Flow cytometry analysis Tissues and cells were prepared as described above for FACS

hieving complete resection. Patients with performance status 2 and distant metastases or gross mediastinal involvement were PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19713189 eligible for enrolment in one of the cisplatin-based chemotherapy trials conducted at the Montpellier University Hospital. Concurrent radiotherapy was proposed to patients with locally advanced disease. Active supportive care, including palliative radiation therapy when needed, was given to patients with advanced stage and poor performance status. Treatment was decided based on clinical and routine biological findings and without knowledge of the level of serum markers. Hence, treatment was not considered as a prognostic variable in this study. 3 / 13 HE4 in Lung Cancer Laboratory methods A blood sample was collected from each patient at diagnosis and serum was separated and stored at -180C until tested. HE4 was measured by using the commercial EIA method. This test is intended for use with serum and is a Danoprevir price solid-phase, non-competitive immunoassay based on the direct sandwich technique using two mouse monoclonal antibodies, 2H5 and 3D8, against two epitopes in the C-WFDC domain of HE4. HE4 present in calibrators or samples is adsorbed to streptavidin-coated microstrips by the biotinylated anti-HE4 antibody 2H5 during the incubation. Strips are then washed and incubated with HRP-labeled anti-HE4 mAB 3D8. After washing, a buffered substrate/chromogen reagent is added to each well. During the enzyme reaction a blue color will develop if the antigen is present. The color intensity is proportional to the amount of HE4 in the sample and is determined using a microplate spectrophotometer at 405 nm after addition of the Stop Solution. Calibration curves were constructed for each assay by plotting the absorbance value versus the concentration of each calibrator. HE4 concentrations in patients’ samples were then calculated based on the calibration curve. The HE4 EIA assay measures H4 concentrations between 15 and 900 pM. We previously validated the test in our laboratory following the COFRAC LAB GTA 04 methodology. The limit of detection was 4 pmol/L and the limit of quantification was 8 pmol/L. The intraand inter-assay coefficients PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19711918 of variation were lower than 10%. Serum neuron-specific enolase was measured by using the solid phase two-site immunoradiometric assay ELSA NSE and serum CYFRA 211 was determined with the IRMA method, as previously described. Other biological variables tested in this study were measured before any treatment and concomitantly to the titrations of tumor markers in a laboratory that implemented good laboratory practice. The upper limit of normal values was 10 000/ml for leukocytes. The lower limits of normal values were 32 g/ l for albumin and 135 mmol/L for serum sodium. Statistical analysis Receiver Operating Characteristic curves were constructed using both patients’ and controls’ serum marker levels in an attempt to establish the sensitivity–specificity relationship for each marker. The areas under the ROC curves were calculated. ROC curve analysis was carried out using the Medcalc statistical software. The sensitivity-specificity relationship was determined using the Youden’s index J, which is the difference between the true positive rate and the false positive rate. The normal distribution of serum HE4 levels was tested using the non-parametric Shapiro–Wilk test for the equality of continuous, one-dimensional variables. For HE4 in the tested populations, the test was significant, thereby rejec