In this study we applied narrow-range peptide IEF to plasma or pleural effusion prior to LC/MS/MS. Two methods for narrow-range IEF were run; IPG strips and free-flow electrophoresis. Data from this ...study was compared with cell line data to evaluate the method performance in body fluids. To test the methods potential in quantitative biomarker discovery studies, plasma and pleural effusion from patients with lung adenocarcinoma (n=3) were compared with inflammatory pleuritis (n=3) using iTRAQ quantification. Using narrow-range IEF on the peptide level we were able to identify and quantify 282 proteins in plasma and 300 proteins in pleural effusion. These body fluid proteomes demonstrated high degree of overlap; however, more proteins significantly differently altered levels related to adenenocarcinoma were found in pleural effusion compared with plasma, suggesting enrichment of lung tissue-related proteins in pleural effusion. Nine proteins were chosen for initial validation with Western blot, and one protein (NPC2) was chosen for further validation using imunohistochemistry. Overall, the quantitative results from IEF/LC/MS/MS showed good correlation with the results from Western blot and imunohistochemistry, showing the potential of this methodology in quantitative biomarker discovery studies.
Abstract Background In a previously published in vitro study based on top-down proteomics we found that the calcium-binding proteins S100A6 and S100A4 were affected by exposure to ionizing radiation ...in a p53-dependent fashion. Both proteins showed post-translational modification changes, and S100A6 also showed increased expression and translocation in response to irradiation. Aim of the present study was to evaluate the expression of S100A6 and S100A4 in non-small-cell lung cancer (NSCLC). Methods S100A6 expression on archival tumor cell lysates from 39 patients with radically resected NSCLC was assessed with SELDI-TOF-MS. S100A6 identity was confirmed using a SELDI-based antibody-capture method on lysates from the A549 lung cancer cell line, cell lysates from two freshly prepared NSCLC samples, four plasma samples and one pleural effusion sample. Immunostainings for S100A6, S100A4 and p53 were performed on tissue microarrays containing 103 stage I surgically resected NSCLC cases and 14 normal lung parenchyma specimens. Results The presence of post-translationally modified S100A6 forms was confirmed with SELDI-MS on enriched tumor cell lysates, as well as in plasma and pleural effusion samples. In addition, high S100A6 peak intensity was associated with longer median survival (35 months vs. 18 months for high and low peak intensity, respectively; p = n.s.). The immunohistochemical analysis showed that 25% of tumors were S100A6 positive. S100A6 expression correlated directly with non-squamous histology ( p < 0.0001) and S100A4 expression ( p = 0.005), and inversely with p53 expression ( p = 0.01). S100A6-positive cases showed a trend of longer survival compared with S100A6-negative cases ( p = 0.07). This difference became significant when the analysis was restricted to p53-negative cases ( n = 72). In this subgroup of patients, whose tumors likely exhibit a functional p53, S100A6 was an independent prognostic factor of improved survival at multivariate analysis (HR 0.49, 95% CI 0.27–0.81, p = 0.017). Conclusions In this study we have validated on clinical material our previous findings on cell lines in terms of S100A6 expression and post-translational modifications pattern in NSCLC. Moreover, the survival results obtained in p53-negative stage I NSCLC cases support the proposed pro-apoptotic function of S100A6 and suggest the hypothesis of a cross regulation between these two proteins.
Abstract Purpose Cytokeratin 18 (CK18) can be used as a serum biomarker for carcinoma cell death, whereas caspase-cleaved (ccCK18) fragments reflect tumour apoptosis. We explored the potential ...diagnostic and prognostic role of circulating CK18 and ccCK18 in patients with non-small-cell lung cancer (NSCLC) in comparison with Cyfra 21.1, a fragment of cytokeratin 19. Methods Subject cohorts consisted of 200 healthy blood donors (HBD), 113 patients with benign lung diseases (BLD) and 179 NSCLC cases. Plasma levels of ccCK18, total CK18 and Cyfra 21.1 were determined with ELISA assays. Results Plasma levels of ccCK18 and total CK18 were higher in the NSCLC group compared to the HBD and BLD cohorts ( p < 0.0001). Using a cut-off of 104 U/L for ccCK18 and 302 U/L for total CK18 (95% specificity in the HBD group) the diagnostic accuracy of both CK18 forms to distinguish between NSCLC and BLD cases was 56%, whereas it was 94% for Cyfra 21.1. Multivariate survival analysis showed that total CK18 was a stronger prognostic factor than both ccCK18 and Cyfra 21.1 (HR 0.64 for low versus high total CK18 levels, 95% confidence interval (CI) 0.50–0.82; p = 0.0004) in the entire NSCLC cohort and in 78 patients with locally advanced or metastatic disease treated with chemoradiotherapy or first-line chemotherapy (HR 0.70 95% CI 0.52–0.94; p = 0.018). Conclusions Cyfra 21.1 is a useful diagnostic biomarker for NSCLC. Total CK18 shows a promising potential as prognostic marker in NSCLC patients, independently of the therapeutical intervention. In contrast, ccCK18 was not of prognostic value in NSCLC, suggesting that tumour necrosis is of particular importance in this disease.
Abstract Several randomized trials have recently investigated the role of maintenance treatment for patients with advanced non-small-cell lung cancer (NSCLC) with responding or stable disease after ...completion of first-line chemotherapy. Maintenance strategy has relevant implications in terms of potential toxicity, logistics and costs, and all of these aspects should be taken into account, together with the magnitude of benefit for the patient. In order to assess the strengths and limitations of available evidence, to help clinical practice, and to suggest priorities for future clinical research, the Italian Association of Thoracic Oncology (AIOT) organized an International Experts Panel Meeting on maintenance treatment of advanced NSCLC, which took place in Sperlonga (Italy) in May 2011. Based on the available evidence, panelists agreed that maintenance therapy represents a treatment option in advanced NSCLC. Maintenance should be discussed with patients not progressed after 4–6 cycles of first-line chemotherapy, who are fit (performance status 0–1) and without persistent chemotherapy-induced toxicity. Patients need to be well informed about potential advantages and disadvantages of accepting additional therapy without a “treatment-free period”. Two different strategies, switch or continuation maintenance, are supported by available evidence. At the moment, there is no direct comparison between switch maintenance and continuation maintenance. For future trials, the panel recommends the use of overall survival as the primary endpoint, with pre-defined second-line treatment. Translational research is essential to identify predictive factors, and should be performed, whenever feasible, in order to achieve treatment optimization with proper patient selection.
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