SciELO - Scientific Electronic Library Online

vol.62 issue8Colorectal anastomotic leakage at the University Hospital of the West Indies an analysis of risk factorsDifferences of ventricular late potential between acute STEMI and NSTEMI patients author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand



  • Have no cited articlesCited by SciELO

Related links

  • Have no similar articlesSimilars in SciELO


West Indian Medical Journal

Print version ISSN 0043-3144

West Indian med. j. vol.62 no.8 Mona Nov. 2013




Diagnostic value of p16 methylation for malignant pleural effusion a meta-analysis


Valor diagnóstico de la metilación p16 en el derrame pleural maligno un meta-análisis



M Li; S-J Guo; Y-C Shen; L-Q Jia; D-D Li; C Wan; F-Q Wen

Department of Respiratory Medicine, West China Hospital of Sichuan University, Chengdu, Sichuan, China and Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, Sichuan University, Chengdu 610041, PR China





OBJECTIVE: To evaluate the overall diagnostic performance of the p16 methylation for diagnosing malignant pleural effusion (MPE).
METHODS: All published literature in English and Chinese were reviewed. Sensitivity, specificity, likelihood ratio and diagnostic odds ratio (DOR) were pooled by using random-effects model or fixed-effects model. Summary receiver operating characteristic (SROC) curve was used to evaluate the overall diagnostic value.
RESULTS: Six studies were included with a total of 378 cases. The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and DOR of p16 methylation in the diagnosis of MPE were 0.41 [95% confidence interval (CI) 0.35, 0.48], 0.97 [95% CI 0.93, 0.99], 9.57 [95% CI 4.53, 20.20], 0.61 [95% CI 0.45, 0.82] and 19.82 [95% CI 8.35, 47.04], respectively. The area under the curve (AUC) was 0.864.
CONCLUSION: Pleural p16 methylation test plays a useful role in the diagnosis of MPE.

Keywords: Malignant pleural effusion, meta-analysis, p16 methylation


OBJETIVO: Evaluar el rendimiento diagnóstico general de la metilación p16 para el diagnóstico del derrame pleural maligno (DPM).
MÉTODOS: Se revisó toda la literatura publicada en inglés y chino. La sensibilidad, especificidad, razón de verosimilitud, y el odds-ratio diagnóstico (DOR) fueron agrupados mediante el modelo de efectos aleatorios o el modelo de efectos. La curva de las características operativas de resumen del receptor (SROC) fue usada para evaluar el valor diagnóstico general.
RESULTADOS: Se incluyeron seis estudios con un total de 378 casos. La sensibilidad, especificidad, razón de verosimilitud positiva (PLR), razón de verosimilitud negativa (NLR) y el DOR de la metilación p16 en el diagnóstico de DPM, fueron 0.41 [95% intervalo de confianza (IC) 0.35 0.48], 0.97 [95% IC 0.93, 0.99], 9.57 [95% IC 4.53, 20.20], [95% IC 0.45, 0.82] 0.61 y 19.82 [95% IC 8.35, 47.04], respectivamente. El área bajo la curva (AUC) fue 0.864.
CONCLUSIÓN: La prueba de metilación p16 pleural desempeña un papel útil en el diagnóstico del DPM.

Palabras claves: derrame pleural maligno, meta-análisis, metilación p16




Pleural effusion is commonly found in patients suffering from different kinds of diseases and sometimes it reveals the malignancy (1). Differentiation of malignant and benign pleural effusion is of great importance because of the significant difference in the treatment and prognosis (malignant pleural effusion (MPE) often means a poor prognosis and more aggressive treatment, such as an chemotherapy). Nevertheless, the establishment of an aetiological diagnosis of pleural effusion is often difficult and challenging. Current methods are far from perfect; they are either invasive or insufficient (2, 3). As a standard method for the diagnosis of MPE, classic cytology findings are positive in only 60% of cases on average, which can be increased slightly by performing closed pleural biopsy (4). As good as the diagnostic accuracy of the thoracoscopy is, this invasive procedure imposes physical and mental stress on the patients and may not be well accepted. Additionally, it is expensive and not available at all facilities (5).

Previous studies have investigated the diagnostic value of many tumour markers but only with limited success (6). Other factors like vascular endothelial growth factor [VEGF] (l) or telomerase have been studied but they are not as good as expected either (8). Therefore, some new effective and efficient pleural markers or methods should be identified to aid in the diagnosis of MPE.

Pl6 gene, an important tumour suppressor gene, plays an important role in regulating the cell cycle, and mutation in p16 increases the risk of developing a variety of cancers (9). Several epigenetic research has demonstrated that aberrant hypermethylation of pl6 gene is highly associated with carcinogenesis (10). Recent publications have also found the presence of promoter hypermethylation of pl6 genes in bodily fluids, including pleural fluid (11). It represents a noninvasive alternative or complementary test. Therefore, an increasing number of studies consider pl6 gene methylation test to be an effective and efficient way to diagnose MPE.

Although the accuracy of pl6 gene methylation detection for the diagnosis of MPE has been extensively studied, the precise diagnostic value remains unclear. This metaanalysis was performed to establish the overall accuracy of pleural p16 methylation detection in the diagnosis of MPE.



Search strategy and study selection

Databases including Medline (using PubMed as the search engine), Embase, Ovid, Web of Science and Cochrane Database (up to March 2012) were searched to identify relevant studies. References of articles were also searched manually. The search terms were "p16", "methylation", "malignant pleural effusion", “sensitivity and specificity" and "accuracy". The languages were limited to English or Chinese. Articles such as conference abstracts, letters to the journal editors and so on were excluded because of the limited data. A study was incorporated into the meta-analysis when both sensitivity and specificity of pl6 for the diagnosis of MPE were provided. Two reviewers (M Li and SJ Guo) independently judged study eligibility when screening the citations. Disagreement was resolved by consensus.

Data extraction and quality assessment

The included literature was evaluated independently by two reviewers (M Li and SJ Guo). The reviewers were blinded to publication details such as the author, the country, the name of the journal, etc. Patient characteristics, test method, cutoff value, sensitivity, specificity and methodological quality were retrieved from the included studies. To assess the trial methodology, we used the QUADAS (quality assessment for studies of diagnostic accuracy) tool (12).

Statistical analysis

We used standard methods recommended for the diagnostic accuracy of meta-analyses (13). We computed the following indices of test accuracy for each study: sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR).

The sensitivity and specificity of each single test threshold identified for each study were used to plot a summary receiver operating characteristic [SROC] (14, 15). Spearman's rank correlation was performed as a test for threshold effect. Heterogeneity was tested using the I2 with significance set at p < 0.05. The average sensitivity, specificity and other related measurements of the studies were calculated by using random-effects model or fixed-effects model (16, ll). If there were enough studies, subgroup analyses would be performed to explore potential between-study heterogeneity (18). Statistical softwares used to perform the analysis were: Stata, version 8.2 (Stata Corporation, College Station, TX), Meta-Test, version 0.6 (New England Medical Center, Boston, MA) and Meta-DiSc for Windows (XI Cochrane Colloquium, Barcelona, Spain).



After a search of the literature, 39 studies concerning p l6 methylation and pleural effusion were selected. Among them, 33 research papers were excluded because they went beyond the scope of the present study or due to unrelated, duplicated or inappropriate data. The remaining six studies were available for the meta-analysis, with a total number of 3l8 patients.

Quality of the literature and study characteristics

In our meta-analysis, the average sample size of the included studies was 65 (range 31 to 81). Diagnosis of MPE was made based on cytological or pathological findings, which are "gold standard". Among the six studies, four studies reported blinded interpretation of pl6 methylation assays independent of the reference standard and four studies were

designed as prospective study. All included studies had 0.41, 95% confidence interval (CI) 0.35, 0.48), while QUADAS scores > 10. The clinical characteristics and other specificity ranged from 0.87 to 1.00 (pooled 0.97, 95% CI information are outlined in Tables 1 and 2.


Diagnostic accuracy

The forest plots of sensitivity and specificity of p16 methylation assays for the diagnosis of MPE are shown in Fig. 1. The sensitivity varied between 0.17 and 0.69 (pooled 0.93, 0.99). The PLR was 9.57 (95% CI 4.53, 20.02), NLR was 0.6 (95% CI 0.45, 0.82) and DOR was 19.82 (95% CI 8.35, 47.04). The I2 values of sensitivity, specificity, PLR, NLR, and DOR were 87.9%, 57.3%, 0.0%, 87.4% and 0.0%, respectively. In the pooled analysis of sensitivity, specificity and PLR, random-effects model was used because the heterogeneity across studies showed significant difference (p < 0.05, Iz > 50%); when analysing the pooled DOR and NLP, fixed-effects model was performed because there was no significant heterogeneity between the studies (p > 0.05, I2 < 50%).



As is shown in Fig. 2, SROC curve was used to summarize overall test performance, which shows the tradeoff between sensitivity and specificity. Q-value is used as a global measure of test efficacy. It is the intersection point of the SROC curve with a diagonal line from the left upper corner to the right lower corner of the ROC space and corresponds to the highest common value of sensitivity and specificity for the test. In the meta-analysis, the maximum joint sensitivity and specificity of our study was 0.799 (the Q-value). The area under the curve (AUC) was 0.869, suggesting that the overall diagnostic value was not as high as expected.





The diagnosis of malignancy in pleural effusions continues to be a challenging clinical problem and traditional methods are limited (4). To find a new and effective diagnostic method for MPE will be of great importance. Detection of p16 methylation has been proposed as an alternative non-invasive way (10).

Our meta-analysis investigated the overall diagnostic performance of pleural effusion p16 methylation assay in the MPE with a specificity of 0.9l (95% CI 0.93, 0.99), indicating a promising role in confirming MPE. In contrast to the higher specificity, the sensitivity was only 0.41 (95% CI 0.35, 0.48) which is not sufficient to exclude non-MPE. Therefore, negative tests do not mean the absence of MPE, and patients with negative p16 methylation results still have a fairly high chance of having MPE. This trade-off has significant clinical implications.

To present a global summary of the test performance, the SROC curve was applied, which indicates the trade-off between sensitivity and specificity (25). The present metaanalysis based on SROC curve has shown the maximum joint sensitivity and specificity (Q value) was 0.199, and the AUC was 0.869, indicating that the overall accuracy was not as high as expected.

Diagnostic odds ratio, the ratio of the odds of positive test results in the diseased relative to the odds of positive test results in the non-diseased, is another indicator of test accuracy. The higher the DOR value, the better the discriminatory tests will perform (26). In our meta-analysis, we have found that the mean DOR was 19.82 (95% CI 8.35, 41.04), suggesting that the test seems to be a useful tool to aid in the diagnosis of MPE.

At the same time, both PLR and NLR are also presented as measures of diagnostic accuracy in our study (26) because likelihood ratios are considered to be more clinically meaningful (21, 28). A PLR value of 9.51 suggests that patients with MPE have an approximately 10-fold higher chance of being assay positive compared with patients without MPE, which is helpful in the clinical practices. On the other hand, a NLR value of 0.61 means that if the assay result was negative, the probability that this patient has MPE is approximately 61%, which is too high to rule out the MPE. In some cases of suspected malignancy and inconclusive initial findings, p16 methylation should be determined prior to the performance of invasive procedures, thereby optimizing the cost-benefit ratio. For patients, especially those who have clinical data suggesting an MPE but negative cytology on analysis of the pleural fluid, it is better to test their level of p16 methylation in pleural effusions. In this situation, a positive result of p16 methylation may indicate the need for biopsy.

It should be emphasized that our meta-analysis still has some limitations although comprehensive search strategy and data extraction were performed. Firstly, we did not include studies published in languages other than English or Chinese, nor did we include unpublished studies or abstracts from conferences, which may have led to publication bias. Secondly, only six studies with 318 cases were included, and the limited number of patients may have influenced the results of the meta-analysis. Because of that, we cannot use QUADAS scores to perform the meta-regression to analyse the effect of study quality on the relative DOR of p16 methylation in the diagnosis of MPE. Effects of other covariates on DOR (cross-sectional design, consecutive or random sampling of patients, blind design, prospective data collection and assay method) were also not analysed for the same reason.

In conclusion, the current evidence suggests that p16 methylation assay in pleural effusion may improve the ability to get the diagnosis of malignancy right although it falls short of perfection. It is helpful to guide the inclusion of patients who may benefit from further invasive pathologic examination.



This study was supported by grants 31171103 and 81230001 from the National Natural Science Foundation of China and grants 06-834 from the China Medical Board of New York to Dr FQ Wen.

Conflict of interest

The authors declare that we have no conflict of interest.



1. Udaya BS, Prakash MD, Reiman HN. Comparison of needle biopsy with cytological analysis for the evaluation of pleural effusion: analysis of 414 cases. Mayo Clin Proc 1985; 60: 158-64.

2. Maskell NA, Butland RJ; BTS guidelines for the investigation of a unilateral pleural effusion in adults. Thorax 2003; 58 (Suppl 2): ii8-ii17. doi:10.1136/thx.58.suppl_2.ii8.

3. Loddenkemper R. Medical thoracoscopy. In: Light RW, ed. Textbook of Pleural Diseases. 5th ed. Baltimore: Lippincott William and Wilkins; 2003: 498-512.

4. Bennett R, Maskell N. Management of malignant pleural effusions. Curr Opin Pulm Med 2005; 11: 296-300.

5. Johanston WW. Approach to the patient. In: Light RW, eds Pleural Diseases. 3rd ed. Baltimore: Lippincott Williams and Wilkins; 1995: 75-82.

6. Liang QL, Shi HZ, Qin XJ, Liang XD, Jiang J, Yang HB. Diagnostic accuracy of tumor markers for malignant pleural effusion: a metaanalysis. Thorax 2008; 63: 35-41.

7. Hamed EA, El-Noweihi AM, Mohamed AZ, Mahmoud A. Vasoactive mediators (VEGF and TNF-alpha) in patients with malignant and tuberculous pleural effusions. Respirology 2004; 9: 81-6.

8. Dikmen G, Dikmen E, Kara M, Sahin E, Dogan P, Ozdemir N. Diagnostic implications of telomerase activity in pleural effusions. Eur Respir J2003; 22: 422-6.

9. Ushijima T, Nakajima T, Maekita T. DNA methylation as a marker for the past and future. J Gastroenterol 2006; 41: 401-7.

10. Baylin SB, Esteller M, Rountree MR, Bachman KE, Schuebel K, Herman JG. Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer. Hum Mol Genet 2001; 10: 687-92.

11. Antony VB, Loddenkemper R, Astoul P, Boutin C, Goldstraw P, Hott J et al. Management of malignant pleural effusions. Eur Respir J 2001; 18: 402-19.

12. Whiting PF, Rutjes AW, Reitsma JB, Bossuyt PM, Kleijnen J. The development of QUADAS: a tool for the quality assessment of studies of diagnostic accuracy included in systematic reviews. BMC Med Res Methodol 2003; 3: 25.

13. Jones CM, Ashrafian H, Skapinakis P, Arora S, Darzi A, Dimopoulos K et al. Diagnostic accuracy meta-analysis: a review of the basic principles of interpretation and application. Int J Cardiol 2010; 140: 138-4. doi: 10.1016/j.ijcard.2009.05.063. Epub 2009 Jul 9.

14. Moses LE, Shapiro D, Littenberg B. Combining independent studies of a diagnostic test into a summary ROC curve: data-analytic approaches and some additional considerations. Stat Med 1993; 12: 1293-1316.

15. Lau J, Ioannidis JP, Balk EM, Milch C, Terrin N, Chew PW et al. Diagnosing acute cardiac ischemia in the emergency department: a systematic review of the accuracy and clinical effect of current technologies. Ann Emerg Med 2001; 37: 453-60.

16. Irwig L, Tosteson AN, Gatsonis C, Lau J, Colditz G, Chalmers TC. Guidelines for meta-analyses evaluating diagnostic tests. Ann Intern Med 1994; 120: 667-76.

17. Vamvakas EC. Meta-analyses of studies of the diagnostic accuracy of laboratory tests: a review of the concepts and methods. Arch Pathol Lab Med 1998; 122: 675-86.

18. Porcel JM, Vives M, Esquerda A, Salud A, Perez B, Rodriquez-Panadero F. Use of a panel of tumor markers (carcinoembryonic antigen, cancer antigen 125, carbohydrate antigen 15-3 and cytokeratin 19 fragments) in pleural fluid for the differential diagnosis of benign and malignant effusions. Chest 2004; 126: 1757-63.

19. Brock MV, Hooker CM, Yung R, Guo M, Han Y, Ames SE et al. Can we improve the cytologic examination of malignant pleural effusions using molecular analysis? Ann Thorac Surg 2005; 80: 1241-7.

20. Benlloch S, Galbis-Caravajal JM, Martín C, Sanchez-Paya J, Rodriguez-Paniagua JM, Romero S et al. Potential diagnostic value of methylation profile in pleural fluid and serum from cancer patients with pleural effusion. Cancer 2005; 15: 1859-65.

21. Katayama H, Hiraki A, Aoe K, Fujiwara K, Matsuo K, Maeda T et al. Aberrant promoter methylation in pleural fluid DNA for diagnosis of malignant pleural effusion. Int J Cancer 2007; 120: 2191-5.

22. Chen ML, Chang JH, Yeh KT, Chang YS, Chang JG. Epigenetic changes in tumor suppressor genes, P15, P16, APC-3 and E-cadherin in body fluid. Kaohsiung J Med Sci 2007; 23: 498-503.

23. Rha S, Lee S, Koo T, Shin BC, Huh JH, Um SJ et al. Diagnosis of malignant pleural effusion by using aberrant methylation of p16 and RARB2. Tuberc Respir Dis (Seoul) 2008; 64: 285-92.

24. Liu DY, Zhou FL, Hu ZJ, Hu HB. Value of detecting p16 gene methylation in the diagnosis of malignant pleural effusion. Med J Southern Medical Univ 2010; 30: 2148-50.

25. Moses LE, Shapiro D, Littenberg B. Combining independent studies of a diagnostic test into a summary ROC curve: data-analytic approaches and some additional considerations. Stat Med 1993; 12: 1293-1316.

26. Glas AS, Lijmer JG, Prins MH, Bonsel GJ, Bossuyt PM. The diagnostic odds ratio: a single indicator of test performance. J Clin Epidemiol 2003; 56: 1129-35.

27. Deeks JJ. Systematic reviews of evaluations of diagnostic and screening tests. In: Egger M, Smith GD, Altman DG, eds. Systematic reviews in health care: meta-analysis in context. London, UK: BMJ Publishing Group; 2001: 248-82.

28. Jaeschke R, Guyatt G, Lijmer J, Meade MO. Diagnostic tests. In: Guyatt G, Rennie D, eds. Users' guides to the medical literature: a manual for evidence-based clinical practice. Chicago, IL: AMA Press; 2002: 121-140.



Dr F-Q Wen
Department of Respiratory Medicine, West China Hospital of Sichuan University
Chengdu, Sichuan, China and
Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, Sichuan University
Chengdu 610041, PR China.