Search

BACKGROUND: Poly (ADP-ribose) polymerase inhibitors combined with immunotherapy have shown antitumour activity in preclinical studies. We aimed to assess the safety and activity of olaparib in combination with the PD-L1-inhibitor, durvalumab, in patients with germline BRCA1-mutated or BRCA2-mutated metastatic breast cancer.METHODS: The MEDIOLA trial is a multicentre, open-label, phase 1/2, basket trial of durvalumab and olaparib in solid tumours. Patients were enrolled into four initial cohorts: germline BRCA-mutated, metastatic breast cancer; germline BRCA-mutated, metastatic ovarian cancer; metastatic gastric cancer; and relapsed small-cell lung cancer. Here, we report on the cohort of patients with breast cancer. Patients who were aged 18 years or older (or aged 19 years or older in South Korea) with germline BRCA1-mutated or BRCA2-mutated or both and histologically confirmed, progressive, HER2-negative, metastatic breast cancer were enrolled from 14 health centres in the UK, the USA, Israel, France, Switzerland, and South Korea. Patients should not have received more than two previous lines of chemotherapy for metastatic breast cancer. Patients received 300 mg olaparib in tablet form orally twice daily for 4 weeks and thereafter a combination of olaparib 300 mg twice daily and durvalumab 1·5 g via intravenous infusion every 4 weeks until disease progression. Primary endpoints were safety and tolerability, and 12-week disease control rate. Safety was analysed in patients who received at least one dose of study treatment, and activity analyses were done in the full-analysis set (patients who received at least one dose of study treatment and were not excluded from the study). Recruitment has completed and the study is ongoing. This trial is registered with ClinicalTrials.gov, NCT02734004.FINDINGS: Between June 14, 2016, and May 2, 2017, 34 patients were enrolled and received both study drugs and were included in the safety analysis. 11 (32%) patients experienced grade 3 or worse adverse events, of which the most common were anaemia (four [12%]), neutropenia (three [9%]), and pancreatitis (two [6%]). Three (9%) patients discontinued due to adverse events and four (12%) patients experienced a total of six serious adverse events. There were no treatment-related deaths. 24 (80%; 90% CI 64·3-90·9) of 30 patients eligible for activity analysis had disease control at 12 weeks.INTERPRETATION: Combination of olaparib and durvalumab showed promising antitumour activity and safety similar to that previously observed in olaparib and durvalumab monotherapy studies. Further research in a randomised setting is needed to determine predictors of therapeutic benefit and whether addition of durvalumab improves long-term clinical outcomes compared with olaparib monotherapy.FUNDING: AstraZeneca.

Background Head and neck squamous cell carcinoma (HNSCC) is composed of a heterogeneous group of tumors arising trough environmental carcinogens or infection by human papillomavirus (HPV). Treatment interventions such as immunotherapy and targeted therapy have shown clinical benefit in HNSCC patients. Despite these encouraging results, resistance to treatment is still observed in the majority of patients. Additionally, clinical effectiveness of treatment options has also been shown to be associated with HPV status. Here we investigate the tumoral and peripheral landscape of HPV(-) vs. HPV(+) head and neck cancers to identify features able to expand treatment options for patients with Viral- and Carcinogen-Driven Head and Neck Cancer. Methods Biopsies and serum samples derived from 502 primary and metastatic HNSCC patients were leveraged for genomic, proteomic and immunochemistry evaluations. Tumor biopsies from HNSCC patients commercially obtained (n=143) or derived from patients enrolled in CP1108 trial (n=19, NCT01693562) were profiled by gene expression. Primary tumor biopsies (N=198) from HNSCC have been assessed by Whole Exome Sequence (WES). Expression of immune markers including CD8, NKp46 was evaluated by immunohistochemistry (IHC) on 186 and 214 tumors biopsies, respectively. The expression of 80 immune related soluble factors was evaluated in serum derived from n=285 patients of HNSCC enrolled in EAGLE (NCT02369874), a randomized, open-label, study assessing Durvalumab and Tremelimumab vs. Standard of Care (SoC). Statistical comparison between HPV(+) vs. HPV (-) samples were conducted using R software. Results Patients with HPV(-) vs. HPV(+) HNSCC were characterized by worse prognosis. Increased levels of immunosuppressive factors including VEGF (p=0.01), IL-8 (p=0.02), IL6 (p=0.07) and macrophages chemo attractive factor CCL4 (p=0.07) was observed in the serum of HPV(-) vs HPV(+) HNSCC patients. In the tumor microenvironment, higher mRNA expression of immune signatures associated with MDSC, Cancer Associated Fibroblast (CAF), and Metalloproteinase (MMP) was observed in HPV(-) vs. HPV (+) HNSCC patients. In contrast, HNSCC HPV(+) patients were characterized by increased mRNA expression of DC signatures and IFNg related genes (i.e. CXCL9). No differential infiltration of T and NK cells (CD8+ and NKp46+) were found in HPV (-) vs. HPV(+) patients. Enrichments of mutations in EGFR, and DNA repair genes (PMS1, POLK, ATM) was observed in HPV(+) patients. On the contrary, enrichments of mutations in TP53 was observed in HPV(-) patients. Conclusions Deep evaluation of tumoral and peripheral landscape of viral- versus carcinogen-driven HNSCC might help understanding differential outcome of treatments regimens in HPV(+) vs HPV (-) HNSCC thus leading to novel therapeutic interventions. Trial Registration NCT01693562,NCT02369874 Ethics Approval The study was approved by Astrazeneca. Consent Patients provided written consent to perform evalutions here described.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

GABA is the main inhibitory neurotransmitter in the mature brain but has the paradoxical property of depolarizing neurons during early development. Depolarization provided by GABAA transmission during this early phase regulates neural stem cell proliferation, neural migration, neurite outgrowth, synapse formation, and circuit refinement, making GABA a key factor in neural circuit development. Importantly, depending on the context, depolarizing GABAA transmission can either drive neural activity, or inhibit it through shunting inhibition. The varying roles of depolarizing GABAA transmission during development, and its ability to both drive and inhibit neural activity, makes it a difficult developmental cue to study. This is particularly true in the later stages of development, when the majority of synapses form and GABAA transmission switches from depolarizing to hyperpolarizing. Here we addressed the importance of depolarizing but inhibitory (or shunting) GABAA transmission in glutamatergic synapse formation in hippocampal CA1 pyramidal neurons. We first showed that the developmental depolarizing-to-hyperpolarizing switch in GABAA transmission is recapitulated in organotypic hippocampal slice cultures. Based on the expression profile of K+-Cl- co-transporter 2 (KCC2) and changes in the GABA reversal potential, we pinpointed the timing of the switch from depolarizing to hyperpolarizing GABAA transmission in CA1 neurons. We found that blocking depolarizing but shunting GABAA transmission increased excitatory synapse number and strength, indicating that depolarizing GABAA transmission can restrain glutamatergic synapse formation. The increase in glutamatergic synapses was activity-dependent, but independent of BDNF signalling. Importantly, the elevated number of synapses was stable for more than a week after GABAA inhibitors were washed out. Together these findings point to the ability of immature GABAergic transmission to restrain glutamatergic synapse formation and suggest an unexpected role for depolarizing GABAA transmission in shaping excitatory connectivity during neural circuit development.

Oncolytic viruses (OV) are defined by their intrinsic or engineered tropism for tumor cells. Understanding the drivers of susceptibility to infection as well as the downstream intracellular signaling and immune activation profile is critical in identifying the patients that will respond to OV therapy. Newcastle Disease Virus (NDV) is a negative stranded RNA avian paramyxovirus, here modified to encode human GM-CSF or IL-12. Patient derived xenografts (PDX) can more accurately reflect the genetic, epigenetic, and architectural heterogeneity of human tumors and were therefore selected to model the in vivo activity of NDV:GM-CSF. Eighty-nine independent models derived from patients with Bladder, Colorectal, Head and Neck, Kidney, Liver, Lung, Ovarian, Pancreatic, and Breast tumors were studied. Mice were dosed intravenously with NDV:GM-CSF, monitored for pharmacodynamic and pharmacokinetic changes 48 hours after the first dose, and followed for tumor growth inhibition. Objective response was observed in 68% of treated mice, with responses observed in all indications. Using total stranded RNAseq, we were able to assess changes in human tumor, murine immune infiltrate, and viral RNA. Results showed that NDV:GM-CSF treatment leads to increases in transcripts of inflammatory chemokines and cytokines such as RANTES and CXCL9, with induction directly correlated to level of viral replication. Additionally, we saw increases in NKp46 and IL-12R, markers of NK cells. To assess if IL-12 would augment immune activation, we treated surgically resected human tumors with NDV:IL-12. NDV:IL-12 was capable of infecting tumor cells, generating IL-12 protein, and driving a strong IFNγ response. Here we show that NDV can infect a wide variety of human tumors across multiple indications and drive a potent immune activation which can be modulated by transgenes. This data provides insight into what types of tumors could be treated with NDV:GM-CSF or NDV:IL-12. Citation Format: Nicholas M. Durham, Kelly McGlinchey, Shannon Burke, Todd Creasy, Nicola Rath, Noel Monks, Ravinder Tammali, Kevin Schifferli, Nick Holoweckyj, Susie Hayes, Emma Jones, Elizabeth J. Kelly, Danielle Carroll, James Harper, Katie Streicher. Newcastle Disease Virus in human derived tumors: Understanding immune recruitment and activation via virally delivered IL-12 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5544.

e12642 Background: Translesion synthesis (TLS) of DNA by specialized polymerases (POLs) and REV1, a scaffold protein that recruits other TLS DNA POLs for nascent strand extension, experimentally enable tumor cells to replicate through DNA damage. TLS POLs are hypothesized to contribute to treatment resistance. Methods: We conducted a discovery gene expression study using NanoString technology with a bespoke codeset enriched for DNA damage response genes and RNA from 12 TN, 10 HER2+ and 29 ER+/HER2- breast cancer patients before neoadjuvant chemotherapy (NACT) and when available, in the matching residual tumor. Eleven patients achieved a pathologic complete response (pCR), 12 had < 1 cm residual disease (partial responders) and 28 patients had > 1cm residual disease (non-responders). RNA counts were normalized and log-transformed for differential expression analyses corrected for false discovery. Results: DNA polymerase Iota ( POLI) was the top overexpressed gene in the pretreatment biopsy of non-responders independent of tumor subtype (p corrected = 0.0002). Expression of REV1 was also significantly higher in non-responders and positively correlated with POLI (r = 0.577, p < 0.0001). Despite a small sample size, pretreatment expression levels of POLI and REV1 were significantly higher in ER+ (n = 38) than ER- (n = 13) tumors (p = 0.0006). When restricted to ER+ tumors, POL1 and REV1 expression levels were significantly higher in pretreatment biopsy of non-responders (n = 25) than partial and complete responders (n = 13). For ER+ patients, response by median POL1/REV1 expression levels are shown in Table. Pre/post treatment POL1 and REV1 expression did not change in patients with residual disease. Conclusions: In discovery, two genes involved in TLS of DNA (POLI and REV1) were found overexpressed in pretreatment biopsies from breast cancers poorly responsive to NACT, independent of tumor subtype. Overexpression was more common among ER+ tumors, which exhibit a high inherent resistance to chemotherapy. If confirmed, expressed levels of POLI and REV1 may identify a subgroup of breast cancers resistant to NACT for which targeted inhibition of mutagenic TLS may be beneficial. [Table: see text]

There has been significant interest in combining anti-PD-1/PD-L1 agents with other clinically active anticancer agents. Gefitinib, a first-generation inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for non-small cell lung cancer (NSCLC) patients with sensitizing EGFR mutations. Durvalumab has demonstrated clinical activity in NSCLC and is approved for Stage III, unresectable NSCLC that has not progressed following platinum-based chemotherapy and radiotherapy. A phase I trial (NCT02088112) combining gefitinib and durvalumab was initiated to establish the safety profile of this combination in tyrosine-kinase inhibitor (TKI)-naive patients with NSCLC containing EGFR-positive sensitizing mutations. As part of this trial, paired tumor biopsies and multiple blood samples were collected for biomarker evaluation. Peripheral blood samples were analyzed for gene expression, cytokine production, immunophenotyping, and circulating DNA (ctDNA). Paired tumor biopsies were evaluated for expression of multiple proteins by immunohistochemistry, including phosphorylated EGFR (pEGFR), PD-L1, CD73, and CD8. We saw ≥50% inhibition of pEGFR in 8 of 17 paired biopsies but no consistent directional changes with PD-L1. We observed known durvalumab-mediated effects such as increases in tumor CD8+ cells (3.7 fold, p=0.011), proliferating CD8+ cells in circulation (2.4 fold, p=0.074), and increased interferon gamma (10.3 fold, p=0.01). A cohort of patients (n=10) was treated with a 28-day gefitinib run-in prior to addition of durvalumab. Sample collection during this period allowed us to evaluate the effects of gefitinib alone on immunologic readouts. While treatment with gefitinib alone resulted in ≥50% reduction of pEGFR in 5 of 8 paired tumor biopsies, its effect on expression of immune-related genes, interferon-related cytokines, and immune subpopulations was minimal. Interestingly, we saw an association between higher baseline tumor PD-L1, pEGFR or CD73 expression and a numerical trend for longer progression-free survival. Finally, analysis of ctDNA showed robust decreases in mutant EGFR could be observed as early as Day 5 of gefitinib treatment, either alone or in combination with durvalumab. Altogether, these results demonstrate that each agent had its expected downstream biologic effects and there was no antagonism between the two agents. We did not observe any novel biologic effects nor any striking synergy for known readouts. New hypotheses (i.e., high PD-L1/pEGFR/CD73) for patient selection biomarkers were identified, although more follow-up is required to determine whether they reflect combination or single-agent treatment. These studies highlight the utility of collecting biomarker samples to confirm target engagement, understand downstream effects, identify patient selection biomarkers, and explore ctDNA biomarkers. Citation Format: Tammie C. Yeh, Helen K. Angell, Vicky Rowlands, Emma V. Jones, Vidalba Rocher Ros, Alison Pritchard, Martine P. Roudier, Nathan Standifer, Mike Kuziora, Philip Martin, Kirill Peskov, Rosie Taylor, J. Carl Barrett, Ben Sidders. Exploratory biomarker analyses of tumor and peripheral blood samples from the phase I durvalumab plus gefitinib trial in EGFR-mutated NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B55.

Glial cell properties dictated by neurons Neurons in the brain coexist with astrocytes, a type of glial cell, which help support many functions of their neighboring nerve cells. Farmer et al. now show that the support goes both ways (see the Perspective by Stevens and Muthukumar). They explored the influence of neurons on two specialized types of astrocytes in the mouse cerebellar cortex. The neurons produced the morphogen known as Sonic Hedgehog. Hedgehog signaling adjusted distinctive gene expression within the two astrocyte cell types. Thus, mature neurons appear to promote and maintain specific properties of associated astrocytes. Science , this issue p. 849 ; see also p. 813

Fixed human brain samples in tissue repositories hold great potential for unlocking complexities of the brain and its alteration with disease. However, current methodology for simultaneously resolving complex three-dimensional (3D) cellular anatomy and organization, as well as, intricate details of human brain cells in tissue has been limited due to weak labeling characteristics of the tissue and high background levels. To expose the potential of these samples, we developed a method to overcome these major limitations. This approach offers an unprecedented view of cytoarchitecture and subcellular detail of human brain cells, from cellular networks to individual synapses. Applying the method to AD samples, we expose complex features of microglial cells and astrocytes in the disease. Through this methodology, we show that these cells form specialized 3D structures in AD that we refer to as reactive glial nets (RGNs). RGNs are areas of concentrated neuronal injury, inflammation and tauopathy and display unique features around β-amyloid plaque types. RGNs have conserved properties in an AD mouse model and display a developmental pattern coinciding with the progressive accumulation of neuropathology. The method provided here will help reveal novel features of the healthy and diseased human brain and aid experimental design in translational brain research.

Astrocytes show a complex structural and physiological interplay with neurons and respond to neuronal activation in vitro and in vivo with intracellular calcium elevations. These calcium changes enable astrocytes to modulate synaptic transmission and plasticity through various mechanisms. However, the response pattern of astrocytes to single neuronal depolarization events still remains unresolved. This information is critical for fully understanding the coordinated network of neuron–glial signaling in the brain. To address this, we developed a system to map astrocyte calcium responses along apical dendrites of CA1 pyramidal neurons in hippocampal slices using single-neuron stimulation with channelrhodopsin-2. This technique allowed selective neuronal depolarization without invasive manipulations known to alter calcium levels in astrocytes. Light-evoked neuronal depolarization was elicited and calcium events in surrounding astrocytes were monitored using the calcium-sensitive dye Calcium Orange. Stimulation of single neurons caused calcium responses in populations of astrocytes along the apical axis of CA1 cell dendrites. Calcium responses included single events that were synchronized with neuronal stimulation and poststimulus changes in calcium event frequency, both of which were modulated by glutamatergic and purinergic signaling. Individual astrocytes near CA1 cells showed low ability to respond to repeated neuronal depolarization events. However, the response of the surrounding astrocyte population was remarkably accurate. Interestingly, the reliability of responses was graded with respect to astrocyte location along the CA1 cell dendrite, with astrocytes residing in the primary dendrite subregion being most responsive. This study provides a new perspective on the dynamic response property of astrocyte ensembles to neuronal activity.

In this study, we examine the T cell receptor (TCR) repertoire in a cohort of Head and Neck Squamous Cell Carcinoma (HNSCC) patients (n=50) at time of diagnosis. The TCR repertoire is indicative of the ability of the body to mount an immune response, with increased productive clonality within the tumor environment suggesting accumulation of tumor antigen specific T cells. Recent studies demonstrate that the intratumoral TCR repertoire is prognostic of response to anti-PD-1 (Tumeh et al., 2014) and anti-CTLA-4 (Page et al., 2016) therapies. Here we examine diversity and clonality of intratumoral T cell β repertoires in HNSCC patients independently and in conjunction with other biomarkers including tumor mutational burden/neoantigen load, expression of inhibitory checkpoints, and characterization of immune cell populations, as well as clinical metadata including HPV status, smoking history, and survival outcomes. The TCRβ repertoire was analyzed from FFPE primary tumor samples through targeted next generation DNA sequencing of the TCRβ loci and examination of the CDR3 sequences to determine clonality of the TCRβ repertoire (1 – Pielou’s evenness). Somatic genomic alterations, tumor mutational burden, and HLA typing were conducted through whole exome sequencing. Inhibitory checkpoint expression and characterization of immune cell populations were conducted through immunohistochemistry, RNA expression, or both methods. Preliminary results indicate productive clonality of the TCR repertoire in the range of 0.03-0.16. In examining HPV+ and HPV- patients, where we hypothesize the presentation of foreign peptides of viral etiology will produce an increase in T cell populations recognizing those neoantigens, we see no difference in either T cell infiltration or productive clonality. However, we observe that PD-L1 status is significantly correlated (p < 0.05) to productive clonality of the tumor infiltrating T cells, with a higher average productive clonality in PD-L1+ patients. Ongoing studies will characterize the landscape of somatic genomic alterations including tumor mutational burden, and gene expression profiles of the tumor microenvironment. Citation Format: Lara McGrath, Elizabeth Maloney, Maria Secrier, Helen Angell, Emma Jones, Brian Dougherty, Daniel Stetson, Patricia McCoon, Andrew Pierce, Craig Barker, Carl Barrett. Intratumoral T cell receptor β repertoire as a biomarker in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B83.

A number of molecular recognition features have been exploited in structure-based design of selective Cathepsin inhibitors.

A series of potent Cathepsin L inhibitors with good selectivity with respect to other cysteine Cathepsins is described and SAR is discussed with reference to the crystal structure of a protein-ligand complex.

The JNK (c-Jun N-terminal kinase)/mitogen-activated protein kinase signalling pathway is a major mediator of stress responses in cells, including the response to DNA damage. DNA damage also causes the stabilization and activation of p73, a member of the p53 family of transcription factors. p73, like p53, can mediate apoptosis by up-regulating the expression of pro-apoptotic genes, including Bax (Bcl2-associated X protein) and PUMA (p53 up-regulated modulator of apoptosis). Changes in p73 expression have been linked to tumour progression, particularly in neuroblastomas, whereas in tumours that feature inactivated p53 there is evidence that p73 may mediate the apoptotic response to chemotherapeutic agents. In the present study, we demonstrate a novel link between the JNK signalling pathway and p73. We use pharmacological and genetic approaches to show that JNK is required for p73-mediated apoptosis induced by the DNA damaging agent cisplatin. JNK forms a complex with p73 and phosphorylates it at several serine and threonine residues. The mutation of JNK phosphorylation sites in p73 abrogates cisplatin-induced stabilization of p73 protein, leading to a reduction in p73 transcriptional activity and reduced p73-mediated apoptosis. Our results demonstrate that the JNK pathway is an important regulator of DNA damage-induced apoptosis mediated by p73.

Specialized postsynaptic structures known as dendritic spines are the primary sites of glutamatergic innervation at synapses of the CNS. Previous studies have shown that spines rapidly remodel their actin cytoskeleton to modify their shape and this has been associated with changes in synaptic physiology. However, the receptors and signaling intermediates that restructure the actin network in spines are only beginning to be identified. We reported previously that the EphA4 receptor tyrosine kinase regulates spine morphology. However, the signaling pathways downstream of EphA4 that induce spine retraction on ephrin ligand binding remain poorly understood. Here, we demonstrate that ephrin stimulation of EphA4 leads to the recruitment and activation of phospholipase Cγ1 (PLCγ1) in heterologous cells and in hippocampal slices. This interaction occurs through an Src homology 2 domain of PLCγ1 and requires the EphA4 juxtamembrane tyrosines. In the brain, PLCγ1 is found in multiple compartments of synaptosomes and is readily found in postsynaptic density fractions. Consistent with this, PLC activity is required for the maintenance of spine morphology and ephrin-induced spine retraction. Remarkably, EphA4 and PLC activity modulate the association of the actin depolymerizing/severing factor cofilin with the plasma membrane. Because cofilin has been implicated previously in the structural plasticity of spines, this signaling may enable cofilin to depolymerize actin filaments and restructure spines at sites of ephrin–EphA4 contact.

e17542 Background: Expression of genes known to be integral to anti-tumour activity may be prognostic for patient outcomes in SCCHN. We hypothesized that broad categories of immune function would be responsible for anti-tumor activity. Methods: A study was conducted using data from patient medical records and analysis of archival tumour samples. Patients were ≥18 years old, diagnosed with SCCHN between 1989 and 2015 (n = 119). We quantified the expression of an annotated list of 71 aggregated genes selected to represent a variety of immunological activities: chemokines and receptors, interferon gamma stimulated genes, immunological checkpoints, HLA class I and II molecules, T cell activation, T cell surface markers, adhesion molecules, cytotoxic granules, immune suppression, inflammation, NK cell associated genes and ‘other functionality’. Results: Using a Cox proportional hazards model starting from consideration of the initial 71 genes and performing 10-fold cross-validation with 100 repetitions, several prognostic gene signatures were derived with overall survival hazard ratios > 2.8 and concordance indexes > 0.7, 95% confidence intervals ranging between [0.709, 0.805]. The signatures remained significantly predictive of outcome when accounting for tumour stage, sex and age of patients, HPV status and smoking habits. A cumulative score per sample taking into account the directionality of association of each gene with outcome and its corresponding expression was derived and this score outperformed tumour staging in distinguishing survival outcomes. The genes CD274 (PD-L1; checkpoints), CD8A (T cell surface markers), CCL11 (chemokines and receptors), GNLY (cytotoxic granules), TNFRSF10A (immune suppression), and EDN1 (other) appeared in all signatures, validating our approach of considering broad categories of functionality to represent the anti-tumor immune system response as an integrated whole. Conclusions: Identification of a prognostic gene signature may have additional utility for evaluating early response to therapy by assessing whether therapy has converted a patient tumour from a prognostically unfavourable to prognostically favourable gene signature status.

Summary Background Excitatory synapses in the CNS are highly dynamic structures that can show activity-dependent remodeling and stabilization in response to learning and memory. Synapses are enveloped with intricate processes of astrocytes known as perisynaptic astrocytic processes (PAPs). PAPs are motile structures displaying rapid actin-dependent movements and are characterized by Ca 2+ elevations in response to neuronal activity. Despite a debated implication in synaptic plasticity, the role of both Ca 2+ events in astrocytes and PAP morphological dynamics remain unclear. Results In the hippocampus, we found that PAPs show extensive structural plasticity that is regulated by synaptic activity through astrocytic metabotropic glutamate receptors and intracellular calcium signaling. Synaptic activation that induces long-term potentiation caused a transient PAP motility increase leading to an enhanced astrocytic coverage of the synapse. Selective activation of calcium signals in individual PAPs using exogenous metabotropic receptor expression and two-photon uncaging reproduced these effects and enhanced spine stability. In vivo imaging in the somatosensory cortex of adult mice revealed that increased neuronal activity through whisker stimulation similarly elevates PAP movement. This in vivo PAP motility correlated with spine coverage and was predictive of spine stability. Conclusions This study identifies a novel bidirectional interaction between synapses and astrocytes, in which synaptic activity and synaptic potentiation regulate PAP structural plasticity, which in turn determines the fate of the synapse. This mechanism may represent an important contribution of astrocytes to learning and memory processes.

Long-lasting changes in synaptic strength, such as those that occur during late-phase long-term potentiation (LTP) and long-term depression (LTD), require new protein synthesis. This new protein synthesis requires signaling through extracellular signal-regulated kinase (ERK) and mammalian target of

The polarization of developing neurons to form axons and dendrites is required for establishing neuronal connections leading to proper brain function. The protein kinase AKT and the MAP kinase scaffold protein JNK-interacting protein-1 (JIP1) are important regulators of axon formation. Here we report that JIP1 and AKT co-localize in axonal growth cones of cortical neurons and collaborate to promote axon growth. The loss of AKT protein from the growth cone results in the degradation of JIP1 via the proteasome, while loss of JIP1 promotes a similar fate for AKT. Reduced protein levels of both JIP1 and AKT in the growth cone can be induced by glutamate and this coincides with reduced axon growth which can be rescued by a stabilized mutant of JIP1 that rescues AKT protein levels. Taken together, our data reveal a collaborative relationship between JIP1 and AKT that is required for axon growth and can be regulated by changes in neuronal activity.

Matricellular proteins are secreted, nonstructural proteins that regulate the extracellular matrix (ECM) and interactions between cells through modulation of growth factor signaling, cell adhesion, migration, and proliferation. Despite being well described in the context of nonneuronal tissues, recent studies have revealed that these molecules may also play instrumental roles in central nervous system (CNS) development and diseases. In this minireview, we discuss the matricellular protein families SPARC (secreted protein acidic and rich in cysteine), Hevin/SC1 (SPARC-like 1), TN-C (Tenascin C), TSP (Thrombospondin), and CCN (CYR61/CTGF/NOV), which are secreted by astrocytes during development. These proteins exhibit a reduced expression in adult CNS but are upregulated in reactive astrocytes following injury or disease, where they are well placed to modulate the repair processes such as tissue remodeling, axon regeneration, glial scar formation, angiogenesis, and rewiring of neural circuitry. Conversely, their reexpression in reactive astrocytes may also lead to detrimental effects and promote the progression of neurodegenerative diseases.

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.

Astrocytes secrete factors that promote neuron survival, synapse formation, and plasticity. Understanding how these factors perform these roles requires a robust in vitro system that can effectively assess the impact of individual glial factors on neuronal properties. A classical approach to studying neuron-glial interactions in vitro uses a system where dissociated embryonic rat neurons are suspended over a feeder layer of rat astrocytes. Here, we describe a useful "sandwich" co-culture system where postnatal mouse hippocampal neurons are grown suspended above a feeder layer of mouse hippocampal astrocytes. We demonstrate that neurons in these cultures remain healthy beyond 3 weeks in vitro and develop more synapses compared to neurons grown without astrocytes. An advantage of this method is that astrocytes and neurons can be prepared separately from postnatal transgenic or knock-out mouse lines allowing one to study, for example, how wild-type neurons develop in the presence of astrocytes from a knock-out mouse line that lacks the expression of a specific astrocyte-secreted factor. We find this culture system to be a convenient and powerful approach to study the contribution of astrocyte-secreted molecules to neuron development.

Astrocytes secrete factors that promote neuron survival, synapse formation, and plasticity. Understanding how these factors perform these roles requires a robust in vitro system that can effectively assess the impact of individual glial factors on neuronal properties. A classical approach to studying neuron-glial interactions in vitro uses a system where dissociated embryonic rat neurons are suspended over a feeder layer of rat astrocytes. Here, we describe a useful "sandwich" co-culture system where postnatal mouse hippocampal neurons are grown suspended above a feeder layer of mouse hippocampal astrocytes. We demonstrate that neurons in these cultures remain healthy beyond 3 weeks in vitro and develop more synapses compared to neurons grown without astrocytes. An advantage of this method is that astrocytes and neurons can be prepared separately from postnatal transgenic or knock-out mouse lines allowing one to study, for example, how wild-type neurons develop in the presence of astrocytes from a knock-out mouse line that lacks the expression of a specific astrocyte-secreted factor. We find this culture system to be a convenient and powerful approach to study the contribution of astrocyte-secreted molecules to neuron development.

Neurons recruit numerous mechanisms to facilitate the development of synaptic connections. However, little is known about activity-dependent mechanisms that control the timing and fidelity of this process. Here we describe a novel pathway used by neurons to regulate glutamate receptors at maturing central synapses. This pathway relies on communication between neurons and astrocytes and the ability of astrocytes to release the factor SPARC (secreted protein, acidic and rich in cysteine). SPARC expression is dynamically regulated and plays a critical role in determining the level of synaptic AMPARs. SPARC ablation in mice increases excitatory synapse function, causes an abnormal accumulation of surface AMPARs at synapses, and impairs synaptic plasticity during development. We further demonstrate that SPARC inhibits the properties of neuronal β3-integrin complexes, which are intimately coupled to AMPAR stabilization at synapses. Thus neuron–glial signals control glutamate receptor levels at developing synapses to enable activity-driven modifications of synaptic strength.

Glial cells play multiple, diverse roles in the central nervous system (CNS), ranging from the basal support of neuronal function to close partnership with the synapse. Growing experimental evidence shows the importance of glia for proper brain functioning and their involvement in injury and disease. Astrocytes are the most intriguing cells among the glial family. It is well known that they provide energetic substrates to neurons, take up neurotransmitters, and maintain ion homeostasis. Recent research has revealed that they can also release gliotransmitters and signaling molecules as well as maintain and regulate the extracellular matrix. Structurally, astrocytic fine processes enwrap synaptic contacts and exhibit remarkable plasticity resulting from crosstalk between these compartments. These features endow astrocytes with the ability to sense neuronal activity and integrate and modulate synaptic transmission, revealing them as a crucial element in mechanisms of synaptic transmission and plasticity. Indeed, astrocyte-synapse interactions are complex and dynamic and are required for normal synaptic physiology and plasticity, as well as for the development and refinement of the neuronal circuits. Although much progress has recently been made in our understanding of the cellular and molecular mechanisms that underlie neuronal-glial interactions, ongoing research is adding new information and new questions regarding the role of glial cells in CNS development, function, and disease. In this special issue we collected research and review articles that focus on glia-synapse interactions with a particular focus on astrocytes. An important role played by these cells in regulating long-term potentiation (LTP) and memory mechanisms in hippocampus is reviewed by Y. Ota et al. In the review “The role of astrocytes in the regulation of synaptic plasticity and memory formation,” the authors presented a summary of receptors and signaling molecules implicated in LTP. In addition, they propose an integrative model describing how astrocytes may modulate LTP at the postsynaptic site. Supported by a growing number of studies, their model confirms the involvement of the glutamatergic, cholinergic, and purinergic pathways in the neuron-astrocyte interactions taking place during synaptic plasticity. Moreover, this cellular interplay implicates also ephrin signaling and cytokines. Finally, Y. Ota and coauthors discuss the central role played by astrocytic calcium and associated gliotransmitters in hippocampal-dependent memory. Although the primary function of astrocytes is to take up glutamate to prevent excitotoxicity, astrocytes are also able to release this neurotransmitter. This is generally well accepted even if the mechanisms of release remain uncertain. In this special issue, a novel mechanism of glutamate release from astrocytes was studied by C. Cali et al. in the manuscript “G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.” They show the role of the proinflammatory mediator prostaglandin E2 (PGE2) in glutamate exocytosis from astrocytes in the intact brain. Inhibition of cyclooxygenase pathway caused a significant reduction in the total number of fusion events of VGLUT1-positive glutamate containing vesicles in astrocytes induced by activation of purinergic and glutamatergic receptors. Prostaglandin-mediated signaling is implicated in the later, slower phase of glutamate release and requires autocrine/paracrine action of PGE2, suggesting a physiological role for this mediator in intercellular communication, in addition to its known role in inflammatory reactions in the brain. Gliotransmitters glutamate and D-serine have been shown to modulate NMDA receptors (NMDAR) at extrasynaptic sites, revealing neuronal NMDAR as active components of glia to neuron communication. In the review “GluN3A: an NMDA receptor subunit with exquisite properties and functions,” L. A. Kehoe et al. discuss recent data on the GluN3A subunit which provides “nonconventional” properties to NMDA receptors. Expression of this subunit in early development helps to shape neuronal networks, but it may also be implicated in different neuropathologies. Interestingly, the presence of GluN3 subunit on perisynaptic astrocytic processes suggests its possible involvement in neuron-glia interactions, although more research is required to elucidate this question. Another mechanism used by astrocytes to regulate intercellular interactions in the CNS is through secretion of matricellular proteins. These proteins are nonstructural molecules that regulate the extracellular matrix and cell-cell interactions. In the paper “Astrocyte-secreted matricellular proteins in CNS remodelling during development and disease,” E. V. Jones and D. S. Bouvier review the roles of matricellular proteins secreted from developing and reactive astrocytes in CNS development, injury, and disease and discuss their potential as therapeutic targets. In the paper “Astrocyte-synapse structural plasticity,” Y. Bernardinelli et al. review the data on plasticity of perisynaptic astrocytic processes (PAPs). The authors discuss electron and optical microscopy data showing the distribution of fine astrocytic processes around synapses and overview growing evidence on PAP structural plasticity. Although the exact mechanisms and roles of this type of astrocytic plasticity are still not clear, recent data has revealed a requirement for neuronal activity and suggests that PAPs may be implicated in the structural support and plasticity of a synapse, control of neurotransmission and intersynaptic crosstalk, energy supply, maintenance of extracellular homeostasis, and integration of synaptic signals. The study of glial structural plasticity (such as PAPs) requires precise quantification of fine processes and their motility. This challenging task is addressed in the paper “Improved method for the quantification of motility in glia and other morphologically complex cells” by M. Sild et al. who propose and describe in detail a new approach to calculate a motility index for cells with complex, dynamic morphologies. In this special issue, only some aspects out of a broad range of topics on synapse-glia interactions are highlighted and discussed. Despite great progress made recently in our understanding of glia and their role in the CNS, there is still a long road ahead. We hope that the data presented in this issue will help the future research in this quickly growing and important field.

Summary The development of neuronal polarity is essential for the determination of neuron connectivity and for correct brain function. The c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) is highly expressed in neurons and has previously been characterized as a regulator of JNK signaling. JIP1 has been shown to localize to neurites in various neuronal models, but the functional significance of this localization is not fully understood [1–4]. JIP1 is also a cargo of the motor protein kinesin-1, which is important for axonal transport [2, 4]. Here we demonstrate that before primary cortical neurons become polarized, JIP1 specifically localizes to a single neurite and that after axonal specification, it accumulates in the emerging axon. JIP1 is necessary for normal axonal development and promotes axonal growth dependent upon its binding to kinesin-1 and via a newly described interaction with the c-Abl tyrosine kinase. JIP1 associates with and is phosphorylated by c-Abl, and the mutation of the c-Abl phosphorylation site on JIP1 abrogates its ability to promote axonal growth. JIP1 is therefore an important regulator of axonal development and is a key target of c-Abl-dependent pathways that control axonal growth.

Background Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Methods Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Results Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Conclusions Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material. Electronic supplementary material The online version of this article (doi:10.1186/s12907-016-0039-3) contains supplementary material, which is available to authorized users.