In the field of cancer research, scientific investigations are based on analysing differences in the secretome, the proteome, the transcriptome, the expression of cell surface molecules, and the deregulation of signal transduction pathways between neoplastic and normal cells. Accumulating evidence indicates a crucial role in carcinogenesis concerning not only stromal cells but also normal cells from target organs and tissue where tumours emerge. The tumour microenvironment (TME) definitively plays an important role in regulating neighbouring cell behaviour. To date, limited attention has been focused upon interactions between cancer cells and normal cells. This review concentrates on the interactions between stromal and healthy cells from the TME in cancer development. In the article, the authors also describe mutations, genes and proteins expression pattern that are involved in tumour development in target organ. IntroductionCancer cell – fibroblast interaction in cancer progressionCancer cell – hepatocyte interaction in cancer progressionCancer cell – lung epithelial cell interaction in cancer progressionInteraction between cancer cells and normal cells of the primary organCancer cell – pleural and peritoneal cells interaction in cancer progressionSummaryIn vitro, these cells form non-haematopoietic adherent cell components from long-term cultures and in vivo make up the microenvironment of haematopoiesis, comprising the set of non-haematopoietic cells from the different haematopoietic sites Cell populations that assist haematopoietic stem cells and its progeny are called stromal cells. Research indicates that the cell environment profoundly affects cancer development. Moreover, it has confirmed the Stephen Paget's ‘seed and soil’ theory from 1889. He postulated that metastases of a particular type of cancer (‘the seed’) often metastasizes to certain sites (‘the soil’) based on the similarity of the environments of the original and secondary tumour sites et al. (2012) proposed the division of the TME stromal component into three general classes: (i) angiogenic vascular cells, (ii) infiltrating immune cells and (iii) cancer-associated fibroblastic cells. These phenotypically, oncologically and morphologically different cell populations interact with each other to ultimately contribute to cancer cells growth, local invasion and metastasis. Fibroblasts are the predominant cells of the stroma and several publications have reported genetic and epigenetic changes in stromal fibroblasts that modulate the expression of many genes encoding growth factors and cytokines inter alia ECM molecules such as fibronectin and tenascin, which influence both cell adhesion and proliferation Hanahan It also bears mentioning that mammalian genomes include a considerable number of endogenous retroviruses (ERVs). These relics of ancestral infectious retroviruses resulted from ancestral germ line infections by exogenous retroviruses which have thereafter been transmitted in a Mendelian fashion. Almost 8% of the human genome comprises ERVs A fundamental understanding of basic pathophysiological processes, for example malignant transformation, can in turn help to better define the targets for clinical intervention. As the cells and most factors from TME are well known, we focus on molecular interactions between healthy cells of the stroma and normal cells surrounding the tumour.Accumulating evidence indicates that CAFs play critical roles in cancer pathogenesis. CAFs are recruited from periacinar cells, circulating marrow-derived progenitors, vessel-associated pericytes, or other tissue-resident mesenchymal stem/progenitor cells via signalling pathways and mechanical stress The impact of CAFs reflects results obtained on a murine model of metastatic breast cancer Tumour growth stops at a diameter of about 1–2 mm unless sufficient blood supply develops The importance of CAFs in tumour progression is highlighted by the fact that they also act as major players in tumour metastasis. Transforming growth factor β (TGF-β) seems to be a major regulator of the processes in which CAFs are involved. TGF-β is a factor which causes recruitment of CAFs by cancer cells et al. demonstrated that reversible EMT may be associated with acquired tumour resistance to the VEGF(Rs) tyrosine kinase inhibitor sunitinib in renal cell carcinoma Epithelial–mesenchymal transition is a process by which epithelial cells gain mesenchymal features causing cancer cell invasiveness, motility and cell stemness In vitro studies have also shown that CAFs secrete matrix metalloproteinases MMP2, MMP9 and urokinase-type plasminogen activator (uPA) et al. have provided evidence that the disseminating tumour cells metastasize to the secondary site together with passenger fibroblasts, from which early signalling originates that contributes to the pre-metastatic niche formation The mesenchymal to amoeboid transition (MAT) process that follows EMT allows cells to glide through ECM barriers is also mediated by CAFs together with endothelial progenitor cells by bidirectional ephrinA1/EphA2 signalling et al. proposed a novel working model of metastatic growth progression based on both paracrine signalling and mechanical impact of the CAF cohort at the tumour–host cell interface Based on previously reported findings, Karagiannis The ‘paracrine’ component of the model postulates that CAFs may secrete paracrine soluble factors at the tumour invasion front, which may affect the various layers of the neoplastic population, generating phenotypic and signalling pathway gradients across the tumour cohort. CAFs may form niches similar to the ones occurring in various physiological and pathophysiological conditions in the human body Indeed, mesenchymal or marrow-derived stromal cells, which constitute a large proportion of the non-neoplastic cells within the TME, constitutively secrete SDF-1/CXCL12 chemokine. CXCL12 secretion by stromal cells attracts cancer cells, acting through its cognate receptor CXCR4, which is expressed by both haematopoietic and non-haematopoietic tumour cells. CXCR4 promotes tumour progression by direct and indirect mechanisms. First, CXCR4 is essential for metastatic spread to organs where CXCL12 is expressed and thereby allows tumour cells to access cellular niches that favour tumour cell survival and growth. Second, stromal-derived CXCL12 itself can stimulate survival and growth of neoplastic cells in a paracrine fashion. Third, CXCL12 can promote tumour angiogenesis by attracting endothelial cells to the TME Enhanced expression of this pro-inflammatory gene indicates that CAFs play an important role throughout the entire process of carcinogenesis, resulting in proliferation promotion, migration and invasion of tumour cells Cancer-associated fibroblasts role in tumour burden has been extensively investigated, thus the knowledge regarding this type of stromal cell is the broadest compared to other types of TME cells. However, the contribution of other cells is also fundamental.In vitro and in vivo studies showed enhanced hepsin expression in breast cancer tissues, which is associated with tumour growth and progression versus bone and lymph node secondary tumours of prostate cancer Hepatic tissue is a primary target for metastases for most disseminating cancers et al. (2012), Klezovitch et al. et al. did not investigate the pathway and molecular mechanism involved in hepsin mediation of hepatic metastasis development. Further investigations would explain the discrepancies in the results obtained between these two groups. Hence, the hepsin level may not be a proper prediction factor for hepatic metastasis.However, data about the role of hepsin in formation of liver metastases remain controversial. The contribution of hepsin in the development of secondary liver tumours is confirmed by the results of studies conducted on prostate cancer models + in the proximal tubule. Similar results have been obtained with in vitro cultured cells In liver metastases of breast cancer, claudin-2 expression was found to be elevated in comparison to primary tumour Colon cancer patients with high levels of metallopeptidase inhibitor 1 (TIMP-1) detected at the time of their initial surgery were found to have a high risk of metachronous liver metastasis and hepatic recurrence following resection of synchronous liver metastasis. Levels of TIMP-1 were found to be significant predictive factors for poor prognosis following liver resection. The authors claim that validation of the obtained results may provide a greater understanding of colon cancer liver metastasis FAM27L gene, whose function is unknown. Thus, it is likely that disruption of the FAM27L gene may play a role in the malignant transformation and/or the metastasis of collateral tumours into the liver versus primary tumours demonstrated that hepatic metastases showed acquisition of genetic aberrations that were not found in their paired primary tumours e.g. losses of chromosomes 4 and 10q and gains of chromosomes 5p and 6p). These genetic changes may be the result of the metastatic process and/or adaption of metastatic cells to the liver microenvironment TWIST1 plays an important role in liver metastasis because of its influence on angiogenesis et al. (2005) demonstrating that TWIST1 overexpression induces in vivo angiogenesis and correlates with chromosomal instability in breast cancer A study of single-nucleotide polymorphisms (SNPs) in colorectal primary tumours with liver metastasis detected recurrent breakpoints at chromosome 17p restricted to the et al. TWIST1 in hepatic secondary tumours, speculating that TWIST1 is involved in an early step of metastasis when it is highly expressed TWIST1 could be explained by the fact that analysis was conducted on samples from already existing liver metastases when the process had already occurred +/H+ exchanger regulating factor 1 (NHERF1) correlated positively with hypoxia inducible factor 1α (HIF-1α) Yet, these findings are in contradiction with the results of Malfettone Research has linked obesity to hepatic metastasis, and insulin-like growth factor-I (IGF-I) plays a key role in the mechanism underlying this phenomenon. Signalling by IGF-I is responsible for establishment of hepatic metastases ITGA3 and ITGB4) and three genes involved in the desmosome complex  Gene expression analysis in adenocarcinoma, squamous cell carcinoma (SCC) and normal bronchus epithelium without metaplasia or dysplasia derived from patients with neoplastic changes revealed 43 differentially expressed genes in vitroThe direct physical contact between normal mesenchymal cells and lung carcinoma cells inhibited growth of malignant cells et al. observed enhanced expression of syndecan-1 (SDC-1) in bronchial carcinoma compared to normal tissue SDC-1 was detected in stromal cells and probably was expressed in epithelial cells on account of their dominance in stroma. Marker analysis suggested a shift of SDC-1 expression from epithelial tumour cells to myofibroblasts within the connective tissue surrounding the tumour cells SDC-1 expression from tumour to stromal cells may be associated with a decreased expression within epithelial tumour cells and it can have important consequences SDC-1 down regulation potentially generates the ability to form a dedifferentiated invasive or migrating tumour cell, mobilization of growth factors from epithelial tumour cell surfaces and ECM and soluble factors needed for the establishment of the cancer-associated non-malignant stroma Mennerich in vitro in a kidney model upon stimulated HA synthase 3 gene expression may be related to pre-malignant changes, including intraluminal invasion and deregulated epithelialization, probably mediated by mitotic spindle orientation defects Hyaluronan (HA) concentration in cells is enhanced under pathological conditions and disturbs the normal cell–cell and cell–ECM interactions in simple epithelia, leading to aberrant epithelial morphogenesis. These morphological abnormalities In vitro observation made by Pistone et al. et al. obtained evidence against these findings et al., revealed that normal epithelial cells inhibit tumour cell proliferation and demonstrated the utility of this co-culture system as a model of early paracrine control of breast cancer One of the factors taking part in this interplay may be IL-15. It has been proposed recombinant IL-15 may kill tumour cells by stopping the blood flow to the tumour and by stimulating white blood cells to kill kidney cancer cells . On the basis of observations made on a renal cancer model, IL-15 initiates the EMT process in tumoural and paratumoural cell lines in contrast to normal cells where it inhibits the drift towards EMT Wnt also shows a link to the inducible nitric oxide synthase (iNOS) and Src kinase In agreement with the cited results presented above, we suggest that it is not the predisposition of the normal tissue which allows for secondary tumour formation, but the ability to properly respond to factors secreted by neoplastic cells that is most crucial here. The response manifests itself in the adjustment of the secretome, the proteome, the transcriptome, the expression of cell surface molecules and the deregulation of signal transduction pathways, all of which favour metastasis.i.e. the primary tumour arising from mesothelial surface, could be considered as a specific model for such interaction. Those have been presented on Figuree.g. its asbestos-related pathogenesis, or the putative role of polioma virus SV40 in enhancing asbestos-related tumourigenesis) make the results achieved so far in this tumour hardly transferable in other solid neoplasms metastasizing to the pleural surface. However, it is also clear in case of this tumour that cancer cells instigate tumour-associated fibroblasts, promoting tumour progression via a malignant cytokine network Limited data are currently available on factors associated with pleural and peritoneal metastasis, even though malignant mesothelioma, in vivo and in vitro inter alia by S100A4 (positively) and E-cadherin (negatively), these are important factors involved in control of invasion and metastasis In matched primary breast cancers and pleural effusions (PE), MMP-2 levels were found to be elevated at metastatic sites CTNNB1 genes encoding a subunit of E-cadherin–β-catenin were up-regulated in the examined cell lines CTNNB1 gene TCT [(Ser) –> TGT (Cys)] was correlated with the formation of metastatic sites CTNNB1 gene favours the formation of pulmonary metastases, while malfunction of the S100A4 gene may prevent from this mechanism. S100A4 also positively regulates MMP-2. Taken together mentioned interactions, also with other described factors, this data emphasize the need for further investigation of MMP-2 signal transduction pathway components to obtain information about molecules mediating pleural and peritoneal metastasis.A large body of evidence supports the calcium-binding protein S100A4 role in control of invasion and metastasis in many invasive tumours. For example, cDNA microarray analysis of peritoneal metastasis in gastric adenocarcinoma indicates that the S100A4 and Observed elevated levels of SDF-1alpha/CXCL12 in pleural malignant exudates may indicate its correlation with metastasis and its possible participation in pleural trafficking in lung cancer et al. demonstrated that genes related to cellular metabolism, aldolase A, sorbitol dehydrogenase, transketolase, and tuberous sclerosis 1 are important in malignant PE pathogenesis The connection between SDF-1α/CXCL12 and glucose metabolism may have an impact on dissemination of malignant cells into pleural space. This is in accordance with results obtained from oligonucleotide microarray on tissue sample cohorts SEMA3B) in promotion of metastasis. Research carried out on human cancerous cells derived from tumours that metastasized to the lung revealed that paradoxically, SEMA3B inhibited growth of primary tumours, but induced metastasis in vivoSEMA3B-expressing tumours exhibited notable defects of pericyte recruitment to blood vessels compared with control xenografts. In tumour cells, SEMA3B expression induced the secretion of IL-8 SEMA3B activity is mediated by NP1-dependent activation of p38, subsequent activation of mitogen-activated protein kinase stimulating p21 protein, which in turn inhibits cancer cell proliferation and IL-8 biosynthesis promoting invasiveness Surprisingly, lung tumours exhibiting pleural invasion also showed elevated expression of apoptosis-related markers: caspase-3 (94.5%), p53 (60%) and bcl-2 (54.5%). As these proteins show rather an anticancer function, their role in metastasis is controversial and unclear ANGPTL4) via the Smad signalling pathway. Upon entering blood circulation and reaching lung capillaries, these cells secrete Angptl4 which disrupts endothelial cell junctions, facilitating more efficient transfer to the lung parenchyma Another cytokine involved in lung metastasis is TGF-β et al. found 54 genes overexpressed in primary breast tumours that promote lung metastases as well et al. have also included intracellular enzymes like COX2 and transcriptional regulators like Id1.Investigation of breast cancer model also revealed a specific gene expression pattern in primary tumours that predestinates them for lung metastasis. Minn Id1 gene encoding a transcriptional regulator and its expression in breast cancer cells has a significant impact on the ability of breast cancer cells to metastasize to the lungs in xenograft models Id1, CXCL1, COX2, EREG, and MMP1 mRNAs increases with lung metastatic ability but is not restricted to aggressive lung metastasis populations. The protein levels were correlated with mRNA levels These genes encode the epidermal growth factor family member epiregulin (EREG), MMP2, MMP1, the chemokine GRO1/CXCL1, the cell adhesion molecule SPARC, and the cell adhesion receptor VCAM1 Research into genes that predestine the development of pleural metastases has led to the finding of a set of genes named Lung Metastasis Signature (LMS). LMS includes 18 genes encoding cell surface proteins and secreted products that affect the interaction of tumour cells with the microenvironment It appears that not only Id1 but also Id3 is involved in this process et al. DSC2, TFCP2L1, UGT8, ITGB8, ANP32E and FERMT1. These encode adhesion molecules that belong to various protein families including desmosome proteins (DSC2), integrins (ITGB8) and focal adhesion molecules (FERMT1); molecules involved in regulation of transcription (TFCP2L1), glycosphingolipid biosynthesis (UGT8) and phosphatase inhibitor activity (ANP32E) In 2008, Landemaine Tumour cells exhibit a selective attachment pattern and growth preference on specific sites within peritoneal tissues It is well-established that the TME affects tumour growth and metastasis


T1 for the decay of nuclear spin magnetization through contact with the thermal molecular environment. Long-lived states, which are correlated quantum states of multiple nuclei, have decay time constants that may exceed T1 by large factors. Here we demonstrate a nuclear long-lived state comprising two 13C nuclei with a lifetime exceeding one hour in room-temperature solution, which is around 50 times longer than T1. This behavior is well-predicted by a combination of quantum theory, molecular dynamics, and quantum chemistry. Such ultra-long-lived states are expected to be useful for the transport and application of nuclear hyperpolarization, which leads to NMR and MRI signals enhanced by up to five orders of magnitude.Nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) are supremely important techniques with numerous applications in almost all branches of science. However, until recently, NMR methodology was limited by the time constant  Many magnetic resonance experiments would benefit from the ability to maintain nuclear spin order for a long time under ambient conditions. Currently, nuclear spin order lifetimes exceeding one hour have only been achieved in the gas phase. It is muT1 by an order of magnitude.methoxy)-7-(([D7]propan-2-yl)oxy)-naphthalene (compound I). 13C labeling of I at both positions 4a and 8a .The relaxation times are significantly shorter when the sample is not degassed, leading to an estimated concentration of 2 mn Figure . Neverthn acetone and the We have found that standard analytical treatments of nuclear singlet relaxation, which are based on rigid molecular tumbling models,–13 fail 13C2-I are summarized in Figure The computational predictions for several mechanistic contributions to singlet relaxation in +), and antisymmetric part (CSA−), which both cause relaxation. Th Th11] Thlaxation., [11],  Co Co11] Corotation. Our inve13C2-I as all other spins (2 H) have low γ and are far from the spin pair. This mechanism is small but significant for 13C2-I in low magnetic field.Intramolecular dipole–dipole relaxation (iDD). The motional modulation of the dipole-dipole couplings between the spin pair and other magnetic nuclei within the same molecule cause singlet relaxation., 13] Th Th13] Th13C2-I, the symmetry-breaking is provided by the small isotropic shift difference, and contributes minimally to singlet relaxation, except at high magnetic fields.Singlet–triplet leakage (STL)., 11] Th Th11] ThIntermolecular relaxation. Nuclear singlet relaxation from dissolved paramagnetic species may be significant. The dataTS−1) as obtained by experiments on the degassed sample, and by simulation. A qualitative agreement (ca. 30 %) is obtained between the simulated relaxation rate constants and the experimental results. The simulated values of TS−1 have confidence limits of ±30 %, which derive mainly from uncertainties in estimates of the chemical shift tensors, the spin-rotation tensors, and the correlation times.Figure 15N2O and noble gases) we anticipate applications to molecular sensing and binding studies. Paramagnetic relaxation caused by dissolved oxygen remains significant but we expect to reduce this by suitable molecular design. We also plan to modify the system to achieve good water solubility. The measured singlet lifetime of more than 10 minutes even without degassing should permit applications in hyperpolarized NMR spectroscopy and MRI.In summary, we have demonstrated a molecular prototype that displays a singlet decay time constant of more than one hour in a room-temperature liquid. We have identified the key relaxation mechanisms responsible for relaxation in this long-time regime and set up a computational methodology for predicting the singlet relaxation rates with good accuracy. The understanding of these mechanisms and the ability to predict singlet relaxation can drive the design of molecules possessing even longer-lived spin states. We anticipate that such systems will be useful for the transport and storage of nuclear hyperpolarization under ambient conditions. Since these molecular systems are amenable to chemical functionalization (unlike


Transfusion of red blood cells (RBC) is the only clinically effective therapeutic approach for treating oxygen transport deficits . However potential shortage of transfusable RBC has been predicted for the near future as a result of an imbalance between supply and demand due to aging population, an increase in the transmission of infectious diseases, limited compatibility of stored stocks, and the requirement for rare blood groups ,2. This Given the potential of stem cells to recapitulate erythropoiesis in vitro under controlled conditions in standard T-flask cultures, we transferred such methodology into stirred tank bioreactors, as the first step towards scaling the bioprocess up to the production of clinically relevant doses. Furthermore, we compared the characteristics of the cells produced in bioreactors to those obtained from traditional manual cultures.The expansion strategy lasted 21 days and consisted of 3 stages based on the use of different media additives: 1) isolation of CD34+ cells from a fresh umbilical cord blood (CB) unit using magnetic beads (on day 0); 2) expansion of CD45+ progenitors up to day 7, using 3 IU/mL Erythropoietin , 100 IU/mL Stem Cell Factor , 5 ng/mL Interleukin-3 , 10 µM hydrocortisone , 330 µg/mL transferrin (Life Technologies), 10 µg/mL insulin (Life technologies), 2 mM L-glutamine (Life Technologies), 5% v/v human serum B -supplemented IMDM (Lonza) medium, and 3) the subsequent maturation and enucleation of erythroblasts into erythrocytes  using EPO/SCF/transferrin/insulin/L-glutamine/hSerB-supplemented media. Cell culture concentration was adjusted at 5x105 cells/mL every 2 days and maintained at 37ºC either in humidified 5% CO2 incubators or in stirred tank bioreactors (Applikon) at 90 rpm.Cells were analyzed by flow cytometry at different culture times for phenotypic expression of specific surface markers using a FACSCalibur flow cytometer (Becton Dickinson). May-Grünwald-Giemsa stainings were performed to identify enucleated cells by microscopy. Erythrocytes were quantified by the Retic-Count assay (Becton Dickinson). Glucose and lactate concentrations in supernatants were determined using an YSI 2700 SELECT automated analyser (Yellow Springs Instruments), as described elsewhere . HaemoglConsiderable progress has been made in the recent years with regard to biological control of the expansion and maturation of erythroid cells with the objective of generating enucleated RBC ex vivo . GiarratDuring erythroid progenitor differentiation, CD45 expression decreased while CD36 expression appeared gradually along the culture time to eventually disappear in mature erythrocytes. The two most mature populations (CD36+CD45- and CD36-CD45-) were analysed for CD71 and CD235a expression at the end of the cultures. High levels of CD71 and CD235a are indicative of the reticulocyte phenotype, while the expression of CD71 is lost in mature erythrocytes. In terms of its clinical use, this does not pose a problem, since it has been demonstrated that reticulocytes fully mature into erythrocytes, once they are injected into the bloodstream [Presence of enucleated cells were confirmed in both culture strategies by May-Grünwald-Giemsa staining. Nucleated cells were removed from the final product by filtering and the final product was composed of a pure population of biconcave in vitro-produced erythrocytes Figure .Kinetic parameters summarized in Table Existing cell culture technologies for RBC production are not easily transferable to large-scale settings and, moreover, cell yields using CB as starting material are far behind those needed in the clinical setting. Hence, it is not likely that the first attempts to generate massive quantities of ex vivo cultured erythrocytes will use this stem cell source. Instead, much work is currently invested on either establishing cell lines or using human embryonic stem cells (hES) and induced pluripotent stem cells (iPS), as unlimited source of HSCs .We demonstrated the feasibility to obtain red blood cells ex vivo from CD34+ umbilical cord blood cells and transfer the expansion strategy from manual cultures to stirred tank bioreactors. Phenotypic characterisation and enucleation rates were similar in manual and bioreactor strategies. Although cells cultured in bioreactors showed lower expansion potential, higher maturation yields were observed. Further work will focus on bioprocess optimisation and scale up to clinically significant doses.This work was supported by the Spanish Cell Therapy Network , and the REDONTAP project was funded by the European Union's Seventh Framework Programme (grant agreement number 229328).


Identify gene expression profiles associated with OA processes in articular cartilage and determine pathways changing during the disease process.Genome wide gene expression was determined in paired samples of OA affected and preserved cartilage of the same joint using microarray analysis for 33 patients of the RAAK study. Results were replicated in independent samples by RT-qPCR and immunohistochemistry. Profiles were analyzed with the online analysis tools DAVID and STRING to identify enrichment for specific pathways and protein-protein interactions.TNFRSF11B and FRZB). Also several inflammatory genes such as CD55, PTGES and TNFAIP6, previously identified in within-joint analyses as well as in analyses comparing preserved cartilage from OA affected joints versus healthy cartilage were among the top genes. Of note was the high up-regulation of NGF in OA cartilage. RT-qPCR confirmed differential expression for 18 out of 19 genes with expression changes of 2-fold or higher, and immunohistochemistry of selected genes showed a concordant change in protein expression. Most of these changes associated with OA severity (Mankin score) but were independent of joint-site or sex.Among the 1717 genes that were significantly differently expressed between OA affected and preserved cartilage we found significant enrichment for genes involved in skeletal development (e.g. We provide further insights into the ongoing OA pathophysiological processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage, which seem relatively consistent and independent of sex or joint. We advocate that development of treatment could benefit by focusing on these similarities in gene expression changes and/or pathways. Osteoarthritis (OA) is a degenerative disease of the joints causing pain and disability for an increasing proportion of the population thereby imposing a large patient and socio-economic burden Initially, expression profiles were established for cartilage from knee OA joints in comparison to healthy joints using only a limited number of genes One of the characteristics of OA is focal loss of articular cartilage, resulting in areas of degradation as well as areas with a relative preservation of cartilage thickness and appearance in the joint. Insight into gene expression specific for the focal areas of cartilage degradation compared to those in preserved areas can provide clues towards dynamic changes of genes and pathways involved in OA pathophysiology independent of confounding factors such as age. Gene expression profiles of cartilage from OA affected and macroscopically preserved areas of the same joint have been determined before, however, in most of these studies limited numbers of donors (4–5 knee joints) were included As part of the ongoing Research Arthritis and Articular Cartilage (RAAK) study we set out to perform genome wide analysis of differential gene expression by comparing 33 pairs of matched OA affected and preserved cartilage samples, originating from the same joint of patients that underwent total joint replacement of either hip or knee. Results provide further insights in the ongoing OA disease processes in cartilage, in particular into differences in macroscopically intact cartilage compared to OA affected cartilage.Participants of the RAAK study provided written informed consent. The ongoing RAAK study and its consent procedure is approved by the institutional ethics review committee .The RAAK study is aimed at the biobanking of joint materials as well as mesenchymal stem cells and primary chondrocytes from patients and controls in the Leiden University Medical Center and collaborating outpatient clinics in the Leiden area. In the current study we used paired preserved and OA affected cartilage samples for 33 donors undergoing joint replacement surgery for primary OA . Characteristics of the donors are shown in At the moment of collection (within 2 hours following surgery) tissue was washed extensively with phosphate buffered saline (PBS) to decrease the risk of contamination by blood. Cartilage was classified macroscopically and collected separately from OA affected and preserved regions around the weight-bearing area of the joint . ClassifValidation was performed by RT-qPCR in 8 sample pairs of the discovery cohort (3 knee and 5 hip) and for replication of the results we included 28 additional matched sample pairs  of similar mean age shown in . SamplinCartilage samples were pulverized using a Retsch MM200 under cryogenic conditions. On average 150 mg of pulverized cartilage was dissolved in 1 ml of Trizol reagent, and mixed vigorously. After addition of 200 µl of chloroform the sample was mixed and centrifuged for 15 minutes . The clear aqueous layer was transferred to a new vial and 1 volume of 70% ethanol/DEPC-treated water was added to precipitate RNA. RNA was collected using Qiagen mini columns according to the manufacturers protocol and quality was assessed using a Bioanalyzer lab-on-a-chip. RNA integrity numbers above 8 were considered suitable for microarray analysis.invitro transcription, amplification, and labeling with biotin-labeled nucleotides  Illumina HumanHT-12 v3 microarrays were hybridized. Sample pairs were randomly dispersed over the microarrays, however each pair was measured on a single chip. Microarrays were read using an Illumina Beadarray 500GX scanner and after basic quality checks using Beadstudio software data were analyzed in R statistical programming language. Intensity values were normalized using the “rsn” option in the Lumi-package and absence of large scale between-chip effects was confirmed using the Globaltest-package in which the individual chip numbers were tested for association to the raw data P>0.05 in more than 50% of the samples) a paired t-test was performed for the remaining 13277 probes comprising 11421 unique genes on all sample pairs while adjusting for chip (to adjust for possible batch effects) and using multiple testing correction as implemented in the “BH” (Benjamini and Hochberg) option in the Limma-package. Analyses for differential expression between OA and healthy and between preserved and healthy cartilage was performed likewise, adjusting in addition for sex and for age.After Gene expression profiles of the samples have been deposited in NCBI’s Gene Expression Omnibus −ΔΔCt method ACTB) expression as internal standard.0.5 µg of total RNA was processed with the First Strand cDNA Synthesis Kit according to the manufacturer’s protocol (Roche Applied Science) and RT-qPCR was performed for the 19 genes showing at least 2-fold expression differences in the microarray analysis  adjacent to the collected area were stained using hematoxilin and eosin (H&E) and toluidine blue. Immunohistochemistry was performed for SERPINE1  without antigen retrieval and for CD55  with heat antigen retrieval  as described previously et alQuantification of OA related cartilage damage was scored by 2 observers (JVMGB and YFMR) according to Mankin P≤0.05 after correction for multiple testing according to Bonferroni were considered significant .Gene enrichment among the 1717 genes showing significant differential expression was performed with the Database for Annotation, Visualization and Integrated Discovery (DAVID) tool Enrichment in protein-protein interactions among the significant genes was analyzed with the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) 9.0 Based on selected publications of genome wide association study meta-analyses P = 4×10−4, paired t-test) and as a result gene expression differences can be directly linked to these differences in Mankin scores.To identify genes with changed expression in response to ongoing OA processes genome wide expression profiles were generated for preserved and OA affected cartilage of the same joint of 33 donors. Characteristics of the donors are shown in TNFAIP6 also known as TSG-6, 2.9-fold up in OA cartilage; P = 4.4×10−8), cytokine receptor-like factor 1 , and Wnt-inhibitor frizzled related protein beta . A notable gene highly up-regulated in OA cartilage was neuronal growth factor .After normalization and correction for multiple testing, significant differential expression between the OA affected and preserved cartilage was identified for 1893 probes, representing 1717 unique genes . Among tCRISPLD1), all genes were significantly different expressed  was significantly less pronounced in knee OA (1.3-fold increase) than in hip OA (2.6-fold increase).Expression profiles of genes with fold-changes of 2 and higher were analyzed for association with Mankin score as a grade of disease severity . Almost CRISPLD1 and COL9A1 in healthy versus preserved cartilage were not significant and appeared to be increased while the expression in preserved versus OA affected cartilage was found to be decreased . Furthermore, of note was the GO-term referring to “extracellular matrix organization” including decorin (DCN) and several collagens . When analyzing for enrichment using the cellular compartment option, most significant GO-term was “extracellular matrix”, and analyzing for molecular function showed genes involved in “copper ion binding” and “glycosaminoglycan binding” to be significantly enriched  in which fibronectin-1 (FN1) seemed to play a central role considering that 6 of the 19 proteins were found to relate to FN1 (Among the 19 genes that were highly changed in OA affected cartilage (at least 2-fold), 3 pathways were significantly enriched with lowest P-value for GO-term “response to wounding” , which ie to FN1 .In addition to differential expression of proteins encoded by genes found in the microarray analysis, immunohistochemical (IHC) staining provides insight in expression pattern and localization of differentially expressed genes in the different cartilage layers. Therefore, as a proof of principal, IHC was performed for SERPINE1 and CD55. P = 2.4×10−4). The expression difference seemed to correlate mostly with toluidine blue staining, and thus with the level of proteoglycan constituents of chondromucin aggregates in the samples (P = 2.9×10−9).Immunohistochemistry for SERPINE1 showed that the protein is expressed in chondrocytes and differential expression between OA and preserved cartilage at the protein level was very pronounced . In OA aCD55 protein expression was most pronounced in the superficial layer, with higher levels in the more OA affected zones of the cartilage, while hardly any CD55 positive cells were detected in the deep layer . The difPdetection≤0.05) and from these, 8 were significantly different between OA and preserved cartilage. Most genes showed only modest expression changes. Of note was differential expression of the HMG-box transcription factor 1 (HBP1) gene, identified in the Rotterdam study P = 2.0×10−3).In order to explore whether genes identified by genome wide association studies are active in cartilage and/or change in response to the OA process, we screened for differential expression of genes originating from recently published large scale meta analyses on OA . SixteenHBA2, HBB, and COL9A1, expression associated with disease severity as determined by scoring according to Mankin CD55 and SERPINE1) was demonstrated by immunohistochemistry.As part of the RAAK study we compared genome wide expression levels between preserved and OA affected cartilage of the same joint from 33 donors. Such a paired study design allows the detection of genes specifically involved in the OA pathophysiological process, independent of inter-individual or age-related confounding factors as also reflected by the highly comparable differential gene expression patterns when stratifying according to joint and sex. After correction for multiple testing 1717 unique genes showed significant differential expression, of which 19 genes had a fold-change of 2 or higher. In an independent paired cartilage sample set, differential expression was confirmed for 18 genes by RT-qPCR. For most of these genes, except CD55PTGES and TNFAIP6TNFRSF11B) gene encoding osteoprotegerin. Very recently we reported in this protein on a gain of function mutation likely causal in a family with early onset OA with chondrocalcinosis We confirmed several genes previously identified in within-joint analyses for OA affected versus preserved cartilage as well as analyses comparing preserved cartilage from OA affected joints versus healthy cartilage such as the inflammatory genes COL9A1 gene that was higher in preserved as compared to healthy cartilage (3.6-fold), but was subsequently decreased in the OA affected cartilage  gene ALDH1A2 was shown to be allele dependent and with decreased expression in OA affected cartilage. Despite this and other recent successes of genome wide association studies PAPPA was most significant (P = 1.1×10−6), positionally localized in close neighborhood of one the arcOGEN genome wide hits: rs4836732 within the ASTN2 gene. The exact linkage disequilibrium across this locus needs to be further explored. We also found HBP1, at the chr7q22 locus, to be differently expressed, although with small effect size in the OA versus preserved comparison (1.1-fold higher in OA affected cartilage). When comparing diseased cartilage  with healthy cartilage we observed a much stronger and opposite direction of effects: healthy versus OA and healthy versus preserved both showed 1.4-fold lower expression , a gene previously identified in GWA as an OA susceptibility gene NGF) is worth mentioning in this respect. Neurotrophin-3 (NT3), another member of the NGF-family of proteins, enhances migration of premyelinating Schwann cells via Dbs/MCF2L NGF was not identified previously by comparing healthy with OA affected cartilage NGF may be more specific for the “late” OA process. Alternatively, selection of druggable targets from early-responsive genes that start changing before damage is irreversible could be more eligible to effectively slow-down or stop the OA process.Although DCN, collagen type 2 A1 (COL2A1), cartilage intermediate layer protein (CILP), and cartilage oligomeric matrix protein (COMP) were amongst the 100 genes with highest levels of expression in the dataset while no bone-specific genes  were identified here.The sample collection is performed by well-trained lab personnel, however, we cannot exclude the possibility of minor contamination with bone tissue. In this respect, it is of note that several cartilage-specific genes  and knee (B) joint with areas of macroscopically preserved (arrow head) and OA affected cartilage . Insets show detail of preserved (right) and OA affected area (left), in A separated by a dashed line .(TIF)Click here for additional data file.Figure S2Individual box plots per status for genes validated by RT-qPCR.(TIF)Click here for additional data file.Figure S3Relative changes in gene expression levels in preserved and OA affected cartilage relative to healthy cartilage for the 19 genes with at least 2-fold difference in the OA versus preserved analysis .(TIF)Click here for additional data file.Table S1Characteristics of OA donors included in the microarray analyses (discovery) and in the replication and characteristics of the healthy donors included in the microarray analysis.(XLSX)Click here for additional data file.Table S2Taqman probes used in the fluidigm RT-qPCR experiment.(XLSX)Click here for additional data file.Table S3Genes significantly differently expressed between OA and preserved cartilage in microarray analysis of 33 paired OA affected and preserved samples .(XLSX)Click here for additional data file.Table S4Genes significantly differently expressed between preserved and healthy cartilage .(XLSX)Click here for additional data file.Table S5Results of the validation of the genes with at least 2-fold significant differential expression between OA affected and preserved cartilage in the microarray analyses .(XLSX)Click here for additional data file.


The aim was to compare the pharmacokinetic properties of artesunate and dihydroartemisinin in the same women: i) pregnant with acute uncomplicated malaria on day 1 and 2, ii) pregnant with convalescent malaria on day 7 and iii) in a healthy state 3 months post-partum on day 1, 2 and 7.Plasmodium falciparum malaria during their second or third trimesters of pregnancy. The same women were restudied 3 months after delivery when fully recovered. Non-compartmental results of the same study have been published previously.Non-linear mixed-effects modelling was used to compare plasma concentration–time profiles of artesunate and dihydroartemisinin over 7 days of treatment following oral and intravenous artesunate administration to pregnant women with uncomplicated Twenty pregnant patients on the Thailand-Myanmar border were studied and 15 volunteered to be restudied 3 months post-partum. Malaria and pregnancy had no effect on the pharmacokinetic properties of artesunate or dihydroartemisinin after intravenous artesunate administration. However, malaria and pregnancy had opposite effects on the absorption of orally administered artesunate. Malaria increased the absolute oral bioavailability of artesunate by 87%, presumably by inhibiting first pass effect, whereas pregnancy decreased oral bioavailability by 23%.The population pharmacokinetic analysis demonstrated opposite effects of malaria and pregnancy on the bioavailability of orally administered artesunate. Lower drug exposures during the second and third trimesters of pregnancy may contribute to lower cure rates and thus the development of drug resistance. Dose optimization studies are required for artesunate containing artemisinin-based combination therapies (ACTs) in later pregnancy. Pregnant patients in their second and third trimesters have lower oral exposures to dihydroartemisinin after oral artesunate dosing which could reduce treatment efficacy and therefore risk the life of the fetus and mother.Acute malaria infections increase the oral exposure to dihydroartemisinin.The relative contributions of malaria and pregnancy on dihydroartemisinin exposure have not been determined.Malaria in pregnancy does not affect the disposition of parenteral artesunate.Malaria alters the bioavailability of oral artesunate, resulting in 87% increased systemic drug exposure during the acute phase of malaria.Pregnancy alters the bioavailability of oral artesunate, resulting in 23% decreased systemic drug exposure during the second and third trimesters of pregnancy.The effects of pregnancy and malaria are independent and concentrations of artesunate and dihydroartemisinin are therefore approximately 25% lower during malaria infection in later pregnancy compared with post-partum patients with a malaria infection.An extended treatment or dose increase during pregnancy to achieve equivalent exposures in pregnant women compared with that in non-pregnant women has to be studied further.Oral artemisinin-based combination therapy (ACT) and parenteral artesunate are the treatments of choice for uncomplicated and severe malaria, respectively Artesunate is rapidly converted into its active metabolite, dihydroartemisinin, by hydrolysis at gastric pH, by esterases in the blood, and by cytochrome P450 (CYP) 2A6 –1 h–1) compared with aparasitaemic and asymptomatic post-partum women (1.26 l kg–1 h–1) and aparasitaemic and asymptomatic non-pregnant women (1.07 l kg–1 h–1) after a single oral artesunate dose Pregnant women are especially vulnerable to malaria infections Plasmodium falciparum malaria on the Thailand-Myanmar border and re-assess these post-partum A non-compartmental analysis has been published previously Plasmodium falciparum malaria and haematocrit values not lower than 25% were enrolled in the study if they provided written informed consent. Estimated gestational age was measured by dating ultrasound. Post-partum women were followed monthly and the 3 months post-partum visit was postponed in the case of malaria detection or any other illness. Ethical approval for the pharmacokinetic study was obtained from the Faculty of Tropical Medicine, Mahidol University Ethics Committee (TM-IR 029/2005) and the Oxford Tropical Research Ethics Committee (OXTREC 007-05).This study was a re-analysis of a previously published pharmacokinetic study conducted on the North Western border of Thailand and Myanmar in the clinics of the Shoklo Malaria Research Unit from April 2008 until March 2009 –1 intravenous artesunate on admission followed by 4 mg kg–1 oral artesunate for the next 6 days. Group 2 received 4 mg kg–1 oral artesunate on admission, 4 mg kg–1 intravenous artesunate on day 2 followed by 4 mg kg–1 oral artesunate for the next 5 days. The same women were followed-up monthly for 3 months during the post-partum period before receiving the same dose regimen and sampling schedule as during the pregnant period. Intravenous and oral artesunate were manufactured by the Guilin Pharmaceutical Factory, Guangxi in China and repackaged in Thailand by Atlantic Pharmaceuticals. The drugs used for treatment  during the pregnancy visit were set aside for their post-partum visit in an air-conditioned pharmacy in Mae Sot.Patients were assigned to either group 1 or 2 and all drug intake was completed under supervision. Group 1 received 4 mg kgg for 7 min at 4 °C. Plasma samples were stored in liquid nitrogen until transfer to −80 °C for storage. After completion of the study, all samples were transferred on dry ice to the Department of Clinical Pharmacology, Bangkok, Thailand for drug quantification. The laboratory is a participant in the QA/QC proficiency testing programme supported by the Worldwide Antimalarial Resistance Network (WWARN) Venous blood samples (2 ml) were drawn on day 1 and on day 2 at 0, 5, 15, 30, 60, 120, 180, 240 and 360 min or 0, 15, 30, 60, 90, 120, 180, 240 and 360 min after intravenous or oral administration, respectively. Additional venous blood samples were also drawn on day 7 at 0, 60, 120, 240 and 360 min after dose in all patients. Samples were collected in pre-chilled tubes containing sodium fluoride/potassium oxalate as anticoagulant. Samples were inverted five to six times and stored on wet ice until centrifugation at 2000 –1 and 2 ng ml–1 for artesunate and dihydroartemisinin, respectively.Artesunate and dihydroartemisinin were quantified using liquid chromatography-tandem mass-spectrometry nonmem 7.2 on a windows 7 operating system was used to perform non-linear mixed-effects modelling. The objective function value (OFV), computed as minus twice the log likelihood of the data, was used to evaluate competing models. A significant improvement between two hierarchical models after inclusion of one additional parameter (one degree of freedom) required at least a 3.84 drop in OFV (P = 0.05). Goodness-of-fit diagnostics and physiological plausibility were also considered during the model building process.Plasma concentrations were converted into molar units and modelled simultaneously as their natural logarithms. n = 1–10] and first pass effect of artesunate), distribution , variability (between subject and between dose/visit occasion variability) and residual error  models were considered. Also, a previously described semi-mechanistic liver compartment model was evaluated to describe artesunate and dihydroartemisinin pharmacokinetics simultaneously A drug-metabolite model was developed to fit artesunate and dihydroartemisinin data simultaneously after oral and intravenous administration under the assumption that artesunate is completely metabolized into dihydroartemisinin The best performing structural model was carried forward to covariate model building, where all available oral and intravenous data were used. Bodyweight was tested as an allometric function on the volume of distribution (power fixed to 1), elimination and inter-compartmental clearance parameters (power fixed to either 3/4 or 2/3) to correct for differences in bodyweight between the pregnancy and post-partum visit.A step-wise covariate model building procedure for all possible covariate-parameter relationships was not practically possible due to the complexity and consequently long run times of the final base model. Therefore, only physiologically plausible covariates were tested on parameters which could affect the systemic exposure of artesunate or dihydroartemisinin.th percentile) or if patients had a fever (>37.5 °C) and tachycardia (>100 beats min–1). The patients were classified as having mildly severe disease if none of the above conditions was met. The disease severity count status was calculated as the sum of fulfilled severity criteria used for mildly/moderately unwell allocation.The disease effect was assessed on bioavailability, first pass effect of artesunate, artesunate elimination clearance and dihydroartemisinin elimination clearance using a variety of descriptors  during acute malaria on day 1 and day 2 of the study. Patients were considered as having moderately severe disease if plasma creatinine, blood urea nitrogen or liver function tests, including bilirubin , aspartate aminotransferase or alanine aminotransferase were raised  as a continuous covariate (linear relationship) were assessed on bioavailability, first pass effect of artesunate, artesunate elimination clearance and dihydroartemisinin elimination clearance.post hoc parameter estimates th, 50th and 95th percentiles overlaid with the 5th, 50th and 95th percentiles of the observed data.The robustness of the parameter estimates from the final model was assessed by relative standard errors derived from a bootstrap using 100 resampled datasets. Eta and epsilon shrinkage was calculated to assess the reliability of diagnostic plots, individual parameter estimates and n = 500) were performed using parameter estimates from the final population pharmacokinetic model. Three different phases within the study design were evaluated to assess the exposure, a typical pregnant patient with acute Plasmodium falciparum malaria (39–64 kg), a typical pregnant woman in the convalescence phase of Plasmodium falciparum malaria (39–64 kg) and a typical post-partum healthy volunteer (37–62 kg). Furthermore, a dose-optimization (n = 500 simulations) was performed using the final model for oral artesunate  in order to evaluate the required dose to achieve similar exposures in pregnant and post-partum patients (40–60 kg) with acute uncomplicated Plasmodium falciparum malaria.To visualize the impact of the different covariates, simulations . Polymerase chain reaction genotyping showed that two out of the four Plasmodium falciparum recurrent infections were novel and two were recrudescent infections. Seventeen women gave birth (mean [range] of 39.2 [35.6–41.5] weeks) to healthy singleton babies and three women were lost to follow-up.Twenty pregnant women with uncomplicated A structural two compartment disposition model for both artesunate and dihydroartemisinin provided a satisfactory fit to the data Figure with no Bodyweight was implemented using allometry on all clearance (power fixed to 3/4) and volume parameters (power fixed to 1) and resulted in a minor improvement of the model fit (ΔOFV = −1.49). Disease status was a significant (ΔOFV = −66.4) categorical covariate affecting oral bioavailability . Pregnancy as a categorical covariate on either oral bioavailability (23% lower bioavailability in pregnant patients) or dihydroartemisinin elimination clearance (27% higher in pregnant patients) significantly improved the model fit  and was superior compared with trimester and estimated gestational age as covariates, which were unstable and did not significantly improve the model fit. Pregnancy as a categorical covariate on oral bioavailability was carried forward since covariate modelling on intravenous data only could not detect a pregnancy effect on elimination clearance. This was also in agreement with results from the non-compartmental analysis, which could not detect differences in exposure between pregnant and post-partum women after intravenous administration th and 5th percentiles of early artesunate concentrations after intravenous and oral artesunate were substantially over and under estimated in the visual predictive check .post hoc estimates, using the final model, showed altered artesunate and dihydroartemisinin exposures as a result of both disease and pregnancy  (post hoc estimates of FART 12.6% and FDHA 71.2%) compared with the convalescence phase at day 7 (post hoc estimates of FART 6.19% and FDHA 38.4%) and healthy status (post hoc estimates of FART 7.62–8.67% and FDHA 49.8–49.6%)  Table. Thus, tia Figure, D.th day of treatment was similar to that in the same women when restudied post-partum as healthy volunteers. Thus, the combination of the study design and the population-based mechanistic analysis methodology enabled the dissection of the disease and pregnancy effects. Artesunate and dihydroartemisinin pharmacokinetics were unaffected by malaria and pregnancy after intravenous artesunate administration which is in agreement with the previous results from a non-compartmental analysis This study in which each patient was studied twice, first when pregnant with acute (days 1 and 2) or convalescent malaria (day 7) and second when post-partum as healthy volunteers  allowed for a quantification of the independent contributions of pregnancy and malaria on the pharmacokinetics of dihydroartemisinin. This assumed that the disease status of pregnant patients on the 7Disposition pharmacokinetics after intravenous administration were satisfactorily described using two distribution compartments. However, a one compartment distribution model was adequate if only oral artesunate and dihydroartemisinin data were modelled. These findings were in agreement with previous studies and indicate that the distribution phase of artesunate and dihydroartemisinin is attenuated by the absorption phase after oral administration A relatively increased exposure of artesunate and dihydroartemisinin during the acute malaria phase on day 1 and 2  compared with the convalescence phase (at day 7) and post-partum volunteers could be explained by a reduced first pass effect of artesunate. The unbalanced sampling schedule (days 1 and 2 dense sampling and day 7 sparse sampling), might bias the results but this is unlikely to result in falsely detected disease effects as the finding is in agreement with previous studies vs. 23%) irrespectively of parameterization. Both the non-compartmental and model-based analysis of intravenous data showed no pregnancy effect on the elimination clearance of artesunate or dihydroartemisinin. Artesunate and dihydroartemisinin are highly-extracted drugs (hepatic extraction ratio > 0.7). Under the assumption of the well-stirred model, hepatic elimination clearance, and thus intravenous exposure, is therefore not affected to a large degree by changes in intrinsic clearance. However, the first pass effect is proportional to the intrinsic clearance for high-extracted drugs. Pregnancy was therefore retained as a categorical covariate on the absolute bioavailability of oral artesunate in the final model. Estimated gestational age as a continuous covariate was not superior to pregnancy as a categorical covariate which reflects the relatively small sample size (i.e. 10 patients in the second and 10 patients in the third trimester). An additional benefit of implementing pregnancy as a categorical covariate  is the simplified dose adjustments needed for pregnant women with malaria.The bioavailability of artesunate decreased by 23% during pregnancy compared with the post-partum phase 3 months later (after compensating for the disease effect). Pregnancy had a statistically greater impact on the elimination clearance of dihydroartemisinin compared with the bioavailability of artesunate, but the effect size was similar (27% –1 h and 1580 [1310–2050] ng ml–1 h) were substantially lower compared with non-pregnant patients with acute uncomplicated malaria (279–386 ng ml–1 h and 3854–4680 ng ml–1 h) The decreased bioavailability in pregnant women is in agreement with previous findings where dihydroartemisinin exposure decreased by 42% in pregnant women compared with a non-pregnant control group –1 h–1) compared with non-pregnant women (1.07 [0.53–1.89] l kg–1 h–1) The magnitude of the pregnancy effect found in this study (23% lower exposure) is comparable but slightly lower than other studies (37.5% – ≥ 50% lower exposure) in silico dose comparison performed in the current analysis which simulated pregnant and post-partum patients would not have been possible with a non-compartmental analysis. The comparison of exposure was focused on dihydroartemisinin pharmacokinetics as artesunate was considered a pro-drug, contributing only a small proportion of the overall antimalarial activity. Variability in the simulated exposures may be underestimated due to the high eta-shrinkage on certain parameters (38.7%–86.6%) although the medians should remain accurate. High eta-shrinkage might result in an over-prediction of the fraction of pregnant patients displaying an exposure above the lower limit of the therapeutic window. Similarly it may result in under-prediction of toxic side effects. However, adverse events with artesunate-dihydroartemisinin are not expected since doses up to 6 mg kg–1 (which would mean a 50% dose increase) have proved to be safe in patients The In conclusion, malaria (87% increase) and pregnancy (23% decrease) showed opposite independent effects on the absolute oral bioavailability of artesunate. Further dose optimization studies are needed in this vulnerable group to achieve equivalent oral exposures in pregnant and non-pregnant patients with malaria and thereby minimize the risk of treatment failures and the development of drug resistance.The Wellcome Trust is a UK-based medical research charity and is independent of all drug companies.It has no financial links with the manufacturers of either the diagnostic tests or the drugs used in this study. The other authors declare no conflict of interest.We sincerely thank the pregnant women for their co-operation in completing this study. We thank also the medical, nursing, midwifery, laboratory, logistic and administrative staff from SMRU and Mahidol Oxford Research Unit (MORU) in Bangkok. The drug assays were supported by the Malaria in Pregnancy consortium, which is funded through a grant from the Bill and Melinda Gates Foundation to the Liverpool School of Tropical Medicine. This investigation was part of the Wellcome Trust Mahidol University-Oxford Tropical Medicine Research Programme supported by the Wellcome Trust of Great Britain.RM, AP, MR, HT, NH, NZ, ND, NW, JT and FN planned and conducted the clinical study. WH performed the drug analysis and FK and JT conducted the pharmacokinetic analysis. FK and JT drafted the manuscript. All authors reviewed the manuscript critically for important intellectual content and approved the final version.


Metastatic squamous cell carcinoma (SCCA) of the anal canal is a rare malignancy for which no standard treatment algorithm exists. To determine the best approach, all patients diagnosed with metastatic SCCA of the anal canal treated at a single institution were evaluated for choice of chemotherapy and treatment outcome. A retrospective study from January 2000 to May 2012 was conducted. Electronic medical records were reviewed for diagnosis of metastatic SCCA of the anal canal. All patients were treatment naïve for metastatic disease and completed all radiographic imaging at our institution. The purpose of this study was to evaluate outcomes among patients who received systemic chemotherapy and if appropriate were referred for multidisciplinary intervention . Seventy-seven patients fulfilled eligibility criteria. Forty-two patients (55%) received 5-fluorouracil (5-FU) + cisplatin (PF); 24 patients (31%) received carboplatin + paclitaxel (CP); 11 patients (14%) received an alternative regimen. After a median follow-up of 42 months, the median progression-free survival (PFS) for all patients was 7 months; the median overall survival (OS) was 22 months. Thirty-three patients (43%) underwent multidisciplinary management for metastatic disease resulting in a median PFS of 16 months (95% CI: 9·2 −22·8) and median OS of 53 months (95% CI: 28·3 – 77·6). Systemic chemotherapy provides durable survival for patients with surgically unresectable metastatic SCCA of the anal canal. Multidisciplinary management for select patients with metastatic disease effectively improves survival and should be considered whenever possible. Currently very little peer-reviewed literature exists regarding treatment of patients with metastatic squamous cell carcinoma of the anal canal. The purpose of this study, which is presently the largest of its kind, was to evaluate treatment outcomes among patients receiving multidisciplinary therapy and to evaluate the benefit of systemic chemotherapy as a treatment option which is being validated in the InterAACT trial. Our findings indicate that multidisciplinary management may provide an improvement in overall survival.Carcinoma of the anal canal is a rare malignancy, representing only 2% of all digestive system cancers in 2014. Yet, the incidence of anal cancer continues to rise in the United States by 2% per year. Often considered a malignancy only associated with chronically immunosuppressed conditions, such as human immunodeficiency virus (HIV/AIDS) infection or transplant immunosuppression, it is commonly under recognized in non-immunosuppressed individuals. An estimated 7,210 new cases of anal carcinoma will be diagnosed in the United States, resulting in 950 deaths in 2014 [The majority of patients with SCCA of the anal canal present with locally advanced disease but can be treated effectively with concurrent chemoradiation therapy reserving abdominal perineal resection (APR) only for surgical salvage therapy , 3. CurrExtrapelvic metastases are diagnosed in only 5% of patients at the time of initial diagnosis, with metastatic disease occurring in 10%–20% of patients following treatment for locally advanced disease. Locally recurrent pelvic disease or distant disease can contribute significantly to patient morbidity and mortality as a result of local tumor effects such as pain, sacral involvement, symptomatic bulky necrotic lymphadenopathy, and destructive anal canal involvement.Due to the low incidence of anal carcinoma and the rarity of the development of metastatic disease, there have been no completed prospective clinical trials exclusively for metastatic anal carcinoma. On the basis of anecdotal case reports and small retrospective case series , 18–21, The purpose of this study was to evaluate treatment outcomes among patients receiving systemic chemotherapy followed by multidisciplinary management or palliative systemic chemotherapy for metastatic SCCA of the anal canal.Seventy-seven patients met eligibility criteria for the purpose of this study. Baseline patient characteristics are described in Table The majority of tumor histology was moderate (34%) or poorly differentiated (51%). Frequent sites of metastatic disease included distant lymph nodes (33%), pelvis (31%), liver (25%), lungs (15%) and bone (8%).Forty-two patients (55%) received 5-FU + cisplatin (PF); and twenty-four patients 31%) received carboplatin + paclitaxel (CP) as first line treatment; other therapy was provided in 11 patients (14%),  vs. 4 months (95% CI: 1·7 – 6·3), respectively, although this difference was not statistically significant. The response rate for the PF regimen also appeared more favorable  underwent surgical resection of the metastatic site or RFA and 14 (42%) underwent chemoradiation, (Table 8% underwp = 0·02) and multivariate analysis (p = 0·04) for increased overall survival.Of those patients who proceeded to receive multidisciplinary management for intent of cure, the median PFS was significantly longer at 16 months (95% CI: 9·2 – 22·8) compared to those patients receiving palliative systemic chemotherapy, with a PFS of only 5 months (95% CI: 3·5 – 6·5), p < 0·001. The difference in OS was also longer with a median OS of 53 months (95% CI: 28·3 – 77·6) compared to those undergoing palliative systemic chemotherapy, whose median OS was 17 months (95% CI: 13·9 – 20·1), p < 0·001 . Furthermore, the NCCN guidelines suggest there is “no evidence to support surgical intervention” in metastatic anal cancer patients. Any consideration of additional types of chemotherapy for these patients has largely been informally extrapolated from the treatment of more common metastatic squamous cell carcinomas such as head and neck, lung, and cervical carcinoma [To our knowledge, this is the largest study to describe treatment outcomes in patients diagnosed with metastatic SCCA of the anal canal. Prior to our analysis the choice of chemotherapy has been 5-FU + cisplatin despite the fact that there is “limited data” . Due to the size of the study and retrospective nature of our analysis we were unable to differentiate a true difference between the two chemotherapy regimens for patients with unresectable disease. Chemotherapy dose intensity and dose delays also may not have been fully captured unless the treatment was provided directly at MDACC. The majority of patients were HIV- so the findings may not be generalizable to HIV+ patients.We believe there are strengths to our analysis. A single institution analysis results in uniformity in care, permitting a more informative median PFS and OS evaluation than previously demonstrated in small case series. Furthermore, systemic chemotherapy supplemented by a multi-modality approach to optimize patient management provided patients an impressive median OS of 53 months which far exceeds previously reported median overall survival rates of 8–12 months in smaller case studies.Though we are in the era of biologic agents or targeted therapies  the use of biologics was rarely provided in conjunction with chemotherapy (N = 2) due to patient provider concerns for reimbursement and due to the lack of prospective data for this patient population. However, recent phase III data indicate that cetuximab has a role in unresectable head and neck carcinomas in conjunction with radiation therapy and also for locally advanced, surgically unresectable, non-small cell lung carcinoma for response and overall survival , 31, 32.It has been previously reported that detectable human papilloma virus (HPV) has a favorable prognosis in head and neck cancer for disease-free and overall survival , 36. A sIt is presumed the incidence of anal carcinoma will continue to gradually increase over the next several years. Given the rising incidence of anal carcinoma, it is likely more patients will be diagnosed with advanced disease. Globally, HPV remains the most common sexually transmitted disease . DespiteWith the support of the National Cancer Institute, the International Rare Cancer Initiative (IRCI) recognizes the global impact of anal carcinoma and the need to identify the most appropriate treatment regimen for metastatic SCCA of the anal canal. InterAACT (NCT02051868) is the first prospective randomized phase II study ever to be conducted in metastatic SCCA of the anal canal patients and will compare 5-FU + cisplatin versus carboplatin + paclitaxel. The trial is now open to enrollment ; HIV+ paTo date, there is no established prospectively evaluated chemotherapy algorithm for metastatic squamous cell carcinoma of the anal canal. Based on our findings, 5-FU + cisplatin or carboplatin + paclitaxel regimens have activity in the treatment of metastatic SCCA of the anal canal warranting further analysis. More importantly, multidisciplinary management  should be considered for select patients whenever appropriate to improve their overall outcomes.Patients with metastatic SCCA of the anal canal treated at MD Anderson Cancer Center (MDACC) between January 1, 2000 and May 1, 2012 were identified. All patients were required to be treatment naïve to systemic chemotherapy for metastatic disease but may have received prior definitive chemoradiation therapy for their locally advanced primary tumor. All patients were reviewed by the MDACC colorectal surgical oncology team and were determined to not be amenable to surgical salvage with an abdominal perineal resection (APR). All patients were recommended to proceed with systemic chemotherapy. Medical records were reviewed for patient demographics, tumor characteristics, and clinical outcomes including: progression-free survival (PFS), overall survival (OS), response rate (RR), prior history of sexually transmitted diseases (STDs) or immunosuppressed state (if documented), histology, chemotherapy regimen, subsequent lines of therapy, and treatment intervention. All patients were provided treatment recommendations by an MD Anderson physician but were allowed to receive their systemic chemotherapy at an outside institution for added convenience. All patients were required to have all radiographic imaging including a CT scan of the chest, and CT or MRI of the abdomen, and pelvis at our facility as well as follow up. If systemic treatment was provided at an outside institution, corresponding physician notes were utilized for verification of toxicity, and response to the recommended treatment regimen. If the patient had demonstrated partial response/stable disease from their systemic chemotherapy, the patient was referred for multidisciplinary treatment recommendations to either surgical oncology or radiation oncology for consultation.For accuracy, we limited the sample to include only those patients who were initially evaluated with treatment recommendations provided at MDACC plus at least 1 follow-up visit with a physician or a mid-level provider with restaging diagnostic imaging completed at MDACC. Patients with prior or concurrent malignancies within 5 years of diagnosis, except squamous or basal cell skin carcinomas, were excluded.Two treatment categories were created: 1) Palliative systemic chemotherapy was defined for all patients with diffuse metastatic disease not amenable to surgical or radiation oncology intervention, or 2) Multidisciplinary management inclusive of those patients that had received systemic chemotherapy but who were determined by the multidisciplinary team to potentially benefit from treatment intervention .OS was defined as date of diagnosis of metastatic disease until date of death or last follow-up. PFS was defined as date of initiation of therapy until date of documented progression. Follow-up was performed by review of clinic notes and contact with corresponding outside oncologist. Death and overall survival were also determined by use of the social security death index (SSDI) search, which was corroborated with institutional medical records and the tumor registry of MDACC. The distribution of each continuous variable was summarized by its mean, standard deviation and range. The distribution of each categorical variable was summarized in terms of its frequencies and percentages. Log rank tests were used to compare each time-to-event variable (OS and PFS) between groups using the Kaplan-Meier method. This analysis was approved by the institutional review board at MDACC and a waiver of consent was granted.


Further, we hypothesised that any differences in COMT−/− mice would be resolved following treatment with Sildenafil, a treatment which rescues fetal growth. Targeted, quantitative comparisons of serum metabolic profiles of pregnant Nos3−/−, COMT−/− and C57BL/6J mice were made using a kit from BIOCRATES. Significant differences in 4 metabolites were observed between Nos3−/− and C57BL/6J mice (p < 0.05) and in 18 metabolites between C57BL/6J and COMT−/− mice (p < 0.05). Following treatment with Sildenafil, only 5 of the 18 previously identified differences in metabolites (p < 0.05) remained in COMT−/− mice. Metabolomic profiling of mouse models is possible, producing signatures that are clearly different from control animals. A potential new treatment, Sildenafil, is able to normalize the aberrant metabolomic profile in COMT−/− mice; as this treatment moves into clinical trials, this information may assist in assessing possible mechanisms of action.Preeclampsia (PE) and fetal growth restriction (FGR) are serious complications of pregnancy, associated with greatly increased risk of maternal and perinatal morbidity and mortality. These complications are difficult to diagnose and no curative treatments are available. We hypothesized that the metabolomic signature of two models of disease, catechol-O-methyl transferase (COMT Together, these complications are a leading cause of maternal and perinatal morbidity and mortality346Preeclampsia (PE) and fetal growth restriction (FGR) are complications of pregnancy, which together affect around 10% of pregnancies worldwide1−/−) mouse and the catechol-O-methyl transferase knockout (COMT−/−) mouse. Nitric oxide (NO) has been observed to play a significant role in pregnancy, specifically by mediating some of the maternal cardiovascular adaptations necessary to ensure adequate placental perfusion9−/− mouse during pregnancy has highlighted significant abnormalities when compared with wild-type counterparts. Key observations, which recapitulate some of the clinical signs of disease, include abnormal uterine artery remodeling1413The development of new therapeutic agents for these complications of pregnancy has been significantly impaired by both a limited ability to diagnose PE/FGR, as well as a lack of suitable animal models with which to test new treatments. The etiology of these conditions is complex, and likely multi-factorial, although increased uterine artery resistance is commonly observed in both cases−/− mouse during pregnancy have demonstrated maternal hypertension and increased proteinuria at late gestationCatechol-O-methyl transferase (COMT) is one of the enzymes involved in the metabolism of 17β-estradiol to 2- and 4-methoxyestradiol−/− and Nos3−/− mice), as well as C57BL/6J control mice. We hypothesized that differences in the metabolomic profiles of COMT−/− and Nos3−/− mice, in comparison with control mice, would be demonstrated.Given the multifactorial etiology of PE and FGR, there are advantages to studying multiple animal models of disease with differing pathophysiological mechanisms. The aim of the study detailed here was to analyze the metabolomic signature of two mouse models of PE/FGR  was then performed on metabolites that demonstrated significant differences in serum concentration. A scatter plot of the first and second canonical variates  describe−/− mice treated with Sildenafil citrate was initially compared with that of untreated C57BL/6J control mice. Differences in only 5 of the 18 significantly altered metabolites persisted . In comparison, the concentration of phosphatidylcholine diacyl C34:2  model in rats, for instance, demonstrates increased blood pressure and proteinuria along with systemic maternal endothelial dysfunction, all of which are characteristic of PE212224A significant hurdle in the development of new treatments has been the lack of appropriate models with which to test their efficacy. Animals do not develop PE −/− mouse contains a null mutation of the enzyme endothelial nitric oxide synthase (Nos3) gene−/− mouse exhibits an abnormal cardiovascular phenotype, including mild hypertension−/− mouse demonstrates a number of changes in its adaptations to pregnancy when compared with C57BL/6J control mice. For instance, the pregnant dams are hypertensive, both before and during pregnancy131415−/− mice, surprisingly only a small number of metabolites were significantly altered when the serum metabolic profile was compared to that of the C57BL/6J control mouse (32−/− miceThe Nos3ol mouse  online. −/− mouse. Catechol-O-methyl transferase (COMT) converts, amongst other substrates, 17β-estradiol to 2-methoxyestradiol (2-ME). This model was firstly developed by Gogos and colleagues and has been widely used as a model of psychiatric disorders−/− mouse model. These include late gestational hypertension, proteinuria and glomerular endotheliosis−/− mice−/− mouse−/− mouse displayed a different serum metabolomic signature when compared with the Nos3−/− mouse, as well as the C57BL/6J control mouse (The second model utilized in this study was the COMTol mouse .−/− compared with control mice. This significant decrease was noted in short-chain (C2), medium-chain (C6) and amino acid-derived  acylcarnitines. As acylcarnitine concentrations are a direct reflection of the rate of fatty acid and amino acid oxidation, this could indicate reduced activity in these energy-producing pathways.A significant number of metabolites were affected by knockout of the COMT gene . Those a38−/− mice17et al. (2015) determined a significant reduction in the expression of a number of genes in placental tissue obtained from cases of FGR compared with normal pregnanciesThe placenta is an extremely metabolically active tissue, and any alterations in energy production could have significant effects on its development or function. Given that placental dysfunction appears to be key in mediating the phenotype observed in COMT−/− mice with Sildenafil citrate resulted in a normalization of umbilical artery Doppler waveforms and an increase in fetal growth−/− mice with Sildenafil citrate also resulted in the normalization of the concentration of all but one of the acylcarnitines. There are numerous studies that have demonstrated the ability of Sildenafil treatment to increase mitochondrial biogenesis and activity in cell lines44Previously, we have observed that treatment of COMT−/− compared with C57BL/6J mice. Kynurenine is a metabolite of the amino acid tryptophan, and used in the production of niacin. Interestingly, niacin has been associated for many years with cardiovascular health. While initial benefits of niacin were believed to be associated with improved lipid profiles−/− mice in this study may be reflected in a reduction in niacin production, which in turn may contribute to altered plasma lipid profiles and/or impaired endothelial function. COMT−/− mice demonstrate a significant increase in placental resistance, which is reduced subsequent to Sildenafil treatment. The increased placental resistance may be attributable to increased vasoconstriction in the uteroplacental circulation, which is then ameliorated by Sildenafil. It is not known if niacin is vasoactive in the uteroplacental circulation and therefore contributing to these changes in placental resistance; in vitro studies should be performed to investigate the importance of this compound in this particular vascular bed.Kynurenine was also observed to be significantly reduced in samples from COMT−/− mice; these compounds are major structural components of plasma lipoproteins and biological membranes−/− mice compared with controls, it appears there may be significant alterations in lipid metabolism pathways, which could result in an increased production of ceramides. Interestingly, nitric oxide is able to prevent ceramide-mediated apoptosis in a cGMP-dependent mannerFinally, significant increases in the concentrations of sphingolipids and glycerophospholipids were observed in COMTInterestingly, the metabolites found to be altered in this model are from similar classes to those observed to be altered in plasma and placental samples taken from women who went on to develop FGRFurther, recent analysis of placental gene expression profiles observed that one of the pathways significantly altered in PE was that of lipid metabolismThis study is the first to apply metabolomics to mouse models of preeclampsia and fetal growth restriction. It has illustrated that metabolomic profiling of mouse models of PE and FGR is able to not only produce a phenotypic signature, which is clearly distinguishable from controls, but is also characteristic of underlying pathophysiological mechanisms. Further, the ability of Sildenafil citrate to increase fetal growth was reflected in alterations of the metabolomic signature.The altered metabolomic profile in the models of disease was found to bear similarities with the altered profile observed in women who later developed preeclampsia or fetal growth restriction. These similarities serve to further validate these models as appropriate – not only for further investigation of potential mechanisms of disease, but also for use to test potential new treatments. As a paucity of appropriate animal models has been a factor in the development of curative treatments, this is an extremely important development and highlights the potential role of metabolomic profiling in developing new therapeutic strategies. Further, additional investigation of metabolites found to be altered in these models may provide information regarding both the pathophysiological mechanisms, and the potential mechanisms of action of therapeutic strategies.Guide for the Care and Use of Laboratory Animals .All protocols were approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee in accordance with the Canadian Council on Animal Care and conformed to the −/−, Nos3−/− and C57BL/6J mice (C57BL/5J mice are the background strain of both knockout models) of 8–12 weeks of age were used. They were mated nightly with males of a corresponding genotype. The morning a vaginal plug was detected was denoted as gestational day (GD) 0.5. A subgroup of COMT−/− and C57BL/6J mice were treated with Sildenafil citrate; Sildenafil citrate . All the serum samples were analyzed using the protocol described in the AbsoluteIDQ user manual. Briefly, serum samples, obtained from pregnant mice at the end of gestation, were thawed on ice, vortexed and then centrifuged at 13,000x g. 10 μL of each serum sample was loaded onto the center of the filter on the upper 96-well kit plate and dried in a stream of nitrogen. Subsequently, 20 μL of a 5% solution of phenyl-isothiocyanate was added for derivatization. After incubation, the filter spots were dried again using an evaporator. Extraction of metabolites was then achieved by adding 300 μL methanol containing 5 mM ammonium acetate. The extracts were obtained by centrifugation into the lower 96-deep well plate, followed by a dilution step with kit MS running solvent. Mass spectrometric analysis was performed on an API4000 Qtrap® tandem mass spectrometry instrument  equipped with a solvent delivery system. The samples were delivered to the mass spectrometer using a reverse phase HPLC column  followed by a direct flow injection (DI) assay . The Biocrates MetIQ software was used to control the entire assay workflow, from sample registration to automated calculation of metabolite concentrations to the export of data into other data analysis programs. A targeted profiling scheme was used to quantitatively screen for known small molecule metabolites using multiple reaction monitoring, neutral loss and precursor ion scans.A targeted quantitative metabolomics approach was applied to analyze serum samples using a commercially available direct flow injection and LC-MS/MS kit from BIOCRATES Life Sciences AG (Austria). This kit, in combination with an ABI 4000 Q-Trap (Applied Biosystems/MDS Sciex) mass spectrometer, can be used for the targeted identification and quantification of up to 180 different endogenous metabolites including amino acids, acylcarnitines, biogenic amines, glycerophospholipids, sphingolipids and sugars. The method used combines the derivatization and extraction of analytes, their separation via reverse-phase PLC and the selective mass-spectrometric detection using multiple reaction monitoring (MRM) pairs. Isotope-labeled internal standards and other internal standards were integrated in the kit plate filter for metabolite quantification. The Absolute−/−, Nos3−/− and C57BL/6J mice, and then COMT−/− and C57BL/6J mice following Sildenafil citrate treatment) were not normally distributed, the differences in metabolic profile distributions were evaluated, per metabolite, using the nonparametric Kruskal-Wallis one-way analysis of variance by ranks test, and for significant metabolites, pairwise group comparisons were performed using the Wilcoxon rank-sum test. Comparisons were considered statistically significant if p-values lower than 0.05 were obtained. Canonical Variate Analysis (CVA)http://www.mathworks.com/). Initially, results from COMT−/− and Nos3−/− mice were compared with C57BL/6J mice, to determine what, if any, differences were present in the metabolomic signature of different models compared with that of their background strain. A comparison between the two models, COMT−/− and Nos3−/−, was also made to determine if the differing pathophysiological mechanisms were reflected in changes in the altered metabolomic signature. COMT−/− mice treated with Sildenafil were compared with untreated C57BL/6J mice to determine the ability of Sildenafil to normalize the metabolomic signature in comparison with ‘normal’ control mice; treated C57BL/6J mice were also included in the event that Sildenafil treatment may alter metabolite concentration in normal mice.As the raw data for each measured metabolite from each of the groups .


More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent  Cancer cells frequently contain chromosomal rearrangements that lead to the formation of fusion genes expressed in the cell . These fin situ hybridization (FISH), a technique in which selected regions of chromosomes are fluorescently labeled through the hybridization of specific oligonucleotide probes. Aclinician must then visually identify two chromosomal regions that have rearranged in a known pattern. FISH has many problems as a technique including being a difficult, low throughput procedure, required knowledge of both gene fusion partners, low spatial resolution, the availability of the necessary fluorescent probes and the need for highly trained personnel to decide if a fusion event has taken place [Gene fusions are typically identified in cells by fluorescent en place . These dhttp://cancergenome.nih.gov/)  has been used to identify gene fusions that are transcribed into RNAs within various cells . SoftwarIn order to test the ability of the Ovation Fusion Panel Target Enrichment System assay to effectively target RNAs of interest, we ran the assay using 100 ng of total RNA from the H2228, lung adenocarcinoma cell line to see what percentage of sequencing reads were derived from the targeted genes. The resulting library was sequenced on an Illumina MiSeq with paired end 75bp reads. 92.5% of reads mapped back to the genome with 96% of those reads mapping to the targeted RNAs. These results indicate a strong selection for targeted RNAs by the Ovation Fusion Panel Target Enrichment System. Comfortable that we were selecting almost exclusively for the sequences we were interested in, we next looked to identify gene fusions.ALK gene that is fused to EML4 and the other allele is fused to PTPN3. In our targeting panel we have probes targeting ALK and EML4, but not PTPN3 so this served as a good test for identifying fusion transcripts for which only one of the genes was directly targeted. The sequencing data were analyzed for the presence of gene fusions , which only requires the hybridization of a single primer in order to obtain sequencing information from the downstream RNA. H2228 cells carry one allele of the ions see  and we wBCR-ABL and NUP214-XKR3. 100 ng of UHR total RNA was used to prepare sequencing libraries with Ovation Fusion Panel Target Enrichment System. Libraries were sequenced on either the Illumina MiSeq or HiSeq and analyzed with SOAPFuse [BCR-ABL and NUP214-XKR3 fusion transcripts. In addition, we also identified an FGFR1-WHSC1L1 gene fusion, which has been reported as a recurring fusion in breast cancer [MLLT10 fused to PICALM. Having identified a set of gene fusions in UHR RNA, we compared the number of fusion reads in these genes detected by SOAPFuse in our datasets to those of a publically available RNA-Seq dataset that consists of 125M paired end sequencing reads [BCR-ABL fusion. The 1.6M targeted sequencing reads has fewer reads for NUP214-XKR3, which is expected because our targeting panel only contains probes for NUP214 and not XKR3. The standard RNA-Seq does not contain any detectable reads for FGFR1-WHSC1L1 nor any reads for MLLT10-PICALM, while the targeted sequencing approach described here identified both of these fusion events. Both of these previously undescribed fusions were verified by RT-PCR and Sanger sequencing. In summary, by using a targeted sequencing approach, we were able to identify known gene fusion events in UHR RNA while using only 1.3% of the sequencing reads necessary to identify approximately the same number of fusion reads in a standard RNA sequencing experiment. In addition we were able to identify previously undocumented gene fusion events using the targeted approach that were not identified in standard RNA sequencing, even though the targeted sequencing had 14–78X fewer sequencing reads. The inability of standard RNA sequencing to identify fusion transcripts in a cell line that has been studied in detail suggests that even samples for which deep RNA sequencing data already exists should be tested again specifically for the presence of fusion transcripts.We next wanted to compare our results to those of standard RNA-Seq experiments to test how many sequencing reads were necessary to identify gene fusion events when using the Ovation Fusion Panel Target Enrichment System relative to a non-targeted RNA-Seq approach. Universal Human Reference RNA (UHR) contains RNA from ten different cancer cell lines. Many standard RNA-Seq experiments have been performed using UHR RNA and these experiments have identified a number of gene fusion events . Our 401SOAPFuse . Our init cancer , but hasATP5L-MLL and EIF4E3-FOXP1. The EIF4E3-FOXP1 fusion . This common procedure is known to fragment nucleic acids making it difficult to retrieve sequence data from them. We extracted RNA from HCC1937 primary breast ductal carcinoma cells that had been formalin-fixed and paraffin-embedded . Briefly, utilizing the contents of the Ovation Fusion Panel Target Enrichment System kit, 50 or 100 ng of total RNA was reverse transcribed into cDNA. The unpurified cDNA was then made double stranded by the addition of a DNA polymerase. The double stranded cDNA (ds-cDNA) underwent end repair to blunt the ends followed by barcoded adapter ligation. Ligated samples were bead purified and hybridizated with targeting probes starting at 95°C followed by a slow ramp down to 60°C overnight. Following probe annealing, a DNA polymerase was added to the mixture which was heated to 72°C to extend the probes through the target region  was a gift from Susana Ortiz, M.D., Ph.D. . Universal Human Reference RNA (UHR) (Agilent Technologies) and Human Brain RNA (Ambion) were purchased and used without modification. IRB approval was not required as the human RNA samples used were reference samples obtained from cadavers and purchased from a commercial source. No identifying patient information was obtained by the authors. FFPE sections of cell line HCC1937 were purchased from Alamak Bioscience. 10 micron sections were cut and RNA was extracted from these samples with the Recoverall Total Nucleic Acid Extraction Kit (Ambion). Total RNA was used as input into the Ovation Fusion Panel Target Enrichment System to generate targeted sequencing libraries following the manufacturer’s recommended protocol in the User Guide and described in gion see . UnextenResulting sequences were analyzed in the following ways: 1. To determine the reads that were derived from targeted RNA sequences, forward reads were mapped to the Hg19 reference genome using STAR v2.4.0d  only allFor publically available UHR RNA-Seq data, we downloaded SRX333353  and for Selected putative gene fusions were verified by designing PCR primers around the predicted fusion site. Reverse transcription PCR was used to amplify the predicted fusion gene junctions from the same starting RNA material as was used for sequencing. Resulting PCR products were purified and Sanger sequenced to verify the fusion junction. 200 ng of total RNA input was used in the Ovation cDNA Module For Target Enrichment to generate the cDNA used for the PCR reaction (included as part of the Ovation Fusion Panel Target Enrichment kit). PCR was performed in 50 ul reactions using standard Taq buffer, 0.5mM dNTPs and standard Taq Polymerase (NEB) with 0.4 uM each primer. The PCR reaction was carried out with the following program: 95°C, 3 minutes, followed by 30 cycles of 95°C, 15 seconds, 57°C, 20 seconds and 72°C, 10 seconds. See 401 genes were selected for inclusion in the initial gene fusion targeting panel. All refseq exons were input into the proprietary probe selection software with the goal of designing at least two probes targeting each exon. For exons smaller than 300 bases probes were designed so that the 3’ end of the probe would hybridize as near as possible to the known exon boundary, with one probe designed to hybridize to each strand of the double stranded cDNA. For exons larger than 300 bases, additional probes were tiled across the exon at 300 base intervals in order to maximize coverage across these exons. Placing probes near the exon boundaries ensures that extension products will cross at least one exon-exon junction, thus increasing the probability of extending across a gene fusion junction. These design parameters resulted in the generation of 17,999 unique targeting probes across 5769 exons.S1 Table(TXT)Click here for additional data file.S2 TableForward and reverse primer sequences used in RT-PCR validation. For Sanger sequences, | denotes the breakpoint between sequence aligning to one gene versus the other in the fusion pair.(TXT)Click here for additional data file.