Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.     

18F-2-deoxy-2-fluoro-D-glucose positron emission tomography, computed tomography, and magnetic resonance imaging for the detection of experimental colorectal liver metastases

During the treatment of colorectal liver metastases, evaluation of treatment efficacy is of the utmost importance for decision making. The aim of the present study was to explore the ability of preclinical imaging modalities to detect experimental liver metastases. Nine male Wag/Rij rats underwent a laparotomy with intraportal injection of CC531 tumor cells. On days 7, 10, and 14 after tumor induction, sequential positron emission tomography (PET), computed tomography (CT), and magnetic resonance imaging (MRI) scans were acquired of each rat. At each time point, three rats were euthanized and the metastases in the liver were documented histologically. Topographically, the liver was divided into eight segments and the image findings were compared on a segment-by-segment basis with the histopathologic findings. Sixty-four liver segments were analyzed, 20 of which contained tumor deposits. The overall sensitivity of PET, CT, and MRI was 30%, 25%, and 20%, respectively. For the detection of tumors with a histologic diameter exceeding 1 mm (n = 8), the sensitivity of PET, CT, and MRI was 63%, 38%, and 38%, respectively. The overall specificity of PET, CT, and MRI was 98%, 100%, and 93%, respectively. This study showed encouraging detectability and sensitivity for preclinical imaging of small liver tumors and provides valuable information on the imaging techniques for designing future protocols.     

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac^–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g^–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity. Correspondence to: R. F. M.van Gorcom     

Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice.

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.     

People with multiple tattoos and/or piercings are not at increased risk for HBV or HCV in The Netherlands

Background

Although published results are inconsistent, it has been suggested that tattooing and piercing are risk factors for HBV and HCV infections. To examine whether tattooing and piercing do indeed increase the risk of infection, we conducted a study among people with multiple tattoos and/or piercings in the Netherlands who acquired their tattoos and piercings in the Netherlands and/or abroad.

Methods

Tattoo artists, piercers, and people with multiple tattoos and/or piercings were recruited at tattoo conventions, shops (N = 182), and a biannual survey at our STI-outpatient clinic (N = 252) in Amsterdam. Participants were interviewed and tested for anti-HBc and anti-HCV. Determinants of HBV and HCV infections were analysed using logistic regression analysis.

Results

The median number of tattoos and piercings was 5 (IQR 2–10) and 2 (IQR 2–4), respectively. Almost 40% acquired their tattoo of piercing abroad. In total, 18/434 (4.2%, 95%CI: 2.64%–6.46%) participants were anti-HBc positive and 1 was anti-HCV positive (0.2%, 95%CI: 0.01%–1.29%). Being anti-HBc positive was independently associated with older age (OR 1.68, 95%CI: 1.03–2.75 per 10 years older) and being born in an HBV-endemic country (OR 7.39, 95%CI: 2.77–19.7). Tattoo- and/or piercing-related variables, like having a tattoo or piercing in an HBV endemic country, surface percentage tattooed, number of tattoos and piercings etc., were not associated with either HBV or HCV.

Conclusions

We found no evidence for an increased HBV/HCV seroprevalence among persons with multiple tattoos and/or piercings, which might be due to the introduction of hygiene guidelines for tattoo and piercing shops in combination with the low observed prevalence of HBV/HCV in the general population. Tattoos and/or piercings, therefore, should not be considered risk factors for HBV/HCV in the Dutch population. These findings imply the importance of implementation of hygiene guidelines in other countries.     

Coxsackievirus Cloverleaf RNA Containing a 5′ Triphosphate Triggers an Antiviral Response via RIG-I Activation

Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5′ triphosphate (5′ppp)-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3), a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5′ppp. We show here that a 5′ppp-containing cloverleaf (CL) RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand.     

Phosphorus intensity determines short-term P uptake by pigeon pea (<Emphasis Type="Italic">Cajanus cajan</Emphasis> L.) grown in soils with differing P buffering capacity

Phosphorus (P) uptake by plant roots depends on P intensity (I) and P quantity (Q) in the soil. The relative importance of Q and I on P uptake is unknown for soils with large P sorption capacities because of difficulties in determining trace levels of P in the soil solution. We applied a new isotope based method to detect low P concentrations (<20 μg P l⁻¹). The Q factor was determined by assessment of the isotopically exchangeable P in the soil (E-value) and the I factor was determined by measurement of the P concentration in the pore water. A pot trial was set up using four soils with similar labile P quantities but contrasting P buffering capacities. Soils were amended with KH₂PO₄ at various rates and pigeon pea (Cajanus cajan L.) was grown for 25 days. The P intensity ranged between 0.0008 and 50 mg P l⁻¹ and the P quantity ranged between 10 and 500 mg P kg⁻¹. Shoot dry matter (DM) yield and P uptake significantly increased with increasing P application rates in all soils. Shoot DM yield and P uptake, relative to the maximal yield or P uptake, were better correlated with the P concentration in the pore water (R ² = 0.83–0.90) than with the E-value (R ²=0.40–0.53). The observed P uptakes were strongly correlated to values simulated using a mechanistic rhizosphere model (NST 3.0). A sensitivity analysis reveals that the effect of P intensity on the short-term P uptake by pigeon pea exceeded the effect of P quantity both at low and high P levels. However, DM yield and P uptake at a given P intensity consistently increased with increasing P buffering capacity (PBC). The experimental data showed that the intensity yielding 80% of the maximal P uptake was 4 times larger in the soil with the smallest PBC compared to the soil with the largest PBC. This study confirms that short-term P uptake by legumes is principally controlled by the P intensity in the soil, but is to a large extent also affected by the PBC of the soil. Section Editor: N. J. Barrow     

The role of DNA methylation,nucleosome occupancy and histone modifications in paramutation

Paramutation is the transfer of epigenetic information between alleles that leads to a heritable change in expression of one of these alleles. Paramutation at the tissue‐specifically expressed maize (Zea mays) b1 locus involves the low‐expressing B′ and high‐expressing B‐I allele. Combined in the same nucleus, B′ heritably changes B‐I into B′. A hepta‐repeat located 100‐kb upstream of the b1 coding region is required for paramutation and for high b1 expression. The role of epigenetic modifications in paramutation is currently not well understood. In this study, we show that the B′ hepta‐repeat is DNA‐hypermethylated in all tissues analyzed. Importantly, combining B′ and B‐I in one nucleus results in de novo methylation of the B‐I repeats early in plant development. These findings indicate a role for hepta‐repeat DNA methylation in the establishment and maintenance of the silenced B′ state. In contrast, nucleosome occupancy, H3 acetylation, and H3K9 and H3K27 methylation are mainly involved in tissue‐specific regulation of the hepta‐repeat. Nucleosome depletion and H3 acetylation are tissue‐specifically regulated at the B‐I hepta‐repeat and associated with enhancement of b1 expression. H3K9 and H3K27 methylation are tissue‐specifically localized at the B′ hepta‐repeat and reinforce the silenced B′ chromatin state. The B′ coding region is H3K27 dimethylated in all tissues analyzed, indicating a role in the maintenance of the silenced B′ state. Taken together, these findings provide insight into the mechanisms underlying paramutation and tissue‐specific regulation of b1 at the level of chromatin structure.     

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.     

Non-rigid deformation to include subject-specific detail in musculoskeletal models of CP children with proximal femoral deformity and its effect on muscle and contact forces during gait

To account for proximal femoral deformities in children with cerebral palsy (CP), subject-specific musculoskeletal models are needed. Non-rigid deformation (NRD) deforms generic onto personalized bone geometry and thereby transforms the muscle points. The goal of this study was to determine to what extent the models and simulation outcomes in CP patients differ when including subject-specific detail using NRD or Magnetic Resonance Imaging (MRI)-based models. The NRD models slightly overestimated hip contact forces compared to MRI models and differences in muscle point positions and moment arm lengths (MALs) remained, although differences were smaller than for the generic model.