A61
Fast and accurate method of microsatellite instability detection in FFPE tissue using high melting analysis (HRM)
Rashmi Seth1, Wakkas Fadhil2, Kirsten Mckay3, Jenny Bell3, Philip Tannier3, Fiona Macdonald3, Mohammad Ilyas2
1Nottingham University Hospital, QMC Campus, Nottingham, UK, 2Nottingham University, Nottingham, UK, 3West Midlands Regional Genetics Laboratory, Birmingham, UK
Background
Identification of microsatellite instability (MSI) phenotype is relevant and important as a screening and prognostic tool in colorectal cancer. Tumours that display microsatellite instability (MSI) have better prognosis, react differently to therapy and histologically, they are heterogeneous in appearance. The current panel of MSI markers used in a diagnostic laboratory in UK are BAT25, BAT 26, BAT40, D17S250, D2S123, D5S346 and D8S255 and the detection method involves amplification and capillary gel electrophoresis which can make it unsuitable for routine high through-put use. The aim of this study was to explore the suitability of HRM technique for the diagnostic detection of MSI status using only a panel of 2 markers BAT25 and BAT26 in DNA obtained from formalin fixed paraffin embedded (FFPE) tissue.
Method
New primers for BAT-25 and BAT-26 were designed and optimised for use in this technique. Genomic DNA from 29 colorectal cancer cell lines with known MSI status were tested using this protocol. There was 100% concordance. The method was further tested blindly by analysing 24 well characterised clinical samples with known MSI status obtained from using the above panel of 7 markers. The protocol was finally used to screen 43 samples from 37 CRC cases.
Results
The assay detection limit determined using a mixture of mutant alleles with a wild type genomic DNA and was found to be 2.5%. The intra- and inter-assay variation was negligible using these primers. Correlation with known samples was 96 % (23/24) concordance. Fifty samples from 37 CRC cases were screened and (2.5%) were found to be mutant for BAT-25 and (3.0%) for BAT-26.
Conclusion
This study optimized the parameters for using high resolution melting technique for MSI on FFPE tissue and may facilitate its implementation in high through-put diagnostic purposes.
Acknowledgements
Grants from NUH charities and University of Nottingham is gratefully acknowledged
