SummaryTumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based “liquid biopsies”.

BackgroundMedulloblastoma is the most common malignant brain tumor in children. Despite recent improvements in cure rates, prediction of disease outcome remains a major challenge and survivors suffer from serious therapy-related side-effects. Recent data showed that patients with WNT-activated tumors have a favorable prognosis, suggesting that these patients could be treated less intensively, thereby reducing the side-effects. This illustrates the potential benefits of a robust classification of medulloblastoma patients and a detailed knowledge of associated biological mechanisms.Methods and FindingsTo get a better insight into the molecular biology of medulloblastoma we established mRNA expression profiles of 62 medulloblastomas and analyzed 52 of them also by comparative genomic hybridization (CGH) arrays. Five molecular subtypes were identified, characterized by WNT signaling (A; 9 cases), SHH signaling (B; 15 cases), expression of neuronal differentiation genes (C and D; 16 and 11 cases, respectively) or photoreceptor genes (D and E; both 11 cases). Mutations in β-catenin were identified in all 9 type A tumors, but not in any other tumor. PTCH1 mutations were exclusively identified in type B tumors. CGH analysis identified several fully or partly subtype-specific chromosomal aberrations. Monosomy of chromosome 6 occurred only in type A tumors, loss of 9q mostly occurred in type B tumors, whereas chromosome 17 aberrations, most common in medulloblastoma, were strongly associated with type C or D tumors. Loss of the inactivated X-chromosome was highly specific for female cases of type C, D and E tumors. Gene expression levels faithfully reflected the chromosomal copy number changes. Clinicopathological features significantly different between the 5 subtypes included metastatic disease and age at diagnosis and histology. Metastatic disease at diagnosis was significantly associated with subtypes C and D and most strongly with subtype E. Patients below 3 yrs of age had type B, D, or E tumors. Type B included most desmoplastic cases. We validated and confirmed the molecular subtypes and their associated clinicopathological features with expression data from a second independent series of 46 medulloblastomas.ConclusionsThe new medulloblastoma classification presented in this study will greatly enhance the understanding of this heterogeneous disease. It will enable a better selection and evaluation of patients in clinical trials, and it will support the development of new molecular targeted therapies. Ultimately, our results may lead to more individualized therapies with improved cure rates and a better quality of life.

We have assembled arrays of approximately 2,400 BAC clones for measurement of DNA copy number across the human genome. The arrays provide precise measurement (s.d. of log2 ratios=0.05-0.10) in cell lines and clinical material, so that we can reliably detect and quantify high-level amplifications and single-copy alterations in diploid, polyploid and heterogeneous backgrounds.

Purpose: To determine the prevalence and prognostic value of mismatch repair (MMR) status and its relation to BRAF mutation (BRAF MT ) status in metastatic colorectal cancer (mCRC). Experimental Design: A pooled analysis of four phase III studies in first-line treatment of mCRC (CAIRO, CAIRO2, COIN, and FOCUS) was performed. Primary outcome parameter was the hazard ratio (HR) for median progression-free survival (PFS) and overall survival (OS) in relation to MMR and BRAF. For the pooled analysis, Cox regression analysis was performed on individual patient data.Results

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes.

The spatial organisation of the genome in the nucleus has a role in the regulation of gene expression. In vertebrates, chromosomal regions with low gene-density are located close to the nuclear periphery. Correlations have also been made between the transcriptional state of some genes and their location near the nuclear periphery. However, a crucial issue is whether this level of nuclear organisation directly affects gene function, rather than merely reflecting it. To directly investigate whether proximity to the nuclear periphery can influence gene expression in mammalian cells, here we relocate specific human chromosomes to the nuclear periphery by tethering them to a protein of the inner nuclear membrane. We show that this can reversibly suppress the expression of some endogenous human genes located near the tethering sites, and even genes further away. However, the expression of many other genes is not detectably reduced and we show that location at the nuclear periphery is not incompatible with active transcription. The dampening of gene expression around the nuclear periphery is dependent on the activity of histone deacetylases. Our data show that the radial position within the nucleus can influence the expression of some, but not all, genes. This is compatible with the suggestion that re-localisation of genes relative to the peripheral zone of the nucleus could be used by metazoans to modulate the expression of selected genes during development and differentiation.

The proteasome inhibitor bortezomib is a novel anticancer drug that has shown promise in the treatment of refractory multiple myeloma. However, its clinical efficacy has been hampered by the emergence of drug-resistance phenomena, the molecular basis of which remains elusive. Toward this end, we here developed high levels (45-to 129-fold) of acquired resistance to bortezomib in human myelomonocytic THP1 cells by exposure to stepwise increasing (2.5-200 nM) concentrations of bortezomib. Study of the molecular mechanism of bortezomib resistance in these cells revealed (1) an Ala49Thr mutation residing in a highly conserved bortezomib-binding pocket in the proteasome ␤5-subunit (PSMB5) protein, (2) a dramatic overexpression (up to 60-fold) of PSMB5 protein but not of other proteasome subunits including PSMB6, PSMB7, and PSMA7, (3) high levels of cross-resistance to ␤5 subunit-targeted cytotoxic peptides 4A6, MG132, MG262, and ALLN, but not to a broad spectrum of chemotherapeutic drugs, (4) no marked changes in chymotrypsin-like proteasome activity, and (5) IntroductionThe ubiquitin proteasome system (UPS) facilitates the degradation of ubiquitin-tagged intracellular proteins, many of which play a regulatory role in cell proliferation, cell survival, and signaling processes. [1][2][3] As such, proteasome inhibitors have been recognized as a new generation of chemotherapeutic agents and antiinflammatory drugs. [4][5][6][7][8][9][10][11][12][13] The boronic dipeptide bortezomib (PS341, Velcade) is the first proteasome inhibitor that has been approved for the treatment of refractory multiple myeloma. 6,14 Bortezomib is a reversible inhibitor that targets primarily the ␤5-subunit (PSMB5) subunit/chymotrypsin-like activity of the 26S proteasome and to a somewhat lesser extent also caspase-like activity harbored by the ␤1 (PSMB6) proteasome subunit. At higher concentrations, bortezomib inhibits trypsin-like proteolytic activity facilitated by ␤2 (PSMB7) proteasome subunits. [15][16][17] Despite promising clinical activity, some patients with multiple myeloma failed to respond to bortezomib therapy. 18 Moreover, the efficacy for bortezomib may differ between tumor types. 6,[19][20][21] Whether these observations are related to common mechanisms of drug resistance frequently seen for anticancer 22 or anti-inflammatory drugs 23 is largely unknown. However, their characterization is of key importance as it may pave the way for the overcoming of drug resistance, thereby enhancing the efficacy of this new class of proteasome-targeted drugs.One mode of primary resistance to bortezomib is conveyed by constitutively high levels of heat shock protein 27. 24 In the context of acquired resistance, studies aimed at delineating the mechanism of acquired resistance to the tripeptidyl aldehyde proteasome inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal) revealed 2 possible molecular mechanisms: (a) enhanced cellular efflux via the multidrug resistance (MDR) transporter P-glycoprotein (Pgp; ABCC1) 25 or multidrug resistance-related pro...

Functional biomolecules, including small noncoding RNAs (ncRNAs), are released and transmitted between mammalian cells via extracellular vesicles (EVs), including endosome-derived exosomes. The small RNA composition in cells differs from exosomes, but underlying mechanisms have not been established. We generated small RNA profiles by RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes. A comprehensive bioinformatics and statistical analysis revealed nonrandomly distributed subsets of microRNA (miRNA) species between B cells and exosomes. Unexpectedly, 3' end adenylated miRNAs are relatively enriched in cells, whereas 3' end uridylated isoforms appear overrepresented in exosomes, as validated in naturally occurring EVs isolated from human urine samples. Collectively, our findings suggest that posttranscriptional modifications, notably 3' end adenylation and uridylation, exert opposing effects that may contribute, at least in part, to direct ncRNA sorting into EVs.