Rare conditions are sometimes ignored in biomedical research because of difficulties in obtaining specimens and limited interest from fund raisers. However, the study of rare diseases such as unusual cancers has again and again led to breakthroughs in our understanding of more common diseases. It is therefore unsurprising that with the development and accessibility of next-generation sequencing, much has been learnt from studying cancers that are rare and in particular those with uniform biological and clinical behavior. Herein, we describe how shotgun sequencing of cancers such as granulosa cell tumor, endometrial stromal sarcoma, epithelioid hemangioendothelioma, ameloblastoma, small-cell carcinoma of the ovary, clear-cell carcinoma of the ovary, nonepithelial ovarian tumors, chondroblastoma, and giant cell tumor of the bone has led to rapidly translatable discoveries in diagnostics and tumor taxonomies, as well as providing insights into cancer biology. The history of biomedical research has repeatedly shown that with the advent of new methodologies, the study of rare, but clinically well-defined disease entities has led to the generation of a disproportionate amount of knowledge. For instance in the eighteenth century, the peculiar scrotal skin cancers of adolescent males with a history of occupation as chimney sweeps led Percivall Pott to describe one of the earliest associations of workplace hazards and cancer . His stuforme fruste tumors. Forme fruste refers to an attenuated manifestation of disease. We recognize that these tumors are true neoplasms and in many cases, cancers. However, they do not have the genomic baggage and heterogeneity due to genomic instability as seen in common cancers and it is this attenuated genomic phenotype that makes them such tractable and useful targets for genomic research. In this review, we will go over recent sequencing studies of some forme fruste tumors which led to discoveries of profound importance.It has long been clear that mutations play a critical role in the development of cancer. However, many common cancers are both biologically and clinically complicated and their mutational landscape reflects such complexity , 6. On tE. coli or by an incredibly large number of separate PCR reactions. However, with massively parallel sequencing platforms, the first shortcoming was overcome by reversible fluorescent nucleotide addition and imaging (used in Illumina platforms) or through monitoring nucleotide addition via ion detection (used in Ion Torrent platforms (Life Technologies)) both achieved by cyclic manipulation of polymerase or ligase enzymes [Ever since the discovery of DNA and its association with human cancer, scientists and clinicians have dreamt of the possibility to scrutinize it base by base. The ability to sequence DNA, which quickly developed into a robust method by Sanger sequencing , was a s enzymes . Moreove enzymes   as well as the WTR1-CAMTA1 fusion in epithelioid hemangioendotheliomas (EHEs).Pathognomic mutations allow a more specific definition of a previously established histological diagnosis. Below, we will discuss the cases of the forme fruste cancer: cytogentics had shown a more stable genome compared to other ovarian tumors [MYC, TP53, ERBB2, or RAS family and outcomes in GCTs [FOXL2 [FOXL2 had been indicated to have an oncogenic role in any tumor, and the first example of a novel disease-defining pathognomonic driver mutation being discovered using massively parallel sequencing.GCTs are rare, constituting only about 5 % of ovarian tumors . Until 2n tumors , and then tumors . There w in GCTs . Based o in GCTs . After a in GCTs . The onls [FOXL2 . The ress [FOXL2 . This stFOXL2 have already become apparent [Although FOXL2 is known to be critical for the development of ovaries and is one of the early differentiation markers , somaticapparent , 21, andapparent . This fiapparent . Hence, WWTR1 to CAMTA1 and establish it as a specific event in EHE [WWTR1 and CAMTA1 [Another example of a disease-defining mutation came with EHE, a rare tumor that can present diagnostic challenges. The tumor is a vascular sarcoma with epithelial-looking cells that show vascular differentiation with positivity for platelet endothelial cell adhesion molecule and CD34 . Throught in EHE . In a vat in EHE . Part oft in EHE , 27. Thid CAMTA1 .WWTR1  encodes a transcriptional coactivator containing the WW domain. This domain, which is named as such because of the two conserved tryptophans, mediates specific protein-protein interactions and has been implicated in Hippo signaling, a critical pathway in regulating organ size and keeping proliferation in check. WWTR1 is phosphorylated by lats tumor suppressor kinases (LATS2) which are key components of Hippo signaling and this in turn leads to binding by 14-3-3 proteins which lead to cytoplasmic localization and hence inactivation of WWTR1 [of WWTR1 . Interesof WWTR1 .forme fruste tumors have led to new classification and subtype establishments. Three examples are discussed as follows.In addition to identifying pathognomonic mutations by deep sequencing a few samples, sequencing studies of JAZF1, a transcriptional repressor, with members of the polycomb complex including SUZ12, PHF1, and EPC1 [YWHAE and the FAM22 family [WWTR1-CAMTA1 fusion in EHEs, this karyotype information greatly aided the analysis of the next-generation sequencing data such that, by the use of just one sample, they were able to focus on the YWHAE-FAM22A translocation event, later showing that in cases missing this particular fusion, YWHAE was fused to homologs of FAM22A such as FAM22B [YWHAE-FAM22 translocations into the classification of endometrial stromal sarcomas [Endometrial stromal sarcoma (ESS) is a malignancy of the uterus that had been previously linked with recurrent fusions: the fusion of and EPC1 , 31. Yet2 family . As in es FAM22B . This wos FAM22B . In factsarcomas .EWSR1-ETS fusions of the top candidate in the differential diagnosis, Ewing sarcoma. Four index cases were used for RNA-seq with fusion analyses, and out of these strong evidence for fusion transcripts was seen in two cases: one that had an atypical Ewing fusion of FUS-FEV and another with a completely novel fusion of exon 15 of BCOR to exon 5 of CCNB3 [BCOR-CCNB3 fusion. Microarray expression profiling of ten such cases showed that these tumors had a different profile than other tumors in the differential diagnosis such as Ewing sarcoma, and hence, a whole new bone sarcoma was established [BCOR is thought to encode a ubiquitously expressed protein with a role in repression of transcription through epigenetic mechanisms and in mesenchymal stem cell function [CCNB3 expression is restricted to testis and the encoded protein is a cyclin expressed during spermatogenesis [BCOR fused CCNB3 in fibroblast lines leads to increased proliferative capacity [An exemplary case of NGS defining a new pathology came through the study of peculiar small round cell bone sarcomas that lacked the of CCNB3 . The autablished . BCOR isfunction . On the ogenesis . The ectcapacity .SMO hotspot mutation, and the mandibular tumors have BRAF hotspot mutations [NF2 mutation whereas those in the anterior and medial regions do not [Another recent study established that ameloblastomas, rare benign tumors of the jaw thought to originate from ameloblasts , have diutations . Althougs do not . Even inThe study of rare tumors has also expanded our knowledge about cancer pathways. We will focus on recent findings of recurrent mutations in chromatin remodelers, microRNA processors, and histones.ARID1A, a member of the already established SWI/SNF chromatin remodeling complex were found [ARID1A gene and led to its inactivation, thus suggesting that this gene may function as a tumor suppressor. Although other core members of the SWI/SNF complex had been linked to cancer previously (SMARCB1 and SMARCA4 are known to have lost expression in a variety of tumors), this study showed that noncanonical members of the SWI/SNF complex could also play important roles in tumorigenesis. Furthermore, lack of evidence for mutations in other members of the complex hinted at a context-specific tumor suppressor role for the individual members of the SWI/SNF complex. Additionally, Wiegand and colleagues also showed that the mutation was present in precursor atypical endometriotic lesions of the tumor, and thus was likely an early driver of ovarian clear-cell carcinoma. ARID1A mutations were later found in a variety of other more common types of cancer including gastric adenocarcinomas [Clear-cell ovarian carcinomas are the second most common type of ovarian cancer  and untire found , 45. Thercinomas  and colorcinomas .SMARCA4, was mutated in an inactivating fashion in the majority of cases, and almost all tumors of this specific diagnosis stained negatively for SMARCA4’s protein product BRG1 [SMARCA4 have been described in more common cancers such as lung adenocarcinomas [SMARCA4 in almost all cases emphasized the driver role of SMARCA4 loss. Another example of a critical driver role of SWI/SNF mutations came through the NGS study of familial multiple spinal meningiomas [NF2 or SMARCB1 mutations, germline SMARCE1 mutations were identified through exome sequencing. Again, the protein was lost in the tumor samples but not in normal tissue, thus suggesting a classic Kundson biallelic inactivation and a tumor suppressor role of SMARCE1.Small-cell carcinoma of the ovary of the hypercalcemic type (SCCOHT) is another rare but genetically stable tumor  that wasuct BRG1 –52. Althrcinomas , they ocingiomas . In famiIt should be noted that the reason for disease specificity of SWI/SNF member mutations and indeed the steps in tumorigenesis associated with their loss are not clear. SWI/SNF is thought to regulate the expression of many genes and interacts with many critical cancer pathways from cell cycle regulation to hedgehog and Wnt signaling. Indeed, it has been suggested that perhaps the remaining complex, which still assembles without the mutated members, might act as an oncoprotein and drive tumorigenesis . Thus, mDICER1 were identified in the rare familial pleuropulmonary blastoma–family tumor and dysplasia syndrome [DICER1 came from the study of nonepithelial ovarian tumors [DICER1 were identified across nonepithelial ovarian tumor types and were most predominantly seen in Sertoli-Leydig cell tumors [DICER1 has been previously associated with worse prognosis in breast cancer [DICER1 can drive cancer through the combination of loss of one allele and a functionally deficient protein, this is an aberration of the classic two-hit hypothesis [DICER1 in nonepithelial ovarian tumors does not fit traditional tumor suppressor or oncogene models. Rather, there seems to be a mix of the two models involved in tumorigenesis. There is an inherited inactivation of one copy of the genes, and the remaining allele is not totally inactivated somatically, which would be lethal in most cells rather is hypomorphic via hotspot mutations  [H3F3B also seemingly encodes the same histone 3.3 protein; however, mutations in this gene were not identified in DIPGs or glioblastomas. In a seminal study, Behjati et al. described H3F3A driver mutations in another tumor type: chondroblastomas [H3F3B mutations in giant cell tumors of bone [H3F3A or H3F3B depending on tumor type. Since there is no expression difference between these genes in giant cell bone tumor versus chondroblastomas, temporal expression, for instance at the time of tumor formation, is a possibility suggested by the authors [Another prime example of insights into cancer biology comes from the identification of mutations in histones in  (DIPGs)  and pedi (DIPGs) . Histone (DIPGs) –68. Intelastomas . Additio of bone . Chondro authors . The aboforme fruste tumors but ended up revealing a complex genome with various tumorigenic mechanisms identified in different cases, unlike the more consistent drivers seen in the tumor types described above [It should be noted that rare tumors with homogenous clinical behavior are not always easy to study, and the examples used above are success stories that have benefitted from the relative ease of interpreting NGS results when the tumors are truly simple genomically. Embryonic rhabdomyosarcomas have clinical and morphologic features of ed above , 71. Simforme fruste tumors have been particularly informative in deep sequencing studies, expanding on our knowledge of cancer biology in a resource-efficient manner. Here, we have cited several successful examples of recent findings that have lead to the discovery of pathognomonic mutations, the establishing new subtypes and classifications, such as the case of high-grade ESS, and providing insight into mechanisms of cancer formation such as findings of SWI/SNF and microRNA processing gene abnormalities. This is not to say that such discoveries are not possible in more common, genetically complex cancers, but in forme fruste tumors, the reduced complexities in the genome allows for identification of driver oncogenic events with the use of very few samples. Part of the reason for success in studying these tumors can also be attributed to the fact that they have tended to be understudied and not so much is known about them. However, their rarity comes with the challenge of a lack of banked samples appropriate for the nucleic acid extractions needed for deep sequencing. Recent advancements in sequencing technologies mean that formalin-fixed paraffin-embedded tissues can now be also used for deep sequencing, and hence, some of the challenges in studying forme fruste tumors are already being overcome [Yet, as a whole, overcome , 72.forme fruste tumors, in particular biphasic cancers like synovial sarcoma, can again provide models that may well prove easier to study. We believe that with all these developing methodologies, rare tumors can be a source of breakthroughs that give clearer answers at lower cost, with fewer samples needed to make discoveries.The focus of this review has been on next-generation sequencing and its role as a molecular microscope helping define tumors in a new way. However, sequencing technologies and associated analytic capacities are advancing at a rapid rate. The ability to study clonal evolution and diversity, which has been successfully utilized in the breast cancer field for instance , can be


Systemic autoimmune diseases are a group of heterogeneous disorders caused by both genetic and environmental factors. Although numerous causal genes have been identified by genome-wide association studies (GWAS), these susceptibility genes are correlated to a relatively low disease risk, indicating that environmental factors also play an important role in the pathogenesis of disease. The intestinal microbiome, as the main symbiotic ecosystem between the host and host-associated microorganisms, has been demonstrated to regulate the development of the body’s immune system and is likely related to genetic mutations in systemic autoimmune diseases. Next-generation sequencing (NGS) technology, with high-throughput capacity and accuracy, provides a powerful tool to discover genomic mutations, abnormal transcription and intestinal microbiome identification for autoimmune diseases. In this review, we briefly outlined the applications of NGS in systemic autoimmune diseases. This review may provide a reference for future studies in the pathogenesis of systemic autoimmune diseases. Since the inception of cyclic array-based next-generation sequencing (NGS) in 2005 Systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), ankylosing spondylitis (AS), and Sjögren’s syndrome (SS) are typical systemic autoimmune diseases, which affect multiple organs and exhibit inherited susceptibility. Multiple causal genes have been identified that influence the development of autoimmune disorders by genome-wide association studies (GWAS) 14–1015 microorganisms, colonizing the human intestinal tract shortly after birth, and remaining there throughout an individual’s life. Both the quantities and the species composition of intestinal microbiota are closely related to human health The human intestinal contains a vast and diverse microbial ecosystem, consisting of 10The value of NGS has been demonstrated in identifying susceptibility genes associated with systemic autoimmune diseases MALT1 and ACT1With the development of NGS technology, WGS, WES, and RNA-seq are widely used to study the genetic mutations and gene expression . The mosIRF2 polymorphisms to SLE in an Asian population, Kawasaki et al. re-sequenced the IRF2 genes using the 454 sequencing platform. The WGS results demonstrated that the rs66801661A gene variant could independently contribute to SLE. Additionally, it was observed that both rs66801661 and rs6233999 variants were correlated with transcriptional activation of IRF2IRF2 correlation with SLE depended on other factors, demonstrating the power of NGS in validation of previously-identified genes. In addition, traditional sequencing did not allow for sufficient analytical accuracy for small sample size, while NGS can facilitate scientists in profiling transcription of disease-related genes in small sample populations. For instance, IRF5 gene has been confirmed as a genetic risk factor for SLE by RNA-seq IRF5 transcription unit by RNA-seq from six patients and three healthy donors confirmed that the IRF5 transcription profile differed in SLE patients compared to the controls. The abnormal IRF5 transcriptional signature was determined by IRF5-SLE risk haplotype SLE is a complex and heterogeneous disease involving both genetic and environmental factors. Multiple susceptibility genes have been identified to be associated with SLE. However, the correlation of some genes with SLE needs to be validated. NGS technology provides scientists with a good opportunity to evaluate the validity of previously-identified genes. In a study to evaluate the contribution of Lactobacillus reuteri and Turicibacter spp. than those ingested neutral pH water (NW). The relative proportions of some bacterial species such as Ruminococcus gnavus, Trichodesmium hildebrandtii, Hydrocarboniphaga daqingensis, and Polaribacter butkevichii were higher in AW recipients than in NW recipients even in the pre-disease stage Although human genetic factors have attracted most attention, there is emerging evidence supporting that the bacterial flora contribute to the pathogenesis of SLE. Culture-independent, high-throughput NGS technologies enable us to investigate the function of the gut microbiome in diseases. Sequencing of the 16S rRNA gene variable region using NGS technology has been used to distinguish and classify bacteria. For instance, 16S rRNA gene sequencing in combination with NGS was used to assess fecal microbial profiles of SLE patients and matched controls, revealing some significantly-decreased Firmicutes families and a lower Firmicutes/Bacteroidetes ratio in SLE individuals compared to controls CDR3) and immunoglobulin heavy chain (IgH) gene segment V4–34 enrichment were associated with RA severity RA is a chronic complex genetic autoimmune disorder characterized by synovial inflammation and erosion of bone and cartilage, which affects around 1% of the world’s population IL2RA and IL2RB genes in RA patients in a European project. WES studies identified a missense variant (rs699738) and a non-coding variant (rs624988), which are believed to contribute to the risk of RA development PLB1 gene locus and two independent non-coding variants (rs116018341 and rs116541814) close to PLB1 gene were identified to be associated with the risk of RA development, indicating that PLB1 is a susceptibility gene for RA NGS is also very powerful in assessing variants that occur at low frequencies. A group of rare non-synonymous variants was found in the Prevotella copri at the level of family abundances in new-onset untreated RA (NORA) patients. Such increase in P. copri is strongly correlated with disease progression. Interestingly, with increased amount of Prevotella, there is a concomitant reduction in abundance of Bacteroides in RA patients Haemophilus spp. was found to decrease in RA individuals, whereas abundance of Lactobacillus salivarius showed a significant increase in active RA patients Scher et al. sequenced V1 and V2 region of 16S rDNA on the Roche 454 platform by shotgun sequencing. They found a significant increase in IL21R gene, which is associated with MS CYP27B1 gene, which can cause loss of gene function, resulting in vitamin D deficiency-induced MS. The CYP27B1 gene was also identified as a MS causative gene TYK2 gene encoding tyrosine kinase 2. Interestingly, rs34536433, which was previously reported to be associated with MS, was not found as a risk-factor for MS in this study MS is a chronic autoimmune disease of the central nervous system (CNS) involving dysfunction of the blood–brain barrier in association with demyelination and axonal damage + T cells are intrathecally enriched in MS patients only Epstein–Barr virus (EBV) has been reported to play an important role in the pathogenesis of MS Polysaccharide A (PSA) produced by intestinal Bacteroides fragilis has been discovered to protect against the disease symptoms in the experimental autoimmune encephalomyelitis (EAE) model Clostridium perfringens type B was found to enhance nascent MS lesion formation Many studies have also shown that gut microbiota plays an important role in the pathogenesis of MS. Paraprevotella and Bacteroides vulgatus in Tg rats further confirmed that the HLA-B27 is associated with an altered gut microbiome In addition to SLE, MS, and RA, NGS has also been applied in the studies related to other systemic autoimmune diseases, including AS and SS. AS is an autoimmune disorder with chronic inflammation affecting both the spine and joints. Previous studies showed that the dysbiosis of the gut microbiome in AS is correlated with the presence of human leukocyte antigen B27 (HLA-B27), which is an AS disease-risk factor SS is another complex autoimmune disease that causes exocrine gland deficiency along with symptoms of dryness with unknown etiology. Abnormal miRNA expression patterns could serve as the potential biomarker for SS The rapid development of NGS has contributed significantly to both basic and clinical studies. The discovery of causative gene loci has given us the chance to unravel the factors involved in pathogenesis of diseases. Many disease-associated genes, which were previously missed due to the limitation of sequencing technology, have been identified by NGS. Gene identification is an important step in the discovery of the pathogenesis of the complicated autoimmune diseases. As mentioned above, NGS has facilitated validation of the association of previously-identified disease-related gene loci with autoimmune diseases. In addition, NGS enables researchers to combine previously-identified gene linkage with new sequencing data, allowing the discovery of previously-undetected linkage signals or pathways. NGS has also resulted in the correction of some previously-controversial linkages of susceptibility genes with diseases. Although WES is currently the main method for detecting rare variants in systemic autoimmune diseases, the dropping cost of WGS will allow for further identification of non-coding variants related to the risk of diseases. In addition to basic studies, application of NGS will also significantly improve the genetic diagnosis of autoimmune diseases, by providing clinicians with more accurate genetic information of the patients and assisting them in the formulation of a much more personalized and precise treatment regimen.Increasing evidence has demonstrated the role of the gut microbiota in systemic autoimmunity Epigenetic markers are closely related to the intestinal homeostasis and can be analyzed by NGS technologies including RNA-seq, ChIP sequencing, and DNA methylation sequencing. Due to limitations of traditional sequencing technologies, there are barely any reports using integrative analysis to study the inter-relationship between genomic mutations, cytokine expression, and the gut microbiome in autoimmune diseases. With the development of NGS technology, a comprehensive analysis that will aid in the understanding of the pathogenesis of systemic autoimmune diseases involving genetic factors, microbiome community, and cytokine network in autoimmunity is now possible. The genetic pedigrees in systemic autoimmune diseases will provide us with ideal candidates to study the correlation between genetic mutations and the alteration of the gut microbiome using NGS.+ system, which can reach up to 1000 bp read length, overcoming short read limitations Although NGS technology has been widely used in autoimmune disease studies, there are some limitations. NGS technology is primarily based on short reads and produces large amounts of sequencing data. This is a big challenge for data analyses. It is also inevitable that NGS will bring in some errors, including the lack of detection on low frequency mutations due to the shorter lengths and repeat base sequences. Roche has developed a new GS FLXThe authors have declared no competing interests.


Chronic organ injury leads to fibrosis and eventually organ failure. Fibrosis is characterized by excessive synthesis, remodeling, and contraction of extracellular matrix produced by myofibroblasts. Myofibroblasts are the key cells in the pathophysiology of fibrotic disorders and their differentiation can be triggered by multiple stimuli. To develop anti-fibrotic therapies, it is of paramount importance to understand the molecular basis of the signaling pathways contributing to the activation and maintenance of myofibroblasts. Several signal transduction pathways, such as transforming growth factor (TGF)-β, Wingless/Int (WNT), and more recently yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) signaling, have been linked to the pathophysiology of fibrosis. Activation of the TGF-β1-induced SMAD complex results in the upregulation of genes important for myofibroblast function. Similarly, WNT-stabilized β-catenin translocates to the nucleus and initiates transcription of its target genes. YAP and TAZ are two transcriptional co-activators from the Hippo signaling pathway that also rely on nuclear translocation for their functioning. These three signal transduction pathways have little molecular similarity but do share one principle: the cytosolic/nuclear regulation of its transcriptional activators. Past research on these pathways often focused on the isolated cascades without taking other signaling pathways into account. Recent developments show that parts of these pathways converge into an intricate network that governs the activation and maintenance of the myofibroblast phenotype. In this review, we discuss the current understanding on the signal integration between the TGF-β, WNT, and YAP/TAZ pathways in the development of organ fibrosis. Taking a network-wide view on signal transduction will provide a better understanding on the complex and versatile processes that underlie the pathophysiology of fibrotic disorders. ThIn the past, signaling cascades were often studied in isolation, i.e., a ligand signals through its receptor and mediates the nuclear accumulation of one or several transcription factors to modulate target gene expression. This view changed since recent advances suggest that these cascades are in fact organized into complex signaling networks which, dependent on the cellular and environmental context, govern cell function and fate in fibrotic disorders. This inter-pathway communication allows for increased versatility and fine tuning of cellular responses, which may explain the variety of phenotypes found in fibrotic disorders. The aim of this review is to discuss the current understanding on the signal integration between the TGF-β, WNT, and YAP/TAZ pathways in the development of fibrosis. We will start with a short overview of the three pathways, and extend our discussion with a detailed view on how these pathways connect at multiple levels of signal transduction in the context of myofibroblast function and fibrosis. Finally, we touch upon the challenges and considerations in the design of anti-fibrotic therapies, with focus on the cross-talk between the three signal transduction cascades.The TGF-β superfamily of growth factors consists of multiple proteins that govern a wide range of physiological processes, such as stem cell pluripotency, cell fate determination, proliferation, and differentiation. In humans, over 30 members of the TGF-β superfamily have been documented, including TGF-βs, activins, inhibins, nodal, growth/differentiation factors (GDFs), and bone morphogenetic proteins (BMPs). In this review, we focus mainly on the canonical signaling through TGF-β1, since considerable evidence exists for its role in fibrosis   and latent TGF-β-binding proteins (LTBPs) in the so-called large latent complex (LLC) . Upon inin vivo accompanied with an increased expression of TGF-β target genes and decreased levels of the inhibitory Smad6 and Smad7. Despite a tremendous body of experimental work, the mechanisms underlying Smad-induced fibroblast activation are incompletely understood, as both activation and inhibition of Smads can promote fibrogenesis, dependent on the context  as Wingless and in the mouse as Int1, together termed WNT in mammals, canonical WNT signaling comprises the molecular interactions leading to the nuclear translocation of β-catenin . The vaPAI1. Whether the interactions between YAP and Smad7 are of importance in this process remain to be elucidated.One of the examples, through which other pathways interact with Smad recycling describes the temporal regulation by CDK8/9 and GSK3. First CDK8/9 phosphorylate Smad1 on Ser206 and Ser214, which allows binding with YAP and simultaneously triggers phosphorylation by GSK3 on Thr202 and Ser210. The latter phosphorylation events cause YAP to dissociate and Smurf2 to bind with Smad1, and initiate ubiquitination . AlthougRemarkable progress in both biology and pharmacology has led to advances in the development of anti-fibrotic therapies. Many of these therapies aim to target the usual suspects such as ligands and receptors of the TGF-β and WNT signaling cascades using antagonistic antibodies or small-molecule inhibitors. Although the therapeutic efficacy in animal models proves promising , trials COL1A1 may be negatively regulated (The small-molecule intracellular inhibitor LY2157299 specifically targets the kinase pocket of the type I receptor of the TGF-β cascade without inhibiting the type II receptor and thereby attenuates Smad2/3-dependent transcription of target genes . Howeveregulated . Howeveregulated , 78. As egulated . VertepoThe disadvantages of pathway-wide molecular inhibitors challenge the scientific community to develop specific targeting strategies against intracellular processes and protein–protein interactions. The increasing insight in the molecular cross-talk between signaling cascades adds new possibilities in drug development. Additionally, by focusing on the elucidation of the crystal structures of protein complexes, we can pursue the rational design of novel small molecular inhibitors to interfere at the cross-roads of signal transduction cascades.in vivo. Broadening our view on signal transduction will provide a better understanding of how a limited set of growth factors is able to govern the complex processes that underlie the physiology and pathology of fibrotic disorders.Recent advancements in the field of TGF-β, WNT, and YAP/TAZ signaling have revealed that these signaling entities do not act alone. The notion that pathway components can have multiple and even opposed functions within one cell partly explains how the inhibition of a single molecular target often does not result in the desired therapeutic effect. This does not only add to the mere understanding of fibrotic processes, but also promotes the necessity to develop highly specific small-molecule intracellular inhibitors that act on protein–protein interactions at the cross-roads of signaling cascades. It should be noted that several of the studies described in this review used artificial ectopic expression of the proteins investigated. This may introduce artifacts that can influence the activity and functionality of the signaling cascades involved. Thus, more detailed studies in representative models for fibrosis focusing on endogenous proteins are required to completely understand the molecular cross-talk BP, RB, and MB designed the manuscript. BP and MB collected literature and BP drafted the manuscript. All authors critically discussed and revised the content of the manuscript and had final approval of the manuscript in its present form.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.


In this regard, the lamprey spinal central pattern generator (CPG) stands out as a well-characterized, conserved vertebrate model of a neural network (Smith et al., 2+ signaling in spinal neuron dendrites orchestrate oscillations that drive network behavior.Within neural networks, synchronization of activity is dependent upon the synaptic connectivity of embedded microcircuits and the intrinsic membrane properties of their constituent neurons. Synaptic integration, dendritic Ca Thus, to explore the idea that active dendritic properties are at the core of this behavior, we examine in detail the lamprey spinal network and draw from other areas of dendritic research to enhance our understanding of what occurs at the level of the dendritic synapse to generate behavior.Orchestration of neuronal activity within networks is integral to correct execution of behavior. Synchronization between groups of neurons is an organizational feature of many neural networks found in the central nervous systems of invertebrates , or even actual movement, to observed locomoting animals. Despite this similarity, it is crucial to understand how physiological patterns of synaptic input and intrinsic membrane electrodynamics generate rhythmic behaviors.Vertebrate locomotion is initiated and maintained by evolutionarily conserved serial pathways originating in the forebrain  of individual VHNs in isolated spinal cords to undergo repetitive oscillations that are in-phase with the ipsilateral VR of the corresponding hemi-segment  during NMDA-TTX oscillations causes the cell to become similarly trapped at a depolarized mV. Thus, Ca2+ is necessary to hyperpolarize the cell from the depolarized state. Ca2+ activates myriad Ca2+-dependent proteins. In particular, VHNs contain Ca2+-dependent K+ channels  and the entering Ca2+ activates KCa2 channels to cause an afterhyperpolarization  NMDAR activation depolarizes VHNs; (2) increasing NMDAR conductance by ejecting Mg2+ from the pore; (3) causing further depolarization and Ca2+ entry via the NMDAR as the mV plateaus; (4) which activates KCa2 channels to hyperpolarize the cell; and (5) ending the depolarized plateau to repolarize the cell where it can repeat the cycle  is unknown. Do many dendrites receive synchronous input from their various synaptic partners? Does input occur in discrete spatial locations? The location and timing of synaptic input is crucial for the transmission of potentials arriving at the soma, which will greatly influence neuronal output  to show synchronized increases in intracellular Ca2+ (Ca2+i). In contrast, local Ca2+ signaling domains  in dendrites can occur following neurotransmitter receptor activation , but also from VGCCs following depolarization from local synaptic potentials , is a large determinant for the extent of spread of Ca2+ because diffusion is restricted at the spine neck -driven VGCC activation in dendrites , NMDARs and metabotropic glutamate receptors (mGluRs) may be activated in the postsynaptic compartment. AMPARs are responsible for fast depolarization, and can locally activate nearby VGCCs to cause Ca2+ entry. Local depolarization, or depolarization induced from bAPs can alleviate Mg2+ block of the NMDAR, facilitating Ca2+ influx during concurrent and subsequent release of glutamate at that synapse    will profoundly impact cell firing rates, dendritic integration, and processing both in real-time during individual cycles of locomotor activity, but also in the long-term. The molecular complexing of Ca2+ sources to secondary effector proteins, like KCa2 in lamprey, will consequently impact spike-timing through activation of the mAHP , alone or within a cocktail of monoamines, can activate locomotion and fictive locomotion in many preparations  as well as from RS command neurons (Buchanan and Grillner, Ca2 channel activation.Thus, the serotonergic system in the spinal cord plays a crucial role in modulating the output of the spinal network. While these results, whether mediated by pre- (Schwartz et al., in vivo are not physiologically activated by a tonic and diffuse release of glutamate. Instead, the release of neurotransmitter and subsequent receptor binding is exquisitely targeted to discrete postsynaptic loci with temporal precision. The physiological activation of NMDARs in any circuit is almost entirely mediated by the synaptic release of glutamate. This will only occur at synapses, and only following presynaptic release of glutamate at those synapses. This constrains the activation of NMDARs spatially and temporally, as well as the KCa2 channels that are subsequently activated.In all vertebrates, 5-HT and glutamate applied exogenously can initiate and influence locomotor-like activity. While it is remarkable that systemic drug application can reliably produce ethologically relevant locomotor patterns in lamprey (Sigvardt et al., Xenopus larval tadpoles, there may be little specificity in anatomical connections early in development (Li et al., While pharmacological activation of the spinal network is presumably far from physiological, it has remained to be demonstrated just how distinct this artificial induction is from supraspinal control of descending command neurons and subsequent spinal CPG activation. It is important to note that generating rhythmic activity and appropriate phase coupling has many theoretical solutions (Wallén et al., in vivo is even less certain than glutamate because exogenous application of these modulators over an artificially and pharmacologically activated network merely compounds errors and cannot match physiological release. Indeed, monoamine cocktails with glutamate agonists evoke spinal network activity (Rossignol et al., Furthermore, our understanding of how monoamines in general and 5-HT in particular act 2+i while Ca2+ oscillations are synchronized throughout the dendrites of a single neuron (Bacskai et al., 2+ entry, whether NMDARs or VGCCs, may become obscured. This may allow KCa2 channels to couple to Ca2+ microdomains as opposed to nanodomains implied by their physiological BAPTA sensitivity. Indeed, in VHNs dialyzed with EGTA, oscillation plateau progressively lengthen immediately following whole-cell access, on a time course equivalent to the diffusion of dyes to the most distal dendrites (Alpert and Alford, 2+ buffering into the distal dendrites where Ca2+ oscillations are largest (Viana di Prisco and Alford, 2+ from binding KCa2 channels to cause the repolarization (Alpert and Alford, 2+ diffusing greater distances from its site of entry can, under certain circumstances, activate KCa2 channels, which then contribute to the repolarization. Nevertheless, even under these non-physiological conditions of bath applied NMDA, cells dialyzed with BAPTA displayed immediate, severely impaired repolarization (Alpert and Alford, Bath-applied NMDA leads to a large increase in baseline Ca2+ entry to bind KCa2 channels in Ca2+ microdomains may be an artifact of bath-applied NMDA and the robust increase in intracellular Ca2+, which may also cause Ca2+-induced Ca2+ release from internal stores. However, it was recently demonstrated that this KCa2 channel conductance is physiologically activated by synaptically driven NMDAR-mediated Ca2+ entry (Alpert and Alford, Ca2 channels, then it should follow that there is complementary patterning dendritic Ca2+ entry which drives the activation of KCa2 channels important for rhythm generation. However, we know very little about the spatiotemporal pattern of dendrite activation during behavior and how synaptic input is integrated in real time to impact cell output.The ability of NMDAR-induced Ca2+ dynamics in active networks are crucial to understanding how physiological patterns of synaptic input are integrated in real time to shape the cellular output and have only recently been investigated. With new advances in genetically encoded Ca2+ indicators (Muto et al., in vivo 2-photon microscopy, it is now becoming possible to “watch dendrites in action” and correlate their activity to sensory input and behavioral output (Dombeck et al., Several recent advances have made it possible to begin to assess how dendrites integrate incoming synaptic information within an active, behaving network. Dendritic spatiotemporal CaThe 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.


Juvenile dermatomyositis (JDM) is a rare rheumatic disorder, often associated with poor functional outcome because of the high incidence of diffuse calcinosis in skeletal muscles, especially in case of late diagnosis.To present the case report of the late-diagnosed JDM with severe calcinosis in a child with disease onset at 9 months.We have observed a female patient suffering from JDM since 9 months.2/week till presence time, IVIG 1,5 g/kg monthly during the first 6 months and quarterly next two year. The treatment provided excellent results with rapid normalization of muscle strength, regression of skin and vascular lesions. The physical activity of the child and a relief of an inflammation led to the good development of muscle mass and displacement of calcifications to the surface of the skin with their localization in the larger conglomerates with its occasional spontaneous emptying.The disease started after revaccination against diphtheria and pertussis from symptoms of fever and muscle involvement (thickness and weakness) following a series of scheduled vaccinations  clinical symptoms worsened, there were the typical skin changes, muscle weakness progressed, there were sporadic calcifications (8 months after disease onset), but the diagnosis was not established as a child experienced in primary care clinics. She was examined in our clinic after 3 years of disease onset when JDM was first diagnosed. The patient had fever, proximal muscle weakness, gait disturbance, typical cutaneous lesions , slight enzymes elevation, nail fold changes by capillaroscopy and extensive diffuse calcinosis with significant muscle atrophy and lipodystrophy. The patient received oral prednisolone 1 mg/kg/day with slowly dose tapering and full cancellation after 4 years, methotrexate10 mg/mUnusual onset of JDM at a very early age, probably triggered by numerous vaccinations and lack of awareness about the disease general pediatricians contributes a significant delay in the diagnosis. But on the other hand there is an opportunity of good outcome of myopathy and calcinosis in infants with JDMNone declared.


O-sulfatases (Sulf1 and Sulf2) or by heparanase, an endo-glycosidase that cleaves the HS polymers releasing smaller fragments from HSPG complexes. Numerous studies have demonstrated how these enzymes actively influence cancer cell proliferation, signaling, invasion, and metastasis. The activity or expression of these enzymes has been reported to be modified in a variety of cancers. Such observations are consistent with the degradation of normal architecture and basement membranes, which are typically compromised in metastatic disease. Moreover, recent studies elucidating the requirements for these proteins in tumor initiation and progression exemplify their importance in the development and progression of cancer. Thus, as the influence of the tumor microenvironment in cancer progression becomes more apparent, the focus on targeting enzymes that degrade HSPGs highlights one approach to maintain normal tissue architecture, inhibit tumor progression, and block metastasis. This review discusses the role of these enzymes in the context of the tumor microenvironment and their promise as therapeutic targets for the treatment of cancer.Heparan sulfate proteoglycans (HSPGs) are an integral and dynamic part of normal tissue architecture at the cell surface and within the extracellular matrix. The modification of HSPGs in the tumor microenvironment is known to result not just in structural but also functional consequences, which significantly impact cancer progression. As substrates for the key enzymes sulfatases and heparanase, the modification of HSPGs is typically characterized by the degradation of heparan sulfate (HS) chains/sulfation patterns via the endo-6- Heparan sulfate (HS) chains are an important component of the extracellular matrix (ECM) and they facilitate a number of important biological processes in health and disease. Incorporated into heparan sulfate proteoglycans (HSPG), they comprise a significant fraction of the complex and dynamic ECM and contribute to the biological roles of this medium. The functions of HSPG are varied: the maintenance of a 3D structural matrix for cell attachment; in certain cases the formation of barrier structures (basement membranes); the provision of a reservoir of biological signaling molecules; acting as a cofactor for signaling molecules binding to their receptors –3. TheseThis review will focus upon the roles of these three enzymes in modifying HS in the tumor microenvironment to facilitate processes that support or repress cancer growth and spread. The importance of heparanase, Sulf1, and Sulf2 will also be demonstrated by exploring their association with poor patient prognosis in clinical studies. Finally, the relevant therapeutic developments in the area will be summarized.There is only one heparanase gene known in mammals (HPSE in humans) and it expresses a 65 kDa precursor polypeptide that is enzymatically inactive. Proteolytic excision by cathepsin L of a 48 amino acid peptide yields an active heterodimer comprising 8 and 50 kDa subunits. Although the structures of precursor and active heparanase are unknown, it has been postulated that the excised linker sequence occludes the active site based on sequence analysis and that once removed the active site is revealed allowing catalysis to proceed . SequencN- and 6-O sulfated  and are processed by a furin-type protease to produce the mature heterodimer comprising 75 and 50 kDa subunits connected by disulfide linkages . Unlike most other sulfatases, which are intracellular and function in the catabolism of sulfated molecules, Sulf1 and Sulf2 are extracellularly targeted and function by selectively removing 6-linkages , 22. Altlinkages . SignifiHeparanase, Sulf1, and Sulf2 have been shown, by virtue of their enzymatic modification of the ECM, to affect the signaling of a number of proteins that are important drivers of tumor growth or progression. In some instances, the signaling function of heparanase may be involved in driving these changes, but it is accepted that heparanase cleavage of HS also plays a significant role in modulating signal transduction. These three enzymes accomplish this modulation in signaling in a number of ways  and the Sulfs (pH 7–8) preclude them from being at their most active in the same microenvironment compartment, or at the same time. The low extracellular pH associated with tumor-driven hypoxia has the consequence of stimulating heparanase-mediated activation of pro-angiogenic growth factors leading to a replenishment of the tumor microenvironment –63. InteFor some time, the involvement of heparanase in the metastatic extravasation of tumor cells and invasion of immune cells has been known. We will not cover in detail the work describing the importance of heparanase in these processes but will instead refer the interested reader to the excellent reviews that have already summarized this data , 6, 66. The transition of epithelial cells from their polarized state to non-polarized multipotent mesenchymal cells is an important process during metastasis. Recent studies examining EMT of kidney tubule epithelial cells into myofibroblasts show that heparanase is involved in driving this process by promoting FGF2 signaling . The mecGiven the role of FGF2 in heparanase-mediated EMT, it may be discovered that Sulfs also have roles in regulating EMT due to their interactions with this growth factor. In support of this, there is a recent finding that suppression of Sulf1, among other proteins, was associated with activation of EMT in HCC cells via increased signaling through the Akt and ERK pathways, which would be expected after Sulf1-mediated FGF2 inhibition was alleviated .Reports identifying an association of heparanase, Sulf1, and/or Sulf2 expression or protein levels from clinical samples have been discussed in extensive detail in many excellent reviews , 74–76. It has been postulated that heparanase and HSPGs act synergistically within the tumor microenvironment to enhance tumor growth and the enzymatic versus non-enzymatic roles of heparanase in cancer have been discussed elsewhere . Althougn = 11) reduced heparanase levels by 64% after 2 weeks of treatment with gemcitabine (Overexpression of heparanase in pancreatic adenocarcinomas (PDAC) was identified several years ago when investigators discovered that this may facilitate cancer cell invasion and enhancement of metastatic dissemination . Postopecitabine . More recitabine .in vivo . Also, there was a direct correlation between the level of FGF2 protein and MVD in HCC tissue suggesting that heparanase enhances growth, invasion, and angiogenesis while FGF2 is a potent angiogenic factor for HCC . InteresAlthough Sulf2 is purported to exhibit an oncogenic effect in HCC , in contRates of positive expression for heparanase mRNA in gastric cancer tissues  compared with adjacent normal tissue  was first reported by Endo and colleagues in 2001 . A folloHigher expression of Sulf1 and Sulf2 compared to normal mucosa has been reported in gastric cancer, with the expression of Sulf1 significantly correlated with higher recurrence rates and worse overall survival in patients. Multivariate analysis revealed that Sulf1 is an independent prognostic factor and lymph node metastasis predictive factor in these patients . HoweverStrong heparanase expression in primary tumors and lymph nodes was initially reported to be associated with prolonged disease-free progression in head and neck squamous cell carcinoma, HNSCC . IntriguThe role of heparanase and HS in breast cancer has been discussed comprehensively in a recent review  but emerBoth Sulf1 and Sulf2 were shown to be overexpressed at gene and protein levels in non-metastatic invasive ductal carcinomas although no significant differences could be detected in metastatic tumors . HoweverO sulfation of heparan sulfate due to increased Sulf1 activities can also be recapitulated or mimicked by downregulation of 6-O-sulfotransferases (HS6ST1 and HS6ST2). More recently, it has been shown that downregulation of HS6ST1 reduced the signaling of HS-binding EGF signaling leading to reduced expression of FGF 1, IL6, and IL8  , 126. WiFor further examples of the studies that detail the clinical association studies with heparanase, readers are referred to several reviews , 74, 75.Of particular interest in the field is the role of heparanase in myeloma because its overexpression in the bone marrow environment was associated with a shorter event-free survival of patients with newly diagnosed myeloma treated with high-dose chemotherapy and stem cell transplantation . HeparanIn mononuclear cells derived from various leukemias, heparanase mRNA was expressed in 14 of 15 acute myeloid leukemia (AML) samples. In contrast, cells derived from all 33 chronic lymphoblastic leukemia, all 7 non-Hodgkin’s lymphoma, 7 of 8 chronic myeloid leukemia, and 6 of 8 acute lymphoblastic leukemia patients showed no detectable expression of the heparanase RNA. Heparanase protein was detected primarily within the cytoplasm of AML cells, indicating that the enzyme is produced and stored within the cytoplasm of myeloid cells, with limited expression on the cell surface . The lowPichia holstii, NRRL Y-2448, which is a non-cleavable structural mimic of HS .Heparan sulfate mimetics or glycol-split heparins are considered the most common therapeutic approach to create novel oncology agents with some others currently in early stage clinical development. PG545 is a synthetic, potent competitive inhibitor of heparanase  demonstra growth . Ronepara growth . Oligomma growth , but hasApart from HS mimetics, small molecule inhibitors, and neutralizing antibody programs were developed by various companies including Oxford Glycosciences , 159, Imin vitro (Recent discovery stage reports have identified new putative heparanase inhibitors but these continue to be classified as heparin derivatives or HS mimetics. TD4-143-1, which is a selectively sulfated tetrasaccharide containing unsubstituted glucosamine residues, which inhibited heparanase activity and suppressed invasion of breast cancer cells in vitro . Althougin vitro . Neverth50 values in the range of 1.2–2.6 μg/mL, which is comparable to that of heparanase (In considering Sulf1 or Sulf2 as drug targets, the main consideration is the increasing evidence implicating the Sulfs in cancer and whether these are responsible for augmenting cancer cell growth or inhibiting it , 165. Thparanase . Althougparanase . In gastparanase . In factparanase . These dparanase .in situ progression to invasive ductal carcinoma in vivo (Conversely, silencing of Sulf2 expression in breast cancer cells attenuated ductal carcinoma  in vivo . Moreove in vivo . Consist in vivo . Taken tThe modification of HSPG in the tumor microenvironment modulates a variety of processes that are important in cancer growth and spread. Three enzymes – heparanase, Sulf1 and Sulf2 – play crucial roles in regulating HSPG functioning in this compartment and the up- and down-regulation of these proteins in a large variety of cancers demonstrates how important they are in promoting or repressing malignancy. While the involvement of heparanase in progressing tumors is well-documented, both at the mechanistic level and in clinical observations, the roles of the Sulfs are not so clear, particularly for Sulf1. This clarity is reflected in the more advanced stage of clinical development that anti-heparanase therapeutics has reached in comparison to Sulf inhibitors. Resolution of the pro- and anti-tumor properties of the Sulfs and how these relate to tumor-stromal relationships, primary and metastatic lesion interactions, and other aspects of tumor growth is required before the development of anti-Sulf therapeutics can proceed with confidence. Nevertheless, there is a substantial body of data describing the importance of heparanase, Sulf1, and Sulf2 in modulating HS functioning in cancer, which highlights the already well founded view that targeting the ability of tumors to modify their microenvironment in order to promote growth and spread represents a solid therapeutic development pathway.Keith Dredge and Edward Hammond are employees of Progen Pharmaceuticals and are actively involved in the development of one of the experimental agents discussed in this review, PG545. The other co-authors report no conflicts of interest.


Enumeration and molecular characterization of circulating tumor cells isolated from peripheral blood of patients with cancer can aid selection of targeted therapy for patients, monitoring of response to therapies and optimization of drug development, while also providing valuable information about intratumoral heterogeneity. With the advent of therapies that target the specific molecular aberrations driving a cancer, the traditional concept of ‘one treatment fits all’ is evolving to ‘one patient, one treatment’. Furthermore, as tumors adapt to the selection pressures of serial targeted drugs through the evolution of drug-resistant clones, their genomic landscape changes over time, and hence treatments need to be tailored accordingly . Thus, tThe endeavor for precision medicine demands that each patient be molecularly characterized and that robust and validated ‘real-time’ assays are made in order to evaluate tumor evolution. Although the study of tumor biopsies or surgical specimens remains the ‘gold standard’ for molecular characterization in clinical trials testing a biomarker ,3, singlCirculating tumor cells (CTCs), which are shed into the bloodstream from solid tumors, are relatively rare, representing only one in more than a million blood cells . PatientCTCs provide the opportunity to assess the biological features of cancer repeatedly during the evolution of the disease, enabling clinicians to react quickly to treat the patient with the most suitable specific targeted therapy.Here, we review the clinical studies to date on the use of CTCs as biomarkers of cancer development and discuss the promise of CTC analysis to guide clinical decision-making and to aid targeted drug development.Methods to capture CTCs from blood rest on their differential physical or immunologic characteristics. The basis for affinity-binding systems used for CTC ‘enrichment’ is the selection of cells expressing certain antigens, such as epithelial cell-adhesion molecules (EpCAMs), and the discard of those cells expressing antigens that are known to be absent on epithelial cells but expressed by other blood cells, such as leukocyte-expressed CD45. Alternatively, CTCs can be isolated based on their distinct physical (size or deformability) or electromagnetic properties -11. The In order to implement a biomarker into clinical practice, it is crucial to obtain analytical validation  and evidence of the clinical significance of any results obtained. In 2011, the semi-automated CellSearch system (Janssen Diagnostics) became the first US Food and Drug Administration-cleared assay for quantitative analysis of CTCs, based on its high reproducibility  and the ex vivo CTC isolation [There are several promising capture platforms in development; a separate article in this special issue comprehensively reviews the advances in isolation and characterization of CTCs . Potentisolation ,21. ThisThe number of CTCs present in the bloodstream of patients with metastatic cancer is prognostic for overall survival in several tumor types, with robust evidence reported for prostate, breast and colorectal cancer -14.P < 0.0001). CTC counts were better indicators of survival than levels of prostate-specific antigen (PSA) , the prognostic value of baseline CTC counts was first assessed in the IMMC38 trial, a study including 164 patients about to start first-line chemotherapy . A high count of CTCs at baseline (defined as ≥5 CTCs in 7.5 ml blood) was associated with a significantly shorter survival compared with having low counts at baseline  and overall survival . In a multivariate analysis including several molecular and clinical prognostic factors, CTC counts were a strong independent prognostic factor [A study by Cristofanilli established the prognostic value of CTC counts in advanced breast cancer. Patients with metastatic breast cancer were tested for CTCs at the time of starting a new line of treatment . Patients with ≥5 CTCs per 7.5 ml blood had the worst progression-free survival  and progression-free survival  [In the setting of metastatic colorectal cancer, the prognostic significance of CTCs was prospectively evaluated in 430 patients before starting a new line of chemotherapy. Patients were stratified based on CTC levels of ≥3 versus <3 per 7.5 ml blood. Patients in the ‘unfavorable prognosis’ group had shorter overall survival  . Pilati  0.0002) .Clinical validation of CTC counts as prognostic biomarkers is being pursued in advanced stages of other tumor types, with promising results reported in lung ,27, melaMoreover, CTCs have also been demonstrated to inform prognosis in earlier stages of the disease. A large study analyzed their presence in 735 patients with colorectal cancer undergoing surgery with curative intent. CTC enrichment selected cells expressing carcinoembryonic antigen, cytokeratin 19, cytokeratin 20 and/or CD-133. Time to relapse and overall survival was significantly poorer for those patients with detectable CTCs .In early stages of breast cancer, CTCs were detected in 21.5% of 2,026 patients after surgery and before the start of adjuvant chemotherapy. The presence of CTCs in this population was an independent prognostic factor for disease-free and overall survival .Overall, CTC enumeration is a strong prognostic tool in patients with advanced cancer and can help in patient stratification. In earlier stages of cancer, there is high hope that CTC enumeration could monitor residual disease after radical treatment, although the counts in this population would be generally low, and the analysis would require a platform with very high sensitivity.Changes in CTC counts as a result of anticancer treatment have also been shown to be a reliable marker of response to treatment in different clinical settings. In mCRPC, the predictive value of changes in CTC counts and the correlation with survival have an enhanced relevance owing to the challenge of evaluating the response to treatment by standard radiological parameters as most of the metastatic disease is confined to the bones ,34. The Similarly, conversion from an unfavorable to favorable CTC count after 4 weeks of treatment (using a threshold of 5 CTC per 7.5 ml blood) is a predictive biomarker of response to first-line therapy for metastatic breast cancer . Moreover, drops in CTC counts upon treatment correlated with improved overall survival . CTCs ofThe value of CTC counts on treatment as an independent marker of response is also well established in metastatic colorectal cancer . The stuAlmost every study judging CTC counts as response markers in different cancer types has simplified the enumeration to a dichotomist variable (high or low count) by using particular thresholds in different tumor types instead of considering CTC counts as a continuous variable; a combined analysis of CTC counts from 111 patients with metastatic breast cancer and 185 with mCRPC who participated in the IMMC-01 and IMMC-38 clinical trials ,24 suppoCTCs represent a valuable resource for studying the molecular underpinnings of cancer in individual patients. CTC analysis can support the delivery of precision anticancer treatments, as specific biomarkers of response to targeted drugs can be appraised.in situ hybridization have been used to describe the variability between CTCs, tumor metastasis and primary prostatic cancers in copy-number aberrations for the androgen receptor and presence of TMPRSS2-ERG fusions and loss of the tumor suppressor gene PTEN (encoding phosphatase and tensin homolog) [Genomic- and proteomic-based assays can be used to analyze CTCs and detect the presence or absence of key signaling oncogenic aberrations. For instance, cytogenetic studies based on fluorescence homolog) .in situ hybridization studies, of how relative changes during treatment in the expression of epithelial and mesenchymal markers in CTCs from 11 patients with metastatic breast cancer correlated with response and prognosis [An example of how transcriptome studies in CTC analysis can be clinically applicable was the assessment, by RNA rognosis .As a result of recent progress in next-generation sequencing techniques, it is now also possible to perform whole-genome and transcriptome amplification from single cells, such as CTCs . Lohr anAs cancer subclonal composition and driver mutation landscapes can change through the outgrowth of drug-resistant subclones after exposure to systemic therapy, it might also be important to assess whether predictive biomarkers are present at the specific time of starting a new line of targeted treatment, contrary to testing biopsies taken at earlier stages of the natural history of the disease; a study on 254 patients with breast cancer was able to detect overexpression of human epidermal growth factor receptor 2 (HER2) in CTCs in almost one-third of patients who had no HER2 overexpression in the primary tumor . This fide novo , suggestive of temporal evolution and a putative acquired mechanism of resistance to therapy [A proof-of-principle study in lung cancer detected activating mutations in the gene encoding the epidermal growth factor receptor (EGFR) in CTCs from 11 out of the 12 patients tested; these patients were receiving EGFR-targeting tyrosine kinase inhibitors, and interestingly some of these mutations in CTCs had emerged  therapy . Analogo therapy -52 suppoIdeally, patients would start a targeted treatment, and molecular indicators of response or resistance would be assessed in CTCs and/or other circulating biomarkers repeatedly over the course of treatment, guiding clinicians regarding when to stop or switch the anticancer treatment. A recent and successful example of this strategy is a study that serially characterized CTCs from 58 patients with mCRPC while receiving either enzalutamide or abiraterone to study mechanisms of resistance; quantitative reverse-transcription polymerase chain reaction was utilized to interrogate the presence of splice variants of the androgen receptor genes in CTCs. Antonarakis and colleagues demonstrated that the splice variant 7 of the androgen receptor (AR-V7) is a predictive factor of enzalutamide or abiraterone failure and, more interestingly, that the appearance of AR-V7 during the course of treatment in patients with no AR-V7 expression at baseline might be a mechanism of acquired resistance to these drugs .VHL gene and loss of heterozygosity of chromosome 3p were the only aberrations detected in all analyzed regions from each tumor, suggesting that these were early founder aberrations, whereas other molecular aberrations, including mutations in members of the phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway  amenable to be targeted with specific drugs, were heterogeneous within individual tumors. Genetic ITH has also been identified across many other solid tumor types such as primary breast, gastric, bladder, prostate and pancreatic cancers, where various degrees of genetic ITH, ranging from intermixed subclones to spatially separated subclones, have been described [Genetic intra-tumor heterogeneity (ITH) has been discovered across a wide range of solid tumor types. For example, exome sequencing of multiple tumor regions from clear-cell kidney tumors, the commonest type of renal cancer, revealed that, on average, only one-third of the somatic mutations and DNA copy-number aberrations detected were present in every region that was analyzed from an individual tumor . Mutatioescribed -58.Integrating ITH in individualized treatment selections remains an unmet clinical need in cancer medicine. Sequencing studies on CTCs allow heterogeneous mutations to be captured, enabling a more detailed picture of ITH and subclonal evolution in a single patient with a minimally invasive approach. It is envisioned that the analysis of subclonal heterogeneity could help clinicians to understand why cancer patients might not respond homogeneously in different metastases to treatment with a targeted drug and might eventually guide treatment selection.Using CTCs for ITH studies facilitates the inference of subclonal structures. Genomic-profiling studies of individual CTCs isolated from patients with metastatic colorectal cancer using the CellSearch platform showed how the presence of some genomic aberrations in CTCs was indicative of their subclonal origin from specific areas of the original tumor, through array-comparative genomic hybridization and multiplexed targeted sequencing of 68 genes relevant to colorectal cancer . CTCs alPharmacodynamics (PD) studies are crucial in modern drug development to provide evidence for proof-of-mechanism in early-phase clinical trials and support ‘go/no go’ decisions about further development of new compounds. Minimally invasive access to CTCs from patients offers a unique opportunity to monitor the PD effect of drugs in phase I clinical trials .Assessment of PD biomarkers in CTCs can be performed repeatedly over the course of treatment, which represents an advantage compared with the study of tumor biopsies. To serve as an example, Wang and colleagues developed a quantitative assay to monitor changes in nuclear levels of the DNA damage marker histone variant γH2AX in CTCs that they then tested in samples from 15 patients with different cancer types participating in diverse phase I trials . Among tVariations in CTC counts after drug exposure can also provide an easy and rapid readout of PD effects: the phase I trials of ARQ197 (a selective inhibitor of the hepatocyte growth factor receptor c-MET) in patients with different tumor types and EZN4176 (a second-generation antisense oligonucleotide to exon 4 of the androgen receptor) in prostate cancer assessed the changes in CTC counts in patients in parallel to the determination of the optimal doses of the drugs ,64 Figu. Precisen = 16) and metastatic (n = 16) breast cancer, reporting high expression levels of phospho-PI3K and phospho-AKT in CTCs from both populations. These results open the door to implementing such assays for patient selection in trials testing drugs against this important oncogenic signaling pathway, as has been done previously with tumor-tissue-based assays [CTC analysis in early clinical trials can also be used to assess putative predictive biomarkers of response to novel targeted treatment and to provide proof-of-mechanism of novel compounds and a definition of biologically active doses. Hotspot mutations in PIK3CA were identified in CTCs from patients with either colorectal or breast cancer in two different studies ,67. Mored assays .CTC analysis is a validated method to study tumor characteristics and aid clinical decision-making. This minimally invasive approach facilitates repetitive analysis over time. Analysis can be purely quantitative, which has been proven to deliver prognostic and predictive information at different stages of cancer, or can involve the assessment of molecular biomarkers in CTCs. It is crucial that the novel isolation platforms currently in development are analytically and clinically validated to ensure biomarker integrity.One caveat for CTC studies is whether the whole burden of disease contributes equally to the CTC pool or whether some subclones of the disease might be under-represented or absent in CTCs. Additionally, it is also important to remember that the CellSearch system and other enrichment platforms based on epithelial markers might fail to recognize cells shed from non-epithelial cancers such as melanomas or sarcomas, in which disease-specific technologic approaches and composite selection criteria might be needed , or cellMolecular characterization of CTCs to guide rational treatment selection has direct applicability in clinical practice; it can help to overcome the inability of single biopsies to portray accurately the genomic landscapes of cancers and the limitations of multi-metastasis biopsy approaches. The possibility of regularly re-assessing evolving cancers is extremely useful for on-going treatment stratification and understanding primary and secondary drug-resistance.Nevertheless, the exact role of CTC counts in routine clinical practice remains yet to be defined. On the one hand, the costs and complexity associated with the enrichment and enumeration process (including kits and operator time) are still high. On the other, a first clinical trial evaluating the benefit from an early switch on systemic treatment guided by changes in CTC counts after the first cycle of treatment in metastatic breast cancer recently failed to demonstrate significant benefit for this strategy compared with maintaining treatment until progression based on traditional endpoints . However


The steady and dramatic increase in the incidence of Alzheimer's disease (AD) and the lack of effective treatments have stimulated the search for strategies to prevent or delay its onset and/or progression. Since the diagnosis of dementia requires a number of established features that are present when the disease is fully developed, but not always in the early stages, the need for a biological marker has proven to be urgent, in terms of both diagnosis and monitoring of AD. AD has been shown to affect peripheral blood mononuclear cells (PBMCs) that are a critical component of the immune system which provide defence against infection. Although studies are continuously supplying additional data that emphasize the central role of inflammation in AD, PBMCs have not been sufficiently investigated in this context. Delineating biochemical alterations in AD blood constituents may prove valuable in identifying accessible footprints that reflect degenerative processes within the Central Nervous System (CNS). In this review, we address the role of biomarkers in AD with a focus on the notion that PBMCs may serve as a peripheral laboratory to find molecular signatures that could aid in differential diagnosis with other forms of dementia and in monitoring of disease progression. The prevalence of dementia has increased globally, most noticeably in the ageing populations of the developed world. Alzheimer's disease (AD) is the most common type of dementia (60% of cases). Individuals affected by AD are 5.4 million in the United States and more than 33.9 million worldwide . MoreoveThere is general agreement that the epidemiological impact of dementia can be reduced by detecting and treating classical vascular risk factors since different studies provide evidence in favour of a coexistence of vascular and degenerative components in its pathogenesis .In western countries vascular dementia (VD) is the second most common cause of dementia after AD among the elderly. A meta-analysis of the European studies on the incidence of dementia showed that VD constitutes 17.6% of all dementias . In Euroβ) peptides stemming from the sequential cleavage of a transmembrane precursor named amyloid precursor protein (APP).AD is characterized by neurofibrillary tangles (NFT) and extracellular amyloid deposits. The former are composed of intraneuronal aggregates of hyperphosphorylated tau proteins and the latter are made of amyloid-beta . The fact that the collection of these biological fluids is significantly less invasive than biopsies is an important practical issue when studying neurodegenerative disorders like AD .It has been reported that the sensitivity ) of an “ideal” biomarker to detect AD should be at least 85%. Similarly, the specificity ) in differentiating AD patients from controls and from patients with other forms of dementia should be at least 75% .Despite the enormous advances in modern medicine, the diagnosis of AD remains largely clinical, based on patient history/examination, neuropsychological testing, and imaging techniques. Unfortunately the clinical diagnosis of AD suffers from limitations in that it only allows us to speak of probable or possible AD  with a 9With the expansion of current knowledge on AD and the increasing availability of technical tools, there is an emerging need for the development of accurate biochemical and imaging tests that support the diagnosis , 15. In As to the more prevalent sporadic cases of AD, the need for a biological marker has proven to be urgent, for both the diagnosis and monitoring of the disease , 17.Indeed, an ideal biomarker for AD would assist in the identification of preclinical disease, early disease diagnosis, staging of disease progression, and response to treatment . Early dOver the past decade, biomarker discovery has become a rapidly advancing area of AD research.With the development of structural, functional, and molecular techniques, neuroimaging is increasingly being employed as a diagnostic and prognostic tool in AD. Quantitative magnetic resonance imaging (MRI) is used to assess neurodegenerative changes in AD, which include reducing whole brain volume and cortical thickness associated with ventricular enlargement . Early dβ42 and increased CSF levels of total tau and phosphotau [β42 ratio instead of either single biomarker, and this is reflected in an increase in sensitivity and specificity to 86% and 97%, respectively [β42 and tau as the most sensitive and specific diagnostic and predictive biomarkers for AD.In view of the close relationship of the CSF with the brain and spinal cord, it is believed that the composition of this fluid may reflect biochemical changes in the CNS and thus provide information on the pathological changes occurring in neurodegenerative disorders . Multiplosphotau . Importaectively . Moreoveectively , 28. TheIt should however be remarked that although neuroimaging and CSF biomarkers seem to be the most promising, they also carry some limitations. They are generally expensive to perform routinely and lumbar puncture is invasive and often unpleasant. Moreover a large variability exists in the literature as to CSF biomarker diagnostic accuracies and cut-offs, hampering or delaying their everyday application in the clinical setting , 30 and Also, it is worth noting that the process of biomarker discovery involves many critical steps including study design, sample preparation, protein and peptide separation and identification, and bioinformatics and data integration issues that must be carefully controlled before achieving independent confirmation and validation.Lastly, patient age is an important confounding factor in these biomarker studies and could explain some of the variability in published diagnostic accuracies and cut-offs . Indeed In the human brain several cell types are responsible for initiating and amplifying a specific inflammatory response. In AD signs of an inflammatory activation of microglia and astroglia are present both inside and outside amyloid deposits. Cell cultures and animal models suggest an interactive relationship between inflammatory response activation, reduced neuronal functioning, and amyloid deposition. Furthermore cells associated with extracellular plaques within AD brains can produce a variety of cytokines, chemokines, and other related proteins that influence plaque and tangle formation .β expression and metabolism, and maintain a chronic intracerebral acute phase protein secretion, which in turn favours formation of Aβ oligomers [There is strong evidence that inflammation exacerbates neuronal loss , 34. In ligomers .α (TNF-α), and interleukin-8 (IL-8), as well as complement components and acute phase proteins [On the other hand, microglial activation leads to an increased brain expression of major histocompatibility complex type II and an increased secretion of proinflammatory cytokines and chemokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor-proteins .β-induced microglial/macrophage inflammatory responses and modify the microglial activity surrounding amyloid neuritic plaques [α, and MCP-1 in differentiated human monocytes and, above all, IL-10 causes dose-dependent inhibition of the IL-6 secretion induced by Aβ in these cells and in murine microglia [A “cytokine cycle” has been proposed where  the anti plaques . Such cyicroglia .Accordingly, several reports make it appears that the risk of AD is substantially influenced by polymorphisms in the promoter region and other untranslated regions, of genes encoding inflammatory mediators. Alleles that favour an increased or decreased expression of inflammatory mediators are more frequent in patients with AD than in controls .β has also been shown to induce a phagocytic response in microglia, suggesting a neuroprotective defense mechanism [Aechanism . This isechanism .Despite these findings, clinical trials of nonsteroidal anti-inflammatory drugs (NSAIDs) in AD patients have been disappointing .Nowadays it remains the need for a reliable, minimally invasive, and inexpensive biomarker for dementia, leading many to investigate peripheral blood. Blood collection is simple, inexpensive, and less invasive than lumbar puncture, allowing for repeated sampling. Approximately 500 mL of CSF is absorbed into the blood daily  and therAD also affects PBMCs that are defined as any blood cell with a round nucleus . These blood cells are a critical component of the immune system which provide defence against infection and respond to intruders. The lymphocyte population consists of CD4+ and CD8+ T cells, B cells and natural killer cells, CD14+ monocytes, basophils, neutrophils, eosinophils, and dendritic cells.α production, associated with an increased risk of AD, in elderly controls [Although studies are continuously providing additional data that emphasize the central role of inflammation in AD, PBMCs have not been sufficiently investigated in this context. Indeed, only scant studies have used PBMCs to measure cytokine release, showing a significantly different production of these inflammatory components in AD and MCI subjects compared to controls , 45 Fig as well controls .Delineating biochemical alterations in AD blood constituents may enable the identification of accessible footprints that mirror degenerative processes within the CNS.Moreover PBMCs could reflect inflammatory mechanisms in a more specific way compared with the serum/plasma, and PBMC-associated biomarkers could thus provide novel insight into the pathogenesis of AD.In the following paragraphs we discuss the potential of PBMCs to serve as a peripheral laboratory to find molecular signatures in AD that could aid both in the differential diagnosis with other forms of dementia and in the monitoring of disease progression. cis-/trans-isomerase Pin1 is a cytosolic protein that isomerizes the peptide bond of a phosphorylated serine or a phosphorylated threonine followed by a proline (pSer-/pThr-Pro). Pin1 catalyzes the cis-/trans-isomerization of its substrates, consequently potentiating the accessibility of the phosphate residue for further dephosphorylation by protein phosphatases such as the protein phosphatase PP2A. Alternatively, the binding of Pin1 to other highly phosphorylated substrates can repress their dephosphorylation by calcineurin. Therefore, through isomerization of pSer-/pThr-Pro, Pin1 regulates the function or degradation of a growing number of proteins including transcription factors and cytoskeletal, mitotic, or proapoptotic proteins [The peptidyl-prolylproteins .Pin1 consists of 2 functional domains. The binding domain corresponds to the amino-terminal region consisting of a group IV WW domain (Trp-Trp domain) that specifically binds to pSer-/pThr-Pro motifs. The carboxyl-terminal region is the catalytic domain . Pin1 suβ, and PP2A, that are involved not only in the cell cycle but also in the phosphorylation state of Tau [Phosphorylation of proteins is a key signalling mechanism in diverse of physiological and pathological processes. Pin1-catalysed conformational changes can have profound effects on phosphorylation signalling by regulating a spectrum of target activities. Interestingly, Pin1 deregulation is implicated in a number of conditions, notably ageing and age-related diseases, including cancer and AD. Pin1 is overexpressed in most human cancers; it activates numerous oncogenes or growth enhancers and also inactivates a large number of tumour suppressors or growth inhibitors. By contrast, ablation of Pin1 prevents cancer but eventually leads to premature ageing and neurodegeneration. Recent studies have demonstrated the reemergence within the brain of cell cycle proteins as patients progress from MCI into AD. Pin1 plays an important role in regulating the activity of key proteins, such as CDK5, GSK3-e of Tau .Indeed, Pin1 facilitates tau dephosphorylation  and reguIt has been reported that Pin1 activity is repressed by oxidation in AD –58 and tIt should be remarked that the expression and activity of Pin1 are tightly regulated at a transcriptional level and that a Pin1 gene polymorphism (−842G/C) has been found to be associated with reduced levels of Pin1 in blood cells and with an increased risk for AD in an Italian cohort .Interestingly, a depletion of the soluble form of Pin1 has been described in neurons from AD subjects , 65 and In particular, in PBMCs from LOAD we observed a significant increase in Pin1 gene expression together with a significant decrease in gene promoter methylation .This latter finding holds particular relevance, since so far little is known about epigenetic patterns in AD. Moreover, epigenetic mechanisms have already been proposed as markers of AD in PBMC-derived DNA  and it h16 phosphorylation levels of Pin1 were lower than in controls , which ultimately blocks the binding of transcription factors causing chromatin compaction and gene silencing .The influence of regulatory small RNAs and microRNAs on gene transcription is also increasingly recognized as a key mechanism of epigenetic gene regulation .Indeed, microRNAs (miRNAs), small regulatory RNAs in cells, probably constitute one of the most investigated extracellular RNAs in body fluidsand the levels of certain miRNAs in the circulation correlate well with different pathological conditions  –81.Epigenetic mechanisms are important in cell growth and differentiation . EpigeneEpigenetics is destined to change across the lifespan. In fact a loss of global DNA methylation and promoter hypermethylation of several specific genes occurs during ageing.In particular ageing-associated DNA hypermethylation occurs predominantly in genes involved in the development of anatomical structures, organs, and multicellular organisms and in the regulation of transcription.This phenomenon may be considered a new aspect of the age remodeling process, a continuous adaptation of the body to the deteriorative changes occurring over time. However, it is not clear how relevant these epigenetic changes are in the context of functional changes in gene expression .Inappropriate epigenetic changes are associated with many diseases including cancer , Rett syEnvironmental signals can trigger epigenetic responses and may be an important mechanism by which environmental exposures are associated with disease . FurtherEpigenetic epidemiology is the study of the effects of heritable epigenetic changes on the occurrence and distribution of diseases in populations . This reEvidence for the role of epigenetics in AD pathogenesis can be found in human studies of various tissues, in animal models, and in cell cultures –102. GloDiscordant data have been reported on specific epigenetic modifications of tau- and amyloid-processing genes. On the one hand an altered regulation was reported across multiple brain regions –105, andHuman postmortem case-control studies have demonstrated global hypomethylation in the entorhinal cortex of AD subjects  and in tAn AD case-control study in the postmortem human parietal lobe cortex has revealed a differential regulation of several miRNAs, including miR-204, miR-211, and miR-44691 .Age-matched AD cases have been found to exhibit an increased neuronal global phosphorylation of histone 3 relative to controls, as determined by immunolabeling in the hippocampus, and such histone modification suggests mitotic activation .β production was increased [In experiments where neuroblastoma cells were cultured under low folate and vitamin B12 conditions, PSEN1 and BACE1 were hypomethylated, mRNA expression of BACE1 and PSEN1 was significantly induced, and Ancreased .An additional study using human neuroblastoma cells and male rat brain tissue reports that APP mRNA expression is repressed by thyroid hormone (T3) sensitive histone modifications .The study of gene regulation in blood cells from living patients offers the possibility to go through the whole history of the disorder  in a more comprehensive perspective, compared to postmortem studies which allow only pinpoint assessment.It is important to note that PBMCs may also be a useful model of epigenetic gene regulation in the brain . In factFor instance, our group has investigated the role of DNA methylation in the PBMCS from LOAD subjects compared to controls and has demonstrated an altered Pin1 gene expression and promoter methylation , as detaAlso, by comparing DNA methylation of Faah and Alox5 promoters we found a direct correlation between these two genes , 115.It has been shown that oxygenation of the FAAH substrates by lipoxygenase activity modulates recognition of these molecules by their protein targets , with poThese results might suggest that a parallel increase of FAAH and 5-LOX expression in AD patients could evoke a sustained inflammatory condition, thus reinforcing neurodegeneration , 115.This finding in peripheral cells is in agreement with previous results in postmortem AD brains , where FRecently, there has been considerable interest in exploring the therapeutic potential of anti-inflammatory agents to prevent, treat, or slow down the progression of AD . HoweverTaken together, these lines of research converge towards the notion that novel anti-inflammatory targets may provide a safer strategy for the prevention and the treatment of AD. In such scenario PBMCs stand out as potential peripheral markers of disease within the CNS.Nutritional alterations have been linked to the epigenetic modulation of some AD-related genes and seem to play a role in AD pathology. There is also evidence in favour of the epigenetic modulation of genes involved in the pathways activated by some dietary factors, both in ageing and disease, further supporting the involvement of epigenetic mechanisms in AD. A number of dietary elements have been reported to be either risk or protective factors for the development of AD. These include fat, fatty acids, antioxidants, fish, vitamins, alcohol, and, more recently, caffeine . In vitro and animal studies have provided convincing data on caffeine's neuroprotective effects against and in the presence of AD pathology [The neuroprotective effect of caffeine consumption on AD pathology is currently emerging from both basic and epidemiological studies . In vitrathology –126. HumHowever, due to the conflicting results from some longitudinal studies, there is no consensus about the role of caffeine in the onset of AD –128.Caffeine is one of the most consumed psychoactive drugs and acts mostly by blocking adenosine receptors . The pur1 and A2A (A2A) receptors, the low-affinity A2B receptor, or the low-abundance A3 receptor [The physiological responses to Ado take place as a result of the binding and activation of different transmembrane receptors: the high-affinity Areceptor .1 is inhibitory Gi-coupled, A2A is excitatory Gs-coupled, thereby decreasing and increasing cAMP levels, respectively [2+ channels and the phospholipase C pathway. Through these actions and by modulating both the release and the uptake of different neurotransmitters, the balance between the activation of adenosine A1 and A2A receptors allows the fine tuning of synaptic transmission and plasticity in the hippocampus [These receptors are G-protein coupled receptors that regulate, in opposite directions, the second messenger cAMP; while Aectively . The actpocampus .2A is present in a wide variety of tissues, including the nervous system and the peripheral immune system, where they display different levels of expression: significant levels in neurons and peripheral cells (lymphocytes and neutrophils) and lower levels in glial cells [2A in different tissues are consistent with the sophisticated, multifaceted neurochemical, and molecular effects of the Ado system. On the basis of in vitro [ in vivo [2A, through complex mechanisms which are still poorly understood [In particular Aal cells . The difin vitro , 136 and in vivo  studies,derstood –141, pladerstood , 143.2A is able to prevent Aβ-induced synaptotoxicity in animal models and cell cultures [Moreover it has been demonstrated that Acultures  and it hcultures , 146.2A on brain cells [2A alleviates the long-term burden of brain disorders such as ischaemia, epilepsy, Parkinson's disease, or AD [2A can protect the CNS against several insults, including ischemia and excitotoxins [Contrasting data have been reported so far on the beneficial/detrimental effects of Ain cells . The bloe, or AD , 149. Ontotoxins , 150.2A contributes to coronary endothelial dilatation in mice [In the periphery A in mice , can inh in mice , and pre in mice .2A gene expression and density in the PBMCs of patients with amnestic MCI (a-MCI), multiple cognitive domain MCI (mcd-MCI), outright AD, VD, and controls. We found that A2A expression is upregulated in the peripheral cells of a-MCI but not AD subjects, supporting an involvement of the Ado system in the early stages of AD [2A expression is lower in the PBMCs of subjects with VD than AD, highlighting its possible relevance as a biomarker that may help differentiate two forms of dementia that are often closely associated (We recently investigated Aes of AD . We alsosociated .2A possesses a moderate degree of sensitivity and specificity for identifying VD patients from a heterogeneous group composed of VD and AD patients. The lower A2A expression in VD compared to AD subjects seems to suggest a differential role of the Ado system in these dementias [Indeed, ROC analysis data showed that Aementias .2A, may explain its different expression in these pathological conditions as well as in the ageing process, as already mentioned [The methylation of the ADORA2A promoter gene, which codes for Aentioned .2A represents the main Ado receptor involved in inflammation and it is interesting to note that also other inflammatory biomarkers are differently expressed in VD and AD subjects, such as alpha1-globulin and alpha2-globulin in the serum [Moreover, Ahe serum  and C3a he serum .2A levels in VD could be a defence mechanism since it has been demonstrated that pharmacologic inactivation or genetic deletion of A2AR reduces neuronal injury after global and focal cerebral ischemia in many animal models [2A may play an important but differential role in both types of dementia: its upregulation in the preclinical stages of AD could counterbalance the existing inflammatory state and its downregulation in VD could reflect the effects of A2A on the brain vasculature [2A could serve as a biomarker in the differential diagnosis between VD and AD.On the other hand the decreased Al models , 160. Frculature . It can Peripheral cells and in particular PBMCs seem to directly participate to neurodegenerative processes. They play critical roles in immune response, metabolism, and communication with other cells as already pointed out many years ago . MoreoveThe substantial evidence in favour of the notion that PBMCs provide a window into the CNS holds particular relevance in neurodegenerative disorders in which, unlike most other diseases, the affected tissue is not directly accessible to evaluation. On a final note, it should be mentioned that the value of biochemical dysfunctions in PBMCs as mirrors of CNS defects appears to extend well beyond dementia.For instance, FAAH and other elements of the endocannabinoid system show alterations in the blood that resemble those within the CNS in a broad spectrum of clinical conditions including Parkinson's and Huntington's disease, multiple sclerosis, schizophrenia, minor depression, and headache .Nowadays we do not know if PBMCs biomarkers are better or worse than the CSF biomarkers. Our study is only a preliminary study, instead multiple studies have examined CSF to establish sensitivity and specificity of CSF biomarkers. Moreover, despite these many studies, a large variability exists in the literature as to CSF biomarker diagnostic accuracies and cut-offs. As biomarker discovery in PBMCs is an ongoing process and PBMCs biomarkers are still immature, we need further analysis to enlarge design population.It will be also of relevance the possibility to utilize intracellular biomarkers in specific blood cell subpopulations. In fact the differences in between subjects could also be due to different composition of their PBMCs pools, even if separating PBMCs into subpopulations would not permit the cell-cell interactions required for activation of lymphocytes.Finally, we assume that the combination of peripheral and CSF markers may be utilized to categorize patients since early stages of dementia and to understand mechanisms underlying dementia.