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Mismatch Repair Deficiency Testing

Mismatch Repair Deficiency Testing. Kenneth J. Bloom, MD, FCAP Chief Medical Officer, Clarient, a GE Healthcare Company. Colorectal Cancer. 3 rd leading cancer and 2 nd leading cause of cancer death in males and females 150,000 new cases and 55,000 deaths in USA in 2006

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Mismatch Repair Deficiency Testing

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  1. Mismatch Repair Deficiency Testing Kenneth J. Bloom, MD, FCAP Chief Medical Officer, Clarient, a GE Healthcare Company

  2. Colorectal Cancer • 3rd leading cancer and 2nd leading cause of cancer death in males and females • 150,000 new cases and 55,000 deaths in USA in 2006 • Most CRCs are preceded by adenomas • Screening • (Currently - FOBT, Endoscopy) • (Future - Virtual Colonoscopy, Stool DNA) • Overall 60% 5 yr. survival • 93% if detected early (Stage I) • 8% after distant metastasis (Stage IV)

  3. Adenoma HGD Metastasis Normal Adenoma Carcinoma Micro-adenoma Kinzler-Vogelstein-Fearon • Sequential steps in APC pathway • At least 7 mutations • After APC the order is variable APC 5q21 KRAS 12p DCC/DPC4 18q21 p53 17p13 Other

  4. Pathways to CRC 3 Possible Pathways AND not linear Chromosomal Instability 50-70% MSS sporadic CRC FAP Microsatellite Instability 15% MSI sporadic CRC HNPCC CpG Island Methylator Phenotype (CIMP) 20-35% APC p53 MMR MLH1 MSH2 TGFβRII, IGF2R, BAX Tend to have KRAS mutation Tend to have BRAF mutation EXCEPT in HNPCC

  5. APC Chromosomal Instability Pathway APC, 5q21, tumor suppressor gene and a “gatekeeper” Signal transduction Cell proliferation Mediation of intercellular adhesion Chromosomal stability Regulation of cell cycle and apoptosis Cell migration Stabilization of cytoskeleton

  6. B-Catenin complex p p B-Catenin B-Catenin APC APC - B-Catenin Interaction Nucleus Binds to TCF (T-cell factor) which triggers transcription of genes that promote cell division Serine/Threonine kinase E-Cadherin Degradation

  7. Sporadic MSS CRC • The majority of CRCs (50-70% of CRC) • MSS, often aneuploid (chromosomal instability) • Left>right sided, M>F • Common phenotype (dirty necrosis, tumor budding, no TILs) • Initial APC somatic mutation • Often KRAS mutation (unresponsive to Anti-EGFR therapy) • Often p53 mutation (usual stage dependant prognosis)

  8. HNPCC • 2-4% of CRCs • Phenotype mimics sporadic CRC, precursor adenoma • MSI-H, often diploid • Germline mutation in MMR (MSH2, MLH1) • No BRAF mutation • Rare or no p53 mutation • Better prognosis, but poor response to 5-FU • R>L • Microscopic features include • Tumor infiltrating lymphocytes (TIL) • Crohn’s-like lymphoid reaction • Poor differentiation • Mucinous change

  9. MSI-H Sporadic CRC • 12-15% of CRCs • Precursor is sessile serrated adenoma • MSI-H, often diploid • Methylation of MLH-1 promoter gene • Often BRAF mutation • Rare or no p53 mutation • R>L, F>M • Better prognosis, but poor response to 5-FU • Microscopic phenotype is the same as HNPCC TIL, mucinous, poor differentiation • No need for prophylactic colectomy or hysterectomy • No response to Anti-EGFR therapy

  10. CpG Island Methylator Pathway (CIMP) Pathway • 20-35% cases of CRC • MSS (or MSI-L) • Methylation of CpG islands (includes some MHL-1, TP16 and others) • Generalized increase in de novo methylation • Often diploid • Rare or no p53 mutation • Precursor serrated adenoma • Often BRAF mutation, some also have KRAS mutation • Frequent family history (but no germline mutation) • R>L, F>M • Generally no TILs (unlike MSI-H) tumors • Poor prognosis compared to MSI CRCs • But responsive to 5-FU

  11. Microsatellite Instability Pathway • Errors in DNA occur constantly during replication • Mis-Match-Repair system identifies, excises and corrects the errors “Caretaker” genes that do not initiate tumor but cause rapid progression • MMR defect is identified by studying DNA microsatellites (tri and tetranucleotide repeat sequences of unknown function distributed throughout the human genome) • Similar short sequences also occur within some tumor suppressor genes (TGFβRII, IGF2R, BAX), which then may become mutated

  12. 1 base pair mismatch 3 base pairs mismatch • MSH2 protein is the “scout” identifying an error has occurred. • Heterodimeric complex is formed with • MSH6 if a single base pair mismatch • MSH3 if there is a 2-8 nucleotide insertion or deletion.

  13. 1 base pair mismatch 3 base pairs mismatch MSH2/MSH6 complex repairs 1 base pair mismatch MSH2/MSH3 complex repairs 3 base pair mismatch

  14. 1 base pair mismatch 3 base pairs mismatch MLH1/PMS1 complex is recruited to excise the mismatched nucleotides

  15. The MMR System Vilar E et al. Nat Rev Clin Onc (2010) 7: 153

  16. Genes Affected in dMMR Hewish M et al. Nat Rev Clin Onc (2010) 7: 197-208

  17. Microsatellite Instability (MSI) BAT25 Mononucleotide BAT26 Mononucleotide D5S346 Dinucleotide D2S123 Dinucleotide D17S250 Dinucleotide MSS (stable) = 0/5 MSI-L (low grade) = 1/5 MSI-H (high grade) = 2 or >/5

  18. Microsatellite Instability (MSI)

  19. Microsatellite Instability (MSI) Unstable Stable Unstable Unstable

  20. Microsatellite Instability by IHC MSH-2 Wild-type and Mutated (likely HNPCC) MLH-1 Wild-type and Mutated (can be sporadic or HNPCC)

  21. IHC Findings in dMMR Vilar E et al. Nat Rev Clin Onc (2010) 7: 153

  22. Problems with IHC as a Surrogate • Mutation involves other than MLH-1, MSH-2 • Mutations which encode a nonfunctional protein • Missense • Frameshift • Splice

  23. Rarely Nonsense Mutations Can Occur Splice mutation Frameshift mutation Known MLH1 germline mutation Wahlberg et al. Cancer Research (2002)

  24. Testing for MSI Gatalica Z and Torlakovic E. Familial Cancer 2008;7:15-26

  25. Genes Affected in dMMR Hewish M et al. Nat Rev Clin Onc (2010) 7: 197-208

  26. Reasons for assessing dMMR • Better classification of colon tumors • Prognostic implications • Predictive implications • Rule out germline mutation • Select patients for further testing

  27. Survival Differences dMMR Ward et al. Gut (2001)

  28. Impact of dMMR on CRC Management

  29. Novel Strategy for dMMR Hewish M et al. Nat Rev Clin Onc (2010) 7: 197-208

  30. Effect of 5-FU in dMMR Vilar E et al. Nat Rev Clin Onc (2010) 7: 153

  31. Effect of 5FU in dMMR Hewish M et al. Nat Rev Clin Onc (2010) 7: 197-208

  32. 5-FU induced death in intact MMR

  33. Evidence of 5-FU Resistance Cell line with methylation of MLH1 - MSI Resistant to 5FU Demethylated cell line- MSS Sensitive to 5FU MSI closely related to CIMP group, ?MGMT methylation

  34. Effect of Irinotecan in dMMR Hewish M et al. Nat Rev Clin Onc (2010) 7: 197-208

  35. “Oh, if only it were so simple.”

  36. Thank You!

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