Synaptic Vesicle Protein 2 and Vesicular Monoamine Transporter 1 and 2 Are Expressed in Neuroblastoma.

Neuroblastoma (NB), the most common extracranial cancer in childhood, exhibits neuroendocrine (NE) differentiation. Two well-established NE markers, chromogranin A (CgA) and synaptophysin (syn), are used in the histopathological diagnostics. Our aims were to explore if the NE markers synaptic vesicle protein 2 (SV2) and vesicular monoamine transporter 1 (VMAT1) and 2 (VMAT2) also are expressed in human NB and if so, evaluate their usefulness in NB histopathological diagnostics. Tumor specimens from 21 NB patients, before and/or after chemotherapy, were immunostained for CgA, syn, SV2, VMAT1, and VMAT2. Clinical data was extracted from patients' records. SV2 was highly expressed in NB, as was CgA while syn was less frequently expressed compared to the other two. Both VMATs were expressed in several NB, VMAT2 in more cases than VMAT1 and its expression was similar to syn. Chemotherapy did not affect the immunoreactivity in an obvious way. SV2 was highly expressed in NB and can thus be useful marker in NB diagnostics. VMAT1 and VMAT2 were also expressed in NB but similar to syn less reliable as tumor markers.

Diagnostic ultrasound-guided cutting needle biopsies in neuroblastoma: A safe and efficient procedure.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood and accounts for 15% of deaths in pediatric oncology. Apart from the clinical stage at diagnosis, molecular factors are important for the characterization of the tumor and for decision on adequate treatment. Pretreatment diagnosis and molecular profiling are based on analysis of a tumor sample, obtained either by fine needle aspiration cytology (FNAC), cutting needle biopsy or open surgical biopsy. The method used depends on local tradition and routines. Ultrasound-guided cutting needle biopsy (UCNB) has been used at the Uppsala University Hospital since 1988 for diagnosis of pediatric solid tumors. PROCEDURES: Medical records of 29 patients with NB who underwent pretreatment, diagnostic, ultrasound-guided needle biopsy were reviewed. Information extracted from the patients' records included: age at diagnosis, gender, tumor site, clinical stage, molecular profiling made on biopsies (e.g. MYCN status, ploidy and chromosomal aberrations), and UCNB complications (i.e. bleeding, pain, or anesthesiologic complications). RESULTS: A total of 34 UCNBs were performed in the 29 patients. Repeated biopsies were done in three patients. UCNB was diagnostic in 90% (26/29). A complete molecular profiling was obtained in all UCNBs after 2008. Two patients (7%) developed a significant bleeding and two (7%) needed analgesics following UCNB. Neither infection nor tumor growth in the needle tract was observed. There were no anesthesiologic complications. CONCLUSIONS: UCNB is reasonably safe in patients with NB and usually gives a sufficient amount of tumor tissue for a histological diagnosis, molecular profiling, and biobank storage.

Chromogranin A and neuron-specific enolase in neuroblastoma: Correlation to stage and prognostic factors.

Chromogranin A (CgA) and neuron specific enolase (NSE) are important markers in adult neuroendocrine tumors (NET). Neuroblastoma (NB) has certain neuroendocrine properties. The aim of this study was to correlate blood concentrations of CgA, chromogranin B (CgB), and NSE to prognostic factors and outcome in children with NB. Blood samples from 92 patients with NB, 12 patients with benign ganglioneuroma (GN), 21 patients with non-NB solid tumors, 10 patients with acute leukemias, and 69 healthy children, were analyzed. CgA concentrations were higher in neonates vs. children older than one month in the control group (p < 0.0001), and in neonates with NB vs. the control group (p < 0.01). CgA and NSE concentrations were higher in patients with stages 3 and 4 disease (p < 0.05 and p < 0.05), in patients having tumors with amplification of MYCN (p < 0.05 and p < 0.001), or chromosome 1 p deletion (p < 0.05 and p < 0.05). NSE correlated to the tumor size at diagnosis (p < 0.001) and to tumor related death (p < 0.01) in NB. CgA and NSE concentrations were elevated in patients with NB and especially in those with advanced disease. Both CgA and NSE correlated to genetic markers, while only NSE correlated to primary tumor size and outcome in NB. We found that CgA and NSE are clinically valuable tumor markers in NB and they merit prospective clinical evaluations as such.

Local delivery of bupivacaine in the wound reduces opioid requirements after intraabdominal surgery in children.

BACKGROUND: Local anaesthetic infusions into the surgical wound have been shown to reduce postoperative pain and the need for opioids in adults. In children, it was found to be safe and efficacious following sternotomy and orthopaedic surgery. The aim of this study was to evaluate the need for opioids postoperatively in children receiving wound catheters delivering either bupivacaine or saline following one of three defined abdominal or bladder procedures. METHODS: Prospective, randomized, double-blind, placebo controlled study. Thirty-three children, 6 months of age to 13 years of age, undergoing elective surgery for enterostomy closure, open gastrostomy or ureteral reimplantation were randomized to receive bupivacaine or saline wound infusions for 72 h postoperatively. All patients received acetaminophen orally or rectally for every 6 h. Breakthrough pain was treated with morphine bolus doses of 0.05 mg/kg or infusions if more than three morphine doses were required. Pain scores were assessed every 3 h. Outcome measures were morphine dosages, return to full oral intake and length of hospital stay. RESULTS: On the first postoperative day, patients with bupivacaine infusions had significantly less need for morphine (1.3 ± 1.3 SD doses) compared to those receiving saline infusions (3.1+/2.5 SD doses, p < 0.05). No difference was seen during postoperative day two or three. There was no significant difference between the groups regarding time to full oral intake and time to discharge. CONCLUSIONS: Continuous infusion of bupivacaine in the abdominal wound was effective in reducing postoperative pain in children. It significantly reduced the need for additional opioids and can be considered for postoperative pain management in children.

Differentiated expression of somatostatin receptor subtypes in experimental models and clinical neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a solid tumor of childhood originating from the adrenal medulla or sympathetic nervous system. Somatostatin (SS) is an important regulator of neural and neuroendocrine function, its actions being mediated through five specific membrane receptors. The aim of this study was to investigate the expression of the different somatostatin receptors (SSTRs) in NB tumor cells that may form targets for future therapeutic development. PROCEDURE: Tumor specimens from 11 children with stage II-IV disease were collected before and/or after chemotherapy. Experimental tumors derived from five human NB cell lines were grown subcutaneously in nude mice. Expression of SSRTs, the neuroendocrine marker chromogranin A (CgA) and SS was detected by immunohistochemistry using specific antibodies. RESULTS: SSTR2 was detected in 90%, SSTR5 in 79%, SSTR1 in 74%, SSTR3 in 68% whereas SSTR4 was expressed in 21% of the clinical tumors. The experimental tumors expressed SSTRs in a high but variable frequency. All clinical tumors showed immunoreactivity for CgA but not for SS. CONCLUSION: The frequent expression of SSTRs indicates that treatment with unlabeled or radiolabeled SS analogs should be further explored in NB.

Metronomic administration of the drug GMX1777, a cellular NAD synthesis inhibitor, results in neuroblastoma regression and vessel maturation without inducing drug resistance.

High-risk neuroblastoma is a rapidly growing tumor with a survival rate below 50%. A new treatment strategy is to administer chemotherapeutic drugs metronomically, i.e., at lower doses and frequent intervals. The aim of the study was to investigate the effects of GMX1777, a chemotherapeutic drug affecting cellular energy metabolism, in a mouse model for high-risk neuroblastoma. Female SCID mice were injected s.c. with MYCN-amplified human neuroblastoma cells and randomized to either treatment with GMX1777 or vehicle. In some animals, treatment was discontinued allowing tumor relapse. Treatment response was evaluated using the pediatric preclinical testing program (PPTP). Immunohistochemistry and qRT-PCR was performed on tumor cryosections to investigate the microscopic and molecular changes in tumors in response to GMX1777. Despite an increase in vessel density, tumor regression and a high group response score according to PPTP criteria was induced by GMX1777 without inducing drug resistance. Treatment resulted in inhibition of tumor cell proliferation, vessel maturation, reduced hypoxia, increased infiltration of MHC class II negative macrophages and expansion of the nonvascular stromal compartment. Decreased stromal VEGF-A and PDGF-B mRNA in response to treatment together with the structural data suggest a "deactivation" or "silencing" of the tumor stroma as a paracrine entity. In conclusion, GMX1777 was highly efficient against high-risk neuroblastoma xenografts through modulation of both the tumor cell and stromal compartment.

The angiogenic growth factors HGF and VEGF in serum and plasma from neuroblastoma patients.

AIM: To determine whether concentrations of the angiogenic growth factors hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGF-A) correlate with clinical and genetic markers in samples taken at diagnosis in children with neuroblastoma (NB). PATIENTS AND METHODS: Heparin plasma (P-) and serum (S-) samples of healthy controls (n=73, mean age +/- SD 3.5+/-2.1; females/males: 23/50) and patients with NB (n=62; 2.2+/-1.8; 26/36) were collected between 1988 and 1999. Clinical data included age at diagnosis, gender, stage, outcome, amplification of the oncogene MYCN, loss of heterozygosity at the short arm of chromosome 1 (1p LOH) and ploidy. RESULTS: HGF and S-VEGF-A were elevated in NB as compared to controls (38/62 patients, p<0.0001 and p<0.05, Mann-Whitney U test). HGF concentrations were higher in high-stage (stage 3-4) as compared to low-stage (stage 1-2) disease (p<0.01). P-HGF was elevated in patients with 1p LOH (p<0.01), MYCN amplification (p<0.001) and di- or tetraploidy (p<0.001). S-HGF concentration was elevated in patients MYCN-amplified tumors only. Plasma and S-HGF concentrations were higher in the deceased group (p<0.05), but not P or S-VEGF-A. CONCLUSION: This study showed that concentrations of HGF and S-VEGF-A are elevated in patients with NB. Furthermore, HGF and S-VEGF-A concentrations correlate to higher stage disease and HGF correlates to genetic markers known to indicate a poor outcome. These observations imply that HGF and VEGF-A have biological roles in NB and suggest the possibility of interference with HGF or VEGF-A signaling as a therapeutic strategy.

Regression of orthotopic neuroblastoma in mice by targeting the endothelial and tumor cell compartments.

BACKGROUND: High-risk neuroblastoma has an overall five-year survival of less than 40%, indicating a need for new treatment strategies such as angiogenesis inhibition. Recent studies have shown that chemotherapeutic drugs can inhibit angiogenesis if administered in a continuous schedule. The aim of this study was primarily to characterize tumor spread in an orthotopic, metastatic model for aggressive, MYCN-amplified neuroblastoma and secondarily to study the effects of daily administration of the chemotherapeutic agent CHS 828 on tumor angiogenesis, tumor growth, and spread. METHODS: MYCN-amplified human neuroblastoma cells (IMR-32, 2 x 10(6)) were injected into the left adrenal gland in SCID mice through a flank incision. Nine weeks later, a new laparotomy was performed to confirm tumor establishment and to estimate tumor volume. Animals were randomized to either treatment with CHS 828 (20 mg/kg/day; p.o.) or vehicle control. Differences between groups in tumor volume were analyzed by Mann-Whitney U test and in metastatic spread using Fisher's exact test. Differences with p < 0.05 were considered statistically significant. RESULTS: The orthotopic model resembled clinical neuroblastoma in respect to tumor site, growth and spread. Treatment with CHS 828 resulted in tumor regression (p < 0.001) and reduction in viable tumor fraction (p < 0.001) and metastatic spread (p < 0.05) in correlation with reduced plasma levels of the putative tumor marker chromogranin A (p < 0.001). These effects were due to increased tumor cell death and reduced angiogenesis. No treatment-related toxicities were observed. CONCLUSION: The metastatic animal model in this study resembled clinical neuroblastoma and is therefore clinically relevant for examining new treatment strategies for this malignancy. Our results indicate that daily scheduling of CHS 828 may be beneficial in treating patients with high-risk neuroblastoma.

The bisphosphonate, zoledronic acid reduces experimental neuroblastoma growth by interfering with tumor angiogenesis.

BACKGROUND: Zoledronic acid is a new member of the bisphosphonate (BP) class of compounds, a family of closely related synthetic molecules originally derived from the naturally occurring pyrophosphate. These compounds that are potent inhibitors of bone resorption, have been shown to reduce the growth of several cancer cell lines in vitro, and can act as inhibitors of angiogenesis. The angiogenesis inhibitor TNP-470, a synthetic analogue of the fungal antibiotic fumagillin, has been shown to inhibit the growth of multiple tumors in vivo, and is currently in Phase II clinical trials for cancer. MATERIALS AND METHODS: The effects of daily subcutaneous (s.c.) administration of zoledronic acid (0.1 mg/kg) were compared with those of TNP-470 (15 mg/kg/day and 30 mg/kg every other day, s.c.) in a nude mouse xenograft model for the childhood cancer, neuroblastoma (NB). RESULTS: Zoledronic acid reduced the tumor growth by 33% whereas TNP-470 was less effective and reduced the tumor growth by 26% and 11% for animals treated with 15 mg/kg/day and 30 mg/kg every other day, respectively. Analysis of angiogenesis showed a significant reduction of the number of vessels per grid and in vessel length in all the treatment groups. CONCLUSION: Zoledronic acid shows tumoristatic and angiostatic properties that might be beneficial in the treatment of solid tumors such as neuroblastoma.

Angiogenesis can be reduced without significant reduction of tumor growth.

BACKGROUND: High risk neuroblastoma (NB) patients have an overall five-year survival of approximately 50%, indicating the need for new treatment strategies, such as angiogenesis inhibition. MATERIALS AND METHODS: The angiogenesis inhibitor TNP-470 (30 mg/kg, every other day, subcutaneously) was given to nude mice with subcutaneous human neuroblastoma xenografts. The plasma concentrations of the angiogenesis stimulators, i.e. vascular endothelial growth factor A (VEGF-A), fibroblast growth factor 2 (FGF-2) and hepatocyte growth factor (HGF), were assayed longitudinally. Angiogenesis, proliferation and apoptosis were quantified on tumor tissue slides. RESULTS: Upon treatment with TNP-470, angiogenesis was significantly inhibited by the reduction of length and surface area of vessels per tumor volume, without having significant effect on tumor growth, tumor cell proliferation or apoptosis. Plasma concentrations of VEGF-A per tumor volume were significantly increased upon treatment. CONCLUSION: Angiogenesis inhibition must reach a threshold before significant tumor cell apoptosis and a reduction of the tumor growth rate occur.

The anti-VEGF antibody bevacizumab potently reduces the growth rate of high-risk neuroblastoma xenografts.

Neuroblastoma (NB) is a rapidly growing, well-vascularized childhood cancer that often presents with metastases. The overall five-year survival in NB is approximately 45% despite multimodality treatment, and therefore there is a clinical need for new therapeutic strategies. NB frequently overexpresses the angiogenic factor VEGF (vascular endothelial growth factor). The aim of this study was to investigate the effect of bevacizumab (Avastin, Genentech/Roche), a humanized anti-VEGF-A antibody, on NB growth in three different xenograft models, chosen to resemble high-risk NB. The human NB cell lines SK-N-AS, IMR-32 and SH-SY5Y, which are poorly differentiated and overexpress VEGF-A, were injected s.c. in immunodeficient mice. Bevacizumab was given intraperitoneally twice weekly at 5 mg/kg body weight, starting at a tumor volume of 0.3 mL. Bevacizumab significantly (p < 0.01-0.05) reduced NB growth in vivo without toxicity by causing a 30-63% reduction of angiogenesis, but had no effect on NB cell survival in vitro. Serum concentrations of VEGF-A increased two- to six-fold during bevacizumab therapy which did not result in faster tumor growth compared with control animals. Based on our experimental data we suggest consideration of bevacizumab in treatment of high-risk NB that does not respond to conventional therapy and that overexpresses VEGF.

The congenital pouch colon can be used for vaginal reconstruction by longitudinal splitting.

Congenital pouch colon (CPC) is a rare malformation in which the distal part of a shortened colon forms a dilated pouch. It is associated with an anorectal malformation. We report 2 patients with CPC, one with a cloaca and one with vestibular fistula and vaginal atresia. It is the first description of CPC, vestibular fistula, and vaginal atresia. The purpose of this report was to demonstrate that the pouch can be split longitudinally--in analogy with Bianchi's intestinal lengthening procedure [Bianchi A. Intestinal loop lengthening: a technique for increasing small intestinal length. J Pediatr Surg 1980;15:145-51]--to create a vagina and to reconstruct the anorectum with preserved blood supply.

Digoxin inhibits neuroblastoma tumor growth in mice.

BACKGROUND: Epidemiological data suggest a more favorable outcome of breast carcinoma in women taking cardiac glycosides. This study investigated whether digoxin could inhibit tumor growth in mice. MATERIALS AND METHODS: Tumor growth experiments were done in mice grafted with the neuroblastoma cell lines SH-SY5Y, Neuro-2a, colonic cancer cells (LS174T) or Lewis lung cancer cells (LLC). Angiogenesis inhibition was investigated in vitro on fibroblast growth factor-2 (FGF-2)-stimulated bovine endothelial cell (BCE) growth and in vivo in the chick chorioallantoic membrane (CAM) assay. RESULTS: SH-SY5Y and Neuro-2a grafts were inhibited by 44% (p=0.008) and 19% (p=0.007), respectively, whereas the colonic cancer xenografts and LLC syngrafts were less responsive. The neuroblastoma specificity was confirmed in vitro. Digoxin also inhibited angiogenesis in the CAM assay and the BCE cell survival in vitro was 50% at 53 ng/ml. CONCLUSION: Our data suggest that digoxin may be a specific neuroblastoma growth inhibitor and an unspecific inhibitor of angiogenesis.

The selective class III/V receptor tyrosine kinase inhibitor SU11657 inhibits tumor growth and angiogenesis in experimental neuroblastomas grown in mice.

Vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. SU11657 (SUGEN) is a selective multitargeted tyrosine kinase inhibitor with antitumor and antiangiogenic activity exerted by targeting PDGF receptors (PDGFR), VEGF receptors (VEGFR), stem cell factor receptor (c-KIT), and FMS-related tyrosine kinase 3. Oral administration of SU11657 at 40 mg x kg(-1) x d(-1) to athymic mice resulted in significant growth inhibition of a panel of s.c. human neuroblastoma xenografts, namely, fast-growing SK-N-AS, MYCN- amplified IMR-32, and SH-SY5Y, by 90, 93.8, and 88%, respectively, and was well tolerated. All of the cell lines expressed VEGFR-2, PDGFR-beta, and c-KIT protein in the tumor cell and endothelial cell compartment by immunohistochemistry, and the expression decreased during therapy. Plasma concentrations of VEGF-A, PDGF-BB, and stem cell factor increased per milliliter of tumor volume at days 10, 18, and 20 of therapy. Furthermore, SU11657 reduced tumor angiogenesis by 63-96%. Our experimental data suggest that the angiogenesis inhibitor SU11657 may be beneficial in the treatment of rapidly growing and highly vascularized solid tumors of childhood, such as neuroblastoma. In summary, the class III/V receptor tyrosine kinases and their ligands are implicated in angiogenesis, tumor cell proliferation, and cell survival, and it seems reasonable to determine whether interference with these pathways can suppress neuroblastoma growth or not.

The minimal active domain of endostatin is a heparin-binding motif that mediates inhibition of tumor vascularization.

Endostatin constitutes the COOH-terminal 20,000 Da proteolytic fragment of collagen XVIII and has been shown to possess antiangiogenic and antitumorigenic properties. In the present study, we have investigated the role of the heparin-binding sites in the in vivo mechanism of action of endostatin. The majority of the heparin binding is mediated by arginines 155/158/184/270 in endostatin, but there is also a minor site constituted by arginines 193/194. Using endostatin mutants lacking either of these two sites, we show that inhibition of fibroblast growth factor-2-induced angiogenesis in the chicken chorioallantoic membrane requires both heparin-binding sites. In contrast, inhibition of vascular endothelial growth factor-A-induced chorioallantoic membrane angiogenesis by endostatin was only dependent on the minor heparin-binding site (R193/194). These arginines were also required for endostatin to inhibit fibroblast growth factor-2- and vascular endothelial growth factor-A-induced chemotaxis of primary endothelial cells. Moreover, we show that a synthetic peptide corresponding to amino acids 180-199 of human endostatin (which covers the minor heparin-binding site) inhibits endothelial cell chemotaxis and reduces tumor vascularization in vivo. Substitution of arginine residues 193/194 for alanine attenuates the antiangiogenic effects of the peptide. These data show an essential role for heparin binding in the antiangiogenic action of endostatin.

Gene therapy of prostate cancer with the soluble vascular endothelial growth factor receptor Flk1.

A variety of novel therapeutic approaches have emerged recently for the treatment of human cancers. We have coupled two of these therapeutic approaches, gene therapy and antiangiogenic therapy and tested them in two murine prostate cancer models Recombinant adenovirus encoding the ligand-binding ectodomain of the VEGF receptor 2 (Flk1) fused to an Fc domain was administered to SCID mice carrying orthotopic human LNCaP tumors as well as to transgenic (TRAMP) mice with spontaneous prostate tumors. Ad Flk1-Fc injection reduced tumor growth by 66% for orthotopic LNCaP tumors and by 42% for spontaneous tumors in TRAMP mice. Microvessel density in the primary tumors was reduced by 68% and 40% in the two models respectively. A decrease in microvessel density was also observed in lymphatic metastases in Ad Flk1-Fc-treated TRAMP mice and was correlated with a decrease in the frequency of regional metastases in the treated animals. Survival time was also extended in the Ad Flk1-Fc-treated TRAMP mice relative to the control-treated animals. Our results suggest that adenoviral delivery of soluble Flk1 receptor can reduce vascular density and prostate tumor growth and prolong survival time in orthotopically implanted tumors as well as in spontaneous prostate tumors in transgenic animals.