Abstract
BackgroundAnalysis of high microsatellite instability (MSI-H) phenotype in colorectal carcinoma (CRC) is important for evaluating prognosis and choosing a proper adjuvant therapy. Although the conventional MSI analysis methods such as polymerase chain reaction (PCR) fragment analysis and immunohistochemistry (IHC) show high specificity and sensitivity, there are substantial barriers to their use.MethodsIn this study, we analyzed the MSI detection performance of three molecular tests and IHC. For the molecular tests, we included a recently developed peptide nucleic acid probe (PNA)-mediated real-time PCR-based method using five quasi-monomorphic mononucleotide repeat markers (PNA method) and two conventional PCR fragment analysis methods using NCI markers (NCI method) or five quasi-monomorphic mononucleotide repeat markers (MNR method). IHC analysis was performed with four mismatch repair proteins. The performance of each method was validated in 166 CRC patient samples, which consisted of 76 MSI-H and 90 microsatellite stable (MSS) CRCs previously diagnosed by NCI method.ResultsOf the 166 CRCs, 76 MSI-H and 90 MSS CRCs were determined by PNA method. On the other hand, 75 MSI-H and 91 MSS CRCs were commonly determined by IHC and MNR methods. Based on the originally diagnosed MSI status, PNA showed 100% sensitivity and 100% specificity while IHC and MNR showed 98.68% sensitivity and 100% specificity. When we analyzed the maximum sensitivity of MNR and PNA method, which used the same five markers, PNA method could detect alterations in all five mononucleotide repeat markers in samples containing down to 5% MSI-H DNAs, whereas MNR required at least 20% MSI-H DNAs to achieve the same performance.ConclusionsBased on these findings, we suggest that PNA method can be used as a practical laboratory test for the diagnosis of MSI.
Highlights
Analysis of high microsatellite instability (MSI-H) phenotype in colorectal carcinoma (CRC) is important for evaluating prognosis and choosing a proper adjuvant therapy
We aimed to evaluate the MSI detection performance of various MSI detection methods, including the following: a recently developed peptide nucleotide acid probe (PNA)-mediated real-time polymerase chain reaction (PCR)-based MSI test using five quasi-monomorphic mononucleotide repeat markers (PNA method) and two conventional PCR fragment analysis methods, PCR fragment analysis with two mononucleotide and three dinucleotide repeat markers proposed by the National Cancer Institute (NCI method) [8], and PCR fragment analysis with the same mononucleotide repeat (MNR) markers as Peptide nucleotide acid (PNA) method (MNR method) as well as IHC method
Case no. 20 was diagnosed as High microsatellite instability (MSI-H) by NCI and MNR, but no loss of protein expression was detected by IHC (Additional file 1: Table S1 and Additional file 2: Figure S1)
Summary
Analysis of high microsatellite instability (MSI-H) phenotype in colorectal carcinoma (CRC) is important for evaluating prognosis and choosing a proper adjuvant therapy. The conventional MSI analysis methods such as polymerase chain reaction (PCR) fragment analysis and immunohistochemistry (IHC) show high specificity and sensitivity, there are substantial barriers to their use. The development of a broad range of CRCs can be explained by the multistep carcinogenesis model and high microsatellite instability (MSI-H) resulting from. There are several laboratory tests for determining MSI status, including polymerase chain reaction (PCR)-based analysis of MSI markers and immunohistochemistry (IHC) staining of MMR proteins [7]. Conventional PCR-based MSI interrogation requires complicated steps and additional equipment and shows low sensitivity for samples with a small proportion of tumor cells. A more accurate and simple MSI test strategy is needed
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