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COMMENTS TO THE AUTHOR:

Reviewer #1: THELANCET-D-12-05192 Not Knudson's retinoblastomas: one hit cancer initiated by the MCYN change

Statistical review

Comments for the authors

The authors present a complex set of analyses to identify a new non-RB1 mutation non-familial retinoblastoma. The statistical methods seem appropriate but they could do with better and clearer explanation. There are a number of statistical / methodological issues arising, as follows:

Major

1. As a non expert on the subject matter expert (statistical reviewer) I found this a difficult paper to follow. What would have helped would be a clear explanation of how the various analyses - the mutation analyses, the genome copy analyses, the protein expression studies, the RNA gene expression analyses, and the age at diagnosis analyses - logically integrated into demonstrating the authors hypothesis. Obviously it would not be appropriate or feasible to give a primer in this complex methodology - but it would help the general reader to e.g. have a figure indicating how the various analyses build to the overall conclusion, and what subsets of the data are being used at each step?

To clarify the presentation of our strong data indicating that we have identified a new form of retinoblastoma, and that the RB1-/- and RB1+/+MYCNA retinoblastoma are separate entities, we have developed a graphic illustration of the cumulative data and probabilities in support of this hypothesis (new figure 4G). The reader can find all the details to validate this high level interpretation in the webappendix data and tables.

2. The conclusion that 'Since these aggressive tumours may rapidly become extraocular, removal of the eye of young children with unilateral non-familial retinoblastoma is important' - to what extent was this claim directly supported by evidence from these data as presented - this wasn't obvious?

We have amended the wording in the abstract (quoted above) to 'Since these aggressive (MYCNA) tumours may rapidly become extra ocular, removal of the eye of these young children with unilateral non-familial retinoblastoma is important'

In the revised manuscript:

p15, paragraph 2: "Young age at diagnosis of unilateral retinoblastoma frequently suggests heritable retinoblastoma, and is often considered a reason to try to cure retinoblastoma without removing the eye. 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 brain27." and p.12 paragraph 3 "One RB1+/+MYCNA retinoblastoma (Amsterdam) had already invaded the optic nerve past the cribriform plate at age 11 months, a feature of aggressive disease (figure 4F)" and page 14, paragraph 3, "RB1+/+MYCNA retinoblastomas diagnosed soon after birth are already large" showing aggressiveness of these tumors. In clinical data examined at the Toronto site MYCNA tumors are described as “large”, “bulky”, “massive”, and another tumor described as “focally invading head of optic nerve” at seven months.

Patients with RB1+/+MYCNA tumors are the youngest at age of diagnosis (median 4.5 months), younger even than bilateral patients with heritable retinoblastoma (median 12 months). However, unlike heritable retinoblastoma with a constitutional RB1 mutation, the other eye is not believed to be at risk of developing a tumor. Page 12, paragraph 3, and p.15 paragraph 2 "The unilaterally affected patients with RB1+/+MYCNA tumours in this study were cured by removal of their affected eye with no adverse outcomes; none developed retinoblastoma in the other eye.” There is no foreseeable mechanism for MYCN amplification to be a constitutional event, or to be determined by a heritable mutation.

However, there appears to be significant risk of these large, aggressive tumors becoming extra-ocular. We suggest that serious consideration be given to removal of the eye for a patient with isolated unilateral retinoblastoma, and a large tumor, in a child less that 6 months of age. In addition enucleation will allow accurate diagnosis (RB1-/- or RB1+/+MYCNA tumor) and optimize the overall care of the child.

3. Abstract 'We calculated a 22% chance that a child diagnosed less than six months old with unilateral non-familial retinoblastoma would have an RB1+\+MCYNA tumour' - this calculation needs to be laid out explicitly - I'm probably missing something obvious but I could not reproduce this number?

Page 11, of the manuscript, bottom of the page refers the reader to Table S4, which shows how this was calculated. We have revised the calculation, and made table S4 as explicit as possible so the basis for the calculation is clear. The revised estimate is that a child ≤ 6 months of age at diagnosis of unilateral retinoblastoma has a 19% chance to have an RB1+/+MYCNA tumour. This revision has threewo corrections over the original: tumor E29 was overlooked as RB1+/+ in the orginal 400 Essen tumors, and the denominator was incorrect (previously used formula #RB1+/+MYCNA/#RB1-/-,

where the correct formula is

#RB1+/+MYCNA /(#RB1+/+MYCNA + #RB1-/-)., and the Dutch total number of tumors increased from 33 to 47 tumors, since now all tumors for which RB1 mutation analysis was performed are included compared to only tumors were in addition MLPA analysis was performed.

4. There is very little context set for the 5 national cohorts that comprise the analysis data set - there is no mention at all of the 5 countries until we get to the tables, no detail given to understand what might be the similarities or differences within and across countries - you feel this is essential detail if we are going to get any insight into how generalisable these findings are likely to be?

Results page 8, paragraph 2, now describes the start of Toronto collaboration with RB1 testing labs in France, Germany, and New Zealand, which confirmed similar proportions of RB1+/+ tumors, and similar proportions of RB1+/+ MYCNA

tumors, demonstrating that the RB1+/+ tumor findings are likely to be generalisable. The similar proportions of RB1+/+ MYCNA tumors in various ethnic populations is in agreement with the known lack of geographic/ethnic variation in RB incidence. In Discussion page x, we describe an anecdotal discovery of RB1+/+ MYCNA disease in the biopsy of an orbit retinoblastoma metastasis in Kenya while reviewing 60 pathology slides, supporting that this a widespread

On page 8 bottom of paragraph 2, we describe meeting with scientists from an Amsterdam RB1 testing lab who had independently discovered and characterized three RB1+/+ MYCNA tumors . The fact that the Amsterdam lab had made the same discovery of MYCNA tumors and reached essentially the same conclusions as the Toronto lab, provides confirmingation of the findingsreproducibility. , and demonstrates that the findings are generalisable.

ThisWe also show that different methods of analyzing the MYCNA tumors yielded the same results in the different centres supporting generalisablity of our observations.

Under "Methods", Mutation Analysis: we have expanded our description of methods: All labs used promoter methylation testing and sequencing for RB1 mutation detection, and polymorphic RB1 intragenic and linked microsatellite

analysis to determine tumor zygosity. Toronto and French labs used QM-PCR for RB1 exon copy-number analysis; , and the other labs used MLPA for this.

Page 9, paragraph 2, we have amended to read: "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."

and page 9, parag 4 now reads "We studied DNA from 48 unilateral retinoblastomas by aCGH10 and 3 (Amsterdam) by SNP analysis" (for genome wide copy-number analysis) (. also showndata in Table S5).

This shows that different methods of analyzing the MYCNA tumors yielded the same results, hence they are generalisable.

Minor

5. I did not get a clear understanding of this 'two hit' and 'one hit' model for the Initiation of the cancer, and probably as a result was struggling to see its relevance? Isn't it possibly easier to just say the authors have uncovered another gene (MYCN oncogene amplified) that is implicated in the cancer initiation?

The historical perspective that cancer is a genetic disease starts with ……

Etc

We have shortened the sections discussing one-hit, two hit (page 12, paragraph 1, and page 14, paragraph 3)., since not particularly important to our findings.

Amended page 14, first sentence of paragraph 3, omitting words "one hit", in reference to MYCN amplification

The point we tried to make was simply that the MYCNA tumors do not fit the classic curve for time to diagnosis for unilateral sporadic tumors (as described by Knudson), caused by two independent somatic mutations in the tumor suppressor gene RB1. (The two mutations can also be a mutation on one allele, then loss of heterozygosity as a second independent event). This "two-hit" curve (for time to diagnosis), where both gene alleles acquire mutations independently, contrasts to the one-hit curve generated for tumors of patients already carrying a constitutional RB1 mutation on one allele. The very early age of diagnosis for the large MYCNA tumors does not fit with the later ages of diagnosis nor the age of diagnosis curve generated for other sporadic unilateral retinoblastomas. The early age may fit well with action of an amplified MYCN oncogene. This does not pre-suppose that the MYCN amplification was caused by one event; it most likely resulted from a series or cascade of events, such as double strand breaks,fusions etc. resulting in MYCN amplification, but simply supports that the timing of events were not limited by the rate-limiting random mutation of both alleles of a tumor suppressor gene.

Amended sentence p14, paragraph 3: ."How the MYCN amplification is initiated and whether MYCN amplification alone suffices to initiate these retinoblastoma remains to be formally demonstrated".

6. For example, page 11 'our similar analysis of 79 patients with unilateral RB1-/- ... fit a two hit model ... and as expected did not fit the one hit model describing heritable retinoblastoma' - the authors should give appropriate statistical details here e.g. How was lack of fit measured? Figure 4B seems to be only an informal graphical impression - is that correct?

.full explanation of figure 4B is in the webappendix. These are not graphical impression, but the 1 hit and 2 hit curves of the real data. The numbers of samples are correct, but the data points over lap with ages at diagnosis are identical for more that one child. Etc….i will respond in detail

7. The authors should explicitly discuss the small number of events that is driving this analysis - yes, they have assembled a hugely impressive 1000+ cases, but there are only 28 RB1+\+ tumours and only 14 of these had the MYCN oncogene amplification. What prospect is there mod confirming these findings in other, independent data sets?

The findings in the Dutch cohort were detected independently with different techniques, we joined our data together later in the study. Since the Dutch cohort is an independent data set it validates the results of the other 4 cohorts. Therefore we strongly believe this is a finding applicable to retinoblastoma tumors worldwide.

Page 13, paragraph1 amended: to read " Despite the low incidence of RB1+/+MYCNA retinoblastoma, RB1+/+MYCNA retinoblastoma was independently discovered and characterized in the Toronto and Amsterdam labs, with different patient cohorts, and using different technologies, thus validating the findings."

8. The unilateral vs bilateral issue was a bit confusing - the introduction mentions that around 90% of subjects develop bilateral disease, but these 1054 were unilateral, and elsewhere subsets are selected that are then obviously unilateral - so how then do these findings relate to bilateral disease?

This was an issue with clarity of the wording in background. In the text it had stated “A child with a heterozygous, constitutional RB1 mutation is predisposed to retinoblastoma (~90% bilateral)” . This means that 90% patients with a germline RB1 mutation are bilateral.

We have re-written the background paragraph to indicate that 40% of all retinoblastoma is bilateral and 60% unilateral: page 5, paragraph 1. Bilateral patients all carry a constitutional RB1 mutation that predisposes to multiple tumors.

The tumors described in our study are from 1054 non-familial unilateral retinoblastoma patients. Our study compares RB1-/- sporadic unilateral tumors

carrying two identified RB1 mutations, with unilateral RB1+/+ sporadic unilateral tumors where no RB1 mutations are detected.

9. Page 14 - '... Using birth as a surrogate for tumour initiation may not be applicable' - so what did the authors do by way of sensitivity type analyses to investigate this e.g. at one extreme use date of first known tumour, or perhaps randomly impute a date between this and date of birth to see what effect this had?

Knudson’s analysis used date of birth as a the presumed time of tumor initiation. For non-heritable unilateral retinoblastoma with both RB1 alleles in the tumor mutant, development of tumors statistically extrapolates quite well to tumor initiation at or after birth. The children with RB1+/+ MYCNA tumors are diagnosed with already large tumors much younger, so it is obvious that their tumors start well before birth. Analysis for mechanisms of tumor initiation (one vs two “hits”) was not possible since there is no way to measure age at tumor initiation.

Reviewer #2: Rushlow and colleagues describe that the MYCN oncogene appears to be causally associated with retinoblastoma.   The authors describe that 2.7% of unilateral non-familial retinoblastomas have no evidence for RB1 mutation, and half of these have evidence for amplification of the MYCN oncogene.   They estimate that 22% of children with unilateral non-familial retinoblastoma have a RB1+ MYCN amplified tumor.   These results provide evidence suggesting that the activation of a single oncogene can be responsible for retinoblastoma.  

This is a well written and highly interesting study.    I think the results are transparently presented and will be well received.   Most of my comments are for consideration of the authors about some of the implications of the work, that perhaps could have been commented on briefly.

Thank you for your careful reading and understanding of our manuscript. We have improved the manuscript as indicated below with your good advice.

These results are highly provocative and interesting. They suggest that MYCN amplification is equivalent to RB-/-. The authors argue that this suggests that retinoblastoma can be caused by a "single" hit. However, this conclusion is perhaps a little misleading. Amplification of the MYCN locus is unlikely to be caused by a single mutation event, such as the deletion of a genomic locus. But, rather likely to be caused by more than one genomic event that selects for MYCN amplification that would likely include a double strand DNA break, fusion and repeat breaks and fusions. This does not detract from the significance of their results. But, MYCN amplification is probably single activated oncogene, but this does not occur through a single hit.  

Because of limited relevance to our findings, we have shortened the sections discussing one-hit, two hit (page 12, paragraph 1, and page 14, paragraph 3).

Amended sentence p14, parag 3: ."How the MYCN amplification is initiated and whether MYCNamplification alone suffices to initiate these retinoblastoma remains to be formally demonstrated".

Amended page 14, first sentence of paragraph 3, omitting words "one hit", in reference to MYCN amplification

The point we tried to make was simply that the MYCNA tumors do not fit the classic curve for time to diagnosis for unilateral sporadic tumors (as described by Knudson), caused by two independent somatic mutations in the tumor suppressor gene RB1. (The two mutations can also be a mutation on one allele, then loss of heterozygosity as a second independent event). This "two-hit" curve (for time to diagnosis), where both gene alleles acquire mutations independently, contrasts to the one-hit curve generated for bilateral tumors of patients already carrying a constitutional RB1 mutation on one allele. The very early age of diagnosis for the large MYCNA tumors does not fit with the later ages of diagnosis nor the age of diagnosis curve generated for other sporadic unilateral retinoblastomas. The early age may fit well with action of an amplified MYCN oncogene. This does not pre-suppose that the MYCN amplification was caused by one event; it most likely resulted from a series or cascade of events, such as double strand breaks,fusions etc. resulting in MYCN amplification, but simply supports that the timing of events were not limited by the rate-limiting random mutation of both alleles of a tumor suppressor gene.

The Knudson hypothesis that retinoblastoma is initiated by one somatic “hit” (in persons with already one constitutional hit (RB1 mutation) indicates that one event is “rate limiting”, ie, initiates the process for the cancer to arise. We have previously shown that this event is not sufficient, since retinoma is not cancer, but already has loss of both RB1 alleles and also specific further genomic changes.{Dimaras, 2008 #11066} The adjacent cancer has increased magnitude of these later events.

While it is clear that the detailed mechanism of genomic amplification can be broken down into multiple steps, its impact on an embryonic cell can be considered mathematically as a single “event”. The genomic copy number stability of the RB1+/+ MYCNA tumors in comparison to the RB1-/- tumors would support that progressive genomic change is at a low rate, but we would expect progressive changes will be occurring as in any cancer. However, these changes do not alter the primacy of the initiating event, MYCN amplification.

The authors do not provide any explanation for the significance of MYC as the "hit" they have found associated with retinoblastoma? There are several possibilities that could be considered. The notion that MYC can initiate rapid tumorigenesis in an immature differentiative context has been describe previously experimentally. MYC has been found to be associated with other pediatric malignancies e.g. hepatoblastoma and T-cell

acute lymphocytic leukemia. There is evidence that MYC and RB are complimentary in cancer formation. The MYC and RB pathways are related. The authors do not consider the significance of why MYC was found and how this may relate to RB.

Yes, the pathway for unregulated MYCN to provoke proliferation would be expected to work through functional inactivation of pRB. We can add one sentence to this effect…..

Finally, one of the hallmarks of pathophysiology of retinoblastoma is that restoration of RB can revert the neoplastic properties of the cancer cells. This manuscript begs the question, would suppression of MYC in a MYC-amplified retinoblastoma do the same? I would not expect the authors to perform this experiment, but it is curious that they do not consider that their results suggests that the MYC pathway may be a important target for some patients with retinoblastoma. The authors do not appear to describe the potential diagnostic and therapeutic significance of there results.

While experimental replacement of a functional RB1 gene may slow growth of retinoblastoma, it does not revert neoplastic properties, since the cancer cells have accumulated many progressive changes that actually drive the malignant proliferation.

We have performed preliminary experiments to knock down MYCN expression and the RB1+/+MYCNA tumor cells rapidly die, and have added a sentence to this effect on page 14, parag 4.

We discuss the exciting potential that anti-MYCN therapy may have a powerful role in children with metastatic RB1+/+MYCNA tumors page 15,parag 1 . For intraocular disease, enucleation is presently the only way to diagnose RB1+/+MYCNA tumors, so anti-MYCN therapy for intraocular tumors is unlikely, as discussed page 15, parag 1. Also these tumours are unlikely to be recognized early enough, since they are already large at very young age.

Reviewer #3: In their manuscript titled "Not Knudson's Retinoblastoma: One-Hit Cancer Initiated by the MYCN Oncogene?" Rushlow et al. explore the possibility that a small subset of human retinoblastomas initiate with MYCN amplification and lack any RB1 mutations. This is clearly a very provocative and potentially important hypothesis and it is essential that every effort is made with current technology to exclude the possibility that the investigators simply missed the RB1 mutations in the ~2% of patient tumors. This is not only essential for the field of retinoblastoma research but it is also of particular importance for the families of children with retinoblastoma as Rushlow and colleagues make very specific and far-reaching conclusions about the clinical significance of their findings. The study is very straightforward. Dr. Gallie is one of the world's leaders in RB1 gene sequencing and she has done so since the discovery of the first human tumor suppressor gene was made

by Weinberg and colleagues in 1986. Many human retinoblastoma tumors and blood samples are sent to Dr. Gallie for genetic analysis on a fee-for-service basis and she provides a much needed clinical service for patients and their families around the world. In this role, she has access to a large number of human retinoblastoma tumors and blood samples and she has assembled an international team for this study. The analysis of the RB1 gene utilizes well-established and clinically certified assays including Sanger DNA sequencing, QM-PCR and MLPA. They report that among 1054 unilateral tumors analyzed in this cohort, there are only 28 (2.7%) with no RB1 mutation. This is a remarkable finding in and of itself. Dr. Gallie's genetic analysis protocols and standard operating procedures are so robust that they only miss 2.7% of mutations. In addition to Dr. Gallie's dedication and well-established protocol for RB1 gene analysis, the other contributing factor for this remarkable success rate for RB1 gene analysis is the unique feature of retinoblastoma. It is essentially a solid tumor that grows into a semi-liquid environment. Thus, pathologists can often acquire a sample that has minimal contamination of surrounding normal cells. Indeed, this is a major challenge for most solid tumors that contain macrophages, B and T cells, vascular endothelial cells and other tumor associated fibroblasts and surrounding normal cells. Based on extensive literature of histopathological analysis of human retinoblastomas it appears that well isolated vitreal tumors are likely to only contain vascular endothelial cells and blood cells such as WBCs, tumor associated macrophages etc. Pathologists can therefore isolate tumor from the vitreous of eyes with advanced stage disease with relatively little contamination of surrounding retina or other normal cells that can be associated with tumors. Indeed, the data suggests that this can be done in 97% of patient's tumors, which is remarkable. However, several decades of research on retinoblastoma tumors has shown that they can occasionally contain some retinal contamination that may contribute to a slightly higher percentage of normal cells. Or, there may be a slight enrichment of macrophages or other inflammatory cells or particularly high numbers of tumor associated endothelial cells. Unfortunately, given the pliant nature of the tumors in the vitreous, it is virtually impossible to determine if the area where the tumor was sampled as a fresh specimen for the preparation DNA for genotyping had high levels of the contaminating normal cells. Indeed, it is virtually impossible to study the H&E sections of the remainder of the globe to infer anything about the purity of the tumor in the fresh isolate used for DNA analysis.

Therefore, it is impossible to know anything about the tumor purity of the samples analyzed in this extensive cohort. It would not be surprising if a small subset of those samples had higher than normal levels of normal contamination. This would be especially true if different pathologists or surgeons obtained the samples from multiple centers over several years of clinical practice.

While the conclusion put forward by Dr. Gallie and colleagues that 2.7% of retinoblastomas initiate with MYCN amplification and do not sustain RB1 loss is important and intriguing, they have failed to rule out the very likely possibility that those 2.7% of patients simply have slightly more normal cell contamination in their tumor specimen used for DNA extraction and the RB1 mutations were below the limit of detection of the assay.

We find no RB1 mutation in 2.8% of tumors, but both no RB1 mutation and MYCN amplification in 1.4% (Table 1).

It is inconceivable that normal cell contamination could mask an RB1 mutation but leave 30-121x copies of MYCN undiluted. The actual MYCN amplification would have to be 4 fold the copy-numbers reported in our study (i.e. up to 480 copies per cell) in the presence of significant normal cell contamination that would dilute a 50% DNA mutation down to 12%. We discuss this further below.

Indeed, recent whole genome sequencing analysis of a wide variety of human solid tumors puts the normal cell contamination at 20-50%. Even for retinoblastoma, the normal cell contamination is over 20% for well-isolated samples. The limit of detection for Sanger sequencing is often cited to be about 30%. This means that the polymorphism or mutation must occur in 30% of the DNA strands sequenced to be able to detect it by Sanger sequencing even with manual review of the tracings.

Ideally, each RB1 mutation is only present in 50% of DNA strands because each hit is independent so a small amount of normal cell contamination could obscure the mutation by Sanger sequencing or other methods with similar sensitivities. Dr. Gallie can identify RB1 mutations in 95% of patient's tumors and this is remarkable because it tells us that the tumors are clonal, virtually all the cells have the same genetic lesions and the normal cell contamination is such that the mutant allele is still represented in 30% of the DNA strands.

However, those 5% that are not detected may be due to the normal cell contamination for the tumor or germline mosaicism for the blood, which is well documented in the RB field. Dr. Gallie suggests that the chance of finding a tumor with two wild type RB1 alleles is 5%x5% or 0.25%. However, this assumes that the inability to detect mutations in 5% of patient's tumors is due to technical difficulty. If it was due to normal cell contamination, it would affect both alleles equally and be 5% overall. Thus, their discovery that 2.7% of human retinoblastomas have no RB1 mutations is perfectly aligned with this hypothesis that these tumors have a slightly higher level of normal cell contamination.

In our collective experience in the molecular study of retinoblastoma in our labs, there is very little normal cell contamination in retinoblastoma specimens. That is not due to the tumor being in vitreous, since the minority of studied specimens come from vitreous, but rather are true samples of the solid primary tumor. Whenever there is a homozygous (due to LOH) mutation, there is no signal from normal RB1, whatever technique is used, clearly shown in routine clinical testing.

There are a few predictions that can be made based on this reasonable alternative hypothesis that is not considered by Dr. Gallie. First, one would predict that the MYCN amplifications would still be present. This is because MYCN amplifications that typically occur as double minutes often occur at very high copy number that can exceed 50 copies per cell or even 100 copies per cell. A slight increase in normal cell contamination would not significantly reduce the proportion of tumors with such MYCN contamination. Indeed, this is what they report in their manuscript. The second

prediction is that the frequency of other copy number changes would be lower in those tumors with normal RB. Remarkably, this is exactly what they report. The reason is that the excess normal cells would also mask those changes. A gain of a single copy of a large or small chromosomal region would be masked by the normal cell contamination.

Thus, the most logical interpretation of the data is that the 2.7% of tumors with MYCN gain and RB1+/+ alleles are simply tumors with slightly more normal cell contamination and they missed the mutations. The presence of full length RB1 protein in IHC or immunoblot for one of the samples does not preclude this model. Indeed, there was a recent report (Zhang et. al. Nature, 2012) showing that one allele of RB1 was inactivated by a splice site mutation that led to an in frame deletion of 3 AA in the conserved B-domain of the pocket for the first hit and DNA hypermethylation for the second hit. Using the assays in this manuscript, the tumor would appear to have full length phosphorylated RB1 in IHC and immunoblot and if the normal cell contamination were slightly higher, they would not have detected the mutation.

Response: Although we concede that one or more of the less studied RB1+/+ tumours WITHOUT MYCNA amplification in our study may indeed be examples where tumors were very small, and the DNA was extracted primarily from normal cells, so that any tumor mutations may be undetectable, we strongly dispute that any of the large, well-characterized RB1+/+ MYCNA tumors in our report have any significant levels of normal cell contamination (NCC). In support of this: The statement of the reviewer that for retinoblastoma the NCC is over 20% in

well-isolated samples, is not backed by clinical data collected in RB1 testing. In fact, the incidence of significant normal cell contamination is very small. Routine analysis of microsatellites within and flanking RB1 nicely shows that almost all retinoblastomas with loss of heterozygosity (LOH) are essentially free of NCC. (NCC can be clearly seen, if present, by low levels of the “lost” allele in microsatellite analysis). In addition, any presence of normal cell contamination at levels of 15% or more can be clearly seen in sequence tracings. In fact, in over 600 unilateral tumors tested at the Toronto lab, only 1-2% showed significant levels of NCC , sufficient to interfere with interpretation of results; in these cases examination of paperwork and follow-up with clinicians has shown either that the tumor submitted was very tiny (i.e.not visible by eye) and/ or the patient was treated by rounds of chemotherapy, prior to enucleation. In contrast, as described in the manuscript, MYCNA tumors are large at a very young age and have not had any previous treatment. Review of extraction data for the seven Toronto+ MYCNA tumors, shows no instance where any MYCNA tumor was noted to be small; pathology reports for the MYCNA tumors describe them as large, bulky, or massive. In all cases, enucleation of the large tumors was performed within 16 months of birth and, (where data is available) within days of diagnosis, with no prior chemotherapy. There is no basis to suspect significant NCC in these samples.

The H&E staining of these tumors also shows a very pure tumor population of homogenous cells.

Copy numbers for KIF14 (1q), DEK and E2F3 (6p), CDH11 (16q) and MYCN (2p) were determined by QM-PCR for all the MYCNA tumors. This data is shown below for one of the Toronto MYCNA tumors. In T14, CDH11 (16q) shows two copies in blood and 1.13 copies in tumor due to loss of one copy. Significant levels of NCC would bring this copy# close to 2.Since this is very close to 1 copy, this represents a loss of one copy of 16q in the tumor, with no evidence of significant NCC.

Patient# Type MYCN copy#

KIF14 DEK E2F3 CDH11

T14 Tumor 49 2.18 2.33 2.18 1.13

blood 2.34 1.83 1.93 1.81 2.19

In addition figure 2A and Table 7, clearly show genomic losses for MYCNA tumors at 1p, 2p,4p, 7p,8p, 10q,11p, 11q ,16q, and 17p, losses that would not be apparent if tumors were diluted down significantly by NCC

The Toronto lab also tests each tumor by an allele-specific PCR screen (AS-PCR) (Rushlow et al, , Hum Mut, 2008), a test for eleven common recurrent RB1 mutations; these eleven mutations make up over 22% of all RB1 tumor mutations identified. The AS-PCR assay detect a mutation present on one allele, even in the presence of one part tumor to 49 parts contaminating normal cells. None of the six Toronto MYCNA tumors tested by AS-PCR showed any low-level mutations by this assay. Significantly, since introduction of the AS-PCR screen in 2006, although low-level mosaicism has frequently been detected in blood, out of an estimated 200-300 tumors screened, only one tumor showed a low level of one of these eleven mutations ; in that sample, the M1 and M2 events had already been identified and an M3 event was identified by AS-PCR as present on one allele in about 20% of the tumor cells, low enough to be difficult to detect by sequence analysis. The rarity of finding such low level mutations in tumor supports the scarcity of RB tumors with significant amounts of normal cell contamination.

The Amsterdam lab estimated tumor cellularity from the SNP array profiles of paired tumor and normal samples using the qpure software tool (described in the recent paper “qPure: A tool to estimate tumor cellularity from genome-wide single-nucleotide polymorphism profiles, Song et al., PLOSone, Volume 7, Issue 9, 2012). Tumor cellularity was not significantly different when comparing 3 RB+/+ MYCNA tumors to 3 RB-/- tumors (2-sample t-test, p = 0.93).

The strongest evidence against the suggestion that the RB1+/+ MYCNA tumors harbour undetected RB1 mutations is that 14/28 of the RB1+/+ tumors show very high level MYCN amplification , not seen in any of 100 RB1-/- tumors. It is extremely unlikely that the only tumors with high level MYCN amplification are the exact same tumors with so much NCC that their RB1 mutations cannot be detected. Although we strongly dispute significant NCC in the MYCNA samples, even if there was enough NCC to dilute out the supposed RB1 mutations, this would not negate the observation that of almost 100 RB1-/- tumors , there was no observed MYCN amplification, but 14/28 RB1+/+ tumors showed high level MYCN amplification as well as distinct characteristics such as very early age of diagnosis, distinct histology and aggressive growth. The tumor cells in these normal-cell contaminated samples would have to show even higher levels of MYCN amplification (i.e. as high as 360-480 copies of MYCN per cell) in order to overcome the 3-4 fold proposed dilution caused by normal cell contamination. The reviewer’s suggestion implies that for some unknown biological reason, only retinoblastoma tumors with high levels of NCC contamination show high-level MYCN amplification. Given that NCC , when it occurs, can be attributed to technical considerations of sample size, the purported link between normal cell contamination and MYCN amplification is even less likely.

There are several key experiments that must be performed to support the conclusion of this manuscript:

[1)] 1) Deep sequencing (Next-Gen sequencing) must be performed for the introns and exons of the RB1 gene in the samples believed to have no RB1 mutations to rule out the possibility that they have mutations but are below the limit of detection. One suitable platform may be liquid capture of all introns and exons of RB1 with significant read depth (500-1000 fold) to ensure that there are no minor alleles of RB1 mutations. This technology is now routine and very cost effective so there is no reason it cannot be performed on all the RB1+/+ samples in their cohort. In fact, multiple samples can be analyzed using this technology for less money than Dr. Gallie charges for genetic analysis for a single tumor in her clinical diagnostic lab.

Firstly, as discussed above, there is no evidence at all for an increased contamination with normal cells in the MYCN tumors compared with Rb-/- tumors. There is thus no need to perform Next Generation Sequencing (NGS) to rule out this possibility.

Secondly, we appreciate that NGS is the way to go for diagnostics in the future. However, we tend to disagreefind no evidence with the reviewer that this strategy is at present routine andor very cost effective.

We have in fact submittedin two of our labs used a variety of samples for Next-Gen sequencingNGS using a couplecouple oseveralf platforms but it isthisdiscovered that NGS is no quick solution for RB1 mutation analysis. turned out to be not as easy as suggested. With each pipeline, it is still necessary to look in the “dustbin”. Our experiences with NGS parallel

recent reports comparing state-of-the-art sequencing technologies (see e.g. ). In any event, NGS has not yielded novel mutations sofar.

However, we are primarily interested in Next-Gen sequencing for our RB1+/+ tumors without MYCNA. We have no doubt that MYCN amplifcation is the driving force in the MYCNA tumors. Also there would be no reason to believe that deep intronic mutations in RB1 or low levels of typical RB1 mutations would lead to a sub-group of retinoblastoma with very early age of diagnosis and unique histology.

2) Transcriptome sequencing is another method that can confirm two key observations. First, it can determine if there are any splice mutations including formation of cryptic splice sites that are missed by Dr. Gallie's analysis. Second, it can prove that both alleles are expressed and that one is not silenced by DNA hypermethylation. This must be done on all of the RB1+/+ samples

1) The only splice mutations we could have missed with current standard diagnostics are the deep-intronic mutations; standard splice mutations would be detected since the exon-intron boundaries are routinely sequenced. However, deep intronic mutations are extremely rare in RB-/- retinoblastoma (Zhang et al, “Patterns of Missplicing Caused by RB1 Gene Mutations…”, Hum Mut, 2008.). Since the MYCN samples do not show LOH, each tumor would have to carry two independent deep intronic changes, making the chance even smaller. Since no additional material was available from the vast majority of clinical RB1+/+ samples, RNA studies could only be performed on a limited number of samples. The expression studies performed suggest the presence of full-length RB1 transcripts and functional RB1 protein in the MYCN cells.

An additional argument against the splice variant theory from the reviewer stems from the unique biology (e.g. histology) and clinical behaviour of the MYCN tumors (e.g. age of onset), which is clearly distinct from RB-/- tumors, including those harbouring known deep-intronic RB1 mutations.

2) I t is questionable whether transcriptome sequencing would reveal in individual cases whether the transcript has been derived from one allele or two, since the RB1 transcript is almost entirely free of sequence variants. Importantly, promoter methylation testing has been performed on DNA from each of the RB1+/+ tumors and all showed normal unmethylated promoters, excluding this mechanism of RB1 inactivation.

3) SNP analysis must be performed in addition to the aCGH presented in the paper. The reason is that the second hit can be copy number neutral LOH, which would not be detected by aCGH, or FISH. This must be done for all of the RB1+/+ samples.

Response: SNP analysis to determine presence of LOH at RB1 is irrelevant for the majority of labs participating in this study since they routinely use microsatellite analysis to determine the presence of LOH (loss of heterozygosity). The German, French, New Zealand, Dutch, and Toronto clinical labs used microsatellite analysis, in particular using polymorphic short tandem repeats (STRs) in RB1 intron 2 (D13S153) and intron 20 (RB1.20), as well as nearby flanking STRs, and determined that all the RB1+/+ tumors in this study were heterozygous at all informative loci in and near RB1. In the manuscript we state that none of the RB1+/+ tumors in the study showed loss of heterozygosity at RB1.

This is an important point since this implies that if these tumors were caused by RB1 mutations then each RB1 allele must be inactivated by a separate independent undetected RB1 mutation, and the probability of this is very small. We have added a sentence to Methods describing how the absence of LOH at RB1 was determined at each international site. In addition, SNP analyses was performed for 3 of the RB+/+MYCNA tumors (from Amsterdam) and there was no (copy number neutral) LOH detected for the RB1 locus. (Supplementary Figure 3).

4) IHC should be done for the entire cohort of 48 tumors used for the aCGH for both RB1 and MYCN. These slides must be read by at least 2 independent pathologists blinded to the genotype data. There is central pathology review in the COG retinoblastoma group for clinical trials and I am confident that those pathologists could review these data.

It is unclear what such experiments would be contributing. If to assess the presence of pRb by staining, the experiments would not confirm or deny the RB1 mutations, which can be non-functional and still have positive staining.Furthermore tumor staining interrogates endstages, which may differ from tumor initiation stages.

5) The authors never correlated MYCN amplification with increased expression. The IHC may help, but additional gene expression analysis and protein analysis should be performed. Indeed, several RB gene expression array datasets have been published and those data are publicly available. It is possible to analyze those data as this paper would suggest that a subset of tumors should have very high MYCN expression and that would correlate with MYCN gain.

In figure 3F MYCN mRNA expression is depicted for several RB+/+ MYCN amplified tumors, where it is clearly seen that tumors with high MYCN amplification show increased expression. (MYCN DNA copy numbers are indicated above the bars with MYCN mRNA expression.)

6)Dr. Gallie needs to perform a series of experiments whereby a known tumor sample is serially diluted with normal DNA to establish the exact level of normal cell contamination that can be tolerated in her assay. Those data should be present with the original Sanger traces in the supplemental data.

The reviewer suggests a series of experiments where a sample with a known RB1 mutation is serially diluted with normal DNA to establish the highest level of NCC that still allows detection of the mutation, also called the limit of detection. In the Toronto lab, the limit of detection has been determined in blinded studies, for purposes of lab certification. For copy number changes detected by QM-PCR or for mutations detected by sequence analysis, the Toronto lab can detect a copy number change present on one tumor allele in the presence of 30% tumor and 70% normal cell contamination (i.e. 15% of the DNA).

In addition, since 2006, the Toronto lab has tested each unilateral tumor by an allele-specific PCR screen (Rushlow et al, “Detection of Mosaic RB1 Mutations in Families with Retinoblastoma”, Hum Mut, 2008), a test for eleven common recurrent RB1 mutations; the eleven mutations make up over 22% of all RB1 mutations identified. The AS-PCR assay can detect mutant levels as low as 1% for each of the eleven mutations, so could detect a mutation present on one allele, even in the presence of one part tumor to 49 parts contaminating normal cells. Significantly, since introduction of the AS-PCR screen in 2006, although low-level mosaicism has frequently been detected in blood, we have seen only one tumor (out of approximately 200-300 tumors screened by AS-PCR) with a very low level of one of these eleven mutations; in that sample, the M1 and M2 events had already been identified and an M3 event was identified by AS-PCR as present on one allele in about 20% of the tumor cells, weakly detectable by sequence analysis. The rarity of finding such low level mutations in tumor supports the scarcity of RB tumors with large amounts of normal cell contamination.

In the routine clinical performance of tests to detect low level mosaics in blood, we dilute tumor samples with the identified AS-PCR mutation 1 in 50 with normal DNA as mutant controls in the testing.

7)In order to directly demonstrate the 95% detection of RB1 mutations in this particular cohort, Dr. Gallie should present all first hits and second hits for every tumor in this cohort. It will include point mutations, translocations, indels, splice site mutations, LOH, copy number neutral LOH and DNA hypermethylation. This is essential to establish the proportion of tumors in their cohort with detected 1 hit and 2 hits. Those with no hits are described but the others are not.

We have added the mutation names to table S5; The RB1+/- tumors show an RB1 mutation on only one allele; RB1-/- tumors show mutations on both alleles. As of October 10, 2012, after analyzing clinical samples for 1299 different families, our current procedures identified RB1 mutations sufficient for complete diagnosis in 596 of 621 (96.0%) bilateral probands, in 616 of 642 (96.0%) tumor from unilateral probands with no known family history, and in 34 of 36 (94.4) unilateral probands with prior family history of retinoblastoma.”

There are several places in the manuscript that require clarification or a more balanced presentation:

1) The title is not appropriate for a scholarly research manuscript. It is more appropriate for a review article or research highlight. I suggest changing the title to something more appropriate for Lancet.

2) Throughout the authors conclude that MYCN amplification is sufficient to drive retinoblastoma. Without whole genome sequencing, they cannot rule out other point mutations. This conclusion is not supported by the data.

No doubt there will be many other changes throughout the genome, most of uncertain significance, but nothing could challenge the common features of the MYCN amplified tumors, the most important of which is dramatic amplification of the MYCN oncogene.

We do not conclude that MYCN amplification is sufficient; we do indicate that there is a high likelihood that MYCN amplification is the rate limiting event initiating these tumors in very young children, whose cancers must start a significant time before birth.

3) The statistics need to be reviewed by an independent biostatistician and human geneticist who can take into account the possibility of normal cell contamination.

The statistics are completely described in the web appendix.

Normal cell contamination is addressed fully above; in summary, retinoblastoma tumors have very little normal cell contamination compare to other cancers. The high level MYCNA is the strongest evidence that these tumors are not normal cells.

4) The expression of MYCN protein needs to be shown with the whole protein blot not just the MW for MYCN.

It is not clear why the reviewer wants the whole protein blot for MYCN to be shown. It is obvious that it is the appropriate band that is shown, since the negative and positive controls, neuroblastoma cell lines without and with MYCN amplification, show no and high MYCN expression respectively.

5) It is not clear why the sample with MYCN amplification in the immunoblot doesn't have more MYCN protein than Y79, which does not have MYCN gain to the best of my knowledge from published FISH analysis.

The Y79 cell line used in our analysis indeed has amplification of the MYCN gene (53 copies, depicted in Figure 3F and Suppl Table 5) – this is why there are elevated levels of MYCN protein present in these cells, as opposed to the cell line WERI-Rb1, which possesses 2 copies of the MYCN gene (Suppl Table S5)

In addition, MYCN amplification in the Y79 cell line has been published previously by Xu et al. (Cell Volume 137 Issue 6, 2009 pages 1018-1031, Figure S12)

Reviewer #4: This is a very interesting study describing the novel finding of MYCN amplification in 50% of unilateral non-familial retinoblastomas without RB1 tumor mutations, promoter methylation or LOH at the RB1 locus. Through a very impressive collaborative effort the authors succeeded in collecting a large number of cases of this rare tumor. Using a combination of DNA sequencing, quantitative multiplex PCR, MLPA and RB1 promoter methylation analysis Rushlow et al identified retinoblastomas with RB1 aberrations (RB-/-) and those without (RB+/+). RB1 aberrations such as mutations, promoter methylation and LOH were found in the large majority of tumors, while no such aberrations in RB1 were found in 2.7% of these tumors. Of the 30 tumors without RB1

aberrations, 16 were found to have amplification of MYCN and this latter subset was found to present at a significantly younger age, with fewer copy number changes and more aggressive histology than those with RB1

aberrations. The authors thus conclude that MYCN amplification may initiate RB1+/+MYCNA retinoblastoma despite normal RB1 genes. The findings reported in this manuscript are novel and potentially important because they challenge the two-hit hypothesis in development of retinoblastoma. However, there are a few issues that are not completely clear about this conclusion.

Major:

1) First of all, the data showing that the RB1 genes are normal in the RB1+/+MYCNA tumors are not entirely convincing. For example, in Figure 3B, why is there decreased protein expression of RB1 in the RB1+/+MYCNA cell line A3, which is supposed to have intact RB?  

Figure 3B has been re-done.

Moreover, in Fig 3C, RB1 RNA expression in the E7 and P5 RB1+/+MYCNA tumors is less than in fetal and adult retina tissue (FR and AR respectively).   It is not clear why this is so, when supposedly these tumors have intact RB1.   Also, in Fig. 3F: the levels of RB1 mRNA in the RB1+/+MYCNA tumors (P5, E7, RB522) appear to be lower than that in fetal and adult retinal tissue.   This is seen in the quantification shown in Table S8, where RB1 mRNA expression in the RB1+/+MYCNA tumors is lower than that in either fetal or adult retina (although of course, not as low as in RB1-/- tumors).   This brings up the question of whether, even in the absence of mutation, LOH or promoter methylation, RB1 expression be decreased in

RB1+/+MYCNA tumors by miRNAs or competing endogenous RNAs? Did the authors make any attempts to determine differential regulation of miRNAs in these tumors? Could it be possible that even a small decrease in RB1 levels may be sufficient to induce a second hit i.e. MYCN amplification? This needs to be discussed in the paper.  

We do agree with the reviewer that RB protein and mRNA levels appear to be lower in RB+/+MYCNA tumors than in fetal and adult retina tissuein comparison to RB-/-, non MYCNA tumors. THE ABSOLUTE LEVELS OF PRB ARE DEPENDENT ON MANY FACTORS; THERE IS NO REASON THINK THAT THE TUMORS THAT ARE CLONES ARISING FROM AND UNKNOWN DEVELOPING RETINAL CELL WILL HAVE THE SAME LEVELS OF EXPRESSION AS FR OR AR. THE ONLY POINT IS THAT THEY HAVE A NORMAL SIZED MESSAGE AND PROTEING.

TO However the sole purpose of theis figure(s) was to demonstrate that RB+/+ tumors do express intact RB1 protein and mRNA, in comparison to RB-/- tumors which do not express any detectable protein, and very low mRNA. Thus investigations into the mechanism of differential RB1 levels between these two tumor types, although scientifically justified, are beyond the scope of this body of work that is to document a new type of RB+/+ retinoblastoma. As the reviewer suggested, this does not preclude the discussion of possible scenarios explaining this small decrease in RB1 levels in MYCNA tumors. This phenomenon has indeed been previously documented in neuroblastoma cell lines, where a study

by Lasorella and colleagues found a similar decrease in RB1 protein in neuroblastoma cell lines with MYCN amplification in comparison to non-amplified cell lines (Lasorella et al, Nature 2000). It is well known that MYCN amplification induces overexpression of the target gene Id2, which in turn inactivates the RB1 protein and induces cellular proliferation (Lasorella et al, Nature 2000). Although the authors do not investigate this discrepancy, there could be a possibility that ID2, being a transcriptional inhibitor, could inhibit proper transcription of the RB1 gene. This scenario remains to be investigated in both neuroblastoma and retinoblastoma. The reviewer also raises the possibility that RB1 gene expression could be affected by MYCN-regulated miRNAs or competing RNAs. We have not conducted such analyses, however it is worth mentioning that these may indeed affect RB1 expression in MYCNA tumors, and remain to be investigated. It is also well documented that pRB deficiency induces genomic instability (Manning and Dyson, Nat Rev Cancer 2012). Whether the small decrease in pRB protein exhibited in MYCNA tumors is sufficient to induce genomic instability and specific amplification of MYCN gene to the exclusion of other genomic changes (as demonstrated in this paper) is unlikely, as many studies (including ours) have observed multiple types of chromosomal aberrations in response to complete pRB depletion or inactivation (Dyson, Genes&Dev, 2010). In this study of almost 100 RB1-/- tumors, many had mutations that would cause small and/or large decreases in pRB protein but none showed MYCN amplification of over ten copies.

Regarding Figure 3B, The pRb expression in the RB+/+ MYCN amplified cell line is compared to lymphoblast and glioblastoma cell lines, these cell lines are obtained from different cell types which can explain the minor difference in expression level.

2) What was the survival outcome of the patients with MYCN amplified RB+/+ tumors compared with that of patients with RB+/+ tumors without MYCN amplification? In general, do patients with intact RB1 have a better outcome than those without? Adding this information would enhance the quality of this manuscript.

. Page 12, para 3, and p.15 para.2 "The unilaterally affected patients with RB1+/+MYCNA tumours in this study were cured by removal of their affected eye with no adverse outcomes". Some of the MYCNA patients in the study are now young adults. Unfortunately we have no information on outcome for the RB+/+ tumors without MYCN amplification, at this time. Because testing in some cases was done many years ago, it is time-consuming and sometimes impossible to locate all patients for follow-up.

3) Most striking is the very early age suggesting a cooperative effect of MYCN with other genes or with minimal loss of RB1. Do patients with RB+/+ tumors and no MYCN amplification present at a significantly younger age than those with RB1-/- tumors?

Patients with RB+/+ tumors but no MYCN amplification present at the same median age (23 months) as patients with RB-/- tumors (24 months) or with RB+/-

tumors (24 months). These data are in agreement with the mean age of diagnosis in a previous study of unilateral retinoblastomas (Shuler, Weber et al 2005)

4) Figure 3A: The difference in RB1 staining on IHC is not easily seen. Can the difference be quantified? Color figures may show the difference better. Is the pRB1 IHC staining in normal retina in the adult or fetal retina? Also MYCN staining in normal retina and RB-/- tumors needs to be included for comparative purposes. Although the middle panels showing pRB and MYCN staining in the RB1+/+MYCNA tumors is labeled as 50 µM, the MYCN stained panel looks smaller, please double check these magnifications.

FOR DAB STAINING COLOR DOES NOT CHANGE THE IMAGE QUALITY OVER GREY SCALE.

We agree with the reviewer that the differential MYCN IHC staining is difficult to appreciate in black/white images. Color images will therefore be incorporated into the revised manuscript. Magnifications have also been verified and corrected, and we will include MYCN staining in RB-/- tumors. However, normal retinal MYCN staining has already been included, as denoted by the asterisk adjacent to the T25 tumor in Figure 3A, middle panel.

**NOTE: As I had mentioned prior to submission, according to supplementary Table S5, T76 is actually an RB+/- tumor – this is most likely why we are seeing some RB1 staining in Fig 3A.

THE STAINING IN T76 IS THE NORMAL CELLS LIKE BLOOD VESSELS AND INFLAMATORY CELLS

I dug up a couple of RB tumors with MYCN staining that we could get Dr. Halliday to scan. I WOULD LIKE TO SEE THSE….

5) Figure 3B: This figure is not clear - the legend states that two bands of un-phosphorylated and phosphorylated pRB in RB1+/+MYCNA cell line A3 is shown - it is unclear where the total RB1 protein band and where the phosphorylated RB is. There should be two blots showing total RB and pRB. Also, what does V stand for? Is this a loading control?   If so, why is this not even?

There is a new figure for 3B which is much clearer, and clarifies all questions for the reviewer.

6) Figure 3B: Furthermore, the legend states "(B) Western blot shows the characteristic two bands of un-phosphorylated and phosphorylated pRB in RB1+/+MYCNA cell line A3, normal lymphoblasts (nLB, HSC-93) and glioblastoma cell line (GB, T98G), while the two RB1-/- cell lines (WERI-Rb1 and Y79) show no full length pRb". Some of the mentioned cell lines (e.g. HSC-93 and T98G) are not shown - this should be addressed.

Dorsman group?HSC-93 is the normal lymphoblast cell line (nLB) and T98G is the Glioblastoma cell line, so they are both indicated in the figure. We have however replaced Figure 3B with a western blot including fetal retina as a control, which we believe is a more appropriate control. Protein from fetal retina was not available at the time the original western blot was performed, therefore the

lymphoblast and glioblastoma cell lines were used initially.There is a new figure for 3B which is much clearer, and clarifies all questions for the reviewer.

7) .Fig. 3D: why is there strong MYCN expression (equivalent to the A3 RB1+/+MYCNA cell line) in the Y79 cell line when it is labeled as RB1-/-?

The Y79 cell line that was employed in our studies, although possessing a RB1-/- status, has acquired amplification of the MYCN gene over many years in culture. We indicate the specific copy number measured in Figure 3F as 53, hence the strong MYCN mRNA and protein expression depicted in our study.

Indeed, most RB1-/- retinoblastoma express significant levels of MYCN since they arise from neuroblasts. Levels of MYCN expression do not generally correlate with DNA amplification, and it is likely that the oncogenic driving force of amplification is loss of regulation, not absolute levels of protein.

Minor:

1) Figure 1A: please label X axis. RB1 copy number needs to be included in the plot. Also in the RB1+/+MYCNA tumor, the bimodal distribution of MYCN copy number is not evident, this should be shown.

The X-axis has been labeled as requested,

All the RB1+/+ tumors show two copies of RB1, by definition. Information on RB1 copy number of all the other tumors in the study is available in table S5 in the mutation data. A sample showing del P-27 in the mutation list indicates loss of one copy of the RB1 gene; If both alleles show delP-27, then both copies of RB1 are lost. We do not see the value of adding RB1 copy numbers to the box plots in figure 1A. The purpose of Fig 1A is to highlight differences in post-RB1 genomic copy numbers of genes on 1q, 6p, 16q and 2p in RB1+/+ tumors vs. tumors with RB1-/- or RB1+/- mutation status. Cursory examination of the data in Table S5 does not show any obvious association between RB1 copy number (within RB1-/- or RB1+/- groups) and copy numbers of genes on 6p, 2p, 1q and 16q, but such examination would be the subject of another study and not relevant to the data presented here.

The bimodal distribution of the RB1+/+ tumors appears clear in fig 1A. The median is the horizontal line within each small box. For the RB1+/+ MYCNA samples (top box) the median is 54 copies of MYCN and for the RB1+/+ without MYCN (second box down), the median is 3.09 copies of MYCN, showing bimodal distribution for the RB1+/+ group. We have added information re the median MYCN copy number value for each sub-group to figure 1A legend .

2) Figure 3: the legend does not correspond to the designated panels. Fig. 3D should be labeled C according to the legend.

Figure 3 has been re-done

3) Figure 4A: inadequately labeled, what do the individual symbols stand for? Also please label the X-axis.

The labels had been inadvertently moved to the back ground; the figures are now fixed.

4) Figure 4B: a higher magnification image would improve the quality of this figure.

WHAT IS HE TALKING ABOUT? FIG 4B IS AGE DX CURVES.

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