55 research outputs found
Microfluidic extraction and digital quantification of circulating cell-free DNA from serum
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Shallow Whole-Genome Sequencing from Plasma Identifies FGFR1 Amplified Breast Cancers and Predicts Overall Survival.
Background: Focal amplification of fibroblast growth factor receptor 1 (FGFR1) defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate FGFR1 copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for FGFR1 amplification by FISH, and positive cases were confirmed with a microarray platform (OncoscanTM). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the FGFR1 status. Results: Tissue-based analyses identified FGFR1 amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% (n = 12) showed concordance for FGFR1 status between tissue and cfDNA. In one case, we were able to detect a high-level FGFR1 amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Conclusions: Screening for FGFR1 amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials
Quantifying HER-2 expression on circulating tumor cells by ACCEPT
Circulating tumor cells (CTCs) isolated from blood can be probed for the expression of treatment targets. Immunofluorescence is often used for both the enumeration of CTC and the determination of protein expression levels related to treatment targets. Accurate and reproducible assessment of such treatment target expression levels is essential for their use in the clinic. To enable this, an open source image analysis program named ACCEPT was developed in the EU-FP7 CTCTrap and CANCER-ID programs. Here its application is shown on a retrospective cohort of 132 metastatic breast cancer patients from which blood samples were processed by CellSearch (R) and stained for HER-2 expression as additional marker. Images were digitally stored and reviewers identified a total of 4084 CTCs. CTC's HER-2 expression was determined in the thumbnail images by ACCEPT. 150 of these images were selected and sent to six independent investigators to score the HER-2 expression with and without ACCEPT. Concordance rate of the operators' scoring results for HER2 on CTCs was 30% and could be increased using the ACCEPT tool to 51%. Automated assessment of HER-2 expression by ACCEPT on 4084 CTCs of 132 patients showed 8 (6.1%) patients with all CTCs expressing HER-2, 14 (10.6%) patients with no CTC expressing HER-2 and 110 (83.3%) patients with CTCs showing a varying HER-2 expression level. In total 1576 CTCs were determined HER-2 positive. We conclude that the use of image analysis enables a more reproducible quantification of treatment targets on CTCs and leads the way to fully automated and reproducible approaches
Sexual dimorphism in parental imprint ontogeny and contribution to embryonic development
The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution
In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline
Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation.
Caractérisation génomique et fonctionnelle de l'empreinte parentale par le modÚle murin mutant pour Dnmt3L
PARIS7-BibliothĂšque centrale (751132105) / SudocSudocFranceF
Ăvolution de lâempreinte parentale chez les mammifĂšres
Lâempreinte parentale ou empreinte gĂ©nomique impose un mode de reproduction sexuĂ© obligatoire chez les mammifĂšres. Ce phĂ©nomĂšne rĂ©sulte de lâexpression mono-allĂ©lique et monoparentale dâun groupe de gĂšnes. Ce mĂ©canisme de rĂ©gulation gĂ©nique concerne les mammifĂšres vivipares comme les euthĂ©riens et, dans une moindre mesure, les marsupiaux. Les mammifĂšres ovipares, ou monotrĂšmes, ne semblent pas dotĂ©s dâempreinte parentale. Cette restriction phylogĂ©nĂ©tique conforte lâhypothĂšse selon laquelle lâĂ©mergence de lâorgane placentaire aurait imposĂ© une pression sĂ©lective pour lâacquisition de lâempreinte. Ces arguments physiologiques ont Ă©tĂ© plus rĂ©cemment Ă©tayĂ©s par des arguments gĂ©nomiques, grĂące au sĂ©quençage du gĂ©nome de lâĂ©trange ornithorynque, un des rares monotrĂšmes encore existants. De nombreuses coĂŻncidences temporelles et fonctionnelles existent entre lâapparition de lâempreinte et lâaccumulation dâĂ©lĂ©ments transposables. Les analyses comparatives systĂ©matiques du gĂ©nome de diffĂ©rentes espĂšces de mammifĂšres, classiques et exotiques, sont essentielles Ă notre connaissance de lâĂ©mergence et de lâĂ©volution de lâempreinte
Circulating extracellular vesicles provide valuable protein, but not DNA, biomarkers in metastatic breast cancer
Abstract Detection of cellâfree circulating tumour DNA (ctDNA) and cancerâspecific extracellular vesicles (EVs) in patient blood have been widely explored as nonâinvasive biomarkers for cancer detection and disease follow up. However, most of the protocols used to isolate EVs coâisolate other components and the actual value of EVâassociated markers remain unclear. To determine the optimal source of clinicallyârelevant circulating biomarkers in breast cancer, we applied a size exclusion chromatography (SEC) procedure to analyse separately the content in nucleic acids of EVâenriched and EVâdepleted fractions, in comparison to total plasma. Both cellular and mitochondrial DNA (cellDNA and mtDNA) were detected in EVârich and EVâpoor fractions. Analysing specific mutations identified from tumour tissues, we detected tumourâspecific cellular alleles in all SEC fractions. However, quantification of ctDNA from total plasma was more sensitive than from any SEC fractions. On the other hand, mtDNA was preferentially enriched in EV fractions from healthy donor, whereas cancer patients displayed more abundant mtDNA in total plasma, and equally distributed in all fractions. In contrast to nucleic acids, using a Multiplexed beadâbased EVâanalysis assay, we identified three surface proteins enriched in EVs from metastatic breast cancer plasma, suggesting that a small set of EV surface molecules could provide a disease signature. Our findings provide evidence that the detection of DNA within total circulating EVs does not add value as compared to the whole plasma, at least in the metastatic breast cancer patients used here. However, analysis of a subtype of EVâassociated proteins may reliably identify cancer patients. These nonâinvasive biomarkers represent a promising tool for cancer diagnosis and realâtime monitoring of treatment efficacy and these results will impact the development of therapeutic approaches using EVs as targets or biomarkers of cancer
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