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Not Knudson’s Retinoblastoma:

One-Hit Cancer Initiated by the MYCN Oncogene?

Diane E Rushlow, 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, 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, R Pang, MA, C Massey, MSc,H Dimaras, PhD, P C Boutros,

PhD, W Halliday, MD, Prof B L Gallie, MD); 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

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

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]

2

mailto:[email protected]


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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, immunohistochemistry, and gene

expression, 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. 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

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 cancers. But since these aggressive tumours may rapidly

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become extra-ocular, removal of the eye of these young children with unilateral non-familial

retinoblastoma is important.

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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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. A

child with bilateral retinoblastoma carries a heterozygous, constitutional RB1 mutation and is

predisposed to retinoblastoma; one additional hit, in which the second RB1 allele is damaged,

initiates retinoblastoma, and/or other cancers later in life. Approximately 60% of children have

unilateral retinoblastoma. Most non-familial unilateral retinoblastomas arise when both RB1 alleles

are damaged in developing retina, but 15% carry a heritable, constitutional RB1 mutation. Accepted

dogma is that damage to both RB1 alleles (RB1-/-) is required for 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.5-7 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 new sub-type of retinoblastoma has immediate

diagnostic, genetic counselling, and therapeutic implications.

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Methods (see webappendix for details)

Clinical samples

Tumours, blood, and clinical data were provided for identification of RB1 mutations for clinical

care of children and their families. Research Ethics Board approvals for the 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 to tumours 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.

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.

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RNA gene expression studies

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

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 compared and analysed to assess the

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 role in study design, data collection, data analysis, data

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. 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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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% an 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 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.

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 and no loss of heterozygosity (LOH) at RB1 (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 RB1+/+ tumours in

which they had found no detectable RB1 mutations or promoter methylation, no RB1 LOH, and no

detectable normal cell contamination (Table 1). After analysis was complete, we learned that the

Amsterdam lab 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. Later, retinoblastoma T101 was predicted clinically and pathologically

to be an RB1+/+MYCNA tumour (figure 4C), then shown to have 40 copies of MYCN, and is included

in some analyses.

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Since our standard is 95% sensitivity to find an RB1 mutation in samples expected to carry one,5-

7 the probability of finding no RB1 mutations in a tumour with no LOH at the RB1 locus 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) (table 1). This suggested that

some RB1+/+ tumours might originate by a mechanism other than 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 S5). Most

significantly, 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), called

RB1+/+MYCNA retinoblastoma (tables 1, S5, 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 the normal two MYCN copies.

In comparison to RB1-/- retinoblastoma, the 16 RB1+/+MYCNA tumours showed reduced frequency

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

KIF14 (19% vs. 62%; p = 0·002), DEK and E2F3 (6% vs. 57%; p = 0·0002) and loss of tumour

suppressor gene CDH11 (13% vs. 56%; p = 0·002) (tables S5, S6).

We studied DNA from 48 unilateral retinoblastomas by aCGH10 and 3 by SNP analysis

(Amsterdam) (14 RB1+/+MYCNA, 12 RB1+/+, and 25 RB1-/-(+)) (tables S5, S7, 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 (table S6).9 The RB1+/+MYCNA retinoblastomas also showed overall

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significantly fewer altered bp and aCGH clones than the RB1-/- retinoblastomas (p = 0·033) (figure

2C, D, table S7).

The amplicons of the 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 (figure 2B,

S2, table S7). 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, S3).

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

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, S4).

RB1+/+MYCNA retinoblastomas expressed pRB. Primary RB1+/+MYCNA retinoblastomas and the

usual normal retinal cell types17 stained for C-terminal (figure 3A) and N-terminal (data not shown)

epitopes of the RB1 protein (pRB), while RB1-/-(+) tumours stained weakly for pRB, depending on

the RB1 mutation. Western blot on cell lines A3 and RB522, derived from RB1+/+MYCNA primary

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retinoblastoma, showed both phosphorylated and un-phosphorylated, full-length pRB18 (figures 3B,

S5). 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

(figure 3C, 3E, table S8). In contrast, 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, and were detected in fetal retina and primary RB1+/+MYCNA retinoblastoma (figure 3C, E),

and RB1-/- retinoblastomas. RB1+/+MYCNA tumours showed reduced expression of the oncogene

KIF14,9 in contrast to normal fetal retina and RB1-/- primary retinoblastomas and cell lines with high

KIF14 expression (figure 3E, 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, three RB1+/+MYCNA, and four RB1-/- primary retinoblastomas (figure

3D).

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).

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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 mutation 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, with undifferentiated cells

with large, prominent, multiple nucleoli, and necrosis, apoptosis, and little calcification, similar to

other MYCNA embryonic tumours, such as neuroblastoma21 (figures 4C, S5). The Flexner-

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

(figure 4D) were not present.

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

young age of these children (figure 4C, S6). 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 disease 23,24 (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 the 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 that may instead be 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 different technologies.

We also identified 14 RB1+/+ retinoblastomas without MYCN amplification. Although we lacked

tumour for gene expression studies, aCGH showed genomic gains and losses distinct from either

RB1-/- or RB1+/+MYCNA retinoblastomas. In particular, these RB1+/+ retinoblastomas showed

increased gain of 19p and q, 17p and q, 2p, and the telomeric end of 9q. The RB1+/+ group may be

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

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

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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, and pRB

with strong 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

phosphorylated and un-phosphorylated pRb, indicating normal functional pRb18.

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

In retinal development, N-myc protein, together with E2f proteins, activates cyclins to inactivate

pRb by phosphorylation; presumably unregulated MYCN expression associated with high level

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

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in rapid death of RB1+/+MYCNA cell lines. We predict that children less than one year of age with

extra-ocular retinoblastoma23 around the world, 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 40 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 S4), if size of tumour is

also considered, 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.

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

MYCN copy-number assists 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 in 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.

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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 apparently

driven by MYCN oncogene amplification. This newly recognised form of retinoblastoma has

immediate clinical implications for patients. RB1+/+MYCNA retinoblastoma can only be 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

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

failure to cure these aggressive oncogene-driven retinoblastomas.

16

http://www.ncbi.nlm.nih.gov/pubmed/


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: 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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19


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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21


Figure 3: RB1+/+MYCNA tumours express pRb and MYCN

(A) Compared to normal patterns of expression in the adjacent retina, RB1+/+MYCNA retinoblastoma

stained positive for N-Myc protein and pRb (C-terminus antibody), while RB1+/- tumour stained

weakly for N-Myc and pRb; T, tumour; INL, inner nuclear layer retina. (B) Western blot shows the

characteristic two bands of hypophoshorylated (active) pRB and phosphorylated (inactive) ppRB in

fetal retina (17 weeks), RB1+/+MYCNA cell line A3, and neuroblastoma cell lines SH-SY5Y and

BE(2)-M17, while the two RB1-/- cell lines (WERI-Rb1 and Y79) show no full length pRb. Low

expression of MYCN protein is detected in fetal retina (week 17) and WERI-Rb-1, while high

expression of MYCN protein is detected in A3, Y79 and BE(2)-M17 (with MYCN amplification),

but not in SH-SY5Y (no MYCN amplification). (C) Primary RB1+/+MYCNA retinoblastomas express

full-length RB1, MYCN and Ki67 transcripts (end point RT-PCR); Ki67 mRNA indicates

proliferation; TBP endogenous control. (D) Expression of retinal progenitor cell marker CRX and

cone cell maker X-arrestin in human fetal retina, human adult retina, primary RB1+/+MYCNA

tumours, and primary RB1-/- tumours with between two and ten MYCN copies; end point RT-PCR;

TBP endogenous control. (E) RB1, MYCN and KIF14 mRNA expression in human fetal (FR) and

(HR) adult retina, RB1+/+MYCNA, RB1-/-, or RB1+/- primary tumours and RB1-/-cell lines; real-time

RT-PCR, triplicate measurements normalized against GAPDH, relative to FR; MYCN DNA copy-

numbers in italics; #, not done; *Y79 has a homozygous RB1 del exons 2 to 6 that results in

increased expression of shortened RB1 mRNA. Cell lines Y79 and RB381 have MYCN

amplification.

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Figure 4: RB1+/+MYCNA tumours in very young children are clinically distinct

(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, fits a two-hit curve (blue)

but not a one-hit curve (red) for unilateral non-familial RB1-/- disease, as expected; for

RB1+/+MYCNA retinoblastoma, 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

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 shown to be 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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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. 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 analysis on age of diagnosis data. Roseline

Godbout discovered and characterised the first RB1+/+MYCNA tumor with MYCN amplification and

normal pRb (RB522) long before anyone believed her; she provided the cell line and data

characterising including the included Western blot. 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 aCGH experiments. Paul C Boutros

performed detailed and novel analysis of the aCGH data, and statistical analyses throughout the

project. Dietmar Lohmann performed literature search, provided RB1 mutation analysis, and

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contributed to figure construction and development of concepts. Josephine C Dorsman coordinated

the Amsterdam study, 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 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 image 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

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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