Detection of microsatellite instability by fluorescence multiplex polymerase chain reaction

Abstract

We have created a clinical molecular diagnostic assay to test for microsatellite instability (MSI) at multiple loci simultaneously in paraffin-embedded surgical pathology colon resection specimens. This fluorescent multiplex polymerase chain reaction (PCR) assay analyzes the five primary microsatellite loci recommended at the 1997 National Cancer Institute-sponsored conference on MSI for the identification of MSI or replication errors in colorectal cancer: Bat-25, Bat-26, D2S123, D5S346, and D17S250. Amplicon detection is accomplished by capillary electrophoresis using the ABI 310 Genetic Analyzer. Assay validation compared 18 specimens previously assessed by radioactive PCR and polyacrylamide gel electrophoresis detection to results generated by the reported assay. Germline and tumor DNA samples were amplified in separate multiplex PCR reactions, sized in separate capillary electrophoresis runs, and compared directly to identify novel length alleles in tumor tissue. A concordance of 100% between the two modalities was achieved. The multiplex assay routinely detected a subpopulation of 10% tumor alleles in the presence of 90% normal alleles. A novel statistical model was generated that corroborates the validity of using results generated by analysis of five independent microsatellites to achieve a single overall MSI diagnosis. The assay presented is superior to standard radioactive monoplex PCR, polyacrylamide gel electrophoretic analysis, primarily due to the multiplex PCR format.

Figure 1.

A: Comparison of results generated by analysis of the Bat-25 locus in cell lines L670 (normal) and Vaco 670 (tumor) using the radioactive PAGE format (left panel, autoradiograph) and the fluorescent monoplex format (right panel, electropherograms). Arrowheads indicate the predominant amplicon size bands (peaks) at 121 bases for Normal (L670), and arrows indicate the predominant amplicon size bands (peaks) at 112 bases for Tumor (Vaco 670). Note the stutter bands surrounding the predominant peak and the absence of germline allele peaks in the tumor cell line. The asterisk to the right of the autoradiograph is the Phi-X174 HinfI-digested band at 118 bases. B: The complete fluorescent multiplex electropherograms of cell lines L670 (normal) and Vaco 670 (tumor) are shown. The red tracing is the GS 500 size standard labeled with Tamara (100, 139, 150, 160, 200, and approximately 245 bases). In the top panel, normal germline alleles are seen where the alleles from one locus (D2S123) are indicated (219 and 225 bases) labeled with Tet (green). The blue tracing represents the D5S346 (2 alleles, 100 and 107 bases) and D17S250 (1 allele, 147 bases) loci labeled with 6-Fam, the green tracing represents Bat-26 (1 allele, 115 bases) labeled with Tet, and the black tracing Bat-25 (1 allele, 121 bases) labeled with Hex. The bottom panel is the fluorescent multiplex electropherogram for the matched tumor cell line Vaco 670. Again designated are the shifted alleles at D2S123 (211 and 213 bases). There are also shifts at all of the other four loci: D5S346 (102 and 109 bases), D17S250 (139 and 147 bases), Bat-26 (103 bases), and Bat-25 (112 bases). C: The complete fluorescent multiplex electropherograms of a representative normal tumor pair. The alleles for D2S123 are designated where the patient’s normal sample revels homozygosity at 208 bases. The tumor shows the germline allele in addition to novel length alleles at 198 with its accompanying stutter bands. Novel length alleles can be seen at each of the other four loci.

Figure 2.

Titration of labeled forward Bat-26 primer into the reverse, unlabeled primer. A: The 1:1 ratio of labeled forward primer to reverse primer produces a significant number of false amplification products indicated by arrows, and appropriate size amplicon (115 bases); arrowhead. B: The 1:15 forward to reverse primer dilution gives the best overall specific product to false amplicon ratio. Here, minimal background contaminant peaks are identified (arrows), and the peak height of the specific 115 base product (arrowhead) is still robust. C: The 1:40 primer dilution is entirely adequate for the Bat-26 locus, with essentially no false product detected, and specific amplicon is well within the linear range of the instrument. Other loci with less robust amplification cannot be not visualized well (insufficient amplification) at this level of forward primer dilution (data not shown). D: The 1:100 labeled forward primer dilution produces no detectable amplicons. The other peaks represent internal size standard as above.

Figure 3.

Determination of the limit of detection of the assay as demonstrated by detection of 10% Vaco 670 (tumor) DNA mixed into 90% L670 (normal) DNA at the Bat-25 locus. The predominant normal (germline) allele is indicated by the bold arrowhead, while the shifted (tumor) allele is indicated by the bold arrow. The Tamara labeled size standard is indicated by broken arrows, sizes as in Figure 1 .

Figure 4.

Statistical modeling of multiple independent tests to establish a diagnosis. A: Varying the number of loci analyzed determines the accuracy of MSI test results. The informativity of each of the microsatellites is kept constant at 70%, while the number of loci analyzed is varied from 3 to 7. Dotted bars, MSI-High; striped bars, MSI-Low; solid bars, MSS. The justification for a constant informativity is based on the overlapping 95% confidence intervals for the informativity of each individual locus (Bat 25: mean 0.73, CI = 0.45–0.92; Bat 26: mean 1.00, CI = 0.78–1.00; D2S123: mean 0.67, CI = 0.30–0.93; D5S346: mean 0.67, CI = 0.38–0.88; D17S250: mean 0.56, CI = 0.21–0.86). B: Varying the informativity determines the accuracy of MSI test results. The number of microsatellite loci is kept constant at five, while the informativity of each of the microsatellite markers analyzed is varied from 50 to 100%. Bar designations are as in A.