Background Early diagnosis and continuous monitoring are necessary for an efficient management of cervical cancers (CC). Liquid biopsy, such as detecting circulating tumor DNA (ctDNA) from blood, is a simple, non-invasive method for testing and monitoring cancer markers. However, tumor-specific alterations in ctDNA have not been extensively investigated or compared to other circulating biomarkers in the diagnosis and monitoring of the CC. Therfore, Next-generation sequencing (NGS) analysis with blood samples can be a new approach for highly accurate diagnosis and monitoring of the CC. Method Using a bioinformatics approach, we designed a panel of 24 genes associated with CC to detect and characterize patterns of somatic single-nucleotide variations, indels, and copy number variations. Our NGS CC panel covers most of the genes in The Cancer Genome Atlas (TCGA) as well as additional cancer driver and tumor suppressor genes. We profiled the variants in ctDNA from 24 CC patients who were being treated with systemic chemotherapy and local radiotherapy at the Jeonbuk National University Hospital, Korea. Result Eighteen out of 24 genes in our NGS CC panel had mutations across the 24 CC patients, including somatic alterations of mutated genes ( ZFHX3– 83%, KMT2C- 79% , KMT2D- 79%, NSD1–67%, ATM- 38% and RNF213 –27%). We demonstrated that the RNF213 mutation could be used potentially used as a monitoring marker for response to chemo- and radiotherapy. Conclusion We developed our NGS CC panel and demostrated that our NGS panel can be useful for the diagnosis and monitoring of the CC, since the panel detected the common somatic variations in CC patients and we observed how these genetic variations change according to the treatment pattern of the patient.
Background/Aim: Non-invasive biomarker detection using DNA from cell-free circulating DNA (cfDNA) and circulating tumor cells (ctcDNA) are emerging as they can be used for early diagnosis, prognosis and therapeutic target selection for cancer. However, cfDNA and ctcDNA from the same patient have not yet been compared extensively on how different the genetic characteristics of the two are in terms of the overlap between them. Materials and Methods: The performance of a customized NGS panel was used to compare the variants found in the 20 pairs of cfDNA and ctcDNA from gynecological cancer patients. Results: A genetic variant analysis revealed that there were only nine common overlapping variants out of 63 between the cfDNA and ctcDNA pairs, while 31 and 22 were unique to cfDNA and ctcDNA, respectively. Conclusion: A combinatory analysis of both cfDNA and CTCs from cancer patients can improve the sensitivity of liquid biopsies. These results are expected to provide better genetic target information for guiding clinical strategies for cancer. Tumor cells can acquire mutations even during cancer therapies and can be selected for survival. Therefore, repeated monitoring of genetic mutations is important for tracking the condition of cancer patients. Genetic mutation profiles can be obtained by panel-based next generation sequencing (NGS) on tissue biopsy samples. Such profiles can provide ample information for choosing suitable drugs or chemotherapy. However, repeated solid tissue biopsies are invasive and inconvenient (1, 2). On the other hand, liquid biopsies including cell-free DNA (cfDNA), circulating tumor cells (CTCs) and tumor-derived exosomes from apoptotic or necrotic cells can be non-invasive and can also be sufficient enough to provide core information on the primary tumors or metastatic sites (3-5). For instance, one of the FDA approved commercial kits, Roche's Cobas plasma EGFR detection test V2 (US-IVD), utilizes cfDNA to detect a single mutation. In the case of next generation sequencing (NGS) application, Guardant360 now allows monitoring and aftercare of patients by screening cfDNA derived mutations for targeted therapies. As observed in many commercial products, liquid biopsy is an emerging field for real-time monitoring of tumor heterogeneity and predicting clinical behaviors of cancer in replace of tissue biopsy (6).Currently, early detection of cancer using circulating tumor cells in patient's blood is limited by the small number of CTCs and the amount of blood drawn, and the sensitivity in isolating CTCs from normal tissue cells. By simply increasing the volume of blood, a higher amount of circulating tumor DNA (ctDNA) can be obtained. Guardant, for example, requires a total 40 ml of blood to be drawn. Although this is an easy way to overcome the low amount issue, collecting a large blood volume is difficult in most clinical settings. Increasing the assay sensitivity is another approach to detect low amounts of ctDNA. Several methods, such as droplet digital PCR (ddPCR) and Beads, Emulsions, 6595
The determination of microRNA (miRNA) levels in biomaterials has become important for understanding their biological functions and for the diagnosis of various diseases. An effective extraction method is needed for maximizing the recovery of miRNAs from cells, while minimizing RNA degradation during the extraction because miRNAs present only approximately 0.01 % of total RNA. In this study, we used Triton X-100 (TX-100) to improve the extraction efficiency of miRNAs with TRIzol® reagent, which is a commonly used commercial microRNA isolation kit. The concentration of TX-100 and the incubation time after the addition of TX-100 were optimized to maximize the extraction efficiency. The extraction recovery by a combination of TX-100 and TRIzol® reagent was approximately 1.9-fold greater than that by the TRIzol® reagent alone. We have established a very effective extraction method for the extraction of low-abundance miRNAs in biological samples for the determination of miRNA levels in biomaterials.
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