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Comparative Study
. 2019 Jan 28;19(1):106.
doi: 10.1186/s12885-019-5306-0.

Droplet digital PCR as an alternative to FISH for MYCN amplification detection in human neuroblastoma FFPE samples

Dinesh Babu Somasundaram  1 , Sheeja Aravindan  2 , Zhongxin Yu  3 , Muralidharan Jayaraman  2   4 , Ngoc T B Tran  3 , Shibo Li  5 , Terence S Herman  1   2 , Natarajan Aravindan  6   7   8
Affiliations
Comparative Study

Droplet digital PCR as an alternative to FISH for MYCN amplification detection in human neuroblastoma FFPE samples

Dinesh Babu Somasundaram et al. BMC Cancer. .

Abstract

Background: MYCN amplification directly correlates with the clinical course of neuroblastoma and poor patient survival, and serves as the most critical negative prognostic marker. Although fluorescence in situ hybridization (FISH) remains the gold standard for clinical diagnosis of MYCN status in neuroblastoma, its limitations warrant the identification of rapid, reliable, less technically challenging, and inexpensive alternate approaches.

Methods: In the present study, we examined the concordance of droplet digital PCR (ddPCR, in combination with immunohistochemistry, IHC) with FISH for MYCN detection in a panel of formalin-fixed paraffin-embedded (FFPE) human neuroblastoma samples.

Results: In 112 neuroblastoma cases, ddPCR analysis demonstrated a 96-100% concordance with FISH. Consistently, IHC grading revealed 92-100% concordance with FISH. Comparing ddPCR with IHC, we observed a concordance of 95-98%.

Conclusions: The results demonstrate that MYCN amplification status in NB cases can be assessed with ddPCR, and suggest that ddPCR could be a technically less challenging method of detecting MYCN status in FFPE specimens. More importantly, these findings illustrate the concordance between FISH and ddPCR in the detection of MYCN status. Together, the results suggest that rapid, less technically demanding, and inexpensive ddPCR in conjunction with IHC could serve as an alternate approach to detect MYCN status in NB cases, with near-identical sensitivity to that of FISH.

Keywords: FISH; Immunohistochemistry; MYCN amplification; Neuroblastoma; ddPCR.

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Conflict of interest statement

Ethics approval and consent to participate

All protocols were approved by the University of Oklahoma Health Sciences Center Institutional Review Board with permission for the research use of de-identified specimens (OUHSC IRB #6207). All experiments were performed in accordance with the University of Oklahoma Health Sciences Institutional Review Board guidelines and regulations for the protection of human subjects.

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Fluorescence in situ hybridization (FISH) for MYCN amplification in human NB specimens. Representative microphotographs of MYCN FISH analysis showing (a) negative amplification (non-amplified) of human MYCN gene with the ratio of MYCN (red signals, indicated by yellow arrowheads) to AFF3 (green signals, indicated by white arrowheads) obviously 1 (2R2G), (b) positive amplification of human MYCN gene with amplified signal of 2 + R2G, and (c) positive MYCN amplification signal appearing as a homogenously stained region and/or double minutes containing numerous signals
Fig. 2
Fig. 2
Immunohistochemistry for MYCN protein expression in human neuroblastomas. Representative microphotographs of MYCN IHC staining showing (a) completely negative (IHC0), (b) weak/faint nuclear positivity (IHC1+), (c) moderate nuclear positivity (IHC2+), and, (d) strong nuclear immunoreactivity (IHC3+) in FFPE sections from NB cases. Insert: Representative staining patterns shown in 40x magnification
Fig. 3
Fig. 3
Analysis of MYCN amplification by droplet digital PCR in human NB specimens. Representative one dimensional ddPCR plots for (a) MYCN and (b) reference gene RNAseP showing side-by-side comparison of MYCN copy number variations in MYCN amplified and non-amplified NB cases. MYCN was read in blue (FAM) channel, while RNASeP was read in green (HEX) channel. Each point represents a single droplet, which is scored as positive (colored and above the threshold intensity, as indicated by the pink line) or negative (grey, below the threshold line), depending on the fluorescent amplitude. (c) Representative (from amplified and non-amplified cases) two-dimensional scatter plots constructed with overlaid ddPCR data of the reference RNAseP (HEX) and MYCN (FAM) showing droplets containing no template (lower left, black), droplets containing only MYCN template (upper left, blue), droplets containing only reference RNAseP template (lower right, green), and droplets containing both MYCN and RNAseP templates (upper right, brown)
Fig. 4
Fig. 4
Comparison of ddPCR and IHC MYCN results with FISH data from the FFPE tissue samples from 79 neuroblastoma cases. Interleaved scatter-plot showing concordance (and discordance) in MYCN amplification status assessment by ddPCR and IHC compared with FISH analysis. A total of 79 neuroblastoma cases with known MYCN status (14 amplified and 65 non-amplified) assessed by FISH as a part of clinical diagnosis were included in the analysis
Fig. 5
Fig. 5
Inter-comparison of MYCN amplification status data from ddPCR, IHC, and FISH analyses of FFPE tissue samples from 33 NB cases. Interleaved scatter-plot showing concordance (and discordance) levels in MYCN amplification status measures between ddPCR, IHC, and FISH analyses. A total of 33 neuroblastoma cases with unknown MYCN status were included in the analysis. FISH was performed on 10 cases, four with known MYCN status (two amplified and two non-amplified) and six from the cohort of unknown status
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