The clinical utility of circulating tumor DNA (ctDNA) monitoring has been shown in tumors that harbor highly recurrent mutations. Leiomyosarcoma represents a type of tumor with a wide spectrum of ...heterogeneous genomic abnormalities; thus, targeting hotspot mutations or a narrow genomic region for ctDNA detection may not be practical. Here, we demonstrate a combinatorial approach that integrates different sequencing protocols for the orthogonal detection of single-nucleotide variants (SNV), small indels, and copy-number alterations (CNA) in ctDNA.
We employed Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for the analysis of SNVs and indels, together with a genome-wide interrogation of CNAs by Genome Representation Profiling (GRP). We profiled 28 longitudinal plasma samples and 25 tumor specimens from 7 patients with leiomyosarcoma.
We detected ctDNA in 6 of 7 of these patients with >98% specificity for mutant allele fractions down to a level of 0.01%. We show that results from CAPP-Seq and GRP are highly concordant, and the combination of these methods allows for more comprehensive monitoring of ctDNA by profiling a wide spectrum of tumor-specific markers. By analyzing multiple tumor specimens in individual patients obtained from different sites and at different times during treatment, we observed clonal evolution of these tumors that was reflected by ctDNA profiles.
Our strategy allows for the comprehensive monitoring of a broad spectrum of tumor-specific markers in plasma. Our approach may be clinically useful not only in leiomyosarcoma but also in other tumor types that lack recurrent genomic alterations.
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Mutational analysis guides therapeutic decision making in patients with advanced‐stage gastrointestinal stromal tumors (GISTs). We evaluated three targeted next‐generation sequencing (NGS) assays, ...consecutively used over 4 years in our laboratory for mutational analysis of 162 primary GISTs: Agilent GIST MASTR, Illumina TruSight 26 and an in‐house developed 96 gene panels. In addition, we investigated the feasibility of a more comprehensive approach by adding targeted RNA sequencing (Archer FusionPlex, 11 genes) in an attempt to reduce the number of Wild Type GISTs. We found KIT or PDGFRA mutations in 149 out of 162 GISTs (92.0%). Challenging KIT exon 11 alterations were initially missed by different assays in seven GISTs and typically represented deletions at the KIT intron 10‐exon 11 boundary or large insertions/deletions (>24 base pairs). Comprehensive analysis led to the additional identification of driver alterations in 8/162 GISTs (4.9%): apart from BRAF and SDHA mutations (one case each), we found five GISTs harboring somatic neurofibromatosis type 1 (NF1) alterations (3.1%) and one case with an in‐frame TRIM4‐BRAF fusion not reported in GIST before. Eventually, no driver alteration was found in two out of 162 GISTs (1.2%) and three samples (1.9%) failed analysis. Our study shows that a comprehensive targeted NGS approach is feasible for routine mutational analysis of GIST, thereby substantially reducing the number of Wild Type GISTs, and highlights the need to optimize assays for challenging KIT exon 11 alterations.
The majority of prostate cancers are hormone-dependent at diagnosis highlighting the central role of androgen signalling in this disease. Surprisingly, most forms of castration-resistant prostate ...cancer (CRPC) are still dependent on the androgen receptor (AR) for survival. Therefore, the advent of new AR-targeting drugs, such as enzalutamide, is certainly beneficial for the many patients with metastatic CRPC. Indeed, this compound provides a substantial survival benefit-but it is not curative. This Perspectives article describes the different ways through which cancer cells can become resistant to enzalutamide, such as AR truncation and other mutations, as well as by-pass of the AR dependence of prostate cancer cells through expression of the glucocorticoid receptor. The clinical relevance of these mechanisms and emerging questions concerning new therapeutic regimens in the treatment of metastatic CRPC are being discussed.
► All nuclear receptors bind monomeric or dimeric 5′-AGAACA-3′ or 5′-AGGTCA-3′ hexamers. ► High throughput definition of binding sites confirm the classical DNA binding rules. ► Dimerization of ...steroid receptors on DNA is asymmetrical. ► The DNA sequence can control a nuclear receptor through allosteric signaling.
The gene family of nuclear receptors is characterized by the presence of a typical, well conserved DNA-binding domain. In general, two zinc coordinating modules are folded such that an α-helix is inserted in the major groove of the DNA-helix displaying a sequence similar to one of two hexameric consensus motifs. Both zinc molecules coordinate four cysteines. Although the DNA-binding domains as well as the hormone response elements are very similar, each nuclear receptor will affect transcription of a specific set of target genes. This is in part due to some important receptor-specific variations on the general theme of DNA interaction.
For most nuclear receptors, the DNA-binding domain dimerizes on DNA, which explains why most hormone response elements consist of a repeat of two hexamers. The hexamer dimers can be organized either as direct, inverted or everted repeats with spacers of varying lengths. The DNA can be bound by homodimers, heterodimers and for some orphan receptors, as monomer.
Another key element for DNA binding by nuclear receptors is the carboxy-terminal extension of the DNA-binding domain extending into the hinge region. This part not only co-determines sequence specificity, but also affects other functions of the receptors like nuclear translocation, intranuclear mobility and transactivation potential. Moreover, allosteric signals passing through towards other receptor domains, explain why to some extent, the DNA elements can also be considered as controlling ligands.
ABSTRACT
Androgens have well‐established anabolic actions on skeletal muscle, although the direct effects of the androgen receptor (AR) in muscle remain unclear. We generated satellite cell‐specific ...AR‐knockout (satARKO) mice in which the AR is selectively ablated in satellite cells, the muscle precursor cells. Total‐limb maximal grip strength is decreased by 7% in satARKO mice, with soleus muscles containing ~10% more type I fibers and 10% less type IIa fibers than the corresponding control littermates. The weight of the perineal levator ani muscle is markedly reduced (–52%). Thus, muscle AR is involved in fiber‐type distribution and force production of the limb muscles, while it is a major determinant of the perineal muscle mass. Surprisingly, myostatin (Mstn), a strong inhibitor of skeletal muscle growth, is one of the most androgen‐responsive genes (6‐fold reduction in satARKO) through direct transcription activation by the AR. Consequently, muscle hypertrophy in response to androgens is augmented in Mstn‐knockout mice. Our finding that androgens induce Mstn signaling to restrain their own anabolic actions has implications for the treatment of muscle wasting disorders.—Dubois, V., Laurent, M. R., Sinnesael, M., Cielen, N., Helsen, C., Clinckemalie, L., Spans, L., Gayan‐Ramirez, G., Deldicque, L., Hespel, P., Carmeliet, G., Vanderschueren, D., and Claessens, F. A satellite cell‐specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle. FASEB J. 28, 2979–2994 (2014). www.fasebj.org
High-level amplification of MDM2 and other genes in the 12q13-15 locus is a hallmark genetic feature of well-differentiated and dedifferentiated liposarcomas (WDLPS and DDLPS, respectively). ...Detection of this genomic aberration in plasma cell-free DNA may be a clinically useful assay for non-invasive distinction between these liposarcomas and other retroperitoneal tumors in differential diagnosis, and might be useful for the early detection of disease recurrence. In this study, we performed shallow whole genome sequencing of cell-free DNA extracted from 10 plasma samples from 3 patients with DDLPS and 1 patient with WDLPS. In addition, we studied 31 plasma samples from 11 patients with other types of soft tissue tumors. We detected MDM2 amplification in cell-free DNA of 2 of 3 patients with DDLPS. By applying a genome-wide approach to the analysis of cell-free DNA, we also detected amplification of other genes that are known to be recurrently affected in DDLPS. Based on the analysis of one patient with DDLPS with longitudinal plasma samples available, we show that tracking MDM2 amplification in cell-free DNA may be potentially useful for evaluation of response to treatment. The patient with WDLPS and patients with other soft tissue tumors in differential diagnosis were negative for the MDM2 amplification in cell-free DNA. In summary, we demonstrate the feasibility of detecting amplification of MDM2 and other DDLPS-associated genes in plasma cell-free DNA using technology that is already routinely applied for other clinical indications. Our results may have clinical implications for improved diagnosis and surveillance of patients with retroperitoneal tumors.
The Tet 5-methylcytosine dioxygenases catalyze DNA demethylation by producing 5-hydroxymethylcytosine and further oxidized products. Tet1 and Tet2 are highly expressed in mouse pluripotent cells and ...downregulated to different extents in somatic cells, but the transcriptional mechanisms are unclear. Here we defined the promoter and enhancer domains in Tet1 and Tet2. Within a 15-kb "superenhancer" of Tet1, there are two transcription start sites (TSSs) with different activation patterns during development. A 6-kb promoter region upstream of the distal TSS is highly active in naive pluripotent cells, autonomously reports Tet1 expression in a transgenic system, and rapidly undergoes DNA methylation and silencing upon differentiation in cultured cells and native epiblast. A second TSS downstream, associated with a constitutively weak CpG-rich promoter, is activated by a neighboring enhancer in naive embryonic stem cells (ESCs) and primed epiblast-like cells (EpiLCs). Tet2 has a CpG island promoter with pluripotency-independent activity and an ESC-specific distal intragenic enhancer; the latter is rapidly downregulated in EpiLCs. Our study reveals distinct modes of transcriptional regulation at Tet1 and Tet2 during cell state transitions of early development. New transgenic reporters using Tet1 and Tet2 cis-regulatory domains may serve to distinguish nuanced changes in pluripotent states and the underlying epigenetic variations.
The LNCaP and C4-2B cell lines form an excellent preclinical model to study the development of metastatic castration-resistant prostate cancer, since C4-2B cells were derived from a bone metastasis ...that grew in nude mice after inoculation with the LNCaP-derived, castration-resistant C4-2 cells. Exome sequencing detected 2188 and 3840 mutations in LNCaP and C4-2B cells, respectively, of which 1784 were found in both cell lines. Surprisingly, the parental LNCaP cells have over 400 mutations that were not found in the C4-2B genome. More than half of the mutations found in the exomes were confirmed by analyzing the RNA-seq data, and we observed that the expressed genes are more prone to mutations than non-expressed genes. The transcriptomes also revealed that 457 genes show increased expression and 246 genes show decreased expression in C4-2B compared to LNCaP cells. By combining the list of C4-2B-specific mutations with the list of differentially expressed genes, we detected important changes in the focal adhesion and ECM-receptor interaction pathways. Integration of these pathways converges on the myosin light chain kinase gene (MLCK) which might contribute to the metastatic potential of C4-2B cells. In conclusion, we provide extensive databases for mutated genes and differentially expressed genes in the LNCaP and C4-2B prostate cancer cell lines. These can be useful for other researchers using these cell models.
The clinical heterogeneity of prostate cancer (PCa) makes it difficult to identify those patients that could benefit from more aggressive treatments. As a contribution to a better understanding of ...the genomic changes in the primary tumor that are associated with the development of high-risk disease, we performed exome sequencing and copy number determination of a clinically homogeneous cohort of 47 high-risk PCas. We confirmed recurrent mutations in SPOP, PTEN and TP53 among the 850 point mutations we detected. In seven cases, we discovered genomic aberrations in the TET1 (Ten-Eleven Translocation 1) gene which encodes a DNA hydroxymethylase than can modify methylated cytosines in genomic DNA and thus is linked with gene expression changes. TET1 protein levels were reduced in tumor versus non-tumor prostate tissue in 39 of 40 cases. The clinical relevance of changes in TET1 levels was demonstrated in an independent PCa cohort, in which low TET1 mRNA levels were significantly associated with worse metastases-free survival. We also demonstrate a strong reduction in hydroxymethylated DNA in tumor tissue in 27 of 41 cases. Furthermore, we report the first exploratory (h)MeDIP-Seq analyses of eight high-risk PCa samples. This reveals a large heterogeneity in hydroxymethylation changes in tumor versus non-tumor genomes which can be linked with cell polarity.
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