PURPOSE: Circulating free DNA (cfDNA) in plasma is promising to be a surrogate for tumor tissue DNA. However, not all epidermal growth factor receptor (EGFR) mutations in tumor tissue DNA has been detected in matched cfDNA, at least partly due to inefficient cfDNA extraction method. The purpose of this study was to establish an efficient plasma cfDNA extraction protocol. MATERIALS AND METHODS: The yield of plasma cfDNA extracted by our modified phenol-chloroform (MPC) method from non-small-cell lung cancer (NSCLC) patients was compared with that by QIAamp MinElute Virus Spin kit (Qiagen kit) as control, using the Wilcoxon rank-sum test. TaqMan quantitative polymerase chain reaction (qPCR) assays were used to quantify the plasma cfDNA extracted. Both Mutant-enriched PCR (ME-PCR) coupled sequencing and DxS EGFR mutation test kit were used to evaluate the impact of extraction method on EGFR mutation analysis. RESULTS: MPC method extracted more plasma cfDNA than Qiagen kit method (p=0.011). The proportion of longer fragment (> or =202 bp) in cfDNA extracted by MPC method was significantly higher than by Qiagen kit method (p=0.002). In the sequencing maps of ME-PCR products, a higher mutant peak was observed on plasma cfDNA extracted by MPC method than by Qiagen kit method. In DxS EGFR mutation test kit results, plasma cfDNA extracted by MPC method contained more tumor-origin DNA than by Qiagen kit method. CONCLUSION: An improved plasma cfDNA extraction method of MPC is provided, which will be beneficial for EGFR mutation analysis for patients with NSCLC.
Base Sequence ; Carcinoma, Non-Small-Cell Lung/*genetics ; Chloroform ; DNA Mutational Analysis/*methods ; DNA, Neoplasm/*blood/*isolation & purification ; Genetic Testing/methods ; Humans ; Lung Neoplasms/*genetics ; Molecular Sequence Data ; Phenol ; Polymerase Chain Reaction/methods ; Receptor, Epidermal Growth Factor/*genetics

OBJECTIVE: To investigate the efficacy of submucosal resection by CO2 laser in the treatment of recurrent laryngeal papilloma and the effect on prognosis. METHOD: A total of 11 patients diagnosed as recurrent laryngeal papilloma were included in this review. Papilloma was marked before operation and checked under fibro-laryngoscope. Papilloma was resected completely including the submucosal tissure with CO2 laser or microequipment. In widespread papilloma, false membrane in raw surface were cleared 7-10 days after operation. Surgical specimens (including membrane) were detected by routine pathology, HPV typing and immunohistochemical pathologic examination. The patients were checked once a month in the first 3 months after operation, and then once for every 3 months. Once the hoarseness and other symptoms aggravated or the disease was recurrent, the patients were treated immediately. RESULT: HPV viral DNA was found in 10/11 cases, with HPV11 (7/11 cases) and HPV6 (3/11 cases). Cases with regards to follow-up, from 6 months to 1 year, 3 cases were followed up 1 year after operation, without recurrence. Five patients including 2 children were followed up 6 to 12 months after operation, without recurrence. Two children underwent 2 or 3 operations, were followed-up more than 6 months withouting recurrence. CONCLUSION Papilloma submucosal resection could decrease postoperative recurrence and is worth to be further investigated.
Child ; DNA, Viral ; blood ; Human papillomavirus 11 ; isolation & purification ; Human papillomavirus 6 ; isolation & purification ; Humans ; Laryngeal Neoplasms ; diagnosis ; surgery ; Laryngoscopes ; Lasers, Gas ; Neoplasm Recurrence, Local ; Papilloma ; diagnosis ; surgery ; virology ; Papillomavirus Infections ; diagnosis ; surgery ; Postoperative Period ; Prognosis ; Respiratory Tract Infections ; diagnosis ; surgery