title: Clinical validation of a customized multiple signature microarray for breast cancer.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of General Surgery, Singapore General Hospital, Singapore.
authors.forename: Benita K T
authors.initials: BK
authors.lastname: Tan
authors.forename: Lay Keng
authors.initials: LK
authors.lastname: Tan
authors.forename: Kun
authors.initials: K
authors.lastname: Yu
authors.forename: Puay Hoon
authors.initials: PH
authors.lastname: Tan
authors.forename: Ming
authors.initials: M
authors.lastname: Lee
authors.forename: Lang Hiong
authors.initials: LH
authors.lastname: Sii
authors.forename: Chow Yin
authors.initials: CY
authors.lastname: Wong
authors.forename: Gay Hui
authors.initials: GH
authors.lastname: Ho
authors.forename: Allen W Y
authors.initials: AW
authors.lastname: Yeo
authors.forename: Pierce K H
authors.initials: PK
authors.lastname: Chow
authors.forename: Heng Nung
authors.initials: HN
authors.lastname: Koong
authors.forename: Wei Sean
authors.initials: WS
authors.lastname: Yong
authors.forename: Dennis T H
authors.initials: DT
authors.lastname: Lim
authors.forename: London L P J
authors.initials: LL
authors.lastname: Ooi
authors.forename: Khee Chee
authors.initials: KC
authors.lastname: Soo
authors.forename: Patrick
authors.initials: P
authors.lastname: Tan
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality; D018450:Disease Progression; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human