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

BACKGROUND: In-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. RESULTS: Cytological 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. CONCLUSION: The 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.

Hemoptysis and coughing up of staples as a late complication of volume reduction surgery for emphysema.

Three patients with very similar clinical symptoms, i.e. hemoptysis and coughing up of staples some time after volume-reducing surgery, are described. There was no deterioration in lung function, nor in the patient's well-being, which could be ascribed to the coughing up of the staples. Thus, staples can some time after volume reducing surgery erode out into the bronchi and be coughed up, often with some small amounts of blood. This has no clinical significance other than the disturbing symptoms. Possibly, the calf pericardium used as strenghtener of the suture lines will with time cause an immunological reaction, causing destruction of the foreign material and thereby dislodging the staples.

[Lung volume reduction surgery must be performed on strict indications, Swedish experiences confirm report on negative results in some patients]. / Lungvolymreducerande kirurgi måste ske på korrekta indikationer. Svenska erfarenheter bekräftar rapport om negativ effekt hos vissa patienter.

The National Emphysema Treatment Trial showed a 30 day mortality of 16% after lung-volume-reduction surgery in high risk patients with emphysema and forced expiratory volume in one second and either carbon monoxide diffusing capacity no more than 20 per cent of their predicted value or homogenous emphysema on computed tomography. There was no mortality in medically treated patients. Seven of 90 patients treated for emphysema with lung-volume-reduction surgery at the Karolinska Hospital in Stockholm had a reduced lung function according to these criteria. Two of the high risk patients (29%) died within one month of the operation compared to two of the remaining 83 patients (2%). Lung-volume-reduction surgery is not a therapeutic option for patients with emphysema and a severely reduced lung capacity but can be performed with a low risk to alleviated symptoms and improve the functional capacity if localised areas for the surgical resection can be identified.

[A chest tube can be inserted safely utilizing standard technique. Minithoracotomy should not be performed routinely]. / Toraxdrän läggs säkert med standardteknik. Minitorakotomi bör inte vara rutin.

The National Swedish Board of Health and Welfare has concluded that a minithoracotomy should be used for the insertion of a chest tube. Our opinion is that a less than 2 cm skin incision followed by blunt dissection through the chest wall is equally safe provided that the anatomical landmarks of the safe triangle are identified and the lung is not adherent to the chest wall. A prerequisite for safe treatment is an examination of the chest x-ray, supervision of chest tube function and the patient. Chest tube malfunction or complications should be managed urgently.

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.

Detection of Chlamydia pneumoniae on cytospin preparations from bronchoalveolar lavage in COPD patients and in lung tissue from advanced emphysema.

Chronic obstructive pulmonary disease (COPD) is associated with smoking but other etiological factors contribute. Chlamydia pneumoniae is an obligate intracellular bacterium causing both acute and chronic respiratory tract infections. Studies have revealed an association between chronic C. pneumoniae infection and COPD, asthma and lung cancer but there have been difficulties detecting C. pneumoniae in the bronchial tree. Cytospin slides prepared from bronchoalveolar lavage (BAL) fluid from 14 patients with COPD, 10 healthy smokers (S) and 7 non smokers (NS) were analyzed with a fluorescein isothiocyanate labeled monoclonal antibody to C. pneumoniae. Lung tissue from 24 patients with advanced emphysema who had undergone lung volume reduction surgery (LVRS) was examined with immunohistochemistry for C. pneumoniae. Archived serum samples for detection of specific C. pneumoniae antibodies by microimmunofluorescence were available for 30 of the BAL subjects and 11 of LVRS patients. C. pneumoniae elementary body like structures were found in 29% of cytospin specimens from COPD patients, 14% of NS and 10% of HS. C. pneumoniae was detected in lung tissue in 8%. COPD patients had higher titres of IgG and IgA than NS and S. There was no association between occurrence of C. pneumoniae in BAL fluid and antibody titres. In conclusion, the assays used for detection of C. pneumoniae in lung tissue are feasible, and could be adapted in adequately powered studies to further confirm an association between C. pneumoniae infection and COPD.

Right thoracotomy for saphenous vein graft aneurysm causing hemoptysis.

Hemoptysis and septicemia 6 years after coronary artery bypass surgery in a 56-year-old man was caused by a 5-cm vein graft pseudoaneurysm bleeding into the right pleura and lung parenchyma. The graft was approached through a right thoracotomy. The patient suffered postoperative neuromyopathy, but fully recovered within 6 months without muscular, respiratory, or cardiac symptoms.