Background: Molecular profiling of circulating tumor DNA (ctDNA) is a helpful tool for cancer treatment indication or for the early detection of relapse. In patients with advanced lung adenocarcinoma cancers (NSCLC), a subset can be cured by immunotherapy, radiotherapy, and/or chemotherapy combined regimens, or targeted therapies depending on their ctDNA molecular profile. But clinical interpretation of ctDNA negative result remains often challenging. Taking advantage that cell-free DNA in association with nucleosomes are released into the bloodstream upon cell death, the characterization of both may give a benefit to patient management. Indeed, dysregulations of epigenetic modifications, such as histone methylation, are found to play a key role in tumorigenesis of different cancers. However, the concentration of circulating nucleosomes in blood, as a biomarker of the contributive value of ctDNA molecular profiling in patient management at diagnosis or during patient follow-up have not previously been investigated. Results: Significantly elevated concentrations of H3K27Me3-nucleosomes were found in NSCLC plasmas at diagnosis and during the follow-up of patients compared to healthy donors (median: 24ng/mL; 16.9ng/mL vs 8ng/mL, p-value < 0.0001, respectively). Interestingly, by combining H3K27Me3 level and ctDNA molecular profile, we found that 25.5% of the patients had high levels of H3K27Me3 (above 22.5 ng/mL) and no somatic alteration detected at diagnosis. This strongly supports the presence of non-mutated ctDNA in the corresponding plasma. During patient follow-up, H3K27Me3 level was lower in ctDNA-negative group compared to ctDNA-positive group (medianctDNA- = 13.4 ng/mL vs medianctDNA+ = 26.1 ng/mL, respectively, p-value < 0.0001). In 41.8% of the samples, no somatic mutation and low level of H3K27Me3-nucleosomes were observed suggesting molecular indicator of treatment response. In contrast, high H3K27Me3-nucleosome levels were found in 15.1% of the samples despite no somatic mutations being detected allowing the identification of disease progression from 43.1% to 58.2% over molecular profiling only. Conclusion: H3K27Me3-nucleosome level is proposed to be a useful biomarker of the contributive value of ctDNA molecular profiling at diagnosis by greatly improving the confidence in the negative molecular result in cfDNA in lung cancer. In addition, H3K27Me3-nucleosome could be a promising biomarker for Molecular Residual Disease monitoring in NSCLC during or after treatment.

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