Methylation and mutational analysis of circulating tumour DNA in non-small cell lung cancer using short and long read sequencing technologies

Abstract

To this day, non-small cell lung cancer remains one of the malignancies with the highest mortality rates in the Nordic countries. Since the introduction of targeted molecular therapy and immunotherapy, treatment outcomes have greatly improved, however, issues still remain. Patients are often diagnosed at late stages of the disease and not all patients respond to treatment. Mutational and methylation signatures in specific genes have been introduced as potential biomarkers to predict and monitor treatment response. Further, it has been shown that these biomarkers can be identified in cell-free DNA extracted from blood samples. In this thesis, a long read sequencing technology, capable of detecting both mutational and methylation information, was set up and used in parallel with two short read sequencing technologies, one utilizing enzymatic methyl sequencing to find methylation signatures and one using standard Illumina sequencing to detect somatic variants. This was done in an effort to evaluate the methods and find potential biomarkers in cell-free DNA from non-small cell lung cancer patients, that could be used to predict and monitor treatment response, as well as the spread of metastases. The results show that, while more optimization is needed to enable proper analysis of differential methylation, methylation signatures could be visualised using long read sequencing. The methylation patterns found were also consistent with the patterns found using the enzymatic methyl sequencing, and some regions were identified were certain samples showed differential methylation patterns. Somatic variants were also identified but all results require further analysis and evaluation before conclusions can be drawn with regard to potential biomarkers. The conclusion of the study was that, while the long read sequencing method require additional time before it is perfected, both long and short read sequencing can be used to detect differential methylation. The results also suggest the possibility of detecting potential biomarkers.