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Clinical Cancer Research 13, 3499-3506, June 15, 2007. doi: 10.1158/1078-0432.CCR-07-0237
© 2007 American Association for Cancer Research
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Human Cancer Biology

Prominent Microvascular Proliferation in Clinically Aggressive Neuroblastoma

Radhika Peddinti1, Rana Zeine2, Dragos Luca5, Roopa Seshadri1, Alexandre Chlenski2, Kristina Cole4, Bruce Pawel4, Helen R. Salwen2, John M. Maris4 and Susan L. Cohn3

Authors' Affiliations: 1 Department of Pediatrics, Children's Memorial Hospital; 2 Robert H. Lurie Comprehensive Cancer Center, Northwestern University; 3 Institute for Molecular Pediatric Sciences, University of Chicago, Chicago, Illinois; 4 Department of Pediatrics, Children's Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; and 5 Department of Pathology, Children's Hospitals and Clinics of Minnesota, Minneapolis, Minnesota

Requests for reprints: Susan L. Cohn, Clinical Sciences, Institute for Molecular Pediatric Sciences, University of Chicago, 5841 Maryland Avenue, MC 4060, Room N114, Chicago, IL 60637. Phone: 773-702-2571; Fax: 773-834-1329; E-mail: scohn{at}peds.bsd.uchicago.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Tumor vasculature is disorganized and glomeruloid microvascular proliferation (MVP) has been identified as a poor prognosticator in some adult cancers. To determine the clinical significance of MVP, including glomeruloid MVP in neuroblastoma, we initially examined vessel architecture in tumor sections from 51 children diagnosed at Children's Memorial Hospital (CMH) and subsequently evaluated 154 neuroblastoma tumors on a tissue microarray constructed at Children's Hospital of Philadelphia (CHOP).

Experimental Design: H&E sections were examined for the presence of structurally abnormal vessels and further characterized by immunostaining for CD31 and von Willebrand factor to highlight endothelial cells and {alpha}-smooth muscle actin for pericytes. Tumors with thickened walls containing a complete layer of hypertrophic endothelial cells plus additional layers of vascular mural cells were classified as MVP positive. Associations between MVP and established clinicopathologic features and outcome were assessed.

Results: In both series, MVP was significantly associated with Schwannian stroma-poor histology (CMH, P = 0.008; CHOP, P < 0.001) and decreased survival probability (CMH, P = 0.017; CHOP, P = 0.014). In the CHOP series, MVP was associated with high-risk group classification (P < 0.001), although this association was not seen in the smaller CMH cohort.

Conclusions: The association between MVP and poor outcome provides further support for the concept that angiogenesis plays an important role in determining the biological behavior of neuroblastoma tumors. Our results also indicate that angiogenesis is regulated differently in Schwannian stroma-rich versus stroma-poor neuroblastoma tumors. Further studies investigating the activity of angiogenic inhibitors in children with clinically aggressive stroma-poor neuroblastoma are warranted.

Neuroblastoma, the most common extracranial solid tumor in children, is unique among pediatric cancers for its broad spectrum of clinical behavior (1). This tumor is composed of two main cell types, neuroblastic/ganglion cells and Schwann cells, and the amount of Schwannian stroma strongly affects prognosis (2). Favorable outcome is associated with tumors with abundant Schwannian stroma. In addition to tumor histology, various genetic and biological factors have been shown to correlate with outcome. However, the cellular mechanisms that underlie the clinical variability observed in neuroblastoma remain largely unknown. It is widely accepted that malignant solid tumors must acquire a new blood supply for their progressive growth to a clinically relevant size and for metastasis (3, 4). In contrast to normal vessels, tumor vasculature tends to be disorganized with vessels that are dilated and tortuous and have uneven diameters and excessive branching (5). Microvascular proliferation (MVP) ranges from slight to extreme degrees. Focal proliferative buddings of endothelial cells resembling a renal glomerulus [glomeruloid MVPs (GMP)] have been described in some types of adult cancers, and several studies have shown an association between the presence of GMP and shorter survival (6–8). Wesseling et al. (9) have shown by electron microscopy that the process of MVP involves not only endothelial cell proliferation but also a significant contribution from pericytes. In the WHO grading of astrocytic neoplasms, GMP is a criterion for increasing the tumor grade (10). However, the clinical significance of MVP has not been evaluated previously in neuroblastoma or other pediatric cancers.

There is significant evidence that angiogenesis contributes to the aggressive behavior of neuroblastoma tumors. In retrospective studies, high vascular density has been correlated with poor clinical outcome (11, 12). Furthermore, high levels of angiogenesis activators have been detected in clinically aggressive neuroblastoma tumors (13). Conversely, increased levels of endogenous inhibitors of angiogenesis are present in Schwannian stroma-rich tumors that are associated with favorable outcome (14, 15). Preclinical studies have also shown that neuroblastoma growth can be inhibited by agents that target blood vessels (16–20). However, no correlation between vascular variables and survival was seen in a study reported by Canete et al. (21). The conflicting results most likely reflect differences in techniques used to measure vessel number, a difficulty encountered in reconciling the results of studies of other solid tumors, such as breast cancer (22).

Because the architecture of tumor blood vessels is distinct from normal vasculature, we hypothesized that structurally abnormal vessels would be identified in neuroblastoma tumors that are highly angiogenic and clinically aggressive. To test this hypothesis, we initially evaluated vessel structure in neuroblastoma tumors from 51 children diagnosed at Children's Memorial Hospital (CMH) in Chicago. In this cohort, MVP was significantly associated with stroma-poor histology and decreased survival. These findings were confirmed using a neuroblastoma tissue microarray (TMA) constructed at the Children's Hospital of Philadelphia (CHOP) that contained 154 tumor samples. Our results indicate that angiogenesis plays a critical role in neuroblastoma pathogenesis and suggest that the process is regulated differentially in Schwannian stroma-rich versus stroma-poor tumors.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and tumor specimens. The CMH patients were selected based on the availability of adequate full tissue sections from the primary tumor. Sections from 51 primary neuroblastoma tumors or ganglioneuromas were obtained from CMH at the time of diagnosis, before administration of chemotherapy. Patients were diagnosed between 1986 and 2005. Medical records were reviewed to obtain information about patient age, sex, tumor stage, histology, MYCN gene status, and outcome. The CMH Institutional Review Board approved this study.

Histology sections of 154 different neuroblastoma tumors on a TMA constructed at CHOP were also examined (one to four cores per tumor). All the samples used were from the initial biopsy or surgery before administration of chemotherapy. The cores were linked to clinical information, including patient age, stage, sex, histology, MYCN status, and outcome. The CHOP Institutional Review Board approved this study.

For both patient cohorts, tumors were staged according to the International Neuroblastoma Staging System (23). MYCN gene status was determined in the Children's Oncology Group Neuroblastoma Reference Laboratory using fluorescence in situ hybridization (24). Tumors were histologically classified as favorable or unfavorable histology according to the criteria described by Shimada et al. (2).

Tissue microarray. The TMA was constructed from formalin-fixed, paraffin-embedded archival tissue specimens accessioned at CHOP from 1974 to 2004. All tumors were reviewed by a pediatric pathologist (B. Pawel). One to four samples (0.6 mm cores) of representative tumor tissue from each case and normal control tissues were included using a manual arrayer (Beecher Instruments, Inc.).

Histologic evaluation. H&E-stained slides were examined histologically for Schwannian stroma and neuroblast differentiation. The entire tissue section was evaluated from one block for each of 51 tumors from the CMH series. Of these, 33 were neuroblastomas, 13 were ganglioneuroblastomas of which 9 were intermixed and 4 were nodular, and 5 were ganglioneuromas. One to four cores sampled on the TMA were assessed for 154 tumors adequate for judging vascular architecture, of which 118 were neuroblastomas, 24 were ganglioneuroblastomas of which 13 were intermixed and 11 were nodular, and 12 were ganglioneuromas. Without knowledge of the patient's stage, MYCN status, or clinical course, the tumors were evaluated for morphology of blood vessels, hemorrhage, and necrosis. Blood vessels were classified into two types according to the vessel wall structure by two independent reviewers (R. Peddinti and R. Zeine). On H&E, sections showing vessels with thickened walls containing a complete layer of hypertrophic endothelial cells plus additional layers of vascular mural cells were classified as MVP positive. The degree of MVP varied from slight to GMP, which was defined as florid proliferation of small vessels with the formation of complex glomeruloid structures (25). Tumor sections that contained only thin walled vessels with no more than one layer of flat, spindle-shaped endothelial cells were classified as MVP negative. Special care was taken to avoid confusing tangentially cut vessels. MVP noted within lymph nodes or in intense inflammatory infiltrates was excluded from analysis. Arteries and veins were also excluded from analysis.

Immunohistochemistry. The structure of the tumor vasculature was further evaluated by examination of adjacent sections from all CMH cases stained by immunohistochemistry for endothelial cell marker, CD31, and for pericytes with {alpha}-smooth muscle actin ({alpha}-SMA). The TMA was stained for von Willebrand factor to highlight endothelial cells. Sections (4 µm) were deparaffinized and heat-induced antigen retrieval was carried out in a steamer for 20 min in citrate buffer (Target Retrieval Solution (pH 6), DakoCytomation) for von Willebrand factor and CD31 and for 20 min in Target Retrieval Solution (pH 9; DakoCytomation) for {alpha}-SMA. Subsequently, slides were immersed in peroxidase block solution (DakoCytomation) and then incubated for 1 h at room temperature with the following primary antibodies: monoclonal mouse anti-human CD31 (clone JC70A, DakoCytomation) at 1:40 dilution, polyclonal rabbit anti-human von Willebrand factor (DakoCytomation) at 1:40 dilution, and monoclonal mouse anti-human {alpha}-SMA (clone 1A4, DakoCytomation) at 1:50 dilution. The EnVision+/Horseradish Peroxidase antimouse and antirabbit detection systems (DakoCytomation) were used to visualize antibody binding sites with 3,3'-diaminobenzidine (DakoCytomation) as a chromogen. Sections were counterstained with Gill's hematoxylin.

For hypoxia-inducible factor (HIF)-1{alpha}, antigen retrieval was done in pH 6 citrate buffer in a pressure cooker. Primary mouse monoclonal antibody to HIF-1{alpha} (ESEE122; Novus Biologicals, Abcam) was visualized using the Catalyzed Signal Amplification System according to the manufacturer's instructions (DakoCytomation).

Immunofluorescence. Sections were deparaffinized and heat-induced antigen retrieval was carried out in a steamer for 20 min in citrate buffer pH 6. Nonspecific staining was blocked by preincubation in PBS containing 10% donkey serum. Primary antibodies for anti-CD31 (platelet/endothelial cell adhesion molecule 1, M-20, Santa Cruz Biotechnology) and anti-{alpha}-SMA (clone 1A4, DakoCytomation) were used at 1:100 dilution. Immunocomplexes were visualized with corresponding FITC-donkey anti-mouse and R-PE donkey anti-goat–labeled secondary antibody (Jackson ImmunoResearch Laboratories).

Statistical analysis. Associations of MVP with various known clinicopathologic prognostic factors of neuroblastoma were analyzed using the {chi}2 or Fisher's exact tests. All degrees of MVP, including GMP, were considered MVP positive. CMH and CHOP cohorts were analyzed separately due to smaller size of the tissue sections on the TMA from the CHOP series. Ganglioneuromas were included only in analyses related to stroma histology. Patients were stratified into two risk groups based on stage, age, and MYCN status. The non–high-risk group included patients with nonamplified MYCN stage 1, 2, and 3 tumors and infants with stage 4 and 4s neuroblastoma that lacked MYCN amplification. Patients with stage 3 MYCN-amplified tumors and children older than 1 year of age with stage 4 disease were classified as high risk. Survival estimates were described using the Kaplan-Meier method, and survival curves were compared among clinical and biological subgroups using the log-rank test. Five-year overall survival estimates are reported with corresponding SE. Cox proportional hazards regression analysis was used to test the association between risk factors and survival. For the CMH cohort, sample size limited the analyses to single predictor models. The best two-predictor model was selected based on the score statistic for the CHOP cohort. Statistical analyses were conducted using SAS statistical software version 9.1 and S-Plus version 6.2.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Clinical and biological characteristics of the patient cohorts. Tables 1 and 2 list the clinical characteristics of CMH and CHOP cohorts. In the CMH series, 15 patients had high-risk disease and 31 patients had non–high-risk disease. Overall survival was 82% ± 6 with a median follow-up time of 5 years and 10 months. The CHOP series consisted of 38 high-risk and 104 non–high-risk patients, and the overall survival was 84% ± 3.


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  		Table 1. Clinical and biological characteristics of the CMH cohort

 

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  		Table 2. Clinical and biological characteristics of the CHOP cohort

 
MVP is associated with widely disseminated disease and high-risk group classification. Table 3 shows the relationships between MVP and stage, age, MYCN status, and risk group classification. In the CHOP series, statistically significant associations between MVP and stage (P = 0.008), MYCN amplification (P = 0.006), and risk group classification (P < 0.001) were seen. However, MVP did not correlate with age (P = 0.82). In the smaller CMH series, the correlation between MVP and stage was not significant (P = 0.6), and MVP was seen in only 5 of the 10 MYCN–amplified tumors. Interestingly, four of these five patients have died, whereas there were no deaths in the subset of patients with MYCN-amplified tumors that lacked MVP (n = 5).


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  		Table 3. Associations between clinical factors and MVP in CMH and CHOP cohorts

 
MVP is associated with Schwannian stroma-poor histology. The presence of MVP was significantly associated with Schwannian stroma-poor histology in the CMH series (P = 0.008) and CHOP series (P < 0.001). All of the Schwannian stroma-dominant ganglioneuromas (CMH, n = 5; CHOP, n = 12) had thin-walled vessels with no evidence of MVP (Fig. 1A-C ). Immunohistochemistry showed a single layer of CD31-positive endothelial cells (Fig. 1B) and good coverage by {alpha}-SMA–positive pericytes (Fig. 1C). Similarly, blood vessels in the Schwannian stroma-rich ganglioneuroblastoma intermixed tumors were thin walled and MVP was not seen (CMH, n = 6; CHOP, n = 13; Fig. 1D-F and Fig. 2, left ).


Figure 1
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  		Fig. 1. Representative sections of human neuroblastoma (NB), ganglioneuroblastoma (GNB), and ganglioneuroma (GNR) stained with H&E (A, D, G, J, M, and P), CD31 (B, E, H, K, N, and Q), and {alpha}-SMA (C, F, I, L, O, and R). Blood vessels are thin walled in Schwannian stroma-dominant ganglioneuroma (A-C) and Schwannian stroma-rich ganglioneuroblastomas (D-F). In contrast, blood vessels are structurally abnormal and show MVP in Schwannian Stroma-poor regions of nodular ganglioneuroblastoma (G-I) and neuroblastoma (J-R). CD31 and {alpha}-SMA immunostaining highlight the endothelial cells and pericytes, respectively. Magnifications, x400 (A-Q) and x600 (R).

 

Figure 2
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  		Fig. 2. Sections of human ganglioneuroblastoma (left) and neuroblastoma (right) double stained for CD31 (red) and {alpha}-SMA (green). Thin vessels showing flat, spindle-shaped endothelial cells and one layer of pericytes (left). MVP seen in vessels with enlarged endothelial cells and multiple layers of {alpha}-SMA–positive vascular mural cells (right). Magnification, x100 (left and right).

 
In contrast, MVP was detected in 65% of the Schwannian stroma-poor tumors, and the incidence was inversely correlated with neuroblast differentiation. In the CMH series, all four (100%) undifferentiated tumors had MVP. MVP was seen in 6 of the 11 (54.5%) differentiating neuroblastomas (Fig. 1J-L) and11 of the 16 (68.7%) poorly differentiated tumors (Fig. 1M-O). Of the 118 neuroblastoma tumors analyzed on the TMA, all eight (100%) of the undifferentiated neuroblastoma had evidence of MVP. Fifty-three of the 82 (64.6%) poorly differentiated tumors and 17 of the 28 (60.7%) differentiating neuroblastomas had MVP. Similarly in the CMH series, all four (100%) undifferentiated tumors had MVP. MVP was seen in 6 of the 11 (54.5%) differentiating neuroblastomas (Fig. 1J-L) and 11 of the 16 (68.7%) poorly differentiated tumors (Fig. 1M-O). Both immunohistochemical and immunofluorescent staining with {alpha}-SMA revealed at least one but sometimes multiple layers of pericytes (Fig. 1L and O and Fig. 2, right). In the CMH series, GMP was extensive in seven of the differentiating and poorly differentiated neuroblastomas, and staining with {alpha}-SMA revealed a prominent pericytic component (Fig. 1L and O). In undifferentiated neuroblastoma tumors, a slightly different pattern of vasculature was noted characterized by continuous networks of MVP (Fig. 1P-R). Tiny microvessels were lined by endothelial cells that were small and weakly positive for CD31 (Fig. 1Q). {alpha}-SMA–positive pericytes were also detected in close proximity to the microvessels in undifferentiated neuroblastoma tumors (Fig. 1R).

The difference in the vascular architecture was most prominent in the composite ganglioneuroblastomas of the nodular type. There were five ganglioneuroblastoma nodular tumors in the CMH series for which both stroma-rich and stroma-poor regions were analyzed. Of the 10 nodular ganglioneuroblastomas in the CHOP series, cores from 6 tumors had only stroma-rich tissue, whereas cores from 4 were stroma-poor areas. In both series, vessels were MVP negative in Schwannian stroma-rich regions and MVP positive in Schwannian stroma-poor areas (Fig. 1G-I). Pericytes were present but provided poor coverage in the vessels with MVP as highlighted by the anti-{alpha}-SMA antibody.

MVP is spatially related to regions of necrosis. Interestingly, necrosis was not detected in tumors that lacked MVP, whereas 12 of 17 (70%) of neuroblastoma tumors with MVP exhibited frank necrosis in close proximity to the abnormal vessels. The neuroblasts surrounding the necrotic areas and leading up to the MVP exhibited a pseudopalisading pattern (Fig. 3A ). In nodular ganglioneuroblastomas, frank necrosis was not observed, although hypocellular and acellular islands of neuropil were noted in the vicinity of MVP within the Schwannian stroma-poor regions. Further evidence of hypoxic-ischemic changes, including nuclear pyknosis, hypereosinophilic cytoplasm, and ghost cells, were also noted in the populations of differentiating neuroblasts closest to the abnormal vessels. In response to hypoxia, tumor cells commonly adapt by up-regulating HIF-1{alpha}, a major regulator of the proangiogenic factor vascular endothelial growth factor. To investigate whether the HIF-1{alpha}–dependent mechanism had a role in the induction of MVP, we stained representative sections from four neuroblastoma tumors that had extensive necrosis for HIF-1{alpha}. In all four neuroblastoma tumor sections, there was focal up-regulation of nuclear HIF-1{alpha} positivity in the neuroblasts intervening between necrosis and MVP (Fig. 3B).


Figure 3
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  		Fig. 3. Sections of human neuroblastoma stained with H&E (A) and HIF-1{alpha} (B). Blood vessels with MVP are seen adjacent to the areas of necrosis (A). Up-regulation of HIF-1{alpha} is seen in the area of necrosis with adjoining blood vessels exhibiting MVP. N, necrosis; T, tumor cells; V, blood vessels. Magnifications, x50 (A) and x600 (B).

 
MVP is associated with poor outcome. MVP correlated strongly with poor outcome in both cohorts. In the CMH series, MVP was seen in the tumors from all seven children who died from disease. The 5-year survival in the cohort of children with tumors that lacked MVP (n = 20) was 100% (Fig. 4B ). In contrast, a survival rate of 70% ± 9.6 was seen in the subset of patients (n = 26) with tumors with MVP (P = 0.017). Of the 12 patients with stage 4 disease, 5 died, and all had tumors with evidence of MVP. Similarly, in the CHOP series, 18 of the 22 (81.8%) patients who died had tumors with MVP (Fig. 4D). The 5-year survival in the groups with (n = 83) and without (n = 60) MVP was 77.9% ± 4.7 versus 94.7% ± 2.9, respectively (P = 0.014). Of the 27 patients with stage 4 disease in the CHOP series, there were 10 deaths, and 9 (90%) of these patients had tumors with MVP.


Figure 4
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  		Fig. 4. Kaplan-Meier analysis of the two cohorts of neuroblastoma patients. A, overall survival in CMH series. B, survival by MVP in CMH series (P = 0.017). C, overall survival in CHOP series. D, survival by MVP in CHOP series (P = 0.014).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Tumor vessels are frequently disorganized and tortuous due to dysregulated angiogenesis. Structural abnormalities, such as GMP, are characteristic of an angiogenic tumor phenotype and are associated with poor prognosis in some types of cancer (6–8). However, much less is known about the role of angiogenesis in pediatric cancers, and to our knowledge, this is the first study evaluating the clinical significance of MVP in neuroblastoma. We initially examined tumor MVP in tumor sections from 51 patients from a single institution and then confirmed our findings using a TMA that contained 154 tumor samples. Other series have shown an overall survival in patients with neuroblastoma in the United States to be 57% (51-63%; ref. 26). Thus, the overall survival of both study groups was more favorable than expected (CMH series, 82% ± 6; CHOP series, 84% ± 3), most likely due to a bias away from high-risk patients for whom tumor sections were not available because the diagnosis was based on bone marrow studies. However, as expected, worse outcome was associated with established unfavorable prognostic clinical and biological factors, including advanced stage disease and MYCN amplification. In both study cohorts, we also found that MVP was significantly associated with decreased survival.

Angiogenesis has been extensively studied in various adult cancers, and it is well established that microvessel density is associated with prognosis in many types of neoplasms (27–29). Much less is known about angiogenesis in pediatric cancers, but there is evidence that angiogenesis also plays a critical role in the pathogenesis of neuroblastoma and Wilms' tumors (11–13). We showed previously that high vascular index correlated with MYCN amplification, metastases, and poor outcome (11). Advanced-stage neuroblastoma has also been associated with high levels of angiogenic stimuli and {alpha}vß3 and {alpha}vß5 integrins, both markers of active angiogenesis (13, 30). However, vascular variables were not predictive of survival in a cohort of neuroblastoma patients analyzed by Canete et al. (21). These investigators used a computerized system to assess CD34-stained sections in the richest vascular area. The contrasting studies highlight the effect different techniques can have in quantifying vessel number and show how difficult obtaining reproducible results can be. In our study, all degrees of MVP were identified readily in H&E as well as immunostained neuroblastoma tumor sections, first on the CMH samples and then reproduced in the smaller TMA cores, suggesting that assessing tumors for MVP may prove to be an effective method for identifying an angiogenic phenotype in neuroblastoma tumors.

In glioblastoma multiforme, where glomeruloid microvessel proliferations and MVP were first described, a spatial relationship between GMP and necrosis is observed (31, 32). Furthermore, the hypoxic conditions associated with necrosis leads to up-regulation of HIF-1{alpha}, a major regulator of the proangiogenic factor vascular endothelial growth factor, and chaotic blood vessel growth (33). In our study of neuroblastoma tumors, we also found a spatial relationship between MVP and necrosis, and nuclear HIF-1{alpha} expression in neuroblasts closest to the necrotic areas in the four tumor sections we evaluated was seen (Fig. 3B). Recently, Holmquist-Mengelbier et al. (34) have reported an association between high levels of HIF-2{alpha} expression and poor outcome in neuroblastoma. However, in contrast to our results, only low to undetectable HIF-1{alpha} staining was seen in well-vascularized areas of the tumors. The reasons for the conflicting results are unclear but may indicate that different HIF-{alpha} proteins may be present in highly vascularized versus necrotic regions of the tumor. Similar to other types of cancers, HIF-1{alpha} stimulates vascular endothelial growth factor mRNA and protein expression in neuroblastoma cells (35). Interestingly, recent studies indicate that serum-derived growth factors, insulin-like growth factor-1, and high levels of brain-derived neurotrophic factor and its tyrosine kinase receptor TrkB, also stimulate HIF-1{alpha} and vascular endothelial growth factor expression in neuroblastoma cells (36).

With the exception of composite ganglioneuroblastomas (nodular), high rates of survival are associated with neuroblastic tumors with abundant Schwannian stroma, suggesting that Schwann cells are capable of influencing neuroblastoma tumor biology (37). In support of this hypothesis, Schwann cells are known to produce neurotrophic factors as well as a spectrum of angiogenesis inhibitors (14, 15, 38). Furthermore, Schwann cell–conditioned medium is capable of inducing neuroblastoma differentiation in vitro (39), and we have shown that infiltrating mouse Schwann cells can induce differentiation and inhibit angiogenesis in human neuroblastoma xenografts in vivo (40). The current study indicates that vessel structure is also influenced by Schwann cells as MVP was not detected in any of the Schwannian stroma-dominant ganglioneuromas or Schwannian stroma-rich intermixed ganglioneuroblastomas. In contrast, structurally abnormal blood vessels were seen in 65% of the stroma-poor neuroblastomas. Similarly, in the nodular ganglioneuroblastomas, prominent MVP was seen in the Schwannian stroma-poor areas, whereas only thin vessels were present areas of the tumor that were Schwannian stroma rich.

Recently, significantly increased survival has been reported in patients with colon, breast, and lung cancer following treatment with antiangiogenic agents in combination with chemotherapy (41, 42). Emerging evidence suggests that antiangiogenic therapy can normalize blood vessel architecture leading to more efficient drug delivery to the tumor (43, 44). Our results correlating MVP with poor survival in children with neuroblastoma provide further rationale for using antiangiogenic strategies in this cohort of patients. Additional clinical studies testing the activity of angiogenic inhibitors alone or in combination with cytotoxic therapy in children with clinically aggressive neuroblastomas are warranted.


   Footnotes
 
Grant support: Clinical Oncology Research Training grant T32 CA079447, NIH grants R01 NS049814 and P01 CA97323, the Neuroblastoma Children's Cancer Society, Friends for Steven Pediatric Cancer Research Fund, the Elise Anderson Neuroblastoma Research Fund, Neuroblastoma Kids, Alex's Lemonade Stand Foundation, and The Robert H. Lurie Comprehensive Cancer Center, NIH, National Cancer Institute Core grant 5P30CA60553.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 2/ 2/07; revised 3/14/07; accepted 3/29/07.


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 Materials and Methods
 Results
 Discussion
 References
 

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