Potential Early Markers for Breast Cancer: A Proteomic Approach Comparing Saliva and Serum Samples in a Pilot Study

Abstract

Breast cancer is the second leading cause of death for women in the United States, and early detection could offer patients the opportunity to receive early intervention. The current methods of diagnosis rely on mammograms and have relatively high rates of false positivity, causing anxiety in patients. We sought to identify protein markers in saliva and serum for early detection of breast cancer. A rigorous analysis was performed for individual saliva and serum samples from women without breast disease, and women diagnosed with benign or malignant breast disease, using isobaric tags for relative and absolute quantitation (iTRAQ) technique, and employing a random effects model. A total of 591 and 371 proteins were identified in saliva and serum samples from the same individuals, respectively. The differentially expressed proteins were mainly involved in exocytosis, secretion, immune response, neutrophil-mediated immunity and cytokine-mediated signaling pathway. Using a network biology approach, significantly expressed proteins in both biological fluids were evaluated for protein-protein interaction networks and further analyzed for these being potential biomarkers in breast cancer diagnosis and prognosis. Our systems approach illustrates a feasible platform for investigating the responsive proteomic profile in benign and malignant breast disease using saliva and serum from the same women.

Keywords: benign; breast cancer; diagnostic; malignant; potential biomarkers; prognostic; protein–protein interaction network; proteomics; saliva; serum.

Figure 1

GO classification and enrichment analysis of significantly expressed proteins in saliva samples of B/N, M/N and M/B groups. (A) GO classification in biological processes, cellular components and molecular functions. Top 10 enriched Reactome pathways for (B) B/N, (C) M/N and (D) M/B groups.

Figure 2

GO classification and enrichment analysis of significantly expressed proteins in serum samples of B/N, M/N and M/B groups. (A) GO classification in biological processes, cellular components and molecular functions. Top 10 enriched Reactome pathways for (B) B/N, (C) M/N and (D) M/B groups.

Figure 3

PPI networks for significant proteins in saliva and serum in B/N, M/B and M/N groups. Proteins colored in red are up-regulated and green colored proteins are down-regulated in expression.

Figure 4

Protein ratios in B/N and M/N groups compared for common proteins identified in iTRAQ analysis. Box plots for significant proteins in 6–8 saliva and serum samples are displayed.

Figure 5

KM plots estimating patients’ survival based on significant proteins in B/N, M/B and M/N groups for breast cancer indicating p-value of the log-rank test and HR for each curve. The censoring samples are shown as “+” marks. Horizontal axis represents time to event.