In this study, we performed high-resolution array comparative genomic hybridization with an array of 4153 bacterial artificial chromosome clones to assess copy number changes in 44 archival breast cancers. The tumors were flow sorted to exclude non-tumor DNA and increase our ability to detect gene copy number changes. In these tumors, losses were more frequent than gains, and gains in 1q and loss in 16q were the most frequent alterations. We compared gene copy number changes in the tumors based on histologic subtype and estrogen receptor (ER) status, i.e., ER-negative infiltrating ductal carcinoma, ER-positive infiltrating ductal carcinoma, and ER-positive infiltrating lobular carcinoma. We observed a consistent association between loss in regions of 5q and ER-negative infiltrating ductal carcinoma, as well as more frequent loss in 4p16, 8p23, 8p21, 10q25, and 17p11.2 in ER-negative infiltrating ductal carcinoma compared with ER-positive infiltrating ductal carcinoma (adjusted P values < 0.05). We also observed high-level amplifications in ER-negative infiltrating ductal carcinoma in regions of 8q24 and 17q12 encompassing the c-myc and c-erbB-2 genes and apparent homozygous deletions in 3p21, 5q33, 8p23, 8p21, 9q34, 16q24, and 19q13. ER-positive infiltrating ductal carcinoma showed a higher frequency of gain in 16p13 and loss in 16q21 than ER-negative infiltrating ductal carcinoma. Correlation analysis highlighted regions of change commonly seen together in ER-negative infiltrating ductal carcinoma. ER-positive infiltrating lobular carcinoma differed from ER-positive infiltrating ductal carcinoma in the frequency of gain in 1q and loss in 11q and showed high-level amplifications in 1q32, 8p23, 11q13, and 11q14. These results indicate that array comparative genomic hybridization can identify significant differences in the genomic alterations between subtypes of breast cancer.

Flow cytometric analyses of DNA content and proliferative fractions have been found to be important prognostic indicators in a variety of human tumors. However, variability in reported results and interlaboratory differences in single‐parameter DNA measurements have impeded the broader use of this methodology. Multiparameter DNA analysis, especially that which allows the ploidy and cell cycle measurements to be targeted specifically to tumor cells, may improve the quality and reliability of these measurements. Cytokeratin labeling simultaneously with DNA allows the identification of the malignant epithelial cells within tumor samples that have been microdissected for tumor enrichment and, thus, can increase the accuracy of tumor ploidy and cell cycle measurements. There are a limited number of reports of cytokeratin labeling of paraffin‐extracted cells, and results with standard preparation procedures can be highly variable. We have developed an improved technique for cytokeratin labeling of paraffin‐extracted cells that is based on predigestion of tissue with collagenase prior to brief pepsin digestion. This two‐step enzymatic digestion produces a superior cytokeratin vs. DNA bivariate analysis, with increased intensity and greater uniformity of cytokeratin labeling. This method should increase the accuracy of ploidy and proliferation measurements from paraffin‐embedded tissue both for retrospective studies and in clinical settings in which only fixed tissue is available. © 1996 Wiley‐Liss, Inc.

Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However, the technology to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can enrich for populations of cells present in various tumor tissues, but it is not easily automated or performed rapidly, and there are tissues in which cells of interest cannot be readily isolated based on morphologic criteria alone. Here we describe a protocol for efficient RNA and DNA isolation from flow cytometrically purified whole epithelial cells from primary tissue. The aqueous reagent, RNAlater, which preserves RNA, allows immunolabeling and purification of whole epithelial cells by flow sorting without special instrument preparation to reduce RNase activity. We used real-time PCR to determine RNA quality afterflow sorting. High-quality RNA and DNA suitable for expression and genotype analysis can be readily obtained from flow cytometrically purified populations of neoplastic cells from human tissues.

Analysis of allelic loss in archival tumor specimens is constrained by quality and quantity of tissue and by technical limitations on the number of chromosomal sites that can be efficiently evaluated in conventional analyses using polymorphic microsatellite markers. Newly developed array-based assays have the potential to yield genome-wide data from small amounts of tissue but have not been validated for use with routinely processed specimens. We used the Affymetrix HuSNP assay, composed of 1494 single nucleotide polymorphism sites, to compare allelic loss results obtained from both formalin-fixed and frozen breast tissue samples. Tumor cells were separated from normal epithelia and nonepithelial cells by dissection and bivariate cytokeratin/DNA flow sorting; normal breast cells from the same patient served as constitutive normal. Allele results from the HuSNP array averaged 96% reproducibility between duplicates and were concordant between the fixed and frozen normal samples. We also analyzed DNA from the same samples after whole-genome amplification (primer extension preamplification). Although overall signal intensities were lower, the genotype data from the primer extension preamplification material was concordant with genomic DNA data from the same samples. Results from genomic normal tissue DNA averaged informative single nucleotide polymorphism at 379 (25%) loci genome-wide. Although data points were clustered and some segments of chromosomes were not informative, our data indicated that the Affymetrix HuSNP assay could provide an efficient and valid genome-wide analysis of allelic imbalance in routinely processed and whole genome-amplified pathology specimens.

Flow cytometric analyses of DNA content and proliferative fractions have been found to be important prognostic indicators in a variety of human tumors. However, variability in reported results and interlaboratory differences in single-parameter DNA measurements have impeded the broader use of this methodology. Multiparameter DNA analysis, especially that which allows the ploidy and cell cycle measurements to be targeted specifically to tumor cells, may improve the quality and reliability of these measurements. Cytokeratin labeling simultaneously with DNA allows the identification of the malignant epithelial cells within tumor samples that have been microdissected for tumor enrichment and, thus, can increase the accuracy of tumor ploidy and cell cycle measurements. There are a limited number of reports of cytokeratin labeling of paraffin-extracted cells, and results with standard preparation procedures can be highly variable. We have developed an improved technique for cytokeratin labeling of paraffin-extracted cells that is based on predigestion of tissue with collagenase prior to brief pepsin digestion. This two-step enzymatic digestion produces a superior cytokeratin vs. DNA bivariate analysis, with increased intensity and greater uniformity of cytokeratin labeling. This method should increase the accuracy of ploidy and proliferation measurements from paraffin-embedded tissue both for retrospective studies and in clinical settings in which only fixed tissue is available.