Abstract A21: Proteomic analysis of residual Pap test fluid: Novel source for detection of ovarian cancer biomarkers
Authors: Boylan Kristin L.M. , Geller Melissa A. , Griffin Timothy J. , Pambuccian Stefan E. , Afiuni Somi , Skubitz Amy P.N. 
Published: August 2014

Abstract
Biomarkers that can be used to screen women in the general population for ovarian cancer have yet to be identified. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. The liquid-based Pap test consists of collecting cervical cells from the external cervical os and placing them into an alcohol-based fixative. The cells are stained and examined by a pathologist to identify the presence of premalignant and malignant cells. It has been reported that ovarian cancer cells can be identified in the liquid-based Pap test fluid. We hypothesize that proteins shed by ovarian cancer cells can be detected in the Pap test fluid by mass spectrometry (MS)-based proteomic techniques. This biospecimen source is ideal for the discovery phase of biomarker development, since it is readily available at the time of a Pap test, derived from a site near the ovarian cancer, and may not contain the high abundance proteins found in blood that could mask a potential biomarker.
In this study, we developed a sample preparation and MS methodology to document the protein profile in residual Pap test fluid. The residual fluid from discarded Pap tests from women over the age of 55 with normal cytology was used to optimize the methodology for sample preparation prior to analysis by MS. The cells were removed by centrifugation and the protein concentration of the cell-free supernatant was determined. The protein composition of individual samples was visualized by separating the proteins on SDS-PAGE gels followed by silver staining. The presence of serum proteins in the residual Pap test fluid was examined by western immunoblot using an antibody against human serum albumin. In order to determine the number of peptides (and proteins) present in the Pap test fluid, samples were precipitated with acetone. The proteins were trypsinized and the resulting peptides were fractionated followed by analysis with tandem MS.
The average volume of the residual Pap tests was 1.5 ml and the average protein concentration was 160 ug/ml. By western blot, we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. The number of peptides and proteins present in residual Pap fluid was determined by searching the MS data against the Human Unipro database. Over 300 proteins were identified in samples pooled from 40 patients with normal cytology. When the proteins were classified according to cellular localization and biological function, we found over half of the proteins identified were extracellular or plasma membrane proteins. The largest category of biological function identified was for proteins involved in immunity and defense; including several mucin proteins, such as MUC16 (CA125) which are characteristic of the healthy cervico-vaginal proteome. Approximately 10% of the proteins identified were components of the blood. Using a modification of this method, we have also analyzed the proteins from individual residual Pap test samples by tandem MS, and were able to identify over 1,000 proteins.
From these preliminary studies, we conclude that residual Pap test fluid contains a sufficient amount of protein for analysis by MS. Studies to perform MS-based proteomic analysis of proteins from residual Pap test fluid of ovarian cancer patients are in progress.
Citation Format: Kristin L.M. Boylan, Melissa A. Geller, Timothy J. Griffin, Stefan E. Pambuccian, Somi Afiuni, Amy P.N. Skubitz. Proteomic analysis of residual Pap test fluid: Novel source for detection of ovarian cancer biomarkers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A21.