MYCN Copy Number Detection in Neuroblastoma
FISH (Fluorescence In Situ Hybridization) is the preferred method for detecting MYCN copy number in neuroblastoma, as it provides superior sensitivity and accuracy compared to PCR, particularly when tumor cell content is low or heterogeneous. 1
Primary Recommendation: FISH as Gold Standard
The NCCN guidelines explicitly recommend FISH as one of the acceptable methods for assessing MYCN amplification status, which must be performed in all neuroblastomas and neuroblastoma nodules of ganglioneuroblastoma nodular tumors. 1 While the guidelines also mention that next-generation sequencing (NGS) is increasingly preferred when available, FISH remains a validated and widely used standard approach. 1
Why FISH Outperforms PCR
Superior Detection in Low Tumor Content Samples
- FISH can accurately detect MYCN amplification even when tumor cells comprise ≤30% of the specimen, whereas Southern blot analysis (the traditional molecular method similar to PCR-based approaches) fails in these scenarios. 2
- This is critical because clinical specimens often contain significant contamination from normal peripheral blood, bone marrow, or stromal cells. 2
Cell-by-Cell Analysis Capability
- FISH allows analysis of multiple individual interphase nuclei of tumor cells, providing direct visualization of MYCN copy number in each cell regardless of surrounding normal tissue. 2
- This single-cell resolution enables detection of the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma, which PCR-based methods cannot reliably identify. 2
Minimal Tissue Requirements
- FISH can be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies, making it ideal for limited biopsy specimens. 2
Clinical Context and Prognostic Importance
Critical Risk Stratification Factor
- MYCN amplification is the strongest independent prognostic risk factor in neuroblastoma and overrides all other prognostic factors, mandating high-risk treatment except for completely resected L1 tumors. 1, 3, 4
- Accurate MYCN detection is indispensable for selecting appropriate treatment, as patients with MYCN amplification have significantly worse outcomes. 5, 6
Definition of Amplification
- MYCN amplification is defined as ≥10 copies of the gene. 5
- Patients with 3-9 copies (MYCN gains) have intermediate outcomes, while those with ≥10 copies have the poorest prognosis. 5
- Even in favorable-stage (stage A, B, or Ds) neuroblastoma, MYCN amplification dramatically worsens outcomes, with 7-year event-free survival dropping from 91% to 50%. 6
Alternative and Complementary Methods
Next-Generation Sequencing (NGS)
- NGS is increasingly recommended when available, as it can simultaneously evaluate MYCN amplification, segmental chromosomal aberrations, and actionable mutations (such as ALK alterations) in a single assay. 1
- NGS is particularly valuable when tissue is limited, as it provides comprehensive molecular profiling. 1
- However, the NGS approach must enable robust assessment of copy number status to be reliable for MYCN detection. 1
Methods NOT Recommended for MYCN Detection
- PCR (particularly RT-PCR) is NOT the standard method for MYCN copy number assessment. The INRG consensus guidelines discuss RT-PCR only for detecting minimal residual disease using tyrosine hydroxylase (TH) mRNA as a target, not for MYCN amplification. 1
- Southern blot analysis, while historically used, has been superseded by FISH due to its inability to detect amplification in specimens with low tumor content. 2
Practical Implementation
For routine clinical practice, order FISH for MYCN amplification on all newly diagnosed neuroblastoma specimens. 1 If your institution has validated NGS with robust copy number assessment capability, this can replace FISH and provide additional therapeutic targets. 1 Ensure results are available before initiating therapy, as MYCN status fundamentally determines risk classification and treatment intensity. 3, 4