The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx).

The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS.

Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21(Cip1) expression and hampers tumour cell growth in vitro and in vivo.

Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21(Cip1) level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.

Toxicity of high-dose chemotherapy with etoposide, thiotepa and CY in treating poor-prognosis Ewing's sarcoma family tumors: the experience of the Bambino Gesù Children's Hospital.

We report the toxicity of high-dose chemotherapy (HDC) based on etoposide, thiotepa and CY (ETC) in children with poor-prognosis Ewing's sarcoma family tumors (ESFTs). A total of 26 patients with high-risk ESFT (metastasis or axis localization or tumor volume >200 ml or necrosis <95%) were reviewed. The conditioning was based on etoposide (600 mg/m(2)), thiotepa (750 mg/m(2)) and CY (120 mg/kg) followed by autologous BM or PBSC rescue. The conditioning regimen was well tolerated, without any toxic deaths. The median time from transplant to a neutrophil count of >0.5 x 10(9)/l was 10 days (range 6-27) and 22.5 days (range 9-114) for a plt count of >50 x 10(9)/l. Oral mucositis was recorded in 20 patients, grade 1/2 in 19 and grade 3 in the last patient. Diarrhea grade 1/2 was recorded in four patients and grade 1/2 liver toxicity in four patients. Sepsis was documented in four cases and skin toxicity in three. Lung and tubular toxicity, respectively, were reported in one patient each. We conclude that the ETC regimen presented a limited and manageable toxicity. Further studies would confirm the role of ETC in high-risk ESFT.

[Injuries: preventive approach and progress of injuries in the construction of the line B1 of the underground of Rome]. / Infortuni: approccio preventive e andamento infortunistico nella costruzione della linea B1 della Metropolitana di Roma 2005/2010.

The line B1 is a branch of the existing Metro line B in Rome. The route is long about 5 km, is completely underground and involves the construction of four new stations: Annibaliano, Libia /Gondar, Conca d'Oro and Jonio. The line will have a capacity of transport of 24,000 people/hour in each direction. The works started in 2006 involve about 500 workers. The report provides a statistical analysis of the events that occurred in the period 2005/2010 and aims to introduce the starting and management of this study, also on the basis of the "Operating procedures" issued by the acquisition of OSHAS 18001 certification from the agent of Metro B) / R.I.M.A.T.I. This analysis aims to provide to supervisors, to social security institutions and to workers, a usefull analysis tool in the prevention of the monitored events.

Metastatic retinoblastoma: single institution experience over two decades.

BACKGROUND: Metastatic spread in retinoblastoma is a rare occurrence in developed countries but still associated with a poor prognosis. PATIENTS AND METHODS: Medical records of all metastatic retinoblastoma diagnosed during a 20-year period were retrospectively reviewed. RESULTS: Six patients out of 104 presented a metastatic disease with an incidence at diagnosis of 2%. Three had a metastatic disease at diagnosis, one patient a trilateral retinoblastoma and two a metastatic spread after enucleation. All but one were sporadic retinoblastoma. Central nervous system (CNS) involvement was reported in five patients, while one patient had an intraorbital lesion, and bone and bone marrow spread. Different treatment strategies were administered based on local treatment plus chemotherapy and radiotherapy with or without high-dose chemotherapy. An ifosfamide/carboplatin/etoposide regimen was administered in three patients resulting in a partial response. Out of six patients, four died, and two patients are alive at 60 and 63 months from diagnosis. Both children with a long follow-up were treated with high-dose chemotherapy. All but one of the patients with CNS involvement died; the survivor was a patient with pineal involvement. CONCLUSION: This retrospective review confirms a curable strategy based on local treatment and conventional plus high-dose chemotherapy. Patients with CNS involvement remain incurable.

POLICRYPS structures as switchable optical phase modulators.

We report on the electrically controlled optical phase modulator behavior of light sculptured periodic structures made of polymer slices alternated to films of well aligned Liquid Crystals (POLICRYPS). Arbitrarily polarized light normally incident on the structure experiences a birefringence that depends on the anisotropy of the composite liquid crystalline material and on the geometrical cell parameters. The sample behaves as a retardation plate in good agreement with the Jones matrices formalism. Birefringence tuning is obtained by applying a suitable voltage, while a negligible birefringence variation is detected by increasing the incidence power. This makes POLICRYPS structures suitable as switchable phase retarders for high power laser beams.

Temozolomide in resistant or relapsed pediatric solid tumors.

PURPOSE: We report the off-label study aimed at investigating the use of temozolomide (TMZ) as single agent in relapsed or resistant pediatric solid tumors. The drug was administered at the dose of 215 mg/m2/day x 5 days or 180 mg/m2/day x 5 days in patients with prior craniospinal irradiation (CSI) or autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: Fifty two patients, median age 127.6 months, with resistant or relapsed solid tumors were enrolled. Tumor types were: neuroblastoma (NB; n = 17), medulloblastoma (MB; 8), brain stem glioma (BSG; 8), extraosseous Ewing's sarcoma/peripheral neuroectodermal tumor (EOES; 4), Ewing's sarcoma (ES; 4), anaplastic astrocytoma (AA; 3), rhabdomyosarcoma (RMS; 2), ependymoma (EP; 2), cerebral primitive neuroectodermal tumor (cPNET; 2), hepatocarcinoma (HC; 1), and osteosarcoma (OS; 1). All patients were pre-treated. Two outpatient courses were administered, with a median of 4.8 courses/pt. RESULTS: Objective response-rate (CR + PR + MR) in our series was 13.4% (1.9% CR, 3.8% PR, and 7.7% MR), SD occurred in 38.4% of patients and 48% had PD. The median survival was 7.8 months (range 1-37) and median time to progression was 3.4 months (range 1-20); these data were significantly correlated with histology and previous nitrosureas administration in multivariate analysis. Haematological toxicity grade 3-4 (mainly thrombocytopenia) was observed in 21.4% of administered courses, nausea was reported in 3.1% and respiratory distress in 0.7%. CONCLUSION: Oral TMZ was well tolerated in children with resistant or relapsed solid tumors and showed activity in NB and CNS tumours refractory to standard chemotherapy.

Ifosfamide/carboplatin/etoposide (ICE) as front-line, topotecan/cyclophosphamide as second-line and oral temozolomide as third-line treatment for advanced neuroblastoma over one year of age.

Children affected by advanced neuroblastoma have a discouraging prognosis, but intensive induction chemotherapy may increase the complete response rate. The combination of ifosfamide, carboplatin and etoposide (ICE) was used for the first time as front-line regimen in patients with stage 4 neuroblastoma over the age of 1 y. Similarly, second-line treatment for children with relapsed neuroblastoma, particularly after high-dose chemotherapy, has been unsatisfactory. The combination of topotecan and cyclophosphamide was studied in resistant or relapsed solid tumors. Furthermore, there is a need for effective palliative treatment in patients failing therapy. Temozolomide, a new dacarbazine analog with optimal oral bioavailability, is being used in an ongoing phase II study as an alternative to oral etoposide. Seventeen patients with stage 4 neuroblastoma have entered the ICE study; 15/16 (94%) major responses after induction were observed and 6/16 (37%) evaluable patients are disease free after a median of 51 mo. Twenty-one patients with relapsed/refractory disease (of whom 13 neuroblastomas) entered the topotecan/cyclophosphamide study: 7/21 (33%) patients responded. Forty-one patients entered the temozolomide study (of whom 16 had neuroblastomas): stable disease and symptom relief were obtained in 15/30 (50%) evaluable patients. Intensive induction with ICE resulted in a faster response with high response rate; a larger study with longer follow-up is needed to confirm a survival advantage. Second-line treatment was effective in obtaining remissions, some of them long lasting. Third-line treatment did not elicit measurable responses in neuroblastoma, but achieved prolonged freedom from disease progression and excellent palliation in several patients.

111 renal neoplasms of childhood: a clinicopathologic study.

PURPOSE: The aim of this study was to perform a clinicopathologic evaluation of a single pediatric institution renal tumor series. Most patients were treated within the frame of 3 consecutive SIOP trials, which included preoperative chemotherapy as their main feature. METHODS: Medical records and diagnoses of 111 patients were reviewed. The association of pathologic features with outcome was investigated by means of the Kaplan-Meier method, the Cox model, and a logistic multivariate analysis. Comparison among different trial results was carried out. RESULTS: In 98 patients (88%), nephroblastoma was diagnosed, followed by 6 adult-type renal tumors, 3 cystic nephromas, 2 mesoblastic nephromas, and 2 clear cell sarcomas. For nephroblastoma, a statistically significant correlation between grade and both disease-free survival rate and 5-year survival rate, and between stage and overall survival rate was shown. Lymph node involvement, local relapse, nephrogenic rests, and older age at presentation appeared to be less important prognostic factors. Tumor spillage was very sensitive to chemo or radiotherapy. No significant difference in outcome was observed among different trials. CONCLUSIONS: Wilms' tumor was the most frequent neoplasm and resulted in a 5-year cure rate of 90%. Clinical course was influenced mainly by diffuse anaplasia and, to a minor extent, by lymph node involvement. Because some tumors followed an unpredictable course, it is likely that also other biological factors played a significant role.

Bloodstream infections in children with cancer: a multicentre surveillance study of the Italian Association of Paediatric Haematology and Oncology. Supportive Therapy Group-Infectious Diseases Section.

A one-year prospective, multicentre surveillance study on aetiology, main clinical features and outcome of bloodstream infections in children with cancer was conducted in 18 paediatric haematology centres belonging to the Italian Association for Paediatric Haematology and Oncology. A total of 191 bloodstream infections were reported during the study period. Of them, 123 (64%) occurred in neutropenic and 68 (36%) in non-neutropenic patients. Gram-positive cocci caused 45% (85/191) of the episodes, gram-negative rods 41% (78/191), and fungi 9% (18/191). The remaining 5% (10/191) of the episodes were poly-microbial infections. A total of 204 pathogens were isolated (46% gram-positive cocci; 44% gram-negative rods; and 10% fungi). The aetiologic distribution was similar among neutropenic and non-neutropenic patients. A correlation between the infection and the presence of an indwelling central venous catheter was found in 20% (23/114) of the episodes among neutropenic patients and in 55% (23/62) among non-neutropenic patients. Gram-negative micro-organisms were isolated in an unusually high proportion of catheter-related infections (48%). The overall mortality rate from any cause within 30 days from the first positive blood culture was 11%, and was higher among patients who were neutropenic at the onset of the infection than among those who were not neutropenic (15 versus 4%, P = 0.03). In addition, the mortality was significantly higher in recipients of bone marrow transplantation than in patients with acute leukaemia or solid tumour (21, 11 and 6%, respectively) and was also higher in fungaemias and poly-microbial infections (22 and 30%) than in single gram-positive and gram-negative bacteraemias (11 and 6%).

Italian guidelines for the management of infectious complications in pediatric oncology: empirical antimicrobial therapy of febrile neutropenia.

The Italian Association for Paediatric Haematology and Oncology prepared a guideline document aimed at unifying and rationalising as much as possible the management of febrile neutropenia in children with cancer, because of the potential impact of these procedures on hospital costs and on the development of antibiotic resistance. Before starting anti-infective therapy, at least 2 blood cultures, a throat swab, urine-culture, and cultures from any suspected infected site, should be performed. Routine chest X-rays at onset of febrile neutropenia are probably not necessary, in absence of respiratory signs. At the present time, the safer option probably remains the combination of a beta-lactam and an aminoglycoside, and treating febrile neutropenia outside of hospital should be considered an investigational approach. The choice of the most appropriated regimen for each institution should be based also on the local bacteriological statistics and patterns of bacterial resistance. Antibiotic toxicity and cost should be other important factors. Every subsequent addition or substitution of antibiotics should be based on objective signs of clinical deterioration. The only accepted empirical modification is empirical antifungal therapy, while the empirical addition of a glycopeptide antibiotic cannot be recommended.

Spindle cell (Kaposiform) hemangioendothelioma with Kasabach-Merritt syndrome in an infant: successful treatment with alpha-2A interferon.

A two-month-old infant developed a vascular tumor of the right flank which upon biopsy proved to be a spindle cell hemangioendothelioma. The increased capillary bed characterizing the neoplasm caused a severe thrombocytopenia together with a consumption coagulopathy (Kasabach-Merritt syndrome). The patient, who was dependent on platelet transfusions, improved quickly after interferon alpha-2a was given at the dosage of 3,000,000 U/m2, with resolution of the Kasabach-Merritt syndrome after three weeks and a 75% decrease of the tumor volume within three months of treatment.

Sporadic acute lymphocytic leukemia arising in a patient with neurofibromatosis and xanthogranulomatosis.

We present a case report of a child who developed acute lymphoblastic leukemia, neurofibromatosis, optic glioma, and xanthogranulomatosis. This complex is unusual, not previously described, and appears to be a coincidence of different diseases. The importance of this case is that it may offer a clue to the genetic base of neurofibrosis syndromes including leukemic associations.

Role of deferoxamine in tumor therapy.

Several studies are consistent with the hypothesis that available iron may have some role in promoting tumor cell growth with different biological mechanisms. For this reason, several studies have been carried out to demonstrate the antitumor activity of deferoxamine (DFO), an iron chelator with a high affinity for ferritin-bound iron. In particular, the effects of DFO have been studied in patients with neuroblastoma, where ferritin is in part tumor derived and high concentrations correlate with poor outcome. To date, in vitro and in vivo studies demonstrating the antitumor effects of DFO are very promising, but further investigations are required to establish an exact role for DFO in the treatment of cancer.

[123I]MIBG scintigraphy in neuroblastoma: a comparison between planar and SPECT imaging.

The aim of the present study was to assess the utility of SPECT imaging with [123I]MIBG in patients with neuroblastoma (NB). Twenty-two children were studied (11 males and 11 females: age range: newborn to 11 years). A total of 39 studies were performed in different clinical phases. Both planar (at 24 and occasionally 48 hours) and SPECT (at 24 hours) imaging were performed in all cases. Planar studies gave a sensitivity of 87.5% (evidence of the disease in 21 of 24 studies performed in children with known NB lesions) and a specificity of 93.3% (true negative results in 14 of 15 studies performed in disease-free patients). In the same patients SPECT gave a sensitivity of 95.8% (23 of 24 positive studies and a specificity equal to that of planar scanning. In 5 of 21 studies positive at planar scanning SPECT showed 9 additional lesions. In conclusion, [123I]MIBG SPECT imaging compared to planar scanning can demonstrate a greater number of lesions. Its use seems to be indicated mainly in cases in which diagnostic assessment is difficult, such as small residual tumors or in the follow up of children no longer in therapy, in whom it can lead to an early diagnosis of relapse.

D-CECaT as preoperative chemotherapy for unresectable neuroblastoma in children over one year of age.

The role of intensive pre- and postoperative chemotherapy in unresectable nonmetastatic neuroblastoma is still controversial. A preoperative regimen that included deferoxamine, cyclophosphamide, etoposide, carboplatin and thiotepa (D-CECaT) was evaluated in 10 children over one year of age at diagnosis, and this was followed by surgery and postoperative chemotherapy. After four courses of D-CECaT, the response rate was 9/10 with 3 complete responses, 6 partial responses and 1 minor response. Severe but transitory myelosuppression was the major toxic effect. Complete remission by combined D-CECaT chemotherapy and surgery was obtained in 9/10 patients, while 1 case achieved complete remission only with postoperative chemotherapy. All children are disease-free with a median follow-up of 30.5 months (range: 1+ to 50+). This intensive treatment was effective in both standard- and high-risk unresectable NB. However, whether a less intensive approach and fewer courses can also give similar results in standard-risk cases warrants further study.

Deferoxamine followed by cyclophosphamide, etoposide, carboplatin, thiotepa, induction regimen in advanced neuroblastoma: preliminary results. Italian Neuroblastoma Cooperative Group.

Based upon phase I and II studies of deferoxamine alone and in combination with cytotoxic agents cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CECaT), we initiated a single arm multicentre trial in 1992 for advanced neuroblastoma. 57 of 65 patients who entered the trial were evaluable. Following 4 courses of the D-CECaT, almost all the patients underwent surgery. Toxicity was moderate and mainly reversible myelosuppression. The post-surgically defined responses in stage 3 high risk, stage 4 moderate risk and stage 4 high risk patients included 24 complete responses, 26 partial responses, and 3 minor responses, and 4 patients had progressive disease. These patients are being followed to determine the impact of this programme on their overall survival.

Peripheral blood stem cells in children with solid tumors. Part I. Feasibility and application.

A method for collecting peripheral blood mononuclear cells following mobilizing chemotherapy in pediatric patients is described. The critical elements of the method included temporary heparinization of the patient to reduce citrate overload, and limiting extracorporeal circulation to 15% of the patient's blood volume using packed red blood cells and albumin. A median of 0.9 x 10(8) mononuclear cells/kg per collection were harvested in 40 collections from eight patients with only one episode of fever and chills. Peripheral blood stem cells were reinfused into six of these patients with refractory/recurrent pediatric tumors after intensive chemotherapy. Bone marrow reconstitution followed with a mean of 30 days (19-38) for absolute neutrophils and 48 days (32-275+) for platelets. Previous chemotherapy did not appear to affect peripheral blood stem cell efficacy in reconstituting chemotherapy-ablated bone marrow.

Deferoxamine, cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CECaT): a new cytoreductive chelation-chemotherapy regimen in patients with advanced neuroblastoma.

Thirteen patients with Stage III (3 patients) or Stage IV (10 patients) neuroblastoma were treated with a new iron chelation-cytotoxic therapy regimen. Deferoxamine given for five consecutive days followed by 3 days of cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CECaT) caused moderate to severe myelotoxicity. In 39 courses there were four episodes of sepsis; platelet and packed red blood cell transfusions were required in 72% and 82% of courses, respectively. Mild nausea and vomiting occurred in 52% of courses. Objective responses after two courses were observed in 12 of 13 patients. Three of four partial responses were achieved in previously treated relapsed patients, and seven of eight complete responses (four of which were surgically documented) were achieved in previously untreated patients. This cytoreduction regimen appears to be an improvement over other initial induction regimens and may be worth testing in larger populations.