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SPARC, FOXP3, CD8 and CD45 correlation with disease recurrence and long-term disease-free survival in colorectal cancer.

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SPARC, FOXP3, CD8 and CD45 Correlation with Disease Recurrence and Long-Term Disease-Free Survival in Colorectal Cancer Angela Chew1,2, Paul Salama3, Anneli Robbshaw1, Borut Klopcic1,2, Nikolajs Zeps3,4,5, Cameron Platell3,6, Ian C. Lawrance1,2* 1 Centre for Inflammatory Bowel Diseases, Fremantle Hospital, Fremantle, Western Australia, Australia, 2 School of Medicine and Pharmacology, University of Western Australia, Fremantle, Western Australia, Australia, 3 School of Surgery, University of Western Australia, Nedlands, Western Australia, Australia, 4 Radiation Oncology, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia, 5 St John of God Pathology, Subiaco, Western Australia, Australia, 6 St John of God Colorectal Service, Subiaco, Western Australia, Australia Abstract Background: SPARC is a matricellular protein involved in tissue remodelling, cell migration and angiogenesis, while forkhead box P3 (FOXP3) protein functions as a transcription factor involved in immune cell regulation. Both SPARC and FOXP3 can play an anti-tumorigenic role in cancer progression. The aim was to determine if SPARC, FOXP3, CD8 and CD45RO expression levels are associated with colorectal cancer (CRC) stage, disease outcome and long-term cancer-specific survival (CSS) in stage II and III CRC. Methods and Findings: SPARC expression was initially assessed in 120 paired normal and stage I-IV CRCs. Subsequently, approximately 1000 paired patient samples of stage II or III CRCs in tissue microarrays were stained for SPARC, FOXP3, CD8 or CD45RO. Proportional hazards modelling assessed correlations between these markers and clinicopathological data, including disease outcome and cancer specific survival (CSS). Both SPARC and FOXP3 expression were significantly greater in CRC than normal colon (p,0.0001). High SPARC expression correlated with good disease outcome ($60 mths without disease recurrence, p = 0.0039) and better long-term CSS in stage II CRC (,0.0001). In stage III CRC, high SPARC expression correlated with better long-term CSS (p,0.0001) and less adjuvant chemotherapy use (p = 0.01). High FOXP3 correlated with a good disease outcome, better long-term CSS and less adjuvant chemotherapy use in stage II (p,0.0037, ,0.0001 and p = 0.04 respectively), but not in stage III CRC. High CD8 and CD45RO expression correlated with better disease outcome in stage II CRC, and better CSS, but the differences were not as marked as for SPARC and FOXP3. Conclusions: These data suggest that high SPARC and FOXP3 are associated with better disease outcome in stage II CRC and may be prognostic indicators of CSS. Further assessment of whether these markers predict patients at high risk of recurrence with stage II CRC and functional studies of these effects are underway Citation: Chew A, Salama P, Robbshaw A, Klopcic B, Zeps N, et al. (2011) SPARC, FOXP3, CD8 and CD45 Correlation with Disease Recurrence and Long-Term Disease-Free Survival in Colorectal Cancer. PLoS ONE 6(7): e22047. doi:10.1371/journal.pone.0022047 Editor: Xin-yuan Guan, The University of Hong Kong, China Received January 10, 2011; Accepted June 15, 2011; Published July 26, 2011 Copyright: ß 2011 Chew et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research was undertaken with the support from the Fremantle Hospital Medical Research Foundation and the National Health and Medical Research Council (NHMRC# 458755). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Ian.Lawrance@uwa.edu.au characteristics of the tumour, through the identification of biomarkers that predict disease extent, may provide a more accurate means of correlating cancer stage with disease outcome, and allow application of the most appropriate treatments for individual patients. Tumour infiltrating T lymphocytes that express CD8 and CD45RO have been shown to correlate with better CRC outcomes[7,8,9,10]. The presence of a high density of CD8+ T cells is also associated with the absence of tumour invasion, an earlier disease stage and improved patient survival[11,12]. High CD45RO+ T cell densities in lymph node metastasis and tumours of CRC also associate with less tumour invasiveness, lower disease stage and a better patient survival[9,12]. Two further potential biomarkers of interest in the pathogenesis and prognosis of CRC are Secreted Protein Acidic and Rich in Cysteine (SPARC) and Introduction Colorectal cancer (CRC) is highly prevalent in western populations and one of the leading causes of cancer morbidity and mortality worldwide[1,2]. Excluding non-melanoma skin cancer, CRC is the most common cancer in Australia and the second most common cause of death due to malignant disease[3,4]. The American Joint Committee on Cancer (AJCC) developed a commonly used staging system to determine patient prognosis. There is, however, overlap in the 5-year survival rates between stages II and III CRC[5,6]. Stage III cancers have demonstrated lymphatic spread whilst stage II cancers have tumours localised to the intestinal mucosa. Some of the stage II tumours, however, may also have micrometastases that go undetected. In view of this, consideration of the biological PLoS ONE | www.plosone.org 1 July 2011 | Volume 6 | Issue 7 | e22047 SPARC and FOXP3 in Colorectal Cancer forkhead box P3 (FOXP3) as they are also both highly expressed in malignant diseases and have been demonstrated to significantly correlate with disease outcome. SPARC is a matricellular glycoprotein that is involved in tissue remodelling, wound repair, cell migration and angiogenesis[13,14,15,16]. It is expressed in numerous tissues, especially in damaged tissues where extracellular matrix (ECM) remodelling is necessary in response to injury[17]. High SPARC expression has been associated with enhanced tumour growth, metastasis and poor disease prognosis in various malignancies including melanoma, breast and oesophageal cancers[6,18,19]. SPARC has also been implicated in colonic polyp development and CRC tumour progression through the regulation of cycloxygenase-2 (COX2)[20] and transforming growth factor beta-1 (TGFb-1)[21]. These are involved in regulation, and breakdown, of the ECM surrounding tumour cells. In ovarian cancers, however, high SPARC expression levels are associated with a good prognosis[22]. This correlation is a result of SPARC’s ability to downregulate macrophage recruitment to the tumour site and the production and activity of interleukin (IL)-6, prostanoids and matrix metalloproteinases to decrease the associated inflammation[23]. FOXP3 is also a biological marker of interest and is the key transcription factor controlling regulatory T cell (Treg) development and function. Tregs suppress the activation of the immune system, thereby maintaining immune system homeostasis and tolerance to self-antigens. In doing so, however, these cells can suppress the anti-tumour immunological response by reducing cytotoxic T cell activity by direct cell-to-cell contact, or through the release of cytokines[24]. A direct link between the presence of Tregs and the progression of ovarian cancer has been demonstrated, where tumour FOXP3+ Tregs suppressed tumour specific immunity and contributed to reduced survival[25]. In breast cancer, an increase in the Treg population, both in peripheral blood and tumour tissue, was also reported and a recent study demonstrated a correlation between intratumoural infiltration of FOXP3+ Tregs in breast cancer and the risk of late relapse[26]. Given the central contribution of FOXP3 to Treg function, the expression of FOXP3 by tumour cells may represent a novel mechanism by which cancers suppress the immune system to escape destruction. In this study we investigated SPARC and FOXP3 in stage II and III CRC tissue and compare their prognostic value against the known CRC prognostic markers CD8 and CD45RO that are expressed by tumour infiltrating T lymphocytes. These have been shown to correlate with a better CRC disease outcome[7,8,9,10]. The presence of a high density of CD8+ T cells is associated with the absence of tumour invasion, an earlier disease stage and improved patient survival[11,12]. The aims of this study were to determine if SPARC and FOXP3 expression levels are associated with CRC stage, disease outcome and long-term cancer-specific survival in stage II and III CRC and to compared the prognostic value of these two markers against the known prognostic CRC markers CD8 and CD45RO. Patient details Demographic information was collected retrospectively on all patients from pathology records, and information on diseasespecific survival was obtained from the Cancer Registry of Western Australia. All patients had a minimum follow-up of 60 months or until death. Information on the use of adjuvant chemotherapy with fluorouracil/leucovorin-based regimens following surgery was obtained from hospital medical records. Patients were considered to have had a good disease outcome if there was no disease recurrence within 60 months of diagnosis, while patients who had disease recurrence within 60 months of diagnosis, or had died from their disease, were considered to have had a poor disease outcome. Initial SPARC assessment SPARC levels were initially assessed in histological sections of AJCC stage I to IV CRC tissue to determine if there was an association with CRC and in which cells SPARC was expressed. This included samples (n = 120) collected from a 464 patient cohort diagnosed with CRC between 1996 and 2002. Tissue microarray (TMA) Construction of the TMAs used in this study has previously been described[27]. The TMAs consisted of approximately 1000 cases each of stage II and III CRC archival tissue samples from patients who were diagnosed with CRC during the period of 1990 to 1999 at the Sir Charles Gairdner Hospital. Each case consisted of two 1mm diameter tumour tissue cores and an additional 1mm diameter core taken from histologically normal colon mucosa. Assessment of SPARC expression was undertaken on 233 patients with AJCC stage II and 125 AJCC stage III CRCs. T cell density analysis of FOXP3, CD8 and CD45RO was derived from a larger patient cohort of the same archived AJCC stage II and III CRC samples that has previously been described[28]. Four micrometer thick formalin-fixed paraffin embedded sections from the TMAs had high temperature antigen retrieval with 1 mM EDTA (pH 8) and 10 mM citrate buffer (pH 6). Nonspecific staining was blocked. Primary antibodies anti-SPARC (1:200; Hematologic Technologies Inc, Vermont, USA), anti-CD8 (clone C8/144B, ready to use; DakoCytomation, Heverlee, Belgium), CD45RO (clone UCHL1, ready to use; DakoCytomation) and anti-FOXP3 (1:100; Abcam, Cambridge, MA, USA) were used with an overnight incubation at 4uC. After washing, the sections were incubated with 1:2 diluted biotinylated link universal antibody and streptadvidin-HRP solutions (Universal LSAB+ kit, DakoCytomation Carpinteria, CA) for 30 minutes each. Colour was developed by incubating in 3,3-diaminobenzidine (DAB) solution (Substrate Chromogen System, DakoCytomation, Carpinteria, CA) and sections were counterstained with Mayers haemotoxylin and coverslipped with Depex. Staining without primary antibody was used as a negative control. Pictures were taken using a digital camera (Nikon DS-L1) in bright field on an inverted microscope (Nikon TE2000-U). Tissue microarrays were scanned using the Aperio ScanScope XT Digital Slide Scanner (Aperio Technologies, CA) located at the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia Materials and Methods Ethics statement Ethical approval for this research was obtained from the Human Research Ethics Committee of the Southern Metropolitan Area Health Services and from the Sir Charles Gairdner Hospital Human Research Ethics Committee. All patients providing tissue for the tissue microarray signed a consent form prior to surgical removal of the intestinal cancer to allow for this research to be undertaken. PLoS ONE | www.plosone.org Assessment of SPARC expression The role of SPARC in the pathogenesis of different tumours is highly contextual. In both CRC, and normal colonic tissue, SPARC was noted to be almost exclusively expressed by vimentinpositive stromal mesenchymal cells. Its expression was not 2 July 2011 | Volume 6 | Issue 7 | e22047 SPARC and FOXP3 in Colorectal Cancer positive cells (cells per square millimetre). Individual cores were examined and annotated to ensure that only normal colonic epithelium or viable tumour tissue was included in the area of analysis. observed in the malignant cells and was seen in only a very small number of normal epithelial cells. SPARC expression within malignant and control tissue, therefore, needed to be controlled for the amount of stromal tissue present. Serial tissue sections were taken and stained for SPARC and the specific mesenchymal cell marker, vimentin (anti-vimentin; 1:30; Novocastra Laboratories Ltd). The vimentin-positive cells were morphologically identified by an independent histopathologist as stromal mesenchymal cells. SPARC positive areas were determined using a positive pixel count algorithm (ImageScope v8.0, Aperio Technologies, CA), which measures the staining intensity of the positive, strongpositive and negative pixels (Figure 1). SPARC expression was only measured in regions containing vimentin-positive cells and the ratio of SPARC to vimentin staining for each TMA core was used to control for the stromal tissue content within each sample. All SPARC results are presented as a ratio of SPARC to vimentin. Statistical analysis Results were expressed as mean 6 SEM. Student’s unpaired Ttest and Kruskal-Wallis analysis with Dunn’s post tests were used to compare SPARC expression and T-cell density between the different groups, and a P value of ,0.05 was considered statistically significant. SPARC expression and T-cell densities were classified as high or low in relation to the median value as shown in Salama et al, 2008[28]. All statistical analyses and graphs were created using Graphpad PrismH 4.0 software package for Windows PC (Graphpad Software, San Diego, CA, USA). The Kaplan-Meier product limit estimate of survival was used to calculate long-term cancer-specific survival. The event variable was death from CRC (as defined by coding on the patient’s death certificate). All survival times were calculated from the date of histological diagnosis of CRC until either an event occurred or they remained alive at 1st March 2008. Assessment of FOXP3, CD8 and CD45RO expression The measurement of T-cell density has been previously reported[28] and used image analysis software (Imagescope v8.0, Aperio) to evaluate the number of FOXP, CD8 and CD45RO Figure 1. Tissue microarray analysis. SPARC and vimentin staining from a single patient. Images demonstrate serial DAB-stained tissue sections for both (A) SPARC and (C) vimentin and computer analysed images of the respective DAB staining (B and C). Blue represents DAB-negative pixels (dark areas), orange DAB-positive pixels and red strongly DAB-positive pixels (light areas). SPARC expression was selectively measured only in the regions of the tissue that were also vimentin-positive. doi:10.1371/journal.pone.0022047.g001 PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e22047 SPARC and FOXP3 in Colorectal Cancer outcome, SPARC expression was significantly greater in patients with a good disease outcome in stage II CRC (p = 0.0039; Figure 3C), however, no statistical difference was observed between the groups in stage III CRC (Figure 3D). Results Stromal SPARC expression in normal colonic tissue vs. stage I to IV CRC SPARC levels were examined in 120 paired normal colonic and AJCC stages I to IV CRC. SPARC expression was significantly greater in all cancers compared to the corresponding normal colonic tissue (Figure 2A). It was also significantly greater in CRC stages II to IV compared to stage I, however, there were no differences between stages II to IV (Figure 2B). When patients with stage II and III CRC were divided into those with a good outcome (no disease recurrence within 5 years of diagnosis) and a poor outcome (disease recurrence within 5 years of diagnosis or death from disease), patients with a good disease outcome in both stage II and III had higher SPARC expression in the primary tumour, but these findings were not significantly different (Figure 2C). T-cell FOXP3, CD8 and CD45RO expression in CRC Examining T cell density using TMAs of a 967 cohort, FOXP3 was significantly higher in CRC tissue compared to normal colonic mucosa (p,0.002, Figure 4A and Figure 5). In contrast, CD8 and CD45RO expression levels were significantly lower in normal colonic mucosa (p,0.0001, Figure 4B and C) consistent with previously findings. When dividing the samples into stages II or III, FOXP3 was significantly greater in stage II compared to stage III (p,0.002, Figure 4D). Similarly, CD8 and CD45RO expression levels were significantly greater in patients with stage II CRC compared to stage III (p,0.0001 for both, Figure 4E and F). When comparing these markers with disease outcome, FOXP3 was significantly greater in patients with a good compared to poor outcome in stage II CRC (p = 0.0037; Figure 6A), whilst no statistical differences were observed in stage III CRC (Figure 6B). These results were similar for CD8 and CD45RO (p = 0.0195 and 0.0054 respectively, Figure 6C, D, E and F). Stromal SPARC expression in stage II and equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | www.ploscompbiol.org 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study [30]. The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step [31], raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps [31]) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee [29], or a slightly open conformation as found in Jimenez-Useche et al. [30]. Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | www.ploscompbiol.org 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to por la lectura a través de la lectura de la prensa. La educación en los medios las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio adquiere pleno derecho y entidad en la sección sexta titulada «competencias sociales y cívi- técnico de los aparatos. cas» que indica que «los alum- nos deberán ser capaces de juz- gar y tendrán espíritu crítico, lo que supone ser educados en los las programaciones oficiales, ya que, a lo largo de un medios y tener conciencia de su lugar y de su influencia estudio de los textos, los documentalistas del CLEMI en la sociedad». han podido señalar más de una centena de referencias a la educación de los medios en el seno de disciplinas 4. Un entorno positivo como el francés, la historia, la geografía, las lenguas, Si nos atenemos a las cifras, el panorama de la las artes plásticas : trabajos sobre las portadas de educación en medios es muy positivo. Una gran ope- prensa, reflexiones sobre temas mediáticos, análisis de ración de visibilidad como la «Semana de la prensa y publicidad, análisis de imágenes desde todos los ángu- de los medios en la escuela», coordinada por el CLE- los, reflexión sobre las noticias en los países europeos, MI, confirma año tras año, después de 17 convocato- información y opinión rias, el atractivo que ejerce sobre los profesores y los Esta presencia se constata desde la escuela mater- alumnos. Concebida como una gran operación de nal (2 a 6 años) donde, por ejemplo, se le pregunta a complementariedad entre la escuela y los profesiona- los niños más pequeños si saben diferenciar entre un les de los medios, alrededor del aprendizaje ciudada- periódico, un libro, un catálogo, a través de activida- no de la comunicación mediática, este evento moviliza des sensoriales, si saben para qué sirve un cartel, un durante toda una semana un porcentaje elevado de periódico, un cuaderno, un ordenador si son capa- centros escolares que representan un potencial de 4,3 ces de reconocer y distinguir imágenes de origen y de millones de alumnos (cifras de 2006). Basada en el naturaleza distintas. Podríamos continuar con más voluntariado, la semana permite desarrollar activida- ejemplos en todos los niveles de enseñanza y práctica- des más o menos ambiciosas centradas en la introduc- Páginas 43-48 ción de los medios en la vida de la escuela a través de la instalación de kioscos, organización de debates con profesionales y la confección por parte de los alumnos de documentos difundidos en los medios profesionales. Es la ocasión de dar un empujón a la educación en medios y de disfrutarlos. Los medios –un millar en 2006– se asocian de maneras diversas ofreciendo ejemplares de periódicos, acceso a noticias o a imágenes, proponiendo encuentros, permitiendo intervenir a los jóvenes en sus ondas o en sus columnas Esta operación da luz al trabajo de la educación en medios y moviliza a los diferentes participantes en el proyecto. 5. La formación de los docentes La formación es uno de los pilares principales de la educación en los medios. Su función es indispensable ya que no se trata de una disciplina, sino de una enseñanza que se hace sobre la base del voluntariado y del compromiso personal. Se trata de convencer, de mostrar, de interactuar. En primer lugar es necesario incluirla en la formación continua de los docentes, cuyo volumen se ha incrementado desde 1981 con la aparición de una verdadera política de formación continua de personal. Es difícil dar una imagen completa del volumen y del público, pero si nos atenemos a las cifras del CLEMI, hay más de 24.000 profesores que han asistido y se han involucrado durante 2004-05. 5.1. La formación continua En la mayoría de los casos, los profesores reciben su formación en contextos cercanos a su centro de trabajo, o incluso en este mismo. Después de una política centrada en la oferta que hacían los formadores, se valora más positivamente la demanda por parte del profesorado, ya que sólo así será verdaderamente fructífera. Los cursos de formación se repartieron en varias categorías: desde los formatos más tradicionales (cursos, debates, animaciones), hasta actividades de asesoramiento y de acompañamiento, y por supuesto los coloquios que permiten un trabajo en profundidad ya que van acompañados de expertos investigadores y profesionales. Citemos, por ejemplo en 2005, los coloquios del CLEMI-Toulouse sobre el cine documental o el del CLEMI-Dijon sobre «Políticos y medios: ¿connivencia?». Estos coloquios, que forman parte de un trabajo pedagógico regular, reagrupan a los diferentes participantes regionales y nacionales alrededor de grandes temas de la educación en medios y permiten generar nuevos conocimientos de aproximación y una profundización. Páginas 43-48 Hay otro tipo de formación original que se viene desarrollando desde hace menos tiempo, a través de cursos profesionales, como por ejemplo, en el Festival Internacional de Foto-periodismo «Visa para la imagen», en Perpignan. La formación se consolida en el curso, da acceso a las exposiciones, a las conferencias de profesionales y a los grandes debates, pero añade además propuestas pedagógicas y reflexiones didácticas destinadas a los docentes. Estas nuevas modalidades de formación son también consecuencia del agotamiento de la formación tradicional en las regiones. Los contenidos más frecuentes en formación continua conciernen tanto a los temas más clásicos como a los cambios que se están llevando a cabo en las prácticas mediáticas. Así encontramos distintas tendencias para 2004-05: La imagen desde el ángulo de la producción de imágenes animadas, el análisis de la imagen de la información o las imágenes del J.T. La prensa escrita y el periódico escolar. Internet y la información en línea. Medios y educación de los medios. 5.2 La formación inicial La formación inicial está aun en un grado muy ini- cial. El hecho de que la educación en medios no sea una disciplina impide su presencia en los IUFM (Institutos Universitarios de Formación de Maestros) que dan una prioridad absoluta a la didáctica de las disciplinas. En 2003, alrededor de 1.400 cursillistas sobre un total de 30.000 participaron en un momento u otro de un módulo de educación en medios. Estos módulos se ofrecen en función del interés que ese formador encuentra puntualmente y forman parte a menudo de varias disciplinas: documentación, letras, historia-geografía Estamos aún lejos de una política concertada en este dominio. La optativa «Cine-audiovisual» ha entrado desde hace muy poco tiempo en algunos IUFM destinada a obtener un certificado de enseñanza de la opción audiovisual y cine. Internet tiene cabida también en los cursos de formación inicial, recientemente con la aparición de un certificado informático y de Internet para los docentes, dirigido más a constatar competencias personales que a valorar una aptitud para enseñarlos. 6. ¿Y el futuro? El problema del futuro se plantea una vez más por la irrupción de nuevas técnicas y nuevos soportes. La difusión acelerada de lo digital replantea hoy muchas cuestiones relativas a prácticas mediáticas. Muchos Comunicar, 28, 2007 47 Comunicar, 28, 2007 Enrique Martínez-Salanova '2007 para Comunicar 48 trabajos que llevan el rótulo de la educación en medios solicitan una revisión ya que los conceptos cambian. La metodología elaborada en el marco de la educación en medios parece incluso permitir la inclinación de la sociedad de la información hacia una sociedad del conocimiento, como defiende la UNESCO. En Francia, se necesitaría unir las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio técnico de los aparatos. Los avances recientes en el reconocimiento de estos contenidos y las competencias que supondrían podrían permitirlo. Referencias CLEMI/ACADEMIE DE BORDEAUX (Ed.) (2003): Parcours médias au collège: approches disciplinaires et transdisciplinaires. Aquitaine, Sceren-CRDP. GONNET, J. (2001): Education aux médias. Les controverses fécondes. Paris, Hachette Education/CNDP. SAVINO, J.; MARMIESSE, C. et BENSA, F. (2005): L’éducation aux médias de la maternelle au lycée. Direction de l’Enseignement Scolaire. Paris, Ministère de l’Education Nationale, Sceren/CNDP, Témoigner. BEVORT, E. et FREMONT, P. (2001): Médias, violence et education. Paris, CNDP, Actes et rapports pour l’éducation. – www.clemi.org: fiches pédagogiques, rapports et liens avec les pages régionales/académiques. – www.ac-nancy-metz.fr/cinemav/quai.html: Le site «Quai des images» est dédié à l’enseignement du cinéma et de l’audiovisuel. – www.france5.fr/education: la rubrique «Côté profs» a une entrée «education aux médias». – www.educaunet.org: Programme européen d’éducation aux risques liés à Internet. dResedfeleexliobnuetsacón Páginas 43-48
SPARC, FOXP3, CD8 and CD45 correlation with disease recurrence and long-term disease-free survival in colorectal cancer.
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SPARC, FOXP3, CD8 and CD45 correlation with disease recurrence and long-term disease-free survival in colorectal cancer.

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