A useful ultrasound score to select thyroid nodules requiring fine needle aspiration in an iodine-deficient area.

BACKGROUND: The high prevalence of thyroid nodules in iodine-deficient areas is a practical problem because of the large number of patients requiring fine needle aspiration (FNA) to detect malignant nodules. AIM: To obtain an ultrasound (US) score for predicting malignant nodules and reduce the number of unnecessary and expensive FNA. SUBJECT AND METHOD: All nodules observed from September 2001 to March 2006 were evaluated by US: echostructure, echogenicity, halo, microcalcifications and ratio between antero-posterior and transversal diameters (AP/TR). Two thousand six hundred and forty-two consecutive patients underwent US-guided FNA for a total of 3645 nodules. RESULTS: Logistic regression analysis showed a potent predictive role for solitary nodules and absence/ incomplete halo (p=0.000). A significant predictive role for microcalcifications and AP/TR ratio was also observed. A 10-point score was constructed using the standardized regression coefficient. Nodules with US score or=5.5 had a frequency of malignancy of 0.4, 1.1 and 5.6% (p<0.001), respectively. Nodules with >or=5.5 US score were characterized by a 66% sensitivity and a 76% specificity compared to the diagnostic values of single parameters which were either sensitive or specific. CONCLUSIONS: According to our data, we suggest FNA for nodules reaching a >5.4 US score, whereas a clinical judgement should be used for the intermediate category nodules. When the score is lower than 2.5 we do not recommend FNA. The practical use of this US score can help reduce unnecessary and expensive FNA in iodine-deficient areas.

Intraoperative autologous blood recovery in prostate cancer surgery: in vivo validation using a tumour marker.

BACKGROUND AND OBJECTIVE: Intraoperative autologous blood recovery during radical retro pubic prostatectomy has the potential of contamination with tumour cells. Its safety is proved by similar survival rates between allogeneic and autologous transfusion to oncology patients without standardization. Silencing of the gene encoding pi class of gluthatione-S transferase is a specific and sensitive molecular marker for prostate cancer, because it is present in more than 90% of prostate tumours. Using such tumour marker, we aimed to demonstrate that viable tumour cells could be eliminated using leucodepletion filters followed by irradiation. MATERIALS AND METHODS: Fifty patients with pi class of gluthatione-S transferase promoter hypermethylation in their primary prostate tumours were included in the analysis. Peripheral blood samples were collected during anaesthetic induction and recovered blood was collected throughout the surgery and then submitted to washing, leucoreduction and irradiation. Samples were analysed stepwise for the presence of promoter hypermethylation using real-time methylation-specific polymerase chain reaction. RESULTS: Positive hypermethylation was found in recovered blood (two samples), recovered and washed blood (three samples), and recovered washed and filtered blood (two samples). After filtration and irradiation of the recovered blood, this marker could not be detected in any of the cases analysed, suggesting the absence of viable tumour cells. CONCLUSION: Even though the risk of disseminating tumour cells in prostate cancer surgery by intraoperative autologous blood recovery is not yet fully established, no tumour-specific gene amplification was found after the association of blood filtration and irradiation, suggesting a significant reduction of such risk.

Fine needle aspiration cytology of thyroid nodules: conventional vs thin layer technique.

OBJECTIVE: Liquid-based cytology using the thin layer technique has recently been introduced in thyroid fine needle aspiration cytology together with or in substitution of direct smears, but its usefulness is still controversial and relatively few studies have been published in this field. The aim of the present study was to compare the results obtained from conventional smears with those from thin layer smears. DESIGN: In 3875 thyroid nodules, a double cytologic sampling was taken in randomized order, to prepare conventional or thin layer smears. MAIN OUTCOME: The diagnoses agreed in 2934 (75.7%) cases and disagreed in 941 (24.3%). The analysis of discordant data showed there were fewer non-diagnostic cases in the thin layer smears (377 vs 541, p<0.001) whereas in conventional smears there were more cases positive for carcinoma (27 vs 4, p<0.001). The cytohistologic correlation was available for 194 cases and showed that conventional smears had a greater capacity for revealing carcinomas (44 vs 31). Finally, diagnoses based on conventional smears were more sensitive than thin layer smears (93.6% vs 65.9%) whereas specificity was constant. CONCLUSIONS: From our experience, the conventional smear offers a greater possibility of diagnosis when suspecting malignancy or diagnosing malignancy cases, whereas thin layer smears significantly reduce the number of non-diagnostic cases. For this reason, we suggest combining the two techniques in routine cytologic diagnosis.

Extended intrathecal methotrexate may replace cranial irradiation for prevention of CNS relapse in children with intermediate-risk acute lymphoblastic leukemia treated with Berlin-Frankfurt-Münster-based intensive chemotherapy. The Associazione Italiana di Ematologia ed Oncologia Pediatrica.

PURPOSE: To assess the effect of treatment intensification and that of extended intrathecal methotrexate substitution for cranial irradiation in intermediate-risk acute lymphoblastic leukemia (ALL) children treated with a Berlin-Frankfurt-Münster (BFM)-based intensive chemotherapy. PATIENTS: Three hundred ninety-six children with non-B-ALL were enrolled onto the Associazione Italiana di Ematologia ed Oncologic Pediatrica (AIEOP) ALL 88 study. Standard risk (SR) included patients with low tumor burden (BFM risk index [RI], < 0.8); intermediate risk (IR) were patients with an RI > or = 0.8 but less than 1.2; and high risk (HR) were those with an RI > or = 1.2 or CNS involvement at diagnosis. The treatment schedule was a modified version of the ALL-BFM 86 study. CNS-directed treatment consisted of high-dose methotrexate (HD-MTX; 5 g/m2 for four courses) plus intrathecal methotrexate (IT-MTX; nine doses); IR patients additionally received extended IT-MTX (nine doses during continuation therapy); cranial irradiation was given only to HR patients. RESULTS: Of the 375 (94.7%) children who achieved remission, 1.3% had an adverse event other than relapse. The estimated event-free survival (EFS) at 6 years was 66.6% (SE 2.4) overall; 80.7% (4.5) in the SR patients, 77.5% (3.9) in the IR patients, and 54.5% (3.7) in the HR patients. Relapse occurred in 107 children (27.0%). Isolated CNS relapse occurred in 20 children (5.0%): 5 (6.3%) in the SR group, 1 (0.8%) in the IR group, and 14 (7.1%) in the HR group. The estimated 6-year CNS leukemia-free survival was 94.6% (1.2) overall: 93.5% (2.8) in the SR group, 99.1% (0.9) in the IR group, and 92.3% (2.0) in the HR group. CONCLUSION: Cranial irradiation may be omitted safely in IR ALL patients treated with BFM-based intensive chemotherapy when extended intrathecal chemotherapy is given. Because the CNS disease control was less complete in the SR group, these data challenge the effectiveness of HD-MTX for protection from CNS disease and support the protective role of extended intrathecal chemotherapy.

Isolation and characterization of the chicken cystatin-encoding gene: mapping transcription start point and polyadenylation sites.

A 6.9-kb fragment containing coding sequences for chicken egg white cystatin (CsnEW) was isolated from a chicken genomic library using the CsnEW cDNA as a probe. The gene is approximately 2.4 kb in length; it contains three exons, two introns and two polyadenylation signals. The exon-intron arrangement corresponds exactly with those of other members of the Csn superfamily. The sequence of the 5' flanking region contains two SP1-binding sites and a high G+C content suggestive of a housekeeping gene. All tissues studied express CsnEW mRNA; Northern analysis showed that CsnEW mRNA levels are most abundant in lung and least abundant in liver and spleen. Mapping of the 3' end of the CsnEW mRNA isolated from brain tissue resolved two CsnEW mRNA species. The larger transcript resulting from the use of the second polyadenylation signal was more abundant than the smaller transcript. Determination of the transcription start point (tsp) of CsnEW mRNA by primer extension and RNase protection assays showed that CsnEW mRNA from a number of chicken tissues was approximately 40-50 nucleotides shorter than that predicted from the CsnEW cDNA isolated from chicken oviduct.

Outcome after cessation of therapy in childhood acute lymphoblastic leukaemia. The Associazione Italiana Ematologia ed Oncologia Pediatrica (AIEOP).

A total of 2192 children with acute lymphoblastic leukaemia who had reached cessation of therapy in complete remission were followed for a median time of 52 months after treatment suspension. Of the 485 relapses observed, 62.3% occurred in the first year off therapy and 68.9% involved the bone marrow. Eight relapses were reported more than 5 years (62-143 months) after treatment withdrawal. Males fared worse than females consistently, experiencing 1.5 times more relapses (P < 0.0001). Thirteen patients died in continuous complete remission, 5 because of non-neoplastic central nervous system complications. There were 11 second solid malignancies, 8 of them in the central nervous system; 9 subjects presented an haematopoietic malignancy after ALL. The projected event-free survival at 8 years is 73%. Twenty-two of the 171 young adults (age > 20 years) were married and 16 have had 21 healthy children. Twenty-four per cent of patients experienced an unfavourable event. Relapses accounted for 93% of failures. Central nervous system late effects and second malignancies were the major causes of non-leukaemic morbidity and mortality.

Localization of cystatin mRNA in chicken brain by in situ hybridization.

The cystatins are a superfamily of proteins that inhibit the lysosomal cysteine proteinases, cathepsins B, H, and L. Members of this superfamily have been found in all tissues and biological fluids analyzed. Previous studies have shown that chicken cystatin mRNA is abundant in brain tissue. In this study, a definitive localization of chicken cystatin mRNA in chicken brain was determined by in situ hybridization. Chicken cystatin mRNA was heavily concentrated in the secretory epithelial cells of the choroid plexus. The rest of the brain failed to show a hybridization signal above that of the control sense strand probe, even after long exposures. We conclude that chicken cystatin is synthesized predominantly by the specialized secretory epithelial cells of the choroid plexus and secreted into the cerebrospinal fluid (CSF). We postulate that chicken cystatin functions to regulate proteinase activity in the CSF and therefore may function as a protective factor for the cellular elements of the central nervous system.

Glucose utilization in islets of hyperglycemic rat models with impaired glucose-induced insulin secretion.

Under most experimental conditions islet glucose metabolism is well-correlated with short-term glucose-induced insulin secretion. Two hyperglycemic rat models (neonatal streptozotocin and glucose infusion) have been previously found to have markedly impaired insulin responses to glucose, and the glucose utilization of islets isolated from these models was therefore studied to see if reduced glucose metabolism might be related to the secretory abnormalities. It was found that glucose utilization in the islets of the two models was similar or higher than in comparable control islets. These data suggest that the secretory defect of these models, which is presumably induced by chronic hyperglycemia, is at a step in the secretion process distal to glucose metabolism.

Promotion of neurite outgrowth by protein kinase inhibitors and ganglioside GM1 in neuroblastoma cells involves MAP kinase ERK1/2.

To investigate mechanisms of neurite outgrowth, murine Neuro-2a neuroblastoma cells were exposed to ganglioside GM1 in the presence or absence of specific protein kinase inhibitors. Isoquinolinesulfonamide (H-89), an inhibitor of cyclic AMP dependent protein kinase A (PKA), and bisindolylmaleimide I (BIM), which inhibits protein kinase C, each stimulated neurite outgrowth in a dose-dependent manner in the absence of exogenous GM1. Minimally effective (threshold) concentrations of H-89 or BIM potentiated outgrowth when they were used in combination with GM1. To search for a shared component in the mechanisms of GM1, H-89 and BIM, phosphorylation of ERK1/2 was examined. Inhibition of the activation of extracellular signal regulated kinases (ERK1/2) by U0126, prevented neuritogenesis of Neuro-2a by all the three agents. Pretreatment of serum-depleted Neuro-2a cultures with GM1 or BIM enhanced ERK1/2 phosphorylation when the serum level was restored to 10%. In contrast, H-89 did not alter the serum-mediated response. In cells exposed to GM1 or BIM without additional serum, a transitory decrease in ERK phosphorylation occurred. These data suggest that GM1 influences two neuritogenic pathways, one modulated by PKC and the other regulated by PKA. Therefore, GM1 may have the potential to stimulate alternate pathways resulting in outgrowth.

Acute changes in blood-CSF barrier permselectivity to serum proteins after intrathecal methotrexate and CNS irradiation.

Ten children affected by acute lymphoblastic leukaemia without CNS involvement were treated with a CNS prophylaxis protocol. Intrathecal methotrexate and CNS irradiation (60Co) administered at different times both induced an increase in blood-CSF barrier permeability to serum proteins (albumin, IgG, alpha 2 macroglobulin). The relationship between permeability coefficients of proteins was analysed by theoretical porous or vesicular blood-CSF barrier models. The analysis indicated that both therapeutic procedures affect endothelial pinocytosis. An increase in radius of pinocytotic vesicles from 400 to 1500 A seemed the most relevant change. The damage of endothelial barrier permselectivity could be involved in acute and late delayed toxic effects of intrathecal methotrexate and of CNS irradiation.

Ganglioside GM1 alters neuronal morphology by modulating the association of MAP2 with microtubules and actin filaments.

In previous studies, we demonstrated that the exogenous ganglioside GM1 increased the complexity of the microtubular network and level of tubulin, selectively changed the distribution of microtubule associated protein-2 (MAP2) immunoreactivity from the perikarya to distal neuritic processes and increased immunogold label of MAP2 in the subplasmalemmal cytoplasm, neuritic filopodia and growth cones of Neuro-2a neuroblastoma cells. Since these areas are rich in actin filaments, our data suggested that MAP2 may be associated with microfilaments in the early stages of ganglioside-induced neuritogenesis. To determine if GM1 alters neuronal morphology by facilitating the interaction of actin and MAP2, we examined the immunolocalization of these two proteins with confocal and electron microscopy. We found that along with the redistribution of MAP2 from perikaryal to neuritic regions, there was parallel redistribution of actin. The uniform subplasmalemmal actin meshwork was disrupted in areas of processes and filopodia with a redistribution of actin to these areas in close association with MAP2. Our present results suggest that gangliosides enhance neuritogenesis by redistributing actin as well as MAP2 to processes and filopodia thereby facilitating their interaction. The association of MAP2 with actin filaments is likely to be an early step in ganglioside-mediated filopodia formation.

GM1 enhances the association of neuron-specific MAP2 with actin in MAP2-transfected 3T3 cells.

The ganglioside GM1 is a glycosphingolipid which enhances process formation of several neuronal lines and potentiates some growth factor-mediated responses. Previously we have shown that 24 h exposure of Neuro 2a cells to GM1 mobilized the neuron-specific microtubule-associated protein, MAP2, away from microtubule-rich areas to areas of neurite sprouting where MAP2 was more closely associated with the subcortical actin network. To examine the role of GM1 in fostering the shift of the association of MAP2 from tubulin to actin, NIH 3T3 cells were co-transfected with pHook-1, which expresses a surface antigen, and a construct expressing MAP2. Transfected cells were selected with magnetic beads coated with a hapten that binds to the expressed surface antigen and treated with 150 microg/ml GM1 for 18-24 h. Actin and MAP2 or tubulin and MAP2 were immunolocalized and examined with confocal microscopy. MAP2 was found throughout the cytoplasm as well as associated with actin filaments. As observed previously with Neuro 2a, GM1 treatment of transfected fibroblasts redistributed the MAP2 away from direct association with microtubules to peripheral areas where the association of MAP2 with actin was enhanced. GM1 did not induce neurite-like processes in MAP2-transfected cells. Treatment with cytochalasin B, which is reported to result in process formation, also did not induce neurite-like processes. These studies suggest that GM1's ability to mobilize MAP2 and promote its association with actin is not restricted to neurons.

Pancreatic islets of variable size--insulin secretion and glucose utilization.

Glucose metabolism and insulin secretion were studied in isolated rat pancreatic islets of different sizes and the amount of tissue was quantitated by the measurement of DNA. It was found that larger islets (140-210 ng DNA/islet) utilized more glucose (based on the conversion of 3H-5-glucose to [3H]20) per ng of DNA than islets containing less DNA (60-120 ng/islet). However, the insulin secreted per ng of DNA in response to a given glucose concentration was the same in islets of all sizes. Also, the islet insulin and glucagon content when expressed in terms of DNA did not depend upon islet size. Thus, although glucose utilization rates expressed as a function of islet DNA content were greater in larger islets, no such relationship was found for glucose-induced insulin release or insulin and glucagon content.

Absolute structure-factor measurements of an Al-Pd-Mn quasicrystal.

A number of X-ray reflections from an icosahedral quasicrystal Al-Pd-Mn have been measured with great accuracy on an absolute basis by making use of Bragg-case diffraction. Since the specimen had high crystal quality, the dynamical theory was used for analyzing the results and to extract structure factors from measured integrated intensities. Good agreement was found between theory and experiment for strong reflections. Anomalous transmission was found to be strong in the 'good' regions of the quasicrystalline specimen and it was measured on an absolute basis, but the small residual strains present in the specimen prevented an accurate comparison between theory and experiment. A detailed discussion is presented on the parameters that mostly affect anomalous transmission in relationship to the adopted structural model.

Decreased activity of cathepsins L + B and decreased invasive ability of PC3 prostate cancer cells.

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L + B activity, and invasion through a Matrigel matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L + B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.

Matrigel invasion by the prostate cancer cell lines, PC3 and DU145, and cathepsin L+B activity.

Cathepsins L and B are lysosomal cysteine proteinases whose activities and cellular location are altered in many types of cancers and cancer cell lines. Cathepsins L and B play an unspecified role in cancer invasion and metastasis. The purpose of our study was to determine whether cathepsins L and B are important for the ability of two prostate cancer cell lines, PC3 and DU 145, to invade the basement membrane-like preparation, Matrigel. Exposure of PC3 and DU145 to the irreversible cysteine proteinase inhibitor, E64, decreases the invasive ability of DU145, but not PC3. PC3 and DU145 were treated with the phorbol ester analogue, phorbol 12-myristate 13-acetate (PMA), a known tumor promoter that activates protein kinase C and contributes to the metastatic phenotype. PMA increased secreted cathepsin L+B activity and the invasive ability of PC3 and DU145; co-exposure to E64 and PMA decreased both cathepsin L+B activity and invasion. We conclude that DU145 requires cathepsin L+B activity more than PC3 for the invasion of the Matrigel. When the amount of secreted cathepsin L+B activity is increased by PMA treatment, however, PC3 becomes dependent on cathepsin L+B for invasion. Our study demonstrates that modulation of the amount of secreted cathepsin L+B activity influences the invasive phenotype of PC3 and DU145.

Undifferentiated (embryonal) sarcoma of the liver in childhood: results of a retrospective Italian study.

The clinical features and the treatment of undifferentiated (embryonal) sarcoma of the liver in 8 patients younger than 19 years old were analyzed. All these cases were registered in the retrospective multicentric study on childhood malignant tumors of the liver, conducted between 1983 and 1985 by the Italian Association of Pediatric Hematology Oncology. The age of the patients ranged from 94 to 190 months (median = 113.5 months); all children were males. An abdominal mass was the common presenting features. Abnormalities in hemogram and common liver tests were rarely reported. Angiography revealed various degrees of vascularization in these tumors. Two patients achieved a surgical complete remission (CR) at diagnosis; one patient achieved surgical CR after primary chemotherapy with vincristine, adriamycin, cyclophosphamide and 5-fluorouracil, which reduced the tumor volume and permitted surgical resection. Two of these patients are still in CR at 14 and 60 months after diagnosis; the third patient died of liver failure without evidence of recurrence 6 months after diagnosis. All of the other patients, who never achieved CR, died of disease. One was lost to follow-up, and one surgical death occurred. Reports of childhood undifferentiated sarcoma are reviewed.

[Massive therapy and transplant of autologous bone marrow in childhood lymphomas and solid tumors. State of the art and perspectives]. / Terapia massiva e trapianto di midollo autologo nei linfomi e tumori solidi pediatrici. Stato dell'arte e prospettive.

The role of massive therapy followed by autologous bone marrow transplantation (ABMT) in pediatric tumours is reviewed. The rationale of such an approach based on dose-response relationship in clinical oncology, the usefulness of total body irradiation (TBI) as a part of conditioning regimens, and phase I and II trials with high dose drug combinations are discussed. The results of ABMT in lymphomas, sarcomas, neuroblastomas and other tumours are critically analyzed with special emphasis on prognostic factors, differences among conditioning regimens, intensive therapy morbidity and marrow purging procedures. Finally future perspectives and questions still unsolved are briefly summarized.

[Prognostic factors in non-Hodgkin's lymphoma in childhood]. / Fattori prognostici dei linfomi non-H. nell'infanzia.

38 children with Non-Hodgkin Lymphoma (age 15 months - 17 years; 27 males and 11 females) have been treated between 1974 and 1982. They have been divided in two different groups: a Good Prognosis group for patients with complete resectable disease and a Poor Prognosis group including patients with mediastinal, bone marrow or CNS involvement or with diffuse and non completely resectable localization. In the Good Prognosis group there were 100% Complete Remission, 12.5% Local Relapses and 12.5% deaths. 88% of patients are alive at 8 1/2 years follow-up. In the Poor Prognosis group there were 83% Complete Remission, 50% Relapses (8 in the first year, 2 in the second and 5 in the third and no more in the next years) and 60% Deaths; 30% of patients are off-therapy with a survival of 40% at 8 1/2 years follow-up. Among the 19 patients with mediastinal involvement there were 84.2% Complete Remission, 68.4% Relapses, 63.1% Deaths and 26.6% off therapy patients. Among the 19 patients with mediastinal involvement there were 84.2% Complete Remission, 68.4% Relapses, 63.1% Deaths and 26.6% off therapy patients. Survival is 70% for the group without mediastinal involvement and 35% for the group with mediastinal involvement. Burkitt-type Lymphoma has a survival of 30% in contrast to the 60% survival for all the others histological types. In summary we conclude that the distinction between Good Prognosis and Poor Prognosis groups, on the basis of a clinical stage involvement and Burkitt histology have an important role for prognosis of Non-Hodgkin Lymphoma in children.

[Association of infantile nephroblastomatosis and Wilms' tumor: discussion of a clinical case and therapeutic problems, analysis of the literature]. / Associazione della nefroblastomatosi infantile e tumore di Wilms: discussione di un caso clinico e delle problematiche terapeutiche, analisi della letteratura.

This is a case report of bilateral nephroblastomatosis in a 19 month child, who underwent a unilateral nephrectomy and chemotherapy. Further review of the nephrectomy specimen and biopsies of the contralateral kidney revealed mature features of nephrogenic rests progressing to Wilms' tumor. We have reviewed the literature and discuss the presentation and different therapeutic approaches.