Proteomic alterations in early stage cervical cancer

Laser capture microdissection (LCM) allows the capture of cell types or well-defined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by “all-or-nothing” analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive “all-or-nothing” way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction Monitoring (PRM). Ingenuity Pathway Analysis using correction for multiple testing by permutation resulted in two networks that were differentially regulated in early stage cervical cancer compared with healthy tissue. From these networks, we learned that specific tumor mechanisms become effective during the early stage of cervical cancer

Proteomic alterations in early stage cervical cancer

Laser capture microdissection (LCM) allows the capture of cell types or welldefined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by "all-ornothing" analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive "all-or-nothing" way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction

Analysis of human serum by liquid chromatography–mass spectrometry: Improved sample preparation and data analysis

Discovery of biomarkers is a fast developing field in proteomics research. Liquid chromatography coupled on line to mass spectrometry (LC–MS) has become a powerful method for the sensitive detection, quantification and identification of proteins and peptides in biological fluids like serum. However, the presence of highly abundant proteins often masks those of lower abundance and thus generally prevents their detection and identification in proteomics studies. To perform future comparative analyses of samples from a serum bank of cervical cancer patients in a longitudinal and cross-sectional manner, methodology based on the depletion of high-abundance proteins followed by tryptic digestion and LC–MS has been developed. Two sample preparation methods were tested in terms of their efficiency to deplete high-abundance serum proteins and how they affect the repeatability of the LC–MS data sets. The first method comprised depletion of human serum albumin (HSA) on a dye ligand chromatographic and immunoglobulin G (IgG) on an immobilized Protein A support followed by tryptic digestion, fractionation by cation-exchange chromatography, trapping on a C18 column and reversed-phase LC–MS. The second method included depletion of the six most abundant serum proteins based on multiple immunoaffinity chromatography followed by tryptic digestion, trapping on a C18 column and reversed-phase LC–MS. Repeatability of the overall procedures was evaluated in terms of retention time and peak area for a selected number of endogenous peptides showing that the second method, besides being less time consuming, gave more repeatable results (retention time: <0.1% RSD; peak area: <30% RSD). Application of an LC–MS component detection algorithm followed by principal component analysis (PCA) enabled discrimination of serum samples that were spiked with horse heart cytochrome C from non-spiked serum and the detection of a concentration trend, which correlated to the amount of spiked horse heart cytochrome C to a level of 5 pmol cytochrome C in 2 µl original serum.

Implementing an advanced laparoscopic procedure by monitoring with a visiting surgeon

Study Objective: To investigate the feasibility of safely implementing a total laparoscopic hysterectomy (LH) in established gynecologists' practices with on-site coaching and monitoring of the learning curve by an experienced visiting surgeon. Design: Multicenter prospective feasibility and implementation study (Canadian Task Force classification II-2). Setting: Eleven general gynecologists in 8 hospitals (1 university hospital and 7 regional hospitals) participated. Patients: Laparoscopic hysterectomy was performed in 83 patients during the learning curve, and in 83 patients after the learning curve. Interventions: During the learning curve, an experienced visiting laparoscopist was available for coaching during each LH. A competence score was marked on an Objective Structured Assessment of Technical Skills (OSATS) form. Complications were recorded intraoperatively and postoperatively for 6 weeks after surgery in all patients. Measurements and Main Results: Nine of 11 gynecologists reached the competence score of at least 28 points during the study, from January 2005 to January 2007. A major complication occurred in 3 of 83 LH procedures (4%) performed during the learning curve, and in 5 of 83 LH procedures (6%) performed after the learning curve (p = .72). Conclusion: The concept of a visiting surgeon for on-site coaching and monitoring of established gynecologists during the learning curve of an advanced laparoscopic procedure using Objectively Structured Assessment of Technical Skills is feasible. According to the observed complication rate during and after the learning curve, on-site coaching is a useful tool when implementing a new laparoscopic technique in established gynecologists' practices. Journal of Minimally Invasive Gynecology (2010) 17, 771-778 (C) 2010 AAGL. All rights reserved