Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress
Clinical Collections
Advances in Alzheimer’s

Keynote webinar | Spotlight on medication adherence

LIVE: Thursday 27th June 2024, 18:00-19:30 (CEST)
Join our expert panel to discover why you need to understand the drivers of non-adherence in your patients, and how you can optimize medication adherence in your clinics to drastically improve patient outcomes.
ADVANCED SEARCH
Log in
Top
BMC Cancer
Published in: BMC Cancer 1/2019

Open Access 01-12-2019 | Fluorescence in Situ Hybridization | Research article

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

Authors: Dinesh Babu Somasundaram, Sheeja Aravindan, Zhongxin Yu, Muralidharan Jayaraman, Ngoc T. B. Tran, Shibo Li, Terence S. Herman, Natarajan Aravindan

Published in: BMC Cancer | Issue 1/2019

Login to get access

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.
Literature
1.
Marc TG, Gurney JG, Smith MA, Olshan AF: Sympathetic nervous system tumors. Cancer Incidence and Survival among Children and Adolescents: United States SEER Program 1975–1995, National Cancer Institute, Bethesda, 1999, NIH Pub. No. 99–4649(ICCC IV):65–72.
2.
Gurney JG, Smith MA, Ross JA. Cancer among infants. Cancer Incidence and Survival among Children and Adolescents: United States SEER Program 1975–1995. Bethesda: National Cancer Institute; 1999, NIH Pub. No. 99–4649 (XII). p. 149–56.
3.
Society AC: Cancer Facts & Figures 2017. Atlanta: American Cancer Society 2017, 1 (1):1–56.
4.
5.
Morgenstern DA, Baruchel S, Irwin MS. Current and future strategies for relapsed neuroblastoma: challenges on the road to precision therapy. J Pediatr Hematol Oncol. 2013;35(5):337–47.CrossRef
6.
Smith MA, Seibel NL, Altekruse SF, Ries LA, Melbert DL, O'Leary M, Smith FO, Reaman GH. Outcomes for children and adolescents with cancer: challenges for the twenty-first century. J Clin Oncol. 2010;28(15):2625–34.CrossRef
7.
Maris JM, Hogarty MD, Bagatell R, Cohn SL. Neuroblastoma. Lancet. 2007;369(9579):2106–20.CrossRef
8.
Matthay KK, Reynolds CP, Seeger RC, Shimada H, Adkins ES, Haas-Kogan D, Gerbing RB, London WB, Villablanca JG. 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(7):1007–13.CrossRef
9.
Cole KA, Maris JM. New strategies in refractory and recurrent neuroblastoma: translational opportunities to impact patient outcome. Clin Cancer Res. 2012;18(9):2423–8.CrossRef
10.
Santana VM, Furman WL, McGregor LM, Billups CA. Disease control intervals in high-risk neuroblastoma. Cancer. 2008;112(12):2796–801.CrossRef
11.
Simon T, Berthold F, Borkhardt A, Kremens B, De Carolis B, Hero B. Treatment and outcomes of patients with relapsed, high-risk neuroblastoma: results of German trials. Pediatr Blood Cancer. 2011;56(4):578–83.CrossRef
12.
Lau L, Tai D, Weitzman S, Grant R, Baruchel S, Malkin D. Factors influencing survival in children with recurrent neuroblastoma. J Pediatr Hematol Oncol. 2004;26(4):227–32.CrossRef
13.
Berthold F, Hero B, Breu H, Christiansen H, Erttmann R, Gnekow A, Herrmann F, Klingebiel T, Lampert F, Muller-Weihrich S, et al. The recurrence patterns of stages I, II and III neuroblastoma: experience with 77 relapsing patients. Ann Oncol. 1996;7(2):183–7.CrossRef
14.
Garaventa A, Parodi S, De Bernardi B, Dau D, Manzitti C, Conte M, Casale F, Viscardi E, Bianchi M, D’Angelo P, et al. Outcome of children with neuroblastoma after progression or relapse. A retrospective study of the Italian neuroblastoma registry. Eur J Cancer. 2009;45(16):2835–42.CrossRef
15.
Weiss B, Vora A, Huberty J, Hawkins RA, Matthay KK. Secondary myelodysplastic syndrome and leukemia following 131I-metaiodobenzylguanidine therapy for relapsed neuroblastoma. J Pediatr Hematol Oncol. 2003;25(7):543–7.CrossRef
16.
London WB, Castel V, Monclair T, Ambros PF, Pearson AD, Cohn SL, Berthold F, Nakagawara A, Ladenstein RL, Iehara T, et al. Clinical and biologic features predictive of survival after relapse of neuroblastoma: a report from the international neuroblastoma risk group project. J Clin Oncol. 2011;29(24):3286–92.CrossRef
17.
Matthay KK, Maris JM, Schleiermacher G, Nakagawara A, Mackall CL, Diller L, Weiss WA. Neuroblastoma. Nat Rev Dis Primers. 2016;2:16078.CrossRef
18.
Thompson D, Vo KT, London WB, Fischer M, Ambros PF, Nakagawara A, Brodeur GM, Matthay KK, DuBois SG. Identification of patient subgroups with markedly disparate rates of MYCN amplification in neuroblastoma: a report from the international neuroblastoma risk group project. Cancer. 2016;122(6):935–45.CrossRef
19.
Huang M, Weiss WA. Neuroblastoma and MYCN. Cold Spring Harbor perspectives in medicine. 2013;3(10):a014415.CrossRef
20.
21.
Cohn SL, London WB, Huang D, Katzenstein HM, Salwen HR, Reinhart T, Madafiglio J, Marshall GM, Norris MD, Haber M. MYCN expression is not prognostic of adverse outcome in advanced-stage neuroblastoma with nonamplified MYCN. J Clin Oncol. 2000;18(21):3604–13.CrossRef
22.
Brodeur GM, Fong CT. Molecular biology and genetics of human neuroblastoma. Cancer Genet Cytogenet. 1989;41(2):153–74.CrossRef
23.
Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science. 1984;224(4653):1121–4.CrossRef
24.
Seeger RC, Brodeur GM, Sather H, Dalton A, Siegel SE, Wong KY, Hammond D. Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med. 1985;313(18):1111–6.CrossRef
25.
Yue ZX, Huang C, Gao C, Xing TY, Liu SG, Li XJ, Zhao Q, Wang XS, Zhao W, Jin M, et al. MYCN amplification predicts poor prognosis based on interphase fluorescence in situ hybridization analysis of bone marrow cells in bone marrow metastases of neuroblastoma. Cancer Cell Int. 2017;17:43.CrossRef
26.
Moreau LA, McGrady P, London WB, Shimada H, Cohn SL, Maris JM, Diller L, Look AT, George RE. Does MYCN amplification manifested as homogeneously staining regions at diagnosis predict a worse outcome in children with neuroblastoma? A Children's oncology group study. Clin Cancer Res. 2006;12(19):5693–7.CrossRef
27.
Favrot MC, Ambros P, Schilling F, Frappaz D, Combaret V, Berthold F, Dominici C, Erttmann R, Esteve J, Jenkner A, et al. Comparison of the diagnostic and prognostic value of biological markers in neuroblastoma. Proposal for a common methodology of analysis. SENSE group. Ann Oncol. 1996;7(6):607–11.CrossRef
28.
Barroca H, Carvalho JL, da Costa MJ, Cirnes L, Seruca R, Schmitt FC. Detection of N-myc amplification in neuroblastomas using southern blotting on fine needle aspirates. Acta Cytol. 2001;45(2):169–72.CrossRef
29.
Crabbe DC, Peters J, Seeger RC. Rapid detection of MYCN gene amplification in neuroblastomas using the polymerase chain reaction. Diagnostic molecular pathology : the American journal of surgical pathology, part B. 1992;1(4):229–34.CrossRef
30.
Huddart SN, Mann JR, McGukin AG, Corbett R. MYCN amplification by differential PCR. Pediatr Hematol Oncol. 1993;10(1):31–4.CrossRef
31.
Anderson J, Gibson S, Williamson D, Rampling D, Austin C, Shipley J, Sebire N, Brock P. Rapid and accurate determination of MYCN copy number and 1p deletion in neuroblastoma by quantitative PCR. Pediatr Blood Cancer. 2006;46(7):820–4.CrossRef
32.
Misra DN, Dickman PS, Yunis EJ. Fluorescence in situ hybridization (FISH) detection of MYCN oncogene amplification in neuroblastoma using paraffin-embedded tissues. Diagnostic molecular pathology : the American journal of surgical pathology, part B. 1995;4(2):128–35.CrossRef
33.
Squire JA, Thorner P, Marrano P, Parkinson D, Ng YK, Gerrie B, Chilton-Macneill S, Zielenska M. Identification of MYCN copy number heterogeneity by direct FISH analysis of neuroblastoma preparations. Molecular diagnosis : a journal devoted to the understanding of human disease through the clinical application of molecular biology. 1996;1(4):281–9.CrossRef
34.
Narath R, Lorch T, Rudas M, Ambros PF. Automatic quantification of gene amplification in clinical samples by IQ-FISH. Cytometry B Clin Cytom. 2004;57(1):15–22.CrossRef
35.
Bhargava R, Oppenheimer O, Gerald W, Jhanwar SC, Chen B. Identification of MYCN gene amplification in neuroblastoma using chromogenic in situ hybridization (CISH): an alternative and practical method. Diagnostic molecular pathology : the American journal of surgical pathology, part B. 2005;14(2):72–6.CrossRef
36.
Hiyama E, Hiyama K, Yokoyama T, Ishii T. Immunohistochemical analysis of N-myc protein expression in neuroblastoma: correlation with prognosis of patients. J Pediatr Surg. 1991;26(7):838–43.CrossRef
37.
Wang LL, Teshiba R, Ikegaki N, Tang XX, Naranjo A, London WB, Hogarty MD, Gastier-Foster JM, Look AT, Park JR, et al. Augmented expression of MYC and/or MYCN protein defines highly aggressive MYC-driven neuroblastoma: a Children's oncology group study. Br J Cancer. 2015;113(1):57–63.CrossRef
38.
Wu YC, Chang IC, Wang CL, Chen TD, Chen YT, Liu HP, Chu Y, Chiu YT, Wu TH, Chou LH, et al. Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan. PLoS One. 2013;8(8):e70839.CrossRef
39.
Le Quesne J, Maurya M, Yancheva SG, O'Brien M, Popat S, Wotherspoon AC, de Castro DG, Nicholson AG. A comparison of immunohistochemical assays and FISH in detecting the ALK translocation in diagnostic histological and cytological lung tumor material. J Thorac Oncol. 2014;9(6):769–74.CrossRef
40.
Eastmond DA, Schuler M, Rupa DS. Advantages and limitations of using fluorescence in situ hybridization for the detection of aneuploidy in interphase human cells. Mutat Res. 1995;348(4):153–62.CrossRef
41.
Heredia NJ, Belgrader P, Wang S, Koehler R, Regan J, Cosman AM, Saxonov S, Hindson B, Tanner SC, Brown AS, et al. Droplet digital PCR quantitation of HER2 expression in FFPE breast cancer samples. Methods. 2013;59(1):S20–3.CrossRef
42.
Aravindan S, Ramraj SK, Somasundaram ST, Aravindan N. Novel adjuvants from seaweed impede autophagy signaling in therapy-resistant residual pancreatic cancer. J Biomed Sci. 2015;22:28.CrossRef
43.
Khan FH, Pandian V, Ramraj S, Aravindan S, Herman TS, Aravindan N. Reorganization of metastamiRs in the evolution of metastatic aggressive neuroblastoma cells. BMC Genomics. 2015;16:501.CrossRef
44.
Khan FH, Pandian V, Ramraj SK, Aravindan S, Natarajan M, Azadi S, Herman TS, Aravindan N. RD3 loss dictates high-risk aggressive neuroblastoma and poor clinical outcomes. Oncotarget. 2015;6(34):36522–34.CrossRef
45.
Khan FH, Pandian V, Ramraj S, Natarajan M, Aravindan S, Herman TS, Aravindan N. Acquired genetic alterations in tumor cells dictate the development of high-risk neuroblastoma and clinical outcomes. BMC Cancer. 2015;15:514.CrossRef
46.
Schwab M, Varmus HE, Bishop JM. Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture. Nature. 1985;316(6024):160–2.CrossRef
47.
Corvi R, Amler LC, Savelyeva L, Gehring M, Schwab M. MYCN is retained in single copy at chromosome 2 band p23-24 during amplification in human neuroblastoma cells. Proc Natl Acad Sci U S A. 1994;91(12):5523–7.CrossRef
48.
Schwab M. Human neuroblastoma: amplification of the N-myc oncogene and loss of a putative cancer-preventing gene on chromosome 1p. Recent results in cancer research Fortschritte der Krebsforschung Progres dans les recherches sur le cancer. 1994;135:7–16.PubMed
49.
Chen QR, Bilke S, Wei JS, Whiteford CC, Cenacchi N, Krasnoselsky AL, Greer BT, Son CG, Westermann F, Berthold F, et al. cDNA array-CGH profiling identifies genomic alterations specific to stage and MYCN-amplification in neuroblastoma. BMC Genomics. 2004;5:70.CrossRef
50.
Tonini GP, Boni L, Pession A, Rogers D, Iolascon A, Basso G, Cordero di Montezemolo L, Casale F, Pession A, Perri P, et al. MYCN oncogene amplification in neuroblastoma is associated with worse prognosis, except in stage 4s: the Italian experience with 295 children. J Clin Oncol. 1997;15(1):85–93.CrossRef
51.
Wang Y, Tsang JYS, Cui Y, Cui J, Lin Y, Zhao S, Law PTW, Cheung SY, Ng EKO, Tse GMK, et al. Robust and accurate digital measurement for HER2 amplification in HER2 equivocal breast cancer diagnosis. Sci Rep. 2017;7(1):6752.CrossRef
52.
Kinugasa H, Nouso K, Tanaka T, Miyahara K, Morimoto Y, Dohi C, Matsubara T, Okada H, Yamamoto K. Droplet digital PCR measurement of HER2 in patients with gastric cancer. Br J Cancer. 2015;112(10):1652–5.CrossRef
53.
Li X, Liu Y, Shi W, Xu H, Hu H, Dong Z, Zhu G, Sun Y, Liu B, Gao H, et al. Droplet digital PCR improved the EGFR mutation diagnosis with pleural fluid samples in non-small-cell lung cancer patients. Clin Chim Acta. 2017;471:177–84.CrossRef
54.
Nadauld L, Regan JF, Miotke L, Pai RK, Longacre TA, Kwok SS, Saxonov S, Ford JM, Ji HP. Quantitative and sensitive detection of Cancer genome amplifications from formalin fixed paraffin embedded tumors with droplet digital PCR. Transl Med (Sunnyvale). 2012;2(2). https://​doi.​org/​10.​4172/​2161-1025.​1000107.
55.
Zhu Y, Lu D, Lira ME, Xu Q, Du Y, Xiong J, Mao M, Chung HC, Zheng G. Droplet digital polymerase chain reaction detection of HER2 amplification in formalin fixed paraffin embedded breast and gastric carcinoma samples. Exp Mol Pathol. 2016;100(2):287–93.CrossRef
56.
Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011;83(22):8604–10.CrossRef
57.
Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. HER-2/neu protein expression in breast cancer evaluated by immunohistochemistry. A study of interlaboratory agreement. Am J Clin Pathol. 2000;113(2):251–8.CrossRef
Metadata
Title
Droplet digital PCR as an alternative to FISH for MYCN amplification detection in human neuroblastoma FFPE samples
Authors
Dinesh Babu Somasundaram
Sheeja Aravindan
Zhongxin Yu
Muralidharan Jayaraman
Ngoc T. B. Tran
Shibo Li
Terence S. Herman
Natarajan Aravindan
Publication date
01-12-2019
Publisher
BioMed Central
Published in
BMC Cancer / Issue 1/2019
Electronic ISSN: 1471-2407
DOI
https://doi.org/10.1186/s12885-019-5306-0

Other articles of this Issue 1/2019

BMC Cancer 1/2019 Go to the issue

Research article

Determination of the androgen receptor status of circulating tumour cells in metastatic breast cancer patients

Research article

Distinct signatures of lung cancer types: aberrant mucin O-glycosylation and compromised immune response

Research article

Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas

Research article

miR-140-5p could suppress tumor proliferation and progression by targeting TGFBRI/SMAD2/3 and IGF-1R/AKT signaling pathways in Wilms’ tumor

Research article

Red blood cell distribution width and platelet counts are independent prognostic factors and improve the predictive ability of IPI score in diffuse large B-cell lymphoma patients

Research article

MUC16 impacts tumor proliferation and migration through cytoplasmic translocation of P120-catenin in epithelial ovarian cancer cells: an original research
Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras
Prof. Fabrice Barlesi
Developed by: Springer Medicine
Image Credits