Diagnostic value of pleural fluid carbohydrate antigen 242 for malignant pleural effusion

Introduction

Malignant pleural effusion (MPE) refers to pleural effusion caused by primary pleural tumors or malignant tumors metastasising to the pleura, with the most common etiologies being lung cancer and breast cancer, which collectively account for 53% of all MPE cases (1-3). The differential diagnosis of MPE is challenging because pleural effusion is not a pathognomonic sign of MPE, as various benign diseases, such as tuberculous pleurisy and heart failure (HF), can also induce pleural effusion (3). The gold standard for MPE diagnosis remains pleural biopsy or cytological examination of the effusion. Although cytology is minimally invasive and provides rapid results, its sensitivity is suboptimal (approximately 58.2%), and its yield is susceptible to the experience of operators and observers (4). Consequently, patients with negative cytology results and a high probability of MPE still require a pleural biopsy. However, pleural biopsy is an invasive procedure that may lead to complications such as bleeding and infection (5,6).

The detection of tumor markers in pleural effusion can aid in the diagnosis of MPE (7). Its clinical significance lies in improving diagnostic efficiency. Specifically, elevated tumor marker levels indicate a high probability of MPE, and thus encourage pleural biopsy, or a repeated biopsy and a repeated effusion cytology when the diagnosis cannot be made after initial assessments (8,9). Although multiple tumor markers are currently available for diagnosing MPE, their overall accuracy remains far below the expectations of clinicians and patients (7,10,11). Therefore, continued research and development of new tumor markers are necessary to provide clinicians with more diagnostic tools and to establish the basis for multi-index combinations.

In 1989, carbohydrate antigen 242 (CA242), a sialylated carbohydrate chain antigen with a molecular weight ranging from 200 to 1,000 kDa, was discovered in the serum of patients with pancreatic cancer (12). Studies have shown that CA242 is expressed at very low levels in healthy individuals and is only minimally expressed in secretory glycoproteins of specific epithelial cells, such as the pancreatic ductal epithelium and colonic mucosa, where it participates in biological processes such as maintaining cell adhesion or mediating specific signaling pathways (12,13). Current research on CA242 as a tumor marker has focused primarily on its diagnostic and prognostic value in gastrointestinal cancers, such as pancreatic cancer (14) and colorectal cancer (15). However, studies investigating the role of CA242 in the diagnosis of MPE are scarce. A prior study enrolled 57 cases of MPE, 30 cases of tuberculous pleural effusion (TPE), and determined effusion CA242 with enzyme-linked immunosorbent assay (ELISA), and found that MPE patients had higher CA242 than TPE (16). Nevertheless, its findings were limited by its small sample size, single-centre retrospective design, and insufficient inclusion of broad aetiologies such as HF and parapneumonic pleural effusion (PPE), which may compromise accuracy (16). To address these limitations, this study expanded the sample size, comprehensively incorporated diverse aetiologies of pleural effusion, and re-evaluated the diagnostic accuracy of CA242 for MPE by measuring CA242 levels using electrochemiluminescence combined with receiver operating characteristic (ROC) curve analysis and decision curve analysis (DCA). We present this article in accordance with the STARD reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1185/rc).

Methods

Clinical trials registration

This study was prospectively registered at the Chinese Clinical Registry (www.chictr.org.cn). The registration number is ChiCTR1800017449, and the registration date is July 30, 2018.

Participants

Our study subjects were derived from the SIMPLE cohort (17). We prospectively recruited participants from Hohhot, China, at the Affiliated Hospital of Inner Mongolia Medical University (Hohhot cohort, from September 2018 to July 2021). The inclusion criterion for participants was an undiagnosed pleural effusion. The following participants were excluded: (I) participants with a history of pleural effusion within the last three months and whose corresponding aetiology was clear; (II) participants aged <18 years; (III) participants who were pregnant; (IV) participants with insufficient pleural fluid samples; (V) participants who developed pleural effusion during hospitalization; and (VI) participants with trauma- or surgery-induced pleural effusion. After admission, the patient’s pleural fluid was collected in a tube without anticoagulants. The sample was centrifuged (1,200 g, 10 min) within four hours of collection, and the supernatant was aliquoted and stored at temperatures between −80 and −70 ℃.

Ethics statement and informed consent

This study received approval from the ethics committees at the Affiliated Hospital of Inner Mongolia Medical University (No. 2018011). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from all participants.

Diagnostic criteria

MPE was diagnosed through pleural fluid cytology or a pleural biopsy. For patients with negative cytology results who were unable or unwilling to undergo pleural biopsy, MPE was clinically determined by combining objective evidence of a primary tumor (e.g., histopathologically or cytologically confirmed malignancy), malignancy-related clinical features (e.g., imaging findings of pleural nodules/thickening or pulmonary masses or bloody/exudative effusion with rapid progression), and exclusion of other benign causes of pleural effusion. Cardiogenic pleural effusion was diagnosed on the basis of a comprehensive evaluation of chest imaging features, clinical manifestations, laboratory tests [e.g., elevated N-terminal pro-B-type Natriuretic Peptide (NT-proBNP) levels], and a significant reduction in effusion volume following standardized anti-HF therapy. TPE was confirmed by positive Mycobacterium tuberculosis culture in the pleural fluid, acid-fast bacilli staining, or pleural biopsy. For patients lacking microbiological evidence but presenting high-probability of TPE [e.g., lymphocyte-predominant exudate with adenosine deaminase >35 U/L (18)], clinical diagnosis was supported by positive M. tuberculosis nucleic acid amplification testing and improved response to antituberculosis therapy. PPE was diagnosed via positive pleural fluid bacterial culture, antibiotic treatment response, imaging characteristics (e.g., loculation), or pleural biopsy. Two independent physicians reviewed medical records to assign diagnoses, with discrepancies resolved through discussion. To avoid bias, clinicians were blinded to the pleural fluid CA242 levels during diagnostic adjudication.

Pleural fluid CA242 and routine biochemical index assays

We recorded the levels of glucose, adenosine deaminase, leukocytes, lactate dehydrogenase, and protein in the patients’ pleural fluid from their medical records. We used the MAGLUMI 2000 (Shenzhen New Industries Biomedical Engineering Company, China) electrochemiluminescence analyser to detect CA242. As claimed by the manufacturer, the limit of blank (LoB), limit of detection (LoD), and limit of quantification (LoQ) (19) of this analyzer are less than 0.5, 0.7, and 0.8 U/mL, and the intra- and inter-day imprecision were 8% and 10%, respectively. The laboratory technicians who determined CA242 levels were unaware of the subjects’ clinical details.

Statistical analysis

Continuous data are presented as medians with interquartile ranges (IQR), while categorical data are shown as absolute numbers and percentages. We utilized the Kolmogorov-Smirnov test to assess the normality of the continuous data. The Mann-Whitney U test was applied for comparisons between two groups, and the Kruskal-Wallis H test was used for comparisons among more than two groups. The Chi-squared test was employed to compare categorical variables. Additionally, ROC curve analysis was conducted to evaluate the diagnostic accuracy of MPE (20). DCA was also performed to assess the net clinical benefit of the assay (21). All statistical analyses and graphical representations were conducted using R and GraphPad Prism 10. P<0.05 was considered statistically significant.

Results

Characteristics of the participants

A total of 170 patients were initially recruited. Among them, 11 were excluded because of uncertain pleural effusion, 2 withdrew informed consent, and 4 were excluded because of a history of malignancy. A total of 153 patients were included in the final analysis, comprising 66 patients with MPE and 87 patients with benign pleural effusion (BPE). The baseline characteristics of these patients were detailed in our prior study (7).

Differences in pleural fluid CA242 levels between patients with MPE and patients with BPE

As shown in Figure 1, the median (IQR) pleural fluid CA242 level was 12.22 (10.88–26.89) U/mL in MPE patients and 10.98 (10.13–11.86) U/mL in BPE patients. The CA242 levels in the pleural fluid was significantly greater in MPE patients than in BPE patients (P<0.001).

Figure 1 Differences in pleural CA242 levels between MPE and BPE patients in the Hohhot cohort. The Mann-Whitney U test used for Hohhot cohort. ****, P<0.001. BPE, benign pleural effusion; CA242, carbohydrate antigen 242; MPE, malignant pleural effusion.

We further investigated whether the aetiology of pleural effusion influenced pleural fluid CA242 levels. Figure 2A illustrates, the pleural fluid CA242 levels varied significantly across the benign aetiologies (P<0.001). Figure 2B shows no statistically significant differences in pleural fluid CA242 levels among MPE patients with underlying malignancies, including small-cell lung cancer, non-small-cell lung cancer, and other tumor types (P=0.28).

Figure 2 Pleural CA242 in patients with different types of pleural effusion in the Hohhot cohort. (A) CA242 level in BPE. (B) CA242 level in MPE caused by SCLC, NSCLC, and other MPE. The Kruskal-Wallis H test used for Hohhot cohort. Other MPE, other types of malignant pleural effusion. Others, other types of pleural effusion. BPE, benign pleural effusion; CA242, carbohydrate antigen 242; HF, heart failure; NSCLC, non-small cell lung cancer; PPE, parapneumonic pleural effusion; SCLC, small cell lung cancer; TPE, tuberculous pleural effusion.

Diagnostic performance of pleural fluid CA242 for MPE

Figure 3 shows the ROC curve of pleural fluid CA242 as a tumor marker for diagnosing MPE. In the study cohort, the area under the ROC curve (AUC) for pleural fluid CA242 in diagnosing MPE was 0.71 [95% confidence interval (CI): 0.63–0.80]. At a cut-off value of 11.46 U/mL, the sensitivity was 0.67 (95% CI: 0.55–0.77), and the specificity was 0.68 (95% CI: 0.58–0.77). Notably, when the pleural fluid CA242 threshold was set to 20 U/mL, the specificity for diagnosing MPE reached 1.00 (95% CI: 0.98–0.10), although the sensitivity decreased to 30% (95% CI: 0.21–0.42%).

Figure 3 Receiver operating characteristic curve of pleural CA242 for malignant pleural effusion. AUC, area under the curve; CA242, carbohydrate antigen 242; CI, confidence interval.

The DCA curve for pleural fluid CA242 as a tumor marker for diagnosing MPE (Figure 4) showed moderate yet consistent separation from the reference lines, indicating an intermediate net clinical benefit.

Figure 4 Decision curve analysis of pleural CA242. The X-axis represents the probability of MPE, which is calculated by a logistic regression model. The Y-axis represents the benefit of CA242 determination in patients with undiagnosed pleural effusion (21). CA242, carbohydrate antigen 242; MPE, malignant pleural effusion.

Discussion

The main findings of this study are as follows: (I) pleural fluid CA242 levels were significantly higher in patients with MPE than in those with BPE; (II) pleural fluid CA242 concentrations varied significantly across BPE patients with different etiologies; (III) pleural fluid CA242 demonstrated moderate diagnostic accuracy for MPE and net clinical benefit; and (IV) when pleural fluid CA242 levels exceeded 20 U/mL, the specificity for diagnosing MPE reached 100%. These results indicate that CA242 has moderate diagnostic value for MPE.

As a second study to evaluate the diagnostic performance of pleural fluid CA242 in differentiating MPE from BPE, our research offers several advantages over previous studies (16). First, we are the first to apply DCA to assess the net clinical benefit of pleural fluid CA242 testing. Net benefit and diagnostic accuracy are distinct concepts: diagnostic accuracy reflects the consistency between a diagnostic test and the reference standard in distinguishing disease from nondisease, but it does not reflect the test’s impact on clinical outcomes (21). Incorporating a diagnostic test into clinical workflows may benefit patients (e.g., true positives) or harm them (e.g., false positives) (22). How doctors value the benefit of a true positive and the harm of a false positive is determined by the threshold they adopt. For more details, please refer to a guideline proposed by Vicker AJ, the developer of DCA (22). Using DCA, we found that the decision curve for pleural fluid CA242 was consistently above the reference line, further confirming its diagnostic value. Notably, the net benefit of CA242 seems inferior to that of carcinoembryonic antigen (CEA), which has been introduced in our previous work (7). However, the specificity of CA242 was 1.00 at the threshold of 20 U/mL, supporting CA242 as a useful diagnostic marker for MPE. Second, while prior studies included only TPE patients, our study incorporated diverse etiologies, such as PPE and HF. Thus, our cohort is more representative, and the findings are more generalizable.

Numerous tumor markers, including CEA, cytokeratin 19 fragment (CYFRA 21-1), carbohydrate antigen 125 (CA125), and carbohydrate antigen 15-3 (CA15-3), have been investigated for MPE diagnosis (23). Previous studies have suggested that these markers typically achieved a sensitivity of approximately 0.50 and a specificity of >0.90 (24-26). The ROC curve of CA242 revealed that when the specificity of CA242 reached 90%, its sensitivity was approximately 40%. These findings suggest that the diagnostic accuracy of CA242 may be slightly inferior to that of conventional tumor markers such as CEA. Our previous study demonstrated that the AUC for pleural fluid CEA was 0.89, further supporting the conclusion that CA242 has poorer overall diagnostic performance than CEA does. While the AUC serves as an indicator of the overall accuracy of a diagnostic method, its clinical interpretability remains less straightforward (27,28). For tumor markers in MPE diagnosis, the threshold corresponding to 100% specificity is critical, as levels above this cut-off can definitively diagnose MPE, and pleural biopsy can be avoided if the patient is not amenable to active oncologic treatment (29). Previous studies identified thresholds such as CEA >50 ng/mL in nonturbid pleural fluid (30), CA50 >15 IU/mL (7), and CA72-4 >21 U/mL (11) for definitive MPE diagnosis. Our finding that pleural fluid CA242 >20 U/mL also confirms MPE diagnosis, expanding the understanding of tumor markers in this context. However, the sensitivity is only around 30% at this threshold, indicating that many MPE patients will be missed if CA242 is used alone. Further studies are needed to verify whether the combination of CA242 and other tumor markers can improve the diagnostic accuracy.

There are several limitations in this study. First, the sample size was small, and the mono-center design. Second, due to the cross-sectional design, we were unable to explore the prognostic value of pleural fluid CA242 in MPE patients. Third, we used frozen pleural fluid samples for CA242 measurement, but the long-term stability of pleural fluid CA242 in these frozen samples is still uncertain. In our previous study on CEA stability in pleural fluid, we revealed minimal effects after 1–3 years of storage at −80 ℃ to −70 ℃ (31). Fourth, serum CA242 levels were not assessed, as serum tumor markers generally exhibit lower diagnostic performance than pleural fluid markers do (8). Fifth, not all MPE patients were pathologically confirmed, although they were strictly diagnosed based on comprehensive clinical criteria (including imaging evidence, fluid characteristics, and exclusion of other benign causes). This diagnostic criterion may introduce potential bias. We categorized these patients as MPE because the representativeness of the cohort would be impaired if these patients were removed from data analyses.

Conclusions

In conclusion, our study demonstrates that pleural fluid CA242 has moderate diagnostic value for MPE and provides net clinical benefit. A pleural fluid CA242 level >20 U/mL can definitively diagnose MPE. Future prospective, large-scale, multi-center studies are warranted to validate these findings.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1185/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1185/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1185/prf

Funding: This work was supported by the Foundation of Inner Mongolia Medical University (No. YKD2022MS019), The Key Project of the Department of Education of Inner Mongolia Autonomous Region (No. NJZZ23008) and Inner Mongolia Medical University Affiliated Hospital Talent Training Project-Sailing Series (to Y.N.X.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1185/coif). The authors report that this work was supported by The Foundation of Inner Mongolia Medical University (No. YKD2022MS019), The Key Project of the Department of Education of Inner Mongolia Autonomous Region (No. NJZZ23008), Inner Mongolia Medical University Affiliated Hospital Talent Training Project-Sailing Series (to Y.N.X.). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study received approval from the ethics committees at The Affiliated Hospital of Inner Mongolia Medical University (No. 2018011). This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from all participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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