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Clinical validation of a customized multiple signature microarray for breast cancer

ABSTRACT: PURPOSE: Current histopathologic systems for classifying breast tumors require evaluation of multiple variables and are often associated with significant interobserver variability. Recent studies suggest that gene expression profiles may represent a promising alternative for clinical cancer classification. Here, we investigated the use of a customized microarray as a potential tool for clinical practice. EXPERIMENTAL DESIGN: We fabricated custom 188-gene microarrays containing expression signatures for three breast cancer molecular subtypes [luminal/estrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2), and "basaloid"], the Nottingham prognostic index (NPI-ES), and low histologic grade (TuM1). The reliability of these multiple-signature arrays (MSA) was tested in a prospective cohort of 165 patients with primary breast cancer. RESULTS: The MSA-ER signature exhibited a high concordance of 90% with ER immunohistochemistry reported on diagnosis (P < 0.001). This remained unchanged at 89% (P < 0.001) when the immunohistochemistry was repeated using current laboratory standards. Expression of the HER2 signature showed a good correlation of 76% with HER2 fluorescence in situ hybridization (FISH; ratio > or =2.2; P < 0.001), which further improved to 89% when the ratio cutoff was raised to > or =5. A proportion of low-level FISH-amplified samples (ratio, 2.2-5) behaved comparably to FISH-negative samples by HER2 signature expression, HER2 quantitative reverse transcription-PCR, and HER2 immunohistochemistry. Luminal/ER+ tumors with high NPI-ES expression were associated with high NPI scores (P = 0.001), and luminal/ER+ TuM1-expressing tumors were significantly correlated with low histologic grade (P = 0.002) and improved survival outcome in an interim analysis (hazard ratio, 0.2; P = 0.019). CONCLUSION: The consistency of the MSA platform in an independent patient population suggests that custom microarrays could potentially function as an adjunct to standard immunohistochemistry and FISH in clinical practice.

ORGANISM(S): Homo sapiens

PROVIDER: GSE7422 | GEO | 2008/07/23

SECONDARY ACCESSION(S): PRJNA97855

REPOSITORIES: GEO

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Project description:Breast cancer is a heterogeneous disease comprised of at least five major subtypes. Luminal subtype tumors confer a more favourable patient prognosis, which is in part, attributed to the Estrogen Receptor-alpha (ER) positivity and anti-hormone responsiveness of these tumors. Expression of the forkhead box transcription factor, FOXA1, also correlates with the luminal subtype and patient survival, but is present in a subset of ER-negative tumors. Similarly, FOXA1 is consistently expressed in luminal breast cancer cell lines even in the absence of ER. In contrast, basal breast cancer cell lines do not express FOXA1, and loss of FOXA1 in luminal cells increases migration and invasion, characteristics of the basal subtype. To delineate an ER-independent role for FOXA1 in maintaining the luminal phenotype, and hence a more favourable prognosis, we performed cDNA microarray analyses on luminal FOXA1-positive, ER-positive (MCF7, T47D) and FOXA1-positive, ER-negative (MDA-MB-453, SKBR3) cell lines in the presence or absence of transient FOXA1 silencing. This resulted in three FOXA1 transcriptomes: (1) a luminal-signature (consistent across cell lines), (2) an ER-positive signature (restricted to MCF7 and T47D) and (3) an ER-negative signature (restricted to MDA-MB-453 and SKBR3). Use of Gene Set Enrichment Analyses (GSEA) as a phenotyping tool revealed that FOXA1 silencing resulted in a transcriptome shift from luminal to basal gene expression signatures. FOXA1 binds to both luminal and basal genes within luminal breast cancer cells, suggesting that it not only transactivates luminal genes, but also represses basal-associated genes. From these results we conclude that FOXA1 controls plasticity between basal and luminal cells, playing a dominant role in repressing the basal phenotype, and thus tumor aggressiveness, in luminal breast cancer cells. Although it has been proposed that FOXA1-targeting agents may be useful for treating luminal tumors, these data suggest that this approach may promote transitions toward a more aggressive cancer. FOXA1 siRNA treated breast cell lines compared directly to nonspecific siRNA treated cell lines using Agilent 4X44 microarrays.

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Clinical validation of a customized multiple signature microarray for breast cancer

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