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The use of dietary supplements (DS) is increasing worldwide. There is limited evidence of their intake level and mode of consumption in association with the Greek population’s dietary and lifestyle habits. Adults (n 4011, 1873 males and 2138 females) aged > 18 years old living in Greece were included in the 2013–2014 National Health and Nutrition Survey – HYDRIA. A dietary supplement user (DSU) was defined as anyone who reported one or more DS on either a Food Propensity Questionnaire, two 24-h dietary recalls, or a questionnaire completed during the blood sample collection examination. DS use was examined according to socio-economic, anthropometric and lifestyle characteristics and the participants’ health and dietary status. DS use was reported by 31 % of the population (40 % women and 22 % men), and it was higher among individuals living in urban areas, men with good self-reported health status and women with a chronic medical condition and higher consumption of fruits. The types of DS more frequently reported were multivitamins with minerals (5·4 %), Ca (5·3 %), multivitamins (4·7 %) and Fe (4·6 %). MVM supplements were preferred by men, while Ca was more frequently reported by women and participants with low education levels. Plant- and oil-based supplement use was below 5 %. Whether DS intake benefits health must be explored. It should also be assessed if dietary supplement intake is as efficient as food intake.

Background: This study aimed to investigate the food and macronutrient intake of the population in Greece and evaluate its adherence to the Greek traditional Mediterranean diet. Methods: Adults over 18 years old (n = 4011) were included from the 2013–2014 National Health and Nutrition survey—HYDRIA. Dietary intake was collected using two 24-h recall interviews and a nonquantitative food frequency questionnaire. Macronutrient intakes were calculated using an updated version of the Greek FCT. Results: Only 28.3% of the adult population had high adherence to the Greek traditional Mediterranean diet, with a higher percentage (39.7%) observed for participants over 65 years compared to those under 65 years (25.5%). Differences in adherence to the MD were observed among the four geographical regions in Greece. Younger adults had a higher intake of meat, cereals, alcoholic and nonalcoholic beverages, and sugar products than older individuals who consumed more vegetables, fruits, legumes, dairy, fish, and lipids (mainly from olive oil). Adults do not meet the international dietary recommendations for the intake of several foods and macronutrients. Conclusions: The adult Greek population, especially younger people, has headed away from the Greek traditional Mediterranean diet. These observations indicate potential detrimental consequences in terms of morbidity and mortality.

BACKGROUND: The role of allium vegetables on gastric cancer (GC) risk remains unclear. METHODS: We evaluated whether higher intakes of allium vegetables reduce GC risk using individual participant data from 17 studies participating in the "Stomach cancer Pooling (StoP) Project", including 6097 GC cases and 13,017 controls. Study-specific odds ratios (ORs) were pooled using a two-stage modelling approach. RESULTS: Total allium vegetables intake was inversely associated with GC risk. The pooled OR for the highest versus the lowest study-specific tertile of consumption was 0.71 (95% confidence interval, CI, 0.56-0.90), with substantial heterogeneity across studies (I(2) > 50%). Pooled ORs for high versus low consumption were 0.69 (95% CI, 0.55-0.86) for onions and 0.83 (95% CI, 0.75-0.93) for garlic. The inverse association with allium vegetables was evident in Asian (OR 0.50, 95% CI, 0.29-0.86) but not European (OR 0.96, 95% CI, 0.81-1.13) and American (OR 0.66, 95% CI, 0.39-1.11) studies. Results were consistent across all other strata. CONCLUSIONS: In a worldwide consortium of epidemiological studies, we found an inverse association between allium vegetables and GC, with a stronger association seen in Asian studies. The heterogeneity of results across geographic regions and possible residual confounding suggest caution in results interpretation.

BACKGROUND: Evidence from epidemiological studies on the role of tea drinking in gastric cancer risk remains inconsistent. We aimed to investigate and quantify the relationship between tea consumption and gastric cancer in the Stomach cancer Pooling (StoP) Project consortium. METHODS: A total of 9438 cases and 20,451 controls from 22 studies worldwide were included. Odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) of gastric cancer for regular versus non-regular tea drinkers were estimated by one and two-stage modelling analyses, including terms for sex, age and the main recognised risk factors for gastric cancer. RESULTS: Compared to non-regular drinkers, the estimated adjusted pooled OR for regular tea drinkers was 0.91 (95% CI: 0.85-0.97). When the amount of tea consumed was considered, the OR for consumption of 1-2 cups/day was 1.01 (95% CI: 0.94-1.09) and for >3 cups/day was 0.91 (95% CI: 0.80-1.03). Stronger inverse associations emerged among regular drinkers in China and Japan (OR: 0.67, 95% CI: 0.49-0.91) where green tea is consumed, in subjects with H. pylori infection (OR: 0.68, 95% CI: 0.58-0.80), and for gastric cardia cancer (OR: 0.64, 95% CI: 0.49-0.84). CONCLUSION: Our results indicate a weak inverse association between tea consumption and gastric cancer.

Objective This study aimed to evaluate and quantify the relationship between coffee and gastric cancer using a uniquely large dataset from an international consortium of observational studies on gastric cancer, including data from 18 studies, for a total of 8198 cases and 21 419 controls. Methods A two-stage approach was used to obtain the pooled odds ratios (ORs) and the corresponding 95% confidence intervals (CIs) for coffee drinkers versus never or rare drinkers. A one-stage logistic mixed-effects model with a random intercept for each study was used to estimate the dose-response relationship. Estimates were adjusted for sex, age and the main recognized risk factors for gastric cancer. Results Compared to never or rare coffee drinkers, the estimated pooled OR for coffee drinkers was 1.03 (95% CI, 0.94-1.13). When the amount of coffee intake was considered, the pooled ORs were 0.91 (95% CI, 0.81-1.03) for drinkers of 1-2 cups per day, 0.95 (95% CI, 0.82-1.10) for 3-4 cups, and 0.95 (95% CI, 0.79-1.15) for five or more cups. An OR of 1.20 (95% CI, 0.91-1.58) was found for heavy coffee drinkers (seven or more cups of caffeinated coffee per day). A positive association emerged for high coffee intake (five or more cups per day) for gastric cardia cancer only. Conclusions These findings better quantify the previously available evidence of the absence of a relevant association between coffee consumption and gastric cancer.

PURPOSE The aim of this study was to investigate the effect of oral factors on adherence to the Mediterranean diet in an older population METHODS: 130 persons over 60 years visiting Open Care Community Centers for Older People participated in this study. Oral interviews recorded demographic and sociomedical information, subjective oral complaints, and dental habits. Adherence to Mediterranean diet was assessed using the MDI_BNC4H index (range: 0-14). An oral examination was performed, and evaluation of the masticatory performance was carried out using a two-color chewing gum that was digitally analysed. RESULTS The mean age of the study participants was 73.9 ± 8.5 years. The score of adherence to the Mediterranean diet ranged from 3 to 9 (5.6 ± 1.4). 58 participants used removable prostheses, while 20 used a pair of complete dentures. Univariate analyses revealed that the parameters that negatively significantly, or marginally significantly, affected the level of adherence to the Mediterranean diet were lower masticatory performance (p = 0.050), larger number of drugs per day (p = 0.056), higher BMI (p = 0.043) and smoking (p = 0.053). The multivariable analysis revealed that lower adherence to the Mediterranean diet was significantly associated with higher BMI (p = 0.047) and lower masticatory performance (p = 0.050). CONCLUSIONS Increased masticatory performance was an independent predictor of better adherence to the Mediterranean diet in an older population.

The aim of this study is to evaluate the changes in the nutritional behavior of the Greek EPIC (European Prospective Investigation into Cancer and Nutrition) cohort participants regarding the consumption of basic food groups, during a 14-year period (1997&ndash, 2011). In the Greek segment of the EPIC cohort study (EPIC-Greece), the changes in dietary habits of 23,505 participants regarding several food items/groups (vegetables, legumes, fruits, nuts, dairy, cereal, meat, fish/seafood, olive oil) were recorded repeatedly over time and compared to the baseline assessment (1994&ndash, 1997), using a short, qualitative, follow-up questionnaire. Descriptive statistics were used to study the trends in nutritional behavior over time and ordinal logistic regression models to study the associations between the ordered responses of the questionnaire and sociodemographic and health factors. More participants reported an increase rather than a decrease in the consumption of vegetables, fruits, fish/seafood, whilst the inverse was observed for dairy products, nuts, cereals, and meat. No prevailing trend was noted for legumes and olive oil. Factors such as being female and having high education relate to more positive (healthy) changes in nutritional behavior. There seems to be primarily a change to a more healthy nutritional behavior of the EPIC-Greece participants over the follow-up period, with different participant subgroups presenting different degrees of nutritional changes.

Several studies have reported associations of hypertension with cancer, but not all results were conclusive. We examined the association of systolic (SBP) and diastolic (DBP) blood pressure with the development of incident cancer at all anatomical sites in the European Prospective Investigation into Cancer and Nutrition (EPIC). Hazard ratios (HRs) (95% confidence intervals) were estimated using multivariable Cox proportional hazards models, stratified by EPIC-participating center and age at recruitment, and adjusted for sex, education, smoking, body mass index, physical activity, diabetes and dietary (in women also reproductive) factors. The study included 307,318 men and women, with an average follow-up of 13.7 (standard deviation 4.4) years and 39,298 incident cancers. We confirmed the expected positive association with renal cell carcinoma: HR = 1.12 (1.08-1.17) per 10 mm Hg higher SBP and HR = 1.23 (1.14-1.32) for DBP. We additionally found positive associations for esophageal squamous cell carcinoma (SCC): HR = 1.16 (1.07-1.26) (SBP), HR = 1.31 (1.13-1.51) (DBP), weaker for head and neck cancers: HR = 1.08 (1.04-1.12) (SBP), HR = 1.09 (1.01-1.17) (DBP) and, similarly, for skin SCC, colon cancer, postmenopausal breast cancer and uterine adenocarcinoma (AC), but not for esophageal AC, lung SCC, lung AC or uterine endometroid cancer. We observed weak inverse associations of SBP with cervical SCC: HR = 0.91 (0.82-1.00) and lymphomas: HR = 0.97 (0.93-1.00). There were no consistent associations with cancers in other locations. Our results are largely compatible with published studies and support weak associations of blood pressure with cancers in specific locations and morphologies.

Background & Aims There is an unclear association between intake of fish and long-chain n-3 polyunsaturated fatty acids (n-3 LC-PUFAs) and colorectal cancer (CRC). We examined the association between fish consumption, dietary and circulating levels of n-3 LC-PUFAs, and ratio of n-6:n-3 LC-PUFA with CRC using data from the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Methods Dietary intake of fish (total, fatty/oily, lean/white) and n-3 LC-PUFA were estimated by food frequency questionnaires given to 521,324 participants in the EPIC study; among these, 6291 individuals developed CRC (median follow up, 14.9 years). Levels of phospholipid LC-PUFA were measured by gas chromatography in plasma samples from a sub-group of 461 CRC cases and 461 matched individuals without CRC (controls). Multivariable Cox proportional hazards and conditional logistic regression models were used to calculate hazard ratios (HRs) and odds ratios (ORs), respectively, with 95% CIs. Results Total intake of fish (HR for quintile 5 vs 1, 0.88; 95% CI, 0.80–0.96; Ptrend = .005), fatty fish (HR for quintile 5 vs 1, 0.90; 95% CI, 0.82–0.98; Ptrend = .009), and lean fish (HR for quintile 5 vs 1, 0.91; 95% CI, 0.83–1.00; Ptrend = .016) were inversely associated with CRC incidence. Intake of total n-3 LC-PUFA (HR for quintile 5 vs 1, 0.86; 95% CI, 0.78–0.95; Ptrend = .010) was also associated with reduced risk of CRC, whereas dietary ratio of n-6:n-3 LC-PUFA was associated with increased risk of CRC (HR for quintile 5 vs 1, 1.31; 95% CI, 1.18–1.45; Ptrend < .001). Plasma levels of phospholipid n-3 LC-PUFA was not associated with overall CRC risk, but an inverse trend was observed for proximal compared with distal colon cancer (Pheterogeneity = .026). Conclusions In an analysis of dietary patterns of participants in the EPIC study, we found regular consumption of fish, at recommended levels, to be associated with a lower risk of CRC, possibly through exposure to n-3 LC-PUFA. Levels of n-3 LC-PUFA in plasma were not associated with CRC risk, but there may be differences in risk at different regions of the colon.

Funder: Centre International de Recherche sur le Cancer; doi: http://dx.doi.org/10.13039/100008700, Purpose: The evidence from the literature regarding the association of dietary factors and risk of prostate cancer is inconclusive. Methods: A nutrient-wide association study was conducted to systematically and comprehensively evaluate the associations between 92 foods or nutrients and risk of prostate cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC). Cox proportional hazard regression models adjusted for total energy intake, smoking status, body mass index, physical activity, diabetes and education were used to estimate hazard ratios and 95% confidence intervals for standardized dietary intakes. As in genome-wide association studies, correction for multiple comparisons was applied using the false discovery rate (FDR < 5%) method and suggested results were replicated in an independent cohort, the Netherlands Cohort Study (NLCS). Results: A total of 5916 and 3842 incident cases of prostate cancer were diagnosed during a mean follow-up of 14 and 20 years in EPIC and NLCS, respectively. None of the dietary factors was associated with the risk of total prostate cancer in EPIC (minimum FDR-corrected P, 0.37). Null associations were also observed by disease stage, grade and fatality, except for positive associations observed for intake of dry cakes/biscuits with low-grade and butter with aggressive prostate cancer, respectively, out of which the intake of dry cakes/biscuits was replicated in the NLCS. Conclusions: Our findings provide little support for an association for the majority of the 92 examined dietary factors and risk of prostate cancer. The association of dry cakes/biscuits with low-grade prostate cancer warrants further replication given the scarcity in the literature.

Importance: Soft drinks are frequently consumed, but whether this consumption is associated with mortality risk is unknown and has been understudied in European populations to date. Objective: To examine the association between total, sugar-sweetened, and artificially sweetened soft drink consumption and subsequent total and cause-specific mortality. Design, Setting, and Participants: This population-based cohort study involved participants (n = 451 743 of the full cohort) in the European Prospective Investigation into Cancer and Nutrition (EPIC), an ongoing, large multinational cohort of people from 10 European countries (Denmark, France, Germany, Greece, Italy, the Netherlands, Norway, Spain, Sweden, and the United Kingdom), with participants recruited between January 1, 1992, and December 31, 2000. Excluded participants were those who reported cancer, heart disease, stroke, or diabetes at baseline; those with implausible dietary intake data; and those with missing soft drink consumption or follow-up information. Data analyses were performed from February 1, 2018, to October 1, 2018. Exposure: Consumption of total, sugar-sweetened, and artificially sweetened soft drinks. Main Outcomes and Measures Total mortality and cause-specific mortality. Hazard ratios (HRs) and 95% CIs were estimated using multivariable Cox proportional hazards regression models adjusted for other mortality risk factors. Results: In total, 521 330 individuals were enrolled. Of this total, 451 743 (86.7%) were included in the study, with a mean (SD) age of 50.8 (9.8) years and with 321 081 women (71.1%). During a mean (range) follow-up of 16.4 (11.1 in Greece to 19.2 in France) years, 41 693 deaths occurred. Higher all-cause mortality was found among participants who consumed 2 or more glasses per day (vs consumers of

Alcohol consumption is associated with higher risk of breast cancer (BC); however, the biological mechanisms underlying this association are not fully elucidated, particularly the extent to which this relationship is mediated by sex hormone levels. Circulating concentrations of estradiol, testosterone, their free fractions and sex-hormone binding globulin (SHBG), were examined in 430 incident BC cases and 645 matched controls among alcohol-consuming postmenopausal women nested within the European Prospective Investigation into Cancer and Nutrition. Mediation analysis was applied to assess whether individual hormone levels mediated the relationship between alcohol intake and BC risk. An alcohol-related hormonal signature, obtained by Partial Least Square (PLS) regression, was evaluated as a potential mediator. Total (TE), natural direct (NDE) and natural indirect effects (NIE) were estimated. Alcohol intake was positively associated with overall BC risk and specifically with estrogen receptor positive tumours with respectively TE=1.17(95%CI: 1.01,1.35) and 1.36(1.08,1.70) for a 1-SD deviation increase of intake. There was no evidence of mediation by sex steroids or SHBG separately except for a weak indirect effect through free estradiol where NIE=1.03(1.00,1.06). However, an alcohol-related hormonal signature negatively associated with SHBG and positively with estradiol and testosterone, was associated with BC risk (OR=1.25 (1.07,1.47)) for a 1-SD higher PLS score, and had a statistically significant NIE accounting for a mediated proportion of 24%. There was limited evidence of mediation of the alcohol-BC association by individual sex hormones. However, a hormonal signature, reflecting lower levels of SHBG and higher levels of sex steroids, mediated a substantial proportion of the association. This article is protected by copyright. All rights reserved.

Age-related changes that could affect the biologic features of mesenchymal stromal cells (MSC), such as a decrease in proliferation and osteoblast differentiation capacity and an increase of senescence markers and apoptosis, have been reported recently. The aim of this study was the evaluation of age-related characteristics and the correlation of age with the functional properties of MSC.The doubling time (DT), colony-forming unit–fibroblast (CFU-F) colonies and surface antigen expression of MSC isolated from bone marrow (BM) of children (C-MSC) were compared with those from adults (A-MSC). The expression of Oct-4 and Nanog transcripts and the relative telomere length were evaluated in both groups.DT values were lower in C-MSC compared with A-MSC, and a higher CFU-F count was observed in children. However, the expression of Oct-4 and Nanog did not differ between C-MSC and A-MSC and was not correlated with the proliferative capacity. The telomere length was significantly higher in C-MSC compared with A-MSC.These data suggest that children's BM-derived MSC could be a more advantageous source of these cells for tissue engineering and cell therapy.

Objectives: Mesenchymal stromal cells (MSC) represent a novel cellular candidate in the field of transplantation and tissue regeneration. Their clinical application requires their in vitro expansion. The aim of this study was to assess the effect of conditions that would favour apoptosis, and of long-term expansion, on the characteristics of MSC from children. Materials and methods: Bone marrow mononuclear cells were cultured for 10 passages (P1–P10). Expression of CD105, CD146, CD95 and apoptosis by 7-amino-actinomycin D staining were evaluated. CFU-F and cell doubling time (DT) were assessed in every passage. Cell-cycle study was performed at P2 and P6. Results: CFU-F decreased from 38 ± 3.7 at P2 to 9.6 ± 3.2 per 10 MSC/cm2 at P10 and DT increased from 1.93 ± 0.1 (P2) to 6.1 ± 2.45 days (P10). A low percentage of apoptotic (dead) cells was detected at P2 and this did not change until P10. Cells at P2 were at G0/G1 phase, but in advanced passages more cells were in an active state. Induction of apoptosis (addition of anti-Fas agonist antibody) using standard culture conditions, showed a minor effect on MSC survival. Serum deprivation of MSC (up to 72 h) revealed no substantial apoptotic effect while cells retained their tri-lineage differentiation capacity. Conclusions: We conclude that MSC from children retain their functional characteristics throughout serial passages and remain stable under conditions that usually cause apoptosis. These features render MSC, especially those of early passages, optimal candidates for use in clinical applications.

The effect of the underlying disease and chemotherapy on megakaryopoiesis has not been extensively studied in children with acute lymphoblastic leukemia (ALL) during and at the end of therapy. Using a serum-free assay, we assessed the megakaryocyte (Mk) colony formation in vitro from bone marrow mononuclear cells of 25 children with ALL during chemotherapy and shortly after the cessation of it. Twelve children with solid tumors without bone marrow involvement and cord blood from 10 full-term normal vaginal deliveries were used as controls. A significant reduction in the number of Mk colonies was observed at diagnosis of ALL, and Mk colony formation remained lower than controls throughout the different phases of leukemia treatment. Our study suggests that defects in megakaryopoiesis of children with ALL in long-term remission may persist during chemotherapy and at least shortly after the end of it.

Background: Adherence to a Mediterranean diet (MD) has been quantified through various indexes that rely on full-length questionnaires, but their application in data collected with brief questionnaires has not been systematically investigated. Objective: We aimed to evaluate the ability of the commonly used MD score (MDS) to classify individuals according to their adherence to an MD when applied to data collected with brief questionnaires. Methods: We assessed the diet of 200 participants from a Greek national health survey with the use of 2 instruments: 1) a validated, detailed food-frequency questionnaire (FFQ) estimating grams per day of intake of individual foods and 2) a selection of 19 questions from the Baseline Nutrition Credits4Health (BNC4H) electronic platform questionnaire that assessed servings with subjective serving sizes. We calculated the MDS_FFQ (referent) and MDS_BNC4H indexes from each questionnaire and estimated their correlation and the percentage of study participants who were ranked in an identical tertile in both indexes. We repeated the analyses for 2 additional indexes defined with criteria different from the MDS [Mediterranean Diet Index (MDI)_BNC4H and Mediterranean Diet Assessment Score (MEDAS)_ BNC4H]. Results: Spearman correlation coefficients for the MDS_FFQ were 0.31 with the MDS_BNC4H, 0.24 with the MDI_BNC4H, and 0.23 with the MEDAS_BNC4H. The proportion of participants ranked into the same adherence level as the referent MDS_FFQ was 50% for the MDS_BNC4H (weighted κ = 0.27) and lower for the other indexes. The use of medians as cutoffs (as in the MDS_FFQ) had low discriminative ability when applied to servings per day (MDS_BNC4H) in some MDS components, leading to comparability problems in the range of values between the 2 indexes. Conclusions: Our findings highlight certain issues that need to be considered when applying pre-existing MD indexes in settings with different dietary assessments. Given the widespread use of electronic platforms for dietary assessment, our results may offer further insight into designing brief, simplified questionnaires that aim to estimate MD adherence with easily quantifiable scores.

Cord blood (CB) has long been regarded as an easily accessible source of hematopoietic progenitors suitable for transplantation, but its efficiency as a source of mesenchymal stromal cells (MSC) remains controversial. The aim of this study was to assess CB as a potential source of MSC, to determine the optimal culture requirements for CB MSC expansion and to compare their functional and immunophenotypic characteristics with bone marrow (BM) MSC from children.Mononuclear cells from 18 full-term CB samples and 23 BM samples from children were set in culture under MSC-inducing conditions. Their immunophenotypic characteristics were assessed by flow cytometry and their differentiation potential was evaluated.Isolation of CB MSC was achieved in 25% of the samples cultured under optimal conditions: high initial cell concentration, fetal calf serum (FCS) enrichment of the culture medium, high FGF-2 concentration and high sample volume. Isolated CB MSC were morphologically similar to the ones derived from BM, but appeared late in culture. An adherent cell layer was formed and reached confluency in 34 days (passage 1; P1) and needed 55 days subsequently (from P1 to P2). CB MSC retained their characteristics for two successive passages. Immunophenotypic analysis showed no expression of CD34 and varying expression of CD45, ranging from 0% to 17.83%, and CD105, from 49% to 83%. CFU-F colonies developed in one case.These findings suggest that CB cannot be considered a sufficient source of MSC for clinical use, although easily accessible. Further research should aim for alternative sources.

Mesenchymal stromal cells (MSC) have become the focus of cellular therapeutics but little is known regarding bone marrow (BM) MSC derived from children. As MSC constitute part of BM stroma, we examined their properties in children with hematologic diseases.BM MSC from children with non-malignant hematologic disorders and acute lymphoblastic leukemia (ALL) were isolated and expanded. MSC were immunophenotypically characterized and their functional characteristics were assessed by CFU-F assay and cell doubling time calculation. Their ability for trilineage differentiation was verified by molecular and histochemical methods. Apoptosis was evaluated and clonal analysis was performed.MSC were isolated from BM of all groups. They acquired the mesenchymal-related markers from the first passage, with a simultaneous decrease of hematopoietic markers. A very low percentage of apoptotic cells was detected in all passages. The proliferative and clonogenic capacity did not differ among groups, with the exception of ALL at diagnosis, in which they were defective. Histochemical and molecular analysis of differentiated MSC yielded characteristics for adipocytes, osteoblasts and chondrocytes. Clonal analysis in a number of BM samples revealed a highly heterogeneous population of cells within each clone.MSC from BM of children with hematologic disorders, with the exception of ALL at diagnosis, can be isolated in sufficient number and quality to serve as a potential source for clinical applications.

Abstract 4853 Fas/FasL is a key pathway of cellular apoptosis. Fas receptor is expressed on membranes of both normal and neoplastic cells while, Fas ligand (FasL) mainly on activated T-lymphocytes. Fas-FasL abnormalities have been detected in malignancies and autoimmune diseases and implicated in resistance to treatment. The aim of the study was the determination of the levels of Fas, FasL and their coexpression in bone marrow cells of children with acute lymphoblastic leukaemia (ALL) at diagnosis, during the course of treatment and the comparison with relevant levels in other hematological and neoplastic diseases. Expression levels of Fas and FasL were determined with flow cytometry in children with ALL at diagnosis (ALLd, n=13), on day 15 of treatment (d15, n=6), on day33 (ALLd33, n=6), during consolidation (ALLhr, n=12) and at the end of therapy (ALLet, n=7) as well as in children with Langerhans Histiocytosis (LCH, n=4), cytopenias (Cytp, n=8) and solid tumors without bone marrow infiltration at diagnosis (STd, n=5) and on therapy (STther, n=6). Results: The lowest levels of Fas expression were detected at diagnosis of ALL and were gradually statistical significantly increased until remission of day 33 (ALLd vs ALLd33: 8.02±1.94 vs 24.04±6.11, p=0.035). At consolidation Fas levels were found to be decreased compared to day33 (16.1±4.18) and were again increased at the end of therapy (ALLd vs ALLet: 8.02±1.94 vs 24.96±7.95, p=0.024). On the contrary, FasL levels were gradually decreased and finally increased to similar to diagnosis levels at the end of treatment (ALLd: 4.59±1.41, ALLet: 5.89±1.99). In solid tumors at diagnosis Fas levels were similar to the ones while on chemotherapy (STd vs STth:16.04±2.2 vs 15.19±6.4). The highest FasL levels were detected in the group of STd with the relevant levels on treatment being lower in comparison (STd vs STther: 10.91±3.32 vs 2.92±0.79, p=0.052). In LCH both Fas and FasL levels were found to be as low as at ALL diagnosis. In cytopenias no significant difference was observed between groups for either Fas (11.05±4.49) or FasL (3.01±0.62). As for Fas+FasL+ coexpression no difference was evident between ALLd, ALLet and STd or STther [ALLd (0.73±0.38), ALLet (0.64±0.17), STd (0.68±0.095), STther (0.66±0.38)]. The lowest coexpreesion levels were observed in the group of cytopenias with statistical significant difference compared to STd (0.68±0.095 vs 0.26±0.08, p=0.031). In conclusion, at diagnosis of ALL Fas levels were expressed in lowest levels that were found to be gradually increased at remission and at the end of treatment. This finding probably correlates with the apoptotic process of the leukemia clone and possibly with response to treatment. Disclosures: No relevant conflicts of interest to declare.

Mesenchymal stromal cells (MSC) have attracted considerable interest in both the scientific and clinical fields because of their potential to regenerate tissues and support hematopoiesis. Although laboratory isolation of MSC is established, the lack of an MSC specific marker and their low frequency in bone marrow necessitates their in vitro expansion in order to obtain a sufficient cell number for applications. During this process their characteristics may be altered and cells may acquire oncogenic properties or abnormal proliferative capacity. We investigated the effect of in vitro expansion on MSC, isolated from BM of children with idiopathic thrombocytopenic purpura (ITP, n=10) and autoimmune neutropenia (AIN, n=6). MSC were maintained in culture for a number of serial passages (P1–P6). We studied the expression profile of tumor suppressor genes (p53, p16, Rb) and the oncogene (H-Ras) along with hTERT, involved in malignant transformation at different passages. The cell line HUVEC was used as normal control for adherent cells and the M5 as positive control for hTERT expression. P2-MSC and P6-MSC (n=9) were grown on soft agar to assess their anchorage dependence. P2-MSC showed normal, relative to GAPDH, expression of p53, p16, Rb and H-Ras (ITP: 0.37, 0.2, 0.53, 0.64 respectively and AIN: 0.41, 0.27, 0.62, 0.75 respectively) compared to HUVEC cells (0.5, 0.23, 0.73, 0.93, respectively) which remained stable upto P6 (ITP P2vsP6: p= 0.63, p= 0.63, p= 0.82, p=0.33, AIN P2vsP6: p= 0.85, p= 0.07, p= 0.14, p=0.07). In all samples, hTERT expression was undetectable both at P2 and P6, and moreover none of the samples showed anchorage independent growth. These findings suggest that expression profile of oncogenes and tumor suppressor genes does not change during in vitro expansion of MSC. Importantly, the similar transcript levels between MSC and HUVEC, the absence of hTERT expression and anchorage independent growth suggest that MSC may not be transformed for a number of passages. In conclusion, although in vivo models are required, such properties encourage MSC use in clinical practice and should be considered as candidates for therapeutic application.

Mesenchymal Stem Cells (MSCs) are multipotent progenitor cells within the bone marrow (BM) capable of differentiating into various tissue specific cells including adipocytes (A), osteoblasts (O), chondrocytes (C), myoblasts, hepatocytes and possibly neuronal tissue. MSCs form an integral part of the BM stroma, have immunomodulatory functions and play an important role in the support of hematopoeisis. Their multipotentiality and ease of ex vivo expansion has raised great interest in the clinical use of MSCs for tissue repair and gene therapy. In order to evaluate if malignant and non malignant hematological diseases quantitatively and qualitatively affect BM derived MSCs, bone marrow from children with Acute Lymphoblastic Leukemia (ALL diagnosis n=9, different stages of treatment n=14, end of therapy n=9), Idiopathic Thrombocytopenic Purpura (ITP, n=12), autoimmune neutropenia (n= 9) and control patients (solid tumors without bone marrow involvement, n=19) was harvested and the mononuclear cell (MNC) fraction isolated. MSCs were expanded in aMEM supplemented with 10% selected FCS, characterized and compared in terms of their phenotypic characteristics, clonogenicity and ability to differentiate into A, O and C lineages. MNCs at day 0 of culture expressed high levels of CD34, CD45, CD29 and CD44, and very low levels of CD14, CD105 and CD90. Expression of hematopoietic markers (CD34, CD45 and CD14) on cells at passage 1 (P1) and thereafter progressively diminished while expression of CD29, CD44, CD90 and CD105 increased approaching 100%. High clonogenicity was observed in all samples at all passages as shown by the presence of CFU-F colonies (>50 cells) with the exception of ALL samples at diagnosis which showed impaired proliferation and clonogenicity that returned to normal at the following stages of treatment till the end of therapy. At P2 or P3, MSCs were differentiated towards the A, O, and C lineages by using specific induction media. Differentiation was assessed by histochemistry (Oil red O for A, von Kossa and Alkaline phosphatase (ALP) for O and Alcian Blue for C) and RT-PCR (LPL and aP2 for A, osteoprotegerin, osteocalcin and ALP for O, aggrecan and Col II for C). P2 or P3 MSCs from all groups exhibited bi- or tri-lineage differentiation. These results indicate that blood diseases of childhood do not affect the characteristics of MSCs and therefore could have clinical uses particularly in hematopoietic reconstitution following allotransplantation.