Astatotilapia burtoni. We manipulated the number and arrangement of egg-spots on the anal fins of males, or removed them entirely, and tested (1) female preference with visual contact only using egg-traps, (2) female preference with free contact using paternity testing with microsatellites and (3) male aggression. We found that females did not prefer males with many egg-spots over males with fewer egg-spots and that females tended to prefer males without egg-spots over males with egg-spots. Importantly, males without egg-spots sired clutches with the same fertilization rate as males with egg-spots. In male aggression trials, however, males with fewer egg-spots received significantly more attacks, suggesting that egg-spots are an important signal in intrasexual communication.Color and pigmentation patterns of animals are often targets of sexual selection because of their role in communication. Although conspicuous male traits are typically implicated with intersexual selection, there are examples where sex-specific displays play a role in an intrasexual context, e.g. when they serve as signals for aggression level and/or status. Here, we focus on the function of a conspicuous male ornament in the most species-rich tribe of cichlid fishes, the haplochromines. A characteristic feature of these ca. 1500 species are so-called egg-spots in form of ovoid markings on the anal fins of males, which are made up of carotenoid based pigment cells. It has long been assumed that these yellow, orange or reddish egg-spots play an important role in the courtship and spawning behavior of these maternal mouth-brooding fishes by mimicking the eggs of a conspecific female. The exact function of egg-spots remains unknown, however, and there are several hypotheses about their mode of action. To uncover the function of this cichlid-specific male ornament, we used female mate choice experiments and a male aggression test in the haplochromine species  Particularly in the latter mode, mate choice is often based on visual ornaments, although color traits can serve with respect to both inter- and intrasexual communication and, hence, inter- and intrasexual selection vice versaSince the publication of Charles R. Darwin's second most famous book ‘The Descent of Man’ in 1871 Color and pigmentation patterns seem to play a central role in the explosively radiating cichlid fish species in the East African Great Lakes in general, and in the haplochromine cichlids in particular Egg-spots appear to be important in the courtship and spawning behavior of haplochromines Astatotilapia elegans there were no differences in fertilization rates between males without and males with intact egg-spots, which at least partly contradicted the mimicry hypothesis. On the other hand, males with intact egg-spots fertilized twice as many clutches compared to males without egg-spots. Further mate choice trials revealed that females always chose males with egg-spots and preferred males with four egg-spots over males with one egg-spot. In Pseudotropheus aurora (now Maylandia aurora), females spawned more frequently with males displaying more egg-spots and male egg-spot number correlated significantly with the number of fertilized clutches Pseudotropheus lombardoi (now Maylandia lombardoi), a Lake Malawi cichlid in which males display a single egg-spot, females preferred males with one egg-spot over males with an artificially added second one P. lombardoi and that egg-spots may be linked to species recognition.Wickler de novo by animals  Previous hypotheses regarding the function of egg-spots involve female choice as the main explanation for the maintenance of this conspicuous male trait (see above). Interestingly, however, essential sequences of courtship behavior like quivering and lateral display are also used in male-male interactions. When males fight, which happens frequently in territorial haplochromines, they quiver, move back and forward and attack sideways Astatotilapia burtoni, we conducted three experiments. First, females had a choice based on visual cues only between two size-matched males differing in egg-spot number  and one trial with manipulated numbers of egg-spots (experiment 1.2)). Second, we performed a female four-way choice experiment in a partial partition set-up  or towards one of the males with an artificial difference in egg-spot number (experiment 1.2), as the actual egg-laying activity towards a male appears to be a better predictor of female preference than e.g. time-spent  between the four males with distinct anal fin phenotypes. Obviously, there is no effect of number and arrangement of egg-spots on fertilization rate, as the number of fertilized eggs was 100% in nearly every clutch, which is in line with previous experiments in A. burtoni. Multiple paternity is rather common in haplochromines. A study on several lekking rock- and sand-dwelling species from Lake Malawi, for example, uncovered multiple paternity in almost every brood A. burtoni (at least under laboratory conditions), but that its frequency is low (10 out of 68 broods) compared to the natural situation in Lake Malawi cichlids.The genotyping of offspring and candidate parents in the four-way choice experiments allowed us to test for extra-pair fertilization in A. burtoni do not serve as recognition pattern (at least on short distances) or attraction signal for females, and that they do not maximize fertilization rates. Still, egg-spots are present in most haplochromine species, indicating an important function additionally to the one in intersexual selection. It has previously been suggested that the honesty and genetic variance of a trait are actually easier maintained through male-male competition than through female choice A. burtoni that the attack readiness of males was influenced by specific body patterns - e.g. the head pattern in form of a black bar increases and the orange patch on the cheek decreases number of bites from a competitor Taken together, our female mate choice experiments demonstrate that egg-spots in Thorichthys meeki (formerly known as Cichlasoma meeki), for example, became more violent when the ornament in form of an eye-spot had been removed Pundamilia, the advantage of more intensely colored males to win a fight (under white light) vanished under green light conditions masking the carotenoid-based red coloration A. burtoni appear to exert a similarly intimidating effect on the competitor during threatening and fighting.There are, in fact, other examples from cichlid fishes where coloration or pigmentation signals have an intimidating effect in male combats. Fights in the North American cichlid Females of several species are known to prefer males with increased carotenoid coloration, which indicates health status: in sticklebacks, for example, the red belly coloration functions as a threat signal in intrasexual competition but also in female mate choice Taken together, the involvement of egg-spots in female choice Astatotilapia burtoni, a maternal mouthbrooder, is a generalist living in the estuaries and affluent river systems of Lake Tanganyika, East Africa. As is typical for polygynous mating systems, the species shows sexual dimorphism: males are larger, more intensively colored and their egg-spots are much more pronounced and show, in contrast to female egg-spots, a hyaline circle, which is characteristic for ‘true egg-spots’ A. burtoni is member of a group of riverine haplochromines that are the sister group to the species flock of Lake Victoria region, and, together with the latter, the sister group to the Lake Malawi species assemblage The cichlid species The female test animals were kept in a pure female tank (100×50×50 cm) and the males in mixed-sex stock tanks (100×50×50 cm), from which they were transferred into smaller individual tanks before testing. All tanks provided standardized conditions of constant water temperature of 26°C, pH 7, and a 12:12 h light:dark cycle. Flake food was fed twice a day and frozen artemia was given once a day. Our aquaria strain population, which was used in most of the experiments, originated from an inbred line. The wild caught specimens used in the four-way choice experiment were imported from the Kalambo region in Zambia in 2009.All laboratory mate choice experiments were performed at the Zoological Institute of the University of Basel under the permission of the Cantonal Veterinary Office, Basel, Switzerland . Manipulations on anal fin egg-spots were performed under clove oil anesthesia (2–3 drops clove oil per liter water).e.g.versus males with fewer egg-spots . In the second experiment (experiment 1.2) artificial variation was created through experimental manipulations with dry ice to entirely remove all egg-spots . As a treatment control, both competitors of a trial were freeze-branded, with one male being treated directly on the egg-spots while the other one was treated below the egg-spots. Males of a male pair had similar territorial body coloration and were size-matched , therefore they did not differ in size nor weight but in the number of anal fin egg-spots ) was recorded. The percentage of eggs per clutch that a female laid in front of each male was calculated. Due to the fact that often all eggs were laid exclusively next to one male, causing zero inflation in the data, we coded the data as 1 if the male with egg-spots received more than 50% of the eggs and 0 if it received 50% or less of the eggs laid by the female compared to the male with fewer egg-spots (experiment 1.1) or without egg-spots (experiment 1.2). We applied generalized linear mixed models (GLMMs) with a logistic link function  or one week (in experiment 1.2 in which recovery from the freeze branding treatment was necessary) to acclimate to the outer tanks and to become territorial . Then a gravid female  no egg-spots; (2) half the amount of egg-spots ; (3) half the amount of egg-spots ; (4) all egg-spots present , their corresponding mothers and the putative fathers were used for paternity testing with at least five available un-linked microsatellite markers using a multiplex approach (Qiagen multiplex kit). We used the following markers: Abur82 xl genetic analyzer and sized in comparison to LIZ 500(-250) (ABI) internal size standard. Genotypes were determined manually using the Genemapper software . With this procedure the father of each fry could be determined and it became apparent if multiple paternity occurred. Similarly as in experiment 1, we coded the data as 1 if the three males with egg-spots sired 75% or more of the eggs and 0 if the male without egg-spots sired 25% or more of the eggs. The three replicates were analyzed separately using binomial tests with a probability of 0.75 to check if the three egg-spot bearing males had a benefit and therefore received a higher number of clutches.Three replicates with four males each and constantly 12 to 20 females were conducted; males were matched by size and weight  and swapThis set up consisted of a tank (60×30×30 cm) containing a shelter for the focal male (n = 13) and two transparent plastic cylinders , one for each stimulus male . The focTable S1Measurements taken from test animals. (A) Experiment 1.1 and 1.2. (B) Experiment 2. (C) Experiment 3.(PDF)Click here for additional data file.


To estimate, from the perspective of the Chinese healthcare system, the economic outcomes of five different first-line strategies among patients with metastatic renal cell carcinoma (mRCC).A decision-analytic model was developed to simulate the lifetime disease course associated with renal cell carcinoma. The health and economic outcomes of five first-line strategies  were estimated and assessed by indirect comparison. The clinical and utility data were taken from published studies. The cost data were estimated from local charge data and current Chinese practices. Sensitivity analyses were used to explore the impact of uncertainty regarding the results. The impact of the sunitinib patient assistant program (SPAP) was evaluated via scenario analysis. The base-case analysis showed that the sunitinib strategy yielded the maximum health benefits: 2.71 life years and 1.40 quality-adjusted life-years (QALY). The marginal cost-effectiveness  gained via the sunitinib strategy compared with the conventional strategy was $220,384  and $16,993 . In general, the results were sensitive to the hazard ratio of progression-free survival. The probabilistic sensitivity analysis demonstrated that the sunitinib strategy with SPAP was the most cost-effective approach when the willingness-to-pay threshold was over $16,000.Our analysis suggests that traditional cytokine therapy is the cost-effective option in the Chinese healthcare setting. In some relatively developed regions, sunitinib with SPAP may be a favorable cost-effective alternative for mRCC. Renal cell carcinoma (RCC), the most common type of kidney cancer, accounts for about 3% of all human malignancies. It is estimated that nearly 30% of the patients with RCC have distant metastatic disease at presentation and that half of those with localised disease subsequently develop metastases during the course of their disease Recently, targeted pharmacological therapies  have been developed for RCC treatment In the current study, we evaluated the long-term economic outcomes of five first-line mRCC regimens based on clinical practice and recommendations: With the R software package , we used a previously developed Markov model to simulate the transition of a cohort of individuals with mRCC based on the clinical course Although multiple agents for mRCC have been evaluated in clinical trials, the most commonly used first-line strategies are interferon-alfa, interleukin-2, sunitinib and bevacizumab Four types of parameters were inputs for the model: transition probabilities, which reflect the probabilities of moving between health states at each cycle; event proportions, which govern the ratios of events; direct medical costs, which were estimated based on health resource consumption; and health state utilities, which project the health-related quality of life for different health states. These data were derived from the published literature or from local health systems.Cost-effectiveness ratios were calculated to evaluate the outcomes of the different strategies. The main health outcomes were presented by quality-adjusted life-year (QALY). The results were presented as an incremental cost-effectiveness ratio (ICER).A decision-analytic Markov model was used to compare the lifetime clinical progression, costs and utilities of treating mRCC with sunitinib, bevacizumab plus interferon-alfa, interferon-alfa, interleukin-2 and interleukin-2 plus interferon-alfa. Three discrete health states reflecting different characteristics of the disease were identified: progression-free survival (PFS), progressed survival (PS) and death. The structure of the model is shown in We performed a literature search in the following electronic databases for the pivotal clinical trials pertaining to mRCC treatments: PubMed, EMBASE, CINAHL, AltHealthWatch, the Cochrane Library, and the National Library of Science and Technology. The search covered the periods from the databases inceptions to the end of July 2011. However, no clinical trial directly comparing these five strategies was identified. Therefore, an indirect comparison of key clinical trials was performed. Clinical effectiveness data, including HR (hazard ratio), were separately extracted from the four multicenter, randomised-controlled clinical trials that had interferon-alfa as a common comparator; each of these trials constituted level 1 evidence The AVOREN Trial randomised 649 patients with mRCC to receive interferon-alfa 2a and bevacizumab (n = 327) or placebo and interferon-alfa 2a (n = 322). The median PFS was significantly longer in the bevacizumab plus interferon-alfa group than in the control group . The median OS was also longer, although the difference was not statistically significant  In the sunitinib trial, 750 mRCC patients were randomly assigned to sunitinib or to interferon-alfa. The median PFS in the sunitinib arm (11 months) was significantly longer than in the interferon-alfa arm (5 months), corresponding to an HR of 0.42 . The median OS was greater in the sunitinib arm than in the interferon-alfa arm  The MRC RE04/EORTC GU 30012 trial enrolled 1,006 advanced mRCC patients who were randomly allocated (1 to 1) by minimisation to receive interferon-alfa alone or combination therapy with interferon-alfa, interleukin-2, and fluorouracil. After a median follow-up of nearly 37 months, the median OS was 18.8 months for the patients receiving interferon-alfa versus 18.6 months for those receiving the combination therapy . There was no evidence that the median PFS differed between the treatment groups  The clinical trial reported by Groupe Francais d'Immunotherapie enrolled 425 mRCC patients who were randomly assigned to receive interleukin-2, interferon-alfa alone or both. There was no significant difference in OS among the three groups. However, the PFS in the combined cytokine treatment group was significantly higher than in the other two groups (P = 0.01) active strategies = )HR. All HR data is presented in et alIndirect comparisons of the five strategies were conducted using a hypothetical average interferon-alfa (reference) survival rate. Weibull survival models were fitted to the Kaplan-Meier PFS and OS data for interferon-alfa. The estimated Weibull parameters, scale (λ) and shape (γ), and their SEs and correlation coefficients are shown in A Chinese healthcare system perspective was used to estimate the costs in the current study. Direct medical costs, such as first and second-line treatment-related medicines, radiological and laboratory examinations, management of serious adverse events (SAEs), physician visits, and BSC in the terminally ill, were included. Indirect costs  were not included 2) daily for 5 days, once every 3 weeks. Sorafenib (a second-line treatment) was assumed to be administered orally at 400 mg twice per day. To estimate the dosage of the therapeutic agents, we assumed that a typical patient weighed 65 kg and had a height of 1.64 m, resulting in a body-surface area (BSA) of 1.72 m2. We assumed that the unused drugs in the open vials were discarded.The drug costs associated with each strategy were estimated according to the following schedules. The interferon-alfa was assumed to be administered by subcutaneous injection three times per week in first cycle at 3 MU/dose in the first week, 6 MU/dose in the second week, and 9 MU/dose thereafter. The subsequent cycles involved three 9-MU/dose injections. The sunitinib was assumed to be administered orally at 50 mg once daily for 4 weeks, followed by 2 weeks off treatment. Bevacizumab 10 mg/kg or placebo was assumed to be administered intravenously every 2 weeks. Interleukin-2 was assumed to be administered intravenously as 18 MU×body-surface area  showed the preferred first-line strategies for mRCC when accounting for a range of cost-per-QALY thresholds. The CEAC plot shows that when no patient assistant program was offered, interleukin-2 achieved a nearly 89% likelihood, when the threshold level was $13,290 . When thNew targeted therapies for the first-line treatment of mRCC have increased survival rates and improved quality of life. Nevertheless, the widespread use of new targeted therapies comes with a dramatic increase in health care costs. A cost-effectiveness evaluation of the recommended first-line therapies in a health resource–limited setting is necessary to balance the economic burden with the health benefits. Using indirect comparison and decision-analytic modelling techniques, we estimated the cost-effectiveness of five first-line mRCC strategies over a lifetime horizon from the perspective of the Chinese healthcare system.Our results suggest that targeted therapies as first-line treatment for mRCC provide more health benefits than do traditional cytokine therapies. Although the enhanced survival benefits resulting from the targeted therapies were not significant, the prolonged PFS survival improved the benefit of the targeted therapies over cytokines, which is an important reason why they are now covered by some developed countries If the sunitinib patient assistance program were available to patients from poor regions, the sunitinib strategy might be the optimal alternative option when the threshold of the willingness-to-pay is greater than $16,000. HoweverAt present, bevacizumab is also an alternative opion for mRCC in China, so the physicians, decision makers or patients would make the choice between sunitinib and bevacizumab. Our results showed sunitinib provides more health benefits for lower costs than bevacizumab plus interferon-alfa, regardless of the SPAP. Our findings are consistent with a recently published study based on the perspectives of the healthcare systems of the US and Sweden, which used indirect comparisons of survival data. The study showed that sunitinib as a first-line option for mRCC was a cost-effective alternative to bevacizumab plus interferon-alfa, because sunitinib was more effective and less costly The current study has some limitations. Using a Weibull distribution to extrapolate the outcomes beyond the time horizon of the trial follow-ups was an inevitable limitation of this analysis. The one-way sensitive analysis showed that the HR of the PFS and OS had important impacts on the final result. The short median follow-ups of the pivotal clinical trials did not provide enough survival data for a comparison with the median survival that was estimated by the model. At the same time, we did not consider the potential benefits of many of the uncertainties surrounding long-term survival rates, such as new therapeutic agents for second-line therapy, which might improve survival and life quality. Our results could be updated when long-term data are available. However, no randomised controlled trial has yet determined the long-term mRCC outcomes of first-line, second-line, or third-line therapies to the point of death. Therefore, it may be difficult to accurately measure the benefits of first-line therapy in future analyses, suggesting that a modelling technique may be the only realistic alternative.Another potential limitation was the choice the Chinese healthcare system as our baseline perspective, which led to only direct medical costs being included in the model. Considering a societal perspective, which adds the additional burden of disease on families and caregivers and other indirect costs, may increase the costs associated with mRCC. As such, oral medications , prolonged PFS  and reduced toxicity may produce more favourable results. There is no well-established method for incorporating such societal perspectives when measuring the impact of cost-effectiveness of first-line therapies for mRCC.Because of the absence of head-to-head trials for the five first-line strategies for mRCC, an indirect comparison was used in the present analysis, which was another inevitable limitation. Similar cohort characteristics for the five strategies were assumed in our indirect comparison, and the results of the indirect comparison were imputed into the analytical model. Nevertheless, when no direct data is available, indirect comparisons using robust methods are accepted by many authors worldwide. Future research should directly compare the clinical efficacy of these strategies, especially those of different targeted therapies.Other important limitations of the current economic analysis should be considered. In particular, we did not fully explore other therapeutic strategies for mRCC treatment, such as temsirolimus, everolimus and pazopanib, because they are still awaiting approval from the State Food and Drug Administration of China In the Chinese healthcare setting, a representative health resource–limited region, traditional cytokine therapy is the cost-effective option. When the threshold is higher than $16,000, sunitinib might be a cost-effective therapy option compared to bevacizumab plus interferon-alfa, interferon-alfa, interleukin-2 and interleukin-2 plus interferon-alfa, based on its superior PFS benefit and Patient Assistance Program.


During respiratory viral infections host injury occurs due in part to inappropriate host responses. In this study we sought to uncover the host transcriptional responses underlying differences between high- and low-pathogenic infections.From a compendium of 12 studies that included responses to influenza A subtype H5N1, reconstructed 1918 influenza A virus, and SARS coronavirus, we used meta-analysis to derive multiple gene expression signatures. We compared these signatures by their capacity to segregate biological conditions by pathogenicity and predict pathogenicity in a test data set. The highest-performing signature was expressed as a continuum in low-, medium-, and high-pathogenicity samples, suggesting a direct, analog relationship between expression and pathogenicity. This signature comprised 57 genes including a subnetwork of chemokines, implicating dysregulated cell recruitment in injury.Highly pathogenic viruses elicit expression of many of the same key genes as lower pathogenic viruses but to a higher degree. This increased degree of expression may result in the uncontrolled co-localization of inflammatory cell types and lead to irreversible host damage. The threat of a highly lethal viral pandemic remains large in the 21st century. In 2003 SARS-coronavirus (CoV) appeared in Asia and then spread globally, causing greater than 40% mortality in individuals over 60 years of age [Injury to the host during respiratory viral infections such as influenza now appears to be the result of inappropriate host responses . DerivinWhat host responses are the hallmarks of high pathogenicity? To approach this question we assembled a compendium of host transcriptional responses to low-, medium-, and high-pathogenic infections  and applied a battery of meta-analysis techniques. Using these techniques we identified signatures of either a digital nature (oppositely expressed in LPIs and HPIs with respect to mock infections) or an analog nature (expressed in a continuum from LPIs to HPIs). We then compared these signatures on the basis of their capacity to predict the outcome of an independent transcriptome experiment. The most accurate signature was analog in nature and implicated excessive chemokine expression and cell recruitment in the development of lethal respiratory infections.We assembled a compendium of microarray data from 12 studies that measured host transcription in mouse lungs following infection with respiratory viruses , and the degree to which LPI-, MPI-, and HPI-associated conditions clustered proximally to conditions of the same pathogenicity and distally from conditions of other pathogenicities was compared. We also compared the ordering and overall separation of the pathogenicity clusters, both by visual inspection and by percentage variance explained. We reasoned that a more effective signature would result in greater separation of pathogenicities oriented along a dimension that also explained a higher proportion of the total variance . Using aWe also compared signatures by their capacity to generate sample expression profiles that correlated with other profiles of the same pathogenicity. We reasoned that a more effective signature would result in stronger correlations  among similar pathogenicities and weaker correlations (lower PCCs) among dissimilar pathogenicities. On a scatter plot of PCCs to exemplar LPI and HPI profiles, more samples would appear in two quadrants, with more LPI samples in quadrant II (LPI-high/HPI-low) and more HPI samples in quadrant IV (LPI-low/HPI-high) where quadrants are defined by an equal partitioning of the PCC scatter plot plane at . Using all of the genes in the native compendium, LPI and HPI samples appeared primarily in one quadrant, high for both LPI and HPI Figure . This inWe then tested the capacity of each signature to predict the pathogenicity of a data set external to the compendium. Specifically we utilized a microarray data set measuring transcription in the lungs of mice infected with either swine-origin influenza A virus (SOIV) subtype H1N1 strain CA/04  or the mouse-adapted variant MA1 CA/04  at days 1, 3, and 5 post-infection . PCC scatter plots were generated using the test set samples and the same LPI and HPI exemplars as above  indicated that a number of pathways may have been altered during HPIs. Genes up-regulated in HPI were associated with inflammation, apoptosis, cell signaling, and cell proliferation, while genes down-regulated in HPI were associated with cytochrome P450-related enzymatic activity. Protein-protein interactions linked many of these genes together indicating possible cooperative effects  and chemokine activity . A network showing known interactions among analog signature gene products highlighted cooperativity between the inflammatory/interferon-response pathway and chemokines such as CXCL10  and compared this proportion to those expected by chance or observed among all genes. Assuming an equal distribution of genes among pathogenicity groups, we expected 33% to display one of these two patterns by chance. By comparison we observed that 43% of all genes in the compendium displayed these patterns Figure . HoweverIFIT3, CXCL10, IIGP1, and IGTP, consistent with the prominence of these genes in functional and network analyses . Previous studies have applied meta-analysis to infection data but focused on more general features of disease. For example, Jenner and Young analyzed the transcriptomes of various host species  infected by bacteria or viruses and identified a signature of approximately 500 genes . This sia priori expectation of which signature would produce the best outcome in terms of these criteria.In contrast to previous studies, our goal was to determine a gene signature specific for HPIs. We therefore applied an ensemble of meta-analysis methods and derived multiple signatures which we compared by multiple criteria including the capacity to predict the outcome of a test data set. Each method had a different statistical rationale and could be expected to identify different features of the compendium. We had no SPP1) and RANTES (CCL5) and the chemokine receptor CCR2. In previous studies mice lacking SPP1 and CCL5 were found to clear influenza infection with no adverse effects [CCR2 survived infection by a mouse-adapted influenza A virus that killed wild-type mice [The 74-gene signature derived using Fisher's summary statistic showed a modest capacity to separate samples into pathogenicity groups and to predict test set pathogenicity. A number of the genes in this signature have known connections to the immune response and the outcome of respiratory infections, e.g., genes for the chemokines osteopontin . In this case the signature appeared to provide a continuously variable signal that matched with output, a characteristic of analog signals. This finding also suggests that high- and low-pathogenic infections may result in the expression of the same key genes but with different kinetics. In particular HPIs may result in increased expression of signature gene products beyond the capacity of the host to cope, resulting in irreversible damage.The best performing signature, however, comprised 57 genes derived using the fold change-based CXCL9), IP-10 (CXCL10), and I-TAC (CXCL11) which all serve as ligands for the receptor CXCR3 [CCL7) and MCP-4 (CCL13). CCR3 is the major receptor expressed on eosinophils and has previously been shown to have a role in the promotion of lung inflammation [Genes of the analog signature largely differed from those in the digital signatures but overlapped at the pathway level. For example, chemokine genes were present in both Fisher's statistic-derived and analog signatures. However, the analog signature displayed additional coherence, encoding multiple chemokines for the same receptor, namely MIG  has been found to cleave influenza hemagglutinin directly resulting in non-infectious virus particles [SFTPA1) and surfactant-associated protein D (SFTPD) maintain pulmonary structure but have also been found to inhibit infectivity by mechanisms that remain to be elucidated [SCGB1A1, SCGB3A1, and SCGB3A3. Interestingly many secretoglobins are expressed specifically in the lung epithelium and may contribute to lung repair following damage [SCGB1A1) is secreted by bronchiolar Clara cells and postulated to have a role in reducing airway inflammation, though its exact function remains to be elucidated [In addition our signatures also identified genes that were down-regulated during HPI, relative to either mock infection or LPI. For example the Fisher's statistic-derived signature included several genes whose products may normally help to resolve infection. Hepsin  and impaired lung protection (down-regulated in HPI) in the events leading to lethality in mice. The different meta-analysis methods all aimed to identify pathology-relevant gene expression, and concordance at the pathway level offered a degree of cross-validation. However, our results also demonstrate that the methods identified different features of the data and argue for the application of multiple methods in future studies when the most predictive features of the data are not known in advance.Finally our finding that pathogenicity corresponds to the expression levels of a defined set of genes may help to inform future therapies. Altering the outcome of infection may not require opposite regulation to be achieved in particular pathways; instead, adequate tempering or delay of those pathways may be sufficient. For example a large number of potential CXCR3 antagonists have been developed recently in the context of other inflammatory diseases . These mA literature search for microarray data pertaining to respiratory viral infections in mouse models was performed, and candidate data sets were identified. Data were included in the compendium if they were derived from experiments on wild-type mice, as this ensured normal development. We also required that raw data be measured on whole-genome array platforms. Data were sourced from either in-house or external studies Table . In the 2-ratios (infected/mock-infected), and one-channel data were converted to a two-channel-like format where mock-infection intensities were averaged prior to ratio. For meta-analysis based on direct comparison of intensities, two-channel data were separated into individual channels, and inter-array normalization was performed using NeONORM [Data from Agilent platforms were obtained from Agilent Feature Extraction software and pre-processed in GeneSpring GX 11  using median-background subtraction on a per-study basis. Data from the Affymetrix platform were pre-processed in GeneSpring GX 11 using the MAS5 algorithm. Quality control was restricted to probe-level filtering. Probes with present flags in all of the technical replicates for at least one biological condition in a given study were retained in the data set for that study. For ratio-based meta-analysis, two-channel data were reformatted as log NeONORM . Additiot-tests were used to identify genes up- and down-regulated relative to mock infection, i.e., for the null hypotheses H0: log2 (infected/mock) ≤ 0 and H0: log2 (infected/mock) ≥ 0, respectively. p-values for each directionality (up- or down-regulation) and pathogenicity (HPI or LPI) were combined according to Fisher's summary statistic S = -2 ∑ log p [S was determined by a permutation test whereby p-values were shuffled 1000 times among biological conditions, S was re-derived each time, and the resulting S values constituted the null distribution. An FDR-corrected p-value of 0.05 was used as a threshold for significance. Genes that were found to be both up- and down-regulated for a given pathogenicity (HPI or LPI) were excluded from further analysis. The signature comprised genes found to be oppositely regulated in HPI and LPI.We applied the Fisher's summary-statistic method based on the previous application of Rhodes et al. . Our imp ∑ log p . The siget al. identifies modules of co-expressed genes from microarray data based on co-expression within the compendium and co-membership in previously annotated gene sets [http://genomica.weizmann.ac.il) using the log-ratio compendium and default settings. Modules were tested for associations with HPI and LPI outcomes, and the signature comprised the largest module found to be oppositely regulated in HPI and LPI.The module-mapping method of Segal ene sets . We usedz-test was used to compute the statistical significance of log2 (HPI/LPI) values according to intra- and inter-group variability. Statistically significant log2 (HPI/LPI) values were then ranked by size, and genes with log2 (HPI/LPI) < -1.6 or > 2 were retained. The cut-offs were selected based on a histogram of fold-changes over all statistically significantly differentially expressed genes.Intensity-based measurements were used to test for differential expression in all HPIs vs. all LPIs. Specifically a fold-change-based 10 of the geometric mean of the hypergeometric test p-values within the cluster [p < 0.05.We used NIH DAVID  and Ingenuity Pathways Analysis  for further analysis of meta-analysis results. DAVID identifies GO terms and other functional annotations enriched in gene lists . An enri cluster . We asseSTC planned the study, performed the research related to the compendium assembly and processing and digital gene signature derivation, and drafted the manuscript. NT performed the research related to the analog gene signature derivation. DG contributed to the interpretation of the data. AB drafted portions of the manuscript. MGK conceived of the study design and helped to draft the manuscript. All authors read and approved the final manuscript.Figure S1. Hierarchical clustering identifying gene clusters oppositely regulated across various conditions in the compendium. Shown are log2-ratios of intensities in infected to mock-infected samples for genes whose ratios were non-zero across all the measurements in the compendium. The two clusters of interest are boxed in yellow and enumerated to the right of the heat map. Heat maps were generated using the heatmap2 function from the gplots package in R statistical environment with clustering by Euclidean distance and the complete linkage method.Click here for fileFigure S2. Derivation of digital gene signatures. (A) The 74-gene signature comprised 44 and 30 genes derived from the intersection of four parent gene sets: those up-regulated in HPI ∩ down-regulated in LPI and those down-regulated in HPI ∩ up-regulated in LPI. Each parent gene set was derived using Fisher's summary statistic following one-tailed t-tests on each biological condition in the compendium. Each intersection was found to represent a significantly larger proportion of its two parent gene sets than expected by chance . (B) Module map resulting from applying Genomica to the log-ratio compendium. Module up-regulation in a given condition is indicated in red, and module down-regulation in green. (C) Expression of Module 5 comprising 265 genes. HPI-associated arrays are indicated in purple, LPI-associated arrays in blue. Values shown are consistent with the overall pattern of module expression in those arrays in which the module is significantly expressed. Module 5 completely subsumed Module 6 and was used in subsequent analysis.Click here for fileTable S1. Digital signature genes by Fisher's summary-statistic.Click here for fileTable S2. Digital signature genes by module-mapping.Click here for filez-testTable S3. Analog signature genes by fold change-based .Click here for fileFigure S3. Characterization of the 74-gene digital signature of pathogenicity by networks of known interactions. Genes present in the signature are indicated in gray shapes. (A) For the 44-gene subset up-regulated in HPIs and down-regulated in LPIs. (B) For the 30-gene subset down-regulated in HPIs and up-regulated in LPIs.Click here for fileFigure S4. Expression levels of select analog signature genes in HPI, MPI, and LPI conditions. These genes met the criterion of being expressed from greatest to least or from least to greatest (inset) by pathogenicity. Error bars represent standard errors of the means across all HPI, MPI, or LPI conditions in the compendium.Click here for file


Multiple mechanisms responsible for endocrine resistance have been proposed, including deregulation of various components of the estrogen receptor (ER) pathway, alterations in cell cycle and cell survival signaling molecules, and the activation of escape pathways. Dysregulation of miRNA expression has been associated with experimental and clinical endocrine therapy resistance. miRNAs are pivotal to understanding the complex biological mechanism of endocrine resistance, and may serve as novel candidate predictive and prognostic surrogates and therapeutic targets. This review focuses on current progress concerning the roles of miRNAs in endocrine resistance, and discusses the challenges and opportunities for implementing miRNA-based assays and treatment for patients with endocrine-resistant breast cancer.Current endocrine therapies for females with estrogen receptor-positive breast cancer have facilitated substantial improvements in outcomes. The effectiveness of endocrine therapy is limited by either initial  IntroductionBrief overview of the mechanisms of endocrine resistance in breast cancermiRNA: Biogenesis and functionmiRNAs and breast cancerRole of miRNAs in the endocrine resistance of breast cancerFuture prospectsConclusion1.de novo and acquired resistance. Significant efforts have been undertaken to resolve the molecular mechanisms that lead to endocrine resistance. miRNAs are evolutionarily conserved short non-coding RNA genes that were first identified over a decade ago . Current endocrine therapies for females with estrogen receptor (ER)-positive breast cancer have led to substantial improvements in outcomes . ER-targcade ago –4. Accorcade ago ,6, and hcade ago . miRNAs cade ago , there hcade ago ,10. miRNThis review addresses efforts to understand how the miRNA profile is altered upon the development of resistance; the critical regulatory role of miRNAs in conferring resistance to commonly used endocrine agents; and how these differentially expressed miRNAs may serve as prognostic and predictive markers and novel therapeutic targets for overcoming endocrine resistance.2.The term endocrine therapy is applied to breast cancer treatments that target the ER by blocking receptor binding with an antagonist or by depriving the tumor of estrogen. The ER, which has nuclear (genomic) and non-nuclear (non-genomic) functions, is the predominant driver of the majority of types of breast cancer . With atRegardless of these significant advances in the treatment of patients with initially hormone-sensitive breast cancer, resistance to all forms of endocrine therapy remains a major clinical issue; a significant proportion of patients experience recurrence caused by intrinsic or acquired resistance to endocrine agents within 15 years . At presAlthough knowledge of the mechanism of endocrine resistance is developing, a more detailed understanding of the molecular mechanisms and regulatory pathways involved in breast cancer cells is required to improve the design of novel anti-tumor drugs and inform the selection of optimal therapeutic strategies.3.Caenorhabditis elegans in the laboratory studies of Victor Ambros and Gary Ruvkun (C. elegans nematode (http://www.microrna.org/)  . As theyna.org/) .It is well-recognized that miRNAs are involved in the regulation of multiple cellular processes, including proliferation, apoptosis, cell-cycle regulation and differentiation . Aberran4.In the last 5 years, miRNA studies of breast cancer have represented a valuable area of research, having resulted in new knowledge on the molecular basis of the disease, tools for molecular classification, new markers with diagnostic and prognostic relevance, as well as the identification of novel breast cancer-predisposing genes . It has et al analyzed the systematic expression of 217 miRNAs from 334 samples (including breast cancer) and observed that the miRNA profiles were informative, reflecting the developmental lineage and differentiation state of the tumors, with a general downregulation of miRNAs noted in the tumors compared with normal tissues  of ∼4.3 kb, which has been reported to reduce mRNA stability and comprise evolutionarily conserved miRNA target sites, enabling it to be regulated by miRNAs.et al  were downregulated by E2 in various ER-positive breast tumor cell lines  functional studies in vitro are implemented to validate the predicted regulatory roles of the miRNAs via ectopic expression and downregulation; and iv) to support the significance of the studies performed in cell lines, clinical data are analyzed and in vivo animal studies are conducted to further confirm the association between miRNAs and endocrine resistance.The model of research  in the met al investigated the role of miRNAs in acquiring resistance to tamoxifen by comparing the miRNA profiles of tamoxifen-resistant with tamoxifen-sensitive MCF-7 breast cancer cell lines by microarray analysis  8.8 . Agreement in the direction of anticipated regulation was detected for 12 putative targets. Pyruvate decarboxylase regulator (PDCD4), B-cell CLL/lymphoma 2 (BCL2), cytochrome P450 1B1 (CYP1B1) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3) were revealed to be differentially expressed between the two cell lines. However, the role of the interaction between the target genes and miRNAs for endocrine resistance  activities, p53 cell cycle checkpoint function and phosphatase and tensin homolog (PTEN) tumor suppressor activity. Overall, these results suggest that miRNA-342 modulates the expression of genes involved in tamoxifen-mediated tumor cell apoptosis and cell cycle progression .et al observed the significant overexpression of miRNA-181b, -221 and -222 in tamoxifen-resistant MCF-7 cells. In addition, anti-miRNA-222 or -181b, in combination with tamoxifen, suppressed the growth of tamoxifen-resistant xenografts in mice through tissue inhibitor of metalloproteinase 3 (TIMP3), which is a common target of miRNA-221, -222 and -181b. Downregulation of TIMP3 enabled the growth of tamoxifen-resistant cells by alleviating its inhibitory effect on a disintegrin and metallopeptidase domain 10 (ADAM10) and ADAM17, which are essential for tamoxifen-resistant cell growth. Furthermore, the authors indicated that TIMP3 and the miRNAs may modulate EGF-induced MAPK and AKT phosphorylation. Based on these findings, the authors proposed that miRNA-221, -222 and -181b facilitate growth factor signaling in tamoxifen-resistant breast cancer by down-regulating TIMP3, and corresponding anti-miRNAs may be utilized to render these tumors responsive to tamoxifen , long-term estrogen-deprived (LTEDaro) and tamoxifen-resistant cell lines. Subsequently, the authors focused on miRNA-128a, which was hormone-responsive and differentially overexpressed in letrozole-resistant cell lines. Furthermore, TGF-β receptor 1 (TGFβR1) and SMAD2 were confirmed to be targets of miRNA-128a. The inhibition of endogenous miRNA-128a resulted in resensitization of the letrozole-resistant lines to the growth inhibitory effects of TGF-β  and tamoxifen indicates that the mechanisms of resistance to these endocrine therapy agents are not identical . Masri eof TGF-β .HER2Δ16  expression promotes endocrine resistance in HER2/ERα-positive breast tumors via the suppression of miRNA-15a/16, which targets BCL-2 . The autThe 14-3-3 family member and conserved protein, 14-3-3 ζ, is upregulated by tamoxifen, and this increased expression is correlated with early disease recurrence . A studyin vivo selection system was used against an miRNA library. ER-positive MCF-7 cells were initially infected with the pooled library and subsequently, the cells were orthotopically injected into female nude mice depleted of estrogen. Further studies highlighted the enrichment of miRNA-101, which alone is able to promote estrogen-independent growth and lead to tamoxifen resistance without targeting the ER. Finally, the direct suppression of membrane associated guanylate kinase (Magi-2) by miRNA-101 was demonstrated to provide a molecular link for miRNA-101-mediated activation of Akt through the suppression of PTEN activity. Given these findings, the authors suggested that miRNA-101 may serve as a biomarker for the subpopulation of ER-positive patients with breast cancer that are intrinsically resistant to tamoxifen  associated with clinical benefit and prolonged time to progression (TTP) in 235 patients with ER-positive tumors, who were administered tamoxifen as the first-line therapy for the treatment of metastatic disease . This inHowever, another study using global miRNA analysis was performed on 152 ER-positive primary tumors from high-risk patients with breast cancer, who had received adjuvant tamoxifen as monotherapy and half of which had developed distant recurrence. Based on the large sample size, there was no single miRNA profile that was predictive of the outcome following adjuvant tamoxifen treatment in a broad cohort of ER-positive patients with breast cancer .Regardless of the notable achievements in the previous studies concerned with the role of miRNAs in endocrine resistance . This process is theoretically similar for the downregulated miRNAs, and the restoration of these miRNAs may be another important therapeutic approach (available as miRNA mimics).Strategies for regulating miRNA expression in endocrine resistant patients include the normalization of deregulated miRNAs and targeting specific miRNAs that may allow for the prevention or resensitization of resistance to hormonal therapies. Antisense oligonucleotides have been shown to block the function of miRNAs in numerous studies –77. Nota tissues . These rwww.clinicaltrials.gov). However, before miRNAs become part of molecular therapies, further studies are required to determine their roles in patients, as the majority of the current findings are indirect, in vitro or both. With continuous research on the biological characteristics of miRNAs and the delivery systems, we propose that such strategies are likely to become available in the near future.At present, clinical trials that utilize the miRNA approach to overcome endocrine resistance are not yet available (7.Differentially expressed miRNAs of endocrine-resistant patients with breast cancer may serve as potential biomarkers for endocrine-resistant tumors. Furthermore, the determination of the target genes/pathways of deregulated miRNAs further enhances the understanding of the mechanism of endocrine resistance and facilitates the development of novel targeted therapeutic agents. In addition to altered expression, the miRNAs themselves may be predictive of drug resistance development and utilized as biomarkers in the personalized clinical management of breast cancer.Although significant progress has been made in clarifying the functional contribution of miRNAs to the modification of endocrine resistance in breast cancer, we are still in the infancy of our understanding. The limitations of the current studies are likely to be overcome by future continuous research via strategic selection and utilization of an already robust and expanding array of analytical tools. Further well-designed preclinical and clinical studies in this field are required. These scientific endeavors should lead to significant developments in the future management of endocrine resistance in patients with breast cancer. We propose that dynamic monitoring of circulating miRNAs through the course of endocrine therapy may facilitate the process.


Growth charts based on data collected in different populations and time periods are key tools to assess children’s linear growth. We analyzed the impact of geographic factors and the secular trend on height-for-age charts currently used in European populations, developed up-to-date European growth charts, and studied the effect of using different charts in a sample of growth retarded children.P<0.0001).In an international survey we obtained 18 unique national height-for-age charts from 28 European countries and compared them with charts from the World Health Organization (WHO), Euro-Growth reference, and Centers of Disease Control and Prevention (CDC). As an example, we obtained height data from 3,534 children with end-stage renal disease (ESRD) from 13 countries via the ESPN/ERA-EDTA registry, a patient group generally suffering from growth retardation. National growth charts showed a clear secular trend in height (mean height increased on average 0.6 cm/decade) and a North-South height gradient in Europe. For countries without a recent (>1990) national growth chart novel European growth charts were constructed from Northern and Southern European reference populations, reflecting geographic height differences in mean final height of 3.9 cm in boys and 3.8 cm in girls. Mean height SDS of 2- to 17-year-old ESRD patients calculated from recent national or derived European growth charts  was significantly lower than when using CDC or WHO growth charts  (Differences between height-for-age charts may reflect true population differences, but are also strongly affected by the secular trend in height. The choice of reference charts substantially affects the clinical decision whether a child is considered short-for-age. Therefore, we advocate using recent national or European height-for-age charts derived from recent national data when monitoring growth of healthy and diseased European children. Age- and sex-specific growth charts are essential clinical tools to monitor the adequacy of children’s longitudinal growth.The impact of the choice of growth charts is even more important to consider when analyzing longitudinal growth of children from different countries, a task of increasing importance with the emergence of multinational pediatric registries and clinical trials. We recently encountered this problem when studying longitudinal growth in European children with end-stage renal disease within the framework of the ESPN/ERA-EDTA Registry. Highly diverse populations, major regional variation in the tempo of growth, and the availability of several national growth charts of variable actuality in Europe illustrate the challenge of applying adequate reference methods to a heterogeneous population.In November 2010, we conducted a survey among pediatric centers in 32 European countries in order to obtain information on height-for-age charts used in clinical practice. A response was obtained from 28 countries (88%). Eighteen countries used a unique height-for-age chart based on data from their own country. The Euro-Growth reference included longitudinal growth data up to 36 months of age from a sample of twelve European countries. The WHO growth standards consist of longitudinal growth measurements from birth to 24 months, followed by cross-sectional data till the age of 5 years based on data from six countries around the globe.To replace older national height-for-age charts and to provide reference tables for those countries in which national growth charts are unavailable, we developed two sets of European reference charts for height accounting for the secular trend as well as for the North-South gradient in height. Data on pediatric end-stage renal disease patients were collected within the framework of the ESPN/ERA-EDTA Registry and included date of birth, sex, treatment modality  at start of renal replacement therapy, and changes in treatment modality. For the present study, we included patients younger than 18 years, who started renal replacement therapy during the period 1995–2010. The most recent height measurement available in the registry was included for: Belgium, Czech Republic, Finland, France, Italy (2–17 years old), Lithuania, Norway, Romania, Russia, Slovakia, Spain, Switzerland, and the United Kingdom.The most recent height measurement of each patient was converted to length/height-for-age standard deviation scores (SDS) using recent national growth charts whenever available. For countries lacking recent national charts SD scores were calculated using the European height-for-age charts constructed in this study. The mean SD scores for patients from separate European countries as well as for all countries combined were then compared with the length SDS calculated from WHO growth standards and Euro-Growth reference for 0–1 year olds and with height SDS according to CDC and WHO growth charts for 2–17 year old children. CDC growth charts in the 0–1 year old group were not included in the comparison since for this age group the use of the WHO growth standards is recommended. To determine whether children were short-for-age we used a length/height SDS cut-off value of <−2.All statistical analyses were carried out using SAS version 9.2 .The height distribution differed substantially between growth charts. Most national height-for-age charts showed higher mean heights than those according to the Euro-Growth reference and the WHO growth standards for one-year-old children, or according to CDC and WHO growth charts for 5, 10 and 18 year old children. Mean height differences among 1 year old children were only marginal and not significant.We tested whether these differences in mean heights were related to the secular trend in height by plotting the midyear of the survey in which growth chart height data were collected against the mean height for the four selected age groups . Recent Besides the secular trend in height, growth charts also varied due to differences in study design (in-and exclusion criteria) and due to modeling techniques used to construct the charts . BetweenP<0.05). To allow for this geographical height gradient and to correct for the secular trend we developed two separate height-for-age charts for Northern and Southern European children  based on CDC growth charts were considered eligible for subsidized rhGH treatment. Theoretically, 1% of the Australian children should meet this criterion. However, only 0.5% of the Australian children had a height SDS below the 1st percentile of the CDC growth charts. The issue raised here is not only of anthropological interest, but has also important clinical implications. Since clinical decision-making such as the indication for growth hormone or other growth promoting therapies Finally, in international growth or registry studies, when comparing linear growth data from different ethnic or geographic sources, international charts do not represent local growth appropriately due to population differences in height. Nevertheless, in these studies, the newly constructed height-for-age charts for Northern and Southern European populations might serve as appropriate reference charts because these better approach geographical height differences compared to one single international growth chart (e.g. CDC or WHO growth chart).A possible limitation of our study is that the overview of national growth charts in this study may be incomplete, as we only included those charts which are applied in pediatric nephrology practice, a discipline in which growth retardation is relatively common. The choice of national growth charts for studying height could be slightly different for other medical disciplines. Moreover, the European height-for-age charts were not smoothed using the standard LMS method As the original height-for-age charts on which our European charts are based did not provide information on gestational age and neither on linear growth of children from non-Western immigrants, our growth charts might not be applicable to small for gestational age babies or children from ethnic minorities living in Europe. However, several reference charts correcting for gestational age We found considerable differences in mean heights among different growth charts with height SDS in children differing depending on the reference chart used. The differences are likely due to the secular trend in height as well as to geographical differences in height. Therefore, we developed new height-for-age charts for Northern and Southern European countries. When monitoring longitudinal growth of European children, we propose to use recent national growth charts. However, if these are lacking we suggest that height-for-age charts for Northern and Southern Europe based on recent national charts, like ours, are preferable to other national or international height-for-age charts.Appendix S1A) Growth charts constructed for Northern European boys; B) Growth charts constructed for Northern European girls; C) Growth charts constructed for Southern European boys; D) Growth Charts constructed for Southern European girls.(DOC)Click here for additional data file.


The accumulation of senescent stromal cells in aging tissue changes the local microenvironment from normal to a state similar to chronic inflammation. This inflammatory microenvironment can stimulate the proliferation of epithelial cells containing DNA mutations which can ultimately lead to cancer. Using geriatric skin as a model, we demonstrated that senescent fibroblasts also alter how epithelial keratinocytes respond to genotoxic stress, due to the silencing of IGF-1 expression in geriatric fibroblasts. These data indicate that in addition to promoting epithelial tumor growth, senescent fibroblasts also can promote carcinogenic initiation. We hypothesized that commonly used therapeutic stromal wounding therapies can reduce the percentage of senescent fibroblasts and consequently prevent the formation of keratinocytes proliferating with DNA mutations following acute genotoxic (UVB) stress. Sun-protected skin on the lower back of geriatric human volunteers was wounded by dermabrasion and the skin was allowed to heal for three months. In geriatric skin, we found that dermabrasion wounding decreases the proportion of senescent fibroblasts found in geriatric dermis, increases the expression of IGF-1, and restores the appropriate UVB response to epidermal keratinocytes in geriatric skin. Therefore, dermal rejuvenation therapies may play a significant role in preventing the initiation of skin cancer in geriatric patients. Cancer is an age-dependent disease in most mammalian species; in humans over 50% of cancers are found in people over 70 years of age . RecentlNMSC has the highest incidence rate of all cancers worldwide, including an estimated 2 million newly diagnosed patients in the United States this year alone -12. Althafter the age of 18 [in vitro and in vivo [in vitro at the time of UVB-irradiation, surviving keratinocytes can continue to proliferate with the potential of converting the damaged DNA into initiating carcinogenic mutations [in vivo [The primary environmental factor that influences the development of skin cancer is exposure to the spectrum of ultraviolet wavelengths found in sunlight. Furthermore, as we age our chances of developing NMSC greatly increases so that at age 65 we have a 50% chance of acquiring a NMSC -16. In fge of 18 , indicatge of 18 . Recent ge of 18 -21 basedge of 18 -23. Thisge of 18 -26. In age of 18 . Previou in vivo . If the utations . Recent [in vivo . Because[in vivo . This neexogenous IGF-1 [endogenous IGF-1 levels and restore the appropriate UVB response in geriatric skin. We assayed whether ablation of both the epidermis and papillary dermis by dermabrasion could upregulate IGF-1 expression in geriatric skin and restore the appropriate UVB response. The successful development of the prophylactic therapies as described here could have a major impact on how NMSCs can be prevented in susceptible individuals.Given our findings that the lack of endogenous IGF-1  in geriaus IGF-1 , these sin vitro until they reach replicative senescence have historically been identified by their expression of senescence-associated β-galactosidase [in vivo.Normal human fibroblasts that are continually cultured tosidase . Similartosidase . Howevertosidase -32. To din vivo by increased expression of DDR markers . Dermabasion also reversed the aging-associated atrophy of the papillary dermis . The loss of senescent cells in dermabraded skin can also be observed by assaying for DDR markers. In contrast to the abundant expression of DDR markers in senescent fibroblasts of control geriatric dermis, DDR-positive fibroblasts are not detected in dermabraded geriatric dermis  of UVB. Twenty-four hours post-irradiation (the time required to clear TD lesions), the UVB-irradiated areas were biopsied and the basal layer keratinocytes were assayed for co-expression of proliferation (Ki67) markers and UVB-induced DNA damage  that the effectiveness of dermabrasion was due to the removal of previously initiated carcinogenic keratinocytes. However, if this hypothesis was true, other ablative procedures should be just as successful in treating NMSC, but they are not. Our studies suggest a new mechanism by which dermabrasion can prevent NMSC carcinogenesis which focuses on its effect on dermal fibroblasts. The accumulation of senescent fibroblasts in geriatric dermis alters the susceptibility of epidermal keratinocytes to accumulate and fix UVB-induced mutations in their genomes. Furthermore, senescent fibroblasts have been shown to provide an enhanced environment for the growth of carcinogenically initiated epithelial cells via their upregulation of inflammatory cytokines -24. TherGeriatric volunteers were recruited from patients treated at Indiana University dermatology clinics. These studies were approved by the Indiana University School of Medicine Institutional Review Board and subjects have signed approved consent forms. Specific requirements for inclusion/exclusion from these studies can be found in the 2 of UVB. In Fitzpatrick Skin Types I and II, this dose of UVB is sufficient to cause a minimal erythematous reaction. Permanent marker was used to outline the areas of skin that was irradiated. Twenty-four hours following UVB exposure, photographs were taken of the skin to document the extent of the UVB reaction. The irradiated skin, as well as unirradiated adjacent skin, was removed by punch biopsy, .Prior to treatment, a region of sun-protected hip/buttock skin was photographed. Next, an approximately 5 × 5 cm area of the subject's lower hip/buttock skin was isolated and anesthetized with xylocaine anesthesia. Under sterile conditions the localized area of skin was then abraded with sterile, coarse (#60) sandpaper down to the mid dermis, with complete removal of all epidermis and superficial dermis. The wounded area was bandaged with moist, occlusive dressings and the volunteer was instructed to change the dressing twice daily until the wound is re-epithelized in 1-2 weeks. Approximately three months later (˜Day 90 +/− 7 days) the volunteer returned to the clinic and a localized area 1 × 1 cm of either dermabrasion or untreated normal skin on the opposite hip/buttock was irradiated with dose of 350 J/mThe epidermal response to UVB irradiation was assayed as previously described . BrieflyStatistical analyses were done by two-tailed Student's t test. Statistical significance was defined as p < 0.05 unless otherwise noted in the figure legend.Four additional figures and specific protocols for quantitative reverse-transcription PCR, immunofluorescence, growth of human fibroblasts, and senescence-associated β-galactosidase assays can be found in the


Patients were eligible if they filled a prescription for latanoprost or travoprost-Z between October 2006 and Q2 2008 (prescription date = index date) AND were continuously enrolled 6 months prior through 12 months after the index date AND had any open-angle glaucoma or ocular hypertension diagnosis within 90 days prior to the index date AND did not have an ocular surface disease diagnosis during the 180 days prior to the index date AND if they had not had a prescription for the index agent in the 180 days prior to the index date. Time to incidence of new coding for ocular surface disease in the first year post-index was estimated with a composite endpoint: diagnosis of dry eye or ocular infection by ICD-9-CM or Current Procedural Terminology code OR by prescription for cyclosporine ophthalmic emulsion or ocular antibiotics.In all, 15,933 patients were treated with latanoprost and 7670 with travoprost-Z. Over 1 year, 4.3% of latanoprost and 4.5% of travoprost-Z patients were identified with dry eye (p = 0.28), and 10.9% and 11.1%, respectively, were identified with an ocular infection (p = 0.79). The 1-year incidence of new coding for ocular surface disease also was similar across treatments .The retrospective analysis of three large prescription databases revealed that open-angle glaucoma and ocular hypertension patients newly treated with latanoprost preserved with BAK or travoprost-Z preserved with SofZia did not differ statistically in rates of dry eye, ocular infection, or ocular surface disease (either dry eye or ocular infection) during the first year post-index. Claims-based analyses are limited by nonrandomization and the inability to account for over-the-counter use or samples. Ocular surface disease is an umbrella term that encompasses both dry eye disease  and non-dry eye disease  . A quest®, an ionic buffered system composed of boric acid, propylene glycol, sorbitol, and zinc chloride, found no statistically or clinically significant between-treatment differences with regard to ocular adverse events or tolerability; the prevalence of dry eye was 1.7% with travoprost-Z versus 2.0% with travoprost  codes and/or prescriptions.Condition identifiers are provided in Table The derivation of the analysis population is summarized in Table Frequencies of patients in the latanoprost and travoprost-Z groups diagnosed with dry eye, ocular infection, or ocular surface disease during 1 year of follow-up were tabulated separately and compared using Cochran-Mantel-Haenszel tests stratified by claims database and by indicator of condition. Kaplan-Meier and Cox proportional hazards survival analyses of time to occurrence of dry eye, ocular infection, or ocular surface disease were performed. The statistical significance of between-group differences in time to occurrence of each endpoint was assessed using the Cox proportional hazards model that included treatment, age, gender, Charlson Comorbidity Index , geograThe analysis set included 15,933 patients prescribed latanoprost and 7670 patients prescribed travoprost-Z. Patients prescribed the two prostaglandins were similar with regard to gender and geographic region; a larger proportion of latanoprost-treated patients was diagnosed with ocular hypertension, and, on average, those in the latanoprost group were older and had a higher score on the Charlson Comorbidity Index , and 10.9% and 11.1%, respectively, were identified with an ocular infection  and the European Pharmacopoeia ,21. SofZWhile preservative-free ocular hypotensive eye drops generally have been associated with fewer side effects -42 and bThe importance of determining the clinical impact of alternative preservatives in ocular hypotensive eye drops reflects the need to reduce the risk of ocular infection while limiting side-effects such as dry eye that might reduce patient adherence and persistence. Although we found no statistically significant difference in the 1-year incidence of ocular surface disease in more than 23,000 patients treated with either latanoprost or travoprost-Z, other studies -48 have As with all research, the present study has both strengths and limitations. Primary strengths include the large sample size. However, this was a retrospective analysis in which patients were not randomized, a basic limitation of all claims-based analyses. Patient use of over-the-counter products and samples as well as off-label use of topical corticosteroids to treat dry eye were not reflected in the database. The proportion of patients recommended to use artificial tears cannot be determined using a database analysis. Because this was a claims-based analysis rather than a clinical trial, we could not directly measure dry eye syndrome but instead measured the addition of a second code for one of many ocular surface diseases; we cannot estimate the frequency with which physicians omitted a relevant second code or patients added an artificial tear supplement without the knowledge of the physician. Identifying dry eye using CPT and ICD-9-CM codes may select for more severe cases of the condition. It is possible that the populations of patients using the two compounds could have been different since travoprost-Z has been specifically marketed and may have been chosen in some individuals who might already have mild, non-coded ocular surface disease prior to 180 days or who had a history of ocular surface symptoms that were not specifically coded for in the study. Not accounted for were any diagnosis that was made >180 days prior to the index date and that might not have been coded for as the subject developed glaucoma or the physician only coded for one, not two diseases which is quite common. In addition, although a glaucoma specialist (GFS) reviewed and approved the codes used to identify the clinical endpoints evaluated, other specialists might argue for the inclusion of other identifiers and/or the exclusion of some used herein; to our knowledge, there is no widely accepted set of identifiers for the general conditions of dry eye, ocular infection, or ocular surface disease.Results of the present retrospective analysis of three large prescription databases suggest that open-angle glaucoma and ocular hypotensive patients newly treated with latanoprost with BAK or with travoprost-Z containing SofZia do not differ statistically in rates of coding for dry eye, ocular infection, or ocular surface disease (either dry eye or ocular infection), during the first year post-index. Prospective, randomized, adequately powered comparisons of similarly effective ocular hypotensive agents with different preservatives are needed to more definitively answer the question of the clinical relevance of alternative preservative formulations.Dr. Schwartz was a paid consultant to Pfizer Inc in connection with the design and conduct of the study, including development of the manuscript. Dr. Fain, Dr. Mardekian, and Mr. Kotak are employees of Pfizer Inc. The study was supported by Pfizer Inc, New York, New York, USA.All authors participated in the study design, interpretation of data, and critical revision of the manuscript for important intellectual content, and all read and approved the final manuscript. SK provided study supervision, and JM facilitated acquisition and analysis of data from databases.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2415/11/14/prepub


Importantly, these studies demonstrated that DM, independent of the presence of CAD, leads to selective up-regulation of arginase-1. Arginase-1 seems to play an important role in limiting l-arginine availability in the close proximity of eNOS in vessels of DM patients. This brief review examines recent clinical studies demonstrating the pathological role of vascular arginase-1 in human diabetes. Whether arginase-1, which is crucial in the synthesis of various fundamental polyamines in the body, will represent a potent therapeutic target for prevention of DM-associated CAD is still debated.Coronary artery disease (CAD) remains the leading cause of death in the Western societies. Diabetes mellitus (DM) is one of the highly prevalent diseases, which remarkably accelerates the development of CAD. Experimental evidence indicates that decreased bioavailability of coronary endothelial nitric oxide (NO) contributes to the development of CAD in DM. There are recent studies showing that a selective impairment of NO synthesis occurs in coronary arteries of DM patients, which is mainly due to the limited availability of endothelial NO synthase (eNOS) precursor,  Diabetes mellitus is associated with an increased incidence of cardiovascular diseases accounting for significant morbidity and mortality in the diabetic population. Coronary artery disease (CAD) is one of the key manifestations of diabetes-associated vascular disease, a pathology, which predisposes diabetic patients to myocardial ischemia. The underlying mechanism(s) of CAD remain incompletely understood in human diabetes, so that effective preventive therapeutic strategies cannot be adopted in diabetic patients.The coronary flow-reserve, as defined by the ratio of coronary flow under maximal agonist-induced vasodilation to coronary flow under resting conditions, is reduced in diabetic patients, even in the absence of significant stenosis of epicardial coronary arteries . Nemes ePrevious studies have shown that animals with experimental insulin resistance and diabetes exhibit a reduced NO-mediated, agonist-induced dilation of cerebral, mesenteric, coronary, and skeletal muscle microvessels –16. Stud−). ONOO− has numerous detrimental effects in the cardiovascular system and plays a crucial role in the development of diabetes-induced vascular pathology  is essential for NO synthesis  precursor, l-arginine into the forearm of diabetic patients prevented ischemia reperfusion-induced endothelial dysfunction in the brachial artery  responsible for the reduced vascular availability of BH4 is not entirely understood in diabetes. It has been shown that ONOO− directly interacts and reduces the level of BH4, as it has greater affinity for BH4 than that of ascorbic acid and glutathione  on patients with non-obstructive coronary disease and found a markedly improved coronary vasodilator response to ACh  study, patients with type 2 diabetes had a significantly lower l-arginine availability than patients without diabetes (l-arginine treatment (8.3 g/day for 21 days) improved endothelial dilator function and increased insulin sensitivity in patients with type 2 diabetes , therefore the conclusion regarding the selective up-regulation of arginase-1 in coronary arteries of diabetic patients is severely limited. In a very recent study by Shemyakin et al. the independent impact of diabetes in affecting arginase-1 expression and its functional consequence was evaluated . Thus, the functional role for arginase-2 in the development of vasomotor dysfunction cannot be entirely excluded. In this regard, a previous study has found that increased expression of arginase-2 leads to decreased NO synthesis in pulmonary endothelial cells of patients with pulmonary arterial hypertension  leading to selective up-regulation of arginase-1 in coronary arterioles in diabetic patients remains elusive. One obvious possibility is the known action of insulin, which suppresses expression and activity of enzymes of urea synthesis pathway. Since insulin signaling is impaired in type 2 diabetes (patients likely to exhibit insulin resistance) it is possible that the failure of insulin regulatory action contributes to up-regulation of arginase-1. In the clinical setting diabetic patients are on insulin sensitizing and oral anti-diabetic medication or commonly take insulin. For instance, in the study by Shemyakin et al. 31 and 56% of patients with CAD plus diabetes were on insulin and biguanides/sulfonylureas, respectively . In our In summary, recent findings, as highlighted in this brief review underline the need for the investigations exploring the underlying mechanisms responsible for up-regulated arginase in human diabetes. Clinical studies emphasize the importance of those investigations that strive to elucidate the vascular effects of specific arginase inhibitors, including their long-term efficacy and safety in diabetic patients with CAD.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BCR-ABL quantification shows that a status of undetectable molecular residual disease (UMRD) is obtained in a significant minority of patients. However, it remains unclear whether these patients are definitively cured of their leukemia. Imatinib mesylate withdrawal trials have demonstrated the rapid appearance of the malignant clone in the majority of the patients whereas some patients remain in a state of UMRD. It has clearly been demonstrated that the most primitive stem cells are refractory to all TKIs used in clinical practice. In addition, long-term dormancy is one of the most fundamental characteristics of hematopoietic stem cells. In this context, we have recently undertaken a systematic analysis of the bone marrow stem cell compartment in several patients in durable UMRD. We have demonstrated the long-term persistence of a considerable amount of BCR-ABL-expressing stem cells, even in the absence of relapse. The phenomenon of long-term leukemic stem cell dormancy is of major importance in CML and one of the key questions in cancer biology in general. We discuss, here, the potential mechanisms, including intrinsic and microenvironmental factors, that control the response of leukemic stem cells (LSCs) to targeted therapies and potential novel strategies currently in progress with a curative intent. Moreover, we propose a molecular evaluation of the residual LSC compartment in selected patients in order to develop rational TKI-cessation strategies in CML.Targeted therapies of chronic myeloid leukemia (CML) using tyrosine kinase inhibitors (TKI) have profoundly changed the natural history of the disease with a major impact on survival. Molecular monitoring with  Chronic myeloid leukemia (CML) is a major model of oncogenesis involving primitive hematopoietic stem cells (HSCs). A unique genetic alteration, the t9;22)q34;q11) translocation that gives rise to the Philadelphia chromosome (Ph1) generates the BCR-ABL fusion oncoprotein with deregulated tyrosine kinase (TK) activity [1 translo2q34;q11)During the last three decades, substantial progress has been accomplished in the therapy of the disease. Allogeneic stem cell transplantation (SCT) was the first potentially curative therapy for CML patients in chronic phase . HoweverOver the last ten years, small compounds such as tyrosine kinase inhibitors (TKIs) have been developed for targeted therapy of CML . The intin vivo persistence of leukemic stem cells (LSCs).In addition to mechanistic aspects of TKI-resistance due to the structure of the target oncoprotein, a novel level of complexity, inherent to the hierarchical nature of CML stem cells has been identified. This refers to the resistance of the most primitive HSCs to all TKIs currently used, because of three principal causes: their quiescence , the higBCR-ABL-expressing hematopoietic stem cells in the context of anti-CML treatment. We analyze the question of the importance of LSCs in the maintenance of the leukemia and the development of resistance to anti-leukemia therapies. Finally, we discuss the notion of LSC persistency and the relationship between the concept of cure and the requirement of targeting leukemic stem cells.In the present research perspective, we focus on BCR and ABL genes were identical in each cell lineage of a same patient strengthened this concept. Although the involvement of T-cells has been rarely reported in CML, the occurrence of T-lymphoid blast crisis has been clearly documented [BCR-ABL gene transfer experiments into both murine and human HSCs suggested that the expression of this oncogene was sufficient to initiate CML [CML was the first malignancy in which the concept of single primitive LSC origin was demonstrated using X-chromosome inactivation studies . The fincumented , suggestcumented ,25 but ncumented . Finallyiate CML .BCR-ABL does not impede the mature cell production is not clear at the present time. However, the unperturbed C/EBP (CAAT/enhancer binding protein)-alpha expression in chronic phase might be involved in this event [in vitro behavior as their cell surface markers are very similar to normal HSCs, which are enriched in cell populations sorted by CD34+CD38−CD71−, and/or Thy1+ markers [As for normal hematopoietic stem cells, CML stem cells (in the chronic phase of the disorder) have the ability to self-renew and to differentiate in committed progenitors, which produce the mature hematopoietic cells. Why and how the expression of is event . CML ste markers . Classic markers  and even markers .in vitro as compared to normal cells was shown by LTC-IC experiments [Although the expansion of the myeloid cell compartment occurs in the marrow, CFU-C activity is highly increased in peripheral blood at diagnosis . A defeceriments . Thus, teriments . Analyseeriments ,36. Theseriments  undoubteLong-term preservation of a normal hematopoiesis is based on the crucial property of HSCs to escape from the cell cycle and to remain quiescent in the bone marrow microenvironment (or niche). The quiescence phenomenon refers to a reversible cell cycle arrest (G0 phase), whereas dormancy concept represents the metabolic state of quiescent cells. Although not exactly synonymous, quiescence and dormancy are now used indifferently in human biology. One of the major characteristics of CML stem cells is the presence of highly quiescent primitive stem cells, which can be present at diagnosis in peripheral blood . The majInteractions between receptors expressed by HSCs and ligands produced by marrow niches have been extensively studied . The ostBCR-ABL-expressing CML cells secreted high levels of osteopontin, a component of the hematopoietic niche capable to induce HSC quiescence [in vitro, this effects being reversed by the use of IM [BCRABL-expressing cell line model, it has been suggested that the expression of CXCR4 under the influence of IM, could generate a migration of the cells escaping apoptosis, towards bone marrow niches, this “homing” inducing quiescence and CML stem cell survival [In CML, the BCR-ABL oncoprotein decreases the adhesion of LSCs to the bone marrow environment through constitutive phosphorylation of several focal adhesion-associated molecules . Howeveriescence   but the notion of intrinsic abnormalities of the niche in CML has not been extensively studied. Despite the fact they are not a part of the malignant Ph1 clone, MSCs from CML patients are abnormal and secrete large amounts of PlFG , which promotes marrow angiogenesis and CML proliferation . Therefond generation inhibitors) demonstrated great efficacy in most chronic phase CML patients, data from first line IM trials show that 15% of patients resist to the therapy [in vitro and in vivo as observed in IM-cessation trials.Although TKI therapy  in CD34+CD38− cell populations and their LTC-IC-derived progeny [+ cells from CML patients using subcloning-PCR [Early clinical observations in CML patients have suggested that ABL-TK mutations can occur in Ph1 stem cells. We have demonstrated,  progeny . These rning-PCR . Consequning-PCR . We cleaning-PCR .Overall, these data suggest that primitive quiescent CML stem cells (with or without ABL-K mutations) are protected from targeted therapies under the influence of cell-autonomous pathways or external cues such as niche-driven mechanisms, leading to the accumulation of a genuine leukemic reservoir. In addition, recent data concerning the “oncogene-independence” of these cells suggest that TKIs alone, although extremely efficient in the bulk of the disease burden, will not theoretically lead to a complete eradication of the disease. The current development of strategies targeting CML leukemic stem cells using specific pathways is based on this concept.Many signaling pathways are involved in the basic properties of HSCs, some of them could be specific of LSCs. Consequently they represent a target for potential therapy against CML stem cells ,67.The canonical inter-connected Wnt/β-catenin, Sonic Hedgehog and Notch signaling pathways play crucial roles in the maintenance of stem cell functions. Wnt/β-catenin pathway is required for survival and long-term renewal of CML stem cells ,69. In tIn vitro, the suppression of AHI-1 over-expression was related to a reduction of LSC growth autonomy. Moreover, AHI-1 was found to interact not only with BCR-ABL but also with the JAK2/STAT5 pathway. In another field, it has been shown that the inhibition of the Hsp90 protein (a chaperone for BCR-ABL) could target CML stem cell [Arachidonate 5-lipoxygenase (Alox5) has been shown to be indispensable for the survival, proliferation and differentiation of CML stem cells. In addition, the inhibition of Alox5 specifically impairs LSC functions and does not affect normal HSCs . The AHItem cell . Finallytem cell ,80. Howetem cell .BCR-ABL mRNA transcripts (assessment of the molecular response). International recommendations have determined the appropriate periods of testing in order to ascertain the status of the response  [The hematological follow-up of CML patients on TKI treatment is classically based on blood cell counts (estimation of the hematologic response), cytogenetic analysis by the determination of the percentage of Ph1-positive metaphases  and quantification of arnings) . Moreovearnings) .BCR-ABL mRNA transcripts. Laboratory protocols are now properly standardized [BCR-ABL/“control gene” ratio is determined with usually ABL as the control gene. The main objective of these collaborative studies was to establish a concordant major molecular response (MMR) value corresponding to a BCR-ABL/ABL ratio of 0.1%. Moreover, for TKI good responders, RT-qPCR remains the ultimate test to follow the residual disease. It must be underlined that the limit of detection of BCR-ABL quantification protocols is around 10−5 (BCR-ABL/ABL ratio ~ 0.001%). The regular quantification of the BCR-ABL mRNA transcript level, evaluated by a BCR-ABL/ABL ratio every 3 months allows the determination of kinetics of CML response throughout the disease monitoring.RT-qPCR is the most sensitive test for the quantification of dardized -86 and idardized . A BCR-ABCR-ABL/ABL ratios, the estimated total-body leukemic cell burden, the CCyR, and the MMR . It is noticeable that the non-detection of BCR-ABL mRNA transcript does not mean the complete eradication of leukemic cells since up to one million CML hematopoietic cells could persist in the patient's body in the UMRD context.A schematic representation of decreasing CML residual disease has been proposed . It inteR Figure . The absBCRABL mRNA transcripts determined during molecular monitoring of CML patients. For most patients responding to imatinib, a biphasic BCR-ABL/ABL exponential decrease is typically observed. Mathematical modeling of such observations related to the hematopoietic differentiation process (from hematopoietic stem cells to mature cells) has been elaborated [BCRABL mRNA decline curves (3-4 log reduction) could be consistent with the eradication of differentiated mature CML cells (fast slope) and leukemic progenitors (slow slope). Therefore, residual leukemic cells in the UMRD context are likely to correspond to CML stem cells. Using the same model, it was recently suggested that sustained TKI therapy might reduce (in part and in some cases) the leukemic stem cell burden [These models are based on the kinetics of aborated ,90. The l burden . In addil burden , the eral burden ,93, and l burden . HoweverData from patients treated with allogeneic SCT, which is considered as the only “curative” therapy of CML, show that late relapses can occur up to 15 years after transplants. An unbalanced relationship between LSCs and graft versus leukemia effect, which might be operational in a fraction of LSC, could be the cause of late relapses . ConcernBCR-ABL-expressing LSC was not evaluated in these early studies. It is important to note that, before the TKI era, IFN-α therapy has induced in a few patients a prolonged CCyR that have persisted after the discontinuation of the treatment [in vivo mouse models are suggestive of its potential to activate quiescent hematopoietic stem cells [For IFN-α therapy, clinical experiences have shown that a minority of CML patients achieved a long-term period of CCyR. In this population, it has been reported that some patients who stopped their treatment maintained a durable CCyR . In thisreatment . Thus, sreatment . Furtherem cells . A two-sem cells .in vivo existence of LSCs believed to be responsible for the molecular relapse, but also address the issue of whether the patients who did not relapse are definitely cured or not.More recently, several clinical pilot studies of imatinib cessation for patients who achieved sustained UMRD have been published -107. OveBCR-ABL mRNA transcript in the peripheral blood by RT-qPCR and/or nested RT-PCR [BCRABL rearrangement in some patients in UMRD after IM therapy or SCT demonstrated the presence of residual leukemic cells not detected by RT-PCR experiments [BCR-ABL positive DNA was detected in peripheral blood. In addition, the stem cell compartment was not analyzed. Moreover, RT-qPCR experiments on purified sorted cells from CML patients treated with imatinib showed the elimination of CML progenitors within one year but the retention of the LSC population [UMRD or complete molecular remission have been defined as non-detection of d RT-PCR . This aseriments ,109. Thepulation .BCR-ABL rearrangement could persist unexpressed in dormant cells [in situ hybridization (FISH) on CD34+ fraction [+ CD38− cells [Ten years ago, , we postulated that the nt cells . The pernt cells . More rent cells -115. Thefraction ,114, CD38− cells  populatiBCR-ABL-expressing LSCs persist in these populations has been unexplored so far. We have analyzed the presence of BCR-ABL-expressing hematopoietic stem cells in bone marrow samples from six CML patients with undetectable BCR-ABL transcript in their peripheral blood for more than 3 years [BCR-ABL mRNA by RT-qPCR and controlled by nested RT-PCR. Using this strategy, we detected a variable percentage of BCR-ABL-expressing stem cells in all patients analyzed. Interestingly, these primitive HSCs did not produce any detectable BCR-ABL transcript in the peripheral blood, suggestive of their quiescent status in vivo, even in patients in whom the anti-leukemic therapy had been stopped for > 11 years. It is noticeable that a greater percentage of BCR-ABL-expressing stem cells was observed in patients previously treated by IFN-α alone as compared with those treated by IM after IFN-α failure. These data could be indicative of a more effective eradication of the stem cell compartment in the latter case, but they must be confirmed on a larger series of patients. Finally, the major involvement of marrow LSCs in these patients  suggest the presence of a highly active inhibitory process in the bone marrow niches, maintaining these stem cells in a long-term dormancy state.The UMRD status can now be obtained in CML patients treated with TKI and, have also been observed in historical cohorts, in a minority of patients treated with IFN-α. The question of whether  3 years . The exp 3 years ,116. AppTKI therapy has dramatically improved the overall survival in CML and a minority of patients achieved a sustained state of UMRD. In this context, for clinicians, patients and socio-economical reasons, the recurrent question remains the discontinuation of TKI therapies. Clinical data show that half of the patients will develop a molecular relapse within a few months after imatinib cessation. Despite the fact that the decision of IM-discontinuation was done without the assessment of the LSC content in these patients, the leukemic stem cells persistency with active stem cells is highly probable in these patients. However, in the remaining 40% of patients who did not relapse, the evaluation of the LSC persistency might be of major interest. Consequently, in the context of molecular remission, the characterization of LSCs by functional assays could be considered in clinical practice. Overall, these data raise some critical questions.BCR-ABL mRNA transcripts in CFU-Cs and LTC-ICs by RT-qPCR that appears more adapted than FISH to analyze large amount of hematopoietic colonies. To ensure the reliability of the test, RT-qPCR experiments have to be carefully controlled at each step of the amplification process (presence of positive and negative controls). However, the method can underestimate the number of BCR-ABL-expressing stem cells because of to the small number of hematopoietic cells analyzed  and a variable transcription of BCR-ABL in Ph1 hematopoietic progenitors [BCR-ABL-expressing progenitors). In the presence of negative CFU-C and LTC-IC assays, in vivo xenogeneic transplantations in immunodeficient NOG (NOD/Shi-scid/IL-2Rγnull) mice can be considered. In the light of clinical data and stem cell tests, it remains to be established whether the persistency of residual BCRABL-expressing stem cells (cycling or quiescent) in CML patients in sustained UMRD represents or not a potential risk of disease recurrence.We have developed a screening procedure based on the detection of genitors . To be mBCR-ABL-expressing clonogenic and LTC-IC-derived progenitors in patients with sustained UMRD might confirm the presence of a significant quiescent CML stem cell compartment. Moreover, it has been shown that the most primitive LSCs are intrinsically refractory to all TKIs used in clinical practice. In addition, TKIs might cause an osteogenic niche homing of LSCs increasing the quiescent CML stem cell burden. In this context, the use of IFN-α (to awake dormant LSCs) could be of interest. Conversely, some studies hypothesize that long-term TKI therapy  could significantly reduce the malignant stem cell compartment. It has also been reported that it was not necessary to kill the last leukemic cell [Before discussing this issue, we have to precise the notion of cure in CML. We can distinguish the “clinical cure” defined by the absence of relapse from the “biological cure” defined by the absence of leukemic cells (including LSCs). This assertion is supported by our data showing that some patients who appeared clinically “cured” had persistent LSCs. The detection of mic cell . NeverthTheoretically, to eradicate a leukemic process, it is mandatory to eliminate the last leukemic stem cell; this might be the definitive objective in CML . NowadayBCR-ABL-expressing stem cells in CML patients with sustained UMRD induced by 2nd generation TKIs must be performed, as these drugs generate molecular responses more rapidly than imatinib.Despite the great success of TKI therapies, many points have to be clarified such as the mechanisms of long-term leukemic stem cell dormancy, the role of an immunomodulatory effect of the treatments, the relation between CML stem cells and the bone marrow microenvironment. Moreover, the analysis of