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 z2.2; P < 0.001), which further improved to 89% when the ratio cutoff was raised to z5. 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.Carcinoma of the breast is a major cause of worldwide morbidity and mortality in females (1). Two important factors in clinical breast cancer classification include determining the estrogen receptor (ER) and the human epidermal growth factor receptor 2 (HER2) status of tumors because both ER and HER2 are prognostic biomarkers (2, 3) and important predictive markers of treatment response to antihormonal and trastuzumab (HER2 monoclonal antibody) therapy, respectively (4, 5). At present, routinely used histopathologic methods such as ER immunohistochemistry and HER2 fluorescence in situ hybridization (FISH) are known to be associated with a large degree of variability. For example, inaccuracies of up to 20% have been reported for HER2 testing in both standard clinical settings and clinical trials (6) due to discrepancies in preanalytic protocols (7) and variations between different observers (8) and laboratories (9, 10). Other limitations of current assays include their reliance on meas...

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