· web viewof molecular pathology, canterbury health laboratories christchurch, new zealand (a...

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,

[email protected]

2

mailto:[email protected]


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

Gallie Brenda, 27/10/12,

Change in numbers due to corrected Netherlands denominator


TO CLARIFY AS SUGGESTED THE HIGH LEVEL OF MYCNDNA COPY NUMBER


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


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


WORDS MODIFIED FOR CLARITY

Administrator, 21/10/12,

DAMAGE TO THE ALLELES IS MORE ACCURATE THAN LOSS


CLARIFIES USE OF “HIT” FOR REVIEWERS; “HIT” IS THE COMMON TERM USED TO DESCRIBE THE RATE-LIMITING DRIVER MUTATIONS TOWARDS CANCER, SINCE THE 1950’S LITERATURE.


TO CLARIFY, WE HAVE ENTERED THE PROPORTIONS OF UNI AND BILATERAL RB. : REVIEWER 1 MISINTERPRETED AND THOUGHT THAT 90% OF RETINOBLASTOMA WAS BILATERAL


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


TEXT ADDED TO CLARIFY TECHNOLOGIES USED at each site


REVIEWER 3: EXPT 3 SUGGESTS THAT SNP ANALYSES ARE NECESSARY TO DETECT LOH.. ALL SITES USED MICROSATELLITES TO DETERMNINE WHETHER OR NOT LOSS OF HETEROZYGOSITY (LOH) WAS PRESENT IN THE RB1 +/+ TUMORS.


REVIEWER 1 POINT 4: WE HAVE ADDED MORE INFORMATION ABOUT ABOUT SIMILARITES AND DIFFERENCES BETWEEN EACH LAB METHODOLGY IN THE FIVE LABS


TO CLARIFY THAT INDIVIDUAL INFORMED CONSENTS ARE NOT REQUIRED BY OUR RESEARCH ETHICS BOARDS FOR THIS KIND OF RESEARCH.


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


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


RECALCULATED THE OVERALL PROBABILITY THAT THE FREQUENCY OF +/+ TUMOR SWAS THE SAME IN ALL SITES.


RE REVIEWER #1-POINT 7 “WITH THE SMALL #S OF MYCNA TUMORS, WHAT PROSPECT IS THERE TO CONFIRM THE DATA IN OTHER INDEPENDENT DATA SETS: IN FACT THIS HAS ALREADY BEEN CONFIRMED IN EACH CENTER, AND INDEPENDENTLY DISCOVERED IN THE AMSTERDAM LAB.


AS PER REVIEWER 1, TO DESCRIBE INITIAL FINDINGS THAT LED TO INTERNATIONAL COLLABORATION, AND TO INTRODUCE THE COUNTRIES WHO COLLABORATED. SEE ALSO METHODS FOR DESCRIPTION OF MUTATION ANALYSIS IN LAB FROM EACH COUNTRY.


PROVIDED DETAIL ABOUT THE SEQUENCE OF EVENTS LEADING TO COLLABORATION


RE: REVIEWER’S 3 COMMENTS THAT NCC MIGHT HAVE CAUSED NO RB1 MUTATION TO BE FOUND IN THE RB1 +/+ MYCNA TUMORS: WE CLARIFY THAT REAL DATA COLLECTED DURING ROUTINE RB1 TESTING SHOWS THAT THE INCIDENCE OF NORMAL CELL CONTAMINATION OR MUTATIONAL MOSAICISM IN RB TUMORS IS VERY LOW, AND ASSOCIATED WITH EXTREMELY SMALL TUMORS OR PRIOR CHEMOTHERAPY.


EXPANDED ON MUTATION FINDINGS IN TUMORS


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


WE HAVE SIMPLIFIED THE DESCRIPTION OF THE DATA


RE-WORDED TO CLARIFY WHERE BLOOD WAS AVAILABLE


WE HAVE SIMPLIEFIED THE TABLE S5 AND THE DESCRIPTION OF THE DATA.


Reference webappendix? For stats?? Check rules

Diane Rushlow, 22/10/12,

SPECIFIED WHAT TYPE OF ANALYSIS WAS DONE AT EACH SITE


THE NUMBERS INCREASED FROM 28 TO 29 WHEN SAMPLE E29 WAS RECOGNIZED TO BELONG IN THE ORIGINAL DATA SET.


OVERALL NUMBERS INCREASED WHEN 14 NETHERLANDS SAMPLES WERE RECOGNIZED TO HAVE BEEN NOT INCLUDED BY ERROR. (SEE RESPONSE TO REVIEWERS)


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


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


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


SHORTENED THIS SECTION, SINCE REVIEWER 1 QUESTIONED RELEVANCE OF ONE-HIT, TWO HIT., ALSO, WITH TO-DAY’S ABILITY TO ACTUALLY SEE RB1 (AND OTHER GENE) MUTATIONS, IS OBVIOUS THAT BOTH ALLELES OF THE TUMOR SUPPRESSOR GENE ARE INACTIVATED IN THESE TUMORS. WE INCLUDE THIS ANALYSIS TO PUT OUR PAPER INTO HISTORICAL CONTEXT.


WE HAVE CLARIFIED TABLE S4 AS SUGGESTED BY REVIEWER…. AND CORRECTED THE NUMBERS.


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

Diane Rushlow, 25/10/12,

MOVED THIS UP FROM LATER PART OF THE DISCUSSION INORDER TO COMPLETE DISCUSSION OF THE NON-MYCN +/+ TUMORS BEFORE THE MYCN TUMORS ARE DISCUSSED FURTHER


ADDRESSING REVIEWER 1’S COMMENT RE PROSPECT OF CONFIRMING THE FINDINGS INDEPENDENTLY ELSEWHERE


NUMBERS UPDATED AS DESCRIBED


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


RE-ARRANGED THE SENTENCES OF THE ORIGINAL DISCUSSION FROM HERE ON TO DEAL FIRST WITH POSSIBLE TREATMENT THRU MYCN KNOCKDOWN, THEN ENUCLEATION, THEN TESTING FOR MYCN


Deleted”one-hit”As pointed out by reviewers 1 and 2, MYCN amplification is likely to be cause by a series of events. To call it one hit is miss-leading


ALTHOUGH WE SHOW ONLY A3 CELL LINE, two others ALSO SHOW BOTH PRb FORMS.


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


WE HAVE CLARIFIED TABLE S4 AS SUGGESTED BY REVIEWER…. AND CORRECTED THE NUMBERS.


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

Tim Corson, 23/10/12,

Really? Hasn’t the concept of “RB2” been around for many years? Is it not in the literature anywhere?

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


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


TABLE 1 REVISED TO INCLUDE P VALUE FOR THE NETHERLANDS.

Berber Mol, 22/10/12,

I have two note that I recently received all the tumor mutation data and we actually have screened 47 tumors with the following results: 41 RB-/-, 3 RB +/- and 3RB+/+ MYCNA. For this paper I included only the samples for which I had performed MLPA, but if we want to include the p-value I think we should add the other patients since that will lowel the proportion to 6.4 % and will not be significant.


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.

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19


Figure 2:Fewer genomic copy-number alterations in RB1+/+MYCNA than RB1-/-tumours

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

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

occurring copy-number variations. The RB1+/- MYCNA tumour T33 shows loss of most of 13q; this

may not be an initiating event. (B) The minimal amplicon of 513 kbp is defined by two MYCNA

tumours (pink band); MYCNcopy-number by QM-PCR, red italics; aCGH individual probes, green

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

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

numbers observed, box bounds first and third quartiles, and horizontal line within the box represents

the median. (D) Fewer aCGH clones are altered in RB1+/+ and RB1+/+MYCNA, than RB1-/-tumours;

each class has more unique clones altered than in common.

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21


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

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

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


REVISED WITH INCLUSION OF NEW WESTERN BLOTS ILLUSTRATING THE PHOSPHORYLATION OF pRb.


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


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


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


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