Identification of tumor antigens that elicit a humoral immune response in breast cancer patients' sera by serological proteome analysis (SERPA)
Introduction
Breast cancer is a major health problem and one of the leading causes of death among women worldwide. Its incidence is steadily rising in developing countries. In Tunisia, the incidence of breast cancer is approximately 19 new cases per 100,000 women per year [1]. Invasive carcinomas represent 70–80% of all breast cancer and among these, infiltrating ductal carcinomas (IDCA) are the most aggressive forms and have a poor prognosis [2].
Up to now, poor diagnosis of breast cancer is due in great part to a lack of specific biomarkers of this disease. Therefore, there is much interest in identifying useful pathological markers that can help not only in detection but also for typing and treatment. Currently, the search for specific cancer-related alterations largely focus upon clinically relevant biologically fluids such as serum. Indeed, it is well known that serum may contain at very low levels peptides or proteins that are aberrantly shed or secreted from necrotic or apoptotic cells in response to a disease [3]. Interestingly, tumor antigens released in the bloodstream by cancer cells may induce a humoral immune response and generate autoantibodies that could be useful for cancer screening, diagnosis, as well as, in immunotherapy [4]. Although the mechanisms by which these antigens elicit autoantibody formation in cancer remain largely unknown, the level of expression of a protein, post-translational modifications, an abnormal processing of antigenic cellular proteins resulting in the creation of new epitopes, as well as, an unusual localization of the protein in the tumor have been suggested to trigger such humoral autoimmune responses in cancer [5].
Until now, autoimmunity has been reported against a number of intracellular and surface antigens in patients with different kinds of tumors. Using proteomics approaches, autoantibodies against triosephosphate isomerase, superoxide dismutase, annexins I and II and PGP9.5 have been reported as useful markers for screening and diagnosis of human lung cancer [6]. Autoantibodies against HSP70 and peroxiredoxin showed significantly higher frequency immunoreaction in patients with hepatocellular carcinomas [7]. Using a similar approach, autoantibodies against peroxiredoxin VI have been reported as potential diagnostic markers in esophageal squamous cell carcinomas [8]. A number of autoantigens in breast cancer patient sera have also been reported using serological screening of tumor-derived cDNA expression libraries and phage display libraries [9]. Using proteomics-based methods, autoantibodies against an oncogenic protein named RS/DJ-1 have been reported as a useful diagnostic marker in breast cancer [10].
The goal of the current study was to apply a serological proteomics-based approach to the identification of novel tumor antigens that may induce a humoral immune response in sera from patients with infiltrating ductal carcinomas of the breast. Cellular proteins from MCF-7 tumor breast cell line were separated by 2-DE, transferred onto PVDF membranes and then sera from breast cancer patients and controls were screened individually by western blot analysis for antibodies that react against separated proteins. Tumor antigens eliciting a humoral immune response by sera from cancer patients were isolated and subsequently identified by mass spectrometry. Using this methodology a number of tumor antigens were found at a significantly higher frequency for eliciting IgG-based reactivity in breast cancer patients compared with control sera. Based on these findings, these biomarkers are interesting candidates that may have utility in breast cancer screening and diagnosis.
Section snippets
Serum samples
Patients and controls were selected from the same population living in the middle coast of Tunisia. Sera were obtained at the time of diagnosis prior to any therapy from 40 patients with histologically diagnosed breast cancer at the department of gynecology at Sousse Hospital after informed consent was given. Tumors classified as infiltrating ductal carcinomas were pathologically staged according to the tumor-node-metastasis classification system of UICC. Histological grade was assessed
Results
In the current study, we have used a serological proteome approach that combines two-dimensional gel electrophoresis and western blotting for the detection of autoantibodies in the sera of patients with breast cancer. Sera obtained at the time of diagnosis from 40 breast cancer patients and 42 matched healthy subjects were investigated for the presence of IgG-based immunoreactivity against MCF7 cytosolic proteins. For this purpose, proteins issued from a tumor cell line MCF-7 were separated by
Discussion
In the current study, we have used an immunoproteomic approach named SERPA to identify tumor antigens that elicit a humoral immune response in patients with breast cancer. Using this methodology, we detected twenty six immunoreactive proteins against which sera from newly diagnosed patients with infiltrating ductal carcinomas exhibited reactivity. These protein spots were targeted by mass spectrometry and details of this identification are summarized in Table 1. In terms of biological
Acknowledgements
This work was supported by le Ministère de l'Enseignement Supérieur et de la Recherche Scientifique, le Ministère de la Santé Publique de la République Tunisienne and by the Centre National de Recherche Scientifique (Strasbourg, France) and the DGRST-CNRS funding program.
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Both authors contributed equally to the study.