Clinical Applications of Next-Generation Sequencing in Cancer Diagnosis

Our data showed that increased risk of malignancy, especially lymphomas, was observed in Korean IgG4-RD patients.

Molecular diagnostics is a mandatory component of modern clinical oncology. The most known examples of molecular diagnostic procedures include the detection of hereditary cancer syndromes and the analysis of somatic drug-sensitizing mutations in protein kinases. Advances in cancer research as well as the development of new technologies led to emergence of new trends in this area of medicine. The invention of next generation sequencing (NGS) has a potential to dramatically change the landscape of molecular diagnostics. NGS allows to significantly improve the efficiency and availability of genetic testing for hereditary cancers as well as to undertake comprehensive tumor mutation profiling to guide the therapy choice. Tumors usually change their properties during therapeutic intervention. Monitoring of these properties is important for proper selection of further treatment options. So-called liquid biopsy is essential for this purpose, as it allows to detect key molecular features of the tumors by a non-invasive approach. There is an increasing popularity of ex vivo tumor models, which allow to cultivate tumor cells and to select the therapy based on the results of drug sensitivity tests.

Background: Next Generation Sequencer (NGS) is a powerful tool for a high-throughput sequencing of human genome. It is important to ensure reliability and sensitivity of the sequence data for a clinical use of the NGS. Various cancer-related gene panels such as Oncomine™ or NCC OncoPanel have been developed and used for clinical studies. Because these panels contain multiple genes, it is difficult to ensure the performance of mutation detection for every gene. In addition, various platforms of NGS are developed and their cross-platform validation has become necessity. In order to create mutant standards in a defined background, we have used CRISPR/Cas9 genome-editing system in HEK 293 T/17 cells. Results: Cancer-related genes that are frequently used in NGS-based cancer panels were selected as the target genes. Target mutations were selected based on their frequency reported in database, and clinical significance and on the applicability of CRISPR/Cas9 by considering distance from PAM site, and off-targets. We have successfully generated 88 hetero-and homozygous mutant cell lines at the targeted sites of 36 genes representing a total of 125 mutations. Conclusions: These knock-in HEK293T/17 cells can be used as the reference mutant standards with a steady and continuous supply for NGS-based cancer panel tests from the JCRB cell bank. In addition, these cell lines can provide a tool for the functional analysis of targeted mutations in cancer-related genes in the isogenic background.