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ESO-ESSO Masterclass in colorectal cancer surgery Genetic and epigenetic analysis of sporadic colon cancer-Decision & trea...
plan <ul><li>Stop press  Aspirin treatment </li></ul><ul><li>CIN and MIN </li></ul><ul><li>Kras and BRAF </li></ul><ul><li...
December 11 th   2008;359:2567-2578 CAPP2: 1009 Lynch  syndrome recruits Gail Barker Anne-Marie Gerdes Julie Coaker Lynn R...
CAPP2: Per protocol analysis (primary endpoint CRC after 2 years treatment)  significantly fewer colorectal cancers [submi...
CAPP2 per protocol analysis All Lynch syndrome cancers HR  0.48 (0.28-0.83)p= 0.008 IRR 0.44 (0.26-0.75)p=0.002
Toronto outline 100 CRC InSiGHT meeting 2005 15 MSI-H 85 MSS 2 HNPCC 13 sporadic 1  MLH1 1  MSH2 1 FAP 1 MAP
Normal epithelium Adenoma Carcinoma Metastases APC, K-ras 12p, DCC 18q, p53 17p, …….. 85 % 15 % CIN MSI FAP HNPCC hMLH1, h...
Molecular Classification of Colorectal Cancer Jass, Histopathology 2007, 50, 113–130 FAP Less  responsive To 5FU? Gallinge...
Colorectal Cancer Genetics & 5-FU Ribic CM NEJM 2003 MSS MSI Hazard Ratio 0.69 (0.50-0.94)   p=0.02 2.17 (0.84-5.55)   p=0...
 
<ul><ul><li>BRAF is a kinase encoding gene from the RAS/RAF/MAPK pathway </li></ul></ul><ul><ul><li>The BRAF-V599E hotspot...
SEQUENOM TM  MALDI TOF MASS SPECTROMETER Matrix Assisted Laser Desorption/Ionisation Time of Flight mass spectrometry Lase...
KRAS in colorectal cancer <ul><li>23 KRAS activating mutations </li></ul><ul><li>Codons 12, 13, 61 and 146 </li></ul><ul><...
T G G A C C G T T C A A C - ve - ve - ve - ve - ve - ve - ve Polymerase - ve - ve - ve - ve - ve - ve - ve C
% resistance change A C T G A C T Time = ACTACT
QuantuMDx technology
Epigenetics <ul><li>“ The chemical and physical code superimposed on the genome that influences gene activity without alte...
Cytosine methylation at CpG islands CpG m CpG N N HO NH 2 1 2 3 4 5 6 CH 3 5-methylcytosine cytosine Normal biallelic expr...
A  C A T   C A C G T   G A TT A C A   Sodium bisulphite (NaHS0 3)  conversion of genomic DNA Genomic DNA Treatment with so...
Constitutional epimutation of  MLH1 <ul><li>Soma-wide mono-allelic promoter methylation </li></ul><ul><li>Detected by the ...
Classic example of epimutation carrier <ul><li>Sri Lankan male, Patient YT </li></ul><ul><li>CRC (ascending) 18y  </li></u...
Tissue-restricted  MSH2  epimutation caused by terminal deletion of  EPCAM <ul><li>Mosaic methylation of  MSH2  promoter -...
Dr Magnus von Knebel Doeberitz
TGT   .   AAA  . AAA . AAA . A CG   . TGC . TGG . CTA . GCT . GA......... C K K K T C W L A . . . TGT .   AAA . AAA . AAA ...
ELISA reactivity reveals immune response to Neopeptides in Lynch syndrome patients with  Previous cancer and in MMR mutati...
Distribution of FSP-specific responses in patients and controls Schwitalle et al., Gastroenterology 2008
T-cells infiltrating MSI-H CRC recognize MSI-induced FSP Exemplary results derived from a MSI-H CRC-patient  Schwitalle et...
Truncating mutations in  ß2m  are more common with tumour progression but absent from mets Kloor et al Int J Cancer 2007 U...
ANVIL <ul><li>A nti   N eopeptide  V accine  I n  L ynch syndrome </li></ul><ul><li>Stimulate cell based immunity to a pan...
Aspirin inhibits IL-4 making cancer stem cells more vulnerable to chemotherapy
summary <ul><li>CIN and MIN: a valuable basic division </li></ul><ul><li>Kras and BRAF: pivotal to targeting cetuximab and...
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MCC 2011 - Slide 6

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MCC 2011 - Slide 6

  1. 1. ESO-ESSO Masterclass in colorectal cancer surgery Genetic and epigenetic analysis of sporadic colon cancer-Decision & treatment Professor Sir John Burn MD FRCP FRCPCH FRCOG FMedSci Institute of Human Genetics, Newcastle University, UK Institute of Human Genetics Newcastle University Centre for Life, Newcastle UK
  2. 2. plan <ul><li>Stop press Aspirin treatment </li></ul><ul><li>CIN and MIN </li></ul><ul><li>Kras and BRAF </li></ul><ul><li>CIMP </li></ul><ul><li>Epimutation </li></ul><ul><li>“ trapped” Darwinian selection </li></ul><ul><li>immunoprevention </li></ul>
  3. 3. December 11 th 2008;359:2567-2578 CAPP2: 1009 Lynch syndrome recruits Gail Barker Anne-Marie Gerdes Julie Coaker Lynn Reed John Mathers Tim Bishop 600mg aspirin for up to 4 yrs had no effect
  4. 4. CAPP2: Per protocol analysis (primary endpoint CRC after 2 years treatment) significantly fewer colorectal cancers [submitted] HR 0.45 (0.22-0.93) p = 0.03 IRR 0.41 (0.20-0.84) p= 0.01
  5. 5. CAPP2 per protocol analysis All Lynch syndrome cancers HR 0.48 (0.28-0.83)p= 0.008 IRR 0.44 (0.26-0.75)p=0.002
  6. 6. Toronto outline 100 CRC InSiGHT meeting 2005 15 MSI-H 85 MSS 2 HNPCC 13 sporadic 1 MLH1 1 MSH2 1 FAP 1 MAP
  7. 7. Normal epithelium Adenoma Carcinoma Metastases APC, K-ras 12p, DCC 18q, p53 17p, …….. 85 % 15 % CIN MSI FAP HNPCC hMLH1, hMSH2, TGF-ß RII, Bax, TCF4, ACVRII, Caspase 5 ………. The Genomic Pathogenesis of Colorectal Cancer
  8. 8. Molecular Classification of Colorectal Cancer Jass, Histopathology 2007, 50, 113–130 FAP Less responsive To 5FU? Gallinger group Ribic CM et al NEJM 2003 ;349:247-57,
  9. 9. Colorectal Cancer Genetics & 5-FU Ribic CM NEJM 2003 MSS MSI Hazard Ratio 0.69 (0.50-0.94) p=0.02 2.17 (0.84-5.55) p=0.10 Courtesy Steve Gallinger
  10. 11. <ul><ul><li>BRAF is a kinase encoding gene from the RAS/RAF/MAPK pathway </li></ul></ul><ul><ul><li>The BRAF-V599E hotspot mutation is found in 40% of sporadic MSI-H tumours and 0% of HNPCC tumours </li></ul></ul><ul><ul><li>Presence of V599E mutations excludes diagnosis of HNPCC </li></ul></ul>MSI and BRAF
  11. 12. SEQUENOM TM MALDI TOF MASS SPECTROMETER Matrix Assisted Laser Desorption/Ionisation Time of Flight mass spectrometry Laser Flight path Detector Time of flight Sequenom chip
  12. 13. KRAS in colorectal cancer <ul><li>23 KRAS activating mutations </li></ul><ul><li>Codons 12, 13, 61 and 146 </li></ul><ul><li>BRAF V600E </li></ul>KRAS c.38G>A; p.13Gly>Asp KRAS c.35G>T; p.12Gly>Val Wild type KRAS c.436G>A; p.146Ala>Thr KRAS c.1799T>A; p.600Val>Glu
  13. 14. T G G A C C G T T C A A C - ve - ve - ve - ve - ve - ve - ve Polymerase - ve - ve - ve - ve - ve - ve - ve C
  14. 15. % resistance change A C T G A C T Time = ACTACT
  15. 16. QuantuMDx technology
  16. 17. Epigenetics <ul><li>“ The chemical and physical code superimposed on the genome that influences gene activity without altering the DNA sequence ” . </li></ul>Adapted from Jane Qiu, Nature (2006) 441:143-145 C G CH 3 CH 3 CH 3 C G Cytosine Methylated cytosine Megan Hitchins N N HO NH 2 1 2 3 4 5 6 N N HO NH 2 1 2 3 4 5 6 CH 3
  17. 18. Cytosine methylation at CpG islands CpG m CpG N N HO NH 2 1 2 3 4 5 6 CH 3 5-methylcytosine cytosine Normal biallelic expression Biallelic methylation – eg cancer cells N N HO NH 2 1 2 3 4 5 6 TF TF
  18. 19. A C A T C A C G T G A TT A C A Sodium bisulphite (NaHS0 3) conversion of genomic DNA Genomic DNA Treatment with sodium bisulphite Unmethylated sequence Methylated sequence A T A T T A C G T G A TT A T A A T A T T A T G T G A TT A T A
  19. 20. Constitutional epimutation of MLH1 <ul><li>Soma-wide mono-allelic promoter methylation </li></ul><ul><li>Detected by the presence of MLH1 methylation in peripheral blood DNA </li></ul><ul><li>Transcriptional silencing of the affected allele </li></ul><ul><li>Normal gene sequence </li></ul>CH 3 CH 3 CH 3 CH 3 MLH1 -93G>A 655A>G X TF G A G A G G G
  20. 21. Classic example of epimutation carrier <ul><li>Sri Lankan male, Patient YT </li></ul><ul><li>CRC (ascending) 18y </li></ul><ul><li>No family history </li></ul><ul><li>No germline mutation </li></ul><ul><li>MSI-H (all 5 markers) </li></ul><ul><li>IHC loss of MLH1 & PMS2 </li></ul>Soma-wide hemiallelic methylation of MLH1 by clonal bisulphite sequencing Goel et al. Int J Cancer (2010) in press PBL Hair follicles Saliva Buccal Methylated CpG Unmethylated CpG
  21. 22. Tissue-restricted MSH2 epimutation caused by terminal deletion of EPCAM <ul><li>Mosaic methylation of MSH2 promoter - predominant in epithelial tissues (eg colonic mucosa) </li></ul><ul><li>MSH2 is mutation-negative </li></ul><ul><li>Linked deletion of the final exon(s) of EPCAM, which abolish the transcription termination (polyadenylation) signal </li></ul><ul><li>Dominant inheritance: large kindreds with CRC showing MSH2-loss </li></ul>EPCAM MSH2 1 5 3 4 2 6 8 7 9 1 5 3 4 2 6 “ Fusion transcript” Normal allele Deletion of EPCAM 3’ 17kb
  22. 23. Dr Magnus von Knebel Doeberitz
  23. 24. TGT . AAA . AAA . AAA . A CG . TGC . TGG . CTA . GCT . GA......... C K K K T C W L A . . . TGT . AAA . AAA . AAA . CGT . GCT . GGC . TAG . CTG . A..... STOP C K K K R A G TGT . AAA . AAA . AAC . GTG . CTG . GCT . AGC . TGA.... frameshift mutations cause (A) 9 (A) 8 repeat length (A) 10 C K K K V L A S STOP <ul><li>loss of function </li></ul><ul><li>generation of cancer specific peptides </li></ul>Slippage in coding microsatellites generates predictable novel peptides
  24. 25. ELISA reactivity reveals immune response to Neopeptides in Lynch syndrome patients with Previous cancer and in MMR mutation carriers patients healthy controls mutation carriers
  25. 26. Distribution of FSP-specific responses in patients and controls Schwitalle et al., Gastroenterology 2008
  26. 27. T-cells infiltrating MSI-H CRC recognize MSI-induced FSP Exemplary results derived from a MSI-H CRC-patient Schwitalle et al. Gastroenterology 2008
  27. 28. Truncating mutations in ß2m are more common with tumour progression but absent from mets Kloor et al Int J Cancer 2007 UICC IV, M1: No truncating mutation in CRC with metastasis ß2m mutation frequency is signif. lower in M1 vs M0 MSI CRC ?Intact ß2m needed for metastasis formation in MSI-H CRC UICC III 9/22 (40.9%) UICC IV 2/26 (7.7%) p=0.01 Only silent mutations or single AA exchanges, w/o functional loss of ß2m
  28. 29. ANVIL <ul><li>A nti N eopeptide V accine I n L ynch syndrome </li></ul><ul><li>Stimulate cell based immunity to a panel of neopeptides predicted in important “tumour” genes using replication deficient chimp adenovirus & MVA as vectors </li></ul><ul><li>Framework 7 proposal with Riccardo Cortese of Okairos ltd </li></ul>
  29. 30. Aspirin inhibits IL-4 making cancer stem cells more vulnerable to chemotherapy
  30. 31. summary <ul><li>CIN and MIN: a valuable basic division </li></ul><ul><li>Kras and BRAF: pivotal to targeting cetuximab and differentiating Lynch syndrome from sporadic MSI high tumours </li></ul><ul><li>CIMP: CpG island methylation phenotype is a marker of gene silencing </li></ul><ul><li>Epimutation: a rare cause of multiple cancers </li></ul><ul><li>“ trapped” Darwinian selection: B2M mutations allow local growth but suppress mets </li></ul><ul><li>Immunoprevention: a new approach to avoidance of MIN cancers </li></ul>

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