Supplementary webappendix This webappendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors. Not Knudson’s Retinoblastoma: One Hit Cancer Initiated by the MYCN Oncogene? Rushlow, D. E., Kennett, J. Y., Mol, B. M. et al. Lancet, submitted. Table of Contents Supplementary Methods........................................2 Tissue Samples....................................................2 Mutation Analyses.................................................2 Quantitative Multiplex PCR (QM-PCR)...............................2 Table S1........................................................2 Multiplex Ligation-Dependent Probe Amplification (MLPA)...........3 Single Nucleotide Polymorphism Studies (SNP)......................3 aCGH Genome Copy Number Analyses..................................3 Immunohistochemistry..............................................3 Western blot......................................................3 Cell Culture......................................................3 Reverse Transcriptase (End-Point) PCR (RT-PCR)....................4 Table S2........................................................4 Quantitative Real-Time PCR........................................4 Table S3. TaqMan® gene assay locations..........................4 Statistical Analyses..............................................4 Age of Diagnosis Distribution Analyses............................4 Table S4.............................Error! Bookmark not defined. Supplementary Results Tables.................................6 Table S5A. Genomic Gain and Loss. Unilateral RB1 +/+ tumours.........6 Table S5B. Genomic Gain and Loss. Unilateral RB1 +/- tumours........7 Table S5C. Genomic Gain and Loss. Unilateral RB1 -/- tumours.........8 Table S5D. Copy number data for the heat map in figure 1B. RB1 +/+ and RB1 -/- tumours.....................................................10 Table S5E. Copy number data for figure 1B. RB1 -/- tumours..........11 Table S6. Comparisons of frequencies of genomic copy-number changes .................................................................13 Table S7A. aCGH alterations by sample............................14 Table S7B. aCGH alterations by sample............................21 Table S7C. Copy number alterations detected by SNP array analyses. .................................................................28 Table S8. RB1 +/+ MYCN A tumours express RB1, high MYCN and low KIF14.. .29 Supplementary Figures.......................................30
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Supplementary webappendix
This webappendix formed part of the original submission and has been peer reviewed.We post it as supplied by the authors.
Not Knudson’s Retinoblastoma: One Hit Cancer Initiated by the MYCN Oncogene?Rushlow, D. E., Kennett, J. Y., Mol, B. M. et al. Lancet, submitted.
Table S3. TaqMan® gene assay locations........................................................................................................4Statistical Analyses.................................................................................................................................... 4Age of Diagnosis Distribution Analyses..............................................................................................4
Table S4...........................................................................................................Error! Bookmark not defined.
Supplementary Results Tables................................................................................................... 6Table S5A. Genomic Gain and Loss. Unilateral RB1+/+ tumours...................................................6Table S5B. Genomic Gain and Loss. Unilateral RB1+/- tumours...................................................7Table S5C. Genomic Gain and Loss. Unilateral RB1-/- tumours....................................................8Table S5D. Copy number data for the heat map in figure 1B. RB1+/+ and RB1-/- tumours.10Table S5E. Copy number data for figure 1B. RB1-/- tumours......................................................11Table S6. Comparisons of frequencies of genomic copy-number changes...........................13Table S7A. aCGH alterations by sample............................................................................................14Table S7B. aCGH alterations by sample............................................................................................21Table S7C. Copy number alterations detected by SNP array analyses...................................28Table S8. RB1+/+MYCNA tumours express RB1, high MYCN and low KIF14.............................29
Supplementary Figures.............................................................................................................. 30Figure S2: RB1+/+MYCNA tumours have narrow amplicons.........................................................30Figure S3: SNP copy number in RB1+/+MYCNA retinoblastoma..................................................31Figure S4: Large prominent nucleoli in RB1+/+MYCNA tumours................................................32
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Supplementary Methods
Tissue SamplesTumours, blood and clinical data were provided for genetic diagnosis of RB1 mutations for clinical care. Research Ethics Boards (REBs) approvals for the use of de-identified data and tissues, after clinical analysis was completed, are on file at each participating site. Dr. R. Bremner (Toronto Western Hospital Research Institute, University Health Network) provided fetal retina mRNA samples with approval of the UHN REB.
Mutation AnalysesMutation analysis for RB1 (Gen bank accession # L11910) included DNA sequencing, copy number detection by QM-PCR or MLPA (MRC-Holland), and RB1 promoter methylation analysis.1
Quantitative Multiplex PCR (QM-PCR)Copy numbers for KIF14, DEK, E2F3, CDH11, and MYCN were determined with gene-specific primers2, and additional gene-specific confirmatory primer sets (table S1), using the Qiagen Multiplex PCR kit and 19 amplification cycles. Each assay included internal controls from 15q and 12q. We established cut-off values for loss and gain, as illustrated in figure S1, using boxplots.
Gene/Exon Primers Primer Sequence Expected Size (bp)
MYCN2-exon2MYC 5x2q cga cgt ggt cac tgt gga g
316MYC 3x2q acg ctc act gtc ctc cga g
KIF KIF 5x24-Cy5 gac aaa tca agc act att tac tc 224KIF 3x24 gct tac agt tat ctt gac ttt c
DEK DEK 5x7-Cy5 tta tgc agc cat tgc cga aat c 278DEK 3x7 ttc tgg ctg tac ctt ata gca g
E2F3 E2F3 5x7-Cy5 cag act tgg ctt caa cca ac 253E2F3 3x7 aag tct tcc acc agt ggg ag
ACVRL1 exon 8 Ex8seq3 cct gtg tag ggt ggg ccc t 303ALK5-E8 tgt ttc tct cag tcc cca cc
Table S1. QM-PCR control primers & additional QM-PCR primers for MYCN, KIF14, DEK and E2F3. The initial primers in use were those cited in Bowles et al. 20072. This table shows additional primer sets used for confirmations.
Figure S1. Separation between normal and abnormal QM-PCR control results determined gain and loss cut-off values. On each boxplot, the vertical line marks maximum and minimum copy-numbers. The box bounds second and third quartiles (50% of the data points); horizontal line within the box represents the median. Gain defined as ≥2·50 copies for KIF14, ≥ 2·60 for DEK, E2F3 and MYCN, and CDH11 loss ≤1·60. A second MYCN primer set (MYCN2) was used to confirm borderline and increased MYCN copy number values.
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Multiplex Ligation-Dependent Probe Amplification (MLPA)MLPA for the Amsterdam samples was performed with the SALSA MLPA kit P252-B1 Neuroblastoma (MRC-Holland, The Netherlands) according to the manufacturer’s instructions. Fragment analysis was performed with GeneMarker software (Softgenetics, USA), DNA copy number ratios of tumour samples were computed using the matched blood sample or a pool of reference samples. Threshold for gains was a ratio of ≥ 1·3, < 5 and for high-level amplification, ≥ 5. Averaging the ratios from different probes for the same gene and multiplying the ratio by 2 subsequently calculated absolute copy number.
Single Nucleotide Polymorphism Studies (SNP) SNP array experiments for the Amsterdam samples were performed at ServiceXS (ServiceXS B.V., Leiden, The Netherlands) using the HumanOmni1-Quad BeadChip (Illumina, Inc., San Diego, CA, U.S.A), according to the manufacturer's instructions. The BeadChip images were scanned on the iScan system and the data were extracted into Illumina’s GenomeStudio software v2010·1. The software’s default settings were used with the cluster file as developed by Illumina for genotype calling. Results were subsequently exported and copy number analysis was performed with Genomic Segmentation algorithm of Partek Genomic Suite (Partek Inc., St. Louis, MO, U.S.A.). Paired analysis was performed with DNA from blood from the corresponding patients as reference.
aCGH Genome Copy Number AnalysesSub-megabase resolution tiling path BAC array comparative genomic hybridization (CGH) platform (BC Cancer Research Centre) comprised 26,363 overlapping elements, with an effective resolution of 79 kbp3,4. Test and reference (male) genomic DNA was competitively co-hybridized to the array5. aCGH images (Array-WoRx charge-coupled device (CCD)-based scanner, Applied Precision, Issaquah, WA) were analysed with Softworx software (Applied Precision), normalized with CGH Normalize suite6. aCGH confirmed 94% of the QM-PCR-detected gene changes indicating excellent technical reproducibility.
ImmunohistochemistryParaffin-embedded sections were stained for pRB-N-terminus, 1:200 (554136, BD Pharmingen); pRB-C-terminus, 1:200 (sc-50), and MYCN, 1:100 (sc-56729, both Santa Cruz)7,8. Human pRB-N-terminus immunoreactivity was detected using Immunopure DAB substrate kit (Pierce); human pRB-C-terminus and N-Myc immunoreactivity using Alexa™ 488 Streptavidin conjugate from Molecular Probes. DAPI was used to visualize nuclei. Slides were mounted using the DAKO-Cytomation Fluorescent Mounting Medium and visualized using a Leica DMLB microscope.
Western blotCell lines were harvested and washed twice with ice-cold PBS. Cells were lysed with Giordano buffer (50mM Tris pH 7.5, 250mM NaCl, 0,1% Triton X-100, 5 mM EDTA) supplemented with phosphatase- and protease inhibitors (Roche). Protein was quantified with Bio-Rad protein assay (Bio-Rad). Lysates were denatured at 70°C and loaded on a 3%-8% NuPAGE Tris-Acetate gradient gel (Invitrogen). After electrophoresis, proteins were transferred to an Immobilon-P membrane (Millipore). The membranes were blocked with 5% fat-free milk in TBS-T (10 mM Tris-HCL pH 7.5, 150 mM NaCl, 0.1% Tween 20) and incubated with primary antibodies against pRb clone 4H1, 1:2000 (Cell Signaling), MYCN clone NMYC-1, 1:1000 (Novus Biologicals), α-Tubulin clone B-5-1-2, 1:10.000 (Santa Cruz) and Vinculin clone H-10, 1:1000 (Santa Cruz). After incubation with horseradish peroxidise-labelled secondary antibodies (Dako Diagnostics), proteins were visualised with a ECL detection kit (Amersham Pharmacia Biotech).
Cell CultureAll cell lines were grown in a humidified 37°C incubator with 5% CO2 in their respective media with 100 U/ml penicillin and 0·1 mg/ml streptomycin (Invitrogen). Retinoblastoma cell lines were cultured in Iscove’s MDM (Invitrogen) with 10% FBS Gold (PAA Laboratories), 10 mg/L insulin (Sigma-Aldrich) and 0·0004% (v/v) β-mercaptoethanol; neuroblastoma cell lines (BE(2)-M17 and SH-SY5Y) in Dulbecco’s modified Eagle medium: Nutrient Mixture F-12 (DMEM/F-12) (1:1) GlutaMAXTM with 15 % FBS and 0.1 mM MEM Non-Essential Amino Acids (al Gibco), HSC-93 in RPMI 1640 (Gibco) with 10 % FBS (Gibco); and glioblastoma T98G in DMEM containing 1 g/L D-Glucose (Gibco) with 10% FBS (Gibco).
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Reverse Transcriptase (End-Point) PCR (RT-PCR)TRIzol (Invitrogen) extracted total RNA was reverse transcribed using random primers and SuperScript II RT (Invitrogen), then amplified (annealing temperature 60˚C) using KOD DNA polymerase (Novagen) following manufacturer’s instructions. Table S2.
Table S2. Primer sequences and expected product sizes used in end-point reverse transcriptase-PCR analysis.
Quantitative Real-Time PCRcDNA was used in a 7900HT Fast Real-Time PCR system with Universal PCR Master Mix. TaqMan® Gene Expression Assays (Applied Biosystems) (table S3). The ΔΔCt method built into the SDS 2·2 software (Applied Biosystems) determined mean relative gene expression in triplicate, normalized to GAPDH.
Statistical AnalysesA p value of 6×10-45 was obtained using a “Chi-squared goodness-of-fit test”, using default probabilities of p1=0·0025 for RB1+/+ and p2=0·9975 for RB1-/- samples, with 28/1054 (0·026) samples identified as RB1+/+, indicating a negligible chance that all RB1+/+ tumours observed were due to undetected RB1 mutations in two independent RB1 tumour alleles (Results, table 1). Frequencies of specific genomic copy number changes between the groups were evaluated using Fisher’s exact test (table S6). Total bp altered per region was summed for each tumour showing that RB1+/+MYCNA tumours have fewer genome alterations than RB1-/- tumours (p=0·033; t-test with Welch’s adjustment). Normalized intensity values were processed using the Circular Binary Segmentation algorithm, with data filtered to identify only high-quality (>3 confirmatory probes), and suitably sized (>25 kbp) regions of clear differential signal relative to normal (|mean-signal| > 0·1) (figure 2C). Separate terms were fitted to patient groups using a general linear model. A contrast matrix was used to identify the number of differentially abundant probes relative to normal samples, with an empirical Bayes moderation of standard error and a false-discovery rate adjustment for multiple testing (figure 2D).
Age of Diagnosis AnalysesWe compared age of diagnosis between groups (table S4A). There is strong evidence to suggest that age of diagnosis is related to the patient group (p< 0.0001). Pair-wise comparisons using the Wilcoxon rank-sum test indicated that patients in the RB1-/- group tend to be older at diagnosis than those with RB1+/+MYCNA tumours (respective median age 24.0 months (range 1.9 – 109.0) versus 4.5 months (range 1.0 – 16.0) (p<0.0001). There is also strong evidence that patients in the RB1+/+ group tend to be older at diagnosis than those with RB1+/+MYCNA tumours (respective median ages 21.5 months (range 8.0 – 83.0) versus 4.5 months (range 1.0 – 16.0) (p<0.0001). In contrast, there is no significant difference in the age at diagnosis of the patients with RB1-/- and RB1+/+ tumours (p = 0.94).We also calculated the risk that a child would have an RB1+/+MYCNA tumor depending on age of diagnosis (table S4B). The proportion of the 79 children with RB1-/- tumors in this study diagnosed less than or equal to 12, 8, 6 and 4 months of age was determined. We extrapolated to estimate the number children diagnosed in each age group with RB1-/- tumours, out of the total 1024 patients in the overall study. For each age group, we then
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calculated the % of children with RB1+/+MYCNA tumours. For children with unilateral non-familial retinoblastoma less than or equal to 6 months of age, 18.6% are predicted to have RB1+/+MYCNA tumours.
Age at Diagnosis
group N MedianRange IQR*
p-value**Minimum Maximum 25th Pctl 75th Pctl
RB1+/+MYCNA 17 4.5 1 16 3.5 10
<0.0001RB1+/+ 14 21.5 8 83 15 47
RB1-/- 79 24 1.9 109 14.5 38*IQR = interquartile range (the middle 50% of the values fall in this range).**P-value from the Kruskal Wallis test.
Table S4A. Comparison of groups by age at diagnosis.
Unilateral RB1-/- Present study (n=1054**)
% study patients MYCNANo MYCNA MYCNA***
n 79 1038 100% 16 100%
mean AGE Dx 30.1 6.3
median AGE Dx 24.0 4.5
# ≤ 12 mo 15 19.0% 197* 19% 14 88% 6.6%
# ≤ 8 mo 5 6.3% 66 6% 10 63% 13.2%
# ≤ 6 mo 3 3.8% 39 4% 9 56% 18.6%
# ≤ 4 mo 2 2.5% 26 3% 6 38% 18.6%
*extrapolated from % date of diagnosis measured for 79 patients.**number of unilateral non-familial patients studied in five centers (Table 1).***RB1+/+(-)MYCNA including T33.
Table S4B. Calculation of % of children with unilateral non-familial retinoblastoma predicted to have RB1+/+MYCNA tumors, by age of diagnosis.
To provide evidence supporting the idea that ages at diagnosis for bilateral cases were from a one-hit distribution, Knudson estimated the parameter k in the equation ln S = -kT (where S is the fraction of cases not yet diagnosed and T is age at diagnosis) through a simple linear no-intercept regression analysis in which the empirical values of S were regressed on age at diagnosis9. This analysis assumes an exponential distribution for age at diagnosis. He then plotted the empirical values versus age at diagnosis along with the best fitting line from the regression analysis, showing that the fitted line provided a good fit to the empirical values. For unilateral cases, he estimated the parameter k in the equation ln S = -kT2 (assuming a Weibull distribution with shape parameter of 2, the distribution derived by Burch10 for describing ages at diagnosis that result from a ‘two-mutation’ mechanism) and plotted the data points along with the best fitting curve. Therefore, for both the RB1-/ and RB1+/+MYCNA cases, we estimated k under both the one-hit and two-hit assumptions and plotted the data points along with both of the best fitting curves on the same plot, in order to compare the relative fit of the data to the two assumptions (figure 4B).
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Supplementary Results TablesTable S5A. Genomic Gain and Loss. Unilateral RB1+/+ tumours.
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Table S5B. Genomic Gain and Loss. Unilateral RB1+/- tumours.
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Table S5C. Genomic Gain and Loss. Unilateral RB1-/- tumours
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Table S5D. Copy number data for the heat map in figure 1B. RB1+/+ and RB1-/- tumours.
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Table S5E. Copy number data for figure 1B. RB1-/- tumours.
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Table S6. Comparisons of frequencies of genomic copy-number changes
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Table S7A. aCGH alterations by sample.
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Table S7B. aCGH alterations by sample.
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Table S7C. Copy number alterations detected by SNP array analyses.
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Table S8. RB1+/+MYCNA tumours express RB1, high MYCN and low KIF14.
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Supplementary FiguresFigure S2: RB1+/+MYCNA tumours have narrow amplicons.
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Figure S3: SNP copy number in RB1+/+MYCNA retinoblastoma.
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Figure S4: Large prominent nucleoli in RB1+/+MYCNA tumours.
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Supplementary References1. Zeschnigk M, Lohmann D, Horsthemke B. A PCR test for the detection of hypermethylated alleles at the retinoblastoma locus [letter]. Journal of medical genetics 1999;36:793-4.2. Bowles E, Corson TW, Bayani J, et al. Profiling genomic copy number changes in retinoblastoma beyond loss of RB1. Genes Chromosomes Cancer 2007;46:118-29.3. Ishkanian AS, Malloff CA, Watson SK, et al. A tiling resolution DNA microarray with complete coverage of the human genome. Nat Genet 2004;36:299-303.4. Watson SK, deLeeuw RJ, Horsman DE, Squire JA, Lam WL. Cytogenetically balanced translocations are associated with focal copy number alterations. Human genetics 2007;120:795-805.5. Kennett JY, Watson SK, Saprunoff H, Heryet C, Lam WL. Technical demonstration of whole genome array comparative genomic hybridization. J Vis Exp 2008.6. Khojasteh M, Lam WL, Ward RK, MacAulay C. A stepwise framework for the normalization of array CGH data. BMC Bioinformatics 2005;6:274.7. Marchong MN, Chen D, Corson TW, et al. Minimal 16q genomic loss implicates cadherin-11 in retinoblastoma. Mol Cancer Res 2004;2:495-503.8. Dimaras H, Khetan V, Halliday W, et al. Loss of RB1 induces non-proliferative retinoma: increasing genomic instability correlates with progression to retinoblastoma. Hum Mol Genet 2008;17:1363-72.9. Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Science, USA 1971;68:820-3.10. Burch PR. Natural and Radiation Carcinogenesis in Man. I. Theory of Initiation Phase. Proc R Soc Lond B Biol Sci 1965;162:223-39.
