We investigated whether tumour markers carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), and cytokeratin 19 fragment (CYFRA 21-1) in pleural effusions and serum can be used to distinguish pleural effusion aetiology.
During the first thoracentesis, we measured pleural fluid and serum tumour marker concentrations and calculated the pleural fluid/serum ratio for patients diagnosed with pleural effusion, using electrochemiluminescence immunoassays. Receiver operating characteristic (ROC) analysis was carried out and the Hanley and McNeil method was used to test the significance of the difference between the areas under ROC curves (AUCs). In order to detect which tumour marker best discriminates between malignant and non-malignant pleural effusions and to establish the predictive value of those markers, discriminant function analysis (DFA) and logistic regression analysis were utilized.
Serum tumour markers CYFRA 21-1 and NSE as well as pleural NSE were good predictors of pleural effusion malignancy and their combined model was found statistically significant (Chi-square = 28.415, P < 0.001). Respective ROC analysis showed significant discrimination value of the combination of these three markers (AUC = 0.79).
Serum markers showed superiority to pleural fluid markers in determining pleural fluid aetiology. Serum CYFRA 21-1 and NSE concentrations as well as pleural fluid NSE values had the highest clinical value in differentiating between malignant and non-malignant pleural effusions. The combination of these three markers produced a significant model to resolve pleural effusion aetiology.
Pleural effusion is the accumulation of liquid in the pleural cavity and, in most cases, is considered a result of a systemic or intrathoracic process. This pathological entity is a frequent problem in pulmonology and, though its incidence varies with clinical background, 90% of all pleural effusions are attributed to congestive heart failure, malignant processes, and pneumonia (
The main issues regarding pleural effusions are the differentiation of exudates and transudates and the accurate determination of effusion aetiology (
When referring to the effusion aetiology, non-malignant pleural effusions are twice as common as malignant effusions and have diverse causes and manifestations, making them a diagnostic challenge (
The objective of the present study was to determine whether tumour-related biomarkers in pleural effusions and serum can be used to distinguish malignant and non-malignant pleural effusions. Our hypothesis was that tumour markers are a worthy substitute of the presence of malignant cells in pleural fluid as malignancy markers. We investigated four tumour markers in Croatian patients: carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cancer antigen 125 (CA-125), and cytokeratin 19 fragment (CYFRA 21-1).
During the first thoracentesis, a total of 110 pleural fluid and serum samples were collected prospectively from 110 consecutive patients diagnosed with pleural effusion and admitted to the Department of Pulmonology at Clinical Hospital Centre in Rijeka, Croatia, between November 2013 and November 2014. Measurements were made at the time of admission to the Clinical Hospital Centre. After excluding 10 patients with unknown pleural effusion origin, 100 patients formed the study group. The 10 excluded patients were not followed up because 7 patients refused additional treatment and/or diagnostics and 3 suffered a fatal outcome (
Flowchart showing the patient recruitment process
Informed consent was obtained from all patients included in the study. All procedures were performed in accordance to the ethical standards of the institutional and national research committee, and the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study protocol was authorized by the ethics committee of the relevant Clinical Hospital Centre.
Pleural effusion was diagnosed after thorough anamnesis, physical examination (lung percussion and auscultation), and chest x-ray. Pleural effusion was confirmed by thoracentesis.
Blood was sampled by venipuncture of antecubital veins. Pleural fluid was collected by a needle inserted between the seventh and ninth rib spaces, between the posterior axillary line and midline (thoracentesis). Both blood and pleural fluid were collected in 7 mL biochemical tubes without anticoagulants (BD vacutainer systems, Plymouth, UK). Blood and pleural specimens were centrifuged at a relative centrifugal force (RCF) of 1917xg for 10 minutes and the supernatant was analysed immediately. Blood specimens were tested at least 30 minutes after sampling, whereas pleural fluid samples were analysed immediately without previous storage. Tumour marker concentrations were determined by electrochemiluminescence immunoassays on the Cobas® e601 analyser (Roche-Diagnostics, Mannheim, Germany). The readers of the tests and reference standards were professionals, blind to the results of the other test.
Transudative and exudative pleural effusions were differentiated by Light’s criteria: effusion/serum protein ratio > 0.5, effusion/serum LD ratio > 0.6, effusion LD activity greater than two-thirds the upper limit of the laboratory’s reference range for serum LD (
If malignant cells were detected in a smear of the pleural effusion sediment using the May-Grünwald-Giemsa staining method under a light microscope, the effusion was recognized as malignant. Pleural effusions from patients with previously diagnosed malignant illness were also considered malignant if other diseases were excluded as the cause. This method of diagnosing malignant pleural effusions was the usual clinical procedure used for defining malignant pleural effusions and regarded as the reference standard (
Statistical analyses were performed using MedCalc software version 12.7.0.0 (Mariakerke, Belgium) and Statistica version 13 (Dell Inc., Tulsa, USA). Variables were tested for normality using a Kolmogorov-Smirnov test. Continuous variables with normal distribution were expressed as mean ± standard deviation (SD) and those with non-normal distribution as median and interquartile range (IQR). To test the difference in tumour marker concentrations between malignant and benign pleural effusions, we used the non-parametric Mann-Whitney U-test because the variables were not normally distributed. To determine the clinical usefulness of tumour markers in the studied group and assess the performance of tumour markers in distinguishing malignant and non-malignant pleural effusions, the marker values were reviewed by a receiver operating characteristic (ROC) analysis. The area under the ROC curve (AUC) serves as an overall measure of a biomarker/diagnostic test’s accuracy (
The diagnostic accuracy of each tumour marker and its pleural fluid/serum ratio was determined by sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-) and the AUC. We used Hanley and McNeil’s method for pairwise comparison of ROC curves in order to test the difference between the AUCs. P < 0.05 was considered significant.
In order to detect the tumour markers which best discriminate between malignant and non-malignant pleural effusions, discriminant function analysis (DFA) was employed. Logistic regression analysis was performed in order to establish the predictive value of those markers.
We calculated the required sample size using MedCalc software assuming that the desired statistical power of the test is 90% with a confidence level of 95% and the ratio of sample sizes in the negative and positive groups is 1. We wanted to show that the expected AUC of 0.7 for a particular test is significant from the null hypothesis value of 0.5. The required sample size was calculated to be 82. The present study had a slightly larger sample size to ensure that we would have enough results if some of the patients were excluded later.
The study group consisted of 73% males (73 male and 27 females). The median age of the study group was 71 years (range 22 - 92 years). In 88 patients (88%), we found a unilateral pleural effusion, whereas 12 (12%) patients were diagnosed with a bilateral effusion. The time interval between defining the pleural effusion as malignant or non-malignant and the determination of tumour marker concentrations was approximately 7 days. No adverse events occurred as a result of venipuncture or thoracentesis.
According to Light’s criteria, pleural exudates made up 71% (N = 71) of the study group; the other 29% (N = 29) were pleural transudates (
malignant | 55 / 71 |
parapneumonic | 7 / 71 |
asbestosis | 4 / 71 |
rib fracture | 1 / 71 |
heart failure | 1 / 71 |
liver cirrhosis | 1 / 71 |
glomerulonephritis | 1 / 71 |
hemopneumothorax | 1 / 71 |
heart failure | 22 / 29 |
liver cirrhosis | 3 / 29 |
chronic renal failure | 2 / 29 |
asbestosis | 1 / 29 |
rib fracture | 1 / 29 |
Primary tumour sites in malignant pleural effusions were distributed as follows: lung (N = 17), pleura (N = 15), ovary (N = 4), colon (N = 4), stomach (N = 3), breast (N = 2), prostate (N = 2), malignant disease of unknown origin (N = 2), skin (N = 1), soft tissues (N = 1), pancreas (N = 1), oesophagus (N = 1), hypopharynx (N = 1), and lymph nodes (N = 1). The non-malignant or benign pleural effusion group (N = 45) consisted of all patients with pleural transudates or non-malignant pleural exudates, who were then compared to the patients diagnosed with malignant pleural effusions (N = 55). The median age of the patients with malignant pleural effusions was 70 (48 – 90) years; whereas the median age of the patients with non-malignant pleural effusions was 73 (22 – 92) years.
The gender distribution was 39 males and 16 females in the malignant group and 34 males and 11 women in the non-malignant pleural effusion group. The concentrations of tumour markers CEA, NSE, CA-125, and CYFRA 21-1 in pleural fluid (P), serum (S) and their pleural fluid/serum ratios (R) in the malignant and non-malignant patient group are presented in
(P) CA-125, mU/mL | 814.3 (215.8 - 1107.0) | 732.7 (278.1 - 984.6) | 0.368 |
(S) CA-125, mU/mL | 175.3 (42.5 - 299.5) | 166.8 (62.2 - 303.5) | 0.967 |
(R) CA-125 | 3.9 (1.9 - 8.2) | 3.8 (1.7 - 7.5) | 0.819 |
(P) CEA, µg/mL | 3.0 (1.0 - 92.6) | 0.9 (0.3 - 1.6) | < 0.001 |
(S) CEA, µg/mL | 3.0 (1.6 - 6.7) | 2.4 (1.3 - 3.6) | 0.070 |
(R) CEA | 0.9 (0.5 - 7.6) | 0.4 (0.2 - 0.7) | < 0.001 |
(P) NSE, ng/mL | 10.8 (5.3 - 31.0) | 4.4 (2.2 - 11.2) | < 0.001 |
(S) NSE, ng/mL | 19.4 (14.5 - 27.0) | 12.7 (10.7 - 17.4) | < 0.001 |
(R) NSE | 0.6 (0.3 - 1.3) | 0.3 (0.2 - 0.1) | 0.047 |
(P) CYFRA 21-1, ng/mL | 57.5 (22.4 - 161.9) | 13.0 (6.3 - 48.6) | < 0.001 |
(S) CYFRA 21-1, ng/mL | 7.9 (3.6 - 14.9) | 2.2 (1.4 - 4.4) | < 0.001 |
(R) CYFRA 21-1 | 7.7 (2.8 - 27.4) | 4.0 (2.6 - 24.0) | 0.484 |
Data are presented as median and interquartile range (IQR). P – pleural fluid concentration. S – serum concentration. R - pleural fluid/serum ratio. P < 0.05 was considered statistically significant. |
The ROC analyses of the mentioned tumour markers (determined in pleural fluid, serum, and as pleural fluid/serum ratio) are shown in
(P) CA-125, mU/mL | > 844.2 | 49.1 |
66.7 |
64.3 |
51.7 |
0.55 (0.45 – 0.65), |
1.47 | 0.76 |
(S) CA-125, mU/mL | ≤ 50.5 | 30.9 |
82.2 |
68.0 |
49.3 |
0.50 (0.40 – 0.60), |
1.74 | 0.84 |
(R) CA-125 | > 0.92 | 87.3 |
20.0 |
57.1 |
56.2 |
0.51 (0.41 – 0.62), |
1.09 | 0.64 |
(P) CEA, |
> 2.2 | 56.4 |
88.9 |
86.1 |
62.5 |
0.75 (0.65 – 0.83), |
5.07 | 0.49 |
(S) CEA, |
> 3.9 | 38.2 |
84.4 |
75.0 |
52.8 |
0.61 (0.50 – 0.70), |
2.45 | 0.73 |
(R) CEA | > 0.56 | 69.1 |
82.2 |
82.6 |
68.5 |
0.80 (0.71 – 0.874), |
3.89 | 0.38 |
(P) NSE, |
> 4.9 | 81.8 |
57.8 |
70.3 |
72.2 |
0.70 (0.60 - 0.79), |
1.94 | 0.31 |
(S) NSE, |
> 13.3 | 81.8 |
57.8 |
70.3 |
72.2 |
0.73 (0.63 – 0.82), |
1.94 | 0.31 |
(R) NSE | > 0.32 | 72.7 |
53.3 |
65.6 |
61.5 |
0.62 (0.52 – 0.71), |
1.56 | 0.51 |
(P) CYFRA 21-1, ng/mL | > 14.1 | 83.6 |
55.6 |
69.7 |
73.5 |
0.72 (0.62 – 0.80), |
1.88 | 0.29 |
(S) CYFRA 21-1, ng/mL | > 3.5 | 76.4 |
71.1 |
76.4 |
71.1 |
0.79 (0.69 – 0.86), |
2.64 | 0.33 |
(R) CYFRA 21-1 | > 7.62 | 50.9 |
66.7 |
65.1 |
52.6 |
0.54 (0.44 – 0.64) |
1.53 | 0.74 |
P – pleural fluid concentration. S – serum concentration. R - pleural fluid/serum ratio. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and area under the curve (AUC) are presented as percentage with corresponding 95% confidence intervals. (LR+) - positive likelihood ratio. (LR-) - negative likelihood ratio. P < 0.05 was considered statistically significant. |
Receiver operating characteristics (ROC) curves for all parameters investigated in pleural fluid, serum and their ratios. P – pleural fluid concentration. S – serum concentration. R - pleural fluid/serum ratio.
After pairwise comparison of all 12 ROC curves, significant differences were shown between AUCs of (S) CYFRA 21-1, (R) CEA, (P) CEA, (S) NSE, (P) NSE, (P) CYFRA 21-1, while no difference was found comparing AUCs of (P) CA-125, (R) CA-125, (S) CA-125 and (R) CYFRA 21-1 (data not shown). These results show that there are significant differences between diagnostic values of these tumour markers. However, there was no statistically significant difference between AUCs for tumour markers (S) CYFRA 21-1, (R) CEA, (P) CEA, (S) NSE, (P) NSE, (P) CYFRA 21-1 (data not shown) and therefore it cannot be concluded that any of them had greater diagnostic accuracy in distinguishing malignant from non-malignant pleural effusions. In order to better explain these results, DFA was performed.
Sets of four tumour markers concentrations measured in pleural fluid and serum, as well as their ratios were analysed with respect to their discriminatory ability, and the results presented in
(P) CA-125 |
0.918 |
1.136 |
0.289 |
2.431 | 0.053 |
(S) CA-125 |
0.854 |
0.254 |
0.616 |
4.139 | 0.004 |
(R) CA-125 |
0.952 |
0.434 |
0.512 |
1.318 | 0.269 |
P – pleural fluid concentration. S – serum concentration. R - pleural fluid/serum ratio. P < 0.05 was considered statistically significant. Wilks' lambda test is used to test which variables significantly contribute in discriminant function analysis. F-value is associated with the respective partial Wilks' lambda. P-value is associated with the respective F-value. |
(S) CYFRA 21-1 | 0.096 | 0.041 | 5.507 | 0.019 | 28.415 | < 0.001 | 0.79 | 0.69 - 0.86 |
(S) NSE | 0.122 | 0.037 | 10.976 | < 0.001 | ||||
(P) NSE | 0.025 | 0.012 | 4.544 | 0.033 | ||||
P – pleural fluid concentration. S – serum concentration. R - pleural fluid/serum ratio. 95% CI – 95% confidence intervals. P < 0.05 was considered statistically significant. b - logistic regression coefficient and its standard error SE (b). The statistical significance of individual regression coefficients is tested using the Wald Chi-square statistic. If P < 0.05 then the variable contributes significantly to the outcome prediction. AUC – area under the ROC curve. |
Positive (> 3.5 µg/mL) | 42 | 14 | 56 |
Negative (≤ 3.5 µg/mL) | 13 | 31 | 44 |
Total | 55 | 45 | 100 |
Positive (> 13.3 ng/mL) | 45 | 19 | 64 |
Negative (≤ 13.3 ng/mL) | 10 | 26 | 36 |
Total | 55 | 45 | 100 |
Positive (> 4.9 ng/mL) | 45 | 19 | 64 |
Negative (≤ 4.9 ng/mL) | 10 | 26 | 36 |
Total | 55 | 45 | 100 |
Determining the diagnostic utility of tumour markers in pleural effusions, especially CEA, NSE, CA-125, and CYFRA 21-1, has many advantages with regard to both cost and the non-invasive approach, which is why we dedicated the entirety of this study to the analysis of these four markers. Targeted analysis of tumour markers in pleural effusions is acceptable, as the price of the test is not high. A cytomorphological finding of malignant cells in pleural effusions and immunocytochemical analysis can be used as alternative tests for greater diagnostic accuracy. Although these tumour markers exhibit great specificity, the low sensitivity of each individual marker limits their diagnostic value, and a few studies suggested they be used along with other pleural effusion tests for greater diagnostic accuracy (
The value of a tumour marker lies in its ability to exclude or confirm the diagnosis of malignancy. In the first case, the marker with greatest sensitivity, NPV, and LR- should be used; in the second case (to confirm the diagnosis), tumour markers with the highest specificity, PPV, and LR+ should be used. One marker can be great for confirming diagnosis but not so good or very bad at excluding the disease and
Studies of the diagnostic value of tumour markers in pleural effusions are not rare, but they have not had definitive and clear results (
The median pleural fluid concentrations for the investigated tumour markers were higher in malignant effusions than benign effusions, with the exclusion of CA-125. This can be explained by the fact that pleural effusions are a filtrate of blood plasma and, therefore, the values of tumour markers do not significantly differ between pleural effusions and serum. Mechanisms involved in the production of pleural effusions are numerous, the most common of which are increased hydrostatic pressure in lung capillaries, reduction of colloid-osmotic blood pressure, increased capillary permeability in the lungs, decreased pressure in the pleural space, lymphatic vessel obstruction in the thorax, and increased pressures in the systemic vein bloodstream. The values of individual tumour markers depend on the dominating mechanism involved in the development of the pleural effusion. Pleural fluid CA-125 concentrations deviate from the other three markers tested in this study because CA-125 is also elevated in the serum of patients suffering from non-malignant diseases (
The pleural fluid/serum ratio for CEA had one of the highest AUC values (
Another valuable diagnostic marker according to the present study is CYFRA 21-1, but primarily considering the serum values. The AUC for serum CYFRA 21-1 was 0.79 and was proven by discriminant function analysis to be one of the best discriminators regarding pleural fluid aetiology. Logistic regression analysis also discovered serum CYFRA 21-1 to be one of the three best predictors of pleural effusion malignancy, according to this study. In a previous study by Korczynski
NSE is currently the most reliable tumour marker in the diagnosis, prognosis, and follow-up of small cell lung cancer (SCLC), even though increased NSE concentrations have been reported in non-small cell lung cancer (NSCLC) (
Although CA-125 was discovered 30 years ago, the Food and Drug Administration (FDA) still recommends it to monitor the response to therapy in patients with epithelial ovarian cancer and to detect residual or recurrent disease in patients who have undergone first-line therapy and who would be considered for second-look procedures (
The present study has some limitations. Although the patients were included as a consecutive series, the fact that the study was performed in the Department of Pulmonology has the consequence of exposure to more diseases related to lung pathology. In addition, the number of patients included in this study is representative of the size of the medical facility where the study was performed. Furthermore, the present study does not evaluate a larger number of different tumour markers (
The advantage of this study is that it is a real-time study conducted in a Croatian population; differences exist in the expression of particular tumour markers in different races and populations due to genetic susceptibility.
In conclusion, we have shown that certain tumour markers have a higher diagnostic value than others in Croatian patients. Serum markers were superior to pleural fluid markers in determining pleural fluid aetiology, according to this study. Serum CYFRA 21-1 concentrations, serum and pleural NSE values were found to be most significant predictors of pleural effusion malignancy and the combination of these three entities produced a significant model for distinguishing malignant from non-malignant pleural effusions. Further research in other populations and larger study groups is needed to determine whether measuring tumour markers in pleural effusions is appropriate in general.
None declared.