Assessment of EGFR and K-ras mutations in fixed and fresh specimens from transesophageal ultrasound-guided fine needle aspiration in non-small cell lung cancer patients

In non-small cell lung cancer (NSCLC) patients, somatic EGFR and K-ras mutations predict therapeutic effectiveness and resistance, respectively, to EGFR tyrosine kinase inhibitors (TKIs). Transesophageal ultrasound-guided fine needle aspiration (EUS-FNA) is a validated technique for diagnosis and staging of NSCLC. In the present study, we compared the feasibility and reliability of EGFR and K-ras gene mutation analysis in fixed and fresh mediastinal lymph nodes and extra-lymph nodal samples obtained by EUS-FNA in patients suspicious for NSCLC. Thirty-six patients were enrolled into the study. For each patient, DNA was extracted from both fresh samples and fixed cytological smears. Exons 18-21 of EGFR and exon 2 of K-ras were amplified by PCR and mutation status was determined by direct sequencing and pyrosequencing. All cases were eligible for analysis. NSCLC was diagnosed in 32 patients (25 adenocarcinomas and 7 squamous cell carcinomas) and 4 patients were free of malignancy. Of the 25 patients with adenocarcinoma, EGFR mutations were detected in 2 (8%) fresh tumor samples and in 3 (12%) fixed cytological smears. K-ras mutations were detected in 8 (32%) fresh samples, and in 9 (36%) fixed cytological smears. Fixed and stained cytological samples seem to be more reliable than fresh material for molecular analysis.     

The effect of repeated administrations of granulocyte colony stimulating factor for blood stem cells mobilization in patients with progressive supranuclear palsy,corticobasal degeneration and multiple system atrophy

Granulocyte colony stimulating factor (GCSF) may boost physiological stem cell repair system in patients with cerebral lesions. Atypical parkinsonisms (PSP, CBD, MSA) are characterized by rapidly progressive course without significant benefit from current therapies.     

An overnight switch to ropinirole therapy in patients with Parkinson's disease

Patients with Parkinson's disease (n = 68) switched from pergolide or bromocriptine to ropinirole overnight (dose equivalence ratios 1:6 and 10:6, respectively). The activities of daily living score for the Unified Parkinson's Disease Rating Scale (UPDRS) was significantly improved 4 weeks after the bromocriptine-ropinirole switch. All other UPDRS scores, including that for the side-effect component, were not significantly different after either switch. Overnight switching may be a safe therapeutic approach that may reduce hospitalisation and related socio-economic costs.     

Lanreotide-induced modulation of 5-fluorouracil or mitomycin C cytotoxicity in human colon cancer cell lines: a preclinical study

The effect on growth of the long-acting somatostatin analogue lanreotide (LAN), alone or in combination with 5-fluorouracil (5-FU) and mitomycin C (MIT), was investigated in three human colon cancer lines. Cell survival inhibition induced by LAN alone, as evaluated by sulforhodamine B assay, ranged from 20% to 40% as a function of cell line and concentration. The IC50, the concentration inhibiting cell survival by 50%, was never reached. The antiproliferative effect produced by a 48 h exposure to 5-FU or MIT was synergistically enhanced in all cell lines by a subsequent 48 h exposure to LAN. The synergistic interaction was not related to specific cell cycle perturbations or to the somatostatin receptor 2 (sst2) mRNA abundance. In conclusion, our study seems to indicate that LAN is a potentially useful modulating agent for enhancing 5-FU and MIT activity in colorectal cancer patients.     

Antibodies to human recombinant tissue transglutaminase may detect coeliac disease patients undiagnosed by endomysial antibodies

BACKGROUND: The screening and diagnosis of coeliac disease have been simplified by the advent of new serological tools. AIM: To assess the clinical utility of a newly developed kit for antibodies to human recombinant tissue transglutaminase (hu-anti-tTG) in a large population of patients undergoing intestinal biopsy for suspected intestinal disorders. METHODS: We evaluated 426 serum samples from consecutive adult patients (250 from untreated coeliac disease patients and 176 from individuals in whom a diagnosis of coeliac disease had been excluded), obtained at the time of intestinal biopsy. Samples were tested for immunoglobulin A (IgA) hu-anti-tTG by enzyme-linked immunoabsorbent assay, IgA endomysial antibodies (EmA) by indirect immunofluorescence and IgA and IgG antigliadin antibodies by enzyme-linked immunoabsorbent assay. A sub-group of samples was also assessed for a guinea-pig-based anti-tissue transglutaminase. RESULTS: According to the cut-off for hu-anti-tTG, the sensitivity, specificity and positive and negative predictive values were 91%, 96%, 97% and 87%, respectively. Simultaneous determination of EmA showed values of 86%, 100%, 100% and 83% for the same parameters. Although 19 coeliac disease patients (7.6%) were negative for EmA and hu-anti-tTG, both tests rendered superior statistical values to antigliadin antibody tests. At diagnosis, IgA deficiency was detected in 11 patients, but both assays were able to detect samples with mild to moderate deficiency. The comparison of hu-anti-tTG with EmA showed excellent concordance between the tests (kappa statistic, 0.85). Discordance was observed in 20 samples from coeliac disease patients (8%) and in nine samples from controls (5%). Fifteen samples had an EmA-negative but hu-anti-tTG-positive serology, and five showed the converse pattern. Comparison of human recombinant and guinea-pig tests showed concordant results in 96% of cases. CONCLUSIONS: The quantitative determination of hu-anti-tTG type IgA using a commercial enzyme-linked immunoabsorbent assay kit was highly sensitive and specific for the detection of coeliac disease. Our results in a large population of patients with a clinical condition suggestive of the disorder demonstrated that the test can be used to detect a substantial number of patients otherwise unrecognized by IgA EmA.     

Docetaxel and gemcitabine activity in NSCLC cell lines and in primary cultures from human lung cancer

The activity of the following drugs was investigated in two established NSCLC cell lines: docetaxel, gemcitabine, vinorelbine, paclitaxel, doxorubicin (0.01, 0.1, 1 microg ml(-1)), cisplatin, ifosfamide (1, 2, 3 microg ml(-1)) and carboplatin (2, 4, 6 microg ml(-1)). The cytotoxic activity was evaluated by the sulphorhodamine B assay. The two most active drugs, docetaxel and gemcitabine, used singly and in association, were investigated as a function of treatment schedule. The sequence docetaxel-->gemcitabine produced only a weak synergistic interaction in RAL but a strong synergism in CAEP cells. The synergistic interaction increased in both cell lines after a 48-h washout between the drug administrations. Flow cytometric analysis showed that in docetaxel-->gemcitabine sequence, docetaxel produced a block in G2/M phase and, after 48 h, provided gemcitabine with a large fraction of recovered synchronized cells in the G1/S boundary, which is the specific target phase for gemcitabine. Conversely, simultaneous treatment induced an antagonistic effect in both cell lines, and the sequential scheme gemcitabine-->docetaxel produced a weak synergistic effect only in RAL cells. Moreover, the synergistic interaction disappeared when washout periods of 24 or 48 h between two drug administrations were adopted. The synergistic activity of docetaxel-->48-h washout-->gemcitabine was confirmed in 11 of 14 primary cultures, which represents an important means of validating experimental results before translating them into clinical practice.     

In vitro IL-6 production by EBV-immortalized B lymphocytes from young and elderly people genotyped for -174 C/G polymorphism in IL-6 gene: a model to study the genetic basis of inflamm-aging

In the present investigation we analysed Interleukin 6 (IL-6) in vitro production by Epstein-Barr virus (EBV)-immortalized B lymphocytes established from 43 subjects, 15 young people and 28 elderly people, including 18 centenarians, after 3, 6, 9, 24, 48 and 72 h of culture. The subjects were genotypized for the C to G transition at nucleotide -174 of IL-6 gene promoter (-174 C/G) and were classified as C allele carriers (C+) and non-carriers (C-). We found that: (i) the interindividual difference in in vitro IL-6 production was wider in elderly individuals in respect to young individuals, leading to different coefficient of variation in the two groups; (ii) the -174 C/G polymorphism had an age-related effect on IL-6 in vitro production. Only among C- people, cells from elderly subjects produced significant higher level of IL-6 than cells from young subjects. These data are consistent with our previous results regarding the IL-6 serum levels in a large group of people of different age, including centenarians. Thus, the EBV-immortalized B lymphocytes can be considered a useful in vitro model for studying the genetic control of IL-6 production and its changes with age.     

Heat shock response by EBV-immortalized B-lymphocytes from centenarians and control subjects: a model to study the relevance of stress response in longevity

'Successful aging', i.e. the ability to attain old age in relatively good health, is believed to be related to the capability to cope with different environmental stresses. Independently of their specific differentiation, all body cells respond to hyperthermia and other stresses with the production of Heat Shock Proteins (HSPs) that play an important role in cell survival. We investigated the heat shock response in B-lymphoid cell lines from 44 centenarians and 23 younger subjects, by studying both HSP70 synthesis and cell survival after hyperthermic treatment. Interestingly, no significant difference could be found between the two age groups as far as HSP70 synthesis was concerned; moreover, cell lines from centenarians appeared to be less prone to heat-induced apoptosis than lines from younger controls. These results, which are in contrast with previous findings showing an age-related decrease of the HSP70 synthesis and of hyperthermic response, corroborate the above mentioned hypothesis that the biological success of centenarians is due to the preservation of the capability to cope with stresses. An A/C polymorphism identified in the promoter region of HSP70-1 gene had been previously shown to affect the probability to attain longevity in females. To investigate if this effect was related to any influence of this polymorphism on HSP70 protein synthesis the correlation between A/C polymorphism and protein synthesis was investigated. We found that cells from AA centenarian females displayed a lower synthesis of HSP70.     

Flow-cytometric determination of tumor cells in lymph nodes

In solid tumors, metastasis occurs through the dissemination of tumor cells in the bloodstream and the lymphatic system. In particular, lymph node infiltration gives useful prognostic information and represents one of the most important factors for selecting the type of clinical treatment in disease management. Furthermore, the analysis of lymph node infiltration has become important for identifying patients with breast cancer or malignant melanoma who may be candidates for regional lymph node dissection. Tumor cells in lymph nodes are currently identified in tissue sections using morphological and immunohistochemical analyses, but these approaches are time-consuming, and micrometastases may escape detection. The aim of the present study was to define the potential of a flow cytometric (FCM) determination based on cell size and autofluorescence to shorten the time required for lymph node analysis. The sensitivity of the FCM approach, defined on mixtures of tumor cells from established cell lines and peripheral blood lymphocytes (PBL(s)) at different concentrations, was 1 tumor cell/1,000 PBL(s). FCM analysis was performed on 89 lymph nodes, 29 from breast, 41 from lung and 19 from colon cancer patients. Agreement between FCM and morphological results, used as gold standard, was observed in 83% of the cases, and there was a 90% sensitivity to the FCM approach for each tumor type. Disagreement was observed for 15 lymph nodes and was due, in the majority of cases (80%), to FCM-positive and morphologically negative results. A large number of patients and a more accurate pathological examination of consecutive histological sections of lymph nodes are needed to further evaluate the validity of the FCM approach.