Microsatellite Instability in Sporadic Colorectal Cancer: A Retrospective Study

Kimberley SlowtherTrainee 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%

Rare Syndromes

<1%

FAP 1%

HNPCC 5%

Familial 19%

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

BRAFSerine-threonine specific kinase40% sporadic MSI-H tumoursNot present in HNPCC Common mutation (90%) is V600E

KRASGTPase90% mutations in codons 12 and 1330-40% sporadic MSS CRC40% HNPCC

Regulates growth, differentiation and apoptosis

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

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

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 microsatelliteanalysis 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

Results: Microsatellite Status and Age

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

Plus-minus values are means ±SD.

Total

MSI-H

MSS + MSI-L

P valu

e

Mean age (y ± SD)

69±14

66±17

70±13 0.36

Results: Microsatellite Status and Gender

More female samples demonstrated MSI-H but this was not statistically significant

TotalMSI-

HMSS + MSI-

LP

value

Gender (%)

Male 36

(49)5 (7) 31 (43)

0.26Female

37 (51)

9 (12) 28 (38)

Results: Microsatellite Status and Tumour Differentiation

MSI-H tumours more poorly differentiated than MSS or MSI-L tumours

TotalMSI-

HMSS + MSI-

L

Differentiation (%)

Poor 10 (15) 6 (9) 4 (6)

Moderate

49 (71) 7 (10) 42 (61)

Well 10 (15) 0 (0) 10 (15)

Results: Microsatellite Status and Tumour Site

MSI-H associated with localisation of tumour to the proximal colon

TotalMSI-

HMSS + MSI-

LP

value

Cancer Site (%)

Proximal

34 (44)

11 (14)

23 (30)0.011

Distal 43 (56)

4 (5) 39 (51)

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

Total

MSI-H

MSS + MSI-L

KRAS mutation (%)

11 1 (9) 10 (91)

BRAF mutation (%)

22

(100)0

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