Combined assay of Circulating Tumor DNA and Protein Biomarkers for early noninvasive detection and prognosis of Non-Small Cell Lung Cancer

Abstract

Purpose: Early diagnosis of lung cancer is critical to curtailing cancer-related deaths. We aimed to develop a highly sensitive assay for the analysis of circulating tumor DNA (ctDNA) to detect non-small cell lung cancer (NSCLC) in the early stages. Materials and Methods: We detected EGFR and KRAS mutations in paired plasma and tumor tissue samples from 147 NSCLC patients. Of these, EGFR/KRAS ctDNA mutations and protein biomarkers were comparatively analyzed in 87 individuals. In addition, tissue samples of 20 patients were subjected to repeat multi-gene detection, and pre-and post-operative paired samples of 28 patients were subjected to multi-gene detection. Clinical information was obtained to complement the prognostic value of the combined assay results and post-operative new ctDNA mutation status. Results: EGFR/KRAS mutations were highly consistent in ctDNA and tumor DNA. Combining the detection of EGFR and KRAS mutations in ctDNA with the detection of protein biomarkers increased cancer detection sensitivity to 74.7% (65/87). None of the healthy controls tested positive using the combined assay (100% specificity). Combined assay results independently associated with recurrence-free survival. Post-operative new ctDNA mutation status independently associated with overall survival and recurrence-free survival. Conclusion: The detection of ctDNA may be exploited for early diagnosis of NSCLC, as highlighted by the developed assay. Further, the combined assay results and post-operative new ctDNA mutation status are promising prognostic indicators in NSCLC patients.

Impact of Preanalytical and Analytical Methods on Cell-Free DNA Diagnostics

“…Biomarkers associated with different diseases have been successfully detected in cfDNA, such as copy number variations, single nucleotide polymorphisms, cfDNA integrity, and epigenetic modifications including cfDNA methylation ( Vaissière et al, 2009 ; Vaca-Paniagua et al, 2015 ; Trigg et al, 2018 ; Lee et al, 2019 ; Constâncio et al, 2020 ; Li S. et al, 2020 ; Rodriguez-Casanova et al, 2021 ; Yin et al, 2021 ). In particular cfDNA quantity, integrity, and methylation have shown themselves to be the most prominent diagnostic parameters ( Trigg et al, 2018 ; Mbaye et al, 2019 ; Lampignano et al, 2020 ; Lo et al, 2021 ; Vandekerkhove et al, 2021 ; Zhang et al, 2021 ).…”

Prognostic Value of Postoperative Circulating Tumor DNA in Patients With Early- and Intermediate-Stage Hepatocellular Carcinoma

“…Indeed, low ctDNA concentration in early-stage cancers is a nonnegligible limitation of ctDNA analysis in early detection of relapse (35). Combination with other protein markers has been proved to improve the sensitivity and specificity of ctDNA for predicting prognosis (36,37). This prospective study evaluated the clinical utility of ctDNA for detection of MRD in HCC.…”

The use of minimal residual disease in thoracic oncology: Gaps between promises and the on‐the‐ground reality of daily practice

“…an approach that combines analyses of cf-DNA mutations and protein biomarkers, such as those used in the CancerSEEK method. 29,30…”

“…New technologies based largely on ultra‐deep sequencing should be able to improve the sensitivity of MRD detection in the near future, especially for the quantitation of very rare somatic mutations. Different approaches are being developed, including (i) the detection of combinations of cf‐DNA mutations and methylation, 25,26 (ii) the fragmentation approach, which detects the size of the cf‐DNA, 27 (iii) an approach that combines variant calling with analysis of cf‐DNA fragment lengths, 28 and (iv) an approach that combines analyses of cf‐DNA mutations and protein biomarkers, such as those used in the CancerSEEK method 29,30 …”