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Discovery of MYCN-amplified retinoblastoma with functional

retinoblastoma protein in very young children

Diane E Rushlow, Berber M Mol,* Jennifer Y Kennett,* Stephanie Yee,* Sanja Pajovic,

Brigitte L Thériault, Nadia L Prigoda-Lee, Clarellen Spencer, Helen Dimaras, Timothy W

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

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); Eugene and

Marilyn Glick Eye Institute, Departments of Ophthalmology, Biochemistry and Molecular

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Gallie Brenda, 16/01/13,

TITLE CHANGE –

MYCN amplified retinoblastoma with normal pRb

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]


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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 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 good care.

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 The 40% of children who have bilateral

retinoblastoma carry a heterozygous, constitutional RB1 mutation and are predisposed to

retinoblastoma; one additional hit, damages the second RB1 allele 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). The 60% of children with

unilateral non-familial retinoblastoma usually have both RB1 alleles damaged only in developing

retina, but 15% of them carry a heritable, constitutional RB1 mutation. Accepted dogma is that

disruption of both RB1 alleles (RB1-/-) initiates all retinoblastoma.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

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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 in clinical care of

children and their families. Research Ethics Board approvals to use 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


immunoblotting with phospho-specific 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, analysis, 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

from unilateral probands with no known family history. In 3-4% of tumours only one RB1 mutation

is identified, and in 1·6%, 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+/+) may include translocations, deep intronic mutations, or

mutations in unknown RB1 regulatory regions.

In clinical work in Toronto, we found seven unilateral retinoblastoma tumours with no RB1

mutations, no promoter methylation, and no 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 (table 1). After

initial studies, we learned that the Amsterdam 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 labs. Retinoblastoma T101, predicted

clinically and pathologically to be an RB1+/+MYCNA tumour (figure 4C), had 40 copies of MYCN,

and is included in some analyses.

Since our standard sensitivity to find 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) (table 1). This suggested that some RB1+/+

tumours might originate by a mechanism other than Knudson’s two RB1 mutations.

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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 S6). 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) (p <0·0001) (tables 1, S6, 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 S6, S7). SNP analysis indicated that the level of

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

(table S2).

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 S6,

S8, 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 S7).9 The RB1+/+MYCNA

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

retinoblastomas (p = 0·033) (figure S3, table S8).

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)

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ADDED SENTENCE TO ACCOMMODATE REVIEWER 3


with 9·2 copies of MYCN, were narrow, spanning 1·1 to 6·3 Mbp encompassing MYCN (figures S4,

5, table S8). 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 spanning 513 kbp, contained 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, S5).

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 2, 4A, S4, S6).

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

retinoblastomas and the expected retinal cell types expressed pRb (ganglion cells, specific inner

nuclear cells, and cone photoreceptors)17 and showed nuclear staining for C-terminal (figure 3A, S6)

and N-terminal (data not shown) epitopes, while RB1-/- and RB1+/- tumours were negative or stained

weakly, depending on their RB1 mutations. Western blots and immunoprecipitation in 3

RB1+/+MYCNA cell lines (A3, RB522, T101) derived from RB1+/+MYCNA primary retinoblastomas,

demonstrated functional nuclear pRb that was full-length (figures S6, S7), normally hypo- and

hyperphosphorylated (figure 3B, S7), and bound to E2F1 (figure 3C), the major target of pRb in

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control of the cell cycle.18RB1 transcripts were detected by end-point and real-time RT-PCR in the 3

RB1+/+MYCNA primary retinoblastomas for which mRNA was available (figures 3D, S8, table S9).

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. MYCN and KI67 transcripts (indicative of proliferation) were at

low levels in adult retina, at higher 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 3A, D, S8A, table S8). RB1+/+MYCNA tumours showed reduced

expression of the oncogene KIF14,9 in contrast to normal fetal retina, and RB1-/- primary

retinoblastomas and cell lines which over-express KIF14 (figure 3F, table S9).

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 RB1+/+MYCNA and RB1-/- primary retinoblastomas (figure S8B).

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 S5A & S6). 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 S5B).

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

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


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).

RB1+/+MYCNA tumours showed distinctive histology, 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, S6). The Flexner-Wintersteiner

rosettes22 and nuclear molding characteristic of prototypic RB1-/- retinoblastoma (figure 4D) were

absent.

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

young age of these children (figures 4C, E, S7, S9). 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. The cell lines still showed no RB1 mutations and maintained the

MYCN amplification of the primary, confirming minimal normal cell contamination of the primary

tumour. 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

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 functional pRb, 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 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 DNA copy-number changes, that are less frequent in RB1+/+MYCNA retinoblastomas.

Are RB1+/+MYCNA tumours truly retinoblastomas? RB1+/+MYCNA retinoblastomas arise in, 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 strong nuclear pRb antibody staining. Three RB1+/+MYCNA cell lines

expressed full length RB1 mRNA, and inactive hyperphosphorylated and active

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.

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

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INSERTED IN RESPONSE TO REVIEWER #3


NEW DATA RE: PRB FUNCTION


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

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.

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

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

other eye and other cancers throughout life.

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

gene mutations (summary 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

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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Figure Legends

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 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.

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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.

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Figure 4: Clinical features of children with RB1+/+MYCNA tumours

(A) Children with RB1+/+MYCNA retinoblastoma are diagnosed significantly

younger than children with RB1-/- tumours (p<0·0001, Wilcoxon rank sum test).

(B) The Knudson plot of proportion not yet diagnosed vs age at diagnosis, using

birth as a surrogate for initiation, showing a two-hit curve (blue) and a one-hit

curve (red) for children with RB1-/- tumours or RB1+/+MYCNA tumours;

scatterplot does not distinguish identically aged children. (C) Fundus image of

a large RB1+/+MYCNA unilateral tumour, extending from optic nerve (white

arrow) to anterior border of retina (double arrows) in a 4 month-old child with

characteristic calcification on ultrasound, and round nuclei with prominent

large multiple nucleoli on pathology, in comparison to (D) RB1-/- tumour

showing classic Flexner-Wintersteiner rosettes and nuclear molding;

hematoxylin-eosin staining. (E) RB1+/+MYCNA retinoblastoma in an 11 month-old

child (A2) with extra-ocular extension into the optic nerve (arrows) (2·5x,

hematoxylin-eosin staining). (F) In comparison, in 3·5 month-old child with

heritable RB1-/- retinoblastoma, a small tumour is present in the inner nuclear

layer of the retina on optical coherent tomography (OCT). (G) Schema of data

establishing RB1+/+MYCNA retinoblastoma as a novel disease; months (m), data

figures (f) and tables (t) indicated in grey on left.

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Contributors

Diane E Rushlow recognized the initial connection between MYCNA amplification and RB1

mutation status, performed literature search and QM-PCR analysis and supervised RB1 mutation

analysis, coordinated collaborations with the other sites and was the major contributor to manuscript

preparation. Berber M Mol recognized the RB1 and MYCN mutation status of the Amsterdam

samples by MLPA and SNP array analysis, performed immunohistochemistry, Western blots and

co-immunoprecipitations. Jennifer Y Kennett performed aCGH and analysed aCGH data. Stephanie

Yee performed analysis of aCGH data, the MYCNA alignment, immunohistochemistry and reverse

transcriptase PCR. Sanja Pajovic performed literature search, reverse transcriptase PCR and

immunohistochemistry. Brigitte L Thériault performed literature search and RNA expression

studies. Nadia L Prigoda-Lee performed literature search, statistical analysis and contributed to

figure and manuscript preparation. Clarellen Spencer performed immunohistochemistry. Helen

Dimaras and Timothy W Corson performed literature searches, assisted in data analysis and

conceptualization of discussion, and contributed to figure and manuscript preparation. Renee Pang

performed statistical and bioinformatic analyses on the aCGH data. Christine Massey performed

statistical analyses. Roseline Godbout discovered and characterised the first tumour with MYCN

amplification and normal pRb (RB522) (now RB1+/+MYCNA) long before anyone believed her; she

provided the cell line and data and Western blot showing functional pRb. Zhe Jiang and Eldad

Zacksenhaus performed Western blots to demonstrate functional phosphorylated pRb. Katherine

Paton and Annette C Moll provided clinical images and material, and conceptual discussion. Claude

Houdayer and Anthony Raizis provided RB1 mutation analysis, and clinical features. William

Halliday recognized and characterized the unique histological features of the RB1+/+MYCNA

retinoblastomas and prepared digital images for publication. Wan L Lam supervised Jennifer

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Kennet and the aCGH experiments. Paul C Boutros performed detailed and novel analysis of the

aCGH data, and statistical analyses throughout the project, and supervised Renee Pang. Dietmar

Lohmann performed literature search, provided RB1 mutation analysis, and contributed to figure

construction and development of concepts. Josephine C Dorsman coordinated the Amsterdam

study, and, with Berber Mol who she supervised, recognized the RB1 and MYCN mutation status of

the Amsterdam samples. Brenda L Gallie supervised overall, performed literature search, provided

critical guidance on all components of the project, and contributed extensively to figure and

manuscript preparation. All authors contributed to manuscript preparation.

Conflicts of interest

BLG was part-owner of Solutions by Sequence. All other authors declare that they have no conflicts

of interest.

Acknowledgments

This study was conducted with the support of the Ontario Institute for Cancer Research to PCB

through funding provided by the Government of Ontario. BMM was funded by a grant from

CCA/V-ICI/ Avanti-STR (to JCD, J. Cloos and ACM), the Dutch research was also funded in part

by KIKA (JCD, H. te Riele, J. Cloos, ACM). SY was funded by the Vision Science Research

Program of the University Health Network and the University of Toronto. RP was funded in part by

a Great West Life Studentship from Queen’s University School of Medicine. We thank Leslie

MacKeen for the montage of RetCam images in figure 3B. We thank Dr. Valerie White of U.

British Columbia for providing clinical and pathological details and images. We thank Cynthia

Vandenhoven for the clinical images in figure 4F. We thank members of the VU University

Medical Center/The Netherlands Cancer Institute, Institut Curie, Institut für Humangenetik, Toronto

retinoblastoma teams and other, wise colleagues for useful discussions. We thank the children and

families who donated tissues for these studies for the benefit of future families.

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