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, 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); Service de Génétique Oncologique,
Institut Curie and Université Paris Descartes Paris, France (C Houdayer, PhD); Department of
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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,
2
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
Word count 300/300
Summary
Background Retinoblastoma is the childhood retinal cancer that defined tumour suppressor genes.
By analysing age of diagnosis, Knudson proposed that two “hits” initiate retinoblastoma, later
attributed to mutation of both alleles of the retinoblastoma suppressor gene, RB1, in tumours.
Persons with hereditary retinoblastoma carry a heterozygous constitutional RB1 mutation; one
additional hit initiates retinoblastoma or other cancers. Non-hereditary retinoblastoma arises when
both RB1 alleles are damaged in developing retina (RB1-/-).
Methods Our international collaboration determined the proportion of 106854 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% (298/106854) of unilateral non-
familial retinoblastomas. Surprisingly, half of these had high-level MYCN oncogene amplification
(>10 copies), while no RB1-/- tumours showed MYCN amplification. RB1+/+MYCNA tumours had
fewer overall genomic copy-number changes and distinct, aggressive histology. Amplification of
the MYCN-encompassing region was the only change on array comparative genomic hybridization
in one RB1+/+MYCNA tumour. 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 2218·4%
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, these children and their families
are believed at population risk to develop other cancers. Since these aggressive tumours may
3
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
rapidly 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.
4
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
Word count 3061?/3000
Background
Retinoblastoma set the paradigm for tumour suppressor genes, with Knudson’s classic hypothesis
predicting that two rate-limiting hits initiate this childhood eye cancer.1 The two hits were later
attributed to the retinoblastoma gene (RB1).2 Approximately 40% of retinoblastoma is bilateral. 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/ (~90% bilateral) 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 lossboth 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.
The undetected RB1 mutations in blood likely include low-level mosaicism,5 translocations, or deep
intronic mutations. 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, fewer 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.
5
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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 for
de-identified use of archival tissue and data, the Toronto and Essen patients provided additional
informed consent for use of de-identified samples in research.
Mutation analyses
Standard of care analyses that identify 95% of RB1 (Gen bank accession #L11910) mutant alleles5-7
were applied to tumours at each collaborating site, including DNA sequencing, quantitative
multiplex PCR (QM-PCR) or Multiplex Ligation-Dependent Probe Amplification (MLPA), 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) (table S1, figure S1),
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.
6
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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.
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 (tables S2, S3).
Age of Diagnosis analysis
Ages at diagnosis vs. proportion not yet diagnosed were analysed by Weibull distributions and least
squares methodology to assess the minimal number of events for tumour initiation. Likelihood of
children having RB1+/+MYCNA tumours at different ages of diagnosis was estimated (table S4).
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.
7
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
Results
The Toronto lab identifies both tumour mutations (or promoter methylation) in >95% of tumours
(as of October 10, 2012, 616 of 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 is believed to account for many of the 5% "no mutation
found" blood samples from bilaterally affected patients.5 However, RB1 sequence, sensitive allele-
specific PCR screens, and microsatellite analysis make it clear that any level of mutational
mosaicism or significant normal cell contamination in retinoblastoma tumours is rare, and if noted,
is usually associated with extremely small tumours, or chemotherapy prior to enucleation. The
undetected RB1 mutations in fully tested tumours (RB1+/+ ) were believed to include translocations,
deep intronic mutations, or mutations in unknown RB1 regulatory regions.
In our clinical work in Toronto, we (DER, BLG) had found seven unilateral retinoblastoma samples
with no RB1 mutations and no loss of heterozygosity (LOH) at RB1. We investigated these tumours
further, using QM-PCR to measure copy-numbers of representative genes at 6p, 1q, 16q and 2p that
are characteristically gained or lost in retinoblastoma tumours. To our surprise, 5/7 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, no RB1 promoter methylation, and no LOH at
RB1 (Table 1). The proportions of RB1+/+ and RB1+/+ MYCNA tumours from each of the four centres
was similar. After our analysis was complete, we discovered that the Amsterdam lab had
independently characterized three RB1+/+MYCNA tumours. We report the combined data after
statistical analysis showing that the frequency was not different (p = 0.08) among the five clinical
labs. An additional Toronto RB1+/+MYCNA tumour (T101) found later is included in some analyses.
8
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
Our clinical assays establish a standard of 95% sensitivity to find an RB1 mutation in samples
expected to carry an RB1 mutation.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%), about 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 the copy numbers of known 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) (two-tailed t-test with
Welch’s adjustment for heteroscedasticity) (table S5). Most significantly, MYCN copy-number in
the 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 blood was available and 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 (Toronto) and 3 by SNP
(Amsterdam) analysis (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
9
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
characteristic of RB1-/-retinoblastomas (table S6).9 The RB1+/+MYCNA retinoblastomas also showed
overall significantly fewer altered bp and aCGH clones than the RB1-/- retinoblastomas (p = 0·033)
(figure 2C, D, table S7).
The amplicons of the 14 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 36 tumours tested by aCGH, 24 unilateral tumours showed no gain or loss at MYCN, and
12 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 are 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 also 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 cell types in normal adjacent retina17 stained for both C-terminal (figure 3A) and N-terminal
(data not shown) epitopes of the RB1 protein (pRB), while RB1-/-(+) tumours to stained weakly for
10
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
pRB, depending on the RB1 mutations. Western blot on cell line A3, derived from RB1+/+MYCNA
primary retinoblastoma, showed both phosphorylated and un-phosphorylated, full-length pRB18
(figure 3B). Three RB1+/+MYCNA primary retinoblastomas for which mRNA was available,
expressed full-length 2·8 kbp RB1 transcripts at levels comparable to fetal retina, using end-point
and real-time RT-PCR (figure 3C, 3E, table S8). In contrast, RB1-/- retinoblastomas expressed low
levels of RB1 transcript.
RB1+/+MYCNA primary retinoblastomas (but not adjacent retina) (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 detected
in fetal retina, primary RB1+/+MYCNA retinoblastoma, and RB1-/- retinoblastomas (as expected for
embryonal neuronal tumours), but were at very low levels in adult retina (figure 3C). RB1+/+MYCNA
tumours showed reduced expression of the oncogene KIF14,9 in contrast to normal fetal retina, and
to the high KIF14 expression in RB1-/- primary retinoblastomas and cell lines (figure 3E, table S8).
RB1+/+MYCNA tumors expressed embryonic retinal cell markers consistent with a retinal origin. The
mRNAs of cone cell marker X-arrestin19 and CRX, a marker of retinal and pineal lineage tumours
strongly expressed in RB1-/- retinoblastoma, but not in neuroblastoma,20 were expressed in fetal
retina, human adult retina, and three RB1+/+MYCNA and four RB1-/- primary retinoblastomas (figure
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 S4, S5). Our data predict that 18.4% of children diagnosed with non-familial,
11
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
unilateral retinoblastoma at age six months or younger will have RB1+/+MYCNA retinoblastoma
(table S4).
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 is consistent with
Knudson’s model and fits a two-hit curve, representative of two independent mutation events in a
tumour suppressor gene, not a one-hit curve (figure 4B). Similar analysis of age at diagnosis vs.
proportion not yet diagnosed for RB1+/+MYCNA tumours was inconclusive: while the data points for
twelve children less than 10 months old approximate the calculated one-hit curve, the age of
diagnosis for the older children deviate better align with the two-hit curve (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, S4). They did not show the
Flexner-Wintersteiner rosettes22 and nuclear molding of prototypic RB1-/- retinoblastoma (figure
4D).
Clinically, the RB1+/+MYCNA retinoblastomas were large and invasive, considering the very young
age of these children (figure 4C, S5). 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 (figure 4E). However, all the
children in this study with RB1+/+MYCNA tumors were cured by removal of their affected eye with
no adverse outcomes; none developed retinoblastomas in the other eye.
Discussion
Knudson’s analysis of retinoblastoma established the basis to understand how normal genes
suppress cancer,1 leading to the identification of the RB1 tumour suppressor gene,2 widely assumed
12
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
to initiate all retinoblastoma. We now show a previously unrecognized type of retinoblastoma with
no detectable RB1 mutations (RB1+/+) and no LOH at RB1, but instead high-level, focal
amplification of the MYCN oncogene, aggressive behaviour, and very young age of diagnosis.
Our international study of 1,068 unilateral retinoblastomas allowed recognition of a distinct
RB1+/+MYCNA subtype comprising 1·4% of unilateral, non-familial retinoblastomas. At least two
previously described tumours may be also examples of RB1+/+MYCNA retinoblastomas,23 although
RB1 genetic status was not defined. Despite the low incidence of RB1+/+MYCNA retinoblastoma,
RB1+/+MYCNA tumors 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
tumor tissues for gene expression studies, aCGH showed genomic gains and losses distinct from
those in either RB1-/- or RB1+/+MYCNA retinoblastomas. In particular, these RB1+/+retinoblastomas
showed increased incidence of gain on 19p and q, 17p and q, 2p, and at the telomeric end of 9q. The
RB1+/+ group is expected to be heterogeneous and merits further study.
Our paper highlights the use of molecular diagnoses to identify novel malignancies that previously
eluded histopathological recognition. Although pathologists have not previously recognized
RB1+/+MYCNA retinoblastoma as distinct, these tumours resemble large nucleolar neuroblastomas
with MYCN-amplification and poor outcome.21 Like neuroblastomas with MYCN-amplification,14
the RB1+/+MYCNA tumors showed less complex patterns of 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 MYCNA tumours may also allow less time for
genomic alterations to accumulate. Although whole genome sequencing has recently suggested that
point mutations (other than in the RB1 gene) are few in RB1-/-retinoblastoma,24 loss of RB1 has been
shown to induce mitotic changes and lagging chromosomes,25 leading to genomic instability. The
RB1 loss in retinoblastoma and pre-malignant retinoma8,16 is associated with specific commonly
13
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
DNA copy-number changes, that are less frequent in RB1+/+MYCNA retinoblastomas with intact RB1
genes.
Are RB1+/+MYCNA tumours truly retinoblastoma? RB1+/+MYCNA retinoblastomas fit the basic
definition of retinoblastoma as ‘a blast cell tumour arising from the retina’, likely arise in
developing retina and express markers of embryonic retina.19,20,26 Our study demonstrated the
presence of intact RB1 genes and pRB in primary RB1+/+MYCNA retinoblastomas, which stained
strongly for N- and C-terminal antibodies to pRb. 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, the lack of RB1 mutations, and the
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 the identification of one RB1+/+MYCNA tumour in which MYCN amplification was the
only genomic copy-number change. The RB1+/+MYCNA retinoblastomas are already large in very
young children, so they likely initiate much earlier in fetal development than RB1-/- retinoblastomas.
When tumours are anticipated because an infant is known to carry the RB1-/- allele of a parent,
tumors are found when very small (figure 4F). How MYCN-amplification is initiated, and whether
MYCN-amplification alone suffices to initiate these retinoblastoma remains to be formally
demonstrated.
It has been shown that RB1-/- retinoblastoma cell lines are sensitive to MYCN knockdown, an effect
that may be more significant in RB1+/+MYCNA retinoblastomas.29 In retinal development, N-myc
protein activates cyclins that inactivate pRB by phosphorylation, presumably also how unregulated
MYCN expression associated with high level amplification promotes cell division.27 The relative
genomic stability of RB1+/+MYCNA retinoblastomas suggests that anti-N-Myc therapeutic agents28
may avoid emergence of drug resistance acquired through progressive genomic rearrangements in
14
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
RB1-/- retinoblastoma. Our preliminary MYCN knock down experiments resulted in rapid death of
RB1+/+MYCNA cell lines. We predict that the children less than one year of age with extra-ocular
retinoblastoma29 around the world, might have RB1+/+MYCNA retinoblastomas and benefit from anti-
MYCN therapy. This idea is consistent with the anecdotal observation during review of 40
retinoblastoma pathology slides in Kenya, of RB1+/+MYCNA histology in an orbital recurrence, later
shown to have 40 copies of MYCN.
Young age at diagnosis of unilateral retinoblastoma is frequently interpreted as an indication of
heritable retinoblastoma, and is often considered 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. One RB1+/+MYCNA retinoblastoma showed early significant optic nerve invasion,
a predictor of high mortality through tumour invasion into brain.30 The patients with RB1+/+MYCNA
tumours in our study had large, aggressive tumors. At similar young ages, the usual hereditary
RB1-/- tumors 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 turn out to be clinically predictable, facilitating prompt
removal of these eyes with good outcomes for the children.
were cured by removal of their affected eye with no adverse outcomes.
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 tumor, especially for
children less than 12 months old, determining MYCN copy-number in tumor assists in 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.
15
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
Our findings challenge the long-standing dogma that all retinoblastomas are initiated by RB1 gene
mutations (figure 4GPanel). 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 suggestion of 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, could may incur high treatment morbidity
and failure to cure these aggressive oncogene-driven retinoblastomas.
16
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
Tables
Table 1. Frequency of RB1+/+ unilateral retinoblastoma at five international diagnostic labs.
TestSite 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%
NewZealand 30 2 6.7% 1 3.3%
TheNetherlands 3347 3 96.14% 3 6.49.1%
Total 105468 29 2.87% 15# 1.41.4%
*Fisher'sexacttestsindicatesthatpercentageofunilateraltumorswithRB1+/+isnotrelatedtosite(p=0.08).
Pair-wiseproportiontest:frequencyofRB1+/+tumoursineachsitecomparedtoallothersites.
**AlargerpercentageofNetherlandspatientswere RB1+/+ comparedtoCanadianpatients(p=0.03).
#OneadditionalsubsequentRB1+/+MYCNA tumor(T101)andoneRB1+/-MYCNA(T33)tumoursweresubsequentlyincludedforatotalof17includedin
someanalyses.s
.
17
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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 tumors, 54 copies; 14
RB1+/+tumors, 3 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.
18
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
19
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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.
20
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
21
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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+/- tumor stained
weakly for N-Myc and pRB. (B) Western blot shows the characteristic two bands of
unphoshorylated and phosphorylated pRB 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 indicated 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 RB+/-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.
22
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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 non-heritable 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 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 4 month-old child with hereditary RB1-/- retinoblastoma, a tiny tumor (obscuring the
choroidal pattern) above the optic disc is revealed in the inner nuclear layer of the retinal on optical
coherent tomography (OCT). (G) Schema of data establishing RB1+/+MYCNA retinoblastoma as a
novel disease; data figures (f) and tables (t) indicated in grey on left.
23
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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. 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 contributed to figure construction and development of concepts.
Josephine C Dorsman coordinated the Amsterdam study, recognized the RB1 and MYCN mutation
24
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
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 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.
25
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
REFERENCES
1. Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Science, USA. 1971;68(4):820-3. 2. Friend SH, Bernards R, Rogelj S, Weinberg RA, Rapaport JM, Albert DM, Dryja TP. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986 Oct 16-22;323(6089):643-6. 3. Cavenee WK, Hansen MF, Nordenskjold M, Kock E, Maumenee I, Squire JA, Phillips RA, Gallie BL. 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 Aug 15;129C(1):23-8. 5. Rushlow D, Piovesan B, Zhang K, Prigoda-Lee NL, Marchong MN, Clark RD, Gallie BL. Detection of mosaic RB1 mutations in families with retinoblastoma. Human mutation. 2009 May;30(5):842-51. 6. Lohmann D, Gallie B, Dommering C, Gauthier-Villars M. Clinical utility gene card for: Retinoblastoma. European journal of human genetics : EJHG. 2011 Mar;19(3). 7. Houdayer C, Gauthier-Villars M, Lauge A, Pages-Berhouet S, Dehainault C, Caux-Moncoutier V, Karczynski P, Tosi M, Doz F, Desjardins L, Couturier J, Stoppa-Lyonnet D. Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF. Human mutation. 2004 Feb;23(2):193-202. 8. Dimaras H, Khetan V, Halliday W, Orlic M, Prigoda NL, Piovesan B, Marrano P, Corson TW, Eagle RC, Jr., Squire JA, Gallie BL. Loss of RB1 induces non-proliferative retinoma: increasing genomic instability correlates with progression to retinoblastoma. Hum Mol Genet. 2008 May 15;17(10):1363-72. 9. Corson TW, Gallie BL. One hit, two hits, three hits, more? Genomic changes in the development of retinoblastoma. Genes Chromosomes Cancer. 2007 Apr 16;46(7):617-34. 10. Ishkanian AS, Malloff CA, Watson SK, DeLeeuw RJ, Chi B, Coe BP, Snijders A, Albertson DG, Pinkel D, Marra MA, Ling V, MacAulay C, Lam WL. A tiling resolution DNA microarray with complete coverage of the human genome. Nat Genet. 2004 Mar;36(3):299-303. 11. Watson SK, deLeeuw RJ, Horsman DE, Squire JA, Lam WL. Cytogenetically balanced translocations are associated with focal copy number alterations. Human genetics. 2007 Feb;120(6):795-805. 12. Myllykangas S, Bohling T, Knuutila S. Specificity, selection and significance of gene amplifications in cancer. Seminars in cancer biology. 2007 Feb;17(1):42-55. 13. O'Neill S, Ekstrom L, Lastowska M, Roberts P, Brodeur GM, Kees UR, Schwab M, Bown N. MYCN amplification and 17q in neuroblastoma: evidence for structural association. Genes Chromosomes Cancer. [Case Reports]. 2001 Jan;30(1):87-90. 14. Mosse YP, Diskin SJ, Wasserman N, Rinaldi K, Attiyeh EF, Cole K, Jagannathan J, Bhambhani K, Winter C, Maris JM. Neuroblastomas have distinct genomic DNA profiles that predict clinical phenotype and regional gene expression. Genes Chromosomes Cancer. 2007 Oct;46(10):936-49. 15. Chen D, Gallie BL, Squire JA. Minimal regions of chromosomal imbalance in retinoblastoma detected by comparative genomic hybridization. Cancer Genet Cytogenet. 2001;129(1):57-63.
26
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
16. Sampieri K, Amenduni M, Papa FT, Katzaki E, Mencarelli MA, Marozza A, Epistolato MC, Toti P, Lazzi S, Bruttini M, De Filippis R, De Francesco S, Longo I, Meloni I, Mari F, Acquaviva A, Hadjistilianou T, Renieri A, Ariani F. Array comparative genomic hybridization in retinoma and retinoblastoma tissues. Cancer Sci. 2009 Mar;100(3):465-71. 17. Spencer C, Pajovic S, Devlin H, Dinh QD, Corson TW, Gallie BL. Distinct patterns of expression of the RB gene family in mouse and human retina. Gene Expr Patterns. 2005 Jun;5(5):687-94. 18. Buchkovich K, Duffy LA, Harlow E. The retinoblastoma protein is phosphorylated during specific phases of the cell cycle. Cell. [Research Support, U.S. Gov't, P.H.S.]. 1989 Sep 22;58(6):1097-105. 19. Murakami A, Yajima T, Sakuma H, McLaren MJ, Inana G. X-arrestin: a new retinal arrestin mapping to the X chromosome. FEBS letters. [Comparative Study Research Support, Non-U.S. Gov't]. 1993 Nov 15;334(2):203-9. 20. Terry J, Calicchio ML, Rodriguez-Galindo C, Perez-Atayde AR. Immunohistochemical Expression of CRX in Extracranial Malignant Small Round Cell Tumors. Am J Surg Pathol. 2012 Aug;36(8):1165-9. 21. 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. 22. Flexner S. A peculiar glioma (neuroepithelioma?) of the retina. Johns Hopkins Hosp Bull. 1891;2:115. 23. Lillington DM, Goff LK, Kingston JE, Onadim Z, Price E, Domizio P, Young BD. High level amplification of N-MYC is not associated with adverse histology or outcome in primary retinoblastoma tumours. British journal of cancer. 2002 Sep 23;87(7):779-82. 24. Zhang J, Benavente CA, McEvoy J, Flores-Otero J, Ding L, Chen X, Ulyanov A, Wu G, Wilson M, Wang J, Brennan R, Rusch M, Manning AL, Ma J, Easton J, Shurtleff S, Mullighan C, Pounds S, Mukatira S, Gupta P, Neale G, Zhao D, Lu C, Fulton RS, Fulton LL, Hong X, Dooling DJ, Ochoa K, Naeve C, Dyson NJ, Mardis ER, Bahrami A, Ellison D, Wilson RK, Downing JR, Dyer MA. A novel retinoblastoma therapy from genomic and epigenetic analyses. Nature. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]. 2012 Jan 19;481(7381):329-34. 25. Manning AL, Longworth MS, Dyson NJ. Loss of pRB causes centromere dysfunction and chromosomal instability. Genes & development. [Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't]. 2010 Jul 1;24(13):1364-76. 26. Kobayashi M, Takezawa S, Hara K, Yu RT, Umesono Y, Agata K, Taniwaki M, Yasuda K, Umesono K. Identification of a photoreceptor cell-specific nuclear receptor. Proc Natl Acad Sci U S A. [Research Support, Non-U.S. Gov't]. 1999 Apr 27;96(9):4814-9. 27. Chen D, Pacal M, Wenzel P, Knoepfler PS, Leone G, Bremner R. Division and apoptosis of E2f-deficient retinal progenitors. Nature. 2009 Dec 17;462(7275):925-9. 28. Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele DA, Bergeron L, Sims RJ, 3rd. Targeting MYC dependence in cancer by inhibiting BET bromodomains. Proceedings of the National Academy of Sciences of the United States of America. 2011 Oct 4;108(40):16669-74. 29. Dimaras H, Kimani K, Dimba EA, Gronsdahl P, White A, Chan HS, Gallie BL. Retinoblastoma. Lancet. [Research Support, Non-U.S. Gov't]. 2012 Apr 14;379(9824):1436-46. 30. Chantada GL, Casco F, Fandino AC, Galli S, Manzitti J, Scopinaro M, Schvartzman E, de Davila MT. Outcome of patients with retinoblastoma and postlaminar optic nerve invasion. Ophthalmology. 2007 Nov;114(11):2083-9.
27
MYCN ONCOGENE-INITIATED RETINOBLASTOMA
28