Fig 1.
A) Overview of the development cohort. A total of 185 colorectal cancer (CRC) samples were analyzed. Forty of these samples were tested on a total of three extraction method and two different real-time PCR instruments. Thirty of the samples were analyzed in a total of five laboratories (1; Locarno, 2; Legnano, 3; Novara, 4; Candiolo, and 5; Terni). B) The validation cohort covered 127 CRC samples distributed in four laboratories.
Fig 2.
Principle of MicroSight® MSI analysis using universal reference.
Four stable and four unstable tumor samples were analyzed using a universal reference for NR24 locus. Applying a temperature shift makes it possible to differentiate unstable from stable samples naturally differing in lengths. A) Normalized HRM curves without temperature shift. B) Difference plot without temperature shift. Threshold was set at -0.04 RFU. C) Normalized HRM curves with temperature shift at 0.1 RFU. D) Difference plot with temperature shift at 0.1 RFU. Threshold was set at -0.04 RFU. MSI: microsatellite instability; HRM: high resolution melt; RFU: relative fluorescence unit.
Fig 3.
Boxplot over stable and unstable cases for the development cohort (n = 185).
The difference in RFU for the tumor sample compared to either universal or paired normal samples are plotted for each of the five microsatellite loci. Stable cases represented by light blue boxes and unstable cases by light red boxes as indicated by the figure legends. A) Difference using paired normal and tumor samples. B) Difference using tumor samples and a universal reference. RFU: relative fluorescence unit.
Table 1.
MicroSight® MSI agreement to the PCR fragment analysis.
Fig 4.
Results from analysis of paired samples on BAT25 using three extraction methods and two real-time PCR instruments.
Forty CRC samples were extracted using the QIAamp method, BasePurifier™, and BaseRelease™ and analyzed using the MyGo Pro. The samples extracted with BasePurifier™ were re-analyzed using a BaseTyper™ real-time PCR instrument.
Fig 5.
Correlation plot of unstable cases (n = 5) using paired samples.
The x-coordinates for the points are the difference of RFU obtained in the reference laboratory (laboratory 1) and the y-coordinate is the difference in RFU obtained in the validation laboratories (laboratory 2–5). A linear fit is applied to the points and the Pearson correlation coefficient (R), and p-values are shown. RFU: relative fluorescence unit.
Table 2.
A 3x3 table covering all four validation laboratories (original MSI status) compared to MicroSight® MSI using paired samples.
Table 3.
Agreement of MSI status in validation cohort from four laboratories.