Discovery of MYCN -amplified retinoblastoma with functional retinoblastoma protein in very young children Not Knudson’s Retinoblastoma: One-Hit Cancer Initiated by the MYCN Oncogene ? Diane E Rushlow, Berber M Mol,* Jennifer Y Kennett,* Berber M Mol,* 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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Discovery of MYCN-amplified retinoblastoma with functional
retinoblastoma protein in very young childrenNot Knudson’s
Retinoblastoma:
One-Hit Cancer Initiated by the MYCN Oncogene?
Diane E Rushlow, Berber M Mol,* Jennifer Y Kennett,* Berber M Mol,* Stephanie Yee,*
Sanja Pajovic, Brigitte L Thériault, Nadia L Prigoda-Lee, Clarellen Spencer, Helen
(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.
Figure 2: Fewer genomic copy-number alterations in RB1+/+MYCNA than
RB1-/-tumours
(A) aCGH on 12 RB1+/+MYCNA (including T33), 12 RB1+/+, 13 RB1+/-, and 11 RB1-/-tumours; gains,
right; losses, left; minimal commonly gained/lost regions in RB1-/- tumours boxed; *normally
occurring copy-number variations. The RB1+/- MYCNA tumour T33 shows loss of most of 13q; this
may not be an initiating event. (B) The minimal amplicon of 513 kbp is defined by two MYCNA
tumours (pink band); MYCNcopy-number by QM-PCR, red italics; aCGH individual probes, green
bars. (C) Boxplot of bp altered shows fewer changes in RB1+/+MYCNA than RB1-/-tumours (p =
0·033; t-test with Welch’s adjustment); vertical line marks the maximum and minimum copy-
numbers observed, box bounds first and third quartiles, and horizontal line within the box represents
the median. (D) Fewer aCGH clones are altered in RB1+/+ and RB1+/+MYCNA, than RB1-/-tumours;
each class has more unique clones altered than in common.
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MYCN amplified retinoblastoma with normal pRb RETINOBLASTOMA
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MYCN amplified retinoblastoma with normal pRb RETINOBLASTOMA
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.
Figure 3: RB1+/+MYCNA tumours express pRb and MYCN
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MYCN amplified retinoblastoma with normal pRb RETINOBLASTOMA
(A) h
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MYCN amplified retinoblastoma with normal pRb RETINOBLASTOMA
Figure 4: Clinical features of children with RB1+/+MYCNA tumours in very
young children are clinically distinct
(A) Children with RB1+/+MYCNARB1+/+ MYCNA retinoblastoma are
diagnosed significantly younger than children with RB1-/-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, fits showing a two-hit curve (blue) but notand a
one-hit curve (red) for children with unilateral non-familial RB1-/-RB1-/-
diseasetumours, as expected; or for RB1+/+MYCNA RB1+/+ MYCNA
retinoblastomatumours, the data points for the 12 children younger than 10
months most closely approximate the one-hit curve (red), but those diagnosed
at older ages deviate toward the two-hit curve; scatterplot does not
distinguish identically aged children. (C) Fundus image of an a large
RB1+/+ MYCNA 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-/-RB1-/- tumour showing classic Flexner-
Wintersteiner rosettes and nuclear molding; hematoxylin-eosin
staining. (E) RB1+/+ MYCNA 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-/-RB1-/- retinoblastoma, a small tumour a small
tumour is present in the inner nuclear layer of the retinashown to be in the
inner nuclear layer of the retina on optical coherent tomography (OCT).
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(G) Schema of data establishing RB1+/+MYCNA 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 RETINOBLASTOMA
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, experiments and performed SNP array data
analysis,performed immunohistochemistry, imaging and Western blots and co-
immunoprecipitations. Jennifer Y Kennett performed aCGH and analysed aCGH data. Berber M
Mol determined MYCN status by MLPA experiments and performed SNP array data analysis,
immunohistochemistry imaging and Western blots. 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 analyseis on age of diagnosis data. Roseline
Godbout discovered and characterised the first RB1+/+MYCNA tumortumour with MYCN
amplification and normal pRb (RB522) (now RB1+/+ MYCNA ) long before anyone believed her; she
provided the cell line and data and the Western blot showing functional proteinpRb. 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
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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 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, and supervised Berber Mol. 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 is 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. 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. 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). 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, Toronto retinoblastoma teams and other, wise
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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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