approximately 15% of lung cancer patients present with a pleural effusion at the time of initial diagnosis , and 50% develop a pleural effusion later in their disease course .
pleural effusions can occur in patients with lung carcinomas of all histological types ; however , they are more common in those with adenocarcinoma ( 40% of all pleural effusion cases ) .
metastatic malignant pleural effusion ( mpe ) due to lung cancer has a poor prognosis ; mpe has been reclassified as m1a disease because its outcome is equivalent to that of distant metastasis .
lung cancer patients suspected of having a pleural effusion undergo a chest x - ray and contrast - enhanced computed tomography to confirm the presence of fluid in the pleural cavity and to evaluate the pleura for nodularity or thickening , which may suggest metastasis .
image - guided aspiration of the pleural fluid is often performed ; however , the diagnosis of mpe by pleural fluid cytology has a sensitivity of only 60% with repeated large - volume ( > 50 ml ) thoracentesis .
therefore , it would be useful to identify biomarkers in the pleural fluid that can be used to improve diagnostic sensitivity .
a systemic review found that carcinoembryonic antigen ( cea ) showed good specificity ( 94% ) but poor sensitivity ( 54% ) for the diagnosis of mpe .
in addition , several studies examined different combinations of biomarkers in an attempt to improve diagnostic accuracy . thus , there is a need to identify alternative noninvasive diagnostic biomarkers to improve the diagnosis of pleural effusions .
micrornas ( mirnas ) are a family of endogenous small ( approximately 22 nucleotides in length ) non - coding and functional rnas that regulate the expression of many genes
. aberrant expression of mirnas occurs in many types of cancer , and several mirnas function as tumor suppressor genes or oncogenes .
recently , circulating extracellular mirnas ( in serum , plasma , pleural fluid , ascites , and other body fluids ) were identified as noninvasive biomarkers for the diagnosis , prognosis , and prediction of responses to cancer treatment .
the present study examined the expression levels of three circulating mirnas ( mir-134 , mir-185 , and mir-22 ) in pleural effusion fluid ; these mirnas are deregulated in lung cancer .
mir-134 regulates cell proliferation , apoptosis , and migration , and inhibits epithelial to mesenchymal transition ( emt ) in non - small cell lung cancer ( nsclc ) ; mir-185 suppresses the proliferation in lung adenocarcinoma cell lines and induces cell cycle arrest at the g1 phase ; mir-22 inhibits the growth and invasion of lung cancer by downregulating the expression of erbb3 .
given the diagnostic and prognostic potential of mir-134 , mir-185 , and mir-22 in lung cancer , we investigated the diagnostic significance of these mirnas in patients with benign pleural effusion ( bpe ) and lung adenocarcinoma - associated mpe ( la - mpe ) and compared it with that of the most commonly used tumor marker , cea .
we analyzed pleural fluid samples from 87 patients with pleural effusion who visited chungbuk national university hospital and kangwon national university hospital between february 2009 and april 2012 .
pleural effusion was diagnosed as benign based on the clinical context and the absence of malignant cells in at least two separate samples from the same patient .
bpe samples were obtained from patients showing no clinical or radiological evidence of malignancy prior to the study .
la - mpe was confirmed by pathological diagnosis based on the presence of adenocarcinoma cells .
la - mpe samples were obtained from patients showing no evidence of another malignant tumor and either 1 ) positive immunocytochemical staining for thyroid transcription factor-1 in mpe cell blocks , or 2 ) tumors that were histologically and/or clinically diagnosed as primary adenocarcinomas of the lung .
all effusion samples were collected before the start of cancer - directed therapy and were transported to the laboratory within 30 minutes of collection .
the supernatant and sediment from each sample were aliquoted into separate eppendorf tubes and stored at -80 until use .
all patients provided written informed consent to participate in the study , which was reviewed and approved by the institutional review board of chungbuk national university hospital .
rna was extracted from each sample ( 500 l of sample ) using a urine mirna purification kit ( genolution pharmaceuticals inc . ,
the amount of rna extracted from each sample was measured using a nanodrop nd-1000 spectrophotometer ( thermo scientific , wilmington , de ) .
the expression levels of mir-134 , mir-185 , and mir-22 in 42 bpe samples and 45 la - mpe samples were measured by reverse transcription quantitative polymerase chain reaction ( rt - qpcr ) .
isolated mirna ( 100 ng ) was reverse transcribed using a high - capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) according to the manufacturer 's protocol and using a specific mirna primer that was provided with the taqman microrna assay kit ( applied biosystems ) .
quantification of mirna was performed by real - time rt - qpcr using an applied biosystems 7500 fast real - time pcr system along with the taqman microrna assay , taqman universal pcr master mix , and no amperase ung ( applied biosystems ) .
all reactions were run in triplicate and cq data were determined using default threshold settings .
the selection of suitable genes for the relative quantification of mirna expression is essential to avoid inaccurate results and to improve the comparability of gene expression data .
we previously examined the expression of the selected mirnas , two small nuclear rnas ( rnu6b and u6 snrna ) , and two small nucleolar rnas ( rnu44 and rnu48 ) to identify the most stably expressed genes for use as reference genes in pleural effusion samples .
u6 snrna was identified as the most stable reference gene in the bpe and la - mpe samples ; thus , the expression levels of the three mirnas were normalized to those of u6 snrna .
after performing the mirna analyses , additional immunoassays were conducted to measure the levels of cea expression in patients with pleural effusion .
cea levels were measured in an electrochemiluminescence immunoassay using a cobas 8000 e602 analyzer ( roche diagnostics , indianapolis , in ) according to the manufacturer 's instructions .
differences in mir-134 , mir-185 , mir-22 , and cea expression between la - mpe and bpe samples were assessed using the mann - whitney u test .
a receiver operating characteristic ( roc ) curve was plotted and the area under the curve ( auc ) was calculated to evaluate the diagnostic performance of the three mirnas and cea . the best sensitivity
risk scores were assigned to all patients according to a linear combination of the expression level of the mirnas and cea , weighted according to the regression coefficient .
10.4.7.0 ( medcalc , mariakerke , belgium ) software was used to perform the roc analysis .
we analyzed pleural fluid samples from 87 patients with pleural effusion who visited chungbuk national university hospital and kangwon national university hospital between february 2009 and april 2012 .
pleural effusion was diagnosed as benign based on the clinical context and the absence of malignant cells in at least two separate samples from the same patient .
bpe samples were obtained from patients showing no clinical or radiological evidence of malignancy prior to the study .
la - mpe was confirmed by pathological diagnosis based on the presence of adenocarcinoma cells .
la - mpe samples were obtained from patients showing no evidence of another malignant tumor and either 1 ) positive immunocytochemical staining for thyroid transcription factor-1 in mpe cell blocks , or 2 ) tumors that were histologically and/or clinically diagnosed as primary adenocarcinomas of the lung .
all effusion samples were collected before the start of cancer - directed therapy and were transported to the laboratory within 30 minutes of collection .
the supernatant and sediment from each sample were aliquoted into separate eppendorf tubes and stored at -80 until use .
all patients provided written informed consent to participate in the study , which was reviewed and approved by the institutional review board of chungbuk national university hospital .
rna was extracted from each sample ( 500 l of sample ) using a urine mirna purification kit ( genolution pharmaceuticals inc . ,
the amount of rna extracted from each sample was measured using a nanodrop nd-1000 spectrophotometer ( thermo scientific , wilmington , de ) .
the expression levels of mir-134 , mir-185 , and mir-22 in 42 bpe samples and 45 la - mpe samples were measured by reverse transcription quantitative polymerase chain reaction ( rt - qpcr ) .
isolated mirna ( 100 ng ) was reverse transcribed using a high - capacity cdna reverse transcription kit ( applied biosystems , foster city , ca ) according to the manufacturer 's protocol and using a specific mirna primer that was provided with the taqman microrna assay kit ( applied biosystems ) .
quantification of mirna was performed by real - time rt - qpcr using an applied biosystems 7500 fast real - time pcr system along with the taqman microrna assay , taqman universal pcr master mix , and no amperase ung ( applied biosystems ) .
all reactions were run in triplicate and cq data were determined using default threshold settings .
the selection of suitable genes for the relative quantification of mirna expression is essential to avoid inaccurate results and to improve the comparability of gene expression data .
we previously examined the expression of the selected mirnas , two small nuclear rnas ( rnu6b and u6 snrna ) , and two small nucleolar rnas ( rnu44 and rnu48 ) to identify the most stably expressed genes for use as reference genes in pleural effusion samples .
u6 snrna was identified as the most stable reference gene in the bpe and la - mpe samples ; thus , the expression levels of the three mirnas were normalized to those of u6 snrna .
after performing the mirna analyses , additional immunoassays were conducted to measure the levels of cea expression in patients with pleural effusion .
cea levels were measured in an electrochemiluminescence immunoassay using a cobas 8000 e602 analyzer ( roche diagnostics , indianapolis , in ) according to the manufacturer 's instructions .
differences in mir-134 , mir-185 , mir-22 , and cea expression between la - mpe and bpe samples were assessed using the mann - whitney u test .
a receiver operating characteristic ( roc ) curve was plotted and the area under the curve ( auc ) was calculated to evaluate the diagnostic performance of the three mirnas and cea . the best sensitivity
risk scores were assigned to all patients according to a linear combination of the expression level of the mirnas and cea , weighted according to the regression coefficient .
10.4.7.0 ( medcalc , mariakerke , belgium ) software was used to perform the roc analysis .
eighty - seven pleural effusion samples ( 42 bpe and 45 la - mpe ) were analyzed by rt - qpcr .
table 1 shows the baseline characteristics of the patients in the bpe and la - mpe groups .
the median age of the bpe patients was 61 years ( range , 22 to 92 years ) and that of the la - mpe patients was 68 years ( range , 33 to 92 years ) .
the expression levels of mir-134 , mir-185 , and mir-22 in la - mpe samples were significantly lower than in bpe samples ( all p<0.001 , mann - whitney u test ) ( fig .
the median expression levels of mir-134 , mir-185 , and mir-22 were 19.758 ( range , 0.069 to 243.601 ) , 1.229 ( range , 0.015 to 47.948 ) , and 1.074 ( range , 0.001 to 95.377 ) , respectively , in bpe samples and 4.209 ( range , 0.005 to 143.520 ) , 0.008 ( range , 0.000 to 2.852 ) , and 0.008 ( range , 0.000 to 8.562 ) , respectively , in la - mpe samples . of the 87 effusion samples , 77 were available for cea testing .
the median cea expression level was significantly higher in the la - mpe samples than in the bpe samples ( 127 ng / ml ; range , 0.2 to 6,574 ng / ml vs. 1.0 ng / ml ; range , 0.2 to 33.3 ng / ml ) ( p<0.001 ) ( fig .
the diagnostic performance of mir-134 , mir-185 , mir-22 , and cea ( and a combination of these markers ) in differentiating bpe from la - mpe was evaluated by roc curve analysis ( table 2 ) .
cut - off points were determined such that they maximized the sum of sensitivity and specificity .
the cut - off points for mir-134 , mir-185 , mir-22 , and cea were 16.99 , 0.092 , 0.051 , and 7.6 ng / ml , respectively .
the diagnostic accuracy of mir-134 , mir-185 , and mir-22 , as measured by the auc , was 0.721 , 0.882 , and 0.832 , respectively ( fig .
the auc for cea was 0.898 , with a sensitivity of 78.4% and a specificity of 97.5% ( fig .
the predicted probability of being diagnosed with la - mpe from the logit model based on the three mirnas [ logit=(-0.020expression level of mir-134)+(-1.103expression level of mir-185)+(-0.452expression level of mir-22 ) ] was used to construct a roc curve .
the auc for the combined mirnas was 0.893 ( 95% confidence interval [ ci ] , 0.808 to 0.949 ) , with a sensitivity of 73.3% and a specificity of 97.6% ( fig .
. the predicted probability of being diagnosed with la - mpe from the logit model based on a combination of the three mirnas plus cea [ logit=(-0.017expression level of mir-134)+(-1.148expression level of mir-185)+(0.332expression level of mir-22)+(0.127expression level of cea ) ] was also used to construct a roc curve .
the auc for a combination of all four markers was 0.942 ( 95% ci , 0.864 to 0.982 ) , with a sensitivity of 91.9% and a specificity of 92.5% ( fig .
eighty - seven pleural effusion samples ( 42 bpe and 45 la - mpe ) were analyzed by rt - qpcr .
table 1 shows the baseline characteristics of the patients in the bpe and la - mpe groups .
the median age of the bpe patients was 61 years ( range , 22 to 92 years ) and that of the la - mpe patients was 68 years ( range , 33 to 92 years ) .
the expression levels of mir-134 , mir-185 , and mir-22 in la - mpe samples were significantly lower than in bpe samples ( all p<0.001 , mann - whitney u test ) ( fig .
the median expression levels of mir-134 , mir-185 , and mir-22 were 19.758 ( range , 0.069 to 243.601 ) , 1.229 ( range , 0.015 to 47.948 ) , and 1.074 ( range , 0.001 to 95.377 ) , respectively , in bpe samples and 4.209 ( range , 0.005 to 143.520 ) , 0.008 ( range , 0.000 to 2.852 ) , and 0.008 ( range , 0.000 to 8.562 ) , respectively , in la - mpe samples . of the 87 effusion samples , 77 were available for cea testing .
the median cea expression level was significantly higher in the la - mpe samples than in the bpe samples ( 127 ng / ml ; range , 0.2 to 6,574 ng / ml vs. 1.0 ng / ml ; range , 0.2 to 33.3 ng / ml ) ( p<0.001 ) ( fig .
the diagnostic performance of mir-134 , mir-185 , mir-22 , and cea ( and a combination of these markers ) in differentiating bpe from la - mpe was evaluated by roc curve analysis ( table 2 ) .
cut - off points were determined such that they maximized the sum of sensitivity and specificity .
the cut - off points for mir-134 , mir-185 , mir-22 , and cea were 16.99 , 0.092 , 0.051 , and 7.6 ng / ml , respectively .
the diagnostic accuracy of mir-134 , mir-185 , and mir-22 , as measured by the auc , was 0.721 , 0.882 , and 0.832 , respectively ( fig .
the auc for cea was 0.898 , with a sensitivity of 78.4% and a specificity of 97.5% ( fig .
. the predicted probability of being diagnosed with la - mpe from the logit model based on the three mirnas [ logit=(-0.020expression level of mir-134)+(-1.103expression level of mir-185)+(-0.452expression level of mir-22 ) ] was used to construct a roc curve .
the auc for the combined mirnas was 0.893 ( 95% confidence interval [ ci ] , 0.808 to 0.949 ) , with a sensitivity of 73.3% and a specificity of 97.6% ( fig .
2e ) . the predicted probability of being diagnosed with la - mpe from the logit model based on a combination of the three mirnas plus cea [ logit=(-0.017expression level of mir-134)+(-1.148expression level of mir-185)+(0.332expression level of mir-22)+(0.127expression level of cea ) ] was also used to construct a roc curve .
the auc for a combination of all four markers was 0.942 ( 95% ci , 0.864 to 0.982 ) , with a sensitivity of 91.9% and a specificity of 92.5% ( fig .
the present study demonstrated that la - mpe samples showed significantly lower expression of mir-134 , mir-185 , and mir-22 than bpe samples .
this suggests that all three mirnas may be useful diagnostic biomarkers for differentiating between bpe and la - mpe .
in addition , the diagnostic performance of the three combined mirnas ( auc , 0.893 ) was comparable with that of cea ( auc , 0.898 ) .
cea is a useful diagnostic biomarker for patients with pleural effusion ; however , cea is found to confer good specificity but relatively poor sensitivity for the diagnosis of la - mpe .
the combination panel comprising mir-134 , mir-185 , mir-22 , and cea improved the diagnostic value ( auc , 0.942 ) ; in particular , the sensitivity increased to 91.9% .
research on the prevention and early diagnosis of cancer has focused on identifying biomarkers that can help to detect tumors using noninvasive diagnostic methods .
recently studies suggest that the profile of circulating mirnas , as well as tissue mirnas , could be used as a diagnostic biomarker for cancer .
mirnas are found in all body fluids and show distinct compositions in different fluid types .
the stability of mirnas in body fluids may be explained , at least in part , by the finding that they are present in extracellular vesicular structures , called exosomes , which protect them from degradation .
mirna - containing exosomes are found not only in blood , but also in other types of body fluid .
circulating exosomes and tumor cells from patients with lung adenocarcinoma harbor similar mirna signatures , and there appears to be a significant difference in exosomal mirna levels between cancer patients and controls .
therefore , it is possible that pleural fluid obtained from patients that are difficult to diagnose using clinical and radiological evaluation could also be assessed for the presence of these molecular biomarkers .
recently , xie et al .   studied the expression levels of 22 mirnas that were aberrantly expressed in the serum of lung cancer patients ( 82 patients with mpe and 28 patients with bpe ) .
they reported that mpe showed higher expression levels of cell - free mir-24 and mir-30d than bpe .
however , they included pleural effusion samples from heterogenous lung cancer patients and also examined ascites from gastric cancer patients . in a previous study
, we examined the differential expression of circulating mirnas in bpe and la - mpe samples using a peptide nucleic acid - based microarray .
the mirna expression signatures were confirmed by rt - qpcr and cross - validated using an independent sample set .
the results showed that the expression level of mir-198 was significantly lower in la - mpe than in bpe .
for the present study , we selected only three mirnas ( mir-134 , mir-185 , and mir-22 ) that are deregulated in lung cancer and examined their expression in plural effusion samples obtained from patients with bpe and la - mpe by rt - qpcr .
we found that the expression of mir-134 , mir-185 , and mir-22 , was lower in la - mpe than in bpe .
downregulation of certain mirnas in cancer suggests that these mirnas may have tumor suppressor characteristics .
the expression of mir-134 correlates with the invasive potential and emt phenotype of nsclc cells .
functional assays showed that mir-134 inhibits emt in nsclc cells , and forkhead box protein m1 ( foxm1 ) , a potential metastasis promoter , was a direct and functional target of mir-134 .
also , low expression of cell - free mir-134 in mpe is associated with poor survival in nsclc patients .
the extent of the growth suppression induced by these mirnas is similar to that induced by the tumor suppressor mirna , let-7 .
in contrast to its tumor suppressor role , mir-185 plays an oncogenic role in both renal cell carcinoma and colorectal cancers .
the implied involvement of mir-22 in the p53 tumor suppressor network suggests that it is a strong candidate tumor suppressor gene in human colon and liver cancers .
moreover , mir-22 restores the cellular senescence program in cancer cells and acts as a tumor suppressor .
it also shows anti - lung cancer activity both in vitro and in vivo , possibly via the post - transcriptional regulation of erbb3 .
however , mir-22 is also oncogenic in transformed human bronchial epithelial cells induced by anti - benzo[a]pyrene-7,8-diol-9,10-epoxide .
these discordant findings suggest that mirnas may target different genes in different cell types , thereby contributing to distinct biological processes .
first , patients with bpe as the control group were not matched with the la - mpe patients with respect to age and gender , which may have affected the results .
the second limitation of the study was that all of three mirnas expression were downregulated in la - mpe compared with bpe , and the cut - off points for mir-185 and mir-22 were very low .
therefore , the diagnostic performance of these mirnas that have tumor suppressor characteristics could be worse than that of oncogenic mirnas .
the present study showed that the circulating extracellular mir-134 , mir-185 , and mir-22 levels in pleural effusions can be used to differentiate between la - mpe and bpe .
furthermore , a panel comprising all three mirnas showed a diagnostic performance comparable with that of cea .
the combination of all three mirnas plus cea showed considerable clinical value for diagnosing la - mpe , with much better sensitivity than cea alone .
thus , we propose that mir-134 , mir-185 , and mir-22 could be used to develop a minimally invasive screening tool for the diagnostic evaluation of pleural effusions .
 purposethe accurate and timely diagnosis of malignant pleural effusion ( mpe ) in lung cancer patients is important because mpe has a poor prognosis and is classified as stage iv disease . 
 molecular biomarkers for pleural effusion , such as circulating extracellular micrornas ( mirnas ) isolated from pleural fluid , may help in the diagnosis of mpe . 
 the present study examined whether mirnas that are deregulated in lung cancer ( mir-134 , mir-185 , and mir-22 ) can serve as diagnostic markers for lung adenocarcinoma - associated mpe ( la - mpe).materials and methodsreal - time reverse transcription quantitative polymerase chain reaction was used to measure the expression of the three mirnas in samples from 87 patients with pleural effusion comprising 45 la - mpes and 42 benign pleural effusions ( bpes ) . 
 the area under the receiver operating characteristic curve ( auc ) was then used to evaluate the diagnostic performance of each of the three mirnas and compare it with that of the common tumor marker , carcinoembryonic antigen ( cea).resultsthe expression of all three mirnas was significantly lower in la - mpe than in bpe ( p < 0.001 ) . 
 the aucs for mir-134 , mir-185 , mir-22 , and cea were 0.721 , 0.882 , 0.832 , and 0.898 , respectively . combining cea with the three mirnas increased the diagnostic performance , yielding an auc of 0.942 ( 95% confidence interval , 0.864 to 0.982 ) , with a sensitivity of 91.9% and a specificity of 92.5%.conclusionthe present study suggests that the expression levels of circulating extracellular mir-134 , mir-185 , and mir-22 in patients with pleural effusion may have diagnostic value when differentiating between la - mpe and bpe . 
Introduction
Materials and Methods
1. Patients and pleural fluid collection
2. RNA extraction and real-time reverse transcription quantitative polymerase chain reaction
3. Immunoassay for CEA
4. Statistical analysis
Results
1. Patient characteristics
2. Expression levels of miR-134, miR-185, miR-22, and CEA in BPE and LA-MPE
3. Diagnostic performance of miR-134, miR-185, miR-22, and CEA for LA-MPE
Discussion
Conclusion