title: A proteomic signature of ovarian cancer tumor fluid identified by highthroughput and verified by targeted proteomics.
abstract: Tumor fluid samples have emerged as a rich source for the identification of ovarian cancer in the context of proteomics studies. To uncover differences among benign and malignant ovarian samples, we performed a quantitative proteomic study consisting of albumin immunodepletion, isotope labeling with acrylamide and in-depth proteomic profiling by LC-MS/MS in a pool of 10 samples of each histological type. 1135 proteins were identified, corresponding to 505 gene products. 223 proteins presented associated quantification and the comparative analysis of histological types revealed 75 differentially abundant proteins. Based on this, we developed a panel for targeted proteomic analysis using the multiple reaction monitoring (MRM) method for validation of 51 proteins in individual samples of high-grade serous ovarian tumor fluids (malignant) and benign serous cystadenoma tumor fluids. This analysis showed concordant results in terms of average amounts of proteins, and APOE, SERPINF2, SERPING1, ADAM17, CD44 and OVGP1 were statistically significant between benign and malignant group. The results observed in the MRM for APOE were confirmed by western blotting, where APOE was more abundant in malignant samples. This molecular signature can contribute to improve tumor stratification and shall be investigated in combination with current biomarkers in larger cohorts to improve ovarian cancer diagnosis.
  
  
  
  Despite advances in cancer research, ovarian cancer has a high mortality and remains a major challenge due to a number of particularities of the disease, especially late diagnosis caused by vague clinical symptoms, the cellular and molecular heterogeneity of tumors, and the lack of effective treatment. Thus, efforts are directed to better understand this neoplasia, its origin, development and, particularly the identification and validation of biomarkers for early detection of the disease in asymptomatic stage. In the present work, we confirmed by MRM method in individual ovarian tumor fluid samples the regulation of 27 proteins out of 33 identified in a highthroughput study. We speculate that the presence and/or differential abundance observed in tumor fluid is a cooperation primarily of high rates of secretion of such tumor proteins to extra tumor environment that will at the end accumulate in plasma, and also the accumulation of acute-phase proteins throughout the entire body. On top of that, consideration of physiological influences in the interpretation of expression observed, including age, menopause status, route-of-elimination kinetics and metabolism of the tumor marker, coexisting disease, hormonal imbalances, life-style influences (smoking, alcoholism, obesity), among others, are mandatory to enable the selection of good protein tumor marker candidates for extensive validation.
authors.affiliation: Department of Biochemistry and Immunology, FMRP,University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil; Department of Gynecology and Obstetrics, FMRP, University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil; Center for Cell Based Therapy, Hemotherapy Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, 14051-140, Ribeirão Preto, SP, Brazil. Electronic address: aline.poersch@gmail.com.
authors.forename: Aline
authors.initials: A
authors.lastname: Poersch
authors.affiliation: Department of Biochemistry and Immunology, FMRP,University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil; Center for Cell Based Therapy, Hemotherapy Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, 14051-140, Ribeirão Preto, SP, Brazil.
authors.forename: Mariana Lopes
authors.initials: ML
authors.lastname: Grassi
authors.affiliation: Department of Biochemistry and Immunology, FMRP,University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil; Medical School of Barão de Mauá University, Rua Ramos de Azevedo, 423, 14090-180 Ribeirão Preto, SP, Brazil.
authors.forename: Vinícius Pereira de
authors.initials: VP
authors.lastname: Carvalho
authors.affiliation: Department of Biochemistry and Immunology, FMRP,University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil.
authors.forename: Guilherme Pauperio
authors.initials: GP
authors.lastname: Lanfredi
authors.affiliation: Department of Biochemistry and Immunology, FMRP,University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil; Center for Cell Based Therapy, Hemotherapy Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, 14051-140, Ribeirão Preto, SP, Brazil.
authors.forename: Camila de Souza
authors.initials: CS
authors.lastname: Palma
authors.affiliation: Center for Cell Based Therapy, Hemotherapy Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, 14051-140, Ribeirão Preto, SP, Brazil; Department of Cell and Molecular Biology and Pathogenic Bioagents, FMRP, University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil.
authors.forename: Lewis Joel
authors.initials: LJ
authors.lastname: Greene
authors.affiliation: Department of Gynecology and Obstetrics, FMRP, University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil.
authors.forename: Christiani Bisinoto
authors.initials: CB
authors.lastname: de Sousa
authors.affiliation: Department of Gynecology and Obstetrics, FMRP, University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil.
authors.forename: Hélio Humberto Angotti
authors.initials: HHA
authors.lastname: Carrara
authors.affiliation: Department of Gynecology and Obstetrics, FMRP, University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil.
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Candido Dos Reis
authors.affiliation: Department of Biochemistry and Immunology, FMRP,University of São Paulo, Av. Bandeirantes, 3900, 14040-900 Ribeirão Preto, SP, Brazil; Center for Cell Based Therapy, Hemotherapy Center of Ribeirão Preto, Rua Tenente Catão Roxo, 2501, 14051-140, Ribeirão Preto, SP, Brazil.
authors.forename: Vitor Marcel
authors.initials: VM
authors.lastname: Faça
mesh_terms: D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000032:analysis*; D001826:Body Fluids / Q000737:chemistry; D055088:Early Detection of Cancer / Q000379:methods*; D005260:Female; D057166:High-Throughput Screening Assays; D006801:Humans; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000032:analysis*; D010051:Ovarian Neoplasms / Q000737:chemistry* / Q000175:diagnosis* / Q000473:pathology; D040901:Proteomics / Q000379:methods*; D012307:Risk Factors; D012720:Severity of Illness Index
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins
keywords: Biomarkers; Highthroughput proteomics; Multiple reactions monitoring (MRM); Ovarian cancer; Tumor fluid