Malignant Pleural Effusion Samples Research Articles

Exploratory pilot study to characterize the immune landscapes of malignant pleural effusions and their corresponding primary tumors from patients with breast carcinoma and lung adenocarcinoma

Malignant pleural effusion (MPE) is a frequent complication of advanced malignancies. In this pilot study, we characterized the immune landscapes of MPEs, compared them to their primary tumor (PT) samples from breast carcinoma (BC) and lung adenocarcinoma (LADC), and tested the utility of multiplexed image technology in cytological samples. We evaluated the immune contexture of 6 BC and 5 LADC MPEs and their PTs using 3 multiplex immunofluorescence panels. We explored the associations between sample characteristics and pleural effusion-free survival. No MPE samples had positive programmed death-ligand 1 expression in malignant cells, although 3 of 11 PTs has positive programmed death-ligand 1 expression (more than 1% expression in malignant cells). Overall, in LADC samples, cluster of differentiation 3 (CD3)+ T cells and CD3+CD8+ cytotoxic T cells predominated (median percentages for MPEs versus PTs: 45.6% versus 40.7% and 4.7% versus 6.6%, respectively) compared with BC. CD68+ macrophages predominated in the BC samples (medians for MPEs 61.2% versus PTs for 57.1%) but not in the LADC samples. Generally in PTs, CD3+CD8+ forkhead box P3+ T cells and the median distances from the malignant cells to CD3+CD8+Ki67+ and CD3+ programmed cell death protein 1 + T cells correlated to earlier MPE after PT diagnosis. The immune cell phenotypes in the MPEs and PTs were similar within each cancer type but different between BC versus LADC. An MPE analysis can potentially be used as a substitute for a PT analysis, but an expanded study of this topic is essential.

Cell-free DNA methylation analysis as a marker of malignancy in pleural fluid

Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology for the diagnosis of malignant pleural effusion is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. This was a blind, prospective case–control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), indeterminate pleural effusion in subjects with known malignancy or IPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to IPE (p = 0.004). We also noted that the methylation signal was significantly higher in IPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and indeterminate pleural effusion groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.

Comparative analysis of the mutation detection using malignant pleural effusion and blood cell-free DNA samples in lung cancer.

e20518 Background: Malignant pleural effusion (MPE) is an abnormal amount of fluid to collect between the chest wall and the lung, and it denotes an advanced malignant disease process. MPE presents a severe medical condition which can result in breathlessness, pain, cachexia and reduced physical activity. MPE is considered to bear cancer signatures because it contains tumor cells released from malignancies, thus can be applied in high-throughput sequencing for cancer screening. Cell-free DNA (cfDNA) in blood consists of small fragments released from apoptotic cells or cancer cells, and pathological conditions can influence this process. Therefore, detection of cfDNA in peripheral blood can identify abnormalities of individuals in a noninvasive manner. Here, we wonder the difference of MPE and cfDNA in lung cancer-related mutation detection. Methods: In this study, MPE and blood samples from 216 patients with late-stage lung cancer were collected and applied to library construction and next-generation sequencing. All samples were collected during August 2018 to December 2022. We focused on the key mutations in cancer-causing genes and drug treatment-related genes (e.g., EGFR, KRAS, BRAF, ERBB2, etc.). Statistical analysis was conducted to compare the detection results between MPE and cfDNA samples by Fisher’s exact test. Results: Among the 216 patients, 60 patients (60/216 = 27.8%) were identified as negative detection with no mutation detected in cancer-causing genes in MPE and blood samples. Of the remaining 156 pairs of samples, there were 118 (118/216 = 54.6%) pairs showing the positive detection in both MPE and blood samples. 12 patients (12/216 = 5.6%) demonstrated the positive detection in blood samples while negative detection in MPE samples. The other 26 patients (26/216 = 12%) demonstrated the positive detection in MPE but negative detection in blood samples. There is no significant difference in detection rates between two types of samples (p = 0.194). Similarity, we next focused on the mutations in drug treatment-related genes and identified 91 patients (91/216 = 42.1%) as negative detection. There were 85 patients (85/216 = 39.4%) showing the same positive detection both in MPE and blood samples. 13 patients (13/216 = 6%) demonstrated the positive detection in blood samples but negative detection in MPE samples. The other 27 patients (27/216 = 12.5%) demonstrated the positive detection in MPE but negative detection in blood samples. No significant difference in detection rates was observed between two types of samples (p = 0.211). Conclusions: The mutation detection results in MPE and blood samples turned out to be consistent in most conditions, suggesting an alternative application of MPE or blood samples in cancer screening. Nonetheless, it is still valuable to integrate the information in both MPE and blood samples for a higher detection sensitivity.

DNA Methylation Analysis of the SHOX2 and RASSF1A Panel Using Cell-Free DNA in the Diagnosis of Malignant Pleural Effusion.

The differential diagnosis of pleural effusion (PE) is a common but major challenge in clinical practice. This study aimed to establish a strategy based on a PE-cell-free DNA (cfDNA) methylation detection system for the differential diagnosis of malignant pleural effusion (MPE) and benign pleural effusion (BPE). A total of 104 patients with PE were enrolled in this study, among which 50 patients had MPE, 9 malignant tumor patients had PE of indefinite causes, and the other 45 patients were classified as benign controls. The methylation status of short stature homeobox 2 (SHOX2) and RAS association domain family 1, isoform A (RASSF1A) was detected using PE-cfDNA specimens by real-time fluorescence quantitative PCR. Total methylation (TM) was defined as the combination of the methylation levels of SHOX2 and RASSF1A. The electrochemiluminescence immunoassay was applied to evaluate the levels of multiple serum tumor markers. The PE-cfDNA methylation status of either SHOX2 or RASSF1A was much higher in MPE samples than in benign controls. The combination of SHOX2 and RASSF1A methylation in PE yielded a diagnostic sensitivity of 96% and a specificity of 100%, respectively. When compared with the corresponding serum tumor marker detection results, TM showed the highest diagnostic efficiency (AUC = 0.985). Furthermore, the combination of the SHOX2 and RASSF1A methylation panels using PE-cfDNA could apparently improve the differential diagnostic efficacy of BPE and MPE and could help compensate for the deficiency of cytology. Our results indicated that SHOX2 and RASSF1A methylation panel detection could accurately classify BPE and MPE diseases and showed better diagnostic performance than traditional serum parameters. The SHOX2 and RASSF1A methylation detection of PE-cfDNA could be a potentially effective complementary tool for cytology in the process of differential diagnosis. In summary, PE-cfDNA could be used as a promising non-invasive analyte for the auxiliary diagnosis of MPE.

Characterization of the tumour microenvironment phenotypes in malignant tissues and pleural effusion from advanced osteoblastic osteosarcoma patients.

Malignant pleural effusion (MPE) is an adverse prognostic factor in patients with osteoblastic osteosarcoma; however, the cellular contexts of MPE are largely unknown. We performed single-cell RNA-sequencing (scRNA-seq) on 27260 cells from seven MPE samples and 91186 cells from eight osteosarcoma tissues, including one recurrent, one lung metastasis and six primary tumour (PT) samples, to characterize their tumour microenvironment. Thirteen main cell groups were identified in osteosarcoma tumour and MPE samples. Immune cells dominate the cellular contexts in MPE with more T/NK cells and less osteoclasts compared to PT samples. Of T/NK cells, CD8+ GNLY+ , CD8+ KLRC2+ T cells and FCGR3A+ NK cells were enriched in MPE but CD4+ FOXP3+ Tregs were enriched in PT samples. Naïve IGHD+ B and immune regulatory IGHA1+ B cells were largely identified in MPE, whereas bone metabolism-related CLEC11A+ B cells were significantly enriched in osteosarcoma PT. M2-type TAMs, including CLEC11A_TAM, C1QC_TAM and Prolif_TAMs, among myeloid cells were enriched in PT, which may suppress cytotoxicity activities of T cells through multiple ligand-receptor interactions. Mature LAMP3+ DCs were transformed from CD1C+ DC and CLEC9A+ DC sub-clusters when exposure to tumour alloantigens, which may improve T cell cytotoxicity activities on tumour cells under anti-PD-L1 treatments. In further, immune cells from MPE usually present up-regulated glycolysis and down-regulated oxidative phosphorylation and riboflavin metabolism activities compared to those in PT samples. Our study provided a novel cellular atlas of MPE and PT in patients with advanced osteosarcoma, which may provide potential therapeutic targets in the future.

The Detection of Immunity against WT1 and SMAD4P130L of EpCAM+ Cancer Cells in Malignant Pleural Effusion.

Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8+ T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms’ Tumor 1 (WT1) antigen over the disease course (two points at MPE1st and 2nd, two months after MPE1st). Epithelial cell adhesion molecule (EpCAM)+ cancer cells (PD-L1− or T cell immunoglobulin mucin-3, TIM-3−), both PD-1 or TIM-3 positive CD8+ T cells, and CD14+CD68+CD163+TIM-3+ macrophages increased from the MPE1st to MPE2nd. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8+ T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (TCM) of WT1-CTLs was decreased. On the other hand, CD8+ T cells in response to SMAD4P130L, which is homogeneously expressed in EpCAM+ cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8+ T cell response to SMAD4P130L was diminished following remarkably decreased numbers of CD8+ TCM in MPE samples. In conclusion, CD8+ T cells responding to WT1 or SMAD4P130L neoantigen expressed in EpCAM+ pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.

Malignant pleural effusion cell blocks are reliable resources for PD-L1 analysis in advanced lung adenocarcinomas: a concordance study with matched histologic samples.

In lung cancer patients presenting with malignant pleural effusion (MPE), cytology might represent the only source of tumor tissue for diagnosis and predictive biomarker testing. Programmed death ligand 1 (PD-L1) expression in tumor cells is a predictive biomarker for immunotherapy in non-small cell lung carcinomas and is tested using immunohistochemistry. However, knowledge of the validity of PD-L1 testing on MPE samples is limited. We evaluated the feasibility of immunocytochemistry (ICC) for PD-L1 in MPE cell blocks (CBs) and assessed the concordance in expression with patient-matched histologic samples. ICC for PD-L1 was performed on formalin-fixed paraffin-embedded CBs of MPE and patient-matched histologic samples, if available, using the automated Ventana PD-L1 SP263 assay. The tumor proportion score (TPS), based on partial or complete membranous tumor cell staining, was categorized as negative (&lt;1%), low (≥1% to &lt;50%), and high (≥50%). In CBs with any degree of PD-L1 expression, ICC for CD163 highlighting macrophages was performed to exclude nonspecific PD-L1 expression in macrophages. The CB PD-L1 TPS was compared with the TPS obtained from the patient-matched histologic samples. Of 43 MPE CBs available, 25 were positive for PD-L1 (25 of 42; 59%), and 1 sample was inadequate. Of the 11 patient-matched histologic samples tested, the PD-L1 TPS categories were concordant for 10 of the 11 (91% concordance) cases. PD-L1 expression in MPE CBs showed good concordance with expression in histologic samples and is feasible as a source for PD-L1 testing. The concurrent use of CD163 immunostains will aid in the manual assessment of PD-L1 TPS.

EP11.01-002 Malignant Pleural Effusion Cell Blocks Are Reliable Resources for PD-L1 Analysis in Advanced Lung Adenocarcinomas

In lung cancer patients presenting with malignant pleural effusion (MPE), cytology may represent the only source of tumor tissue for diagnosis and predictive biomarker testing. Programmed death ligand-1 (PD-L1) expression in tumor cells is a predictive biomarker for immunotherapy in non-small cell lung carcinomas and is tested by immunohistochemistry. However, there is limited knowledge on the validity of PD-L1 testing on MPE samples. The aim of the study was to evaluate the feasibility of immunocytochemistry (ICC) for PD-L1 in MPE cell blocks (CB) and assess concordance in expression with patient-matched histology samples.

Intrapleural nano-immunotherapy promotes innate and adaptive immune responses to enhance anti-PD-L1 therapy for malignant pleural effusion.

Malignant pleural effusion (MPE) is indicative of terminal malignancy with a uniformly fatal prognosis. Often, two distinct compartments of tumour microenvironment, the effusion and disseminated pleural tumours, co-exist in the pleural cavity, presenting a major challenge for therapeutic interventions and drug delivery. Clinical evidence suggests that MPE comprises abundant tumour-associated myeloid cells with the tumour-promoting phenotype, impairing antitumour immunity. Here we developed a liposomal nanoparticle loaded with cyclic dinucleotide (LNP-CDN) for targeted activation of stimulators of interferon genes signalling in macrophages and dendritic cells and showed that, on intrapleural administration, they induce drastic changes in the transcriptional landscape in MPE, mitigating the immune cold MPE in both effusion and pleural tumours. Moreover, combination immunotherapy with blockade of programmed death ligand 1 potently reduced MPE volume and inhibited tumour growth not only in the pleural cavity but also in the lung parenchyma, conferring significantly prolonged survival of MPE-bearing mice. Furthermore, the LNP-CDN-induced immunological effects were also observed with clinical MPE samples, suggesting the potential of intrapleural LNP-CDN for clinical MPE immunotherapy.

A novel method for the isolation of CD45-positive and CD45-negative cells from malignant pleural effusion

Background: Patient-derived malignant pleural effusion (MPE) samples can be used to identify a patient-specific drug combination since MPE samples are readily available and cost-effective tumor cell sources. However, the isolation of target cancer cells from MPE has been inefficient because MPE samples contain a complex mixture of immune cells, non-cancerous cells, and cancer cells. Hence, new methods need to be developed to effectively isolate target cancer cells from MPE samples that can be used for 3-dimensional (3D) cell culture. Patient-derived in vitro 3D tumor models are expected to facilitate more precise drug treatment.Methods: MPE samples were obtained from Seoul St. Mary’s Hospital, The Catholic University of Korea with consent from patients previously diagnosed with lung adenocarcinoma. We isolated target cells from MPE samples using 2 different Percoll-gradient centrifugation methods.Results: The use of 40% and 75% Percoll-gradient centrifugation led to a clearer separation of CD45-positive (CD45pos) and CD45-negative (CD45neg) cells than the traditional 44% and 67% Percoll-gradient centrifugation method.Conclusion: Our findings strongly suggest that the 40% and 75% Percoll-gradient centrifugation method is more useful for the isolation of CD45pos or CD45neg cells than the previously described Percoll-gradient centrifugation method. Furthermore, our novel method was useful for the isolation of MPE-derived target cancer cells that can be used to construct in vitro patient-specific 3D tumor models.

ALDH1 and SALL4 Expression in Cell Block Samples from Patients with Lung Adenocarcinoma and Malignant Pleural Effusion.

Malignant pleural effusion (MPE) can accompany advanced lung adenocarcinoma. Recent studies suggest that MPE could contain a heterogeneous subpopulation of cells with stem-like properties, such as tumorigenicity and self-renewal, indicating that they could be the source of metastasis. Although previous studies analyzed the correlation between cancer stem cell (CSC) marker expression and clinical outcomes using lung cancer tissues, investigations regarding the association of MPE with CSC marker expression are limited. We performed immunohistochemistry to examine the expression of aldehyde dehydrogenase 1 (ALDH1) and Sal-like 4 (SALL4) in 46 cell block samples of MPE from patients with lung adenocarcinoma. ALDH1-positive and SALL4-positive cancer cells in MPE were detected in 30 (65.2%) and 21 samples (45.7%), respectively. Cluster formation was detected in 26 samples (56.5%). The number of clusters was significantly higher in ALDH1-positive/SALL4-negative samples. SALL4 expression was inversely correlated with the cluster ratio (r = −0.356) and positively associated with the Ki-67 index (r = 0.326), suggesting that MPE cells with high SALL4 expression comprised the proliferative subpopulation. In conclusion, we demonstrated that MPE contains an ALDH1-positive/SALL4-negative subpopulation exhibiting cluster formation and a SALL4-positive proliferative subpopulation.

Fluorescent sensing of nucleus density assists in identifying tumor cells using an AIE luminogen

Nucleus density is an important factor associated with a wide variety of cell behaviors. Mapping nucleus density is essential to study its roles in fundamental biology and pathological conditions, but ideal tools are still lacking. Herein, we report a density-responsive bioprobe (MASPB) for fluorescent sensing of cell nucleus density. MASPB is a small-molecule probe with aggregation-induced emission (AIE) attribute. It can easily penetrate into the nucleus with red fluorescence, whose intensity is correlated to the nuclear condensation. Upon MASPB staining, distinct structures in nucleus between tumor cells and normal cells are clearly revealed. We demonstrated the ability of MASPB to distinguish rare tumor cells from normal cells in malignant pleural effusion samples by virtue of their different cellular distributions, which outshined traditional histochemical stain in contrast and successfully detected tumor cells from all 10 cancer patients. Moreover, the capability of MASPB for fluorescent differentiation of tumor/normal regions in various tissue specimens was also validated. The findings presented herein provide a facile and rapid tool for nucleus density mapping. This could enable a wide range of applications in the fields of basic research and clinical cancer diagnosis.

Detection of Klebsiella pneumoniae cfDNA in pleural fluid and its clinical value

Klebsiella pneumoniae (KP) is an important opportunistic pathogen that can easily cause pneumonia and pleural effusion when body resistance is reduced. However, the positive rate of KP detected from clinical pleural effusion by traditional methods, including bacterial culture, is meager. Therefore, new detection methods are urgently needed to improve the positive detection rate of KP and other bacteria in pleural effusion. Simulated pleural fluid of KP infection was first set up. Then circulating cell-free DNA (cfDNA) was extracted from cultured hydrothorax and detected by fluorescence polymerase chain reaction (PCR) to verify KP cfDNA in the pleural fluid. The specificity, sensitivity, and repeatability of this method are verified by detecting the cfDNAs in pleural effusion, samples of malignant pleural effusion, tuberculous pleural effusion, and other common microbial infections. Finally, this method was compared with three traditional methods, pleural effusion, precipitation DNA, sputum culture, and pleural effusion culture to explore the clinical diagnostic value of this method. KP cfDNA was positive by fluorescence PCR from the simulated KP infected pleural effusion, which confirmed KP cfDNA in pleural effusion. KP cfDNA was positive by fluorescence PCR from the pleural effusion of KP infected patients, while with the same detection method, KP cfDNA in clinical carcinomatous hydrothorax, tuberculosis hydrothorax, and other standard microbial infection samples was negative, which confirmed the method had high specificity, high sensitivity, and reproducibility. Compared with the three traditional methods, this method has a higher positive rate. Compared with the gold standard, sputum bacterial culture, the sensitivity, specificity, positive predictive value, and negative predictive value of this method were 91.67%, 95.45%, 91.7%, and 95.5%, respectively. The detection of cfDNA by fluorescence PCR is possible. Moreover, the positive rate of this method in clinical pleural effusions is high.

Microbiome profile associated with malignant pleural effusion.

IntroductionThere is ongoing research into the development of novel molecular markers that may complement fluid cytology malignant pleural effusion (MPE) diagnosis. In this exploratory pilot study, we hypothesized that there are distinct differences in the pleural fluid microbiome profile of malignant and non-malignant pleural diseases.MethodFrom a prospectively enrolled pleural fluid biorepository, samples of MPE were included. Non-MPE effusion were included as comparators. 16S rRNA gene V4 region amplicon sequencing was performed. Exact Sequence Variants (ESVs) were used for diversity analyses. The Shannon and Richness indices of alpha diversity and UniFrac beta diversity measures were tested for significance using permutational multivariate analysis of variance. Analyses of Composition of Microbiome was used to identify differentially abundant bacterial ESVs between the groups controlled for multiple hypothesis testing.Results38 patients with MPE and 9 with non-MPE were included. A subgroup of patients with metastatic adenocarcinoma histology were identified among MPE group (adenocarcinoma of lung origin (LA-MPE) = 11, breast origin (BA-MPE) = 11). MPE presented with significantly greater alpha diversity compared to non-MPE group. Within the MPE group, BA-MPE was more diverse compared to LA-MPE group. In multivariable analysis, ESVs belonging to family S24-7 and genera Allobaculum, Stenotrophomonas, and Epulopiscium were significantly enriched in the malignant group compared to the non-malignant group.ConclusionOur results are the first to demonstrate a microbiome signature according to MPE and non-MPE. The role of microbiome in pleural effusion pathogenesis needs further exploration.

PD-L1 expression in malignant pleural effusion samples and its correlation with oncogene mutations in non-small cell lung cancer.

BackgroundProgrammed death ligand 1 (PD-L1) tumor proportion score (TPS) is currently widely used for selection of immune therapies in non-small cell lung cancer (NSCLC). Most of samples for PD-L1 expression were obtained from tumor tissue. However, the feasible of malignant pleural effusion (MPE) cytological samples for PD-L1 detection is poorly reported. And the correlation between oncogene mutations and PD-L1 expression based on high-throughput sequencing is rarely studied.MethodsNSCLC MPE cytological samples and partially paired tumor tissue from our institution analyzed for PD-L1 immunohistochemistry (IHC) using the clone SP263 pharmDx kit and evaluated genomic aberrations in all patients using next generation sequencing (NGS).ResultsOne hundred and twenty-three MPE cell blocks and 29 paired tumor tissue were successfully tested for PD-L1 expression. PD-L1 TPS of ≥50% were seen in 18.7% (23/123) of all samples. The accordance of PD-L1 expression in tumor tissue and MPE samples was 86.2% (50% as cut-off value). PD-L1 TPS ≥50% tumors were significantly associated with EGFR wild-type (P=0.007), but, no correlation between other genes and PD-L1 expression. A trend of longer overall survival (OS) was observed in patients with PD-L1 TPS <50% than those TPS ≥50% (20.0 vs. 13.8 months, P=0.057). No difference of tumor mutational burden (TMB) was observed between patients with PD-L1 ≥50% and <50% (8.2/MB and 7.7/MB, P=0.47).ConclusionsOur results suggest that cytological material is feasible for PD-L1 IHC analysis. Gene alterations could partially contribute to select the samples that with different PD-L1 expression. No correlation between the PD-L1 expression and TMB.

Multiplexed molecular profiling of lung cancer with malignant pleural effusion using next generation sequencing in Chinese patients.

Lung cancer is the most common type of cancer and the leading cause of cancer-associated death worldwide. Malignant pleural effusion (MPE), which is observed in ~50% of advanced non-small cell lung cancer (NSCLC) cases, and most frequently in lung adenocarcinoma, is a common complication of stage III–IV NSCLC, and it can be used to predict a poor prognosis. In the present study, multiple oncogene mutations were detected, including 17 genes closely associated with initiation of advanced lung cancer, in 108 MPE samples using next generation sequencing (NGS). The NGS data of the present study had broader coverage, deeper sequencing depth and higher capture efficiency compared with NGS findings of previous studies on MPE. In the present study, using NGS, it was demonstrated that 93 patients (86%) harbored EGFR mutations and 62 patients possessed mutations in EGFR exons 18–21, which are targets of available treatment agents. EGFR L858R and exon 19 indel mutations were the most frequently observed alterations, with frequencies of 31 and 25%, respectively. In 1 patient, an EGFR amplification was identified and 6 patients possessed a T790M mutation. ALK + EML4 gene fusions were identified in 6 patients, a ROS1 + CD74 gene fusion was detected in 1 patient and 10 patients possessed a BIM (also known as BCL2L11) 2,903-bp intron deletion. In 4 patients, significant KRAS mutations (G12D, G12S, G13C and A146T) were observed, which are associated with resistance to afatinib, icotinib, erlotinib and gefitinib. There were 83 patients with ERBB2 mutations, but only two of these mutations were targets of available treatments. The results of the present study indicate that MPE is a reliable specimen for NGS based detection of somatic mutations.

Identification of differentially expressed miRNAs in differentiating benign from malignant pleural effusion

BackgroundTuberculosis pleural effusion (TPE) and malignant pleural effusion (MPE) are very common clinical complications. Considering the totally different prognosis and clinical treatment of TPE and MPE, the accurate and non-invasive diagnosis are very critical for patients with pleural effusion to initiate efficient management and treatment. However, effective clinical biomarkers were rarely explored to distinguish benign from MPE. The purpose of this study is to identify potential miRNAs which can probably be used to differentiate malignant pleural effusion from TPE.ResultsA total of 23 significantly differentially expressed miRNAs were identified in MPE, with 18 up-expressed and 5 down-expressed. And the target genes of the miRNAs mainly involved in the biology process of nervous system, cancer, immune system and metabolic process etc. Three high confident target genes, AGO4, FGF9 and LEF1 can be regulated by miR-195-5p, miR-182-5p and miR-34a-5p respectively. And these genes participate in the canonical pathway of regulation of the Epithelial-Mesenchymal and the biological functions of apoptosis, growth of tumor and cell proliferation of tumor cell lines. Further, RT-PCR validation results based on 64 collected individuals showed that the expression levels of the three miRNAs were 2–5 times higher in MPE samples, which were consistent with the microarray results. In addition, ROC curve analysis demonstrated that the combination of the three miRNAs can achieve higher AUC of 0.93 (p-value< 0.0001) to differentiate MPE from TPE.ConclusionsThe identified miR-195-5p, miR-182-5p and miR-34a-5p can become potential diagnostic biomarkers for MPE with further evidences.

Identification and Single-Cell Analysis of Viable Circulating Tumor Cells by a Mitochondrion-Specific AIE Bioprobe.

Liquid biopsies of cancer via single‐cell molecular profiling of circulating tumor cells (CTCs) are hampered by the lack of ideal CTC markers. In this study, it is reported that TPN, a bioprobe with aggregation‐induced emission (AIE) activity is capable of distinguishing various tumor cells from blood leukocytes based on the difference in cell mitochondria. TPN is a cell‐permeant live‐cell stain that has little effect on cell viability and integrity, enabling single‐cell DNA/RNA analysis with improved efficiency compared with traditional antibody‐based methods. Using TPN labeling, CTCs and CTC cluster are detected in the blood from patients with lung or liver cancer. The capability of TPN to identify rare tumor cells in the malignant pleural effusion samples is also demonstrated. Furthermore, RNA sequencing of single lung CTC identified by TPN is successfully performed. The findings presented here provide an antibody‐free, low‐cost, and nondisruptive approach for detection and genomic characterization of viable tumor cells based on a mitochondria‐targeting AIE luminogen. It might serve as a new tool for monitoring of genomics dynamic of tumor and unraveling the mechanisms of tumor metastasis.

CD45+CD326+ Cells are Predictive of Poor Prognosis in Non-Small Cell Lung Cancer Patients.

The epithelial-to-mesenchymal transition, the major process by which some cancer cells convert from an epithelial phenotype to a mesenchymal one, has been suggested to drive chemo-resistance and/or metastasis in patients with cancer. However, only a few studies have demonstrated the presence of CD45/CD326 doubly-positive cells (CD45/CD326 DPC) in cancer. We deployed a combination of cell surface markers to elucidate the phenotypic heterogeneity in non-small cell lung cancer (NSCLC) cells and identified a new subpopulation that is doubly-positive for epithelial and non-epithelial cell-surface markers in both NSCLC cells and patients' malignant pleural effusions. We procured a total of 39 patients' samples, solid fresh lung cancer tissues from 21 patients and malignant pleural effusion samples from 18 others, and used FACS and fluorescence microscopy to check their surface markers. We also examined the EGFR mutations in patients with known acquired EGFR mutations. Our data revealed that 0.4% to 17.9% of the solid tumor tissue cells and a higher percentage of malignant pleural effusion cells harbored CD45/CD326 DPC expressing both epithelial and nonepithelial surface markers. We selected 3 EGFR mutation patients and genetically confirmed that the newly identified cell population really originated from cancer cells. We also found that higher proportions of CD45/CD326 DPC are significantly associated with poor prognosis. In conclusion, varying percentages of CD45/CD326 DPC exist in both solid cancer tissue and malignant pleural effusion in patients with NSCLC. This CD45/CD326 doubly-positive subpopulation can be an important key to clinical management of patients with NSCLC.

P1.03-04 Use Supernatant of Malignant Pleural Effusion to Identify Driver Mutants and Monitor Response to Targeted Therapy

Malignant pleural effusion (MPE) from patients with non-small cell lung cancer (NSCLC) is useful for genetic testing due to advantages including availability, less-invasiveness, and less-heterogeneity. Generally, cell pellets of MPE are used. This study is to further address whether supernatant of MPE is a suitable source to identify key oncogenic mutants in NSCLC patients and provide evidence for clinical molecular testing. MPE samples from 12 NSCLC patients were centrifuged to obtain supernatants and cell pellets, and DNA were extracted. The DNA samples were analyzed by next generation sequencing (NGS) panels using Illumina HiSeq platform. First, MPE samples with the corresponding cancer tissues were collected from 3 NSCLC patients and analyzed with a 500-gene comprehensive cancer panel. Nine mutants were identified in both the paired MPE and cancer tissue samples. We then analyzed nine more NSCLC patient samples using an 18-gene panel to detect key oncogenic mutants; in total, 8 mutants including EGFR L858R, 19DEL, or T790M were identified in the MPE samples. For all of the 17 mutants from the 12 MPE samples, 10 mutants were observed in both the supernatants and pellets of the matched sample sets, of which more pairs (6 out of 10) had supernatants with higher abundance of mutants than the corresponding cell pellets. Importantly, 7 of the 17 mutants were detected only in the supernatants but not the pellets of the paired MPE samples. These results suggest that supernatant of MPE is a better source to detect key oncogenic mutants of NSCLC. Interestingly, 2 patients had both sensitive and resistant mutants to EGFR tyrosine kinase inhibitor (TKI) detected in supernatants of MPE; both patients had treated with EGFR TKI previously, suggesting the development of TKI resistant mutant and supporting the usage of MPE supernatants in monitoring TKI resistance. This study demonstrates that supernatant of MPE is a suitable source for identifying key oncogenic driver mutants for NSCLC and can also be used to monitor response to targeted therapy. The study provides evidence of using supernatant of MPE as an alternative for molecular testing and thus direct precise targeted therapy and surveillance of the therapy effect.