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Clinical Validation of a Customized Multiple Signature Microarray for Breast Cancer

Authors' Affiliations: ¹ Departments of General Surgery and ² Pathology, Singapore General Hospital; ³ Agenica Research, ⁴ Cellular and Molecular Research, and ⁵ Department of Surgical Oncology, National Cancer Centre; ⁶ Duke-NUS Graduate Medical School; and ⁷ Genome Institute of Singapore, Singapore, Singapore

Requests for reprints: Patrick Tan, Cellular and Molecular Research, National Cancer Centre, 11 Hospital Drive, Singapore 1169610, Singapore. Phone: 65-6-436-8345; Fax: 65-6-226-5694; E-mail: cmrtan{at}nccs.com.sg.

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 2.2; P < 0.001), which further improved to 89% when the ratio cutoff was raised to 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.