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Single nCounter assay for prediction of MYCN amplification and molecular classification of medulloblastomas: a multicentric study

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Abstract

Purpose

Medulloblastoma is the most frequent pediatric malignant brain tumor, and is divided into four main subgroups: WNT, SHH, group 3, and group 4. MYCN amplification is an important medulloblastoma prognostic biomarker. We aimed to molecular classify and predict MYCN amplification in a single assay.

Methods

It was included 209 medulloblastomas from 205 patients (Brazil, Argentina, and Portugal), divided into training (n = 50) and validation (n = 159) sets. A nCounter assay was carried out using a custom panel for molecular classification, with additional genes, including MYCN. nSolver 4.0 software and the R environment were used for profiling and MYCN mRNA analysis. MYCN amplification by FISH was performed in 64 cases.

Results

The 205 medulloblastomas were classified in SHH (44.9%), WNT (15.6%), group 3 (18.1%) and group 4 (21.4%). In the training set, MYCN amplification was detected in three SHH medulloblastomas by FISH, which showed significantly higher MYCN mRNA counts than non-FISH amplified cases, and a cutoff for MYCN amplification was established (\(\overline{X }\) + 4σ = 11,124.3). Applying this threshold value in the validation set, we identified MYCN mRNA counts above the cutoff in three cases, which were FISH validated.

Conclusion

We successfully stratified medulloblastoma molecular subgroups and predicted MYCN amplification using a single nCounter assay without the requirement of additional biological tissue, costs, or bench time.

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Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ACTB:

Actin Beta

CNS:

Central nervous system

DAPI:

4′,6-Diamidino-2-phenylindole

FFPE:

Formalin-Fixed Paraffin-Embedded

FITC:

Fluorescein isothiocyanate

FISH:

Fluorescence in situ hybridization

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

HCL:

Chloridric acid

LDH:

Lactate Dehydrogenase

mRNA:

Messenger RNA

MYCN :

MYCN Proto-Oncogene

SD:

Standard deviation

SHH:

Sonic hedgehog

SSC:

Saline Sodium Citrate

WHO:

World health organization

WNT:

Wingless

t-SNE:

T-distributed stochastic neighbor embedding

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Funding

We want to thank the funding support from Barretos Cancer Hospital and the Public Ministry of Labor (MPT), Campinas (Research, Prevention, and Education of Occupational Cancer, Brazil), and post-doc scholarship for DAM from the National Oncology Care Support Program (PRONON), Brazil. MPT supported LSS.

Author information

Authors and Affiliations

  1. Molecular Oncology Research Center Dr. Paulo Prata, Barretos Cancer Hospital, Barretos, Brazil

    Daniel Antunes Moreno, Luciane Sussuchi da Silva, Bruna Minniti Mançano & Rui Manuel Reis

  2. Molecular Diagnosis Laboratory Dr. Paulo Prata, Barretos Cancer Hospital, Barretos, Brazil

    Maicon Fernando Zanon, Murilo Bonatelli, Flávia Escremim de Paula & Rui Manuel Reis

  3. Pathology Department Dr. Paulo Prata, Barretos Cancer Hospital, Barretos, Brazil

    Marcus de Medeiros Matsushita, Gustavo Ramos Teixeira & Iara Viana Vidigal Santana

  4. Barretos School of Health Sciences Dr. Paulo Prata, Barretos Cancer Hospital, Barretos, Brazil

    Gustavo Ramos Teixeira

  5. Department of Pathology and Forensic Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil

    Fabiano Saggioro & Luciano Neder

  6. Federal University of São Paulo (UNIFESP), São Paulo, Brazil

    João N. Stavale & Suzana Maria Fleury Malheiros

  7. AC Camargo Hospital, São Paulo, Brazil

    Matheus Lima & Glaucia Noeli Maroso Hajj

  8. Pathology Department, Italian Hospital of Buenos Aires, Buenos Aires, Argentina

    Hernan Garcia-Rivello & Silvia Christiansen

  9. Pediatric Oncology Department, Hospital São João, Porto, Portugal

    Susana Nunes & Maria João Gil da Costa

  10. Clinical Hematology Department, Hospital São João, Porto, Portugal

    Maria José Soares

  11. Department of Pathology, Hospital São João, Porto, Portugal

    Jorge Pinheiro

  12. Pediatric Oncology Department, Pediatric Neurosurgery Department, Barretos Cancer Hospital, Barretos, Brazil

    Carlos Almeida Junior & Bruna Minniti Mançano

  13. ICVS/3B’s—PT Government Associate Laboratory, Braga/Guimarães, Portugal

    Rui Manuel Reis

  14. School of Medicine, Life and Health Sciences Research Institute (ICVS), University of Minho, Braga, Portugal

    Rui Manuel Reis

Authors
  1. Daniel Antunes Moreno
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  2. Luciane Sussuchi da Silva
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  3. Maicon Fernando Zanon
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  5. Flávia Escremim de Paula
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  6. Marcus de Medeiros Matsushita
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  7. Gustavo Ramos Teixeira
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  8. Iara Viana Vidigal Santana
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  19. Maria José Soares
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Contributions

DAM (ORCID 0000–0002-0764-447X): Data collection, experimental procedures, data analysis, manuscript writing. LSdS (ORCID 0000–0002-7470–4655): Data analysis, prognostic results discussion, manuscript review. MFZ: FISH experiments, data analysis, and manuscript review. MB: nCounter experiments, manuscript review. FEdP: nCounter experiments, manuscript review. IVVS: Pathological review of tumor samples, results, discussion, manuscript review. GRT: Pathological review of tumor samples, results, discussion, and manuscript review. MdMM: Pathological review of tumor samples, manuscript review. FS: Pathological review of tumor samples, results, discussion, and manuscript review. LNS: Pathological review of tumor samples from Ribeirão Preto Medical School, Ribeirão Preto, São Paulo, Brazil. JNS: Pathological review of tumor samples, results, discussion, and manuscript review. SMFM: Clinical review of cases, results, discussion, and manuscript review. ML: Clinical review of cases, results, discussion, and manuscript review. GNMH: Clinical review of cases, results, discussion, and manuscript review. HG-R (ORCID 0000–0003-2056-464X): Pathological review of tumor samples, results, discussion, and manuscript review. SC: Clinical review of cases, results, discussion, and manuscript review. MJGdC: Clinical review of cases, results, discussion, and manuscript review. SN: Clinical review of cases, results, discussion, and manuscript review. MJS: FISH experiments, data analysis, and manuscript review. JP: Pathological review of tumor samples, results, discussion, and manuscript review. CAJ: Clinical review of cases, results, discussion, and manuscript review. BMM: Clinical review of cases, results, discussion, and manuscript review. RMR (ORCID 0000–0002-9639–7940): Supervisor and project coordinator, results, discussion, manuscript writing, and review.

Corresponding author

Correspondence to Rui Manuel Reis.

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Conflict of interests

The authors of the present study declare that they have no conflicts of interests.

Ethical approval

The institutional review board approved this retrospective study from Barretos Cancer Hospital (CAAE: 59979816.6.1001.5437 Number: 4.719.466).

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Moreno, D.A., da Silva, L.S., Zanon, M.F. et al. Single nCounter assay for prediction of MYCN amplification and molecular classification of medulloblastomas: a multicentric study. J Neurooncol 157, 27–35 (2022). https://doi.org/10.1007/s11060-022-03965-1

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