Topotecan is anti-angiogenic in experimental hepatoblastoma

Background: Advanced hepatoblastoma often is lethal despite current therapies, yet development of novel approaches has been hampered by the lack of biologically relevant models. One new strategy selectively targets endothelium rather than tumor cells using frequently administered, low-dose ([ldquo ]metronome[rdquo ]) chemotherapy. Metronome topotecan has antiangiogenic activity in some experimental tumors. The authors developed a xenograft model of human hepatoblastoma to test the effect of metronome topotecan in this system. Methods: Xenografts resulted from intrarenal injection of cultured human hepatoblastoma cells in athymic mice. Topotecan (0.36 mg/kg/dose) or vehicle was injected intraperitoneally 5 times per week. At week 6, 10 control/treated mice were killed, and remaining animals were maintained without treatment until week 8. Tumor weights were compared by Kruskal-Wallis analysis, and vascular alterations were ascertained by specific immunostaining. Results: Metronome topotecan affected tumor weights in a delayed fashion: weights were diminished significantly only at 8 weeks (treated v control: 6 weeks, 0.59 g v 82 g, P value, not significant; 8 weeks, 1.13 g v 3.82; P [lt ] .02). Decreased vascularity and increased endothelial cell apoptosis were observed in treated xenografts. Conclusions: Metronome topotecan inhibits growth and neovascularization in experimental hepatoblastoma. The durability of this effect is novel and has not been observed in other xenograft tumor models. Cytotoxic targeting of endothelial cells may hold particular promise for therapy of children with advanced hepatoblastoma. J Pediatr Surg 37:857-861.     

Anti-VEGF antibody in experimental hepatoblastoma: suppression of tumor growth and altered angiogenesis

Background: Hepatoblastoma is the most common primary hepatic malignancy of childhood, frequently presenting as advanced disease. Vascular endothelial growth factor (VEGF) is an endothelial mitogen and survival factor critical to growth and angiogenesis in many human cancers. Inhibition of VEGF effectively suppresses tumorigenesis in multiple experimental models. The authors hypothesized that anti-VEGF antibody would alter vascular architecture and impede tumor growth in experimental hepatoblastoma. Methods: The Institutional Animal Care and Use Committee of Columbia University approved all protocols. Xenografts were established in athymic mice by intrarenal injection of cultured human hepatoblastoma cells. Anti-VEGF antibody (100 [mu ]g/dose) or vehicle was administered intraperitoneally 2 times per week for 5 weeks. At week 6, 10 control/treated mice were killed and remaining animals maintained without treatment until week 8. Tumor weights were compared by Kruskal-Wallis analysis, and vascular alterations ascertained by fluorescein angiography and specific immunostaining. Results: Anti-VEGF antibody significantly inhibited tumor growth at 6 weeks (1.85 g +/[minus ] 0.60 control, 0.05 +/[minus ] 0.03 antibody, P [lt ] .0003). In comparison with controls, treated xenografts showed decreased vascularity and dilated surviving vessels with prominent vascular smooth muscle elements. Conclusions: Specific anti-VEGF therapy inhibits neoangiogenesis and significantly suppresses tumor growth in experimental hepatoblastoma. Surviving vasculature displays dilation and increased vascular smooth muscle. Anti-VEGF agents may represent new therapeutic alternatives for children with advanced disease. J Pediatr Surg 38:308-314.     

Combination antiangiogenic therapy: increased efficacy in a murine model of Wilms tumor

BACKGROUND/PURPOSE: Antibody to vascular endothelial growth factor (anti-VEGF) suppresses tumor growth and metastasis in experimental Wilms tumor. However, tumor growth accelerates if antibody is withdrawn. As recently shown, low-dose, frequently administered topotecan, a topoisomerase-1 inhibitor, has anti-angiogenic activity. The authors hypothesized that combined topotecan/anti-VEGF therapy would suppress tumor growth and metastasis more durably than either agent alone. METHODS: Xenografts were induced by intrarenal injection of human Wilms tumor cells in athymic mice (n = 59). Mice were divided into control (n = 10), anti-VEGF (n = 16), topotecan (n = 17), and topotecan plus anti-VEGF (n = 16) groups. All control and half the treated mice were killed at week 6. Remaining ("rebound") mice were maintained without treatment until week 8. Tumor vasculature was mapped by fluorescein angiography/PECAM immunostaining. Endothelial apoptosis was assessed by TUNEL assay. RESULTS: 6 weeks: Tumor weights were reduced significantly in treated mice (P <.003 v control). Seven of ten control and 1 of 25 treated mice displayed lung metastases (P <.003). Rebound tumors were largest in topotecan-only, intermediate in antibody-treated, and smallest in combination-treated mice. Immunostaining and angiography results showed sparse vascularity in treated xenografts. Endothelial apoptosis was observed only in treated tumors. CONCLUSION: Combination low-dose topotecan and anti-VEGF antibody therapy is antiangiogenic and suppresses tumor growth and metastasis in experimental Wilms tumor more durably than either agent alone.     

Synergistic interleukin-18 and low-dose interleukin-2 promote regression of established murine neuroblastoma in vivo

Background/Purpose: Severe systemic toxicities have limited the clinical applications of the potent cytokine, interleukin-2 (IL-2). Recent studies have shown that IL-18 synergizes with IL-2 to enhance cytolytic activity in vitro. Combination therapy allows for IL-2 dose reduction, thus, limiting its toxicity while augmenting natural killer cell activity. The authors hypothesize that IL-18 plus low-dose IL-2 may induce a potent and sustained antitumor response in vivo providing effective immunotherapy for neuroblastoma. Methods: Four groups of A/J mice (n = 28) were inoculated subcutaneously in the right flank with 1 [times ] 10⁶ murine neuroblastoma cells (TBJ). On day 7, 5 consecutive daily peritumoral injections were performed with saline (control), human rIL-2 (30,000 IU), murine IL-18 (1 [mu ]g), or IL-2 plus IL-18. Tumor growth was monitored, and animals with tumor progression were killed on day 21. Seven weeks after the initial treatment, animals with rejected tumors were rechallenged with 5 [times ] 10⁶ cells in the opposite flank. Quantitative data were analyzed by Student's t test. Results: Rapid tumor growth and death was noted in all control animals by 21 days. Complete tumor eradication was seen in 28% of mice treated with IL-2 (P = .03), 42% of mice treated with IL-18 (P [lt ] .05), and 57% of mice treated with of IL-2 plus IL-18 (P [lt ] .05). Despite the initial response, all animals failed rechallenge and developed new or recurrent tumors within 7 to 10 days. Conclusions: Coadministration of low-dose IL-2 plus IL-18 induced a potent primary response to murine neuroblastoma likely caused by activation of natural killer cells in the tumor microenvironment. This combined cytokine therapy strategy was unable to induce sustained immunity to rechallenge. However, dendritic cell vaccination combined with IL-2 plus IL-18 cytokine treatment did allow for the establishment of a complete and durable antitumor response. J Pediatr Surg 38:301-307.     

Clinical significance of intensive surgery with intraoperative radiation for advanced neuroblastoma: does it really make sense?

Purpose: The aim of this study was to evaluate the significance of intensive surgery combined with intraoperative radiation therapy (IORT) in advanced neuroblastoma.Methods: Clinical features and outcome were reviewed in 33 advanced neuroblastoma patients (24 with INSS stage 4, 9 with stage 3), who had surgery (total excision 29, subtotal excision 4) with IORT (10 to 15 Gy) against the primary tumor site.Results: Three patients (8.8%) had relapse at the primary site, all of which arose from the unirradiated area after stem cell transplantation. Among 29 patients with total excision, disease-free survival was obtained in 15 (51.7%) for an average of 6.9 years, which included 5 survivors of 9 patients (55.9%) with amplified N-myc. In contrast, none of 4 patients with macroscopic residual survived. The Kaplan-Meier analysis showed significantly longer survival rates in the patients with total resection compared with those with macroscopic remnants.Conclusions: The intensive surgery with IORT dramatically increased the local eradication and improved the outcome even in advanced neuroblastoma with N-myc amplification. However, long-term survival was not obtained in patients with unresectable residual disease. These results may indicate the key role of surgical eradication in advanced neuroblastoma.     

Effective targeted cytotoxicity of neuroblastoma cells

Background/Purpose: Despite aggressive treatment with surgery, chemotherapy, and radiotherapy, the prognosis for many children with neuroblastoma remains poor. Targeted toxins represent novel cancer therapeutics designed to selectively target and kill cancer cells. The authors have developed a novel fusion toxin, DT5F11, consisting of truncated diphtheria toxin (DT_A) linked to a single chain antibody (sc5F11) targeting the GD₂ antigen found on most neuroblastoma cells. This report describes the construction, expression, and in vitro function of DT5F11. Methods: Utilizing restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis, the prkDTL5F11 plasmid was created by the fusion of distinct coding sequences for a single-chain GD₂ targeting antibody (sc5F11) and truncated diphtheria toxin (DT_A). DH5[alpha ] Escherichi coli-competent cells were transformed with prkDTL5F11; DNA was amplified, isolated, and sequenced. The fusion protein was expressed and assayed by Western blot. Targeted cytotoxicity was analyzed on GD₂-positive (SK-N-AS, IMR-32, SK-N-MC, LAN-1) and GD₂-negative (HeLa) cells. Results: Fluorescent dye[ndash ]labeled cycle sequencing identified the constructed fusion toxin gene. Western blot analysis using a mouse antihuman DT_A antibody showed a 69-kD band identifying the fusion toxin, DT5F11. Targeted cell killing with DT5F11 was seen only in GD₂ positive cells. Conclusions: This study demonstrates creation of a novel fusion toxin with effective GD₂-targeted cellular toxicity. Further investigation of this fusion toxin as a therapeutic agent in the management of neuroblastoma is warranted.     

Effects of irradiated tumor vaccine and continuous localized infusion of granulocyte-macrophage colony-stimulating factor on neuroblastomas in mice

Background/Purpose: Immunomodulatory treatment has been proposed as a feasible strategy for neuroblastoma treatment. In this study, the antitumor effects of a continuous localized subcutaneous infusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) into the injection site of irradiated tumor vaccine used as a source of tumor antigens on mouse neuroblastoma were investigated. Methods: A/J mice were inoculated subcutaneously with wild type neuro-2a neuroblastoma cells and then treated with 5 doses of irradiated tumor vaccine or continuous localized infusion of GM-CSF (1 ng/d or 10 ng/d) via an osmotic minipump. Survival rates and survival times were compared among the groups. Tumor growth rates and animal survival times were followed and compared among different groups. Histologic and immunohistochemical analyses were performed to observe the immune response induced by various treatment strategies. Results: Tumor growth rates were reduced significantly and survival times prolonged significantly by the treatment using tumor vaccine and continuous infusion of 10 ng/d of GM-CSF when compared with the control group (P [lt ] .05). One mouse treated with tumor vaccine and a 10 ng/d infusion of GM-CSF showed tumor regression and long-term survival, and no tumor growth was noted after rechallenge with wild-type neuro-2a cells. In contrast, using tumor vaccine only, or tumor vaccine combined with a 1 ng/d infusion of GM-CSF was less effective than tumor vaccine combined with a 10 ng/d infusion of GM-CSF (P [lt ] .05). Infusion of GM-CSF alone had no antitumor effects. Immunohistologic analyses showed significant CD4+ and CD8+ T cell infiltration of the tumor in the mice treated with tumor vaccine and a 10 ng/d infusion of GM-CSF. Conclusions: The results suggest that an irradiated tumor vaccine combined with continuous localized infusion of GM-CSF may induce a tumor-specific antitumor immune response that can suppress tumor growth and prolong survival. Such a treatment strategy deserves consideration as a possible adjuvant treatment for neuroblastoma. J Pediatr Surg 37:1298-1304.     

Expression of vascular endothelial growth factor and its receptor Flk-1 in human neuroblastoma using in situ hybridization

Background/Purpose: Although angiogenic factors may play an important role in the biology of neuroblastoma, which frequently spreads hematogenously, the mechanism remains unclear. The authors studied tumor progression and invasion from the perspective of angiogenesis and sought to understand the features of this type of tumor. Methods: Thirty-one specimens were resected from patients with neuroblastoma and the expression of vascular endothelial growth factor (VEGF), and its receptor (Flk-1) was examined using immunohistochemistry. The authors looked for correlations among the expressions of VEGF and its receptor with various clinicopathologic factors. In addition, they examined the expression and location of VEGF and Flk-1 mRNA in 10 primary neuroblastoma using in situ hybridization. Results: Both in situ hybridization and immunohistochemistry showed the presence of VEGF expression within the neuroblastoma cells. We found VEGF mRNA in neuroblastoma cells but not vascular endothelial cells according to in situ hybridization. Further, Flk-1 mRNA was present both in neuroblastoma cells and vascular endothelial cells. The level of VEGF expression was higher in unfavorable histology, using the criteria of Shimada, than in favorable histology. Conclusion: The authors suggest that paracrine and autocrine systems are involved in the angiogenesis of neuroblastoma, and the expression of VEGF correlates with the prognosis in neuroblastoma.     

Soluble receptor for advanced glycation endproducts (sRAGE) effectively reduces tumor growth as a single agent and in combination with doxorubicin in a spontaneous mammary tumor model

Introduction: The receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin family interacts with distinct ligands that have been implicated in various pathophysiologic states such as diabetes, atherosclerosis, inflammation as well as tumor growth. We have previously demonstrated that blockade of RAGE in tumors raised in mice from rat C6 glioma cells or murine Lewis lung carcinoma effectively reduced tumor growth, invasion and metastases. Blockade of RAGE has also previously been shown to halt the progression of breast tumors in a murine model that spontaneously produces breast tumors. We hypothesize that the addition of RAGE antagonists to standard chemotherapeutic agents for breast cancer may enhance the effectiveness of monotherapy. Methods: Female MMTV transgenic mice (5-8 weeks) were divided into four groups: (1) Control group-no treatment, (2) doxorubicin (DOX) 2 mg/kg IP weekly x 4 weeks (3) sRAGE 20 μg IP daily x 4 weeks and (4) DOX 2 mg/kg IP weekly x 4 weeks and sRAGE 20 μg IP daily x 4 weeks. The mice were sacrificed and tumors harvested on treatment day 35 and were weighed and measured. Results: There was no difference in tumor weight between the control mice (n = 9) and DOX-treated mice (n = 4) (0.192 g v. 0.193). The administration of sRAGE (n = 7) decreased tumor weight to 0.12 g and the combination of sRAGE and DOX (n = 8) decreased tumor weight by a third (0.192 g v. 0.061 g, p = 0.11). While the mean tumor volume in the control mice was 316.7 mm³, sRAGE decreased tumor volume to 89.65 mm³ and the combination of sRAGE and DOX decreased tumor volume by 6.5 fold (316.7 mm³ vs. 48.08 mm³, p = 0.006). The mean tumor volume in the DOX treated group was 500.9 mm³. Conclusions: RAGE antagonists suppress local tumor growth in transgenic mice with spontaneously occurring cancer in an early intervention model. The addition of sRAGE, a low molecular weight RAGE antagonist, to doxorubicin enhanced the effectiveness of this standard chemotherapy agent.     

Insulin-like growth factor-I protects neuroblastoma against starvation-induced apoptosis and is associated with increased Bcl-2 expression

Background/Purpose: Aggressive neuroblastomas avoid apoptosis and have increased expression of the antiapoptotic protein, Bcl-2. Insulin-like growth factor-I (IGF-I) is mitogenic and may promote tumor survival by inhibiting apoptosis. The authors hypothesize that IGF-I may protect neuroblastoma cells from apoptosis by upregulating their Bcl-2 expression. Methods: Human neuroblastoma cells (IMR-32) are cultured, and 3 experimental groups are established: 1 group with cells cultured in standard growth media (control), 1 with cells grown in serum-depleted media (starvation), and 1 with neuroblastoma cells cultured in starvation media plus IGF-I. The cells are harvested at 14 and 24 hours, and cytospin slides are made. Bcl-2 expression is measured by immunohistochemistry. Apoptosis is detected with the TUNEL method. Results: Bcl-2 expression is decreased 90% in the serum starved neuroblastoma cells. In addition, apoptosis is 150 times higher in the starved neuroblastoma cells. These changes are abrogated by the addition of IGF-I, where apoptosis is decreased 50% and Bcl-2 is 14-fold higher in the IGF-I[ndash ]treated group. These changes are most apparent at 24 hours. Conclusions: IGF-I protects neuroblastoma cells from apoptosis and increases Bcl-2 expression. Growth factors may have a direct role in promoting tumorigenesis by inducing the expression of antiapoptotic proteins by the tumor.     

Stephen L. Gans overseas lecture. Mass screening for neuroblastoma in Japan: lessons learned and future directions

Background/Purpose: Since 1985, a nationwide mass screening program (MS) for neuroblastoma has been conducted for 6-month-old infants throughout Japan, resulting in the detection of more than 1,900 cases of neuroblastoma. The outcome of these patients has been excellent: more than 97% of them are alive. Yet, several reports suggest that the number of advanced-stage neuroblastoma patients over 1 year of age has not changed substantially. The current report focuses on the 15-year experience with MS of the Kyushu Pediatric Oncology Study Group. Methods: The clinical and biological features of neuroblastoms detected (n = 320) and not detected by MS (n = 245) were compared. Regional and national statistics for neuroblastoma before and after 1985 were analyzed using standard epidemiologic measures for the occurrence of disease. Results: The majority of the MS-positive cases were biologically favorable and had an excellent outcome. In contrast, the majority of non-MS patients in whom neuroblastoma later developed had advanced-stage, unfavorable-prognosis tumors. The overall mortality rate of neuroblastoma in the Kyushu area was not improved by MS. Conclusions: The optimal time for screening is the point at which neuroblastomas regressing spontaneously can no longer be detected, but more aggressive disease can be found. A birth cohort study could determine the optimal timing for a second screening. Identification of other new prognostic factors may be required. J Pediatr Surg 37:949-954.     

Wilms' tumor growth is suppressed by antiangiogenic pigment epithelium[ndash ]derived factor in a xenograft model

Background/Purpose: Pigment epithelium[ndash ]derived factor (PEDF), a potent endogenous inhibitor of angiogenesis, is highly expressed in the kidney. The authors postulated that systemic administration of PEDF would decrease Wilms' tumor growth in a xenograft model, and increased renal vascularity would result in a mouse null for PEDF. Methods: Tumors were induced in athymic mice using human anaplastic Wilms' tumor cells. Purified PEDF protein or vehicle was administered for 7 days beginning 2 to 3 weeks after inoculation. Tumors were stained with anti-PEDF and anti[ndash ]Factor VIII antibodies. Mitoses and microvascular density (MVD) were counted per high-power field (hpf). PEDF-null mice were generated on a SV129/C57Bl6 background. Wild-type and null kidneys were assessed for MVD. Results: Mean tumor weight in the 2-week group was 60% less than controls (P [lt ] .05). The MVD and mitotic count in treated tumors were significantly less than controls (P [lt ] .05). PEDF stained strongly in normal kidneys but was minimal to absent in Wilms' tumor. PEDF-null kidneys had increased MVD compared with wild-type (P [lt ] .05). Conclusions: PEDF is expressed strongly in normal murine kidney, and loss of its angioinhibitory activity may contribute to pathologic angiogenesis in Wilms' tumor. Systemic PEDF suppresses WT growth by targeting both the tumor cells and its associated vasculature. J Pediatr Surg 38:336-342.     

Dorsoventral patterning in oesophageal atresia with tracheo-oesophageal fistula: Evidence from a new mouse model

Background/Purpose: The well-established Adriamycin rat model of oesophageal atresia (OA) and tracheo-oesophageal fistula (TOF) complements recently described mouse genetic models in which loss of function mutations in foregut patterning genes, such as Nkx2.1 (Ttf 1), lead to OA/TOF. The authors aimed to integrate the 2 systems by adapting the Adriamycin model to the mouse to study molecular aspects of tracheo-oesophageal development. Methods: Pregnant CBA/Ca mice were injected intraperitoneally with 4 mg/kg of Adriamycin on embryonic days 7.5 and 8.5. Embryos and fetuses of various gestational ages were subjected to morphologic or histologic examination. Sections were stained with H [amp ] E or processed for immunohistochemistry using an antibody specific for Nkx2.1. Results: Tracheo-oesophageal malformations were observed in 47% of Adriamycin-treated embryos. Early foregut development was similar in Adriamycin-exposed and control embryos but, by E11.5, many treated embryos had an undivided oesophago-trachea, which gave rise to the lung buds and a fistula to the stomach. The fistula originated from the dorsal aspect of the undivided tube and was negative for Nkx2.1, or showed only transient Nkx2.1 expression, compared to the strongly positive bronchi ventrally. Conclusions: The Adriamycin model of OA is adaptable to the mouse. In the absence of tracheo-oesophageal separation, the dorsal fistula retains its nonrespiratory commitment suggesting that dorsoventral patterning of foregut development is undisturbed by Adriamycin exposure.     

VEGF upregulates Bcl-2 expression and is associated with decreased apoptosis in neuroblastoma cells

Background/Purpose: Both the expression of Bcl-2 and the amount of vascular endothelial growth factor (VEGF) are increased in neuroblastoma cells cocultured with hepatocytes. The authors hypothesize that VEGF upregulates Bcl-2 expression by the neuroblastoma cells and protects them from apoptotic stimuli. Methods: To determine whether VEGF will induce Bcl-2 expression in neuroblastoma cells, the cells are plated with standard media (control) or media supplemented with VEGF. After 24 hours, Bcl-2 expression is measured. To determine whether VEGF protects neuroblastoma cells from apoptosis, the cells are subjected to tumor necrosis factor alpha (TNF-[alpha ]) or serum starvation to induce apoptosis either with or without VEGF added to the culture media. The cells are collected and apoptosis measured using the deoxynucleotidyltransferase-mediated dUTP neck end labeling (TUNEL) method. Results: VEGF increases Bcl-2 expression by 33% over cells cultured in standard media. Serum starving the tumor cells or adding TNF-[alpha ] significantly increases the percentage of apoptotic cells. The addition of VEGF significantly protects the neuroblastoma cells from the apoptotic effects of both serum starvation and TNF-[alpha ]. Conclusions: VEGF increases the expression of Bcl-2 and also abrogates TNF-[alpha ] and serum starvation[ndash ]induced apoptosis in neuroblastoma cells in vitro. VEGF may promote neuroblastoma survival not only through angiogenesis, but also by altering apoptosis and its regulating proteins.     

Vitamin E succinate decreases lung cancer tumor growth in mice

Background.In vitro studies have shown that Vitamin E Succinate (VES) arrests lung cancer cellular proliferation; however, in vivo studies have not been performed. This study examined in vivo effects of VES on lung cancer. Methods. An in vitro dose-response curve of human A549 lung cancer tumors to VES was established. A549 tumors were established in the right submammary fat pads of athymic nude mice (C57/BL/6J-Hfh11nu). Seven days postinjection, mice were separated into VES and control groups. VES mice (n = 12) underwent daily intraperitoneal (IP) injection of 0.1 Ml VES in polyethylene glycol and dimethysulfoxide (7% DMSO, 93% PEG); control mice (n = 11) were injected with vehicle only. At 27 days, harvested tumors were measured and weighed. Lungs were stained for metastases using hematoxylin-eosin. Tumor volume and weights were compared using a two-sample t test. Tumor growth curves were compared using a mixed model analysis of variance. Results.In vitro studies demonstrated dose-dependent inhibition of A549 cell proliferation by VES (IC₅₀ 18 μg/ml). Final tumor volumes and weights differed significantly between VES and control mice with volumes of 292.9 ± 31.4 mm³ vs.192.6 ± 20.4 mm³ (P = 0.01) and weights of 255.7 ± 37.0 mg versus 168.6 ± 20.0 mg (P = 0.05), respectively. Tumor growth curves differed significantly (P < 0.001). Both groups of mice showed pulmonary metastases. Conclusions. Intraperitoneal VES was associated with decreased A549 tumor volume, weight, and growth; however, despite reduced tumor growth, pulmonary metastases were seen in VES-treated mice. Nonetheless, these results suggest that lung cancer patients may benefit from inclusion in eventual clinical studies using VES.