Lotta Orre

A novel method for sample preparation of fresh lung cancer tissue for proteomics analysis by tumor cell enrichment and removal of blood contaminants

BackgroundIn-depth proteomics analyses of tumors are frequently biased by the presence of blood components and stromal contamination, which leads to large experimental variation and decreases the proteome coverage. We have established a reproducible method to prepare freshly collected lung tumors for proteomics analysis, aiming at tumor cell enrichment and reduction of plasma protein contamination. We obtained enriched tumor-cell suspensions (ETS) from six lung cancer cases (two adenocarcinomas, two squamous-cell carcinomas, two large-cell carcinomas) and from two normal lung samples. The cell content of resulting ETS was evaluated with immunocytological stainings and compared with the histologic pattern of the original specimens. By means of a quantitative mass spectrometry-based method we evaluated the reproducibility of the sample preparation protocol and we assessed the proteome coverage by comparing lysates from ETS samples with the direct lysate of corresponding fresh-frozen samples.ResultsCytological analyses on cytospin specimens showed that the percentage of tumoral cells in the ETS samples ranged from 20% to 70%. In the normal lung samples the percentage of epithelial cells was less then 10%. The reproducibility of the sample preparation protocol was very good, with coefficient of variation at the peptide level and at the protein level of 13% and 7%, respectively. Proteomics analysis led to the identification of a significantly higher number of proteins in the ETS samples than in the FF samples (244 vs 109, respectively). Albumin and hemoglobin were among the top 5 most abundant proteins identified in the FF samples, showing a high contamination with blood and plasma proteins, whereas ubiquitin and the mitochondrial ATP synthase 5A1 where among the top 5 most abundant proteins in the ETS samples.ConclusionThe method is feasible and reproducible. We could obtain a fair enrichment of cells but the major benefit of the method was an effective removal of contaminants from red blood cells and plasma proteins resulting in larger proteome coverage compared to the direct lysis of frozen samples. This sample preparation method may be successfully implemented for the discovery of lung cancer biomarkers on tissue samples using mass spectrometry-based proteomics.

Chronic empyema: importance of preventing complications in the management of pleural effusions

We report a case of chronic empyema in a 63-year-old man with a history of asbestos exposure and alcohol overconsumption. In 2009, he presented with dyspnoea, exudative pleurisy on the right side with no symptoms of infection or malignancy. In 2013, the patient presented with increased dyspnoea and a massive chronic empyema had evolved. Culture of the pleural fluid was positive for Escherichia coli and anaerobic bacteria, and he was treated with antibiotics, chest drainage as well as surgical evacuation. After surgery, as the lung failed to expand, growth of opportunistic bacteria and rising C reactive protein obliged long-time treatment with broad-spectrum antibiotics as well as chest drainage with daily saline flushes. The patient still suffers from fatigue, poor nutritional status and anaemia, and further treatment with chest drainage and antibiotics is planned. Advanced chronic empyema is a difficult condition with poor response to treatment, and diagnostic delay is the main cause of complications.