Immunohistochemical evaluation of molecular radiotherapy target expression in neuroblastoma tissue

  • Jennifer E. Gains
  • Neil J. Sebire
  • Veronica Moroz
  • Keith Wheatley
  • Mark N. GazeEmail author
Original Article



Neuroblastoma may be treated with molecular radiotherapy, 131I meta-Iodobenzylguanidine and 177Lu Lutetium DOTATATE, directed at distinct molecular targets: Noradrenaline Transporter Molecule (NAT) and Somatostatin Receptor (SSTR2), respectively. This study used immunohistochemistry to evaluate target expression in archival neuroblastoma tissue, to determine whether it might facilitate clinical use of molecular radiotherapy.


Tissue bank samples of formalin fixed paraffin embedded neuroblastoma tissue from patients for whom clinical outcome data were available were sectioned and stained with haematoxylin and eosin, and monoclonal antibodies directed against NAT and SSTR2. Sections were examined blinded to clinical information and scored for the percentage and intensity of tumour cells stained. These data were analysed in conjunction with clinical data.


Tissue from 75 patients was examined. Target expression scores varied widely between patients: NAT median 45%, inter-quartile range 25% - 65%; and SSTR2 median 55%, interquartile range 30% – 80%; and in some cases heterogeneity of expression between different parts of a tumour was observed. A weak positive correlation was observed between the expression scores of the different targets: correlation coefficient = 0.23, p = 0.05. MYCN amplified tumours had lower SSTR2 scores: mean difference 23% confidence interval 8% - 39%, p < 0.01. Survival did not differ by scores.


As expression of both targets is variable and heterogeneous, imaging assessment of both may yield more clinical information than either alone. The clinical value of immunohistochemical assessment of target expression requires prospective evaluation. Variable target expression within a patient may contribute to treatment failure.


Immunohistochemistry Lutetium DOTATATE Meta-Iodobenzylguanidine Molecular radiotherapy Neuroblastoma Noradrenalin transporter molecule Somatostatin receptor 



This work was made possible by a generous grant from the Adam Hay Fund of the Children’s Cancer and Leukaemia Group. The Children’s Cancer and Leukaemia Group Tissue Bank is funded by Cancer Research UK. The authors were supported by: the National Institute for Health Research University College London Hospitals Biomedical Research Centre, The Neuroblastoma Alliance and Joining Against Cancer in Kids. The Cancer Research UK Clinical Trials Unit at the University of Birmingham receives core funding from Cancer Research UK.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflicts of interest as follows:

Jennifer E. Gains declares that she has no conflict of interest.

Neil J. Sebire declares that he has no conflict of interest.

Veronica Moroz declares that she has no conflict of interest.

Keith Wheatley declares that he has no conflict of interest.

Mark N. Gaze declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with animals performed by any of the authors.

This article does not contain any studies with human participants performed by any of the authors.

This article does contain a study on human tissue obtained from the Children’s Cancer and Leukaemia Group Tissue Bank. All tissue banking procedures are performed in accordance with the ethical standards of the national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study, or their parent or guardian, prior to tissue banking. This consent also prospectively covered subsequently approved studies on the tissue.


  1. 1.
    Gains J, Mandeville H, Cork N, Brock P, Gaze M. Ten challenges in the management of neuroblastoma. Future Oncol. 2012;8:839–58.CrossRefPubMedGoogle Scholar
  2. 2.
    Pearson AD, Pinkerton CR, Lewis IJ, et al. High-dose rapid and standard induction chemotherapy for patients aged over 1 year with stage 4 neuroblastoma: A randomised trial. Lancet Oncol. 2008;9:247–56.CrossRefPubMedGoogle Scholar
  3. 3.
    Matthay KK, Reynolds CP, Seeger RC, et al. Long-term results for children with high-risk neuroblastoma treated on a randomized trial of myeloablative therapy followed by 13-cis-retinoic acid: A children's oncology group study. J Clin Oncol. 2009;27:1007–13.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    AL Y, Gilman AL, Ozkaynak MF, et al. Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N Engl J Med. 2010;363:1324–34.CrossRefGoogle Scholar
  5. 5.
    Ladenstein R, Pötschger U, Pearson AD, et al. Busulfan and melphalan versus carboplatin, etoposide, and melphalan as high-dose chemotherapy for high-risk neuroblastoma (HR-NBL1/SIOPEN): An international, randomised, multi-arm, open-label, phase 3 trial. Lancet Oncol. 2017;18:500–14.CrossRefPubMedGoogle Scholar
  6. 6.
    Wilson JS, Gains JE, Moroz V, Wheatley K, Gaze MNA. Systematic review of 131I-meta iodobenzylguanidine molecular radiotherapy for neuroblastoma. Eur J Cancer. 2014;50:801–15.CrossRefPubMedGoogle Scholar
  7. 7.
    Gaze MN, Gains JE, Walker C, Bomanji JB. Optimization of molecular radiotherapy with [131I]-meta Iodobenzylguanidine for high-risk neuroblastoma. Q J Nucl Med Mol Imaging. 2013;57:66–78.PubMedGoogle Scholar
  8. 8.
    Gains JE, Bomanji JB, Fersht NL, et al. 177Lu-DOTATATE molecular radiotherapy for childhood neuroblastoma. J Nucl Med. 2011;52:1041–7.CrossRefPubMedGoogle Scholar
  9. 9.
    Glowniak JV, Kilty JE, Amara SG, Hoffman BJ, Turner FE. Evaluation of metaiodobenzylguanidine uptake by the norepinephrine, dopamine and serotonin transporters. J Nucl Med. 1993;34:1140–6.PubMedGoogle Scholar
  10. 10.
    Mairs RJ, Gaze MN, Barrett A. The uptake and retention of metaiodobenzyl guanidine by the neuroblastoma cell line NB1-G. Br J Cancer. 1991;64:293–5.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Matthay KK, Shulkin B, Ladenstein R, et al. Criteria for evaluation of disease extent by 123I-metaiodobenzylguanidine scans in neuroblastoma: A report for the international Neuroblastoma risk group (INRG) task force. Br J Cancer. 2010;102:1319–26.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Lewington V, Lambert B, Poetschger U, et al. 123I-mIBG scintigraphy in neuroblastoma: Development of a SIOPEN semi-quantitative reporting method by an international panel. Eur J Nucl Med Mol Imaging. 2017;44:234–41.CrossRefPubMedGoogle Scholar
  13. 13.
    Taniyama Y, Suzuki T, Mikami Y, Moriya T, Satomi S, Sasano H. Systemic distribution of somatostatin receptor subtypes in human: An immunohistochemical study. Endocr J. 2005;52:605–11.CrossRefPubMedGoogle Scholar
  14. 14.
    Albers AR, O'Dorisio MS, Balster DA, et al. Somatostatin receptor gene expression in neuroblastoma. Regul Pept. 2000;88:61–73.CrossRefPubMedGoogle Scholar
  15. 15.
    Georgantzi K, Tsolakis AV, Stridsberg M, Jakobson A, Christofferson R, Janson ET. Differentiated expression of somatostatin receptor subtypes in experimental models and clinical neuroblastoma. Pediatr Blood Cancer. 2011;56:584–9.CrossRefPubMedGoogle Scholar
  16. 16.
    Schilling FH, Bihl H, Jacobsson H, et al. Combined 111In-pentetreotide scintigraphy and 123I-mIBG scintigraphy in neuroblastoma provides prognostic information. Med Pediatr Oncol. 2000;35:688–91.CrossRefPubMedGoogle Scholar
  17. 17.
    Menda Y, O'Dorisio MS, Kao S, et al. Phase I trial of 90Y-DOTATOC therapy in children and young adults with refractory solid tumors that express somatostatin receptors. J Nucl Med. 2010;51:1524–31.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Strosberg J, El-Haddad G, Wolin E, et al. Phase 3 trial of 177Lu-Dotatate for Midgut Neuroendocrine Tumors. N Engl J Med. 2017;376:125–35.CrossRefPubMedGoogle Scholar
  19. 19.
    Ladenstein R, Weixler S, Baykan B, et al. Ch14.18 antibody produced in CHO cells in relapsed or refractory stage 4 neuroblastoma patients: A SIOPEN phase 1 study. MAbs. 2013;5:801–9.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kramer K, Humm JL, Souweidane MM, et al. Phase I study of targeted radioimmunotherapy for leptomeningeal cancers using intra-Ommaya 131-I-3F8. J Clin Oncol. 2007;25:5465–70.CrossRefPubMedGoogle Scholar
  21. 21.
    Dubois SG, Geier E, Batra V, et al. Evaluation of Norepinephrine transporter expression and Metaiodobenzylguanidine avidity in Neuroblastoma: A report from the Children's oncology group. Int J Mol Imaging. 2012;250834:2012.Google Scholar
  22. 22.
    Volante M, Brizzi MP, Faggiano A, et al. Somatostatin receptor type 2A immunohistochemistry in neuroendocrine tumors: A proposal of scoring system correlated with somatostatin receptor scintigraphy. Mod Pathol. 2007;20:1172–82.CrossRefPubMedGoogle Scholar
  23. 23.
    Carlin S, Mairs RJ, McCluskey AG, et al. Development of a real-time polymerase chain reaction assay for prediction of the uptake of meta-[131I]iodobenzylguanidine by neuroblastoma tumors. Clin Cancer Res. 2003;9:3338–44.PubMedGoogle Scholar
  24. 24.
    Lode HN, Bruchelt G, Seitz G, et al. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of monoamine transporters in neuroblastoma cell lines: Correlations to meta-iodobenzylguanidine (MIBG) uptake and tyrosine hydroxylase gene expression. Eur J Cancer. 1995;31A:586–90.CrossRefPubMedGoogle Scholar
  25. 25.
    Mairs RJ, Livingstone A, Gaze MN, Wheldon TE, Barrett A. Prediction of accumulation of 131I-labelled meta-iodobenzylguanidine in neuroblastoma cell lines by means of reverse transcription and polymerase chain reaction. Br J Cancer. 1994;70:97–101.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Montaldo PG, Raffaghello L, Guarnaccia F, Pistoia V, Garaventa A, Ponzoni M. Increase of metaiodobenzylguanidine uptake and intracellular half-life during differentiation of human neuroblastoma cells. Int J Cancer. 1996;67:95–100.CrossRefPubMedGoogle Scholar
  27. 27.
    More SS, Itsara M, Yang X, et al. Vorinostat increases expression of functional norepinephrine transporter in neuroblastoma in vitro and in vivo model systems. Clin Cancer Res. 2011;17:2339–49.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Raggi CC, Maggi M, Renzi D, et al. Quantitative determination of sst2 gene expression in neuroblastoma tumor predicts patient outcome. J Clin Endocrinol Metab. 2000;85:3866–73.PubMedGoogle Scholar
  29. 29.
    Sestini R, Orlando C, Peri A, et al. Quantitation of somatostatin receptor type 2 gene expression in neuroblastoma cell lines and primary tumors using competitive reverse transcription-polymerase chain reaction. Clin Cancer Res. 1996;2:1757–65.PubMedGoogle Scholar
  30. 30.
    Briganti V, Sestini R, Orlando C, et al. Imaging of somatostatin receptors by indium-111-pentetreotide correlates with quantitative determination of somatostatin receptor type 2 gene expression in neuroblastoma tumors. Clin Cancer Res. 1997;3:2385–91.PubMedGoogle Scholar
  31. 31.
    Kogner P, Borgström P, Bjellerup P, et al. Somatostatin in neuroblastoma and ganglioneuroma. Eur J Cancer. 1997;33:2084–9.CrossRefPubMedGoogle Scholar
  32. 32.
    Orlando C, Raggi CC, Bagnoni L, et al. Somatostatin receptor type 2 gene expression in neuroblastoma, measured by competitive RT-PCR, is related to patient survival and to somatostatin receptor imaging by indium −111-pentetreotide. Med Pediatr Oncol. 2001;36:224–6.CrossRefPubMedGoogle Scholar
  33. 33.
    John M, Meyerhof W, Richter D, et al. Positive somatostatin receptor scintigraphy correlates with the presence of somatostatin receptor subtype 2. Gut. 1996;38:33–9.CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Montaldo PG, Carbone R, Ponzoni M, Cornaglia-Ferraris P. Gamma-interferon increases metaiodobenzylguanidine incorporation and retention in human neuroblastoma cells. Cancer Res. 1992;52:4960–4.PubMedGoogle Scholar
  35. 35.
    Iavarone A, Lasorella A, Servidei T, Riccardi R, Mastrangelo R. Uptake and storage of m-iodobenzylguanidine are frequent neuronal functions of human neuroblastoma cell lines. Cancer Res. 1993;53:304–9.PubMedGoogle Scholar
  36. 36.
    Moyes JS, Babich JW, Carter R, Meller ST, Agrawal M, McElwain TJ. Quantitative study of radioiodinated metaiodobenzylguanidine uptake in children with neuroblastoma: Correlation with tumor histopathology. J Nucl Med. 1989;30:474–80.PubMedGoogle Scholar
  37. 37.
    Hadj-Djilani NL, Lebtahi NE, Delaloye AB, Laurini R, Beck D. Diagnosis and follow-up of neuroblastoma by means of iodine-123 metaiodobenzylguanidine scintigraphy and bone scan, and the influence of histology. Eur J Nucl Med. 1995;22:322–9.CrossRefPubMedGoogle Scholar
  38. 38.
    Brans B, Laureys G, Schelfhout V, et al. Activity of iodine-123 metaiodobenzylguanidine in childhood neuroblastoma: Lack of relation to tumour differentiation in vivo. Eur J Nucl Med. 1998;25:144–9.CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Jennifer E. Gains
    • 1
  • Neil J. Sebire
    • 2
  • Veronica Moroz
    • 3
  • Keith Wheatley
    • 3
  • Mark N. Gaze
    • 1
    Email author
  1. 1.Department of OncologyUniversity College London Hospitals NHS Foundation TrustLondonUK
  2. 2.Department of PathologyGreat Ormond Street Hospital for Children NHS Foundation TrustLondonUK
  3. 3.Cancer Research UK Clinical Trials UnitUniversity of BirminghamBirminghamUK

Personalised recommendations