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Background: Osteosarcoma is an aggressive bone tumor. Itrepresents the principal cause of cancer-associated death in children.Considering the recent findings on the role of iron in cancer, iron chelation has been investigated for its antineoplastic properties in many tumors. Deferasirox is the most used iron chelator compound and in previous studies showed an anticancer effectinhematologic and solid malignancies. Eltrombopag is a Thrombopoietin receptor used in thrombocytopenia, that also binds and mobilize iron. It demonstrated an effect in iron overload conditions and also in contrasting cancer cells proliferation. Objective: We analyzed the effects of Deferasirox and Eltrombopag in Human Osteosarcoma cells, in the attempt to identify other therapeutic approaches for this tumor. Methods: We cultured and treated withDeferasirox and Eltrombopag, alone and in combination, two human osteosarcoma cell lines, MG63 and 143B. After 72h exposure, we performed RTqPCR, Western Blotting, Iron Assay and cytofluorimetric assays to evaluate the effect on viability, apoptosis, cell cycle progression and ROS production. Results: The iron chelating properties of the two compounds are confirmed also in Osteosarcoma, but we did not observe any direct effect on tumor progression. Discussion: We tested Deferasirox and Eltrombopag, alone and in combination, in Human Osteosarcoma cells for the first time and demonstrated that their iron chelating activity does not influence biochemical pathways related to cancer progression and maintenance. Conclusion: Although further investigations on possible effects mediated by cells of the tumor microenvironment could be of great interest, in vitro iron chelation in Osteosarcoma does not impair tumor progression.

Osteosarcoma is an aggressive bone tumor of the pediatric age. It is therefore important to improve conventional therapies (chemotherapy and surgery). Anticancer drugs often cause osteoporosis due to a misbalance of RANK/RANK-L/OPG pathway. Denosumab is a monoclonal antibody with high affinity and specificity to RANK-L, the ligand released by osteoblasts that enhances osteoclasts differentiation and bone resorption. It is used in osteoporosis and in other conditions characterized by bone mass loss. Doxorubicin is a chemotherapic drug used in several kinds of tumors, and also patients treated with it often develop osteoporosis. We investigated the effects of Denosumab alone and in combination with Doxorubicin, in two human osteosarcoma cell lines (MG63 and U-2 OS). We evaluated the effect of these treatments on apoptosis, cell cycle progression, invasion capacity and bone metabolism. We observed for the first time an anti-invasive effect of Denosumab in OS cells and confirmed its anti-osteoporotic activity also in Osteosarcoma. On the other hand, we demonstrate that Denosumab not only does not affect apoptosis and cell cycle progression, but when used in combination with Doxorubicin, it causes an unexpected reduction of its activity. These results indicate that the presence of Denosumab might inhibit the efficacy of the chemotherapic drug. In conclusion, while our results certainly support and confirm the efficacy of Denosumab in Osteoporosis, we discourage the use of Denosumab in addition to conventional chemotherapy in Osteosarcoma, even though, certainly further investigations are necessary to better clarify the clinical role of this monoclonal antibody in cancer.

The improvement of chemotherapy, radiotherapy, and surgical interventions, together with hematopoietic stem cell transplantation, increased childhood cancer survival rate in the last decades, reaching 80% in Europe. Nevertheless, anti-cancer treatments are mainly responsible for the onset of long-term side effects in childhood cancer survivors (CCS), including alterations of the endocrine system function and activity. In particular, the most frequent dysfunction in CCS is a metabolic bone disorder characterized by low bone mineral density (BMD) with increased skeletal fragility. BMD loss is also a consequence of a sedentary lifestyle, malnutrition, and cancer itself could affect BMD, thus inducing osteopenia and osteoporosis. In this paper, we provide an overview of possible causes of bone impairment in CCS in order to propose management strategies for early identification and treatment of skeletal fragility in this population.

Actually, there is still no consensus related to diagnostic and management algorithms in case of head and neck lymphadenopathy in children. The aim of our study was to analyze the causes of head and neck lymphadenopathy in children to determine a systematic diagnostic approach. We enrolled all cases of head and neck lymphadenopathy in children under the age of 18 diagnosed at the Unit of Hemato-Oncology, Pediatric Department of University "Luigi Vanvitelli," Naples, over a 15-year period (January 2003-December 2017). In total, 405 patients (271 males) were enrolled in the study. Thirteen cases due to other causes, were left off the study. Therefore, the study was performed on 392 cases. A total of 220 patients (56.1%) had a history of infection, 66 cases (16.8%) a diagnosis of neoplasia, and 101 (24.9%) cases a diagnosis of reactive inflammatory changes of nonspecific origin. We have observed the following from our study: (1) the acute infections are the most common causes of head and neck lymphadenopathy in the pediatric population; (2) in about a quarter of patients, the lymphadenopathy resulted by nonspecific origin; (3) the supraclavicular nodes should be regarded with a high index of suspicion of malignancy.

Simple Summary Around 80% of children treated for childhood cancer become long-term survivors. Although chemotherapy and radiotherapy improve survival of these patients, they cause a low-grade chronic inflammation (inflamm-aging) which induces premature aging processes and vital organ failure, a condition known as frailty. Understanding frailty’s biological and molecular mechanisms and identifying inflamm-aging key biomarkers in childhood cancer survivors could be useful to facilitate the screening of comorbidities and to understand whether treatments, used to counteract inflamm-aging, may prevent side effects. Abstract Anti-cancer treatments improve survival in children with cancer. A total of 80% of children treated for childhood cancer achieve 5-year survival, becoming long-term survivors. However, they undergo several chronic late effects related to treatments. In childhood cancer survivors a chronic low-grade inflammation, known as inflamm-aging, is responsible for frailty, a condition characterized by vital organ failure and by premature aging processes. Inflamm-aging is closely related to chemotherapy and radiotherapy, which induce inflammation, accumulation of senescent cells, DNA mutations, and the production of reactive oxygen species. All these conditions are responsible for the onset of secondary diseases, such as osteoporosis, cardiovascular diseases, obesity, and infertility. Considering that the pathobiology of frailty among childhood cancer survivors is still unknown, investigations are needed to better understand frailty’s biological and molecular processes and to identify inflamm-aging key biomarkers in order to facilitate the screening of comorbidities and to clarify whether treatments, normally used to modulate inflamm-aging, may be beneficial. This review offers an overview of the possible biological mechanisms involved in the development of inflamm-aging, focusing our attention on immune system alteration, oxidative stress, cellular senescence, and therapeutic strategies.

Osteosarcoma (OS) is the most severe bone malignant tumor, responsible for altered osteoid deposition and with a high rate of metastasis. It is characterized by heterogeneity, chemoresistance and its interaction with bone microenvironment. The 5-year survival rate is about 67% for patients with localized OS, while it remains at 20% in case of metastases. The standard therapy for OS patients is represented by neoadjuvant chemotherapy, surgical resection, and adjuvant chemotherapy. The most used chemotherapy regimen for children is the combination of high-dose methotrexate, doxorubicin, and cisplatin. Considered that the necessary administration of high-dose chemotherapy is responsible for a lot of acute and chronic side effects, the identification of novel therapeutic strategies to ameliorate OS outcome and the patients’ life expectancy is necessary. In this review we provide an overview on new possible innovative therapeutic strategies in OS.

Tumor-associated macrophages and their alternative activation states together with cytokines and growth factors trapped in tumor microenvironment contribute to the progression of OS. In contrast to other tumor types, M2 polarized macrophages, reduce metastasis and improve survival in osteosarcoma patients. Mifamurtide is an immunomodulatory drug given together with standard adjuvant chemotherapy in high-grade osteosarcoma to improve outcome. Macrophages obtained from peripheral blood mononucleated cells of healthy donors and MG63 cells were cultured alone and together, and treated with Mifamurtide. We analyzed the effects of Mifamurtide on macrophage polarization and on MG63 proliferation, migration and differentiation, evaluating the expression of M1/M2 and osteoblast markers and molecules involved in metastasis and proliferation pathways. Our data suggest that Mifamurtide, switching macrophage polarization towards a TAM-like intermediate M1/M2 phenotype, may modulate the delicate balance between pro-inflammatory and immunomodulatory macrophage functions. Moreover, Mifamurtide may inhibit the cellular proliferation and induce the tumor cell differentiation, probably through the down regulation of pSTAT3, pAKT and IL-17R.

The prognosis for children with recurrent or refractory neuroblastoma remains a significant clinical challenge, and currently there are no known curative salvage regimens. In this paper we investigated the effect of imatinib with rapamycin and the chemotherapeutic agents temozolomide and irinotecan. We treated two children with recurrent neuroblastoma with this so called RIST protocol. Both patients, off therapy for 15 and 31 months, respectively are well, and developing normally, without any complications. These findings suggest that a combination regimen of RIST may provide a therapeutic benefit with a favorable toxicity profile to a unfortunate subset of patients with neuroblastoma.

Introduction: Rhabdomyosarcoma is a soft tissue malignant musculoskeletal tumor frequent in children. Biliary duct localization is extremely rare, but it is the most common cause of malignant obstructive jaundice in pediatric patients. Methods: This report describes a series of 10 patients under 18 years of age with biliary tract rhabdomyosarcoma who were enrolled, from 1979 to 2004, in 3 consecutive Italian pediatric cooperative protocols that had been drawn up by the Soft Tissue Sarcoma Committee of the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP). Results: Considering initial and delayed surgery, tumor resection was achieved in 7 cases, 3 complete with free margins (2 liver transplants) and 4 with microscopic residual disease. Chemotherapy was given to all patients and radiotherapy to 3. At present, 5 patients survive in complete remission 90-200 months after diagnosis while 4 died of disease progression or relapse and 1 of liver transplant-related complications. Conclusions: Better outcomes in this series were associated with the feasibility of conservative surgery due to the favorable location of the tumor, in particular in the common bile duct. Chemotherapy and radiotherapy might obviate the need for demolitive surgery or liver transplant, which were linked to worse outcomes in our series.

Background Children with Wilms’ tumor (WT) aged under 24 months (infants) have a better prognosis than older patients. Our aim was to study the epidemiology of this age group, with focus on the modality of diagnosis, tumor size, and association with malformations/syndromes, seeking to understand if any of these factors might be related to prognosis. Patients and methods Infants diagnosed with WT between 2003 and February 2010 were evaluated. A query form was used to collect data on the modality of WT diagnosis (symptomatic or incidental), tumor volume, maximum diameter, site, and stage. Results Data were collected for 117 of 124 WT infants registered. Twenty-four cases had an incidental diagnosis (ID) of renal mass, usually arising from an abdominal ultrasound performed for other reasons, and 93 had been diagnosed based on clinical signs/symptoms. The incidental cohort displayed unifocal disease, mean tumor diameter 5.52 cm, mean tumor volume 84.30 ml, and 14 patients showed associated malformations. Symptomatic patients had mean maximum tumor diameter of 10.18 cm, mean tumor volume of 451.18 ml, and six had associated malformations. Conclusions Our study showed that 20% of the infants had an ID of WT; they had a relatively smaller nonmetastatic tumor and a higher rate of malformations than infants of the symptomatically diagnosed group, but we did not detect any difference in age at diagnosis between the two groups. Conversely, we found a significant difference in the 5-year event-free survival rate (P = 0.018) between infants under 1 year (96%), more frequently associated with congenital malformations, and infants 1–2 years (80%).

Background: Cornelia de Lange syndrome is the prototype for cohesinopathy disorders, which are characterized by defects in chromosome segregation. Kidney malformations, including nephrogenic rests, are common in Cornelia de Lange syndrome. Only one post-mortem case report has described an association between Wilms tumor and Cornelia de Lange syndrome. Here, we describe the first case of a living child with both diseases. Case presentation: Non-anaplastic triphasic nephroblastoma was diagnosed in a patient carrying a not yet reported mutation in NIPBL (c.4920 G > A). The patient had the typical facial appearance and intellectual disability associated with Cornelia de Lange syndrome in absence of limb involvement. The child's kidneys were examined by ultrasound at 2 years of age to exclude kidney abnormalities associated with the syndrome. She underwent pre-operative chemotherapy and nephrectomy. Seven months later she was healthy and without residual detectable disease. Conclusion: The previous report of such co-occurrence, together with our report and previous reports of nephrogenic rests, led us to wonder if there may be any causal relationship between these two rare entities. The wingless/integrated (Wnt) pathway, which is implicated in kidney development, is constitutively activated in approximately 15-20 % of all non-anaplastic Wilms tumors. Interestingly, the Wnt pathway was recently found to be perturbed in a zebrafish model of Cornelia de Lange syndrome. Mutations in cohesin complex genes and regulators have also been identified in several types of cancers. On the other hand, there is no clear evidence of an increased risk of cancer in Cornelia de Lange syndrome, and no other similar cases have been published since the fist one reported by Cohen, and this prompts to think Wilms tumor and Cornelia de Lange syndrome occurred together in our patient by chance.

Kaposiform hemangioendothelioma (KHE) is a rare, locally aggressive, vascular spindle-cell proliferation, with resemblance to Kaposi sarcoma. Usually, this tumor occurs in the skin and the retroperitoneum. We described a girl with a kidney localization and extension into the inferior vena cava and even into the right atrium. The case presented here is unique in 2 ways. First, kidney involvement of KHE has never been described in the literature until now. Second, and most remarkably, extensive tumor thrombosis suggests surgical excision even with cardiopulmonary bypass. The KHE of the kidney is a rare tumor but should be taken into account in the differential diagnosis with other pediatric renal neoplasms.

Background One of the most important adverse prognostic factors for adult renal cell carcinoma (RCC) is the retroperitoneal lymph node involvement. The aim of this article is to study the prognostic significance of local lymph node involvement in pediatric RCC and the role of retroperitoneal lymph node dissection (RLND) at diagnosis. Procedure The series included 16 patients with RCC and lymph nodes involvement registered in the Italian Rare Tumors Pediatric Age (TREP) project, accounting for 26.2% of 61 pediatric RCC observed at AIEOP centers. Results A radical nephrectomy was performed in all cases: at diagnosis in 12 cases, after preoperative chemotherapy (CT) in 4 cases. As a part of the same procedure 9 patients underwent RLND, and 7 received a more limited lymph nodes resection. Five (31.2%) developed disease recurrence 2–34 months after diagnosis (median, 6 months) plus 1 developed progression; 6 patients died, 1 of them from secondary leukemia. Among the nine patients receiving RLND, eight are alive and disease free. This compares with only one patient surviving among the seven receiving a more limited lymph nodes resection. The estimated 25-year PFS and OS rates for all patients were 61.4% (95% CI 33.2–80.5) and 50.8% (95% CI 16.5–77.5), respectively. Conclusions Lymph node involvement is an unfavorable prognostic factor in children with RCC. RLND appears to be a critical factor to improve the outcome. However, when compared to similar adult patients, the outcome in children appears to be better, suggesting that pediatric RCC, or the host, may be critical differences. Pediatr Blood Cancer 2008;51:475–478. © 2008 Wiley-Liss, Inc.

Background Pediatric nonrhabdomyosarcoma soft tissue sarcomas (NRSTS) may rarely occur in visceral tissues, and little is known about their clinical history. The present study retrospectively analyzed a group of patients prospectively registered in Italian pediatric protocols conducted between 1979 and 2004. Methods Inclusion criteria for the study were as follows: a pathological diagnosis of “adult-type NRSTS,” arising at visceral sites (lung-pleurae, liver, kidney, and mesentery-bowel); age under 18 years; no previous treatment except for primary surgery; available clinical data; and written consent. Results Thirty cases with visceral NRSTS were collected and analyzed. Sites of origin were as follows: mesentery-bowel in 12 cases, lung-pleurae in 11, liver in 5, and kidney in 2. According to the Intergroup Rhabdomyosarcoma Study (IRS) surgical grouping system, patients were classified as follows: nine IRS group I, three group II, 12 group III, and six group IV. Patients were treated with a multimodal approach including surgery, radiotherapy, and/or chemotherapy, according to their characteristics. For the series as a whole, the 5-year event-free and overall survival rates were 33.3% and 40.0%, respectively. The IRS group (reflecting the feasibility of initial complete resection) emerged as the main prognostic factor. Survival rates also correlated with tumor size and local invasiveness, histological subtype, and tumor sites (the worst outcome was seen for tumors arising in the lung and pleurae). Conclusions This study confirmed that visceral NRSTS are aggressive tumors carrying a worse prognosis than pediatric NRSTS arising in soft tissues of the extremities. Local treatment remains the main challenge for these tumors.

Neuroblastoma (NB) is a paediatric tumor that arises from neural crest and shows heterogeneous clinical and biological features. The serine-protease high temperature requirement A1 (HtrA1) has a pivotal role in both cell proliferation and differentiation. Here we report the expression and localization of HtrA1 in NB tumor samples to assess HtrA1 role as a possible new biomarker of cellular differentiation in NB patients. HtrA1 protein expression by Western Blot assay was performed in 60 tissue samples of 50 children with NB and 10 children with ganglioneuroblastoma (GNB). HtrA1 was expressed in 56/60 (93.3 %) samples with different expression levels: low levels in 36/56 samples (64.3 %) and high levels in 20/56 (35.7 %). Higher levels were found in 1, 2 and 4s stages (80 %), whereas 3 and 4 stages (20 %) showed a low expression, with a statistically significant difference (p = 0.003). Among not amplified N-MYC group, 28 (60 %) had low/absent expression of HtrA1: seven with recurrent disease and negative outcome and 21 in continuous complete remission (CCR), whereas all samples with high expression of HtrA1 (17/44) were in CCR (p = 0.03). The immunohistochemical analysis showed localization of HtrA1 in differentiated areas higher than in undifferentiated areas where the protein was absent. Moreover, HtrA1 was highly expressed in all GNB samples. In conclusion, the over-expression of HtrA1 is correlated to cellular differentiation grade and stage of NB at diagnosis. Moreover, HtrA1 could represent a new marker of undifferentiation and biological aggressiveness of NB. Neuroblastoma (NB) is a paediatric tumor that arises from neural crest and shows heterogeneous clinical and biological features. The serine-protease high temperature requirement A1 (HtrA1) has a pivotal role in both cell proliferation and differentiation. Here we report the expression and localization of HtrA1 in NB tumor samples to assess HtrA1 role as a possible new biomarker of cellular differentiation in NB patients. HtrA1 protein expression by Western Blot assay was performed in 60 tissue samples of 50 children with NB and 10 children with ganglioneuroblastoma (GNB). HtrA1 was expressed in 56/60 (93.3 %) samples with different expression levels: low levels in 36/56 samples (64.3 %) and high levels in 20/56 (35.7 %). Higher levels were found in 1, 2 and 4s stages (80 %), whereas 3 and 4 stages (20 %) showed a low expression, with a statistically significant difference (p = 0.003). Among not amplified N-MYC group, 28 (60 %) had low/absent expression of HtrA1: seven with recurrent disease and negative outcome and 21 in continuous complete remission (CCR), whereas all samples with high expression of HtrA1 (17/44) were in CCR (p = 0.03). The immunohistochemical analysis showed localization of HtrA1 in differentiated areas higher than in undifferentiated areas where the protein was absent. Moreover, HtrA1 was highly expressed in all GNB samples. In conclusion, the over-expression of HtrA1 is correlated to cellular differentiation grade and stage of NB at diagnosis. Moreover, HtrA1 could represent a new marker of undifferentiation and biological aggressiveness of NB. © 2014 Springer Science+Business Media New York.

High-dose methotrexate (MTX) is a key component of most treatment protocols for childhood and adolescent non-Hodgkin lymphoma (NHL). Recent studies have suggested that the toxicity of antifolate drugs, such as MTX, is affected by inherited single nucleotide polymorphisms (SNPs) in folate metabolizing genes. The aim of our study was to investigate the potential influence of the C677T and A1298C genetic variants of the methylenetetrahydrofolate reductase (MTHFR) gene on the clinical toxicity and efficacy of MTX in pediatric patients with NHL (n = 95) treated with therapeutic protocols Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) LNH-97 and EURO LB-02. We demonstrated that patients with the 677T genotype had an approximately six-fold greater risk of developing hematological toxicity compared with wild-type carriers, especially in the 1 g/m(2) treatment group (p = 0.01). Moreover, we identified a correlation between the risk of relapse and the T genotype: T carriers had reduced disease-free survival compared with wild-type patients (67% vs. 100%). Our data suggest a pharmacogenetic influence on the adverse effects of high-dose MTX in the 1 g/m(2) treatment group.

Purpose To evaluate the prognostic factors in a series of children affected by pleuropulmonary blastoma (PPB). Patients and Methods Clinicopathological findings, treatment, and outcome of 22 PPB cases observed in 13 Italian Associations for Pediatric Hematology and Oncology centers are reported. Clinical data, surgical notes, pathologic findings, and summaries of treatment were taken from the charts and correlated with outcome by standard statistical methods. Results The series included 22 patients (14 males) with a median age of 30.5 months followed up for a median of 22 months (range 2–176 months). In nine patients the PPB developed with lung involvement only. Congenital lung cysts were recorded in five cases. Nine patients had recurrences. Gender, side, tumor size, pre-existing lung cysts, and extent of surgical resection at diagnosis did not significantly affect survival by univariate analysis. Achieving total resection of the tumor at any time of treatment resulted in a significantly better prognosis (P = 0.01), whereas extrapulmonary involvement at diagnosis resulted in a significantly worse prognosis (P = 0.01). Estimated 15-year event-free and overall survival rates were 44 and 49% for all patients, respectively. Conclusions PPB is an aggressive neoplasm. Total resection of PPB performed at any time of treatment appears to provide a better outcome, whereas extrapulmonary involvement at diagnosis worsens the prognosis. Pediatr Blood Cancer 2007;48:318–323. © 2006 Wiley-Liss, Inc.

Wilms tumour (WT), the most frequent malignant childhood renal tumour, shows a high degree of genetic and epigenetic heterogeneity. Loss of imprinting on chromosome 11p15 is found in a large fraction of cases and mutations in a few genes, including WT1, CTNNB1, WTX, TP53 and, more recently, SIX1, SIX2 and micro RNA processing genes (miRNAPGs), have been observed. However, these alterations are not sufficient to describe the entire spectrum of genetic defects underlying WT development. We inspected data obtained from a previously performed genome-wide single nucleotide polymorphism (SNP) array analysis on 96 WT samples. By selecting focal regions commonly involved in chromosomal anomalies, we identified genes with a possible role in WT development, based on the prior knowledge of their biological relevance, including MYCN, DIS3L2, MIR562, HACE1, GLI3, CDKN2A and CDKN2B, PALB2, and CHEK2. The MYCN hotspot mutation c.131C>T was detected in seven cases (7.3%). Full sequencing of the remaining genes disclosed 16 rare missense variants and a splicing mutation. Most of these were present at the germline level. Promoter analysis of HACE1, CDKN2A and CDKN2B disclosed partial methylation affecting HACE1 in a consistent fraction of cases (85%). Interestingly, of the four missense variants identified in CHEK2, three were predicted to be deleterious by in silico analyses, while an additional variant was observed to alter mRNA splicing, generating a functionally defective protein. Our study adds additional information on putative WT genes, and adds evidences involving CHEK2 in WT susceptibility.