DNA In Pleural Fluid Research Articles

Diagnosis of Parapneumonia Pleural Effusion with Serum and Pleural Fluid Cell-Free DNA

Objective As cell-free DNA levels in the pleural fluid and serum of parapneumonic pleural effusion (PPE) patients have not been thoroughly explored, we evaluated their diagnostic potential. Methods Twenty-two PPE and 16 non-PPE patients were evaluated. Serum and pleural fluids were collected, and cell-free DNA was quantified. All biomarkers were assessed for correlation with days after admission. Receiver operating characteristic (ROC) curve analysis was used to determine diagnostic accuracy and optimal cut-off point. Results Nuclear and mitochondrial DNA levels in the pleural fluid and nuclear DNA levels in serum of PPE patients were significantly higher than in those of the non-PPE patients. However, only cell-free DNA levels in pleural fluid correlated with days after admission among PPE patients (r= 0.464, 0.538, respectively). ROC curve analysis showed that nuclear and mitochondrial DNA in pleural fluid had AUCs of 0.945 and 0.889, respectively. With cut-off values of 134.9 and 17.8 ng/ml for nuclear and mitochondrial DNA in pleural fluid, respectively, 96% sensitivity and 81% specificity were observed for PPE diagnosis. Conclusion Nuclear and mitochondrial DNA in pleural fluid possess PPE diagnostic potential and correlated with disease severity. Serum nuclear DNA could also be used to distinguish freshly admitted PPE patients (Day 1) from non-PPE patients, but with less accuracy.

Precision-guided, Personalized Intrapleural Fibrinolytic Therapy for Empyema and Complicated Parapneumonic Pleural Effusions: The Case for the Fibrinolytic Potential.

Complicated pleural effusions and empyema with loculation and failed drainage are common clinical problems. In adults, intrapleural fibrinolytic therapy is commonly used with variable results and therapy remains empiric. Despite the intrapleural use of various plasminogen activators; fibrinolysins, for about sixty years, there is no clear consensus about which agent is most effective. Emerging evidence demonstrates that intrapleural administration of plasminogen activators is subject to rapid inhibition by plasminogen activator inhibitor-1 and that processing of fibrinolysins is importantly influenced by other factors including the levels and quality of pleural fluid DNA. Current therapy for loculation that accompanies pleural infections also includes surgery, which is invasive and for which patient selection can be problematic. Most of the clinical literature published to date has used flat dosing of intrapleural fibrinolytic therapy in all subjects but little is known about how that strategy influences the processing of the administered fibrinolysin or how this influences outcomes. We developed a new test of pleural fluids ex vivo, which is called the Fibrinolytic Potential or FP, in which a dose of a fibrinolysin is added to pleural fluids ex vivo after which the fibrinolytic activity is measured and normalized to baseline levels. Testing in preclinical and clinical empyema fluids reveals a wide range of responses, indicating that individual patients will likely respond differently to flat dosing of fibrinolysins. The test remains under development but is envisioned as a guide for dosing of these agents, representing a novel candidate approach to personalization of intrapleural fibrinolytic therapy.

Pleural fluid DNA integrity index as a diagnostic marker of malignant pleural effusion

The presence of tumour cells in pleural fluid or tissue defines the effusion as malignant. Pleural fluid cytology has a sensitivity of 50–60% which is insufficient for making a clinical diagnosis, ...

Pleural fluid cell-free DNA integrity index to identify cytologically negative malignant pleural effusions including mesotheliomas

BackgroundThe diagnosis of malignant pleural effusions (MPE) is often clinically challenging, especially if the cytology is negative for malignancy. DNA integrity index has been reported to be a marker of malignancy. The aim of this study was to evaluate the utility of pleural fluid DNA integrity index in the diagnosis of MPE.MethodsWe studied 75 pleural fluid and matched serum samples from consecutive subjects. Pleural fluid and serum ALU DNA repeats [115bp, 247bp and 247bp/115bp ratio (DNA integrity index)] were assessed by real-time quantitative PCR. Pleural fluid and serum mesothelin levels were quantified using ELISA.ResultsBased on clinico-pathological evaluation, 52 subjects had MPE (including 16 mesotheliomas) and 23 had benign effusions. Pleural fluid DNA integrity index was higher in MPE compared with benign effusions (1.2 vs. 0.8; p<0.001). Cytology had a sensitivity of 55% in diagnosing MPE. If cytology and pleural fluid DNA integrity index were considered together, they exhibited 81% sensitivity and 87% specificity in distinguishing benign and malignant effusions. In cytology-negative pleural effusions (35 MPE and 28 benign effusions), elevated pleural fluid DNA integrity index had an 81% positive predictive value in detecting MPEs. In the detection of mesothelioma, at a specificity of 90%, pleural fluid DNA integrity index had similar sensitivity to pleural fluid and serum mesothelin (75% each respectively).ConclusionPleural fluid DNA integrity index is a promising diagnostic biomarker for identification of MPEs, including mesothelioma. This biomarker may be particularly useful in cases of MPE where pleural aspirate cytology is negative, and could guide the decision to undertake more invasive definitive testing. A prospective validation study is being undertaken to validate our findings and test the clinical utility of this biomarker for altering clinical practice.

Microsatellite Alterations and Cell-Free DNA Analysis: Could They Increase the Cytology Sensitivity in the Diagnosis of Malignant Pleural Effusion?

The exact diagnosis of malignant pleural effusions (PE) is difficult and often requires combined procedures, because the cytological examination of pleural fluid does not detect tumoral cells in 40% of malignant effusion cases. The aim of this study was to analyze microsatellite alterations (MA) in malignant PE and to determine their diagnostic value as an additional test to cytological examination. The increase in cell-free DNA levels was also evaluated as a signal of probable malignancy. A total of 84 patients with PE were enrolled and underwent PE and whole blood and exhaled breath condensate analyses. Free DNA was measured by spectrophotometer analyses. DNA was extracted from all samples and analyzed for MA, using the microsatellite markers at chromosomes 3p, 12p, 5q, and 17p. The microsatellite analysis of PE exhibited a higher percentage of alterations in malignant PE than in benign PE. In addition to this, cell-free DNA in PE was seen to be significantly more elevated in malignant than in benign PE. The sensitivity of the sole cytology increased considerably when patients showed at least one MA or DNA>4 ng/μL in the PE. In conclusion, it was seen that the combination of the cytological examination with microsatellite analyses and cell-free DNA in pleural fluid could increase the sensitivity of the diagnosis in patients with PE who have a suspected malignancy, obviating the need for other invasive diagnostic procedures.

Aberrant DNA methylation profile in pleural fluid for differential diagnosis of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) usually develops pleural fluid. We investigated the value of DNA methylation in the pleural fluid for differentiating MPM from lung cancer (LC). Pleural fluid was collected from 39 patients with MPM, 46 with LC, 25 with benign asbestos pleurisy (BAP) and 30 with other causes. The methylation of O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a) , ras association domain family 1A (RASSF1A), death-associated protein kinase (DAPK), and retinoic acid receptor β (RARβ) was examined using quantitative real-time PCR. DNA methylation of RASSF1A, p16(INK4a), RARβ, MGMT and DAPK was detected in 12 (30.8%), 3 (7.7%), 11 (28.2%), 0 (0.0%) and five patients (12.8%) with MPM, and in 22 (47.8%), 14 (30.4%), 24 (52.2%), 1 (2.2%) and six patients (13.0%) with LC, respectively. The mean methylation ratios of RASSF1A, p16(INK4a) and RARβ were 0.37 (range 0.0-2.84), 0.11 (0.0-2.67) and 0.44 (0.0-3.32) in MPM, and 0.87 (0.0-3.14), 1.16 (0.0-5.35) and 1.69 (0.0-6.49) in LC, respectively. The methylation ratios for the three genes were significantly higher in LC than in MPM (RASSF1A, P = 0.039; p16(INK4a), P = 0.005; and RARβ, P = 0.002). Patients with methylation in at least one gene were 3.51 (95% confidence interval, 1.09-11.34) times more likely to have LC. Hypermethylation seemed no greater with MPM than with BAP. Extended exposure to asbestos (≧30 years) was correlated with an increased methylation frequency (P = 0.020). Hypermethylation of tumor suppressor genes in pleural fluid DNA has the potential to be a valuable marker for differentiating MPM from LC.

Identification of Bacterial DNA in Noninfectious Pleural Fluid with a Highly Sensitive PCR Method

Background: Bacterial DNA due to bacterial translocation has been identified in noninfectious ascitic fluid samples. Objective: This study investigated the possible presence of bacterial DNA in the pleural fluid of patients with pleural effusions of noninfectious origin, using a highly sensitive PCR-based method. Methods: Pleural fluid samples from 175 patients (average age ± SD: 69 ± 14 years) with noninfectious pleural effusion (62 transudates, 113 exudates) were analyzed. Bacterial DNA was detected using nested PCR with amplification of a fragment of the gene r16S, with 2 amplification protocols, i.e. low sensitivity (10 and 40 cycles) and high sensitivity (40 and 40 cycles). Results: With the less sensitive amplification process, only 1 sample was positive (Haemophilus parainfluenzae in a patient with hepatic hydrothorax). With the highly sensitive nested PCR method, bacterial DNA was identified in the pleural fluid, of both transudative and exudative origin, of 75 of the 175 patients (43%). In cases of isolation of a single bacterium, the more frequent were Escherichia coli, Salmonella enterica and Streptococcus pneumoniae.Conclusions: Regardless of its origin, bacterial DNA can be identified in almost half of noninfectious pleural effusions by using a highly sensitive PCR-based method. The possible clinical significance or prognostic value of these findings deserves to be evaluated.

Aberrant promoter methylation in pleural fluid DNA for diagnosis of malignant pleural effusion

Accumulating evidence implicates epigenetic changes such as hypermethylation in carcinogenesis. We investigated whether DNA methylation of 5 tumor suppressor genes in pleural fluid samples could aid in diagnosis of malignant effusion. In samples from 47 patients with malignant pleural effusions and 34 with nonmalignant effusions, we used a methylation-specific polymerase chain reaction to detect aberrant hypermethylation of the promoters of the DNA repair gene O(6)-methylguanine-DNA methyltransferase (MGMT), p16(INK4a), ras association domain family 1A (RASSF1A), apoptosis-related genes, death-associated protein kinase (DAPK), and retinoic acid receptor beta (RARbeta). Promoter hypermethylation was associated with malignant effusion for MGMT (Odds ratio (OR) = infinity), p16(INK4a) (OR = infinity), RASSF1A (OR = 13.8; CI, 1.71-112), and RARbeta (OR = 3.17; CI, 1.10-9.11), but not for DAPK. Instead, DAPK methylation was associated with the length of smoking (p < 0.05). Patients with hypermethylation of MGMT, p16(INK4a), RASSF1A or RARbeta were 5.68 times more likely to have malignant effusions than patients without methylation (p = 0.008). Methylations per patient were more numerous for lung cancer than nonmalignant pulmonary disease (0.915 vs. 0.206, p < 0.001). Sensitivity, specificity, and positive predictive value of methylation in one or more genes for diagnosis of malignant effusion were 59.6%, 79.4%, and 80.0% respectively. In conclusion, aberrant promoter methylation of tumor suppressor genes in pleural fluid DNA could be a valuable diagnostic marker for malignant pleural effusion.

Potential diagnostic value of methylation profile in pleural fluid and serum from cancer patients with pleural effusion

The objective of this study was to investigate the diagnostic value of methylation profiles for discrimination between malignant and benign pleural effusions. A secondary objective was to examine the concordance of methylation in samples of serum and pleural fluid. The authors used methylation-specific polymerase chain reaction (MSP) analysis to examine the promoter methylation status of 4 genes in patients with pleural effusion: death-associated protein kinase (DAPK), Ras association domain family 1A (RASSF1A), retinoic acid receptor beta (RARbeta), and p16/INK4a. Pleural effusions were collected from 87 patients who had their diagnoses confirmed on cytologic and/or histologic examinations and clinical evolution. Pleural effusions were classified as malignant (n = 53 patients) or benign (n = 34 patients). Methylation was detected in serum from 45.3% of patients with malignant pleural effusions and from 0% of patients with benign pleural effusions, and it was detected in pleural fluid samples from 58.5% of patients with malignant pleural effusions and from 0% of patients with benign pleural effusions (P = .001). The sensitivity of MSP was greater than that of cytologic examination alone (39.1%; P = .001). When MSP was used together with cytologic examination, sensitivity increased to 69.8% (P = .001). Cell-free methylated DNA in pleural fluid can be detected in patients with neoplastic malignancy in a single extraction by thoracocentesis. Adequate management of the extracted pleural fluid can provide a rapid and reliable diagnosis in patients with pleural effusions who have suspected malignancy. MSP, used together with cytologic examination, may obviate the need for other invasive diagnostic tests.

Cell-Free DNA Concentration in Pleural Fluid and Serum: Quantitative Approach and Potential Prognostic Factor in Patients with Cancer and Pleural Effusions

PurposeThe presence of pleural effusions in patients with tumors is often indicative of locally advanced or metastatic disease, and detection of malignancy in effusion samples frequently leads to a disease upstaging. Our purpose was to quantify the DNA in pleural effusion and serum in patients presenting pleural effusion in order to assess the potential prognostic impact. Patients and MethodsThe DNA level was determined by amplifying hRNase P in paired samples of serum and pleural fluid in 70 consecutive patients with cancer showing pleural effusion. A group of 30 patients without cancer was included. The correlation between serum and pleural DNA was calculated. Survival curves according to serum and pleural DNA were analyzed. ResultsMedian DNA concentrations were greater in patients with neoplasia than in patients without malignancy: 105 ng/mL versus 40 ng/mL (P = 0.001) in serum samples, respectively; 93 ng/mL versus 21 ng/mL (P = 0.001) in pleural fluids, respectively. A positive correlation between serum and pleural levels was confirmed (r = 0.3; P < 0.05). Median survival time for patients with serum DNA ≤ 105 ng/mL was 11.03 months in contrast to only 3.63 months for patients with higher values (P = 0.036). Accordingly, median survival time for patients with pleural DNA ≤ 93 ng/mL was 12.3 months versus only 4.63 months in case of higher levels (P = 0.027). ConclusionThis study shows that there is a strong correlation between higher levels of free DNA in pleural fluid or serum and malignancy. Survival is worse for patients with higher DNA levels in serum and pleural fluid.

Identification of epigenetic aberrant promoter methylation in pleural fluid DNA is useful for diagnosis of malignant pleural effusion

9567 Lung cancer and malignant mesothelioma are major cause of pleural effusion, and management of lung cancer patients with pleural effusion requires effective treatment based on timely and accurate diagnosis. Thus, differentiating between malignant and nonmalignant pleural effusions is a critical clinical problem, and conventional methods have proven to be inadequate. Aberrant methylation of CpG islands, one of the major mechanisms to inactivate tumor suppressor genes in human cancers, has been found in lung cancer and other malignancies. Recent studies revealed that detection of aberrant methylation of DNA in serum or BAL might be available to diagnose malignancies. Based on these studies, we investigated to identify aberrant promoter methylation of DNA in pleural effusion and evaluate the usefulness of this approach for differential diagnosis of pleural effusion. We examined methylation status of RARβ, p16INK4a, DAPK, RASSF1A, and MGMT in 81 pleural effusion samples using methylation-specific PCR. Forty-seven patients were given a diagnosis of malignancies (median age 69 years; range 29–87; male/female 35/12). 34 patients were non-malignant pleural effusion. In patients with malignancies, DNA methylation of pleural effusion was detected in 6.4% for MGMT, 21.3% for p16INK4a, 27.7% for RASSF1A, 29.8% for DAPK, and 40.4% for RARβ. Of 47 patients with malignancies, 34 (72.3%) had methylation of at least one gene. The total number of methylation in five genes per patient was significantly higher in malignancies (1.26 genes/patient) than in non-malignant pulmonary diseases (0.56 genes/patient, p<0.001). A crude unconditional logistic regression model correlating methylation status and risk of malignancies showed that the patients with at least one methylated gene showed 3.31 times higher probability of having mlignancies compared to patients without any methylated genes (p = 0.01). These results suggested that patients with methylation have significant high risk for malignancies,and identification of aberrant promoter methylation in pleural effusion DNA could be a useful tool for differential diagnosis of pleural effusion. No significant financial relationships to disclose.

Quantitative analysis of pleural fluid cell-free DNA as a tool for the classification of pleural effusions.

Recently, much interest has been focused on the quantification of DNA in miscellaneous body fluids. In this study, the application is extended to classifying pleural effusions by measuring cell-free DNA in pleural fluid. We recruited 50 consecutive patients with pleural effusions with informed consent. Pleural fluids were centrifuged at 13000 g, with supernatants aliquoted for extraction and analysis of beta-globin DNA sequence by quantitative real-time PCR. Serum and pleural fluid biochemistries were performed to classify pleural effusions using the modified criteria of Light et al. (Ann Intern Med 1972;77:507-13). The ROC curve was plotted to determine the cutoff DNA concentration for classifying pleural fluids as transudates or exudates. Indicators of diagnostic accuracy were calculated for both pleural fluid DNA and modified criteria of Light et al., using the discharge, microbiologic, and histologic diagnoses as the reference standard. The area under the ROC curve was 0.95 [95% confidence interval (CI), 0.84-0.99]. At 509 genome-equivalents/mL, pleural fluid DNA alone correctly classified 46 of 50 pleural effusions with 91% sensitivity (95% CI, 76-98%), 88% specificity (95% CI, 64-98%), and positive and negative likelihood ratios of 7.7 (95% CI, 3.1-19.5) and 0.10 (95% CI, 0.04-0.27), respectively. With the modified criteria of Light et al., 43 of 50 pleural effusions were correctly classified with 97% sensitivity (95% CI, 91-100%) and 67% specificity (95% CI, 45-89%). There were significant correlations between cell-free DNA and both lactate dehydrogenase and total protein in pleural fluid, suggesting their common origin. Pleural fluid DNA concentrations are markedly increased in exudative effusions, making it a potential new tool to evaluate the etiologic causes of pleural effusions.

Evaluation of the Polymerase Chain Reaction Method for Detection of Streptococcus pneumoniae DNA in Pleural Fluid Samples

Streptococcus pneumoniae is the most frequent causative agent of community-acquired pneumonia (CAP); however, an etiologic diagnosis by traditional techniques can be accomplished in only a small percentage of patients with CAP. Pleural fluid is present in approximately 40% of patients with CAP; therefore, we hypothesized that detection of S pneumoniae DNA in pleural fluid by polymerase chain reaction (PCR) may help to increase the rate of diagnosis of pneumococcal pneumonia. A prospective study of cases. A university hospital in Lleida, Spain. One hundred two samples of pleural fluid (51 samples from consecutive adult patients with pneumonia and 51 samples from unselected control subjects) were tested by the nested-PCR method to detect selected pneumolysin gene of S pneumoniae, and the results were compared with those provided by alternative diagnostic methods. PCR in pleural fluid had a diagnostic sensitivity of 78% in patients with pneumococcal pneumonia, with positive results in 2 of 2 patients (100%) and 5 of 7 patients (71%) who had positive or negative pleural fluid culture findings, respectively. PCR results were also positive in 3 of 24 patients (12%) with pneumonia of unknown etiology and negative in all patients with pneumonia due to microorganisms other than S pneumoniae. Thus, the calculated specificity was 93%. Among control subjects, PCR gave positive results in two cases (4%). The nested-PCR test, applied to pleural fluid samples from patients with CAP, showed a sensitivity of 78% and a specificity of 93% in the diagnosis of pneumococcal pneumonia.

Identification of mycobacterium tuberculosis in pleural effusion by polymerase chain reaction(PCR).

Background: By amplifying small amount of DNA, polymerase chain reaction (PCR) can be used for the detection of very small amount of microbial agent, and may be especially useful in certain cases which are difficult to be diagnosed microbiologically or serologically. Tuberculous pleurisy is a disease that can be diagnosed in only 70% of cases by conventional diagnostic tools, and PCR would be a very rapid, easy, and sensitive diagnostic method. Method: The specificity and sensitivity of PCR to detect Mycobacterium tuberculosis DNA were evaluated using various strains of Mycobacteria. To evaluate the diagnostic usefulness of PCR in tuberculous pleurisy, we used PCR to detect Mycobacterium tuberculosis DNA in pleural fluid. The amplification target was 123 base pair DNA, a part of IS6110 fragment, 10~16 copies of which are known to exist per genome. The diagnostic yield of PCR was compared with conventional methods, including pleural fluid adenosine deaminase (ADA) activity. Also, the significance of PCR in undiagnosed pleural effusion was evaluated prospectively with antituberculosis treatment. Results: 1) Using cultured Mycobacterium tuberculosis and other strains, PCR could detect upto 1 fg DNA and specific for only Mycobacterium tuberculosis and Mycobacterium bovis. 2) Using pleural effusions of proven tuberculosis cases, the sensitivity of PCR was 80.0% (16/20), and the specificity 95.0% (19/20). 3) Among 13 undiagnosed, but suspected tuberculous effusion, the positive rate was 60% in 10 improved cases after antituberculosis medications, and 0% in 3 cases of proven malignancy later. 4) Adenosine deaminase level of proven and clinically diagnosed tuberculous pleurisy patients was significantly higher than that of excluded patients, and correlated well with PCR results. Conclusion: We can conclude that PCR detection of Mycobacterium tuberculosis in pleural effusion has acceptable sensitivity and specificity, and could be an additional diagnostic tool for the diagnosis of tuberculous pleurisy.