Breast cancer subtypes identified in genomic studies have different underlying genetic defects. Mutations in the tumor suppressor p53 occur more frequently in estrogen receptor (ER) negative, basal-like and HER2-amplified tumors than in luminal, ER positive tumors. Thus, because p53 mutation status is tightly linked to other characteristics of prognostic importance, it is difficult to identify p53's independent prognostic effects. The relation between p53 status and subtype can be better studied by combining data from primary tumors with data from isogenic cell line pairs (with and without p53 function).The p53-dependent gene expression signatures of four cell lines  were identified by comparing p53-RNAi transduced cell lines to their parent cell lines. Cell lines were treated with vehicle only or doxorubicin to identify p53 responses in both non-induced and induced states. The cell line signatures were compared with p53-mutation associated genes in breast tumors.Each cell line displayed distinct patterns of p53-dependent gene expression, but cell type specific  commonalities were evident. Further, a common gene expression signature associated with p53 loss across all four cell lines was identified. This signature showed overlap with the signature of p53 loss/mutation status in primary breast tumors. Moreover, the common cell-line tumor signature excluded genes that were breast cancer subtype-associated, but not downstream of p53. To validate the biological relevance of the common signature, we demonstrated that this gene set predicted relapse-free, disease-specific, and overall survival in independent test data.In the presence of breast cancer heterogeneity, experimental and biologically-based methods for assessing gene expression in relation to p53 status provide prognostic and biologically-relevant gene lists. Our biologically-based refinements excluded genes that were associated with subtype but not downstream of p53 signaling, and identified a signature for p53 loss that is shared across breast cancer subtypes. The tumor suppressor p53 is mutated in 30% of breast cancers , but ratin vitro studies of gene expression have demonstrated that cell line models of luminal breast cancers show a strong stress response following chemotherapeutic treatment, with notable changes in p53-regulated genes such as p21 (Cip1). The same magnitude of p53-regulated responses was not observed for cell line models of basal-like breast cancer . Kaplan-Meier analysis showed that this signature significantly predicted OS  and RFS (p = 2.6 × 10-7) for the Chang et al. dataset, and disease-specific survival  for the Miller et al. dataset. We also performed multivariate analysis, using the Chang et al. dataset. Controlling for standard clinical predictors , Cox proportional hazards ratios were estimated for both OS  and RFS . Thus, independent of standard clinical predictors the p53 expression classifier significantly predicted both OS (p = 0.006) and RFS (p = 0.006).Kaplan-Meier survival analysis yielded highly significant survival differences between groups from Figure et al.), the gene expression classifier had 82% agreement with sequence-based mutation status. True mutation status data was not available for the Chang et al. data set, but our classifier had 82% agreement with sequence-based mutation status in the Miller et al. data set. We were able to examine the location and type of mutations and compare them to classifier results using the Miller et al. data. Of the 29 mutants incorrectly classified as wildtype, 25 (86%) were either missense mutations or in-frame insertions/deletions. This differs significantly (p = 0.02) from the percentage of mutations that were missense or in-frame among correctly classified mutants (58%). Among the missense tumors, mutations in DNA binding domains of the p53 protein were also significantly more frequent (p = 0.01) in tumors classified as mutant (87%) than wildtype (45%).In our training data set  and are likely to be regulated by multiple oncogenic pathways. Our aim was not to identify a new prognostic signature that improves on previously published signatures. Rather, we aimed to demonstrate that events that are downstream of functional p53 loss are clearly associated with prognostic outcome, and are therefore biologically relevant. The predictive accuracy of p53-dependent gene expression profiles  supportset al. and Miller et al. datasets. This high level of agreement across data sets attests to the fact that the signature is indicative of p53 across a wide range of cell backgrounds. If the signature were merely correlated with proliferation, ER status, or another tumor characteristic, then poor concordance with p53 mutational status would be expected in cross validation. The samples where gene expression and mutation status disagree may represent true differences in the functional p53 pathway. For example, the tumor BC606 was p53 wildtype by sequence but clustered with p53 mutants using the 52 gene classifier. This tumor overexpressed MDM2 mRNA (data not shown), a key negative regulator of p53. Among the false negatives (sequence mutant but wildtype expression signature), our analysis of the Miller et al. data showed that misclassified mutants had higher proportions of mutation types that are likely to be less deleterious (missense mutations and in-frame insertion/deletions).Our data analysis also showed that there was good agreement between mutation status and expression profiles. Using our 52 gene list, there was >80% agreement between p53 mutation status and p53 expression class in both the Sorlie In addition to identifying a stereotyped signature associated with p53 loss, these results demonstrate that the relative importance of p53-regulated functions such as cell cycle control, DNA repair, and apoptosis are subject to significant inter-individual variation. Each cell line displayed a unique p53 response signature. However, similarities according to cell type were also evident. Both of the HMEC-derived cell lines showed a greater response to p53 loss in the untreated state, while the MCF-7 and ZR-75-1 lines showed a stronger p53-regulated signature following DOX treatment. These results extend previous observations [Our data reconfirmed the complex relation between chemosensitivity and p53 status . Previouin vitro setting allowed for control of expression of p53 protein, breast cancer subtype, and p53-inducing events [in vitro approach is limited in that a small number of cell lines can be reasonably examined, representing only a handful of tumors. By combining the in vitro expression data with data from human tumors assayed before and after DOX treatment, we examined a much wider range of individual responses to p53 loss than cell line experiments could reasonably examine, and performed a controlled experiment that cannot be accomplished in humans. Previous studies have characterized p53-responses in breast cancer using gene expression data from tumors and statistical models to try to control the effects of breast cancer heterogeneity. For example, in Miller et al. [A strength of our study was the use of cell line experiments to control a range of variables that influence p53-response. The g events -29. Hower et al. , prolifer et al.  was deriMany of the genes associated with p53 loss in this analysis were of prior interest in breast cancer. For example, GATA3 is involved in growth control and maintenance of the differentiated state in breast epithelial cells and has been hypothesized to play a role in tumorigenesis of ER-positive breast tumors . p21  declare that they have no competing interests.MAT participated in study design, created the isogenic cell line pairs, performed toxicity assays, western blots, and microarrays, performed analysis and interpretation, drafted and revised the manuscript. JIH participated in study design, creation of cell line pairs, interpretation of results, and manuscript revisions. DSO performed survival and gene ontology analyses. XH performed microarrays. KAH participated in toxicity assays and manuscript revisions. CSB participated in interpretation and critical review of the manuscript. CP participated in study design and coordination, supervision of experimental conduct and analysis, interpretation of results, drafting and revision of the manuscript, and approved the final version. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Microsoft Excel spreadsheet (196 kB) containing lists of genes for which expression levels were significantly associated with p53 loss .Click here for fileCluster figure showing parents and RNAi-transductants for all four parent cell lines . Genes included are the 696 genes identified as significant by SAM analyses comparing parents to RNAi-transductants.Click here for file


We show that the allelic composition of the glycolytic enzyme phosphoglucose isomerase(Pgi) has a significant effect on the growth of local populations, consistent with previously reported effects of allelic variation on flight metabolic performance and fecundity in the Glanville fritillary andColias butterflies. The strength and the sign of the molecular effect on population growth are sensitive to the ecological context , which affects genotype-specific gene flow and the influence of migration on the dynamics of local populations. The biological significance of the results forPgi is underscored by lack of any association between population growth and allelic variation at six other loci typed in the same material. In demonstrating, to our knowledge for the first time, that molecular variation in a candidate gene affects population growth, this study challenges the perception that differential performance of individual genotypes, leading to differential fitness, is irrelevant to population dynamics. These results also demonstrate that the spatial configuration of habitat and spatial dynamics of populations contribute to maintenance ofPgi polymorphism in this species.The dynamics of natural populations are thought to be dominated by demographic and environmental processes with little influence of intraspecific genetic variation and natural selection, apart from inbreeding depression possibly reducing population growth in small populations. Here we analyse hundreds of well-characterised local populations in a large metapopulation of the Glanville fritillary butterfly Pgi locus (an enzyme linked with flight metabolic performance) and local population growth within a metapopulation of butterflies.Researchers observe a link between the genetic composition of the The notion that a population's genetic composition influences its ecological dynamics is conceptually deeply rooted in population biology –3, but For gene, which encodes the cGMP-dependent protein kinase, influence foraging behaviour inDrosophila melanogaster larvae and adults. The analogous gene in honeybees influences the regulation of division of labour between forager and nurse bees  for populations with at least six copies ofPgi typed in the autumn 1995 sample and hence a minimum sample size of three larvae from three larval families (hence minimumtN was 3). The material included 139 such populations, but we lacked information for eight populations in 1996 and hence these populations had to be omitted. We added “1” to population size to allow the inclusion of populations that went extinct by 1996 (n = 34). Regional change in population sizes in the surroundings of the focal populationi is defined asi,trendN =i,tS1+/i,tS [i,*S is given byProportional change in population size  was calculated as/Si,t , whereSPgi-f allele in small and large habitat patches using the following results and assumptions. Among the 139 populations in 1995, the average sizes of “small” and “large” populations were 0.10 and 0.89 ha, respectively, using the limit ofA = 0.3 ha between the two size classes  gave the following daily probabilities of laying subsequent clutches, and the average clutch sizes: et al. ; hence wf and non-f females):f females before and after day 14, 0.05 and 0.3; non-f females before and after day 14, 0.05 and 0.15.In the experiment described by Hanski et al. , daily mL =f 0.2,Sf = 0.4, non-L =f 0.05, non-Sf = 0.4. The justifications are as follows. Hanski et al. [f females than old populations [f and non-f females, but it is likely that all butterflies have high emigration rate from very small patches (see [Proclossiana eunomia).Butterflies were assumed to stay in the natal patch until they became mated and had laid their first clutch, after which they emigrated with the following daily probabilities: et al.  found dalations . We do nes , which Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Expression of the tumour suppressor gene p53 was examined in squamous cell carcinoma of the head and neck using two p53 antibodies, PAb 421 and PAb 1801. Elevated p53 expression was found in 67% of the 73 patients investigated. P53 expression was not found to correlate with whether the patient had been previously treated or not, nor any of the clinico-pathological parameters. However a correlation was found between the patients smoking history and positive p53 staining. Six out of seven non-smokers did not express p53 whereas 29 of 37 heavy smokers were found to have elevated p53 expression (P less than 0.005). Also, of a group of ten patients who had given up smoking more than 5 years ago, nine had elevated expression. Epidemiological studies have shown a correlation between heavy smoking and head and neck cancer. The present study indicate a genetic link for this correlation.


Rad51C mutant mice exhibit precocious separation of sister chromatids at metaphase II, a phenotype that reflects a defect in sister chromatid cohesion, not a lack of Holliday junction resolution. Here we discuss a model to explain how a Holliday junction resolution defect can lead to sister chromatid separation in mouse oocytes. We also describe other recent in vitro and in vivo evidence supporting a late role for RAD51C in homologous recombination in mammalian cells, which is likely to be resolution of the Holliday junction.DNA double strand breaks are efficiently repaired by homologous recombination. One of the last steps of this process is resolution of Holliday junctions that are formed at the sites of genetic exchange between homologous DNA. Although various resolvases with Holliday junctions processing activity have been identified in bacteriophages, bacteria and archaebacteria, eukaryotic resolvases have been elusive. Recent biochemical evidence has revealed that RAD51C and XRCC3, members of the RAD51-like protein family, are involved in Holliday junction resolution in mammalian cells. However, purified recombinant RAD51C and XRCC3 proteins have not shown any Holliday junction resolution activity. In addition, these proteins did not reveal the presence of a nuclease domain, which raises doubts about their ability to function as a resolvase. Furthermore, oocytes from infertile  The breaks generated as a result of this cleavage could be sealed by a DNA ligase, resulting in intact resolved products. A search for resolvases in other organisms resulted in the identification of one of the best-characterized resolvases, RuvC of E. coli [E. coli RuvC have been identified like CCE1 in yeast, RuvC in Lactoccocal bacteriophage and A22R in Poxvirus [Exchange of genetic material by homologous recombination during meiosis is essential for generating genetic diversity in living organisms. It also results in the formation of chiasmata, that are the cytological manifestation of crossovers and ensure proper chromosome segregation. Homologous recombination is important for error-free repair of damaged DNA as well . One of  E. coli ,6, which E. coli . RecentlPoxvirus -11. SomePoxvirus ,13. In aPoxvirus -16.Saccharomyces pombe raised hopes that the eukaryotic resolvase has been identified [S. pombe mus81 mutants are infertile due to failed meiotic recombination, which could be rescued by the expression of bacterial RusA resolvase [Saccharomyces cereviciae (Mus81-Mms4), Arabidopsis (AtMUS81/At4g30870) and humans (Mus81-Eme1 or Mms4) [S. pombe mutants, S. cereviciae mus81 mutants are partially fertile, and MUS81-deficient Arabidopsis plants and mice are fully fertile, with no defect in meiotic recombination [Although many resolvases have been identified in prokaryotes, few have been found in eukaryotes. The yeast CCE1 is encoded by a nuclear gene but functions as a resolvase in the mitochondria ,17. Idenentified . It has entified . Geneticesolvase . These sor Mms4) -26. Howebination -23,27,28bination .The idea that RAD51 paralogs may be involved in resolution of HJs came from the observation that the protein fraction with HJ resolution activity contained RAD51C and XRCC3, members of the RAD51-like protein family . This obArabidopsis [Studies in chicken B-lymphocyte DT40 cells have shown that only RAD51 is essential for cell viability, while loss of other RAD51 paralogs does not affect cell survival . Interesbidopsis -40. Deplbidopsis . These fbidopsis ,42. Hereirs3 defective in RAD51C was shown to lack the HJ processing function. Recently, mouse embryonic fibroblasts generated from Rad51c knockout embryos revealed marked reduction in HJ activity [XRCC3, irs1SF, also showed a defect in HJ resolution activity, while mutation in another RAD51 paralog, XRCC2, had no effect on HJ resolution [In a very significant study, Liu et al. demonstrated that RAD51C is required for the resolution of HJs in mammalian cells . After aactivity . Interessolution . AlthougMore recently, in a follow-up study, Liu et al. provide new biochemical evidence to link RAD51C and XRCC3 to the HJ processing function . Using aMlh1-deficient mice, which have a severely reduced number of crossover sites, also showed a marked reduction in RAD51C foci. This observation strengthens the notion that RAD51C foci formation during pachytene depends on the generation of crossovers, which is one of the last steps of homologous recombination. Surprisingly, co-localization of MLH1 and RAD51C foci on the bivalents was not observed. Also, XRCC3 foci were not detected on these bivalents. These observations raise concerns and are currently difficult to explain. The lack of co-localization of RAD51C and MLH1 may be due to temporal differences in their localization at crossover sites. It is interesting that, although no XRCC3 foci were observed on autosomal bivalents, XRCC3 foci, along with RAD51C and MLH1 foci, were present in the pseudoautosomal region of the sex chromosomes, a region on X and Y chromosomes that undergoes an obligatory crossover event. These foci in the pseudoautosomal region were not detected in Mlh1-deficient spermatocytes, supporting their dependence on a crossover event.In addition to the biochemical studies, Liu et al. have presented interesting immunofluorescence data showing the localization of RAD51C on meiotic chromosomes, which further supports its role in late stages of homologous recombination . Since vin vivo model system. In Drosophila, the loss of RAD51C-like protein encoded by spn-D gene results in a meiosis-specific defect and may play a role similar to DMC1 [Arabidopsis revealed that RAD51C is essential for repair of Spo11-induced DSBs during prophase I of meiosis [Rad51c-deficient plant meiocytes fail to synapse and become severely fragmented. These results support a role for RAD51C early in meiotic recombination but do not shed light on its role later in the process.It was surprising that Liu et al. did not detect RAD51C foci during the leptotene and zygotene stages of prophase I, where it is expected to play an important role based on its known function in the early stages of homologous recombination. However, failure to observe RAD51C foci does not rule out its functional importance at this stage. Different antibodies and more sensitive imaging methods may help resolve this discrepancy. In the meantime, it may be interesting to examine the consequence of the loss of these proteins in an  to DMC1 . Recent  meiosis ,46. Homoin vivo, as a defect in the former may result in the arrest of meiocytes. Because RAD51C-deficient mice die during embryogenesis and no suitable meiosis-specific Cre transgenic mouse line is available yet, it is a challenge to generate a suitable mouse model to study the meiotic functions of RAD51C. We recently reported the generation of a hypomorphic allele of Rad51c that results in a reduction of the protein level due to aberrant splicing [in vivo model system to study the role of RAD51C in meiotic recombination. In addition, the meiotic phenotype associated with the loss of RAD51C function is sexually dimorphic, showing an early meiotic defect in males and a late defect in females, which provides a unique opportunity to study the dual function of RAD51C in mouse meiosis.Because RAD51C is predicted to have a dual function, an early and a late role in homologous recombination, it may be difficult to demonstrate the latter function splicing . This abInfertile males revealed an early role for RAD51C in meiosis, marked by the spermatocyte arrest at leptotene and early zygotene stages, reduction of RAD51 foci at leptotene, and persistence of DNA breaks and unsynapsed chromosomes at pachytene. This finding is consistent with the known function of RAD51C in DSB repair but does not provide any evidence to implicate RAD51C in the late steps of the recombination.Brca2 in the gonads have been generated and shown to have a defect in the early stages of meiotic recombination [Brca2 mutant oocytes displayed a wide range of abnormalities, including a 2.5-fold reduction in the number of oocytes that progressed to metaphase II, compared to controls and major defects in chromosome segregation into polar bodies. It will be interesting to determine whether these oocytes display any PSSC defect at metaphase II.Histological examination of the ovaries of mutant females revealed a defect in ovulation, which could be partially rectified by hormonal treatment. The number of oocytes was markedly reduced, and the embryos that developed from these oocytes suffered from severe developmental defects. The reduced number of oocytes suggests that some oocytes may have been lost due to a defect in the early meiotic prophase I. While all oocytes obtained after superovulation progressed normally to metaphase I, they displayed a number of abnormal features at metaphase II. These abnormalities included aneuploidy and broken chromosomes, but most strikingly, precocious separation of sister chromatids  declare that they have no competing interests.


Patients with breast cancer treated with MPA often report an improvement in appetite. Similar appetite stimulation is seen in patients treated with some corticosteroids, but MPA has a potential advantage over these drugs in that it does not exert a catabolic effect. MPA  has therefore been compared with placebo in 60 patients with advanced malignant disease. Twenty-one patients in the MPA group and 20 in the placebo group were receiving chemotherapy. Patients were treated for 6 weeks and were assessed at weeks 0, 3 and 6 for appetite, energy, mood and pain using visual analogue scales. Nutritional status was assessed by the measurement of serum proteins and anthropometrics. Karnofsky score was recorded as a measure of performance status. There was a significant improvement in appetite in the MPA group between weeks 0 (pre-study) and 3 (P = 0.0002) and 0 and 6 (P = 0.015). There was no significant improvement in appetite in the placebo group. Supporting this finding was the significant increase in serum thyroid binding pre-albumin and retinol binding protein in the MPA group between weeks 0 and 3 and 0 and 6 (P = 0.023 and P = 0.039 respectively). These two parameters showed no significant change in the placebo group. There was no change in anthropometric measurements, weight, performance status, energy, mood or pain in either group. These data indicate that there was a significant increase in appetite in anorexic patients with advanced cancer treated with MPA which was reflected in increases in rapid turnover proteins reported to reflect nutritional status. However, this apparent increase in appetite did not result in improved weight, performance status, energy levels, mood or relief of pain. Further studies to investigate the effect of higher doses of MPA are indicated.


The availability of antibodies which recognise p53 protein in paraffin-embedded tissue has created the opportunity to use immunohistochemistry to study the expression of p53 in a wide variety of clinical material. In this paper we have investigated the relationship between the type of fixative and the pattern of p53 staining in mammary carcinoma. Optimal results were obtained from breast tissue fixed in phenol formol saline, methacarn or cold formol saline with positive staining for stabilised p53 protein occurring in 69/95 (73%) cases studied. Care must be taken in the interpretation of these results since positive staining for p53 protein is not always indicative of mutation of the p53 gene. Furthermore, a range of staining patterns is seen in mammary carcinomas, making interpretation difficult. Assessment of staining needs to be standardised in order that different studies can be compared. However, in breast carcinoma, p53 immunohistochemistry appears to give information relating to tumour grade and, independently, to prognosis.


As with miRNAs a decade ago, the scientific community recently understood that lncRNAs represent a new layer of complexity in the regulation of gene expression. Although only a subset of lncRNAs has been functionally characterized, it is clear that they are deeply involved in the most critical physiological and pathological biological processes. This review shows that in ovarian carcinoma, data already available testify to the importance of lncRNAs and that the demonstration of an ever-growing role of lncRNAs in the biology of this malignancy can be expected from future studies. We also underline the importance of their relationship with associated protein partners and miRNAs. Together, the available information suggests that the emerging field of lncRNAs will pave the way for a better understanding of ovarian cancer biology and might lead to the development of innovative therapeutic approaches. Moreover, lncRNAs expression signatures either alone or in combination with other types of markers  could prove useful to predict outcome or treatment follow-up in order to improve the therapeutic care of ovarian carcinoma patients. Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. More than 230,000 new cases are diagnosed each year, at a late dissemination stage in most cases, leading to the death of about 140,000 women . Wh. Whcis  cell line was associated with a loss of cell polarity, as well as with increased capacities of adhesion to the peritoneum. This latter finding may be of some importance as spreading in the intraperitoneal cavity and peritoneal carcinomatosis invasion is a very common feature of ovarian cancer [n cancer .AB073614 is an lncRNA that was recently found to be overexpressed in ovarian cancer versus normal tissue in publicly available databases. The authors also checked for AB073614 expression in cancer tissue versus normal adjacent tissue in a cohort of 75 ovarian cancer patients and observed that AB073614 high expression was correlated with a shorter 5-year overall survival . In vivo, AB073614 downregulation in xenografted mice led to reduced tumor growth and reduced expression of proliferation- and invasion-related proteins (PCNA and MMP2 and MMP9 respectively). In vitro, AB073614 was greatly overexpressed in HO-8910 and OVCAR3 cell lines and its downregulation decreased proliferation and induced cell death in them. In addition, PCNA, MMP2 and MMP9 were also decreased in vitro in response to AB073614 downregulation, as well as members of key signaling pathways such as the phosphorylated forms of AKT and ERK. Although the mechanisms of action of AB073614 remain to be identified, it appears to be a key regulator of critical processes in ovarian carcinoma cells [ma cells .HOST2 is one of the five human ovarian cancer specific transcripts (HOSTs) identified by a SAGE study from a database of 137 SAGE libraries of normal and neoplastic ovarian tissue. Five tags were specifically expressed in ovarian cancer with one of them, HOST2, corresponding to a transcript lacking a coding capacity [myc, hmga2, dicer and imp3 [capacity . HOST2 wand imp3 .LSINCT5 has been shown to be overexpressed in many breast and ovarian cancer cell lines and tumor samples. LSICNT5 down-regulation decreases cell proliferation in breast cancer cell lines. A microarray analysis found that LSINCT5 down-regulation triggered the deregulation of 816 genes, of which 95 were deregulated at least 2-fold. Of those, the down-regulation of CXCR4 was postulated by the authors to be responsible for the effects on cell proliferation, but this remained to be demonstrated experimentally [mentally . In anotmentally . AlthougFAL1 was recently identified by Hu et al. [p21 (CDKN1A), leading to sustained proliferation and reduced senescence. Conversely, down-regulation of FAL1 by siRNA delivery in mice xenografted with ovarian carcinoma cells reduced tumor growth, decreasing cell proliferation and increasing apoptosis.u et al. . This lnPVT1 is an lncRNA located in the 8q24 chromosomal region. This region also harbors myc, which is amplified in 45% of ovarian carcinomas. Guan et al. [et al. [pvt1 and myc duplication in almost every tumor bearing myc amplification. Their analysis included more than 30 000 tumors from the Progenetix dataset and more than 10 000 from TCGA. When all cancers were considered, more than 18% of them presented the duplication of both pvt1 and myc, whereas fewer than 0.5% showed only myc or pvt1 duplicated. In more than 1 000 ovarian cancers from TCGA, more than 45% displayed both pvt1 and myc amplification, with only a few cases (<1%) having only myc or pvt1 duplicated. Interestingly, this study also suggested that the multiple miRNAs encoded in the pvt1 region did not play a significant role in MYC/PVT1-driven tumorigenesis [et al. [pvt1 inactivation impaired the development of tumor xenografts originating from the colon cancer cell line HCT116. This striking discrepancy might be due to the use of tumor cell lines from diverse origins (ovarian or colon cancer cells) and therefore to the existence of different cellular contexts. This underlines the need for further studies on the exact role of the myc/pvt1 couple in ovarian cancer.n et al.  reported [et al.  challengigenesis . In addiigenesis . Howeverigenesis , it woul [et al.  where pvXIST is a major player in the complex mechanism leading to heterochromatinization and subsequent transcriptional inactivation of one of the two X chromosomes in women, a phenomenon necessary for dosage compensation. A role for XIST has been described in several malignancies but its effects are dependent on the cancer type. For example, XIST expression is upregulated in glioblastoma tissue and stem cells and its knockdown exerts tumor suppressive functions. These tumor suppressive effects do not rely directly upon the effects of XIST but on miR-152, which is released from its interaction with XIST. Indeed, miR-152 binds to XIST and this miRNA is able to induce anti-tumor effects comparable to XIST downregulation, supporting a ceRNA relationship between miR-152 and XIST in this pathology [r = 0.653, P = 0.001). In the same study, a reduced expression of XIST in several ovarian cancer cell lines was associated with an increased resistance to paclitaxel. However, no modification in the response to cisplatin was observed in cell lines with reduced XIST expression. [athology . Conversathology . In ovarathology . A reducression. . OverallHOTAIR is an lncRNA expressed from the HOXC locus on chromosome 12. It has been implicated in the proximal and distal orientation during development by inducing the transcriptional silencing of genes in trans in the HOXD locus on chromosome 2 [vs. 61 months), similar to FIGO stage and histological grade [et.al [et.al [in vivo cell growth and/or capacity of adhesion to the peritoneum [mosome 2 . It has mosome 2 . In breamosome 2 . In humamosome 2 . In a coal grade . Howeveral grade . The fire [et.al  includedl [et.al  includedl [et.al . Similarl [et.al . Moreoveritoneum . Interesritoneum . This obritoneum .HOXA11-AS is an lncRNA located in the HOXA locus in chromosome 7 that harbors several coding and non-coding transcripts. HOXA coding genes regulate mullerian duct differentiation and are not expressed in ovarian surface epithelium under normal conditions. However, they are re-expressed in ovarian cancer, including some in a subtype-specific manner. For example, HOXA9 is re-expressed in serous, endometrial and mucinous subtypes, irrespective of grade, whereas HOXA10 is expressed in endometrial and mucinous but not serous subtypes [HOXA locus hosts 3 lncRNAs, namely HOXA10-AS, HOXA11-AS and HOTTIP. A recent study looked for variants of these 3 lncRNAs in a cohort of 1947 ovarian cancer patients (1201 of them of serous subtype) and 2009 control cases. No significant variant was identified, although the authors described an A>T variant in HOXA11-AS that was marginally associated with reduced ovarian cancer risk (p = 0.06). Expression of HOXA11-AS was reduced in ovarian tumor versus matched normal tissue in a cohort of 18 ovarian cancer patients, irrespective of variant status. Plasmid-based expression of HOXA11-AS, either with the “A” or “T” allele, in OVCA-433 and C19 ovarian cancer cell lines reduced proliferation, migration and invasion in vitro, as well as tumor burden in vivo in a xenograft model with C13 cells. Interestingly, expression of the “T” minor allele increased the inhibitory effects of HOXA11-AS in vitro and in vivo, in line with a putative reduced risk for ovarian cancer [trans regulation of distant genes. However, the mechanisms of the HOXA11-AS effects remain unknown, as well as the reason why the “T” allele displays an increased inhibitory effect. In this regard, the A>T variation does not change the secondary structure of HOXA11-AS. In view of this and given the lack of coding potential for HOXA11-AS, understanding why the “A” and “T” variants show significantly different activities could be of importance for further understanding the modes of action of HOXA11-AS.subtypes . The 5-pn cancer . The autANRIL is an lncRNA originating from the 9p21 chromosomal region in the same location as the p14, p15 and p16 genes, which play a central role in cell cycle regulation, apoptosis and senescence. The ANRIL transcript encompasses p14, p15 and p16 and is transcribed antisense to them. SNPs in the anril locus have been associated with a number of pathologies including cardiovascular diseases, diabetes and endometriosis [in vitro in ovarian cancer cell lines through the modulation of MET and MMP3, although the authors did not determine whether or not this regulation is dependent on p14, p15 and p16 [etriosis . ANRIL eetriosis  by epigeetriosis . The resetriosis  and prometriosis . A recen and p16 . It has  and p16 .MEG3 is an imprinted, maternally expressed lncRNA associated with PRC2, most likely via its EZH2 subunit [Meg3 promoter was reported to be methylated and MEG3 expression was absent or reduced in ovarian cancer tissues and cell lines. Its re-expression decreased OVCAR3 cell line growth and proliferation [brca1/2 [meg3 promoter methylation [ subunit . It is e subunit , which i subunit , 91. Its subunit . The Megferation . Interesferation , whereas[brca1/2 . It coulhylation . Howeverhylation . Anotherhylation . This obhylation . Howeverhylation .H19 is one of the most widely studied lncRNA and is at the center of a complex regulatory network involving the implication of miRNAs in diverse ways. H19 expression is restricted to fetal tissue and adult muscle under normal conditions [et al. [cis on the igf2 gene and in trans on the imprinted gene network controlling growth in mice [in vivo [nditions . Apart f [et al. ) through in mice , H19 has in mice  and to b in mice , 101 inc in mice . Alterna[in vivo .et al. in a muscle differentiation model [One role of H19 is through its ability to sponge the family of tumor suppressor let-7 miRNAs, as shown by Kallen on model . In ovaron model , suggestin vivo in mice where H19 overexpression increased the metastatic behavior of lung carcinoma cells [However, the involvement of H19 in cancer biology has been mainly described owing to its role as a precursor for miR-675. This miRNA displays an oncogenic role in cancer cell lines from various origins. The tumorigenic functions of miR-675 are mediated, at least in part, thanks to its ability to target directly the tumor suppressor RB in colorectal cancer  and hepama cells . TherefoBMI1 and EZH2, which are members of the PRC1 and PRC2 complexes respectively, and therefore partners in the action of lncRNAs in the control of gene expression, have been shown to be deregulated in ovarian cancer.BMI1 plays an oncogenic role in epithelial malignancies. It is frequently overexpressed in breast, cervical, endometrial and ovarian cancer [Several lines of evidence show that n cancer , 109. Inn cancer  and is an cancer . InteresEZH2, a methyltransferase subunit of the PRC2 complex, has been shown to be associated with advanced stages of ovarian cancer and to be independently associated with shorter overall survival in ovarian cancer patients [in vitro. In addition, miR-101 was found to target EZH2 directly in ovarian cancer cell lines. MiR-101 is under-expressed in ovarian cancer tissues and its low expression correlates with FIGO stage but not with tumor grade [In vivo, miR-101 restoration leads to the inhibition of growth of ovarian tumor xenografts. In another study using a cohort of patients with ovarian carcinoma, the authors confirmed several observations already made by Semaan et al. [in vivo. Finally, they demonstrated that miR-101 was less expressed in ovarian cancer cell lines resistant to cisplatin than in their sensitive counterparts and that forced miR-101 ectopic expression re-sensitized resistant cells to cisplatin, which is in agreement with the EZH2-induced resistance to cisplatin described in ovarian cancer cells [p21 and to an increase in p21 transcription [Furthermore, an elevated expression of patients . EZH2 knor grade . In vivon et al. . They foer cells . These fcription , which icription .Another interesting miRNA in the context of EZH2 is let-7. In ovarian cancer, most let-7 family members appear to be down-regulated , lncRNAs expression signatures could be used to predict outcome or response to treatment in order to improve the therapeutic care of ovarian carcinoma patients. The recent discovery that miRNAs are present in blood and other body fluids and the potential use of circulating miRNAs as diagnostic or prognostic tools in ovarian carcinoma have opened up new perspectives for the use of easily accessible biomarkers. Regarding lncRNAs, no study has yet reported their presence in serum or plasma from ovarian cancer patients. However, proof of concepts for the meaningful use of lncRNAs present in body fluids has been obtained with the characterization of urinary PCA3 levels as a diagnostic tool in prostate cancer , and cirSince several lncRNAs are overexpressed (See Table Another strategy to modulate lncRNAs activity, when it relies on chromatin modification mediated by PRC1 and PRC2 complexes, is to use the pharmacological inhibitors of these proteins. Specific inhibitors of EZH2 methyltransferase activity such as GSK126, EPZ005687 and EI1 have been recently developed . Therefoet. al. presented an original strategy in which they used a construct of diphtheria toxin under the control of the H19 promoter. The transfection of this plasmid, BC-819, led to the selective killing of malignant H19-overexpressing cells. Local IP administration of the plasmid vectorized with PEI has reached phase I in ovarian carcinoma patients, with no reported toxicity and encouraging preliminary clinical data [Furthermore, the characterization of the genes and pathways modulated by lncRNAs could lead to the identification of potential therapeutic targets for which inhibitors are under development or already available in clinical practice. In addition, the identification of the regulators of lncRNAs expression might provide interesting tools. Using this concept, Mizrahi cal data , 101. ThIn summary, the emerging field of lncRNAs has created new opportunities of investigation for a better understanding of ovarian cancer biology. Although limited to a few examples so far, the huge amount of lncRNAs whose roles and functions have yet to be unraveled holds great promise for major improvements in the understanding and future management of this disease.


Fear is an important emotional reaction that guides decision making in situations of ambiguity or uncertainty. Both recognition of facial expressions of fear and decision making ability can be impaired after traumatic brain injury (TBI), in particular when the frontal lobe is damaged. So far, it has not been investigated how recognition of fear influences risk behavior in healthy subjects and TBI patients. The ability to recognize fear is thought to be related to the ability to experience fear and to use it as a warning signal to guide decision making. We hypothesized that a better ability to recognize fear would be related to a better regulation of risk behavior, with healthy controls outperforming TBI patients. To investigate this, 59 healthy subjects and 49 TBI patients were assessed with a test for emotion recognition  and a gambling task (Iowa Gambling Task (IGT)). The results showed that, regardless of post traumatic amnesia duration or the presence of frontal lesions, patients were more impaired than healthy controls on both fear recognition and decision making. In both groups, a significant relationship was found between better fear recognition, the development of an advantageous strategy across the IGT and less risk behavior in the last blocks of the IGT. Educational level moderated this relationship in the final block of the IGT. This study has important clinical implications, indicating that impaired decision making and risk behavior after TBI can be preceded by deficits in the processing of fear. In social situations human information processing capacity is to a large extent taken up by the processing of emotional information that is crucial for making adequate decisions –3. In siStudies from different fields suggest that the experience of emotions is an important process linked to emotion perception. Theories of ‘embodied cognition’ state that facial emotion perception involves perceptual, somatovisceral, and motoric re-experiencing of the relevant emotion in one's self . In a stA frequently used task to measure risky decision making is the Iowa Gambling Task (IGT) . The IGTThe ethical committee of the University of Groningen approved of the collection of data of healthy volunteers. Assessment of TBI patients was part of the inclusion procedure for a study on behavioral sequelae after TBI, which was approved by the medical ethical committee of the University Medical Center Groningen, the Netherlands. All participants gave informed written consent prior to study inclusion for their information to be used for research, and were treated in accordance with the declaration of Helsinki.The patient group consisted of 49 moderate to severe TBI patients. At the time of assessment, all patients were outpatients who were seen in the sub-acute or chronic stage for clinical follow-up by the trauma neurologist or the rehabilitation physician of the University Medical Center Groningen. Clinicians referred patients for assessment of behavioural and social cognitive deficits.TBI patients were included if they were free from neurological conditions other than TBI, psychiatric conditions and substance abuse pre- or post-injury. Mean age of the 49 patients was 44.6 years . The group consisted of 38 male and 11 female patients. Mean educational level was 5.1  primary school only to 7) university level). There was no significant relationship between age and educational level of TBI patients . For 43 patients data were available concerning the duration of Post Traumatic Amnesia (PTA); the mean PTA duration was 25.9 days (SD 25.1), with a range from 1 to 112 days. For 25 patients Glasgow Coma Scale scores were available, ranging from 3 to 15, with a mean of 8.6 (SD 4.0). Of 42 patients imaging data (MRI or CT) with regard to frontal damage was available: 27 patients had visible lesions to the frontal lobe, while 15 patients did not have frontal lesions. Mean time since injury (TSI) was 104.8 months (SD 103.3), with a broad range of 4 to 402 months.2 = 2.8, p =.10).Normal healthy volunteers participated in the study, matched to patients based on age, gender and educational level. They were recruited through newspaper advertisement and from social networks of the investigators. They were free from alcohol or drug abuse, and had no psychiatric or neurological history. Data were collected from 59 participants, 37 men and 22 women. Mean age of the group was 43.5 years . Mean educational level was 5.4 . Age of control subjects appeared to be negatively related to their educational level . Patients and healthy controls did not differ significantly in age , distribution of educational level  or distribution of gender (XDecision making was assessed using the Iowa Gambling Task (IGT). The current Dutch computerized version of the IGT is an implementation based on the original description . ParticiDeck A and B are ‘risky’ and will eventually lead to a loss of money and deck C and D are ‘safe’ and will lead to profit. The frequency and magnitude of gains and losses differ for each deck and must be learned by the participant during the task. See Cards are drawn 100 times. For each 20 draws, the number of cards drawn from advantageous decks (C+D) are subtracted from the number of cards chosen from the disadvantageous decks (A+B) resulting in 5 net block scores.Recognition of basic emotions  was measured using the Ekman 60 Faces Test from the Facial Expression of Emotion- Stimuli and Tests (FEEST) ,51. The The course of performance in the IGT was analyzed using General Linear Model, Repeated Measures Analyses, with Difference contrast and Group as a Between-subjects variable. Box’ test was used to test the homogeneity of variance-covariance matrices assumption which was always met. When the assumption of sphericity was not met, a Greenhouse-Geisser correction was applied. When variances were equal, Bonferroni post-hoc procedure was used, with unequal variances Games-Howel post-hoc test was applied. When patients and healthy controls were compared, T-tests were used for parametric data and Mann-Whitney U tests for ordinal data. Pearson’s correlation was used for parametric data and Spearman’s rho for ordinal data. The influence of emotion recognition on IGT performance was analyzed with GLM Repeated Measures, with net block scores (CD-AB) and choices B as outcome measures. Fear, Anger, Sadness, Disgust, Happiness, Surprise, Age and Educational level were entered as covariates; Group and Gender were used as Between-subjects-factors. These analyses were subsequently performed with Fear as emotion recognition covariate together with the covariates age and educational level. Group and Gender were entered as Between-subjects-Factors. In analyzing the course of net block scores and the course of choices for deck B in TBI patients, duration of PTA and TSI were entered as covariates, while the presence/absence of frontal lesions was entered as Between-subjects-factor. Alpha levels were two-tailed with p <. 05. All statistical analyses were performed with IBM SPSS Statistics 20.Repeated measure analysis on the 5 net block scores (C+D—A+B) of the IGT showed an overall increase of positive net block scores from block 1 to block 5  = 3.2, p =.02) for both patients and healthy controls  = 0.5, p =.74). Healthy controls and TBI patients differed significantly in their performance on the IGT  = 5.4, p =.02). Healthy controls made more advantageous choices than TBI patients in Block 3  = 3.9, p =.05), while this difference just did not reach significance in block 5  = 3.5, p =.07).Age appeared to influence the course of net block scores in both patients and controls  = 2.6, p =.05; Block*age*group, F =.71, p =.57). Educational level or gender did not significantly interact with course of net block scores  = 1.6, p =.17; Block*gender, F = 1.9, p =.12) in both healthy controls and TBI patients  = 1.2, p =.29), Block*education*group, F =.32, p =.84).In TBI patients, PTA duration  =.34, p =.79), TSI  =.46, p =.71) and the presence of frontal lesions  =.10, p =.96) did not affect the course of net scores from block 1 to block 5. See In healthy controls, women had significantly higher scores on fear recognition than men . Healthy females were significantly better than healthy males in recognizing anger and disgust . In addition, total FEEST scores were significantly higher in healthy females than in healthy males .In healthy controls, higher educational level was significantly related to higher fear recognition , higher anger recognition , higher recognition of disgust  and higher total FEEST score . Age was significantly related to total FEEST score, with higher age related to poorer emotion recognition .In TBI patients, there were no significant differences between men and women in the recognition of the six basic emotions. Age of patients was not significantly related to emotion recognition. Higher educational level was significantly related to higher fear recognition ability  and to poorer recognition of surprise . PTA duration was not significantly related to emotion recognition ability. TSI was not significantly related to emotion recognition, except for a significant positive correlation between TSI and recognition of anger . Mean emotion recognition scores of patients with (n = 27) and without (n = 15) frontal lesions did not differ significantly.Repeated measure analysis on the influence of emotion recognition ability on the course of net block performance (A+B—C+D) in the IGT showed that fear recognition was significantly related to the course of decision making in the IGT  = 3.2, p =.02). Recognition of anger  =.69, p =.58), disgust  =.78, p =.52), happiness  = 1.2, p =.33), sadness  =.44, p =.75), and surprise  =.72, p =.56) were not significantly related to the course of decision making on the IGT. Three way interactions with block, emotions and group were not significant, indicating no differences in the influence of emotion recognition on the course of decision making for healthy controls and TBI patients. Three way interactions with demographical variables , block and emotions showed no significant effects,Both fear recognition and decision making were significantly worse in TBI patients compared to healthy controls. In healthy controls, but not in TBI patients, fear recognition correlated significantly with total profit on the IGT .Fear recognition scores were related to the course of net block performance (A+B—C+D) in the IGT for both healthy controls and TBI patients. GLM repeated measure analyses showed a main effect of block  = 3.3, p =.01), an interaction between fear recognition and the course of net block score  = 5.9, p =.00) but no interaction between course, fear and group  = 1.1, p =.38). Thus, fear recognition ability significantly interacted with the course of choice behavior in the IGT in healthy controls and patients. Post-hoc contrast showed the interaction between fear and choice behavior in the IGT to be significant in block 4  = 4.3, p =.04) and block 5  = 15.2, p =.000). This effect was similar for both groups  =.05, p =.83; Block5 vs previous, Block*Fear*Group, F = 2.1, p =.15). Higher fear recognition ability was related to more advantageous choices in the last blocks of the IGT in both healthy controls and TBI patients.Age was significantly related to the course of net block scores in the IGT for patients and controls. Age, however, did not interact significantly between fear recognition and net block scores in the IGT  = 2.0, p =.10). Educational level was positively related to fear recognition ability in patients. Educational level also interacted with fear recognition and net block scores in the IGT  = 2.8,p =.03), which appeared to be similar for patients and controls. Contrasts showed that this interaction effect was present in the last block of IGT  = 4.6, p =.04). When age was also entered into this model, it did not appear to significantly moderate the relationship between educational level and fear recognition  = 2.0, p =.10).Fear recognition ability was significantly better in female compared to male controls. Gender, however, did not interact significantly between fear recognition and course of IGT net block scores  = 2.1, p =.09). This was found in patients and healthy controls  = 1,5, p =.13).With regard to risky choices, healthy controls and TBI patients differed significantly in the amount of total choices for deck B. See Repeated measure analysis on the influence of emotion recognition ability on the course of choices for deck B in the IGT showed that only fear recognition was significantly related to the course of choices of deck B  = 4.5, p =.002). Recognition of anger  = 1.6, p =.19), disgust  =.45, p =.76), happiness  =.78, p =.53), sadness  =.74, p =.55), and surprise  =.28, p =.88) were not significantly related to the course of choices for deck B on the IGT. Three way interactions with block, separate emotions and group were not significant, indicating no differences in the influence of emotion recognition on the course of choices for deck B for healthy controls and TBI patients.Analyzed as a separate emotion, fear recognition ability significantly interacted with the course of choice behavior of deck B in both patients and controls  = 7.3, p =.000; Block*Fear*Group, F =.54, p =.69). Post-hoc contrast showed that fear recognition interacted with choosing deck B in block 3  = 12.8, p =.001) and block 5 of the IGT  = 13.9, p =.000). This interaction was similar in patients and healthy controls. Further analyses showed a negative correlation in both groups, with higher fear recognition abilities related to choosing deck B less often .Age did not interact significantly with the course of choices for deck B  = 1.3, p =.10), nor with fear recognition and the course of choices for deck B  = 1.1, p =.33). Educational level interacted with the course of choices for deck B  = 2.5, p =.05), but there was no significant three-way-interaction between educational level, fear recognition and the course of choices for deck B  = 2.1, p =.09). Gender was neither related to the course of choices for deck B  = 1.4, p =.25), nor did it show an interaction between fear recognition and course of choices for deck B  = 1.2, p =.32).In TBI patients, the presence of frontal lesions was neither related to the course of choices for deck B  = 0.1, p =.95) nor did it show an interaction between fear recognition and choices for deck B  =.11, p =.95). PTA-duration was not related to the course of choices for deck B  =.29, p =.83). Also, PTA-duration did not interact with fear recognition ability in influencing the course of choices for deck B  = 1.4, p =.25). TSI was neither related to the course of choices for deck B  =.62, p =.60) nor did it interact with fear recognition ability in influencing the course of choices for deck B  =.81, p =.49).This is the first study that investigated and indeed found a relation between facial fear recognition ability and gambling behavior in both healthy subjects and patients with moderate to severe TBI. In both groups, a higher ability to recognize fear, but not a higher ability to recognize other basic emotions, was related to the development of a more advantageous strategy and to the course of risky choices across blocks in the IGT. As expected, patients with TBI were significantly more impaired in recognizing fear from faces than healthy subjects –15. MoreIn both TBI patients and healthy controls decision making on the IGT was related to the ability to perceive fear. This relationship between perception and action is thought to be mediated by the ability to subjectively experience fear ,28. A reIn the present study, we also investigated in TBI patients and healthy controls whether fear recognition influences decision making under ambiguity or whether it is related to more deliberate behavior. Maia and McClelland (2004) identified three levels of awareness in the IGT . At the In the current study, fear recognition was not only related to overall decision making in both groups, but in particular to risky choice behavior at the end of the IGT. In both TBI patients and control subjects we found that fear was explicitly used as a warning signal not to choose disadvantageously. Heilman and colleagues (2010) also found in healthy subjects that explicit use of fear was related to risk behavior in the IGT . In the As expected, we found that TBI patients performed more poorly on the IGT than healthy controls. Originally, the IGT was developed to assess deficits in affective decision making after ventromedial prefrontal cortex (VMPFC) damage . HoweverIn both healthy controls and TBI patients, a negative effect of age on net block performance on the IGT was found. Performance on the IGT is based on localized but also on distributed mental information processes such as memory functioning, which are sensitive to age ,50,72. WAccording to Yechiam and colleagues (2005) different psychological components can underlie poor performance on the IGT, including a motivational tendency to attend to gains, a learning process to attend to recent outcomes and a response tendency with poor consistency of choices . With reThe present study design has some limitations. Fear recognition and risk behavior were measured, with the assumption that a better identification, experience and regulation of fear underlies behavior. It would have been valuable to additionally measure the ability of subjects to experience and appraise fear, either by questionnaires or by physiological measures. This might also give more insight into the specific deficits in the experience of fear in TBI patients. The advantage of the inclusion of an emotion recognition task is, however, that it allows objective measurement. Different from questionnaires, its responses do not depend on patients’ insight, which can also be impaired after TBI. The patient group used in the current study is a selection of patients with moderate to severe injury and behavioral difficulties. It is important to also analyze this relationship in a larger and more heterogeneous sample of TBI patients. The majority of the TBI patients in this study was male, and the inclusion of more female patients is preferred. In a greater sample of male and female patients, gender differences with regard to the influence of fear recognition on decision making can be analyzed in greater detail and with more power. However, male dominance is a characteristic of the TBI population .In conclusion, the most important finding of the present study is that fear recognition is related to deliberate choice and risk behavior in both healthy controls and TBI patients suggesting strongly that deficits in behavior are preceded by deficits in emotion processing. This is a clinically relevant finding because deficits in emotion processing might be the target of research and treatment for TBI patients with risky and inappropriate behavior. So far, only few studies have investigated the effect of training of underlying deficits in emotion processing in TBI patients ,77,78. A


Gastroenteropancreatic neuroendocrine carcinomas (GEP-NECs) are aggressive, rapidly proliferating tumors. Therapeutic response to current chemotherapy regimens is usually short lasting. The aim of this study was to examine the expression and potential clinical importance of immunoreactive p53 protein in GEP-NEC.Tumor tissues from 124 GEP-NEC patients with locally advanced or metastatic disease treated with platinum-based chemotherapy were collected from Nordic centers and clinical data were obtained from the Nordic NEC register. Tumor proliferation rate and differentiation were re-evaluated. All specimens were immunostained for p53 protein using a commercially available monoclonal antibody. Kaplan-Meier curves and cox regression analyses were used to assess progression-free survival (PFS) and overall survival (OS).All tumor tissues were immunoreactive for either one or both neuroendocrine biomarkers (chromogranin A and synaptophysin) and Ki67 index was >20% in all cases. p53 immunoreactivity was only shown in 39% of the cases and was not found to be a prognostic marker for the whole cohort. However, p53 immunoreactivity was correlated with shorter PFS in patients with colorectal tumors  in a univariate analysis as well as to poorer PFS  and OS  in patients with colorectal tumors with distant metastases, a correlation which remained significant in the multivariate analyses.In this cohort of GEP-NEC patients, p53 expression could not be correlated with clinical outcome. However, in patients with colorectal NECs, p53 expression was correlated with shorter PFS and OS. Further studies are needed to establish the role of immunoreactive p53 as a prognostic marker for GEP-NEC patients. Their proliferative capacity is high, with Ki67 proliferation index >20% and/or mitoses >20 per 2 mm2 .GEP-NECs account for approximately 35–55% of all extra-pulmonary NECs. They are mainly located in the esophagus, stomach, pancreas, colon and rectum, but in 30% of cases, they present as tumors of unknown primary location .The present WHO 2010 classification for NENs G3 has been debated for not being optimal as it assumes that all G3 tumors are poorly differentiated. Furthermore, the WHO G3 group includes all tumors with Ki67 index above 20% as one disease entity. In recent publications, the presence of tumors that are well-differentiated, but with Ki67 index >20% and with a better prognosis than poorly differentiated GEP-NECs was demonstrated . HoweverThere is a reported increase in incidence of GEP-NECs over the years, but there is still a lack of effective treatment resulting in persistent poor survival for these patients , 6. In tPlatinum-based chemotherapy has been used as a first-line treatment for GEP-NECs since the nineties and the Nordic, European and North American Societies of neuroendocrine tumors (NETs) recommend combination chemotherapy with cisplatin/carboplatin and etoposide –10. In tTP53 is a known tumor suppressor gene normally present in all human cells and the p53 pathway is usually activated by different types of stress signals due to e.g. DNA damage [TP53 are common and occur in many cancer types, including NEC: 70–100% of tumor cells have been shown to be mutated in high grade pulmonary NECs [TP53 mutations are associated with poorer clinical outcome, treatment resistance and higher degree of metastases in different types of cancer [TP53 have been shown to result in an immunohistochemically detectable expression of the p53 protein; since the mutated protein is not degraded, it accumulates into tumor cell nuclei [A damage . The tumA damage . Mutatioary NECs . Furtherf cancer –15. Mutal nuclei .A few studies have investigated the immunohistochemical expression of p53 protein in carcinomas and most of them have shown heterogeneity in the outcome , 17, 18.We hypothesized that immunohistochemical expression of p53 protein is associated with shorter progression-free survival (PFS) and OS and might be of prognostic relevance in GEP-NEC patients.This cohort included patients diagnosed with poorly differentiated GEP-NEC with a primary tumor located in the gastrointestinal tract or a cancer of unknown primary (CUP). CUP was defined as NEC with predominant abdominal metastases but where no primary tumor could be identified.Tumor specimens were collected retrospectively based on availability from the Nordic NEC Study, resulting in 124 GEP-NEC patients treated with a platinum-based chemotherapy at the Nordic Centers, and diagnosed 1999–2011. Clinical data was obtained from the Nordic NEC register [Formalin-fixed paraffin-embedded (FFPE) material included: 40 needle biopsies, 20 surgical biopsies and 64 surgical specimens. All tumors were immunoreactive (IR) for CgA and/or synaptophysin and all tumors had Ki67 index >20%. An endocrine pathologist (LG) recalculated the frequency of Ki67 IR tumor cells in all tumor samples and the morphological differentiation (well vs. poorly) was re-assessed by a panel of experienced neuroendocrine pathologists.Fifty-seven tumor specimens were retrieved from primary tumors and 67 from metastases. Exclusion criteria were Ki67 index <20% and the diagnose of a mixed adenocarcinoma-neuroendocrine carcinomas (MANEC) based on the WHO definition describing these tumors as having an exocrine and endocrine component where the neuroendocrine component is present in at least 30% of the tumor .Patients were divided into groups depending on location of the primary tumor: esophagus, stomach, pancreas, colon and rectum, or CUP. Four tumors were located in the esophagus, 11 in the stomach, 28 in the pancreas, 31 in colon and 17 in rectum. In 33 cases, the primary tumor was unknown.The clinical variables chosen to be investigated in the statistical analyses included age (median age 60 years), Ki67 index, LDH, therapeutic response  and performance status (according to the Eastern Cooperative Oncology Group consensus (ECOG)).Specimen size was included to evaluate if larger specimens yielded more tumor cells IR for p53. A sub-analysis for PFS and OS was done for patients with distant metastases for each group of primary tumor location.FFPE tissue specimens were cut into approximately 4-µm thick sections and attached to positively charged glass slides .Before immunostaining, the sections were treated in a pressure cooker reaching maximum temperature of 121°C using Tris-HCL buffered saline, pH 9.0 as retrieval solution. The sections were incubated with a primary monoclonal antibody  at room temperature for 30 minutes (dilution 1:100). A polymer-detection system was used  according to manufacturer’s instructions. Diaminobenzidine was used as chromogen.The Ki67 index was calculated in a light-microscope at a magnification of x40, with a square grid in one of the ocular to facilitate the cell counting. At least 2000 tumor cells were counted in the areas with the highest tumor cell proliferation. In small biopsies, containing less than 2000 tumor cells, all tumor cells were counted. The Ki67 index was expressed as the percentage of IR tumor cells.The presence of p53 immunoreactivity was semi-quantitatively estimated (in percent) by assessing the area of IR tumor cells vs the total tumor area by light-microscopy at a magnification of x40. Ten percent or more p53 nuclear IR tumor cells in the tumor area was considered as positive outcome based on the results from a previous study performed with the same antibody .Photographs were taken using a Zeiss Observer Z1 microscope and the Axiovision software .TP53 mutation, was used as a positive control, and omission of the primary antibody was used as a negative control.Colon adenocarcinoma tissue, with a known The defined event was death from any cause. PFS was defined as the time between date of first treatment and date of tumor progression and OS was defined as time from diagnosis of locally advanced or metastatic disease until date of death; or if event was not found, censored at date of last observation.Kaplan-Meier plots were used for PFS and OS analysis, and the log-rank test was used to compare curves separated according to expression of p53. Cox proportional regression was performed for the estimation of hazard ratios (HRs) and confidence intervals (CIs).Spearman correlation was used to assess the correlation of p53 protein expression to clinicopathological variables. For the statistical analyses all variables were dichotomized: p53 IR vs. non-IR, age ≤60 years vs. >60 years, Ki67 ≥55% vs. ≤55% LDH normal vs. high and performance status ECOG 0+1 vs. 2+3.All statistical analyses were performed using IBM SPSS statistics software .Local ethics committees in the Nordic countries from which tissue samples were collected approved the research protocol.The study was approved and the need for consent was waived by the local ethics committee, Regionala etikprövningsnämnden , in Uppsala, Sweden.Of the 124 tumors stained, 39% (n = 48) were p53 IR . The freColon primary was the tumor group with most frequently p53 IR cells followed by pancreas, CUP, rectum, stomach and esophagus. In both the p53 IR and p53 non-IR groups, approximately 80% had distant metastases and Ki67 was >55% in a majority of patients in both groups .Based on the p53 staining, two different patterns could be distinguished: one with scattered cells and the other with densely packed cells. Five of the 48 p53 IR tumors (10%) showed a scattered pattern, where 20–40% of the tumor cells were p53 IR. The remaining 43 (90%) had a homogenous pattern where 60–100% of the tumor cells were IR .Statistical analyses of the whole cohort with all clinical variables dichotomized showed that p53 immunoreactivity was positively correlated with Ki67 with a higher frequency of p53 IR cells for patients with Ki67 index above 55%. The tumor specimen size correlated positively with p53 immunoreactivity in the whole cohort i.e. large specimens were more often IR. There was no correlation between p53 expression and small cell or large cell morphology.For patients with colorectal tumors, a positive correlation was found between p53 immunoreactivity and performance status showing that those with p53 IR tumors had a poorer performance status compared to those with non-IR tumors. For patients with colorectal tumors and distant metastases, p53 immunoreactivity correlated negatively with treatment response. No significant result was obtained for correlation with response from sub-analysis of patients with metastatic primaries in esophagus, the gastric mucosa or pancreatic or in the CUP subgroup.Spearman’s correlations are presented in Kaplan-Meier analysis, dichotomized for p53 immunoreactivity, including all 124 patients did not show any differences in PFS and OS  . In the In univariate analysis, p53 immunoreactivity showed a significant correlation with shorter PFS in colorectal patients  and patients with colorectal tumors and distant metastases showing p53 immunoreactivity had a significantly shorter PFS and OS  compared to patients with non IR- tumors .In the group of colorectal patients with distant metastases, these associations remained significant in multivariate analysis adjusted for age, Ki67 index, LDH and the ECOG for performance status . The ranDCR after platinum-based chemotherapy in this cohort was 67% .In the group of patients with colorectal tumors with distant metastases, a positive correlation was found between p53 immunoreactivity and disease control by chemotherapy . For patThe p53 positive control showed IR tumor cells. When omitting the primary antibody, the immunoreactivity was completely abolished.To our knowledge this is the largest study investigating p53 protein expression as a possible prognostic marker for GEP-NEC patients. We found that patients with colorectal NECs with distant metastases, expressing p53, had a significantly shorter PFS and OS, than those lacking p53 expression. This is in line with the correlation between performance status and response within the same group. Furthermore, the positive correlation between p53 expression and Ki67 indicates that p53 may be a marker for poorer prognosis .In accordance with these findings, others have also suggested that p53 expression might be useful to distinguish between GEP-NECs and tumors belonging to the recently recognized category of well differentiated G3 NENs [Two immunohistochemical staining patterns were observed for p53 expression. One subgroup of tumors exhibited scattered p53 IR tumors cells, where 20–40% of the tumor cells were IR, while another subgroup displayed densely packed tumor cells with 60–100% of tumor cells showing immunoreactivity. This is in accordance with a study of p53 in gastric cancer where similar staining patterns were observed. However, as in this study, no correlation was seen between these patterns and clinicopathological parameters , i.e. thWe found that tumor specimen size correlated positively with p53 protein expression in the way that surgical specimens were more frequently IR for p53 than needle biopsies. This finding is rather unexpected since patients undergoing surgery are more likely to have a better performance status and expected longer OS, and presence of p53 IR tumors is hypothesized to be associated with poorer outcome. One possible explanation might be that biopsies can be less representative than larger specimens.TP53 gene have been observed in a significant number of cases, suggesting it may have a role in NEC development [TP53 gene was confirmed in a recent report, in which next-generation sequencing of 50 cancer-related genes in 23 NEC tumors showed several different mutations, but with the presence of TP53 mutation in the majority of tumors. A high number of tumors with TP53 mutation has also been demonstrated in pancreatic NEC-patients [Genetically, GEP-NECs show chromosomal instability. Alterations in the elopment , 23. FreTP53 mutation in tumors, and consequently, provides a quicker screening method to choose appropriate treatment [Results from a study of ovarian carcinoma demonstrate that p53 expression can be used as an alternate marker for reatment . Howeverreatment .There was a significant correlation between response to chemotherapy and p53 expression in patients with colorectal tumors with distant metastases; patients with a p53 expression had a poorer response to platinum-based chemotherapy. These patients also had a worse prognosis in general, and patients with p53 IR tumors scored lower on the ECOG scale for performance status.In conclusion, p53 expression was correlated with poorer survival and poorer response to chemotherapy in patients with colorectal NECs, especially for those with distant metastases. To confirm the possible prognostic role of p53 in GEP-NECs, additional genetic studies are necessary. These should preferably be prospective and include genotyping to compare genetic information with p53 immunoreactivity and other possible biomarkers such as RB1, ATRX and DAXX to further investigate the relationship to clinical outcomes.


S. cerevisiae has been particularly useful. This seemingly naive model allows the expression of a functional human p53 and thus the assessment of p53 intrinsic transcriptional activity. The aim of this article is to review the various contributions that the budding yeast has made to the understanding of p53, p63 and p73 biology and to envision new possible directions for yeast-based assays in the field of cancer as well as other p53-family-related diseases.Since its discovery in 1979, p53 has been on the forefront of cancer research. It is considered a master gene of cancer suppression and is found mutated in around 50% of all human tumors. In addition, the progressive identification of p53-related transcription factors p63 and p73 as well as their multiple isoforms have added further layers of complexity to an already dense network. Among the numerous models used to unravel the p53 family mysteries,  Saccharomyces cerevisiae has proven to be a precious tool to unravel its mysteries. 37 years after p53 original discovery, this article reviews the various uses of S. cerevisiae and envisions new possible applications for already existing yeast-based assays, as well as the creation of original yeast models dedicated to the study of the p53 family.Mutation of p53 is the most common genetic alteration in human cancers , 2, makiS. cerevisiae took part in the early identification of p53 function. In order to verify the suspected transcription activation role of p53, R.W. O’Rourke et al. tested its ability to activate the transcription of the CAT (chloramphenicol acetyltransferase) reporter gene. The amino-terminal fragment of p53 was indeed able to induce transcription and p53 was thus proposed to be a transcription factor of the tumor suppressive response . Al. Al32]. Although p63 is scarcely found mutated in cancers, mutations in this gene are strongly linked to developmental syndromes such as ADULT  and EEC  . The majBaker's yeast allows the expression of a functional human p53 protein, which is isolated from its natural mammalian partners making it a seemingly ideal model to study p53 intrinsic transcription factor activity and the impact of its mutations.Homo sapiens, Mus musculus, Xenopus laevis, Danio rerio, Drosophila melanogaster and Caenorhabditis elegans) using a firefly reporter system under the control of various p53-REs sequences . Significant variations in sequence specificity, temperature sensitivity or transcriptional potential of p53 were highlighted between these different species [Yeast has also been used to assess the divergence of p53 transcriptional activity throughout evolution. M. Lion et al. compared the transcriptional response of p53 from several species , apoptosis-inducing factor (YNR074C or AIF1), endonuclease G (NUC1) or Bcl2-like proteins (Ybh3p) [NUC1 [S. cerevisiae, p53-WT, but not p53-R248W, induces signs of apoptosis which are associated with the repression of thioredoxin genes (TRX1/2) and the production of high levels of reactive oxygen species [ACT1 which over-expression is known to be toxic in yeast [in levuro may appear limited, it provides interesting elements to the understanding of p53 family transcriptional role. And even if the quest for a p53 orthologue in yeast remains unsuccessful to date, answers may come from digging deeper in the function of the older evolutive member p63, which has been implicated in female germline protection during meiosis in vertebrates [In yeast, the apoptotic machinery seems conserved, since several key elements homologues have been identified that include caspases ( (Ybh3p) . Confirmp) [NUC1 . Moreove species . In addiin yeast , 61. Of in yeast . p53 hasin yeast . The exitebrates . This futebrates , 66 and M. trossulus [Yeast two-hybrid (Y2H) allows the identification of protein-protein interactions and has been used to explore the vast interaction network of p53. Y2H allowed the identification and characterization of some of p53 most prominent partners like Mdm2. Mdm2 is an E3-ligase which is a core regulator of p53 in charge of maintaining cellular p53 levels low in the absence of stress by promoting its ubiquitination and consequently its degradation by the 26S proteasome. Mdm2 was also found to inhibit p53 transcriptional activity by binding to its transactivation domain . Y2H wasrossulus . Two othrossulus . Severalrossulus , BAF60 [rossulus , Ki-1/57rossulus  or influrossulus . In addirossulus , 53BP1 [rossulus  or MBP1 rossulus . More rerossulus . Among trossulus .Due to their late identification, p63 and p73 are not renowned for their large social networks but can be expected to close the gap in a near future. At least two p63 partners have been discovered using Y2H: Stxbp4  and SetdInteractions within the p53 family have also been subjected to Y2H experiments and showed strong homotypic interactions  but much lower heterotypic interactions due to the diversification of their respective tetramerization domains . Howevervia its C-terminal domain in yeast, although no RNA sequence or structure specificity was identified. Specific p53-RNA interaction might indeed be dependent on post-translational modifications of its C-terminal domain that do not occur in yeast [Yeast three-hybrid, a variant of the yeast two-hybrid, allows the detection of interactions between proteins and RNA. Due to the highly basic composition of the p53 C-terminal domain, K.J.-L. Riley et al. looked for possible RNA sequences able to associate with p53. They showed that p53 is indeed able to bind RNA sequences in yeast .in vitro, many have fallen short in vivo [The discovery of drugs targeting p53 remains highly dynamic but in spite of multiple promising candidates identified  in vivo . Two appMdm2 interacts with p53 in yeast and induces a diminution of its transcriptional activity. However Mdm2 does not induce a significant degradation of p53 in yeast, but can bind to its transactivation domain. Mdm2 would thus interfere with transcription cofactors such as 53BP1, through a competitive mechanism. These results indicate that, although most of the p53 pathway is absent from yeast , the intADE2 reporter gene by Firefly or Renilla luciferase reporters allowing a miniaturization of the assay and hence a high throughput. Compounds targeting the p53-Mdm2 interaction (Nutlin and RITA) are also effective in yeast and restore p53 transcriptional activity, thereby validating the yeast model for further screening. In contrast, PRIMA-1, which restores mutant p53 transcriptional activity in vitro, happens to be inactive in yeast [in silico for their ability to bind Mdm2 and then in yeast for their ability to restore p53-dependent growth inhibition. Several candidate drugs were isolated that include pyranoxanthone that turned out to be also active in mammalian cells [In order to use yeast as a model for pharmacological screening, FASAY has once again been adapted by replacing the in yeast . Recentlan cells , α-Mangoan cells . The saman cells  and led an cells . Nutlin-an cells . Unlike an cells .p53 has travelled across numerous models, most of the time with its mammalian companions, and seems to fit in everywhere but it definitely found a second home in baker's yeast. From the early years of p53 characterization to recent high-throughput pharmacological screenings, yeast has proven to be a versatile tool, which helped unraveling p53 mysteries. Now 21 years old, FASAY has undergone various transformations and evolutions to accompany the new questions about p53, p63, p73, their mutants and isoforms and it will probably not stop there as the p53 field is far from drying out. The p53 family remains an “unresolved puzzle”  of whichIn a sense, p53 really recalls the disease it contributes to cause. In the early developments of chemotherapy, the question asked was: is it possible to destroy cancerous cells while preserving the sane ones? With p53 one can wonder: how can we disable its mutants or isoforms while preserving its functional elements? The answer will not come easily, but baker's yeast is likely to help solving this critical issue.


Acetylcholine and dopamine are neurotransmitters that play multiple important roles in perception and cognition. Pharmacological cholinergic enhancement reduces excitatory receptive field size of neurons in marmoset primary visual cortex and sharpens the spatial tuning of visual perception and visual cortical fMRI responses in humans. Moreover, previous studies show that manipulation of cholinergic or dopaminergic signaling alters the spatial tuning of macaque prefrontal cortical neurons during the delay period of a spatial working memory (SWM) task and can improve SWM performance in macaque monkeys and human subjects. Here, we investigated the effects of systemic cholinergic and dopaminergic enhancement on the precision of SWM, as measured behaviorally in human subjects. Cholinergic transmission was increased by oral administration of 5 mg of the cholinesterase inhibitor donepezil, and dopaminergic signaling was enhanced with 100 mg levodopa/10 mg carbidopa. Each neurotransmitter system was separately investigated in double-blind placebo-controlled studies. On each trial of the SWM task, a square was presented for 150 ms at a random location along an invisible circle with a radius of 12 degrees of visual angle, followed by a 900 ms delay period with no stimulus shown on the screen. Then, the square was presented at new location, displaced in either a clockwise (CW) or counterclockwise (CCW) direction along the circle. Subjects used their memory of the location of the original square to report the direction of displacement. SWM precision was defined as the amount of displacement corresponding to 75% correct performance. We observed no significant effect on SWM precision for either donepezil or levodopa/carbidopa. There was also no significant effect on performance on the SWM task  for either donepezil or levodopa/carbidopa. Thus, despite evidence that acetylcholine and dopamine regulate spatial tuning of individual neurons and can improve performance of SWM tasks, pharmacological enhancement of signaling of these neurotransmitters does not substantially affect a behavioral measure of the precision of SWM in humans. Spatial working memory (SWM) refers to the short-term storage of locations of items not currently present in the environment for immediate use. The limits on working memory can be quantified by measuring capacity (the amount of information that can be remembered) as well as precision . In the domain of visual SWM, precision is often quantified as the average distance in the visual field between the encoded location and the location reported during retrieval.Neural correlates of SWM precision have been described in macaque dorsolateral prefrontal cortex (dlPFC). Here, neurons exhibit sustained spiking activity during a delay period between encoding and retrieval, and the magnitude of this activity varies as a function of the remembered location  completed the donepezil study, and 20  completed the levodopa/carbidopa study. One female subject from the donepezil study and two female subjects from the levodopa/carbidopa study were excluded from the analyses because their calculated SWM thresholds were greater than the maximum displacement we tested (described in “Stimuli and Task” section below).Subjects were not enrolled in the study if they reported that they smoked tobacco, were taking any drugs that could affect cholinergic (for the donepezil study) or dopaminergic (for the levodopa/carbidopa study) function, or had a history of substance abuse, heart arrhythmia or heart problems, neurological or psychiatric illness, or liver disease. Because levodopa/carbidopa can cause hypotension, blood pressure was measured just before administration of levodopa/carbidopa (or placebo). Participants were required to have a resting blood pressure reading between 100/60 mmHg and 140/90 mmHg and a pulse rate above 60 bpm to continue in the experiment. Participants’ ages ranged from 18 to 27 (donepezil study) and from 19 to 31 (levodopa/carbidopa study).We employed a double blind within-subject experimental design in which each subject ingested either placebo or an active drug  on different days. Carbidopa was co-administered in order to inhibit peripheral metabolism of levodopa, thereby allowing more levodopa to cross the blood-brain barrier  they did not take during the second session. Participants waited 3 h after ingesting donepezil and 45 min after ingesting levodopa/carbidopa to begin the SWM task, intervals that correspond to the time to reach peak plasma concentration after oral ingestion for each drug . Across all eccentricities, the mean hit rate was 61%, and the mean correct reject rate was 75%. It should be noted that we used a 500 ms stimulus presentation time in this control experiment instead of the 150 ms stimulus duration used in the SWM experiments, as 150 ms is not enough time for the subjects to make an eye movement to the target while it was still being displayed. This 150 ms stimulus duration was selected to discourage eye movements to the stimulus to be remembered during the SWM task.During the SWM experiment, participants were encouraged to take breaks whenever they wanted to, and they communicated this by either withholding their response or informing the experimenter, who would then pause the experiment after the subject’s response. Additionally, the experimenter explicitly asked participants if they wanted to take a break every time they completed 20% of the trials .For the baseline session, the set of displacements was 0.3, 1, 2, 3, 4, 6, 8 and 10 degrees (defined as the polar angle between the encoded stimulus and the probe). Performance was plotted as a function of this displacement angle Figure  to comput(37) = −1.49, p = 0.14). We therefore included all collected data in our analyses.For the pharmacology sessions, displacements ranged from 0.3 to 12 degrees of polar angle, with the intervening displacements at 10%, 30%, and 60% above and below the subject’s threshold (computed from the baseline session). The baseline session had 960 trials, and the pharmacology sessions had 1080 trials each. Due to experimenter error, for a subset of the participants (10 in the donepezil study and seven in the levodopa/carbidopa study), data were not collected at a displacement of 60% above threshold. In order to estimate the effect of this missing data, we removed the 60% above threshold data point from those subjects with a complete data set and then recomputed the thresholds. We found that there was no significant difference between thresholds calculated from the complete data set and those from the data that were missing the 60% above threshold value  = 0.10, p = 0.73) or levodopa/carbidopa (t(17) = 0.49, p = 0.12; Figure To assess stability of SWM precision across multiple testing sessions, we compared threshold displacement (measured in units of degrees of polar angle) for the two pharmacology sessions in each study (acetylcholine and dopamine) using paired t(17) = −0.25, p = 0.81) or between levodopa/carbidopa and placebo (t(17) = 0.80, p = 0.44; Figure We observed no significant difference in SWM precision thresholds between donepezil and placebo (t(17) = 0.46, p = 0.65; levodopa/carbidopa: t(17) = 0.50, p = 0.62; Figure We also examined the effects of cholinergic and dopaminergic enhancement on overall task performance (percent correct) and again observed no significant drug effects (donepezil: t(17) = 1.21, p = 0.24) or levodopa/carbidopa (t(17) = 0.50, p = 0.62) on lapse rate  or levodopa/carbidopa /(SWM placebo threshold + SWM drug threshold)) for each study. This contrast index will have a value of zero when the drug has no effect on displacement threshold, positive values when the drug enhances precision (decreases threshold), and negative values when the drug reduces precision (increases threshold). This correlation was not significant for either donepezil (t(35) = −0.70, p = 0.48) or between the left and right hemifields (t(35) = −1.25, p = 0.21). Lower visual field advantages in perception have often been measured for stimuli on or near the vertical meridian  = 0.71, p = 0.47).Finally, we explored whether SWM precision varies across different locations in the visual field. There is a well-established lower visual field advantage in performance for a variety of visual perception tasks  = 2.24, p = 0.03; Figure t(17) = −1.21, p = 0.23; Figure t(17) = 1.52, p = 0.15; Figure The oblique effect is another well-studied anisotropy in visual perception across visual field locations Appelle, , charactThe purpose of this study was to investigate the effects of cholinergic and dopaminergic enhancement on SWM precision, using the cholinesterase inhibitor donepezil and the dopamine metabolic precursor levodopa, respectively. We found no detectable effects of enhanced acetylcholine and dopamine signaling on either SWM precision or task performance.At the single neuron level, local administration of acetylcholine reduces excitatory receptive field size in marmoset V1  could reveal cholinergic and/or dopaminergic effects on a behavioral measure of SWM precision in humans.The acute dose of donepezil that we used was 5 mg, corresponding to the lowest dose prescribed clinically for daily administration. While it is possible that cholinergic effects on SWM precision would be observed at higher doses of donepezil, previous studies in our lab have documented statistically significant effects of a single dose of 5 mg donepezil on spatial extent of fMRI responses in visual cortex  in the donepezil study and 0.60 degrees (19.1% change from placebo) in the levodopa/carbidopa study in order to produce a significant drug effect at Another consideration is that we used a delay period of 900 ms without a visual mask. In principle, persistence of the sensory response to the stimulus to be remembered could have aided subjects’ performance on the SWM task. However, this type of retinal persistence, often studied in the psychological literature as iconic memory, fades after 300 ms (Sperling, Much of the evidence for cholinergic and dopaminergic effects on spatial tuning of working memory representations comes from studies of macaque dlPFC neurons. Although the SWM task we employed is very similar to that used in the macaque studies, recent evidence from human patients with lesions to dlPFC has raised questions about the homologies between humans and macaques in this region (Mackey et al., These lesion results are supported by a recent transcranial magnetic stimulation study in which disruption of human dlPFC did not affect accuracy of memory-guided saccades (Mackey and Curtis, Our results support a distinction between the limits of spatial resolution in visual cortical neurons and visual perception and the corresponding limits in SWM representations. Although there are clear cholinergic effects on spatial resolution at the level of single neurons (Roberts et al.,  (Harewood et al., We also found no evidence for visual field asymmetries in the precision of SWM, a result that also indicates fundamental differences between the spatial resolution of perception and memory. Previous studies have documented a clear lower visual field advantage in visual crowding tasks (He et al., Thus, even though SWM representations must be derived from perceptual representations to some extent, we have found fundamental dissociations between the spatial resolution of SWM and perception, both in their associated neurochemical mechanisms as well as visual field anisotropies.ANHS, JAC and MAS designed the experiment and gave final approval of this version of the manuscript to be published. ANHS and JAC collected data and created figures for the article. ANHS and MAS created data analysis procedures, interpreted data, and drafted and edited the manuscript.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. The handling editor is currently co-organizing a Research Topic with one of the reviewers AD, and confirms the absence of any other collaboration.