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Liquid biopsy using blood is the next-generation technology in the field of cancer diagnosis. Since the proportion of circulating tumor DNA (ctDNA) in the total cell-free DNA (cfDNA) can be extremely low, it is important to use as much cfDNA input as possible to detect variant alleles by next-generation sequencing (NGS). NGS will lose their detection sensitivity when the input amounts are short due to low concentrations of cfDNA in blood. In this study, we asked whether concentrations of cfDNA in plasma were sufficient in patients across various cancer types including rare cancers such as soft tissue sarcoma for NGS-based liquid biopsies. Using a magnetic bead-based method, we extracted cfDNA in 22 plasma samples from 14 patients with advanced lung, gastrointestinal, head and neck, thymic, unknown primary cancer, and soft tissue sarcoma. Concentrations of cfDNA in plasma were then analyzed. Four samples from four patients with metastatic lung cancer showed cfDNA concentrations at a median of 10.3 ng/mL (1.7-15.6 ng/mL). Their concentrations were comparable to those in literatures. Five samples from three patients with advanced soft tissue sarcoma showed a median of 6.2 ng/mL (3.9-62.8 ng/mL). Concentrations in plasma of the other patients were at a median of 4.8 ng/mL (0-29.9 ng/mL, n = 13). Subsequently, we performed the IonPGM NGS to analyze single-nucleotide variants in ctDNA. This system required the input of 20ng and 5 ng of cfDNA to lower the detection limit at 0.1% and 0.25%, respectively. With the input of 20ng of cfDNA, we were able to detect a single-nucleotide variant in TP53 at an allelic frequency of 0.36% in a patient with metastatic soft tissue sarcoma. Our data showed that 11 out of 14 (79%) cancer patients had sufficient concentrations of cfDNA for the NGS-based liquid biopsy with the detection limit at 0.25%. Thus, liquid biopsy using blood may be a versatile tool for patients across various types of cancer. |
