Regression of cervical intraepithelial neoplasia (CIN) 2-3 to CIN 1 or less is associated with immune response as demonstrated by immunohistochemistry in formaldehyde-fixed paraffin-embedded (FFPE) biopsies. Proteomic analysis of water-soluble proteins in supernatants of biopsy samples with LC-MS (LTQ-Orbitrap) was used to identify proteins predictive of CIN2-3 lesions regression. CIN2-3 in the biopsies and persistence (CIN2-3) or regression (≤CIN1) in follow-up cone biopsies was validated histologically by two experienced pathologists. In a learning set of 20 CIN2-3 (10 regressions and 10 persistence cases), supernatants were depleted of seven high abundance proteins prior to unidimensional LC-MS/MS protein analysis. Mean protein concentration was 0.81？mg/mL (range: 0.55–1.14). Multivariate statistical methods were used to identify proteins that were able to discriminate between regressive and persistent CIN2-3. The findings were validated in an independent test set of 20 CIN2-3 (10 regressions and 10 persistence cases). Multistep identification criteria identified 165 proteins. In the learning set, zinc finger protein 441 and phospholipase D6 independently discriminated between regressive and persistent CIN2-3 lesions and correctly classified all 20 patients. Nine regression and all persistence cases were correctly classified in the validation set. Zinc finger protein 441 and phospholipase D6 in supernatant samples detected by LTQ-Orbitrap can predict regression of CIN2-3. 1. Introduction Among cancers affecting women, cervical cancer has the second highest occurrence worldwide, with an incidence in 2008 of 529,800 cases (14.5% in developed countries and 85.5% in developing countries) and 275,000 estimated deaths [1]. Infection of cervical epithelial cells with high risk human papillomavirus (HPV) is the most important risk factor for development of cervical cancer, as first highlighted by zur Hausen [2]. Noninvasive cervical intraepithelial neoplasia (CIN) precedes the development of invasive cancer and may progress from CIN2-3 to (micro)invasive cancer in 10–25 years on average [3]. Three CIN grades (CIN1, CIN2, and CIN3) are recognized by the World Health Organization to distinguish the degrees of epithelial abnormality and are associated with increasing risks for invasive cancer development. A CIN lesion is, however, not a static event but a dynamic process that can persist and progress but also spontaneously regress [4, 5]. If left untreated, 5–30% of all CIN2-3 lesions (confirmed by a histological punch biopsy) will develop invasive cancer [6].
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