Single Nucleotide Polymorphism Array Analysis of Flow-Sorted Epithelial Cells from Frozen Versus Fixed Tissues for Whole Genome Analysis of Allelic Loss in Breast Cancer

Abstract

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.

High-Resolution Analysis of DNA Copy Number Using Oligonucleotide Microarrays

“…This study demonstrates that the Affymetrix p501 oligonucleotide array coupled with the whole-genome sampling assay (WGSA) generates reproducible SNP-based genotyping data that can be used in a range of genomic applications (Schubert et al 2002;Dumur et al 2003). We have shown that the SNP array can also be used to detect copy number variations in cancer cell lines.…”

Expression of the FAM5C in tongue squamous cell carcinoma

“…Several studies have successfully utilized the Affymetrix 10K SNP Mapping Array to identify consistent LOH regions (1,3,(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). However, there are no previous studies identifying CNA and LOH in the 1q31.1 region which we observed in the present study.…”

“…Several studies have successfully utilized this array for identifying consistent LOH regions in breast cancer (4)(5)(6)(7)(8), bladder cancer (9,10), prostate cancer (11,12), osteosarcoma (13), lung cancer (1,14) and oral SCC-derived cell lines (3,15). However, only a limited number of studies have utilized this assay for identifying consistent LOH regions in human oral SCCs.…”

High-Resolution Mapping of Structural Mutations in Prostate Cancer With Single Nucleotide Polymorphism Arrays

“…Published data by the Sellers and Meyerson groups and by others demonstrate that SNP arrays provide high-resolution maps of LOH when one compares the pattern of heterozygosity in the constitutional germline DNA to the pattern seen in the tumor (Allinen et al, 2004;Dumur et al, 2003;Lieberfarb et al, 2003;Primdahl et al, 2002;Schubert et al, 2002;Wang et al, 2004). More recently, we have developed methods of analyzing homozygous allele frequencies and regions of linkage 4 Award Number W81XWH-05-1-0031 disequilibrium to map regions of LOH without the use of paired normal germline DNA samples (Beroukhim et.…”