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Parental socioeconomic position (SEP) is a known determinant of a child’s health. We aimed to investigate whether a low parental education, as proxy of SEP, has a direct effect on physician-diagnosed asthma, current asthma and current allergic rhinitis in children, or whether associations are mediated by exposure to other personal or environmental risk factors. This study was a secondary data analysis of two cross-sectional studies conducted in Italy in 2006. Data from 2687 adolescents (10–14 years) were analyzed by a path analysis model using generalized structural equation modelling. Significant direct effects were found between parental education and family characteristics (number of children (coefficient = 0.6229, p < 0.001) and crowding index (1.1263, p < 0.001)) as well as with exposure to passive smoke: during pregnancy (maternal: 0.4697, p < 0.001; paternal: 0.4854, p < 0.001), during the first two years of children’s life (0.5897, p < 0.001) and currently (0.6998, p < 0.001). An indirect effect of parental education was found on physician-diagnosed asthma in children mediated by maternal smoking during pregnancy (0.2350, p < 0.05) and on current allergic rhinitis mediated by early environmental tobacco smoke (0.2002; p < 0.05). These results suggest the importance of promotion of ad-hoc health policies for promoting smoking cessation, especially during pregnancy.

Objectives The objective of the study is to analyse how the quality of life of children diagnosed with attention-deficit/hyperactivity disorder (ADHD) impacts the relationship between disease severity and family burden. Method The data collected by a longitudinal, observational study involving 1478 children with ADHD residing in 10 European countries (aged 6 to 18 years) were analysed to evaluate the relationships between ADHD severity, the children's quality of life and family burden. Results The disorder's severity directly and indirectly affected the children's health-related quality of life (HRQoL) and family burden. The degree of family burden was modulated by the children's HRQoL. Conclusions One of the primary causes of the stress experienced by parents of children with ADHD is their perception of the child's reduced HRQoL and not the symptom severity itself. Efforts to minimize symptom severity cannot alone reduce family burden.

Universal salt iodization has been adopted by many countries to address iodine deficiency. More recently, salt-reduction strategies have been widely implemented to meet global salt intake targets of5 g/d. Compatibility of the two policies has yet to be demonstrated. This study compares urinary iodine excretion (UIE) according to 24-h urinary sodium excretion, between South Africa (SA) and Ghana; both countries have implemented universal salt iodization, but in Ghana no salt-reduction legislation has been implemented.Participants from the World Health Organization's Study on Global Ageing and Adult Health Wave 3, with survey and valid 24-h urinary data (Ghana, n = 495; SA, n = 707), comprised the sample. Median 24-h UIE was compared across salt intake categories of5, 5-9 and9 g/d.In Ghana, median sodium excretion indicated a salt intake of 10.7 g/d (interquartile range [IQR] = 7.6), and median UIE was 182.4 µg/L (IQR = 162.5). In SA, both values were lower: median salt = 5.6 g/d (IQR = 5.0), median UIE = 100.2 µg/L (IQR = 129.6). UIE differed significantly across salt intake categories (P0.001) in both countries, with positive correlations observed in both-Ghana: r = 0.1501, P0.0011; South Africa: r = 0.4050, P0.0001. Participants with salt intakes9 g/d in SA did not meet the World Health Organization's recommended iodine intake of 150 µg/d, but this was not the case in Ghana.Monitoring and surveillance of iodine status is recommended in countries that have introduced salt-reduction strategies, in order to prevent reemergence of iodine deficiency.

Background The evaluation of child healthcare is not yet widely explored, especially from a cross-country comparison perspective. The routine adoption of measures by national assessment agencies is under-investigated. Though the guiding principles developed at international level call for a child-centric multi-dimensional evaluation of child care, its feasibility is hampered by the availability of robust and harmonized data. Methods To explore the data availability, international databases (IDBs) were scrutinized and measures dealing with child health-related issues were collated. In parallel, an ad hoc questionnaire was administrated to 30 Country Agents (CAs) to gather measures routinely adopted at local level. To facilitate the comparison of measures, a three-level conceptual map was developed. Results The IDBs yielded at 207 measures that pertained mainly to non-health determinants of health, whereas the 352 measures obtained from CAs focused on process and outcome. A set of 33 common measures that related to immunization, morbidity and mortality were identified. Conclusions A limited set of measures used both in IDBs and at national level identify common areas of concerns that certainly capture crucial issues with child prevention and health outcomes. However, they are far from satisfying a child-centric multi-dimensional approach to the evaluation of child well-being and well-becoming. There is room for improvement at both international and national levels. IDBs should include and harmonize measures that concern the provision of child-centric services and encompass physical, social and mental development. At the national level, efforts towards the inclusion of measures that concern non-health determinants of health should be pursued.

Background The high variability in the types and number of measures adopted to evaluate childcare across European countries makes it necessary to investigate country practices to identify trends in setting national priorities in the assessment of child well-being. Objective This paper intends to investigate country practices under the lens of variability to explore possible trends in setting national priority in the evaluation of childcare. In particular, it analyses variability considering to what extent this depends on the tendency of adopting a broad vision (i.e. selecting measures for a larger variety of aspects) or whether this is influenced by the choice of adopting an in-depth approach (i.e. using more measures to analyse a specific aspect) Methods An ad hoc questionnaire was administered to a national expert in each country and yielded 352 measures. To analyse variability, the breadth in the number of aspects considered was explored using a convergence index, while the depth in the distribution of measures in each aspect was investigated by computing a coefficient of variation. Countries were grouped by adopting a hierarchical clustering approach. Results There is a high variability across countries in the selection of measures that cover different aspects of childcare. Preferences in the distribution of measures are significant even at the domain level and in countries that use a limited number of measures and become more evident at the category and sub-category levels. The statistical analysis clusters countries in four main groups and two outliers. The in-depth distribution of measures focused on a specific aspect shows a homogeneous pattern, with the identification of two main groups of countries. Conclusions A limited set of measures are shared across countries hampering a robust comparison of paediatric models. The selection of measures shows that the evaluation is closely related to national priorities as resulting from the number and types of measures adopted. Moreover, a range of a reasonable number of measures can be hypothesized to address the quality of childcare under a multi-dimensional perspective.

Background New challenges in the medical field of the third millennium emphasise the "humanization of medicine" leading to a redefinition of doctors’ values, limits and roles. The study aims to assess whether there are different personality dimensions of physicians in relation to their perception of professional values and public expectations. Methods A questionnaire on the perception of professional values and the opinion on work in the medical field, work relationships and public expectations was administered to 374 doctors attending Continuing Medical Education courses. Results Two personality dimensions were identified: the first dimension (which we termed "Performance Attainment") is associated preeminently with values of competence, advocacy, confidentiality, spirit of enquiry, integrity, responsibility and commitment; the second dimension (which we called “Personal Involvement”) focuses on concern and compassion. The doctors that have more difficulty accepting judgements on their activity are those who think that “Performance attainment” is less important (β = 6.01; p-value = 0.007). Instead, the doctors who believe “public expectation of the health system” is not high enough, tend to think that “Performance Attainment” is more important (β = -6.08; p-value = 0.024). The less importance is given to the values of "Personal Involvement", the less is the doctor’s perception of having a leading role in respect to other health professionals (β = -2.37; p-value = 0.018). Conclusions Our results demonstrate that there are two different attitudes in terms of recognition and selection of the essential values to better practice the medical profession. Whether the doctors attach more importance to one dimension or the other, they do not differ in our analysis for how they answered the questions about relationships with patients, colleagues or family commitments in the questionnaire, even if they work in different areas. This suggests that in our research there is no single personal attitude that characterizes “a good doctor”.

Background and goals Even though the conceptions and contents of quality of healthcare for children have substantially improved in the last decades, little is known about the routine adoption of measures used at national/regional level to evaluate childcare. This paper intends to investigate country practices in the evaluation of child healthcare providing insights on the priority posed in 30 European/EEA countries. It reports some of the main results achieved during the MOCHA (Models of Child Health Appraised) project. Methods An ad hoc questionnaire was developed and sent to country-specific experts (CAs), appointed for each of the MOCHA’s countries, to gather information on measures adopted to evaluate the quality of child care at country/regional level. Results were analysed and compared on the basis of a two-level hierarchical map of domains that helped the comparison and also allowed the analysis on the breadth of child-centred evaluation of healthcare. The frequency of adoption of the whole set of measures was analyzed in order to investigate whether a similar behavior in terms of sparsity/convergence is present across countries. Moreover, to identify whether a core set of common measures is adopted across countries, we analyzed measures reported by at least 25% of the CAs under the perspective of child-centeredness which may be also expressed by age ranges reported as variable of the measures and disease specificity. Results Among the 27 CAs that completed the questionnaire, 23 countries reported to have a national/regional agency that assesses healthcare systems. 12 of them devote a specific part of the quality assessment to child health care, while the other countries integrate some child specific items within their health system evaluation. In total 352 measures were reported. The collected measures are not equally distributed among countries, as they range from a minimum of 6 measures in Iceland to a maximum of 130 in the UK. Considering the analysis of core measures the most common one reported by 13 countries is the immunization rate, while a two other focal points can be identified in measures related to mortality and natality. Conclusions The analyses confirmed a high variability across countries, both in number and type of measures adopted. Only a limited set of common measures, focused on immunization, mortality and hospitalization Only a limited set of common measures, focused on immunization, mortality and hospitalization, was identified, even if sparsely distributed among a restricted number of countries.

In order to assess the state of health of Europe's children, or to appraise the systems and models of healthcare delivery, data about children are essential, with as much precision and accuracy as possible by small group characteristic. Unfortunately, the experience of the Models of Child Health Appraised (MOCHA) project and its scientists shows that this ideal is seldom met, and thus the accuracy of appraisal or planning work is compromised. In the project, we explored the data collected on children by a number of databases used in Europe and globally, to find that although the four quinquennial age bands are common, it is impossible to represent children aged 0-17 years as a legally defined group in statistical analysis. Adolescents, in particular, are the most invisible age group despite this being a time of life when they are rapidly changing and facing increasing challenges. In terms of measurement and monitoring, there is little progress from work of nearly two decades ago that recommended an information system, and no focus on the creation of a policy and ethical framework to allow collaborative analysis of the rich anonymised databases that hold real-world people-based data. In respect of data systems and surveillance, nearly all systems in European society pay lip service to the importance of children, but do not accommodate them in a practical and statistical sense.

Evaluating primary care for children has not before been undertaken on a national level, and only infrequently on an international level, an adult-focused perspective is the norm. The Models of Child Health Appraised (MOCHA) project explored the evaluation of quality of primary care for children in a nationally comparable way, which recognises the influence of all components of child well-being and well-becoming. Using adult-focused metrics fails to account for children’s physical and psycho-social development at different ages, differences in health and non-health determinants, patterns of disease and risk factors and the stages of the life course. To do this, we attempted to identify comparable measures of child health in the European Union and European Economic Area countries, we aimed to perform a structural equation modelling technique to identify causal effects of certain policies or procedures in children’s primary care and we aimed to identify and interrogate large datasets for key tracer conditions. We found that the creation of comparative data for children and child health services remains a low priority in Europe, and the largely unmet need for indicators covering all the healthcare dimensions hampers development of evidence-based policy. In terms of the MOCHA project objective of appraising models of child primary health care, the results of this specific work show that the means of appraisal of system and service quality are not yet agreed or mature, as well as having inadequate data to fuel them.

Background: There is limited evidence about the prevalence of depression among older people in sub-Saharan Africa, about access to treatment or the potential efficacy of community-based interventions. Objective: Using nationally representative data from the WHO SAGE survey, we examine the prevalence of and factors associated with depression among people aged 50 and over in Ghana. Compare self-reported diagnosis and a symptom algorithm to assess treatment gaps and factors associated with the size of gap. Assess the feasibility of a small community-based intervention specifically for older people. Method: Prevalence and treatment data were taken from the WHO SAGE 2007 survey in Ghana, including 4,725 people aged 50 or over. Outcomes of interest were self-reported depression and diagnosis of depression derived from a symptom-based algorithm. The data were subjected to bivariate and multivariate analysis. In parallel, a pilot intervention was conducted with 35 older people, which included screening by a trained psychiatrist and follow-up group sessions of psychotherapy. Results: The symptomatic algorithm reported an overall rate of 9.2 per cent for the study population, with associations with female sex and older age. The treatment gap for these cases was found to be 83.0 per cent. The implementation of the pilot study was perceived as effective and replicable by stakeholders and there was some evidence of enhanced outcomes for people with mild depression. Conclusions: Large numbers of older people in Ghana experience depression, but very few have access to treatment. There is an urgent need to develop and validate community-based services for older people experiencing this condition.

Background The sweat test is one of the main diagnostic tools used in newborn screening programs and as a confirmatory test, in case of suspect of Cystic Fibrosis (CF). Since sweat chloride (Cl) concentration is also considered an appropriate parameter to explore the efficacy of CFTR modulators in clinical trials, it is crucial to evaluate the biological variability of this test in healthy and pathological conditions. The aim of this pilot study was to determine the intra-individual biological variability of sweat Cl, both in healthy individuals and CF patients and to assess its correlation with diet, season, and menstrual cycle. Methods Thirty-five out of 36 selected subjects (6-18 years) were enrolled by 2 CF care centers and assigned to 3 cohorts: CF, CFTR-related disorder (CFTR-RD) and healthy volunteers. Each participant was subjected to eight sweat tests in different conditions and time of the year. Data were analyzed using linear mixed effects models for repeated measures, taking also into account intra-individual correlations. Results We observed a high intra-individual variability of sweat Cl, with the lowest mean CV% values among CF patients (20.21 in CF, 29.74 in CFTR-RD, and 31.15 in healthy subjects). Gender and diet had no influence on sweat Cl variability, nor had pubertal age and menstrual phase. Conclusion Results of this pilot study confirmed that sweat Cl variability is high in CF patients, although non-CF individuals displayed even higher mean CV% values. Season significantly influenced sweat test values only in CF patients, likely due to changes in their hydration status.

Objectives Reliable assessment of estrogen, progesterone (ER and PR), and HER2 receptor status are essential in breast cancer (BC) treatment. Immunohistochemical methods are limited by intra- and inter-laboratory variability. Furthermore, current methods are not the ideal approach for reproducing the biological continuum of ER, PR, and HER2 receptor levels, due to their intrinsic, semi-quantitative nature, relying in part on subjective interpretation. Methods In the present study, we tested a molecular approach to define ER, PR, and HER2 status in fine-needle-aspirate (FNA) samples from patients with early BC. We performed flow cytometry analysis on 88 FNA specimens from suspect BC patients to determine cellularity. We used quantitative Real Time PCR (QRT-PCR) to assess ER, PR, HER2 status, and qPCR for HER2 gene copy number (GCN). Results ER and PR mRNA levels showed a highly significant correlation with IHC data on surgical samples. qPCR showed greater accuracy than IHC in defining HER2 status. QRT-PCR defined better than IHC the continuous spectrum of the expression of the assessed receptors. Moreover, PCR analysis demonstrated a strict correlation between HER2 status and higher levels of its transcript, correctly stratifying HER2+ and HER2− patients. Finally, there was a strongly significant agreement between HER2 GCN assessed on FNA specimens by qPCR and FISH data obtained on pathological tissue specimens. Conclusions The present results support a comprehensive approach to determine ER, PR, and HER2 status by PCR (QRT-PCR and qPCR) in FNA specimens, with high relevance for therapeutic strategies like neoadjuvant treatment.

Lapatinib, a dual HER2 and EGFR tyrosine kinase inhibitor is highly active in HER2+ breast cancer. However, its efficacy is limited by either primary or acquired resistance. Although mutations in ras genes are rarely found in breast cancer, H-ras overexpression is frequently observed. Moreover, genetic alterations that do not directly involve ras such as Brk amplification, ultimately result in increased ras signaling. Using SKBR3 cells, a HER2+ breast cancer cell line that is naturally devoid of mutations in PI3KCA, PTEN, BRAF, and ras we show that both H-ras overexpression and expression of an oncogenic ras allele (ras V12) reduce susceptibility to lapatinib in analogy to what observed with activating PI3KCA mutations and with a constitutively active form of Akt. Importantly, we found that resistance to lapatinib due to ras overexpression or to ras V12 is overcome by MEK inhibition with U0126, suggesting a key role for the MEK-Erk pathway in ras-induced resistance. Similar results were obtained in BT474 cells, another HER+ breast cancer cell line. Therefore, our data indicate that overexpressed/mutated ras may act as a biological modifier of the response to lapatinib. Combining MEK inhibitors with lapatinib may help overcome this form of resistance and increase the efficacy of lapatinib in these tumors.

We give bounds for the mean square deviation with respect to arbitrary probability measures of the number of integer points in translated or dilated convex bodies. The proofs are based on Fourier analytic methods.

Purpose: Overexpression of antiapoptotic Bcl-2 family members has recently been related to resistance to chemo/radiotherapy in several human malignancies, particularly lymphomas. Hence, innovative approaches bypassing this resistance mechanism are required in the therapeutic approach. This study evaluated whether chemoresistance associated with Bcl-2 and Bcl-xL overexpression would be overcome by activating the death receptor pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in the Jurkat cell model Experimental Design: We made use of genetically modified Jurkat cells to evaluate the effect of Bcl-2 or Bcl-xL overexpression on the cytotoxic effect produced by the anticancer drugs doxorubicin, etoposide, and oxaliplatin and TRAIL. Caspase activation was detected by cleavage of caspase-8 and -3. The mitochondrial transmambrane potential was assessed by staining with DiOC6 and flow cytometry. Caspase activity was blocked by the broad-spectrum caspase inhibitor zVAD-fmk. Results: Bcl-2 and Bcl-xL overexpression but not lack of caspase-8 protects the Jurkat cells from the anticancer drug-induced cytolysis. However, Bcl-2/Bcl-xL Jurkat cells retained some susceptibility to TRAIL-induced cytolysis. A highly synergistic cytotoxic effect of the combination of TRAIL with any of the antiblastic used in this study was detected in the chemoresistant cells. This effect was associated with mitochondrial disassemblage and dependent on caspase activation Conclusions: The combination of TRAIL with conventional anticancer drugs may prove to be useful in the treatment of antiapoptotic Bcl-2 family proteins-expressing malignancies.

To the editor: Nahimana and coworkers have recently reported that the nicotinamide phosphoribosyltransferase (NAMPT) inhibitor APO866 elicited massive cell death in primary leukemia cells and in numerous leukemia/lymphoma cell lines.[1][1] In particular, in 32 primary leukemias (including 12 B-cell

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the expansion of a leukemic stem cell (LSC) clone, carrying a Philadelphia translocation, able to overcome the non-malignant hematopoietic stem cells. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib are gold-standard for CML treatment. Each shows an impressive rate of complete cytogenetic response in chronic phase (CP)-CML. However, relapse and treatment failure are major problems with long-term use of TKIs. A polymerase chain reaction (PCR) assay to detect the mRNA expression of BCR-ABL1 represents the main molecular approach to monitoring response to treatment. However, using this analysis it is currently not possible to prospectively identify patients whose disease will relapse due to LSC reappearance. The aim of our study was to investigate whether the mRNA expression analysis of two Hedgehog (Hh) stemness signaling molecules, Smoothened (SMO) and Patched-1 (PTCH1), could predict upcoming molecular relapse. At the time of diagnosis, patients with high Sokal risk (n = 12) showed higher and lower levels of SMO and PTCH1, respectively (p = 0.0132), compared with patients with different Sokal scores (p = 0.0316 for intermediate risk and p = 0.0340 for low risk). These data suggest that Hh signaling was functionally more active in this risk group at the time of diagnosis. Furthermore, the kinetics of Hh signaling activity during the individual medical history correlated with BCR-ABL1 mRNA level and with upcoming molecular relapse. Also, mutation analysis of BCR-ABL1 revealed that activation of Hh signaling precedes molecular relapse by several months, mostly in patients carrying the gatekeeper mutation T315I. Importantly, in vitro data showed a synergistic effect of chemical inhibitors of Hh signaling and TKIs in both wild-type and resistant (T315I) CML cell lines. Collectively our data show that monitoring Hh pathway activity contemporaneously with BCR-ABL1 mRNA level may improve the chance of early detection of patients who will experience a relapse (mainly in the high Sokal risk group), paving the way for an innovative management of this hematologic malignancy.

Chronic myeloid leukemia (CML) is a hematological disease accounting for about 15-20% of all adult leukemias. The clinical and biologic advances achieved in such a malignancy, represent one of the best successes obtained by translational medicine. Indeed, identification of the fusion oncogene BCR-ABL has allowed using of small molecule inhibitors of its tyrosine kinase activity which, in turn, have literally revolutionized the treatment of CML. Importantly the successfully clinical management was also realized on appropriate diagnosis, disease monitoring as well as early identification of such mutations causing drug resistance. Notably the recent availability of refined laboratory equipments represented by the Next Generation Sequencing (NGS) and genomic analyses has further contributed to gain ground towards the cure of this tumor. These issues are discussed here together with an overview on how patients treated with tyrosine kinase inhibitors should be monitored.

Personalized medicine emphasizes the practice of considering individual patient characteristics as opposed to that centered on standards derived from epidemiological studies which, by definition, do not take into account the variability of individuals within a given population. When applied to oncology, personalized medicine is an even more complex concept because it extends the variability beyond the individual patient to the individual tumor. Indeed, the great genotypic and phenotypic variability (both in primary and metastatic sites of cancer) the development of targeted therapies, and the growing availability of biological assays complicate the scenario of personalized medicine in the oncological field. In this paper we review the results of anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mAb) therapy in metastatic colorectal cancer (mCRC) in the context of tumor biology, delineating the future prospects of patient-tailored medicine in this area. In particular, we deal with EGFR inhibition by Cetuximab, a chimeric mouse human IgG1 mAb, and panitumumab, a fully human IgG2 mAb. We discuss the clinical impact of anti-EGFR mAbs on wild-type (WT) KRAS mCRC, also taking into account the feasibility of novel multi-marker approaches to treatment decision-making, aimed at increasing the predictive power of pre-therapy biomarkers. Experimental topics and fields of ongoing research, such as targeting microRNAs (miRNAs) with novel anticancer drugs and epigenetics in CRC are also addressed.

The present study showed that engagement of CD31 delivers a survival signal in chronic lymphocytic leukaemia (CLL) cells. We describe two groups of CLL, showing different kinetics of apoptosis in vitro and distinct ratios between anti-apoptotic and pro-apoptotic proteins: CLL-I displayed low Bcl-x(L) /Bax and Bcl-2/Bax ratio and underwent rapid apoptosis in vitro; CLL-II had high Bcl-x(L) /Bax and Bcl-2/Bax ratio and were resistant to apoptosis for several days. Nurse-like cells, expressing vimentin, CD68 and CD31 were detected mainly in CLL-II cultures. Of note, CD31 cross-linking, obtained with a specific monoclonal antibody (mAb), induced phosphatidylinositol-3-kinase-dependent Akt phosphorylation and nuclear translocation of the nuclear factor-kBp65 and p52 subunits in both CLL groups, leading to upregulation of Bcl-2 and Bcl-x(L) transcription and increased cell survival. Binding to CD31(+) stable transfectants, could also deliver an anti-apoptotic signal in B cells of both CLL-I and CLL-II, increasing the Bcl-2 and Bcl-x(L) protein content, regardless the expression of CD38. On the other hand, the addition of the F(ab')₂ (that is unable to oligomerize the target molecule) of the anti-CD31 mAb prevented these effects. These data suggest that the CD31 adhesion system may play a role also in vivo in maintaining CLL survival.

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2- hydroxy-1-arylethyl]amine}methyl) pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group = 4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival. (C) 2010 Elsevier Ltd. All rights reserved.

Acquired resistance to imatinib in the advanced phase of chronic myeloid leukemia (CML) has been associated with mutations in the kinase domain (KD) of BCR-ABL. On the contrary, the prognostic implication of KD mutations in early chronic phase (CP) patients at diagnosis before imatinib-based therapy has not yet been established. We have reviewed the status of mutations in 43 patients with early CP-CML on the samples collected at diagnosis. Mutations were identified by direct sequencing (DS) with BidDye Terminator v 1.1. cycle sequencing kit and analyzed with a 3130 ABI capillary electrophoresis system. Eight out 13 (61.5%) high Sokal risk patients showed the following mutations: Y253C, S265R, E255K, F359Y, N374S, E255V, E255V, E255V. Three patients progressed during imatinib and second-line inhibitors and died of blastic phase CML at 23, 33, and 69 months. Another patient with intermediate Sokal risk showed D363G mutation at diagnosis, progressed under imatinib, was allografted and he is now alive in major molecular remission (MMR). No low-risk patient carried KD mutation at diagnosis. In conclusion, KD mutations conferring high-level imatinib resistance are present in patients with de novo CML and in some of them lead to disease progression.

Molecular monitoring of the BCR-ABL1 transcript in chronic myelogenous leukemia (CML) using quantitative real-time PCR (RQ-PCR) can be performed using either bone marrow (BM) or peripheral blood (PB). However, a recent report by Stock et al. [International Journal of Oncology 28 (2006) 1099] questioned the reliability of PB samples for BCR-ABL1 detection as performed by RQ-PCR. We report a study on 114 CML patients who received allogeneic stem cell transplantation (ASCT), and who were monitored by RQ-PCR using paired samples of BM and PB: the total number of determinations was 428, with a median follow-up after transplant of 8 years. BCR-ABL1 transcript was undetectable or0.1%, in 106 (49.57%) and 62 (29%) paired determinations, respectively. BCR-ABL1 was0.1% in 36 (16.8%) paired determinations and was discordant in 10 (4.7%). Agreement between PB and BM results was quantified by the kappa test (k = 0.85; 95% CI 0.76-0.94). This study shows that BCR-ABL1 RQ-PCR monitoring of CML patients after ASCT with PB is concordant with BM in 95.3% of cases, and thus may be used to monitor the disease. This may be relevant when discussing both quality of life issues and the need for post-transplant monitoring with the patient.

Abstract 4732 Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866 mediated Nampt inhibition. As a consequence, impaired proliferation, reduced TNF-a production, ATP shortage, and, finally, autophagic cell demise result. We demonstrate that upregulation of the of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. We relate defective TNF-a production in response to FK866 to impaired SIRT6 activity. Finally, in line with its capacity to interfere with T lymphocyte function and survival, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly sirtuin inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders. Disclosures: No relevant conflicts of interest to declare.

Chromosomal translocations involving the 12q14 band are rarely detected in hematological disorders, and are usually correlated with HMGA2 gene expression. HMGA2 is highly expressed during embryonic cell growth and differentiation, and regulates transcription and chromatin organization, but is rarely detectable in adult tissues. We describe a case of polycythemia vera with a t(12;21)(q14;q22). The 12q14 breakpoint was characterized by fluorescence in situ hybridization analysis using the bacterial artificial chromosome RP11-366L20 containing 3' sequences of the HMGA2 gene. Qualitative and quantitative polymerase chain reaction showed the presence of high levels of HMGA2 gene expression, which were temporarily reduced with hydroxyurea therapy. The present case confirms that involvement of the 12q14 band may be associated with HMGA2 overexpression in chronic Philadelphia chromosome-negative myeloproliferative disease, regardless of the partner chromosome involved in the translocation. Such overexpression may contribute to the pathogenesis of the disease, which otherwise of itself shows a favorable and stable course.

In cancer patients, the ability to detect disseminated tumour cells in peripheral blood or bone marrow could improve prognosis and consent both early detection of metastatic disease and monitoring of the efficacy of systemic therapy. These objectives remain elusive mainly due to the lack of specific genetic markers for solid tumours. The use of surrogate tissue-specific markers can reduce the specificity of the assays and give rise to a clinically unacceptable false-positive rate. Mammaglobin (MAM) and maspin are two putative breast tissue-specific markers frequently used for detection of occult tumour cells in the peripheral blood, bone marrow and lymph nodes of breast cancer patients. In this study, it was evaluated whether MAM and maspin gene expression may be induced in the normal blood and bone marrow cells exposed to a panel of cytokines, including chemotactic factors (C5a, interleukin (IL)-8), LPS, proinflammatory cytokines (TNF-alpha, IL-1beta) and growth factors (IL-3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor). The experimental data show that all cytokines included in the panel, except for IL-8, were able to induce maspin expression; on the contrary, MAM gene was never induced. These results suggest that MAM is more specific than maspin and that the possible interference of cytokines should be taken into account in interpreting molecular assays for detection of isolated tumour cells.

Proteasome inhibitors exhibit antitumor activity against malignancies of different histology. Yet, the mechanisms underlying this effect are poorly understood. Recent evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, possibly reducing their cytotoxicity. These include the Bcl-2 family member Mcl-1, whose down-regulation has been proposed to initiate apoptosis in response to genotoxic stimuli. In this study, we found that proteasome inhibitors release cyotochrome c and second mitochondria-derived activator of caspase (SMAC)/Diablo and trigger the subsequent apoptotic cascade in spite of concomitant Mcl-1 increase. However, our data indicate that subtraction of Mcl-1 during apoptosis, although not required for early release of proapoptotic factors, is probably relevant in speeding up cell demise, since RNA interference-mediated Mcl-1 silencing is lethal in lymphoma cells. Consistent with this, the cytotoxic effects of proteasome inhibitors are enhanced when Mcl-1 increase is impeded. Thus, this study identifies Mcl-1 accumulation as an unwanted molecular consequence of exposure to proteasome inhibitors, which slows down their proapoptotic effects. Pharmacologic or genetic approaches targeting Mcl-1, including therapeutic RNAi, may increase the effectiveness of these compounds. (Blood. 2005;105:3255-3262)

Background: Immunohistochemistry (IHC) and fluorescence-in-situ hybridization (FISH) are the standard methods to assess human epidermal growth factor receptor 2 (HER-2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (Q-PCR) and quantitative reverse transcriptase PCR (qRT-PCR) allow quantitative determination of gene copy number and gene expression on, respectively, DNA and RNA. These molecular methods are available to assess HER-2 amplification/overexpression but their use remains controversial. Here we performed a parallel comparison of these four methods to define their concordance rates and evaluate their relative role in HER-2 status determination. Patients and Methods: HER-2 status was determined by IHC, FISH, Q-PCR and qRT-PCR in a retrospectively assessed cohort of 130 BC patients. The studied set was enriched in cases scoring as 3+ by standard IHC analysis. Tests were performed blindly in parallel. Western blotting was performed on a subset of equivocal cases. Kappa statistics and ROC curves were used as appropriate, and concordance analyses were interpreted according to the ASCO/CAP guidelines. Results: Of the 130 enrolled patients, 47 (36%) were classified as positive and 27 (21%) as equivocal by IHC. With FISH, 50 patients (38%) were HER-2 amplified, while 80 (62%) were not. The overall agreement between FISH and Q-PCR was 98.5% (95% CI, 94.6% to 99.6%) with a k value of 0.97 (95% CI, 0.92 to 1). Assuming FISH as the standard reference, Q-PCR showed a sensitivity of 98% (95% CI, 89.5% to 99.6%) and a specificity of 98.8% (95% CI, 93.3% to 99.8%), with a global accuracy of 98%. The overall agreement between FISH and qRT-PCR was 96.9% (95% CI, 92.3% to 98.8%) with a k value of 0.94 (95% CI, 0.87 to 1). For both comparisons, the observed concordance values were greater than the 95% threshold required by the ASCO/CAP guidelines to validate novel approaches for HER-2 testing. 3% of samples showed HER-2 overexpression only by qRT-PCR analysis, in all cases belonging to the equivocal range as defined by either IHC or FISH. In these patients, WB confirmed higher HER-2 protein levels compared to healthy diploid controls. Conclusions: The high concordance between FISH and both Q-PCR and qRT-PCR supports the use of molecular tests as an alternative to current standard methods. Present results also suggest that qRT-PCR may be able to unequivocally classify patients belonging to the IHC/FISH equivocal range. Non-amplified HER-2 overexpressing BCs may account for the rare trastuzumab-responders observed in phase III trials, where trastuzumab response was assessed in HER-2-amplified vs. non-amplified cases. Citation Format: Gabriele Zoppoli, Anna Garuti, Ilaria Rocco, Claudia Palermo, Gabriella Cirmena, Enrico Carminati, Daniele Friedman, Ludovica Verdun di Cantogno, Anna Sapino, Franco Patrone, Alberto Ballestrero. Agreement of immunohistochemistry, fluorescence in situ hybridization, real-time quantitative polymerase-chain reaction, and quantitative reverse transcriptase PCR for Her-2/Neu status assessment in breast cancer patients: a single center, retrospective m [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 49. doi:10.1158/1538-7445.AM2013-49

Abstract 1215 Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the expansion of a Leukemic Stem Cell (LSC) clone carrying a Philadelphia translocation, which outgrows the non-malignant haematopoietic stem cells. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib, are the gold standard for CML treatment since each one shows an impressive rates of complete cytogenetic and molecular response in chronic phase (CP) CML. However the major problem concerning the final efficacy of TKIs therapy is that the majority of responding CP CML patients have detectable BCR-ABL transcripts which might arise from a population of quiescent CML LSC not effectively targeted by TKIs. Therefore the molecular monitoring not always provide a sufficiently precise evaluation of patients to allow the appropriate choice of clinical interventions. Accordingly, it is necessary to monitor the appearance and increase of LSCs to identify and to treat quickly the fundamental responsible for relapse. Thus, we focused Hedgehog (Hh) signalling which has been proved essential for maintenance of cancer stem cells in myeloid leukaemia. Notably recent studies reported that the expansion of BCR-ABL positive leukemic stem cell is dependent on Hh pathway activation. Here, we analyzed the mRNA levels of Smoothened (SMO), a seven-transmembrane domain receptor protein, and Ptch1, a surface receptor regulator of SMO, in 20 CP-CML patients (8 High, 4 Intermediate and 8 Low Sokal Risk respectively) at diagnosis and during the follow-up. Using RT-PCR, in diagnosis setting, we proved that 60% of patients (bone marrow samples) showed Hh signalling significantly activated compared to CD34+ cells from healthy donors. In detail 75% (6/8) of High Sokal Risk CML patients showed an up-regulation of Smo and a down-regulation of Ptch1 mRNA levels, suggestive of active Hh signalling at diagnosis. Conversely Low and Intermediate Sokal Risk CML patients did not show features of Hh activation. Finally we monitored, during the follow-up, the mRNA levels of Smo and Ptch1 together with BCR-ABL to assess the kind of relationship between these parameters. Interestingly we noticed a direct correlation between the increase of BCR-ABL mRNA levels and signs of Hh activity (Smo mRNA increase level and Ptch1 mRNA decrease level). Characteristically, molecular monitoring highlighted that all patients developing resistance to TKIs treatment, showed a tendency to Hh activation (high and low mRNA levels of Patch1 and Smo respectively) few months before the development of Abl KD mutation (10/10). Typically this last behaviour was more evident in patients developing the gatekeeper mutation T315I. Finally, in accordance with published data, we noted that the pharmacological inhibition of Hh signalling impairs the growth of TKIs resistant human CML cell line BaF/T315I, suggesting a novel treatment option. All our data provide molecular evidence for a role of the stemness pathway to predict quickly both the relapse and the TKIs resistance during CML treatment. Therefore, we propose to use Ptch1 and Smo mRNA levels together with BCR-ABL for the molecular monitoring of CP CML. Disclosures: No relevant conflicts of interest to declare.

Abstract 2733 Poster Board II-709 Introduction: APO866, a reversible inhibitor of nicotinamide phosphoribosyltransferase (Nampt), acts by severely depleting intracellular NAD+ content and thus eliciting mitochondrial dysfunction, ATP depletion, and autophagic cell death. TNF-Related Apoptosis Inducing Ligand (TRAIL) binds to plasma membrane receptors DR4 and DR5 and causes apoptosis through activation of caspase-8 and caspase-10. These, in turn, activate effector caspases either directly or via Bid cleavage and subsequent initiation of the mitochondrial apoptotic pathway. In the present work, we have explored the interaction between APO866 and TRAIL in cellular models of hematological malignancies. Materials and Methods: The lymphoma cell lines Jurkat, PEER, MOLT-4, H9, and Namalwa, primary B-cell chronic lymphocytic leukemia (B-CLL) cells, as well as normal peripheral blood mononuclear cells (PBMCs) were treated with increasing concentrations of APO866, TRAIL, or with 1:1, 1:3, or 1:10 combinations of both agents for 72 hours (48 h APO866 + 24 h TRAIL). Cell death was assessed by propidium iodide staining and flow cytometry (FC). Apoptosis was assessed by Annexin V-propidium iodide staining and FC. Combination indices were calculated according to the Chou and Talalay method. DR5 membrane expression was assessed with a FITC-conjugated anti-DR5 antibody and FC after 12, 24, and 48 h exposure to APO866. Mitochondrial transmembrane potential (Δψm) and cell cycle analysis after single agent or combination treatment were determined by tetramethylrhodamine (TMRE) and Nicoletti staining, respectively, and FC. Intracellular NAD+ and ATP contents were quantified by cyclic assays and HPLC, respectively. The role of autophagy and of caspase activity in the cytotoxic activity of APO866, TRAIL, and of their combination was assessed using the PI3K inhibitors 3-methyladenine, LY2940002, and wortmannin, and the caspase inhibitors Z-VAD-FMK (pan-caspase inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-IETD-FMK (caspase-8 inhibitor), respectively. Results/Discussion: APO866 induced NAD+ depletion, Δψm dissipation, and ATP exhaustion in the absence of caspase activation as detected by caspase-3 cleavage. Accordingly, autophagy but not caspase inhibitors blocked APO866 activity. Vice versa, TRAIL-induced apoptosis was blocked by the caspase inhibitors and enhanced by the PI3K inhibitors. Remarkably, APO866 and TRAIL showed a potent synergistic interaction in lymphoma cell lines and in primary B-CLL cells, consistently exhibiting combination indices below 1 upon exposure to a wide range of drugs concentrations. On the other hand, the two drugs and their combinations were poorly active and failed to show any cooperation in healthy PBMCs, indicating a specific activity against malignant cells. As a metabolic correlate of their interaction, TRAIL enhanced APO866-induced NAD+ depletion and Δψm. dissipation. As a result, lymphoma cells underwent massive ATP depletion and autophagic cell death. No DR5 upregulation at the cell surface of lymphoma cells was observed in response to APO866. Conclusions: APO866 and TRAIL show potent synergistic interaction in preclinical cell models of Non Hodgkin's Lymphoma and B-CLL. Due to the mild and manageable side effects of these two drugs in clinical trials, the addition of TRAIL to APO866 appears as an interesting option for the treatment of refractory hematological malignancies. Disclosures: No relevant conflicts of interest to declare.