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




Systematic antibody generation and validation via tissue microarray technology leading to identification of a novel protein prognostic panel in breast cancer - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Cancer

, 13:175

Translational oncology

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.

KeywordsPrognostic biomarkers Tissue microarray Breast cancer Antibody screening Antibody validation AbbreviationsANLNAnillin

BCABicinchoninic acid

BCSSBreast cancer-specific survival

BGABetween group analysis

AUCArea under the curve

DABDiaminobenzidine

DCISDuctal carcinoma in situ

EREstrogen receptor

FFPEFormalin-fixed paraffin-embedded

Her2Human epidermal growth factor receptor 2

His6ABPHexa-histidine albumin binding protein

HPAHuman protein atlas

HRHazard ratio

HRPHorseradish peroxidase

IDIntensity distribution

IDCInfiltrating ductal carcinoma

IHCImmunohistochemistry

p38MAPKp38 mitogen-activated protein kinase

PBKPDZ-binding kinase

PBS-TPhosphate-buffered saline with 0.1% Tween 20

PDZK1PDZ-Domain Containing 1

PRProgesterone receptor

PrESTProtein epitope signature tag

RIPARadioimmunoprecipitation assay buffer

ROCReceiver operator curve

RFSRecurrence-free survival

SDS-PAGESodium dodecyl sulfate polyacrylamide gel electrophoresis

TBS-TTris-buffered saline with 0.1% Tween 20

TMATissue microarray.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2407-13-175 contains supplementary material, which is available to authorized users.

Patrick C OLeary, Sarah A Penny, Roisin T Dolan, Catherine M Kelly, Karin Jirström and William M Gallagher contributed equally to this work.

Download fulltext PDF



Autor: Patrick C OLeary - Sarah A Penny - Roisin T Dolan - Catherine M Kelly - Stephen F Madden - Elton Rexhepaj - Donal J B

Fuente: https://link.springer.com/







Documentos relacionados