Gene expression profiles from formalin fixed paraffin embedded breast cancer tissue are largely comparable to fresh frozen matched tissue

Background and Methods

Formalin Fixed Paraffin Embedded (FFPE) samples represent a valuable resource for cancer research. However, the discovery and development of new cancer biomarkers often requires fresh frozen (FF) samples. Recently, the Whole Genome (WG) DASL (cDNA-mediated Annealing, Selection, extension and Ligation) assay was specifically developed to profile FFPE tissue. However, a thorough comparison of data generated from FFPE RNA and Fresh Frozen (FF) RNA using this platform is lacking. To this end we profiled, in duplicate, 20 FFPE tissues and 20 matched FF tissues and evaluated the concordance of the DASL results from FFPE and matched FF material.

Methodology and Principal Findings

We show that after proper normalization, all FFPE and FF pairs exhibit a high level of similarity (Pearson correlation >0.7), significantly larger than the similarity between non-paired samples. Interestingly, the probes showing the highest correlation had a higher percentage G/C content and were enriched for cell cycle genes. Predictions of gene expression signatures developed on frozen material (Intrinsic subtype, Genomic Grade Index, 70 gene signature) showed a high level of concordance between FFPE and FF matched pairs. Interestingly, predictions based on a 60 gene DASL list (best match with the 70 gene signature) showed very high concordance with the MammaPrint® results.

Conclusions and Significance

We demonstrate that data generated from FFPE material with the DASL assay, if properly processed, are comparable to data extracted from the FF counterpart. Specifically, gene expression profiles for a known set of prognostic genes for a specific disease are highly comparable between two conditions. This opens up the possibility of using both FFPE and FF material in gene expressions analyses, leading to a vast increase in the potential resources available for cancer research.     

Coordinate induction of PPAR alpha and SREBP2 in multifunctional protein 2 deficient mice

Mice with inactivation of the D-specific multifunctional protein 2 (MFP2), a crucial enzyme of peroxisomal beta-oxidation, develop multiple pathologies in diverse tissues already starting in the postnatal period. Gene expression profiling performed on liver of 2-day-old pups revealed up-regulation of PPAR alpha responsive genes in knockout mice. Surprisingly, also genes involved in cholesterol biosynthesis were markedly induced. Real-time PCR confirmed the induction of PPAR alpha target genes and of HMGCR and SREBP2, both involved in cholesterol synthesis, in lactating and in adult MFP2 knockout mice. In accordance, the rate of cholesterol biosynthesis was significantly increased in liver of knockout mice but the hepatic cholesterol concentration was unaltered. In MFP2/PPAR alpha double knockout mice, up-regulations of SREBP2 and HMGCR were markedly attenuated. These data demonstrate a tight interrelationship between induction of PPAR alpha by endogenous ligands and up-regulation of genes of cholesterol biosynthesis through increased expression of SREBP2.     

Association of glutathione-S-transferase omega haplotypes with susceptibility to chronic obstructive pulmonary disease

Cigarette smoking is the main risk factor for developing the inflammatory lung disease chronic obstructive pulmonary disease (COPD). Differences in susceptibility among smokers have been attributed to a genetic predisposition. A recent publication on the Framingham Heart Study found a strong association of the Asn142Asp SNP in Glutatthione-S-transferase Omega (GSTO) 2 with forced expiratory volume in the first second (FEV₁) and forced vital capacity (FVC). FEV₁ is the main parameter reflecting the degree of airflow limitation in patients with COPD. Therefore the present study was undertaken to investigate whether the Asn142Asp polymorphism in GSTO2 occurs more frequently in patients with COPD than healthy subjects and to replicate the finding that it strongly correlates with FEV₁. Furthermore, the Ala140Asp substitution in GSTO1 was examined. Genotyping was carried out in 195 healthy controls and 355 patients with COPD. The results demonstrate that the Asn142Asp polymorphism in GSTO2 and the GSTO1140Asp/GSTO2142Asp haplotype were associated with increased risk of COPD. However, single-marker and haplotype-based analyses failed to reveal an association between lung function parameters and investigated non-synonymous coding SNPs in the GSTO genes. In conclusion, GSTO2 is a candidate gene for COPD, but is not associated with FEV₁.     

Short distance migrants travel as far as long distance migrants in lesser black‐backed gulls Larus fuscus

Migration strategies differ greatly among and within avian populations. The associated trade‐offs and fitness consequences of diverse strategies and how they persist are pertinent questions in migration research. Migration is a costly endeavour, presumably compensated for by better survival conditions in the non‐breeding area. One way to assess the cost of alternative strategies is to investigate the investment in movement across the entire annual cycle, an assessment made increasingly feasible with improvements in tracking technology. Our study focuses on lesser black‐backed gulls, generalist seabirds that exploit a broad range of resources, exhibit diverse migration strategies and have potentially altered migration strategies in response to human activities and climate change. We used GPS tracking to quantify lesser black‐backed gulls’ movement throughout their annual cycle and compare trade‐offs among four migration strategies. The annual cumulative distance travelled by long distance migrants wintering in west Africa, over 4000 km from their breeding colony, did not differ significantly from individuals of the same breeding colony wintering only a few hundred kilometres away in Great Britain. Short distance migrants returned to the colony first, and long distance migrants returned last. Sex and wing length were not correlated with maximum range, cumulative distance travelled or timing. Individuals spent only a small proportion of their time in flight and spent on average 17% of their time at sea throughout an annual cycle, suggesting a reliance on inland resources for many individuals. Analysing movement throughout the annual cycle can change our perspective and understanding of consequences of different migration strategies. Our study shows that a range of migration strategies coexists and we propose that the long term costs and benefits of these strategies balance out. Diversity in migration strategies may contribute to the resilience of this species in the face of ongoing anthropogenic impact on the environment.     

Low Atmospheric Nitrogen Loads Lead to Grass Encroachment in Coastal Dunes,but Only on Acid Soils

The impact of atmospheric N-deposition on succession from open sand to dry, lichen-rich, short grassland, and tall grass vegetation dominated by Carex arenaria was surveyed in 19 coastal dune sites along the Baltic Sea. Coastal dunes with acid or slightly calcareous sand reacted differently to atmospheric wet deposition of 5–8 kg N ha⁻¹ y⁻¹. Accelerated acidification, as well as increased growth of Carex and accumulation of organic matter, was observed only at acid sites with pH_NaCl of the parent material below 6.0. At sites with slightly calcareous parent material, increased N-deposition had no effect. A trigger for grass encroachment seems to be high acidification in early successional stages to below pH_NaCl 4.0. Metals like Al or Fe become freely available and may hamper intolerant species. At acid sites, N-mineralization increases with elevated N-deposition, which may further stimulate Carex arenaria. Due to high growth plasticity, efficient resource allocation and tolerance of high metal concentrations, C. arenaria is a superior competitor under these conditions and can start to dominate the dune system. Carex-dominated vegetation is species-poor. Even at the moderate N-loads in this study, foliose lichens, forbs and grasses were reduced in short grass vegetation at acid sites. Species indicating these first effects of atmospheric deposition on dry, lichen-rich, short grasslands are identified and recommendations for restoration of grass-encroached sites given.