Exploring the neuroblastoma epigenome:perspectives for the discovery of prognostic biomarkers             Cytometry 2011, ...
Overview Epigenetics - introduction     Introduction     DNA-methylation and histone modifications     The interplay b...
Epigenetics > Introduction          -genetics     Heritable changes to the DNA or histones without      affecting the DN...
Epigenetics > Introduction
Epigenetics > Introduction                             DNA-methylation                             Histone tail modificati...
Epigenetics > DNA-methylation Isolated CG dinucleotides are in most  cases methylated Some regions are CG-rich: “CpG isl...
Epigenetics > DNA-methylation Is a normal phenomenon    Development, differentiation      •   Genes, active only during ...
Epigenetics > DNA-methylation Is a normal phenomenon    Development, differentiation      •   Genes, active only during ...
Epigenetics > DNA-methylation DNA-methylation and cancer            Local                   Global       hypermethylation...
Epigenetics > DNA-methylation Dense methylation in promoter regions  causes transcriptional silencing    Blocked binding...
Epigenetics > Histone modifications Histone modifications    Acetylation       •   Activating (e.g. H3K9, H3K14, H3K18, ...
Epigenetics > Interplay Interplay between DNA-methylation and  histone modifications
Epigenetics > Detection Detection of DNA-methylation
Epigenetics > Detection Bisulfite conversion – MSP    Primerdesign in CG regions
Epigenetics > Detection Detection of histone modifications    Affinity-based       •   Antibodies against modifications ...
Epigenetics > Detection / Prognosis / Prediction Detection / Prognosis / Prediction    Require ‘biomarkers’       •   Ea...
Epigenetics > Detection / Prognosis / Prediction Detection / Prognosis / Prediction    DNA-methylation is not random   ...
Epigenetics > Detection / Prognosis / Prediction Detection / Prognosis / Prediction    Dependent on environmental factor...
Epigenetics > Detection / Prognosis / Prediction (Early) detection – diagnostic    Diagnostic: who    Screening programs
Epigenetics > Detection / Prognosis / Prediction Prognosis    Prognostic: who    Follow-up
Epigenetics > Detection / Prognosis / Prediction Prognosis                                        Biomarker – bad progn. ...
Epigenetics > Detection / Prognosis / Prediction Prognosis    Survival in colorectal cancer
Epigenetics > Detection / Prognosis / Prediction Prediction    Predictive: what    Treatment
Epigenetics > Detection / Prognosis / Prediction Prediction    Predictive: what    Treatment
Epigenetics > Detection / Prognosis / Prediction Prediction                                       Biomarker    Predictiv...
Epigenetics > Detection / Prognosis / Prediction Prediction    Predictive: what    Treatment
Epigenetics > Detection / Prognosis / Prediction Prediction    Chemotherapy respons (MGMT in brain cancer -     temozolo...
Overview Epigenetics - introduction     Introduction     DNA-methylation and histone modifications     The interplay b...
Mapping the neuroblastoma epigenome Neuroblastoma
Mapping the neuroblastoma epigenome Neuroblastoma
Mapping the neuroblastoma epigenome Neuroblastoma                                            Risk factors:               ...
Mapping the neuroblastoma epigenome Neuroblastoma   8 neuroblastoma cell lines      •   CHP902R, CLBGA, IMR32, LAN2, N20...
Mapping the neuroblastoma epigenome Sequencing               Control of fragment sizes with high sensitivity DNA chips Co...
Mapping the neuroblastoma epigenome Sequencing data analysis   Mapping on the human reference genome      •   Input: 45 ...
Mapping the neuroblastoma epigenome Sequencing data analysis
Mapping the neuroblastoma epigenome Sequencing data analysis
Mapping the neuroblastoma epigenome Sequencing data analysis
Mapping the neuroblastoma epigenome Neuroblastoma methylation   Combine several data sources to filter the most    relev...
Mapping the neuroblastoma epigenome Integrated data analysis   Expression results      •   Public expression data from a...
Mapping the neuroblastoma epigenome Integrated data analysis (PCDHB-cluster)
Mapping the neuroblastoma epigenome Integrated data analysis
Mapping the neuroblastoma epigenome
Mapping the neuroblastoma epigenome Samples   89 primary NB samples, 3 prognostic groups     Characteristic             ...
Mapping the neuroblastoma epigenome MSP detection      Roche LightCycler 480 Instrument:     Cq      LC480 melting curv...
Mapping the neuroblastoma epigenome MSP detection    48 assays    96 samples    Total: 4608 MSP reactions    12 384-w...
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
Exploring the neuroblastoma epigenome: perspectives for improved prognosis
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Exploring the neuroblastoma epigenome: perspectives for improved prognosis

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EXPLORING THE NEUROBLASTOMA EPIGENOME: PERSPECTIVES FOR THE DISCOVERY OF PROGNOSISTIC BIOMARKERS
M. Ongenaert, A. Decock, J. Vandesompele, F. Speleman

Center for Medical Genetics, Ghent University, Ghent, Belgium (mate.ongenaert@ugent.be)

Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system cells. Although recently new insights into genes involved in NB have emerged, the molecular basis of neuroblastoma development and progression still remains poorly understood. The best-characterized genetic alterations include amplification of the proto-oncogene MYCN, ALK activating mutations or amplification, gain of chromosome arm 17q and losses of 1p, 3p, and 11q. Epigenetic alterations have been described as well: caspase-8 (CASP8) and RAS-association domain family 1 isoform A (RASSF1A) DNA-methylation are important events for the development and progression of neuroblastoma. In total, there are about 75 genes described as epigenetically affected in NB cell lines and/or NB primary samples.
Most of these methylation markers are found using ‘candidate gene’ approaches and the methylation frequencies are usually very low. In order to find novel methylation markers that can be used for improved prognosis, we applied a whole-genome methylation screen. This technique relies on capturing with the MBD2 protein, containing a methyl-binding domain (MBD), with a very high affinity towards methylated genomic regions. In an initial phase, MBD2-seq was performed on 8 NB cell lines (where we also had micro-array data of, before and after treatment with DAC). As these results are promising, we will explore the complete methylomes of 45 primary NB tumors.
Based on an integrative analysis (re-expression results, expression micro-arrays, MBD2-sequencing on cell lines), 48 MSP (Methylation Specific PCR) assays were tested on 89 primary neuroblastoma patients of different risk categories. The results of this validation study demonstrate the power of epigenetic biomarkers as several assays are informative for prognosis and survival.

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Exploring the neuroblastoma epigenome: perspectives for improved prognosis

  1. 1. Exploring the neuroblastoma epigenome:perspectives for the discovery of prognostic biomarkers Cytometry 2011, Paris 26/10/2011 Maté Ongenaert Center for Medical Genetics Ghent University Hospital, Belgium
  2. 2. Overview Epigenetics - introduction  Introduction  DNA-methylation and histone modifications  The interplay between epigenetics  Applications of epigentics Mapping the neuroblastoma epigenome  Sequencing the neuroblastoma epigenome  Integrated data analysis  Real-time methylation-assays for improved prognosis
  3. 3. Epigenetics > Introduction -genetics  Heritable changes to the DNA or histones without affecting the DNA sequence  A whole range of changes are described • DNA-methylation • Histone tail modifications – Methylation – Acetylation – Phosphorylation – ….  Epigenetic changes are interconnected
  4. 4. Epigenetics > Introduction
  5. 5. Epigenetics > Introduction DNA-methylation Histone tail modifications
  6. 6. Epigenetics > DNA-methylation Isolated CG dinucleotides are in most cases methylated Some regions are CG-rich: “CpG islands”  More than half of the promoter regions have a CpG island  Are not methylated in most cases
  7. 7. Epigenetics > DNA-methylation Is a normal phenomenon  Development, differentiation • Genes, active only during specific stages of the embryonic development  Genomic imprinting • Only one of the parental copies is active  Silencing large chromosomal domains, e.g. X- chromosome • Mosaic X-chromosome in females  Protection against intra-genomic parasites: retrotransposons and other junk in the genome
  8. 8. Epigenetics > DNA-methylation Is a normal phenomenon  Development, differentiation • Genes, active only during specific stages of the embryonic development
  9. 9. Epigenetics > DNA-methylation DNA-methylation and cancer Local Global hypermethylation hypomethylation
  10. 10. Epigenetics > DNA-methylation Dense methylation in promoter regions causes transcriptional silencing  Blocked binding of transcription machinery (physical blockage)  In reality, shows to be more complex  Link between DNA methylation and histone modifications
  11. 11. Epigenetics > Histone modifications Histone modifications  Acetylation • Activating (e.g. H3K9, H3K14, H3K18, H3K56)  Methylation • Repressing (H3K9 – H3K27 – H4K20) • Activating (H3K4 – H3K36 – H3K79)  Phosphorylation • Activating (H3S10)  Ubiquitinilation • Repressing (H2A K119) • Activating (H2B K123)  Sumoylation
  12. 12. Epigenetics > Interplay Interplay between DNA-methylation and histone modifications
  13. 13. Epigenetics > Detection Detection of DNA-methylation
  14. 14. Epigenetics > Detection Bisulfite conversion – MSP  Primerdesign in CG regions
  15. 15. Epigenetics > Detection Detection of histone modifications  Affinity-based • Antibodies against modifications • Enrichment of fragments, bound by antibody  Platforms: • Chip (ChIP-chip) • Seq (ChIP-seq)
  16. 16. Epigenetics > Detection / Prognosis / Prediction Detection / Prognosis / Prediction  Require ‘biomarkers’ • Easy to detect using molecular techniques • Often an ‘early event’ • Suitable biomarker for detection / screening • Can be detected in blood, urine, sputum (non-invasive sampling) • Biomarkers in various cancer types  Beyond tumor detection • Stratification of patient groups • Stage/grading classification • Prognosis (survival, disease-free survival >) • Chemotherapy respons (MGMT in brain cancer – temozolomide >) • Personalized medicine
  17. 17. Epigenetics > Detection / Prognosis / Prediction Detection / Prognosis / Prediction  DNA-methylation is not random  Can be more frequent than mutations
  18. 18. Epigenetics > Detection / Prognosis / Prediction Detection / Prognosis / Prediction  Dependent on environmental factors  Methylation profile of twins (genetically identical)
  19. 19. Epigenetics > Detection / Prognosis / Prediction (Early) detection – diagnostic  Diagnostic: who  Screening programs
  20. 20. Epigenetics > Detection / Prognosis / Prediction Prognosis  Prognostic: who  Follow-up
  21. 21. Epigenetics > Detection / Prognosis / Prediction Prognosis Biomarker – bad progn.  Prognostic: who  Follow-up Biomarker – good progn.
  22. 22. Epigenetics > Detection / Prognosis / Prediction Prognosis  Survival in colorectal cancer
  23. 23. Epigenetics > Detection / Prognosis / Prediction Prediction  Predictive: what  Treatment
  24. 24. Epigenetics > Detection / Prognosis / Prediction Prediction  Predictive: what  Treatment
  25. 25. Epigenetics > Detection / Prognosis / Prediction Prediction Biomarker  Predictive: what  Treatment
  26. 26. Epigenetics > Detection / Prognosis / Prediction Prediction  Predictive: what  Treatment
  27. 27. Epigenetics > Detection / Prognosis / Prediction Prediction  Chemotherapy respons (MGMT in brain cancer - temozolomide)
  28. 28. Overview Epigenetics - introduction  Introduction  DNA-methylation and histone modifications  The interplay between epigenetics  Applications of epigentics Mapping the neuroblastoma epigenome  Sequencing the neuroblastoma epigenome  Integrated data analysis  Real-time methylation-assays for improved prognosis
  29. 29. Mapping the neuroblastoma epigenome Neuroblastoma
  30. 30. Mapping the neuroblastoma epigenome Neuroblastoma
  31. 31. Mapping the neuroblastoma epigenome Neuroblastoma Risk factors: - Age at diagnosis - MYCN amplification - INSS Stage Under- and overtreatment - Molecular biomarkers - 59 gene signature (qPCR) - Methylation biomarkers for improved prognosis
  32. 32. Mapping the neuroblastoma epigenome Neuroblastoma  8 neuroblastoma cell lines • CHP902R, CLBGA, IMR32, LAN2, N206, SHSY5Y, SJNB1, SKNAS  Re-activation after DAC-treatment • Expression: micro-array Affymetrix HGU-133plus2.0  Sequencing • Capture with MBD2 antibody – MBD2 has the highest affinity towards methylated DNA • Multiplex library preparation (MID tags to identify sample) and sequencing • Illumina GAIIx, paired-end sequencing (2x45bp)
  33. 33. Mapping the neuroblastoma epigenome Sequencing Control of fragment sizes with high sensitivity DNA chips Concentration determination of the fragmented DNA with Fluostar Optima plate reader MBD2 immunoprecipitation reaction (MethylCollector Kit)
  34. 34. Mapping the neuroblastoma epigenome Sequencing data analysis  Mapping on the human reference genome • Input: 45 bp ‘sequence tags’ • Output: mapped sequence reads: chromosome-location • Coverage: number of tags at a certain genomic location • In this case: coverage ~ captured DNA fragments ~ MBD2 binding ~ methylation  Peak detection • Compared to the ‘background’, how unusual is the signal I see in a specific region  Peak annotation • Genomic location > Genes / functions / …  Visualisation
  35. 35. Mapping the neuroblastoma epigenome Sequencing data analysis
  36. 36. Mapping the neuroblastoma epigenome Sequencing data analysis
  37. 37. Mapping the neuroblastoma epigenome Sequencing data analysis
  38. 38. Mapping the neuroblastoma epigenome Neuroblastoma methylation  Combine several data sources to filter the most relevant biomarkers out: integrated data analysis  Very specific for biological question  Purpose: prognostic DNA-methylation biomarker (risk groups)  Expression results • High stage vs. low stage • MYCN amplified vs. MYCN non-amplified • High risk vs. low risk  Re-expression results  Methylation capture results
  39. 39. Mapping the neuroblastoma epigenome Integrated data analysis  Expression results • Public expression data from a total of 380 primary NB samples • Three different arrays, two different platforms • Uniform scoring scheme (RankProd analysis - ranking statistics)  Re-expression results • Expression array before and after DAC treatment, 8 NB cell lines • Score assigned (RankProd)  Methylation capture results • MBD2 sequencing in 8 NB cell lines • Score assigned (TSS, p-value peaks, tags/length, FE)
  40. 40. Mapping the neuroblastoma epigenome Integrated data analysis (PCDHB-cluster)
  41. 41. Mapping the neuroblastoma epigenome Integrated data analysis
  42. 42. Mapping the neuroblastoma epigenome
  43. 43. Mapping the neuroblastoma epigenome Samples  89 primary NB samples, 3 prognostic groups Characteristic Classes Count (percentage) HR_DOD 28/89 (31%) Risk Classification HR_Surv 30/89 (34%) LR 31/89 (35%) 1 21/89 (24%) 2 12/89 (14%) INSS Stage 3 17/89 (19%) 4 39/89 (44%) Not amplified (0) 50/89 (56%) MYCN Amplified (1) 39/89 (44%) Age at diagnosis > 12 months 53/89 (60%) (0) Age Age at diagnosis < 12 months 36/89 (40%) (1)
  44. 44. Mapping the neuroblastoma epigenome MSP detection  Roche LightCycler 480 Instrument: Cq  LC480 melting curve analysis: Tm  Caliper LabChip GX: Size  Cq, Tm and size  methylation call (compared to HCT-116 – SssI– methylated control) Controls  HCT-116 / SssI: methylated control  HCT-116 / DKO: unmethylated control  Control primers: ACTB
  45. 45. Mapping the neuroblastoma epigenome MSP detection  48 assays  96 samples  Total: 4608 MSP reactions  12 384-well plates  Pipetting: Tecan robot

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