Characterization of retinoblastoma tumours without RB1 mutations: genomic, gene expression and clinical studies 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, René e e 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 (now Impact Genetics) and the Toronto Western Hospital Research Institute, Campbell Family Cancer Research Institute, Princess Margaret Cancer Centre, University Health Network; Informatics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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Characterization of retinoblastoma tumours without RB1
mutations: genomic, gene expression and clinical studies
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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Tim Corson, 01/23/13,
Can you include n for Fig 1A, too?
Tim Corson, 23/01/13,
Typo in Fig 1A axis label
MYCN amplified retinoblastoma with functional pRbRetinoblastoma without RB1 mutations
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 of retina. (B) Western blots with pRb antibody that
recognizes both hypo- and hyperphosphorylated pRb, phospho-Rb (Ser795)
antibody, and E2F1 N-MycE2F1 antibody. Fetal retina (FR) and neuroblastoma
(NB) lysates included for comparison. (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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Berber, 01/23/13,
Figure 3 B shows E2F1 and murine thymus as controls (S7C shows MYCN and FR and NB) or will the figures be swopped?
Tim Corson, 01/23/13,
Needs to be updated to match the figure; are 3B and 3C going to webappendix?
Tim Corson, 01/23/13,
In Fig parts A & B, protein should be named N-Myc
MYCN amplified retinoblastoma with functional pRbRetinoblastoma without RB1 mutations
Figure 4: Clinical features of children with RB1+/+MYCNA tumours
(A) (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.
Figure 5: Clinical features of RB1+/+ MYCNA tumours.
(A) Fundus image of an RB1+/+ MYCNA unilateral tumor, 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
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Tim Corson, 23/01/13,
I find these arrows very similar
MYCN amplified retinoblastoma with functional pRbRetinoblastoma without RB1 mutations
nuclei with prominent large multiple nucleoli on histopathology, in
comparison to (B) RB1-/- tumour showing classic Flexner-Wintersteiner
rosettes and nuclear moulding; hematoxylin-eosin staining. (C) RB1+/+ MYCNA
retinoblastoma in an 11 month-old child (A2) with extra-ocular extension
into the optic nerve (whiteblack arrows) (2.5x, hematoxylin-eosin staining).
(D) In comparison, in a 3.5 month-old child with heritable RB1-/-
retinoblastoma, a small tumor is present in the inner nuclear layer of the
retina on optical coherent tomography (OCT).
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MYCN amplified retinoblastoma with functional pRbRetinoblastoma without RB1 mutations
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 and performed 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, grew cell lines, 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. Renée 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 Western blot
showing multiple bands of pRb. Zhe Jiang and Eldad Zacksenhaus performed Western blots
specific to phosphorylated pRb. Katherine Paton and Annette C Moll provided and interpreted
clinical data and images. Claude Houdayer and Anthony Raizis provided RB1 mutation analysis,
and clinical data. 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 Kennett and the aCGH experiments. Paul C Boutros performed detailed and
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MYCN amplified retinoblastoma with functional pRbRetinoblastoma without RB1 mutations
novel analysis of the aCGH data, and statistical analyses throughout the project, and supervised
Renéee 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
searches, provided critical guidance on all components of the project, and contributed extensively to
figure and manuscript preparation. All authors contributed to concept development and manuscript
preparation.
Conflicts of interest
BLG was part-owner of Solutions by Sequence and Member of the Board of Retinoblastoma
Solutions, which are now merged into Impact Genetics with BLG as Medical Director. All other
authors declare that they have no conflicts of interest.
Acknowledgments
NCI-NIH grant 5R01CA118830-05 supported the early discovery at the Canadian site (BLG).
Canadian Institutes for Health Research grants (WLL) (MOP-86731, MOP-77903, MOP-110949)
supported the aCGH studies, and MOP-77710 (EZ) supported the pRb phosphorylation studies. The
Canadian Retinoblastoma Society, Hyland Foundation and Toronto Netralya and Doctors Lions
Clubs provided critical funding for additional experiments. The Ontario Ministry of Health and
Long Term Care provided infrastructure. Retinoblastoma Solutions and Solutions by Sequence
(Now merged into Impact Genetics) supported the overall project, data analysis and manuscript
preparation. The German study was made possible by grant DFG (Lo 530/6-2), while UK-Essen
provided infrastructure. The Dutch study was made possible by grants from Avanti-STR (JCD, J.
Cloos and ACM) and KiKa (JCD, H. te Riele, J. Cloos, ACM), while VUmc provided
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MYCN amplified retinoblastoma with functional pRbRetinoblastoma without RB1 mutations
infrastructure. 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 54F. 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 functional pRbRetinoblastoma without RB1 mutations
REFERENCES
1. Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Science, USA 1971; 68(4): 820-3.
2. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 1986; 323(6089): 643-6.
3. Cavenee WK, Hansen MF, Nordenskjold M, Kock E, Maumenee I, Squire JA, et al. Genetic origin of mutations predisposing to retinoblastoma. Science 1985; 228(4698): 501-3.
4. Lohmann DR, Gallie BL. Retinoblastoma: Revisiting the model prototype of inherited cancer. Am J Med Genet 2004; 129C(1): 23-8.
5. Rushlow D, Piovesan B, Zhang K, Prigoda-Lee NL, Marchong MN, Clark RD, et al. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat 2009; 30(5): 842-51.
7. Houdayer C, Gauthier-Villars M, Lauge A, Pages-Berhouet S, Dehainault C, Caux-Moncoutier V, et al. Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF. Hum Mutat 2004; 23(2): 193-202.
8. Dimaras H, Khetan V, Halliday W, Orlic M, Prigoda NL, Piovesan B, et al. Loss of RB1 induces non-proliferative retinoma: increasing genomic instability correlates with progression to retinoblastoma. Human molecular genetics 2008; 17(10): 1363-72.
9. Corson TW, Gallie BL. One hit, two hits, three hits, more? Genomic changes in the development of retinoblastoma. Genes Chromosomes Cancer 2007; 46(7): 617-34.
10. Ishkanian AS, Malloff CA, Watson SK, DeLeeuw RJ, Chi B, Coe BP, et al. A tiling resolution DNA microarray with complete coverage of the human genome. Nat Genet 2004; 36(3): 299-303.
11. Watson SK, deLeeuw RJ, Horsman DE, Squire JA, Lam WL. Cytogenetically balanced translocations are associated with focal copy number alterations. Hum Genet 2007; 120(6): 795-805.
12. Myllykangas S, Bohling T, Knuutila S. Specificity, selection and significance of gene amplifications in cancer. Semin Cancer Biol 2007; 17(1): 42-55.
13. O'Neill S, Ekstrom L, Lastowska M, Roberts P, Brodeur GM, Kees UR, et al. MYCN amplification and 17q in neuroblastoma: evidence for structural association. Genes Chromosomes Cancer 2001; 30(1): 87-90.
14. Mosse YP, Diskin SJ, Wasserman N, Rinaldi K, Attiyeh EF, Cole K, et al. Neuroblastomas have distinct genomic DNA profiles that predict clinical phenotype and regional gene expression. Genes Chromosomes Cancer 2007; 46(10): 936-49.
15. Chen D, Gallie BL, Squire JA. Minimal regions of chromosomal imbalance in retinoblastoma detected by comparative genomic hybridization. Cancer Genet Cytogenet 2001; 129(1): 57-63.
16. Sampieri K, Amenduni M, Papa FT, Katzaki E, Mencarelli MA, Marozza A, et al. Array comparative genomic hybridization in retinoma and retinoblastoma tissues. Cancer Sci 2009; 100(3): 465-71.
17. Spencer C, Pajovic S, Devlin H, Dinh QD, Corson TW, Gallie BL. Distinct patterns of expression of the RB gene family in mouse and human retina. Gene Expr Patterns 2005; 5(5): 687-94.
18. Templeton DJ, Park SH, Lanier L, Weinberg RA. Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and
MYCN amplified retinoblastoma with functional pRbRetinoblastoma without RB1 mutations
nuclear tethering. Proceedings of the National Academy of Sciences of the United States of America 1991; 88(8): 3033-7.
19. Murakami A, Yajima T, Sakuma H, McLaren MJ, Inana G. X-arrestin: a new retinal arrestin mapping to the X chromosome. FEBS Lett 1993; 334(2): 203-9.
20. Terry J, Calicchio ML, Rodriguez-Galindo C, Perez-Atayde AR. Immunohistochemical Expression of CRX in Extracranial Malignant Small Round Cell Tumours. The American journal of surgical pathology 2012; 36(8): 1165-9.
21. Tornoczky T, Semjen D, Shimada H, Ambros IM. Pathology of peripheral neuroblastic tumours: significance of prominent nucleoli in undifferentiated/poorly differentiated neuroblastoma. Pathol Oncol Res 2007; 13(4): 269-75.
22. Flexner S. A peculiar glioma (neuroepithelioma?) of the retina. Johns Hopkins Hosp Bull 1891; 2: 115.
23. Dimaras H, Kimani K, Dimba EA, Gronsdahl P, White A, Chan HS, et al. Retinoblastoma. Lancet 2012; 379(9824): 1436-46.
24. Chantada GL, Casco F, Fandino AC, Galli S, Manzitti J, Scopinaro M, et al. Outcome of patients with retinoblastoma and postlaminar optic nerve invasion. Ophthalmology 2007; 114(11): 2083-9.
25. Lillington DM, Goff LK, Kingston JE, Onadim Z, Price E, Domizio P, et al. High level amplification of N-MYC is not associated with adverse histology or outcome in primary retinoblastoma tumours. Br J Cancer 2002; 87(7): 779-82.
26. Zhang J, Benavente CA, McEvoy J, Flores-Otero J, Ding L, Chen X, et al. A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature 2012; 481(7381): 329-34.
27. Manning AL, Longworth MS, Dyson NJ. Loss of pRB causes centromere dysfunction and chromosomal instability. Genes Dev 2010; 24(13): 1364-76.
28. Kobayashi M, Takezawa S, Hara K, Yu RT, Umesono Y, Agata K, et al. Identification of a photoreceptor cell-specific nuclear receptor. Proceedings of the National Academy of Sciences of the United States of America 1999; 96(9): 4814-9.
29. Chen D, Pacal M, Wenzel P, Knoepfler PS, Leone G, Bremner R. Division and apoptosis of E2f-deficient retinal progenitors. Nature 2009; 462(7275): 925-9.
30. Hook KE, Garza SJ, Lira ME, Ching KA, Lee NV, Cao J, et al. An integrated genomic approach to identify predictive biomarkers of response to the aurora kinase inhibitor PF-03814735. Molecular cancer therapeutics 2012; 11(3): 710-9.