Assessment of the frequency of allelic imbalance in human tissue using a multiplex polymerase chain reaction system

Abstract

Genomic instability can generate chromosome breakage and fusion randomly throughout the genome, frequently resulting in allelic imbalance, a deviation from the normal 1:1 ratio of maternal and paternal alleles. Allelic imbalance reflects the karyotypic complexity of the cancer genome. Therefore, it is reasonable to speculate that tissues with more sites of allelic imbalance have a greater likelihood of having disruption of any of the numerous critical genes that cause a cancerous phenotype and thus may have diagnostic or prognostic significance. For this reason, it is desirable to develop a robust method to assess the frequency of allelic imbalance in any tissue. To address this need, we designed an economical and high-throughput method, based on the Applied Biosystems AmpFlSTR Identifiler multiplex polymerase chain reaction system, to evaluate allelic imbalance at 16 unlinked, microsatellite loci located throughout the genome. This method provides a quantitative comparison of the extent of allelic imbalance between samples that can be applied to a variety of frozen and archival tissues. The method does not require matched normal tissue, requires little DNA (the equivalent of approximately 150 cells) and uses commercially available reagents, instrumentation, and analysis software. Greater than 99% of tissue specimens with >or=2 unbalanced loci were cancerous.

Figure 1

Electropherograms of VIC-labeled amplicons from a matched normal and renal carcinoma sample. PCR was performed and the resulting amplicons resolved as described in Materials and Methods. Only VIC-labeled amplicons are shown. In this particular sample, the D3S1358, THO1, and D2S1338 loci are heterozygous, and D13S317 and D16S539 loci are homozygous. Fluorescence intensity is shown on the y axis, and amplicon size, in bp, is shown on the x axis. The ratios of the fluorescent intensities of each allelic pair of heterozygous loci are shown. Loci with allelic ratios of >1.60 are defined as sites of AI for matched normal (A) or tumor (B) tissue.

Figure 2

Reproducibility and effect of admixtures of matched normal and renal carcinoma DNA on allelic peak height ratios. A: Allelic peak height ratios were determined for 198 heterozygous loci in 16 normal buccal samples. The plot represents the first determination (x axis) and the second determination (y axis). The region defined by the gray shaded box represents all of the loci that were determined not to be a site of AI on both determinations. The labeled points (allelic peak height ratios for both determinations) represent the five loci that were not correctly identified on repeating the experiment. B: The specified admixtures were generated using DNA from a matched pair of normal renal tissue and renal cell carcinoma as shown in Figure 1. Data from the heterozygous D3S1358 locus are shown. The allelic ratios are 1.09 in the normal renal tissue and 2.02 in the renal carcinoma. The best-fit line was generated by linear regression and has a correlation coefficient (R²) of 0.965.

Figure 3

Frequency of AI in normal and tumor tissues. The numbers of sites of AI (ie, 0, 1, ≥2) were determined in 118 samples of normal tissue (A) and in 239 samples of tumor tissue (B). The number of specimens in each tissue set (n) is indicated below the set designation. LN, lymph node; PBL, peripheral blood lymphocytes; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; Endo, endometrial. See Materials and Methods for additional details.