An optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers

Abstract

Purpose: Microsatellite 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 design: We 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.

Results: Amplification 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 > or = 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.

Conclusions: An optimized pentaplex (or triplex) PCR offers a facile, robust, very inexpensive, highly sensitive, and specific assay for the identification of MSI in CRC.

Figure 1. Frequency of allele size distribution for the five pentaplex markers.

A) Allele size distribution (in base pairs) from 213 normal DNA specimens. For each marker, blue shading indicates the adjusted QMVR, while the gray shading indicates the entire range of allelic size obtained from 426 germline alleles. B) Distribution of allele sizes in MMR-deficient (orange) and MMR-proficient (green) CRCs.

Figure 2. Performance characteristics of pentaplex markers based on QMVR, availability of normal DNA, and numbers of markers required to determine MSI in colorectal cancers.

A) The figure illustrates the performance of the pentaplex mononucleotide-repeat maker panel when defining the MSI status of tumor DNA by “QMVR only” (when matched normal DNA was not available) and by “Normal DNA” by subtracting the germline allele lengths from tumor for each tumor. Data in the two panels on the left is from MMR-deficient tumors, while the other two panels on the right represent MMR-proficient CRCs. (* indicates one case with loss of both MLH1 and MSH2). Black squares indicate a tumor positive for allelic variation (i.e., unstable) and white squares indicate a tumor negative for any allele variations (i.e., stable). B) Shows the frequency of MMR-deficient tumors with number of markers displaying allelic variations when data were analyzed from all five pentaplex markers. C) Shows the frequency of MMR-deficient tumors with number of markers displaying allelic variations when data were analyzed from just three pentaplex markers (BAT26, NR21 and NR27).

Figure 3. Correlation between various mono-nucleotide markers in the pentaplex PCR.

A) A scatter-plot matrix demonstrating the pair-wise correlation coefficient (r) between five microsatellite markers in the cohort of MMR-proficient and deficient CRCs. The Y and X-axis denote absolute differences in allele sizes between the tumor DNA and normal DNA. B) The figure shows hierarchical clustering analysis derived from 104 MMR-deficient and 99 MMR-proficient CRCs. The data are presented in matrix format in which the rows represent each CRC and the columns indicate the individual mononucleotide markers. The color scale represents the gradient (green to red) of absolute allele length differences between tumor and germline DNA from QMVR standardized data; green (no differences in allele size between tumor and normal DNA) to red (significant differences in allelic lengths between tumor and normal DNA).

Figure 4. Comparison between pentaplex PCR and NCI panel of markers to determine MMR-deficiency in colorectal cancers.

A) The figure shows the performance comparison between the NCI panel markers and the QMVR optimized pentaplex PCR. Black squares indicate a tumor positive for allelic variation (or unstable) and white squares indicate a tumor negative for any allele variations (or stable). The dinucleotide repeat markers (D2S123, D5S346 and D17S250) are less robust than the mononucleotide repeats for detecting MSI. B) The figure illustrates the frequency of tumors with number of markers displaying allelic variations in MMR-deficient and proficient CRCs. As indicated, pentaplex PCR shows a higher sensitivity and specificity compared to NCI panel of markers, and the distribution of altered markers is unambiguously bimodal.