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

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

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

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

Microsatellite Instability Normal Tumour Normal Tumour

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

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

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 The mean 5-year survival was significantly higher in the subgroup of patients with microsatellite instability (76%) than in the patients with microsatellite stability (54%). Tumours with high-frequency microsatellite instability were also less likely to metastasise to regional lymph I nodes (odds ratio 0·33 [95% CI I 0·21–0·53]) or distant organs (0·49 I [0·27–0·89]), irrespective of the depth of tumour invasion at diagnosis (N Engl J Med 2000: 342; 69–77). For example, patients with stage II or III colorectal cancers with high-frequency microsatellite instability might not need adjuvant chemotherapy,

Treatment of CRC Dictated by stage Chemotherapy Drugs 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 Of particular interest is the evidence of an improvement in survival with chemotherapy for patients with stage II cancer.

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

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

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 Talk about department

Results: Microsatellite Status and Age Total MSI-H MSS + MSI-L P value Mean age (y ± SD) 69±14 66±17 70±13 0.36 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

Results: Microsatellite Status and Gender Total MSI-H MSS + MSI-L P value Gender (%) Male 36 (49) 5 (7) 31 (43) 0.26 Female 37 (51) 9 (12) 28 (38) More female samples demonstrated MSI-H but this was not statistically significant

Results: Microsatellite Status and Tumour Differentiation Total MSI-H MSS + MSI-L Differentiation (%) Poor 10 (15) 6 (9) 4 (6) Moderate 49 (71) 7 (10) 42 (61) Well 0 (0) MSI-H tumours more poorly differentiated than MSS or MSI-L tumours

Results: Microsatellite Status and Tumour Site Total MSI-H MSS + MSI-L P value Cancer Site (%) Proximal 34 (44) 11 (14) 23 (30) 0.011 Distal 43 (56) 4 (5) 39 (51) MSI-H associated with localisation of tumour to the proximal colon

Results: Microsatellite Status and BRAF/KRAS Mutations Total MSI-H MSS + MSI-L KRAS mutation (%) 11 1 (9) 10 (91) BRAF mutation (%) 2 2 (100) 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

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

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

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

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