EZH2 is increased in paediatric T-cell acute lymphoblastic leukemia and is a suitable molecular target in combination treatment approaches.

BACKGROUND: T-cell Acute Lymphoblastic Leukemia (ALL) represents about 10-15 % of pediatric ALL cases. EZH2, one of the components of Polycomb group proteins (PRC2) complex, catalyzes the trimethylation of histone H3 lysine 27 that is associated with transcriptional repression and tumor development. METHODS: We examined the expression levels of PRC2 complex in primary samples of T cells ALL at diagnosis by western blotting and real time PCR. We evaluated the effect of 3-deazaneplanocin-A (DZNep), an EZH2 inhibitor, alone and in combination with Daunoblastine on cell viability, apoptotic death and cell cycle distribution of T cell established Jurkat cell line. RESULTS: EZH2 was expressed in 75 % samples at different extents mainly with high expression level. SUZ12 was expressed in 60 % samples and EED in all samples, respectively. The Kaplan-Meier analysis shows that T-ALL expressing EZH2 had a lower probability of disease-free survival (DFS) compared to T-ALL negative for EZH2 (23 % vs 100 %) (p = 0.01). The EZH2 inhibitor DZNep used in combination with Daunoblastine was synergistic in inducing growth inhibition and increasing the apoptosis in T-ALL Jurkat cells at 48 and 72 h paralleled by EZH2 decreased expression. Moreover, the combination decreased the activity of Erk-1/2 proliferation enzymes with no effects on Akt survival pathway. CONCLUSIONS: The evaluation of EZH2 expression in pediatric T-ALL can be useful in predict the clinical outcome of the patients and EZH2 can be a useful target to improve the efficacy of conventional chemotherapy in this subset of patients with bad prognosis.

Effect of rMnSOD on survival signaling in pediatric high risk T-cell acute lymphoblastic leukaemia.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that defends against oxidative damage due to reactive oxygen species (ROS). A new isoform of MnSOD with cytotoxic activity was recently discovered in liposarcoma cells. Here, we tested the effectiveness of a recombinant form of this isoform (rMnSOD) on leukemic T cells, Jurkat cells, and lymphocytes. Our results confirm that leukemic T cells can internalize rMnSOD and that rMnSOD causes apoptosis of 99% of leukemic cells without showing toxic effects on healthy cells. Using light and electron microscopy, we determined that an rMnSOD concentration of 0.067 µM most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 µM rMnSOD resulted in high expression of the pro-apoptotic protein Bax and low expression of the anti-apoptotic protein Bcl-2 in leukemia cells. Concerning signal transduction pathway no influence was observed after treatment except for Jurkat cells showing a slightly decreased expression of ERK phosphorylation. These results suggest that rMnSOD may be an effective and non-toxic treatment option for T-cell leukemia.

Infections from CVC in the pediatric neoplastic patient. Single institution experience.

AIM: The present clinical study was carried out in order to evaluate in a perspective way the incidence of the infections caused by CVC, the micro-organisms mostly involved in the infectious process, the condition of aplasia in patients when blood cultures show positiveness and the incidence of removals expressed as number of performed removals/number of positive blood culture. METHODS: Between January 2003 and December 2009 452 blood cultures from CVC were carried out on 120 patients affected by acute lymphoblastyic and myelougenous leukemia (38), Hodgkin and non-Hodgkin lymphoma (17) and solid tumors (65), with an average of 65 blood cultures per year showing an average positiveness of 21 cases/year. The blood cultures were performed, in hyperpyrexia, when there was a clinical suspicion of infection from CVC. RESULTS: On 452 blood cultures from CVC carried out (31.4% positive per Gram +, 53.7% per Gram-, 14.9% per miceti) 128 (28.3%) resulted positive, excluding presumed contaminations. They were divided as follows: 21 of Staphylococcus epidermidis (16%), 10 of Escherichia coli (8%), 10 of Klebsiella pneumoniae (8%), 8 of Pseudomonas aeruginosa (6%), 8 of Staphylococcus aureus (6%), 6 of Enterobacter cloacae (5%), 4 of Candida parapsilosis (3%) and 61 of other micro-organisms (48%). It was necessary to perform 27 CVC removals. The micro-organisms most frequently involved in removals of the CVC were finally analyzed and the resulting frequency percentages are: - 85% for Gram- germs; -8% for Gram + germs; -7% for Mycete. CONCLUSION: Our clinical study has confirmed that in pediatric age neoplastic individuals there is a prevalence of CVC-correlated infections from Gram- and an elevated association of removals of the CVC caused by infections from Pseudomonas and Klebsiella, germs more frequently associated to clinical conditions of marked aplastic anemia.

Mortality from second tumour among long-term survivors of retinoblastoma: a retrospective analysis of the Italian retinoblastoma registry.

Survivors of retinoblastoma (Rb) are at high risk of dying from second malignant tumour. The occurrence of second malignant neoplasm (SMN) and related mortality in a cohort of 1111 cases from the Italian Retinoblastoma Registry was analysed, considering the possible role of both genetic and iatrogenic causes. Rb patients had a greater than 10-fold excess in overall mortality compared with the general population (standardized mortality ratio (SMR) 10.73, 95% CI 9.00-12.80). Their excess risk attributable to cancers other than Rb was 14.93 95% CI 10.38-21.49). Survivors of hereditary Rb had an SMR for all causes of 16.25 (95% CI 13.20-20.00), whereas their SMR for all cancers was 25.72 (95% CI 17.38-38.07). Survivors of unilateral sporadic Rb had an SMR of 4.12 from all cancers (95% CI 1.55-10.98) and a much higher excess for overall mortality (SMR 13.34, 95% CI 10.74-16.56). As expected, survivors of hereditary Rb had higher mortality from cancers of the bone (SMR 391.90, 95% CI 203.90-753.20) and soft tissue (SMR 453.00, 95% CI 203.50-1008.40), small intestine (SMR 1375.50, 95% CI 344.00-5499.70), nasal cavity (SMR 13.71, 95% CI 1.93-97.35) and cancers of the brain and central nervous system (SMR 41.14, 95% CI 13.2-127.55).

P-glycoprotein 170 expression and function as an adverse independent prognostic factor in childhood acute lymphoblastic leukemia.

Little is known about the prognostic role of multidrug resistance (MDR) in newly diagnosed childhood acute lymphoblastic leukemia (ALL). P-glycoprotein 170 (MDR1), a cellular drug efflux pump, is thought to be one of the major causes of MDR. The aim of this retrospective study was to evaluate in 85 children with ALL the impact of the MDR1 product of the mdr-1 gene on the achievement of complete remission (CR) and outcome. MDR1 protein expression was performed by immunocytochemistry (ICC), and flow cytometry (FC). MDR1 functional activity was performed by a rhodamine (Rhd)-123 efflux test with or without verapamil. All patients enrolled in our study were treated with AIEOP ALL 91-95 protocols. At diagnosis, 40 patients (47%) expressed MDR1 protein at significant levels, and 45 (53%) were MDR1 negative. Forty-three of the latter patients were also negative for MDR1 function, while 34/40 (85%) patients MDR1 positive preserved the function. Rhd-123 efflux was inhibited by the MDR modulator verapamil in 12/40 (30%) patients. After induction treatment, CR was achieved in 77/85 children (90.6%). All patients who did not achieve CR were MDR1 positive. Twenty-nine patients relapsed, 17 (58.6%) of whom were MDR1 positive. The 10-year overall survival (OS) rate, and disease-free survival (DFS) for MDR1 negative patients compared to MDR1 positive patients were 75.7% versus 54.8%, and 67.5% versus 46%, respectively. The 10-year event-free survival (EFS) rate was significantly higher (67.5% versus 36.8%) in the MDR1 negative group compared with the MDR1 positive population (p=0.001). Multivariate analysis showed that only EFS was independent of age, WBC count, immunophenotype, FAB subtype and prednisone response (p=0.019). Our results, derived from a monocentric study, demonstrate that MDR1 expression in childhood ALL is an independent adverse prognostic factor on outcome, and could be a useful biological marker of response in these patients. Moreover, MDR1 function was also a predictor of response, but only in univariate analysis.

Determination of the in vivo effects of prednisone on Bcl-2 family protein expression in childhood acute lymphoblastic leukemia.

Glucocorticoid resistance is often associated with treatment failure in children with acute lymphoblastic leukaemia (ALL) but the underlying molecular mechanisms are still unclear. In 30 consecutive children with ALL treated with prednisone we determined changes in the expression of Bcl-2, Bax and Bcl-xl proteins in leukemic lymphoblasts and related these to clinical features and rate of prednisone-induced apoptosis. The apoptotic index increased after prednisone therapy in 24 of the 30 patients. At diagnosis, we detected expression of Bcl-2 and Bcl-xl protein in 28 samples, while Bax expression protein was detected in 21 of the 30 patients. Prednisone treatment induced a decrease in Bcl-2 and Bcl-xl levels in 17 and 16 of the 28 patients, respectively, while Bax protein increased in 14 of the 21 patients. Twenty of the 30 patients studied were considered to be good prednisone responders, whereas 10 were poor responders. We observed a statistically significant decrease only for Bcl-xl protein expression in T phenotype ALL, in the poor responder group and in patients with >20000/mm(3) white cell count (WBC) at diagnosis. These data suggest a role of Bcl-xl in the mechanisms of protection of leukemic cells from apoptosis induced by glucocorticoids (GCs).

Left ventricular systolic and diastolic function after anthracycline chemotherapy in childhood.

BACKGROUND: In childhood, late cardiotoxicity is characterized by inappropriately thin wall and consequent increased end-systolic wall stress, but the associations of impaired left ventricular geometry and function occurring under these circumstances need further investigation. HYPOTHESIS: The purpose of this study was to assess anthracycline late effects on the relationships occurring between increased end-systolic stress (ESS) and changes in both M-mode systolic measurements (i.e., endocardial and midwall fractional shortening) and Doppler diastolic indices in the pediatric age. METHODS: The population consisted of 101 children treated with anthracyclines for at least 12 months and 91 healthy children. Using M-mode echocardiography, end-systolic wall stress was calculated as index of afterload, and endocardial and midwall fractional shortening as systolic indices. Doppler transmitral measurements were made as diastolic indices. RESULTS: Patients treated with anthracyclines showed significantly lower relative wall thickness and left ventricular mass index, greater end-systolic wall stress, reduced endocardial and midwall fractional shortening and peak E/A ratio, prolonged deceleration, and isovolumic relaxation times. Direct relationships were found between end-systolic wall stress and both endocardial and midwall shortening. The use of midwall shortening in the relation showed a greater, but not significant increase (from 3 to 6%) in the proportion of patients with depressed systolic function than did endocardial shortening. In the anthracycline group, end-systolic wall stress was also inversely related to relative wall thickness and directly to isovolumic relaxation time. CONCLUSIONS: In childhood, reduced myocardial thickness and increased afterload explain much of systolic and diastolic dysfunction of late anthracycline toxicity. Midwall fractional shortening does not seem to add useful information for identifying subsets of children more prone to the development of heart failure.

Recent advances in the prevention of anthracycline cardiotoxicity in childhood.

The prevention of anthracycline cardiotoxicity is particularly important in children who can be expected to survive for decades after cancer chemotherapy with these agents. The rapid increase in clinical toxicity at doses greater than 550 mg/m(2) of doxorubicin (DOX) has made this dose the limiting one in order to avoid DOX-induced cardiac failure. However, arbitrary dose limitation is inadequate because of variability of individual tolerance. Decreasing myocardial concentrations of anthracyclines (ANT) and their metabolites and schedule modification of administration can reduce anthracycline cardiotoxicity. Anthracycline structural analogues such as epirubicin, idarubicin and mitoxantrone have been used in clinical practice. In addition, the liposomal ANT, which can be incorporated into a variety of liposomal preparations, are a new class of agents that may permit more specific organ targeting of ANT, thereby producing less cardiac toxicity. Much interest has focused on the administration of ANT in conjunction with another agent that will selectively attenuate the cardiotoxicity. As is known, the ANT chelate iron and the DOX-iron complex catalyzes the formation of extremely reactive hydroxyl radicals. Many agents, such as dexrazoxane (DEX), able to remove iron from DOX, have been investigated as anthracycline cardioprotectors. Clinical trials of DEX have been conducted in children and significant short-term cardioprotection with no evidence of interference with antitumor activity has been demonstrated. Whether long-term cardiac toxicity will also be avoided in surviving patients has not yet been determined.

Storage in the yolk platelets of low MW DNA produced by the regressing follicle cells.

The present work was carried out to clarify the nature and origin of the yolk DNA present in vitellogenic oocytes of the lizard Podarcis sicula. Morphological and biochemical evidences indicate that it has an intrafollicular origin, from the apoptotic bodies resulting from follicle cells regression at the end of previtellogenesis. This conclusion is reinforced by the observation that the oocyte membrane, in in vitro experiments, is unpermeable to exogenous DNA. Biochemical evidences reveal that the yolk DNA has a low (200bp) molecular weight and this suggests that it is produced by the endonucleases typically involved in apoptotic DNA laddering. Indeed, immunocytochemical analyses demonstrate that follicle cells contain significant amounts of DNAse I. In immunoblots, carried out during different periods of the ovarian cycle, the enzyme shows a MW of about 33, 66 or 100 kDa thus indicating that its activity in the follicle of Podarcis is modulated by dimerization and/or binding to regulatory factors. Mol. Reprod. Dev. 59: 422-430, 2001.

Treatment of childhood acute lymphoblastic leukemia. Long-term results of the AIEOP-ALL 87 study.

BACKGROUND AND OBJECTIVES: In March 1987 AIEOP started the AIEOP-ALL-87 study, based on the previous AIEOP-ALL-82. The aim of this new study was to evaluate, for all risk groups: a) the efficacy of treatment intensification achieved by adding a fourth drug (daunomycin) in the induction phase and a 3-drug reinduction phase for all risk groups; b) the impact of the addition of three doses of intrathecal methotrexate during cranial radiotherapy and extended exposure to weekly high-dose L-aspariginase during late intensification in high risk patients. We report the long-term results of the AIEOP ALL-87 study. DESIGN AND METHODS: From 1987 to 1991, a total of 632 eligible and evaluable children (age 1 to < or =16 years) with non-B-cell acute lymphoblastic leukemia (ALL), were enrolled and stratified as follows: standard risk (SR, 79 patients, 12.5%) had WBC <10,000/mm3, age > or = 3 and <7 years, and FAB L1 morphology. The high risk (HR, 175 patients, 27.7%) group included patients with WBC > or =50,000/mm3 or FAB L3 morphology or T immunophenotype or acute undifferentiated leukemia (AUL) or leukemia-lymphoma syndrome. All the remaining patients formed the intermediate risk group (IR, 378 patients, 59.8%). All patients received a 4-drug induction therapy; intermediate-dose methotrexate was given to HR patients; cranial radiotherapy was given to IR and HR patients, while SR patients received extended intrathecal methotrexate; all patients received a 3-drug reinduction phase; high dose L-asparaginase (HD-L-ASP; E.Coli, Bayer) was given to HR patients; continuation therapy with 6-mercaptopurine, i.m. methotrexate, and monthly vincristine and prednisone pulses was given to all patients. Treatment duration was 2 years. RESULTS: Six hundred and nineteen patients (97.9%) achieved complete remission. The remission rate was 98.7% in the SR group, 98.1% in the IR group, and 97.1% in the HR group. The overall 10-year survival and event-free survival (EFS) rates (SE) are 74.7% (1.8) and 62.8% (2.0) respectively; EFS rates by risk group are 67.5% (5.5) in SR, 62.8% (2.6) in IR, and 61.9% (3.8) for HR. The 10-year EFS for all eligible patients was 63.9% (1.9). INTERPRETATION AND CONCLUSIONS: When compared to the results of the AIEOP-ALL-82 study, treatment intensification in the ALL-87 study has improved long-term survival and EFS from 66.4% and 53.6% to 74.7% and 62.8%, respectively. Failures were mostly due to marrow or extramedullary relapses suggesting that further treatment intensification, as being used in current therapeutic strategies, is appropriate, although patients relapsing after less intensive treatment may have better chances of rescue. These results, although obtained in a relatively large proportion of patients, in which infants were not included, indicate that the addition of high-dose L-asparaginase to a relatively non-intensive treatment may be of major benefit for HR patients and that the addition of intrathecal methotrexate during CRT, may improve the central nervous system-disease control with a marked reduction of nervous system relapses.

Pleuropulmonary blastoma: management and prognosis of 11 cases.

BACKGROUND: Pleuropulmonary blastoma (PPB) is a rare and aggressive malignant tumor that affects children and adults. This neoplasm is histologically characterized by primitive blastema and a malignant mesenchymal stroma that often demonstrates multidirectional differentiation. Despite the introduction of multimodal therapy, the prognosis of patients with PPB remains poor. METHODS: In the current study the authors reported on PPB cases from a national retrospective search performed in 18 Italian Associations for Pediatric Hematology and Oncology centers. Clinical data, surgical notes, pathologic findings, and summaries of chemotherapy and radiotherapy were obtained from reports and correlated with outcome by standard statistical methods. RESULTS: The series included 11 patients (7 boys and 4 girls) with a median age of 32 months. Respiratory distress was the most common clinical symptom. In three patients the PPB developed from other primary dysplastic diseases: cystic adenomatoid malformation in one case and congenital lung cysts in the other two cases. Five patients experienced disease recurrences (local recurrence in three patients and distant metastasis in two patients, within the central nervous system and an intraocular location, respectively). Patients with a type 2 histologic pattern and/or pleural involvement were found to have a worse outcome compared with patients without such features. Event free survival at 2 years from the time of diagnosis was 45% for all patients. Overall survival at 2 years was 72% for all patients. CONCLUSIONS: PPB is an aggressive neoplasm of early childhood and to the authors' knowledge no adequate therapy has been defined to date for patients with PPB. After making the diagnosis, the main goal of therapy should be radical surgery, even in patients with microscopic residual disease. Because the response to chemotherapy is poor, the authors' experience suggests that chemotherapy should be given with local radiotherapy in the majority of patients.

New site of temporary storage of rRNA in O. mykiss previtellogenic oocytes.

This article describes a new organelle found in the cytoplasm of the growth stage fish oocytes. In particular, we describe its organization at the morphological level and investigate its composition by different cytochemical and immunocytochemical approaches with both light and electron microscope. The conclusion is that the body is a peculiar protein scaffold functioning as a temporary trap for the storage of rRNA in the mid to late growth stage oocytes. Its presence would be related to the reorganization of the mass of amplified rDNA in micronucleoli and to the consequent temporary stop in the rRNA synthesis.

Cardiac toxicity after anthracycline chemotherapy in childhood.

The clinical use of anthracyclines, a family of chemotherapeutic agents with efficacy against many solid tumors and leukemias is limited by unique cumulative dose-limiting cardiotoxicity. Overt heart failure occurs in 4.5% to 7% of patients treated with anthracyclines and the incidence of cardiac function abnormalities increases with the time. Anthracycline-induced congestive heart failure is usually due to permanent changes in the myocardium, changes most consistent with the contractile failure of cardiomyopathy. Although the causes of anthracycline-induced cardiotoxicity are probably many, a large body of evidence points to free-radical-mediated myocyte damage. The risk of developing cardiac heart failure is modified by the presence of certain risk factors that reduce cardiac tolerance to anthracyclines. Age and female gender seem to have an important role in the anthracycline cardiotoxicity. This cardiotoxicity can be divided, on the base of when it started, into acute, subacute and progressive late, chronic form. Various invasive and non-invasive methods have been used to measure the extent of cardiac damage done. Depending on the sensitivity of the method employed, the proportion of hearts found to be damaged has varied widely. Attempts to ameliorate anthracycline cardiotoxicity have been directed toward: 1. decreasing myocardial concentrations of anthracyclines and their metabolites, 2. developing less cardiotoxic analogous, and 3. concurrently administering cardioprotectants to attenuate the effects of anthracyclines on the heart. Much progress has been made in terms of monitoring of clinical and subclinical anthracycline cardiotoxicity, finding alternative schedules, introducing special carriers of anthracyclines and using cardioprotecting agents. It is hoped that with all these effects and with results of ongoing and future trials, we will be able to reduce further or even eliminate anthracycline cardiotoxicity.

Alveolar soft part sarcoma in children and adolescents: A report from the Soft-Tissue Sarcoma Italian Cooperative Group.

BACKGROUND: Alveolar soft part sarcoma (ASPS) is a rare malignant tumor and little is known about its clinical features and management. We report on a series of 19 pediatric patients managed over 20 years. PATIENTS AND METHODS: Primary conservative surgery was performed in all patients and was radical in nine, non-radical in three; seven patients underwent biopsy alone (3 unresectable tumors, 4 metastatic disease). In two cases radical surgery was performed after primary chemotherapy. Radiotherapy was delivered to 8 patients, chemotherapy to 15. RESULTS: After a median follow-up of 74 months, the five-year survival was 80% for the whole series, 91% for patients with localized disease, 100% for patients with tumor < or = 5 cm, and 31% for those > 5 cm; 16 of 19 patients were alive (12 of 12 with grossly-resected tumor in first continuous remission). Chemotherapy achieved two partial remission among seven evaluable patients. CONCLUSIONS: Pediatric ASPS has a more favorable prognosis than its adult counterpart. In this series, tumor size correlates with metastatic disease at onset and is the major factor influencing survival. Surgery is the mainstay of therapy. The effectiveness of adjuvant therapy remains to be established, though radiotherapy may be advisable in cases of inadequate surgery.

Dual or single hepatitis B and C virus infections in childhood cancer survivors: long-term follow-up and effect of interferon treatment.

We conducted a long-term prospective study of 89 cancer survivor children who had acquired hepatitis B virus (HBV) and/or hepatitis C virus (HCV) during treatment for neoplasia, the aim being to evaluate the natural history of the diseases and the effect of interferon (IFN) treatment. Patients were followed up for a median period of 13 years (range, 8 to 20); 46 were infected by HBV, 11 by HCV, and 32 coinfected by HBV and HCV. A spontaneous clearance of hepatitis B surface antigen (HBsAg) occurred more frequently in coinfected patients (19%) than in the HBV-infected (2%; P =.004), with an annual seroconversion rate of 2.1% and 0.2%, respectively (P =.008). Loss of hepatitis Be antigen (HBeAg) occurred in 44% of coinfected and in 28% of HBV-infected patients. Clearance of serum HCV-RNA was observed in 34% and 9%, respectively, of coinfected and HCV-infected patients. Seventeen HBV-infected, 4 HCV-infected, and 16 coinfected patients received alpha-IFN treatment. In the HBV group, 6 patients (35%) cleared serum HBV DNA and seroconverted to anti-HBe; in the HCV-group, none cleared HCV-RNA. In the coinfected group, 1 patient cleared both HBV DNA and HCV-RNA, 6 patients cleared serum HCV-RNA alone, and 1 only HBV DNA and HBeAg. Overall, the diseases showed a mild histological course with no evidence of liver cirrhosis. A reciprocal interference on viral replication between HBV and HCV may occur in coinfected patients. Treatment seems to be effective for selected cases and is justified in view of the uncertain prognosis of the disease in these patients.

Structural and functional modifications of the nucleolus during previtellogenic oocyte growth in the lizard Podarcis sicula.

In the present study we analyse the nature and the functional significance of the spherical and fibrillo-granular structures appearing in the oocyte nucleus of the lizard Podarcis sicula, following the disappearance of the typical nucleolus. By LM and TEM approaches, we demonstrate that the fibrillo-granuli, containing DNA, RNA and nucleolar proteins, are micronucleoli transcriptionally active and that their DNA is probably derived from nucleolar fragmentation. By contrast, we could not explain the origin and role of the so-called spherical bodies, appearing earlier in oocyte growth; these, in fact, do not contain nucleic acids or nucleolar proteins and do not incorporate uridine. Different possible explanations of their significance are discussed.