Circadian rhythm and circulating cell-free DNA release on healthy subjects.

In the last decade, clinical studies have investigated the clinical relevance of circulating cell-free-DNA (ccfDNA) as a diagnostic and prognosis tool in various diseases including cancers. However, limited knowledge on ccfDNA biology restrains its full development in the clinical practice. To improve our understanding, we evaluated the impact of the circadian rhythm on ccfDNA release in healthy subjects over a 24-h period. 10 healthy female subjects underwent blood sampling at 8am and 20 healthy male subjects underwent serial blood sampling (8:00 AM, 9:00 AM, 12:00 PM, 4:00 PM, 8:00 PM, 12:00 AM, 4 AM (+ 1 Day) and 8 AM (+ 1 Day)). We performed digital droplet-based PCR (ddPCR) assays to target 2 DNA fragments (69 & 243 bp) located in the KRAS gene to determine the ccfDNA concentration and fragmentation profile. As control, half of the samples were re-analyzed by capillary miniaturized electrophoresis (BIAbooster system). Overall, we did not detect any influence of the circadian rhythm on ccfDNA release. Instead, we observed a decrease in the ccfDNA concentration after meal ingestion, suggesting either a post-prandial effect or a technical detection bias due to a higher plasma load in lipids and triglycerides. We also noticed a potential effect of gender, weight and creatinine levels on ccfDNA concentration.

A prospective observational study for justification, safety, and efficacy of a third dose of mRNA vaccine in patients receiving maintenance hemodialysis.

The level of protection achieved by the standard two doses of COVID-19 mRNA vaccines in patients receiving maintenance hemodialysis (MHD) remains unclear. To study this we used the French Renal Epidemiology and Information Network (REIN) Registry to compare the incidence and severity of 1474 cases of COVID-19 diagnosed in patients receiving MHD after none, one or two doses of vaccine. Vaccination significantly reduce COVID-19 incidence and severity, but 11% of patients infected after two doses still died. Lack of vaccinal protection in patients naïve for SARS-CoV-2 could be due to defective Tfh response [38% of patients with negative spike-specific CD4+ T-cell interferon gamma release assay] and failure to generate viral neutralizing titers of anti-spike receptor binding domain (RBD) IgGs (63% of patients with titer at or under 997 BAU/ml, defining low/no responders) after two doses of vaccine. To improve protection, a third dose of vaccine was administered to 75 patients [57 low/no responders, 18 high responders after two doses] from the ROMANOV cohort that prospectively enrolled patients receiving MHD vaccinated with BNT162b2 (Pfizer). Tolerance to the third dose was excellent. High responders to two doses did not generate more anti-RBD IgGs after three doses but had more side effects. Importantly, 31 (54%) of low/no responders to two doses reached neutralizing titers of anti-RBD IgGs after three doses. A positive interferon gamma release assay and/or suboptimal titer of anti-RBD IgGs after two doses were the only predictive variables for response to three doses in multivariate analysis. Thus, the standard scheme of vaccination insufficiently protects patients receiving MHD. Anti-RBD IgG and specific CD4+ T-cell response after two doses can guide personalized administration of the third dose, which improves the humoral response of SARS-CoV-2-naïve patients receiving MHD.

The ROMANOV study found impaired humoral and cellular immune responses to SARS-CoV-2 mRNA vaccine in virus-unexposed patients receiving maintenance hemodialysis.

Patients on maintenance hemodialysis (MHD), which are at high risk of infection by SARS-CoV-2 virus and death due to COVID-19, have been prioritized for vaccination. However, because they were excluded from pivotal studies and have weakened immune responses, it is not known whether these patients are protected after the "standard" two doses of mRNA vaccines. To answer this, anti-spike receptor binding domain (RBD) IgG and interferon gamma-producing CD4+ and CD8+ specific-T cells were measured in the circulation 10-14 days after the second injection of BNT162b2 vaccine in 106 patients receiving MHD (14 with history of COVID-19) and compared to 30 healthy volunteers (four with history of COVID-19). After vaccination, most (72/80, 90%) patients receiving MHD naïve for the virus generated at least one type of immune effector, but their response was weaker and less complete than that of healthy volunteers. In multivariate analysis, hemodialysis and immunosuppressive therapy were significantly associated with absence of both anti-RBD IgGs and anti-spike CD8+ T cells. In contrast, previous history of COVID-19 in patients receiving MHD correlated with the generation of both types of immune effectors anti-RBD IgG and anti-spike CD8+ T cells at levels similar to healthy volunteers. Patients receiving MHD naïve for SARS-Cov-2 generate mitigated immune responses after two doses of mRNA vaccine. Thus, the good response to vaccine of patients receiving MHD with a history of COVID-19 suggest that these patients may benefit from a third vaccine injection.

Characterization of Plasma Cell-Free DNA Integrity Using Droplet-Based Digital PCR: Toward the Development of Circulating Tumor DNA-Dedicated Assays.

Background: Cellular-cell free-DNA (ccfDNA) is being explored as a diagnostic and prognostic tool for various diseases including cancer. Beyond the evaluation of the ccfDNA mutational status, its fragmentation has been investigated as a potential cancer biomarker in several studies. However, probably due to a lack of standardized procedures dedicated to preanalytical and analytical processing of plasma samples, contradictory results have been published. Methods: ddPCR assays allowing the detection of KRAS wild-type and mutated sequences (KRAS p.G12V, pG12D, and pG13D) were designed to target different fragments sizes. Once validated on fragmented and non-fragmented DNA extracted from cancer cell lines, these assays were used to investigate the influence of the extraction methods on the non-mutated and mutated ccfDNA integrity reflected by the DNA integrity index (DII). The DII was then analyzed in two prospective cohorts of metastatic colorectal cancer patients (RASANC study n = 34; PLACOL study n = 12) and healthy subjects (n = 49). Results and Discussion: Our results demonstrate that ccfDNA is highly fragmented in mCRC patients compared with healthy individuals. These results strongly suggest that the characterization of ccfDNA integrity hold great promise toward the development of a universal biomarker for the follow-up of mCRC patients. Furthermore, they support the importance of standardization of sample handling and processing in such analysis.