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.     

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.     

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.     

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.     

Feasibility of thoracoscopic repair of esophageal atresia with distal fistula

Backgound/Purpose: Evaluation of the feasibility of thoracoscopic correction of esophageal atresia with distal fistula. Methods: Eight consecutive neonates with esophageal atresia and distal fistula were treated thoracoscopically. Mean birth weight was 3,048 g (range, 2,140 to 3,770). The patients were intubated endotracheally and placed in a [frac34] left prone position. Three cannulae were inserted along the inferior tip of the scapula. CO₂ was insufflated at a pressure of 5mm Hg and a flow of 0.5 L/min. The fistula was either clipped or ligated. The proximal esophagus was opened and an anastomosis was made over a 6F or 8F nasogastric tube with interrupted 5-0 Vicryl. Results: All procedures were completed thoracoscopically without major peroperative complications. The mean operating time was 198 minutes (range, 138 to 250). One patient had a major leak, resulting in a stormy postoperative course, but the leak healed on conservative treatment. This patient and 3 others had stenosis requiring dilatation, respectively, 3, 6, 12, and 1 times. The babies were fed after a median period of 8 days. The median hospital stay was 13 days. Conclusions: Thoracoscopic repair of esophageal atresia with distal fistula is feasible. Larger series are needed to determine the exact place of the thoracoscopic approach.     

Expression of PPAR-gamma is correlated with the clinical course of neuroblastoma

Background/Purpose: Neuroblastoma is a common pediatric tumor of the sympathetic nervous system. Unlike the ones found in older children, the tumors found in patients younger than one year of age often show spontaneous differentiation and regression. Peroxisome proliferator-activated receptor gamma (PPAR-[gamma ]), a member of the nuclear hormone receptor superfamily is expressed in several human cancers. Recently, PPAR-[gamma ] has been reported to be expressed in neuroblastoma, and the agonist of this receptor caused differentiation of neuroblastoma cells. Methods: In this report we studied the expression of PPAR-[gamma ] mRNA, using LightCycler in neuroblastoma samples diagnosed in 17 patients under the age of one year. Results: Twelve samples showed PPAR-[gamma ] mRNA expression. There was no significant difference in the PPAR-[gamma ] mRNA expression based on age, histology, staging, and DNA ploidy. The PPAR-[gamma ] mRNA expression level was significantly correlated with the change in urinary vanillyl mandelic acid (VMA). The neuroblastoma samples resected from patients who showed a decrease in their urinary VMA before the operation showed significantly higher PPAR-[gamma ] expression than those from patients who showed an increase in their urinary VMA before the operation. Conclusions: PPAR-[gamma ] may have played a role in the reduction of VMA and possibly in the regression of early-onset neuroblastoma. J Pediatr Surg 38:205-210.     

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.     

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.     

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.     

Alterations in hepatic microcirculation, mitochondrial redox state and tissue oxygenation after trauma and sepsis

Introduction: Hepatic microcirculatory dysfunction and mismatch of blood supply to metabolic demand play an important role in liver failure after stress. Here, we assessed hepatic microcirculation in response to the vasoregulator Endothelin-1 (ET-1) after femur fracture (FFx) and cecal ligation and puncture (CLP). We hypothesized that sequential stress causes hepatic microcirculatory dysfunction leading to altered mitochondrial redox state and tissue PO₂ (tPO₂). Methods: Male Sprague-Dawley rats (200-300 g, n = 4/groups) underwent sham surgery, FFx, CLP and FFx+CLP. Using intravital microscopy, hepatic microcirculation was assessed by sinusoidal volumetric flow (pL/sec) and perfusion index (PI, pL/sec/200μm). Changes in hepatic mitochondrial redox state and liver tPO₂ were determined by NADH autofluorescence (AU, arbitrary unit) and ruthenium (Ru, AU) fluorescence, respectively. Western blot analysis was used to determine ET_A/ET_B receptor protein expression (AU). Differences among groups were compared using one-way ANOVA followed by Student-Newman-Keuls post hoc test. Results: After 10 minutes of intraportal ET-1 infusion, liver microcirculation was altered in CLP and FFx+CLP groups (Table, values are mean ± SE). Sequential stress also led to increased NADH and Ruthenium fluorescence intensity, indicating a reduction in O₂ availability. Vasoactive receptor proteins ET_A and ET_B were upregulated after injury. Conclusion: Our data suggest that sepsis and sequential stress result in liver microcirculatory disturbances leading to altered global hepatic perfusion, and contribute toward a reduction in O₂ supply. These changes in the liver microcirculation, mediated in part by Endothelin-1, may play an important role in the development of liver failure after inflammatory stress.     

Distinct response of experimental neuroblastoma to combination antiangiogenic strategies

Background: The authors have shown previously that experimental neuroblastoma is partially inhibited (48%) by antivascular endothelial growth factor (anti-VEGF) antibody. The topoisomerase-I inhibitor, topotecan, has been shown to have antiangiogenic activity when administered in a low-dose, high-frequency regimen. We hypothesized that combining topotecan with anti-VEGF would suppress neuroblastoma more effectively than either agent alone. Methods: A total of 10⁶ neuroblastoma cells were implanted intrarenally in athymic mice. Animals received vehicle, topotecan, anti-VEGF, or topotecan plus anti-VEGF (n = 9, 20, 20, 20, respectively). All control and half the treated mice were killed at 6 weeks. Remaining (rebound) mice were maintained without treatment for 3 more weeks. Patterns of vasculature and apoptosis were determined immunohistochemically. Results: Tumor weights at 6 weeks were reduced significantly in topotecan-only (0.07g) and combination-treated animals (0.08 g), compared with controls or anti-VEGF[ndash ]treated mice (1.18 g, 0.53 g; P [lt ] .0007, all). At 9 weeks, rebound tumor weights were greatest in anti-VEGF (2.82 g), intermediate in topotecan (1.82 g), and least in combination-treated animals (1.47 g); however, the only significant difference was between anti-VEGF and combination therapy (P = 0.04). All treated tumors were vascularized sparsely in comparison with controls at 6 weeks, but exhibited brisk neoangiogenesis at 9 weeks. Conclusions: Topotecan either with or without anti-VEGF antibody significantly suppresses neuroblastoma xenograft growth in comparison with controls or anti-VEGF antibody alone. Combining topotecan with anti-VEGF antibody significantly inhibited rebound tumor growth in comparison with anti-VEGF antibody alone. Combination therapy may improve durability of antiangiogenic inhibition of neuroblastoma.     

Intestinal epithelial cells exposed to heat shock stress release a soluble compound that activates the virulence of P. aeruginosa

Introduction. Although there is a significant body of evidence that bacteria activate a variety of pro-inflammatory signals following their interaction with human epithelial cells, there is virtually no information on signaling in the opposite direction. In this study we sought to determine whether host cell factors released by intestinal epithelial cells subjected to stress could signal the opportunistic pathogen, P. aeruginosa, to express a key virulence related protein, the PA-I lectin. Methods. Human intestinal epithelia (Caco-2_bbe) were grown to confluence in HDMEM media. To expose cells to a clinically relevant stress, hyperthermic conditions were created by immersing sealed culture dishes in a 42°C water bath for 23 min, followed by 2 h of recovery at 37°C and 5% CO₂. Control cells were maintained in 37°C at 5% CO_2.. Media were then filtered through a 0.22-μm filter before serial centrifugal membrane filtration using 100, 50, 30, 10, and 3 kDa filters and resuspended in HDMEM. GFP reporter strains of P. aeruginosa carrying a fusion construct for the PA-I lectin were then exposed to the epithelial cell derived filtrates from heat shock stressed Caco-2 cells. Dynamic fluorimetry was used to measure fluorescence (i.e. PA-I activity) over time. To determine if inducing compounds were proteins, fractions capable of activating PA-I were then heat inactivated by heating to 70°C for 30 min and tested against active fractions by the same method. Results. A 30- to 50-kDa media fraction from heat-stressed cells induced a 10-fold increase in PA-I promoter activity over controls, as measured by fluorescence (1082 ± 216%, P < 0.01), while all other fractions showed no effect. Heat inactivation completely eliminated this effect (−156% of control ± 293%, P = NS). Conclusion. Human intestinal epithelial cells release a soluble 30- to 50-kDa compound(s) that can activate a key virulence protein in P. aeruginosa. We speculate that the molecular dialogue between a pathogen and the intestinal epithelium is bidirectional and can be initiated by host stress conditions such as hyperthermia.     

Surgical management for intractable cholangitis in biliary atresia

Background/Purpose: The efficacy of antireflux surgical procedures involving the Roux-en-Y jejunal limb for cholangitis was evaluated retrospectively in patients with biliary atresia (BA). Methods: From July 1993 to December 2001, 41 patients with BA underwent hepatic portojejunostomy with Roux-en-Y reconstruction. Of these patients, 11 had intractable cholangitis that was treated by creation of a value with or without lengthening of the Roux-en-Y limb. Results: Among the 11 patients, the first episode of cholangitis occurred within 6 months after portojejunostomy in 10 patients and at the age of 4 years in one patient. Cholangitis developed at various intervals from once every week to once every 2 months requiring hospitalization each time. All patients underwent valve creation at 2 months to 5 years postoperatively, whereas 2 had an additional lengthening of the limb. Cholangitis resolved completely after surgery in all cases. Two patients underwent liver transplantation, and the third patient died of an unrelated cause. The 8 survivors with native livers are doing well after 1 to 8 years of follow-up. Conclusion: Early surgical intervention could control intractable cholangitis in all patients, both delaying the time of liver transplantation and improving the quality of life.     

Laparoscopy-assisted suction colonic biopsy and intraoperative rapid acetylcholinesterase staining during transanal pull-through for Hirschsprung's disease

Background: It is crucial to identify the exact level of transition to normal ganglion cells in instances of Hirschprung's disease. This report describes a technique for laparoscopy-assisted suction colonic biopsy during transanal pull-through. Methods: Laparoscopy-assisted suction colonic biopsy (SCBx) was used in 12 patients with Hirschsprung's disease affecting the rectosigmoid. Average age was 4.4 [plusmn] 2.1 months with a mean operative weight 6.2 [plusmn] 1.0 kg. The pull-through was performed as the primary operative procedure in 11 patients. Using a 2-team approach (laparoscopic team and transanal team), the site was chosen for transanal suction biopsy and marked externally by the laparoscopic team with a silver clip. Biopsies were processed for ganglion cells and rapid AChE technique. Results: There were no biopsy-induced perforations. Abnormal biopsies were repeated more proximally until ganglion cells were observed. Transanal pull-through was performed and an open full-thickness biopsy performed to confirm the presence of ganglion cells. All procedures were performed successfully. Conclusions: Laparoscopy-assisted SCBx can be used successfully in patients with Hirschsprung's disease affecting the rectosigmoid (80% of cases). The technique, when used with rapid AChE staining, provides accurate identification of the level of normoganglionosis.     

The survivin:Fas ratio is predictive of recurrent disease in neuroblastoma

Background/Purpose: Several clinical and biologic features of neuroblastoma (NB) are used to predict the risk of recurrent disease. The balance between antiapoptotic and proapoptotic factors within a tumor may affect its ability to survive. Survivin is an antiapoptotic factor expressed in highly proliferative NB, whereas Fas is a proapoptotic factor that portends a favorable prognosis. The authors determined whether the ratio of survivin to Fas (S:F ratio) is predictive of recurrent disease in patients with NB. The authors previously have shown the S:F ratio is predictive of recurrent disease in pediatric renal tumors. Methods: The authors quantified the levels of 9 different apoptotic mRNA species using Rnase Protection assay (RPA, Riboquant, PharMingen, San Diego, CA). Twenty-eight primary tumor specimens were evaluated from patients with ganglioneuroma (n = 3), ganglioneuroblastoma (n = 2), and neuroblastoma (n = 23) from tumors of all clinical stages obtained at the time of diagnosis. mRNA levels were calculated as a percentage of L32 for each specimen assayed, and positive expression was assumed to be greater than 10% of L32. Results: Survivin was expressed in 90% of tumors that went on to recur and only in 27.7% of those that were cured. The S:F ratio was significantly greater in tumors that went on to recur (n = 10) compared with those from patients that were cured (n = 18) (median S:F ratio, 3.3 v 0.75; P = .0002, Wilcoxon rank-sum test). A cutoff ratio of 2.3 was highly predictive of tumor recurrence irrespective of clinical stage of disease (area under ROC curve = 0.906). Sensitivty was 80% (CI, 44.4% to 97.5%), specificty was 94.4% (CI, 72.7% to 99.9%), positive predictive value was 88.9% (CI, 51.8% to 99.7%), and negative predictive value was 89.5% (66.9% to 98.7%). Twenty-five of 28 (89.3%) tumor ratios were correct in predicting outcome. Conclusions: The survivin: Fas ratio in primary tumors may be used to predict the risk for recurrent disease in patients with NB. The S:F ratio appears to be a more sensitive predictor of recurrent disease than survivin expression alone. Determining this ratio may not only be helpful in guiding follow-up of patients with NB, but also may aid in stratifying patients for more aggressive therapeutic strategies.