Validation of housekeeping genes for quantitative real-time PCR in in-vivo and in-vitro models of cerebral ischaemia

<p>Abstract</p> <p>Background</p> <p>Studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. The normalization of relative qRT-PCR data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. No validated housekeeping genes have been reported for the relative quantification of the mRNA expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used.</p> <p>The present study aims to determine the expression stability of ten housekeeping genes (Gapdh, β2m, Hprt, Ppia, Rpl13a, Oaz1, 18S rRNA, Gusb, Ywhaz and Sdha) to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models.</p> <p>Results</p> <p>The expression stability of the candidate reference genes was evaluated using the 2^-ΔC'Tmethod and ANOVA followed by Dunnett's test. For the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for Rpl13a and Sdha were found to have significantly different levels of mRNA expression. These different levels were also found in the case of the in-vivo model of pMCAO in rats except for Hprt, Sdha and Ywhaz mRNA, where the expression did not vary. Sdha and Ywhaz were identified by geNorm and NormFinder as the two most stable genes.</p> <p>Conclusion</p> <p>We have validated endogenous control genes for qRT-PCR analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. For normalization purposes, Rpl13a and Sdha are found to be the most suitable genes for the in-vitro model and Sdha and Ywhaz for the in-vivo model. Genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup.</p

Comprehensive analysis of copy number aberrations in microsatellite stable colon cancer in view of stromal component

Background: Somatic copy number aberrations (CNA) are common acquired changes in cancer cells playing an important role in the progression of colon cancer (CRC). This study aimed to perform a characterization of CNA and their impact in gene expression.Methods: CNA were inferred from SNP array data in a series of 99 CRC. CNA events were calculated and used to assess the association between copy number dosage, clinical and molecular characteristics of the tumours, and gene expression changes. All analyses were adjusted for the quantity of stroma in each sample, that was inferred from gene expression data.Results: High heterogeneity among samples was observed, the proportion of altered genome ranged between 0.04 and 26.6%. Recurrent CNA regions with gains were frequent in chromosomes 7p, 8q, 13q, and 20 while 8p, 17p, and 18 cumulated loses. A significant positive correlation was observed between the number of somatic mutations and total CNA (Spearman r=0.42, P=0.006). Approximately 37% of genes located in CNA regions changed their level of expression, and the average partial correlation (adjusted for stromal content) with copy number was 0.54 (inter-quartile range 0.20 to 0.81). Altered genes showed enrichment in pathways relevant for colorectal cancer. Tumours classified as CMS2 and CMS4 by the consensus molecular subtyping showed higher frequency of CNA. Loses of one small region in 1p36.33, with gene CDK11B, were associated with poor prognosis. More than 66% of the recurrent CNA were validated in the TCGA data when analysed with the same procedure. Also 79% of the genes with altered expression in our data were validated in the TCGA.Conclusion: Though CNA are frequent events in MSS CRC, few focal recurrent regions were found. These aberrations have strong effects on gene expression and contribute to deregulate relevant cancer pathways. Due to the diploid nature of stromal cells, it is important to consider the purity of tumour samples to accurately calculate CNA events in CRC

Disease presence in goat flocks on temperate valleys of Argentina’s Northwest

Una encuesta transversal ad hoc se llevó a cabo en veintitrés majadas caprinas correspondientes a unidades productivas (UP) familiares extensivos (UPF, n=19) y semiintensivas (UPL, n=4) dedicadas a la producción de leche en los valles templados de Salta y Jujuy con el objetivo de describir la presencia de enfermedades en sus majadas. Se recabaron datos del manejo general y sanitario en particular. Se procesaron sueros para el diagnóstico de brucelosis por las pruebas tamiz del antígeno bufferado en placa y confirmadas por polarización fluorescente. Paraparatuberculosis, herpesvirus caprino tipo 1 (CaHV-1), neosporosis y toxoplasmosis se utilizó ELISA indirecto y para leptospirosis microaglutinación. Se determinaron los valores de Cu2+, Zn2+, Mg2+ y Ca2+ sérico por espectrofotometría de absorción atómica. En heces, se realizó el recuento de huevos de helmintos por gramo (hpg) y la diferenciación de géneros por coprocultivo. Las UP muestreadas crían en promedio 48,4 ±30,7 (UPF) y 221,3 ±78,7 (UPL) caprinos respectivamente, sin contar los cabritos menores a los 45 días de edad. La cría de las UPF fue extensiva con encierre nocturno y la alimentación sobre pastizal natural mientras que las UPL combinaban pasturas bajo riego con suplementación en base de concentrados. Existen dos períodos de pariciones al año; el principal donde pare casi la totalidad de la majada se localiza desde fines de marzo a fines de septiembre y el segundo período donde pare una menor proporción de cabras se ubica desde septiembre a diciembre. El 94,7% de los productores desparasitaba en forma rutinaria toda la majada y el 75% trataban los piojos. En un 41,2% de las UP se inyectaba mineralizantes-vitamínicos a toda la majada y en solo un 15% vacunaba contra enfermedades Clostridiales. El promedio de la tasa de mortalidad anual fue del 20,1±9,8% y 7,8±3,0% respectivamente en UPF y UPL. En el 100% de las UPF se registró la ocurrencia de abortos, y de estos el 25% presentó retención de placenta. El promedio intramajada de abortos comunicados fue del 14,5 ±11,6%. El 58,8% de las UP declaró tener casos de ectima contagioso. Se registraron un 26,7% cuadros de muertes agudas probablemente por infecciones Clostridiales. En el 93,3% de las UPF se registraron problemas de mastitis. El porcentaje de UP con seroprevalencia positiva a brucelosis, leptospirosis, paratuberculosis, toxoplasmosis, neosporosis y CpHV-1 fue respectivamente de 0%, 0%, 0%, 73,3%, 40% y 87,5%. Los promedios de los hpg de invierno – primavera y los de verano-otoño fueron respectivamente de 965 y 2859 hpg, con Haemonchus y Trichostrongylus y en menor medida Teladorsagia como nematodes predominantes. En el 31,6% de las UP se diagnosticó Fasciolosis. El 66,7% de las UP describió la presencia, a la faena, de quistes hidatídicos. En un 58% de las majadas se describió bocio. Los promedios por UP de cobre, zinc, magnesio y calcio séricos fueron respectivamente 0,59±0,10 ppm, 0,67±0,05 ppm, 2,30±0,29mg/dl y 8,42±1,92 mg/dl. Sin embargo, para el cobre y el calcio los valores promedios difieren (p<0,05) entre las UPF (0,66±0,08 ppm y 9,36 ±1,85 mg/dl) y las UPL (0,54±0,06 ppm y 7,02±0,76 mg/dl). Un 63,6% de las UP dijo tener intoxicación con cegadera (Heterophyllaea pustulata) y de muertes (50%) en animales jóvenes atribuidas al romerillo (Baccharis coridifolia) y en menor medida a la hediondilla (Cestrum parqui). Un 58,8% de los propietarios declararon tener problemas de pérdidas de animales principalmente por pumas. Estos primeros resultados muestran la importancia de llevar a cabo estudios más específicos respecto de la salud de las majadas para incrementar la producción y el bienestar general de las familias productoras.A cross-sectional survey was performed in 23 goat flocks (UP) of family extensive farming units (UPF) and semi-intensive farming units (UPL) from the temperate valleys of Jujuy and Salta provinces, with the aim of describing disease occurrence in goat flocks. Farmers were subjected to an ad hoc survey to gather data on general management and specific health issues. Sera were processed to diagnose brucellosis using the BPA screening test and positive sera were confirmed by FPA test. Paratuberculosis, caprine herpesvirus 1, toxoplasmosis and neosporosis were tested by indirect ELISA, and leptospirosis by microagglutination test. Serum Cu2+, Zn2+, Mg2+ and Ca2+ were determined by atomic absorption spectrophotometer. Fecal samples were taken for eggs counts per gram of feaces (epg) and identifying genera by coproculture. UP had on average 48,4 ±30,7 (UPF) y 221,3 ±78,7 (UPL) goats (not including kids). UPF animals are extensively bred, with nocturnal enclosure, and flock feeding is based on grazing the hills and UPL combined pastures under irrigation with concentrate-based supplementation. Kidding is concentrated in two periods: from late March to late September, when almost all the flock kids, and a second minor kid period that was from September to December. Farmers (94,7%) routinely treat all the flock against parasites and the 71,8% treat against lice. The 41,2% of UP provide all the flock with mineral-vitamin mixtures and only the 15, % vaccinated against clostridium diseases. Mean annual mortality rate per flock was 20,1±9,8% and 7,8±3,0% respectively for UPF and UPL. Abortions were recorded in 100% of the UP, 25% with retained placenta. Mean within-flock reported abortions were 14,5 ±11,6%. Contagious echtyma (orf) was detected in 58,8% of the UP and acute deaths probably due to clostridial infections were recorded in 26,7% of the UP. Mastitis-associated problems were recorded in 93,3% of UP. Positive seroprevalence of brucellosis, leptospirosis, paratuberculosis, toxoplasmosis, neosporosis and CpHV-1, in UP was 0%, 25%, 50%, 42,8%, 88,8%, 85,7%, 12,5% and 0% respectively. Mean epg were 965 and 2859 for winter-spring and summer-autumn respectively, with Haemonchus and Trichostrongylus and secondly Teladorsagia being the prevailing nematodes. Fasciola infections were recorded in 31,6% of the UP. The presence of hydatid cysts in slaughtered animals were indicated by 66,7% of UP. Signs of goiter were described in 58% of the flocks. Mean serum cooper, zinc, magnesium and calcium per UP were 0,59±0,10 ppm, 0,67±0,05 ppm, 2,30±0,29mg/dl and 8,42±1,92 mg/dl respectively. However cooper and calcium was different (p<0,05) between UPF (0,66±0,08 ppm and 9,36±1,85 mg/dl) and UPL (0,54±0,06 ppm and 7,02±0,76 mg/dl). A 63,6% of the UP reported cases of intoxication by cegadera (Heterophyllaea pustulata) and death (50%) were reported and attributed by farmers to the ingestion of romerillo (Baccharis coridifolia) and secondly of hediondilla (Cestrum parqui) by young animals. Some farmers (58.8%) indicated loss of animals mostly due to the attack by pumas. These first results regarding show the importance of conducting specific studies about the health of flocks in order to increase competitiveness in goat production and the welfare of farming families.Fil: Suarez, Víctor Humberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; ArgentinaFil: Dodero, Ana Mariela. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Almudevar, F. M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; ArgentinaFil: Bertoni, E. A.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; ArgentinaFil: Salatin, A. O.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Viñabal, A. E.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Saldaño, R.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; ArgentinaFil: Martínez, G. M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; ArgentinaFil: Micheloud, Juan Francisco. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Salta-Jujuy. Estación Experimental Agropecuaria Salta; ArgentinaFil: Fiorentino, Maria Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Agencia de Extensión Rural Balcarce; ArgentinaFil: Brihuega, Bibiana. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar; ArgentinaFil: Romera, Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias Castelar; Argentin

Clinical value of prognosis gene expression signatures in colorectal cancer: a systematic review

Introduction: the traditional staging system is inadequate to identify those patients with stage II colorectal cancer (CRC) at high risk of recurrence or with stage III CRC at low risk. A number of gene expression signatures to predict CRC prognosis have been proposed, but none is routinely used in the clinic. The aim of this work was to assess the prediction ability and potential clinical usefulness of these signatures in a series of independent datasets. Methods: a literature review identified 31 gene expression signatures that used gene expression data to predict prognosis in CRC tissue. The search was based on the PubMed database and was restricted to papers published from January 2004 to December 2011. Eleven CRC gene expression datasets with outcome information were identified and downloaded from public repositories. Random Forest classifier was used to build predictors from the gene lists. Matthews correlation coefficient was chosen as a measure of classification accuracy and its associated p-value was used to assess association with prognosis. For clinical usefulness evaluation, positive and negative post-tests probabilities were computed in stage II and III samples. Results: five gene signatures showed significant association with prognosis and provided reasonable prediction accuracy in their own training datasets. Nevertheless, all signatures showed low reproducibility in independent data. Stratified analyses by stage or microsatellite instability status showed significant association but limited discrimination ability, especially in stage II tumors. From a clinical perspective, the most predictive signatures showed a minor but significant improvement over the classical staging system. Conclusions: the published signatures show low prediction accuracy but moderate clinical usefulness. Although gene expression data may inform prognosis, better strategies for signature validation are needed to encourage their widespread use in the clinic

Management practices and disease occurrence in goat flocks from Chaco Region in Salta province

Una encuesta transversal ad hoc se llevó a cabo entre abril y noviembre del 2011 en 37 unidades productoras familiares (UPF) del departamento de Rivadavia, provincia de Salta, con el objetivo de describir las prácticas productivas y la presencia de enfermedades en sus majadas caprinas en el Chaco Salteño. Se recabaron datos del manejo general y sanitario en particular. Se procesaron sueros para el diagnóstico de brucelosis por la prueba tamiz BPA y confirmadas por fijación de complemento, para paratuberculosis (PTBC), artritis-encefalitis (CAEV), chlamydiosis, herpesvirus caprino tipo 1 (CaHV-1), fiebre Q y toxoplasmosis se utilizó ELISA indirecto y para leptospirosis la microaglutinación. Se determinaron los valores de Cu2+, Mg2+ y Ca2+ sérico por espectofotómetría de absorción atómica. En heces, se realizó el recuento de huevos de helmintos por gramo (hpg) y la diferenciación de géneros por coprocultivo. Las UPF tuvieron en promedio 165.0± 102.1 caprinos (sin contar cabritos), siendo el 87% cabras madres. Las instalaciones de las UPF fueron muy precarias, en su mayoría solo constaban de corrales de troncos y enramadas con limitada disponibilidad de agua. La cría fue extensiva con encierre nocturno y la alimentación de la majada es en base al ramoneo de hojas y frutos del monte, con escasa oferta de gramíneas. El servicio fue continuo y la parición se concentraba en dos períodos, en abril-junio donde paría casi la totalidad de la majada y una segunda parición que se dividía entre agosto-septiembre (60%) y octubre-noviembre (30%). El destino de la producción caprina de las UPF fue la venta de cabritos y el consumo interno de capones y cabras adultas, aunque un 43.2% ordeñaba en promedio 25.6±18.7 cabras al vender los cabritos. El 61.1% de los productores desparasitaba en forma rutinaria toda la majada en otoño o invierno, generalmente con ivermectina y un 19,6% inyectaba mineralizantes-vitamínicos a toda la majada. El promedio de la tasa de mortalidad anual por majada fue del16.2±12.4%. En el 71.9% de las UPF se registró la ocurrencia de abortos, mayormente con retención de placenta (70%). El promedio intramajada de abortos comunicados es del 15.2±14.8%. Se detectó ectima contagioso en un 71.4% de las UPF y en un 53.1% cuadros de muertes agudas o subagudas probablemente por infecciones clostridiales. En el 96.9% de las UPF se registraron problemas de mastitis, fundamentalmente durante los períodos de lluvias. La seroprevalencia positiva a brucelosis, leptospirosis, paratuberculosis, toxoplasmosis, CpHV-1, CAEV y fiebre Q en las UPF fue respectivamente de 27%, 70%, 60%, 100%, 100%, 10% y 0%. En cuanto a ectoparásitos las UPF destacaron la importancia de las miasis (Cochliomyia hominivorax: 87,5%), de los piojos, (Linognathus spp.: 90.3%), del pique (Tunga penetrans: 77.4%). El promedio de los hpg fue de 714, 536 y 340 para los meses de abril, julio y octubre con Haemonchus y Trichostrongylus como nematodes predominantes. En el 61,3% de las UPF se describió la presencia a la faena de quistes hidatídicos. En un 54.3% de las majadas se describieron signos de ataxia enzóotica en cabritos; El promedio por UPF de cobre, magnesio y calcio séricos fueron respectivamente de 0.49±0.12 ppm, 3.0±0.37mg/d y 8.28±0.21 mg/dl. Se describieron casos de timpanismo y muertes (18.8%) atribuidas por los propietarios a la ingesta de sacha sandia y frutos del chañar. Un 56.3% de los propietarios declararon tener problemas de pérdidas de animales en el monte a causa del ataque en mayor medida de pumas. Estos primeros resultados en cuanto a prácticas productivas, mortandad, pérdidas y presencia de enfermedades y zoonosis, muestran la importancia de llevar a cabo estudios más específicos respecto de la salud de las majadas para incrementar la competitividad en la producción, la salud de los propietarios y por ende el bienestar general de las familias productoras.A cross-sectional survey was performed between April and November 2011 in 37 family farming units (FFU) from Rivadavia department, Salta province, with the aim of describing production practices and disease occurrence in goat flocks in the Chaco region of Salta. Farmers were subjected to an ad hoc survey to gather data on general management and specific health issues. Sera were processed to diagnose brucellosis using the BPA screening test and positive sera were confirmed by complement fixation test. Paratuberculosis (PTBC), caprine arthritis-encephalitis virus (CAEV), Q fever and toxoplasmosis were tested by indirect ELISA, and leptospirosis by microagglutination test. Serum Cu2+, Mg2+ and Ca2+ were determined by atomic absorption spectrophotometer. Fecal samples were taken for counting helminth eggs (epg) and identifying genera by coproculture. FFUs had on average 165.0± 102.1 goats (not including kids), with 87% being does. FFU facilities were very poor and water availability was limited; facilities were mostly composed of corrals enclosed by logs and branches. Animals are extensively bred, with nocturnal enclosure, and flock feeding is based on browsing of woodland leaves and fruits, with low offer of graminoids. Breeding is continuous and kidding is concentrated in two periods: April-June, when almost all the flock kids, and a second period that is subdivided into August-September (60% of kid) and October-November (30% of kid). Goat production of FFUs is devoted to the sale of kids and internal consumption of wethers and adult goats; however, 43.2% of FFUs milks on average 25.6±18.7 goats when kids are sold and does are weaned. Farmers (61.1%) routinely treat all the flock against parasites in autumn and winter, generally with ivermectin, and 19.6% provide all the flock with mineral-vitamin mixtures. Mean annual mortality rate per flock was 16.2±12.4%. Abortions were recorded in 71.9% of the FFUs, mostly with retained placenta (70%). Mean within-flock reported abortions were 15.2±14.8%. Contagious echtyma (orf) was detected in 71.4% of the FFUs and acute or subacute deaths probably due to clostridial infections were recorded in 53.1% of the FFUs. Mastitis-associated problems were recorded in 96.9% of FFUs, mainly during the rainy periods. Positive seroprevalence of brucellosis, leptospirosis, paratuberculosis, toxoplasmosis, CpHV-1, CAEV and Q fever in FFUs was 27%, 70%, 60%, 100%, 100%, 10% and 0%, respectively. The following ectoparasites were indicated as important by FFU farmers: myiasis by Cochliomyia hominivorax (87.5%), lice Linognathus spp. (90.3%) and fleas Tunga penetrans (77.4%). Mean epg was 714, 536 and 340 for April, July and October, respectively, with Haemonchus and Trichostrongylus being the prevailing nematodes. The presence of hydatid cysts in slaughtered animals was indicated by 61.3% of FFUs. Signs of enzootic ataxia in kids were observed in 54.3% of the flocks. Mean serum Cu2+, Mg2+ and Ca2+ per FFU was 0.49±0.12 ppm, 3.0±0.37mg/d and 8.28±0.21 mg/dl respectively. Cases of bloat and death (18.8%) were reported due to distended abdomen and colic which farmers attributed to the ingestion of sacha sandia (Capparis salicifolia) and fruits of chañar Geoffroea decorticans. Some farmers (56.3%) indicated loss of animals in the woodland, mostly due to the attack by pumas. These first results regarding production practices, mortality, production losses and occurrence of diseases and zoonosis show the importance of conducting specific studies about the health of flocks in order to increase competitiveness in goat production, farmers’ health and, therefore, general welfare of smallholder families.Fil: Suarez, Víctor Humberto. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Rosetto, C. B.. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Gaido, A. B.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Salatin, A. O.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Bertoni, E. A.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Dodero, M.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Viñabal, A. E.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Investigación Animal del Chaco Semiárido; ArgentinaFil: Pinto, G.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Brihuega, Bibiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Romera, Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Maidana, Silvina Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentin

Clinical value of prognosis gene expression signatures in colorectal cancer: a systematic review

Introduction: the traditional staging system is inadequate to identify those patients with stage II colorectal cancer (CRC) at high risk of recurrence or with stage III CRC at low risk. A number of gene expression signatures to predict CRC prognosis have been proposed, but none is routinely used in the clinic. The aim of this work was to assess the prediction ability and potential clinical usefulness of these signatures in a series of independent datasets. Methods: a literature review identified 31 gene expression signatures that used gene expression data to predict prognosis in CRC tissue. The search was based on the PubMed database and was restricted to papers published from January 2004 to December 2011. Eleven CRC gene expression datasets with outcome information were identified and downloaded from public repositories. Random Forest classifier was used to build predictors from the gene lists. Matthews correlation coefficient was chosen as a measure of classification accuracy and its associated p-value was used to assess association with prognosis. For clinical usefulness evaluation, positive and negative post-tests probabilities were computed in stage II and III samples. Results: five gene signatures showed significant association with prognosis and provided reasonable prediction accuracy in their own training datasets. Nevertheless, all signatures showed low reproducibility in independent data. Stratified analyses by stage or microsatellite instability status showed significant association but limited discrimination ability, especially in stage II tumors. From a clinical perspective, the most predictive signatures showed a minor but significant improvement over the classical staging system. Conclusions: the published signatures show low prediction accuracy but moderate clinical usefulness. Although gene expression data may inform prognosis, better strategies for signature validation are needed to encourage their widespread use in the clinic

Comprehensive analysis of copy number aberrations in microsatellite stable colon cancer in view of stromal component

Background: Somatic copy number aberrations (CNA) are common acquired changes in cancer cells playing an important role in the progression of colon cancer (CRC). This study aimed to perform a characterization of CNA and their impact in gene expression.Methods: CNA were inferred from SNP array data in a series of 99 CRC. CNA events were calculated and used to assess the association between copy number dosage, clinical and molecular characteristics of the tumours, and gene expression changes. All analyses were adjusted for the quantity of stroma in each sample, that was inferred from gene expression data.Results: High heterogeneity among samples was observed, the proportion of altered genome ranged between 0.04 and 26.6%. Recurrent CNA regions with gains were frequent in chromosomes 7p, 8q, 13q, and 20 while 8p, 17p, and 18 cumulated loses. A significant positive correlation was observed between the number of somatic mutations and total CNA (Spearman r=0.42, P=0.006). Approximately 37% of genes located in CNA regions changed their level of expression, and the average partial correlation (adjusted for stromal content) with copy number was 0.54 (inter-quartile range 0.20 to 0.81). Altered genes showed enrichment in pathways relevant for colorectal cancer. Tumours classified as CMS2 and CMS4 by the consensus molecular subtyping showed higher frequency of CNA. Loses of one small region in 1p36.33, with gene CDK11B, were associated with poor prognosis. More than 66% of the recurrent CNA were validated in the TCGA data when analysed with the same procedure. Also 79% of the genes with altered expression in our data were validated in the TCGA.Conclusion: Though CNA are frequent events in MSS CRC, few focal recurrent regions were found. These aberrations have strong effects on gene expression and contribute to deregulate relevant cancer pathways. Due to the diploid nature of stromal cells, it is important to consider the purity of tumour samples to accurately calculate CNA events in CRC

Comprehensive analysis of copy number aberrations in microsatellite stable colon cancer in view of stromal component

Background: Somatic copy number aberrations (CNA) are common acquired changes in cancer cells playing an important role in the progression of colon cancer (CRC). This study aimed to perform a characterization of CNA and their impact in gene expression.Methods: CNA were inferred from SNP array data in a series of 99 CRC. CNA events were calculated and used to assess the association between copy number dosage, clinical and molecular characteristics of the tumours, and gene expression changes. All analyses were adjusted for the quantity of stroma in each sample, that was inferred from gene expression data.Results: High heterogeneity among samples was observed, the proportion of altered genome ranged between 0.04 and 26.6%. Recurrent CNA regions with gains were frequent in chromosomes 7p, 8q, 13q, and 20 while 8p, 17p, and 18 cumulated loses. A significant positive correlation was observed between the number of somatic mutations and total CNA (Spearman r=0.42, P=0.006). Approximately 37% of genes located in CNA regions changed their level of expression, and the average partial correlation (adjusted for stromal content) with copy number was 0.54 (inter-quartile range 0.20 to 0.81). Altered genes showed enrichment in pathways relevant for colorectal cancer. Tumours classified as CMS2 and CMS4 by the consensus molecular subtyping showed higher frequency of CNA. Loses of one small region in 1p36.33, with gene CDK11B, were associated with poor prognosis. More than 66% of the recurrent CNA were validated in the TCGA data when analysed with the same procedure. Also 79% of the genes with altered expression in our data were validated in the TCGA.Conclusion: Though CNA are frequent events in MSS CRC, few focal recurrent regions were found. These aberrations have strong effects on gene expression and contribute to deregulate relevant cancer pathways. Due to the diploid nature of stromal cells, it is important to consider the purity of tumour samples to accurately calculate CNA events in CRC