N99: Identification of mismatch repair-deficient colorectal cancers using a molecular inversion probe based sequencing assay of short mononucleotide repeats

R. Gallon1, H. Sheth1, C. Hayes1, L. Redford1, G. Alhilal1, A. Miguel Alonso2, S. Moreno Laguna3, M. Arends4, A. Oniscu4, O. O’Brien2, S. Needham5, G.M. Borthwick1, M.S. Jackson1, M. Santibanez-Koref1, J. Burn1

1 – Institute of Genetic Medicine, Newcastle University, UK. 2 – Northern Genetics Service, Newcastle Hospitals NHS Foundation trust, UK. 3 – Servicio de Genetica Medica, Complejo Hospitalario de Navarra, Hospi-tal Virgen del Camino, C/Irunlarrea 4, E-31008 Pamplona, Spain. 4 – Western General hospital, Edinburgh, UK. 5 – Department of Cellular Pathology, Royal Victoria Infirmary, Newcastle Hospitals NHS Foundation Trust, UK.

Aim: UK’s NICE guidelines recommend mismatch repair (MMR) deficiency testing of colorectal cancers (CRCs) to identify Lynch syndrome, a hereditary predisposition for CRC. Current assays – immunohistochemistry and fragment analysis are not amenable to high throughput testing due to manual workflows and results interpretation. We aimed to develop a sequencing-based assay of short mononucleotide repeats (MNR) to assess microsatellite instability (MSI), a biomarker of MMR deficiency, using single molecule-molecular inversion probes (smMIPs), with a view to improving the clinical uptake of MMR deficiency testing.

Method: 24 MNRs, together with BRAF V600E marker, were amplified in multiplex using smMIPs. Amplicons were sequenced using Illumina MiSeq platform, aligned to reference genome hg19, and analysed using R scripts. An MSI classifier was trained on 98 CRCs collected from Edinburgh and Spain, and validated in 98 CRCs collected at Newcastle.

Results: The MSI classifier showed 100% sensitivity and specificity relative to fragment analysis. Frequency of BRAF V600E mutation concurred with previous observations. Analysis cost estimate for the proposed assay is £4.28/sample in contrast to £11.65/sample with fragment analysis.

Conclusions: Our novel MSI assay could streamline CRC molecular diagnostics by providing cheap and high throughput detection of MMR deficiency and improving the identification of Lynch syndrome patients.