Peptides Generated Ex Vivo from Serum Proteins by Tumor-Specific Exopeptidases Are Not Useful Biomarkers in Ovarian Cancer John F. Timms, 1*† Rainer Cramer, 2† Stephane Camuzeaux, 1 Ali Tiss, 2 Celia Smith, 2 Brian Burford, 3 Ilia Nouretdinov, 3 Dmitry Devetyarov, 3 Aleksandra Gentry-Maharaj, 1 Jeremy Ford, 1 Zhiyuan Luo, 3 Alex Gammerman, 3 Usha Menon, 1 and Ian Jacobs 1 BACKGROUND: The serum peptidome may be a valuable source of diagnostic cancer biomarkers. Previous mass spectrometry (MS) studies have suggested that groups of related peptides discriminatory for different cancer types are generated ex vivo from abundant serum pro- teins by tumor-specific exopeptidases. We tested 2 complementary serum profiling strategies to see if sim- ilar peptides could be found that discriminate ovarian cancer from benign cases and healthy controls. METHODS: We subjected identically collected and pro- cessed serum samples from healthy volunteers and patients to automated polypeptide extraction on octadecylsilane-coated magnetic beads and separately on ZipTips before MALDI-TOF MS profiling at 2 cen- ters. The 2 platforms were compared and case control profiling data analyzed to find altered MS peak inten- sities. We tested models built from training datasets for both methods for their ability to classify a blinded test set. RESULTS: Both profiling platforms had CVs of approx- imately 15% and could be applied for high-throughput analysis of clinical samples. The 2 methods generated overlapping peptide profiles, with some differences in peak intensity in different mass regions. In cross- validation, models from training data gave diagnostic accuracies up to 87% for discriminating malignant ovarian cancer from healthy controls and up to 81% for discriminating malignant from benign samples. Diag- nostic accuracies up to 71% (malignant vs healthy) and up to 65% (malignant vs benign) were obtained when the models were validated on the blinded test set. CONCLUSIONS: For ovarian cancer, altered MALDI-TOF MS peptide profiles alone cannot be used for accurate diagnoses. © 2009 American Association for Clinical Chemistry More than 204 000 new cases of ovarian cancer occur yearly (1). The disease is associated with poor progno- sis (2), mainly due to late diagnosis attributable to lack of recognizable symptoms and lack of reliable screen- ing methods. At presentation, 70% of patients ex- hibit disease spread beyond the pelvis [stage III, Inter- national Federation of Gynecology and Obstetrics (FIGO) 4 ]. The main diagnostic tests for symptomatic patients are transvaginal sonography and serum cancer antigen 125 (CA-125). However, CA-125 is also pro- duced by other mesothelium-derived tissues and can be increased in benign conditions and other cancers. False negatives also occur because not all early-stage tumors produce CA-125 (3). Consequently, there are calls for better biomarkers for detecting and diagnosing early-stage ovarian cancer (4). The performance characteristics required of an ovarian cancer biomarker depend on the specific clin- ical use. Markers are currently used clinically along with imaging tests to differentiate benign pelvic masses from cancers before initial treatment in symptomatic women. For this indication, a marker would need to improve on or add to the performance of existing tests, which can achieve a diagnostic sensitivity of 85%–90% for detection of symptomatic ovarian cancer in combi- nation with a specificity of 85%–90% for benign dis- 1 Institute for Women’s Health, University College London, UK; 2 BioCentre and Department of Chemistry, University of Reading, UK; 3 Computer Learning Research Centre, Royal Holloway, University of London, UK. * Address correspondence to this author at: Cancer Proteomics Laboratory, Institute for Women’s Health, University College London, Cruciform Building 1.1.09, Gower St., London, WC1E 6BT, UK. Fax +44-20-7679-6334; e-mail jtimms@wibr.ucl.ac.uk. † J.F. Timms and R. Cramer contributed equally to this work. Received July 7, 2009; accepted November 10, 2009. Previously published online at DOI: 10.1373/clinchem.2009.133363 4 Nonstandard abbreviations: FIGO, International Federation of Gynecology and Obstetrics; CA-125, cancer antigen 125; MS, mass spectrometry; SELDI-TOF, surface-enhanced laser desorption/ionization time-of-flight; MS/MS, tandem MS; UCL, University College London; UCLH, University College London Hospital; UKOPS, United Kingdom Ovarian Cancer Population Study; UKCTOCS, UK Collaborative Trial of Ovarian Cancer Screening; TFA, trifluoroacetic acid; CHCA, -cyano-4-hydroxycinnamic acid; m/z, mass-to-charge; S/N, signal-to-noise ra- tio; C18, octadecylsilane; kNN, k-nearest neighbor algorithm; SVM, support vector machine; ESI, electrospray ionization; UoR, University of Reading. Clinical Chemistry 56:2 262–271 (2010) Proteomics and Protein Markers 262 Downloaded from https://academic.oup.com/clinchem/article/56/2/262/5622506 by guest on 29 March 2023