FISH molecular testing in cytological preparations from solid tumors

Many of the exciting new developments in solid tumor molecular cytogenetics impact classical and molecular pathology. Fluorescence in situ hybridization to identify specific DNA target sequences in nuclei of non-dividing cells in solid neoplasms has contributed to the integration of molecular cytogenetics into cytology in spite of the remarkable promiscuity of cancer genes. Indeed, although it is a low-throughput assay, fluorescence in situ hybridization enables the direct disclosure and localization of genetic markers in single nuclei. Gene fusions are among the most prominent genetic alterations in cancer, providing markers that may be determinant in needle biopsies that are negative or suspicious for malignancy, and may contribute to the correct classification of the tumors. In view of the expanding use of fluorescence in situ hybridization in cytology, future challenges include automated sample evaluation and the specification of common criteria for interpreting and reporting result

Outcome evaluation of school-based alcohol prevention: a European multi-centric trial (EU-Dap study)

Background: Curricula aiming at preventing substance use are widespread in European schools, without formal evaluation of their effectiveness. Promising programmes based on the Comprehensive Social Influence (CSI) model have never been evaluated in Europe. Aim: The overall aim of this thesis was to advance knowledge on the effectiveness of school-based best practice programmes on adolescents’ alcohol use, as well as to elucidate the possible pathways to behavioural changes. Methods: A cluster randomized controlled trial was carried out in 143 schools from seven European countries, involving 7079 students 12-14 years of age. Schools were randomly assigned to either control or to a 12-session standardised curriculum based on the CSI model, taught by trained teachers. Randomisation was blocked within socioeconomic levels of the school neighbourhood. Students were surveyed through a self-completed anonymous questionnaire at baseline, 6 (short term) and 18 (medium term) months thereafter. The effect of the programme on alcohol-related cognitive factors (intention to drink and to get drunk, knowledge on alcohol-related effects, skills to resist pressure to drink alcohol, normative beliefs, expectations and risk perceptions concerning alcohol) was analysed at the short term follow-up, on alcohol consumption and problematic use at the medium term. Additionally, the effect of some class characteristics on programme implementation was studied using the intervention classes as units of analysis. Given the hierarchical structure of the data all data analyses were carried out using multilevel models. Results: The programme was associated with decreased positive expectations towards alcohol (odds ratio (OR)=0.81) and perception of peer drinking (OR=0.79), as well as with increased resistance skills against pro-alcohol pressures (OR=1.21) and knowledge (OR=2.25) at the short term follow-up. One year later the programme was associated with a decreased odds of reporting episodes of drunkenness (OR=0.79) and alcohol-related problematic behaviours (OR=0.78). There was no reduction in the frequency of alcohol use but non-drinkers and occasional drinkers at baseline progressed towards frequent drinking less often in the intervention group than in the control group. Associations were stronger among students from schools located in area of low socio economic level. Some characteristics of the class predicted the level of programme implementation: prevalence of substance use was associated with a decreased odds of implementing the programme in its entirety (OR=0.81), while students’ connectedness was associated with an increased odds of teachers using roleplay (OR=1.52). Conclusions: School curricula based on the CSI model can modify cognitive factors for alcohol use and reduce occurrence of drunkenness and alcohol-related behavioural problems among students from European Countries. These programmes are likely to be particularly useful in socially disadvantaged areas. There was support for the hypothesis that resistance skills, norm perception and positive expectations may mediate the effect of school prevention on behavioural outcomes. Specific organizational strategies such as teachers’ training in class management techniques may be integral to the provision of this type of programmes, in order to improve their implementation

Genetic heterogeneity of HER2 amplification and telomere shortening in papillary thyroid carcinoma

Extensive research is dedicated to understanding if sporadic and familial papillary thyroid carcinoma are distinct biological entities. We have previously demonstrated that familial papillary thyroid cancer (fPTC) cells exhibit short relative telomere length (RTL) in both blood and tissues and that these features may be associated with chromosome instability. Here, we investigated the frequency of HER2 (Human Epidermal Growth Factor Receptor 2) amplification, and other recently reported genetic alterations in sporadic PTC (sPTC) and fPTC, and assessed correlations with RTL and BRAF mutational status. We analyzed HER2 gene amplification and the integrity of ALK, ETV6, RET, and BRAF genes by fluorescence in situ hybridization in isolated nuclei and paraffin-embedded formalin-fixed sections of 13 fPTC and 18 sPTC patients. We analyzed BRAFV600E mutation and RTL by qRT-PCR. Significant HER2 amplification (p = 0.0076), which was restricted to scattered groups of cells, was found in fPTC samples. HER2 amplification in fPTCs was invariably associated with BRAFV600E mutation. RTL was shorter in fPTCs than sPTCs (p < 0.001). No rearrangements of other tested genes were observed. These findings suggest that the association of HER2 amplification with BRAFV600E mutation and telomere shortening may represent a marker of tumor aggressiveness, and, in refractory thyroid cancer, may warrant exploration as a site for targeted therapy

Centrosomal and mitotic abnormalities in cell lines derived from papillary thyroid cancer harboring specific gene alterations

<p>Abstract</p> <p>Background</p> <p>Differentiated thyroid carcinoma offers a good model to investigate the possible correlation between specific gene mutations and chromosome instability. Papillary thyroid neoplasms are characterized by different mutually exclusive genetic alterations, some of which are associated with aneuploidy and aggressive phenotype.</p> <p>Results</p> <p>We investigated the centrosome status and mitotic abnormalities in three thyroid carcinoma-derived cell lines, each maintaining the specific, biologically relevant gene alteration harbored by the parental tumors: <it>RET/PTC1 </it>rearrangement in TPC1; heterozygous and homozygous <it>BRAF^V600E</it>mutation in K1 and in B-CPAP, respectively. B-CPAP cells showed a statistically significant (<it>P </it>< 0.01) higher frequency of abnormal mitotic figures compared to TPC1 and K1 cells.</p> <p>Conclusions</p> <p>Our data indicate that <it>RET/PTC1 </it>oncogenic activity is not related to mitotic chromosome impairment and missegregation whereas, based on the consistent difference in types/frequencies of centrosome and spindle abnormalities observed between K1 and B-CPAP cells, the hetero/homozygous allelic status of <it>BRAF^V600E</it>mutation seems to be not irrelevant in respect to chromosomal instability development.</p

Opioid overdose risk during and after drug treatment for heroin dependence: An incidence density case–control study nested in the VEdeTTE cohort

Introduction and Aims: To corroborate protective effects of a range of drug treatment modalities against overdose mortality risk. Design and Methods: Nested case–control study, with incidence density sampling, selecting controls retrospectively at each case event. Cases and controls came from a sub-cohort of opioid-dependent patients (n = 4444) from two Italian regions (Lazio and Piedmont). From 1998 to 2005, there were 91 overdose deaths (cases) matched to 352 controls. The primary outcome was overdose mortality and the primary exposure was drug treatment: opioid agonist treatment (OAT), opioid detoxification, residential community, psychosocial and other pharmacological treatment. Conditional logistic regression models generated intervention effects comparing mortality risk in and out of treatment, adjusting for confounding variables. Results: Overall, drug treatment reduced overdose mortality risk by 80% [adjusted odds ratio (AOR) 0.18, 95% confidence interval (CI) 0.10–0.33, P < 0.001] compared to being out of treatment. There was a particularly strong protective effect of OAT on overdose mortality (AOR 0.08, 95% CI 0.03–0.23, P < 0.001) compared to being out of treatment. There was evidence of a substantially elevated risk of overdose in the first month of leaving treatment (AOR 23.50, 95% CI 7.84–70.19, P < 0.001) compared to being in treatment. Discussion and Conclusions: The nested case–control design strengthened earlier findings that OAT in Italy has strong protective effects on overdose mortality risk, much stronger than has been previously seen in other Western European settings

Thyrospheres enriched in stem-like cells from B-CPAP thyroid cancer cell line: morphomolecular characterization

Many studies performed over the past years have shown that tumor growth is sustained by a subpopulation of cells with stem-like features (cancer stem cells, CSCs), such as self renewal, multipotency, high migration capacity, drug resistance and aberrant differentiation, but little is known about thyroid tumor CSCs. Among solid tumors, papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer representing up to 80% of thyroid tumors. Isolation and propagation of stem-like cancer cells from established cancer cell lines by sphere forming assay in selective serum-free medium has been extensively reported. We report here the enrichment and morphomolecular characterization of sphere-propagating cells with stem-like properties from the B-CPAP papillary thyroid cancer-derived cell line. Thyrospheres from B-CPAP cells could be propagated up to ten generations. The “stemness” profile was evaluated by functional assays, RT-PCR, western blot, immunocytochemistry. Sphere forming efficiency (SFE) and self renewal increased exponentially at every generation with maximum value at the 8th. Results showed an increase in mRNA expression of stem cell (Oct 3/4, Nanog, ABCG2, Nestin), endodermal (GATA4), tumoral (TP63), and early thyroid differentiated (PAX8, TTF1) markers. A decrease in mRNA expression was observed in late thyroid differentiated marker (TG) along the generation of spheres. Positive staining of Oct3/4, GATA4, Tp63 and TTF1 was also confirmed by immunoblotting and immunocytochemistry. We conclude that thyroid cancer stem/progenitor cell populations are present in the B-CPAP cell line, and that it can represent a model to propagate putative thyroid cancer stem-like cells. Supported by a RAS Grant (Regione Autonoma della Sardegna, P.O.S. FSE 2007-2013, L.R. 7/2007)

Human Enterovirus B: Selective Inhibition by Quinoxaline Derivatives and Bioinformatic RNA-Motif Identification as New Targets

The Enterovirus genus includes many viruses that are pathogenic in humans, including Coxsackie viruses and rhinoviruses, as well as the emerging enteroviruses D68 and A71. Currently, effective antiviral agents are not available for the treatment or prevention of enterovirus infections, which remain an important threat to public health. We recently identified a series of quinoxaline derivatives that were provento be potent inhibitors of coxsackievirus B5, the most common and a very important human pathogen belonging to the enterovirus genus. We have shown how most active derivatives interfere with the earliest stages of viral replication, blocking infection. Considering the broad antiviral spectrum, a very attractive property for an antiviral drug, we aimed to investigate the antiviral activity of the most promising compounds against other Enterovirus species. Here, we investigated the susceptibility of a panel of representatives of Enterovirus genus (enterovirus A71, belonging to A species; coxsackieviruses B4 and B3;echovirus 9, belonging to B species; and enterovirus D68, belonging to D species) to quinoxaline inhibitors. We also tested cytotoxicity and selectivity indices of the selected compounds, as well as their effects on virus yield.We also investigated their potential mechanism of action by a time course assay. In addition, a bioinformatic analysis was carried out to discover potential new conserved motifs in CVB3 and CVB4 compared to the other enterovirus species that can be used as new targets

A mutation screening of oncogenes, tumor suppressor gene TP53 and nuclear encoded mitochondrial complex I genes in oncocytic thyroid tumors.

Background: Thyroid neoplasias with oncocytic features represent a specific phenotype in non-medullary thyroid cancer, reflecting the unique biological phenomenon of mitochondrial hyperplasia in the cytoplasm. Oncocytic thyroid cells are characterized by a prominent eosinophilia (or oxyphilia) caused by mitochondrial abundance. Although disruptive mutations in the mitochondrial DNA (mtDNA) are the most significant hallmark of such tumors, oncocytomas may be envisioned as heterogeneous neoplasms, characterized by multiple nuclear and mitochondrial gene lesions. We investigated the nuclear mutational profile of oncocytic tumors to pinpoint the mutations that may trigger the early oncogenic hit. Methods: Total DNA was extracted from paraffin-embedded tissues from 45 biopsies of oncocytic tumors. High-resolution melting was used for mutation screening of mitochondrial complex I subunits genes. Specific nuclear rearrangements were investigated by RT-PCR (RET/PTC) or on isolated nuclei by interphase FISH (PAX8/PPARγ). Recurrent point mutations were analyzed by direct sequencing. Results: In our oncocytic tumor samples, we identified rare TP53 mutations. The series of analyzed cases did not include poorly- or undifferentiated thyroid carcinomas, and none of the TP53 mutated cases had significant mitotic activity or high-grade features. Thus, the presence of disruptive TP53 mutations was completely unexpected. In addition, novel mutations in nuclear-encoded complex I genes were identified. Conclusions: These findings suggest that nuclear genetic lesions altering the bioenergetics competence of thyroid cells may give rise to an aberrant mitochondria-centered compensatory mechanism and ultimately to the oncocytic phenotype. Keywords: Oncocytic carcinoma, Nuclear mitochondrial complex I subunits, Oncogene mutation analysi

Glutamine Starvation Affects Cell Cycle, Oxidative Homeostasis and Metabolism in Colorectal Cancer Cells

Cancer cells adjust their metabolism to meet energy demands. In particular, glutamine addiction represents a distinctive feature of several types of tumors, including colorectal cancer. In this study, four colorectal cancer cell lines (Caco-2, HCT116, HT29 and SW480) were cultured with or without glutamine. The growth and proliferation rate, colony-forming capacity, apoptosis, cell cycle, redox homeostasis and metabolomic analysis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT), flow cytometry, high-performance liquid chromatography and gas chromatography/mass spectrometry techniques. The results show that glutamine represents an important metabolite for cell growth and that its deprivation reduces the proliferation of colorectal cancer cells. Glutamine depletion induces cell death and cell cycle arrest in the GO/G1 phase by modulating energy metabolism, the amino acid content and antioxidant defenses. Moreover, the combined glutamine starvation with the glycolysis inhibitor 2-deoxy-D-glucose exerted a stronger cytotoxic effect. This study offers a strong rationale for targeting glutamine metabolism alone or in combination with glucose metabolism to achieve a therapeutic benefit in the treatment of colon cancer