ESR1 and EGF genes affects breast cancer risk, tumour characteristics or breast cancer survival.Oestrogen exposure is a central factor in the development of breast cancer. Oestrogen receptor alpha (ESR1) is the main mediator of oestrogen effect in breast epithelia and has also been shown to be activated by epidermal growth factor (EGF). We sought to determine if common genetic variation in the ESR1 and 54 SNPs in EGF in 92 Swedish controls and selected haplotype tagging SNPs (tagSNPs) that could predict both single SNP and haplotype variation in the genes with an R2 of at least 0.8. The tagSNPs were genotyped in 1,590 breast cancer cases and 1,518 controls, and their association with breast cancer risk, tumour characteristics and survival were assessed using unconditional logistic regression models, Cox proportional hazard models and haplotype analysis.We genotyped 157 single nucleotide polymorphisms (SNPs) in ESR1 (between rs3003925 and rs2144025) for association with breast cancer risk (p = 0.001), but the result did not withstand adjustment for multiple comparisons (p = 0.086). A similar region was also implicated by haplotype analyses, but its significance needs to be verified by follow-up analysis.The single tagSNP analysis did not reveal association evidence for breast cancer risk, tumour characteristics, or survival. A multi-locus analysis of five adjacent tagSNPs suggested a region in ESR1 and EGF genes and breast cancer risk, tumour characteristics or survival.Our results do not support a strong association between common variants in the  ESR1 (MIM 133430) and EGF (MIM 131530) genes affecting the function or expression of their respective proteins could thus potentially affect the risk of developing breast cancer, characteristics of the tumour or the risk of dying from the disease.Breast cancer is the most common cancer in women overall worldwide. Oestrogen exposure is a central factor in the development and progression of this cancer -3 and itESR1 gene, yet none have previously been investigated in the EGF gene. As far as we are aware, no attempt to capture the common genetic variation in the ESR1 gene in its entirety has yet been published. One group, who genotyped 17 SNPs in the ESR1 gene, found a decreased risk of breast cancer for carriers of three common haplotypes in the gene and an increased risk for carriers of one common haplotype  + 1 × nodal stage  + 1 × grade  Nodal stage was defined as 1 if there were no lymph node metastases, 2 for a total of 1–3 metastatic nodes, and 3 for more than 3 metastatic nodes. A tumour of high differentiation was assigned grade 1, a tumour of intermediate differentiation grade 2, and a low differentiated tumour was assigned grade 3. We categorised the NPI into two groups: ≤ 4 or > 4. Four is the mean NPI value of the present study. It has also been shown that breast cancer survival decreases rapidly for NPI above 4 .ESR1 and EGF tagSNPs and risk of breast cancer  or the NPI . Adjusting for age (in 5-year age groups) did not affect our results. We estimated the hazard ratio of death due to breast cancer in relation to the genes' tagSNP using Cox proportional hazards models. The tagSNPs were included as covariates in the models either one at a time or in groups of five (codominant main effects only). The latter method was used for detection of association with haplotypes. Although it does not require resolution of gametic phase, tests based on such models can be powerful within regions of strong LD [et al. [We applied unconditional logistic regression models for assessing the association between trong LD . Likelihtrong LD  and taketrong LD , combinitrong LD  that clu [et al. .et al. [et al. [To estimate power in the risk component of the study, we used a method described by Chapman et al. , which aet al.  in a sim [et al. . AnalyseTable More case-related information has been provided in our previous work . The breESR1 and EGF genes are summarised in Table ESR1 and EGF genes were genotyped in 92 randomly selected controls (Supplementary Table 1 (ESR1) and Supplementary Table 2 (EGF) in Additional File ESR1) and 2 (EGF) in Additional File The genotyping results and SNP coverage in the ESR1 and 15 tagSNPs in EGF that could predict the included SNPs and their haplotypes with an R2 of at least 0.8. The tagSNPs were genotyped in all cases and controls  did not reveal any association with breast cancer risk, NPI or breast cancer survival whose statistical significance withstood multiple testing correction. The strongest signal of association with breast cancer risk was obtained by the window analysis including TAGs 26–30 . Within the region, there were seven common haplotypes that accounted for 92% of the chromosomes. Including the expected dosages of common haplotypes and the rare haplotypes (combined into a single variable) as covariates in a logistic regression model with the most common haplotype as reference, gave a global p (likelihood ratio test) of 0.0493 in relation to breast cancer risk , we first tested the association of each tagSNP and then performed a haplotype analysis using a logistic regression sliding-window approach where five adjacent tagSNPs were analysed together (without resolution of gametic phase). The results are summarised in Figure We also explored a sliding-window analysis of haplotypes using a variable window size. Three haplotypes within the region from tagSNP 18 to tagSNP 27 were implicated, showing frequency differences between cases and controls Table . The sigAlso, an analysis within groups of diabetes mellitus, menopausal hormone use or family history furthermore did not reveal any significant evidence for any tagSNP to be associated with breast cancer risk (data not shown).EGF showed association with breast cancer risk, NPI, or breast cancer survival that withstood multiple testing correction  and TAG30 (rs2144025) in the ESR1 gene showed a signal for association with breast cancer risk in the multi-locus analysis of five adjacent tagSNPs, but the result did not withstand multiple testing correction. Interestingly, the suggestive evidence from further haplotype analyses converge to this region, but its significance needs to be determined by follow-up analysis. None of the genotyped SNPs within this region were located in exons  and are thus unlikely to affect ESR1 protein structure. It is still a possibility however that the SNPs themselves, or one or more SNPs in LD with any of the SNPs, may effect the regulation of ESR1 protein expression. In fact, it has been shown that ESR1 protein overexpression is common in breast cancer [EGF gene did not appear to affect the risk of developing breast cancer, developing a tumour with high NPI, or dying from the disease.We had comprehensive SNP coverage of the entire t cancer . Common ESR1 or EGF genetic variation, as it depended on the inability of the respective pathology department to retrieve the samples. The tissue sample availability was therefore random and could not have lead to selection bias. The main concern is that the non-participation of a small number of deceased cases might have reduced the power of our study, especially for the survival analysis. Furthermore, a problem might have arisen since we were not able to genotype seven tagSNPs in ESR1 and one tagSNP in EGF in the tissue samples. If these eight tagSNPs were in fact associated with severe disease, the association with risk of breast cancer death might have been biased towards null in our study since we did not genotype all the severe cases. The fact that the results were not different when we restricted our analyses to the most severe cases among those who donated blood samples indicates that the eight tagSNPs were unlikely to be associated with severe disease.Our study was a well designed, population-based case-control study. Case ascertainment and case survival status were established using the nationwide, high-quality Cancer Registry and Causes of Death Registry in Sweden. Exposure status of the participants was determined using genotyping methods with low error rates from which all results underwent detailed quality control. We sought to obtain tissue samples from the deceased cases and those cases that had declined donation of a blood sample, and were able to obtain the majority of the samples requested. The relative minor lack of tissue accessibility is unlikely to be related to our exposure, In the selection stage of our study, we oversampled cases and controls that were long-term users of menopausal hormones and those that had self-reported diabetes mellitus. In the case of an association between the tagSNPs under study and menopausal hormone use or diabetes mellitus, this oversampling might have caused us to detect an artificial association between the tagSNPs and breast cancer risk. We therefore assessed if the tagSNPs were associated with menopausal hormone use or diabetes mellitus. We found no connection between the factors and conclude that the oversampling is unlikely to have posed a problem in our study.ESR1 gene and breast cancer risk have included only a few polymorphisms in the gene. One study, however, genotyped 17 common SNPs in the ESR1 gene and found three haplotypes to decrease breast cancer risk and one haplotype that increased the risk [ESR1 we found to be associated with breast cancer risk. Two of the haplotypes that showed a protective effect against breast cancer risk (H4 and H6) carried our TAG21  [Most previous publications regarding the the risk . None of H4 and H carried PvuII , XbaI , codon 243  and codon 325  variants are among the most commonly studied polymorphisms in the ESR1 gene. The first two have been suggested in a couple of studies to decrease the risk of endometrial cancer [PvuII might affect breast cancer survival depending on oestrogen receptor status of the tumour [The l cancer  and PvuIe tumour , but no e tumour ,28-37. WESR1 and EGF genes in relation to breast cancer risk, tumour characteristics and breast cancer survival using a comprehensive haplotype tagging analysis. To our knowledge, this is the first systematic association study of these two genes for breast cancer susceptibility and prognosis. We located a region in ESR1 which showed a moderate signal for association with breast cancer risk, but were unable to link common variation in the EGF gene with breast cancer aetiology or prognosis.We analysed common genetic variation in the EGF = epidermal growth factor; ESR1 = (o)estrogen receptor 1; HWE = Hardy-Weinberg equilibrium; LD = linkage disequilibrium; MAF = minor allele frequency; NPI = Nottingham Prognostic Index; PLEM = partition ligation expectation maximisation; SNP = single nucleotide polymorphism; tagSNP = tagging single nucleotide polymorphism; TNM = tumour, nodes, metastasis.The authors declare that they have no competing interests.KE, KC, SW, ETL, PH, KH and JL were involved in planning the study. YLL, YQL and CB administrated the genotyping analysis. KE, HD, YL, AS, KH and JL performed the statistical analysis. KE, KH, PH and JL drafted the manuscript, and all the authors approved the manuscript.Additional file Click here for file


Breast cancers that overexpress the human epidermal growth factor receptor 2 (HER2) are eligible for effective biologically targeted therapies, such as trastuzumab. However, accurately determining HER2 overexpression, especially in immunohistochemically equivocal cases, remains a challenge. Manual analysis of HER2 expression is dependent on the assessment of membrane staining as well as comparisons with positive controls. In spite of the strides that have been made to standardize the assessment process, intra- and inter-observer discrepancies in scoring is not uncommon. In this manuscript we describe a pathologist assisted, computer-based continuous scoring approach for increasing the precision and reproducibility of assessing imaged breast tissue specimens.Computer-assisted analysis on HER2 IHC is compared with manual scoring and fluorescence in situ hybridization results on a test set of 99 digitally imaged breast cancer cases enriched with equivocally scored (2+) cases. Image features are generated based on the staining profile of the positive control tissue and pixels delineated by a newly developed Membrane Isolation Algorithm. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis.A computer-aided diagnostic approach has been developed using a membrane isolation algorithm and quantitative use of positive immunostaining controls. By incorporating internal positive controls into feature analysis a greater Area Under the Curve (AUC) in ROC analysis was achieved than feature analysis without positive controls. Evaluation of HER2 immunostaining that utilized membrane pixels, controls, and percent area stained showed significantly greater AUC than manual scoring, and significantly less false positive rate when used to evaluate immunohistochemically equivocal cases.It has been shown that by incorporating both a membrane isolation algorithm and analysis of known positive controls a computer-assisted diagnostic algorithm was developed that can reproducibly score HER2 status in IHC stained clinical breast cancer specimens. For equivocal scoring cases, this approach performed better than standard manual evaluation as assessed by ROC analysis in our test samples. Finally, there exists potential for utilizing image-analysis techniques for improving HER2 scoring at the immunohistochemically equivocal range. The human epidermal growth factor receptor 2 (HER2) gene on chromosome 17 is amplified in 20% to 30% of breast cancer patients . MoreoveGiven the clinical impact of HER2 overexpression, there has recently been a heightened emphasis on reliably assessing HER2 status in breast cancers -9. GuideIn light of the pressing need for accurate determination of HER2 overexpression, the authors of the above ASCO/CAP study have acknowledged the use of image analysis as a viable approach for assessment. Previous work with image analysis systems have focused on the validation of commercially available technology in the clinic by demonstrating greater accuracy in quantification of HER2 immunohistochemistry when compared to manual interpretation ,11-13, aFurthermore, the cost of performing the analysis may actually serve as a barrier to mainstream adoption of this technology in practice. Skaland et. al. has contributed valiantly in this realm by using readily available (and free) NIH image processing software to analyze images of HER2 stained breast cancer specimens . HoweverIn this manuscript, we describe the design, development and evaluation of a new suite of algorithms to assist in increasing the precision (i.e. number of scoring levels) at which HER2 immunostains are quantified. We provide a detailed description of the workflow and associated processes that are used to evaluate HER2 stained breast slides and compute the HER2 expression score. We describe three clinically based image features used in our system and compare their performance with manual scoring techniques in assessing specimens. The performance results of the computer-assisted quantification algorithms based on the newly developed image features are then compared with manual scoring using Receiver Operator Characteristic (ROC) analysis. In addition, the adequacy of the quantification algorithm is crucially examined in the equivocal scoring ranges in order to demonstrate its applicability in the laboratory. Finally, we discuss the capacity of the system to be optimally calibrated for the specific conditions of the given laboratory. By increasing the precision with which HER2 immunohistochemistry is quantified, we anticipate that breast cancer patients who might benefit from anti-HER2 therapy will be accurately identified, while the remaining subpopulation of patients will be spared a costly and potentially harmful treatment.In order to design a clinically relevant computer-based quantification scheme, we first begin by examining the current methods that are used routinely in the laboratory. Briefly, the pathologist focuses on two primary features to arrive at a HER2 score: the quality of membrane staining (since HER2 is a receptor functioning at the cytoplasmic membrane) and the percent of invasive tumour cells stained Table . In thisThe workflow of the quantification scheme is outlined in Figure The specimens used in these studies consisted of 99 breast cancer cases that had been diagnosed between January 2005 and March 2007 and stored in archives at the Department of Pathology and Laboratory Medicine at Robert Wood Johnson University Hospital, New Brunswick, and N.J. During this period of time it was standard procedure for both assays to be performed. Cases which had received an IHC score of 0 or 1+ were limited in order to enrich the amount of 2+ cases  to approximately 25% of the data. The purpose of this experimental design was that since 2+ cases are considered equivocal, they represent the patient population that would tend to benefit most from the use of computer aided quantification. Specimens with significant mechanical crushing and sectioning artifacts were omitted from the study to arrive at the final 99 cases. Both invasive ductal and lobular carcinomas were included in the experiments.Immunohistochemical staining was performed at the Department of Pathology and Laboratory Medicine at Robert Wood Johnson University Hospital utilizing an automated immunostainer, Ventana BenchMark IHC/ISH system . Ventana PATHWAY HER-2 (clone CB-11) was used for the primary detection of c-erbB-2 antigen in sections of formalin fixed, paraffin embedded tissue. A known 3+, FISH positive control was fixed on each slide along with the patient sample in order to analyze both under identical staining conditions.Manual immunohistochemical grading was performed by a board certified surgical pathologist (P.J.) at Robert Wood Johnson University Hospital according tithe Scoring Guide for the Interpretation of Ventana Pathway HER2 Staining of Breast Carcinomas . BrieflySpecimens were sent to Genzyme Genetics  for FISH analysis and the ratio of discrete signals for the HER2 gene and centromere probe for chromosome 17 (CEP17) was reported. A ratio of HER2 gene/CEP17 ≥ 2.3 was considered positive for HER2 overexpression.A board certified pathologist (P.J.) delineated the region of invasive carcinoma, and then a resident pathologist (M.I.) used a robotic microscope to digitally acquire images at 20× magnification within the specified boundaries. The control from each slide was also digitized at 20× magnification. All images are taken with an Olympus AX70 microscope  equipped with a Prior six-way robotic stage and motorized turret  and Olympus DC330 720-line 3-Chip video camera at 1360 × 1024 pixel resolution and a fixed exposure time of 1/600 s. This allows the digital images to be captured through red, green, and blue channels, and thus, span the entire visible range.The DAB HER2) stained regions were isolated from the hematoxylin counterstain in the digital image of the patient sample and control images through the use of color decomposition  computer with 1.0 GB memory was used for processing each image. The color decomposition algorithm was implemented in Java. All other processing was implemented utilizing Matlab  code. The color decomposition and membrane isolation together took approximately 10–20 seconds per image to complete.Receiver Operator Characteristic curve analysis was used to compare the accuracy of the manual and automated computerized methods for grading IHC. FISH assays were used as an independent means for assessing IHC scores. The area -under- the -curve (AUC) was calculated for each ROC curve utilizing the trapezoidal rule  in orderInstitutional Review Board approval was obtained for this retrospective study through protocol (IRB #5381).Table Two examples of HER2 stained images Fig.  are showMn feature which specifically takes into account the intensity of the positive control.Figure Mn, FISH, and standard manual scoring. It is shown that the image-based system is capable of delineating two groups of patients: those with high Mn scores and gene amplification, and those with low Mn scores and no amplification. The classification of several cases has improved with computer-assisted image analysis (arrows). When image analysis was performed, four IHC 3+ cases (but FISH-) are clustered with the non-amplified group, while a 2+ case with genetic amplification is grouped with other FISH positive cases. Also, it is important to note that, the Mn score for some cases was computed to be beyond a ratio of one, indicating that these patient tissues stained darker than the control tissue, which possibly suggests a greater concentration of HER2 protein than the control tissue.Figure Mn and Mp features with that of manual scoring  is significantly greater than the Mp curve , indicating that quantitatively utilizing the control provides a better diagnostic test than analyzing only patient tissue. When the AUC of the Mn curve was compared to that of manual assessment, no significant difference was found .In order to quantitatively compare these scoring systems, a ROC curve was generated comparing the diagnostic reliability of the ing Fig. . The optMn score was similar to that of manual scoring. Consequently, the percent stained area was incorporated into the Ma feature through an addition of the d/N coefficient. The Ma ROC curve (red line) is compared to the ROC curve generated from manual scoring . More importantly, the difference between the two scoring systems is most pronounced at the clinically crucial point: the equivalent score of 2+. The arrow , note how the computer-assisted scoring system can significantly reduce false positive rates (arrows) for IHC = 2+ cases (p < .05), as Ventana scoring guidelines considers all 2+ cases weakly positive.Lastly, these computer-assisted scoring methods can be engineered to perform optimally on equivocal cases. Fig. 64% of the time, whereas manual scoring was only able to correctly classify 23% of the cases . There was a reduction false positive rate (percentage of FISH negative cases considered FISH positive) from 100% to 31%, as 100% of manually scored 2+ cases are considered by Ventana scoring guidelines to be positive. Lastly, the false negative rate was only 4%.To estimate a robust classification performance of the system on only these 2+ cases, an iterative leave-one-out classification experiment was performed. The cutoff point to determine FISH positivity was based on twenty-one training samples. This cutoff point was chosen as the maximum cutoff point yielding 100% sensitivity (i.e. fix sensitivity at 100% while maximizing specificity). Then the novel case not used in training was scored based on the cut-off score. Correct was defined as either above the cut-off and FISH ≥2.3 (a true positive decision), or below the cut-off score and FISH <2.3 (a true negative). Repeating the experiment 1000× with a randomly chosen test case, we arrived at an estimate of the system's classification performance on 2+ cases. From this experiment, the 2+ test case was correctly classified Mn feature) was shown to be superior to analyzing the patient tissue alone (Mp feature). This improvement is hypothesized to stem from the unique staining conditions of each slide that is reflected in both the patient and control sample. Furthermore, this clinically based combination (Mn) demonstrated an ability to correlate with gene amplification results that was similar to that of manual assessment.In this retrospective study of 99 cases of breast carcinoma, we have demonstrated the processes and methods behind the design, development, and evaluation of an image-based, computer-assisted HER2 immunohistochemistry quantification scheme. The result was an introduction of a membrane isolation algorithm and the quantitative use of positive staining controls. The membrane isolation algorithm in combination with the quantitative use of controls , responses greater than 15 produced the greatest area under the ROC curve using the Mn feature, and thus the k = 15 threshold was selected to produce the results in these experiments.Click here for fileMembrane Isolation Algorithm results using both high and low thresholds. These are the results from the membrane isolation algorithm using 2 different thresholds k = 15, and k = 5. These are specimens which stained less intensely.Click here for fileROC curves using Membrane Isolation Algorithm based on k = 15 and a combination . This is a comparison of ROC curves based on different Membrane Isolation Algorithm (MIA) variations. Since lighter cases showed enhanced membrane isolation detection when lower thresholds were used, a MIA using k = 5 threshold for lighter images and k = 15 for darker images was evaluated (green line). However, this did not improve AUC in comparison to one universal threshold (black line), and consequently, only one threshold (k = 15) was used in the results of this manuscript. It is interesting to note that the combination threshold used had very similar results to manual scoring (blue line).Click here for file


Several recent studies have shown that screen detection remains an independent prognostic factor after adjusting for disease stage at presentation. This study compares the molecular characteristics of screen-detected with symptomatic breast cancers to identify if differences in tumour biology may explain some of the survival benefit conferred by screen detection.A total of 1379 women (aged 50–70 years) with invasive breast cancer from a large population-based case–control study were included in the analysis. Individual patient data included tumour size, grade, lymph node status, adjuvant therapy, mammographic screening status and mortality. Immunohistochemistry was performed on tumour samples using 11 primary antibodies to define five molecular subtypes. The effect of screen detection compared with symptomatic diagnosis on survival was estimated after adjustment for grade, nodal status, Nottingham Prognostic Index (NPI) and the molecular markers.Fifty-six per cent of the survival benefit associated with screen-detected breast cancer was accounted for by a shift in the NPI, a further 3–10% was explained by the biological variables and more than 30% of the effect remained unexplained.Currently known biomarkers remain limited in their ability to explain the heterogeneity of breast cancer fully. A more complete understanding of the biological profile of breast tumours will be necessary to assess the true impact of tumour biology on the improvement in survival seen with screen detection. Breast cancer is the most common female cancer in the UK with more than 45 000 women diagnosed per annum. The National Health Service Breast Screening Programme (NHSBSP) was introduced in 1988 in the UK and around 15 000 breast cancer cases are now detected every year through screening. The NHSBSP initially offered three yearly mammography to women aged 50–65 years, however, since 2004 the upper age limit has been increased to 70 years, with approximately two million women now screened in the UK each year.The widespread introduction of mammographic screening for breast cancer has led to a significant reduction in breast cancer mortality. The policy of offering screening is associated with a 20% reduction in breast cancer mortality and the effect of actually attending screening is likely to be considerably larger . AlthougScreen-detected cancers are more likely to be smaller in size and well differentiated, and are less likely to be associated with regional lymph node involvement . Cases for SEARCH are identified through the Eastern Cancer Registration and Information Centre and details of the study have been published previously  was performed for the following 11 primary antibodies: ER, PR, HER2, CK 5-6, CK14, EGFR, E-cadherin, Ki-67, BCL2, p63 and α-smooth muscle actin (ASMA). Details of the primary antibodies and antigen retrieval methods are given in Tissue microarrays were constructed using a tissue microarrayer  as previously described with a single representative 0.6 mm tissue core taken from each tumour block ). A total score of >2 was defined as positive . All immunohistochemistry was scored by SJD. Details of the scoring methods used and the cut-off values applied to each marker to indicate positive and negative staining are shown in positive .Tumours were classified into five molecular groups based on the expression of ER, PR, HER2, basal cytokeratins and EGFR as follows: subtype 1 =ER+ and/or PR+ and HER2−; subtype 2 =ER+ and/or PR+ and HER2+; subtype 3 (HER2 over-expressing)=ER− and PR− and HER2+; subtype 4 =ER−, PR−, HER2− and CK5/6−, CK14−, EGFR−; subtype 5 =ER−, PR−, HER2− and CK5/6+ and/or CK14+ and/or EGFR+.χ2-test. Cox regression analysis was used to determine the effect of each factor on all-cause mortality after diagnosis. Follow-up was censored on the date of death from any cause, or, if death did not occur, on the date last known alive or at 15 years after diagnosis, whichever came first. Time at risk began on the date of entry into SEARCH to allow for the fact that SEARCH is an ongoing epidemiology study and some cases are recruited after diagnosis .Differences between screen-detected and symptomatic patients regarding categorical variables were assessed using the vs 59% respectively had tumour size ⩽2 cm, P<0.0001). Screen-detected tumours were more likely to have lower histological grade  and less likely to have lymph node involvement . In keeping with these findings, screen-detected tumours were more likely to have favourable NPI categories (P<0.0001). Women with screen-detected tumours were also less likely to receive adjuvant systemic chemotherapy .Baseline clinical and pathology data from the 1379 women included in the analysis are summarised in vs 74%, P<0.0001)) and PR+   and lower rates of Ki-67 positivity  were identified. There was no significant difference in the expression of the basal cytokeratins between the two groups but a higher proportion of tumours detected without screening expressed EGFR .A higher proportion of tumours detected at screening were ER+ (86 P=0.002) . BCL2 wavs 72%, P<0.0001). Furthermore, screen-detected tumours were less likely to belong to the basal-like subtype as defined by either the triple negative phenotype  or the triple negative phenotype and the expression of core basal markers ). The prevalence of luminal B  and HER2-overexpressing subtypes  was similar between the screen-detected and non-screen-detected groups.The distribution of the five defined molecular subtypes was explored in cancers detected at screening and those detected without screening . TumoursP<0.0001; Women with screen-detected tumours had improved overall survival compared to women with tumours detected without screening (P=0.001).When comparing survival according to molecular subtype, the luminal A subtype was associated with the best overall survival and the HER2-overexpressing subtype with the worst . TumoursThe effect of screen detection on survival was then explored in a multivariate analysis after adjustment for tumour characteristics, the NPI, individual molecular biomarker expression and molecular subtype . The effThe Freedman estimate of the proportion of the survival advantage from screen detection attributable to NPI was 56%. As one might argue that histological grade, a component of the NPI, is a biological variable, we also estimated the proportion attributable to size and node status only. This was 53%. Adjustment for the other molecular characteristics in addition to NPI gave attributable proportions ranging from 59 to 66%. Multivariate adjustment for several biological variables did not substantially change the proportion of the screen detection effect explained. Thus, additional adjustment for molecular characteristics explained a further 3–10% of the survival advantage of screen-detected tumours in addition to NPI (6–13% in addition to size and node status). Currently known biomarkers and tumour characteristics still remain limited in their ability to explain the heterogeneity of breast cancer fully. It is likely that a more complete understanding of the biological profile of breast tumours will be necessary to eliminate potential length bias and adjust for its impact on the improvement in survival seen with screen detection.vs symptomatic breast cancers in 1379 women diagnosed with invasive breast cancer in the East of England to identify if differences in tumour biology may explain some of the survival benefit conferred by screen detection. Although differences in the molecular subtype of screen-detected vs symptomatic cancers were identified, the expression of individual molecular biomarkers had minimal effect on the improved outcome associated with screen detection.Women with screen-detected cancers have a strong survival advantage compared to women with symptomatic cancers detected without screening . The majFavourable molecular characteristics have previously been described in screen-detected tumours including higher expression of ER and PR and less frequent expression of HER2 and Ki-67 .vs symptomatic breast cancers, our analysis showed minimal attenuation of the screen-detected survival advantage after adjustment for the expression of individual molecular biomarkers or molecular subtype in multivariate analysis. The Freedman estimates of the proportion of the effect suggest that the NPI explains 56% of the effect, up to a further 10% is explained by the biological variables and more than 30% of the effect remains unexplained. In a recent study by vs symptomatic breast cancers accounted in part for the better outcome of screen-detected cancers, however, the effect of molecular subtype on the survival advantage conferred by screen detection was not assessed in this analysis.Despite the identification of distinct differences in the molecular characteristics of screen-detected vs symptomatic tumours, our analysis indicates that these differences in tumour biology only account for a small proportion of the residual survival benefit. Measurement of tumour attributes is not perfect and there may be some residual variability in survival, which might be better explained by stage and other histological tumour characteristics if these were measured more precisely. In addition, there are prognostic factors that were unavailable to us, such as lymphovascular invasion. Adjustment for these might further explain some of the better survival of screen-detected cases. However, currently known biomarkers and tumour characteristics still remain limited in their ability to explain the heterogeneity of breast cancer fully. It is likely that a more complete understanding of the biological profile of breast tumours will be necessary to eliminate potential length bias and adjust for its impact on the improvement in survival seen with screen detection.The policy of offering mammographic screening has led to a reduction in breast cancer mortality of around 20% . The pop


Hypocrea jecorina (anamorph Trichoderma reesei) using Rapid Subtraction Hybridization (RaSH). Based on these data, we analyzed whether these genes are influenced by ENVOY and if overexpression of ENVOY in darkness would be sufficient to execute its function.In fungi, light is primarily known to influence general morphogenesis and both sexual and asexual sporulation. In order to expand the knowledge on the effect of light in fungi and to determine the role of the light regulatory protein ENVOY in the implementation of this effect, we performed a global screen for genes, which are specifically effected by light in the fungus The cellular functions of the detected light responsive genes comprised a variety of roles in transcription, translation, signal transduction, metabolism, and transport. Their response to light with respect to the involvement of ENVOY could be classified as follows: (i) ENVOY-mediated upregulation by light; (ii) ENVOY-independent upregulation by light; (iii) ENVOY-antagonized upregulation by light; ENVOY-dependent repression by light; (iv) ENVOY-independent repression by light; and (v) both positive and negative regulation by ENVOY of genes not responsive to light in the wild-type. ENVOY was found to be crucial for normal growth in light on various carbon sources and is not able to execute its regulatory function if overexpressed in the darkness.H. jecorina at several cellular processes, and that it has both positive and negative effects. The data also emphasize that ENVOY has an apparently more widespread cellular role in this process than only in modulating the response to light.The different responses indicate that light impacts fungi like  Neurospora crassa [white-collar-1  or whit2 . These 2  exhibits an altered cellulase gene transcription pattern both in the presence and absence of light, thus showing that env1 is directly or indirectly impacting cbh1 gene expression in the darkness [env1, thus suggesting a regulatory feedback being operative.llulases -25. The llulases . Interesdarkness . In addienv1 gene. We will show that light affects transcription of genes of H. jecorina both positively as well as negatively, and that for both effects env1-independent variants are found. In addition, we will show that ENVOY also acts as a light-independent repressor for several genes, is crucial for normal growth in light on several carbon sources, but is not able to fully execute its regulatory function when overexpressed in darkness. Our data suggest a function of ENVOY in coordination of the light signal with other environmental signals, which is comparable to the gating function shown for VVD of Neurospora crassa.Our detection of a light-dependence of cellulase gene transcription of the PAS/LOV domain protein ENVOY raised the question whether there would be more cellular functions in fungi which are controlled by either light and/or ENVOY. To address this question we have performed a genome wide screening for genes regulated by the presence of light, using Rapid Subtraction Hybridization (RaSH). Genes thereby identified were investigated for whether their response would be dependent on a functional H. jecorina QM 9414 pregrown in the dark, and mRNAs from the same strain after subjection to illumination to screen for mRNAs which are more abundant under the latter conditions and thus upregulated by light applying Rapid Subtraction Hybridization [We used mRNAs from dization . To prevConsequently, 300 putatively positive ESTs were isolated and tested by Reverse Northern blotting -29. The env1 upon cultivation after onset of illumination in minimal medium with 1% glycerol as carbon source [N. crassa ccg-2 and NC2B7 , we assessed the response of expression of all genes to light by Northern blotting. This investigation proved that among the genes analyzed, 20 were indeed significantly  upregulated upon illumination. Interestingly, four genes were shown to be actually repressed by light, one of them representing a false positive result of RaSH regarding the aim of the assay, while the others were three of the genes which – because of their roles in signaling – were intentionally included in the analysis. Since we will present these data in a broader context below, they are not given at this place. For three genes neither significant light- nor ENVOY dependent regulation was detected and therefore they are not discussed further. We also noted that the fluctuations in transcript abundance, which were also seen earlier with the light regulatory gene n source  or N. crre 4A in ) also ocTrichoderma reesei genome database v2.0 and retrieved the corresponding full length proteins and the correspondingly annotated gene models. The protein sequences were checked for conserved domains using the NCBI CDD search to allow for assigning a function to the respective gene (Table Gibberella zeae (Fusarium graminearum), Neurospora crassa and Saccharomyces cerevisiae (Table env1 and phr1) encoded ENVOY and photolyase 1, respectively, which have already been reported to be up-regulated by light [rpl7), indicating that illumination results in increased respiratory energy production. Other genes isolated are involved in stress response, such as a toxin efflux pump of the major facilitator superfamily (tre10571), genes involved in protein degradation , nucleotide degradation (IMP pyrophosphatase tre22454) and the thiazole biosynthetic enzyme thi4, the homologue of which is upregulated under stress in Fusarium oxysporum [tmk3, the cross pathway control protein cpc2) were also upregulated by light. The remaining genes encoded proteins of unclear function in the physiology of H. jecorina, such as a predicted porphyromonas-type peptidyl-arginine deimidase (tre45629) which causes citrulinylation of proteins, and a putative CAP20 virulence related protein tre41865, an orthologue of which is involved in appressorium formation and virulence in Colletotrichum gloeosporioides [In order to identify the genes corresponding to the ESTs isolated, we used them to BLAST the ae Table . Four ofby light ,30 and txysporum . Finallyorioides .H. jecorina by light, we now investigated whether they would indeed require the function of env1 for this purpose. To this end, we compared the expression profile of these genes in H. jecorina QM 9414 to that of the env1PAS- strain over a period of 240 min. This strain lacks the PAS-domain of the light regulatory protein ENVOY and shows altered response to light as well as a considerably decreased light tolerance [cpc2, phr1, tmk3, tre39031, tre40105, and tre42719) showed a behaviour which was consistent with the default expectation: a transient upregulation by light, which was not seen in the env1PAS- strain and therefore at least partially regulated env1 , while also showing this upregulation by light, did so also in the env1PAS- mutant. Despite the fact that ENVOY seems to be involved in their regulation due to the altered transcription pattern in env1PAS-their response to light by increased transcription is not exclusively dependent on ENVOY  also exhibited significant upregulation of gene expression upon exposure to light, but this upregulation was even stronger in the env1PAS- mutant, indicating that ENVOY antagonizes this activation in the wild-type strain . Among them thi4 was found to be significantly up-regulated in the env1PAS- strain indicating repression by ENVOY. The other three exhibited significantly lower transcript abundance in the env1PAS- strain and thus are apparently dependent on a function of env1, which is not directly related to light response  to the presence of light  is present in the promoter of the N. crassa light response output gene albino-3 as well as in other light-regulated genes of Neurospora. Deletion of this motif abolished the difference in mRNA levels of al-3 in light and darkness [N. crassa frq-promoter and is known as LRE . Thec stress . Howeverc stress .env1 , it is evident that the two strains differ significantly stronger along the y-axes, thus indicating that light inhibits growth of strain env1PAS-. On the other hand, growth in darkness (with the exception of glycerol) was only very little affected (= both strains occur at similar positions at the x-axes). In the case of utilization of glycogen, whose position in the graph indicates light inhibition in the mutant strain, the results are in perfect agreement with the expression of gph1 . The inhibitory effect of light in the absence of ENVOY is therefore carbon source dependent.Because of the up-regulation of energy metabolism by light in dependency of t strain . The datenv1 would be sufficient to induce transcription of its target genes in darkness, we introduced the env1 open reading frame under the control of the inducible N. crassa qa2-promoter into the H. jecorina parent strain QM9414, resulting in strain 4env1qa+. Strains were grown in constant darkness and expression of env1 was induced by addition of quinic acid to the parent strain and the env1-overexpressing strain. After quinate addition, induction of env1 transcription could be demonstrated . Thus, despite the effect of the lack of functional ENVOY on transcription of phr1, overexpression of ENVOY is not sufficient for upregulation of phr1 in the darkness.In order to find out whether up-regulation of d Figure . To elucenv1 would be sufficient for the up-regulation of tre39031 in darkness and if the effect of the mutation of env1 in env1PAS- . These results are in agreement with the assumption that ENV1 does not directly regulate the transcription of its target genes , but executes its function indirectly by interaction with transcriptional regulators, which are not available or inactive in darkness.As a second model case, we wanted to test whether expression of H. jecorina, which led to the identification of a total of 20 genes which were upregulated and 4 which were downregulated shortly after illumination. In addition, several of these genes were differentially effected by a mutation in the light regulatory protein ENVOY [N. crassa VIVID, is known to be a photoreceptor [et al.[Hypocrea atroviridis (anamorph Trichoderma atroviride) for the screening of genes regulated by light, and identified 30 genes to be upregulated. This corresponds to 2.8% of the genes contained in the array used, and compares well to a value of 3% obtained for a similar study in N. crassa [H. atroviridis [N. crassa [Neurospora and Hypocrea/Trichoderma spp. [phr1) identified in this study was also found by Rosales-Saveedra et al. [H. jecorina and H. atroviridis belong to the same fungal genus. The authors mentioned unpublished data that "several of the genes identified by them were also light upregulated in H. jecorina." This difference may be due to the missing of 80% of the genome in this study. On the contrary, the method of subtraction hybridization used in our study includes the whole transcriptome of the respective conditions to be compared, but from our experience also yields only a subset of all genes regulated under the conditions of interest. Hence we consider the present study complementary to that of Rosales Saveedra et al.[H. atroviridis may not be functional: this assumption is supported by the following findings: first, its N-terminus is truncated at amino acids 1–6; second, it contains two upstream open reading frames close to the ATG , which can profoundly influence the translation of the main ORF [H. atroviridis env1 under several conditions where env1 is strongly transcribed in H. jecorina (data not shown). Taken together, this could reflect a different light regulatory machinery in these two closely related fungi.In this study, we used RaSH to isolate early light-responding genes from in ENVOY , whose creceptor . Besidesr [et al. have recoviridis  were alsrma spp. . It is ta et al. , althougra et al.. Yet anomain ORF . Finallyphr1, which has also been isolated from H. atroviridis, and which plays a role in the protection of genes against UV-light by photoreactivation of cyclobutan dimers of the pyrimidine nucleotides [blu3), which encodes a protein with an endonuclease III-type domain and which could function in excision repair. This gene was not identified in this study. However, another gene identified in this study (tre22454) encodes an ITP triphosphatepyrophosphatase, an enzyme responsible for the degradation of IMP and XMP, which accumulate as a result of cellular degradation of nucleotides which were modified by oxidative stress [H. jecorina involves reactions both against UV-light as well as oxidative stress. This is supported by the upregulation of tre42719 . Yoshida et al. [N. crassa to light evokes an oxidative stress response, in which nucleoside diphosphate kinase 1 plays a essential role e.g. by associating with a G-protein &-subunit for transmission of the light signal [ubi4 (encoding polyubiquitin). The upregulation of an MSF toxin efflux pump (tre10571) with homology to proteins providing tolerance against fungicides in Botrytinia fuckeliana . One of them was the photolyase gene leotides . Rosalesleotides  also repe stress . These da et al.  have shot signal . Finally 3E-130; ,50) and  5E-127; ) upon iloviridis , severalva et al. that expenv1-dependent gene shows intriguing characteristics – tre45629: although the primary structure of the encoded protein is only poorly conserved, it contains all the signature sequences of a peptidyl arginine deiminase, an enzyme which converts arginine residues in proteins to citrulline, thereby altering the positive charge and hence the proteins ability to interact with other proteins and membranes. To the best of our knowledge, the role of this deimination has not yet been investigated in fungi. Moscarello et al.[One light-responsive but not lo et al. have recA. nidulans. This activation is executed by the transcription factor CPC1/CPCA. In the presence of amino acids the pathway is repressed by the transcription factor CPC2/CPCB [H. jecorina cpc1 would also respond to light. Interestingly, cpc1 did not respond to light, but is influenced by the presence of ENVOY. Thus, in Hypocrea jecorina the repressing factor is regulated by light but the activating factor is modulated by ENVOY. The coregulation of cpc1 and hac1 by ENVOY is also interesting in the context that Gcn4p (the S. cerevisiae orthologue of CPC1) is involved in the unfolded protein response [H. jecorina [Another interesting finding was the detection that the cross pathway control protein CPC2 is regulated by light. Cross pathway control (CPC) of amino acid biosynthetic pathways is activated during amino acid starvation and also controls sexual development in PC2/CPCB . We therresponse , and thajecorina . This suN. crassa, VIVID – have been described as proteins modulating the cellular response to light. However, we have shown here that this is only one of several roles which ENVOY apparently plays. Only eleven of the nineteen genes upregulated by light needed the function of env1 for this process. Five other genes showed an upregulation by light independently of env1, and in three genes the light-dependent upregulation was even stimulated in an env1-negative background. The nature of the proteins encoded by these genes did not yet provide us with an explanation for the specific role of these env1-independent and env1-repressed upregulations.Envoy – and particularly its putative counterpart in phr1 and tre39031 – not able to execute its light-dependent function in darkness suggests that the presence of its putative interaction partners is required for a proper function of this regulatory mechanism. Similarly, also for N. crassa White collar-1 (WC-1) Lewis et al.[N. crassa orthologue VIVID [The results of this study point at an involvement of Envoy in the regulation of various cellular processes. Although based on this study we cannot differentiate between direct and indirect influences of ENVOY, it is obvious that this protein plays an important regulatory role at a central junction of signaling pathways. The finding that ENVOY is at least in some cases – as exemplified by the influence on is et al. showed, is et al., ENVOY cue VIVID . In otheThe different responses to light, as demonstrated in this study, stress that light plays a role in several cellular processes of fungi, thereby displaying both positive and negative effects. Our data also emphasize that ENVOY has an apparently more widespread cellular role in this process than only in modulating the response to light. The importance of such a coordinator becomes apparent when it is considered that sunlight causes subsequent changes such as a rise in temperatures, decrease in humidity, and increase in UV light intensity. The adaptation to these environmental cues is of crucial importance in the evolution of every organism.H. jecorina (T. reesei) wild-type strain QM9414 (ATCC 26921) and the env1 recombinant mutant lacking the PAS-domain (env1PAS-[H. jecorina was grown in liquid culture in 1-L Erlenmeyer flasks on a rotary shaker (200 rpm) at 28°C in 200 ml of medium as described by [8 conidia/L  as inoculum in constant darkness and harvested with red safety light or after the time of illumination  as indicated with the figures. Cultivations of wild-type and mutant strain were done in parallel to ensure equal conditions.The (env1PAS-) were usribed by  with 1% E. coli JM109 [li JM109  was usedet al.[The experiment was performed essentially as described by Schmoll et al. accordinet al.. For theEcoRII digestion and purification. The candidates for a more detailed analysis were chosen by visual inspection first, then this decision was cross-checked by quantitative measurements using the BIORAD Geldoc Imaging system  and BIORAD Quantity One software, both for three different expositions of the blot.For the Reverse Northern Hybridization, PCR products were loaded onto duplicate agarose gels and blotted with 0.4 N NaOH onto Hybond N membranes . Hybridization was performed using 2.5 μg of PCR amplified and subsequently radioactively labeled cDNA from tester or driver, respectively, as probes after env1 [Fungal mycelia were harvested by filtration, washed with tap water, frozen and ground in liquid nitrogen. For extraction of DNA, mycelial powder was suspended in buffer A , incubated for 20 min at 65°C, cooled down on ice, mixed with 1 vol. phenol:chloroform:isoamylalcohol 49:49:2 (v/v/v) and centrifuged . Following an extraction with 1 vol. of chloroform:isoamylalcohol 24:1 (v/v), the DNA was precipitated with 1 vol. of isopropanol and washed with 70% (v/v) ethanol. Total RNA was isolated by the guanidinium thiocyanate method ,61. Stanenv1  under thNormalization of gene expression was performed according to the following formula:{(transcript abundance of gene x at time point)/(transcript abundance of gene x in wild-type in darkness)}/{/}. The quantitative measurements were performed using the BIORAD Geldoc Imaging system and BIORAD Quantity One software from different expositions of the respective film. For every set of Northern blots one 18S rRNA hybridization was included as a loading and blotting control and used for quantification of the respective films.T. reesei genome database v2.0 [The most promising candidates for light responsive genes showing clear differential transcription in the Reverse Northern Blot were selected for sequencing. PCR products as used for reverse Northern blotting were sequenced using primer RaSH1R, which binds within pBluescript immediately upstream of the inserts to be analyzed . The resase v2.0 . For thease v2.0 ,65 and fase v2.0 ,66,68. Iet al.[-2 s-1) or in the dark at 28°C, and percent absorbance at 750 nm determined in 12 h intervals between 36 and 72 h. Analyses were repeated at least three times for each strain.Growth rates on selected carbon sources were investigated by means of the Biolog FF MicroPlate™ assay  as described by Druzhinina et al.. Inoculaenv1 into the SmaI-site of the vector pmyx2 [env1 being under the control of the N. crassa qa-2 promoter which can be induced by addition of quinic acid to the culture medium to a final concentration of 0.6%. The resulting construct was transformed into the wild-type strain QM9414. Two positive strains were selected by PCR screening and Southern blotting, pregrown for 24 h on 1% (w/v) glycerol in darkness before adding quinic acid and transcript abundance was analyzed at several time points after addition of quinic acid in constant darkness. The wild-type strain was used as a control in parallel to those strains and was treated equally.For inducible expression of ENV1 we introduced the open reading frame of or pmyx2 , resultiAS performed Northern analyses and bioinformatic analysis of the genes described. CPK participated in the bioinformatic analysis and strategic planning of the work and drafted the manuscript. MAF carried out the BIOLOG-analysis and – together with ISD – performed the analysis of the respective data. MS performed Rapid Subtraction hybridization, Reverse Northern blotting, prepared the ENV1 overexpressing strains, participated in the data analysis and strategic planning of the work and wrote the final version of the manuscript.


Pluripotency, the property of a cell to differentiate into all cellular types of a given organism, is central to the development of stem cell-based therapies and regenerative medicine. Stem cell pluripotency is the result of the orchestrated activation of a complex transcriptional network characterized by the expression of a set of transcription factors including the master regulators of pluripotency Nanog and Oct4. Recently, it has been shown that pluripotency can be induced in somatic cells by viral-mediated expression of the transcription factors Oct3/4, Sox2, Klf4, and c-Myc.Here we show that 5-Aminoimidazole-4-carboxamide-1-b-riboside (AICAR) is able to activate the molecular circuitry of pluripotency in mouse embryonic stem cells (mESC) and maintain Nanog and Oct4 expression in mESC exposed to the differentiating agent retinoic acid. We also show that AICAR is able to induce Klf4, Klf2 and Myc expression in both mESC and murine fibroblasts.AICAR is able to activate the molecular circuitry of pluripotency in mESC and to induce the expression of several key regulators of pluripotency in somatic cells. AICAR is therefore a useful pharmacological entity for studying small molecule mediated induction of pluripotency. Pluripotency, the ability to differentiate into all embryonic tissues, is a defining characteristic of embryonic stem cells and of induced pluripotent stem cells. Understanding how to induce, modulate, and maintain the pluripotent state of mammalian cells is of great importance for the development of critical tools for regenerative medicine. Pluripotency has been shown to be the product of an extended transcriptional network that canWhile viral overexpression of transcription factors has been an invaluable tool to investigate the molecular basis of pluripotency, the presence in reprogrammed cells of viral genomes seems to be a barrier for the implementation of safe regenerative therapies. In light of this, small molecules are currently seen as plausible alternative to induce the expression of the critical transcription factors and, eventually, pluripotency -11.Our laboratory has recently shown that the cell permeable nucleoside AICAR can induce the expression of different members of the Klf family of transcription factors in endothelial cells . Thus, we hypothesized that AICAR could induce Klf2 and Klf4 expression in other cell types and that it could activate the pluripotency transcriptional network. Here we show that AICAR induces Klf2 and Klf4 expression and activates the pluripotency transcriptional network in mESC. This effect is able to antagonize mESC retinoic acid induced differentiation. Moreover, AICAR is able to induce Klf4, Klf2 and Myc expression in mouse embryonic fibroblasts, the prototypical somatic cells used for reprogramming studies. Our data describes a new property of AICAR in modulating mESC pluripotency network, and defines this small molecule as a new potential tool for the pharmacological reprogramming of somatic cells.2 and exposed to 1 mM AICAR for 72 h. Gene expression was measured by total RNA extraction followed by RNA reverse transcription and real-time Taqman quantitative PCR. As seen in Figure The J1 mESC line is a well characterized pluripotent cell line derived from the 129 Sv/J mouse strain. This cell line was used to assess the effects of AICAR on gene expression in pluripotent cells. ES cells were plated at 5000/cmIn order to further assess the effects of AICAR on the pluripotency transcriptional network, mESC were treated with AICAR in the presence of high doses of Retinoic Acid (RA), a well-characterized differentiating agent that induces downregulation of several members of the pluripotency transcriptional network, including Klf4, Klf2, Nanog, Oct4 and Sox2 . As showEmbryonic fibroblasts are the prototypical cells used to study transcription factor induced somatic reprogramming ,18,19. WPluripotency is the product of the activation of a complex transcriptional network that induces expression of genes encoding transcription factors, signal transduction components, and chromatin-modifying enzymes. The pluripotency transcriptional network seems to be the same in both embryonic stem cells and induced pluripotent stem cells (somatic cells reprogrammed to gain pluripotency). While there have been few reports of small molecules that can maintain pluripotency of embryonic stem cells -22, therIn the present work we show that AICAR is able to activate the pluripotency transcriptional network in mESC and to upregulate Klf4, Klf2 and Myc expression in murine fibroblasts. The effect of AICAR as activator of the pluripotency molecular circuitry is clearly shown by the AICAR-mediated induction of the master regulators of pluripotency Nanog, Oct4, Myc and Sox2 and by the AICAR-mediated induction of Klf4 and Klf2, two transcription factors highly expressed in undifferentiated ESCs, which are quickly downregulated upon induction of differentiation. The effOur experiments with MEFs show that AICAR is able to upregulate Klf4, Klf2 and Myc also in somatic cells. In contrast to the effects exerted on mESC, in this context AICAR is not able to induce Oct4, Nanog or Sox2 expression and is therefore not able to activate the pluripotency network. This result is not surprising if we consider that while Klf4 has been suggested to be able to regulate the expression of all the transcription factors required for somatic cell reprogramming , this abAICAR has been previously shown to have a number of effects on cellular function -31 incluOur present data clearly do not provide a mechanism underlying AICAR effects on mESC pluripotency and Klf transcription in fibroblasts. Since AICAR has been previously shown to prevent skeletal myoblasts differentiation via activation of the longevity regulator Sirt1 , it is iCollectively, this work describes for the first time a small molecule that is both able to activate the pluripotency transcriptional network in mESC, and to induce the expression in somatic cells of two of the four transcription factors required for reprogramming. Our study identifies AICAR as a pharmacological space important for the establishment and/or maintenance of induced pluripotent stem cells.The J1 mouse embryonic stem cell line  was gelatin adapted and grown on 1% gelatin coated tissue culture flasks in DMEM  10% FCS, 1× non essential amino acids , 55 μM β-mercaptoethanol , Penicillin 10 units/ml , Streptomycin 10 μg/ml , 1000 U/ml LIF  and split by trypsinization every 2–3 days. The E14 mouse embryonic stem cell  line was cultured in similar conditions but on a feeder layer of mytomycin-treated murine embryonic fibroblasts . E14 mESC were deprived of contaminating embryonic fibroblasts by trypsinization and 45 minutes incubation on non-gelatinized tissue culture flasks prior to plating for exposure to AICAR.Strain CF-1 Mouse embryonic fibroblasts were purchases from Millipore (USA) and cultured in DMEM  10% FCS, Penicillin 10 units/ml , Streptomycin 10 ug/ml .Total RNA was extracted with the ABI 6100 Nucleic Acid PreStation  according to manufacturer's instructions. Reverse transcription of RNA was performed with Multiscribe reverse transcriptase  according to manufacturer's instruction. Real-time Taqman PCR was performed in 20 μl reactions with primers from Applied Biosystems, according to manufacturer's instructions.5-Aminoimidazole-4-carboxamide-1-β-riboside  was dissolved in PBS without calcium at a 100 mM final concentration. All trans-retinoic acid  was dissolved in DMSO at a final concentration of 2.5 mM. To induce mESC differentiation, Retinoic Acid was added to mESC culture media without LIF at a final concentration of 5 micromoles per liter. Media with RA and or AICAR was replaced every 24 hours.Growing cells were washed in PBS, fixed for one minute in 4% PFA PBS, and then incubated for 30 minutes in BM purple alkaline phosphatase (AP) staining solution . The percentage of alkaline phosphatase positive colonies was calculated counting the number of AP+ and AP- cell clusters in three different fields at a 4× magnification.Two way analysis was performed with un-paired t-test assuming samples with equal variance. Three way analysis was performed with single factor ANOVA.LA and GGC designed the experiments. LA and YZ performed the experiments. LA wrote the manuscript under the supervision of GGC.


In these cases, the sequence of the infecting virus can be estimated accurately, enabling inference of both the direction of substitutions as well as distinction between insertion and deletion events. By integrating information across multiple acutely infected hosts, we find evidence of adaptive evolution of HIV-1 env and identify a subset of codon sites that diversified more rapidly than can be explained by a model of neutral evolution. Of 24 such rapidly diversifying sites, 14 were either i) clustered and embedded in CTL epitopes that were verified experimentally or predicted based on the individual's HLA or ii) in a nucleotide context indicative of APOBEC-mediated G-to-A substitutions, despite having excluded heavily hypermutated sequences prior to the analysis. In several cases, a rapidly evolving site was embedded both in an APOBEC motif and in a CTL epitope, suggesting that APOBEC may facilitate early immune escape. Ten rapidly diversifying sites could not be explained by CTL escape or APOBEC hypermutation, including the most frequently mutated site, in the fusion peptide of gp41. We also examined the distribution, extent, and sequence context of insertions and deletions, and we provide evidence that the length variation seen in hypervariable loop regions of the envelope glycoprotein is a consequence of selection and not of mutational hotspots. Our results provide a detailed view of the process of diversification of HIV-1 following transmission, highlighting the role of CTL escape and hypermutation in shaping viral evolution during the establishment of new infections.The pattern of viral diversification in newly infected individuals provides information about the host environment and immune responses typically experienced by the newly transmitted virus. For example, sites that tend to evolve rapidly across multiple early-infection patients could be involved in enabling escape from common early immune responses, could represent adaptation for rapid growth in a newly infected host, or could represent reversion from less fit forms of the virus that were selected for immune escape in previous hosts. Here we investigated the diversification of HIV-1  envelope gene in a cohort of 81 subjects acutely infected with HIV-1 subtype B and found evidence of adaptive evolution, with a proportion of sites that tended to diversify more rapidly than expected under a model of neutral evolution. Several of these rapidly diversifying sites facilitate escape from early cytotoxic immune responses. Interestingly, hypermutation of the virus, brought about by host proteins as a strategy to restrict infection, appeared to be associated with early immune escape. In addition to single base substitutions, insertions and deletions are an important aspect of HIV evolution. We show that insertion and deletion mutations occur evenly across the gene, but are preferentially fixed in the variable loop regions.HIV is a rapidly evolving virus, displaying enormous genetic diversity between and even within infected individuals, with implications for vaccine design and drug treatment. Yet, recent research has shown that most new infections result from transmission of a single virus resulting in a homogeneous viral population in early infection. The process of diversification from the transmitted virus provides information about the selection pressures experienced by the virus during the establishment of a new infection. In this paper, we studied early diversification of the  Mutations that enable escape from the host's immune responses will come under selective pressure, in an order that reflects the timing of the immune responses, underlying mutational rates, and the relative fitness costs of mutations. In addition, escape mutations that occurred in the infecting host, particularly those resulting in a reduction of viral fitness Env is a trimeric glycoprotein composed of three gp120 and gp41 heterodimers that are exposed on the viral surface and are anchored on the viral particle by the transmembrane domains of the gp41 subunit. Viral cell entry is initiated by the binding of gp120 to the CD4 receptor, followed by CCR5 or CXCR4 coreceptor binding et al.The transmission of HIV is associated with a severe population bottleneck, such that, in most cases, new infections result from transmission or expansion of a single virus Phylogenetic trees of the sequences from individual patients had short branch lengths and few internal branches, reflecting the short time since transmission and rapid expansion in the new host. It was not possible to apply standard phylogenetic models of evolution to these data to assess whether diversification of the virus following transmission is primarily a stochastic process, or driven by evolutionary selection pressure, because sequences from individual patients provide too little information to allow statistical discrimination between alternative evolutionary models. However, by considering all patients simultaneously, it was possible to determine whether similar patterns of sequence diversification were repeated either in multiple patients or within the same patient. Shared patterns of rapid diversification at the same codons in early infection can reveal sites that are typically involved in viral adaptation to the new host at the earliest stage of infection. In cases where these sites are associated with specific host immune responses, they shed light on the nature, extent, and timing of the immune responses that most frequently exert selective pressure on the transmitted virus during acute and early infection. However, there is also the possibility that shared mutations across multiple patients may be the result of deviation from the expected mutation rates, so that particular sites with high mutation rates, for example resulting from APOBEC-mediated substitutions, might be mutated in an unexpectedly high proportion of patients.env that tend to diversify most rapidly across recently infected patients, and explored the reasons for rapid diversification at these sites. The sequence of the transmitted founder virus at or near the time of transmission, which had been inferred previously env has previously been reported at transmission compared to chronic infection envelope gene. In this study we found that i) there is evidence of positive selection in early HIV-1 infection, which appears to be driven in many cases by escape from early cytotoxic T lymphocyte (CTL) responses; ii) in several cases CTL escape occurred via mutations in the APOBEC sequence context, suggesting a role for APOBEC in determining the pathway of immune escape; and iii) frameshifting insertions and deletions are evenly spread throughout env but are preferentially fixed in the variable regions. These results provide the most detailed view yet of the process of diversification of the homogeneous viral population following transmission.Using HyPhy env coding sequences from early stages of HIV-1 infection as described in the We fitted models of codon sequence evolution to 2,207 www.hiv.lanl.gov). For fifteen of the patients the env sequences showed an overall elevated rate of G→A mutations in a sequence context associated with APOBEC hypermutation . As this was a cross-sectional study, the sequences from which we infer selective pressure are from the moment of transition, thus only direct experiments can determine which of the variants represent the susceptible form.There were 10 positively selected sites that were located in 5 potential CTL epitopes. An additional site  that was detected as an enriched change in late infection  and a diminished response to the variant KAHDPEVYNVW  is quite likely (p = 0.45). Using this approach, only at one site in the alignment  do we find a significantly greater number of patients sharing a mutation than can be explained by chance .The 12 other sites that were identified cannot be explained by APOBEC mediation and do not occur within mutation clusters and were therefore not investigated as possible CTL epitopes. Two sites  are identified largely because of mutations in patients Z31 and TT31P, where infection may have resulted from multiple closely related strains of the virus, rather than a single viral strain We used a separate phylogenetic method to identify sites that appear to have a different rate of mutation in the initial, compared to later clinical stages of early infection . This anWe assessed by visual inspection whether there was evidence of directional evolution in any of the 24 rapidly evolving sites identified above (i.e. a consistent trend towards replacement of one amino acid by another amino acid across multiple patients). Position 646 (518 in HXB2), which occurs in the fusion peptide of gp41, showed consistent replacement of Methionine with either Valine or Isoleucine  was not significantly different than the expectation under a model of random mutation. We, therefore, found no evidence of a tendency for mutations to disrupt PNGSs in early infection. . We applied a similar approach to determine whether there was an overall enrichment of mutations within epitope-dense regions. In this case, we compared the number of mutations that occur within epitope rich regions  in env but with a bias towards strings of like bases, the compensatory single base indels were not proximal to like bases, were found exclusively in heterogeneous infections, and were all concentrated in the hypervariable V1, V2 and V4 loops . Of the 49 unique insertions, 43 were in-frame, and the other six each had a nearby compensatory mutation. There were many more multiple base deletions among the homogeneous patients; 115 were found, again all unique, with 33 very large deletions  that were presumably lethal. Of the 82 smaller deletions, 27 were in-frame (33%) and 55 were frameshifting. In contrast, in the heterogeneous infections there were 289 deletions resulting from 82 unique patient/deletion combinations. Among these unique deletions 56 were in-frame and 53 of these were in V1, V2 and V4. Another 7 deletions were compensated by nearby indels. The in-frame and out-of-frame multiple base indels were distributed very differently across the genome  the observed diversity can be inferred to have arisen in the newly infected patient, and not in the individual from whom the infection was acquired env, particularly in the variable loops, were masked from the analysis due to ambiguous sequence alignment, sites that confer resistance to neutralizing antibody escape may have been overlooked. Although our results underscore the importance of CTL escape in early infection they do not provide a basis to discount the potential contribution of escape from antibody responses to the evolution of env early in HIV-1 infection.We used HyPhy We used statistical model comparison to make the case that there is evidence of adaptive evolution in HIV-1. To do this we compared the fit to the data of a model in which no sites in the alignment were allowed to evolve with ω>1 to the fit of a model in which a subset of sites were allowed to have ω>1. The latter model had a significantly better fit to the data for the sequences from Fiebig stages I–III and Fiebig stages I–V, suggesting that the virus evolves adaptively in these clinical stages. However, there are two important issues to consider here. First, the fact that the positive selection model did not have a significantly better fit to the data in Fiebig stages I–II does not prove that viral diversification is not shaped by positive selection at this stage of infection. Sequences from stages I–II were highly homogeneous, thus a very small number of mutations from the infecting virus were observed in each patient, severely limiting the power to detect evidence of selection. Secondly, a more general issue is that models of codon evolution used to detect positive selection rely upon several assumptions about the way the sequences are evolving and deviations from these assumptions could lead to false positive results The phylogenetic codon model we applied using HyPhy does not account for recombination, which is known to cause false positive inference of selection env in early infection since this could impact our understanding of the nature of the transmitted virus. It is clear from several studies We tested for evidence of directional change in Discounting sites that may have been affected by APOBEC3G/F hypermutation we found just a single site with significant evidence for directional change. At position 646, (518 in HXB2), which is in the fusion peptide of gp41, we found consistent mutations from Methionine to Valine or Isoleucine. However, at this site, there is just a single mutant sequence observable in each of the nine patients in which the mutation is found, and this seems inconsistent with a mutation that is coming under selection for increased frequency in multiple patients. Furthermore, we do not observe an increased frequency of Methionine in acute sequences compared with chronic sequences at this site (data not shown), as would be expected if the consistent mutations from Methionine to Isoleucine or Valine represented reversion associated with selective viral transmission. A single site was observed to undergo recurrent change in a single subject in Fiebig stage II, and as this is prior to the immune response it may be indicative of rapid reversion. Alternatively, the observation could be explained instead by transmission of two highly related forms, together with recombination either in the donor or soon after transmission.env gene in maintaining the survival of HIV-1 during the earliest stages of infection is of particular interest as the carbohydrate surface establishes the first means of contact between the host and the virus. A previous study env. We did not find evidence of accelerated diversification at potential N-linked glycosylation sites in early infection, which suggests that if escape from neutralizing antibodies is among the major driving forces of HIV-1 evolution at this stage the mechanism is unlikely to be primarily through single base substitutions in glycosylation sites, at least in the portion of env that could be aligned with high confidence.Neutralizing antibody responses drive the evolution of viral escape and are therefore thought to play an important role in shaping the evolutionary changes observed in the envelope gene. Mutations, insertions and deletions, as well as the distribution of glycosylation sites along the envelope gene mediate the ability of the virus to escape from neutralizing antibodies although the relative contribution of each of these is unknown The majority of the rapidly evolving sites that recurred within individuals were clustered and appeared to be under selection for escape from cellular immune responses, and several of these mutations are G→A substitutions in APOBEC3 motifs. We also observed a highly significant enrichment overall for APOBEC3 motifs among the rapidly diversifying sites identified using HyPhy, even when stringent precautions were taken to exclude the 15 subjects that had enrichment of G→A mutations within the APOBEC3 motif. This suggests the possibility that APOBEC3 and imperfect Vif function can play an important biological role in the patterns of mutation found in early infection, and may contribute to determining the first immune escape mutations. Thus base mutation rate may be in some cases making a contribution to determining which immune escape events happen first, along with fitness In our dataset several of the rapidly evolving sites are associated with clusters of mutations that occur in close proximity to one another in the same patient . In someenv, with the exception of single base indels in stretches of like-bases, which may in part be attributable to experimental artifact. On the other hand, in-frame and compensated indels are highly concentrated in 4 hypervariable domains in the heterogeneous infections leading to extensive length variation in V1, V2, V4 and V5. Because this was exclusively found in heterogeneous infections, we assume the length variants most likely arose in the newly infected individual's partner, at a later phase of infection. This indicates that there is no molecular trigger that increases the background rate of indels in the hypervariable regions during reverse transcriptions, and that the concentration of length variation in the hypervariable domains is instead a consequence of selection. Also this data does not provide evidence for selection for length variation during acute infection, suggesting that this may occur during the chronic phase of HIV-1 infection.In this study we have also shown that inactivating indels are distributed evenly through This cross-sectional study has provided a glimpse into the nature of the diversification of HIV-1 following transmission in a population of 81 B clade infected individuals. As expected purifying selection acts on single base mutations at the majority of sites as well as on indels, but there is also evidence of a subset of sites that diversify more rapidly than expected under a model of neutral evolution. Our results suggest that APOBEC3 may play a role in shaping the dynamics of early T-cell immune escape. APOBEC3 motifs are highly enriched among the rapidly diversifying sites. This could be the result of an anomalously high mutation rate giving the appearance of selection pressure when there is none, or a higher mutation rate promoting a particular route of immune escape; the two are not mutually exclusive. Finally early T-cell escape appears to be much more common than reversion. We provide new evidence supporting the idea that concentrated regions of change within an individual during early infection Envelope sequences (n = 3476) from 102 individuals infected with HIV-1 subtype B, were generated using Single Genome Amplification (SGA), as part of a previous study et al.et al.http://www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html) were discarded. Among the patients, 15 had a generalized increase in the rate of G→A mutations in the context of the sequence motif associated with APOBEC hypermutation. In these cases there was an excess of G→A mutations in the APOBEC sequence context across all sequences from the patients and not concentrated in individual severely hypermutated sequences http://www.hiv.lanl.gov/content/sequence/hiv/user_alignments/keele.env gene evolving under the influence of positive Darwinian selection et al.et al.iD, θ are the data at site i in the alignment, the phylogenetic tree and the parameters of the model, respectively. We extend this to data from multiple patients by writing the likelihood asikD and k, respectively. In this formulation ω has the same value at a given site across all within-patient sequence alignments. Following likelihood parameter estimation, posterior probabilities of belonging to each of the site classes of ω were estimated for each site using the naïve empirical Bayes method We used HyPhy To identify sites that showed different patterns of amino acid change comparing samples from earlier versus later Fiebig stages, we included the full dataset in a maximum likelihood tree based on an alignment of all sequences from the 81 homogeneous patients. We excluded extremely hypermutated sequences, but left in the 15 patients that showed overall enrichment for G→A mutations in an APOBEC context. Phylogenetic trees used for this analysis were inferred using PhyML s sequences sampled g generations from a common ancestor is −5et al.n out of L is n changes are split as 1n and 2n at the two sites. In the calculations presented here, we used computer code to generalize these results to the case when each patient had a different number of sequences and generations since the last common ancestor.To calculate the probability of finding mutations repeatedly in multiple patients at a given site in the absence of selection, we assumed an exponential growth model with a large reproductive ratio. In such a case, a small number of sequences sampled from a patient are overwhelmingly likely to have a star phylogeny. Since in a star phylogeny every mutation arises independently, the probability of seeing a position mutated in at least one out of e 2 days  and mutaFor this analysis, all insertions and deletions (indels) in each subject relative to the subject consensus were systematically tracked, and information regarding their location, length, and the precise inserted or deleted fragments, and surrounded bases were retained and analyzed. The data were divided into 4 sets, single base insertions and deletions, and multiple base insertions and deletions, and each was considered separately. For the sequences derived from the 81 homogeneous early infections, the direction of the indel could be ascertained relative to the consensus, which was presumed to be the transmitted strain, and observed indels were assumed to be random events within the study subject. This assumption was supported by the high frequency of frameshifting inactivating substitutions among indels in the homogeneous infections. In contrast, the 21 heterogeneous infections had an excess of indels that were in-frame and thus more likely to be viable, and so probably arose in the donor and were transmitted independently to the recipient.2. PBMCs and peptides were tested in R-10 medium ). Coating, development and reading of ELISpot plates has been described previously 18mer and 9mer peptides were synthesized  to match sequences from patients MEMI, Z13, Z33 and Z36. Cryopreserved PBMC samples were thawed and rested for 2 hours before being placed in the ELISpot plates at 1×105 cells/well. Peptides in the peptide plates were mixed with 1∶1 with PBMC in the ELISpot plate to a final concentration of 2 µg/ml and incubated for 20 hours at 37°C, 5% COet al.2 for 10 days in RAB-10 ) and were stimulated with 1800 U/ml IL-2 (Novartis) on days 3 and 7. On day 10, the STCL were washed three times with PBS (Sigma) and rested for 30 hours in RAB-10 at 37°C, 5% CO2. On day 11, 4×104 cells/well were placed into an IFN-γ ELISpot as described above and stimulated by each individual peptide in triplicate at 2 µg/ml in the presence of RAB-10.This assay has been previously described in Goonetilleke et al.HLA typing was performed by T. Rostron  using the Sequence-Specific Primer (SSP) method adapted from Bunce Cryopreserved peripheral blood mononuclear cells were thawed, rested overnight and stimulated for 6 hours at 37°C, 5% CO2 with 2 µg/ml of individual HIV-1 peptides representing the wild-type and escape sequences of the epitopic regions. The stimulation was conducted in presence of anti-CD28 mAb , anti-CD49d mAb , anti-CD107a-Alexa 680 , 5 µg/ml Brefeldin A (Sigma) and 1 µg/ml Golgi Stop (Pharmingen). After stimulation, the PBMCs were stained with LIVE/DEAD Fixable Violet Dead Cell Stain , anti-CD14-Cascade Blue (M5E2), anti-CD19-Cascade Blue (HIB 19) (to exclude non-viable cells), anti-CD4-Cy5.5-phycoerythrin (PE) (M-T477), anti-CD8-QD705 (RPA-T8), anti-CD27-Cy5-PE (M-T271), anti-CD57-QD605) (NK-1), anti-CD45RO-PE-Texas Red  for 20 minutes at room temperature. PBMC were then fixed and permeabilized with Cytofix/Cytoperm (Pharmingen). PBMC were stained with anti-CD3-Cy7-APC (SK7), anti-IFN-g-fluorescein isothiocyanate (FITC) (B27), anti-IL-2- Allophycocyanin (APC) (MQ1-17H12), anti-TNF-a-Cy7-PE (MAb11) and anti-MIP-1b-PE (D21-1351)  for 45 min at 4°C. After washing and fixation samples were run on a custom built LSRII (BD Biosciences). A minimum of 300,000 total events was acquired using a custom made LSR II flow cytometer . Data analysis was performed using FlowJo 8.8.2 software (TreeStar), Pestle for background subtraction, and Spice for frequency analysis. .‘Memory’ CD8 T-cells are defined as live / lymphocytes / CD3 / CD8 / excluding the CD27hiCD45RO-negative population. Individual and total cytokine responses were considered as positive if the frequency of the total responding memory CD8+ subset was greater than 0.05% after background subtraction.Figure S1http://www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter.html) illustrating different patterns of hypermutation. Green stars show mutations consistent with Apobec hypermutation. Statistical tests of hypermutation were performed using Hypermut (http://www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html). (A) Sequences from a patient showing no evidence of hypermutation. (B) A patient with a single significantly hypermutated sequence (P = 0.003). (C) Sequences from a patient showing an overall elevated rate of hypermutation. No single sequence showed significant evidence of hypermutation; however, after compressing the mutations into a single sequence (using a tool available from the highlighter website) and re-running Hypermut with the compressed sequence, there was a significant elevation in the rate of G→A mutations in the Apobec sequence context (P = 9×10−6).Highlighter plots ((1.46 MB TIF)Click here for additional data file.Table S1Observerd mutations, sequence context within specific patients, and possible alternative explanations besides selection.(0.09 MB XLS)Click here for additional data file.