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.

Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of Lynch syndrome patients.

Heterozygous mutations in PMS2 are involved in Lynch syndrome, whereas biallelic mutations are found in Constitutional mismatch repair-deficiency syndrome patients. Mutation detection is complicated by the occurrence of sequence exchange events between the duplicated regions of PMS2 and PMS2CL. We investigated the frequency of such events with a nonspecific polymerase chain reaction (PCR) strategy, co-amplifying both PMS2 and PMS2CL sequences. This allowed us to score ratios between gene and pseudogene-specific nucleotides at 29 PSV sites from exon 11 to the end of the gene. We found sequence transfer at all investigated PSVs from intron 12 to the 3' end of the gene in 4 to 52% of DNA samples. Overall, sequence exchange between PMS2 and PMS2CL was observed in 69% (83/120) of individuals. We demonstrate that mutation scanning with PMS2-specific PCR primers and MLPA probes, designed on PSVs, in the 3' duplicated region is unreliable, and present an RNA-based mutation detection strategy to improve reliability. Using this strategy, we found 19 different putative pathogenic PMS2 mutations. Four of these (21%) are lying in the region with frequent sequence transfer and are missed or called incorrectly as homozygous with several PSV-based mutation detection methods.

[Agreement between centers on the interpretation of exercise echocardiography]. / Concordancia intercentros en la interpretación de la ecocardiografía de ejercicio.

INTRODUCTION AND OBJECTIVES: A low-to-moderate level of agreement on the interpretation of dobutamine echocardiography has been reported, but there are no similar findings on exercise echocardiography. The objectives of this study were to assess the level of agreement between centers on the use of exercise echocardiography and to evaluate the accuracy of the technique when used in a blinded manner. PATIENTS AND METHOD: Six institutions with experience in exercise echocardiography each sent 25 study results to the other centers. Of these, 15 were positive or negative studies on consecutive patients undergoing coronary angiography, and 10 were on non-diabetic patients who had non-coronary chest pain or were asymptomatic and whose pretest probability of coronary artery disease was < 10%. Each institution evaluated 150 studies: 125 blinded and 25 of their own with knowledge of clinical data. RESULTS: For 116 patients (78%), four or more of the five centers blindly evaluating each study agreed with the positive or negative result. The average kappa coefficient was 0.48 (intercenter range 0.45-0.52). The percentage agreement was higher with three-vessel disease (93%, range 85%-95%), with left anterior descending coronary artery disease (83%, range 80%-86%), and when the referring institution reported baseline dyssynergy (86%, range 82%-90%), dyssynergy in left anterior descending coronary artery territory (81%, range 76%-84%), or a peak wall motion score index > 1.50 (88%, range 85%-90%). When the technique was used blinded to detect > or = 50% coronary narrowing in > or = 1 vessel, its sensitivity, specificity and accuracy were 68%, 66% and 67%, respectively, with wide variability between centers. CONCLUSIONS: There was moderate agreement between centers on the interpretation of exercise echocardiography. When used blinded, the technique's accuracy was lower than that reported when clinical data is known.