Abstract MYCN amplification strongly predicts adverse outcome of neuroblastoma. However, the significance of MYCN expression in the clinical and biological behavior of neuroblastoma has been unclear. To address this question, we first examined the expression of MYCN in combination with TrkA (a favorable prognostic indicator of neuroblastoma) in 91 primary neuroblastoma by quantitative reverse transcription-PCR and investigated the relationship among patient survival, MYCN, and TrkA expressions. Three subsets of neuroblastoma were defined based on MYCN and TrkA expression. Neuroblastoma expressing the highest level of MYCN but little TrkA were MYCN-amplified cases, which had a 5-year survival of 9.3%. Interestingly, MYCN and TrkA expression showed a linear correlation (r = 0.5664, P < 0.00005) in neuroblastoma lacking MYCN amplification, and the 5-year survival of neuroblastoma patients with low MYCN and low TrkA expressions was 63.7%, whereas those with high expression of both had a 5-year survival of 88.1% (P < 0.00005). This nonlinear distribution of disease outcome relative to MYCN expression in neuroblastoma explains why MYCN expression is not predictive of neuroblastoma disease outcome by dichotomous division of the neuroblastoma cohort. However, high-level MYCN expression is associated with favorable outcome in neuroblastoma lacking MYCN amplification. Furthermore, forced expression of MYCN significantly suppresses growth of neuroblastoma cells lacking MYCN amplification by inducing apoptosis and enhancing favorable neuroblastoma gene expression. Collectively, these data suggest that high-level MYCN expression in neuroblastoma lacking MYCN amplification results in a benign phenotype. Thus, the high MYCN expression confers the opposite biological consequence in neuroblastoma, depending on whether or not MYCN is amplified. (Cancer Res 2006; 66(5): 2826-33)

Supplementary Data from The MYCN Enigma: Significance of MYCN Expression in Neuroblastoma

Intra‐operative tumor spill increases the risk of local recurrence of Wilms tumor, and adversely impacts relapse‐free (RFS) and overall survival (OS) rates.

Neuroblastoma is a common pediatric tumor characterized by clinical heterogeneity. Because it is derived from sympathetic neuroblasts, the NTRK family of neurotrophin receptors plays an integral role in neuroblastoma cell survival, growth, and differentiation. Indeed, high expression of NTRK1 is associated with favorable clinical features and outcome, whereas expression of NTRK2 and its ligand, brain-derived neurotrophic factor (BDNF), are associated with unfavorable features and outcome. AZ64 (Astra Zeneca) is a potent and selective inhibitor of the NTRK tyrosine kinases that blocks phosphorylation at nanomolar concentrations. To determine the preclinical activity of AZ64, we performed intervention trials in a xenograft model with NTRK2-overexpressing neuroblastomas. AZ64 alone significantly inhibited tumor growth compared to vehicle-treated animals ( p = 0.0006 for tumor size). Furthermore, the combination of AZ64 with conventional chemotherapeutic agents, irinotecan and temozolomide (irino–temo), showed significantly enhanced anti-tumor efficacy compared to irino–temo alone [( p < 0.0001 for tumor size, p < 0.0005 for event-free survival (EFS)]. We also assessed the combination of AZ64 and local radiation therapy (RT) on a neuroblastoma hindlimb xenograft model, and the efficacy of local RT was significantly increased when animals were treated simultaneously with AZ64 ( p < 0.0001 for tumor size, p = 0.0006 for EFS). We conclude that AZ64 can inhibit growth of NTRK-expressing neuroblastomas both in vitro and in vivo. More importantly, it can significantly enhance the efficacy of conventional chemotherapy as well as local RT, presumably by inhibition of the NTRK2/BDNF autocrine survival pathway.

TrkB acts as an oncogenic kinase in a subset of human neuroblastomas. Lestaurtinib, a multi-kinase inhibitor with potent activity against Trk kinases, has demonstrated activity in preclinical models of neuroblastoma.

Neuroblastomas (NBs) with favorable outcome usually express TrkA, whereas unfavorable NBs frequently express TrkB and its cognate ligand BDNF. P75 (p75LNTR, NGFR, TNFRSF16) binds NGF-related neurotrophins with low affinity and usually is coexpressed with Trk receptors in NBs. Here, we investigated the importance of p75 coexpression with Trk receptors in NBs. We transfected p75 into two Trk-null NB cell lines, SH-SY5Y and NLF that were also engineered to stably express TrkA or TrkB. Cell numbers were compared between single (Trk alone) and double (Trk+p75) transfectants, and proliferation was assessed by flow cytometry. P75 coexpression had little effect on cell growth in Trk NB cells in the absence of ligand, but it increased sensitivity and greatly enhanced the effect of cognate ligand. Exogenous NGF induced greater phosphorylation of TrkA and AKT. This was associated with increased cell number in TrkA/p75 cells compared to TrkA cells (p<0.01), which was due to increased proliferation in TrkA/p75 cells (p<0.05), followed by differentiation. Exogenous BDNF also increased cell number in TrkB/p75 compared to TrkB cells (p<0.01), due to an increase in proliferation, but without differentiation. Coexpression of p75 also increased specificity of Trk-expressing cells to ligand. NT3-induced phosphorylation of TrkA and AKT was reduced in TrkA/p75 cells. NT3-induced phosphorylation of TrkB (as well as AKT and MAPK) was also reduced with p75 coexpression. Our results suggest that p75 plays an important role in enhancing both the sensitivity of Trk receptors to low levels of ligand, as well as increasing the specificity of Trks to their cognate ligands. It also enhances ligand-induced differentiation in TrkA/p75 but not TrkB/p75 cells.

Purpose: Neuroblastoma, a common pediatric tumor of the sympathetic nervous system, is characterized by clinical heterogeneity. The Trk family neurotrophin receptors play an important role in this behavior. Expression of TrkA is associated with favorable clinical features and outcome, whereas TrkB expression is associated with an unfavorable prognosis. We wanted to determine if the Trk-selective inhibitor lestaurtinib had therapeutic efficacy in a preclinical neuroblastoma model. Experimental Design: We performed intervention trials of lestaurtinib alone or in combination with other agents in TrkB-overexpressing neuroblastoma xenograft models. Results: Lestaurtinib alone significantly inhibited tumor growth compared to vehicle-treated animals [P = 0.0004 for tumor size and P = 0.011 for event-free survival (EFS)]. Lestaurtinib also enhanced the antitumor efficacy of the combinations of topotecan plus cyclophosphamide (P < 0.0001 for size and P < 0.0001 for EFS) or irinotecan plus temozolomide (P = 0.011 for size and P = 0.012 for EFS). There was no additive benefit of combining either 13-cis-retinoic acid or fenretinide with lestaurtinib compared to lestaurtinib alone. There was dramatic growth inhibition combining lestaurtinib with bevacizumab (P < 0.0001), but this combination had substantial systemic toxicity. Conclusions: We show that lestaurtinib can inhibit the growth of neuroblastoma both in vitro and in vivo and can substantially enhance the efficacy of conventional chemotherapy, presumably by inhibition of the Trk/brain-derived neurotrophic factor autocrine survival pathway. It may also enhance the efficacy of selected biological agents, but further testing is required to rule out unanticipated toxicities. Our data support the incorporation of Trk inhibitors, such as lestaurtinib, in clinical trials of neuroblastoma or other tumors relying on Trk signaling pathways for survival. Clin Cancer Res; 16(5); 1478–85

Neuroblastoma, the most common and deadly solid tumor in children, exhibits heterogeneous clinical behavior, from spontaneous regression to relentless progression. Current evidence suggests that the TRK family of neurotrophin receptors plays a critical role in these diverse behaviors. Neuroblastomas expressing TrkA are biologically favorable and prone to spontaneous regression or differentiation, depending on the absence or presence of its ligand (NGF) in the microenvironment. In contrast, TrkB-expressing tumors frequently have MYCN amplification and are very aggressive and often fatal tumors. These tumors also express the TrkB ligand (BDNF), resulting in an autocrine or paracrine survival pathway. Exposure to BDNF promotes survival, drug resistance, and angiogenesis of TrkB-expressing tumors. Here we review the role of Trks in normal development, the different functions of Trk isoforms, and the major Trk signaling pathways. We also review the roles these receptors play in the heterogeneous biological and clinical behavior of neuroblastomas, and the activation of Trk receptors in other cancers. Finally we address the progress that has been made in developing targeted therapy with Trk-selective inhibitors to treat neuroblastomas and other tumors with activated Trk expression.

Neuroblastoma, the most common and deadly solid tumor in children, exhibits heterogeneous clinical behavior, from spontaneous regression to relentless progression. Current evidence suggests that the TRK family of neurotrophin receptors plays a critical role in these diverse behaviors. Neuroblastomas expressing TrkA are biologically favorable and prone to spontaneous regression or differentiation, depending on the absence or presence of its ligand (NGF) in the microenvironment. In contrast, TrkBexpressing tumors frequently have MYCN amplification and are very aggressive and often fatal tumors. These tumors also express the TrkB ligand (BDNF), resulting in an autocrine or paracrine survival pathway. Exposure to BDNF promotes survival, drug resistance, and angiogenesis of TrkB-expressing tumors. Here we review the role of Trks in normal development, the different functions of Trk isoforms, and the major Trk signaling pathways. We also review the roles these receptors play in the heterogeneous biological and clinical behavior of neuroblastomas, and the activation of Trk receptors in other cancers. Finally we address the progress that has been made in developing targeted therapy with Trk-selective inhibitors to treat neuroblastomas and other tumors with activated Trk expression.

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Neuroblastoma is a common solid tumor of childhood that is derived from the neural crest. Expression of epidermal growth factor (EGF) receptors (EGFRs) has been associated with enhanced cell growth and aggressive behavior in other tumors. Here, we examined the expression profile of EGFRs in neuroblastoma cell lines and primary tumors. We found that all 13 neuroblastoma cell lines examined expressed EGFR1 (HER1), most at readily detectable levels. Low levels of other human EGFR family receptors were also detected in almost all cell lines. All primary tumors examined expressed readily detectable levels of HER1 and HER3 and lower levels of HER2 and HER4. EGF had a significant effect on the proliferation of neuroblastoma cell lines in vitro. EGF treatment (100 ng/mL) of the cell lines SY5Y and NLF significantly increased cell number (P < 0.01). EGF stimulated more cells to enter S and G2-M phase, as suggested by flow cytometry, indicating that EGF increases cell number by increasing proliferation, with no appreciable change in apoptosis. EGF exposure resulted in receptor autophosphorylation and activation of both the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. Exposure to 0.5 micromol/L ZD1839, a HER1-specific inhibitor, caused a 40% to 50% reduction in the number of SY5Y and NLF cells grown in medium containing 10% fetal bovine serum (P < 0.01). Even at 0.01 micromol/L, ZD1839 inhibited autophosphorylation of HER1 by EGF. At 0.1 micromol/L, it also blocked phosphorylation of AKT, but not MAPK, in NLF cells. Additional studies showed that the PI3K/AKT-specific inhibitor LY294002 had a more profound effect than the MAPK-specific inhibitor U0126 in blocking EGF-induced cell proliferation. This suggests that the PI3K/AKT pathway is the main signaling pathway responsible for the proliferation effects of EGF in neuroblastomas. Our results also indicate that ZD1839 is a potent inhibitor of neuroblastoma cell proliferation; therefore, it may be a useful, biologically based therapeutic agent for these tumors.

Medical and Pediatric OncologyVolume 41, Issue 2 p. 141-144 Topical Topic Commentary: Symposium on new drugs in cancer therapy October 29–November 2, 2001 Audrey E. Evans MD, Corresponding Author Audrey E. Evans MD evansa@email.chop.edu Children's Hospital of Philadelphia, Division of Oncology, Abramson Research Center, Civic Center Boulevard, Philadelphia, PennsylvaniaChildren's Hospital of Philadelphia, Division of Oncology, 902 Abramson Research Center, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318.Search for more papers by this author Audrey E. Evans MD, Corresponding Author Audrey E. Evans MD evansa@email.chop.edu Children's Hospital of Philadelphia, Division of Oncology, Abramson Research Center, Civic Center Boulevard, Philadelphia, PennsylvaniaChildren's Hospital of Philadelphia, Division of Oncology, 902 Abramson Research Center, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318.Search for more papers by this author First published: 19 June 2003 https://doi.org/10.1002/mpo.10259AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume41, Issue2August 2003Pages 141-144 RelatedInformation

Medical and Pediatric OncologyVolume 41, Issue 6 p. 541-544 Topical Topic 14th EORTC-NCI-AACR symposium on “molecular targets and cancer therapeutics,” November 19–22, 2002, Frankfurt, Germany Audrey E. Evans MD, Corresponding Author Audrey E. Evans MD evansa@email.chop.edu University of Pennsylvania School of Medicine, Department of Pediatrics, Children's Hospital of Philadelphia, Division of Oncology, Suite 902 Abramson Research Building, 3615 Civic Center Boulevard, Philadelphia, PennsylvaniaUniversity of Pennsylvania School of Medicine, Department of Pediatrics, Children's Hospital of Philadelphia, Division of Oncology, Suite 902 Abramson Research Building, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318.Search for more papers by this author Audrey E. Evans MD, Corresponding Author Audrey E. Evans MD evansa@email.chop.edu University of Pennsylvania School of Medicine, Department of Pediatrics, Children's Hospital of Philadelphia, Division of Oncology, Suite 902 Abramson Research Building, 3615 Civic Center Boulevard, Philadelphia, PennsylvaniaUniversity of Pennsylvania School of Medicine, Department of Pediatrics, Children's Hospital of Philadelphia, Division of Oncology, Suite 902 Abramson Research Building, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318.Search for more papers by this author First published: 24 October 2003 https://doi.org/10.1002/mpo.10412Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume41, Issue6December 2003Pages 541-544 RelatedInformation

Medical and Pediatric OncologyVolume 38, Issue 1 p. 49-52 Topical Topic Symposium on new drugs in cancer therapy, November 7–10, 2000 Audrey E. Evans MD, Corresponding Author Audrey E. Evans MD Evans@email.chop.edu Professor of Pediatrics University of Pennsylvania School of MedicineDivision of Oncology, The Children's Hospital of Philadelphia, 3400 Civic Center Boulevard, Philadelphia, PA 19104.Search for more papers by this author Audrey E. Evans MD, Corresponding Author Audrey E. Evans MD Evans@email.chop.edu Professor of Pediatrics University of Pennsylvania School of MedicineDivision of Oncology, The Children's Hospital of Philadelphia, 3400 Civic Center Boulevard, Philadelphia, PA 19104.Search for more papers by this author First published: 21 December 2001 https://doi.org/10.1002/mpo.1263AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume38, Issue1January 2002Pages 49-52 RelatedInformation

Background EPH family receptor tyrosine kinases and their ligand ephrins play pivotal roles in development. High-level expression of transcripts encoding EPHB6 receptors (EPHB6), its ligands ephrin-B2 and ephrin-B3 (EFNB2, EFNB3) is predictive of favorable disease outcome of neuroblastoma (NB). When combined with TrkA expression, the expression of EPHB6, EFNB2, or EFNB3 predicts more accurately the disease outcome than each of the four variables alone. Procedure Cox regression and Kaplan-Meier analyses were used to assess the prognostic significance of EPHB6, EFNB2, EFNB3, and TrkA expressions in NB without MYCN amplification. Results High-level expression of EFNB3 or TrkA predicted favorable NB outcome of NB without MYCN amplification (p < 0.03). As found in the general NB population, EPHB6, EFNB2, or EFNB3 expression in combination with TrkA expression was significantly predictive of the disease outcome of normal MYCN NB (p < 0.01). Conclusions EPHB6, EFNB2, and EFNB3 expressions may permit further refinement of the prognostic stratification of NB into favorable and unfavorable groups. Med. Pediatr. Oncol. 35:656–658, 2000. © 2000 Wiley-Liss, Inc.