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PURPOSE: The specific aims of the AIEOP-TW-2003 protocol included prospectively investigating a possible association of tumor loss of heterozygosity with outcomes in children treated for Wilms tumor. MATERIALS AND METHODS: We analyzed 125 unilateral favorable histology Wilms tumors registered between 2003 and 2008 in the Italian cooperative protocol for microsatellite markers mapped to chromosomes 1p, 7p, 11q, 16q and 22q. RESULTS: The 3-year disease-free survival and overall survival probabilities were 0.87 (95% CI 0.81-0.93) and 0.98 (95% CI 0.96-1.0), respectively. Loss of heterozygosity at 1p was significantly associated with a worse disease-free survival (probability 0.67 for patients with and 0.92 for those without 1p loss of heterozygosity, p = 0.0009), as confirmed also by multivariate analysis adjusting for tumor stage and patient age at diagnosis. There was no difference in disease-free survival probability among children with loss of heterozygosity in the other chromosomal regions tested. The worse outlook for children older than 2 years at diagnosis did not seem to be influenced by the loss of heterozygosity patterns considered. CONCLUSIONS: Chromosome 1p loss of heterozygosity seems to be a risk factor for nonanaplastic Wilms tumor, possibly regardless of other clinical factors. Our findings were uninformative regarding loss of heterozygosity in the other chromosomal regions tested.

PURPOSE: We analyzed whether the prognosis can differ among Wilms tumors (WT) labeled as Stage III according to currently adopted classification systems. METHODS AND MATERIALS: Patients with nonanaplastic Stage III WT consecutively registered in two Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) trials (CNR-92, TW-2003) were the subjects in the present analysis. The steady mainstay of therapy was primary nephrectomy, followed by three-drug chemotherapy with vincristine, dactinomycin, doxorubicin, and abdominal radiotherapy (RT). RESULTS: Ninety-nine WT patients met the criteria for classification as Stage III according to a revised version of the National Wilms Tumor Study-3 staging system (51 patients in CNR-92, 48 patients in TW-2003). Regional lymph nodes (LN) were not biopsied in 16 patients. After a median follow-up of 66 months, the 4-year disease-free survival (DFS) and overall survival (OS) rates were 85% ± 4% and 92% ± 3%, respectively, for the whole group. For 38 children with positive LN, the 4-year DFS rate was 73% ± 7%, as opposed to 98% ± 2% for the 45 children with Stage III WT according to the other criteria but with negative biopsied LN (p = 0.001). The subgroup with the worst prognosis consisted of children more than 2 years old with positive LN (DFS 67% ± 8%). A delay between surgery and RT > 30 days had an adverse impact on the abdominal tumor relapse rate. CONCLUSIONS: This study provides further evidence that Stage III tumors with LN metastases might be distinguished from WTs meeting the other criteria for classification as Stage III. The worse outcome of the former may warrant a prospective study on the effects of intensified therapy. A subclassification of Stage III tumors is discussed.

In Podarcis sicula specialized follicle cells send reserve materials to the previtellogenic oocyte via intercellular bridges. Immediately before the onset of vitellogenesis this transferring becomes particularly massive so that the cell volume significantly reduces, meanwhile in the nucleus the morphological alterations typical of apoptosis appear. To clarify why these follicle cells are not simply fully resorbed by the oocyte and to determine whether their DNA is discarded or recycled, we carried out a series of morphological and biochemical investigations. The finding that large macromolecular scaffolds are formed and that these are able to retain the DNA until it is extensively cut by two different endonucleases suggests that regression of the follicle cells is programmed and that the fate of their DNA is strictly controlled. Following its genetical neutralization via fragmentation, the DNA is apparently recycled by being transferred into the oocyte via the intercellular bridges, that, in fact, remain open until the very late stages of cell regression. The small DNA fragments reaching the oocyte cytoplasm would not interfere with meiosis completion but could significantly contribute to the stock of reserve materials to the advantage of the growing oocyte and/or developing embryo.

The rationale of this study was the evaluation of response to chemotherapy in children with advanced neuroblastoma using currently available diagnostic modalities.Iodine-131-metaiodobenzylguanidine (MIBG) imaging and 24-hr urinary vanillylmandelic acid (VMA) measurement were evaluated in 14 patients (7 males, 7 females, age range: 2-68 mo) with advanced neuroblastoma both pre- and postchemotherapy (5.6 +/- 2.8 mo) as well as serum ferritin (FER) and neuron-specific enolase (NSE) levels in 9 and 8 patients, respectively. MIBG images were qualitatively compared in each patient.Prechemotherapy, a total of 39 abnormal foci of MIBG uptake was detected. Postchemotherapy, 15 of these showed unchanged MIBG uptake, 7 had decreased uptake and 17 showed no uptake. In addition, four new abnormal foci of uptake were found. Postchemotherapy, a significant reduction of abnormal MIBG uptake (p0.01) was observed using a lesion-by-lesion analysis. When biochemical and MIBG postchemotherapy changes were compared, a significant relationship was found only between MIBG and VMA results (r = 0.84, p0.01).In postchemotherapy follow-up of children with advanced neuroblastoma, laboratory evaluation using VMA, FER and NSE measurements reflect only the global functional status of the disease, and are not helpful in defining the response of individual tumor lesions to treatment. Conversely, qualitative analysis using MIBG imaging may allow lesion-by-lesion evaluation of the heterogeneity of neuroblastoma response to chemotherapy. In this setting, changes in MIBG uptake are mirrored by the changes in catecholamine production, as measured by VMA levels.

Graft-versus-host disease (GVHD) is a common complication of allogeneic haematopoietic stem cell transplantation (HSCT), but membranous glomerulopathy (MG) has rarely been described as a manifestation of chronic GVHD. We report 2 cases of MG in children who were given allogeneic HSCT. The clinical findings were characterized by edema of the lower extremities and nephrotic proteinuria in one case; hypertension, haematuria and edema with non-nephrotic proteinuria in the other one. Renal biopsy was consistent with MG and appropriate immunosuppressive therapy was prescribed. Both patients achieved complete remission and are alive without renal disease 4 and 2 years after the diagnosis of MG, respectively. The normal levels of albumin and non-nephrotic proteinuria in one of the 2 cases raise the question of whether the real incidence of MG after HSCT is underestimated. Therefore, we strongly suggest regular urine analysis during the follow up of children given HSCT to timely diagnose MG.

BACKGROUND: Teratoma with a malignant somatic component (TMSC) is rare but described in adults, whereas information on pediatric presentation is sparse. PROCEDURE: The Associazione Italiana Ematologia Oncologia Pediatrica identified 14 cases of TMSC. Clinical files and pathology specimens were reviewed. RESULTS: The series (9 female, 5 male) showed the following disease: testis (2), sacrococcygeal (3), ovary (3), retroperitoneum (3), mediastinum (2), and foot soft tissue (1). Distribution of the somatic component was: carcinoma (4), pancreatic neuroendocrine tumor (1), neuroblastoma (3), rhabdomyosarcoma (3), rhabdomyosarcoma plus liposarcoma, chondrosarcoma, neurogenic sarcoma (1), chondrosarcoma plus neuroectodermal sarcoma (1), malignant peripheral nerve sheath tumor (1). Three patients were in stage I, four in stage II, three in stage III, and four in stage IV. All but one patient underwent surgery and only females showed carcinoma components. Nine patients relapsed or progressed and eight died. Six patients are alive and disease-free. Two patients underwent complete resection and four were treated based on transformed histologies. Relapse-free and overall survival rates were 35.7% and 42.8%, respectively (median follow-up, 31 months). CONCLUSIONS: Prognosis for germ cell tumors (GCTs) containing MSC is worse than that for GCTs. The pediatric disease appears to be more heterogeneous in tumor site distribution and MSC histology than in adults. Our series suggests no effects of age, histology, or gender on outcome. Surgery has an essential role in localized disease, with complete resection highly desirable. Chemotherapy optimized for histology should include reagents directed to the somatic malignancy, if chemosensitive. Malignant GCT warrants GCT-directed chemotherapy.

Molecular parameters involved in the prediction of response of childhood acute lymphoblastic leukemia (ALL) are still unclear. We have evaluated the expression and mutational status of p53 and the expression of bcl-xL and bax in a series of 62 consecutive children (median age: 4 years; 38 males and 24 females) affected by de novo ALL. Alterations and overexpression of p53 were uncommon events (9/62, 14.5%) while bcl-xL and bax overexpression were frequent (about 70%). EFS was directly correlated to age < 6 years (p = 0.0178), nonT phenotype (p = 0.0470), WBC at diagnosis ≤ 20000/μl (p = 0.0093), response to induction therapy with prednisone (p = 0.0211) and inversely correlated to mutated p53 or overexpression of p53 (p = 0.0039) and high intensity of Bcl-xL expression (p = 0.0055). OS was directly correlated with age < 6 years (p = 0.0004), female gender (p = 0.0139), nonT phenotype (p = 0.0012), WBC at diagnosis ≤ 20000/μl (p = 0.0187), response to induction therapy with prednisone (p = 0.0211), wild type p53 or low expression of p53 (p = 0.035). When all factors were considered in a stepwise Cox regression analysis, only the good response to PDN (p = 0.013) and the low intensity of Bcl-xL expression (p = 0.001) were independent significant prognostic factors with regard to EFS. Moreover, only age (p = 0.022), gender (p = 0.036) and WBC at the diagnosis (p = 0.050) were independent prognostic factors with regard to OS. Moreover, the mutated status of p53 was statistically correlated to the resistance to the induction therapy with PDN (correlation coefficient: -0.349, p = 0.008). In conclusion, both bcl-x L and bax were frequently expressed at high intensity, but only bcl-xL was an independent predictor of EFS in our series. Moreover, p53 alterations were uncommon and alone not strong independent predictors of outcome, but they were correlated to poor response to therapy with PDN and inversely correlated to EFS and OS in univariate analysis. ©2005 Landes Bioscience.

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.From 1987 to 1991, a total of 632 eligible and evaluable children (age 1 toor =16 years) with non-B-cell acute lymphoblastic leukemia (ALL), were enrolled and stratified as follows: standard risk (SR, 79 patients, 12.5%) had WBC10,000/mm3, ageor = 3 and7 years, and FAB L1 morphology. The high risk (HR, 175 patients, 27.7%) group included patients with WBCor =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.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).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.

AIM: To evaluate the role of postchemotherapy FDG PET and compare it with other predictive factors in paediatric Hodgkin's disease (HD). MATERIALS AND METHODS: In this retrospective study, 98 paediatric patients with HD (enrolled in eight Italian centres) were analysed. Their mean age was 13.8 years (range 5-19 years). A PET scan was performed at the end of chemotherapy and reported as positive or negative on the basis of visual and/or semiquantitative analysis. True outcome was defined as remission or disease on the basis of combined criteria (clinical, instrumental and/or histological) with a mean follow-up period of 25 months. Statistical analyses were performed for the postchemotherapy PET results and other potential predictive factors (age cut-off, stage, presence of bulky masses and therapeutic group) with respect to patient outcome and progression-free survival (PFS). RESULTS: Overall the patients had a mean PFS of 23.5 months (range 4-46 months): 87 achieved remission (88.8%) and 11 showed disease. Of the 98 patients, 17 were positive on postchemotherapy PET . Seven patients (41%) showed disease during follow-up, and relapse occurred in only four out of the 81 patients who were negative on PET (p = 0.0001). Kaplan-Meier analysis demonstrated significant correlations between PFS and the postchemotherapy PET result (p = 0.0001) and a cut-off age at diagnosis of 13.3 years (p = 0.0337), whereas disease stage (p = 0.7404), therapeutic group (p = 0.5240) and presence of bulky masses (p = 0.2208) were not significantly correlated with PFS. Multivariate analysis confirmed a statistically significant correlation with PFS only for the postchemotherapy PET findings (p = 0.0009). CONCLUSION: In paediatric HD, age at diagnosis and postchemotherapy PET results are the main predictors of patient outcome and PFS, with FDG PET being the only independent predictive factor for PFS.

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.

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.

Teratoma is the most common germ cell tumour in childhood; mature (MT) and immature teratomas (IT) are benign tumours, but if they recur, they can be in some cases malignant. The aim of this paper is to evaluate Italian patients with MT and IT enrolled from 1991 to 2001, in a prospective multicentric study. One hundred and eighty-three patients, observed in 15 Italian Centers of Paediatric Oncology and three Paediatric Surgical Units were enrolled. Clinical data, treatment and results were all analysed. Initial evaluation and subsequent follow up included clinical examination, tumour markers and imaging procedures. Surgical resection was recommended for all the tumours. Histology was centrally reviewed and IT was classified as grading 1–3. Chemotherapy (CT) with Vinblastine, D-actinomycin and cyclophosphamide was indicated for extra-testicular IT grade 2 or 3. MT was diagnosed in 127 patients (93 F and 34 M, age 1–192 months, median 24): 58 patients had gonadic tumour (23 testicular, 35 ovaric), 69 extragonadic (45 sacrococcygeal, 11 mediastinic, 7 retroperitoneal, 6 in other sites). A complete resection was performed in 117 patients, a partial resection in eight patients and biopsy in one. IT was diagnosed in 56 patients (34 F, 22 M, age 1–168 months, median 7). The T grading was 1 in 14 cases, 2 in 26, 3 in 16; 28 had gonadic T (17 ovary, 11 testis), 28 extragonadic (sacrococcygeal 19, mediastinic 3, retroperitoneal 2, other sites 4). CT was administered in eight patients; 15/182 patients relapsed (1 in a metastatic site) and in 5/15 the relapse showed malignant histology. Seven MT (5.5%) relapsed (five sacrococcygeal, one retroperitoneal, one mediastinic): surgery at diagnosis had been complete in five and with residual in two; the relapse was malignant in two patients with sacrococcygeal (sc) tumours, who had a complete resection and a partial resection respectively. Eight IT (14.2%) relapsed (four ovary, three sc, one retroperitoneal). The initial surgical resection had been complete in one, with residual in six, and a biopsy had been performed in one. A malignant recurrence occurred in two patients with sc tumours (after partial resection in one and after biopsy + CT in one) and in one patient with ovarian IT after a partial resection. All the patients underwent surgical excision of the recurred mass; CT according to Protocol for Malignant GCT was administered to those who had malignant recurrence; 122/126 patients with MT and 53/56 with IT are alive without disease with a follow up of 8–144 months (median 56). Two patients with malignant relapse (one with sc MT, one with sc IT) died because of the progression of the disease. Another two died due to severe malformations (one MT, one IT) and three were lost to follow up (two MT, one IT). The overall survival (OS) at 10 years is 98% (95% CI 93.9–99.4); the event free survival (EFS) is 90.4% (95 CI 84.8–94.0). At Cox analysis no significant difference in EFS was found regarding age and site of the primary tumour, while females (P = 0.011), patients with grade 1–3 histology (P = 0.025) and patients with incomplete resection appeared at higher risk of death or relapse (P < 0.001), with a seven, three and eightfold increase in risk, respectively. Our data showed that incomplete resection and female gender are important risk factors for relapse or death, more so than IT histology. The number of patients treated with CT is not sufficient to evaluate the efficacy of CT in avoiding malignant relapse.

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).

Background Data on the presence of myocardial abnormalities in long-term Hodgkin disease survivors are contradictory. The purpose of this study was to determine if myocardial performance index (MPI) was capable of discovering cardiac abnormalities. Procedure Echocardiographic evaluation was performed in 31 survivors of Hodgkin disease (mean age 17.0 years), who received doxorubicin as part of chemotherapeutic treatment (median dose 164.8 ± 42.5 mg/m2). Control group comprised 22 healthy subjects (mean age 16.7 years). Results Peak A velocity was increased (P = 0.004) and peak E/A velocity ratio was lower (P = 0.002) in patients compared to controls. Mean isovolumetric contraction time was longer in patients than in controls (P = 0.0001). Ejection time was significantly shorter in patients than in the controls (P = 0.001). Consequently, the MPI was significantly greater in the patients than in the controls (P = 0.0001). Abnormal MPI was found in 25/31 patients (83%). Conclusions The Doppler-derived index of combined systolic and diastolic myocardial performance demonstrates the presence of subtle cardiac abnormalities in the majority of Hodgkin disease survivors. Pediatr Blood Cancer © 2005 Wiley-Liss, Inc.

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 normal 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 ??M most effective on apoptosis induction. Western blot analysis showed that treatment with 0.067 ??M 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.

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 -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.

Clinical, biochemical, and sonographic evaluation of the remaining kidney function and size was performed in 34 patients, 12 males and 22 females, ages 2.1–19.6 years, nephrectomized for Wilms' tumor at least 2 years before (mean 8.6). All patients had normal blood pressure and serum bicarbonates. Two of them had microhematuria, four proteinuria 4 mg/m2/hr, and 11 microalbuminuria (MA) >20 mg/24 hr. Only one patient had reduced creatinine clearance and maximum bipolar length (MBL) as well as kidney volume (KV)

Primary childhood germ cell tumors (GCTs) represent a rare and heterogeneous group of tumors that varies in histologic differentiation, age of presentation and clinical outcome. In malignant neoplasms, apoptosis is a prognostic marker and a predictive factor of response to therapy. Therefore, the study of the expression and mutation of molecules involved in the regulation of apoptosis could be useful in order to both predict the clinical outcome and design self-tailored therapeutic approaches. We retrospectively analysed tissue samples of 54 childhood GCTs. The expression of p53 and BAX protein was assessed by immunohistochemistry (IHC). Moreover, we investigated the presence of mutations in the BAX and p53 genes SSCP-PCR and direct sequencing. IHC analysis of BAX protein expression showed that 14 out of 54 tumors (26%) had no BAX protein expression, in the remaining 40 patients (74%) the intensity of BAX was low in 20 patients (37%) and high/intermediate in 20 (37%). BAX was mutated in 6 patients. p53 was expressed in 43 patients (79.6%), was not detectable in the remaining 11 (20.4%) and mutated in only 3 patients. p53 mutational status and expression were not correlated to the overall survival (OS). On the other hand, both IHC score and mutations for BAX were correlated to sacrococcygeal primary localization. BAX mutations were inversely correlated with OS (p=0.0419) while BAX IHC intensity was directly correlated with OS (p=0.0376). The stratification for histotype showed a direct correlation between BAX IHC and OS in both immature teratoma (p=0.045) and mixed malignant GCT (p=0.010) while the correlation was lost in mature teratoma (p=0.300). These results indicate that both mutations and BAX protein levels are useful molecular biological markers for prognosis and clinical management of pediatric GCT.

The fact that anthracyclines are cardiotoxic seriously narrows their therapeutic index in cancer therapy. The cardiotoxic risk increases with the cumulative dose and may lead to congestive heart failure (CHF) and dilated cardiomyopathy in adults and in children. The prevention of anthracycline-induced cardiotoxicity is particularly important in children who can be expected to survive for decades after being cured of their malignancy. Attempts to reduce anthracycline cardiotoxicity have been directed towards: (i) decreasing myocardial concentrations of anthracyclines and their metabolites by dose limitation and schedule modification; (ii) developing less cardio-toxic analogs; and (iii) concurrently administering cardioprotective agents to attenuate the effects of anthracyclines on the heart. As regards schedule modification, avoidance of anthracycline peak levels may reduce the pathologic and clinical cardiotoxicity, although this has not always been observed. The analogs of doxorubicin, such as idarubicin and epirubicin, have similar cardiotoxicity to that of doxorubicin when given in amounts of equivalent myelotoxicity. Liposomal anthracyclines are a new class of agents that may permit more specific organ targeting, thereby producing less systemic and cardiac toxicity, but more studies are required to assess the advantages, if any, of these preparations over classical anthracyclines. The cardioprotective agent, dexrazoxane, an iron chelator, is highly effective and provides short-term cardioprotection to most patients receiving even the most intensive doxorubicin-containing regimens. Its long-term benefits remain to be determined. In addition, data remain insufficient to make specific recommendations regarding current use of dexrazoxane in children. It is thought that subtle abnormalities, related to anthracycline treatment in childhood, can develop into more permanent myocardial disease resulting in cardiomyopathy, which may progress to CHF. As regards the therapy of patients with anthracycline cardiotoxicity, two different situations have, therefore, to be considered: (i) if the patient presents with cardiac abnormalities, such as a reduction in fractional shortening at echocardiogram, without cardiac symptoms; and (ii) if the patient has CHF. In the presence of CHF, recovery with digitalis-diuretic therapy alone seldom occurs, and in patients who have refractory hemodynamic decompensation, heart transplantation is indicated. In patients with CHF, therapy with ACE inhibitors induces improvement in left ventricular structure and function, but this improvement is transient. Randomized clinical trials are, therefore, necessary to determine the effects of ACE inhibitors in mild-to-moderate left ventricular dysfunction. The beneficial effects of beta-adrenoceptor antagonists (beta-blockers) on cardiac function in heart failure due to anthracyclines seem comparable with those observed in other forms of heart failure with systolic dysfunction. Many drugs are available to treat children with CHF due to anthracycline treatment, but they are only palliative.

Childhood Acute Lymphoblastic Leukemia (ALL) represents approximately 40% of pediatric cancers, but molecular mechanisms involved in the therapeutic resistance of ALL are still unclear. The disregulation of cell cycle could be a mechanism of progression of leukemic blasts and glucocorticoids (GCs), the main pharmacological agent in the treatment of ALL, could affect cell cycle distribution. In our study we have evaluated cell cycle distribution and the expression of several molecules involved in cell cycle regulation in blasts collected from 32 patients with ALL before and 48 h after treatment with GCs. A significant increase of the percentage of ALL blasts in G(0)/G(1) phase was recorded after treatment with GCs in 22 (69%) out of 32 patients and 18 of these patients were also good responders to GC therapy. In these patients an increase of the expression of at least one of the 4 evaluated CDKIs (p15, p16, p21 and p27) was found in 29 out of 32 patients (90.6%) without any change in CDK2 and 4 expression. All patients expressed detectable levels of Rb-1 phosphorylation at the diagnosis. Twenty (63%) patients showed a decrease, while two patients showed an increase of p110 Rb-1 phosphorylation and no changes were detected in the remaining 10 patients after GC therapy. The univariate analysis showed that the reduction of pRb-1 phosphorylation was significantly higher in B-cell lineage patients and in good responders. In conclusion, this is the first report that evaluate the Rb-1 function as predictor of response in childhood ALL and our data suggest that its hypophosphorylation and, consequently, reduced activity correlates with a statistical significance with the responsiveness to GC therapy. These results suggest that Rb-1 can be a useful molecular target for the therapy of this subset of patients.

Background and Aims About 20% of patients with germ cell tumor (GCT) are still resistant to therapy. To investigate which features are present in resistant patients, a multicenter study on GCT in children was undertaken to correlate clinical and laboratory parameters with the outcome. Methods Patients aged less than 16 years, with histologically proven extracranial GCT were included. Results Ninety-five patients (median age 33 months, 45 males) were eligible. The site of the primary tumor was gonadal in 59, extragonadal in 36. The stage was I in 39; II in 5; IIIa (microscopic residue) in 7; IIIb (macroscopic residue) in 16; IIIc (unresectable) in 13; IV in 15. The treatment was surgery alone in 31; surgery plus radiotherapy in 1; chemotherapy ± surgery in 63. Post-chemotherapy resection in 19 (10 complete, 9 partial). The chemotherapy regimen was carboplatin 400 mg/m2/day on days 1, 2; etoposide 150 mg/m2/day on days 1, 2; ifosfamide 1,500 mg/m2/day on days 21, 22; dactinomycin 1.5 mg/m2/day on day 21; vincristine 1.5 mg/m2/day on day 21. Three patients died because of toxicity and two non-responders (to primary chemotherapy), died of progression; among the remaining 90 patients 20 relapsed, 9 are in second remission, 2 are alive with disease, and 9 died of disease progression (one from progression and intracranial hemorrhage). Overall survival was 82.7% and event-free survival: 71.5%. Survival according to: (a) site: testis: 100%; ovary: 88%; sacrococcyx: 69.6%; other sites: 33.3% (P

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.

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.

Chemotherapy is a recognized cause of morphological alterations to the proximal intestine. Lactose malabsorption, the functional consequence of a small intestinal enzymatic derangement, has been shown to play an important role in causing gastrointestinal symptoms in subjects receiving chemotherapy. To establish a rational basis for the exclusion of lactose from the diet and to reduce the risk of developing gastrointestinal symptoms, we conducted a study of lactose absorption in 20 children during cancer chemotherapy. Because lactose is an important nutritional sugar, the tolerance of lactose provided by yogurt was examined. Lactose absorption was investigated by a hydrogen breath test (BT) after oral ingestion of milk (250 ml) containing physiological doses of lactose (12 g). The effect of yogurt supplementation was also tested by BT after meals of yogurt (450 g) also containing physiological doses of lactose (12.1 g). In 11 children, lactose malabsorption was detected by BT during the study before any gastrointestinal symptom revealed this status. Of these 11 children, no gastrointestinal discomfort developed in five receiving a lactose-excluded diet. In contrast, in the six children not restricted in lactose intake, gastrointestinal symptoms were observed 4 to 13 weeks after lactose malabsorption was detected by BT. The findings of our study suggested the usefulness of dietary supplementation with yogurt, a lactose-containing food, in children who developed lactose malabsorption. In fact, all lactose-malabsorbent children showed good lactose absorption and tolerance when tested by yogurt BT.(ABSTRACT TRUNCATED AT 250 WORDS)

Two groups of children with acute lymphoblastic leukemia or non-Hodgkin lymphoma, treated with anthracyclines (ANT), were studied: group I, consisting of 10 patients, with coenzyme Q10 (CoQ) therapy; group II, consisting of 10 patients without CoQ therapy. The ANT cumulative dose was 240 +/- 20.0 mg/m2 in group I and 252.0 +/- 20.1 mg/m2 in group II. Echocardiographic study was performed at the beginning, at the cumulative dose of 180 mg/m2 and at the end of therapy with ANT. Percentage left ventricular fractional shortening (%LVFS) decreased from baseline (40.36 +/- 4.6) to end value (35.82 +/- 5.02) (P < 0.05) in group I; %LVFS decreased from baseline (39.89 +/- 4.37) to end value (33.43 +/- 3.46) (P < 0.002) in group II. Interventricular septum wall thickening decreased only in group II from baseline (46.10 +/- 10.1) to end therapy (27.00 +/- 18.54) (P < 0.01). Septum wall motion abnormalities were detected only in 2 patients of group II. These data demonstrate a protective effect of CoQ on cardiac function during therapy with ANT.

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.

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).

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.

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.

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.