In Situ Analyses of Genome Instability in Breast Cancer

Chin K, de Solorzano CO, Knowles D, Jones A, Chou W, Rodriguez EG, Kuo WL, Ljung BM, Chew K, Myambo K, Miranda M, Krig S, Garbe J, Stampfer M, Yaswen P, Gray JW, and Lockett SJ. In situ analyses of genome instability in breast cancer. Nat Genet 36: 984–8, 2004.

he molecular mechanisms involved in normal cells progressing to invasive metastatic cancer have been well studied in cell culture. These include changes in genome stability and reactivation of telomerase enzyme activity—a critical event that protects the ends of chromosomes, allowing cancer cells to divide indefinitely. Reporting in the September issue of Nature Genetics, my colleagues at the University of California, San Francisco, and I provide new insight into how breast cancer develops by examining the way the genome loses its integrity within the cells in the tumor itself. The study traces the stage-specific evolution of genome instability that occurs during the benign-to-malignant transition in breast cancer. Central to the study was the development of computational algorithms to detect intact, individual cell nuclei from 3D images of thick tissue (20 to 40 μm) and to enumerate the copy numbers of fluorescence in situ hybridization (FISH)–labeled DNA sequences in each nucleus (Figure 1). Calculation of the copy number variation from cell to cell gave a measure of genome instability in the tissue. We found that genome instability was low in benign and hyperplastic tissues, but dramatically increased in ductal carcinoma and invasive cancers (Figure 2). The highest levels occurred at the same time that cells reactivated telomerase and attained the capacity to continue dividing. This raises a new paradigm of episodic, as opposed to progressive, genome instability in the progression to cancer.

Figure 1. A) Surface rendering of the intact cell nuclei in an image showing spatial segregation of near diploid and polyploid cells. The larger nuclei, which had > 2 copies of both a locus in the centromeric region of chromosome 1 and a locus at 20q13, are shown in gray and formed a central cluster surrounded by smaller nuclei with <= 2 copies of both loci, in green. B) Expansion of the region inscribed in panel A showing the individual nuclei with their surface opacities reduced to reveal the fluorescence in situ hybridization (FISH) signals. Cyan dots are the chromosome 1 centromere (1cen) signals, and magenta dots are 20q13 signals. Note that the two green nuclei have two copies of both 1cen and 20q13, whereas the gray nuclei have > 2 copies of both. Raw confocal slices through the nuclei are shown beside each nucleus. Reprinted with permission from Macmillan Publishers Ltd: Nature Genetics 36: 984–8, 2004, online supplemental information, http://www.nature.com/ng/journal/v36/n9/suppinfo/ng1409_S1.html, copyright 2004.

Figure 2. 2D-confocal YO-PRO-1 images and bivariate copy number histograms of chromosome 1 centromere (1cen) and 20q13.2 signals in 3D-images from breast tumors. White bars in each histogram indicate the numbers of cells, with copy numbers specified on the X- and Y-axes. The black bar in each histogram indicates the number of cells with 2 copies each of 1cen and 20q13.2. A) A 2D-confocal YO-PRO-1 image of normal ductal epithelium taken midway through a 30 μm thick tissue section. The white squares (100 μm × 100 μm) indicate regions for which 3D-confocal images were acquired for copy number analysis. B) A bivariate copy number frequency histogram of the number of copies of 1cen and 20q13.2 in the regions indicated in (A). Over 90% of nuclei show two copies of 1cen and 20q13.2. C) A 2D-confocal YO-PRO-1 image of a usual ductal hyperplasia. D) A bivariate frequency histogram of 1cen and 20q13.2 copy numbers measured for the regions indicated in (C). While most cells showed two copies of 20q13.2, 22% of cells had only one copy of 1cen. E) A 2D-confocal YO-PRO-1 image of a ductal carcinoma in situ (DCIS) showing an expanded duct filled with heterogeneous tumor cells. F) A bivariate frequency histogram of 1cen and 20q13.2 copy numbers measured for the regions indicated in panel (E) showing substantial genomic instability. Reprinted with permission from Macmillan Publishers Ltd: Nature Genetics 36: 984–8, 2004, copyright 2004.

Stephen J. Lockett, PhD
Director, NCI/SAIC Image Analysis Laboratory
NCI-Frederick, Bldg. 538/Rm. 157
Tel: 301-846-5515
Fax: 301-846-6552
slockett@ncifcrf.gov