Galectin-3 is a marker of favorable prognosis and a biologically relevant molecule in neuroblastic tumors.

Childhood neuroblastic tumors are characterized by heterogeneous clinical courses, ranging from benign ganglioneuroma (GN) to highly lethal neuroblastoma (NB). Although a refined prognostic evaluation and risk stratification of each tumor patient is becoming increasingly essential to personalize treatment options, currently only few biomolecular markers (essentially MYCN amplification, chromosome 11q status and DNA ploidy) are validated for this purpose in neuroblastic tumors. Here we report that Galectin-3 (Gal-3), a ß-galactoside-binding lectin involved in multiple biological functions that has already acquired diagnostic relevance in specific clinical settings, is variably expressed in most differentiated and less aggressive neuroblastic tumors, such as GN and ganglioneuroblastoma, as well as in a subset of NB cases. Gal-3 expression is associated with the INPC histopathological categorization (P<0.001) and Shimada favorable phenotype (P=0.001), but not with other prognostically relevant features. Importantly, Gal-3 expression was associated with a better 5-year overall survival (P=0.003), and with improved cumulative survival in patient subsets at worse prognosis, such as older age at diagnosis, advanced stages or NB histopathological classification. In vitro, Gal-3 expression and nuclear accumulation accompanied retinoic acid-induced cell differentiation in NB cell lines. Forced Gal-3 overexpression increased phenotypic differentiation and substrate adherence, while inhibiting proliferation. Altogether, these findings suggest that Gal-3 is a biologically relevant player for neuroblastic tumors, whose determination by conventional immunohistochemistry might be used for outcome assessment and patient's risk stratification in the clinical setting.

[Malignant pleural cancers mortality and compensated cases for asbestos related diseases in Lazio municipalities (1980-2001)]. / Mortalità per tumore maligno della pleura e casi indennizzati di malattie asbesto correlate nei comuni del Lazio (1980-2001).

BACKGROUND: Occupational exposure to asbestos has been widely reported in the Region, but a high risk for non-occupational and environmental contaminations have also been documented. OBJECTIVES: To describe the geographical distribution ofpleural cancer deaths and compensated asbestosis cases from 1980 to 2001 in the Lazio Region. METHODS: For each municipality Standardized Mortality Ratios (SMRs) for pleural cancer and Standardized Incidence Ratios (SIRs) for asbestosis were estimated. Expected cases were estimated from age and gender specific rates in Lazio. SatScan software was used to identify clusters and to verf;j their statistical significance. RESULTS: 789 deaths from pleural cancer (495 males and 294 females) occurred in Lazio from 1980 to 2001. The standardized mortality rate per 100.000 inhabitants is 0,74 (0,95 for males and 0,54 for females). The main excess mortality from pleural cancer occurred in the municipalities of Civitavecchia (SMR: 269,9; 95% CI: 164,9 - 416,8), Colleferro (SMR: 304,9; 95% CI: 139,4-578,8) and Rocca Priora (SMR: 379,2; 95% CI: 103,3-970,9). Significant SIRs for compensated asbestosis cases were found in the industrial areas of the Naples-Rome highway and in the shipyard area of Civitavecchia. Nofemale compensated cases were found. The most important clusters were identified in the municipality of Civitavecchia for pleural cancer (p-value = 0,117) and in the Colleferro industrial area for compensated asbestosis cases (p-value = 0,001). CONCLUSIONS: Epidemiological surveillance of incident cases of malignant mesothelioma in the Lazio Region and the investigation of modalities of asbestos exposure are urgently needed for prevention of occupational diseases.

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.

Morphological and molecular assessment of apoptotic mechanisms in peripheral neuroblastic tumours.

Multiple defects in apoptotic pathways have been described in peripheral neuroblastic tumours (NTs). Mitosis-karyorrhexis index (MKI) is a reliable morphological marker identifying favourable and unfavourable NTs. The extent to which apoptotic processes contribute to determine the clinical significance of MKI is still undefined. Apoptosis was investigated in a series of 110 peripheral NTs by comparing MKI to immunohistochemical and molecular apoptotic features. High MKI was found in 55 out of 110 NTs (50%) and was associated with advanced stage (P = 0.007), neuroblastoma (NB) histological category (P = 0.024), MYCN amplification (P < 0.001), and poor outcome (P = 0.011). Overall survival probability was 45% in patients with high MKI compared to 73% in patients with low MKI. In the same 110 NTs, the expression of Bcl-2, Bcl-XL, Bax and Mcl-1 was studied by immunohistochemistry, but no significant associations were found with clinicohistological features. Microarray analysis of apoptotic genes was performed in 40 out of 110 representative tumours. No significant association was found between the expression of apoptotic genes and MKI or clinicohistological features. Proliferative activity was assessed in 60 out of 110 representative tumours using Ki67 immunostaining, but no significant correlations with MKI or clinicobiological features were found. In NTs, the combination of apoptosis and proliferation as expressed by MKI is a significant prognostic parameter, although neither of them is per se indicative of the clinicobiological behaviour and outcome.

Combined use of gemcitabine and radiation in mice.

The aim of this study was to explore, in a murine tumor, if the effectiveness of radiation, in doses and schedules commonly used in clinical practice is potentiated by the combined use of the recently developed drug gemcitabine. Gemcitabine (30-360 mg/kg b.w.) was administered i.p. in female C3D2F1 mice bearing a mammary adenocarcinoma alone or combined with X-rays. Firstly, gemcitabine (single administration) was administered alone or at 20 min, 4 h, and 24 h before X-ray treatments. The significant effect observed only at 24 h time interval, depended on the X-ray dose and not on the gemcitabine dose. Secondly, 4 gemcitabine administrations every 3 days were used in fractionated combined schedules (overall treatment time of 10 days). We studied the relationship among different doses of gemcitabine, alone or combined with 10 daily X-ray treatments (2 Gy/fraction). We observed an interactive effect of gemcitabine up to its threshold dose of 60 mg/kg/fraction. Furthermore, 10 X-ray daily treatments and 4 X-ray treatments every 3 days (total doses 20-40 Gy) were performed with gemcitabine 60 mg/kg/fraction to study the effect of different doses and schedules of X-rays. Tumor growth delays increase with higher X-ray doses, and this occurs more with 4 X-ray treatments than with 10 X-ray treatments. Our results re-affirm the uselessness of high gemcitabine doses, and indicate the effectiveness of combined gemcitabine-radiation fractionated protocols.

Schedule dependent toxicity and efficacy of combined gemcitabine/paclitaxel treatment in mouse adenocarcinoma.

Increased interest in combining drugs with different targets has emerged over recent years. Our study aims at evaluating the effectiveness of combined gemcitabine/paclitaxel treatment taking into consideration doses, schedules, and toxicity. A spontaneous mammary carcinoma was transplanted into the right-hind foot of C3D2F1 mice. Paclitaxel (in doses from 20 to 80 mg/kg b.w.) and gemcitabine (in doses from 30 to 480 mg/kg b.w.) were administered i.p. in single or fractionated treatments. Toxicity and tumor growth delay (TGD) were the endpoints. TGDs for different gemcitabine doses in single administration (120, 240, and 360 mg/kg) overlapped (TGD approximately = 2.5 days). Toxicity was very high in daily administration. Results with gemcitabine alone showed the efficacy of treatments every 3 days. TGDs in fractionated treatments of 60 and 120 mg/kg x 4 were of approximately equals 16 days. Also in this case, tumor growth curves overlapped pointing out the uselessness of the high drug doses. For combined treatments, we used only fractionated protocols, administering gemcitabine every 3 days. Paclitaxel was administered alone in one or two fractions and with different sequences in respect to gemcitabine administration. With 120 mg/kg of gemcitabine all the protocols showed an increased unacceptable toxicity. The best result was obtained administering paclitaxel 40 mg/kg on days 1 and 15 and gemcitabine 60 mg/kg on days 3, 6, 9, and 12 (TGD = 38.2 days). The light toxicity and the high efficacy obtained with this protocol indicate the possible use of gemcitabine/paclitaxel treatment in clinical practice.

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.

Analysis of DNA oxidative damage related to cell proliferation.

In vivo and in vitro cell populations exhibit a different sensitivity and a heterogeneous response to many genotoxic agents. Several studies have been carried out to evaluate the possibility that the different sensitivity of the cells is related to their proliferative status. In this study, the sensitivity of proliferating (P) and quiescent (Q) C3H10T1/2 cells to oxidative damage and their repair capability has been investigated by single cell gel electrophoresis (SCGE) and micronucleus test. Furthermore the possibility to simultaneously detect DNA damage and cell cycle position has been evaluated. Our results showed a dose-related increase of DNA damage in exponential and plateau phase cells treated with hydrogen peroxide (doses ranging between 2.5 and 100 microM). DNA damage was almost completely repaired within 2 h after treatment in both culture conditions. The percentage of cells in the various phases of the cell cycle has been determined by comet assay and by flow cytometry, and a good agreement between the results of the two techniques was found. Untreated exponentially growing cells in G1 phase showed a lower tail moment than S and G2/M cells. The same cell cycle dependence was evidenced in cells treated with low doses of H(2)O(2), while, at the higher doses, all cells showed a similar level of damage. These results confirm the sensitivity of the Comet Assay in assessing DNA damage, and support its usefulness in evaluating cell cycle-related differential sensitivity to genotoxic agents.

Expression of B-myb in neuroblastoma tumors is a poor prognostic factor independent from MYCN amplification.

The transcription factors of the Myb family are expressed in several tissues and play an important role in cell proliferation, differentiation, and survival In this study, the expression of A-myb, B-myb, and c-myb was investigated in a group of 64 neuroblastomas at different dinical stages by a sensitive reverse transcription-PCR tchnique and correlated with patients' survival. All of the myb genes were frequently expressed in neuroblastoma tumors. Interestingly, the expression of B-myb, which was detected in 33 cases, was associated with an increased risk of death (P = 0.027 in a univariate analysis), whereas there was no correlation with A-myb and c-myb expression. In addition, in a multivariate Cox regression analysis that included myb gene expression, MYCN status, age at diagnosis, and tumor staging, MYCN amplification and B-myb expression were independently associated to an increased risk (P < 0.01 and P = 0.015, respectively). In overall survival curves obtained by stratifying the neuroblastoma cases on the basis of MYCN status and B-myb expression, the group of patients without MYCN amplification and positive for B-myb expression had worse survival probability than that without MYCN amplification and nonexpressing B-myb (P < 0.01). In summary, these findings provide the first demonstration that B-myb expression can be a useful prognostic marker in human neuroblastoma. Moreover, B-myb expression has a prognostic value complementary to MYCN amplification and can identify a group of high-risk patients that would not be predicted on the basis of the MYCN status only.

Combined hyperfractionated radiotherapy and protracted infusion chemotherapy in bladder cancer for organ preservation.

PURPOSE: To test an innovative schedule of concurrent protracted intravenous infusion (PVI) of cisplatin (CDDP) and 5-fluorouracil (5-FU) and hyperfractionated radiotherapy (HFRT) with organ-sparing intent in bladder cancer. PATIENTS AND METHODS: Fifty-two patients (pts) were selected to receive an aggressive TURB followed by 2 MCV cycles, and HFRT with concomitant CDDP and 5-FU PVI (33 pts) or HFRT and concomitant CDDP and 5-FU PVI (20 pts). The 5-FU and CDDP doses ranged from 180 to 220 mg/sm/day and from 4 to 6 mg/sm/day, respectively. Radiotherapy was delivered as three 100 cGy fraction per day or two 150 cGy fraction per day to a total dose of 50 Gy to the pelvis and a 20 Gy boost to the bladder. RESULTS: Grade III toxicity in pts who received or not MCV was: rectal tenesmus 12/33 and 0/20, dysuria 6/33 and 4/20, leukopenia 3/33 and 0/20, thrombocytopenia 7/33 and 1/20 pts, respectively. A Grade IV toxicity was observed in 2 pts. Of the 28 evaluable patients treated with MCV, CR were observed in 23 (82%) and PR in 5 cases. Of the 18 evaluable patients treated without MCV, CR were observed in 18 cases (100%). Actually, 65% and 14% of the CR pts treated with or without HCV are alive and free of tumor. CONCLUSIONS: This bladder-sparing treatment shows an acceptable acute and late toxicity, similar to that observed with radiotherapy alone. The high CRs and bladder preservation rates observed deserve further clinical evaluation.

Analysis of lung damage induced by trichloroethylene inhalation in mice fed diets with low, normal, and high copper content.

Copper is both an essential nutrient required for the activity of several enzymes and a toxic element able to catalyze free radical formation. Trichloroethylene (TCE) is a xenobiotic that generates epoxidic intermediates by bioactivation through the cytochrome P-450 system. In this study, the influence of a dietary copper imbalance on the TCE-induced lung damage was investigated. Weaning mice were fed copper-deficient, copper-sufficient, and copper-excessive diets. After 4 wk, mice were exposed for 30 min to 6,500 ppm of TCE and euthanatized 48 hr later. Lung damage in the TCE-treated mice consisted of vacuolations of Clara cells and was quantitatively evaluated by counting the vacuolated cells per micrometer of basal lamina. At the ultrastructural level, vacuolations appeared as the result of hydropic swelling of the endoplasmic reticulum cisternae. The copper-deficient mice presented the highest number of vacuolated Clara cells. These mice also showed alteration of the capillary endothelium and interstitium and decreased pulmonary copper-zinc-superoxide dismutase activity. Occurrence of oxidative stress in lungs of both copper-sufficient and copper-deficient mice following TCE treatment was indicated by a decrease in reduced glutathione and an increase in its oxidized form.

Flow cytometric and immunohistochemical correlations in high incidence human solid tumors.

475 patients with carcinoma at different sites (141 colon-rectum; 102 breast; 50 stomach; 48 kidney; 46 head and neck; 41 bladder; 47 other sites) submitted to surgery have been analyzed after histopathological staging and grading, by flow cytometry (monoparametric DNA content analysis) and immunohistochemistry (p53, c-erbB-2, and PCNA expression). In breast cancer patients the presence of receptors for estrogen (ER) and progesterone (PGR) has also been determined. Flow cytometry-derived parameters were DNA ploidy, fraction of cells in S-phase (SPF), and DNA content heterogeneity (multi-clonal stem cell lines with different DNA index and/or more than one subpopulations with different ploidy levels in different samples from the same tumor). Correlations of the results obtained by the different techniques have been attempted by the non-parametric Spearman's rank correlation approach. Significant associations (P < 0.05) were found between the histopathological, immunohistochemical and flow cytometric parameters considered in some anatomical regions, such as stomach (p53 vs DNA content aneuploidy and vs heterogeneity), colon-rectum (TNM vs p53 and vs heterogeneity), bladder (grading vs DNA content aneuploidy and vs heterogeneity). Tumor heterogeneity proved to be dependent on the number of tumor samples taken. The results of this preliminary assessment will subsequently be compared with the data obtained from a currently ongoing follow-up survey.

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.

4'-Epi-doxorubicin and hyperthermia in a murine mammary carcinoma: influence of the dose and fractionation.

Our aim was to analyse the dose-response rate of 4'-epi-doxorubicin (EP) alone and of EP combined with hyperthermia (HT) treatments in tumor-bearing mice. A spontaneous mammary carcinoma, transplanted into the right foot of female hybrid (C3H/RIxDBA/2J) mice, was used. EP (from 5 to 30 mg/kg) was administered i.p. and local HT (45-60 minutes at 42 or 43 degrees C) was carried out. Mice were treated with EP and/or HT in 1, 2 or 3 doses at 8 day intervals; in the case of 3 HT treatments EP was administered before the first or before each HT session (same EP total dose). When EP was given alone, in 1 or 2 fractions, results showed a clear dose-response relationship: tumor growth delay depended on the total dose only. Combining different EP single doses and 1 HT treatment (43 degrees C), an additive effect and perhaps a synergistic effect at the highest doses was observed. Among all tested combinations, the best results were observed combining 3 HT with only 1 EP treatment.

PCNA/cyclin expression and BrdU uptake define proliferating myosatellite cells during hyperplastic muscle growth of fish (Cyprinus carpio L.).

The possibility of detecting in situ proliferating myosatellite cells during postlarval muscle growth in the carp (Cyprinus carpio, L.) by means of BrdU and PCNA (Cyclin) immunohistochemistry has been evaluated on paraffin embedded sections. Nine subadult stages were defined according to the body length and weight. The fish were injected intraperitoneally with BrdU and fixed one hour later. Adjacent cross sections mounted on glass slides were incubated with monoclonal antiBrdU (1:100) and antiPCNA (1:200) antibody. The proliferative rate, defined as the percentage of labelled cells for each stage, was correlated to the corresponding percentage of small fibers (area less than 200 microns 2) determined by morphometric analysis. Desmin expression, immunocytochemically detected, was aimed at discriminating between the postmitotic and stem myosatellite cells. A low myosatellite cell proliferative activity (2-7%) throughout the carp growth period considered was demonstrated. Quantitative analysis provided evidence that during growth stages in which small fibers are numerous, the myosatellite cell proliferative activity is low and it increases when the small fibers number decreases. It is suggested that an age dependence of myosatellite cell proliferative rate controls the recruitment of new fibers in the carp.

Quality control study of the Italian group of cytometry on flow cytometry cellular DNA content measurements.

A quality control study on DNA flow cytometry, extended to 43 national laboratories, has been carried out by the Italian Group of Cytometry, using defined fixed suspensions of cultured human leukemia K562 cells and human blood lymphocytes. The participating laboratories were allowed to follow their own staining and measurement protocols. Aliquots of cellular suspension had to be measured three times on the same day and two other times on different days. A large heterogeneity of procedures emerged among participants. The average of mean DNA index laboratory values, from 36 laboratories who sent evaluable data, was 1.68, with a range from 1.49 to 1.97. The coefficients of variation ranged from 2.35 to 9.39% and from 2.79 to 8.5% for diploid and aneuploid peaks, respectively. Statistical analysis of the results showed quite good intralaboratory reproducibility, but statistically significant differences were observed among laboratories, for both DNA indices and coefficients of variation. These differences appear to be consistent. For standardization, it is essential that efforts should be made to identify the main sources of variation and to control them.