Abstract
Rationale: Serum mesothelin is a new biomarker for the diagnosis of mesothelioma. Patients with mesothelioma commonly present with pleural effusions. To define the clinical utility of mesothelin quantification in pleural fluid, we assessed its additional value over pleural fluid cytology and its short-term reproducibility and reliability after pleural inflammatory processes, including pleurodesis.
Objectives: To assess the diagnostic role of pleural fluid mesothelin and the effect of common clinical factors that may influence measurement accuracy.
Methods: Mesothelin was quantified in 424 pleural fluid and 64 serum samples by ELISA. Fluid was collected prospectively from 167 patients who presented with pleural effusions for investigation. Serial pleural fluid samples were obtained from patients (n = 33) requiring repeated drainage. Mesothelin levels were also measured in patients (n = 32) prepleurodesis and postpleurodesis.
Measurements and Main Results: Pleural fluid mesothelin concentrations were significantly higher in patients with mesothelioma (n = 24) relative to those with metastatic carcinomas (n = 67) and benign effusions (n = 75): median (interquartile range, 25th−75th percentile) = 40.3 (18.3–68.1) versus 6.1 (1.5–13.2) versus 3.7 (0.0–12.4) nM, respectively, P < 0.0001. Mesothelin measurement was superior to cytological examination in the diagnosis and exclusion of mesothelioma (sensitivity, 71 vs. 35%; specificity, 89 vs. 100%; negative predictive value, 95 vs. 82%, respectively). In patients with “suspicious” cytology, pleural fluid mesothelin was 100% specific for mesothelioma, and in cytology-negative effusions (n = 105) offered a negative predictive value of 94%. Intraindividual reproducibility of pleural fluid mesothelin was excellent: mean (±SD) variation, −0.15 (±8.41) nM in samples collected within 7 days from patients with mesothelioma. Measurements remained reliable after pleurodesis and were not affected by the presence of bacteria.
Conclusions: Pleural fluid mesothelin provides additional diagnostic value relative to cytological examination. Mesothelin measurements are reproducible and not affected by inflammatory pleural processes.
Serum mesothelin is a diagnostic biomarker for mesothelioma. Pleural fluid values are significantly higher than serum levels and may offer additional value over pleural fluid cytology for diagnosis of pleural malignancy. The clinical utility of pleural fluid mesothelin has not yet been defined.
This study demonstrates that pleural fluid mesothelin is a valuable adjunct to cytological examination in patients with an undiagnosed pleural effusion. It is reproducible and not influenced by common inflammatory pleural processes.
Malignant pleural mesothelioma is increasing in incidence rapidly worldwide (2) and in the United Kingdom, kills one patient every 4 hours (3). Most patients with mesothelioma present with a pleural effusion, but diagnosis is often difficult as no clinical or radiological features reliably separate mesothelioma from metastatic carcinomas or benign pleuritis (4). Interpretation of cytology and histology can be challenging, but is essential to establish a diagnosis. Although pleural fluid cytology is effective in identifying adenocarcinomas, it has low sensitivity (∼20%) in diagnosing mesothelioma, as differentiating between normal, reactive, and malignant mesothelial cells is notoriously difficult (5).
Soluble mesothelin–related peptides (or “mesothelin”) are potentially a substantial advance in this diagnostic problem and may be a robust biomarker for mesothelioma (6–13). Serum mesothelin has been approved by the U.S. Food and Drug Administration for monitoring and diagnosis of mesothelioma. Mesothelin is a glycoprotein overexpressed by mesothelioma cells and released into pleural fluid. Hence its measurement in pleural fluid offers a theoretical advantage over serum measurement and has attracted attention as a diagnostic test, with promising results from several studies (7, 8, 11).
The actual clinical utility of pleural fluid mesothelin has not been defined. The practical contribution it makes in establishing a clinical diagnosis, its reproducibility and reliability, and its stability after common pleural inflammatory insults such as pleurodesis and pleural infection have not been determined. This study addresses these questions to define its role in clinical practice. Part of the results of this study has been presented in the form of abstracts in scientific conferences (14–16).
A total of 424 pleural fluid samples were prospectively collected from 209 patients and quantified for mesothelin. All patients were recruited from the Oxford Pleural Unit (Oxford Centre for Respiratory Medicine, Oxford, UK), a tertiary referral center for pleural disease that also admits patients presenting with acute respiratory problems.
Samples were collected over a 13-month period from 167 consecutive patients with pleural effusions being investigated for possible pleural malignancy, who underwent thoracentesis, tube thoracostomy, or thoracoscopy. Patients were excluded if insufficient pleural fluid was available after submitting for necessary clinical tests.
Serial pleural fluid samples were obtained from patients who required repeat pleural aspirations (n = 14) or indwelling pleural catheters (n = 19) for the management of symptomatic recurrent pleural effusions.
Serum and pleural fluid samples were collected from patients (n = 31 and 32, respectively) at baseline and 24–48 hours after pleurodesis with talc slurry (4 g; Novatech, La Ciotat, France) as part of our published trial examining the effect of pleurodesis on inflammatory markers (17).
Mesothelin concentrations were measured on pleural fluid samples (n = 5) at baseline and after inoculation of samples with (1) viable and (2) heat-killed bacteria.
The study was approved by the Mid and South Buckinghamshire and Central Oxford Research Ethics Committees (17). All participants provided written consent.
Pleural fluid samples were collected by aseptic techniques and transferred on ice in standard blood collection tubes containing sodium citrate. Samples were centrifuged at 3,000 rpm for 10 minutes at 4°C, and supernatants were stored at −80°C until assay.
At the time of initial thoracentesis pleural fluid samples were sent to the laboratory for routine clinical investigations. Pleural fluid glucose, pH, lactate dehydrogenase (LDH), and protein were measured as previously reported (18). For cytological examination the pleural fluid was processed by standard clinical methods (19). In brief, samples were centrifuged and direct smears were made from cells resuspended in saline. If the cell pellet was small, the sample was centrifuged and a cytospin slide was prepared. The prepared slides were fixed in alcohol for Papanicolaou staining (20, 21), and air-dried for Giemsa staining. Extra smears or cytospins were made if immunostaining was required (19).
Mesothelin concentrations were measured with a commercially available ELISA (CIS Bio, Gif-sur-Yvette, France) according to the manufacturer's instructions. All samples were encrypted and analyzed in duplicate by an investigator (R.S.) blinded to the patients' diagnoses.
The hospital discharge records were interrogated for the diagnosis of the pleural effusion. The clinical data and investigation results were reviewed independently by two investigators (H.E.D. and Y.C.G.L.) to confirm the discharge diagnosis given by the attending pulmonologists.
The diagnostic criteria were identical to those we previously published (22). In brief, Light's criteria were used to differentiate transudates from exudates (23). A malignant pleural effusion was diagnosed if cancer cells were confirmed in pleural fluid or pleural tissue biopsy by a qualified pathologist, or in a patient with disseminated malignancy and no alternative explanation for an exudative effusion. All cases of mesothelioma were diagnosed histocytologically with immunohistochemical confirmation.
Benign effusions were classified under the following categories after a mean (SD) 14 (4.2)-month period of follow-up:
Effusions of infective etiologies included empyema, parapneumonic effusions (24), and tuberculous effusion. Empyema was diagnosed if thoracentesis yielded frank pus, or bacteria were identified (by Gram stain or culture). Parapneumonic effusions were exudative effusions in the presence of clinical evidence of pneumonia. Tuberculous pleuritis was diagnosed by positive mycobacterial staining/culture of pleural fluid and/or tissue or the presence of caseating granulomas on pleural biopsy.
Fibrinous pleurisy was diagnosed histologically on thoracoscopic biopsy samples with no alternative diagnosis evident on follow-up.
Congestive heart failure was diagnosed if the pleural fluid was a transudate in a patient with clinical features of cardiac failure.
A hepatic hydrothorax was diagnosed in patients with a transudative effusion in the presence of hepatic cirrhosis and ascites (25).
Rheumatoid arthritis–related effusion was diagnosed in the presence of a sterile exudate with high LDH, low pH, and/or glucose in a patient with known rheumatoid arthritis and no features of pleural infection or malignancy.
Traumatic hemothorax was a heavily hemorrhagic effusion that developed after trauma with no alternative explanation.
Chylothorax and pseudochylothorax were diagnosed on the basis of the presence of chylomicrons or cholesterol crystals, respectively, and/or appropriate lipid criteria (19).
We assessed the reliability of pleural fluid mesothelin measurement in the presence of bacteria to exclude any interference in mesothelin quantification. Streptococcus pneumoniae capsular serotype 2 strain, D39 (from J. Brown, London, UK), was cultured as published (26). The bacteria (1.2 × 106 colony-forming units [CFU] in 20 μl of phosphate-buffered saline solution) were added to 1 ml of pleural fluid and incubated for 24 hours at 37°C. Pleural fluid samples (n = 5; three epithelioid mesothelioma, one metastatic breast adenocarcinoma, and one fibrinous pleurisy) known to contain high mesothelin levels were used. As controls, each pleural fluid sample was treated under the same conditions with 20 μl of phosphate-buffered saline only or with heat-treated bacteria (1.2 × 106 CFU) and incubated as described previously. To heat-kill the bacteria, the bacterial stock solution was kept at 60°C for 30 minutes. At the experiment end points, the pleural fluid samples were centrifuged as described and the supernatant was stored at −20°C until assay. Mesothelin levels were quantified as described previously.
All analyses were performed with SPSS software (version 14.0; SPSS, Chicago, IL), and two-sided P values less than 0.05 were regarded as statistically significant. Data of parametric and nonparametric distributions are expressed as means (±SD) or medians (with interquartile range [IQR25–IQR75]), respectively.
Intergroup comparisons were compared by one-way analysis of variance (ANOVA). Nonparametric data were analyzed by ANOVA on-ranks and Dunn's post hoc test. Paired t tests were used to assess change in pleural fluid mesothelin concentration over time and after exposure to bacteria. The Wilcoxon signed-rank test was used to compare differences in mesothelin levels after pleurodesis. Sensitivity and specificity of mesothelin, and the receiver operating characteristic (ROC) curves, were calculated according to standard formulas (27–29). The area under the ROC curve (AUC) was computed with 95% confidence intervals (CIs). Correlations between nonparametric variables were determined using Spearman's rank correlation coefficient (rs).
Pleural fluid samples (n = 167) were collected and classified as malignant (n = 91) or benign (n = 75) (Table 1). One patient withdrew before full investigations had been completed and was excluded. Mesothelioma was diagnosed in 24 cases (11 epithelioid; 4 biphasic and 5 sarcomatoid; 4 cytologically defined).
Mesothelioma (n = 24) | Nonmesothelioma Malignancy (n = 67) | Benign Pleural Effusion (n = 75) | |
|---|---|---|---|
| Age, yr (mean ± SD) | 72 ± 7.5 | 68 ± 15.6 | 71 ± 16.0 |
| Male sex, n (%) | 24 (100) | 29 (43.3) | 59 (78.7) |
| Pleural fluid, median (IQR25–IQR75) | |||
| Protein, g/L | 51 (41–54) | 44 (36–48) | 40 (30–46) |
| LDH,* IU/L | 263 (160–507) | 215 (123–462) | 226 (86–1,067) |
| Glucose, mmol/L | 3.4 (2.1–5.2) | 5.6 (4.5–7.1) | 4.7 (2.1–6.3) |
| pH | 7.31 (7.26–7.37) | 7.40 (7.28–7.43) | 7.28 (7.07–7.47) |
The median pleural fluid mesothelin concentrations were 6.6- and 10.9-fold higher in the mesothelioma group compared with metastatic carcinomas and benign effusions, respectively: median (IQR25–IQR75) values were 40.3 (18.3–68.1) versus 6.1 (1.5–13.2) versus 3.7 (0.0–12.3) nM, respectively (P < 0.0001) (Figure 1).
Figure 1. (A) Pleural fluid mesothelin concentrations in pleural effusions from patients with mesothelioma, other pleural malignancies, and benign disease. (B) Receiver operating characteristic (ROC) curve for differentiating mesothelioma from all other conditions at a pleural fluid mesothelin cutoff of 20 nM (area under the curve, 0.878; 95% confidence interval, 0.807 to 0.948; P < 0.0001). (C) Pleural fluid mesothelin levels in effusions from patients with different histological subtypes of mesothelioma. (D) Comparison between pleural fluid mesothelin concentrations in patients with mesothelioma and other malignant or benign conditions. The dashed line represents a cutoff value of 20 nM for pleural fluid mesothelin concentration. Only one other patient with a pleural effusion of benign etiology (simple parapneumonic effusion) had a pleural fluid mesothelin value greater than 20 nM (82.0 nM). AUC = area under the curve; CHF = congestive heart failure; GI = gastrointestinal origin; MPM = malignant pleural mesothelioma; NS = not significant; QR = interquartile.
Using ROC curve analysis, pleural fluid mesothelin offered an AUC of 0.878 (95% CI, 0.807 to 0.948; P < 0.0001) in its ability to differentiate between patients with mesothelioma and all other diagnoses at an optimal cutoff value of 20 nM. The AUC was 0.684 (95% CI, 0.604 to 0.763; P < 0.0001) when differentiating all malignant and benign effusions.
The diagnostic sensitivity and specificity of pleural fluid mesothelin for distinguishing mesothelioma from all other causes of pleural effusion, at a cutoff value of 20 nM, were 71% (95% CI, 49 to 87%) and 90% (95% CI, 84 to 95%), respectively. The positive and negative predictive values (PPV and NPV) were 53 and 95%, respectively (Table 2).
Sensitivity | Specificity | PPV | NPV | |||||
|---|---|---|---|---|---|---|---|---|
| Mesothelioma | ||||||||
| Cytology positive | 34 | 100 | 100 | 82 | ||||
| Mesothelin positive* | 71 | 90 | 53 | 95 | ||||
| Either cytology or mesothelin positive | 74 | 71 | 31 | 94 | ||||
| Both cytology and mesothelin positive | 35 | 92 | 42 | 89 | ||||
| All malignancies | ||||||||
| Cytology positive | 57 | 100 | 100 | 66 | ||||
| Mesothelin positive | 33 | 97 | 95 | 55 | ||||
| Either cytology or mesothelin positive | 67 | 100 | 100 | 72 | ||||
| Both cytology and mesothelin positive | 23 | 100 | 100 | 53 | ||||
The histological subtype of mesothelioma significantly influenced expression of mesothelin: the median level was highest with epithelioid histology, followed by biphasic and then sarcomatoid subtypes (57.2 [41.7–73.1] vs. 38.8 [5.1–85.7] vs. 16.0 [3.6–24.0] nM, respectively). Pleural fluid mesothelin was positive in 82% of patients with epithelioid tumor but only in 20% with sarcomatoid subtypes; the latter being the most common explanation of “false negative” (four of seven samples) cases.
Adenocarcinomas accounted for 12 of the 13 “false positive” cases. Of the different subtypes of adenocarcinoma, those that metastasized to the pleura from an unknown primary site had the highest incidence of high mesothelin levels (seven of eight cases). On retrospective review, none of these seven patients had clinical or radiological evidence of ovarian or pancreatic cancers (known to have high mesothelin expression) throughout their disease course.
Positive pleural fluid mesothelin levels had a high predictive value (94%) for the effusion being of a malignant nature. The median (IQR25–IQR75) levels of mesothelin in empyemas and congestive heart failure (0.0 [0.0–1.5] and 4.7 [0.0–12.8] nM, respectively) were significantly lower than those of mesotheliomas.
Only 2 of 75 cases (2.7%) of benign disease had elevated mesothelin concentrations. One patient (pleural fluid mesothelin level, 82.0 nM) presented with clinical and radiological features of left lower lobe community-acquired pneumonia and subsequently developed a pleural effusion. Pleural aspiration yielded pleural fluid with a normal pH and glucose concentration of 4.0 mmol/L. A final diagnosis of a simple parapneumonic effusion was made and he recovered with antibiotic therapy. Interval computed tomographic imaging showed no evidence of mesothelioma and he remained well on follow-up at 1 year. The second patient (pleural fluid mesothelin level, 34.5 nM), a 71-year-old male, underwent thoracoscopy for a persistent left pleural effusion. Pleural biopsies revealed fibrous thickening and chronic inflammation. He died with bronchopneumonia 2 months later and his autopsy showed no evidence of pleural malignancy.
There was no significant correlation between pleural fluid mesothelin levels and pleural fluid protein (Spearman's correlation coefficient rs = 0.098, P = 0.223), lactate dehydrogenase (rs = −0.088, P = 0.268), glucose (rs = −0.165, P = 0.040), or pH (rs = 0.007, P = 0.944) values.
We reviewed pleural fluid cytological analyses, requested as part of standard clinical practice, and assessed the potential additional diagnostic value of pleural fluid mesothelin. Cytological examination was requested for 152 of the 166 pleural fluid samples. Fourteen did not have initial cytological testing, as pleural fluid analyses suggested that underlying malignant disease was unlikely, for example, in patients with frank empyema.
Of the specimens analyzed, 47 (31%) were cytology positive, 94 (62%) were cytology negative, and 11 samples were “suspicious” for pleural malignancy. In samples where malignant cells were identified, 17% were mesotheliomas (10.6% epithelioid). The remainder with positive cytology were mostly non–small cell lung carcinoma–related effusions (27.7%) and adenocarcinomas (23.4% from primary carcinomas from the breast, 6.4% gastrointestinal tract, and 12.8% adenocarcinomas of unknown origin).
Other malignancies (n = 6) detected by cytological analysis of the pleural fluid were small cell lung carcinoma, epithelioid hemangioendothelioma, chronic lymphocytic leukemia, endometrial carcinoma, leiomyosarcoma, and ovarian carcinoma.
As a single test, a pleural fluid mesothelin concentration exceeding 20 nM had advantages over pleural fluid cytological examination in the diagnosis and exclusion of mesothelioma (sensitivity, 71 vs. 35%; specificity, 89 vs. 100%, respectively). The NPV of pleural fluid mesothelin for mesothelioma was 95% (vs. 82% for pleural fluid cytology).
Pleural fluid mesothelin levels complemented cytological examination whether the latter was positive, equivocal, or negative. In cases where positive malignant cells were identified (47 of 166 samples investigated), a pleural fluid mesothelin level greater than 20 nM correctly identified all eight cases of mesothelioma, providing useful confirmatory evidence of the diagnosis (sensitivity, 100%; specificity, 72%; NPV, 100%).
In patients with pleural fluid cytology revealing abnormal cells suspicious but not diagnostic of malignancy (n = 11), raised mesothelin levels were found in five cases, all of which were eventually diagnosed as mesothelioma by pleural biopsy (sensitivity, 63%; specificity, 100%). In this group of patients, an elevated pleural fluid mesothelin level provided a 100% specificity value and a 100% PPV for a diagnosis of underlying malignancy (mesothelioma or nonmesothelioma) (Figure 2).
Figure 2. Flow diagram of pleural fluid and mesothelin results. *Cytology not performed, underlying malignancy clinically unlikely; †E = epithelioid mesothelioma; B = biphasic; S = sarcomatoid; the remaining cases were cytologically proven (subtype not defined). MPM = malignant pleural mesothelioma; NPV = negative predictive value; PPV = positive predictive value.
[More] [Minimize]For all patients with negative cytology results (n = 105), the addition of pleural fluid mesothelin to the panel of routine investigations offers further support to the confident exclusion of underlying mesothelioma: A negative pleural fluid mesothelin level combined with negative cytological examination provided a diagnostic specificity of 97% with an NPV of 94% (compared with 100 and 82%, respectively, for cytology alone). For all malignant etiologies of pleural effusion, a diagnostic specificity of 96% was achieved with PPV and NPV values of 77 and 73%, respectively.
A total of 167 samples were collected from 33 patients with malignant pleural effusions (n = 7 mesothelioma). Fluid was obtained when patients attended for clinical review with a mean (SD) of 5 (3) pleural fluid samples provided by each patient. Sequential samples were collected at intervals of 1 to 157 days apart (median, 10 d).
To determine the reproducibility of mesothelin measurements, all paired samples (n = 51) taken within 7 days from the same patient were analyzed. The majority of patients (59%) had elevated pleural fluid mesothelin concentrations at baseline (Day 1). Mesothelin levels did not change significantly (mean [SD] 0.75 [±6.01] nM; P = 0.371) in the overall cohort among the paired samples, although two patients crossed from just above the 20 nM cutoff (20.9 and 22.0 nM) to values below the cutoff. No significant changes were observed when patients with mesothelioma were analyzed separately (−0.15 [±8.41] nM; P = 0.962) (Figure 3).
Figure 3. Change in pleural fluid mesothelin concentration between baseline (Day 1) and a second sample collected within 7 days. Each horizontal dot plot represents an individual patient (n = 51). Solid dots = pleural fluid mesothelin concentration on Day 1; open dots = pleural fluid mesothelin concentration within 7 days.
[More] [Minimize]To assess change in pleural fluid mesothelin concentration during the course of an individual patient's disease, we compared differences between baseline values and measurements from samples obtained at various time points. This was performed to evaluate whether timing of mesothelin measurement affected the diagnostic utility of the assay. In patients with mesothelioma, a significant correlation between mesothelin concentration and time (r = 0.595 [95% CI, 0.112–0.340]; P < 0.0001) was observed. None of these patients underwent surgical treatment or received chemotherapy or other potentially disease-modifying agents, during the period of pleural fluid collection (Figure 4).
Figure 4. Correlation of the change in pleural fluid mesothelin concentration from Day 1 with time in patients with mesothelioma. Each plot represents the mesothelin value of samples taken from individual patients at each time point.
[More] [Minimize]Pleural fluid samples (n = 31) and serum (n = 32) were collected from patients with malignant effusions who underwent talc slurry pleurodesis. Pleural fluid was obtained from 26 of the 31 patients at 24 hours and from 18 patients at 48 hours. Talc pleurodesis induced a statistically, but not clinically, significant decrease in pleural fluid mesothelin levels at 24 hours (median [IQR25–IQR75], −5.8 [−12.9 to −0.5] nM; P = 0.002) and 48 hours (−3.6 [−7.1 to 0.0] nM; P = 0.106) but did not induce any significant change in serum mesothelin concentration (0.0 [−2.1 to 0.0] nM; P = 0.260) (Table 3).
When human pleural fluid was cocultured with pneumococci, no significant difference was observed in pleural fluid mesothelin levels between samples with no bacteria and those with viable bacteria (mean [SD] difference, 1.5 [±3.6] nM; P = 0.938) or heat-killed pneumococci (0.9 [±5.5] nM; P = 0.369). The change in pleural fluid mesothelin values between specimens containing nonviable or viable bacteria was not statistically different (P = 0.166).
This study has assessed the practical utility of pleural fluid mesothelin quantification in one of the largest cohorts reported to date. It is the first to evaluate mesothelin measurement prospectively against standard clinical parameters in the work-up of undiagnosed effusions.
Pleural fluid mesothelin levels were significantly elevated in patients with mesothelioma relative to other malignant or benign pleural diseases (by 6.6- and 10.9-fold, respectively). We showed that pleural fluid mesothelin levels contributed valuable additional information to pleural fluid cytology alone, especially when the latter was inconclusive or “suspicious,” in which case mesothelin offered 100% specificity in establishing the diagnosis of mesothelioma. As one third of patients with mesothelioma fall within this category, integration of the pleural fluid mesothelin assay into current diagnostic pathways may benefit up to 3,000 patients in Western Europe each year (30). Pleural fluid mesothelin also provided a 94% negative predictive value in patients whose fluid cytology was negative, offering support for exclusion of underlying mesothelioma. Mesothelin measurements were reproducible in pleural fluid within individuals and appeared not to be significantly affected by pleural inflammatory processes (pleurodesis in vivo or presence of bacteria ex vivo).
Malignant pleural effusions are common and account for up to 70% of pleural fluid exudates; this diagnosis must be confirmed or excluded in patients presenting with an undiagnosed effusion. Pleural fluid cytology analysis has been incorporated in recommended diagnostic algorithms (19).
The incidence of mesothelioma is rising exponentially in Europe and a dramatic increase is also expected in developing countries where asbestos use is less regulated (31–35). Most patients with mesothelioma present with a unilateral exudative pleural effusion and nonspecific symptoms. Unlike for adenocarcinomas, the yield of pleural fluid cytology for mesothelioma is often low as morphological separation of benign or malignant mesothelial cells is difficult, and no reliable diagnostic immunomarkers exist. Many patients are therefore subjected to invasive procedures (e.g., thoracoscopy or thoracotomy) to secure tissue diagnoses that have known associated morbidities.
Mesothelin is a cell surface protein overexpressed in mesothelioma, especially the epithelioid subtype. The measurement of pleural fluid (instead of blood) mesothelin levels offers theoretical advantages as mesothelin is produced locally and released directly into the pleural cavity. Pleural fluid mesothelin levels are known to be substantially higher (by ∼22-fold) than those of corresponding serum in mesothelioma (11); and its diagnostic accuracy is as good as serum measurement (7, 11). As patients with mesothelioma commonly present with an effusion, the standard investigation of which is thoracentesis and biochemical analysis of the pleural fluid, mesothelin quantification can easily be included. Theoretically, pleural fluid cytological analysis and the measurement of a biomarker for mesothelioma will complement each other. We tested the usefulness of this combination in a prospective “real life” cohort of patients presenting with undiagnosed effusions.
Our optimal cutoff value (20 nM) for pleural fluid mesothelin concentration, and its sensitivity for detection of mesothelioma, concurs with other studies (7, 8, 11). Our data confirmed that, although not a perfect marker, pleural fluid mesothelin compares favorably with clinically utilized serum tumor markers (e.g., prostate-specific antigen and CA-125) (36–41), especially if the cutoff level is set to provide a high specificity. The diagnostic sensitivity of mesothelin for the diagnosis of mesothelioma is high in this cohort of patients. It should, however, be noted that in this study there was a high proportion of mesothelioma cases (reflecting the rising incidence of mesothelioma in the United Kingdom and the tertiary setting from which patients were recruited). Validation in settings of low mesothelioma incidence is required.
Importantly, this study showed that pleural fluid mesothelin can play an important supplementary role in the diagnostic algorithms for mesothelioma to improve clinical practice. As a single test, pleural fluid mesothelin was significantly more useful than pleural fluid cytology in the diagnosis and exclusion of mesothelioma (sensitivity, 71 vs. 35%; specificity, 89 vs. 100%, respectively). The NPV of pleural fluid mesothelin was 95% (vs. 82% for pleural fluid cytology). False-negative cases consisted mainly of sarcomatoid mesothelioma, a subtype accounting for about 10% of all mesotheliomas, which often has low mesothelin expression.
When malignant mesothelioma cells were identified on cytological examination, a raised pleural fluid mesothelin level lends confirmation to the diagnosis. This is important as clinicians are often unwilling to accept a cytological diagnosis of mesothelioma given the recognized difficulties separating benign from malignant mesothelial cells (5).
The additional clinical utility of pleural fluid mesothelin appears to be greatest in patients with “suspicious” pleural fluid cytology. In these patients, an elevated mesothelin level provided a high specificity and PPV (100% in our series) for a diagnosis of underlying mesothelioma, although the numbers (n = 11) of such cases included in this study are small.
Cytological examination is known to have a low pick-up rate for mesothelioma. However, the combination of negative pleural fluid cytology and low pleural fluid mesothelin level provides a NPV of 94% for mesothelioma and 73% for all malignant pleural disease (mesothelioma plus metastatic carcinomas) compared with 82 and 67%, respectively, for cytological analysis alone. This adds confidence to clinicians and patients in cases where pleural fluid cytology is benign.
Pleural fluid concentration of any molecule is a dynamic balance between local production, plasma extravasation, and systemic absorption via pleural lymphatics. Few, if any, studies have examined the reproducibility or longitudinal changes of tumor markers in pleural effusions, as repeated pleural fluid sampling (unlike venipuncture) is difficult.
Our study is the first to show a high reproducibility of mesothelin measurement in pleural fluid—a prerequisite if pleural fluid mesothelin is to be employed for clinical use. The observed short-term variation in mesothelin values when samples collected within 7 days were compared is of little clinical significance (mean increase, 0.75 nM).
Pleurodesis is a standard management strategy for malignant pleural effusions. Talc, the most commonly used agent, acts by creating an acute pleural injury and has been shown to induce denudement of mesothelial cells (42) and apoptosis in mesothelioma cells (43). It is therefore possible that pleurodesis may induce a significant rise in mesothelin in pleural fluid and serum, and influence the role of mesothelin as a disease-monitoring tool. Likewise, bacterial infection usually induces intense pleural inflammation and mesothelial cell injury. However, in our cohort and that of others, patients with empyema had low pleural fluid mesothelin levels (7). This prompted us to investigate whether the presence of bacteria interferes with mesothelin measurement. Although frank empyema is uncommon in mesothelioma, patients often undergo a number of diagnostic and therapeutic pleural procedures with the adherent risk of transient bacterial spread in the pleural space.
Exactly how, on malignant transformation, mesothelial cells up-regulate their expression of mesothelin remains unknown. Our results show that talc, a potent inflammatory stimulus known to induce mesothelial release of a milieu of biological mediators, did not increase mesothelin production. Likewise, the mesothelin level did not correlate with, and could not be predicted by, results of conventional pleural fluid indicators of inflammation, for example, LDH and pH. Taken together, these data suggest that mesothelin is a robust marker for malignant mesothelioma not influenced by talc-induced pleural inflammation and not altered by the presence of bacteria.
Quantification of mesothelin can easily be incorporated into the work-up of pleural effusions. Alternatively, mesothelin is stable in storage and could be measured at a later stage if initial tests were uninformative.
In conclusion, pleural fluid mesothelin provides a valuable adjunct in the diagnostic assessment of patients presenting with pleural effusions, especially when cytological examination is not definitive, and can improve clinical practice. This test is reproducible and not influenced by common inflammatory pleural processes. The combined use of pleural fluid cytology and mesothelin may represent a useful initial test in patients with suspected mesothelioma and warrants further validation.
H.E.D., R.J.O.D., and Y.C.G.L. conceived and designed the study. H.E.D., N.A.M., N.M.R., and Y.C.G.L. enrolled patients and collected and compiled data. R.S.S., S.B., B.L.F., and Y.C.G.L. performed the ELISA and bacterial analyses. H.E.D. and Y.C.G.L. analyzed and interpreted the data. H.E.D. and Y.C.G.L. wrote the report. R.S.S., S.B., N.A.M., N.M.R., R.J.O.D., and B.L.F. commented on and revised the report. All authors approved the final version.
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