P2.11-34 Application of Next-Generation Sequencing for Screening of Sputum Samples

Abstract

Since the efficiency of CT mass-screening was reported in NEJM in 2013, many countries have been actively adopting CT screening for detection of lung cancers, especially peripheral-type lung adenocarcinoma. On the other hand, another mass-screening method, “sputum cytology”, has a very low cancer detection rate and its use has been decreasing worldwide. Nevertheless, sputum is one of the easiest types of sample to collect from patients, and sputum samples are thought to contain not only cancer cells, but also cancer cell-free DNA. The present study examined genomic abnormalities in DNA contained in sputum, and investigated the efficiency of next-generation sequencing (NGS) for detection of lung cancer or identifying patients at high risk. Using the Saccomanno method, we collected sputum samples from 15 patients (5 with squamous cell carcinoma, 5 with adenocarcinoma, and 5 with non-neoplastic conditions). A 1500 μl volume of each sample was centrifuged, and the sediment was resuspended in 180 μl of supernatant. After incubation with protease K at 56ºC for 90 min, DNA was extracted using MagLead 6GC (Precision System Science Co., Ltd., Japan). All DNA samples were subjected to NGS using Ion AmpliSeq Cancer Hotspot Panel v2 (Thermo Fisher Scientific, USA). The median age of the patients overall was 71 years (range, 56–94 years), and that of patients with squamous cell carcinomas, adenocarcinomas and non-neoplastic conditions was 66 years (64-78), 71 years (67-73) and 86 years (56-94), respectively. HRAS and p53 mutations were found only in cancer patients (HRAS 2/10, p53 4/10). There were no significant differences in mutation pattern between squamous cell carcinoma and adenocarcinoma patients. Although mutations of ALK, PIK3CA, PTEN, KRAS, FLT3, and RB1 were frequently found in samples from cancer patients (ALK 5/10, PIK3CA 5/10, PTEN 7/10, KRAS 7/10, FLT3 4/10, and RB1 8/10), such mutations were also present in non-neoplastic conditions. Mutations at the same locus in ALK, PIK3CA, PTEN and RB1 were also found in non-neoplastic conditions (ALK 1/5, PIK3CA 1/5, PTEN 2/5, and RB1 1/5). Seventeen out of 48 gene mutations were found only in non-neoplastic conditions. Detectable mutation patterns differed between cancer and non-neoplastic conditions, but were similar between squamous cell carcinoma and adenocarcinoma. Validation tests will be performed on DNA extracted from more than 400 sputum samples.