Microsatellite Instability Testing

1. Microsatellite Instability Testing Changing Methods and Future Strategy Carly Foster – Genetic Technologist WMRGL

2. HNPCC • Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is an autosomal dominant disorder predisposing to colon cancer • Caused by germline mutations in one of four known mismatch repair (MMR) genes; MLH1, MSH2, MSH6 and PMS2 • MMR proteins → replication error repair (RER)

3. MSI in HNPCC • Microsatellites are regions where the DNA sequence is repeated • Mononucleotide repeat AAAAAAAAA…… • Dinucleotide repeat AGAGAGAGA…… • If MMR protein function is lost mistakes in these sequences are not repaired and microsatellite instability (MSI) can occur • MSI is present in more than 90% of colorectal tumours in HNPCC patients

4. Microsatellite Instability Testing • Previous Method → In House PCR for Beckman Fragment Analysis • New Method → Promega MSI analysis system for ABI Fragment Analysis

5. In House PCR for Beckman Fragment Analysis • DNA is extracted from Normal and Tumour tissue mounted on slides • Analysed for the presence of MSI using 7 microsatellite markers, BAT25, BAT26, BAT40, D2S123, D5S346, D8S255, and D17S250. • These markers have been selected due to their sensitivity to loss of MMR protein function

6. In House PCR for Beckman Fragment Analysis • The PCR is set up on batches of 23 samples plus a negative control in two 96 well plates using the Biomek NX Robot

7. In House PCR for Beckman Fragment Analysis • Once the PCR is complete the products from the two plates are pooled into one plate using the post PCR Biomek NX robot • This reduces the number of wells required for Beckman fragment analysis to 72 (i.e. 3 per sample) • 1.6µl of mix from each well is run along with a 600bp size standard on the Beckman Coulter CEQ 8000 using fluorescent capillary electrophoresis

8. In House PCR for Beckman Fragment Analysis • The data is then processed using the Beckman CEQ8000 software • Data from the Normal and Tumour DNA from each patient for each marker is compiled into one report for comparison – this process is very time consuming

9. Promega MSI Analysis System for ABI Fragment Analysis • The Promega MSI Analysis System allows seven markers to be amplified in a multiplex system – 1 tube per patient • Five mononucleotide repeat markers to determine MSI status • BAT-25, BAT-26, NR-21, NR-24 and MONO-27 • Two highly polymorphic pentanucleotide repeat markers for sample identification • Penta C and Penta D

10. Promega MSI Analysis System for ABI Fragment Analysis • The PCR is currently performed on Normal and Tumour DNA in batches of 16 samples • The PCR products are run on the ABI 3130xl genetic analyser along with a 600 base pair internal lane size standard (ILS 600) • The data from the 3130xl is then processed using the GeneMarker software and a single report per run is produced with one sample per page

11. Promega MSI Analysis System for ABI Fragment Analysis

12. MSI Analysis • Alleles from the tumour sample are compared to those in the normal sample, or assessed independently if no normal sample is available • Any alleles or extra peaks in the tumour sample which do not appear in the normal sample indicate that the marker is unstable • If the profile of the tumour sample matches that of the normal sample then the patient is stable in that marker

13. MSI Analysis RER 7 • PCR amplification of microsatellite loci generates artifacts that appear as smaller peaks 1bp above or below the prominent mononucleotide repeat allele or 2bp in dinucleotide markers • Factors such as loss of heterozygozity (LOH), split peaking and stutter peaks in dinucleotide repeat markers can make interpretation difficult

14. MSI Analysis RER 7

15. MSI Analysis RER 7

16. MSI Analysis RER 7

17. MSI Analysis RER 7

18. MSI Analysis Promega Kit • The MSI markers in the Promega kit are all mononucleotide repeat markers • Markers are also quasi-monomorphic • almost all individuals are homozygous for the same common allele

19. MSI Analysis Promega Kit

20. MSI Analysis Promega Kit

21. MSI Analysis Promega Kit

22. MSI Analysis Promega Kit

23. Comparison of Methods • Poor quality DNA is more obvious in the Promega kit due to the extra pressures of the multiplex system, however the majority of these issues can be overcome by using different dilution factors • The cost of the Promega kit is high however this is negated by switching from Beckman to ABI analysis

24. Comparison of Methods • As the Promega kit contains only mononucleotide markers for the assessment of MSI status this removes the issue of inconclusive results due to split peaks and loss of heterozygosity in the dinucleotide repeat markers in the in house kit

25. Proposed Strategy • As the markers in the Promega kit are quasi-monomorphic this makes determination of MSI status in tumour only samples much easier • Therefore in future only tumour samples will be extracted and tested • If all markers are homozygous or the sample is MSI-high the result will be reported • If one marker is heterozygous or appears unstable the normal sample will be extracted and processed for comparison

26. Extract and test Tumour DNA All markers homozygous 1 marker heterozygous/ unstable 2-5 markers unstable No Result Extract and test Normal DNA Repeat test (e.g. neat) Normal heterozygous - Stable Normal homozygous - Unstable No Result MSS MSI-LOW MSI-HIGH FAIL Proposed Strategy