Discovery of MYCN-amplified retinoblastoma with functional retinoblastoma protein in very young children Diane E Rushlow, Berber M Mol,* Jennifer Y Kennett,* Stephanie Yee,* Sanja Pajovic, Brigitte L Thériault, Nadia L Prigoda-Lee, Clarellen Spencer, Helen Dimaras, Timothy W Corson, Renee Pang, Christine Massey, Roseline Godbout, Zhe Jiang, Eldad Zacksenhaus, Katherine Paton, Annette C Moll, Claude Houdayer, Anthony Raizis, William Halliday, Wan L Lam, Paul C Boutros, Dietmar Lohmann, Josephine C Dorsman, Brenda L Gallie *These authors share second authorship Retinoblastoma Solutions and the Toronto Western Hospital Research Institute, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network; Informatics and Biocomputing Platform, Ontario Institute for Cancer Research; Departments of Hematology/Oncology, Ophthalmology and Visual Science and of Pathology, Hospital for Sick Children; and Departments of Molecular Genetics, Ophthalmology, Medical Biophysics, Pathobiology and Lab Medicine, University of Toronto, Toronto, ON, Canada (D E Rushlow, BSc, S Yee, MSc, 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
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Discovery of MYCN-amplified retinoblastoma with functional
retinoblastoma protein in very young children
Diane E Rushlow, Berber M Mol,* Jennifer Y Kennett,* Stephanie Yee,* Sanja Pajovic,
Brigitte L Thériault, Nadia L Prigoda-Lee, Clarellen Spencer, Helen Dimaras, Timothy W
(brown), and 11 RB1-/- (green) tumours; gains, right, and losses, left of
chromosome; minimal commonly gained/lost regions in RB1-/- tumours boxed;
*normally occurring copy number variations. Tumour T33 shows loss of most of
13q. (B) The minimal amplicon of 513 kbp is defined by two MYCNA tumours
(pink band); MYCN copy number by QM-PCR, red italics; aCGH individual
probes, green bars.
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MYCN amplified retinoblastoma with normal pRb
Figure 3: Expression of RB1 and MYCN
(A) Staining of adjacent retina and RB1+/+MYCNA or RB1 mutant retinoblastoma
for N-Myc protein and pRB (C-terminus antibody); T, tumour; INL, inner nuclear
layer retina. (B) Western blots with pRb antibody that recognizes both hypo-
and hyperphosphorylated pRb, phospho-Rb (Ser795) antibody, and E2F1
antibody. (C) Cell lysates were immunoprecipitated with antibodies to mouse
IgG (negative control), pRb or E2F1, and western blots performed with
antibodies to pRb and E2F. (D) Real-time RT-PCR for RB1, MYCN and KIF14 in
human fetal (FR) and adult (HR) retina, RB1+/+MYCNA, and RB1-/- primary
tumours and cell lines; triplicate measurements normalized against GAPDH,
relative to FR; MYCN DNA copy-numbers in italics; #, KIF14 not done.
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MYCN amplified retinoblastoma with normal pRb
Figure 4: Clinical features of children with RB1+/+MYCNA tumours
(A) Children with RB1+/+MYCNA retinoblastoma are diagnosed significantly
younger than children with RB1-/- tumours (p<0·0001, Wilcoxon rank sum test).
(B) The Knudson plot of proportion not yet diagnosed vs age at diagnosis, using
birth as a surrogate for initiation, showing a two-hit curve (blue) and a one-hit
curve (red) for children with RB1-/- tumours or RB1+/+MYCNA tumours;
scatterplot does not distinguish identically aged children. (C) Fundus image of
a large RB1+/+MYCNA unilateral tumour, extending from optic nerve (white
arrow) to anterior border of retina (double arrows) in a 4 month-old child with
characteristic calcification on ultrasound, and round nuclei with prominent
large multiple nucleoli on pathology, in comparison to (D) RB1-/- tumour
showing classic Flexner-Wintersteiner rosettes and nuclear molding;
hematoxylin-eosin staining. (E) RB1+/+MYCNA retinoblastoma in an 11 month-old
child (A2) with extra-ocular extension into the optic nerve (arrows) (2·5x,
hematoxylin-eosin staining). (F) In comparison, in 3·5 month-old child with
heritable RB1-/- retinoblastoma, a small tumour is present in the inner nuclear
layer of the retina on optical coherent tomography (OCT). (G) Schema of data
establishing RB1+/+MYCNA retinoblastoma as a novel disease; months (m), data
figures (f) and tables (t) indicated in grey on left.
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MYCN amplified retinoblastoma with normal pRb
Contributors
Diane E Rushlow recognized the initial connection between MYCNA amplification and RB1
mutation status, performed literature search and QM-PCR analysis and supervised RB1 mutation
analysis, coordinated collaborations with the other sites and was the major contributor to manuscript
preparation. Berber M Mol recognized the RB1 and MYCN mutation status of the Amsterdam
samples by MLPA and SNP array analysis, performed immunohistochemistry, Western blots and
co-immunoprecipitations. Jennifer Y Kennett performed aCGH and analysed aCGH data. Stephanie
Yee performed analysis of aCGH data, the MYCNA alignment, immunohistochemistry and reverse
transcriptase PCR. Sanja Pajovic performed literature search, reverse transcriptase PCR and
immunohistochemistry. Brigitte L Thériault performed literature search and RNA expression
studies. Nadia L Prigoda-Lee performed literature search, statistical analysis and contributed to
figure and manuscript preparation. Clarellen Spencer performed immunohistochemistry. Helen
Dimaras and Timothy W Corson performed literature searches, assisted in data analysis and
conceptualization of discussion, and contributed to figure and manuscript preparation. Renee Pang
performed statistical and bioinformatic analyses on the aCGH data. Christine Massey performed
statistical analyses. Roseline Godbout discovered and characterised the first tumour with MYCN
amplification and normal pRb (RB522) (now RB1+/+MYCNA) long before anyone believed her; she
provided the cell line and data and Western blot showing functional pRb. Zhe Jiang and Eldad
Zacksenhaus performed Western blots to demonstrate functional phosphorylated pRb. Katherine
Paton and Annette C Moll provided clinical images and material, and conceptual discussion. Claude
Houdayer and Anthony Raizis provided RB1 mutation analysis, and clinical features. William
Halliday recognized and characterized the unique histological features of the RB1+/+MYCNA
retinoblastomas and prepared digital images for publication. Wan L Lam supervised Jennifer
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MYCN amplified retinoblastoma with normal pRb
Kennet and the aCGH experiments. Paul C Boutros performed detailed and novel analysis of the
aCGH data, and statistical analyses throughout the project, and supervised Renee Pang. Dietmar
Lohmann performed literature search, provided RB1 mutation analysis, and contributed to figure
construction and development of concepts. Josephine C Dorsman coordinated the Amsterdam
study, and, with Berber Mol who she supervised, recognized the RB1 and MYCN mutation status of
the Amsterdam samples. Brenda L Gallie supervised overall, performed literature search, provided
critical guidance on all components of the project, and contributed extensively to figure and
manuscript preparation. All authors contributed to manuscript preparation.
Conflicts of interest
BLG was part-owner of Solutions by Sequence. All other authors declare that they have no conflicts
of interest.
Acknowledgments
This study was conducted with the support of the Ontario Institute for Cancer Research to PCB
through funding provided by the Government of Ontario. BMM was funded by a grant from
CCA/V-ICI/ Avanti-STR (to JCD, J. Cloos and ACM), the Dutch research was also funded in part
by KIKA (JCD, H. te Riele, J. Cloos, ACM). SY was funded by the Vision Science Research
Program of the University Health Network and the University of Toronto. RP was funded in part by
a Great West Life Studentship from Queen’s University School of Medicine. We thank Leslie
MacKeen for the montage of RetCam images in figure 3B. We thank Dr. Valerie White of U.
British Columbia for providing clinical and pathological details and images. We thank Cynthia
Vandenhoven for the clinical images in figure 4F. We thank members of the VU University
Medical Center/The Netherlands Cancer Institute, Institut Curie, Institut für Humangenetik, Toronto
retinoblastoma teams and other, wise colleagues for useful discussions. We thank the children and
families who donated tissues for these studies for the benefit of future families.
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MYCN amplified retinoblastoma with normal pRb
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