High Neuropilin-1 Expression Research Articles

Peritumoral Neuropilin-1 and VEGF receptor-2 expression increases time to recurrence in hepatocellular carcinoma patients undergoing curative hepatectomy.

To determined Neuropilin-1 (NRP-1) and vascular endothelial growth factor receptor 2 (VEGFR-2) expression in the tumoral and peritumoral tissues of 214 treatment-naïve HCC patients and its correlation with overall survival (OS) and time to recurrence (TTR). NRP-1 and VEGFR-2 expression were examined by tissue microarray and peritumoral hypoxia by pimonidazole staining and angiogenesis by microvessel density (MVD). OS and TTR were evaluated by Kaplan-Meier analysis and log-rank test. Peritumoral NRP-1 and VEGFR-2 expression were significantly higher than that of the tumoral tissue (p < 0.001 for both), and high peritumoral expression of both factors was negatively associated with tumor size (p < 0.001 for both). Patients with high peritumoral expression of both proteins had the longest median OS (>94.0 months) and TTR (>84.0 months). The multivariate Cox proportional hazards analysis revealed that patients with high peritumoral expression of both NRP-1 and VEGFR-2 were more than 4 times less likely to have recurrence (p = 0.004) and more than 10 times likely to survive (p < 0.001). Peritumoral NRP-1 and VEGFR-2 expression is associated with prolonged TTR and extended OS of HCC patients and both may be useful as predictors of surgical outcome of HCC patients and explored as potential therapeutic targets.

Abstract IA17: Targeting placental growth factor/ neuropilin-1 pathway inhibits growth and spread of medulloblastoma

Abstract Background: Medulloblastoma (MB) is the most common pediatric brain tumor and a heterogeneous disease. Several studies have characterized tumor genetic heterogeneity with the hope of identifying targets for personalized therapy. In fact, inhibitors of Sonic Hedgehog (Shh) - a developmental pathway aberrantly activated in a subset of MBs - are currently being evaluated. Unfortunately, mutations and potential developmental adverse effects in the pediatric population can limit these therapies. We propose targeting the tumor stroma as an efficacious alternative treatment option. In particular, we aim to identify and target crucial cancer cell-stromal cell crosstalk signaling that sustains tumor growth and spread. In this context, we have evaluated the effects of blocking signaling of Placental Growth Factor (PIGF) - a member of the vascular endothelial growth factor (VEGF) family - that is dispensable in development but relevant during disease, in the growth and progression of MB. Here, we show the expression of PIGF and its receptor Neuropilin-1 (NRP-1) across primary tumors; dissect the mechanisms that regulate PIGF secretion and downstream signaling; and demonstrate the dramatic efficacy of specific anti-PIGF and anti-NRP-1 blocking antibodies in three MB models in vivo. Methods: Expression of PIGF and NRP-1 was analyzed in a cohort of 32 surgical samples of pediatric MBs by immunohistochemistry, array-comparative genomic hybridization, and deep-gene sequencing. The correlation between NRP-1 expression and 5-year overall survival was evaluated retrospectively in an independent, clinically annotated cohort of 42 MBs. Human orthotopic xenograft (D283-Med; D341-Med) and spontaneous (Smo/Smo) established tumors were treated with anti-PIGF (Thrombogenics or Genentech) or anti-NRP-1 (Genentech) antibodies. Tumor growth, progression and response to therapy was followed by 1) clinical evaluation of posterior fossa symptoms and body weight; 2) whole-body luminescence and blood GLuc levels; 3) small animal MRI; or 4) Optical Frequency Domain Imaging (OFDI). Results: We report that: 1) PIGF is expressed by more than 90% of primary pediatric MBs, regardless of their genetic subtype or histological classification; and high expression of NRP-1 correlates with poor overall survival of patients; 2) MB cells release Shh ligands that stimulate paracrine production of stromal PIGF by cerebellar granule neurons; 3) PIGF signals through NRP-1 - and independent of vascular endothelial growth factor receptor 1 (VEGFR1) - to activate the Erk/MEK pathway and sustain tumor survival; 4) Blockade of PIGF or NRP-1 with specific antibodies causes dramatic regression of MB, decreases spinal metastatic burden, and prolongs survival in in vivo orthotopic and spontaneous mouse models. Conclusion: This work identifies PIGF as the first common target across MB subgroups and provides insight into the critical role of tumor-stroma interactions. The study demonstrates that targeting PIGF/NRP-1 may provide a safe and efficient treatment option for pediatric MBs and lays the ground for evaluation of anti-PIGF therapy in clinical trials. Reference: M. Snuderl et al, Cell, 152(2):1065-76 (2013). Citation Format: Matija Snurdel, Ana Batista, Nathaniel Kirkpatrick, Carmen Almodovar, Lars Riedemann, Teresa Peterson, Napoleone Ferrara, Michael Klagsbrun, Dan G. Duda, Dai Fukumura, Lei Xu, Peter Carmeliet, Rakesh Jain. Targeting placental growth factor/ neuropilin-1 pathway inhibits growth and spread of medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA17.

Abstract 153: High neuropilin-1 expression on monocytes is positively associated with trastuzumab-mediated antibody-dependent cellular cytotoxicity of the HER2-overexpressing breast cancer cell line

Abstract Purpose Neuropilin-1 (Nrp1) was initially characterized as a guide for migrating cells and axons in developing nervous systems and is essential for the precise formation of neurons and vasculature. However, it was recently reported to also play an important role in the immune system. This study aimed to investigate the role of Nrp1 on monocytes in trastuzumab-mediated antibody-dependent cellular cytotoxicity (ADCC) targeting the HER2-positive human breast cancer cell line SKBR3. In addition, we evaluated the gene expression profile of monocytes expressing high and low levels of Nrp1. Methods Peripheral blood mononuclear cells from healthy volunteers were prepared using BD Vacutainer CPT Cell Preparation Tubes. The cells were stained with fluorescein isothiocyanate-conjugated anti-CD14 and allophycocyanin-conjugated anti-Nrp1 antibodies, and Nrp1high and Nrp1low monocyte subsets were sorted using FACSAria. These cells were used as effector cells in a conventional ADCC assay with SKBR3 as target cells and trastuzumab as the antibody. Target cell cytotoxicity was measured using the LDH cytotoxic test kit. The effector cell to target cell ratios of 20:1 and 10:1 were used. Gene expression analysis was performed by Affymetrix GeneChip microarrays. All study protocols were approved by the Ethics Committee for Clinical Research, Kyoto University Hospital, Kyoto, Japan (authorization number G424). Results The LDH cytotoxic test revealed significantly higher target cancer cell cytotoxicity in sorted Nrp1high monocytes than in Nrp1low monocytes (75% vs. 45%, p&amp;lt;0.01). Trastuzumab-mediated ADCC was significantly suppressed by Nrp1 neutralization antibodies in a dose-dependent manner (Control arm 14.3%, Treatment arm 6%; p&amp;lt;0.01). Gene expression analysis revealed that 308 genes were up-regulated in the Nrp1high population, including monocyte-specific chemokine receptor and hematopoietic transcriptional factor. Conclusion These results suggest that determination of Nrp1 expression can be used to classify monocytes as populations with and without cellular cytotoxic activity. Analysis of Nrp1 expression levels in monocytes can therefore be used to identify HER2-positive breast cancer patients for whom trastuzumab therapy will be less effective. Citation Format: Kosuke Kawaguchi, Eiji Suzuki, Masao Kawashima, Masakazu Toi. High neuropilin-1 expression on monocytes is positively associated with trastuzumab-mediated antibody-dependent cellular cytotoxicity of the HER2-overexpressing breast cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 153. doi:10.1158/1538-7445.AM2014-153

High Neuropilin 1 Expression Was Associated With Angiogenesis and Poor Overall Survival in Resected Pancreatic Ductal Adenocarcinoma

Neuropilin 1 (NRP-1) appears to promote angiogenesis by acting as a coreceptor with vascular endothelial growth factor receptor. We correlated NRP-1 expression with microvessel density (MVD) and overall survival (OS) in human pancreatic ductal adenocarcinomas (PDACs). Neuropilin 1 expression was graded semiquantitatively using immunohistochemistry in patients with resected PDAC. Moreover, MVD was determined with an anti-CD31 antibody staining. Expression of NRP-1 was correlated with MVD and clinicopathologic features in patients with PDAC. Overall survival effects of NRP-1 expression were evaluated by multivariate Cox regression and Kaplan-Meier analyses. High NRP-1 expression was associated with advanced Union for International Cancer Control stage (P = 0.046), T stage (P = 0.031), and lymph node invasion (P = 0.045). Microvessel density was significantly higher in the tumors with high NRP-1 expression than that in the tumors with low NRP-1 expression (mean, 13.9 [SD, 9.1] vs 10.2 [SD, 7.2] per high-power field; P = 0.001). The multivariate Cox regression analysis demonstrated that high NRP-1 expression was independently associated with reduced OS (hazard ratio, 2.10; 95% confidence interval, 1.19-3.70). Neuropilin 1 is highly expressed in PDACs, and high expression of NRP-1 is significantly correlated with angiogenesis, advanced tumor-node-metastasis stage, p T stage, node invasion, and poor postoperative OS.

Investigation of a novel biomarker, neuropilin-1, and its application for poor prognosis in acute myeloid leukemia patients.

According to the previous studies, numerous biomarkers impact on the prognosis of acute myeloid leukemia (AML) and the prediction for AML had been improved tremendously in the past decades. However, accurate risk-stratification at diagnosis or prognosis remained difficult. In order to further investigate the prognosis evaluation biomarker, the transcription or expression of neuropilin-1 (NRP-1) in 87 AML patients and 32 non-malignant controls were examined. Real-time quantitative polymerase chain reaction (RT-PCR) and Western blot were used to detect the NRP-1 expression. Clinical data were collected and analyzed for the 87 AML patients. The results indicated that high NRP-1 expression discriminated the complete remission (CR) rate of AML patients (22.12 % vs. 68.04 % for AML, P < 0.01). De novo AML patients tended to express higher NRP-1 proteins than relapsed AML patients. The overall survival (OS) and relapse-free survival (RFS) rate of the high NRP-1 expression patients decreased significantly compared with the low NRP-1 expression patients (P < 0.001). NRP-1 was revealed to be an independent risk factor for OS in AML (P = 0.003). In conclusion, NRP-1 could predict the shorter OS and RFS rate, and also related with the CR response in AML. Therefore, NRP-1 may act as a more aggressive and promising predictor for the poor prognosis of AML.

NRP-1 expression in bladder cancer and its implications for tumor progression

Neuropilin-1 (NRP-1) overexpression has been reported in a variety of human cancers. However, the role of NRP-1 in bladder cancer (BC) remains unclear. The aim of present study was to analyze NRP-1 protein expression in BC tissues and to assess its prognostic significance for BC. NRP-1 messenger ribonucleic acid (mRNA) and protein expression were determined by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunohistochemistry in specimens of primary cancer and their adjacent noncancerous tissues in BC patients. Additionally, NRP-1 protein expression in 139 archived paraffin-embedded BC samples was analyzed by immunohistochemistry and correlated with clinicopathological characteristics and survival. Student's t test, Spearman's rank correlation, Kaplan-Meier plots, and Cox's proportional hazards regression model were used to analyze the data. By qRT-PCR and immunohistochemistry, the levels of NRP-1 mRNA and protein were significantly higher in BC, compared to that in adjacent noncancerous tissues (P<0.001). High expression of NRP-1 was significantly associated with histologic grade (P=0.016) and tumor stage (P=0.001). Multivariate analysis showed that high expression of NRP-1 was an independent prognostic factor for overall survival. Our study suggests that overexpression of NRP-1 may play an important role in the progression of BC, and NRP-1 expression may serve as a biomarker for poor prognosis for BC.

NRP1 Presented in trans to the Endothelium Arrests VEGFR2 Endocytosis, Preventing Angiogenic Signaling and Tumor Initiation

Neuropilin 1 (NRP1) modulates angiogenesis by binding vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. We examined the consequences when VEGFR2 and NRP1 were expressed on the same cell (cis) or on different cells (trans). In cis, VEGF induced rapid VEGFR2/NRP1 complex formation and internalization. In trans, complex formation was delayed and phosphorylation of phospholipase Cγ (PLCγ) and extracellular regulated kinase 2 (ERK2) was prolonged, whereas ERK1 phosphorylation was reduced. Trans complex formation suppressed initiation and vascularization of NRP1-expressing mouse fibrosarcoma and melanoma. Suppression in trans required high-affinity, steady-state binding of VEGF to NRP1, which was dependent on the NRP1 C-terminal domain. Compatible with a trans effect of NRP1, quiescent vasculature in the developing retina showed continuous high NRP1 expression, whereas angiogenic sprouting occurred where NRP1 levels fluctuated between adjacent endothelial cells. Therefore, through communication in trans, NRP1 can modulate VEGFR2 signaling and suppress angiogenesis.

Prognostic impact of neuropilin-1 expression in children with B-lineage acute lymphoblastic leukemia

Background Neuropilins are transmembrane glycoproteins that act as receptors for vascular endothelial growth factors and are involved in the process of tumor angiogenesis. Objective The aim of this work was to study the prognostic value of neuropilin-1 (NRP-1) expression in children with B-lineage acute lymphoblastic leukemia (ALL). Patients and methods We analyzed NRP-1 expression level in 50 newly diagnosed children with B-lineage ALL and in 20 healthy controls using flow cytometry. Expression of NRP-1 at diagnosis was correlated with standard clinical and laboratory prognostic features. Results The present study revealed a highly significant difference in NRP-1 expression between ALL patients and controls; the mean percentage expression of NRP-1 on bone marrow (BM) blasts of B-ALL patients was 36.86% overall. The highest levels of NRP-1 expression were noted in pre-B-ALL (74.04%) followed by early pre-B-ALL (23.55%) patients, and the lowest expression levels were in mature B-ALL (12.06%) patients, with significant differences between the three subtypes. The expression of NRP-1 was significantly associated with higher white blood cell count, bone marrow blast percentage, and serum lactate dehydrogenase levels at diagnosis. Also, there was a significant association between higher NRP-1 expression and both Philadelphia-positive and CD10-negative ALL types. We found significantly higher levels of NRP-1 expression on BM blasts at diagnosis in patients who relapsed or died compared with those who achieved and maintained complete remission; patients with higher NRP-1 expression had significantly shorter overall survival and disease-free survival than did patients expressing NRP-1 on less than 20% of their BM blasts. Conclusion Our findings suggest that NRP-1 is significantly expressed in children with B-lineage ALL, especially with the pre-B phenotype, and has a bad prognostic impact on the course of this disease.Recommendations We recommend the incorporation of NRP-1 as a prognostic marker in children with B-lineage ALL to offer a chance for intensive therapeutic intervention in patients designated as having poor prognosis, and for studying its role as a potential target for antileukemic and antiangiogenic therapy. Egyptian J Haematol 39:-0 © 2014 The Egyptian Society of Haematology.

Prognostic implication of neuropilin-1 upregulation in human nasopharyngeal carcinoma

ObjectiveAs a receptor for both vascular endothelial growth factors and semaphorin, neuropilin-1 (NRP-1) is reported to be up-regulated in cells of several cancers. However, its roles in human nasopharyngeal carcinoma (NPC) are still unclear. Therefore, the goal of this study was to investigate the expression pattern of NRP-1 in NPC tissues, to clarify the clinical significance of NRP-1 expression in NPC as well as the potential prognostic implication of NRP-1 expression.MethodsImmunohistochemistry was performed to detect the expression of NRP-1 in tumor tissue samples from 266 NPC patients. The association of NRP-1 protein expression with the clinicopathological characteristics and the prognosis of NPC were subsequently assessed.ResultsImmunohistochemical analysis showed that 176 of 266 (66.17%) paraffin-embedded archival NPC biopsies showed high expression of NRP-1, but no non-cancerous nasopharyngeal specimens showed positive expression of NRP-1. In addition, high NRP-1 expression was significantly associated with advanced clinical stage (P = 0.02), positive recurrence (P = 0.001) and metastasis status (P = 0.001) of NPC. Moreover, the NPC patients with higher NRP-1 expression had shorter overall survival, whereas patients with lower NRP-1 expression had better survival (P < 0.001). Furthermore, the multivariate analysis indicated that the overexpression of NRP-1 protein was an independent prognostic factor for overall survival (P = 0.001) in NPC patients.ConclusionThese findings suggest for the first time that NRP-1 upregulation may be a novel biomarker for the prediction of advanced tumor progression and unfavorable prognosis in NPC patients who may benefit from alternative treatment strategy and targeted treatment.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1507827881105018.

SY9-5 - Optimization of Anti-Angiogenic Therapy for Gastrointestinal Cancer

ABSTRACT Bevacizumab (BV) was associated with a survival benefit when combined with chemotherapy in metastatic colorectal cancer (mCRC). Patients who received BV plus standard chemotherapy (FOLFOX/XELOX or FOLFIRI/XELIRI) as the initial treatment of mCRC and were then continued on BV with a different chemotherapy regimen as a second-line agent after their cancer progressed lived significantly longer than patients who received chemotherapy alone in the second-line setting, based on the recent results of the ML18147 study. This is the first trial to confirm the efficacy of continuing on a BV-based regimen after disease progression and to prove the necessity of BV continuation for blocking VEGF. Aflibercept, which is a soluble fusion protein consisting of the extracellular domains of human VEGFR-1,2 linked to a human IgG1 Fc portion, binds all VEGF-A isoforms, VEGF-B, and PlGF. Regorafenib is an oral multi-kinase inhibitor that targets VEGFR, PDGFR and KIT kinases. Both drugs showed survival benefit in other phase III trials. Understanding the underlying mechanisms of resistance to anti-angiogenic therapy will facilitate the optimization of the use of these three active agents in the treatment of mCRC. The global phase III AVAGAST trial did not demonstrate a survival benefit of BV when it was administered with a chemotherapy regimen of capecitabine plus cisplatin (XP) for metastatic gastric cancer. Low tumor neuropilin-1 expression was associated with shorter overall survival (OS) in placebo-treated patients. The addition of BV seems to produce a survival benefit; patients with low tumor neuropilin-1 expression had OS treatment hazard ratio values that were better than those with high neuropilin-1 expression. A similar effect was observed in the NO16966 mCRC study (FOLFOX/XELOX ± BV). In addition, lower baseline plasma VEGF-A was correlated with longer OS. Additional data from the AVAGAST trial revealed different survival outcomes based on the histological and regional subtype of gastric cancer. Although European patients with diffuse type and distal non-diffuse type gastric cancer had the worst prognosis for survival; these individuals were most likely to benefit from the addition of BV to XP. Different approaches to the development of novel agents for each histologic type of gastric cancer treatment are required.

Bevacizumab in Combination With Chemotherapy As First-Line Therapy in Advanced Gastric Cancer: A Biomarker Evaluation From the AVAGAST Randomized Phase III Trial

The AVAGAST study showed that adding bevacizumab to chemotherapy in patients with advanced gastric cancer improves progression-free survival and tumor response rate but not overall survival. To examine the hypothesis that angiogenic markers may have predictive value for bevacizumab efficacy in gastric cancer, AVAGAST included a prospective, mandatory biomarker program. Patients with previously untreated, locally advanced or metastatic gastric cancer were randomly assigned to bevacizumab (n = 387) or placebo (n = 387) in combination with chemotherapy. Blood and tumor tissue samples were collected at baseline. Prespecified biomarkers included plasma vascular endothelial growth factor-A (VEGF-A), protein expression of neuropilin-1, and VEGF receptors-1 and -2 (VEGFR-1 and VEGFR-2). Correlations between biomarkers and clinical outcomes were assessed by using a Cox proportional hazards model. Plasma was available from 712 patients (92%), and tumor samples were available from 727 patients (94%). Baseline plasma VEGF-A levels and tumor neuropilin-1 expression were identified as potential predictors of bevacizumab efficacy. Patients with high baseline plasma VEGF-A levels showed a trend toward improved overall survival (hazard ratio [HR], 0.72; 95% CI, 0.57 to 0.93) versus patients with low VEGF-A levels (HR, 1.01; 95% CI, 0.77 to 1.31; interaction P = .07). Patients with low baseline expression of neuropilin-1 also showed a trend toward improved overall survival (HR, 0.75; 95% CI, 0.59 to 0.97) versus patients with high neuropilin-1 expression (HR, 1.07; 95% CI, 0.81 to 1.40; interaction P = .06). For both biomarkers, subgroup analyses demonstrated significance only in patients from non-Asian regions. Plasma VEGF-A and tumor neuropilin-1 are strong biomarker candidates for predicting clinical outcome in patients with advanced gastric cancer treated with bevacizumab.

Targeting Neuropilin 1 as an Antitumor Strategy in Lung Cancer

Neuropilin 1 (NRP1) is a mediator of lung branching and angiogenesis in embryonic development and angiogenesis in cancer. The role of NRP1 in cancer progression is not fully elucidated. We investigated the role of NRP1 in cancer invasion and tumor angiogenesis, its signaling pathways, prognostic significance, and therapeutic implications. Sixty patients with non-small cell lung cancer (NSCLC) were studied. NRP1 mRNA expression was measured using real-time quantitative reverse-transcription PCR. NRP1 and cancer cell invasion, angiogenesis, and signaling pathways were studied using NRP1 stimulation by vascular endothelial growth factor 165 (VEGF(165)) and NRP1 inhibition by small interfering RNAs (siRNA), soluble NRP1 (sNRP1), and NRP1-inhibition peptides. The NRP1-inhibition peptides were identified using a phage display peptide library. NSCLC patients with high expression of NRP1 had shorter disease-free (P = 0.0162) and overall survival (P = 0.0164; log-rank test). Multivariate analyses showed NRP1 is an independent prognostic factor in overall (HR, 2.37, 95% CI = 1.15 to 4.9, P = 0.0196) and disease-free survival [hazard ratio (HR), 2.38; 95% confidence interval (95% CI), 1.15-4.91; P = 0.0195] of NSCLC patients. Knockdown of NRP1 suppressed cancer cell migration, invasion, filopodia formation, tumorigenesis, angiogenesis, and in vivo metastasis. NRP1 signaling pathways involved VEGF receptor 2 and phosphoinositide-3-kinase (PI3K) and Akt activation. Two potent synthetic anti-NRP1 peptides, DG1 and DG2, which block NRP1 signaling pathways and suppress tumorigenesis, cancer invasion, and angiogenesis, were identified. NRP1 is a cancer invasion and angiogenesis enhancer. NRP1 expression is an independent predictor of cancer relapse and poor survival in NSCLC patients. NRP1 plays a critical role in tumorigenesis, cancer invasion, and angiogenesis through VEGF, PI3K, and Akt pathways. NRP1 may have potential as a new therapeutic target in NSCLC.

Neuropilin-1 promotes unlimited growth of ovarian cancer by evading contact inhibition

Objective. Neuropilin-1 (NRP-1) is a receptor for both semaphorin and vascular endothelial growth factor and is up-regulated in a variety of human cancers. While there are some reports of NRP-1 expression in ovarian neoplasm, those results differ in pattern of its expression and its role in ovarian cancer is still unclear. We sought to investigate the expression pattern and role of NRP-1 in ovarian cancer. Methods. NRP-1 expression was analyzed with eighty-seven ovarian tissue samples by immunohistochemistry and four ovarian cell lines by quantitative RT-PCR and Western blotting. To detect its molecular role in ovarian cancer, WST-1 assay, invasion assay and soft agar assay were performed with or without NRP-1 suppression by the introduction of short hairpin RNAs. Results. NRP-1 expression was found to be enhanced in ovarian cancer compared with ovarian surface epithelium (OSE), benign adenoma and tumors of low malignant potential. In vitro, NRP-1 expression was augmented threefold during malignant transformation of OSE cells with oncogene ras, suggesting an association between NRP-1 and oncogenesis. Suppression of NRP-1 reduced cell proliferation in a dense state, indicating that persistently high expression of NRP-1 in ovarian cancer enhances proliferation through evasion of contact inhibition. Suppression of NRP-1 also decreased cell growth in soft agar and invasion to the extracellular matrix in vitro. Conclusions. These results suggest that NRP-1 is not only associated with oncogenesis, but also with ovarian cancer malignancy, and this molecule is a targeting candidate for the treatment of ovarian malignancies.

Correlation of neuropilin-1 overexpression to survival in acute myeloid leukemia

Neuropilin-1 (NRP-1), a vascular endothelial growth factors and semaphorin receptor functioning as mediator of angiogenesis and neuronal guidance, is expressed by various solid tumors. The importance of NRP-1 in hematological malignancies such as acute myeloid leukemia (AML) remains to be elucidated. Therefore, we determined NRP-1 expression by immunohistochemical analysis of bone marrow biopsies of patients with newly diagnosed, untreated AML. The expression of NRP-1 was significantly increased in AML patients (n = 76; median 12.9 arbitrary units (a.u.)) as compared with controls (n = 38; median 2.75 a.u.). Survival was significantly poorer in patients with high (> median) versus low (< or = median) NRP-1 expression levels with 5-year overall survival rates of 16.9 versus 49.6% (P = 0.050). In conclusion, our data provide evidence of increased NRP-1 expression in AML with significant correlation to survival. Thus, NRP-1 might constitute a promising target for antileukemic and antiangiogenic treatment strategies in AML.

Neuropilin-1 expression in adenocarcinoma and squamous cell carcinoma of the lung is differentially regulated by hypoxia

17152 Background: Neuropilin-1 (NRP-1) is an isoform-specific receptor for VEGF165 and semaphorin3A, initially discovered on migrating neurons. NRP-1 expression has been reported on a number of tumour cell lines in the absence of the other VEGF receptors, where it mediates survival signals. In this study we examined the regulation of NRP-1 by hypoxia, its effect on survival in a panel of lung cancer cell lines and its potential as a biomarker in retrospective human lung tumours. Methods: A549, SK-MES1, H460 and H647 cells were grown in serum depleted media (0.5%) in normoxic or hypoxic (0.1% O2) conditions and screened for NRP-1 expression by western and immunocytochemistry analysis. Cell survival and apoptosis was determined using BrdU and Annexin-V/PI staining respectively following treatment with an antibody to the extracellular NRP-1 domain. A panel of 100 retrospective resected lung tumours and matched normal samples were stained for NRP-1 expression by immunhistochemistry. Results: A549, SKMES-1 and H647 cell lines all expressed NRP-1 and displayed reduced survival following treatment with NRP-1 antibody (1ug/ml) compared to controls (A549 46%, SKMES-1 61%, H647 53%). H460 did not express NRP-1 and no survival inhibition was seen in the cell line (104%). Reduced survival was accompanied by increased apoptosis in all NRP-1 positive cell lines. Hypoxia strongly increased NRP-1 expression in the A549 adenocarcinoma (AC) cell line, while NRP-1 was decreased in SKMES-1 squamous cell carcinoma (SCC) following hypoxia. Neutralisation of NRP-1 had a greater effect in A549 cells under hypoxia (37%), with a lesser effect in SKMES-1 cells (82%). Western analysis of matched frozen normal and lung cancer biopsies showed NRP-1 overexpression in AC and decreased expression in SCC relative to normal. High NRP-1 expression was confirmed in AC and large cell carcinoma by immunohistochemistry, relative to normal. However, SCCs had a lower level of NRP-1 staining, supporting the results by western analysis and following hypoxia in vitro. Conclusions: These results implicate NRP-1 as an important survival pathway in lung cancer. Hypoxia differentially regulated NRP-1 mediated survival implicating this pathway as a potential therapeutic strategy in AC. No significant financial relationships to disclose.