Improved Detection of Microsatellite Instability in Early Colorectal Lesions

<div><p>Microsatellite instability (MSI) occurs in over 90% of Lynch syndrome cancers and is considered a hallmark of the disease. MSI is an early event in colon tumor development, but screening polyps for MSI remains controversial because of reduced sensitivity compared to more advanced neoplasms. To increase sensitivity, we investigated the use of a novel type of marker consisting of long mononucleotide repeat (LMR) tracts. Adenomas from 160 patients, ranging in age from 29–55 years old, were screened for MSI using the new markers and compared with current marker panels and immunohistochemistry standards. Overall, 15 tumors were scored as MSI-High using the LMRs compared to 9 for the NCI panel and 8 for the MSI Analysis System (Promega). This difference represents at least a 1.7-fold increase in detection of MSI-High lesions over currently available markers. Moreover, the number of MSI-positive markers per sample and the size of allelic changes were significantly greater with the LMRs (p = 0.001), which increased confidence in MSI classification. The overall sensitivity and specificity of the LMR panel for detection of mismatch repair deficient lesions were 100% and 96%, respectively. In comparison, the sensitivity and specificity of the MSI Analysis System were 67% and 100%; and for the NCI panel, 75% and 97%. The difference in sensitivity between the LMR panel and the other panels was statistically significant (p<0.001). The increased sensitivity for detection of MSI-High phenotype in early colorectal lesions with the new LMR markers indicates that MSI screening for the early detection of Lynch syndrome might be feasible.</p></div

High concordance between IHC and MSI results using LMRs.

<p>There was 96% (79/82) concordance between MSI results using LMR repeats and loss of MMR expression by IHC. For example, tubular adenoma 267B was unstable at all five markers and lacked MSH2 and MSH6 expression. Note that when MSH2 is lost, the level of binding partner MSH6 is often significantly lower due to reduced stability. The area indicated by the rectangle in the H&E panel is enlarged 2x in each of the lower panels. Size bar for H&E, 500μm.</p

The number of MSI-positive markers per sample was highest for the LMR panel and increased confidence in MSI classification.

<p>The number of markers that were MSI-positive for each of 15 MSI-High samples is given for the three marker panels. The number of unstable markers per sample was significantly higher for the LMR panel compared to the other panels (z-test; p<0.001). MSI analysis with LMRs resulted in MSI-High samples with either 5/5 or 4/5 unstable markers in 80% of cases.</p

Use of LMR markers for MSI analysis typically resulted in larger allele size changes in MSI-High lesions.

<p>(A) Electropherogram of MSH2-deficient tumor sample 267B and matching normal tissue screened for MSI using the experimental LMR panel which shows all five markers were unstable with size shifts of up to 24bp. (B) The average change in tumor allele size of MSI-High lesions was determined for the markers in the LMR panel and the MSI Analysis System. The average size shift was significantly greater in LMR markers (p = 0.0014), which increased confidence in MSI classification. Error bars show standard error of the mean.</p

Characteristics of the 15 samples classified as MSI-High by the LMR panel from 9 patients

<p>^a Personal history of LS-associated cancer. CRC,colorectal cancer; number indicates age in years at diagnosis.</p><p>^b 1^st or 2^nd degree relative with LS-associated cancer. CRC,colorectal cancer; OV,ovarian cancer; EC,endometrial cancer; number indicates age in years at diagnosis; question mark indicates unknown age.</p><p>^c AC, adenocarcinoma; HP, hyperplastic polyp; SSP, sessile serrated polyp; TA, tubular adenoma; TVA, tubulovillous adenoma.</p><p>^d ND, no data. MLH1, loss of MLH1 and PMS2 expression by IHC. MSH2, loss of MSH2 and MSH6 expression by IHC. Normal, intact IHC staining. Germline DNA sequencing information was available for patients 1–3.</p><p>^e BRAF V600E mutation tested by DNA sequencing and/or by ARMS-PCR [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132727#pone.0132727.ref025" target="_blank">25</a>].</p><p>^f Group: Patients were classified as LS if they had a pathogenic germline MMR mutation. Patients were classified as LS probable if tumor was MSI-High and no BRAF V600E mutation was found, plus one of more of the following: loss of expression for MSH2, MSH6 or PMS2 by IHC, colorectal cancer before age 50 or a 1^st or 2^nd degree relative with LS-associated cancer.</p><p>Characteristics of the 15 samples classified as MSI-High by the LMR panel from 9 patients</p

The relative MSI sensitivity of the LMR markers was significantly higher than that of currently used markers.

<p>A total of 15 tumors were classified as MSI-High using the LMR panel, the MSI Analysis System or the NCI panel. The percentage of tumors that were scored as MSI-positive is shown for each individual marker. The relative sensitivity of the LMR panel was significantly higher than that of the MSI Analysis System (p = 0.0012) and the NCI panel (p<0.0038) using the t-test.</p

Comparison of MSI results for three marker panels.

<p>The MSI classifications of tumor samples are given for all samples that were scored for all three panels and positive for at least one marker from the experimental LMR panel (Promega), the MSI Analysis System (Promega) or the NCI panel. Samples were classified as MSI-High when two or more markers out of a panel of five were unstable, MSI-Low when one out of five markers was unstable and MSI stable when there were no unstable markers. The MSI stable samples with intact MMR staining are not shown. Sample 011C is not shown owing to inconclusive results with the LMR panel. *Sample 027B was scored as MSI-Low for the NCI panel because only the dinucleotides were unstable. Tumors in which only dinucleotides are unstable are often misclassified as MSI-High and typically show MMR expression by IHC, as was the case for sample 027B [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132727#pone.0132727.ref007" target="_blank">7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132727#pone.0132727.ref049" target="_blank">49</a>]. +, MSI-positive;-, MSI stable; ND, not done.</p