A proteomic signature of ovarian cancer tumor fluid identified by highthroughput and verified by targeted proteomics
Graphical abstract
Introduction
Ovarian cancer (OC) is the most fatal gynecologic malignancy with 140,000 deaths per year, the sixth most common cancer worldwide among women, and is the leading cause of mortality among gynecological malignancies in developed countries [1], [2]. Due to vague clinical symptoms, it is difficult to detect ovarian cancer at an early stage, in which the 5-year survival rate is approximately 92%. Therefore, most patients will be diagnosed with advanced stage disease with wide peritoneal metastasis (approximately 75% stages III or IV), in which the 5-year survival rate is only 30% [3], [4], [5], [6].
Moreover, OC is a heterogeneous disease at the cellular and molecular levels, and comprises a variety of tumors with diverse histopathological features and biological behavior [7], [8]. Since ovarian cancer cells with various histological types may express tumor markers differently, it is important to use multiple tumor markers to detect all ovarian cancers [9]. On the other hand, and because of such heterogeneity, the study of each histologic type separately, may present the most significant advance in understanding the disease [10]. Tumor markers, including a diversity of substances like cell surface antigens, cytoplasmic proteins, enzymes, hormones, oncofetal antigens, receptors, oncogenes and their products, are assuming a growing role in different aspects of cancer care, whether for diagnosis, screening, prediction or treatment monitoring [11], [12].
The discovery of such tumor markers depends on suitable biological samples and an appropriate methodology. Different types of biological samples can be used in cancer research, each one having advantages and disadvantages. Alternatively, our group and others have also explored the use of intra-tumor fluids, which provides information about the protein expressed by the tissue, enabling the detection of low abundance secreted proteins (compared with human plasma) [10], [13], [14], [15]. Therefore, the use of OC intra-tumor fluids as a sample for tumor marker studies allows the detection of panels of potential targets that will fall into the circulation and could truly represent candidate biomarkers that can be validated in other types of samples, such human plasma.
The increasing interest in applying proteomics methods to study OC have been contributing to assist the understanding the pathogenesis of cancer, elucidating the mechanism of drug resistance and in the development of biomarkers for ovarian cancer detection [10], [14], [16], [17], [18], [19], [20]. Analysis of protein sets, also denoted as molecular signatures, is a promising approach to adequately grasp biological complexity and to efficiently contribute for the diagnose complex diseases, monitor physiological changes and successfully develop new drugs [21]. To contribute to the elucidation of new sets of OC tumor markers, we combined highthroughput and targeted proteomic analysis to discover and verify panels of differentially abundant proteins in ovarian tumor fluid samples (Fig. 1).
Section snippets
Patients and tumor fluid samples
Tumor fluid samples were obtained during the surgery and stored at − 80 °C until analysis, from 10 women with high grade serous ovarian tumor (mean age 51.2, range 36–72), not treated previously with antineoplastic drugs or radiotherapy, and 10 women with benign serous cystadenoma (mean age 49.7, range 37–71). Patients' characteristics are described in Table 1. The patients were recruited at the University Hospital (Ribeirão Preto, São Paulo, Brazil) from the Ambulatory of the Gynecology and
Patient groups
In our study, samples used for the highthroughput and targeted proteomics analysis were very carefully selected and characterized. As aforementioned, histopathological and molecular features are extremely important for the rigorous selection of tumor marker candidates. Thus we evaluated some clinical parameters in our sample such as age, menarche age, menopause status, menopause premature, number of children born alive and number of abortions, and found no statistically significant difference
Conclusion
Tumor ovarian fluid proved to be a challenging sample to study by proteomics due to its similarity to the plasma. However, we believe that the fluid can provide novel information regarding tumor biology. We have succeeded in using the isotopic labeling by alkylation with acrylamide, enabling the identification and quantitation of a significant number of proteins. In addition, using a MRM method, most of the results were confirmed, revealing a wide variability in proteins levels between
Conflict of interest
The authors declare no conflict of interests.
Transparency document
Acknowledgements
The authors gratefully acknowledge the cooperation of all volunteers who participated in this study. This research was supported by FAPESP (Fundação de Amparo à Pesquisa do Estado de são Paulo) (Young Scientist Grant – Proc. No. 2011-0947-1), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico - grants 479934/2011-8, 454703/2014-7), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FAEPA (Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das
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