Abstract OBJECTIVES The study aimed to assess the combination of DNA ploidy analysis (DPA) and the expression of microRNA-21 (miR-21) or microRNA-24 (miR-24) in the detection of malignant pleural effusion (MPE). METHODS In this prospective research, a total of 40 samples (20 benign and 20 malignant effusions), flexural effusion exfoliated cells and cell-free miR-21 and miR-24 were collected. DPA and exfoliative cytology examinations were conducted to diagnose flexural effusion exfoliated cells. Quantitative reverse transcriptase polymerase chain reaction was carried out to measure the expressions of miR-21 and miR-24. Receiver operating characteristic curve and the area under the curve were applied to evaluate the accuracy rate of different diagnostic approaches on MPE. RESULTS In the MPE group, DPA demonstrated a higher rate of accuracy in MPE diagnosis than exfoliative cytology. The expressions of miR-21 and miR-24 were significantly higher in MPE than in benign pleural effusion (P < 0.05). Furthermore, area under the curve, sensitivity and specificity were 0.942, 95% and 90% for the combination of miR-21 and DPA and 0.973, 100% and 80% for the union of miR-24 and DPA, respectively, representing a significant improvement in both accuracy and sensitivity. Therefore, the combination of DPA and miR-21 or miR-24 appears to be a better biomarker for discriminating MPE from benign pleural effusion. CONCLUSIONS The combination of DPA and miR-21 or miR-24 may function as a promising diagnostic tool of MPE. Registration number ChiCTR-TRC-14004719 DNA ploidy analysis, miR-21, miR-24, Malignant pleural effusion, Exfoliative cytology INTRODUCTION Pleural effusion is a common condition of many diseases  and its aetiologies include malignant neoplasms, pulmonary infection, fluid retention states, chylothorax as well as other less common causes . Many diseases with malignant pleural effusion (MPE) are systemic diseases, thus patients cannot benefit from local therapy such as surgical resection or radiotherapy . However, early detection and treatment can dramatically increase the survival rate of patients. Therefore, the rapid diagnosis of MPE biomarkers is vitally important, indicating the existence of malignant tumours. However, the present diagnosis is mainly achieved through complicated clinical management. What’s worse, exfoliative cytology (EC) examination for pleural effusion is often regarded as the initial and the only approach to obtain a diagnosis,  with a poor sensitivity (between 16% and 75%) . Therefore, we attempted to find a more effective and sensitive diagnostic method for earlier confirmation of MPE. MicroRNAs (miRNAs), endogenous non-coding small RNAs, are composed of approximately 22 nucleotides. It has been proved that miRNAs take part in the changes of gene expressions by binding to the 3ʹ untranslated region of targeted messenger RNAs , and the altered expressions of miRNAs are associated with carcinogenesis or carcinostasis . In many research studies, miRNAs were demonstrated as diagnostic biomarkers in MPE, such as miR-21 and miR-24 , and they can also be used in the diagnosis of many other diseases including glioma, ovarian carcinoma  and malignant mesothelioma . Although the diagnostic performance of a single miRNA was not as effective as is desired, there are few research studies combining miRNAs with other methods in MPE diagnosis. DNA ploidy analysis (DPA) is a long-standing technique of early cancer screening generally divided into 2 methods: flow cytometry analysis  and image cytometry analysis . Quantitative measurements of cell-free DNA were previously applied to prenatal diagnosis, acute trauma examination, cancer testing and monitoring of transplantation [12, 13]. Chan et al.  concluded that DPA was an effective way to determine the aetiological causes of pleural effusions, and this has been confirmed by further studies. For instance, Fiegl et al.  used fluorescence in situ hybridization analysis for aneuploidy on tumour cell detection in 2004 and Osterheld et al.  verified the role played by DPA in pleural effusions in 2005. In this study, we aimed to discriminate MPE from benign pleural effusion (BPE) based on the analyses of EC, DPA, miR-21 and miR-24 in pleural effusion and combined several diagnostic methods to detect the best biomarker of MPE. MATERIALS AND METHODS Patients and pleural fluid collection In this prospective study, a total of 40 pleural effusion patients (15 female and 25 male) visiting the Second People’s Hospital of Yueyang between March and September 2015 were randomly divided into 2 groups, MPE group (n = 20) and BPE group (n = 20). All patients were evaluated by EC examination with results confirmed by histological test subsequently, and the diagnoses were then reconfirmed by 2 pathologists . This study was approved by the Second People’s Hospital of Yueyang. Exfoliative cytology examination Before examination, all patients received standard thoracentesis. Ten millilitres of pleural fluid and 2 ml surrounding blood were collected, and the pleural fluid samples were separated into 2 parts. The samples were preserved at indoor temperature and analysed by EC examination within 2 h. DNA ploidy analysis All DNA-stained Feulgen slides  were analysed with the MotiCytometer automatic cell image analysis system (Motic Medical Diagnostic Systems Co., Xiamen, China), and pleural fluid cells were pelleted after centrifugation. After completing the cell preservation solution, Feulgen staining was performed for DPA. On the basis of the automatic analysis, the results illustrated 3 possibilities: First, when DNA index = 1, the cell was diploid (2C). No heteroploid cell or abnormal cell peak existed. Second, when DNA index = 2, the cell was tetraploid (4C). With no visible heteroploid cell, the sample was diagnosed as a case of hyperplasia. Third, when more than 3 cells measured DNA index ≥ 2.5 or when aneuploidy cells were observed, the specimen was diagnosed as positive. Quantitative reverse transcriptase polymerase chain reaction The expression levels of miR-21 and miR-24 were analysed via quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The miRNAs were extracted from the pleural fluid with ABI miRNA extraction kit (Applied Biosystems, Foster City, CA, USA), and their purity and concentration were detected using protein nucleic acid spectrophotometer (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). They were then reverse transcribed to complementary DNAs using RT kit (Tokara, Tokyo, Japan). The primer of miR-21 was 5ʹ-TAGCTTATCAGACTGATGTTGA-3ʹ and that of miR-24 was 5ʹ-TGGCTCAGTTCAGCAGGAACAG-3ʹ. The internal control U6 was 5ʹ-GCTTCGGCAGCACATATACTAAAAT-3ʹ. In qRT-PCR, the relative quantity expressions of 2 miRNAs were calculated using the 2−ΔΔCt method and normalized to the expression of U6 small nuclear RNA (snRNA) (ΔCt=CtmiRNA−CtU6snRNA). All the above experiments were repeated 3 times. Statistical analysis SPSS 18.0 (SPSS Inc., Chicago, IL, USA) and MedCalc v14.8.1 (MedCalc Software, Inc., Mariakerke, Belgium) were used for statistical analyses, and differences between 2 groups were analysed using the t-test. Validation set and cross-validation were conducted to verify the research as shown in Supplementary Material, Fig. S1 and Supplementary Material, Tables S1–S4. The receiver operating characteristic (ROC) curve was used to evaluate the ability of several diagnosis methods in MPE. The area under the curve (AUC), 95% confidence interval (95% CI) and the Youden index (YI) were also calculated to assess the accuracy and diagnostic value of different methods. In addition, the data of DPA, EC and qRT-PCR were used as predictor variables in binary logistic regression. In this study, the P-value <0.05 was regarded as statistically significant, and data were summarized in the form of mean ± standard deviation. YI < 0.6 was regarded as poor diagnostic value, YI∈(0.6, 0.7) was considered as medium value and YI > 0.8 was viewed as being of great value. RESULTS Patient characteristics After histological or cytological confirmation, 40 inpatients with pleural effusion were randomly selected and enrolled in this study, 20 with MPE and 20 with BPE. The basic characteristics of patients and their aetiologies are illustrated in Table 1. Table 1: Patient demographics and their aetiology Items Type of effusion MPE (n = 20) BPE (n = 20) Male/female 12/8 13/7 Age (years) Range 44–70 26–68 Median 60.00 ± 8.026 52.15 ± 13.624 Aetiology (n) Lung adenocarcinoma (9) Pulmonary tuberculosis (9) Lung squamous carcinoma (4) Pneumonia (6) Small-cell lung carcinoma (2) Cardiac failure (3) Breast cancer (2) Cirrhosis of liver (2) Malignant lymphoma (2) Malignant pleural mesothelioma (1) Items Type of effusion MPE (n = 20) BPE (n = 20) Male/female 12/8 13/7 Age (years) Range 44–70 26–68 Median 60.00 ± 8.026 52.15 ± 13.624 Aetiology (n) Lung adenocarcinoma (9) Pulmonary tuberculosis (9) Lung squamous carcinoma (4) Pneumonia (6) Small-cell lung carcinoma (2) Cardiac failure (3) Breast cancer (2) Cirrhosis of liver (2) Malignant lymphoma (2) Malignant pleural mesothelioma (1) BPE: benign pleural effusion; MPE: malignant pleural effusion. Logistic regression analysis The results for DPA, EC examination and qRT-PCR were used as predictor variables in logistic regression analyses. The logit (P) was calculated to detect performance value, ROC curves were plotted as shown in Fig. 1A–D and the regression constants were demonstrated (Table 2). Table 2: The result of logit (P) regression Variables b SE Wald (χ2) P-value EC + DPA EC −0.087 1.32574 21.639 0.9477 DPA 3.526 1.259 0.0047 miR-21 + miR-24 miR-21 0.096 0.053 46.528 0.072 miR-24 0.126 0.059 0.033 DPA + miR-21 DPA 3.111 1.155 34.055 0.007 miR-21 0.077 0.031 0.014 DPA + miR-24 DPA 3.944 1.409 38.224 0.005 miR-24 0.094 0.059 0.111 Variables b SE Wald (χ2) P-value EC + DPA EC −0.087 1.32574 21.639 0.9477 DPA 3.526 1.259 0.0047 miR-21 + miR-24 miR-21 0.096 0.053 46.528 0.072 miR-24 0.126 0.059 0.033 DPA + miR-21 DPA 3.111 1.155 34.055 0.007 miR-21 0.077 0.031 0.014 DPA + miR-24 DPA 3.944 1.409 38.224 0.005 miR-24 0.094 0.059 0.111 b: regression coefficient maximum likelihood estimate; DPA: DNA ploidy analysis; EC: exfoliative cytology; miR-21: microRNA-21; miR-24: microRNA-24; SE: estimated value of standard error; Wald (χ2): Wald test statistical values. Figure 1: View largeDownload slide ROC curve analysis. The receiver operating characteristic plots for DPA, EC and DPA+EC (A); miR-21, miR-24 and miR-21 + miR-24 (B); DPA, miR-21 and DPA + miR-21 (C) and DPA, miR-24 and DPA + miR-24 (D) were used to differentiate malignant pleural effusion from benign pleural effusion. DPA: DNA ploidy analysis; EC: exfoliative cytology; miR-21: microRNA-21; miR-24: microRNA-24. Figure 1: View largeDownload slide ROC curve analysis. The receiver operating characteristic plots for DPA, EC and DPA+EC (A); miR-21, miR-24 and miR-21 + miR-24 (B); DPA, miR-21 and DPA + miR-21 (C) and DPA, miR-24 and DPA + miR-24 (D) were used to differentiate malignant pleural effusion from benign pleural effusion. DPA: DNA ploidy analysis; EC: exfoliative cytology; miR-21: microRNA-21; miR-24: microRNA-24. Diagnostic performances of DNA ploidy analysis and exfoliative cytology DPA showed more accurate MPE diagnosis than EC. As presented in Table 3, among 20 MPE samples DPA detected 17 MPE-positive cases, whereas EC detected only 13 (P < 0.01). In Fig. 1A and Table 4, the ROC curve showed that the AUC of DPA was 0.85 (95% CI 0.702–0.943), whereas EC (95% CI 0.561–0.854) was only 0.725 and the combination of DPA and EC only increased by 0.001 (95% CI 0.703–0.944). Both the sensitivity and specificity of DPA were 85%, whereas the sensitivity and specificity of EC was 55% and 90%, respectively. The joint detection of EC and DPA did not show obvious differences from that of DPA. Further, YI index also illustrated poor diagnostic value of EC. Therefore, we came into the conclusion that the diagnostic performance of DPA was significantly superior to EC. Table 3: Results of EC, DPA, miR-21 and miR-24 in malignant and benign effusions Positive EC Positive DPA miR-21 miR-24 MPE (n = 20) 13 17 69.143 ± 41.233 73.358 ± 80.771 BPE (n = 20) 2 3 15.040 ± 9.515 16.322 ± 13.031 P-value 0.0008 <0.0001 <0.0001 0.0054 Positive EC Positive DPA miR-21 miR-24 MPE (n = 20) 13 17 69.143 ± 41.233 73.358 ± 80.771 BPE (n = 20) 2 3 15.040 ± 9.515 16.322 ± 13.031 P-value 0.0008 <0.0001 <0.0001 0.0054 BPE: benign pleural effusion; DPA: DNA ploidy analysis; EC: exfoliative cytology; miR-21: microRNA-21; miR-24: microRNA-24; MPE: malignant pleural effusion. Table 4: Area under the ROC curve and the best cut-offs with relative to sensitivity and specificity of the 4 diagnosis methods on MPE and their combination Diagnosis AUC 95% CI Sensitivity (%) Specificity (%) Youden index DPA 0.85 0.702–0.943 85 85 0.7 EC 0.725 0.561–0.854 55 90 0.45 DPA + EC 0.851 0.703–0.944 85 85 0.7 miR-21 0.874 0.730–0.957 80 95 0.75 miR-24 0.86 0.714–0.949 80 95 0.75 miR-21 + miR-24 0.875 0.732–0.958 80 95 0.75 DPA + miR-21 0.973 0.864–0.999 95 90 0.85 DPA + miR-24 0.942 0.820–0.991 100 80 0.8 Diagnosis AUC 95% CI Sensitivity (%) Specificity (%) Youden index DPA 0.85 0.702–0.943 85 85 0.7 EC 0.725 0.561–0.854 55 90 0.45 DPA + EC 0.851 0.703–0.944 85 85 0.7 miR-21 0.874 0.730–0.957 80 95 0.75 miR-24 0.86 0.714–0.949 80 95 0.75 miR-21 + miR-24 0.875 0.732–0.958 80 95 0.75 DPA + miR-21 0.973 0.864–0.999 95 90 0.85 DPA + miR-24 0.942 0.820–0.991 100 80 0.8 AUC: area under the curve; CI: confidence interval; DPA: DNA ploidy analysis; EC: exfoliative cytology; ROC: receiver operating characteristic; miR-21: microRNA-21; miR-24: microRNA-24; MPE: malignant pleural effusion. Expression level and diagnostic utility of cell-free miR-21 and miR-24 in pleural effusion The expression levels of miR-21 and miR-24 were significantly increased in MPE in comparison with in BPE, and the diagnostic ability of miR-21, miR-24 as well as the combination of both proved similarly effective. Observation through qRT-PCR for pleural effusion cell-free miRNAs revealed that the expressions of miR-21 and miR-24 were 69.143 ± 41.233 and 73.358 ± 80.771 in MPE compared with 15.040 ± 9.515 and 16.322 ± 13.031 in BPE, respectively, indicating a significant difference (P < 0.05, Table 3). The ROC curves for 2 biomarkers, miR-21 and miR-24, on the diagnostic ability of MPE are plotted in Fig. 1B. The AUC was 0.874 (95% CI: 0.730–0.957) for miR-21 and 0.860 (95% CI: 0.714–0.949) for miR-24 (Table 4). At the best cut-off values, the sensitivity and specificity were 80% and 95%, respectively, for both biomarkers. Additionally, there were only slight differences in diagnostic performance between the combination of 2 biomarkers and either biomarkers individually. The AUC for the combination of 2 biomarkers diagnosis was only 0.875 (95% CI 0.732–0.958), whereas the sensitivity and specificity were 80% and 95%, respectively. Therefore, miR-21 and miR-24 were demonstrated to be ideal biomarkers of MPE. Diagnostic effect of combination DNA ploidy analysis and biomarkers The diagnostic performances of DPA and biomarkers were significantly more effective than that of DPA, miR-21 or miR-24 alone. In Table 4, the results demonstrated that in 4 independent diagnostic methods, the performance of EC was the worst, with an AUC of 0.725 (95% CI 0.561–0.854), sensitivity of 55%, specificity of 90% and YI of 0.45 (indicating poor diagnostic value). Thus, we combined DPA with miR-21 and miR-24 respectively. The diagnostic performance saw clear improvements as a result (Table 4). The AUC for combination of DPA and miR-21 was 0.942 (95% CI 0.820–0.991), the sensitivity was 95% and the specificity was 90% (Fig. 1C). The AUC for combined DPA and miR-24 was greater, 0.973 (95% CI 0.864–0.999, Fig. 1D) and the sensitivity was 100%. The only real shortcoming of the diagnostic indicator was its 80% specificity. All in all, in these two combinations, we witnessed a significant increase in diagnostic performance, which demonstrated that they are exceptionally promising new methods of discriminating MPE from BPE. Aside from this, a validation set was also conducted to verify the research. The results showed that in all independent and combination groups, the combination of DPA + miR-21 and the union of DPA-miR-24 achieved the highest AUC value of 0.97 (95% CI 0.861–0.999) and 0.95 (95% CI 0.831–0.994), with a sensitivity of 95% and 85%, specificity of 85% and 90% and YI index of 0.85 and 0.8, respectively. Thus, these 2 combinations demonstrated the best diagnostic performances, consistent with the previous findings. DISCUSSION In this study, the AUC for DPA, miR-21 and miR-24 was around 0.860 higher than that of EC examination. We combined the diagnostic methods 2 by 2 and made an assessment on the diagnostic value of these combinations. The AUC for the union of miR-21 and miR-24 took was the lowest at 0.875, an increase of only 0.001; however, the diagnostic performance improvements made by the combination of DPA and the 2 miRNAs, respectively, were dramatic. The AUC was 0.942 for the combination of DPA and miR-21 and 0.973 for DPA and miR-24. The key difference in the 2 combinations was that DPA and miR-21 had a higher specificity (95%), whereas DPA and miR-24 had a higher sensitivity (100%). The results demonstrated that DPA, miR-21 and miR-24 appeared to be far better MPE diagnostic indicators than EC and the utility of DPA and miR-21 or miR-24 showed the highest diagnostic value and thus can be considered ideal biomarkers in MPE detection. So far, clinical evaluation, imaging, biochemical analysis, histopathology and biomarkers have all been used in the diagnosis of MPE [16–23]. However, the visual detection ability of cancer cells in effusion samples is heavily dependent on the advanced diagnostic skill and experienced cytopathologists . Histopathology (EC examination) is usually regarded as the ‘golden criterion’ diagnostic tool in MPE. However, as noted in the introduction, the sensitivity of EC examination was only 16–75%, so we used it here as the reference point from which to evaluate the diagnostic performances of other methods, especially the combination of DPA and miR-21 or miR-24. DPA has been proved to be a cancer biomarker in many studies. For example, DNA ploidy is an independent prognostic biomarker in breast invasive ductal carcinoma , a prognostic marker in Stages I and II serous adenocarcinoma of the endometrium , and flow cytometric study of cell cycle and DNA ploidy is useful in diagnosing bilharzial bladder cancer. Further, numerous studies have focused on the diagnosis of pleural effusion through DPA and the combination of DPA and other tumour markers, for example, the value of DNA image cytometry in the diagnosis of malignant pleural effusion . Indeed, DNA aneuploidy has the potential to become an adjunct for the routine cytological diagnosis of effusion . Many miRNAs, including miR-21 and miR-24, have also been shown to be aberrantly expressed in certain cancers and can potentially be used as biomarkers for these cancers. For instance, miR-423-5p as a circulating biomarker for heart failure, serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer , comprehensive microRNA analysis identifying miR-24 and miR-125a-5p as plasma biomarkers for rheumatoid arthritis  and cell-free miR-24 and miR-30d as potential diagnostic biomarkers in malignant effusions . However, little research has been conducted regarding the combination of the expression of miR-21 or miR-24 with DPA or other methods in MPE diagnosis. Here, we made original attempts to combine DPA and potential biomarkers for the diagnosis of MPE. By comparing the results of EC, DPA, miR-21, miR-24, EC + DPA and miR-21 + miR-24, we finally came to the conclusion that the combination of DPA and miR-21 or miR-24 showed the highest diagnostic value, which was consistent with the previous tests. Therefore, we confirmed that DPA and miR-21 or miR-24 can be viewed as a new method of MPE diagnosis. Limitations However, there still existed some limitations in this study, such as the relatively small sample size of only 40 patients which meant the available cross-validation method we used was limited, and there were some lab errors and factors that need further investigation. Therefore, this result should be further confirmed with larger samples to obtain a more reliable conclusion. Additionally, MPE is a common symptom of many diseases, and thus we should undertake further research to find targeted diagnostic methods for particular diseases. Also, the effects of TB and pneumonia in the analysis of DPA and microRNA still require further investigation. Lastly, a prediction model could be built in future studies with larger patient samples. Compliance with ethical standards All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee, as well as with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. CONCLUSION In conclusion, the combination of DPA and miR-21 or miR-24 showed the best diagnostic performance among the methods examined in this research and could be of great value in MPE diagnosis. SUPPLEMENTARY MATERIAL Supplementary material is available at ICVTS online. Conflict of interest: none declared. REFERENCES 1 Shin YM, Yun J, Lee OJ, Han HS, Lim SN, An JY et al. Diagnostic value of circulating extracellular miR-134, miR-185, and miR-22 levels in lung adenocarcinoma-associated malignant pleural effusion. Cancer Res Treat 2014; 46: 178– 85. Google Scholar CrossRef Search ADS PubMed 2 Chan MH, Chow KM, Chan AT, Leung CB, Chan LY, Chow KC et al. 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Published: Mar 1, 2018
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