Table 2 Human clinical trials—observational and anti-NET interventional

VTE in Cancer

VTE prediction in cancer patients

Elevated CitH3 associated with 13% RR increase of VTE.

Elevated cfDNA associated with higher risk of VTE during first 3-6 months

Mauracher et al.⁷⁶

VTE

VTE incidence

D-dimer correlated risk model

Higher CitH3 and NE associated with VTE.

Adding to D-dimer based risk model did not improve AUC

Smith et al.⁶⁹²

Pulmonary Embolism

CitH3

High endogenous thrombin potential, elevated CitH3, prolonged clot lysis time associated with 8x risk of PE-related death

Enhanced NET formation associated with higher early mortality risk

Zabczyk et al.⁶⁹³

STEMI with PCI

Cardiac endpoints

CitH3 independent predictor of endpoint (MI, stroke, stent thrombosis, cardiovascular related death) [HR 3.74, p = 0.042]

Ferre-Vallverdu et al.⁶⁹⁴

Cardiac arrest

Predictive value for 28-day all-cause mortality

Serum cfDNA, citH3, MPO, NE higher in all arrest patients, and significantly higher in nonsurvivor group.

cfDNA, CitH3, nucelosomes on first day after ROSC independent predictors of primary outcome

Li et al.⁶²⁸

Retinal vein occlusion

Biomarker and disease incidence

Plasma cfDNA, MPO-DNA, citH3 increased in RVO cases. Associated with thrombus formation

Wan et al.⁶⁹⁵

ACS/acute ischemic stroke

ACS/AIS Risk

dsDNA concentrations higher in ACS/AIS

ACS risk – TnI, dsDNA concentration

AIS – dsDNA concentration

Lim et al.⁵⁰⁸

Acute Liver Failure

Transplant-specific survival

cfDNA 7.1× higher in ALF

MPO-DNA 2.5× higher in ALF

cfDNA higher in severe disease

MPO-DNA 30% higher in ALF patients who died or required urgent transplant

Positive tissue NETs in 12/18 patient specimens

Meijenfeldt et al.³³⁴

AMI

1-year MACE

Platelet + soluble p-selecting + all NET markers strongest predictor of 1-year MACE [OR 1.94, 95%CI 1.16-3.25]

Hally et al.⁶⁹⁶

Ulcerative Colitis

Disease prognosis prediction

PAD4 expression associated with increasing histopathologic grade (p = 0.001), anatomical disease extent (p = 0.038), lack of therapeutic response (p = 0.046), subjection to radical surgery (p = 0.046)

El Hafez et al.⁶⁹⁷

Antiphospholipid syndrome

Association with thrombosis

Higher levels of circulating MPO-DNA and PAD4 expression. Higher expression in patients with recurrent thrombosis than incident or control (43.8% higher MPO-DNA, 2x higher RNA expression)

Mazetto et al.⁶⁹⁸

Diabetic Foot Ulcer (DFU)

Amputation probability

Serum NET levels higher in DFU group.

NET amputation probability [HR 0.19, p < 0.01]

Ibrahim et al.⁶⁹⁹

DFU

Impaired wound healing

NET specific markers higher in DFU patients than in without.

Tissue elastase increased in wounds with infections and delayed healing.

Significantly lower healing rates and higher amputation rates in highest quartile of CitH3

Yang et al.⁶⁵¹

Lupus Nephritis

Complete remission

Progression to renal impairment at 24 months

Higher NET remnants in SLE

Higher NET levels with active lupus nephritis compared to SLE without nephritis (Elastase p = 0.03, HMGB1-DNA p = 002)

Higher NET remants in proliferative nephritis (Elastase p < 0.0001, HMGB1-DNA p = 0.0003)

Higher NETs with reduced odds of complete remission [Elastase OR 2.34, p = 0.0007, HMGB1 OR 2.61, p = 0.006

Higher NETs with increased risk of progression to severe renal impairment (Elastase OR 2.84 p = 0.006, HMGB1 OR 2.04, p = 0.02)

Whittall-Garcia et al.⁷⁰⁰

COVID-19

Biomarker for prognostication

NET markers elevated in COVID-19

Associated with respiratory support requirement and short-term mortality

Correlated with WBC, inflammatory cytokines, CRP, and markers of coagulation/fibrinolysis

Contribute to immunothrombosis

Ng et al.⁷⁰¹

Streptococcal Bacteremia

Cardiovascular morbidity and mortality

Higher MPO-DNA in bacteremic

Higher MPO-DNA in abscess prone Strep groups (p = 0.02)

Combined WBC counts + MPO-DNA to predict all cause 30d mortality with commensal strep BSI—lowest among patients with neither high MPO-DNA nor abnormal WBC (p = 0.058)

This group has favorable composite outcome of MACE and all-cause mortality (p = 0.026)

Kuo et al.⁷⁰²

COVID-19

Association with MIS-C and CLL (Chilblain-like lesions)

Decreased NET degradation

No NET elevation with asymptomatic infection

Decreased NET levels with Omicron infection compared to other strains

Carmona-Rivera et al.⁷⁰³

Pleural Effusion

Diagnosis and prognostication

Highest NET marker concentration with parapneumonic effusion

CitH3 (R = 0.66) and eDNA (R = 0.73) correlated with LDH (p < 0.001)

Twaddell et al.⁷⁰⁴

Deep surgical site infection

NET index predicting DSSI occurrence after laparotomy

Higher NET formation index (NFI) in DSSI group (p < 0.01)

NFI positively correlated with APACHE II (R = 0.269, p < 0.01) and SOFA score (R = 0.258, p = 0.013)

Higher risk of DSSI with NFI score

NFI AUC 0.912 compared to CRP (0.748) and PCT (0.731)

Duan et al.⁷⁰⁵

Locally Advanced Rectal Cancer

Prognosis and predictive response to Neoadjuvant Therapy (RFS, CR, NCR)

High tissue NET density predicted poor post-operative survival

NETs independent prognostic factor for RFS

Low NET-density LARC had increased CD8 infiltration

High NET density associated with EMT.

High NET density associated with reduced likelihood of complete/near complete response

Zhong et al.⁶³⁵

Colon Adenocarcinoma

Predicting Response to Immunotherapy

NET risk score upregulated in patient samples

Levels correlated with tumor clinicopathological and immune traits

MPO linked to malignancy and poor clinical outcome.

Feng et al.⁷⁰⁶

Breast Cancer

Survival prognosis, treatment response

NET-related lncRNA risk scores

Low risk groups had improved OS

High risk groups enriched in immune-related functions and higher TMB

Response to chemo/immunotherapy related with expression of NET related lncRNA (p < 0.001)

Jiang et al.⁷⁰⁷

High-grade serous ovarian cancer (HGSOC)

Biomarker role in disease diagnosis and management

Higher concentration of cfDNA, citH3, and calprotectin in plasma and peritoneal fluid.

Neoadjuvant treatment reduced NET biomarkers in plasma, less so in peritoneal fluid

Tomas-Perez et al.⁷⁰⁸

Gastric Cancer

Prediction for immune cell infiltration

Low NET score linked to higher MSI-H, mutation load, immune activity.

CSC index and chemotherapeutic treatment sensitivity connected to NET score.

Li et al.⁷⁰⁹

Gastric Cancer

OS Prediction and TME Identification

OS longer in low-risk group (p = 0.005)

Differences in immune infiltration across groups.

NE DNA independent factor affecting OS prognosis (P = 0.006)

Qu et al.⁶¹²

Glioblastoma Multiforme

1-3 year OS prediction

NET signature to form risk groups

High risk group more sensitive to treatment biclutamide, gefitinib, dasatinib

Low risk group poor response to immunotherapy

Sun et al.⁷¹⁰

Pancreatic neuroendocrine tumor

Prediction of post-operative recurrence

Positive expression of tumor NETs with worse RFS (p < 0.05)

Independent prognostic factor for RFS (p < 0.05)

Xu et al.¹⁰⁷

Non-small cell lung cancer

OS prediction

12-NETs lncRNA signature to develop risk score

High risk group with significantly shorter OS (p < 0.0001)

Risk score is independent predictive factor of OS [HR > 1, p < 0.001]

NSCLC cell lines have higher levels of three adverse prognostic NET related lncRNA than normal lung cells

Fang et al.⁷¹¹

Head and Neck Squamous Cell Carcinoma

Prediction of 3 and 5 year clinical outcomes and immunotherapy response

6 NET-related genes to construct high vs low risk model

Higher OS in low risk (p < 0.001)

Higher TMB in high-risk model (p = 0.017)

TMB positively correlated with risk score (R = 0.11, p = 0.019)

Immune therapy more beneficial for low-risk patients (p < 0.001)

Response to anticancer drugs closely correlated with expression of NET related genes (p < 0.001)

Chen et al.⁶¹⁰

Gastric Adenocarcinoma

Diagnostic and prognostic predictive value

NET markers had better diagnostic value than CEA, CA19-9

High level of NETs correlated with lymph node metastases

Blood NET markers inversely correlated with short-term efficacy of first-line treatment

Negative HER2 status associated with higher baseline NETs and worse PFS

Zhang et al.⁶¹¹

Breast Cancer

Association with clinical stages

Higher levels of NE-DNA complexes in regional and distant stages compared to local disease

NETs increase in proportion to disease stage

Rivera-Franco et al.⁷¹²

Head and Neck squamous cell carcinoma

NET-related gene signature prognostic score

Seven NET-related genes to create score signature

Score highly correlated with clinicopathologic and immune traits

NIFK upregulated in HNSCC pateint samples

NIFK required for HNSCC cell proliferation and metastasis

Li et al.⁷¹³

Pancreatectomy

Surgically induced NET formation

CfDNA and CitH3 elevated after pancreatic resection

Increased NET-inducing cytokines post-op

Reduced NETs with robotic approach

Increased NETs in with pancreatic leak

Ivey et al.⁷¹⁴

Generalized malignancy

Peripheral blood biomarker in diagnosis and disease progression

CitH3 and cfDNA distinguishes healthy control and tumor

CitH3/cfDNA increased with clinical stage

Correlation between cfDNA and systemic inflammation related parameters in tumor patients

Did not predict VTE in short-term

Wang et al.⁷¹⁵

Clear cell renal cell carcinoma

NET pathway association with clinicopathologic features, prognosis, prediction of therapeutic benefit

NET clusters A – metabolic pathways, better survival outcome

Cluster C – immune pathways, higher immune score, poorer prognosis

Higher NET scores associated with immune cell infiltration, targeted drug response, immunotherapy benefits

Teng et al.⁷¹⁶

Clear cell renal cell carcinoma

Validation of molecular subtype and survival prognosis

Six NET-related gene signature

Good performance in predicting OS of ccRCC

Signature significantly correlated with pTMN, immune infiltration, TMB, microsatellite instability, drug sensitivity

Quan et al.⁷¹⁷

Breast Cancer

Prediction/prognosis and immunotherapy response

Risk signature model

High risk score associated with poor immunotherapy response and adverse clinical outcomes

Zhao et al.⁷¹⁸

AIS/AMI

NET composition and association with clinical outcome

NETs present in all patients with AIS, and 20.8% patients with AMI.

Abundance of NET in thrombi associated with poor outcome score in AIS, and reduced EF in AMI

Novotny et al.⁷¹⁹

Esophageal Cancer

OS

Leukocytosis associated with decreased OS and DFS.

Leukocytosis resulted in higher intratumoral NET infiltration (p < 0.001)

Higher levels of NET infiltration associated with worse OS and DFS (p < 0.001)

Zhang et al.⁷²⁰

Coronary Artery Disease

Adverse clinical outcomes (unstable angina, stroke, MI, death)

NET markers weakly intercorrelated (R = 0.103, p < 0.001)

Highest quartiles of dsDNA had weakly but significantly elevated hypercoagulability markers (p < 0.001)

Higher dsDNA in groups experiencing clinical endpoint (p 0.019)

Upper 3 quartiles of NETS had OR 2.01 for endpoint (p 0.019)

Langseth et al.³⁶²

Community-Acquired Pneumonia

Primary – time to clinical stability

Secondary length of stay, mortality

Serum NETs associated with 3.8× increased OR of 30-day mortality Elevated serum NETs associated with higher risk for clinical instability, prolonged length of stay and 30-day mortality

Ebrahimi et al.⁷²¹

Appendicitis

Prediction of incidence and outcome

CfDNA (AUC 0.87) and CtiH3 (AUC 0.88) demonstrated excellent predictive power for appendicitis

CitH3 able to distinguish noncomplicated from complicated appendicitis and predict patient outcomes, compared to WBC and CRP

Boettcher et al.⁶²⁷

Primar hepatic malignancy

RFS and OS

High pre-surgery serum NET associated with shorter RFS/OS

RFS: HCC - HR 2.9, CC – HR 3.22

High CitH3 level also predicted shorter RFS/OS

Kaltenmeier et al.⁶⁰⁸

Pancreatic Ductal Adenocarcinoma

PFS, Disease-specific survival

Positive NET expression exhibited poorer PFS and DSS

NET formation is independent prognostic predictor of DSS

PDAC with negative NET staining more likely to benefit from ACT

Chen et al.⁷²²

Surgery

NET formation between mild and severe surgical trauma

Decreased NETosis after severe surgical trauma

Suggesting inducibility of NETs after surgical trauma may be compromised

Huang et al.⁷²³

Age

NET production and activity

Greater NET production in elderly (>65) than adult (20-50) adults.

NETs produced in elderly reduced bactericidal capacity.

Higher NET size in elderly (size of extruded DNA threads)

Sabbatini et al.⁷²⁴

Exercise (HIIT)

NET production

Baseline induction of NETosis greater in older men (p < 0.05)

HIIT reduced induction of NETosis in older men

Vidal-Seguel et al.⁷²⁵

COVID-19 ARDS

Dornase alfa/MPO-DNA complex

Reduced BALF MPO DNA

Improved PF ratio

Improved static lung compliance

In short term

Holliday et al.³⁴⁰

COVID-19

RhDNase-1 with nanoparticulate

Reduced cfDNA

Lee et al.⁶³⁰

COVID-19

RhDNase

Decreased NETs in sputum

Associated wtih recovery and improved oxygenation

Fisher et al.⁷²⁶

Pancreatic cancer

Lidocaine/Circulating NETs

No improvement is OS or DFS

Zhang et al.⁶³³

Pre-diabetes

Metformin/Net components elastase, proteinase-3, histones, dsDNA

Reduced NET components (elastase, proteinase-3, histones, dsDNA).

Better than with other glycemic agents

Menegazzo et al.⁷²⁷

Breast cancer

IV lidocaine/MPO, CitH3, VEGF

Decreased post-op expression of NETosis

Galos et al.⁶³²

ARF after Trauma

Inhaled Dornase Alfa

Enrolling Incidence of moderate to severe ARDS in ventilated trauma patients in ICU

NCT03368092

ACS after PCI

Colchicine

Suppresses NET formation by storing cytoskeletal dynamics

Vaidya et al.⁷²⁸

Breast Cancer

Tamoxifen/Serum NETs, drug resistance, cancer metastasis, comorbidities

Currently enrolling

NCT05056857

Lung cancer

Perioperative lidocaine/dexmedetomidine

Reduced serum MPO

Reduced MMP-3

Ren et al.⁷²⁹