Pleural Effusion Supernatant Research Articles

Pleural effusion supernatant: a reliable resource for cell-free DNA in molecular testing of lung cancer

DNA extracted from malignant pleural effusion (PE) sediments is the traditional source of tumor DNA for predictive biomarker molecular testing (MT). Few recent studies have proposed the utility of cell-free DNA (cfDNA) extracted from effusion cytology centrifuged supernatants (CCS) in MT. The aim of this study was to assess the feasibility and utility of molecular testing on cfDNA extracted from PE CCS in lung cancer patients. The study was of prospective design. All PE CCS were collected and stored. Subsequently, in patients confirmed as primary lung adenocarcinoma (LUAD) and where patient matched effusion sediment/tissue biopsy/plasma was being tested for EGFR mutations, cfDNA extraction and EGFR MT by real-time polymerase chain reaction (qPCR) were performed. Custom panel targeted next-generation sequencing (NGS) (Ion Torrent; Thermo Fisher, Carlsbad, CA) was also performed wherever feasible. Out of 299 PE CCS collected, 20 CCS samples were included in the study. Concordant EGFR mutations were detected in pleural effusion CCS of 10 of 11 (91%) EGFR mutant cases as per qPCR performed on the matched sediment DNA (n = 8), lung biopsy (n = 2), and plasma (n = 1) samples. In 1 positive sample, CCS detected additional EGFR T790M mutation. Among 10 CCS samples also tested by NGS, additional EGFR mutations missed by qPCR were picked up in 2 (2 of 10). Success of mutation detection in CCS cfDNA did not correlate with cfDNA quantity or tumor fraction in sediment. cfDNA from effusion CCS is a reliable and independent source of tumor DNA highly amenable for MT and complement results from other tumor DNA sources for comprehensive mutation profiling in LUAD patients.

Liquid biopsy using ascitic fluid and pleural effusion supernatants for genomic profiling in gastrointestinal and lung cancers

BackgroundPrecision medicine highlights the importance of incorporating molecular genetic testing into standard clinical care. Next-generation sequencing can detect cancer-specific gene mutations, and molecular-targeted drugs can be designed to be effective for one or more specific gene mutations. For patients with special site metastases, it is particularly important to use appropriate samples for genetic profiling. This study aimed to determine whether genomic profiling using ASC and PE is effective in detecting genetic mutations.MethodsTissues, plasma, ascites (ASC) supernatants, and pleural effusion (PE) samples from gastrointestinal cancer patients with peritoneal metastasis and lung cancer patients with pleural metastasis were collected for comprehensive genomic profiling. The samples were subjected to next-generation sequencing using a panel of 59 or 1021 cancer-relevant genes panel.ResultsA total of 156 tissues, 188 plasma samples, 45 ASC supernatants, and 1 PE samples from 304 gastrointestinal cancer patients and 446 PE supernatants, 122 tissues, 389 plasma samples, and 45 PE sediments from 407 lung cancer patients were analyzed. The MSAF was significantly higher in ASC and PE supernatant than that in plasma ctDNA (50.00% vs. 3.00%, p < 0.0001 and 28.5% vs. 1.30%, p < 0.0001, respectively). The ASC supernatant had a higher actionable mutation rate and more actionable alterations than the plasma ctDNA in 26 paired samples. The PE supernatant had a higher total actionable mutation rate than plasma (80.3% vs. 48.4%, p < 0.05). The PE supernatant had a higher frequency of uncommon variations than the plasma regardless of distant organ metastasis.ConclusionASC and PE supernatants could be better alternative samples when tumor tissues are not available, especially in patients with only peritoneal or pleural metastases.

Characteristics of Genomic Alterations in Pericardial Effusion of Advanced Non-small Cell Lung Cancer.

Background: The feasibility and value of pericardial effusion as a liquid biopsy sample for actionable alteration detection in patients with non-small cell lung cancer (NSCLC) has not been adequately investigated. Here, we aim to reveal genomic alterations between pericardial effusion and paired tumor tissue, plasma (plasma cfDNA), and pleural effusion supernatant (PE-cfDNA) based on second-generation sequencing technology. Material and methods: A total of 26 advanced NSCLC patients were retrospectively studied. The following samples were collected and sequenced using two targeted next-generation sequencing panels: pericardial effusion (n = 26), matched tumor tissue (n = 6), plasma (n = 16), and pleural effusion supernatant (n = 5). Results: A total of 10 actionable alterations were identified in pericardial effusion of the NSCLC patients, including MET amplification, EGFR L858R, EGFR T790M, EGFR exon 19 deletion, EGFR L861Q, KRAS G12C, EML4-ALK (exon 18: exon 20) fusion, EML4-ALK (exon 20: exon 20) fusion, EML4-ALK (exon 6: exon 20) fusion, and ERBB2 exon 20 insertion. All these actionable alterations harbored multiple drug-sensitive targets as well as several drug-resistant targets, such as EGFR T790M. Compared to plasma cfDNA of 16 patients, paired pericardial effusion had higher number of actionable alterations (p = 0.08) as well as higher percentage of the population with actionable alterations (p = 0.16). Moreover, 8 out of 10 actionable alterations with single nucleotide variations (SNVs) or insertions/deletions (indels) had a higher variant allele frequency (VAF) in pericardial effusion than plasma cfDNA. In addition, we identified two actionable alterations in paired pericardial effusion, which were absence in PE-cfDNA. Clearly, 2 out of 3 actionable alterations with SNVs/indels in pericardial effusion had a higher VAF than those in PE-cfDNA. Our finding suggested the importance of pericardial effusion in the optimal selection of patients for targeted therapy. Conclusion: Among liquid biopsy specimens from the advanced NSCLC patients, pericardial effusion may be a better candidate for genomic profiling than plasma cfDNA, while it could serve as a supplement to PE-cfDNA in detecting actionable alterations. Therefore, pericardial effusion might provide a new alternative for selection of patients for better treatment management.

Abstract 558: Muti-gene ARMS PCR using both cfDNA and cfRNAin the supernatant of pleural effusion achieves rapid and accuracy driver gene mutations detection

Abstract Background Pleural effusion from patients with advanced non-small cell lung cancer has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging. Methods In this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and FFPE cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR-based 9-gene mutation detection kit. Results In addition to mutations in EGFR, KRAS and HER2 detected in genomic DNA from cell sediments, FFPE cell blocks, and in cfDNA from supernatants, and fusions in ALK detected in RNA from cell sediments and FFPE cell blocks, fusions in ALK were also successfully detected in cfRNA from supernatants. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by FFPE cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment-naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5%, 92.3%) with both 100% for fusions. Based on the results, an optimized driver gene mutations detection procedure of pleural effusion is proposed. With the proposed procedure, an overall sensitivity 85.3% was achieved, with 82.1% for SNV/Indels and 100% for fusions. Conclusions CfDNA and cfRNA derived from pleural effusion supernatant (PES) have been successfully tested with a PCR-based multi-gene detection kit. An optimized procedure could maximize clinical value of testing pleural effusion samples and has good potential for routine clinical application. Citation Format: Xuejing Chen, Kun Li, Zichen Liu, Fei Gai, Guanshan Zhu, Shun Lu, Nanying Che. Muti-gene ARMS PCR using both cfDNA and cfRNAin the supernatant of pleural effusion achieves rapid and accuracy driver gene mutations detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 558.

Next-Generation Sequencing of Cell-Free DNA Extracted From Pleural Effusion Supernatant: Applications and Challenges.

Cell-free DNA (cfDNA) extracted from diverse specimen types has emerged as a high quality substrate for molecular tumor profiling. Analytical and pre-analytical challenges in the utilization of cfDNA extracted from pleural effusion supernatant (PES) are herein characterized in patients with metastatic non-small cell lung carcinoma (NSCLC). Pleural effusion specimens containing metastatic NSCLC were collected prospectively. After ThinPrep® (TP) and cell block (CB) preparation, DNA was extracted from residual PES and analyzed by gel electrophoresis for quality and quantity. Libraries were prepared and sequenced with a targeted next-generation sequencing (NGS) platform and panel clinically validated for plasma specimens. Results were compared with DNA extracted from corresponding FFPE samples that were sequenced using institutional targeted NGS assays clinically validated for solid tumor FFPE samples. Tumor (TC) and overall cellularity (OC) were evaluated. Fourteen specimens were collected from 13 patients. Median specimen volume was 180 mL (range, 35–1,400 mL). Median TC and OC on TP slides and CB sections were comparable. Median extracted DNA concentration was 7.4 ng/μL (range, 0.1–58.0 ng/μL), with >5 ng/μL DNA extracted from 10/14 specimens (71%). Mutations were identified in 10/14 specimens, including 1/3 specimens with median molecular coverage <1,000 reads. The minimal detected allelic fraction was 0.6%. NGS was falsely negative for the presence of one driver mutation. No correlation was identified between sample volume or OC, quality or quantity of extracted DNA, or mutation detection. Despite analytical and pre-analytical challenges, PES represents a robust source of DNA for NGS.

Multigene PCR using both cfDNA and cfRNA in the supernatant of pleural effusion achieves accurate and rapid detection of mutations and fusions of driver genes in patients with advanced NSCLC.

BackgroundPleural effusion from patients with advanced non‐small cell lung cancer (NSCLC) has been proved valuable for molecular analysis, especially when the tissue sample not available. However, simultaneous detection of multiple driver gene alterations especially the fusions is still challenging.MethodsIn this study, 77 patients with advanced NSCLC and pleural effusion were enrolled, 49 of whom had matched tumor tissues. Supernatants, cell sediments, and cell blocks were prepared from pleural effusion samples for detection of driver alterations by a PCR‐based 9‐gene mutation detection kit.ResultsMutations in EGFR, KRAS, and HER2 were detected in DNA and cfDNA, fusions in ALK was detected in RNA and cfRNA. Compared with matched tumor tissue, the supernatant showed the highest overall sensitivity (81.3%), with 81.5% for SNV/Indels by cfDNA and 80% for fusions by cfRNA, followed by cell blocks (71.0%) and the cell sediments (66.7%). Within the group of treatment‐naïve patients or malignant cells observed in the cell sediments, supernatant showed higher overall sensitivity (89.5% and 92.3%) with both 100% for fusions.ConclusionsCfDNA and cfRNA derived from pleural effusion supernatant have been successfully tested with a PCR‐based multigene detection kit. Pleural effusion supernatant seems a preferred material for detection of multigene alterations to guide treatment decision of advanced NSCLC.

Distinct profile of cell-free DNA in malignant pleural effusion of non-small cell lung cancer and its impact on clinical genetic testing.

Cell-free DNA (cfDNA) in supernatant of pleural effusion from advanced NSCLC patients has been proved as surrogate sample detecting therapeutic targets as well as tumor mutation burden (TMB). As recently reported, cfDNA in pleural effusion supernatant is superior to plasma in TMB evaluation. It is reasonable to hypothesize that cfDNA profile in pleural effusion (PE) and plasma might be different. It remains to be elucidated why cfDNA in PE supernatant impacts on genetic analysis. Consequently, the approach dealing with cfDNA from PE supernatant might need to be different from that for plasma cfDNA in order to obtain accurate clinical genetic testing result.Methods: Pleural effusion samples from 32 patients with stage IV lung adenocarcinoma were collected. Supernatant and sediment were processed separately to extract Cell-free DNA as well as sediment DNA (PE-S). cfDNA from pleural effusion was analyzed by Agilent 2100 bioanalyzer. Libraries were prepared by 1) direct use of the total cfDNA without fragmentation step (PE-FL) or 2) use of full-length cfDNA fragmented to 150-250bp (PE-F), 3) use of cfDNA fragments enriched to ~167bp (PE-E167) as well as 4) use of cfDNA fragments larger than 500bp enriched (PE-E500). All samples were subjected to targeted next-generation sequencing (NGS) with a panel of 448 cancer-related genes as well as a panel of 10 NSCLC driver genes.Results: cfDNA were successfully extracted from 30 MPE samples. cfDNA displayed distinct profile in supernatant of malignant pleural effusion from that of plasma cfDNA. No statistical difference in detection of hotspot variations between PE-E167 and PE-F by 448-gene or 10-gene panel. While TMB from PE-F samples was significantly higher than that from PE-E167 and PE-FL. Higher TMB from PE-F was resulted from cancer-unspecific variants with low allele frequency (0.1%-1%) which were mainly introduced by long-fragment cfDNA. Similar genetic profile was observed between paired cfDNA of PE-FL and cfDNA of PE-E167.Conclusion: Long-fragment cfDNA in the PE supernatant will introduce low abundant cancer unrelated variants which leads overestimation of TMB. Paired PE-FL and PE-E167 gave comparable outcomes. Direct use of the total cfDNA without fragmentation step (PE-FL) is recommended for library preparation of NGS testing in clinical practice to exclude interference from long fragments of the cfDNA.

Standardization of pleural effusion-based tumor mutation burden (TMB) estimation using capture-based targeted sequencing

BackgroundTumor mutation burden (TMB) has received considerable attention as a potential predictive biomarker for response to anticancer treatment with immune checkpoint inhibitors (ICIs), and has been increasingly incorporated into clinical practice. Currently, TMB is often determined with tissue biopsies using whole-exome sequencing (WES) or panel-based targeted sequencing. Meanwhile, liquid biopsies such as blood are actively investigated as alternative media, although there is currently no report of the performance of targeted sequencing in assessing TMB using pleural effusion (PE) specimens.MethodsThirty-two patients diagnosed with advanced non-small cell lung cancer (NSCLC) with associated PE were prospectively enrolled (NCT 03546452). Cell-free DNA (cfDNA) from the supernatant of PE was subjected to both WES and capture-based targeted sequencing using various commercially-available panels.ResultsAll five panels assessed in this study demonstrated a good correlation with WES-derived TMB, with correlation coefficients ranging from 0.68–0.81. Two- and three-tier classification systems built on the TMB estimates achieved respective concordance rates of 74% and 63% between classifications based on WES- and panel-derived TMB levels.ConclusionsThis study provides real-world evidence that all panels assessed in this study can be used for TMB evaluation based on PE samples. We also demonstrated that PE can serve as an alternative medium for TMB evaluation. To the best of our knowledge, this is the first study evaluating the potential of PE samples for TMB estimation, thereby providing a basis for establishing future standard protocols.

Molecular Mechanisms of Resistance to Osimertinib from Liquid Biopsies of Plasma and Pleura Effusion in EGFR Mutant NSCLC Patients

In the past decade, tyrosine kinase inhibitors (TKI) have been developed and widely-approved by countries to treat advanced lung cancer patients with sensitizing epidermal growth factor (EGFR) mutations, and substantially improved patients’ prognosis compared to traditional chemotherapies. However, a large proportion of patients who initially experienced ostensibly remarkable therapeutic outcomes still developed inevitable drug resistance eventually, including those received newly developed third-generation TKIs. Previous studies have demonstrated the value of plasma cell-free DNA (cfDNA) in monitoring patients’ response to therapies in lung cancer. However due to several physiological reasons and DNA degradation, DNA released from tumor cell apoptosis in lung was hard to maintain at a high concentration and good quality before being collected from venous blood, leading to the loss of pivotal mutational information. Pleural effusion is a potential substitute to overcome these problems. However, limited studies have been reported to characterize the consistency of genetic alterations found in these two sample types, and due to the modest sample sizes, the mechanisms underlying third-generation TKI resistance remains elusive. Herein we drew 8ml venous blood and collected 20ml pleural effusion by pleural effusion drainage from 8 lung cancer patients, and performed hybrid-capture based next generation sequencing assays on 18 matched plasma cfDNA, pleural effusion cells genomic DNA and pleural effusion supernatant cfDNA samples, we have shown that only sequencing either plasma or pleural effusion sample resulted in the missing of mutations in key cancer drivers. Sequencing both pleural effusion and plasma yielded a more concrete genetic landscape for monitoring tumor response. We have also exhibited that somatic mutations and copy number changes in PI3K/AKT pathway, NOTCH signaling pathway, hedgehog signaling pathway and pathways involved in DNA damage repair might be potential mechanisms underlying TKI resistance, and might serve as candidate targets for developing novel therapies to circumvent patients’ resistance.

A multicenter real-world study of tumor-derived DNA from pleural effusion supernatant in genomic profiling of advanced lung cancer.

BackgroundPleural effusion (PE) is commonly observed in advanced lung cancer. Research has suggested that molecular profiling of PE could be used to detect tumor driver mutations, thus informing clinical decision-making. However, the performance of PE samples in a real-world setting has yet to be examined.MethodsA total of 678 metastatic lung cancer patients with pleural effusion were enrolled in this study. Cohort 1 included 22 patients whose PE and matched plasma samples were simultaneously collected as a pilot study. Cohort 2 comprised 656 patients, from whom 734 samples were collected in a real world setting. These samples were subjected to targeted next-generation sequencing (NGS) of 1,021 cancer-related genes.ResultsPE supernatant was the preferred choice for genetic profiling. While the maximal somatic allele frequency (MSAF) of plasma in patients with M1a stage was significantly lower than that in patients with M1b/c stages (4.4%±9.6% vs. 9.0%±14.1%, P<0.01), the MSAF of PE supernatant was similar between M1a and M1b/c stages. PE supernatant demonstrated higher sensitivity than plasma in detecting actionable mutations in cohort 1 (81.8% vs. 45.5%, P=0.01) as well as in M1a disease (84.7% vs. 42.1%, P<0.01), but not in M1b/c disease, in cohort 2. Known resistant mutations were identified in 72 of the 117 patients who were resistant to first- or second-generation EGFR-TKIs, 22 of the 42 patients who were resistant to osimertinib, and 9 of the 13 patients who were resistant to crizotinib. Remarkably, PE supernatant outperformed plasma in identifying mutations that confer resistance to first- and second-generation EGFR-TKIs (75.4% vs. 29.8%, P<0.001).ConclusionsThis real-world large cohort study verified that PE supernatant had higher sensitivity than plasma for identifying actionable mutations, including resistance mutations. PE supernatant would be preferred by physicians for assessing tumor genomics in advanced lung cancer when tumor tissue is not available.

A multicenter real-world study of tumor-derived DNA from pleural effusion supernatant in genomic profiling of advanced lung cancer.

e21584 Background: Pleural effusion (PE) is commonly observed in advanced lung cancer. Researches have suggested molecular profiling of PE represents a minimally invasive approach of detecting tumor driver mutations for clinical decision making. The objective of this study is to investigate the efficacy and precision of detecting gene alterations in PE samples in the real world setting. Methods: 656 metastatic lung cancer patients with pleural effusion were enrolled in this study. Seven hundred and thirty-two samples, including 351 samples of PE supernatant, 224 plasma, 138 tissue, and 25 PE sediments from these patients were collected and subjected to targeted next-generation sequencing (NGS) of 1021 cancer-related genes in a real world setting. The efficacy of pleural effusion in detecting actionable mutations and identifying resistant mechanisms of targeted therapy were analyzed by comparing different samples. Results: Among the 656 NSCLC patients, 413 were in M1a stage and 243 were in M1b/M1c stage, while 272 were newly diagnosed and 384 was previously treated. When comparing different groups of stage and therapeutic history, PE supernatant was preferred as the choice for those patients (46.6% - 48.2% vs 23.3%-34.8% of plasma vs 16.8%-21.2% of tissue and 0.96%-7.3% of PE sediment). While mutant allele frequency (MAFs) of plasma in patients of M1a stage was significantly lower than that of M1b/c stages, MAFs was similar for PE supernatant. EGFR, KRAS, MET, ALK, BRAF, ERBB2, ROS1, and RET actionable mutations were identified in 60, 12, 9, 7, 6, 3, 2, and 1 of the 118 PE supernatant samples at M1a stage taken before treatment. PE-supernatant demonstrated higher sensitivity than plasma of detecting actionable mutations in M1a disease (84.7% of PE-supernatant vs 42.1% of plasma, p &lt; 0.01) but not in M1b/c stages (80.7% of PE-supernatant vs 86.4% of plasma). Seventy-two of the 117 patients who were resistant to 1st or 2nd generation of EGFR-TKI, 22 of the 42 patients resistant to osimertinib, and 9 of the 13 patients resistant to crizotinib had known resistant mutations identified. Remarkably, PE supernatant outperformed plasma in identifying resistant mutations to 1st/2nd generation EGFR-TKI (75.4% vs 29.8%, p &lt; 0.001). Conclusions: This real world large cohort study verified that genomic profiling of PE-supernatant has higher actionable mutation detection sensitivity than plasma. It offers an alternative approach in assessing tumor genomics in advanced lung cancer when tumor tissue is not available.

Gene Alterations in Paired Supernatants and Precipitates from Malignant Pleural Effusions of Non-Squamous Non-Small Cell Lung Cancer

OBJECTIVE: This study investigated the feasibility of using malignant pleural effusion (MPE) supernatant and paired cell blocks (precipitate) for gene profiling in patients with non-small cell lung cancer (NSCLC) using next-generation sequencing (NGS) technique. METHODS: Stage IV non-squamous NSCLC patients with MPE were eligible in this prospective study and recruited from Zhejiang Cancer Hospital between May 2014 and October 2015. MPE supernatant and paired precipitate sample gene alterations were determined with NGS containing 14 cancer-related genes. Progression free survival (PFS) was evaluated using Kaplan–Meier method and compared using log-rank test. RESULTS: A total of 102 patients were enrolled in the present study. All pleural effusions were confirmed as malignant with cytological smears. A total of 77 paired MPE supernatant and precipitate samples were acquired from the 102 patients. The results revealed that there were no statistically significant differences in the detection rate and maximum allelic fraction between supernatant and precipitate samples (P = 1.0 and P = .6). Collectively, 172 and 158 genomic alterations with 112 shared mutations were identified in supernatant and precipitate samples, respectively. Comparable PFS was found in EGFR mutation patients according to the supernatant and precipitate sample results (14.0 vs.13.9 months, P = .90). CONCLUSIONS: These results demonstrated that MPE supernatants were comparable to precipitate samples for detection of genetic alterations. However, gene mutation heterogeneity was found between these two media types.

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.

The study of pleural effusion supernatant cell-free tumor DNA in tumor mutational burden assessment of advanced lung cancer

Objective: To evaluate the feasibility of cell-free tumor DNA in pleural effusion supernatant for assessing the tumor mutational burden (TMB) of advanced lung cancers. Methods: From December 2016 to August 2018, 34 lung cancer patients (19 males and 15 females) with pleural effusion were enrolled at Zhongshan Hospital, Fudan University. The median age of the patients was 65 (range, 34-85) years. Before systemic or local antitumor therapy, tumor specific mutations in tumor tissue, pleural effusion supernatant, pleural effusion sediment, and plasma samples from these patients were examined using targeted next-generation sequencing, and TMB levels were calculated respectively. Subgroup analysis was based on smoking history and driver mutation status. Statistical differences were determined using SPSS 16.0 software, and individual groups were compared using the one-way analysis of variance (ANOVA) and LSD-t test. Results: The median TMB level of pleural effusion supernatant was 6.23 mutations/Mb, similar to that of tumor tissue (6.23 vs 6.86 mutations/Mb, t=1.174, P=0.245), but significantly higher than that of pleural effusion sediment (2.49 mutations/Mb, t=3.044, P=0.003) and plasma (2.49 mutations/Mb, t=2.464, P=0.016). Compared with tumor tissue in TMB assessment, pleural effusion supernatant had a positive percentage agreement of 52% (9/17), and a negative percentage agreement of 65% (11/17). Subgroup analysis showed that the TMB level was higher in smokers (n=11) than that in non-smokers (n=23, 14.4 vs 5.4 mutations/Mb, t=3.238, P=0.003). Conclusion: For advanced lung cancer patients with pleural effusion, pleural effusion supernatant is a promising substitute to tumor tissue for TMB assessment, which is a potential biomarker for immunotherapy.

Targeted deep sequencing from multiple sources demonstrates increased NOTCH1 alterations in lung cancer patient plasma.

IntroductionTargeted therapies are based on specific gene alterations. Various specimen types have been used to determine gene alterations, however, no systemic comparisons have yet been made. Herein, we assessed alterations in selected cancer‐associated genes across varying sample sites in lung cancer patients.Materials and MethodsTargeted deep sequencing for 48 tumor‐related genes was applied to 153 samples from 55 lung cancer patients obtained from six sources: Formalin‐fixed paraffin‐embedded (FFPE) tumor tissues, pleural effusion supernatant (PES) and pleural effusion cell sediments (PEC), white blood cells (WBCs), oral epithelial cells (OECs), and plasma.ResultsMutations were detected in 96% (53/55) of the patients and in 83% (40/48) of the selected genes. Each sample type exhibited a characteristic mutational pattern. As anticipated, TP53 was the most affected sequence (54.5% patients), however this was followed by NOTCH1 (36%, across all sample types). EGFR was altered in patient samples at a frequency of 32.7% and KRAS 10.9%. This high EGFR/ low KRAS frequency is in accordance with other TCGA cohorts of Asian origin but differs from the Caucasian population where KRAS is the more dominant mutation. Additionally, 66% (31/47) of PEC samples had copy number variants (CNVs) in at least one gene. Unlike the concurrent loss and gain in most genes, herein NOTCH1 loss was identified in 21% patients, with no gain observed. Based on the relative prevalence of mutations and CNVs, we divided lung cancer patients into SNV‐dominated, CNV‐dominated, and codominated groups.ConclusionsOur results confirm previous reports that EGFR mutations are more prevalent than KRAS in Chinese lung cancer patients. NOTCH1 gene alterations are more common than previously reported and reveals a role of NOTCH1 modifications in tumor metastasis. Furthermore, genetic material from malignant pleural effusion cell sediments may be a noninvasive manner to identify CNV and participate in treatment decisions.

Cell-free DNA in the supernatant of pleural effusion can be used to detect driver and resistance mutations, and canguide tyrosine kinase inhibitor treatment decisions.

ObjectivesMolecular profiling of tumours has become the mainstay of diagnostics for metastasised solid malignancies and guides personalised treatment, especially in nonsmall cell lung cancer (NSCLC). In current practice, it is often challenging to obtain sufficient tumour material for reliable molecular analysis. Cell-free DNA (cfDNA) in blood or other bio-sources could present an alternative approach to obtain genetic information from the tumour. In a retrospective cohort we analysed the added value of cfDNA analysis in pleural effusions for molecular profiling.MethodsWe retrospectively analysed both the supernatant and the cell pellet of 44 pleural effusions sampled from 39 stage IV patients with KRAS (n=23) or EGFR (n=16) mutated tumours to detect the original driver mutation as well as for EGFR T790M resistance mutations. Patients were diagnosed with either NSCLC (n=32), colon carcinoma (n=4), appendiceal carcinoma (n=2) or adenocarcinoma of unknown primary (n=1). Samples collected in the context of routine clinical care were stored at the Netherlands Cancer Institute biobank. We used droplet digital PCR for analysis.ResultsThe driver mutation could be detected in 36 of the 44 pleural effusions by analysis of both the supernatant (35 out of 44 positive) and the cell pellet (31 out of 44 positive). In seven out of 20 pleural effusions from patients with EGFR mutation-positive tumours, a T790M mutation was detected. All seven supernatants and cell pellets were positive.ConclusionscfDNA in pleural effusion can be used to detect driver mutations as well as resistance mechanisms like EGFR T790M in pleural effusion with high accuracy and is therefore a valuable bio-source.

Tumor-derived DNA from pleural effusion supernatant as a promising alternative to tumor tissue in genomic profiling of advanced lung cancer.

Pleural effusion (PE) is commonly observed in advanced lung cancer and was suggested to contain both cell-free tumor DNA and tumor cells. Molecular profiling of PE represents a minimally invasive approach of detecting tumor driver mutations for clinical decision making, especially when tumor tissues are not available. The objective of this study is to investigate the efficacy and precision of detecting gene alterations in PE samples to address the feasibility in clinical use.Methods: Sixty-three metastatic lung cancer patients with (n=30, cohort 1) or without (n=33, cohort 2) matched tumor tissues were enrolled in this study. PE and plasma samples of each patient were collected simultaneously. Supernatant and cell precipitate of PE were processed separately to extract cfDNA (PE-cfDNA) and sediment DNA (sDNA). All samples were subjected to targeted next-generation sequencing (NGS) of 416 cancer-related genes.Results: PE supernatants contain more abundant tumor DNA than PE sediments and plasma samples, suggested by higher mutant allele frequencies (MAF) and elevated mutation detection rate in PE-cfDNA (98.4% vs. 90.5% in PE sDNA vs. 87% in plasma cfDNA). In Cohort 1 with matched tumor tissue, tumor mutational burden (TMB) of PE-cfDNA was similar as tumor tissues (6.4 vs. 5.6), but significantly higher than PE sDNA (median TMB: 3.3) and plasma cfDNA (median TMB: 3.4). Ninety-three percent (27 out of 29) of tissue-determined driver mutations were detected in PE-cfDNA, including alterations in ALK, BRAF, EGFR, ERBB2, KRAS, NF1, PIK3CA, and RET, while only 62% were captured in plasma cfDNA. PE-cfDNA also has the highest detection rate of EGFR driver mutations in the full cohort (71% vs. 68% in PE sDNA vs. 59% in plasma cfDNA). Mutation detection from cytological negative and hemorrhagic PE is challenging. Comparatively, PE-cfDNA demonstrated absolute superiority than PE sDNA in such a scenario, suggesting that it is an independent source of tumor DNA and therefore less influenced by the abundance of tumor cells.Conclusion: Genomic profiling of PE-cfDNA offers an alternative, and potentially more meticulous approach in assessing tumor genomics in advanced lung cancer when tumor tissue is not available. Our data further demonstrate that in hemorrhagic or cytologically negative PE samples, PE-cfDNA has higher mutation detection sensitivity than sDNA and plasma cfDNA, and therefore is a more reliable source for genetic testing.

Liquid biopsy using the supernatant of a pleural effusion for EGFR genotyping in pulmonary adenocarcinoma patients: a comparison between cell-free DNA and extracellular vesicle-derived DNA

BackgroundEGFR genotyping in pulmonary adenocarcinoma patients who develop pleural effusions is mostly performed using cytology or cell block slides with low sensitivity. Liquid biopsy using the supernatant of pleural effusions may be more effective because they contain many components released by cancer cells. Extracellular vesicles (EVs) are known to carry oncogenic double-stranded DNA that is considered a notable biomarker. Here, we investigate the efficiency of liquid biopsy using cell-free DNA (cfDNA) and extracellular vesicle-derived DNA (EV-derived DNA) from the supernatant of pleural effusions for EGFR genotyping in patients with pulmonary adenocarcinoma.MethodsFifty pleural effusion samples from patients with pulmonary adenocarcinoma were evaluated. The supernatant, after removing the cell pellet by centrifugation, was used for liquid biopsy, and EVs were isolated from the pleural effusion by ultracentrifugation. EV-derived DNA and cfDNA were extracted separately, and EGFR genotyping was performed by the PNA clamping method.ResultsAmong 32 patients who were EGFR-tyrosine kinase inhibitor (TKI) naïve with a known tissue EGFR genotype, liquid biopsy using EV-derived DNA from the pleural effusion supernatant showed 100% matching results with tissue EGFR genotyping in 19 EGFR mutant cases and detected three additional EGFR mutations in patients with wild-type (WT) tissue. Liquid biopsy using cfDNA from pleural effusion supernatants missed two cases of tissue-based EGFR mutations and found two additional EGFR mutation cases. In 18 patients who acquired resistance to EGFR-TKI, EGFR genotyping using EV-derived DNA from the pleural effusion supernatant detected the T790 M mutation in 13 of 18 (72.2%) patients, and this mutation was detected in 11 (61.1%) patients using cfDNA. By contrast, only three patients were found to present the T790 M mutation when using cell block or cytology slides.ConclusionsLiquid biopsy using the supernatant of pleural effusions showed significantly improved results for EGFR genotyping compared to those using conventional cell block or cytology samples. Liquid biopsy using EV-derived DNA is promising for EGFR genotyping, including T790 M detection in pulmonary adenocarcinoma patients who develop pleural effusions.

High Sensitive and Non-invasive ctDNAs Sequencing Facilitate Clinical Diagnosis And Clinical Guidance of Non-small Cell Lung Cancer Patient: A Time Course Study

Lung cancer is one of leading causes of cancer death all over the world. Non-small cell lung cancer (NSCLC) is the most predominant subtype of lung cancer. Molecular targeting therapy has been shown great success in the treatment of advanced NSCLC. Thus, an easy, sensitive, and specific way of recognizing therapeutic gene targets would help to select effective treatments, to improve physical condition and increase patient survival. In this study, we recruited and followed up a female NSCLC patient, whose plasma ctDNAs (circulating tumor DNAs), blood cell DNAs, psDNAs (pleural effusion supernatant DNAs), and ppDNAs (pleural effusion pellet DNAs), were collected and analyzed over periodic time points by methods of next generation sequencing (NGS), droplet digital PCR (ddPCR), and Amplification Refractory Mutation System (ARMS). In addition, pleural effusion pellets were stained by IHC (immunohistochemistry). The investigation results showed that EGFR L858R mutation was recognized by methods of NGS, ddPCR, and ARMS, while EGFR T790M mutation was only identified by methods of NGS and ddPCR but not ARMS, indicating that ARMS as an auxiliary clinical diagnostic method, is less sensitive and less reliable than NGS and ddPCR. In summary, the non-invasive and sensitive way of collecting ctDNAs for NGS and/or ddPCR screenings offers patients new diagnosis and therapeutic options.

Abstract 641: Cell free DNA in the supernatant of pleural effusion can detect driver and resistance mutations and can guide TKI treatment decisions

Abstract Introduction: Molecular profiling of tumors has become the mainstay of diagnostics for metastasized solid malignancies and guides personalized treatment, especially in non-small cell lung cancer (NSCLC). In current practice it is often challenging to obtain sufficient tumor material for reliable molecular analysis. Cell free (cf) DNA in blood or other bio-sources could present an alternative approach to obtain genetic information from the tumor. In a retrospective cohort we analyzed the added value of cfDNA analysis in pleural effusions for molecular profiling. Methods: We retrospectively analyzed both the supernatant and the cell pellet of 44 pleural effusions sampled from 39 patients with KRAS (23) or EGFR (16) positive tumors for the original driver gene mutation as well as for EGFR T790M resistance mutations. Patients were diagnosed with either NSCLC (32), colon carcinoma (4), appendiceal carcinoma (2) or adenocarcinoma of unknown primary (1). Samples collected in the context of routine clinical care were stored in the NKI-AVL biobank. We used Bio-Rad QX200 droplet digital PCR for analysis. Results: The original driver gene mutation could be detected in 37 of the 44 pleural effusions by analysis of both supernatant (35/44 positive) and cell pellet (29/44 positive). In 7 out of 20 pleural effusions from patients with EGFR mutation positive tumors, a T790M mutation was detected. All 7 supernatants were positive as were 5 of the 7 cell pellets. The EGFR T790M mutation was confirmed in all supernatants (4/4) and in 3 of the 4 cell pellets sampled from patients with T790M positive tumors (4). Conclusions: Cell free DNA in pleural effusion proved to be a valuable bio-source and can be used to detect driver gene mutations as well as resistance mechanisms like EGFR T790M in pleural effusion. Citation Format: Karlijn Hummelink, Mirte Muller, Dorothe Linders, Vincent van der Noort, Petra Nederlof, Sjaak Burgers, Gerrit Meijer, Michel van den Heuvel, Daan van den Broek, Kim Monkhorst. Cell free DNA in the supernatant of pleural effusion can detect driver and resistance mutations and can guide TKI treatment decisions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 641.