Sequencing-based microsatellite instability testing using as few as six markers for high-throughput clinical diagnostics.

Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer-predisposition, and can be used to predict response to immunotherapy. Here, we present a single-molecule molecular inversion probe and sequencing-based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward-forward stepwise selection was used to identify a 6-marker subset of equal accuracy to the 24-marker panel. Assessment of assay detection limits showed that the 24-marker panel is marginally more robust to sample variables than the 6-marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

Fennoscandian distribution of an important parasite of cervids, the deer ked (Lipoptena cervi), revisited.

The deer ked [Lipoptena cervi (L. 1758) (Dipt., Hippoboscidae)] is a blood-sucking ectoparasite of cervids. The species has been resident in Sweden for more than two centuries, whereas in Finland ( approximately 50 years) and Norway ( approximately 30 years), it has established itself relatively recently. L. cervi may cause serious health problems in its natural hosts, act as a vector for zoonotic diseases, and pose a socioeconomic threat to forest-based activity. In this paper, we review the distribution and former expansion of the species in Fennoscandia. The current distribution of L. cervi appears bimodal, and the geographical range expansion of the species shows notable differences across Fennoscandia. The western population in Norway and Sweden has its northern edge of range at respective latitudes of 61 degrees N and 62 degrees N, whereas the eastern population in Finland reaches 65 degrees N. The future expansion of L. cervi is dependent on several interdependent extrinsic and intrinsic factors. International multidisciplinary collaboration is needed to achieve a synthesis on the factors affecting expansion rates and to understand the effects of L. cervi on wildlife, human health, and the rural societies of Fennoscandia.

A novel panel of short mononucleotide repeats linked to informative polymorphisms enabling effective high volume low cost discrimination between mismatch repair deficient and proficient tumours.

Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate, and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7-12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI.