National clinical study

DETECT: Early detection of lung cancer by blood samples - A prospective national observational study

The study investigates the use of aberrantly methylated circulating tumor DNA (ctDNA) and protein biomarkers in blood and bronchial wash fluid (BWF) as potential diagnostic tools in lung cancer.

The project received funding in 2020

Abstract

Impact/relevance 
The study investigates the use of aberrantly methylated circulating tumor DNA (ctDNA) and protein biomarkers in blood and bronchial wash fluid (BWF) as potential diagnostic tools in lung cancer. If the new approach has a high negative likelihood ratio, it could be used to eliminate suspicion of lung cancer without any invasive procedures. It is quick and simple to perform with minimal discomfort to the patient and could mean less redundant diagnostic and potentially harmful procedures for patients. If the approach has a high positive likelihood ratio, it could be used to aid clinical decision making especially regarding small (< 10 mm) indeterminate nodules and stage I lung cancer.

Background 
Lung cancer is the leading cause of cancer deaths all over the Western world, including Denmark. The disease is often diagnosed at an advanced stage and at present, improved survival relies on earlier diagnosis. The diagnostic workup for lung cancer is based on imaging and pathology. The main challenge is to discriminate small, early cancers from benign nodules. Fine needle aspiration has a complication rate of 10-15%. The current methods are by no means sufficient, and there is an obvious need for new approaches.

Recently, a major international study focused on 4 proteins (CA125, carcinoembryonic antigen (CEA), cytokeratin fragment (CYFRA21-1), and precursor form of surfactant protein B (pro-SFTPB) in serum (Guida F et al., JAMA Oncol 2018). The results indicated that a risk score based on these markers holds significant diagnostic information with an AUC of 0.83.

ctDNA holds several advantages over tissue tests, being easily accessible with little discomfort to the patients. Aberrant methylation occurs in almost all malignant tumors and a number of studies have suggested a potential for diagnosis and screening (Weiss G et al., J Thorac Oncol 2017). Our group has developed an analysis of methylated Homeobox A9 (HOXA9), an important factor in cell proliferation. Malignant lung tumors only shed small amounts of DNA into the circulation complicating the use of ctDNA in the diagnostic setting. We have therefore used BWF, which may represent a better starting material. Our group conducted a “proof of principle” study with 250 patients referred on suspicion of lung cancer. Preliminary results suggest a sensitivity, specificity, and positive predictive value of 73%, 82%, and 86%, respectively (Wen S et al., ASCO 2020). Thus, the analysis may have potential as an adjunct diagnostic tool.

Aim 
To investigate how protein biomarkers (CA125, CEA, CYFRA 21-1) and ctDNA (methylated HOXA9) can contribute to the diagnostic process in lung cancer.

Methods 
Methylated HOXA9: DNA is extracted from 4 ml plasma or 10 ml BWF with the QiaSymphony purification system (Qiagen). DNA is bisulfite converted as recommended by the manufacturer (Zymo Research). The converted DNA is analyzed with a methylation specific assay and control assay using the BioRad® Droplet Digital PCR (ddPCR) system QX100.

Protein biomarkers: The protein biomarkers CA125, CEA, and CYFRA 21-1 are analyzed by a multiplex Luminex assay. Each protein is detected using both biotinylated detection antibodies and streptavidin conjugated to fluorochromes. Results are quantified on the Bio-Plex 200® analyzer.

Expected outcome 
The results from this study will contribute to our knowledge on how ctDNA can aid the diagnostic process in lung cancer. By enrolling a large number of patients, we will be able to make subgroup analyses according to stage and optimize the analysis parameters to identify early stage lung cancer. We hope that the results can guide a future clinical trial in order to gather further experience and eventually implement the method in routine clinical practice. The ultimate goal is to be able to diagnose lung cancer at an early stage with minimally invasive procedures.

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