The sixth “Melanoma Bridge Meeting” took place in Naples, Italy, December 1st–4th, 2015. The four sessions at this meeting were focused on: (1) molecular and immune advances; (2) combination therapies; (3) news in immunotherapy; and 4) tumor microenvironment and biomarkers. Recent advances in tumor biology and immunology has led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS) of cancer patients. Immunotherapies in particular have emerged as highly successful approaches to treat patients with cancer including melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC), bladder cancer, and Hodgkin’s disease. Specifically, many clinical successes have been using checkpoint receptor blockade, including T cell inhibitory receptors such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and the programmed cell death-1 (PD-1) and its ligand PD-L1. Despite demonstrated successes, responses to immunotherapy interventions occur only in a minority of patients. Attempts are being made to improve responses to immunotherapy by developing biomarkers. Optimizing biomarkers for immunotherapy could help properly select patients for treatment and help to monitor response, progression and resistance that are critical challenges for the immuno-oncology (IO) field. Importantly, biomarkers could help to design rational combination therapies. In addition, biomarkers may help to define mechanism of action of different agents, dose selection and to sequence drug combinations. However, biomarkers and assays development to guide cancer immunotherapy is highly challenging for several reasons: (i) multiplicity of immunotherapy agents with different mechanisms of action including immunotherapies that target activating and inhibitory T cell receptors ; adoptive T cell therapies that include tissue infiltrating lymphocytes (TILs), chimeric antigen receptors (CARs), and T cell receptor (TCR) modified T cells; (ii) tumor heterogeneity including changes in antigenic profiles over time and location in individual patient; and (iii) a variety of immune-suppressive mechanisms in the tumor microenvironment (TME) including T regulatory cells (Treg), myeloid derived suppressor cells (MDSC) and immunosuppressive cytokines. In addition, complex interaction of tumor-immune system further increases the level of difficulties in the process of biomarkers development and their validation for clinical use. Recent clinical trial results have highlighted the potential for combination therapies that include immunomodulating agents such as anti-PD-1 and anti-CTLA-4. Agents targeting other immune inhibitory  or immune stimulating  receptors on T cells and other approaches such as adoptive cell transfer are tested for clinical efficacy in melanoma as well. These agents are also being tested in combination with targeted therapies to improve upon shorter-term responses thus far seen with targeted therapy. Various locoregional interventions that demonstrate promising results in treatment of advanced melanoma are also integrated with immunotherapy agents and the combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for melanoma patients’ population. This meeting’s specific focus was on advances in immunotherapy and combination therapy for melanoma. The importance of understanding of melanoma genomic background for development of novel therapies and biomarkers for clinical application to predict the treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into personalized-medicine approach for treatment of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma. We also discussed the requirements for pre-analytical and analytical as well as clinical validation process as applied to biomarkers for cancer immunotherapy. The concept of the fit-for-purpose marker validation has been introduced to address the challenges and strategies for analytical and clinical validation design for specific assays. The Cancer Genome Atlas (TCGA) identified four genetically defined subtypes of cutaneous melanoma: BRAF mutant, RAS mutant, NF1 mutant, and Triple Wild-Type. Mutations in each of the driver genes , contribute to deregulation of the mitogen activating protein kinase (MAPK/ERK) pathway, leading to uncontrolled cell growth. The most common subtype found was the BRAF subtype with 52% of cutaneous melanoma tumors harboring BRAF somatic mutations. Additional frequently affected molecular pathways identified through the TCGA analysis include the PI3K/AKT/mTOR , cell cycle regulators , P53 , and epigenetic regulation  pathways  is a TNF family member that interacts with lymphotoxin receptor (LTR)3 and herpesvirus entry mediator (HVEM) expressed on stromal cells and T cells, respectively, and functions in T-cell responses. LIGHT systems also plays an important role in regulating expression of genes crucial for innate and adaptive defenses to pathogens and may contribute to immune tolerance in control of autoimmune diseases. LIGHT exhibits potent, CD28-independent costimulatory activity for T cell priming and expansion leading to enhanced T cell immunity against tumors by increasing tumor T cell infiltration and/or increased autoimmunity . TargetiV600E melanoma cells upon treatment with BRAF inhibitors, and with chemotherapeutic agents. Furthermore the mAb W9 defined Grp94 epitope has a restricted distribution in normal tissues.It is generally assumed that molecules have to be expressed on plasma cell membrane in order to be recognized by the corresponding antibodies in viable cells. This assumption has excluded the use of intracellular molecules  as potential targets of antibody-based immunotherapy for the treatment of malignant diseases. This dogma has been challenged, since there is growing evidence that intracellular molecules may migrate to the plasma cell membrane in malignant cells. By panning a human phage display antibody library with a cultured human melanoma cell line we have isolated the single chain Fv(scFv) W9 which recognizes an extracellular epitope of the intracellular chaperone glucose-regulated protein of 94,000 daltons which is a member of the heat shock protein 90 family (Grp94). This molecule, which is a member of the heat shock protein 90 family plays an important role in the biology of malignant cells because of its asssociation with components of signaling pathways involved in cell proliferation, survival and migration . The mAbTherefore, Grp94 represents an attractive target for antibody-based immunotherapy of various types of solid tumors. The in vitro and in vivo anti-tumor activity of mAb W9 was investigated in a malignant melanoma model. The results indicate that mAb W9 inhibits the growth of melanoma cells. The antibody induces apoptosis as well as inhibits several signaling pathways . The anti-tumor activity of mAb W9 is enhanced by the small molecule LDE225, an inhibitor of the sonic hedgehog homolog (SHH) pathway. Furthermore, mAb W9 delays the development of BRAF inhibitor resistance in melanoma cells with mutant BRAF . Lastly,Intralesional therapy is a promising approach in melanoma treatment; especially for cutaneous metastases of metastatic melanoma that constitute a major clinical problem and that are accessible to injection in a high percentage of patients. The aim of this strategy is not only local control of melanoma, but potentially developing a systemic effect by stimulating an immune response after tumor injection.A variety of oncolytic viruses are being tested in clinical trials including adenovirus, vaccinia, herpes, reovirus, Seneca Valley virus and coxsackievirus . LocoregNDV has a potential as anti-cancer agent because it readily infects the majority of cancer cells because of ubiquitous expression of the viral receptor  and it is strong inducer of type I interferon and dendritic cells maturation. Clinical trials with systemically-administered NDV in humans demonstrated safety and durable clinical benefit in different cancer types. The durability of the clinical benefit suggested the strong immune component responsible for the treatment efficacy. Furthermore, NDV infection in animal model upregulated MHC and co-stimulatory molecules on the surface of tumor cells and delayed distant B16-F10 tumor growth, although only few complete regressions were observed. On the contrary, combination therapy of NDV transfection and CTLA-4 blockade lead to rejection of the locally administered with the virus and distant B16-F10 tumors and long-term survival in mice. In addition, the treatment induced inflammatory responses in distant tumors. Anti-tumor response of NDV and CTLA-4 blockage combination therapy is dependent on CD8 T cells, NK cells, and type I and II interferons .Another unanswered question is whether there is a role for oncolytic virus therapies in patients who fail checkpoint inhibitors treatment. Coxsackievirus A21 (CVA21) is a naturally occurring Picornavirus, which CVA21 displays potent oncolytic activity in both in vitro cancer cells cultures and in vivo xenografts in mouse models of human cancers which exhibit high level of surface ICAM-1 expression. CVA21 rapidly replicates, rupturing the cells to release progeny virus particles and tumour antigens. Progeny virus continues the cycle by infecting and lysis of new cells, whereas tumor antigens activate the immune response . CAV21 hThe main intralesional agent currently in phase III trials is Talimogene laherparepvec (T-VEC) that is a HSV-1-derived oncolytic virus that has both a local effect (tumor cell lysis) and, to a lesser degree, a systemic effect (tumor-specific immune response) –68 T-VECCombining T-VEC that promotes release of tumor-derived antigens with an immune checkpoint inhibitor that improves T cell responses such as ipilimumab significantly enhances efficacy of the combination as compared to either therapy alone. The phase 1b portion of a phase 1b/2 combination study (NCT01740297) completed enrollment and met its primary objective with no dose limiting toxicities and objective response rate (ORR) of 56% . Also thIn conclusion, intralesional approaches may be applicable in melanoma treatment thanks to their local activity and the ability to trigger a systemic immune effect. Combination of locoregional therapy using oncolytic viruses with systemic immunotherapy such as checkpoint therapy appears to be safe and multiple agents and combinations are already in clinic or are in pre- and clinical development. Also, expanding the definition of “injectable lesion” to liver, deep lymph nodes and other locations would increase the efficacy of such therapies. However, further clinical studies and biomarkers are needed for optimal selection of patients for treatment with these agents to combine with other immunotherapy interventions and to improve the outcome of such strategies.Immune checkpoint blockade with agents such as ipilimumab, pembrolizumab, and nivolumab has dramatically changed the outlook for patients with metastatic melanoma. Several studies have investigated the combination of various checkpoint blocking antibodies such as the combination of ipilimumab and nivolumab and the combination of ipilimumab and pembrolizumab. Questions remain about how to best combine or sequence these agents.In a phase 2 study testing the combination of nivolumab + ipilimumab versus ipilimumab as first-line treatment for patients with advanced melanoma, the combination of nivolumab and ipilimumab compared to ipilimumab alone demonstrated a higher objective response rate by RECIST 1.1  and progression free survival . In the The overall survival benefits of the combination compared to single agent PD-1 are not yet known. Nonetheless, the long-term overall survival rates in the phase I study of the combination of nivolumab + ipilimumab are impressive with 68% of patients alive at 3-years. The most important question, however, is whether there will be an overall survival advantage of the combination upfront versus single agent PD-1 first-line with ipilimumab (or the combination) as second-line therapy. Overall survival data from the phase 3 checkmate 067 study will be critical and determining the rate of cross-over to other immune checkpoint blocking antibodies in patients initially treated with single agent PD-1 and ipilimumab will be critical in this analysis.Ideally there would be a biomarker to select the combination for patients who are most likely to benefit. Unfortunately, no biomarker, including PD-L1, is yet ready to be used for selecting patients for combination immunotherapy versus single agent PD-1 or ipilimumab. This remains a highly active area of research.Locoregional interventions can markedly improve overall survival in stage IV melanoma, can contribute to palliative care to improve quality of life or to decrease symptoms as well as it can be combined with other therapy approaches including immunotherapy or target therapy. Numerous technical advances in diagnostic methodologies e.g., intraoperative ultrasound imaging (IOUS), magnetic resonance imaging (MRI), positron emission tomography (PET) and PET-computed tomography (PET/CT) provide 3-D tools for better diagnosis, staging and monitoring of the treatment of cancer including surgery.Despite the major advances offered by new systemic therapies, surgery of stage IV melanoma remains an important therapeutic tool that can be used to rapidly and safely resolve localized disease. The rational for surgical resection as first option in stage IV melanoma is based on several factors. Single lesions are best treated by surgery while studies have that shown complete resection is possible in 25% of stage IV patients (M1a through M1c inclusive) . The surFor example, overall survival for patients with distant metastases (M1a) recurrence treated with surgery and with or without systemic medical therapy (SMT) versus SMT alone was retrospectively compared in the first Multicenter Selective Lymphadenectomy Trial (MSLT-I). Although all patients who underwent surgery had favorable survival , those with M1a metastases did particularly well. These patients had a median survival of greater than 60 months with surgery with or without SMT versus 12.4 months with SMT alone. These outcomes are superior to those on many patients with stage III metastases .Surgery may also have an important role in combination with immunotherapy. Indeed, the removal of lesions that may be resistant to treatment with ipilimumab may improve outcomes for some patients. Pathological evaluation of the excised tissue is important to assess the presence of immune-infiltrate. In several cases, analysis of the excised tissue has revealed the presence of a diffuse immune infiltration which correlated with the outcome of these patients . Also, sSurgery is very useful in well selected patients with stage IV melanoma and is associated with good outcomes and, in particular, combined modality approaches are likely to be most successful. Overall patients’ selection is paramount in order to identify patients most likely to benefit from surgery. Furthermore, clinical and translational studies are required to determine optimal combinations and treatment algorithms, but it appears that surgery should continue to play a prominent role.Adjuvant therapies can have a fundamental role in improving the survival of melanoma patients diagnosed at earlier operable stages but continue to be at a high-risk of death from melanoma relapse (AJCC stages IIB-III). Three major meta-analysis studies of all randomized controlled trials (RCTs) of IFNα have supported its impact on relapse free survival (RFS) and overall survival (OS) of these patients . TherefoThe results of a preplanned interim analysis of the phase III adjuvant bevacizumab study in patients with high risk melanoma at (AVAST-M) trial were reported by Corrie et al. . This trNeoadjuvant therapy has improved the outcome of patients with multiple different solid tumors, including head and neck, breast, bladder, esophageal, and rectal cancers –87. BenePatients with locoregionally advanced but surgically operable melanoma continue to carry a high risk of relapse and death despite the best available standard management approaches. Neoadjuvant studies targeting this patient population tested chemotherapy with temozolomide and biochemotherapy (BCT), in which BCT demonstrated high tumor response rates but was eventually abandoned with the failure of BCT to deliver survival benefits in randomized trials of metastatic disease. Smaller neoadjuvant immunotherapy studies with IFNα and ipilimumab have yielded promising clinical activity and important mechanistic insights and biomarker findings. Newer targeted and immunotherapeutic agents and combinations currently are being translated into the neoadjuvant setting at an accelerated pace and carry significant clinical promise. In drug development, the neoadjuvant approach allows access to blood and tumor tissue before and after initiation of systemic therapy, which allows for the conduct of novel mechanistic and biomarker studies in the circulation and the tumor microenvironment. Such studies may guide drug development and allow for the discovery of predictive biomarkers selected on the basis of their capacity to classify patients according to the degree of benefit from treatment or the risk for significant toxicity.18F]-fludeoxyglucose PET/CT parameters at baseline (T0) and at the first scan after two doses of ipilimumab (T1) were unable to predict the risk of recurrence after surgery (at the significance level of 0.05). The number of lesions at T1 showed a trend towards predicting a higher chance of disease recurrence (p = 0.06). The median RFS was 11 months . Biomarker and mechanistic data nested within this study have supported the immunotherapeutic predictive value of the proinflammatory tumor microenvironment as measured by mRNA expression and CD8 T cell density at the tumor invasive margin [Neoadjuvant ipilimumab was tested in locoregionally advanced melanoma to evaluate safety and to define markers of activity and toxicity in the blood and tumor of patients at baseline and early on-treatment times . Patiente margin . Other se margin . OngoingAmong other ongoing targeted neoadjuvant combination studies are trials with targeted therapy agents vemurafenib/cobimetinib  and dabrafenib/trametinib . However, resistance ultimately develops in the majority of patients with metastatic disease, and several resistance mechanisms have been well characterized. Future studies optimizing MAPK pathway inhibition (targeting ERK and CDK4/6 as monotherapy and in combinations) and combinations that target alternate pathways implicated in mediating resistance , may take advantage of the neoadjuvant approach. Combination studies with immunotherapy  that are underway in patients with metastatic disease also may be transitioned into the neoadjuvant setting, supported by the hypothesis that the high response rates seen with BRAF/MEK inhibitors can be transformed into high durable response rates with immunotherapy , 91.Other approaches for neoadjuvant therapy are intralesional approaches described earlier that have been shown to be relatively safe and well tolerated, with evidence of local and potential bystander/distant antitumor clinical activity that appears to be most promising with T-VEC at this time. This approach may provide a neoadjuvant therapeutic platform that can be combined with other immune-activating agents, including cytokines and checkpoint inhibitors. Combination studies of T-VEC with anti-CTLA4 and anti–PD-1 antibodies are underway in metastatic disease, and at least one neoadjuvant study with T-VEC monotherapy is planned in resectable regionally advanced melanoma (NCT02211131).In conclusion, neoadjuvant therapy has the potential to improve the outcomes—including survival, surgical resectability, local control, and organ preservation—of patients with locoregionally advanced melanoma. Other advantages are the ability to evaluate the clinical and pathologic responses and the potential to identify immunologic and histologic correlates of tumor response. Access to tumor tissue before and after neoadjuvant therapy may allow a better understanding of the antitumor mechanisms of action that may enable more selective application of therapeutic agents to those patients who are more likely to benefit. Neoadjuvant immunotherapy with HDI and ipilimumab have yielded several important findings, and multiple studies involving newer immunotherapeutic and targeted agents and combinations are underway. Neoadjuvant therapy in melanoma continues to be investigational and should only be pursued in the context of a clinical trial .The anti-CTLA-4 ipilimumab is an example of long-term survival benefits with the use of immunooncology compounds. Anti-CTLA-4 has shown to improve survival in melanoma patients and also to improve the long-term survival rate , 94. IndAnti PD-1 agent nivolumab has been shown to improve overall survival and progression-free survival, as compared with dacarbazine, in previously untreated patients who had metastatic melanoma and that was significant in patients without a BRAF mutation . More reMultiple mechanisms of melanoma-induced immune escape contribute to the failure of the spontaneous or vaccine-induced immune T cell responses to promote tumor regression in humans. In particular, a number of inhibitory pathways play a critical role in impeding T cell responses to tumor antigens (TAs), including PD-1, T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) receptor, B- and T-lymphocyte attenuator (BTLA) and T-cell immunoreceptor with Ig and ITIM domains (TIGIT). These inhibitory receptors (IRs) are expressed by TA-specific CD8+ T cells in the tumor microenvironment (TME) while their respective ligands are expressed by antigen-presenting cells (APCs) and tumor cells. In patients with advanced melanoma, TA-specific CD8+ T cells present in the periphery and at tumor site co-express multiple IRs . CirculaAmong IRs, TIGIT appears to represent an interesting target for the next generation of immune checkpoint blockade for several reasons. First, it is highly expressed by the majority of CD8+ TILs together with PD-1. Second, the TIGIT ligands, CD155/PVR, and CD112, are highly expressed in the TME by melanoma cells and APCs. Third, it has been shown that dual PD-1/TIGIT blockade augments the expansion and function of human TA-specific CD8+ T cells in vitro and promotes tumor rejection in animal models . InteresStrategies aimed at changing the tumor microenvironment (TME) include direct injection into tumor, checkpoint inhibition and vaccine/adjuvants. Early phase 2 trial data presented at the 2015 ASCO demonstrated by multi-spectral analysis that Coxsackievirus A21 induces both immune cell infiltration and up-regulation of immune response genes in the micro-environment of melanoma lesions. Similarly, adoptive immunotherapy with PD-1-deficient CD4+ tumor-specific T cells has been shown to augment therapeutic efficacy of tumor-specific CD8 T cells , presumaAlternative approaches to change the TME include cancer vaccines. Short-lived proteins (SLiPs) and defective ribosomal products (DRiPs) are at the center of the MHC class I antigen processing pathway, linking immunosurveillance of viruses and tumors to mechanisms of specialized translation and cellular compartmentalization. Since SLiPs and DRiPs, are thought to represent the majority of epitopes presented by tumor cells and enable the immune system to rapidly detect alterations in cellular gene expression with great sensitivity , they reRecently, this approach was applied in clinical trials for patients with NSCLC and prostate cancer. DPV-001 is an off-the-shelf allogeneic DRibble vaccine developed from two human cancer cell lines ). The vaccine is composed of DC-targeted microvesicles containing natural agonists for TLR 2, 3, 4, 7 and 9, 15 DAMPs and more than 170 proteins overexpressed by the average NSCLC. Preliminary data from a phase II trial of cyclophosphamide with DPV-001 alone or with GM-CSF or imiquimod for adjuvant treatment of definitively treated stage IIIa or IIIb NSCLC showed that DPV-001 induced and boosted broad anti-cancer immunity in every patient . This imT cells engineered to express high affinity T cell receptors or chimeric antigen receptors (CAR) have been successful for treating cancer and hematological malignancies. CD19- and CD22-CAR T cell therapy is currently being used children with acute lymphocytic leukemia (ALL), disialoganglioside GD2-CAR T cell therapy to treat children with osteosarcoma and neuroblastoma and B cell maturation antigen (BCMA)-CAR T cells to treat adults with multiple myeloma. A recent clinical trial of CD19-CAR T cell therapy revealed a 70% clinical complete response rate among twenty pediatric B-Cell ALL patients. Grade 4 cytokine release syndrome occurred in 14% of patients but all toxicities were reversible and prolonged B-cell aplasia did not occur . Althoug6 transduced T cells/kg) [CAR T cells are manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates which are enriched for lymphocytes prior to initiating the T cell culture and gene transfer. Potential causes for T cell expansion failure included, poor quality T cells  and poor quality of the apheresis product (due to inhibition by contaminating cells). A review of 43 CD19-and 11 GD2-CAR T cell manufacturing records at one academic center found that for CD19-CAR T cells the yield of transduced cells was highly variable and 4 out of 28 CD19-CAR T cells products failed to meet transduced T cell dose criteria (1 or 3 × 10ells/kg) . FurtherThese results suggest that some autologous PBMC concentrates collected from patients with ALL, sarcoma or neuroblastoma contain large quantities of monocytic and granulocytic myeloid derived suppressor cells that inhibit T cell expansion. Furthermore, aggressive depletion of PBMCs of monocytes and granulocytes improves T cell expansion.Biomarkers can help with clinical decisions making for example predictive biomarkers to select patients who have a high likelihood of response to immunotherapy drugs. Other categories of biomarkers can serve different role in immune oncology (IO) as: (i) biomarkers before diagnosis that can be used for risk assessment and screening; (ii) at diagnosis biomarkers can assist with staging, grading, and therapy selection; and (iii) biomarkers also can be used to select additional therapy or for rational design of combination therapies or (iv) monitor for recurrent disease .Immuno-therapeutic drug development now requires novel biomarker approaches considering the increasing number of immunotherapy agents available for the treatment of advanced cancers and the percentage of patients not responding to immune checkpoint inhibitors. Drug-specific predictive biomarkers would improve, at baseline for patient selection, benefit/risk and ultimately effectiveness. Also, biomarkers guiding biology-based combinations as well as optimal dose-schedule are still an unmet need.Sources of inter-patient variability in IO include host germline polymorphisms in immune-regulatory genes, somatic alterations in tumor cells and environmental factors. Due to the complexity of the immune response and tumor biology it is unlikely that a predictive biomarker based on a single analyte will be very informative. Integrated model measuring different parameters including host, cancer and microbiota would be needed to predict with high accuracy which patient will benefit from which approach, single agent or combination.24 different immune cell types in PBMC or tissueimmunologic functionstumor specific antigens such as CT antigenshousekeeping genes to facilitate sample to sample normalizationMolecular barcoding is a proprietary (Nanostring) technology platform based on a single molecule fluorescent barcoding that allows for direct, digital, multiplexed measurements of gene expression from low amount of RNA without need for amplification with high precision and sensitivity (<1 copy per cell). It also has been optimized for formalin fixed paraffin embedded (FFPE) tissues/biopsies. Measurements are performed by the nCounter analysis instrument using ad hoc software that provides automatic quality control (QC), normalization and different visualization tools. Application of this technology to immune-oncology has been already demonstrated as (PAM50) gene signature panel showed prognostic value in breast cancer and received clearance by FDA under 510(k) regulation. Pancancer immune profiling panel provides a multiplexed gene expression probes designed to quantitate770 genes which fall under 4 categories:Nanostring platform has been used to study the relationship between immune gene signatures and clinical response to PD-1 blockade with pembrolizumab in patients with advanced solid tumors , 124. ImTwo broad categories of tumor escape based on cellular and molecular characteristics of the tumor microenvironment have been recently suggested. One major subset shows a T cell-inflamed phenotype consisting of infiltrating T cells, a broad chemokine profile and a type I interferon signature indicative of innate immune activation. These tumors appear to resist immune attack through the dominant inhibitory effects of immune system-suppressive pathways. Most immunotherapy responders including those treated with anti-PD1 have this phenotype. The other major phenotype lacks this T cell-inflamed phenotype and appears to resist immune attack through immune system exclusion or ignorance. These two major phenotypes of tumor microenvironment may require distinct immunotherapeutic interventions for maximal therapeutic effect . If checIn order to evaluate the mutational landscape and antigenic repertoire, malignant melanoma samples from TCGA were segregated based on T cell-inflamed gene signature. Expression of differentiation antigens and cancer-germline antigens was found to be comparable in T cell signature-high versus-low patients. Similarly, the overall mutational load (non-synonymous mutations) is comparable in T cell signature-high versus -low samples. Also, T cell signature-high and -low patients have equal patterns of predicted HLA-A0201 binding peptides. On the other hand, a minimal representation of so-called “tetrapeptide” sequences were observed in T cell signature-high versus -low tumors.The second unanswered questions is, if overall antigen density is just as high in non-T cell-inflamed tumors, what is molecular explanation for absence of immune infiltrate ? A modelBifidobacterium spp. correlated with antigen-specific T cell responses. Oral administration of Bifidobacterium mix to tumor-bearing TAC recipients improved tumor-specific immunity and response to αPD-L1 mAb. It is possible to conclude that T cell-inflamed tumor microenvironment may serve as a predictive biomarker for response to immunotherapies and the lack of the T cell-inflamed tumor microenvironment phenotype does not appear to be due to lack of antigens [The importance of commensal bacteria in explaining the different T cell-inflamed versus non-inflamed tumor microenvironments is based on their ability to shape systemic immunity. Jackson (JAX) and Taconic (TAC) mice exhibit robust versus weak anti-tumor immune responses and accumulation of intratumoral T cells. The difference between TAC and JAX mice was transferrable by co-housing or fecal transplant. The administration of JAX feces led to significantly improved tumor control and increased frequency of circulating antigen-specific T cells. The reciprocal transfer had little effect, consistent with cohousing experiments. Finally, the abundance of antigens .Mechanisms of PD-L1 expression may be either innate or adaptive, and the relative contribution of each mechanism varies by tumor type and even within tumor types. In melanoma, the adaptive resistance mechanism predominates . PD-L1 mIn addition, to the different mechanisms underlying PD-L1 expression, some of the variation reported in the predictive value of PD-L1 as a biomarker may be attributable to the different immunohistochemical (IHC) assays used for detection. The Blueprint Project is currently underway to characterize potential differences between the marketed diagnostic assays using the 28–8, 22C3, SP263, and SP142 monoclonal antibodies. When these four antibodies were compared in a laboratory derived test (rather than the marketed diagnostic assays), strong correlations were seen in levels of PD-L1 detection in melanoma samples. Most of the observed variation was attributable to geographic heterogeneity between different tumor sections as opposed to antibody performance. Next steps will likely focus on the reconciliation of the different marketed diagnostic assays amongst each other and with the standardization of evolving laboratory derived tests.Other single markers have been nominated as predictors of response to anti-PD-1/PD-L1 therapies, including CD8 density and mutational load. The TCGA dataset was used to explore the relationship between PD-L1, a cytotoxic gene signature (CYT), mutational load and survival in patients with metastatic melanoma. Increasing values for all three parameters were associated with improved survival. Notably, CYT and PD-L1 expression were highly interdependent, while mutational load was not directly related to the presence of an inflamed tumor phenotype. Future biomarker panels will undoubtedly incorporate multiple parameters assessed in surgical pathology specimens, and studies focused on the integration and prioritization of distinct parameters beyond PD-L1 expression are currently underway.The impact of the advances of the last few years on patients is tremendous. However, responses are heterogeneous and are not always durable. There is a critical need to better understand who will benefit from therapy, and this may best be accomplished through a deep molecular and immune analysis in samples from patients on therapy. In order to gain insight into response and resistance to targeted therapy for melanoma, serial biopsies  were performed in patients on BRAF inhibitors, pre-treatment, on-treatment, and at progression time points. Multiple molecular mechanisms of resistance have been identified. Oncogenic mutations contribute to tumor escape via multiple mechanisms. This is certainly the case in melanoma, where over half of patients have oncogenic mutations in the BRAF gene. Mutations in this gene lead to constitutive signaling though the MAPK pathway, with several deleterious effects, including uncontrolled proliferation, resistance to apoptosis, increased angiogenesis, invasion and metastasis, as well as immune evasion. By blocking oncogenic BRAF it is possible to abrogate the effects, actually making tumors more immunogenic. Immune mechanisms of response and resistance to targeted therapy were also identified, demonstrating that treatment with targeted therapy leads to a more favorable tumor microenvironment with increased melanoma antigens and CD8+ T cells and decreased immunosuppressive cytokines and VEGF. There is also an increase in expression of the immunomodulatory molecule PD-L1, suggesting a possible immune mechanism of resistance to therapy. These favorable immune changes are not likely to be directly related to increased melanoma antigen expression, but to overall changes in the microenvironment . InvestiThe hypothesis that combining targeted therapy and immune checkpoint blockade would enhance responses to therapy was then tested in a murine model and synergy was observed with delayed tumor outgrowth and prolonged survival when mice were treated with BRAF targeted therapy and PD-1 blockade, compared to either therapy alone .Building on this data, the efforts were focused on better understanding responses to immune checkpoint blockade. To do this, a deep tissue-based analysis in longitudinal tumor samples from patients on immune checkpoint blockade  was performed. Then a deep molecular and immune profiling of these tumors was carried out to investigate whether molecular and immune “signatures” exist in pre-treatment, and early on-treatment samples of patients receiving CTLA-4 and PD-1 blockade may be predictive of response. Results demonstrated that immune signatures in early on-treatment tumor biopsies on PD-1 blockade were highly predictive of response . It is hThe rationale of studying myeloid cells and tumor exosomes for assessing and targeting immunosuppression is based on the evidence that tumors, through the release of systemic factors into the blood stream, can influence bone marrow myelopoiesis and promote the release of altered myeloid cells that can then feed the cancer site with immunosuppressive effects. The major population involved in this process is represented by myeloid-derived suppressor cells (MDSC) expressing either granulocytic or monocytic markers in both mice and humans, and exerting a pleiotropic suppression on antitumor T cells and NK cells through complex pathways. MDSC infiltrate tumor site where they can differentiate into tumor associated macrophages (TAM2) and sustain chronic inflammation. The immunosuppressive activity of monocytes from melanoma patients was investigated and CD14+ monocytes were found to exert immunosuppressive activity increased by 96 kDa heat shock protein (HSPPC-96)/GM-CSF vaccine, thus permitting to identify a new subset of MDSC potentially expandable by the administration of GM-CSF—based vaccines in metastatic melanoma patients .Several studies have shown that tumor exosomes deliver information not only between tumor cells but also to other cell types, including different immune cell components. Exosomes are endosomal-derived nanovesicles released by most cells types, including tumor cells, and principally involved in intercellular communication in disease. There is increasing evidence that these extracellular vesicles (EVs), when released by malignant cells, may contribute to cancer progression by influencing different immune cell types, likely blunting specific T cell immunity and skewing innate immune cells toward a pro-tumorigenic phenotype. Because of this function and the ability to deliver molecular signals modulating neo-angiogenesis and stroma remodeling, tumor exosomes are believed to play a role in tumor progression by establishing metastatic niche . ExosomeExosomes might be also involved in the generation of MDSC and in the cell-independent miRNA biogenesis and in the promotion of tumorigenesis. However, the question whether there is any link between tumor exosomes and MDSC and whether exosomes have the ability to mediate immune suppression remains unanswered. Preliminary data demonstrating the existence of this cross-talk  showed that systemic injection of melanoma exosomes leads to MDSC induction in several immune sites including the bone marrow . These dIt is therefore possible that the melanoma lesions release larger vesicles that target CD14+ cells and are responsible for the increased frequency of MDSC. Also, as MDSC specific miRNAs are present at higher levels in plasma of melanoma patients, they can serve as a plasma surrogate of MDSC activity and a potential plasma biomarker of myeloid dysfunctions and as a target for immunomodulation in melanoma patients. These data open new potential routes to the use of MDSC-specific miRNAs as biomarker and therapeutic target in clinical cancer setting.The availability of immune prognostic factors will help to optimize adjuvant therapies in stage III metastatic melanoma. More than 125 immune parameters were investigated by flow cytometry and on paired blood and tumor specimen in 39 stage III melanoma patients. Results demonstrated that T cell exhaustion markers correlate best with prognostic clinical parameters and there is a high correlation between immunophenotypic biomarkers in blood and tumor. High frequencies of CD45RA+ CD4+ and CD3− CD56— tumor-infiltrating lymphocytes appear to be independent prognostic factors of short progression-free survival (PFS). Also, regulatory Treg within tumor-infiltrating lymphocytes are indicative of dismal prognosis, specifically in BRAF mutated melanoma. High natural toxicity receptor NKG2D expression on CD8+ tumor-infiltrating lymphocytes, low level of Treg, and low PD-L1 expression on circulating T cells predicted prolonged overall survival in the multivariate Cox analysis model .Chemokines are critical regulators of leukocyte trafficking and immune functions. The expression patterns of nine homing receptors (CCR/CXCR) in naive and memory CD4+ and CD8+ T lymphocytes in 57 patients with metastatic melanoma (MM) with various metastatic sites were retrospectively analyzed to evaluate whether T cell CCR/CXCR expression correlates with intratumoral accumulation, metastatic progression, and/or overall survival (OS). Expression of homing receptor on lymphocytes strongly correlated with metastatic dissemination. Polyfunctional inflammatory cells such as CD8+ CCR6+ TEM and CD4+ CLA+ CCR10+ TEM that are TH2 cells present in blood are associated with bad prognosis in stage IV metastatic melanoma. Whereas CD8+ CCR9+ TN and CD4+ CXCR3+ TEM cells in blood are associated with more favorable prognosis in stage IV metastatic melanoma. Finally, in mice, CCR9/CCL25 and CXCR3 play complementary roles in natural immunosurveillance of cancer . These dBacteroides species of gut microbiota. Link between CTLA-4 blockade, intestinal damage, dysbiosis, anti-microbial immune responses and anti-cancer immunity and rejection have been demonstrated [The role of intestinal dysbiosis should also be considered in response to immunotherapy in melanoma. The antitumor effects of CTLA-4 blockade depends on distinct nstrated . Enterotnstrated .In 2014 the Society for Immunotherapy of Cancer (SITC) convened an Immune Biomarkers Task Force to review the state of art of existing assays and technologies, identify challenges for further success and provide recommendations for developing biomarkers for immunotherapy. The charge to one of the Working Group was to address pre-analytical and analytical as well as clinical and regulatory aspects of the validation process as applied to biomarkers for cancer immunotherapy.Biomarker assay validation process can be separated into several continuous steps; assessment of basic assay performance ; characterization of the performance of the assay with regard to its intended use ; and validation in clinical trials that ensures that the assay performs robustly according to predefined specifications (fit-for-purpose) and facilitates the establishment of definitive acceptance criteria for clinical use . A typical analytical validation plan involves several steps in which the assay must be optimized for multiple parameters: (i) sample-related , (ii) assay-related ; and (iii) data-related  .An important step in biomarker validation is the evaluation of pre-analytical factors that may affect assay performance due to specimen-related variability. For immunotherapies, there may be a need to monitor ex vivo immune responses in phenotypical or functional assays, which require high-quality samples to ensure reliable analytic output. To ensure that optimal pre-analytic processing regimens are followed, standard operating procedures (SOPs) for controlling specific biomarker development steps are essential.Preanalytical processing of blood and analyte stability can be affected by the sample collection process including anticoagulants used for blood draws, freezing/thawing, time between collection and testing, and storage conditions before processing. Tissue based biomarkers can be measured on freshly frozen (FF) tumor samples or formalin fixed paraffin embedded (FFPE) tissue. FFPE tissue blocks are often available as archival materials as part of bio-banked samples for conventional immunohistochemistry (IHC). IHC is a multi-step diagnostic process that requires standardized conditions for tissue collection, fixation and processing, preparation of the IHC slide, and interpretation of the staining results. IHC based assays remain an important test as companion diagnostics (CDx) to assess antigen expression on diagnostic or surgical specimens for selecting patients and predicting patient-response to specific targeted therapy , and more recently PD-L1 as companion diagnostic for pembrolizumab treatment of NSCLC patients .General guidelines, including analyte stability and laboratory quality control, for performing analysis of tissue-based molecular biomarkers have been published .Next generation sequencing (NGS) tests for tumor mutation analysis, similar to other complex molecular diagnostic tests, should demonstrate adequate analytical and clinical performance .It should follow SOPs that specifically address materials and procedures including patient’s sample type, method of DNA extraction, as well as technical metrics for DNA quantification and quality, which can negatively impact on sensitivity and reproducibility of the assay .Analytical Validation involves confirming that the assay used for the biomarker measurement has established: (i) Accuracy, (ii) Precision, (iii) Analytical sensitivity, (iv) Analytical specificity, (v) Reportable range of test results for the test system, (vi) Reference intervals  with controls and calibrators, (vii) Harmonized analytical performance if the assay is to be performed in multiple laboratories, (viii) Establishment of appropriate quality control measures. Depending on the particular category an assay can require distinct type of validation. Definite quantitative assays make use of calibrators and a regression model to calculate absolute quantitative values for unknown samples. The reference standard must be well defined and should be a representative of the biomarker. This type of assay can be accurate and precise. In relative-quantitative assays, reference calibrators can be used; however, because standards are not fully representative of the biomarker, assay precision can be validated, while the accuracy of the assay can only be estimated. Validation and maintaining reproducibility of multiparametric assays is much more challenging considering the number of analytic variables associated with high content assays , mutational load, TCR sequencing). The capacity of high throughput platforms, such as NanoString or flow based analysis SCNP, enable multi-dimensional analysis of the immune system. Instead of detecting a single or limited number of molecular targets, assays are able to detect tens to hundreds of distinct molecular features simultaneously .The post-analytical phase of biomarker evaluations involves data interpretation of the assay results. Dichotomous variables are relatively straightforward to incorporate into calculations of data sensitivity and specificity. However, most variables in measurement of immune response are continuous, resulting in variability with respect to analytical performance criteria and clinical relevance of the assay, e.g., cut-off points for clinical decision making. Essentially, a cut-off for classifying a sample as positive or negative needs to be determined empirically by correlating results with clinical outcomes in a clinical trial exploring efficacy of a drug.As high throughput methods became widely available there is a need for computational methodologies for interpretation of the complex data for biological and clinical implications. Algorithms to develop multimodal signatures integrating various types of molecular tumor data  with TME factors that reflect the complex biomarker information require the development of multifactorial classifiers/algorithms.The final stage in the development of a biomarker predictive of clinical outcome or response is the assessment of its clinical validity and utility through the application of the analytically validated assay within a clinical trial, with multiple design options depending on the intended use of the test and availability of specimens from previous clinical trials.Clinical validity relates to the observation that the predictive assay reliably divides the patient population(s) of interest into distinct groups with different expected outcomes to a specific treatment. The criteria for validation are defined by the nature of the question that the biomarker is intended to address . A predictive biomarker needs to demonstrate the association with a specific clinical endpoint  in pre-treatment samples from patients that have been treated or exposed to a uniform treatment intervention. For example, the programmed cell death-1 protein ligand (PD-L1) immunohistochemistry (IHC 22C3 pharmDx) test was approved as a CDx to pembrolizumab (anti-PD-1 mAb inhibitor from Merck) as a single agent in second-line non-small cell lung cancer (NSCLC) .There are multiple steps for biomarker clinical validation that encompass important elements of clinical study design and data analysis, including statistical assessments that rely on samples collected from prospective clinical trials or from archived samples that are well annotated with relevant clinical information. Clinical validation i.e., assessment of the test’s correlation with clinical outcome and the amount of improvement in patient outcomes its adoption would entail. The clinical sensitivity and specificity of the assay must be demonstrated through robust receiver operating characteristics (ROC) curves that provide support for the cut points, established using appropriate statistical analysis to identify responders versus non-responders. Statistically, several approaches can be applied for clinical validation of an assay. Internal validation can be achieved by using a study population that reflects the target population in which the test will be used. The study population is divided into two independent groups of specimens. One of these groups is the “training set”, i.e., the set of samples used to identify and characterize the “biomarker”  or to build a mathematical model or algorithm (in case of multi-variate assays). The second sample group is “the validation set” that is used to test whether the external validity of the biomarker/model is maintained in a sample cohort—independent from the training set.There are three basic phase III design options that are frequently considered for assessing the ability of a biomarker to identify a subgroup of patients who will benefit from  a new therapy. These are classified broadly into three categories: (i) The enrichment design; (ii) The stratified design; and (iii) The strategy design.A clinical trial to evaluate the clinical utility of an omics test should be conducted with the same rigor as a clinical trial to evaluate a new therapy. This includes development of a formal protocol clearly detailing pre-specified hypotheses, study methods, and a statistical analysis plan. In some instances, a candidate predictive test for an existing therapy can be evaluated efficiently by using a prospective-retrospective design, in which the test is applied to archived specimens from a completed trial and the results are compared with outcome data that have already been collected. The “retrospective” aspect of this design requires that the assay can in fact be performed reliably on stored specimens .In conclusion, immunotherapies have emerged as the most promising class of drugs to treat patients with cancer with diverse tumor types, however many patients do not respond to these therapies. Therefore, determining which patients derive clinical benefit from immune checkpoint agents remains an important clinical question and efforts to identify predictive markers of response are ongoing. The analytical and clinical validation of predictive biomarkers require appropriate clinical studies in which the evaluation of the clinical utility of the biomarker is a pre-specified endpoint of the study. A variety of study designs have been proposed for this purpose. Although, the randomized biomarker stratified design provides the most rigorous assessment of biomarker clinical utility, other study designs might be acceptable depending on the clinical context.


In this study we compared the effects of two different surgical procedures for closure of adult atrial septal defect (ASD) on postoperative P-wave changes.max, Pmin and P-wave dispersions (Pd).Patients who underwent cardiac surgery for secundum type ASD closure were evaluated retrospectively. Seventy-two patients with primary repair of ASD and 29 patients with pericardial patch plasty repair were compared according to Pmax) and minimum P-wave duration (Pmin) were statistically significant. There was no statistically significant difference between post- and pre-operative Pd values. In the comparison between group 1 and group 2 in terms of postoperative P-wave changes  there was no statistically significant difference.In each group, the increases in postoperative maximum P-wave duration (PComparing patch plasty and primary repair for the surgical closure of ASD in the early to mid-postoperative period, no difference was found and both surgical procedures can be performed in adult ASDs. Atrial septal defect (ASD) is one of the most common congenital heart defects in adulthood. It can be repaired percutaneously or surgically, depending on the defect size. Primary surgical repair or patch plasty (closure with patch) are the two surgical ASD closure techniques used depending on diameter of the defect.max) and increased P dispersion (Pd) may be pioneer indicators of disturbance of the inter-atrial conduction pathway and atrial fibrillation in ASD patients.2Atrial arrhythmias may develop in repaired and unrepaired ASD patients. The inter-atrial conduction pathway may influence this, and postoperative arrhythmias may develop due to increased size of the right atrium in unrepaired patients, or tension in the suture line in the postoperative period.Many different studies have been carried out for P-wave changes following ASD closure surgeries. These studies evaluated the effects of percutaneous or surgical closure techniques on the P wave after defect repair.The study design was approved by the institutional review board. Patients who underwent surgical ostium secundum type ASD closure between the years 2004 and 2014 in the cardiovascular surgery clinic of the GATA Haydarpasa Training Hospital were included in the study. Patients with primum ASD and patients with cardiac pathologies requiring additional surgical treatment were excluded from the study.Twelve-lead surface electrocardiograms were collected from patients’ records who underwent surgical ostium secundum type ASD closure. The 101 patients were divided into two groups  accordinSeventy-two patients without increased atrial stretch and with a small- to medium-sized defect diameter were evaluated as suitable for primary surgical repair (group 1) and they underwent primary surgical repair for ASD closure. Twenty-nine patients with a larger defect diameter and/or patients with accompanying sinus venosus type ASD (group 2) underwent the patch plasty technique for ASD closure. The demographic data of the patients in each group and their pre- and postoperative five- to seven-day and three-month 12-lead surface electrocardiogram P waves were compared.All surgeries were carried out under general anaesthesia. A median sternotomy, mini-thoracotomy and mini-sternotomy were performed in 69, 14 and 18 patients, respectively . CardiopCardiopulmonary bypass was established and following cardioplegic arrrest, 32°C blood–body temperature was provided. Following right atriotomy, the defect was evaluated in the presence of pre-operative echocardiographic findings. In group 1, the defect was closed with a primary continuous suture technique with 4/0 prolene. In group 2, the defect was closed by means of a fresh autologous pericardial patch using a continuous suture technique with 4/0 prolene.Pre-operative, fifth- and seventh-day postoperative and threemonth 12-lead surface electrocardiograms were provided frompatients’ records, which were obtained at a paper speed of 50 mm/s with 1-mV/cm standardisation. Electrocardiograms were scanned for evaluation of the P waves. Electrocardiograms exhibiting P waves at least nine in 12 derivations were analysed for the existing P waves.4max whereas the shortest P wave was Pmin. The difference between Pmax and Pmin was the P-wave dispersion (Pd) (Pd = Pmax – Pmin).Measuring the length of the P waves using Photoshop® (Adobe), the longest P wave was denoted as PU- and Student’s t-tests. A p-value ≤ 0.05 was considered statistically significant.The 5.0 version of the GraphPad Prism program was used for statistical analysis. Data are shown as mean ± standard deviation. Postoperative P-wave changes of the patients in group 1 and 2 were compared with pre-operative values and the differences were evaluated. Continuous variables were compared using Mann–Whitney Pre-operative demographic characteristics of the two groups were similar. Demographic characteristics and transthoracic\ echocardiographic data of our patients are shown in max was significantly increased in the five to seven days postoperatively, and Pmax was still significantly longer three months after the procedure   .max and Pd extension develops as a result of prolongation of the atrial depolarisation time.d and Pmax reflect non-homogeneous and discontinuous sinus stimulation and may be predictors of atrial fibrillation.2The presence of ASD causes volume overload and increased stretch induces right heart dilatation and dysfunction. In these patients, PIn the literature, P-wave changes have been compared in repaired and unrepaired ASDs, or in repaired ASDs by surgical or percutaneous means. There is no study comparing the effects of two different surgical techniques on the P wave. In our study, two different surgical ASD closure techniques were compared in terms of P-wave analyses using primary repair and pericardial patch plasty techniques.The incidence of dysrhythmia increases with increasing age in patients with unrepaired ASD.9max and Pmin values increased but Pd values did not change. Increase in Pmax and Pmin values in the third month may have been related to early remodelling, however because of the unchanged Pd, we believe that the risk of arrhythmia will not increase in the long term.In our study, we compared the effects of two surgical techniques on atrial conduction pathways and remodelling caused by inflammatory fibrosis, by analysis of the P wave. In both primary repair and patch plasty groups, Pd value decreased in patients who had regressed right atrial dilatation. However, Pmax and Pd were longer in patients with persistent atrial dilatation.dvalues in patients with permanent atrial dilatation support the notion that Pdmay be used as a non-invasive parameter for prediction of atrial arrhythmia.In the postoperative period, the Pd values. By contrast, Thilen et al.13Our findings showed that the risk of atrial arrhythmia was similar in both primary closure and patch plasty techniques, since there was no difference between Pmaxvalues had increased, Pd values did not change and there was no arrhythmia. From our results, we believe that in repair of ASD in adults, the Pdvalue could be a more meaningful predictor for the risk of arrhythmia.In our study, we were able to obtain three-month follow-up records of the patients. During the follow up, although Pmax and Pd, thus decreasing the risk for atrial fibrillation in older patients.16The age of the patient at ASD closure may be a risk factor for the development of arrhythmia in the follow-up period.d value did not change after surgical repair and the rate of returning normal atrial size may have been higher since most of our patients were young adults.In our study we investigated 101 patients who had had a diagnosis of ASD. Ninety of them were younger than 25 years and their mean age was 21.9 ± 2.8 years. None of the patients had arrhythmia before the surgery and none had atrial arrhythmia during the three-month follow-up period. In our patients, the Pd was more meaningful in the early period.The ASD closure technique did not affect the P wave in a comparison of different percutaneous techniques, or surgical repair and percutaneous techniques in previous studies.d value or cause changes.We compared primary closure and patch plasty techniques as two different surgical techniques, to analyse the effects on the P wave, and there were no differences between the two surgical techniques. When considering the anatomy of the right atrial electrophysiological conduction pathway, there are no significant conduction pathways into the atrial septum. We believe that different surgical closure techniques do not affect the PIn long-term follow up after ASD closure, increase in Pd values could be a sign of atrial arrhythmia.max and Pd values in the first three months continue in the long term and whether these changes led to arrhythmias in the follow-up period.There are limitations to this study. The majority of patients were young and they were in the second and third decades oftheir lives. After three months’ postoperative period, we had limited access to long-term follow-up records, because they were carried out in their homelands. We need long-term results to assess whether the increased PIn this study we compared patch plasty and primary repair for the surgical closure of ASD in the early to mid-postoperative period. No differences were found between the methods in terms of postoperative P-wave changes, and we concluded that both surgical procedures can be performed in adult ASDs.


N2O administered alone produces EEG spectral characteristics that are distinct from most hypnotics. Furthermore, EEG frequency-derived trends before and after clinically relevant time points vary depending on N2O concentration. Remifentanil typically increases low frequency and decreases high frequency activity in the EEG, but how it influences N2O’s EEG effect is not known. Previous attempts to characterize EEG signals of patients anesthetized with N2O using frequency-derived measures have shown conflicts and inconsistencies. Thus, in addition to determining the spectral characteristics of this unique combination, we also test whether a newly proposed characterization of time-delayed embeddings of the EEG signal tracks loss and recovery of consciousness significantly at clinically relevant time points. We retrospectively investigated the effects of remi and N2O on EEG signals recorded from 32 surgical patients receiving anesthesia for elective abdominal surgeries. Remifentanil and N2O (66%) were co-administered during the procedures. Patients were tested for loss and recovery of response (ROR) to verbal stimuli after induction and upon cessation of anesthesia, respectively. We found that the addition of remifentanil to N2O anesthesia improves the ability of traditional frequency-derived measures, including the Bispectral Index (BIS), to discriminate between loss and ROR. Finally, we found that a novel analysis of EEG using nonlinear dynamics showed more significant differences between states than most spectral measures.Nitrous oxide (N BIS is a spectral measure generated by the BIS brain monitor technology used to monitor anesthetic depth, but does not perform well during N2O anesthesia  who were undergoing anesthesia for elective intraabdominal procedures classified as status 1–3 by the American Society of Anesthesiologists. We retrospectively reviewed the database and analyzed data under a separate Stanford Approved IRB. Patients were not pregnant or breast feeding and were within 35% of ideal body weight. Patients with history of substance abuse, seizures, severe respiratory disease, renal or hepatic dysfunction, congestive heart failure or symptomatic ischemic heart disease were excluded from the study . After LOR, succinylcholine was administered 1 mg/kg intravenous dose prior to intubation. After the surgical procedure was completed and N2O and remi were discontinued, ROR was determined when patients made purposeful response to verbal stimuli.A detailed description of surgical procedures and anesthetic administration have been previously described . Eight patients were excluded because we could not extract consistent artifact-free clips in their electroencephalograms (EEGs) around our time points of interest (discussed below). Patients ranged in age from 28 years old to 75 years old (mean age of 53 years old with a standard deviation of 13).An A-1000 EEG monitor  was attached to the patients using adhesive electrodes prior to starting the anesthetic as per the manufacturer’s instructions. EEGs were recorded at At1  referenced to Fpz Figure  at 128 Hn = 29) for both time periods of interest (22 patients were the same for both LOR and ROR groups). Three of the authors  visually inspected all EEG data for each patient to ensure clips selected for subsequent analysis were free of noise and artifacts.Twenty second artifact-free clips occurring within 2 min before and after LOR and within 2 min before and 4 min after ROR were manually selected for subsequent analysis Figure . A largen = 32) as we wanted to test if our measures were sensitive to the difference between anesthetic states once the patients were already anesthetized. We made comparisons for these deeply anesthetized periods and LOR and ROR events in the same cases.Since we had blood concentrations of remifentanil during the procedure, we took 20 s clips within 2 min prior to the measured sample of the highest concentration of remifentanil in each case to represent a point at which patients were most anesthetized (High-Remi). The average measured remi concentration was 12.7 ± 6.2 ng /ml for these clips (an outlier blood concentration for 1 patient was removed). For this condition, we used cases that had either LOR and ROR clips  and total power from our multitaper spectral analysis. Total power results were converted to decibels. To make our results comparable to previous work, we calculated SEF95 and total power both for the full range of frequencies available given our acquisition system sampling rate and with a band-limited range between 0 Hz and 30 Hz. In addition, we calculated the percentage of total power for individual frequency bands per condition. The percentage of total power was used because previous research , we were able to obtain the BIS and EMGlow values corresponding to the same 20 s clips we used for our analysis. BIS values represent an index provided by the manufacturer that is intended to guide clinicians during anesthesia titration. The EMGlow value is another value generated by the manufacturer that gives the absolute power in the 70–110 Hz range. The manufacturer implies that this represents EMG activity; however, as it is used here  it could also be interpreted as high gamma  of the EEG signal as previously described . Given the well-established differences in LOR and ROR brain states  are from Wilcoxon Signed Rank Tests with Bonferroni correction. We found that full spectrum SEF95 showed highly significant differences between pre- and post- LOR (p < 0.0001). Lesser but still significant differences between pre- and post-ROR , but a significant difference between High-Remi and pre-ROR . In this range, we did not observe a significant difference between post-LOR and High-Remi . We observed a significant increase in the High-Remi to pre-ROR , but not a significant change between pre- and post-ROR .To quantify these observations, we first computed the spectral edge frequency (SEF95) for each of our time points, including the period of highest remifentanil concentration that we considered to be our most deeply anesthetized state (High-Remi). We show our results for our full spectrum in Figure p) are again from post Wilcoxon Signed Rank Tests with Bonferroni corrections. We found a significant difference between pre- and post-LOR full spectrum total power , but High-Remi and pre-ROR were significantly different . In the band-limited (0–30 Hz) total power calculation we observed a significant decrease in total power between pre- and post-LOR , but no significant difference between pre- and post-ROR . We did not observe a significant difference between post-LOR and High-Remi , but did observe a significant difference between High-Remi and post-ROR . We then calculated the percentage of total power in band-limited ranges. We found significant increases in delta activity (0.1–4 Hz) between pre- to post-LOR  of increasing delta pre- to post-ROR , but a significant increase between High-Remi and pre-ROR during recovery from anesthesia . In the theta band (4–9 Hz) we observed no significant changes in the activity except for a significant increase in theta in the pre-ROR clip . We observed a significant decrease in the alpha band (9–12 Hz) between pre- and post-LOR , but a significant increase in alpha between High-Remi and pre-ROR . Beta (15–30 Hz) and gamma (30–60 Hz) were the frequency bands in which we saw significant changes during the pre- and post-LOR period , but did see a significant increase in percentage of beta between pre-ROR and High-Remi . In the gamma band we observed a decrease in the percentage of gamma from pre- to post-LOR .To investigate the specific frequency bands that underlie these differences we calculated and compared the total power between these anesthetic conditions. We show results for LOR and ROR before and after transitions Figure  and repoR Figure . There w Figures . In the p < 0.0001) and again significantly decreased from post-LOR to High-Remi (p < 0.0001). We observed increased BIS values from High-Remi to pre-ROR (p < 0.0001) and again significant increases in BIS values pre- to post-ROR upon recovery (p = 0.0006). This trend in BIS numbers is what is expected from the manufacturer’s manual during a typical timeline for anesthesia.Since EEG data were recorded on an A-1000 EEG monitor we were able to obtain the BIS numbers corresponding to the same 20 s clips that we were analyzing; BIS numbers were averaged over the 20 s. We made the following comparisons to see how well BIS was able to identify differences in anesthetic depth: pre- and post- LOR, post-LOR and High-Remi, High-Remi and pre-ROR, and pre- and post-ROR Figure . We founp < 0.0001), and again from post-LOR to High-Remi . EMGlow values then increased from High-Remi to pre-ROR  and again from pre- to post-ROR .In addition to the BIS value, the A-1000 EEG monitor also outputs a value that it labels EMGlow which is said to be the absolute power in the 70–110 Hz range and related to EMG activity. EMGlow is calculated from the same EEG scalp electrodes used to record the EEG activity. We extracted these numbers as well from the same time as the 20 s clips and averaged the values. We found EMGlow numbers were significantly different in these comparisons. Values are reported as median  and significance was assessed using Wilcoxon Signed Rank Test with Bonferroni correction. EMGlow values significantly decreased from pre- to post-LOR . However, despite the attractors already having been flattened pre-LOR, we still observed significantly more flattening and more ellipsoidal shapes post-LOR  constructed from time-delayed embeddings of the 20 s EEG clips at 5 ms delays  and a further significant decrease in between post-LOR to High-Remi . Additionally, we observed a significant increase between High-Remi and pre-ROR  as well as a further significant increase from pre- to post-ROR . Clearly the attractors distinguished all of the different anesthetic states reliably.As described above, we quantified this shape change by fitting the 3D attractors with ellipsoids and measuring the ratio between the lengths of the minor and major axes (radius ratio). We observed a significant decrease in the radius ratio between pre- to post-LOR . We characterized the spectral changes that occurred during these periods as well as the discriminability of the spectral edge frequency, BIS values, and our new radius ratio analysis that quantifies the shape changes we observe in point clouds constructed from time-delay embeddings of the EEG signal.We analyzed effects of the unique combination of Nb50) with 66% N2O  min prior to ROR and in the other two-thirds of patients remi was discontinued 3.4 (±2) min after ROR. This means that this significant increase in theta activity prior to recovery does not appear to be affected by the presence of remi even after the patient has recovered their response to verbal commands.Previous EEG examinations of NWe expected to see a significant decrease in delta and increase in beta power between pre- and post-ROR given the significant fluctuations in the opposite directions that we observed during pre- and post-LOR. We can formulate several hypotheses as to why we did not observe this in our data. The first is the confluence of effects from the agents administered. We observe increases in the percentage of delta activity at LOR that was maintained at High-Remi, similar to what has been described previously about remi influences on EEG activity  was given an average of 26 (±13) min prior to the onset of the anesthesia. The influence of midazolam on the EEG activity (namely increases in beta power) at the dose administered can last ~1–2 h after administration; however, the increase in beta power caused by midazolam rapidly declines after administration (Veselis et al., 2O, reported that the BIS number does not change with LOR due to the maintenance of high frequency content in the EEG post anesthetic induction (Yamamura et al., 2O and remifentanil together. This is likely due to the effects of remifentanil increasing the lower frequency power and, more importantly, decreasing the power in higher frequency bands (Lozito et al., 2O administration alone (Rampil et al., 2O administration may also improve the ability of other monitoring algorithms, such as entropy, to detect LOR and ROR in surgical patients (Anderson and Jakobsson, Previous attempts to use the BIS to determine depth of anesthesia, using N2O administration increasing EMGlow (Rampil et al., One issue with our data, and indeed of any EEG measurement, is muscle contamination. Visually inspected clips were manually selected to rule out any contamination from large muscle artifacts such as eye blinks, teeth-chattering, and eye rolls. However, muscle activity is a pernicious and precarious issue in EEG as electrical activity generated from facial and neck muscles can extend from the 20 Hz to 200 Hz frequency ranges (Shackman et al., 2O and remi in human surgical patients. For this study, we wanted to determine whether shape changes could be observed with time-delayed embeddings. The nuances of our frequency measures indicate that it would be useful for future work to test the contribution of different frequency bands to the embedding structure. We are confident that the results are not due to muscle artifact as similar changes have been observed in intracranial recordings from rats (MacIver and Bland, Previous work has demonstrated that 3D time-delayed embeddings of the EEG signal transition from spherical balls to flatter shapes as patients lose consciousness during surgical procedures, and return to spherical configurations upon recovery (Watt and Hameroff, 2O anesthesia improves performance of spectral and BIS values to distinguish anesthetic states when compared to previous work. We also observed similar shape changes in three-dimensional embeddings of EEG activity; whereby, these embeddings change from spheres during awake states to elongated ellipsoids during anesthetized states. We introduced a novel characterization of these shape changes, the radius ratio, which significantly distinguished between anesthetic states better than most spectral measures and as well as the BIS.We have shown that addition of remifentanil to NMBM, DRD and SLE designed the work. DRD acquired the data. CMD organized the anesthesia records for analysis. SLE, MBM and NTO analyzed the data. SLE wrote the manuscript. All authors contributed to the intellectual content with rounds of review. All authors approved the final version of the manuscript for publication.DRD is a consultant for Masimo Inc. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SH and handling Editor declared their shared affiliation.


Papillary thyroid carcinoma (PTC) is the most frequent type of malignant thyroid tumor. Several lncRNA signatures have been established for prognosis prediction in some cancers. However, the prognostic value of lncRNAs has not been investigated in PTC yet. In this study, we performed genome-wide analysis of lncRNA expression profiles in a large cohort of PTC patients from The Cancer Genome Atlas and identified 111 differentially expressed lncRNAs between tumor and non-tumor samples and between recurrent and recurrence-free samples. From the 111 differentially expressed lncRNAs, four independent lncRNA biomarkers associated with prognosis were identified and were integrated into a four-lncRNA signature which classified the patients of training dataset into the high-risk group and low-risk group with significantly different overall survival . The prognostic value of the four-lncRNA signature was validated in the independent testing dataset. Multivariate analysis indicated that the four-lncRNA signature was an independent prognostic predictor. Moreover, identified four lncRNA biomarkers demonstrates good performance in predicting disease recurrence with AUC of 0.833 using leave one out cross-validation. Our study not only highlighted the potential role for lncRNAs to improve clinical prognosis prediction in patients with PTC and but also provided alternative biomarkers and therapeutic targets for PTC patients. Thyroid cancer is the ninth most common cancer and is the most prevalent cancer among endocrine tumors . The incet al. also identified thousands of significantly differentially expressed lncRNAs in PTC relative to noncancerous thyroid tissue [Recent advancements in the genome and transcriptome analysis revealed that protein-coding genes only comprised ∼2% of the human genome sequences, whereas a substantial fraction of the human genome can be transcribed into non-coding RNAs (ncRNAs) . Long nod tissue . These sThe purpose of this study is to identify novel lncRNA biomarkers closely associated with the survival and recurrence of PTC patients from The Cancer Genome Atlas (TCGA), and to assess the prognostic value of novel lncRNA biomarkers for predicting survival and recurrence in patients with PTC.We first performed differentially expressed analysis to identify potential lncRNAs associated with prognosis in PTC. Compared with the normal samples, we found that there were 77 differentially expressed lncRNAs . Among them, 22 lncRNAs were up-regulated and 55 lncRNAs were down-regulated in PTC. Hierarchical clustering of 246 PTC patients in the entire TCGA dataset according to the expression patterns of these 77 differentially expressed lncRNAs suggested that these up-regulated and down-regulated lncRNAs could effectively discriminate tumor and non-tumor samples . As showed in Figure RP11-536N17.1, RP11-508M8.1, AC026150.8 and CTD-2139B15.2) as independent lncRNA biomarkers that were correlated with prognosis by multivariate Cox proportional hazards regression analysis (P<0.05). Table These differentially expressed lncRNAs were subjected to univariate Cox proportional hazard regression analysis to identify lncRNAs, whose expression level could be significantly correlated with patient survival. We identified a set of six lncRNAs (p<0.05) that was significantly correlated with patient survival. We then selected four lncRNAs (RP11-536N17.1) +  +  + ). Using the median lncRNA signature score as risk cutoff value, the lncRNA signature classified 246 PCT patients of training dataset into the high-risk group (n=123) and low-risk group (n=123) with significantly different survival time   Figure . FurtherTo confirm the survival prediction power of the lncRNA signature, we validated the predictive ability of the lncRNA signature in the testing dataset. By using the same risk score formula and risk cutoff value derived from the training dataset, the patients of the testing dataset were classified as high-risk n=128) or low-risk (n=118) according to the lncRNA signature. Patients in the high-risk group had significantly shorter median survival time than those in the low-risk group  Figure . In a un8 or low-To assess whether the prognostic ability of the lncRNA signature in survival prediction is independent of other clinical factors of the patients with PTC, we performed multivariate Cox regression analysis including lncRNA signature, age, stage and gender as covariables. The results from the training dataset showed that the lncRNA signature  and gender  was significantly correlated with overall survival of the patients with PTC  procedure. Results of LOOCV procedure showed that the four-lncRNA classifier achieved an overall predictive accuracy of 91.7% with a sensitivity of 87.5% and a specificity of 92.3%. The discriminatory performance of the four-lncRNA classifier, evaluated by calculating the AUC and DOR, revealed that the AUC was 0.833  is the most common form of well-differentiated thyroid cancer, and the most common form of thyroid cancer. Although the mean survival rate after 10 years is higher than 90% and most of PTC patients have favorable prognosis especially among patients up to 45 years of age, approximately 5∼20% of patients faced the risk of disease recurrence and suffer aggressive and lethal outcomes. Hence, the use of prognostic biomarkers has become increasingly relevant with the potential goal of improving PTC patient management and making tailored therapeutic decisions. Previous studies establishing the utility of molecular biomarkers in the management of PTC have focused on protein-coding genes or miRNAs in order to identify high-risk patients for more aggressive management. In recent years, increasing evidence have shown that lncRNAs function as a big player in gene regulation and tend to be expressed in a more cell- and tissue-type manner than protein-coding genes, demonstrating the intrinsic advantages of lncRNAs as diagnostic and prognostic biomarkers in cancers . TherefoRP11-536N17.1, RP11-508M8.1, AC026150.8 and CTD-2139B15.2) was constructed to form lncRNA signature which was closely related to survival of patients with PTC. The prognostic value of this signature was verified in the training dataset of 246 PTC patients and in an independent testing dataset of 246 PTC patients. Furthermore, the prognostic value of the four-lncRNA signature was independent of clinical factors by performing multivariable Cox regression analysis. We further evaluated the clinical relevance of the four independent lncRNA biomarkers as a predictor in the risk evaluation of recurrence using SVM algorithm. Our findings demonstrated the feasibility and potential power of the four independent lncRNA biomarkers in predicting the risk of recurrence.Several groups have identified thousands of significantly differentially expressed lncRNAs in PTC relative to noncancerous thyroid tissue. In one lncRNA profiling study in three pairs of PTC and adjacent noncancerous samples, 675 lncRNAs were found to be differentially expressed and 8 randomly selected differentially expressed lncRNAs were validated using quantitative polymerase chain reaction (qPCR) . AnotherSome limitations should be acknowledged for this study. First, roles of four lncRNA biomarkers in PTC pathogenesis are presently unclear. Additional experimental investigations of these four lncRNAs should be conducted to improve our understanding of the biological behavior of three four lncRNAs in PTC. Second, PTC patients used in this study were obtained from a single source (TCGA) and randomly assigned to training and testing datasets for the discovery and validation. So, prognostic lncRNA biomarkers identified here should be validated in independent external PTC datasets. Despite these drawbacks, our study highlighted the potential role for lncRNAs to improve clinical prognosis prediction in patients with PTC. As pointed and discussed in some recent papers , the webRP11-536N17.1, RP11-508M8.1, AC026150.8 and CTD-2139B15.2) were identified, which can be used an independent prognostic marker to robustly predict survival and recurrence of patients with PTC. With further independent validation studies in prospective cohorts, these identified lncRNAs might serve as alternative biomarkers and therapeutic targets for PTC patients.In conclusion, the present study performed genome-wide analysis for lncRNA expression in a large cohort of PTC patients from TCGA and showed altered expression patterns between tumor and non-tumor samples and between recurrent samples and recurrence-free samples. A lncRNA signature comprising four lncRNAs (https://cancergenome.nih.gov/). All PTC patients were randomly divided into two distinct sets of equal size: the 246-patient training dataset for discovery purpose and the 246-patient testing dataset for validation purpose. There was no significant difference in clinical and pathological characteristics between patients in the training dataset and those in the testing dataset. Detailed clinical and pathological of patients with PTC used in this study was shown in Table Clinical and pathological data of 496 patients with PTC were retrieved from The Cancer Genome Atlas (TCGA) (http://ibl.mdanderson.org/tanric/_design/basic/download.html) [Genome-wide lncRNA expression data of 496 PTC samples and 59 normal samples with PTC were obtained from the TANRIC database (ad.html) . BrieflyA paired student t-test was used to identify differentially expressed lncRNAs between normal samples and PTC samples or between samples with recurrence and samples without recurrence. LncRNAs with an adjusted P-value <0.05 after Benjamini and Hochberg correction were considered as differentially expressed lncRNAs. Hierarchical clustering of the expression values of differentially expressed lncRNAs was performed with R software using the metric of euclidean distance and complete linkage.Univariate Cox analysis was used to investigate the relationship between the continuous expression level of each lncRNAs and survival. Those lncRNAs with p-value <0.05 were selected as candidate lncRNA biomarkers. Then those candidate lncRNA biomarkers were fitted in the multivariate Cox analysis to identify independent lncRNA biomarkers. Finally, a lncRNA signature was constructed by the linear combination of the expression levels of independent lncRNA biomarkers with the multivariate Cox regression coefficient as the weight. This lncRNA signature could calculate an expression-based risk score for each patient and classify patients into high-risk group and low-risk group using the median risk score from the training datasetKaplan-Meier survival curves and log-rank tests were used to assess the differences in survival time between the high-risk and low-risk patients. Univariate and multivariate analyses with Cox proportional hazards regression for survival were performed on the individual clinical variables with and without the lncRNA signature in the training dataset and testing dataset. Hazard ratios (HR) and 95% confidence intervals (CI) were calculated.For classification of samples with recurrence or samples without recurrence, independent lncRNA biomarkers were integrated to form classifier using support vector machine (SVM) with the sigmoid kernel . An unbi


Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Here, we conducted immunocytochemistry experiments to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. Our data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain.Circadian clocks consist of molecular negative feedback loops that coordinate physiological, neurological, and behavioral variables into “circa” 24-h rhythms. Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged  Temperature was maintained at 25 ± 1°C with a 12:12 h light/dark cycle with fluorescent light of luminous energy of 8 ± 2 μmol m−2s−1. We used mated males in all experiments to minimize differences in lifespan, which may vary with sex and mating status. Males were aged in groups of 50 in polypropylene cages  inverted over 35 mm petri dish  containing 15 mL of diet. Diet dishes were replaced every 2–3 days. Young (day 5) and old (day 55) males expressing nuclear GFP in specific glial cell subtypes were obtained by crossing w;UAS-GFP with nuclear localization signal (Bloomington Drosophila Stock Center stock 4775) males with females carrying GAL4 drivers expressing in the following glia types: perineurial glia, NP6293-GAL4  22 and ZT10 which correspond to high and low levels of PER protein in wild-type flies, respectively . The p- and t-values and the degrees of freedom (df) from these measurements are provided in the results section and in the figure captions.In order to quantify the relative fluorescence of PER signal at ZT10 and ZT22 in each glia subtype, images were reviewed and maximum intensity projections were created using ZEN 2012 software (Zeiss). Due to their location on the outer surface of the adult Drosophila brains . Subperineurial glial cells were visualized via moody-GAL4 driver line .Surface glia consist of two distinct glial subtypes namely perineurial and subperineurial glia. Perineurial glial cells were labeled by crossing UAS-GFP to NP2222-GAL4 or NP577-GAL4 .Cortex glia surround neuronal cell bodies that reside underneath the surface glia. To visualize cortex glia cells, we used two drivers, 0 Figure . Analysimz0709-GAL4 .Neuropil glia consists of two morphologically distinct subtypes, the ensheathing glia and astrocytes. Ensheathing glia were visualized via  Figures . At ZT10 Figures . The intNP6520-GAL4 driver was observed in the medulla segment of the optic lobe  indicating that PER levels in these glial cells are reduced as the function of age similar to other glia types discussed above.Another group of glial cells marked with GFP via  Figures . Based o Figures . Cell nu Figures . Neverthalrm-GAL4 driver  via two independent methods was shown to alter circadian locomotor activity whereas expression of clock-associated kinase CK1ε in astrocytes was sufficient to lengthen the period of PER oscillations (Tso et al., We determined that the astrocyte glia in the central brain in the vicinity of the pacemaker neurons do not express PER. These results are consistent with a previous report that the astrocytes of the central brain are PER/TIM negative (Suh and Jackson, Drosophila glia. In the lamina of the fly visual system, glial cells show rhythmic changes in volume coordinated with the volume changes in the photoreceptor-contacting interneurons (Gorska-Andrzejak, +/K+-ATPase, which is in high abundance in glia and its rhythmic expression is per-dependent, as per01 mutants lack rhythmic expression of this subunit (Gorska-Andrzejak et al., Gclc, a rhythmically expressed component of the rate-limiting enzyme in glutathione synthesis (Chow et al., cycle via loco-GAL4>cycRNAi was sufficient to significantly decrease rhythmicity of Gclc expression (Chow et al., Little is known about clock-controlled output processes in per or cryptochrome via RNAi failed to alter the free-running behavior of adult Drosophila (Ng et al., Drosophila glial cell functions are required for behavioral rhythmicity (Jackson, The ubiquitous and rhythmic expression of PER in glia reported here opens more questions regarding the functional significance of glial clocks. The role of glia in locomotor activity rhythms is not clear. Pan-glial knockdown of the circadian clock genes Jackson, . FinallyJackson, .per mRNA expression is higher in the heads of old flies compared to young while protein levels are decreased in old (Kuintzle et al., One of the important findings of our study is that, with the exception of the central brain neuropil glia, all PER expressing glial subtypes display significant age-related decline in PER protein levels. These data are consistent with the age-related decrease in PER protein reported by Western blot in whole heads (Luo et al., Our detailed analysis of PER expression suggests that circadian clock may function in several glial subtypes. These data should facilitate future functional analysis of glial circadian clocks and their roles in homeostasis of the nervous system. The age-related decline in PER protein expression in various glial subtypes may provide new ways of investigating the physiological processes that decline with age.This study was conceived, analyzed, and written by DL and JG. All experiments were performed by DL.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.