Solid Tumor In Infants Research Articles

Secondary T-cell lymphoblastic lymphoma in a child after anticancer therapy for neuroblastoma: clinical case

Neuroblastoma (NB) is the most common extra-cranial solid tumor in infants with the most heterogeneous clinical course to compare with other malignant diseases. Due to intensive multimodal anticancer treatment there are an increased number of survivors and issues related to long-term effects are becoming increasingly important. One of them is the risk of secondary malignant neoplasms. This article represents a clinical case of T-cell lymphoblastic leukemia in a child aged 2 years and 5 months who received combined antitumor therapy for NB with an intermediate risk group under the age of one year. We observed literature data to investigate the incidence of second malignant neoplasms in patients with NB for the period from 1948 to 2018 and analyzed risk factors.

Epigenome-wide association study reveals CpG sites related to COG of neuroblastoma

Background. Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children. Its variable location and complex pathogenesis make NB hard for early diagnosis and risk classification. Methodology. We analyzed the methylation data of 236 samples from patients with NB in Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. Kaplan–Meier survival analysis was used for comparing overall survival of NB patients in different groups. Epigenome-wide association study (EWAS) was conducted to screen CpGs significantly associated with NB patients’ Children’s Oncology Group (COG). Logistic regression method was used for constructing a model to predict NB patients’ COG. Results. NB patients in low COG showed significantly superior prognosis than those in high COG. A total of seven CpG sites were found closely related to COG. Logistic regression model based on those CpGs showed superior performance in separating NB patients in different COGs. Conclusions. The present study highlights the important role of DNA methylation in NB development, which might provide evidence for treatment decisions for children NB.

MicroRNA-143 functions as a tumor suppressor in neuroblastoma by targeting bone morphogenetic protein-7

IntroductionNeuroblastoma (NB) is one of the most common types of extracranial malignant solid tumor in infants and children. MicroRNA-143 (miR-143) was reported to be dysregulated in several human cancers. However, the function and mechanism of miR-143 in NB are still unclear. The purpose of this study was to explore the biological functions of miR-143 in NB progression.Material and methodsThe expression of miR-143 in 32 NB tissue samples and 4 NB cell lines was detected by quantitative real-time PCR. Then, bioinformatics analysis combined with luciferase reporter assay were used to identify the target gene of miR-143 in SK-N-SH cells. Cell proliferation, migration and invasion assay were performed to investigate the roles of miR-143 in NB progression.ResultsOur study revealed that miR-143 was significantly downregulated in NB tissues as well as cell lines. Bone morphogenetic protein-7 (BMP7) was a direct target of miR-143 in NB cells and inversely associated with the expression of miR-143 in NB tissues. Overexpression of miR-143 inhibited NB cell proliferation, migration and invasion through targeting BMP7.ConclusionsOur data for the first time demonstrated that miR-143 functions as a tumor suppressor in NB by directly targeting BMP7, and also suggest miR-143 as a potential thera­peutic target for NB patients in the future.

Epidemiology and prognostic factors of infantile malignant solid tumor

As an important part of childhood tumors, infantile malignant solid tumor is relatively rare, which has some common characteristics of childhood cancer as well as its own particularity.Most infantile tumors are embryo-derived tumors, whose pathogenesis is related to developmental biology, genetics and environmental factors.The distribution of constituent ratio of infantile malignant tumors is different from that of older children, and the epidemiological data statistics can be influenced by the imperfect children tumor registration system, population migration, birth defects and perinatal factors, environmental exposure, gender, race and other factors.Heterogeneity is one of the prominent features of malignant solid tumor in infants. The experience gained from treatment of older children cannot be directly extrapolated to infants.The principle of treating infantile malignant solid tumors should take into account both the effectiveness and the long-term impact on infant growth.Early diagnosis and appropriate treatment mean that the prognosis of most infants with malignant solid tumor to be better than that of older children.Therefore, it is necessary to manage these tumors as a separate entity.The advance of epidemiology and prognostic factors of infantile malignant solid tumors at home and abroad was reviewed in this paper. Key words: Infant; Malignant; Solid tumor; Incidence rate; Constituent ratio; Prognosis

Lymphatic Leakage after Surgery for Neuroblastoma: A Rare Complication?

Neuroblastoma is the most common extracranial solid tumor in infancy. It is responsible for around 15% of all oncological deaths during childhood. Due to its retroperitoneal location, neuroblastoma is invasively growing directly in and around the lymphatic duct. Consecutively, lymphatic leakage (LL) after surgery for neuroblastoma is a known complication. The purpose of this study is the investigation of frequency and impact of this complication. Between February 2003 and December 2016, 204 patients with neuroblastoma received surgical treatment in our department. A retrospective analysis for macroscopical extent of resection, duration of drainage postsurgery, maximum amount of fluid drained in 24 hours, MYCN amplification status, therapeutic options for LL, follow-up status, and overall survival was performed. A total of 40% of patients (82/204) showed LL to some extent. In patients with MYCN amplification, LL was seen significantly more often than in patients without MYCN amplification status (p = 0.019). LL was also significantly correlated with extent of surgery (p = 0.005). Follow-up status and overall survival were significantly inversely associated with LL (p = 0.004 and p = 0.0001). LL was self-limiting in all cases. There was a trend toward shorter duration of LL if either no special therapy was chosen or total parenteral nutrition (TPN) was administered (p = 0.0603). We show that LL in neuroblastoma is a common complication of tumor resection and occurring more often than anticipated. Since, in our study cohort, all cases of LL were self-limiting, we question the indication for invasive therapy besides supporting measures.

Perfusion-guided sonopermeation of neuroblastoma: a novel strategy for monitoring and predicting liposomal doxorubicin uptake in vivo.

Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, and imposes significant morbidity and mortality in this population. The aggressive chemoradiotherapy required to treat high-risk NB results in survival of less than 50%, yet is associated with significant long-term adverse effects in survivors. Boosting efficacy and reducing morbidity are therefore key goals of treatment for affected children. We hypothesize that these may be achieved by developing strategies that both focus and limit toxic therapies to the region of the tumor. One such strategy is the use of targeted image-guided drug delivery (IGDD), which is growing in popularity in personalized therapy to simultaneously improve on-target drug deposition and assess drug pharmacodynamics in individual patients. IGDD strategies can utilize a variety of imaging modalities and methods of actively targeting pharmaceutical drugs, however in vivo imaging in combination with focused ultrasound is one of the most promising approaches already being deployed for clinical applications. Over the last two decades, IGDD using focused ultrasound with “microbubble” ultrasound contrast agents (UCAs) has been increasingly explored as a method of targeting a wide variety of diseases, including cancer. This technique, known as sonopermeation, mechanically augments vascular permeability, enabling increased penetration of drugs into target tissue. However, to date, methods of monitoring the vascular bioeffects of sonopermeation in vivo are lacking. UCAs are excellent vascular probes in contrast-enhanced ultrasound (CEUS) imaging, and are thus uniquely suited for monitoring the effects of sonopermeation in tumors.Methods: To monitor the therapeutic efficacy of sonopermeation in vivo, we developed a novel system using 2D and 3D quantitative contrast-enhanced ultrasound imaging (qCEUS). 3D tumor volume and contrast enhancement was used to evaluate changes in blood volume during sonopermeation. 2D qCEUS-derived time-intensity curves (TICs) were used to assess reperfusion rates following sonopermeation therapy. Intratumoral doxorubicin (and liposome) uptake in NB was evalauted ex vivo along with associated vascular changes.Results: In this study, we demonstrate that combining focused ultrasound therapy with UCAs can significantly enhance chemotherapeutic payload to NB in an orthotopic xenograft model, by improving delivery and tumoral uptake of long-circulating liposomal doxorubicin (L-DOX) nanoparticles. qCEUS imaging suggests that changes in flow rates are highly sensitive to sonopermeation and could be used to monitor the efficacy of treatment in vivo. Additionally, initial tumor perfusion may be a good predictor of drug uptake during sonopermeation. Following sonopermeation treatment, vascular biomarkers show increased permeability due to reduced pericyte coverage and rapid onset of doxorubicin-induced apoptosis of NB cells but without damage to blood vessels.Conclusion: Our results suggest that significant L-DOX uptake can occur by increasing tumor vascular permeability with microbubble sonopermeation without otherwise damaging the vasculature, as confirmed by in vivo qCEUS imaging and ex vivo analysis. The use of qCEUS imaging to monitor sonopermeation efficiency and predict drug uptake could potentially provide real-time feedback to clinicians for determining treatment efficacy in tumors, leading to better and more efficient personalized therapies. Finally, we demonstrate how the IGDD strategy outlined in this study could be implemented in human patients using a single case study.

Primary cerebral neuroblastoma in young adult

Neuroblastoma is an embryonal malignancy of the sympathetic nervous system arising from pluripotent sympathetic cells (neuroblasts). It is the most common extracranial solid tumor in infants. Although it can arise anywhere along sympathetic chain, it is extremely unusual for neuroblastoma to present as intracranial lesion and that too in a young adult. Here, we describe a 24-year-old male presented with complaints of headache and retro-orbital pain. Magnetic resonance imaging (MRI) of the brain revealed a left occipital lobe mass lesion. He underwent left parieto-occipital craniotomy and near-total excision. Postoperative MRI showed minimal residual lesion. Initial histopathological examination was reported as a malignant round cell tumor. However, block and slide review with immunohistochemistry markers was suggestive of neuroblastoma. He received adjuvant radiation of 50.4 Gy in 28 fractions, followed by eight cycles of adjuvant chemotherapy with lomustine and cisplatin. At 3-year follow-up, MRI showed post-operative gliosis in left parietal lobe with no evidence of recurrence.

1219P - Maternal immunization against ALK as a weapon to fight neuroblastoma

BackgroundNeuroblastoma (NB) is the most common extracranial solid tumor in infancy. Some germline and somatic mutations associated with NB have been identified as activating mutations of the anaplastic lymphoma kinase (ALK) oncogene. Because of the nature of NB, which can occur in the early post-natal life or even during fetal life, we evaluated the efficacy of a DNA vaccine against ALK in a spontaneous preclinical model of NB, harboring ALKF1174L mutation in association with MYCN amplification (ALKF1174L/MYCN mice) by means of maternal immunization (MI). MethodsMYCN transgenic females were immunized against ALK by DNA vaccination by using a plasmid coding for the extracellular and trans-membrane domains of human ALK (ALK-ECTM) followed by electroporation, and then they were mated with an ALKF1174L-transgenic male. In ALKF1174L/MYCN offspring, the presence of abdominal, cervical and paraspinal tumors has been evaluated and quantified by Magnetic Resonance Imaging. The humoral immune response induced against ALK in the mothers and their offspring, as well as the presence of immune-complexes containing ALK, have been evaluated by ELISA. ALK expression in tumor tissue was assessed by Western blot. ResultsPre-birth immunization against ALK leads to an extended survival time and to a lower tumor growth kinetic in ALKF1174L/MYCN offspring born from ALK-ECTM-vaccinated mothers (ALK-ECTM offspring) as compared to controls born from control empty vector-vaccinated mothers. Maternally derived anti-ALK antibodies were successfully transferred from mothers to newborns. Moreover, anti-ALK IgM were found in the sera of five- and six-week old ALK-ECTM offspring, suggesting the induction of the pups’ own immune response against ALK. This effect could be due to the breast milk-mediated transfer of immune-complexes containing ALK, found in the milk of vaccinated mothers and in their offspring sera. Finally, MI against ALK induces a decrease in ALK expression in ALK-ECTM offspring’ tumor tissue. ConclusionsOverall, these results indicate that MI against ALK induces an active immunization against this oncoantigen in the offspring, impairing tumor development and enhancing survival time in a preclinical model of NB. Legal entity responsible for the studyDepartment of Molecular Biotechnology and Health Sciences. FundingAssociazione Italiana per la ricerca sul Cancro. DisclosureAll authors have declared no conflicts of interest.

Abstract 2881: Antiproliferative effect of copper complex of clotam in neuroblastoma cell lines

Abstract Neuroblastoma (NB) is the most common extracranial solid tumor in infants and young children. NB often metastasizes to lymph nodes and bone marrow. When compared to low or intermediate risk tumors, high risk neuroblastoma (HRNB) is more aggressive with low (40-50%) 5 year survival rates. Furthermore, current intensive treatment methods induce higher toxicity and side effects. In search of identifying less-toxic agents to induce anti-cancer activity in NB cells, our group reported the anti-cancer activity of a non-steroidal anti-inflammatory drug, Clotam (TA) using HRNB cell lines potentially inhibiting the transcription factor, Specificity protein 1 (Sp1) and an inhibitor of apoptosis protein, survivin. Recently, our laboratories tested a copper complex of TA (Cu-TA) using 12 cell lines and found that Cu-TA’s IC50 values were 30-80% lower when compared to TA. In this study, we investigated the cytotoxicity of SH-SY 5Y, LA1-55n and SMS-KCNR cells in the presence of vehicle (Dimethyl sulfoxide) or TA or Cu-TA using CellTiter-Glo kit and determined the IC50 values using SigmaPlot software. The effect of TA or Cu-TA on apoptosis and cell cycle phase distribution was evaluated by flow cytometry using Annexin-V and Propidium Iodide kits respectively. The characterization and stability of the Cu-TA was also determined. SH SY5Y and SMS-KCNR cells were treated with IC50 values of TA or Cu-TA and the expression of Sp1 and survivin was measured by qPCR and Western blot analysis. The results showed that while both TA and Cu-TA inhibited NB cell growth, Cu-TA’s IC50 values were 60-70% less than TA. The inhibition of NB cells with Cu-TA was accompanied by induced apoptotic cell population and cell cycle arrest at G2M phase. Western blot and qPCR results confirmed that Cu-TA inhibited the expression of both Sp1 and survivin with predominant effect on SMS-KCNR cells. These results demonstrate that Cu-TA is more effective than TA for inhibiting Sp1 and survivin in NB cells, which was consistent with the anti-proliferative activity against NB cells. Since TA and Cu-TA are less toxic than standard chemotherapeutic agents, employing these agents for the treatment of pediatric malignancies could be useful. Eventually, combination of Cu-TA and chemotherapeutic agents will be tested. If Cu-TA sensitizes NB cells to chemotherapy, it will help to address the issues associated with the side effects of chemotherapy. Citation Format: Yazmin Hernandez, Sarah Sarah Grebennikov, Emily Zakhary, Ashni Dudhia, Rajasekhar Maram, Diego Casanova, Umesh T. Sankpal, W. Paul Bowman, Deondra T. Brown, Jaya Chhabra, Alvin A. Holder, Riyaz M. Basha. Antiproliferative effect of copper complex of clotam in neuroblastoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2881.

Abstract 2616: A novel iron-chelating agent reduces MYC transcription via E2F gene family regulation

Abstract Childhood neuroblastoma, a disease of the sympathetic nervous system, is the most common solid tumour of infancy. Amplification of the MYCN oncogene, which occurs in approximately 25% of patients with neuroblastoma, is one of the most powerful independent prognostic markers for this disease. MYCN belongs to the Myc gene family that codes for transcription factors often aberrantly expressed in both solid and liquid malignancies. However, despite their importance in adult and childhood malignancies, Myc oncoproteins have to date proven to be ‘undruggable’. Using a chemical library of 34,000 compounds we aimed to identify novel MYCN inhibitors, and among a number of molecules identified, M606 was found to markedly reduce MYCN protein levels as well as its downstream targets in MYCN-amplified SK-N-BE(2)-C neuroblastoma cells. Bioinformatic analysis of signalling pathways affected by M606 using FACTORIAL™ technology (Attagene Inc) demonstrated that this compound inhibits Myc-mediated transcription and simultaneously activates the HIF1α pathway. MYCN/MYC protein and mRNA stability were unaltered whereas their mRNA levels were significantly decreased. Metabolomics analysis suggested that the mechanism by which M606 inhibits MYCN transcription involves its capacity to chelate iron and, in this regard, M606-mediated inhibition of MYCN was reversed by the addition of iron. Progressive deletions of the MYCN promoter in luciferase reporter assays identified a minimal promoter region responsive to M606 that contains two E2F sites. Microarray assays performed on SK-N-BE(2)-C cells after M606 treatment showed significant changes in the expression level of the E2F gene family. Chromatin immune precipitation (ChIP) and gel shift (EMSA) assays revealed a reduction in E2F binding at the MYCN promoter region after M606 treatment. Moreover, western blot analysis showed that the RB protein, which is involved in E2F binding and transcriptional activation, becomes inactive due to hypophosphorylation following M606 treatment. On the other hand, M606 lost its inhibitory activity on a pRB-deficient osteosarcoma cell line SAOS-2, underlying the importance of the RB/E2F axis for M606 function. In conclusion, M606 is a novel Myc inhibitor and iron chelator that directly downregulates MYCN/MYC transcription via the E2F signalling pathway, providing a potentially valuable therapeutic approach in the treatment of cancers overexpressing Myc oncoproteins. Citation Format: Emanuele Valli, Chengyuan Xue, Leanna Cheung, Laura Gamble, Ruby Pandher, Simone Di Giacomo, Catherine Burkhart, Natalia Fedtsova, Francesca Ferrucci, Sergei Makarov, Thomas Telfer, Rachel Codd, David Scott, Giovanni Perini, Andrei Osterman, Andrei Gudkov, Murray Norris, Michelle Haber. A novel iron-chelating agent reduces MYC transcription via E2F gene family regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2616.

MYCN is amplified during S phase, and c‑myb is involved in controlling MYCN expression and amplification in MYCN‑amplified neuroblastoma cell lines.

Neuroblastoma derived from primitive sympathetic neural precursors is a common type of solid tumor in infants. MYCN proto-oncogene bHLH transcription factor (MYCN) amplification and 1p36 deletion are important factors associated with the poor prognosis of neuroblastoma. Expression levels of MYCN and c-MYB proto-oncogene transcription factor (c-myb) decline during the differentiation of neuroblastoma cells; E2F transcription factor 1 (E2F1) activates the MYCN promoter. However, the underlying mechanism of MYCN overexpression and amplification requires further investigation. In the present study, potential c-Myb target genes, and the effect of c-myb RNA interference (RNAi) on MYCN expression and amplification were investigated in MYCN-amplified neuroblastoma cell lines. The mRNA expression levels and MYCN gene copy number in five neuroblastoma cell lines were determined by quantitative polymerase chain reaction. In addition, variations in potential target gene expression and MYCN gene copy number between pre- and post-c-myb RNAi treatment groups in MYCN-amplified Kelly, IMR32, SIMA and MHH-NB-11 cell lines, normalized to those of non-MYCN-amplified SH-SY5Y, were examined. To determine the associations between gene expression levels and chromosomal aberrations, MYCN amplification and 1p36 alterations in interphases/metaphases were analyzed using fluorescence in situ hybridization. Statistical analyses revealed correlations between 1p36 alterations and the expression of c-myb, MYB proto-oncogene like 2 (B-myb) and cyclin dependent kinase inhibitor 1A (p21). Additionally, the results of the present study also demonstrated that c-myb may be associated with E2F1 and L3MBTL1 histone methyl-lysine binding protein (L3MBTL1) expression, and that E2F1 may contribute to MYCN, B-myb, p21 and chromatin licensing and DNA replication factor 1 (hCdt1) expression, but to the repression of geminin (GMNN). On c-myb RNAi treatment, L3MBTL1 expression was silenced, while GMNN was upregulated, indicating G2/M arrest. In addition, MYCN gene copy number increased following treatment with c-myb RNAi. Notably, the present study also reported a 43.545% sequence identity between upstream of MYCN and Drosophila melanogaster amplification control element 3, suggesting that expression and/or amplification mechanisms of developmentally-regulated genes may be evolutionarily conserved. In conclusion, c-myb may be associated with regulating MYCN expression and amplification. c-myb, B-myb and p21 may also serve a role against chromosome 1p aberrations. Together, it was concluded that MYCN gene is amplified during S phase, potentially via a replication-based mechanism.

Abstract 3191: The MDM2 inhibitor CGM097 synergizes with the BET inhibitor OTX015 to induce cell death in neuroblastoma cells

Abstract Background: Neuroblastoma (NB) is the most common extracranial solid tumor in infants and children, with amplification of the oncogene MYCN being a hallmark of high-risk disease and poor prognosis. Most NB tumors initially respond to chemotherapy; however, over half of these cases relapse, resulting in chemoresistant disease. In this stage of NB there is evidence of p53 inactivation, which suggests a role for p53 in chemo-sensitivity. Although highly mutated in other cancer types, p53 mutations in NB are rare. Previous data have shown that amplification or deregulation of MDM2, a negative regulator of p53, may be the more common mechanism of p53 suppression and chemoresistance in NB. MYCN and MDM2 have been shown to interact and contribute to NB growth and disease progression. In vitro treatment of NB cells with MDM2 inhibitors has shown promise in increasing the expression of p53, leading to a decrease in proliferation, and increasing apoptosis. BET (Bromodomain and Extra-Terminal domain) inhibitors have also been shown to be effective in treating NB cells in vitro, decreasing MYCN expression, and resulting in increased apoptosis and differentiation. We hypothesize that the combination treatment of a MDM2 inhibitor (CGM097) with a BET inhibitor (OTX015) will result in greater p53 activation and a subsequent synergistic increase in NB cell death. Methods: Single-agent IC50 values for both CGM097 and OTX015 were obtained for the established NB cell lines SMS-KCNR, SH-SY5Y, BE(2)-C, and CHLA-90 using Calcein AM and Cell Titer-Glo cell viability assays after 48 hours of treatment. Drug combination studies were performed to evaluate synergism between CGM097 and OTX015 in the treatment of NB cells. IncuCyte ZOOM live cell imaging was used for kinetic monitoring of apoptosis with single-agent and combination drug treatment in NB cells. Western blot analysis was used to determine protein levels of apoptosis (Caspase-3) and MYCN/p53 pathway targets (MYCN, MDM2 and p53). Results: IC50 values for the four established NB cell lines ranged from 0.384 µM-13.5 µM and 7.25 µM-42.79 µM for CGM097 and OTX015, respectively. The combination treatment exhibited synergistic effects in wild-type p53 NB cells, with supporting data obtained from IncuCyte imaging tracking real-time cell death. IncuCyte showed induction of caspase-mediated apoptosis confirmed by Western blot analysis of Cleaved Caspase 3. Pathway analysis shows the importance of wild-type p53 for treatment effect. Conclusion: This study indicates that the combination of CGM097 and OTX015 synergistically decreases viability in NB cells with wild-type p53 expression. Further in vitro and in vivo work will be necessary to elucidate the mechanisms behind this interaction and in vivo efficacy. Citation Format: Tyler P. Maser, Joseph W. Zagorski, Austin J. Goodyke, Elizabeth A. VanSickle, Jeffrey P. Bond, Giselle L. Saulnier Sholler. The MDM2 inhibitor CGM097 synergizes with the BET inhibitor OTX015 to induce cell death in neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3191.

MicroRNAs in Neuroblastoma: Biomarkers with Therapeutic Potential.

Neuroblastoma is the most common extracranial solid tumor in infancy. The majority of children have a disseminated disease at diagnosis with bone marrow as the most common site of metastasis. Although several prognostic factors have been defined (i.e. age, stage, histology, recurrent genetic anomalies), the identification of non-invasive biomarkers for disease follow-up and therapy monitoring is indeed still a clinical need. Aberrant regulation of microRNAs (miRNAs) expression has been implicated in several malignancies. In this mini-review, we describe the recent findings about miRNAs in neuroblastoma, both in the tumor and circulation, with particular focus on those involved in tumor progression and drug resistance. Furthermore, we will discuss the use of specific miRNAs as potential therapeutic tools in this tumor. Several miRNAs have been identified to be down- or up-regulated in primary tumors and have been associated with MYCN amplification, differentiation, dissemination and chemoresistance. Little evidence is available in the literature about circulating miRNAs which are of particular interest as potential biomarkers for liquid biopsy. Identification of body-fluid markers for non-invasive diagnosis, risk stratification, treatment monitoring and tumor follow-up, is gaining growing interest, especially in the pediatric field. miRNAs are suitable candidates as biomarkers in neuroblastoma but further investigations are needed to expand knowledge regarding their role in this malignancy to design specific approaches of miRNAs-mediated therapies.

Pediatric Solid Tumors of Infancy: An Overview.

1. Wendy Allen-Rhoades, MD, FAAP* 2. Sarah B. Whittle, MD, MS, FAAP* 3. Nino Rainusso, MD, FAAP* 1. *Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine and Texas Children's Hospital Cancer and Hematology Centers, Houston, TX * Abbreviations: AFP: : α-fetoprotein ASCT: : autologous stem cell transplant CT: : computed tomography FDA: : Food and Drug Administration MRI: : magnetic resonance imaging OMS: : opsoclonus myoclonus syndrome VIP: : vasoactive intestinal peptide 123I-MIBG: : 123I-meta-iodo-benzylguanidine Pediatricians should recognize the role of age, genetic factors, and syndromes that predispose to the development of certain pediatric solid tumors. Many symptoms of common childhood illnesses that progress or do not resolve in a timely manner should require a detailed evaluation and prompt referral to a cancer specialist. After completing this article, readers should be able to: 1. Recognize the presenting signs and symptoms of pediatric solid tumors (eg, abdominal mass, constipation, shortness of breath, back pain, bone pain, fever, and hypertension). 2. Identify the signs and symptoms of retinoblastoma, neuroblastoma, hepatoblastoma, and Wilms tumor. 3. Recommend genetic evaluation and close disease surveillance for patients with certain solid tumors or particular predisposing conditions. 4. Recognize general aspects of the multidisciplinary treatment approach in children with retinoblastoma, neuroblastoma, hepatoblastoma, and Wilms tumor. Pediatric solid tumors are a group of nonhematologic, extracranial cancers that occur during childhood. This heterogeneous group of tumors represents approximately 40% of all pediatric cancers (Fig 1). Many pediatric solid tumors are referred to as embryonal or developmental cancers because they arise in young children or adolescents as a result of alterations in the processes of organogenesis or normal growth. In this review, we address common symptoms developed in children diagnosed as having malignant solid tumors and offer a general description of the most common pediatric solid tumors in infants and young children. Common malignant solid tumors in adolescents will be addressed in a separate article. Figure 1. Percentage distribution of pediatric cancers and solid tumors in children …

Abstract A119: Nanocarriers with a camptothecin-mitocan co-drug suppress tumor growth and extend survival in models of chemosensitive and chemoresistant neuroblastoma

Abstract Background: Neuroblastoma (NB) ranks as the most common solid tumor in infants and is responsible for 15% of all childhood cancer deaths. Chemotherapeutics used as a first-line treatment in high-risk NB patients are inherently toxic and ultimately ineffective. High-risk NB patients have <50% survival rate, and a particular challenge is posed by a group of “ultrahigh”-risk patients who show no response to therapy. With the goal of developing a clinically viable treatment strategy for high-risk NB, this study focused on an experimental approach integrating novel mutual prodrugs (co-drugs) and biodegradable nanocarriers. Nanocarrier-mediated co-drug delivery is designed to take advantage of enhanced accumulation and retention of sub-100 nm-sized nanoparticles (NP) in the tumor, followed by controlled release and in situ activation of the co-drug. Toward this goal, we developed a series of reversibly hydrophobized co-drugs exhibiting stable encapsulation in NP and activated hydrolytically to produce in one step 1) a potent camptothecin analog that inhibits topoisomerase I (SN-38) and 2) a pro-oxidant “helper” agent of a redox-silent tocol family of mitocans acting on the mitochondrial respiratory chain of cancerous cells. The effectiveness of co-drug loaded NP was evaluated in vitro and in orthotopic models of MYCN-amplified, chemosensitive, and drug-resistant NB. Methods and Results: A series of SN38-tocol conjugates constructed with progressively increasing hydrolytic activation rates were synthesized in high yields via direct coupling of the components. Co-drug loaded biodegradable NP (average size: 85±36 nm) were formulated using a nanoprecipitation approach adapted for producing sub-100 nm-sized nanocarriers. In comparative in vitro studies, a co-drug designed with a phenolic ester linkage was found to be most effective, achieving profound and sustained growth inhibition of MYCN-amplified NB cells, both chemo-naïve and chemoresistant (IMR-32 and BE(2)-C cells derived at diagnosis and during relapse after chemotherapy, respectively) under conditions modeling different levels of exposure experienced by NB cells within the tumor. Phenolic carbonate and aliphatic ester designs were notably less efficient. In an in vivo model of chemosensitive disease (IMR-32 orthotopic xenograft), nanocarriers with phenolic ester co-drug administered over 4 weeks (10 mg/kg, once a week) induced tumor regression and completely inhibited tumor growth over a 26-week period. This co-drug/nanocarrier formulation tested against chemoresistant NB (BE(2)-C orthotopic xenograft) potently suppressed tumor growth and extended animal survival up to 7 weeks, in comparison to irinotecan, which was only marginally effective (event-free survival of 3 weeks vs. 2 weeks in "no treatment" and drug-free NP groups). Conclusions: Camptothecin-mitocan co-drugs can be rationally designed and optimized to enhance their performance in the context of nanocarrier-based therapy of aggressive NB. The co-drug/nanocarrier combination strategy, whose feasibility and effectiveness against both chemosensitive and drug-resistant NB was demonstrated in the present preclinical studies, holds promise as a new therapeutic strategy for high-risk NB as well as for other solid tumors currently lacking effective treatment options. Citation Format: David Guerrero, Ivan S. Alferiev, Ferro Nguyen, Peng Guan, Venkatadri Kolla, Danielle Soberman, Ilia Fishbein, Robert J. Levy, Garrett M. Brodeur, Michael Chorny. Nanocarriers with a camptothecin-mitocan co-drug suppress tumor growth and extend survival in models of chemosensitive and chemoresistant neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A119.

Abstract 2479: Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation

Abstract Background: Neuroblastoma is the most common solid tumor of infancy and third leading cause of cancer death in children. It has one of the lowest survival rates of all pediatric cancers with less than 50% survival in “high risk” disease. Curcumin is the active compound of the yellow spice turmeric. Curcumin induces apoptosis and inhibits proliferation, angiogenesis, invasion and metastasis in many human cancer cells. The mechanism of cytotoxicity in neuroblastoma is unclear. Objectives: In this study we investigated the effects of curcumin on SMS-KCNR and CHLA-20 human neuroblastoma cells. Methods: SMS-KCNR and CHLA-20 neuroblastoma, cells were treated with increasing concentrations of curcumin. Cell viability was determined by Alamar Blue assays. Real-time PCR analysis of ER stress markers and apoptotic pathway genes was performed. Western Blotting was also performed to examine the downstream signaling pathways. Measurement of endogenous sphingolipids was performed by LC/MS. Sphingolipid pathway enzyme activities were also determined. Results: Curcumin was cytotoxic to both cell lines at 10 and 20 uM concentrations. PARP cleavage was noted at 24 hours, but cleavage of caspases 3, 8 and 9 was not observed. Treatment with the pancaspase inhibitor z-VAD did not reverse the cytotoxicity in curcumin treated cells, confirming that curcumin induced cell death was caspase-independent. Since perturbation in complex sphingolipid levels is associated with apoptosis, LC/MS measurement of endogenous sphingolipids was performed and showed increases in both dihydroceramides and ceramides. The increases in endogenous dihydroceramides, indicated that the dihydroceramide desaturase (DEGS-1) enzyme was inhibited. DEGS-1 activity was inhibited in-situ in a dose dependent manner in SMS-KCNR cells. There was no change in the mRNA or protein levels of DEGS-1, supporting that curcumin inhibited this enzyme indirectly. Next, the mechanism of ceramide generation was investigated by measuring the activity of sphingomyelin synthase (SMS) glycosylceramide synthase (GCS), acid ceramidase, neutral ceramidase, acid sphingomyelinase and neutral sphingomyelinase (SMase). At 6hrs, curcumin downregulated SMS activity by 30% and 54% GCS activity by 40% and 42% at concentrations of 10 and 20uM respectively. Curcumin has been demonstrated to induce ROS generation. Pre-treatment with the antioxidants N-acetylcysteine or glutathione abrogated curcumin mediated apoptosis and sphingolipid generation in SMS-KNCR cells. Furthermore, curcumin mediated SMS and GCS inhibition was blocked by these antioxidants. Conclusions: ROS plays a key role in sphingolopid and curcumin induced-cytotoxicity in neuroblastoma cells. Modulation of sphingolipid signaling pathways may provide a more effective and novel approach for the treatment of pediatric solid tumors. Curcumin is a potential novel therapy for neuroblastoma. Citation Format: Mehrdad Rahmaniyan, Li Li, Julio Garcia, A A. Qudeimat, Jacqueline M. Kraveka. Curcumin mediated apoptosis in human neuroblastoma cells via ROS and sphingolipid generation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2479.

Abstract B18: Role of sphingolipids and ER stress in curcumin mediated cytotoxicity In human neuroblastoma cells

Abstract Background: Neuroblastoma is the most common solid tumor of infancy and third leading cause of cancer death in children. It has one of the lowest survival rates of all pediatric cancers with less than 50% survival in “high risk” disease. Curcumin is the active compound of the yellow spice turmeric. Curcumin induces apoptosis and inhibits proliferation, angiogenesis, invasion and metastasis in many human cancer cells. The mechanism of cytotoxicity in neuroblastoma is unclear. Objectives: In this study we investigated the effects of curcumin on SMS-KCNR and CHLA-20 human neuroblastoma cells. Methods: SMS-KCNR and CHLA-20 neuroblastoma, cells were treated with increasing concentrations of curcumin. Cell viability was determined by Alamar Blue assays. Real-time PCR analysis of ER stress markers and apoptotic pathway genes was performed. Western Blotting was also performed to examine the downstream signaling pathways. Measurement of endogenous sphingolipids was performed by LC/MS. Sphingolipid pathway enzyme activities were also determined. Results: Curcumin was cytotoxic to both cell lines at 10 and 20 uM concentrations. PARP cleavage was noted at 24 hours, but cleavage of caspases 3, 8 and 9 was not observed. Treatment with the pancaspase inhibitor z-VAD did not reverse the cytotoxicity in curcumin treated cells, confirming that curcumin induced cell death was caspase-independent. ER stress markers of activation of the unfolded protein response were then examined and increase in the expression of CHOP (9 fold and 16 fold) and GRP78 (3 and 2 fold) mRNA levels was noted at 6 and 24 hours respectively. Increases in protein levels of CHOP and GRP-78 were also observed. ER stress may play the main role in curcumin induced-cytotoxicity in neuroblastoma. Since perturbation in complex sphingolipid levels is associated with ER stress and apoptosis, LC/MS measurement of endogenous sphingolipids was performed and showed increases in both dihydroceramides and ceramides. The increases in endogenous dihydroceramides, indicated that the dihydroceramide desaturase (DEGS-1) enzyme was inhibited. DEGS-1 activity was inhibited in-situ in a dose dependent manner in SMS-KCNR cells. There was no change in the mRNA or protein levels of DEGS-1, supporting that curcumin inhibited this enzyme indirectly. Next, the mechanism of ceramide generation was investigated by measuring the activity of sphingomyelin synthase (SMS) glycosylceramide synthase (GCS), acid ceramidase, neutral ceramidase, acid sphingomyelinase and neutral sphingomyelinase (SMase). At 6hrs, curcumin downregulated SMS activity by 30% and 54% GCS activity by 40% and 42% at concentrations of 10 and 20uM respectively. Conclusions: ER stress plays a key role in curcumin induced-cytotoxicity in neuroblastoma cells. Modulation of sphingolipid signaling pathways may provide a more effective and novel approach for the treatment of pediatric solid tumors. Curcumin is a potential novel therapy for neuroblastoma. Citation Format: Mehrdad Rahmaniyan, Li Li, Amr Qudeimat, Julio Garcia, Jacqueline M. Kraveka. Role of sphingolipids and ER stress in curcumin mediated cytotoxicity In human neuroblastoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B18.

Lack of Associations between XPC Gene Polymorphisms and Neuroblastoma Susceptibility in a Chinese Population.

Neuroblastoma is one of the most malignant solid tumors in infants and young children. No more than 40% of neuroblastoma patients can survive for longer than five years after it has been diagnosed. XPC protein is a pivotal factor that recognizes DNA damage and starts up the nucleotide excision repair (NER) in mammalian cells. This makes up the first group to defend against the cancer. Previous studies have identified that XPC gene polymorphisms were associated with various types of cancer. However, the associations between XPC gene polymorphisms and neuroblastoma risk have not yet been studied. We investigated the associations between three XPC gene polymorphisms (rs2228001 A>C, rs2228000 C>T, and rs2229090 G>C) and neuroblastoma risk with 256 neuroblastoma patients and 531 healthy controls in a Chinese Han population. Odds ratios and 95% confidence intervals were used to access the association between these three polymorphisms and neuroblastoma risk. No significant association was detected between these three polymorphisms and neuroblastoma risk in the overall analysis as well as in the stratification analysis. These results suggest that none of these three polymorphisms may be associated with the risk of neuroblastoma in the Chinese Han population.

Abstract B51: Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection

Abstract Introduction: Neuroblastoma (NB) is the most common extracranial solid tumor of infants. We constructed a tissue microarray (TMA) including 47 diagnostic NB cases and performed immunohistochemical (IHC) staining for a number of biomarkers. Differential IHC staining was identified between duplicate tissue cores for a proportion of NB cases. Differential IHC staining occurs frequently in cancer specimens, and can be due to technical factors, tissue artefacts, or underlying biological differences between tumor sub-regions. As laser capture microdissection (LCM) allows isolation of cells from formalin-fixed paraffin-embedded (FFPE) tissue slides, we applied LCM to FFPE NB samples, to investigate the basis of differential IHC staining. Material and Methods: NSE, NB84, ALK, TPD52 and MGMT proteins were detected within diagnostic NB TMA cores and whole NB sections using IHC. Cells were isolated from FFPE sections using LCM, and genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Extracted genomic DNA was quantified using a Nanodrop spectrophotometer, and DNA quality was assessed by a Bioanalyzer 2100. All DNA samples were tested using PCR with TPD52 and ALK primers. Results including clinical and sample variables were analysed using SPSS version 19. Results: Of 47 NB cases within the TMA, 35 cases were represented by duplicate tissue cores, whereas the remaining 12 cases were represented by single cores. IHC analysis of 5 biomarkers showed visually different IHC staining in tumour tissue between duplicate tissue cores in 15/35 (43%) cases. Whole tissue sections from 6 of these 15 cases were then examined, which reproduced differential IHC staining. Isolation of NB cells by LCM was successfully performed after substantial optimisation. A total of 14 regions from 8 NB cases were microdissected, producing DNA yields ranging from 0.12 - 4.05 pg/ µm2 tissue. There were no significant associations between total yields of extracted DNA, and microdissected tissue areas, or tissue sample ages. All DNA samples were successfully amplified by TPD52 but not by ALK PCR primers. Conclusions: Genomic DNA can be reliably extracted from microdissected FFPE NB tissues, although only one of 2 genes could be successfully PCR amplified. Downstream assays of DNA extracted from FFPE samples may therefore have particular design requirements. Further application of this approach may help improve our understanding of the level of tumour heterogeneity present in NB samples at diagnosis. Citation Format: Le M. Thwe, Laurence C. Cantrill, Daniel R. Catchpoole, Loretta Lau, Jennifer A. Byrne. Extraction and analysis of genomic DNA from formalin-fixed paraffin-embedded neuroblastoma samples following laser capture microdissection. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B51.

Abstract 4380: The miR-15 family members are therapeutic candidates to treat chemoresistant neuroblastomas

Abstract Neuroblastoma (NBL) is the most common solid tumor in infants and accounts for approximately 15% of cancer deaths in children. Neuroblastomas are very heterogeneous, with prognosis ranging from spontaneous regression to aggressive clinical course, and often become refractory to all current forms of treatment. While low and intermediate-risk patients have good prognosis, high-risk patients have poor prognosis and need intense chemotherapy. Despite the aggressive treatment, 60-70% of patients will die, mainly due to resistance to therapy and metastasis. Owing to the multiple different mechanisms that confer NBL resistance to therapy, targeting single components of a pathway may not suffice to restore chemosensitivity. In this respect, it is desirable to find molecules that can regulate multiple cellular processes or different component of the same pathways, thereby overcoming MDR and improving the therapeutic response. Our strategy is aimed to use microRNAs (miRNAs) to target the oncogenic properties of MDR neuroblastomas and render them vulnerable to treatment. The mRNA expression of genes related to multidrug resistance was analyzed in multiple neuroblastoma data sets. The 3′UTR of the genes differentially expressed in the highest-risk neuroblastoma group (Stage 4, MYCN amplified) was scanned with miRNA-binding sites prediction algorithms (TargetScan, PicTar, miRANDA). MicroRNAs predicted to regulate the expression of 2 or more overexpressed genes were selected for functional validation. Thirty-six miRNAs mimetics were transfected in two different chemoresistant neuroblastoma cell lines and effects on cell proliferation were evaluated. We have found a substantial number of genes implicated in the DNA damage response deregulated in high-risk NBL. MicroRNA-binding prediction algorithms revealed that most of them could potentially be regulated by microRNAs. Functional analysis in chemoresistant NBL cell lines identified several microRNAs that regulate proliferation, the best of which were the miR-15 family members. The overexpression of miR-497 reduces de proliferation of chemoresistant NBL cell lines and induces apoptotic cell death only in MYCN-amplified cell lines. Moreover, conditional overexpression of miR-497 in neuroblastoma xenografts led to a reduction in tumor growth. CONCLUSIONS: A substantial number of genes implicated in the MDR phenotype are deregulated in high-risk NBL and can be simultaneously targeted with miRNAs, providing a new therapeutic approach for chemoresistant neuroblastomas. This work is supported by Instituto de Salud Carlos III (CP11/00052, RD12/0036/0016 and PI11/00740) cofinanced by the European Regional Development Fund (ERDF) and Asociación Española Contra el Cáncer-Junta de Barcelona. Citation Format: Aroa Soriano, Laia Paris-Coderch, Luz Jubierre, Ana Almazán-Moga, Carla Molist, Josep Roma, Soledad Gallego, José Sánchez de Toledo, Miguel F. Segura. The miR-15 family members are therapeutic candidates to treat chemoresistant neuroblastomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4380. doi:10.1158/1538-7445.AM2014-4380