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. 2012 Dec;228(4):586-95.
doi: 10.1002/path.4092. Epub 2012 Oct 12.

A Robust Genomic Signature for the Detection of Colorectal Cancer Patients With Microsatellite Instability Phenotype and High Mutation Frequency

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Free PMC article

A Robust Genomic Signature for the Detection of Colorectal Cancer Patients With Microsatellite Instability Phenotype and High Mutation Frequency

Sun Tian et al. J Pathol. .
Free PMC article

Abstract

Microsatellite instability (MSI) occurs in 10-20% of colorectal tumours and is associated with good prognosis. Here we describe the development and validation of a genomic signature that identifies colorectal cancer patients with MSI caused by DNA mismatch repair deficiency with high accuracy. Microsatellite status for 276 stage II and III colorectal tumours has been determined. Full-genome expression data was used to identify genes that correlate with MSI status. A subset of these samples (n = 73) had sequencing data for 615 genes available. An MSI gene signature of 64 genes was developed and validated in two independent validation sets: the first consisting of frozen samples from 132 stage II patients; and the second consisting of FFPE samples from the PETACC-3 trial (n = 625). The 64-gene MSI signature identified MSI patients in the first validation set with a sensitivity of 90.3% and an overall accuracy of 84.8%, with an AUC of 0.942 (95% CI, 0.888-0.975). In the second validation, the signature also showed excellent performance, with a sensitivity 94.3% and an overall accuracy of 90.6%, with an AUC of 0.965 (95% CI, 0.943-0.988). Besides correct identification of MSI patients, the gene signature identified a group of MSI-like patients that were MSS by standard assessment but MSI by signature assessment. The MSI-signature could be linked to a deficient MMR phenotype, as both MSI and MSI-like patients showed a high mutation frequency (8.2% and 6.4% of 615 genes assayed, respectively) as compared to patients classified as MSS (1.6% mutation frequency). The MSI signature showed prognostic power in stage II patients (n = 215) with a hazard ratio of 0.252 (p = 0.0145). Patients with an MSI-like phenotype had also an improved survival when compared to MSS patients. The MSI signature was translated to a diagnostic microarray and technically and clinically validated in FFPE and frozen samples.

Keywords: Colorectal cancer; deficient mismatch repair system; gene expression; genomic signature; microsatellite instability; mutation frequency; prognosis.

Figures

Figure 1
Figure 1
(A) A 64-gene expression signature for identification of colorectal cancer samples with MSI, MSI-like and MSS phenotypes. The MSI signature read-outs (index) are shown for 276 tumour samples (cohort A): red, relative up-regulation; green, down-regulation. Standard hospital-based MSI assessment is indicated in the middle bars, together with the BRAF V600E mutation status: light grey, MSS or BRAF wild-type, dark grey, MSI or BRAF mutation. (B) ROC curve and AUC of the signature read-out on validation cohort B. (C) ROC curve and AUC of the MSI signature on validation cohort D (PETACC-3 study). The optimal sensitivity and specificity (with a sensitivity of at least 0.9 and sum of sensitivity and specificity is maximal) is indicated in grey.
Figure 2
Figure 2
MSI and MSI‐like samples classified by the 64‐gene signature show an increased mutation frequency. Seventy‐three colorectal tumour samples were sorted according to their MSI‐signature index; the middle bar shows standard hospital‐based MSI assessment when available (light grey, MSS; dark grey, MSI) and the right barplot show the mutation frequency (% of genes mutated) of each sample in the ‘cancer kinome’ (615 genes).
Figure 3
Figure 3
Prognostic value of the 64‐gene MSI signature in 263 stage II colorectal cancer (cohorts B and C combined). (A) Kaplan–Meier (KM) survival curves for samples classified as MSI (MSI and MSI‐like combined) and MSS by the gene signature; (B) KM curves for samples classified as MSI, MSI‐like and MSS by the gene signature. p values are based on log‐rank test.
Figure 4
Figure 4
Relative gene expression levels (log10 scale) of thymidylate synthase (TYMS) in samples classified as MSS, MSI and MSI‐like by the 64‐gene signature. Samples classified as MSI showed a significant higher expression of TYMS compared to samples classified as MSS (p < 1e‐18, Student's t‐test) Samples classified as MSI‐like also showed a significantly higher expression of TYMS compared to MSS (p = 3.9e‐13, Student's t‐test).
Figure 5
Figure 5
Reproducibility and precision of the 64‐gene signature. (A) Replicate analysis of 53 tumour samples shows a very high correlation in signature index. (B) Stability of the MSI signature read‐out for three representative diagnostic samples across a time period of 20 consecutive days. In both panels, the classification threshold (MSI vs MSS) is indicated by the dashed line.

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