Pierluigi Altavista

Radiosensitization by oxaliplatin in a mouse adenocarcinoma: influence of treatment schedule

PURPOSE The aim of our study was to investigate if oxaliplatin (1-OHP) could be used as a radiosensitizer in vivo. MATERIALS AND METHODS Experiments were performed in mice (C3D2F1) bearing a transplanted mammary carcinoma in a foot. Drugs, 1-OHP and cis-diammine-dichloro-platinum (CDDP), were administered i.p. Results were analyzed in terms of tumor growth delay (TGD). RESULTS 1-OHP and CDDP were tested in single doses of 6 and 10 mg/kg body weight. Administration of either 1-OHP or CDDP produced a significant TGD but only with the dose of 10 mg/kg. Single dose combined X-ray (10 Gy) and 1-OHP (6 and 10 mg/kg) treatments were performed with different sequences and time intervals (1 h, 4 h, and 24 h). All TGDs of these combined treatments were uniform among themselves (indicating that sequence and time interval did not influence the results), and did not depend on the drug dose. In X-ray (10 and 20 Gy) and 1-OHP (6 and 10 mg/kg) combined treatment, the TGDs increased only with X-ray dose. Different 1-OHP administration schedules were performed for fractionated experiments: two treatments every 4 days. The least toxic protocol (1-OHP total dose from 6 to 14 mg/kg) was selected for combined treatments with 10 daily X-ray treatments of 2 Gy. A clear drug dose-effect relationship was observed in those treatments with 1-OHP doses from 10 to 14 mg/kg. CONCLUSION Although low-dose 1-OHP did not induce a TGD when administered alone, in combined protocols it increased X-ray efficacy.

Predictions of mortality from pleural mesothelioma in Italy: A model based on asbestos consumption figures supports results from age-period-cohort models

Italy was the second main asbestos producer in Europe, after the Soviet Union, until the end of the 1980s, and raw asbestos was imported on a large scale until 1992. The Italian pattern of asbestos consumption lags on average about 10 years behind the United States, Australia, the United Kingdom and the Nordic countries. Measures to reduce exposure were introduced in the mid‐1970s in some workplaces. In 1986, limitations were imposed on the use of crocidolite and in 1992 asbestos was definitively banned. We have used primary pleural cancer mortality figures (1970–1999) to predict mortality from mesothelioma among Italian men in the next 30 years by age‐cohort‐period models and by a model based on asbestos consumption figures. The pleural cancer/mesothelioma ratio and mesothelioma misdiagnosis in the past were taken into account in the analysis. Estimated risks of birth cohorts born after 1945 decrease less quickly in Italy than in other Western countries. The findings predict a peak with about 800 mesothelioma annual deaths in the period 2012–2024. Results estimated using age‐period‐cohort models were similar to those obtained from the asbestos consumption model.

Exposure to Radiofrequency Radiation (900 MHz, GSM signal) does not Affect Micronucleus Frequency and Cell Proliferation in Human Peripheral Blood Lymphocytes: An Interlaboratory Study

Abstract Scarfì, M. R., Fresegna, A. M., Villani, P., Pinto, R., Marino, C., Sarti, M., Altavista, P., Sannino, A. and Lovisolo, G. A. Exposure to Radiofrequency Radiation (900 MHz, GSM signal) does not Affect Micronucleus Frequency and Cell Proliferation in Human Peripheral Blood Lymphocytes: An Interlaboratory Study. Radiat. Res. 165, 655–663 (2006). The objective of this study was to investigate whether 24 h exposure to radiofrequency electromagnetic fields similar to those emitted by mobile phones induces genotoxic effects and/or effects on cell cycle kinetics in cultured human peripheral blood lymphocytes. The effect of 900 MHz exposure (GSM signal) was evaluated at four specific absorption rates (SARs, 0, 1, 5 and 10 W/kg peak values). The exposures were carried out in wire patch cells under strictly controlled conditions of both temperature and dosimetry, and the induction of genotoxic effects was evaluated in lymphocyte cultures from 10 healthy donors by applying the cytokinesis-block micronucleus assay. Positive controls were provided by using mitomycin C. Two research groups were involved in the study, one at ENEA, Rome, and the other at CNR-IREA, Naples. Each laboratory tested five donors, and the resulting slides were scored by both laboratories. Following this experimental scheme, it was also possible to compare the results obtained by cross-scoring of slides. The results obtained provided no evidence for the existence of genotoxic or cytotoxic effects in the range of SARs investigated. These findings were confirmed in the two groups of five donors examined in the two laboratories and when the same slides were scored by two operators.

Impact of a Single Nucleotide Polymorphism in the MDM2 Gene on Neuroblastoma Development and Aggressiveness: Results of a Pilot Study on 239 Patients

Purpose: MDM2 is a key negative regulator of p53 activity, and a single nucleotide polymorphism (SNP309, T>G change; rs 2279744) in its promoter increases the affinity for the transcription factor SP1, enhancing MDM2 expression. We carried out a pilot study to investigate the effect of this polymorphism on development and behavior of neuroblastoma, an extracranial pediatric tumor with unfrequent genetic inactivation of p53. Experimental Design: We genotyped the MDM2-SNP309 alleles of tumor DNA from 239 neuroblastoma patients and peripheral blood DNA from 237 controls. In 40 of 239 neuroblastomas, the MDM2-SNP309 alleles were also genotyped in peripheral blood DNA. Data were analyzed by two-sided Fishers exact test, log-rank test, and Kaplan-Meier statistics. Where appropriate, data are reported with 95% confidence intervals (CI). Results: The frequency of both the T/G and G/G genotypes or the G/G or T/G genotype only was higher in neuroblastoma DNA samples than in controls: 60.3% (95% CI, 54.1-66.5) versus 47.3% (95% CI, 40.9-53.6), 30.4% (95% CI, 22.4-37.8) versus 15.0% (95% CI, 9.2-20.7), and 52.0% (95% CI, 45.0-59.9) versus 41.9% (95% CI, 35.3-48.5), respectively; Two-Sided Fishers Exact Test P values were 0.006, 0.003, and 0.048, respectively; Odds ratios were 1.69 (95% CI, 1.18-2.43), 2.45 (95% CI, 1.37-4.39) and 1.51 (95% CI, 1.02-2.22), respectively. A significant association (P = 0.016) between heterozygous (T/G)/homozygous (G/G) genotypes at SNP309 and advanced clinical stages was also shown. Homozygous/heterozygous SNP309 variant carriers had a shorter 5-year overall survival than patients with the wild-type allele (P = 0.046; log-rank test). A shorter overall survival in patients with heterozygous/homozygous SNP309 was also observed in the subgroups with age at diagnosis >1 year and adrenal primary tumor (P = 0.024 and P = 0.014, respectively). Conclusions: Data from this pilot study suggest that the MDM2 G/G and T/G-SNP309 alleles are markers of increased predisposition to tumor development and disease aggressiveness in neuroblastoma. However, additional studies with larger patient cohorts are required for a definitive assessment of the clinical relevance of these data.

Sperm DNA fragmentation induced by DNAse I and hydrogen peroxide: an in vitro comparative study among different mammalian species.

Sperm DNA damage may have adverse effects on reproductive outcome. Sperm DNA breaks can be detected by several tests, which evaluate DNA integrity from different and complementary perspectives and offer a new class of biomarkers of the male reproductive function and of its possible impairment after environmental exposure. The remodeling of sperm chromatin produces an extremely condensed nuclear structure protecting the nuclear genome from adverse environments. This nuclear remodeling is species specific, and differences in chromatin structure may lead to a dissimilar DNA susceptibility to mutagens among species. In this study, the capacity of the comet assay in its two variants (alkaline and neutral) to detect DNA/chromatin integrity has been evaluated in human, mouse, and bull sperm. The hypothesis that chromatin packaging might influence the amount of induced and detectable DNA damage was tested by treating sperm in vitro with DNAse I, whose activity is strictly dependent upon its DNA accessibility. Furthermore, hydrogen peroxide (H2O2) was used to assess whether spermatozoa of the three species showed a different sensitivity to oxidative stress. DNAse I-induced damage was also assessed by the sperm chromatin structure assay and the TUNEL assay, and the performances of these two assays were compared and correlated with the comet assay results. Results showed a different sensitivity to DNAse I treatment among the species with human sperm resulting the most susceptible. On the contrary, no major differences among species were observed after H2O2 treatment. Furthermore, the three tests show a good correlation in revealing sperm with DNA strand breaks.

Clinical Significance of CXC Chemokine Receptor-4 and c-Met in Childhood Rhabdomyosarcoma

Purpose: The CXC chemokine receptor-4 (CXCR4)/stromal-derived factor-1 and c-Met/hepatocyte growth factor axes promote the metastatic potential of rhabdomyosarcoma cell lines in experimental models, but no data are available on their role in rhabdomyosarcoma tumors. The expressions of CXCR4 and c-Met were evaluated in primary tumors and isolated tumor cells in marrow, and were correlated with clinicopathologic variables and survival. Experimental Design: Forty patients with recently diagnosed rhabdomyosarcoma were retrospectively enrolled. CXCR4 and c-Met expression was investigated in primary tumors by immunohistochemistry, in isolated marrow-infiltrating tumor cells using double-label immunocytology. Results were expressed as the mean percentage of immunostained tumor cells. Results: CXCR4 and c-Met were expressed in ≥5% of tumor cells from 40 of 40 tumors, with 14 of 40 cases showing ≥50% of immunostained tumor cells (high expression). High CXCR4 expression correlated with alveolar histology (P = 0.006), unfavorable primary site (P = 0.009), advanced group (P < 0.001), marrow involvement (P = 0.007), and shorter overall survival and event-free survival (P < 0.001); high c-Met expression correlated with alveolar histology (P = 0.005), advanced group (P = 0.04), and marrow involvement (P = 0.02). In patients with a positive diagnosis for isolated tumor cells in marrow (n = 16), a significant enrichment in the percentage of CXCR4-positive (P = 0.001) and c-Met–positive (P = 0.003) tumor cells was shown in marrow aspirates compared with the corresponding primary tumors. Conclusions: CXCR4 and c-Met are widely expressed in both rhabdomyosarcoma subtypes and, at higher levels, in isolated marrow-infiltrating tumor cells. High levels of expression are associated with unfavorable clinical features, tumor marrow involvement and, only for CXCR4, poor outcome. In rhabdomyosarcoma, CXCR4 and c-Met represent novel exploitable targets for disease-directed therapy.

Prenatal Exposure to Non-ionizing Radiation: Effects of WiFi Signals on Pregnancy Outcome, Peripheral B-Cell Compartment and Antibody Production

Abstract During embryogenesis, the development of tissues, organs and systems, including the immune system, is particularly susceptible to the effects of noxious agents. We examined the effects of prenatal (in utero) exposure to WiFi signals on pregnancy outcome and the immune B-cell compartment, including antibody production. Sixteen mated (plug-positive) female mice were assigned to each of the following groups: cage control, sham-exposed and microwave-exposed (WiFi signals at 2.45 GHz, whole body, SAR 4 W/kg, 2 h/day, 14 consecutive days starting 5 days after mating). No effects due to exposure to WiFi signals during pregnancy on mating success, number of newborns/mother and body weight at birth were found. Newborn mice were left to grow until 5 or 26 weeks of age, when immunological analyses were performed. No differences due to exposure were found in spleen cell number, B-cell frequency or antibody serum levels. When challenged in vitro with LPS, B cells from all groups produced comparable amounts of IgM and IgG, and proliferated at a similar level. All these findings were consistently observed in the female and male offspring at both juvenile (5 weeks) and adult (26 weeks) ages. Stress-associated effects as well as age- and/or sex-related differences were observed for several parameters. In conclusion, our results do not show any effect on pregnancy outcome or any early or late effects on B-cell differentiation and function due to prenatal exposure to WiFi signals.

Flow cytometric DNA ploidy, p53, PCNA, and c-erbB-2 protein expressions as predictors of survival in surgically resected gastric cancer patients†

In order to determine retrospectively the impact of some cytometric and immunohistochemical parameters on the overall survival of gastric cancer patients treated with surgery alone, paraffin-embedded tumor samples from 137 gastric carcinoma patients undergoing curative resection from 1987-1993 were analyzed by flow cytometry (FCM) and immunohistochemistry (p53, c-erbB-2, and PCNA expression). FCM-derived parameters were DNA ploidy and fraction of S-phase cells (SPF). Multiple regression analysis was applied to determine the prognostic significance of the conventional clinicopathologic findings together with the flow cytometric and immunohistochemical parameters on overall survival. When all parameters were entered simultaneously into the Cox regression model, stage and DNA ploidy (DNA index >1.35) clearly emerged as the only independent prognostic factors. When the stages were analysed separately, the independent prognostic factors resulted DNA ploidy in early stages (I-II) and grading in stage IIIA tumors. For stage IIIB tumors, no independent prognostic factor was found. These results indicate that the DNA ploidy pattern is a valuable predictor of survival in curatively resected gastric cancer patients, especially when less advanced tumors are taken into consideration.

Quality control study of the Italian group of cytometry on flow cytometry DNA content measurements: II. Factors affecting inter‐ and intralaboratory variability

A multicentric national quality control study has been organized under the auspices of the Italian Group of Cytometry to find a possible influence of some procedural steps in DNA flow cytometry measurements on DNA index (DI) values and to identify the main parameters affecting the interlaboratory variability. To 40 participating laboratories we provided suspensions containing unknown mixture of different cell types: an homogeneous thymocyte population used to check instrument linearity; one mixture composed of two cell types characterized by DI = 1.00 and 1.10; and another composed of three different cell types with relative DIs of 1.00, 1.26, and 1.62, respectively. Possible effects due to staining protocols were studied, allowing the participants to stain cellular DNA according to the procedure routinely adopted in each laboratory, in addition to a standardized procedure with a fixed PI solution. As far as the influence of instrument linearity on DI values is concerned, we did not find any correlation with the DI variability observed, even if the use of a standardized staining protocol could lead to a sensible gain in interlaboratory DI reproducibility. Twenty-five of 40 (65%) laboratories were able to discriminate the near-diploid subpopulation, and a coefficient of variation of less than 4% was the minimum condition necessary to recognize the DI = 1.1 population. In samples containing two aneuploid subpopulations, 25 of 35 (71.4%) laboratories showed a high reproducibility with the standard staining protocol and 22 of 38 (57.9%) with the free staining protocol. However, a sensible improvement in interlaboratory reproducibility emerged with respect to the previous trial.

Retrospective mortality cohort study of Italian workers compensated for silicosis.

Objectives: To estimate cause specific mortality in a large cohort of Italian workers compensated for silicosis. Methods: The cohort included 14 929 subjects (14 098 men and 831 women) compensated for silicosis between 1946 and 1979, alive on 1 January 1980, and resident in Tuscany (a region of central Italy with 3 547 000 inhabitants). Mortality follow up ranged from 1980 to 1999. Vital status and the causes of death were determined by linkage with the regional mortality registry and with the national mortality database. The cohort mortality rates were compared to the rates of the local reference population. SMRs and their 95% confidence intervals were computed assuming a Poisson distribution of the observed deaths. Specific SMR analyses were performed according to the level of disability, the year of compensation assignment, and the job type. Results: A significant excess mortality was observed in male silicotics for cancer of the lung, trachea, and bronchus and cancer of the liver, respiratory diseases (silicosis, asbestosis, antracosilicosis, and other pneumoconiosis), and for tubercolosis. Statistically significant mortality excess was observed in female silicotics for respiratory diseases (specifically silicosis and other pneumoconiosis) and tuberculosis. Analyses for period of compensation assignment showed a twofold increased SMR for biliary tract cancer among female workers and for liver cancer among male workers compensated before 1970. Conclusions: The excess mortality from respiratory tract cancers and respiratory tract diseases detected in Italian compensated silicotics are in agreement with previous epidemiological studies. Although the twofold increased risk for liver cancer among males is suggestive of a possible association with silica dust exposure, the finding needs to be confirmed.