Association of pronounced elevation of NET formation and nucleosome biomarkers with mortality in patients with septic shock.

BACKGROUND: Understanding the mechanisms underlying immune dysregulation in sepsis is a major challenge in developing more individualized therapy, as early and persistent inflammation, as well as immunosuppression, play a significant role in pathophysiology. As part of the antimicrobial response, neutrophils can release extracellular traps (NETs) which neutralize and kill microorganisms. However, excessive NETs formation may also contribute to pathogenesis, tissue damage and organ dysfunction. Recently, a novel automated assay has been proposed for the routine measurement of nucleosomes H3.1 (fundamental units of chromatin) that are released during NETs formation. The aim of the present study was to measure nucleosome levels in 151 septic shock patients (according to sepsis-3 definition) and to determine association with mortality. RESULTS: The nucleosome H3.1 levels (as determined by a chemiluminescence immunoassay performed on an automated immunoanalyzer system) were markedly and significantly elevated at all-time points in septic shock patients compared to the control group. Immunological parameters indicated tremendous early inflammation (IL-6 = 1335 pg/mL at day 1-2) along with marked immunosuppression (e.g., mHLA-DR = 3853 AB/C and CD4 = 338 cell /µL at day 3-4). We found significantly positive correlation between nucleosome levels and organ failure and severity scores, IL-6 concentrations and neutrophil count. Significantly higher values (day 1-2 and 3-4) were measured in non-survivor patients (28-day mortality). This association was still significant after multivariate analysis and was more pronounced with highest concentration. Early (day 1-2) increased nucleosome levels were also independently associated with 5-day mortality. At day 6-8, persistent elevated nucleosome levels were negatively correlated to mHLA-DR values. CONCLUSIONS: This study reports a significant elevation of nucleosome in patients during a one-week follow-up. The nucleosome levels showed correlation with neutrophil count, IL-6 and were found to be independently associated with mortality assessed at day 5 or 28. Therefore, nucleosome concentration seems to be a promising biomarker for detecting hyper-inflammatory phenotype upon a patient's admission. Additional investigations are required to evaluate the potential association between sustained elevation of nucleosome and sepsis-induced immunosuppression.

Circulating H3K27 Methylated Nucleosome Plasma Concentration: Synergistic Information with Circulating Tumor DNA Molecular Profiling.

The molecular profiling of circulating tumor DNA (ctDNA) is a helpful tool not only in cancer treatment, but also in the early detection of relapse. However, the clinical interpretation of a ctDNA negative result remains challenging. The characterization of circulating nucleosomes (carrying cell-free DNA) and associated epigenetic modifications (playing a key role in the tumorigenesis of different cancers) may provide useful information for patient management, by supporting the contributive value of ctDNA molecular profiling. Significantly elevated concentrations of H3K27Me3 nucleosomes were found in plasmas at the diagnosis, and during the follow-up, of NSCLC patients, compared to healthy donors (p-value < 0.0001). By combining the H3K27Me3 level and the ctDNA molecular profile, we found that 25.5% of the patients had H3K27Me3 levels above the cut off, and no somatic alteration was detected at diagnosis. This strongly supports the presence of non-mutated ctDNA in the corresponding plasma. During the patient follow-up, a high H3K27Me3-nucleosome level was found in 15.1% of the sample, despite no somatic mutations being detected, allowing the identification of disease progression from 43.1% to 58.2% over molecular profiling alone. Measuring H3K27Me3-nucleosome levels in combination with ctDNA molecular profiling may improve confidence in the negative molecular result for cfDNA in lung cancer at diagnosis, and may also be a promising biomarker for molecular residual disease (MRD) monitoring, during and/or after treatment.

The cytosolic 5'-nucleotidase cN-II lowers the adaptability to glucose deprivation in human breast cancer cells.

The cytosolic 5'-nucleotidase cN-II is a highly conserved enzyme implicated in nucleotide metabolism. Based on recent observations suggesting additional roles not directly associated to its enzymatic activity, we studied human cancer cell models with basal or decreased cN-II expression. We developed cancer cells with stable inhibition of cN-II expression by transfection of shRNA-coding plasmids, and studied their biology. We show that human breast cancer cells MDA-MB-231 with decreased cN-II expression better adapt to the disappearance of glucose in growth medium under normoxic conditions than cells with a baseline expression level. This is associated with enhanced in vivo growth and a lower content of ROS in cells cultivated in absence of glucose due to more efficient mechanisms of elimination of ROS. Conversely, cells with low cN-II expression are more sensitive to glucose deprivation in hypoxic conditions. Overall, our results show that cN-II regulates the cellular response to glucose deprivation through a mechanism related to ROS metabolism and defence.

TET2 exon 2 skipping is an independent favorable prognostic factor for cytogenetically normal acute myelogenous leukemia (AML): TET2 exon 2 skipping in AML.

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p<10-4). TET2E2S and FLT3-ITD, but not age or NPM1 mutation status were independent prognostic factors for DFS and event-free survival (EFS), while TET2E2S was the sole prognostic factor that we identified for overall survival (OS). In both the intermediate-1 and favorable ELN genetic categories, TET2E2S remained significantly associated with prolonged survival. There was no correlation between TET2E2S status and outcomes in 34 additional AML patients who were unfit for IC. Therefore our results suggest that assessments of TET2 exon 2 splicing status might improve risk stratification in CN-AML patients treated with IC.

Oncogene- and drug resistance-associated alternative exon usage in acute myeloid leukemia (AML).

In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.