Table 1.
Demographic data, comorbidity and tumor types of study participants.
Fig 1.
Elevations of plasma H3Cit and cfDNA in patients with advanced cancer.
(A) Quantification of H3Cit showed increased levels in patients with advanced cancer compared to both healthy individuals and severely ill patients without known cancer. No significant difference in the levels of plasma H3Cit between severely ill patients without known cancer and healthy individuals were observed. (B) Plasma levels of H3Cit were significantly higher in patients with adenocarcinomas compared to patients with tumors of other histopathologies and (C) in patients with spread cancer compared to patients with localized tumors. (D) The levels of plasma cfDNA were significantly higher in cancer patients compared to both healthy individuals and severely ill patients without known cancer. The levels were also significantly higher in severely ill patients without known cancer compared to healthy individuals. (E) A significant positive correlation was found between plasma levels of H3Cit and cfDNA in cancer patients and in severely ill patients without known cancer. Lines represent medians with IQR. Groups were compared with the Mann-Whitney U test. Significance of correlation was analyzed with Pearson correlation coefficient after log transformed data to obtain a normal distribution. NS P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Fig 2.
Cancer-associated neutrophil activation correlates with circulating H3Cit and cfDNA.
(A) Peripheral neutrophil count was higher in cancer patients compared to severely ill patients without known cancer (dotted line represents upper reference interval). (B) Flow cytometry analysis showed a higher number of neutrophils positive for intracellular H3Cit in cancer patients compared to both healthy individuals and severely ill patients without known cancer. No significant difference was observed between severely ill patients without known cancer and healthy individuals. (C-D) Plasma NE (C) and MPO (D) were equally elevated in cancer patients and severely ill patients without known cancer compared to healthy individuals. (E) Plasma MPO-DNA complexes were significantly higher in cancer patients compared to both healthy individuals and severely ill patients without known cancer. The levels were also significantly higher in severely ill patients without known cancer compared to healthy individuals. (F) H3Cit and cfDNA correlated positively with NE, MPO and MPO-DNA complexes in cancer patients. (G) Similar correlations were seen in severely ill patients without known cancer, with the exception of correlations between H3Cit and NE and MPO. Lines represent medians with IQR. Groups were compared with the Mann-Whitney U test. Significance of correlation was analyzed with Pearson correlation coefficient after log transformed data to obtain a normal distribution. NS P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Fig 3.
Inflammatory cytokines are elevated and correlate with H3Cit in patients with advanced cancer.
(A-E) Plasma levels of IL-8, IL-6, TNFα, IL-1β, and G-CSF were all significantly higher in cancer patients compared to healthy individuals, as well as to severely ill patients without known cancer, with the exception of G-CSF which was similarly elevated in cancer patients and severely ill patients without known cancer. (F) Plasma levels of H3Cit correlated to plasma levels of IL-8 and IL-6, but weaker or no correlations were found to TNFα, IL-1β and G-CSF. (G) Multivariable regression confirmed the predictive influence of plasma cfDNA, NE, MPO, MPO-DNA complexes, IL-8, and IL-6 on plasma H3Cit levels (p<0.001). VIP, variable influence on projection. Lines represent medians with IQR. Groups were compared with the Mann-Whitney U test. Significance of correlation was analyzed with Pearson correlation coefficient after log transformed data to obtain a normal distribution. NS P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.
Table 2.
Correlations between plasma H3Cit and inflammatory cytokines in severely ill patients without known cancer.
Fig 4.
Prognostic value of high levels of plasma H3Cit, NE, IL-8 and IL-6 in patients with advanced cancer.
Kaplan-Meier curves and survival analyses with the log rank test obtaining HR were performed to assess associations between laboratory markers and short-term (100 days) mortality. Patients with plasma H3Cit (A) levels above the 75th percentile presented with a 2-fold increased risk for short-term mortality compared with patients with plasma levels below the 75th percentile (P = 0.02). Increased levels of cfDNA (B) lacked prognostic significance (P = 0.24). Increased levels of NE (C) displayed a similar prognostic significance as high levels of plasma H3Cit (P<0.001). High levels of MPO (D) and MPO-DNA complexes (E) lacked prognostic significance (P = 0.39). The inflammatory cytokines IL-8 (F) and IL-6 (G) were also strongly associated with short-term mortality (P = 0.001 and <0.001 respectively). There was no significant association between high levels of TNFα (H), IL-1β (I) and G-CSF (J) and short-term mortality (P = 0.10, 0.12 and 0.27 respectively).