Variation in patterns of gene expression can result from modifications in the genome that occur without a change in the sequence of the DNA; such modifications include methylation of cytosine to generate 5-methylcytosine (5mC) resulting in the generation of heritable epimutation and novel epialleles. This type of non-sequence variation is called epigenetics. The enzymes responsible for generation of such DNA modifications in mammals are named DNA methyltransferases (DNMT) including DNMT1, DNMT2 and DNMT3. The later stages of oxidations to these modifications are catalyzed by Ten Eleven Translocation (TET) proteins, which contain catalytic domains belonging to the 2-oxoglutarate dependent dioxygenase family. In various mammalian cells/tissues including embryonic stem cells, cancer cells and brain tissues, it has been confirmed that these proteins are able to induce the stepwise oxidization of 5-methyl cytosine to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and finally 5-carboxylcytosine (5caC). Each stage from initial methylation until the end of the DNA demethylation process is considered as a specific epigenetic mark that may regulate gene expression. This review discusses controversial evidence for the presence of such oxidative products, particularly 5hmC, in various plant species. Whereas some reports suggest no evidence for enzymatic DNA demethylation, other reports suggest that the presence of oxidative products is followed by the active demethylation and indicate the contribution of possible TET-like proteins in the regulation of gene expression in plants. The review also summarizes the results obtained by expressing the human TET conserved catalytic domain in transgenic plants. It is accepted as the fifth nucleotide base in mammalian and other genomes It has been widely demonstrated that modification of plant genomes by an array of epigenetic marks is involved in the regulation of plant growth and development Cytosine methylation primarily occurs at CG dinucleotide sites, although CNG and CNN sites can also be significantly methylated in plants This review was undertaken in order to assess the evidence relating to the possible presence of oxidised derivatives of 5mC with plant genomes, and to consider the hypothesis that plants contain equivalents to the TET-like enzymes present in animals.2.It has been suggested that the epigenetic information including DNA methylation of the epigenome can be maintained and also be translated through the dynamic activity of DNA methylases (writers), demethylases (erasers) and also reader proteins. Collectively, these proteins are responsible for the recognition and interpretation of the information in both mammals and plants In terms of their function, all TET family members have an active role in regulating the transcription of genes Nanog gene It has also been demonstrated that TET1 has a role in maintaining the stem cell state in embryonic stem cells via interaction with the promoter of the 3.TET genes in mammals and other metazoa have been extensively studied Tet genes in association with a low level of 5hmC was reported. In summary, all this evidence, together with additional studies has revealed the significant role of 5hmC in embryogenesis The importance of 5hmC and the ubiquitous occurrence of Medicago truncatula shows the ability of plants to remodel their landscape of DNA 5mC across gene structures under salinity stress In plants, many studies have been also conducted to investigate the genome-wide distribution of 5mC, its effect on gene expression and its biological function in terms of morphological characteristics and adaptation to unfavourable environment. For example, one study on DNA methylation of Arabidopsis including DEMETER (DME), REPRESSOR OF SILENCING1 (ROS1), DEMETER-LIKE2 (DML2), and DEMETERLIKE3 (DML3) are involved in active reversion of cytosine methylation As regards the corresponding phenomenon of demethylation in plants, a subfamily of helix-hairpin-helix-Gly/Pro/Asp (HhH-GPD) DNA glycosylases in It has been stated that DME is essential during seed development as a consequence of its involvement in DNA demethylation. It is found to be required in one of the female gametes in regulating active reversion of DNA methylation before fertilization In contrast to the situation in metazoa, there is limited evidence related to the presence of 5hmC in plants; this has resulted in a controversial debate about the definitive existence of 5hmC as well as functional counterparts of TET-like enzymes 4.TET enzymes are members of the oxoglutarate/iron dependent dioxygenases (2-OG)-dioxygenases, which are one of the most functionally diverse super-families of non-haem enzymes. They are distributed in a wide range of prokaryotes and eukaryotes, and are involved in many different important biological activities including biosynthesis of plant products and antibiotics, posttranslational modification, DNA/RNA damage repair and metabolism of lipids AtGA20ox1 in an Arabidopsis mutant reduced internode length and subsequently stem height Oryza sativa) and barley (Hordeum vulgare) containing mutations  in the cereal GA20ox2, have been selected by breeders for lodging resistance and subsequently increased yield For example, within plants one sub-class of these enzymes comprise anthocyanidin synthase, flavanone 3β-hydroxylase and flavonol synthase A. thaliana. This superfamily of dioxygenases includes a group of important catalytic enzymes named Prolyl 4-hydroxylases (P4H) that are found in mammals and plants ATP4H1-ATP4H13, have been described in ArabidopsisThe presence of more than 130 dioxygenase genes has been reported in the genome of 5.Arabidopsis are mutated; the methylation status was only affected at some specific loci Previous studies confirmed that glycosylases can directly remove 5mC from the DNA backbone and subsequently might create an abasic site as a part of the BER pathway To determine whether oxidation products of 5mC are present in plant DNA, different methods with various sensitivities and specifications have been used. A series of studies Arabidopsis at a frequency 0.07% of total cytosines, suggesting that this derivative results from passive loss of DNA methylation following DNA replication The detection of 5hmC has been reported in leaves and flowers of Moreover, significant enrichment of 5hmC in heterochromatin regions, particularly in transposable element (TE)-related genes, has been identified in rice. This result led researchers to suggest a significant association between chromatin structure and levels of 5hmC A. thaliana, Lycopersicon esculentum, Ginkgo biloba, Platycladus orientalis, Zea mays, and Oryza sativaArabidopsis thalianaIn line with this suggestion, one comprehensive study used chemical derivatization coupled with liquid chromatography/Tandem mass spectrometry analysis to determine oxidative products of 5mC in samples from plants including Tet1, 2, 3 genes in mammals Tet genes is that there are no oxidised products of 5mC. However, previous data on the presence of oxidative derivatives of 5mC led to the conclusion that there must be another family of protein(s) responsible for the production of DNA modifications and its biochemistry may be similar to that of the TET protein family because of the presence of the intermediate and terminal oxidation products such as 5hmC (see section below).Although oxidative products of 5mC have been detected in plants, as previously mentioned, there is no evidence to confirm the involvement of plant enzymes equivalent to the TET family proteins as described in mammals. Again as mentioned previously, there is evidence for some functional redundancy for 6.ArabidopsisAfter the discovery of the functional role of human TET proteins it became of immediate interest to examine the possible activity of such proteins when expressed in plants. The first such published study was that in which the C-terminal catalytic domain of the human Tet3 gene, under the control of the constitutive 35S promoter, was transformed into Three other groups have also reported results from similar FLOWERING WAGENINGEN (FWA) gene in Arabidopsis CACT1 transposon and thereby generated targeted demethylation. In addition, they developed a CRISPR/dCAS9-based targeted demethylation The second group who expressed human TET domains in plants used a construct in which a section of the Arabidopsis ubiquitin10 promoter mediated expression of the human TET1 catalytic domain (TETcd) fused to an artificial zinc finger (ZF) designed to target the promoter of the Brassica oleracea shown to be highly methylated in the progeny of a microspore-derived doubled haploid The third group who used this approach generated a plasmid containing the catalytically inactive SpCas9 joined to human TET3cd (aa 850–1795) In combination, these TET-based molecular tools demonstrate the possibility of generating new epialleles of genes of interest, as well as reactivating expression of silenced genes, transgenes or transposons Taken together, these four studies provide definitive evidence that the human TET catalytic domains are active when expressed in plants and they may be capable of generating additional epigenetic marks by oxidation of 5mC and thereby producing stably inherited phenotypic variation.7.The present absence of direct functional evidence for TET-like enzyme activity in plants, as discussed above, raises the question about how a search for such evidence should be best conducted. There are a number of options, which include the following. First, all the plant 2-OG oxygenases with known or unknown substrate(s) could be overexpressed in a heterologous system, purified, and then tested on a range of synthetic DNA and RNA A second question raised by any search for TET-like enzymes is whether the oxidative function of these enzymes might be affected by evolutionarily non-related enzymes. Such enzymes would fall under the definition being non-homologous , isofunctional proteins 8.Arabidopsis. The second group accepts the evidence for the presence of 5mC derivatives generated from active demethylation in a process similar to that in the mammalian pathway. Hopefully, the increasingly sophisticated analytical and molecular techniques will be able to resolve this important issue.Despite the extensive experimental evidence outlined above, several important questions regarding the biological function of Tet-like proteins in plants remain unanswered. Are there any TET equivalents in plants? To what extent are dioxygenase proteins containing conserved domains similar to those of TETs involved in oxidation of 5mC in plants? This remains a controversial issue that has divided scientists into two main groups. The first group strongly supports the conclusion that there is no biologically relevant 5hmC in plants and particularly


The Hypothalamic-Pituitary-Adrenal (HPA) axis has an important role in maintaining the physiological homeostasis in relation to external and internal stimuli. The HPA axis dysfunctions were extensively studied in neuroendocrine disorders such as depression and chronic fatigue syndrome but less so in hepatic cholestasis, cirrhosis or other liver diseases. The HPA axis controls many functions of the liver through neuroendocrine forward signaling pathways as well as negative feedback mechanisms, in health and disease. This review describes cell and molecular mechanisms of liver and HPA axis physiology and pathology. Evidence is presented from clinical and experimental model studies, demonstrating that dysfunctions of HPA axis are correlated with liver cholestatic disorders. The functional interactions of HPA axis with the liver and immune system in cases of bacterial and viral infections are also discussed. Proinflammatory cytokines stimulate glucocorticoid (GC) release by adrenals but they also inhibit bile acid (BA) efflux from liver. Chronic hepatic inflammation leads to cholestasis and impaired GC metabolism in the liver, so that HPA axis becomes depressed. Recently discovered interactions of GC with self-oscillating transcription factors that generate circadian rhythms of gene expression in brain and liver, in the context of GC replacement therapies, are also outlined. Activation of hypothalamus-pituitary-adrenal (HPA) axis is an essential component of stress response driving the production of glucocorticoids  necessary to mediate the adaptation to changes in environmental  and internal  conditions. HPA axis is a complex neuroendocrine system that functions synergistically with the locus coeruleus/norepinephrine autonomic nervous system in response to stress and also in a circadian rhythm . HypothaThe HPA axis dysfunctions were largely studied in relation to disorders such as depression, chronic fatigue syndrome (CFS) and fibromyalgia (FM), but less so in hepatic cholestasis, cirrhosis or other liver diseases . BecauseLiver is a vital organ with complex physiology and essential functions including: (i) regulation of glucose and lipid metabolism and homeostasis; (ii) bile acid (BA) synthesis, traffic to gallbladder, intestine, and reabsorption from the enterohepatic circulation into the liver; (iii) metabolic processing and excretion of xenobiotic toxicants and drugs; (iv) serum protein synthesis; (v) coagulation, fibrinolysis and platelet production.+, K+, HCO3−, An important physiologic role of the liver involves the synthesis of BA, formation, secretion and traffic of bile to the intestine, as well as reabsorption and recirculation of it throughout the enterohepatic circulation . Bile isc ratios , 11. The+-taurocholic acid co-transporting polypeptide or Slc10a1) mediates absorption of BA from blood circulation into the liver  impaired hepatocellular bile transport; (ii) bile duct pathology; (iii) bile flow obstructions due to gall stones or malignancies , 42. TheMost forms of cholestasis are related to dysregulation of genes involved in bile secretion from liver into the biliary tract, or in bile transport from gallbladder to the intestine, or in the uptake of BA from enterohepatic circulation. Cholastasis and jaundice were found in both hereditary and acquired dysfunctions related to biliary production and circulation between liver and intestine. Hereditary hepatobiliary disorders characterized by cholestasis, include progressive familial intrahepatic cholestasis (PFIC) types 1–5, benign recurrent intrahepatic cholestasis (BRIC) 1–2, intrahepatic cholestasis of pregnancy (ICP), Low Phospholipid Associated Cholelithiasis or Gallbladder Disease 1. They are caused by defects in hepatobiliary transporters, structural proteins of canalicular ducts or in BA-sensing receptors such as FXR and TGR5 , 43. TheInterestingly, most of these diseases are treated successfully with corticosteroids together with statins, cholestyramine or ursodeoxycholic acid (UDCA), suggesting that stimulation of HPA axis can improve cholestatic conditions . HereditThe acquired cholestatic liver diseases include dysfunctions of hepatobiliary production and transport of the bile, caused by biliary duct obstruction due to gall stones or malignancies, or by medications and medical procedures such as liver transplant , 31.+ as cofactor, and the reduction of cortisone using NADPH as cofactor and its activity direction is dictated by the NADP+/NADPH ratio. The 41 kD type 2 enzyme 11β-HSD2, has much higher affinity for cortisol than 11β-HSD1 and catalyzes only the oxidation of cortisol to cortisone using NAD+ cofactor. Loss of function mutations of 11β-HSD2 were correlated with hypercortisolism and congenital adrenal hyperplasia  receives stimuli which are internal and external to the body, and in response sends signals to all organs to ensure metabolic and physiological homeostasis. The HPA axis controls many functions of the liver through neuroendocrine forward signaling pathways as well as negative feedback mechanisms as illustrated in Figure erplasia , 58 as werplasia . An alteerplasia . 5α-R1 aogenesis . In an eogenesis . In concClinical reports indicate that cholestatic jaundice in infancy may be associated with hypoglycemia and GC deficiency due to various causes including dysfunctions of pituitary or adrenal glands. Isolated glucocorticoid deficiency (IGD) is a rare but potentially lethal hereditary disease caused by loss of function mutations of ACTH receptor gene . IGD-affAssociation of liver cholestasis and cirrhosis with dysfunctions of HPA axis was also reported in adult patients. Low levels of cortisol, indicating a dysfunction of the HPA axis, have been reported in patients with end-stage liver disease , 71. Thein vivo  is a rare condition in which the endogenous cortisol secretion is excessive and out of the HPA axis control due to abnormal secretion of ACTH from a pituitary tumor . A cliniCorticosteroids play an important role in the management of many inflammatory conditions but their systemic long-term use is often associated with serious side effects leading to osteoporosis, osteonecrosis, HPA axis suppression, weight gain and even alterations of brain-related functions –89. Cort−/− mice  indicated that the increased intrahepatic bile duct mass, cholangiocyte proliferation and the overall liver fibrosis were significantly reduced by corticosterone treatment, with better results in males than in females  and demonstrated that there is an impaired activation of the HPA axis in cholestatic animals in response to various types of stress including chemical  or immun females .A strong correlation of the liver disease-related fatigue with an impaired function of the HPA axis was also demonstrated by studies of locomotive behaviors of cholestatic rats , 98. AniIn vitro experiments proved that dexamethasone increased BA synthesis in cultured rat hepatocytes by induction of cholesterol 7-α hydroxylase  for example, demonstrated that IL-1 was able to induce secretion of hormones within the HPA axis acting as immunoregulator with role in signaling between the immune and neuroendocrine systems . Numeroucontrols . InteresCytokines have multiple functions including signaling the initiation of the immune response to liver injury, with progression to proinflammatory phase for injury containment, followed by tissue repair during an anti-inflammatory phase . In hepaInflammation-induced cholestasis can be caused by LPS (lipopolysaccharide) and endotoxin released by bacteria in various microbial infections . In respDuring 1980's several immunomodulating therapies such as IL-2 and INF-α were considered for standard medical treatments of disorders including cancer , infectious diseases  and neural degeneration  . HoweverIFN-α has been shown to be effective in the treatment of various forms of cancer and infectious diseases. INF-α has numerous neuroendocrine effects within the HPA axis where it exhibits concentration-dependent biphasic activation and inhibition of the HPA axis  123). I. I123). +-taurocholate transport protein, or Ntcp/Slc10a1) resulting in cholesterol-gallstone development and disruption of systemic BA circulation  and modulate gene expression. The main BA-binding NR is FXR (farnesoid X receptor) which is activated by BA and downregulates genes involved in BA biosynthesis and transport , 131. BAeductase . In vivoes of GC . To confes of GC . It was es of GC . While tes of GC . The cores of GC . Moreovees of GC . These des of GC . Taken tIn regard with brain vs. serum concentrations of BA in hepatic cholestasis models, it was demonstrated that a very large (7–20-fold) increase of systemic BA concentration in rats with BDL-induced cholestasis, resulted in 25–30% increase in the brain BA content . In ratsCortisol has a circadian rhythm with rising concentration in the early hours of the morning, peaking on waking and declining over the day with low concentrations in the evening and night . Once esThe circadian system consists of a central clock located in SCN, and peripheral clocks ubiquitously expressed in all tissues . The cenSince there are obvious links of HPA axis activity and circadian rhythm regulators with liver functions, the pathology in one system may lead to disruption in another. Many forms of hepatic cholestasis are treated with GC replacement therapy which has beneficial but also negative side effects in long term treatments. Future studies on the crosstalk of GR with clock proteins in the regulation of GC-controlled genes will improve GC-based therapies.In summary, clinical reports and experimental model studies demonstrate a strong association of cholestatic liver disorders with dysfunctions of the HPA axis. Several physiological functions connect the liver with the HPA axis: (i) a neuroendocrine signaling pathway along the HPA axis regulates the hepatic functions according to environmental and stress conditions; (ii) metabolic pathways of cholesterol synthesis in the liver, and cholesterol transport from liver to the adrenal glands for production of GC and other adrenal hormones, affect the HPA axis functions; (iii) the interaction of both liver and HPA axis with the immune system is critical for constant surveillance against injury factors, as well as initiation and progression of tissue repair; (iv) increase of bile acids in the systemic circulation due to liver cholestasis has a major impact on the brain and especially the HPA axis function. Any dysfunction of the liver, HPA or immune system can result in impaired function of the other connected systems. Due to this close interconnection, therapies of cholestatic liver diseases need to address and correct the HPA axis dysregulation in addition to solving the hepatic problems.The interactions between HPA axis and the liver are very intricate and still not completely understood. Thus, aspects of liver dysfunction having effects on HPA axis and how therapies directed to the liver could help alleviate disorders related to HPA axis dysregulation, are still to be investigated . Also, mIt was reported that despite elevated cortisol levels during critical illness, there is a tissue resistance to GC which is believed to occur due to insufficient GC-mediated anti-inflammatory response . This tyAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pazopanib is the first and only tyrosine kinase inhibitor currently approved for the treatment of multiple histological subtypes of soft tissue sarcoma (STS). Initially developed as a small molecule inhibitor of vascular endothelial growth factor receptors, preclinical work indicates that pazopanib exerts an anticancer effect through the inhibition of both angiogenic and oncogenic signaling pathways. Following the establishment of optimal dosing and safety profiles in early phase studies and approval for the treatment of advanced renal cell carcinoma, pazopanib was investigated in STS. A landmark phase III randomized study demonstrated improved progression-free survival with pazopanib compared to that with placebo in pretreated patients with STS of various subtypes. The efficacy of pazopanib in specific STS subtypes has been further described in real-world-based case series in both mixed and subtype-specific STS cohorts. At present, there are no clinically validated predictive biomarkers for use in selecting patients with advanced STS for pazopanib therapy, limiting the clinical effectiveness and cost-effectiveness of the drug. In this review, we summarize the preclinical and clinical data for pazopanib, outline the evidence base for its effect in STS and explore reported studies that have investigated putative biomarkers. After receiving marketing authorization for the treatment of metastatic renal cell carcinoma (mRCC), pazopanib became the first (and currently only) TKI licensed for the treatment of multiple subtypes of advanced soft tissue sarcoma (STS). This approval was based on the results of a double-blind, placebo-controlled randomized phase III trial that demonstrated significant prolongation of progression-free survival (PFS) in patients with pretreated advanced STS who received pazopanib6. However, despite this evidence of an antitumor effect, no significant difference in overall survival (OS) was observed between pazopanib and placebo-treated patients. The failure of PFS gain to translate to OS benefit has adversely influenced the cost assessment of pazopanib for this indication, leading to funding limitations in certain health economies worldwide9. There is currently a poor understanding of the clinical mechanisms of pazopanib response and resistance and an unmet need for predictive biomarkers that are able to prospectively select the subgroup of STS patients most likely to benefit from pazopanib, thus improving the clinical efficiency of the drug. In this review, we summarize the preclinical and early clinical development of pazopanib, explore the evidence for efficacy in STS and outline reported data resulting from efforts to identify biomarkers for pazopanib response.Pazopanib is an oral multitarget tyrosine kinase inhibitor (TKI) with a clinical antitumor effect that is thought to be exerted through its selective inhibition of vascular endothelial growth factor receptor (VEGFR)-mediated angiogenesis, as well as its direct blockade of growth-promoting receptor tyrosine kinases (RTKs), including platelet-derived growth factor receptors (PDGFRs), fibroblast growth factor receptors (FGFRs), and KIT10. After identification of an initial lead compound as an inhibitor of VEGFR2 by using a direct kinase activity assay, chemical optimization was undertaken to increase the inhibitory potency and improve the pharmacokinetic (PK) profile in mouse models. Optimized molecules were also shown to have inhibitory potency against VEGFR1 and VEGFR3, as well as other closely-related RTKs, including PDGFRB, KIT, FGFR1, and colony-stimulating factor 1 receptor (CSF1R). This study and further reports on the kinase inhibitory profile of pazopanib are summarized in Fig. 13. Pharmacodynamic (PD) studies of the biological activity of the drug were performed in a range of in vitro and in vivo assays3. Treatment of cultured human umbilical vein endothelial cells (HUVECs) with pazopanib resulted in potent inhibition of VEGF-mediated phosphorylation of VEGFR2, with associated inhibition of HUVEC proliferation. In vitro inhibition of ligand-mediated phosphorylation of KIT and PDGFRB by pazopanib was also shown in human lung cancer and foreskin fibroblast cells, respectively, although the drug had no effect on proliferation in an unspecified panel of tumor cells. Inhibition of angiogenesis was demonstrated in a mouse model of ocular angiogenesis and subcutaneous implantation of a ligand-containing Matrigel plug. Administration of oral pazopanib to immunocompromised mice was associated with dose-dependent inhibition of growth of established xenografts of human colorectal, melanoma, prostate, breast, kidney, and breast cancer cell lines. A steady-state plasma concentration was shown to provide optimal in vivo inhibition of VEGFR2 phosphorylation, angiogenesis, and xenograft growth, with a strong correlation between inhibition of VEGFR2 kinase activity and antitumor effect in xenografts.Pazopanib was identified through the chemical screening of compounds for inhibition of VEGFR2, a key mediator of tumor angiogenesis5. This effect on myeloma cells correlated with downregulation of several cancer-related genes involved in pathways including cytokine and chemokine signaling, cell cycle, and insulin receptor pathways, as well as upregulation of proapoptotic genes. Pazopanib has been shown across a series of studies to act as a pan-RAF inhibitor and to exert an anticancer effect through inhibition of MAPK pathway signaling in cancer cells in the absence of a demonstrable antiangiogenic effect11. In in vivo and xenograft models of a brain-tropic Her2-positive, BRAF-mutated breast cancer cell line, pazopanib prevented the growth of brain metastasis in association with reduced MAPK pathway activation but no change in markers of angiogenesis. A later study in the same breast cancer xenograft model showed that the inhibition of brain metastasis growth by pazopanib was accompanied by a reduction in the number of PDGFRB-expressing, metastasis-associated astrocytes, suggesting a possible role for pazopanib-mediated therapeutic modulation of the tumor microenvironment (TME)12. In follow-on studies, a panel of breast cancer and melanoma cell lines with varying BRAF mutational status were used in orthotopic xenograft models that were then treated with pazopanib13. Here, xenografts with either wildtype or exon-11-mutated BRAF showed significant sensitivity to pazopanib and a corresponding reduction of MAPK pathway activation in tumor cells and reduced angiogenesis.Further preclinical evidence has indicated that the antitumor effect of pazopanib may not solely be mediated by inhibition of angiogenesis but may also be mediated through a direct effect on tumor cells. A study investigating the effect of pazopanib in human multiple myeloma models found that, in addition to the inhibition of endothelial cell proliferation and in vivo tumor angiogenesis, the drug also had direct antiproliferative and proapoptotic effects on tumor cells and xenograftsCollectively, these preclinical data demonstrate that pazopanib is a potent inhibitor of several key kinases involved in angiogenic and oncogenic pathways, with an antitumor effect that is mediated by both antiangiogenic and direct anticancer cell activity.14. PK assessment identified that steady-state exposure was achieved at doses of 800 mg or more as a once daily oral dose. In line with toxicities observed with other antiangiogenic TKIs, hypertension was the most common adverse event (grade 3 in 25%), followed by diarrhea, hair depigmentation, nausea, anorexia, and fatigue. Proteinuria was the most common laboratory abnormality (any grade seen in 52% of patients), followed by a range of cytopenias and blood biochemistry disturbances, which were grade 1 and 2 in the large majority of affected patients. As no maximally tolerated dose was identified, an oral dose of 800 mg once daily was selected for further studies because doses > 800 mg did not increase drug exposure.Based on these preclinical findings of antitumour effects and proposed optimal dosing, a phase I trial of pazopanib was performed, with 43 patients enrolled in an initial dose-escalation phase and a further 20 patients in a subsequent dose-expansion phasePD analyses in this phase I study demonstrated that plasma VEGF concentrations increased by more than three-fold in ~50% of treated patients following drug initiation. In a subset of patients who underwent dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), 7/12 (58%) patients were seen to have a >50% reduction in tumor blood flow at Day 8 of treatment, and 10/11 (91%) at Day 22. The incidence of hypertension was associated with higher trough drug levels on Day 22 of therapy, suggesting that hypertension may act as a PD marker of pazopanib activity.15. All patients who underwent DCE-MRI evaluation of tumor vascular dynamics demonstrated decreases in tumor blood flow and permeability, while two objective partial responses ) and stable disease of >6 months in eight patients (seven with sarcomas) were observed.Assessment of preliminary clinical activity in this study recorded a partial response by RECIST criteria in three patients (two with mRCC and one with pancreatic adenocarcinoma), while stable disease of at least 6 months duration was observed in 14 patients—of note, among these were two patients with chondrosarcoma, one with leiomyosarcoma (LMS), and one with a gastrointestinal stromal tumor (GIST). A further phase I trial to assess PK and PD in 53 patients aged 2–22 years was also undertaken and demonstrated a similar toxicity profile to that seen in adult patients, with one patient with occult brain metastasis experiencing intracranial bleeding17. These studies established pazopanib as a standard of care in mRCC while also providing further data on drug toxicity, confirming severe hypertension as a commonly encountered adverse effect, as well as significant neutropenia and/or liver enzyme derangement in ~10% of patients.Based on these phase I data, pazopanib was deemed to be a safe and generally well-tolerated drug with an optimal oral dose of 800 mg once daily. Early evidence of clinical efficacy prompted further development in mRCC, a cancer with a well-described central role of angiogenesis in tumor development. Subsequent randomized phase III trials in mRCC demonstrated superior PFS with pazopanib vs. placebo in pretreated patients and noninferior disease control and survival. In addition, pazopanib showed favorable quality-of-life outcomes compared to sunitinib, another antiangiogenic TKI already approved for first line treatmentAs a result of the evidence of durable disease stabilization seen in 4/9 patients with sarcomas treated with pazopanib in the initial phase I trial, further development of the drug in these diseases was pursued.18. Although this study was nonrandomized, patients were prospectively stratified into one of four histologically defined strata: LMS, liposarcoma (LPS), synovial sarcoma (SS) and a heterogeneous ‘other’ stratum. The primary efficacy endpoint was the progression-free rate at 12 weeks of treatment (12wPFR), with predefined rates of 40% and 20% determined as thresholds reflecting drug activity or inactivity19. For each stratum, an initial enrollment of 17 evaluable patients was to be treated at 800 mg once daily until disease progression, unacceptable toxicity, or withdrawal of consent. If 4/17 patients experienced nonprogression at 12 weeks, that stratum was expanded to a total accrual of 37 patients, and if 11 or more out of 37 patients were progression-free, this would indicate that further investigation of pazopanib in that stratum would be warranted.A noncomparative phase II trial of pazopanib was undertaken by the European Organization for Research and Treatment of Cancer (EORTC) Soft Tissue and Bone Sarcoma Group (STBSG) in patients with advanced intermediate or high-grade STS with confirmed disease progression, who were either ineligible for cytotoxic chemotherapy or who had received fewer than three prior cytotoxic agents for advanced diseaseA total of 142 patients were treated within the study. In the LPS arm, only 3 of the first 17 patients showed nonprogression at 12 weeks, and thus, recruitment to this cohort was terminated due to lack of activity. The three other arms continued to complete the second stage of recruitment. Included among the 44 patients recruited into the ‘other subtypes’ stratum were small numbers of malignant peripheral nerve sheath tumors (MPNSTs), vascular sarcomas, fibrohistiocytic or fibroblastic sarcomas, solitary fibrous tumors, rhabdomyosarcoma and GIST, as well as 19 tumors of unspecified differentiation. Four out of 142 patients were excluded from the efficacy analysis due to lack of measurable disease, change of diagnosis to GIST on central histopathology review or lack of objective disease progression prior to commencement of pazopanib. A further two patients were not evaluable due to resection of the target lesion or withdrawal due to coronary heart disease, resulting in a total of 136 patients evaluable for efficacy.19.Although accrual of the LPS arm was stopped after the first stage, two patients who had initially been categorized into one of the other three strata were found on central histopathology review to have LPS. As both of these patients met the 12-week progression-free endpoint, the overall 12wPFR in the LPS arm was 26% (5/19 patients). In the LMS, SS and ‘other’ strata, 12wPFR was 44% (18/41 patients), 49% (18/37), and 39% (16/41), respectively. An objective partial response was seen in 9/136 patients (6.6%): 5 SS, 3 ‘other’, and 1 LMS. Of these patients, 4  experienced disease progression between 253 and 503 days after treatment initiation, while the other 5  were still progression free at 415–812 days. The median PFS was 80, 91, 161, and 91 days, and the median OS was 197, 354, 310, and 299 days in the LPS, LMS, SS, and ‘other’ strata, respectively. These PFS and OS rates compared favorably to the historic controls for LMS, SS, and other subtype cohorts6. In this international, multicenter trial, adult patients with progressing advanced STS were randomized on a 2:1 basis to receive pazopanib 800 mg once daily or placebo until disease progression, unacceptable toxicity or patient withdrawal. On-trial crossover to pazopanib was not permitted for patients who progressed on placebo. Eligibility criteria stipulated that patients must have received between 1 and 4 lines of previous systemic therapy, including an anthracycline, for advanced disease. Most common histological subtypes of STS were eligible for enrollment—notably, based on the earlier phase II evidence of limited activity, LPS was not included in this phase III trial. PFS was the primary endpoint, with a sample size designed to provide 95% power to detect a 15% difference in the progression-free rate at 6 months, corresponding to a hazard ratio of 0.63. This sample size would also provide 90% power to detect HR 0.67 for OS, which was included in secondary endpoints along with toxicity and quality-of-life measures.Building on the results of the EORTC phase II study, the STBSG, in collaboration with GlaxoSmithKline (the manufacturer of pazopanib), undertook the Pazopanib explored in SofT-Tissue Sarcoma (PALLETTE) study, a double-blinded, placebo-controlled phase III RCTp < 0.0001). There was no significant difference in OS . Posttrial systemic treatment was received by 49% and 63% of patients in the pazopanib and placebo arms, respectively. Respective best objective responses as determined by an external review in the pazopanib and placebo arms were partial response in 6% and 0%, stable disease in 67% and 38%, and disease progression/death in 24% and 62% of patients, respectively. Safety and toxicity data were broadly in keeping with earlier pazopanib studies, with fatigue, hypertension, diarrhea, and anorexia among infrequently experienced grade 3–4 toxicities. A small excess in decreased left ventricular ejection fraction, thromboembolic events, and pneumothorax was observed in the pazopanib arm. One of eight on-treatment deaths in the pazopanib arm—a patient who died of multiorgan failure—was possibly related to the study drug. Subsequently, an exploratory health-related quality-of-life analysis reported that the scores for general health status did not significantly differ between pazopanib and placebo-treated patients, while specific toxicity-related measures related to diarrhea, anorexia, nausea, fatigue, and role functioning favored the placebo20.Between October 2008 and February 2010, 369 patients were randomized, with 246 allocated to pazopanib and 123 allocated to placebo. In the analysis of the intention-to-treat cohort, after a median follow-up of 25 months, a clinically significant 3-month improvement in median PFS was seen with pazopanib  demonstrated similar levels of PFS and OS benefit to the overall study population21. There was no overall difference in the type or severity of pazopanib-related toxicity within the Japanese subcohort and overall study population, although a higher rate of dose reduction and lower average daily dose was experienced by Japanese patients. The lower pazopanib exposure but equivalent efficacy seen in the Japanese cohort potentially suggests contrasting PK profiles between different ethnic groups. However, given that pazopanib dose reduction within these trials was at investigator discretion, it is possible that the more frequent dose reductions reflect regional variations in practice. A similar comparison of TKI safety in patients with mRCC treated in an RCT with pazopanib or sunitinib reported similar levels of drug exposure but a distinct pattern and severity of adverse events in Asian versus non-Asian subgroups22. Meanwhile, a phase I study of pazopanib in a Japanese population reported a similar PK profile to the initial phase I23. Although these data do not provide a consistent picture of ethnic and geographical variation in the PK and PD profile of pazopanib, recognized differences in the metabolism of many cancer and noncancer drugs between different ethnic groups indicate the potential for ethnic background to contribute to interindividual variation in pazopanib exposure and toxicity25.A subsequent post hoc analysis of the Japanese subcohort from the PALETTE study 26. Compared to patients with uterine sarcomas who received placebo within the PALETTE study, patients randomized to pazopanib had a significantly longer median PFS  and median OS .A further post hoc subgroup analysis of data from the PALETTE trial, in combination with data from the preceding phase II, focused on patients with uterine sarcomas treated with pazopanib  were consistent with efficacy data for other STS subtypes from the PALETTE study. However, particularly given that the EORTC pazopanib phase II study did not distinguish between the clinical and biological diversity encompassed within the principle LPS subtypes29, the precise role of pazopanib and other antiangiogenic agents in LPSs remains to be defined.Several other prospective and retrospective series provide additional evidence for the efficacy of pazopanib, both in unselected cohorts of mixed histological subtype, as well as diagnosis-specific series with a variable focus on epithelioid sarcoma, SFT, DSRCT, chondrosarcomas, and vascular sarcomas. In addition, data have been reported for 211 patients of mixed STS subtypes treated with pazopanib in an international expanded access program that was conducted following the PALETTE trial33. While several of these combinations have been associated with unacceptable toxicity and/or insufficient activity in phase I studies, others have progressed to examining efficacy in STS-enriched cohorts. Due to the lack of convincing evidence of incremental activity of such combinations, further detail of these studies is not explored in this review.In an attempt to improve the generally modest activity of pazopanib in unselected advanced STS, a number of reported and ongoing trials have investigated the combination of the drug with various cytotoxic chemotherapy regimens or targeted agents, such as histone deacetylase inhibitors36. The absence of a demonstrated OS benefit in an unselected STS population has adversely affected cost-effectiveness appraisals of pazopanib, resulting in limited access to the drug in several health economies9. The ability to prospectively identify patients with advanced STS who are most likely to benefit from pazopanib would aid clinical decision making, increase the clinical and cost-effectiveness of the drug and improve patient experience and survival outcomes. However, as summarized below and in Table The effect of pazopanib in STS is evidenced by the improvement in PFS without associated deterioration in quality of life compared to placebo in a randomized phase III study, as well as reports of postapproval experience in ‘real world’ settings. However, the benefit of pazopanib treatment to individual patients is highly variable and, in the context of infrequent objective tumor responses, often difficult to confirm. Furthermore, despite the RCT evidence of an antitumor effect in an otherwise poor-prognosis patient group with limited treatment options, no significant difference in OS was seen between pazopanib-treated and placebo-treated patients. It has not been established why a significant PFS gain did not translate into an OS benefit; proposed explanations have included a lack of statistical power, the confounding effect of subsequent posttrial treatments or a potential ‘rebound’ effect, wherein initial treatment with pazopanib may select for or induce a more aggressive subsequent phenotype or, alternatively, where accelerated progression is encountered after the cessation of treatment18. Within the PALETTE study cohort, multivariate analysis identified good performance status and lower histological grade as factors associated with improved outcome, both of which are well-established prognostic factors in STS regardless of pazopanib exposure6. Predictive analysis did not detect a significant interaction between histological subtype and pazopanib benefit, with improved PFS with pazopanib vs. placebo seen in all three histological subgroups . Other baseline characteristics, including gender, ethnicity, and extent of prior treatment, were not found to be predictive for OS37.Preplanned and post hoc analyses of the EORTC phase II and III trials of pazopanib have been performed in an attempt to identify patient or tumor characteristics that enrich for drug benefit. As discussed above, LPS was identified as a potentially insensitive histological subtype within the phase II study, but subsequent analysis and other studies call into question whether there are subsets of patients with LPS who may respond to treatmentn = 344) were included in a retrospective analysis that sought to identify baseline factors associated with a good outcome following treatment38. Here, the authors defined PFS ≥ 6 months and OS ≥ 18 months as long-term response and survival, respectively, with 36% of patients showing long-term response, 34% showing long-term survival, and 22% showing both long-term response and survival. Descriptive and multivariate analyses again identified tumor grade and performance status as having prognostic relevance for PFS and OS, as was the case with baseline hemoglobin levels. There was no preponderance for any histological subgroup in the long-term responder or survivor patient groups. These findings, consistent with results from other case series summarized in Table Patients who received pazopanib within the EORTC phase II and phase III trials and who met PALETTE-eligibility criteria  -based criteria that account for a reduction in tumor density that reflects tumor cell death have been developed by Choi et al.39. These criteria have been demonstrated to be more sensitive for TKI benefit than RECIST criteria in GIST and have shown promise for other STS subtypes in terms of specificity in predicting pathological response and favorable outcome in patients treated with cytotoxic chemotherapy42. Durable disease control with pazopanib has been reported in patients with SFT who had SD as the best response by RECIST but PR by Choi criteria44. Following these observations, a prospective, single arm phase II trial of pazopanib in advanced SFT performed at centers in Italy, France, and Spain assessed the overall response by Choi criteria as its primary endpoint45. Objective responses by Choi criteria were seen in 18/35 (51%) of treated patients, compared to 2/35 (6%) by RECIST. While Choi criteria and RECIST-based nonprogressing patients exhibited similarly favorable OS, Choi criteria were better than RECIST for identifying patients with progressive disease (PrD) and poor OS , and the median OS in patients identified as PrD by RECIST but PR by Choi was 24 months. These data suggest that Choi criteria may better discriminate between patients with or without beneficial responses to pazopanib in the treatment of SFT. Further information on the utility of Choi criteria, as well as functional MRI and fluorodeoxyglucose positron emission tomography (FDG-PET), in predicting pazopanib benefit will be provided by a now-completed window-of-opportunity study of preoperative treatment in localized STS (NCT01543802)46. The potential utility of FDG-PET-CT in this scenario is also informed by small studies that have demonstrated an association with either a decreased maximum standardized uptake value (SUVmax) during therapy and durable disease control or the absence of a SUVmax response and lack of clinical benefit48. Given that Choi criteria, PET-CT, and functional MRI lack validation in this setting and face issues of interobserver reproducibility, further development of these imaging approaches as a tool for accurate early detection of pazopanib benefit is required. However, these modalities remain promising avenues for distinguishing between responding patients who are likely to benefit from ongoing therapy and those without a response who would be better served by a change of therapy.Data from the PALETTE study indicate that the PFS gain seen with pazopanib is associated with a minor increase in the rate of radiological objective response compared to placebo (6% vs. 0%) but a much larger increase in stable disease rates (67% vs. 38%)50, giving rise to the possibility that variability in drug exposure levels may, at least in part, explain the variability in outcome in pazopanib treatment of advanced STS. A prospective cohort study of patients with mRCC or advanced STS/GIST treated with TKIs including pazopanib demonstrated that dose optimization in response to suboptimal trough drug levels was successful in attaining subsequent adequate trough levels, indicating that dose monitoring and adjustment could represent a path to improved clinical effectiveness of these drugs51.Numerous PK factors can influence systemic drug exposure, including oral absorption and metabolism, drug–drug interactions, food–drug interactions and patient characteristics such as age, gender, and bodyweight, which may be related to variations in the volume of distribution. As such, high interpatient variability of plasma exposure to oral anticancer drugs, including pazopanib, may affect drug efficacy and treatment outcome. Retrospective analyses of clinical trial data on the use of pazopanib in the treatment of mRCC and advanced GIST have reported associations between higher trough levels of plasma pazopanib concentration and longer PFS53. A later analysis of other on-target toxicities within the same cohort found no association between the incidence of drug-induced proteinuria, hypothyroidism, or cardiotoxicity with survival54. These findings are consistent with data from a single center 26-patient case series that found no significant association between suboptimal trough levels of pazopanib and worse PFS following treatment, indicating that drug exposure alone is unable to account for variation in pazopanib effect55.Hypertension is a frequently occurring on-target side effect of pazopanib and correlates with the PK degree of drug exposure. As such, the use of hypertension as a biomarker for adequate drug dosing and/or treatment effect has been pursued. However, as has been seen with the use of pazopanib in mRCC, the combined analysis of prospective patient data from the EORTC phase II and phase III trials has indicated that the development of hypertension during pazopanib therapy is not associated with improved PFS or OS56. They found that patients who were coadministered GAS for >80% of the duration of their pazopanib therapy had significantly shorter PFS and OS compared to patients who received pazopanib and took either no or less frequent concomitant GAS. These associations were found to be independent of other prognostic factors by multivariate Cox analyses. No association between GAS and survival outcome was found in patients who received placebo, suggesting a causative interaction between GAS use and reduced benefit from pazopanib. However, such conclusions should be tempered by the limitations of the post hoc, retrospective design of this study, wherein correlative PK data were not available, no formal testing for an interaction between GAS exposure and pazopanib benefit compared to placebo was performed, and no association between GAS use and on-target pazopanib toxic effects was observed.In light of the established PK effect of gastric acid suppression (GAS) on many anticancer TKIs, including pazopanib, Mir et al. recently reported a further analysis of the EORTC trial cohorts that investigated a possible association between the concomitant use of proton pump inhibitors or histamine H2-receptor antagonists and pazopanib efficacy57. However, the retrospective nature of many of these studies and the lack of interaction between biomarker and treatment effects in prospective studies have limited the extent to which such investigations inform the search for predictive, rather than prognostic, biomarkers for pazopanib. An exception to this situation was seen in the analysis of prospectively collected pretreatment blood samples taken from patients with mRCC treated in either a noncomparative phase II or randomized phase III study of pazopanib58. After initially screening for the association of 17 CAFS with outcome in the phase II cohort, seven candidates were then taken forward for validation in the phase III cohort. Among these, interleukin-8 (IL-8) and osteopontin were validated as negative prognostic, but not predictive, biomarkers for PFS. However, the effect of interleukin-6 (IL-6) levels on post pazopanib outcome was shown to significantly interact with pazopanib exposure, indicating that while patients with an increased baseline IL-6 had a worse prognosis than those with low IL-6, the former was the group in which pazopanib treatment delivered the most benefit. Despite this high-level evidence for baseline circulating IL-6 levels for pazopanib in mRCC, this biomarker has not impacted routine practice so far, for reasons that include the limited analytical replicability of IL-6 assays and the lack of validation in an independent prospective cohort. Other studies have investigated tumor-based pazopanib biomarkers, including retrospective assessment of gene expression-based mRCC molecular subgroups, hypoxia-inducible factor (HIF) levels or von Hippel–Lindau tumor suppressor gene (VHL) mutational status in tissue series. These studies have indicated prognostic associations of the investigated markers, but due to their retrospective design and lack of validation, their predictive utility remain unproven60.Due to the inhibitory activity of pazopanib against a number of molecular mediators of tumor angiogenesis and the relative ease of using multiplexed antibody-based assays to assess for protein levels in blood, biomarker research has so far largely focused on investigating circulating angiogenic factors (CAFs). This is especially true in mRCC, a disease where dysregulated angiogenic pathways are known to contribute significantly to tumor development, and for which pazopanib and other TKIs that target angiogenic mediators are established standards of care. These studies have identified a varying repertoire of CAFs or cytokines whose baseline level or change in response to therapy have been associated with differential outcomes following TKI therapy61. These findings are consistent with the role of pazopanib as an antiangiogenic agent and an association of various CAFs with poor post pazopanib survival, but the impact of the study is limited by high reported false discovery rates combined with lack of validation in independent cohorts.Several studies have reported baseline biomarkers with putative predictive associations with pazopanib efficacy in advanced STS patients. Sleijfer et al. examined the serum levels of cytokine and angiogenic factors in a cohort of 85 patients from the EORTC phase II trial and demonstrated that increased baseline plasma levels of proangiogenic hepatocyte growth factor (HGF) and basic nerve growth factor (bNGF) were associated with worse PFS in pazopanib-treated patients62. Blood samples collected at pretreatment baseline and after 50 days of therapy were used to assess the association between NLR and pazopanib outcome in 333 patients treated with pazopanib within the EORTC phase II and III studies63. While elevated NLR at baseline was a poor prognostic marker regardless of treatment with pazopanib or placebo, no observed pattern of change in NLR between baseline and Day 50  was seen to have any association with PFS or OS. In contrast, in a smaller study of 25 patients treated with pazopanib in several Japanese centers, a decrease in NLR from baseline to Week 4 of therapy showed a highly significant association with improved PFS, while baseline NLR had no prognostic association64. This study used a higher cut-off to define high and low NLR patients  and did not provide a definition as to what constituted a significant change in NLR during therapy, potentially contributing to the discrepancy with the EORTC cohort analysis. Regardless, beyond the consistently observed prognostic association of NLR in STS and other cancers, its role as a potential predictive biomarker for pazopanib appears to have little promise.The ratio of neutrophils to lymphocytes (NLR) in the circulation of patients can serve as an easy-to-measure marker of systemic inflammatory state in cancer patients. High NLR has been shown to be a negative prognostic marker in multiple solid tumor types65. TP53 and RB1 were the only two genes found to be altered in >20% , with all detected mutations of TP53 predicted to confer loss-of-function . While RB1 mutational status had no association with post-pazopanib outcome, patients with TP53-mutated tumors were shown to have significantly longer PFS than those with TP53 wild-type tumors. These data represent the only currently reported tumor-based candidate biomarker for pazopanib in advanced STS and have yet to be validated. The biological basis of any association between TP53 function and pazopanib response remains to be determined.In a study by Koehler et al. next-generation sequencing (NGS) was used to sequence 405 cancer-related genes in pretreatment tumor samples from a retrospective cohort of 19 patients with advanced STS treated with antiangiogenic agents (18 pazopanib and 1 sunitinib)66. These cell lines were shown to express phosphorylated PDGFRA and FGFR1, both kinase targets of pazopanib. Dual pharmacological inhibition or genetic silencing using RNA interference (RNAi) of these targets led to a synergistic increase in tumor cell apoptosis. Acquired pazopanib resistance was then derived in these cells through culture in the presence of an escalating drug dose. Comparison of molecular profiles between parental and resistant cell lines identified significant downregulation of PDGFRA but maintenance of FGFR1 expression and activation. These cells were no longer dependent upon PDGFRA signaling but remained sensitive to FGFR1 inhibition, indicating that loss of PDGFRA could serve as a marker of acquired pazopanib resistance that could potentially be therapeutically targeted with inhibitors of FGFR1. In a follow-up study, further comparative mass spectrometry-based phosphoproteomic characterization of these paired pazopanib-sensitive and pazopanib-resistant cells identified increased activity in cytoskeletal regulatory pathways and downregulation of histone deacetylase activity in pazopanib-resistant cells67. Elsewhere, immunoblot and antibody array-based comparative proteomic profiling was performed on four different synovial cell lines, three of which were sensitive to pazopanib, with the other showing primary pazopanib resistance68. Increased expression of PDGFRB and phospho-activation of tyrosine kinases, including FGFR3 and VEGFR1, were shown to be unique to the resistant cell line, while RNAi-mediated silencing of PDGFRB, MET, and protein tyrosine kinase 2 beta (PTK2B) reduced viability of the pazopanib-resistant cells. Collectively, these studies indicate that the relative pazopanib sensitivity of sarcoma cells is reflected and possibly determined by differential expression and phosphorylation of RTKs and downstream pathway signaling proteins. While translational correlates of these preclinical findings are lacking, there is an indication that investigating the expression levels of molecular targets of pazopanib within tumor tissue may yet identify biomarkers for drug effect.Given that pazopanib selectively inhibits several growth-promoting RTKs, the expression levels of these targets in tumor cells are also attractive candidates for evaluation as predictive biomarkers. While translational studies of tumor-based expression of RTKs in pazopanib-treated STS are lacking, several preclinical studies have assessed the association between RTK expression and pazopanib effect. In one such study, screening of 14 cell lines representing eight different sarcoma subtypes identified that only the two malignant rhabdoid tumor (MRT) cell lines displayed pazopanib sensitivity71. Assessment for differential treatment outcomes between such biologically classified STS subgroups within pazopanib-treated cohorts represents a worthwhile avenue for biomarker research.The successful clinical development of pazopanib as a treatment for advanced STS has addressed the longstanding and ongoing need for effective novel agents for these rare diseases. However, reported clinical trial data indicate that only a minority of patients within the indicated STS population will receive benefit from treatment and that the duration of benefit may in many cases be short. Reported data from subgroup analyses of prospective pazopanib studies have so far failed to identify baseline clinicopathological characteristics that enrich for pazopanib benefit. Moreover, the small number of translational studies that have investigated circulating or tumor-based biomarkers have yet to provide reproducible and validated candidate biomarkers. A growing volume of biomolecular profiling data indicate the existence of biological intrinsic subgroups within individual STS histotypes. In addition, biological traits such as increased chromosomal instability can be demonstrated in a proportion of tumors across multiple STS subtypes and are associated with shared clinical phenotypesThe further development and validation of putative imaging surrogate markers for survival would provide better discriminatory trial endpoints and assist in the early detection of clinical effects. At present, it is unclear by what precise mechanism or mechanisms pazopanib exerts its anticancer effect in STS and whether this effect varies between and within different STS subtypes.Furthermore, even in patients showing an unequivocal initial benefit from treatment, the development of pazopanib resistance is ubiquitous. A greater understanding of the mechanisms of drug effect and primary and secondary drug resistance is required to inform patient selection and the development of novel combinatorial regimens in which pazopanib might be paired with other small molecule inhibitors, cytotoxic drugs, or potential immunotherapeutic approaches. A greater degree of preclinical and translational research is required to reveal the therapeutic and resistance mechanisms of pazopanib in disease-specific contexts.


After hospital-based acute care therapy, with or without percutaneousor surgical intervention, patients are discharged and receive advice for long-termtherapy. However, some patients develop symptoms again in the short-term course and arereadmitted to the hospital due to clinical as well as non-clinical causes and withclinical and other non-clinical consequences.1Acute ischemic syndromes  may occur 2 Additional studiescompared not-for-profit hospitals (15.7%) with proprietary hospitals (16.6%) regardingreadmission rates in the first month of discharge without demonstrating an associationof hospital ownership with the success in programs devoted to decrease readmission rate;the results of such interventions were not statistically different between the two typesof hospitals.3 Socioeconomic and ethnicfactors were systemic influences without overt manifestations in a specifichospital.4 In many instances,the readmission rate was studied as a surrogate marker of quality of care;7the heterogeneity of patients and medical conditions made a clear association with theindex admission elusive and not easily predicted.5Hospital readmissions deserve studies in different settings and have been reported inmedical literature. In one report, the readmission rate after one year in patients olderthan 65 years surviving myocardial infarction was 49.9% in 4.767 hospitals in UnitedStates of America between 2008 and 2010; the readmission rate, as well as mortality,were higher in the first months after discharge and decreased thereafter over 12months.8 of a hospital-based sample of 536eligible patients with acute coronary syndrome admitted to three private hospitals and onepublic hospital to scrutinize variables associated with re-admissions. They observed the readmissionrate was high  and the mortality of the readmitted patients was alsohigh - 7%. Acute coronary syndromes and heart failure were the leading causes ofhospital readmission, mainly in patients admitted to private hospitalfacilities,8 probably relatedto characteristics of the access. The authors recognize that a hospital-based studysample without full follow-up data are limitations that may stimulate following theprevious suggestion to remain vigilant2for health deterioration after discharge from in-hospital treatment, emphasizing theimportance of continuous medical advice, support, and therapy. A previous study with asmall sample  in the same region of the city of the current studysuggested low adherence of patients to medical therapy as a significant influence on thefrequency of readmissions and mortality.9In this issue, colleagues from Aracaju  report the studyFinally, further studies and interventions are to be stimulated in other cities anddifferent clinical settings, either hospital-based or at community medical facilities,taking into consideration local challenges and opportunities to make therapy moresuccessful by preventing clinical deterioration that need hospital re-admissions after aperiod of in-hospital treatment of acute ischemic syndromes.


Precision oncology aims to offer the most appropriate treatments to cancer patients mainly based on their individual genetic information. Genomics has provided numerous valuable data on driver mutations and risk loci; however, it remains a formidable challenge to transform these data into therapeutic agents. Transcriptomics describes the multifarious expression patterns of both mRNAs and non-coding RNAs (ncRNAs), which facilitates the deciphering of genomic codes. In this review, we take breast cancer as an example to demonstrate the applications of these rich RNA resources in precision medicine exploration. These include the use of mRNA profiles in triple-negative breast cancer (TNBC) subtyping to inform corresponding candidate targeted therapies; current advancements and achievements of high-throughput RNA interference (RNAi) screening technologies in breast cancer; and microRNAs as functional signatures for defining cell identities and regulating the biological activities of breast cancer cells. We summarize the benefits of transcriptomic analyses in breast cancer management and propose that unscrambling the core signaling networks of cancer may be an important task of multiple-omic data integration for precision oncology. First, DNA variations may not reveal the actual activities of the corresponding biological pathways. In some cases, the master signaling pathways may be deregulated without any observed genomic alterations, and such cases are probably ignored in genomic analyses. Furthermore, even though the genetic variants have been discovered in genomics, it is often difficult to discriminate between “passenger” and “driver” mutations. Functional genomics needs to be replenished by large-scale data derived from other omic platforms. Transcriptomics is the most frequently used method for unscrambling genomic information e.g., microRNAs (miRNAs), can be enriched for better coverage Transcriptomic analyses have been frequently utilized for exploring prospective biomarkers and potential therapeutic targets for human cancers TNBC is exceedingly heterogeneous, poorly differentiated and highly metastatic e.g., bicalutamide, an AR antagonist) are undergoing clinical trials Using massively parallel mRNA sequencing, numerous transcripts that are differentially expressed between TNBC and non-TNBC have been identified BRCA-mutated breast cancer Genomic analyses have uncovered a rapidly growing number of genetic variants that may participate in cancer initiation and progression. However, two intractable challenges remain. On one hand, genomic analyses fail to distinguish between the “driver” mutations that are critical for pathogenesis and the “passenger” incidents that occur coincidentally. On the other hand, hundreds of unanticipated synthetic lethal (SL) interactions are hidden in cancerous abnormalities. SL interactions refer to gene pair relationships in which the separate inactivation of either gene does not affect the viability of cancer cells, but joint inactivation is lethal i.e., small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs). siRNAs are applied to achieve transient and short-term gene silencing, whereas vector-based shRNAs enable stable and long-term gene silencing. Both siRNAs and shRNAs can be used in array-based screening or in pooled formats. To circumvent the aforementioned limitations of genomic studies, loss-of-function RNAi screening technology has been widely adopted to define the functional genes that are necessary for cancer cells and to disclose SL relationships in exploring novel therapeutic options for cancer treatment PPMID and SP1 significantly reduces the viability of two TNBC cell lines and increases their sensitivity to paclitaxel. When combined with paclitaxel, both CCT007093 and mithramycin, the respective chemical inhibitors of protein phosphatase Mg2+/Mn2+ dependent 1D (PPMID) and specificity protein 1 (SP1), suppress the growth of the paclitaxel-resistant TNBC cells HER2 based on previous meta-analyses and discovered that both NR1D1 and PBP are novel survival factors essential for HER2+ breast cancer cells + breast cancer. Subsequently, Marotta et al. further expand the number of breast cancer candidate genes and find that the IL-6/JAK2/Stat3 axis is significantly activated in CD44+CD24− breast cancer cells RAD21, EIF3H, CHRAC1, TANC2, and GNASIn RNAi screening, a small-scale array can be used to selectively suppress the up-regulated genes that have been detected in previous transcriptomic analyses or the genes that are differentially expressed among different cancer subtypes PTEN and TTK genes ERBB3 and CCND1 are frequently amplified in breast cancer, whereas some skeletal system morphogenesis-related genes, such as PDGFRA, ACVR2B, TGFBR2, DLG1, FGFR1, and FGFR2, are highly-expressed in osteosarcoma BRD4 is a putative targeted option for luminal breast cancer and PIK3CA mutations probably determine the resistance to bromodomain and extra-terminal domain (BET)-inhibitors The development of large-scale RNAi libraries has enabled non-biased genome-wide loss-of-function screening The majority of human genome, approximately 98%, is transcribed into ncRNAs The expression of some miRNAs is up-regulated in TNBC and these miRNAs may function as tumor promoters to increase the proliferation and/or invasion of TNBC cells. This type of miRNAs is thus termed as oncomiRs, which include miR-146a/146b According to the critical roles of miRNAs in cell function and fate determination, a “Helm” model has been proposed to describe miRNAs as functional signatures for precisely characterizing cell identities in temporal-spatial specific status that primarily depends on the abundances of different miRNAs and the balance between these miRNAs and their corresponding targets Transcriptomic analyses have provided massive amounts of information on the gene expression patterns in breast cancer. For clinical applications, the mRNA expression profiles can be employed to classify TNBC into unique molecular subtypes and to propose reliable therapeutic targets. Loss-of-function RNAi screening can be performed to discover the driver mutations and the SL partners of these inactivated genes for exploring novel targeted options. Moreover, an increasing number of miRNA are detected to be differentially expressed in breast cancer, many of which play critical roles as tumor promoters (oncomiRs) or tumor suppressors (anti-oncomiRs). The applications of transcriptomics in breast cancer are summarized in With the development of high-throughput sequencing technologies and computational analysis tools, it has become much easier to obtain and decipher enormous datasets that are relevant to different biological layers of human cancers besides genomic and transcriptomic studies. Epigenomic studies reveal the architecture of epigenetic alterations in human genes, including DNA methylation and chromatin modifications e.g., the capabilities of proliferation, metastasis, immune evasion, and energy metabolism) in different malignant cells.The generation of multi-omic data has become an addictive routine for cancer studies. However, an intractable puzzle arises, that is, it is becoming increasingly intricate to assimilate the rapidly growing number of “big data”, as mentioned by Dr. Weinberg We suppose that the core signaling networks that are derived from hundreds of unique cancer-related signaling transduction pathways may be the appropriate candidates to unravel Weinberg’s puzzle. These networks reflect the cell identities, including the varieties of biological capabilities and the temporal–spatial status in cell differentiation, and may be used as functional classification criteria for the characterization of different subtypes of human cancers. Accordingly, pathway-targeted drugs and therapeutic strategies may be precisely designed to redress the deregulated networks. The integration of multi-omic data into the core signaling transduction channels that determine the development of cancers may be a potential direction for us to go out from Weinberg’s puzzle of the biological “big data” and helps to provide accurate guidelines for diagnosis and management for cancer patients .Breast cancer is well-classified using both cellular and molecular features. Titanic efforts have been made to acquire a great deal of multi-omic data that display diverse biological signatures of the development of breast cancer, particularly in TNBC, which has not been well-characterized and for which no targeted drugs are yet available. It is high time for us to take advantage of these rich resources to identify the core signaling networks in TNBC and we are filled with hope that this most difficult-to-treat cancer will be precisely targeted in the near future.The authors declare no competing interests.


Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are common and significant morbidities of prematurely born infants. These diseases have in common altered and pathologic vascular formation in the face of incomplete organ development. Therefore, it is reasonable to question whether factors affecting angiogenesis could have a joint pathogenic role for both diseases. Inhibition or induced expression of a single angiogenic factor is unlikely to be 100% causative or protective of either of BPD or ROP. It is more likely that interactions of multiple factors leading to disordered angiogenesis are present, increasing the likelihood of common pathways in both diseases. This review explores this possibility by assessing the evidence showing involvement of specific angiogenic factors in the vascular development and maldevelopment in each disease. Theoretical interactions of specific factors mutually contributing to BPD and ROP are proposed and, where possible, a timeline of the proposed relationships between BPD and ROP is developed. It is hoped that future research will be inspired by the theories put forth in this review to enhance the understanding of the pathogenesis in both diseases. Premature infants are at risk of developing bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) that despite advances in neonatal care and neonatal research, remain significant causes of morbidity and mortality in preterm infants. BPD is characterized by altered alveolarization and reduced and abnormal alveolar microvascular development and remodeling in the premature lung . ROP is It is likely that cellular changes that cannot be documented using current diagnostic tools take place in the premature lung and retina before the point in time when BPD and ROP can be clinically diagnosed. These early events may provide clues to the existence of molecular pathways common to the pathological course of both BPD and ROP and identify common mechanisms in their initiation and/or progression. This review focuses on mediators of angiogenesis important for lung and eye development and the pathogenesis of BPD and ROP. Most of the work in this field addresses promoters of angiogenesis. However, inhibitors of angiogenesis are also important in development and disease and these are also reviewed. Table BPD and ROP share the major risk factors of perinatal inflammation and exposure to oxidative stress , 2, 4–6.Abnormal retinal vessel development after preterm birth occurs in a biphasic pattern in ROP Figure . The firNumerous studies show that vascular endothelial growth factor (VEGF) dysregulation has a major role in both BPD and ROP pathogenesis , 15–21. This change from decreased to elevated VEGF expression in ROP has also been demonstrated by Bhandari et al. and others in some animal models of oxygen-induced lung injury and in human studies , 24. In Endogenous VEGF antagonists are also coming to attention as potential contributors to the development of BPD. The endogenous VEGF antagonist soluble fms-like tyrosine kinase-1 (sFlt-1) is present in amniotic fluid and is markedly increased with chorioamnionitis and pre-eclampsia, where it mediates maternal pregnancy-related vascular disease. Postulating that sFlt-1 from pregnancy participates in the development of BPD in the premature infant, Wallace et al.  developeGiven the essential need for regulated VEGF expression in both ROP and BPD, investigators have expressed the concern that treatment of ROP with intra-vitreous injection of anti-VEGF therapy may adversely impact lung vascular development via unintended systemic side effects . FurtherT polymorphism had a 71% increase in promoter activity, suggesting that a consequential increase in VEGF activity would be observed  as a risk factor for BPD or ROP. One candidate SNP is the VEGF 460C/T allele on chromosome 6. Haplotypes containing this Cobserved . While nF levels . In anotF levels . The autF levels , 31. AltF levels . These VOne of the effects of altered VEGF levels in BPD and ROP is the impact on activation of the type 2 VEGF receptor  that stimulates endothelial nitric oxide synthase (eNOS) production in endothelial cells . ENOS isin vitro studies evaluating NO treatment, meta-analyses of studies using inhaled NO therapy in preterm neonates for prevention or treatment of BPD show a lack of benefit  via the placenta and amniotic fluid. IGF-1 serum concentration increases significantly throughout the third trimester. Preterm birth then prevents this normal third trimester rise in serum IGF-1, and IGF-1 continues to drop with extra-uterine advancing gestational age , 46 Fig. In contA slower rise in serum IGF-1 levels in the first 4 weeks following premature birth is an independent risk factor for ROP . In phasSerum free IGF-1 levels are impacted by pregnancy associated plasma protein A (PAPP-A), a metalloproteinase that enhances dissociation of IGF-1 from insulin-like growth factor binding proteins 2, 4, and 5  increasing bioavailable IGF-1  Figure . PAPP-A in utero gestational age but can decrease postnatally after preterm birth (Insulin-like growth factor binding protein 3 (IGFBP-3) is a major regulator of free IGF-1 levels. It also has independent IGF-1-like activity. Figure rm birth . IGFBP-3rm birth  linking rm birth . This efrm birth . IGFBP-3rm birth . Adequatrm birth .Premature infants with established ROP have lower serum IGFBP-3 levels than infants with no ROP . IGFBP-3Overall these studies suggest that a systemic increase in IGFBP-3, potentially achieved through improved nutrition, could prevent or slow the progression of BPD by increasing eNOS - mediated NO production in lung microvascular endothelial cells, by downregulating inflammation and reducing IFNγ and activation of IFNγ targets, while promoting IGF-1 beneficial effects Figures , 3. SimiAngiopoietins are critical modulators of physiologic and pathologic neovascularization. Their production is intimately connected with that of VEGF . Both anAng2 counteracts Ang1 by decreasing tyrosine phosphorylation of Tie2. This promotes destabilization of endothelial cell junctions to enable both vascular leakage and sprouting of new vessels , 55 Fig. Ang2, tThe temporal patterns of Ang1 and Ang2 expression levels in BPD and ROP are similar Figure , 55. InfEndothelial cells have the capacity to alter the balance of activity between Ang1 and Ang2. Proteolytic cleavage of the extracellular domain of Tie2 releases soluble Tie2 (sTie2) from the endothelial cell membrane Figure . BindingIt is possible that decreasing sTie2 concentrations by inhibiting the proteolytic cleavage of the extracellular domain would be beneficial in the treatment of BPD and ROP. This would shift the Ang1/Ang2 ratio toward Ang1 activity, driving stabilization of developing vessels while maintaining neovascular formation via Ang2, thereby reducing susceptibility to oxygen effects on vessel development. Finally, higher intra-amniotic levels of Ang2 were found in women presenting with intra-amniotic inflammation, suggesting that decreasing inflammation during pregnancy could also reduce Ang2 activity in the premature infant  Figures, 4.The transforming growth factor beta (TGFβ) family members signal through dimers of TGFβ type I and II receptors, and are vital for regulation of cell growth, differentiation, migration, and extracellular matrix protein production, events that all play important roles in the developing fetus and newborn, including the regulation of vascular development . A large2 0.75). This degree of hyperoxia reduced the levels of retinal TGFβ1 and VEGFR1 by one third. Administration of TGFβ1 following 17 h of hyperoxia exposure protected retinal vascular development. These TGFβ1-treated mice retained more retinal capillaries than controls. TGFβ1 also induced retinal pericyte αSMA expression. Pericytes in the retinal vascular bed express αSMA. Specifically, pericytes of mature retinal vessels all express αSMA, but only a fraction of vascular pericytes express αSMA while neovascularization of the retina is in process. It is important to note that retinal vessels whose pericytes express αSMA are resistant to oxygen-induced vessel loss , likely through its essential role in regulating the formation of retinal photosensitive visual pigment and not from promoting the completion of retinal vasculogenesis , 85. In Thus, like the clinical studies of BPD, currently the outcomes of studies of vitamin A supplementation to reduce ROP are also disappointing. How vitamin A treatment can better ameliorate the development of both BPD and ROP is not yet clear. Questions on dose timing, dose size, delivery method and optimal therapeutic formulation need to be further studied. In addition, complementing vitamin A with other therapies such as rhSOD3 or nitric oxide may enhance both short and long-term clinical outcomes. In support of this concept, a recent retrospective study suggested that vitamin A and nitric oxide synergistically improved pulmonary and neurodevelopmental outcomes for extremely preterm infants (birth weight <749 g) . UnfortuThrombospondin-1 is part of a family of matricellular calcium binding extracellular glycoproteins that regulate cell-cell and cell-matrix interactions and is mainly localized to small and medium sized vascular structures , 87. TheIn studying the effects of thrombospondin-1 in ROP, newborn Sprague Dawley rats were exposed to a 24-h cycle of 50 and 10% oxygen for 2 weeks to reproduce the retinal hyperoxia and hypoxia stages of ROP. When this exposure was followed by injection with thrombospondin-1 immediately or 3 days after transfer to room air, there was a thrombospondin-1 dose-dependent decrease in retinal neovascularization in the rats who were injected immediately after transfer to room air compared to control rats . While vPigment Epithelium-Derived Factor (PEDF) is a neuro-protective growth factor with significant anti-angiogenic properties; thus, its mechanism of action in BPD or ROP is of interest. PEDF is named for the retinal pigment epithelium cell layer where it was originally identified. Subsequent studies showed that PEDF is produced by epithelial cells in numerous organs and directly counteracts VEGF-induced neovascularization . A mousePEDF levels were elevated during the avascular stage of developing ROP in the retinas of neonatal rat pups cycled daily between hyperoxia and hypoxia  Figure . The rat2 exposure. Mice with a genetic depletion of MMP2 expression have reduced retinal neovascularization in a hyperoxia model of ROP, consistent with continued activity of PEDF secondary to a loss of its breakdown  and platelet derived growth factor (PDGF) were not discussed because they likely have regulatory effects on several processes besides angiogenesis. Nevertheless, these also should be considered as potential candidates for mechanistic links between BPD and ROP –103. We This review has explored the possibility of a mechanistic overlap in the development of BPD and ROP through the process of abnormal regulation of angiogenesis. No single angiogenic or angiostatic factor can fully account for the pathogenesis of either disease, yet many factors appear involved in some capacity. Further, current literature does not allow connecting the vascular factors discussed into one road map for the lung and eye alone or together for a map for normal or abnormal vasculogenesis in both organs. However, this review brings together information that may be useful for identifying potential candidates to consider for therapies to simultaneously decrease BPD and ROP development. It also emphasizes the need for further work to more fully elucidate the roles and timing of many of these factors in BPD and ROP. Finally, although not explicitly discussed in this review, the current studies of BPD and ROP in human and animal models are hampered by the lack of a clear consensus regarding the optimal animal model, including the specific approach to oxygen exposure, lack of a model of both diseases simultaneously, as well as the lack of agreement regarding the best clinical definitions of both diseases –121. UnfMV is Co-senior author with HN and MV contributed to the development of the topic for the review, aided, and advised AS in research and critical review of the literature, preparation, and organization of the manuscript. AS researched and provided the significant body of material reviewed and aided in the decision of data and information to be included in the manuscript as well as playing a significant role in preparing and revised the manuscript. CD aided in preparation, writing, and revision of the manuscript and contributed ideas on topics to be included in the review. HN is Co-senior author with MV and significantly contributed to the critical review of the literature to be included in the manuscript, as well as to guidance in preparation and organization of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.