Lung Adenocarcinoma-associated Malignant Pleural Effusion Research Articles

Heterogeneity of cancer stem cell-related marker expression is associated with three-dimensional structures in malignant pleural effusion produced by lung adenocarcinoma.

Cancer stem cells have been described in lung adenocarcinoma-associated malignant pleural effusion. They show clinically important features, including the ability to initiate new tumours and resistance to treatments. However, their correlation with the three-dimensional tumour structures in the effusion is not well understood. Cell blocks produced from lung adenocarcinoma patients' pleural effusion were examined for cancer stem cell-related markers Nanog and CD133 using immunocytochemistry. The three-dimensional cancer cell structures and CD133 expression patterns were visualized with tissue-clearing technology. The expression patterns were correlated with tumour cell structures, genetic variants and clinical outcomes. Thirty-nine patients were analysed. Moderate-to-strong Nanog expression was detected in 27 cases (69%), while CD133 was expressed by more than 1% of cancer cells in 11 cases (28%). Nanog expression was more homogenous within individual specimens, while CD133 expression was detected in single tumour cells or cells within small clusters instead of larger structures in 8 of the 11 positive cases (73%). Although no statistically significant correlation between the markers and tumour genetic variants or patient survival was observed, we recorded seven cases with follow-up specimens after cancer treatment, and four (57%) showed a change in stem cell-related marker expression corresponding to treatment response. Lung adenocarcinoma cells in the pleural effusion show variable expression of cancer stem cell-related markers, some showing a correlation with the size of cell clusters. Their expression level is potentially correlated with cancer treatment effects.

Use of Platelet Parameters in the Differential Diagnosis of Lung Adenocarcinoma-Associated Malignant Pleural Effusion and Tuberculous Pleural Effusion.

Background Both malignant pleural effusion (MPE) and tuberculous pleural effusion (TPE) are common etiologies of pleural effusion; the present study was conducted to establish the diagnostic value of platelet parameters in the differential diagnosis of MPE and TPE. Methods This retrospective study enrolled patients with lung adenocarcinoma-associated MPE and TPE. Platelet parameter data, including platelet count (PLT), mean platelet volume (MPV), plateletcrit (PCT), platelet distribution width (PDW), and platelet-larger cell ratio (P-LCR), were collected. Principal component analysis and multiple logistic regression modelling were carried out to assess the diagnostic value of these platelet parameters. Results The MPE group and the TPE group enrolled 270 and 433 patients, respectively. Demographic characteristics of patients were more female and higher age in the MPE group. MPV, PDW, and P-LCR were significantly higher in MPE patients, while PLT and PCT were significantly higher in TPE patients. Principal component analysis generated two principal components (PCs) based on above platelet parameters. After adjusting for confounding factors including gender and age, multiple logistic regression showed positive association between PC1 and MPE. Conclusion Platelet parameters were potential biomarkers in distinguishing lung adenocarcinoma-associated MPE from TPE. A patient with lower PLT and PCT and higher MPV, PDW, and P-LCR was more likely to be diagnosed as the former. Principal component analysis and multiple logistic regression performed well in improving multicollinearity, adjusting confounding factors, and identifying important risk factors for MPE.

Microarray expression profile and analysis of circular RNA regulatory network in malignant pleural effusion

ABSTRACT Malignant pleural effusion (MPE) is a common complication of lung cancer. Accumulating evidence has suggested that circular RNAs (circRNAs) play important roles in oncogenesis and progression of cancer. However, the expression pattern of circRNAs in MPE remains largely unknown and awaits investigation. The study was designed to elucidate the potential roles of differentially expressed circRNAs in MPE. Herein, we detected a total of 1350 differentially expressed circRNAs and 1727 differentially expressed mRNAs in lung adenocarcinoma-associated malignant pleural effusion (LA-MPE) compared with tuberculous pleural effusion (TPE) by Clariom D Human Microarray. Among the top 5 up-regulated circRNAs (hsa_circ_0067705, hsa_circ_0025542, hsa_circ_0072793, hsa_circ_0084927, and hsa_circ_0085386), four were verified significantly up-regulated in LA-MPE by qRT-PCR and hsa_circ_0085386 had an increasing trend. CircRNA-miRNA-mRNA network for the top 5 up-regulated circRNAs was constructed and pathway analysis indicated that the enriched mRNA targets involved in PI3K-Akt signaling pathway, Axon guidance, Regulation of actin cytoskeleton and Rap1 signaling pathway were potentially regulated by these aberrantly expressed circRNAs. We generated specific circRNA profiles in LA-MPE for the first time. And analysis of circRNA regulatory network could provide evidence that circRNAs are important in MPE development because they participate in cancer-related pathways by sequestering miRNAs. Our findings suggested that aberrantly expressed circRNAs may be involved in the development of LA-MPE.

Exosomal MicroRNA Expression Profiling in Patients with Lung Adenocarcinoma-associated Malignant Pleural Effusion

Cytological analysis for diagnosing malignant pleural effusion associated with lung adenocarcinoma (Ad-MPE) shows limited sensitivity and novel diagnostic biomarkers are needed. The aim of this study was to evaluate the profile of four microRNAs (miRNAs) in exosomes in Ad-MPE and benign (non-neoplastic) pleural effusion (BPE). A total of 56 pleural effusion samples from patients with lung adenocarcinoma and benign diseases were collected. Exosomal miRNA expressions were evaluated using real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). The expression levels of miR-182 and miR-210 were significantly higher in the Ad-MPE than in the BPE samples. The receiver operating characteristics curve analyses of miR-182 and miR-210 for diagnosis of Ad-MPE yielded areas under the receiver operating characteristic curves of 0.87 and 0.81, respectively. These miRNA signatures may have a diagnostic potential for differentiating Ad-MPE from BPE.

EGFR Mutation Status in Lung Adenocarcinoma-Associated Malignant Pleural Effusion and Efficacy of EGFR Tyrosine Kinase Inhibitors.

PurposeMalignant pleural effusions (MPEs) are often observed in lung cancer, particularly adenocarcinoma. The aim of this study was to investigate epidermal growth factor receptor (EGFR) mutation status in lung adenocarcinoma-associated MPEs (LA-MPEs) and its correlation with efficacy of EGFR tyrosine kinase inhibitor (TKI) therapy.Materials and MethodsSamples comprised 40 cell blocks of pathologically-confirmed LA-MPEs collected before the start of EGFR TKI therapy. EGFR mutation status was re-evaluated by peptide nucleic acid clamping and the clinical outcomes of EGFR TKI‒treated patients were analyzed retrospectively.ResultsEGFR mutations were detected in 72.5% of LA-MPE cell blocks (29/40). The median progression-free survival for patients with EGFR mutations in LA-MPEs was better than that for patients with wild-type EGFR (7.33 months vs. 2.07 months; hazard ratio, 0.486; 95% confidence interval, 0.206 to 1.144; p=0.032). The objective response rate (ORR) of 26 patients with EGFR mutations in LA-MPEs among the 36 patients with measurable lesions was 80.8%, while the ORR of the 10 patients with wild-type EGFR in LA-MPEs was 10% (p < 0.001). Among the 26 patients with EGFR mutations in LA-MPEs, the ORR of target lesions and LA-MPEs were 88.5% and 61.5%, respectively (p=0.026).ConclusionEGFR mutation status in cell blocks of LA-MPEs confirmed by pathologic diagnosis is highly predictive of EGFR TKI efficacy. For patients with EGFR mutations in LA-MPEs, the response to EGFR TKIs seems to be worse for pleural effusions than for solid tumors.

Diagnostic value of tumor markers for lung adenocarcinoma-associated malignant pleural effusion: a validation study and meta-analysis.

Pleural effusion is one of the most common complications of lung adenocarcinoma and is diagnostically challenging. This study aimed to investigate the diagnostic performance of carcinoembryonic antigen (CEA), cytokeratin fragment (CYFRA) 21-1, and cancer antigen (CA) 19-9 for lung adenocarcinoma-associated malignant pleural effusion (MPE) through a validation study and meta-analysis. Pleural effusion samples were collected from 81 lung adenocarcinoma-associated MPEs and 96 benign pleural effusions. CEA, CYFRA 21-1, and CA19-9 were measured by electrochemiluminescence immunoassay. The capacity of tumor markers was assessed with receiver operating characteristic curve analyses and the area under the curve (AUC) was calculated. Standard methods for meta-analysis of diagnostic studies were used to summarize the diagnostic performance of CEA, CYFRA 21-1, and CA19-9 for lung adenocarcinoma-associated MPE. The pleural levels of CEA, CYFRA 21-1, and CA19-9 were significantly increased in lung adenocarcinoma-associated MPE compared to benign pleural effusion. The cut-off points for CEA, CYFRA 21-1, and CA19-9 were optimally set at 4.55ng/ml, 43.10μg/ml, and 12.89 U/ml, and corresponding AUCs were 0.93, 0.85, and 0.81, respectively. The combination of CEA, CYFRA 21-1, and CA19-9 increased the sensitivity to 95.06%, with an AUC of 0.95. Eight studies were included in this meta-analysis. CEA showed the best diagnostic performance with pooled sensitivity, specificity, positive/negative likelihood ratio, and diagnostic odds ratio of 0.75, 0.96, 16.01, 0.23, and 81.49, respectively. The AUC was 0.93. CEA, CYFRA 21-1, and CA19-9 play a role in the diagnosis of lung adenocarcinoma-associated MPE. The combination of these tumor markers increases the diagnostic accuracy.

Diagnostic performance of CD66c in lung adenocarcinoma-associated malignant pleural effusion: comparison with CEA, CA 19-9, and CYFRA 21-1

Various tumour markers have been evaluated in malignant pleural effusions, but not CD66c. This study evaluated the diagnostic ability of CD66c in lung adenocarcinoma-associated malignant pleural effusions (LA-MPEs) and compared it with other known tumour markers. Forty-seven cases of LA-MPE and 52 cases of benign pleural effusions were collected. The levels of CD66c, CEA, CA 19-9, and CYFRA 21-1 were measured by enzyme immunoassay. The expression of CD66c, CEA, and CA 19-9 in cell blocks was measured by immunocytochemistry. CEA had the best diagnostic values, with a sensitivity of 87.2% and specificity of 92.3%. Both CD66c and CA 19-9 showed the highest specificity of 98.1%, with sensitivities of 63.8% and 55.3%, respectively. CYFRA 21-1 had a sensitivity of 83.0% and specificity of 76.9%. CEA combined with CA 19-9 reached a sensitivity of 91.5% and a specificity of 98.1%. The sensitivities of immunocytochemical staining for CD66c, CEA, and CA 19-9 were 72.5%, 75%, and 40%, respectively. CD66c showed a diagnostic performance comparable to CYFRA 21-1 and CA 19-9 by enzyme immunoassay. Immunocytochemical study showed that CD66c and CEA were more sensitive than CA19-9. Both studies support CD66c as a potential tumour marker to differentiate LA-MPE from benign effusions.

Abstract 4358: MicroRNA-198 inhibits non-small cell lung cancer migration and invasion through targeting OTX1 and VCP

Abstract Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for the majority of cases. In previous studies, microRNA-198 (miR-198) was shown to be significantly downregulated in lung adenocarcinoma-associated malignant pleural effusion and suggested as a diagnostic biomarker. Herein, we show that miR-198 regulates migration and invasion of NSCLS cells by directly targeting orthodenticle homeobox 1 (OTX1) and valosin containing protein (VCP). Expression of miR-198 suppressed migration and invasion of NSCLS cells, whereas inhibition of miR-198 expression increased the invasive efficiency of the cancer cells. MiR-198 directly targets 3′UTR of OTX1 and VCP, thus regulates their protein level. Furthermore, we found reduced levels of miR-198 in human NSCLS tissue samples compared to adjacent normal tissues. Our findings indicate that miR-198 regulates NSCLC cell migration and invasion, partially via the down-regulation of OTX1 and VCP. Thus, miR-198 may represent a potential therapeutic target for NSCLC intervention. Citation Format: Jin-Ah Kim, Junguk Yang, Yeong Nang Jo, Chang Woo Lee, Ok-Jun Lee, Song-Yi Choi, Jong Soon Kang, Jieun Yun. MicroRNA-198 inhibits non-small cell lung cancer migration and invasion through targeting OTX1 and VCP. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4358. doi:10.1158/1538-7445.AM2014-4358

189P - Diagnostic Performance of Cd66C in Lung Adenocarcinoma-Associated Malignant Pleural Effusion: Comparison with Cea, Ca 19-9, and Cyfra 21-1

ABSTRACT Aim: Various tumor markers have been evaluated to diagnose malignant pleural effusions, but not CD66c, a member of the CEACAM family. This study evaluated the diagnostic ability of CD66c in lung adenocarcinoma-associated malignant pleural effusions (LA-MPEs) and compared with other known tumor markers (CEA, CA 19-9, and CYFRA 21-1). Methods: Pleural effusions were collected from 47 patients with LA-MPE, and from 52 patients with benign conditions. The levels of CD66c, CEA, CA 19-9, and CYFRA 21-1 were measured by enzyme immunoassay. The expression of CD66c, CEA, and CA 19-9 in effusion cell blocks was measured by immunocytochemical staining. Results: The levels of all tumor markers were significantly higher in LA-MPE than in BPE (p Conclusions: CD66c showed a diagnostic performance comparable to CYFRA 21-1 and CA 19-9 by enzyme immunoassay. Immunocytochemical study showed that CD66c and CEA were more sensitive than CA19-9. Both studies support CD66c as a potential tumor marker to differentiate LA-MPE from benign effusions. Disclosure: All authors have declared no conflicts of interest.

Identification of suitable reference genes for the relative quantification of microRNAs in pleural effusion.

Circulating cell-free microRNAs (miRNAs) are potential biomarkers of cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is widely used in miRNA expression studies. The aim of this study was to identify suitable reference genes for RT-qPCR analyses of miRNA expression levels in pleural effusion. The expression levels of candidate reference miRNAs were investigated in 10 benign pleural effusion (BPE) and 10 lung adenocarcinoma-associated malignant pleural effusion (LA-MPE) samples using miRNA microarrays. The expression levels of candidate reference miRNAs, together with those of U6 small nuclear RNA (snRNA), RNU6B, RNU44 and RNU48 small RNAs, in 46 BPE and 45 LA-MPE samples were validated by RT-qPCR, and were analyzed using the NormFinder and BestKeeper algorithms. The impact of different normalization approaches on the detection of differential expression levels of miR-198 in BPE and LA-MPE samples was also assessed. As determined by the miRNA microarray data, five candidate reference miRNAs were identified. Following RT-qPCR validation, U6 snRNA, miR-192, miR-20a, miR-221, miR-222 and miR-16 were evaluated using the NormFinder and BestKeeper software programs. U6 snRNA and miR-192 were identified as single reference genes and the combination of these genes was preferred for the relative quantification of miRNA expression levels in pleural effusion. Normalization of miR-98 expression levels to those of U6 snRNA, miR-192 or a combination of these genes enabled the detection of a significant difference between BPE and LA-MPE samples. Therefore, U6 snRNA and miR-192 are recommended as reference genes for the relative quantification of miRNA expression levels in pleural effusion.

Diagnostic Value of Circulating Extracellular miR-134, miR-185, and miR-22 Levels in Lung Adenocarcinoma-Associated Malignant Pleural Effusion.

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.

Abstract 4165: Combination of cell-free miR-198, carcinoembryonic antigen, and CYFRA 21-1 as a diagnostic biomarker for lung adenocarcinoma-associated malignant pleural effusion.

Abstract Circulating cell-free microRNAs (miRNAs) have been identified as potential noninvasive biomarkers in cancer. The aim of this study was to identify miRNAs that are differentially expressed between benign pleural effusion (BPE) and lung adenocarcinoma-associated malignant pleural effusion (LA-MPE). We have also analyzed the expression of carcinoembryonic antigen (CEA) and cytokeratin fragment (CYFRA) 21-1, which are the most commonly used tumor markers in pleural effusion. Cell-free miRNA expression was investigated in 107 patients with pleural effusion. Microarrays were used to screen 160 miRNAs in a discovery set comprising 20 effusion samples (10 BPEs and 10 LA-MPEs). The PANArrayTM miRNA expression profiling kit (PANAGENE, INC. Daejeon, Korea) was used to identify candidate miRNAs in the discovery set. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the profiling results obtained for the discovery set and those obtained for a validation set comprising 42 BPEs and 45 LA-MPEs. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of the identified miRNAs, CEA, and CYFRA 21-1. Microarray profiling showed that miR-198 was significantly downregulated in LA-MPE compared with BPE (P = 0.002). The miRNA microarray analysis results were confirmed by qRT-PCR (P &amp;lt; 0.001) using the validation set. miR-198 showed an AUC of 0.887, with a sensitivity of 71.1% and a specificity of 95.2%. The AUC for CEA was 0.898, with a sensitivity of 78.4% and a specificity of 97.5%. The AUC for CYFRA 21-1 was 0.836, with a sensitivity of 78.4% and a specificity of 82.5%. The diagnostic performance of miR-198 was comparable with that of CEA, but better than that of CYFRA 21-1. The AUC for all three markers combined was 0.926 (95% confidence interval, 0.843-0.973) with a sensitivity of 89.2% and a specificity of 85.0%. The present study shows that expression of miR-198 was significantly downregulated in LA-MPE compared with BPE, and combination of cell-free miR-198, CEA and CYFRA 21-1 can be used to differentiate LA-MPE from BPE with a high degree of accuracy. These results suggest that miR-198 in pleural effusions may be a potential diagnostic biomarker for discriminating between BPE and LA-MPE. Citation Format: Hye Sook Han, Jieun Yun, Ok-Jun Lee. Combination of cell-free miR-198, carcinoembryonic antigen, and CYFRA 21-1 as a diagnostic biomarker for lung adenocarcinoma-associated malignant pleural effusion. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4165. doi:10.1158/1538-7445.AM2013-4165

Downregulation of cell‐free miR‐198 as a diagnostic biomarker for lung adenocarcinoma‐associated malignant pleural effusion

Circulating cell-free microRNAs (miRNAs) are potential cancer biomarkers. The aim of this study was to identify miRNAs that are differentially expressed between benign pleural effusion (BPE) and lung adenocarcinoma-associated malignant pleural effusion (LA-MPE). The expression level of cell-free miRNA was investigated in 107 patients with pleural effusion. Microarrays were used to screen 160 miRNAs in a discovery set comprising 20 effusion samples (ten BPEs and ten LA-MPEs). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to validate the profiling results obtained for the discovery set and those obtained for a validation set comprising 42 BPEs and 45 LA-MPEs. The area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic performance of the identified miRNAs and other common tumor markers, such as carcinoembryonic antigen (CEA) and cytokeratin fragment (CYFRA) 21-1. Microarray profiling showed that miR-198 was significantly downregulated in LA-MPE compared with BPE (p = 0.002). The miRNA microarray analysis results were confirmed by qRT-PCR (p < 0.001) using the validation set. The AUCs for miR-198, CEA and CYFRA 21-1 in the validation set were 0.887, 0.898 and 0.836, respectively. The diagnostic performance of miR-198 was comparable with that of CEA, but better than that of CYFRA 21-1. The AUC for all three markers combined was 0.926 (95% confidence interval, 0.843-0.973) with a sensitivity of 89.2% and a specificity of 85.0%. The present study suggests that cell-free miR-198 from patients with pleural effusion might have diagnostic potential for differentiating LA-MPE from BPE.

Diagnostic value of Her-2/neu, Cyfra 21-1, and carcinoembryonic antigen levels in malignant pleural effusions of lung adenocarcinoma

Cytology fails to detect neoplastic cells in 40-50% of cases of malignant pleural effusion, a condition that frequently accompanies lung adenocarcinoma. Published reports of diagnostic sensitivity of various tumour markers are inconsistent, and optimal cut-off points have not been determined. This study aimed to evaluate the ability of three markers to discriminate lung adenocarcinoma-associated malignant pleural effusion (LAC-MPE) from benign effusion. Pleural effusion samples were collected from 41 patients with LAC-MPE, and from 93 with various benign conditions. The diagnostic sensitivity and specificity for Her-2/neu, Cyfra 21-1, and carcinoembryonic antigen (CEA) were evaluated. Cut-off points for these markers are optimally set at 3.6 microg/L, 60 microg/L, and 6.0 microg/L, respectively. Her-2/neu, Cyfra 21-1, and CEA vary in their diagnostic accuracy to differentiate LAC-MPE from benign pleural effusion: 79.85%, 88.81%, and 94.03%, respectively. CEA combined with Cyfra 21-1 increases diagnostic sensitivity to 97.6%, with a specificity of 91.4%. With appropriate cut-off points, CEA currently provides the best diagnostic accuracy. Combining CEA with Cyfra 21-1 increases diagnostic sensitivity to nearly 100%. The results of the present study may help clinicians decide whether to obtain a cytological/histological specimen by invasive means to investigate a possible diagnosis of malignancy.

DIAGNOSTIC ACCURACY AND CUTOFF POINTS OF HER-2/NEU, CYFRA 21-1, AND CEA IN LUNG ADENOCARCINOMA-ASSOCIATED MALIGNANT PLEURAL EFFUSIONS

PURPOSE: Cytology fails to detect neoplastic cells in approximately 40–50% of malignant pleural effusions, which commonly accompany lung adenocarcinomas. Published reports of the diagnostic sensitivity of various tumor markers are inconsistent. Reports on Her-2/neu in lung adenocarcinoma-associated malignant pleural effusion, for example, differ markedly (72% vs. 10%). In addition, optimal cutoff points have not been determined. To clarify contrasting reports and maximize diagnostic efforts, we evaluated the diagnostic sensitivity, specificity and optimal cutoff points for Her-2/neu, Cyfra 21-1 and carcinoembryonic antigen (CEA) in distinguishing lung adenocarcinoma-associated malignant from benign pleural effusions.