Breast cancer presenting in women aged ≤ 35 years has a more aggressive behaviour, and\ud thus poorer prognosis, which is thought to be due to differences in the tumour biology of these tumours compared to those from older women.\ud The aim of this study was to examine the expression of 9 novel gene targets (A-kinase anchor protein-1 (AKAP1), Acidic protein rich in leucines (APRIL), CCAAT enhancer binding protein alpha (C/EBPα), Damage-specific DNA binding protein 2 (DDB2), Granulin, Nuclear Receptor Coactivator 3 (NCOA3), Retinoic acid receptor Responder 3 (RARRES3), Retinoblastoma binding protein 4 (RBBP4), and Transforming Growth Factor beta Induced (TGFβI) identified previously by a cDNA microarray in breast cancers and female controls by RT-qPCR, western blotting, and immunohistochemistry.\ud Six breast cell lines, 9 samples of organoids from reduction mammoplasty tissues, and 35 tumour tissues (20 cases >35 years, 15 cases ≤35 years in age) were analysed for the expression of the nine target genes using real time quantitative RT-PCR. Of the nine target genes investigated, five showed differences between normal and cancers ≤35, or between breast cancers ≤35 and those >35 years. NCOA3 and RARRES3 showed elevated levels of mRNA in breast cancers ≤35 years compared to those >35 years (p= 0.001 and p=0.002 respectively). Compared to the normal breast, TGFβI showed a reduced level of mRNA expression in both younger and older cases (p= 0.026 and p=0.001 respectively), while DDB2 and C-EBPα showed a reduced level of mRNA expression in younger group only (p=0.002 and p=0.001 respectively).\ud NCOA3 protein expression examination using western blotting found high levels in the\ud ER+ve cell lines MCF-7, ZR-75-1 and T47-D with a weak expression in ER−ve cell lines\ud HBL-100 and MDA-MB-468. RARRES3 protein expression was found in 4 breast cell lines\ud (HBL-100, MDA-MB-468, MCF-7, and ZR-75-1). IHC found expression of NCOA3 in\ud younger and older tumours including ER+ve and ER−ve cases.\ud This study identifies NCAO3 and RARRES3 as potential markers for breast cancers in\ud younger women, but the data need confirmation in a larger series of cases
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