Soluble Neuropilin-1 Is Elevated in Sepsis and Correlates with Organ Dysfunction and Long-Term Mortality in Critical Illness.

Critical illness and sepsis may cause organ failure and are recognized as mortality drivers in hospitalized patients. Neuropilin-1 (NRP-1) is a multifaceted transmembrane protein involved in the primary immune response and is expressed in immune cells such as T and dendritic cells. The soluble form of NRP-1 (sNRP-1) acts as an antagonist to NRP-1 by scavenging its ligands. The aim of this study was to determine the value of sNRP-1 as a biomarker in critical illness and sepsis. We enrolled 180 critically ill patients admitted to a medical intensive care unit and measured serum sNRP-1 concentrations at admission, comparing them to 48 healthy individuals. Critically ill and septic patients showed higher levels of sNRP-1 compared to healthy controls (median of 2.47 vs. 1.70 nmol/L, p < 0.001). Moreover, sNRP-1 was also elevated in patients with sepsis compared to other critical illness (2.60 vs. 2.13 nmol/L, p = 0.01), irrespective of disease severity or organ failure. In critically ill patients, sNRP-1 is positively correlated with markers of kidney and hepatic dysfunction. Most notably, critically ill patients not surviving in the long term (one year after admission) showed higher concentrations of sNRP-1 at the time of ICU admission (p = 0.036), with this association being dependent on the presence of organ failure. Critically ill and septic patients exhibit higher serum concentrations of circulating sNRP-1, which correlates to organ failure, particularly hepatic and kidney dysfunction.

IFN-ß reduces NRP-1 expression on human cord blood monocytes and inhibits VEGF-induced chemotaxis.

Type I interferons (IFNs) inhibit angiogenesis, the sprouting of new blood vessels, during tissue development, remodeling, and tumor growth. One of the major targets type I IFNs inhibit are circulating monocytes, which promote vascular development by secreting growth factors, chemokines, and proteases. This study tested the hypothesis that IFN-ß directly inhibits monocyte chemotaxis towards VEGF. We were interested in looking at chemotaxis towards VEGF because VEGF is known to create a pro-angiogenesis environment by acting as a stimulator and chemotactic factor for endothelial cells and monocytes. Here, we demonstrate that IFN-ß, a type I IFN, downregulates neuropilin-1 (NRP-1) expression by human monocytes and inhibits chemotaxis induced by vascular endothelial growth factor (VEGF), a NRP-1 ligand. Together, the data suggest that IFN-ß directly downregulates NRP-1 expression in monocytes, thus inhibiting monocyte chemotaxis toward a VEGF enriched environment.

Maternal plasma soluble neuropilin-1 is downregulated in fetal growth restriction complicated by abnormal umbilical artery Doppler: a pilot study.

OBJECTIVES: Placental expression of neuropilin-1 (NRP1), a proangiogenic member of the vascular endothelial growth factor receptor family involved in sprouting angiogenesis, was recently discovered to be downregulated in pregnancies with fetal growth restriction (FGR) and abnormal umbilical artery (UA) Doppler. Soluble NRP1 (sNRP1) is an antagonist to NRP1; however, little is known about its role in normal and FGR pregnancies. This study tested the hypotheses that, first, sNRP1 would be detectable in maternal circulation and, second, its concentration would be upregulated in FGR pregnancies compared to those with normal fetal growth and this would correlate with the severity of the disease as assessed by UA Doppler. METHODS: This was a prospective case-control pilot study of 40 singleton pregnancies (20 FGR cases and 20 uncomplicated controls) between 24 + 0 and 40 + 0 weeks' gestation followed in an academic perinatal center from January 2015 to May 2017. FGR was defined as an ultrasound-estimated fetal weight < 10th percentile for gestational age. The control group was matched to the FGR group for maternal age and gestational age at assessment. Fetal ultrasound biometry and UA Doppler were performed using standard protocols. Maternal plasma sNRP1 measurements were performed using a commercially available ELISA. RESULTS: Contrary to the study hypothesis, maternal plasma sNRP1 levels were significantly decreased in FGR pregnancies as compared to those with normal fetal growth (137.4 ± 44.8 pg/mL vs 166.7 ± 36.9 pg/mL; P = 0.03). However, there was no significant difference in sNRP1 concentration between the control group and FGR pregnancies that had normal UA Doppler. Plasma sNRP1 was downregulated in FGR pregnancies with elevated UA systolic/diastolic ratio (P = 0.023) and those with UA absent or reversed end-diastolic flow (P = 0.005) in comparison to FGR pregnancies with normal UA Doppler. This suggests that biometrically small fetuses without hemodynamic compromise are small-for-gestational age rather than FGR. CONCLUSIONS: This study demonstrated a significant decrease in maternal plasma sNRP1 concentration in growth-restricted pregnancies with fetoplacental circulatory compromise. These findings suggest a possible role of sNRP1 in modulating fetal growth and its potential as a biomarker for FGR. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Serum concentrations of neuropilin-1 in women with endometriosis.

Objective of this work is to investigate, for the first time, serum concentration of neuropilin-1 (NRP-1), aiming to evaluate its diagnostic performance in endometriosis and usability as a potential non-invasive serum marker of endometriosis. Two hundred women were treated laparoscopically. After laparoscopic surgery women were divided into two groups: 120 women diagnosed with endometriosis and 80 healthy women (control group). Blood samples were taken from all women undergoing laparoscopy half an hour before the induction of anesthesia, for the purpose of collection of serum. The level of NRP-1 in serum was assayed by a standardised sandwich enzyme-linked immunosorbent assay. Differences between endometriosis and healthy control group in NRP-1 levels were significant. All values were significantly and several times higher in patients group, p < .001. After receiver operating characteristic analysis, the area under curve was 0.97 (95% confidence interval: 0.941 to 0.989, p < .0001) at 11 µg/L cut-off level for NRP-1. Preliminary threshold values for NRP-1 in serum were assumed to serve as diagnostic parameters with sensitivity of 99.3% and specificity of 97.8%. Serum concentration of NRP-1 can be considered as a potentially good laboratory diagnostic, non-invasive marker for endometriosis.

Establishment of a bead-based duplex assay for the simultaneous quantitative detection of Neuropilin-1 and Neuropilin-2 using xMAP technology and its clinical application.

BACKGROUND: Neuropilins (Nrps) are a new type of broad-spectrum tumor marker. Currently, a method for accurate simultaneous quantification of Nrps is not available. We aimed to develop a bead-based and duplexed flow cytometric assay that could be used for accurate and simultaneous quantification of Nrp1 and Nrp2 for scientific research or clinical diagnosis. METHODS: We coupled anti-human Nrp1-11# mAb and anti-human Nrp2-C3 mAb to magnetic beads 18# and 25#, respectively. Capturing antibodies and detecting antibodies were then combined to detect Nrps by a bead-based Luminex assay, which was subsequently applied to quantify Nrps in clinical serum samples. RESULTS: The results showed that the detection value of Nrps ranged from 10 to 100 000 pg/mL for Nrp1 and from 25 to 100 000 pg/mL for Nrp2. The detection sensitivity reached 10 pg/mL for Nrp1 and 24.8 pg/mL for Nrp2. Intra-assay variances ranged from 1.0% to 2.6% for Nrp1 and from 2.9% to 4.0% for Nrp2, and interassay variances ranged from 1.5% to 6.4% for Nrp1 and from 4.2% to 8.1% for Nrp2. The Nrp1 and Nrp2 recoveries were 96.6%-103.6% and 95.6%-102.3%, respectively. Irrelevant antigens had no interference in the paired-detection system, and the mean fluorescence intensity (MFI) values were stable for months. CONCLUSION: A bead-based, duplexed flow cytometric assay (xMAP® technology) was developed to detect Nrp1 and Nrp2. The assay provided rapid, high-throughput results and was much more sensitive, specific, reproducible, and stable than existing assays. In addition, this assay could be applied in early-stage cancer screening, tumor malignancy analysis, and prognosis assessment.

Characterization of a sandwich ELISA for quantification of total human soluble neuropilin-1.

BACKGROUND: Neuropilin-1 (NRP1) is a highly interactive molecule that exists as transmembrane and soluble isoforms. Measurement of circulating levels of soluble NRP1 (sNRP1) in human serum and plasma has proven to be difficult due to present matrix interferences and due to the lack of a reliable technique. METHODS: We developed a highly specific and sensitive sandwich ELISA assay for total sNRP1 quantification in peripheral blood, and we validated the test according to ICH guidelines. The linear epitopes of the employed polyclonal and monoclonal anti-human NRP1 antibodies were mapped with microarray technology. We included a sample pre-treatment step with guanidine hydrochloride (GuHCl) to release sNRP1 from existing interferants. RESULTS: The ELISA assay which is calibrated with sNRP1 isoform 2 and covers a calibration range from 0.375 to 12 nmol/L detects sNRP1 in human serum and plasma (heparin, EDTA, and citrate). Multiple linear epitopes recognized by the polyclonal coating antibody are distributed over the whole sNRP1 sequence. The monoclonal detection antibody binds to a linear epitope which is in the N-terminal region of the a1 domain of human sNRP1. Assay parameters like precision (intra-assay: 6%), dilution linearity (95%-115%), specificity (98%), and spike recovery (81%-109%) meet the international standards of acceptance. CONCLUSION: Our novel sandwich ELISA provides a reliable tool for the quantitative determination of total human sNRP1. The assay detects free and previous ligand-bound total NRP1.

Neuropilin-1/CD304 Expression by Flow Cytometry in Pediatric Precursor B-Acute Lymphoblastic Leukemia: A Minimal Residual Disease and Potential Prognostic Marker.

Flow cytometry (FCM) is used for quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) through discriminating leukemic B-lymphoblasts from normal B-cell precursor counterparts "hematogones." Neuropilin-1 (NRP-1)/CD304 is a vascular endothelial growth factor receptor implicated in the progression of hematological malignancies. We evaluated NRP-1/CD304 as MRD and prognostic marker in pediatric precursor B-ALL using FCM. Seventy children with precursor B-ALL and 40 control children were enrolled. CD304 percentage and fluorescence intensity were significantly higher in precursor B-ALL at diagnosis compared with controls. In total, 28 of 70 (40%) precursor B-ALL patients at diagnosis were CD304 (group A), whereas 42/70 (60%) patients were CD304 (group B). Group A showed higher incidence of lymphadenopathy and TEL-AML1 fusion gene than group B. CD304 was reevaluated in group A patients at day 28 postinduction chemotherapy which revealed 12/28 (42.9%) patients with persistent CD304 expression (MRD; group A1) and 16/28 (57.1%) patients who turned CD304 (MRD; group A2). At diagnosis, group A1 showed lower incidence of TEL-AML1 fusion gene and higher risk stratification than group A2. NRP-1/CD304 expression by FCM is efficient in discriminating leukemic B-lymphoblasts from hematogones, a stable leukemia-associated phenotype for MRD monitoring, and a putative poor prognostic marker in pediatric precursor B-ALL.

Utility of serum neuropilin-1 and angiopoietin-2 as markers of hepatocellular carcinoma.

This study aimed to assess the diagnostic value of two serum angiogenetic markers neuropilin-1 (NRP-1) and angiopoietin-2 (ANG-2) in patients with hepatocellular carcinoma (HCC) and their relation to tumor characteristics. 149 subjects were recruited and divided into 50 patients with recently diagnosed HCC, 49 patients with cirrhosis on top of hepatitis C virus infection, and 50 healthy subjects. Serum NRP-1 and ANG-2 were estimated by ELISA. Alpha-fetoprotein (AFP) levels were measured using fluorescence immunoassay. Serum NRP-1 and ANG-2 levels were significantly higher in patients with HCC (2221.8±1056.6 pg/mL and 3018.5±841.4 pg/mL) than healthy subjects (219.3±61.8 pg/mL and 2007.7±904.8 pg/mL) and patients with cirrhosis (1108.9±526.6 pg/mL and 2179.1±599.2 pg/mL), respectively. In multivariate logistic regression analysis, NRP-1 and AFP were the only independent factors of HCC development and correlated positively with each other (r=0.781, p<0.001). Receiver operating characteristic curve analysis showed that the area under the curve (AUC) of NRP-1 was higher than that of ANG-2 in discriminating HCC from patients with cirrhosis (0.801 vs 0.748, p=0.250) and healthy subjects (0.992 vs 0.809, p<0.001). The AUC of NRP-1 was detected to be increased (0.994) when combined estimation with AFP was performed. Elevated serum NRP-1 and ANG-2 levels were detected in patients with HCC with tumor numbers >3, tumor size ≥5 cm, tumor stages B/C according to the Barcelona Clinic Liver Cancer staging system, vascular invasion, and distant metastasis. In conclusion, NRP-1 is a potential serological marker for HCC diagnosis and is better than ANG-2. It is feasible to be estimated in combination with AFP to enhance its diagnostic power. High serum NRP-1 and ANG-2 levels are associated with advanced HCC tumor characteristics.

Soluble Neuropilin-1 is an independent marker of poor prognosis in early breast cancer.

BACKGROUND: Neuropilin-1 (NRP-1) is a transmembrane protein that acts as a multifunctional non-tyrosine kinase receptor with an established role in development and immunity. NRP-1 also regulates tumor biology, and high expression levels of tissue NRP-1 have been associated with a poor prognosis. Recently, ELISA-based quantification of soluble NRP-1 (sNRP-1) has become available, but little is known about the prognostic value of sNRP-1 in malignancies. MATERIALS AND METHODS: We measured sNRP-1 in the serum of 509 patients with primary early breast cancer (BC) at the time of diagnosis using ELISA. RESULTS: Mean serum values of sNRP-1 were 1.88 ± 0.52 nmol/l (= 130.83 ± 36.24 ng/ml). SNRP-1 levels weakly correlated with age, and were higher in peri- and postmenopausal patients compared to premenopausal patients, respectively (p < 0.0001). Low levels of sNRP-1 were associated with a significant survival benefit compared to high sNRP-1 levels at baseline (p = 0.005; HR 1.94; 95%CI 1.23-3.06). These findings remained significant after adjustment for tumor stage including lymph node involvement, grading, hormone receptor, HER2 status, and age (p = 0.022; HR 1.78; 95%CI 1.09-2.91). CONCLUSION: Our findings warrant further investigations into the prognostic and therapeutic potential of sNRP-1 in BC.

Serum and tissue expression of neuropilin 1 in precancerous and malignant vocal fold lesions.

OBJECTIVES: The study was designed to evaluate the tissue expression of NRP-1 and serum level of sNRP-1 in the same patients with intraepithelial laryngeal lesions or early staged laryngeal cancer to identify the clinical significance of these biomarkers in the diagnosis of laryngeal lesions. MATERIAL AND METHODS: A prospective analysis of tissue was performed on specimens and blood samples from 49 patients, who were admitted for surgical resection due to suspicious vocal fold lesions and were diagnosed as non-dysplasia, low-grade dysplasia, high-grade dysplasia and invasive cancers. RESULTS: ELISA was conducted on 48 blood samples. The minimum level of sNRP-1 was 0.15 ng/ml and maximum- 37.71 ng/ml. The Kruskal-Wallis one-way analysis of variance revealed no differences in sNRP-1 levels between different histopathological stages of vocal fold lesions (p = 0.234). IHC was conducted in 49 tissue samples. The evaluated mean scores of NRP-1 tissue expression were compared to histopathological stage of the lesion. The Kruskal-Wallis one-way analysis of variance revealed no differences in NRP-1 tissue expression between different histopathological stages of vocal fold lesions (p = 0.536). The correlation of tissue NRP-1 expression and serum levels of NRP-1 within analyzed group was insignificant. The Spearman's rank correlation coefficient was 0.076 (p = 0.606). CONCLUSIONS: The NRP-1 tissue expression and serum levels are unlikely to be a prognostic factor for identification of laryngeal dysplasia or early stage laryngeal cancer. Further studies investigating biomolecules involved in laryngeal carcinogenesis are necessary.

Expression and Clinical Significance of Neuropilin-1 in Patients With Multiple Myeloma.

BACKGROUND: Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor A (VEGFA), and has been reported to be overexpressed in several malignancies. Since angiogenesis plays an important role in pathogenesis of multiple myeloma (MM) and the role of NRP1 in MM has not been studied yet, we characterized the expression of NRP1 in this disease. MATERIALS AND METHODS: The expression level of NRP1 was measured in 140 patients newly diagnosed with MM and 28 healthy controls by flow cytometry and quantitative reverse transcriptase polymerase chain reaction. RESULTS: Expression of NRP1 was significantly reduced on plasma cells (median=2.05%) compared to that on B-cells (median=10.05%, p<0.0001) in bone marrow of patients with MM. In MM, the expression of NRP1 was high on plasmacytoid dendritic cells (median=85.85%) and low on regulatory T-cells (median=0.6%). CONCLUSION: In MM, NRP1 is regulated differentially as compared to other B-cell malignancies at both the RNA and protein level.

Gene expression of semaphorin-3A, semaphorin-7A, neuropilin-1, plexin-C1, and ß1 integrin in treated-multiple sclerosis patients.

OBJECTIVE: Recently, members of the semaphorin family have received major attention in various medical fields, especially autoimmunity. In this study, we selected semaphorin-3A (Sema3A), semaphorin-7A (Sema7A), and their receptors to determine the possible relationship between these molecules and multiple sclerosis (MS). METHOD: We measured the gene expression of Sema3A, Sema7A, neuropilin-1 (NP-1), plexin-C1, and ß1 integrin in the blood samples of relapsing-remitting multiple sclerosis (RRMS) patients, treated with high-dose interferon-ß1a (IFN-ß1a), low-dose IFN-ß1a, IFN-ß1b, and glatiramer acetate (GA) via quantitative real-time polymerase chain reaction (qRT-PCR) assay, and then, compared the results of treatment-naive patients with the healthy controls. RESULTS: The gene expression of Sema3A (P = 0.02), NP-1 (P < 0.001), and plexin-C1 (P < 0.01) significantly decreased in the treatment-naive group, compared to the healthy controls. Sema3A significantly increased in all treated patients, compared to the treatment-naive patients (P < 0.001). However, expression of NP-1 (P < 0.001), plexin-C1 (P < 0.001), and ß1 integrin (P < 0.05) only increased in patients receiving high-dose IFN-ß1a, IFN-ß1b, and GA. Expression of Sema7A increased in only two groups of patients treated with IFN-ß1b (P < 0.001) and GA (P = 0.018), without any significant decrease in the treatment-naive group, compared to the healthy controls (P > 0.05). CONCLUSION: Our findings confirm that the presence of Sema3A, Sema7A, and their receptors can play critical roles in the treatment of MS patients. Therefore, they can be potential target molecules for MS treatment in the future.

Neuropilin 1 (NRP1) is a novel tumor marker in hepatocellular carcinoma.

BACKGROUND: TEA domain transcription factor (TEAD) has an oncogenic role in hepatocellular carcinoma (HCC). However, whether a membrane protein can serve not only as a tumor marker that reflects TEAD function but also as a therapeutic target that stimulates tumorigenesis in HCC remains unknown. METHODS: Tissue NRP1 was measured using immunohistochemistry. Cell viability, colony formation and caspase3/7 activity were assessed using MTT, soft agar and caspase 3/7 Glo assays, respectively. Serum NRP1 was examined using ELISA and Western blotting. RESULTS: NRP1 expression was up-regulated by TEAD. We also identified a conserved TEAD-binding motif in the NRP1 promoters, which was essential for the TEAD-NRP1 interaction. NRP1 was upregulated in HCC tissues and cell lines, and knockdown of NRP1 inhibited the transformative phenotypes of HCC cells. Notably, the concentrations of serum NRP1 in the HCC patients were much higher than those of hepatitis B, hepatitis C, cirrhosis, breast cancer, colon cancer, gastric cancer and lung cancer patients. Moreover, serum NRP1 was significantly associated with AFP, γ-GT, Alb, bile acid, ALT, AST, ALP and pre-Alb. The area under the receiver operating characteristic curve (AUC-ROC) for serum NRP1 was 0.971, presenting better diagnostic performance compared to AFP. CONCLUSIONS: NRP1 is a novel tumor marker in HCC.

Evidence for a Derangement of the Microvascular System in Patients with a Very Early Diagnosis of Systemic Sclerosis.

OBJECTIVE: To investigate whether patients with a very early diagnosis of systemic sclerosis (VEDOSS) may already present circulating markers and in vitro signs of microvascular dysfunction. METHODS: Serum samples were obtained from 55 patients with systemic sclerosis (SSc), 25 patients with VEDOSS, and 55 matched healthy controls (HC). Serum levels of pan-vascular endothelial growth factor (VEGF) and soluble neuropilin-1 (sNRP-1) were measured by ELISA. Human dermal microvascular endothelial cells (H-MVEC) were cultured and stimulated with SSc, VEDOSS, and HC sera. Protein expression of NRP-1 was analyzed by Western blotting, cell proliferation by 5'-bromodeoxyuridine assay, migration capacity by wound-healing assay, and capillary-like tube formation by Matrigel assay. RESULTS: Serum levels of pan-VEGF were increased in patients with VEDOSS and SSc versus HC (p = 0.05 and p = 0.003, respectively). Serum levels of sNRP-1 were significantly reduced in patients with VEDOSS and SSc compared with controls (p = 0.012 and p = 0.027, respectively). NRP-1 expression was decreased in H-MVEC stimulated with VEDOSS sera (p < 0.001 vs HC). Proliferation was reduced in H-MVEC stimulated either with VEDOSS or SSc sera in comparison with HC sera (p = 0.015 and p = 0.043, respectively). Wound healing was compromised in H-MVEC stimulated with VEDOSS and SSc sera versus HC sera (p < 0.01 for both). Capillarogenesis was decreased in H-MVEC stimulated with VEDOSS sera (p < 0.01) and SSc sera (p < 0.001) compared with cells stimulated with HC sera. CONCLUSION: Similar to patients with SSc, patients with VEDOSS already present biological signs of endothelial dysfunction. Our data demonstrate that VEDOSS sera significantly modify endothelial cell behavior and impair the angiogenic potential of the microvascular system.

Neuropilin-1 Associated Molecules in the Blood Distinguish Poor Prognosis Breast Cancer: A Cross-Sectional Study.

Circulating plasma and peripheral blood mononuclear (PBMCs) cells provide an informative snapshot of the systemic physiological state. Moreover, they provide a non-invasively accessible compartment to identify biomarkers for personalized medicine in advanced breast cancer. The role of Neuropilin-1 (NRP-1) and its interacting molecules in breast tumor tissue was correlated with cancer progression; however, the clinical impact of their systemic levels was not extensively evaluated. In this cross-sectional study, we found that circulating and tumor tissue expression of NRP-1 and circulating placental growth factor (PlGF) increase in advanced nodal and metastatic breast cancer compared with locally advanced disease. Tumor tissue expression of NRP-1 and PlGF is also upregulated in triple negative breast cancer (TNBC) compared to other subtypes. Conversely, in PBMCs, NRP-1 and its interacting molecules SEMA4A and SNAI1 are significantly downregulated in breast cancer patients compared to healthy controls, indicating a protective role. Moreover, we report differential PBMC expression profiles that correlate inversely with disease stage (SEMA4A, SNAI1, PLXNA1 and VEGFR3) and can differentiate between the TNBC and non-TNBC tumor subtypes (VEGFR3 and PLXNA1). This work supports the importance of NRP-1-associated molecules in circulation to characterize poor prognosis breast cancer and emphasizes on their role as favorable drug targets.

BATON-CRC: A Phase II Randomized Trial Comparing Tivozanib Plus mFOLFOX6 with Bevacizumab Plus mFOLFOX6 in Stage IV Metastatic Colorectal Cancer.

PURPOSE: Tivozanib, a selective inhibitor of VEGFR-1, -2, and -3, plus mFOLFOX6 in an advanced gastrointestinal cancer phase Ib study had encouraging antineoplastic activity and a tolerable safety profile. This randomized, open-label, phase II trial of tivozanib/mFOLFOX6 versus bevacizumab/mFOLFOX6 in patients with previously untreated metastatic colorectal cancer (mCRC) evaluated tivozanib activity versus bevacizumab. EXPERIMENTAL DESIGN: Treatment-naïve patients received mFOLFOX6 every 2 weeks of each 28-day cycle plus either tivozanib orally 1.5 mg once daily for 21 days or bevacizumab intravenously 5 mg/kg every 2 weeks. Investigator-assessed progression-free survival (PFS) was the primary endpoint; some secondary endpoints included safety, overall survival, overall response rate (ORR), duration of response, time to treatment failure, and biomarker subgroup analyses. RESULTS: A prespecified interim futility analysis demonstrated that the futility boundary for superiority of tivozanib/mFOLFOX6 over bevacizumab/mFOLFOX6 for PFS in the intent-to-treat population was crossed; median PFS was 9.4 versus 10.7 months [HR = 1.091; confidence interval (CI), 0.693-1.718; P = 0.706]. Tivozanib/mFOLFOX6 resulted in PFS and ORR comparable with bevacizumab/mFOLFOX6; interim analyses biomarker results revealed no significant PFS association. Post hoc final analyses demonstrated a potential difference in tivozanib-specific PFS in patients with low neuropilin-1 (NRP-1), but not in patients with high NRP-1. Tivozanib/mFOLFOX6 was tolerable and adverse events were comparable with both bevacizumab/mFOLFOX6 and previous tivozanib studies. CONCLUSIONS: The efficacy of tivozanib/mFOLFOX6 was comparable with but not superior to bevacizumab/mFOLFOX6 in patients with previously untreated mCRC. Since data from the prespecified interim analysis did not demonstrate superiority, this resulted in discontinuation of the study. The safety and tolerability profile of tivozanib/mFOLFOX6 was consistent with other tivozanib trials. NRP-1 is a potential predictive biomarker for tivozanib activity, but these results require further validation. Clin Cancer Res; 22(20); 5058-67. ©2016 AACR.

Quantitation of Circulating Neuropilin-1 in Human, Monkey, Mouse, and Rat Sera by ELISA.

Neuropilin-1 (NRP1) is a single spanning transmembrane glycoprotein that acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factors. Naturally occurring soluble NRP1 isoforms containing partial extracellular domain (ECD) have been reported. In addition to soluble NRP1, full-length NRP1 ECD has also been identified in human and animal sera. Here, we describe primate and rodent NRP1 ELISAs that measure total circulating NRP1 including soluble NPR1 and NRP1 ECD in human, monkey, mouse, and rat sera.

Circulating soluble neuropilin-1 in patients with early cervical cancer and cervical intraepithelial neoplasia can be used as a valuable diagnostic biomarker.

OBJECTIVE: To investigate soluble neuropilin-1 (sNRP-1) in circulating and NRP-1 protein in cervical tissues from patients with cervical cancer or cervical intraepithelial neoplasia (CIN). METHODS: sNRP-1 was measured in 64 preoperative patients and 20 controls. NRP-1 protein in cervical tissue was detected in 56 patients and 20 controls. RESULTS: Both sNRP-1 and NRP-1 proteins were correlated with stage. sNRP-1 presented a high diagnostic ability of cervical cancer and CIN, with a sensitivity of 70.97% and a specificity of 73.68%. CONCLUSIONS: sNRP-1 in circulating can serve as a possible valuable diagnostic biomarker for cervical cancer and CIN.

Pharmacokinetic and pharmacodynamic analysis of circulating biomarkers of anti-NRP1, a novel antiangiogenesis agent, in two phase I trials in patients with advanced solid tumors.

PURPOSE: MNRP1685A is a monoclonal antibody to neuropilin-1 (NRP1). We evaluated blood-based pharmacodynamic biomarkers of MNRP1685A in two phase I studies to assess exposure/response relationships to inform target dose and regimen selection. EXPERIMENTAL DESIGN: The phase I studies evaluated escalating doses of MNRP1685A as a single agent or in combination with bevacizumab. Plasma placental growth factor (PlGF), VEGF, and circulating NRP1 (cNRP1) were evaluated at multiple time points using meso-scale discovery (MSD) assays and ELISA, respectively. Plasma PlGF was also measured in a phase I/II trial of bevacizumab in metastatic breast cancer (AVF0776). The association between PlGF and MNRP1685A dose was described by a sigmoid E(max) model. cNRP1 and MNRP1685A PK profiles were described using a two-target quasi-steady state (QSS) model. RESULTS: A dose- and time-dependent increase in plasma PlGF and cNRP1 was observed in all patients treated with MNRP1685A. PK/PD analysis showed that bevacizumab and MNRP1685A had an additive effect in elevating PlGF. Predictions based on the two-target QSS model showed that the free drug concentration to maintain greater than 90% saturation of membrane NRP1 (mNRP1) and cNRP1 is about 8 µg/mL. CONCLUSION: These data show that MNRP1685A inhibits the VEGF pathway in humans as assessed by an increase in plasma PlGF. MNRP1685A seems to enhance bevacizumab-mediated VEGF pathway blockade, as showed by an increase in the magnitude of PlGF elevation when combined with bevacizumab. PK/PD analysis of biomarkers in the phase I population allowed identification of doses at which apparent maximal pathway modulation was observed.

Identification of circulating neuropilin-1 and dose-dependent elevation following anti-neuropilin-1 antibody administration.

Neuropilin-1 (NRP1) acts as a co-receptor for class 3 semaphorins and vascular endothelial growth factor and is an attractive angiogenesis target for cancer therapy. In addition to the transmembrane form, naturally occurring soluble NRP1 proteins containing part of the extracellular domain have been identified in tissues and a cell line. We developed ELISAs to study the existence of circulating NRP1 and to quantify it in serum. As measured by ELISAs, circulating NRP1 levels in mice, rats, monkeys and humans were 427 +/- 77, 20 +/- 3, 288 +/- 86 and 322 +/- 82 ng/ml (mean +/- standard deviation; n > or = 10), respectively. Anti-NRP1(B), a human monoclonal antibody, has been selected from a synthetic phage library. A 4-fold increase in circulating NRP1 was observed in mice receiving a single dose of 10 mg/kg anti-NRP1(B) antibody. In rats and monkeys receiving single injections of anti-NRP1(B) at different dose levels, higher doses of antibody resulted in greater and more prolonged increases in circulating NRP1. Maximum increases were 56- and 7-fold for rats and monkeys receiving 50 mg/kg anti-NRP1(B), respectively. In addition to the soluble NRP1 isoforms, for the first time, a approximately 120 kDa circulating NRP1 protein containing the complete extracellular domain was detected in serum by western blot and mass spectrometry analysis. This protein increased more than the putative soluble NRP1 bands in anti-NRP1(B) treated mouse, rat and monkey sera compared with untreated controls, suggesting that anti-NRP1(B) induced membrane NRP1 shedding.