Detection Of Copy Number Variations In The Circulating Free DNA Of Early Stage Lung Cancer Patients

BackgroundLung cancer is the most commonly diagnosed cancer and the leading cause of cancer death all around the world. Approximately 85% of them are non-small cell lung cancer, including adenocarcinoma and squamous cell carcinoma. After low-dose CT being widely used in the screening of high risk patients, the death rate decreases significantly, though it brings other problems—overdiagnosis and overtreatment.Nowadays, tissue biopsy is still considered to be the gold standard in current tumor diagnosis, which provides materials for genotyping, treatment assessment and prognostic evaluation. However, sampling tumor tissue has significant inherent limitations. Tumor tissue is a single snapshot in time, and thus is subject to selection bias resulting from tumor heterogeneity. It is also difficult to obtain, since the invasive procedure may increase the risk of patients.The detection of circulating free DNA (cfDNA) is becoming the hotspot in cancer diagnosis recently. And the cancer-related cfDNA is called circulating tumor DNA (ctDNA).Most of the current cfDNA research are focusing on late stage tumors. As for early stage tumors, the concentration of cfDNA and the allele fraction of ctDNA are extremely low, due to the low tumor burdens. Therefore, the detection and assessment is quite difficult in early stage tumors. With the development of next generation sequencing (NGS) technology, the sensitivity and specificity of ctDNA detection both improved a lot, providing more opportunities of ctDNA detection for early diagnosis.ObjectiveThe detection of cfDNA copy number variations (CNVs), which is based on the next generation sequencing (NGS) technology, mostly focus on late stage tumors. The objective of our study is to detect CNVs in cfDNA of early stage lung cancer patients, to compare the differences between lung cancer patients and patients with benign pulmonary disease, both in tissue and plasma, and thus trying to raise a new method of early cancer detection.Materials and Methods1. Patients recruitmentEight patients were included in our study, with four lung cancer patients and four patients with benign pulmonary disease. These patients all underwent pulmonary lesion resection in Department of Thoracic Surgery in Peking Union Medical College Hospital (PUMCH). The FFPE and blood samples were collected following the rules.2. Whole genome sequencingAccording to the workflow of whole genome sequencing, we finished purification of both the DNA of tumor samples and plasma, the preparation of DNA library, the quality control, pooling and sequencing samples, and bioinformatics analysis.3. Data analysis After quality control and mapping, we use Control-FREEC software to do the CNVs calculations.ResultsThe CNVs in patients of benign pulmonary disease were all undetectable, both in tumor tissues and plasma. Neither can we detect the CNVs in three lung cancer patients of stage IA. Significant CNVs is detected in the lung cancer patient of stage IB. But CNVs in cfDNA and the corresponding tumor tissue DNA did not appear to correlate very well in some genomic regions. One positive CNVs region was located on the short arm of chromosome 10.ConclusionOur study demonstrate that CNVs in cfDNA can be detected using next generation sequencing in early lung cancer patients. The high sensitivity and specificity of the detection platform is very important. More patients and controls should be included in future researches to further exploration and analysis.