Optimization of the liquid biopsy workflow

Subtitle:from research to clinical practice

Liquid biopsy, consisting of the analysis of several biomarkers in the blood, is a minimally invasive technique to assess the molecular landscape of the tumor. The development of highly sensitive analysis platforms has enabled the analysis of several of these biomarkers, such as circulating cell-free (tumor) DNA (cfDNA / ctDNA) and RNA (cfRNA / ctRNA). However, the lack of standard operating procedures has limited its implementation in a clinical setting. In this PhD dissertation we have focused on identifying key steps to compose an optimized liquid biopsy workflow. As even highly sensitive techniques are dependent on the number of genome equivalents, we started by evaluating several cfDNA isolation kits. We selected the most optimal isolation kit for use throughout this dissertation. Next, we assessed the generation of cfDNA and cfRNA by several centrifugation protocols in both standard EDTA tubes and stabilizing blood collection (Streck) tubes. We demonstrated that the selected centrifugation protocol is dependent on the downstream analysis. Another important aspect of the liquid biopsy workflow is the time-to-processing of blood samples and the choice of blood collection tube (BCT). We performed a systematic review to evaluate the effect of delayed processing of blood samples collected in different (specialized) BCTs on cfDNA. Furthermore, we confirmed these findings in a small study. Next, we performed a multicenter study to investigate the influence of several (pre-) analytical variables on cfDNA-based liquid biopsy. In total, we analyzed 549 plasma samples of 234 non-small cell lung cancer (NSCLC) patients. Based on these findings, we have composed an optimal liquid biopsy workflow to ensure successful cfDNA analysis. Lastly, we further highlighted the potential of liquid biopsy in a case report. We clearly demonstrated the advantage of combining clinical data with molecular information from tissue and liquid biopsy to provide patients with personalized treatment. To conclude, in this PhD dissertation we constructed an optimized workflow for plasma-based liquid biopsy. The combination of several biomarkers is becoming more important. Hence, our work on both cfDNA and cfRNA will aid in the standardization and further implementation of liquid biopsy in routine clinical setting. Furthermore, future research will reveal whether this workflow is stringent enough to also enable urine- or sputum-based liquid biopsy. Thereby providing patients with a completely non-invasive tool for real-time follow-up.

Publication year:2019

Keywords:Doctoral thesis

Accessibility:Open