Circulating H3K27 Methylated Nucleosome plasma concentration: a synergic information with ctDNA Molecular Profiling

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. ### Competing Interest Statement J.C., N.H., R.R., G.R. and M.H. are employees of Belgian Volition SRL. ### Funding Statement The authors thank AstraZeneca and Belgian Volition for financial support. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Patients were recruited from the pulmonology department at Lyon University Hospital from 2015 to 2022. Study number: Etude RNIPH 22-5065 NUCLEO_CIRCAN Scientific and Ethics Committee of Lyon University Hospital gave ethical approval for this work : number 22-5065. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data presented in this study are available in the Additional Files and from the corresponding author: lea.payen-gay@chu-lyon.fr