nature template - pc word 97 · web viewinterpretation amplification of the mycn oncogene may...

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

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, S Pajovic, PhD, B L Thériault, PhD, N L

Prigoda-Lee, MSc, C Spencer, BSc, Z Jiang, BSc, E Zacksenhaus, PhD, R Pang, MA, C Massey,

MSc, H Dimaras, PhD, P C Boutros, PhD, W Halliday, MD, Prof B L Gallie, MD); Departments of

Clinical Genetics, Ophthalmology and Pediatric Oncology/Hematology, VU University

Medical Center Amsterdam, Amsterdam, The Netherlands (B M Mol, MSc, A C Moll, MD, Prof J

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Gallie Brenda, 16/01/13,

TITLE CHANGE –

MYCN amplified retinoblastoma with normal pRb RETINOBLASTOMA

C Dorsman, PhD); British Columbia Cancer Research Centre and Departments of

Ophthalmology and Pathology & Laboratory Medicine, University of British Columbia,

Vancouver, BC, Canada (J Y Kennett, MSc, K Paton, MD, Prof W L Lam, PhD); Departments of

Clinical Genetics, Ophthalmology and Pediatric Oncology/Hematology, VU University

Medical Center Amsterdam, Amsterdam, The Netherlands (B M Mol, MSc, A C Moll, MD, Prof J

C Dorsman, PhD); Eugene and Marilyn Glick Eye Institute, Departments of Ophthalmology,

Biochemistry and Molecular Biology, Indiana University School of Medicine Indianapolis,

Indiana, USA (Timothy W Corson, PhD); University of Alberta, Cross Cancer Institute,

Edmonton, Alberta (Prof Roseline Godbout, PhD); Service de Génétique Oncologique, Institut

Curie and Université Paris Descartes Paris, France (C Houdayer, PhD); Department of

Molecular Pathology, Canterbury Health Laboratories Christchurch, New Zealand (A Raizis,

PhD); and Institut für Humangenetik, Universitätsklinikum, Essen, Germany (Prof D Lohmann,

MD)

Correspondence to Dr Brenda L. Gallie, Campbell Family Cancer Research Institute, Princess Margaret

Cancer Centre, University Health Network, Rm 8-415, 610 University Ave, Toronto, ON, M5G 1M9, Canada,

[email protected]

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mailto:[email protected]


Word count 297/300

Summary

Background Retinoblastoma is the childhood retinal cancer that defined tumour suppressor genes.

By analysing age of diagnosis, Knudson proposed that two “hits” initiate retinoblastoma, later

attributed to mutation of both alleles of the retinoblastoma suppressor gene, RB1, in tumours.

Persons with hereditary retinoblastoma carry a heterozygous constitutional RB1 mutation; one

additional hit initiates retinoblastoma and/or other cancers. Non-hereditary retinoblastoma arises

when both RB1 alleles are damaged in developing retina (RB1-/- ).

Methods We analysed clinical data, genomic copy-number, histology, RB1 gene expression and

protein function, and retinal gene expression, in unilateral non-familial retinoblastomas with no

evidence of RB1 mutations (RB1+/+ ), compared to RB1-/- tumours.

Findings We found that 2·7% (29/1068) of unilateral non-familial retinoblastomas had no evidence

of RB1 mutations (RB1+/+ ). Surprisingly, half of the RB1+/+ tumours had high-level MYCN oncogene

amplification (28 to 121 copies), while no RB1-/- primary tumours showed MYCN amplification

(p<0·0001). RB1+/+ MYCNA cell lines expressed functional RB1 protein. RB1+/+ MYCNA tumours had

fewer overall genomic copy-number changes and distinct, aggressive histology. MYCN

amplification was the sole copy-number change detected in one RB1+/+ MYCNA retinoblastoma.

Median age at diagnosis of RB1+/+ MYCNA tumours was 4·5 months, compared to 24 months for

non-familial unilateral RB1-/- retinoblastoma. Children diagnosed with unilateral non-familial large

retinoblastoma at six months of age or less have >18% chance of RB1+/+ MYCNA tumour.

Interpretation Amplification of the MYCN oncogene may initiate RB1+/+ MYCNA retinoblastoma

despite normal RB1 genes. Although they are young, it is unlikely that these children carry a

hereditary risk for other retinoblastoma or cancers. Since these tumours may rapidly become

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extraocular, removal of the eye of young children with potential large RB1+/+ MYCNA retinoblastoma

is recommended.

Funding NCI-NIH; CIHR; Canadian Retinoblastoma Society; Hyland Foundation; Ontario

Ministry of Health and Long Term Care; Toronto Netralya and Doctors Lions Clubs; and

Foundations Avanti-STR and KiKa.

Word count 298/300

Summary

Background Retinoblastoma is the childhood retinal cancer that defined tumour suppressor genes.

By analysing age of diagnosis, Knudson proposed that two “hits” initiate retinoblastoma, later

attributed to mutation of both alleles of the retinoblastoma suppressor gene, RB1, in tumours.

Persons with hereditary retinoblastoma carry a heterozygous constitutional RB1 mutation; one

additional hit initiates retinoblastoma or other cancers. Non-hereditary retinoblastoma arises when

both RB1 alleles are damaged in developing retina (RB1-/-).

Methods Our international collaboration determined the proportion of 1068 unilateral non-familial

retinoblastomas with no evidence of RB1 mutations despite high-sensitivity assays. We analysed

clinical data, genomic copy-number changes, histology, RB1 gene expression and protein function,

comparing RB1+/+and RB1-/- tumours.

Findings No evidence of RB1 mutation (RB1+/+) was found In 2·7% (29/1068) of unilateral non-

familial retinoblastomas. Surprisingly, half of these had high-level MYCN oncogene amplification

(28 to 121 copies), while no RB1-/- primary tumours showed MYCN amplification. . Analysis of three

revealed (EZ – otherwise sounds like only three RB+/+ tumor show functional RB)RB1+/+MYCNA

tumours had fewer overall genomic copy-number changes and distinct, aggressive histology. MYCN

amplification was the sole copy-number change detected in one RB1+/+MYCNA retinoblastoma.

Median age at diagnosis of RB1+/+MYCNA tumours was 4·5 months, compared to 24 months for

non-familial unilateral RB1-/- retinoblastoma. We calculate an 18·4% chance that A child diagnosed

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with unilateral non-familial retinoblastoma at six months of age or less will have an RB1+/+MYCNA

tumour.

Interpretation Amplification of the MYCN oncogene may initiate RB1+/+MYCNA retinoblastoma

despite normal RB1 genes. Despite their young age at diagnosis, it is very unlikely that these

children carry a hereditary risk for more other cancers. But since these aggressive tumours may

rapidly disseminate become extra-ocular, removal of the eye of these young children with unilateral

non-familial RB1+/+ MYCNA retinoblastoma is important/ - or recommended.

Funding NCI-NIH; CIHR; Canadian Retinoblastoma Society; Hyland Foundation; Ontario

Ministry of Health and Long Term Care; Toronto Netralya and Doctors Lions Clubs; and

Foundations Avanti-STR and KiKa.

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Word count 2998/3000

Background

Retinoblastoma set the paradigm for tumour suppressor genes, with Knudson’s classic hypothesis

predicting that two rate-limiting hits initiate this childhood eye cancer.1 The two hits were later

attributed to the retinoblastoma gene (RB1).2 Approximately 40% of retinoblastoma is bilateral.

AThe 40% of children who have with bilateral retinoblastoma carries carry a heterozygous,

constitutional RB1 mutation and is are predisposed to retinoblastoma; one additional hit, in

whichdamages the second RB1 allele is damaged and initiates retinoblastoma, and/or other cancers

later in life. In 70% of tumours, the second hit involves loss of the normal allele with duplication of

the mutant allele (loss of heterozygosity, LOH). Approximately The 60% of children have with

unilateral non-familial retinoblastoma usually have. Most non-familial unilateral retinoblastomas

arise when both RB1 alleles are damaged only in developing retina, but 15% of them carry a

heritable, constitutional RB1 mutation. Accepted dogma is that damage todisruption of both RB1

alleles (RB1-/-) is required forinitiates all retinoblastoma development.2-4

The heterozygous mutant RB1 allele is identifiable in blood of 95% of bilaterally affected

persons. RB1 mutations or promoter methylation, are detected on both alleles (RB1-/-) in 95% of

unilateral retinoblastomas.5, 6 The possibility that some unilateral retinoblastomas with no detectable

RB1 mutations occur by an independent mechanism has not been previously explored.

We report the first identification of unilateral retinoblastomas with normal RB1 alleles and high-

level MYCN gene amplification (MYCNA). These unilateral RB1+/+MYCNA retinoblastomas are

characterized by distinct histology, few of the genomic copy-number changes characteristic of

retinoblastoma, and very early age of diagnosis. This newly recognized sub-type of retinoblastoma

has immediate diagnostic, genetic counselling, and therapeutic implications.

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Methods (details in webappendix)

Clinical samples

Tissues and clinical data were provided for identification of RB1 mutations for in clinical care of

children and their families. Research Ethics Board approvals for theto use of de-identified data and

tissues after clinical analyses, are on file at each participating site. Although not required, the

Toronto and Essen patients provided additional informed consent.

Mutation analyses

Standard of care analyses that identify 95% of RB1 (Gen bank accession #L11910) mutant alleles5-7

were applied at each site, including DNA sequencing, quantitative multiplex PCR (QM-PCR) or

Multiplex Ligation-Dependent Probe Amplification (MLPA), and RB1 promoter methylation

testing. Intragenic and closely-linked RB1 microsatellite markers were used to determine zygosity

of the RB1+/+ tumours.

Genome copy-number analyses

Genomic copy-numbers of five genes were determined by QM-PCR (Toronto) or MLPA and single

nucleotide polymorphism (SNP) analyses (Amsterdam). Sub-megabase resolution array

comparative genomic hybridization (aCGH) or SNP array were used to assess overall genomic

copy-numbers and normal cell contamination.

Protein expression studies

Paraffin-embedded sections of retinoblastomas and adjacent normal retinas were stained for full-

length pRb protein (antibodies targeting N- and C-terminal pRb) and N-Myc.8 Western blots were

performed on RB1+/+MYCNA, RB1-/- and control cell lines. Function of pRB was assessed by

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METHODOLOGY FOR DETECTION OF NCC: SEE RESPONSE TO REVIEWERS


immunostainingblotting with phosphor- specific for phosphorylated pRb antibody and co-

immunoprecipitation of pRb and its major effector molecule, E2F1.

RNA gene expression studies

Expression of RB1, MYCN, and genes reflecting the proliferative status and retinal derivation of

tumours were assessed using End-Point Reverse Transcriptase PCR (RT-PCR) and/or Quantitative

Real-Time PCR.

Age of Diagnosis analysis

Ages at diagnosis vs. proportion not yet diagnosed were analysed to assess minimal number of

events for tumour initiation. Likelihood of children having RB1+/+MYCNA tumours at different ages

of diagnosis was estimated.

Role of the funding sources

The sponsors of the study had no roles in study design, data collection, data analysis, data or

interpretation, or writing of the manuscript. No author was paid to write this article. All authors had

full access to all data in the study; the corresponding author (BLG) had final responsibility for the

decision to submit for publication. NCI-NIH grant 5R01CA118830-05 supported the early

discovery at the Canadian site. Canadian Institutes for Health Research grants (MOP-86731, MOP-

77903, MOP-110949) supported the aCGH studies, and MOP-77710 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. Solutions by Sequence supported

the overall project, data analysis and manuscript preparation. The Dutch study was made possible

by Avanti-STR and KiKa, while VUmc provided infrastructure.

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NEW EXPERIMENT TO ASSESS FUNCTION.


Results

The Toronto lab identifies both tumour mutations (or promoter methylation) in >95% of tumours

(as of October 10, 2012, 616/642, 96.0%) from unilateral probands with no known family history.

In 3-4% of tumours an only one RB1 mutation is identified on only one allele, and in 1·6% of

tumours, no RB1 mutation is found. Low-level mosaicism may account for the 5% "no mutation

found" blood samples from bilaterally affected patients.5 However, undetected RB1 mutations in

fully tested tumours (RB1+/+) were are believed to include translocations, deep intronic mutations,

or mutations in unknown RB1 regulatory regions. Unlike most cancers, normal cell contamination

of retinoblastoma tumours is rare, and detectable by RB1 sequence, sensitive allele-specific PCR,

and microsatellite analysis (EZ – polish sentence – undetectable/detactable…). Rarely, significant

normal cell contamination is associated with very small tumours.

In our clinical work in Toronto, we (DER, BLG) found seven unilateral retinoblastoma tumours

with no RB1 mutations, no promoter methylation, and no LOH at RB1 LOH (RB1+/+). We used QM-

PCR to measure in RB1+/+ tumours, copy-numbers of genes at 6p, 1q, 16q and 2p that are

characteristically gained or lost in RB1-/- retinoblastoma tumours.9 To our surprise, 5/7 RB1+/+

tumours showed dramatic MYCN oncogene amplification (MYCNA). To validate this observation,

we collaborated with RB1 clinical laboratories in Germany, France, and New Zealand, to study

similar RB1+/+ tumours in which they had found no detectable RB1 mutations or promoter

methylation, no RB1 LOH, and low normal cell contamination (tTable 1). After analysis was

completeinitial studies, we learned that the Amsterdam lab group had independently discovered

three RB1+/+MYCNA tumours. We report our combined data after statistical analysis showed that

frequencies were not different (p = 0·.08) among the five clinical labs. Retinoblastoma T101,

predicted clinically and pathologically to be an RB1+/+MYCNA tumour (figure 4C), was later shown

to havehad 40 copies of MYCN, and is included in some analyses.

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Since our standard detection sensitivity >95% to find an the RB1 mutation in blood of bilaterally

affected persons is >95%,5-7 the probability of finding no RB1 mutations in a tumour with no RB1

LOH is equivalent to the probability of missing two independent RB1 mutations in one sample

(0·05 x 0·05) or 0·25%. By combining our data on 1068 unilateral non-familial tumours, we

identified 29 RB1+/+tumours (2·7%), 10-fold more than expected (p = 6 x 10 -45) (ttable 1). This

suggested that some RB1+/+ tumours might originate by a mechanism other than Knudson’s two RB1

mutations.

To characterize copy numbers of genes commonly gained or lost in retinoblastomas,9 we used

QM-PCR (Toronto) or MLPA/SNP (Amsterdam) analyses. MYCN copy-number was elevated in

27/30 (90%) RB1+/+ vs. 60/93 (65%) RB1-/- retinoblastomas (p = 3·4 x 10-4) (table S65). No RB1-/-

primary tumours showed over >10 MYCN copies. In comparison, 16/30 (53%) RB1+/+ tumours

showed high-level MYCN amplification (28 to 121 copies of MYCN), (RB1+/+ MYCNA

retinoblastoma) MYCN copy-number in RB1+/+ tumours showed bimodal distribution, with 16/30

(53%) RB1+/+ tumours showing high-level MYCN amplification (28 to 121 copies of MYCN),

henceforth referred to as RB1+/+MYCNA retinoblastoma ((p <0·0001) (tables 1, S65, figure 1). The

remaining 14 RB1+/+ tumours showed between 2 and 10 MYCN copies. For ten children with

RB1+/+MYCNA tumours, normal DNA from available blood showed two MYCN copies.

Compared to RB1-/- retinoblastoma, the 16 RB1+/+MYCNA tumours showed lower frequency of

copy-number changes in four other genes characteristic of retinoblastoma: gain of oncogenes KIF14

(62% vs. 19%; p = 0·002), DEK and E2F3 (57% vs. 6%; p = 0·0002) and loss of tumour suppressor

gene CDH11 (56% vs. 13%; p = 0·002) (tables S65, S76). SNP analysis indicated that the level of

normal cell contamination in the RB1+/+ MYCNA tumours was low and similar to RB1-/- tumours

(table S2).

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ADDED SENTENCE TO ACCOMMODATE REVIEWER 3


To probe for other genomic gains/losses, we studied DNA from 48 unilateral retinoblastomas by

aCGH10 and 3 by SNP analysis (Amsterdam) (14 RB1+/+MYCNA, 12 RB1+/+, 25 RB1-/-(+)) (tables S65,

S87, figure 2A). None of the RB1+/+MYCNA retinoblastomas showed any evidence of copy-number

changes or translocations11 at the RB1 locus. Except for MYCN copy-number, aCGH (figure 2A)

confirmed a reduced frequency in RB1+/+MYCNA retinoblastomas of the specific genomic copy-

number changes characteristic of RB1-/- retinoblastomas (figure 1, table S76).9 The RB1+/+MYCNA

retinoblastomas also showed overall significantly fewer altered bp and aCGH clones than the RB1-/-

retinoblastomas (p = 0·033) (figure 2C, DS3, table S87).

MYCN amplicons of 14 (11 by aCGH, 3 by SNP analysis) RB1+/+MYCNA retinoblastomas, as

well as one RB1+/- tumour (T33) with 73 copies of MYCN, and one RB1-/- primary tumour (RB381)

with 9·2 copies of MYCN, were narrow, spanning 1·1 to 6·3 Mbp encompassing MYCN (figures

2BS4, 5, S2, table S87). Importantly, the sole copy-number change detected in one RB1+/+MYCNA

retinoblastoma (E4) was 48 copies of 2p24·.2-24·.3 encompassing MYCN. The minimal common

amplicon defined by RB1+/+(-) MYCNA retinoblastomas T33 and P2 spanned 513 kbp containing only

MYCN. RB1+/+MYCNA retinoblastomas T5 and P2 also defined a minimal common amplicon

including only MYCN. Of the remaining 35 tumours tested by aCGH, 24 unilateral tumours showed

no gain or loss at MYCN, and 11 had moderate gain spanning a broad region of at least 28 Mbp of

chromosome 2p, too large to meet the definition of amplification.12

Three (23%) RB1+/+MYCNA tumours showed 17q21·.3-qter or 17q24·.3-qter gain; two

RB1+/+MYCNA tumours showed 11q loss. Both regions are commonly altered in neuroblastoma,13, 14

but rare in RB1-/- retinoblastoma (present and published data15, 16). Other changes in RB1+/+MYCNA

retinoblastomas not often seen in RB1-/- retinoblastoma were gains at 14q and 18q, and losses at 11p

(figures 2A, S53).

Retinoblastoma T33 (RB1+/-) showed high-level MYCN amplification and loss of one copy of

most of 13q, including RB1; we suspect that amplification of MYCN initiated proliferation, followed

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by 13q deletion. Since T33 showed numerous characteristics of RB1+/+MYCNA tumours, we included

T33 with MYCNA retinoblastomas in many analyses (figures 2A & B, 4A, S2, S4S4, S6).

RB1+/+MYCNA retinoblastomas expressed functional RB1 protein (pRB). pRB. Primary pRB. The

RB1+/+MYCNA retinoblastomas and the expected usual normal retinal cell types expressing pRb

(ganglion cells, specific inner nuclear cells, and cone photoreceptors)17 stained showed nuclear

staining for C-terminal (figure 3A, S6) and N-terminal (data not shown) epitopes of the RB1 protein

(pRB), pRB, while RB1-/- and RB1+/- tumours were negative or stained weakly for pRB, depending

on their RB1 mutations. Western blots and immunoprecipitation on of in 3 RB1+/+ MYCNA cell lines

(A3, and RB522, T101) derived from RB1+/+MYCNA primary retinoblastomas, showed demonstrated

functional nuclear pRb that was both phosphorylated and un-phosphorylated, full-length (figures

S6, S7), normally hypo- and hyperphosphorylated (figure 3B, S7), and bound to pRBE2F1 (figure

3C), the major target of pRb in control of the cell cycle.18{Sahin, 2010 #16727;Templeton, 1991

#18635} {Buchkovich, 1989 #17829} (figures 3B, S5).Full-length 2·8 kbp RB1 transcripts were

detected by end-point and real-time RT-PCR in the 3 Three RB1+/+MYCNA primary retinoblastomas

for which mRNA was available, end-point and real-time RT-PCR showed full-length 2·8 kbp RB1

transcripts at levels comparable to fetal retina (figures 3C, 3E3D, S8,, table S98). In contrast, most

RB1-/- retinoblastomas expressed low RB1 transcript levels.

RB1+/+MYCNA primary retinoblastomas (figure 3A) and three derived cell lines (data not shown)

stained strongly for N-Myc protein. RB1+/+MYCNA retinoblastomas showed increased N-Myc

protein and transcripts compared to RB1-/- primary retinoblastomas and fetal retina (figure 3B, C, E,

table S8). MYCN and MKI67 transcripts (indicative of proliferation) were at low levels in adult

retina, at and were detectedhigher levels in fetal retina and RB1-/- retinoblastomas without MYCN

amplification, and at very high levels in primary RB1+/+MYCNA retinoblastoma and RB1-/- cell lines

with MYCN amplification (figure 3AC, E, F, table S8). , and RB1-/- retinoblastomas. RB1+/+MYCNA

tumours showed reduced expression of the oncogene KIF14,9 in contrast to normal fetal retina, and

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CHANGE NEW DATA: FIG 3B-D, S4, S5; page 10.


RB1-/- primary retinoblastomas and cell lines with which over-express high KIF14 expression (figure

3FE, table S8).

RB1+/+MYCNA tumours expressed embryonic retinal cell markers consistent with a retinal origin.

The mRNAs of cone cell marker X-arrestin19 and CRX, a marker of retinal and pineal lineage

tumours, (strongly expressed in RB1-/- retinoblastoma but not in neuroblastoma,20) were expressed in

fetal retina, human adult retina, and three RB1+/+MYCNA, and four RB1-/- primary retinoblastomas

(figure 3DS6).

Children with RB1+/+MYCNA retinoblastomas were much younger at diagnosis than children with

unilateral RB1-/- retinoblastomas. The median age at diagnosis of 17 children (with 16 RB1+/+MYCNA

and one RB1+/-MYCNA [(T33]) tumours) was 4·5 months, significantly younger than children with

unilateral sporadic RB1-/- (24·0 months, p<10-4) or RB1+/+ (21·5 months, p<10-4) retinoblastomas

(figure 4A, tables S4A & S5). We estimate that 18·.4% of children diagnosed with non-familial,

unilateral retinoblastoma at age six months or younger will have RB1+/+MYCNA retinoblastoma

(table S4B).

Analysis of age at diagnosis vs. proportion not yet diagnosed led Knudson to propose that two

hits initiate retinoblastoma.1 Our data from 79 unilaterally affected RB1-/- patients was consistent

with Knudson’s model and fit a two-hit curve, representative of two independent mutational events

in a tumour suppressor gene (figure 4B). Similar analysis for RB1+/+MYCNA tumours was

inconclusive: while the data points for twelve children diagnosed before 10 months approximated

the calculated one-hit curve, ages for the older children deviated (figure 4B).

On histological examination, RB1+/+MYCNA tumours were distinctive, displaying undifferentiated

cells with large, prominent, multiple nucleoli, and necrosis, apoptosis, and little calcification,

similar to other MYCNA embryonic tumours, such as neuroblastoma21 (figures 4C, S45). The

Flexner-Wintersteiner rosettes22 and nuclear molding characteristic of prototypic RB1-/-

retinoblastoma (figure 4D) were absent.

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Clinically, the RB1+/+MYCNA retinoblastomas were large and invasive, considering the very

young age of these children (figure 4C, E, and S76). Three RB1+/+MYCNA retinoblastomas (RB522,

T101, and A3) from enucleated eyes grew rapidly into cell lines, unlike the RB1-/- retinoblastomas

that grow poorly in tissue culture. One RB1+/+MYCNA retinoblastoma had already invaded the optic

nerve past the cribriform plate at age 11 months, a feature of aggressive disease23, 24 (figure 4E).

However, all the children in this study with RB1+/+MYCNA tumours were cured by removal of their

affected eye with no adverse outcomes; none developed retinoblastomas in their other eye.

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Discussion

Knudson’s analysis of retinoblastoma was fundamental to the concept that normal genes suppress

cancer,1 leading to the identification of the RB1 tumour suppressor gene,2 widely assumed to initiate

all retinoblastoma. We now describe a previously unrecognized type of retinoblastoma with no

evidence of RB1 mutations and with afunctional pRb, that likely initiated by amplification of the

MYCN oncogene.

Our international study of 1,068 unilateral retinoblastomas revealed a distinct RB1+/+MYCNA

subtype comprising 1·4% of unilateral, non-familial retinoblastomas. At least two previously

described tumours may also be RB1+/+MYCNA retinoblastomas,25 although RB1 genetic status was

not defined. Despite the low incidence of RB1+/+MYCNA retinoblastoma, RB1+/+MYCNA tumours

were independently discovered and characterized in the Toronto and Amsterdam labs, with different

patient cohorts and technologies.

We also identified 14 RB1+/+ retinoblastomas without MYCN amplification. aCGH showed

genomic gains and losses distinct from either RB1-/- or RB1+/+MYCNA retinoblastomas, but we

lacked RB1+/+ tumours for gene expression or protein studies. In particular, these RB1+/+

retinoblastomas showed increased frequent gain of 19p and q, 17p and q, 2p, and the telomeric end

of 9q. The RB1+/+ group appears heterogeneous and merits further study.

Our paper highlights how molecular diagnostics can identify novel malignancies that elude

histopathological recognition. Although RB1+/+MYCNA retinoblastomas were not previously

recognized as distinct, they resemble large nucleolar neuroblastomas with MYCN-amplification and

poor outcome.21 Like MYCN-amplified neuroblastomas,14 RB1+/+MYCNA tumours showed less

complex genomic copy-number alterations than tumours without MYCN amplification, suggesting

that MYCNA may be the critical driver of malignancy. However, the early age of diagnosis of

RB1+/+MYCNA tumours may allow less time to accumulate genomic alterations. Although whole

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genome sequencing recently suggested that point mutations (other than in the RB1 gene) are few in

RB1-/- retinoblastoma,26 loss of RB1 induces mitotic changes and lagging chromosomes,27 leading to

genomic instability. RB1 loss in retinoblastoma and pre-malignant retinoma8, 16 is also associated

with specific common DNA copy-number changes, that are less frequent in RB1+/+MYCNA

retinoblastomas.

Are RB1+/+MYCNA tumours truly retinoblastoma? RB1+/+MYCNA retinoblastomas arise in

developing retina and express markers of embryonic retina, meeting the definition of retinoblastoma

as ‘a blast cell tumour arising from the retina’.19, 20, 28 We demonstrate intact RB1 genes in primary

RB1+/+ MYCNA retinoblastomas, with and pRB with strong nuclear N- and C-terminal pRb antibody

staining, in primary RB1+/+MYCNA retinoblastomas. The Three cell lines derived from

RB1+/+MYCNA tumours expressed full length RB1 mRNA, and inactive hyperphosphorylated and

active un-hypophosphorylated pRb that binds to E2F1, indicating normal functional pRb18. The

possibility of normal cell contamination masking an RB1 mutations in the RB1+/+ MYCNA tumours is

ruled out by the cell lines that maintain the RB1+/+ genotype of the primary tumour, the very high

level MYCN amplification, histology with the only normal cells being vascular, and strong

histological and biochemical evidence that pRb is functional.

The very early presentation of RB1+/+MYCNA retinoblastomas, lack of RB1 mutations, functional

pRb and high MYCN-amplification in a relatively copy-number stable genome, suggests that these

retinoblastomas arise by somatic MYCN oncogene amplification in a retinal progenitor cell. This is

supported by one RB1+/+MYCNA tumour in which MYCN amplification was the only identified

genomic copy-number change. The RB1+/+MYCNA retinoblastomas are already large in very young

children, so they likely initiate earlier in retinal development than RB1-/- retinoblastomas. In

children with an RB1 mutation of equivalent age, the tumours are usually much smaller (figure 4F).

How MYCN-amplification is initiated, and whether MYCN-amplification alone suffices to initiate

these retinoblastomas remains to be formally demonstrated.

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INSERTED IN RESPONSE TO REVIEWER #3


NEW DATA RE: PRB FUNCTION


In retinal development the N-myc protein can support cell division in the absence of activating

E2fs; presumably unregulated MYCN expression associated with high- level gene amplification in

RB1+/+MYCNA tumours promotes cell division indirectly through inactivation of pRb.29 The relative

genomic stability of RB1+/+MYCNA retinoblastomas suggests that anti-N-Myc therapeutic agents30

may avoid emergence of drug resistance acquired through progressive genomic rearrangements in

RB1-/- retinoblastoma. Our preliminary MYCN knock down experiments resulted in rapid death of

RB1+/+MYCNA cell lines. We predict that children less than one year of age with extra-ocular

retinoblastoma around the world23, may have RB1+/+MYCNA retinoblastomas that might benefit from

anti-N-Myc therapy. This idea is consistent with our (BG) anecdotal observation during review of

retinoblastoma pathology slides in Kenya, of RB1+/+MYCNA histology in an orbital recurrence,

confirmed to have 40 copies of MYCN.

Young age at diagnosis of unilateral retinoblastoma is frequently interpreted as an indication of

heritable retinoblastoma, and is currently cited as a reason to try to cure the cancer without

enucleation. However, attempts to salvage an eye with a large RB1+/+MYCNA retinoblastoma could

be dangerous; in our study, prompt removal of the unilateral affected eyes was curative with no

adverse outcomes. The young patients with RB1+/+MYCNA tumours in our study had large,

aggressive tumours, including invasion into the optic nerve. At similar young ages, the usual

hereditary RB1-/- tumours are very much smaller, detected only by active surveillance (figure 4F).

While our data predict that 18·.4% of children diagnosed with non-familial, unilateral

retinoblastoma at age six months or younger will have RB1+/+MYCNA retinoblastoma (table S4B), if

size of tumour size is also considered, the risk will be higher, and RB1+/+MYCNA tumours may be

clinically predictable, facilitating prompt removal of these eyes with good outcomes for the

children.

Standard care for unilateral non-familial retinoblastoma is identification of the RB1 mutant

alleles in tumour, and examination of blood to determine whether either mutant allele is germline.

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Our study suggests that when no RB1 mutation is detected in a retinoblastoma tumour, determining

MYCN copy-number may assist on-going care. The diagnosis of RB1+/+MYCNA retinoblastoma

strongly suggests non-hereditary disease with normal population risks for retinoblastomas in the

other eye and other cancers later inthroughout life.

Our findings challenge the long-standing dogma that all retinoblastomas are initiated by RB1

gene mutations (figure 4G). RB1+/+MYCNA retinoblastoma provides an intriguing contrast to

classical retinoblastoma, of immediate importance to patients.

Panel: Research in Context

Systematic Review

We systematically searched the published, peer-reviewed literature on PubMed

(http://www.ncbi.nlm.nih.gov/pubmed/) using the search terms “retinoblastoma”, “initiation” and

“genetics”; “retinoblastoma tumour genetics”; “retinoblastoma development”; and “retinoblastoma

initiation”. We reviewed publications with a main focus on genetic initiation and development of

human retinoblastoma. We found no data that challenged Knudson’s 1971 conclusion that two rate-

limiting events1, later shown to be loss of both RB1 gene alleles,2-5, 7 are essential but not necessarily

sufficient for development of retinoblastoma. We found no data to suggest another form of

retinoblastoma.

Interpretation

Our collaborative studies identify a previously unrecognized disease: retinoblastoma with normal

RB1 genes, apparently driven by MYCN oncogene amplification. This newly recognised form of

retinoblastoma has immediate clinical implications for patients. RB1+/+MYCNA retinoblastomas are

diagnosed by molecular study of the tumour after removal of the eye of very young children with

unilateral non-familial disease. The children with RB1+/+MYCNA retinoblastoma and their relatives

are predicted to be at normal population risk for other cancers. Attempts to salvage the eye on the

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assumption of heritable disease in young children may incur high treatment morbidity and failure to

cure these aggressive oncogene-driven retinoblastomas.

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Tables

Test Site Total RB1+/+ Proportion RB1+/+Number

RB1+/+MYCNA

Proportion

RB1+/+MYCNA

Canada 441 7 1·6% 5 1·1%

Germany 400 12 3·0% 4 1·0%

France 150 5 3·3% 2 1·3%

New Zealand 30 2 6·7% 1 3·3%

The Netherlands 47 3 6·4% 3 6·4%

Total 1068 29 2·7% 15# 1·4%

*Fisher's exact tests indicates that percentage of unilateral tumours with RB1+/+ is not related to site (p = 0·08).

Table 1. Frequency of RB1+/+ unilateral retinoblastoma at five international diagnostic labs

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Figures

Figure 1: Copy numbers of genes commonly altered in RB1-/-

retinoblastoma

(A) Box-plot of genomic copy-numbers determined by QM-PCR for indicated

genes in unilateral retinoblastoma, grouped by RB1 mutation status. T33 is an

outlier of the RB1+/- group, showing an RB1+/+ MYCNA -like profile. Gray line, 2-

copies. (B) Heat-map for copy number for the profile genes (red, increased, and

blue, decreased copy-number; gray, 2 copies; white, not tested; n, number in

each group). Recently discovered RB1+/+ MYCNA tumours not in (A) are indicated

by black triangles.

Figure 1: The 5-gene copy number signature of retinoblastoma

(A) Box-plot of genomic copy-numbers determined by QM-PCR for indicated genes in unilateral

retinoblastoma, categorized by RB1 mutation status. On each boxplot, vertical line marks the

maximum and minimum copy-numbers observed while the box bounds second and third quartiles,

and horizontal line within the box represents the median (11 RB1+/+MYCNA tumours, median 54

MYCN copies; 14 RB1+/+tumours, median 3·1 MYCN copies). T33 is an outlier of the RB1+/-group,

showing an RB1+/+MYCNA-like profile; gray line, 2-copies. (B) Heat-map for copy-number by QM-

PCR for the profile genes (red, increased, and blue, decreased copy-number; gray, 2 copies; white,

not tested; n, number in each group) including more recently discovered RB1+/+MYCNA tumours

(black triangles) not included in (A). (C) MYCN copy-number assessment in 30 RB1-/- and 3 RB1+/+

tumours studied by MLPA, showing high MYCN copy-number in the RB1+/+ tumours.

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Figure 2: Genomic copy number alterations

(A) aCGH on 12 RB1+/+ MYCNA (including T33) (red), 12 RB1+/+ (blue), 13 RB1+/-

(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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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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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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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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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.

6. Lohmann D, Gallie B, Dommering C, Gauthier-Villars M. Clinical utility gene card for: Retinoblastoma. Eur J Hum Genet 2011; 19(3).

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

34

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10111213141516171819202122232425262728293031323334353637383940414243444546


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 TumorTumours. The American journal of surgical pathology 2012; 36(8): 1165-9.

21. Tornoczky T, Semjen D, Shimada H, Ambros IM. Pathology of peripheral neuroblastic tumortumours: 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.

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