Pancreatic ductal adenocarcinoma (PDAC) is the most prevalent form of pancreatic cancer and carries the worst prognosis of all common cancers. Five-year survival rates have not surpassed 6% for some decades and this lack of improvement in outcome urges a better understanding of the PDAC-specific features which contribute to this poor result. One of the most defining features of PDAC known to contribute to its progression is the abundance of non-tumor cells and material collectively known as the stroma. It is now well recognized that the different non-cancer cell types, signalling molecules, and mechanical properties within a tumor can have both tumor-promoting as well as –inhibitory effects. However, the net effect of this intratumour heterogeneity is not well understood. Heterogeneity in the stromal makeup between patients is even less well established. Such intertumour heterogeneity is likely to be affected by the relative contributions of individual stromal constituents, but how these contributions exactly relate to existing classifications that demarcate intertumour heterogeneity in PDAC is not fully known. In this review, we give an overview of the available evidence by delineating the elements of the PDAC stroma and their contribution to tumour growth. We do so by interpreting the heterogeneity at the gene expression level in PDAC, and how stromal elements contribute to, or interconnect, with this. The most common form of pancreatic cancer (PC) is pancreatic ductal adenocarcinoma (PDAC), which represents over 85% of all PC cases. PDAC is ranked as the fourth leading cause of cancer-related deaths, with a median 5-year survival rate of 3% in Europe ,2,3,4. IOn a histological level, one of the most remarkable features of PDAC is a dense stroma. The stroma can constitute up to 90% of the tumour bulk and is characterized by extensive fibrosis, lack of vascularization, immune infiltrates, and hypoxia . This fiThe interaction between stromal and pancreatic cancer cells that together form the tumour microenvironment (TME) plays a crucial role in PDAC development and progression. Two distinct stroma subtypes have been identified that are independent of tumour subtype and impact on patient survival . TherefoThe development of PDAC is thought to be a sequential process that relies on the accumulation of mutations. PDAC tumours can evolve from pre-malignant precursor lesions; mucinous cystic neoplasms (MCN), intraductal papillary mucinous neoplasms (IPMN) and pancreatic intraepithelial neoplasia (PanIN). PanINs are the most common and widely studied precursor lesions. These lesions are classified into three grades based on their morphology, ranging from low grade PanIN-1A/PanIN1B to advanced grade PanIN3s . ElementActivating mutations in KRAS are a hallmark of PDAC, and are found in nearly 95% of cases ,16. The Whole genome sequencing and copy number variation (CNV) analyses have been used to classify PDAC into four subtypes by patterns of structural genetic variation. These 4 subtypes range from stable, locally rearranged, scattered, to an unstable subtype, all referring to the degree of structural variations present in the genome . At a loSeveral signalling pathways in PDAC contribute to the development and maintenance of the stroma. One of the most prominent is the role of the Hedgehog (Hh) pathway in PDAC progression, as was demonstrated in 2003 . Later, The sum of the complex contributions of the many deregulated genetic, epigenetic and signalling pathways in PDAC, as well as any extracellular influences, can be best captured at the mRNA level. Unsupervised classification methods of the transcriptome have aimed to classify pancreatic cancer based on their expression patterns ,20,32,33These subtypes are the result of inter- and intra-tumour heterogeneity, and are the sum of differences in morphology, cellular composition, genetic makeup, and many other levels of biological information that exist between or within cancers, respectively. This heterogeneity can have consequences for the diagnosis of a tumour, as well as for the treatment. The onset of intra-tumour heterogeneity for tumour cells is thought to evolve from a cancer stem cell model or a clonal evolution model . The canThese models are typically tested in tumour cell models only, and the impact of the stroma is normally not taken into account. In order to take the stromal contributions into consideration we must know the constituents of the stroma.The ECM normally functions to maintain tissue integrity by providing structural and biochemical support to cells. The ECM is constantly remodelled by enzymes like matrix metalloproteases (MMPs) and tissue inhibitor metalloproteinases (TIMPs). The ECM can be modified in abundance and composition, and this is important for example in wound healing. In PDAC both the pancreatic cancer cells and the stromal cells act on the ECM by the secretion of structural components and modifying enzymes ,37. In hThe main component of the ECM is collagen I, which promotes cell adhesion, proliferation and increased migration via α2β1 integrins by disrupting cell-cell adhesion contacts in normal tissue ,39. The snail and LEF-1 [Decreased expression of E-cadherin in tumour cells is initiated by collagen I signalling via the upregulation of Smad-interacting protein 1 and activation of focal adhesion kinase (FAK) ,41. The nd LEF-1 ,44. All nd LEF-1 .Laminins are heterotrimeric glycoproteins that consist of three chains, the α-, β- and γ-chain. They contribute to cell attachment and differentiation, mainly via integrin receptors . FibroneDetachment of the ECM in normal tissue induces apoptosis by cytochrome-c release and subsequent caspase activation. Laminin and fibronectin both contribute to the resistance against this cytochrome-c induced apoptosis and increase PDAC tumour cell survival by inhibiting caspase activity . BesidesProteoglycans are small heavily glycosylated proteins, and function to bind ECM components like collagens. Proteoglycans can be expressed by tumour cells as well as stellate cells. Glypican-1 is a proteoglycan overexpressed in PDAC tumour cells, involved in tumorigenicity . ExosomeHyaluronic acid (HA) is a glycosaminoglycan. It is formed in the plasma membrane instead of the Golgi membranes, allowing for large HA chains to be formed . The molThe ECM has been found to reduce the efficacy of chemotherapeutic agents against tumour cells in vitro ,59. The Clinical trials targeting ECM proteins have shown promising results: Combining hyaluronidase (HA degrading enzyme) to gemcitabine increases the overall survival of patients compared to single treatment, though this treatment was only beneficial in patients with high levels of HA . An indiThe normal vasculature is relatively quiescent, and the endothelial cells (ECs) that line blood vessels only divide about once every couple years . Blood vThe formation of new blood vessels is called angiogenesis. The secretion of angiogenic factors and proteolytic enzymes in the TME ensures the formation of new blood vessels. Key regulators of the initiation of angiogenesis are cytokines, growth factors and hypoxia. The regulation of angiogenesis is unclear and very complex in pancreatic tumours. Many pro- and anti-angiogenic factors are produced in the TME.Various cell types in cancer tissue such as tumour cells, PSCs, and infiltrated immune cells respond to hypoxia by the secretion of factors that act on angiogenesis. For instance, pancreatic cancer cells secrete large amounts of the anti-angiogenic endostatin in response to hypoxia, further contributing to a hypoxic environment . In contThe effect of PSCs on angiogenesis is heterogeneous within tumours, as we can conclude from the fact that the pan- and juxtatumoral-stroma show a difference in vascularization. The juxtatumoral-stroma is the stroma in close proximity to the tumour cells mostly defined as the <100 µm from the tumour cells, whereas the pan-stroma comprises the rest of the stroma. The juxtatumoral stroma is hypovascularized due to tumour cell-secreted endostatin whereas the pan stroma, often associated with the invasive front of a tumour, presents a higher density of blood vessels, as was shown by the analysis of tissue microarrays . In vitrThe lack of vascularization not only deprives the tumour of oxygen but also nutrients, causing metabolic stress. The metabolism of a tumour cell needs to be altered, as other nutrient sources need to be used. KRAS mutated cells are able to survive metabolic stress for instance by the uptake of extracellular proteins and lipids by scavenging . AnotherThe cellular response to severe hypoxic conditions is mediated by hypoxia inducible factor 1α (HIF-1α), to help cells to cope with cellular stress ,83. HIF-Hypoxia is a mediator of chemoresistance and exerts its effect via several mechanisms. Hypoxia is reported to affect chemosensitivity through Hh pathway upregulation, stimulation of NF-κB signalling, or ERK1/2 signalling and reduction of chemotherapy-induced ROS ,91,92,93CAFs are the major cellular component in the PDAC stroma and play a key role in PDAC development and progression ,97. CAFsIn the healthy pancreas PSCs comprise 4–7% of the pancreatic parenchymal cells and are characterized by expression of desmin, vimentin, nestin and glial fibrillary acidic protein (GFAP) . The maiDuring activation, quiescent PSCs lose their vitamin A lipid droplets and start expressing α-SMA . The actCompelling evidence supports the existence of an essential interaction between PDAC tumour cells and PSCs that results in an increased tumour growth and metastases ,109. TumIn preclinical mouse experiments, targeting the stroma with Hh pathway inhibitors and PDAC tumour cells with chemotherapy enhanced the survival time of mice . Phase IActivated PSCs play an important role in chemoresistance, either by a mechanical protective role of the PSCs, or through secreted proteins. Addition of PSC-conditioned medium in vitro protects tumour cells from gemcitabine treatment or radiotherapy . This prAs 90% of the tumour bulk can be occupied by the stroma, and this stroma is mainly occupied by PSC-derived CAFs, these cells determine the fate of PDACs tumours to a great extent. An example of this stromal influence is evident from the analysis by Moffitt et al. [Immune cells are attracted to inflammatory signals and normally function to protect the body from infectious diseases caused by pathogens. The balance between pro- and anti-tumorigenic factors, produced in the TME, determine the function of infiltrated immune cells . DependiMacrophages are essential in tissue homeostasis and defence against foreign pathogens. Resident macrophages secrete a wide range of chemokines and cytokines, triggering immune cell recruitment . MonocytResident and infiltrated macrophages present within the TME are called tumour-associated macrophages (TAMs) and have been identified as one of the most abundant immune infiltrated cells in PDAC . High amTherapeutic strategies aiming to reprogram TAMs into an effective anti-tumour activity have started to emerge. Low dose irradiation in xenotransplant mouse models reprogrammed M2-like to M1-like macrophages, which resulted in effector T-cell recruitment . SimilarNeutrophils are another first line of immunity, characterized by their ability to phagocytose, release granules containing lytic enzymes, and to produce ROS . NeutropThe neutrophil to lymphocyte ratio (NLR) is a prognostic factor that compares the absolute neutrophil and lymphocyte count pre-operatively in blood samples, wherein a high NLR is associated with poor prognosis . NeutropThe second line, or the so-called adaptive immune response is mediated by Lymphocytes. Lymphocytes that infiltrate the tumour are called tumour-infiltrated lymphocytes (TILs). Different subsets of TILs are present in the TME. Th1 and cytotoxic T cell functionality are generally impaired whereas the function of Th2 and Treg cells are enhanced. In accordance with this, PDAC patients with high Th1 and cytotoxic T cell counts have a good prognosis . Tumour TIL differentiation into Tregs is regulated by immunosuppressive ligands, mainly expressed by activated PSCs, like TGF-β, IL-10 and IL-2 . Tumour Studies identifying prognostic markers have found many markers for immune system components that positively correlate with survival. In contrast, high macrophage infiltration, especially Th2 type macrophage infiltrations, are associated with a worse outcome . These cOur understanding of the biology that underlies the growth and progression of tumours has benefited enormously from the identification of gene expression-based subtypes, determined by both supervised and unsupervised classification methods. A large number of studies have employed supervised clustering to classify gene expression profiles using clinical parameters such as survival and metastatic propensity ,156,157.n = 27) [n = 34) and by combining the transcription profiles from another study (n = 36). Three prognostic subtypes were identified: classical, quasi-mesenchymal (QM) and exocrine-like, that could be identified by a classifier called PDAssigner. The classical subtype is one that is defined by high expression of adhesion specific and epithelial genes and has the best survival. The QM subtype showed higher expression of mesenchymal genes, and was associated with the poorest prognosis. The exocrine subtype was associated with the expression of digestive enzyme related genes. This subtype however was not found in cancer cell lines, and this has led to some discussion on how tumour-specific the exocrine subtype is.As the first to classify PDAC into gene expression based subtypes, Collisson et al. were hindered by the paucity of pancreatic tissue available for sequencing (n = 27) . This wap = 0.001). The molecular gene signatures were compared to the PDAssigner from Collisson et al. and subtype 3 had similar defining differentially expressed genes as the exocrine subtype, arguing in favour of the existence of the exocrine subtype. No comparisons were shown to correlate type 1 and 2 subtypes to the PDAssigner.This classification of 96 PDAC samples resulted in three gene expression-based subtypes . The subTo delineate the tumour-specific gene expression in PDAC, Moffitt and colleagues incorporated primary tumour, metastatic tumour and normal samples . By perfUsing a set of 27 PDAC patients, two distinct GEPs (Gene Expression Profiles) were identified via PCA and hierarchical clustering . GEP-A wFor this study 118 tumour PDAC samples and 13 normal pancreas samples were sequenced . The setExpression analysis on 96 tumours was performed by RNA-Seq in addition to the genomic analyses on 456 samples . UnsuperCombined genomic, transcriptomic and proteomic analyses of 150 PDAC patient samples were performed with whole exome sequencing (WES), RNAseq, miRNAseq, DNA methylation and reverse-phase protein array (RPPA) . GenomicAs apparent from the classification described by Moffitt et al. the stroma in PDAC can show substantial differences in activation status and contribute significantly to bulk tumour heterogeneity and patient outcome. In this review, we have given an overview of the non-tumour cell populations that contribute to intertumour heterogeneity in bulk PDAC tissue, and summarized this in We hypothesize that the stromal influences on bulk tumour heterogeneity are based on three principles: (1) A differential composition of the stroma, in which “the stroma” is just the sum of the gene expression of these individual components. For example, a relatively high fraction of immune cells infiltrated in a tumour will yield an “immune” subtype tumour, whereas a paucity of (myo)fibroblasts is likely to result in a “classical” or canonical/epithelial subtype. As the tumour host and thus the stroma are genetically homogenous, a differential cellular composition of the stroma is likely to be the result of differential instruction from the genetically instable tumour cells. One observation that argues against this is that Moffitt’s activated stroma subtype does not specifically associate with the classical or basal subtype. (2) It is also plausible that within defined stromal cell populations, such as the fibroblasts, the crosstalk with tumour cells has yielded a very distinct gene expression pattern for that population of cells that is as heterogeneous between patients as the tumour cell compartment is. Again, this heterogeneity likely originates from early events in the tumour cells that impact on the stromal cells (for instance a SMAD4 mutation ), but itFrom the above, a “chicken or the egg” causality dilemma is apparent. Do the genetically driver tumour cells first instruct their surroundings, attracting their own specific TME, and thus determine their own fate? This would mean that the TME initially has a low heterogeneity and that during the development of the tumour, the heterogeneity of the TME evolves along. Or is the establishment of the TME stochastically coordinated, in other words; the TME largely determines its own fate by chance, and subsequently of the tumour cells as well. If a complex crosstalk loop is indeed required throughout tumour progression, stochastic events in the stroma could be given the chance to exert long-lasting and significant effects. The complex interactions between stromal cell populations allow and support such cross talk. For instance, an additional layer of complexity in the stroma comes from the differential regulation of vascularization under the influence of PSCs, which are instructed by tumour cells. In turn, these effects on vascularization are likely to impact on immune infiltration further adding to heterogeneity.A more daring hypothesis is that the development of the stroma is largely uncoupled from the tumour compartment, and that the interactions within the stroma are sufficient to generate heterogeneity that is measurable at the whole tumour level. Along those lines, more systemic patient characteristics could initiate stroma heterogeneity; for instance, if a given patient has high numbers of circulating mesenchymal stem cells this could result in a very fibroblast-rich tumour that will likely classify as “mesenchymal”. A similar scenario could be envisioned for immune cells and the resulting tumour bulk subtype. Experimental models that allow longitudinal assessment of intertumour heterogeneity from onset (PanIN) to late stage disease to test these hypotheses should yield much needed insight.Concluding, the classification of PDAC at the bulk tumor gene expression level holds promise for clinical decision-making but this will take time and rigorous validation. Perhaps it could be argued that, given the important contributions of stromal cells to these classifications and also the abundance of this tissue in PDAC, we should investigate treatments that specifically consider these stromal contributions to tumor biology, and importantly, the heterogeneity that exists in this compartment. This notion is supported by the advent of clinical trials using stroma targeting agents , but the


Retinoblastoma is the most common intraocular malignancy occurring in children. It can metastasize to the regional lymph nodes, central nervous system and distant organs usually the bones and bone marrow and very rarely to the soft tissue. Here, we report a case of unilateral retinoblastoma in a 4-year-old girl accompanied by a large metastasis of the parotid and submandibular glands that developed about 6 months previously and gradually increased in size 5 months after enucleation of the left eye.A 4-year-old girl with a history of unilateral retinoblastoma presented with a large, painful and worsening mass (about 20 × 23 cm) of the left side of the neck. Following surgery, the orbital tumour was completely resected, and the large tumour invasion range in the left side of the neck was not resected completely. Histopathological examination revealed retinoblastoma of the orbit and the parotid and submandibular glands. After chemotherapy and additional local radiotherapy on the parotid and submandibular glands, the tumour was inactive and stable.Delayed detection and inappropriate management contribute to poor outcomes. Fundus examinations, education regarding the early signs of RB, and optimization of the therapeutic strategy for RB may play important roles in ocular health. Retinoblastoma is the most common intraocular malignancy in children, with a reported incidence ranging from 1 in 15,000 to 1 in 18,000 live births –5. The fA 4-year-old girl was admitted to our hospital in November 2014 with a large, painful and worsening mass (about 20 × 23 cm) on the left side of the neck Fig. . DependiA systemic workup including bone marrow biopsy and cerebrospinal fluid (CSF) analysis was also carried out. The bone marrow was negative for tumour deposits of malignant round cells. The CSF cytology was within normal limits. Based on this, a diagnosis of stage IVa metastatic retinoblastoma (a distant metastatic retinoblastoma without CNS involvement) was made. The systemic condition of the child was poor.A one-stage surgery was conducted immediately. The orbital tumour was completely resected in December 2014. Since the patient had a large tumour invasion range in the left side of the neck not suitable for complete resection, the tumour was resected as much as possible while retaining organ functions. Histopathological examination revealed orbital retinoblastoma and a large intraparotid mass. The immunohistochemical staining results were as follows: Syn(++), β-micprotin(++), Ki-67, CD56(++), NSE(++), Rb(++), MyoD1(++), Fli-1(+), S-100(-), CgA(-), CD99(-), CD57(-), vimentin(-), EMA(-), myogenin(-), Pax-5(-), CK18(-), LCA(-), and desmin(-). Metastasis of the parotid and submandibular glands was confirmed, and the patient underwent systemic evaluation for any other site of metastasis; however, there was no other organ involvement.The patient was started on a 3-weekly high-dose chemotherapy protocol with vincristine, etoposide and carboplatin (VEC regime). Following six cycles, the left orbital tumour had regressed completely, there was a small residual tumour in the left side of the neck, and additional local radiotherapy was performed on the parotid and submandibular glands Fig. . The girRetinoblastoma is the most commonly diagnosed malignancy in children. The worldwide incidence of retinoblastoma has been reported to be 1 in 18,000 to 30,000 live births. It accounts for 3% of all childhood malignant tumours in developed countries. Metastasis of retinoblastoma is also rare in the developed world, with a reported incidence ranging from 4.8% in the United States to 5.8% in the United Kingdom, where early diagnosis achieves a cure for most children . HoweverRetinoblastoma can metastasize to the regional lymph nodes, CNS and abdominal organs. The most common site of metastasis is the intracranial region, accounting for approximately 50% of cases . Bone isJubran et al. suggested four patterns of metastatic retinoblastoma, each having different clinical features and outcomes: trilateral retinoblastoma, regional metastasis, extension into CNS and distant metastasis . CarbajaOptic nerve involvement is the most important risk factor for CNS metastasis, whereas extrascleral invasion is reported to be the most significant risk factor for distant metastasis as the tumour gains access to vascular and lymphatic channels outside the eye . Other rA definitive cure for intraocular retinoblastoma with no possibility of useful vision is achieved by removal of the eye before the tumour spreads , 29. HowThe prognosis remains relatively poor for patients whose disease disseminates into the CNS and those with distant metastatic disease . There iA definitive cure for intraocular retinoblastoma with no possibility of useful vision is achieved by removal of the eye before the tumour spreads. Additional adjuvant chemotherapy and radiotherapy followed by appropriate surgery are advised after operation to reduce metastasis. When the tumour extends beyond the globe into the orbit or metastasizes to a distant site, a combination of radiation therapy and high-dose chemotherapy should be used. Delayed detection and inappropriate management contribute to poor outcomes. Fundus examinations, education regarding the early signs of RB, and optimization of the therapeutic strategy for RB may play important roles in ocular health. Early detection, accurate diagnosis, and active intervention are conducive to control of retention of patients’ vision. A better referral system and/or an education programme for referring physicians are needed to prevent such cases in the future.


The advent of rapid and inexpensive sequencing technology allows scientists to decipher heterogeneity within primary tumours, between primary and metastatic sites, and between metastases. Charting the evolutionary history of individual tumours has revealed drivers of tumour heterogeneity and highlighted its impact on therapeutic outcomes.Scientists are using improved sequencing technologies to characterise and address the challenge of tumour heterogeneity, which is a major cause of resistance to therapy and relapse. Heterogeneity may fuel metastasis through the selection of rare, aggressive, somatically altered cells. However, extreme levels of chromosomal instability, which contribute to intratumour heterogeneity, are associated with improved patient outcomes, suggesting a delicate balance between high and low levels of genome instability.We review evidence that intratumour heterogeneity influences tumour evolution, including metastasis, drug resistance, and the immune response. We discuss the prevalence of tumour heterogeneity, and how it can be initiated and sustained by external and internal forces. Understanding tumour evolution and metastasis could yield novel therapies that leverage the immune system to control emerging tumour neo-antigens. In his 1958 essay, Foulds  cancer eRecent technological advances have allowed scientists to study tumour complexity in more detail. Following exome sequencing on multiple spatially separated samples obtained from primary carcinomas and metastatic sites, intratumour heterogeneity and parallel evolution of subclonal driver events was uncovered –7. MultiIntratumoral heterogeneity exists in many forms, from somatic coding and non-coding alterations to epigenetic, transcriptomic and post-translational modifications , 17. IntCahill and Vogelstein  discusseMost normal diploid cells negatively select against chromosomal instability (CIN). This is partially mediated by p53, which inhibits cell propagation after genome instability , 33. CINBCL9L promote tolerance of chromosome missegregation events, propagation of aneuploidy and genetic heterogeneity [In tumours, selection might favour the mitigation of excessive CIN to prevent cell autonomous lethality. Partial dysfunction of anaphase-promoting complex/cyclosome (APC/C) lengthens mitosis, allowing more time for correction of impending chromosome segregation errors. This permits tumour cells to fine-tune CIN during tumour evolution, navigating the delicate equilibrium between cell death as a result of too much or too little genomic instability . In coloogeneity . This toogeneity .Once cells have undergone genome doubling (GD), propagation of aneuploidy follows as the tolerance of GD allows cells to endure continuing CIN . One mecThe study of intratumour heterogeneity has yielded novel findings about the timing and evolutionary drivers of metastasis. Metastasis is a multistep process consisting of local invasion, intravasation, survival in the circulation, extravasation and distant colonisation , 47.Two prominent models of metastasis are the linear and parallel progression models –48. BothIn colorectal, breast, pancreatic, renal, and prostate cancer, multiregional sequencing supports both metastatic models . Both moThese data suggest metastasis is complex and diverse, and that tumour heterogeneity is critical in the metastatic process. Since more work is required to understand tumour heterogeneity in late stage tumours and metastases, in depth efforts are now examining post-mortem tumour heterogeneity. The UK national PEACE autopsy  program, may help to decipher the complexity cancer evolutionary processes at death.It was recognised in the 1970s that heterogeneous subclones within tumours contain differing capacities for growth and metastatic ability –57. EarlDrosophila melanogaster [D. melanogaster model of epithelial tumour formation to show that overexpression of both epidermal growth factor receptor (EGFR) and miR-8 in wing imaginal disc cells results in supercompetitive cells that engulf those surrounding them. These supercompetitive cells drive tumourigenesis and metastasis, whereas cells overexpressing either EGFR or miR-8 alone do not. Competition between normal and oncogenic tissues was also uncovered: in D. melanogaster, imaginal epithelial cells activate nonapoptotic JNK signalling in response to oncogenic mutant cells [D. melanogaster studies demonstrated cooperation between cells with oncogenic RasV12 mutations and cells lacking the scribbled gene (scrib-/-) to promote growth and invasion of the RasV12 mutant cells [Growing interest in tumour heterogeneity and subclonal interactions led to the development of a useful model for studying subclonal competition and cooperation in nogaster . Eichenlnogaster  used a Dnt cells . This lent cells . Growingnt cells , 63.high) melanoma cells cooperated with poorly invasive (MITFlow) cells to invade away from the primary site through solid tissue [high cells around the primary tumour was critical for this co-invasion of MITFhigh and MITFlow cells [Xenotransplant models have been used to understand interactions between human tumour subclones. In a glioblastoma multiforme mouse xenotransplant model, a minor mutant EGFR subpopulation enhanced tumourigenicity of the entire tumour. This occurred via a paracrine mechanism that promoted growth in wtEGFR cells within the tumour, illustrating the advantages of heterogeneity . In a zed tissue . The proow cells . HeterogPolyak and colleagues  used a mThese xenograft studies provide insights into subclonal interaction dynamics, demonstrating their complexity, and that they are involved in many aspects of tumour evolution including tumorigenesis, vascularisation, invasion and metastasis.Advances in molecular biology have allowed a deeper study of subclonal cooperation in cancer mouse models. Autochthonous models permit long-term study of clonal dynamics in a specific genetic context .V12, and the mixed population endowed mesenchymal cells with metastatic ability [In a mouse model of small cell lung cancer (SCLC), tumours were composed of distinct cell populations with either neuroendocrine or mesenchymal markers . Both ce ability .HrasmutWntlow and luminal HraswtWnt1high subclones, which cooperated to maintain tumour propagation [Cleary and colleagues  used a mpagation . AlthougIn a p53-null mouse model of basal-like breast cancer, two tumour cell populations were identified, one expressing mesenchymal markers , and another defined as tumour-initiating cells (TICs) . When boMouse models have revealed how subpopulations within tumours cooperate to promote tumour growth, maintenance and metastasis. Although not fully representative of the clinical presentation of disease in patients, these autochthonous models show the diverse ways in which subclones can interact and affect tumour progression. These studies suggest that to more effectively treat heterogeneous tumours, we must first understand the dynamics between different populations within a tumour, and how targeted treatment changes these interactions.de novo.Therapeutic resistance often develops in the advanced setting following therapeutic targeting of clonal alterations, as subclones containing somatic events driving resistance can pre-exist or be generated In non-small cell lung cancer (NSCLC) a low frequency subclone with MET amplification was selected for during treatment with EGFR tyrosine kinase inhibitors (TKI), allowing for development of a resistant MET- amplified tumour . The EGFIn patients with HER2-positive breast cancer, Janiszewska et al.  discoverKRAS clones emerging during treatment with EGFR-specific antibodies declined during treatment breaks [In patients with colorectal cancer, mutant t breaks . Engelmat breaks  demonstrt breaks .These studies demonstrate that heterogeneous tumours develop resistance when specific clones are targeted, allowing pre-existing or newly evolved subclones to emerge.Competition between subclones is frequently revealed during and following therapy exposure. Keats and collaborators  illustraIs intratumour heterogeneity associated with worse patient outcomes? In premalignant Barrett’s oesophagus, clonal diversity is an important predictor of tumour progression . By adapIn breast, ovarian, gastric and NSCLC, there is a paradoxical relationship between CIN and prognosis. The worst outcomes are in tumours with intermediate CIN, while those with extreme CIN scores had an improved outcome , 82. TheIn a recent study of 12 different cancer types , mortaliPTEN and RB1, and the other enriched in oncogenes, including MYC, and in putative regulatory elements of genes such as ESR1, with direct effects on transcription [In breast cancer, enriched areas of tandem duplications previously thought to be unimportant passengers, were shown to be potentially important for tumourigenesis . These tcription . These eEmerging evidence suggests that intratumour heterogeneity may also influence the anti-tumour immune response. Rapid advances in cancer genome sequencing have enabled scientists to decipher the impact of somatic coding alterations upon immune recognition and surveillance. Non-silent mutations generate neoantigens that can be recognised by the immune system , 89. BotIn patients with melanoma, whole exomes from pre-treatment melanoma tumour biopsies and matching germline tissue samples were examined to determine if neoantigen burden affects responses to antibodies directed against cytotoxic T lymphocyte-associated antigen-4 (CTLA4) . OverallRizvi and colleagues  exploredWe investigated whether melanoma and NSCLC sensitivity to PD-1 and CTLA-4 treatment was enhanced in tumours where tumours had a high clonal neoantigen burden. Considering a combination of neoantigen clonality and neoantigen burden allowed us to better discriminate responder from non-responder patients than either metric alone . These fMultiple laboratories are now focusing on a deeper understanding of immune evasion and resistance to immunotherapy. One study  uncovereT cell exhaustion is another mechanism of immune evasion. This state of T cell dysfunction arises during chronic antigen exposure, which is defined by poor effector function, sustained expression of inhibitory receptors, and an abnormal transcriptional state –103. In Alterations in the genes encoding interferon receptor-associated Janus kinase 1 (JAK1) or 2 (JAK2), along with deletion of the wild-type allele, lead to defects in pathways involved in interferon receptor signalling and in antigen presentation. This results in resistance to PD-1 blockade in melanoma . Loss ofWe have begun to witness how tumour heterogeneity affects tumour progression and metastasis, and the tumour immune response. A deeper understanding of how the immune system can be leveraged to tackle clonal alterations within tumour cells is required, to identify high-risk tumour subclones that might be extinguished prior to, or at the time of, metastatic seeding. Longitudinal characterisation of tumour evolution using advanced sequencing technology is important, as will be characterisation of the distinct mechanisms of tumour heterogeneity from tumour diagnosis through to progression and death. These studies may open avenues to attenuate tumour heterogeneity in minimal residual disease when disease burden is low, or to target multiple clonal mutations present in every cell simultaneously to limit the acquisition of therapy resistance.As tumour heterogeneity is arguably the major force behind tumour progression, evolution and metastasis, insight into the clonal complexity of individual tumours and its contribution to progression, immune evasion and exhaustion may be critical to the development of more effective cancer therapeutics.


The temporal resolution of sub-relativistic ultrafast electron diffraction (UED) is generally limited by the radio frequency (RF) phase and amplitude jitter of the RF lenses that are used to compress the electron pulses. We theoretically show how to circumvent this limitation by using a combination of several RF compression cavities. We show that if powered by the same RF source and with a proper choice of RF field strengths, RF phases, and distances between the cavities, the combined arrival time jitter due to RF phase jitter of the cavities is cancelled at the compression point. We also show that the effect of RF amplitude jitter on the temporal resolution is negligible when passing through the cavity at a RF phase optimal for (de)compression. This will allow improvement of the temporal resolution in UED experiments to well below 100 fs. I.010 modeA successful method to improve the temporal resolution in sub-relativistic pump-probe ultrafast electron diffraction (UED) experiments is the use of a resonant radio frequency (RF) cavity in the TMHowever, RF phase instabilities in the synchronization system lead to variations in the arrival time of the electron bunches, thus limiting the temporal resolution of UED experiments to a few 100 fs.010 cavities, depending on the velocity chirp of the incoming electron beam. If powered by the same RF source and with a proper choice of RF field strengths, phases, and distances between the cavities, the combined phase jitter is cancelled at the compression point. The effect of amplitude instabilities can be minimized by operating the compression cavity at a RF phase for optimal (de)compression. In this way, the temporal resolution can be improved substantially.This paper theoretically describes how to eliminate the RF phase jitter using two or three TMboth phase and amplitude fluctuations of the RF field in the compression cavities. Finally  the on-axis longitudinal electric field amplitude, ω the angular frequency, and zp an electron acquires by traveling through an RF cavity is given bye the electron charge, E0 = E(0) the electric field strength at the center of the cavity, zv the average speed of the electron bunch, and f of a such a cavity is given bym the electron mass and v ≈z v.The principle of using resonant RF cavities as longitudinal lenses for sub-relativistic UED is an established technique which is described in Refs. zp of the electron pulse passing through the cavity is zero if the center of the bunch passes through the center of the cavity when the RF electric field goes through zero, i.e., Equation ε, and the focal length of the lens. This leads to arrival time fluctuations δt at a distance d from the cavity,RF phase variations both RF phase and amplitude fluctuations at some point behind the two cavities. For optimal (de-)compression, i.e., ε are a second order effect.Equation ω = 2π⋅3 GHz and f = d, we find phaseδt ≈ 110 fs. Solid state RF amplifiers are commercially available with a RF amplitude stability of δP = 5 × 10−4, which results in an electric field amplitude stability of ampδt = phaseδt⋅ε  ≈  28 as. Clearly, the amplitude contribution to the arrival time jitter is negligible, owing to it being a second order effect. The validity of Eq. We can illustrate this with a numerical example: state-of-the-art synchronization by an RF phase locked loop system has a typical residual phase RF phase jitter ε  =  0) position of the waist. The Courant-Snyder wτ the pulse length at the longitudinal waist and RF amplitude fluctuations also cause the longitudinal focus to shift position, thus resulting in bunch length fluctuations at the nominal  longitudinal emittance ε < 0.1, which is easily achievable.For 100 keV electrons, A.We will now first derive how to longitudinally compress an electron pulse by using a two lens focussing system, as is illustrated in Fig. f1 < 0. This lens stretches the electron bunch; the second lens is a positive lens with a focal length f2 > 0. This lens is used to compress the electron bunch, as illustrated in Fig. lensd. The longitudinal divergence of the incoming electron beam is parameterized by the length d0, which is the distance of the focal point with respect to the position of the first lens if The first lens is a negative lens with focal length d0 > 0 corresponds to a converging beam which is longitudinally focused a distance d0 behind the first cavity, as is schematically indicated in Fig. d0 < 0 represents an diverging electron beam which originates from a beam waist a distance d0 before the first lens.focusL with respect to the second lens  the position of the focal point becomesIn the case that the first lens collimates the incoming converging beam  due to the first cavity (lens 1) is given bySimilarly, the arrival time jitter at a distance jitterL behind the second cavity (lens 2) due to the second cavity is given byThe arrival time jitter at a distance jitterL behind the second cavity (lens 2) when δt1L + δt2L = 0 which shows that both phase and amplitude ε variations cancel in first order. The point where there is no jitter is given byjitterL > 0.There will be no arrival time jitter at a distance jitterL has to overlap with the longitudinal focal point focusL. These points overlap when the following equation holds:d0 > lensd since Eq. f2 < lensd – f1. The point where the pulse length is independent of RF phase fluctuations for all electrons inside a bunch is then given byTo improve the temporal resolution of UED experiments, the no-jitter point d0 > lensd > 0). This means that in order to cancel the phase jitter in an RF focussing system with two cavities, we need an already focussing electron beam. This is the case for an electron beam which is extracted from a longitudinally extended source such as a laser cooled gas.This means that overlapping the focal point and the no-jitter point is only possible for beams with negative energy chirp  with a two lens focussing system, as schematically indicated in Fig. d0 = 700 mm, lensd = 200 mm, and f1 = −1000 mm.Subsequently, we simulated the elimination of the arrival time jitter in the longitudinal focus by using two RF cavities. According to theory Sec. , we can f2, ranging from f2 = 750 mm to f2 = 1000 mm in steps of 50 mm. The dashed lines indicate the positions of the first and second cavities. The figure shows that we enter the first cavity with a negatively chirped bunch. The first cavity defocusses (f1 < 0) the electron bunch; the front gets accelerated and the back decelerated. The second cavity focusses (f2 > 0) the electron beam.Figure f2 = 750 mm to f2 = 1000 mm in steps of 50 mm. At certain positions behind the second cavity, the arrival time difference cancels out. The dashed lines again indicate the positions of the first and second cavities.Figure jitterL. Similarly, we can determine the focal point focusL from Fig. jitterL (circles) and focusL (squares) as a function of the focal length f2. The solid black curve was calculated using Eq. ε  =  0. The solid grey curve was calculated using Eq. ε  =  0. The theoretical curves perfectly describe the simulations. At the position where the longitudinal focus and the zero jitter point intersect, we find a longitudinal waist that is insensitive to arrival time jitter due to RF phase fluctuations.From Fig. ε  =  0 arrival time as a function of the relative amplitude variations ε. The circles represent the simulated results and the solid black curve was calculated using Eq. Figure ε. The circles represent the simulation results and the solid black line was calculated by using Eq. Finally, Fig. We therefore conclude that the analytical theory perfectly agrees with realistic charged particle simulations.IV.filterf of the jitter that can be removed is limited by the time it takes an electron to travel between the cavities: filterf ≈ 200 MHz per meter distance between the cavities.The highest frequency gunδt at a distance d from the gun due to relative beam energy fluctuations In this paper, we have assumed that the arrival time of the electron bunch at the first RF cavity does not vary in time. Average longitudinal energy fluctuations will result in additional arrival time jitter at the first cavity and thus at the longitudinal focus, limiting the temporal resolution. This will be the limiting factor on the temporal resolution if the arrival time jitter due to RF phase fluctuations is completely cancelled. The arrival time jitter d = 1 m, an electron beam energy of 100 keV and relative energy fluctuations gunδt = 23 fs. This is easily achievable for DC photoguns.As an example, at a distance gunδt scales with gunδt = 15 fs for the same conditions as used above.For 1 MeV electron guns, the arrival time fluctuations due to gun jitter will be even lower since This shows that our method should improve the temporal resolution of UED experiments significantly for both sub-relativistic and relativistic UED experiments.V.010 mode. If powered by the same RF amplifier and with specific values for the distances between the cavities, the focal lengths, and the RF phases, the RF jitter can be canceled at the position of the longitudinal focus. If the incoming electron bunch is longitudinally converging, i.e., with a negative chirp, a set of minimally two RF cavities is required. When the incoming bunch is longitudinally diverging, i.e., with a positive chirp, a set of minimally three cavities is required. The analytical theory results are confirmed by charged particle simulations. This means that we can improve the temporal resolution of UED experiments to well below 100 fs by creating a jitter free longitudinal focus allowing both phase and amplitude variations.We have theoretically shown that we can eliminate RF phase jitter in an RF bunch compression system by using a set of two or three RF cavities operated in the TM


Scientific Data 4:170052 doi: 10.1038/sdata201752 (2017); Published 25 April 2017; Updated 1 August 2017The financial support for this Data Descriptor was not properly acknowledged. The Acknowledgements section should have recognised a grant from the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning of the Government of South Korea (grant no. 2017R1A2B2006032).


Drosophila melanogaster and its associated powerful genetic tools have become a very attractive model system to study tumour-intrinsic and non-tumour-derived processes that mediate tumour development in vivo. In this review, we will summarize recent work on Drosophila as a model system to study cancer biology. We will focus on the interactions between tumours and their microenvironment, including extrinsic mechanisms affecting tumour growth and how tumours impact systemic host physiology.The study of cancer has represented a central focus in medical research for over a century. The great complexity and constant evolution of the pathology require the use of multiple research model systems and interdisciplinary approaches. This is necessary in order to achieve a comprehensive understanding into the mechanisms driving disease initiation and progression, to aid the development of appropriate therapies. In recent decades, the fruit fly Despite being the most studied human disease, cancer remains a leading cause of mortality worldwide. Nearly 1 in 6 deaths in 2015 was attributable to cancer, according to the World Health Organization, with an increase of 70% of new cases projected within the next two decades [ in vivo studies of non-tumour autonomous processes difficult to accomplish in conventional whole animal model systems.The organismal implications and ultimate outcome of tumour burden in patients are undoubtedly determined by a combination of tumour-intrinsic mechanisms and interactions between tumours and proximal, as well as distal tissues –4. WhileDrosophila melanogaster remains the most powerful genetic model in research. During the last decades, the development of various tumour models, including leukaemia, neuroblastoma, glioblastoma, colorectal, and ovarian cancer, has made the fruit fly an attractive in vivo model system to decipher tumour intrinsic  and extrinsic  molecular mechanisms mediating tumour growth and metastasis [ scribble-group of tumour suppressors genes , which encode key components of the basolateral polarity complex [ Ras or Src, drives tumour cell proliferation, spreading to distant tissues [ scrib-group genes as tumour suppressors in Drosophila, research has provided growing evidence that these models are directly relevant to human conditions. Indeed, scrib and dlg proteins are known targets of several oncogenic viruses, such as Human Papillomavirus, the main agent of cervical cancers. These viruses induce the degradation of the polarity complex proteins, comprising a key part of the process of malignant transformation in these conditions [ scrib has also been shown to work as a tumour suppressor in human breast, liver, skin, and lung cancers [ Drosophila homolog, scrib also cooperates with the Ras oncogene to promote tumour cell invasion [tastasis , 10. Suc complex . These m tissues , 13. Durnditions , 14, 15. cancers –19. The  cancers  and hepa cancers  and is a cancers . Moreoveinvasion , 23. Drosophila that have shed light into how extrinsic signals influence tumours, as well as mechanisms that mediate the systemic impact of tumours in the host. We focus on new findings highlighting the influence of immunity and metabolism in cancer progression and cancer-related disorders.Here, we discuss recent discoveries in Drosophila has allowed the discovery of molecular mechanisms mediating both pro- and antitumoural immunity. In contrast to mammals, which possess both innate and adaptive immunity, Drosophila only relies on innate immunity to fight against pathogens and tumours. Additionally, while mammals have numerous types of white blood cells, the cellular arm of Drosophila innate immunity includes only three main cells types—plasmatocytes, lamellocytes, and crystal cells—commonly called haemocytes. Only plasmatocytes have been currently reported to be associated with tumours [ Drosophila and mammals [Work in mammals has highlighted the immune system as a key component of the tumour microenvironment (TME), which plays a critical role in defining tumour outcome. While early studies on cancer patients support anticancer activity of the immune system , recent  tumours ; however mammals –29.α) is a major proinflammatory cytokine produced within the TME, which was originally characterised for its ability to induce tumour death [α's discovery led to great expectations for its use as a therapeutic target for cancer. However, further experiments have revealed a dual role for TNF-α as both an anti- and protumour factor [α's antagonistic actions were poorly understood. However, recent research in Drosophila has highlighted some key molecular aspects underlying this dual action of the cytokine. Drosophila possesses a single TNF-α homolog called Eiger (Egr) [ Drosophila tumour models through the use of mutants of the scrib-group of tumour suppressors genes. Egr expression is induced in tumours and tumour-associated immune cells [α, which is detected in tumour cells as well as macrophages and T lymphocytes [Tumour Necrosis Factor alpha (TNF-ur death . Consistr factor . The moler (Egr) , 33, whoer (Egr) . The impne cells , 36, mucphocytes . Given tphocytes , 37. scrib, lgl, or dlg mutant cells generated in imaginal discs, delaminate, and are mostly removed from the epithelia through cell competition [ Egr specifically within haemocytes, highlighting a conserved non-tumour autonomous anticancer function of Egr in Drosophila [α receptor Grindelwald (Grnd) in scrib mutant cells suppressed their removal from the epithelia [α is conserved in mammals, as TNF-α induces cell death through TNFR1 and subsequent JNK signalling activation [ Drosophila, including immune response proteins, may uncover new mechanisms involved in the elimination of cancer cells from a healthy tissue [ scrib-group genes, where neoplastic tumours develop from the whole imaginal disc, also show dependency from haemocyte-derived Egr to trigger JNK activation and tumour cell death [α-dependent inflammation in Drosophila models of cancer.Experimental evidence showed that immune cell-derived Egr has antitumoural activity. Patches ofpetition , 37–40. osophila , 36 . Ras is a conserved proto-oncogene mutated in many cancer types, with a 16% overall incidence rate in all analysed human tumours [ Drosophila, clones of cells mutated for scrib-complex proteins and overexpressing Rasv12 fail to be eliminated by surrounding epithelial cells. Instead, they form neoplastic tumours that can invade distant tissues [ scrib mutant clones, cooperation with Rasv12 in these clones diverts the function of JNK pathway activation toward tumour cell proliferation and invasion [ scrib, Rasv12 mutant cells to grow and invade. Strikingly, transplantation of Egr-wild-type immune cells could rescue the progression of scrib, Rasv12 tumours, as well as JNK activation, providing the final demonstration that Drosophila TNF shares protumour effects with its mammalian counterpart [ scrib, Rasv12 tumours and by data showing that Grnd knockdown in those tumours also disrupts their growth and invasive properties [scrib, Rasv12 tumours [v12-driven tumour progression requires the activation of Caspases, which function as tumour promoters. This mechanism is suggested to be one of the key mediators of the switch of Egr from an antitumour to a protumour cytokine by Ras. The protumoural function of TNF-α produced by immune cells is highly reminiscent to the one described in mammalian systems. In a mouse model of skin carcinogenesis where loss of TNF-α suppresses tumour formation [α competent mice is sufficient to restore tumour formation. However, this effect appears to be indirectly mediated through TNF-α-dependent regulation of T-cell number [α-dependent antitumoural responses in flies and humans comes from work on Kras-dependent intrahepatic cholangiocarcinoma. In this context, TNF-α produced by Kuppfer cells (liver-specific myeloid cells) drives preneoplastic lesions through JNK signalling pathway activation [In contrast to the described antitumoural functions, tumours . In Dros Ras Rasv. Ras is  tissues . While Jinvasion , 49. In nterpart  , a Syntaxin involved in the fusion of endocytic vesicles to the early endosome, is dependent on Grnd but escapes the need for Egr [ avl tumours produced high levels of Wingless (Wg) protein, which is a known target of JNK pathway activation and a key driver of compensatory proliferation, which is linked to cancer progression [ Drosophila showed that Wg dependent tumours proliferate independently of the TME and TNF-α/Grnd [v12 and hyperactivation of the nonreceptor tyrosine kinase Src, which also feature Wg overexpression [α. The expression of growth factors and activation of downstream signalling pathways in epithelial tissues in general and in Drosophila imaginal discs in particular are usually restricted to certain tissue locations [ Drosophila identified the presence of “tumour hot-spots.” Tumour hot-spots are defined as locations within tissues where neoplastic mutations are more likely to result in successful tumoural growths capable of invading normal tissues and it is a process involving differential activation of JAK/STAT signalling [α in this context.The demonstration of antagonistic actions of TNF- for Egr . Interesgression , 56. It F-α/Grnd . Similarpression , are larocations , 60. Recgnalling . It is l scrib-group mutants is the loss of epithelial cell polarity. In tumours lacking lgl, knockdown of the JNK pathway rescues loss of cell polarity [α may be a determinant in the loss of cell polarity in tissues carrying these neoplastic transformations. Indeed, Egr regulates asymmetric localisation of determinants of asymmetric division, Miranda and Prospero, in neuroblasts, supporting a role for Egr in cell polarity determination [α-dependent loss of cell polarity has been reported upon induction of chronic inflammation in the mouse intestine [α-dependent EMT increases lung cancer metastasis [α and cell polarity could also be the driving force for TNF-α's protumour effect on Rasv12 expressing cells, as Ras hyperactivation facilitates the prosurvival function of JNK signalling.A key phenotypic feature ofpolarity . Loss ofpolarity , 64. Givmination . Interesntestine . Likewistastasis . This poβ) signalling family, can promote intestinal stem cell (ISC) proliferation in response to infection [ Drosophila interleukin homologs that function as ligands of the JAK/STAT pathway [ scrib mutant larvae Upd 3 produced by the tumour induces JAK/STAT activation in the immune tissues (fat body and haemocytes), leading to a positive feedback loop that increases Upd 3 levels in haemocytes, which is required for JAK/STAT-induced proliferation of haemocyte and subsequent tumour suppression [ scrib/Rasv12 tumour, where it cooperates with JNK to promote growth and metastasis [ Drosophila blood cell neoplasia [The discovery of other immune-derived cytokines may have implications on their role in cancer progression through the TME. Haemocyte-derived Dpp, the fly homolog of Bone Morphogenetic Protein 2/4 (BMP2/4), a member of the Transforming Growth Factor beta , and JAK/STAT signalling [ Spaetzle (spz) in haemocytes or by removing Egr from tumours, suggesting that Egr produced by the tumour promotes Spz production by haemocytes, which in turn activates the Toll pathway in the fat body [α-dependent activation of systemic Toll signalling is an important component of a nonautonomous tumour suppressor program  [ Drosophila models. These findings demonstrate the drastic effect that the perturbation of host metabolism by extrinsic factors can have on tumour growth, how tumours exert systemic effects on distant tissues, and how the genetic properties of the tumour itself are critical in mediating this crosstalk.Obesity and type 2 diabetes are common comorbidities in modern society and are characterised by systemic insulin resistance and hyperglycaemia. These conditions are associated with an increased risk of developing cancer and are a risk factor for cancer mortality –94. Insuondition . In thisondition . Tumoursondition  that actondition . Under nt (slif) . Interest (slif) . Interest (slif) , which it (slif) –101. Thi Drosophila models and those found in vertebrates. Preexisting obesity and diabetes promoted tumour growth in a rat cancer model [ Drosophila cancer models involving diet and obesity are therefore particularly relevant to the human condition, as the protumour effects demonstrated in the contexts of these studies appear to be conserved in higher organisms, and the mediating factors are environmental influences that are very common in developed societies. The studies discussed here highlight new aspects of tumour physiology, suggesting that tumours are direct competitors to host tissues for nutrients and are frequently able to outcompete them for access to metabolic resources through various means  that drive the onset of microenvironmental autophagy are not yet fully defined; however, ROS signalling is an excellent candidate for further investigation , a secreted insulin-signalling antagonist that functions by direct binding to Dilp2 [ ImpL2 in the tumour was sufficient to reduce the systemic insulin resistant phenotype and thus partially rescue the wasting phenotypes observed in peripheral tissues, without impacting the growth of the tumour [ Drosophila cancer models in the field of tumour-microenvironment interactions. Research into cachexia is an emergent field, and the identification of a tumour-derived factor that mediates a systemic effect on host tissue metabolism is an important example of the ability of Drosophila models to recapitulate and dissect complex phenotypes. Interestingly, autophagy is one of the main mechanisms of tissue degradation during cancer cachexia [ Drosophila models discussed here represent a good opportunity to answer some of these important open questions.Two independent models of cancer cachexia have shown the utility ofresearch , 120. Boto Dilp2 . These sto Dilp2 –126 (Fige tumour , 120. Thcachexia , 128. Thcachexia . Togethecachexia , 130. Th Drosophila is a relevant model for studying cancer and its interactions with the TME, with many parallels to orthologous vertebrate conditions. Research utilising Drosophila as a model system has shown that immune and metabolic processes induced in a nonautonomous manner by the presence of the tumour are sufficient to feed back to the tumour and alter its characteristics. This can be shown well in the studies of microenvironmental autophagy, which is induced in the TME by the tumour, and serves to support tumour growth and metastasis [The studies discussed here demonstrate thattastasis , 104, antastasis , 46 (Fig Drosophila. Work in human cell culture and mouse models has demonstrated that tumours can alter host immunity via directly influencing immune cell metabolism. Lactic acid secreted by the tumour into the TME changes macrophages metabolism, polarising them towards a tumour-promoting state [Given the effects observed in response to the tumour there are likely to be interactions between the immune system and metabolism in this context. Indeed, nutrient restriction in larvae inhibits TOR signalling in the fat body, leading to increased levels of circulating Egr. Egr binds to insulin-producing cells in the brain and suppresses the production and secretion of progrowth Dilp2 and Dilp5 . As prevng state , 133. Thng state . Anotherng state , 87. Drosophila cancer models represent an excellent basis for the study of the roles these factors may play, both individually and combined together, and how nonautonomous signalling inputs might influence both tumour and host tissue responses. There are likely to be inevitable questions about the ability of simple Drosophila tumour models with one or two genetic drivers to fully recapitulate the complexity of tumour burden in higher animals, including human patients. However, the simplicity of these models is likely to prove advantageous when attempting to dissect the contributing roles of the multiple interacting factors that comprise tumour-TME interactions. There is also interesting work on the generation of Drosophila “avatars,” fly lines that can generate close homologs of tumours from specific patients, including the numerous genetic aberrations that drive a particular type of tumour in humans [ Drosophila cancer models, in order to investigate whether these discoveries still hold in a more complex tumour setting, including tumour-TME responses.However, there is a lack of comprehensive understanding as to how different factors such as diet and metabolism, immune responses, and tumours interact and cooperate or synergise when presented together. This is often the case in the human condition, for example, in the case of a cancer patient with diabetes.n humans . Such av Drosophila models represent an excellent platform for the continued investigation of these complex interactions, thanks to the multiple advantages of the model system. Low genetic redundancy, powerful genetic tools, and the possibility of tightly controlling not only the genetics of the tumour but also various aspects of the tumour micro- and macroenvironment render Drosophila a strong paradigm for further work into these complex interactions that impact human health and disease.The mechanisms mediating the crosstalk between tumours and local and distal tissues are still being uncovered. Improving the understanding of the signalling pathways that may link together the complex interactions between host metabolism, immunity, and tumour growth is an essential aspect towards the unravelling of such crosstalk.


Varicella zoster virus (VZV) causes varicella and herpes zoster. These vaccine preventable diseases are common globally. Most available data on VZV epidemiology are from industrialised temperate countries and cannot be used to guide decisions on the immunization policy against VZV in Africa. This systematic review aims to review the published data on VZV morbidity and mortality in Africa.All published studies conducted in Africa from 1974 to 2015 were eligible. Eligible studies must have reported any VZV epidemiological measure . For inclusion in the review, studies must have used a defined VZV case definition, be it clinical or laboratory-based.Twenty articles from 13 African countries were included in the review. Most included studies were cross-sectional, conducted on hospitalized patients, and half of the studies used varying serological methods for diagnosis. VZV seroprevalence was very high among adults. Limited data on VZV seroprevalence in children showed very low seropositivity to anti-VZV antibodies. Co-morbidity with VZV was common.There is lack of quality data that could be used to develop VZV control programmes, including vaccination, in Africa.CRD42015026144.PROSPERO 2015: The online version of this article (10.1186/s12879-017-2815-9) contains supplementary material, which is available to authorized users. Varicella zoster virus (VZV), a member of the herpesviridiae family, is responsible for two distinct disease entities: varicella (chicken pox) and herpes zoster (shingles). VZV is a ubiquitous virus and both diseases of varicella and herpes zoster occur commonly throughout the world , 2. PrimFollowing the primary infection, VZV remains dormant in the host. With decreased immunity, as seen in elderly persons or with human immunodeficiency virus (HIV)-associated immunosuppression, VZV reactivates causing herpes zoster, a painful vesicular rash with a dermatomal distribution. A common complication of herpes zoster is post-herpetic neuralgia, which can result in significant morbidity. Eye involvement, including uveitis with the risk of blindness, can also occur. Occasionally herpes zoster may present without a rash (zoster sine herpete) and VZV neurological complications can sometimes occur in the absence of a rash . The epiMolecular epidemiological analysis of VZV shows some evidence of geographical segregation, which may partly be explained by climatic conditions . The VZVSafe and effective vaccines against both varicella and herpes zoster exist . The varThe vaccine (live attenuated VZV) used against herpes zoster is similar to that for varicella, but the herpes zoster vaccine has 14-fold more plaque-forming units of the attenuated virus per dose , 10. As In Africa, there are several other vaccine preventable diseases (VPD’s) associated with a greater public health burden , 12, comFor Africa to design effective strategies that can mitigate the VZV-associated disease burden, it is crucial to understand the epidemiology of both varicella and herpes zoster on the continent. Most of the available and published VZV epidemiology data come from settings such as Europe and North America . This epPrior to this review, there has been no synthesised literature on VZV epidemiology in Africa. Our review aimed to address this gap in knowledge by describing the epidemiology of VZV in Africa, taking into account both varicella and herpes zoster. Because HIV/AIDS is prevalent in Africa, we were also interested in assessing the impact of the HIV/AIDS epidemic on the epidemiology of varicella and herpes zoster.We registered and published a detailed description of the methods used for this systematic review .Studies were eligible for inclusion in this review if they reported the epidemiology of VZV in any age group, were conducted in any African country and reported any outcome of interest as defined in our protocol . An addiClinical description: An acute illness with diffuse maculopapulovesicular rash, without other apparent cause.Laboratory criteria: Isolation of varicella virus from a clinical specimen, or varicella antigen detected by direct fluorescent antibody test, or varicella-specific nucleic acid detected by polymerase chain reaction (PCR), or significant rise in serum anti-varicella IgG antibody level by any standard serologic assay.For varicella, included studies must have stated a case definition; failure to do which would result in exclusion. The case definition considered for varicella is the one used by the Centres for Disease Control and Prevention (CDC) is :ClinicalFor herpes zoster, included studies must have stated the clinical signs , as it has been shown to be pathognomonic .Incidence or prevalence of varicella or herpes zosterProportion of varicella or herpes zoster cases requiring hospitalizationMortality associated with varicella or herpes zosterProportion of varicella or herpes zoster morbidity or mortality associated with HIV/AIDSDetails of the literature search strategy are provided in the protocol . We searCross-sectional, cohort and intervention studies were eligible for inclusion. All studies published from January 1974 to September 2015 were included without language restrictions. For non-English articles, Google translator software was used to translate to English, to enable us conduct a preliminary screening based on titles or abstracts. Subsequently, if the record needed further scrutiny, we contacted a scientist who is a native speaker of the language that needed translation to English. For articles in French that appeared likely eligible for inclusion, we sought translation support from a native speaker within our network of collaborators.The first (HSH) and the second (LHA) authors screened the literature search outputs using titles and abstracts. In addition, study setting, study design, methods as well as study outcomes were evaluated. The two authors then independently read through the full text of all potentially eligible studies to assess if inclusion criteria were met. Discrepancies between the two authors were resolved through discussion and consensus, with the assistance of the last author (BMK). The bibliographies of the included studies were screened for any other relevant studies. No additional studies were included in this review from the reference list of the selected studies. The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) flow diagram Fig.  illustraData were independently extracted from the included studies by two reviewers, and recorded on a predesigned form. For more information on the data collection, see the protocol ., [We adapted the risk of bias and quality assessment tool developed by Hoy et al.,  and modi,  for prevThe extracted data were heterogeneous and therefore, meta-analysis was not feasible.Narrative reporting was used to describe the data from the studies. The findings of this systematic review are reported according to the PRISMA guidelines .The complete raw data-set is available as a supplementary file  included in the final analysis , 20% in rural areas (n = 4) while 10% in both rural and urban areas (n = 2). 10% of the studies were conducted in peri-urban areas. One study was partially conducted in an urban area and a refugee camp [From the 20 studies, a total population of 92,613 participants were studied. Two large surveillance studies from Guinea-Bissau , 23 contgee camp  while thgee camp .). Across the included studies, variable case definitions for VZV disease were used, and these can be broadly categorised into two types: serological and clinical. Only two (10%) studies combined both serological and clinical criteria for the VZV case definition. According to our results, the available data from the 13 (65%) studies on the burden of VZV disease in Africa is mostly from adult populations.We evaluated the reported incidence, prevalence, hospitalization and mortality data from the included studies Table  and 4. ). Using population registers to estimate the denominator, ncidences of 441 per 100,000 and 3420 per 100,000 were reported by Poulsen et al., 2002 and 2005 respectively [, 2002 were bacterial skin infections (43.6%), cough (23.6%), conjunctivitis (4%) and diarrhoea (1.8%). Poulsen et al., 2005 reported a case-fatality rate of 0.13%. Poulsen et al., 2002 did not report mortality.Of the 20 included studies, only 3 (15%) reported incidence of VZV. Two of the three incidence studies reported varicella in Guinea Bissau, and these studies were published by the same authors and used a combination of serology and clinical tests to diagnose VZV Table . Using ectively , 23. As ectively  and 4.4 ectively , 23 resp, 2015 diagnosed herpes zoster clinically and reported an annual incidence of 751 per 100,000 in Ugandan HIV positive patients [3.The study by Rubaihayo et al.patients . Median There were eight (40%) studies that reported prevalence of VZV-associated disease quantified by viral detection using PCR. Although expensive and requiring greater technical expertise and resources, PCR tests, whether quantitative or qualitative, show superior sensitivity and specificity compared to antibody detection tests . Of the , 1985 who indicated that a 0.15 regression value of the EIA was the cut-off positivity value [Evaluation of antibodies to VZV  was reported in ten studies Fig. . IgG proty value . The assty value ) to 100%ty value ). In gen, 2009 compared the VZV seroprevalence in healthy HIV uninfected blood donors (45%) with symptomatic HIV patients (57.2%), and found the odds ratio for having VZV antibodies was 1.6  [For these results, we focused on the studies that used a laboratory detection of the VZV in quantifying the burden Table . Our lim1.1–2.6) .Table 3H, 2015 assessed the trends of opportunistic infections among HIV-infected persons in Uganda and found that the VZV incidence was 1340 per 100,000 at the start of the study in 2002 in the pre-ART era, compared to the incidence of 330 per 100,000 in 2013 in the ART era [Nine (45%) of the 20 included studies evaluated VZV burden in HIV infected populations. As an example, Rubaihayo et al. ART era .Only one study reported VZV seropositivity (IgG) data in pregnant women and found it to be 80.9% in Tunisia . One stuMost co-morbidities diagnosed alongside varicella or herpes zoster required hospitalization  Table . This maSeveral studies commented on the poor patient history of previous VZV infection, possibly due to recall bias , and patCrowded living conditions fuel the spread of VZV, and thus affect VZV seroprevalence. VZV seropositive status was associated with a higher number of individuals per household in rural South Africa . A studyA risk of bias and quality assessment was conducted independently by two raters using a modified assessment tool adapted from Hoy et al. . ThereafUsing the average score of the two independent raters, 8 studies had a score of 6–8 points, suggesting that they were at a moderate risk of bias, while 12 studies had a score of 8–10 points, implying that they were a low risk of bias.We successfully conducted a systematic review on the epidemiology of VZV in Africa. A high but variable burden of VZV-associated disease was found. In this review, we observed high VZV seroprevalence (IgG) in adults. This review identified limited VZV seroprevalence data in children which was insufficient to compare with data from adults. One study by Adamani et al. showed relatively low rates of detectable antibodies in children . In contAlthough this review identified limited data on VZV in children, increasing age, even in adults was found to be associated with increased VZV seropositivity. A South African study reported that only a moderately high proportion of young adults had detectable VZV antibodies, compared to all individuals at 60 years of age in the same setting . The VZV, who reported VZV seroconversion in Asian tropical countries peaking in adolescence and adulthood [Climatic conditions are known to influence the transmission of VZV . Africa dulthood . In contdulthood .Exposure to VZV is cumulative over time and seroconversion, therefore, is expected to increase with age. Seropositivity depends on what antibody level cut offs were used, and were unfortunately not reported for most studies in this review. Understanding the age of primary VZV infection in a given setting is crucial in the design of an optimal vaccination strategy. The only two studies that looked at varicella incidence (both from Guinea-Bissau) reported high incidence rates in both children and adults , 23. TheClimatic conditions vary greatly between and within African countries, and, therefore, the data from Guinea-Bissau cannot be extrapolated to the whole continent. In addition, high levels of household overcrowding might negate the effect of a tropical climate on VZV transmission . There wImportant risk factors in the development of herpes zoster include advanced age and altered cell mediated immunity, as seen in malignancy, immunosuppressive treatment and HIV infection . HIV pos, study where the VZV incidence dramatically decreased from the pre-ART era, compared to the ART era [The widespread and increasing availability of antiretroviral treatment (ART) in Africa  will lik ART era . These f ART era . The dev ART era . Taken t ART era . The impOptimal vaccination programmes against VZV are effective in mitigating the disease burden . There aOur review is limited by a paucity of data on VZV in Africa. Between 1974 and 2015, only 20 studies from 13 countries met the inclusion criteria for our review. The countries that contributed to the review may not be representative of the continent as a whole . As our Effective vaccines for both varicella and herpes zoster exist. We observed low and high levels of VZV seropositvity in children and adults respectively. The observations are worrisome, particularly in the context of a high HIV burden and an ageing population in Africa, as it suggests some primary exposure to VZV may be happening in adulthood. Primary VZV infection is more severe in adults than children. The resultant increased risk of morbidity and mortality from VZV could be mitigated by vaccines. Where universal varicella vaccination may not be feasible, such as in most African countries, vaccination of high risk individuals, such as healthcare workers, immunocompromised patients and their household contacts has been suggested . But impAdditional file 1: Table S1.Varicella in Africa – raw dataset. Data extracted from the included studies. (XLSX 44 kb)Additional file 2: Table S2.Bias Scoring Agreement. Interrater agreement of risk of bias and quality assessment. (DOCX 11 kb)


Birmingham, United KingdomInformations: Lorraine RichardsonPhone: +447711132946Fax: 01296 733823lorrainerichardson1@btinternet.comE-mail: www.bismics.org.ukSite: New Delhi, IndiaInformations: Dr. Kunal SarkarPhone: +919830080006kunal.cardiac@gmail.comE-mial: www.icc2016.inSite: London, United KingdomInformations: Alison ChanPhone: 02072903937Fax: 02072903992cardiothoracic@rsm.ac.ukE-mail: www.rsm.ac.uk/events/cth01Site: Tradate, ItalyInformations: Sue HesfordPhone: + 44 1392 430671Fax: + 44 1392 430671sue@ests.org.ukE-mail: www.ests.orgSite: Windsor, United KingdomInformations: EACTShttp://www.eacts.org/academy/2016-programme/Site: Beverly, United StatesInformations: Rebecca LawPhone: 760-720-2263Fax: 760-720-6263rlaw@promedicacme.comE-mail: http://promedicacme.com/Site: Hong Kong S.A.R., ChinaInformations: Division of Cardiothoracic Surgery, Department of Surgery TheChinese University of Hong KongPhone: (852) 2632 2629/2632 3586Fax: (852) 2637 7974enb2016@surgery.cuhk.edu.hkE-mail: www.surgery.cuhk.edu.hk/enb2016Site: Leeds, United KingdomInformations: Sue Hesfordsue@ests.org.ukE-mail: www.ests.orgSite: Windsor, United KingdomInformations: EACTShttp://www.eacts.org/academy/2016-programme/Site: Windsor, United KingdomInformations: EACTShttp://www.eacts.org/academy/2016-programme/Site: Lecce, ItalyInformations: Gaetano Di RienzoPhone: +39/3496398344Fax: +39/0832661608gaetanodirienzo1@gmail.comE-mail: chirurgiatoracicalecce.itSite: Tel Aviv, IsraelInformations: Paragon Israel (Dan Knassim)Phone: +972-3-5767730/7secretariat@icimeeting.comE-mail: http://2016.icimeeting.com/Madrid, SpainInformations: David MathewsPhone: 7025085200eurocardiology@cardiologyconference.orgE-mail: http://cardiology.conferenceseries.com/europe/Site:


Perineural invasion is an underrecognized route of metastatic spread along the nerve bundles within the nerve sheath into the surrounding tissues. It hinders the ability to establish local control as tumour cells can traverse along nerve tracts well beyond the extent of any local invasion rendering them inoperable and unresectable. Perineural invasion is a marker of poor prognosis. Oral submucous fibrosis with oral cancer constitutes a clinicopathologically distinct disease. Our case highlights an enigmatic presentation of oral submucous fibrosis and its coexistence with oral cancer presenting with unusual neurological disturbance of the inferior alveolar nerve and facial nerve and diffuse widening of the mandibular canal. The objective of this case report is to enumerate the significance of perineural invasion in determining the course of the disease and necessitate the need for future studies that can shed light on molecular mediators and pathogenesis of perineural spread. The mandibular canal widening is an unusual presentation related only to a few pathologic conditions affecting the lower jaw. Loss of cortical bone surrounding the mandibular canal appears as wide radiolucency on a radiograph, inferred as canal widening . GeneralPerineural invasion (PNI) is considered as a distinct third mode of tumour metastasis for oral squamous cell carcinoma (OSCC) together with lymphatic and blood vessel invasion . It can A 48-year-old man with a swelling on the right side of his face below the lower lip reported to our Department of Oral and Maxillofacial Surgery. He presented with a six-month history of nonhealing ulcer in the right side of the buccal mucosa with an extraoral draining sinus and dysphagia for one month .In addition, he also presented with a two-month history of inability to close his right eye and deviation of the corner of the mouth to the left side followed by numbness in the lower lip and chin region. General health status of the patient and blood and urine analyses were unremarkable. Extraoral examination confirmed the classical signs of lower motor neuron type facial nerve palsy which included absence of wrinkles on the forehead, lagophthalmos of the right eye positive bells sign, flattening of the nasolabial fold , and devIntraoral examination revealed an erosive lesion approximately 6 × 2 cm in size, extending from the angle of the mouth on the right side anteriorly up to the retromolar trigone posteriorly. The floor was covered with a pseudomembranous slough, with rolled edges and erythematous margins. On palpation, there was induration, tenderness, and the presence of fibrous bands . Soft, dThe panoramic radiograph revealed the presence of generalized bone loss with diffuse uniform enlargement of the mandibular canal, starting from the mandibular foramen to the mental foramen . SphericIncisional biopsy of the right buccal mucosa confirmed the clinical diagnosis of squamous cell carcinoma of right gingival-buccal sulcus. Histopathology sections revealed pleomorphic tumour cells with individual cell keratinization and dense peritumoural inflammatory response. Representative histological section demonstrating PNI (Modified Liebig Type A Classification) and infiltration of the epineurium was also seen , 10 FigThe treatment plan included full-thickness, wide local excision of buccal mucosa, segmental mandibulectomy, and modified radical neck dissection preserving internal jugular vein, spinal accessory nerve, followed by reconstruction with the free fibula graft using reconstruction plate. The resected specimen showed well differentiated squamous cell carcinoma with margins and nodes free of tumour. Adjuvant radiotherapy was planned considering the PNI.Perineural invasion is well defined when at least 33% of the circumference of the nerve is surrounded by tumour cells. The biologic mechanism of PNI pathogenesis remains elusive with postulations that it relates to reciprocal signaling interactions and the intrinsic capacity of tumour cells to retort to signals within the peripheral nerve and promote invasion .PNI is “a tropism of tumour cells for nerve bundles in the surrounding tissues” . It is oP < .0001).PNI is an important predictor for outcome of patients with SCC of the oral cavity and oropharynx. It is significantly associated with tumour differentiation, lymph node metastasis, depth of invasion, locoregional recurrence, and distant metastasis. Rahima et al.  concludeThe centrifugal and centripetal propagation of squamous cell carcinoma along the perineural space can demonstrate intracranial spread and jeopardise the available treatment options.PNI in adenoid cystic carcinoma is well recognized; however, it should also be corroborated in oral squamous cell carcinoma specimens that have of late demonstrated PNI. The presence of PNI can connote a late stage disease and is a hallmark of subclinical invasion that can necessitate the need for aggressive resection, coincident management of neck lymph nodes, and the addition of adjuvant therapy. Targeted drug therapy for PNI in oral cancer is still at its infancy and can be a boon in the years to come. Patients with oral cancer often undergo adjuvant radiation for pathologically high risk features including positive nodal disease, extracapsular spread, and positive tumour margins. Excluding these high risk features, PNI is an independent risk factor necessitating the need for adjuvant radiation .Widening of the mandibular canal is a classical sign of neurofibromatosis but it has been unusual so far in oral cancer. Intraosseous schwannomas may sometimes cause distension of the inferior alveolar canal; they typically produce well-defined unilocular radiolucency mimicking an odontogenic cyst or a tumour. The other causes of mandibular canal widening like perineuroma, multiple endocrine neoplasia syndrome type 2b, vascular leiomyoma, arteriovenous malformation, and traumatic neuroma have been reported in the literature .The persistent paraesthesia of lower lip and chin region is a warning sign of a tumour or a malignancy compressing or invading the nerve.Sphenopalatine ganglionJunction of the chorda tympani and the lingual nerveParotid gland along the auriculotemporal branch of the mandibular nerveKoivisto et al. (2016) [Facial nerve and the trigeminal nerve are assumed to form a synapse at three strategic locations:et al. 20 [16] advThe prognostic utility of PNI with respect to the diameter of the nerves is debated with conflicting results being reported from different series of cases. It has been postulated that the stroma of the perineural sheath promotes tumour growth, with the perineural space as a conduit for tumour growth. In view of this large diameter, nerves closer to the surgical margins may be associated with recurrence . ContrarOral squamous cell carcinoma often presents with a nonhealing exophytic/endophytic ulcer fixed to the underlying skin or the mucosa. This case differs from its usual presentation by its aggressive nature, multiple neural invasion, synchronization of submucous fibrosis and oral cancer, and diffuse widening of the mandibular canal. PNI is an independent risk factor for occult metastasis along with depth of invasion, size of primary tumour, differentiation, and immunosuppression. Further studies are warranted to elucidate its molecular biology of pathogenesis, histopathological pattern of PNI, and its significance in the prognosis. With a better understanding of the mechanisms involved, we can progress to develop therapeutic agents that can target this form of intriguing tumour spread.


The establishment of a regional testicular tumour study group is described with emphasis on the importance of a local facility for tumour marker measurements by a fully integrated laboratory. Advantages over larger scale studies are suggested.