Abstract P2-02-04: Circulating protein biomarker profile for inflammatory breast cancer using a multiplexed proximity extension assay
Authors: Cohen EN , Jayachandran G , Gao H , Tin S , Alvarez RH , Valero V , Lim B , Woodward WA , Ueno NT , Reuben JM 
Published: February 2018

Abstract
Background: Expression of cancer related genes and proteins in clinical specimens are the mainstay of personalized targeted therapy; however, a diagnostic signature for inflammatory breast cancer (IBC) remains elusive. In this study, we employed a blood-based, non-invasive, sensitive technology to map biomarkers in patients with IBC at the protein level. Proximity Extension Assay consists of a harmonious blending of immunoassay and PCR to amplify protein expression signal, thereby enabling multiplexing with small sample input (1 μl). Other multi-platform assays require a large amount of clinical material, multi-step sample processing and complicated data analysis.
Materials and methods: Serum samples (n= 159) from patients with primary IBC (IBC, n= 30), metastatic IBC (MIBC, n= 54), locally advanced breast cancer (LABC, n= 24) and metastatic breast cancer (MBC, n= 27) were prospectively collected from subjects prior to starting a new therapy (treatment naive) or a new line of therapy between 2009 and 2012. Sera from 24 healthy normal donors (HD) were included in the analysis for comparison. The samples were analyzed using two panels: Proseek Multiplex Oncology II and Proseek Multiplex Inflammation I (Olink Proteomics, Uppsala, Sweden) for simultaneous detection of 92 human protein biomarkers in each panel. In the assay, each protein biomarker is detected by a matched pair of antibodies coupled to unique DNA-tags. Upon binding to the proteins, the correctly hybridized DNA-tags form an amplicon that can be measured by PCR.For initial analysis, sample populations were compared using the Mann-Whitney-U test.
Results: In comparison with HD sera, sera of breast cancer patients had 41 proteins from the oncology panel and 28 from the inflammation panel that were significantly higher, whereas 5 from the inflammation panel were significantly lower. From the inflammation panel, 11 proteins (PD-L1, IL-2, IL-7, IL-18, uPA, CCL4, CCL23, CXCL9, CXCL10, CXCL11 and TNF-alpha) showed significant differential expression between IBC and non-IBC derived samples (irrespective of metastatic status); for each marker, levels were higher in IBC than in non-IBC. In contrast, 9 proteins from the oncology panel (CRNN, CTSV, ERBB4, FR-gamma, ITGAV, MIA, PODXL, SCF and SEZ6L) were differentially expressed; however, each of these proteins was higher in non-IBC than in IBC. Among the aforementioned proteins, CCL4, IL-2, IL-7, PD-L1, TNF-alpha, uPA, CRNN, CTSV, FR-gamma, ITGAV, MIA, SCF and SEZ6L did not differentiate cancer and HD, but were uniquely characteristic of the IBC vs non-IBC comparison.
Conclusion: These preliminary data suggest that it is possible to distinguish between cancer patients and healthy normal donors, and also to delineate between IBC and non-IBC patients based on expression of serum proteins. Validation of this serum protein signature is planned in a larger patient cohort.
Citation Format: Cohen EN, Jayachandran G, Gao H, Tin S, Alvarez RH, Valero V, Lim B, Woodward WA, Ueno NT, Reuben JM. Circulating protein biomarker profile for inflammatory breast cancer using a multiplexed proximity extension assay [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-04.