Not Knudson’s Retinoblastoma:

One-Hit Cancer Initiated by the MYCN Oncogene?

Diane E. Rushlow, B.Sc.1, Jennifer Y. Kennett, M.Sc.*, Berber M. Mol, M.Sc.*,

Stephanie Yee, M.Sc.*, Sanja Pajovic, Ph.D2,,4 , Renee Pang, M.A.,

Brigitte L. Thériault, Ph.D2, Nadia L. Prigoda-Lee, M.Sc. 1,2,4, Clarellen Spencer, B.Sc. 2, 4

Helen Dimaras, Ph.D5, Timothy W. Corson, Ph.D, Christine Massey, M.Sc. 3,

Katherine Paton, M.D., Annette C. Moll, M.D., Claude Houdayer, Ph.D,

William Halliday, M.D.8,9, Anthony Raizis, Ph.D, Wan L. Lam,

Ph.D, Paul C. Boutros, Ph.D6, Dietmar Lohmann, Ph.D, Josephine C. Dorsman, Ph.D, &

Brenda L. Gallie, M.D.1, 2,4,5,6,9

*Co-second authors

1Retinoblastoma Solutions and the Toronto Western Hospital Research Institute, 2Campbell Family Cancer Research

Institute and Ontario Cancer Institute, 3Department of Biostatistics, 4Princess Margaret Hospital – all University Health

Network, in Toronto, ON, Canada; 5Department of Molecular Genetics, 6Departments of Ophthalmology, 7Medical

Biophysics, 8Pathobiology and Lab Medicine– 9all University of Toronto, in Toronto, ON, Canada; British Columbia

Cancer Research Centre (J.Y.K., W.L.L) and Departments of Ophthalmology (K.P.) and Pathology & Laboratory

Medicine (J.Y.K, W.L.L), – all University of British Columbia, in Vancouver, BC, Canada; Departments of Clinical

Genetics (B.M.M, J.C.D), Ophthalmology (A.C.M.) and Pediatric Oncology/Hematology (B.M.M), VU University

Medical Center Amsterdam – all in Amsterdam, The Netherlands; Informatics and Biocomputing Platform, Ontario

Institute for Cancer Research (R.P., P.C.B.) in Toronto, ON, Canada; Departments of Hematology/Oncology (H.D.), of

Ophthalmology and Visual Science (H.D., B.L.G.) and of Pathology (W.H), Hospital for Sick Children – in Toronto,

ON, Canada; Eugene and Marilyn Glick Eye Institute, Departments of Ophthalmology and of Biochemistry and

Molecular Biology, Indiana University School of Medicine (T.W.C.) – in Indianapolis, Indiana, USA; Service de

Génétique Oncologique, Institut Curie and Université Paris Descartes (C.H.) – in Paris, France; Department of

Molecular Pathology, Canterbury Health Laboratories (A.R.) – in Christchurch, New Zealand; and Institut für

Humangenetik, Universitätsklinikum (D.L.) – in Essen, Germany.

Address reprint requests to Dr. Gallie at the Campbell Family Cancer Research Institute and Ontario Cancer

Institute, University Health Network, Rm. 8-415, 610 University Ave., Toronto, ON, M5G 1M9, Canada, or at

brenda@gallie.ca; or to Ms. Rushlow at Retinoblastoma Solutions, Rm. MP 13-302 MP, Toronto Western

Hospital Research Institute, University Health Network, 399 Bathurst Street, Toronto, ON, M5T 2S8, Canada.

2

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

Dr. Rushlow and colleagues present convincing data that a small subset of human retinoblastomas

is driven not by biallelic inactivation of RB1, but rather by amplification of the MYCN oncogene.

This observation, made possible only by an impressive international collaborative effort, alters a

long held paradigm that RB1 inactivation is pathognomonic for retinoblastoma. This is an important

observation, as well as the clinical and biological correlative analyses in this manuscript that

phenotypes “Rb+/+, MYCN amp” tumors as unilateral aggressive neoplasms occurring in very

young infants.

Word count 266/300

SUMMARY

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

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

attributed to loss of both alleles of the retinoblastoma suppressor gene, RB1 in the tumors. Children

with hereditary retinoblastoma carry heterozygous constitutional RB1 mutations, so only one hit is

required to develop retinoblastoma and other cancers. Non-hereditary retinoblastoma is considered

to arise when both RB1 alleles are lost in developing retina.

Methods In an international collaboration, we determined the proportion of 1054 unilateral non-

familial retinoblastoma tumors that had no evidence of RB1 mutations when studied by high

mutation detection sensitivity assays. We analysed clinical data, genomic copy-number changes,

histology, immunohistochemistry, array CGH or SNP analysis, and gene expression.

Findings No evidence of RB1 mutation (RB1+/+) was found in 2% (30/1054) of unilateral

retinoblastoma. Surprisingly, half of these had high-level MYCN oncogene amplification. Compared

to RB1-/- retinoblastomas, RB1+/+MYCNA tumors had fewer genomic copy-number changes and

distinct, aggressive histology. Median age at diagnosis of RB1+/+MYCNA tumors was 4·5 months,

compared to 24 months for children with non-familial unilateral RB1-/- retinoblastoma. Children less

3

than six months at diagnosis of unilateral non-familial retinoblastoma have a 22% chance to have a

RB1+/+MYCNA tumor.

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

the presence of normal RB1 genes. Since these aggressive tumors may rapidly become extraocular,

diagnosis by removal of the eye of young children with unilateral non-familial retinoblastoma is

important. Despite their young age, these children and their families are at normal population risk to

develop other cancers.

Funding NCI-NIH; Canadian Retinoblastoma Society; Hyland Foundation; Ontario Ministry of

Health and Long Term Care; Toronto Netralya and Doctors Lions Clubs; and the Foundation KiKa.

4

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

Word count 2802/3000

Background

Retinoblastoma set the paradigm for tumor suppressor gene-driven cancers with Knudson’s classic

hypothesis predicting that only two rate-limiting hits initiate this childhood eye cancer.1 The two

hits were later attributed to the retinoblastoma gene (RB1).2 Children with RB1 heterozygous

constitutional mutations are predisposed to retinoblastoma (~90% in both eyes, bilateral) and other

cancers later in life; the second RB1 allele is affected in each tumor. Most children with non-

familial unilateral disease have normal constitutional RB1 alleles, but 15% carry a germline RB1

mutation that usually is not inherited. It is widely accepted that loss of both functional RB1 alleles

(RB1-/-) is an absolute requirement for retinoblastoma development.2-4

A mutant RB1 allele has been identified in 95% of blood samples of bilaterally affected persons,

and in both alleles (RB1-/-) of 95% of retinoblastoma tumors, using multiple assays.5, 6 Loss of RB1

is associated with a characteristic “signature” of genomic gain and loss of other specific gene

regions in RB1-/- retinoblastomas.7, 8 Similar changes in these genes are already manifest in RB1-/-

retinoma, the benign, non-proliferative precursors to retinoblastoma, and increase in magnitude in

the adjacent malignant retinoblastoma.9-11 The 5% of children with bilateral retinoblastoma with no

detectable RB1 mutations in blood likely carry low-level mosaic mutations,6 translocations, or deep

intronic mutations which are not detectable by conventional testing. However, mosaicism cannot

explain the 5% of non-familial unilateral retinoblastoma tumors that show no RB1 gene

abnormalities. The possibility that retinoblastomas with no detectable RB1 mutations occur by an

independent mechanism has not been explored.

We report the first identification of retinoblastoma with normal RB1 and high-level MYCN gene

amplification (MYCNA). These RB1+/+MYCNA retinoblastomas are characterized by unilateral retinal

5

cancer with retinal progenitor-like features, presenting very young. Histology, relative genomic

copy number and high-level MYCN are distinct from RB1-/- retinoblastoma. This new sub-type of

retinoblastoma has immediate diagnostic, genetic counselling, and therapeutic implications.

Methods

Clinical samples

Tumors, blood, and clinical data were provided for genetic diagnosis of RB1 mutations for clinical

care of the children and their families. Research Ethics Board approvals for the use of de-identified

data and tissues after clinical analysis was completed, are on file at each participating site.

Mutation analyses

Standard of care analysis techniques that identify 95% of RB1 (Gen bank accession # L11910)

mutant alleles6, 12 was applied to all tumor samples, including DNA sequencing, quantitative

multiplex PCR (QM-PCR)5 or MLPA (MRC-Holland), and RB1 promoter methylation analysis13

(see webappendix for details).

Genome copy number analyses

Genomic copy numbers of KIF14, DEK, E2F3, CDH11, and MYCN were determined by QM-PCR,7

or single nucleotide polymorphism (SNP) array experiments (see webappendix for details). Sub-

megabase resolution array comparative genomic hybridization (aCGH) platform (BC Cancer

Research Centre) with an effective resolution of 79 kbp14, 15 or SNP array experiments were used to

assess whole genome copy number of the tumors (see webappendix for details).

6

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

Immunohistochemistry

Paraffin-embedded sections were stained for pRB-N-terminus, 1:200 (BD Pharmingen); pRB-C-

terminus, 1:200, and MYCN, 1:100 (both Santa Cruz).9, 16 Human pRB-N-terminus

immunoreactivity was detected using Immunopure DAB substrate kit (Pierce); human pRB-C-

terminus and N-Myc immunoreactivity using Alexa™ 488 Streptavidin conjugate from Molecular

Probes. DAPI was used to visualize nuclei. Slides were mounted using the DAKO-Cytomation

Fluorescent Mounting Medium and visualized using a Leica DMLB microscope.

Role of the funding sources

NCI-NIH; Canadian Retinoblastoma Society; Hyland Foundation; Ontario Ministry of Health and

Long Term Care; Toronto Netralya and Doctors Lions Clubs; and the Foundation KiKa

Results

To survey a patient cohort with high power, we formed an international collaboration of five RB1

clinical testing laboratories. We defined RB1+/+ disease as non-familial unilateral retinoblastoma

with no detectable tumor RB1 mutations, no promoter methylation, and no loss of heterozygosity at

the RB1 locus. Our routine assays set the clinical standard of 95% sensitivity to detect RB1

mutations,6, 12, 17 giving a 0·25% chance that two independent RB1 mutant alleles in a tumor are

undetected. In total, 1054 unilateral non-familial retinoblastomas were analysed and 28 RB1+/+

tumors (2·7%) were observed, about 10-fold more than expected by chance (p = 6 x 10-45) (table 1).

The Dutch cohort served partly as independent validation using complementary DNA diagnostics

and other analyses. Subsequent to the initial survey, we found two additional RB1+/+ retinoblastoma

tumors that were included in further analyses.

Quantitative multiplex PCR (QM-PCR) or Multiplex Ligation-dependent Probe Amplification

(MLPA) were used to characterize gene copy number (figure 1). Intriguingly, MYCN copy number

7

was dramatically elevated in RB1+/+ relative to RB1-/- tumors (p = 3·4 x 10-4) (two-tailed t-test with

Welch’s adjustment for heteroscedasticity). Of the 30 RB1+/+ tumors, 16 (53%) showed 28 to 121

copies of the MYCN locus (RB1+/+MYCNA); three showed the normal two copies and 11 showed

gain (1·7 to 9 copies) of MYCN (figure 2, table 1). Two copies of MYCN were present in blood of

the ten children with RB1+/+MYCNA tumors for whom blood DNA was available.

We measured copy-number of four other genes characteristic of RB1-/- retinoblastoma8 in 30 RB1+/+

and 96 RB1-/- non-familial unilateral tumors (figure 1, tables S1, S2). The 16 RB1+/+MYCNA tumors

showed reduced frequency of these specific genomic copy-number changes, compared to RB1-/-

tumors: gain of oncogenes KIF14 (19% vs. 62%; p = 0·002), DEK and E2F3 (6% vs. 57%; p =

0·0002) and loss of tumor suppressor gene CDH11 (13% vs. 56%; p = 0·002).

These genomic changes were validated by aCGH14, 18: 94% of the QM-PCR-detected gene changes

were confirmed by aCGH, indicating excellent technical reproducibility ( Table 1). Analysis of

aCGH (figure 2A) confirmed a reduced frequency in RB1+/+MYCNA tumors of the specific genomic

copy number changes characteristic of RB1-/- tumors (gain at chromosomes 1q, 6p, and 2p; and loss

of chromosomes 13q and 16q), (reviewed in Corson, et al.8). The RB1+/+MYCNA tumors also showed

overall significantly fewer altered bp and aCGH clones than the RB1-/- tumors (p = 0·033; t-test with

Welch’s adjustment) (figure 2C, D).

As known for MYCN amplification in neuroblastoma and other tumors, the amplicons in the 14

RB1+/+MYCNA tumors tested by aCGH or SNP array, as well as one RB1+/- tumor (T33) with 73

copies of MYCN and one RB1-/- primary tumor (RB381) with 9·2 copies of MYCN, were narrow,

spanning 1·1 to 6·3 Mbp encompassing MYCN (figure 3A, B, and figure S1 and Tables S1, S3 in

webappendix). Importantly, the sole copy number change detected in one RB1+/+MYCNA tumor (E4)

was 48 copies of the 2p24.2-24.3 region encompassing MYCN. The minimal common amplicon

defined by primary RB1+/+(-)MYCNA tumors P2 and T33 spanned 513 kbp (15,919,161-16,432,161)

8

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

containing only the MYCN gene (15,998,134-16,004,580) (figure 2B). In contrast, no RB1+/+ or

RB1-/- unilateral tumor showed MYCN amplification: 24/36 unilateral tumors had normal MYCN

copy-number and 12/36 had moderate gain spanning a broad region of at least 28 Mbp of 2p, too

large to qualify to be considered amplification.

Three of 14 (23%) RB1+/+MYCNA samples analyzed by aCGH or SNP array showed 17q21.3-qter or

17q24.3-qter gain; two RB1+/+MYCNA samples showed 11q loss. Both these regions are commonly

altered in high-risk neuroblastoma,19-22 but are rare in RB1-/- retinoblastoma (present and published

data10, 23). Other changes in RB1+/+MYCNA tumors not often seen in RB1-/- retinoblastoma were gains

at 14q, gains at 18q, and losses at 11p (figure 2A, and Fig S2 in webappendix).

The RB1 mutant allele in the RB1+/- tumor, T33, with high level MYCNA, showed deletion of most of

13q (unlike the usual RB1-/- mutant alleles), and shared numerous characteristics of RB1+/+MYCNA

tumors (figures 2A, 3A, B, and figures S1 and S3 in webappendix). We conclude that the loss of

one RB1 allele from T33 may be a late event, not initiating.

RB1+/+ tumors expressed full-length pRB, supporting the conclusion that they have normal RB1

genes. Primary RB1+/+ tumors and expected cell types in normal adjacent retina24 stained positively

for both N-terminal and C-terminal epitopes of the RB1 protein (pRB), while RB1-/- tumors failed to

stain for pRB (figure 1A). The three RB1+/+ primary tumors from which mRNA was available,

expressed full-length 2·8 kbp RB1 transcripts at levels comparable to fetal retina, using end-point

and real-time reverse-transcriptase PCR (RT-PCR) (figure 3B, D, and Table S4 in webappendix). In

contrast, RB1-/- tumors and cell lines with RB1 null mutations expressed extremely low levels of

RB1 transcripts (figure 3D, Table S4 in webappendix).

RB1+/+MYCNA primary tumors (but not adjacent retina) (figure 3A) and three cell lines derived from

primary RB1+/+MYCNA tumors (RB522, T101, A3) stained strongly for N-Myc protein (data not

shown). RB1+/+MYCNA tumors showed 3 to 9-fold increased MYCN transcripts compared to fetal

9

retina (figure 3B, D, Table 4 in the appendix). MYCN and MKI67 transcripts (encoding Ki-67,

indicative of proliferation) were detected in fetal retina, RB1-/- tumors as expected for embryonal

neuronal tumors25 and primary RB1+/+MYCNA retinoblastoma, but were at very low levels in adult

retina (figure 3B).

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

Neural progenitor cell marker CRX26, 27 and cone cell marker X-arrestin28, 29 mRNA was expressed

in fetal retina, human adult retina, three RB1+/+MYCNA primary tumors, and four RB1-/- tumors with

genomic gain or normal copies of MYCN (figure 3C). Several other markers indicated differences

between RB1-/- and RB1+/+MYCNA tumors, consistent with the gene copy number studies.

RB1+/+MYCNA tumors showed markedly reduced expression of KIF14 in contrast to normal fetal

retina and the high KIF14 expression in RB1-/- primary retinoblastoma tumors and cell lines (figure

3D, and Table S4 in webappendix).

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

tumors (figure 4A, B). The median age at diagnosis of 17 children (16 with RB1+/+MYCNA tumors,

one with RB1+/-MYCNA, T33) 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) tumors (figure 4A, Table

S1A in webappendix). These data predict that 22% of children presenting with non-familial,

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

webappendix page #).

Analysis of age at diagnosis vs proportion of children not yet diagnosed led Knudson to propose

that two-hits initiate retinoblastoma1; these hits were later shown to be loss of both RB1 alleles.2, 30, 31

Our similar analysis of 79 patients with unilateral RB1-/- tumors (age range 1.9 to 109 months) fit a

two hit model only when we excluded children diagnosed over 60 months (the oldest children

included in Knudson’s original analysis were 60 months) and as expected, did not fit the one-hit

10

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

model describing heritable retinoblastoma. For children with RB1+/+MYCNA tumors, age at

diagnosis vs proportion not yet diagnosed fit both one-hit and two-hit models poorly (See

webappendix page #).

On clinical examination, the RB1+/+MYCNA tumors were indistinguishable from unilateral RB1-/-

retinoblastoma (figure 4C). However, on histological examination, RB1+/+MYCNA tumors contained

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

calcification, common in other MYCNA embryonic tumors, such as neuroblastoma32 (figure 4D, and

figure S3 in webappendix). They were distinctly different from the prototype RB1-/- retinoblastoma,

missing the typical Flexner-Wintersteiner rosettes33 (figure 4E).

Three RB1+/+MYCNA primary tumors (RB522, T101, and A3) extracted from enucleated eyes and

placed in tissue culture, grew rapidly into cell lines, unlike the usual RB1-/- retinoblastoma that is

very hard to grow in tissue culture. One RB1+/+MYCNA tumor had invaded the optic nerve past the

cribriform plate when diagnosed at age 11 months, a feature of aggressive tumors (figure 4F). None

of the patients had any abnormalities apart from the unilateral tumors, and none has developed

tumors in his/her other eye.

11

Panel: Research in Context

Systematic Review

Knudson 2hit; age at dx non-familial

Studies showing 95% of tumors RB1-/-

MYCNA in neuroblastoma, prognosis, stable genome and yet poor prognosis

Our definition of RB1+/+ tumors

Discovery of RB1+/+MYCNA

Interpretation

Defined novel type of retinoblastoma previously unrecognized

Young babies, aggressive disease, dx on removal of eye

If unrecognized, incorrect assumption of hereditable disease because of young age at dx

An attempt to salvage eye with RB1+/+MYCNA disease could incur high treatment morbidity and risk

of failure for aggressive oncogene-driven tumor.

12

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

Discussion

Knudson’s analysis of retinoblastoma established the basis to understand how normal genes

suppress cancer,1 leading to the identification of the RB1 tumor suppressor gene named for the

disease (RB1),2 widely assumed to initiate all retinoblastoma. We now show a previously

unrecognized type of retinoblastoma with no detectable RB1 mutations (RB1+/+) and no loss of

heterozygosity at RB1, but instead high-level focal amplification of the MYCN oncogene, aggressive

behaviour, and very young age of onset. This discovery has immediate diagnostic, genetic

counselling, and therapeutic implications.

Our large-scale international study of 1,054 unilateral retinoblastomas allowed recognition of this

distinct RB1+/+MYCNA subtype comprising 1-2% of unilateral, non-familial retinoblastomas. At

least two previously described tumors may be also examples of RB1+/+MYCNA.34 However, RB1

genetic status was not defined. Notably, our proposal of a discrete retinoblastoma subtype was

validated through independent observations in different patient cohorts using different technologies.

Our paper also highlights the importance of molecular diagnoses as a means to identify novel

malignancies that previously eluded histopathological recognition. Although pathologists have not

previously recognized RB1+/+MYCNA retinoblastoma as distinct, they histologically resemble large

nucleolar neuroblastomas, with MYCNA and very poor outcome.32

Like neuroblastomas with MYCNA,21 the RB1+/+MYCNA retinoblastoma showed overall less complex

patterns of genomic copy-number alterations than those without MYCN amplification, suggesting

that MYCNA may be the critical driver of malignancy. Genomic instability for specific DNA copy

changes has been associated with RB1 loss in both retinoblastoma and its benign precursor,

retinoma.9 On the other hand, point mutations (other than in the RB1 gene) are few in RB1-/-

13

retinoblastoma, shown recently by whole genome sequencing of four RB1-/- retinoblastomas.35 RB1

loss has been shown to induce mitotic changes and lagging chromosomes.36 This is consistent with

our data: the normal RB1 protein may support a stable genomic copy-number in the RB1+/+MYCNA

tumors despite the high level MYCNA.

c. Any data that could be added to show that Rb is functional in these tumors

(phosphorylation status of Rb or activation of E2F target genes) would be very supportive of the

overall conclusions of this manuscript. This is important in order to understand if Rb is functionally

inactivated in this rare subset of tumors via defects in cyclins, cyclin dependant kinases, or

perhaps even via downstream effects of MYCN.

2. The manuscript appropriately focuses on the subset of Rb +/+ tumors with MYCN

amplification, but also needs to address what might be the oncogenic driver in the Rb +/+ tumors

without MYCN amplification.

Are RB1+/+MYCNA tumors truly retinoblastoma? Strictly speaking, the word retinoblastoma defines

‘a blast cell tumor arising from the retina’. RB1+/+MYCNA tumors fit this basic definition, arising in

developing retina and expressing markers of embryonic retina26-29 not expressed in neuroblastoma.37

The very young age of diagnosis of RB1+/+MYCNA tumors combined with expression of early retinal

and neural progenitor-like and stem cell markers suggests that MYCN amplification occurs in a

somatic cell during fetal retinal development. Whether amplification of MYCN suffices to initiate

retinoblastoma remains to be formally demonstrated; this proof is expected to be difficult since

RB1+/+MYCNA oncogenesis may start in mid-gestation, much earlier than even hereditary RB1-/-

tumors.

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

in the tumor, and examination of blood to determine if the child carries either mutant allele in the

germline. Our study suggests that when no RB1 mutation is detected, testing for MYCN copy

number, especially when the patient is <12 months of age would be important in on-going care. The

14

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

diagnosis of RB1+/+MYCNA retinoblastoma strongly suggests non-hereditary disease with normal

population risks for retinoblastoma in the other eye and other cancers later in life. Delaying

enucleation of the unilaterally affected eye (Combined with a widespread trend to attempt to save

eyes with retinoblastoma, young age at diagnosis of unilateral retinoblastoma is frequently assumed

to suggest heritable retinoblastoma. However, attempts to salvage an eye with RB1+/+MYCNA

retinoblastoma could be very dangerous and ineffective.

The very early presentation of RB1+/+MYCNA tumors, the lack of RB1 mutations, and the high

MYCN amplification in an otherwise copy-number stable genome suggests that these tumors may

arise by one hit: somatic MYCN amplification in a retinal progenitor cell. Because RB1+/+MYCNA

tumors diagnosed very soon after birth are already large, we suggest that they initiate a longer time

before birth that RB1-/- tumors, so that Knudson’s analysis using birth as a surrogate for tumor

initiation is not applicable, confounding precise determination of how many hits initiate

RB1+/+MYCNA tumors (See webappendix page 3). Aggressive growth of RB1+/+MYCNA tumors may

also contribute to the very early diagnosis, and further confound the analysis.

The unilaterally affected patients with RB1+/+MYCNA tumors in this study were cured by removal of

their affected eye with no adverse outcomes. One RB1+/+MYCNA tumor showed significant optic

nerve invasion, a predictor of high mortality through tumor invasion into brain.38 Of note, more

successful establishment of three RB1+/+MYCNA cell lines may relate to aggressive properties (data

not shown). A higher proportion of extraocular retinoblastoma in developing countries, where

diagnosis is frequently delayed, may be RB1+/+MYCNA tumors.39

The relative genomic stability of RB1+/+MYCNA tumors suggests that anti-Myc therapeutic agents40-

42 may not evoke the resistance to therapy acquired by genomic rearrangements. It has been shown

that RB1-/- retinoblastoma cell lines are sensitive to MYCN knock-down,43 an effect that may be

more significant in RB1+/+MYCNA retinoblastoma.

15

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

mutations. Although a one-hit onset is not formally proven (and may not be provable in humans),

RB1+/+MYCNA retinoblastoma provides an intriguing counterpart to classical retinoblastoma of

immediate importance to ocular oncologists and their patients.

16

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

Supported by Grants from the Canadian Retinoblastoma Society, the NCI-NIH grant 5R01CA118830-04, the

annual Keene Plant Sale, Toronto Netralya Lions Club and Doctors Lions Club and the Ontario Ministry of

Health and Long Term Care (OMOHLTC). The views expressed do not necessarily reflect those of the

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

P.C.B. through funding provided by the Government of Ontario. S.Y. was funded by the Vision Science

Research Program of the University Health Network and the University of Toronto. R.P. was funded in part

by a Great West Life Studentship from Queen’s University School of Medicine. B.M.M. was funded by a

grant from CCA/V-ICI/ Avanti-STR (J.C.D., J. Cloos and A.C.M.), the Dutch research was also funded in

part by KIKA (J.C.D., H. te Riele, J. Cloos, A.C.M.). The funders had no role in study design, data collection

and analysis, decision to publish, or preparation of the manuscript. 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 Dr. Rod Bremner and Dr. Marek Pacal of the Toronto

Western Research Institute for providing antibodies for immunocytochemistry. 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.

17

TABLES

Table 1 | Frequency of RB1+/+ unilateral retinoblastoma at five international sites.

Test Site Total RB1+/+Proportion

RB1+/+P-value*

Number

RB1+/+MYCNA

Proportion of RB1+/+

with MYCNA

Canada 441 7 1·6% 0·09 5 71%

Germany 400 11 2·8% >0·99 3 27%

France 150 5 3·3% 0·77 2 40%

New Zealand 30 2 6·7% 0·39 1 50%

The Netherlands 33 3 9·1% 0·057 3 100%

Total 1054 28 2·7% 14 50%

*Pair-wise proportion test: frequency of RB1+/+ tumors in each site compared to all other sites.

18

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

FIGURE LEGENDS

Figure 1. RB1+/+MYCNA tumours express pRB and MYCN.

(A) RB1+/+MYCNA retinoblastoma stained positive for pRB (C-terminus and N-terminus antibodies)

and MYCN protein; controls, normal retina and adjacent normal retina (*). RB-/- tumor is negative.

(B) In primary RB1+/+MYCNA retinoblastomas full-length RB1, MYCN and Ki67 transcripts are

shown by end point RT-PCR; Ki67 mRNA indicated proliferation; TBP endogenous control. (C)

Expression of retinal progenitor cell marker CRX and cone cell maker X-arrestin in human fetal

retina, human adult retina, primary RB1+/+MYCNA tumors, and primary RB1-/- tumors with genomic

gain or two MYCN copies; end point RT-PCR of total RNA; positive control, TBP. (D) RB1, MYCN

and KIF14 mRNA expression in human fetal (FR) and (HR) adult retina, RB1+/+ MYCNA tumors

(red) and RB1-/- (brown) or RB+/- (green) primary tumors 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 del exons 2 to 6 that results in increased expression of

shortened, non-functional RB1 mRNA and protein.

Figure 2: Copy Numbers of the 5-Gene Signature of Retinoblastoma.

(A) Box-plot of genomic copy numbers of indicated genes in unilateral retinoblastoma categorized

by defined RB1 gene status; 50% of the RB1+/+ tumors show high level amplification of MYCN

(RB1+/+MYCNA) and few copy number changes in the other genes usually gained or lost in RB1-/-

tumors; tumor T33 is an outlier of the RB1+/- group, showing an RB1+/+MYCNA-like profile; gray

line, 2-copies; on each boxplot, the 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. (B) Copy number heat-map for the profile genes (red, increased and blue,

decreased copy number; gray, 2 copies; white, not tested; n, number in each group, including five

more recently discovered RB1+/+MYCNA tumors (black triangles) not included in (A). (C) MYCN

19

copy number assessment in 30 RB1-/- and three RB1+/+ tumors studied by MLPA, showing high

MYCN copy number in the RB1+/+ tumors.

Figure 3: Fewer genomic copy number alterations in RB1+/+MYCNA than RB1-/- tumors

(A) aCGH on 12 RB1+/+MYCNA (including T33), 12 RB1+/+, 13 RB1+/-, and 11 RB1-/- tumors; gains,

right; losses, left; minimal commonly gained/lost regions in RB1-/- tumours boxed; *normally

occurring copy number variations. The RB1+/- MYCNA tumor 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

tumors (pink band); MYCN copy number by QM-PCR, red italics; aCGH individual probes, green

bars. (C) Boxplot of bp altered shows fewer changes in RB1+/+MYCNA than RB1-/- tumors (p =

0·033; t-test with Welch’s adjustment); vertical line marks the maximum and minimum copy-

numbers observed, box bounds second 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-/-

tumors; each tumor class has more unique clones altered than in common.

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

(A) Children with RB1+/+MYCNA tumours 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 uses birth as a surrogate for initiation, a good assumption for RB1-/-

disease, but a poor assumption for RB1+/+MYCNA disease; best fitting curves modeling the

relationship between age at diagnosis and the fraction of cases not yet diagnosed, based on

maximum likelihood models assuming a Weibull distribution under assumption that ages at

diagnosis have Weibull distribution with shape parameter = 1 (red lines) (exponential distribution)

Weibull distribution with shape parameter = 2 (blue lines); actual data points (proportion of ages

greater than a particular age) are shown. (C) Fundus image of an RB1+/+MYCNA unilateral tumour in

a four month-old child shows characteristic calcification on ultrasound. (D) Round nuclei with large

20

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

prominent multiple nucleoli shown on primary pathology, in comparison to (E) RB1-/- tumour

showing classic retinoblastoma pathology: Flexner-Wintersteiner rosettes and nuclear molding;

hematoxylin-eosin staining. (E) RB1+/+MYCNA tumor in an 11 month-old child shows extraocular

extension into the optic nerve (white arrows) (2·5x, hematoxylin-eosin staining).

21

REFERENCES

REF count 43/40

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 (New York, NY. 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. Richter S, Vandezande K, Chen N, Zhang K, Sutherland J, Anderson J, et al. Sensitive and

efficient detection of RB1 gene mutations enhances care for families with retinoblastoma. American

journal of human genetics. 2003; 72(2): 253-69.

6. Rushlow D, Piovesan B, Zhang K, Prigoda-Lee NL, Marchong MN, Clark RD, et al.

Detection of mosaic RB1 mutations in families with retinoblastoma. Human mutation. 2009; 30(5):

842-51.

7. Bowles E, Corson TW, Bayani J, Squire JA, Wong N, Lai PB, et al. Profiling genomic copy

number changes in retinoblastoma beyond loss of RB1. Genes Chromosomes Cancer. 2007; 46(2):

118-29.

8. 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.

22

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

9. 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. Hum Mol Genet. 2008; 17(10): 1363-72.

10. 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.

11. Amenduni M, Livide G, Ariani F, Renieri A. Retinoma and Retinoblastoma: Genomic

Hybridisation. Tumors of the Central Nervous System DOI: 101007/978-94-007-4213-0_10; 2012.

p. 93-102.

12. Lohmann D, Gallie B, Dommering C, Gauthier-Villars M. Clinical utility gene card for:

Retinoblastoma. European journal of human genetics : EJHG. 2011; 19(3).

13. Zeschnigk M, Lohmann D, Horsthemke B. A PCR test for the detection of hypermethylated

alleles at the retinoblastoma locus [letter]. Journal of medical genetics. 1999; 36(10): 793-4.

14. 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.

15. Watson SK, deLeeuw RJ, Horsman DE, Squire JA, Lam WL. Cytogenetically balanced

translocations are associated with focal copy number alterations. Human genetics. 2007; 120(6):

795-805.

16. Marchong MN, Chen D, Corson TW, Lee C, Harmandayan M, Bowles E, et al. Minimal 16q

genomic loss implicates cadherin-11 in retinoblastoma. Mol Cancer Res. 2004; 2(9): 495-503.

17. 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. Human mutation. 2004; 23(2): 193-202.

23

18. Watson SK, deLeeuw RJ, Ishkanian AS, Malloff CA, Lam WL. Methods for high

throughput validation of amplified fragment pools of BAC DNA for constructing high resolution

CGH arrays. BMC Genomics. 2004; 5(1): 6.

19. 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.

20. Bown N, Lastowska M, Cotterill S, O'Neill S, Ellershaw C, Roberts P, et al. 17q gain in

neuroblastoma predicts adverse clinical outcome. U.K. Cancer Cytogenetics Group and the U.K.

Children's Cancer Study Group. Medical and pediatric oncology. 2001; 36(1): 14-9.

21. 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.

22. Attiyeh EF, London WB, Mosse YP, Wang Q, Winter C, Khazi D, et al. Chromosome 1p

and 11q deletions and outcome in neuroblastoma. The New England journal of medicine. 2005;

353(21): 2243-53.

23. 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.

24. 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.

25. Squire J, Goddard AD, Canton M, Becker A, Phillips RA, Gallie BL. Tumour induction by

the retinoblastoma mutation is independent of N-myc expression. Nature. 1986; 322(6079): 555-7.

24

MYCN ONCOGENE-INITIATED RETINOBLASTOMA

26. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, et al. Identification of

human brain tumour initiating cells. Nature. 2004; 432(7015): 396-401.

27. Seigel GM, Hackam AS, Ganguly A, Mandell LM, Gonzalez-Fernandez F. Human

embryonic and neuronal stem cell markers in retinoblastoma. Molecular vision. 2007; 13: 823-32.

28. Craft CM, Whitmore DH, Wiechmann AF. Cone arrestin identified by targeting expression

of a functional family. J Biol Chem. 1994; 269(6): 4613-9.

29. Murakami A, Yajima T, Sakuma H, McLaren MJ, Inana G. X-arrestin: a new retinal arrestin

mapping to the X chromosome. FEBS letters. 1993; 334(2): 203-9.

30. Godbout R, Dryja TP, Squire J, Gallie BL, Phillips RA. Somatic inactivation of genes on

chromosome 13 is a common event in retinoblastoma. Nature. 1983; 304(5925): 451-3.

31. Cavenee WK, Dryja TP, Phillips RA, Benedict WF, Godbout R, Gallie BL, et al. Expression

of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature. 1983; 305(5937): 779-

84.

32. Tornoczky T, Semjen D, Shimada H, Ambros IM. Pathology of peripheral neuroblastic

tumors: significance of prominent nucleoli in undifferentiated/poorly differentiated neuroblastoma.

Pathol Oncol Res. 2007; 13(4): 269-75.

33. Flexner S. A peculiar glioma (neuroepithelioma?) of the retina. Johns Hopkins Hosp Bull.

1891; 2: 115.

34. 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. British journal of cancer. 2002; 87(7): 779-82.

35. 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.

25

36. Manning AL, Dyson NJ. RB: mitotic implications of a tumour suppressor. Nat Rev Cancer.

2012; 12(3): 220-6.

37. Kobayashi M, Takezawa S, Hara K, Yu RT, Umesono Y, Agata K, et al. Identification of a

photoreceptor cell-specific nuclear receptor. Proc Natl Acad Sci U S A. 1999; 96(9): 4814-9.

38. 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.

39. Dimaras H, Kimani K, Dimba EAO, Gronsdahl P, White A, L. CHS, et al. Retinoblastoma.

The Lancet. 2012; In press.

40. Chanthery YH, Gustafson WC, Itsara M, Persson A, Hackett CS, Grimmer M, et al.

Paracrine signaling through MYCN enhances tumor-vascular interactions in neuroblastoma. Sci

Transl Med. 2012; 4(115): 115ra3.

41. Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele DA, et al. Targeting

MYC dependence in cancer by inhibiting BET bromodomains. Proceedings of the National

Academy of Sciences of the United States of America. 2011; 108(40): 16669-74.

42. Soucek L, Whitfield J, Martins CP, Finch AJ, Murphy DJ, Sodir NM, et al. Modelling Myc

inhibition as a cancer therapy. Nature. 2008; 455(7213): 679-83.

43. Xu XL, Fang Y, Lee TC, Forrest D, Gregory-Evans C, Almeida D, et al. Retinoblastoma has

properties of a cone precursor tumor and depends upon cone-specific MDM2 signaling. Cell. 2009;

137(6): 1018-31.

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