Microsatellite Instability in Sporadic Colorectal Cancer: A Retrospective Study

1. Microsatellite Instability in Sporadic Colorectal Cancer: A Retrospective Study Kimberley Slowther Trainee Project West Midlands Regional Genetics Laboratory

2. Colorectal Cancer • 35,000 diagnosed per year • Treatment and prognosis depend upon tumour stage

3. Causes of CRC Sporadic 75% Familial 19% Rare Syndromes <1% HNPCC 5% FAP 1%

4. Genetic Pathways in CRC • Microsatellite Instability (MSI-H) • Deficiency in DNA mismatch repair (MMR) • HNPCC (MSH2, MLH1, MSH6, PMS2) • 15% sporadic (hypermethylation of MLH1 promoter) • Chromosomal Instability (CIN) • APC, TP53, KRAS, Loss of Heterozygosity, aneuploid DNA content • CpG Island Methylator Phenotype (CIMP) • Methylation of CpG islands in promoter regions of tumour suppressor genes • Sporadic CRCs demonstrating MSI are a subset

5. Normal Normal Tumour Tumour Microsatellite Instability

6. Comparison of Sporadic MSI-H with HNPCC Tumours • Both are MSI-H and proximally located • BUT Sporadic MSI-H:- • Greater predilection for the proximal colon • Higher frequency of BRAF mutations • Lower frequency of KRAS mutations • More age related • More common in females • Originate from serrated polyps • HNPCC tumours originate from adenomas

7. RAS-RAF-MEK-ERK Pathway Regulates growth, differentiation and apoptosis KRAS • GTPase • 90% mutations in codons 12 and 13 • 30-40% sporadic MSS CRC • 40% HNPCC BRAF • Serine-threonine specific kinase • 40% sporadic MSI-H tumours • Not present in HNPCC • Common mutation (90%) is V600E

8. MSI and Prognosis • MSI in CRC is a positive prognostic indicator • Better five-year rate of overall survival than MSS tumours • Less likely to metastasise • Why? • Enhanced mutation rate induces a burden not compatible with tumour cell survival • Abnormal peptides produced elicit antitumour immune responses that limit tumour growth

9. Treatment of CRC • Dictated by stage • Stage I: surgery alone • Stage II, III+IV: adjuvant therapy • Less clear cut with Stage II • Patients reviewed on individual basis • Chemotherapy Drugs • 5-fluorouracil/folinic acid (5FU/FA) to Stage II and III patients • 5FU/FA and Oxaliplatin to Stage IV and fitter Stage II and III patients

10. MSI and Chemotherapy • Resistance of MSI-H CRC to 5FU is well documented • MSS patients have increased survival with 5FU • MSI-H patients do not have improved survival following treatment • Why have 5FU treatment if there is no benefit? • Oxaliplatin therapy not affected by loss of MMR • Information about MSI status could impact treatment:- • Decision of whether or not to opt for adjuvant therapy • Allocation patients to oxaliplatin therapy

11. Project Aim Investigate whether microsatellite analysis of sporadic colorectal cancer would be a valuable service to offer in future in order to tailor patient treatment

12. Methods • MSI status of patients with locally advanced, treatable sporadic (absence of family history) CRC • 41 – Department of Surgery Epithelial Research Group • 32 – Department of Histopathology • 7 microsatellite markers • MSI-H if instability present at 2 or more markers • MSI-L if instability present at 1 marker • Analysed MSI data in relation to:- • Age at diagnosis • Gender • BRAF exon 15 mutation • KRAS codon 12 and 13 mutation (41/73) • Tumour site • Tumour differentiation

13. Results: Microsatellite Status and Age Plus-minus values are means ±SD. • On average patients demonstrating MSI in their tumours were younger but this was not statistically significant • Of the 14 patient samples demonstrating MSI, 7 were older than 70 at diagnosis • Usually opt not to be treated with adjuvant therapy; MSI status could be used to help make this decision

14. Results: Microsatellite Status and Gender • More female samples demonstrated MSI-H but this was not statistically significant

15. Results: Microsatellite Status and Tumour Differentiation • MSI-H tumours more poorly differentiated than MSS or MSI-L tumours

16. Results: Microsatellite Status and Tumour Site • MSI-H associated with localisation of tumour to the proximal colon

17. Results: Microsatellite Status and BRAF/KRAS Mutations • KRAS mutations and BRAF mutations were mutually exclusive • MSS and MSI-L tumours had a higher frequency of KRAS mutations • MSI-H tumours higher frequency of BRAF mutations

18. Discussion • What are the benefits of MSI analysis for sporadic CRC? • Improved patient care • MSI-H not given unnecessary treatment (5-FU) • MSI-H allocated to more effective treatments (Oxaliplatin) • ? Cost Benefit • Reduced chemotherapy? • Perhaps not all tumours were sporadic • Ethical Implications • Possibility that HNPCC patients included • Only 14% MSI-H samples contained BRAF mutations • Recommend that patients are counselled

19. Future Developments • Prospective study • Treatment vs no treatment • ? Unethical • Complicated by wide use of oxaliplatin • Oxaliplatin vs 5FU • In the future just offer oxaliplatin therapy to MSI-H patients

20. Conclusion • Techniques available to put a system into place to offer MSI analysis of sporadic colorectal tumours • Tailor patient treatment depending upon tumour stage, microsatellite status and age

21. Acknowledgements • West Midlands Regional Genetics Laboratory • Fiona Macdonald • Jennie Bell • Kerry Wall • University of Birmingham Department of Surgery Epithelial Research Group • Dion Morton • Germaine Caldwell • University Hospital of Birmingham Histopathology Department • Philippe Taniere • Brendan O’Sullivan • Graham Caine