HNPCC & FAP – SIMILARITIES & DIFFERENCES

1. HNPCC & FAP – SIMILARITIES & DIFFERENCES Elaine Whitfield

2. Colon Cancer • Colon cancer is the third most common form of cancer and the second leading cause of cancer-related death in the Western world & causes 655,000 deaths worldwide per year. • Lifetime risk in UK is 1 in 30 • Hereditary in 5-10% of cases • HNPCC causes 5% of all colorectal cancer • FAP causes 1% of all colorectal cancer

3. HNPCC Germline mutation in mismatch repair genes Assoc with MSI AD with reduced penetrance ~80% lifetime risk of colon cancer ↑risk of endometrial, ovary, stomach cancer Ave age of diagnosis = 50s Polyps rarely seen Slow tumour initiation & rapid progression FAP Germline mutation in APC gene – Tumour suppressor Assoc with LOH AD fully penetrant ~100% lifetime risk of colon cancer if untreated CHRPE & soft tissue & desmoid tumours Ave age of diagnosis = 39 100s – 1000s polyps Rapid tumour initiation and slow progression Disease Characteristics

4. Inheritance & Genes Responsible • HNPCC • Autosomal dominant inheritance with reduced penetrance (80% colon, 60% endometrial lifetime risk) • Genes mutated – MSH2 (60%), MLH1 (30%), MSH6 (5%) & PMS1 & PMS2 (<2%) – all genes involved in DNA mis-match repair, found on various chromosomes • 100s mutations identified (233 in MLH1 – majority point mutations esp. missense, nonsense & splicing) • Cause cancer by allowing more mutations to accumulate

5. Inheritance & Genes Responsible • FAP • Autosomal dominant inheritance with ~100% penetrance of colon cancer without surgery • Gene mutated- APC (Adenomatous Polyposis Coli) gene – tumour suppressor gene 5q22 • Many different mutation identified – most truncating (80%) • LOH for APC  adenoma (cells partially escape cell cycle control & divide adenoma), require further mutations in oncogene such as RAS  late adenoma, then DCC mutation  late stage adenoma, then p53 mutation  cancer • APC mutations also recessive at the cellular level

6. Normal Protein Function • HNPCC • Mismatch repair pathway proteins • Identify and remove single nucleotide mismatches or insertion and deletion loops • At least 5 proteins involved four of which can cause HNPCC (MSH3 not involved in HNPCC) • Mlh1 dimerises with Pms2 to co-ordinate binding of other proteins • Msh2 forms a heterodimer with either Msh6 or Msh3 which identifies the mismatches – clamp model

7. Normal Protein Function • FAP • APC protein product is a tumour suppressor • APC forms a complex with glycogen synthase kinase 3b (GSK-3b) which targets β-catenin a protein involved in cell adhesion & signalling • APC protein maintains normal apoptosis and may decrease cell proliferation through its regulation of β -catenin • Abnormal APC leads to high levels of cytosolic β -catenin which binds to transcription factors and may activate oncogenes • Also contributes to chromosome stability

8. Disease Diagnosis • HNPCC • Diagnosis – Amsterdam II criteria • 3+ relatives with HNPCC related cancers (1 who is a 1st degree) • 2 successive generation affected • 1+ diagnosed <50 yrs • Exclusion of FAP • 78 % sensitivity

9. Disease Identification & Progression - MSI • HNPCC • Microsatellite Instability (MSI) or molecular testing required for accurate diagnosis • Bethesda guidelines developed to identify individuals whose tumours are candidates for MSI • MSI – microsatellites are particularly susceptible to acquiring errors when mismatch repair gene function is impaired • Panel of 5 markers used: MSI high = >30% show instability, MSI low <30% and MSI stable if 0% (more markers can be used) also RER+ or RER- • Can perform MSI on polyp biopsy but limited material and slightly lower rate of MSI (different markers for different tissues) also lower MSI in endometrial carcinomas

10. Microsatellite Instability

11. Mutation Screening • HNPCC • MLH1 & MSH2 account for 90% of mutations, MSH6 ~5%, PMS1 & PMS2 <2% • Mutation scanning – DGGE, SSCP, dHPLC or direct sequencing. MLPA – up to 5% MLH1 & 20% MSH2 mutations are deletions • Targeted sequencing if familial mutation known or if belong to ethnic group characterised by high frequency of founder mutations (Finnish, Danish, Ashkenazi) • Mutation specific database search to investigate pathogenicity of sequence variation detected • Immunohistochemical analysis of mismatch repair proteins in tumour can determine which gene is involved in the pathogenesis by detecting protein expression – use as first screen?

12. Disease Diagnosis • FAP • Clinically diagnosed in an individual with >100 colorectal adenomatous polyps or <100 polyps and a relative with FAP • Other clinically diagnostic criteria – CHRPE, soft tissue tumours, desmoid tumours

13. Molecular Diagnosis • FAP • Full gene sequencing will detect ~90% mutations • Del / Dup ~10% - MLPA • ~80% of mutations are truncating so can use PTT for mutation scanning • Hypermethylation of APC promoter – additional mechanism • Immunohistochemical assessment of APC protein expression to detect APC status regardless of mechanism of gene inactivation – also suitable for archival tumour material

14. Loss of Heterozygosity • Relies on individual being heterozygous for markers close to APC locus & comparison between normal & tumour tissue

15. HNPCC MSH2 mutations greater risk for extracolonic tumours MSH6 tumours – low MSI, later age of onset FAP Intra-familial variation depending on location of mutation 5’ & 3’ & exon 9 mutations = Attenuated FAP (fewer polyps & later age of onset) Codon 1309 earlier age and more polyps Genotype-Phenotype Correlations

16. HNPCC Variants • Muir-Torre – colon cancer & sebaceous skin neoplasms – MSH2 mutations more common than MLH1 • Turcot syndrome – colorectal cancer in addition to tumours of central nervous system (can be due to MMR gene – MLH1/ PMS2 or APC gene mutation) • Early-onset haematological malignancy, brain tumours, HNPCC-associated tumours, and signs of neurofibromatosis type 1– homozygous MMR gene mutation – MLH1, MSH2 PMS2 – carrier consanginous parents

17. FAP Variants • Attenuated FAP – fewer polyps, later age of onset • Turcot syndrome

18. Monitoring and Treatment • HNPCC • Colonoscopy every one to two years from age of 20-25 or ten years before earliest diagnosis in family • If colon cancer detected full colectomy is recommended • Prophylactic removal of the uterus and ovaries may be considered after childbearing is complete

19. Monitoring and Treatment • FAP • Sigmoidoscopy (less invasive than colonoscopy) every one to two years from age ten to twelve • Annual colonoscopy once polyps detected • Colectomy once 20 to 30 adenomas have occurred

20. MYH- Associated Polyposis • Phenotypically similar to FAP and attenuated FAP but inherited in Autosomal Recessive manner • MYH MutY human homologue gene – chr 1p32.1-34.3 • Occassionally biallelic mutations found in individuals with no polyps • If no APC mutation is detected in FAP consider MYH – analysis of pedigree consistent with AR inheritance? • 1/50 carrier frequency - ?increased risk for carriers • Important for counselling purposes as siblings at 25% risk, whereas FAP & HNPCC 50% risk to all first degree relatives

21. MYH- Associated Polyposis • MYH protein is a base excision repair glycosylase involved in the repair of DNA damage • If MYH is dysfunctional can cause mutations in APC & KRAS • Dutch study of 170 CC patients found MYH mutations in 24% of APC & HNPCC mutation negative patients referred for testing • All mutations point mutations, no rearrangements detected by Southern blotting • High level of breast cancer in females – 18%, significantly higher than Dutch population risk – BRCA1 & 2 also involved in base excision repair

22. Additional References • European Journal of Human Genetics (2008) 16, 62–72; Kruger et alHomozygous PMS2 germline mutations in two families with early-onset haematological malignancy, brain tumours, HNPCC-associated tumours, and signs of neurofibromatosis type 1 • Clinical Chemistry 49:4 552–561 (2003) Bonk et al Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry-based Detection of Microsatellite Instabilities in Coding DNA Sequences: A Novel Approach to Identify DNA-Mismatch Repair-deficient Cancer Cells • Gene Tests