Nature template - PC Word 97 · Web viewLow expression of MYCN protein is detected in fetal retina (week 17) and WERI-Rb-1, while high expression of MYCN protein is detected in

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 299/300

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 1054 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 2.87% (298/1054) 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 a 1922%

chance that a child diagnosed less than six months oldwith 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, 21/10/12,

NUMBER CHANGED FROM 28 TO 29, BECAUSE TUMOUR E29 IS NOW RECOGNIZED TO BELONG IN THE ORIGINAL SET, NOT ADDITIONAL AS PREVIOUSLY DESCRIBED.


WE HAVE RECALCULATED AND PROVIDE A REVISED, MORE CLEAR, TABLE S4.


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 3152/3000

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 by

local REBs for de-identified use of archival tissue and data, 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, and the use of intragenic and closely-linked RB1 microsatellite

markers 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.8Western 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 inat 6p, 1q, 16q and 2p,

characteristically gained or lost in retinoblastoma. 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, and collected DNA from RB1+/+

tumours, tested in these centres, that had no detectable RB1 mutations, no RB1 promoter

methylation, and no LOH at RB1 (tTable 1). The proportions of RB1+/+ and RB1+/+ MYCNA tumours

from each of the four centres was similar. After our studyanalysis was complete, we discovered that

anthe Amsterdam lab had independently characterized three RB1+/+ MYCNA tumours. We include

their data, and data from an additional Toronto RB1+/+ MYCNA tumour (T101) found after the initial

studyanalysis, in this report.

8


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


Clinical assays in our labs 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 1054 unilateral

non-familial tumours, we identified 29 RB1+/+tumours (2·8%), 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). 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) (table S6, S5).

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, figure2A).

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.

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.


PAIR-WISE COMPARISON IS DESCRIBED IN TABLE 1, SO DELETED FROM THIS TEXT.

Tim Corson, 23/10/12,

I really think these should be replaced with regular decimal points, or at least used consistently!


characteristic of RB1-/-retinoblastomas.9 The RB1+/+MYCNA retinoblastomas also showed overall

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

with Welch’s adjustment) (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 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, gains at 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, 2B, 4A, S2, S4).

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

usual cell types in normal adjacent retina17 stained for both N-terminal (figure 3A) and C-terminal

(data not shown) epitopes of the RB1 protein (pRB), while RB1-/-(+) tumours failed to stain for pRB.

10


Western blot on cell line A3, derived from RB1+/+ MYCNA primary retinoblastoma, showed both

phosphorylated and un-phosphorylated, full-length pRBWestern blot on three cell lines derived

from RB1+/+MYCNA primary retinoblastomas 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 MYCN protein and transcripts compared to RB1-/- primary retinoblastomas and

fetal retina (figure3D, 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 3CB).

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

3ED, table S8).

RB1+/+MYCNAtumors expressed embryonic retinal cell markers consistent with a retinal origin. The

mRNAs of cone cell marker X-arrestin19 and CRX, a marker of retinal and pineal lineage tumours

strongly expressed in RB1-/- retinoblastoma, but not in neuroblastoma,20 were expressed in fetal

retina, human adult retina, three RB1+/+MYCNA primary retinoblastomas, and four RB1-/-

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 (16 RB1+/+MYCNA

tumours, one 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) retinoblastomas

11


(figure 4A, table S5). Our data predict that 1922% of children diagnosed with non-familial,

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 with unilateral RB1-/-tumours (age range 1·9 to 109 months), using birth as a

surrogate for cancer initiation, fita two hit model only when we excluded children diagnosed older

than 60 months (the oldest children included in Knudson’s analysis were 60 months) and as

expected, did not fit the one-hit model describing heritable retinoblastoma. OurRB1-/-data points fits

a two-hit curve, representative of two independent mutation events in a tumour suppressor gene, not

a one-hit curve. We similarly analysed age at diagnosis vs. proportion not yet diagnosed for

RB1+/+MYCNA tumours: whileage at diagnosis vs proportion not yet diagnosed fit both one-hit and

two-hit models poorly, perhaps reflecting that the majority of these tumours initiated before birth.

thewhile the data points of the twelve children less than 10 months old fall close to the calculated

one-hit curve, the age of diagnosis for some of the older children deviate away from it, falling on

either side of the predicted curve (figure 4B).

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

RB1-/- retinoblastomas (figure 4C). However, on histological examination, RB1+/+MYCNA tumours

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

and little calcification, similar to other MYCNA embryonic tumours, such as neuroblastoma21 (figures

4D, S43). They were distinctly different from prototype RB1-/- retinoblastoma, missing the typical

Flexner-Wintersteiner rosettes22 (figure 4E).

Three RB1+/+MYCNA primary retinoblastomas (RB522, T101, and A3) grew rapidly into cell lines,

unlike the usual RB1-/-retinoblastomas that grow poorly in tissue culture. One RB1+/+MYCN A

retinoblastoma had already invaded the optic nerve past the cribriform plate at age 11 months, a

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.


feature of aggressive disease (figure 4F). The unilaterally affected patients with RB1+/+ MYCNA

tumours in this study had large tumors and early age of diagnosis, and 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

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 onset.

Our 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 tumours may be also examples of RB1+/+MYCNA retinoblastomas,23 although

RB1 genetic status was not defined. Notably, our discovery of a discrete retinoblastoma subtype was

validated through independent observations in different patient cohortsDespite 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, thus validatingcorroborating the findings.

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

frozen tumours to perform expression studies, aCGH of DNA showed genomic gains and losses

distinct from those seen in either RB1-/-or RB1+/+MYCNA retinoblastomas. In particular, these

RB1+/+retinoblastomas showed increased incidence of gain on 19p and q, 17 p and q, 2p, and at the

telomeric end of 9q. The RB1+/+ group is expected to be heterogeneous; mechanisms may include

translocations of oncogenes leading to tumourigenesis. This group merits further study.

13


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


RE REVIEWER #4-POINT2; THIS EMPHASIZES GOOD OUTCOME OF MYCNA PATIENTS POST-ENUCLEATION


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, early age of diagnosis for MYCNA tumours may also factor into the reduced

copy-number alterations, since alterations have less time to accumulate. Although whole genome

sequencing has recently shown 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 specific recurrent DNA copy-number changes

associated with RB1 loss in retinoblastoma and retinoma8,16 are less frequent in RB1+/+MYCNA

retinoblastomas with intact RB1 genes.

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

defines ‘a blast cell tumour arising from the retina’. RB1+/+MYCNA retinoblastomas fit the basic

definition of retinoblastoma as ‘a blast cell tumour arising from the retina’, since they 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 RB1+/+ MYCNA cell line A3 showed 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 one hit: somatic MYCN oncogene amplification in a retinal progenitor cell.

This is supported by the identification of one RB1+/+MYCNA tumour where the only genomic copy-

14


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.


We don’t actually show this; can we say “likely arise”?


number change we detected was amplification of the MYCN region. Because RB1+/+MYCNA

retinoblastomas diagnosed soon after birth are already large, they likely initiate much earlier in fetal

development than RB1-/- retinoblastomas, possibly even earlier than hereditary retinoblastomas, so

Knudson’s analysis using birth as a surrogate for tumour initiation may not be applicable. In

addition, the aggressive growth ofthese retinoblastomas may confound determination of how many

hits initiate RB1+/+MYCNAtumours, using Knudson's analysis.How the 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 The relative genomic stability of

RB1+/+ MYCNA retinoblastomas suggests that anti-Myc therapeutic agents28 may not evoke the

resistance to therapy acquired by genomic rearrangements in RB1-/- retinoblastoma. We have

performed preliminary experiments to knock down MYCN expression and observed rapid death of

RB1+/+ MYCNA cell lines. Globally, many children less than one year of age with extra-ocular

retinoblastoma30 might have RB1+/+MYCNA retinoblastomas and benefit from anti-MYCN therapy.

For intraocular disease, enucleation is presently the only way to diagnose RB1+/+MYCNA tumors, so

anti-MYCN therapy for intraocular tumours is unlikely.

Young age at diagnosis of unilateral retinoblastoma frequently suggests heritable retinoblastoma,

and is often considered a reason to try to cure retinoblastoma without enucleation. However,

attempts to salvage an eye with RB1+/+MYCNA retinoblastoma could be dangerous. One

RB1+/+ MYCNA retinoblastoma showed early significant optic nerve invasion, a predictor of high

mortality through tumour invasion into brain.27 The patients with unilateral RB1+/+ MYCNA tumours

in this study 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

15


THESE EXPERIMENTS ARE BEYOND THE SCOPE OF THIS PAPER, WHICH SHOWS THAT THE RB1+/+MYCNA TUMORS ARE DISTINCT FROM RB1-/-RETINOBLASTOMA


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


SHORTENED SECTION RE KNUDSON'S CURVE, NOT PARTICULARLY RELEVANT (SE REVIEWER #1)


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 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.

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

mutations (Panel). 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

16


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/


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

failure to cure these aggressive oncogene-driven retinoblastomas.

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

RB1+/+MYCNA

Canada 441 7 1.6% 0.06 5 1.1%

Germany 400 12 3.0% 0.31 4 1.0%

France 150 5 3.3% 0.22 2 1.3%

New Zealand 30 2 6.7% 0.06 1 3.3%

The Netherlands 3347 3 96.14% 0.03** 3 6.49.1%

Total 105468 29 2.87% 15# 1.41.4%

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

**A larger percentage of Netherlands patients were RB1+/+ compared to Canadian patients (p = 0.03).

# One additional RB1+/+MYCNA and one RB1+/-MYCNA (T33) tumours were subsequently included for a total of 17 included in some analyses

.

18

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.

Berber Mol, 24/10/12,

Initially, for the Dutch patients, only the patients were included were MLPA was performed. For completeness we added al unilateral non-familial rb tumors for which RB1 mutation analyses was performed.


Figures

Figure 1:The 5-gene copy number signature of retinoblastoma

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

by RB1 mutation status: 11 of the original 25 RB1+/+tumours showed high-level amplification of

MYCN (RB1+/+MYCNA) (median MYCN copy-number: 54), and few copy-number changes in the

other genes typically gained or lost in retinoblastoma. Median MYCN copy of the other 14 (non

MYCNA) RB1+/+ tumors was 3.09. T33 is an outlier of the RB1+/-group, showing an RB1+/+MYCNA-

like profile; gray line, 2-copies; 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.(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+/+MYCNAtumours (black triangles) not included in (A). (C)

MYCN copy-number assessment in 30 RB1-/-and 3 RB1+/+ tumours studied in Amsterdam, showing

high MYCN copy-number in the RB1+/+tumours.

19


In response to reviewer 4, we have included median values for MYCN copy number for the RB1+/+ and RB1+/+ MYCN tumors, showing the bi-modal distribution. Also clarified that fig 1A only shows the original 25 RB1+/+ tumors without the additional 5 found later.


20


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 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-/-

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

21


22


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

(A) RB1+/+MYCNA retinoblastomas stained positive with pRB N-terminus antibody and for MYCN

protein; controls, normal retina and adjacent normal retina (*); RB-/-tumour is negative for 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 RB1+/+ MYCNA cell lines A3, Y79 and BE(2)-M17 (with

MYCN amplification), but not in SH-SY5Y (which has 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.

23


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


C-terminus staining not shown


24


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) Using our data, tThe Knudson plot of proportion

not yet diagnosed vs. age at diagnosis, using birth as a surrogate for initiation, fits the two-hit curve

(blue) but not the one-hit curve (red) for RB1-/- disease, as expected. F; for RB1+/+MYCNA

retinoblastoma, while the data points of the twelve children less than 10 months of age fall close to

the calculated one-hit curve (red), the age of diagnosis for some of the older children deviate away

from it, falling on either side of the predicted curve (data points for identical ages overlap). (C)

Fundus image of an RB1+/+MYCNA unilateral tumour in a 4 month-old child shows characteristic

calcification on ultrasound. (D) Round nuclei with large 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. (F)

RB1+/+MYCNAretinoblastoma in an 11 month-old child shows extra-ocular extension into the optic

nerve (white arrows) (2.5x, hematoxylin-eosin staining).

25


26


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 features 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

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

27


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.

28


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. Schuler A, Weber S, Neuhauser M, Jurklies C, Lehnert T, Heimann H, Rudolph G, Jockel KH, Bornfeld N, Lohmann DR. Age at diagnosis of isolated unilateral retinoblastoma does not distinguish patients with and without a constitutional RB1 gene mutation but is influenced by a parent-of-origin effect. Eur J Cancer. 2005 Mar;41(5):735-40. 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.

29


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. 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. 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. Xu XL, Fang Y, Lee TC, Forrest D, Gregory-Evans C, Almeida D, Liu A, Jhanwar SC, Abramson DH, Cobrinik D. Retinoblastoma has properties of a cone precursor tumor and depends upon cone-specific MDM2 signaling. Cell. 2009 Jun 12;137(6):1018-31. 30. 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.

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Documents

Nature template - PC Word 97 · Web viewLow expression of MYCN protein is detected in fetal retina (week 17) and WERI-Rb-1, while high expression of MYCN protein is detected in