Close monitoring of treatment response of patients with metastatic Non-Small Cell Lung cancer using liquid biopsy with focus on ctDNA methylation and ctRNA PD-L1 status

Purpose:
This study aims to improve treatment strategies of advanced lung cancer patients by monitoring ctDNA methylation pattern in plasma before and during treatment. Moreover, levels of ctRNA PD-L1 and ctDNA mutations will be measured. By comparing ctDNA methylation, ctDNA and PD-L1 ctRNA we expect to obtain a better understanding of the potential of these biomarkers as prognostic and predictive tools.

Background:
Lung cancer is the leading cause of cancer related death worldwide. The prognosis is often poor and in Denmark, the 5-year survival is 1% for metastatic disease. NSCLC is a heterogenic group of lung cancers and treatment choices of advanced lung cancer are limited. Lack of efficient monitoring of tumor response during treatment of the disease is a challenge. Today, CT scanning is the only assessment of treatment response. Precise and more accessible methods for measurement of treatment response are needed. Liquid biopsy measuring ctDNA has a wide potential in many aspects of cancer diagnostics like screening for the disease, assessing response to treatment, monitoring tumor progression, and detection of recurrent disease. Furthermore, liquid biopsy can be used when tissue biopsy or re-biopsy is not available for detection of targetable mutations or secondary resistance mutations.

Epigenetic changes such as DNA methylation is a common alteration in lung cancer, often associated with inactivation of tumorsuppressor genes by addition of a methyl group to cytosine of CpG dinucleotides. Multiple studies has shown ctDNA methylation of promoter regions of tumorsuppressor genes as a marker for early detection of lung cancer and as a prognostic biomarker for metastatic NSCLC. With this study, we will investigate the methylation patterns for a large panel of genes in plasma during treatment of NSCLC patients.

Additionally, cfRNA levels of PD-L1 in plasma will also be monitored as part of the study. Patients with advanced NSCLC being positive for PD-L1 are often more sensitive for immunotherapy, resulting in a longer progressions-free survival and overall survival compared to NSCLC patients treated with chemotherapy alone. A recent study showed that PD-L1 status changed in a group of NSCLC during treatment indicating the relevance of measuring PD-L1 ctRNA during treatment using liquid biopsy.

Experimental design:
This is a prospective explorative method study. Patient population (approx. 200 patients) includes patients diagnosed with advanced NSCLC being candidates for chemotherapy, immunotherapy or a combination. Plasma samples are collected at time of diagnosis, before each treatment cycle and at time of progression. Furthermore, at time of diagnosis and at time of disease progression a tissue biopsy is taken. Tissue samples will be screened for mutations, DNA methylation and PD-L1 status. For screening purpose of tissue, NGS and Agena’s MassARRAY System will be used. Monitoring ctDNA methylation and ctDNA levels of specific mutations will be performed using droplet digital PCR. Digital droplet PCR will be used as well for monitoring PD-L1 ctRNA status in plasma.

End points:
The goal of this study is to monitor therapy response using liquid biopsies and thereby early detection of ineffective treatment. By combining and comparing 1) levels of ctDNA, 2) ctDNA methylation patterns and 3) measurement of PD-L1 of ctRNA, this study will contribute to enable optimal clinical treatment monitoring and assignment of precision medicine in advanced lung cancer.

To our knowledge, this is the first study to monitor ctDNA methylation in advanced NSCLC for complete first line treatments. We intent that results from this study will form the basis for an intervention study.

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Christina Søs Auður Andersen

PhD Student