A Subset of Retinoblastoma Lacking RB1 Gene Mutations Have High-Level MYCN Gene Amplification

A Subset of Retinoblastoma Lacking RB1 Gene Mutations have High-level MYCN Gene Amplification

Stephanie Yee

A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Molecular Genetics University of Toronto

A Subset of Retinoblastoma Lacking RB1 Gene Mutations With High-Level MYCN Gene Amplification

Stephanie Yee

Master of Science

Department of Molecular Genetics University of Toronto

2010 Abstract

Retinoblastoma is the prototype genetic cancer caused by mutations disrupting the RB1 tumor suppressor gene. Following loss of RB1, retinoblastoma acquires further genetic changes in a characteristic set of oncogenes and tumor suppressors including gains of the oncogenes KIF14,

DEK, E2F3, and MYCN and loss of the tumor suppressor CDH11. The constellation of genetic changes is the postulated genetic pathway leading to retinoblastoma. However, advances in molecular diagnostic testing for RB1 gene mutations allows detection of at least one RB1 mutation in 98% of unilateral retinoblastomas leaving 2% of cases with undetectable RB1 mutations (RB1+/+ retinoblastoma). RB1+/+ retinoblastomas have high-level MYCN gene amplification (>30 copies) and few other genetic changes. In addition, RB1+/+ retinoblastoma present earlier than conventional RB1-/- retinoblastoma and show histologic features similar to

MYCN-amplified neuroblastoma. Altogether, this study describes a distinct genetic subset of retinoblastoma characterized by wild-type RB1 gene and high-level MYCN gene amplification.

Acknowledgments

I would like to express my sincerest gratitude to my supervisor Dr. Brenda L. Gallie for her guidance, encouragement and support throughout the project. I am grateful to Dr. Sanja Pajovic who has been a mentor to me and has provided assistance in numerous ways throughout this thesis. I thank Clarellen Spencer for her technical assistance in the laboratory. I thank my laboratory colleagues Dr. Ying Guo, Dr. Ghada Kurban, Dr. Brigitte Theriault, Tim To, Christine Yurkowski and Dr. Helen Dimaras for their friendship and support and for making my time in the lab a memorable experience.

I would like to thank Diane Rushlow, Jennifer Kennett, Dr. Paul Boutros and Anthony Mak for their technical and intellectual contributions.

The generous support from the Vision Science Research Program Graduate Student Scholarship is greatly appreciated.

Last but not least, I thank the people in my life who have given me years of unwavering love and support; my parents Kim Hook Yee and Beng Cheng Yee, my sister Sylvia Yee and Klint Ramdass.

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Table of Contents

Acknowledgments ...... iii

Table of Contents ...... iv

List of Tables ...... vii

List of Figures ...... viii

List of Appendices ...... ix

List of Abbreviations ...... x

Chapter 1 ...... 1

1 Introduction ...... 1

1.1 Retinoblastoma ...... 1

1.2 Current Retinoblastoma Treatment ...... 2

1.3 Molecular function of pRB ...... 2

1.4 Inactivation of pRB ...... 3

1.4.1 RB1 gene mutations ...... 3

1.4.2 Inactivation of pRB or RB pathway members ...... 4

1.5 Retinal Development ...... 5

1.6 Genomic changes in retinoblastoma ...... 5

1.6.1 1q Gain ...... 6

1.6.2 6p Gain ...... 7

1.6.3 16q Loss ...... 8

1.6.4 2p Gain ...... 8

1.7 MYCN amplification in neuronal tumors ...... 10

1.8 Genomic changes in MYCN-amplified neuroblastomas ...... 10

1.9 MYCN amplicon ...... 12

1.10 MYCN gene structure and expression ...... 13

1.11 MYCN protein and functions ...... 14

1.11.1 MYCN protein ...... 14

1.12 MYCN amplification in transgenic murine model of neuroblastoma ...... 16

Chapter 2 ...... 18

2 Characterization of RB1+/+ retinoblastoma ...... 18

2.1 Introduction ...... 18

2.2 Hypothesis ...... 19

2.3 Thesis Aims and Rationale ...... 19

2.3.1 Frequency of RB1+/+ retinoblastoma ...... 19

2.3.2 Characterize genomic profile of RB1+/+ retinoblastomas ...... 20

2.3.3 Determine mRNA and protein levels of RB1 and MYCN genes ...... 20

2.3.4 Analysis of clinical and pathological features of RB1+/+ retinoblastomas with MYCN amplification ...... 20

2.3.5 Determine the effect of MYCN silencing in MYCN-amplified retinoblastoma ..... 20

2.3.6 Designing a Mycn-overexpressing lentivirus ...... 20

2.4 Materials and Methods ...... 21

2.4.1 Samples ...... 21

2.4.2 RB1 gene mutation testing ...... 21

2.4.3 Gene-specific QM-PCR ...... 21

2.4.4 Sub-megabase resolution tiling array comparative genomic hybridization ...... 23

2.4.5 SMRT aCGH data analysis ...... 24

2.4.6 Statistics ...... 24

2.4.7 RT-PCR ...... 25

2.4.8 Immunohistochemistry ...... 25

2.4.9 Lentivirus production ...... 26

2.4.10 Lentivirus titration ...... 26

2.4.11 Proliferation assay ...... 27

2.4.12 Construction of Mycn-overexpression lentivirus ...... 27

2.4.13 Transduction of retinal explants ...... 27

2.4.14 Western blot analysis ...... 28

2.5 Results ...... 29

2.5.1 Frequency of RB1+/+ retinoblastoma across four independent sites ...... 29

2.5.2 Genomic profile of RB1+/+ retinoblastomas ...... 29

2.5.3 Expression of RB1 and MYCN mRNA transcripts and protein in RB1+/+ retinoblastomas ...... 41

2.5.4 Clinical features of RB1+/+ retinoblastomas ...... 42

2.5.5 Functional consequence of MYCN silencing in retinoblastoma with high levels of MYCN ...... 45

2.5.6 Construction of a Mycn-overexpression lentivirus ...... 46

Chapter 3 ...... 48

3 Discussion ...... 48

3.1.1 RB1+/+ MYCNA retinoblastoma is observed in independent clinical samples ...... 48

3.1.2 RB1+/+ MYCNA: a novel genetic subset of retinoblastoma ...... 48

3.1.3 MYCN-driven tumorigenesis ...... 49

3.1.4 Chromosome 8;13 translocation ...... 50

3.1.5 MYCN copy number as a rapid screen for RB1+/+MYCNA retinoblastoma ...... 51

3.1.6 Targeting MYCN ...... 51

3.1.7 Future directions ...... 52

References ...... 55

Appendices ...... 67

List of Tables

Table 1 Samples used for QM-PCR ...... 23

Table 2 List of primer sequences and expected product sizes used in RT-PCR analysis ...... 25

Table 3 Frequency of RB1+/+ retinoblastomas across four sites ...... 29

Table 4 Frequencies of M3-Mn changes in RB1+/+ versus RB1-/- retinoblastomas ...... 32

Table 5 Summary of retinoblastoma histopathological features in RB1+/+ MYCNA retinoblastomas ...... 44

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List of Figures

Figure 1 M3-Mn profile of M3-Mn copy number in 139 primary retinoblastomas and 6 cell lines ...... 31

Figure 2 Summary of chromosomal changes for 47 primary retinoblastomas, 5 retinoblastoma cell lines and 1 neuroblastoma cell line, IMR32 ...... 33

Figure 3 Number of CNAs per retinoblastoma tumors ...... 34

Figure 4 Number of aberrant base pairs in the different subtypes of retinoblastoma ...... 35

Figure 5 Whole genome tiling path array CGH karyogram of RB1+/+MYCNA retinoblastoma FA793 ...... 36

Figure 6 Specific amplification of the MYCN locus in RB1+/+ MYCNA RB1348 ...... 37

Figure 7 The minimal MYCN amplicon ...... 39

Figure 8 RB381 der(8)t(8;13)(q21.2;q14.12) ins(13;8)(q14; q21.2-q23.3) translocation ...... 40

Figure 9 Expression of pRB and MYCN in primary human retinoblastoma and normal retina .. 42

Figure 10 Age of diagnosis of 11 RB1+/+ MYCNA retinoblastomas ...... 43

Figure 11 Large prominent nucleoli in two RB1+/+MYCN A retinoblastomas, RB2237 and NZ499J ...... 45

Figure 12 MYCN shRNA knockdown in Y79 retinoblastoma cells ...... 46

Figure 13 Lentiviral overexpression of Mycn in HEK293 cells ...... 47

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List of Appendices

Table 6 Copy numbers of M3-Mn genes in retinoblastomas as measured by QM-PCR ...... 67

Table 7 SMRT aCGH alterations by sample ...... 71

List of Abbreviations

aCGH Array comparative genomic hybridization

ACVRL-1 Activin A receptor type II-like 1

ALK Anaplastic lymphoma kinase

ANOVA Analysis of variance

ATF Activating transcription factor 1

BAC Bacterial artificial chromosome bHLH Basic helix-loop-helix

BIM Bcl-2 interacting mediator of cell death

Bp Base pairs

BSA Bovine serum albumin

CAN NUP214, nucleoporin 214kDa

CASP9 Caspase 9, apoptosis-related cysteine peptidase

CDH8 Cadherin 8, type 2

CDH11 Cadherin 11, type 2, OB-cadherin (osteoblast)

CDH13 Cadherin 13, H-cadherin (heart)

Cdk Cyclin-dependent kinase cDNA Complementary DNA

CGH comparative genomic hybridization

ChIP Chromatin immunoprecipitation

CMV Cytomegalovirus

CNA Copy number alteration

CNV Copy number variation

CpG Cytosine next to Guanine

Cy3 Cyanine 3

Cy5 Cyanine 5

DAB 3,3´-diaminodbenzidine

DAPI 4’, 6-diamidino-2-phenylindole dCTP deoxycytidine triphosphate

DDX1 DEAD (Asp-Glu-Ala-Asp) box polypeptide 1

DEK DEK oncogene (DNA binding)

DFFA DNA fragmentation factor, 45kDa, alpha polypeptide

DFMO alpha-difluoromethylornithine

DM Double minute

DMEM Dulbecco’s modiﬁed Eagle's medium

DNA Deoxyribonucleic acid

E2F E2F transcription factor

ECL Electrochemiluminescence

EDTA Ethylenediaminetetraacetic acid

EGFP Enhanced green fluorescence protein

ESD Esterase D

ETAA16 Ewing tumor-associated antigen 16

Ets V-ets erythroblastosis virus E26 oncogene homolog 1

FAM49A Family with sequence similarity 49, member A

FAM84A Family with sequence similarity 84, member A

FBS Fetal bovine serum

GSK3 Glycogen synthase kinase 3

H3 Histone cluster 3, H3

HEK293 Human embryonic kidney 293

HEK293T Human embryonic kidney 293 SV40 large T-antigen

HLH Helix-loop-helix

HPV Human papilloma virus

HSR Homogeneously staining region

ID2 Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein

IE1 Intermediate early 1

INL Inner nuclear layer

IRES Internal ribosomal entry site kb Kilobase pairs kDa Kilodalton

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Ki67 Antigen identified by monoclonal antibody Ki-67

KIF14 Kinesin family member 14

M1 Mutation1

M2 Mutation 2

M3-Mn Mutation 3-n

Mad MAX dimerization protein 1

MAP Small G protein signaling modulator 3

MAX MYC associated factor X

Mb Megabase pairs mCMV Murine cytomegalovirus

MDM2 Mdm2 p53 binding protein homolog

MEIS Meis homeobox miR microRNA

MLPA Multiplex ligation-dependent probe amplification

MRG Minimal region of gain mRNA Messenger ribonucleic acid

Mnt MAX binding protein

Mxi MAX interactor 1

Myc v-myc myelocytomatosis viral oncogene homolog (avian)

MYCN v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (avian)

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MYCNA High-level MYCN amplification (>10 copies)

MYCNOS N-myc opposite strand

NAG Neuroblastoma amplified sequence

NAHR Nonallelic homologous recombination

ODC1 Ornithine decarboxylase 1

ONL Outer nuclear layer

P0 Postnatal day 0 p19ARF cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4), alternate reading frame p21 cyclin-dependent kinase inhibitor 1A p27 cyclin-dependent kinase inhibitor 1B p53 Tumor protein p53 p107 Retinoblastoma-like 1 p130 Retinoblastoma-like 2

Pax Paired-box 1

PBS Phosphate-buffered saline

PCAN1 Gene differentially expressed in prostate

PRC1 Protein-regulating cytokinesis 1 pRB Retinoblastoma protein

PP1 Protein phosphatase 1

PCR Polymerase chain reaction

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PNA Peptide nucleic acid

QM-PCR Quantitative multiplex-polymerase chain reaction

RB Retinoblastoma

RB1 Retinoblastoma 1

REXOIL1 REX1, RNA exonuclease 1 homolog (S. cerevisiae)-like 1

RIPA Radioimmunoprecipitation assay buffer

RNA ribonucleic acid

RPE Retinal pigment epithelium rRNA Ribosomal RNA

RT-PCR Reverse transcriptase PCR

S phase Synthesis phase

SDS Sodium dodecyl sulfate shRNA Short hairpin RNA siRNA Small interfering RNA

SMRT Sub-megabase resolution tiling

SP1 SP1 transcription factor

SSC Saline sodium citrate

SSTR2 Somatostatin receptor 2

TAg SV40 large T antigen

TBP TATA binding protein

TBS Tris-buffered saline

TH Tyrosine hydroxylase tRNA Transfer RNA

UL97 Tegument serine/threonine protein kinase

Wnt Wingless-type MMTV integration site family

WPRE Woodchuck hepatitis post-transcriptional regulatory element

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Chapter 1 1 Introduction 1.1 Retinoblastoma

Retinoblastoma is a childhood cancer of the eye that affects approximately 1:18000 live birth children (Devesa 1975). In the two-hit hypothesis, Alfred Knudson correctly postulated that retinoblastoma is caused by at least two mutational events (Knudson 1971). The first clues to the location of the mutated gene came from studies of chromosome 13q deletion syndrome in which affected children presented with retinoblastoma along with developmental defects (Lele, Penrose et al. 1963; Grace, Drennan et al. 1971; Wilson, Towner et al. 1973). Linkage between retinoblastoma and the esterase D gene (ESD) narrowed the search to chromosomal band 13q14 (Sparkes, Sparkes et al. 1980; Connolly, Payne et al. 1983). Restriction endonuclease mapping identified different restriction fragment length polymorphisms of DNA isolated from chromosome 13 (Cavenee, Dryja et al. 1983; Dryja, Rapaport et al. 1986). This was followed by cloning of a DNA fragment present in many tumor types but missing in retinoblastomas and osteosarcomas which led to the discovery of the gene we now know as RB1 (Friend, Bernards et al. 1986; Lee, Bookstein et al. 1987).

Retinoblastoma can present either in one eye (unilateral) or both eyes (bilateral). About 60% of patients are unilaterally affected with sporadic disease i.e. no family history. About 40% of patients are bilaterally affected, often with multifocal tumors in both eyes. In general, bilateral patients are diagnosed earlier than unilaterally affected patients with median ages of 11 and 22 months respectively. In the unilateral form of the disease, two mutations to RB1 occur in a susceptible retinal cell. In the bilateral form of the disease, one mutation is either inherited or occurs de novo in a germ cell and the second mutation is acquired in the somatic retinal cell. Individuals with a germline RB1 mutation have a lifetime susceptibility to second primary tumors such as osteosarcoma (Matsunaga 1980; Draper, Sanders et al. 1986; Marees, Moll et al. 2008).

1.2 Current Retinoblastoma Treatment

Since retinoblastoma can be a fatal disease if metastasis occurs, the first goal of treatment is to save the patient’s life and secondly to salvage vision. In developed countries it is often detected early and the survival rate is > 95% (Chintagumpala, Chevez-Barrios et al. 2007). Treatment given depends on whether one or both eyes are affected. In unilateral cases, the patient is often cured by enucleation or removal of the affected eye. In bilateral cases, a variety of treatments are available, including enucleation, external beam therapy, cryotherapy, laser photocoagulation, thermotherapy, brachytherapy and systemic chemotherapy (Lin and O'Brien 2009). Although current treatments are successful in curing retinoblastoma, many patients experience serious side effects from chemotherapy and radiation therapy. In cases where external beam radiation is used, there is a significant increased risk of secondary tumors later in life. There remains a need for treatments with less toxicity and this might be achieved through targeting specific molecular targets. The oncogene MYCN is gained in 16% of primary retinoblastomas (Bowles, Corson et al. 2007). Tonelli et al developed a peptide that specifically targeted the MYCN transcript and found that growth in neuroblastoma cells could be inhibited (Tonelli, Purgato et al. 2005). Thus, there is potential to direct therapies that specifically inhibit MYCN-driven tumorigenesis. Though developing targeted therapies specific for secondary genomic changes may treat only a small subset of retinoblastoma patients, it may be one way to reduce side effects associated with general systemic therapies given today.

1.3 Molecular function of pRB

The RB1 gene was the first tumor suppressor to be discovered. It is a principal regulator of the cell cycle and also has roles in differentiation, apoptosis and senescence (van den Heuvel and Dyson 2008). pRB, along with the proteins p107 and p130, belong to a family of proteins containing a pocket domain. Together they work at different times in the cell cycle to coordinate the expression of S phase genes by binding to different targets, most notably the E2Fs. There are at least 8 different E2Fs in the mouse. E2F1, E2F2 and E2F3a are activators of gene transcription and E2F3b, E2F4 and E2F5 repress transcription by recruitment of chromatin modifying enzymes. The E2F C-terminal domain mediates binding to the pocket domains of the pRB family proteins. Each pRB family protein interacts with different subsets of E2Fs, and they have overlapping but unique roles in cell cycle control. For example, pRB mainly associates

3 with E2F1, E2F2, and E2F3a. When cyclin dependent kinases phosphorylate pRB, a conformational shift in pRB results in the release of the E2Fs allowing them to bind to promoters of S phase genes. The functional inactivation of pRB due to mutation or inactivation by binding to cellular or viral proteins can result in uncontrolled cell cycling, failed differentiation and apoptosis.

1.4 Inactivation of pRB

1.4.1 RB1 gene mutations

The RB1 gene is located on chromosome 13q14.2 and is composed of 27 exons distributed along 183 kb of genomic sequence. At its 5’ end, lies a promoter with a CpG island that is normally unmethylated. The promoter has sequences recognized by transcription factors such as Sp1, ATF but does not contain TATA or CAAT motifs. In addition, the RB1 promoter has an E-box (Martelli, Cenciarelli et al. 1994) which can be recognized by the Myc family of transcription factors. Patients who are heterozygous at the RB1 locus can develop tumors in one eye or both eyes (variable expressivity) and in rare cases none at all (reduced penetrance) since it is due to chance that a second mutation will occur in the other normal allele (Lohmann and Gallie 2004). However, not all of the phenotypic variations can be accounted for by stochastic effects and it is now known that the penetrance and phenotypes vary in part due to the nature of the predisposing mutation (Lohmann and Gallie 2004). The RB1 gene does not contain any hot spots for mutations and all classes of mutations can be detected in retinoblastomas and are distributed throughout the gene (Richter, Vandezande et al. 2003). The majority of germline mutations are null mutations which include whole gene and exonic gene deletions, splice mutations and nonsense mutations (Richter, Vandezande et al. 2003). Nonsense mutations make up the majority of the null mutations in both bilateral and unilateral tumors (Richter, Vandezande et al. 2003). In some genetic diseases, the phenotype varies depending on the location of the stop codon, however, in retinoblastoma nonsense mutations result in no transcript being produced, presumably by nonsense mediated decay (Frischmeyer and Dietz 1999; Wen and Brogna 2008). In a heterozygous cell, this results in only transcripts produced by the normal allele. Aberrant splicing is the second most important class of mutations and is caused by point mutations affecting splice acceptor or donor sequences in intronic and exonic sequences. Splice mutations in set splice sites can lead to premature stop codons or exon skipping resulting in complete

4 penetrance. However, splice mutations in less conserved sequences are more likely to result in incomplete penetrance (Lohmann and Gallie 2004). Missense mutations are in-frame changes to nucleotide sequence that result in substitution of an amino acid residue. The majority of missense mutations (81%) occur in the A/B “pocket” domain (Richter, Vandezande et al. 2003) essential for interaction of pRB with E2F transcription factors (DiCiommo, Gallie et al. 2000). Missense mutations often result in incomplete penetrance because some mutant alleles retain partial activity (Otterson, Chen et al. 1997).

Richter et al developed a highly sensitive set of molecular tests to determine RB1 gene mutations (Richter, Vandezande et al. 2003). The tests consists of sequencing of all 27 exons and promoter, quantitative-multiplex PCR (QM-PCR) to detect gains or deletions in the promoter and exons, promoter methylation assay and allele-specific PCR to detect 11 point mutations that recur with significant frequency (Richter, Vandezande et al. 2003). Using this method, both mutations can be identified in >95% of bilateral tumors and at least one mutation can be identified in >98% (94%, both mutations identified and 4.8% one mutation identified) of unilateral tumors leaving 1.6% of unilateral retinoblastoma with no evidence of mutations (Rushlow, Piovesan et al. 2009).

1.4.2 Inactivation of pRB or RB pathway members

Aside from mutations to the RB1 gene sequence, there are many ways to inactivate the wild-type protein, pRB. The holoenzyme protein phosphatase 1, PP1, binds to and dephosphorylates pRB restoring its cell cycle negative regulatory function at mitotic exit (Vietri, Bianchi et al. 2006). The loss of any of the catalytic subunits of PP1 could result in the deactivation of pRB. Likewise, overexpression of proteins that phosphorylate and inactivate pRB such as cyclin D/cdk4/6 and cyclin E/cdk2 could lead to absence of pRB function. When overexpressed the cellular protein, inhibitor of differentiation 2, ID2, can inhibit pRB by binding to and sequestering pRB away from its normal binding partners (Iavarone, Garg et al. 1994). Genomic gain or overexpression of genes suppressed by pRB such as E2F transcription factors can ultimately lead to progression through the cell cycle and unrestrained proliferation.

Viral proteins such as human papillomavirus (HPV) E7 and human cytomegalovirus UL97 can bind and destabilize or hyperphosphorylate pRB (Kamil, Hume et al. 2009). However, a recent screen was performed to look for the presence of several pRB-inactivating DNA tumor viruses

5 which included subtypes of HPV, adenovirus and others in 40 RB1-/- retinoblastomas but could not find any evidence of viral sequences in the tumors (Gillison, Chen et al. 2007). Hence, viruses may not play a role in the development of retinoblastoma.

1.5 Retinal Development

Eye development begins in the 18-day embryo with outpocketing of the forming neural tube to form two optic grooves on either side. The optic grooves grow larger to become optic vesicles which make contact with the surface ectoderm. Together the surface ectoderm and the optic vesicle invaginate to form the lens placode and optic cup respectively. The lens placode fuses with itself separating from the surface ectoderm to become the lens vesicle and later the lens. The optic cup has two layers separated by a lumen, called the intraretinal space. The outer layer gives rise to the retinal pigmented epithelia (RPE) and the inner layer gives rise to the neural retina (Chow and Lang 2001). Starting from the layer bordering the intraretinal space, the neural retina gives rise to the rod and cone photoreceptors whose cell bodies make up the outer nuclear layer (ONL) followed by bipolar, horizontal, amacrine, and Müller cells which make up the inner nuclear layer (INL) (Dyer and Cepko 2001). Internal to INL is the ganglion cell layer which also includes some amacrine cells.

1.6 Genomic changes in retinoblastoma

Following loss of RB1 a specific set of genomic losses and gains drives benign non-proliferative retinomas into malignant retinoblastomas (Dimaras, Khetan et al. 2008). Early cytogenetic studies identified gains of chromosome 1q, 2p and 6p and loss of 16q to be the most common abnormalities in retinoblastoma tumors (Kusnetsova, Prigogina et al. 1982; Squire, Gallie et al. 1985; Pogosianz and Kuznetsova 1986). Using the higher resolution of comparative genomic hybridization several groups confirmed that these changes occurred frequently (Mairal, Pinglier et al. 2000; Chen, Gallie et al. 2001; Herzog, Lohmann et al. 2001; Lillington, Kingston et al. 2003; van der Wal, Hermsen et al. 2003; Zielinski, Gratias et al. 2005). A summary of these six studies showed that gain of 1q, 2p and 6p and loss of 16q occurred in 53%, 34%, 54% and 32% of retinoblastomas respectively (Corson and Gallie 2007).

Each of the chromosomal regions of gain or loss contained many genes. To differentiate true oncogenes and tumor suppressors from the “passengers,” i.e. genes that were gained or loss due

6 to close proximity to the “driver” or causative gene, differential overexpression or decreased expression of genes in minimal regions of gain or loss in tumor versus normal tissue respectively was assessed. To determine the minimal overlapping regions of gain and loss, copy numbers of sequence tagged sites spanning evenly across the chromosomal regions of interest were measured using QM-PCR or real-time PCR. Eventually a peak or “hotspot” was found to be the most common site gained or lost in the sample of primary tumors. This approach narrowed the search to a few genes. Expression of these genes was then assessed at the mRNA and protein levels in tumor versus normal tissue to identify the overexpressed potential oncogenes or under expressed potential tumor suppressor genes. In the next sections, the candidate genes found on each of the above mentioned chromosomes will be discussed.

1.6.1 1q Gain

1.6.1.1 KIF14

Using the QM-PCR approach described above, KIF14 was identified as a target of 1q gain (Corson, Huang et al. 2005). KIF14 is a mitotic kinesin motor protein that interacts with microtubule bundling protein PRC1 (protein-regulating cytokinesis 1) and citron kinase and has an essential role in regulating cytokinesis (Gruneberg, Neef et al. 2006). Corson et al reported that out of 14 genes in 1q32 minimal region of gain only KIF14 was overexpressed at 341-fold higher compared to normal human retina (Corson, Huang et al. 2005). In addition, KIF14 was gained frequently not only in retinoblastoma but in other cancers including breast, lung and medulloblastoma. Higher levels of KIF14 mRNA expression in breast cancer correlated with more aggressive tumors (Corson and Gallie 2006). siRNA-mediated knockdown of KIF14 in a cervical and non-small cell lung cancer cells resulted in decreased proliferation and ability to form colonies in soft-agar (Corson, Zhu et al. 2007). KIF14 knockdown in ovarian cancer cells lead to similar results and overexpression of KIF14 in ovarian cancer cells significantly increased proliferation and soft-agar colony formation (Brigitte Theriault, personal communication).

1.6.2 6p Gain

1.6.2.1 DEK and E2F3

Early karyotypic studies showed that the 6p isochromosome is one of the most common regions of genomic gains in retinoblastoma (Squire, Gallie et al. 1985). QM-PCR analysis of 70 retinoblastoma tumors was used to narrow down the minimal region of gain on 6p to a 0.6-Mb size region at 6p22 (Chen, Pajovic et al. 2002). Through the study of expression level of mRNA and protein levels of 6 genes in the 6p22 MRG the oncogenes DEK and E2F3 were identified as targets of 6p22 gain since they were the only 2 genes to show overexpression in tumor compared to normal adjacent retina (Orlic, Spencer et al. 2006). In addition, 3 out 4 retinoblastoma cell lines showed increased copy number of DEK and E2F3 genes due to isochromosome 6p formation and the cell lines that showed further rearrangements on 6p shared the common translocation breakpoint located at 6p22 (Paderova, Orlic-Milacic et al. 2007). DEK is a nuclear protein (Kappes, Burger et al. 2001) that binds to chromatin and is involved in modifying DNA structure through the introduction of supercoils (Kappes, Scholten et al. 2004). It is highly expressed in proliferating cells and its phosphorylation status oscillates with the cell cycle peaking during G1 phase (Kappes, Damoc et al. 2004). In acute myeloid leukemia it is involved in a fusion gene called DEK-CAN resulting from a t(6;9) translocation (von Lindern, Breems et al. 1992), however the transforming ability of this fusion gene is debated as overexpression of DEK-CAN failed to inhibit differentiation of myeloid precursor cell line (Boer, Bonten-Surtel et al. 1998). Nevertheless, DEK is frequently overexpressed in other types of tumor cells as well including hepatocellular carcinoma (Kondoh, Wakatsuki et al. 1999), melanoma (Grottke, Mantwill et al. 2000) and acute myeloid leukemia (Casas, Nagy et al. 2003).

E2F3 is an important cell cycle gene. The E2F3 locus encodes two protein products E2F3a and E2F3b through two alternate promoters (Leone, Nuckolls et al. 2000). The expression patterns of E2F3a and E2F3b contrast each other during the cell cycle with E2F3a expressed in proliferating cells and peaking during G1 and E2F3b expressed at a constant level during the cell cycle (Leone, Nuckolls et al. 2000). E2Fs can be activators or repressors of gene transcription. E2F3a is considered an activator which controls DNA synthesis and cell cycle progression genes (Humbert, Verona et al. 2000). E2F3b is considered a repressor which has been shown to interact with pRB in quiescent cells (Leone, Nuckolls et al. 2000). One of the genes E2F3b represses is the p19ARF tumor suppressor gene which activates the p53 pathway (Aslanian,

Iaquinta et al. 2004). E2F3 overexpression works in concert with inactivation of the RB pathway. A study by Hurst et al showed that 6p22.3 amplification and E2F3 overexpression were always associated with loss of pRB expression in bladder cancer (Hurst, Tomlinson et al. 2008). These data suggest that DEK and E2F3 play important roles in cell proliferation and may be potential targets in retinoblastoma treatment.

1.6.3 16q Loss

1.6.3.1 CDH11

CDH11 (Cadherin 11) is a member of the cadherin family of molecules. They are cell-cell adhesion molecules that have important roles in a wide variety cellular functions including cell polarity, cell signaling, most notably through the β-catenin-Wnt pathway and regulation of growth factor signaling. Chromosome 16q is lost in 32% of retinoblastomas (Corson and Gallie 2007). Using a combination of loss of heterozygosity and QM-PCR analyses, the minimal region of loss was narrowed to a 2.62 Mb region at 16q22 (Marchong, Chen et al. 2004). The 16q22-24 region harbours a cluster of cadherin genes including CDH8, CDH11 and CDH13. Marchong et al demonstrated that a sequence tagged site, WI5835, located in intron 2 of the CDH11 gene was lost in 54% of retinoblastomas and that 91% of retinoblastomas with loss of this marker also had reduced or no expression of the CDH11 protein (Marchong, Chen et al. 2004). The study also showed that advanced transgenic murine SV40 large T antigen–induced (TAg) retinoblastoma tumors displayed a loss of Cdh11 mRNA transcript in contrast to smaller earlier tumors which still expressed Cdh11 protein thus supporting the hypothesis that CDH11 loss promotes progression (Marchong, Chen et al. 2004). In a more recent study, the same authors used TAg Cdh11 null mice to show that Cdh11 loss caused larger tumors and higher levels of programmed cell death than in mice with normal Cdh11 alleles, suggesting that Cdh11 functions as a tumor suppressor by promoting apoptosis in tumor cells (Marchong, Yurkowski et al. 2009, submitted).

1.6.4 2p Gain

1.6.4.1 MYCN

MYCN (v-myc avian myelocytomatosis viral-related oncogene, neuroblastoma-derived), located on chromosome 2p24.3 is thought to be the major target of 2p gain and amplification in

9 retinoblastoma, neuroblastoma and several other neuroectodermal cancers. It was first identified in the early 1980s by Khol et al who cloned a gene with sequence homology to the oncogene c- myc from neuroblastoma cell lines (Kohl, Kanda et al. 1983). MYCN amplification commonly manifests as extrachromosomal DNA units called double minutes, DM (Kohl, Kanda et al. 1983), or intrachromosomal tandem repeats called homogeneously staining regions (HSR) (Amler and Schwab 1989). Its role as an oncogene was supported by studies showing amplification occurred in advanced metastatic stages of neuroblastoma (Brodeur, Seeger et al. 1984; Brodeur, Azar et al. 1992; Chan, Gallie et al. 1997). Regardless of whether MYCN occurs as DMs or HSRs, there is no difference in survival outcome, and amplification of MYCN in either form is associated with poor prognosis in neuroblastoma (Moreau, McGrady et al. 2006). In retinoblastoma MYCN amplification was first observed in primary tumors and the retinoblastoma cell line Y79 by Lee et al (Lee, Murphree et al. 1984). Many retinoblastomas highly express MYCN (Squire, Goddard et al. 1986) and 3% of primary tumors and 29% of retinoblastoma cell lines have MYCN genomic amplification (Bowles, Corson et al. 2007) suggesting MYCN amplification gives the cell a proliferative advantage.

1.6.4.2 ID2

The gene ID2 is a potential target of 2p gain and is located at 2p25. It is a member of the HLH family of transcription factors and is a transcriptional target of MYCN. Although ID proteins contain the HLH domain they lack the basic domain required for DNA binding and therefore act as dominant negative antagonists of bHLH proteins by sequestering them in non-functional complexes. The ID proteins are also known as inhibitors of differentiation because the bHLH proteins that ID proteins bind to, such as Ets and Pax, are transcription factors that regulate differentiation. Interestingly, Id2 was found to bind specifically to the hypophosphorylated form (active) of pRB and both of its related proteins p107 and p130 and could reverse the growth suppressive activities of pRB, p107 and p130 (Iavarone, Garg et al. 1994; Lasorella, Iavarone et al. 1996). Its role in tumorigenesis was further demonstrated when it was shown that the Id2-null mutation could prevent the formation of pituitary tumors in Rb1+/- mice (Lasorella, Rothschild et al. 2005). ID2 may represent a possible means by which MYCN can exert its pRB inhibitory action. The significance of ID2 in neuroblastoma however is controversial. On one hand, clinical studies fail to find a correlation between ID2 overexpression and MYCN expression or survival and thus that evidence suggests it lacks prognostic significance (Alaminos, Gerald et al.

2005). On the other hand, functional studies using MYCN-targeting silencing RNA in neuroblastoma cell lines show that ID2 is regulated by MYCN (Woo, Tan et al. 2008). Thus how ID2 contributes to tumorigenesis in neuroblastoma and retinoblastoma remains to be fully defined.

1.7 MYCN amplification in neuronal tumors

MYCN amplification occurs in tumors of neuroectodermal origin. In addition to retinoblastoma and neuroblastoma, other cancers include glioblastoma (Hui, Lo et al. 2001), medulloblastoma (Bayani, Zielenska et al. 2000; Fruhwald, O'Dorisio et al. 2000), rhabdomyosarcoma (Barr, Duan et al. 2009), and small cell lung carcinoma (Nau, Brooks et al. 1986; Dietzsch, Lukeis et al. 1994; Salido, Arriola et al. 2009). In neuroblastoma, MYCN amplification occurs in 25-30% of primary tumors (Fix, Lucchesi et al. 2008) and correlates strongly with advanced stages and indicates poor prognosis (Brodeur, Seeger et al. 1984; Brodeur, Azar et al. 1992; Chan, Gallie et al. 1997; Fix, Lucchesi et al. 2008). In many cases, MYCN amplifications occur in the form of DMs or HSRs (Bown 2001; Moreau, McGrady et al. 2006). In the large nucleolar neuroblastoma subset, MYCN amplification is associated with distinct histology characterized by large prominent nucleoli (Tornoczky, Semjen et al. 2007). Large prominent nucleoli are significantly associated with poor prognosis in neuroblastoma (Ambros, Hata et al. 2002). In retinoblastoma, the prognostic significance of MYCN amplification is not as clear since retinoblastomas with high level MYCN amplification do not seem to show adverse histology nor do the patients show worse survival (Lillington, Goff et al. 2002). It is important to note, however, that treatment of retinoblastoma has > 95% cure rate (Chintagumpala, Chevez-Barrios et al. 2007) largely due to enucleation prior to extension of tumor outside the eye, precluding outcome analysis. In addition, MYCN amplification in neuronal tumors is often accompanied by other genomic changes, most commonly 1p36 loss and/or 17q gain. Overall, MYCN-amplified tumors have a less complex pattern genomic copy number alterations compared to other low or high risk neuroblastoma subtypes (Mosse, Diskin et al. 2007).

1.8 Genomic changes in MYCN-amplified neuroblastomas

Unlike retinoblastoma, neuroblastoma is a very heterogeneous disease with outcomes ranging from spontaneously regressing to aggressive metastatic with poor prognosis. This heterogeneity is reflected in the pattern of genetic changes in the different subtypes of neuroblastoma. In the

11 past few years, array comparative genomic hybridization (aCGH) technology has been used to characterize genomic changes and stratify stage and outcomes (Mosse, Diskin et al. 2007; Fix, Lucchesi et al. 2008; Janoueix-Lerosey, Schleiermacher et al. 2009). The most aggressive forms of disease are divided into two classes, with MYCN amplification and without. In neuroblastoma, 1p36 deletion is strongly associated with MYCN amplification, however, 1p deletion also occurs in high-risk tumors without MYCN amplification (Chen, Bilke et al. 2005; Janoueix-Lerosey, Schleiermacher et al. 2009; Lavarino, Cheung et al. 2009). 10q loss occurs in 53% of MYCN-amplified neuroblastoma (Mosse, Diskin et al. 2007). It had been speculated that amplification of MYCN would be associated with higher genomic instability (Schwab 1999) but surprisingly, recent findings show that MYCN-amplified neuroblastomas tend to have fewer genomic changes in comparison to other subtypes including high risk neuroblastomas without MYCN amplification (Chen, Bilke et al. 2005; Mosse, Diskin et al. 2007). Gains of 17q are a common change across all neuroblastomas (Mosse, Diskin et al. 2007; Janoueix-Lerosey, Schleiermacher et al. 2009). There is a strong inverse relationship between 11q deletion and neuroblastomas without MYCN amplification (Guo, White et al. 1999; Chen, Bilke et al. 2005; Lavarino, Cheung et al. 2009). Despite identification of common regions of copy number alterations, few candidate genes have been identified aside from MYCN. Candidate targets of 1p loss have been suggested but most have since been rejected (White, Maris et al. 1995; Grenet, Valentine et al. 1998). Abel et al proposed apoptotic pathway genes CASP9 and DFFA as candidate targets of 1p36 loss since the two genes are located within the minimal region of loss (Abel, Sjoberg et al. 2002). However, even though higher stage neuroblastoma showed a slight decrease in expression of both genes compared to lower stage neuroblastoma, the study failed to show functional evidence that either CASP9 or DFFA were definitive targets of 1p36 loss. Recently, Wei et al identified a microRNA miR-34a, located on 1p36 that directly targeted MYCN (Wei, Song et al. 2008); exogenous expression of miR-34a in neuroblastoma cell lines with MYCN-amplification decreased proliferation by increasing apoptosis. For 17q gain, the gene SSTR2 was proposed as a potential target but no correlation between 17q gain and SSRT2 expression was found, nor were any mutations found in the gene in neuroblastoma tumors (Abel, Ejeskar et al. 1999). Four independent groups identified ALK, anaplastic lymphoma kinase, as the cause of hereditary neuroblastoma in 2008 (Chen, Takita et al. 2008; George, Sanda et al. 2008; Janoueix-Lerosey, Lequin et al. 2008; Mosse, Laudenslager et al. 2008). Mosse et al demonstrated linkage to chromosomal bands 2p23-24 in neuroblastoma pedigrees (Mosse,

Laudenslager et al. 2008) and Chen et al showed that ALK was a target of recurrent gains and amplification on 2p. All four groups found activating somatic mutations in high risk neuroblastomas and provided functional evidence that ALK had transforming ability (Chen, Takita et al. 2008; George, Sanda et al. 2008; Janoueix-Lerosey, Lequin et al. 2008; Mosse, Laudenslager et al. 2008). However, mutations and gains of ALK only account for a small percentage of neuroblastomas and continued analysis of genomic changes is needed to identify more candidate oncogenes and tumor suppressors.

1.9 MYCN amplicon

The MYCN amplicon ranges in size from 100kb to >1Mb (reviewed in Schwab 2004). The amplicon is arranged in tandem repeats of DNA segments with the intact MYCN coding region in the central location (Amler and Schwab 1989; Pandita, Godbout et al. 1997). Sequence analysis has not detected mutations within the MYCN gene (Stanton, Schwab et al. 1986; Ibson and Rabbitts 1988) therefore it is likely that the increased gene dosage of the wild type gene is what contributes to tumorigenesis. MYCN is often co-amplified with other neighboring genes. The two most frequently co-amplified genes are DDX1 and NAG at 65% and 20-40% of MYCN- amplified neuroblastomas, respectively (Scott, Board et al. 2003; Weber, Imisch et al. 2004). Co-amplification of these genes has also been documented in retinoblastoma (Godbout and Squire 1993) and other neuronal cancers (Fruhwald, O'Dorisio et al. 2000; Barr, Duan et al. 2009; Hodgson, Yeh et al. 2009). DDX1 is located 400kb telomeric to MYCN and NAG just telomeric to DDX1 (Amler, Schurmann et al. 1996; Scott, Board et al. 2003). There have been conflicting reports as to the prognostic significance of DDX1 co-amplification. A few groups have reported that co-amplification of DDX1 in high risk MYCN-amplified neuroblastomas correlate with a more favorable outcome and higher survival rate within the MYCN-amplified group (Weber, Imisch et al. 2004; Kaneko, Ohira et al. 2007). However, De Preter et al provided evidence that DDX1 co-amplification is only coincidental due to proximity to MYCN and that there is no significant correlation to better event free or overall survival (De Preter, Speleman et al. 2005). Despite these contradicting reports on DDX1, it is important to note that MYCN has been the only consistent gene on the amplicon and that none of the co-amplified genes have been reported to amplify independent of MYCN.

1.10 MYCN gene structure and expression

The MYCN gene was first identified in neuroblastoma cells and was so named due to its similarity in nucleotide and protein sequence to the well characterized oncogene MYC (Kohl, Kanda et al. 1983). Like MYC, MYCN is made up of three exons; the first exon contributes to a long 5’ untranslated region and the second and third exons make up the coding regions sharing an overall 32% amino acid sequence identity with MYC (Stanton, Schwab et al. 1986). MYCN transcription gives rise to two forms of mRNA resulting from use of two separate promoters each with a different first exon (Stanton and Bishop 1987). Both MYCN mRNAs are unstable and have short half lives of approximately 15 minutes (Stanton and Bishop 1987).

Despite the homology of MYCN and MYC, their patterns of gene expression differ spatially and temporally. Both are expressed in proliferating cells; however, MYCN is expressed almost exclusively in embryonic tissue whereas MYC is expressed in proliferating cells of both embryonic and adult tissues. In the developing mouse embryo, Mycn and Myc have complementary patterns of expression; Mycn is expressed mainly in neural tissues and myc is expressed in proliferating cells that do not express Mycn (Hurlin, Queva et al. 1997; Hurlin 2005). Human developing brain normally expresses levels of MYCN that are comparable to expression from 150 gene copies (Grady, Schwab et al. 1987) thus it has been speculated that high levels of MYCN protein in tumors with normal copy number of MYCN may reflect the cell of tumor origin or undifferentiated state of the tumor (Squire, Goddard et al. 1986). Expression declines when cells become differentiated (Martins, Zindy et al. 2008). MYCN amplification does not always translate to high expression levels (Matthay 2000; Tang, Zhao et al. 2006). High expression of MYCN transcript in neuroblastomas without amplification does not indicate poor prognosis (Tang, Zhao et al. 2006). However, several explanations have been proposed to reconcile discordance between genomic copy number and levels of expression. Matthay et al noted that discrepancies could be a result of the use of different methods of quantification such as northern blot, reverse transcriptase PCR, real-time PCR immunohistochemistry and Western blot or that clinical factors such as stage, age and treatment protocols were not always consistent within and between studies (Matthay 2000). In an effort to explain the MYCN expression paradox, a recent study proposed that high levels of the antisense transcript of MYCN, MYCNOS, could contribute to decreased MYCN expression in neuroblastomas with high levels of MYCN transcript (Jacobs, van Bokhoven et al. 2009). This group showed that MYCNOS expression

14 correlated with advanced disease but overexpression of MYCNOS in MYCN-amplified neuroblastoma cell line, IMR32, did not decrease levels of MYCN mRNA ruling out the mechanism of RNA interference. Further analysis will be required to determine of the relationship between amplification and gene expression.

1.11 MYCN protein and functions

1.11.1 MYCN protein

MYCN is a member of the basic helix-loop-helix (bHLH) family of transcription factors. The MYCN gene encodes two protein products with apparent molecular weights of 65 and 67 kDa that localize in the nucleus (Ramsay, Stanton et al. 1986). The C-terminal contains a basic domain which binds DNA and an HLH domain which mediates dimerization with other HLH domain-containing proteins such as MAX (Wenzel and Schwab 1995). MYCN can act as a transcriptional activator when bound to MAX or a repressor when bound to Mnt, Mxi, Mad or other cofactors. MYCN-MAX heterodimers recognize conserved sequences called E-boxes. The N-terminus contains the four evolutionarily conserved myc boxes which together make up the transactivation domain (Cowling and Cole 2006). The N-terminus also contains phosphorylation sites for casein kinase II (Hamann, Wenzel et al. 1991) as well as phosphorylation sites for MAP kinase and GSK3 (Henriksson, Bakardjiev et al. 1993).

1.11.1.1 MYCN and RB pathway

MYCN regulates genes involved in cell proliferation and is thought to control cell cycle genes, however, the precise mechanism remained elusive until recently. Woo et al showed that MYCN controls S phase genes (Woo, Tan et al. 2008). Through the use of MYCN silencing RNA, the authors demonstrated that inactivation of MYCN in amplified neuroblastoma cell lines resulted in increase of p27 and decrease of cell cycle genes E2F1, E2F2, and CDK6 as well as the differentiation gene ID2. E2F promoters have E-boxes that MYC can bind to. ChIP analysis showed that E2F1 recruitment to E2F elements of target genes is dependent on the binding of MYC to E-boxes of E2F promoters (Leung, Ehmann et al. 2008). This provides evidence of a direct link between MYC and transition from G1/G0 to S phase. MYCN can also drive proliferation independent of E2Fs.

In the developing murine retina, cells with triple knock out of E2f 1, 2 and 3 can retain the ability to divide (Chen 2009, in press). Proliferation is only inhibited in a quadruple knock out of four genes E2f1, 2, 3 and Mycn, indicating that Mycn provides a compensatory or redundant cell division promoting mechanism (Chen 2009, in press). Mycn-mediated cell proliferation in the absence of E2fs is accomplished through maintenance of E2f targets and down regulation of Cdk1a and c. MYCN was recently shown to directly upregulate a cluster of miRNAs 17-5p-92 which inhibit the cyclin-dependent kinase inhibitor p21 (Fontana, Fiori et al. 2008). p21 lies upstream of pRB and negatively regulates the cell cycle by inactivating Cdk2-cyclin E complexes that inhibit pRB through phosphorylation. Fontana et al showed that ectopic expression of miRNA 17-5p-92 in neuroblastoma cells increased proliferation and downregulated p21 and that primary neuroblastomas with MYCN-amplification also had upregulation of miRNA 17-5p-92 coupled with low p21 expression. In addition, miRNA 17-5p- 92 also downregulated expression of the pro-apoptotic protein BIM (Bcl-2 interacting mediator of cell death) shutting down the apopotic pathway (Fontana, Fiori et al. 2008).

1.11.1.2 Other MYCN functions

While MYCN can induce proliferation and cell cycle progression, its overexpression also strongly activates apoptosis (Hogarty 2003). This opposing function of MYCN is likely a protective mechanism and indeed, high levels of MYCN protein in neuroblastoma cells without MYCN amplification has been shown to inhibit proliferation and induces apoptosis (Peirce and Findley 2009). Consequently, in order for the tumor cell to survive, a balance must be struck between MYCN-driven proliferation and MYCN-induced cell death. The tumor suppressor p53 can induce apoptosis, cell cycle arrest and DNA repair mechanisms in response to a variety of cell stress. In an unstressed cell, p53 is kept inactive mainly by E3 ubiquitin ligase MDM2 which targets p53 for degradation by the proteasome. MDM2 was recently identified as a transcriptional target of MYCN (Slack, Chen et al. 2005). MYCN was shown to bind to E boxes of the MDM2 promoter and when MYCN was inhibited in amplified neuroblastoma cells, resultant decrease in MDM2 was accompanied by stabilization of p53 (Slack, Chen et al. 2005). The same group confirmed their in vitro findings in vivo in a follow-up study. Mdm2+/- MYCN+/+ mice had significantly delayed tumor development and lower overall incidence of tumors (Chen, Lin et al. 2009). p19Arf which suppresses Mdm2 was found to be epigenetically

16 silenced in Mdm2+/- mice suggesting that reduction of the Mdm2 inhibitor is another mechanism by which MYCN circumvents induction of apoptosis.

The MYC family of proteins is considered to be weak activators of gene transcription yet it is estimated that they activate 15% of all human genes (Patel, Loboda et al. 2004; Dang, O'Donnell et al. 2006). In an effort to reconcile this apparent paradox, Cotterman et al, studied the effects of MYCN on chromatin regulation and found that a surprising 90-95% of histone H3 acetylation and methylation marks were dependent on MYCN expression (Cotterman, Jin et al. 2008). Using ChIP coupled with array technology, the group found that MYCN bound extensively to the entire genome and predicted that there were an estimated ~20 000-40000 MYCN binding sites with 40% of sites at least 10kb away from transcriptional start sites (Cotterman, Jin et al. 2008). This indicated that MYCN can not only regulate genes as a transcription factor but it can indirectly activate transcription of potentially thousands of genes by opening up large stretches euchromatin to transcription. Altogether, the evidence presented above shows that MYCN can exert its proliferative effect in a wide variety of ways ranging from the manipulation of the multiple arms of the RB and apoptotic pathways as well acting as a general transcriptional stimulus in the cell.

1.12 MYCN amplification in transgenic murine model of neuroblastoma

Functional evidence that MYCN overexpression can initiate neuroblastoma tumors came when Weiss et al used the tyrosine hydroxylase promoter to target a Mycn transgene to neural crest cells in mice (Weiss, Aldape et al. 1997). The TH-MYCN mice developed tumors with similar histopathology and expression of neuronal markers consistent with human neuroblastoma and CGH analysis of tumors showed genomic changes accompanying tumor progression occurred in the regions syntenic to those often gained and lost in human neuroblastomas, such as gains of 11 and loss of 17 (Weiss, Aldape et al. 1997; Cheng, Cheng et al. 2007). Further characterization of the TH-MYCN mice showed that the transgene specifically became amplified as the disease progressed (Hansford, Thomas et al. 2004; Cheng, Cheng et al. 2007). Taken together, these data provide evidence that overexpression of MYCN can initiate tumorigenesis and gives good justification that a similar model in which MYCN overexpression in the retina will be a useful tool to dissect the mechanism of MYCN tumorigenesis in retinoblastoma.

1.12.1.1 Role of MYCN in retinal development

Recently, it was shown that MYCN plays an important role in the coordination of growth of the murine retina. Transgenic mice lacking Mycn had a smaller but properly proportioned eyes compared to their littermates with normal Mycn (Martins, Zindy et al. 2008). The authors showed that in mice, the level of cyclin-dependent kinase inhibitor p27 expression was increased and that the small eye phenotype could be rescued by also knocking out p27, an inhibitor of pRB phosphorylation/inactivation thus demonstrating that Mycn acts through the RB pathway.

Chapter 2 2 Characterization of RB1+/+ retinoblastoma

N.B.: Diane Rushlow, Jennifer Kennett, Paul Boutros and Anthony Mak contributed to part of the work presented in this chapter. Diane Rushlow provided the M3-Mn gene-specific QM-PCR copy numbers depicted in figure 1 and table 6 (Appendices), Jennifer Kennett at Dr. Wan Lam’s laboratory performed the sub-megabase resolution tiling array comparative genomic hybridization and assisted in the data analysis, Paul Boutros assisted in the statistical analysis depicted in Tables 3 and 4 and Anthony Mak assisted in the construction of the Mycn- overexpression lentiviral construct depicted in figure 13. I would also like to acknowledge Tim Corson for providing the SKY analysis in figure 8.

2.1 Introduction

Genetic screening is performed to identify RB1 mutations in order to diagnose retinoblastoma earlier and to provide genetic counseling to families. However, genetic testing is a difficult task for 2 reasons: (1) the RB1 gene is large and made up of 27 exons distributed over 183 kb of genomic sequence and has a promoter containing a normally unmethylated CpG island; (2) almost all mutations are unique and scattered along the entire gene with no real hot spots. To date a sensitive and efficient series of molecular tests have been developed. Mutation screening consists of QM-PCR of all 27 exons and the core promoter to detect copy number changes, sequencing of the core promoter and 27 exons as well as 25 intronic nucleotides flanking each exon, and testing for hypermethylation of the RB1 core promoter (Richter, Vandezande et al. 2003). Currently, sensitivity for detecting both mutations in bilateral patients is 95% (443/467) and the remaining 5% are predicted to be mosaic in blood (Rushlow, Piovesan et al. 2009). In unilateral patients 94% (413/441) of patients have both mutations identified in retinoblastoma tumor, 4.8% (21/441) have one mutation identified and in 1.6% (7/441) of cases, no mutations can be detected. This 1.6% of unilateral retinoblastomas also does not show loss of heterozygosity; hence, they will be referred to as RB1+/+ retinoblastomas and will be the focus of this research project (See Table 3).

Retinoblastoma is caused by inactivating mutations on both alleles of the RB1 gene. However, recent work by Dimaras et al, demonstrated that these first two mutations (M1 and M2) cause a

19 benign precursor called retinoma and further mutational events termed M3-Mn are required for progression to malignant retinoblastoma (Dimaras, Khetan et al. 2008). Using techniques such as karyotype analysis, metaphase comparative genomic hybridization (CGH) and aCGH, retinoblastoma was shown to display a specific constellation of genomic changes (Squire, Gallie et al. 1985; Chen, Gallie et al. 2001; Corson and Gallie 2007; Sampieri, Amenduni et al. 2009). Candidate M3-Mn genes have been characterized, including oncogenes KIF14 on 1q (Corson, Huang et al. 2005), MYCN on 2p (Bowles, Corson et al. 2007), E2F3 and DEK on 6p (Orlic, Spencer et al. 2006), and potential tumor suppressor CDH11 on 16q (Marchong, Chen et al. 2004). A QM-PCR was developed to profile retinoblastoma M3-Mn progressive genomic changes, including the MYCN gene. QM-PCR results showed that M3-Mn changes occur frequently in retinoblastomas. In RB1-/- unilateral retinoblastomas, the oncogenes KIF14, MYCN, DEK and E2F are gained at frequencies of 50%, 15%, 40% and 70% and the tumor suppressor CDH11 is lost at a frequency of 45% (Bowles, Corson et al. 2007). When gene-specific QM- PCR was used to profile the RB1+/+ retinoblastomas, it was discovered that they showed a completely different genomic profile than RB1-/- retinoblastomas (Rushlow and Gallie, personal communication). First, a high proportion of RB1+/+ retinoblastomas (57%, 4/7) showed high- level MYCN amplification (33-121 gene copies) whereas out of 70 RB1-/- unilateral retinoblastomas tested, none showed copy numbers greater than 10 copies. Second, RB1+/+ showed few M3-Mn genomic alterations characteristic of retinoblastoma. These observations led to the hypothesis that in addition to the two known genetic forms of retinoblastoma, both of which are caused by RB1 mutations, there may be a third form of retinoblastoma in which no mutations to RB1 are required.

2.2 Hypothesis

RB1+/+ retinoblastomas represent a previously unrecognized subset of retinoblastoma that have a genetic signature distinct from conventional RB1-/- retinoblastomas.

2.3 Thesis Aims and Rationale

2.3.1 Frequency of RB1+/+ retinoblastoma

To confirm that RB1+/+ retinoblastomas were not isolated cases limited to the Toronto subset of tumors, collaborations with three other RB1 testing centers were set up to collect a larger number

20 of samples. Once collected, statistical analysis was performed to determine frequency of RB1+/+ retinoblastomas across four RB1 gene testing centers.

2.3.2 Characterize genomic profile of RB1+/+ retinoblastomas

Gene-specific QM-PCR was performed on samples from the three other RB1 testing sites to determine whether they shared the M3-Mn retinoblastoma genomic signature of RB1-/- or RB1+/+ retinoblastomas with the Toronto subset. Following gene-specific QM-PCR analysis, sub- megabase resolution tiling aCGH (SMRT aCGH) was used to profile the entire genomes of each RB1+/+ retinoblastoma compared to RB1-/- retinoblastomas.

2.3.3 Determine mRNA and protein levels of RB1 and MYCN genes

The levels of RB1 and MYCN mRNA and protein were confirmed for two reasons: (1) to determine whether full-length RB1 transcript and protein were expressed consistent with RB1+/+ status and (2) to determine the levels at which they were expressed, particularly whether levels of MYCN transcript and protein correlated with the amplified genomic status of the tumor.

2.3.4 Analysis of clinical and pathological features of RB1+/+ retinoblastomas with MYCN amplification

Clinical features such as age of diagnosis and histology were assessed to determine whether or not RB1+/+ retinoblastoma showed the characteristic features of RB1-/- retinoblastoma.

2.3.5 Determine the effect of MYCN silencing in MYCN-amplified retinoblastoma

The MYCN-amplified retinoblastoma cell line Y79 was treated with MYCN-targeting shRNA lentivirus to determine the effect on proliferation rate.

2.3.6 Designing a Mycn-overexpressing lentivirus

A lentivirus overexpressing Mycn was developed for the purpose of assessing the effect of high- levels of Mycn in vivo. The lentivirus will potentially be used for injection into the murine retina to determine whether exogenous expression of Mycn can initiate tumors.

2.4 Materials and Methods

2.4.1 Samples

Analysis was performed on 410 DNA samples from primary retinoblastoma tumors of probands with sporadic unilateral retinoblastoma collected at Retinoblastoma Solutions, Toronto, Ontario, Canada. The Research Ethics Boards of the Wellesley Hospital, the Hospital for Sick Children, the University Health Network, and the University of Toronto approved research use of tumor material with parental consent. Additional samples were collected from 3 other sites: Essen, Germany; Paris, France and Christchurch, New Zealand (Table 1).

2.4.2 RB1 gene mutation testing

For samples from Toronto, DNA was extracted using the Gentra PuregeneTM kit (now Qiagen, Mississauga, ON). Samples were primarily submitted as clinical samples for RB1 mutation detection, and were screened for RB1 mutations or epigenetic changes using QM-PCR of all 27 exons and the core promoter to detect copy number changes, sequencing of the core promoter and 27 exons (as well as 25 intronic nucleotides flanking each exon), and testing for hypermethylation of the RB1 core promoter (Richter, Vandezande et al. 2003; Rushlow, Piovesan et al. 2009) .

Tumors samples from each of the three additional sites had been tested for any changes in the RB1 gene, including sequence analysis, testing for whole or multi-exon copy number changes using either QM-PCR or MLPA (MRC-Holland), methylation of the RB1 promoter, and for loss of heterozygosity at RB1 using microstaellite analysis (Raizis, Schmitt et al. 1995; Stirzaker, Millar et al. 1997; Raizis, Clemett et al. 2002; Schouten, McElgunn et al. 2002; Houdayer, Gauthier-Villars et al. 2004; Schüler, Weber et al. 2005; Mitter, Rushlow et al. 2009).

2.4.3 Gene-specific QM-PCR

After completion of RB1 mutation screening, gene-specific primers were used in a QM-PCR reaction to determine genomic copy number for KIF14 (1q32.1), DEK (6p22) E2F3 (6p22), and CDH11 (16q22). Copy number of these four genes were determined in 91 tumor DNA samples collected from all four sites with both mutations identified (12 of these previously reported in Bowles, Corson et al. 2007), in 27 tumor DNA samples with no RB1 tumor mutation identified (RB1+/+), one tumor reported to be bilateral with no RB1 tumor mutation identified (RB522) and

20 tumors with only one RB1 tumor mutation identified (RB1+/-) (see Table 1). Copy number for MYCN (2p24.3) were determined using a second QM-PCR reaction and MYCN specific primers (Bowles, Corson et al. 2007), in 70 primary tumors with both RB1 mutations identified, in 21 tumors with one tumor mutation identified (RB1+/-), and in 27 tumors with neither RB1 mutation identified (RB1+/+) .

Gene-specific primers were used for KIF14, DEK, E2F3, CDH11, and MYCN, previously described (Bowles, Corson et al. 2007). Each reaction tube contained 7.5µl of Qiagen Multiplex PCRTM 2X Master Mix (Qiagen, Mississauga), 0.2 µl of gene-specific primer pool (12.5-25ng/µl of each primer ), 0.5 µl of C4 control primers, 0.3 µl of ALK-1 exon 5 control primers, 3.5 µl of water and 3 µl of DNA at 30ng/µl. Cycling required 15 minutes at 95ºC to activate the hot- start enzyme followed by 19 cycles of 94 ºC for 30 seconds, 60 ºC for 1’30 seconds, 72 ºC for 1’30 seconds, and a final extension of 10 minutes at 72 ºC. One primer of each pair was Cy5.0 labeled; product peak sizes were quantified using Visible Genetics’TM sequencers (Siemens) and GeneObjectsTM 3.1 software.

Primers for two internal control fragments were included in each assay: a 329-bp fragment (C4) from exon 4 of the retinaldehyde-binding protein (chromosome 15) and a 198-bp product from exon 5 of the ACVRL-1 (ALK-1) gene (chromosome 12). One internal control peak was set to 2 copies and the ratios of the other peak heights to the control peak were compared to ratios obtained for normal two-copy DNA from blood samples to establish copy number for each gene. The second internal control peak in each assay acted as a check and was expected to give close to two copies to verify that a DNA sample was amplifying consistently and that there was no significant degradation of the DNA. Each run included at least four normal control samples and two samples previously characterized as showing gain/loss for each of the genes of interest. Tumor samples showing gain or loss were confirmed by repeat analysis.

Table 1 Samples used for QM-PCR Test Site # of RB1-/- # of RB1-/- # of RB1+/- # of RB1+/- # of RB1+/+ tested for tested for tested for tested for tested for KIF14, DEK, MYCN copy KIF14, DEK, MYCN copy KIF14, DEK, E2F3, CDH11 number E2F3, CDH11 number E2F3, CDH11 copy number copy number and MYCN copy number Toronto, 69 48 16 17 8* Canada Essen, 12 12 4 4 12 Germany Paris, France 10 10 0 0 5 Christchurch, 0 0 0 0 2 New Zealand Total 91 70 20 21 27 *This number includes RB522 which was originally diagnosed as bilateral. 2.4.4 Sub-megabase resolution tiling array comparative genomic hybridization

The array platform, comprised of 26,363 overlapping elements, was manufactured on site, as previously described (Ishkanian, Malloff et al. 2004; Watson, deLeeuw et al. 2007). The effective resolution of the array is 79 kb (Ishkanian, Malloff et al. 2004). Briefly, 200 ng of test and reference (single male) DNA were separately labeled with Cyanine-3 and Cyanine-5 dCTPs Using the BioPrime DNA labeling system (Invitrogen, Burlington, Ontario, Canada). DNA probes were then pooled and unincorporated nucleotides were removed with a YM-30 Microcon centrifugation tube (Millipore). Next, 100 μg of Cot-1 DNA (Invitrogen) was added and the entire mixture was precipitated. This material was then re-suspended in a 45 μl cocktail consisting of DIG Easy hybridization solution (Roche), sheared herring sperm DNA (Sigma- Aldrich), and yeast tRNA (Calbiochem). Probe denaturing and blocking steps followed at 85°C and 45°C for 10 minutes and for one hour respectively. Subsequently, the probe mixture was applied to the surface of the array, coverslips were applied, and arrays were incubated at 45°C for 36 hours. Slides next underwent five agitating washes in 0.1× SSC, 0.1% SDS at 45°C (each wash ~5 min). Rinses with 0.1× SSC followed, then drying by centrifugation.

2.4.5 SMRT aCGH data analysis

CGH array images were obtained with the Array-WoRxCCDscanner (Applied Precision, Issaquah,WA) at a resolution of 10 mm with median intensity channel normalization. Image analysis was performed with the Softworx software suite (Applied Precision). The raw data was normalized for spatial and printing intensity bias with CGH Normalize suite (Khojasteh, Lam et al. 2005). Data was imported into SeeGH (Chi, DeLeeuw et al. 2004) a program allowing electronic representation visualization, multiple alignment, and copy number annotation of the data. To minimize the potential noise due to dust or scratches on the array, all results were screened using the variance between the duplicate spots. A clone was not included in analysis if the variance exceeded 0.075. Additionally, the calculated signal to background ratio of 10 for each spot was used to omit any spots.

Breakpoint boundaries were determined by the end sequence position of the BAC clone on either side of the breakpoint. When a BAC clone exhibited a ratio that was an intermediate value of the two flanking copy number ratio levels, the clone was considered to contain the breakpoint. When such a clone did not exist, the two flanking clones were considered to encompass the breakpoint event. Breakpoints at the centromeres were indiscernible due to their repetitive DNA and subsequent incomplete mapping, which prohibits precise loci determination. Segmentation in the samples were analyzed with the CNA Hmmer algorithm (Shah, Xuan et al. 2006).

2.4.6 Statistics

Comparisons of the frequency of mutations in RB1+/+ and RB1-/- patients were made using a two- tailed proportion test with Yates' continuity correction, as implemented in the R statistical environment (v2.7.2).

The Mann-Whitney rank sum test was used to make pair-wise comparisons of number of CNAs and aberrant base pairs between all combinations of the four retinoblastoma subsets RB1+/+MYCNA (MYCN > 10 copies), RB1+/+ (2-copy MYCN), RB1+/-, and RB1-/- and cell lines were made using the the GraphPad Prism software (v5.02).

Comparisons of age of diagnosis were made using a one-way analysis of variance (ANOVA) using the GraphPad Prism software (v5.02).

2.4.7 RT-PCR

Total RNA was extracted from fetal, adult and primary retinoblastoma samples using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA concentration was measured using the NanoDrop-1000 spectrophotometer (Thermo Scientific). For cDNA synthesis, 1μg of total RNA was reverse transcribed using random primers (Invitrogen) and SuperScript II Reverse Transcriptase (Invitrogen) at 42°C for 50 minutes. The reaction was inactivated by heating at 70°C for 15 minutes. The resulting cDNA library was used in end-point PCR gene expression analyses in a reaction mixture consisting of 200 μM dNTPs, 2.5 mM MgSO4, 0.5 μM each of forward and reverse primers, 0.5 U KOD hot start DNA polymerase (Novagen), 1x PCR buffer (Novagen) and 1 μl of product from cDNA synthesis, in a final volume of 25 μl. PCR was performed using the RoboCycler Gradient 96 thermal cycler (Stratagene). Primers are listed in Table 2. Cycling conditions are as follows: 2 minutes at 94°C, next 30 cycles of amplification (30 seconds 94°C, 30 seconds 65°C and 1 minute 30 seconds 72°C) and lastly 10 minutes extension at 72°C.

Table 2 List of primer sequences and expected product sizes used in RT-PCR analysis Gene Primer Sequence Expected Size (bp)

RB1 5’-ATGCCGCCCAAAACCCCCCGAAAA-3’ 2787 5’-TCATTTCTCTTCCTTGTTTGAGGT-3’ MYCN 5’-CACAAGGCCCTCAGTACCTC-3’ 283 5’-TCTTCTGTGGGGGTGCAT-3’ Ki67 5’-GCTAAAACATGGAGATGTAAT-3’ 631 5’-ATTTTGGTCTTGACTTACGC-3’ TBP 5’-ACAACAGGCTGCCACCTTAC-3’ 743 5’-GCTGGAAAACCCAACTTCTG-3’

2.4.8 Immunohistochemistry

Formalin-fixed, paraffin-embedded sections of human retina and retinoblastoma were studied. Slides were re-hydrated by incubating two times 10 minutes each in xylene, two times 5 minutes each in 100% ethanol, once for 2 minutes in 95%, 70%, and 50% ethanol, followed by 5 minute incubation in TBS. For antigen retrieval, sections were treated with 0.1% trypsin for 5 min at 37°C or heated in PBS citrate for 17 min in a pressure cooker prior to incubation with primary antibody. Slides were then incubated in 5% Triton-X for 10 minutes at room temperature. Blocking was carried out for 30 minutes at room temperature in TBS with 10% DAKO Protein

Block (DAKO-Cytomation), 1% BSA and 0.05% Tween-20. Sections were stained for pRB-N- terminus, 1:200 (BD Pharmingen, Missisauga, ON), pRB-C-terminus, 1:200 (Santa Cruz) and MYCN 1:100 (Santa Cruz) in TBS with 1% BSA, 0.05% Tween-20 and 10% Antibody Diluent (DAKO-Cytomation), followed by three washes in TBS with 0.1% BSA and 0.05% Tween-20. Human pRB-N-terminus immunoreactivity was detected using Immunopure DAB substrate kit (Pierce). Human pRB-C-terminus and MYCN immunoreactivity was detected using fluorescent staining by Alexa™ 488 Streptavidin conjugate from Molecular Probes. DAPI was used to visualize nuclei of cells. Slides were mounted using the DAKO-Cytomation Fluorescent Mounting Medium. Slides were visualized using a Zeiss LSM510 confocal microscope (Zeiss, Toronto, Canada)

2.4.9 Lentivirus production

Bacterial stocks of MYCN-targeting lentiviral Mission® shRNA vectors were purchased from Sigma-Aldrich. The pLKO.1-shRNA lentiviral plasmid vector DNA was isolated according to standard phenol-chloroform isolation procedure. 8 X 105 human embryonic kidney 293T (HEK 283T) cells were plated in 10 cm plates in 10 ml of growth media with antibiotics and incubated overnight for 24 hours at 37°C in 5% CO2. The next day, the cells were transfected with pLKO.1-shRNA lentiviral plasmid vector along with Pax2 and MD2.G packaging vectors using the Lipofectamine 2000® transfection reagent (Invitrogen) for shRNA lentivirus and FuGENE® 6 transfection reagent (Roche) for the Mycn-overexpression vector according to manufacturer’s instructions. From this point onwards, the cells were incubated in an incubator reserved only for virus work. After 18 hours of incubation, the media was replaced with 30% serum growth medium. At 48 and 72 hrs after transfection, virus was harvested by collecting media from cells. Debris was spun down and the supernatant was aliquoted and frozen at -70°C. Media was replaced on the cells after 48 and incubated again overnight for the 72 hour harvest.

2.4.10 Lentivirus titration

HEK293 cells were seeded onto 6-well dishes at a density of 2 x 104/ml. The next day, cells were transduced with serial dilutions of virus (10-2 to 10-6) in DMEM 10% FBS media in triplicate and incubated overnight. The next day, virus was removed and cells were supplemented with 2 ml of fresh media and incubated overnight. The next day, media was changed to DMEM with 1 μg/ml of puromycin. Media was changed every 3-4 days for 14 days.

Cells were then rinsed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes. Following rinsing with PBS two times, cells were stained with crystal violet stain (0.1% crystal violet powder in 10% ethanol) and rinsed 6 times with double distilled water. Number of viral colonies were counted and averaged across the triplicate wells to give transforming units per ml which was multiplied by the volume (1 ml) to give the titer.

2.4.11 Proliferation assay

1 x 106 Y79 retinoblastoma cells were transduced with a 1/20 dilution of the undiluted virus stock of each of the 5 MYCN Mission® shRNA lentiviral particles as well as the empty pLKO- puro lentivirus in T75 flasks and incubated overnight. The next day, virus was removed and fresh RB media (500 Iscove’s medium, 89.4 ml Fetal Clone III serum, 5ml 100X penicillin- streptomycin, 2.38 μl β-mercaptoethanol, and 596 μl 10mg/ml insulin) was replaced. Forty-eight hours after transduction, media was replaced with RB media containing 1μg/ml puromycin. Media was changed every 3-4 days for one week. Cells were then plated at a density of 2 X 104 cells/well in 24-well plates in triplicate for each of the 6 viruses in 7 sets. The number of cells in each well was counted everyday for 7 days and the triplicate counts were averaged.

2.4.12 Construction of Mycn-overexpression lentivirus

Mycn cDNA was PCR-amplified from mouse fetal retina cDNA library using the following primers: forward 5’-CGAACCCATGCCCAGCTGCA-3’ and reverse 5’- GAAACGTTAGCAAGTCCGA-3’. The amplified Mycn product was cloned into the StrataClone™ PCR cloning vector according to manufacturer’s instructions. Mycn cDNA was sequenced and verified and further subcloned into the pSY series of lentiviral vectors (Figure 13). Lentiviral particles were then produced as described above. HEK293 cells were transduced with undiluted virus stock with polybrene at a concentration of 8μg/ml to test the expression of Mycn protein. Media was changed 24 hours post-infection and fresh growth media was replaced every 2 days for 4 days at which time, cells were scraped and lysed with RIPA buffer. The lysed cell debris was spun down and supernatant collected for western blot analysis.

2.4.13 Transduction of retinal explants

Transduction of explants has been previously described (DiCiommo, Duckett et al. 2004). Mice were treated in accordance with the Canadian Council on Animal Care and with approval from

28 the University Health Network Animal Care Committee. Briefly, to test expression of Mycn in murine retinal explants, P0 B-6 mice (Ontario Cancer Institute) were sacrificed and retinas were dissected and placed on a cell culture membrane (Millipore) in explant media (DMEM/F-12 supplemented with 5% FBS, insulin (5μg/mL), pyruvate, and glutamate for 24 hours. To transduce explants, a sterile 1ml pipette tip was cut to 0.5 cm at the base and placed around the retina, 200 μl of virus stock was placed in the pipette tip barrier and incubated overnight at 37°C,

5% CO2. The next day, virus was removed and replaced with fresh media and media was changed every 48 hrs for 5 days. The retinal explants were then harvested by removing media and fixed with 4% paraformaldehyde on ice for 1 hour. The paraformaldehyde was removed and replaced with 70% ethanol overnight at 4°C. The retinal explants were then embedded in paraffin and slides were cut for immunohistochemistry analysis of Mycn and EGFP protein expression.

2.4.14 Western blot analysis

Total protein was extracted from harvested cell pellets. Samples were mixed with cold Triton-X buffer (1% Triton-X, 20mM Tris (pH 7.5), 150mM NaCl, 1mM EDTA, 1X Roche complete protease) and incubated for 20 minutes at 4°C. After centrifugation at 12,600x g for 15 minutes, supernatants were recovered and protein concentrations were determined using a Bradford protein assay (Bio-Rad, Hercules, CA). Proteins (30μg) were separated by 4-20% gradient pre- cast SDS-PAGE (Lonza, Rockland, ME) at 120V for 2 hours and transferred to a polyvinylidene fluoride membrane (Bio-Rad). The membrane was blocked [5% Blotto (Bio-Rad)] overnight and incubated with either rabbit polyclonal anti-MYCN (NCM II 100, Santa Cruz) (1:200) or rabbit isotype IgG control. X-ray film (Kodak) was used to detect chemiluminescence generated using ECL reagent (GE Healthcare).

2.5 Results

2.5.1 Frequency of RB1+/+ retinoblastoma across four independent sites

To establish the frequency of RB1+/+ retinoblastomas over a larger set of patients, retinoblastoma samples were collected from three other RB1 testing centers performing similar testing to Retinoblastoma Solutions, Toronto (Table 3). Of a total of 400, 152, and 30 unilateral retinoblastomas screened for RB1 mutations at the Institute für Humangenetik, Essen, Germany, the Institut Curie, Paris, France and the Christchurch School of Medicine, Christchurch, New Zealand, 12 (3%), 5 (3%) and 2 (7%) respectively were found to be RB1+/+ (Table 3). These were similar frequencies to the Toronto data set (P = 0.168, 0.475 and 0.269 pair-wise proportion test) and all 3 sets of RB1+/+ retinoblastomas were used in gene-specific QM-PCR analysis (Table 3 and see Aim 2.1 below). In total 26 RB1+/+ retinoblastomas out of 992 tumors tested were collected from 4 independent sites as part of a multi-site analysis. RB1+/+ retinoblastomas occur at a frequency similar between all sites thus justification could be made to pool them into one set making the frequency of RB1+/+ retinoblastomas 2.6% across 4 independent sites.

Table 3 Frequency of RB1+/+ retinoblastomas across four sites Test Site Total Number of Number of Number of P-value (pair- Number of Unilateral RB1-/- and RB1+/+ wise RB1+/+ retinoblastomas RB1+/- (Freq.) proportion MYCNA test) Canada 441 434 7 (2%) 4 Germany 400 388 12 (3%) P=0.168 3 France 152 147 5 (3%) P=0.475 2 New Zealand 30 28 2 (7%) P=0.269 1 Total 1023 997 26 (3%) 10

2.5.2 Genomic profile of RB1+/+ retinoblastomas

2.5.2.1 Copy number changes in M3-Mn genes in RB1+/+ retinoblastomas

Several studies have shown that, following the initial two mutations to RB1, further characteristic genomic gains and losses (M3-Mn) are common in retinoblastoma including the gain of oncogenes KIF14, MYCN, DEK, and E2F3, and loss of the tumor suppressor CDH11 (Marchong, Chen et al. 2004; Corson, Huang et al. 2005; Orlic, Spencer et al. 2006; Bowles, Corson et al.

2007; Dimaras, Khetan et al. 2008). A previously described gene-specific QM-PCR method (Bowles, Corson et al. 2007) was used to measure copy number changes of the above genes in the RB1+/+ and RB1-/- retinoblastoma subsets (Table 6, Appendices). Gain is defined as 2.5 to 10 copies, loss as less than 1.5 copies and amplification as more than 10 copies (Figure 1). Figure 1 shows the copy numbers of M3-Mn genes in the 26 RB1+/+, 20 RB1+/-, 91 RB1-/- retinoblastomas and 6 cell lines. With the larger sample sizes available for the current analysis, it was found that gains of KIF14, MYCN, DEK, and E2F3, and loss of CDH11 are more frequent in RB1-/- retinoblastomas than previously thought (Bowles, Corson et al. 2007) as they were gained and lost at frequencies of 61%, 64%, 57%, 58% and 64% respectively. In RB1+/+ retinoblastomas, however, it was found that gains in KIF14, DEK, and E2F3, and loss of CDH11, were much less frequent at 23%, 34%, 25%, and 19% respectively compared to RB1-/- retinoblastomas (Table 4). However, four tumors MC945, RB1348 and MA94 did show gains of KIF14, DEK, and E2F3, and RB1700 showed loss of CDH11 (see Figure 1 and Table 6 in Appendices). Most striking was the frequent occurrence of high-level amplification of the MYCN gene (MYCNA) in the RB1+/+ retinoblastomas (10/26; 38%) of the RB1+/+ samples showed MYCN genomic copy numbers ranging from 38 to 121 (see Table 6 Appendices). This was in stark contrast to and significantly different from RB1-/- retinoblastomas where, although low level gain of 3-5 copies of MYCN was frequently observed and occasionally moderate gain of up to 9 copies of MYCN, MYCN amplification of over ten copies was never seen in any of the 91 RB1-/- unilateral tumor samples tested (P-value 9.55 x 10-9).

Of the 21 RB1+/- unilateral tumor DNA samples tested for MYCN copy number, one sample, RB2285, showed high level MYCN amplification. The one non-germline RB1 mutation found in this sample was deletion of one copy of most of the 13q arm (see Whole genomic profiling below), including RB1. This sample showed high similarity to the RB1+/+ MYCN A subset, including a very early age of diagnosis (4 months). Thus it is hypothesized that the loss of 13q in this tumor may be a secondary event and that this sample may in fact belong in the RB1+/+ MYCN A set.

RB522 was originally diagnosed to be from a bilaterally affected child, however, since the clinical evidence of bilateral retinoblastoma is not definitive (two small white retinal areas were ablated with cryotherapy, no images recorded), no RB1 mutations were found in either tumor nor blood sample of the patient and the M3-Mn profile including high-level MYCN amplification are

31 similar to the RB1+/+ MYCNA retinoblastomas, this sample has been subsequently included in the analysis as an RB1+/+ retinoblastoma.

Figure 1. M3-Mn profile of M3-Mn copy number in 139 primary retinoblastomas and 6 cell lines. The unilateral tumors are grouped by RB1 mutation status, RB1+/+, RB1+/-, RB1-/-. Each row represents an individual tumor or cell line while across the top is the genes KIF14, DEK, E2F3, CDH11, and MYCN. Pink indicates gain; green amplification; blue loss; white no change; and gray copy number not determined. For copy numbers of each gene see Table 6 in Appendices.

Table 4 Frequencies of M3-Mn changes in RB1+/+ versus RB1-/- retinoblastomas MYCN MYCN Amplificati KIF14 DEK E2F3 CDH11 Gain on (2.5-10) (>10) France 0/5 1/5 1/5 0/5 2/5 2/5 RB1+/+ 6/10 6/10 6/10 6/10 9/10 0/10 RB1-/- Germany 3/17 9/17 7/17 3/17 10/17 3/17 RB1+/+ 5/11 6/11 6/11 5/11 11/11 0/11 RB1-/- Toronto 3/8 1/8 0/8 2/8 2/8 4/8 RB1+/+ 45/70 41/70 42/70 38/70 25/91 0/91 RB1-/- New Zealand 1/2 0/2 0/2 1/2 1/2 1/2 RB1+/+ NA NA NA NA NA NA RB1-/- Pooled 7/32 11/32 8/32 6/32 15/32 10/32 RB1+/+ 56/91 53/91 54/91 49/91 45/112 0/112 RB1-/- P-Value 2.57 x 10-4 0.034 1.71 x 10-3 1.25 x 10-3 0.635 9.55 x 10-9

2.5.2.2 Whole genomic profiling of RB1+/+ retinoblastomas

DNA copy number alterations (CNA) are present in almost all tumor cells. CNAs can range in sizes from a few kilobases to whole chromosomal arm deletions or amplifications. Thus sub- megabase resolution tiling array CGH (SMRT aCGH) (Ishkanian, Malloff et al. 2004) was used to profile and identify CNAs in 49 primary retinoblastomas and 22 of corresponding blood samples and 6 cell lines. The 49 primary retinoblastomas included 11 RB1+/+ MYCNA, 13 RB1+/+, 15 RB1+/-, 10 RB1-/- (Figure 2). The 6 cell lines included 5 retinoblastoma cell lines (RB247, RB383, RB1021, WERI and Y79) and one neuroblastoma cell line, IMR32 (Figure 2). Both Y79 and IMR32 are well characterized and long known to have MYCN amplification (Reid, Albert et al. 1974; Schwab, Ellison et al. 1984). A complete list of alterations and sizes of each CNA is listed in Table 7 Appendices. CNAs were divided into two categories: whole chromosomal arm changes and segmental DNA copy changes (Figure 2).

Figure 2. Summary of chromosomal changes for 47 primary retinoblastomas, 5 retinoblastoma cell lines and 1 neuroblastoma cell line, IMR32. Samples are grouped into RB1+/+MYCNA, RB1+/+, RB1+/-, RB1-/-, RB1-/- bilateral and Cell lines. A blue box indicates the presence of at least one segmental change on the chromosome arm and a red box represents a whole arm alteration. Case numbers are listed to the left and chromosomal regions are listed at the top.

CNAs are a contiguous segment of aberrant DNA as defined by the algorithm CNA HMMer that is separated by normal DNA or a CNA in the opposite direction. The size of each CNA can be measured as the number of aberrant base pairs contained in the altered DNA segment. RB1+/+ MYCNA retinoblastomas did not show a significant difference in number of CNAs per tumor when pair-wise comparisons were made between RB1+/+, RB1+/-, and RB1-/- retinoblastomas (Mann-Whitney test, P<0.05) except for when compared to the retinoblastoma and neuroblastoma cell lines (Mann-Whitney test, P value = 0.012) (Figure 3).

Figure 3. Number of CNAs per retinoblastoma tumors. Horizontal lines represent the mean while vertical bars represent standard error of the mean. Breakpoints were determined by CNA Hmmer. X and Y chromosomes were excluded from the analysis.

However, when pair-wise comparisons of number of aberrant base pairs between tumor types were performed it was found that there was a significant difference between RB1+/+ MYCNA and RB1-/- retinoblastomas (Mann-Whitney test, P value = 0.0433) and between RB1+/+MYCNA and the cell lines (P value = 0.0160). Overall, RB1+/+MYCNA retinoblastomas had fewer aberrant base pairs in their genomes than RB1-/- retinoblastomas and cell lines (Figure 4) consistent with reports that MYCN-amplified tumors are more genomically stable (Chen, Bilke et al. 2005; Mosse, Diskin et al. 2007). Figure 5 shows a typical RB1+/+ MYCNA retinoblastoma with few genomic alterations.

Figure 4. Number of aberrant base pairs in the different subtypes of retinoblastoma. Breakpoints were determined by CNA Hmmer. X and Y chromosomes were excluded from the analysis.

In the 11 RB1+/+ MYCNA retinoblastomas, chromosome 1q was gained in 3 (27%) samples, chromosome 6p was gained in 1 (9%) sample and chromosome 16q was lost in 4 (36%) samples, however loss of 16q in RB2237 occurred at 16q24.1-qter and did not encompass CDH11. None of the 11 RB1+/+ MYCNA retinoblastomas had rearrangements on chromosome 13. In contrast to RB1+/- and RB1-/- retinoblastomas with low-level gain of the whole 2p arm, RB1+/+ retinoblastomas showed a small, highly amplified region specifically at the MYCN locus (Figure 6).

Figure 5. Whole genome tiling path array CGH karyogram of RB1+/+MYCNA retinoblastoma FA793. RB is shown. Each dark blue dot on the karyogram represents the average signal ratio for an individual BAC clone from the array. Clones were plotted vertically against known chromosomal position. Log2 signal intensity ratios for each clone were plotted horizontally, with colored vertical lines denoting log2 signal ratios from -0.5 to 0.5. Where the signal intensity ratio equals zero (purple line), equivalent DNA copy number between the sample and the reference DNA was inferred. DNA copy number increases were indicated by log2>0 (red line) and losses indicated by log2<0 (green line). MYCN amplification is magnified in the orange box.

Figure 6. Specific amplification of the MYCN locus in RB1+/+ MYCNA RB1348. This is contrasted by low-level whole 2p gain seen in RB1777 (RB1+/-).

In the 13 RB1+/+ with 2-copy number MYCN, chromosome 1q was gained in 5 (38%) samples, chromosome 6p was gained in 5 (38%) samples, chromosome 16q was lost in 1 (8%) samples and chromosome 13q was normal in 11 samples. Two RB1+/+ retinoblastomas with 2-copy number MYCN, MC561 and MC887, showed loss of chromosome 13 that did not encompass RB1 but involved 13q32.1-ter and 13q21.1-ter, respectively, both telomeric to RB1. In the 16 RB1+/- retinoblastomas chromosome 1q was gained in 4 (27%) samples, chromosome 6p was gained in 7 (44%) samples and chromosome 16q was lost in 1 (7%) samples. One RB1+/- retinoblastoma sample RB2285 showed MYCN amplification but normal copy numbers in chromosomes 1q, 6p and 16q and normal copy number in the rest of the 2p arm. The one non- germline RB1 mutation found in this sample consisted of a deletion of one copy of most of the 13q arm, including RB1. This sample showed high similarity to the RB1+/+ samples with MYCN amplification, including an early age of diagnosis (4 months). It is hypothesized that the loss of 13q in this tumor may be a secondary event and that this sample may in fact belong in the RB1+/+ group with MYCN amplification. In the 10 RB1-/- retinoblastomas analyzed, chromosome 1q was

38 gained in 5 (50%) samples, chromosome 6p was gained in 5 (50%) samples, chromosome 16q was lost in 8 (80%) samples and rearrangements and deletions of chromosome 13 occurred in 4 (40%) consistent with their deleted RB1 gene status (data not shown). In the 5 retinoblastoma cell lines chromosome 1q was gained in 4 (80%), chromosome 6p was gained in 3 (60%), chromosome 16q was lost in 3 (60%) and 2p was gained in 4 (80%). Y79 and IMR32 showed amplification of the MYCN locus but normal copy of the rest of the 2p arm. IMR32 showed gain of 1q, gain of 6p and consistent with a previous report (Spieker, van Sluis et al. 2001) had a second amplicon on chromosome 2p14 which included only two genes, MEIS and ETAA16.

2.5.2.3 Minimal MYCN amplicon in RB1+/+ MYCNA retinoblastomas

To determine the size of the minimal MYCN amplicon, SeeGH software was used to perform a multiple alignment of 14 samples that showed amplification of MYCN. These included 11 primary RB1+/+ retinoblastomas (RB1348, RB1700, RB2237, RB2285, RB2532, MA72, MA94, MC945, FA337, FA793, NZ499J, and RB522), one RB1+/- retinoblastoma RB2285, and the Y79 retinoblastoma and IMR32 neuroblastoma cell lines (Figure 7). The minimal amplification spans 448 kb, located on chromosome 2, cytoband 2p24.3, between BAC clones RP11-451A14 and RP11-463P22, and between base pairs 15703698 and 16152619. The minimal region of amplification was bounded by primary sample RB2285 and Y79 and contains only the MYCN gene (see Figure 7 dark blue box). Excluding the two cell lines and using only the primary retinoblastomas to determine the minimal region of overlap, the region was bounded by samples RB2285 and FA337 (see Figure 7 pink box). This region was 513 kb in size and still only contained MYCN. Both of the minimal regions of overlap excluded neighboring genes such as NAG, DDX1, FAM49A and FAM84A. The NAG and DDX1 genes are commonly co-amplified with MYCN in neuroblastomas (Weber, Imisch et al. 2004), so the exclusion of NAG and DDX1 is significant since the importance of co-amplified surrounding genes has been disputed in a number of neuroblastoma studies (Squire, Thorner et al. 1995; Weber, Imisch et al. 2004; De Preter, Speleman et al. 2005; Weber, Starke et al. 2006; Kaneko, Ohira et al. 2007).

Figure 7. The minimal MYCN amplicon. The minimal region of amplification contains only the MYCN oncogene within cytoband 2p24.3, based on genomic amplification in 11 RB1+/+MYCNA retinoblastomas, one RB1+/- primary retinoblastomas, and two cell lines, Y79 and IMR32. It spans 448kb. It lies between BAC clones RP11-451A14 and RP11-463P22, and between base pairs 15703698 and 16152619. The dark blue line highlights the smallest minimal amplicon including the two amplified cell lines Y79 and IMR32 and is bounded by primary retinoblastoma RB2285 and the cell line Y79. The pink band underneath indicates the common region of gain in the primary retinoblastomas that is bounded by RB2285 and FA337.

2.5.2.4 Detection of translocation breakpoints

Translocation is a chromosomal aberration caused by a recombination event. Translocations can be balanced, resulting in no net loss or gain of DNA, or unbalanced resulting in duplication or loss of DNA. Translocations are common in precancerous and progressing tumor cells and can not only cause loss or gain of DNA but can cause silencing of genes or form fusion genes that lead to tumorigenesis. Thus the precise identification of chromosomal breakpoints is important in the characterization of events that could contribute to oncogenesis. Recently it was shown that

40 translocations that appeared balanced by cytogenetic characterization methods such as spectral karyotyping (SKY) were discovered on SMRT aCGH to be associated with focal DNA CNAs that could pinpoint with higher resolution the location of the breakpoint (Watson, deLeeuw et al. 2007). The cell line derived from primary retinoblastoma RB381 was previously characterized using SKY (Corson, personal communication) and it was determined that despite detecting no mutation in RB1 by the methods described above, a portion of chromosome 13 was found to be translocated to chromosome 8 der(8)t(8;13)(q21;q14) ins(13;8)(q14;?q?q) however the exact location of the second translocation could not be identified. By SMRT aCGH on the original primary tumor DNA from RB381, the breakpoints for der(8)t(8;13)(q21;q14) can now be confirmed and pinpointed to their chromosomal sub-bands der(8)t(8;13)(q21.2;q14.12) and the breakpoint for the chromosome 8 insertion into the chromosome 13q14.12 can be determined to be at sub bands 8q21.2-23.3 ins(13;8)(q14; q21.2-q23.3) (Figure 8).

Figure 8. RB381 der(8)t(8;13)(q21.2;q14.12) ins(13;8)(q14; q21.2-q23.3) translocation. A. (left) SeeGH karyogram of chromosome 8. (center) SKY data (Tim Corson, unpublished data) for the translocation. (right) SeeGH karyogram of chromosome 13. Red lines indicate breakpoints corresponding to both SMRT array and SKY data (Tim Corson, unpublished data). Yellow line indicates REXO1L1 gene. Green line indicates RB1 gene. B. CNA loss of 0.5 Mb at locus 8q21.2. C. CNA loss of 0.6 Mb at locus 8q23.3. D. CNA loss of 0.9 Mb at locus 13q14.2.

The gene REXO1L1 is located in the small region of loss at chromosome 8q21.2 breakpoint and is a known site of copy number variation (CNV), in humans and chimpanzees (Perry, Tchinda et al. 2006). CNVs are naturally occurring structural variations that can be found across populations and are not usually pathologic however it is postulated that they can contribute to phenotypic variation and inherent susceptibility to diseases. Indeed it was found that 13 retinoblastomas and RB247 cell line from all four types of RB1 mutation status groups showed CNV of the REXO1L1 gene. Blood DNA for RB381 did not show deletion at 8q21.2. However, array CGH showed that tumor sample RB2052 had complex rearrangements on chromosomes 13 and 8 while the corresponding blood had a normal karyogram except for deletion of REXO1L1 in blood. In addition, 3 other samples RB2532, RB2903, RB2589 that showed REXO1L1 deletion in tumor also had deletion in blood DNA consistent with the observation that REXO1L1 CNV is a common in the population. However, whether it predisposes to chromosomal rearrangement in retinoblastoma needs to be further investigated.

2.5.3 Expression of RB1 and MYCN mRNA transcripts and protein in RB1+/+ retinoblastomas

Whether RB1+/+ genomic status corresponded with expression of mRNA and full-length retinoblastoma protein (pRB) in the tumor (Figure 9) was verified next. To do this, primers were designed that targeted the RB1 and MYCN transcripts and reverse transcriptase PCR was used to analyze levels of the transcript in two RB1+/+ retinoblastomas for which mRNA was available. The expected 2.8 kb coding-containing transcript of RB1 was detected in two of the primary RB1+/+ retinoblastomas compared to normal retina and MYCN transcript was detected in fetal retina and two primary RB1+/+ retinoblastoma but not adult retina as expected (Figure 9A). Ki67 transcript, indicative of proliferation, was expressed abundantly in fetal retina and in the two primary tumors but not in adult retina. To confirm the presence of full-length pRB protein, formalin-fixed, paraffin-embedded slides from three primary RB1+/+ retinoblastomas (RB2237 is shown) were stained with two different pRB antibodies, recognizing an N-terminus and the other a C-terminus epitope (Figure 9B). All three tumors stained positively with both pRB antibodies suggesting that full-length pRB was expressed. The tumor but not adjacent retina stained strongly with MYCN antibody confirming that tumors amplified for MYCN expressed abundant protein (Figure 9C).

Figure 9. Expression of pRB and MYCN in primary human retinoblastoma and normal retina. A. RT-PCR was performed using primers that spanned the entire 2.8kb coding region of RB1 and primers spanning the 283 bp coding region of MYCN to determine if RB1+/+ MYCNA retinoblastomas MA94 and FA793 expressed full-length RB1 transcript and to look for presence of MYCN transcript. Normal fetal and adult retinas were included as positive controls. Presence of Ki67 mRNA transcript in fetal and both RB1+/+ MYCNA retinoblastomas indicates proliferation while absence of Ki67 mRNA was expected in non-proliferative adult retina. TBP housekeeping gene was included as a loading control. B and C. Paraffin sections of RB1+/+ MYCNA retinoblastoma RB2237 were stained with pRB C-terminus, pRB N-terminus and MYCN antibodies. Positively staining cells are indicated by green fluorescent or DAB (brown) staining. Scale bar in b is 50 µm in c is 100 µm. 2.5.4 Clinical features of RB1+/+ retinoblastomas

The median age at diagnosis of the 12 RB1+/+ MYCNA including RB2285 (RB1+/-) was 6 months (Figure 10). This is significantly younger than the median ages of diagnosis of 15 RB1+/+ with 2-copy MYCN, 10 RB1+/- and 147 RB1-/- retinoblastomas randomly selected from the Toronto data set which were diagnosed at 23, 24, and 24 months respectively (P-value 0.0023, one-way ANOVA). A previous study also reported a median age of diagnosis of 23 months in unilateral retinoblastomas (Schüler, Weber et al. 2005).

Four enucleated RB1+/+ MYCNA retinoblastomas RB2237, RB522, NZ499, and MA94 were stained with hematoxylin and eosin and assessed for histological features of retinoblastoma (Table 5). In addition, two pathology reports were available for RB1348 and RB1700. None of the tumors had Flexner-Wintersteiner rosettes in the sections analyzed, which are specific for retinoblastoma and seen in 70% of cases (Poulaki and Mukai 2009). All of the tumors were described as large filling most of the eye, showed undifferentiated cells, large areas of necrosis and calcification. Apoptosis was present in RB2237, RB522, NZ499 and, MA94.

Of note is that RB2237, RB522 and NZ499 displayed large prominent nucleoli (Figure 11), a feature that is atypical of retinoblastoma but common in other neuroectodermal or embryonal type tumors such as neuroblastoma (Tornoczky, Semjen et al. 2007). Large nucleolar neuroblastomas are associated with a high incidence of MYCN amplification and are also largely undifferentiated (Tornoczky, Semjen et al. 2007).

Figure 10. Age of diagnosis of 11 RB1+/+ MYCNA retinoblastomas. Samples include RB2973 (RB1+/-) (circle), 15 RB1+/+ retinoblastomas with 2-copy MYCN (square), 10 RB1+/- retinoblastomas (triangle) and 147 RB1-/- retinoblastomas (diamond). Lines represent medians.

Table 5 Summary of retinoblastoma histopathological features in RB1+/+ MYCNA retinoblastomas Pathologic RB1348 RB1700 RB522 RB2237 MA94 NZ499 RB2903 features (report (report (2-copy only) only) MYCN ) Flexner------+ Wintersteiner rosettes Homer-Wright NS + - + - - - Pseudorosettes ------Retinoma ------Mitotic figures NS NS + + + + + Necrosis + + + + + + + Calcification + + + + + + + Vitreous NS NS + + - + + seeding Nuclear NS NS - - - - + moulding Apoptosis NS NS + + + + + Optic nerve - - NS - - - - involvement Choroid and + + - - - + - Sclera involvement Anterior NS ------segment Large NS NS + + - + - prominent nuclei NS, Not scored

Figure 11. Large prominent nucleoli in two RB1+/+MYCNA retinoblastomas, RB2237 and NZ499. RB1+/- retinoblastoma RB2903 is shown for contrast.

2.5.5 Functional consequence of MYCN silencing in retinoblastoma with high levels of MYCN

The retinoblastoma cell line Y79 has genomic amplification of MYCN and expresses high levels of MYCN protein and mRNA. Gene-specific QM-PCR indicated that Y79 has 53 copies of MYCN. To determine whether decreased MYCN expression would affect the growth and cellular fate of RB cell lines with MYCNA amplification, 5 different shRNA-expressing viruses (Mission shRNA system, Sigma) targeting the human MYCN transcript were used to transduce Y79 cells. All 5 shRNA vectors could efficiently knockdown MYCN expression in Y79 cells (Figure 12A). Four out of 5 Y79 clones stably transduced with shRNA vectors showed decreased growth rate compared to Y79 cells transduced with empty vector (Figure 12B).

A B

Figure 12. MYCN shRNA knockdown in Y79 retinoblastoma cells. A) Western blot showing MYCN and β-tubulin protein expression in Y79 cells infected with virus vector carrying the empty PLKO construct or one of 5 different shRNAs targeting different regions of the MYCN transcript. B) Growth curve of Y79 cells infected with empty pLKO vector or 5 shRNAs targeting MYCN. For each day and each shRNA, three wells (24-well plate) were counted and averaged. Error bars show standard deviation of triplicate samples.

2.5.6 Construction of a Mycn-overexpression lentivirus

Similar to the TH-MYCN murine model for MYCN-amplified neuroblastoma (Weiss, Aldape et al. 1997), it is hypothesized that overexpression of MYCN in the mouse retina during early development would result in tumor growth. To determine if overexpression of Mycn is sufficient to initiate tumors during murine retinal development, a Mycn-overexpression lentiviral construct was developed for injection into eyes of P0 mice of various genetic backgrounds. Lentiviral delivery of the oncogene driven by a strong viral promoter, human cytomegalovirus (CMV), was chosen since it is not known which cell type in the retina is the tumor-initiating cell in RB1+/+ MYCNA retinoblastoma. Three Mycn-expression vectors; all containing a truncated human CMV promoter to drive Mycn expression and two vectors that co-expressed EGFP were constructed (Figure. 13A). Mycn cDNA was cloned from fetal mouse cDNA and the sequence was verified in the lentiviral plasmids. HEK293 cells were infected with the three viruses followed by western blot analysis to determine level of Mycn expression. Mycn protein was detected in infected HEK293 cells by all three constructs (Fig. 13B).

Figure 13. Lentiviral overexpression of Mycn in HEK293 cells. A) pSY-hCMV-Mycn-F (pJLM backbone, truncated human CMV, mouse Mycn, C-terminal Flag), pSY-hCMV-MycnF-IRES- WPRE (pJLM backbone, truncated human CMV, mouse Mycn, C-terminal Flag, internal ribosomal entry site, enhanced green fluorescent protein, woodchuck hepatitis post- transcriptional regulatory element), pSY- hCMV- MycnF-EGFP-WPRE (pJLM backbone, truncated human CMV, mouse Mycn, C-terminal Flag, human phosphoglycerate kinase promoter, enhanced green fluorescent protein, woodchuck hepatitis post-transcriptional regulatory element). B) Expression of Mycn protein in lentivirus infected HEK293 cells. Only cells infected with pSY-hCMV-MycnF, pSY-hCMV-MycnF-IRES-WPRE, and pSY-hCMV-MycnF- EGFP-WPRE lentivirus constructs expressed murine Mycn protein. Cells were either untreated, infected with pJLM-eGFP-Flag control and pJLM empty vectors or pSY-hCMV-MycnF lentiviral constructs. Cells were harvested lysed and western blot analysis was performed using anti- Mycn antibody and anti-β-tubulin (loading control).

To test whether these viruses could infect the murine retina, P0 murine retinal explants were dissected and infected with lentivirus followed by immunohistochemistry to determine level and pattern of expression of Mycn protein. Fluorescence was detected in cells infected by virus with EGFP; however, Mycn protein was not detected in paraffin embedded sections (data not shown). This may indicate that the human CMV promoter may not drive Mycn expression in murine cells. Replacement of the CMV promoter with the murine CMV intermediate early promoter 1 (mCMV IE1) is underway since the mCMV IE1 promoter has been shown to drive strong expression of exogenous proteins in a variety of cell types (Dorsch-Hasler, Keil et al. 1985).

Chapter 3 3 Discussion

3.1.1 RB1+/+ MYCNA retinoblastoma is observed in independent clinical samples

The RB1+/+ MYCNA retinoblastoma subset was initially identified in clinical retinoblastoma samples at Retinoblastoma Solutions when mutations could not be identified in 7/441 unilateral retinoblastoma tumors. Four (57%) of these samples showed high-level MYCN amplification with no other M3-Mn gene copy changes. In this study, collaborations were set up with three other centers from around the world, each independently performing similar clinical RB1 gene testing, to collect data for a total of 1023 unilateral retinoblastomas. It was determined that each center had a similar frequency of RB1+/+ MYCNA retinoblastomas and overall, the frequency of RB1+/+ MYCNA retinoblastomas was 3%. It is expected that future studies involving samples to be collected from Africa, China and India will yield a similar frequency of RB1+/+ MYCNA retinoblastomas.

3.1.2 RB1+/+ MYCNA: a novel genetic subset of retinoblastoma

This study showed that RB1+/+ MYCNA retinoblastomas display a very different genetic signature to the well-characterized RB1-/- retinoblastomas. They possess fewer copy number gains and losses in the M3-Mn genes, KIF14, DEK, E2F3, and CDH11. Instead, 7/11 RB1+/+ MYCNA retinoblastomas showed normal copy numbers for those genes. Mutation analysis for each of the M3-Mn genes was not performed in this study, thus there remains a possibility that activating or inactivating point mutations exist in the potential oncogenes and tumor suppressor genes respectively, however there has been no evidence to date suggesting that M3-Mn genes are mutated in retinoblastoma.

With a larger sample set combined with optimized protocol for gene-specific QM-PCR for M3- Mn genes as previously described (Bowles, Corson et al. 2007), it was observed that the frequencies of M3-Mn changes in RB1-/- retinoblastomas are actually higher than previously thought. Of note is that the frequencies of DEK and E2F3 gains were reported as 40% and 70% respectively however in this study, in almost every sample where gain of DEK was observed the

49 same level gain was observed in E2F3 which is consistent with the fact that both genes are within close proximity to each other on chromosome 6p22.

Consistent with the observations that RB1+/+ MYCNA retinoblastomas display fewer M3-Mn gene-specific changes, whole genomic profiling showed that overall, RB1+/+ MYCNA retinoblastomas had fewer altered base pairs than RB1-/- retinoblastomas further supporting that they are a distinct genetic subset of retinoblastoma and are more genomically stable. This observation is consistent with the array CGH study by Mosse et al showing that neuroblastoma samples with MYCN amplification had overall less complex genome-wide pattern of CNAs (Mosse, Diskin et al. 2007).

It was also confirmed that the minimal amplified region contained only the MYCN gene. These results suggest that MYCN-amplification drives tumorigenesis in this unique subset of retinoblastomas that may be similar to neuroblastomas with high-level MYCN amplification. In further support that RB1+/+ MYCNA retinoblastomas are similar to neuroblastomas with MYCN- amplification, it was shown that RB1+/+ MYCNA retinoblastoma histology was uncharacteristic of conventional retinoblastoma but similar to large nucleolar neuroblastomas, most notably they shared the feature of multiple large prominent nucleoli. Overexpression of MYCN in neuroblastoma cells strongly upregulates genes involved in ribosome biogenesis (Boon, Caron et al. 2001). rRNAs and genes involved in ribosome biogenesis reside in nucleoli, which provides an explanation for the increased nucleoli size in MYCN-amplified cells.

3.1.3 MYCN-driven tumorigenesis

Several lines of evidence in this thesis and other studies support the hypothesis that MYCN- amplification drives tumorigenesis. This study showed that RB1+/+ MYCNA retinoblastomas expressed high levels of MYCN transcript. RB1+/+ MYCNA retinoblastomas displayed histology that suggest an activated cell state most likely involving increased ribosome biogenesis and proliferation. These tumors presented at a much earlier age than RB1-/- retinoblastomas suggesting that they may initiate earlier, grow faster and/or may be more aggressive. In vitro, this study showed that inhibition of MYCN in the highly proliferative and MYCN-amplified retinoblastoma cell line, Y79, significantly decreased proliferation. This study also showed that fetal retina, but not adult retina, expressed high levels of MYCN transcript (see Figure 9A) consistent with studies that show normal fetal retina and brain express high levels of MYCN

(Squire, Goddard et al. 1986; Grady, Schwab et al. 1987; Martins, Zindy et al. 2008). Given that amplification of a MYCN transgene expressed in a precursor neuronal cell occurs in the TH- MYCN murine model, it can be hypothesized that perhaps it is a normal precursor retinal cell expressing high levels of MYCN that gives rise to the MYCN-amplified retinoblastoma cell. Failure to downregulate MYCN and differentiate during embryogenesis may give this cell a proliferative advantage. Due to as yet unknown factors, this cell may begin to amplify MYCN providing a selective advantage, giving rise to a larger and earlier presenting tumor than a tumor initiated by loss of RB1. Future in vivo studies involving the Mycn overexpression in the murine retina with wild-type Rb1 may validate this model of MYCN-driven tumorigenesis.

3.1.4 Chromosome 8;13 translocation

In this study, it was found that a focal deletion of the gene REXO1L1 was associated with translocation of chromosome 8 to chromosome 13 in one sample RB381 and that many retinoblastoma samples regardless of RB1 status exhibited CNV at this gene, mostly in the form of deletion and in two cases it was gained. CNV at REXO1L1 is common in both chimpanzees and humans (Perry, Tchinda et al. 2006). Though most CNVs are common and usually considered benign variations in the human genome, they can be associated with complex genetic diseases (Zhang, Gu et al. 2009). Due to the inherent instability at those regions, they can be hotspots for genomic structural rearrangements. Nonallelic homologous recombination (NAHR) is one mechanism postulated to cause CNVs. It is the process by which paralogous genes, different genes which share highly similar DNA sequences, realign and crossover during meiosis or mitosis resulting in deletion or fusion of genes. NAHR occurring on separate chromosomes can result in chromosomal translocations (Lupski 1998). In this study, tumor sample RB381 showed deletion of REXO1L1 at 8q21.2 but normal in blood. However, it was observed that 3 samples in which REXO1L1 was deleted in both tumor and blood DNA. It is tempting to speculate that this common CNV could be a potential recombination hotspot that when combined with other factors could predispose an individual to t(8;13) translocations. To investigate this hypothesis further, a larger sample set of tumor and corresponding blood DNA combined with cytogenetic analyses would be required.

3.1.5 MYCN copy number as a rapid screen for RB1+/+MYCNA retinoblastoma

This study has shown that retinoblastomas with high-level MYCN amplification are a distinct subset characterized by the absence of RB1 mutations and lack of the retinoblastoma genetic signature. Based on these observations, it may be possible to quickly distinguish RB1+/+MYCNA retinoblastomas from RB1-/- retinoblastomas by performing a rapid quantitative-PCR screen for MYCN copy number as an initial test before performing standard RB1 gene testing. In conjunction with clinical data: age of diagnosis and histology of tumors, the identification of samples with high level MYCN amplification would suggest that an RB1 mutation might not be found. Functional assays such as immunohistochemistry to show the presence of pRB might be performed before the full RB1 gene testing. In addition, identified RB1+/+ MYCNA patients could benefit from MYCN-specific therapies that may become available in the future.

3.1.6 Targeting MYCN

Several groups have developed various methods that inhibit MYCN. Ornithine decarboxylase 1 (ODC1) is a rate limiting enzyme in the polyamine synthesis pathway and bona fide target of MYCN (Hogarty, Norris et al. 2008). Using a MYCN reporter construct consisting of a luciferase reporter driven by the ornithine decarboxylase (ODC1) promoter, Lu et al developed a chemical screen for small molecules that could inhibit MYCN/MAX dimerization in a neuroblastoma cell line and successfully identified 8 compounds that could reduce luciferase activity by at least 50% (Lu, Pearson et al. 2003). A few groups have targeted ODC1 directly using the Odc inhibitor alpha-difluoromethylornithine (DFMO) with the reasoning that polyamine synthesis is essential for cell growth and proliferation (Hogarty, Norris et al. 2008; Rounbehler, Li et al. 2009). MYCN has also been suggested to be a potential target for immunotherapy. In a very different approach, Himoudi et al proposed that MYCN would be a good “candidate antigen” to elicit a specific and sustained immune response since it satisfies three essential criteria; it is expressed highly in tumor tissue, it is virtually undetectable in normal tissue and is required for tumorigenesis and will likely not be downregulated by the tumor cell (Himoudi, Yan et al. 2008). The authors performed a proof-of-principle experiment to show that MYCN is indeed immunogenic. T cell lines from normal blood donors could be stimulated to kill MYCN expressing cells and importantly, cytotoxic T-cells specific for MYCN could be generated from a neuroblastoma patient with advanced disease indicating that the generation of a MYCN peptide

52 vaccine is a potential avenue for therapy (Himoudi, Yan et al. 2008). Using yet another approach, an anti-gene peptide nucleic acid (PNA) was developed to inhibit MYCN transcription (Tonelli, Purgato et al. 2005). The PNA consisted of an antisense sequence targeting the second exon of MYCN conjugated to a peptide with a nuclear localization signal. The researchers developed the PNA in an effort to overcome the limitation that antisense oligonucleotides are rapidly degraded. They showed that the anti-gene PNA could inhibit MYCN transcription and protein production and could inhibit growth of neuroblastoma cells (Tonelli, Purgato et al. 2005). Altogether, these studies indicate there is great interest in developing a MYCN-specific therapy that could one day be used to treat RB1+/+ MYCNA retinoblastomas.

3.1.7 Future directions

3.1.7.1 RB1+/+ retinoblastomas

By the most current methods, mutations in the RB1+/+ retinoblastomas could not be detected. Though it was shown that RB+/+ MYCNA retinoblastomas were a distinct subset, it cannot be ruled that there may be deep intronic mutations, promoter mutations and translocations that are not detectable by current technologies. With the advent of next generation sequencing, a future study could resequence the entire RB1 gene in all RB1+/+ retinoblastomas to look for such mutations. This approach has the potential to first further delineate RB+/+ MYCNA retinoblastomas from RB1+/+ retinoblastomas with 2-copy MYCN grouping them as either RB1-/- or an entirely different subset with no RB1 mutations or MYCN amplification and secondly find the second mutations in RB1+/- retinoblastomas that would categorize them as RB1-/- retinoblastomas.

3.1.7.2 Functional status of pRB in RB1+/+ MYCNA retinoblastomas

This study showed that RB1+/+ MYCNA retinoblastoma express full-length pRB, however it has yet to be determined whether the pRB expressed is functional. The p16 is a potent inhibitor of CDK4, which phosphorylates and inactivates pRB. Overexpression of p16 therefore has the effect of activating pRB and arresting cells in G1 phase. The cell line RB522 is derived from a primary tumor in the mid-1980s and was identified to be RB1+/+ MYCNA (Godbout and Squire 1993). These results were confirmed in this current study. To test whether pRB is functions normally in RB1+/+ MYCNA retinoblastomas, a future experiment can be set up such that p16 is exogenously expressed in RB522 cell line and pRB phosphorylation and cell cycle status

53 assayed. If pRB is hypophosphorylated and G1 cell cycle arrest is induced, this would indicate the presence of functional pRB and would suggest that MYCN induces proliferation independent of the RB pathway.

3.1.7.3 Determine effect of MYCN amplification in extraocular retinoblastoma

This study has provided new insight to the role of MYCN in retinoblastoma which can perhaps be applied to other neuroectodermal tumors, especially neuroblastoma where MYCN amplification has prognostic significance. In retinoblastoma, it is not clear whether MYCN amplification causes more aggressive disease since in developed nations, a high cure rate (>95%) is achieved by surgically removing the affected eye (Chintagumpala, Chevez-Barrios et al. 2007). However in developing nations where metastasis is more prevalent, extraocular tumors can be analyzed for MYCN amplification to determine if there is a correlation with more aggressive disease. In conjunction with the clinical analysis of extraocular tumors in developing nations, an in vivo experiment can be set up to test whether Mycn amplification causes more aggressive disease and extraocular spread by injecting the eyes of nude mice with either the WERI cell line which does not cause metastasis (Chevez-Barrios, Hurwitz et al. 2000), or the RB522 cell line, which this study has identified as RB1+/+ MYCNA.

3.1.7.4 In vivo model of MYCNA in mice with intact pRB

It is expected that MYCN overexpression alone will initiate tumors in the murine retina similar to the TH-MYCN murine model of neuroblastoma (Weiss, Aldape et al. 1997). A lentiviral MYCN- overexpression vector was developed for the purpose of injection into the murine retina in this study. When human embryonic kidney cells were transduced with virus, Mycn protein was detected compared to cells transduced with an empty control virus. However, when murine retinal explants were transduced no protein expression was detected indicating that the human CMV promoter was incompatible with murine cells. This experiment highlights an important obstacle in the development of RB+/+MYCNA retinoblastoma model. It is important to find a promoter that can achieve expression in the majority of cell types since it is not known which cell in the retina is the tumor initiating cell. Future experiments must first replace the human CMV promoter with a promoter that can drive Mycn expression to develop the model. Three potential promoters are initially proposed: the Pax 6 α-enhancer, which has been used to drive

54 expression of Cre recombinase in Rbf/f; p107-/- mice, the enhancer that drives expression in SV40 large Tag retinoblastoma mouse model (Windle, Albert et al. 1990) and is active in Müller Glia cells and finally the PCAN1 promoter which is expressed in the neural retina (Cross, Reding et al. 2004). Once a promoter is selected and the model developed, tumor growth, histology of tumors and CGH could be performed to determine whether syntenic regions to human retinoblastoma are altered. This model could be used to validate therapies for MYCN-amplified retinoblastoma as well as other tumors of neuroectodermal origin.

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Appendices

Table 6 Copy numbers of M3-Mn genes in retinoblastomas as measured by QM-PCR Sample AOD RB1 KIF14 DEK E2F3 CDH11 MYCN (months) RB1348 9 +/+ 3.92 2.23 2.30 1.77 38 RB1700 7 +/+ 2.18 2.33 2.18 1.13 49 RB2237 1 +/+ 2.14 2.24 2.11 2.13 76 RB2532 16 +/+ 2.07 2.40 1.99 1.83 73 MA72 4.5 +/+ 1.86 1.66 2.13 1.90 48 MA94 4.3 +/+ 2.14 2.85 2.98 2.47 43 MC945 12.5 +/+ 3.13 2.74 2.34 1.61 57 FA337 12 +/+ 2.16 2.29 2.11 2.38 121 FA793 3 +/+ 2.05 2.15 2.01 2.09 54 NZ499J 10 +/+ 2.37 2.04 1.73 1.93 93 RB522 2 +/+ 2.62 2.01 2.12 2.09 33 RB2285 4 +/- 2.10 1.75 1.71 2.17 73 Total 2/10 (20%) 2/10 (20%) 1/10 (10%) 1/10 (10%) 10/10 (100%) RB818 38.5 +/+ 1.96 2.31 2.39 2.20 2.02 RB2583 56.5 +/+ 2.25 1.98 2.05 1.88 3.17 MA43 83 +/+ 2.55 2.66 2.33 1.20 2.59 MA89 47 +/+ 2.06 1.76 2.25 2.54 2.12 MC140 24.5 +/+ 3.20 7.82 5.98 1.55 5.65 MC336 23 +/+ 2.04 3.95 3.42 1.68 4.48 MC385 17 +/+ 2.20 3.00 2.90 1.85 4.06 MC431 8.5 +/+ 2.38 2.60 2.46 2.08 4.25 MC561 18 +/+ 2.29 2.17 2.03 1.84 3.00 MC887 45.5 +/+ 1.97 2.32 2.04 2.80 4.21 MC972 10.5 +/+ 2.12 4.36 3.66 1.81 3.34 FA319 20 +/+ 2.15 3.50 3.27 1.82 2.99 FA448 8 +/+ 2.02 1.81 1.91 1.93 1.67 FA502 63 +/+ 1.81 1.63 1.66 1.87 3.35 NZ945 15 +/+ 3.44 2.25 2.06 1.16 3.72 Total 2/16 (15%) 7/16 (44%) 5/16 (31%) 3/16 (19%) 11/16 (69%) Total 4/26 (15%) 9/26 (35%) 6/26 (23%) 4/26 (15%) 10/26 RB1+/+MYCNA (38%) and RB1+/+ Toronto, Canada and Essen, Germany RB2903 9 +/- 2.19 2.30 2.02 1.91 1.77 RB 3132 NI +/- 3.29 2.74 2.29 1.58 3.90 RB 2285 4 +/- 2.10 1.75 1.71 2.17 73 RB 374 NI +/- 1.90 2.18 2.00 1.68 2.10 RB 1451 NI +/- 2.05 2.46 2.30 1.99 1.60 RB 1466 NI +/- 1.92 2.85 2.33 1.81 3.25 RB 1777 27 +/- 2.73 3.80 3.49 1.83 4.39

Sample AOD RB1 KIF14 DEK E2F3 CDH11 MYCN (months) RB 1790 20 +/- 2.39 1.80 1.94 2.01 3.34 RB 1962 NI +/- 3.09 2.50 2.20 1.57 4.27 RB 2625 49 +/- 2.27 4.60 4.20 2.05 3.05 RB 2733 30 +/- 2.90 3.00 3.12 1.06 2.42 RB 1530 54 +/- 3.65 3.21 3.12 1.32 4.21 RB 1979 96 +/- 2.40 2.79 5.11 2.48 6.85 RB 2780 20 +/- 2.36 2.31 2.08 2.10 2.75 RB 2854 12 +/- 1.84 2.17 2.02 1.74 3.45 RB 3100 15 +/- 3.03 3.50 3.55 2.16 4.78 MA41 NI +/- 2.23 1.70 2.36 2.36 2.42 MA49 NI +/- 3.32 3.28 3.87 1.95 3.07 MA80 NI +/- 1.73 1.99 2.15 2.03 2.41 MC951 NI +/- 2.48 2.25 2.15 1.96 4.30 Toronto, CANADA RB1436 NI -/- NA NA NA NA 3.47 RB613 NI -/- NA NA NA NA 1.60 RB1545 NI -/- 3.03 2.24 2.26 1.01 6.80 RB381 NI -/- 2.57 5.31 4.67 1.20 7.70 RB2589 NI -/- 2.18 2.20 2.21 1.10 2.02 RB2631 53.7 -/- 3.10 4.11 3.82 1.45 1.69 RB2641 NI -/- 2.95 2.87 2.92 1.03 1.66 RB2647 15.7 -/- 2.87 2.35 2.17 1.51 2.03 RB2683 27 -/- 3.40 5.29 5.54 1.45 1.70 RB2687 NI -/- 2.35 3.66 3.52 1.82 2.18 RB2306 59.9 -/- 3.89 1.62 1.95 1.01 2.30 RB2699 NI -/- 2.16 2.06 2.13 1.78 2.90 RB2686 NI -/- 2.78 2.60 2.76 2.13 2.74 RB2639 NI -/- 2.35 2.35 2.24 2.44 3.40 RB2637 45.1 -/- 2.61 4.62 3.77 1.17 2.70 RB2708 NI -/- 3.15 2.90 3.50 1.41 1.48 RB2598 9.4 -/- 2.20 3.92 3.33 1.77 2.60 RB2838 18.4 -/- 2.70 2.21 2.47 1.69 3.57 RB2582 NI -/- 2.28 1.93 1.70 2.16 3.45 RB2274 NI -/- 2.64 4.86 5.27 1.61 9.58 RB2934 NI -/- 2.16 2.94 3.24 1.98 3.98 RB2820 22.9 -/- 2.84 3.68 4.08 1.25 2.14 RB2960 50.6 -/- 4.78 7.25 5.72 1.50 3.59 RB2280 NI -/- 2.07 2.56 1.98 2.20 2.21 RB2391 1.2 -/- 2.73 1.66 2.58 1.17 2.08 RB1796 NI -/- 2.26 2.08 1.89 0.92 1.86 RB1707 NI -/- 3.07 2.95 3.41 0.81 1.99 RB1738 NI -/- 2.27 2.40 2.11 2.25 3.47 RB1760 NI -/- 3.26 3.13 3.53 1.00 2.05 RB2527 32.5 -/- 1.96 1.61 2.20 2.35 1.80 RB1519 NI -/- 1.70 2.88 3.09 1.06 6.07 RB2327 NI -/- 1.83 1.14 1.72 1.07 4.48 MO13 NI -/- 4.09 3.27 2.69 1.03 4.99 MO15 NI -/- 2.98 2.14 2.10 1.85 3.21 MO21 NI -/- 2.69 2.27 2.27 1.49 2.17

Sample AOD RB1 KIF14 DEK E2F3 CDH11 MYCN (months) MO33 NI -/- 2.67 2.36 2.06 1.31 3.41 MO37 NI -/- 2.66 3.89 3.38 2.20 2.76 MO38 NI -/- 3.48 2.81 2.91 1.86 3.38 MO39 NI -/- 2.81 4.47 3.66 1.44 3.58 MO41 NI -/- 3.00 4.81 5.26 1.17 2.32 MO45 NI -/- 2.83 3.42 3.91 1.48 3.57 MO46 NI -/- 2.46 2.39 2.50 1.72 4.67 MO49 NI -/- 3.82 3.37 3.31 1.25 3.42 MO50 NI -/- 2.10 1.94 1.87 1.04 2.34 MO637 NI -/- 2.19 3.79 3.74 1.92 1.96 RB1575 NI -/- 2.97 2.64 2.92 0.96 NA RB2437 14.8 -/- 3.18 2.84 3.22 1.00 NA RB2621 31 -/- 2.86 4.24 3.77 1.00 NA RB2651 NI -/- 3.03 7.06 6.14 2.61 NA RB2669 NI -/- 3.02 2.17 4.14 2.10 NA RB2670 33.3 -/- 3.29 3.76 4.17 1.10 NA RB2667 NI -/- 2.76 2.00 1.96 1.84 NA RB2674 25.4 -/- 4.68 2.51 2.61 1.16 NA RB2671 NI -/- 5.20 5.40 3.23 1.79 NA RB2675 8.5 -/- 2.83 NA 2.10 1.06 NA RB2646 NI -/- 3.43 3.08 3.35 1.03 NA RB2676 10.4 -/- 2.15 2.79 2.80 2.08 NA RB2680 NI -/- 2.87 4.19 3.65 1.93 NA RB2661 22.6 -/- 3.54 1.85 1.73 1.13 NA RB2630 6.7 -/- 2.45 3.63 2.84 1.69 NA RB2599 NI -/- 4.46 5.91 5.48 1.01 NA RB2672 50.2 -/- 3.46 NA 2.19 0.68 NA RB2591 7.9 -/- 1.91 2.33 2.16 1.72 NA RB2253 NI -/- 2.57 2.65 2.67 2.30 NA RB2284 NI -/- 2.76 2.25 2.47 1.59 NA RB2389 9.8 -/- 2.41 2.16 2.36 2.09 NA RB2396 13.2 -/- 2.38 2.56 2.55 2.01 NA RB2409 NI -/- 3.16 3.42 3.20 1.16 NA RB3110 16.5 -/- 2.17 3.22 2.86 1.59 NA Essen, GERMANY MB109 NI -/- 3.00 2.22 1.82 1.10 2.70 MB190 NI -/- 2.10 4.45 3.66 0.98 2.72 MB209 NI -/- 3.30 5.00 3.87 1.71 3.17 MB213 NI -/- 2.03 2.25 1.97 1.78 5.97 MB429 NI -/- 2.17 1.98 1.75 1.00 3.59 MB449 NI -/- 2.11 2.25 1.77 1.94 3.91 MB456 NI -/- 2.06 1.94 1.67 1.89 3.53 MB486 NI -/- 1.83 2.93 2.66 1.29 3.50 MB607 NI -/- 2.63 3.13 2.80 1.31 3.35 MB703 NI -/- 3.96 6.44 5.89 1.68 3.52 MC480 NI -/- 2.04 4.47 4.60 2.03 2.21 Paris, FRANCE FB014 NI -/- 2.98 4.54 4.00 0.99 2.70

Sample AOD RB1 KIF14 DEK E2F3 CDH11 MYCN (months) FB103 NI -/- 2.18 2.26 2.02 1.63 2.60 FB162 NI -/- 2.11 2.43 2.04 1.78 2.30 FB204 NI -/- 2.54 4.38 3.54 1.16 3.18 FB307 NI -/- 3.49 5.95 4.96 1.25 3.18 FB327 NI -/- 4.09 1.77 2.03 1.01 2.86 FB343 NI -/- 2.60 3.63 3.06 0.82 2.94 FB539 NI -/- 2.15 3.51 3.51 1.75 2.73 FB809 NI -/- 3.17 5.04 3.49 0.85 7.36 FB987 NI -/- 2.94 2.57 2.50 2.32 3.74 Total 54/89 50/87 52/89 49/89 43/67 (61%) (57%) (58%) (55%) (64%) NI, No information. NA, Not assessed.

Table 7 SMRT aCGH alterations by sample Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 1q arm N0510I18 N0068F13 142647117 246833917 104187 Gains 2p24.3 N0451A14 N0065N17 15703698 16825910 1122 2p24.3 N0091E09 N0788G01 14051860 15454023 1402 RB1348 2p24.2 N0701F16 N0631D03 16693859 17748109 1054 Deletions 10q25.2-ter N0466I19 N0091E02 114682303 135262317 20580 16q arm N0180E11 N0163C18 45081598 86413580 41332 2p24.3-p24.2 N0451A14 N0723P04 15703698 18180341 2477 4q33-q35.2 N0132L10 N0555D07 170864319 191239174 20375 Gains 18q21.1 N0776B03 N0774B22 42660453 42846788 186 RB1700 19q13.31 N0653D16 N0160A19 47715841 48626174 910 Deletions 11p and q N0182E22 M2013A02 79527 134436514 134357 16q N0708O13 F0600M14 44997310 88699594 43702 Gains 2p24.3 N0231J10 N0701F16 14410329 16915017 2505 RB2237 10q26.2-ter N0317I13 N0106C07 129057100 135271097 6214 Deletions 16q24.1-q24.3 N0150H19 F0600M14 83749375 88699594 4950 2p25.1-25.3 N0158D10 N0641J22 87030 11754223 11667 2p24.3-p24.2 N0571E19 N0102G08 14565626 17282900 2717 Gains 2p24.21-2q35.1 N0597D07 N0267H19 21369662 223774942 202405

14q21.3-q32.33 N0016G17 M2011A05 45462518 106302057 60840 18q21.1 N0776B03 N0093N16 42660453 42968870 308 RB2532 1p36.11 N0335G20 N0157K08 25454993 25639555 185 2p24.3 N0005H04 N0316B08 11750125 14632932 2882 Deletions 2p24.1-p24.2 N0149C19 N0452B12 17417308 21011770 3594 2q36.1-ter N0060D20 N0321A15 224111722 242359512 18247 8q21.2 N0509F16 M2067O20 86620140 87055825 436 1q arm N0510I18 N0068F13 142647117 246833917 104187 2p24.2-24.3 N0091E09 N0554B24 14051860 18350885 4299 6p24.1-25.3 N0812K10 N0805G18 71610 13047861 13041 Gains 7q31.33-ter N0618G22 N0083D03 123744612 158783389 35039 RB522 11q14.1-q24.1 N0444N24 N0381C13 77769745 122631670 44862 17q21.31-ter M2245G16 N0196O11 39583819 78615238 39031 11p13-ter N0182E22 M2270H09 79527 32378741 32299 Deletions 17p-13.3 N0411G07 N0189D22 415552 18114697 17699 MA72 Gains 2p24.2-24.3 N0723F23 N0422A06 14978618 17691585 2713 2p24.3 N0619O15 N0631D03 15374591 17177195 1803 Gains 6p21.1-ter N0812K10 N0323A09 71610 44642253 44571 MA94 14q22.1-ter M2075E15 M2011A05 48962539 106302057 574340 Deletions 11q14.1-ter N0671D11 M2013A02 83754905 134436514 50682 1q21.1-q41 N0510I18 N0503C11 142647117 212366365 69719 2p25.1-ter N0463H16 N0005H04 79317 11931175 11852 Gains 2p24.2-24.3 N0220H05 N0631D03 13269910 17177195 3907 2p12-2p24.1 N0452B12 N0543B23 20842473 80228525 59386 MC945 8p22-ter N0418D21 N0533K07 30472 18942661 18912 16q12.1-ter N0242N20 F0600M14 50079561 88699594 38620 Deletions 7p11.2-13.3 N0411G07 N0064J19 415552 21191548 20776 7q12 N0342F22 N0722D15 31583727 33809137 2225

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 2p24.3-25.1 N0686G09 N0062M03 10097116 16432161 6335 14q31.3-ter N0557O19 N0012F16 90694710 106218118 15523 Gains 17q21.31-ter N0419E16 N0196O11 40291911 78615238 38323 FA337 19p13.11-ter N0009F15 N0715L15 189657 19436075 19246 8p21.1-ter N0521M14 N0418D21 30472 26980569 26950 Deletions 8q21.2 N0509F16 N0639P04 86620140 87082985 463 2p24.2-24.3 N0541K19 N0554B24 15467410 18350885 2883 FA793 Gains 13q32.1-ter N0517L15 N0226B11 96024440 114103214 18079 2p24.2-24.3 N0723F23 M2305P22 14978618 18851300 3873 Gains 7q34-ter N0119F21 N0083D03 142897566 158783389 15886 18q22.1-22.3 N0607G19 N0399L12 60553398 67415521 6862 1p35.3-ter N0045C18 N0026P17 38264 29279256 29241 2p24.3 N0571E19 N0592G02 14565626 14892009 326 NZ499J 2p24.2 N0424F04 N0118G07 18945057 19624269 679 Deletions 4p14-15.1 N0325H01 N0260B15 30463631 37236400 6773 8q21.2 N0509F16 N0639P04 86620140 87082985 463 16q22.1-23.1 N0598D24 N0594G15 65200179 78178406 12798 17p-17q12 N0411G07 N0592L16 415552 34833159 34418 RB818 Deletions 8q21.2 N0509F16 N0639P04 86620140 87082985 463 1p34.2-ter N0045C18 N0350G05 38264 40504667 40466 1q21.1-23.2 N0026E04 N0646D10 143217450 160958046 17741 1q32.1-32.2 N0017B07 N0345I23 198807950 208088220 9280 1q42.12-44 N0014D01 N0332D17 223882340 245744383 21862 2p23.2-ter N0651P03 N0371D08 148491 28477624 28329 4p16.1-16.3 N0071F05 N0640N05 358140 8437036 8079 6p21.1-ter N0328C17 N0323A09 177604 44642253 44465 6q24.3-ter N0117P04 N0159J07 160357898 170880179 10522 9q33.2-ter N0147E03 N0668B20 125161993 140237228 15075 Gains 17p13.3-q21.3 N0411G07 N0607H13 415552 46208463 45793 17q22-24.2 N0695B13 N0120M18 52856819 64137803 11281 17q24.3-ter N0353I13 N0196O11 68705146 78615238 9911 MA43 19p13.11-ter N0110A24 N0715L15 134914 19436075 19301 19q arm N0719O04 N0493D23 33507965 63696484 30189 20p12.3-ter N0640A09 M2130I11 60370 5961085 5901 20p11.21-12.1 N0176D18 N0269F15 13794775 26013217 12218 21q22.2-ter N0017J10 N1000I21 40973387 46940213 5967 22q arm N0423L23 N0040G15 15935029 49569190 33643 3p26.1-ter N0385A18 N0810H01 38685 8163492 8125 3p22.1-24.3 N0015O03 N0092J20 16938767 43774860 26836 4p16.1-q34.3 N1338A24 N0231C10 8655779 181250916 172595 Deletions 8q arm N0691F16 N0639O03 46999570 146236298 99237 9q21.1-ter N0632I19 N0143M01 22027 32152961 32131 14q11.1-23.2 N0404K10 M2285E05 18071243 61866126 43795 16q12.1-21 N0708O13 F0600M14 44997310 88699594 43702

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 1p34.1-ter N0379K15 N0802K22 95421 44425691 44330 6p21.1 N0380E17 N0375K06 41356867 44392522 3036 9p13.2-13.3 N0284F01 N0644E22 33879013 37940151 4061 9q33.3-ter N0205K06 N0668B20 128256342 140237228 11981 11p15.5 N0182E22 N0474E20 79527 1930170 1851 17p13.1-ter N0634P19 N0657A11 415552 10130238 9715 Gains MA89 17q12-21.33 N0342F22 N0021I09 31583727 46560288 14977 17q22-ter N0468D03 N0196O11 52252199 78615238 26363 19p and q arms N0519F09 N0493D23 27679 63696484 63669 20q13.31 N0267N05 N0476I15 54593535 62434320 7841 21q22.3 N0891L10 N1000I21 42282192 46940213 4658 22q12.3 N0452N11 N0040G15 33919042 49569190 15650 Deletions 8q21.2 N0038K03 M2067O20 86757546 87055825 299 1p36.13 N0777P08 N0148H11 16665329 16891342 266 1q arm N0510I18 N0068F13 142647117 246833917 104187 2p arm N0463H16 N0785H17 79317 91633812 91554 MC140 Gains 6p arm N0812K10 N0325M17 71610 58872610 58801 10q26.2-ter N0223P11 N0106C07 128657820 135271097 6613 13q34-ter N0412K14 N0226B11 110600643 114103214 3503 21q22.3 N0447A17 N0457P07 44437000 46926492 2489 2p13.3-ter N0463H16 N0482J04 79317 70893187 70814 6p arm N0812K10 N0325M17 71610 58872610 58802 9q33.3-ter N0661B09 N0350O14 127160749 139220879 12060 Gains MC336 19p13.11-ter N0744L24 N0657O13 189657 18874065 18684 20q11.21-13.2 N0620H13 N0694L10 29986634 51672430 21686 20q13.31-ter N0231B02 N0134L13 55955329 62416964 6461 Deletions 20q13.2 N0474C21 N0262B23 53355489 53684580 329 1q21.1-25.3 N0510I18 N0804A08 142647117 186845681 44199 Gains 1q32.1-ter N0119D06 N0059M10 199215670 246789440 47574 MC385 2p24.3-ter N0463H16 N0733B22 79317 74792843 74714 Deletions 2p12-13.1 N0123I06 N0755O06 74988529 83668734 8680 MC431 Gains 8q21.2 N0038K03 F0574H12 86757546 86973008 215 1q arm N0510I18 N0068F13 142647117 246833917 104187 Gains MC561 13q32.1-ter N0504C17 N0226B11 96334426 114103214 17769 Deletions 22q12.3-ter N0564B15 N0040G15 31062997 49569190 18506 10p13-15.1 N0453H02 N0606G04 6485574 17251721 10766 10q11.21-23.1 N0770F09 N0470J18 42409119 86380414 43971 13q21.1-ter N0114F16 N0226B11 53055272 114103214 61048 Gains 17q23.1-ter M2001K22 N0196O11 55060077 78615238 23555 18p and q arms N0059I11 N0565D23 37518 76103181 76066 MC887 19p arm N0009F15 N0717E18 189657 24391802 24202 3p arm N0385A18 M2185K04 38685 90584932 90546 4q21.1-22.1 N0077J09 N0737D22 76868319 90108053 13240 Deletions 8q21.2 N0038K03 F0574H12 86757546 86973008 215 10p15.1-ter N0797F08 N0284M10 65726 4908726 4843 13q14.3 N0435C23 N0715B19 52101811 52938369 837 6p arm N0812K10 N0325M17 71610 58872610 58801 9q33.3-ter N0121C13 N0668B20 126474147 140237228 13763 Gains MC972 19p13.11-ter N0519F09 N0715L15 27679 19436075 19408 19q12-ter N0109B11 N0493D23 36871853 63696484 26825 Deletions 8q21.2 N0038K03 F0574H12 86757546 86973008 215

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name FA448 No alterations 5q13.2 N0155O16 N0313J05 69070334 70698370 1628 FA502 Gains 15q13.3 N0336F16 N0732H03 30112611 30621426 509 20q13.2 N0006L15 N0790B05 52483586 54304290 1821 1q arm N0510I18 N0068F13 142647117 246833917 104187 Gains NZ945 15q13.3 N0336F16 N0732H03 30112611 30621426 509 Deletions 16q arm N0708O13 F0600M14 44997310 88699594 43702 13q14.3-21.1 N0470H04 N0435C23 50664473 52278216 1614 13q21.32 N0583P19 N0731A24 66046052 66805219 759 Gains 13q32.1 N0432C10 N0080B03 94004743 94578199 573 13q32.1 N0638C04 N0297E16 96178610 96782355 604 15q13.3 N0336F16 N0732H03 30112611 30621426 509 5q23.2 N0619L15 N0009E08 124791998 125696404 904 8q21.2 N0509F16 M2067O20 86620140 87055825 436 13q13.1 RB2903 N0379M14 N0584M10 32574544 33186613 612 13q13.3-14.13 N0624H01 N0164I01 35802418 46132877 10330 13q14.13 N0651I05 N0307O11 59901013 60620577 720 Deletions 13q21.2 N0164E20 N0640A08 66882861 67681892 799 13q21.32 M2026N21 N0440F07 70875970 71173005 297 13q21.33 N0298G13 N0026J21 76201583 77651205 1450 13q22.3 N0133E12 N0459D15 93005803 93348854 343 13q31.3 N0411G07 N0687M21 415552 13940898 13525 17p12-ter 2p24.2-24.3 N0674F13 N0631D03 15919161 17177195 1258 Gains RB2285 16p12.3-ter N0766H16 N0164A06 73492 18186964 18113 Deletions 13q13.3-ter N0336L17 M2323L19 35268823 113989403 78721 RB374 Deletions 13q14.13 N0071H01 N0454H21 46787216 53074718 6288 5q11.2-21.3 N0619H18 N0099I23 53825498 109768296 55943 RB1466 Deletions 13q13.3-14.11 N0051K07 N0316D04 38740354 43148508 4408 1q arm N0510I18 N0068F13 142647117 246833917 104187 2p arm N0463H16 N0447F08 79317 89958830 89880 Gains 6p arm N0812K10 N0325M17 71610 58872610 58801 14q arm N0643D12 M2011A05 21377600 106302057 84924 19p13.2 N0282G19 N0203K06 8665783 8832847 167 13q14.11 N0756F10 N0131B13 39917249 40286991 370 13q13.11 N0632G17 N0467G16 42205257 42694359 489 RB1777 13q14.2 N0192F23 N0602C22 47196559 48889915 1693 13q21.1-14.3 N0435C23 N0458G10 52101811 52701353 600 Deletions 13q21.2 N0750I15 N0675B18 58083246 58915551 832 13q21.31 N0109J06 N0586C17 60883541 61395239 512 13q21.33 N0626I10 M2026N21 69505102 71053759 1549 13q22.3 N0598D17 N0203P02 76679454 77764214 1085 13q31.1 N0398A22 N0467P19 85215055 86720366 1505 1q arm N0510I18 N0068F13 142647117 246833917 104187 2p23.1-ter N0463H16 N0450L18 79317 31455726 31376 RB1790 Gains 10p arm N0797F08 N0787P11 65726 39116217 39050 17q21.22-ter N0472H05 N0196O11 44810989 78615238 33804

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 10q arm N0496O18 N0106C07 41753627 135271097 93517 13q12.11 N0717M17 N0064F04 19491702 20304995 813 13q12.3 N0058M19 N0706H02 28785074 29739221 954 13q14.11 N0110C17 N0267N09 41349120 43792169 2443 Deletions 13q14.2 N0685I15 N0795F23 46601076 47823050 1222 13q21.2-32.1 N0616E14 N0621G18 53146615 96075230 42929 13q33.1 N0624M01 N0681O22 102099748 107271825 5172 17p arm N0411G07 N0399C02 415552 22128721 21713 22q13.1-ter N0806D02 N0040G15 36789448 49569190 12780 2p and q N0463H16 N0321A15 79317 242359512 242280 5q14.1-pter N0348B13 N0043N06 102972 79524609 79422 6p25.3-p22.3 N0812K10 N0159C08 71610 20622576 20551 Gains 13q31.3-q34 N0487A02 N0226B11 90908618 114103214 23195

15q11.2 N0607H20 N0034K18 18273500 19941698 1668 RB2625 19q12-13.11 N0738N05 N0306G07 32864434 40136267 7172 20p and q N0766B22 N0476I15 67103 62434320 62367 1p34.3-p11.2 N0799L22 N0115N23 43367715 121064448 77697 Deletions 6p22.3-qter N0648P12 N0113J06 20726090 170851849 150126 13q14.11-q31.3 N0350A18 N0035H02 39817963 90730242 50912 14q13.1-q21.3 N0345M21 N0634H08 34291680 46587232 12296 1q21.1-25.3 N0510I18 N0703I24 142647117 183125425 40478 1q32.1-ter N0051H18 N0068F13 199695815 246833917 47138 2p and q N0463H16 N0321A15 79317 242359512 242280 6p12.3-ter N0812K10 N0734G07 71610 48046066 47974 Gains 7p21.1-ter N0669C22 M2245C05 40844 18783666 18743 13q N0563G05 N0226B11 18014607 114103214 96089 19p and q N0519F09 N0493D23 27679 63696484 63669 RB1530 20p and q N0766B22 N0476I15 67103 62434320 62367 3p and q N0038B22 N0192L23 16865 199240276 199223 4q N0365H22 N0555D07 52388942 191239174 138850 9p and q N0143M01 N0668B20 22027 140237228 140215 Deletions 12p and q M2094C14 M2140B24 16595 132289487 132273 14q N0404K10 M2011A05 18071243 106302057 88231 16p and q N0568F01 F0600M14 74714 88699594 88625 22q M2177M20 N0040G15 14440103 49569190 35129 6p23 N0144A19 N0810G03 15608023 16239152 631 6p22.2-23 N0597G24 N0006N23 19267498 24574853 5307 6p22.1 N0600F15 N0313H11 26943704 27628148 684 Gains 6p21.31 N0043G08 N0528P20 33689655 35027691 1338 12q24.21-24.22 N0749J02 N0612H13 112884293 116443044 3559 12q24.31-ter N0387F15 N0503G07 122580551 131701422 9121 18p and q N0683L23 N0565D23 17653 76103181 76086 RB1979 8p and q N0091J19 N0639O03 304176 146236298 145932 9p and q N0143M01 N0668B20 22027 140237228 140215 12p-q24.21 N0574G08 N0666G06 38722 112890869 112852 Deletions 12q24.22- N0791N06 N0197N18 116308201 122155363 5847 q24.31 N0012E03 M2012N23 44251042 73915995 29665 13q14.12 N0095I09 N0558B22 18459439 100221959 81763 15q arm 13q31.3-ter N0618L13 N0226B11 93475025 114103214 20628 RB2780 Gains 19p13.2 N0282G19 N0203K06 8665783 8832847 167

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 1q arm N0510I18 N0068F13 142647117 246833917 104187 Gains 2p arm N0463H16 N0785H17 79317 91633812 91554 4q28.3-ter N0090M18 N0555D07 140693364 191239174 50546 4q28.3 N0685M01 N0745L13 131288820 133157751 1869 RB3100 5q21.2-ter N0358E06 N0324K20 103000264 180730036 77730 Deletions 6p arm N0812K10 N0325M17 71610 58872610 58801 8q21.2 N0509F16 N0639P04 86620140 87082985 463 13q14.13-14.2 N0417P21 N0685I15 46257295 46771856 515 1p34.1-ter N0045C18 N0767N06 38264 45939116 45901 6p21.1-ter N0812K10 N0375K06 71610 44392522 44321 9q33.2-ter N0498E02 N0035I18 123966918 140185674 16219 Gains MA41 17q12-ter N1330O06 N0196O11 31801769 78615238 46813 19p and q N0657O13 N0493D23 189657 63696484 63507 22q N0437O02 N0620A14 15930262 45478948 29549 Deletions 13q14.3 N0790J06 N0746C24 49769922 77566504 27797 1q32.1-ter N0133P06 N0068F13 198475140 246833917 48359 6p21.1-ter N0812K10 N0323A09 71610 44642253 44571 9q33.3 N0258M22 N0668B20 126850503 140237228 13387 17q12-21.33 N0208D07 N0167K20 30340614 46634199 16294 17q22 N0016A07 N0013C05 52448493 63066270 10618 Gains 17q25.1-ter N0647F02 N0196O11 69684458 78615238 8931 MA49 19p and q N0657O13 N0493D23 189657 63696484 63507 20q N0702M08 N0151C05 29644114 35444952 5801 20q13.12 N0014E17 N0260O01 43271693 50137940 6866 21q22.2-ter N0035C04 N1000I21 42127232 46940213 4813 22q N0437O02 N0040G15 15930262 49569190 33639 Deletions 13q14.3-21.1 N0572P15 N0196C09 49941858 55373857 5432 2p21-ter N0371D08 N0749C14 148491 46586091 46438 13q22.2-ter N0639I16 N0226B11 75781463 114103214 38322 Gains 18p11.32-q11.2 N1035E02 N0009E17 18338 22399113 22381 19p12-ter N0657O13 N0468G14 189657 20746786 20557 1p32.1-31.1 N0010A17 N0794G09 59319690 77102399 17783 MA80 4p16.2-qter N1235J08 N0555D07 3576555 191239174 187663 8p and q N0418D21 N0639O03 30472 146236298 146206 Deletions 9q33.2-pter N0143M01 N0804N08 22027 122729099 122707 13q13.1-13.2 N0045L14 N0090F05 32165473 34512960 2347 13q21.31-21.32 N0108P18 N0816F07 60620617 65283696 4663 16p13.3-q23.3 N0063H12 N0813D14 4772802 82400842 77628 2p25.1-25.3 N0129I01 N0517E08 5022521 9267587 4245 Gains 2p24.2-24.3 N0541K19 M2123N14 15467410 18028443 2561 7q21.1-36.1 N0634B10 F0620M21 77767314 153591479 75824 2p N0119K02 N0788G01 9249270 15454023 6205 RB381 8q21.2 N0509F16 N0639P04 86620140 87082985 463 Deletions 13q14.12 N0564M19 N0164H01 45178511 46121109 943 13q32.3 N0044I07 N0418I10 100614576 101707600 1093 19p13.2 N0282G19 N0203K06 8665783 8832847 167 6p arm N0328C17 N0325M17 177604 58872610 58695 16q N0708O13 F0600M14 44997310 88699594 43702 RB1336 Gains 19p-q13.42 N0009F15 N0066H23 189657 59985032 59795 20p and q N0640A09 N0476I15 60370 62434320 62374 21q N0073I15 N0457P07 14327625 46926492 32599

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name RB1740 Deletions 17p12-ter N0411G07 N0687M21 415552 13940898 13525 1q32.1 N0165E10 N0595K11 200361154 205125953 4765 4p16.1-ter N0335H03 N0238O15 39105 9220751 9182 4p15.31-15.33 M2185J18 N0013H04 15392919 19998505 4606 4p15.1 N0092M20 N0103P18 29923977 31661464 1737 4p13-14 N0142P03 N0384C20 38295574 43297704 5002 4q13.3-21.1 N0632A23 N0641E03 73533568 78634666 5101 4q31.3-32.1 N0509I22 N0071A06 154805443 156270117 1465 6p and q N0812K10 N0113J06 71610 170851849 170780 8p21.2-21.3 N0116M17 N0795G08 23526552 26272272 2746 8p11.23-8p12 N0745K06 N0156L03 36910075 38058325 1148 RB2052 Gains 13q11-12.11 N0563G05 N0506L23 18014607 20335446 2321 13q12.11-12.12 N0273F15 N0499O19 21528172 24215466 2687 13q13.3 N0162F21 N0718A20 35606696 38322375 2716 13q14.3 N0630I06 N0655C11 51289896 52555670 1266 13q22.1-22.2 N0441J10 N0010A23 73593525 74860115 1267 13q33.3-ter N0014G15 N0226B11 109310260 114103214 4793 18p and q N0683L23 N0565D23 17653 76103181 76086 19p and q N0519F09 N0493D23 27679 63696484 63669 20 p and q N0766B22 N0476I15 67103 62434320 62367 21q N0675D22 N0457P07 21358972 46926492 25568

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 4p16.1 N0063P01 N0065O09 10208020 10941759 734 4q13.1 N0371B02 N0484B23 60325977 65150139 4824 4q21.1-21.3 N0638G02 N0272G21 79651029 87642586 7992 4q26 N0111L14 N0122K18 115365486 119579104 4214 4q28.1 N0728C08 N0427N08 125060157 127718213 2658 4q32.1 N0423E20 N0295F08 159055244 161426872 2372 4q34.1-34.2 M2220L14 N0520H06 174532621 177417393 2885 4q34.3-35.1 N0442N05 N0692E14 181621943 184692674 3071 4q35.1 N0099L17 N0472G22 185082952 186877885 1795 4q35.1-35.2 N0571N01 N0016L12 186855084 189851441 2996 8p23.3-ter N0111E15 N0521J16 156973 3647702 3491 8p21.3-22 N0711D03 N0652H24 23535579 16420058 7116 8p12-8p21.2 N0521M14 N0051J09 26980569 30264501 3284 8p12 N0608P11 N0654O09 32968313 34431087 1463 8p11.21 N0745M10 N0117K13 41575316 42689304 1114 8q11.22-11.23 N0818C13 N0669G08 52218489 53575828 1357 8q12.1 N0022E14 N0314N12 57031842 58582905 1551 Deletions 8q12.1 N0661A03 N0051L11 59655134 60743597 1088 8q21.11 N0503K13 N0064K14 75247524 78302251 3055 8q21.2-21.3 N0509F16 N0023B03 86620140 93815239 7195 8q22.3-23.1 N0109C19 N0659A24 103542026 109058233 5516 8q23.3-24.13 N0096J05 N0047A23 116012122 122584169 6572 8q24.22 N0238O02 N0213I02 132286383 133463867 1177 8q24.3 N0265N12 N0792G19 140919525 142491552 1572 10p and q N0797F08 N0106C07 65726 135271097 135205 13q12.13-12.3 N0662C02 N0629E24 24792776 29602194 4809 13q13.3-14.11 N0050D16 N0025N03 38419312 39705077 1286 13q14.13-14.3 N0417P21 N0686G10 46257295 50401135 4144 13q21.1-21.2 N0685E08 M2012K04 53909558 58001888 4092 13q21.33-22.1 N0521B14 N0709B02 68182005 73283703 5102 13q31.1-32.1 N0605B09 N0747M20 78322396 95902058 17580 13q33.1-33.3 N0790J08 N0359M07 104048543 109061691 5013 15q arm M2200G17 N0558B22 19970520 100221959 80251 16p and q N0568F01 F0600M14 74714 88699594 88625 6p N0157M05 N0159J07 65779919 170880179 105100 Gains 17q24.3-ter N0075H13 N0196O11 66875850 78615238 11739 20q arm N0559K10 N0476I15 29294627 62434320 33140 1p N0045C18 N0385C11 38264 120345972 120308 8q21.2 N0509F16 M2067O20 86620140 87055825 436 RB2589 8q24.13-ter N0636H23 N0639O03 123692681 146236298 22544 Deletions 10q24.31-ter F0628D17 N0091E02 102443708 135262317 32819 16p13.2-ter N0766H16 N0107G06 73492 9954815 9881 16q12.2-ter N0533J12 F0600M14 52309608 88699594 36390 17p arm N0411G07 N0399C02 415552 22128721 21713 1p arm N0510I18 N0059M10 142647117 246789440 104142 6p arm N0059N17 N0325M17 351266 58872610 58521 13q14.2-ter N0563G05 N0685I15 18014607 46771856 28757 RB2631 Gains 13q14.3-21.1 N0157B12 N0715B19 51626286 52938369 1312 13q31.1-32.3 N0514P01 N0813H05 89114601 98902788 9788 13q33.2-ter N0111G22 N0226B11 105217625 114103214 8886 14q24.3 N0306K22 M2011A05 74286435 106302057 32016

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 8p23.1-ter N0418D21 N0802F15 30472 11564386 11534 13q14.2-14.3 N0438K10 N0509N17 47560571 51364049 3803 Deletions 16p and q N0773G07 N0665E03 235519 88686715 88451 17p N0411G07 N0728E14 415552 21322111 20907 20p and q N0649H22 N0134L13 6122936 62416964 56294 1q N0510I18 N0059M10 142647117 246789440 104142 6p N0537J16 N0325M17 367602 58872610 58505 Gains 9q N0088D03 N0668B20 66405147 140237228 73832 13q N0563G05 N0226B11 18014607 114103214 96089 RB2641 16q N0046B20 N0665E03 84705552 88686715 3981 1p35.3 N0414L23 N0467D18 608751 29444707 28836 Deletions 14q N0521F15 N0046B20 62108060 84867117 22759 19p and q N0081I08 N0265J21 902662 58496669 57594 1q N0510I18 N0059M10 142647117 246789440 104142 Gains 5p N0597A21 M2220G19 350757 45985067 45634 RB2647 3q26.1-ter N0593A09 M2110L16 167465632 199131621 31666 Deletions 16q N0584H05 N0665E03 45532097 88686715 43155 1q N0114B18 N0068F13 149888055 246833917 96946 6p N0812K10 N0325M17 71610 58872610 58801 Gains 7q32.1-ter N0475K08 N0083D03 127479042 158783389 31304 14q21.3 N0279J20 N0012F16 47251765 106218118 58966 RB2683 1p35.1pter N0045C18 N0114D07 38264 34244419 34206 13q13.3-21.31 N0336L17 N0109J06 35268823 61051693 25783 Deletions 16p and q N0773G07 N0665E03 235519 88686715 88451 17p13.2 N0411G07 N0220M19 415552 5373014 4957 1q23.2-ter N0297K08 N0059M10 159374906 246789440 87415 Gains 9q N0211E19 N0644H13 70091012 139839547 69749 16q11.2-12.1 N0471D09 N0545E02 45229845 47619108 2389 1p35.3-ter N0776O18 N0333N08 307737 30171718 29864 RB2306 5q31.3-ter N0035N12 N0324K20 140231805 180730036 40498 8p and q N0091J19 N0639O03 304176 146236298 145932 Deletions 16p13.11-ter N0568F01 N0103G05 74714 15025243 14951 16p11.2-12.3 N0813D06 N0590H03 18938492 33871612 14933 16q N0001F10 N0665E03 47811144 88686715 40876 1q N0510I18 N0059M10 142647117 246789440 104142 2p N0420M07 N0785H17 264456 91633812 91369 5p and q N0811I15 N0324K20 72312 180730036 180658 6p N0059N17 N0325M17 351266 58872610 58521 Gains 9p31.2-ter N0069E16 N0668B20 109899366 140237228 30338 RB247 11p15.1-ter N0182E22 N0583F24 79527 19004570 18925 17q21.31-ter N0135M15 N0196O11 40812466 78615238 37803 18p11.31 N0683L23 N0297J24 17653 6128065 6110 8q21.2 N0509F16 F0574H12 86620140 86973008 353 Deletions 15q26.1 N0267B15 N0558B22 88814205 100221959 11408 1p21.3-22 N0212D05 N0263K19 150227817 153541655 3314 2p N0371D08 N0495B16 148491 82715436 82567 5q31.1-33.2 N0254M05 N0096J04 132519214 152951910 20433 RB383 Gains 6p N0812K10 N0325M17 71610 58872610 58801 13q N0563G05 N0226B11 18014607 114103214 96089 22q N0619K17 N0040G15 22778020 49569190 26791

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 5q33.2-ter N0565D08 N0324K20 153948314 180730036 26782 Deletions 18p N0059I11 N0390I06 37518 15399258 15362 1q N0510I18 N0059M10 142647117 246789440 104142 7q N0665G16 N0083D03 80719268 158783389 78064 Gains 14q22.3 N0118M18 M2011A05 55340193 106302057 50962 21q21.2-ter N0705P12 N0457P07 25638123 46926492 21288 5q11.2-13.2 N0143O12 N0808I04 55718971 68938439 13219 RB1021 5q33.2-ter N0096J04 N0324K20 152764659 180730036 27965 10p N0592K16 F0547M11 286972 39012541 38726 Deletions 11p N0063A07 N0699N10 621027 50692538 50072 11q14.3-23.3 N0798B05 N0152O02 91188732 118910577 27722 16q N0471D09 F0600M14 45229845 88699594 43470 19q13.2-ter F0497A07 N0493D23 46383400 63696484 17313 1q21.1-23.3 N0510I18 N0578G06 142647117 162556579 19909 1q24.3-24.3 N0448B23 N0118D10 170841028 178664532 7824 1q31.3-32.2 N0124P19 N0002P02 192123480 206526445 14403 1q41 N0101K19 N0514C19 213850669 219396428 5546 1q41-42.3 N0381O10 N0739C15 219492439 233391573 13899 2p12-ter N0463H16 N0543B23 79317 80228525 80149 Gains 3p22.3-ter N0038B22 N0384L08 16865 32142422 32126 5p14.3 N0811I15 N0638C22 209225 18922411 18713 7p15.3-ter M2245C05 N1151M13 40844 25288541 25248 13q12.12-12.2 N0294L03 N0624L24 23076054 27970959 4895 13q31.1-ter N0089A14 N0226B11 82412127 114103214 31691 17q12-ter N0032H06 N0196O11 35262985 78615238 43352 21q22.13-ter N0777J19 N0457P07 37641258 46926492 9285 1q24.1-24.3 N0713G21 N0341B16 164981867 169464909 4483 WERI 1q25.2-25.3 N0796G10 N0450F15 178395125 181243272 2848 1q31.1-31.2 M2245J12 N0272B08 185734306 191129912 5396 1q41-42.13 N0638A13 N0638A13 219624020 228145219 8521 3q28-ter N0456E14 N0192L23 190215218 199240276 9025 5q11.2-12.1 N0008N21 N0593B07 55340491 62741971 7401 10p N0109N22 N0797F08 65726 35812347 35747 10q11.23 N0423G02 N0640E12 50166065 54430559 4264 Deletions 13q12.3-13.1 N0057H24 N0645A10 27927907 31278920 3351 13q13.3-14.11 N0289J04 N0039L22 35991523 43637987 7646 13q14.2-21.33 N0795F23 N0465K12 47624175 70603880 22980 13q22.2-31.1 N0715H21 N0103P23 75329929 82201310 6871 16q23.2-ter N0474K20 F0600M14 78991561 88699594 9708 18q22.1-22.3 N0607G19 N0798A24 60553398 68679304 8126 21p N0376P20 N0259G22 9721646 10197104 475 21q11.2-21.2 N0429H22 N0709D12 13270806 24687098 11416 2p24.3 N0619O15 N0463P22 15175256 16152619 977 7q35-ter N0564O04 N0083D03 147151220 158783389 11632 11q24.1-ter N0381C13 M2013A02 122460473 134436514 11976 Gains 13q21.32-ter N0001L24 N0226B11 64741928 114103214 49361 Y79 18q12.3-21.1 M2032O09 N0002E13 41713136 46266399 4553 18q23 N0531M16 N0703M10 71676600 72686216 1010 21q22.11-ter N0004F08 N0457P07 32359411 46926492 14567 5p13.3-15.33 N0128A03 M2335O24 4054083 32497485 28443 Deletions 16q11.2-12.2 N0180E11 N0070N16 45081598 54541026 9459

Sample Locus Start clone End clone Base pair start Base pair end Size (kb) Name 1p31.3-33 N0682P18 N0185E18 51504350 62616624 11112 1q21.1-25.3 N0510I18 N0522B03 142647117 182897083 40250 1q32.1-ter N0133P06 N0332D17 198475140 245744383 47269 2p24.3-24.3 N0571E19 N0427M01 14565626 17376543 2811 Gains 2q14 N0678O18 N0812M06 66473520 67573409 1100 IMR32 6p21.1-21.22 N0065L23 N0061H05 30666043 44747012 14081 15q N0137P24 N0584I15 59763464 91451479 31688 17q N0606M07 N0196O11 37680398 78615238 40935 20q-ter N0004O09 N0476I15 29737772 62434320 32697 1p33-ter N0379K15 N0670L22 95421 50599578 50504 Deletions 16q22.2-ter N0113E03 N0655C18 69787193 85754878 15968