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 [3
]. Our results are also consistent with previous preclinical studies demonstrating a correlation between NET protein levels and MIBG uptake into myocardial cells [29
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 [18
]. 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
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. Our results do not address these alternative possibilities and will require further study.
We were unable to replicate previous findings demonstrating an association between NET mRNA expression and clinical MIBG avidity [4
]. Several explanations may account for this discrepancy. 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
]. In one previous study, 5 of 11 patients with negative PCR for NET mRNA nevertheless had positive MIBG scans [4
]. 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
]. In addition, response rates after high-dose 131
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 131
I-MIBG therapy [32
]. The association between MYCN
status and clinical MIBG avidity will be investigated further in a future analysis by our group.