Leiden, Netherlands
Leiden, Netherlands

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ter Braak B.,Leiden University | Siezen C.L.,Medicines Evaluation Board MEB | Lee J.S.,Tel Aviv University | Rao P.,ServiceXS | And 5 more authors.
Breast Cancer Research | Year: 2017

Background: The insulin-like growth factor 1 (IGF1) signaling axis plays a major role in tumorigenesis. In a previous experiment, we chronically treated mice with several agonists of the IGF1 receptor (IGF1R). We found that chronic treatment with insulin analogues with high affinity towards the IGF1R (IGF1 and X10) decreased the mammary gland tumor latency time in a p53R270H/+WAPCre mouse model. Frequent injections with insulin analogues that only mildly activated the IGF1R in vivo (glargine and insulin) did not significantly decrease the tumor latency time in this mouse model. Methods: Here, we performed next-generation RNA sequencing (40 million, 100bp reads) on 50 mammary gland tumors to unravel the underlying mechanisms of IGF1R-promoted tumorigenesis. Mutational profiling of the individual tumors was performed to screen for treatment-specific mutations. The transcriptomic data were used to construct a support vector machine (SVM) classifier so that the phenotypic characteristics of tumors exposed to the different insulin analogue treatments could be predicted. For translational purposes, we ran the same classifiers on transcriptomic (micro-array) data of insulin analogue-exposed human breast cancer cell lines. Genome-scale metabolic modeling was performed with iMAT. Results: We found that chronic X10 and IGF1 treatment resulted in tumors with an increased and sustained proliferative and invasive transcriptomic profile. Furthermore, a Warburg-like effect with increased glycolysis was observed in tumors of the X10/IGF1 groups and, to a lesser extent, also in glargine-induced tumors. A metabolic flux analysis revealed that this enhanced glycolysis programming in X10/IGF1 tumors was associated with increased biomass production programs. Although none of the treatments induced genetic instability or enhanced mutagenesis, mutations in Ezh2 and Hras were enriched in X10/IGF1 treatment tumors. Conclusions: Overall, these data suggest that the decreased mammary gland tumor latency time caused by chronic IGF1R activation is related to modulation of tumor progression rather than increased tumor initiation. © 2017 The Author(s).


PubMed | University of Amsterdam, Federal University of Minas Gerais, Leiden University, University of Groningen and 3 more.
Type: | Journal: PeerJ | Year: 2016

The integration of invasive species into native food webs represent multifarious dynamics of ecological and evolutionary processes. We document incorporation of Prunus serotina (black cherry) into native insect food webs. We find that P. serotina harbours a herbivore community less dense but more diverse than its native relative, P. padus (bird cherry), with similar proportions of specialists and generalists. While herbivory on P. padus remained stable over the past century, that on P. serotina gradually doubled. We show that P. serotina may have evolved changes in investment in cyanogenic glycosides compared with its native range. In the leaf beetle Gonioctena quinquepunctata, recently shifted from native Sorbus aucuparia to P. serotina, we find divergent host preferences on Sorbus- versus Prunus-derived populations, and weak host-specific differentiation among 380 individuals genotyped for 119 SNP loci. We conclude that evolutionary processes may generate a specialized herbivore community on an invasive plant, allowing prognoses of reduced invasiveness over time. On the basis of the results presented here, we would like to caution that manual control might have the adverse effect of a slowing down of processes of adaptation, and a delay in the decline of the invasive character of P. serotina.


Vrijenhoek T.,University Utrecht | Kraaijeveld K.,Leiden University | Elferink M.,University Utrecht | De Ligt J.,Radboud University Nijmegen | And 44 more authors.
European Journal of Human Genetics | Year: 2015

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care. © 2015 Macmillan Publishers Limited All rights reserved.


Pontillo C.,Mosaiques Diagnostics GmbH | Pontillo C.,Charité - Medical University of Berlin | Filip S.,Charité - Medical University of Berlin | Filip S.,Academy of Athens | And 8 more authors.
Proteomics - Clinical Applications | Year: 2015

CE-MS is applied in clinical proteomics for both the identification of biomarkers of disease and assessment of biomarkers in clinical diagnosis. The analysis is reproducible, fast, and requires only small sample volumes. However, successful CE-MS analysis depends on several critical steps that can be consolidated as follows: (i) proper sample preparation and fractionation, (ii) application of suitable capillary coating and appropriate CE-MS interfaces, to ensure the reproducibility and stability of the analysis, and (iii) an optimized clinical and statistical study design to increase the chances for obtaining clinically relevant results. In this review, we cover all these aspects, and present several examples of the application of CE-MS in clinical proteomics. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Jewell R.,St James's Hospital | Conway C.,St James's Hospital | Mitra A.,St James's Hospital | Randerson-Moor J.,St James's Hospital | And 15 more authors.
Clinical Cancer Research | Year: 2010

Purpose: To use gene expression profiling of formalin-fixed primary melanoma samples to detect expression patterns that are predictive of relapse and response to chemotherapy. Experimental Design: Gene expression profiles were identified in samples from two studies (472 tumors). Gene expression data for 502 cancer-related genes from these studies were combined for analysis. Results: Increased expression of DNA repair genes most strongly predicted relapse and was associated with thicker tumors. Increased expression of RAD51 was the most predictive of relapse-free survival in unadjusted analysis (hazard ratio, 2.98; P = 8.80 × 10-6). RAD52 (hazard ratio, 4.73; P = 0.0004) and TOP2A (hazard ratio, 3.06; P = 0.009) were independent predictors of relapse-free survival in multivariable analysis. These associations persisted when the analysis was further adjusted for demographic and histologic features of prognostic importance (RAD52 P = 0.01; TOP2A P = 0.02). Using principal component analysis, expression of DNA repair genes was summarized into one variable. Genes whose expression correlated with this variable were predominantly associated with the cell cycle and DNA repair. In 42 patients treated with chemotherapy, DNA repair gene expression was greater in tumors from patients who progressed on treatment. Further data supportive of a role for increased expression of DNA repair genes as predictive biomarkers are reported, which were generated using multiplex PCR. Conclusions: Overexpression of DNA repair genes (predominantly those involved in double-strand break repair) was associated with relapse. These data support the hypothesis that melanoma progression requires maintenance of genetic stability and give insight into mechanisms of melanoma drug resistance and potential therapies. ©2010 AACR.


De Ruijter T.C.,Maastricht University | De Hoon J.P.,Maastricht University | Slaats J.,Maastricht University | De Vries B.,Maastricht University | And 8 more authors.
Laboratory Investigation | Year: 2015

Current genome-wide methods to detect DNA-methylation in healthy and diseased tissue require high-quality DNA from fresh-frozen (FF) samples. However, well-annotated clinical samples are mostly available as formalin-fixed, paraffin-embedded (FFPE) tissues containing poor-quality DNA. To overcome this limitation, we here aimed to evaluate a DNA restoration protocol for usage with the genome-wide Infinium HumanMethylation450 BeadChip assay (HM-450K). Sixty-six DNA samples from normal colon (n=9) and breast cancer (n=11) were interrogated separately using HM-450K. Analyses included matched FF/FFPE samples and technical duplicates. FFPE DNA was processed with (FFPEr) or without a DNA restoration protocol (Illumina). Differentially methylated genes were finally validated in 24 additional FFPE tissues using nested methylation-specific PCR (MSP). In summary, β-values correlation between FFPEr duplicates was high (ρ=0.9927 (s.d. ±0.0015)). Matched FF/FFPEr correlation was also high (ρ=0.9590 (s.d. ±0.0184)) compared with matched FF/FFPE (ρ=0.8051 (s.d. ±0.1028). Probe detection rate in FFPEr samples (98.37%, s.d. ±0.66) was comparable to FF samples (99.98%, s.d. ±0.019) and substantially lower in FFPE samples (82.31%, s.d. ±18.65). Assay robustness was not decreased by sample archival age up to 10 years. We could also demonstrate no decrease in assay robustness when using 100 ng of DNA input only. Four out of the five selected differentially methylated genes could be validated by MSP. The gene failing validation by PCR showed high variation of CpG β-values in primer-binding sites. In conclusion, by using the FFPE DNA restoration protocol, HM-450K assays provide robust, accurate and reproducible results with FFPE tissue-derived DNA, which are comparable to those obtained with FF tissue. Most importantly, differentially methylated genes can be validated using more sensitive techniques, such as nested MSP, altogether providing an epigenomics platform for molecular pathological epidemiology research on archived samples with limited tissue amount. © 2015 USCAP, Inc All rights reserved.


PubMed | Leiden University, ServiceXS, Maastricht University and RWTH Aachen
Type: Journal Article | Journal: Laboratory investigation; a journal of technical methods and pathology | Year: 2015

Current genome-wide methods to detect DNA-methylation in healthy and diseased tissue require high-quality DNA from fresh-frozen (FF) samples. However, well-annotated clinical samples are mostly available as formalin-fixed, paraffin-embedded (FFPE) tissues containing poor-quality DNA. To overcome this limitation, we here aimed to evaluate a DNA restoration protocol for usage with the genome-wide Infinium HumanMethylation450 BeadChip assay (HM-450K). Sixty-six DNA samples from normal colon (n=9) and breast cancer (n=11) were interrogated separately using HM-450K. Analyses included matched FF/FFPE samples and technical duplicates. FFPE DNA was processed with (FFPEr) or without a DNA restoration protocol (Illumina). Differentially methylated genes were finally validated in 24 additional FFPE tissues using nested methylation-specific PCR (MSP). In summary, -values correlation between FFPEr duplicates was high (=0.9927 (s.d. 0.0015)). Matched FF/FFPEr correlation was also high (=0.9590 (s.d. 0.0184)) compared with matched FF/FFPE (=0.8051 (s.d. 0.1028). Probe detection rate in FFPEr samples (98.37%, s.d. 0.66) was comparable to FF samples (99.98%, s.d. 0.019) and substantially lower in FFPE samples (82.31%, s.d. 18.65). Assay robustness was not decreased by sample archival age up to 10 years. We could also demonstrate no decrease in assay robustness when using 100ng of DNA input only. Four out of the five selected differentially methylated genes could be validated by MSP. The gene failing validation by PCR showed high variation of CpG -values in primer-binding sites. In conclusion, by using the FFPE DNA restoration protocol, HM-450K assays provide robust, accurate and reproducible results with FFPE tissue-derived DNA, which are comparable to those obtained with FF tissue. Most importantly, differentially methylated genes can be validated using more sensitive techniques, such as nested MSP, altogether providing an epigenomics platform for molecular pathological epidemiology research on archived samples with limited tissue amount.

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