Copy number alteration analysis for neuroblastoma using droplet digital polymerase chain reaction

Abstract

Background: Neuroblastoma (NB) is a malignant tumor derived from the neural crest. MYCN amplification is a well-known adverse molecular prognostic factor for NB. Genome copy number alterations (CNAs) such as chromosome (Chr) 11q deletion, 1p deletion, and 17q gain are associated with a poor prognosis. Fluorescence in situ hybridization (FISH) and Southern blotting analysis are frequently used to detect MYCN amplification. Although comparative genomic hybridization (CGH) and single-nucleotide polymorphism (SNP) chip arrays can easily detect CNAs, these methods are impractical for clinical use due to their cost and run time. Consequently, genome copy number analysis using digital droplet PCR has become widely used to monitor CNAs. Methods: In this study, we used digital droplet polymerase chain reaction to detect MYCN amplification and Chr 11q CNA, which was used for risk stratification according to the International Neuroblastoma Risk Group classification system. We compared the results with data from SNP chip arrays in seven NB cell lines and eight primary NB samples. Results: Digital droplet PCR assays successfully detected MYCN amplification and 11q CNA. The results were very consistent with those obtained by SNP chip assay. Conclusions: Digital droplet PCR can be conducted more rapidly than FISH or Southern blotting. Accordingly, it should be useful for on-site clinical applications aimed at detecting CNAs in NB and performing risk stratification promptly after diagnosis.

17q Gain in Neuroblastoma: A Review of Clinical and Biological Implications

“…Although this method is not currently used in clinical practice, it could be useful for detecting NB in broad screening protocols because of 17q gain's frequency [79]. Ishii et al developed a rapid droplet digital PCR method to detect 11q in NB samples, but they suggested the same technology could be reliably used for analyzing 17q [80]. FISH is frequently used in cytological laboratories to detect chromosome amplifications, deletions, and translocations.…”

Quantitative assessment of copy number alterations by liquid biopsy for neuroblastoma

“…Recently, droplet digital PCR (ddPCR) is being utilized as a robust method to detect copy number alterations with superior accuracy, also in MYCNamp detection of tumor tissues. [25][26][27][28] However, there are only limited reports regarding quantitative ddPCR analysis of MYCN copy number in plasma cfDNA. 12,17 In this study, we attempted a quantitative analysis of MYCNamp and 11qLOH, and also confirmed the utility of liquid biopsy in NB, by demonstrating presence of sufficient amount of tumor-derived cfDNA in plasma of NB patients.…”

“…Recently, droplet digital PCR (ddPCR) is being utilized as a robust method to detect copy number alterations with superior accuracy, also in MYCNamp detection of tumor tissues 25–28 . However, there are only limited reports regarding quantitative ddPCR analysis of MYCN copy number in plasma cfDNA 12,17 .…”