Identification of protein biomarkers for cervical cancer using human cervicovaginal fluid.

OBJECTIVES: Cervicovaginal fluid (CVF) can be considered as a potential source of biomarkers for diseases of the lower female reproductive tract. The fluid can easily be collected, thereby offering new opportunities such as the development of self tests. Our objective was to identify a CVF protein biomarker for cervical cancer or its precancerous state. METHODS: A differential proteomics study was set up using CVF samples from healthy and precancerous women. Label-free spectral counting was applied to quantify protein abundances. RESULTS: The proteome analysis revealed 16 candidate biomarkers of which alpha-actinin-4 (p = 0.001) and pyruvate kinase isozyme M1/M2 (p = 0.014) were most promising. Verification of alpha-actinin-4 by ELISA (n = 28) showed that this candidate biomarker discriminated between samples from healthy and both low-risk and high-risk HPV-infected women (p = 0.009). Additional analysis of longitudinal samples (n = 29) showed that alpha-actinin-4 levels correlated with virus persistence and clearing, with a discrimination of approximately 18 pg/ml. CONCLUSIONS: Our results show that CVF is an excellent source of protein biomarkers for detection of lower female genital tract pathologies and that alpha-actinin-4 derived from CVF is a promising candidate biomarker for the precancerous state of cervical cancer. Further studies regarding sensitivity and specificity of this biomarker will demonstrate its utility for improving current screening programs and/or its use for a cervical cancer self-diagnosis test.

Comprehensive proteomic analysis of human cervical-vaginal fluid using colposcopy samples.

BACKGROUND: Cervical-vaginal fluid (CVF) plays an important role in the prevention of gynecological infections, although little is known about the contribution of CVF proteins to the immunity of the lower female genital tract. In order to analyze the protein composition of human CVF, we used CVF samples that are routinely collected during colposcopy, but are usually discarded. Since these samples are available in large quantities we aimed to analyze their usefulness for proteomics experiments. The samples were analyzed using different prefractionation techniques (ultrafiltration and C4(RP)-LC protein separation) followed by C18(RP)-LC peptide separation and identification by MALDI-TOF-TOF mass spectrometry. To determine the reproducibility of this proteomics platform we analyzed three technical replicates. Using spectral counting, protein abundances were estimated in a semiquantitative way. We also compared the results obtained in this study with those from previous studies derived from patients with different physiological conditions in order to determine an overlapping protein set. RESULTS: In total, we were able to identify 339 proteins in human CVF of which 151 proteins were not identified in any other proteomics study on human CVF so far. Those included antimicrobial peptides, such as human beta-defensin 2 and cathelicidin, which were known to be present in CVF, and endometrial proteins such as glycodelin and ribonucleoprotein A. Comparison of our results with previously published data led to the identification of a common protein set of 136 proteins. This overlapping protein set shows increased fractions of immunological and extracellular proteins, confirming the extracellular immunological role of CVF. CONCLUSION: We demonstrated here that CVF colposcopy samples can be used in proteomics experiments and hence are applicable for biomarker discovery experiments. The delineation of an overlapping set of proteins that is identified in most proteomics studies on CVF may help in the description of a reference proteome when performing proteomics studies on human CVF.