Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer

Abstract

BackgroundAlthough 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.MethodsWe 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.ResultsAntibody 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).ConclusionsWe 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.

Knockdown of ANLN by lentivirus inhibits cell growth and migration in human breast cancer

“…ANLN is found to be expressed at high level in human breast cancer cells and associated with survival rate [12,17]. In this study, we confirmed that ANLN is highly expressed in breast cancer tissues.…”

“…The complex clinical behaviors caused by underlying molecular mechanisms impede the study of its therapy [15,16]. Most recently, a systematic antibody generation and validation via tissue microarray technology revealed 3 candidate prognostic markers for human breast cancer, ANLN, PDZ-Binding Kinase (PBK), and PDZDomain Containing 1(PDZK1) [17]. High PBK, high ANLN, and low PDZK1 expression are associated with decreased recurrence-free survival and breast cancer-specific survival.…”

Monoclonal antibodies in immunodiagnostic assays: a review of recent applications

“…Although, a considerable variability has been attributed to selection and validation of primary antibodies, quality controls and lack of proper assay optimization (O Leary et al, 2013). The public health significance of most zoonotic parasites especially those transmitted through the consumption of infective agents in tissues of infected animals such as Toxoplasma gondii and Trichinella spirali sis alarming (Chharba & Singla 2009).…”

Evaluation of Cross-Sample Contamination in Tissue Microarrays by Polymerase Chain Reaction

“…[2][3][4][5][6][7] This technology can become fundamental in exploring the human proteome considering the information acquired from human genome efforts [17][18][19] since it can provide a means to explore, in a rapid and costeffective manner, the huge number of antibodies being generated from the non-redundant protein database. In addition, both proteomic and genomic data can be simultaneously obtained from the same specimen.…”

“…In addition, both proteomic and genomic data can be simultaneously obtained from the same specimen. 19 However, to meet this challenge and even more importantly its reliability in diagnostic and research applications, it is of paramount importance to ascertain the absence of cross-sample contamination, due to the use of the same needle in consecutive coring. To our knowledge, this particular issue, to date, has not yet been thoroughly reported.…”