High-resolution copy number analysis of paraffin-embedded archival tissue using SNP BeadArrays

Abstract

High-density SNP microarrays provide insight into the genomic events that occur in diseases like cancer through their capability to measure both LOH and genomic copy numbers. Where currently available methods are restricted to the use of fresh frozen tissue, we now describe the design and validation of copy number measurements using the Illumina BeadArray platform and the application of this technique to formalin-fixed, paraffin-embedded (FFPE) tissue. In fresh frozen tissue from a set of colorectal tumors with numerous chromosomal aberrations, our method measures copy number patterns that are comparable to values from established platforms, like Affymetrix GeneChip and BAC array-CGH. Moreover, paired comparisons of fresh frozen and FFPE tissues showed nearly identical patterns of genomic change. We conclude that this method enables the use of paraffin-embedded material for research into both LOH and numerical chromosomal abnormalities. These findings make the large pathological archives available for genomic analysis, which could be especially relevant for hereditary disease where fresh material from affected relatives is rarely available.

Figure 1.

Exploration of BeadArray signal properties. (A) Example of the distributions of the green (Cy3) and red (Cy5) fluorescent signals from a normal sample. The combined intensity (sum of both alleles of a probe) is shown in black. Note that the combined signal is not simply the addition of both signals because of the reciprocal relationship between the alleles. (B) Simulation of the distribution of single channel signal intensities derived from SNPs with zero (red), one (orange), and two (blue) copies of an allele. The frequency for each allele type was taken from the red signal of panel A. The separate distributions were modeled as Gaussian distributions with mean = copy number and SD = copy number × 0.4 + 0.15. The black line indicates the combined signal distribution plot. (C) Scatter plot of raw intensities for homozygous (red and green) and heterozygous (yellow) SNPs. (D) Histogram of raw intensities in polar coordinates. The red line indicates the most prevalent angle for homozygous Cy5-labeled SNPs; the green line, homozygous Cy3-labeled SNPs.

Figure 2.

Effects of background estimation and sample normalization. The effects on amplitude (top panel) and variability (bottom panel) of different preprocessing and normalization strategies on the same data set are shown. The symbols indicate different copy number states: ◇, blood; ◽, tumor, normal; ×, tumor, loss; +, tumor, gain. The middle panel indicates the settings. BG, background estimation method: raw, no background estimation; min, minimum intensity in sample; mode, mode of intensities in sample; angmode, mode of angle in polar coordinates near the quadrants. Qnt: Q indicates quantile normalization between channels of a sample. GT: All, use all loci for normalization; Het, use heterozygous loci to calculate normalization factor. pGCS, proportion of relative gene call score. Use only loci with GCS higher than value to calculate normalization factor.

Figure 3.

Comparison of platforms for calculation of copy numbers for tumor T106. Smoothed values are plotted along idiograms. Red, GeneChip; green, BAC array; blue, BeadArray. Below each idiogram, gray bars indicate heterozygous SNPs in the corresponding normal sample. Loci that are not heterozygous in the tumor are shown as black bars.

Figure 4.

Chromosomal plots from BeadArrays. Each panel depicts the smoothed copy number as a continuous line and the 10th and 90th percentiles as dashed lines. The unsmoothed copy number values are shown as dots. (A,B) X- and Y-chromosome from leukocyte DNA. Red, 44 male; blue, 106 female; cyan, 108 male; green, 514 female. (C–E) Comparisons of copy numbers. The green lines depict FFPE tumor tissue, and the red lines depict fresh frozen tumor tissue. Bars below the plot indicate heterozygous SNPs in the corresponding normal sample. At black bars, the SNP has switched to homozygosity in the tumor. Physical loss is called when the upper percentile line drops below 2; gain is called when the lower percentile line exceeds 2. (C) Chromosome 17 in tumor 106. (D) Chromosome 5 in tumor 44. Blue line, FFPE BAC array; cyan line, fresh frozen BAC array. (E) Chromosome 2 in tumor 514.

Figure 5.

Analysis of 22 colorectal carcinomas. Validation of the application was demonstrated on a series of 22 colorectal carcinomas. Each column in the plot shows a genome-wide overview of the numerical changes in red (gain) and blue (loss) on the left and probes with LOH on the right. The common patterns of chromosomal instability (Diep et al. 2006), such as physical loss on chromosomes 4 and 18, gain of chromosomes 13 and 20, and copy neutral LOH on chromosomes 4, 5, 10, and 17 can all be identified from this series.