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A Subset of Retinoblastoma Lacking RB1 Gene Mutations have High-level MYCN Gene Amplification
by
Stephanie Yee
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Molecular Genetics University of Toronto
© Copyright by Stephanie Yee 2010
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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.
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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
v
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
vi
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
vii
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
viii
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
ix
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
x
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
xi
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
xii
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
xiii
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)
xiv
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
xv
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
xvi
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
1
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).
2
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).
7
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,
8
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.
10
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,
12
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.
13
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.
15
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.
17
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.
18
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.
21
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
22
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.
23
Table 1 Samples used for QM-PCR
Test Site # of RB1-/-
tested for KIF14, DEK,
E2F3, CDH11 copy number
# of RB1-/- tested for
MYCN copy number
# of RB1+/- tested for
KIF14, DEK, E2F3, CDH11 copy number
# of RB1+/- tested for
MYCN copy number
# of RB1+/+ tested for
KIF14, DEK, E2F3, CDH11
and MYCN copy number
Toronto, Canada
69 48 16 17 8*
Essen, Germany
12 12 4 4 12
Paris, France 10 10 0 0 5
Christchurch, New Zealand
0 0 0 0 2
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.
24
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).
25
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’ 5’-TCATTTCTCTTCCTTGTTTGAGGT-3’
2787
MYCN 5’-CACAAGGCCCTCAGTACCTC-3’ 5’-TCTTCTGTGGGGGTGCAT-3’
283
Ki67 5’-GCTAAAACATGGAGATGTAAT-3’ 5’-ATTTTGGTCTTGACTTACGC-3’
631
TBP 5’-ACAACAGGCTGCCACCTTAC-3’ 5’-GCTGGAAAACCCAACTTCTG-3’
743
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
26
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.
27
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).
29
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 Unilateral
retinoblastomas
Number of RB1-/- and
RB1+/-
Number of RB1+/+ (Freq.)
P-value (pair-wise
proportion test)
Number of RB1+/+ MYCNA
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.
30
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.
32
Table 4 Frequencies of M3-Mn changes in RB1+/+
versus RB1-/-
retinoblastomas
KIF14 DEK E2F3 CDH11 MYCN Gain
(2.5-10)
MYCN Amplificati
on (>10)
France RB1+/+
RB1-/-
0/5 6/10
1/5 6/10
1/5 6/10
0/5 6/10
2/5 9/10
2/5 0/10
Germany RB1+/+
RB1-/-
3/17 5/11
9/17 6/11
7/17 6/11
3/17 5/11
10/17 11/11
3/17 0/11
Toronto RB1+/+
RB1-/-
3/8 45/70
1/8 41/70
0/8 42/70
2/8 38/70
2/8 25/91
4/8 0/91
New Zealand RB1+/+
RB1-/-
1/2 NA
0/2 NA
0/2 NA
1/2 NA
1/2 NA
1/2 NA
Pooled RB1+/+
RB1-/-
7/32 56/91
11/32 53/91
8/32 54/91
6/32 49/91
15/32 45/112
10/32 0/112
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).
33
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.
34
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.
35
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).
36
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.
37
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).
39
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.
41
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).
42
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.
43
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.
44
Table 5 Summary of retinoblastoma histopathological features in RB1+/+
MYCNA retinoblastomas
Pathologic features
RB1348 (report only)
RB1700 (report only)
RB522 RB2237 MA94 NZ499 RB2903 (2-copy MYCN )
Flexner-Wintersteiner
rosettes
- - - - - - +
Homer-Wright NS + - + - - -
Pseudorosettes - - - - - - -
Retinoma - - - - - - -
Mitotic figures NS NS + + + + +
Necrosis + + + + + + +
Calcification + + + + + + +
Vitreous seeding
NS NS + + - + +
Nuclear moulding
NS NS - - - - +
Apoptosis NS NS + + + + +
Optic nerve involvement
- - NS - - - -
Choroid and Sclera
involvement
+ + - - - + -
Anterior segment
NS - - - - - -
Large prominent
nuclei
NS NS + + - + -
NS, Not scored
45
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).
46
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).
47
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).
48
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
50
(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.
51
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.
55
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67
Appendices
Table 6 Copy numbers of M3-Mn genes in retinoblastomas as measured by QM-PCR
Sample AOD (months)
RB1 KIF14 DEK E2F3 CDH11 MYCN
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 RB1
+/+MYCN
A
and RB1+/+
4/26 (15%) 9/26 (35%) 6/26 (23%) 4/26 (15%) 10/26 (38%)
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
68
Sample AOD (months)
RB1 KIF14 DEK E2F3 CDH11 MYCN
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
69
Sample AOD (months)
RB1 KIF14 DEK E2F3 CDH11 MYCN
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
70
Sample AOD (months)
RB1 KIF14 DEK E2F3 CDH11 MYCN
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 (61%)
50/87 (57%)
52/89 (58%)
49/89 (55%)
43/67 (64%)
NI, No information. NA, Not assessed.
71
Table 7 SMRT aCGH alterations by sample
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
RB1348
Gains 1q arm 2p24.3
N0510I18 N0451A14
N0068F13 N0065N17
142647117 15703698
246833917 16825910
104187 1122
Deletions
2p24.3 2p24.2 10q25.2-ter 16q arm
N0091E09 N0701F16 N0466I19 N0180E11
N0788G01 N0631D03 N0091E02 N0163C18
14051860 16693859 114682303 45081598
15454023 17748109 135262317 86413580
1402 1054 20580 41332
RB1700 Gains
2p24.3-p24.2 4q33-q35.2 18q21.1 19q13.31
N0451A14 N0132L10 N0776B03 N0653D16
N0723P04 N0555D07 N0774B22 N0160A19
15703698 170864319 42660453 47715841
18180341 191239174 42846788 48626174
2477 20375 186 910
Deletions
11p and q 16q
N0182E22 N0708O13
M2013A02 F0600M14
79527 44997310
134436514 88699594
134357 43702
RB2237 Gains 2p24.3 N0231J10 N0701F16 14410329 16915017 2505
Deletions 10q26.2-ter 16q24.1-q24.3
N0317I13 N0150H19
N0106C07 F0600M14
129057100 83749375
135271097 88699594
6214 4950
RB2532
Gains
2p25.1-25.3 2p24.3-p24.2 2p24.21-2q35.1 14q21.3-q32.33 18q21.1
N0158D10 N0571E19 N0597D07 N0016G17 N0776B03
N0641J22 N0102G08 N0267H19 M2011A05 N0093N16
87030 14565626 21369662 45462518 42660453
11754223 17282900 223774942 106302057 42968870
11667 2717 202405 60840 308
Deletions
1p36.11 2p24.3 2p24.1-p24.2 2q36.1-ter 8q21.2
N0335G20 N0005H04 N0149C19 N0060D20 N0509F16
N0157K08 N0316B08 N0452B12 N0321A15 M2067O20
25454993 11750125 17417308 224111722 86620140
25639555 14632932 21011770 242359512 87055825
185 2882 3594 18247 436
RB522
Gains
1q arm 2p24.2-24.3 6p24.1-25.3 7q31.33-ter 11q14.1-q24.1 17q21.31-ter
N0510I18 N0091E09 N0812K10 N0618G22 N0444N24 M2245G16
N0068F13 N0554B24 N0805G18 N0083D03 N0381C13 N0196O11
142647117 14051860 71610 123744612 77769745 39583819
246833917 18350885 13047861 158783389 122631670 78615238
104187 4299 13041 35039 44862 39031
Deletions 11p13-ter 17p-13.3
N0182E22 N0411G07
M2270H09 N0189D22
79527 415552
32378741 18114697
32299 17699
MA72 Gains 2p24.2-24.3 N0723F23 N0422A06 14978618 17691585 2713
MA94 Gains
2p24.3 6p21.1-ter 14q22.1-ter
N0619O15 N0812K10 M2075E15
N0631D03 N0323A09 M2011A05
15374591 71610 48962539
17177195 44642253 106302057
1803 44571 574340
Deletions 11q14.1-ter N0671D11 M2013A02 83754905 134436514 50682
MC945
Gains
1q21.1-q41 2p25.1-ter 2p24.2-24.3 2p12-2p24.1
N0510I18 N0463H16 N0220H05 N0452B12
N0503C11 N0005H04 N0631D03 N0543B23
142647117 79317 13269910 20842473
212366365 11931175 17177195 80228525
69719 11852 3907 59386
Deletions
8p22-ter 16q12.1-ter 7p11.2-13.3 7q12
N0418D21 N0242N20 N0411G07 N0342F22
N0533K07 F0600M14 N0064J19 N0722D15
30472 50079561 415552 31583727
18942661 88699594 21191548 33809137
18912 38620 20776 2225
72
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
FA337
Gains
2p24.3-25.1 14q31.3-ter 17q21.31-ter 19p13.11-ter
N0686G09 N0557O19 N0419E16 N0009F15
N0062M03 N0012F16 N0196O11 N0715L15
10097116 90694710 40291911 189657
16432161 106218118 78615238 19436075
6335 15523 38323 19246
Deletions 8p21.1-ter 8q21.2
N0521M14 N0509F16
N0418D21 N0639P04
30472 86620140
26980569 87082985
26950 463
FA793 Gains 2p24.2-24.3 13q32.1-ter
N0541K19 N0517L15
N0554B24 N0226B11
15467410 96024440
18350885 114103214
2883 18079
NZ499J
Gains 2p24.2-24.3 7q34-ter 18q22.1-22.3
N0723F23 N0119F21 N0607G19
M2305P22 N0083D03 N0399L12
14978618 142897566 60553398
18851300 158783389 67415521
3873 15886 6862
Deletions
1p35.3-ter 2p24.3 2p24.2 4p14-15.1 8q21.2 16q22.1-23.1 17p-17q12
N0045C18 N0571E19 N0424F04 N0325H01 N0509F16 N0598D24 N0411G07
N0026P17 N0592G02 N0118G07 N0260B15 N0639P04 N0594G15 N0592L16
38264 14565626 18945057 30463631 86620140 65200179 415552
29279256 14892009 19624269 37236400 87082985 78178406 34833159
29241 326 679 6773 463 12798 34418
RB818 Deletions 8q21.2 N0509F16 N0639P04 86620140 87082985 463
MA43
Gains
1p34.2-ter 1q21.1-23.2 1q32.1-32.2 1q42.12-44 2p23.2-ter 4p16.1-16.3 6p21.1-ter 6q24.3-ter 9q33.2-ter 17p13.3-q21.3 17q22-24.2 17q24.3-ter 19p13.11-ter 19q arm 20p12.3-ter 20p11.21-12.1 21q22.2-ter 22q arm
N0045C18 N0026E04 N0017B07 N0014D01 N0651P03 N0071F05 N0328C17 N0117P04 N0147E03 N0411G07 N0695B13 N0353I13 N0110A24 N0719O04 N0640A09 N0176D18 N0017J10 N0423L23
N0350G05
N0646D10 N0345I23 N0332D17 N0371D08 N0640N05 N0323A09 N0159J07 N0668B20 N0607H13 N0120M18 N0196O11 N0715L15 N0493D23 M2130I11 N0269F15 N1000I21 N0040G15
38264 143217450 198807950 223882340 148491 358140 177604 160357898 125161993 415552 52856819 68705146 134914 33507965 60370 13794775 40973387 15935029
40504667 160958046 208088220 245744383 28477624 8437036 44642253 170880179 140237228 46208463 64137803 78615238 19436075 63696484 5961085 26013217 46940213 49569190
40466 17741 9280 21862 28329 8079 44465 10522 15075 45793 11281 9911 19301 30189 5901 12218 5967 33643
Deletions
3p26.1-ter 3p22.1-24.3 4p16.1-q34.3 8q arm 9q21.1-ter 14q11.1-23.2 16q12.1-21
N0385A18 N0015O03 N1338A24 N0691F16 N0632I19 N0404K10 N0708O13
N0810H01 N0092J20 N0231C10 N0639O03 N0143M01 M2285E05 F0600M14
38685 16938767 8655779 46999570 22027 18071243 44997310
8163492 43774860 181250916 146236298 32152961 61866126 88699594
8125 26836 172595 99237 32131 43795 43702
73
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
MA89 Gains
1p34.1-ter 6p21.1 9p13.2-13.3 9q33.3-ter 11p15.5 17p13.1-ter 17q12-21.33 17q22-ter 19p and q arms 20q13.31 21q22.3 22q12.3
N0379K15 N0380E17 N0284F01 N0205K06 N0182E22 N0634P19 N0342F22 N0468D03 N0519F09 N0267N05 N0891L10 N0452N11
N0802K22 N0375K06 N0644E22 N0668B20 N0474E20 N0657A11 N0021I09 N0196O11 N0493D23 N0476I15 N1000I21 N0040G15
95421 41356867 33879013 128256342 79527 415552 31583727 52252199 27679 54593535 42282192 33919042
44425691 44392522 37940151 140237228 1930170 10130238 46560288 78615238 63696484 62434320 46940213 49569190
44330 3036 4061 11981 1851 9715 14977 26363 63669 7841 4658 15650
Deletions 8q21.2 N0038K03 M2067O20 86757546 87055825 299
MC140 Gains
1p36.13 1q arm 2p arm 6p arm 10q26.2-ter 13q34-ter 21q22.3
N0777P08 N0510I18 N0463H16 N0812K10 N0223P11 N0412K14 N0447A17
N0148H11 N0068F13 N0785H17 N0325M17 N0106C07 N0226B11 N0457P07
16665329 142647117 79317 71610 128657820 110600643 44437000
16891342 246833917 91633812 58872610 135271097 114103214 46926492
266 104187 91554 58801 6613 3503 2489
MC336 Gains
2p13.3-ter 6p arm 9q33.3-ter 19p13.11-ter 20q11.21-13.2 20q13.31-ter
N0463H16 N0812K10 N0661B09 N0744L24 N0620H13 N0231B02
N0482J04 N0325M17 N0350O14 N0657O13 N0694L10 N0134L13
79317 71610 127160749 189657 29986634 55955329
70893187 58872610 139220879 18874065 51672430 62416964
70814 58802 12060 18684 21686 6461
Deletions 20q13.2 N0474C21 N0262B23 53355489 53684580 329
MC385 Gains
1q21.1-25.3 1q32.1-ter 2p24.3-ter
N0510I18 N0119D06 N0463H16
N0804A08 N0059M10 N0733B22
142647117 199215670 79317
186845681 246789440 74792843
44199 47574 74714
Deletions 2p12-13.1 N0123I06 N0755O06 74988529 83668734 8680
MC431 Gains 8q21.2 N0038K03 F0574H12 86757546 86973008 215
MC561 Gains
1q arm 13q32.1-ter
N0510I18 N0504C17
N0068F13 N0226B11
142647117 96334426
246833917 114103214
104187 17769
Deletions 22q12.3-ter N0564B15 N0040G15 31062997 49569190 18506
MC887
Gains
10p13-15.1 10q11.21-23.1 13q21.1-ter 17q23.1-ter 18p and q arms 19p arm
N0453H02 N0770F09 N0114F16 M2001K22 N0059I11 N0009F15
N0606G04 N0470J18 N0226B11 N0196O11 N0565D23 N0717E18
6485574 42409119 53055272 55060077 37518 189657
17251721 86380414 114103214 78615238 76103181 24391802
10766 43971 61048 23555 76066 24202
Deletions
3p arm 4q21.1-22.1 8q21.2 10p15.1-ter 13q14.3
N0385A18 N0077J09 N0038K03 N0797F08 N0435C23
M2185K04 N0737D22 F0574H12 N0284M10 N0715B19
38685 76868319 86757546 65726 52101811
90584932 90108053 86973008 4908726 52938369
90546 13240 215 4843 837
MC972 Gains
6p arm 9q33.3-ter 19p13.11-ter 19q12-ter
N0812K10 N0121C13 N0519F09 N0109B11
N0325M17 N0668B20 N0715L15 N0493D23
71610 126474147 27679 36871853
58872610 140237228 19436075 63696484
58801 13763 19408 26825
Deletions 8q21.2 N0038K03 F0574H12 86757546 86973008 215
74
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
FA448 No alterations
FA502 Gains 5q13.2 15q13.3 20q13.2
N0155O16 N0336F16 N0006L15
N0313J05 N0732H03 N0790B05
69070334 30112611 52483586
70698370 30621426 54304290
1628 509 1821
NZ945 Gains
1q arm 15q13.3
N0510I18 N0336F16
N0068F13 N0732H03
142647117 30112611
246833917 30621426
104187 509
Deletions 16q arm N0708O13 F0600M14 44997310 88699594 43702
RB2903
Gains
13q14.3-21.1 13q21.32 13q32.1 13q32.1 15q13.3
N0470H04 N0583P19 N0432C10 N0638C04 N0336F16
N0435C23 N0731A24 N0080B03 N0297E16 N0732H03
50664473 66046052 94004743 96178610 30112611
52278216 66805219 94578199 96782355 30621426
1614 759 573 604 509
Deletions
5q23.2 8q21.2 13q13.1 13q13.3-14.13 13q14.13 13q21.2 13q21.32 13q21.33 13q22.3 13q31.3 17p12-ter
N0619L15 N0509F16 N0379M14 N0624H01 N0651I05 N0164E20 M2026N21 N0298G13 N0133E12 N0411G07
N0009E08 M2067O20 N0584M10 N0164I01 N0307O11 N0640A08 N0440F07 N0026J21 N0459D15 N0687M21
124791998 86620140 32574544 35802418 59901013 66882861 70875970 76201583 93005803 415552
125696404 87055825 33186613 46132877 60620577 67681892 71173005 77651205 93348854 13940898
904 436 612 10330 720 799 297 1450 343 13525
RB2285 Gains
2p24.2-24.3 16p12.3-ter
N0674F13 N0766H16
N0631D03 N0164A06
15919161 73492
17177195 18186964
1258 18113
Deletions 13q13.3-ter N0336L17 M2323L19 35268823 113989403 78721
RB374 Deletions 13q14.13 N0071H01 N0454H21 46787216 53074718 6288
RB1466 Deletions 5q11.2-21.3 13q13.3-14.11
N0619H18 N0051K07
N0099I23 N0316D04
53825498 38740354
109768296 43148508
55943 4408
RB1777
Gains
1q arm 2p arm 6p arm 14q arm 19p13.2
N0510I18 N0463H16 N0812K10 N0643D12 N0282G19
N0068F13 N0447F08 N0325M17 M2011A05 N0203K06
142647117 79317 71610 21377600 8665783
246833917 89958830 58872610 106302057 8832847
104187 89880 58801 84924 167
Deletions
13q14.11 13q13.11 13q14.2 13q21.1-14.3 13q21.2 13q21.31 13q21.33 13q22.3 13q31.1
N0756F10 N0632G17 N0192F23 N0435C23 N0750I15 N0109J06 N0626I10 N0598D17 N0398A22
N0131B13 N0467G16 N0602C22 N0458G10 N0675B18 N0586C17 M2026N21 N0203P02 N0467P19
39917249 42205257 47196559 52101811 58083246 60883541 69505102 76679454 85215055
40286991 42694359 48889915 52701353 58915551 61395239 71053759 77764214 86720366
370 489 1693 600 832 512 1549 1085 1505
RB1790 Gains
1q arm 2p23.1-ter 10p arm 17q21.22-ter
N0510I18 N0463H16 N0797F08 N0472H05
N0068F13 N0450L18 N0787P11 N0196O11
142647117 79317 65726 44810989
246833917 31455726 39116217 78615238
104187 31376 39050 33804
75
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
Deletions
10q arm 13q12.11 13q12.3 13q14.11 13q14.2 13q21.2-32.1 13q33.1 17p arm 22q13.1-ter
N0496O18 N0717M17 N0058M19 N0110C17 N0685I15 N0616E14 N0624M01 N0411G07 N0806D02
N0106C07 N0064F04 N0706H02 N0267N09 N0795F23 N0621G18 N0681O22 N0399C02 N0040G15
41753627 19491702 28785074 41349120 46601076 53146615 102099748 415552 36789448
135271097 20304995 29739221 43792169 47823050 96075230 107271825 22128721 49569190
93517 813 954 2443 1222 42929 5172 21713 12780
RB2625
Gains
2p and q 5q14.1-pter 6p25.3-p22.3 13q31.3-q34 15q11.2 19q12-13.11 20p and q
N0463H16 N0348B13 N0812K10 N0487A02 N0607H20 N0738N05 N0766B22
N0321A15 N0043N06 N0159C08 N0226B11 N0034K18 N0306G07 N0476I15
79317 102972 71610 90908618 18273500 32864434 67103
242359512 79524609 20622576 114103214 19941698 40136267 62434320
242280 79422 20551 23195 1668 7172 62367
Deletions
1p34.3-p11.2 6p22.3-qter 13q14.11-q31.3 14q13.1-q21.3
N0799L22 N0648P12 N0350A18 N0345M21
N0115N23 N0113J06 N0035H02 N0634H08
43367715 20726090 39817963 34291680
121064448 170851849 90730242 46587232
77697 150126 50912 12296
RB1530
Gains
1q21.1-25.3 1q32.1-ter 2p and q 6p12.3-ter 7p21.1-ter 13q 19p and q 20p and q
N0510I18 N0051H18 N0463H16 N0812K10 N0669C22 N0563G05 N0519F09 N0766B22
N0703I24 N0068F13 N0321A15 N0734G07 M2245C05 N0226B11 N0493D23 N0476I15
142647117 199695815 79317 71610 40844 18014607 27679 67103
183125425 246833917 242359512 48046066 18783666 114103214 63696484 62434320
40478 47138 242280 47974 18743 96089 63669 62367
Deletions
3p and q 4q 9p and q 12p and q 14q 16p and q 22q
N0038B22 N0365H22 N0143M01 M2094C14 N0404K10 N0568F01 M2177M20
N0192L23 N0555D07 N0668B20 M2140B24 M2011A05 F0600M14 N0040G15
16865 52388942 22027 16595 18071243 74714 14440103
199240276 191239174 140237228 132289487 106302057 88699594 49569190
199223 138850 140215 132273 88231 88625 35129
RB1979
Gains
6p23 6p22.2-23 6p22.1 6p21.31 12q24.21-24.22 12q24.31-ter 18p and q
N0144A19 N0597G24 N0600F15 N0043G08 N0749J02 N0387F15 N0683L23
N0810G03 N0006N23 N0313H11 N0528P20 N0612H13 N0503G07 N0565D23
15608023 19267498 26943704 33689655 112884293 122580551 17653
16239152 24574853 27628148 35027691 116443044 131701422 76103181
631 5307 684 1338 3559 9121 76086
Deletions
8p and q 9p and q 12p-q24.21 12q24.22-q24.31 13q14.12 15q arm
N0091J19 N0143M01 N0574G08 N0791N06 N0012E03 N0095I09
N0639O03 N0668B20 N0666G06 N0197N18 M2012N23 N0558B22
304176 22027 38722 116308201 44251042 18459439
146236298 140237228 112890869 122155363 73915995 100221959
145932 140215 112852 5847 29665 81763
RB2780 Gains 13q31.3-ter 19p13.2
N0618L13 N0282G19
N0226B11 N0203K06
93475025 8665783
114103214 8832847
20628 167
76
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
RB3100
Gains 1q arm 2p arm 4q28.3-ter
N0510I18 N0463H16 N0090M18
N0068F13 N0785H17 N0555D07
142647117 79317 140693364
246833917 91633812 191239174
104187 91554 50546
Deletions
4q28.3 5q21.2-ter 6p arm 8q21.2 13q14.13-14.2
N0685M01 N0358E06 N0812K10 N0509F16 N0417P21
N0745L13 N0324K20 N0325M17 N0639P04 N0685I15
131288820 103000264 71610 86620140 46257295
133157751 180730036 58872610 87082985 46771856
1869 77730 58801 463 515
MA41 Gains
1p34.1-ter 6p21.1-ter 9q33.2-ter 17q12-ter 19p and q 22q
N0045C18 N0812K10 N0498E02 N1330O06 N0657O13 N0437O02
N0767N06 N0375K06 N0035I18 N0196O11 N0493D23 N0620A14
38264 71610 123966918 31801769 189657 15930262
45939116 44392522 140185674 78615238 63696484 45478948
45901 44321 16219 46813 63507 29549
Deletions 13q14.3 N0790J06 N0746C24 49769922 77566504 27797
MA49 Gains
1q32.1-ter 6p21.1-ter 9q33.3 17q12-21.33 17q22 17q25.1-ter 19p and q 20q 20q13.12 21q22.2-ter 22q
N0133P06 N0812K10 N0258M22 N0208D07 N0016A07 N0647F02 N0657O13 N0702M08 N0014E17 N0035C04 N0437O02
N0068F13 N0323A09 N0668B20 N0167K20 N0013C05 N0196O11 N0493D23 N0151C05 N0260O01 N1000I21 N0040G15
198475140 71610 126850503 30340614 52448493 69684458 189657 29644114 43271693 42127232 15930262
246833917 44642253 140237228 46634199 63066270 78615238 63696484 35444952 50137940 46940213 49569190
48359 44571 13387 16294 10618 8931 63507 5801 6866 4813 33639
Deletions 13q14.3-21.1 N0572P15 N0196C09 49941858 55373857 5432
MA80
Gains
2p21-ter 13q22.2-ter 18p11.32-q11.2 19p12-ter
N0371D08 N0639I16 N1035E02 N0657O13
N0749C14 N0226B11 N0009E17 N0468G14
148491 75781463 18338 189657
46586091 114103214 22399113 20746786
46438 38322 22381 20557
Deletions
1p32.1-31.1 4p16.2-qter 8p and q 9q33.2-pter 13q13.1-13.2 13q21.31-21.32 16p13.3-q23.3
N0010A17 N1235J08 N0418D21 N0143M01 N0045L14 N0108P18 N0063H12
N0794G09 N0555D07 N0639O03 N0804N08 N0090F05 N0816F07 N0813D14
59319690 3576555 30472 22027 32165473 60620617 4772802
77102399 191239174 146236298 122729099 34512960 65283696 82400842
17783 187663 146206 122707 2347 4663 77628
RB381
Gains 2p25.1-25.3 2p24.2-24.3 7q21.1-36.1
N0129I01 N0541K19 N0634B10
N0517E08 M2123N14 F0620M21
5022521 15467410 77767314
9267587 18028443 153591479
4245 2561 75824
Deletions
2p 8q21.2 13q14.12 13q32.3 19p13.2
N0119K02 N0509F16 N0564M19 N0044I07 N0282G19
N0788G01 N0639P04 N0164H01 N0418I10 N0203K06
9249270 86620140 45178511 100614576 8665783
15454023 87082985 46121109 101707600 8832847
6205 463 943 1093 167
RB1336 Gains
6p arm 16q 19p-q13.42 20p and q 21q
N0328C17 N0708O13 N0009F15 N0640A09 N0073I15
N0325M17 F0600M14 N0066H23 N0476I15 N0457P07
177604 44997310 189657 60370 14327625
58872610 88699594 59985032 62434320 46926492
58695 43702 59795 62374 32599
77
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
RB1740 Deletions 17p12-ter N0411G07 N0687M21 415552 13940898 13525
RB2052 Gains
1q32.1 4p16.1-ter 4p15.31-15.33 4p15.1 4p13-14 4q13.3-21.1 4q31.3-32.1 6p and q 8p21.2-21.3 8p11.23-8p12 13q11-12.11 13q12.11-12.12 13q13.3 13q14.3 13q22.1-22.2 13q33.3-ter 18p and q 19p and q 20 p and q 21q
N0165E10 N0335H03 M2185J18 N0092M20 N0142P03 N0632A23 N0509I22 N0812K10 N0116M17 N0745K06 N0563G05 N0273F15 N0162F21 N0630I06 N0441J10 N0014G15 N0683L23 N0519F09 N0766B22 N0675D22
N0595K11 N0238O15 N0013H04 N0103P18 N0384C20 N0641E03 N0071A06 N0113J06 N0795G08 N0156L03 N0506L23 N0499O19 N0718A20 N0655C11 N0010A23 N0226B11 N0565D23 N0493D23 N0476I15 N0457P07
200361154 39105 15392919 29923977 38295574 73533568 154805443 71610 23526552 36910075 18014607 21528172 35606696 51289896 73593525 109310260 17653 27679 67103 21358972
205125953 9220751 19998505 31661464 43297704 78634666 156270117 170851849 26272272 38058325 20335446 24215466 38322375 52555670 74860115 114103214 76103181 63696484 62434320 46926492
4765 9182 4606 1737 5002 5101 1465 170780 2746 1148 2321 2687 2716 1266 1267 4793 76086 63669 62367 25568
78
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
Deletions
4p16.1 4q13.1 4q21.1-21.3 4q26 4q28.1 4q32.1 4q34.1-34.2 4q34.3-35.1 4q35.1 4q35.1-35.2 8p23.3-ter 8p21.3-22 8p12-8p21.2 8p12 8p11.21 8q11.22-11.23 8q12.1 8q12.1 8q21.11 8q21.2-21.3 8q22.3-23.1 8q23.3-24.13 8q24.22 8q24.3 10p and q 13q12.13-12.3 13q13.3-14.11 13q14.13-14.3 13q21.1-21.2 13q21.33-22.1 13q31.1-32.1 13q33.1-33.3 15q arm 16p and q
N0063P01 N0371B02 N0638G02 N0111L14 N0728C08 N0423E20 M2220L14 N0442N05 N0099L17 N0571N01 N0111E15 N0711D03 N0521M14 N0608P11 N0745M10 N0818C13 N0022E14 N0661A03 N0503K13 N0509F16 N0109C19 N0096J05 N0238O02 N0265N12 N0797F08 N0662C02 N0050D16 N0417P21 N0685E08 N0521B14 N0605B09 N0790J08 M2200G17 N0568F01
N0065O09 N0484B23 N0272G21 N0122K18 N0427N08 N0295F08 N0520H06 N0692E14 N0472G22 N0016L12 N0521J16 N0652H24 N0051J09 N0654O09 N0117K13 N0669G08 N0314N12 N0051L11 N0064K14 N0023B03 N0659A24 N0047A23 N0213I02 N0792G19 N0106C07 N0629E24 N0025N03 N0686G10 M2012K04 N0709B02 N0747M20 N0359M07 N0558B22 F0600M14
10208020 60325977 79651029 115365486 125060157 159055244 174532621 181621943 185082952 186855084 156973 23535579 26980569 32968313 41575316 52218489 57031842 59655134 75247524 86620140 103542026 116012122 132286383 140919525 65726 24792776 38419312 46257295 53909558 68182005 78322396 104048543 19970520 74714
10941759 65150139 87642586 119579104 127718213 161426872 177417393 184692674 186877885 189851441 3647702 16420058 30264501 34431087 42689304 53575828 58582905 60743597 78302251 93815239 109058233 122584169 133463867 142491552 135271097 29602194 39705077 50401135 58001888 73283703 95902058 109061691 100221959 88699594
734 4824 7992 4214 2658 2372 2885 3071 1795 2996 3491 7116 3284 1463 1114 1357 1551 1088 3055 7195 5516 6572 1177 1572 135205 4809 1286 4144 4092 5102 17580 5013 80251 88625
RB2589
Gains 6p 17q24.3-ter 20q arm
N0157M05 N0075H13 N0559K10
N0159J07 N0196O11 N0476I15
65779919 66875850 29294627
170880179 78615238 62434320
105100 11739 33140
Deletions
1p 8q21.2 8q24.13-ter 10q24.31-ter 16p13.2-ter 16q12.2-ter 17p arm
N0045C18 N0509F16 N0636H23 F0628D17 N0766H16 N0533J12 N0411G07
N0385C11 M2067O20 N0639O03 N0091E02 N0107G06 F0600M14 N0399C02
38264 86620140 123692681 102443708 73492 52309608 415552
120345972 87055825 146236298 135262317 9954815 88699594 22128721
120308 436 22544 32819 9881 36390 21713
RB2631 Gains
1p arm 6p arm 13q14.2-ter 13q14.3-21.1 13q31.1-32.3 13q33.2-ter 14q24.3
N0510I18 N0059N17 N0563G05 N0157B12 N0514P01 N0111G22 N0306K22
N0059M10 N0325M17 N0685I15 N0715B19 N0813H05 N0226B11 M2011A05
142647117 351266 18014607 51626286 89114601 105217625 74286435
246789440 58872610 46771856 52938369 98902788 114103214 106302057
104142 58521 28757 1312 9788 8886 32016
79
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
Deletions
8p23.1-ter 13q14.2-14.3 16p and q 17p 20p and q
N0418D21 N0438K10 N0773G07 N0411G07 N0649H22
N0802F15 N0509N17 N0665E03 N0728E14 N0134L13
30472 47560571 235519 415552 6122936
11564386 51364049 88686715 21322111 62416964
11534 3803 88451 20907 56294
RB2641
Gains
1q 6p 9q 13q 16q
N0510I18 N0537J16 N0088D03 N0563G05 N0046B20
N0059M10 N0325M17 N0668B20 N0226B11 N0665E03
142647117 367602 66405147 18014607 84705552
246789440 58872610 140237228 114103214 88686715
104142 58505 73832 96089 3981
Deletions 1p35.3 14q 19p and q
N0414L23 N0521F15 N0081I08
N0467D18 N0046B20 N0265J21
608751 62108060 902662
29444707 84867117 58496669
28836 22759 57594
RB2647 Gains
1q 5p
N0510I18 N0597A21
N0059M10 M2220G19
142647117 350757
246789440 45985067
104142 45634
Deletions 3q26.1-ter 16q
N0593A09 N0584H05
M2110L16 N0665E03
167465632 45532097
199131621 88686715
31666 43155
RB2683
Gains
1q 6p 7q32.1-ter 14q21.3
N0114B18 N0812K10 N0475K08 N0279J20
N0068F13 N0325M17 N0083D03 N0012F16
149888055 71610 127479042 47251765
246833917 58872610 158783389 106218118
96946 58801 31304 58966
Deletions
1p35.1pter 13q13.3-21.31 16p and q 17p13.2
N0045C18 N0336L17 N0773G07 N0411G07
N0114D07 N0109J06 N0665E03 N0220M19
38264 35268823 235519 415552
34244419 61051693 88686715 5373014
34206 25783 88451 4957
RB2306
Gains 1q23.2-ter 9q 16q11.2-12.1
N0297K08 N0211E19 N0471D09
N0059M10 N0644H13 N0545E02
159374906 70091012 45229845
246789440 139839547 47619108
87415 69749 2389
Deletions
1p35.3-ter 5q31.3-ter 8p and q 16p13.11-ter 16p11.2-12.3 16q
N0776O18 N0035N12 N0091J19 N0568F01 N0813D06 N0001F10
N0333N08 N0324K20 N0639O03 N0103G05 N0590H03 N0665E03
307737 140231805 304176 74714 18938492 47811144
30171718 180730036 146236298 15025243 33871612 88686715
29864 40498 145932 14951 14933 40876
RB247
Gains
1q 2p 5p and q 6p 9p31.2-ter 11p15.1-ter 17q21.31-ter 18p11.31
N0510I18 N0420M07 N0811I15 N0059N17 N0069E16 N0182E22 N0135M15 N0683L23
N0059M10 N0785H17 N0324K20 N0325M17 N0668B20 N0583F24 N0196O11 N0297J24
142647117 264456 72312 351266 109899366 79527 40812466 17653
246789440 91633812 180730036 58872610 140237228 19004570 78615238 6128065
104142 91369 180658 58521 30338 18925 37803 6110
Deletions 8q21.2 15q26.1
N0509F16 N0267B15
F0574H12 N0558B22
86620140 88814205
86973008 100221959
353 11408
RB383 Gains
1p21.3-22 2p 5q31.1-33.2 6p 13q 22q
N0212D05 N0371D08 N0254M05 N0812K10 N0563G05 N0619K17
N0263K19 N0495B16 N0096J04 N0325M17 N0226B11 N0040G15
150227817 148491 132519214 71610 18014607 22778020
153541655 82715436 152951910 58872610 114103214 49569190
3314 82567 20433 58801 96089 26791
80
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
Deletions 5q33.2-ter 18p
N0565D08 N0059I11
N0324K20 N0390I06
153948314 37518
180730036 15399258
26782 15362
RB1021
Gains
1q 7q 14q22.3 21q21.2-ter
N0510I18 N0665G16 N0118M18 N0705P12
N0059M10 N0083D03 M2011A05 N0457P07
142647117 80719268 55340193 25638123
246789440 158783389 106302057 46926492
104142 78064 50962 21288
Deletions
5q11.2-13.2 5q33.2-ter 10p 11p 11q14.3-23.3 16q 19q13.2-ter
N0143O12 N0096J04 N0592K16 N0063A07 N0798B05 N0471D09 F0497A07
N0808I04 N0324K20 F0547M11 N0699N10 N0152O02 F0600M14 N0493D23
55718971 152764659 286972 621027 91188732 45229845 46383400
68938439 180730036 39012541 50692538 118910577 88699594 63696484
13219 27965 38726 50072 27722 43470 17313
WERI
Gains
1q21.1-23.3 1q24.3-24.3 1q31.3-32.2 1q41 1q41-42.3 2p12-ter 3p22.3-ter 5p14.3 7p15.3-ter 13q12.12-12.2 13q31.1-ter 17q12-ter 21q22.13-ter
N0510I18 N0448B23 N0124P19 N0101K19 N0381O10 N0463H16 N0038B22 N0811I15 M2245C05 N0294L03 N0089A14 N0032H06 N0777J19
N0578G06 N0118D10 N0002P02 N0514C19 N0739C15 N0543B23 N0384L08 N0638C22 N1151M13 N0624L24 N0226B11 N0196O11 N0457P07
142647117 170841028 192123480 213850669 219492439 79317 16865 209225 40844 23076054 82412127 35262985 37641258
162556579 178664532 206526445 219396428 233391573 80228525 32142422 18922411 25288541 27970959 114103214 78615238 46926492
19909 7824 14403 5546 13899 80149 32126 18713 25248 4895 31691 43352 9285
Deletions
1q24.1-24.3 1q25.2-25.3 1q31.1-31.2 1q41-42.13 3q28-ter 5q11.2-12.1 10p 10q11.23 13q12.3-13.1 13q13.3-14.11 13q14.2-21.33 13q22.2-31.1 16q23.2-ter 18q22.1-22.3 21p 21q11.2-21.2
N0713G21 N0796G10 M2245J12 N0638A13 N0456E14 N0008N21 N0109N22 N0423G02 N0057H24 N0289J04 N0795F23 N0715H21 N0474K20 N0607G19 N0376P20 N0429H22
N0341B16 N0450F15 N0272B08 N0638A13 N0192L23 N0593B07 N0797F08 N0640E12 N0645A10 N0039L22 N0465K12 N0103P23 F0600M14 N0798A24 N0259G22 N0709D12
164981867 178395125 185734306 219624020 190215218 55340491 65726 50166065 27927907 35991523 47624175 75329929 78991561 60553398 9721646 13270806
169464909 181243272 191129912 228145219 199240276 62741971 35812347 54430559 31278920 43637987 70603880 82201310 88699594 68679304 10197104 24687098
4483 2848 5396 8521 9025 7401 35747 4264 3351 7646 22980 6871 9708 8126 475 11416
Y79
Gains
2p24.3 7q35-ter 11q24.1-ter 13q21.32-ter 18q12.3-21.1 18q23 21q22.11-ter
N0619O15 N0564O04 N0381C13 N0001L24 M2032O09 N0531M16 N0004F08
N0463P22 N0083D03 M2013A02 N0226B11 N0002E13 N0703M10 N0457P07
15175256 147151220 122460473 64741928 41713136 71676600 32359411
16152619 158783389 134436514 114103214 46266399 72686216 46926492
977 11632 11976 49361 4553 1010 14567
Deletions 5p13.3-15.33 16q11.2-12.2
N0128A03 N0180E11
M2335O24 N0070N16
4054083 45081598
32497485 54541026
28443 9459
81
Sample Name
Locus Start clone End clone Base pair start Base pair end Size (kb)
IMR32 Gains
1p31.3-33 1q21.1-25.3 1q32.1-ter 2p24.3-24.3 2q14 6p21.1-21.22 15q 17q 20q-ter
N0682P18 N0510I18 N0133P06 N0571E19 N0678O18 N0065L23 N0137P24 N0606M07 N0004O09
N0185E18 N0522B03 N0332D17 N0427M01 N0812M06 N0061H05 N0584I15 N0196O11 N0476I15
51504350 142647117 198475140 14565626 66473520 30666043 59763464 37680398 29737772
62616624 182897083 245744383 17376543 67573409 44747012 91451479 78615238 62434320
11112 40250 47269 2811 1100 14081 31688 40935 32697
Deletions 1p33-ter 16q22.2-ter
N0379K15 N0113E03
N0670L22 N0655C18
95421 69787193
50599578 85754878
50504 15968
![Catalog No. XIM-MA001 Anti c-Myc [9E10]search.cosmobio.co.jp/cosmo_search_p/search_gate2/... · Anti c-Myc [9E10] Catalog No. XIM-MA001 Background: c-Myc is a very strong proto-oncogene](https://img.dokumen.tips/doc/110x75/5fe818d3580d03273c7554a0/catalog-no-xim-ma001-anti-c-myc-9e10-anti-c-myc-9e10-catalog-no-xim-ma001.jpg)
