Computational expression deconvolution in a complex mammalian organ

BACKGROUND: Microarray expression profiling has been widely used to identify differentially expressed genes in complex cellular systems. However, while such methods can be used to directly infer intracellular regulation within homogeneous cell populations, interpretation of in vivo gene expression data derived from complex organs composed of multiple cell types is more problematic. Specifically, observed changes in gene expression may be due either to changes in gene regulation within a given cell type or to changes in the relative abundance of expressing cell types. Consequently, bona fide changes in intrinsic gene regulation may be either mimicked or masked by changes in the relative proportion of different cell types. To date, few analytical approaches have addressed this problem. RESULTS: We have chosen to apply a computational method for deconvoluting gene expression profiles derived from intact tissues by using reference expression data for purified populations of the constituent cell types of the mammary gland. These data were used to estimate changes in the relative proportions of different cell types during murine mammary gland development and Ras-induced mammary tumorigenesis. These computational estimates of changing compartment sizes were then used to enrich lists of differentially expressed genes for transcripts that change as a function of intrinsic intracellular regulation rather than shifts in the relative abundance of expressing cell types. Using this approach, we have demonstrated that adjusting mammary gene expression profiles for changes in three principal compartments – epithelium, white adipose tissue, and brown adipose tissue – is sufficient both to reduce false-positive changes in gene expression due solely to changes in compartment sizes and to reduce false-negative changes by unmasking genuine alterations in gene expression that were otherwise obscured by changes in compartment sizes. CONCLUSION: By adjusting gene expression values for changes in the sizes of cell type-specific compartments, this computational deconvolution method has the potential to increase both the sensitivity and specificity of differential gene expression experiments performed on complex tissues. Given the necessity for understanding complex biological processes such as development and carcinogenesis within the context of intact tissues, this approach offers substantial utility and should be broadly applicable to identifying gene expression changes in tissues composed of multiple cell types

Genomic analysis of early murine mammary gland development using novel probe-level algorithms

We describe a novel algorithm (ChipStat) for detecting gene-expression changes utilizing probe-level comparisons of replicate Affymetrix oligonucleotide microarray data. A combined detection approach is shown to yield greater sensitivity than a number of widely used methodologies including SAM, dChip and logit-T. Using this approach, we identify alterations in functional pathways during murine neonatal-pubertal mammary development that include the coordinate upregulation of major urinary proteins and the downregulation of loci exhibiting reciprocal imprinting

Characterization of adjacent breast tumors using oligonucleotide microarrays

BACKGROUND: Current methodology often cannot distinguish second primary breast cancers from multifocal disease, a potentially important distinction for clinical management. In the present study we evaluated the use of oligonucleotide-based microarray analysis in determining the clonality of tumors by comparing gene expression profiles. METHOD: Total RNA was extracted from two tumors with no apparent physical connection that were located in the right breast of an 87-year-old woman diagnosed with invasive ductal carcinoma (IDC). The RNA was hybridized to the Affymetrix Human Genome U95A Gene Chip(®) (12,500 known human genes) and analyzed using the Gene Chip Analysis Suite(®) 3.3 (Affymetrix, Inc, Santa Clara, CA, USA) and JMPIN(®) 3.2.6 (SAS Institute, Inc, Cary, NC, USA). Gene expression profiles of tumors from five additional patients were compared in order to evaluate the heterogeneity in gene expression between tumors with similar clinical characteristics. RESULTS: The adjacent breast tumors had a pairwise correlation coefficient of 0.987, and were essentially indistinguishable by microarray analysis. Analysis of gene expression profiles from different individuals, however, generated a pairwise correlation coefficient of 0.710. CONCLUSION: Transcriptional profiling may be a useful diagnostic tool for determining tumor clonality and heterogeneity, and may ultimately impact on therapeutic decision making