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ABSTRACT: Lipid added as rapeseed or palm oil to the diet of dairy goats over 8 mo of one lactation alters fat secretion and milk fatty acid (FA) and protein composition. In this study, we examined the contribution of mammary gene expression to these changes and included 30 multiparous goats of Norwegian dairy goat breed for a 230-d experimental period, with indoor feeding from 1 to 120 d in milk (DIM), mountain grazing from 120 to 200 DIM, and indoor feeding from 200 to 230 DIM. After an initial period (1–60 DIM) when the control diet was given to all goats, the animals were subdivided into 3 groups of 10 goats. Treatments (60–230 DIM) were basal concentrate (control) alone or supplemented with either 8% (by weight) hydrogenated palm oil enriched with palmitic acid (POFA) or 8% (by weight) rapeseed oil (RSO). Milk was sampled individually from all animals throughout lactation, at 60, 120, 190, and 230 DIM for milk yield and composition. On d 60, 120, 190, and 230, mammary tissue was collected by biopsy to measure mRNA abundance of 19 key genes. None of the 19 genes involved in milk protein, apoptosis, lipid metabolism, transcription factors, and protein of the milk fat globule membrane, as measured by mRNA abundance, were affected by the lipid supplements, although POFA increased milk fat content, and POFA and RSO affected milk FA composition. Over the experimental period (120–230 DIM), the mRNA abundance of 13 of the 19 studied genes was affected by lactation stage. For some genes, expression either gradually increased from 120 to 230 DIM (CSN2, CASP8, CD36, GLUT4) or increased from 120 to 200 and then remained stable (XDH), or decreased (CSN3, G6PD, SREBF1, PPARG1) or increased only at 230 DIM (SCD1, SCD5, ELF3). For a second group of genes (CSN1, LALBA, FABP3, FASN, LPL, MFGE8), expression was stable over the lactation period. Our results suggest that factors other than gene expression, such as substrate availability or posttranscriptional regulation of these genes, could play an important role in the milk fat and FA responses to dietary fat composition in the goat. In conclusion, mammary gene expression in goats was more regulated by stage of lactation than by the dietary treatments applied.

ABSTRACT: In this study, we tested a response function comprising responses in milk to changes in organic matter digestibility of silages and concentrate supply. We studied the effect of changes in silage digestibility and concentrate supply on milk yield, feed intake, body weight, and methane production using 60 Norwegian Red cows. The experiment was a complete randomized block design comprising 3 periods. The pre-experimental period lasted 20 d and all the cows were fed a common silage for ad libitum intake and concentrate according to yield. Next, response period 1 lasted 17 d and the cows were divided into 2 treatments, where a low-digestible silage (LDS) was fed to half of the cows, and the other half were fed a high-digestible silage (HDS). Both groups were fed silage for ad libitum silage intake. Concentrate was optimized according to the yield and type of silage offered. In this period, the effect of silage was evaluated using a mixed model, including the results from pre-experimental period, with parity as a covariate and animal as a random effect. In response period 2, which lasted 20 d, the concentrate level was evaluated by dividing the silage digestibility treatments further into 3 subgroups. Concentrate was increased by 2 kg of dry matter (DM) per day, decreased by 2 kg of DM/d, or remained unchanged. In response period 1, silage treatments were optimized to obtain similar yields and resulted in a lower concentrate offer to HDS treatment. However, the HDS treatment showed a 3.0 kg of DM/d higher total feed intake due to a higher than expected silage intake. This resulted in 3.5 kg higher energy-corrected milk (ECM). Methane emissions were similar between silage treatments, but HDS showed lower methane per kilogram of DM due to its higher intake. The effect of concentrate supply level and interaction with silage digestibility was evaluated using mixed models, including the results for response period 1, with parity as a covariate and animal as a random effect. The reduction in concentrate offer by 2 kg/d in response period 2 was compensated for by increased 1.3 kg of DM/d of silage intake for HDS, resulting in similar intake (22.1 kg of DM/d and 21.7 kg of DM/d without and with concentrate reduction, respectively) and ECM yields (29.4 and 29 kg of ECM without and with concentrate reduction, respectively). However, concentrate offer reduction could not be compensated for by increased silage intake for LDS and resulted in lower milk yields (27.5 kg of ECM). Increased concentrate showed a higher marginal ECM response (kg of ECM per kg of additional concentrate intake) for LDS (1.8 vs. 3.3 kg of ECM for HDS and LDS, respectively). Thus, the drop in milk yields could be compensated for by increased concentrate offers if LDS are fed. Total methane production increased with increased concentrate intake, regardless of silage digestibility. Methane emissions per unit of milk were affected by total DM intake rather than by changes in silage digestibility and concentrate level. The results of this study are based on short-term periods and could show differences if study periods were longer; the results should be interpreted accordingly.

Four formulations of concentrate feeds, three contrasting qualities of grass silages, and mixtures of the silages (55%) and concentrates (45%, dry weight) were tested for in vitro fermentation kinetics, in vitro dry matter degradation (IVDMD), utilizable crude protein (uCP), and metabolizable energy (ME) values. The concentrates were pelleted control concentrate for dairy cows (CONT-P); pelleted alkaline concentrate with ammoniated cereal grains (ALKA-P); mash form concentrate with ALKA-P main ingredients but with feed-grade urea and barley replacing ammoniated cereal grain (UREA-M); and mash form of ALKA-P ingredients prior to alkalization (ALKA-M). The grass silages were early cut, late cut, and a mixture (1:1) of early and late cut. The objectives were to test if the feeds differed in the tested parameters within each feed category and assess the modulatory effect of concentrate feeds on the grass silage fermentation characteristics in the mixed diets. No interaction effects of the concentrate feeds by silage quality were observed for the tested parameters in the mixed diets. For concentrates, the pelleted diets were higher ( p < 0.05) in IVDMD and molar proportion of propionate but lower in butyrate. The ALKA-P produced the highest estimated uCP ( p < 0.01). For silages, uCP, ME, total short-chain fatty acids (VFAs), and molar proportions of propionate and branched-chain VFAs decreased ( p < 0.05) with increasing stage of maturity. In conclusion, the ALKA-P could match the CONT-P in uCP and ME values and fermentation characteristics. Results for silages and their mixtures with concentrates highlight the importance of silage quality in dietary energy and protein supply for ruminants.

Mitigating enteric methane (CH 4 ) emissions is crucial as ruminants account for 5% of global greenhouse gas emissions. We hypothesised that less frequent harvesting, use of crops with lower WSC concentration, ensiling at low crop dry matter (DM) and extensive lactic acid fermentation would reduce in vitro CH 4 production. Timothy (T), timothy + red clover mixture (T + RC) or perennial ryegrass (RG), cut either two or three times per season, was wilted to 22.5% or 37.5% DM and ensiled with or without formic acid-based additive. Silages were analysed for chemical composition and fermentation products. In vitro CH 4 production was measured using an automated gas in vitro system. Methane production was, on average, 2.8 mL/g OM lower in the two-cut system than in the three-cut system (P < 0.001), and 1.9 mL/g OM lower in T than in RG (P < 0.001). Silage DM did not affect CH 4 production (P = 0.235), but formic acid increased CH 4 production by 1.2 mL/g OM compared to the untreated silage (P = 0.003). In conclusion, less frequent harvesting and extensive silage fermentation reduce in vitro CH 4 production, while RG in comparison to T resulted in higher production of CH 4 ., (© 2023. The Author(s).)

The current study assessed the effects of red macroalgae Asparagopsis taxiformis (AT)-included as an enteric methane inhibitor-in dairy cow diets on feed intake and eating-rumination behaviour. Fifteen early lactating Norwegian Red dairy cows were offered ad libitum access to drinking water and a total mixed ration (TMR) composed of 35% concentrate feed and 65% grass silage on a dry matter (DM) basis. The experiment lasted for 74 days with the first 22 days on a common diet used as the covariate period. At the end of the covariate period, the cows were randomly allocated into one of three dietary treatments: namely, 0% AT (control), 0.125% AT and 0.25% AT in the TMR. The TMR was offered in individual feed troughs with AT blended in a 400 g ( w / w ) water-molasses mixture. Eating-rumination behaviour was recorded for 11 days using RumiWatchSystem after feeding the experimental diets for 30 days. The 0.25% AT inclusion significantly reduced the DM intake (DMI). Time (min/d) spent on eating and eating in a head-down position increased with the increasing AT level in the diet, whereas rumination time was not affected. The greater time spent on eating head-down with the 0.25% AT group resulted in a significantly higher chewing index (min/kg DMI). Estimated saliva production per unit DMI (L/kg DMI, SE) increased from 10.9 (0.4) in the control to 11.3 (0.3) and 13.0 (0.3) in the 0.125% and 0.25% AT groups, respectively. This aligned with the measured ruminal fluid pH (6.09, 6.14, and 6.37 in the control, 0.125% AT and 0.25% AT groups, respectively). In conclusion, either the level of the water-molasses mixture used was not sufficient to mask the taste of AT, or the cows used it as a cue to sort out the AT. Studies with relatively larger numbers of animals and longer adaptation periods than what we used here, with varied modes of delivery of the seaweed may provide novel strategies for administering the additive in ruminant diets.

Lipid added as rapeseed or palm oil to the diet of dairy goats over 8 mo of one lactation alters fat secretion and milk fatty acid (FA) and protein composition. In this study, we examined the contribution of mammary gene expression to these changes and included 30 multiparous goats of Norwegian dairy goat breed for a 230-d experimental period, with indoor feeding from 1 to 120 d in milk (DIM), mountain grazing from 120 to 200 DIM, and indoor feeding from 200 to 230 DIM. After an initial period (1-60 DIM) when the control diet was given to all goats, the animals were subdivided into 3 groups of 10 goats. Treatments (60-230 DIM) were basal concentrate (control) alone or supplemented with either 8% (by weight) hydrogenated palm oil enriched with palmitic acid (POFA) or 8% (by weight) rapeseed oil (RSO). Milk was sampled individually from all animals throughout lactation, at 60, 120, 190, and 230 DIM for milk yield and composition. On d 60, 120, 190, and 230, mammary tissue was collected by biopsy to measure mRNA abundance of 19 key genes. None of the 19 genes involved in milk protein, apoptosis, lipid metabolism, transcription factors, and protein of the milk fat globule membrane, as measured by mRNA abundance, were affected by the lipid supplements, although POFA increased milk fat content, and POFA and RSO affected milk FA composition. Over the experimental period (120-230 DIM), the mRNA abundance of 13 of the 19 studied genes was affected by lactation stage. For some genes, expression either gradually increased from 120 to 230 DIM (CSN2, CASP8, CD36, GLUT4) or increased from 120 to 200 and then remained stable (XDH), or decreased (CSN3, G6PD, SREBF1, PPARG1) or increased only at 230 DIM (SCD1, SCD5, ELF3). For a second group of genes (CSN1, LALBA, FABP3, FASN, LPL, MFGE8), expression was stable over the lactation period. Our results suggest that factors other than gene expression, such as substrate availability or posttranscriptional regulation of these genes, could play an important role in the milk fat and FA responses to dietary fat composition in the goat. In conclusion, mammary gene expression in goats was more regulated by stage of lactation than by the dietary treatments applied., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

In this study, we tested a response function comprising responses in milk to changes in organic matter digestibility of silages and concentrate supply. We studied the effect of changes in silage digestibility and concentrate supply on milk yield, feed intake, body weight, and methane production using 60 Norwegian Red cows. The experiment was a complete randomized block design comprising 3 periods. The pre-experimental period lasted 20 d and all the cows were fed a common silage for ad libitum intake and concentrate according to yield. Next, response period 1 lasted 17 d and the cows were divided into 2 treatments, where a low-digestible silage (LDS) was fed to half of the cows, and the other half were fed a high-digestible silage (HDS). Both groups were fed silage for ad libitum silage intake. Concentrate was optimized according to the yield and type of silage offered. In this period, the effect of silage was evaluated using a mixed model, including the results from pre-experimental period, with parity as a covariate and animal as a random effect. In response period 2, which lasted 20 d, the concentrate level was evaluated by dividing the silage digestibility treatments further into 3 subgroups. Concentrate was increased by 2 kg of dry matter (DM) per day, decreased by 2 kg of DM/d, or remained unchanged. In response period 1, silage treatments were optimized to obtain similar yields and resulted in a lower concentrate offer to HDS treatment. However, the HDS treatment showed a 3.0 kg of DM/d higher total feed intake due to a higher than expected silage intake. This resulted in 3.5 kg higher energy-corrected milk (ECM). Methane emissions were similar between silage treatments, but HDS showed lower methane per kilogram of DM due to its higher intake. The effect of concentrate supply level and interaction with silage digestibility was evaluated using mixed models, including the results for response period 1, with parity as a covariate and animal as a random effect. The reduction in concentrate offer by 2 kg/d in response period 2 was compensated for by increased 1.3 kg of DM/d of silage intake for HDS, resulting in similar intake (22.1 kg of DM/d and 21.7 kg of DM/d without and with concentrate reduction, respectively) and ECM yields (29.4 and 29 kg of ECM without and with concentrate reduction, respectively). However, concentrate offer reduction could not be compensated for by increased silage intake for LDS and resulted in lower milk yields (27.5 kg of ECM). Increased concentrate showed a higher marginal ECM response (kg of ECM per kg of additional concentrate intake) for LDS (1.8 vs. 3.3 kg of ECM for HDS and LDS, respectively). Thus, the drop in milk yields could be compensated for by increased concentrate offers if LDS are fed. Total methane production increased with increased concentrate intake, regardless of silage digestibility. Methane emissions per unit of milk were affected by total DM intake rather than by changes in silage digestibility and concentrate level. The results of this study are based on short-term periods and could show differences if study periods were longer; the results should be interpreted accordingly., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The objective of this experiment was to examine how supplements of rapeseed oil or palm oil fatty acids would affect milk production and composition, body lipid stores, and energy balance in 30 multiparous goats of Norwegian dairy goat breed. The experiment lasted 230 d, with 1 to 120 d in milk (DIM) for indoor feeding (P1), 120 to 200 DIM for mountain grazing (P2), and 200 to 230 DIM for indoor feeding (P3). Grass silage was fed according to appetite during indoor feeding periods. After an adjustment period (1-60 DIM) when the control diet was given to the goats, the animals were subdivided into 3 groups of 10 goats. Treatments (60-230 DIM) were (1) basal concentrate (control; no added fat); (2) control concentrate with 8% (added on air-dry basis) hydrogenated palm oil enriched with palmitic acid (POFA); and (3) control concentrate with 8% (added on air-dry basis) rapeseed oil (RSO). Individual energy balances based on energy intake and milk production were estimated on 10, 30, 60, 90, 120, 200, and 230 DIM. At the same times, body weight (BW), body condition score (BCS), body mass index, and body tissue stores using computed tomography were monitored. Silage intake was depressed by POFA throughout the experimental period. Reduced BW and body mass index were observed in the POFA and RSO groups, whereas no effect on BCS or body composition was observed throughout lactation. Generally, a minor decrease in BW was observed from 10 to 120 DIM (only 0.6 kg on average) and the total amount of body lipid was reduced by 4.4 kg. During the mountain grazing period, a further reduction in body lipid stores (2.7 kg) was observed, and BW was reduced by 3.9 kg in the same period. The goats mobilized, on average, 72% of their fat reserves during the first 200 DIM. In this period, dietary fat supplementation did not reduce the mobilization of adipose tissue but resulted in greater milk fat yield (2 kg more, on average, compared with the control group). Milk yield was not affected by POFA or RSO supplementation. Milk fat content was higher in the POFA group than in the control and RSO groups. Milk protein and lactose contents were not affected by lipid supplements. In late lactation, a rapid accumulation of fat deposits followed the intense mobilization during the grazing period. Dietary lipid supplements had no effect on milk fat yield at this stage. Milk production depends heavily on the ability to mobilize body lipid stores, and neither POFA nor RSO supplements at rates used in our study affected this mobilization., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer remains unsettled. We aimed to assess the prognostic value of this phenotype analyzing a total of 1126 tumor samples obtained from two Norwegian consecutive colorectal cancer series. CIMP status was determined by analyzing the 5-markers CAGNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 by quantitative methylation specific PCR (qMSP). The effect of CIMP on time to recurrence (TTR) and overall survival (OS) were determined by uni- and multivariate analyses. Subgroup analyses were conducted according to MSI and BRAF mutation status, disease stage, and also age at time of diagnosis (<60, 60-74, ≥75 years). Patients with CIMP positive tumors demonstrated significantly shorter TTR and worse OS compared to those with CIMP negative tumors (multivariate hazard ratio [95% CI] 1.86 [1.31-2.63] and 1.89 [1.34-2.65], respectively). In stratified analyses, CIMP tumors showed significantly worse outcome among patients with microsatellite stable (MSS, P < 0.001), and MSS BRAF mutated tumors (P < 0.001), a finding that persisted in patients with stage II, III or IV disease, and that remained significant in multivariate analysis (P < 0.01). Consistent results were found for all three age groups. To conclude, CIMP is significantly associated with inferior outcome for colorectal cancer patients, and can stratify the poor prognostic patients with MSS BRAF mutated tumors., (© 2017 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Impaired quality due to a high content of free fatty acids (FFA) and off-flavors has caused challenges in the development of Norwegian goat milk products. The present study aimed to examine the effect of lipid-supplemented concentrates on milk fat content, fatty acid composition, FFA, lipoprotein lipase activity, sensory properties, and size of milk fat globules of goat milk. Thirty goats assigned to 3 experimental groups were fed different concentrates from 60 d in milk (DIM) until late lactation (230 DIM). The diets were (1) control concentrate (no added fat); (2) control concentrate with 8% (added on air-dry basis) hydrogenated palm oil enriched with palmitic acid (POFA); and (3) control concentrate with 8% (added on air-dry basis) rapeseed oil (RSO). The POFA group produced milk with the highest fat content, and fat content was positively correlated with the mean size of milk fat globules. Goats in the RSO group had a higher content of long-chain and unsaturated fatty acids, whereas milk from goats in the POFA group had a higher content of palmitic and palmitoleic acids (C16:0 and C16:1 cis). The control group produced milk with a higher content of short-, medium-, odd-, and branched-chain fatty acids compared with the 2 other groups. The content of FFA in milk was low in early and late lactation and peaked in mid lactation (90 DIM). A high content of FFA was correlated with poor sensory properties (tart/rancid flavor). The RSO group produced milk with lower content of FFA and off-flavors in mid lactation and a higher proportion of unsaturated fatty acids. Therefore, replacement of palm oil with rapeseed oil as a lipid source in dairy goat feed would be favorable., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Colorectal cancer (CRC) cell lines are widely used pre-clinical model systems. Comprehensive insights into their molecular characteristics may improve model selection for biomedical studies., Methods: We have performed DNA, RNA and protein profiling of 34 cell lines, including (i) targeted deep sequencing (n = 612 genes) to detect single nucleotide variants and insertions/deletions; (ii) high resolution DNA copy number profiling; (iii) gene expression profiling at exon resolution; (iv) small RNA expression profiling by deep sequencing; and (v) protein expression analysis (n = 297 proteins) by reverse phase protein microarrays., Results: The cell lines were stratified according to the key molecular subtypes of CRC and data were integrated at two or more levels by computational analyses. We confirm that the frequencies and patterns of DNA aberrations are associated with genomic instability phenotypes and that the cell lines recapitulate the genomic profiles of primary carcinomas. Intrinsic expression subgroups are distinct from genomic subtypes, but consistent at the gene-, microRNA- and protein-level and dominated by two distinct clusters; colon-like cell lines characterized by expression of gastro-intestinal differentiation markers and undifferentiated cell lines showing upregulation of epithelial-mesenchymal transition and TGFβ signatures. This sample split was concordant with the gene expression-based consensus molecular subtypes of primary tumors. Approximately ¼ of the genes had consistent regulation at the DNA copy number and gene expression level, while expression of gene-protein pairs in general was strongly correlated. Consistent high-level DNA copy number amplification and outlier gene- and protein- expression was found for several oncogenes in individual cell lines, including MYC and ERBB2., Conclusions: This study expands the view of CRC cell lines as accurate molecular models of primary carcinomas, and we present integrated multi-level molecular data of 34 widely used cell lines in easily accessible formats, providing a resource for preclinical studies in CRC.

To investigate the feasibility of milk fatty acids as predictors of onset of luteal activity (OLA), 87 lactations taken from 73 healthy Norwegian Red cattle were surveyed over 2 winter housing seasons. The feasibility of using frozen milk samples for dry-film Fourier transform infrared (FTIR) determination of milk samples was also tested. Morning milk samples were collected thrice weekly (Monday, Wednesday, Friday) for the first 10 wk in milk (WIM). These samples had bronopol (2-bromo-2-nitropropane-1,3-diol) added to them before being frozen at -20°C, thawed, and analyzed by ELISA to determine progesterone concentration and the concentrations of the milk fatty acids C4:0, C14:0, C16:0, C18:0, and cis-9 C18:1 as a proportion of total milk fatty acid content using dry-film FTIR, and averaged by WIM. Onset of luteal activity was defined as the first day that milk progesterone concentrations were >3 ng/mL for 2 successive measurements; the study population was categorized as early (n=47) or late (n=40) OLA, using the median value of 21 DIM as the cutoff. Further milk samples were collected 6 times weekly, from morning and afternoon milkings, these were pooled by WIM, and one proportional sample was analyzed fresh for fat, protein, and lactose content by the dairy company Tine SA, using traditional FTIR spectrography in the wet phase of milk. Daily energy-balance calculations were performed in 42 lactations and averaged by WIM. Animals experiencing late OLA had a more negative energy balance in WIM 1, 3, 4, and 5, with the greatest differences been seen in WIM 3 and 4. A higher proportion of the fatty acids were medium chained, C14:0 and C16:0, in the early than in the late OLA group from WIM 1. In WIM 4, the proportion of total fatty acid content that was C16:0 predicted late OLA, with 74% sensitivity and 80% specificity. The long-chain proportion of the fatty acids C18:0 and cis-9 C18:1 were lower in the early than in the late OLA group. Differences were greatest in WIM 4 and 5. Differences in concentrations of cis-9 C18:1 were seen between the groups from WIM 1. No relationship was seen between OLA and milk concentrations of either protein or fat, or between OLA and the milk fat:protein ratio. The differences in milk fatty acid proportions between the 2 groups are most likely related to differences in energy balance. The study shows that frozen milk samples can be tested for fatty acids by FTIR spectroscopy and that FTIR spectroscopy of milk can be used to provide real-time information about cow reproductive function., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Colorectal cancer is a global health challenge with high incidence rate and mortality. The patients' prognosis is strongly associated with disease stage and currently there is a need for improved prognostic and predictive biomarkers. In this study, novel colorectal cancer-specific transcript structures were nominated from whole transcriptome sequencing of seven colorectal cancer cell lines, two primary colorectal carcinomas with corresponding normal colonic mucosa and 16 normal tissues. The nominated transcripts were combined with gene level outlier expression analyses in a cohort of 505 colorectal cancers to identify biomarkers with capacity to stratify colorectal cancer subgroups. The transcriptome sequencing data and outlier expression analysis revealed 11 novel colorectal cancer-specific exon-exon junctions, of which 3 were located in the gene VNN1. The junctions within VNN1 were further characterized using rapid amplification of cDNA ends (RACE) and the prevalence of the subsequently characterized novel transcript, VNN1-AB, was investigated by real-time RT-PCR in 291 samples of miscellaneous origins. VNN1-AB was not present in any of the 43 normal colorectal tissue samples investigated, but in 5 of the 6 polyps, and 102 of the 136 (75%) colorectal cancers. We have identified a novel transcript of the VNN1 gene, with an organ-confined complete specificity for colorectal neoplasia., (© 2014 UICC.)

Genes with altered DNA methylation can be used as biomarkers for cancer detection and assessment of prognosis. Here we analyzed the methylation status of a colorectal cancer biomarker panel (CNRIP1, FBN1, INA, MAL, SNCA, and SPG20) in 97 cancer cell lines, derived from 17 different cancer types. Interestingly, the genes were frequently methylated also in hematological cancer types and were therefore subjected to analyses in primary tumor samples from the major types of non-Hodgkin lymphomas (NHL) and in healthy controls. In total, the genes CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 were methylated in 53%, 23%, 52%, 69%, 97%, and 92% of the tumor samples, respectively, and were unmethylated in all healthy controls. With the exception of a single tumor sample, a correct prediction of lymphoma or normal sample was made in a blinded analysis of the validation series using a combination of SNCA and SPG20. The combined ROC-curve analysis of these genes resulted in an area under the curve of 0.999 (P = 4.2 × 10(-18)), and a sensitivity and specificity of 98% and 100%, respectively, across the test and validation series. Interestingly, the promoter methylation of CNRIP1 was associated with decreased overall survival in diffuse large B-cell lymphoma (DLBCL) (P = 0.03).   In conclusion, our results demonstrate that SNCA and SPG20 methylation might be suitable for early detection and monitoring of NHL. Furthermore, CNRIP1 could potentially be used as a prognostic factor in DLBCL.

Epigenetic alterations of gene expression are important in the development of cancer. In this study, we identified genes which are epigenetically altered in major lymphoma types. We used DNA microarray technology to assess changes in gene expression after treatment of 11 lymphoma cell lines with epigenetic drugs. We identified 233 genes with upregulated expression in treated cell lines and with downregulated expression in B-cell lymphoma patient samples (n = 480) when compared to normal B cells (n = 5). The top 30 genes were further analyzed by methylation specific PCR (MSP) in 18 lymphoma cell lines. Seven of the genes were methylated in more than 70% of the cell lines and were further subjected to quantitative MSP in 37 B-cell lymphoma patient samples (diffuse large B-cell lymphoma (activated B-cell like and germinal center B-cell like subtypes), follicular lymphoma and Burkitt`s lymphoma) and normal B lymphocytes from 10 healthy donors. The promoters of DSP, FZD8, KCNH2, and PPP1R14A were methylated in 28%, 67%, 22%, and 78% of the 36 tumor samples, respectively, but not in control samples. Validation using a second series of healthy donor controls (n = 42; normal B cells, peripheral blood mononuclear cells, bone marrow, tonsils and follicular hyperplasia) and fresh-frozen lymphoma biopsies (n = 25), confirmed the results. The DNA methylation biomarker panel consisting of DSP, FZD8, KCNH2, and PPP1R14A was positive in 89% (54/61) of all lymphomas. Receiver operating characteristic analysis to determine the discriminative power between lymphoma and healthy control samples showed a c-statistic of 0.96, indicating a possible role for the biomarker panel in monitoring of lymphoma patients.

Cell lines are invaluable biomedical research tools, and recent literature has emphasized the importance of genotype authentication and characterization. In the present study, 24 out of 27 cell line identities were confirmed by short tandem repeat profiling. The molecular phenotypes of the 24 colon cancer cell lines were examined, and microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) were determined, using the Bethesda panel mononucleotide repeat loci and two epimarker panels, respectively. Furthermore, the BRAF, KRAS and PIK3CA oncogenes were analyzed for mutations in known hotspots, while the entire coding sequences of the PTEN and TP53 tumor suppressors were investigated. Nine cell lines showed MSI. Thirteen and nine cell lines were found to be CIMP positive, using the Issa panel and the Weisenberger et al. panel, respectively. The latter was found to be superior for CIMP classification of colon cancer cell lines. Seventeen cell lines harbored disrupting TP53 mutations. Altogether, 20/24 cell lines had the mitogen-activated protein kinase pathway activating mutually exclusive KRAS or BRAF mutations. PIK3CA and PTEN mutations leading to hyperactivation of the phosphoinositide 3-kinase/AKT pathway were observed in 13/24 cell lines. Interestingly, in four cell lines there were no mutations in neither BRAF, KRAS, PIK3CA nor in PTEN. In conclusion, this study presents molecular features of a large number of colon cancer cell lines to aid the selection of suitable in vitro models for descriptive and functional research.

The three main types of urological cancers are mostly curable by surgical resection, if early detected. We aimed to identify novel DNA methylation biomarkers common to these three urological cancers, potentially suitable for non-invasive testing. From a candidate list of markers created after gene expression assessment of pharmacologically treated cell lines and tissue samples, two genes were selected for further validation. Methylation levels of these genes were quantified in a total of 12 cancer cell lines and 318 clinical samples. PCDH17 and TCF21 methylation levels provided a sensitivity rate of 92% for bladder cancer, 67% for renal cell tumors and 96% for prostate cancer. Methylation levels were significantly different from those of cancer free individuals (n = 37) for all tumor types (p < 0.001), providing 83% sensitivity and 100% specificity for cancer detection. Although in urine samples the sensitivity was 60%, 32% and 26% for bladder, renal, and prostate tumors, respectively (39% overall), absolute specificity was retained. We identified novel and highly specific methylation markers common to the three main urological cancers. However, additional efforts are required to increase the assay's sensitivity, enabling the simultaneous non-invasive screening of urological tumors in a single voided urine analysis.

Purpose: To identify a panel of epigenetic biomarkers for accurate bladder cancer (BlCa) detection in urine sediments., Experimental Design: Gene expression microarray analysis of BlCa cell lines treated with 5-aza-2'-deoxycytidine and trichostatin A as well as 26 tissue samples was used to identify a list of novel methylation candidates for BlCa. Methylation levels of candidate genes were quantified in 4 BlCa cell lines, 50 BlCa tissues, 20 normal bladder mucosas (NBM), and urine sediments from 51 BlCa patients and 20 healthy donors, 19 renal cancer patients, and 20 prostate cancer patients. Receiver operator characteristic curve analysis was used to assess the diagnostic performance of the gene panel., Results: GDF15, HSPA2, TMEFF2, and VIM were identified as epigenetic biomarkers for BlCa. The methylation levels were significantly higher in BlCa tissues than in NBM (P < 0.001) and the cancer specificity was retained in urine sediments (P < 0.001). A methylation panel comprising GDF15, TMEFF2, and VIM correctly identified BlCa tissues with 100% sensitivity and specificity. In urine samples, the panel achieved a sensitivity of 94% and specificity of 100% and an area under the curve of 0.975. The gene panel could discriminate BlCa from both healthy individuals and renal or prostate cancer patients (sensitivity, 94%; specificity, 90%)., Conclusions: By using a genome-wide approach, we have identified a biomarker panel that allows for early and accurate noninvasive detection of BlCa using urine samples., (©2010 AACR.)

Purpose: The purpose of this study was to identify genetic aberrations contributing to clinical aggressiveness of malignant peripheral nerve sheath tumors (MPNSTs)., Patients and Methods: Samples from 48 MPNSTs and 10 neurofibromas were collected from 51 patients with (n = 31) or without (n = 20) neurofibromatosis type 1 (NF1). Genome-wide DNA copy number changes were assessed by chromosomal and array-based comparative genomic hybridization (CGH) and examined for prognostic significance. For a subset of 20 samples, RNA microarray data were integrated with the genome data to identify potential target genes., Results: Forty-four (92%) MPNSTs displayed DNA copy number changes (median, 18 changes per tumor; range, 2 to 35 changes). Known frequent chromosomal gains at chromosome arms 8q (69%), 17q (67%), and 7p (52%) and losses from 9p (50%), 11q (48%), and 17p (44%) were confirmed. Additionally, gains at 16p or losses from 10q or Xq identified a high-risk group with only 11% 10-year disease-specific survival (P = .00005). Multivariate analyses including NF1 status, tumor location, size, grade, sex, complete remission, and initial metastatic status showed that the genomic high-risk group was the most significant predictor of poor survival. Several genes whose expression was affected by the DNA copy number aberrations were identified., Conclusion: The presence of specific genetic aberrations was strongly associated with poor survival independent of known clinical risk factors. Conversely, within the total patient cohort with 34% 10-year disease-specific survival, a low-risk group was identified: without changes at chromosomes 10q, 16p, or Xq in their MPNSTs, the patients had 74% 10-year survival.

Progesterone profiles in Norwegian Red cows were categorized, and associations between the occurrence of irregularities in the profiles and the commencement of luteal activity were investigated. The cows were managed in 3 feeding trials from 1994 to 2001 and from 2005 to 2008 at the Norwegian University of Life Sciences. The cows were followed from calving, and the milk samples collected represented 502 lactations from 302 cows. Milk samples for progesterone analysis were taken 3 times weekly from 1994 throughout 1998 and from 2005 to 2008 and 2 times weekly from 1999 to 2001. Commencement of luteal activity was defined as the first day of 2 consecutive measurements of progesterone concentration >or=3 ng/mL not earlier than 10 d after calving. Delayed ovulation type I was defined as consistently low progesterone concentration, <3 ng/mL for >or=50 d postpartum. Delayed ovulation type II was defined as prolonged interluteal interval with milk progesterone measurements <3 ng/mL for >or=12 d between 2 luteal phases. Persistent corpus luteum (PCL) type I was defined as delayed luteolysis with milk progesterone >or=3 ng/mL for >or=19 d during the first estrous cycle postpartum. Persistent corpus luteum type II was defined as delayed luteolysis with milk progesterone >or=3 ng/mL for >or=19 d during subsequent estrous cycles before first artificial insemination. Delayed ovulation type I was present in 14.7%, delayed ovulation type II in 2.8%, PCL type I in 6.7%, and PCL type II in 3.3% of the profiles. Commencement of luteal activity was related to milk yield, parity, PCL type I, and the summated occurrence of PCL type I and II. The least squares means for the interval to commencement of luteal activity were 24.2 d when PCL type I and II were present and 29.5 d when PCL type I and II were absent. The likelihood of pregnancy to first service was not affected in cows with a history of PCL when artificial insemination was carried out at progesterone concentrations <3 ng/mL (i.e., during estrus); however, cows that had experienced PCL were more likely to be inseminated during a luteal phase. The occurrence of delayed ovulation and PCL in Norwegian Red cows was less than that reported in most other dairy populations.

Background: Tumor-derived aberrantly methylated DNA might serve as diagnostic biomarkers for cancer, but so far, few such markers have been identified. The aim of the present study was to investigate the potential of the MAL (T-cell differentiation protein) gene as an early epigenetic diagnostic marker for colorectal tumors., Methods: Using methylation-specific polymerase chain reaction (MSP) the promoter methylation status of MAL was analyzed in 218 samples, including normal mucosa (n = 44), colorectal adenomas (n = 63), carcinomas (n = 65), and various cancer cell lines (n = 46). Direct bisulphite sequencing was performed to confirm the MSP results. MAL gene expression was investigated with real time quantitative analyses before and after epigenetic drug treatment. Immunohistochemical analysis of MAL was done using normal colon mucosa samples (n = 5) and a tissue microarray with 292 colorectal tumors., Results: Bisulphite sequencing revealed that the methylation was unequally distributed within the MAL promoter and by MSP analysis a region close to the transcription start point was shown to be hypermethylated in the majority of colorectal carcinomas (49/61, 80%) as well as in adenomas (45/63, 71%). In contrast, only a minority of the normal mucosa samples displayed hypermethylation (1/23, 4%). The hypermethylation of MAL was significantly associated with reduced or lost gene expression in in vitro models. Furthermore, removal of the methylation re-induced gene expression in colon cancer cell lines. Finally, MAL protein was expressed in epithelial cells of normal colon mucosa, but not in the malignant cells of the same type., Conclusion: Promoter hypermethylation of MAL was present in the vast majority of benign and malignant colorectal tumors, and only rarely in normal mucosa, which makes it suitable as a diagnostic marker for early colorectal tumorigenesis.

Molecular markers that could stratify prostate cancer patients according to risk of disease progression would allow a significant improvement in the management of this clinically heterogeneous disease. In the present study, we analyzed the genetic profile of a consecutive series of 51 clinically confined prostate carcinomas and 27 benign prostatic hyperplasias using comparative genomic hybridization (CGH). We then added our findings to the existing literature data in order to perform a meta-analysis on a total of 294 prostate cancers with detailed CGH and clinicopathological information, using multivariate statistical methods that included principal component, hierarchical clustering, time of occurrence, and regression analyses. Whereas several genomic imbalances were shared by organ-confined, locally invasive, and metastatic prostate cancers, 6q and 10q losses and 7q and 8q gains were significantly more frequent in patients with extra-prostatic disease. Regression analysis indicated that 8q gain and 13q loss were the best predictors of locally invasive disease, whereas 8q gain and 6q and 10q losses were associated with metastatic disease. We propose a genetic pathway of prostate carcinogenesis with two distinct initiating events, namely, 8p and 13q losses. These primary imbalances are then preferentially followed by 8q gain and 6q, 16q, and 18q losses, which in turn are followed by a set of late events that make recurrent and metastatic prostate cancers genetically more complex. We conclude that significant differences exist in the genetic profile of organ-confined, locally invasive, and advanced prostate cancer and that genetic features may carry prognostic information independently of Gleason grade.

It was recently shown by flow and static cytometry that a large sub-group of follicular adenomas of the thyroid--fetal/embryonal adenomas--display an aneuploid phenotype. It was also shown that thyroid lesions with a DNA content within the triploid range were either fetal adenomas or follicular carcinomas with a fetal adenoma growth pattern. Follicular tumours with growth patterns other than the so-called fetal adenoma-like pattern were usually diploid or near-diploid. In an attempt to clarify the pattern of chromosomal imbalances in follicular tumours, comparative genomic hybridization (CGH) analysis was performed in a series of 18 follicular neoplasms (ten fetal/embryonal and four common follicular adenomas and four minimally invasive follicular carcinomas). For each tumour, the DNA content was determined by flow cytometry and, in some cases, also by static cytometry. Finally, the copy number of selected chromosomes was determined by interphase fluorescence in situ hybridization (FISH) using centromere probes. With the exception of the single diploid fetal adenoma, all fetal adenomas displayed several DNA copy number changes, with frequent gains of several chromosomes, which were found to be either tetrasomic or trisomic by FISH. This genetic pattern was also present in the single case of follicular carcinoma with aneuploidy and fetal adenoma-like growth pattern. Follicular adenomas other than fetal adenomas, and the remaining follicular carcinomas, showed more losses than gains of chromosomes. These results suggest that follicular tumourigenesis may follow at least two pathways: one characterized by prominent aneuploidy and numerous gains, in which the tumours display a fetal adenoma-like growth pattern; and another accompanied by less obvious aneuploidy or even quasi-diploidy and dominant chromosome losses, in which the tumours display a common follicular architecture., (Copyright 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

In cancer biology, cell lines are often used instead of primary tumors because of their widespread availability and close reflection of the in vivo state. Cancer is a genetic disease, commonly caused by small- and large-scale DNA rearrangements. Therefore, it is essential to know the genomic profiles of tumor cell lines to enable their correct and efficient use as experimental tools. Here, we present a comprehensive study of the genomic profiles of 20 colon cancer cell lines combining conventional karyotyping (G-banding), comparative genomic hybridization (CGH), and multicolor fluorescence in situ hybridization (M-FISH). Major differences between the microsatellite instability (MSI) and chromosome instability (CIN) cell lines are shown; the CIN cell lines exhibited complex karyotypes involving many chromosomes (mean: 8.5 copy number changes), whereas the MSI cell lines showed considerably fewer aberrations (mean: 2.6). The 3 techniques complement each other to provide a detailed picture of the numerical and structural chromosomal changes that characterize cancer cells. Therefore, 7 of the cell lines (Colo320, EB, Fri, IS2, IS3, SW480, and V9P) are here completely karyotyped for the first time and, among these, 5 have not previously been cytogenetically described. By hierarchical cluster analysis, we show that the cell lines are representative models for primary carcinomas at the genome level. We also present the genomic profiles of an experimental model for tumor progression, including 3 cell lines (IS1, IS2, and IS3) established from a primary carcinoma, its corresponding liver- and peritoneal metastasis from the same patient. To address the question of clonality, we compared the genome of 3 common cell lines grown in 2 laboratories. Finally, we compared all our results with previously published CGH data and karyotypes of colorectal cell lines. In conclusion, the large variation in genetic complexity of the cell lines highlights the importance of a comprehensive reference of genomic profiles for investigators engaged in functional studies using these research tools.

Background: The widespread use of prostate-specific antigen (PSA) testing to screen for prostate carcinoma has led to significant overdiagnosis, due to the frequent detection of indolent malignancies on PSA screening. The detection of abnormal PSA levels typically is followed by ultrasound-guided needle biopsy. Therefore, in an effort to identify genetic markers that augment the information provided by standard histopathologic classification, the authors tested the feasibility of using these minute biopsy samples for genomic profiling via chromosome banding analysis and comparative genomic hybridization (CGH)., Methods: Ultrasound-guided needle biopsy specimens obtained preoperatively from 35 patients with prostate carcinoma were analyzed via chromosome banding analysis (after short-term culturing) and CGH. The findings of these analyses then were analyzed for potential correlations with clinicopathologic parameters., Results: Chromosome banding analysis and CGH were possible in 34 and 33 of the 35 study specimens, respectively. Combined analysis revealed aberrations in 69% of all samples investigated. Copy number losses occurred most commonly at 8p (58% of all abnormal specimens), 16q (42%), and 13q (37%), whereas the only gains detected in more than 1 specimen were those that occurred at 8q (37%). Genomic imbalances and losses at 16q were significantly associated with more poorly differentiated subtypes of prostate carcinoma (P = 0.048 and P = 0.019, respectively), whereas gains at 8q and losses at 16q were significantly correlated with clinically advanced disease (P = 0.048 for the finding of a gain at 8q together with a loss at 16q; P = 0.01 for the finding of either aberration alone)., Conclusions: The authors conclude that genomic analysis of suspected prostate carcinoma specimens obtained via ultrasound-guided needle biopsy is feasible. Thus, it may be possible to use genetic markers to obtain diagnostic and/or prognostic information that is useful in the making of preoperative decisions regarding prostate carcinoma management.

Background: Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths in the Western world, and despite the fact that metastases are usually the ultimate cause of deaths, the knowledge of the genetics of advanced stages of this disease is limited. In order to identify potential genetic abnormalities underlying the development of local and distant metastases in CRC patients, we have, by comparative genomic hybridization, compared the DNA copy number profiles of 10 primary carcinomas, 14 local recurrences, 7 peritoneal carcinomatoses, and 42 liver metastases from 61 CRC patients., Results: The median number of aberrations among the primary carcinomas, local recurrences, carcinomatoses, and liver metastases was 10, 6, 13, and 14, respectively. Several genetic imbalances, such as gains of 7, 8q, 13q, and 20, and losses of 4q, 8p, 17p, and 18, were common in all groups. In contrast, gains of 5p and 12p were more common in the carcinomatoses than in other stages of the disease. With hierarchical cluster analysis, liver metastases could be divided into two main subgroups according to clusters of chromosome changes., Conclusions: Each stage of CRC progression is characterized by a particular genetic profile, and both carcinomatoses and liver metastases are more genetically complex than local recurrences and primary carcinomas. This is the first genome profiling of local recurrences and carcinomatoses, and gains of 5p and 12p seem to be particularly important for the spread of the CRC cells within the peritoneal cavity.

Desmoid tumors are benign neoplasms of the fibromatosis group. Data on their acquired chromosomal changes are sparse and, therefore, we wanted to ascertain what genomic losses and gains these tumors may have incurred. DNA was extracted from a total of 26 formalin-fixed, paraffin-embedded desmoid tumors followed by comparative genomic hybridization (CGH) and interphase fluorescence in situ hybridization (I-FISH) analyses. Ten of 12 informative tumors were normal by CGH; the two abnormal ones had loss of chromosome 6 and loss of 6q and gain of chromosome 20, respectively. I-FISH analyses with an alpha-satellite probe specific for chromosome 8 of 26 desmoids, including one tumor that by karyotyping had +i(8)(q10), showed no evident abnormalities. An explanation for the relatively high frequency of genomically normal tumors by CGH seen in this study may be sought in the fact that as many as 10 of the 12 informative tumors were abdominal desmoids, a subset of tumors also previously found to exhibit genomic changes only rarely. It is therefore possible that abdominal desmoids might be non-neoplastic tumors or neoplastic tumors with genetic changes too small to be discovered by CGH, whereas desmoid tumors from other locations exhibit detectable genomic changes at a significantly higher frequency.

The majority of genetic studies of colorectal carcinogenesis have focused on changes found in primary tumors. Despite the fact that liver metastases are a leading cause of colorectal cancer deaths, the molecular genetic basis of the advanced disease stages remains poorly understood. We performed comparative genomic hybridization (CGH) on 17 liver metastases from colorectal carcinomas and compared the quantitative profile with the qualitative profile previously obtained with chromosome banding. An average of 12.6 aberrations per tumor was found by CGH. Chromosome 18 and chromosome arms 4q, 8p, and 17p were most frequently lost, whereas chromosomes 7 and 20 and chromosome arms 6p, 8q, and 13q were most frequently gained. We compared the chromosome banding and CGH data after converting the karyotypes into net copy number gains and losses. Ten tumors showed agreement between the findings of the two techniques, whereas five tumors did not (in two cases, no mitotic cells were obtained for banding analysis). All five discordant cases had a "simple" abnormal or normal karyotype, but revealed multiple changes by CGH. A likely explanation for this discrepancy is that in vitro growth before G-banding selected against the cancer cells. Interestingly, by comparing the CGH profiles of the "complex" vs. the "simple"/normal karyotype groups, deletion of 8p and gain of 16q were seen more frequently in the former group. The liver metastases had the same aberrations as seen in primary colorectal carcinomas, summarized in a literature survey. However, these aberrations were seen more frequently in liver metastases, which may be attributable to increased genetic instability., (Copyright 2003 Wiley-Liss, Inc.)

In order to investigate the genetics of testicular germ cell tumors (TGCTs), we examined 33 TGCTs, including 15 familial/bilateral and 18 sporadic tumors, using comparative genomic hybridization. The frequencies of the histological subtypes were comparable between the two groups. Gains of the whole or parts of chromosome 12 were found in 30 tumors (91%). Furthermore, increased copy number of the whole or parts of chromosomes 7, 8, 17, and X, and decreased copy number of the whole or parts of chromosomes 4, 11, 13, and 18 were observed in > or = 50% of the tumors. Sixteen smallest regions of overlapping changes were delineated on 12 different chromosomes. The chromosomal copy numbers of familial/bilateral and sporadic TGCTs were comparable, suggesting similar genetic pathways to disease in both groups. However, significant differences were observed between the two main histological subgroups. Gains from 15q and 22q were associated with seminomas (P = 0.005 and P = 0.02, respectively), whereas gain of the proximal 17q (17q11.2-21) and high-level amplification from chromosome arm 12p, and losses from 10q were associated with nonseminomas (P < 0.001, P = 0.04, and P = 0.03, respectively)., (Copyright 2002 Wiley-Liss, Inc.)

Malignant ovarian germ cell tumors (OGCTs) include immature teratomas (ITs), dysgerminomas (DGs), endodermal sinus tumors (ESTs), choriocarcinomas, and embryonal carcinomas. Knowledge about the genetic changes associated with malignant OGCT development is sparse. We therefore analyzed 25 OGCTs (12 DGs, 4 ESTs, and 9 ITs) for gains and losses by comparative genomic hybridization. In total, more gains than losses were observed, and the number of alterations ranged from 0-20 per tumor. The average number of changes among DGs, ESTs, and ITs was 10, 6, and 1.4, respectively. The most common changes in DGs were gains from chromosome arms 1p (33%), 6p (33%), 12p (67%), 12q (75%), 15q (42%), 20q (50%), 21q (67%), and 22q (58%); gains of the whole of chromosomes 7 (42%), 8 (42%), 17 (42%), and 19 (50%); and losses from 13q (58%). Two of three DGs with a gonadoblastoma component showed gains of 3p21 and loss of 5p, whereas none of the nine pure DGs had these changes, suggesting that they might be characteristic either of gonadoblastoma or of DG developing from a gonadoblastoma. Gain of 12p and gain from 1q were seen in three of four ESTs, whereas gains from 3p, 11q, and Xp and loss from 18q were each found in two tumors. Five of the ITs revealed changes (range, 1-4 changes/tumor), with gains from 1p, 16p, 19, and 22q each being found in two tumors. We conclude that ovarian DGs and ESTs seem to develop via the same genetic pathways that are already known for testicular germ cell tumors. On the other hand, ITs do not exhibit gain of 12p and also typically show fewer changes than other malignant OGCTs, indicating that they arise via different pathogenetic mechanisms.