Yanfei Wang1, Zhen Liang2, Yong Liu3, Furong Kou1, Danfeng Jiang1, Yanqun Zheng1, Wei Liu1, Budong Zhu1. 1. Daycare Center, Peking University Cancer Hospital and Institute, Beijing 100142, China. 2. Department of Thoracic Surgery I, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Peking University Cancer Hospital and Institute, Beijing 100142, China. 3. ComWin Biotech Co., Ltd, Beijing 102206, China.
Abstract
BACKGROUND: Malignant plural effusion (MPE) is one of the most common specimen for liquid biopsy gene detection. This study aims to explore a method for isolating tumor cells from large volume of MPE and evaluate its efficacy and application prospect in gene detection. METHODS: Pleural effusions (>500 mL) from 20 advanced lung cancer patients were obtained by effusion drainage and used to isolate tumor cells with cell separation media Percoll and Ficoll. Cell number and purity were calculated. DNA was extracted from the supernatant (etDNA), total cells and isolated tumor cells of pleural effusion (ETC-DNA) to detect the mutation of tumor-related genes by next-generation sequencing. RESULTS: The median number of cells isolated from malignant pleural effusion was 8.50×10⁴ (interquel range: 9.25×10³-3.75×10⁵), 85.50%±5.80% of the cells were identified as tumor cells. The detection rates of epidermal growth factor receptor (EGFR) gene mutation of etDNA, total cell DNA and ETC-DNA were 70.00%, 50.00% and 70.00%, reseparately, while the median EGFR mutation abundance in 3 components was 16.05% (4.78%-43.06%), 1.09% (0.00%-2.39%), and 33.02% (18.50%-76.70%), respectively. ETC-DNA had good consistency with tissue DNA (P>0.999, kappa=1.000) and etDNA (P>0.999, kappa=1.000). ETC-DNA inclined to have higher EGFR mutation than etDNA, but the result was not statistically significant. CONCLUSIONS: Our method can isolate large amount of tumor cells from a large volume of malignant pleural effusion with high purity. Using ETC-DNA as specimen improves the efficacy of gene detection, thus is worth further study.
BACKGROUND:Malignant plural effusion (MPE) is one of the most common specimen for liquid biopsy gene detection. This study aims to explore a method for isolating tumor cells from large volume of MPE and evaluate its efficacy and application prospect in gene detection. METHODS:Pleural effusions (>500 mL) from 20 advanced lung cancerpatients were obtained by effusion drainage and used to isolate tumor cells with cell separation media Percoll and Ficoll. Cell number and purity were calculated. DNA was extracted from the supernatant (etDNA), total cells and isolated tumor cells of pleural effusion (ETC-DNA) to detect the mutation of tumor-related genes by next-generation sequencing. RESULTS: The median number of cells isolated from malignant pleural effusion was 8.50×10⁴ (interquel range: 9.25×10³-3.75×10⁵), 85.50%±5.80% of the cells were identified as tumor cells. The detection rates of epidermal growth factor receptor (EGFR) gene mutation of etDNA, total cell DNA and ETC-DNA were 70.00%, 50.00% and 70.00%, reseparately, while the median EGFR mutation abundance in 3 components was 16.05% (4.78%-43.06%), 1.09% (0.00%-2.39%), and 33.02% (18.50%-76.70%), respectively. ETC-DNA had good consistency with tissue DNA (P>0.999, kappa=1.000) and etDNA (P>0.999, kappa=1.000). ETC-DNA inclined to have higher EGFR mutation than etDNA, but the result was not statistically significant. CONCLUSIONS: Our method can isolate large amount of tumor cells from a large volume of malignant pleural effusion with high purity. Using ETC-DNA as specimen improves the efficacy of gene detection, thus is worth further study.
Authors: Virginia Espina; Julia D Wulfkuhle; Valerie S Calvert; Amy VanMeter; Weidong Zhou; George Coukos; David H Geho; Emanuel F Petricoin; Lance A Liotta Journal: Nat Protoc Date: 2006 Impact factor: 13.491
Authors: James Che; Albert J Mach; Derek E Go; Ish Talati; Yong Ying; Jianyu Rao; Rajan P Kulkarni; Dino Di Carlo Journal: PLoS One Date: 2013-10-28 Impact factor: 3.240