metaiodobenzylguanidine ( mibg ) is an agent that is specifically taken up by sympathetic nervous system tissues , including neuroblastoma tumors .
i - mibg plays an essential role in the diagnostic evaluation of patients with neuroblastoma .
in addition , high - dose i - mibg therapy is an important part of the treatment of patients with relapsed or refractory neuroblastoma . the norepinephrine transporter ( net ; encoded by slc6a2 gene )
is thought to be the primary transporter responsible for specific active cellular uptake of mibg .
several studies have demonstrated that neuroblastoma cell lines that lack net mrna expression fail to accumulate mibg [ 46 ] .
net mrna levels appear to correlate in vitro with extent of mibg uptake [ 68 ] .
moreover , a range of cells that do not typically accumulate mibg can be engineered to do so by transfection of the net gene [ 917 ] .
additional studies in neuroblastoma and other neuroendocrine tumors have suggested that vesicular monoamine transporters ( vmats ) and organic cation transporters ( octs ) may also play a role in mediating uptake of mibg [ 1820 ] .
approximately 10% of patients with neuroblastoma have tumors that do not accumulate mibg on the basis of negative diagnostic i - mibg scans .
one small study utilized rt - pcr to evaluate net gene expression in 6 neuroblastoma tumors from patients with negative baseline mibg diagnostic scans .
none of these tumors had detectable net mrna , while 90% of the 48 mibg - avid tumors had detectable net transcripts .
the correlation between clinical mibg uptake with net protein expression and mrna expression of other putative mibg transporters has not been studied .
the primary aim of the current study was to determine the association between tumor net mrna and protein expression with mibg avidity in patients with neuroblastoma .
secondary aims included evaluation of the association of slc6a2 gene polymorphisms with mibg avidity , association of mrna expression for other membrane transporters with mibg avidity , as well as correlation of net protein expression levels with clinical features .
patients were selected from children 's oncology group ( cog ) protocols a3961 and a3973 for the treatment of intermediate - risk or high - risk neuroblastoma , respectively [ 22 , 23 ] . for the primary analyses focused on net expression ,
all patients with institutional report of mibg nonavid tumors and available tumor mrna were included ( n = 29 ) .
an unselected group of patients with institutional report of mibg avid tumors and available tumor mrna was also included ( n = 54 ) to achieve the desired sample size for the primary analyses . for evaluation of net genotype ,
patients from a3961 and a3973 who had been included in a previous large - scale neuroblastoma genome - wide association project were included [ 24 , 25 ] .
patients were treated according to protocol therapy , as previously described [ 22 , 23 ] .
all laboratories performing rt - pcr , immunohistochemistry , and genotyping were blinded to patient characteristics , including mibg avidity .
all participants or legal guardians provided consent for use of submitted tissue at the time of initial enrollment onto a cog or legacy group neuroblastoma biology study .
the cog neuroblastoma biology committee and the ucsf committee on human research approved the study .
patients underwent diagnostic i- or i - mibg whole body scans at the time of study entry onto either clinical trial a3973 or a3961 .
scans were obtained according to local institutional practice and coded as mibg avid or nonavid by the treating investigator .
baseline diagnostic mibg scans were available for central review for 27 patients with high - risk disease treated on protocol a3973 who also had tumor mrna available for analysis .
the primary clinical endpoint for the current study , mibg avidity , is based upon the results of central review in these 27 patients .
central review was independent of the current study and preceded determination of tumor transporter expression levels for the current study .
investigator assessment of mibg avidity was used only for evaluation of correlation of net gene polymorphisms with mibg avidity .
neuroblastoma tumor material from patients participating in the cog neuroblastoma biology study was submitted frozen to the neuroblastoma tumor bank at the cog biopathology center ( columbus , oh ) . for tumors with a minimum of 60% neuroblastoma tumor in the submitted material
, rna was extracted using invitrogen life technologies trizol reagent ( total rna isolation reagent ; grand island , ny ) .
mrna quality was assessed using either rna integrity number ( rin ) or ratio of absorbance at 260 and 280  nm ( a260/a280 ) .
reverse transcriptase pcr ( rt - pcr ) was performed with applied biosystem 's ( abi ) high capacity reverse transcription kit using 2  g of total rna as per manufacturer instructions .
quantitative real - time pcr ( q - pcr ) reactions were conducted with taqman fast universal master mix ( abi ) in 384-well reaction plates using 10  ng of cdna per reaction .
commercial taqman probe and primer sets for net ( assay i d : hs01567441_m1 ) , oct-1 ( assay i d : hs00427552_m1 ) , oct-2 ( assay i d : hs00533907_m1 ) , oct-3 ( assay i d : hs01009568_m1 ) , vmat-1 ( assay i d : hs00915193_m1 ) , vmat-2 ( assay i d : hs00161858_m1 ) , mate-1 ( assay i d : hs00217320_m1 ) , gapdh ( assay i d : hs99999905_m1 ) , and pgk1 ( assay i d : hs99999906_m1 ) were purchased from abi .
reactions were run on an abi prism 7900ht , and the thermal cycling conditions were 95c for 20 seconds followed by 60 cycles of 95c for 3 seconds and 60c for 30 seconds .
results using either control were well correlated ( r = 0.91 ) such that only the results normalized to pgk1 are presented .
expression vector containing net cdna was used as a positive control and demonstrated that net expression levels in the tumor tissue were within the linear range of the standard curve .
all samples were tested in triplicate , and the result reported is the mean of three separate experiments .
paraffin - embedded tumor material was prepared using standard methods at each treating institution and submitted to the cog neuroblastoma tumor bank .
each of the formalin fixed paraffin embedded glass slides was stained with a commercially available net antibody ( net17 - 1 ; mab technologies ; stone mountain , ga ) at a 1  :  1000 dilution .
antigen retrieval was performed in a pressure cooker with 0.01  m - citrate buffer at a ph of 7.6 .
slides were blocked using the avidin biotin blocking kit ( sp-2001 ; vector laboratories ; burlingame , ca ) .
the secondary antibody used was a biotinylated anti - mouse igg 1  :  200 dilution ( vector ) .
slides were then incubated per manufacturer protocol with an avidin biotin complex ( vector ) .
lastly , the slides were incubated with diaminobenzidine using a high - sensitivity substrate chromogen system ( dako ; carpinteria , ca ) and counterstained with hematoxylin .
staining results were assessed by one pathologist ( bp ) using a four point scoring system : 0 + = tumor cells display complete lack of staining ; 1 + = tumor cells show faint cytoplasmic staining ; 2 + = tumor cells show intermediate cytoplasmic staining ; 3 + = tumor cells display intense cytoplasmic staining ( see supplemental figure 1 for representative sections in suplementary material available online at doi:10.1155/2012/250834 . ) the percentage of cells positive for net by ihc was also determined ( supplemental figure 1 ) .
a net protein expression composite score was derived by multiplying the percentage of tumor cells expressing net by the intensity score .
germline dna was isolated and genotyped as previously described . in brief , the illumina humanhap550 beadchip was used to genotype samples at over 550,000 single nucleotide polymorphisms ( snps ) . from this larger dataset ,
all snps in the net gene ( slc6a2 ) locus were chosen for analysis in the current study .
while no snps were excluded due to low minor allele frequencies , only 4 snps had minor allele frequencies < 10% .
given the sample size and nonnormal distribution of transporter levels , the wilcoxon rank sum test was used to compare transporter expression levels between patients with mibg avid versus mibg nonavid tumors .
the fisher exact test was used to compare net protein intensity category between patients with mibg avid vs. mibg nonavid tumors .
the wilcoxon rank sum test was also used to compare net protein levels between groups defined by categorical clinical characteristics other than mibg avidity .
kaplan - meier methods were used to estimate event - free survival ( efs ) from time of entry on to clinical trial ( a3961 or a3973 ) to disease progression , relapse , death from any cause , or second malignancy .
differences in efs based on median net protein expression levels were assessed using the log - rank test .
genotyping data were coded based on the number of copies of the minor allele present at each position evaluated in the net gene .
binomial tests for equality of proportions were used to evaluate statistical associations using a dominant genetic model , while logistic regression was used to assess an additive genetic model . to adjust for multiple testing , the r library    q - value    that converts p values into corresponding false discovery rates   was used .
the characteristics of the remaining 82 patients who form the main analytic cohort are shown in table 1 .
thirty - one patients had intermediate - risk and 51 patients had high - risk neuroblastoma .
sixty - four percent of patients in this selected cohort had mibg uptake on scan by institutional report .
of the 82 patients with available tumor mrna , 27 patients had centrally reviewed mibg scans .
of the 82 patients with available tumor mrna , 61 also had paraffin embedded tumor available for net immunohistochemistry . of these 61 patients ,
23 patients had centrally reviewed mibg scans . based on strong preclinical data implicating net as the primary transporter responsible for mibg uptake
the distribution of net tumor mrna expression according to centrally - reviewed mibg avidity is shown in figure 1 .
this distribution demonstrates extensive overlap in net expression between patients with and without mibg avid tumors .
the median net expression level for the 19 patients with mibg avid tumors was 12.9% ( range 1.673.7% ) , while the median net expression level for the 8 patients with mibg nonavid tumors was 5.9% ( range 0.6110.0% ; p = 0.31 ) .  
germline genotype data for 26 snps ( supplemental table 1 ) in the slc6a2 gene encoding net were available in 325 patients treated on protocols a3961 and a3973 who were included in a previous genome - wide association study [ 24 , 25 ] .
for this analysis only , investigator assessment of mibg avidity , rather than central review assessment of mibg avidity , was used as the clinical outcome of interest .
there was no correlation between mibg avidity and any of the snp genotypes , using either dominant or additive genetic models ( supplemental table 1 ) . since neither net mrna expression nor slc6a2 genotype correlated with mibg avidity
of the 82 patients in our overall cohort , adequate archival tumor material was available in 61 cases .
of these 61 patients , 23 had centrally reviewed mibg scans ( 15 mibg avid and 8 mibg nonavid ) .
the percent of tumor cells with net protein expression correlated with mibg avidity . specifically , the median percent expression was 50% ( range 0100% ) in mibg avid patients compared to 10% ( range 080% ) in mibg nonavid patients ( p = 0.027 ) .
however , these two groups showed extensive overlap in the distribution of the percent of tumor cells expressing net , including one mibg - avid patient with 0% net expression ( figure 2(a ) ) .
patients with mibg avid tumors had a trend towards higher net intensity scores compared to patients with mibg nonavid tumors ( p = 0.06 ) .
only 1 of 8 patients with centrally reviewed mibg nonavid tumors had a net protein intensity score 2 .
we also evaluated a composite score of percent of tumor cells with net protein expression and intensity score . this composite score correlated with mibg avidity .
specifically , the median composite score was 100 ( range 0300 ) in mibg avid patients compared to 10 ( range 0240 ) in mibg nonavid patients ( p = 0.023 ; figure 2(b ) ) .
we next evaluated whether net mrna expression levels correlated with net composite protein expression score .
a scatterplot revealed little correlation between mrna and protein expression in 61 patients with both measurements ( correlation coefficient = 0.22 ; p = 0.09 ; supplemental figure 2 ) .
we also categorized mrna expression as above and below the group median and evaluated potential differences in net composite protein score .
the median composite protein score for patients with high net mrna expression was 130 ( range 0300 ) compared to 90 ( range 0300 ) for patients with low net mrna expression ( p = 0.15 ) .
we next evaluated whether net protein expression composite score correlates with patient clinical characteristics ( table 3 ) .
one of the strongest findings was that patients with mycn nonamplified tumors showed significantly higher net protein expression compared to patients with mycn amplified tumors ( p < 0.001 ) .
patients with intermediate - risk disease and localized tumors also had significantly higher net protein expression . in order to evaluate the prognostic impact of net protein expression composite score , a kaplan - meier curve of estimated efs
outcomes did not differ by log - rank test ( p = 0.69 ; data not shown ) .
while our results demonstrate differential net protein expression between patients with mibg avid versus nonavid tumors , we observed substantial overlap in the extent of net protein expression between these groups . in order to evaluate
if other cation transporters might also be responsible for mibg uptake , we compared mrna expression for the following transporters between patients with mibg avid and nonavid tumors : oct1 ; oct3 ; mate1 ; vmat1 ; vmat2 . while the median vmat1 and vmat2 expression levels were higher in mibg avid tumors , none of these differences were statistically significant ( p > 0.10 for all comparisons ; table 4 ) .
we report for the first time a positive association between clinical mibg tumor avidity and net protein expression by neuroblastoma cells .
this finding supports the critical role of net in mediating specific active uptake of mibg into neuroblastoma cells .
our results are also consistent with previous preclinical studies demonstrating a correlation between net protein levels and mibg uptake into myocardial cells .
while we observed a significant association between mibg avidity and net protein expression , we also noted overlap in net protein expression between patients with mibg avid and mibg nonavid tumors .
specifically , our cohort includes both patients with high net protein expression and mibg nonavid tumors as well as patients with low net protein expression and mibg avid tumors .
given that mibg avidity was centrally assessed by independent review , misclassification of mibg avidity is unlikely to account for these findings .
our genotyping results suggest that individual variations in the structure of the slc6a2 gene are also unlikely to account for these results . instead
, our findings raise the possibility that mibg uptake may be influenced by factors in addition to net protein .
it is possible that tumors with low net protein expression may accumulate mibg via other transporters .
candidate transporters include octs and vmats as these transporters have been implicated in mibg uptake in other neuroendocrine tumors [ 1820 ] .
our evaluation of oct and vmat mrna expression did not reveal any statistically significant associations with mibg avidity , including subset analysis focusing only on patients with low net protein expression and mibg avid tumors ( data not shown ) .
given the trend showing higher vmat1 and vmat2 mrna median levels in mibg avid tumors , future studies will focus on tumor vmat protein levels .
likewise , it is possible that tumors with high net protein expression that fail to accumulate mibg may have increased expression of mibg efflux transporters that account for low net uptake of mibg .
while mate-1 mrna expression did not correlate with mibg avidity , evaluation of protein levels of mate-1 and other efflux transporters may be informative .
alternatively , other physiologic parameters , such as tumor vascularity and ph , may impede distribution of mibg to the tumor cell membrane .
we were unable to replicate previous findings demonstrating an association between net mrna expression and clinical mibg avidity .
first , our study relied on archived tumor mrna obtained within the context of cooperative group trials .
as such , degradation of mrna may have resulted in a false negative result , though our mrna quality data may argue against this point .
our findings suggest that immunohistochemistry for net protein may be a more practical approach for evaluating net expression in future cooperative group studies and in clinical practice .
second , as discussed above , it is possible that the association between mibg avidity and either net protein or mrna expression is imperfect .
we note that in previous studies of net mrna levels as predictors of clinical mibg uptake , cases of clinical mibg avidity in the setting of low net mrna expression have been reported [ 4 , 30 ] .
in one previous study , 5 of 11 patients with negative pcr for net mrna nevertheless had positive mibg scans .
third , it is possible that , as we observed , net mrna expression does not correlate with net protein expression in human neuroblastoma tissue , perhaps through posttranslational modification of net protein .
one of our secondary analyses yielded the previously unreported association between mycn amplification and low net protein expression .
other statistically significant clinical correlations with lower net protein expression ( patients with high - risk disease and patients with metastatic disease ) may be driven by this association with mycn amplification . as an exploratory secondary analysis unadjusted for multiple statistical testing , it is also possible that this association is a chance finding and therefore requires replication by other groups .
we note that one previous report did not detect a difference in clinical mibg avidity according to tumor mycn status .
in addition , response rates after high - dose i - mibg therapy do not appear to differ between patients with mycn amplified and mycn nonamplified tumors , though all patients were required to have mibg avid tumors to receive i - mibg therapy .
the association between mycn status and clinical mibg avidity will be investigated further in a future analysis by our group .
we have demonstrated that neuroblastoma tumor cell net protein expression correlates with clinical mibg avidity and also with tumor mycn status
. additional work , including gene and protein expression profiling efforts , will need to investigate other mechanisms of mibg uptake in cases with low net protein expression .
future studies may also focus on the correlation between tumor net protein expression and response to targeted radiotherapy with high - dose i - mibg .
 
 purpose . 
 123i - metaiodobenzylguanidine ( mibg ) is used for the diagnostic evaluation of neuroblastoma . 
 we evaluated the relationship between norepinephrine transporter ( net ) expression and clinical mibg uptake . 
 methods . quantitative reverse transcription pcr ( n = 82 ) and immunohistochemistry ( ihc ; n = 61 ) were performed for neuroblastoma net mrna and protein expression and correlated with mibg avidity on diagnostic scans . 
 the correlation of net expression with clinical features was also performed . results . 
 median net mrna expression level for the 19 mibg avid patients was 12.9% ( range 1.673.7% ) versus 5.9% ( range 0.6110.0% ) for the 8 nonavid patients ( p = 0.31 ) . 
 median percent net protein expression was 50% ( range 0100% ) in mibg avid patients compared to 10% ( range 080% ) in nonavid patients ( p = 0.027 ) . 
 mycn amplified tumors had lower net protein expression compared to nonamplified tumors ( 10% versus 50% ; p = 0.0002 ) . 
 conclusions . 
 net protein expression in neuroblastoma correlates with mibg avidity . 
 mycn amplified tumors have lower net protein expression . 
 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions