title: Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer.
abstract: BACKGROUND
  Although omic-based discovery approaches can provide powerful tools for biomarker identification, several reservations have been raised regarding the clinical applicability of gene expression studies, such as their prohibitive cost. However, the limited availability of antibodies is a key barrier to the development of a lower cost alternative, namely a discrete collection of immunohistochemistry (IHC)-based biomarkers. The aim of this study was to use a systematic approach to generate and screen affinity-purified, mono-specific antibodies targeting progression-related biomarkers, with a view towards developing a clinically applicable IHC-based prognostic biomarker panel for breast cancer.
  
  
  METHODS
  We examined both in-house and publicly available breast cancer DNA microarray datasets relating to invasion and metastasis, thus identifying a cohort of candidate progression-associated biomarkers. Of these, 18 antibodies were released for extended analysis. Validated antibodies were screened against a tissue microarray (TMA) constructed from a cohort of consecutive breast cancer cases (n = 512) to test the immunohistochemical surrogate signature.
  
  
  RESULTS
  Antibody screening revealed 3 candidate prognostic markers: the cell cycle regulator, Anillin (ANLN); the mitogen-activated protein kinase, PDZ-Binding Kinase (PBK); and the estrogen response gene, PDZ-Domain Containing 1 (PDZK1). Increased expression of ANLN and PBK was associated with poor prognosis, whilst increased expression of PDZK1 was associated with good prognosis. A 3-marker signature comprised of high PBK, high ANLN and low PDZK1 expression was associated with decreased recurrence-free survival (p < 0.001) and breast cancer-specific survival (BCSS) (p < 0.001). This novel signature was associated with high tumour grade (p < 0.001), positive nodal status (p = 0.029), ER-negativity (p = 0.006), Her2-positivity (p = 0.036) and high Ki67 status (p < 0.001). However, multivariate Cox regression demonstrated that the signature was not a significant predictor of BCSS (HR = 6.38; 95% CI = 0.79-51.26, p = 0.082).
  
  
  CONCLUSIONS
  We have developed a comprehensive biomarker pathway that extends from discovery through to validation on a TMA platform. This proof-of-concept study has resulted in the identification of a novel 3-protein prognostic panel. Additional biochemical markers, interrogated using this high-throughput platform, may further augment the prognostic accuracy of this panel to a point that may allow implementation into routine clinical practice.
authors.affiliation: UCD School of Biomolecular and Biomedical Science, UCD Conway Institute of Biomolecular and Biomedical Science, University College Dublin, Dublin 4, Ireland.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: O Leary
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Penny
authors.forename: Roisin T
authors.initials: RT
authors.lastname: Dolan
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Kelly
authors.forename: Stephen F
authors.initials: SF
authors.lastname: Madden
authors.forename: Elton
authors.initials: E
authors.lastname: Rexhepaj
authors.forename: Donal J
authors.initials: DJ
authors.lastname: Brennan
authors.forename: Amanda H
authors.initials: AH
authors.lastname: McCann
authors.forename: Fredrik
authors.initials: F
authors.lastname: Pontén
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Radoslaw
authors.initials: R
authors.lastname: Zagozdzon
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Duffy
authors.forename: Malcolm R
authors.initials: MR
authors.lastname: Kell
authors.forename: Karin
authors.initials: K
authors.lastname: Jirström
authors.forename: William M
authors.initials: WM
authors.lastname: Gallagher
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor / Q000032:analysis*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism* / Q000401:mortality / Q000473:pathology; D002352:Carrier Proteins / Q000032:analysis / Q000096:biosynthesis*; D003285:Contractile Proteins / Q000032:analysis / Q000096:biosynthesis*; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D007150:Immunohistochemistry; D053208:Kaplan-Meier Estimate; D008565:Membrane Proteins; D008875:Middle Aged; D020929:Mitogen-Activated Protein Kinase Kinases / Q000032:analysis / Q000096:biosynthesis*; D020411:Oligonucleotide Array Sequence Analysis; D011379:Prognosis; D016016:Proportional Hazards Models; D046888:Tissue Array Analysis
chemical_list: D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D002352:Carrier Proteins; D003285:Contractile Proteins; D008565:Membrane Proteins; C484659:PDZK1 protein, human; C095886:anillin; D020929:Mitogen-Activated Protein Kinase Kinases; C409022:PDZ-binding kinase
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