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Background: Previously we found that SPAG5 gene was amplified/gained in 20-35% of HER2+BC. Herein, we investigated the prognostic and predictive significance of SPAG5 (mRNA, protein) expression in 1726 HER+BC patients with median follow-up >5 years. Methods: Analysis of SPAG5 mRNA (cDNA array expression) and protein (immunohistochemistry) and their association with distant relapse risk (DRR) were determined in 446 and 642 cases of HER+2 early stage BC in which 36% and 40% of them had received adjuvant Herceptin (H) + Anthracycline based (AC) + Taxane (T); respectively. In 33% and 31% of SPAG5 mRNA cohort and 21% and 38% of SPAG5 protein cohort had received adjuvant (Adj) AC+T or chemotherapy (CT) naïve; respectively. The association between SPAG5 expression (mRNA, protein) and both pCR and DRR after receiving neoadjuvant chemotherapy (Neo-Adj-CT) were evaluated in 476 and 162 patients with HER2+ locally advanced BC; respectively. Neo-Adj AC+T+HER2 targeting (Herceptin, Herceptin+ Lapatinib or Lapatinib) and AC+/-T has been prescribed to 51% and 49% of mRNA cohort whereas the 45% and 55% of the protein expression cohort has received Neo-Adj AC+T+H and AC+/-T; respectively. Findings: In patients with SPAG5 mRNA overexpression (+;> median), those who had received AC+/-T Neo-Adj-CT alone achieved similar pCR to those who had received AC+T+HER2 targeting Neo-Adj (38% vs., 37%; OR (95% CI): 1.0 (0.6–1.6), p=0.923) in either ER- (46% vs., 52%; OR (95% CI): 1.3 (0.5–3.0), p=0.58) or ER+ subgroups (25% vs., 26%; OR (95% CI): 1.1 (0.4–3.4), p=0.88). Whereas in patients with low SPAG5 mRNA (-), those who had received AC+T+HER2 targeting Neo-Adj had achieved 2.5 fold increased in pCR compared to those who received AC+/-T alone (47% vs., 26%; OR (95% CI): 2.5 (1.4–4.4), p=0.001) in either ER- (60% vs., 31%; OR (95% CI): 3.4 (1.7–6.7), p Conclusion: SPAG5 expression could help in selecting patients who would benefit from both HER2-targeting agents and or AC-CT. Therefore patients with SPAG5- expression could avoid unnecessary AC-CT whereas those with SPAG5+ could receive a shorter Herceptin course. Citation Format: Abdel-Fatah TM, Ball GR, Ellis IO, Chan A, Chan SY. Sperm associated antigen 5 (SPAG5) predicts pathological complete response (pCR) and distant relapse risk to HER2 targeting agents and anthracycline based chemotherapy in HER2 positive (HER2+) breast cancer (BC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-11-08.

Introduction: SPAG5 has been found to be involved in the functional and dynamic regulation of mitotic spindles, and to be essential for chromosome segregation fidelity. Recently we found by using neural network and pathways analysis of a gene expression array data that SPAG5 was among top 10 ranked genes out of 48,000 of transcripts, that accurately predicted worse clinical outcome based on a 10-fold external cross-validation analysis with an average classification accuracy of >99.999%. Moreover we found that 5% of BC showed amplification of SPAG5 locus at chromosome 17q11.2 and SPAG5 mRNA expression levels displayed a statistically significant correlation with its copy number. Methods: In the current study the molecular and clinicopathological features of SPAG5 expression and its effect on management of BC have been investigated in 2800 BC patients with primary operable invasive BCs constituted four cohorts: 1) A series of 1650 BC patients received adjuvant endocrine and/or CMF chemotherapy according to NPI.2) A series of 256 BC received adjuvant anthracycline-based chemotherapy (ATC-CT)3) A series of 140 primary BC HER2+ patients treated with ATC-CT+ Herceptin 4) To validate SPAG5 as a predictor factor for ATC-CT, 260 patients with locally advanced primary breast cancer treated with neoadjuvant ATC-CT were included and the pathological complete response (pCR) was used to evaluate the response to chemotherapy. Immunohistochemical staining was performed using Anti–SPAG5 rabbit polyclonal (HPA022479; Sigma). Results: i) By using dual immunoflurescent in BC cell lines, co-expression of SPAG5 and proliferating cell nuclear antigen (PCNA) was detected in 4 out of 5 of the breast cancer cell lines screened (MCF7, T47D, MDA468 and MDA231) providing evidence for the importance of SPAG5 in cell proliferation. ii) 20% of breast cancer showed SPAG5 protein overexpression. SPAG5 overexpression showed a statistically significant association with ER−, PR−, triple negative phenotype, high grade tumour, high ki67, basal like phenotype and epithelial mesenchymal transition phenotype, p53 mutation and absence of DNA repair genes (BRCA1, ATM and XRCC1); p values Conclusion: SPAG5 is an important novel gene implicated in the survival of BC cells and its protein expression is an independent predictor for ATC- CT. SPAG5 may provide new avenues for the discovery of new predictive marker to guide therapeutic intervention. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-07-18.

Breast cancer is a clinically heterogeneous disease, making it imperative to use biomarkers, such as the estrogen receptor (ER) and human epidermal growth factor 2 (HER2), to stratify patients into specific prognostic groups. Biomarker stratification provides patients with more accurate diagnoses, predictions of clinical outcomes, and offers targeted therapeutic options. SPAG5 (Sperm Associated Antigen 5) is involved in the maintenance of spindle-pole integrity, efficient chromosomal alignment, and cell proliferation. Its role in cell division may be a cancer susceptibility factor. Low expression of SPAG5 has been correlated with good prognosis and absence of metastases in ER+ breast cancer. However, its relationship to other molecular subtypes of breast cancer and its functional role in the disease are relatively unknown. The purpose of this study was to investigate the role of SPAG5 in breast cancer through (i) microarray analysis and (ii) correlating its expression with invasiveness, a factor involved in disease progression. In part (i), SPAG5 expression in breast cancers was evaluated by using five Affymetrix, three Agilent and one Illumina microarray datasets. The associations between SPAG5 expression and various tumor pathologies were analyzed via log2 expression ratios. Pathologies that were investigated included the ER+ subtype, HER2 overexpression, lymph node metastasis, and breast cancer stem cell-like/undifferentiated tumors. In part (ii), SPAG5 expression across five breast cancer epithelial cell lines of varying invasive ability (MDA-MB-231, MDA-MB-157, BT549, MCF-7, and CAMA1) was investigated. The cells were cultured at normal conditions and lysed using the CytoBusterTM protein extraction reagent. The supernatants were collected by microcentrifugation at 16,000 rcf at 4°C for 5 minutes. Western blotting was completed using standard techniques. From the microarray analysis, overexpression of SPAG5 showed strong associations with HER2 overexpression, ER-negative and triple-negative cancers, high grade tumors, metastasis, and poor clinical outcomes in a total of 1595 breast cancers. Via immunoblotting, it was shown that higher levels of SPAG5 accompanied highly invasive cell lines (MDA-MB-231, MDA-MB-157, and BT549) and lower levels occurred in weakly invasive cells (MCF-7 and CAMA1). These results suggest that SPAG5 is a biomarker of malignancy and invasiveness and may be a novel therapeutic target for the prevention of metastasis. Stratification into specific prognostic groups using SPAG5 as a biomarker could lead to early detection and intervention of breast cancers likely to metastasize. It is possible that treatments targeting the modulation of SPAG5 expression levels may also aid in improving clinical outcomes. Ongoing functional analysis of SPAG5 will elucidate its role in breast cancer and metastasis and aid in further characterization of SPAG5. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3303.