Abstract
PurposeMicrosatellite instability (MSI) is used to screen colorectal cancers (CRC) for Lynch Syndrome, and to predict outcome and response to treatment. The current technique for measuring MSI requires DNA from normal and neoplastic tissues, and fails to identify tumors with specific DNA mismatch repair (MMR) defects. We tested a panel of five quasi-monomorphic mononucleotide repeat markers amplified in a single multiplex PCR reaction (pentaplex PCR) to detect MSI.Experimental DesignWe investigated a cohort of 213 CRC patients, comprised of 114 MMR-deficient and 99 MMR-proficient tumors. Immunohistochemical (IHC) analysis evaluated the expression of MLH1, MSH2, PMS2 and MSH6. MSI status was defined by differences in the quasi-monomorphic variation range (QMVR) from a pool of normal DNA samples, and measuring differences in allele lengths in tumor DNA.ResultsAmplification of 426 normal alleles allowed optimization of the QMVR at each marker, and eliminated the requirement for matched reference DNA to define MSI in each sample. Using ≥2/5 unstable markers as the criteria for MSI resulted in a sensitivity of 95.6% (95% CI = 90.1–98.1%) and a positive predictive value of 100% (95% CI = 96.6%–100%). Detection of MSH6-deficiency was limited using all techniques. Data analysis with a three-marker panel (BAT26, NR21 and NR27) was comparable in sensitivity (97.4%) and positive predictive value (96.5%) to the five marker panel. Both approaches were superior to the standard approach to measuring MSI.ConclusionsAn optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.
Highlights
Microsatellite instability (MSI), which is defined as the accumulation of insertion-deletion mutations at short repetitive DNA sequences is a characteristic feature of cancer cells with DNA mismatch repair (MMR) deficiency [1]
It was later recognized that MSI occurs in,12% of sporadic colorectal cancer (CRC) occurring in patients that lack germline MMR mutations, and MSI in these patients is due to promoter methylation-induced silencing of the MLH1 gene expression [4]
MMR-Deficient and MMR-Proficient CRCs To determine the accuracy of a pentaplex PCR system for detecting MMR deficient CRCs, we investigated a cohort of 213 CRCs which comprised of 114 MMR-deficient and 99 MMRproficient tumors
Summary
Microsatellite instability (MSI), which is defined as the accumulation of insertion-deletion mutations at short repetitive DNA sequences (or ‘microsatellites’) is a characteristic feature of cancer cells with DNA mismatch repair (MMR) deficiency [1]. Inactivation of any of several MMR genes, including MLH1, MSH2, MSH6 and PMS2, can result in MSI. MSI was shown to correlate with germline defects in MMR genes in patients with Lynch syndrome (LS), where .90% of colorectal cancer (CRC) patients exhibit MSI [2,3]. It was later recognized that MSI occurs in ,12% of sporadic CRCs occurring in patients that lack germline MMR mutations, and MSI in these patients is due to promoter methylation-induced silencing of the MLH1 gene expression [4]. Determination of MSI status in CRC has clinical use for identifying patients with germline defects predisposing to MMR-deficiency. MSI status has prognostic and therapeutic implications, because MSI CRCs typically have a better prognosis, and these cancers are less responsive to 5FU-based adjuvant chemotherapy [5]
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