High throughput sequence analysis of EMAST (elevated microsatellite alterations at selected tetranucleotide repeats) in Lynch and non-Lynch tumours

Leigh Betts, Elena Stepannikova, Richard Gallon, Rachel Jones, Christine Hayes, Peter Gawthorpe, Shaun Prior, Harsh Sheth, Neil Rajan, Gillian Borthwick, John Burn, Mauro Santibanez-Koref, Michael Jackson

Newcastle University, Newcastle upon Tyne, United Kingdom


Objectives: Microsatellite instability testing, to assess tumour mismatch repair (MMR) status, is most sensitive and specific using mononucleotide repeats (MNRs, e.g. 1,2). However, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) has a higher reported frequency across multiple tumour types than MNR instability, and has been associated with defects in the MSH3 MMR gene, inflammation, and poor prognosis (reviewed in 3). Despite these associations, EMAST detection has not been standardised. Here, we investigate the feasibility of detecting EMAST using high throughput sequencing.


Methods:  Molecular inversion probes (4) were developed for 15 novel tetranucleotide repeats (7-11 units) and 4 established markers (MYCL1, D20S85, D8S321, D9S242). These were amplified and sequenced in 157 human cancers, comprising 92 unselected and 65 Lynch Syndrome (LS) tumours, and compared to paired blood and unrelated normal controls to detect EMAST. Both fresh and formalin-fixed paraffin-embedded (FFPE) material was used.


Results: (a) EMAST was identified in both MMR deficient and MMR proficient tumours using paired normal and unpaired analyses, and was correlated with MNR instability. (b) Similar to MNR instability, EMAST was higher in LS colorectal than in LS endometrial cancers. (c) However, a major impact of sample processing (FFPE) and storage was detected.


Conclusion: High throughput sequencing can quantify EMAST in tumours, and may be used to further explore its clinical relevance. However, due to the impact of FFPE, sample processing will have to be accounted for. It remains to be established if in vivo instability can be distinguished from that introduced by sample processing.

Abstract references

  1. Bacher JW et al. (2004) Disease Markers. 20(4-5):237-50.
  2. Gallon R et al. (2019) Human Mutation. 40(5):649-655.
  3. Carethers JM (2017) Current Colorectal Cancer Reports. 13(1):73-80.
  4. Hiatt JB et al. (2013) Genome Research. 23(5):843-854.