Molecular Profiling for Supernatants and Matched Cell Pellets of Pleural Effusions in Non–Small-Cell Lung Cancer
2020; Elsevier BV; Volume: 22; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2020.01.011
ISSN1943-7811
AutoresChan Xiang, Mingfei Huo, Shengji Ma, Lianying Guo, Ruiying Zhao, Haohua Teng, Jie Zhang, Yuchen Han,
Tópico(s)Lung Cancer Diagnosis and Treatment
ResumoPleural effusion (PE) is commonly observed in advanced lung cancer patients. Cell-free total nucleic acid (cfTNA) isolated from cancer patients' plasma has allowed noninvasive tumor genome analyses; however, there are limited studies of detection and characterization of cfTNA in PE. Herein, we included 47 advanced non–small-cell lung cancer patients with PE, who had lung cancer driver mutations tested on tumor tissue specimens either at diagnosis or during disease progression. The supernatant and cell pellet of each PE were evaluated for molecular profiles in parallel on an Ion Torrent next-generation sequencing platform. Somatic mutations were detected in 89.1% supernatant cfTNA, but in only 54.3% of cell pellets. The overall concordance rate between supernatants and formalin-fixed, paraffin-embedded cell pellets at the mutation level was 53.3%. By contrast, 41.7% and 5.0% of somatic alterations were detected in supernatants and cell pellets, respectively. Furthermore, joint analysis of supernatants and cell pellets from PE showed a high concordance (88.3%) of variant detection with their respective tumor tissue specimens. Low-frequency T790M mutations in three cases (0.29%, 0.41%, and 1.56%) were detected in supernatants but not in the matched cell pellets or tumor tissues. In conclusion, pleural effusion–derived cfTNA can effectively be used in clinical practice for molecular analysis by next-generation sequencing, even in cases where corresponding cell pellets or tumor tissues yield insufficient material. Pleural effusion (PE) is commonly observed in advanced lung cancer patients. Cell-free total nucleic acid (cfTNA) isolated from cancer patients' plasma has allowed noninvasive tumor genome analyses; however, there are limited studies of detection and characterization of cfTNA in PE. Herein, we included 47 advanced non–small-cell lung cancer patients with PE, who had lung cancer driver mutations tested on tumor tissue specimens either at diagnosis or during disease progression. The supernatant and cell pellet of each PE were evaluated for molecular profiles in parallel on an Ion Torrent next-generation sequencing platform. Somatic mutations were detected in 89.1% supernatant cfTNA, but in only 54.3% of cell pellets. The overall concordance rate between supernatants and formalin-fixed, paraffin-embedded cell pellets at the mutation level was 53.3%. By contrast, 41.7% and 5.0% of somatic alterations were detected in supernatants and cell pellets, respectively. Furthermore, joint analysis of supernatants and cell pellets from PE showed a high concordance (88.3%) of variant detection with their respective tumor tissue specimens. Low-frequency T790M mutations in three cases (0.29%, 0.41%, and 1.56%) were detected in supernatants but not in the matched cell pellets or tumor tissues. In conclusion, pleural effusion–derived cfTNA can effectively be used in clinical practice for molecular analysis by next-generation sequencing, even in cases where corresponding cell pellets or tumor tissues yield insufficient material. Lung cancer is the most common cancer and the leading cause of cancer-related mortality in China and worldwide.1Chen W. Zheng R. Baade P.D. Zhang S. Zeng H. Bray F. Jemal A. Yu X.Q. He J. Cancer statistics in China, 2015.CA Cancer J Clin. 2016; 66: 115-132Crossref PubMed Scopus (14096) Google Scholar,2Torre L.A. Bray F. Siegel R.L. Ferlay J. Lortet-Tieulent J. Jemal A. Global cancer statistics, 2012.CA Cancer J Clin. 2015; 65: 87-108Crossref PubMed Scopus (23566) Google Scholar Development of molecular targeted therapy has brought numerous benefits to non–small-cell lung cancer (NSCLC) patients with targetable driver mutations. Testing these mutations has become a standard procedure before treatment. In current clinical practice, genotyping tumor tissue represents the gold standard for guiding the selection of personalized therapy and monitoring resistance mutations for advanced NSCLC patients.3Lindeman N.I. Cagle P.T. Aisner D.L. Arcila M.E. Beasley M.B. Bernicker E.H. Colasacco C. Dacic S. Hirsch F.R. Kerr K. Kwiatkowski D.J. Ladanyi M. Nowak J.A. Sholl L. Temple-Smolkin R. Solomon B. Souter L.H. Thunnissen E. Tsao M.S. Ventura C.B. Wynes M.W. Yatabe Y. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology.J Thorac Oncol. 2018; 13: 323-358Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar However, optimal mutation detection is hindered by complications associated with biopsy procedure, tumor heterogeneity, and limited sensitivity of test method. Biopsy procedure is invasive, and it is challenging to obtain sufficient tumor biopsy specimens needed for clinical molecular testing. Furthermore, it is common for mutations, such as epidermal growth factor receptor (EGFR) T790M, to be missed in tumor samples because of tumor heterogeneity. Last, detecting low-frequency mutations is also limited by sensitivity and specificity of test approaches. Recently, liquid biopsy using cell-free total nucleic acids [cfTNAs; including cell-free DNA (cfDNA) and cell-free RNA], which are released by apoptotic or necrotic tumor cells to blood or other bodily fluids, has provided a noninvasive approach. It overcomes the spatial bias of tissue samples by capturing the global heterogeneous tumor genome from a single fluid sample.4Corcoran R.B. Chabner B.A. Application of cell-free DNA analysis to cancer treatment.N Engl J Med. 2018; 379: 1754-1765Crossref PubMed Scopus (412) Google Scholar Pleural effusion (PE) is one of potential biosources of tumor-derived bodily fluids for analyzing the genetic alterations, especially as approximately 15% to 25% NSCLC patients exhibit malignant PE when they are first evaluated.5Morgensztern D. Waqar S. Subramanian J. Trinkaus K. Govindan R. Prognostic impact of malignant pleural effusion at presentation in patients with metastatic non-small-cell lung cancer.J Thorac Oncol. 2012; 7: 1485-1489Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar,6Porcel J.M. Gasol A. Bielsa S. Civit C. Light R.W. Salud A. Clinical features and survival of lung cancer patients with pleural effusions.Respirology. 2015; 20: 654-659Crossref PubMed Scopus (117) Google Scholar In those cases, cell pellet specimens, including cell block or cytology, are used for the routine pathologic diagnosis and molecular testing. However, the tumor cellularity is often inadequate in clinical settings, which has stimulated interest in analyses using surrogate supernatant cfDNA. In previous studies, liquid biopsy using the supernatant cfDNA of PE showed promising results for the detection of individual point mutations, especially EGFR mutations.7Kimura H. Fujiwara Y. Sone T. Kunitoh H. Tamura T. Kasahara K. Nishio K. EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib.Br J Cancer. 2006; 95: 1390-1395Crossref PubMed Scopus (111) Google Scholar, 8Soh J. Toyooka S. Aoe K. Asano H. Ichihara S. Katayama H. Hiraki A. Kiura K. Aoe M. Sano Y. Sugi K. Shimizu N. Date H. Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer.Int J Cancer. 2006; 119: 2353-2358Crossref PubMed Scopus (95) Google Scholar, 9Zhang X. Zhao Y. Wang M. Yap W.S. Chang A.Y. Detection and comparison of epidermal growth factor receptor mutations in cells and fluid of malignant pleural effusion in non-small cell lung cancer.Lung Cancer. 2008; 60: 175-182Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 10Jian G. Songwen Z. Ling Z. Qinfang D. Jie Z. Liang T. Caicun Z. Prediction of epidermal growth factor receptor mutations in the plasma/pleural effusion to efficacy of gefitinib treatment in advanced non-small cell lung cancer.J Cancer Res Clin Oncol. 2010; 136: 1341-1347Crossref PubMed Scopus (61) Google Scholar, 11Liu X. Lu Y. Zhu G. Lei Y. Zheng L. Qin H. Tang C. Ellison G. McCormack R. Ji Q. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.J Clin Pathol. 2013; 66: 1065-1069Crossref PubMed Scopus (125) Google Scholar, 12Yeo C.D. Kim J.W. Kim K.H. Ha J.H. Rhee C.K. Kim S.J. Kim Y.K. Park C.K. Lee S.H. Park M.S. Yim H.W. Detection and comparison of EGFR mutations in matched tumor tissues, cell blocks, pleural effusions, and sera from patients with NSCLC with malignant pleural effusion, by PNA clamping and direct sequencing.Lung Cancer. 2013; 81: 207-212Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 13Lin J. Gu Y. Du R. Deng M. Lu Y. Ding Y. Detection of EGFR mutation in supernatant, cell pellets of pleural effusion and tumor tissues from non-small cell lung cancer patients by high resolution melting analysis and sequencing.Int J Clin Exp Pathol. 2014; 7: 8813-8822PubMed Google Scholar, 14Liu D. Lu Y. Hu Z. Wu N. Nie X. Xia Y. Han Y. Li Q. Zhu G. Bai C. Malignant pleural effusion supernatants are substitutes for metastatic pleural tumor tissues in EGFR mutation test in patients with advanced lung adenocarcinoma.PLoS One. 2014; 9: e89946Crossref PubMed Scopus (39) Google Scholar, 15Husain H. Nykin D. Bui N. Quan D. Gomez G. Woodward B. Venkatapathy S. Duttagupta R. Fung E. Lippman S.M. Kurzrock R. Cell-free DNA from ascites and pleural effusions: molecular insights into genomic aberrations and disease biology.Mol Cancer Ther. 2017; 16: 948-955Crossref PubMed Scopus (63) Google Scholar, 16Shin S. Kim J. Kim Y. Cho S.M. Lee K.A. Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.Clin Chem Lab Med. 2017; 55: 1962-1969Crossref PubMed Scopus (33) Google Scholar, 17Kawahara A. Fukumitsu C. Azuma K. Taira T. Abe H. Takase Y. Murata K. Sadashima E. Hattori S. Naito Y. Akiba J. A combined test using both cell sediment and supernatant cell-free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients.Cytopathology. 2018; 29: 150-155Crossref PubMed Scopus (25) Google Scholar, 18Lee J.S. Hur J.Y. Kim I.A. Kim H.J. Choi C.M. Lee J.C. Kim W.S. Lee K.Y. 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.BMC Cancer. 2018; 18: 1236Crossref PubMed Scopus (55) Google Scholar, 19Hummelink K. Muller M. Linders T.C. van der Noort V. Nederlof P.M. Baas P. Burgers S. Smit E.F. Meijer G.A. van den Heuvel M.M. van den Broek D. Monkhorst K. Cell-free DNA in the supernatant of pleural effusion can be used to detect driver and resistance mutations, and can guide tyrosine kinase inhibitor treatment decisions.ERJ Open Res. 2019; 5 (00016-2019)Crossref PubMed Scopus (18) Google Scholar Commonly used technologies in these studies include direct sequencing7Kimura H. Fujiwara Y. Sone T. Kunitoh H. Tamura T. Kasahara K. Nishio K. EGFR mutation status in tumour-derived DNA from pleural effusion fluid is a practical basis for predicting the response to gefitinib.Br J Cancer. 2006; 95: 1390-1395Crossref PubMed Scopus (111) Google Scholar,8Soh J. Toyooka S. Aoe K. Asano H. Ichihara S. Katayama H. Hiraki A. Kiura K. Aoe M. Sano Y. Sugi K. Shimizu N. Date H. Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer.Int J Cancer. 2006; 119: 2353-2358Crossref PubMed Scopus (95) Google Scholar,11Liu X. Lu Y. Zhu G. Lei Y. Zheng L. Qin H. Tang C. Ellison G. McCormack R. Ji Q. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.J Clin Pathol. 2013; 66: 1065-1069Crossref PubMed Scopus (125) Google Scholar and PCR-based assay, such as high-resolution melting analysis,13Lin J. Gu Y. Du R. Deng M. Lu Y. Ding Y. Detection of EGFR mutation in supernatant, cell pellets of pleural effusion and tumor tissues from non-small cell lung cancer patients by high resolution melting analysis and sequencing.Int J Clin Exp Pathol. 2014; 7: 8813-8822PubMed Google Scholar amplification refractory mutation system,11Liu X. Lu Y. Zhu G. Lei Y. Zheng L. Qin H. Tang C. Ellison G. McCormack R. Ji Q. The diagnostic accuracy of pleural effusion and plasma samples versus tumour tissue for detection of EGFR mutation in patients with advanced non-small cell lung cancer: comparison of methodologies.J Clin Pathol. 2013; 66: 1065-1069Crossref PubMed Scopus (125) Google Scholar,14Liu D. Lu Y. Hu Z. Wu N. Nie X. Xia Y. Han Y. Li Q. Zhu G. Bai C. Malignant pleural effusion supernatants are substitutes for metastatic pleural tumor tissues in EGFR mutation test in patients with advanced lung adenocarcinoma.PLoS One. 2014; 9: e89946Crossref PubMed Scopus (39) Google Scholar peptide nucleic acid–clamping PCR,8Soh J. Toyooka S. Aoe K. Asano H. Ichihara S. Katayama H. Hiraki A. Kiura K. Aoe M. Sano Y. Sugi K. Shimizu N. Date H. Usefulness of EGFR mutation screening in pleural fluid to predict the clinical outcome of gefitinib treated patients with lung cancer.Int J Cancer. 2006; 119: 2353-2358Crossref PubMed Scopus (95) Google Scholar,12Yeo C.D. Kim J.W. Kim K.H. Ha J.H. Rhee C.K. Kim S.J. Kim Y.K. Park C.K. Lee S.H. Park M.S. Yim H.W. Detection and comparison of EGFR mutations in matched tumor tissues, cell blocks, pleural effusions, and sera from patients with NSCLC with malignant pleural effusion, by PNA clamping and direct sequencing.Lung Cancer. 2013; 81: 207-212Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar,18Lee J.S. Hur J.Y. Kim I.A. Kim H.J. Choi C.M. Lee J.C. Kim W.S. Lee K.Y. 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.BMC Cancer. 2018; 18: 1236Crossref PubMed Scopus (55) Google Scholar allele-specific PCR test,16Shin S. Kim J. Kim Y. Cho S.M. Lee K.A. Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.Clin Chem Lab Med. 2017; 55: 1962-1969Crossref PubMed Scopus (33) Google Scholar and droplet digital PCR.19Hummelink K. Muller M. Linders T.C. van der Noort V. Nederlof P.M. Baas P. Burgers S. Smit E.F. Meijer G.A. van den Heuvel M.M. van den Broek D. Monkhorst K. Cell-free DNA in the supernatant of pleural effusion can be used to detect driver and resistance mutations, and can guide tyrosine kinase inhibitor treatment decisions.ERJ Open Res. 2019; 5 (00016-2019)Crossref PubMed Scopus (18) Google Scholar These technologies differ significantly in terms of sensitivity, specificity, and coverage of detected mutations. Although these studies indicate that supernatant cfDNA of PE has great value for molecular testing, its use in multiplexed molecular profiling has not yet been fully investigated. Next-generation sequencing (NGS) techniques, which have been developed to comprehensively and precisely characterize the genomic landscape, have also been tailored for cfDNA.20Leary R.J. Kinde I. Diehl F. Schmidt K. Clouser C. Duncan C. Antipova A. Lee C. McKernan K. De La Vega F.M. Kinzler K.W. Vogelstein B. Diaz Jr., L.A. Velculescu V.E. Development of personalized tumor biomarkers using massively parallel sequencing.Sci Transl Med. 2010; 2: 20ra14Crossref PubMed Scopus (416) Google Scholar, 21Forshew T. Murtaza M. Parkinson C. Gale D. Tsui D.W. Kaper F. Dawson S.J. Piskorz A.M. Jimenez-Linan M. Bentley D. Hadfield J. May A.P. Caldas C. Brenton J.D. Rosenfeld N. Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA.Sci Transl Med. 2012; 4: 136ra168Crossref Scopus (920) Google Scholar, 22Murtaza M. Dawson S.J. Tsui D.W. Gale D. Forshew T. Piskorz A.M. Parkinson C. Chin S.F. Kingsbury Z. Wong A.S. Marass F. Humphray S. Hadfield J. Bentley D. Chin T.M. Brenton J.D. Caldas C. Rosenfeld N. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA.Nature. 2013; 497: 108-112Crossref PubMed Scopus (1308) Google Scholar, 23Adalsteinsson V.A. Ha G. Freeman S.S. Choudhury A.D. Stover D.G. Parsons H.A. et al.Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors.Nat Commun. 2017; 8: 1324Crossref PubMed Scopus (364) Google Scholar They also showed satisfactory sensitivity and specificity in EGFR mutation detection of plasma cfDNA compared with other platforms.24Xu T. Kang X. You X. Dai L. Tian D. Yan W. Yang Y. Xiong H. Liang Z. Zhao G.Q. Lin S. Chen K.N. Xu G. Cross-platform comparison of four leading technologies for detecting EGFR mutations in circulating tumor DNA from non-small cell lung carcinoma patient plasma.Theranostics. 2017; 7: 1437-1446Crossref PubMed Scopus (68) Google Scholar Recently, capture-based targeted sequencing on the Illumina (San Diego, CA) platform was applied to molecular profiling for supernatant cfDNA of PE from NSCLC patients. It showed that supernatant of PE reveals a more comprehensive mutation spectrum than the matched plasma.25Guo Z. Xie Z. Shi H. Du W. Peng L. Han W. Duan F. Zhang X. Chen M. Duan J. Lin J. Chen X. Lizaso A.A. Han-Zhang H. He J. Yin W. Malignant pleural effusion supernatant is an alternative liquid biopsy specimen for comprehensive mutational profiling.Thorac Cancer. 2019; 10: 823-831Crossref PubMed Scopus (29) Google Scholar The present study investigated the feasibility of molecular profiling using supernatants from PE in comparison with cell pellet–based genotyping in NSCLC patients by Oncomine Lung cfTNA Research Assay and Oncomine Focus Assay on Ion Torrent NGS platform (all from Thermo Fisher Scientific, Waltham, MA). The detection concordance for somatic alterations between combined supernatants and cell pellets of PE and tissue samples was also compared. This is the first time investigating and comparing molecular prolife between matched supernatants and cell pellets from PE in parallel using amplicon-based sequencing on the Ion Torrent platform. A total of 47 NSCLC patients presenting with PE from January 2018 to March 2019 at Shanghai Chest Hospital (Shanghai, China) were included in this research. In clinical practice, these patients had molecular testing performed on tumor tissues with clinically validated diagnostic assays, including amplification refractory mutation system (Amoy-Dx, Xiamen, China), capture-based NGS (Burning Rock Biotech Ltd, Guangzhou, China), or Ventana ALK (D5F3) CDx Assay (Roche Diagnostics, Indianapolis, IN), for the driver mutations. The patients' clinical characteristics are summarized in Table 1. All cases were diagnosed by at least two pathologists (H.T. and Y.H.). All patients had lung adenocarcinoma, with 12 central type and 35 peripheral type. The average age for these patients was 60.4 years, ranging from 35 to 83 years, with 27 males and 20 females. Among them, 14 were smokers and 33 were nonsmokers. Twenty-three patients had received targeted therapy before PE sampling. Information on PE appearance was extracted from procedure notes available in the electronic medical record by pulmonary physician who had performed the thoracentesis. Two groups were separated on the basis of fluid appearance: 26 cases with hemorrhagic PE and 21 cases with clear PE. The detailed clinicopathologic profiles are listed in Supplemental Table S1. The supernatant fluid and cell pellet of PE of each patient were collected. Informed consent was obtained from all subjects, and the present study was approved by the Ethics Committee of Shanghai Chest Hospital.Table 1Clinical Characteristics of Selected NSCLC PatientsCharacteristicPatients, n (total N = 47)Patients, %Age, years Mean (SD)60.4 (11.8) Median (range)62.0 (35–83)Sex Male2757.4 Female2042.6Smoking status Never3370.2 Ever1429.8Major tumor location RLL/RML/RUL2757.4 LLL/LUL2042.6Imageological type Central1225.5 Peripheral3574.5Histology Adenocarcinoma47100.0Targeted therapy received Yes2348.9 No2451.1PE appearance Hemorrhagic PE2655.3 Clear PE2144.7LLL, left lower lobe; LUL, left upper lobe; NSCLC, non–small-cell lung cancer; PE, pleural effusion; RLL, right lower lobe; RML, right middle lobe; RUL, right upper lobe. Open table in a new tab LLL, left lower lobe; LUL, left upper lobe; NSCLC, non–small-cell lung cancer; PE, pleural effusion; RLL, right lower lobe; RML, right middle lobe; RUL, right upper lobe. According to the workflow shown in Figure 1, PE was centrifuged at 2000 × g for 10 minutes at 4°C within 1 to 4 hours after collection. A total of 10 mL supernatant was transferred to a new tube, and centrifuged at 16,000 × g for 10 minutes at 4°C to remove additional cellular debris. A total of 3 to 6 mL cell-free supernatant was carefully separated, and the approximate hemoglobin concentration in mg/dL of supernatant was evaluated; then, it was immediately stored at −80°C until cfTNA extraction. During the first spin, for hemorrhagic PE, the cell pellets were generated with 100 mL substance, whereas for clear PE, the pellets were generated with at least 200 mL substance. Then, the cell pellets were used for pathologic diagnosis, tumor cellularity assessment, and DNA/RNA extraction by formalin-fixed, paraffin-embedded (FFPE) specimens, according to the standard histopathologic procedures. Cell-free TNA was extracted from supernatant of PE using the MagMax Cell-Free Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. DNA/RNA from the cell pellet of PE was extracted by RecoverAll Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific) following its manual. Nucleic acid yield and quality were assessed by A260/A280 ratio on Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific) and Qubit dsDNA/RNA HS Assay Kit on Qubit 3.0 fluorometer (Thermo Fisher Scientific). Nucleic acid fragments were assessed by Agilent High Sensitivity DNA/RNA Kit on Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). For each patient, 20 to 50 ng of cfTNA was used for cfTNA library preparation with Oncomine Lung Cell-Free Total Nucleic Acid Research Assay (Thermo Fisher Scientific), which enables the detection of >169 hot spot mutations across 12 genes frequently identified in NSCLC. In addition, 10 to 15 ng of DNA/RNA from FFPE specimen of each patient was used for DNA/RNA library preparation with Oncomine Focus Assay (Thermo Fisher Scientific), targeting 52 key solid tumor genes, 11 of which were covered by the cfTNA panel, including ALK, BRAF, EGFR, ERBB2, KRAS, MAP2K1, MET, NRAS, PIK3CA, RET, and ROS1. The concentration of each library was then determined by real-time quantitative PCR with the Ion Library Quantitation Kit (Thermo Fisher Scientific) in triplicate reactions on 7900HT Fast Real-Time PCR Instrument (Thermo Fisher Scientific). Individual bar-coded cfTNA library was diluted to 100 pmol/L and mixed equally while DNA and RNA libraries from FFPE specimens were diluted to 100 pmol/L, and combined at an 80:20 (DNA library/RNA library) ratio. The template was prepared on the Ion OneTouch 2 System, enriched on Ion OneTouch ES System, and then loaded onto the Ion 530 or 540 Chip and sequenced on Ion Torrent GeneStudio S5 Sequencer (Thermo Fisher Scientific). Data from the Ion GeneStudio S5 runs was primarily processed using the Torrent Suite software version 5.10 (Thermo Fisher Scientific). A sequencing coverage of 2500× or 25,000× was the minimum coverage required to detect the variations for the DNA/RNA library or cfTNA library, respectively. After alignment to the hg19 human genome reference, the VariantCaller plugin was applied using the Lung cfNA or Focus panel target regions as a reference. The Ion Reporter software version 5.10 (Thermo Fisher Scientific) was used to filter and annotate all DNA and RNA variations. All detected variants were manually reviewed with the Integrative Genomics Viewer version 2.4 (Broad Institute, Cambridge, MA) to filter out possible strand bias and sequencing errors. The limit of detection (LOD) values of variation allele frequency (VAF) for supernatant and FFPE cell pellet were 0.1% and 5%, respectively. To evaluate supernatant cfTNA-based liquid biopsy and FFPE cell pellet genotyping of PE by NGS panel testing, we included a total of 47 advanced-stage NSCLC patients with PE, who had tissue driver gene genotyping either at diagnosis or during disease progression (Table 1 and Supplemental Table S1). Pleural effusion from each patient was collected and separated to cell-free supernatant and cell pellet. Then, amplicon-based sequencing was performed on supernatant and FFPE cell pellet samples via the Ion Torrent platform using Oncomine Lung cfTNA and Oncomine Focus FFPE panels, respectively (Figure 1). In general, PE can be characterized into hemorrhagic (bloody) and clear (nonbloody) PE based on its appearance.26Ozcakar B. Martinez C.H. Morice R.C. Eapen G.A. Ost D. Sarkiss M.G. Chiu H.T. Jimenez C.A. Does pleural fluid appearance really matter? the relationship between fluid appearance and cytology, cell counts, and chemical laboratory measurements in pleural effusions of patients with cancer.J Cardiothorac Surg. 2010; 5: 63Crossref PubMed Scopus (13) Google Scholar There are two major impacts that the blood cells affected molecular profiling of PE. The detection of somatic variants in cell pellets could be underestimated because of the small amount of neoplastic cells embedding into a large number of normal blood cells. Variant detection in cfTNA from supernatant might also be impacted by genomic DNA contamination due to hemolysis. Thus, PE appearance from each patient was first evaluated after collection, with hemorrhagic in 26 cases and clear in 21 cases. After two runs of centrifugation, approximate hemoglobin concentration in supernatant was further evaluated, according to Figure 1. Three supernatants isolated from hemorrhagic PE were high in hemoglobin concentration (Case 3/4/25; >100 mg/dL), suggesting hemolysis. The tumor cellularity of each FFPE cell pellet was evaluated by two pathologists (H.T. and R.Z.). Forty-seven samples included 10 cases negative for tumor cells, 15 cases with 10% tumor cells. Detailed information about PE appearance, hemoglobin concentration of supernatants, PE input, tumor cellularity, nucleic acids, and library yields is listed in Supplemental Table S2. Specifically, 46 cfTNAs were isolated from pleural fluid supernatants. The median cfTNA yield was 65.8 ng (range, 5.9 to 1510.0 ng) in 1 mL supernatant of PE. The size pattern of the cfDNA from PE was similar to cfDNA from plasma, with double-stranded fragments of approximately 150 to 200 bp and a minor peak between 300 and 400 bp in length (Figure 1). Three of them showed contamination of genomic DNA because of hemolysis in hemorrhagic PE (Case 3/4/25). In addition, 40 genomic DNAs (range, 42.0 to 2706.0 ng) and 37 RNAs (range, 45.0 to 3600.0 ng) were successfully extracted from FFPE cell blocks. Furthermore, 46 cfTNA libraries were prepared, but only 38 DNA libraries and 28 RNA libraries have been constructed successfully from the respective FFPE cell blocks (Table 2). PE appearance or tumor cellularity in cell pellets had no significant correlation with cfTNA level (Figure 2, A and B ) and the success rates of cfTNA extraction and library preparation from supernatants (Figure 2, C and D). However, low abundance (≤10%) or absence of tumor cells might reduce the success rates of DNA/RNA extraction and library preparation from FFPE cell pellets of PE (Figure 2, C and D).Table 2Success Rates of Nucleic Acid Extraction and Library Preparation between Supernatants and FFPE Cell Pellets from PEsVariablePE-supernatant, n (%)PE–cell pellet, n (%)cfTNAcfTNA libraryDNADNA libraryRNARNA libraryPE appearance Hemorrhagic PE (N = 26)26 (100.0)26 (100.0)21 (80.8)20 (80.8)19 (73.1)13 (50.0) Clear PE (N = 21)20 (95.2)20 (95.2)19 (90.5)18 (85.7)18 (85.7)15 (71.4)Tumor cellularity, % 0 (N = 10)9 (90.0)9 (90.0)7 (70.0)6 (60.0)6 (60.0)5 (50.0) 1–10 (N = 15)15 (100.0)15 (100.0)11 (73.3)11 (73.3)9 (60.0)3 (20.0) >10 (N = 22)22 (100.0)22 (100.0)22 (100.0)21 (95.5)22 (100.0)20 (90.9)Total (N = 47)46 (97.9)46 (97.9)40 (85.1)38 (80.9)37 (78.7)28 (59.6)cfTNA, cell-free total nucleic acid; FFPE, formalin fixed, paraffin embedded; PE, pleural effusion. Open table in a new tab cfTNA, cell-free total nucleic acid; FFPE, formalin fixed, paraffin embedded; PE, pleural effusion. Collectively, the supernatants from pleural effusions showed higher success rates in terms of nucleic acid extraction (cfTNA, 97.9%, 46/47) and library construction (cfTNA libraries, 97.9%, 46/47) than the matched cell pellets (DNA, 85.1%, 40/47; RNA, 78.7%, 37/47; DNA libraries, 80.9%, 38/47; RNA libraries, 59.6%, 28/47) even when the tumor cellularity was extremely low in some pleural effusions (Table 2). Next, somatic alterations in the matched supernatants and FFPE cell blocks from PE were examined, and their results were compared with those of paired tissue samples, which were used as references. If additional somatic variants were detected in supernatants or cell pellets beyond the paired tissue samples, ortho
Referência(s)