Selection Of Subpopulations Research Articles

Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling.

Metastasis of cancer cells involves multiple steps, including their dissociation from the primary tumor and invasion through the endothelial cell barrier to enter the circulation and finding their way to distant organ sites where they extravasate and establish metastatic lesions. Deficient contact inhibition is a hallmark of invasive cancer cells, yet surprisingly the vascular invasiveness of commonly studied cancer cell lines is regulated by the density at which cells are propagated in culture. Cells grown at high density were less effective at invading an endothelial monolayer than cells grown at low density. This phenotypic difference was also observed in a zebrafish model of vascular invasion of cancer cells after injection into the yolk sac and extravasation of cancer cells into tissues from the vasculature. The vascular invasive phenotypes were reversible. A kinome-wide RNAi screen was used to identify drivers of vascular invasion by panning shRNA library transduced non-invasive cancer cell populations on endothelial monolayers. The selection of invasive subpopulations showed enrichment of shRNAs targeting the LATS1 (large tumor suppressor 1) kinase that inhibits the activity of the transcriptional coactivator YAP in the Hippo pathway. Depletion of LATS1 from non-invasive cancer cells restored the invasive phenotype. Complementary to this, inhibition or depletion of YAP inhibited invasion in vitro and in vivo. The vascular invasive phenotype was associated with a YAP-dependent up-regulation of the cytokines IL6, IL8, and CXCL1, 2, and 3. Antibody blockade of cytokine receptors inhibited invasion and confirmed that they are rate-limiting drivers that promote cancer cell vascular invasiveness and could provide therapeutic targets.

Current View on Osteogenic Differentiation Potential of Mesenchymal Stromal Cells Derived from Placental Tissues.

Mesenchymal stromal cells (MSC) isolated from human term placental tissues possess unique characteristics, including their peculiar immunomodulatory properties and their multilineage differentiation potential. The osteogenic differentiation capacity of placental MSC has been widely disputed, and continues to be an issue of debate. This review will briefly discuss the different MSC populations which can be obtained from different regions of human term placenta, along with their unique properties, focusing specifically on their osteogenic differentiation potential. We will present the strategies used to enhance osteogenic differentiation potential in vitro, such as through the selection of subpopulations more prone to differentiate, the modification of the components of osteo-inductive medium, and even mechanical stimulation. Accordingly, the applications of three-dimensional environments in vitro and in vivo, such as non-synthetic, polymer-based, and ceramic scaffolds, will also be discussed, along with results obtained from pre-clinical studies of placental MSC for the regeneration of bone defects and treatment of bone-related diseases.

Adaptive graph-based multiple testing procedures.

Multiple testing procedures defined by directed, weighted graphs have recently been proposed as an intuitive visual tool for constructing multiple testing strategies that reflect the often complex contextual relations between hypotheses in clinical trials. Many well-known sequentially rejective tests, such as (parallel) gatekeeping tests or hierarchical testing procedures are special cases of the graph based tests. We generalize these graph-based multiple testing procedures to adaptive trial designs with an interim analysis. These designs permit mid-trial design modifications based on unblinded interim data as well as external information, while providing strong family wise error rate control. To maintain the familywise error rate, it is not required to prespecify the adaption rule in detail. Because the adaptive test does not require knowledge of the multivariate distribution of test statistics, it is applicable in a wide range of scenarios including trials with multiple treatment comparisons, endpoints or subgroups, or combinations thereof. Examples of adaptations are dropping of treatment arms, selection of subpopulations, and sample size reassessment. If, in the interim analysis, it is decided to continue the trial as planned, the adaptive test reduces to the originally planned multiple testing procedure. Only if adaptations are actually implemented, an adjusted test needs to be applied. The procedure is illustrated with a case study and its operating characteristics are investigated by simulations.

Adaptive Sequential Testing for Multiple Comparisons

We propose a Markov process theory-based adaptive sequential testing procedure for multiple comparisons. The procedure can be used for confirmative trials involving multi-comparisons, including dose selection or population enrichment. Dose or subpopulation selection and sample size modification can be made at any interim analysis. Type I error control is exact.

Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania.

Author SummaryVariations in the copy number of DNA segments account for a substantial amount of genome diversity of most organisms. DNA amplification, a contributor to copy number variation, can occur in response to various stresses or after altered growth conditions, leading to extensive and often reversible genetic variation. DNA amplification in the parasite Leishmania occurs outside the normal chromosomes and arises by DNA rearrangements involving homologous repeated sequences. We show here that such repeated sequences are widespread in the Leishmania genome and that most of the Leishmania genome is subject to stochastic gene rearrangements mediated by these low-copy repeat sequences. Thus, although cells in the population have a common core genome, many individual cells will differ from the rest of the population by carrying one or more distinct extrachromosomal amplicon. Upon selection with either drugs or culture conditions, a subpopulation can emerge where the amplicon copy number per cell increases, and this clone of cells can then expand to dominate the population. We propose that Leishmania uses adaptive gene amplification at a genome-wide scale as one strategy to adapt to a changing environment.

Genetic variation and the potential response to selection on leaf traits after habitat degradation in a long-lived cycad

Rapid evolution may be common in human-dominated landscapes where environmental changes are severe. We used phenotypic selection analyses and a marker-based method to estimate genetic variances and covariances to predict the potential response to selection in populations of a long-lived cycad recently exposed to drastic environmental changes. Patterns of selection in adult fecundity showed that different traits were under directional selection in subpopulations from native-undisturbed habitats and the novel degraded-forest habitat. Plants from a native-habitat subpopulation tend to maximize fitness through larger leaf area or smaller specific leaf area (SLA). In contrast, larger leaf production increased fitness in a degraded-habitat subpopulation, and canopy openness appears to be a major agent of selection for this trait. Leaf production and SLA showed significant additive genetic variance and no genetic trade-offs with examined traits, suggesting that these traits can respond to selection. Directional selection coefficients and heritability values were large, therefore significant phenotypic changes between subpopulations in few generations are possible. These results suggest that recent environmental change can result in strong directional selection in subpopulations of this cycad, and that these subpopulations have the potential to diverge at the genetic level in leaf traits after anthropogenic habitat degradation.

Comparison of vaccine subpopulation selection, viral loads, vaccine virus persistence in trachea and cloaca, and mucosal antibody responses after vaccination with two different Arkansas Delmarva Poultry Industry -derived infectious bronchitis virus vaccines.

Factors responsible for the persistence of Arkansas Delmarva Poultry Industry (ArkDPI)-derived infectious bronchitis vaccines in commercial flocks and the high frequency of isolation of ArkDPI-type infectious bronchitis viruses in respiratory cases are still unclear. We compared dynamics of vaccine viral subpopulations, viral loads, persistence in trachea and cloaca, and the magnitude of infectious bronchitis virus (1BV)-specific antibody induction after vaccination with two commercial ArkDPI-derived Arkansas (Ark) serotype vaccines. One of the vaccines (coded vaccine B) produced significantly higher vaccine virus heterogeneity in vaccinated chickens than the other vaccine (coded A). Chickens vaccinated with vaccine B had significantly higher viral loads in tears at 5 days postvaccination (DPV) than those vaccinated with vaccine A. Vaccine B also induced a significantly higher lachrymal immunoglobulin M response at 11 DPV, an earlier peak of IBV-specific lachrymal immunoglobulin A, and higher serum antibodies than vaccine A. In addition, a significantly higher proportion of birds vaccinated with vaccine B had vaccine virus detected in the trachea at 20 DPV than those vaccinated with vaccine A. Furthermore, the virus detected at 20 DPV in most of the chickens vaccinated with vaccine B was a single specific subpopulation (subpopulation 4) selected from multiple vaccine subpopulations detected earlier at 5 and 7 DPV in the same chickens. On the other hand, a higher proportion of chickens vaccinated with vaccine A had virus detected in cloacal swabs at 20 DPV. Thus we found differences in mucosal antibody induction and selection and persistence of vaccine viruses between two ArkDPI-derived vaccines from different manufacturers. The higher vaccine virus heterogeneity observed in chickens vaccinated with vaccine B compared with those vaccinated with vaccine A may be responsible for these differences. Thus the high frequency of Ark IBV viruses in the field may be due to the inherent ability of some ArkDPI-derived vaccine viruses to be selected and persist in vaccinated chickens. Vaccine virus persistence may offer genetic material for recombination or may undergo mutations with the potential to result in increased virulence.

Carcinoembryonic antigen expression level as a predictive factor for response to 5-fluorouracil in colorectal cancer

Carcinoembryonic antigen (CEA) expression has been shown to protect cancer cell lines from apoptosis and anoikis. The aim of this study was to further elucidate the role of CEA expression on resistance to anticancer drugs in human colorectal cancer (CRC). We transfected CEA negative CRC cell line SW742 as well as CHO cells to overexpress CEA and their chemoresistance were assessed by MTT assay. In comparison to the parental cell lines, transfected cells had significantly increased resistance to 5-fluorouracil (5-FU). The results also showed a direct correlation between the amount of cellular CEA protein and 5-FU resistance in CEA expressing cells. We found no significant difference in sensitivity to cisplatin and methotrexate between CEA-transfected cells and their counter parental cells. We also compared the association between CEA expression and chemoresistance of 4 CRC cell lines which differed in the levels of CEA production. The CEA expression levels in monolayer cultures of these cell lines did not correlate with the 5-FU resistance. However, 5-FU treatment resulted in the selection of sub-populations of resistant cells that displayed increased CEA expression levels by increasing drug concentration. We analyzed the effect of 5-FU in a 3D multicellular culture generated from the two CRC cell lines, LS180 and HT29/219. Compared with monolayer culture, CEA production and 5-FU resistance in both cell lines were stimulated by 3D growth. In comparison to the 3D spheroids of parental CHO, we observed a significantly elevated 5-FU resistance in 3D culture of the CEA-expressing CHO transfectants. Our findings suggest that the CEA level may be a suitable biomarker for predicting tumor response to 5-FU-based chemotherapy in CRC.

On-site education of VEGF-recruited monocytes improves their performance as angiogenic and arteriogenic accessory cells

Adult neovascularization relies on the recruitment of monocytes to the target organ or tumor and functioning therein as a paracrine accessory. The exact origins of the recruited monocytes and the mechanisms underlying their plasticity remain unclear. Using a VEGF-based transgenic system in which genetically tagged monocytes are conditionally summoned to the liver as part of a VEGF-initiated angiogenic program, we show that these recruited cells are derived from the abundant pool of circulating Ly6C(hi) monocytes. Remarkably, however, upon arrival at the VEGF-induced organ, but not the naive organ, monocytes undergo multiple phenotypic and functional changes, endowing them with enhanced proangiogenic capabilities and, importantly, with a markedly increased capacity to remodel existing small vessels into larger conduits. Notably, monocytes do not differentiate into long-lived macrophages, but rather appear as transient accessory cells. Results from transfers of presorted subpopulations and a novel tandem transfer strategy ruled out selective recruitment of a dedicated preexisting subpopulation or onsite selection, thereby reinforcing active reprogramming as the underlying mechanism for improved performance. Collectively, this study uncovered a novel function of VEGF, namely, on-site education of recruited "standard" monocytes to become angiogenic and arteriogenic professional cells, a finding that may also lend itself for a better design of angiogenic therapies.

Adaptive enrichment with subpopulation selection at interim: Methodologies, applications and design considerations

There is a growing interest in pursuing adaptive enrichment for drug development because of its potential to achieve the goal of personalized medicine. There are many versions of adaptive enrichment proposed across many disease indications. Some are exploratory adaptive enrichment and others aim at confirmatory adaptive enrichment. In this paper, we give a brief overview on adaptive enrichment and the methodologies that are growing in statistical literature. A case example is provided to illustrate a regulatory experience that led to drug approval. There were two design elements used for adaptation in this case example: population adaptation and statistical information adaptation. We articulate the challenges in the implementation of a confirmatory adaptive enrichment trial. The challenges include logistic aspects on the appropriate choice of study population for adaptation and the ability to follow the pre-specified rules for statistical information or sample size adaptation. We assess the consistency of treatment effect before and after adaptation using the approach laid out in Wang et al. (2013). We provide the rationales for what would be an appropriate treatment effect estimate for reporting in the drug label. We discuss and articulate design considerations for adaptive enrichment among a dual-composite null hypothesis, a flexible dual-independent null hypothesis and a rigorous dual-independent null hypothesis.

Emergencia de la resistencia a colistina en Klebsiella pneumoniae. Caracterización microbiológica y epidemiológica de aislamientos productores y no productores de carbapenemasa de tipo KPC

Sixty-four colistin-resistant Klebsiella pneumoniae isolates recovered from clinical specimens from 57 patients admitted to Hospital de Clinicas Jose de San Martin during the period 2010–2012 were studied to describe the microbiological and epidemiological characteristics and factors associated with the emergence of colistin-resistance. Fiftyfour colistin-susceptible K. pneumoniae isolates from the same period were also included in the study. The genetic relatedness among the isolates was studied by a PCR assay. Fifty percent of the resistant isolates were KPC-2 producers, 45.3% were ESBL producers and 4.7% only showed resistance to aminopenicilins. All KPC-producers (resistant and susceptible to colistin) were genotipically indistinguishable except for one, whereas the presence of 7 clonal types, which were different from the ones identified in the colistinsusceptible isolates, were detected among ESBL producers. The previous use of colistin was the main factor associated with the acquisition of resistance, and in the case of non-KPC producers the stay in ICU was another significant factor observed. Colistin resistance emerged in our hospital in the year 2010, reaching 3% in nosocomial isolates and maintaining this rate in successive years, due to the selection of resistant subpopulations in the epidemic clonal type in KPC-producers and due to the dispersion of colistinresistant clonal types in non-KPC producing-isolates.

Synergistic Activity of Colistin plus Rifampin against Colistin-Resistant KPC-Producing Klebsiella pneumoniae

Infections caused by carbapenem-resistant KPC-producing Klebsiella pneumoniae are responsible for high rates of mortality and represent a major therapeutic challenge, especially when the isolates are also resistant to colistin. We used the checkerboard method to evaluate the synergistic activity of 10 antibiotic combinations against 13 colistin-resistant KPC-producing K. pneumoniae isolates (colistin MIC range of 8 to 128 mg/liter). Colistin plus rifampin was the only combination that demonstrated consistent synergistic bacteriostatic activity against 13/13 strains tested, reducing the colistin MIC below the susceptibility breakpoint (MIC ≤ 2 mg/liter) in 7/13 strains at rifampin concentrations ranging from 4 to 16 mg/liter. Bactericidal synergistic activity was also documented for 8/13 tested strains. Other antimicrobial combinations with carbapenems, gentamicin, and tigecycline showed variously synergistic results. Colistin plus rifampin also exhibited bacteriostatic synergistic activity against 4/4 colistin-susceptible KPC-producing K. pneumoniae isolates (colistin MIC range of 0.5 to 2 mg/liter) and 4/4 ertapenem-resistant extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae isolates (ertapenem MIC range of 16 to 32 mg/liter). Collectively, our data suggest that colistin plus rifampin is the most consistently synergistic combination against KPC-producing K. pneumoniae isolates, including colistin-resistant strains. Colistin-rifampin combinations may have a role in the treatment of multidrug-resistant K. pneumoniae and may possibly slow the selection of heteroresistant subpopulations during colistin therapy.

Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model

Ertapenem resistance is increasing in Enterobacteriaceae. The production of extended-spectrum β-lactamases (ESBLs) and reduced expression of outer membrane porins are major mechanisms of resistance in ertapenem-resistant Klebsiella pneumoniae. Less is known of ertapenem resistance in Escherichia coli. The aim of this study was to explore the impact of ESBL production in E. coli on the antibacterial activity of ertapenem. Two E. coli strains, with and without ESBL production, were exposed to ertapenem in vitro for 48 h at concentrations simulating human pharmacokinetics with conventional and higher dosages. Isolates with non-susceptibility to ertapenem (MICs 0.75-1.5 mg/L) were detected after five of nine time-kill experiments with the ESBL-producing strain. All of these isolates had ompR mutations, which reduce the expression of outer membrane porins OmpF and OmpC. Higher dosage did not prevent selection of porin-deficient subpopulations. No mutants were detected after experiments with the non-ESBL-producing strain. Compared with other experiments, experiments with ompR mutants detected in endpoint samples showed significantly less bacterial killing after the second dose of ertapenem. Impaired antibacterial activity against E. coli with ESBL production and ompR mutation was also demonstrated in time-kill experiments with static antibiotic concentrations. The combination of ESBL production and porin loss in E. coli can result in reduced susceptibility to ertapenem. Porin-deficient subpopulations frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics. Inappropriate use of ertapenem should be avoided to minimize the risk of selection of ESBL-producing bacteria with reduced susceptibility to carbapenems.

Authors′ Reply

Background Although in the general population circulating vascular progenitor cell levels have been implicated in the homeostasis of the vascular wall through differentiation into endothelium and/or smooth muscle cells, it has not yet been assessed in HIV-infected patients. We herein investigated the number of progenitor cell subpopulations in HIV-infected patients and its relationship to carotid intima-media thickness (c-IMT). Methods Blood samples were collected from 200 HIV-infected patients and CD34/KDR, CD34/VE-cadherin, and CD14/Endoglin progenitor cells were identified by flow cytometry. c-IMT was determined by ultrasonography. A group of 27 healthy subjects was used as control group. Results In our population (20 ART-naive patients and 180 treated patients), traditional cardiovascular risk factors were not found predictive of vascular progenitor cell levels. However, antiretroviral therapy (ART)-treatment was identified as the main predictive value for low CD34/KDR cells and high CD14/Endoglin cells after adjustment by cardiovascular risk factors (age, sex, hypertension, diabetes, and hyperlipidaemia) and HIV-related characteristics (HIV duration and ART treatment). Low levels of circulating CD34/KDR or CD34/VE-cadherin endothelial progenitor cells tended to be associated with increased c-IMT. However, a positive association was found between CD14/Endoglin cells and c-IMT. Low number of CD34/KDR cells was also associated with the longest exposure to nucleoside reverse transcriptase inhibitors and/or protease inhibitors. Conclusions ART exposure is the main predictor of circulating vascular progenitor cell levels. However, their levels are only partially associated with high c-IMT in HIV-infected patients. ART has already been found to have proatherogenic effect, but our data first describe its relationship with vascular progenitor cells and c-IMT.

Babesia bovis biological clones and the inter-strain allelic diversity of the Bv80 gene support subpopulation selection as a mechanism involved in the attenuation of two virulent isolates

The virulence phenotype of Babesia bovis subpopulations was evaluated using biological clones derived from the high-virulence BboS2P and the low-virulence BboR1A strain and two original virulent isolates, BboL15 and BboL17, multiplied extensively in vitro or attenuated by successive passages in splenectomized calves. The virulence phenotype was assessed both by inoculation of normal Holstein adult steers and by analyses of polymorphic fragments of the single-copy Bv80 gene as a subpopulation marker. BboS2P and its nine derived clones contained a single 750bp fragment with identical nucleotide sequences and numbers of repeats. A single fragment of approximately 850bp was observed in BboR1A and its derived clones (Ca3B1, Ca2B1). Ca3B1 and Ca2B1 were differentiated by a stable deletion of 15 contiguous nucleotides in the Bv80 allele of Ca3B1. Both alleles were identified in the parental strain. Original isolates BboL15 and BboL17 contained two Bv80 fragments of different sizes. Interestingly, the heavy and light fragments persisted in the in vivo-attenuated strains and the virulent in vitro-multiplied strains, respectively. Despite the inter-strain allelic diversity of the Bv80 gene, the fragments had identical nucleotide sequences and numbers of repeats compared to their respective parental Bv80 genes. The high-virulence and low-virulence phenotypes remained unchanged after they were multiplied in vitro. In conclusion, the polymorphic B. bovis Bv80 gene, was a useful marker for differentiating subpopulations with different phenotypes. The brevity of the procedure to isolate one parasite from the original isolate or strain before in vitro cloning and the fact that the continuous in vitro multiplication did not modify the virulence phenotype of B. bovis clones strongly suggest that the in vivo-attenuated subpopulations existed in the original isolates before they were selected by passages in splenectomized calves.

Reduced Acquisition and Overgrowth of Vancomycin-Resistant Enterococci and Candida Species in Patients Treated With Fidaxomicin Versus Vancomycin for Clostridium difficile Infection

Fidaxomicin causes less disruption of anaerobic microbiota during treatment of Clostridium difficile infection (CDI) than vancomycin and has activity against many vancomycin-resistant enterococci (VRE). In conjunction with a multicenter randomized trial of fidaxomicin versus vancomycin for CDI treatment, we tested the hypothesis that fidaxomicin promotes VRE and Candida species colonization less than vancomycin. Stool was cultured for VRE and Candida species before and after therapy. For patients with negative pretreatment cultures, the incidence of VRE and Candida species acquisition was compared. For those with preexisting VRE, the change in concentration during treatment was compared. The susceptibility of VRE isolates to fidaxomicin was assessed. Of 301 patients, 247 (82%) had negative VRE cultures and 252 (84%) had negative Candida species cultures before treatment. In comparison with vancomycin-treated patients, fidaxomicin-treated patients had reduced acquisition of VRE (7% vs 31%, respectively; P < .001) and Candida species (19% vs 29%, respectively; P = .03). For patients with preexisting VRE, the mean concentration decreased significantly in the fidaxomicin group (5.9 vs 3.8 log10 VRE/g stool; P = .01) but not the vancomycin group (5.3 vs 4.2 log10 VRE/g stool; P = .20). Most VRE isolates recovered after fidaxomicin treatment had elevated fidaxomicin minimum inhibitory concentrations (MICs; MIC90, 256 µg/mL), and subpopulations of VRE with elevated fidaxomicin MICs were common before therapy. Fidaxomicin was less likely than vancomycin to promote acquisition of VRE and Candida species during CDI treatment. However, selection of preexisting subpopulations of VRE with elevated fidaxomicin MICs was common during fidaxomicin therapy.Clinical Trials Registration. NCT00314951.

Comparative minimum inhibitory and mutant prevention drug concentrations of enrofloxacin, ceftiofur, florfenicol, tilmicosin and tulathromycin against bovine clinical isolates of Mannheimia haemolytica

Mannheimia haemolytica is the most prevalent cause of bovine respiratory disease (BRD) and this disease accounts for 75% of morbidity, 50–70% of feedlot deaths and is estimated to cost up to $1 billion dollars annually in the USA. Antimicrobial therapy is essential for reducing morbidity, mortality and impacting on the financial burden of this disease. Due to the concern of increasing antimicrobial resistance, investigation of antibacterial agents for their potential for selecting for resistance is of paramount importance. A novel in vitro measurement called the mutant prevention concentration (MPC) defines the antimicrobial drug concentration necessary to block the growth of the least susceptible cells present in high density (≥107 colony forming units/ml) bacterial populations such as those seen in acute infection. We compared the minimum inhibitory concentration (MIC) and MPC values for 5 antimicrobial agents (ceftiofur, enrofloxacin, florfenicol, tilmicosin, tulathromycin) against 285 M. haemolytica clinical isolates. The MIC90/MPC90 values for each agent respectively were as follows: 0.016/2, 0.125/1, 2/≥16, 8/≥32, 2/8. Dosing to achieve MPC concentrations (where possible) may serve to reduce the selection of bacterial subpopulations with reduced antimicrobial susceptibility. The rank order of potency based on MIC90 values was ceftiofur>enrofloxacin>florfenicol=tulathromycin>tilmicosin. The rank order of potency based on MPC90 values was enrofloxacin>ceftiofur>tulathromycin>florfenicol≥tilmicosin.

Genome-wide scan with nearly 700,000 SNPs in two Sardinian sub-populations suggests some regions as candidate targets for positive selection.

This paper explores the genetic structure and signatures of natural selection in different sub-populations from the Island of Sardinia, exploiting information from nearly 700,000 autosomal SNPs genotyped with the Affymetrix Genome-Wide Human SNP 6.0 Array. The genetic structure of the Sardinian population and its position within the context of other Mediterranean and European human groups were investigated in depth by comparing our data with publicly available data sets. Principal components and admixture analyses suggest a clustering of the examined samples in two significantly differentiated sub-populations (Ogliastra and Southern Sardinia), as confirmed by AMOVA (F(ST)=0.011; P<0.001). Differentiation of these sub-populations was still evident when they were pooled together with supplementary Sardinian samples from HGDP and compared with several other European, North-African and Near Eastern populations, confirming the uniqueness of the Sardinian genetic background. Moreover, by applying several statistical approaches aimed at assessing differences at the SNP level, the highest differentiated genomic regions between Ogliastra and Southern Sardinia were thus investigated via an extended haplotype homozygosity (EHH)-based test to point out potential selective sweeps. Using this approach, 40 genomic regions were detected, with significant differences between Ogliastra and Southern Sardinia. These regions were subsequently investigated using a long-range haplotype test, which found significant REHH values for SNPs rs11070188 and rs11070192 in the Ogliastra sub-population. In the light of these results and the overlap of the different computed statistics, the region encompassing these loci can be considered a strong candidate to have undergone selective pressure in Ogliastra.

Abstract 4605: Neurotrophin system expression in human lung cancer stem cells

Abstract The availability of in vitro and in vivo systems which reproduce the natural heterogeneity of spontaneous human cancers is of critical importance to assess the potential of new combination treatments and to establish better correlations between molecular signatures and response to therapy. We have focused our attention on the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases and have recently reported that Malignant Pleural Effusions (MPEs) represent an excellent source to culture a wide variety of Lung AdenoCa cells from different donors (Mancini et al, PlosOne, 2011). Reproducible protocols for rapid cancer cell isolation from MPEs and growth in adherent and spheroid conditions have been established in our lab. In order to avoid long periods of adaptations in cell culture that may lead to the selection of subpopulation of cells with particular growth advantages all samples are banked at early passages, on average one month following MPE collection. Furthermore we have observed that these cells can achieve efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells. We believe to have a set up solid system for the study of AdenoCa biology. Recently, we have started to study the expression of neurotrophins (NTs) in cultured lung adenocarcinoma cells from malignant pleural effusion. Using enzyme-linked immunosorbent assays in cancer stem cells and adherent cells from the same patients we have detected nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), and their levels are higher as compared with adhesion and spheroid cells. We have documented, using molecular biology and immunocytochemistry, a different expression of the NTs and their cognate receptors, in lung cancer stem cells in comparison with differentiated cancer cells. Furthermore, the expression on these peptides resembles the previously documented pattern demonstrated in human embryonic stem cells. These data further support the possible role of NTs in cancer cell behavior. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4605. doi:1538-7445.AM2012-4605

Antiangiogenic therapy—evolving view based on clinical trial results

Antiangiogenic therapies that target VEGF or its receptors have become a mainstay of cancer therapy in multiple malignancies. However, the clinical efficacy of these agents is less than originally anticipated and, in most settings, requires the addition of cytotoxic chemotherapy suggesting that, as for other targeted therapies, VEGF inhibitors will require selection of patient subpopulations to achieve maximal clinical benefit. Without the identification and use of predictive biomarkers for VEGF-targeted agents, and other agents that target the vasculature, further improvements in current clinical outcomes are unlikely. Exciting new data presented in 2011 at the ESMO conference showed that retrospective evaluation of plasma concentrations of VEGF-A predicted progression-free survival and/or overall survival benefit from bevacizumab in phase III trials in certain tumour types; prospective evaluation of the assay is required. This endeavour should be followed by further biomarker research, requiring inter-laboratory collaboration and high-quality, adequately powered clinical trials.