How best to assess the quality of life in long-term survivors after surgery for NSCLC? Comparison between clinical predictors and questionnaire scores.

BACKGROUND: The determinants and predictors of QOL in lung cancer survivors who have received surgery remain defined vaguely and still debated. We evaluate clinical, surgical, and pulmonary function characteristics as predictors of QOL in long-term lung cancer survivors who received surgery. METHODS: Quality of life was evaluated 5 years after surgery in 67 lung cancer patients using the European Organization for Research and Treatment of Cancer (EORTC) QOL Core Questionnaire, its lung cancer-specific module QLQ LC-13, and the Hospital Anxiety and Depression Scale questionnaire. Preoperative clinical, surgical, and pathologic data were matched with the questionnaire scores. RESULTS: Sex was associated with role functioning and symptoms, with males more often reporting fatigue and pain, appetite loss, coughing, and hemoptysis (P < .05). Lower education was associated with better cognitive functioning (P < .05). Symptoms were worse for younger patients and for those with major comorbidity. Histology marginally influenced the global health status (P < .10) and the cognitive functioning (P < .05). Patients receiving complementary therapy more easily suffered from fatigue and insomnia (P < .05), and to a lesser extent from nausea and vomiting, constipation, and stress related to financial difficulties (P < .10). Higher values of forced expiratory volume at the first second (FEV(1)) and forced vital capacity (FVC) were significantly (P < .05) associated with a lower frequency of nausea and vomiting and appetite loss, while low percentage levels of FEV(1) and FVC were associated with lower global function and a greater severity of specific and nonspecific symptoms (P < .10 and P < .05). CONCLUSIONS: Several preoperative features, particularly those reflecting pulmonary function, were moderately associated with QOL in long-term survivors and may be useful to address therapeutic strategies in lung cancer patients after surgery.

Human population studies with the exfoliated buccal micronucleus assay: statistical and epidemiological issues.

OBJECTIVES: Micronucleus (MN) assay in buccal exfoliated cells is a minimally invasive method for monitoring genetic damage in human populations. Statistical and epidemiological issues related to the design and analysis of studies based on this biomarker are addressed. METHODS: A systematic review of recent literature on the buccal MN assay has been carried out to provide a state-of-the-art evaluation of how critical topics such as control for confounding, sample size and statistical power, number of cells scored, endpoint selection, and statistical modelling, are considered. In addition, a meta-analysis has been performed to estimate the impact of most common confounders on MN frequency, and to provide a baseline value of MN frequency in the control population. RESULTS: A total number of 63 eligible studies were included in the analysis. Age (98.4%), gender (85.7%), and smoking habit (90.5%) were the most commonly studied confounders. Univariate statistics were estimated in most studies while multivariate analysis was applied only in the 47.6%. Baseline MN frequency in controls was 1.10/1000 cells (95% confidence interval 0.70-1.72), and the relative increment in subjects exposed to genotoxic agents or affected by disease correlated with similar observations in lymphocytes (R(2)=0.74). A minimum number of 4000 cells is recommended to reduce the variability of the MN mean estimates, in contrast with the current practice of scoring only 2000 cells (81% of studies). Poisson or Negative Binomial are the preferred statistical models when more than 2000 cells are scored. Studies scoring smaller numbers of cells should consider the use of statistical models taking into account the excess of zeros, e.g., the Zero Inflated Poisson (ZIP) models. CONCLUSIONS: The quality of papers published on the buccal MN assay can be substantially improved, with better consideration of basic issues such as power analysis, control for confounding, choice of the statistical model, and the number of cells to be scored.

State of the art survey of the buccal micronucleus assay--a first stage in the HUMN(XL) project initiative.

The study of DNA damage in exfoliated buccal cells is a minimally invasive method for monitoring populations for exposure to genotoxic agents. The presence of micronuclei (MN) and other nuclear anomalies within these cells has been shown to be associated with genetic defects in genome maintenance, accelerated ageing, genotoxic damage and some degenerative diseases. To identify important information gaps regarding these biomarkers, a new initiative was launched within the framework of the HUman MicroNucleus (HUMN) collaborative programme, the HUMN(XL) project ('XL' designating eXfoLiated cell). An invitation to join the project was sent out together with a questionnaire to all laboratories that have published on the buccal micronucleus assay. Overall, 188 messages were delivered and 58 laboratories from 25 countries agreed to participate (43 contributing data). The questionnaire was designed to collect methodological information regarding the laboratory's performance of the assay and to assess the extent and type of epidemiological data that are routinely collected. The results provide an overview of the most commonly used methods for buccal cell collection and preparation, slide preparation, staining, scoring criteria and an evaluation of epidemiological data, including demographics, genetic background, gender, health status, occupation, exposure, lifestyle and dietary habit. According to this survey, a potential base of 15 103 subjects can be included in future pooled analyses. A number of protocol discrepancies emerged, implying that method standardization is a major priority. The results of this survey will contribute to (i) identify technical and epidemiological key variables that impact on buccal MN frequency in human populations, (ii) drive the design of future intra- and interlaboratory validation studies and (iii) determine the role of MN frequency and other biomarkers, in monitoring genomic damage and predicting cancer and other degenerative diseases.

A novel Bim-BH3-derived Bcl-XL inhibitor: biochemical characterization, in vitro, in vivo and ex-vivo anti-leukemic activity.

BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-X(L), involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-X(L), we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic.

Normalization of low-density microarray using external spike-in controls: analysis of macrophage cell lines expression profile.

BACKGROUND: The normalization of DNA microarrays allows comparison among samples by adjusting for individual hybridization intensities. The approaches most commonly used are global normalization methods that are based on the expression of all genes on the slide and on the modulation of a small proportion of genes. Alternative approaches must be developed for microarrays where the proportion of modulated genes and their distribution are unknown and they may be biased towards up- or down-modulated trends. RESULTS: The aim of the work is to study the use of spike-in controls to normalize low-density microarrays. Our test-array was designed to analyze gene modulation in response to hypoxia (a condition of low oxygen tension) in a macrophage cell line. RNA was extracted from controls and cells exposed to hypoxia, mixed with spike RNA, labeled and hybridized to our test-array. We used eight bacterial RNAs as source of spikes. The test-array contained the oligonucleotides specific for 178 mouse genes and those specific for the eight spikes. We assessed the quality of the spike signals, the reproducibility of the results and, in general, the nature of the variability. The small values of the coefficients of variation revealed high reproducibility of our platform either in replicated spots or in technical replicates. We demonstrated that the spike-in system was suitable for normalizing our platform and determining the threshold for discriminating the hypoxia modulated genes. We assessed the application of the spike-in normalization method to microarrays in which the distribution of the expression values was symmetric or asymmetric. We found that this system is accurate, reproducible and comparable to other normalization methods when the distribution of the expression values is symmetric. In contrast, we found that the use of the spike-in normalization method is superior and necessary when the distribution of the gene expression is asymmetric and biased towards up-regulated genes. CONCLUSION: We demonstrate that spike-in controls based normalization is a reliable and reproducible method that has the major advantage to be applicable also to biased platform where the distribution of the up- and down-regulated genes is asymmetric as it may occur in diagnostic chips.

Synthesis of photoactivable inhibitors of osteoclast vacuolar ATPase.

Amides of (2Z,4E)-5-[(5,6-dichloroindol-2-yl)]-2-methoxy-N-[3-[4-[3-(carboxymethoxy)phenyl)] piperazin-1-yl]propyl]-2,4-pentadienamide (1) and of 5-(5,6-dichloro-2-indolyl)-2-methoxy-2,4-pentadienoic acid (2) are strong inhibitors of the vacuolar ATPase located on the plasma membrane of osteoclasts. In order to understand which V-ATPase subunit is involved in the interaction with these novel inhibitors, analogues containing a photoactivable group and an iodine atom were designed. A series of alcohols or amines containing the photoactivable trifluoroaziridinophenyl or benzophenone moiety and an iodine atom were linked to the above acids via an ester or amide group. These compounds could be thereafter used as a radioactive photoprobe to label the protein. Whereas the compounds containing the photoactivable groups maintained good inhibitory activity, the introduction of the bulky iodine atom was generally detrimental, decreasing potency significantly. Better results were obtained by linking 3-(4-aminopiperidinomethyl)-3'-iodobenzophenone to 3-ethoxy-4-(2-(5,6-dichlorobenzimidazolyl))benzoic acid to give the corresponding amide 27, that inhibited vacuolar ATP-ase with a IC(50)=140 nM. The feasibility of introducing a radioactive 125I atom was ascertained by exchanging the iodine with a tributylstannyl group, that was again substituted by iodine.