A preliminary study to compare cfDNA levels in lung cancer cases and high risk controls to evaluate the role of cfDNA levels in early lung cancer detection


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Fiona Taylor1,Shobha Silva2,Barbara Ottolini3,James Bradford2,Penella Woll2,Dawn Teare2,Jacqui Shaw3,Cox Angela2
1Weston Park Hospital,2University of Sheffield,3University of Leicester

Abstract

Background

Tumour DNA is shed into the blood stream from tumour cells resulting in raised levels of circulating cell-free DNA (cfDNA). Studies evaluating cfDNA levels report conflicting results due to methodological and patient differences.  To evaluate cfDNA levels as a biomarker of early lung cancer detection we compared levels between lung cancer cases and high risk controls aged 50-75 years defined by a Liverpool Lung Cancer risk score ≥ 2.5 and low risk controls defined by score  <2.5. 

Method

Blood from participants in the Sheffield ReSoLuCENT study was collected and processed according to standard protocol to obtain plasma for cfDNA analysis. Plasma was stored at -80 °C prior to cfDNA extraction with the Circulating Nucleic Acid kit (Qiagen).  Extracted cfDNA was quantified by determining the level of GAPDH by SYBR green quantitative PCR.  Targeted sequencing was performed on a subset of cases with the Ion AmpliSeq Colon/Lung panel v2.  

Results

The median cfDNA levels of lung cancer cases (N=52) were significantly higher than high risk (N=30) and low risk controls (N=10) (7.25 ng/ml vs 4.63 ng/ml p=0.015 and 4.34 ng/ml p=0.006 Mann Whitney). However, there was no difference in the median cfDNA levels between early stage cancer (stage I-IIIA, N=21) and high risk controls (N=30) (4.74 ng/ml vs 4.63 ng/ml, p=0.73 Mann Whitney).  ROC curve analysis gave an area under the curve of 0.53.  Performance status, disease stage, gender and cfDNA levels were significantly associated with time until death in multivariate analysis.  There was no difference in cfDNA levels between samples with somatic mutations detected with targeted sequencing (N=6) and samples without mutations detected (N=14) (12.3 ng/ml and 11.7 ng/ml p=0.60 Mann Whitney).   

Conclusion

CfDNA levels do not distinguish between early lung cancer cases and high risk controls. Further investigation of cfDNA levels to aid early lung cancer detection is therefore not recommended.