Abstract
Background
Some studies have assessed the diagnostic value of SHOX-2 methylation for malignant pleural effusion (MPE), but their findings were inconsistent. This study aimed to conduct a systematic review and meta-analysis to evaluate the diagnostic accuracy of SHOX-2 methylation for MPE.
Methods
We searched the PubMed and Web of Science databases to identify studies involving SHOX-2 methylation in diagnosing MPE. The last search date was in December 2024. We evaluated the quality of the included studies using the revised Quality Assessment of Diagnostic Accuracy Studies tool-2 (QUADAS-2). A bivariate model was used to pool the sensitivity, specificity, and their 95% confidence intervals (CIs) of SHOX-2 methylation. The overall diagnostic accuracy of SHOX-2 methylation was assessed by a summary receiver operating characteristic (sROC) curve. The publication bias was analyzed using Deek’s test.
Results
Six studies with 613 MPE patients and 723 benign pleural effusion (BPE) patients were included. The pooled sensitivity (95% CI) and specificity (95% CI) of SHOX-2 methylation were 0.64 (0.36–0.85) and 0.96 (0.92–0.98), respectively. The area under the sROC curve was 0.96 (95% CI: 0.94–0.97). There was no publication bias across the eligible studies.
Conclusions
SHOX-2 methylation has a moderate diagnostic value for MPE.
Keywords: SHOX-2 methylation, malignant pleural effusion (MPE), sensitivity, specificity, meta-analysis
Highlight box.
Key findings
• SHOX-2 methylation had a pooled sensitivity of 0.64 and a specificity of 0.96 for diagnosing malignant pleural effusion (MPE).
What is known and what is new?
• Studies indicated that SHOX-2 methylation in plasma is a diagnostic biomarker for lung cancer. What’s more, many studies have shown that SHOX-2 methylation in pleural fluid is useful for diagnosing MPE.
• This is the first meta-analysis evaluating the diagnostic accuracy of SHOX-2 methylation for MPE. We systematically reviewed all eligible studies and assessed their quality. The pooled sensitivity and specificity of SHOX-2 methylation were 0.64 and 0.96, respectively.
What is the implication, and what should change now?
• SHOX-2 methylation can be used to estimate the risk of MPE in patients with undiagnosed pleural effusion.
• Further studies are needed to address the diagnostic role of SHOX-2 in combination with other diagnostic tools.
Introduction
Pleural effusion is a common clinical sign that can be caused by various diseases (1). An epidemiological survey indicates that there are approximately 1.5 million new cases of pleural effusion each year in the United States (2). Pleural effusion is primarily categorized into benign pleural effusion (BPE) and malignant pleural effusion (MPE) (1). BPE primarily develops as a result of conditions such as pneumonia, tuberculosis, and heart failure (3). MPE is formed primarily by metastasis of the primary pleural tumor (4). Non-small cell lung cancer and breast cancer together account for approximately 50–60% of MPE; mesothelioma, renal cancer, and lymphoma also account for MPE (5). MPE indicates the late stage of cancer and a substantially poor outcome. Obtaining a definitive diagnosis of MPE is crucial because it is the prerequisite for MPE management (6).
The gold standard for MPE is effusion cytology and pleural biopsy (7,8). Cytology has a 100% specificity for diagnosing MPE, but the overall diagnostic sensitivity is approximately 60% (9,10). Pleural biopsy has a high diagnostic yield, but it is invasive (7,11-13) and can cause bleeding and infection-related complications (14). In contrast, biomarkers in pleural fluid are easy to perform and minimally invasive (13-15), which represent an alternative diagnostic tool (15,16).
SHOX, also known as short stature homeobox genes, is a group of genes closely related to bone formation, and mutations or deletions in SHOX genes can lead to poor bone development (17). The human SHOX-2 gene belongs to the SHOX gene family (17,18). It is essential to the development of proximal bones of the limbs, the humerus, and the femur (19). In addition, SHOX-2 participates in the development of the circulatory system (20). Studies implies that SHOX-2 methylation in plasma can be used as a diagnostic biomarker for lung cancer (21,22). Because plasma is a source of pleural fluid, and more than half of MPE is caused by lung cancer, it is reasonable to hypothesize that SHOX-2 methylation is a potential diagnostic marker for MPE. Indeed, in recent years, many studies have shown that SHOX-2 methylation in pleural fluid is useful for diagnosing MPE (23-28). However, the results varied, and to our knowledge, no systematic review and meta-analysis have investigated the diagnostic value of SHOX-2 methylation for MPE. Therefore, this systematic review used meta-analysis to pool the findings from available studies, aiming to establish robust evidence of the diagnostic role of SHOX-2 for MPE. We present this article in accordance with the PRISMA-DTA reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-686/rc).
Methods
Search strategy and databases
We searched PubMed and Web of Science databases. The last search date was in December 2024. The search algorithm in the PubMed was (“SHOX protein, human”[nm] OR “Short Stature Homeobox Protein”[nm] OR “Short Stature Homeobox Protein”[MESH] OR “SHOX2” OR “Short Stature Homeobox 2” OR “SHOX-2” OR “SHOX 2” OR “DNA Methylation”[MeSH]) AND (“Pleural Effusion”[MESH] OR “Pleural Effusions*” OR “Pleural fluid” OR “Pleur*”). The search formula for the Web of Science database was (“SHOX protein, human” OR “Short Stature Homeobox Protein” OR “Short Stature Homeobox Protein” OR “SHOX2” OR “Short Stature Homeobox 2” OR “SHOX-2” OR “SHOX 2” OR “DNA Methylation”) AND (“Pleural Effusion*” OR “Pleural fluid” OR “Pleur*”). Manual search with the references in the searched literature was also performed to identify potential studies.
Inclusion and exclusion criteria
The inclusion criteria for this systematic review and meta-analysis were: (I) the study provides the accuracy of SHOX-2 methylation in pleural fluid for diagnosing MPE; (II) the study reports sensitivity and specificity, allowing us to create a two-by-two table. The exclusion criteria were: (I) non-English publications; (II) animal studies; (III) reviews, conference abstracts, and commentaries.
Two systematic reviewers independently screened the retrieved studies. In the first round, they excluded obviously irrelevant studies by reviewing the titles and abstracts. The second round involved reading the full texts to verify eligibility.
Data extraction and quality assessment
We extracted the following data from the eligible studies: First author, year of publication, country, type of study, SHOX-2 assay, sample sizes of MPE and BPE, types of patients involved in MPE and BPE, diagnostic criteria for MPEs, the area under the curve (AUC) of SHOX-2 methylation, sensitivity and specificity, and cut-off values. A two-by-two table was constructed according to sensitivity, specificity, and the sample sizes of MPE and BPE. The two-by-two table includes true positive (TP), false positive (FP), false negative (FN), and true negative (TN).
We assessed the quality of the included studies using the revised Quality Assessment Tool for Diagnostic Accuracy Studies tool-2 (QUADAS-2) (29). Two system reviewers independently conduct a quality assessment. Any disagreements were resolved by consensus.
Statistical analysis
We calculated the pooled specificity and sensitivity of SHOX-2 methylation with a bivariate model (30). We assessed the diagnostic accuracy of SHOX-2 methylation for MPE with a summary receiver operating characteristics (sROC) curve (31). The inconsistency index (I2) was used to reflect the magnitude of heterogeneity across studies (32). A Fagan plot was used to calculate positive predictive value (PPV) and negative predictive value (NPV) at different prevalence rates. The Deek’s test was used to assess the presence of publication bias across all eligible studies (33). All statistical analyses were performed with Stata 18.0 (Stata Corp LP, College Station, TX, USA).
Results
Characteristics of the included studies
Figure 1 is a flowchart of the study selection. Six studies were included, with 613 MPEs and 723 BPEs. Table 1 shows the characteristics of the eligible studies. Four studies were from China (23-26), two were from Germany (27,28). Two studies were retrospective (27,28), two studies were prospective (24,26), and the type of data collection was not reported in the remaining two studies (23,25). All studies used cytology or pleural biopsy to diagnose MPE (23-28).
Figure 1.
A flowchart of the study selection.
Table 1. Characteristics of the eligible studies.
| Author | Year | Country | Study design | SHOX-2 assay | MPE/BPE | MPE | BPE | MPE reference standard |
|---|---|---|---|---|---|---|---|---|
| Zhang et al. (23) | 2024 | China | NR | MS-PCR | 45/35 | LC, SCC, MPM | PPE, TPE, others | Biopsy, cytology |
| Zhang et al. (25) | 2023 | China | NR | MS-PCR | 50/45 | LC, OC, Ly, MPM, SCC, CA, EC | PPE, TPE, HF | Biopsy, cytology, histopathology |
| Zhong et al. (24) | 2023 | China | Prospective | MS-PCR | 104/110 | LC, BC, GC, Ly, others | PPE, TPE, others | Biopsy, cytology, histopathology |
| Liang et al. (26) | 2022 | China | Prospective | MS-PCR | 100/48 | LC, BC, GC, OC, MPM, others | PPE, TPE, others | Biopsy, histological |
| Ilse et al. (27) | 2013 | Germany | Retrospective | MS-PCR | 276/443 | LC, BC, GC, MPM, Ly, others | IF, HF, others | Biopsy, cytology |
| Dietrich et al. (28) | 2013 | Germany | Retrospective | MS-PCR | 38/42 | LC, BC, OC, Ly, others | PPE, IF, HF, others | Biopsy, cytology, histological |
BC, breast cancer; BPE, benign pleural effusion; CA, cystadenocarcinoma; EC, esophageal cancer; GC, gastric cancer; HF, heart failure; IF, infectious disease; LC, lung cancer; Ly, lymphoma; MPE, malignant pleural effusion; MPM, malignant pleural mesothelioma; MS-PCR, methylation-specific polymerase chain reaction; NR, not reported; OC, ovarian cancer; PPE, parapneumonic pleural effusion; SCC, thymic squamous cell carcinomas; SHOX-2, short stature homeobox 2; TPE, tuberculous pleural effusion.
Quality assessment
Table 2 shows the quality assessment of the included studies. Three studies were high risk regarding patient selection (24,27,28). Two studies were labelled as unknown in terms of patient selection because they did not report the type of data collection (prospective or retrospective) (23,25). Three studies had high risk in the index domain because they used data-driven thresholds to define positive SHOX-2 (25-27). The flow and timing domain of five studies was labelled as high risk because not all patients were included (23,25-28).
Table 2. Quality assessment of the eligible studies.
| Author | Risk of bias | Applicability concerns | ||||||
|---|---|---|---|---|---|---|---|---|
| Patient selection | Index test | Reference standard | Flow and timing | Patient selection | Index test | Reference standard | ||
| Zhang et al. (23) | NR | Low | Low | High | Low | Low | Low | |
| Zhang et al. (25) | NR | High | Low | High | Low | Low | Low | |
| Zhong et al. (24) | High | Low | Low | Low | Low | Low | Low | |
| Liang et al. (26) | Low | High | Low | High | Low | Low | Low | |
| Ilse et al. (27) | High | High | Low | High | Low | Low | Low | |
| Dietrich et al. (28) | High | Low | Low | High | Low | Low | Low | |
NR, not reported.
Diagnostic accuracy of SHOX-2 methylation for MPE
Table 3 summarizes the diagnostic accuracy of SHOX-2 methylation for MPE. The AUC of SHOX-2 methylation in all the eligible studies ranged from 0.65 to 0.96, the sensitivity ranged from 0.18 to 0.93, and the specificity ranged from 0.91 to 1.
Table 3. Diagnostic accuracy of SHOX-2 methylation in the eligible studies.
| Authors | AUC (95% CI) | Cut-off | Sensitivity (95% CI) | Specificity (95% CI) | TP | FP | FN | TN |
|---|---|---|---|---|---|---|---|---|
| Zhang et al. (23) | 0.96 (0.90–1.00) | 10 | 0.93 (0.81–0.98) | 0.94 (0.80–0.99) | 42 | 2 | 3 | 33 |
| Zhang et al. (25) | 0.96 (NR) | 10 | 0.90 (0.77–0.96) | 1.00 (0.90–1.00) | 45 | 0 | 5 | 45 |
| Zhong et al. (24) | 0.65 (NR) | 9 | 0.61 (NR) | 0.91 (NR) | 63 | 10 | 41 | 100 |
| Liang et al. (26) | 0.71 (0.61–0.80) | 9 | 0.56 (NR) | 0.96 (NR) | 56 | 2 | 44 | 46 |
| Ilse et al. (27) | 0.72 (0.68–0.76) | 7.5 | 0.40 (0.34–0.46) | 0.96 (0.94–0.98) | 109 | 17 | 167 | 426 |
| Dietrich et al. (28) | NR | 10 | 0.18 (NR) | 1.00 (NR) | 7 | 0 | 31 | 42 |
AUC, area under the curve; CI, confidence interval; FN, false negative; FP, false positive; NR, not reported; SHOX-2, short stature homeobox 2; TN, true negative; TP, true positive.
Figure 2 is a forest plot of SHOX-2 methylation for diagnosing MPE. The pooled sensitivity (95% confidence interval, 95% CI) and specificity (95% CI) were 0.64 (0.36–0.85) and 0.96 (0.92–0.98), respectively. The I2 for sensitivity and specificity were 0.96 and 0.86 (P<0.001 for both), respectively. The positive likelihood ratio (PLR) was 15.50 (7.40–32.70), and the negative likelihood ratio (NLR) was 0.38 (0.18–0.78). The diagnostic odds ratio (DOR) was 41 [12, 146].
Figure 2.
A forest plot for diagnosing MPE by SHOX-2 methylation in pleural fluid. CI, confidence interval; df, degrees of freedom; I2, inconsistency index; MPE, malignant pleural effusion; NR, not reported; Q, Cochran’s Q statistic.
Figure 3 shows the sROC curve of SHOX-2 methylating in the pleural fluid. The AUC of the sROC was 0.96 (0.94–0.97).
Figure 3.
sROC curve of SHOX-2 methylation in the diagnosis of MPE. AUC, area under the ROC curve; MPE, malignant pleural effusion; ROC, receiver operating characteristic; SENS, sensitivity; SPEC, specificity; sROC, summary receiver operating characteristic.
The Fagan plot of SHOX-2 methylation is shown in Figure 4. When the prevalence of MPE is 24%, the probability of a participant with a positive SHOX-2 methylation is 83%.
Figure 4.
Fagan diagram of SHOX-2 methylation for MPE. LR, likelihood ratio; MPE, malignant pleural effusion.
Publication bias
The funnel plot showed no significant publication bias across eligible studies (P=0.15, Figure 5).
Figure 5.
The funnel plot assessment of potential publication bias. ESS, effective sample size.
Discussion
To our knowledge, there are no systematic reviews or meta-analyses that investigate the diagnostic accuracy of SHOX-2 methylation for MPE. This meta-analysis included a total of 1,336 patients from six studies. The findings revealed a pooled sensitivity of 0.64 (95% CI: 0.36–0.85) for SHOX-2 methylation in diagnosing MPE, indicating a 36% chance of missed diagnoses. The pooled specificity was found to be 0.96 (95% CI: 0.92–0.98), suggesting a low probability of misdiagnosis. Additionally, the AUC for the summarized receiver operating characteristic curve (sROC) was 0.96 (95% CI: 0.94–0.97). There was no evidence of publication bias among the studies included in the analysis. These results demonstrate that SHOX-2 methylation is an auxiliary diagnostic marker for MPE.
Sensitivity and specificity are crucial indicators of diagnostic accuracy tests; however, their values are threshold dependent (34,35). In contrast, the AUC of the sROC is not affected by the cut-off value. It, therefore, provides a comprehensive picture of the overall accuracy of a diagnostic tool (36). The AUC value ranges from 0.5 to 1.0, with higher values indicating more accurate diagnostic methods. This study revealed that the AUC of SHOX-2 methylation for the MPE was 0.96 (95% CI: 0.94–0.97), suggesting its high overall diagnostic accuracy for detecting MPE. Additionally, the DOR combines the strengths of sensitivity and specificity, making it a reliable indicator of diagnostic performance (37). The DOR ranges from zero to positive infinity. Higher values indicate better diagnostic performance (37). Our meta-analysis revealed a DOR of 41 [12, 146], suggesting that positive SHOX-2 methylation is 41 times over BPE. The PLR and NLR are important indicators used to confirm or exclude the target disease. A PLR greater than 10 and an NLR less than 0.1 is generally considered strong evidence to confirm or exclude a diagnosis (38,39). Our study found SHOX-2 methylation had a PLR of 15.50 (7.40–32.70) and an NLR of 0.38 (0.18–0.78), suggesting that methylation of the SHOX-2 gene can assist in the diagnosis of MPE, and its diagnostic value should be interpreted in the clinical context.
So far, numerous studies have investigated the diagnostic performance of conventional biomarkers for MPE, such as carcinoembryonic antigen (CEA), cancer antigen (CA) 15-3, CA 19-9, CA 125, cytokeratin fragment (CYFRA), and neuron-specific enolase (NSE) (15,40). These studies have shown that the pooled sensitivities of these biomarkers for MPE are around 0.50, and the pooled specificities are around 0.90 (41). Our meta-analysis revealed that the SHOX-2 methylation exhibited a pooled sensitivity of 0.64 and a pooled specificity of 0.96 for diagnosing MPE. Compared to conventional biomarkers, the SHOX-2 methylation showed slightly improved sensitivity and specificity. Therefore, SHOX-2 methylation may serve as a more promising tumor biomarker. Someone may ask the role of SHOX-2 methylation determination because no soluble markers in pleural fluid can adequately confirm MPE, and pleural biopsy is unavoidable for nearly all patients with undiagnosed pleural effusion. However, we believe that SHOX-2 methylation determination is of value because it can be used to estimate the risk of MPE before pleural biopsy, and positive findings encourage further pleural biopsy, even repeated biopsy if the initial findings are inconclusive (42).
We noted that three of the included studies also assessed the diagnostic accuracy of SHOX-2 in combination with Ras Association Domain Family 1 Isoform A (RASSF1A) methylation for diagnosing MPE, and two revealed improved diagnostic accuracy (23-25). In addition, one study investigated the combination of SHOX-2 and the septin gene family (SEPT) gene methylation for the diagnosis of MPE, and the combination improved the diagnostic sensitivity (28). Future studies still need to investigate the diagnostic accuracy of SHOX-2 in combination with multiple markers, signs, and symptoms.
We used the QUADAS-2 tool to evaluate the quality of the included studies and found some weaknesses in the study design of previous studies (29). Most studies did not include all participants in the final analyses, which may bias the findings. For example, two studies showed that SHOX-2 methylation was undetectable in some participants, suggesting that the SHOX-2 methylation assay needs to be improved (27,28). Some studies categorized patients with pleural effusion into MPE, BPE, and undetermined pleural effusion, and only MPE and BPE were included in the analysis. Excluding participants with undetermined pleural effusion may impair the representativeness of the participants, thus biasing the findings. There was also an inappropriate exclusion in some studies, which may have affected participants’ representativeness. Finally, the cut-off in most studies was data-driven, which may overestimate the diagnostic accuracy of SHOX-2 methylation (43).
This study represents the first meta-analysis of pleural fluid SHOX-2 for diagnosing MPE; however, it has limitations. First, the number of included studies and the overall sample size were relatively small, indicating that the precision of the results needs to be improved. Additionally, the small number of included studies does not allow us to study the sources of heterogeneity with meta-regression or subgroup analyses. Still, the included studies have some weaknesses in study design, which may affect the reliability of their findings, as well as the findings of this meta-analysis. Therefore, further studies with rigorous design are needed to reevaluate the diagnostic accuracy of SHOX-2 methylation.
Conclusions
This meta-analysis suggests that pleural fluid SHOX-2 methylation has a moderate diagnostic value for MPE. Due to the limited number of included studies, the precision of the study needs to be further improved. In addition, further studies with rigorous design are needed to address the diagnostic accuracy of SHOX-2 methylation in the future.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
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.
Footnotes
Reporting Checklist: The authors have completed the PRISMA-DTA reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-686/rc
Funding: This work was supported by the Foundation for Central Government Guiding Local Government’s Science and Technology Development (No. 2022ZY0203).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-686/coif). The authors have no conflicts of interest to declare.
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