th - negative patients ( 4.0% ) . lower 5-year probabilities of pfs and os were observed in th - positive patients than in th - negative patients ( pfs : 63.86.9% vs. 94.72.1% , p < 0.001 ; os : 70.2 6.7% vs. 92.42.5% , p < 0.001 ) ( fig . 2a ) . in analysis confined to only high - risk patients , the 5-year probabilities of pfs and os remained lower in th - positive patients ( pfs : 55.78.2% vs. 89.65.8% , p=0.001 ; os : 61.48.1% vs. 87.26.1% , p=0.064 ) ( fig . however , in low- and intermediate - risk patients , no difference in the 5-year probabilities of pfs and os was observed between th - positive patients and th - negative patients ( pfs : 96.81.9% vs. 90.08.7% , p=0.447 ; os : 100% vs. 94.52.5% , p=0.725 ) . among high - risk patients , there was no difference in the 5-year probabilities of pfs and os according to the hdct regimen ( tm - tbi vs. i - mibg - tm ) ( pfs : 63.38.8% vs. 78.76.6% , p=0.184 ; os : 70.08.4% vs. 76.7 6.4% , p=0.745 ) . in analysis confined to only th - positive high - risk patients , there was also no difference in survival rates according to the hdct regimen ( pfs : 50.012.5% vs. 61.2 10.5% , p=0.523 ; os : 56.312.4% vs. 67.09.3% , p=0.923 ) . positive th expression in pb at diagnosis was an independent factor for pfs ( hazard ratio , 6.35 ; 95% confidence interval , 2.10 to 19.25 ; p=0.001 ) . the results were similar when the analysis was confined to high - risk patients ( hazard ratio , 9.02 ; 95% confidence interval , 2.06 to 39.50 ; p=0.004 ) ( table 4 ) . among th - positive patients , a higher expression level of th was associated with a worse outcome ( above median level vs. below median level : 52.9 10.0% vs. 75.78.8% , p=0.075 for 5-year pfs ; 55.99.6% vs. 89.25.9% , p=0.047 for 5-year os ) ( fig . a total of 210 patients ( 118 boys and 92 girls ) were evaluated for the presence of th mrna in pb at diagnosis . the median age at diagnosis was 22 months ( range , 0 to 232 months ) and median follow - up duration was 51 months ( range , 1 to 122 months ) . at diagnosis , 134 patients were older than 12 months , 103 patients had metastatic tumors including four stage 4s tumors , 90 had histologically unfavorable tumors , and 34 had mycn amplified tumors . among 89 patients with high - risk tumors , 30 patients used tm - tbi regimen in the second hdct / auto - sct and the remaining 59 patients used i - mibg - tm regimen . th expression was positive in pb at diagnosis in 60 of 210 patients ( 28.6% ) . the median expression level ( th / abl ratio ) of th mrna transcript detected in pb was 1,58010 ( range , 210 to 3,711,86410 ) . positive th expression was associated with high - risk features ( i.e. , advanced stage , older age , unfavorable pathology , and mycn amplification ) . the levels of serum ldh , ferritin , nse , and urine vma were also higher in th - positive patients ( table 2 ) . among 60 th - positive patients , 50 had metastatic tumors and the remaining 10 had localized tumors ( stage 1 in three , stage 2 in two , and stage 3 in five patients ) . among the th - positive patients , a higher th expression level was associated with advanced stage and high - risk tumor , but not with older age (  12 months ) or mycn amplification ( fig . relapse or progression occurred in 19 of 60 th - positive patients ( 31.7% ) , compared with only six of 150 th - negative patients ( 4.0% ) . lower 5-year probabilities of pfs and os were observed in th - positive patients than in th - negative patients ( pfs : 63.86.9% vs. 94.72.1% , p < 0.001 ; os : 70.2 6.7% vs. 92.42.5% , p < 0.001 ) ( fig . 2a ) . in analysis confined to only high - risk patients , the 5-year probabilities of pfs and os remained lower in th - positive patients ( pfs : 55.78.2% vs. 89.65.8% , p=0.001 ; os : 61.48.1% vs. 87.26.1% , p=0.064 ) ( fig . however , in low- and intermediate - risk patients , no difference in the 5-year probabilities of pfs and os was observed between th - positive patients and th - negative patients ( pfs : 96.81.9% vs. 90.08.7% , p=0.447 ; os : 100% vs. 94.52.5% , p=0.725 ) . among high - risk patients , there was no difference in the 5-year probabilities of pfs and os according to the hdct regimen ( tm - tbi vs. i - mibg - tm ) ( pfs : 63.38.8% vs. 78.76.6% , p=0.184 ; os : 70.08.4% vs. 76.7 6.4% , p=0.745 ) . in analysis confined to only th - positive high - risk patients , there was also no difference in survival rates according to the hdct regimen ( pfs : 50.012.5% vs. 61.2 10.5% , p=0.523 ; os : 56.312.4% vs. 67.09.3% , p=0.923 ) . positive th expression in pb at diagnosis was an independent factor for pfs ( hazard ratio , 6.35 ; 95% confidence interval , 2.10 to 19.25 ; p=0.001 ) . the results were similar when the analysis was confined to high - risk patients ( hazard ratio , 9.02 ; 95% confidence interval , 2.06 to 39.50 ; p=0.004 ) ( table 4 ) . among th - positive patients , a higher expression level of th was associated with a worse outcome ( above median level vs. below median level : 52.9 10.0% vs. 75.78.8% , p=0.075 for 5-year pfs ; 55.99.6% vs. 89.25.9% , p=0.047 for 5-year os ) ( fig . a number of genetic markers have been investigated for detection of minimal levels of nb cells [ 13 - 17 ] . viprey et al .   reported that the th , dcx , and phox2b genes are the best candidates for detection of circulating nb cells . corrias et al . , who investigated the advantages of measuring multiple molecular targets over a single molecular target , reported that combinations of multiple targets did not add to the predictive power of th analysis alone . gilbert et al .   also reported that th might be the one of the most useful molecular targets . in this context , in the current study we measured th mrna alone for molecular detection of minimal disease in all patients who were newly diagnosed with nb . positive th expression in pb at diagnosis was associated with high - risk features and worse outcome . more importantly , positive th expression in pb at diagnosis was an independent poor prognostic factor for pfs . current staging systems use conventional diagnostic modalities such as ct , mri , bm biopsy , and various radioisotope scans . patients are stratified into risk groups based on these staging systems and patients within each risk group are treated uniformly . however , our study showed that outcomes within the high - risk group differ according to th expression in pb at diagnosis . previous studies also reported that positive th expression was a poor prognostic indicator in metastatic nb . these findings suggest that th expression at diagnosis might be a significant factor in future methods of risk stratification for nb . some studies examined the clinical implication of positive th expression in non - metastatic nb . yanez et al .   reported an association of positive th expression in pb at diagnosis with a higher probability of relapse in patients with non - metastatic nb . these findings suggest that some patients with localized tumors ( by conventional diagnostic modalities ) might have disseminated micrometastatic disease and therefore might benefit from upstaging and more aggressive treatment . however , in the current study , because only one of 10 th - positive patients with localized tumor experienced relapse , the significance of th expression in patients with localized tumors was unclear . further studies including more patients are needed to evaluate the prognostic importance of th expression in the pb of patients with localized tumors . among th - positive patients , higher th expression was associated with advanced stage and high - risk tumor , but not with older age or mycn amplification , and a higher th expression level at diagnosis was associated with a worse outcome . yanez et al .   reported that a high level of th mrna in pb was a significant independent predictor of event - free survival and os when detected at diagnosis , post - induction therapy , and at the end of treatment . they proposed inclusion of th mrna monitoring throughout the disease course by rt - qpcr as a standard method of disease evaluation that would be helpful in predicting differences in outcome and identify children for whom current treatment is failing or insufficient . the european hr - nbl1/siopen study defined patients with very high th expression as ultrahigh - risk . these ultrahigh - risk patients showed very poor outcome among the group of high - risk nb patients ( pfs , 0% ) , most of whom underwent single hdct / auto - sct following short induction treatment . the authors suggested that patients with very high th expression are candidates for alternative treatment strategies . in the current study , the prognosis of patients with high th expression was also poorer than for those with low th expression , but was not dismal . the relatively better outcome in our patients with high th expression compared to those in the european hr - nbl1/siopen study might be attributable to the longer induction treatment and more intensive consolidation treatment using tandem hdct / auto - sct . in conclusion , positive th expression in pb at diagnosis of neuroblastoma is associated with high - risk features and is an independent prognostic factor for poor pfs . results of the current study suggest that treatment intensity should be tailored according to th expression in pb at diagnosis .