Comparison of Bisulfite Pyrosequencing and Methylation-Specific qPCR for Methylation Assessment.

Different methodological approaches are available to assess DNA methylation biomarkers. In this study, we evaluated two sodium bisulfite conversion-dependent methods, namely pyrosequencing and methylation-specific qPCR (MS-qPCR), with the aim of measuring the closeness of agreement of methylation values between these two methods and its effect when setting a cut-off. Methylation of tumor suppressor gene p16/INK4A was evaluated in 80 lung cancer patients from which cytological lymph node samples were obtained. Cluster analyses were used to establish methylated and unmethylated groups for each method. Agreement and concordance between pyrosequencing and MS-qPCR was evaluated with Pearson's correlation, Bland-Altman, Cohen's kappa index and ROC curve analyses. Based on these analyses, cut-offs were derived for MS-qPCR. An acceptable correlation (Pearson's R2 = 0.738) was found between pyrosequencing (PYRmean) and MS-qPCR (NMP; normalized methylation percentage), providing similar clinical results when categorizing data as binary using cluster analysis. Compared to pyrosequencing, MS-qPCR tended to underestimate methylation for values between 0 and 15%, while for methylation >30% overestimation was observed. The estimated cut-off for MS-qPCR data based on cluster analysis, kappa-index agreement and ROC curve analysis were much lower than that derived from pyrosequencing. In conclusion, our results indicate that independently of the approach used for estimating the cut-off, the methylation percentage obtained through MS-qPCR is lower than that calculated for pyrosequencing. These differences in data and therefore in the cut-off should be examined when using methylation biomarkers in the clinical practice.

Relevance of matrix metalloproteases in non-small cell lung cancer diagnosis.

BACKGROUND: The need for novel biomarkers that could aid in non-small cell lung cancer (NSCLC) detection, together with the relevance of Matrix Metalloproteases (MMPs) -1, -2, -7, -9 and -10 in lung tumorigenesis, prompted us to assess the diagnostic usefulness of these MMPs and the Tissue Inhibitor of Metalloproteinase (TIMP) -1 in NSCLC patients. METHODS: Markers were evaluated in an initial study cohort (19 NSCLC cases and 19 healthy controls). Those that better performed were analyzed in a larger sample including patients with benign lung diseases. Serum MMPs and TIMP-1 were determined by multiplexed immunoassays. Logistic regression was employed for multivariate analysis of biomarker combinations. RESULTS: MMPs and TIMP-1 were elevated in the serum of NSCLC patients compared to healthy controls. MMP-1, -7 and -9 performed at best and were further evaluated in the sample including benign pathologies, corroborating the superiority of MMP-9 in NSCLC discrimination, also at early-stage NSCLC. The optimal diagnostic value was obtained with the model including MMP-9, gender, age and smoking history, that demonstrated an AUC of 0.787, 85.54% sensitivity and 64.89% specificity. CONCLUSION: Our results suggest that MMP-9 is a potential biomarker for NSCLC diagnosis and its combined measurement with other biomarkers could improve NSCLC detection.

Validation of the portable Air-Smart Spirometer.

BACKGROUND: The Air-Smart Spirometer is the first portable device accepted by the European Community (EC) that performs spirometric measurements by a turbine mechanism and displays the results on a smartphone or a tablet. METHODS: In this multicenter, descriptive and cross-sectional prospective study carried out in 2 hospital centers, we compare FEV1, FVC, FEV1/FVC ratio measured with the Air Smart-Spirometer device and a conventional spirometer, and analyze the ability of this new portable device to detect obstructions. Patients were included for 2 consecutive months. We calculate sensitivity, specificity, positive and negative predictive value (PPV and NPV) and likelihood ratio (LR +, LR-) as well as the Kappa Index to evaluate the concordance between the two devices for the detection of obstruction. The agreement and relation between the values of FEV1 and FVC in absolute value and the FEV1/FVC ratio measured by both devices were analyzed by calculating the intraclass correlation coefficient (ICC) and the Pearson correlation coefficient (r) respectively. RESULTS: 200 patients (100 from each center) were included with a mean age of 57 (± 14) years, 110 were men (55%). Obstruction was detected by conventional spirometry in 73 patients (40.1%). Using a FEV1/FVC ratio smaller than 0.7 to detect obstruction with the Air Smart-Spirometer, the kappa index was 0.88, sensitivity (90.4%), specificity (97.2%), PPV (95.7%), NPV (93.7%), positive likelihood ratio (32.29), and negative likelihood ratio (0.10). The ICC and r between FEV1, FVC, and FEV1 / FVC ratio measured by the Air Smart Spirometer and the conventional spirometer were all higher than 0.94. CONCLUSION: The Air-Smart Spirometer is a simple and very precise instrument for detecting obstructive airway diseases. It is easy to use, which could make it especially useful non-specialized care and in other areas.

Highly Sensitive Marker Panel for Guidance in Lung Cancer Rapid Diagnostic Units.

While evidence for lung cancer screening implementation in Europe is awaited, Rapid Diagnostic Units have been established in many hospitals to accelerate the early diagnosis of lung cancer. We seek to develop an algorithm to detect lung cancer in a symptomatic population attending such unit, based on a sensitive serum marker panel. Serum concentrations of Epidermal Growth Factor, sCD26, Calprotectin, Matrix Metalloproteinases -1, -7, -9, CEA and CYFRA 21.1 were determined in 140 patients with respiratory symptoms (lung cancer and controls with/without benign pathology). Logistic Lasso regression was performed to derive a lung cancer prediction model, and the resulting algorithm was tested in a validation set. A classification rule based on EGF, sCD26, Calprotectin and CEA was established, able to reasonably discriminate lung cancer with 97% sensitivity and 43% specificity in the training set, and 91.7% sensitivity and 45.4% specificity in the validation set. Overall, the panel identified with high sensitivity stage I non-small cell lung cancer (94.7%) and 100% small-cell lung cancers. Our study provides a sensitive 4-marker classification algorithm for lung cancer detection to aid in the management of suspicious lung cancer patients in the context of Rapid Diagnostic Units.

Serum calprotectin, CD26 and EGF to establish a panel for the diagnosis of lung cancer.

Lung cancer is the most lethal neoplasia, and an early diagnosis is the best way for improving survival. Symptomatic patients attending Pulmonary Services could be diagnosed with lung cancer earlier if high-risk individuals are promptly separated from healthy individuals and patients with benign respiratory pathologies. We searched for a convenient non-invasive serum test to define which patients should have more immediate clinical tests. Six cancer-associated molecules (HB-EGF, EGF, EGFR, sCD26, VEGF, and Calprotectin) were investigated in this study. Markers were measured in serum by specific ELISAs, in an unselected population that included 72 lung cancer patients of different histological types and 56 control subjects (healthy individuals and patients with benign pulmonary pathologies). Boosted regression and random forests analysis were conducted for the selection of the best candidate biomarkers. A remarkable discriminatory capacity was observed for EGF, sCD26, and especially for Calprotectin, these three molecules constituting a marker panel boasting a sensitivity of 83% and specificity of 87%, resulting in an associated misclassification rate of 15%. Finally, an algorithm derived by logistic regression and a nomogram allowed generating classification scores in terms of the risk of a patient of suffering lung cancer. In conclusion, we propose a non-invasive test to identify patients at high-risk for lung cancer from a non-selected population attending a Pulmonary Service. The efficacy of this three-marker panel must be tested in a larger population for lung cancer.

Evaluation of pleural effusion sCD26 and DPP-IV as diagnostic biomarkers in lung disease.

In this study, we measured ADA and DPP-IV enzymatic activity and sCD26 concentration in 150 pleural effusion (PE) samples and tested for correlations between these and other cellular and biochemical measures. We found that DPP-IV in particular might improve the specificity (but not the sensitivity) of the ADA test for diagnosis of pulmonary tuberculosis, since half of the false ADA positive results in non-tuberculous PE were also DPP-IV positive. A percentage of patients with malignant PE were sCD26 or DPP-IV positive; however, some patients with benign PE also tested positive. As a pattern associated with DPP-IV (but not the CD26 protein) was observed in PE, we searched for a finding that might increase the value of these biomarkers for diagnosis of malignancy. The observed pattern was related to the presence of leukocytes, as indicated by correlations with the cell count, and to a band of 180 kDa, detected by immunoblotting.

Levels of PEDF in pleural effusions from lung adenocarcinoma and benign disease patients.

Anti-tumor properties assigned to PEDF, beside its role as an inhibitor of angiogenesis, make it a promising candidate in the search of new biomarkers for malignancy. In this study levels of PEDF were investigated in pleural effusions from lung adenocarcinoma and benign inflammatory disease patients. The mean PEDF concentration in the malignant group was slightly superior to that in patients suffering benign diseases (4.59 µ g/mL vs 3.97 µg/mL), although the difference did not reach statistical significance (P 0.166). Pleural effusion PEDF levels were not related to gender, age, smoking habit or pleural effusion size. We also investigated the possible relationship of PEDF levels in pleural effusion regarding clinicopathological features. Correlations were found for monocytes (P 0.010) and polymorphonuclear leukocytes (P 0.023) with PEDF levels in pleural effusion of malignant origin.